Effects of LNP-Bry on latent virus expression and induction of CD69.

<p>(A) Representative flow cytometry plots of GFP expressing J-Lat 10.6 cells incubated with 10 nM bryostatin-2 or brostain-2 loaded LNP (LNP-Bry). (B) Percentage GFP+ cells was measured after incubation of LNP-Bry or bryostatin-2 with J-Lat 8.4 cells for 16 hr. (C) Percentage GFP+ cells was measured after incubation of LNP-Bry or bryostatin with J-Lat 10.6 cell lines for 16 hr. (D) Representative flow cytometry plots displaying the synergistic effect of 2 mM sodium butyrate (NaBut) in conjunction with LNP-Bry in J-Lat 8.4 cells. (E) J-Lat 8.4 cells were incubated with a fixed dose of NaBut (2 mM) and increasing dose of bryostatin-2 or LNP-Bry. (F) Representative flow cytometry plots displaying CD69 induction in primary resting CD4+ cells after incubation with LNP-Bry or bryostatin-2 for 16 hr. (G) Percentage CD69+ cells as detected by flow cytometric analysis. (H) Fold increase in CD69 mean fluorescence intensity was measured after incubation with increasing amounts of LNP-Bry or bryostatin-2 in primary CD4+ cells. (I) Flow cytometry plots displaying the CD4+/CD8+ cell profiles from mock or NL4-3 infected SCID-hu (Thy/Liv) implants. (J) In the presence of 1 µM raltegravir, CD4 single-positive thymocytes were stimulated <i>ex-vivo</i> with anti-CD3/CD28 beads, 10 nM bryostatin-2 or 10 nM LNP-Bry for 48 hr and assayed for intracellular Gag protein by flow cytometry. Error bars indicate the standard deviation of triplicate data points and are representative of at least 2 experiments. * p<0.01 as compared with media only (infected non-stimulated cultures) or LNP-con (non-drug loaded nanoparticles) in a paired t-test.</p

Lipid nanoparticle (LNP) characterization and uptake in various cell types.

<p>(A) LNP were synthesized and characterized for their size by dynamic light scattering. (B) The membrane stain wheat germ agglutinin (WGA) visualized in blue was used with fluorescent microscopy in order to visualize the uptake of LNP-FITC (green) after a 16 hr incubation with HeLa cells. (C) WGA visualized in red was used to observe LNP-FITC (green) uptake in primary macrophages. (D) CEM cells were visualized by phase contrast images with LNP (green) using fluorescent microscopy. (E) LNP uptake in CEM cells was dose and energy dependent as detected by fold increase in FITC mean fluorescent intensity (MFI) using flow cytometry. (F) LNP uptake in CEM cells also increased over time as detected by flow cytometry.</p

Simultaneous incorporation of the protease inhibitor nelfinavir (Nel) and bryostatin-2 (Bry) into the lipid nanoparticles (LNP-Bry-Nel) can both activate latent virus expression and inhibit viral spread.

<p>(A) CEM cells were infected with HIV_NL4-3 and the cells were incubated for 3 days in the presence of various drug combinations including LNP-Bry-Nel. Viral p24 protein in the culture supernatant was measured by ELISA. (B) LNP-Bry-Nel was further tested for its ability to activate latent virus in J-Lat 10.6 cells as measured by induction of GFP expression. Error bars indicate the standard deviation of triplicate data points and are representative of at least 2 experiments. Media represents untreated infected cultures and LNP-con represents non-drug loaded nanoparticles.</p

Targeting LNP to primary CD4+ cells and activation of latent virus expression.

<p>(A) Schematic representation showing construction of the LNP-CD4 (anti-CD4 coated nanoparticles) and LNP-Iso (isotype control antibody coated nanoparticles) with bryostatin-2 incorporation. (B) Fluorescent microscopy image displaying the targeting of LNP-CD4 (green) to CD4+ cells (red) while minimizing cellular association with CD8+ cells (violet) in the same culture of total peripheral blood mononuclear cells (PBMCs) after a 30 min exposure. (C) Fluorescent microscopy image displaying the lack of targeting by LNP-Iso (green) to CD4+ cells (red) or CD8+ cells (violet). (D) Quantification of LNP-CD4 uptake in PBMCs using flow cytometry (% cells CD4+ with increase in FITC-LNP uptake) after a 30 min exposure. (E) Representative flow cytometry plots of CD4+ and CD8+ cells analyzed for the induction of the early activation marker CD69 after incubation with CD4 targeting nanoparticles for 16 hr. (F) Bar graphs illustrating the fold increase in CD69 MFI in CD4+ and CD8+ cells after incubation with LNP-CD4 Bry. (G) LNP-CD4 Bry was further tested for its ability to stimulate latent virus expression in J-Lat 8.4 cells. (H) <i>Ex vivo</i> CD4 single-positive thymocytes isolated from infected SCID-hu (Thy/Liv) implants were incubated with LNP-CD4 Bry and viral p24 protein present in the culture supernatants was analyzed after 48 hr. Error bars indicate the standard deviation of triplicate data points and are representative of at least 2 experiments. * p<0.01 in a paired t-test as indicted in (F) and as compared with media only or LNP-con in (G) and (H).</p

In vivo activation of latent HIV with a synthetic bryostatin analog effects both latent cell "kick" and "kill" in strategy for virus eradication

