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Non-Human Primate Models

for Evaluation of
Anthrax and Plague Vaccines

Robert L. Sherwood, PhD


Director, Applied Life Sciences & Toxicology

Lovelace Respiratory Research Institute


2425 Ridgecrest Drive SE, Albuquerque, NM 87108
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OVERVIEW
• Disease models
• Why NHP?
• Microbiological characterization
• Bioaerosol characterization
• Animal models
• NHP Anthrax efficacy
• NHP Plague efficacy
• Summary

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Bacillus anthracis
• Bacillus anthracis is very large, Gram-positive, sporeforming
rod, 1 - 1.2µm in width x 3 - 5µm in length.
• The bacterium can be cultivated in ordinary nutrient medium
under aerobic or anaerobic conditions.
• Member of the Bacillus cereus family
– Includes B. cereus, B. thuringiensis, and B. anthracis

Gamma phage lysis


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Bacillus anthracis
• Causative agent of anthrax
– Cutaneous (skin contact with infected material)
• Skin infection begins as a raised itchy bump
that resembles an insect bite but within 1-2
days develops into a vesicle and then a
painless ulcer, usually 1-3 cm in diameter, with
a characteristic black necrotic (dying) area in
the center.
• Edema or swelling of the surrounding tissues
may develop and lymph glands in the adjacent
area may swell.
• About 20% of untreated cases of cutaneous
anthrax will result in death.
– Intestinal (ingestion of infected meat)
• Symptoms include nausea, loss of appetite,
vomiting, fever are followed by abdominal
pain, vomiting of blood, and severe diarrhea.
• Intestinal anthrax results in death in 25% to
60% of cases.
– Inhalation (breathing spores)
• Symptoms of the common cold progressing to
severe breathing problems and shock
• Inhalation anthrax is usually fatal 5797-4
Yersinia pestis

• Gram-negative coccobacilli
– Nonmotile, nonhemolytic
– Capsule
• Member of Enterobacteriacea
– Facultative aerobe
• Carries 3 plasmids
– 9.5 kb pPla
• Plasminogen activator
– 64 kb pCD1 (pYV or pCad)
• Yop proteins
– 100-100 kb pMT1 (pFra)
• Fraction 1 capsule antigen

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Y. pestis (causative agent of Plague)

bubo

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Why NHP?
• FDA “Animal Rule”
– Title 21, Subchapter D, Part 314, Subpart I – Approval of New
Drugs When Human Efficacy Studies are Not Ethical or Feasible
– Requires the following:
• Adequate and well-controlled animal studies that
establish that the drug product is reasonably likely to
produce clinical benefit in humans
• Reasonably well-understood pathophysiological
mechanism
• Effect is demonstrated in more than one animal species
expected to react with a response predictive for humans
• The animal study endpoint is clearly related to the
desired benefit in humans
• The data on the kinetics and pharmacodynamics allows
selection of an effective dose in humans

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Why NHP?

• Animal model correlation to human disease is required


– Multiple models may be necessary
– Monkey will probably be closest correlate to human

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Microbiological Characterization
• The bacteriology of the microbial challenge needs to be well
understood and well characterized
• Does your microbial growth method recover optimally?
• What samples are going to be analyzed? Liquid, tissue?
Recovery from tissue? Clumping?
• Are appropriate samples analyzed to assess purity and titer?
• What is the process? Fresh growth vs. frozen?
• Is there an effect on results from small variations in target dose?
• Working from a well characterized stock? Seed stock, working
stock. Known titers, purity, genetic stability.

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TSA vs BHI

Percent Recovery of Yersinia


pestis on Solid Media
120%
% Recovery

100%
80%
60%
40%
20%
0%
TSA TSA + Nutrient BHIA
sheep Agar
blood

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Descriptive Method

incubate Set up sprays


or Perform
aerosols
centrifuge

Count
plates
incubate Recover spray
& AGI
suspensions
Serial dilutions
and plating
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Final Method for Y. pestis NHP Challenge
• Inoculate TBAB slants with loop of frozen stock suspension
• Incubate at 28ºC for 48 – 96 hrs
• Harvest growth from TBAB slants into 1% peptone (2 mL per slant)
• Centrifuge and resuspend in 4 ml 1% peptone
• Take OD of suspension to estimate titer
• Compare OD to standard curve
• Dilute microbial suspension to dose in BHI
• Set up sprays with 10 mL BHI of dose suspension (weigh vials)
• Set up impingers with 20 mL of BHI + antifoam (weigh vials)
• Perform sprays
• Reweigh spray and impinger vials to determine loss
• Titer viable CFU in 1:10 dilutions of sterile 1% peptone
• Plate on TSA and incubate for 48-72 hrs at 28ºC
• Count colonies
• Calculate dose

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Bioaerosol Characterization
• Very important to understand effects of aerosolization on
microbial agent
• May require some form of protection (high protein medium)
• Humidity requirement?
• Collison nebulizer efficiently creates a uniform microbial
aerosol; it also efficiently inactivates microbes
• Need to understand the operating parameters of the bioaerosol
system so that target challenge doses can be achieved
reproducibly

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Bioaerosol Set Up
3X HEPA filter

HEPA-
filtered air
AGI

Collison
nebulizer

16 L Head-only NHP whole body


Exposure plethysmography box
Chamber
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NHP Head-only Exposure Chamber

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Bioaerosol Chamber

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Target vs. Predicted SprayConcentrations

8.00

7.00

6.00

5.00
Log10 CFU

4.00

3.00

2.00

1.00

0.00
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Log10 CFU

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Effect of Spray Conc. On Spray Factor

