Study On Biological Properties Of HIV Isolates And Function Of HIV Specific Cd8~+T Cells In Primary HIV Infection

Objective:After the host is infected with human immunodeficiency virus?HIV?,the virus continues to replicate,leading to target cell depletion and eventually development of AIDS?AIDS?.HIV is prevalent worldwide.According to the UNAIDS report,about35 million people are living with HIV-1/AIDS.Although it has been contained in many regions of the world,it still can not be cured completely,seriously endangering human health.Primary HIV infection is the initial stage that immune system responds to virus after HIV invades the body,characterized by the high level the virus replication.Early HIV infection with the body's immune response to the interaction of disease prognosis has far-reaching influence.It is very important to clarify the early immune response and its influence on HIV vaccine research.Antiviral response mediated CD8⁺T cells play a central role in adaptive immune responses.It is reported specific CD8⁺T cell were detected in the early HIV infection?after infection 25-32 days?,far earlier than neutralizing antibodies,which was the earliest anti-HIV response.CD8⁺T cells response were reported to be associated with the viral load decline and related to virus set point and CD4⁺T cells decrease.The research suggests anti-HIV response of CD8⁺T cells in the early stages changed,the effector function of CD8⁺T cells may result the different disease progression HIV infected persons.At present,the mechanism that CD8⁺T cells control virus in the early stage and the loss of function in the chronic infection remains unclear.“Polyfunctional T cells”is a hot topic in immunology research in the recent years.Polyfunctionality of CD8⁺T cells may delay the disease progression in chronic HIV infection.In the early stage of infection,functional profile of CD8⁺T cells may be different from that of chronic infection.Guido Ferrari,etc.in 7 cases of HIV infections found functional CD8⁺T cells have oligofunctionality,suggested that multifunction are not necessary factors of the selection pressure of the virus.At present,functional characteristics of CD8⁺T cells and its relationship with the capacity to control the virus remain controversial,the changes of CD8⁺T cells effect function from the early stage to the chronic infection is uncertain.Model of T cells exhaustion in chronic virus infection is established by lymphocytic choriomeningitis virus?LCMV?,which suggests the exhaustion of T cells is one of the caused that virus replication is difficult to control in the chronic virus infection.In the HIV infection,up-regulation of PD-1 was report to associate to the T cells exhaustion.Reports on rhesus monkeys infected by Simian Immunodeficiency Virus?SIV?suggested blocking of PD-1 specific T cells could enhance the T cell response.In the HIV infection,association between the CD8⁺T cell exhaustion and its control of the virus is not clear,especially CD8⁺T cell exhaustion in the early stages has not been reported.In the HIV infection,the"ultimate effect function"of the specific CD8⁺T cells is to clear HIV infected cells and suppresses HIV replication,which is closely related to target cells,the antigen,and HLA of the hosts.The assessment of CD8⁺T cell function is of critical importance.Asier Saez-Cirion etc.founded a method to evaluate the ultimate effect function of CD8⁺T cells based on HIV isolates and suppress HIV replication of the"ultimate effect function".The basic goal of this research is to figure out the dynamics of“ultimate effector function”of CD8⁺T cells and its relationship with the functional profile.For this purpose,we are going to use a novel assay to evaluate HIV-specific CD8⁺T-cell responses?"ultimate effect function"?,which takes advantage of autologous isolates from HIV infected persons,and its relationship with the functional profile of CD8⁺T cells,including the markers of“polyfunctionality”and exhaustion associated inhibitory receptors,which may found basis for development of HIV vaccine and strategy for prevention and control HIV.Materials and Methods:1.Study populationPrimary HIV infection among MSM population in the Liaoning were studied.Inclusion criteria:meet any items of the following 3 criteria.?1?Seroconversion