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

HIV elimination from reservoirs – viral dynamics during suppressive ART Fabian Otte Klimkait Lab DBM Basel Arevir 05.05.2018

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 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 2 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 ( 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? 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 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 3 3 Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions. J. Virol. 90, 7142–58 (2016).

1. First results: MACS CD4-Selection Integrin ß7-Selection 4

2. tSNE (t-distributed stochastic neighborhood embedding) Titration of infectious HIV expressing cell clone (HUT4-3) into HIV negative • Lymphocyte cell line (SupT1) 1:100 5

2. HIV Hut4-3 titration (T-cell lineage) (T-cell lineage) 6

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 Bader 1 ) soon avialable (proposal re-submission) • FACS sorts on fixed (coming soon) • Detailed FACS (sorts) for deep HIV Immuno characterisation of characterized patients 1 (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 7

Appendix: tSNE considerations 1 Lymphocytes > Singlets (5’000 events/patient) 8

2. tSNE considerations 2 (GALT) (LN) (CTL) (B-cell) (GALT) Use Gag intra-cellular staining (ICS) on HIV infected (T-cell lineage) • activated/unactivated cells + cell surface lypmh homing marker stain, to identify which cell identity (T H 17) has most Gag translation (implemetnting potential viral intactness) Difficulty: only patients with high proviral loads will • have enough Gag production, which can be 9 analysed by FACS!

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

Appendix: viral intactness Hiener et al 2017, Cell (FLIPS) 1 Bruner et al 2016, Nat Med 2 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) 11 2 Bruner, K. M. et al. Defective proviruses rapidly accumulate during acute HIV-1 infection. Nat Med 22, 1043–1049 (2016).

Appendix: FLIPS 12

Appendix: histoCAT B. Bodenmiller et al 2017, Nature Methods 13

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) 14