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Topic review HIV eradication
 

Topic review HIV eradication

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focus on CCR5 knockout strategies using Zinc-finger nucleases

focus on CCR5 knockout strategies using Zinc-finger nucleases

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    Topic review HIV eradication Topic review HIV eradication Presentation Transcript

    • HIV cure and eradication Rationales and Options Pana Tanjararak, MD
    • Introduction
      • Despite significant reduction in morbidity and mortality following combination antiretroviral therapy (cART) cannot eradicate HIV
      • Long lived latently infected cells and residual viral replication
      • Anatomical reservoirs
        • GI tract
        • Lymphoid tissue
        • Central nervous system
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Why do we need a cure for HIV?
      • Full life expectancy not been restored
      • In a prospective study of 3,990 HIV-infected in Denmark,
        • HIV patient reaching the age of 70 was 50% that of uninfected population controls
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • Incidence morbidity also elevated despite successful cART due to complex interactions
        • Drug toxicity
        • Persistent inflammation
        • Risk behaviours
      • Financial resource to support life long Rx
      Why do we need a cure for HIV? Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Functional or sterilizing cure
      • 2 potential strategies for cure
      • Infectious diseases model
        • Elimination of all HIV infected cells in all compartments and sanctuaries and plasma HIV RNA < 1 copy/ml (Sterilizing cure)
      • Cancer model
        • Long term health in the absence of treatment, with low level viremia < 50 copies/ml. (Functional cure)
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Sterilizing cure: Elimination of HIV following BMT
      • The recent case report of a German patient with AML
        • BMT from a donor who carried a 32 base pair deletion in the CCR5 gene
        • The only current example of a sterilizing cure
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • Stop cART
      • HIV RNA remained at <1 copy/ml.
      • No HIV-DNA or HIV-RNA were detected
        • Multiple biopsies of GI tract
        • Brain biopsy
        • Analysis of CSF
        • Bone marrow
      Sterilizing cure: Elimination of HIV following BMT Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • > 45 months post stopping cART and HIV is still not detected
      • Reconstitution of circulating and mucosal CD4+ T-cells that did not express CCR5
      • Mutant CCR5+ mucosal macrophages were detected early post transplantation in the GI tract but at later timepoints,
      Sterilizing cure: Elimination of HIV following BMT Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Sterilizing cure: Elimination of HIV following BMT
      • The patient’s PBMC were permissive to CXCR4, patients CD4+ T-cells were not resistant to HIV
      • Potential factors leading to the elimination of long lived reservoirs in this patients (Cells that express CCR5)
        • Specific chemotherapy
        • Total body irradiation
        • Low grade graft versus host disease  eliminating the capacity for any residual replication by removing target cells that express CCR5.
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • Strategy of using BMT with a CCR5 mutant donor not a realistic cure for HIV given the toxicity of the treatment
      • Need comprehensively study to fully understand how and why HIV was eliminated
      Sterilizing cure: Elimination of HIV following BMT Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Functional cure: elite controllers (EC)
      • Long term HIV-RNA < 50 copies/ml (absence of Rx)
      • Role of genetics, virus and immune response in EC
      • HLA Class 1 genes
      • Effective cytolytic CD8+T-cell response in blood which has been associated with enhanced activity of the T-box transcription factor t-bet
      • Increased production of IL-21
      • Strong HIV specific CD4+ and CD8+ T-cell responses were also identified in mucosal tissue from EC
      • The innate immune system may also be important with enhanced activity of myeloid dendritic cells
      • Effective immune response, perhaps via vaccination, may be one strategy to achieve a functional cure.
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Functional cure: elite controllers (EC)
      • Some elite controllers do not bear the protective alleles HLA B27/ HLA B57
      • Mechanisms other than enhanced T-cell immunity have also been explored
      • No evidence currently that activated CD4+ T-cells from EC patients resistant to HIV
      • In contrast to Pts on cART with HIV RNA <50 copies/ml, there is evolution in HIV RNA sequences in EC in approximately 7% of EC, CD4+ T-cells decline over time
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • EC potentially be the best candidates to test strategies  sterilizing cure
        • Low total number of infected cells
        • Robust HIV-specific immune responses
      Functional cure: elite controllers (EC) Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Measuring latently infected cells and the ‘‘reservoir’’ in vivo
      • The major reason why HIV cannot be cured is the persistence of HIV in a latent form in different cellular and anatomical reservoirs.
