Sue Charlton presents analytical methods for assessing the efficacy of anthrax vaccines. A number of assays have been developed including macrophage cell lysis assays, toxin and antibody ELISAs, toxin neutralization assays, mouse potency tests, and rabbit challenge models. These assays have been used to evaluate traditional and novel anthrax vaccines and generate data to support regulatory submissions. Clinical studies on humans have also evaluated the immune response to booster vaccinations and how it is affected by time since the last booster. The various efficacy tests together provide a toolbox to characterize different anthrax vaccines and their ability to induce a protective immune response.

1. Efficacy Assessment of Anthrax
Vaccines
Sue Charlton

2. Abstract
There are currently two anthrax vaccines licensed for human use; the UK
anthrax vaccine precipitated and the US BioThrax anthrax vaccine
adsorbed. A number of second and third generation vaccines are also under
development.
We have developed a tool box of in vivo and in vitro tests to evaluate the
efficacy of traditional and novel vaccines. The tests include macrophage cell
lysis, antigen and antibody ELISAs for the three toxin components, a FRET
assay for LF activity, lethal toxin neutralisation, mouse potency assays and
rabbit efficacy challenge model. The assays (except for FRET) have been
validated and data generated used to support regulatory submissions.
These tests have been used to characterise a number of vaccines.
2 Efficacy Assessment of Anthrax Vaccines

3. Outline of talk
Anthrax vaccines
Vaccine efficacy
• Efficacy vs potency?
• Efficacy and the product lifecycle
• Issues
Analytical methods to support efficacy testing of anthrax vaccines
Example data
Conclusions
3 Efficacy Assessment of Anthrax Vaccines

4. Anthrax vaccines
Licenced
vaccines
AVP
AVA
Live attenuated
vaccines
rPA based
vaccines
rPA “lite”
rPA “plus”
Others
Novel delivery
mechanisms
DNA vaccines
Capsule based
Spore based
4 Efficacy Assessment of Anthrax Vaccines

5. Vaccine efficacy…..what is it?
“The percentage reduction in disease incidence in a
vaccinated group compared to an unvaccinated group”
“Efficacy is best measured in a double-blind,
randomized, controlled trials”
Potency testing is performed to demonstrate the
capacity of the product to confer protective immunity
5 Efficacy Assessment of Anthrax Vaccines

6. Efficacy testing and the product
(vaccine) lifecycle
6 Efficacy Assessment of Anthrax Vaccines
Phase3
Phase2
Phase1
INDapplication
Pre-clinical
• animal models
• assay
development
• process
formulation
ValidationDiscovery
Discovery (3-5 years) Development (5-10 years)
• Develop manufacturing process
• Formulation / stability
• GMP manufacture
• Regulatory submission
• Approval for sale
• Product Support

7. Issues/Challenges
7 Efficacy Assessment of Anthrax Vaccines
Anthrax vaccines present issues that impact on regulatory compliance
& efficacy testing
• Population not normally exposed to hazard
• Field trials after accidental or hostile exposure not usually feasible
• Unusual route of infection
• Need to respond rapidly
• ‘Normal’ licensure may present unacceptable risk
– Takes too long
– Cannot conduct human challenge/protection studies
– Surrogate markers (correlates of protection)?
• Phase III clinical trials not possible
• Animal rule
• Combination vaccines

8. Analytical Tool Box
Characterisation, Evaluation, Efficacy, Release/Stability
8 Efficacy Assessment of Anthrax Vaccines
• MCLA
• Antigen ELISAs (PA, LF, EF) & MSD
• Functional assays (eg FRET for LF)
Antigen content & activity of vaccines
• Challenge models (Rabbit, NHP)
• Histopathology / bacterial burden / clinical signs
• Non-challenge based potency tests (MPT)
Efficacy
• Antibody ELISA
• LT neutralisation (TNA) – human, mouse, guinea pig,
NHP, rabbit
• Cell mediated immunity (Flow cytometry / RT-PCR)
Immunogenicity

9. Anthrax toxin:
mode of action
9 Efficacy Assessment of Anthrax Vaccines
Image from Qiagen

10. 10
Plated onto flat bottomed,
96-well cell culture plates
(>70% viability) 17-19hr at 37 ºC(+/- 2 ºC),
5% CO2 (+/- 1% CO2)
J774A.1 mouse
macrophage cells
Add MTT (dye internalised by active mitochondria)
3hrs (+/- 10mins) at 37 ºC (+/- 2 ºC), 5% CO2 (+/- 1% CO2)
Add solubilising buffer (cell lysis and solubilisation of MTT)
1hr (+/- 10mins) at 37 ºC (+/- 2 ºC), 5% CO2 (+/- 1% CO2)
Read plates at 570nm
Shake for 30mins
The reference curve is
used as a control to
monitor assay
performance
Dilute 2 fold
Cells >80% confluent
Efficacy Assessment of Anthrax Vaccines
Macrophage Cell LysisAssay
10

