University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
11/18/08 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
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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
•As reported last month, vgrG1 vgrG2 Schuh4 strain
inoculated into mice intranasally:
•Groups of 5 mice inoculated with 103, 104, 105, 106 CFU
•All groups survived inoculation 30 days p.i.= highly
attenuated
•vgrG1 vgrG2 vaccinated mice were challenged with Schuh4
i.n. (81 CFU) 30 days p.i.
•No protective efficacy (next slide), all vaccinated mice
succumbed to infection similar to unvaccinated control
•We have now constructed 3 different FPI mutations in Schuh4
(iglC, iglD, and vgrG), with similar results: high degree of
attenuation, no evidence of protective efficacy via i.n. route
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Ftt vgrG1::L1.LtrB vgrG2::L1.LtrB mutant strain virulence
and protective efficacy:
vgrG1 vgrG2
CFU (intranasal route)
Survival
(30 days)
Schuh4
Challenge
(81 CFU i.n.)
PBS
5/5
0/5
1.9 X 103
5/5
0/5
1.9 X 104
5/5
0/5
1.9 X 105
5/5
0/5
1.9 X 106
5/5
0/5
N.A.
Wildtype Shuh4 (76 CFU)
0/5
The Ftt vgrG1 vgrG2 mutant is highly attenuated, but not protective
via the intranasal route
Will still be evaluated by different routes of administration,
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different animal model (rat)
Reminder:
•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):
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∆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
6
•Creation of a C-term nadM Schuh4 mutant:
•The LD50 of nadM Ftn mutant is ~105 CFU, we
anticipate nadM Schuh4 mutant to be similarly
attenuated (i.e. approximately 4-5 logs).
•Ft NadM structure has been solved (Huang et al. Structure
16:196-209)
NMNAT domain (~1-185)
Nudix domain (~200-315)
•N-terminus predicted to be essential for NAD biosynth.
•C-terminus not essential (mutants derived in Ftn)
•We will target C-terminal domain
•We have ordered primers for Targetron inactivation
of Schuh4 nadM (FTT0386), for insertion between
nucleotides 602-603 (after aa 200)
•We will construct Targetron plasmid with these primers
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
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•We already created LVS and Schuh4 recA mutants, evaluated
for attenuation/protective efficacy (reported previously)
•We will create Schuh4 iglC recA mutant, but since our BSL-3
lab is currently closed for recertification, and thus we could not
proceed on construction of this strain, we decided to use this
time to evaluate attenuation/protective efficacy of two mutants
discussed at TVDC annual meeting:
• FTT0584, FTT0748: required for inhibition of ASC/casp1
(replicate normally in macs, induce high apoptosis)
(Weiss et al. PNAS 104:6037-6042)
•We have complete ordered library of transposon mutants in
every non-essential gene of Ftn
•We used a Tn mutant of FTT0548 (FTN0757; hypothetical)
and a Tn mutant of FTT0748 (FTN0720; txn regulator)
•Tn insertion sites were verified by PCR with Tn- and genespecific primers.
•FTN0757 and FTN0720 mutants evaluated for virulence: 9
•Groups of mice were inoculated intranasally with
FTN0720 and FTN0757 mutants (200 and 534 CFU)
•Both strains attenuated, only one mouse died from
FTN0720 infection, and no mice died from FTN0757
•FTN0757 appears slightly more attenuating, although
accurate LD50 will require more experimentation
•Will report vaccine efficacy next month
strain
Dose of
Inoculum
(CFU
i.n.)
D1
FTN0720
200
6/6
6/6 6/6
5/6
5/6
FTN0757
534
5/5
5/5 5/5
5/5
5/5
5/5
5/5 5/5
5/5
5/5
PBS
D2
D3
D8
D11
survival
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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
Evaluate the protective efficacy of KKT10 (ΔiglD
of SCHU S4) vaccination against intradermal
SCHU S4 challenge
Mice were vaccinated intradermally or orally with
single dose of KKT10 (103 CFU) and challenged
i.d. with either 20 or 100 CFU of SCHU S4 three
weeks after the immunization. Control mice were
mock-vaccinated with PBS. Survival was
monitored daily.
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% Survival
100
80
60
Mock 20 CFU
Mock 100 CFU
KKT10 20 CFU
KKT10 100 CFU
40
20
0
0
1
2
3
4
5
Days after challenge
6
7
8
Fig. 1. Protective efficacy of KKT10 (ΔiglD of SCHU S4) immunization against SCHU S4 infection. BALB/c mice
(5 per group) were immunized intradermally (i.d.) with 10 3 CFU of KKT10 or PBS and i.d. challenged with lethal
dose of F. tularensis SCHU S4 strain (20 or 100 CFU). Mice were monitored for survival rate.
