Transcript UTSA TVDC TechCall minutes 12-18-07

University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
12/18/07 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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During Nov 2007, the BSL-3 laboratory was shut down for
mandatory annual repairs, testing, and recertification.
The laboratory was back “on-line” as of 11/14/07
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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 working on creating two different mutant
Schuh4 strains: iglD and vgrG
•We are utilizing Tulatron technique
•Splicing by overlap PCR performed to generate fragment
to insert vgrG target sites (2 different sites chosen; 30/31
and 81/82) into tulatron vector
•One apparent correct clone for each vgrG target site
Identified, will be sequenced to verify
•Each will be transformed into Schu4 to generate
vgrG mutant
•iglD tulatron cloning ongoing, clones to insert into two
sites (30/31 and 255/256) in the process of being
constructed
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We have also been working on system to remove
one copy of the FPI to facilitate mutagenesis in Schuh4:
•Step 1: insert FRT site into pdpA in one FPI
(pdpA1::FRTKanR or pdpA2::FRTKanR)
We have accomplished this.
• Step 2 : Construction of pdpD::FRTermC vector
We appear to have constructed the correct clone:
FRT ermC
pdpD3’
pdpD5’
Legend:
BamHI / Not I Digested
1. 1 Kb Ladder ( Invitrogen )
1 Kb
Indicates
Correct
fragment
1
2
3
4
5
6
7
8
2. Uncut pKEK1042
3. pKEK1042
9.0
4. C1 pKEK1042+5Õ
pdpD
5. C2 pKEK1042+5Õ
pdpD
1.0
6. C4 pKEK1042+5Õ
pdpD
7. C9 pKEK1042+5Õ
pdpD
8. Uncut C2
pKEK1042 +5Õ
pdpD
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•We will need to move this entire construct into
a pUC-based plasmid, and then transform Schu4
in which we have already inserted the other FRT site
into pdpA (reported previously)
•Expression of the FLP recombinase in these strains will
result in excision of entire FPI
(documented in UTSA TVD notebook #1 and #5)
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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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•Splicing by overlap PCR performed to generate fragment
to insert into tulatron vector, two targets chosen for
recA gene (720/721 and 840/841)
•Fragments cloned into tulatron vector (all appear correct):
•One each (lanes 4 and 10) sequenced, confirmed
to be correct, will be utilized to transform Schuh4
(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
Determine the LD50 of Ft subsp. novicida
uvrBpdpD double mutant
Groups of BALB/c mice (female, 4-6 weeks)
were intranasally (i.n.) challenged with 105, 106
or 107 CFU of ΔuvrBpdpD and monitored daily.
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% Survival
100
80
60
40
20
% Body weight
0
110
105
100
95
90
85
80
0
4
8
12
16
20
105 CFU
106 CFU
107 CFU
0
2
4
6
8
10
12
14
Days post-challenge
Fig.1. Survival of mice infected with Ft subsp. uvrBpdpD double mutant.
Groups of BALB/c mice (female, 6-week old) were challenged intra-nasally with
3 doses (105, 106, and 107 CFU) of ΔuvrBpdpD to determine LD50 of this strain.
Results: Mice infected with 105 CFU of the bacteria exhibited 80% survival, while animals infected with 10 6 CFU and 107
CFU maintained a 40% survival rate respectively. The LD50 of ΔuvrBpdpD in the intranasal infection model (BALB/c
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mice) was determined to be between 105 and 106 CFU
Milestone #50A: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Results Update
Monitor Ft subsp. novicida ΔuvrBpdpD replication and
dissemination in mice after intranasal challenge
BALB/c mice were challenged with ΔuvrBpdpD mutant (105
CFU) intranasally. Lungs, liver, spleen, and lymph nodes were
collected from the infected mice at day 3, 7 and 14 after
challenge. Numbers of bacteria in each organ were determined
by dilution plating .
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9
7
5
Lung
Spleen
*
Log 10 CFU / Organ
3
1
9
7
Spleen
Spleen
Liver
Spleen
C
Spleen
*
Lymph nodes
5
*
3
1
3
7
14
3
Days after Challenge
7
14
Fig. 2 Kinetic growth and clearance of Ft novicida ΔuvrpdpD in target
organs after i.n. vaccination. Bacterial burdens were determined from lungs,
liver and spleen of individual mouse and from pooled lymph nodes at each
time point (3 mice per time point). Numbers (*) of mice without detectable
bacterial burden are indicated.
