Tissue Reservoirs

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Tissue Reservoirs Melissa Churchill HIV Neuropathogenesis Laboratory, Centre for Biomedical Research, Burnet Institute. Departments of Medicine and Microbiology, Monash University

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Melissa Churchill HIV Neuropathogenesis Laboratory, Centre for Biomedical Research, Burnet Institute. Departments of Medicine and Microbiology, Monash University. Tissue Reservoirs. Outline. What is a tissue reservoir Types of tissue reservoirs - PowerPoint PPT Presentation

Transcript of Tissue Reservoirs

Page 1: Tissue Reservoirs

Tissue Reservoirs

Melissa Churchill HIV Neuropathogenesis Laboratory, Centre for Biomedical Research, Burnet Institute.Departments of Medicine and Microbiology, Monash University

Page 2: Tissue Reservoirs

What is a tissue reservoir

Types of tissue reservoirs

The central nervous system as a tissue reservoir

Eradication and the CNS reservoir

Highlights from the NeuroAIDS pre conference symposium: July 18th AMREP.

Outline

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Types of tissue reservoirs

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GI tract is a significant viral reservoir on HAART

Chun et al., J Infect Dis 2008; Yukl et al., J Infect Dis 2010

8 HIV+ subjects on ART, VL <50 c/ml for a median 5 years

HIV DNA measured from cell isolates and normalized by FACS data for CD4+ Tcells.

Significantly more HIV DNA was found in all tissues analysed relative blood

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Preferential infection of central memory T cells that express CCR6 and CXCR3 in HIV-infected patients on ART may contribute to a larger

reservoir in rectal tissueAnderson JA et al., 20th International AIDS Conference, LBPE05

All patients on ART for >3 years

Blood (leukapharesis)

Significant in the integrated HIV DNA in CD4+ T-cells in rectal tissue Vs blood and LN CD4+ T-cells

preferential infection of rectal CD4+ T cells compared blood and LN CD4+ T cells

Significant in integrated HIV DNA in blood central memory CD4+ T-cells expressing both CCR6 and CXCR3 relative to the 3 other subsets

preferential infection of central memory CD4+ T-cells in the blood that express both CCR6 and CXCR3

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Rectal tissue enriched with CCR6+ CXCR3+ memory CD4+ T-cells

Rectal Tissue

% C

D3+

CD

4+

CD

45R

A-

Rectal tissue harbours a high % of memory CD4+ T-cells expressing both CCR6 and CXCR3

Taken together with the finding that in blood memory CD4+ T-cells expressing both CCR6 and CXCR3 also harbour increased levels of integrated DNA

may explain why rectal tissue has a larger integrated DNA reservoir that blood.

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Types of tissue reservoirs

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The CNS as viral reservoir The CNS is a potential but controversial viral reservoir of

HIV Potential: during the course of disease HIV enters the

CNS.

Controversial: no direct evidence that in virally suppressed patients the CNS contains HIV infected cells harboring competent pro-viral genomes.

However, there is a significant amount of indirect evidence from numerous studies to suggest, that at least in some HIV infected virally suppressed patients, the CNS may be a viral reservoir

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Key questions relevant to the CNS compartment as a tissue reservoir

Are cells of the CNS infected with HIV-1, in sufficient numbers, and contain integrated pro-viral genomes during suppressive cART? Is there evidence of ongoing persistent replication and immune

activation within the CNS in the presence of suppressive cART?

Do HIV-1 infected cells in the CNS have mechanisms in place to allow for the virus to escape from the biochemical decay processes or immune mechanisms and persist for long period of times, ie long lived cells (latency) Do cells of the CNS have regulatory mechanisms that facilitate a

latent infection?

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Are cells of the CNS infected with HIV and

contain an integrated pro-viral genome?

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Macrophages: major sites of productive infection resident microglia perivascular microglia and macrophages, meningeal macrophages, choroid plexus macrophages (Hickey, 1999a).

Astrocytes: infection non-productive and restricted(Takahashi et al, 1996; Sharer et al., 1996; An et al, 1999)

CNS targets of HIV infection

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Do CNS cells from symptomatic HIV-1 infected patients do contain integrated HIV-1 DNA?

