University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
3/18/08 tech call
1
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
2
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
3
Milestone #49: Construction of vgrG, iglD F. tularensis subsp.
tularensis strain
•We are working on creating two different mutant
Schuh4 strains: iglD and vgrG
•vgrG targetron transformants have been isolated,
primary transformants screened first for evidence of
Insertion (targetron-specific and vgrG-specific primers:
Lane 2 Schuh4
Lanes 3-14,
Representative
Transformants
•This demonstrates the presence of the targetron
insertion in vgrG within these strains.
4
•Screen of vgrG targetron transformants with vgrGspecific primers shows that these are mixed colonies,
i.e. there’s a mixture of wt and mutant vgrG genes:
insertion
wt vgrG
•We are performing further colony purification and
screening to isolate pure colonies with only
mutated vgrG
5
•We have constructed two iglD tulatrons, (30/31 and
(255/256)
•Results of 255|256 transformation into Schuh4:
•Transformant colonies screened by intron-specific
and iglD-specific primers reveals that transformants
have targetron insertions in iglD:
Lane2 (top and bottom) Schuh4
Lanes 3-10 transformants, top
is intron and upstream primer,
bottom is same transformants with
intron and downstream primer.
Further PCR screen with iglD-specific primers indicates
that these are mixed colonies (I.e. contain both wt and
mutant vgrG), we are currently performing colony
6
purification to isolate “pure” colonies.
Additional work in milestone:
•Isolating and digesting chromosomal DNA for
Southern blot for iglC1 iglC2 Schuh4 mutant,
necessary to prove no additional insertions in
chromosome.
•PCR amplification and digestion of DpdpD::FRTermC
construct to clone into pUC118, which will then
be transformed into Schuh4 DpdpA::FRTKan,
This is part of strategy to remove one copy of
FPI in Schuh4 (discussed previously).
(documented in UTSA TVD notebook #1 and #5)
7
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
8
•LVS transformed with recA targetron, colonies
screened for recA mutation:
•We’ve already identified colonies with “pure”
recA mutation (i.e. not mixed colonies)
•Single recA colony chosen, then grown at
37°C to remove targetron plasmid
•PCR (below) verifies recA LVS mutant is correct
•We are now going to test this mutant for
virulence in mice (will be reported next month)
(documented in UTSA TVD notebook #2)
9
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
10
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
11
Milestone #50A: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Results Update
Measure intramacrophage (J774) replication of Ft
subsp. tularensis SCHU S4 iglC mutant
Murine macrophage cell line (J774) were seeded in
a 96-well plate overnight and infected with the
SCHU S4 wild type, or mutants (iglC,  mglA)
or the F. novicida (U112)  iglC mutant. Numbers
of viable bacteria in macrophages were measured
at 3 hr and 24 hr post-infection.
12
6
3h
24 h
CFU (Log1 0)
5
4
3
2
1
0
WT
mglA
SCHU S4
iglC
iglC
U112
Fig. 1. Intramacrophage survival of iglC mutant. Murine macrophage cell line (J774)
were infected with the iglC mutant or its parental strain (SCHU S4) using an inoculum of
10 MOI. Two previously characterized mutants, mglA of SCHU S4 and iglC of F.
novicida (U112), with minimal growth in macrophage were also assayed for
comparison. Numbers of viable bacteria in macrophages were measured at 3hrs and 24
hrs post-infection.
Results: Unlike its parental strain the SCHU S4 iglC mutant had minimal increases in replication at 24 hr. This
attenuation in intramacrophage replication is comparable to that seen in the iglC F. novicida mutant. The
replication seen in both iglC mutants is higher than that of the mglA of SCHU S4 mutant.
13
Milestone #50B: Characterization of protective immunity against
pulmonary tularemia via intra-gastric LVS vaccination
Results Update
Evaluate the protective efficacy of F. tularensis
SCHU S4 iglC vaccination against wild type
SCHU S4 challenge
Groups of BALB/c mice (female, 4-6 weeks)
were immunized with 103 CFU of  iglC
intragastrically (i.g.) or intradermally (i.d.). Sera
and fecal pellets were collected at day 21 after
immunization and assayed for anti-  iglC
specific antibody titers.
14
5000
i.g.
i.d.
Ab Titer
4000
3000
2000
1000
0
Total Ab
IgG1
IgG2a
Results: Mice immunized with iglC by either the i.d. or i.g. route induced significant amount of serum
antibody. Further IgG isotyping analyses of the sera indicated i.g. immunization of iglC resulted in producing
comparable titers of IgG1 and IgG2a, while i.d. vaccination induced dominant IgG2a antibody response.
15
0.50
A 414
0.40
0.30
0.20
0.10
0.00
IgA
IgM
IgA
iglC
IgM
Moc k
.iglC specific secretory IgA in the GI tract
Results: Intragastric immunization also induced measurable anti-
16
Table1. Protective efficacy of SCHU S4 iglC mutant against homologous wild type challenge
Route of
Vaccinatio
n
Route of
Challeng
e
i.d.
i.n.
i.d.
i.g.
i.n.
i.d.
