Transcript UTSATVDTechCall2-19-08 final minutes

University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
2/19/08 tech call
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Active milestones during last reporting period:
Milestone #49B: Construction of iglD, vgrG F. tularensis subsp.
tularensis strain
Milestone #50: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Milestone #52: Create recA mutants in F. tularensis subsp. tularensis
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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 iglA, iglB
mutagenesis plasmids
Mate into Schuh4,
select for transconjugate,
Counterselect for mutant
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Milestone #49: Construction of vgrG, iglD F. tularensis subsp.
tularensis strain
•We are working on creating two different mutant
Schuh4 strains: iglD and vgrG
•We are utilizing Tulatron technique
•We have constructed two different vgrG Tulatron
vectors, these target two different sites within vgrG
(30|31and 81|82)
•Each has been transformed into Schuh4 to generate
vgrG mutant
•Transformants have been isolated, primary transformants
screened first for native length vgrG gene:
Lanes 2&3 Schuh4
Lanes 4-11
Representative
Transformants
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•We could not detect insertion in initial screen, however
more diagnostic screen of intron-specific primer plus
vgrG primer will identify insertion in mixed colonies.
•Currently performing re-streaking to segregate
mutant colonies, also additional PCR screens.
•We appear to have constructed two iglD tulatrons,
(30/31 and 255/256)
Example shown, 30|31
Construct, tulatron gives
3 digested fragments,
Smaller size of largest
Fragment in lanes 5-9
Indicate correct clones
We’re transforming each into Schuh4 to isolate iglD
mutant
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(documented in UTSA TVD notebook #1 and #5)
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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•Tulatron vectors created to target two different
sites within recA, transformed first into LVS
•(Ping still waiting for BSL3 access)
•LVS transformant clones screened to identify recA
mutants at 840|841:
•Lanes
arethe potential
clones
showing
with
tulatron
Lane2 2-5
to lane5 were
Rec A mutants
with the PCR recA
produ ct at about
1500bp,
whereas about
630bp yield for wild type LVS (lane 6). Since the two primers flank ed RecA gene, we c ould be sur e that
the retargetedlane
intron RNA6
hadis
beenLVS
inserted into
RecA of LVS. There
was a very weak
band at about
the
Insertion,
parent,
primers
flank
recA
same size(600bp) as wt LVS (lane6) on lane5. It was probable that LVS with insertion mixed with LVS
the plasmid inside but no insertion happening.
•We with
are
now streaking at higher temperature (37°C)
to facilitate loss of tulatron plasmid
(documented in UTSA TVD notebook #2)
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Milestone 50-A
Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
F. novicida uvrA, uvrB
Double mutant
F. novicida uvrA+pdpD
F.novicida uvrB+pdpD
iglA, iglB, iglC, iglD
In vitro Growth
In vivo Bacterial Burden
LD50 determination
In vitro Growth
In vivo Bacterial Burden
LD50 determination
Red: completed
Green: in progress
Blue: Steps in the milestone
LVS uvrA, uvrB
F. tularensis Schu4 iglC
In vitro Growth
In vivo Bacterial Burden
LD50 determination
Further immunological characterization
based on initial screen
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Milestone 50-B
Characterization of protective immunity against
pulmonary tularemia via intra-gastric LVS vaccination
Duration and limits of
protective efficacy
Correlates of humoral
and cellular immunity
Survival 1, 2, 3 months
Vaccination/boost strategy
Bacterial dissemination
Histological analyses
CD4+ and CD8+ T cell
responses
Serum antibody responses
Secreted, BAL antibody
responses
Red: completed
Green: in progress
Blue: Steps in the milestone
Contribution of cell
mediated and
humoral immunity
CD4+, CD8+, B cell depletion
vaccination/challenge
KO mice vaccination/challenge
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Milestone #50A: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Results Update
Measure intramacrophage (J774) replication of Ft
subsp. tularensis (SCHU S4) iglC
We have expanded frozen bacterial stocks of the
wild type SCHU S4, and iglC mutant for these
analyses.
