Transcript UTSATVDTechCall7-15-08 no minutes

University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
7/15/08 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 constructed iglD1 iglD2 Schuh4 strain (KKT10),
described in previous reports
•KKT10 inoculated into mice intranasally to determine
level of attenuation, this was performed at end of June,
and experiment still ongoing.
•Preliminary results (will be reported in full detail in next
month’s report):
•Groups of 5 mice inoculated with 104, 105, 106, 107 CFU
•All groups still alive at 6 days p.i., still appear healthy
•Control mice inoculated i.n. with 102 Schuh4 succumbed
to infection on days 4 and 5.
•Preliminary conclusion: iglD1 iglD2 Schuh4 strain highly
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attenuated for virulence by i.n. route in mice.
•We began construction of vgrG Schuh4 mutant again
•We transformed Schuh4 with vgrG targetron plasmid
•Plenty of KanR colonies isolated
•Screening of colonies revealed the presence of
Targetron insertion within vgrG, using intron-specific
and gene-specific oligos:
transformant
colonies
1 2 3 4 5 6 7 8
1 Kb
Legend:
1. 1 Kb Ladder
2. KKT1
3. C13 VgrG
4. C15 VgrG
1.5
5. C17 VgrG
0.5
6. C21 VgrG
These strains all show evidence
of targetron insertion into
vgrG
7. C23 VgrG
8. C24 VgrG
parent
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Screening of colonies with oligos that flank vgrG demonstrate
That very little insertion is present in the colonies (i.e. these
are “mixed” colonies of mostly wildtype with a little mutant)
transformant
parent
colonies
1 Kb
1 2 13 14 15 16 17 18 19 20 21 22
Legend:
1. 1 Kb Ladder
Clone 17
2. KKT1
1.5
3. C1 VgrG
13. C11 VgrG
0.5
4. C2 VgrG
14. C12 VgrG
5. C 3 VgrG
15. C 13 VgrG
6. C 4 VgrG
16. C 14 VgrG
7. C 5 VgrG
17. C 15 VgrG
8. C 6 VgrG
18. C 16 VgrG
9. C 7 VgrG
19. C 17 VgrG
10. C 8 VgrG
20. C 18 VgrG
11. C 9 VgrG
21. C 19 VgrG
12. C 10 VgrG
22. C 20 VgrG
1.5
0.5
1 2 3 4 5 6 7 8
9 10 11 12
parent transformant
colonies
•Clone 17 was cycled for growth
in liquid media, then colonies pooled
and screened
•Evidence of insertion, still no “pure”
colonies, we continue to purify&screen
•We are looking for a shift
to a larger size (~1800 bp)
due to insertion of 900 bp
targetron
•Clone 17 appeared to have
detectable 1800 bp
fragment, so we chose this
clone for further purification:
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parent
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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•In previous reports, we have created LVS recA strain
and shown it is highly attenuated in mice:
i.p., LD50 >103 CFU (LVS LD50 ~10 CFU)
•We have also created Schuh4 recA strain (KKT11)
and shown it is fully virulent at high dosage in mice:
i.n., LD50 <105 CFU (Schuh4 LD50 ~10 CFU)
We tested the virulence of Schuh4 recA mutant infected at lower
i.n. dose into mice, to determine if there is any attenuation:
Inoculum
KKT11
Wt Schu S4
PBS
Route of
Inoculation
I.n.
I.n.
I.n.
Inoculation
Dose
(CFU)
206
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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
The Schuh4 recA mutant is SLIGHTLY attenuated by the i.n. route,
1/6 infected animals survived at 206 CFU inoculum.
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•We are also creating a plasmid to express bacterial luciferase
in F. tularensis
•The luciferase gene cluster luxCDABE from Photorhabdus
luminescens is active at 37°C.
•Last month we reported isolation of 7 kb luxCDABE EcoRI
fragment from pUTminiTn5kmlux and ligation with pKEK843
(Ft plasmid with groEL promoter)
•We only obtained 4 colonies from transformation, none of
which were correct.
•We also tried to clone lux fragment into pUC118, screened
19 colonies, none were correct.
