Kelly Thomas presentation on "KT efficacy of anthrax vaccines in rabbits"

Efficacy Assessment of Anthrax

Vaccines and Therapeutics in

Rabbits

Kelly Thomas

2

Human Anthrax Vaccines

There are two main types of anthrax vaccine for human use:

1. A cell free filtrate that contains antigenic proteins which are adsorbed or

precipitated using an aluminium-based adjuvant and have been obtained

from cultures of attenuated, avirulent strains of Bacillus anthracis. The

principal active ingredient is the Protective Antigen (PA) component of the

anthrax toxin complex. These vaccines include Anthrax Vaccine Adsorbed

(AVA) and Anthrax Vaccine Precipitated (AVP).

• AVA, adsorbed onto aluminium hydroxide

• AVP, precipitated onto aluminium potassium phosphate

2. A live attenuated vaccine containing spores from attenuated strains of B.

anthracis. The vaccine is administered as a two-dose regimen in the skin of

the shoulder by scarification.

http://www.who.int/vaccine_safety/initiative/tools/Anthrax_Vaccine_rates_information_sheet.pdf

Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits





Next generation vaccines

3 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits

Live spores engineered to

express relevant antigens

Inactivated spores plus

other components

e.g. PA

Capsule

Antigen delivery methods

(microspheres, nanoemulsion, nanoparticles,

liposomes)

Live vectors e.g.

Salmonella, lactobacillus,

viruses

Adjuvants (AVA)

Plant-based vaccines

Next generation

vaccines:

rPA based


rPA vaccines

Subunit & other components

Fusion proteins

PA-LF

PA/LF mutant strains

PA functional domain mutants

DNA vaccine

Animal rule (FDA)


Approval Of New Drugs / Biological Products

When Human Efficacy Studies Are Not Ethical

Or Feasible

21 CFR Part 314 / 601

• Medical countermeasures for treatment / prevention of serious or life-

threatening conditions caused by exposure to lethal or permanently

disabling toxic biological, chemical, radiological or nuclear substances

• Efficacy studies cannot be conducted

• Safety evaluation is still required

Guidance for Industry, Product Development Under the Animal Rule. May 2014 & October 2015

Animal rule (FDA)


Approval based on evidence of

effectiveness from studies in animals

• Safety must have been established

• Adequate well-controlled animal studies to

establish benefit in humans

Requirements:

• Understanding of mechanism of toxicity and prevention/reduction by product

• Demonstrated in well-characterised animal model/in more than one animal

species predictive of human situation

• Animal study endpoint relates to desired benefit in humans

• Data allow the selection of effective human dose

Guidance for Industry, Product Development Under the Animal Rule. May 2014 and October 2015

Rabbit efficacy challenge model

• Most commonly used/accepted anthrax model (GUP & PEP)

• Recommended standardised schedule for testing Anthrax Vaccines for non-

inferiority to AVA

• NZW rabbits, 20-32 per group

• 2 vaccinations of 1/16 of a human dose of AVA

• Challenge with 200 LD50s of B. anthracis Ames via aerosol route

• Secondary readout serology (TNA NF50) day 0, 14, 28, 35, 42, 56, 69

7

Day 0 14 28 35 42 56 69,70


Development of reagents and assays • Challenge spores:

• Production

• Characterisation

• LD50 estimation

• Assay reagent generation:

• Rabbits vaccinated with anthrax vaccine

• Blood samples processed into large volumes of serum

• Assay development:

• Cell-based TNA adapted to measure immune responses in rabbits

• ELISA to measure antibody titres in rabbits

• New assays to study cell-mediated immunity

Efficacy of Anthrax Vaccines in rabbit models 8 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits

Rabbit efficacy challenge model

GUP Application – AVP & AVA

• 2 dose regime replicated initially

• 20 rabbits vaccinated with 1/16 dose AVP

• 20 rabbits vaccinated with 1/16 dose AVA

• 4 rabbits vaccinated with saline


Day 0 14 28 35 42 56 70 84

Pre-challenge ear bleeds

• Serum:

