University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
12/15/09 tech call
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Active milestones during last reporting period:
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
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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
Generate, optimize
mutant strain construction
in Schuh4
Transform into iglC,
vgrG, iglD (other)
Schuh4 strains,
isolate mutants
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Breaking down restriction barriers in Schuh4:
•(last month) We constructed targetron plasmid to inactivate
second restriction enzyme (Ftt-specific) FTT0523 (pKEK1282)
•We transformed pKEK1282 into Schuh4; screened
transformants for presence of insertion in FTT0523:
PCR with external primers show that the eight
colonies tested (lanes 2-9) have “pure” insertion
in FTT0523 (compare to Schuh4 in lane 10).
We subsequently removed the plasmid by growth at 37°C,
resulting strain KKT28 is Schuh4 FTT0523 strain, will
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be tested for transformation efficiency.
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:
•(last month) plasmid targeting lpxF transformed into Schuh4
strain, transformants screened for presence of insertion in lpxF.
•We continue to cycle transformants to obtain pure lpxF mutant:
Cycle 3
PCR with external primers shows the
presence of insertion (arrow), but also
presence of wildtype lpxF in all colonies.
Note increasing prevalence of insertion from
cycle 3 to cycle 4. We continue to cycle
to obtain pure lpxF mutant.
Cycle 4
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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
Evaluation of an SCHU S4 restriction enzyme (FTT0523)
mutant KKT-28
FTT0523 functions as a restriction enzyme for defragmenting the
foreign DNA entering into SCHU S4 strain. Mutation of this gene
allows stabilization of the foreign plasmid. Murine macrophage cell
line (J774) were infected with KKT-28 or its parental strain (SCHU
S4) using an inoculum of 10 or 100 MOI. Numbers of viable
bacteria in macrophages were measured at 3 hr and 24 hr postinfection.
Intramacrophage Replication of KKT28
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
Evaluation of the cellular response of Fischer 344 rats
following intratracheal vaccination with Francisella subsp.
novicida iglB.
Fischer 344 rats were vaccinated i.t. with 107 CFU of the iglB
strain or mock vaccinated with PBS and rested for 14 days to ensure
enough time to develop a cellular response. Single cell suspensions
from spleens and cervical lymph nodes were recalled in vitro for 72
hours with UV-inactivated iglB and the unrelated antigen HEL.
Cellular Response of Fischer 344 rats Following Intratracheal
Vaccination with Francisella subsp. novicida iglB.
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 +FTT1579 Schuh4 double
mutant.
2. Test mutants for transformation efficiency.
Milestone #54:
1. Continue construction of lpxF Schuh4 mutant.
2. Construct atpC targetron plasmids, transform
Schuh4.
Continued on following slide
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Plan for following month: Milestone #53-A&B:
53A: Bacterial dissemination profile following oral and
intratracheal U112 vaccination and subsequent SCHU S4
challenge in Fischer 344 rats.
53B: Antibody responses in Fischer 344 rats following oral
KKF235 (iglB of U112) immunization.
Additional points:
Description of deliverables completed for each active milestone:
Milestone 52: Schuh4 recA, iglC1 iglC2 recA, FTT1579, FTT523
strains
Milestone 53: None at this time
Milestone 54: None yet, in process
List of relevant publications from the past month
None
MSCR status
MS 50: UNM reviewing the MSCR from UTSA (HR sent to BG 11/19/09)
MS 49: UTSA writing MSCR (MS 49 was scientifically done 11/17/09)
MS 51: UTSA reviewing revised MS51 MSCR (BG sent edits on 12/4/09)
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Action Items
• Freyja asked UTSA to perform minimal
comparison experiments between the mouse
and rat models.
• UTSA will work on the 3 MSCRs.
• Freyja: try to review UTSA rat model paper
by 12/18/09. Patrick Sanz already reviewed
it. (completed 12/17/09 and communicated
to UTSA)
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