Transcript UTSA Tech Call 2009.08.18

University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
8/18/09 tech call
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Active milestones during last reporting period:
Milestone #49: Construction of nadM, FTT0748 F. tularensis subsp.
tularensis strains
Milestone #52: Create recA mutants in F. tularensis subsp. tularensis
Milestone #53: Immune characterization of F. tularensis subsp.
tularensis mutant strains
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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
•Despite a lot of work, we have been unsuccessful in constructing
a nadM (Cterm) Schuh4 mutant via nadM targetron plasmid
(long story, see previous reports)
•We decided to pursue an alternate strategy of moving nadM Tn
insertion from Ft novicida into Ft tularensis via pJS84 plasmid
•Obtained KanR Schuh4 transformants with pJS84nadM::Tn
construct, are screening these now:
Kan PCR
pJC84
nadM KanR
clone
Ftn
KanR
nadM pJC84 clone
nadM
Very few transformants. PCR analysis
indicates lack of KanR gene in nadM
in KanR transformants (left A).
KanR transformant shows very light PCR
signal for plasmid origin, indicating it
had been transformed with plasmid
(right B).
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Disappointing. This is last month of this milestone,
we are attempting one last transformation with pJC84nadM
plasmid, also inserting a mob (transfer) gene into this
plasmid to increase numbers of transformants.
We may not be successful in generating a Schuh4 nadM
mutant.
But…
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Construction of FTT0748 F. tularensis subsp. tularensis strain:
•We constructed FTT0748 Schuh4 strain.
•We identified correct mutant, and cured of targetron plasmid
by growth at 37°C.
•We have inoculated this mutant strain into mice via intranasal
inoculation, and will report next month on results of whether
this strain is attenuated for virulence.
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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:
•Last month we reported identification of insertion in
FTT1579 (restriction enzyme) in U112, this was done to “test”
plasmid while BSL3 lab was inaccessible.
•We successfully generated FTT1579 U112 mutant (KKF378).
•We transformed targetron plasmid into Schuh4, screened
98 mutants via PCR, identified two transformants with
“pure” insertion in FTT1579 gene:
gene
primers
#1
#2
Gene + intron
primers
#1
#2
Wt KKF Wt
Schuh4 378 Schuh4
PCR screening with primers flanking
FTT1579 show clones #1 and #2
are “pure” colonies with targetron
insertions (left).
PCR screening with gene + intron
Primer shows presence of targetron
within FTT1579 (right).
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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
Evaluate the protective efficacy of intranasal KKT-23
(ΔrecAiglC of SCHU S4) vaccination against intranasal F.
tularensis challenge
Mice (C57BL/6) were intranasally vaccinated with KKT-23
(3x106 CFU), rested for 30 days, and challenged i.n. with
187 CFU of SCHU S4
All mice vaccinated with KKT-23 or mock treated with PBS
succumbed to SCHU S4 pulmonary challenge. This is a
similar result to our previous protective efficacy study using
the single iglC SCHU S4 mutant, which provided no
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protection against SCHU S4 challenge
Milestone #53A: Immunologic characterization of defined
F. tularensis mutants
Results Update
Analyze the antibody profiles of mice vaccinated i.n. vs
orally with KKF235 (Ft subsp. novicida iglB)
Mice (C57BL/6) were intranasally vaccinated with KKT-23
(3x106 CFU), rested for 30 days, and challenged i.n. with
187 CFU of SCHU S4
Sera, intestinal and respiratory fluids were obtained from
KKF235 immunized mice at day 28 after vaccination. AntiKKF235 specific antibody titer in serum (total Ig, IgG1,
IgG2a), intestinal fluids (IgA, IgM) and BAL fluids (total
Ig, IgG1, IgG2a, IgA, IgM) were determined by ELISA
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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
Measure the cellular response of Fisher 344 rats
following LVS vaccination
Groups of F344 rats (3 rats per group) were
vaccinated either orally or intradermally (I.D.)
with 107 CFU of LVS in PBS or mock vaccinated
orally with PBS alone and rested for 14 days.
Rats were then sacrificed, spleens and cervical
lymph nodes were removed, and single cell
suspensions were made. Cells were then
incubated in the presence of UV-inactivated LVS
(103, 104, 105 CFU), or the unrelated antigen
hen egg lysozyme (HEL), for 72 hours. Culture
supernatants were then collected and analyzed
for the presence of IFN- by cytokine ELISA.
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Plan for following month:
Milestone #16: completed.
Milestone #39: completed.
Milestone #48: completed.
Milestone #43: completed.
Milestone #50: completed.
Milestone #51: completed.
Milestone #49:
1. Continue construction of nadM Schuh4 mutant.
2. Measure virulence of FTT0748 Schuh4 mutant in mice.
Milestone #52:
1. Construction of FTT0523 (restriction enzyme #2)
Schuh4 mutants.
Continued on following slide
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Plan for following month: Milestone #53-A&B:
53A:
(1) Evaluate the intramacrophage growth of KKT 19 (FTT_1579)
mutant
53B:
(1) Determine cellular response of Fisher 344 rats following F.
novicida challenge
(2) Determine bacterial dissemination following F. novicida
challenge
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