WO2022217373A1 - 一种降低人免疫缺陷病毒感染的病毒设定点及重建细胞免疫的因子和药物应用 - Google Patents

一种降低人免疫缺陷病毒感染的病毒设定点及重建细胞免疫的因子和药物应用 Download PDF

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WO2022217373A1
WO2022217373A1 PCT/CN2021/086313 CN2021086313W WO2022217373A1 WO 2022217373 A1 WO2022217373 A1 WO 2022217373A1 CN 2021086313 W CN2021086313 W CN 2021086313W WO 2022217373 A1 WO2022217373 A1 WO 2022217373A1
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cells
vmip
tcm
hiv
infection
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French (fr)
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利时雨
孙晗笑
王玉哲
邓健善
刘姝婷
钟志颖
耿承旭
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利时雨
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention belongs to the basic research field of viral macrophage inflammatory protein vMIP-II in the prevention and treatment of inflammation and SIV/HIV infection. Efficiently dissociates HIV antigen-receptor trimer complexes in the initial stage of infection, thereby lowering the viral set point; and induces cluster differentiation8 thymus-dependent lymphocytes (CD8 + T cells) to dephosphorylate into long-lived central memory cells (CD8+ T cells) + Tcm) new mechanism of action.
  • CD8 + T cells cluster differentiation8 thymus-dependent lymphocytes
  • CD8+ T cells long-lived central memory cells
  • CD8 + T cells are a subset of T cells that play an important role in disease defense, primarily against viruses and tumor cells.
  • Memory CD8 + T cells circulating in the blood and present in lymphoid organs are an important component of long-lived T cell immunity. This is because CD8 + T cells are activated mainly by recognizing antigenic peptides presented by MHC I molecules through the T cell receptor (TCR) on their surface, and activated CD8 + T cells will undergo 90% to 95% apoptosis in the contraction phase , only a small fraction formed antigen-specific memory CD8 + T cells.
  • TCR T cell receptor
  • memory CD8 + T cells are still able to rapidly effect effector clearance of antigen upon re-exposure to pathogens, but also share many of the same properties as the naive cells, including pluripotency and the ability to migrate to the lymph nodes and spleen.
  • memory cells embody features shared by both naive and effector cells, leading to a long-standing debate around whether memory T cells develop from effector cells or directly from naive cells.
  • Memory T cells achieve circulation between peripheral blood and lymphoid tissues by expressing or not expressing chemokine receptor CCR7 and vascular L-selectin CD62 (CD62L), and are divided into central memory T cells (Central memory T cells, Tcm) and effector memory T cells (Tem).
  • Tcm cells express CCR7 and CD62L, and are mainly distributed in immune organs and lymph nodes in peripheral tissues. When stimulated by antigens again, they can rapidly divide, proliferate and differentiate; Tem cells express CCR7 and CD62L at low levels or do not express, mainly in non-lymph Tissues and organs, involved in the systemic circulation, can migrate to peripheral inflammatory tissues to develop immediate effector functions.
  • Tissue-resident memory T cells do not express CCR7 and CD62L, but highly express CD69 and/or CD103 and do not participate in the systemic circulation.
  • CD8 + T cells differentiate into effector cells when endogenous peptides are presented on antigen-presenting cells (APCs) by MHC class I molecules. This process requires co-stimulatory molecules, such as the interaction of CD80/86, and cytokine signaling typically provided by DCs and activated CD4 + T cells.
  • naive specific CD8 + T cells Once naive specific CD8 + T cells are activated, an efficient response requires clonal expansion and formation of primary effector cells capable of recognizing peptides from virally infected or tumor cells, resulting in direct killing via perforin, granzyme and Fas/FasL interactions Dead antigen-carrying cells.
  • cytokines with antimicrobial effects such as TNF- ⁇ and IFN- ⁇
  • chemokines MIP-1 ⁇ / ⁇ and RANTES
  • Viral macrophage inflammatory protein-II is a human chemokine small molecule protein encoded by the Kaposi's sarcoma herpes virus (KSHV) K4 gene, which interacts with human CC chemokine macrophage inflammatory proteins. Protein I has high homology in amino acid sequence. vMIP-II can interact with its receptors by utilizing the structural framework similar to other chemokines. Studies have shown that vMIP-II is a broad-spectrum chemokine receptor inhibitor and has the ability to bind a variety of human chemokines. It has the ability of receptor subfamily, and can competitively inhibit the binding of HIV to co-receptors CCR5, CXCR4, CCR3, etc.
  • KSHV Kaposi's sarcoma herpes virus
  • Chemokine receptors contain serine/threonine at the carboxyl terminus of their intracellular domain, which can be phosphorylated to couple with G proteins and participate in the regulation of signal transduction pathways such as MAPK, JAK-STAT, and NF- ⁇ B.
  • MAPK MAPK
  • JAK-STAT phosphorylated to couple with G proteins and participate in the regulation of signal transduction pathways
  • NF- ⁇ B In mammals, NF- ⁇ B is tightly bound to the inhibitory protein I ⁇ B and exists in the cytoplasm in an inactive state. Under the stimulation of inflammatory signals (such as TNF- ⁇ , LPS, IL-1, etc.), IKK can be activated.
  • IKK catalyzes the phosphorylation of I ⁇ B, and then I ⁇ B can be labeled by ubiquitin and then transported to the proteasome for degradation, releasing free NF- ⁇ B. After that, free NF- ⁇ B can be translocated into the nucleus through the nuclear pore complex under the mediation of nuclear translocation sequence, and play a role in the transcription of a variety of inflammation and immune-related genes (including IL-1 ⁇ and TNF- ⁇ , etc.). Regulation.
  • a large number of cell surface integrins aggregate to activate the G protein coupled to the cytoplasm, causing rapid mobilization of Ca 2+ and further activation of phosphorylated mitogens Protein kinase (MAPK), focal adhesion kinase (FAK), protein kinase C, guanosine triphosphatase and other tyrosine kinases, a variety of tyrosine kinase pathways mediate signal transduction, recombine intracellular skeleton proteins, produce Chemotaxis, causing the movement of target cells, participates in various physiological and pathological processes in vivo.
  • MAPK Protein kinase
  • FK focal adhesion kinase
  • protein kinase C protein kinase C
  • guanosine triphosphatase and other tyrosine kinases a variety of tyrosine kinase pathways mediate signal transduction, recombine intracellular skeleton proteins,
  • CCR7 is a surface marker of central memory CD8 + T cells and exists on the surface of CD8 + T cells, when vMIP-II binds to it, it prevents CCR7 from binding to high-affinity ligands CCL21 and CCL19, possibly triggering dephosphorylation effect.
  • the CD4 molecule on the surface of T lymphocytes is the receptor of HIV.
  • the HIV envelope protein gp120 binds to the CD4 molecule on the cell membrane, the conformational change of gp120 exposes gp41.
  • gp120-CD4 interacts with the chemokine CXCR4 on the surface of the target cell.
  • CXCR5 binds to form a CD4-gp120-CXCR4/CXCR5 trimolecular complex.
  • gp41 acts as a bridge, using its own hydrophobic effect to mediate the fusion of the viral envelope and the cell membrane, eventually causing the cell to be destroyed.
  • HIV-1 epitopes from incoming virions pass exogenous in primary human dendritic cells MHC-I pathway presentation, and to a lesser extent in macrophages, leads to cytotoxic T lymphocyte activation in the absence of viral protein synthesis. It is suggested that HIV exogenous antigen peptide can stimulate the activation and proliferation of specific CD8 + T cells.
  • vMIP-II can promote the clonal proliferation of HIV-specific CD8 + T cells by using receptor inhibitors such as vMIP in the acute phase of SIV, so as to achieve The purpose of lowering the viral set point early in infection, delaying viral progression.
  • vMIP-II can also block most chemokine receptors to improve the effect.
  • Immunocompetence of CD8 + T cells we intend to use rhesus monkeys infected with simian HIV SIVmac251 as a model experimental object to observe the effect of vMIP on the distribution of effector CD8 + T subsets, and to detect the effect of vMIP-II binding to CCR7 on the dephosphorylation pathway of target cells. transcription factors to verify whether effector CD8 + T cells can be dephosphorylated into long-lived memory CD8 + T cells.
  • the present invention successfully constructs a rhesus monkey SIV infection model and finds that vMIP-II can effectively dissociate the HIV antigen-receptor trimer complex in the early stage of HIV infection, thereby reducing the new action mechanism of the virus set point; CD8 + T cells that proliferate into CD8 + Tcm cells from dephosphorylation under vMIP-II treatment and study the new mechanism of vMIP-II-induced dephosphorylation of effector CD8 + T cells into long-lived memory CD8 + Tcm cells, so that it can be used in anti-inflammatory drugs. HIV/SIV virus and anti-tumor adoptive immunity and inflammatory response play a role in the prevention or/and treatment.
  • the method for constructing a rhesus monkey SIV infection model of the present invention is to inoculate Mamu-A*01 positive rhesus monkeys with SIVmac251 at a dose of 50 TCID 50 intravenously, and use an ultra-sensitive branched DNA amplification assay to conduct viral load analysis to determine Construction of a rhesus monkey SIV infection model.
  • the present invention conducts vMIP-II intervention treatment through rhesus monkey SIV infection model, studies the effect of vMIP-II on lymphoid tissue, CD8 + T cells, CD8 + Tcm cells and CD8 + Tem cells, and studies the effects of vMIP-II on CD8 + T cells, CD8 + Tcm cells, and CD8 + Tcm cells.
  • Cells and CD8 + Tem cells were sorted by flow cytometry to determine the ratio of CD8 + Tcm cells and CD8 + Tem cells.
  • the vMIP-II provided by the present invention can effectively dissociate the HIV antigen-receptor trimer complex in the early stage of HIV infection, reduce the virus set point and induce the dephosphorylation of CD8+ T cells into Tcm cells, which can promote lymphoid tissue proliferation, Enlarge the germinal center of lymph node, make CD8 + Tcm cells proliferate, reduce the effect of inflammatory response, and thus have a protective effect on the body's immunity.
  • vMIP-II can significantly reduce the viral load in the acute phase of HIV infection and promote the generation of specific effector CD8 + T cells through the dynamic study of fluorescently labeled gp120 antigen peptides and a series of experiments such as immunoblotting, and vMIP-II CD4 + T cells can be protected during the acute phase of HIV infection by dissociating the gp120-CD4-CCR5 trimer on HIV target cells, blocking the entry of viral RNA and releasing free gp120 protein.
  • the vMIP-II provided by the present invention can effectively dissociate the HIV antigen-receptor trimer complex in the early stage of HIV infection, reduce the virus set point and induce the dephosphorylation of CD8 + T cells into Tcm cells, which can induce CD8 + Tcm cells
  • Proliferation, gene sequencing showed that the differentially expressed genes between the proliferating cells and CD8 + T cells were mainly enriched in surface chemokine receptors CCR7, CXCR4, CXCR5 and CX3CR1 and phosphorylation pathway-related genes.
