Robert L. Sherwood, Ph.D.

8/8/2019 Robert L. Sherwood, Ph.D.

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5797-1

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





5797-3

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



5797-4

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



5797-5

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



5797-6

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.





5797-11

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



5797-14

Bioaerosol Set Up

Collison

nebulizer

16 L Head-only

Exposure

Chamber

NHP whole body

plethysmography box

AGI

HEPA-

filtered air

3X HEPA filter



5797-15

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



5797-19

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)



5797-27

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)



5797-28

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



5797-29

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=



5797-30

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





5797-32

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



5797-33

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



5797-34

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



Lov e lac e™

Robert L. Sherwood, Ph.D.

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