University of Texas San Antonio
F. tularensis attenuated vaccine strain construction and evaluation
10/6/08 Annual TVDC Meeting
Progress on milestones to date:
Milestone #16: Create luciferase expressing F. tularensis LVS
COMPLETE 4/30/06
Milestone #39: Creation of uvrA and uvrB mutant F.
tularensis subsp. novicida strains
COMPLETE 8/31/06
Milestone #43: Creation of uvrA and uvrB mutant F.
tularensis subsp. holarctica (LVS) strains
COMPLETE 8/31/07
Milestone #48: Characterize uvrA and uvrB mutant F.
tularensis subsp. novicida strains
COMPLETE
Milestone #51: Construction of F. tularensis subsp. novicida
uvrB + pdpD, iglA, iglB, iglC, iglD strains.
COMPLETE
Milestone #49: Construction of mutant F. tularensis subsp.
tularensis strains
ONGOING
Milestone #50: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
ONGOING
Milestone #52: Construction of mutant F. tularensis subsp.
tularensis strains containing recA mutations
ONGOING
Our laboratory is focusing on the generation and characterization
of live attenuated F. tularensis subsp. novicida, tularensis, and
LVS strains for their vaccine potential
We are performing targeted disruption of various genes, including:
vgrG
iglABCD
These are virulence genes located in pathogenicity
island, they have high potential to attenuate strain
recA
This gene facilitates genetic recombination; its
inactivation will stabilize potential vaccine strain
We have spent a considerable amount of time developing
and optimizing techniques for genetic manipulation of
Francisella tularensis with SUCCESS!
Major accomplishments have included:
1. New optimized targeted mutagenesis of Ft novicida
2. New plasmid-based mutagenesis of Ft holarctica (LVS)
3. New technique to mutagenize Ft tularensis and Ft holarctica
“Tulatron”
4. Progress in removing 1 copy of FPI from Ft tularensis
•RNA loops EBS1 and EBS2
basepair with specific sequences
in target
•LtrA recognizes flanking sequences
•EBS1 and EBS2 can be “retargeted”
to recognize sites in target gene
•LtrA site preferences within target gene
are identified by Targetron computer
algorithm, which designs appropriate oligos
to retarget intron to your gene
Tulatron contains:
1. Ft promoters to drive
Antibiotic resistance
Intron RNP
2. Ft ori
3. ts mutation
4. Ec ori
5. KanR
6. lacZa “stuffer”
Tulatron has worked to simultaneously inactivate both copies
of iglC, iglD, and vgrG found in duplicated FPI in Ftt.
We have successfully inactivated iglC1 + iglC2, iglD1 + iglD2,
and vgrG1 + vgrG2
IglC is most upregulated protein during intramacrophage growth.
IglC, IglD, VgrG are required for phagosome escape, intramac.
growth
VgrG is involved in secretion of proteins into mac. cytosol
Ftt iglC1::L1.LtrB iglC2::L1.LtrB mutant strain virulence:
Survival
(30
days)
Schuh4
Challenge
7.5 X 101
5/5
0/5
9.0 X 102
5/5
0/5
9.0 X 103
5/5
0/5
9.0 X 104
5/5
0/5
9.0 X 105
5/5
0/5
PBS control
N.A.
0/5
CFU (intranasal route)
(210 CFU
i.n.)
survival
The Ftt iglC1 iglC2 mutant is highly attenuated, but not protective
via the intranasal route (slight delay to death at higher dose vacc.)
Ftt iglD1::L1.LtrB iglD2::L1.LtrB mutant strain virulence:
CFU (intranasal route)
Survival
(30 days)
Schuh4
Challenge
(778 CFU
i.n.)
survival
4.8 X 103
5/5
0/5
4.8 X 104
5/5
0/5
4.8 X 105
5/5
0/5
4.8 X 106
5/5
0/5
PBS control
N.A.
0/5
The Ftt iglD1 iglD2 mutant is highly attenuated, but not protective
via the intranasal route
We are testing vgrG1 vgrG2 mutant in mice…
Ftt vgrG1::L1.LtrB vgrG2::L1.LtrB mutant strain virulence:
(preliminary, day 17 post inoculation)
CFU (intranasal route)
Survival
(17 days)
1.9 X 103
5/5
1.9 X 104
5/5
1.9 X 105
5/5
1.9 X 106
5/5
Wildtype Shuh4 (76 CFU)
0/5
Schuh4
Challenge
??