  • the vMIP-II that induces the dephosphorylation of CD8 + T cells into CD8 + Tcm cells has the following mechanism: when vMIP-II is treated, CD8 + T cells mainly down-regulate signaling pathways related to phosphorylation, including low expression CD8 + T cell G protein level, reducing cellular Ca 2+ concentration and mitochondrial membrane potential, etc., make CD8 + T cells undergo metabolic reprogramming, and by inhibiting phosphorylation-related genes GNAT1, PI3K, ERK, AKT, BCL-2 make CD8 + T cell phosphorylated proteins ERK1/2 and Akt are down-expressed, thereby dephosphorylating CD8 + T into CD8 + Tcm cells and promoting the proliferation of CD8 + Tcm cells.
  • the present invention provides a vMIP-II action mechanism that can effectively dissociate the HIV antigen-receptor trimer complex in the early stage of HIV infection, reduce the virus set point and induce the dephosphorylation of CD8 + T cells into Tcm cells.
  • the invention can be used to prepare a drug for treating HIV/SIV infection and AIDS, and provides a new means for the prevention or/and treatment of adoptive immunity against virus and tumor and verification of response.
  • vMIP-II can block the entry of viral RNA and release free gp120 protein by dissociating the gp120-CD4-CCR5 trimer on HIV target cells in the acute phase of HIV infection. , thereby protecting CD4 + T cells.
  • Free gp120 protein can be captured by APC and presented to CD8 + T cells to differentiate into specific effector CD8 + T cells, enhance the body's cellular immunity, and clear infected cells, so vMIP-II is both CD4 + T cells
  • Cell protectors are also stimulators of CD8 + T cells, thereby reducing the early viral set point, reducing the viral latent pool, and significantly delaying the progression of AIDS.
  • vMIP-II also promotes the proliferation of CD8 + Tcm cells, which is associated with the co-action of the chemokine receptors CCR7, CXCR4, CXCR5 and CX3CR1 on the surface of CD8 + T cells.
  • CD8 + T cells Upon vMIP-II treatment, the chemokine receptors CCR7, CXCR4, CXCR5 and CX3CR1 on the surface of CD8 + T cells are blocked, resulting in downregulation of phosphorylation-related signaling pathways, including low expression of CD8 + T cell G protein levels, decreased cell Ca 2+ concentration and mitochondrial membrane potential, etc., make CD8 + T cells undergo metabolic reprogramming, and dephosphorylate CD8 + T cells to CD8 + Tcm through low expression of phosphorylated genes GNAT1, PI3K, ERK, AKT, BCL-2 cells, thereby promoting the proliferation of CD8 + Tcm cells.
  • vMIP-II can promote lymphoid tissue hyperplasia in vivo, and the germinal center in the middle of lymph node enlarges.
  • the discovery of the mechanism of action of vMIP-II provides a new strategy for the development of HIV/SIV-infected AIDS drugs, as well as a new approach for adoptive immunotherapy against viruses and tumors.
  • Figure 1 is the pathological histological section of rhesus monkey lymphoid tissue hyperplasia after 13 weeks of continuous intravenous injection of vMIP-II (800 ⁇ g/kg) in Example 1 (Note: A is the pathological section of the lymph node follicle; B is the pathological section of the thymus follicle; C is the spleen White pulp pathological section).
  • FIG. 4 is a flow cytometry analysis diagram of CD8 + T cells in different dose groups treated with infection for 18 days in Example 1.
  • FIG. 4 is a flow cytometry analysis diagram of CD8 + T cells in different dose groups treated with infection for 18 days in Example 1.
  • Figure 5 is the number of CD4 cells with labeled gp120 antigen peptide on the cell surface analyzed by flow cytometry at 24h in Example 1.
  • A SIVmac251-infected positive non-treatment control group; B, 50 ⁇ g/kg vMIP-II dose treatment group; C, 200 ⁇ g/kg vMIP-II dose treatment group; D, 800 ⁇ g/kg vMIP-II dose treatment group; E, AZT +3TC treatment group.
  • Figure 6 shows the interaction between CCR5, CD4 and gp120 by immunoblotting in Example 1.
  • Each lane is: 1. SIV-infected positive untreated control group (group I); 2. 50 ⁇ g/kg vMIP-II treatment group (group II); 3. 200 ⁇ g/kg vMIP-II treatment group (group III); 4. 800 ⁇ g/kg vMIP-II treatment group (group IV); 5. AZT+3TC treatment group (group V).
  • Figure 7 shows the phenotype of PBMC effector CD8 + T cells in Example 1 (Control is a control group not infected with SIVmac251; SIVmac251 is a test group infected with SIVmac251).
  • Figure 8 shows the expression of chemokine receptors of CD8 + T cells in PBMC effectors of Example 1 (Control is a control group not infected with SIVmac251; SIVmac251 is a test group infected with SIVmac251).
  • Figure 9 shows the expression of homing receptors for PBMC effector CD8 + T cells in Example 1.
  • FIG. 10 is the MA map of differentially expressed genes in Example 1.
  • Figure 11 shows the KEGG pathway analysis in Example 1 (Note: the longer the bar, the higher the enrichment; the shorter the bar, the lower the enrichment).
  • Figure 13 shows the effect of vMIP-II on the expression of intracellular G protein detected by Western blot in Example 1.
  • Figure 14 shows the effect of Example 1 vMIP-II on intracellular calcium flux.
  • FIG. 15 is the flow cytometry analysis of peak calcium flux in Example 1.
  • Figure 16 shows that the mitochondrial membrane potential of effector CD8 + T cells is decreased by vMIP-II in Example 1.
  • FIG. 17 is the overall level of phosphorylation of effector CD8 + T cells in Example 1.
  • Figure 18 is the effect of Example 1 vMIP-II on MAPK/ERK, Akt phosphorylation pathway.
  • Example 1 vMIP-II inhibits HIV infection and promotes proliferation by reducing the CD4 + T virus set point Effector mechanism of dephosphorylation of CD8 + T cells into CD8 + Tcm cells
  • SIVmac251 propagates in human peripheral blood mononuclear cells (PBMC). Viral titer titration showed that inoculated SIV contained approximately 50 TCID50/ml in human PBMC.
  • vMIP-II stock solution for injection and lyophilized powder for injection vMIP-II antigen standard (physical and chemical reference substance) was developed by the Institute of Genomic Medicine of Jinan University and passed the national drug and biological product test.
  • vMIP-II monoclonal antibody, monkey CD3, CD4, CD8 monoclonal antibodies were purchased from American R&D Company.
  • Anti-CD8 antibody-APC allophycocyanin, allophycocyanin
  • anti-CD3 antibody-FITC fluorescein isothiocyanate, fluorescein isothiocyanate
  • anti-CD4 antibody-ECD anti-CCR7 antibody-PE (phycoerythrin, phycoerythrin), etc.
  • rhesus monkeys were inoculated intravenously with SIVmac251 at a dose of 25 TCID 50 .
  • 20 Mamu-A*01 positive rhesus monkeys were randomly divided into 5 groups (4 in each group). The first group was the untreated control group infected with SIVmac251; the second group was the 50 ⁇ g/kg dose group; the third group was the 200 ⁇ g/kg dose group; the fourth group was the 800 ⁇ g/kg dose group; the fifth group was the treatment control group.
  • the untreated control group was intravenously injected with the same dose of normal saline; the II, III, and IV groups were intravenously injected with the corresponding dose of vMIP-II (prepared by our laboratory, expressed and purified in E. coli); the treatment control group was intravenously injected (AZT+3TC) (Glaxo, AZT 100 mg/kg, 3TC 50 mg/kg). Treatments for all groups were administered individually by group starting on the day of infection with SIV-mac251, once a day for two consecutive weeks. Then the drug was stopped for one week, and the drug was repeated for two weeks on the 6th to 7th week.
  • peripheral blood of 5 groups of rhesus monkeys was collected at 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 84, 98, and 119 days after SIVmac251 infection.
  • PBMCs were separated by gradient centrifugation as described above, and stored at -80 °C; -80°C.
  • DNA was extracted from PBMC cells according to the instructions of the QIAampDNA MiniKit (Qiagen). DNA was dissolved in 100 ⁇ L of sterilized water, and the DNA concentration was measured at A 260 (three duplicate wells were made for each duplicate tube), and the average value was taken.
  • Lymphoid tissue includes lymph nodes, spleen, thymus, and mucosa of the small intestine.
  • Plasma samples were collected from the upper layer following Ficoll density gradient centrifugation of cryopreserved plasma. Viral RNA levels were measured using an ultrasensitive branched DNA amplification assay (Bayer Diagnostics, Berkeley, CA). The lower limit of detection was 200 copies per milliliter.
  • the specific peptide p11C (CTPYDINQM) was synthesized by New England Peptide LLC, and the Mamu-A*01/p11C tetrameric complex was prepared as described.
  • the PE-conjugated Mamu-A*01/p11C complex was combined with anti-CD8-FITC (Becton Dickinson, San Jose, CA), anti-CD4-ECD (Beckman Coulter, Miami, FL) and anti-rhesus CD3-APC ( Dako, Glostrup, Denmark) to stain peripheral blood mononuclear cells (PBMCs) of all animals.
  • PBMCs peripheral blood mononuclear cells
  • Sorting was performed on a Coulter EPICS Elite ESP (Beckman Coulter) located in the dedicated BSL-3 area to obtain individual CD8 + T lymphocyte subsets for each animal.
  • the sorter is electronically set to achieve >98% enrichment of selected cell subsets.
  • Negative controls consisting of SIVmac251 infected Mamu-A*01 negative or uninfected Mamu-A*01 positive animals were included in each flow cytometry analysis.
  • the effector CD8 + T cells with higher purity in the sorted effector CD8 + T cells were suspended in RPMI-1640 medium containing 10% fetal bovine serum, and gp120 antigen peptide was added at the same time, and then evenly spread in 96 wells plate (the number of cells per well is 1 ⁇ 10 5 ); the cell culture plate was cultured in a cell culture incubator at 5% CO 2 , saturated humidity and 37°C.
  • gp120 antigen peptide will combine with CD4 and the receptor CXCR4 or CCR5 on the surface of the target cell membrane to form a complex, and under the stimulation of gp120 antigen peptide, CD8 + T cells proliferate stably, so we dynamically study gp120 antigen peptide by fluorescent labeling method , to determine the mechanism of action that induces proliferation of CD8 + T cells.
  • the CD4 + T cells sorted from peripheral blood were disrupted by sonication, centrifuged for 10 min, the supernatant was collected, and the pellet was discarded. The supernatant was subjected to SDS-PAGE electrophoresis. After obtaining electrophoresis bands, the bands corresponding to gp120, CD4, and CCR5 were cut out and transferred to the membrane. The membrane was stained with 1 ⁇ Ponceau red dye for 5 minutes. After washing, the membrane was dried and used. . After soaking the membrane with TBS from bottom to top, transfer it to a plate containing blocking solution, and shake it on a destaining shaker at room temperature to block for 1 h.
  • RNA-seq library The total RNA was enriched with Oligo (dT), the RNA was randomly broken into 200 nt, the random primer hexamer was reverse transcribed into cDNA, end repaired, A was added, and PCR amplification was performed after adding a linker. The construction of the augmented library was implemented.
  • the sample library is mixed according to the Illumina standard to prepare a Cluster: one end of the replication chain is fixed on the chip, and the other end is randomly complementary to another nearby primer and fixed to form a "bridge".