within 3months;?2?the HIV antibody negative but HIV RNA positive;?3?Uncertainty result of the Western Blot antibody test and HIV antibody is positive when follow-up.Subjects were divided into 2 groups according to coreceptor switch within 3 years post infection.EDTA anticoagulation venous blood was collected before antiviral treatment.All respondents were informed agreement signed.2.CD4⁺T cells countsCD4⁺T cell counts were measured using TriTEST CD4FITC/CD8PE/CD3PerCP reagent with 20?l anticoagulated whole blood.After incubation for 15 minutes in the dark at room temperature,FACS lying solution was added and then incubated.CD4⁺T cell counts and corresponding ratios were obtained by flow cytometer analysis with FACS MULTISET software.3.Viral load assayPlasma HIV RNA was quantified with Amplicor HIV Monitor test,version 1.5?Roche Diagnostics,Rotkreuz Switzerland?by an ultra-sensitive modification.4.Virus isolationPBMCs were isolated from blood of the HIV infected patients and healthy donors using Ficoll-Paque gradient.The patients'PBMCs were co-cultured with PHA-stimulated PBMCs from healthy donors at a 1:1 ratio.The cell cultures were maintained for 5-20weeks in R10(RPMI 1640 medium containing 10%fetal bovine serum?FBS?,1%penicillin and streptomycin?PS?and 20U/ml recombinant human interleukin-2?IL-2?,the culture media were refreshed twice a week.The culture supernatants were collected for detection of HIV p24 production using an enzyme-linked immunosorbent assay?ELISA?kit?Vironostika HIV-1 Microelisa system,BioMérieux,France?.5.p24 antigen detectionThe culture supernatants were collected for detection of HIV p24 production using an enzyme-linked immunosorbent assay?ELISA?kit?Vironostika HIV-1 Microelisa system,BioMérieux,France?according to the operating manual strictly.6.Determination of replicative capacity of HIV primary isolatesPHA-stimulated PBMCs from healthy donors were inoculated with HIV isolates containing 5 ng p24/ml equivalent viruses.The cell cultures were maintained for 19weeks in R10(RPMI 1640 medium containing 10%FBS,1%PS and 20U/ml IL-2,the culture media were refreshed twice a week.The culture supernatants were collected for detection of HIV p24 production using an ELISA kit?Vironostika HIV-1 Microelisa system,BioMérieux,France?.7.Determination of tropism phenotypeViral co-receptor usage was determined using GHOST?3?cell lines expressing CCR5 or CXCR4.Briefly,1×105 of GHOST?3?Hi5 cells expressing CCR5 and GHOST?3?CXCR4 cells expressing CXCR4 were seeded in a 12 well plate in DMEM containing 10%FBS and incubated overnight.On the following day,the cells were infected with HIV isolates containing 5 ng p24/ml equivalent viruses and 10?g/ml DEAE for 2.5 hrs.Then the unbound viruses were removed and the cells were cultured in fresh media for 2 days.HIV infection of the cells was monitored by flow cytometric analysis after trypsinizing the culture.X4/R5 dual tropic HIV SF33 were included as controls in each experiment.Green cells were identified by a microscope and GFP positive cells percentage was determined by flow cytometry.8.RNA extraction and cDNA synthesisRNA from primary isolates was extracted with QIAamp Viral RNA Mini Kit?Qiagen,German?according to the manufacture's recommendations.RNA was reverse transcribed into cDNA with a reverse transcriptase kit?TAKARA,Japan?.C2-V5 region of HIV env gene were amplified withouter primers and followed by nested PCR with inner primers.PCR products were sequenced by BGI Company?Beijing?.9.Co-receptor usage predictionCo-receptor usage predictions based on V3 sequences were performed by Geno2pheno?http://coreceptor.bioinf.mpi-inf.mpg.de/index.php?with a false-positive rate of 10%and the Position-Specific Scoring Matrix?PSSM?approach?http://ubik.microbiol.washington.edu/computing/pssm/?.In addition,a combination of criteria from the 11/25 and net charge rules was performed.10.Capacity of CD8+T cells to inhibit autologous HIV isolates replication inautologous CD4+T cellsConcentrate PBMCs?typically to 10⁷ cells per ml?in chilled separation buffer after centrifugation at 400g for 5 min and perform positive selection of CD4⁺cells with an anti-CD4⁺antibody coupled to magnetic beads as recommended by the manufacturer.Collect positive fraction as CD4⁺T cells,cultured