sue charlton presntation on "efficacy assessment of anthrax vaccines"
TRANSCRIPT
Efficacy Assessment of Anthrax Vaccines
Sue Charlton
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.
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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
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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
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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
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Efficacy testing and the product (vaccine) lifecycle
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Phase 3
Phase 2
Phase 1
IND
application
Pre-clinical • animal models • assay development • process formulation
Validation Discovery
Discovery (3-5 years) Development (5-10 years)
• Develop manufacturing process • Formulation / stability
• GMP manufacture
• Regulatory submission • Approval for sale • Product Support
Issues/Challenges
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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
Analytical Tool Box Characterisation, Evaluation, Efficacy, Release/Stability
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• 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
Anthrax toxin: mode of action
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Image from Qiagen
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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 Lysis Assay
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Dilution
1 10 100 1000 100000
0.5
1
1.5Graph#1
4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2Plot#1 (ReferenceP1: Dilution vs Values) 0.108 2.54 45 1.36 0.996Plot#2 (Sample 1: Dilution vs Values) 0.0995 3.72 55.3 1.35 0.995Plot#3 (Sample 2: Dilution vs Values) 0.114 3.61 70.6 1.35 0.997Plot#4 (Sample 3: Dilution vs Values) 0.104 3.33 74.7 1.35 0.998Plot#5 (Sample 4: Dilution vs Values) 0.115 4.07 103 1.32 0.999
__________Curve Fit Option - Fixed Weight Value
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MCLA – generation of a reportable value
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Toxin Neutralisation Assay (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
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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%)
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Efficacy Assessment of Anthrax Vaccines
TNA – generation of a reportable value
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Picture of NF50
Dilution
OD
Efficacy Assessment of Anthrax Vaccines
Rabbit study data
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14121086420
100
80
60
40
20
0
Days-to-Death
Perc
ent *
*2.09
MedianTable of Statistics
123
Group
Survival Plot for Days to death
Censoring Column in CensoringActuarial Method
Mouse potency test (MPT)
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Immunise mice
Collect serum
Analyse in TNA
Determine potency
relative to reference
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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.
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Potency Test output
Human clinical study: Assessment of the effect of prior AVP vaccination on the immune response to booster AVP vaccination.
• Group A – received annual boosters as per schedule
• Group B – delayed booster recipients (not received AVP for 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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Responses: change from baseline
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Responses: absolute values
TNA responses – time since last booster
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Conclusions
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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)
Acknowledgments
• Bassam Hallis
• Kelly Thomas
• Hannah Cuthbertson
• Emily Hughes
• Lorna McInroy
• Debbie Powell
• Pamela Proud
• Chris Neil
• Kim Steeds
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• Mary Matheson
• Anna England
• Simon Funnell
• Irene Taylor
• Graham Hatch
• Hugh Dyson (DSTL)