  • the formed bridge single-stranded uses the surrounding primers as amplification primers, and is amplified on the surface of the chip to become double-stranded, and then denatured to form a single-stranded, then a bridge will be formed again, and the next round of amplification can be performed. After several times of amplification, each single molecule can be amplified in a large amount to form a Cluster.
  • the obtained data results are removed according to the quality control standards to remove short sequences containing adapters, remove short sequences with a ratio of N > 10%, and also remove low-quality short sequences.
  • the final data (Q30> 85%) is used for subsequent analyze.
  • KEGG is a database resource for understanding and utilizing high-level functions of biological systems such as cells, organisms and ecosystems, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental techniques (http:/ /www.genome.jp/kegg/) for mining information.
  • Statistical enrichment analysis of differentially expressed genes in the KEGG pathway was performed using KOBAS software. Analysis of whether the differentially expressed genes are over-presented in a certain pathway is the Pathway enrichment analysis of the differentially expressed genes, and the enrichment degree of the Pathway is analyzed by the enrichment factor (Enrichment Factor).
  • RNA-Seq data we selected some differentially expressed genes and performed relative fluorescence quantitative PCR analysis on their expression levels.
  • the sorted effector CD8 + T cells and CD8 + Tcm cells were placed in a 5% CO 2 cell incubator and cultured at 37 °C for 24 h.
  • Total RNA from effector CD8 + T cells and Tcm cells was extracted according to the operating instructions of the cell total RNA extraction kit, and then cDNA synthesis was performed using the SuperScript TM Preamplification System for First Strand cDNA Synthesis Kit. All primer sequences of the fluorescence quantitative PCR reaction are listed in Table 1, and the reaction was carried out in a fluorescence quantitative PCR instrument: MiniOpticon TM (BIO-RAD, Laboratories, Inc.
  • the total reaction volume was 25.0 ⁇ L, including 2.0 ⁇ L of total RNA, 8.5 ⁇ L of RNase-free H 2 O, 0.5 ⁇ L of forward and reverse primers (10 mM/L), 12.5 ⁇ L of 2 ⁇ One-step SYBR RT-PCR Buffer III, TaKaRa Ex Taq HS (5U/ ⁇ L) 0.5 ⁇ L, and PrimeScript RT Enzyme Mix II 0.5 ⁇ L.
  • PCR reaction conditions were 95 °C for 5 min, 95 °C for 30 s, 60 °C for 45 s, and 72 °C for 45 s, a total of 33 cycles. All experiments were performed in 3 independent biological replicates. Standard relative transcript levels were estimated using the 2- ⁇ Ct method.
  • the effector CD8 + T cells obtained by sorting the PBMCs of animals in the 800 ⁇ g/kg dose group were disrupted by sonication, centrifuged for 10 min, the supernatant was collected, and the precipitate was discarded.
  • the supernatant was subjected to SDS-PAGE electrophoresis to obtain electrophoresis bands, and the bands corresponding to Tcm and CCR7 were cut out and transferred to the membrane, and the membrane was stained with 1 ⁇ Ponceau staining solution for 5 min. After washing, the membrane was air-dried for use. After soaking the membrane with TBS from bottom to top, transfer it to a plate containing blocking solution, and shake it on a destaining shaker at room temperature to block for 1 h.
  • PHA polyhydroxyalkanoate
  • the effector CD8 + T&Tcm cell subsets were significantly dephosphorylated. Therefore, we took the effector CD8 + T cells and CD8 + Tcm cells mixed subsets for Bio-Plex suspension chip system detection. Verify the overall level of phosphorylation changes.
  • the experimental procedure was carried out according to the instructions of Bio-Plex pro assay.
  • the effector CD8 + T cells isolated from PBMCs of animals in the 800 ⁇ g/kg dose group were starved for 1 h in 0.1% bovine serum RPMI 1640 medium, stimulated with or without CCL21 (200 ng/ml), and washed with cold PBS for 3 days after collection. Second, after cell lysis, total protein was extracted, quantified by Bradford method, denatured by heat, and the same amount of protein was loaded for SDS-PAGE. Electroporated to PVDF membrane and blocked overnight with 5% nonfat dry milk in TBS.
  • Akt and ERK1/2 (1:1000) primary antibodies were added and incubated at room temperature for 2 h, TTBS (TBS with 1 ⁇ Tween-20) was added to wash the membrane, goat anti-rabbit IgG The secondary antibody (1:2000) was incubated at room temperature for 1 h. After washing the membrane, the membrane was subjected to ECL autoradiography and exposed to Kodak film. Taking Akt and ERK2 as reference, the band absorbance value was analyzed by image analysis software GELpro3.0.
  • lymphoid organs had obvious hyperplasia, and showed a dose-dependent effect, namely: the 50 ⁇ g/kg dose group had less proliferation; the 200 ⁇ g/kg dose group lymph node lymphoid follicle hyperplasia; 800 ⁇ g/kg dose group
  • lymph node lymphoid follicles, spleen white pulp, thymus gland, and small intestinal mucosal lamina intestinal lymphoid follicles all showed hyperplasia, and the latter was accompanied by obvious central enlargement, as shown in Figure 1.
  • the degree of hyperplasia was significantly reduced in the second week of the recovery period.
  • Plasma viral load was monitored throughout the study or until death of each animal. As shown in Figure 2, although all 20 rhesus monkeys displayed variable plasma viral loads, the peaks of acute plasma viremia were comparable, and the 50 ⁇ g/kg, 200 ⁇ g/kg and 800 ⁇ g/kg dose groups showed comparable The measured values of plasma viral load were generally lower than those in the plasma of the untreated control group, and the plasma viral load of the animals in the 50 ⁇ g/kg, 200 ⁇ g/kg and 800 ⁇ g/kg dose groups was dependent on vMIP-II, But slightly higher than the AZT + 3TC treatment control group. Plasma viral load results showed that the administration of chemokine receptor inhibitors, especially vMIP-II, helped control SIV replication in vivo.
  • Tetramer-positive CD8 + T cells were detected using MHC class I tetramer and fluorescence-activated cell sorting staining. As expected, Mamu-A*01/p11C tetramer-bound cells were undetectable ( ⁇ 0.1%) in Mamu-A*01 positive animals prior to infection ( Figure 3).
  • PBMCs from rhesus monkeys in the 50 ⁇ g/kg, 200 ⁇ g/kg and 800 ⁇ g/kg dose groups showed a significant increase in the level of Mamu-A*01/p11C tetramer-bound CD8 + T cells, and the There was a positive relationship between proliferation and vMIP-II treatment dose, which was consistent with the results obtained by flow cytometry analysis (Fig. 4).
  • the level of tetramer-bound CD8 + T cells was lower, with only about 2% CD8 + T cells, and the number of CD8 + T cells in the AZT+3TC group was slightly lower. lower than the untreated control group.
  • chemokine receptor inhibitor vMIP-II can control SIV replication by maintaining the expansion of SIVmac251 epitope-specific CD8 + T cells. It should be noted, however, that the observed expansion of the virus-specific T cell population does not necessarily represent the division of individual cells, but rather a measure of a net increase in cell number.
  • gp120-CD4 binds to the receptor on the surface of the target cell membrane, CXCR4 or CCR5 forms a complex.
  • CXCR4 or CCR5 forms a complex.
  • vMIP-II can dissociate the gp120-CD4-CCR5 trimer to form free gp120 protein, which is captured by APC cells and presented to CD8 + T cells to stimulate the production of effector T cells.
  • APC cells After Western blot analysis (Fig. 6), under the detection of anti-Flag specific antibody, the trimer band was observed in group I, and the trimer band in groups II, III, IV and V was obviously weakened or even absent. .
  • Rhesus monkey PBMC were taken to prepare single cell suspension, and after staining, the expression levels of effector CD8 + T cell surface molecules CD44, CD69, CD103, CD62L and CCR7 were detected by flow cytometry.
  • the results showed that after SIVmac251 infection, rhesus monkey PBMC effector CD8 + T cells highly expressed CD62L (94.6%), CD45RO (90.2%), CCR7 (79.8%), and low expressed CD44 (7.9%), CD69 (3.3%) ( Figure 7).
  • RNA-seq sequencing was used to detect the differential expression of effector CD8 + T&Tcm cell subset genes in the 800 ⁇ g/kg vMIP-II treatment group and the vMIP-II treatment group without treatment. Through comparison, a total of 79 significantly different genes were screened according to the screening criteria. (fold difference ⁇ 2, p ⁇ 0.01), including 48 genes that were significantly up-regulated and 31 genes that were significantly down-regulated (Figure 10).
  • vMIP-II is mainly involved in the regulation of effector CD8 + T cell apoptosis and phosphorylation pathway in the immune response of the body infected with SIVmac251 virus.
  • the abundant enrichment of differentially expressed genes in signaling pathway molecules and cytoplasm also illustrates the above point of view from another aspect.
  • phosphorylation pathway, TCA cycle and regulation of apoptosis are the three GO-terms with the most significant enrichment of differentially expressed genes. Sorted in descending order from small to small, and screened the differential genes with a multiple of difference>3 (ie P-value ⁇ 0.001; log 2 Ration>1.5), and finally identified GNAT1, PI3K, ERK, AKT, NF- ⁇ B, BCL-2, FAS , PUMA, BAX, p53AIP1 these 10 genes as the key target genes of our follow-up research.
  • PI3K, ERK, AKT, and BCL-2 were significantly decreased, while the expressions of NF- ⁇ B, BAX, FAS, PUMA, and p53AIP1 were significantly increased, which was consistent with the RNA-seq sequencing results.
  • vMIP-II Compared with the normal control group, vMIP-II not only could not significantly increase the calcium ion concentration in effector CD8 + T cells, but also significantly reduced the calcium ion concentration in effector CD8 + T cells (P ⁇ 0.05). At the same time, compared with the positive control group, pretreatment of cells with vMIP-II could significantly decrease the calcium ion concentration induced by Fractalkine (P ⁇ 0.01, Figures 14 and 15).
  • Figure 16 shows the effect of vMIP-II on mitochondrial membrane potential of effector CD8 + T cells.
  • the JC-1 fluorescent dye aggregates in the mitochondrial matrix and produces red fluorescence when the mitochondrial membrane potential is high; when the three-dimensional membrane potential of the cell line decreases, JC-1 begins to become monomer and produces green fluorescence.
  • the mitochondrial membrane potential of effector CD8 + T cells isolated from rhesus monkeys in the 800 ⁇ g/kg dose group was analyzed. Compared with the control group, the mitochondrial membrane potential of effector CD8 + T cells after vMIP-II treatment decreased significantly (P ⁇ 0.01).
  • rhesus effector CD8 + T cells were starved for 1 h in 0.1% bovine serum medium, stimulated with or without CCL21 (200 ng/ml), and the negative control group not treated with vMIP-II was used as the control.
  • the protein expressions of phosphorylated ERK1/2 and Akt were detected at 60 min of stimulation, respectively.
  • Figure 18 shows that high levels of phosphorylated ERK1/2 and Akt can be detected in CD8 + Tcm cells only under the action of CCL21.
  • the expression levels of phosphorylated ERK1/2 and Akt were significantly reduced. This indicated that vMIP could inhibit the expression of phosphorylated ERK1/2 and Akt proteins, and vMIP-II could antagonize the activation of ERK1/2 and Akt by CCL21.