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Milestone #50A: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Results Update
Evaluate the protective efficacy of F. tularensis SCHU S4 iglC
vaccination against wild type SCHU S4 challenge
Groups of BALB/c mice (female, 4-6 weeks) have been
immunized with 103 CFU of iglC intragastrically (i.g.) or
intradermally (i.d.). Mice treated with PBS were used as a
mock-control. These mice will be challenged i.n.or i.d. with
two doses of SCHU S4. Animals will be monitored for survival
and weight loss.
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Milestone #50B: Characterization of protective immunity against
pulmonary tularemia via intra-gastric LVS vaccination
Results Update
Measure bacterial dissemination in various
tissues at early time points following
intragastric LVS immunization
BALB/c mice were challenged with LVS (~103
CFU) intragastrically. Lungs and trachea were
collected from the infected mice at 30 min and
at days 1, 2, 3 and 5 after challenge (3 mice per
time point). Liver, spleen, cervical and
mesenteric lymph nodes were collected from
the infected mice at days 1, 2, 3, 5 and 7 after
challenge. Numbers of bacteria in each organ
were determined by dilution plate counting.
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CFU Per Organ
A
10
6
10
10
6
5
10
5
10
4
10
4
10
3
10
3
Trachea
30
Min
1
2
3
5
Da y s Afte r In o c u l a ti o n
Lungs
30
Min
1
2
3
5
Da y s Afte r In o c u l a ti o n
Results: As shown in Fig. 2a, there were no detectable bacteria present in either the trachea or lungs within 30
minutes or 1 day post-inoculation, with gradual increases of viable bacteria recovered from days 2 to 5 in the
lungs
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CFU Per Organ
B
10
6
5
10
5
10
4
10
4
10
3
10
3
10
6
10
6
10
5
10
5
10
4
10
4
10
3
10
3
10
6
10
Liver
CLN
1
2
3
5
7
Spleen
MLN
1
2
3
5
7
Day s After Inoc ulation
Fig.2. Kinetic growth and clearance of Ft LVS in target organs after intragastric vaccination. (A) Bacterial burdens
were determined in the trachea and lungs per individual mouse. (B) Bacterial burdens were determined in the liver
and spleen per individual mouse and cervical and mesenteric lymph nodes per group of mice. Dashed lines
indicate level of detection.
Results: As shown in Fig. 2b, elevated numbers of bacteria were not present in the liver and spleen until day 5
after inoculation which is consistent with results reported previously (Nov. 2007). There were minimal bacteria
recovered from both cervical and mesenteric lymph nodes after challenge.
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Plan for following month:
Milestone #16: completed.
Milestone #39: completed.
Milestone #48: completed.
Milestone #43: completed.
Milestone #51: completed.
Milestone #49:
1. Identify iglD tulatron insertion within transformed Schuh4.
2. Identify vgrG Schuh4 mutant among transformants.
3. Construct pdpD::FRT in pUC-based vector
Milestone #52:
1. Remove tulatron plasmid from recA LVS,
Assay LVS recA for virulence traits (macs, mice)
2. transform into Schuh4, isolate recA mutant.
Continued on following slide
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Plan for following month:
Milestone #50-A&B:
50A: (1) Measure intramacrophage (J774) replication of Ft subsp. tularensis (SCHU S4) iglC.
(2) Evaluate the protective efficacy of F. tularensis SCHU S4 iglC vaccination against wild type
SCHU S4 challenge:
(3) Evaluate the protective efficacy of intragastric F. novicida iglB vaccination against SCHU S4
intranasal and intradermal challenge in C57BL mice.
50B: (1) Evaluate the protective efficacy of intragastric LVS vaccination against Francisella type A
SCHU S4 intranasal challenge at 8 weeks after either a single vaccination or after receiving a
secondary booster dose.
(2) Analyze antigen-specific cytokine production in spleens and lymph nodes at both 2 and 4 weeks
after intragastric inoculation with LVs.
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Action items
UTSA Will show UNM and NIAID the successful vgrG
insertion data next month.
• UTSA Will report next month on the iglD transformations
into SCHUS 4.
• UTSA is performing side by side comparison with
intentional lung infection vs intentional GI infection;
• UTSA will try PCR to detect the delivery of bacteria to
lungs, trachea (at 30 min) and to the liver at 2 days
• UTSA should make luciferase expressing plasmid using
the LUX so don’t need to worry about substrate getting to
the bacteria
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