Bob Palermo Group Meeting October 13, 2008 WaNPRC.

Bob Palermo

Group Meeting

October 13, 2008

Katze

LabKatzeKatze

LabLabWaNPRC

KatzeLab

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

KatzeLab

Contemporary View of Research Efforts in Primate Functional Genomics and Systems Biology.

2006 - P51 Division of Functional Genomics and Infectious Disease.

Macaque transcriptome

Macaque microarray

Macaque proteome

Transcriptional profiling

Quantitative protein profiling

Rhesus genome sequence

DNA RNA

Macaque EST sequences

Protein

AMT tag databases

Comprehensive picture of virus infection and immunity

Function

Immunologic assays

Innate and adaptive immunity

Macaque

Virus infection models

Clinical dataHistopathology

Animal Models Genomics ProteomicsProtective Immunity

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.

KatzeLab

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 4 Day 0

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Day 7 Day 2

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Day 1

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Day -14

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Day 3

KatzeLab

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.

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PBMCGALT

lymph node

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0PBMCGALT

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14PBMCGALT

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24PBMCGALT

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21PBMCGALT

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17PBMCGALT

lymph node

Group

42· PBMC· GALT· lymph node

· lung· liver· bone marrow

· brain· lung

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PBMCGALT

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PBMCGALT

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PBMCGALT

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3PBMCGALT

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7PBMCGALT

lymph node

10PBMCGALT

lymph node

0PBMCGALT

lymph node

14PBMCGALT

lymph node

24PBMCGALT

lymph node

21PBMCGALT

lymph node

17PBMCGALT

lymph node

Group

42· PBMC· GALT· lymph node

· lung· liver· bone marrow

· brain· lung

KatzeLab

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.

KatzeLab

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 SIVmac251 model

O + INAd-HIV/SIV

ITAd-HIV/SIV

IMBoost

IMBoost

0 12 24 36 44 wks

SHIV89.6P

Ad5 Immunogens Boost

Group I Ad5hr-HIVenv89.6P + Ad5hr-SIVgag239 + Ad5hr-SIVnef239Δ1-14 No boost (PBS)

Group II Ad5hr-HIVenv89.6P + Ad5hr-SIVgag239 + Ad5hr-SIVnef239Δ1-14 HIV89.6Pgp140ΔCFI in MPL-SE

Group III Ad5hr-HIVenv89.6P + Ad5hr-SIVgag239 + Ad5hr-SIVnef239Δ1-14 HIV peptomer in PBS

Group IV Ad5hr-ΔE3 3 MPL-SE 3 PBS only

KatzeLab

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-apoptoticAKT1 and BAX nodes

B cell fateBLC6 node

B cell and T cellReceptor Signaling

LAT and BLNK Nodes

KatzeLab

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-12 6 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

0 2 4 6 83

4

5

6

7

8ControlSIVSIV + IL12SIV + IL15SIV + RantesSIV + Rantes/IL15

Weeks

RN

A c

op

ies/

ml

KatzeLab

Heterogeneity observed in arrays, reflective of biological heterogenity?Group 2: Immunogens + IL-12 DNA

4389439744004404

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.

KatzeLab

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.

Table 1. Incidence (95% CI) of HIV Infection MITT population (males). From the presentation by Dr. Mike Robertson, HVTN Conference, Nov. 7, 2007

Baseline Ad5 titer

Vaccine V

Placebo P

Relative Incidence (V:P)

≤ 18 4.0 (2.5, 6.3) 4.0 (2.5, 6.2) 1.0 (0.5, 2.0)

19 – 200 4.4 (1.9, 8.8) 2.2 (0.6, 5.5) 2.1 (0.6, 9.3)

201 – 1000 6.1 (3.3, 10.2) 3.0 (1.2, 6.2) 2.0 (0.8, 5.9)

> 1000 4.4 (1.8, 9.2) 1.2 (0.2, 4.5) 3.5 (0.7, 35.0)

≤ 18 4.0 (2.5, 6.3) 4.0 (2.5, 6.2) 1.0 (0.5, 2.0)

> 18 5.1 (3.4, 7.3) 2.2 (1.2, 3.8) 2.3 (1.1, 4.7)

≤ 200 4.2 (2.8, 6.0) 3.5 (2.3, 5.2) 1.2 (0.7, 2.1)

> 200 5.4 (3.3, 8.2) 2.3 (1.0, 4.3) 2.4 (1.0, 5.8)

Overall 4.6 (3.4, 6.1) 3.1 (2.1, 4.3) 1.5 (0.9, 2.4)

Is increasing Ad5 titer associated with increased incidence in

vaccinees, or decreased incidence in placebos?

KatzeLab

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.

KatzeLab

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.

KatzeLab

Depletion of Abundant Proteins in Plasma

• Challenge of dynamic range in plasma– often quoted concentration range for proteins of 109

– 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) Ladder2) Filtered Human plasma3) Human flow-through4) Human elution 35) M.fl. 20uL filtered plasma6) M.fl. 20uL flow-through (some Ig breakthrough?7) M.fl. 20uL wash 18) M.fl. 20uL elution 39) M.fl. 10uL filtered plasma10) M.fl. 10uL flow-through11) M.fl. 10uL wash 112) M.fl. 10uL elution 3

Recheck on Protein A column

Comparable results with rhesus and pig-tailed plasma samples

KatzeLab

Systems Biology Contract

• 400 Microarray comparisons and 200 TaqMan Array comparisons per year.

• What else need we say?

KatzeLab

KatzeLab

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.

KatzeLab


• 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

KatzeLab

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

A B


KatzeLab

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

KatzeLab

H5N1 VN1203 Tx91

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.

KatzeLab

Evolving Infrastructure with use of Open-Source Tools

KatzeLab

LabKey Server – Open Source Databasefor Shared Access via Web Interface

Access control

Folder like organization.Hot links to project areas

KatzeLab


• Example: NIDA Project Area contains interface to database of clinical data associated with functional genomics study of hepatitis C-associated liver fibrosis

Process andworkflow tracking

Search interfaces

KatzeLab


• 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.

KatzeLab

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

KatzeLab

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

Bob Palermo Group Meeting October 13, 2008 WaNPRC.

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Transcript of Bob Palermo Group Meeting October 13, 2008 WaNPRC.