Transcript UNM TVDC UTSATVDTech slides&Minutes 061907 final

University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
6/19/07 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 constructed uvrA::Kan construct, mated into LVS,
could not maintain antibiotic pressure
•We designed Targetron primers to target two different
potential sites within uvrA
•PCR performed, target sites inserted into Tulatron vector
to create two different uvrA Tulatron plasmids.
•We have stored these plasmids, since it was decided
that uvrA LVS mutant not necessary; however, if there
is free time we will go ahead and transform LVS with
plasmids and isolate uvrA LVS mutant anyways.
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•We have chosen PepO as our secreted protein to
tag with Tcell epitope SIINFEKL, this is a protease that
is the only protein characterized to be secreted in Ft
•We have constructed a Ft plasmid expressing
PepO-FLAG for other purposes.
•We have designed SIINFEKL complimentary oligos
to replace FLAG tag with SIINFEKL in this plasmid,
we have ligated into this plasmid and have identified
clones containing PepO-SIINFEKL (both in E. coli
expression vector pBAD24 and Ft expression vector)
(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 pdpD, 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
FLP-mediated excision of one FPI copy
• Schuh4 contains two identical copies of FPI, which has
genes targeted for deletion in this milestone.
• To facilitate strain construction in Schuh4, we are pursuing
Strategy to remove one copy of FPI in Schuh4
• This is “value-added” technology, we already developed
the components and have verified that it works in Ft
novicida.
• The strategy is to insert FLP recombinase recognition target
sites in both ends of the same FPI, then express FLP
recombinase to catalyze deletion of intervening DNA
(seen on following slide).
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FLP-mediated excision of FPI
pdpD iglA B C D
pdpB
pdpC
pdpA
FLP Recombinase
FLP Recombinase-mediated
recombination
Ft-FLP
“scar”
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• We already constructed pdpA::FRT Kan in Ft novicida,
We amplified the DNA from this strain containing 1000 bp
flanking homology
• This region was cloned into pUC plasmid containing
ermC, potential clone isolated, sent for sequencing.
• The potential correct clone was transformed into Schuh4,
KanR colonies isolated.
• Several clones grown in liquid media, then plated and
screened for KanR, ErmS (phenotype of correct strain).
A number of these colonies identified, currently being screened
Construction of mutagenesis vector
• We are re-constructing mutagenesis vector (R6K ori,
FTgroELp to drive antibiotic and sacB expression) for
mutagenesis.
• First step is PCR amplification to remove native promoter
for sacB, and introduce better MCS
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Currently screening clones.
Mutagenesis of iglC in Schuh4:
1. (low copy) vector
•iglC,plasmid transformed into AmpS Schuh4
strain, AmpR colonies isolated.
•AmpR was weak, colonies screened did not have
evidence of iglC.
•We need to re-engineer this plasmid with Ft promoter
to drive AmpR and repeat.
2. (Value added) Targetron
•We have constructed Targetron vector to target
knockout of iglC, and have successfully achieved
knockout of both copies of iglC in LVS
•We have transformed iglC Targetron into Schuh4,
first time poor transformation efficiency only gave
one colony.
•We have repeated and achieved many colonies,
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currently screening for iglC mutant.
•We are constructing iglD Targetron plasmids for
inactivation of iglD in Schuh4 as well.
•We have PCR amplified 2 different iglD targets
and are in the process of ligating into Targetron
vector
(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
•iglB::ermC chromosomal DNA prepared from Ft
novicida iglB::ermC strain, cryotransformed into
uvrB::Kan strain, ErmR colonies selected
•Colonies in process of being screened. This is
slightly different than previous strain constructs,
which were transformed to KanR
•Have not identified mutant yet, will transform iglB::ermC
mutant with uvrB::Kan DNA next.
(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 uvrBiglC 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 uvrBiglC. 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 30 days of vaccination.
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Results: uvrBiglC -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.
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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 uvrBiglC mutant after vaccination
Blood was collected from the PBS- and ΔuvrBiglC - immunized
mice at day 14 and day 28 after priming. Specific antiΔuvrBiglC total antibody titer as well as IgG1 and IgG2a
isotypes were determined by ELISA.
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10
8
8
Total Ab
Day 14
Titer (x1000)
6
6
Day 28
4
4
2
2
0
0
5
5
IgG1
4
4
3
3
2
2
1
1
0
0
PBS
105
106
ΔuvrBiglC
107
0
IgG2a
PBS
105
106
107
ΔuvrBiglC
Fig. 2. Humoral response to ΔuvrBiglC immunization. BALB/c mice were
intranasally immunized with 105, 106 or 107 CFU of the ΔuvrBiglC mutant or PBS
alone as mock vaccination. Sera were collected 2 weeks and 4 weeks after
immunization and used to determine titers of anti- ΔuvrBiglC specific antibody.
Results: Mice immunized with ΔuvrBiglC produced significant amounts of specific serum total antibody (at
day 14 after priming for all vaccination doses. The titers were increased at day 28 after priming (2 days before
bacterial challenge). Isotyping analyses indicated both Th1 (IgG2a) and Th2 (IgG1)- type antibodies were 18
produced in mice after the ΔuvrBiglC immunization.
Plan for following month:
Milestone #16: completed.
Milestone #39: completed.
Milestone #48: completed.
Milestone #43:
1. Test PepO-SIINFEKL tag for expression in E. coli
and Ft by Western blot
Milestone #49A:
1. Screen Schuh4 clones with iglC Targetron vector
for inactivation of both copies of iglC
2. Create and test iglD Targetron vector
3. Verify pdpA::FRT Shuh4 mutant, work on pdpD::FRT
insertion into these strains.
Milestone #51:
1. Finish uvrB::Kan iglB::ermC double mutant
Continued on following slide
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Plan for following month:
Milestone #50:
 Determine the LD50 of Ft subsp.
novicida uvrBiglD double mutant
 Monitor Ft subsp. novicida ΔuvrBiglD
replication and dissemination in mice
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UTSA 6/19 call: Action Items
•
Action:Bernard will be in Malaysia for the next UTSA tech call and
the time zone is 14 hrs ahead. For 7/17 meeting, Bernard will give his
slides to Karl in advance.
• Action: Barbara said Bernard and Karl’s group can arrive on Sunday
night for the 2007 TVDC Annual Meeting and drive up in one rented
car to Santa Fe together to stay at the Lodge at Santa FE, using the $83
government rate.
• Action item: added 7/5/07- Bernard will vaccinate mice intragastrically
with an unrelated enteropathic organism (e.g. salmonella or listeria)
and then challenge with SCHU S4 to show that the intragastric
protection is specific, rather than non-specific. UTSA would not
expect the salmonella or listeria vaccination to protect against SCHU
S4.
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