• TNA

• Analysis in anti-PA, anti-LF and anti-EF ELISA

• Whole heparinised blood used to develop CMI assays:

• Flow cytometry – CD4, CD8, CD45

• Stimulate lymphocytes with rPA or ConA, RT-PCR for cytokines

• Whole blood cell count and viability, NC200 image cytometer

• Baseline blood streak

10 Efficacy of Anthrax Vaccines in rabbit models – Kelly Thomas


Post-challenge samples • Samples to calculate presented dose

• Ear bleed at onset of clinical signs?

• Animals monitored for clinical observations, temperature, weight

• 2ml ear bleeds taken where possible

• Serum stored – trigger to treat ECL assay under development

• Post-mortem blood streak – samples taken after all deaths (as a result of

challenge, euthanasia at humane endpoint or at scheduled cull). Allows cause

of death confirmation



Necropsy • Carried out on all survivors

• Carried out for animals that died mid-study where feasible

• Blood:

• Serum – for TNA and ELISA

• Bacteriology

• Lungs and lymph nodes:

• Bacteriology

• Histopathology

• Spleens:

• Bacteriology

• Histopathology

12



0

0.2

0.4

0.6

0.8

1

0 7 14 21 28 35 42 49 56 63 70

NF 5

0

Study day

Median pre-challenge TNA NF50s

AVP

AVA



14

Post-challenge survival

Days post-challenge

0 2 4 6 8 10 12 14

% s

urv

ival

0

20

40

60

80

100

AVP

AVA

Saline

AVP

AVA

Saline





0

20

40

60

80

100

0 2 4 6 8 10 12 14

Clin

ical

sco

re a

s %

of

max

imu

m p

oss

ible

sco

re

Time (days post-challenge)

Clinical score

AVP

AVA

Saline

Blood & tissues • Bacteriology

• Blood and spleen of survivors negative for bacteria

• Bacteria enumerated in blood of some animals that died - highest level in control.

Some vaccinated animals positive, some negative

• All lungs contained bacteria. Highest in controls

• Highest bacterial counts in control spleen, some others positive

• Lymph nodes (where available) all <LOD

• Histology

• Control animal showed high level of bacteraemia, some lesions in spleen and lung

• More severe lesions in vaccinated animals

• More tissues in AVA group classified as ‘within normal limits’



• Study design modified to 3 doses and included generic rPA vaccine

• 10 rabbits vaccinated with 1/16 dose AVP

• 10 rabbits vaccinated with 1/16 dose AVA

• 10 rabbits vaccinated with 10mg rPA with alum

• 4 rabbits vaccinated with saline

17

Day 0 28 56 70 98 112


3 dose GUP study – AVP, AVA & rPA

0

0.4

0.8

1.2

1.6

2

0 14 28 42 56 70 84 98

TNA

NF 5

0

Study day

Median pre-challenge NF50s

AVP

AVA

rPA&alum




3 dose GUP study – AVP, AVA & rPA Post-challenge survival

Days post-challenge

0 2 4 6 8 10 12 14

% s

urv

ival

0

20

40

60

80

100

AVP

AVA

rPA&alum

Saline



0

20

40

60

80

100

0 2 4 6 8 10 12 14

Clin

ical

sco

re a

s %

of

max

imu

m p

oss

ible

sco

re

Time (days post challenge)

Clinical score

AVP

AVA

rPA&alum

Saline

Blood & tissues

• Bacteriology:

• Bacteria in blood of controls. Most survivors negative

• Spleens of control animals all positive. Survivors negative

• Lungs of control animals contained highest level of bacteria. Some survivors’ lungs

positive, some negative

• Histopathology:

• Visible bacilli in most control spleens and lungs, not survivors

• Moderate lesions of disease in control spleens, absent/milder in survivors

• Moderate inflammation in lungs of controls. Some survivors showed distinct lesions,

limited/no inflammation in most survivors – especially rPA & alum group

• Most survivors showed tissue hyperplasia/cell infiltration - reactive/protective?



