University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
4/21/09 tech call
1
Active milestones during last reporting period:
Milestone #49: Construction of nadM, FTT0748 F. tularensis subsp.
tularensis strains
Milestone #50: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Milestone #52: Create recA mutants in F. tularensis subsp. tularensis
Milestone #53: Create recA mutants in F. tularensis subsp. tularensis
2
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
3
Milestone #49: Construction of nadM, FTT0748 F. tularensis subsp.
tularensis strain
•We are in the process of constructing a nadM (Cterm)
Schuh4 mutant via nadM targetron plasmid
•We’ve already screened colonies by PCR that verified
insertion in nadM, we’re now cycling to isolate pure mutant
WT
transformants
WT transformants
external primers to nadM reveals
presence of larger size (insertion)
fragment in pool, we are now up
to 7 and 8 cycles. Still no pure
mutant.
This is unusual, we’ve never
experienced this before. We will
continue to cycle until our
alternative strategy is ready to go.
4
Construction of nadM F. tularensis subsp. tularensis strain by
an alternate strategy:
•Since cycling of targetron transformants hasn’t yielded
pure mutants yet (after 7-8 cycles), we are pursuing
an alternate strategy simultaneously.
•We are cloning Tn insertion from nadM Ft novicida mutant
(this mutant was already characterized in previous reports)
into Ftt mutagenesis plasmid pJC84 (J. Celli).
•nadM::Tn5 from Ftn nadM was cloned into pGEM-T
•We are now cloning this into pJC84
•Have screened colonies, do not have correct construct yet.
5
Construction of FTT0748 F. tularensis subsp. tularensis strain:
•We are constructing FTT0748 Schuh4 strain.
•We ordered oligos to target this gene by Targetron,
PCR amplification of fragment and subsequent cloning
into Targetron plasmid (pKEK1140):
clones
parent
clones
In correct clones, BglII
fragment will decrease from
3.8 to 3.4 kbp, all clones
Appear correct.
We have sent off the clone
in lane 9 for sequencing,
will proceed with this
targetron if sequence is
correct.
6
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
7
Construction of a iglC1 iglC2 recA mutant
•Strain KKT5 (iglC1 iglC2) was transformed with recA
targetron vector (pKEK1186), colonies were isolated,
screened for recA disruption; strain with recA insertion
detected and isolated (last period).
•Targetron plasmid was removed from this strain by growth at
37°C, then colonies checked to ensure insertion remained
at recA:
“WT”
All colonies maintain
colonies
colonies
recA insertion.
We have successfully
created iglC1 iglC2 recA
Schuh4 strain, designated
KKT23.
8
Breaking down restriction barriers in Schuh4:
•Ftt has restriction/modification systems that inhibit the
introduction of non-Ftt DNA, this inhibits genetic manipulation
of Ftt.
•Inactivation of restriction enzymes will “break” the restriction
barrier and facilitate easy transformation; this has been
accomplished with Ft novicida (J. Bact. 2008. 190:7830).
•We have identified two intact restriction enzyme genes in
Ftt: FTT1579 (shared with Ftn) and FTT0523 (unique to Ftt)
•We will inactivate each gene utilizing Targetron and test
for transformation.
•We chose two sites in FTT1579 (849/850 and 1254/1255),
ordered targetron oligos, performed PCR:
We successfully amplified both
targets, are now cloning each into
targetron vector (pKEK1140).
9
Milestone 50-A
Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
F. novicida uvrA, uvrB
Double mutant
F. novicida uvrA+pdpD
F.novicida uvrB+pdpD
iglA, iglB, iglC, iglD
In vitro Growth
In vivo Bacterial Burden
LD50 determination
In vitro Growth
In vivo Bacterial Burden
LD50 determination
Red: completed
Green: in progress
Blue: Steps in the milestone
LVS: uvrA, uvrB
Schu4: iglC, iglD,
vgrG,
In vitro Growth
In vivo Bacterial Burden
LD50 determination
Further immunological characterization
based on initial screen
10
Milestone #50A: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Results Update
Evaluate the protective efficacy of oral KKF235
(ΔiglB of U112) vaccination against Type B
(OR96-0246 strain)
Mice were orally given a single dose (103 CFU) of
KKF235, LVS or mock-treated (PBS), rested for
30 days, and challenged intranasally with 34 CFU
of Type B. Mice were monitored daily.
