Mario H. Skiadopoulos Presentation on "Evaluation of the Antibody Threshold of Protection Conferred by a NextGeneration Anthrax Vaccine Candidate Adjuvanted with the Immunostimulatory CPG 7909 TLR9 Agonist" at Biology of Anthrax, Tampa 2016

1. Pre-Exposure Anthrax
Prophylaxis Rabbit Studies
refine the TNA Threshold
of Protection for the
BioThrax Vaccine
The Biology of Anthrax Conference
15 November – 18 November 2016
Mario H. Skiadopoulos, PhD
Biodefense Division, Emergent BioSolutions

2. Pre-Exposure Anthrax Prophylaxis Rabbit
Studies Refine the TNA Threshold of
Protection for BioThrax® (Anthrax Vaccine
Adsorbed) (AVA)
 BioThrax licensure for post-exposure prophylaxis
against anthrax
 Licensure under the FDA Animal Rule pathway
 Logistic regression for estimating probability of
survival
 Factors that can affect the logistic regression and
TNA threshold estimation
2
Anthrax
Vaccine

3. 3
BioThrax vaccine is FDA-approved for the
Prevention of Anthrax
 Licensure in 1970 based partly on field efficacy
trial (Brachman et al., 1962) and animal studies
 Indicated for active immunization for anthrax:
– IM administration
– Primary series: 0, 1, 6 months
– Boosters: 12, 18 months, and annual thereafter
 Used by DoD for pre-exposure prophylaxis (PrEP)
 Stockpiled for civilian biodefense preparedness for
post-exposure prophylaxis (PEP)
Anthrax
Vaccine

4. BioThrax Licensure
 BioThrax vaccine is a sterile product, made from cell-free filtrates of
cultures of a strain of a non-pathogenic strain of B. anthracis. The vaccine
is prepared from the solution that results after the filtration of the culture
of anthrax bacteria and is adjuvanted with Alhydrogel® adjuvant 2%
(Aluminum hydroxide gel)
 BioThrax vaccine is the only FDA-licensed vaccine available for protection
against anthrax disease. It is indicated for the active immunization of
individuals between the ages of 18 and 65 who are at high risk of exposure
 BioThrax vaccine was licensed in 2015 under the FDA Animal Rule for use
in post-exposure prophylaxis of disease following suspected or confirmed
B. anthracis exposure, when administered in conjunction with
recommended antibacterial drugs
4
Anthrax
Vaccine

5. 5
Licensure Strategy for PEP
 Licensure under Animal Rule:
– Demonstrated immunogenicity and efficacy in animals
– Identified TNA threshold of protection based on TNA titer associated with
protection
– Demonstrated ability of vaccine to generate protective TNA titers in humans
 Non-Clinical Studies:
– Passive transfer studies in rabbits & NHPs  demonstrated that TNA can
confer protection
– Post-exposure studies in rabbits  demonstrated added value over
antibiotics
– Pre-exposure prophylaxis studies in rabbits and NHPs  identified TNA
threshold of protection
Post-Exposure
Prophylaxis

6. Animal Models for Anthrax Vaccines
6
Animal
Models
New Zealand White rabbit
Cynomolgus macaque

7. 7
Animal Efficacy Models Used to Address
the Requirements of the Animal Rule
PrEP (pre-exposure prophylaxis)PEP (post-exposure prophylaxis) Passive transfer
Role of
Model
Demonstrates added value
of vaccine over antibiotics
alone, in a post-exposure
setting
Establishes correlation
between pre-challenge TNA
titer and probability of
survival
Demonstrates that
neutralizing antibody alone
is capable of protection
Value in
Deriving
Correlate of
Protection
Limited:
Due to dynamic nature of
the model
Complicated because
immune response is due to
both vaccination and
infection
Most appropriate:
Immune response is due
to vaccination only
Protection conferred by
circulating antibody and
memory response
Timing of challenge
simulates residual spore
exposure in humans
Limited:
Protection conferred by
circulating antibody only
Overestimates protective
antibody titer vs. active
immunization scenario
Post-Exposure
Prophylaxis