in IL-2 R15 medium.Adjust the concentration of separated CD4⁺T cells in the IL-2 R15 medium.25?l CD3CD28beads/106 cells were added to active CD4⁺T cells.Cells were observed under microscopy every day and counted 2 times weekly count cell.When cell concentration was greater than 2 x 106 cells/ml,concentration were readjusted to 1 x 106 cells/ml.After every 8 days,magnetic beads bounded cells were removed and 25?l CD3CD28beads/106 cells were added again.Collect the cells in the log phases.Concentrate the negative cellular fraction from separation of CD4⁺T cells in chilled separation buffer after centrifugation at 400g for 5 min,and perform indirect magnetic cell sorting of untouched CD8⁺T cells with a CD8⁺T-cell enrichment kit as recommended by the manufacturer.Collect negative fraction as CD8⁺T cells and cultured in cytokine free medium.Add 100?l of CD4⁺T cells to wells in a 96-well round-bottom plate.Allow three wells for noninfected in vitro control,three wells for HIV infected in vitro CD4⁺T cells,three more wells for coculture with CD8⁺T cells at a 1:1 ratio.Add 100?l of CD8⁺T cells at 106 cells per ml to each of three wells containing 105 CD4⁺T cells for coculture at a 1:1 CD4⁺/CD8⁺T cell ratio.Centrifuge the plate at 400g for 5 min at room temperature and remove 100?l of the supernatant from CD4⁺/CD8⁺T cell coculture-containing wells.Add 100?l IL-2 medium to each of three wells destined as noninfected in vitro controls.Dilute viral stocks to a 2×assay viral dose in IL-2 medium and add 100?l of viral suspension to the three wells containing CD4⁺T cells and to wells containing CD4⁺/CD8⁺T cell cocultures.Centrifuge plate at 1,200g for 1 h at 22°C in a refrigerated centrifuge to improve infection efficiency.Incubate plate for 1 h at 37°C in a humidified incubator under 5%CO2.Remove 190?l of supernatants using an eight-channel pipette,add 190?l of culture medium and centrifuge at 400g for 5 min at room temperature.Repeat twice.Use 190?l of IL-2 medium after the last wash.Incubate plate at 37°C in a humidified incubator under 5%CO2.At day 4 after infection,centrifuge the plate at 400g for 5 min at room temperature,collect the supernatant for p24 detection.Virus inhibition was calculated as reduction in p24 from wells containing CD8⁺T cells as compared with control wells lacking CD8⁺T cells.11.Determination of CD8⁺T cells functional profile.Collect the cells in the experiment 10 at 4 days after the infection.Add CD107a-FITC,CD28CD49d 1?l/tube,37?,5%CO2 incubation 15 min,add Golgi Stop 0.7 u l/106 cells and Golgi Plug 1?l/106 cells.37?,5%CO2 incubation 6 HRS.Wash cells using PBS at 1500 r/min centrifuge 10 min.Mix cells well and split the same amount into three tubes.The surface staining:Add CD8-PerCP,to each tube,add HLA-DR-APC and PD-1-APC to 2 tubes of the three.Stain at 4?for 30 min avoid light.Wash with cold PBS,centrifuge at 1500 r/min 10 min.Add 250?l Fixation/Permeabilization solution,incubate for 10 min at room temperature avoid light,add 2 ml BD Perm/Wash buffer,centrifuge at 1700 r/min for 7 min.Intracellular staining:add Ki-67-PE to the HLA-DR-APC tube,add IFN gamma APC and TNF?to the CD107a-FITC tube,incubate at room temperature avoid light for 25 min.Add 2 ml BD Perm/Wash buffer to wash the unbounded antibody.Supernatant was dumped and cells were resuspended,300?l 1%formaldehyde were added to fix the cells,LSR II flow cytometry were used for acquisition.BD FACSDiva software were used to analyze surface markers including PD-1,HLA-DR,CD107a and intracellular antigen including Ki-67,IFN?and TNF?expressed on CD8⁺T cells.12.Statistical analysisThe GraphPad Prism5 software were used to describe the replication kinetic curve,p24 antigen peak value and the area under the curve were calculated.The SPSS17software was used in analysis.The rate of decline in CD4⁺T-cell counts per month was calculated,with decreases being evaluated by means of linear mixed-effect models.Survival analyses were used for analyzing the expected time duration until coreceptor switch.Mean±standard deviation were used.Spearman Rank Correlation was used to correlation analysis.Statistical probability with double side inspection,P<0.05 is statistical significance.Results:1.Virus isolationAccording to the inclusion criteria,a total of 59 patients