1. 1 University of Texas San Antonio Update on F. tularensis attenuated vaccine strain construction and evaluation TVD Team 2/19/08 tech call

2. 2 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

3. 3 Milestone 49 Creation of mutant F. tularensis subsp. tularensis strains C. Construct  iglA,  iglB mutagenesis plasmids Transform into Schuh4, select for transconjugate, Counterselect for mutant Verify mutants, Pass on to Milestone 50 Mate into Schuh4, select for transconjugate, Counterselect for mutant Red: completed Green: in progress Blue: Steps in the milestone B. Construct  vgrG,  iglD mutagenesis plasmids A. Construct  iglC mutagenesis plasmid(s) Mate into Schuh4, select for transconjugate, Counterselect for mutant

4. 4 Milestone #49: Construction of vgrG, iglD F. tularensis subsp. tularensis strain We are working on creating two different mutant Schuh4 strains: iglD and vgrG We are utilizing Tulatron technique We have constructed two different vgrG Tulatron vectors, these target two different sites within vgrG (30|31and 81|82) Each has been transformed into Schuh4 to generate vgrG mutant Transformants have been isolated, primary transformants screened first for native length vgrG gene: Lanes 2&3 Schuh4 Lanes 4-11 Representative Transformants

5. 5 We’re transforming each into Schuh4 to isolate iglD mutant (documented in UTSA TVD notebook #1 and #5) We could not detect insertion in initial screen, however more diagnostic screen of intron-specific primer plus vgrG primer will identify insertion in mixed colonies. Currently performing re-streaking to segregate mutant colonies, also additional PCR screens. We appear to have constructed two iglD tulatrons, (30/31 and 255/256) Example shown, 30|31 Construct, tulatron gives 3 digested fragments, Smaller size of largest Fragment in lanes 5-9 Indicate correct clones

6. 6 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 Red: completed Green: in progress Blue: Steps in the milestone

7. 7 Tulatron vectors created to target two different sites within recA, transformed first into LVS (Ping still waiting for BSL3 access) LVS transformant clones screened to identify recA mutants at 840|841: Lanes 2-5 are clones showing recA with tulatron Insertion, lane 6 is LVS parent, primers flank recA We are now streaking at higher temperature (37°C) to facilitate loss of tulatron plasmid (documented in UTSA TVD notebook #2)

8. 8 Milestone 50-A Immunologic characterization of F. tularensis subsp. novicida, subsp. tularensis, and LVS strains F. novicida uvrA, uvrB Double mutant In vitro Growth In vivo Bacterial Burden LD 50 determination F. novicida uvrA+pdpD F.novicida uvrB+pdpD iglA, iglB, iglC, iglD In vitro Growth In vivo Bacterial Burden LD 50 determination Further immunological characterization based on initial screen 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

9. 9 Milestone 50-B Characterization of protective immunity against pulmonary tularemia via intra-gastric LVS vaccination Duration and limits of protective efficacy Survival 1, 2, 3 months Vaccination/boost strategy Bacterial dissemination Histological analyses Correlates of humoral and cellular immunity Contribution of cell mediated and humoral immunity 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

10. 10 Milestone #50A: Immunologic characterization of F. tularensis subsp. novicida, subsp. tularensis, and LVS strains Results Update Measure intramacrophage (J774) replication of Ft subsp. tularensis (SCHU S4) iglC We have expanded frozen bacterial stocks of the wild type SCHU S4, and iglC mutant for these analyses.

11. 11 Milestone #50A: Immunologic characterization of F. tularensis subsp. novicida, subsp. tularensis, and LVS strains Results Update Evaluate the protective efficacy of F. tularensis SCHU S4 iglC vaccination against wild type SCHU S4 challenge Groups of BALB/c mice (female, 4-6 weeks) have been immunized with 10 3 CFU of iglC intragastrically (i.g.) or intradermally (i.d.). Mice treated with PBS were used as a mock-control. These mice will be challenged i.n.or i.d. with two doses of SCHU S4. Animals will be monitored for survival and weight loss.

12. 12 Milestone #50B: Characterization of protective immunity against pulmonary tularemia via intra-gastric LVS vaccination Results Update Measure bacterial dissemination in various tissues at early time points following intragastric LVS immunization BALB/c mice were challenged with LVS (~10 3 CFU) intragastrically. Lungs and trachea were collected from the infected mice at 30 min and at days 1, 2, 3 and 5 after challenge (3 mice per time point). Liver, spleen, cervical and mesenteric lymph nodes were collected from the infected mice at days 1, 2, 3, 5 and 7 after challenge. Numbers of bacteria in each organ were determined by dilution plate counting.

13. 13 Results: As shown in Fig. 2a, there were no detectable bacteria present in either the trachea or lungs within 30 minutes or 1 day post-inoculation, with gradual increases of viable bacteria recovered from days 2 to 5 in the lungs

14. 14 Results: As shown in Fig. 2b, elevated numbers of bacteria were not present in the liver and spleen until day 5 after inoculation which is consistent with results reported previously (Nov. 2007). There were minimal bacteria recovered from both cervical and mesenteric lymph nodes after challenge.

15. 15 Plan for following month: Milestone #16: completed. Milestone #39: completed. Milestone #48: completed. Milestone #43: completed. Milestone #51: completed. Milestone #49: 1.Identify iglD tulatron insertion within transformed Schuh4. 2.Identify vgrG Schuh4 mutant among transformants. 3.Construct pdpD::FRT in pUC-based vector Milestone #52: 1.Remove tulatron plasmid from recA LVS, Assay LVS recA for virulence traits (macs, mice) 2. transform into Schuh4, isolate recA mutant. Continued on following slide

16. 16 Plan for following month: Milestone #50-A&B: 50A: (1) Measure intramacrophage (J774) replication of Ft subsp. tularensis (SCHU S4) iglC. (2) Evaluate the protective efficacy of F. tularensis SCHU S4 iglC vaccination against wild type SCHU S4 challenge: (3) Evaluate the protective efficacy of intragastric F. novicida iglB vaccination against SCHU S4 intranasal and intradermal challenge in C57BL mice. 50B: (1) Evaluate the protective efficacy of intragastric LVS vaccination against Francisella type A SCHU S4 intranasal challenge at 8 weeks after either a single vaccination or after receiving a secondary booster dose. (2) Analyze antigen-specific cytokine production in spleens and lymph nodes at both 2 and 4 weeks after intragastric inoculation with LVs.

17. 17 Action items UTSA Will show UNM and NIAID the successful vgrG insertion data next month. UTSA Will report next month on the iglD transformations into SCHUS 4. UTSA is performing side by side comparison with intentional lung infection vs intentional GI infection; UTSA will try PCR to detect the delivery of bacteria to lungs, trachea (at 30 min) and to the liver at 2 days UTSA should make luciferase expressing plasmid using the LUX so don’t need to worry about substrate getting to the bacteria