We are also in process of removing one copy of FPI from
Ftt so that we can mutagenize other copy, by recombinasemediated excision, this will facilitate easy mutagenesis
of the second FPI:
pdpD iglA B C D
FRT site
IN PROGRESS
pdpC
insertion of FRT
sites via allelic
exchange
pdpB
pdpA
FRT site
DONE
Some of the things we have learned about genetic
manipulation of F. tularensis:
Use of Ft promoter to drive antibiotic resistance
is essential (both ermC and KanR work well)
Amount of flanking homology is critical for Ftt and Fth
(>=1 kbp)
Second recombination (loss of plasmid) does not
occur at high enough frequency without counterselection
Tulatron works well in Ftt and Fth
Unmarked deletions extremely difficult in Ftt, marked
deletions/insertions have been successful
Milestone #52: Construction of mutant F. tularensis subsp.
tularensis strains containing recA mutations
recA is required for recombination/DNA repair in bacteria.
The introduction of a recA mutation will stabilize defined
mutant strains of F. tularensis
Because of the role of recA in DNA repair, it may also be
required for survival under stressful conditions (i.e. in vivo)
We have created recA mutants of LVS and Schuh4:
recA attenuates LVS virulence (i.p. route)
recA has little effect on Schuh4 virulence (i.n. route)
LVS recA mutant infected i.p. into mice
Route of
Inoculation
I.P.
Mock
Inoculation Survival Rate Route of
Dose
(D30)
Chall enge
(CFU)
1300
7/7
I.P.
I.P.
Chall enge Survival Rate
Dose
D6
D30
(CFU)
700
7/7
7/7
110
0/5
LVS recA highly attenuated i.p., LD50 >103 CFU (LVS LD50 ~10 CFU)
Schuh4 recA mutant infected i.n. into mice
Inoculum
Route of
Inoculation
KKT11
Wt Schu S4
I.n.
I.n.
Inoculation
Dose
(CFU)
105
175
Survival Rate
D1
D2
D3
D4
D5
D6
5/5
5/5
5/5
5/5
5/5 0/5
5/5 5/5 4/5 0/5
No evidence of Schuh4 recA attenuation at high i.n. dose
Inoculum
KKT11
Wt Schu S4
PBS
Route of
Inoculation
I.n.
I.n.
I.n.
Inoculation
Dose
(CFU)
206
34
Survival Rate
D1
D2
D3
D4
D5
D6
D7
D8
D30
6/6
5/5
5/5
6/6
5/5
5/5
6/6
5/5
5/5
6/6
5/5
5/5
6/6
5/5
5/5
2/6
1/5
5/5
2/6
0/5
5/5
1/6
1/6
5/5
5/5
Slight attenuation of Schuh4 recA at low i.n. dose LD50 <102 CFU
Milestone 50
Summary 50A: Immunologic Characterization of F. tularensis subsp.
novicida, subsp. tularensis, and LVS strains
We evaluated the protective efficacy of additional mutants against
Francisella infection and host immune responses induced by the
vaccination.
 Mutants: 4 mutants were studied: DuvrBpdpD (U112), DiglB (U112)
DiglC (SCHU S4), and DiglD (SCHU S4).
 Virulence: All mutants are attenuated in mice via intranasal (i.n.) challenge.
 Immune responses: All mutants stimulated significant amounts of
antigen-specific antibody.
 Protective efficacy:
DuvrBpdpD protect mice against U112 challenge via i.n. immunization.
DiglB provided partial protection against SCHU S4 challenge
via oral immunization.
Level of Attenuation of Defined Francisella mutants
LD50 (Log10) i.n.
ΔuvrBpdpD
(U112)
ΔiglB
(U112)
ΔiglC
(SCHU)
ΔiglD
(SCHU)
5-6
>7
>7
>7
 ΔiglC and ΔiglD of SCHU S4 were highly attenuated and showed minimal
or no replication inside macrophage (J774) when compared to the parental
strain (increased about 1000 fold from 3h to 24h post-infection).
Profile of Antigen-Specific Antibody Response after Immunization
Vaccination
IgG(H+L)
IgG1
IgG2a
sIgA
ΔuvrBpdpD
(U112)
i.n. (105)
BALB/c
2000
700
2500
ΔiglB
(U112)
Oral (103)
C57BL
2600
2200
1600
+++
(7 fold to
mock)
ΔiglC
(SCHU)
Oral (103)
BALB/c
2300
1700
1700
+
(2 fold to
mock)
i.d. (103)
BALB/c
3600
600
2500
i.n. (105)
BALB/c
2400
2500
<200
ΔiglD
(SCHU)
Protective efficacy: Protection Provided by Vaccination via
Different Routes
Mutant
DuvrBpdpD
(U112)
DiglB
(U112)
Immunization
(dose/route)
Challenge
(strain/dose/route)
% Survival
(at day 28)
103 / i.n.
BALB/c
U112 / 1000 CFU / i.n.
100
103 / oral
C57BL
SCHU / 20 CFU / i.n.
66.7
SCHU / 20 CFU / i.d.
16.7
103 / oral
C57BL
103 / oral
C57BL boost
DiglC
(SCHU)
103 / i.d.
