| Objective: Acquired immune deficiency syndrome(AIDS)is one of the major infectious diseases that seriously endanger human health.Antiretroviral therapy(ART),which is widely used in the world,can effectively inhibit the replication of the virus in Human Immunodeficiency Virus(HIV)infected patients,reduce the plasma viral load to an extremely low level and maintain it for a long time.Changes of plasma viral RNA levels in HIV infected patients during the application of ART can be divided into two stages.The first stage of viral RNA level declines rapidly,accompanied by a large number of deaths of activated CD4+ T cells carrying actively replicating viruses;the second stage of viral RNA level is slowly decline to a very low level and maintain it,this decrease may be related to the longer life span of the infected cells and their clonal expansion.However,if the treatment is interrupted,within 2-8 weeks,the viral load of almost all infected persons will quickly rebound to the level before treatment.The root cause is in the early stage of HIV-1 infection,a virus reservoir mainly composed of resting memory CD4+ T cells is formed.Therefore,the virus reservoir is the fundamental reason why HIV-infected patients cannot stop the drug,and it is also the main obstacle to the functional cure of AIDS.At present,it is believed that HIV reservoirs can be divided into two categories.One is the latent reservoir: cells that carry a complete viral genome but do not express viral transcripts or proteins.The other type is active reservoir: cells with transcriptional activity that can still produce HIV RNA,protein or virus particles after long-term antiviral treatment.The formation mechanism of this type of reservoir is unclear,so it cannot be routinely tested.With the emergence of London patient and Berlin patient,the significance of dynamic reservoir has attracted more attention.The recently identified HIV host restriction factor TRAB domain-containing protein 2A(TRABD2A)can degrade the translated viral structural protein Gag in resting CD4+ T cells,thereby inhibiting the assembly and release of progeny viruses on the cell membrane.This mechanism provides a new idea for the formation of HIV dynamic reservoir,that is,due to the existence of certain host inhibiting factors,the replication of the virus is stagnated at a certain stage and cannot be successfully released to form a reservoir.With technological innovations,methods for detecting the size of the reservoirs are emerging in endlessly.However,due to the extremely low proportion of reservoir cells in the overall cell,the virus in patients with long-term treatment often has mutations and defects,and there are also reservoirs in some difficult-to-detect tissues,the existing reservoir detection methods are not available.How to reflect the truly clinically significant rebound virus is one of the main obstacles of AIDS research,and it is also a challenging frontier scientific issue in the world.Based on the inhibition of HIV release by cell membrane metalloprotease TRABD2 A in resting CD4+T cells,this study clarified the relationship between cellular membrane metalloprotease TRABD2 A and reservoir cells for the first time,and used humanized TRABD2 A monoclonal antibody to achieve reservoir cells release.The release of virus in the resting state is expected to indirectly calculate the size of the reservoir through the amount of virus released.The results of this study provide new ideas for the development of new detection methods for virus reservoirs,and provide more reliable guidance for clinical medication and treatment.Methods: 1.Research Object A total of 60 samples were collected in this study,including 2 healthy donors(healthy group),50 HIV-infected patients who received antiretroviral therapy(ART group),and 8 HIV-infected patients who did not receive antiretroviral therapy(noART group).Inclusion criteria for the healthy group: 1)Age: 18 to 55 years old;2)Experiments proved to be negative for HIV antigen and antibody.Inclusion criteria for ART group: 1)Age: 18 to 55 years old;2)HIV antibody positive after confirmation;3)HIV-1 infected persons have received antiretroviral therapy for more than 1 year,and the HIV-1 RNA level is below the limit of detection for at least 6 months,and the number of CD4+T cells is greater than 350/ml.Inclusion criteria for the no-ART group: 1)Age: 18 to 55 years old;2)HIV antibody positive after confirmation;3)Never received antiretroviral treatment.The exclusion criteria for the above three groups of study subjects are: 1)combined infection,active autoimmune disease,fever of unknown origin;2)combined with severe or unstable heart,lung,liver,kidney and hematopoietic system diseases,including activities Hepatitis HBV,HCV,etc.;3)Have taken other experimental drugs,immunomodulators or Chinese herbal medicines during antiretroviral treatment;5)Pregnant or lactating women;6)Currently taking drugs,having severe mental or neurological diseases,mental disability cannot communicate effectively with doctors.Volunteers participating in this study have given informed consent and signed an informed consent form.This study has been approved by the Ethics Committee of