University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
3/16/10 and 3/17/10 tech call
1
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
2
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
3
Increasing recombination frequency in Schuh4:
•Bacteriophage l encodes proteins (l Red recombinase) that
enhance recombination into bacterial chromosome
•This has been exploited in other bacteria to enhance
recombination for genetic construction (e.g. PNAS 97:6640)
•We are adapting this system for use in Ftt Schuh4:
•PCR amplification of lRed genes from pKD46, and
subsequent ligation into Ft plasmid pKEK1140:
Three colonies from ligation
Screened by PCR with lRed
Primers, all give expected 2.2
Kbp fragment
•We have cloned lRed genes into Ft plasmid behind Ft promoter,
We will next test to see if recombination into chromosome 4
has been enhanced.
We are also testing requirement of tryptophan biosynthesis
for F. tularensis virulence:
•Chlamydia trachomatis, an obligate intracellular organism,
must be able to synthesize tryptophan for full virulence:
•IFNg effectively reduces intracellular Trp to starve Trpintracellular organisms, C. trachomatis trpB mutants
attenuated in IFNg-treated cells (e.g. Mol Micro 49:1347)
•Since Ft is intracellular, and IFNg inhibits Ft growth, we
investigated importance of Trp biosynthesis:
•trpBA in operon, trpA encodes second-to-last step in
Trp biosynthesis, trpB encodes last step in Trp biosynthesis
•trpB mutants are Trp auxotrophs
•trpA mutants unable to make indole, if no other pathway
for indole production, then they are also Trp auxotrophs
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•Evaluation of virulence of trpB and trpA mutants of
Ft novicida (intranasal, ~100 CFU, Balb/C mice).
•The trpB mutant is attenuated for virulence, the trpA mutant
is not. The trpB mutant is a Trp auxotroph, the trpA mutant is not
(not shown).
•This is a promising mutation! In combination with other
mutations may be excellent for live vaccine candidate.
•We will characterize in IFNg +/- mice, macrophages, and
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Introduce into Schuh4 as well.
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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Milestone #54: Construction of mutant F. tularensis subsp.
tularensis strains
Inactivation of lpxF, atpC in SchuhS4:
•(last month) plasmid targeting lpxF transformed into Schuh4
strain, transformants screened for presence of insertion in lpxF.
•We decided to discontinue cycling, as agreed upon during
last phone conference, due to lack of enrichment for pure lpxF
mutant, despite >6 cycles.
•(last month) we created atpC SchuhS4 mutant
•We tested for virulence in Balb/C mice via i.n. route
(following page):
•No significant attenuation of atpC SchuhS4 mutant, all
groups of mice died, even at lowest inoculum (682 CFU)
Conclusion: this is not a good attenuating mutation for a live
vaccine strain, there is no evidence that the atpC SchuhS4
strain is attenuated.
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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
Infectivity of KKT-29 (SCHU S4 restriction enzyme [FTT0523 and
FTT1579] double mutant) in vitro
Murine macrophage cell line (J774) were infected with KKT-29 or its
parental strain (SCHU S4) using an inoculum of 10 or 100 MOI.
Numbers of viable bacteria in macrophages were measured at 3hrs
and 24 hrs post-infection.
Intramacrophage replication of SCHU S4 FTT0523/FTT1579
double mutant (KKT-29)
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
Cellular response in Fischer 344 rats following oral vaccination
with F.t. novicida wild-type or DiglB strains
Fischer 344 rats were vaccinated orally with 105 CFU of U112, 107
CFU of the DiglB strain or mock vaccinated with PBS and rested
for 14 days to ensure development of a cellular response. Spleens,
cervical and mesenteric lymph nodes were collected and single cell
suspensions were recalled in vitro for 72 hours with UV-inactivated
homologous antigen or the unrelated antigen HEL. Supernatants
were collected to analyze for the presence of IFN-g by ELISA.
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. Challenge trpB Ftn surviving mice with wt Ftn
2. Test trpA/B mutants in IFNg treated macrophages
for intracellular growth
3. Test lRed-expressing Ft for enhanced recombination.
Milestone #54:
1. Create targetron plasmids to inactivate FTT1103
(lipoprotein identified by Stulik as important for virulence)
Continued on following slide
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Plan for following month: Milestone #53-A&B:
53A: Intramacrophage replication of F. novicida FTN 0771
(protein disulfide isomerase) and FTN 1159 (g-glutamyl
transpeptidase) mutant strains
53B: Isolation of F344 primary alveolar macrophages for
Francisella intramacrophage replication
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
The manuscript “The Fischer 344 Rat Reflects Human Susceptibility to
Pulmonary Francisella Challenge and Provides a New Platform for
Virulence and Protection Studies” was accepted for publication in PLoS
ONE
MSCR status
MS 49: UTSA writing MSCR 49 (MS 49 was scientifically done 11/17/09;
Crystal Lauriano will write)
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)
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Action Items
• Heather will vaccinate rats with iglB F novicida strain and perform
recall experiment with SCHU S4
• when UTSA has a tryp B mutant in SCHU S4, UTSA will compare
attenuation with the atpC mutant at less than 100 CFU and with the
tryp B mutant at 10-100 CFU dose.
• Karl/Bernard will email pdf of newly published rat model article to
Barbara. Barbara will email it to NIAID
• Karl will work with Crystal Lauriano to write the MS49 MSCR and to
complete the MS51 MSCR.
• Barbara will email Crystal an example of a final, NIAID accepted
UTSA MSCR, to aid her writing. (done 3/17/10, including sending MS
49 MSCR template and instructions)
• Barbara will email Patrick a prioritization of 4 MSCR reviews (MS 11,
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33,46,50) (done 3/17/10)