WO2019019597A1 - 一种CD4 +T细胞来源的exosomes及其应用 - Google Patents

一种CD4 +T细胞来源的exosomes及其应用 Download PDF

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WO2019019597A1
WO2019019597A1 PCT/CN2018/074894 CN2018074894W WO2019019597A1 WO 2019019597 A1 WO2019019597 A1 WO 2019019597A1 CN 2018074894 W CN2018074894 W CN 2018074894W WO 2019019597 A1 WO2019019597 A1 WO 2019019597A1
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exo
vaccine
cells
exosomes
cell
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王胜军
陆健
田洁
吴静
王运刚
马洁
马斌
许化溪
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江苏大学
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Definitions

  • the invention relates to a CD4 + T cell-derived exosomes and application thereof, belonging to the field of cell biology, molecular biology or clinical application, in particular to the preparation and biological function of CD4 + T exo isolated from mouse spleen and The application in strengthening humoral immunity in mice.
  • Exosomes is a bilayer membrane vesicle with a diameter of 30-110 nm. It originates from the early endosomes in the cell and can be released into the extracellular environment and exert biological effects after fusion with the plasma membrane. Studies have shown that almost all living cells secrete exosomes and are widely present in various body fluids. Exosomes can carry the protein, DNA and RNA components of the source cells, making them less susceptible to degradation by the external environment, and is beneficial to the biological functions of the relevant active ingredients. Exosomes are internalized by receptor cells by means of macrocytosis, lipid rafts and receptor-mediated endocytosis. Internalized exosomes can regulate various biological functions of recipient cells and communicate between cells. Play an important role.
  • Exosomes have an advantage over the cells from which they are derived when performing biological functions. Taking dendritic cell-derived exosomes as an example, the surface can carry more effector molecules, such as MHC class II molecules, which are 10-100 times more abundant than cells. Therefore, exosomes can carry more effector molecules and exert stronger biological functions.
  • exosomes are widely present in various body fluids, indicating that they are well tolerated in humans.
  • Exosome-encapsulated drugs maintain a long half-life in the body and improve efficacy. More importantly, the exosomes prepared in vitro are stable in nature, can be stored for a long time at -80 ° C, and have no response to related immunosuppressive molecules, showing their great superiority in future clinical applications.
  • Hepatitis B is a disease caused by hepatitis B virus (HBV), which is mainly caused by inflammatory lesions of the liver, and can cause damage to multiple organs and even liver cancer. Hepatitis B is widely prevalent in all countries of the world. In recent years, the incidence of hepatitis B has increased significantly. At present, the prevention of hepatitis B in China is mainly through the injection of hepatitis B vaccine, but the immunization is weak and non-immune after the immunization and after immunization. Vaccine adjuvants, as a substance that enhances the body's adaptive immune response, have been researched for decades, but at present, there are few adjuvants that have been certified for use in humans.
  • HBV hepatitis B virus
  • Aluminum adjuvants can effectively induce body fluids. Immune response, but does not work for cellular immunity. Emulsion adjuvants can cause side effects such as inflammatory reactions, granuloma, and ulcers at the injection site, and are rarely used clinically. Therefore, it is particularly necessary to study a new adjuvant that can safely and effectively enhance the immune response induced by the hepatitis B recombinant protein vaccine and make it have clinical therapeutic potential.
  • T cell-derived exosomes can also be endocytosed by dendritic cells or B cells, regulating the function of antigen-presenting cells and affecting T cell responses.
  • CD4 + T cells play an important role in the adaptive immune response of the body as a helper T cell. Whether the exosomes from which they are derived can also play a role in promoting the immune response of the body. It has been confirmed that M1 polarized macrophages can be used as immunopotentiators for cancer vaccines, showing exosomes in vaccine immunization. Important role, and the use of exosomes derived from CD4 + T cells in vaccine immunization has not been reported.
  • CD4 + T Exo is a typical biconcave disc-like membrane structure under electron microscope. Flow cytometry revealed that CD4 + T cell surface molecules CD4 and CD25 were expressed on the surface. WB detection showed that the exosomes expressed CD63 and did not express calcium. The connexin, Calnexin, meets the criteria for exosome identification. Compared with other CD4 + T vesicle secretion, biological characteristics of CD4 + T Exo clearer. CD4 + T Exo activates B cells in vitro, promotes B cell proliferation and secretes more antibodies.
  • CD4 + T Exo In vivo application of antigen-specific CD4 + T Exo enhances humoral immunity in sheep red blood cells (SRBC) and hepatitis B vaccine-immunized mice, producing more specific antibodies. At the same time, it can also increase the proportion of spleen CD8 + T cells and Th2 cells in mice immunized with hepatitis B vaccine, and enhance cellular immunity in mice. Therefore, CD4 + T Exo is a natural immunomodulator that is stable in the body and has strong applicability as a novel immunoadjuvant.