Results: No protection was conferred by i.d. immunization with KKT10
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100
% Survival
80
Mock 20 CFU
Mock 100 CFU
KKT10 20 CFU
KKT10 100 CFU
60
40
20
0
0
1
2
3
4
5
Days after challenge
6
7
Fig. 2. Protective efficacy of KKT10 (ΔiglD of SCHU S4) immunization against SCHU S4 infection.
Mice (8 per group) were immunized orally with 103 CFU of KKT10 or PBS and intradermally
challenged with lethal dose of F. tularensis SCHU S4 strain (20 or 100 CFU). Mice were monitored for
survival rate.
Results: No protection was conferred by oral immunization with KKT10
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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+, Depletion
vaccination/challenge
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Milestone #50B: Characterization of protective immunity against
pulmonary tularemia via intra-gastric LVS vaccination
Results Update
Analyze the cellular responses to mice vaccinated
orally with LVS at 8 and 12 weeks after
vaccination.
Mice were vaccinated orally with 103 CFU of LVS
or mock vaccinated with PBS alone. At either 8 or
12 weeks after immunization, spleens were
collected, single cells were made and incubated in
the presence of increasing amounts of UVinactivated LVS for 72 hours. Splenocytes were
also cultured in the presence of the unrelated
antigen HEL or media alone as controls. At the end
of the culture period, supernatants were collected
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and analyzed for IFN-g production
Mo ck (PBS) I.G.
LVS I.G.
B 4
ia
ed
EL
10 4
M
S
LV
S
ia
ed
M
H
S
10 5
10 4
LV
S
LV
S
LV
H
<31.250
10 5
<31.250
S
1
LV
1
10 3
2
EL
2
10 3
3
IFN- g (ng/mL )
3
12 Weeks
LV
8 Weeks
A 4
Fig.3. Cellular responses to LVS IG vaccination. Groups of mice (3 mice/group) were inoculated
IG with 103 CFU OF LVS. At either 8 weeks (A) 12 weeks (B) after vaccination, spleens were
collected, single cells were prepared and incubated in the presence of LVS, and supernatants
were analyzed for IFN-g production.
Results: Cells from mice collected at 8 weeks after LVS vaccination produced significant amounts of IFN-g when cultured with
higher doses of LVS when compared to mock vaccinated mice or to cells cultured with HEL. As seen in Figure 3B, IFN-g was also
produced by cells collected at 12 weeks similarly to the 8 week timepoint. Collectively the levels of antigen-specific IFN-g wane
from week 2 till week 12 (observation period).
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Milestone #50B: Characterization of protective immunity against
pulmonary tularemia via intra-gastric LVS vaccination
Results Update
Survival after LVS Oral vaccination and CD8+ 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 200 mg of
neutralizing anti-CD8 antibody, prepared from the
hybridoma cell line TIB-210 (ATCC), at days -2, 1, 0 and every subsequent third day after intranasal
challenge with 140 CFU of SCHU S4.
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LVS Vac c
100
LVS Vac c /Rat Ig
LVS Vac c /anti-CD8 Ab
Moc k Vac c
% Survival
80
60
40
20
0
0
5
10
15
20
25
30
Day s After Challenge
Fig.5. Protective efficacy of LVS intragastric immunization followed by anti-CD8 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 200mg of either anti-CD8
antibody or rat IgG as a control (day -2, -1, 0 and every subsequent third day), or given no treatment. On
day 0, all mice were challenged with 140 CFU of SCHU S4 and monitored daily for survival.
Results: LVS vaccinated mice which received either rat IgG or no treatment exhibited a median level of survival (50%) following
type A challenge. Mice which received the anti-CD8 antibody showed early symptoms of disease and complete mortality by day 11
post challenge. All mock vaccinated mice succumbed to infection by day 6 after challenge. These results indicate that CD8+ T-cells
may play a role in clearance of infection following oral vaccination with LVS.
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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. Continue 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. Continue evaluation of FTN0720 and FTN0757 for
vaccine efficacy
Continued on following slide
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Plan for following month: Milestone #50-A&B:
50A:.
(1) Measure humoral responses after KKT10 (iglD mutant of
SCHU S4) oral immunization and complete protective efficacy
of KKT10 oral immunization against intranasal SCHU S4
challenge.
(2) Measure intramacrophage growth of SCHU S4 vgrG mutant.
50B:
(1) Evaluation of protective efficacy of LVS oral vaccination
against SCHU S4 challenge at 8 week post vaccination.
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Action Items
• Barbara: requested clarification on the proposed budget for the MS53
changes in scope. Will the $241,857 direct, original MS 53 budget be
maintained with the change in scope of work under MS53? Heather
will review and call Barbara
• Barbara: will further discuss UTSA’s proposed MS 53 changes with
Rick and start the subcontract modification for the change in scope of
work.
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