University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
8/19/08 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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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 constructed iglD1 iglD2 Schuh4 strain (KKT10),
described in previous reports
•KKT10 inoculated into mice intranasally to determine
level of attenuation.
•Groups of 5 mice inoculated with 103, 104, 105, 106 CFU
•All groups still alive 30 days p.i.
•Control mice inoculated i.n. with 102 Schuh4 succumbed
to infection on days 4 and 5.
•iglD1 iglD2 Schuh4 strain highly attenuated for virulence
by i.n. route in mice.
•Vaccinated mice were challenged with Schuh4 i.n.
30 days p.i., results will be reported next month.
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•Construction of vgrG Schuh4 mutant:
•Last report we showed evidence of targetron insertion
in vgrG, continued to passage and screen for
mutant with targetron insertions in both vgrG genes:
•Latest screening of promising colonies shows
Presence of targetron insertion in every colony,
and increasing intensity of “mutant” vgrG:
This screen is with intron and
parent Transformant colonies
vgrG primers, shows every
colony has targetron in vgrG
This screen is with vgrG
primers, shows presence of
mutant and wildtype vgrG gene
parent
We are close! One more passage
And screen should result in “clean”
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vgrG1 vgrG2 mutant Schuh4 strain
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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•In previous reports, we have created LVS recA strain
and shown it is highly attenuated in mice:
i.p., LD50 >103 CFU (LVS LD50 ~10 CFU)
•We have also created Schuh4 recA strain (KKT11)
and shown it is fully virulent at high dosage in mice:
i.n., LD50 <105 CFU (Schuh4 LD50 ~10 CFU)
•Last month we showed that Schuh4 recA strain shows
slight attenuation at low dosage (1/6 survived at 206 CFU)
We challenged the single recA -vaccinated animal with a lethal
low dose of Schuh4 i.n. (136 CFU) at 30 days post-vaccination:
Group of
mice
Route of
challenge
Dose of
challenge(CFU)
KKT11
PBS
I.n.
I.n.
136
136
D1
1/1
5/5
Surviva l rate
D2 D3 D4 D5
1/1 1/1 1/1 0/1
5/5 5/5 5/5 1/5
D6
0/5
This result suggests that recA Schuh4 mutant does not provide
protection against wildtype challenge; however, recA is still an
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important mutation to stabilize constructed defined vaccine strains
•We are also creating a plasmid to express bacterial luciferase
in F. tularensis
•The luciferase gene cluster luxCDABE from Photorhabdus
luminescens is active at 37°C.
•We have cloned luxCDABE into low-copy vector pWSK30,
and tested resulting clone for luciferase activity via luminometer.
•The resulting plasmid, pKEK1193, was positive for luciferase
activity, demonstrating that gene cluster was active.
•We isolated luxCDABE fragment and cloned into Ft expression
vector pKEK843, screened resulting clones by luminometer:
•Lane 2 is luciferase-positive clone, digested
with XhoI and XcmI, gives expected 3
Fragments (10, 4.5, 0.6) vs. 2 frags from
Parent pKEK843 (4.5, 3.2) in lane 3
•Plasmid designated pKEK1194, will
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be transformed into Ftn, LVS
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
Schu4: iglC, iglD,
pdpD, vgrG,
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
Measure intramacrophage (J774) survival of
SCHU S4 iglD mutant
Murine macrophage cell line (J774) were seeded in
a 96-well plate overnight and infected with the
iglD mutant (designated KKT-10) or its parental
strain (SCHU S4) using an infection dose of 10 or
100 MOI. Numbers of viable bacteria in
macrophages were measured at 3 hr and 24 hr postinfection.
10
6
3h
24 h
5
4
3
2
1
SCHU S4
KKT-10
SCHU S4
10 MOI
KKT-10
100 MOI
Fig. 1. Intramacrophage survival of SCHU S4 iglD mutant. Murine macrophage cell line (J774) were infected with the
iglD mutant or its parental strain (SCHU S4) using an inoculum of 10 or 100 MOI. Numbers of viable bacteria in
macrophages were measured at 3hrs and 24 hrs post infection.
