Kelly Thomas presentation on "KT efficacy of anthrax vaccines in rabbits" at Biology of Anthrax, Tampa 2016

1. Efficacy Assessment of Anthrax
Vaccines and Therapeutics in
Rabbits
Kelly Thomas

2. 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

3. 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

4. Next generation
vaccines:
rPAbased
4 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
rPA
vaccines
Subunit & other
components
Fusion
proteins
PA-LF
PA/LF
mutant
strains
PA
functional
domain
mutants
DNA
vaccine

5. Animal rule (FDA)
5 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
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

6. Animal rule (FDA)
6 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
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

7. 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
Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits

8. 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 models8 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
Rabbit efficacy challenge model

9. GUPApplication –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
9 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
Day 0 14 28 35 42 56 70 84

10. 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
GUPApplication –AVP &AVA

11. 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
11 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
GUPApplication –AVP &AVA

12. 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
GUPApplication –AVP &AVA
Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits

13. 0
0.2
0.4
0.6
0.8
1
0 7 14 21 28 35 42 49 56 63 70
NF50
Study day
Median pre-challenge TNA NF50s
AVP
AVA
13 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
GUPApplication –AVP &AVA

14. 14
Post-challenge survival
Days post-challenge
0 2 4 6 8 10 12 14
%survival
0
20
40
60
80
100
AVP
AVA
Saline
AVP
AVA
Saline
GUPApplication –AVP &AVA
Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits

15. 15 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
GUPApplication –AVP &AVA
0
20
40
60
80
100
0 2 4 6 8 10 12 14
Clinicalscoreas%ofmaximumpossiblescore
Time (days post-challenge)
Clinical score
AVP
AVA
Saline

16. 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’
16 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
GUPApplication –AVP &AVA

17. • 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
Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
3 dose GUP study –AVP,AVA& rPA

18. 0
0.4
0.8
1.2
1.6
2
0 14 28 42 56 70 84 98
TNANF50
Study day
Median pre-challenge NF50s
AVP
AVA
rPA&alum
18 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
3 dose GUP study –AVP,AVA& rPA

19. 19 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
3 dose GUP study –AVP,AVA& rPA
Post-challenge survival
Days post-challenge
0 2 4 6 8 10 12 14
%survival
0
20
40
60
80
100
AVP
AVA
rPA&alum
Saline

20. 20 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
3 dose GUP study –AVP,AVA& rPA
0
20
40
60
80
100
0 2 4 6 8 10 12 14
Clinicalscoreas%ofmaximumpossiblescore
Time (days post challenge)
Clinical score
AVP
AVA
rPA&alum
Saline

21. 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?
21 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
3 dose GUP study –AVP,AVA& rPA

22. • 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
PEPApplication –AVP &AVA
Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits

23. 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
Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
PEPApplication –AVP &AVA

24. 24
Post-challenge survival
Days post-challenge
0 5 10 15 20 25 30
%survival
0
20
40
60
80
100
AVP
AVA
Antibiotics
Saline
AVP & Antibiotic
AVA & Antibiotic
Antibiotic
Saline
PEPApplication –AVP &AVA
Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits

25. 25 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
PEPApplication –AVP &AVA
0
20
40
60
80
100
0 5 10 15 20 25 30
Clinicalscoreas%ofmaximumpossiblescore
Time (days post challenge)
Clinical score
AVP & Antibiotic
AVA & Antibiotic
Antibiotic
Saline

26. 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
26 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits
PEPApplication –AVP &AVA

27. 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 Footer27 Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits

28. 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
Efficacy Assessment of Anthrax Vaccines and Therapeutics in Rabbits