2014 “Towards an HIV Cure” symposium Melbourne Induction and Clearance of Latent HIV Infection: an Ex-Vivo Assessment of Immune Effectors using Cells from ART-treated Patients DM Margolis1, C Garrido1, JM Sung1, S Lam2, R Bateson1, B Allard1, N Dahl1, CR Cruz2, P Castillo-Caro3, MT Ngo3, J Kuruc1, A Crooks1, CM Rooney3, CM Bollard2, and NM Archin1 1University of North Carolina Chapel Hill, NC; 2Children’s National Medical Center, Washington, DC; 3Baylor College of Medicine, Houston, Texas

Model systems to assess clearance Primary cell systems Cytotoxic T cells NK cells ADCC antibodies Immunotoxins Selective apoptosis inducers

Why Ex-vivo Expanded CTLs Bypasses impaired immune response Precisely controlled quantity & timing of administration CTLs can persist Safe and effective for treatment of viral infections in oncology patients As complex as the immune system is and given how profound the depth and breadth of immune exhaustion in chronic HIV infection is, there are many avenues to approaching the problem of enhancing the immune system in order to clear latent HIV infection; This work focuses on ex-vivo expanded CTLs for proof of concept work as this approach bypasses many of the obstacles to a robust immune response that occur in the face of chronic HIV infection, including dysfunctional and impaired DC function, CD4 help and CD8 proliferation; additionally, we can precisely control the timing of administration-which will be an important consideration if combining with a latency reactivating agent, which may have its own impact (transient or otherwise) on the immune system, as well as control over the number and type of CTLs that are ultimately infused AM Leen, et al. Nat Med. 2006 Bollard, et al. Blood. 2007

Ex vivo Expansion of HIV-specific T cells ART to prevent In vitro spread ++ HIV-CTLs T cell Immature DC PBMC freeze HXTCs IL-7 IL-15 IL-2 IL-12 IL-15 Here you can see the method for generating the ex-vivo expanded HIV-CTLs; T cells derived from patient PBMCs undergo stimulation with matured autologous DC and then irradiated PHAblasts that have been loaded with multiple overlapping consensus clade b peptides for gag, pol, and nef in order to minimize the impact of CTL escape variants; This is followed by a third and final round of stimulation with irradiated loaded K562 cells that have been modified to overepress costimulatory molecules to really amp up the numbers; The use of the modified, irradiated K562 cells has been found to be safe for clinical use; Using this method, CTls were expanded by anywhere from 37 to 300 fold PHAblast CD80/86 4-1BBL CD32 + K562 Mature DC + gag/ pol/ nef Cath Bollard, DC Children’s Clio Rooney, Baylor and PACT

HXTCs are a Mixture of Phenotypes 82% CD8+ T cells 19% CD4+ T cells 80% EM T cells 13% CM T cells IFNγ Release to Cognate Peptides

HXTCs Inhibit Productively Infected Cells CD8 deplete patient PBMCs Activate 2-3 days Superinfect w/ virus via spinoculation + autologous unexpanded CD8 or expanded HIV-CTLs (or no effector control) Co-culture x 7 days Media change q3-4 days Supernatant harvested for p24 ELISA wash Plate in triplicate HIV-CTLs To measure the antiviral activity of the HIV-CTLs, we modified a viral inhibition assay well described in the literature; HIV-CTLs, or as a comparator unexpanded autologous CD8s, or as a control, media only are added to autologous targets that had been superinfected with JR-CSF; Yang, et al. JID 2012 Freel, et al. J Virol. 2012

HXTCs Inhibit Autologous Reservoir Virus HIV p24 (% of no effector at day 7) E:T E:T E:T E:T