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Abstract

vMIP-Ⅱ在HIV感染初期有效解离HIV抗原-受体三聚体复合物而降低病毒设定点(急性感染后建立的稳定的病毒载量)并诱导CD8 +T细胞去磷酸化为Tcm细胞的应用，属于生物技术领域。通过恒河猴SIV感染模型研究发现vMIP-II可以在HIV感染的早期通过解离HIV-靶细胞上的gp120-CD4-CCR5三聚体，释放出游离的gp120蛋白，以保护CD4 +T细胞。进而游离的gp120蛋白可以被APC捕获，并且呈递给CD8 +T细胞，使其分化成为特异性效应CD8 +T细胞，并使CD8 +Tcm含量明显增多。同时发现Tcm增殖是vMIP-Ⅱ封闭CD8 +T趋化因子受体而低表达G蛋白，降低胞内Ca 2+浓度和线粒体膜电位、抑制磷酸化的相关基因，使磷酸化蛋白ERK1/2和Akt低表达，从而使CD8 +T磷酸化信号减弱，发生代谢重编程而转化为Tcm。因此，vMIP-Ⅱ这种阻止病毒RNA进入靶细胞的新型作用机制，为HIV/SIV感染艾滋病的药物研发提供全新策略，为抗病毒与抗肿瘤的过继性免疫治疗提供新的靶点，具有重要的临床应用价值。

Description

一种降低人免疫缺陷病毒感染的病毒设定点及重建细胞免疫的因子和药物应用 技术领域
本发明属病毒病毒巨噬细胞炎性蛋白vMIP-Ⅱ在防治炎症和SIV/HIV感染等方面进行的基础研究领域，更具体地说，本发明涉及病毒巨噬细胞炎性蛋白vMIP-Ⅱ在HIV感染初期有效解离HIV抗原-受体三聚体复合物，从而降低病毒设定点；并且诱导群集分化8胸腺依赖性淋巴细胞(CD8 +T细胞)去磷酸化为长寿命中枢记忆细胞(CD8 +Tcm)的新作用机制。
背景技术
CD8 +T细胞是T细胞的亚群，其在疾病防御中具有重要作用，主要针对病毒和肿瘤细胞。在血液中循环并存在于淋巴器官中的记忆性CD8 +T细胞是长寿命T细胞免疫的重要组成部分。这是由于CD8 +T细胞主要通过其表面的T细胞受体(TCR)识别由MHCⅠ分子递呈的抗原肽而被活化，活化后的CD8 +T细胞会在收缩阶段凋亡90％～95％，仅小部分形成具有抗原特异性的记忆CD8 +T细胞。这些记忆CD8 +T细胞在再次暴露于病原体时仍能迅速发挥效应作用清除抗原，但也有许多与初始细胞相同的特性，包括多能性和迁移到淋巴结和脾脏的能力。因此，记忆细胞体现了初始细胞和效应细胞两者共有的特征，由此而引发了一场围绕记忆T细胞是从效应细胞发展还是直接从初始细胞发展的长期争论。
记忆T细胞通过表达或不表达趋化因子受体CCR7和血管L-选择素CD62(CD62L)来实现在机体外周血和淋巴组织间的循环，分为中央记忆性T细胞(Central memory T cells，Tcm)和效应记忆性T细胞(Effector memory T cells，Tem)。一般情况下Tcm细胞表达CCR7和CD62L，主要分布于外周组织免疫器官和淋巴结，当再次受抗原刺激时可迅速分裂增殖和分化；Tem细胞低水平表达或不表达CCR7和CD62L，主要存在于非淋巴组织和器官，参与周身循环，可迁移至外周炎症组织发生速发性效应功能。组织定居记忆性T细胞(Tissue-resident memory T cells，Trm)则不表达CCR7和CD62L，高表达CD69和/或CD103且不参与体循环。
在HIV感染期间，感染的发病机制和HIV进展为艾滋病的速度与感染早期的病毒控制有关。CD8 +T细胞表现出的特异性反应对于建立早期、有效和持续的对病毒的控制，防止严重的免疫改变和器官功能障碍至关重要，包括由CD8 +T细胞应答介导，包括细胞毒性和非细胞毒性的抗病毒响应。当初始CD8 +T细胞在抗原呈递细胞(APC)通过I类MHC分子呈现内源肽时，会分化为效应细胞。这个过程需要共刺激分子，例如CD80/86的相互作用，以及通常由DC和活化的CD4 +T细胞提供的细胞因子信号。一旦初始特异性CD8 +T细胞被激活，有效反应需要克隆扩增并形成能够识别来自病毒感染或肿瘤细胞的肽的初级效应细胞，导致通过穿孔素，颗粒酶和Fas/FasL的相互作用直接杀死携带抗原的细胞。此外，还可以释放具有抗微生物作用的细胞因子，如TNF-α和IFN-γ，以及趋化因子(MIP-1α/β和RANTES)。所有这些机制都有助于清除改变的细胞。
病毒巨噬细胞炎症蛋白-Ⅱ(vMIP-Ⅱ)是由卡波氏肉瘤疱疹病毒(KSHV)K4基因编码的一种人趋化 因子小分子蛋白，与人CC类趋化因子巨噬细胞炎性蛋白Ⅰ在氨基酸序列上有较高的同源性。vMIP-Ⅱ可以利用与其他趋化因子相似的结构骨架与其受体进行相互结合作用，已有研究证明，vMIP-Ⅱ是广谱的趋化因子受体抑制剂，具有结合多种人趋化因子受体亚族的能力，并能竞争性抑制HIV与靶细胞上共受体CCR5、CXCR4、CCR3等的结合，以阻止病毒进入靶细胞，具有抗HIV感染的作用。趋化因子受体其胞内区的羧基末端含丝氨酸/苏氨酸，可发生磷酸化，从而与G蛋白相偶联，参与MAPK、JAK-STAT、NF-κB等信号转导通路的调控。在哺乳动物中，NF-κB与抑制性蛋白IκB紧密结合，以无活性状态存在于细胞质当中。在炎性信号的刺激下(如TNF-α、LPS、IL-1等)，IKK可被激活。接着IKK催化IκB发生磷酸化，进而IκB可被泛素标记，从而被转运到蛋白酶体发生降解，释放出游离的NF-κB。之后，游离的NF-κB便可通过核孔复合物在核转位序列的介导下转位入核，对多种炎症与免疫相关基因(包括IL-1β和TNF-α等)的转录发挥调控作用。
因此，在配体与表达于细胞表面趋化因子受体结合后，细胞表面整合素大量聚集，激活耦联于细胞浆内的G蛋白，引起Ca 2+的快速动员以及进一步磷酸化分裂原活化蛋白激酶(MAPK)、黏着斑激酶(FAK)、蛋白激酶C、鸟苷三磷酸酶等酪氨酸激酶，多种酪氨酸激酶通路介导信号转导，重新组合细胞内的骨架蛋白，产生趋化作用，引起靶细胞运动，参与体内多种生理和病理过程。由于CCR7是中央记忆CD8 +T细胞的表面标记物，而且存在于CD8 +T细胞表面，因此当vMIP-Ⅱ与其结合时，会阻碍CCR7与高亲和力配体CCL21和CCL19结合，可能引发去磷酸化效应。
在之前的研究中，证实了用广谱受体抑制剂vMIP-Ⅱ抑制了HIV共同受体时，特别是CXCR4，可以促进体内SIV感染的恒河猴中抗原特异性CD8 +T细胞亚群的克隆扩增，这可以有效抑制病毒复制并增强急性SIV期间的免疫应答，因此趋化因子受体如CXCR4或CCR5的抑制剂vMIP-Ⅱ可能有助于增强抗原特异性CD8 +T细胞的克隆扩增能力从而抑制SIV感染。
T淋巴细胞表面的CD4分子是HIV的受体，通过HIV的囊膜蛋白gp120与细胞膜上的CD4分子结合后，gp120构像改变使gp41暴露，同时gp120-CD4与靶细胞表面的趋化因子CXCR4或CXCR5结合形成CD4-gp120-CXCR4/CXCR5三分子复合物。gp41在其中起着桥的作用，利用自身的疏水作用介导病毒囊膜与细胞膜融合，最终造成细胞被破坏。在早先的另一项研究中(MHC-I限制性表达HIV-1病毒粒子抗原，无病毒复制)，显示来自进入的病毒粒子的HIV-1表位通过原代人树突细胞中的外源MHC-I途径呈递，并且在较低程度上呈现在巨噬细胞中，导致在没有病毒蛋白质合成的情况下细胞毒性T淋巴细胞活化。提示了HIV外源抗原肽可以刺激特异性CD8 +T细胞的激活和增殖。
根据上述研究，并且鉴于SIV和HIV的同源性，我们设计在SIV急性期，通过使用vMIP等受体抑制剂，观察vMIP-II是否可以促进HIV特异性CD8 +T细胞的克隆增生，从而达到在感染早期降低病毒设定点的目的，延缓病毒的进展。探讨vMIP抑制HIV感染的新机制。
本实验室前期研究发现重组vMIP具有明显抑制病毒进入靶细胞和保护靶细胞免受病毒感染的作用，其表明vMIP可能是通过某种机制抑制细胞内SIVmac病毒的产生。因此我们进一步研究了重组vMIP-Ⅱ对食蟹猴免疫系统的影响，结果表明长期大剂量注射重组vMIP-Ⅱ具有刺激食蟹猴免疫系统， 并促进CD8 +T细胞增生和增加CD8 +T细胞功能的作用。但重组vMIP-Ⅱ是如何抑制病毒进入靶细胞、如何促进CD8 +T细胞增生的分子机制尚不清楚。
根据vMIP-Ⅱ对CCR5和CXCR4共受体的封闭性，以及CCR7与CCR5和CXCR4同属于CC类趋化因子受体，所以我们推测vMIP-Ⅱ也能封闭多数趋化因子受体，以提高效应CD8 +T细胞的免疫能力。因此，我们拟以猴艾滋病病毒SIVmac251感染恒河猴为模型实验对象，观察vMIP对效应CD8 +T的亚群分布影响，并通过检测vMIP-Ⅱ与CCR7结合对靶细胞去磷酸化途径所影响的转录因子，以验证效应CD8 +T细胞是否可以去磷酸化转化为长寿记忆CD8 +T细胞。
发明内容
本发明成功构建恒河猴SIV感染模型发现可以vMIP-Ⅱ在HIV感染初期有效解离HIV抗原-受体三聚体复合物，从而降低病毒设定点的新作用机制；同时分选出能在vMIP-Ⅱ治疗下由去磷酸化增殖为CD8 +Tcm细胞的CD8 +T细胞并研究vMIP-Ⅱ诱导效应CD8 +T细胞去磷酸化为长寿记忆CD8 +Tcm细胞的新作用机制，使其在抗HIV/SIV病毒与抗肿瘤的过继性免疫以及炎症反应的预防或/和治疗中发挥作用。
本发明构建恒河猴SIV感染模型的方法，是用50TCID 50剂量的SIVmac251静脉注射接种Mamu-A*01阳性恒河猴，并使用超灵敏分支DNA扩增测定法进行病毒载量分析，以确定恒河猴SIV感染模型的构建。
本发明通过恒河猴SIV感染模型进行vMIP-Ⅱ干预治疗，研究vMIP-Ⅱ对淋巴组织、CD8 +T细胞、CD8 +Tcm细胞、CD8 +Tem细胞的影响，对CD8 +T细胞、CD8 +Tcm细胞和CD8 +Tem细胞进行流式细胞仪分选，以确定CD8 +Tcm细胞和CD8 +Tem细胞的比例。
本发明提供的可以在HIV感染初期有效解离HIV抗原-受体三聚体复合物而降低病毒设定点并诱导CD8+T细胞去磷酸化为Tcm细胞的vMIP-II能促使淋巴组织增生，使淋巴小结生发中心扩大，并使CD8 +Tcm细胞增殖，降低炎症反应的作用，从而对机体免疫产生保护作用。