      • HIV latency occurs in resting CD4+ T-cells either as pre-integration or post-integration latency
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Measuring latently infected cells and the ‘‘reservoir’’ in vivo
      • The major reservoir of cells that harbour post-integration latency in vivo are resting
        • Central memory CD4+ T-cells
        • (CD45RA- CCR7+ CD27+)
        • Transitional memory CD4+ T-cells
        • (CD45RA- CCR7- CD27+)
      • Latent infection can established in other long lived cells including
        • Naive T-cells
        • Bone marrow progenitor cells
        • Thymocytes
        • Astrocytes
        • Other cells such as monocyte/macrophages can support long lived low level productive infection
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • Latently infected cells can be detected in both blood and tissue, including
        • GI tract
        • Genital tract
        • CNS
      • When activated, latently infected T-cells can release viral particles or became productively infected T-cells
      • When treatment is stopped  viral rebound
      Measuring latently infected cells and the ‘‘reservoir’’ in vivo Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Replication competent virus – cell associated
      • The gold standard used to measure the frequency of resting CD4+ T cells carrying latent but replication competent virus is based on co-culture of
        • Highly purified resting CD4+ T cells from Pts
        • PBMC from an HIV negative donor
      • The major limitation of using this technique in large multi-site clinical trials is
        • The need for large blood volume
        • Often requiring leukapheresis
        • The assay is labor intensive, has a wide coefficient of variation and cannot be performed with tissue biopsies
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • HIV-DNA – unintegrated, integrated and circular
      • Within infected cells HIV-DNA can exist as
        • Linear nonintegrated forms
        • Circular forms
        • Integrated provirus (in effective cART)
      • used technique to quantify the number of cells that contain integrated virus is called Alu-LTR PCR
      • To improve sensitivity of this assay, many published methods use nested PCR, with the second round of amplification having both primers within the HIV LTR
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • HIV-DNA – unintegrated, integrated and circular
      • One popular and widely used technique to quantify the number of cells that contain integrated virus is called Alu-LTR PCR
      • To improve sensitivity of this assay, many published methods use nested PCR , with the second round of amplification having both primers within the HIV LTR
      • This approach increases sensitivity but also increases the complexity of the assay and multiple replicates and a large number of controls are needed to enhance the accuracy and reproducibility of this assay
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • HIV-DNA – unintegrated, integrated and circular
      • The quantification of 2-LTR circles that are episomal forms of nonintegrated HIV-DNA containing two copies of the long terminal repeat is also a useful tool to quantify residual viral replication in patients on cART.
      • 2-LTR circles are produced following ongoing infection of a cell and have a relatively short half life, therefore detection of 2-LTR circles is generally considered to be a surrogate marker of ongoing viral replication, rather than a marker of the number of residual infected cells
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • HIV-DNA – unintegrated, integrated and circular
      • Total HIV-DNA quantifies integrated, and nonintegrated DNA as well as latent and defective virus.
      • There is a strong correlation between total HIV-DNA and integrated HIV-DNA in patients on cART and therefore cell associated HIV-DNA is likely to be a good surrogate marker of the total number of latently infected cells
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • In a recent small study, 3 of 7 patients examined had an excess of unintegrated DNA compared with integrated DNA suggesting that total HIV-DNA may not be an ideal way to quantify the reservoir in all patients .
      • Quantification of total cell associated HIV DNA is the most feasible tool to evaluate the frequency of infected cells in large scale clinical trials and cohorts.
      HIV-DNA – unintegrated, integrated and circular Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • HIV RNA - plasma
      • The quantification of low level viremia using an ultrasensitive PCR-based assay that can measure down to 1 copy/ml
      • Using this assay, over 80% of patients on cART have detectable viremia at around 3–5 copies/ml
      • There are several limitations to this assay.
        • The assay requires a large volume of plasma (7 ml)
        • In 10% of patients, amplification of the viral sequence is not possible
        • It is currently unclear whether this low level virus is indeed replication competent and how this virus is related to rebound virus following treatment cessation.
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • HIV RNA - plasma
      • When HIV RNA is < 50 copies/ml the sequence of plasma virus over time is very stable and shares little homology with the sequence of HIV derived from resting CD4+ T-cells or monocytes
      • Further work is needed to understand the source of low level viremia and whether this virus contributes to viral rebound.
