Transcript UTSATVDTechCall8 21 07PtI

University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
8/21/07 tech call
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Active milestones during last reporting period:
Milestone #43: Creation of uvrA and uvrB mutant F.
tularensis subsp. holarctica (LVS) strains
Milestone #49A: Construction of iglC F. tularensis subsp.
tularensis strain
Milestone #50A: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Milestone #51: Construction of F. tularensis subsp. novicida
uvrB + pdpD, iglA, iglB, iglC, iglD strains.
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Red: completed
Green: in progress
Blue: Steps in the milestone
Milestone 43
Creation of uvrA and uvrB mutant F.
tularensis subsp. holarctica (LVS) strains
Construct uvrB::Kan
mutagenesis plasmid
Mate into LVS,
select for transconjugate,
Counterselect for mutant
Added on:
Construct SIINFEKL-tagged
secreted protein for
expression in F. tularensis
Construct uvrA::Kan
mutagenesis plasmid
Mate into LVS,
select for transconjugate,
Counterselect for mutant
Verify mutants,
Pass on to Milestone 50
Construct PepO-SIINFEKL
plasmid
Transform into LVS,
Confirm expression
Pass on to Cerus
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Milestone #43: Creation of uvrA and uvrB mutant F.
tularensis subsp. holarctica (LVS) strains
•We have created uvrB::Kan LVS strain (documented
in previous reports)
•We also previously created uvrA Targetron vector which we
had stored away.
•This was transformed into LVS, and colonies
with uvrA Targetron insertion identified, verified
by sequencing.
•We removed TempS plasmid from
LVS uvrA::Targetron strain and verified uvrA::Targetron
Insertion (following page).
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2
3
4
5
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7 8 9 10
1.0kb
0.5kb
1.5kb
1.0kb
0.5kb
1.1kb ladder
2.Positive c otrol
3.W ild typ e LVS
4.C olony2
5.C olony 3
6.C olony 13
7.C olony 21
8.C olony 24
9.C olony 27
10.1kb ladder
11.1kb ladder
12.Positive c ontrol
13.W ild type LVS
14.C olony 2
15.C olony 3
16.C olony 13
17.C olony 21
18.C olony 24
19.C olony 27
20.1kb ladder
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Top gel: PCR with intron primer + uvrA primer, lanes 4-9
uvrA::Targetron LVS colonies, lane 3 wt LVS
Bottom gel: PCR with uvrA primers, lanes 14-19
uvrA::Targetron LVS colonies, lane 13 wt LVS
Both PCR reactions confirm uvrA::Targetron LVS strains 5
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75KD
50kd
Lane1: with1.5 glucose
1.Overnight culture for
pkek1169
2.1hr culture for pkek1169
3.2hrs culture for pkek1169
4.3hrs culture for pkek1169
5.4hrs culture for pkek1169
6.Overnight culture for
pkek1169
7.DH5  E.coli.
Lane2-6: with 1.5 Arabinose
Construction of SIINFEKL-tagged protein:
•We constructed PepO-SIINFEKL plasmids, both in E. coli
and Ft expression vectors, sequencing confirmed
these were correct.
•We have visualized PepO-SIINFEKL in pBAD plasmid
in E. coli by Western blot with -SIINFEKL monoclonal
(U. Mass)
Above: lane 1, E.c. with pBAD-PepO-SIINFEKL grown in
glucose (represses pBAD promoter), lanes 2-6 same
grown in arabinose (induces pBAD promoter) at 1, 2, 3, 4h
and overnight, lane 7 E.c. with no plasmid.
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Construction of SIINFEKL-tagged protein:
•We have been unable to detect PepO-SIINFEKL
expressed from Ft plasmid in E. coli, even though
sequencing confirms construct is correct.
•We are attempting to transform this plasmid into LVS,
so far unsuccessful
•If we achieve transformation, we will test with Western
to confirm expression, then send to Cerus.
(documented in UTSA TVD notebook #2)
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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 #49A: Construction of iglC F. tularensis subsp.
tularensis strain
•We had transformed iglC Targetron vector into Schuh4,
achieved many colonies.
•We achieved a number of colonies that appear to have
insertion in iglC (no wildtype band visible, suggesting
Inactivation of both iglC genes, found in FPI-I and FPI-II).
•To distinguish between FPI’s, we use specific primers
located outside FPI (long-range PCR, very difficult)
A. Outside FPI-II + intron
Primers
B. Outside FPI-II + iglC
Primers
Lane 2 Schuh4
Lanes 3-5 individual iglC::
Targetron mutants
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Conclusion: FPI-II iglC has targetron insertion
•PCRs with FPI-I have not been as clear, we’ve
had difficulty with long range PCR at this locus
•We are performing Western blot analysis in these
strains, we have IglC-specific antisera, to confirm
that BOTH copies of iglC have insertions
•We are also doing animal infections, if both copies
of iglC are inactivated, the strain will be attenuated,
if only one it will be virulent.
We have also been working on system to remove
one copy of the FPI to facilitate mutagenesis in Schuh4:
•This is “value-added” technology, we already developed
the components and have verified that it works in Ft
novicida. (described in last month’s conference call).
•Step 1: insert FRT site into pdpA in one FPI
(pdpA1::FRTKanR or pdpA2::FRTKanR
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• We transformed with pdpA::FRTKanR plasmid, we
have identified Schuh4 strains that appear to have
pdpA::FRT-KanR, now the question is, which FPI?
