Tissue Reservoirs
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Transcript of Tissue Reservoirs
Tissue Reservoirs
Melissa Churchill HIV Neuropathogenesis Laboratory, Centre for Biomedical Research, Burnet Institute.Departments of Medicine and Microbiology, Monash University
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
Types of tissue reservoirs
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
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
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.
Types of tissue reservoirs
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
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?
Are cells of the CNS infected with HIV and
contain an integrated pro-viral genome?
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
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).
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.
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+
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
Ongoing replication in the CNS in the
presence of cART
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
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
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
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
Ongoing replication in the CNS in the
presence of cART: Immune activation
and biomarkers
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)
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
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
Latency in the CNS
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)
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
Latency in the CNS: responsiveness to
transcriptional activators
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
The majority of transcriptional activators are non-toxic in CNS cells
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)
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)
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:
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
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
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
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