Transcript UTSA Tech Call 2009.11.17

University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
11/17/09 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 constructed Schuh4 strain with insertion in
FTT1579 (restriction enzyme), then removed
Targetron plasmid = KKT19
•We constructed targetron plasmid to inactivate
second restriction enzyme (Ftt-specific) FTT0523 (pKEK1282)
•We transformed pKEK1282 into Schuh4; screened
transformants for presence of insertion in FTT0523:
PCR with internal +
external primers show
that all five transformants
tested (lanes 1-5) have
insertion in FTT0523.
We will obtain pure
mutant by further culture
and screening.
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:
•We constructed a targetron plasmid for inactivation of lpxF
(Lipid A modification enzyme; pKEK1311)
•pKEK1311 transformed into Schuh4 strain, transformants
screened for presence of insertion in lpxF:
PCR with internal + external primers
show that all four transformants
tested (lanes 3-6) have insertion in
lpxF. (upper figure, no fragment in
Schuh4 wt lane 2)
PCR with external primers show
presence of insertion in lpxF in all strains
(arrow), but wt lpxF still present as well
(seen in control Schuh4 lane 2). 6
We continue to cycle and screen.
B
6 7
Legend:
1. 1 Kb ladder
2. KKT1 (wt)
3. 1311-1 (LpxF)
4. 1311-2 (LpxF)
5. 1311-3 (LpxF)
6. 1311-4 (LpxF)
7. pKEK1311 (LpxF plasmid)
1 2 3 4 5 6 7
0.8 Kb
rimers
EBS Univ. + Forward Primers
Legend:
1
2.0 Kb
0.5 Kb
2
3 4
5 6
7
8 9 10 11 12 13 14
1.
2.
3.
4.
5.
6.
7.
1 Kb Ladder
KKT1 (wt)
1311-3A
1311-3B
1311-3C
1311-3D
1311-3E
8. 1311-3E
9. 1311-3F
10. 1311-4A
11. 1311-4B
12. 1311-4C
13. 1311-4D
14. pKEK1311
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
Cellular cytokine recall responses to i.n. and oral KKF235
(iglB of F. novicida) vaccination
C57BL/6 mice were intranasally or orally vaccinated with KKF235
(104 CFU). Spleen, cervical lymph nodes (CLNs), and mesenteric
lymph nodes (MTLs) were collected at day 14 after vaccination,
single cells were made and reacted with UV-inactivated KKF235
cells or HEL (an unrelated antigen). Frequency of IFN-g secreting
cells were measured by ELISPOT. Age matched naïve mice were
used as a mock control.
Cellular cytokine recall responses to i.n. and oral KKF235
(iglB of F. novicida) vaccination
Interpretation of results: Vaccination with F. novicida iglB induces significant amounts of antigenspecific IFN-g in the spleen and cervical and mesenteric lymph nodes. There is no appreciable
difference in this response between mice vaccinated orally or intranasally, thus there must be another
mechanism related to the superior protection elicited from oral vaccination in these mice.
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
Bacterial dissemination following LVS vaccination
and SCHU S4 challenge
Fischer 344 rats were vaccinated either orally or intradermally with
LVS, or mock vaccinated with PBS, and rested for 30 days. Rats
were then challenged intratracheally with SCHU S4 and lungs,
livers and spleens were collected at several time points after
challenge to determine effect of vaccination on bacterial numbers at
the site of infection and dissemination of organisms to systemic
tissues.
Bacterial Dissemination Following LVS Vaccination/
SCHU S4 Challenge
Interpretation of results: Following SCHU S4 challenge, bacterial numbers in lungs, livers and
spleens were similar in all groups at 1 and 3 days after challenge but were lower in LVS vs. mock
vaccinated rats by 5 days after challenge. There was no appreciable difference between bacterial
numbers in mice vaccinated by the oral or intradermal route. This data suggests that LVS vaccination,
regardless of route of inoculation, does not stop dissemination of bacteria, but rather controls
replication within the organs following dissemination.
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. Construction of FTT0523 (restriction enzyme #2)
single and double (+FTT1579) Schuh4 mutants.
Milestone #54:
1. Construction of lpxF Schuh4 mutant.
2. Construct atpC targetron plasmids, transform
Schuh4.
Continued on following slide
13
Plan for following month: Milestone #53-A&B:
53A:
(1) Intramacrophage replication of SCHU S4 FTT_0523
mutant strain
53B:
(1) Cellular responses following vaccination of Fischer 344
rats with F. novicida iglB
Additional points:
Description of deliverables completed for each active milestone:
Milestone 52: Schuh4 recA, iglC1 iglC2 recA, FTT1579 strains
Milestone 53: None at this time
Milestone 54: None yet, in process
List of relevant publications from the past month
None
MSCR status
Heather working on MS 50 MSCR
15
Action Items
• UTSA will follow some vaccinated/ infected rats
longer (to normal death day ~ day 8), even during
organ dissemination studies designed to look at
early events in days 1-5.
• Heather: will email MS 50 MSCR to Barbara as
soon as the MSCR’s file image sizes are reduced
(UTSA emailed MS 50 MSCR at 2mb to Barbara
on 11/19/09)
• Note added after call: UTSA will submit the MS
49 MSCR, as this milestone is 100% completed. 16
What is a reasonable timeline for submission?