Study On The Effect Of HIV-1 Latency-reversing Drug Combinations Reversing Latency HIV-1 Virus In CD4~+T Cell Memory Subsets

Objective: The use of highly effective antiretroviral therapy(HAART)significantly reduces the morbidity and mortality of hiv-infected individuals.However,although HAART treatment can prevent HIV-1 replication,but it does not eliminate the HIV-1 virus that is latent in cells.The cause of this phenomenon is the presence of the HIV Reservoir.Resting CD4+T cells are considered to be the largest and most important latent reservoirs in the body,which is the main obstacle to the removal of HIV.Resting CD4+ T cells contained the naive T cell subset(TNA),the central memory T cell subset(TCM),the effector memory T cell subset(TEM),stem memory T cell subset(TSCM),transitional memory T cell subset(TTM),etc.The research by Wightman.F.et al.found that TCM,TEM,TSCM and TTM are important latent reservoirs of HIV-1 virus.Currently,the "Shock and Kill" therapy is often used to eliminate the latent infection of HIV-1 virus,and the latency reversing agents(LRA)are used to activate the latent hiv-1 virus,which can be recognized and eliminated by the body's immune system and drugs.How to effectively activate latent virus,especially the effective activation of viruses in different reservoirs,becomes the key to clear the latent virus.The activation effect of the LRA combinations has been shown to be better than the activation effect of the drug alone.Latest research adopts Quanti Gene Flow RNA Assay the new technical note two PKC agonist(Bryostatin-1 and Ingenol-3-Angelate)activate different subsets repository,speculated that other categories LRA to the activation effect of the latent infection HIV-1 virus in different subsets is different.At present the research on latent reversal agent to activate the virus repository more concentrated in PBMC and in resting CD4+ T cells.The studies of the LRA combinations active the latent infection HIV-1 virus in memory CD4+ T cell subsets has not been reported.It is clear that LRA can selectively select drugs to improve the activation efficiency of different CD4+ T cell memory subsets.This study is going to study activation effect of HIV reservoirs in memory CD4+ T cell subsets among people who have taken effective HAART treatment.Explore the activation effect of different latency reversing agentsused alone and in combinations in memory CD4+ T cells subsets HIV reservoirs and its influence on cell activation and apoptosis,thus to provide evidence for clinical choose targeted drugs,improve the efficiency of activation.Methods:1.Study subjects.This study selected 12 patients infected with HIV.Among them,6 patients were HIV-1 infected without HAART treatment;6 patients were HIV-1 infected with HAART treatment,and six healthy subjects were selected.2.Peripheral blood mononuclear cells(PBMC)were extracted and counted.Lymphocyte separation fluid was used to extract individual nuclear cells in peripheral blood through density gradient centrifugation.3.Negative selection of CD4+ T cells.The CD4+ T Cell enrichment kit of Stem Cell was used to select CD4+ T cells from PBMC.4.The establishment of an experimental method for latent infection of HIV RNA in CD4+ T cell subsets(TCM,TEM,TNA)was detected by the FISH-FLOW method.The b DNA probe was designed and optimized according to the total sequence of 39 strains isolated from the red ribbon outpatient department of the first affiliated hospital of China medical university.Through the in-situ hybridization and fluorescence amplification of HIV-1 RNA in the untreated CD4+ T cells,the experimental data were finally detected by flow cytometry.5.The effect of latency reversing agents(LRA)on cell death and apoptosis.Each LRA is divided into high,medium and low concentrations.In healthy people PBMC suspension respectively add high,medium and low concentration of Ingenol-3-Angelate,Panobinostat,Romidepsin,Prostratin,Bryostatin-1,JQ,PHA/IL-2 and DMSO,blank control group,after incubated for 24 hours,marked with 7 aad and Annexin V for cell death and apoptosis,testing with BD FACSCanto ? flow cytometry,and use Flowjo V10 software for analysis.6.Effects of laten cy reversing agents(LRA)on cell activation.In healthy people PBMC suspension respectively add Ingenol-3-Angelate 10 nm,Panobinostat 30 nm,Romidepsin 40 nm,Prostratin 300 nm,Bryostatin-1 10 nm,JQ-1 1 um,DMSO,PHA/IL-2 and blank control group,culture for 24 hours and marked with CD69 as