11. Dilution
1 10 100 1000 10000
0
0.5
1
1.5
Graph#1
4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2
Plot#1 (ReferenceP1: Dilution vs Values) 0.108 2.54 45 1.36 0.996
Plot#2 (Sample 1: Dilution vs Values) 0.0995 3.72 55.3 1.35 0.995
Plot#3 (Sample 2: Dilution vs Values) 0.114 3.61 70.6 1.35 0.997
Plot#4 (Sample 3: Dilution vs Values) 0.104 3.33 74.7 1.35 0.998
Plot#5 (Sample 4: Dilution vs Values) 0.115 4.07 103 1.32 0.999
__________
Curve Fit Option - Fixed Weight Value
11 Efficacy Assessment of Anthrax Vaccines
MCLA– generation of a reportable value
11

12. Toxin NeutralisationAssay (TNA)
Functional ability of antisera, containing antibodies to anthrax
lethal toxin components (PA and/or LF), to specifically
protect J774A.1 cells against Bacillus anthracis lethal toxin
cytotoxicity
Characterisation of the immune response to anthrax
vaccination – human, mouse, rabbit, NHP and guinea pig
12 Efficacy Assessment of Anthrax Vaccines12

13. Toxin Neutralisation Assay Format
Prepare dilutions of test sera
Add to LT (1µg/ml rPA and 0.2µg/ml rLF) and
pre-incubate (30mins ±5mins)
Add to cells (>80% confluent)
Add MTT (dye internalised by active mitochondria)
Add 100 μl/well solubilising buffer
(cell lysis and solubilisation of MTT)
Read plates at 570nm
Shake for 30mins (±5mins)
17- 20 hrs
37 ºC (±2ºC), 5% CO2 (±1%)
9x104 cells/well
(>70% viability)
J774A.1
Day 1
(DMEM, 10%FBS,
L-Glutamine, Pen/Strep)
1 2 3 4 5 6 7 8 9 10 11 12
A-
H
QC
(1)
T1
(1)
T2
(1)
Ref
(1)
T3
(1)
T4
(1)
T1
(2)
T2
(2)
Ref
(2)
T3
(2)
T4
(2)
QC
(2)
3hrs (±5min) 37 ºC (±2ºC), 5% CO2 (±1%)
1hrs (±5min) 37 ºC (±2ºC), 5% CO2 (±1%)
13 Efficacy Assessment of Anthrax Vaccines

14. TNA– generation of a reportable value
14
Picture of NF50
Dilution
OD
Efficacy Assessment of Anthrax Vaccines

15. Rabbit study data
15 Efficacy Assessment of Anthrax Vaccines
14121086420
100
80
60
40
20
0
Days-to-Death
Percent
*
*
2.09
Median
Table of Statistics
1
2
3
Group
Survival Plot for Days to death
Censoring Column in Censoring
Actuarial Method

16. Mouse potency test (MPT)
16 Efficacy Assessment of Anthrax Vaccines
Immunise
mice
Collect
serum
Analyse in
TNA
Determine
potency
relative to
reference

17. 17 Efficacy Assessment of Anthrax Vaccines
Mouse Potency Test
• GMP compliant, validated relative potency (RP) assay to replace
challenge assays for release and stability testing of Anthrax Vaccines.
• The in vivo phase, where mice are vaccinated on Days 0 and 14 with
the prepared doses and bled on Day 28.
• The in vitro phase, where mouse serum is tested in the mouse Toxin
Neutralisation Assay (mTNA).
• A dose dilution series is used, with 10 CD1 mice per dose for the test
batches and reference batch.
• The dose response of the test batches is compared to the reference
batch generating a relative potency (RP) value.

18. 18 Efficacy Assessment of Anthrax Vaccines
Potency Test output

19. Human clinical study:
Assessment of the effect of priorAVPvaccination on the immune
response toboosterAVPvaccination.
• GroupA– received annual boosters as per schedule
• Group B – delayed booster recipients (not receivedAVPfor at
least 2 years)
• Subjects received booster on day 1
• Blood samples taken on days 1, 8, 15, 29 and 120
• anti-PA, anti-LF, anti-EF IgG
• TNA
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20. Responses: change from baseline
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21. 21 Efficacy Assessment of Anthrax Vaccines
Responses: absolute values

22. TNAresponses – time since last booster
22 Efficacy Assessment of Anthrax Vaccines

23. Conclusions
23 Efficacy Assessment of Anthrax Vaccines
Established GUP & PEP protocols in rabbits and NHPs
• Primary endpoint is survival
• Immune responses (ELISA & TNA)
• Bacterial and spore counts in blood and tissues
• Clinical parameters
• Time to death
An assay “tool box” is required to support efficacy
studies
• Toxin quantitation (ELISA, MSD)
• Toxin activity (MCLA)

24. Acknowledgments
• Bassam Hallis
• Kelly Thomas
• Hannah Cuthbertson
• Emily Hughes
• Lorna McInroy
• Debbie Powell
• Pamela Proud
• Chris Neil
• Kim Steeds
24 Efficacy Assessment of Anthrax Vaccines
• Mary Matheson
• Anna England
• Simon Funnell
• Irene Taylor
• Graham Hatch
• Hugh Dyson (DSTL)