  • SRBC sheep red blood cells
  • hepatitis B vaccine-immunized mice producing more specific antibodies.
  • CD4 + T Exo is a natural immunomodulator that is stable in the body and has strong applicability as a novel immunoadjuvant.
  • CD4 + T Exo As a micro-vesicle secreted by a cell, CD4 + T Exo is not repelled in the body, is stable, has a long half-life, and is targeted. It can be stored for a long time in vitro and the preparation technology is mature. It can be used as a new adjuvant in clinical practice, and can reach the goal of “low toxicity, high efficiency,” and has good clinical application prospects.
  • the object of the present invention is to overcome the technical defects of hepatitis B immunization or treatment in the prior art, and to provide a CD4 + T cell-derived exosomes (CD4 + T Exo) which expresses the surface molecule CD4 of CD4 + T cells. And CD25, a complex of subcellular structures. It has the possibility of activating B cells possessed by CD4 + T cells, and therefore its role in enhancing the adaptive immune response of the body, in order to find a novel immunoadjuvant method.
  • the present invention firstly provides an exosomes derived from CD4 + T cells, which are circular or elliptical biconcave disc-shaped microcapsule structures having a complete envelope, a low electron density component in the cavity, and a predominantly distributed particle size. At 30-110 nm.
  • the CD4 + T Exo in the present invention is a membrane-type vesicle containing a cell membrane lipid component derived from a cell, and has a surface molecule similar to that derived from the cell, and functions similarly to CD4 + T cell-activated B cells. It has many advantages such as stable storage in vitro and in vivo immune tolerance.
  • the invention provides an application of CD4 + T cell derived exosomes in preparing a vaccine adjuvant
  • the vaccine is an infectious disease vaccine adjuvant, and further, the vaccine is a hepatitis B vaccine adjuvant.
  • the invention also provides an application of CD4+ T cell derived exosomes in preparing a vaccine
  • the vaccine is an infectious disease vaccine, and further, the vaccine is a hepatitis B vaccine.
  • the present invention also provides a vaccine adjuvant comprising a hepatitis B vaccine adjuvant, the adjuvant comprising exosomes derived from CD4 + T cells.
  • the present invention also provides a vaccine comprising a hepatitis B vaccine comprising exosomes derived from CD4 + T cells.
  • the CD4 + T Exo described in the present invention has an effect of activating B cells in vitro, promoting B cell proliferation, and promoting antibody production.
  • In vivo application can play an immune-enhancing role, promoting SRBC-immunized mice to produce more SRBC antibodies and enhance humoral immune responses.
  • New adjuvants offer new ways.
  • the present invention uses magnetic beads to sort CD4 + T cells, which is faster than the flow sorting technique, has better cell activity, and can be operated in large quantities, laying a foundation for its application and related biological function research.
  • CD4 + T Exo has the following characteristics: a diameter of 30-110 nm, and a genetic material such as protein and mRNA, microRNA, and the like.
  • the proteins containing CD63 and ⁇ -actin were detected by Western blot, and the proteins CD4 and CD25 which were consistent with the surface molecules of CD4 + T cells were detected by flow cytometry.
  • CD4 + T Exo can directly activate B cells in vitro and promote the expression of B cell surface activation molecules CD86 and MHC II.
  • CD4 + T Exo can promote the proliferation of B cells in vitro, and can also promote the production of total IgG antibodies and antigen-specific IgG antibodies of B cells, in order to study the interaction between body T/B cells and adaptive immunity.
  • the regulation of response provides new ideas.
  • CD4 + T Exo can enhance the immune effect of rabbit red blood cells (SRBC) in Babl/c mice, promote the body to produce more sheep red blood cell-specific IgG antibodies, and enhance the humoral immune response of the body.
  • SRBC rabbit red blood cells
  • the CD4 + T Exo as an adjuvant has little side effect in the present invention, and can significantly increase the proportion of CD19 - CD138 + plasma cells in the bone marrow of Babl/c mice.
  • Enhance the immune effect of hepatitis B vaccine promote the body to produce more hepatitis B surface antigen antibody (HbsAb), enhance the humoral immune response of mice, and also increase the proportion of CD8 + T cells and Th2 cells in mouse spleen, and promote cellular immune response.
  • HbsAb hepatitis B surface antigen antibody
  • Its preparation and application provide a new way for the preparation of novel immunological adjuvants.
