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Tissue Reservoirs Melissa Churchill HIV Neuropathogenesis Laboratory, Centre for Biomedical Research, Burnet Institute. Departments of Medicine and Microbiology, Monash University Slide 2 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 18 th AMREP. Outline Slide 3 Types of tissue reservoirs Slide 4 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, VLSlide 5 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 tissue Anderson 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 Slide 6 Rectal tissue enriched with CCR6+ CXCR3+ memory CD4+ T-cells Rectal Tissue % CD3+ CD4+ CD45RA- 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. Slide 7 Types of tissue reservoirs Slide 8 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 Slide 9 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? Slide 10 Are cells of the CNS infected with HIV and contain an integrated pro-viral genome? Slide 11 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 Slide 12 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). Slide 13 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 GAPDH HIV-1 env sequences detected in astrocytes and macrophages. Slide 14 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 GFAPCD68 HIV- HIV+ Slide 15 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 Slide 16 Ongoing replication in the CNS in the presence of cART Slide 17 Ongoing replication during cART: asymptomatic CSF viral persistence Eden et al., JID 2010 In patients on suppressive cART (Plasma VL50cps/ml) in CSF using a standard assay Letendre at al., (CROI 2009) 300 patients with suppressed plasma VL ( Slide 18 Single genome sequencing demonstrates CSF HIV distinct from plasma during suppressive therapy Dahl et al., JID, 2014. Blood CSF 0.0005 0.02 * * * * * Trees rooted with pre-suppresion plasma sequence. *Significant bootstrap value Grey/black pre-ART sequences Colors on ART sequences: interval 1, 2, 3, 4 Subject 7027 Chronic Infection Subject 9058 Primary Infection Slide 19 Indirect evidence of ongoing replication during cART: symptomatic CSF escape Patient 2000 Plasma VLSlide 20 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 Slide 21 Ongoing replication in the CNS in the presence of cART: Immune activation and biomarkers Slide 22 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) Slide 23 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 treatment Baseline 10 20 10 30 40 50 60 nmol/L Serum Neopterin 10 20 10 30 40 50 60 >4yrs treatment Baseline nmol/L CSF Neopterin Neopterin (nmol/l) CSF HIV-1 RNA (copies/ml) CSF Neopterin Slide 24 Garvey et al, AIDS, 2014. Anthony et al, J Neuropathol Exp Neurol, 2005 Yilmaz 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 Slide 25 Latency in the CNS Slide 26 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) Slide 27 Latency in the CNS: animal models IV SIV Innoculation 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 Slide 28 Latency in the CNS: responsiveness to transcriptional activators Slide 29 Do transcriptional activators activate HIV in primary CS cells? NameTypeCNS penetration Potenc y (ACH2) PanobinostatHDACi (Pan)?52x RomidepsinHDACi (Class I) -/+9x VorinostatHDACi (Pan)+++7x EntinostatHDACi (Class I) -/+53x HMBATat mimetic++25x DisulfiramAkt signalling+++4x JQ-1BRDi++7x ChaetocinHMTi?25x Slide 30 The majority of transcriptional activators are non-toxic in CNS cells Slide 31 Transcriptional activators are non-toxic in CNS cells NameTypeResting T cells MDMSVGPFAActivation conc. PanobinostatHDACi (Pan)24053473435783199260 RomidepsinHDACi (I)110222908273417540 VorinostatHDACi (Pan)>50000 1738>500001000 EntinostatHDACi (I)14385>50000 ND500 HMBATat mimetic31438>50000 ND50000 DisulfiramAkt signalling10847>50000 10000 JQ-1BRDi>50000 500 ChaetocinHMTi14126312819100 LD 50 values (nM) Slide 32 Transcriptional activators are non-toxic in CNS cells NameTypeResting T cells MDMSVGPFAActivation conc. PanobinostatHDACi (Pan)24053473435783199260 RomidepsinHDACi (I)110222908273417540 VorinostatHDACi (Pan)>50000 1738>500001000 EntinostatHDACi (I)14385>50000 ND500 HMBATat mimetic31438>50000 ND50000 DisulfiramAkt signalling10847>50000 10000 JQ-1BRDi>50000 500 ChaetocinHMTi14126312819100 LD 50 values (nM) Slide 33 KpnI XbaI env gag/pol RRE nef Luciferase CMV LTR U3 R U5 R 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: Slide 34 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 Slide 35 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 Slide 36 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 Slide 37 Acknowledgements Burnet Institute HIV Neuropathogenesis Lachlan Gray Daniel Cowley Wan Jun Cheng Hung On Anne Ellet Steve Wesselingh HIV Molecular Pathogenesis Paul Gorry Michael Roche HIV Molecular Interactions Gilda Tachedjian Alfred Hospital Sharon Lewin, Hao Lu Michael Moso Fiona Wightman Kirby Institute Stuart Turville Peter Doherty Institute Damian Purcell Jonathan Jacobsen Johns Hopkins Justin McArthur Carlos Pardo St Vincents Hospital Sydney Bruce Brew Gilles Guillemin Yale University Serena Spudich APP1051093 APP1009533 NIHU19A1096109 R21 MH100954-02