University of Texas San Antonio
F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
10/8/07 Annual Meeting
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Progress on milestones to date:
Milestone #16: Create luciferase expressing F. tularensis LVS
COMPLETE 4/30/06
Milestone #39: Creation of uvrA and uvrB mutant F.
tularensis subsp. novicida strains
COMPLETE 8/31/06
Milestone #43: Creation of uvrA and uvrB mutant F.
tularensis subsp. holarctica (LVS) strains
COMPLETE 8/31/07
Milestone #48: Characterize uvrA and uvrB mutant F.
tularensis subsp. novicida strains
COMPLETE
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Milestone #49: Construction of mutant F. tularensis subsp.
tularensis strains
ONGOING
Milestone #50: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
ONGOING
Milestone #51: Construction of F. tularensis subsp. novicida
uvrB + pdpD, iglA, iglB, iglC, iglD strains.
COMPLETE
Milestone #52: Construction of mutant F. tularensis subsp.
tularensis strains containing recA mutations
ONGOING (just started 9/15/07)
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Our laboratory is focusing on the generation and characterization
of live attenuated F. tularensis subsp. novicida, tularensis, and
LVS strains for their vaccine potential
We are performing targeted disruption of various genes, including:
uvrA
uvrB
These genes are involved in DNA repair following
UV damage, their inactivation facilitates KBMA
technique of Cerus
pdpD
iglABCD
These are virulence genes located in pathogenicity
island, they have high potential to attenuate strain
recA
This gene facilitates genetic recombination; its
inactivation will stabilize potential vaccine strain
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We have spent a considerable amount of time developing
and optimizing techniques for genetic manipulation of
Francisella tularensis with SUCCESS!
Major accomplishments have included:
1. New optimized targeted mutagenesis of Ft novicida
2. New plasmid-based mutagenesis of Ft holarctica (LVS)
3. New technique to mutagenize Ft tularensis and Ft holarctica
“Tulatron”
4. Progress in removing 1 copy of FPI from Ft tularensis
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Milestone 39 & 51: Creation of uvrA, uvrB and uvrB + pdpD, iglA, iglB,
iglC, and iglD Ft novicida strains
Optimized strain construction in Ft novicida:
1. Optimized antibiotic resistance
2. Optimized splicing by overlap extension
3. Universal priming sites allow easy exchange of resistance genes
4. Non-polar nature of insertion mutations
PUBLISHED!
Liu J., Zogaj, X., Barker, J. R., Klose, K. E. 2007. Construction of
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targeted insertion mutations in Francisella tularensis subsp. novicida. Biotechniques
Milestone #43: Creation of uvrB mutant F.tularensis subsp. holarctica
(LVS) strains
•We have created new low copy vector for mutant construction in
LVS/Schuh4, pKEK1090, this is conjugative, conditionally replicative,
with FT groELp to drive sacB (counterselective marker) and CmR
•uvrB::Kan was inserted into this plasmid
•Successfully created uvrB::Kan LVS strain with this plasmid
groELp
MCS
sacB
CmR
oriR6K
mob
MCS= NotI
ApaI
PstI
MluI
XhoI
XbaI
NdeI
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Milestone #43 & 49: Creation of mutant Ft subsp. tularensis and holarctica
strains
We have adapted Targetron Group II intron mutagenesis system
to work at high efficiency in F. tularensis (value added!)
•Group II introns consist of
RNP (RNA molecule + LtrA
protein)
•Target specific sites by basepairing
of two loops in RNA molecule
plus flanking sequences
recognized by LtrA
•RNA is spliced into target site,
reverse transcribed by LtrA
Yao and Lambowitz, AEM 73:2735
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•RNA loops EBS1 and EBS2
basepair with specific sequences
in target
•LtrA recognizes flanking sequences
•EBS1 and EBS2 can be “retargeted”
to recognize sites in target gene
•LtrA site preferences within target gene
are identified by Targetron computer
algorithm, which designs appropriate oligos
to retarget intron to your gene
Tulatron contains:
1. Ft promoters to drive
Antibiotic resistance
Intron RNP
2. Ft ori
3. ts mutation
4. Ec ori
5. KanR
6. lacZa “stuffer”
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Tulatron works to simultaneously inactivate both copies
of iglC found in duplicated FPI! (data from LVS)
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Ftt iglC1::L1.LtrB iglC2::L1.LtrB mutant strain virulence:
CFU (intranasal route)
7.5 X 101
9.0 X 102
9.0 X 103
9.0 X 104
9.0 X 105
PBS control
Survival
(30 days)
Schuh4
Challenge
(210 CFU i.n.)
survival
5/5
5/5
5/5
0/5
0/5
0/5
5/5
5/5
N.A.
