Slide 1 ASU TVDC Progress Report 6/24/08 Kathryn F. Sykes and Stephen A. Johnston Completed Milestones: 25 and 32, 33, 34 Active Milestones: 26, 28, 35 Currently Inactive Milestones: 30, 36-38

Slide 2 MILESTONE 26 Gray: (sub )milestone title Red: completed Green: in progress or inactive

Slide 3 Milestone 26 ML26-1: Completed. ORFs are efficiently generated using oligo and gene designs established in ML #25 LEEs are specifically and efficiently assembled from construct designs established in ML #25 ML26-2: Completed. We observe improved yields of polypeptide from linear template in a continuous-exchange cell-free “feed” system All components and parameters have been titrated to optimize yield and minimize cost Bacterial in vitro transcription/translation system is optimized for HTP application.

ML26-3 : Develop protein purification protocols Slide 4

ML26.3 Previous Status Proper conformation appears to be critical for efficient separation of cross-reacting molecules from polypeptides by: size filtration acetone precipitation affinity (Ni-binding) purification Several folding-independent purification methods have been developed Several methods to improve folding have been developed. Several depletion methods have been developed as an alternative to polypeptide purification Slide 5

Slide 6 ML26-3. Update

Imidazole wash titration: LVS cross-reactivity of E. coli proteins nonspecifically bound to Ni beads 50% Naïve+ 50% DO11.10 LVS vaccinated No template Ova 20mM 50mM 100mM 200mM 500mM 20mM 50mM 100mM 200mM 500mM Mass spec analysis of bead-bound proteins showed similar composition after either 20mM or 500mM wash Slide 7

Conclusion Proteins are nonspecifically stuck to Ni beads. Imidazole wash concentration has no effect on cross-reacting material. Imidazole titration elutes ova polypeptide as expected at ~100mM. Slide 8

Pre-clear IVT lysates with anti-LVS antisera 50% Naïve+ 50% DO11.10 LVS vaccinated LVS immunized sera incubated with protein G beads for 1 hour (load beads with anti- LVS) Beads were washed with PBS E.coli lysate (pre-reaction) from IVT kit was incubated with IgG bound protein G beads for another hour (capture cross-reacting E. coli proteins) Supernatant from beads (anti LVS depleted material) was tested in T-cell assay 1: No pre clearing of IVT lysate, 2: 10ul, 3: 20ul, 4: 50ul, 5: 100ul protein G beads used for pre-clearing Slide 9

Conclusion Depleting IVT lysates of components that bind anti- LVS antibodies reduces cross-reactivity. This result confirms that we are observing specific cross-reactivity between Francisella and E. coli antigens. However we are presumably depleting conserved translation machinery Slide 10

Slide 11 Testing NEB IVT kit for cross- reactive components NEB pure IVT kit contains only essential components for transcription and translation This limits the diversity of E.coli proteins exposed to T-cells Testing depletion of E. coli IVT proteins post-reaction Removal of trxn/ranslational machinery after polypeptide production will not interfere with synthesis However, any FTU polypeptide with homology to E. coli will be depleted.

Post-IVT reaction depletion of E.coli proteins from OVA polypeptide prep NEB PURE INVITROGEN 50% Naïve+ 50% DO11.10 LVS Vaccinated : IVT as is 2: 10ul E.coli IgG bound protein G beads 3: 50ul E.coli IgG bound protein G beads 4: 100ul E.coli IgG bound protein G beads IVT synthesized Ovalbumin polypeptide was incubated with indicated amounts of protein G beads with attached anti-E.coli IgG The supernatant was used in T-cell assay Slide 12

Conclusion NEB pure appears to be similarly immune- stimulating relative to standard lysate composiiton High variability in samples This will be repeated with uniform bead pipetting method Slide 13

Sensitivity of Rabbit Retic IVT on Protein G beads in T-cell assay IVT as isProtein G beads Ova FTU901 FTU % Naïve+ 50% DO11.10 LVS Vaccinated Slide 14

Conclusion In positive control samples, the bead-bound ova polypeptide stimulated a few more T cells relative to. However, numbers are generally low (antigen amount is low) and not discernable above background in bead-bound IVT negative control sample. Slide 15

His/Thio-double tag LEE Construct ORF 6-His T7-Term 6-His Thio. T7-Pro Thio. T7-Term N-terminal: Thioredoxin tag C-Terminal: His tag Slide 16

IVT thio fusion proteins bead bound via Thio or His tag in T-cell assay G-α-Thio G-α-His Ni 50% Naïve+ 50% DO11.10 LVS Vaccinated No Template Ova FTU901 FTU1695 Slide 17

Conclusions The nickel NTA (nickel chelated Nitrilotriacetic Acid ) is significantly more “sticky”, with respect to the cross-reacting lysate proteins, than either the anti-His or Thio antibody. The His antibody is stickier than the Thio antibody Use of the thio tag appears to be the a clean approach (no or very low amounts of nonspecifically bound IVT components.) Slide 18