with primary infection of CRF01_AE subtype entered the study.The primary infection was predominantly Han MSM,and the median time to enter the study was 31 days after infection?IQR:25-59?.A follow-up of 59 infected patients was followed by a median follow-up of 3.2 years?IQR 1.9-4.4?and a cumulative follow-up of 188.2 person-years.We found that up to 45?76.3%?of the 59 patients with primary CRF01_AE subtypes had CD4 cell counts that fell below 350 cells/?L during follow-up,with a median time of 0.6 years?IQR 0.2-2.7?.The linear mixed model showed a slope of CD4 cell decline of-0.10?CD4/month.This result demonstrated MSM infected with HIV-1 CRF01_AE subtype in Northeast China showed rapid disease progression.2.Viral tropism is associated with disease progression.In the present study,single-genome amplification?SGA?method was used to amplify the plasma virus C2-V5 region of 59 patients with primary infection.After sequencing,the four most widely used algorithms in the world were used to predict the genotype of viral tropism.A total of 458 SGA sequences were obtained from 59 patients with PHI?including 31 sequences obtained in the early stage of the laboratory?,and the net charge rule was predicted by Geno2pheno?FPR=10%,5%,2%?,PSSMx4r5,PSSMsinsi and 11/25.The number of R5 viruses was 360?78.8%?,412?89.9%?,442?96.5%?,442?96.5%?,446?97.4%?,and 450?98.2%?.Of the 59 infected individuals,Geno2pheno?FPR=10%,5%,2%?predicted that only carried R5 viruses were 41,51,and 55?69.5%,86.4%,93.5%?.And the other three algorithms,the predicted numbers were 54,57,56?91.5%,96.6%,95.0%?.For the primary infected person,49 strains of primary virus isolates were obtained,of which 48 were R5 virus?98.0%?and only 1 was X4/DM strain?2.0%?.The genotype and phenotype results suggest that the R5 strain is predominantly prevalent in the primary infection of the CRF01_AE subtype.The primary infected persons were followed up for phenotypic phenotypic testing.It was found that in the first 1-4 years after infection,4,11,2,1 cases of tropic transformation occurred,including 1 case of primary infection.At the time of infection with X4/DM tropism.After 3 years of follow-up,a total of 19 infected patients were censored because of treatment,loss of follow-up and other reasons.Survival analysis showed that 7.9%of infected patients developed tropic switch in the first year after infection,and 92.1%of infected persons mainly carried R5 strain.However,by the second year of infection,the number of people who switched was increased to 32.8%.As of the third year after infection,39.5%of CRF01_AE subtypes infected with MSM had undergone tropic transformation.CRF01_AE infected patients were divided into switch group and non-switch group by switch into X4/DM within 3 years post infection,and the rate of CD4 cell decline between the two groups was compared by linear mixed model.The results showed that the rate of CD4⁺T cell decline in the switch group was significantly higher than that in the non-switch group?P<0.001?,adjusted for age,baseline CD4⁺T cell level and HLA-B*44?previous results suggest HLA-B*44 was related to protect patients from rapid disease progression?,the difference was still significant.To further elucidate the relationship between the rate of decline in CD4⁺T cells after switch in HIV-infected individuals,we further performed a sensitivity analysis.The CD4⁺T cell count before switch was removed in the switch group;in the non-switch group,the CD4⁺T cell count before the infection was removed for 482 days?the median infection time at which tropism switch occurred?;The rate of change in CD4⁺T cell count in switch group was10 cells/?L/month?IQR:1.8-19.6?per month,and the rate of non-switch group was decreased by 5 cells/?L/month?IQR:3.2-6.3?per month.The linear mixed model results showed that the rate of CD4⁺T cell count decline in the swtich group was significantly higher than that in the non-switch group?P=0.003?.Our data suggest that early tropism swtich within 3 years after infection is closely related to the rapid decline of CD4⁺T cells in MSM infected with CRF01_AE subtype.3.Correlation between viral replication and disease progression in patients withprimary infectionEighteen patients with primary infection whose p24 concentration was higher than20ng/mL