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. 2021 Aug 10;95(17):e0081621.
doi: 10.1128/JVI.00816-21. Epub 2021 Aug 10.

TLR1/2 Agonist Enhances Reversal of HIV-1 Latency and Promotes NK Cell-Induced Suppression of HIV-1-Infected Autologous CD4+ T Cells

Siqin Duan  1 , Xinfeng Xu  1 , Jinshen Wang  1 , Liwen Huang  1 , Jie Peng  2 , Tao Yu  2 , Yang Zhou  3 , Kui Cheng  1 , Shuwen Liu  1   4
Affiliations

TLR1/2 Agonist Enhances Reversal of HIV-1 Latency and Promotes NK Cell-Induced Suppression of HIV-1-Infected Autologous CD4+ T Cells

Siqin Duan et al. J Virol. .

Abstract

The complete eradication of human immunodeficiency virus type 1 (HIV-1) is blocked by latent reservoirs in CD4+ T cells and myeloid lineage cells. Toll-like receptors (TLRs) can induce the reversal of HIV-1 latency and trigger the innate immune response. To the best of our knowledge, there is little evidence showing the "killing" effect of TLR1/2 agonists but only a small "shock" potential. To identify a new approach for eradicating the HIV latent reservoir, we evaluated the effectiveness of SMU-Z1, a novel small-molecule TLR1/2 agonist, in the "shock-and-kill" strategy. The results showed that SMU-Z1 could enhance latent HIV-1 transcription not only ex vivo in peripheral blood mononuclear cells from aviremic HIV-1-infected donors receiving combined antiretroviral therapy but also in vitro in cells of myeloid-monocytic origin targeting the NF-κB and mitogen-activated protein kinase pathways. Interestingly, the activation marker CD69 was significantly upregulated in natural killer (NK) cells, B cells, and monocytes 48 h after SMU-Z1 treatment. Furthermore, SMU-Z1 was able to activate T cells without global T cell activation, as well as increasing NK cell degranulation and gamma interferon (IFN-γ) production, which further block HIV-1-infected CD4+ lymphocytes. In summary, the present study found that SMU-Z1 can both enhance HIV-1 transcription and promote NK cell-mediated inhibition of HIV-1-infected autologous CD4+ T cells. These findings indicate that the novel TLR1/2 agonist SMU-Z1 is a promising latency-reversing agent (LRA) for eradication of HIV-1 reservoirs. IMPORTANCE Multiple in vivo studies showed that many LRAs used in the shock-and-kill approach could activate viral transcription but could not induce killing effectively. Therefore, a dual-function LRA is needed for elimination of HIV-1 reservoirs. We previously developed a small-molecule TLR1/2 agonist, SMU-Z1, and demonstrated that it could upregulate NK cells and CD8+ T cells with immune adjuvant and antitumor properties in vivo. In the present study, SMU-Z1 could activate innate immune cells without global T cell activation, induce production of proinflammatory and antiviral cytokines, and enhance the cytotoxic function of NK cells. We showed that SMU-Z1 displayed dual potential ex vivo in the shock of exposure of latently HIV-1-infected cells and in the kill of clearance of infected cells, which is critical for effective use in combination with therapeutic vaccines or broadly neutralizing antibody treatments aimed at curing AIDS.

Keywords: HIV-1 reservoirs; NK cells; TLR2; latency-reversing agents; shock and kill.

