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Tissue Reservoirs

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

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Tissue Reservoirs

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

  2. Outline • 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.

  3. Types of tissue reservoirs

  4. GI tract is a significant viral reservoiron HAART • 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 Chun et al., J Infect Dis 2008; Yukl et al., J Infect Dis 2010

  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 tissueAnderson JA et al., 20th International AIDS Conference, LBPE05 Blood (leukapharesis) • Significant in the integrated HIV DNA in CD4+ T-cells in rectal tissue Vsblood 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 All patients on ART for >3 years

  6. Rectal tissue enriched with CCR6+ CXCR3+ memory CD4+ T-cells Rectal Tissue • 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. % CD3+ CD4+ CD45RA-

  7. Types of tissue reservoirs

  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

  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?

  10. Are cells of the CNS infected with HIV and contain an integrated pro-viral genome?

  11. CNS targets of HIV infection • 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)

  12. CNS cells contain integrated HIV proviral genomes 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. • (Churchill et al., 2006).

  13. 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. Churchill et al 2009, Annals of Neurology

  14. HIV DNA detected in CNS cells in pre-smptomatic HIV patients pre-ART GFAP CD68 • 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- HIV+ HIV+ Thompson et al, Am J Pathology, 2011.

  15. Independent and distinct evolution of HIV-1 occurs in the CNS compartment: evidence of infection and replication • 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)

  16. Ongoing replication in the CNS in the presence of cART

  17. Ongoing replication during cART: asymptomatic CSF viral persistence 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) 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. Slide courtesy of Steve Deeks

  18. 0.0005 0.02 Single genome sequencing demonstrates CSF HIV distinct from plasma during suppressive therapy Blood CSF Grey/black – pre-ART sequences Colors – on ART sequences: interval 1, 2, 3, 4 Subject 9058 Primary Infection Subject 7027 Chronic Infection * * * * * Trees rooted with pre-suppresion plasma sequence. *Significant bootstrap value Dahl et al., JID, 2014.

  19. 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 CSF WBC = 26 cells/ul* hand tremor, ataxia, slurred speech, aphasia CSF HIV RNA Lamivudine Abacavir Lopinavir/r Latest CD4 308 cells/ul * Normal CSF WBC < 5 cells/ul Peluso et al., AIDS, 2012. Also: Canestri et al., Clin Infect Diseases, 2010. Slide courtesy of A/Prof Serena Spudich

  20. Indirect evidence of ongoing replication during cART: symptomatic CSF ‘escape’ 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 Slide courtesy of A/Prof Serena Spudich

  21. Ongoing replication in the CNS in the presence of cART: Immune activation and biomarkers

  22. Ongoing replication in CNS during cART: Biomarkers (NFL) 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

  23. Indirect evidence of ongoing replication during cART: ongoing immune activation Serum Neopterin 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 CSF Neopterin nmol/L 60 60 50 50 40 40 Baseline >4yrs treatment 30 30 CSF Neopterin Neopterin (nmol/l) 20 20 10 10 10 10 Baseline >4yrs treatment nmol/L CSF HIV-1 RNA (copies/ml) • Hagberget al., AIDS Res and therapy 2010, Eden et al., JID 2007. Yilmezetal., 2008 JAIDS

  24. Persistent CNS immune activation on ART / HIV- 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. Garvey et al, AIDS, 2014. Anthony et al, J NeuropatholExpNeurol, 2005 Yilmaz et al., J Acquir Immune DeficSyndr, 2008 Courtesy of Serena Spudich

  25. Latency in the CNS

  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;, Desplatset al., 2013 Neurology)

  27. Latency in the CNS: animal models Day 0 12 175 • 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 IV SIV Innoculation Examination • Treatment • no ART • saquinavir/atazanavir, integrase inhibitor • atazanavir, integrase inhibitor Zink et al, 2010, JID

  28. Latency in the CNS: responsiveness to transcriptional activators

  29. Do transcriptional activators activate HIV in primary CS cells?

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

  31. Transcriptional activators are non-toxic in CNS cells LD50 values (nM)

  32. Transcriptional activators are non-toxic in CNS cells LD50 values (nM)

  33. Generation of VSV-G pseudotypedlentiviral LTR-Luc virus: RRE gag/pol nef U5 env R Luciferase CMV KpnI nef pGBFM.nefFluc Ori LTR U3 R U5 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 XbaI

  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

  35. Summary • There is strong data to suggest persistentHIV 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

  36. 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?’

  37. Acknowledgements 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 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 R21 MH100954-02 NIHU19A1096109 APP1051093 APP1009533

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