hiv cure: “shock & kill” in siv macaque model ... · hiv cure: “shock & kill” in...
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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
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SIV Macaque Model of AIDS & CNS Disease
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Innate Immune & Cytokine Changes – Acute Infection Brain
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cART Reduces Virus Replication & CNS DiseaseViral DNA in Brain and Tissues
Zink et al., JID, 2010
Tenofovir / Saquinavir / Atazanavir / Merck L-870812
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Latently Infected SIV Resting CD4+ T Cells by QVOA
Dinoso et al., JV, 2009
Plasma Tissues
QVOA parallels HIV quantitative viral outgrowth assay
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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
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Deeks. S., Nature - 2012
cART+ Shock Kill No ViralSpread
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Ingenol derivatives activate latent HIVin cell lines & resting CD4+ T cells from ART suppressed patients
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Ingenol-B Reactivates HIV & SIV in resting CD4+ T cells
Siliciano Lab Van Lint Lab
Clements Lab
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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
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Plasma Virus Load – Ingenol-B & Vorinostat
Untreated
Ingenol-B + Vorinostat
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Activation of CD69 on CD4+ & CD8+ Lymphocytes
No increase in cytokines in plasma: consistent with NO “cytokine storm”as in acute HIV/SIV infection
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Activation of CD69 CD4+ & CD8+ Lymph Nodes & Spleen
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Decrease in Resting CD4+ T cell Reservoir
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CSF Virus– Ingenol-B & Vorinostat
Untreated
Ingenol-B + Vorinostat
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Increased CCL2, Neopterin & NFL in CSF
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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
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• 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
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S. Weitgrefe & A. Haase
In Situ Hybridization of Brain
165 RNA copies/ml CSF
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Phylogenetic Analyses of Env V1 Region in Pt2Virus in Plasma & CSF – DNA in Brain and Peripheral Tissues
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Phylogenetic Analyses of Env V1 Region in Pt3 & P4Virus in Plasma & CSF – DNA PBMC
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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?
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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
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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.