University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
6/30/09 tech call
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Active milestones during last reporting period:
Milestone #49: Construction of nadM, FTT0748 F. tularensis subsp.
tularensis strains
Milestone #50: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Milestone #52: Create recA mutants in F. tularensis subsp. tularensis
Milestone #53: 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 nadM,
FTT0748
mutagenesis plasmids
Mate into Schuh4,
select for transconjugate,
Counterselect for mutant
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Milestone #49: Construction of nadM, FTT0748 F. tularensis subsp.
tularensis strain
•We are in the process of constructing a nadM (Cterm)
Schuh4 mutant via nadM targetron plasmid
•We’ve already screened colonies by PCR that verified
insertion in nadM, we’re still cycling to isolate pure mutant
WT
transformants
After 10 cycles, external primers
to nadM revealed single clone with
only larger size (arrow=insertion)
fragment, appears to (finally) be pure
mutant.
We will now remove targetron plasmid
from this clone.
WT transformants
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Construction of nadM F. tularensis subsp. tularensis strain by
an alternate strategy:
•cloning Tn insertion from nadM Ft novicida mutant
into Ftt mutagenesis plasmid pJC84 (J. Celli).
•nadM::Tn5 from Ftn nadM cloned into pJC84:
Correct
pattern
Correct
pattern
We obtained
correct clone
(BHI and PstI
digests
shown) with
Ftn nadM::Tn
cloned into
pJC84
We will now transform pJC84 Ftn nadM::Tn into Schuh4
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Construction of FTT0748 F. tularensis subsp. tularensis strain:
•We are constructing FTT0748 Schuh4 strain.
•We transformed FTT0748 targetron plasmid into Schuh4,
obtained KanR colonies.
•screening for correct mutant reveals 5/10 already appear
to be pure mutants:
wt transformants
wt transformants
FTT 0748 primers
Intron + FTT 0748 primers
We will now remove targetron plasmid from select strain
via growth at 37°C
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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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Breaking down restriction barriers in Schuh4:
•We have identified two intact restriction enzyme genes in
Ftt: FTT1579 (shared with Ftn) and FTT0523 (unique to Ftt)
•Inactivation of FTT1579: we chose two target sites in gene
(849/850 and 1254/1255), constructed targetron plasmids
(last progress report).
•The 849/850 targetron was transformed into U112 to test
for efficacy (due to limited access to BL3 last month):
Screening with primers flanking
FTT1579 did not reveal evidence
of targetron insertion in 20 colonies,
We will screen more, also try other
targetron (1254/1255), and transform
Schuh4 next month.
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Milestone 53A
Immunologic characterization of defined
F. tularensis mutants
Strains from milestone #52
And #54
In vitro growth
In vivo bacterial burden
LD50 determination
Red: completed
Green: in progress
Blue: Steps in the milestone
F. tularensis rec A
recAiglC
In vitro growth
In vivo bacterial burden
LD50 determination
Further immunological characterization
based on initial screen
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Milestone #53A: Immunologic characterization of defined
F. tularensis mutants
Results Update
Measure the intramacrophage growth of KKT11 (a Schu S4
recA mutant), KKT5 (a Schu S4 iglC mutant) and KKT23
(a Schu S4 recAiglC mutant)
Murine macrophage cell line (J774) was seeded in a 96-well
plate (2x105/well) overnight and then infected with 10 MOI
of various Schu S4 mutants. Numbers of viable bacteria in
macrophages were measured at 3 hr and 24 hr postinfection.
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Log1 0 C FU / w el
l
7
6
3h
24h
5
4
3
2
1
Sc hu S4
rec A
iglC
rec AiglC
Fig.1. Intramacrophage growth of Schu S4 recA, iglC and recAiglC mutants. Murine macrophage cell line (J774) were infected with
mutants or their parental strain (Schu S4) using an inoculum of 10 MOI. Numbers of viable bacteria in macrophages were measured at 3
hr and 24 hrs post-infection.
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Milestone 53-B
Characterization of protective immunity against
pulmonary tularemia via oral vaccination in the F344 rat model
Characteristics of oral
vs. i.d. vaccination of
LVS/survival
Correlates of humoral
and cellular immunity
of LVS vaccination
Protective efficacy of
2 attenuated SCHU S4
strains
Intramacrophage survival
Vaccination/challenge
Bacterial dissemination
Histological analyses
CD4+ T cell
responses
Serum antibody responses
Secreted, BAL antibody
responses
Intramacrophage survival
vaccination/challenge
antibody responses
Bacterial dissemination and
histology
Red: completed
Green: in progress
Blue: Steps in the milestone
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Milestone #53B: Characterization of protective immunity against
pulmonary tularemia via oral vaccination in the F344 rat model
Results Update
Determination of intestinal and respiratory antibody
profiles following LVS vaccination of Fisher 344
rats.
Groups of F344 rats (6 rats per group) were
vaccinated either orally or intradermally with 107
CFU of LVS or mock vaccinated orally with PBS
and rested for three weeks. Fresh fecal pellets and
bronchoalveolar lavage fluid were collected and
supernatants were analyzed for LVS-specific
antibodies by ELISA.
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Fig. 2. Intestinal antibody profile following LVS vaccination. Groups of F344 rats
were vaccinated with 107 CFU of LVS either orally or intradermally and rested for
21 days. Fecal samples were collected and analyzed for LVS-specific antibodies.
Results: As shown in Fig. 2, neither vaccination route induced significant antigen-specific antibodies compared to mock-vaccinated
animals.
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Fig. 3. Respiratory antibody profile following LVS vaccination. Groups of F344
rats were vaccinated either orally or intradermally and rested for 21 days.
Bronchilar lavage fluid was collected and analyzed for LVS specific antibodies
Results: As shown in Fig. 3, LVS vaccination by either route induced antigen-specific total antibody, IgG2a, IgG2b, IgA and IgM.
Neither vaccination route induced any IgG1 antibody.
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Milestone #53B: Susceptibility of Fisher 344 rats to F. novicida
pulmonary challenge
Results Update
Determination of virulence of Francisella novicida
U112 following intratracheal challenge of F344 rats.
Groups of F344 rats (4 rats per group) were
challenged intratrachealy with either 102 or 105 CFU
of U112 in PBS. Rats were monitored daily for
morbidity and mortality.
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Fig. 4. Survival and weight loss following F. novicida U112 challenge. Groups of
F344 rats were challenged intratrachealy with either 102 or 105 CFU of F.
novicida. Rats were monitored daily for survival and weight loss.
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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. Continue construction of nadM (C-term) Schuh4 mutant.
2. Continue construction of FTT0748 Schuh4 mutant.
Milestone #52:
1. Continue construction of FTT1579 and FTT0523
Schuh4 mutants.
Continued on following slide
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Plan for following month: Milestone #53-A&B:
53A:
(1) Analyze the antibody profiles of mice immunized with the F.
tularensis recAiglC mutant (KKT23) at day 28 after vaccination.
(2) Evaluate the protective efficacy of i.n. KKF23 vaccination
against pulmonary Schu S4 challenge
53B:
(1) Survival of LVS vaccinated F344 rats following F. tularensis
SCHU S4 challenge
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Action Items
• Heather will show rats’ antibody response to F novicida
primary infection.
• Heather will look at F novicida dissemination to rat
organs, with i.t. primary F novicida challenge.
• Heather could do total protein to normalize the relative
levels of immunoglobulin classes and subclasses in rat
bronchoalveolar lavages
• Karl will design technically feasible experiment to prove
that the recA mutation stablizes other mutations in SCHU
S4.
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