CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

Conclusion: Despite limited reservoir establishment with early cART, profound depletion of CD4+ T cells, including those potentially harboring virus, had no measurable impact on off-cART viral rebound. These findings underscore the potency that will be required of reservoir reducing strategies to meaningfully reduce total body reservoir size. 350 SIV PERSISTS IN LYMPHOID TISSUES DESPITE ALEMTUZUMAB-INDUCED CD4+ T CELL DEPLETION Afam Okoye 1 , Colleen Xu 1 , Mukta Vaidya 1 , Derick M. Duell 1 , William B. Brantley 1 , Alejandra Marenco 1 , Yoshinori Fukazawa 1 , Haesun M. Park 1 , Thomas A. Rasmussen 2 , Jeffrey D. Lifson 3 , Michael K. Axthelm 1 , Steven G. Deeks 4 , Louis J. Picker 1 , Sharon R. Lewin 5 1 Oregon Health and Sciences University, Portland, OR, USA, 2 Aarhus University Hospital, Aarhus, Denmark, 3 Leidos Biomedical Research, Inc, Frederick, MD, USA, 4 University of California San Francisco, San Francisco, CA, USA, 5 Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia Background: Alemtuzumab (ATM) is a lymphocyte-depleting humanized anti- CD52 monoclonal antibody used for the treatment of multiple malignancies and licensed for relapsing-remitting multiple sclerosis. Here, we evaluated antiretroviral therapy (cART). We hypothesized that ATM-induced depletion and reconstitution in the presence of cART may significantly reduce SIV persistence. Methods: RMwere initially screened for CD52 expression on erythrocytes using an agglutination assay to minimize any risk of hemolysis. Nine RM were intravenously inoculated with SIVmac239 and after 12 days received cART (tenofovir/emtricitabine/dolutegravir). Once sustained virus suppression (<15 RNA copies/ml) was achieved, 6 RM received intravenous doses of ATM at 5mg/ Kg on days 0, 7, 14 and 29. Three RM did not receive ATM (controls). Lymph node (LN) and gastrointestinal tract (GIT) tissue was collected at 10 and 20 weeks. SIV DNA and RNA were quantified by qPCR/qRT-PCR and markers of immune activation by flow cytometry. Results: ATM induced a rapid and profound depletion in circulating lymphocyte populations, including T cells, NK cells, B cells and monocytes, including a significant depletion in CD4+memory and naive T cells in the blood (>95%) and some depletion in LN (~50%). CD4+ T regulatory cells were also significantly depleted (>90%). T cell reconstitution was associated with a massive burst in memory T cell proliferation as measured by Ki67, which peaked around 3-4 weeks post-ATM and followed by a gradual recovery of all T cell subsets in blood. After 7 months, naïve, central, and effector memory CD4+ T cell subsets were 51%, 60% and 100% of pre-ATM levels, respectively. Post- ATM, but while still on cART, plasma SIV RNA remained detectable but below 15 copies/ml with only 1 of 6 RM showing blips above 100 SIV RNA copies/ml. At 10 weeks post-ATM, total SIV DNA in peripheral blood mononuclear cells (PBMC) decreased from a mean of 2.4 to 1.4 log copies per 10e6 cells (p=0.03); however, SIV DNA in peripheral LN and GIT remained unchanged. Conclusion: Although ATM can significantly reduce SIV DNA levels in the PBMC of SIV-infected RM on cART, low level viremia persists. The minimal effect on SIV DNA in LN and GIT suggests either limited depletion in those tissues or a rapid reconstitution at those sites post-ATM, perhaps by expanded clones. 351 ∆CCR5 ANTI-HIV CAR T CELLS ENGRAFT AND PERSIST IN SHIV-INFECTED PIGTAIL MACAQUES Thor A. Wagner 1 , Christopher Peterson 2 , Brenda Seymour 3 , Taylor Mesojednik 3 , Jeb English 3 , Bryan Sands 1 , Courtnee Clough 1 , Karsten Eichholz 2 , Larry Corey 2 , Hans-Peter Kiem 2 , David J. Rawlings 1 1 University of Washington, Seattle, WA, USA, 2 Fred Hutchinson Cancer Research Center, Seattle, WA, USA, 3 Seattle Children’s Research Institute, Seattle, WA, USA Background: Persistence of HIV-infected cells with replication competent provirus is the primary barrier to HIV cure. To eradicate HIV-infected cells, we developed chimeric antigen receptors (CAR) that utilize single chain variable fragments (scFv) from broadly neutralizing monoclonal antibodies, and protected the CAR+ cells from HIV infection by disrupting CCR5. To test this approach in vivo, we treated SHIV-infected pigtail macaques with autologous CCR5-disrupted(∆CCR5), VRC07-523-based CAR+ T cells. Methods: Six pigtail macaques were leukapheresed prior to infection with the CCR5-tropic SHIV-1157ipdN4. 12 weeks after SHIV infection and one week after immunoconditioning with cyclophosphamide, ∆CCR5 CAR+ T cells were infused in the absence of antiretroviral therapy. Four animals received VRC07-523- whether ATM can deplete long-lived latently infected CD4+memory T cells in SIV-infected rhesus macaques (RM) on suppressive combination

