University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
10/20/09 tech call
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Active milestones during last reporting period:
Milestone #49: Construction of nadM, FTT0748 F. tularensis subsp.
tularensis strains (finished this milestone)
Milestone #52: Create recA mutants in F. tularensis subsp. tularensis
Milestone #53: Immune characterization of F. tularensis subsp.
tularensis mutant strains
Milestone #54: Construction of mutant F. tularensis subsp.
tularensis strains (Just started)
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Red: completed
Green: in progress
Blue: Steps in the milestone
Milestone 49
Creation of mutant F. tularensis
subsp. tularensis strains
A. Construct iglC
mutagenesis plasmid(s)
Transform into Schuh4,
select for transconjugate,
Counterselect for mutant
B. Construct vgrG, iglD
mutagenesis plasmids
Mate into Schuh4,
select for transconjugate,
Counterselect for mutant
Verify mutants,
Pass on to Milestone 50
C. Construct nadM,
FTT0748
mutagenesis plasmids
Mate into Schuh4,
select for transconjugate,
Counterselect for mutant
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Milestone #49: Construction of nadM, FTT0748 F. tularensis subsp.
tularensis strain
•We were ultimately unsuccessful in constructing
a nadM (Cterm) Schuh4 mutant via nadM targetron plasmid
(see previous reports); we identified mutant but could not
purify, suggesting this mutation is lethal.
•We successfully constructed FTT0748 Schuh4 strain.
•We inoculated FTT0748 strain into mice via intranasal
Inoculation
•This strain is NOT attenuated for virulence.
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Red: completed
Green: in progress
Blue: Steps in the milestone
Milestone 52
Creation of recA mutant F.
tularensis subsp. tularensis mutant strains
Construct recA
mutagenesis plasmid
Transform into Schuh4,
isolate mutant
Verify mutants,
Pass on to Milestone 50
Transform into iglC,
vgrG, iglD (other)
Schuh4 strains,
isolate mutants
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Breaking down restriction barriers in Schuh4:
•We constructed Schuh4 strain with insertion in
FTT1579 (restriction enzyme), then removed
Targetron plasmid = KKT19
•We are now constructing targetron plasmid to inactivate
second restriction enzyme (Ftt-specific) FTT0523
•Have ordered primers, PCR-amplified fragment, and cloned
into the targetron vector.
•Are proceeding with making double mutant (FTT1579 + FTT523)
FTT523 will also be inactivated in Schuh4 (wt) strain
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Red: completed
Green: in progress
Blue: Steps in the milestone
Milestone 54
Creation of mutant F. tularensis
subsp. tularensis strains
Construct lpxF, atpC, 3 other
mutagenesis plasmids
Mate into Schuh4,
select for transconjugate,
Counterselect for mutant
Verify mutants,
Pass on to Milestone 50
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Milestone #54: Construction of mutant F. tularensis subsp.
tularensis strains
Inactivation of lpxF, atpC in SchuhS4:
•We identified potential target sites within lpxF and atpC genes
•We ordered primers for construction of targetron plasmid
•We PCR-amplified fragments for each (lpxF and atpC), and
are cloning each into the targetron vector.
•Once constructed, we will transform into Schuh4 (wt) strain
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Milestone 53A
Immunologic characterization of defined
F. tularensis mutants
Strains from milestone #52
And #54 : nadM, ipxF, atpC
In vitro growth
In vivo bacterial burden
LD50 determination
Red: completed
Green: in progress
Blue: Steps in the milestone
F. tularensis rec A
recAiglC
In vitro growth
In vivo bacterial burden
LD50 determination
Further immunological characterization
based on initial screen
Milestone #53A: Immunologic characterization of defined
F. tularensis mutants
Results Update
Cellular cytokine recall responses to i.n. and oral KKF235
(iglB of F. novicida) vaccination
C57BL/6 mice were intranasally or orally vaccinated with KKF235
(104 CFU). Spleen, cervical lymph nodes (CLNs), and mesenteric
lymph nodes (MTLs) were collected at day 14 after vaccination,
single cells were made and reacted with UV-inactivated KKF235
cells or HEL (an unrelated antigen). Frequency of IL-2 secreting
cells were measured by ELISPOT. Age matched naïve mice were
used as a mock control.
Cellular cytokine recall responses to i.n. and oral
KKF235 (iglB of F. novicida) vaccination
Milestone 53-B
Characterization of protective immunity against
pulmonary tularemia via oral 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
Red: completed
Green: in progress
Blue: Steps in the milestone
Milestone #53B: Characterization of protective immunity against
pulmonary tularemia via oral vaccination in the F344 rat model
Results Update
Intracellular replication of Francisella Strains
We have previously reported the replicative ability of Francisella
strains in bone marrow derived macrophages from F344 rats. Since
the highly virulent SCHU S4 strain was unable to replicate in these
cells, we investigated the ability of both Type B, which did replicate
in BMDM, and SCHU S4 to replicate in primary hepatocytes.
Intracellular Replication Within F344 Hepatocytes
Plan for following month:
Milestone #16: completed.
Milestone #39: completed.
Milestone #48: completed.
Milestone #43: completed.
Milestone #50: completed.
Milestone #51: completed.
Milestone #49: completed.
Milestone #52:
1. Construction of FTT0523 (restriction enzyme #2)
single and double (+FTT1579) Schuh4 mutants.
Milestone #54:
1. Obtain lpxF and atpC targetron plasmids, transform
Schuh4.
Continued on following slide
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Plan for following month: Milestone #53-A&B:
53A:
(1) Comparison of cell mediated response by i.n. and oral
KKF235 (iglB of U112) immunization using IFN-g
ELISPOT analyses
53B:
(1) Bacterial dissemination profile following oral and
intradermal LVS vaccination and subsequent SCHU S4
challenge
Action Items
• Barbara: email all 5 institutions to convey
that the monthly technical slides will
replace the monthly technical reports.
• Bernard: will submit MS 50 MSCR to
Barbara/UNM
• UTSA: will focus released efforts on
publications, MSCRs and scientific efforts
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