ASU TVDC Technical Report 6/22/10 Kathryn F. Sykes and Stephen A. Johnston and Phillip Stafford Completed Milestones: 25, 26, 28* and 32, 33, 34, 35, 36 Active Milestones: UNM 29*/ASU28*, ASU 37/38 Currently Inactive Milestones: 30 Slide 1 MILESTONE 28 Build SCHU S4 proteome Gray: (sub)milestone title Yellow: Re-opened Build ORF expression library corresponding to proteome Generate complete protein-fragment library Array protein-fragments for T cell stimulation assays Re-opened Re-opened Re-opened Slide 2 Generation of polypeptide antigens for UNM T cell assays SOP for detecting T cell stimulation with synthetic (IVTT) antigens Production of antigens (ASU) Production Assay development (UNM) T cell proliferation IFNg ELISpot assay Screening (UNM) Identification of stimulatory proteins & peptides Confirmation Assay optimization using ivt proteins Slide 3 Evaluating alternative IVTT 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 IFNgamma ELISPOT assays. • We determined that background was low using either bead. • A second test was designed to test specificity of stimulated response, against IglC1-immune rat splenocytes. • Large scale synthesis of IglC 1 (putative positive)and ASFV053 (negative control) was repeated on both small MyOne (1micron) and large M-280 (2.8 micron)beads. Slide 4 New Preps of IglC1 and ASFV053 on the two bead formats 0.25x 0.75x 0.25x BSA, ug 2 1 0.5 ASFV053 IVT-rx 250150100755037kDa 37- 33kDa 2520- MyOne MyOne 10- M-280 MyOne 15M-280 Precision Plus Standard (Bio-Rad) kDa 0.75x 4 0.75x iglC1 IVT-rx Slide 5 Production of LEE Templates for New IVTT FTT Library • New vs. old prep of genomic FTT was compared in PCRs. • Titrating optimal new template amount for PCRs. Slide 6 Evaluation of new Schu-S4 genomic DNA preparation (May 2010) as a template for ORF amplification L 1 2 3 4 5 1 2 FTT1008A 3 4 5 L 1 2 FTT1800A 3 4 5 1 2 FTT1001A 3 4 5 FTT1016A bp 1000 – 500 – 250 – bp 1000 – 750 – PCR by-products 500 – 250 – PCR by-products L 1 2 3 4 FTT0549A 5 1 2 3 4 5 L FTT0633A 1 2 3 4 FTT0511A 5 1 2 3 4 5 FTT1215B bp 1000 – bp 1000 – 750 – 500 – 250 – 500 – 250 – PCR by-products PCR by-products L - 1kb DNA Step Ladder (Promega) 1 - 4 ng “new” gDNA prep template (May 2010) 2 - 9 ng “new” gDNA prep template (May 2010) 3 - 22 ng “new“ gDNA prep template (May 2010) 4 - 100 ng “new” gDNA prep template (May 2010) 5 - 60 ng “old” gDNA prep template (March 2006) Slide 7 Interpretations • ORF PCR products are considerably cleaner; there are few to no byproducts. • Significantly less DNA is required. As little as 4ng of starting genomic DNA was sufficient to amplify most ORFs, at high yields and acceptable specificity. • However,100ng of starting template appears to be least likely to generate the lower migrating by product Slide 8 Testing parameters for optimal LEE construction • Assembly of LEEs from ORFs generated with old vs. new gDNA. • Methods of purifying ORFs before assembly • Amount of PCA template for final production step Slide 9 Overlapping PCR optimization for LEE template assembling with potentially problematic ORFs AMP 12 11 12 11 12 13 14 AMP 11 12 11 12 AMP AMP AMP AMP GP GP 1 step PCA Test 3 AMP AMP AMP AMP EP UP GP AMP Test 2 GP AMP EP UP UP GP AMP EP GP AMP EP UP Test 1 1kb DNA Step Ladder (Promega) GP GP AMP AMP 1kb DNA Step Ladder (Promega) bp 10005001 2 3 4 7 8 14 13 14 14 14 2 AMP AMP AMP AMP AMP AMP AMP AMP 1 2 AMP GP GP AMP AMP AMP AMP GP GP AMP AMP 2 step PCA EP UP GP AMP EP 6 4 UP GP AMP 3 UP 2 GP AMP EP GP AMP EP UP 1 5 10005009 5 6 7 10 1 2 4 5 7 2 1 2 1 2 3 4 3 4 3 4 3 4 3 4 8 8 - “Old” (March 2006) gDNA template for ORF amplification - GP – gel purified products for PCA; AMP – AMPure purified products for PCA ; EP – ethanol precipitated PCR products for PCA; UP – un-purified PCR products for PCA - 1step PCA: 15x cycles (Ta=58C) for LEE (P+O+T) - 1/5 PCA template for PCR 1 - “Old” (March 2006) gDNA template for ORF amplification - AMPure purified products