Mock
i.n.
i.d.
Challeng
e Dose
(CFU)
%
Survival
(at day14)
Median
survival
(days)
KaplanMeier
Survival
analysis
20
0
6.5
*
100
16.7
5.5
**
20
16.7
8
**
100
33.3
11
**
20
0
5
100
0
4
20
0
5.5
**
100
0
6
**
20
0
5
100
0
4
20
0
4
100
0
4
* p<0.05 compared to mock vaccinated group
** p<0.01 compared to mock vaccinated group
.iglC specific secretory IgA in the GI tract
Results: Intragastric immunization also induced measurable anti-
17
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 LVS at 8 weeks
after vaccination
Mice were vaccinated intragastrically with 103
CFU LVS or mock immunized with PBS alone. At
8 weeks after inoculation, blood was collected and
sera were prepared. Some mice received a second
booster dose of 103 CFU LVS I.G. Blood was
collected from these mice three weeks after booster
vaccination dose and sera were prepared. Specific
anti-LVS total antibody titers were determined by18
ELISA.
20000
LVS IG 8 wk
10000
LVS IG 8 wk Plus IG Boos t
50% Binding T iter
Moc k (PBS)
1000
100
LVS
HEL
Results: Mice immunized with LVS I.G. retain high total antibody titers 8 weeks after vaccination and mice
which received a second dose of LVS at 8 weeks after initial vaccination also exhibited elevated titers.
19
Milestone #50B: Characterization of protective immunity against
pulmonary tularemia via intra-gastric LVS vaccination
Results Update
Analyze antigen-specific cellular responses to mice
vaccinated intragastrically with LVS at 2 and 4
weeks after vaccination.
Mice were vaccinated I.G. with 103 CFU of LVS
IG or mock vaccinated with PBS alone. At either 2
or 4 weeks after immunization, spleens were
collected, single cells were made and incubated in
the presence of increasing amounts of UVinactivated LVS for 72 hours. At the end of the
culture period, supernatants were collected and
analyzed for IFN-g production
20
Mo ck (PBS) I.G.
LVS I.G.
B 8
2 Weeks
A 14
4 Weeks
IFN- g (ng/mL )
12
6
10
8
4
6
4
2
2
ia
ed
M
EL
H
10 5
S
LV
S
LV
S
LV
10 4
10 3
ia
ed
M
EL
H
10 5
S
LV
LV
S
10 3
S
LV
10 4
<31.250
<31.250
Results: Cells from mice collected at 2 weeks after LVS vaccination produced high levels of IFN-g which
increased when cultured with higher doses of LVS. Little to no IFN-g was produced by cells from mock
vaccinated mice, or by cells cultured with HEL. Moreover IFN-g was also produced by cells collected at
214
weeks after vaccination, although at a lower level than at 2 weeks after vaccination.
Plan for following month:
Milestone #16: completed.
Milestone #39: completed.
Milestone #48: completed.
Milestone #43: completed.
Milestone #51: completed.
Milestone #49:
1. Isolate pure iglD1 iglD2 Schuh4 mutant.
2. Isolate pure vgrG1 vgrG2 Schuh4 mutant.
3. Perform Southern blot on iglC1 iglC2 Schuh4 mutant.
4. Construct pdpD::FRT in pUC-based vector
Milestone #52:
1. Assay LVS recA strain for virulence traits (macs, mice)
2. Transform recA targetron into Schuh4, isolate recA mutant.
Continued on following slide
22
Plan for following month: Milestone #50-A&B:
50A: (1) Continue monitoring the survival and weight loss of the
iglC-immunized/SCHU S4-challenged mice in the ongoing
experiment.
(2) Evaluate the protective efficacy of intragastric F. novicida iglB
vaccination against SCHU S4 intranasal and intradermal challenge.
50B: (1) Evaluate the protective efficacy of intragastric LVS
vaccination against Francisella type A SCHU S4 intranasal
challenge at 8 weeks after either a single vaccination or after
receiving a secondary booster dose. We will initiate this experiment
and results are expected to be reported three month later.
(2) Measure LVS dissemination to target organs early after
intragastric immunization by PCR.
23
Action Items
• Bernard will email the next vaccination and challenge
protocols to Barbara in order to share with NIAID.
• NIAID wants to look at UTSA rationale before next
vaccination and protection experiment within the next 1 to
1.5 months for prime boost strategy
• Barbara check that Julie Wilder has UTSA’s uv LVS
antigen prep and ELISA protocols (BG has Bernard’s
antigen prep protocol but not Bernard’s ELISA protocol.)
• Bernard: email ELISA protocol to Barbara Griffith
• Karl and Terry discuss anesthesia method for rats
24