Fate of HIV-1 infected cells under suppressive ART Fabian Otte - - PowerPoint PPT Presentation

Fabian Otte Molecular Virology DBM Basel AREVIR cologne 03.05.2019

Fate of HIV-1 infected cells under suppressive ART

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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

Regoes, R. R. & Bonhoeffer, S. The HIV coreceptor switch: a population dynamical perspective. doi:10.1016/j.tim.2005.04.005

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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
• Patients presenting as R5 at

beginning of cART stay R5

• In 80% of patients no increase in X4

viral frequency

• 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 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).

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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

1Guadalupe, M. et al. T-Cell Depletion in Gut Lymphoid Tissue during Primary Human Immunodeficiency Virus Type 1 Infection and Substantial Delay in Restoration

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

Therapy naÏve LTNP HIV neg Therapy naÏve LTNP HIV neg

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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

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 Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions. J. Virol. 90, 7142–58 (2016).

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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

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

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).

Bruner et al 2016 3 Ho et al 2013 2 Baxter et al 2018 1 “Detectability”

• f viral

reservoir

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Proviral enrichment and lymphoid homing

Anti-CD19- Biotin Anti-Biotin- beads

CD19 CD4+ CD4- CD8 CD4- CD4+

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)? Adapted from Miltenyi Biotec

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MACS: gut (Integrin ß7) vs LN (CCR7)

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Identification of the viral reservoir

1) Staining 2) Data Acquisition and Analysis (tSNE)

• 2. ICS GAG
• 1. Surface +

Env staining Surface HIV-1 Env and intracellular Gag expression Activation 36-40h

HIV-1 chronically infected cell line

HUT4-3: chronically infected cell line SupT1: uninfected lymphpcyte cell line, susceptible to HIV-1 DP DN

Lymphocyte infection

HIV-1 “Mal” : Subtype D, R5-tropic HIV-1 “Bru” : Subtype B, X4-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

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Summary: Identification of the viral reservoir

1) Staining 2) Data Acquisition and Analysis (tSNE) 3) Patient cell sorting

• 1. Sort

RNA seq, FLIP seq

• 2. ICS GAG
• 1. Surface + Env

staining Surface HIV-1 Env and intracellular Gag expression Activation 36-40h

I. Viral compartmentalization (X4 vs R5) II. Viral integration, transcriptome and proviral landscape

• III. Lymphoid homing tropism

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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

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Acknowledgements

Molecular Virology Lab

• Prof. Thomas Klimkait

Rahel Bircher Jennifer Brown Silvia Caimi Nina Marty Siro Ellenberger Benjamin Schwob Ulrike Seeburg Lorena Urda

PACommitte:

• Prof. C. Dehio (Biozentrum),
• Dr. M. Stöckle (USB)

FACS Core Facility DBM DBM Basel

• Dr. J. Spagnuolo

Uni-Klinik Köln Florian Klein Rolf Kaiser

• Dr. C. Wiethe (Biolegend)

Andrin Wacker, Alain Hirschy (Miltenyi)

Thanks ks for yo your attention!

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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