0/5
0/5
0/5
The Ftt iglC mutant is highly attenuated, but not protective
via the intranasal route
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We are also in process of removing one copy of FPI from
Ftt so that we can mutagenize other copy, by recombinasemediated excision, this will facilitate easy mutagenesis
of the second FPI:
pdpD iglA B C D
FRT site
IN PROGRESS
pdpC
insertion of FRT
sites via allelic
exchange
pdpB
pdpA
FRT site
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DONE
FLP-mediated excision of FPI
pdpD iglA B C D
pdpB
pdpC
pdpA
FLP Recombinase
FLP Recombinase-mediated
recombination
Ft-FLP
“scar”
This process works, we’ve already accomplished this in Ft novicida
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(value added technology)
Some of the things we have learned about genetic
manipulation of F. tularensis:
Use of Ft promoter to drive antibiotic resistance
is essential (both ermC and KanR work well)
Amount of flanking homology is critical for Ftt and Fth
(>=1 kbp)
Second recombination (loss of plasmid) does not
occur at high enough frequency without counterselection
Tulatron works well in Ftt and Fth
Unmarked deletions extremely difficult in Ftt, marked
deletions/insertions have been successful
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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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Protective Efficacy of F. novicida Mutants Against Pulmonary Tularemia
uvrAuvrB
iglB
uvrBiglA
uvrBiglC
uvrBiglD
<1
>7
>7
>7
>7
Weight
change(%)
80*-100
97-109
98-111
93-106
93-106
Protection
(%)
n.d.
100
100
100
100
Weight
change(%)
n.d.
93-106
95-105
93-107
95-105
LD50 (Log10)
* Mice that lost greater than 20% body weight were euthanized
 iglB, uvrBiglA, uvrBiglC, and uvrBiglD were highly attenuated with LD50
greater than 107 CFU and did not cause significant weight loss in infected mice
 Immunization with iglB, uvrBiglA, uvrBiglC, or uvrBiglD (105-107 CFU)
protected mice against lethal intranasal F. novicida wild-type challenge (100 LD50)
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Replication and Dissemination of F. novicida Mutants in
Mice after Intranasal Challenge (106 CFU)
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Lungs
iglB
uvrBiglA
uvrBiglC
uvrBiglD
Liver
6
(2) (1)
CFU (log10)
4
(1)
(2) (1)
(2)
2
<1
8
(0)
(0)(0)
(0)
Lymph nodes
Spleen
(N) number of mice
with detectable bacteria
6
(2)
4
2
<1
(0) (0)
3
7
14
3
7
14
Days after challenge
 All mutants replicated in the primary infection site and disseminated to secondary
target organs.
 Two-week after immunization, bacteria are still detectable in most examined tissues
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Humoral Response to Defined F. novicida Strains
Specific Ab
Titer
iglB
uvrBiglA
uvrBiglC
uvrBiglD
Total Ab
2842
2167
7145
1708
IgG1
2921
2408
2215
752
IgG2a
2433
2039
2997
1039
 Immunization of mice with either iglB, uvrBiglC, uvrBiglD, or uvrBiglA
(106 CFU) induced significant amount of antibody. Isotyping analyses indicated
both Th1 (IgG2a) and Th2 (IgG1)- type antibodies were produced
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Challenges/Problems:
1. LVS manipulation (generation of uvrA and uvrB mutations)
took considerably longer than anticipated, this milestone (43)
was extended. We were eventually successful.
2. Milestone 43 was also extended to incorporate an
additional (unrelated) deliverable: a secreted protein
with a MHC-I tag from OVA (SIINFEKL). We chose
PepO (secreted protease), constructed PepO-SIINFEKL
expressing plasmid, confirmed expression in LVS and Ftn,
and sent to Cerus.
3. Genetic manipulation of Schuh4 (MS49) took a long time to
perfect, with multiple simultaneous strategies. We were
eventually successful.
4. We substituted vgrG Schuh4 mutant for pdpD mutant,
based on results in Ftn demonstrating that pdpD is not
an essential virulence gene, but vgrG is.
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Plans for next six months:
Milestone #16: completed.
Milestone #39: completed.
Milestone #43:completed
Milestone #48:completed
Milestone #51:completed
Milestone #49:
Creation of iglD and vgrG Ftt mutant strains
Milestone #50:
Immune characterization of F. tularensis mutant strains
Milestone #52:
Construction of recA Schuh4 mutant
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