11
100
80
LVS
PBS (mock)
60
% Survival
40
20
0
100
0
5
10
15
20
25
30
KKF235
PBS (mock)
80
60
40
20
0
0
5
10
15
20
25
30
Days post challenge
Fig. 1. Protective efficacy of KKF235 (iglB mutant of U112) immunization against Type B F. tularensis infection. Mice were mock
vaccinated with PBS or orally immunized with 103 CFU of KKF235 or LVS and i.n. challenged with lethal dose (34 CFU) of OR96-0246
strain. Mice were monitored for survival.
Results: KKF235 vaccinated mice exhibited a significant prolonged median survival time of 9.5 days when compared to 6 days for
the PBS mock vaccinated mice. These mice exhibited a 17% survival. All LVS vaccinated mice completely survived the bacterial
challenge.
12
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
13
Milestone #53B: Characterization of protective immunity against
pulmonary tularemia via oral vaccination in the F344 rat model
Results Update
Replication of F. novicida U112 and F. tularensis
SCHU S4 within rat bone marrow derived
macrophages.
Bone marrow derived macrophages were seeded in
96-well culture plates. Cells were infected with
either F. novicida or F. tularensis SCHU S4 at 10 and
100 MOI for 2 hours. Cells were then pulsed with
Gentamicin for 1 hour. Cells were lysed at 3, 24, 48,
or 72 hours after infection and serial dilutions of
lysate were plated.
14
F. nov ic ida
F. tularens isSCHU S4
10 MOI
10
7
10 6
10
6
10 5
10
5
10 4
10
4
10 3
10
3
10 2
10
2
10
1
CFU
10 7
10 1
3
24
48
72
100 MOI
3
24
48
72
Hours After Inoc ulation
Fig. 1. Intramacrophage growth of F. novicida and SCHU S4 in rat BMDM. Primary
bone marrow derived macrophages derived from Fisher 344 rats were infected
with F. novicida U112 or F. tularensis SCHU S4 at either 10 or 100 MOI. Cells
were lysed and viable bacteria were counted at 3, 24, 48 and 72 hours after
infection.
Results: As shown in Figure 1, there was uptake and replication observed with F. novicida. In contrast, there seem to be minimal
uptake and replication seen with SCHU S4.
15
Plan for following month:
Milestone #16: completed.
Milestone #39: completed.
Milestone #48: completed.
Milestone #43: completed.
Milestone #51: completed.
Milestone #49:
1. Continue construction of nadM (C-term) Schuh4 mutant.
2. Continue construction of FTT0748 Schuh4 mutant.
Milestone #52:
1. Continue construction of FTT1579 and FTT0523
Schuh4 mutants.
Continued on following slide
16
Plan for following month: Milestone #53-A&B:
53A:
(1) Intramacrophage replication of SCHU S4 RecA and SCHU S4
RecA/IglC mutant
53B:
(1)Assess the need for opsonization of SCHU S4 to replicate within
F344 bone marrow derived macrophages
(2) Serum antibody titers of F344 rats following either oral or
intradermal LVS vaccination
17
Action Items
•
•
•
•
•
UTSA will send the mutant gene list /table to UNM with the monthly technical report
due on 5/7/09. This was an action item from the UTSA site visit.
Bernard will make new flow chart slide for MS53B for SCHU S4 and iglA double
mutant.
UNM: NMFTA1 Type A clinical isolate. UNM/Terry will send this strain to UTSA
UTSA: OR96-0246 Type B isolate. UTSA/Bernard will send this strain to UNM.
NOTE AFTER CALL: On 4/17/09 ,Barbara sent requested MS 51 MSCR revisions to
Jeff Barker and requested return by 4/30/09.
18