Nanoparticle delivery of molecular agents to tackle HIV latency
Antiretroviral therapy (ART) is currently only capable of controlling HIV-1 replication but not completely eradicating virus from HIV-infected individuals. Even after prolonged therapy, if treatment is stopped, virus rebounds. Many reservoirs of replication competent HIV-1 virus have been identified but the most important are long lived latently infected CD4+ T cells. One approach toward tackling HIV-1 latency is the activation of HIV viral production by latency reversal agents (LRAs) such as Histone deacetylase inhibitors, HDACi, Bromodomain Inhibitors and PKC agonists. Virus production will lead to virus mediated cytolysis or clearance through immune recognition (often called “Shock and Kill”). However, LRAs are not specific for HIV, have off target effects and lack sufficient potency to eliminate infected T cells. Nanoparticle delivery systems possess several advantages over more traditional drug delivery methods but there are challenges for uptake of nanoparticles in resting T-cells, the major reservoir of latent HIV.
Preliminary work in our laboratories has investigated whether biocompatible polymer nanoparticles could be successfully delivered to a CD4+ T cell line. Nanoparticles <300nm were successfully taken up by CD4+ T cells. Using a LIVE/Dead violet stain that binds to the amine groups in nanoparticles, we could distinguish particles within and on the outside of cells.
In this project, we aim to develop biodegradable biopolymer nanoparticles to deliver latency reversing agents to resting CD4+ T cells. We will systematically determine the effects of size on nanoparticle uptake and will demonstrate the location of the nanoparticle using imaging flow cytometry and confocal microscopy. We will then load these nanoparticles with the HDACi romidepsin and PKC agonist bryostatin and determine the effects on latency reversal compared to free drug. Finally, we will optimise nanoparticles delivery of HIV RNA to resting CD4+ T cells to determine the effects of HIV RNA on the innate immune signalling pathway.
Nucleic acid sensing by T cells during HIV-1 infection
Receptors of the innate immune system sense foreign molecules and structures such as the highly conserved microbe-associated molecular patterns (MAMPs) like sugars, lipids or proteins, which occur only on microbes like bacteria, fungi or parasites. Since viruses use the biosynthesis machinery of the infected (host) cell itself, they do not incorporate completely foreign molecules. Therefore, viruses are predominantly recognised by unusual localisation, structure or modification of their nucleic acids. Stimulation of innate nucleic acid sensing immune receptors trigger cell-autonomous antiviral defence mechanisms, which may also include apoptosis, and secretion of cytokines and chemokines, which lead to alarming of neighbouring cells and attracting immune cells, in this way initiating adaptive immune responses.
The human immunodeficiency virus 1 (HIV-1) is a lentivirus causing Acquired Immune Deficiency Syndrome (AIDS) with fatal outcome in humans if left untreated. A small cohort of patients, so called elite controllers, are able to limit progression of AIDS in absence of antiretroviral therapy (ART) due to their capacity to raise a rapid and sustained IFN-a/b response in dendritic cells, indicating that the antiviral IFN-a/b response also interferes with HIV-1 replication. Although, ART which directly target different processes during HIV replication has led to a substantial reduction in morbidity and mortality, the major barrier to curing HIV is the long term persistence of latently infected resting memory T-cells in HIV-infected patients. Such latently infected T cells express HIV-1 RNA, which was described to stimulate nucleic acid receptors in principle, but have no upregulated IFN-a/b induced genes, raising the question, if present RNA sensor pathways (e.g. RIG-I) are suppressed in those cells by viral or endogenous epigenetic regulation. Although nucleic acid receptors are well studied in somatic and myeloid cells, their presence and function in T cells is poorly defined. The aim of this project is to analyse the response of T cells to nucleic acid receptor stimulation and if HIV-1, especially in latently infected T cells (which express viral RNA), counteracts such viral recognition mechanisms. Conversely, the effect of transfected immunostimulatory nucleic acids on re-activation of latently HIV-1 infected cells will be analysed.