University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
1/22/08 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
•We are utilizing Tulatron technique
•We have constructed two different vgrG Tulatron
vectors, these target two different sites within vgrG
•Each has been transformed into Schuh4 to generate
vgrG mutant
•Transformants have been isolated, are currently
being screened for presence of insertion
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•iglD tulatron cloning ongoing, clones to insert into two
sites (30/31 and 255/256) in the process of being
constructed, previous attempt unsuccessful
•This time, process started from scratch, ligations
appear to have been successful, currently
identifying correct clone.
•We’re also working on cloning pdpD::FRT construct
For insertion into Schuh4 chromosome.
(documented in UTSA TVD notebook #1 and #5)
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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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•Tulatron vectors created to target two different
sites within recA, sequenced and correct.
•Both were transformed first into LVS to perfect
screening technique (while Ping waited for BSL3
access)
•LVS transformant clones currently being screened
to identify recA mutants
(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
F. tularensis Schu4 iglC
In vitro Growth
In vivo Bacterial Burden
LD50 determination
Further immunological characterization
based on initial screen
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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 #50A: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Results Update
Evaluate the protective efficacy of the Ft subsp.
novicida uvrBpdpD mutant
Groups of BALB/c mice (female, 4-6 weeks) were
intranasally (i.n.) challenged with 105 CFU of
ΔuvrBpdpD. The immunized mice were challenged
with 1000 CFU of F. novicida (~100 LD50) by the
i.n. route after 30 days of vaccination.
10
100
% Survival
80
uv rBpdpD
PBS
60
40
20
0
0
4
8
12
16
20
% Body weight
110
105
100
95
90
85
80
0
2
4
6
8
10
12
14
Days after challenge
Fig. 1. Protective efficacy of ΔuvrBpdpD immunization against F. novicida infection.
BALB/c mice were immunized intra-nasally with 105 CFU of ΔuvrBpdpD or PBS and
i.n. challenged with lethal dose of F. novicida (1000 CFU). Mice were monitored for
survival rate and weight change.
Results: ΔuvrBpdpD-vaccinated mice were highly protected against subsequent pulmonary challenge with F. novicida. No
significant loss of body weight was observed in the protected animals. As expected PBS-treated mock-vaccinated mice
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succumbed by day 5.
Milestone #50A: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Results Update
Analyze the antibody profiles of mice immunized with the Ft
novicida uvrBpdpD mutant after vaccination
Blood was collected from the ΔuvrBpdpD (105 CFU)immunized mice at day 14 and day 28 after priming. Specific
anti-ΔuvrBpdpD total antibody titer as well as IgG1 and IgG2a
isotypes were determined by ELISA.
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4
Total Ab
IgG1
Day 14
Day 28
IgG2a
Titer (x1000)
3
2
1
0
PBS
uvrBpdpD
PBS
uvrBpdpD
PBS
uvrBpdpD
Fig. 2. Humoral response to ΔuvrBpdpD immunization. BALB/c mice were
intranasally immunized with 105 CFU of the ΔuvrpDpD mutant or PBS alone as
mock vaccination. Sera were collected 2 weeks and 4 weeks after immunization
and used to determine titers of anti- ΔuvrBpdpD specific antibody.
Results: Specific serum total antibody in mice immunized with ΔuvrBpdpD was detectable at day 14 and increased by
day 28 after priming. Isotyping analyses indicated both Th1 (IgG2a) and Th2 (IgG1)- type antibodies were produced
in mice after the ΔuvrBpdpD immunization. However, the presence of dominant IgG2a isotype implied strong Th1type immune responses in the ΔuvrBpdpD-immunized mice.
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Milestone #50B: Characterization of protective immunity against
pulmonary tularemia via intra-gastric LVS vaccination
Results Update
Evaluate the protective efficacy of intragastric
LVS vaccination against F. tularensis SCHU S4
challenge
Groups of BALB/c mice (female, 4-6 weeks)
were intragastrically (i.g.) immunized with 1600
CFU of LVS. Mice treated with PBS were used
as a mock-control. The immunized mice were
challenged 3 weeks later with either 100 or 500
CFU of SCHU (100 or 500LD50) by the i.n.
route.
