Dynamic Changes Of Cellular HIV-1DNA And Cytokine Levels In Highly Active Antiretroviral Therapy In Chinese HIV Infected Individuals

The introduction of highly active antiretroviral therapy (HAART) for the teatment of human immunodeficiency virus (HIV) infection lead to a significant decrease of mortality and transmission of Acquied immunodeficiency symdrom (AIDS).HAART can effectively restrain HIV replication, reduce serum viral load and increase CD4cell level, restore the immune function in patients with AIDS. However, HIV cannot be eliminated thoroughly by HAART.A major obstacle to HIV eradication is the ability of the virus to remain latent in a subpopulation of the cells it infects. Latently infected cells can escape the viral immune response and persist for long periods of time, despite the presence of successful HAART。A viral reservoir can be difined as a cell type or anatomical site where a replication competent form of the virus persists for a long time than in the main pool of actively replicating virus. There are several components of the cellular reservoirs among which the resting CD4+T cells, mononuclear cells and macrophages, dendritic cells, NK cells and CD8+T cells, etc.And there were reports that CD4/CD8double positive T cells and CD4/CD8double negative T cells may be HIV reservoir. This study includes two parts. The first one which assesses the HIV-1proviral DNA in NK cells,peripheral T lymphocytes and monocytes in15patients who receiving HAART for4-7years (mean62.45±13.53months).The second one which is a two years perspective study assesses the dynamic changes of the HIV-1proviral DNA in NK cells、peripheral T lymphocytes and monocytes and the dynamic changes of cykines levers in40HIV/AIDS patients. And analysis of the correlation between the CD4+T cell count and quantification of HIV-1proviral DNA; correlation between quantification of HIV-1RNA and quantification of HIV-1proviral DNA; correlation between quantification of cytokines level and quantification of HIV-1proviral DNA. Our aim is to find if HIV-1DNA detection can be a prognostic marker for disease progression and response to therapy, independent of other parameters. And this study is a groundwork for explore new strategy of HIV-1elimination. PART I The dynamic changes of Cellular HIV-1DNA quantification during highly active antiretroviral therapy in Chinese HIV infected individualsObjective:The aim of this study was to verify that NK cells were one type of the virus reservoir cells, and to observe the dynamic changes of three types of viral reservoir cells (CD3-CD56+NK cells,CD3+T lymphocytes and CD14+monocytes),and its relationship with main immunological parameters (peripheral CD4+cell count) and the virological parameters (plasma HIV RNA level) in Chinese HIV-infected patients receiving highly-active antiretroviral treatment (HAART) for2years,and the characteristic of HIV-1proviral DNA as a marker for disease progression and response to therapy.Method:A group of15chronic HIV-1-infected adults who received4-7years-HAART(mean62.45±13.53months) participated in this study for crosssectional study.40chronic HTV-1-infected adults (mean37.8±10years old) initiated HAART between June2009and December2010were enrolled in this study. The patients didn’t occur with opportunistic infection or other diseases which might influence the immune response during the study period. After obtaining the ethical approval,40milliliter peripheral whole blood was obtained from each patient at baseline (0month),6,12,18and24months. The amount of T cell subgroup was detected with flow cytometry, the NK cells, T lymphocytes and monocytes were separated from peripheral blood mononuclear cells (PBMC) using microbead sorting method (CD3+,CD14+and CD56+antibody microbeads), and DNA was extracted with human DNA kit. Real-time fluorescent quantitative PCR was used to detect the serum HIV RNA, HIV DNA purified from the three types of cells at baseline (0month),6,12,18and24months. Graphpad prism5software was used to analyze the data collected.Result:The results of15patients with4-7-year-HAART:plasma HIV-1RNA of all the patients are undetectable;There are6patients in15were detected HIV-1DNA in T lymphocyte (mean2.03±0.481ogcopies/106cells),4patients in15were detected HIV-1DNA in Nk cells (mean1.82±0.32logcopies/106cells),2patients in15were detected HIV-1DNA in monocytes (meanl.75±0.19logcopies/10cells).The results of40patients in2-years perspective study: Generally we observed an increase of the CD4count accompanied