Results: As expected, the wild type SCHU S4 replicated rapidly inside macrophages within a day using MOI of 10 or 100. However,
minimal replication of KKT-10 was observed (either at 10 or 100 MOI).
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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 #50B: Characterization of protective immunity against
pulmonary tularemia via intra-gastric LVS vaccination
Results Update
Monitor Ft subsp. tularensis SCHU S4 replication
and dissemination in mice after intragastric LVS
vaccination
BALB/c mice were vaccinated orally with LVS
(103 CFU) or mock (PBS) vaccinated. Mice were
rested for three weeks and then challenged
intranasally with ~130 CFU of SCHU S4. Lungs,
livers and spleens were collected from these mice
at day 1, 3 and 4 after challenge (5 mice per time
point). Numbers of bacteria in each organ were
determined by dilution plating.
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LVS
M oc k (PBS)
CFU per Organ
1 09
1 09
Lungs
Liver
1 09
1 08
1 08
1 08
1 07
1 07
1 07
1 06
1 06
1 06
1 05
1 05
1 05
1 04
1 04
1 04
1 03
1 03
1 03
1
3
4
Spleen
1
1
3
4
Days A fter SCHU S4 Challenge
3
4
Fig. 1. Kinetics of bacterial growth and clearance in target organs following LVS and mock (PBS) I.G. vaccination and
subsequent F.t. SCHU S4 challenge. Bacterial burdens were determined in lungs, livers and spleens of individual mice (5 mice
per time point).
Results: There were low numbers of bacteria present in the lungs of both LVS and mock-vaccinated mice and minimal bacteria in the
spleens and livers at day 1 after challenge. By day 3 after challenge, there were high numbers of bacteria present in mock (PBS)
vaccinated mice while there were still very low levels of bacteria in the tissues of all LVS vaccinated mice. At day 4 after challenge,
at a time point right before mice normally succumbed to infection, bacterial numbers were as high in the organs of mock vaccinated
mice while there remained reduced numbers of bacteria in LVS vaccinated mice.
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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. Isolate pure vgrG1 vgrG2 Schuh4 mutant.
2. Complete challenge of iglD1 iglD2 Schuh4 vaccinated mice.
3. Continue working on pdpD::FRT plasmid.
Milestone #52:
1. Transform Ft plasmid luxCDABE into U112, LVS
2. Test for luciferase expression.
Continued on following slide
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Plan for following month: Milestone #50-A&B:
50A:.
(1) Measure humoral responses after KKT-10 (iglD mutant of
SCHU S4) intranasal immunization.
(2) Assays for cell-mediated immune responses after F. novicida
iglB oral immunization.
50B:
(1) Survival after LVS I.G. vaccination and CD4+ T cell
depletion/SCHU S4 challenge.
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Quality Assurance Issues from Karl Klose 8/19/08 after call:
1. Pipettors calibrated frequently, last calibration date:
8/1/08
2. Major pieces of equipment on service contract:
Sorvall RC5B, ABI RealTime PCR, BioRad FPLC,
Genepix Microarray Reader
3. All genetic constructs confirmed by DNA sequencing
both strands, typically with IDT (Houston TX)
4. BSL-3 laboratory undergoes quality assurance and
recertification once/year (last date 11/6-14/07. This involved
complete decontamination, recertification of biosafety cabinets,
eyewash and shower, balance of air flow, verification of
alarms and security, change of Hepa filters, re-sealing of
animal holding room ceiling. Records on file with UTSA 17
Safety office.
Action Items
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QA: Bernard will provide report at 10/21/08 UTSA tech call
Barbara included Karl’s QA slide after the 8/19 tech call.
Karl & Bernard: UTSA: the Material Transport and Storage plan is due to UNM on 9/2;
UTSA can use UNM’s plan as an example to facilitate developing the UTSA plan
Karl is willing to present the specific mutants on day 2 of the annual meeting.
Karl and Bernard will review MS budgets (53 and 54) and determine if funds can be
shifted to the intragastric plan for an additional year on MS 50.
Karl: will send Lux 5 gene cluster constructs in LVS and F novicida to UNM, when the
transformations are completed and verified.
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