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • HIV RNA – cell associated
      • Measurement of cell associated HIV RNA, includes quantification of extracellular or cell associated unspliced (US) and multiply spliced (MS) RNA
      • In latently infected cells, one would expect no extracellular HIV RNA if ongoing viral replication has been blocked
      • However, ongoing production of virus from latently infected cells will produce both US and MS RNA.
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • HIV RNA – cell associated
      • Following cART in patients with chronic infection, extracellular HIV RNA rapidly declines while US RNA persists in approximately 70–80% of patients
      • MS-RNA is critical for the production of tat, nef and rev , which are all required for efficient production of full length US RNA
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • HIV RNA – cell associated
      • In latently infected cells, there is a block in nuclear export of MS RNA and inefficient production of US RNA
      • Therefore, detection of specific types of MS RNA differs in patients with productive infection and following suppressive cART
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Blood or tissue?
      • The highest concentration of HIV DNA and cell associated US HIV RNA in patients on cART is found in tissues such as lymphoid or GI tract tissue
      • HIV DNA and RNA in the GI tract 10 times that in blood in patients on cART
      • In a recent study of anatomical reservoirs in RT-SHIV infected macaques on cART
      • The largest pool of infected cells (measured by cell associated HIV-DNA and US RNA)
        • Lymphoid tissue (including spleen and lymph node)
        • GI tract
      • Minimal residual infected cells were detected in the
        • CNS
        • Reproductive tract
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Blood or tissue?
      • It is likely that factors that maintain and or allow for the establishment of latency may differ in blood and tissues
      • Therefore it is critical that for new interventions aimed at eradication, if at all possible, quantification of latently and productively infected cells, should include tissue as well as blood
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Current and future strategies: advantages, disadvantages
      • Treatment intensification
        • There have been a number of studies that have looked at the effect of treatment intensification on residual virus, in patients receiving cART.
        • These studies have included the addition of agents to an already suppressive regimen
          • Enfuvirtide
          • Protease inhibitors (ritonavir boosted atazanavir or lopinavir)
          • Raltegravir
        • No study demonstrated decline in low-level viremia IUPM or cell associated HIV DNA
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • In addition, 2 small non randomised studies showed no significant decline in residual virus infection in the GI tract following intensification with raltegravir or in the CSF following intensification with maraviroc, lopinavir/ritonavir or enfurvitide
      Current and future strategies: advantages, disadvantages Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • A recent small, nonrandomised intensification study with maraviroc, a CCR5 antagonist
        • This is the only intervention to date that has shown a decrease in the number of latently infected cells (measured by IUPM)
        • The mechanism for how maraviroc may be working is unclear
      Current and future strategies: advantages, disadvantages Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • Early treatment
        • Cell associated HIV DNA and HIV US RNA, decreases to a significantly lower level
        • In a recent longitudinal study
          • Patients who initiated cART during very early acute infection and stayed on cART for a prolonged period
          • 5/32 (16%) patients following cessation of cART, HIV RNA was maintained at < 50 copies/ml for a median of 77 months off cART
      Current and future strategies: advantages, disadvantages Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • Early treatment
        • Contrast to many other reports of viral rebound in nearly all patients following cessation of cART, even when initiated during acute infection
        • Role of very early treatment requires further investigation
      Current and future strategies: advantages, disadvantages Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • Elimination of latently infected T-cells via induction of virus production
      • Induce virus production from latently infected cells  productively infected cell die
        • JAK-STAT pathway
        • Activation of NF-kB: prostratin
        • Enhance histone acetylation: histone deacetylase inhibitors (HDACi)
        • Inhibit DNA methylation: methylation inhibitors
        • Combination strategies
      Current and future strategies: advantages, disadvantages Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • JAK-STAT pathway
        • IL-7 is a cytokine that can effectively induce productive infection from latently infected resting CD4+T-cells
        • IL-7 is currently undergoing clinical trials (ERAMUNE)
      Current and future strategies: advantages, disadvantages Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • Activation of NF-kB: prostratin
        • Promote T-cell activation and HIV transcription in vitro
        • The large diversity of latently infected T-cell subsets may differ in their capacity to proliferate, and/or uptake of these drugs
        • Not yet been assessed for safety and toxicity
      Current and future strategies: advantages, disadvantages Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • Enhance histone acetylation: histone deacetylase inhibitors (HDACi)
