1. Bob Palermo Group Meeting October 13, 2008 WaNPRC

2. K atze L ab Present Project Activities (in no particular order) P51 Division of Functional Genomics and Infectious Disease R24 “Development of a Primate Genomics Resource” – macaque proteomics R21 – Gene Expression Profiling of AIDS Vaccine Strategies Simian Vaccine Evaluation Unit (“SVEU”) Merck Step Study LSDF Sample Preparation Pipeline SysBio Contract – Transcriptional Analysis Core

3. K atze L ab Contemporary View of Research Efforts in Primate Functional Genomics and Systems Biology. 2006 - P51 Division of Functional Genomics and Infectious Disease. Fig. 2. The four Cores of the Division of Functional Genomics and Infectious Disease (Animal Models, Genomics, Proteomics, and Protective Immunity) provide the virus infection models and the clinical, genomic, proteomic, and immunologic resources needed to perform comprehensive analyses on nonhuman primates. EST: expressed sequence tag; AMT: accurate mass and time.

4. K atze L ab P51 Division of Functional Genomics and Infectious Disease. 2007 Macaque Influenza Model using highly pathogenic H5N1 virus (VN1203), reassortants of the 1918 virus, contemporary low path comparator. –Supportive role for genomics studies that were under the 1918 Flu Grant –Now leading a proteomics follow-up that is being supported under the P51 Present Division Activities – new macaque influenza model –evaluating impact of primary and secondary infection (Secondary infection will allow assessment of adaptive immune response) –study using a H3N2 from a recent outbreak. –study has a very extensive immunology component requiring protocols for sorting several lymphocyte populations for both phenotypic analysis, functional assays and microarrays (and proteomics if we can stretch them). DAY OF INOCULATION Day 4Day 0 *** Day 7 Day 2 * Day 1 * Day -14 * Day 3

5. K atze L ab 2008 - R24 renewal to cultivate the field of macaque proteomics. From relationship with Smith Lab (PNNL), and early efforts to apply proteomics in an influenza model.

6. K atze L ab R24 Timeline Issues with the commencement of this grant….. We are accumulating the tissues for the baseline characterization of the target specimens (Thanks Eric!) The IACUC protocol for the animal model is approved. Funding for animal model was supposed to come from Primate Center. However, this is proving problematic and support is being sought from the SVEU.

7. K atze L ab R21 – Gene Expression Profiling of AIDS Vaccine Strategies Ad vectors target mucosal sites. Ad serotypes available with little pre-existing immunity in human population Ad5 host-range mutant can replicate in macaque Previously shown to be highly effective in an SIV mac251 model O + IN Ad-HIV/SIV IT Ad-HIV/SIV IM Boost IM Boost 0 12243644 wks SHIV 89.6P

8. K atze L ab R21 - Network Diagram of Immune-Associated Genes Reveals a Cluster of Anti-apoptotic Expression Changes Genes distinguishing Day 0, Group II (prime boost) from Group IV (empty vector) BCR TCR Pro-survival Anti-apoptotic AKT1 and BAX nodes B cell fate BLC6 node B cell and T cell Receptor Signaling LAT and BLNK Nodes

9. K atze L ab SVEU Study P147 – Electroporated DNA Vaccines with Accompanying Expression of Macaque Immunological Adjuvants Group 1: pCSIV DNAs: gag, pol, env 6 animals Group 2: pCSIV DNAs: gag, pol, env + pmacIL-126 animals Group 4: Controls (naïve and/or empty pCmac plasmid controls)6 animals Group 5: pCSIV DNAs: gag, pol, env + pmacRANTES 5 animals Reminder about the samples on the arrays. Fresh PBMCs from day immediately prior to challenge –overnight treatment –stimulation with overlapping peptide pool of SIV pol OR –mock stimulation with media only (genetically matched comparator for ratios) Arrays are then comparison of transcription profiles of pol-stimulated PBMCs to mock

10. K atze L ab Heterogeneity observed in arrays, reflective of biological heterogenity? Group 2: Immunogens + IL-12 DNA 4389 4397 4400 4404 This appearance of heterogeneity in the arrays is not a priori an issue. However, we are not seeing much tendency for the arrays to spontaneously organize by groups or virological response.

11. K atze L ab Transcriptional profiling of archived PBMCs to characterize vaccine and placebo recipients as a function of baseline Ad5 titers PARENT PROTOCOL: HVTN502 Primary hypotheses: The observed differences in incidence of HIV-1 infection between placebo and vaccine groups, as a function of increased baseline Ad5 titers, reflects underlying immunological distinctions that will be reflected in differences in global gene expression profiles. Is increasing Ad5 titer associated with increased incidence in vaccinees, or decreased incidence in placebos?