Ex-Vivo Latency Clearance Assay: A modified quantitative viral outgrowth Assay CTL Expansion CD8 negative selection PBMCs CD8 HXTCs No Effectors or or Resting CD4 Negative selection CD8, CD14, CD16, CD19, CD56, glycophorin A Cx with ARVs 24H Induction PHA/IL2 or VOR Add CTLs Co Cx 24H Add Allo Feeders CoCx x2 Measure p24 at Day 15 To do this, we modified the standard quantitative outgrowth assay-outlined below-in which resting CD4 cells are isolated and activated with PHA/IL-2- to include the addition of effector cells-either the ex=vivo expanded HIV-CTLs, or autologous unexpanded CD8s as a comparator, or as a control, no effectors/just media are added and allowed to co-culture with the reactivated CD4 cells for 24 hours prior to addition of allogeneic CD8 depleted PBMCs to amplify any residual infection; wash wash Media changes q3-4 days Plate: 0.5-1x106/well, 12 wells group

HXTCs Clear Infected Cells Emerging from Latency PHA Stimulation VOR Induction 425 532 250 20 40 60 80 100 120 % viral recovery No Effectors CD8 HXTC 1 2 3 4 5 6 7 8 #wells positive (out of 12 total) Et 1:10 Et 1:10 Black dot is pt 492 with all 3 Some with only Nef response suppressed p <0.03 Wilcoxon signed rank p <0.02 t test

VOR does not Impair CD8 Antiviral Activity at Physiogically Relevant Exposures Viral inhibition assay at E:T ratio 1:1 B. Control 24 48 72 20 40 60 80 100 No CD8 Control 335nM 500nM 1000nM No VOR HIV p24 (ng/ml) hours Patient 532

Why NKs to target residual HIV Crucial innate immune effectors against viral infections Do not recognize specific antigens nor require prior antigen sensitization Function is balanced by inhibitory and activating receptors Kill cells by release of granzymes and perforins, which causes apoptosis NK cells may help control HIV-1 infection Memory NK cells may also provide a more effective antiviral response NK function may be augmented by clinically applicable cytokines

VIRAL INHIBITION ASSAY Unstimulated NKs PBMCs Negative isolation NK cells IL-2 /IL-15 stimulated NKs No effectors Negative isolation ≠ E:T ratios Super-infection (autologous reservoir virus) CD4+T cells HIV-CD4+ cells +/- effectors PHA 24h 7 days HIV-p24

VIRAL INHIBITION ASSAY † † † † † † CD4+ Targets only 1:1 1:10 1:100 1:1 1:10 1:100 1:1 1:10 1:100 NK cells IL2-stimulated NK cells IL15-stimulated NK cells †: p <0.01

LATENCY CLEARANCE ASSAY PBMCs Negative isolation NK cells Resting CD4+T cells ARV 24h Stimulation CD4+T cells +/- effectors Add feeders Unstimulated NKs IL-2 stimulated NKs Measure number of positive wells for p24 at day 15 24h No effectors Compare number of positive wells with/without effectors

LATENCY CLEARANCE ASSAY PHA reactivation VOR reactivation VOR Positive trends but more assays needed

IMPACT OF VOR ON NK FUNCTION NKs were treated with or without 335 nM VOR overnight For degranulation cells 4 hours in the presence of K562 cell targets VOR Viral inhbition assays Degranulation Accepted 270 registrations, approximately 270 people attended over the two days. 20 oral abstracts accepted, of which 8 were LB’s. 52 poster abstracts were accepted = total of 72 abstracts 51 full and partial scholarships were offered. VOR did not impair NK cytotoxicity or antiviral activity

Summary Polyclonal HXTCs with activity against multiple peptides can be generated in clinically relevant numbers HXTCs and cytokine-treated NK cells show enhanced antiviral activity Using both lab strain JR-CSF virus and autologous reservoir virus NK cells and HXTCs reduce viral recovery from resting CD4 cells reactivated with PHA/IL-2, demonstrating a potential to clear latent HIV infection ex-vivo Preliminary results also show reduction in viral recovery from resting CD4 cells reactivated with VOR, and no adverse effect on function at relevant VOR exposures

Children’s National Medical Center Juila Sung MD Carolina Garrido Pavon, PhD Natalia Soriano, PhD Nancie Archin, PhD Noelle Dahl Rosalie Bateson Brigette Allard Joann Kuruc, MSN RN Amanda Crooks Cynthia Gay, MD, MPH Children’s National Medical Center Catherine Bollard, MD Sharon Lam Special thanks to the HIV+ volunteers