Using laser capture microdissection of autopsy brain tissues to purify pure astrocytes and macrophages/microglia, integrated genomes identified using alu-PCR

Integrated pro-viral genomes detected in macrophages/ microglia and astrocytes, not neurons.

CNS cells contain integrated HIV proviral genomes

(Churchill et al., 2006).

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Churchill et al 2009, Annals of Neurology

Extent of infected CNS tissue infection

Are substantial numbers of cells infected?

Using laser capture microdissection and triple nested multiplex PCR for V3 and GAPDHHIV-1 env sequences detected in astrocytes and

macrophages.

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Thompson et al, Am J Pathology, 2011.

Laser capture microdissection of brain cells of pre-asymptomatic HIV infected patients off treatment

HIV-1 DNA detected in in perivascular macrophages, microglial cells and astrocytes

Astrogliosis (GFAP) and microglial cell activation (CD68) in HIV-infected patients

HIV DNA detected in CNS cells in pre-smptomatic HIV patients pre-ART

GFAP CD68

HIV-

HIV+

HIV+

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HIV-1 sequence compartmentalization CNS and non CNS (Ohagen et al 2003, Thomas et al 2007, Harrington et

al., 2009, Schnell et al., 2009, Sturdevant et al., 2012)

different brain regions (Smit et al 2001)

different cell types (Thompson, Churchill et al, Annals Neurol. 2004)

at the level of Env, Nef, Tat and the LTR

Functional compartmentalization CNS derived envelopes have an enhanced ability to utilise

low levels of CD4 permitting m-tropism. more open conformation, exposing CD4 binding domain sensitivity to CD4 binding site n-Abs than matched viruses

from blood (Gorry et al., 2002, Peters et al., 2004 & 2008, Thomas et al., 2007, Dunfee et al., 2009)

Independent and distinct evolution of HIV-1 occurs in the CNS compartment: evidence of infection

and replication

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Ongoing replication in the CNS in the

presence of cART

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Ongoing replication during cART: asymptomatic CSF viral persistence

Eden et al., JID 2010 In patients on suppressive

cART (Plasma VL<50cps/ml) 10% of patients had detectable

VL (>50cps/ml) in CSF using a standard assay

Letendre at al., (CROI 2009) 300 patients with suppressed

plasma VL (<50 cps/ml) using an ultra sensitive assay 40% of patients has CSF viral

escape with more than 2 cps/ml in the CSF.

Drug CSF viral escape,

n(%)EFV 4 (15 %)

LPV/r 1 (5 %)ATV/r 2 (10 %)TDF 3 (9 %)ABC 4 (22 %)ZDC 0 (0 %)3TC 4 (10 %)FTC 3 (12 %)

CROI 2014 UPDATES-Rate of asymptomatic CSF escape:- 13% in UK (Abstract 442)- 10.3% in Italy (Abstract 443)- 23% in Sweden (Abstract 445)CSF escape relates to depression incidence (Abstract 33)

Slide courtesy of Steve Deeks

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Single genome sequencing demonstrates CSF HIV distinct from plasma during suppressive therapy

Dahl et al., JID, 2014.

Blood CSF

0.00050.02

*

*

**

*

Trees rooted with pre-suppresion plasma sequence.*Significant bootstrap value

Grey/black – pre-ART sequencesColors – on ART sequences: interval 1, 2, 3, 4

Subject 7027Chronic Infection

Subject 9058Primary Infection

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Indirect evidence of ongoing replication during cART: symptomatic CSF ‘escape’

Patient 2000 Plasma VL

<50cps/ml for 8 yrs,

presents with onset of HAND

found elevated levels of virus in CSF

Peluso et al., AIDS, 2012. Also: Canestri et al., Clin Infect Diseases, 2010.

