University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
10/21/08 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 constructed iglD1 iglD2 Schuh4 strain and vgrG1
vgrG2 Schuh4 strain (described previously).
•Inoculated both into mice intranasally to determine
level of attenuation.
•Groups of 5 mice inoculated with 103, 104, 105, 106 CFU
•All groups alive 30 days p.i.
•Control mice inoculated i.n. with ~102 Schuh4 succumbed
to infection on days 4 and 5.
•iglD1 iglD2 and vgrG1 vgrG2 Schuh4 strains highly
attenuated for virulence by i.n. route in mice.
•iglD1 iglD2 vaccinated mice were challenged with Schuh4
i.n. 30 days p.i., no protection (next slide).
•vgrG1 vgrG2 vaccinated mice to be challenged this week
with Schuh4, results will be reported next month.
4
Ftt iglD1::L1.LtrB iglD2::L1.LtrB mutant strain virulence:
CFU (intranasal route)
Survival
(30 days)
Schuh4
Challenge
(778 CFU
i.n.)
survival
4.8 X 103
5/5
0/5
4.8 X 104
5/5
0/5
4.8 X 105
5/5
0/5
4.8 X 106
5/5
0/5
PBS control
N.A.
0/5
The Ftt iglD1 iglD2 mutant is highly attenuated, but not protective
via the intranasal route
5
Ftt vgrG1::L1.LtrB vgrG2::L1.LtrB mutant strain virulence:
(preliminary, day 30 post inoculation)
CFU (intranasal route)
Survival
(30 days)
1.9 X 103
5/5
1.9 X 104
5/5
1.9 X 105
5/5
1.9 X 106
5/5
Wildtype Shuh4 (76 CFU)
0/5
Schuh4
Challenge
??
(occuring
this week)
The Ftt vgrG1 vgrG2 mutant is highly attenuated, we do not yet
know if it is protective via the intranasal route
6
•nadM: required for NAD synthesis, sole means in Ft
•nadM gene has two domains: N-term (NAD synth)
C-term ADP-ribosyl pyrophosphatase (Nudix), involved
in NAD recycling (other activities as well)
•We have found that C-term nadM mutation attenuates
Ft novicida (intranasal inoculation):
6
∆nadM 8.9 x 103
5
4
3
WT 1.1 x
2
∆nadM 8.9 x 105
103
1
nadM/pnadM 9.7 x 103
0
1
2
3
4
5
6
7
8
9
7
•This is “value added” vaccine candidate identification.
•Based on discussion at annual TVDC meeting, the
iglA and iglB Schuh4 mutants proposed for this
milestone are likely to have same efficacy as iglC and
iglD Schuh4 mutants.
•We propose to substitute the creation of a C-term nadM
Schuh4 mutant in place of the creation of iglA Schuh4
mutant originally planned.
•Deliverable will be nadM (C-terminal) Schuh4 mutant
instead of iglA1 iglA2 Schuh4 mutant, with caveat that
this is based on the viability of nadM (C-terminal)
Schuh4 mutant (if the mutant is not viable, we will need
to substitute another attenuated mutant).
•We have several other potential attenuated Ftn
candidates being screened, we will substitute iglB
Schuh4 with one of these at a later date.
8
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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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
Route of
Inoculation
KKT11
Wt Schu S4
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
Inoculum
KKT11
Wt Schu S4
PBS
Route of
Inoculation
I.n.
I.n.
I.n.
Inoculation
Dose
(CFU)
206
34
Survival Rate
D1
D2
D3
D4
D5
D6
D7
D8
D30
6/6
5/5
5/5
6/6
5/5
5/5
6/6
5/5
5/5
6/6
5/5
5/5
6/6
5/5
5/5
2/6
1/5
5/5
2/6
0/5
5/5
1/6
1/6
5/5
5/5
Slight attenuation of Schuh4 recA at low i.n. dose LD50 <102 CFU
10
•We have created a plasmid to express bacterial luciferase
in F. tularensis
•The luciferase gene cluster luxCDABE from Photorhabdus
luminescens is active at 37°C.