Results: There was heightened replication of the organism in the lungs within the first 7 days post-challenge, with reduction
observed at day 14. The bacteria rapidly disseminated to lymph nodes, liver and spleen as early as 3 day after intranasal infection.
At day 14, significant numbers of bacteria can still be found in the lungs, liver, spleen and lymph nodes from at least two of the
three infected mice. Dissemination of ΔuvrBpdpD from lungs to lymph nodes and liver was faster than our previously
characterized ΔuvrBiglA (see August 2007 report), and ΔuvrBiglC (April 2007 report). Bacterial loads in the target organs of
ΔuvrBpdpD-infected mice are also higher than uvrBigl mutants (ΔuvrBiglA,B,C,D). In summary, ΔuvrBpdpD is moderately
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attenuated but more virulent than uvrBigl mutants.
Milestone #50B: Characterization of protective immunity against
pulmonary tularemia via intra-gastric LVS vaccination
Results Update
Monitor LVS replication and dissemination in
mice after intragastric immunization
BALB/c mice were challenged with LVS (1600
CFU) intragastrically. Lungs, liver, spleen, and
lymph nodes were collected from the infected
mice at day 1, 2, 3, 5, 7 and 14 after challenge
Numbers of bacteria in each organ were
determined by dilution plating. .
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CF U Per Organ
10 8 Lungs
10 7
10 6
10 5
10 4
10 3
10 8 Spleen
10 7
10 6
10 5
10 4
10 3
1 2 3
10
10
10
10
10
10
8
Liv er
7
6
5
4
3
10 8 Ly mph Nodes
10 7
10 6
10 5
10 4
10 3
1 2 3 5 7
5 7 14
Days After Inoculation
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Fig. 3 Kinetic growth and clearance of Ft LVS in target organs after i.g.
vaccination. Bacterial burdens were determined from lungs, liver, spleen and
lymph nodes of individual mouse (3 mice per time point).
Results: There was heightened replication of the organism in the lungs within the first 7 days post-inoculation,
with decreased numbers of viable bacteria recovered at day 14. Elevated numbers of bacteria were not present
in the liver and spleen until day 5 after inoculation, after which bacterial numbers in the spleen decline until
day 14 while they persist in the livers. Low numbers of bacteria were detected in the lymph nodes.
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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. Construct iglD tulatron vector.
2. Transform vgrG tulatron vector into Schuh4.
3. Move pdpD::FRT construct into pUC-based vector
Milestone #52:
1. Transform recA tulatron vector into Schuh4.
Continued on following slide
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Plan for following month:
Milestone #50-A&B:
1.
Evaluate the protective efficacy of the Ft subsp. novicida
uvrBpdpD mutant. Groups of vaccinated mice will be challenged
i.n. with Ft subsp. novicida. Animals will be monitored for
survival and weight loss.
2.
Analyze the antibody profiles of mice immunized with the Ft
novicida uvrBpdpD mutant at day 14 and 28 after vaccination.
3.
Evaluate the protective efficacy of intragastric LVS vaccination.
Groups of vaccinated mice will be challenged i.n. with two doses of
Ft subsp. tularensis SCHU S4 at three weeks after inoculation.
Animals will be monitored for survival and weight loss.
4.
Analyze the serum, fecal and respiratory antibodies of mice
vaccinated intragastrically with LVS at 21 days after immunization.
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Action Items
•
•
•
•
•
Karl/Bernard- UTSA has essentially exhausted studying the uvr mutants, and have
adhered to the schedule. UTSA plans to publish on the uvr mutants as UTSA has
accumulated ample data on these mutants.
Bernard- will do a BAL (Broncheoalveolar lavage) on the animals immediately after IG
vaccination to be sure that the mice are not aspirating the bacteria into the lungs and
effectively getting a direct lung infection of LVS.
Bernard- After IG vaccination, UTSA will determine the bacterial burden in mesenteric
lymph nodes and not the cervical lymph nodes as has been performed to date.
Karl will send UNM a copy of the Tulatron paper before it is submitted for publication
as a courtesy. The tulatron system was developed under UTSA PO1, and is "value
added" technology to the TVDC. After developing the Tulatron, UTSA moved the
technology into the TVDC and it has greatly facilitated contract work. However, the
paper is describing the development of this system, and there is no overlap with the
contract. Therefore it doesn't require approval by NIH, will not be attributed to the
contract.
Barbara soon will be emailing UTSA regarding possible dates for the annual site visit to
UTSA for spring 2008
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