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% Survival
100
80
1600 CFU LVS I.G./500 CFU SCHU S4
40
Mock (PBS) I.G./100 CFU SCHU S4
20
0
% Weight Loss
1600 CFU LVS I.G./100 CFU SCHU S4
60
110
105
100
95
90
85
80
0
0
5
2
10
15
20
25
4
6
8 10 12
Days After Challenge
30
14
Fig. 3. Protective efficacy of LVS intragastric immunization followed by F.
tularensis SCHU S4 challenge. Groups of BABL/c mice were immunized I.G. with
1600 CFU LVS or PBS and rested for 3 weeks. Mice were then challenged i.n.
with SCHU S4 (100 or 500 CFU) and monitored daily for survival and weight loss.
Results: LVS-vaccinated mice were highly protected against subsequent pulmonary challenge with SCHU S4
and exhibited no appreciable weight loss. PBS-treated mock-vaccinated mice succumbed to the infection by
day 6 after challenge.
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Milestone #50B: Characterization of protective immunity against
pulmonary tularemia via intra-gastric LVS vaccination
Results Update
Analyze the antibody profiles of mice
intragastrically immunized with F. LVS.
Blood, feces and bronchalveolar lavage fluid
(BAL) was collected from the PBS- and LVS
(1600 CFU)- immunized mice at day 21 after
vaccination. Specific anti-LVS total antibody
titer, as well as IgG1, IgG2a, and IgA isotypes
for serum, IgA and IgM isotypes for fecal
samples, and IgG1, IgG2a, IgA and IgM
isotypes for BAL, were determined by ELISA.
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20000
10000
LVS
50% Binding Titer
Mock (PBS)
LVS/HEL
Mock (PBS)/HEL
1000
100
Total Ab
IgG1
IgG2a
IgA
Fig. 4. Humoral responses to intragastric LVS immunization. Groups of
BALB/c mice were vaccinated I.G. with 1600 CFU of LVS or PBS as a
control. Sera were collected 3 weeks later and analyzed to determine titers
for anti-LVS specific antibodies.
Results: Mice immunized i.g. with LVS produced high titers of LVS-specific total, IgG1 and IgG2a antibodies,
but did not produce any measurable IgA. No LVS-specific antibody was detected in mice mock-vaccinated with
PBS.
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LVS
Mock (PBS)
LVS/HEL
Mock (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. 5. Humoral responses to intragastric LVS immunization. Groups of
BALB/c mice were vaccinated I.G. with 1600 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.
Results: Intragastrically immunized mice produce a high amount of LVS-specific IgA in the G.I. tract, while
only producing a small amount of total antibody and almost no IgM. Little to no LVS-specific antibody was
detected in mice mock-vaccinated with PBS.
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2.00
Total Ab
1.50
O.D.
1.50
O.D.
2.00
IgA
1.00
1.00
1.00
0.50
0.50
0.50
0.00
0.00
0.00
2.00
2.00
IgG1
1.50
O.D.
O.D.
1.50
1.00
IgM
1.50
O.D.
2.00
IgG2a
LVS
Mock (PBS)
LVS/HEL
Mock (PBS)/HEL
1.00
0.50
0.50
0.00
0.00
Fig. 6. Humoral responses to intragastric LVS immunization. Groups of
BALB/c mice were vaccinated I.G. with 1600 CFU of LVS or PBS as a
control. Bronchoalveolar lavage fluid was collected 3 weeks later and
analyzed to determine titers for anti-LVS specific antibodies.
Results: , Mice vaccinated i.g with LVS produced specific antibodies in the BAL including total antibody, IgA,
IgM, IgG1 and IgG2a. No LVS-specific antibody was detected in mice mock-vaccinated with PBS.
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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. Finish iglD tulatron vectors and transform Schuh4.
2. Identify vgrG Schuh4 mutant among transformants.
3. Construct pdpD::FRT in pUC-based vector
Milestone #52:
1. Identify recA mutant in LVS, transform into Schuh4.
Continued on following slide
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Plan for following month:
Milestone #50-A&B:
1. (A) Measure intramacrophage (J774) replication
of Ft subsp. tularensis (SCHU S4) iglC defined
mutant
2. (A) Evaluate the protective efficacy of intragastric
F. novicida iglB vaccination against Francisella
type A (SCHU S4) and type B (OR96-0246)
challenge.
3. (B) Completion of bacterial dissemination studies
following intragastic LVS immunization,
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Action Items
• Bernard: share uvrB paper with UNM and NIAID
90 days before submission to the journal. (time is
defined in the first modification to the UTSA subcontract)
• Karl: determine status of MS completion reports
for MS 39, 48, 43 and 51. Revisions to MS 39
and 48 were sent to Jeff Barker in Dec 2007.
• Bernard: For IG vaccination studies, work to
determine whether CFU delivered to the lungs
may impact the interpretation of the IG protection
results.
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