TLR7 agonists induce transient viremia and reduce the viral reservoir in SIV-infected rhesus macaques on antiretroviral therapy

Abstract

Antiretroviral therapy (ART) can halt HIV-1 replication but fails to target the long-lived latent viral reservoir. Several pharmacological compounds have been evaluated for their ability to reverse HIV-1 latency, but none has demonstrably reduced the latent HIV-1 reservoir or affected viral rebound after the interruption of ART. We evaluated orally administered selective Toll-like receptor 7 (TLR7) agonists GS-986 and GS-9620 for their ability to induce transient viremia in rhesus macaques infected with simian immunodeficiency virus (SIV) and treated with suppressive ART. In an initial dose-escalation study, and a subsequent dose-optimization study, we found that TLR7 agonists activated multiple innate and adaptive immune cell populations in addition to inducing expression of SIV RNA. We also observed TLR7 agonist-induced reductions in SIV DNA and measured inducible virus from treated animals in ex vivo cell cultures. In a second study, after stopping ART, two of nine treated animals remained aviremic for more than 2 years, even after in vivo CD8⁺ T cell depletion. Moreover, adoptive transfer of cells from aviremic animals could not induce de novo infection in naïve recipient macaques. These findings suggest that TLR7 agonists may facilitate reduction of the viral reservoir in a subset of SIV-infected rhesus macaques.

Fig. 1. Lymphocyte activation and transient plasma viremia in SIV-infected monkeys on ART after GS-986 treatment (study 1)

Macaques (n = 4; blue) were treated with escalating doses of GS-986 over the 14 weeks of the study. Dose concentrations were 0.1 and 0.2 mg/kg, and then five consecutive doses of 0.3 mg/kg were given orally every other week. The control group received formulation vehicle only (n = 6, red). (A) Activation of lymphocyte subsets in the peripheral blood of rhesus macaques treated with escalating doses of GS-986 was monitored by flow cytometric detection of CD69. Expression of CD69 was measured in CD4⁺ T cells, CD8⁺ T cells, NK cells, and B cells. CD69 expression was measured at pre–GS-986 dosing (0 hours) and then at 24, 48, 72, and 168 hours after GS-986 treatment. The absolute differences in percent CD69 expression by cells from time of treatment (baseline), for each of seven doses, are shown. Bar height indicates mean difference. Error bars indicate SEM. (B) Number of SIV RNA copies (expressed as log₁₀ copies/ml) in plasma of SIV-infected rhesus macaques on ART after a single oral dose of TLR7 agonist every other week.

Fig. 2. Effect of TLR7 agonist on cell-associated viral DNA in CD4⁺ T memory cells from SIV-infected rhesus macaques on ART (study 1)

PBMCs, inguinal LNMCs, and GMMCs were collected from rhesus macaques before the administration of the TLR7 agonist GS-986 (pre-TLR7) and 1 week after the seventh dose of GS-986 (post-TLR7). Mononuclear cells were isolated from each tissue and sorted into four populations of CD4⁺ T cells: naïve, central memory (CM), transitional memory (TM), and effector memory (EM). Memory T cell subsets were then pooled. SIV DNA in the pooled memory T cell populations is expressed as log₁₀ copies per 10⁶ cells. Threshold of detection, as indicated by the dotted line, is <3 copies per 10⁶ cells.

Fig. 3. The kinetics of SIV RNA rebound in the plasma of SIV-infected rhesus macaques after ART cessation (study 1)

Log₁₀ SIV RNA copies/ml in plasma was assessed in SIV-infected rhesus macaques for days 1 to 84 after ART cessation. Median log₁₀ SIV RNA copies/ml for GS-986–treated (blue) and control vehicle-treated (red) animals is shown relative to the value on the day that ART was stopped (day 0).

Fig. 4. Activation of effector memory T cells by TLR agonists (study 2)

Activation of naïve (CD95⁻CD28⁺), central memory (CD95⁺CD28⁺), and effector memory (CD95⁺CD28⁻) T cells for CD4⁺ (A) and CD8⁺ (B) T cell populations in the peripheral blood of SIV-infected rhesus macaques on ART is shown. Rhesus macaques were treated with vehicle (red), GS-986 (0.1 mg/kg; blue), GS-9620 (0.05 mg/kg; purple), or GS-9620 (0.15 mg/kg; black). T cell activation was measured by flow cytometric detection of CD69. CD69 expression within each T cell subset was measured at the time of the first dose (baseline) and at 24, 48, and 72 hours (doses 1 to 7 only) after the first dose (time points indicated by black arrows on x axis). Data for doses 1, 3, 5, and 7 are shown on the left, and data for doses 11 to 19 are shown on the right. Changes in CD69 expression are shown as absolute difference in percent from baseline for naïve, central memory, and effector memory T cell subsets.