<div><p>The ability of HIV to establish a long-lived latent infection within resting CD4+ T cells leads to persistence and episodic resupply of the virus in patients treated with antiretroviral therapy (ART), thereby preventing eradication of the disease. Protein kinase C (PKC) modulators such as bryostatin 1 can activate these latently infected cells, potentially leading to their elimination by virus-mediated cytopathic effects, the host’s immune response and/or therapeutic strategies targeting cells actively expressing virus. While research in this area has focused heavily on naturally-occurring PKC modulators, their study has been hampered by their limited and variable availability, and equally significantly by sub-optimal activity and <i>in vivo</i> tolerability. Here we show that a designed, synthetically-accessible analog of bryostatin 1 is better-tolerated <i>in vivo</i> when compared with the naturally-occurring product and potently induces HIV expression from latency in humanized BLT mice, a proven and important model for studying HIV persistence and pathogenesis <i>in vivo</i>. Importantly, this induction of virus expression causes some of the newly HIV-expressing cells to die. Thus, designed, synthetically-accessible, tunable, and efficacious bryostatin analogs can mediate both a “kick” and “kill” response in latently-infected cells and exhibit improved tolerability, therefore showing unique promise as clinical adjuvants for HIV eradication.</p></div

HIV Proteins Block the Antiviral Signalling of IPS-1.

<p>(A) IPS-1 overexpression was used to induce IFN-β reporters. HEK 293T cells were cotransfected with expression plasmids for different HIV protein products and the levels of IFN-β luciferase were determined. Reporter activation in A-C was measured, and graphed in relative light units normalized to cotransfected Renilla luciferase (designated RLUs). * are values with p<1x10^-4 as compared to IPS-1 alone. (B) HEK 293T cells were co-transfected with IPS-1 and vector control, <i>Vpu</i> or <i>Nef</i> and an ISRE reporter. * are values with p<1x10^-3 as compared to IPS-1 alone. (C) HEK 293T cells were co-transfected with IPS-1 and vector control, <i>Vpu</i> or <i>Nef</i> and a κB reporter. * are values with p<1x10^-3 as compared to IPS-1 alone. (D) CEM cells were stably transduced with either Vpu or Nef. The cells were then infected with VSV that expresses GFP upon active infection. After 48 hours, supernatants were collected and monitored by ELISA for the amount of secreted IFN. * are values with p<1x10^-4 as compared to vector infected cells. (E) In addition, cells were collected from the infection in D and assayed for GFP levels, an indicator of VSV replication in the culture.</p

Modulation of Type I Interferon Affects Different Aspects of HIV Pathogenesis.

<p>1. HIV infection of a target CD4+ T cell allows for proviral integration. 2. Normally HIV RNA is recognized by RIG-I and should signal through IPS-1. However, as we show here, Vpu and Nef lead to the degradation of IPS-1. 3. Ablation of primary IFN release even in the presence of viral growth allows for enhanced virion production. 4. Increased levels of HIV in the microenvironment allows for increased recognition by pDCs. 5. This leads to increased signalling in the pDC even though these cells do not support efficient HIV replication. 6. Increased signalling leads to subsequent release of increased IFN levels. 7. Increased IFN leads to upregulation of TRAIL on pDCs and decrease in bystander CD4+ T cell proliferation and activation.</p

HIV Infection Could But Does Not Induce IFN.

<p>(A) CEM T cells were infected with either HIV or VSV and after 48 hours the levels of IFN-α in supernatants were determined by ELISA. (B) Lysates from purified HIV virions were transfected along with an IFN-β reporter into HEK 293T cells. Lysates were treated as indicated. IFN-β reporter activation was measured, and graphed in relative light units normalized to cotransfected Renilla luciferase (designated RLUs).</p

Vpu and Nef are Required for Disruption of IFN Signalling by HIV Infection.

<p>(A) A mutant of HIV was constructed lacking expression of the Vpu and Nef gene products. CEM T cells were infected with wildtype (NL4.3) or the mutant (Delta Vpu/Nef) and after 48 hours the level of intracellular p24 was analysed by flow cytometry to verify equal infection levels. (B) Protein levels of STAT1, IRF3 and IPS-1 were analysed via western blotting of cell pellets from the infection in B. (C) The level of p24 in the supernatant of infected cells was monitored by ELISA over the three days of infection. (D) The level of IFN-α was quantitated via ELISA of the supernatant of the infected cells from B. The increase in the IFN-α released from the mutant compared to the wildtype has a p value <0.00001. (E) Primary CD4+ T cells were infected with wildtype (NL4.3) or the mutant (Delta Vpu/Nef) for three days and the level of p24 in the supernatant was analysed by ELISA to monitor viral infection. (F) The level of IFN-α was quantitated via ELISA of the supernatant of the infected cells from E.</p

Induction of CD69 expression by compounds in primary CD4+T cells.

<p><b>A)</b> Isolated CD4+ T cells from healthy donors were stimulated for 24h with the indicated compound and then analyzed for CD69 expression by flow cytometry. Error bars represent ±1 Standard Error (N = 3 different primary cell donors). * Indicates p = 0.05 for all tested compounds in untreated vs. 1000 nM treatment conditions (1-sided Wilcoxon rank sum test). <b>B)</b> EC₅₀ comparison showing correlation between HIV latency activation and induction of CD69 expression by PKC activators. Data for prostratin analogs are from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006575#ppat.1006575.ref018" target="_blank">18</a>], and data for bryostatin analogs are from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006575#ppat.1006575.ref026" target="_blank">26</a>] and this study. Data were analyzed by linear regression. The p-value reflects evidence against the null hypothesis of no effect (i.e. that the regression coefficient is zero). The p-value being below 0.05 suggests that CD69 activation is significantly related to latency activation.</p