Transcript UTSA Tech Call 2009.07.21

University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
7/21/09 tech call
1
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
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 were in the process of constructing a nadM (Cterm)
Schuh4 mutant via nadM targetron plasmid
•Last month, we identified “pure” nadM mutant after 11 cycles (!),
however, upon subsequent culturing, this mutant was lost
and the wildtype profile again took over the progeny clones:
Starting
mutant
WT
clones
Clearly there is selective pressure
against obtaining this particular
targetron insertion in nadM and
we will no longer pursue this
strategy, and rather exclusively pursue
the alternate strategy with the Tn
insertion in pJC84 (pKEK1305). We
Sequenced, confirmed it is correct,
will transform Schuh4 this month. 4
Construction of FTT0748 F. tularensis subsp. tularensis strain:
•We are constructing FTT0748 Schuh4 strain.
•Last month identified potential targetron insertions in FTT0748
in Schuh4
•We chose single clone and performed further culturing
to obtain pure culture:
colonies
wt
Intron + nadM primers
nadM primers
We now have FTT0748 mutant, we will remove targetron
plasmid via growth at 37°C, then infect mice to test for5
attenuation
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
6
Breaking down restriction barriers in Schuh4:
•Last month we reported transformation of targetron plasmid
to inactivate FTT1579 (restriction enzyme) in U112, but no
insertions recovered upon initial screen.
•We performed further cycling (4X) and screening, and have now
obtained many colonies with insertion in FTT1579:
wt
Screening with primers flanking
FTT1579 show many “pure” colonies
with targetron insertions.
Since targetron works and BSL3 is
now free, we will transform
Schuh4 next month.
7
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
Analyze the antibody profiles of mice intranasally
immunized with KKT23 (Ft subsp. tularensis recAiglC)
Blood was collected from PBS- and KKT23 (3X103, 3X104,
3X105, 3X106 CFU) - immunized mice at day 28 after
vaccination. Anti-KKT23 specific total serum antibody, as
well as IgG1 and IgG2a isotype titers was determined by
ELISA.
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
Protective efficacy of LVS vaccination followed by
F. tularensis SCHU S4 pulmonary challenge in
Fisher 344 rats.
Groups of F344 rats (6 rats per group) were
vaccinated either orally or intradermally with 107
CFU of LVS or mock vaccinated orally with PBS
and rested for four weeks. Rats were then challenged
intratracheally (I.T.) with 104 CFU of SCHU S4 and
monitored daily for weight loss and survival.
Fig. 2. Protective efficacy of LVS vaccination against SCHU S4 challenge in Fisher 344 rats.
Groups of F344 rats were vaccinated either orally or I.D. with 107 CFU of LVS and rested for 4
weeks. Rats were then challenged with 104 CFU of SCHU S4 and monitored daily for survival and
weight loss.
Milestone #53B: Characterization of pulmonary F. novicida U112
challenge in the F344 rat model
Results Update
Determination of antigen-specific serum antibody
profile following intratracheal (I.T.) challenge of
Fisher 344 rats with F. novicida U112
Groups of F344 rats (4 rats per group) were
challenged intratrachealy with either 102 or 105 CFU
of U112 in PBS and rested for 4 weeks. Blood was
collected and sera were prepared for determination of
U112-specific total antibody, IgG1 and IgG2a by
ELISA.
Fig. 3. Antigen-specific serum antibody profile following F. novicida U112 pulmonary challenge of
Fisher 344 rats. Groups of F344 rats were challenged I.T. with either U112 or mock challenged
with PBS and rested for 4 weeks. Sera were then prepared and analyzed for U112-specific
antibodies.
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 (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) Evaluate the protective efficacy of i.n. KKF23 vaccination
against pulmonary Schu S4 challenge
53B:
(1) Determine Cellular response of Fisher 344 rats following both
oral and intradermal LVS vaccination