1. 1 University of Texas San Antonio Update on F. tularensis attenuated vaccine strain construction and evaluation TVD Team 5/19/10 tech call

2. 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. 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. 4 Increasing recombination frequency in Schuh4: Bacteriophage encodes proteins ( Red recombinase) that enhance recombination into bacterial chromosome We previously made plasmid for expressing Red recombinase in Francisella pKEK1327 (March) We also showed that this plasmid stimulates recombination in Ftn (April) We will now test in Ftt First step: transform into KKT29 (Schuh4 with restrict. mutations) We will next test to see if recombination into Ftt chromosome has been enhanced. Ten colonies from transformation screened by PCR with Red primers, 9/10 give expected 2.2 Kbp fragment (lanes 2-6, 10-13), similar to control plasmid (lane 7)

5. 5 We are also testing requirement of tryptophan biosynthesis for F. tularensis virulence: We previously showed that trpB, but not trpA mutant of Ftn is attenuated for virulence in mice We previously tested growth of trpB mutant in IFN  -treated J774 cells, no difference between wt and trpB growth Here we tested for growth of trpA and trpB mutants in BMDM: trpA and trpB mutants recovered at lower levels w/o IFN  at 24, 48 h trpB not recovered at 48 h w/ IFN  We need to repeat experiment to show significant difference at 48 h

6. 6 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

7. 7 Creation of attenuated Schuh4 strains: We previously showed creation of two targetron plasmids to inactivate FTT1103 (lipoprotein involved in Ftt virulence) We transformed both plasmids into Schuh4: Only targetron targeted to 466 gave positive PCR with intron-specific and FTT1103-specific primers (lanes 4-10) indicating insertion in FTT1103 Several colonies chosen and cycled further: Primers spanning FTT1103 gene should show larger fragment with insertion (compared to wt, lane 2), insertion can be seen (e.g. lane 6), more cycling is required to obtain pure mutant.

8. 8 We are also targeting FTT1181 (ggt), a gamma-glutamyl peptidase that contributes to virulence of Ftt We performed PCR to generate fragments to inactivate ggt by targetron: Fragments designed to target 398|399 and 601|602 were generated by PCR (lanes 2 & 3), these will next be ligated into targetron plasmid and transformed into Schuh4

9. 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

10. Milestone #53A: Immunologic characterization of defined F. tularensis mutants Results Update Evaluation of F. novicida lipoprotein (FTN_0771) and  - glutamyltranspeptidase (FTN_01159) mutant strains as tularemia vaccines FTN_0771, an ortholog of SCHU S4 FTT1103, is a hypothetical lipoprotein and shares some similarity with DsbA proteins, which are proteins that catalyze disulfide bond formation. FTN_1159, an orthology of SCHU S4 FTT1181, is a  -glutamyltranspeptidase (GGT) that involves in the metabolism of γ-glutamyl-containing peptides. To assess their virulence in mouse, we intranasally challenged BALB/c mice (6 per group) with escalating doses (10 3 – 10 6 cfu) of  ftt0771 and  ftn1159, and monitored mice daily for their weight loss and survival. Results showed that these two mutants might be as virulent as wild type U112 in pneumonic tularemia mouse model (LD 50 < 1000 CFU). However, the virulence of SCHU S4 homologous mutants remains to be evaluated.

11. LD 50 of Δftt1159 and Δftn0771 determined using an intranasal challenge mouse model

12. 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

13. Milestone #53B: Characterization of protective immunity against pulmonary tularemia via oral vaccination in the F344 rat model Results Update Groups of Fischer 344 rats (6 rats per group) were vaccinated orally with 10 6 CFU of U112, 10 7 CFU of the  iglD strains or mock vaccinated with PBS and rested for 30 days. Rats were then challenged intratracheally with 10 4 CFU of SCHU S4 and monitored daily for morbidity and mortality. Evaluation of protective efficacy of oral vaccination with the  iglD mutant of both F. tularensis subsp. novicida and subsp. tularensis against intratracheal SCHU S4 challenge in Fischer 344 rats

14. Oral vaccination with F. tularensis subsp. novicida U112, U112  iglD and SCHU S4  iglD protected rats against subsp. tularensis SCHU S4 intratracheal challenge

15. 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.Test trpA/B mutants in IFN  treated BMDM for intracellular growth 3. Test Red-expressing Ftt for enhanced recombination. Milestone #54: 1.Create targetron plasmids to inactivate FTT1181 (ggt) 2.Continue cycling FTT1103 targetron transformants, isolate pure mutant Continued on following slide

16. Plan for following month: Milestone #53-A&B: 53A: Intramacrophage replication of the F.t. novicida FTN_1734 and FTN_0109 mutants. These two proteins were identified as dominant sero-reactive antigens from the Felgner’s immuno-array data set. 53B: Humoral responses to oral F.t. novicida and tularensis  iglD vaccination in the Fischer 344 rat

17. 17 Additional points: Description of deliverables completed for each active milestone: Milestone 52: Schuh4 recA, iglC1 iglC2 recA, FTT1579, FTT523, FTT1579 + FTT523 strains Milestone 53: None at this time Milestone 54: Schuh4 atpC strain List of relevant publications from the past month MSCR status MS 49: UTSA writing MSCR 49 (MS 49 was scientifically done 11/17/09; Crystal Lauriano will write and is due in first week of June 2010 ) 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; Crystal Lauriano revising Jeff Barker’s MSCR; was due the first week of May 2010; overdue)

18. Action Items UTSA will try titrating the IFN gamma in the BMDM growth of the Tryp mutants to enhance sensitivity. Bernard will look at the weight loss data on the one Mock survivor (slide 14) and add the clinical signs like weight loss to the tech call minutes Bernard: will do QC on rat lung deposition as UNM does. UTSA will confirm the deposition approximately 45min to 1 hr after IT challenge, by homogenizing a few lungs and plate them out to enumerate the deposited challenge dose. Crystal will submit the MS 51 MSCR, which is overdue as of first week of May 2010, or else another UTSA scientist will complete the MS51 MSCR and submit to UNM. 18

19. 19 Comparison of weight loss between groups of rats orallyvaccinated with either PBS or F. tularensis subsp. novicida U112, and subsequently challenged intratracheally with subsp. tularensis SCHU S4 * * Rats survived from SCHU S4 challenge * * * *