        • latency can be reversed
        • Viral production can be activated by promoting histone acetylation
        • Modify gene expression by changing the acetylation state of histones, leading to enhanced transcription from multiple genes including from the HIV LTR
      Current and future strategies: advantages, disadvantages Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • Enhance histone acetylation: histone deacetylase inhibitors (HDACi)
        • In cancer cells, HDACi induce cell death and cell cycle arrest of rapidly dividing malignant cells and many HDACi are now in advanced clinical development for the treatment of different cancers
        • HDACi treatment of latently infected cells lines
          • valproic acid
          • MCT1
          • MCT3
          • Oxamflatin
          • Vorinostat
          • panobinostat
        • Preferential apoptosis in cells producing virus
      Current and future strategies: advantages, disadvantages Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • Enhance histone acetylation: histone deacetylase inhibitors (HDACi)
        • A theoretical risk of HDACi is that they will induce activation of other retroviruses and/or DNA viruses
          • including CMV, HBV and JC viruses
      Current and future strategies: advantages, disadvantages Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Current and future strategies: advantages, disadvantages
      • Inhibit DNA methylation: methylation inhibitors
        • The methylation inhibitor 5-aza-deoxycytadine (decitabine) is a nucleoside analogue that promotes DNA cytosine methylation
        • Rx of MDS
        • Similar effect to HDACi in promoting HIV transcription in vitro (only in a subset of latently infected resting CD4+T-cells)
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Current and future strategies: advantages, disadvantages
      • Inhibit DNA methylation: methylation inhibitors
        • Potency was greatest when used in combination with other drugs such as prostratin and an HDACi
        • Histone methyltransferase inhibitors (HMTis)
          • Also active in latently infected cells
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Current and future strategies: advantages, disadvantages
      • Combination strategies
        • a combination of strategies, for example SAHA + prostratin appears to have greatest potency in promoting HIV transcription, at least in vitro
        • Most of the studies that evaluate a combination approach have been performed in latently infected T-cell and monocytic cells lines and it is currently unknown whether this approach will also enhance potency in latently infected primary T-cells
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Boosting immunity to HIV: therapeutic vaccination
      • Induction of T-cell immunity via vaccination may potentially generate a functional cure
      • Therapeutic vaccination in patients receiving cART has not been successful
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • In one of these studies
        • treatment interruption resulted in a significantly shorter time to viral rebound following therapeutic vaccination compared with placebo
      Boosting immunity to HIV: therapeutic vaccination Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • The animals who received the polyICLC vaccine had significantly elevated titres of neutralising antibodies compared to the control groups suggesting that the generation of neutralising antibodies may be important in preventing viral rebound following cessation of cART
      Boosting immunity to HIV: therapeutic vaccination Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Making cells resistant to HIV
      • Mimic HIV eradication in the HIV-infected German patient include gene therapy to reduce expression of CCR5
      • Successfully perform in mice through the introduction of a zinc finger nuclease into hematopoietic progenitor cells, which effectively disrupts the gene coding for CCR5 in all daughter cells
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Making cells resistant to HIV
      • Reduction in the expression of CCR5 in a subset of transplanted cells.
      • Following HIV infection of these mice, there was a selective advantage for the CCR5-/- cells which subsequently increased, HIV RNA remained low and CD4+ T-cells were preserved in both blood and tissue
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Making cells resistant to HIV
      • A recent phase 2 study
        • Demonstrated that infusion of autologous T-cells transduced with a zinc finger nuclease was safe in a small study of 6 HIV-infected patients on cART
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Making cells resistant to HIV
      • An alternative approach is to use RNA-based gene therapy to reduce CCR5 expression, as well as specifically inhibit HIV replication
      • The investigators demonstrated that this procedure was safe and that the transduced genes persisted in a subset of cells for 24 months
      • Although wide spread use of these therapies is many years away, these results are encouraging for the possible development of a gene therapy based treatment strategy that may achieve a functional cure.
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Moving toward clinical trials to test for eradication
      • Animal models
        • Trialed in macaques to determine efficacy
        • Limited number of antiretroviral agents that are active to control of SIV RNA to <50 copies/ml for a prolonged period of time.
        • High cost
        • Finally most detailed analyses of factors that modify HIV transcription have been performed in vitro models of latent HIV infection in human cells and it is unclear whether this can be translated to SIV infection.
