1. Slide 1 ASU TVDC Technical Report Kathryn F. Sykes and Stephen A. Johnston Completed Milestones: 25, 26, 28* and 32, 33, 34, 35, 36 Active Milestones: UNM 29*/ASU28*, ASU 37/38 Currently Inactive Milestones: 30

2. Slide 2 MILESTONE 28 Gray: (sub)milestone title Yellow: Re-opened Build SCHU S4 proteome Build ORF expression library corresponding to proteome Generate complete protein-fragment library Re-opened Array protein-fragments for T cell stimulation assays Re-opened

3. Generation of polypeptide antigens for UNM T cell assays Slide 3 SOP for detecting T cell stimulation with synthetic (IVTT) antigens T cell proliferation IFN  ELISpot assay Assay optimization using ivt proteins Production Assay development (UNM) Production of antigens (ASU) Identification of stimulatory proteins & peptides Screening (UNM) Confirmation

4. Testing new protein capture beads In our initial test of the small vs. large diameter capture beads, immune cells from LVS-immunized NHP were used for the IFN-gamma ELISPOT assays. We determined that background was low using either bead. Slide 4

5. ELIspot: two bead comparison Slide 5

6. ELIspot Plate Layout: two bead data IglCSFV053 1 uMMyONEM280MyONEM280 0.2 uMMyONEM280MyONEM280 0.04 uMMyONEM280MyONEM280 FF LVS neatFF LVS 1:5FF SCHU S4 neatFF SCHU S4 1:5 Slide 6 Experimental Design: IFN  ELISpot plate layout

7. Preparing for Specificity Test for Improved Capture Beads Objective: Repeat large scale synthesis of IglC 1 and ASFV053 (negative control) on both small MyOne (1micron) and large M280 (2.8 micron)beads. Last month these preps were tested against LVS-immunized NHP immune cells. These antigens will next be evaluated at UNM using immune cells from IglC1-immunized rats. Slide 7

8. 33 kDa - 10 - 15 - 20 - 25 - 37 - 50 - 75 - 100 -250 - 150 kDa Precision Plus Standard (Bio-Rad) 0.5xIVT-rx ASFV053 BSA, ug AMPure PureLink 0.5 1 2 MyOne M-280 New Preps of IglC1 and ASFV053 on the two bead formats is complete These bead-bound antigens (17ug of each) have been sent to UNM

9. Evaluation of new PCR purification system for LEE templates prior to IVTT reactions Slide 9 AMPure is better

10. Production of LEE Templates for IVTT Assessing quality and quantity of stored 2008 LEE templates and oligo primers A 2006 prep of genomic FTT was used to evaluate the stored primers. Even amplifications that did not work well originally (QC neg) work well now, with titrated amounts of template. Slide 10

11. New Amplification of the QC positive vs. negative templates in order to evaluate stored FTT primers

12. Slide 12 MILESTONE 37/38 Gray: (sub)milestone title Green: open Generate and purify mg quantities of 12 selected FTT proteins Transcriptome Ag discovery Proteome Ag discovery Active Live vector-based Ag discovery Active

13. Slide 13 MILESTONE 35 (cont’d) Array hybridations with mouse RNAs from virulent Schu 4 infection & RT PCR confirmation of candidates Virulent Schu 4 SamplesRT-PCR Confirmations Initial samples Dose-Response of Infection Completed Gray: (sub )milestone title Red: completed Green: in progress Slide 13 Multi datatype analysis Phase 1 – Protein array, expression (qRT-PCR and array), western, ELIspot

14. Slide 14 MILESTONE 36 Final integration of expression data and informatics analysis Gray: (sub )milestone title Red: completed Green: in progress Slide 14

15. UNM/DVC/NIAID 5/24/10 Rick Lyons : UNM is in the discovery phase of correlates primarily in rats (Fisher 344) and cynomolgus macaque primates. Data: o Functional: Elispot (IFN gamma) detecting responses from lymph nodes from LVS vaccinated cynos vs. FT protein library synthesized by ASU o Transcriptome: FTT genes expressed during first 24 hrs of host infection in mice and rats. o Host antibody response to LVS vaccination and/or SCHU S4 challenge: Felgner arrays run on UNM TVDC sera from rats, mice, humans and NHP o Western data: DVC kindly ran UNM LVS immunized rat sera on their FTT 2D protein Western blots UNM TVDC is looking across species and across different assays, for correlates and potential vaccine candidates.

16. Presented by Phillip Stafford, PhD 5/24/2010 Multidata type analysis

17. Many methods exist for analysis of multiple datatypes. 1) Data fusion (low level): combines data from multiple sources to produce new data limitation: difficult, produces non-linear biases, may amplify the inherent error 2) Data mining (high level): extracts patterns in data without hypotheses or direction (unsupervised) limitation: unsupervised mining requires high precision and nearly zero data bias to identify patterns 2a) Literature-based data mining: uses discovered knowledge to query existing data which may be novel or different from literature limitation: only previously discovered facts can be used

18. Multidatatype analysis Many methods exist for analysis of multiple datatypes. 3) Hypothesis testing (high level, supervised): extracts conclusions dataset by dataset, resulting in interpretations across a broad swatch of biology limitation: when using high stringency, important information can be excluded 4) Hypothesis projection (high level, supervised): extracts conclusions using hypotheses, and projects results onto other datasets limitation: caution when interpreting the results, an expert in each field must review the interpretation 5) Binary clustering, decision tree: examines each dataset for a valid ‘yes/no’ answer, then combines answers into a final interpretation limitation decision tree: must have a solid set of answerable questions before beginning limitation binary clustering: must be able to transform data to binary reliably and sensibly before clustering limitation binary clustering: results may not make biological sense

19. Example: 1) FTT list from Western data for Rat provides proteins that have high binding to protected sera 2) This list is then projected onto Protein Array data, a similar datatype but orthogonal 3) The consensus of these data are projected onto Expression data to detect the relative abundance of transcripts during infection 4) These data are then cross-referenced to literature-based data (pathogenicity island) Protein array - rat Western ‘hits’ Overlap between literature, western, ELISpot genes Transcriptome data Western data 1234