本发明通过荧光标记的gp120抗原肽的动态研究和免疫印迹等系列实验发现，vMIP-II可以明显降低HIV感染急性期的病毒载量并促进特异性效应CD8 +T细胞的生成，并且vMIP-II可以在HIV感染的急性期通过解离HIV靶细胞上的gp120-CD4-CCR5三聚体，阻碍病毒RNA的进入并释放出游离的gp120蛋白，从而保护CD4 +T细胞。
本发明提供的可以在HIV感染初期有效解离HIV抗原-受体三聚体复合物而降低病毒设定点并诱导CD8 +T细胞去磷酸化为Tcm细胞的vMIP-II可引起CD8 +Tcm细胞增殖，基因测序显示该增殖细胞与CD8 +T细胞的差异表达基因主要富集于表面趋化因子受体CCR7、CXCR4、CXCR5和CX3CR1和磷酸化通路相关基因。
本发明提供的诱导CD8 +T细胞去磷酸化为CD8 +Tcm细胞的vMIP-Ⅱ具有如下机制：当vMIP-Ⅱ治疗时，CD8 +T细胞主要通过下调与磷酸化相关的信号通路，包括低表达CD8 +T细胞G蛋白水平、降低细胞Ca 2+浓度和线粒体膜电位等，使CD8 +T细胞发生代谢重编程，并通过抑制磷酸化相关 基因GNAT1、PI3K、ERK、AKT、BCL-2使CD8 +T细胞磷酸化蛋白ERK1/2和Akt低表达，从而使CD8 +T去磷酸化为CD8 +Tcm细胞，促进CD8 +Tcm细胞增殖。
本发明提供了一种可以在HIV感染初期有效解离HIV抗原-受体三聚体复合物而降低病毒设定点并诱导CD8 +T细胞去磷酸化为Tcm细胞的vMIP-II作用机制，其可用于制备治疗HIV/SIV感染艾滋病的药物，为抗病毒与抗肿瘤的过继性免疫以及验证反应的预防或/和治疗提供新的手段。
相对于现有技术，本发明首次发现，vMIP-II可以在HIV感染的急性期通过解离HIV靶细胞上的gp120-CD4-CCR5三聚体，阻碍病毒RNA的进入并释放出游离的gp120蛋白，从而保护CD4 +T细胞。游离的gp120蛋白可以被APC捕获，并且呈递给CD8 +T细胞，使其分化成为特异性的效应CD8 +T细胞，增强机体的细胞免疫，清除受感染的细胞，因此vMIP-II既是CD4 +T细胞的保护剂，也是CD8 +T细胞的刺激因子，并由此降低了早期的病毒设定点，减少了病毒潜伏库，显著地延缓了艾滋病的进展。vMIP-Ⅱ还能促使CD8 +Tcm细胞增殖，该增殖与CD8 +T细胞表面的趋化因子受体CCR7、CXCR4、CXCR5和CX3CR1的共同作用相关。当vMIP-Ⅱ治疗时，CD8 +T细胞表面的趋化因子受体CCR7、CXCR4、CXCR5和CX3CR1封闭，引起与磷酸化相关的信号通路下调，包括低表达CD8 +T细胞G蛋白水平、降低细胞Ca 2+浓度和线粒体膜电位等，使CD8 +T细胞发生代谢重编程，并通过低表达磷酸化基因GNAT1、PI3K、ERK、AKT、BCL-2使CD8 +T细胞去磷酸化为CD8 +Tcm细胞，从而促进CD8 +Tcm细胞增殖。vMIP-Ⅱ在体内可以促使淋巴组织增生，淋巴小结中间的生发中心增大。vMIP-Ⅱ的作用机制的这一发现为HIV/SIV感染艾滋病的药物研发提供了全新策略，也为抗病毒与抗肿瘤的过继性免疫治疗提供新的手段。
附图说明
图1为实施例1 vMIP-Ⅱ(800μg/kg)连续静脉注射13周后恒河猴淋巴组织增生病理组织切片图(注：A为淋巴结滤泡病理切片；B为胸腺滤泡病理切片；C为脾白髓病理切片)。
图2为实施例1不同vMIP-Ⅱ剂量组对血浆病毒载量的影响(注：与未治疗对照组相比，*P＜0.05；**P＜0.01；Mean±SD，n＝4)。
图3为实施例1感染后不同时间点vMIP-Ⅱ诱导CD8 +T细胞增殖的折线图(注：与未治疗对照组相比，*P＜0.05；**P＜0.01；Mean±SD，n＝4)。
图4为实施例1对感染治疗18天不同剂量组CD8 +T细胞的流式细胞仪分析图。
图5为实施例1 24h时通过流式细胞术分析细胞表面带有标记的gp120抗原肽的CD4细胞数量。A、SIVmac251感染的阳性非治疗对照组；B、50μg/kg vMIP-II剂量治疗组；C、200μg/kg vMIP-II剂量治疗组；D、800μg/kg vMIP-II剂量治疗组；E、AZT+3TC治疗组。
图6为实施例1免疫印迹观察CCR5、CD4和gp120之间的相互作用。各个泳道分别是：1、SIV感染的阳性未治疗对照组(Ⅰ组)；2、50μg/kg vMIP-Ⅱ治疗组(Ⅱ组)；3、200μg/kg vMIP-Ⅱ治疗组(Ⅲ组)；4、800μg/kg vMIP-Ⅱ治疗组(Ⅳ组)；5、AZT+3TC治疗组(Ⅴ组)。
图7为实施例1 PBMC效应CD8 +T细胞的表型(Control为未用SIVmac251感染的对照组； SIVmac251为用SIVmac251感染的试验组)。
图8为实施例1PBMC效应CD8 +T细胞趋化因子受体表达情况(Control为未用SIVmac251感染的对照组；SIVmac251为用SIVmac251感染的试验组)。
图9为实施例1 PBMC效应CD8 +T细胞归巢受体表达情况。
图10为实施例1差异表达基因MA图。
图11为实施例1 KEGG通路分析(注：bar越长，富集度越高；bar越短，富集度越低)。
图12为实施例1差异表达基因的qRT-PCR验证结果(注：上述数值均为Mean±SD，n＝3；与未用vMIP-Ⅱ治疗组相比，**P＜0.01和***P＜0.001)。
图13为实施例1 Western blot检测vMIP-Ⅱ对胞内G蛋白表达的影响。
图14为实施例1 vMIP-Ⅱ对细胞内钙流的影响。
图15为实施例1流式细胞术分析钙流峰值。
图16为实施例1 vMIP-Ⅱ导致效应CD8 +T细胞线粒体膜电位下降。
图17为实施例1效应CD8 +T细胞磷酸化总体水平。
图18为实施例1 vMIP-Ⅱ对MAPK/ERK、Akt磷酸化通路的影响。
具体实施方式
以下结合实施例，对本发明进行进一步详细说明。
实施例1 vMIP-II通过降低CD4 +T病毒设定点抑制HIV感染并促进增殖的效应CD8 +T细胞去磷酸化为CD8 +Tcm细胞的作用机制研究
1材料与方法
1.1实验材料
实验动物：SPF级Mamu-A*01健康成年中国恒河猴20只，5～9kg，雌雄各半，购自北京中国医学科学院医学生物科学研究所。血清测试呈SIV、SRV、STLV-1和HBV反应阴性。采用如前所述的基于序列特异性引物聚合酶链反应(PCR)的MHC分型方法，以确定恒河猴中存在MAMU-A*01主要组织相容性复合体(MHC)Ⅰ类等位基因。本动物实验参照“实验动物护理和使用指南”(美国国家科学院出版社，华盛顿特区出版)中规定的标准进行。
病毒和细胞：SIVmac251在人外周血单个核细胞(PBMC)中增殖。病毒滴度滴定显示接种的SIV在人PBMC中大约包含50TCID 50/ml。
实验试剂：vMIP-Ⅱ注射用原液和冻干粉针剂，vMIP-Ⅱ抗原标准品(理化对照品)由暨南大学基因组药物研究所研制并通过国家药品和生物制品检定。vMIP-Ⅱ单克隆抗体、猴CD3、CD4、CD8单克隆抗体购自美国R&D公司。抗CD8抗体-APC(allophycocyanin，别藻蓝蛋白)、抗CD3抗体-FITC(fluorescein isothiocyanate，异硫氰酸荧光素)、抗CD4抗体-ECD、抗CCR7抗体-PE(phycoerythrin，藻红蛋白)等，均购自Biolegend公司；Real-time PCR Master Mix试剂盒(日本TOYOBO公司)；BD FACS流式细胞仪、荧光定量PCR系统(美国Bio-Rad公司)；人CC趋化因子受体7(CCR7)ELISA试剂盒； 1640完全培养基、二硫苏糖醇(DTT)；1mmol/L乙二胺四乙酸(EDTA)等。
1.2 SIV感染和动物分组给药方法
所有动物以氯胺酮7mg/kg麻醉后，用25TCID 50剂量的SIVmac251静脉注射接种恒河猴。在感染前，将Mamu-A*01阳性恒河猴20只随机分为5组(每组4只)。第Ⅰ组为SIVmac251感染的未治疗对照组；第Ⅱ组为50μg/kg剂量组；第Ⅲ组为200μg/kg剂量组；第Ⅳ组为800μg/kg剂量组；第Ⅴ组为治疗对照组。其中未治疗对照组静脉注射同等剂量的生理盐水；Ⅱ、Ⅲ、Ⅳ组分别单次静脉注射相应剂量的vMIP-Ⅱ(由本实验室制备，在大肠杆菌中表达并纯化)；治疗对照组静脉注射(AZT+3TC)(Glaxo，AZT 100mg/kg，3TC 50mg/kg)。所有组别的治疗均在用SIV-mac251感染的当天开始按照分组分别给药，每天一次，连续两周。然后停药一周，第6～7周重复用药两周。
1.3猴PBMC、血浆的制备和PBMC中DNA的抽提
在SIVmac251感染后的3、7、14、21、28、35、42、49、56、63、70、84、98、119天取5组恒河猴的外周血。将5组猴外周血样品经EDTA处理后，如前所述的用梯度离心法分离PBMCs，于-80℃储存；血浆分离按常规方法自收集后3h内的外周血样品中制备，冻存于-80℃。按QIAampDNAMiniKit(Qiagen)试剂盒说明书抽提PBMC细胞DNA。DNA溶于100μL的灭菌水中，于A 260测定DNA浓度(每个复管做三复孔)，取平均值。
1.4淋巴组织病理学检查
全面细致观察并记录淋巴组织的肉眼变化并作常规病理切片检查。淋巴组织包括淋巴结、脾脏、胸腺和小肠黏膜。
1.5病毒载量分析
将冻存的血浆在Ficoll密度梯度离心后从上层收集血浆样品。使用超灵敏分支DNA扩增测定法(Bayer Diagnostics，Berkeley，CA)测量病毒RNA水平。检测下限为200拷贝每毫升。
1.6四聚体染色和CD4 +T细胞与CD8 +T细胞计数和分选
特异性肽p11C(CTPYDINQM)由New England Peptide LLC合成，并且如所述那样制备Mamu-A*01/p11C四聚体复合物。将PE偶联的Mamu-A*01/p11C复合物与抗CD8-FITC(Becton Dickinson，San Jose，CA)、抗CD4-ECD(Beckman Coulter，Miami，FL)和抗恒河猴CD3-APC(Dako，Glostrup，Denmark)一起使用，对所有动物外周血单个核细胞(PBMCs)进行染色。在位于专用BSL-3区域的Coulter EPICS Elite ESP(Beckman Coulter)上进行分选，以分别获得各个动物的CD8 +T淋巴细胞亚群。该分选器通过电子方式设置，以达到>98％的所选细胞亚群的富集。在每次流式细胞术分析中均包括由SIVmac251感染的Mamu-A*01阴性或未感染的Mamu-A*01阳性动物组成的阴性对照。
