Penn Mental Health AIDS Research Center

Core E: Laboratory and Biobehavioral Marker Core

May 9, 2019

Core Director: Steven D. Douglas, M.D.

Co-Director: Ruben C. Gur, Ph.D.

Core E: Specific Aims

Aim 1 The Core E Laboratory performs assays of body fluids including serum, plasma and CSF markers related to HIV/AIDS, psychiatric disease, and comorbidities.

Aim 2 Core E utilizes novel assays generating “big data” output such as RNAseq, SOMAscan and CyTOF available at UPenn and CHOP to identify new biomarkers related to HIV/AIDS, psychiatric disease, and comorbidities.

Aim 3 Core E, in collaboration with the Center for Neuroimaging in Psychiatry and the Brain Behavior Lab, perform state of the art assessments of brain-behavior function in normative and clinical populations.

Aim 4 The Core E maintains a specimen BioBank, which includes plasma, serum, cells, and genomic DNA.

Core E: Services

Routine Pilot Panel

Immune/Inflamm Immune Inflamm Stress Neurodamage Cardio Psychiatric Fractalkine MIP-1α IFNγ hsCRP Cortisol NFH Fractalkine Cortisol

IFN-γ sCD14 CD4/CD8 LPS NFL sICAM IL-6

IL-1β sCD163 Neopterin sVCAM TNFαIL-6 sICAM sCD163 hsCRPIL-8 sVCAM IFN-γ

IL-10 TNFα CD4/CD8MCP-1 TNFα-R1

Custom panels available upon request

Analysis of Viral Reservoirs

• QVOA - Quantitative Viral outgrowth assay

• Single copy assay for residual viremia

• Total HIV-1 DNA in PBMC

• Total HIV-1 DNA in purified resting CD4+ T cells

• Integrated HIV-1 DNA

• HIV-1 DNA and RNA in biopsy samples (rectal, lymph nodes)

• Total HIV-1 RNA in PBMC or CD4+ T cells

New Aim 2:

• SOMAscan• RNAseq• CyTOF• ELLA• SiMOA• Bioinformatics (CHRI informatics)

Assays generating “big data” output to identify new biomarkers related to

HIV/AIDS, psychiatric disease, and comorbidities

Kraemer et.al.,PLoS One. 2011;6(10):e26332.

SOMAscan

SOMAscan: Pathway Map

Pilot study with SOMAscan, unique protein biomarker discovery tools, which measure 1,300 plasma proteins including biomarkers of inflammation and other indicators of

monocyte/macrophage polarization, as part of U01MH090325 - Anti-HIV Neuroimmunomodulatory Therapy with Neurokinin-1 Antagonists

Principles of RNA sequencing

Bauer JW et al. (2009) Gene-expression profiling in rheumatic disease: tools and therapeutic potential

Nat Rev Rheumatol doi:10.1038/nrrheum.2009.503

http://tucf-genomics.tufts.edu/home/faq

Effect of SP treatment on gene expression of non-classical compared to classical monocytes

DifferentialGene Expression

Shiroguchi et al. PNAS 2012

SP treatment FACS sorting RNA sequencing

Differentially Expressed Genes in non-classical

monocytes compared to classical monocytes

Differentially Expressed Genes in SP treated

monocytes compared to control monocytes

Effect of SP treatment on gene expression of monocytes compared to controls

CyTOF

Citrus analysis of NK cell abundance reveals phenotypic differences after cytokine treatment.Cytometry Part B: Clinical CytometryJulia Fukuyama, Dara M. Strauss-Albee, Susan Holmes, Catherine A. BlishVolume 92, Issue 1, pages 57-67, 20 JAN 2017

Mass Cytometry Analytical Approaches Reveal Cytokine‐Induced Changes in

Natural Killer Cells

CyTOF

Ella Assay: Advantages

• High reproducibility across multiple labs

• Less hands-on, more automated

• Ability to measure endogenous levels at high and low concentrations

• Small sample volume

• Multiplexing without cross-reactivity

Ella

SiMOA Assay: Advantages

• Utilizes Single Molecule Array technology to deliver femtogram per milliliter (fg/ml) level sensitivity for measurement of low-abundance biomarkers in serum, plasma and other matrices

• Unique immunoassay technology provides robust detection for various single analytes and select multiplex panel options

• High precision service offers accurate and reproducible data with < 10% inter- and intra-assay CVs

• Over 50 analytes available including IL-17A, TNF-alpha, IL-23, IP-10, TRAIL, IL-6, IL-10 and IL-12p70

SiMOA

SiMOA

0 50 100 150 200 2500

2

4

6

NHP

CSF

plas

ma

Pearson r 0.9362R square 0.8764

0 2000 4000 6000 8000 100000

5

10

15

20

25

Human

CSF

plas

ma

Pearson r 0.7912R square 0.6259

Comparison of plasma and CSF levels of neurofilamentsmeasured by conventional ELISA (CSF) and plasma (Simoa) in humans and NHPs

Core E: Pilot Support

• Year 1-Core E supported 4 of the 6 Pilot Projects-Epperson, Kayser, Langleben,

McGuire

• Year 2-Core E supported 5 Pilot Projects-Arnold, Coviello, Dowshen, McLean, Thase

• Year 3-Core E supported 5 Pilot Projects-Gibbons, Greeson, Kolson, Scott, Weljie

• Year 4-Core E supported 4 Pilot Projects-Ashare, Berrettini, DeBiasi, Gross

• Year 5-Core E supported 3 Pilot Projects-Anderson, Hill/Lazar, Wood

• Year 6-Core E supported 3 Pilot Projects-Brooks, Colon-Rivera/Montaner, Deo

• Year 7-Core E is planning to support 3 Pilot Projects-Spitsin/Benton, Bien-

Gund/Ashare/Schnoll/Gross, Kornfield

Core E: Goals• Measure novel biomarkers

• Develop assays to detect HIV DNA and RNA for reservoir

quantitation

• Assess non-cytolytic NK function interferon gamma production (UO1

HIV SSRI Depression)

• Assess the reliability and validity of the Penn Computerized

Neurocognitive Battery (CNB) for the diagnosis of HAND

• Proteomics (SOMAscan), RNASeq, CyTOF, ELLA, SIMOA

Future Directions

• Testing new bioanalytic platforms • Single cell RNAseq• Alternative splicing • Supporting new collaborations and pilot

projects – IFI – Microbiome

Reverse Transcription and Nucleocapsid

David S. Goodsell 2015 (http://hive.scripps.edu/resources.html)

HIV Life Cycle

Core E: ServicesPeripheral Blood Leukocyte Cell Phenotypes

T cell phenotypes:

Naïve T cells

Central memory T cells

Effector memory T cells

Effector T cells

Stem cell-like T cells

Core E: ServicesPeripheral Blood Leukocyte Cell Phenotypes

Monocyte and NK Cell phenotypes

Classical monocytes

Intermediate monocytes

Non-classical monocytes

Typical NK cells

NK cells precursor-like

.Gold et.al.,N Biotechnol. 2012 15;29(5):543-9.

SOMAscan