derived CAR+ ∆CCR5 T cells and two animals received GFP+ cells as a control. Engineered T cells were tracked in the peripheral blood and various tissues using flow cytometry, and confirmed by PCR amplification and sequencing of the CAR and the CCR5 locus. One CAR treated animal underwent necropsy after two weeks, the rest underwent necropsy six weeks after CAR T cell treatment. Results: A median of 88% (range 74-90%) of the engineered T cells expressed either the CAR or GFP transgene. 71% (range 44-78%) of CCR5 alleles were disrupted in the CAR T cell product, as measured by an endonuclease re- cleavage assay. Cells were expanded ex vivo 20.5 fold (range 11-30). Animals were treated with 67 million (range 28-90 million) CAR+ or control cells per kg. Compared to the GFP control animal, all four CAR treated animals had detectable populations of CAR+ T cells in the bone marrow, PBMC, spleen, and lung at necropsy, as measured by flow cytometry and PCR-based assays. Conclusion: This is one of the first attempts to study HIV-specific, infection resistant CAR T cell therapy in nonhuman primates. We successfully engineered nonhuman primate T cells with high-levels of CAR expression and CCR5 disruption. CAR+ T-cells persisted in vivo at levels consistent with cell proliferation. We are working to evaluate the antiviral effect of the CAR T cells and to determine if the CAR T cells that persist are functional. Based on initial data, methods to improve the persistence and distribution of infection-resistant CAR T cells may be important. Helen Wu , Benjamin Burwitz, Shaheed Abdulhaqq, Christine Shriver-Munsch, Tonya Swanson, Alfred Legasse, Katherine Hammond, Jason Reed, Mina Northrup, Stephanie Junell, Gabrielle Meyers, Richard Maziarz, Jeffrey Stanton, Jonah Sacha Oregon Health and Sciences University, Portland, OR, USA Background: Timothy Brown remains in cART-free HIV remission following allogeneic hematopoietic stem cell transplant (HSCT), but attempts to recapitulate his cure have been unsuccessful. We recently established a nonhuman primate model of fully MHC-matched allogeneic HSCT to investigate the mechanism of cure in Timothy Brown. In this model, a reduced intensity conditioning (RIC) regimen consisting of chemotherapy, CD3 depletion, and total body irradiation (TBI) prior to HSCT results in durable, full multi-lineage donor chimerism in SIV-naïve HSCT recipients. Here, we sought to investigate (1) if similar results could be achieved without TBI, and (2) the impact of allogeneic HSCT on SIV reservoir size in cART-suppressed, SIV-infected recipients. Methods: Allogeneic HSCT was performed with fully MHC-matched Mauritian cynomolgus macaque (MCM) donor-recipient pairs, including two fully cART- suppressed, SIV-infected recipients. Mobilized peripheral stem cells collected from donors by leukapheresis were transplanted into recipients following RIC with or without TBI. Donor engraftment was monitored by Illumina sequencing of single nucleotide polymorphisms. Immune subset reconstitution was assessed longitudinally by flow cytometric phenotyping and complete blood counts. Results: We performed two HSCTs without TBI, both resulting in low T cell donor chimerism (<15%). The first, SIV-naïve recipient experienced incomplete T cell rebound resulting in polyoma virus reactivation and euthanasia. The second, cART-suppressed, SIV-infected recipient stabilized post-HSCT, and experienced a reduction in lymph node-associated SIV DNA despite incomplete T cell donor chimerism. Adding back TBI for HSCT of a second cART-suppressed, SIV-infected recipient resulted in high levels of donor chimerism in whole blood (>95%) and T cells (~80%) within 30 days of HSCT (see figure). Subsequent donor lymphocyte infusion increased blood and tissue T cell donor chimerism levels to nearly 100%, but led to development of clinical graft-versus-host disease (GVHD) and euthanasia. At necropsy, SIV DNA was below the limit of detection in lymph nodes, constituting a ~3 log reduction from pre-HSCT levels. Conclusion: These data demonstrate that TBI is critical to achieving high levels of T cell donor chimerism post-HSCT in MCM, and that while HSCT immune conditioning decreases the viral reservoir size, GVHD is associated with enhanced reservoir clearance. This model facilitates future studies of HSCT- mediated HIV cure.

352 FULLY MHC-MATCHED ALLOGENEIC HSCT IN SIV-INFECTED, ART- SUPPRESSED MACAQUES

Poster Abstracts

CROI 2018 123