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Figures

FIG 1
FIG 1
SMU-Z1 enhances HIV-1 transcription in latent T cells and PBMCs. (A) Percentages of cells positive for TNF-α, IL-1β, and IL-6 in CD14+ monocytes from HIV-negative and HIV-positive donors (n = 5 for each group). UT, untreated. (B) Representative chart of analysis by flow cytometry of cytokines in monocytes treated or untreated with 1 μM SMU-Z1. (C) Fold change by ELISA of supernatant TNF-α produced in THP1 and TLR2-knockout THP1 cells stimulated with SMU-Z1. (D) Flow chart of J-Lat cells cocultured with PBMCs treated with or without SMU-Z1 in a Transwell chamber. (E) Percentages of GFP-positive J-Lat A2 cells (left) and J-Lat 10.6 cells (right) cocultured with PBMCs from three healthy individuals and treated with graded concentrations of SMU-Z1, as determined by flow cytometry. (F) Percentages of GFP-positive J-Lat A2 cells cocultured with PBMCs and PBMCs without monocytes and treated or untreated with 10 μM SMU-Z1 (n = 6). (G) HIV-1 Gag mRNA levels measured in PBMCs from six aviremic patients that were treated with 1 μM SMU-Z1 for 48 h, calculated by RT-PCR (n = 6). Each symbol corresponds to a different donor, and horizontal lines represent the means. Data were analyzed by one-way ANOVA with the Geisser-Greenhouse correction for multiple comparisons or the Wilcoxon matched-pairs signed-rank test. The P values are defined as follows: *, P < 0.05; **, P < 0.01, compared to control. NS, not significant.
FIG 2
FIG 2
SMU-Z1 reactivates latent HIV-1 in U1 cells. (A and B) U1 cells were treated with SMU-Z1 at concentrations ranging from 0.1 to 8 μM for 48 h (A) and for different time points within 48 h (B), and p24 protein levels were evaluated by Western blotting. (C) HIV Gag mRNA was examined by RT-PCR. (D) ELISA was used to measure p24 in the supernatant of U1 cells at 48 h. (E) TLR2 protein expression was determined by flow cytometry. (F and G) The fold change of HIV-1 p24 in TLR2-knockdown cells stimulated with 1 μM SMU-Z1 was detected by Western blotting (F) and ELISA (G). Data are reported as means ± SEMs from three independent experiments and were analyzed by the Mann-Whitney test or one-way ANOVA with the Geisser-Greenhouse correction for multiple comparisons. The P values are defined as follows: **, P < 0.01; ***, P < 0.001, compared to control.
FIG 3
FIG 3
GO and KEGG analyses identify key pathways affected by SMU-Z1 in patients receiving ART. (A) Volcano plot showing upregulation and downregulation of DEGs between the SMU-Z1-treated group and the untreated group. (B) Heat map showing unsupervised clustering of DEGs within the two groups. UT, untreated. (C) Enrichment analyses for GO terms (referred to as pathways) (left) and KEGG pathways (right) of DEGs. (D) U1 cells were treated with 1 μM SMU-Z1 at the indicated time points, and protein levels of p-IκBα, IκBα, p-p65, p-p38, p38, p-JNK, JNK, p-ERK, and ERK were detected by Western blotting; GAPDH was used as the loading control. ImageJ was used for analysis of relative expression levels. One representative experiment from three independent experiments is shown.
FIG 4
FIG 4
SMU-Z1 activates immune cells and upregulates expression of TLR2. PBMCs from HIV-negative and HIV-positive donors were treated with or without 1 μM SMU-Z1 for 48 h (n = 6 in each group). Cell markers were analyzed by flow cytometry with unstained and FMO controls. UT, untreated. (A) Representative chart of analysis of CD69- and TLR2-positive cells among monocytes and lymphocytes. (B) Percentages of CD69-positive monocytes, NK cells, and B cells (left) and T cells (right) from HIV-negative and HIV-positive donors. (C) Percentages of TLR2-positive monocytes, NK cells, and B cells (left) and T cells (right) from HIV-negative and HIV-positive donors. Data represent means ± SEMs. The Wilcoxon matched-pairs signed-rank test was used. NS, not significant; *, P < 0.05.
FIG 5
FIG 5
SMU-Z1 promotes NK cell degranulation and IFN-γ production. PBMCs from HIV-negative and HIV-positive individuals were either left untreated or treated with 1 μM SMU-Z1 (n = 9 or 8 each group). Cells were then cocultured with K562 cells before analysis by flow cytometry. (A) Gating strategy and flow chart for analyzing CD107a and IFN-γ expression on CD56+ NK cells, with untreated (UT) and IL-15 conditions as negative and positive controls, respectively. (B) CD107a expression (left), intracellular IFN-γ expression (middle), and simultaneous expression of both CD107a and IFN-γ (right) in NK cells, as assessed by surface or intracellular staining between HIV-negative and HIV-positive groups. (C) Comparison of CD107a and IFN-γ production in NK cells from HIV-negative and HIV-positive donors. (D) Supernatant IFN-γ levels from PBMCs treated with or without SMU-Z1 for 48 h, as measured by ELISA (left), and comparison of supernatant IFN-γ levels from PBMCs from HIV-negative or HIV-positive donors (right). Each dot corresponds to one donor. The blue squares indicate cells from HIV-negative donors, the red circles indicate cells from HIV-positive donors, and the lines represent the means. Data were evaluated using the Wilcoxon matched-pairs signed-rank test or the Mann-Whitney test. NS, not significant.
FIG 6
FIG 6
SMU-Z1 inhibition of HIV-1 replication is mediated by PBMCs and NK cells. (A) Supernatant p24 levels from PBMCs infected with HIV-1 strains in the presence of 1 μM SMU-Z1 (n = 8). Each color represents a unique donor. (B) Longitudinal evaluation of p24 protein expression over 7 days from one donor treated with the indicated concentrations of SMU-Z1. One-way ANOVA with the Geisser-Greenhouse correction for multiple comparisons was used. *, P < 0.05; **, P < 0.01. (C) HIV-1 Gag mRNA expression in PBMCs assessed by RT-PCR at day 5 (n = 4). (D) Flow chart of the coculture assay with NK cells and T cells from PLWH. (E) Supernatant p24 from infected NK cells-T cells assessed by ELISA (n = 6) (Wilcoxon matched-pairs signed-rank test) (left) and intracellular p24 from CD4+ T cells analyzed by flow cytometry (n = 5) (Mann-Whitney test) (right). Lines represent the means. Each dot corresponds to one donor. Data were analyzed by the Wilcoxon matched-pairs signed-rank test and one-way ANOVA with the Geisser-Greenhouse correction for multiple comparisons.
FIG 7
FIG 7
SMU-Z1 analogs reactivate latent HIV-1 in U1 cells. (A) Chemical structures of SMU-Z1 and its analogs. (B) Intracellular and supernatant p24 levels in U1 cells treated with SMU-Z40, Z37, and Z25 at the indicated concentrations for 48 h, measured by Western blotting (upper) and ELISA (lower). (C) Supernatant p24 expression in U1 cells stimulated with SMU-Z31 or Z12 at graded concentrations. Data represent the means ± SEMs of three independent experiments, and one-way ANOVA was used for data analysis. (D) 50% effective concentration (EC50) values of SMU-Z1 and its analogs in activating TLR2 signaling in HEK-Blue-hTLR2 cells. The P values were defined as follows: **, P < 0.01; ***, P < 0.001, compared to control.
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