Nanodepots Encapsulating a Latency Reversing Agent and Broadly Neutralizing Antibody Enhance Natural Killer Cell Cytotoxicity Against an in vitro Model of Latent HIV

Abstract

Joshua Ghofrani,1,2 Allan Bowen,2 Jie Chen,2 Preethi Bala Balakrishnan,2 Allison B Powell,1,2 Kondareddy Cherukula,2 Conrad Russell Y Cruz,2,3 R Brad Jones,4 Rebecca M Lynch,5 Elizabeth E Sweeney,2,6 Rohan Fernandes1,2,7 1The Institute for Biomedical Sciences, School of Medicine and Health Sciences, George Washington University, Washington, DC, USA; 2The George Washington Cancer Center, The George Washington University, Washington, DC, USA; 3Center for Cancer and Immunology Research, Children’s National Hospital, Washington, DC, USA; 4Division of Infectious Diseases, Department of Medicine, Weill Cornell Medicine, New York, NY, USA; 5Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA; 6Department of Biochemistry and Molecular Medicine, School of Medicine and Health Sciences, George Washington University, Washington, DC, USA; 7Department of Medicine, School of Medicine and Health Sciences, George Washington University, Washington, DC, USACorrespondence: Rohan Fernandes, 800 22nd St. NW, Science and Engineering Hall, Suite 8410, Washington, DC, 20052, USA, Tel [phone], Email [email protected]: Current antiretroviral therapies (ART) for human immunodeficiency virus (HIV) are not curative, as the virus persists in latent reservoirs, requiring lifelong adherence to ART and increasing the risk of co-morbidities. “Shock and kill” approaches to reactivate HIV from latent reservoirs followed by administration of anti-HIV drugs represent a promising strategy for eradicating latent HIV. To achieve effective shock and kill, we describe a strategy to eradicate the HIV reservoir that combines latency reversing agents (LRAs), broadly neutralizing antibodies (bnAbs), and natural killer (NK) cells. This strategy utilizes a polymer nanodepot (ND) that co-encapsulates the LRA and bnAb to reactivate latent infection and elicit enhanced cytotoxicity from co-administered NK cells.Methods: Poly(lactic-co-glycolic acid) (PLGA) NDs were synthesized using the nanoprecipitation method to co-encapsulate an LRA (TNF-α) and a bnAb (3BNC117) (TNF-α-3BNC117-NDs). ACH-2 cells were used as a cellular model of latent HIV infection. An NK92 subline, genetically modified to constitutively express the Fc receptor CD16, was administered to ACH-2 cells in combination with TNF-α-3BNC117-NDs. ACH-2 cell death and extracellular p24 were measured via flow cytometry and ELISA, respectively.Results: Stable PLGA NDs co-encapsulated TNF-α and 3BNC117 with high efficiencies and released these agents in physiological conditions. NK92 phenotype remained similar in the presence of TNF-α-3BNC117-NDs. TNF-α released from NDs efficiently reactivated HIV in ACH-2 cells, as measured by a 3.0-fold increase in the frequency of intracellular p24 positive cells. Released 3BNC117 neutralized and bound reactivated virus, targeting 57.5% of total ACH-2 cells. Critically, TNF-α-3BNC117-NDs significantly enhanced NK92 cell-mediated killing of ACH-2 cells (1.9-fold) and reduced extracellular levels of p24 to baseline.Conclusion: These findings suggest the therapeutic potential of our novel ND-based tripartite strategy to reactivate HIV from latently infected cells, generate an HIV-specific site for bnAb binding, and enhance the killing of reactivated HIV-infected target cells by NK92 cells.Keywords: latent HIV reservoirs, latency reversing agent, broadly neutralizing antibody, shock and kill strategies, NK cell therapy, PLGA nanodepots, TNF-α, 3BNC117, ACH-2 cells

Keywords

• latent hiv reservoirs
• latency reversing agent
• broadly neutralizing antibody
• shock and kill strategies
• nk cell therapy
• plga nanodepots
• tnf-α
• 3bnc117
• ach-2 cells