1.7 CD8 +Tcm细胞分选
之前的研究表明，HIV可以通过其囊膜上的糖蛋白gp120与靶细胞膜表面的CD4分子结合，导致gp120构象改变，并且gp120-CD4结合靶细胞膜表面的受体CXCR4或CCR5会形成复合物，因此我们在实验中添加gp120抗原肽进行刺激。然后对从各组动物PBMC中分选获得的效应CD8 +T细胞 进行抗体标记(抗CD8抗体-APC、抗CD45RA抗体-FITC和抗CCR7抗体-PE)，然后立即用流式细胞术检测CD8 +Tcm细胞和CD8 +Tem细胞所占的比例。根据实验结果确定vMIP-Ⅱ治疗下效应CD8 +T细胞与CD8 +Tcm比例最高的剂量组，然后确定将该组动物用于后续验证实验。
1.8效应CD8 +T细胞培养
将分选出来的各组效应CD8 +T细胞中纯度较高的效应CD8 +T细胞悬浮于含10％胎牛血清的RPMI-1640培养基中，同时加入gp120抗原肽，然后均匀铺于96孔板中(每孔细胞数为1×10 5)；将细胞培养板置于5％CO 2、饱和湿度和37℃的细胞培养箱中培养。
1.9荧光标记的gp120抗原肽的动态研究
根据gp120抗原肽会与CD4和靶细胞膜表面的受体CXCR4或CCR5结合形成复合物，而且在gp120抗原肽的刺激下，CD8 +T细胞稳定增殖，因此我们通过荧光标记的方法动态研究gp120抗原肽，以确定引起CD8 +T细胞增殖的作用机制。我们将抗原肽gp120通过FITC标记修饰，加入到先前所制备的各组外周血单个核细胞(PBMCs)中，分别在培养0h、24h、48h，通过流式细胞分选出带标记的CD4 +T细胞和CD8 +T细胞，通过荧光显微镜观察荧光标记的情况。
1.10免疫印迹法
从外周血分选所得的CD4 +T细胞经超声破碎后，离心10min，收集上清液，弃沉淀。将上清液进行SDS-PAGE电泳，获得电泳条带后，剪取gp120、CD4、CCR5对应的条带转膜，将膜用1×丽春红染液染5min，水洗后将膜晾干备用。将膜用TBS从下向上浸湿后，移至含有封闭液的平皿中，室温下脱色摇床上摇动封闭1h。将一抗用TBST稀释至适当浓度并加至膜上；室温下孵育1～2h后，用TBST在室温下脱色摇床上洗两次，每次10min；再用TBS洗一次，10min。接着准备二抗稀释液并与膜接触，室温下孵育1～2h后，用TBST在室温下脱色摇床上洗两次，每次10min；再用TBS洗一次，10min，进行化学发光反应。将胶片进行扫描或拍照，用凝胶图象处理系统分析目标带的分子量和净光密度值。
1.11 PBMC效应CD8 +T细胞表型测定
分别采集上述确定的剂量组和对照组恒河猴PBMC 2ml，EDTA抗凝后加入淋巴细胞分离液，密度梯度离心后收集中间悬浮的细胞，用免疫荧光抗体分别标记CD3、CD4、CD8、CD44、CD45、CD69，及趋化因子受体CCR3、CCR4、CCR5、CCR7、CXCR2、CXCR3、CXCR4、CXCR5、XCR1、CX3CR1，归巢受体α4β7、CD62L。然后分别孵育免疫荧光抗体20min，加入破膜剂后继续孵育5min，最后在流式细胞仪上测定。
1.12 RNA-seq
转录组测序由上海康成生物科技有限公司完成。测序平台为Illumina Hiseq 2500 V4，测序模式为125PE，样品为800μg/kg vMIP-Ⅱ治疗组的效应CD8 +T&Tcm细胞亚群的RNA-seq文库。RNA-seq文库的构建：总RNA经过利用Oligo(dT)富集RNA，将RNA随机打断成200nt，随机引物六聚体反转录成cDNA，进行末端修复，加A，加接头后PCR扩增实现文库的构建。
按照Illumina标准进行样本文库混合，制备Cluster：复制链其一端固定在芯片上，另一端随机与附近的另一个引物互补被固定，形成“桥”。形成的桥单链以周边的引物为扩增引物，在芯片的表面扩增而变成双链，然后经过变性而形成单链，于是又会再次形成桥，即可进行下一轮的扩增反应，扩增若干次后，每个单分子就都能得到大量扩增，形成Cluster。
将得到的数据结果按照质量控制标准去掉包含接头的短序列，去掉N的比例>10％的短序列，同时也去掉低质量的短序列，最终得到的数据(Q30>85％)，用于后续分析。
1.13基因差异表达分析
800μg/kg vMIP-Ⅱ治疗组和未用vMIP-Ⅱ治疗组的效应CD8 +T&Tcm细胞亚群样品的基因差异表达分析采用DESeq软件进行。结果的P值均使用Benjamini和Hochberg的方法控制错误发现率并进行调整。调整后的P值<0.01且差异表达倍数＞2(|log2|＞1)即被DESeq筛选出并标记为差异表达基因。
差异表达基因Gene Ontology(http://www.geneontology.org/)功能富集分析采用GOseq R软件包进行。KEGG是了解和利用生物系统如细胞、生物体和生态系统的高级功能的数据库资源，从分子水平信息，特别是由基因组测序和其他高通量实验技术产生的大规模分子数据集(http://www.genome.jp/kegg/)中挖掘信息。KEGG通路中差异表达基因的统计富集分析采用KOBAS软件进行。分析差异表达基因在某一通路上是否过出现(over-presentation)即为差异表达基因的Pathway富集分析，并利用富集因子(Enrichment Factor)分析Pathway的富集程度。
1.14荧光定量PCR
为了验证RNA-Seq数据的准确性，我们挑选部分差异表达基因，对其表达量进行了相对荧光定量PCR分析。将分选出的效应CD8 +T细胞和CD8 +Tcm细胞置于5％的CO 2细胞培养箱中，37℃下培养24h。根据细胞总RNA提取试剂盒操作说明进行效应CD8 +T细胞和Tcm细胞总RNA提取，然后使用SuperScript TM Preamplification System for First Strand cDNA Synthesis试剂盒进行cDNA合成。荧光定量PCR反应的所有引物序列列于表1中，反应按照操作手册说明的方法在荧光定量PCR仪：MiniOpticon TM(BIO-RAD,Laboratories，Inc.USA)系统中进行。反应总体积25.0μL，包括总RNA 2.0μL，RNase-free H 2O 8.5μL，正向和反向引物(10mM/L)各0.5μL，2×One-step SYBR RT-PCR Buffer Ⅲ 12.5μL，TaKaRa Ex Taq HS(5U/μL)0.5μL，以及PrimeScript RT Enzyme Mix Ⅱ 0.5μL。PCR反应条件为95℃ 5min，95℃ 30s，60℃ 45s，72℃ 45s，共33个循环。所有实验均进行3次独立的生物学重复。标准的相对转录水平用2 -ΔΔCt法估算。
表1 荧光定量PCR反应的引物序列
Figure PCTCN2021086313-appb-000001
Figure PCTCN2021086313-appb-000002
1.15 Western blot检测G蛋白α的表达
将800μg/kg剂量组动物PBMC分选所得的效应CD8 +T细胞经超声破碎后，离心10min，收集上清液，弃沉淀。将上清液进行SDS-PAGE电泳，获得电泳条带后，剪取Tcm、CCR7对应的条带转膜，将膜用1×丽春红染液染5min，水洗后将膜晾干备用。将膜用TBS从下向上浸湿后，移至含有封闭液的平皿中，室温下脱色摇床上摇动封闭1h。将G蛋白α一抗用TBST稀释至适当浓度并加至膜上；室温下孵育1～2h后，用TBST在室温下脱色摇床上洗两次，每次10min；再用TBS洗一次， 10min。接着准备二抗稀释液并与膜接触，室温下孵育1～2h后，用TBST在室温下脱色摇床上洗两次，每次10min；再用TBS洗一次，10min，进行化学发光反应。将胶片进行扫描或拍照，用凝胶图象处理系统分析目标带的分子量和净光密度值。
1.16钙流实验
将5mg/L聚羟基脂肪酸酯(PHA)加入从800μg/kg剂量组动物PBMC分选所得的效应CD8 +T细胞悬液，于37℃、50ml/L CO 2中培养24h，按实验分组分别加入Fractalkine或vMIP-Ⅱ，4℃处理30min，加入Fluo-3(6×10 -3mol/ml)探针，室温避光孵育30min后用PBS洗涤，过滤上样流式细胞仪，激发波长488nm，检测波长530nm，每管样品检测1×10 4个细胞，获取数据用CELLQuest分析结果。
1.17流式细胞术检测细胞线粒体膜电位水平
取1×10 6个细胞，重置于0.5ml细胞培养液中，加入0.5ml JC-1染色工作液，颠倒数次混匀。细胞培养箱中37℃孵育20min。在孵育期间，按照每1ml JC-1染色缓冲液(5×)加入4ml蒸馏水的比例，配置适量的JC-1染色缓冲液(1×)，并放置于冰浴。孵育结束后，600g 4℃离心3～4min，沉淀细胞，弃上清。再加入1ml JC-1染色缓冲液(1×)重悬细胞，600g 4℃离心3～4min，沉淀细胞，弃上清。再加入适量JC-1染色缓冲液(1×)重悬后，立刻使用流式细胞仪检测分析。
1.18 Bio-Plex悬液芯片系统检测效应CD8 +T细胞的磷酸化总体水平
根据基因芯片结果显示，效应CD8 +T&Tcm细胞两细胞亚群存在明显的去磷酸化，因此，我们取效应CD8 +T细胞和CD8 +Tcm细胞混合亚群进行Bio-Plex悬液芯片系统检测，以验证其磷酸化总体水平变化情况。
