UNM Overview
&
Update on Fischer 344 Rat Model
UNM Team
Rick Lyons, Terry Wu, Jason Zsemlye, Gloria Statom,
Amanda DuBois, Gopi Mara-Koosham, Julie Hutt,
1
Key Achievements
5 Evaluation of small animal models (Fischer 344 rats)
• Small LVS vaccination dose (~103 cfu s.c.) sufficient for protection
against i.t. challenge
• Variable susceptibility to s.c. challenge: MID ≤ 10 cfu, LD50  105 cfu
•
•
•
Rapid and severe weight loss and death
Gradual weight loss and recovery
No weight loss
– Similar observations reported in human studies
10 Evaluation of new vaccine candidates
• Vaccinated mice and rats with Lm-based vaccine from Aduro/Cerus
11 Characterization of small animal model (Fischer 344 rats)
• LVS vaccination dependent on T cells
• Passive immunization
•
•
•
Protection 2-3 logs lower than LVS vaccination (immune serum & purified immune IgG)
Intermediate phenotype between naïve and LVS-vaccinated rats: bacteria growth
kinetics, histology and cytokines (prelim)
Protection dependent more on CD8 than CD4 T cells (prelim)
2
Key Achievements
12/13 Assays for detecting relevant immune responses
• Developed and optimized antibody ELISA and IFNg ELISpot assay
capable of distinguishing vaccinated and unvaccinated individuals
19 Interactions between human alveolar macrophages and F.
tularensis
• TNF and IFNg have no effect on SCHU S4 growth in human alveolar
macrophages or macrophage viability/recovery
21 Correlates of protection
• 48 h prestimulation of human PBMC with inactivated LVS induced IFNg
and reduced SCHU S4 growth – analogous to Elkins assay
35 Optimization of RNA isolation and hybridization conditions
• Provided ASU with RNA and DNA as needed
No activity on MS14, 17, 18, 29 because precedents have not been
completed
3
Problems, Deviations, & Resolutions
5
Inconsistent protection of mice by LVS
• Test protection after modifying culture conditions, including CO2
11 Inability to verify CD4 T cell depletion because antibody is nondepleting
• Working on functional measure of inactivation
12/13 Inability to extract and quantify protein content in heat-killed or
formalin fixed bacterial preparations
• B-PER bacterial lysis, sonication, and beat-beater. No linear
relationship between live bacteria number and protein content
• RIPA and alterative approach to normalize
4
Next 6 Months
10 Evaluation of new vaccine candidates
• Challenge mice/rats vaccinated with Lm-based vaccines and repeat if
necessary
11 Characterization of small animal model
• Characterize T cell responses in LVS-vaccinated and passively
immunized rats – Intracellular cytokine, ELISpot, depletion, adoptive
transfer, etc
• Characterize the mechanism responsible for passive immunity
12/13 Assays for detecting relevant immune responses
• Develop micro-agglutination assay
• Continue efforts to normalize antigen preparations
5
Next 6 Months
21 Correlates of protection
• Increase number of vaccinated and unvaccinated human samples
• Compare humans before and after LVS vaccination
• Activate MS 18 and 20 to determine the role of specific cellular responses
and effector mechanisms
• Develop analogous assay in Fischer rats
29 Correlates of protection
• Repeat/verify results with remaining vaccinated NHP?
6
Characterization
of
Fischer 344 Rat Model
7
Summary of Fischer 344 Rat Model
• Very susceptible to respiratory tularemia caused by
inhalation of SCHU S4
• Protected by multiple routes of LVS vaccination
• 2-3 log greater protection than mice
• Vaccination does not prevent infection or dissemination
but limited bacterial growth and pathology
• Clear similarities to historical human vaccine studies
8
Humoral Immunity Against Tularemia
• Hyperimmune serum used to successfully treat
tularemia patients
• Immune serum protected mice against i.n.
challenge with LVS and SCHU S4
• IgA-/- & mMT mice less protected against i.n.
SCHU S4 challenge after oral vaccination
• Limit of protection in mice <200 SCHU
9
Can Fischer 344 rats be used as a model
to study humoral immunity in vaccinated
humans?
