Developments in Tularemia Animal
Models
UNM
Alexandra Scrymgeour
Gloria Statom
Jason Zsemlye
Terry Wu
C. Rick Lyons
1
Active milestones
5
Evaluation of small animal models
•
•
Fischer 344 rat is potentially a good model
Eliminated Hartley guinea pigs from further evaluation
12/13 Assays for detecting relevant immune responses
•
•
19
IFNg Elispot assay may be better than proliferation assay for mouse model
Developing the IFNg Elispot and proliferation assays for the rat model
Interaction between human alveolar macrophages and F. tularensis
•
Developing culture conditions
21 T cell-induced macrophage killing of intracellular bacteria
•
•
Assay developed in mouse model for killing LVS; working on SCHU S4
Developing killing assay for the rat model
27 Optimization of T cell assays to define vaccine candidates
•
Testing IFNg Elispot as alternative to T cell proliferation assay for screening
34 Optimization of RNA isolation and hybridization conditions
2
Fischer 344 rats
3
It all started here…
4
Comparison of human and rat vaccine studies
Species
Sensitivity to respiratory
SCHU S4 challenge
Sensitivity to LVS vaccination
Vaccine-induced protection against aerosol
or i.n. SCHU S4 challenge
Scarification
(s.c. / i.d.)
Pulmonary
(Aerosol / i.n.)
Scarification
vaccination
Pulmonary
vaccination
Human
High
MID = 10
40-60% mortality
Low
Low
(> 108 LVS)
Protected against
< 2 x 103 SCHU S4
Protected against
2.5 x 104 SCHU S4
Fischer 344
Rats
High
LD50 ~ 180
100% mortality
Low
(> 5 x 107 LVS)
Low
(> 2 x 107 LVS)
Protected against
> 104 SCHU S4
Protected against
> 104 SCHU S4
Species
Innate immunity
Humoral immunity
Cell mediated immunity
Human
Macrophages, neutrophils,
type II alveolar epithelial
cells, TNF
IgM, IgA, IgG appear 14 d after
vaccination and persist for
years
CD4 & CD8 T cell proliferation
and IFNg production appear
14 d after vaccination and
persists for years
Fischer 344
Rats
???
???
???
5
Method of pulmonary infection
6
RC Molthen. J Am Assoc Lab Anim Sci. 2006 Jan;45(1):88-93.
Tracking pulmonary delivery in rats
7
Sensitivity of naïve Fischer 344 rats to i.t.
SCHU S4 challenge
8
Clinical signs and scoring
(-)
(+/-)
(+)
(++)
(+++)
Active, bright, alert, and responsive to cage movement
Active, alert and responsive to cage movement, slight
piloerection, slight porphyrin secretion around eyes
Slightly decreased activity, alertness and responsiveness to
cage movement, clear piloerection, eyes open but with slight
porphyrin secretion
Definite decreased activity, moves slowly to cage movement ,
very ruffled coat, rapid and shallow breathing, hunched over,
huddle in group, eyes half closed with large amount of
porphyrin secretion
Inactive and unresponsive to cage movement, very ruffled
coat, weight loss, wobbly, difficulty walking, labored breathing,
eyes closed, isolated from group
9
Sensitivity of naïve Fischer 344 rats to
various routes of LVS vaccination
Route of LVS
vaccination
4 wk survival
Dose
ratio
(CFU/rat) (# survived/total)
i.t.
2.0 x 107
28/30
i.d.
5.0 x 107
30/30
s.c.
5.0 x 107
30/30
10
Kinetics of LVS proliferation, dissemination
and clearance after s.c. vaccination
Log10 mean bacterial load ± SD
(No. infected tissue/total)
Day p.i.
