HIV elimination from reservoirs viral dynamics during suppressive - - PowerPoint PPT Presentation

Fabian Otte Klimkait Lab DBM Basel Arevir 05.05.2018

HIV elimination from reservoirs – viral dynamics during suppressive ART

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Observations

• In early infection patients harbour 80-90 % R5 viruses
• In absence of cART X4 increases concomitant with transition to AIDS in 50% of patients
• under successfull cART 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 ( encompasing >50% of IS) gets depleted during acute phase of HIV infection and never

recovers 1

• total body HIV burden almost solely in GALT after

therapy 2

• no recombination of X4 and R5 virus 3

☛ Where does the viral reservoir hide?

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 3 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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• 1. First results: MACS

CD4-Selection Integrin ß7-Selection

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• Titration of infectious HIV expressing cell clone (HUT4-3) into HIV negative

Lymphocyte cell line (SupT1) 1:100

• 2. tSNE (t-distributed stochastic neighborhood

embedding)

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• 2. HIV Hut4-3 titration

(T-cell lineage) (T-cell lineage)

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Outlook 1

• Widen MACS approach to CCR7+/- or ß7+/- selection + CD4+/-

selection –> further implementations for proviral reservoir?

• tSNE (HIV Gag):

Patients > 10‘000 HIV DNA/10^6 cells: 18 (10 K Metzner Zurich; 8 Bader1) soon avialable (proposal re-submission)

• FACS sorts on fixed (coming soon)
• Detailed FACS (sorts) for deep HIV Immuno characterisation of

characterized patients1 (Prof. Protzer/ Dr. Dr. B Bengsch)

• BD Melody in BSL-3**: currently 2-way sorting possible (soon upgrade to 4-way)

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

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Appendix: tSNE considerations 1

Lymphocytes > Singlets (5’000 events/patient)

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• 2. tSNE considerations 2
• Use Gag intra-cellular staining (ICS) on HIV infected

activated/unactivated cells + cell surface lypmh homing marker stain, to identify which cell identity has most Gag translation (implemetnting potential viral intactness)

• Difficulty: only patients with high proviral loads will

have enough Gag production, which can be analysed by FACS!

(GALT) (LN) (CTL) (B-cell) (GALT) (T-cell lineage) (TH17)

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Appendix: X4 vs R5

• In early infection patients harbour 80-90 % R5 viruses
• In absence of cART 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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Hiener et al 2017, Cell (FLIPS) 1 Bruner et al 2016, Nat Med 2

Appendix: viral intactness

1 Hiener, B. et al. Identification of Genetically Intact HIV-1 Proviruses in Specific CD4 + T Cells from Effectively Treated Participants. Cell Rep. 21, 813–822 (2017) 2 Bruner, K. M. et al. Defective proviruses rapidly accumulate during acute HIV-1 infection. Nat Med 22, 1043–1049 (2016).

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Appendix: FLIPS

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Appendix: histoCAT

• B. Bodenmiller et al 2017, Nature Methods

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Appendix: Codex

• 1. Goltsev, Y. et al. Deep profiling of mouse splenic architecture with CODEX multiplexed
• imaging. bioRxiv 203166 (2018). doi:10.1101/203166 (http://dx.doi.org/10.1101/203166)