Specific Elimination of Latently HIV-1 Infected Cells Using HIV-1 Protease-Sensitive Toxin Nanocapsules

Abstract

Anti-retroviral drugs suppress HIV-1 plasma viremia to undetectable levels; however, latent HIV-1 persists in reservoirs within HIV-1-infected patients. The silent provirus can be activated through the use of drugs, including protein kinase C activators and histone deacetylase inhibitors. This "shock" approach is then followed by "kill" of the producing cells either through direct HIV-1-induced cell death or natural immune mechanisms. However, these mechanisms are relatively slow and effectiveness is unclear. Here, we develop an approach to specifically target and kill cells that are activated early in the process of virus production. We utilize a novel nanocapsule technology whereby the ricin A chain is encapsulated in an inactive form within a polymer shell. Specificity for release of the ricin A toxin is conferred by peptide crosslinkers that are sensitive to cleavage by HIV-1 protease. By using well-established latent infection models, J-Lat and U1 cells, we demonstrate that only within an HIV-1-producing cell expressing functional HIV-1 protease will the nanocapsule release its ricin A cargo, shutting down viral and cellular protein synthesis, and ultimately leading to rapid death of the producer cell. Thus, we provide proof of principle for a novel technology to kill HIV-1-producing cells without effects on non-target cells.

Fig 1. The formation of ricin A nanocapsules (n-ricinA).

A) Illustration of the synthesis, delivery and function of HIV-1 PR-sensitive nanocapsules: I) self-assembly of positive monomer A, hydrophilic monomer B and HIV-1 PR-sensitive crosslinker C on the surface of ricin A molecules; II) formation of HIV-1 PR-sensitive n-ricinA; III) delivery; IV) reactivation by drugs; V) specific release upon intracellular HIV-1 PR and VI) killing only the reactivated cells. B) TEM image of n-ricinA (Scale bar = 100 nm). C) Particle size distributions and D) surface charge (zeta potential) of native ricinA and n-ricinA. E) Stability of n-ricinA in PBS at 4°C for two weeks. Size of n-ricinA was monitored by Dynamic light scattering.

Fig 2. Minimal non-specific cytotoxicity of n-ricinA.

Dead cells of J-Lat were removed by MACS^® LS columns. Dead-cell-removed J-Lat cells were transduced with 1 μM of native ricinA, n-ricinA_BIS (ricin A nanocapsule with non-degradable crosslinker), n-BSA (BSA (bovine serum albumin) nanocapsule with HIV-1 PR cleavable peptide as crosslinker), and antiCD4-ricinA (immunotoxin anti-CD4 conjugated ricinA), respectively. Cell cytotoxicity was determined with CytoTox-Glo^TM cytotoxicity kit (Promega) using a plate reader. Cytotoxicity was calculated by dividing the dead cell luminescence by the total cell luminescence. Error bars: Mean±SD.

Fig 3. Specific release of n-ricinA to HIV-1 PR protein treatment in vitro.

A) SDS-PAGE of ricin A released from nanocapsules with protease treatment. Samples were treated with or without 50 nM protease (HIV-1 PR or HIV-2 PR) in protease working condition at 37°C for 2 hours, and applied to SDS-PAGE, followed by Coomassie Brilliant Blue staining. B) Knockdown of luciferase in HIV-1-infected 293-Affinofile cell by shLuc DNA cassette released from HIV-1-PR sensitive DNA nanocapsules with different ratios (A-3:0; B-2:1; C-1:2 and D-0:3) of HIV-1-PR-senstive to non-degradable crosslinker. Error bars: Mean±SD.

Fig 4. Rapid killing of reactivated HIV-1 infected cells by n-ricinA.

A) A time course of HIV-1 PR expression in J-Lat after prostratin reactivation. A FRET peptide substrate of HIV-1 PR was added to cell lysate to monitor HIV-1 PR activity with the fluorescence signal at 490 nm. B) A time course of the relative cell numbers of J-Lat after prostratin reactivation without transduction, with transduction of n-ricinA, or with incubation of antiCD4-ricinA. Error bars: Mean±SD.

Fig 5. Specific elimination of J-Lat cells by n-ricinA after reactivation.

A) Specific elimination of J-Lat cells with reactivation of provirus. J-Lat cells and control Jurkat expressing BFP were co-cultured at a 1:1 ratio. Cells were transduced with n-ricin, n-ricinA_GDMA, antiCD4-ricinA, or ricinA for 4 hours. Cells were then treated with 10 μM prostratin (Pro) for reactivation of provirus. With reactivation, three populations, reactivated J-Lat (EGFP+), control Jurkat (BFP+), and unreactivated Jurkat cells (EGFP-BFP-), were clearly separated by fluorescence. The cells with Indinavir sulfate (IDV) treatment were pretreated with 10 μM IDV for 18 hours before transduction. B) Relative cell numbers of these three populations in J-Lat cells were further analyzed both on Day 1 and Day 2 after reactivation. C) The production of virus from transduced J-Lat cells one day after reactivation. One million cells were cultured in 500 μL medium with 10 μM of prostratin, and p24 in the culture supernatant were quantitated by ELISA at 12 hours and 24 hours after reactivation. Error bars: Mean±SD.

Fig 6. Specific elimination of U1 cells by n-ricinA after reactivation.

A) Two days after 10 μM prostratin reactivation, levels of intracellular p24 antigen were analyzed by flow cytometry. B) Relative cell numbers of U1 cells transduced with ricinA, n-ricinA and antiCD4-ricinA on Day 1 and Day 2 after prostratin reactivation. Error bars: Mean±SD.

Fig 7. Elimination of latently infected primary T cells by n-ricinA after reactivation.

A) 2x10⁶/mL of naïve CD4+ T cells were infected with 2000 ng/mL of wild type HIV-1 NL4-3 and cultured with 5 ng/mL of IL-7 and 5 ng/mL of TGFβ-1 for 2 weeks to obtain the latently infected primary T cells. Cells were then transduced with nanocapsule for 4 hours with or without 1μg/mL of CD3 and CD28 antibody reactivation for three days, levels of intracellular p24 antigen in latently infected primary T cells were stained by KC57-RD1 and analyzed by flow cytometry. B) Intracellular p24 levels of latently infected primary T cells with or without reactivation were measured based on flow cytometry results. Data are presented as the mean fluorescence intensity of p24 staining. C) The production of virus p24 from reactivated primary T cells in culture supernatant three days after reactivation. Mock: cells without virus infection. Control: infected cells not transduced with n-ricinA. Error bars: Mean±SD.