•We are now cloning into low-copy vector pWSK30, will
then move to Ft expression plasmid (to be reported next month)
(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
Schu4: iglC, iglD,
pdpD, 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
Histological analyses of lungs from F. novicida
iglB-vaccinated and SCHU S4-challenged mice
In our last monthly report, we showed the efficacy
of the prime/boost vaccination with iglB. To
evaluate the histological changes in the protected
mice, lungs were collected 35 days after challenge,
fixed in formalin, and embedded in paraffin. Thin
sections (5 mm) of the embedded tissues were
prepared and stained with hematoxylin and eosin.
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4x
10x
20x
Naive
iglB
Fig. 1. Lung histology after F. novicida iglB vaccination and SCHU S4 challenge. Groups of C57BL/6 mice were vaccinated orally
with 103 CFU of iglB and challenged intranasally with SCHU S4 (50 CFU) 3 weeks after the boost. Lungs were collected from
survivors at day 35 after the challenge. Lung sections were stained with hematoxylin and eosin.
Results: At day 35 post-challenge, lungs of iglB-vaccinated and SCHU S4-challenged mice appeared mostly
devoid of inflammation, although small pockets of mononuclear cell infiltrates around some bronchioles still could
be seen
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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 #50B: Characterization of protective immunity against
pulmonary tularemia via intra-gastric LVS vaccination
Results Update
Perform histological analyses on tissues at varying
time points after LVS intragastric immunization
and subsequent SCHU S4 challenge
BALB/c mice were vaccinated intragastrically with 103 CFU
LVS or mock immunized with PBS alone. Four weeks later,
mice were challenged intranasally with 100 CFU SCHU S4.
At various time points after challenge (days 45 and 60), mice
were sacrificed and lungs were collected. Briefly, 10%
neutral buffered formalin was injected in to the lungs via the
trachea wherein lungs were removed and set overnight in
formalin for fixation. Tissues were then imbedded in paraffin
and sliced in 5 mm sections and placed on slides, 3 sections
per slide. Every fourth slide was stained with hematoxylin
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and eosin and visualized using light microscopy.
LVS
Mock
(PBS)
45 Days Post
Challenge
LVS
60 Days Post Challenge
4X
20X
Fig. 2. Lung histology after LVS vaccination and SCHU S4 challenge. Groups of BALB/c mice
were vaccinated I.G. with 103 CFU of LVS or PBS as a control. Mice were challenged 3 weeks
later with 100 CFU SCHU S4 i.n. and lungs were collected at varying time points after challenge.
Lung sections were stained with hematoxylin and eosin.
Results: As reported in May, 2008, LVS vaccinated mice at 30 days after challenge still displayed minimal amounts of inflammatory
cellular infiltrates. Therefore, we looked at two further time points to ensure that inflammation had cleared completely. As shown in
Fig. 2, at 45 days post-challenge, lungs of vaccinated and challenged mice appeared with minimal inflammation. However, there
were still small pockets of mononuclear cell infiltrates around some bronchioles. At 60 days post-challenge, immunized-challenged
lungs were completely clear and comparable to lungs of naïve mice. Therefore, while SCHU S4 i.n. challenged LVS vaccinated mice
might display a slightly prolonged inflammatory response, by 2 months after challenge the lungs of these mice appear to have
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completely recovered.
Plan for following month:
Milestone #16: completed.
Milestone #39: completed.
Milestone #48: completed.
Milestone #43: completed.
Milestone #51: completed.
Milestone #49:
1. Isolate pure vgrG1 vgrG2 Schuh4 mutant.
2. Continue working on pdpD::FRT plasmid.
Milestone #52:
1. Continue to evaluate Schuh4 recA mutant for virulence/
protective efficacy in animals
2. Work on cloning luxCDABE into Ft expression plasmid.
Continued on following slide
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Plan for following month: Milestone #50-A&B:
50A:.
(1) Measure intramacrophage (J774) replication of Ft subsp.
tularensis (SCHU S4) iglD mutant.
50B:
(1) Bacterial dissemination in oral LVS-vaccinated BALB/c mice
after SCHU S4 challenge.
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