Transcript UTSA

University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
7/20/10 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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We are 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
•We also showed (last month) that the trpB mutant was defective
for growth in activated macrophages (i.e. treated with IFN-g) but
not in untreated macrophages.
•This suggests that the trpB mutant would not be attenuated
for virulence in IFNgR knockout mice
•We performed virulence assay in wildtype C6BL and IFNgR-/Mice of trpA, trpB, and wildtype strains (next slide).
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•The trpB mutant is attenuated for virulence in the wildtype mice
but not in the IFNgR-/- mice (same inoculum for both, 10 CFU
via intranasal route). Wildtype and trpA mutants are not
attenuated for virulence.
•We will repeat intramacrophage experiment in BMM from both
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wildtype and IFNgR-/- mice +/- 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) and FTT1181 (gamma glutamyl peptidase
shown to be important for cysteine acquisition) via targetron
•We are still screening FTT1103.
•Last month, we showed we had created FTT1181 targetron
plasmid.
We transformed into Schuh4, screened colonies:
Results of screening colonies with
primers that span FTT1181 gene
show presence of insertion (arrow),
one clone looks pure (lane 25)
We will further purify this clone,
and remove targetron plasmid
(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.
UTSA conducted pulmonary challenge experiments to assess the virulence of the
Dftn0109 and Dftn1734 mutants in mice. BALB/c mice were challenged
intranasally with escalated numbers (102 to 105 cfu ) of Dftn0109 or Dftn1734
to determine 50% lethal dose. Results showed that the ftn0109 mutant was highly
attenuated with a LD50 greater than 105 cfu when infected mice i.n., in contrast
to the virulent Dftn1734 , which had a LD50 less than 100 cfu.
F. novicida ftn1734 mutant is virulent in
intranasally challenged mice
F. novicida ftn0109 mutant is highly
attenuated in intranasally challenged mice
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
UTSA is evaluating the protective immunity of subcutaneous (S.Q.) F. novicida
U112 vaccination in the Fischer 344 rat model.
Groups of rats (6 rats per group) were vaccinated S.Q. with increasing doses (5
X 101, 5 X 102, 5 X 104 CFU) of the F. novicida wild-type strain U112 and
monitored for signs of inflammation. Results suggest that subcutaneous
vaccination of the F. novicida U112 wild-type strain may cause only slight local
inflammation at the highest administered doses and does not cause any apparent
illness in the Fischer 344 rat model.
S.Q. injection:
 Dose: 5 X 101, 5 X 102, 5 X 104 CFU of U112.
 Site: An area of approximately 1 ½” in diameter was shaved on the back
of the rats, below the neckline and the inoculums was injected between the
shoulder blades.
Inflammation:
 Two lower vaccination doses (5 X 101, 5 X 102 CFU): no sign of local
inflammation was ever displayed.
 Highest dose (5 X 104 CFU): three of the six rats exhibited a slight,
diffuse rash within ¾” from the injection site which appeared 1-2 days
after vaccination. The rash remained the same size for 2 days, after
which it became visibly fainter with all signs of the inflammation
completely gone by 5-6 days after vaccination.
Morbidity:
 None of the rats in any vaccination group lost weight or showed any
other outward signs of illness following S.Q. U112 vaccination.
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-/- macrophages
2. Exchange KanR in lRed-expressing plasmid for
ermC.
Milestone #54:
1. Isolate FTT1181 (ggt) mutant, test for virulence in mice
2. Continue cycling FTT1103 targetron transformants,
isolate pure mutant
Continued on following slide
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Plan for following month: Milestone #53-A&B:
53A: Humoral responses of mice induced by intranasal
Dftn0109 vaccination
53B: Replication of the F. novicida U112 and F. tularensis
SCHU S4 strains within primary alveolar macrophages
obtained from Fischer 344 rats
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 by October 28, 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; will be revised by Aug 31, 2010)
Action Items
• UTSA will email draft manuscripts to UNM
for UNM and NIAID review, prior to UTSA
submission of the manuscript to a journal.
(For Trp B and other work).
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