were infected with CD8 deleted PBMCs in vitro to detect the viral replication ability.The replication kinetics curves showed that the p24 concentration of the primary isolates increased gradually?day5-day15?,plateau?day15-day22?and decreased?day22-day31?after infection with PBMC in healthy individuals,and the median time to reach the peak was 5.50 log10 pg/mL?IQR:5.45-31?at 19 days?IQR:15-22 days?.5.56log10pg/mL,AUC was 6.63 log10pg/mL.day?IQR:6.54-6.73 log10pg/mL.day?.Primary infections were divided into two groups according to the level of viral replication AUC within 19 days.Primary infections with AUC higher than the median were those with high replication ability,while those with AUC lower than the median were those with low replication ability.The linear mixed model showed no significant difference in the rate of CD4 decline between the 2 groups of primary infections?P>0.05?.4.The ability of CD8⁺T cells to inhibit HIV replication in primary infectedpersons with different outcome.There was a significant positive correlation between the inhibition rate of CD8⁺T cells and the absolute count of CD4?r=0.762,P=0.004?and a negative correlation with the viral load?r=0.045,P=0.894?.According to the ability of CD8⁺cells to inhibit auto-virus,the patients with primary infection were divided into two groups.The CD8⁺cells whose ability to inhibit auto-virus was higher than the median of the high group,and the lower group was the low group.The decrease rate of CD4⁺cell count in the high group was significantly lower than that in the low inhibition group?P=0.001?.Kaplan-Meier survival analysis showed that the mean time for CD4⁺cell absolute count to decrease to 350 cells/ml in patients with low inhibitory ability was 272?±93 days??95%CI,90-454 days?,and that for those with high inhibitory ability,the mean time for CD4⁺T cell absolute count to decrease to 350 cells/ml was 1042?±36 days??95%CI,972-1112days?.Day.The mean time for CD4⁺T cell count to decrease to 350 cells/ml in HIV patients with low inhibitory capacity was significantly shorter than that in HIV patients with high inhibitory capacity?log rank₂=11.46,P=0.001?.Our data suggest that the ability of CD8⁺T cells to inhibit autologous viruses is negatively related to the rapid decline of CD4⁺T cells infected with MSM.5.CD8⁺T cell exhaustion associated inhibitory receptor PD-1 expression inprimary HIV infection.PD-1 positive CD8⁺T cell percentage was negatively related to the ability of CD8⁺T cells to restrain HIV replication?r=-0.748,p=0.005?,and negatively related to the CD4⁺T cell absolute count?r=-0.72,p=0.008?,and had a positive tendency of relationship with viral load?p=0.247?.6.Capacity of cytokine secretion of HIV specific CD8⁺T cell in primary HIVinfection.IFN?positive CD8⁺T cell percentage was negatively related to the ability of CD8⁺T cells to restrain HIV replication,as well as the TNF?positive CD8⁺T cells?p>0.05?.7.Proliferation potential of HIV specific CD8⁺T cell in primary HIV infection.HLA-DR positive CD8⁺T cell percentage was negatively related to the ability of CD8⁺T cells to restrain HIV replication,and Ki-67 positive CD8⁺T cell percentage was negatively related to the ability of CD8⁺T cells to restrain HIV replication?p>0.05?.8.Degranulation of HIV specific CD8⁺T cell in primary HIV infection.CD107a positive CD8⁺T cell percentage was not significantly related to the ability of CD8⁺T cells to restrain HIV replication?p=0.106?.Conclusion1.R5 virus is mainly prevalent in MSM with CRF01_AE infection.Nearly 40%of the patients infected with CRF01_AE experienced corecepter switch within 3 years of infection,which was closely related to the rapid decrease of CD4 cell count in CRF01_AE infected patients.2.HIV specific CD8⁺T cell response that inhibits autologous virus replication in primarily infected patients.The results showed that the inhibition rate of autologous virus was associated with disease progression at the early stage of infection.The results showed that the expression of PD-1 in CD8⁺T cells was negatively correlated with the ability to inhibit the replication of autologous viruses in HIV-infected patients in China,suggesting that the exhaustion of CD8⁺T cells mediated by PD-1 may be an important factor in the progression of HIV-infected patients.