Characteristics Of HIV-1 Latent Reservoir In Peripheral Blood With Early HIV-1 Infection And HAART

BackgroundAcquired Immune Deficiency Syndrome(AIDS)isa highly hazardous infectious disease caused by human immunodeficiency virus(HIV)infection.In recent years,AIDS has shown a trend of sustained growth which has become increasingly popular around the world and seriously threatened human life.It has become a serious public health and social problem.With the progress of society,people have a deeper understanding of strategies and methods in AIDS treatment.Highly active antiretroviral therapy(HAART)is currently the most effective treatment for AIDS.It can effectively control the replication of the virus in the human body,reduce the viral load in peripheral blood of HIV-1 infected patients,and increase CD4+T lymphocytes.The level will enable AIDS patients to rebuild their immune function,reduce the incidence of opportunistic infections and tumors,and greatly improve the patient’s prognosis.However,due to the adherence of AIDS patients and viral resistance mutations,especially the persistence of HIV-1 latent reservoir in patients,once the antiretroviral treatment is stopped,the proviral DNA integrated into the host genome is activated in the latent reservoir.The HIV-1 virus will rebound within 3-4 weeks,resulting in failure of antiretroviral therapy.Therefore,the HIV-1 latent reservoir is currently the most important obstacle to the cure of AIDS.How to accurately detect and eliminate the HIV-1 latent reservoir is a hot and difficult issue in the study of HIV-1.HIV-1 latent reservoir refers to the hidden cell sites and anatomical sites of HIV-1 virus under the body’s immune response and antiretroviral treatment.Its characteristics are stable integration,no expression,activation and production of new viruses after drug withdrawal.Resting memory CD4+T lymphocytes are the major sites for HIV-1 latent reservoir.The persistence of HIV-1 latent reservoir is the biggest obstacle to the discontinued treatment of antiretroviral drugs.Early diagnosis of HIV-1 infection is essential to prevent and control the disease Patients may also benefit from early treatment.The functional cure of HIV-1 refers to the suppression of HIV-1 DNA in the latent reservoir after interruption of HARRT to below the detection level,and the state of the body’s immune function is basically normal.This is the goal pursued by clinical AIDS patients for treatment.At present,it will take some time for HIV-1 infected patients to really achieve a functional cure.This study focused on the existence of HIV-1 latent reservoir in early infected persons with HIV-1,and whether there are differences between HIV-1 chronically infected individuals and whether HAART treatment affects HIV-1 latent reservoir.In addition,the correlation between the HIV-1 latent reservoir and T-lymphocyte subsets,HIV-1 viral load was analyzed.Through the study of HIV-1 latent reservoir in peripheral blood lymphocyte,we provide further experimental basis to explore the functional cure of HIV-1/AIDS.Part Ⅰ Study of Early HIV-1 Infection Phase on HIV-1 Latent Reservoir in Peripheral BloodObjectiveTo analyze the difference of the HIV-1 latent reservoir between two groups of early infected and chronically infected patients,and to explore the impacts of HIV-1infection time on the latent reservoir of HIV-1 infected patients.MethodsThis study was conducted on HIV-1 infected patients in AIDS hospital inpatient areas or specialist clinics inGuangzhou Eighth People’s Hospital.All HIV-1infections were confirmed by the HIV antibody validation laboratories in Western Blot(WB).None of the patients received antiretroviral therapy.The antibody affinity assay was used to detect the HIV-1 LAg-Avidityenzyme immunoassay(LAg-Avidit EIA).The principle of the LAg-Avidit EIA method was that the ability of human HIV-1 specific antibodies produced by HIV-1 to recognize and bind to the corresponding antigen increases with the duration of infection.From the above cases,early HIV-1 infections and chronic HIV-1 infections were identified.22 cases were early HIV-1 infections and 22 cases were chronic HIV-1 infections.Peripheral blood mononuclear cells(PBMCs)were collected and isolated from all patients by density gradient centrifugation.HIV-1 RNA in peripheral blood of patients were detected by COBAS TaqMan automatic viral load detection system.The peripheral blood HIV-1RNA and the integrated DNA in lymphocytes of all patients were detected by real-time fluorescence quantitative PCR.CD3+/CD4+/CD8+T cell subpopulations were detected by flow cytometry.Statistical Analysis of Data was used by SPSS16.0Software.Measurement data of non-normal distribution in the two groups of patients expressed as M(Q25,Q75)using Wilcoxon rank sum test.Graphs was used by GraphPad Prism 5 software,P<0.05 indicated statistically significant difference.ResultsCompared with the HIV-1 integrated DNA in PBMCs of the early infected group and the