the First Affiliated Hospital of China Medical University.2.Extraction of PBMCs and isolation of CD4+T cells Use blood collection tubes containing EDTA anticoagulant to collect peripheral blood from healthy controls or HIV-infected individuals.After gently mixing,Ficoll was used for density gradient centrifugation of peripheral blood to separate PBMCs.Isolation of CD4+ T cells: Use Easy SepTM Human CD4+ T Cell Enrichment Kit(STEMCELL Technologies)to sort CD4+T lymphocytes.3.PBMCs and CD4+T cells culture and activation Use RPMI1640 medium containing 10% inactivated FBS and 100U/ml penicillin-streptomycin to culture PBMCs and CD4+T cells at rest.When activating cells,add 50U/ml Interleukin-2(IL-2)to RPMI1640 medium containing 10% inactivated FBS,100U/ml penicillin-streptomycin,and Dynabeads(?) Human TActivator CD3/CD28(Thermo Fisher)was added at a ratio of 25?l/106 cells.4.RNA interference technology in PBMCs and CD4+T cells Use Nucleofector 2b electroporation instrument and Human T Cell NucleofectorTM Kit(Lonza)to perform si RNA knockdown on primary cells,set program U-14,and follow the instructions.5.Treatment of primary cells with different TRABD2 A inhibitors The cells were plated into a 24-well plate at a density of 2 million/ml,and 5?g/ml monoclonal antibody or different doses of 1,10-phenanthroline were added respectively,and the activated cultured cells were used as a positive control.After adding the treatment,continue to incubate in a 37°C incubator for 24 h or 48 h.6.Luciferase activity detection Use Passive Lysis Buffer(Promega)to lyse TZM-bl cells,then add luciferase substrate(Promega)to the lysate,and detect luciferase activity.The specific operation is carried out in accordance with the instructions.7.Ultra-sensitive p24 detection Collect 200?l of the processed culture supernatant,centrifuge at 10000 g for 5min,take the supernatant and submit it to Quanterix for testing.According to the manufacturer's instructions,the detection limit of the ultrasensitive digital ELISA is 0.01–0.04 pg/ml.8.RT-q PCR Total cell RNA was extracted by TRIzol reagent.Use Prime ScriptTM RT reagent Kit with g DNA Eraser(Perfect Real Time)(TAKARA)to remove DNA doped in RNA and reverse transcribe RNA into c DNA.Use TB Green(?) Premix Ex TaqTM II(TAKARA)kit for fluorescence real-time quantification PCR.9.Flow Cytometry Before cell processing,use cell trace to stain the cells,and the specific operation steps follow the instructions.After the cell processing is completed,transfer the cells to a flow tube,wash twice with FACS Buffer(PBS containing 2% FBS),and add surface antibodies: ?CD69-PE(BD Biosciences),?CD25-BB515(BD Biosciences),?HLA-DR-APC(BD Biosciences),stain for 25 minutes on ice in the dark,add FACS Buffer and centrifuge to remove the flow dye,then test on the machine.10.SDS-PAGE electrophoresis Add the corresponding buffer to the sample and boil it at 100°C for 5 minutes to obtain a protein sample that can be used for SDS-PAGE electrophoresis.Use SDSPAGE gel preparation kit(KGI Bio)to prepare 12% gel.Fix the gel plate on the electrophoresis instrument,add the electrophoresis solution and load the sample.Separate gel electrophoresis at 100 V for 30 min,and concentrate gel at 150 V until it reaches the bottom of the gel.Cut the glue from the glue plate and stain with Coomassie Brilliant Blue for 10 minutes.After dyeing,wash off the dyeing solution with water,and decolorize in the decolorizing solution until the glue becomes transparent and clear bands are visible.Place the decolorized glue on the BioRed Chemi DocTM XRS+ instrument to take pictures. 11.Statistical analysis Use Flowjo X for flow analysis and graphing,and use SPSS19.0 and Graph Pad Prism8.0 software for data analysis and graphing.The measurement data was analyzed by unpaired T test,and P<0.05 was considered as statistically different.*P<?0.05,**P<0.01,***P<0.001,****P<0.0001Results 1.Research on the relationship between TRABD2 A and the cell reservoir of ART patients According to previous research results,in ex vivo experiments,TRABD2 A,which is highly expressed in resting CD4+T cells,can inhibit virus assembly and release by degrading Gag protein.When the antiviral function of TRABD2 A is inhibited,the virus can be released.Therefore,we isolated PBMCs and CD4+T cells from long-term ART treated patients to study the role of TRABD2 A in reservoir cells.First,the RNA interference technology was used to knock down the TRABD2 A gene in cells to decrease its expression.It was found that after knocking down TRABD2 A,the amount of virus in the culture supernatant increased significantly.However,since the activation of resting cells can also cause a large amount of virus release,we have tested the activation and proliferation markers of the cells,and the results show that the knockdown of TRABD2 A by RNA interference technology does not cause cell activation.This proves that inhibition of TRABD2 A can cause virus release in PBMCs and CD4+T cells of patients with long-term treatment.Since TRABD2 A is a metalloprotease,we choose 1,10-phenanthroline,a non-specific divalent metal ion chelating agent,to treat the cells and observe the virus release.The results showed that