Transcript UTSA

University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
5/19/10 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
•We previously made plasmid for expressing lRed recombinase
in Francisella pKEK1327 (March)
•We also showed that this plasmid stimulates recombination
in Ftn (April)
•We will now test in Ftt
•First step: transform into KKT29 (Schuh4 with restrict. mutations)
Ten colonies from transformation
screened by PCR with lRed
primers, 9/10 give expected 2.2
Kbp fragment (lanes 2-6, 10-13),
similar to control plasmid (lane 7)
•We will next test to see if recombination into Ftt chromosome
has been enhanced.
4
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
•We previously tested growth of trpB mutant in IFNg-treated
J774 cells, no difference between wt and trpB growth
•Here we tested for growth of trpA and trpB mutants in
BMDM:
trpA and trpB mutants
recovered at lower
levels w/o IFNg at
24, 48 h
trpB not recovered
at 48 h w/ IFN g
We need to repeat
experiment to show
significant difference
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at 48 h
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
6
Creation of attenuated Schuh4 strains:
•We previously showed creation of two targetron plasmids
to inactivate FTT1103 (lipoprotein involved in Ftt virulence)
•We transformed both plasmids into Schuh4:
Only targetron targeted to 466 gave
positive PCR with intron-specific
and FTT1103-specific primers
(lanes 4-10) indicating insertion in
FTT1103
Several colonies chosen and cycled
further:
Primers spanning FTT1103 gene should
show larger fragment with insertion
(compared to wt, lane 2), insertion can
be seen (e.g. lane 6), more cycling is
required to obtain pure mutant.
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•We are also targeting FTT1181 (ggt), a gamma-glutamyl
peptidase that contributes to virulence of Ftt
•We performed PCR to generate fragments to inactivate
ggt by targetron:
Fragments designed to target 398|399 and 601|602
were generated by PCR (lanes 2 & 3), these
will next be ligated into targetron plasmid and
transformed into Schuh4
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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_0771) and gglutamyltranspeptidase (FTN_01159) mutant strains as tularemia
vaccines
FTN_0771, an ortholog of SCHU S4 FTT1103, is a hypothetical lipoprotein and
shares some similarity with DsbA proteins, which are proteins that catalyze
disulfide bond formation.
FTN_1159, an orthology of SCHU S4 FTT1181, is a g-glutamyltranspeptidase
(GGT) that involves in the metabolism of γ-glutamyl-containing peptides.
To assess their virulence in mouse, we intranasally challenged BALB/c mice (6
per group) with escalating doses (103 – 106 cfu) of Dftt0771 and Dftn1159, and
monitored mice daily for their weight loss and survival. Results showed that
these two mutants might be as virulent as wild type U112 in pneumonic
tularemia mouse model (LD50 < 1000 CFU). However, the virulence of SCHU
S4 homologous mutants remains to be evaluated.
LD50 of Δftt1159 and Δftn0771 determined
using an intranasal challenge mouse model
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 protective efficacy of 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
challenged intratracheally with 104 CFU of SCHU S4 and
monitored daily for morbidity and mortality.
Oral vaccination with F. tularensis subsp. novicida U112, U112 DiglD
and SCHU S4 DiglD protected rats against subsp. tularensis SCHU S4
intratracheal challenge
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 IFNg treated BMDM
for intracellular growth
3. Test lRed-expressing Ftt for enhanced recombination.
Milestone #54:
1. Create targetron plasmids to inactivate FTT1181 (ggt)
2. Continue cycling FTT1103 targetron transformants,
isolate pure mutant
Continued on following slide
15
Plan for following month: Milestone #53-A&B:
53A: Intramacrophage replication of the F.t. novicida
FTN_1734 and FTN_0109 mutants. These two proteins
were identified as dominant sero-reactive antigens from
the Felgner’s immuno-array data set.
53B: Humoral 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 try titrating the IFN gamma in the BMDM growth of
the Tryp mutants to enhance sensitivity.
• Bernard will look at the weight loss data on the one Mock
survivor (slide 14) and add the clinical signs like weight loss to
the tech call minutes
• Bernard: will do QC on rat lung deposition as UNM does.
UTSA will confirm the deposition approximately 45min to 1 hr
after IT challenge, by homogenizing a few lungs and plate them
out to enumerate the deposited challenge dose.
• Crystal will submit the MS 51 MSCR, which is overdue as of first
week of May 2010, or else another UTSA scientist will complete
the MS51 MSCR and submit to UNM.
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Comparison of weight loss between groups of rats
orallyvaccinated with either PBS or F. tularensis subsp.
novicida U112, and subsequently challenged
intratracheally with subsp. tularensis SCHU S4
*
*
**
*
* Rats survived from SCHU S4 challenge
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