•Plasmid designated pKEK1194, transformed into LVS
Bioluminescence of LVS/Lux operoon
•LVS transformants
measured for light
production (left)
•Transformant
colony 3 (arrow)
gave highest light
production, frozen
and sent to UNM
250000
150000
S e ries 1
100000
50000
0
Co
lo
ny
1
Co
lo
ny
9
Co
lo
ny
8
Co
lo
ny
7
Co
lo
ny
6
Co
lo
ny
5
Co
lo
ny
4
Co
lo
ny
3
Co
lo
ny
2
Co
lo
ny
1
LV
S
w
t
Tm
in
iT
n5
k
m
lu
x
0
11
pU
Reactive Light Unit
200000
Sample
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
12
Milestone #50A: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Results Update
Measure humoral responses after KKT10 (iglD
mutant of SCHU S4) intranasal immunization
BALB/c mice were intranasally immunized with
KKT10 (104, 105, 106, or 107) or PBS as mock
control. Animals were bled at day 28 after
vaccination. Specific anti-KKT10 total antibody
titer as well as IgG1 and IgG2a isotypes were
determined by ELISA.
13
3000
Mock/P BS
10 4 CFU
Ab Titer
2500
10 5 CFU
10 6 CFU
2000
10 7 CFU
1500
1000
500
0
Total Ab
IgG1
IgG2a
Fig. 1. Humoral response to KKT10 (SCHU S4 ΔiglD) immunization. BALB/c mice were intranasally
immunized with 104, 105, 106 or 107 CFU of KKT10 or PBS alone as mock vaccination. Sera were collected
4 weeks after immunization and used to determine titers of anti-KKT10 specific antibody.
Results: Isotyping analyses indicated only Th2 (IgG1)- type antibodies were detected in mice after the KKT10
immunization via the intranasal route. No KKT10- specific antibody was detected in mice mock-vaccinated with
PBS at day 28 after immunization. All tested serum samples showed no reactivity to the unrelated HEL protein (
HEL data not shown).
14
Milestone #50A: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Results Update
Assays for cell-mediated immune responses after oral
KKF235 (iglB of F. novicida) immunization using
cellular cytokine recall assay
BALB/c mice were immunized orally with KKF235
(103 CFU) or mock immunized with PBS. Spleens,
cervical lymph nodes, and mesenteric lymph nodes
were collected from mice at day 14 after
immunization, single cells were made and stimulated
with UV-inactivated KKF235 (104 or 105 bacteria) or
HEL (hen egg lysozyme) as control. Supernatant were
analyzed for the production of IFN-g, IL-2 and IL-12).
15
40
Spleen
Mock/P B S
K K F235
15
10
IL-2 (pg/ml)
IFN-g (ng/ml)
20
IL-12 (pg/ml)
104
500
CLN
400
300
200
100
0
20
0
105
IL-12 (pg/ml)
0
30
10
5
500
CLN
104
105
MLN
400
300
200
100
104
105
104
105
UV-KKF235
0
104
105
104
105
UV-KKF235
Fig. 2. Cellular cytokine recall responses to oral KKF235
immunization. Spleen, cervical lymph nodes (CLN), and
mesenteric lymph nodes (MLN) were collected from KKF235
or PBS-mock vaccinated mice, single cells were made and
reacted with UV-inactivated KKF235 cells. Cytokines were
measured from supernatants at 72 h after additions of
stimuli.
Results: Antigen-specific cytokine productions were only found in KKF235 primed cells, but not in any examined
PBS-mock immunized samples. High amount of IFN-γ was induced in KKF235-primed spleen cells recalled with
UV-inactivated KKF235 (104 and 105 CFU). Significant amount of IL-2 can be detected in KKF235-primed
cervical lymph nodes, and the IL-12 was induced in both primed cervical and mesenteric lymph nodes. On the
other hand, a minimal amount of IL-4 (Th-2 type cytokine) was detected in all examined samples (data not 16
shown).