as the concentration of 1,10-phenanthroline increased,the amount of progeny virus released by the cells also increased.That is,1,10-phenanthroline inhibited the function of TRABD2 A in a dose-dependent manner in the reservoir cells.Since TRABD2 A is a transmembrane protein,we used hybridoma technology to prepare murine TRABD2 A monoclonal antibodies.In the experiment,the use of monoclonal antibodies to block TRABD2 A can cause the release of intracellular viruses without changing the cells' proliferation and activation state.This proves that inhibiting the function of TRABD2 A in the reservoir cells can cause the release of the virus in the reservoir.2.Synthesis of TRABD2 A humanized monoclonal blocking antibody with clinical application value First,the recombinant human TRABD2 A protein was synthesized as the screening antigen of the phage library.We use phage display technology to prepare humanized monoclonal antibodies.First,the recombinant human TRABD2 A protein was synthesized as the screening antigen of the phage library.The recombinant TRABD2 A plasmid was synthesized by double-enzyme digestion of the TRABD2A-201 plasmid and vector,then recovery and connection.The recombinant plasmid was transformed and amplified in competent cells,and IPTG was added to induce protein expression.After the protein was expressed,Tris-HCl and urea were used for purification to obtain human full-length recombinant TRABD2 A protein.Our selected phage libraries HDB 169 and HDB 323 are constructed from the same set of donors with variable CDRH3 genes,the constructed phage storage capacity is 1012.After panning with recombinant human TRABD2 A protein,Fab-positive high-titer phage strains were obtained for antigen-antibody binding experiment verification,and phage with strong binding ability was selected for gene sequencing,then TRABD2 A humanized light chain and heavy chain genes were obtained.Analysis of the sequencing results showed that the antibody gene has one single open reading frame and does not contain stop codons,and after comparison,the variable region has typical antibody characteristics.After inserting the antibody gene into the vector,the protein is expressed in the cell and purified to obtain the humanized TRABD2 A monoclonal antibody.The monoclonal antibody produced by this method is fully humanized,with low immunogenicity and strong binding force,and can be used for subsequent experimental studies in cells.3.Study on the effect of TRABD2 A humanized m Ab on HIV reservoir In this part,we used the newly synthesisd humanized TRABD2 A monoclonal antibody to block TRABD2 A in PBMCs and CD4+ T cells of untreated patients and long-term ART-treated patients whose viral load was below the limit of detection,so that it cannot exert antiviral function.Through the supernatant detection of hypersensitive p24,infection of TZM-bl reporter cells and viral load determination of the RNA in the supernatant,it can be found that TRABD2 A can inhibit virus production in patients' resting PBMCs and CD4+T lymphocytes.Blocking the TRABD2 A in cells can cause the obvious release of the virus.And it can be seen from the luciferase results of TZM-bl cells that the released virus contains progeny viruses with infectious ability.At the same time,PBMCs and CD4+T cells treated with TRABD2 A humanized monoclonal antibody were tested for cell proliferation and activation.It can be seen that the cell surface activation marker CD25/CD69/HLA-DR did not change,and the cell trace staining results showed that the cells did not amplification.This shows that the virus released from the cell comes from the assembly and release of the virus itself in the cell,rather than the virus replication caused by cell activation.Cell RNA is extracted for RT-q PCR to detect the transcription level of the virus Gag,and RNA-seq has performed to analyze the overall genome transcription level of the cell.It was found that the transcription levels of virus and cell genes remained unchanged.When using healthy human PBMCs to dilute the patient reservoir cells in a gradient,it can be seen that the amount of virus released has a linear relationship with the number of reservoir cells.All above results can prove that inhibiting the metalloprotease TRABD2 A by humanized monoclonal antibody to induce virus release in reservoir cells is a feasible,safe and convenient method.Conclusions 1.In the reservoir cells of long-term treatment patients,TRABD2 A can limit the release of progeny viruses.Using different methods to inhibit the function of TRABD2 A can cause virus release from reservoir cells without changing the state of cell proliferation and activation.2.Synthesize a humanized TRABD2 A monoclonal blocking antibody that has high affinity with TRABD2 A protein and can be used in clinical applications.3.The use of humanized antibodies to block TRABD2 A in the patients' cells can cause virus release without changing the resting state of the cells and the transcription level of the whole cell genome.The amount of virus released from the reservoir caused by this method is positively correlated with the number of reservoir cells. |
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