University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
6/15/10 tech call
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Active milestones during last reporting period:
Milestone #52: Create recA mutants in F. tularensis subsp. tularensis
Milestone #53: Immune characterization of F. tularensis subsp.
tularensis mutant strains
Milestone #54: Construction of mutant F. tularensis subsp.
tularensis strains
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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
Generate, optimize
mutant strain construction
in Schuh4
Transform into iglC,
vgrG, iglD (other)
Schuh4 strains,
isolate mutants
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Increasing recombination frequency in Schuh4:
•Bacteriophage l encodes proteins (l Red recombinase) that
enhance recombination into bacterial chromosome
•We transformed pKEK1327 (Ft plasmid expressing lRed into
KKT29 (Schuh4 with restrict. mutations) (last month)
•We tested to see if we could recover transformants following
transformation with Ft novicida chromosomal DNA from
a iglI::ermC strain.
•We recovered no colonies.
•Problem: we are only allowed 2 antibiotic resistance markers
in Ftt (Kan, Erm) and lRed plasmid has KanR, therefore
we can only use Erm to test function of lRed in Ftt, our only
Erm marked genes are in Ftn
•Solution: change lRed plasmid to ErmR, then test in Ftt with
Schuh4 DNA derived from Tn (KanR) mutant (kindly supplied
by Brad Jones).
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•We will switch out KanR for ErmR in pKEK1327
We are also testing requirement of tryptophan biosynthesis
for F. tularensis virulence:
•We previously showed that trpB, but not trpA mutant of Ftn
is attenuated for virulence in mice
•The trpB mutant is a Trp auxotroph, whereas the trpA
mutant is not (growth in Chamberlin’s +/- Trp)
•IFNg stimulation reduces intracellular Trp, thus likely explaining
the attenuation of the trpB mutant.
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•Last month we assayed for growth in BMDM +/- IFNg
•We repeated intramac assay for growth of trpA and trpB
mutants in BMDM +/- IFNg:
trpA and trpB mutants
recovered at lower
levels w/o IFNg at
48 h
trpB not recovered
at 48 h w/ IFN g
•Results similar to last experiment: trpB mutant shows
significant defect compared to WT and trpA at 48 h + IFNg
•We will test for virulence of trpB in IFNgR-/- mice, and then
for growth in IFNgR-/- macrophages +/- IFNg
Red: completed
Green: in progress
Blue: Steps in the milestone
Milestone 54
Creation of mutant F. tularensis
subsp. tularensis strains
Construct lpxF, atpC, 3 other
mutagenesis plasmids
Mate into Schuh4,
select for transconjugate,
Counterselect for mutant
Verify mutants,
Pass on to Milestone 50
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Creation of attenuated Schuh4 strains:
•We are working at inactivating FTT1103 (a lipoprotein involved
in Ftt virulence) via targetron
•We previously transformed plasmid into Schuh4, are currently
screening transformants:
Results of screening colonies with primers that span FTT1103
gene show presence of insertion (arrow), but also presence of
wildtype gene (lower band); more cycling/screening necessary.
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•We are also targeting FTT1181 (ggt), a gamma-glutamyl
peptidase that contributes to virulence of Ftt
•We ligated fragments targeted to FTT1181
(positions 398 and 602) into targetron vector, screened
potential clones:
All clones correct (3-12), show
smaller fragment than controls
(lanes 2, 13), we sent off two for
sequencing (correct).
We have already transformed into
Schuh4, obtained colonies, results
will be discussed next month.
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Milestone 53A
Immunologic characterization of defined
F. tularensis mutants
Strains from milestone #52
And #54 : nadM, ipxF, atpC..
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
Milestone #53A: Immunologic characterization of defined
F. tularensis mutants
Results Update
Evaluation of F. novicida lipoprotein FTN_0109 and FTN_1734 mutant
strains as tularemia vaccines
These two proteins were identified as dominant sero-reactive antigens in the
Felgner’s immuno-array data set. FTN_1734 is a putative lipoprotein with an
unknown function and FTN_0109 is a novel protein of unknown function that
may be associated with the outer membrane. FTN_0109 has been shown to be
strongly attenuated and reduced for growth in Drosophila melanogaster.
UTSA applied an intramacrophage growth assay to evaluate the virulence of
Dftn0109 and Dftn1734 in vitro. Murine macrophage cell line (J774) were
infected with mutant strains or their parental strain (U112) using an inoculum of
10 or 100 MOI. Results suggest that Dftn0109 and Dftn1734 are moderately
attenuated for growth within macrophages. In vivo challenge experiments are
needed to further assess the virulence of the Dftn0109 and Dftn1734 mutants.
Intramacrophage uptake and replication of F.
novicida mutants Δftn0109 and Δftn1734
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
Milestone #53B: Characterization of protective immunity against
pulmonary tularemia via oral vaccination in the F344 rat model
Results Update
Evaluation of humoral responses elicited by oral vaccination with
the DiglD mutant of both F. tularensis subsp. novicida and subsp.
tularensis against intratracheal SCHU S4 challenge in Fischer 344
rats
Groups of Fischer 344 rats (6 rats per group) were vaccinated orally
with 106 CFU of U112, 107 CFU of the DiglD strains or mock
vaccinated with PBS and rested for 30 days. Rats were then bled
and sera were analyzed for antigen-specific antibodies by ELISA
Oral vaccination with F. tularensis subsp. novicida U112, U112 DiglD
and SCHU S4 DiglD elicit significant antigen-specific antibody
responses
Plan for following month:
Milestone #16: completed.
Milestone #39: completed.
Milestone #48: completed.
Milestone #43: completed.
Milestone #50: completed.
Milestone #51: completed.
Milestone #49: completed.
Milestone #52:
1. Test trpA/B mutants in IFNgR-/- mice
2. Exchange KanR in lRed-expressing plasmid for
ermC.
Milestone #54:
1. Characterize Schuh4 transformed with targetron against
FTT1181 (ggt)
1. Continue cycling FTT1103 targetron transformants,
isolate pure mutant
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Continued on following slide
Plan for following month: Milestone #53-A&B:
53A: Determination of LD50 of F. t. novicida FTN_1734 and
FTN_0109 mutants in a pulmonary challenge murine
model.
53B: Cellular responses to oral F.t. novicida and tularensis
DiglD vaccination in the Fischer 344 rat
Additional points:
Description of deliverables completed for each active milestone:
Milestone 52: Schuh4 recA, iglC1 iglC2 recA, FTT1579, FTT523, FTT1579 +
FTT523 strains
Milestone 53: None at this time
Milestone 54: Schuh4 atpC strain
List of relevant publications from the past month
MSCR status
MS 49: UTSA writing MSCR 49 (MS 49 was scientifically done 11/17/09;
Crystal Lauriano will write and is due in first week of June 2010 )
MS 50: NIAID reviewing as of 3/4/10 (UNM sent MS50 MSCR, 8 SOPs and 2 published
references on 3/4/10)
MS 51: UTSA reviewing revised MS51 MSCR (UNM sent edits on 12/4/09; Crystal
Lauriano revising Jeff Barker’s MSCR; was due the first week of May 2010; overdue)
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Action Items
• UTSA will add error bars to the slide
number 6 data in the tech call minutes.
• Crystal will email Barbara with firm
timelines for the two MSCRs (49 and 51)
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