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • Recent work has however demonstrated that infection of rhesus macaques with SIVmac239 that contains the HIV- 1 RT from clone HXBc2 (RT-SHIV) treated with tenofovir, emtricitabine and efavirenz appears to be a very promising model of suppressive cART
      Moving toward clinical trials to test for eradication Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • These animals had detectable but low level viremia of 2-58 copies/ml consistent with suppressive cART in humans
      • Blood-liver-thymus (BLT) mouse that can be efficiently infected with R5 HIV
        • HIV RNA declines in response to ART
      Moving toward clinical trials to test for eradication Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Clinical trial
      • First
        • Measuring the reservoir is complex
        • Require large volumes of cells
        • Invasive
        • No assays have yet been standardised across multiple laboratories
        • Unknown what assay will best predict the likelihood of viral rebound following cessation of cART
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Clinical trial
      • Second
        • Most studies small, non-randomised studies
        • Need for larger randomised studies
      • Third
        • Careful consideration of the risk benefit
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
      • Finally
        • There remains a need for more multidisciplinary studies including the use of mathematical models to study the dynamics of reservoirs taking in account half-life of the subsets of infected cells and their homeostatic proliferation
      Clinical trial Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • Conclusion
      • There are multiple barriers to the eradication of HIV infection and despite some recent significant advances in vitro models of latency
      • Better animal models
      • Identification of several compounds that can reverse latency in vitro
      • There is still a need for more research
      Sharon R Lewin and Christine Rouzioux. AIDS 2011; 25: 000-000. Infectious diseases unit, The Alfred, Monash University Melbourne, Australia. Laboratory of virology, Hopital Necker, Universite Paris Descartes, Paris, France.
    • HIV Cure and Eradication Chemokine Receptor 5 Knockout Strategies Using Zinc-finger Nuclease Rongpong Plongla, MD
      • Typically 110 nm in diameter
      • containing two single strands of RNA
      • Viral genome 10 kb
      Marvin S Reitz Jr., and Robert C Gallo. In Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, 7th ed. 2009
    • The Life Cycle of Human Immunodeficiency Virus Type 1 Marvin S Reitz Jr and Robert C Gallo. In Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, 7th ed. 2009 Joan stephenson. JAMA. 2002;287(13):1635-1637 .
    • Mechanism of HIV entry Jose’ A Este’ , Amalio Telenti. Lancet 2007; 370: 81–88 Retrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autnoma deBarcelona, Badalona, Spain (J A Este PhD) and Institute of Microbiology, University Hospital Centre, University of Lausanne, Lausanne, Switzerland (A Telenti MD)
      • Glycoprotein 120, the viral-envelope protein, binds to the CD4 molecule on susceptible cells, but cell entry requires the presence of a coreceptor.
      • The coreceptor for macrophage-tropic non–syncytia-forming strains is CCR5 , a surface chemokine receptor.
        • Such viruses have recently been renamed R5 viruses to reflect their coreceptor requirement
        • whereas T-cell–tropic, syncytia-inducing viruses, which require CXCR4 for entry, are termed X4 viruses.
      • Langerhans' cells, tissue dendritic cells-- the earliest target of the virus, express CCR5 but may not express CXCR4, the coreceptor required for the entry of X4 viral isolates.
      James O. Kahn, M.D., and Bruce D. Walker, M.D. N Engl J Med 1998; 339:33-39 From the AIDS Program, San Francisco General Hospital and the University of California, San Francisco (J.O.K.), and Partners AIDS Research Center, Massachusetts General Hospital and Harvard Medical School, Boston (B.D.W.)
      • This may explain why R5 viruses are the predominant strains transmitted during acute HIV-1 infection.
      • This receptor pattern also explains why persons who are homozygous for a 32-bp deletion in CCR5 (CCR5Δ32) are relatively resistant to infection with the usual R5 strains, although rare cases of transmission of X4 viruses have recently been reported in such persons.
      • Irrespective of the route of transmission, R5 viruses are preferentially transmitted over X4 viruses, and acute infection is almost exclusively associated with R5 strains.
        • The basis of this preferential selection is unclear, but many barriers to infection by X4 viruses have been proposed.
        • R5 viruses also predominate during most of the chronic stage of the disease.
      • X4 variants eventually emerge in 40–60% of HIV-1-positive individuals.
