TLR1/2 Agonist Enhances Reversal Of HIV-1 Latency And Promotes NK Cell-Induced Elimination Of HIV-1-Infected T Cells

Human immunodeficiency virus type 1(HIV-1)can be controlled by combined antiretroviral therapy(cART)and viral loads in the peripheral blood are below the detection limit.However,complete eradication of HIV and achievement of a functional cure is prevented by the major obstacle of the latent HIV-1 reservoirs,which exist in peripheral blood and lymphoid tissues.Numerous latency-reversing agents(LRAs)have been investigated since the "shock and kill" strategy was proposed,and only a small pool of candidates have been studied or are undergoing in clinical trials.Most of these LRAs could activate viral transcription but could not induce "killing" effectively.Therefore,a dual function LRA is needed for elimination of latent reservoirs.Up to now,none of these first-generation LRAs,including histone deacetylase inhibitors(HDACi)have been shown to substantially reduce the size of HIV reservoirs.Meanwhile,the second generation of LRAs such as TLR agonists have attracted much attention with potential for activating innate antiviral immune response,among which TLR7 and TLR9 agonists have been mainly studied in clinical trials.However,to the best of our knowledge,there is no prior evidence for the anti-HIV-1 potential of TLR1/2 agonists.We previously developed a small molecule TLR1/2 agonist,SMU-Zl,and demonstrated that it could upregulate murine NK cells and CD8+T cells with immune adjuvant and anti-tumor properties in vivo.The present study focused on the potential and mechanism of SMU-Z1 in the "shock and kill" strategy.HIV-1 latent cell models and human peripheral blood mononuclear cells(PBMCs)were used for in vitro experiments.Firstly,a series of TLR2 agonists were found to activate latent HIV-1 in U1 cells of myeloid-monocytic origin,from which the best active one was screened to be SMU-Z1,which was able to target TLR2 to promote p24 expression,and knockdown of TLR2 resulted in the loss of SMU-Z1-mediated activation of HIV-1.Secondly,SMU-Z1 promotes PBMC and THP1 cells to produce inflammatory factor TNF-?,which could reduce in THP1 cells after knocking down TLR2.Moreover,SMU-Z1 can promote monocytes to reactivate latent HIV-1 in latent T cells indirectly and in PBMC from aviremic HIV-1-infected donors.In order to evaluate the mechanism,RNA-sequences analysis on PBMC and U1 cells was performed respectively,and SMU-Z1-induced activation of NF-?B and MAPK pathways was confirmed by western blot.We then explored the effects of SMU-Z1 on human immune cells,activation of PBMC derived from healthy and HIV-1-infected donors was analyzed by flow cytometry.The results showed that the compound was able to activate NK cells,B cells,monocytes,and CD4+T cells without global T cell activation.Furthermore,we analyzed the TLR2 expression in cell subsets and found that SMU-Z1 can up-regulate the expression of TLR2 in NK and B cells.Moreover,SMU-Z1 enhanced the cytotoxic function of NK cells by promoting the production of granzymes CD107a and IFN-?.In addition,ELISA and RT-qPCR results showed that SMU-Z1 can inhibit the HIV-1 replication in PBMC.To further explore the effect of SMU-Z1 on NK cells,NK cells from PLWH were isolated and co-cultured with HIV infected CD4+T cells,ELISA and Flow cytometry showed that SMU-Z1 can promote NK cell-mediated inhibition of HIV-1-infected autologous CD4+T cells.And CCK-8 and apoptosis assay showed that SMU-Z1 has a relatively low cytotoxicity.In summary,the present study found that SMU-Z1 displayed dual potential in the"shock" of exposure of HIV-1 latently infected cells and in the "kill" of reduction the production of HIV-1 infected cells through an innate immune response.Further investigation revealed that this small molecular is more stable in blood than the classical TLR2 agonist Pam3CSK4,which is critical for effective use in combination with therapeutic vaccines or broadly neutralizing antibody treatments.Taken together,our study provides an excellent candidate LRA aimed to the "shock and kill" strategy for elimination of the HIV reservoir,suggesting that SMU-Z1 could be further developed as potential therapeutics for HIV/AIDS and lays a theoretical foundation for future clinical study.