Slide courtesy of A/Prof Serena Spudich

LamivudineAbacavirLopinavir/r

Latest CD4 308 cells/ul

hand tremor, ataxia, slurred speech, aphasia

CSF HIV RNA

CSF WBC = 26 cells/ul*

* Normal CSF WBC < 5 cells/ul

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Genotyping of the CSF virus revealed resistance to almost all drugs in regime Suggested ongoing replication and evolution in the CNS away from the virus

detected in the plasma and fully suppressed Adjustment of the the drug regiment resulted in resolution of signs and

symptoms

Indirect evidence of ongoing replication during cART: symptomatic CSF ‘escape’

Slide courtesy of A/Prof Serena Spudich

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Ongoing replication in the CNS in the

presence of cART: Immune activation

and biomarkers

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Krut at al 2014 (PLos One) Presence of light subunit of the

neurofilament protein (NFL) in CSF (major structural component of mylelinated axons)

Although significantly lower in cART patients that in untreated controls, still significantly elevated over uninfected controls

Suggestive of ongoing axonal injury

Ongoing replication in CNS during cART: Biomarkers (NFL)

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Neopterin (marker of macrophage/microglia activation) is an indicator of immune activation in blood and CNS Levels of Neopterin correlate

with CSF RNA copies

Following 4 years of treatment in patients resulting in full viral suppression

blood serum levels resolve to levels similar to uninfected controls,

CSF levels fail to resolve

Indirect evidence of ongoing replication during cART: ongoing immune activation

Hagberg et al., AIDS Res and therapy 2010, Eden et al., JID 2007. Yilmezet al., 2008 JAIDS

>4yrs treatmentBaseline

10

20

10

30

40

50

60

nmol

/L

Serum Neopterin

10

20

10

30

40

50

60

>4yrs treatmentBaseline

nmol

/L

CSF Neopterin

Neo

pter

in (

nmol

/l)

CSF HIV-1 RNA (copies/ml)

CSF Neopterin

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Garvey et al, AIDS, 2014.Anthony et al, J Neuropathol Exp Neurol, 2005Yilmaz et al., J Acquir Immune Defic Syndr, 2008

Positron emission tomography (PET): Increased brain PK11195 uptake (specific for activated microglia) in 7 HIV subjects on 3.6 years suppressive ART.

Brain pathology: Excess activated microglia (CD68+ cells) in HIV-infected individuals on > 1.5 years suppressive ART.

Persistent CNS immune activation on ART

/ HIV-

Courtesy of Serena Spudich

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Latency in the CNS

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in vitro latency

in vitro studies have demonstrated that infection of astrocytes is non productive/latent (Brack-Werner et al, 1992; Neumann et al; 1995, Gorry et al, 1999)

one of the major blocks to viral production in astrocytes is at the level of transcription (Shahabuddin, Volsky et al. 1992, Swingler, Easton et al. 1992, Ludvigsen, Brack-Werner et al. 1996, Niikura, et al,. 1996, Ensoli, Wang et al., 1997)

in vitro studies have demonstrated persistent HIV-1 can be reactivated in human fetal glial cells using TNF alpha and interlukin-1 beta (Tornatore et al., 1991)

Elevated levels of COUP-TF biding protein CTIP2(Bcl11B) in HIV+ ‘latent’ microglia involved in the recruitment of histone demethylases to the LTR (Le Douce et al, 2012;, Desplats et al., 2013 Neurology)

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Latency in the CNS: animal models

IV SIVInnoculation

Day 0 12 175

Treatment• no ART• saquinavir/atazanavir, integrase inhibitor• atazanavir, integrase inhibitor

Examination

Despite a decrease HIV-1 RNA (and GP41) in the CNS there was no decrease in the level of HIV-1 DNA in the CNS following treatment

Zink et al, 2010, JID

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Latency in the CNS: responsiveness to

transcriptional activators

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Do transcriptional activators activate HIV in primary CS cells?

