1 University of Texas San Antonio Update on F. tularensis attenuated vaccine strain construction and evaluation TVD Team 3/16/10 and 3/17/10 tech call

2 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

3 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 Generate, optimize mutant strain construction in Schuh4

4 Increasing recombination frequency in Schuh4: Bacteriophage encodes proteins ( Red recombinase) that enhance recombination into bacterial chromosome This has been exploited in other bacteria to enhance recombination for genetic construction (e.g. PNAS 97:6640) We are adapting this system for use in Ftt Schuh4: PCR amplification of Red genes from pKD46, and subsequent ligation into Ft plasmid pKEK1140: We have cloned Red genes into Ft plasmid behind Ft promoter, We will next test to see if recombination into chromosome has been enhanced. Three colonies from ligation Screened by PCR with Red Primers, all give expected 2.2 Kbp fragment

5 We are also testing requirement of tryptophan biosynthesis for F. tularensis virulence: Chlamydia trachomatis, an obligate intracellular organism, must be able to synthesize tryptophan for full virulence: IFN  effectively reduces intracellular Trp to starve Trp- intracellular organisms, C. trachomatis trpB mutants attenuated in IFN  -treated cells (e.g. Mol Micro 49:1347) Since Ft is intracellular, and IFN  inhibits Ft growth, we investigated importance of Trp biosynthesis: trpBA in operon, trpA encodes second-to-last step in Trp biosynthesis, trpB encodes last step in Trp biosynthesis trpB mutants are Trp auxotrophs trpA mutants unable to make indole, if no other pathway for indole production, then they are also Trp auxotrophs

6 Evaluation of virulence of trpB and trpA mutants of Ft novicida (intranasal, ~100 CFU, Balb/C mice). The trpB mutant is attenuated for virulence, the trpA mutant is not. The trpB mutant is a Trp auxotroph, the trpA mutant is not (not shown). This is a promising mutation! In combination with other mutations may be excellent for live vaccine candidate. We will characterize in IFNg +/- mice, macrophages, and Introduce into Schuh4 as well.

7 Milestone 54 Creation of mutant F. tularensis subsp. tularensis strains Verify mutants, Pass on to Milestone 50 Red: completed Green: in progress Blue: Steps in the milestone Construct lpxF, atpC, 3 other mutagenesis plasmids Mate into Schuh4, select for transconjugate, Counterselect for mutant

Inactivation of lpxF, atpC in SchuhS4: (last month) plasmid targeting lpxF transformed into Schuh4 strain, transformants screened for presence of insertion in lpxF. We decided to discontinue cycling, as agreed upon during last phone conference, due to lack of enrichment for pure lpxF mutant, despite >6 cycles. (last month) we created atpC SchuhS4 mutant We tested for virulence in Balb/C mice via i.n. route (following page): Milestone #54: Construction of mutant F. tularensis subsp. tularensis strains

9 No significant attenuation of atpC SchuhS4 mutant, all groups of mice died, even at lowest inoculum (682 CFU) Conclusion: this is not a good attenuating mutation for a live vaccine strain, there is no evidence that the atpC SchuhS4 strain is attenuated.

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 LD 50 determination Further immunological characterization based on initial screen F. tularensis rec A recAiglC In vitro growth In vivo bacterial burden LD 50 determination Red: completed Green: in progress Blue: Steps in the milestone

Milestone #53A: Immunologic characterization of defined F. tularensis mutants Results Update Infectivity of KKT-29 (SCHU S4 restriction enzyme [FTT0523 and FTT1579] double mutant) in vitro Murine macrophage cell line (J774) were infected with KKT-29 or its parental strain (SCHU S4) using an inoculum of 10 or 100 MOI. Numbers of viable bacteria in macrophages were measured at 3hrs and 24 hrs post-infection.

Intramacrophage replication of SCHU S4 FTT0523/FTT1579 double mutant (KKT-29)

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 Intramacrophage survival Vaccination/challenge Bacterial dissemination Histological analyses Correlates of humoral and cellular immunity of LVS vaccination Protective efficacy of 2 attenuated SCHU S4 strains Red: completed Green: in progress Blue: Steps in the milestone CD4 + T cell responses Serum antibody responses Secreted, BAL antibody responses Intramacrophage survival vaccination/challenge antibody responses Bacterial dissemination and histology

Milestone #53B: Characterization of protective immunity against pulmonary tularemia via oral vaccination in the F344 rat model Results Update Fischer 344 rats were vaccinated orally with 10 5 CFU of U112, 10 7 CFU of the  iglB strain or mock vaccinated with PBS and rested for 14 days to ensure development of a cellular response. Spleens, cervical and mesenteric lymph nodes were collected and single cell suspensions were recalled in vitro for 72 hours with UV-inactivated homologous antigen or the unrelated antigen HEL. Supernatants were collected to analyze for the presence of IFN-  by ELISA. Cellular response in Fischer 344 rats following oral vaccination with F.t. novicida wild-type or  iglB strains

15 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.Challenge trpB Ftn surviving mice with wt Ftn 2.Test trpA/B mutants in IFN  treated macrophages for intracellular growth 3. Test Red-expressing Ft for enhanced recombination. Milestone #54: 1.Create targetron plasmids to inactivate FTT1103 (lipoprotein identified by Stulik as important for virulence) Continued on following slide

Plan for following month: Milestone #53-A&B: 53A: Intramacrophage replication of F. novicida FTN 0771 (protein disulfide isomerase) and FTN 1159 (  -glutamyl transpeptidase) mutant strains 53B: Isolation of F344 primary alveolar macrophages for Francisella intramacrophage replication

17 Additional points: Description of deliverables completed for each active milestone: Milestone 52: Schuh4 recA, iglC1 iglC2 recA, FTT1579, FTT523, FTT FTT523 strains Milestone 53: None at this time Milestone 54: Schuh4 atpC strain List of relevant publications from the past month The manuscript “The Fischer 344 Rat Reflects Human Susceptibility to Pulmonary Francisella Challenge and Provides a New Platform for Virulence and Protection Studies” was accepted for publication in PLoS ONE MSCR status MS 49: UTSA writing MSCR 49 (MS 49 was scientifically done 11/17/09; Crystal Lauriano will write) MS 50: NIAID reviewing as of 3/4/10 (UNM sent MS50 MSCR, 8 SOPs and 2 published references on 3/4/10) MS 51: UTSA reviewing revised MS51 MSCR (UNM sent edits on 12/4/09)

Action Items Heather will vaccinate rats with iglB F novicida strain and perform recall experiment with SCHU S4 when UTSA has a tryp B mutant in SCHU S4, UTSA will compare attenuation with the atpC mutant at less than 100 CFU and with the tryp B mutant at CFU dose. Karl/Bernard will pdf of newly published rat model article to Barbara. Barbara will it to NIAID Karl will work with Crystal Lauriano to write the MS49 MSCR and to complete the MS51 MSCR. Barbara will Crystal an example of a final, NIAID accepted UTSA MSCR, to aid her writing. (done 3/17/10, including sending MS 49 MSCR template and instructions) Barbara will Patrick a prioritization of 4 MSCR reviews (MS 11, 33,46,50) (done 3/17/10) 18