Spleen
Liver
Lung
1
4.2 ± 0.7 (6/6)
3.7 ± 0.2 (5/6)
2.5 ± 0.2 (3/6)
2
4.5 ± 0.2 (6/6)
4.3 ± 0.4 (6/6)
3.1 ± 0.4 (5/6)
3
4.0 ± 0.6 (5/6)
4.0 ± 0.6 (6/6)
3.5 ± 0.6 (4/6)
4
4.1 ± 0.1 (6/6)
3.5 ± 0.3 (6/6)
2.5 ± 0.6 (2/6)
5
3.8 ± 0.3 (6/6)
2.7 ± 0.3 (3/6)
2.8 ± 0.4 (4/6)
6
3.5 ± 0.2 (6/6)
0 (0/6)
2.1 (1/6)
14
0 (0/6)
0 (0/6)
0 (0/6)
Inoculum = 3.7 x 107 CFU/rat
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Sero-conversion after s.c. LVS vaccination
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Vaccination protects Fischer 344 rats
against i.t. SCHU S4 challenge
13
Vaccination protects Fischer 344 rats against
i.t. SCHU S4 challenge: experiment 2
14
SCHU S4 proliferation and dissemination in
naïve and vaccinated Fischer 344 rats
Mean Log10 CFU/organ ± SD
Naïve
Day
Lung
0
4.2 ± 0.3
1
7.3 ± 0.1
2.5 (1/4)
1.9 ± (1/4)
2
8.2 ± 0.2
5.0 ± 0.6
4.7 ± 0.7
7.4 ± 0.1
4.5 ± 0.3
3
8.5 ± 0.3
7.2 ± 0.2
7.1 ± 0.4
7.4 ± 0.1
4.5 ± 1.0.2
4
8.9 ± 0.3
7.9 ± 0.3
8.4 ± 0.3
7.7 ± 0.7
5.1 ± 0.3
6.8 ± 0.4
5.0 ± 0.2
4.6 ± 0.3
6
6.0 ± 0.8
3.9 ± 0.7
4.1 ± 0.8
21
2.9 ± 0.3
0
0
42
0
0
0
5
Liver
s.c. LVS vaccinated
Spleen
Lung
Liver
Spleen
2.2 ± 0.3
0
4.2 ± 0.1
Died
n = 4-5/group
15
Frequency of IFNg-producing, LVSspecific splenocytes
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Summary of the Fischer 344 rat model
•
•
•
•
Extremely sensitive to i.t. SCHU S4 challenge (LD50 = 180)
Can be vaccinated s.c., i.d. or i.t. with large doses of LVS
Vaccination increases resistance to SCHU S4 by 3- 4 logs
Vaccination limits SCHU S4 proliferation, dissemination and
histopathology
• Vaccination increases frequency of LVS-specific T cells
• The best model so far for human disease, but more
characterization required
17
Problems and resolutions
5
Evaluation of small animal models
•
•
Problem: Occasionally missing pulmonary infection
Resolution: Quantum dots or surgical infection
12/13 Assays for detecting relevant immune responses
•
•
Problem: Unreliable T cell proliferation results
Resolution: Testing IFNg Elispot as an alternative
19 Interaction between human alveolar macrophages and F. tularensis
•
•
Problem: Accessibility to human volunteers
Resolution: School started and monocyte-derived macrophages
21 T cell-induced macrophage killing of intracellular bacteria
•
•
Problem: Generating/maintaining macrophage monolayer
Resolution: Received help from Karen Elkins
27 Optimization of T cell assays to define vaccine candidates
•
•
Problem: Unreliable T cell proliferation results
Resolution: Testing IFNg Elispot as an alternative
34 Optimization of RNA isolation and hybridization conditions
•
Problems: None
18
Next six months…
5
Evaluation of small animal models
•
Detailed characterization of the rat model for comparison with humans,
including roles of CD4 and CD8 T cells, B cells and antibodies, cytokines
12/13 Assays for detecting relevant immune responses
•
•
Optimize IFNg Elispot assay for the mouse model
Develop and evaluate IFNg Elispot and T cell proliferation assays for the
rat model
19 Interaction between human alveolar macrophages and F. tularensis
•
•
•
Establish conditions for working with human alveolar macrophages and
monocyte-derived macrophages
Characterize the growth of LVS and SCHU S4 in macrophages
Determine the effect of IFNg
19
Next six months…
21 T cell-induced macrophage killing of intracellular bacteria
•
•
•
Develop killing assay in the mouse model using SCHU S4
Develop assay in the rat model
Develop assay using macrophages and T cells from vaccinated
volunteers
27 Optimization of T cell assays to define vaccine candidates
•
Test usefulness of the IFNg Elispot assay for screening candidate
peptide and protein vaccines from ASU
34 Optimization of RNA isolation and hybridization conditions
•
Provide ASU with RNA and DNA as needed
20
Questions after presentation
•
•
•
•
•
•
Karl: why vaccinate intratracheal rather than intranasal?
Rick: turvinates in rats cause a volume issue; can’t get enough volume
down their lungs via the intranasal route. 50ul in mouse goes down to
the lungs but 50ul in rats doesn’t get down into the lungs. Some rats
even drowned with 50ul intranasally.
SAJ: are the T cells cd 8 or cd4 involved in the rat? Terry responded
that UNM hasn’t done the experiment yet.
Rick: Guinea Pig is very resistant to LVS and LVS provided no
protection to SCHU S4 challenge. Rat is giving a little bigger window
for vaccination and protection than the mouse gave. Rick doesn’t like
any naïve controls surviving a lethal SCHU S4 challenge. Consistently
down in 80-90% survival which is reasonable. Fisher 344 are inbred
rats. UNM still has work to do on the rat model but is promsing.
Karl: Has UNM used nose aerosol exposures yet?
Chuck: One can do nose only in the rats. It may be easier to do aerosol
with nose, rather than intratracheal in rats. It maybe faster to give 1
dose to 100 rats by aerosol cone than intratracheal infection.
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