University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
5/19/09 tech call
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Active milestones during last reporting period:
Milestone #49: Construction of nadM, FTT0748 F. tularensis subsp.
tularensis strains
Milestone #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
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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
•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 still cycling to isolate pure mutant
WT
transformants
external primers to nadM reveals
presence of larger size (insertion)
fragment in pool, we are now up
to 8 and 9 cycles. Still no pure
mutant.
We will continue to cycle until our
alternative strategy is ready to go.
WT transformants
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Construction of nadM F. tularensis subsp. tularensis strain by
an alternate strategy:
•we are pursuing an alternate strategy involving
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 (still).
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Construction of FTT0748 F. tularensis subsp. tularensis strain:
•We are constructing FTT0748 Schuh4 strain.
•We have constructed the correct Targetron plasmid to
knockout this gene (confirmed by sequencing).
•We have transformed this plasmid into Schuh4, obtained
KanR colonies.
•We will report screening for correct mutant in next report.
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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:
•Ftt has restriction/modification systems that inhibit the
introduction of non-Ftt DNA, this inhibits genetic manipulation
of Ftt.
•We have identified two intact restriction enzyme genes in
Ftt: FTT1579 (shared with Ftn) and FTT0523 (unique to Ftt)
•Inactivation of FTT1579: we chose two target sites in gene
(849/850 and 1254/1255)
•Performed ligation with PCR products into pKEK1140, screened c
849/850
pKEK
1140
1254/1255
We successfully constructed
Targetrons to 849/850 and
1254/1255 (seen by slightly
Smaller fragment; arrow), these
were confirmed by sequencing.
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Construction of a iglC1 iglC2 recA mutant
•We successfully created Schuh4 iglC1 iglC2 recA strain
last reporting period.
•This period we concentrated on breaking down restriction
barriers of Schuh4 to facilitate genetic manipulation.
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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
Milestone #53A: Immunologic characterization of defined
F. tularensis mutants
Results Update
Determine the LD50 of F. tularensis recA (KKT-11) and
recAiglC (KKT-23) double mutant
Groups of C57/Bl6 mice were intranasally challenged with
75 or 320 CFU of KKT-11 (SCHU S4 recA mutant) and
3X103-3X106 CFU of KKT-23 (SCHU S4 recAiglC double
mutant). Mice were monitored daily.
% Surv iv al
100
80
75 CFU
320 CFU
60
40
20
0
% O riginal Body Weight
0 .0 0
110
105
100
95
90
85
80
0
2 .0 0
2
4 .0 0
6 .0 0
8 .0 0
1 0 .0 0
4
6
8
10
Days after Infection
1 2 .0 0
1 4 .0 0
12
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Fig.1. Survival of mice infected with SCHU S4 recA (KKT-11) mutant. Groups of C57BL/6 mice were challenged intranasally with
either 75 or 320 CFU of KKT-11 and monitored for survival and weight loss..
% Survival
100
80
3x10 3 CFU
3x10 4 CFU
3x10 5 CFU
3x10 6 CFU
60
40
% O riginal Body Weight
20
00
2
4
2
4
6
8
10
12
14
6
8
10
Days after infection
12
14
120
110
100
90
80
0
Fig.2. Survival and weight loss of mice infected with SCHU S4 recAiglC (KKT-23) double mutant. Groups of C57BL/6 mice were
challenged intranasally with escalating inocula (3x103 , 3x104 , 3x105 and 3x106 CFU) of KKT-23.
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
Replication of F. holarctica LVS and F. tularensis
SCHU S4 within rat bone marrow derived
macrophages.
Bone marrow derived macrophages were seeded in 96-well culture
plates at a density of 2 X 105 cells per well and allowed to adhere over
night. Cells were infected with fresh overnight cultures of either F.
holarctica LVS or F. tularensis SCHU S4 at 100 MOI for 2 hours.
Cells were then pulsed with Gentamicin for 1 hour to kill any
remaining extracellular bacteria, after which they were incubated at 37
degrees C in either fresh media, or media containing 10% homologous
rat serum. Cells were lysed at 3, 24, 48, or 72 hours after infection and
serial dilutions of lysate were plated on TSA plates to enumerate
intracellular bacteria. Additionally, culture supernatants from each
timepoint were plated to determine if some bacteria were being
released in to the media.
Figure 1. Rat BMDM phagocytosis of LVS and SCHU S4. F344 bone marrow derived macrophages
were infected with overnight cultures of either LVS or SCHU S4 at 100 MOI. At indicated time points,
serial dilutions of culture supernatants and cell lysates were plated for bacterial enumeration.
Results: As shown in Figure 1, 2-3 logs of both LVS and SCHU S4 were taken up at three hours after infection. At 24 hours, there
was a slight decrease of intracellular LVS and a minimal increase of intracellular SCHU S4 while 3-4 logs of either bacteria were
present within the culture supernatant. The numbers of intracellular organisms continued to decrease throughout the time course with
only minimal numbers recovered at 72 hours while the numbers of bacteria in supernatant remained constant. It was also found that
the presence of homologous serum did not have an effect on either bacterial uptake or replication.
Milestone #53B: Characterization of protective immunity against
pulmonary tularemia via oral vaccination in the F344 rat model
Results Update
Determination of LVS-specific serum antibodies
following LVS vaccination of F344 rats.
Groups of female F344 rats (6 rats per group) were
vaccinated either orally or intradermally with 107
CFU of LVS in PBS or mock vaccinated (PBS
alone). Four weeks later, rats were bled and sera
were prepared. LVS-specific antibody titers were
determined by ELISA.
Figure 2. Serum antibody titers after LVS vaccination of F344 rats. Groups of female F344
rats were vaccinated either orally or intradermally with 107 CFU of LVS in PBS or mock
vaccinated with PBS alone. Four weeks later, rats were bled and serum analyzed for LVS-specific
antibody. Results represented as 50% end-point titers.
Results: As shown in Figure 2, vaccinated rats exhibited high total antibody titers which were predominantly of the IgG2a isotype
while there was no detectable IgG1 response. There was no significant difference between antibody titers of rats vaccinated orally or
intradermally.
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
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Plan for following month: Milestone #53-A&B:
53A:
(1) Intramacrophage replication of SCHU S4 RecA and SCHU S4
RecA/IglC mutant
(2) Analyze the antibody profiles of mice immunized with the F.
tularensis recAiglC mutant at day 28 after vaccination
53B:
(1) Survival analyses of F344 rats following oral/ID LVS
vaccination and SCHU S4 challenge
(2) Respiratory and intestinal antibody titers of F344 rats following
either oral or ID LVS vaccination
Action Items
•UTSA’s timeline for updating the Summary of Mutants chart is within the
week (so by 5/22/09). Heather said that UTSA gathered the data and just
needs to enter it into the chart, in Excel.
•UTSA will make a decision on the NadM gene mutants in SCHU S4- has
been challenging to purify the mutant strain away from the wildtype SCHU
S4.
•Karl reminded that UNM will share a NM type A strain with UTSA and
UTSA will share a type B strain with UNM. Action: Crystal and Terry will
coordinate. 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.
•Mice vaccinated with double mutant recA/iglC SCHU S4 will be
challenged with wildtype SCHU S4.
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