12. K atze L ab HVTN STEP Microarray Project – Study Design Vaccine regimen: treatments at t=0, 4 weeks, 24 weeks Specimens from week 8 (4 weeks after second treatment). Uninfected patient pool (less restricted sample access). Matching demographics of age and race to male patent pool that constituted 82 of 83 infecteds as of Nov. 2007. Samples are viably frozen PBMCs 120 samples: 60 vaccinees, 60 placebo; distributed with 15 per quartile of Ad5 titer Assessing gene expression differences as a consequence of baseline Ad5 titers. Assessing gene expression differences as a consequence of treatment.

13. K atze L ab Life Sciences Discovery Fund Sample Preparation Pipeline A program and platform for biomarker discovery, centered on indentifying protein biomarkers present in the blood of HCV-infected patients with or at risk of liver fibrosis. LSDF UW Milestones and sample processing requirements at UW Platform for semi-automated processing of plasma or serum samples, for depleting the top 12 most abundant proteins from this biological fluid –commercial affinity column for this purpose. Define methods, sample pipeline, and quality metrics. Year 1 – complete processing of 60 samples (500 μL samples) Year 2 – complete processing of 300 samples Year 3 – continued sample processing for additional studies, either as immediate follow-on to the fibrosis study, or possibly in other disease indications.

14. K atze L ab Depletion of Abundant Proteins in Plasma Challenge of dynamic range in plasma –often quoted concentration range for proteins of 10 9 –comparison to Mt. Everest (from 9000 meters down to 9 microns) Preferred method for depletion from human plasma: IgY-12 –Mix of 12 polyclonal avian antibodies against human albumin, IgG, IgM, IgA, transferrin…) 1 2 3 4 5 6 7 8 9 10 11 12 IgY-12 gel: 1) Ladder 2) Filtered Human plasma 3) Human flow-through 4) Human elution 3 5) M.fl. 20uL filtered plasma 6) M.fl. 20uL flow-through (some Ig breakthrough? 7) M.fl. 20uL wash 1 8) M.fl. 20uL elution 3 9) M.fl. 10uL filtered plasma 10) M.fl. 10uL flow-through 11) M.fl. 10uL wash 1 12) M.fl. 10uL elution 3 Recheck on Protein A column Comparable results with rhesus and pig-tailed plasma samples

15. K atze L ab Systems Biology Contract 400 Microarray comparisons and 200 TaqMan Array comparisons per year. What else need we say?

16. K atze L ab

17. K atze L ab Our infrastructure supports statistical methods and functional analysis tools to gain insight in the key pathways and genes. Resolver Example - Genomics Analysis of Lung Tissue Influenza study comparing high-path H5N1 virus, 1918 reassortants and low-path contemporary H1N1 virus. 36 total macaques across 4 time points, 2 biological replicates per time point.

18. K atze L ab Resolver Example - Genomics Analysis of Lung Tissue Sophisticated statistical algorithms focus on the key differences in a study. List management tools allow ready comparison across numerous experiments ANOVA, t-test or Wilcoxon methods with multiple test corrections “Broadcast” between experiments List management and Venn diagram tool

19. K atze L ab A- Genes induced in a lethal but not in a nonlethal infection. B- Genes induced in a nonlethal but not in a lethal infection. H5N1 Tx91 3/5 1918 2/6 1918 AB Resolver Example - Genomics Analysis of Lung Tissue

20. K atze L ab Genomics Analysis of Lung Tissue – Functional Analysis Tools Ability to see what pathways/networks are perturbed for a given set of conditions Visualization across all multiple experiments

21. K atze L ab H5N1 VN1203Tx91 Genomics Analysis of Lung Tissue – Functional Analysis Tools Visualization of immune response functions, from genes identified by ANOVA, comparing expression levels in a high path vs. low path infection.

22. K atze L ab Evolving Infrastructure with use of Open-Source Tools

23. K atze L ab LabKey Server – Open Source Database for Shared Access via Web Interface Access control Folder like organization. Hot links to project areas

24. K atze L ab LabKey Server – Open Source Database for Shared Access via Web Interface Example: NIDA Project Area contains interface to database of clinical data associated with functional genomics study of hepatitis C-associated liver fibrosis Process and workflow tracking Search interfaces

25. K atze L ab LabKey Server – Open Source Database for Shared Access via Web Interface Views of data can be customized Web interface inherently browsable, and affords the option to drill to underlying records or associated material from differing studies.

26. K atze L ab LabKey Server – Public Data Access from Web Portals Lab Website is built from conventional HTML, more suitable for text intermixed with extensive graphics and more guided browsing. Links into LabKey Pages

27. K atze L ab LabKey Server – Public Data Access from Web Portals Katze LabKey Server(s) – evolving to handle an expanding scope of data types Increased demand for serving structured collections of data Query and browsing capacity for public exploration Drill to Publication- Associated Microarray Data