Name Type CNS penetration

Potency(ACH2)

Panobinostat

HDACi (Pan) ? 52x

Romidepsin HDACi (Class I) -/+ 9xVorinostat HDACi (Pan) +++ 7xEntinostat HDACi (Class I) -/+ 53xHMBA Tat mimetic ++ 25xDisulfiram Akt signalling +++ 4xJQ-1 BRDi ++ 7xChaetocin HMTi ? 25x

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The majority of transcriptional activators are non-toxic in CNS cells

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Transcriptional activators are non-toxic in CNS cells

Name Type Resting T cells

MDM SVG PFA Activation conc.

Panobinostat HDACi (Pan) 2405 34734 3578 31992 60

Romidepsin HDACi (I) 1102 22908 2734 175 40

Vorinostat HDACi (Pan) >50000 >50000 1738 >50000 1000

Entinostat HDACi (I) 14385 >50000 >50000 ND 500

HMBA Tat mimetic 31438 >50000 >50000 ND 50000

Disulfiram Akt signalling 10847 >50000 >50000 >50000 10000

JQ-1 BRDi >50000 >50000 >50000 >50000 500

Chaetocin HMTi 14 1263 128 19 100

LD50 values (nM)

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Transcriptional activators are non-toxic in CNS cells

Name Type Resting T cells

MDM SVG PFA Activation conc.

Panobinostat HDACi (Pan) 2405 34734 3578 31992 60

Romidepsin HDACi (I) 1102 22908 2734 175 40

Vorinostat HDACi (Pan) >50000 >50000 1738 >50000 1000

Entinostat HDACi (I) 14385 >50000 >50000 ND 500

HMBA Tat mimetic 31438 >50000 >50000 ND 50000

Disulfiram Akt signalling 10847 >50000 >50000 >50000 10000

JQ-1 BRDi >50000 >50000 >50000 >50000 500

Chaetocin HMTi 14 1263 128 19 100

LD50 values (nM)

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KpnI

XbaI

env

gag/pol RRE

nef

nef

LuciferaseCMV

LTR U3

RU5

RU5

Ori

pGBFM.nefFluc

293T cells were co-transfected with pGBFM.nefFluc (lentiviral LTR-Luc vector), pVSV-G (VSV-G Env vector), pRSV-Rev (HIV-1 Rev vector), and pMDLg/pRRE (HIV-1 Gag, Pol vector) using lipofectamine 2000. Cells were media changed 6 hours later and virus stocks harvested 48 hours post-transfection

Generation of VSV-G pseudotyped lentiviral LTR-Luc virus:

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Transcriptional activators activate LTR in PFA and MDM

Romidepsin, JQ-1 and Panobinostat were the most potent PFA (11-, 9- and 7-fold, respectively) MDM, same trend but lower magnitude (4-, 3- and 2-fold, respectively)

Vorinostat, HMBA, Disulfiram, and Chaetocin showed minimal LTR activation

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There is strong data to suggest persistent HIV replication can occur in the CNS during suppressive cART

The CNS by nature presents unique and extensive challenges.

How do we treat activation in the CNS?What are the targets?Do antiretroviral drugs penetrate the CNS?Do antiretroviral function in CNS?Immune control?Biomarkers?

Assuming a CNS reservoir exists current ‘cure’ strategies are likely to result in replication in the CNS

Summary

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Tissue reservoirs are currently a major barrier to the eradication/cure of HIV-1

identification and characterisation of all viral reservoirs of HIV-1 including the CNS

Importantly‘Do cells within CNS cells, during suppressive cART,

contain a competent integrated HIV-1 genome?’

Summary

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Acknowledgements

Burnet InstituteHIV NeuropathogenesisLachlan GrayDaniel CowleyWan Jun ChengHung OnAnne ElletSteve WesselinghHIV Molecular PathogenesisPaul GorryMichael Roche

HIV Molecular InteractionsGilda Tachedjian

Alfred HospitalSharon Lewin, Hao LuMichael MosoFiona Wightman

Kirby InstituteStuart Turville

Peter Doherty InstituteDamian PurcellJonathan Jacobsen

Johns HopkinsJustin McArthurCarlos Pardo

St Vincents Hospital SydneyBruce BrewGilles Guillemin

Yale UniversitySerena Spudich

APP1051093APP1009533

NIHU19A1096109

R21 MH100954-02