University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
2/17/09 tech call
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Active milestones during last reporting period:
Milestone #49: Construction of nadM 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 nadM,
FTT0748
mutagenesis plasmids
Mate into Schuh4,
select for transconjugate,
Counterselect for mutant
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Milestone #49: Construction of nadM, FTT0748 F. tularensis subsp.
tularensis strain
•We are in the process of constructing a nadM (Cterm)
Schuh4 mutant via nadM targetron plasmid
•We’ve already screened colonies by PCR that verified
insertion in nadM, we’re now cycling to isolate pure mutant
WT
transformants
transformants
external primers to
nadM reveals
presence of larger
size (insertion) fragment
in pool, some are after
1, 2, and 3 cycles.
Sequencing of internal +
external primer PCR
product confirmed
insertion in nadM
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Construction of nadM F. tularensis subsp. tularensis strain by
an alternate strategy:
•Since cycling of targetron transformants hasn’t yielded
pure mutants yet (after 3 cycles), we decided to pursue
an alternate strategy simultaneously.
•We will move the Tn insertion in nadM Ft novicida mutant
(this mutant was already characterized in previous reports)
Into Ft tularensis
•We amplified nadM::Tn5 and flanking regions from Ftn nadM
strain, and cloned into pGEM-T (TA vector)
•This will be further cloned into pJC84 (Schuh4 mutagenesis
plasmid from J. Celli).
Cleavage of clones with SalI
reveal insertion of nadM::Tn5
into this vector (size of insert
is close to size of vector)
5
Construction of FTT0748 F. tularensis subsp. tularensis strain:
•As discussed previously, we will also construct FTT0748
Schuh4 strain.
•We ordered oligos to target this strain by Targetron,
will report construction of this plasmid in following report.
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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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Construction of a iglC1 iglC2 recA mutant
•Strain KKT5 (iglC1 iglC2) was transformed with recA
targetron vector (pKEK1186), colonies isolated,
screened for recA disruption:
transformants
recA
mutant
transformants
All clones have
recA insertion,
all (except #6)
are already pure
mutant!
WT recA
Targetron plasmid will be removed from one of
these clones via growth at 37°C for final strain
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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,
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 humoral responses after KKT13 (vgrG
mutant of SCHU S4) oral immunization
C57BL/6 mice were orally immunized with
KKT13 (103 CFU) or PBS (mock control). Sera
and fecal pellets were collected at day 21 after
immunization and assayed for anti-KKT13 specific
antibody titers by ELISA.
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A.
B.
3000
0.50
KKT13
KKT13
Mock (PBS)
0.40
2000
Titer
A405
0.30
0.20
1000
0.10
0
0.00
Ig(H+L) IgG1
IgG2a
IgA
IgM
IgA
IgM
Fig.1. Mucosal immune responses induced by KKT13 (vgrG of SCH S4) oral
immunization. Mice were immunized with 103 CFU of KKT13 or mock vaccinated
with PBS. Sera (A) and fecal pellets (B) were collected 3 weeks after
immunization, and assayed for anti-KKT13 specific antibody.
Results: Mice vaccinated with KKT13 induced significant amounts of antigen-specific total serum antibody as shown in Fig. 1A.
Further IgG isotyping analyses of the sera indicated oral immunization of KKT13 resulted in producing both Th1- (IgG2a) and Th2(IgG1) type antibodies. Oral immunization also induced measurable anti-KKT13 specific secretory IgA in the prepared fecal pellet
samples (Fig. 1B.). No KKT13- specific antibody was detected in mice mock-vaccinated with PBS at day 21 after immunization. All
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tested samples showed no reactivity to the unrelated HEL protein (data not shown).