  • Figure 1 shows the purity results of CD4 + T cells sorted by immunomagnetic beads method by flow cytometry (FACS);
  • Figure 2 shows the morphology of CD4 + T Exo observed by electron microscopy
  • Figure 3 shows the expression of CD4 + T Exo surface molecules by flow cytometry
  • Figure 4 shows the results of Western blot analysis of proteins contained in CD4 + T Exo
  • Figure 5 shows the surface of spleen B cells of mice immunized with OVA by CD4 + T Exo (N-Exo) extracted from the spleen of normal mice and CD4 + T Exo (OVA-Exo) extracted from the spleen of OVA-immunized mice by flow cytometry.
  • the effect of activated molecules
  • Figure 6 shows the effect of N-Exo and OVA-Exo (50 ⁇ g) on the proliferation of spleen B cells in OVA-immunized mice;
  • A is the result of observation under electron microscope, and B is the CFSE staining method for detecting CD4 from different sources.
  • + T Exo results in the effect of B cell proliferation.
  • Figure 7 is a graph showing the effect of N-Exo and OVA-Exo on total IgG production in vitro by spleen B cells of OVA-immunized mice;
  • Figure 8 shows the effect of N-Exo and OVA-Exo on the production of OVA-specific IgG antibody in spleen B cells of OVA-immunized mice by ELISA; it can be seen that CD4 + T Exo from different sources can promote OVA-specific IgG in B cells in a dose-dependent manner in vitro. antibody.
  • Figure 9 is an ELISA for the detection of CD4 + T Exo (N-Exo) extracted from the spleen of normal mice and CD4 + T Exo (S-Exo) extracted from the spleen of SRBC-immunized mice. Effect, Figure 9A is a flow chart of the experimental method of N-Exo and S-Exo intervention in SRBC-immunized mice; Figure 9B shows the amount of SRBC-specific IgG antibody in peripheral blood serum of SRBC-immunized mice after treatment with different sources of CD4 + T Exo Impact;
  • Figure 10 is an ELISA for detecting the adjuvant effect of CD4 + T Exo (N-Exo) extracted from the spleen of normal mice and CD4 + T Exo (V-Exo) extracted from the spleen of hepatitis B vaccinated mice on mice vaccinated against hepatitis B;
  • 10A is a flow chart of an experimental method for N-Exo and V-Exo intervention in mice vaccinated with hepatitis B;
  • FIG. 10B is an effect of N-Exo and V-Exo treatment on the amount of HbsAb produced in peripheral blood serum of mice vaccinated with hepatitis B;
  • Figure 11 shows the effect of flow detection on the proportion of plasma cells in the spleen and bone marrow of mice vaccinated with hepatitis B after treatment with different sources of CD4 + T Exo;
  • Figure 12 shows the effect of flow detection on the ratio of Th2 cells in the spleen of mice vaccinated with hepatitis B after treatment with different sources of CD4 + T Exo;
  • Figure 13 shows the effect of flow detection on the ratio of CD8 + T cells in the spleen of mice vaccinated with hepatitis B after treatment with different sources of CD4 + T Exo.
  • Example 1 Sorting of CD4 + T cells and preparation of culture supernatant
  • SRBC Sheep red blood cell
  • Ovalbumin (OVA) mice immunized: 100 ⁇ g of OVA was immersed in 100 ⁇ L of PBS, and an equal amount of complete Freund's adjuvant was ground into a water-in-oil state, and 6-8 w female Babl/c mice were injected subcutaneously into the right back. After 7 days, the mice were incubated with double OVA in PBS and then boosted subcutaneously on the contralateral side. On the 9th day, the mice were sacrificed to isolate spleen cells.
  • OVA Ovalbumin
  • Hepatitis B vaccine immunization In the 6-8w female Babl/c mice, the left hind thigh muscle was injected with hepatitis B vaccine (Huabei Pharmaceutical Jintan Biotechnology Co., Ltd.) 100 ⁇ L (including 2 ⁇ g of HbsAg), and the 14th day was contralateral thigh muscles. The dose was boosted once and the mice were sacrificed on day 17 to isolate spleen cells.
  • hepatitis B vaccine Humanabei Pharmaceutical Jintan Biotechnology Co., Ltd.
  • CD4 + T cells 1 ⁇ 10 6 CD4 + T cells were collected in EP tubes, resuspended in 1 mL PBS, centrifuged at 5 ° C, 500 g for 5 min, and the remaining 100 ⁇ L of PBS was discarded; resuspended and added 0.5 ⁇ L anti-mouse CD4 fluorescent antibody was incubated at 4 °C for 30 min; resuspended in 1 mL PBS, centrifuged at 500 °g for 5 min at 5 °C, and the supernatant was discarded; resuspended in 200 ⁇ L PBS, and the expression of cell surface molecules was detected by flow cytometry. As shown in Fig. 1, the purity of the sorted CD4 + T cells was determined by FACS, and the purity was as high as 95% or more.