        • X4 variants have been associated with expanded cell tropism, an increased virus replication rate, faster disease progression, and the onset of AIDS.
        • Whether the emergence of X4 phenotype is a cause or consequence of HIV disease progression remains controversial.
      Jose’ A Este’ , Amalio Telenti. Lancet 2007; 370: 81–88 Retrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autnoma deBarcelona, Badalona, Spain (J A Este PhD) and Institute of Microbiology, University Hospital Centre, University of Lausanne, Lausanne, Switzerland (A Telenti MD)
    • HIV Tropism and Disease Progression Kuhmann SE, et al. J Viral Entry. 2005;1:4-16. Moore JP, et al. AIDS Res Hum Retroviruses. 2004;20:111-126. Time Since Seroconversion HIV-1 RNA Level Current assays’ X4 detection limit Weeks Years R5 virus X4 virus R5 Infection R5 + X4 Infection
    • Jose’ A Este’ , Amalio Telenti. Lancet 2007; 370: 81–88 Retrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autnoma deBarcelona, Badalona, Spain (J A Este PhD) and Institute of Microbiology, University Hospital Centre, University of Lausanne, Lausanne, Switzerland (A Telenti MD)
      • R5 HIV-1 strains are commonly
      • transmitted and persist during the course of infection but might evolve to dual tropic (D) or mixed (M) virus populations, and to X4 phenotype at late stages of disease.
      • Of treatment-naive individuals
      • 12–19% have D/M viral populations
      • < 1% are infected with X4 viruses.
      • Of treatment-experienced
      • 22–48% have D/M viral populations
      • 2–4% are infected with X4 viruses.
    • CCR5 Wild Type and CCR5  32 CCR5 wild type   CCR5  32 2 normal copies 1 copy of  32 2 copies of  32 wt/wt wt/  32  32 /  32 Standard disease progression Delayed disease progression “ Resistant” to HIV infection Normal Heterozygous Homozygous Liu R, et al. Cell. 1996;86:367-367. Samson M, et al. Nature. 1996;382:722-725. Dean M, et al. Science. 1996;273:1856-1862. Huang Y, et al. Nat Med.1996;2:1240-1243. Michael NL, et al. Nat Med. 1997;3:1160-1162. Eugen-Olsen J, et al. AIDS. 1997;11:305-310.
    • Patients Homozygous or Heterozygous for CCR5  32
      • Homozygous
        • ~ 1% of white population [1]
        • Lack CCR5 molecules on CD4+ cell surface [2,3]
          • Resistant to R5 viruses: natural resistance to HIV acquisition
          • Susceptible to infection with X4 virus
        • Relatively normal immune function
      • Heterozygous
        • 10% to 15% of white population [1]
        • Fewer CCR5 molecules on CD4+ cell surface [4]
        • Normal immune function [2]
      1. McNicholl JM, et al. Emerg Infect Dis. 1997;3:261-271. 2. Liu R, et al. Cell. 1996;86:367-367. 3. Samson M, et al. Nature. 1996;382:722-725. 4. Wu L, et al. J Exp Med. 1997;185:1681-1691.
    •  
    • < 6hr 1 day
      • The first phase involves the rapid decay of the cells that produce the vast majority of the plasma virus.
      • These appear to be activated CD4+ T cells.
      • In the productively infected state, these cells have a very short half-life, surviving only about 1 day before dying from viral cytopathic effects or host cytolytic effector mechanisms.
      • a second, slower phase of decay.
      • This reflects the turnover of a minor population of virus producing cells.
      • These may be infected macrophages, which are more resistant to the cytopathic effects of infection or CD4+ T cells that are in a state of partial activation.
      • These cells turn over with a half-life of about 2 weeks.
