University of Texas San Antonio
Update on F. tularensis attenuated vaccine strain construction and evaluation
TVD Team
7/17/07 tech call
1

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.
2

Red: completed
Green: in progress
Blue: Steps in the milestone
Milestone 43
Added on:
Creation of uvrA and uvrB mutant F.
tularensis subsp. holarctica (LVS) strains
Construct SIINFEKL-tagged secreted protein for expression in F. tularensis
Construct
 uvrB::Kan mutagenesis plasmid
Construct
 uvrA::Kan mutagenesis plasmid
Construct PepO-SIINFEKL plasmid
Mate into LVS, select for transconjugate,
Counterselect for mutant
Mate into LVS, select for transconjugate,
Counterselect for mutant
Transform into LVS,
Confirm expression
Verify mutants,
Pass on to Milestone 50 Pass on to Cerus
3

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.
4

• We designed SIINFEKL complimentary oligos to replace FLAG tag with SIINFEKL in PepO-FLAG plasmids (one for E. coli expression, one for Ft expression),
• We identified PepO-SIINFEKL plasmids, both in E. coli and Ft expression vectors), sequencing confirmed these were correct.
• We attempted to visualize PepO-SIINFEKL in E. coli by Western blot with anti-SIINFEKL monoclonal (U. Mass), but failed to detect--control OVA antigen also failed to detect.
• Will repeat Western, also transform LVS with Ft plasmid
5

• We also previously created uvrA Targetron vector which we had stored away.
• In free time, this was transformed into LVS, and colonies with uvrA Targetron insertion identified, verified by sequencing.
• We are currently attempting to remove plasmid from
LVS uvrA ::Targetron strain, this will be characterized and passed on to Cerus when finished.
(documented in UTSA TVD notebook #2)
6

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) B. Construct
 vgrG,
 iglD mutagenesis plasmids
C. Construct
 iglA,
 iglB mutagenesis plasmids
Transform into Schuh4, select for transconjugate,
Counterselect for mutant
Mate into Schuh4, select for transconjugate,
Counterselect for mutant
Verify mutants,
Pass on to Milestone 50
Mate into Schuh4, select for transconjugate,
Counterselect for mutant
7

Milestone #49A: Construction of iglC F. tularensis subsp.
tularensis strain
This past month we concentrated on Targetron-mediated mutagenesis of iglC in Schuh4:
• We had constructed Targetron vector to target knockout of iglC , and had successfully achieved knockout of both copies of iglC in LVS (value-added)
• We transformed iglC Targetron vector into Schuh4, achieved many colonies.
• Intron (~900 bp) is designed to insert into iglC sequence between bp 427 and 428.
• Utilizing iglC primers at beginning and end of the gene, we can detect if intron inserted into iglC by shift in size.
• We achieved a number of colonies that have insertion in iglC (next slide)
8

WT iglC
3 Schuh4 Targetron transformants, appear to have insertion in iglC
• Note that transformants appear to have 900 bp insertion in iglC , as predicted for Targetron insertion
• Also note there is NO WT-length iglC in transformants, suggesting both copies of iglC have been inactivated.
• We are currently amplifying both copies of iglC by longrange PCR in these strains to verify that both iglC copies are inactivated.
9

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 .
• 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).
• We have identified Schuh4 strain with pdpA ::FRT-Kan R , this is in FPI-1, we have candidates with pdpA ::FRT-Kan R in
FPI-2 that are currently being analyzed
10

pdpD iglA B C D pdpC pdpB pdpA
FLP Recombinase
FLP Recombinase-mediated recombination
“scar”
Ft-FLP
11

• Construction of mutagenesis vector
• We have constructed mutagenesis vector (R6K ori, sacB ) for Schuh4 mutagenesis, we have removed native promoter for sacB and introduced better MCS
• We are now working to insert FTgroELp to drive antibiotic and sacB expression.
(documented in UTSATVD Notebook 3).
12

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
Construct
 iglB::ermC mutagenesis plasmid
Transform into U112, isolate mutant
Transform into pdpD, iglA, iglB, iglC, iglD novicida strains, isolate mutant
Verify mutants,
Pass on to Milestone 50
13