10
Passive Immunization Protects Rats Against a Low
Dose SCHU S4 challenge
11
Passively Immunized Rats Exhibits Transient
Weight Loss and Mild Clinical Signs
12
Passively Immunization Does not Decrease
Initial Bacterial Growth
13
Summary
The protection of Fischer 344 rats by passive
immunization is consistent with historical data on
the effective treatment of human tularemia patients
with immune sera
14
Milestone 53-B
Characterization of protective immunity against pulmonary
tularemia via oral and intradermal vaccination in the F344 rat model
Characteristics of oral
vs. i.d. vaccination of
LVS/survival
Correlates of humoral
and cellular immunity
of LVS vaccination
Protective efficacy of
2 attenuated SCHU S4
strains
Intramacrophage survival
Vaccination/challenge
Bacterial dissemination
Histological analyses
CD4+ T cell
responses
Serum antibody responses
Secreted, BAL antibody
responses
Intramacrophage survival
vaccination/challenge
antibody responses
Bacterial dissemination and
Histology if warranted
Red: completed
Green: in progress
Blue: Steps in the milestone
15
Key Achievements
1) We have evaluated the ability of Francisella strains to
replicate within bone marrow-derived macrophages
from F344 rats
 Bone marrow culturing conditions are able to produce 95%
pure macrophage population
 Determined intramacrophage replication profiles of F. novicida,
LVS, F. holarctica and F. tularensis
 Francisella strains induce differential nitric oxide production in
bone marrow-derived macrophages
 F. holarctica and F. tularensis are able to replicate within
primary hepatocytes from F344 rats
16
Key Achievements
2) We have examined the protective capabilities of LVS
oral vs. intradermal vaccination against SCHU S4
pulmonary challenge
 Both oral and intradermal LVS vaccination induce elevated
levels of antigen-specific IFN-g
 Both routes of vaccination induce high serum, intestinal and
respiratory antibody titers
 Both routes of vaccination provide 100% protection against
pulmonary challenge with 104 CFU SCHU S4
17
Key Achievements
3) We have investigated the in vivo susceptibility of
F344 rats to F. novicida pulmonary challenge
 F344 rats are highly resistant to F. novicida intratracheal
challenge
 F. novicida challenge induces high cellular and humoral
responses
 F. novicida vaccination is highly protective against pulmonary
challenge with 104 CFU SCHU S4
18
Protective Efficacy of Oral vs.
Intradermal LVS Vaccination Against
SCHU S4 Pulmonary Challenge
19
Examine Protective Efficacy of Oral vs. Intradermal
LVS Vaccination Against SCHU S4 Pulmonary
Challenge
Cellular Response
Humoral Response
20
Examine Protective Efficacy of Oral vs. Intradermal
LVS Vaccination Against SCHU S4 Pulmonary
Challenge
Intestinal Antibodies
BAL Antibodies
21
Examine Protective Efficacy of Oral vs. Intradermal
LVS Vaccination Against SCHU S4 Pulmonary
Challenge
22
in vivo Susceptibility of F344 Rats to
Pulmonary F. novicida Challenge
23
Investigate in vivo Susceptibility of F344 Rats to
Pulmonary F. novicida Challenge
24
Bacterial Dissemination at Early Time Points
Following F. novicida Pulmonary Challenge
25
Investigate in vivo Susceptibility of F344 Rats to
Pulmonary F. novicida Challenge
26
Efficacy of F. novicida Vaccination Against
Pulmonary SCHU S4 Challenge
27
Work Plans for the Coming Six Months
Milestone 53B
 Further investigate alternative cell types for intracellular
replication
 Evaluate bacterial dissemination following SCHU S4
challenge of LVS vaccinated rats
 Perform histological analysis of LVS vaccinated rats
following SCHU S4 challenge
 Evaluate both F. novicida and F. tularensis attenuated
mutants for protective efficacy against SCHU S4
challenge
28
10/5/09: Action Items
• Action: UTSA may want give same doses
of heat killed bacteria vs. live bacteria, to
discriminate a shock response from other
types of immune responses.
29