      14 day
    • J Allergy Clin Immunol 2008; 122: 22-8 • Latently infected resting memory CD4+ T cells are the best characterized latent reservoir for HIV-1. • Less than 1 cell per 1,000,000 resting CD4+ T cells from patients on HAART harbor latent HIV-1 provirus. • Sequence of latent proviruses does not evolve, which suggests no ongoing viral replication. • Discontinuation of HAART allows viral relapse from latent reservoir. • Patients successfully treated with HAART for longer than 10 years exhibit no appreciable decrease in the size of the latent reservoir. • The persistence of latently infected memory CD4+ T lymphocytes precludes their elimination by HAART alone for the lifetime of the patient. • Other drug-insensitive reservoirs, including brain, macrophages, and hematopoietic stem cells, may also exist. • Latency is likely established and maintained by numerous blocks at multiple steps in the HIV-1 replicative pathway, which potentially complicates eradication strategies. Douglas D. Richman.* David M. Margolis, Martin Delaney, Warner C. Greene, Daria Hazuda, Roger J. Pomerantz. Science 6 March 2009: Vol. 323 no. 5919 pp. 1304-1307 . University of California San Diego
    • Can We Do Better Than HAART?
    •  
    • The Berlin Patients
      • A 40-year-old white man with newly diagnosed acute myeloid leukemia (FAB M4 subtype, with normal cytogenetic features)
      • HIV-1 infection > 10 years
        • TDF/FTC/EFV for the previous 4 year
        • CD4 T-cell count was 415 per cubic millimeter, and HIV-1 RNA was not detectable
        • Web position specific scoring matrix (WebPSSM), and geno2pheno bioinformatic software: not found X4 variant
        • The ultradeep sequencinganalysis revealed a proportion of 2.9% for the X4 and dual-tropic variants combined.
      N Engl J Med 2009;360:692-8.
    • allogeneic stem-cell transplantation with CD34+ peripheral-blood stem cells from an HLA-identical donor who had been screened for homozygosity for the CCR5 delta32 allele.
      • no active, replicating HIV could be detected 20 months after HAART had been discontinued
      • HIV-1 virus could not be detected in peripheral blood, bone marrow, or rectal mucosa, as assessed with RNA and proviral DNA PCR assays.
      N Engl J Med 2009;360:692-8. Cobas AmpliPrep–TaqMan HIV assay system (Roche).
      • The loss of anti-HIV, virus-specific, interferon- γ–producing T-cells during follow-up suggests that HIV antigen stimulation was not present after transplantation.
      N Engl J Med 2009;360:692-8. + - D14 D625 Immunosuppressive treatment has been stopped 38 months
      • demonstrate successful CD4 T-cell reconstitution at the systemic level as well as in the largest immunologic organ after CCR5  32/  32 SCT
      Blood 2011; 117: 2791-99. effector memory cells (EM), central memory cells (CM), recent thymic emigrants (RTE), and central naïve cells (CN)
      • and in addition provide evidence for the reduction in the size of the potential HIV reservoir over time : Brain, Liver, Colon
      • Although the recovered CD4 T cells are susceptible to infection with X4 HIV, the patient remains without any evidence of HIV infection for more than 3.5 years after discontinuation of ART.
      • Immunoblot analysis revealed a continuing decline of HIV specific antibodies thereafter demonstrating the process of serodeconversion:
        • HIV core-directed antibodies (p17, p24) disappeared completely
        • the serum level of antibodies against the HIV envelope (gp41, gp120) further decreased.
      Blood 2011; 117: 2791-99.
    • Paula Cannon, CROI2011
    • CCR5 knockout strategies
      • However, the risks associated with allogeneic transplantation and the impracticality of obtaining sufficient numbers of matched CCR5Δ32 donors mean that broader application of this approach will require methods for generating autologous CCR5−/− cells.
      • Various gene therapy approaches to block CCR5 expression are being evaluated CCR5 -specific
        • Ribozymes, siRNAs, Intrabodies
        • The targeted cell populations include both mature T cells and CD34+ HSPCs.
        • Loss of CCR5 in HSPCs appears to have no adverse effects on hematopoiesis
      Holt N, etal. Nat Biotechnol. 2010 Aug;28(8):839-47.
    • An alternative approach: the use of engineered ZFNs to permanently disrupt the CCR5 open reading frame .
      • A significant advantage of this approach is that permanent gene disruption can result from only transient ZFN expression.
      • disruption of CCR5 in HSPCs is likely to provide a more durable anti-viral effect and to give rise to CCR5−/− cells in both the lymphoid and myeloid compartments that HIV-1 infects
      Holt N, etal. Nat Biotechnol. 2010 Aug;28(8):839-47.
    • ZFNs: designer restriction enzymes
      • Contain 2 linked domain
        • DNA-binding zinc finger protein
          • Artificial array of zinc finger peptides confer sequence-specific DNA-binding properties
          • a string of ‘zinc-finger motifs’, each a stretch of around 30 amino acids, stabilized by a zinc ion, that binds to a particular three base
      • DNA sequence.