  • the sorted CD4 + T cells were inoculated into a 24-well plate of CD3 pre-coated plates at 2 ⁇ 10 6 cells/well, and 1 mL of 10% FBS 1640 medium was resuspended, and 2 ⁇ g/mL CD28 was added for 24 h stimulation.
  • the collected CD4 + T cell supernatant was centrifuged at 300 ° C for 20 min at 4 ° C, and the supernatant was collected; the filtrate was collected through a 0.22 ⁇ m filter; and the filtrate was transferred to a MWCO 100 kDa ultrafiltration centrifuge tube (Millipore). Centrifuge at 1500 g for 30 min and collect the concentrate in the inner tube.
  • step (2) Extract CD4 + T exo with exoQuick-TCTM exosome kit purchased from SBI:
  • the concentrate obtained in step (1) is mixed with exoQuick-TCTM exosome reagent at a volume ratio of 5:1, shaken, and placed at 4 ° C for 12 h. Above; centrifuge at 30 °C for 10 min at 4 ° C, discard the supernatant, and collect the precipitate, which is CD4 + T Exo.
  • the prepared exosomes were dissolved in PBS, dispensed into EP tubes, and stored at -80 ° C for subsequent experiments.
  • CD4 + T Exo extracted from spleen cells of SRBC-immunized mice is simply referred to as S-Exo
  • CD4 + T Exo extracted from spleen cells of OVA-immunized mice is abbreviated as OVA-Exo, from hepatitis B.
  • the CD4 + T Exo extracted from the spleen cells of the vaccine-immunized mice is abbreviated as V-exo
  • CD4 + T exo extracted from the spleen cells of normal mice is simply referred to as N-Exo.
  • CD4 + T Exo Observation of CD4 + T Exo morphology by transmission electron microscopy: Take 20 ⁇ L of CD4 + T Exo suspension and add it to a copper mesh with a diameter of 3 mm, let stand for 2 min at room temperature; gently blot the liquid with filter paper and add 2 of pH 6.8. The % phosphotungstic acid solution was on the copper mesh, negatively dyed for 1 min; the filter paper was blotted and dried, and dried under an incandescent lamp, and observed under a transmission electron microscope. The results are shown in Fig. 2. Under the transmission electron microscope, the CD4 + T Exo is a circular or elliptical biconcave disc-shaped microcapsule structure with a complete envelope and a low electron density component in the cavity. The diameter is mainly distributed at 30-110 nm.
  • B cell surface activation molecules CD86, CD80, CD40 and MHCII molecules magnetic beads sorted OVA immunized mouse spleen CD19 + B cells: isolation of mouse spleen cells as in Example 1 step (2), 500 ⁇ L Resuspend, add 10 ⁇ L anti-CD19biotin antibody to 10 7 CD19 + B cells; incubate for 30 min on ice, mix once every 10 min; add 10 mL PBE, resuspend, centrifuge at 500 g for 5 min, discard the supernatant, leave 100 ⁇ L, mix well Add 10 ⁇ L of anti-biotin microbeads, place on ice, incubate for 30 min, mix once every 10 min; add 10mLPBE, mix, centrifuge at 5°C, 500g for 5min, discard the supernatant, add 500 ⁇ L PBE, and prepare cell suspension; The sorting column was placed on a VarioMACS sorter and rinsed with 3 mL of PBE; the cell
  • each well was 200 ⁇ L, and cultured with RPMI-1640 medium containing 10% fetal bovine serum, pH 7.2, cultured at 37 ° C, 5% CO 2 for 48 h; centrifuged at 500 g for 5 min, discarded the supernatant; resuspended in 100 ⁇ L PBS, added anti-small Rat fluorescent antibody and isotype control; incubate at 4 °C for 30 min, resuspend in 1 mL PBS, centrifuge at 500 g for 5 min, discard the supernatant; resuspend in 200 ⁇ L PBS, and check on the machine.
  • OVA-Exo or N-Exo significantly enhanced the expression of CD86 and MHC class II molecules in spleen B cells of OVA-immunized mice compared with PBS control group (P ⁇ 0.05, P ⁇ 0.001).
  • OVA-Exo is more potent than N-Exo, but has no effect on the expression of CD80 and CD40, and OVA-Exo activates B cells more effectively.
  • CFSE staining was used to detect the proliferation of B cells: pre-warmed 0.1% BSA PBS and resuspended CD19 + B cells to a final concentration of 1 ⁇ 10 6 cells/mL; 2.5 mM CFSE was added and incubated at 37 ° C for 10 min.
  • the total volume was 200 ⁇ L; cultured at 37 ° C, 5% CO 2 for 96 h; centrifuged at 300 g for 10 min, the supernatant was discarded, and resuspended in 200 ⁇ L PBS, and the proliferation peak of CFSE in each well was detected by flow cytometry.