Fig. 5. Transient plasma viremia induced by TLR7 agonist dosing of SIV-infected macaques on ART (study 2)

SIV-infected rhesus macaques on ART were treated with vehicle (n = 2; red), GS-986 (0.1 mg/kg; n = 3; blue), GS-9620 (0.05 mg/kg; n = 3; purple), or GS-9620 (0.15 mg/kg; n = 3; black) over the 23 weeks of the study. Plasma viral RNA in vehicle-treated or TLR7 agonist–dosed animals is shown. Black arrows indicate the timing of each TLR7 agonist dose. The horizontal dotted line represents the limit of detection of plasma viremia, which was 50 SIV RNA copies/ml.

Fig. 6. Changes in cell-associated viral DNA in mononuclear cells isolated from tissues of SIV-infected macaques treated with TLR7 agonists (study 2)

(A) Mononuclear cells were isolated from tissues of animals treated with either vehicle (red), GS-986 (0.1 mg/kg; blue), GS-9620 [0.05 mg/kg (purple) or 0.15 mg/kg (black)]. Mononuclear cells were sorted into four populations of CD4⁺ T cells: naïve, central memory, transitional memory, and effector memory, and then memory T cell subsets for GMMCs were pooled to increase sensitivity for SIV DNA analysis. SIV DNA is expressed as log₁₀ copies per 10⁶ CD4⁺ memory T cells at the time of administration of the TLR7 agonist (pre-TLR7) and a week before stopping ART (post-TLR7). (B) SIV DNA (log₁₀ copies per 10⁶ cells) in each subpopulation of CD4⁺ T cells from TLR7-treated rhesus macaques pre- and post-TLR7 agonist treatment. The threshold of detection, as indicated by the horizontal dotted line, is <3 copies per 10⁶ CD4⁺ T cells. Changes in SIV DNA between pre- and post-TLR7 treatment were compared using a Wilcoxon matched paired test. (C) Results of an ex vivo mitogen-stimulated virus production assay. PBMCs or LNMCs (post-19th dose only) were isolated from macaques before the administration of the first dose of TLR7 agonist (pre-TLR7) and then after the 10th and 19th doses of the TLR7 agonist (post-TLR7). Cells were grown in basal culture medium supplemented with 10 mM raltegravir, with or without concanavalin A (ConA; 5 μg/ml) for 6 days. The number of SIV RNA copies/ml of supernatant on day 6 is shown. (D) SIV plasma RNA rebound kinetics in SIV-infected macaques after cessation of ART. The viral rebound kinetics after stopping ART were assessed in groups of animals treated with either vehicle control (n = 2; red), GS-986 (0.1 mg/kg; n = 3; blue), GS-9620 (0.05 mg/kg; n = 3; purple), or GS-9620 (0.15 mg/kg; n = 3; black). Log plasma virus RNA was assessed between days 1 and 210 after ART cessation.

Fig. 7. Number of CD8⁺ lymphocytes and plasma SIV RNA kinetics after in vivo CD8⁺ cell depletion

(A) CD8⁺ lymphocytes were depleted in vivo by infusion of monoclonal antibody MT807R1, and numbers of CD8⁺ cells per microliter of whole blood were monitored by flow cytometry for four macaques (two aviremic and two viremic). Log plasma virus RNA was assessed between days 0 and 35 after CD8⁺ cell depletion. (B) Four SIV-naïve rhesus macaques served as recipients for adoptive transfer of cells from two donor rhesus macaques in remission. Left: In the first transfer, about 50 million frozen PBMCs and LNMCs from donor animals 177-10 and 344-10, isolated after the initiation of ART but before TLR7 agonist treatment, were used for adoptive transfer. For each donor rhesus macaque (177-10, black; 344-10, blue), PBMCs and LNMCs were thawed, combined, and then infused into two naïve rhesus macaques. Right: In the second transfer, about 120 million fresh PBMCs and LNMCs from each donor were isolated at 448 days after cessation of ART. PBMCs and LNMCs were combined and then infused into two additional naïve rhesus macaques. Log plasma virus RNA was assessed between days 0 and 28 after adoptive transfer.