8. 88
Rabbit PreP Study: Survival is Dose-Dependent
Group N Vaccine
Immunization
Schedule
(days)
Challenge
(day)
Survivors
/Total
Percent
Survival
1 24 BioThrax 1:4 0, 28 70 24/24 100
2 24 BioThrax 1:16 0, 28 70 23/24 96
3 24 BioThrax 1:64 0, 28 70 15/24 63
4 24 BioThrax 1:256 0, 28 70 3/24 13
5 12 Alhydrogel 0, 28 70 0/24 0
Emergent Study 646-N107247: Pre-Exposure Immunization and Challenge Study in NZW Rabbits
BioThrax
Rabbit Studies

9. 9
Immunization of Animals with Various Dilutions
of Vaccine Stratifies the Immune Response Prior
to Challenge
0.01
0.1
1
10
100
0 14 28 42 56 70
TNANF50(GMT+95%CI)
Study Day
1:4
1:16
1:64
1:256
Adjuvant
Anthrax
Challenge
BioThrax
Rabbit Studies

10. 10
LLOQ
TNA titers of Survivors and Non-Survivors
Preceding B. anthracis Exposure
BioThrax
Rabbit Studies

11. 11
Pre-Challenge (day 69) TNA Titer
Correlates with Survival in Rabbits
Emergent Study 646-N107247: Pre-Exposure Immunization and Challenge Study in NZW Rabbits
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0.01 0.1 1 10
TNA Titer (NF50)
ProbabilityofSurvival
96 animals included
in logistic
regression analysis
Dashed lines
indicate 95% CI
BioThrax
Rabbit Studies

12. The BioThrax TNA threshold was set using
data from Rabbit Study 646
12
0.56
n = 96
BioThrax
Rabbit Studies
Rabbit pre-exposure prophylaxis study 646 was
used to set the TNA threshold of protection of
BioThrax for PEP
0.56

13. TNA Threshold of Protection for AVA
Immunized NHPs
13
BioThrax
NHP Study
0.290.29

14. 14
BioThrax PEP

15. Rabbit Pre-exposure Prophylaxis
Immunogenicity and Efficacy Studies
Using BioThrax Vaccine
Since the completion of Study 646 in 2007, a substantial body of additional data has been generated
using different BioThrax (AVA) lots in 5 different studies:
15
Study 2396 (Initial B55 POC study)
B12 AVA FAV 373A
Group 5 (1:4) n=24
Group 6 (1:16) n=24
Group 7 (1:64) n=24
Group 8 (1:256) n=24
Total: 96
Study 2453 Pilot Efficacy Study 1:
B12 AVA lot 373A
Group 4 (1:16) n=24
Group 5 (1:32) n=24
Group 6 (1:64) n=16
Total: 64
Study 2454 Pilot Efficacy Study 2:
B12 AVA FAV373A
Group 1 (1:32) n=48
Group 5 (1:16) n=48
Total: 96
Study 2455 Pilot GLP Efficacy Study 3:
B12 AVA lot FAV423
Group 1 (1:16) n=32
Group 2 (1:32) n=32
Total: 64
B12 AVA lot FAV424
Group 3 (1:16) n=32
Group 6 (1:32) n=32
Total: 64
Study 3237 Pivotal, GLP, blinded study
B12 AVA FAV423
Group 1 (1:64) n=16
Group 4 (1:4) n=16
Group 6 (1:16) n=60
Group 7 (1:32) n=60
Total: 152
Additional Rabbit
PrEP Studies

16. GLP and blinded rabbit PrEP study 16
TNA Threshold of Protection for AVA
Immunized Rabbits
BioThrax
TNA Threshold
0.17
n=152