  • the OVA-Exo and N-Exo treatment groups showed obvious proliferation groups under the light microscope, and the OVA-Exo treatment group had a larger value group.
  • CFSE staining also confirmed that CD4 + T Exo can promote the proliferation of B cells in vitro.
  • the internal ELISA instrument measures the OD value.
  • 10 ⁇ g of OVA-Exo and N-Exo did not significantly enhance the spleen B cells of OVA-immunized mice in the absence of any B cell activators.
  • Levels of OVA-specific IgG antibodies were produced, and when the dose was increased to 50 ⁇ g, CD4 + T Exo from different sources significantly enhanced the level of OVA-specific IgG antibodies produced by B cells (P ⁇ 0.01).
  • the OVA-Exo treatment group was more capable of producing OVA IgG antibodies in vitro from B cells.
  • Adjuvant effect of CD4 + T Exo on SRBC as antigen-immunized mice 6-8w female Babl/c mice were used to construct SRBC immunized mouse model, which were divided into PBS control group, N-Exo treatment group and S-Exo treatment group. , 5 in each group. On day 0 and day 9, 5% of SRBC was injected intraperitoneally, while 50 ⁇ g of the corresponding CD4 + T Exo, PBS was injected as a control. Blood was taken from the tail vein on the 16th, 23rd, 30th, 40th, and 50th days, and the serum anti-SRBC IgG content in the peripheral blood of the mice was measured and expressed by the OD value (Fig. 9A).
  • mice in S-Exo-treated mice gradually increased in serum levels over time, and were greater than the N-Exo treatment group and the control group, with the largest difference 50 days after the initial immunization. And statistically significant (P ⁇ 0.05).
  • N-Exo treatment there was no significant difference in SRBC-specific IgG antibody levels in peripheral blood serum of SRBC-immunized mice compared with PBS control group, that is, only antigen-specific CD4 + T Exo was able to In vivo promotes specific antibody levels in peripheral blood of SRBC-immunized mice (Fig. 9B).
  • Adjuvant effect of CD4 + T Exo on mice immunized with hepatitis B vaccine 6-8w female Babl/c mice were used to construct a mouse model of hepatitis B vaccine immunization, which was divided into PBS control group, N-Exo treatment group and V-Exo treatment group. , 5 in each group.
