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. 2017 Sep 21;13(9):e1006575.
doi: 10.1371/journal.ppat.1006575. eCollection 2017 Sep.

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

Matthew D Marsden  1 , Brian A Loy  2 , Xiaomeng Wu  3 , Christina M Ramirez  4 , Adam J Schrier  2 , Danielle Murray  5 , Akira Shimizu  2 , Steven M Ryckbosch  2 , Katherine E Near  2 , Tae-Wook Chun  5 , Paul A Wender  2 , Jerome A Zack  1   3
Affiliations

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

Matthew D Marsden et al. PLoS Pathog. .

Abstract

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 in vivo tolerability. Here we show that a designed, synthetically-accessible analog of bryostatin 1 is better-tolerated in vivo 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 in vivo. 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.

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Conflict of interest statement

I have read the journal’s policy and the authors of the manuscript have the following competing interests: Neurotrope Biosciences and BryoLogyx have licensed some of this technology from Stanford University (P.A.W.), the former for the treatment of neurological disorders such as Alzheimer's disease and the latter for use in HIV/AIDS eradication and cancer immunotherapy. P.A.W is a consultant to both companies and a co-founder of BryoLogyx.

Figures

Fig 1
Fig 1. A synthetic bryostatin analog exhibits pan-PKC binding selectivity similar to bryostatin 1.
A) Structure of bryostatin 1. B) Structure of bryostatin analog SUW133. C) & D) Affinities of bryostatin 1 and SUW133 in binding to conventional and novel PKC isoforms.
Fig 2
Fig 2. Activation of HIV from latency in patient-derived cells.
CD4+ T cells from ART-treated patients (described in S1 Table) were isolated and exposed to SUW133 or other stimuli before quantification of cell-free virion-associated viral RNA levels in the culture supernatant. A) Resting CD4+ T cells from 6 aviremic patients were stimulated for 36h with media alone or 50 nM of SUW133, p value was calculated using a 2-sided Wilcoxon rank sum test. B) CD4+ T cells from an additional 3 ART-treated, aviremic patients were subjected to stimulation for 48h with the indicated compounds. Using a rank sum test (for independence), SUW133 induced significantly higher (p = 0.03) amounts of virus production versus all other stimuli except maximal T cell stimulation (anti-CD3 + anti-CD28 costimulation).
Fig 3
Fig 3. Induction of CD69 expression by compounds in primary CD4+T cells.
A) 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) EC50 comparison showing correlation between HIV latency activation and induction of CD69 expression by PKC activators. Data for prostratin analogs are from [18], and data for bryostatin analogs are from [26] 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.
Fig 4
Fig 4. In vivo stimulation in C57bl6 mice.
A) In vivo toxicity and bioactivity (CD69 upregulation in splenocytes) of bryostatin 1 at different doses of compound in C57/bl6 mice. The “% survival” indicates the percent of mice that survived the 17-24h overnight time course. Error Bars = SEM, *p = 0.05 (One-sided Wilcoxon rank sum test of treatment versus control [0 dose]). The number of mice (N) used for each comparison is given at the top of the figure. B) Comparative bioactivity and tolerability of bryolog SUW133 in C57/bl6 mice. Statistics and labels are as described in part A. C) Time course (12h, 24h, 30h, 3 days, and 9 days post-stimulation) of CD69 activation in splenocytes following in vivo administration of SUW133. Each data-point represents a different mouse (3–6 mice per group. *p = 0.05 (One-sided Wilcoxon rank sum test of treatment versus control).
Fig 5
Fig 5. Bryolog induces stimulation of human cells in vivo.
Humanized BLT mice were injected with SUW133 and the blood and spleen evaluated 24h later for human cells expressing activation markers. A) % CD69+ cells or CD25+ cells in different human T cell subsets in peripheral blood. B) % CD69+ cells or %CD25+ cells in different human T cell subsets in spleen. N = 3 mice/group control and 4 mice/group SUW133. *p<0.05 (Wilcoxon rank sum test).
Fig 6
Fig 6. Synthetic bryolog can induce expression of latent HIV in vivo in humanized mice.
A) Experimental scheme: Humanized BLT mice were infected with HIV strain NL-HA, which expresses the HA epitope on the surface of productively-infected cells (including those induced to express from latency). Infected mice were treated with ART (emtricitibine, tenofovir disoproxil fumarate, and raltegravir) for 3–5 weeks to suppress viral loads. Mice were then bled to obtain a baseline data point for HA expression and treated with a single dose of SUW133. After an overnight incubation, mice were sacrificed and the frequency of HIV-expressing human cells re-assessed in blood and spleen. B) Example flow cytometry plot showing low numbers of HA+ (HIV-expressing) human cells in the peripheral blood of ART-suppressed mice. C) Peripheral blood staining (from the same animal show in in panel B) 17h after administration of SUW133, showing increase in HA+ (HIV-expressing) human cells. D) Percentage of HA+ human cells in the peripheral blood before and after administration of SUW133 (each line represents an individual mouse). E) Percentage of human HA+ (HIV-expressing) cells in spleen of unstimulated ART-treated infected animals or those that received the indicated dose of SUW133 (each data-point represents an individual mouse). P-values were determined using a 2-sided Wilcoxon sign rank test for paired data.
Fig 7
Fig 7. Death of cells reactivated from latency in vivo.
A) Example staining of HA+ (HIV-expressing) versus HA-negative cells using “Ghost Red”, which stains dead or dying cells with disrupted membranes. B) Percentage of Ghost Red bright (dead/dying) cells in the HA-negative versus HA+ (HIV-expressing) cells in spleen. C) Mean fluorescent intensity of Ghost Red in HA-negative versus HA+ cells in spleen. Each data-point represents an individual mouse. P-values were assessed using a 2-sided Wilcoxon rank sum test for independent data.
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