for PCA only - 1 step PCA: 15x cycles (Ta=58C) for LEE (P+O+T) - 2 step PCA: 10x cycles (Ta=53C) for LEE (P+O) + 10x cycles (Ta=58C) for LEE (PO+T) - 1/25 PCA template for PCR - “New” (May 2010) gDNA template for ORF amplification - GP – gel purified products for PCA; AMP – AMPure purified products for PCA - black - 160 fmole, blue – 320 fmole, purple – 480 fmole of each component (P,O,T) in PCA - red - 160 fmole ORF with byproduct after AMPure purification - 1 step PCA: 10x cycles (Ta=58C) for LEE(P+O+T) - 1/50 PCA template for PCR Genes: 1- FTT1001A; 2- FTT1068A; 3- FTT0511A; 4- FTT1215B; 5- FTT0639A; 6- FTT1329A; 7- FTT0877A; 8-FTT1712A; 9- FTT0162A; 10- FTT1589A; 11- FTT1008A; 12- FTT1800A; 13- FTT0549A; 14- FTT0633A Slide 10 Continued: Evaluation of quality of different re-array and dilution primer sets from 2007-2008 to produce potentially problematic ORFs - 5 and 10 uM primer and primer-mix subsets significantly degraded during storage and can not be use for ORF amplification. - 50 uM original primer stocks and 0.8 uM primer-mix subsets had demonstrated decent quality during test-ORF amplification. However both subsets are partial and can cover amplification of 70-80% of the targeted (problematic) ORFs. Slide 11 Evaluation of primer quality from 50 uM stock plate subset 1 2 3 4 5 6 7 8 bp 1000500- PCR by-products Genes: 1- FTT1166A; 2- FTT0478B; 3- FTT1401A; 4- FTT0451A; 5- FTT0611A; 6-FTT1036B; 7- FTT0420A; 8-FTT1715A Slide 12 Overlapping PCR optimization for LEE template assembling with potentially problematic ORFs 3 AMP AMP 7 AMP UP AMP AMP UP AMP UP AMP UP AMP UP AMP UP AMP UP Test 4 bp 15001000- 500- 1 2 3 4 5 PCA in 50 ul 6 4 5 PCA in 30 ul - “New” (May 2010) gDNA template for ORF amplification - UP – un-purified PCR products (P,O,T )for PCA AMP – AMPure purified products (P,O,T) for PCA - 160 fmole of each component (P,O,T) in PCA - 1 step PCA: 10x cycles (Ta=58C) for LEE(P+O+T) - 1/50 or 1/30 PCA template for PCR (20x cycles) Genes: 1- FTT1166A; 2- FTT0478B; 3- FTT1401A; 4- FTT0451A; 5- FTT0611A; 6-FTT1036B; 7- FTT0420A Slide 13 Additional Points • Description of deliverables completed for each active milestone: – MS 35: Microarray results with qPCR verification – MS 36: Set of 9 proteins for testing as vaccine candidates • List of relevant publications from the past month • ASU reports no TVDC publications in May 2010 • MSCR status – MS33: NIAID provided review on 6/2/10; ASU edited 10 SOPs; NIAID MSCR comments under review at UNM on 6/17/10. – MS34: Is overdue; At ASU site visit, Mitch planned to submit to UNM by 3/31/10 Wed. – MS35: Mitch- completed work; ASU will write MSCR (5/31/10- overdue) – MS36: Mitch- completed work; ASU will write MSCR (5/31/10) – note, MSCR36 doesn’t include additional analysis ; overdue Slide 14 MILESTONE 37/38 Generate and purify mg quantities of 12 selected FTT proteins Gray: (sub)milestone title Green: open Transcriptome Ag discovery Proteome Ag discovery Live vector-based Ag discovery Active Active Active Slide 15 Construction of bacterial expression construct for IglC1 in pET32b pJet2.1 TOPO bp 3000- 1000640- iglC1 500- BamHI and HindIII digest of iglC1 sequence-perfect pJet2.1/blunt and pCR-BluntII-TOPO clones Slide 16 MILESTONE 36 Final integration of expression data and informatics analysis Gray: (sub )milestone title Red: completed Green: in progress Felgner and Expression and Western Analysis Slide 17 Just received annotations for protein array data, compiling data for data mining to come. Early visualizations are presented here. TOPO pJet2.1 Slide 18 First Look: Felgner Array Data NHP TOPO Early analysis: pJet2.1 FTT1282, FTT1776c are two commonly high (multi-species) genes highly expressed in expression in mouse. Slide 19 First Look: Felgner Array Data TOPO Human pJet2.1 Slide 20 First Look: Felgner Array Data TOPO Mouse pJet2.1 Slide 21 First Look: Felgner Array Data TOPO Rat pJet2.1 Vaccine and Naïve and doses are intermingled Slide 22