University of Texas San Antonio
Update on F. tularensis attenuated vaccine strain construction and evaluation
TVD Team
11/20/07 tech call
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Active milestones during last reporting period:
Milestone #49B: Construction of iglD, vgrG F. tularensis subsp.
tularensis strain
Milestone #50: Immunologic characterization of F. tularensis subsp. novicida , subsp. tularensis , and LVS strains
Milestone #52: Create recA mutants in F. tularensis subsp. tularensis
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During the past month (spanning Oct-Nov 2007), our
BSL-3 laboratory was shut down for mandatory annual repairs, testing, and recertification.
The preparation and actual shut-down took several weeks, this has affected all of our experiments, most of which rely on BSL-3 access.
The laboratory was back “on-line” as of 11/14/07
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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) B. Construct
 vgrG,
 iglD mutagenesis plasmids
C. Construct
 iglA,
 iglB mutagenesis plasmids
Transform into Schuh4, select for transconjugate,
Counterselect for mutant
Mate into Schuh4, select for transconjugate,
Counterselect for mutant
Verify mutants,
Pass on to Milestone 50
Mate into Schuh4, select for transconjugate,
Counterselect for mutant
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Milestone #49: Construction of vgrG, iglD F. tularensis subsp.
tularensis strain
• In experiments documented up until now, Schuh4 iglC1::Ll.LtrB iglC2 :: Ll.LtrB
created utilizing the
Targetron technology
• Previously, we showed that iglC Schuh4 strain inoculated up to 10 5 CFU intranasally caused no death in mice
• We challenged these “vaccinated” mice with 400 CFU
Schuh4, there was NO significant protection.
• Thus the iglC Schuh4 strain failed to show protection
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• We are now working on creating two different mutant
Schuh4 strains: iglD and vgrG
• We are utilizing Tulatron technique (vector below left)
• We have ordered primers for iglD (shown previously) and vgrG (below right)
• Splicing by overlap PCR performed to generate fragment to insert into tulatron vector, cloning for correct construct currently taking place
FTT1347_ 30/31a-IBS
AAAACTCGAGATAATTATCCTTA GCTGCCGTGATG GTGCGCCCAGATA GGGTG
FTT1347_ 30/31a-EBS1d
CAGATTGTACAAATGTGGTGATA ACAGATA AGTCGTGATGATTA ACTT ACCTTTCTTT
GT
FTT1347_ 30/31a-EBS2
TGAACGCAAGTTTCTAATTTCGATTGCAGCTCGATA GAGAAAGTGTCT
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We have also been working on system to remove one copy of the FPI to facilitate mutagenesis in Schuh4:
• Step 1: insert FRT site into pdpA in one FPI
(
 pdpA1 ::FRTKan R or
 pdpA2 ::FRTKan R )
We have accomplished this.
• Step 2 : Construction of
 pdpD ::FRT ermC vector
• We are currently working on finishing this plasmid, two of three fragments are already cloned, we are cloning the third, this will then be used to move
Mutation into
 pdpA ::FRTKan R strains
(documented in UTSATVD Notebook 3).
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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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• We are utilizing Tulatron technique
• We have ordered primers for recA
• Splicing by overlap PCR performed to generate fragment to insert into tulatron vector, cloning for correct construct currently taking place
(documented in UTSA TVD notebook #2)
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Milestone 50-A
F. novicida uvrA , uvrB
Double mutant
Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis, and LVS strains
F. novicida uvr A+ pdpD
F.novicida uv rB+ pdpD iglA, iglB, iglC, iglD
LVS uvrA, uvrB
F. tularensis Schu4 iglC
In vitro Growth
In vivo Bacterial Burden
LD
50 determination
Red: completed
Green: in progress
Blue: Steps in the milestone
In vitro Growth
In vivo Bacterial Burden
LD
50 determination
In vitro Growth
In vivo Bacterial Burden
LD
50 determination
Further immunological characterization based on initial screen
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Milestone 50-B
Characterization of protective immunity against pulmonary tularemia via intra-gastric LVS vaccination
Duration and limits of protective efficacy
Correlates of humoral and cellular immunity
Contribution of cell mediated and humoral immunity
Survival 1, 2, 3 months
Vaccination/boost strategy
Bacterial dissemination
Histological analyses
Red: completed
Green: in progress
Blue: Steps in the milestone
CD4 + and CD8 + T cell responses
Serum antibody responses
Secreted, BAL antibody responses
CD4 + , CD8 + , B cell depletion vaccination/challenge
KO mice vaccination/challenge
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Milestone #50A: Immunologic characterization of F. tularensis subsp. novicida , subsp. tularensis , and LVS strains
Results Update
Evaluate the protective efficacy of the Ft subsp. novicida uvrBiglA mutant as a vaccine candidate
Groups of BALB/c mice (female, 4-6 weeks) were intranasally (i.n.) immunized with 10 5 , 10 6 or 10 7 CFU of ΔuvrBiglA . Mice treated with
PBS were used as a mock-control. The immunized mice were challenged with 1000
CFU of F. novicida (~100 LD
50
) by the i.n. route after 30 days of vaccination.