Set-up to test Sensitivity of an antigen in the T-cell assay Recombinant ovalbumin and FTU proteins were purified (bound and eluted) from affinity column (Ni) and quantified Four different concentrations prepared from stock and used for T-cell assay were 0.1ug/ml, 1.0ug/ml, 5.0 ug/ml, and 10ug/ml (0.5ng, 5ng, 25ng, and 50ng) 100ul of the eluant was used to bind Ni beads, Protein G beads with anti-his , or left in the PBS buffer Slide 19

R:\GeneVac\FTU\Contr act\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT Coomassie gels Ova FTU1695 FTU901 Ova FTU901 FTU % Naïve+ 50% DO11.10 LVS vaccinated PBS ProG Ni : 0.1ug/ml, 2: 1.0 ug/ml, 3: 5.0 ug/ml, 4: 10.0 ug/ml Purified antigen Slide 20

Comparison of antigen (OVA) presentations in T-cell assay Slide 21

Slide 22 Conclusions The protein G beads with attached antibody provide higher sensitivity than Ni beads in T cell assay. Our assay sensitivity is sufficient to specifically detect ~5ug/ml of antigen

Next steps 1.Generate magnetic beads with covalently attached anti-Thioredoxin to increase antigen binding capacity. Protein G beads have limited Fc binding capacity. 2.Capture FTU IVT Thio-fusion polypeptides using these beads (during and after rxn) and evaluate in T-cell assays 3.Mice have been immunized with IVT lysate. We will collect sera to use as reagent in post-IVT reaction clearing step. IVT bound beads will also be used to generate antisera reagent for depletion step. This will reduce loss of FTU polypeptides with E. coli homology. Cleared samples will be tested for cross-reactivity in T-cell assay Slide 23

Dynabeads M-280 Tosylactivated Hydrophobic, monodisperse magnetic particles (2.8 m in diameter) with p- toluene-sulfonyl (tosyl) groups, further surface activation is not required. Allows easy coupling of antibodies with optimal orientation for affinity purification of proteins. Covalent coupling to primary amino- or sulphydryl groups in proteins/peptides Slide 24

Slide 25 MILESTONE 28 Gray: (sub)milestone title Red: inactive Green: in progress Build SCHU S4 proteome Build ORF expression library corresponding to proteome Active Generate complete protein-fragment library Preparing plan Array protein- fragments into measurable pools For T cell stimulation Inactive

Next Steps The last protocol decisions with respect to IVT sample clearing/purifying are becoming independent of ORF and polypeptide production. In parallel with optimizing the sample preclearing method and finalizing bead protocol for affinity tag binding, we propose initiating library production

Slide 27 MILESTONE 35 Array hybridations with mouse RNAs from virulent Schu 4 infection & RT PCR confirmation of candidates Virulent Schu 4 Samples Initial samples Dose-Response of Infection RT-PCR Confirmations To Be Determined Gray: (sub )milestone title Red: completed Green: in progress

Slide 28 Previous Status Two biological replicates of a dose response challenge Handled independently Positive correlation between dose responses Positive correlation between experiments Repeat amplifications were performed for reproducibility studies

LAPT of in vitro grown SCHU S4 Slide 29

qPCR Assessment of FTT901 (Tul4) Slide 30

FTT901 Standard Curve Slide 31

Slide 32 Conclusions Problems with in vitro produced RNA Losses after RNAeasy purification Poor amplification Poor labeling qPCR Initial runs show good differentials with genomic samples

Slide 33 Upcoming Transcriptome Goals Q-PCR validation of the hits Primers are designed now for 7 genes Time Course Experiment Repeat Rat and Mice Challenge with 104 SCHU S4 organisms Harvest 1,3,5,7 and 24 hours Parallel cultures in Chamberlain’s medium

Action Items Terry will repeat the ELIspot assay again with the IVT thio fusion proteins bead bound via thio or His tag or Ni ASU/UNM could use 2 concentrations for each antigen in the ElIspot T cell assay to show a dose effect over the background. Rick wants ASU/UNM to perform the T cell ELIspot assay with NHP lymph node T cells from vaccinated primates available in approximately 10 days, and then 35 to 42 days. Alex and Terry: develop a written protocol for the use of the NHP lymph node cells with many ASU IVT products purified by different approaches. Include the use of lysates, different purification strategies and give the IVT’s to Terry to use in the NHP hylar lymph node assays detected by Elispot ASU can prepare the plan for making the LEES etc for the protein library generation, but should not implement the plan yet. Rick wants to see the next confirming experiment by ASU/UNM in the mouse one more time, before starting the mass LEE production. Don’t implement the plan until one more confirmatory mouse experiment is done. Terry, send twice as much RNA from next SCHU S4 growth in Chamberlains to ASU Next ASU tech call is 7/22/08 4 th Tuesday in July. Monthly technical reports are due on 7/7/2008. Slide 34