17. 17
TNA Threshold of Protection for AVA from
Selected Rabbit Efficacy Studies
BioThrax
TNA Threshold
0.170.18
n = 152n = 96
Pivotal, blinded GLP studyPilot GLP study

18. 18
o A total of five additional GUP studies were completed between 2012 and 2015 for the B55 program, using AVA
manufactured
in B12
o TNA threshold of protection analysis for each of the five GUP studies indicated that 70% probability of survival was
associated with an NF50 between 0.16 and 0.24
o A meta-analysis of all study data from B12 AVA immunized rabbits indicates that the threshold of protection associated
with a 70% probability of survival is 0.21 NF50
BioThrax
TNA Threshold TNA threshold of protection for AVA
immunized rabbits
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0.01 0.1 1 10 100
TNA (NF50)
ProbabilityofSurvival
646
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0.01 0.1 1 10 100
TNA (NF50)
ProbabilityofSurvival
All AVA(B12) Studies
0.56 0.21
n = 96 n = 632

19. TNA threshold of protection for AVA
immunized rabbits
19
• A TNA titer of 0.56 is associated nearly 100% protection
BioThrax
TNA Threshold
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0.01 0.1 1 10 100
TNA (NF50)
ProbabilityofSurvival
646
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0.01 0.1 1 10 100
TNA (NF50)
ProbabilityofSurvival
All AVA(B12) Studies
0.56 0.56

20. 20
Several factors may have accounted for the differences
observed between the original determination and subsequent
estimations of the TNA threshold of protection
Logistic Regression

21. R a b b it S tu d y 3 2 3 7
TNA(NF50)
N
o
n
-S
u
rv
iv
o
rs
S
u
rv
iv
o
rs
0 .0 1
0 .1
1
1 0
1 0 0
37n 115n
L L O Q
21
Comparison of Pre-challenge TNA titers
for AVA Immunized Survivors vs Non-
Survivors from Two Pivotal GLP Studies
R a b b it S tu d y 6 4 6
TNA(NF50)
N
o
n
-S
u
rv
iv
o
rs
S
u
rv
iv
o
rs
0 .0 1
0 .1
1
1 0
1 0 0
31n 65n
L L O Q
In Study 646, 8 animals with titers above ~ 0.3 did not survive
Very few to no animals with titers above ~ 0.3 died in any of the subsequent GUP studies
1)
BioThrax
TNA Threshold

22. R a b b it S tu d y 3 2 3 7
TNA(NF50)
N
o
n
-S
u
rv
iv
o
rs
S
u
rv
iv
o
rs
0 .0 1
0 .1
1
1 0
1 0 0
37n 115n
L L O Q
22
R a b b it S tu d y 6 4 6
TNA(NF50)
N
o
n
-S
u
rv
iv
o
rs
S
u
rv
iv
o
rs
0 .0 1
0 .1
1
1 0
1 0 0
31n 65n
L L O Q
A ‘gap’ in TNA values between the LLOQ (0.086) and ~ NF50 = ~0.3 (survivors or non-survivors)
may also have contributed to an overestimation of the threshold of protection in Study 646
2)
Comparison of Pre-challenge TNA titers
for AVA Immunized Survivors vs Non-
Survivors from Two Pivotal GLP Studies
BioThrax
TNA Threshold

23. 23
The Slope of the Curve is Affected by the
TNA Values in the Analysis
BioThrax
TNA Threshold
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0.01 0.1 1 10 100
TNA (NF50)
ProbabilityofSurvival
646
In the original Study 646, the combined effect of multiple deaths at
relatively high TNA titers, as well as the relatively few TNA values
between ~LLOQ and ~0.3 had a net effect of decreasing the slope
and increasing the TNA NF50 value associated with 70 % survival
0.56