Milestone 53-B
Characterization of protective immunity against
pulmonary tularemia via oral vaccination in the F344 rat model
Characteristics of oral
vs. i.d. vaccination of
LVS/survival
Correlates of humoral
and cellular immunity
of LVS vaccination
Protective efficacy of
2 attenuated SCHU S4
strains
Intramacrophage survival
Vaccination/challenge
Bacterial dissemination
Histological analyses
CD4+ T cell
responses
Serum antibody responses
Secreted, BAL antibody
responses
Intramacrophage survival
vaccination/challenge
antibody responses
Bacterial dissemination and
histology
Red: completed
Green: in progress
Blue: Steps in the milestone
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Milestone #53B: Characterization of protective immunity against
pulmonary tularemia via oral vaccination in the F344 rat model
Results Update
Measure replication of F. novicida U112 and LVS
within rat bone marrow derived macrophages
Bone marrow derived macrophages were prepared
from F344 rats, seeded in 96-well culture plates at a
density of 2 X 105 cells per well. Cells were infected
with either F. novicida or LVS at 10 and 100 MOI for
2 hours and intramacrophage replication performed
at 3, 24, 48, or 72 hours.
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F. nov ic ida
LVS
10 MOI
10
6
10 5
10
5
10 4
10
4
10 3
10
3
10 2
10
2
10 1
10
1
CFU
10 6
3
24
48
72
100 MOI
3
24
48
72
Hours After Inoc ulation
Fig. 1. Intramacrophage growth of F. novicida and LVS in rat BMDM. Primary
bone marrow derived macrophages derived from Fisher 344 rats were infected
with F. novicida U112 or F. holarctica LVS at either 10 or 100 MOI. Cells were
lysed and viable bacteria were counted at 3, 24, 48 and 72 hours after infection.
Results: As shown in Figure 1, there was an initial uptake of 102-103 CFU of F. novicida at 3 hours followed by 1-2 logs of
replication by 24 hours post-infection. The levels of replication were relatively maintained from 24 to 72 hours. In contrast, LVS
uptake within rat macrophages were greatly diminished with minimal replication seen within rat macrophages.
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Milestone #53B: Characterization of protective immunity against
pulmonary tularemia via oral vaccination in the F344 rat model
Results Update
Comparison of intramacrophage Francisella
replication using bone marrow derived macrophages
from the Fisher 344 and the Lewis strain
This experiment was performed as previously
described at an MOI of 100.
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F. nov ic ida
LVS
F344 Rat
10
6
10 5
10
5
10 4
10
4
10 3
10
3
10 2
10
2
10 1
10
1
CFU
10 6
3
24
48
72
Lewis Rat
3
24
48
72
Hours After Inoc ulation
Fig. 2. Intramacrophage growth of F. novicida and LVS in rat BMDM. Primary
bone marrow derived macrophages derived from Fisher 344 and Lewis rats were
infected with F. novicida U112 or F. holarctica LVS at 100 MOI. Cells were lysed
and viable bacteria were counted at 3, 24, 48 and 72 hours after infection.
Results: As shown in Figure 2, the replication profile for both strains was highly comparable and followed the same
trend as in figure 1. F. novicida replicated favorably within these cells, while there was minimal replication seen with
LVS.
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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. Continue construction of nadM (C-term) Schuh4 mutant.
2. Begin construction of FTT0748 Schuh4 mutant.
Milestone #52:
1. Finish construction of iglC recA Schuh4 mutant
Continued on following slide
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Plan for following month: Milestone #50-A&B:
50A:.
(1) Evaluation of protective efficacy of intranasal KKF235 (iglB
of U112) vaccination against Francisella Type B (OR96-0246
strain) challenge.
53B:
(1) Perform phagocytosis assay of F. holarctica and F. tularensis
SCHU S4 with F344 rat bone marrow derived macrophages.
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Action Items
•
•
Notes after meeting: UTSA and UNM need to compare data with LVS and
SCHU S4 infections of rat macrophages. The two groups maybe performing
the assay slightly differently , leading to the differences in results. Rick asked
Terry to pull the data together and share it with Bernard.
Bernard- will email Bernard’s oral immunization Pub Med publication and
reference to Barbara
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