• Study design based on previous publications

• Rabbits challenged with 200 LD50s Ames spores

• 30 rabbits received 50mg/kg levofloxacin once a day for 7 days from day 0

• 10 rabbits vaccinated with 1/16 dose AVP on day 0 and day 7

• 10 rabbits vaccinated with 1/16 dose AVA on day 0 and day 7

• 10 rabbits received saline injections on day 0 and day 7

• 4 controls

22

Day 0 7 16 21 30

PEP Application – AVP & AVA


Samples • Enumeration of challenge material

• Ear bleeds taken on day 0, 7, 16 and 21 from all remaining animals

• TNA/ELISA

• ECL assay

• Streak plates

• Necropsy carried out on all survivors on day 30 (or some mid-study deaths):

• Serum

• Blood for bacteriology

• Spleen and lungs for bacteriology and histology

23

Dilution

OD



24

Post-challenge survival

Days post-challenge

0 5 10 15 20 25 30

% s

urv

ival

0

20

40

60

80

100

AVP

AVA

Antibiotics

Saline

AVP & Antibiotic

AVA & Antibiotic

Antibiotic

Saline





0

20

40

60

80

100

0 5 10 15 20 25 30

Clin

ical

sco

re a

s %

of

max

imu

m p

oss

ible

sco

re

Time (days post challenge)

Clinical score

AVP & Antibiotic

AVA & Antibiotic

Antibiotic

Saline

Blood & tissues

• Bacteriology:

• Blood streak plates positive from most premature deaths. Negative for survivors

• Lungs contained bacteria when animals died mid-study. Lungs of survivors

low/negative

• Bacteria in spleens of most mid-study deaths, survivors negative

• Histopathology:

• Visible bacteria in tissues of some premature deaths

• Sporadic lung, spleen and lymph node lesions. Generally worst in control animals,

moderate in antibiotic group – milder in vaccinated groups

• Different changes seen in vaccinated animals compared to controls/antibiotic only

– some may be protective response rather than disease related



Rabbit studies - summary • Rabbit model – widely used to study AV efficacy in US

• Developed gold standard rabbit model in-house at PHE

• Efficacy of range of Anthrax Vaccines demonstrated and compared

• Supplementary information gathered on tissue pathology & bacterial burden

• Developed assays to study immune responses/correlates of protection and for

rapid toxin detection – important for well-characterised model

• Modified GUP schedule as appropriate for test vaccine (can also modify route)

• Adapted model to show PEP benefits (can apply to new and existing vaccines)

Presentation title - edit in Header and Footer 27 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits

Acknowledgements

Vaccine Research Group • Sue Charlton

• Bassam Hallis

• Phillip Brown

• Ines Vitoriano

• Nadina Wand

• Anna Pang

• Kim Steeds

• Debbie Powell

• Ross Fothergill

• Emma Kennedy

• Emily Hughes

• Rebecca Cobb

• Ester Schallinger

• Charlotte Ford

• Marilyn Aram

• Tom Slocombe

• Shea Creaven

• Lara Mason

• Jessica Fretwell

• Julia Sung

• Simon Eastham

28

Immunoassay Group • Mary Matheson

Biodefense and Preclinical

Evaluation Group • Julia Vipond

• Simon Funnell

• Helen Shuttleworth, Kerry Godwin,

Nicola Jones

Influenza Research Group • Karen Gooch, Jennifer Logue, Kathryn

Ryan

Biological Investigations Group • Irene Taylor

• Graham Hatch

• Susan Ryrie


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Kelly Thomas presentation on "KT efficacy of anthrax vaccines in rabbits"