24. 24
BioThrax
TNA Threshold The Slope of the Curve is Affected by the
TNA Values in the Analysis
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0.01 0.1 1 10 100
TNA (NF50)
ProbabilityofSurvival
3237
In the original Study 646, the combined effect of multiple deaths at
relatively high TNA titers, as well as the relatively few TNA values
between ~LLOQ and ~0.3 had a net effect of decreasing the slope
and increasing the TNA NF50 value associated with 70 % survival
0.17

25. PA-based anthrax vaccines have a unique TNA
threshold of protection depending on the antigen-
adjuvant components
25
Anthrax
Vaccine TNA
Threshold

26. AVA B55 pivotal rabbit study
26
AV7909 pilot GP study 2 rPA 7909 pilot GP study 1
TNA Titers of Survivors and Non-Survivors
Preceding B. anthracis Exposure
Anthrax
Vaccine TNA
Threshold

27. Addition of CpG to BioThrax Vaccine Significantly
Reduces the TNA Threshold Required for
Protection
27
n=138
0.063
Anthrax
Vaccine TNA
Threshold
This threshold has been confirmed in 2 guinea pig and 2 NHP studies, including a total of 307 animals
0.08
Reference Standard BMI526 was used;
RS AVR801 converted TNA value is 0.08 NF50

28. A Recombinant-PA Based Anthrax Vaccine Candidate
Alhydrogel Adjuvant and CpG Has a Significantly
Higher TNA Threshold of Protection
n=111
Reference Standard BMI526 was used; AVR801 converted TNA value is 0.74 NF50
0.94
Anthrax
Vaccine TNA
Threshold

29. Antigen-Adjuvant Combinations Affect the
TNA Threshold Associated with Protection
29
Anthrax
Vaccine TNA
Threshold
AVA AV7909 rPA7909
0.08 0.740.21
NF50 associated with a
70% probability of survival:
AVA B55 pivotal rabbit study AV7909 pilot GP study 2 rPA 7909 pilot GP study 1

30. 30
Anthrax
Vaccine TNA
Threshold
o The antigens and adjuvants used to elicit the immune response can have a direct
impact on the quality of the immune response and affect:
 the circulating TNA level present after immunization
 antibody avidity
 B-cell repertoire
o Study design is critical:
 A pilot and confirmatory study should be conducted
 The immune response must be stratified from very low to high titers:
appropriate vaccine dilutions must be used
 A pilot study can inform which dilutions to use
 An appropriate number of animals must be used in order for the threshold
estimation to be robust
Conclusions

31. Acknowledgments
31
Non-Clinical Development
Jeffry Shearer
Vladimir Savransky
Tanya Nelson
Former members:
Boris Ionin
Nina Malkevich
Regulatory
Brenda Wolling
Former members:
Brett Pleune
Danielle Craig
CDC
Jarad Schiffer for SAS logistic regression program
Clinical Development
Robert Hopkins
Victor Montalvo-Lugo
Battelle Biomedical Research Center
Gloria Sivko
Dan Sanford
Lisa Henning
Greg Stark
Statistics
Na Li
Grace Lin
Yukon Wu
These programs have been funded in part with federal funds from the Biomedical Advanced Research and Development
Authority (BARDA), Department of Health and Human Services, under contracts no. HHSO100200700037C;
HHSO100201000034C; HHSO100201000035C; HHSO100201000059C; National Institute of Allergy and Infectious
Diseases (NIAID) Grant No. 1U01AI082224; U01 AI078169-02; BARDA/NIAID Contract No. HHSN272200800051C;
HHSO100201500004C;Defense Advanced Research Projects Administration (DARPA; Contract No. DAAD1903C0002)
BioThrax® and any and all Emergent BioSolutions Inc. brand, product,
service and feature names, logos and slogans are trademarks or registered
trademarks of Emergent BioSolutions Inc. or its subsidiaries in the United
States or other countries. All rights Reserved.
Alhydrogel® is a trademark of Brenntag Nordic A/S
Public Limited Company. All other brand, product,
service and feature names of trademarks are the
property of their respective owners.