University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
6/17/08 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 are working on creating two different mutant
Schuh4 strains: iglD and vgrG
•iglD transformants from previous month were promising,
looked like only mutated iglD, no wildtype-length gene
•Several of these were evaluated by Western blot with
anti-IglD antisera:
IglD can be detected in wildtype
Schuh4, but not in any of the
potential mutants, confirming they
are defective in both iglD genes.
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•iglD mutant strains were grown at 37°C to remove
targetron plasmid:
mutant
WT
PCR with iglD flanking primers
confirmed lack of wildtype
iglD gene in the two strains
frozen away (vertical arrows),
these strains have also lost
targetron plasmid.
•Thus, we have successfully created Schuh4 iglD1 iglD2 strain,
this strain will be evaluated for virulence parameters in the
next months.
•We are also working on creating vgrG Schuh4 mutant, we have
not identified “pure” mutant, and are beginning again with
transformation of Schuh4 with vgrG targetron plasmid.
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(documented in UTSA TVD notebook #1 and #5)
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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•Last month, we reported the identification of Schuh4
recA mutant in transformed colonies.
•Pure recA colony was grown at 37°C to remove
targetron plasmid:
targetron + recA primers
recA primers
PCR with internal
and external recA
mutant primers indicates
WT strains are correct,
frozen away as
KKT11 (vertical arrow),
•We thus have successfully created Schuh4 recA strain
•The Schuh4 recA strain and the LVS recA strain
were evaluated for virulence in mice.
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LVS recA mutant infected i.p. into mice
Route of
Inoculation
I.P.
Mock
Inoculation Survival Rate Route of
Dose
(D30)
Chall enge
(CFU)
1300
7/7
I.P.
I.P.
Chall enge Survival Rate
Dose
D6
D30
(CFU)
700
7/7
7/7
110
0/5
LVS recA highly attenuated i.p., LD50 >103 CFU (LVS LD50 ~10 CFU)
Schuh4 recA mutant infected i.n. into mice
Inoculum
KKT11
Wt Schu S4
Route of
Inoculation
I.n.
I.n.
Inoculation
Dose
(CFU)
105
175
Survival Rate
D1
D2
D3
D4
D5
D6
5/5
5/5
5/5
5/5
5/5 0/5
5/5 5/5 4/5 0/5
No evidence of Schuh4 recA attenuation at high i.n. dose;
LD50 <105 CFU (Schuh4 LD50 ~10 CFU)
We will test virulence of Schuh4 recA mutant infected at lower
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i.n. dose into mice, in case inoculum was too high to see attenuation
•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.
7 kb luxCDABE EcoRI
fragment from
pUTminiTn5kmlux
EcoRI digested
pKEK843 (Ft plasmid
with groEL promoter)
These fragments are being ligated, transformants will
be evaluated next report.
(documented in UTSA TVD notebook #2)
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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,
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
Evaluate the protective efficacy of intragastric F.
novicida iglB vaccination (prime and one boost)
against SCHU S4 intranasal challenge in C57BL
mice
Mice were given intragastrically a single or a booster dose of
 iglB (103 CFU) and challenged intranasally with 50 or CFU
of SCHU S4 three weeks after the final immunization.
Survival was monitored daily.
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100
Moc k
Prime
Prime/ Boos t
80
60
40
20
0
0
3
6
9
12
15
18
21
Fig. 1. Protective efficacy of F. novicida ΔiglB immunization against F. tularensis infection. C57BL/6 mice (10
per group) were intragastrically (i.g.) primed or primed and boosted with 103 CFU of ΔiglB and challenged
intranasally with F. tularensis SCHU S4 strain (50 CFU) three weeks after the final immunization. Mice were
monitored for survival rate. Mice received PBS i.g. were used as mock control.
Result: Single i.g. vaccination with ΔiglB did not protect C57BL/6 mice against i.n. SCHU S4 challenge (50 CFU), however, survival
rate increased to 40% when mice received a booster dose (Fig. 1). All mock control mice were succumbed to SCHU S4 infection by
day 6.
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Milestone #50A: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Results Update
Evaluate the humoral responses (prime/boost) after
intragastric F. novicida iglB vaccination
Groups of C57BL mice (female, 4-6 weeks) were
primed or primed and boosted with 103 CFU of
 iglB intragastrically. Sera and fecal pellets were
collected at day 21 after immunization and assayed
for anti-  iglB specific antibody titers.
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A
4000
B
1.00
Total Ab
IgG1
3000
0.80
IgG2a
0.60
2000
0.40
1000
0
0.20
0.00
Prime
Prime/ Boos t
Prime
Prime/ Boos t Prime Prime/ Boos t
IgA
IgM
Results:Mice received single immunization of iglB induced significant amount of serum antibody as shown in Fig. 2A. Further IgG
isotyping analyses of the sera indicated i.g. immunization of  iglB resulted in producing comparable titers of IgG1 and IgG2a.
Intragastric immunization also induced measurable anti- iglB specific secretory IgA in the prepared fecal pellet samples (Fig. 2B.).
