Transcript UTSA

University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
2/16/10 tech call
1
Active milestones during last reporting period:
Milestone #52: Create recA mutants in F. tularensis subsp. tularensis
Milestone #53: Immune characterization of F. tularensis subsp.
tularensis mutant strains
Milestone #54: Construction of mutant F. tularensis subsp.
tularensis strains
2
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
Generate, optimize
mutant strain construction
in Schuh4
Transform into iglC,
vgrG, iglD (other)
Schuh4 strains,
isolate mutants
3
Breaking down restriction barriers in Schuh4:
•We have constructed single insertion mutations in two
different restriction enzymes in Schuh4: FTT1579 (KKT19)
and FTT0523 (KKT28) (previous reports) and now have
constructed a double mutant (FTT1579 + FTT0523; KKT29)
•We tested the single mutants and double mutants for
transformation efficiency:
•We have succeeded! The double mutant is ~20-fold increased
for transformation efficiency
4
Red: completed
Green: in progress
Blue: Steps in the milestone
Milestone 54
Creation of mutant F. tularensis
subsp. tularensis strains
Construct lpxF, atpC, 3 other
mutagenesis plasmids
Mate into Schuh4,
select for transconjugate,
Counterselect for mutant
Verify mutants,
Pass on to Milestone 50
5
Milestone #54: Construction of mutant F. tularensis subsp.
tularensis strains
Inactivation of lpxF, atpC in SchuhS4:
•(last month) plasmid targeting lpxF transformed into Schuh4
strain, transformants screened for presence of insertion in lpxF.
•We continue to cycle transformants to obtain pure lpxF mutant:
PCR with external primers shows the
presence of insertion (arrow), but also
presence of wildtype lpxF in all colonies.
We continue to cycle
to obtain pure lpxF mutant.
Inactivation of atpC in SchuhS4:
•atpC mutant was attenuated for virulence in Ft novicida
(Kraemer et al IAI 77:232); mutant had LD50>100 CFU via
aerosol, replicated but was eventually cleared from liver
and spleen.
•We created atpC mutant last month, have now removed
targetron plasmid to create KKT32:
external primers show pure mutant
atpC in most colonies, one was chosen,
plasmid removed by growth at 37°C,
and named KKT32
external primers
•We will test atpC mutant strain for virulence in mice.
7
Milestone 53A
Immunologic characterization of defined
F. tularensis mutants
Strains from milestone #52
And #54 : nadM, ipxF, atpC
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
Evaluation of a SCHU S4 atpC (FTT0065)
mutant strain KKT-32
AtpC is the epsilon subunit of FoF1-ATP synthase. Murine
macrophage cell line (J774) were infected with KKT-32 or its
parental strain (SCHU S4) using an inoculum of 10 or 100 MOI.
Numbers of viable bacteria in macrophages were measured at 3 hr
and 24 hr post-infection.
Replication of SCHU S4 atpC mutant (KKT-32) within
macrophage is comparable to the wild type
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
Evaluation of protective efficacy of oral vaccination with F.
tularensis subsp. novicida wild-type strain U112 or mutant DiglB
strain against intratracheal SCHU S4 challenge in Fischer 344 rats
Groups of Fischer 344 rats (6 rats per group) were vaccinated orally
with 105 CFU of U112, 107 CFU of the DiglB strain or mock
vaccinated with PBS and rested for 30 days. Rats were then
challenged intratracheally with 104 CFU of SCHU S4 and
monitored daily for morbidity and mortality.
Oral vaccination with F. tularensis subsp. novicida U112 and DiglB
protected rats against subsp. tularensis SCHU S4 intratracheal challenge
Plan for following month:
Milestone #16: completed.
Milestone #39: completed.
Milestone #48: completed.
Milestone #43: completed.
Milestone #50: completed.
Milestone #51: completed.
Milestone #49: completed.
Milestone #52:
1. Test trpA/trpB Ftn mutants for attenuation.
2. Create Ft plasmid to express lRed proteins for
enhanced recombination.
Milestone #54:
1. Continue (finish?) construction of lpxF Schuh4 mutant.
2. Test atpC Schuh4 mutant for virulence in mice.
Continued on following slide
14
Plan for following month: Milestone #53-A&B:
53A: Intramacrophage replication of the SCHU S4 restriction
enzyme double mutant strain KKT-29
53B: Cellular responses by oral F.t. novicida DiglB vccination
in Fischer rat 344
Additional points:
Description of deliverables completed for each active milestone:
Milestone 52: Schuh4 recA, iglC1 iglC2 recA, FTT1579, FTT523
strains
Milestone 53: None at this time
Milestone 54: None yet, in process
List of relevant publications from the past month
None
MSCR status
MS 49: UTSA writing MSCR 49 (MS 49 was scientifically done 11/17/09;
Crystal Lauriano will write the MS 49 MSCR)
MS 50: UNM reviewing v 0.4 MSCR 50 (UTSA sent edits on 1/21/10;
NIAID will review the 8 SOPs with the MSCR)
MS 51: UTSA reviewing revised MS51 MSCR (BG sent edits on 12/4/09)
16
Action Items
• UTSA will pursuit the lpxF mutant in SCHU S4
till end of Februry 2010 and cease if unsuccessful.
• Note after call: UTSA will email final, accepted
rat model paper to UNM, after journal accepts the
edits. UNM will send to NIAID.
• Karl and Bernard: email potential site visit dates to
Barbara, for the period of 3/29/2010 to end of
April 2010. UNM will develop a mutually
possible date for the UTSA site visit.
17