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100
80
60
40
20
10
10
5
6
10 7
PBS
0
110
0
105
100
95
90
85
80
0
4 8 12 16 20
2 4 6 8 10 12 14
Days after challenge
Fig. 1. Protective efficacy of ΔuvrBiglA immunization against F. novicida infection.
BALB/c mice were immunized intra-nasally with 3 doses (10 5 , 10 6 , and 10 7 CFU) of
ΔuvrBiglA or PBS and i.n. challenged with lethal dose of F. novicida (1000CFU). Mice were monitored for survival rate and weight change.
Results : ΔuvrBiglA -vaccinated mice were highly protected against subsequent pulmonary challenge with F. novicida . No significant loss of body weight was also observed in the protected animals. As expected PBS-treated mock-vaccinated mice succumbed by day 4.
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Milestone #50A: Immunologic characterization of F. tularensis subsp. novicida , subsp. tularensis , and LVS strains
Results Update
Analyze the antibody profiles of mice immunized with the Ft novicida uvrBiglA mutant after vaccination
Blood was collected from the PBS- and ΔuvrBiglA - immunized mice at day 14 and 28 after priming. Specific antiΔuvrBiglA total antibody titer as well as IgG1 and IgG2a isotypes were determined by ELISA.
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1
500
3
Total Ab
2
Day 14
Day 28
3 IgG1
2
3
2
IgG2a
1
500
1
500
PBS 10 5 10 6
ΔuvrBiglA
10 7 PBS 10 5 10 6
ΔuvrBiglA
10 7
Fig. 2. Humoral response to ΔuvrBiglA immunization. BALB/c mice were intranasally immunized with 10 5 , 10 6 or 10 7 CFU of the
ΔuvrBiglA mutant or PBS alone as mock vaccination. Sera were collected 2 weeks and 4 weeks after immunization and used to determine titers of anti-
ΔuvrBiglA specific antibody.
Results : mice immunized with ΔuvrBiglA produced measurable amounts of specific serum total antibody (at day 14 after priming with the two higher vaccination doses). The titers were increased at day 28 after priming with all three doses (2 days before bacterial challenge). Isotyping analyses indicated both Th1 (IgG2a) and
Th2 (IgG1)- type antibodies were produced in mice after the specific antibody was detected in mock-vaccinated mice.
ΔuvrBiglA immunization. No ΔuvrBiglA 15

Plan for following month:
Milestone #16 : completed.
Milestone #39 : completed.
Milestone #48: completed.
Milestone #43 : completed.
Milestone #51: completed.
Milestone #49 :
1. Construct iglD tulatron vector.
2. Construct vgrG tulatron vector.
3. Construct pdpD ::FRT vector
Milestone #52:
1. Construct recA tulatron vector.
Continued on following slide
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Plan for following month:
Milestone #50-A&B :
1. Determine the LD
50 mutant of Ft subsp. novicida uvrBpdpD double
2. Monitor Ft subsp. novicida Δ uvrBpdpD replication and dissemination in mice via intranasal inoculation
3. Monitor LVS replication and dissemination in mice via intragastric inoculation.
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Quality Assurance Issues:
1. Pipettors calibrated frequently, last calibration date:
9/18/07
2. Major pieces of equipment on service contract:
Sorvall RC5B, ABI RealTime PCR, BioRad FPLC,
Genepix Microarray Reader
3. All genetic constructs confirmed by DNA sequencing both strands, typically with IDT (Houston TX)
4. BSL-3 laboratory underwent quality assurance and recertification from November 6-14, 2007. This involved complete decontamination, recertification of biosafety cabinets, eyewash and shower, balance of air flow, verification of alarms and security, change of Hepa filters, re-sealing of animal holding room ceiling. Records on file with UTSA
Safety office.
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• Action: Karl will have Jeff review and return revised
MSCR 16, 39 and 48 and 3 SOPs to Barbara within a week (11/27/07). UTSA also needs to generate MSCR for
MS 43 and 51.
• Action: Bernard will begin working on SOPs and sections of the MS 50 MSCR (milestone completion report)
• UNM technical call is on 11/28 (noon-1pm MT) and others are invited to join
• Action: Marlene is away the week of 11/26 but Vicki will attend the 11/27 ASU call and the 11/28 UNM calls
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