T-Pharmacytes for the Targeted Eradication of HIV Reservoirs
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1 T-Pharmacytes for the Targeted Eradication of HIV Reservoirs Walker Lab Rachel O Connor Alicja Trocha Dan Karel Irvine Lab Stefanie Mueller Humanized Mouse Core Vlad Vrbanac Andy Tager Todd Allen Darrell Irvine Bruce Walker Clinical Samples Colin Kovacs Erika Benko Mario Ostrowski Altor Biosciences Hing Wong Emily Jeng
2 Viral Reservoirs as a Barrier to HIV Eradication Antiretroviral therapy (ART) suppresses HIV viremia to undetectable levels but fails to eradicate infection Why? Infection is established in long-lived memory CD4+ T-cells half-life = 44 weeks
3 Flush and Kill Approach to HIV Eradication Latently infected cell
4 Flush and Kill Approach to HIV Eradication Latently infected cell Latency-reversing drug Present HIV peptides In MHC-I Productively infected cell
5 Flush and Kill Approach to HIV Eradication HIV-specific CTL Recognize/kill Infected target Productively infected cell
6 Flush and Kill Approach to HIV Eradication HIV-specific CTL Recognize/kill Infected target
7 T-Pharmacytes CTL conjugated to drug-loaded nanoparticles Conjugate HIV-specific CTL to nanoparticles loaded with latency-reversing drugs T-Pharmacytes given by adoptive immunotherapy
8 Mathias Stephan
9 Characterization of T-Pharmacytes Developed to provide cytokine support to adoptively-transferred CTL (nanoparticles loaded with IL-15 superagonist IL-15SA) T-Pharmacytes: Traffic normally and accumulate at sites of antigen expression in murine tumor models Exhibit unimpaired killing of target cells in vitro Expand and persist in vivo dramatically better than normal CTL (when loaded with IL-15SA) Eradicate established melanoma tumors where normal CTL failed Stephan et al. Nat. Med, 2010
10 IL-15SA Reverses HIV Latency from Patient Samples Purified resting CD4+ T-cells treated with IL-15SA or anti-cd3/cd28 beads p=0.004 p=0.027
11 IL-15SA enhances survival/function (autocrine) IL-15SA Containing NP HIV-specific T- pharmacyte IL-15SA induces HIV expression (paracrine) Latently infected cell HIV-specific CTL kills productively infected cell
12 Latency Reversal by T-Pharmacytes Resting primary CD4+ T-cells Infected with luciferase HIV
13 RLU (HIV-Luciferase) IL-15SA-loaded T-Pharmacytes Reverse HIV Latency Mock HIV-NL4-3-Gluc Free Drug T-Pharmacytes
14 CTL Reservoir Recognition Assay Target CD4+ T-cells: Infected or uninfected Resting or activated HIV-Gag-specific or CMV-specific (neg cntrl) CD8+ T cell clone Co-culture Readout IFN-g in supernatants by ELISA
15 Latency Reversal Recognition Assay (drugs washed out)
16 Day 7 co-culture T-Pharmacytes with latently-infected targets
17 Testing T-Pharmacyte Eradication Strategy in Humanized Mouse Model Long durations of treatment are required to establish latent reservoirs in vivo Problematic for non-human primate studies due to high cost Problematic for existing humanized mouse models as animals progress to graft-versus host disease Can we develop a more rapid humanized mouse model?
18 hucd4-nsg Model 50x10 6 CD4 + T-cells from ARV-treated patients (contains natural HIV reservoir) Autologous T-pharmacytes or controls (CTL only, etc) NSG mouse Two weeks with ARVs Cells home to lymphoid tissues and undergo homeostatic proliferation Two weeks with ARVs Stop ARVs Monitor time to viral rebound
19 hucd4 NSG model
20 hucd4 NSG model Reconstitute mice with CD4+ T-cells from HIV-infected subject rebound of endogenous HIV Rebound not observed with cells from 5/6 elite controllers
21 hucd4 NSG model HIV replication occurs in lymphoid tissue in hucd4 NSG model Brown HIV-Gag
22 hucd4 NSG model ARV Suppression Pediatric formulations of AZT, 3TC, NVP given in drinking water
23 hucd4-nsg Adoptive Transfer Experiments #1 no ARVs 50x10 6 expanded CD4 + T-cells from HIV-infected patient + exogenous NL4-3 virus 1.5x10 6 Autologous T-pharmacytes or controls (CTL only, etc) Two days (no ARVs) Monitor Viral Load NSG mouse
24 hucd4-nsg Adoptive Transfer Experiment #1 Utilized HIV-Gag-SLYNTVATL-specific CTL clone with potent in vitro killing of NL4-3 infected target cells T-Pharmacytes conjugated to nanoparticles loaded with 1.38mg/ml of the IL-15SA ALT-803 (Altor Biosciences)
25 hucd4-nsg Adoptive Transfer Experiments no ARVs 50x10 6 CD4 + T-cells from A02+ HIV-uninfected subject + exogenous HIV JRCSF Two days (no ARVs) 2.5x10 6 Allogeneic HIV-Gag-SL9-spec T-pharmacytes or controls (CTL only, etc) Monitor Viral Load NSG mouse
26 hucd4-nsg Adoptive Transfer Experiment #2
27 hucd4-nsg Adoptive Transfer Experiment #2 Day 17 Day 81
28 Conclusions T-Pharmacytes carrying the IL-15 superagonist ALT-803 can reverse HIV latency in vitro and exert antiviral effects in vivo The hucd4-nsg mouse is a promising model for eradication studies including adoptive transfer approaches Ongoing studies aim to characterize how the reservoir is shaped by homeostatic proliferation upon transfer into NSG mouse Adoptive transfers of T-Pharmacytes into hucd4-nsg mice in the context of ARVs are planned. Does this result in reduction or eradication of reservoirs?
29 Bershteyn et al. Soft Matter (2008); Moon et al. Nat. Mater. (2011) ICMVs I
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