Fate of HIV-1 infected cells under suppressive ART Fabian Otte Molecular Virology DBM Basel AREVIR cologne 03.05.2019
Infection without therapy • In early infection patients harbor 80-90 % R5 viruses • In absence of therapy X4 proportion in 50% of patients increases concomitant with transition to AIDS 2 Regoes, R. R. & Bonhoeffer, S. The HIV coreceptor switch: a population dynamical perspective. doi:10.1016/j.tim.2005.04.005
Infection wi with therapy • In early infection patients harbor 80-90 % R5 viruses • In absence of therapy X4 increases concomitant with transition to AIDS in 50% of patients • under successfull therapy X4 viruses are markedly reduced 1 o Patients presenting as R5 at beginning of cART stay R5 o In 80% of patients no increase in X4 viral frequency o MOTIVATE Study 2 ☛ Synergy of cART and recovering immune system that readily detects X4 (less glycosylation) 1 Bader, J. et al. Therapeutic immune recovery prevents emergence of CXCR4-tropic HIV-1. Clin. Infect. Dis. ciw737 (2016). doi:10.1093/cid/ciw737 3 2 Westby, M. et al. Emergence of CXCR4-Using Human Immunodeficiency Virus Type 1 (HIV-1) Variants in a Minority of HIV-1-Infected Patients following Treatment with the CCR5 Antagonist Maraviroc Is from a Pretreatment CXCR4-Using Virus Reservoir. J. Virol. 80, 4909–4920 (2006).
Observations: GALT major HIV reservoir • GALT ( encompasing >50% of IS) gets depleted during acute phase of HIV infection and does not recover 1 • total body SIV burden almost solely in GALT after therapy 2 Therapy naÏve LTNP HIV neg Therapy naÏve LTNP HIV neg 1 Guadalupe, M. et al. T-Cell Depletion in Gut Lymphoid Tissue during Primary Human Immunodeficiency Virus Type 1 Infection and Substantial Delay in Restoration 4 following Highly Active Antiretroviral Therapy. J. Virol. 77, 11708–11717 (2003). 2 Estes, J. D. et al. Defining total-body AIDS-virus burden with implications for curative strategies. Nat. Publ. Gr. (2017). doi:10.1038/nm.4411
Observations: Compartmentalization • GALT ( encompasing >50% of IS) gets depleted during acute phase of HIV infection and never recovers 1 • provirus stable after initial decay 3 • no recombination of X4 and R5 virus 4 3 Besson, G. J. et al. HIV-1 DNA Decay Dynamics in Blood During More Than a Decade of Suppressive Antiretroviral Therapy. doi:10.1093/cid/ciu585 5 4 Zhou, S., Bednar, M. M., Sturdevant, C. B., Hauser, B. M. & Swanstrom, R. Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage 5 Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions. J. Virol. 90, 7142–58 (2016).
Hypothesis I. The differences in tropism (X4 vs R5) leads to district compartmentalization (e.g. due to half-life, immune clearance) , assessable by homing properties of the CD4 T cells II. GALT-homing plays a key role in HIV persistence, and gut-associated lymphatic cells represent an important reservoir for HIV-persistence III. GALT depletion is a continuous consequence of viral pressure (and therefore dependent on the „GALT homing viral tropism“) ☛ which cells are involved in these “viral dynamics during suppressive therapy” & which respective lymphoid tissue is involved in reservoir formation, stability and viral clearance 6 Buggert et al. Everything in its right place - resident memory CD8 + T cell immunosurveillance of HIV infection. Current Opinion in HIV and AIDS: 2019-03-V14-2 p93-99
HIV viral reservoir Baxter et al 2018 1 Ho et al 2013 2 “Detectability” of viral reservoir Bruner et al 2016 3 1 Baxter AE et al. Beyond the replication-competent HIV reservoir: transcription and translation-competent reservoirs. Retrovirology201815:18 2 Ho, Y. C. et al Replication-Competent Noninduced Proviruses in the Latent Reservoir Increase Barrier to HIV-1 Cure 3 Bruner, K. M. et al. Defective proviruses rapidly accumulate during acute HIV-1 infection. Nat Med 22, 1043–1049 (2016).
Proviral enrichment and lymphoid homing CD4- Anti-CD19- CD8 Biotin CD19 CD4- CD4+ CD4+ Anti-Biotin- Adapted from Miltenyi Biotec beads Arbitrarily chosen SHCS surplus material: Depletion of unrelevant cells: CD8 (CTL), CD19 (B-cells) -> negative control • Enrichment for cells of interest: CD4 + vs CD4 - (Vpu,Nef), CCR7 + vs CCR7 - , • Integrin ß7 + vs ß7 - Proviral loads of sorted cells • ☛ first indications of proviral enrichment (of intact viruses)? 8
MACS: gut (Integrin ß7) vs LN (CCR7) 9
Identification of the viral reservoir 1) Staining Surface HIV-1 Env and intracellular Gag expression 1. Surface + Activation Env staining 36-40h 2. ICS GAG 2) Data Acquisition and Analysis (tSNE) 10
HIV-1 chronically infected cell line DP HUT4-3: chronically infected cell line SupT1: uninfected lymphpcyte cell line, susceptible to HIV-1 DN
Lymphocyte infection HIV-1 “Bru” : Subtype B, X4-tropic HIV-1 “Mal” : Subtype D, R5-tropic Cells Supernatant
CD4+ T-cell infection HIV-1 “Bru”: Subtype B, X4-tropic HIV-1 “ALA”: Subtype B, X4-tropic Cells Supernatant
tSNE visualization of patient cells
Summary: Identification of the viral reservoir 1) Staining Surface HIV-1 Env and intracellular Gag expression 1. Surface + Env staining Activation 36-40h 2. ICS GAG 2) Data Acquisition and Analysis (tSNE) 3) Patient cell sorting I. Viral compartmentalization (X4 vs R5) II. Viral integration, 1. Sort RNA seq, FLIP seq transcriptome and proviral landscape 15 III. Lymphoid homing tropism
Outlook • tSNE: Patients > 10‘000 HIV DNA/10^6 cells: 3-9X10^6 cryo-preserved cells • FACSorts of viable patient cells for deep HIV Immuno characterisation of patients (UZH: BSL-3, 4-way) to characterize reactivated virus by SGS/MIP-sequencing • CyTOF experiments with Dr. Bengsch using PBMC and/or biopsies • C. Apetrei, I. Pandrea: SIV experiments in simian models regarding X4- vs R5-tropic infection • HIV+ Lymph nodes/GALT biopsies for CODEX imaging 16
Acknowledgements PACommitte : Prof. C. Dehio (Biozentrum), Dr. M. Stöckle (USB) Molecular Virology Lab Prof. Thomas Klimkait Rahel Bircher FACS Core Facility DBM Jennifer Brown Silvia Caimi Nina Marty DBM Basel Siro Ellenberger Dr. J. Spagnuolo Benjamin Schwob Ulrike Seeburg Lorena Urda Uni-Klinik Köln Florian Klein Rolf Kaiser Dr. C. Wiethe (Biolegend) Andrin Wacker, Alain Hirschy (Miltenyi) Thanks ks for yo your attention! 17
Now is your chance! We urgently need patient cells of either untreated, newly diagnosed or patients with recent treatment initiation We would be very happy to collaborate Thanks 18
Recommend
More recommend