Milestone #51: Creation of uvrB + pdpD, iglA, iglB, iglC, iglD mutant F. tularensis subsp. novicida strains
•  uvrB ::Kan DNA transformed into
 iglB :: ermC strain, Kan R colonies isolated.
• Kan R colonies screened for
 uvrB ::Kan mutation, several confirmed to contain mutation by PCR.
• Double mutant strain will be confirmed by additional
PCR as well as intramacrophage growth, frozen.
(documented in UTSATVD Notebook 2).
14

Milestone 50
F. novicida uvrA , uvrB
Double mutant
Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis, and LVS strains
F. novicida uvr A+ pdpD
F.novicida uv rB+ pdpD iglA, iglB, iglC, iglD
LVS uvrA, uvrB
F. tularensis Schu4 iglC
In vitro Growth
In vivo Bacterial Burden
LD
50 determination
Red: completed
Green: in progress
Blue: Steps in the milestone
In vitro Growth
In vivo Bacterial Burden
LD
50 determination
In vitro Growth
In vivo Bacterial Burden
LD
50 determination
Further immunological characterization based on initial screen
15

Milestone #50A: Immunologic characterization of F. tularensis subsp. novicida , subsp. tularensis , and LVS strains
Results Update
Determine the LD
50 of Ft uvrBiglD double mutant subsp. novicida
Groups of BALB/c mice (female, 4-6 weeks) were intranasally (i.n.) challenged with 10 5 , 10 6 or 10 7 CFU of ΔuvrBiglD . Animals were monitored for morbidity and mortality.
16

100
80
60
40
10 5
10 6
10 7
20
0
0 2 4 6 8 10 12 14 16 18 20
110
105
100
95
90
85
80
0 2 4 6 8 10 12 14
Days post-challenge
Fig.1
. Survival of mice infected with Ft subsp. uvrBiglD double mutant. Groups of BALB/c mice (female, 6-week old) were challenged intra-nasally with 3 doses (10 5 , 10 6 , and 10 7 CFU) of ΔuvrBiglD to determine LD
50 of this strain.
Results : there is no mortality observed at any given challenge dose, indicating the high degree of attenuation with this organism. No significant weight loss of infected mice was also observed. The LD
50 of ΔuvrBiglD in the intranasal infection model (BALB/c mice) was greater than 10 7 CFU.
17

Milestone #50A: Immunologic characterization of F. tularensis subsp. novicida , subsp. tularensis , and LVS strains
Results Update
Monitor Ft subsp. novicida ΔuvrBiglD replication and dissemination in mice after intranasal challenge
BALB/c mice were challenged with ΔuvrBiglD mutant (10 6
CFU) intranasally. Lungs, liver, spleen, and lymph nodes were collected from the infected mice at day 3, 7 and 14 after challenge (3 mice per time point). Numbers of bacteria in each organ were determined by dilution plating.
18

10
8
6
4
Lungs
2
1
6
4
Spleen
Liver
Lymph node
* *
2
1
3 7 14 3
Days post-challenge
7 14
Fig. 2 Kinetic growth and clearance of Ft novicida ΔuvrBiglD 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 slight reduction noted at day 14. Numbers of bacteria in the liver, spleen and lymph node are lower than that in the lungs and are consistent throughout the observed period. 19

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
(again) and Ft by Western blot
Milestone #49A :
1. Screen Schuh4 clones with iglC Targetron vector for inactivation of both copies of iglC by PCR
2. Create and test iglD Targetron vector
3. Verify pdpA ::FRT Shuh4 mutant, work on pdpD ::FRT insertion into these strains.
Milestone #51:
1. Analyze
 uvrB ::Kan igl double mutants for intramac growth, send double mutants to Cerus.
Continued on following slide
20

Plan for following month:
Milestone #50 :
 Evaluate the protective efficacy of the Ft subsp. novicida uvrBiglD mutant as a vaccine candidate
 Analyze the antibody profiles of mice immunized with the Ft novicida uvrBiglD mutant at day 14 and 28 after vaccination
21

• Action : Karl will send SIINFEKL to Justin for testing in T cell assays, if Western repeat doesn’t work
• Action: Karl will send uvrA LVS to Justin, when it is ready.
• Action: Barbara will provide UTSA’s gastric plan to
NIAID by 8/14, in preparation for the call with NIAID on
8/21
• Action: Theresa/Karl will respond to Barbara’s proposed reallocation of funds among UTSA’s completed/nearly completed milestones to fund the gastric plan testing to be added to MS 50.
22