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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
Analyze the serum and fecal antibody isotypes of
mice intragastrically immunized with LVS at 12
weeks after vaccination
Mice were vaccinated intragastrically with 103 CFU LVS or
mock immunized with PBS alone. At 12 weeks after
inoculation, blood and feces were collected. Some mice
received a second booster dose of 103 CFU LVS I.G. Blood
and fecal pellets were collected from these mice three weeks
after booster vaccination dose. Specific anti-LVS total
antibody titers, as well as IgG1, IgG2a, and IgA isotypes for
serum and Ig (H+L), IgA and IgM isotypes for fecal samples,
were determined by ELISA.
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20000
LVS 12 wk
10000
LVS 12 wk + Boost
Mock (PBS)
50% Binding Titer
LVS/ HEL
Mock (PBS)/ HEL
1000
100
Total Ab
IgG1
IgG2a
IgA
Fig. 1. Humoral responses to intragastric LVS immunization at 12 week timepoint. Groups of BALB/c mice were vaccinated
I.G. with 103 CFU of LVS or PBS as a control. Sera were collected 12 weeks later and analyzed to determine titers for anti-LVS
specific antibodies. Some mice were given a second boost dose of LVS I.G. at 12 weeks and sera was collected 3 weeks later.
Results: As shown in Fig. 1, mice immunized with LVS I.G. have decreased total antibody titers at 12 weeks when compared to
analyses performed at 8 weeks after vaccination. Mice which received a second dose of LVS at 12 weeks after initial vaccination had
comparable titers.
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LVS 12 wk
LVS 12 wk + Boos t
Moc k (PBS)
LVS/HEL
M oc k (PBS)/HEL
O.D. (630 nm)
0.80
Ig (H+L)
2.00
IgA
0.20
0.60
1.50
0.15
0.40
1.00
0.10
0.20
0.50
0.05
0.00
0.00
0.00
IgM
Fig. 2. Humoral responses to intragastric LVS immunization at 12 week timepoint. Groups of BALB/c mice were vaccinated I.G. with 103 CFU
of LVS or PBS as a control. Fecal samples were collected 3 weeks later and analyzed to determine titers for anti-LVS specific antibodies. Some
mice were given a second boost dose of LVS I.G. at 12 weeks and fecal samples were collected 3 weeks later.
Results: As shown in Fig. 2, mice retain high levels LVS-specific IgA in the G.I. tract at 12 weeks after vaccination. There was
comparable levels of IgA in the boosted mice. Similar to the 3 and 8 week time points, there were minimal amounts of LVS-specific
total antibody and almost no IgM isotype.
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Milestone #50B: Characterization of protective immunity against
pulmonary tularemia via intra-gastric LVS vaccination
Results Update
Perform histological analyses on tissues at varying
time points after LVS intragastric immunization
and subsequent SCHU S4 challenge
BALB/c mice were vaccinated intragastrically with 103 CFU
LVS or mock immunized with PBS alone. Four weeks later,
mice were challenged intranasally with 100 CFU SCHU S4.
At various time points after challenge (3, 15 and 30 days),
mice were sacrificed and lungs were collected. Briefly, 10%
neutral buffered formalin was injected in to the lungs via the
trachea wherein lungs were removed and set overnight in
formalin for fixation. Tissues were then imbedded in paraffin
and sliced in 5 mm sections and placed on slides, 3 sections
per slide. Every fourth slide was stained with hematoxylin
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and eosin and visualized using light microscopy.
Results: As shown in Fig. 3, at three days post-infection, mock immunized lungs displayed minimal inflammatory cellular
infiltration. In contrast, lungs from LVS vaccinated mice displayed focal peri-bronchiolar and peri-vascular mononuclear cell
infiltration, which appear to be predominantly composed of lymphocytes. At 15 days after challenge, lungs from vaccinated mice
showed a greater degree of peri-bronchiolar and peri-vascular lymphocytic infiltration, including the presence of bronchiolar and
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vascular cuffing by the inflammatory cells. At 30 days after challenge, lung sections from vaccinated mice displayed minimal
inflammatory cellular infiltrates. Mock vaccinated mice succumb to infection by day 5 after challenge.
Plan for following month:
Milestone #16: completed.
Milestone #39: completed.
Milestone #48: completed.
Milestone #43: completed.
Milestone #51: completed.
Milestone #49:
1. Evaluate iglD1 iglD2 Schuh4 mutant for virulence.
2. Isolate pure vgrG1 vgrG2 Schuh4 mutant.
3. Continue working on pdpD::FRT plasmid.
Milestone #52:
1. Test Schuh4 recA mutant for virulence at lower inoculum
2. Work on cloning luxCDABE into Ft expression plasmid.
Continued on following slide
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Plan for following month: Milestone #50-A&B:
50A:.
(1) Measure intramacrophage (J774) replication of Ft subsp.
tularensis (SCHU S4) iglD mutant
50B:
(1) Survival after LVS I.G. vaccination and SHU S4 challenge at 8
weeks after immunization .
(2) Histological analyses of lungs of LVS vaccinated mice at 45 and
60 days after challenge to check for the presence of long-term
pathology.
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Action Items
• Rick/Terry: will share with Bernard, the
results of the comparative i.n. LVS
vaccination results with mice from different
vendors
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