Sue Charlton presntation on "Efficacy assessment of anthrax vaccines"

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.

2 Efficacy Assessment of Anthrax Vaccines

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


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


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


Efficacy testing and the product (vaccine) lifecycle


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


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


• 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


Image from Qiagen

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


Macrophage Cell Lysis Assay

10

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


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

12 Efficacy Assessment of Anthrax Vaccines 12

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


TNA – generation of a reportable value

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Picture of NF50

Dilution

OD


Rabbit study data


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)


Immunise mice

Collect serum

Analyse in TNA

Determine potency

relative to reference


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.


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


Responses: change from baseline



Responses: absolute values

TNA responses – time since last booster


Conclusions


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


• Mary Matheson

• Anna England

• Simon Funnell

• Irene Taylor

• Graham Hatch

• Hugh Dyson (DSTL)

Science

Sue Charlton presntation on "Efficacy assessment of anthrax vaccines"