  • 100 ⁇ L of hepatitis B vaccine (containing 2 ⁇ g of HbsAg) was injected into the thigh muscle of the mice, and 50 ⁇ g of the corresponding exosomes were injected into the tail vein while inoculation, and PBS was used as a control.

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Abstract

提供了一种CD4 + T细胞来源外泌体（exosomes）及其在制备传染病疫苗和制备用于加强疫苗免疫效果的新型佐剂中的用途，该外泌体命名为CD4 +T exo。CD4 +T exo在体外能促进B细胞增殖和激活，并产生更多的抗体。CD4 +T exo在体内能够作为佐剂加强绵羊红细胞免疫小鼠的免疫效果，对乙肝疫苗免疫的小鼠也能够起到加强效应，促进外周血产生更多的乙肝表面抗体。

Description

一种CD4 +T细胞来源的exosomes及其应用 技术领域
本发明涉及一种CD4 +T细胞来源的exosomes及其应用，属细胞生物学、分子生物学领域或临床应用领域，具体涉及小鼠脾脏分离的CD4 +T exo的制备、生物学功能研究及其在加强小鼠体液免疫中的应用。
背景技术
Exosomes是一种直径30-110nm的双层膜囊泡，起源于细胞内的早期内体，与质膜融合后能够释放到细胞外环境中并发挥生物学作用。研究表明几乎所有的活细胞都能分泌exosomes，并且广泛存在于各种体液中。Exosomes能够携带来源细胞的蛋白质、DNA及RNA等成分，使之不易受外界环境降解，有利于相关活性成分发挥生物学功能。通过巨胞饮作用，脂质筏和受体介导的内吞作用等方式，exosomes被受体细胞内化，内化的exosomes可以调控受体细胞的多种生物学功能，在细胞间交流过程中发挥重要作用。Exosomes在免疫应答、凋亡、血管生成、炎症反应及肿瘤发生发展等过程中的作用均有报道。研究表明在发挥生物学功能时，exosomes比其来源细胞更有优势。以树突状细胞来源外泌体为例，其表面能够携带更多的效应分子，比如MHC II类分子，其含量是细胞的10-100倍。因此，外泌体能够携带更多的效应分子，发挥的更强的生物学功能。除此之外，外泌体作为一种天然的脂质体，广泛存在人体各种体液，说明其在人体内有良好的耐受性。而外泌体包裹的药物在体内能够保持一个较长的半衰期从而提高疗效。更重要的是，体外制备好的外泌体性质稳定，在-80℃可长期保存，且对相关免疫抑制分子无反应，显示出了其在今后临床应用上的巨大优越性。
乙型病毒性肝炎是由乙肝病毒(HBV)引起的、以肝脏炎性病变为主，并可引起多器官损害甚至导致肝癌的一种疾病。乙肝广泛流行于世界各国，近年来乙肝发病率呈明显增高的趋势。现阶段我国对乙型肝炎的预防主要是通过注射乙型肝炎疫苗,但接种免疫过程中和接种免疫后会出现免疫较弱和不免疫现象。而疫苗佐剂作为一种提高机体适应性免疫应答的物质，尽管其研究已经有几十年的历史，但是目前，已认证应用于人类的佐剂仍寥寥无几，铝佐剂能有效的诱导体液免疫应答，但对细胞免疫不起作用。而乳剂类佐剂会导致注射部位的炎症反应、肉芽肿、溃疡等副作用，很少用于临床。因此，研究能够安全有效地增强乙肝重组蛋白疫苗诱导的机体免疫应答，使之具有临床治疗潜力的新型佐剂就显得尤为必要。
研究表明活化的T细胞能够释放更多的外泌体，该外泌体能能够携带TCR/CD3复合物、Fas配体以及APO2配体等相关分子介导免疫调控。而T细胞来源的外泌体也能够被树突状细胞或B细胞内吞，调控抗原提呈细胞的功能进而影响T细胞反应。CD4 +T细胞作为辅助性T细胞在机体适应性免疫应答中发挥重要的作用。其来源的外泌体是否也能够起到促进机体免疫应答的作用呢，目前已有研究证实M1极化的巨噬细胞可作为癌症疫苗的免疫增强剂，显示了外泌体在疫苗免疫中的重要作用，而有关于CD4 +T细胞来源的外泌体在疫苗免疫中的应用尚未见报道。
研究发现CD4 +T Exo在电镜下成典型的双凹圆盘状膜结构，流式检测发现其表面表达CD4 +T细胞表面分子CD4以及CD25，WB检测显示该外泌体表达CD63，不表达钙联接蛋白Calnexin，符合外泌体的鉴定标准。与CD4 +T分泌的其他囊泡相比，CD4 +T Exo的生物学特性更为清晰。CD4 +T Exo能够体外激活B细胞，促进B细胞的增殖以及分泌更多抗体。体内应用抗原特异性的CD4 +T Exo能够加强绵羊红细胞(SRBC)以及乙肝疫苗免疫小鼠的体液免疫，产生更多的特异性抗体。同时也能够提高乙肝疫苗免疫小鼠脾脏CD8 +T细胞以及Th2细胞的比例，增强小鼠体内细胞免疫。因此，CD4 +T Exo作为一种在机体中稳定存在的天然免疫调节剂，在作为一种新型的免疫佐剂方面具有较强的应用性。