BALB/c
103 / oral
BALB/c
PBS/mock
10
SCHU / 50 CFU / i.n.
40
SCHU / 20 CFU/ i.n.
0
SCHU / 20 CFU / i.d.
16.7
SCHU / 20 CFU / i.n.
0
SCHU / 20 CFU / i.d.
0
SCHU / 20, 50 CFU / i.n.
0
Work Plan for the Coming Six Months
 Evaluate SCHU S4 DiglD as a vaccine candidate:
1. Protective efficacy of oral immunization against SCH S4 challenge
2. Immune responses induced by oral immunization
 Evaluate Ft tularensis (SCHU S4) vgrG, pdpD mutants as vaccine
candidates when mutants are generated
Summary 50B: Characterization of Protective Immunity
against Pulmonary Tularemia via oral LVS Vaccination
 Mice immunized orally with LVS (103 CFU) produced significant amounts of
antigen-specific IFN-g .
 Mice immunized orally with LVS produced significant levels of intestinal IgA
and high levels of antigen specific antibody in the serum and bronchoalveolar
lavage fluid (BAL).
 Mice immunized orally with LVS continued to produce high levels of antigen
specific total antibody in the serum at 12 weeks after vaccination. Mice that
were given a second booster dose of LVS after either eight or twelve weeks had
somewhat higher total serum antibody titers three weeks after the booster dose.
 Oral immunization with LVS (103 CFU) was highly protective (80-100%
survival) against subsequent SCHU S4 challenge (100 and 500 CFU) at 3
weeks after vaccination.
Summary 50B: Characterization of Protective Immunity
against Pulmonary Tularemia via oral LVS Vaccination
 Oral LVS vaccination leads to a reduction in bacterial replication following
pulmonary SCHU S4 challenge.
 Oral LVS vaccination induces the initiation of a cellular influx early into lungs
following SCHU S4 challenge.
 After oral LVS vaccination, mice which were treated with an anti-CD4 depleting
antibody at time of SCHU S4 challenge were found to be more susceptible to
infection than mice which were treated with a control Ig.
Profile of Cellular and Humoral Responses Following Oral LVS
Immunization
Cellular Responses (IFN-g)
Recall Against 104 LVS
Recall Against 105 LVS
2 Weeks
9.645 ng/ml
11.378 ng/ml
4 Weeks
.208 ng/ml
6.545 ng/ml
8 Weeks
.077 ng/ml
1.561 ng/ml
12 Weeks
<.031 ng/ml
1.125 ng/ml
Humoral Responses
Serum
(50% Binding Titer)
Total Ab
IgG1
IgG2a
IgA
IgM
4 weeks
5081
4552
8949
<100
<100
8 weeks
7086
1858
6934
<100
<100
12 weeks
3354
1741
4635
<100
<100
Oral LVS Vaccination Reduces Bacterial Replication and Increases
Cellular Infiltration into the Lungs Following SCHU S4 Challenge
Protective Efficacy of Oral LVS Vaccination Against Pulmonary SCHU S4
Challenge Following CD4+ T-cell Depletion
Localization to M-cells
Mock
M cells
LVS
M-cherry LVS
Nuclei
Challenges/Problems:
1. Milestone 43 was extended to incorporate an
additional (unrelated) deliverable: a secreted protein
with a MHC-I tag from OVA (SIINFEKL). We chose
PepO (secreted protease), constructed PepO-SIINFEKL
expressing plasmid, confirmed expression in LVS and Ftn,
and sent to Cerus.
2. Milestone 52 was modified to include the creation of a Ft plasmid
to express bacterial luciferase in F. tularensis. The original luciferase
plasmid we created in a completed milestone contained firefly
luciferase; it functioned correctly, but firefly luciferase was not optimal
for use in animals (as determined by UNM; requirement for substrate
and suboptimal temperature)
3. We substituted vgrG Schuh4 mutant for pdpD mutant,
based on results in Ftn demonstrating that pdpD is not
an essential virulence gene, but vgrG is. We will discuss other potential
attenuating mutations in tomorrow’s session.
Plans for next six months:
Milestone #16: completed.
Milestone #39: completed.
Milestone #43:completed
Milestone #48:completed
Milestone #51:completed
Milestone #49:
Creation of iglA and iglB Ftt mutant strains
Milestone #50:
Immune characterization of F. tularensis mutant strains
Milestone #52:
Construction of recA + iglC/iglD/vgrG Schuh4 mutants
Work Plan for the Coming Six Months
 Evaluate long term protective immunity to SCHU S4 challenge following
oral LVS immunization
 Begin to evaluate the oral LVS vaccination model in the Fisher 344 rat
- Assess primary LVS challenge including survival and bacterial
dissemination
- Humoral and cellular responses to oral LVS inoculation
- Protective efficacy of oral LVS vaccination against
intratracheal SCHU S4 challenge