实验流程按照Bio-Plex pro assay说明书进行。(1)打开Bio-Plex System系统并校正。真空吸板机的真空度调节为1～2Hg压力，用96孔板盖住。(2)将标准品稀释后加入96孔板中，然后加入微珠。轻轻盖上封板膜和铝簿，使每个小孔完全遮住。室温下以1100rpm/s下振动30s，再以600rpm/s下振动30min。(3)振动结束后，以100μL Bio-Plex Assay Buffer润湿滤板，轻轻打开封板膜和铝簿，用100μL Bio-Plex wash buffer洗2次，吸去buffer，使之完全吸净以防止交叉污染。(4)每孔加入50μL标准品，轻轻盖上封板膜和铝薄，使每个小孔完全遮住。室温下以1100rpm/s下振动30s，再以600rpm/s下振动30min。然后将封板膜和铝薄打开，过程中要避免溅出，真空抽去buffer，再100μL Bio-Plex wash buffer洗2次，吸去buffer，使之完全吸净以防止交叉污染。(5)漩涡振动检测抗体数秒，每孔加入detection antibodies 25μL，轻轻盖上封板膜和铝簿，使每个小孔完全遮住。室温下以1100rpm/s下振动30s，再以600rpm/s下振动30min。然后将封板膜和铝薄打开，过程中要避免溅出，真空抽去buffer，再100μL Bio-Plex wash buffer洗2次，吸去buffer，使之完全吸净以防止交叉污染。(6)漩涡混合Streptavidin-PE荧光色素，每孔加入50μL，轻轻盖上封板膜和铝簿，使每个小孔完全遮住。室温下以1100rpm/s下振动30s，再以600rpm/s下振动30min。然后将封板膜和铝薄打开，过程中要避免溅出，真空抽去buffer，再100μL Bio-Plex wash buffer洗2次，吸去buffer，使之完全吸净以防止交叉污染。(7)每孔加入125μL Bio-Plex assay buffer微珠重悬，轻轻用密封袋盖住， 1100rpm/s室温摇床30s后立即放入仪器中进行读盘检测。
1.19 Western blot检测磷酸化蛋白表达
将800μg/kg剂量组动物PBMC分选所得的效应CD8 +T细胞在0.1％牛血清RPMI 1640培养液饥饿1h，加入或不加CCL21(200ng/ml)进行刺激，收集细胞后用冷PBS洗3次，细胞裂解后抽提总蛋白，Bradford法定量，加热变性，等量蛋白上样行SDS-PAGE。电转至PVDF膜，用TBS配制的5％脱脂奶粉封闭过夜。加入兔抗磷酸化Akt(1:1000)、Akt(1:1000)、ERK1/2(1:1000)一抗室温孵育2h，TTBS(TBS加入1‰Tween-20)洗膜，羊抗兔IgG二抗(1:2000)室温孵育1h，洗膜后经ECL放射自显影，曝光于Kodak胶片。以Akt和ERK2为参照，用图像分析软件GELpro3.0对条带吸光度值进行分析。
1.20统计分析
使用Prism软件在三个或更多个生物学重复样品上测定除WGBS分析之外的所有体内和体外研究的统计学显著性。使用双尾t检验确定P值。*P<0.05；**P<0.01。
2结果与分析
2.1 vMIP-Ⅱ长期静脉注射对恒河猴血液指标的影响
在观察期间，各组动物的一般状况、体温、心电图、尿常规和血液生化指标均无明显异常。外周血成分检查显示：低剂量组动物各项指标均在正常范围内；与阴性对照组相比，中、高剂量组动物外周血淋巴细胞总数和分类在给药6、13周时显著性增高(P<0.05)，恢复期2周中剂量组恢复至正常范围，如表1所示。
表1 vMIP-Ⅱ连续给药13周期间恒河猴外周血淋巴细胞总数的改变(Mean±SD,n＝4,10 9L -1)
Figure PCTCN2021086313-appb-000003
注：*表示与阴性对照组相比，P＜0.05；**表示与阴性对照组相比，P＜0.01。
3.2 vMIP-Ⅱ长期静脉注射引起的恒河猴淋巴器官组织学改变
vMIP-Ⅱ连续静脉注射13周后，我们发现淋巴器官出现明显的增生，且呈现剂量依赖效应，即：50μg/kg剂量组增生较少；200μg/kg剂量组淋巴结淋巴滤泡增生；800μg/kg剂量组动物淋巴结淋巴滤泡、脾白髓、胸腺、小肠黏膜固有层淋巴滤泡均出现增生，后者并伴有较明显的中心扩大，见图1。恢复期第2周与第13周相比，增生程度显著性减轻。
3.3 vMIP-Ⅱ对血浆病毒载量的影响
在整个研究期间或直至各动物死亡时监测血浆病毒载量。如图2所示，虽然所有20只恒河猴均 显示出可变的血浆病毒载量，但急性血浆病毒血症的峰值相当，而且50μg/kg、200μg/kg和800μg/kg剂量组动物的血浆病毒载量测定值均普遍低于未治疗对照组动物血浆中的载量，并且50μg/kg、200μg/kg和800μg/kg剂量组动物的血浆病毒载量与vMIP-Ⅱ呈依赖关系，但略高于AZT+3TC治疗对照组。血浆病毒载量结果显示趋化因子受体抑制剂，尤其是vMIP-Ⅱ的施用有助于控制体内SIV复制。
3.4 vMIP-Ⅱ对四聚体阳性CD8 +T淋巴细胞亚群扩增的影响
使用MHCⅠ类四聚体和荧光激活细胞分选染色检测四聚体阳性CD8 +T细胞。正如所料，感染前Mamu-A*01阳性动物的Mamu-A*01/p11C四聚体结合细胞检测不到(<0.1％)(图3)。但感染后，来自50μg/kg、200μg/kg和800μg/kg剂量组恒河猴的PBMC显示Mamu-A*01/p11C四聚体结合CD8 +T细胞水平显着增加，且CD8 +T细胞的增殖与vMIP-Ⅱ治疗剂量存在正比关系，与流式细胞仪图谱分析所得结果一致(图4)。相反，在未治疗对照组和AZT+3TC组动物中，四聚体结合CD8 +T细胞的水平较低，CD8 +T细胞仅为2％左右，且AZT+3TC组动物CD8 +T细胞数量略低于未治疗对照组。表明AZT+3TC引起病毒载量降低并不是通过增殖CD8 +T细胞来实现的。这些数据表明趋化因子受体抑制剂vMIP-Ⅱ可通过保持SIVmac251表位特异性CD8 +T细胞的扩增来控制SIV复制。然而，应该注意的是，观察到的病毒特异性T细胞群的扩增不一定代表单个细胞的分裂，而是细胞数净增加的量度。
3.5 CD8 +Tcm和CD8 +Tem细胞纯度和所占比例分析
在感染治疗18天时，对上述使用MHCⅠ类四聚体和荧光激活细胞染色分选所得的各组效应CD8 +T细胞分别进行抗体标记，分别检测其中CD8 +Tcm细胞和CD8 +Tem细胞所占的比例。结果表明(表2)，在未用vMIP-Ⅱ治疗时，CD8 +Tcm细胞含量较少，仅占2.33％；但在vMIP-Ⅱ治疗的Ⅱ、Ⅲ和Ⅳ组中，CD8 +Tcm细胞发生明显的增殖，并存在剂量依赖性。而且，我们发现CD8 +Tcm细胞增殖的同时，CD8 +T细胞的纯度在增加，而CD8 +Tem细胞的含量呈现出减少的趋势。这些数据显示出gp120抗原肽刺激可诱导CD8 +T细胞增殖，并进一步分化为CD8 +T细胞，而且我们发现效应CD8 +Tcm细胞的增殖与效应CD8 +T细胞存在某种必然的联系，但这些均还需作进一步的研究。因此，我们认为vMIP-Ⅱ可以诱导效应CD8 +T细胞向CD8 +Tcm细胞分化，以增强细胞和机体的免疫。
表2 感染治疗18d后CD8 +T细胞的纯度以及CD8 +Tcm和CD8 +Tem所占的比例(％.Mean±SD.n＝4)
Figure PCTCN2021086313-appb-000004
注：与未治疗对照组相比，*P＜0.05
3.8 gp120抗原肽的动态研究
根据HIV通过其囊膜上的糖蛋白gp120与靶细胞膜表面的CD4分子结合，导致gp120构象改变，暴露出被其掩盖的另一种糖蛋白gp41，gp120-CD4结合靶细胞膜表面的受体CXCR4或CCR5形成复合物。我们拟在体外研究gp120通过与CD4或靶细胞膜表面的受体CXCR4或CCR5形成复合物来达到治疗HIV的目的。因此我们在体外动态观察了gp120抗原肽。
在荧光显微镜下观察到，在0h时，五个分组中多数CD4 +T细胞上均有荧光标记的gp120抗原肽；24h时(如图5所示)，Ⅰ组中被标记的CD4 +T细胞荧光增强，数量增多；Ⅱ、Ⅲ、Ⅳ和Ⅴ组中被标记的CD4 +T细胞荧光减弱，数量减少。与此同时，在0h时，大部分CD8 +T细胞上无荧光标记的gp120抗原肽；24h时，Ⅰ组出现微弱荧光且被标记的CD8 +T细胞数量较少；Ⅱ、Ⅲ、Ⅳ和Ⅴ组出现较强荧光且被标记的CD8 +T细胞数量较多，随着时间进展，Ⅰ组荧光保持微弱且数量无明显变化，Ⅱ、Ⅲ、Ⅳ和Ⅴ组荧光逐渐增强且数量增加。
3.9免疫印迹结果
vMIP-Ⅱ可以使gp120-CD4-CCR5三聚体解离，从而形成游离的gp120蛋白，被APC细胞捕获后呈递给CD8 +T细胞，刺激其产生效应T细胞。经过Western blot分析(图6)，在anti-Flag特异性抗体检测下，Ⅰ组中观察到三聚体条带，Ⅱ、Ⅲ、Ⅳ和Ⅴ组中三聚体条带明显变弱甚至不存在。结果更一步确定vMIP可以促使APC捕获游离的gp120抗原肽，刺激效应CD8 +T细胞的形成。
3.6 PBMC效应CD8 +T细胞的表型特征
取恒河猴PBMC制备单细胞悬液，染色后通过流式细胞仪检测效应CD8 +T细胞表面分子CD44、CD69、CD103、CD62L和CCR7的表达水平。结果显示，SIVmac251感染后，恒河猴PBMC效应CD8 +T细胞高表达CD62L(94.6％)、CD45RO(90.2％)、CCR7(79.8％)，低表达CD44(7.9％)、CD69(3.3％)(图7)。该结果说明PBMC效应CD8 +T细胞主要为中枢记忆型T细胞，即Tcm细胞。
3.7 PBMC效应CD8 +T细胞趋化因子受体和归巢受体的表达情况
基于趋化因子受体和归巢受体的表达与细胞募集密切相关，本实验采用流式细胞术对SIVmac251感染恒河猴PBMC效应CD8 +T细胞的趋化因子受体和归巢受体进行检测。结果显示与对照组相比，感染组恒河猴PBMC效应CD8 +T细胞高表达趋化因子受体CXCR5、CXCR4、CX3CR1和CCR7，具有统计学差异(图8)。归巢受体α4β7和L-selectin在PBMC效应CD8 +T细胞的表达相对较低，并且与对照组相比没有显著的统计学差异(图9)。这说明PBMC效应CD8 +T细胞主要是通过趋化因子受体募集到PBMC的而不是通过归巢受体。
3.10 RNA-seq测序检测结果
3.10.1组间差异表达基因
我们应用RNA-seq测序检测800μg/kg vMIP-Ⅱ治疗组和未用vMIP-Ⅱ治疗组的效应CD8 +T&Tcm细胞亚群基因的差异表达，通过对比，根据筛选标准共筛选出79个显著差异基因(差异倍数≥2，p＜0.01)，其中包括48个基因显著上调，31个基因显著下调(图10)。
