Janice E. Clement, Lucio Gama

The Retrovirus Laboratory

NeuroHIV 2015, Matera, Italy

October 10, 2015

HIV CURE: “Shock & Kill” in SIV Macaque Model: Reactivation of Virus in CSF & Plasma

SIV Macaque Model of AIDS & CNS Disease

Innate Immune & Cytokine Changes – Acute Infection Brain

cART Reduces Virus Replication & CNS DiseaseViral DNA in Brain and Tissues

Zink et al., JID, 2010

Tenofovir / Saquinavir / Atazanavir / Merck L-870812

Latently Infected SIV Resting CD4+ T Cells by QVOA

Dinoso et al., JV, 2009

Plasma Tissues

QVOA parallels HIV quantitative viral outgrowth assay

Zink et al., JID, 2010

Viral RNA Viral DNA

Residual Viral RNA (<30 copies/ug RNA)NO difference in viral DNA

Bra

in S

IV R

NA

(lo

g10

co

py

eq./

ug

RN

A

In situ RNA Hybridization in Brain

HAART Reduces Virus Replication & CNS Disease –Viral DNA Reservoir Persists

Deeks. S., Nature - 2012

cART+ Shock Kill No ViralSpread

Ingenol derivatives activate latent HIVin cell lines & resting CD4+ T cells from ART suppressed patients

Ingenol-B Reactivates HIV & SIV in resting CD4+ T cells

Siliciano Lab Van Lint Lab

Clements Lab

0 200 400 60010-1

101

103

105

107

109

days p.i.

SIV

RN

A c

op

y e

q/m

LPlasma Viral Load

Spleen biopsy Spleen biopsy

+ Ing-B

Ingenol-B oral dose 0.4mg/kg daily 45 d

Ingenol-B oral dose 0.6mg/kg daily 12 dVorinostat 6 mg/kg 4 doses 12 d

Long-term cART Suppressed SIV Macaques Shock – Ingenol-B & Vorinostat

Untreated

Ingenol-B + Vorinostat

Plasma Virus Load – Ingenol-B & Vorinostat

Untreated

Ingenol-B + Vorinostat

Activation of CD69 on CD4+ & CD8+ Lymphocytes

No increase in cytokines in plasma: consistent with NO “cytokine storm”as in acute HIV/SIV infection

Activation of CD69 CD4+ & CD8+ Lymph Nodes & Spleen

Decrease in Resting CD4+ T cell Reservoir

CSF Virus– Ingenol-B & Vorinostat

Untreated

Ingenol-B + Vorinostat

Increased CCL2, Neopterin & NFL in CSF

RNA was quantified by dd-PCR and normalized to the ug of RNA used in the assay.

SIV RNA in Tissues from Ingenol-B Vorinostat Treated Macaques

• SIV-infected macaques treated suppressed with long-term ART >6 months.• Release of macaques from ART, resulted in reactivation of SIV in plasma

PT3 at 4d plasma virus 216 copies of SIV RNA/ml; CSF virus 168 copies of SIV RNA/ml.

PT4 at 5 d plasma virus 225 copies of SIV RNA/ml; CSF virus <10 copies of SIV RNA/ml.

Reactivation of SIV in Suppressed Macaques Removed ART

S. Weitgrefe & A. Haase

In Situ Hybridization of Brain

165 RNA copies/ml CSF

Phylogenetic Analyses of Env V1 Region in Pt2Virus in Plasma & CSF – DNA in Brain and Peripheral Tissues

Phylogenetic Analyses of Env V1 Region in Pt3 & P4Virus in Plasma & CSF – DNA PBMC

Conclusions

• SIV Macaque experiments provided proof-of-concept for “Shock & Kill”:• Shock: There was increase in viral load – both plasma & CSF• Kill: There was decay of latently-infected resting CD4+ T cells

in blood

• A combination of Latency Reversing Agents reactivated SIV in plasma and CSF in macaques suppressed for >500 days:• In situ hybridization detection of SIV in brain.• Cytokine increase in CSF and not plasma• Increase in NFL in CSF • Increased expression of CD69 on CD4+ and CD8+ T cells in blood.• NO Cytokine storm in plasma

• Reactivation in brain accompanied by CNS symptoms – caveat for CURE?

Collaborators

U19 AI 096113

Johns Hopkins SOM

Lucio Gama

Joseph Mankowski

Kelly Pate

M. Christine Zink

Robert Adams

Robert Siliciano

Gregory Laird

Kai Deng

Janet SilicianoIndustry Support

Merck

Gilead

Bristol-Myers Squibb

NIH Support

NIMHNINDS NIAID

University of Rio de Janiero

Luis Pianowski

University of Wisconsin

Shelby O’ Connor

University of Minnesota

Ashley Haase

Steve Weitgrefe

Is Latency & Reactivation of HIV in Macrophages a Deterrent to HIV CURE?

• Currently, human trials with CURE therapeutic approaches examine only plasma for HIV reactivation. CSF should also be tested for virus or plasma examined for CNS Biomarkers of activation – NFL, sCD163?

• Research on HIV latency and reactivation should include cells of myeloid lineage, multiple tissue macrophages – brain, spleen, liver...

• In vitro models used to screen drugs candidates should includehuman macrophage that accurately mirror a variety of tissue macrophages.

• HIV CURE therapy may need to include neuroprotective/anti-inflammatory agents to protect brain & other tissues from virus reactivation in CD4+ T cells and myeloid cells.