indicators of cell activation,testing by BD FACSCanto ? flow cytometry,and use Flowjo V10 software for analysis.7.The detection of latency reversing agents(LRA)activated latent HIV virus in CD4+ T cell subsets(TCM,TEM,TNA).Extraction the peripheral blood mononuclear cells of HAART treatment patients(selection criteria such as the aforementioned),using StemCell CD4+ T Cell enrichment kit for negative selection of CD4+ T cells from PBMC.Respectively to join in the CD4+ T cells suspension Ingenol-3-Angelate,Panobinostat,Romidepsin,Prostratin,Bryostatin-1,PHA/IL-2,blank control group and drug combination: Bryostatin-1 + Romidepsin,Bryostatin-1 + Panobinostat,Bryostatin-1 + Ingenol-3-Angelate,Prostratin + Romidepsin,Prostratin + Panobinostat,Prostratin + Ingenol-3-Angelate,cultivate after 24 hours,by means of FISH-FLOW activation of HIV-1 RNA in situ hybridization,HIV-1 fluorescent signal amplifier,ultimately testing through the BD FACSCanto ? FLOW cytometry,and use Flowjo V10 software for analysis.8.The absolute value of CD4+ T cells.The absolute value of CD4+ T cells was obtained through the BD FACS Calibur flow cytometry and Multi SET software analysis.9.HIV viral load detection.The method of real-time fluorescence quantitative PCR was used to detect the viral load of Roche's COBAS Taq Man System.10.Statistical analysis.Statistical analysis and mapping were performed through SPSS19.0 and Graph Pad Prism7 software.The median of the data was indicated,and non-parametric test was used for comparison between groups.Mann-whitney test was used for correlation analysis,and P<0.05 was considered statistically significant.Results:1.An experimental method was established to evaluate the effect of LRA on HIV activation of CD4+ T cell subsets(TCM,TEM,TNA)by the FISH-FLOW method.In this paper,we successfully established the in-situ hybrid-flow detection technique for CRF01_AE subtype in China,and tested the HIV RNA in CD4+ T cells of different untreated HIV patients.2.Effects of latency reversing agents on cell death,apoptosis and activation.Most LRA did not cause the obvious death and apoptosis of PBMCs,and did not promote cell activation.Besides ingenol-3-Angelate and Bryostatin-1,the survival rate of cells was reduced to about 70%.PKC agonist drugs have the effect of promoting cell activation.According to the influence of LRA on cell death,apoptosis and activation,the optimal drug concentration of LRA used to activate HIV in CD4+ T cells was determined.3.LRA single drug and drug combinations active latent virus in CD4+ T cells among effective treatment HIV infected patients.In a single drug,ingenol-3-Angelate has the best activation effect on latent HIV infection.In combinations,Bryostatin-1 + Romidepsin,Prostratin + Romidepsin,Prostratin + Ingenol-3-Angelate and Prostratin + Panobinostat,the HIV activation of CD4+ T cells was better.4.LRA single drug and drugcombinations active latent virus in CD4+ T cell subset among effective treatment HIV infected patients.Combined application of Prostratin + Ingenol-3-Angelate,Prostratin + Romidepsin was better for the HIV activation in TNA subset.The combined application of Prostratin + Ingenol-3-Angelate,Bryostatin-1 + Romidepsin,Prostratin + Romidepsin was better for the HIV activation in TCM subset.The combined application of Bryostatin-1 + Romidepsin was better for the HIV activation in TEM subset.5.Bryostatin-1 and Romidepsin have an effect on the composition of different subsets in HIV RNA+ cells when applied separately and in combination.Conclusion:1.To establish and optimize the in-situ hybridization of CRF01_AE subtype virus strains,which are mainly popular in China,can effectively detect the relevant HIV RNA in cells.2.This study used LRA with appropriate concentration to promote HIV transcription,and no obvious toxicity to CD4+ T cells.3.Ingenol-3-Angelate is the best to activate HIV in single drug stimulation.In the combination of drugs,Bryostatin-1 + Romidepsin,Prostratin + Romidepsin,activates HIV better than Romidepsin.Prostratin + Ingenol-3-Angelate activates HIV better than Prostratin.Prostratin + Panobinostat activates HIV better than Panobinostat.4.The combined application of Prostratin + Romidepsin was superior to Prostratin or Romidepsin in the activation of HIV in TCM subset.5.Compared with the single drug,the combination of Bryostatin-1 + Romidepsin has a certain effect on the composition of different subsets of CD4+ T cells that are positive for HIV RNA.