3.10.2差异表达基因GO本体分析
我们对效应CD8 +T&Tcm细胞亚群进行分析，在800μg/kg vMIP-Ⅱ治疗组和未用vMIP-Ⅱ治疗组之间确定了97个显著差异基因，对这些基因进行GO本体分析，结果显示(表3、4、5)：这些基因的生物过程(Biological process)主要集中在细胞分化(Cell differentiation)、细胞凋亡(Apoptosis)、细胞定位和代谢(Cell localization and metabolism)以及磷酸化过程(Phosphorylation process)等；细胞组分(Cellular component)主要集中在线粒体(Mitochondrion)、细胞质(Cytoplasm)、内质网(Endoplasmic reticulum)、膜组分(Membrane component)和细胞器(Organelle)等；分子功能(Molecular function)主要包括信号通路分子(Signaling pathway molecule)、核酸结合(Nucleic acid binding)、跨膜受体调节蛋白(Transmembrane receptor regulatory protein)、序列特异性DNA绑定(Sequence-specific DNA binding)和转录因子活性(Transcription factor activity)等。由此可知vMIP-Ⅱ在感染SIVmac251病毒的机体免疫反应中主要参与调控效应CD8 +T细胞凋亡和磷酸化途径分化为CD8 +Tcm细胞的相关过程，尤其是对磷酸化信号通路的影响最为显著，同时差异表达基因在信号通路分子和细胞质的大量富集，也从另一方面说明了上述观点。
表3 差异表达基因的GO分析-生物过程富集分析
Figure PCTCN2021086313-appb-000005
表4 差异表达基因的GO分析-细胞组分富集分析
Figure PCTCN2021086313-appb-000006
Figure PCTCN2021086313-appb-000007
表5 差异表达基因的GO分析-分子功能富集分析
Figure PCTCN2021086313-appb-000008
3.10.3差异表达基因KEGG通路分析结果
我们对差异基因进行了在线KEGG生物通路注释，发现它们主要富集与细胞凋亡通路、能力代谢(TCA循环)通路、磷酸化通路以及一些细胞信号通路等(图11)。通过综合分析效应CD8 +T细胞和CD8 +Tcm细胞在机体中的成分和比例以及基因芯片通路分析，我们得出差异表达基因最富集的通路是磷酸化信号通路。
3.10.4确定候选差异表达基因
根据GO本体分析的结果，磷酸化途径、TCA循环和调控细胞凋亡是差异表达基因最显著富集的三个GO-term，我们将这3组生物过程中涉及基因的差异表达倍数按照从大到小进行降序排列，依次筛选差异倍数＞3(即P-value＜0.001；log 2Ration＞1.5)的差异基因，最后确定了GNAT1、PI3K、ERK、AKT、NF-κB、BCL-2、FAS、PUMA、BAX、p53AIP1这10个基因作为我们后续研究的重点目标基因。
3.11 qRT-PCR反向验证
我们对这10个基因用qRT-PCR进行反向验证，结果如图12所示，相比未用vMIP-Ⅱ治疗的效应CD8 +T&Tcm细胞组，用800μg/kg vMIP-Ⅱ治疗组中GNAT1、PI3K、ERK、AKT、BCL-2的表达量均明显减少，而NF-κB、BAX、FAS、PUMA、p53AIP1的表达明显增加，这与RNA-seq的测序结果一致。
3.12 vMIP-Ⅱ对效应CD8 +T细胞内G蛋白受体α的影响
通过基因芯片和qRT-PCR，我们的结果显示vMIP-Ⅱ治疗组可影响效应CD8 +T细胞/CD8 +Tcm细胞的G蛋白受体α。因此，我们进行Western blot检测效应CD8 +T细胞内G蛋白受体α的表达情况，以确定vMIP-Ⅱ对效应CD8 +T细胞内G蛋白受体α是否有影响。根据Western blot结果(图13)，与未用vMIP-Ⅱ治疗组相比，vMIP-Ⅱ治疗后的效应CD8 +T细胞内G蛋白受体α的表达明显抑制，表明vMIP-Ⅱ存在时，能抑制效应CD8 +T细胞内的G蛋白受体α的表达。
3.13 vMIP-Ⅱ对钙离子信号通路的影响
相对于正常对照组，vMIP-Ⅱ不仅不能明显诱导效应CD8 +T细胞内钙离子浓度的升高，反而明显诱导效应CD8 +T细胞内钙离子浓度降低(P＜0.05)。同时，相对于阳性对照组，vMIP-Ⅱ预处理细胞可使Fractalkine诱导的钙离子浓度明显下降(P<0.01，图14、15)。
3.14 vMIP-Ⅱ对效应CD8 +T细胞线粒体膜电位的影响
vMIP-Ⅱ对效应CD8 +T细胞线粒体膜电位的影响见图16。JC-1荧光染料聚集在线粒体的基质中，在线粒体膜电位较高时，产生红色荧光；当细胞线立体膜电位下降时，JC-1开始变为单体，并产生绿色荧光。对从800μg/kg剂量组恒河猴分选出的效应CD8 +T细胞进行线粒体膜电位分析，与对照组相比，vMIP-Ⅱ治疗后的效应CD8 +T细胞线粒体膜电位下降极显著(P＜0.01)。
3.15 Bio-Plex悬液芯片系统检测磷酸化总体水平
如图17所示，在未用vMIP-Ⅱ治疗组和用800μg/kg vMIP-Ⅱ治疗组的效应CD8 +T细胞磷酸化总体水平中，两组之间的差异有统计学意义(P＜0.01)。因此，vMIP-Ⅱ可引起效应CD8 +T细胞磷酸化总体水平下降，即发生去磷酸化，与RNA-seq所得结果一致。
3.16磷酸化通路分析
自800μg/kg剂量组恒河猴效应CD8 +T细胞在0.1％牛血清培养液饥饿1h，加入或不加CCL21(200ng/ml)刺激，以未用vMIP-Ⅱ治疗的阴性对照组作对照，分别于刺激60min检测磷酸化ERK1/2、Akt蛋白表达。图18结果显示，只在CCL21作用下，CD8 +Tcm细胞可检测到高水平磷酸化ERK1/2和Akt。而当在vMIP-Ⅱ存在时，磷酸化ERK1/2和Akt表达水平显著降低。这表明vMIP会抑制磷酸化ERK1/2和Akt蛋白的表达，而且vMIP-Ⅱ能够拮抗CCL21对ERK1/2和Akt的活化。

Claims (8)

  1. 一种可以在HIV感染初期有效解离HIV抗原-靶细胞受体三聚体复合物，从而降低病毒设定点的解离因子vMIP-II，其特征在于，所述药物可以保护CD4 +T细胞并刺激效应CD8 +T细胞的生成，从而降低SIV/HIV感染初期受感染的CD4 +T细胞数量，控制感染进程。还包括vMIP-Ⅱ的可药用的盐和酯、选择性取代的类似物或者包含vMIP-Ⅱ的一种或多种化合物的组合，还包括vMIP-Ⅱ的有活性的基因分子、肽段及其衍生物，或者其有活性的基因分子、肽段及衍生物在药学上可接受的介质或载体。
  2. 权利要求1所述的一种可以在HIV感染初期有效解离HIV抗原-受体三聚体复合物，从而降低病毒设定点的解离因子vMIP-II，其特征在于，所述vMIP-II可以通过解离CD4细胞膜上gp120-CD4-CCR5蛋白三聚体，并释放出游离的gp120蛋白从而阻碍病毒RNA的进入，游离出的gp120蛋白被APC捕获并呈递给CD8 +T细胞，刺激细胞毒性淋巴细胞的生成，起到保护CD4 +T靶细胞和增加抗原特异性CD8 +T细胞的作用。
  3. 权利要求1所述的一种诱导CD8 +T细胞去磷酸化为CD8 +Tcm细胞的病毒巨噬细胞炎性蛋白vMIP-Ⅱ，其特征在于，所述的病毒巨噬细胞炎性蛋白vMIP-Ⅱ包括可诱导CD8 +T细胞去磷酸化为CD8 +Tcm细胞的vMIP活性成分。
  4. 根据权利要求2、3所述的一种解离因子vMIP-II在预防和治疗HIV感染初期/急性期或治疗发病过程中的二次感染药物中的应用，其特征在于可以在HIV感染初期或者发病期的靶细胞二次感染中刺激机体效应CD8 +T细胞的生成，保护机体CD4 +T靶细胞的，降低受感染细胞数量，以增强细胞免疫，清除受感染细胞，减少病毒潜伏库。
  5. 权利要求3所述能诱导CD8 +T细胞去磷酸化为CD8 +Tcm细胞的病毒巨噬细胞炎性蛋白vMIP-Ⅱ的应用，其特征在于，所述的vMIP-Ⅱ作用机制包括vMIP-Ⅱ通过封闭CD8 +T细胞表面趋化因子受体CCR7、CXCR4、CXCR5和CX3CR1，以降低其下游信号通路的靶标分子。
  6. 权利要求1、3所述一种可以在HIV感染初期有效解离HIV抗原-受体三聚体复合物而降低病毒设定点并诱导CD8 +T细胞去磷酸化为Tcm细胞的 vMIP-II，其特征在于，vMIP-Ⅱ在恒河猴SIV感染模型中能促使淋巴组织增生，淋巴小结生发中心扩大，病毒载量降低，CD8 +Tcm细胞增殖。
  7. 权利要求5所述vMIP-Ⅱ诱导CD8 +T细胞去磷酸化为CD8 +Tcm细胞的作用机制，其特征在于，vMIP-Ⅱ封闭CD8 +T细胞的CCR7、CXCR4、CXCR5和CX3CR1通过下调其与磷酸化相关的信号通路，包括低表达CD8 +T细胞G蛋白水平、降低细胞Ca 2+浓度和线粒体膜电位等，使CD8 +T细胞发生代谢重编程，并通过抑制磷酸化相关基因GNAT1、PI3K、ERK、AKT、BCL-2使CD8 +T细胞磷酸化蛋白ERK1/2和Akt低表达，从而使CD8 +T发生重编程而去磷酸化为CD8 +Tcm细胞，促进CD8 +Tcm细胞增殖。
  8. 一种可以在HIV感染初期有效解离HIV抗原-受体三聚体复合物而降低病毒设定点并诱导CD8 +T细胞去磷酸化为Tcm细胞的vMIP-II，其特征在于，包括有效剂量的作为活性成分的用于抗HIV/SIV感染艾滋病、抗炎症反应和抗肿瘤过继性免疫治疗的药物成分的应用。
PCT/CN2021/086313 2021-04-11 2021-04-11 一种降低人免疫缺陷病毒感染的病毒设定点及重建细胞免疫的因子和药物应用 WO2022217373A1 (zh)

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