CD4 +T Exo作为一种细胞分泌的的微囊泡，在机体中不被排斥，稳定存在，半衰期长，且具有靶向性。体外能够长期保存，制备技术成熟。使其能够作为一种新型佐剂应用于临床，可到达“低毒性，高效能，”的目的，具有良好的临床应用前景。
发明内容
本发明的目的在于克服现有技术中乙肝免疫或治疗的技术缺陷，创新性的提供一种CD4 +T细胞来源的exosomes(简称CD4 +T Exo)，该exosomes表达CD4 +T细胞的表面分子CD4以及CD25，是一种亚细胞结构的复合物。其具有CD4 +T细胞所具备的活化B细胞的可能性，因此研究其在加强机体适应性免疫应答中的作用，以期寻求一种新型免疫佐剂的方法。
本发明首先提供一种CD4 +T细胞来源的exosomes，所述exosomes为圆形或椭圆形的双凹圆盘形微囊结构，有完整包膜，腔内为低电子密度成分，粒径主要分布在30-110nm。
本发明中的CD4 +T Exo是含有来源细胞胞膜脂质成分的膜性囊泡，具有与其来源细胞相似的表面分子，发挥类似CD4 +T细胞活化B细胞的功能。且具有体外稳定保存， 体内免疫耐受等细胞不具备的众多优点。
本发明提供一种CD4 +T细胞来源的exosomes在制备疫苗佐剂中的应用，所
述疫苗为传染病疫苗佐剂，更进一步的，所述疫苗为乙肝疫苗佐剂。
本发明还提供一种CD4+T细胞来源的exosomes在制备疫苗中的应用，所
述疫苗为传染病疫苗，更进一步的，所述疫苗为乙肝疫苗。
本发明还提供一种疫苗佐剂，所述疫苗佐剂为乙肝疫苗佐剂，所述佐剂含有CD4 +T细胞来源的exosomes。
本发明还提供一种疫苗，所述疫苗为乙肝疫苗，所述疫苗含有CD4 +T细胞来源的exosomes。
本发明中所述的CD4 +T Exo具有体外激活B细胞，促进B细胞增殖以及促进抗体产生的作用。体内应用可发挥免疫加强作用，促进SRBC免疫小鼠产生更多的SRBC抗体，加强体液免疫应答。促进乙肝疫苗接种小鼠产生更多的乙肝表面抗原抗体(HbsAb)，同时也能够提高乙肝疫苗接种小鼠脾脏中CD8 +T细胞以及Th2细胞的比例，促进细胞免疫应答，为今后研发安全又有效的新型佐剂提供了新途径。
与现有技术相比较，本发明的有益效果体现在如下几个方面：
(1)本发明使用磁珠分选CD4 +T细胞，与流式分选技术相比速度更快，细胞活性更好，且能够大批量操作，为其应用和相关生物学功能研究奠定基础。
(2)本发明制备CD4 +T Exo的过程与传统的差速离心法制备exosomes相比，本专利的制备过程耗时短，操作方便，便于大剂量提取。
(3)本发明中CD4 +T Exo，具有如下特征：直径为30-110nm，内部包裹蛋白质和mRNA、microRNA等遗传物质。经Western blot检测含有CD63和β-actin蛋白分子，流式细胞术检测表面表达与CD4 +T细胞表面分子一致的蛋白CD4以及CD25。
(4)本发明中CD4 +T Exo体外能够直接激活B细胞，促进B细胞表面活化分子CD86以及MHC II的表达。
(5)本发明中CD4 +T Exo体外能够促进B细胞增殖，同时也能促进B细胞总IgG抗体以及抗原特异性IgG抗体的产生，为研究机体T/B细胞间的相互作用以及适应性免疫应答的调控提供了新思路。
(6)本发明中CD4 +T Exo体内能够加强Babl/c小鼠绵羊红细胞(SRBC)免疫效果，促进机体产生更多的绵羊红细胞特异性IgG抗体，加强机体体液免疫应答。
(7)相较于传统铝佐剂或者乳剂类佐剂而言，本发明中CD4 +T Exo作为佐剂副 作用小，且能够显著提高Babl/c小鼠骨髓中CD19 -CD138 +浆细胞的比例，加强乙肝疫苗免疫效果，促进机体产生更多的乙肝表面抗原抗体(HbsAb)，增强小鼠体液免疫应答，同时也能够提高小鼠脾脏中CD8 +T细胞以及Th2细胞的比例，促进细胞免疫应答。其制备和应用为新型免疫佐剂的制备提供了新途径。
附图说明
图1为流式细胞术(FACS)检测免疫磁珠法分选的CD4 +T细胞的纯度结果；
图2为电镜观察CD4 +T Exo的形态；
图3为流式细胞术检测CD4 +T Exo表面分子的表达结果；
图4为Western blot检测CD4 +T Exo所含蛋白的结果；
图5为流式细胞术检测正常小鼠脾脏提取的CD4 +T Exo(N-Exo)和OVA免疫小鼠脾脏中提取的CD4 +T Exo(OVA-Exo)对OVA免疫小鼠脾脏B细胞表面活化分子的影响；图6为N-Exo和OVA-Exo(50μg)对OVA免疫小鼠脾脏B细胞增殖的作用结果；图中A为电镜下观察的结果，B为CFSE染色法检测不同来源CD4 +T Exo对B细胞增殖的作用结果。
图7为ELISA检测N-Exo和OVA-Exo对OVA免疫小鼠脾脏B细胞体外产生总IgG抗体的影响；