Actual CFU vs. SprayFactor

-3
2 3 4 5 6 7 8 9 10 Avg. Spray
-4
Factor =
1.42x10-6
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Spray Factor (Log10)

-6

-7

-8

-9
Log10 CFU in Spray

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NHP Models
• Cynomolgus macaques (Macaca fascicularis)
• Cyno does not necessarily equal a cyno
– Chinese cyno
– Indonesian cyno
– Vietnamese cyno
– Mauritius cyno (less genetic diversity)
– Need to indicate origin of animal in model development
because the origin may have an effect on results

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Anthrax and Plague NHP Model Overview
Anthrax
Lethargy, Diarrhea,
Drop in Respiration changes,
Body Temp Stop eating

Infectious Death
challenge
(250 LD50)
Bacteremia

Plague
Lethargy, Diarrhea,
Increase body Stop eating
temp

Infectious
challenge Bacteremia Death
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Anthrax Data

• Combined data from 3 separate passive transfer


studies
– Ab infusion 1 hr pre-challenge
• Pulmonary challenge (approx. 250 LD50 doses)
• 15 days of observation
• Daily bleeds for bacteremia
• Tissue load at euthanasia (day 3 to 15)

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Survival Time of Treated NHP Exposed to Plague
Aerosol Challenge

120

100
PBS 0
% Survival

80 MAb A 10
MAb B 10
60
MAb C 10
40 MAb B 2
MAb B 1
20

0 10
4

12

14
0

2
D

D
Study Day

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NHP Anthrax Time to Death
14

12

10
PBS 0
Time (Days)

Mab A 10
8
Mab B 10
Mab C 10
6 Mab B 2
Mab B 1
4

0
N= 12 5 4 11 5 5

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NHP Anthrax Bacteremia

4.0
3.5
3.0 PBS 0
2.5 Mab A 10
Log10 CFU

Mab B 10
2.0 Mab C 10
1.5 Mab B 2
Mab B 1
1.0
0.5
0.0
D0 D1 D2 D3 D4 D5 D6 D9 Term
Days

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NHP Anthrax Tissue Load

9.000
8.000
7.000
PBS 0
6.000
Mab A 10
Log10 CFU/g

5.000 Mab B 10
Mab C 10
4.000
Mab B 2
3.000 Mab B 1
2.000
1.000
0.000
Spleen Liver Trach LN Lung

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Anthrax Summary

• Animals with early bacteremia succumb quickly


• Bacteremia indicates vegetative microbes escaping host defense
• Tissue loads can reach very high levels of vegetative microbes
in as little as 2-3 days
• Spore counts in the lung decrease slowly over time, but may
take a long time to drop to levels that the host defense can
manage
• Antibody pretreatment can result in significant survival from
pulmonary anthrax challenge (Mab C @ 10 mg had highest
survival)

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Plague Data

• Combined data from 3 vaccine efficacy studies


– 1, 2, or 3 challenges
– + / - adjuvant
– Low (25 µg) or high (250 µg) dose
• Pulmonary challenge (approx. 75 LD50 doses)
• 15 days of observation post infection
• Daily bleeds for bacteremia (Days 2, 3, 4, 5, 6, Term)
• Tissue load at euthanasia (Day 3 to 15)

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Survival Time of Vaccinated NHP Exposed to Plague
Aerosol Challenge

120

100 Ag+Adj

80 Ag A + Adj 250 1
% Survival

Ag A + Adj 250 2
60
Ag A + Adj 250 3
40 Ag A + Adj 25 3
20 Ag A 250 3

0
10
0

12

14
2
D

Study Day D

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NHP Plague Time to Death

14
12
10 Ag+Adj
Ag A +Adj250 1
8 Ag A +Adj250 2
Ag A +Adj250 3
6
Ag A +Adj25 3

4 Ag A 250 3

2
0
N= 16 5 5 20 3 3

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NHP Plague Bacteremia
7.0
6.0
Ag+Adj 0 3
5.0
Ag A + Adj 250 1
Log10 CFU

4.0 Ag A + Adj 250 2


Ag A + Adj 250 3
3.0
Ag A + Adj 25 3
2.0 Ag A 250 3
1.0
0.0
D0
D1
D2

D5
D6
D7
D3
D4

0
4
D1
D1
Days

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NHP Plague Tissue Load

10.000
9.000
8.000
7.000 Ag+Adj 0 3
Ag A + Adj 250 1
6.000
Ag A + Adj 250 2
5.000
Log10 CFU/g

Ag A + Adj 250 3
4.000 Ag A + Adj 25 3
3.000 Ag A 250 3

2.000
1.000
0.000
Spleen Liver TrachLN Lung

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Plague Summary
• Plague bacteremia indicates early break out from host defense
• Lower bacteremia correlates with better survival prognosis
• Tissue loads can reach very high bacterial concentrations in 4-5
days
• Lower bacteremia also correlates with lower tissue loads
• 3 challenge doses > 2 doses > 1 dose
• Adjuvant was required for optimal host response

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Summary

• Pulmonary anthrax and plague in Cynomolgus macaques have


many similarities to human disease
• Therapies that impact spread of vegetative anthrax have
improved results in NHP anthrax model
• Therapies that decrease septicemia of plague have improved
results in NHP plague model

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Acknowledgments

• Vidadi Yusibov, PhD (Fraunhofer USA)


• NIAID
• LRRI
– Microbiology group
• Trevor Brasel, PhD
• Liz Zinter
• Rebecca Wisecup
– Bioaerosol group
• Ed Barr
• Steve Storch
– Toxicology Group
• Ron Couch, PhD
• Michelle Valderas, PhD
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Lovelace

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