with a decrease of the viral load during HAART. After HAART initiation, the HIV-1DNA showed a significant decrease in NK cells,T lymphocytes and monocytes. However this decrease was different between cells. In the time of HAART0month,6 months,12months,18months and24months, the HIV-1DNA load in NK cells was3.40±0.79,2.51±0.53,2.40±0.43,2.18±0.42,2.02±0.42log copies/106cells; the HIV-1DNA load in T lymphocytes respectively was3.71±0.67,2.99±0.51,2.49±0.42,2.23±0.48,2.11±0.36logcopies/106cells; the HIV-1DNA load in monocytes respectively was2.92±0.78,2.04±0.29,2.03±0.22,2.01±0.27,1.97±0.10log copies/106cells. The HIV-1DNA from NK cells, T lymphocytes and monocytes correlated positively with the HIV RNA while NK cells and T lymphocytes correlated negatively with the CD4cell count. However we did not find significant correlation between the CD4cell count and the HIV-1DNA in monocytes. The comparison between HIV DNA in NK cells and T lymphocytes showed a positive correlation but there were neither correlation nor positive correlation between NK cells and monocytes.Conclusion:Our results conducted on Chinese HIV infected individuals confirm the inhibitory effect of HAART on the HIV-1viral reservoirs cells. More over we found that NK cells was an important HIV cellular reservoir besides T lymphocytes and monocytes. The HIV-1DNA decreased faster in NK cells and monocytes than in lymphocytes and there was a positive correlation of the HIV-1DNA between NK cells and T lymphocytes. HIV-1DNA in these three types of cells correlated positively with HIV-1RNA suggesting the possibility to use of HIV-1DNA for monitoring the disease progression when the HIV-1RNA are under the detection limit. These findings suggest that, T lymphocytes may be the main long lasting HIV reservoir. All the data are the first time reported in Chinese HIV-1infected individuals. PART Ⅱ The correlation between quantification of Cellular HIV-1DNA and cytokines during highly active antiretroviral therapy in Chinese HIV infected individualsObjective:The aim of this study was to observe the dynamic changes of Th1cytokines(IL-2,IFN-y). Th2cytokines (IL-4、IL-10) and IL-15during HAART,and its relationship with the HIV-1DNA load in NK cells,T lymphocytes and monocytes,and to set a groundwork for exploring new strategy of HIV-1elimination.Method:40chronic HIV-1-infected adults (mean37.8±10years old) initiated HAART between June2009and December2010were enrolled in this study and the normal group also has40individuals. The patients didn’t occur with opportunistic infection or other diseases which might influence the immune response during the study period. After obtaining the ethical approval,40milliliter peripheral whole blood was obtained from each patient at baseline (0month),6,12,18and24months. The amount of T cell subgroup was detected with flow cytometry, the NK cells, T lymphocytes and monocytes were separated from peripheral blood mononuclear cells (PBMC) using microbead sorting method (CD3+, CD14+and CD56+antibody microbeads), and DNA was extracted with human DNA kit. Real-time fluorescent quantitative PCR was used to detect the serum HIV RNA, HIV DNA in the three types of cells at baseline (0month),6,12,18and24months. Enzyme Linked Immunosorbent Assay(ELISA) was used to detect the plasma level of IL-2,IFN-yIL-4and IL-10,and QuantiGlo ELISA was used to detect the plasma level of IL-15in the normal group and HIV-1infected patients at HAART baseline (0month),6,12,18and24months. Graphpad prism5software was used to analyze the data collected.Result:IL-2, IFN-y and IL-15showed a significant increase while the IL-4and IL-10showed a significant decrease after HAART initiation. and IL-2and IL-15correlated positively with CD4cell count and HIV-1DNA in T lymphocytes while correlated negatively with HIV-1RNA after HAART initiation; also, IL-15correlated positively with HIV-1DNA in NK cells.however, IFN-γ, IL-4and IL-10did not show any correlation with CD4cell count,HIV-1RNA or HIV-1DNA in NK cells,T lymphocytes and monocytes. We also notice that IL-2and IL-15in patients who has a more rapid decrease of HIV-1DNA in T lymphocytes are significant higher than the rest.Conclusion:In this study we found a close relationship between the increase of the CD4cell count and the IL-2, IL-15,also a positive correlation between HIV-1DNA in T lymphocytes and IL-2,IL-15; a positive correlation between HIV-1DNA in NK cells and IL-15.It reveals that IL-2and IL-15may take part in the immune therapy of HIV-1reservoir elimition.