robert l. sherwood, ph.d
TRANSCRIPT
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Non-Human Primate Models
for Evaluation of
Anthrax and Plague Vaccines
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
Robert L. Sherwood, PhD
Director, Applied Life Sciences & Toxicology
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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 bumpthat resembles an insect bite but within 1-2days develops into a vesicle and then apainless ulcer, usually 1-3 cm in diameter, witha characteristic black necrotic (dying) area inthe center.
• Edema or swelling of the surrounding tissuesmay develop and lymph glands in the adjacentarea may swell.
• About 20% of untreated cases of cutaneousanthrax will result in death.
– Intestinal (ingestion of infected meat)
• Symptoms include nausea, loss of appetite,vomiting, fever are followed by abdominalpain, vomiting of blood, and severe diarrhea.
• Intestinal anthrax results in death in 25% to60% of cases.
– Inhalation (breathing spores)
•
Symptoms of the common cold progressing tosevere breathing problems and shock
• Inhalation anthrax is usually fatal
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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 NewDrugs 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 toproduce clinical benefit in humans
• Reasonably well-understood pathophysiologicalmechanism
• 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 thedesired benefit in humans
• The data on the kinetics and pharmacodynamics allowsselection 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 wellunderstood 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, workingstock. Known titers, purity, genetic stability.
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Descriptive Method
incubate
centrifuge
Set up spraysor Perform
aerosols
Recover spray& AGI
suspensionsSerial dilutions
and plating
Countplates
incubate
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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 microbialaerosol; it also efficiently inactivates microbes
• Need to understand the operating parameters of the bioaerosol
system so that target challenge doses can be achievedreproducibly
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Bioaerosol Set Up
Collison
nebulizer
16 L Head-only
Exposure
Chamber
NHP whole body
plethysmography box
AGI
HEPA-
filtered air
3X HEPA filter
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NHP Head-only Exposure Chamber
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Bioaerosol Chamber
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Target vs. Predicted SprayConcentrations
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Log10 CFU
L o g 1 0 C F U
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Effect of Spray Conc. On Spray Factor
Actual CFU vs. SprayFactor
-9
-8
-7
-6
-5
-4
-3
2 3 4 5 6 7 8 9 10
Log10 CFU in Spray
S p r a
y F a c t o r ( L o g 1 0 )
Avg. SprayFactor =
1.42x10-6
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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 developmentbecause the origin may have an effect on results
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Anthrax and Plague NHP Model Overview
Infectiouschallenge(250 LD50)
Death
Bacteremia
Anthrax
Plague
Death
Infectious
challenge(50 LD50)
Bacteremia
Lethargy, Diarrhea,Stop eatingIncrease body
temp
Drop inBody Temp
Lethargy, Diarrhea,Respiration changes,
Stop eating
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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 PlagueAerosol Challenge
0
20
40
60
80
100
120
D 0
D 2
D 4
D 6
D 8
D 1 0
D 1 2
D 1 4
Study Day
% S
u r v i v a l
PBS 0
MAb A 10
MAb B 10
MAb C 10
MAb B 2
MAb B 1
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NHP Anthrax Time to Death
0
2
4
6
8
10
12
14
T i m e ( D a y s
)PBS 0
Mab A 10
Mab B 10
Mab C 10
Mab B 2
Mab B 1
12 5 11 5 54N=
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NHP Anthrax Bacteremia
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
D0 D1 D2 D3 D4 D5 D6 D9 Term
Days
L o g 1 0 C F
U
PBS 0
Mab A 10
Mab B 10Mab C 10
Mab B 2
Mab B 1
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NHP Anthrax Tissue Load
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
8.000
9.000
Spleen Liver Trach LN Lung
L o g 1 0 C F U / g
PBS 0
Mab A 10
Mab B 10
Mab C 10
Mab B 2
Mab B 1
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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 microbesin as little as 2-3 days
• Spore counts in the lung decrease slowly over time, but maytake 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 highestsurvival)
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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 PlagueAerosol Challenge
0
20
40
60
80
100
120
D 0
D 2
D 4
D 6
D 8
D 1 0
D 1 2
D 1 4
Study Day
%
S u r v i v
a l
Ag+Adj
Ag A + Adj 250 1
Ag A + Adj 250 2
Ag A + Adj 250 3
Ag A + Adj 25 3
Ag A 250 3
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0
2
4
6
810
12
14
Ag+AdjAg A +Adj250 1
Ag A +Adj250 2
Ag A +Adj250 3
Ag A +Adj25 3
Ag A 250 3
NHP Plague Time to Death
16 5 5 320 3N=
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NHP Plague Bacteremia
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
D 0
D 1
D 2
D 3
D 4
D 5
D 6
D 7
D 1 0
D 1 4
Days
L o g 1 0
C F U
Ag+Adj 0 3
Ag A + Adj 250 1
Ag A + Adj 250 2
Ag A + Adj 250 3
Ag A + Adj 25 3
Ag A 250 3
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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 4-5days
• 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 havemany 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 improvedresults 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