Update on Fischer 344 Rat Model
UNM Team
Rick Lyons, Terry Wu, Jason Zsemlye, Gloria Statom,
Alexandra Scrymegeour, Amanda DuBois,
Gopi Mara-Koosham, Julie Hutt,
1
Active Milestones
5 Evaluation of small animal models
• Rats from NCI and Harlan were equally sensitive to SCHU S4
• Developed bronchopneumonia
• Statistical analyses indicated 6 or 8 rats per group and104 per rat
11 Develop GLP-like SOP for small animal model
• Improved inoculation with quantum dots for tracking pulmonary delivery
13 Assays for detecting relevant immune responses
• Optimized IFNg ELISpot assays for mouse, rats, & NHP
14 Assays in vaccinated humans
• Just started, not much data yet
• Activation of macrophage killing mechanisms by LVS vaccinees T cells
17 Analyses of cellular and humoral immunity in vaccinated humans
and animals
• Passive immunization protected rats but not mice against i.t. SCHU S4 2
challenge
Active Milestones
19 Interactions between human alveolar macrophages and F. tularensis
• Cytokine responses after LVS and SCHU S4 infection
21 Correlates of protection
• Developed assay to measure activation of macrophage killing mechanisms
by immune T cells in mouse, working on rats
• Detection of multifunctional T cells in NHP
27 Optimization of IFNg ELISpot for identification of protein vaccine
candidates
• Helped ASU identify strategy for producing and purifying ivt protein vaccine
candidates
35 Optimization of RNA isolation and hybridization conditions
• Provided ASU with RNA and DNA as needed
3
High Impact Findings
1.
Fischer 344 rats from NCI and Harlan have similar
sensitivity to SCHU S4 infection
2.
Lung histopathology in Fischer 344 rats similar to
Cynomolgus macaque but different from BALB/c mice
3.
Passive immunization with immune rat serum is
protective against SCHU S4 for rats but not mice
4.
Statistical analyses indicated 6 (max 8) rats sufficient
to detect significant effect by vaccines
LVS Vaccination Protected Fischer 344
Rats Against i.t. SCHU S4 Challenge
NCI and Harlan Rats Have Similar
Sensitivity to SCHU S4
6
Variability and Group Size
• n > 90 for statistical significance due to
large variation in natural resistance to
SCHU challenge
Downs, C. M., L. L. Coriell, G. B. Pinchot, E. Maumenee, A. Klauber, S. S.
Chapman, and B. Owen. 1947. Studies on Tularemia: I. The Comparative
Susceptibility of Various Laboratory Animals. J Immunol 56:217-228.
Olsufiev, N. G., O. S. Emelyanova, and T. N. Dunayeva. 1959. Comparative study
of strains of B. tularense in the old and new world and their taxonomy. J Hyg
Epidemiol Microbiol Immunol 3:138-49.
7
Proportional Hazard Regression
Analyses of Fischer 344 rats
Unvaccinated
0
0
Estimated Survival (%)
20
40
60
80
Estimated Survival (%)
20
40
60
80
100
100
Intratracheal
2
4
6
8
10
Days Post-Innoculation
12
2
Intradermal
100
100
Estimated Survival (%)
20
40
60
80
Estimated Survival (%)
20
40
60
80
2
10 CFU
4
10 CFU
0
4
6
8
10
Days Post-Innoculation
12
Subcutaneous
0
2
4
6
8
10
Days Post-Innoculation
12
2
6
10 CFU
8
10 CFU
4
6
8
10
Days Post-Innoculation
12
8
Results of Statistical Analyses
• i.d. and s.c. are equally protective and both are slightly
better than i.t. LVS vaccination
• Future challenge dose should be 104 SCHU S4 for best
confidence for 90% of naïve rats to die but 90% of
vaccinated rats to survive
• If the vaccine candidate is as effective as LVS, then 6
rats per group is sufficient to detect a significant
improvement over unvaccinated rats
• If vaccine is not as effective as LVS (using i.t. LVS
vaccination as reference), then 8 per group is the
maximum number required to detect a significant
protective effect
9
Bronchopneumonia in SCHU S4
Infected Rats
10
Bronchopneumonia (Rats) vs. Vasculitis (Mice)
11
Reports of Effective Passive Immunization
• Foshay, L. 1940. Tularemia: A summary of certain
aspects of the disease including methods for early
diagnosis and the results of serum treatment in 600
patients. Medicine:1.
• Foshay, L. 1946. A comparative study of the treatment
of tularemia with immune serum, hyperimmune serum,
and streptomycin. Am. J. Med. 1:180
• Foshay, L., I. Ruchman, and P. S. Nicholes. 1947.
Antitularense Serum: Correlation between Protective
Capacity for White Rats and Precipitable Antibody
Content. J Clin Invest 26:756-60.
Immune Rat Serum Did Not Protect BALB/c
Mice Against Intranasal SCHU S4 Challenge
LVS Challenge
SCHU Challenge
Naive
Normal serum
Immune serum
100
Percent survival
Percent survival
100
75
50
25
0
75
50
25
0
0
5
10
15
Days p.i.
20
25
0
5
10
Days p.i.
13
Passive Immunization Protects
Against i.t. SCHU S4 challenge
Treatment
Unvaccinated
LVS vaccinated
Normal serum
Immune serum
Survival Ratio
(No. alive/total)
0/6
6/6
0/6
6/6
Implications
Much better characterized model
1. Model is robust (NCI vs. Harlan)
2. Reasonable group size (proportional hazard
regression)
3. Optimal challenge dose 104 SCHU S4/rat
4. Similar lung pathogenesis as Cynomolgus
macaque (and humans?) (histopathology)
5. Similar protective effect of humoral immunity
as humans?
15
Problems & Corrective Actions
• Loss of protection in LVS-vaccinated mice
but not rats
– Testing new LVS and SCHU S4 stocks in
mice
– Currently using 10-fold higher LVS
vaccination dose to protect against 10-fold
lower SCHU S4 dose
• Low macrophage yield from whole blood
for activation assay
– Reduce scale
Next 6 Months
5 Evaluation of small animal models
• Complete MSCR
11 Develop GLP-like SOP for small animal model
• Complete & test SOPs for the rat
13 Assays for detecting relevant immune responses
• None
14 Assays in vaccinated humans
• Activation of macrophage killing mechanisms by LVS vaccinees T cells
17 Analyses of cellular and humoral immunity in vaccinated humans
and animals
• Continue studies of cellular (CD4/8 depletion) and humoral (passive
transfer) immunity in rats
• Start work with CD4/8 T cells from human LVS vaccinees
17
Next 6 Months
19 Interactions between human alveolar macrophages and F. tularensis
• Cytokine responses after LVS and SCHU S4 infection
21 Correlates of protection
• Continue assay development to functional measure of T cell function in rats
and humans
• Continue multifunctional T cells in NHP
27 Optimization of IFNg ELISpot for identification of protein vaccine
candidates
• Screen protein vaccine candidates from ASU
35 Optimization of RNA isolation and hybridization conditions
• Provide ASU with RNA and DNA as needed
18