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
17
Milestone #50B: Characterization of protective immunity against
pulmonary tularemia via intra-gastric LVS vaccination
Results Update
Survival after oral LVS vaccination and CD4+ T
cell depletion/F. t. subsp. tularensis SCHU S4
challenge
Groups of Balb/c mice (8 mice/group) were
vaccinated orally with 103 CFU of LVS or mock
vaccinated (PBS) and rested for three weeks. One
group of mice were treated IP with 250mg of
neutralizing anti-CD4 antibody, grown up by the
hybridoma cell line GK1.5 (ATCC), at days -2, -1,
0 and every subsequent third day after intranasal
challenge with 80 CFU of F.t. SCHU S4. One
group of mice received IP injections of rat IgG2b
18
and another group received no treatment.
% Survival
A 100
% Body Weight
B
80
Moc k (PBS)
60
LVS
40
LVS/Rat IgG2b
20
0
110
105
100
95
90
85
80
LVS/anti-CD4 Ab
0
5
0
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 anti-CD4 antibody treatment and F.
tularensis SCHU S4 challenge. Groups of BALB/c mice (8 mice per group) were immunized IG with 103
CFU of LVS and rested for three weeks. Mice were then treated IP with 250 mg of either anti-CD4 antibody
or rat IgG2b as a control (day -2, -1, 0 and every subsequent third day), or given no treatment. On day 0, all
mice were challenged with 80 CFU of SCHU S4 and monitored daily for (A) survival and (B) weight loss
Results: Mice which received either rat IgG2b or no treatment exhibited a high level of survival (87%). Mice
which received the anti-CD4 antibody showed delayed effects of bacterial challenge and started to die by day 7
after challenge. After CD4 depletion, 37% of these mice were still surviving. As expected, all mock vaccinated
mice succumbed to infection by day 6 after challenge.
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Anti-CD4 Depletion profile
0.0%
0.4%
0.5%
17.6%
0.6%
Fig. 4. in vivo depletion of CD4+ T-cells. BALB/c mice
were given IP injections of 250mg of either anti-CD4
antibody or rat IgG2b as a control at D -2, -1, 0 and 3. On
day 4, single cell suspensions of splenocytes were
stained with APC-CY7 labeled CD4 antibody and
fluorescence was measured by flow cytometry.
20
Plan for following month:
Note: Annual shut-down and recertification of UTSA
BSL-3 lab scheduled to occur Nov. 9-15, this may delay some
of the plans for the following month.
Milestone #16: completed.
Milestone #39: completed.
Milestone #48: completed.
Milestone #43: completed.
Milestone #51: completed.
Milestone #49:
1. Begin construction of nadM (C-term) Schuh4 mutant.
2. Continue working on pdpD::FRT insertion in Schuh4.
Milestone #52:
1. Begin construction of iglC recA Schuh4 mutant
2. Screen several potential attenuated Ftn mutants for
protective efficacy (to identify novel vaccine candidates)
Continued on following slide
21
Plan for following month: Milestone #50-A&B:
50A:.
(1) Measure humoral responses after KKT10 (iglD mutant of SCHU
S4) oral immunization.
(2) Evaluation of protective efficacy of KKT10 after oral
immunization against SCHU S4 challenge
50B:
(1) Survival after LVS oral vaccination and CD8+ T cell
depletion/SCHU S4 challenge
22
Action Items
•
•
•
•
•
•
Bernard will test the Ftt iglD1:iglD2 mutant by the intragastric route in the
future, to look for protection
Karl will do experiments to prove that the C terminal NadM mutant will be
viable in SCHU S4.
Terry Wu will test the pKEK1194 LVS- luciferase strain for light production
at UNM
Jeff Barker will write the MS51 MSCR before he defends his thesis and
leaves the Klose lab in a couple of months.
Karl got Barbara’s email of 10/10 and will work on the tech plan and budget
for the new mutants (e.g. nadM), no later than when Theresa C returns from
medical leave (around 11/1).
MS 49: will open with the rat model and will add new deliverables. No
overall total budget changes will result. Heather is working on the budget
reallocations for Karl. Won’t start the rat model until BSL3 is back up again
after shutdown, later in November
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