Transcript UTSATVDTechCall12-16-08 minutes final

University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
12/16/08 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
Note: annual BSL-3 lab shutdown and recertification occurred
during this last period, the laboratory resumed operations
mid-November.
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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 nadM, 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 have 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
•We are now in the process of constructing a nadM (Cterm)
Schuh4 mutant
•Targetron oligonucleotides designed to inactivate nadM
between nucleotides 602 and 603.
•PCR product ligated into pKEK1140:
1 Kb
1
4.0
1.5
1.0
2
3 4 5
6 7 8 9 10
Legend
1. 1 Kb ladder
2. Uncut C1 1140+NadM
3. KEK1140
4. C1 1140+NadM
5. C2 1140+NadM
6. C3 1140+NadM
7. C4 1140+NadM
8. C5 1140+NadM
9. C6 1140+NadM
10. Uncut KEK1140
Several clones gave
correct pattern, including
clone 1 (lane 4),
compared to parent
pKEK1140 (lane 3).
We’ve sent for
sequencing
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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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•We utilized the last period to evaluate attenuation/protective
efficacy of two mutants discussed at TVDC annual meeting:
• FTN0757, FTN0720: required for inhibition of ASC/casp1
(replicate normally in macs, induce high apoptosis)
(Weiss et al. PNAS 104:6037-6042)
•FTN0757 and FTN0720 Ft novicida mutants evaluated for
virulence via intranasal route:
FTN0757
(534 CFU)
% Survival Rate
100
FTN0720
(200 CFU)
75
N
R5330
D
ose 200 C
FU I . N
50
N
R7241
D
ose 534 C
FU I . N
25
N=6 or
5
PBS
I.N
Both strains
attenuated.
0
1
2
3
4
5
6
7
8
14 20 26 33
Da y a fte r I.N. i n fe c ti o n
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•Mice inoculated with FTN0720 and FTN0757 were
challenged with wildtype U112 (242 CFU) intranasally.
•Both sets of immunized mice were fully protected
against challenge (PBS vaccinated mice succumbed)
Group
of Mice
NR5330
NR7241
PBS
Dose of
Inoculum(CFU)
242
242
242
Route of
Inoculation
i.n.
i.n.
i.n.
D1
5/5
5/5
5/5
D2
5/5
5/5
5/5
Surviva l Rate
D3
D4
5/5
5/5
5/5
5/5
5/5
2/5
D5
5/5
5/5
0/5
D6
5/5
5/5
•We recommend substituting FTT0748 (FTN0720) for
iglB in Milestone 49
•We will work on iglC recA Schuh4 double mutant in
the coming period.
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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
Measure humoral responses after KKT10 (iglD
mutant of SCHU S4) oral immunization
BALB/c mice were orally immunized with KKT10
(103 CFU) or PBS (mock control). Sera and fecal
pellets were collected at day 21 after immunization
and assayed for anti-KKT10 specific antibody
titers by ELISA .
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A.
5000
B.
0.30
K K T10
K K T10
Mock/P B S
IgA
IgA
1000
A405
T iter
0.20
0.10
100
Ig(H+L) IgG1 IgG2a
0.00
IgM
IgM
Fig.1. Humoral immune responses induced by KKT10 (iglD of SCH S4) oral immunization. Mice were
immunized with 103 CFU of KKT10 or mock vaccinated with PBS. Sera (A) and fecal pellets (B) were collected 3
weeks after immunization, and assayed for anti-KKT10 specific antibody.
Results: Mice received single immunization of KKT10 induced significant amount of antigen-specific total serum antibody (Ig(H+L)) as shown in Fig. 1A.
Isotyping analyses of the sera indicated oral immunization with KKT10 resulted in production of high titers of IgG1. Oral immunization also induced low but
measurable anti-KKT10 specific secretory IgA in the prepared fecal pellet samples (Fig. 1B.).
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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 oral KKT10
vaccination against SCHU S4 intranasal challenge
BALB/c mice were given orally a single dose of
KKT10 (103 CFU), rested for 32 days, and
challenged intranasally with either 80 or 400 CFU
of SCHU S4.
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100
80
Moc k /PBS 80 CFU
% survival
Moc k /PBS 400 CFU
KKT10 80 CFU
60
KKT10 400 CFU
40
20
0
0
2
4
6
8
10
Day s after c hallenge
Fig. 2. Protective efficacy of KKT10 immunization against F. tularensis infection. BALB/c mice were
orally immunized with 103 CFU of KKT10 or PBS and i.n. challenged with lethal dose of F. tularensis
SCHU S4 strain (80 or 400 CFU). Mice were monitored for survival rate.
Results: All KKT10- and PBS/mock- vaccinated mice succumbed to SCHU S4 challenge by day 9. However, between the groups of mice challenged with 400
CFU of SCHU S4, the KKT10 immunized mice had a prolonged median-time-to-death of 6.5 days compared to 4 day for the PBS mock immunized mice.
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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
Determine long-term protective efficacy of oral
LVS vaccination against Ft subsp. tularensis
SCHU S4 challenge
We have previously conducted a study which showed that
administration of a primary dose of LVS orally followed by a
secondary boost vaccination, was able to extend the length of
protection against 100 (80% survival) and 500 (60% survival)
CFU of SCHU S4. (Ray et al., manuscript in review) In this
experiment, we wished to increase the challenge dose to test
the limits of this extended protection. BALB/c mice were
vaccinated orally with LVS (103 CFU) or mock (PBS)
vaccinated. Mice were rested for eight weeks. Some mice
were given a second oral dose of LVS (103 CFU) and rested
for an additional four weeks. All mice were challenged
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intranasally with 1200 CFU of F.t. SCHU S4.
LVS 8 wk/ SCHU S4 challenge
100
LVS 8 wk/ boost / SCHU S4 challenge
Mock (PBS)/ SCHU S4 challenge
% Surv iv al
80
60
40
20
0
0
5
10
15
20
25
30
Day s After Challenge
Fig. 3. Long term protective efficacy of oral LVS vaccination followed by SCHU S4 challenge.
BALB/c mice were vaccinated orally with 103 CFU of LVS or mock (PBS) vaccinated. Mice were
rested for 8 weeks. Some mice were given a second vaccination (103 CFU) and rested for an
additional 4 weeks. Mice were challenged with 1200 CFU SCHU S4 and monitored for survival
Results: All of the mice that only received one oral vaccination dose died by day 7 after challenge. However, mice that received a second oral vaccination
boost, exhibited 34% survival at this large lethal challenge inoculation. All of the mock (PBS) vaccinated mice died by day 5 after challenge as expected.
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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
Continued on following slide
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Plan for following month: Milestone #50-A&B:
50A:.
(1) Evaluate intramacrophage growth of SCHU S4 vgrG mutant
Evaluate the protective efficacy of oral KKT13 SCHU S4 vgrG
mutant vaccination against SCHU S4 intranasal challenge
50B: Submilestone-complete
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