chronically infected group,the HIV-1 DNA amount in PBMCs of the chronically infected group was much higher than that in PBMC of the early infected group.The difference was statistically significant(Z=-2.770,P=0.006).In the early infected group,CD4+T lymphocytes was 258.50(139.25,373.25)cells/ul,CD8+T lymphocytes was 1000.00(633.50,1921.75)cells/ul,the ratio of CD4/CD8 was 0.27(0.08,0.41)and HIV-1 RNA was 6.7×10~5(9.37×10~4,1.39×10~6)copies/ul.In the chronically infected group,CD4+T lymphocytes was 57.50(18.50,231.75)cells/ul,CD8+T lymphocytes was 699.50(344.25,782.25)cells/ul,the ratio of CD4/CD8was 0.09(0.05,0.32)and HIV-1 RNA was 1.45×10~5(5.89×10~4,5.04×10~5)copies/ul.CD4+T,CD8+T lymphocytes in the two groups were statistically significant(Z=-2.70,-2.82,P=0.007,0.005).There was no significant difference in the ratio of CD4/CD8+T lymphocytes and HIV-1 RNA between the two groups(Z=-1.69,-1.69,P=0.091,0.091).In early infected group,there was no correlation between HIV-1 integrated DNA in PBMCs and CD4+T lymphocyte count,CD8+T lymphocyte count,CD4/CD8+T lymphocyte ratio,and HIV-1 RNA(r=-0.267,-0.026,-0.190,-0.169,P>0.05).In the chronic infection group,there was no correlation between HIV-1 integrated DNA in PBMCs and CD4+T lymphocyte count,CD8+T lymphocyte count,CD4/CD8+T lymphocyte ratio,and HIV-1 RNA(r=0.291,0.133,0.113,0.114,P>0.05).In the early infected group,there was a negative correlation between HIV-1 RNA and CD4+T lymphocyte counts,CD4/CD8+T lymphocyte ratio(r=-0.478,-0.591,P=0.024,0.005),and there was no correlation between HIV-1 RNA and CD8+T lymphocyte counts(r=-0.036,P=0.876).In the chronic infection group,there was a negative correlation between HIV-1 RNA and CD4+T lymphocyte counts,CD4/CD8+T lymphocyte ratio(r=-0.704,-0.615,P=0.001,0.004),and there was no correlation between HIV-1RNA and CD8+T lymphocyte counts(r=-0.296,P=0.205).Conclusions1.The presence of HIV-1 integrative DNA in PBMCs of early infected HIV-1patients indicates that the HIV-1 latent reservoir has been established early in the HIV-1 infection.With the increase of infection time,the capacity of HIV-1 latent reservoir increases.2.HIV-1 viral load in the early infected patients is still at a high level and the mechanism of T lymphocyte immune responseat this time has begun to start.PartⅡImpacts of Highly Active Antiretroviral Therapy on HIV-1 Latent Reservoir in Peripheral BloodObjectToprovide evidence for exploring HIV functional cure by observing the changes of HIV-1 latent reservoir in peripheral blood lymphocyte subsets of HIV-1infected patients before and after HAART.MethodsThis study was conducted on HIV-1 infected patients in AIDS hospital inpatient areas or specialist clinics in Guangzhou Eighth People’s Hospital(21 patients with HAART-na?ve and 22 patients with HAART).PBMCs were collected and isolated from all patients by density gradient centrifugation.CD4+T cells,CD8+T cells and non-T cells in PBMCs were sorted by magnetic bead cell sorting.HIV-1 RNA in peripheral blood of patients were detected by COBAS TaqMan automatic viral load detection system.The integrated DNA in three classes of lymphocyte subsets were detected by real-time fluorescence quantitative PCR.CD3+/CD4+/CD8+T cell subpopulations were detected by flow cytometry.Statistical Analysis of Data was used by SPSS16.0 Software.Measurement data of non-normal distribution in the two groups of patients expressed as M(Q25,Q75)using Wilcoxon rank sum test.Graphs was used by GraphPad Prism 5 software,P<0.05 indicated statistically significant difference.ResultsIntegrated HIV-1 DNA in CD4+T cells were higher than that in CD8+T cells and non-T cells both in HAART-na?ve group and HAART group.The difference was statistically significant(Z=-5.549、-5.547、-5.360、-5.493,P<0.000);There was significant difference in the integrated HIV-1 DNA of non-T cells(Z=-3.326,P=0.001)and no significant difference in CD4+T cells and CD8+T cells(Z=-1.871、-1.360,P=0.061、0.174)between HAART-na?ve group and HAART group.In HAART-na?ve group,integrated HIV-1 DNA in CD4+T cells were positive correlated with HIV RNA(r=0.594,P=0.005),negatively correlated with CD4 cells count and CD4/CD8 ratio(r=-0.547、-0.482,P=0.010、0.027)and not correlated with CD8 cells count(r=-0.102,P=0.668).There was no correlation between the integrated HIV-1 DNA in CD8+T cells and HIV RNA、CD4 cells count、CD8 cells count、CD4/CD8 ratio in HAART-na?ve group(r=0.281、0.043、0.134、0.145,P>0.05).So was the integrated HIV-1 DNA in non-T cells(r=0.274、0.218、-0.015、0.041,P>0.05).There was no correlation between the integrated HIV-1 DNA in three classes of lymphocyte subsets and CD4 cells count、CD8 cells count、CD4/CD8 ratio in HAART group(r=-0.042、-0.333、0.208,P>0.05;r=0.167、0.002、0.024,P>0.05;r=0.078、0.216、0.261,P>0.05).Conclusions1.HIV-1 latent reservoir mainly exists in peripheral blood CD4+T cells,a small amount in the presence of CD8+T cells and non-T cells.2.The HIV-1 latent reservoir in CD4+T cells could be minished by HAART.