• This involves long-range PCR with primers outside
FPI-I and FPI-II coupled with Kan primers (difficult,
these are extremely long amplifications).
Left: pdpA primer + outside
FPI-I (should amplify all)
Right: Kan primer + outside
FPI-I (should only amplify
Insertions in FPI-I)
Lane 2: Schuh4
Lanes 4-7, potential pdpA::FRT
KanR clones
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Conclusion: 3 clones likely pdpA1 insertion, 1 clone maybe pdpA2
• Construction of pdpD::FRTermC vector
This will be Step 2 in removal of one copy of FPI
• We are currently working on finishing this plasmid,
two of three fragments are already cloned, we are
cloning the third.
(documented in UTSATVD Notebook 3).
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Red: completed
Green: in progress
Blue: Steps in the milestone
Milestone 51
Creation of uvrB + pdpD, iglA, iglB, iglC,
and iglD mutant F.
tularensis subsp. novicida double mutant strains
Construct uvrB::Kan
mutagenesis plasmid
Transform into pdpD, iglA,
iglB, iglC, iglD novicida
strains, isolate mutant
Verify mutants,
Pass on to Milestone 50
Construct iglB::ermC
mutagenesis plasmid
Transform into U112,
isolate mutant
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Milestone #51: Creation of uvrB + pdpD, iglA, iglB, iglC, iglD
mutant F. tularensis subsp. novicida strains
•uvrB iglA, uvrB iglC, uvrB iglD, and uvrB pdpD double
mutant Fn strains tested for intramacrophage growth
•uvrB iglA, uvrB iglC, and uvrB iglD strains defective, as
predicted, uvrB pdpD strain not defective, as predicted
•uvrB iglB strain being verified by sequence, will be tested
once verified
(documented in UTSATVD Notebook 2).
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Milestone 50
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 #50A: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Results Update
Evaluate the protective efficacy of the Ft subsp.
novicida uvrBiglD mutant as a vaccine
candidate
Groups of BALB/c mice (female, 4-6 weeks)
were intranasally (i.n.) immunized with 105, 106
or 107 CFU of ΔuvrBiglD. Mice treated with
PBS were used as a mock-control. The
immunized mice were challenged with 1000
CFU of F. novicida (~100 LD50) by the i.n.
route after 21 days of vaccination.
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100
105 CFU
106 CFU
107 CFU
PBS
% Survival
80
60
40
20
0
0
6
12
18
24
30
% Body weight
110
105
100
95
90
85
80
0
2
4
6
8
10
12
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Days after challenge
Fig. 1. Protective efficacy of ΔuvrBiglD immunization against F. novicida infection.
BALB/c mice were immunized intra-nasally with 3 doses (105, 106, and 107 CFU) of
ΔuvrBiglD or PBS and i.n. challenged with lethal dose of F. novicida (1000CFU). Mice
were monitored for survival rate and weight change.
Results: ΔuvrBiglD-vaccinated mice were highly protected against subsequent pulmonary challenge with F. novicida. No
significant loss of body weight was also observed in the protected animals. As expected PBS-treated mock-vaccinated
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mice succumbed by day 6.
Milestone #50A: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Results Update
Analyze the antibody profiles of mice immunized with the Ft
novicida uvrBiglD mutant after vaccination
Blood was collected from the PBS- and ΔuvrBiglDimmunized mice at day 21 after priming. Specific antiΔuvrBiglD total antibody titer as well as IgG1 and IgG2a
isotypes were determined by ELISA.
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3000
PBS
uvrBiglD 10 5
uvrBiglD 10 6
uvrBiglD 10 7
Ab Titer
2500
2000
1500
1000
0500
500
00
Total Ab
IgG1
IgG2a
Fig. 2. Humoral response to ΔuvrBiglD immunization. BALB/c mice were
intranasally immunized with 105, 106 or 107 CFU of the ΔuvrBiglD mutant or PBS
alone as mock vaccination. Sera were collected 3 weeks after immunization and
used to determine titers of anti- ΔuvrBiglD specific antibody.
Results: mice immunized with ΔuvrBiglD produced significant amounts of specific serum total antibody.
Isotyping analyses indicated both Th1 (IgG2a) and Th2 (IgG1)- type antibodies were produced in mice after
the ΔuvrBiglD immunization. No ΔuvrBiglD- specific antibody was detected in mice mock-vaccinated with
PBS at day 21 after immunization. All tested serum samples showed no reactivity to the unrelated HEL
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protein.
Plan for following month:
Milestone #16: completed.
Milestone #39: completed.
Milestone #48: completed.
Milestone #43:
1. Transform PepO-SIINFEKL Ft plasmid into LVS, test for
expression by Western blot.
2. Construct new Ft PepO-SIINFEKL plasmid from E.c.
PepO-SIINFEKL construct, which expresses protein
Milestone #49A:
1. Western blot analysis and animal infections with iglC::
Targetron Schuh4 strain to confirm both iglC genes mutated
2. Verify pdpA::FRT Shuh4 mutant, work on pdpD::FRT
insertion into these strains.
Milestone #51:
1. Finish analysis of uvrB::Kan iglB double mutant,
send double mutants to Cerus.
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Continued on following slide
Plan for following month:
Milestone #50:
 Determine the LD50 of Ft subsp.
novicida uvrBiglA double mutant.
 Monitor Ft subsp. novicida ΔuvrBiglA
replication and dissemination in mice.
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