Transcript UTSA TVD TechCall minutes 041508

University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
4/15/08 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
•iglD transformants from last time showed presence
of insertion, we are in process of isolating “pure
strain with only mutated iglD genes:
1 Kb
Legend:
1 2 13 14 15 16 17 18 19 20 21 22
1. 1Kb Ladder
Screening of clones with
“outside” iglD primers, should
yield only larger (mutated) fragment
(arrow) and no smaller (wildtype)
fragment
2. KKT1
3.0
A
1.0
3.0
B
1.0
13. 2D4A clone
lac2 14. 2D4B clone
3. 2D3A clone
15. 2D4C clone
4. 2D3B clone
16. 2D4D clone
5. 2D3C clone
17. 2D4E clone
6. 2D3D clone
18. 2D4F clone
7. 2D3E clone
19. 2D4G clone
8. 2D3F clone
20. 2D4H clone
9. 2D3G clone
21. 2D4I clone
10. 2D3H clone
22. 2D4J clone
__
11. 2D3I clone
12. 2D3J clone
1 2 3 4 5 6 7 8 9 10 11 12
•One clone looks promising (lower), others still appear
to be mixed wt + mutant. We will continue and isolate
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“pure” mutant, and check for mutation of both iglD copies
•vgrG targetron transformants with vgrGspecific primers indicated insertions, and clones are
Being cycled and screened, we still have mixed colonies,
i.e. there’s a mixture of wt and mutant vgrG genes.
•We are performing further colony purification and
screening to isolate pure colonies with only
mutated vgrG
Additionally:
•Performed Southern blot for iglC1 iglC2 Schuh4 mutant,
We saw no additional insertions in chromosome, the
Individual iglC genes could not be resolved, due to frag size.
•We constructed pdpD::FRTermC and transformed
into Schuh4 pdpA::FRTKan,to remove one copy of
FPI in Schuh4 (discussed previously); results will be
discussed next month.
(documented in UTSA TVD notebook #1 and #5)
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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 constructed LVS recA mutant (discussed
previously)
•LVS recA mutant is attenuated for virulence:
1000 CFU delivered intraperitoneally to 5 mice
Failed to kill any, while 100 CFU WT LVS was
100% lethal to 5 mice.
•We are now working on constructing the Schuh4
recA mutant.
•We’ve transformed, appear to have “pure” colonies:
•We’ll remove plasmid and test for virulence
(documented in UTSA TVD notebook #2)
Colonies with
insertion
Schuh4
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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
F. tularensis Schu4 iglC
In vitro Growth
In vivo Bacterial Burden
LD50 determination
Further immunological characterization
based on initial screen
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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 #50A: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Results Update
Evaluate the protective efficacy of intragastric F.
novicida iglB vaccination against SCHU S4
intranasal and intradermal challenge
Groups of C57BL mice (female, 4-6 weeks) were
immunized with 103 CFU of iglB intragastrically.
Sera and fecal pellets were collected at day 21 after
immunization and assayed for anti-iglB specific
antibody titers.
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5000
Titer
4000
3000
2000
1000
0
Total Ab
IgG1
IgG2a
Fig.1. Humoral responses induced by F. novicida iglB immunization. Mice
were vaccinated with 1000 CFU of the iglB-null mutant by the intragastric
route. Sera were prepared 3-week after immunization, and assayed for antiiglB specific antibody.
Results: Mice immunized with iglB induced significant amount of serum antibody. Isotyping analyses of the sera
indicated i.g. immunization of iglB resulted in comparable titers of IgG1 and IgG2a.
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1.00
1.0
A405
0.80
0.8
0.60
0.6
0.4
0.40
0.2
0.20
0
0.00
IgA
IgM
 iglB
IgA
IgM
Mock
Fig.2. Mucosal immune responses induced by F. novicida iglB
intragastric immunization. Mice were vaccinated with 103 CFU
of the iglB-null mutant and fecal pellets were collected 3-week
after immunization, and assayed for anti-iglB specific
antibody. Mice received PBS are used as mock control.
Results: Intragastric immunization also induced measurable anti-iglB specific secretory IgA in the prepared fecal
pellet samples.
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Antigen
Route of Challenge
% Survival (@ day 21)
iglB
i.n.
66.7 %
i.d.
16.7 %
i.n.
16.7 %
i.d.
16.7%
PBS/mock
Results: These mice were challenged i.n. or i.d. with 20 CFU of SCHU S4 3-week after vaccination. Animals were
monitored for survival. The iglB-immunized and i.n.-SCHU-challenged mice had a 66.7% survival rate while
only 16.7% PBS/mock immunized mice survived by day 21 post-infection.
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Milestone #50B: Characterization of protective immunity against
pulmonary tularemia via intra-gastric LVS vaccination
Results Update
Measure the presence of LVS in various tissues at
early time points after i.g. immunization by PCR
Groups of BALB/c mice (3 per time point) were
immunized i.g. with 103 CFU of LVS. At 24, 48
and 72 hours after vaccination, mice were
sacrificed and the lungs, livers and spleens were
snap frozen. We have optimized our protocol for
extracting genomic DNA from the tissues and will
be performing the PCR on the samples.
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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. Transform pdpD::FRT plasmid into pdpA::FRT strain,
isolate mutant.
Milestone #52:
1. Challenge LVS recA infected mice with LVS
2. Isolate Schuh4 recA mutant, test for virulence.
Continued on following slide
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Plan for following month: Milestone #50-A&B:
50A:.
(1) Evaluate the protective efficacy of intragastric F. novicida iglB
vaccination (prime and one boost) against SCHU S4 intranasal
and intradermal challenge.
50B:
(1) Measure LVS dissemination to target organs early after
intragastric immunization by PCR.
(2) Analyze the antibody profiles for serum and fecal samples taken
at both eight and twelve weeks after LVS i.g. immunization
either with or without a second booster dose of LVS.
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Action Items
• No new action items from Tech call
• UTSA will proceed with the planned
experiments
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