        • DNA-cleaving domain
          • Endonuclease domain of type 1 restriction enzyme FokI
          • Not specific
          • must dimerize to achieve efficient DNA cleavage
      Paula Cannon and Carl June. Current Opinion in HIV and AIDS 2011; 6: 74-79. Molecular Microbiology & Immunology, University ofSouthern California Keck School of Medicine, Los Angeles, California and Department of Pathology andLaboratory Medicine, University of PennsylvaniaSchool of Medicine, Philadelphia, Pennsylvania, USA Flavobacterium okeanokoites
    • Flavobacterium okeanokoites
    • Double-strand break
    • Fyodor D. Urnov 1 , Edward J. Rebar 1 , Michael C. Holmes 1 , H. Steve Zhang 1 & Philip D. Gregory Nature Reviews Genetics 11, 636-646 (September 2010)
    • Paula Cannon and Carl June. Current Opinion in HIV and AIDS 2011; 6: 74-79. Molecular Microbiology & Immunology, University ofSouthern California Keck School of Medicine, Los Angeles, California and Department of Pathology andLaboratory Medicine, University of PennsylvaniaSchool of Medicine, Philadelphia, Pennsylvania, USA
    • Paula Cannon, CROI2011
    • Pre-clinical testing using humanized mice
      • optimized the delivery of CCR5- specific ZFNs to human CD34+ HSPCs (nucleofection, viral vectors)
      • transplanted the modified cells into non-obese diabetic/severe combined immunodeficient/ interleukin-2r γ null (NOD/SCID/IL2r γ null; NSG) mice, which support both human hematopoiesis and HIV-1 infection.
      • Infected the mice with a CCR5-tropic strain of HIV-1
      Nat Biotechnol. 2010 Aug;28(8):839-47.
    • Nat Biotechnol. 2010 Aug;28(8):839-47.
    • Nat Biotechnol. 2010 Aug;28(8):839-47.
    • Paula Cannon, CROI2011
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    • Paula Cannon, CROI2011
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    • Paula Cannon and Carl June. Current Opinion in HIV and AIDS 2011; 6: 74-79. Molecular Microbiology & Immunology, University ofSouthern California Keck School of Medicine, Los Angeles, California and Department of Pathology andLaboratory Medicine, University of PennsylvaniaSchool of Medicine, Philadelphia, Pennsylvania, USA
    • Clinical Trials
    • SB-728
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    • Nat Biotech 2008
    • Nat Biotech 2008 25% CCR5 modification
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    • About 50% of the human population has antibodies !
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    • 6%, 3%, 1%, 2% and 2% at 14 days
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    • Fever, Fatigue
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    • Treatment interruption
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      • “ This isn’t for mass production or fully ready for prime time,” cautioned Scott Hammer, an AIDS researcher at Columbia University and a vice chair of the conference.
      • “ This is early work that takes molecular biology into the clinic. Like everything else in science and HIV, it’s an early and important finding, but we shouldn’t be raising the flag to say we’ve solved the problem yet.”
      BMJ 2011; 342:d1373
    • many questions remain unanswered:
      • What will happen long-term with the newly engrafted CD4 cells? Will they provide a survival or comorbidity benefit in these patients? (Note: Patients in this study will be followed for life.)
      • Will this approach help control HIV replication in patients who stop antiretrovirals after long-term HIV suppression?
      • How can we ethically ask patients to enroll in studies that require a structured treatment interruption?
      • Will institutional review boards be willing to approve these kinds of studies in the future?
      • Do people do better with more than one infusion? What is the best number of cells to ensure optimum immunological response?
      • Will this approach help control HIV replication in treatment-naive patients who have a detectable viral load and who are not taking HIV antiretrovirals? Will these cells still have a survival advantage when challenged with untreated HIV?
      • Will these gene-modified cells have a survival and activity advantage against HIV across the board? Will HIV viral load be controlled well enough for people to stop using antiretrovirals -- or, as stated in the question above, allow them to avoid antiretrovirals entirely?
      • What will the cost of this procedure be?
      • What happens to those with pre-exposure to the adenovirus vector who cannot respond to this type of zinc finger nuclease delivery method?
    • Thank You