Transcript UTSA

University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
1/19/10 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)
•To make double mutant (FTT1579 + FTT0523), we transformed
pKEK1282 (targeted to FTT0523) into KKT19; screened
transformants for presence of insertion in FTT0523:
Internal+external primers
external primers
Internal + external primers show insertion in FTT523 in all five colonies
(lanes 2-6, left, compare to parent in lane 7), and external primers show
only mutant FTT523 in all colonies except one (lanes 2-6, right,
compare to parent in lane 7).
•We will remove plasmid, then test both single and double
mutant strains 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 targetron plasmid to inactivate atpC.
•Plasmid was transformed into Schuh4:
external primers
Internal + external primers show insertion in
atpC in all four colonies (lanes 3-6, right,
compare to Schuh4 in lane 2), and
external primers show almost pure mutant
atpC in all four colonies (lanes 3-6, left,
compare to Schuh4 in lane 2).
Internal+external primers
•We will remove plasmid, then 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
Bacterial replication in different lobes of lungs in SCHU S4 challenged
Fischer 344 rats following oral vaccination with Francisella subsp.
novicida wild-type strain U112
F344 rats were vaccinated orally with 105 cfu of U112 or mock
vaccinated with PBS and rested for one month before challenging
intratracheally with SCHU S4 (103 CFU). On days 1, 3, and 5 postchallenge, 1-3 rats were sacrificed from each group and lungs were
collected and separated by lobe as shown Fig.1. Lobes were
homogenized and serially diluted; dilutions were plated to
determine bacterial burdens in each lobe.
Designation of the lobes of Fischer 344 rat lungs
Time course of bacterial replication in lobes of F344 rats
following oral U112 vaccination and i.t. SCHU S4 challenge
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
Humoral response in Fischer 344 rats following oral vaccination
with F.t. novicida wild-type or DiglB strains
Fischer 344 rats were vaccinated orally with 105 CFU of U112, 107
CFU of the DiglB strain or mock vaccinated with PBS and rested
for 28 days to ensure development of a humoral response. (Fig 3)
Blood was collected and prepared sera were analyzed for antigenspecific antibodies by ELISA. (Fig 4) Fresh fecal pellets were
collected in protease inhibitor and supernatants were analyzed for
the presence of antigen-specific antibodies by ELISA.
Antigen-specific serum antibody response
Antigen-specific fecal antibody response
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. Finish FTT0523 +FTT1579 Schuh4 double
mutant.
2. Test mutants for transformation efficiency.
Milestone #54:
1. Continue construction of lpxF Schuh4 mutant.
2. Finish atpC Schuh4 mutant, test for virulence.
Continued on following slide
16
Plan for following month: Milestone #53-A&B:
53A: Intramacrophage replication of SCHU S4 atpC mutant
53B: Survival of Fischer 344 rats following oral F.t. novicida
DiglB vaccination and SCHU S4 challenge
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
UTSA submitted the manuscript “The Fischer 344 Rat Reflects
Human Susceptibility to Pulmonary Francisella Challenge
and Provides a New Platform for Virulence and Protection
Studies” to PLoS One for review
MSCR status
MS 50: UTSA reviewing v 0.4 MSCR 50 (BG sent edits on 12/22/09;
requested UTSA edits v by 1/15; NIAID will review the 8 SOPs with the MSCR)
MS 49: UTSA writing MSCR 49 (MS 49 was scientifically done 11/17/09;
Crystal Lauriano will write the MS 49 MSCR)
18
MS 51: UTSA reviewing revised MS51 MSCR (BG sent edits on 12/4/09)
Action Items
• Crystal Lauriano will write the MS 49
MSCR
• Bernard will email the UTSA BSL3 Lab
Manager posting to Barbara at UNM, who
will route to the Lyons lab team at UNM.
• UTSA will send 3 researchers to
USAMRIID for the LVS vaccination rather
than 5 people due to funding
19