图8为ELISA检测N-Exo和OVA-Exo对OVA免疫小鼠脾脏B细胞产生OVA特异性IgG抗体的影响；可见不同来源CD4 +T Exo均能够体外剂量依赖性促进B细胞产生OVA特异性IgG抗体。
图9为ELISA检测正常小鼠脾脏提取的CD4 +T Exo(N-Exo)和SRBC免疫小鼠脾脏中提取的CD4 +T Exo(S-Exo)对绵羊红细胞(SRBC)免疫小鼠的佐剂效应，图9A为N-Exo和S-Exo干预SRBC免疫小鼠的实验方法流程图；图9B为不同来源CD4 +T Exo处理后对SRBC免疫小鼠外周血血清中SRBC特异性IgG抗体产生量的影响；
图10为ELISA检测正常小鼠脾脏提取的CD4 +T Exo(N-Exo)和乙肝疫苗接种小鼠脾脏中提取的CD4 +T Exo(V-Exo)对乙肝疫苗接种小鼠的佐剂效应；图10A为N-Exo和V-Exo干预乙肝疫苗接种小鼠的实验方法流程图；图10B为N-Exo和V-Exo处理对乙肝疫苗接种小鼠外周血血清中HbsAb产生量的影响；
图11为流式检测不同来源CD4 +T Exo处理后对乙肝疫苗接种小鼠脾脏以及骨髓中浆细胞比例的影响；
图12为流式检测不同来源CD4 +T Exo处理后对乙肝疫苗接种小鼠脾脏中Th2细胞比例 的影响；
图13为流式检测不同来源CD4 +T Exo处理后对乙肝疫苗接种小鼠脾脏中CD8 +T细胞比例的影响。
具体实施方式
下面结合具体实施例对本发明的技术方案作进一步描述，但本发明并不限于这些实施例。
实施例1：CD4 +T细胞的分选及培养上清制备
(1)构建免疫小鼠模型：Babl/c小鼠(江苏大学动物实验中心)分三组进行免疫处理。
绵羊红细胞(SRBC)小鼠免疫：5％SRBC腹腔注射至6-8w雌性Babl/c小鼠(江苏大学动物实验中心)，7天后再次注射加强免疫一次，第9天处死小鼠分离脾脏细胞。
卵清白蛋白(OVA)(Sigma)小鼠免疫：100μgOVA融于100μLPBS，加等量完全弗氏佐剂磨成油包水状态，右侧背部皮下多点注射6-8w雌性Babl/c小鼠，7天后用双倍OVA融于PBS后于对侧背部皮下多点加强免疫一次，第9天处死小鼠分离脾脏细胞。
乙肝疫苗小鼠免疫：于6-8w雌性Babl/c小鼠左后大腿肌肉注射乙肝疫苗(华北制药金坦生物技术股份有限公司)100μL(含2μg的HbsAg)，第14天对侧大腿肌肉等剂量加强一次，第17天处死小鼠分离脾脏细胞。
(2)分离脾细胞：造模最后一天，眼球放血法处死上述不同免疫小鼠模型，无菌摘取脾脏；在0.22μm的筛网中磨碎脾脏，过滤并将悬液4℃、500g离心5min，弃上清；细胞沉淀中加入5mL ACK裂解液裂解红细胞，混匀，静置5min；加等量RPMI-1640培养液终止，4℃、500g离心5min，弃上清；计算细胞数量。
(3)磁珠分选CD4 +T细胞：每10 8个脾细胞用500μL PBE重悬后加入40μL L3T4Microbeads(德国美天旎生物技术有限公司)，混匀，4℃放置20min，每5min混匀一次；加入10mL PBE液，混匀，将悬液4℃、500g离心5min，弃上清；加入500μLPBE，混匀；将MACS分选柱置于VarioMACS分选器上，用3mL PBE润洗1次；加入细胞悬液，待最后一滴悬液流出后，用3mL PBE洗涤分选柱3次；将分选柱撤移出分选器，加入5mL PBE，推动柱栓，冲柱两次，将结合于分离柱上的细胞压出，收集细胞悬液，即为CD4 +T细胞。
(4)CD4 +T细胞的纯度鉴定：收集1×10 6个CD4 +T细胞于EP管中，1mL PBS重悬，4℃、500g离心5min，弃上清剩余100μL的PBS；重悬后加0.5μL抗小鼠CD4荧光抗体，4℃孵育30min；1mL PBS重悬后4℃、500g离心5min，弃上清；加入200μL  PBS重悬，流式细胞仪检测细胞表面分子表达情况，结果如图1所示，通过FACS测定分选的CD4 +T细胞纯度，纯度高达95％以上。
实施例2：CD4 +T Exo制备及蛋白浓度测定
(1)将分选后的CD4 +T细胞按照2×10 6个细胞/孔接种于CD3预包板的24孔板中，1mL10％FBS 1640培养液重悬，加入2μg/mL CD28刺激24h收集上清，将收集的CD4 +T细胞上清于4℃、300g离心20min，收集上清；过0.22μm的滤器，收集滤液；再将滤液转移至MWCO 100kDa的超滤离心管(Millipore)中，1500g离心30min，收集内管中的浓缩液。
(2)用购自SBI公司的exoQuick-TCTM exosome试剂盒提取CD4 +T exo：步骤(1)中所得的浓缩液与exoQuick-TCTM exosome试剂按体积比5:1混合，震荡，4℃放置12h以上；4℃、1000g离心30min，弃上清，收集沉淀物，即为CD4 +T Exo。将制备的exosomes用PBS溶解，分装至EP管中，-80℃保存，用于后续试验。为描述方便，本发明中把从SRBC免疫小鼠脾脏细胞提取出来的CD4 +T Exo简称为S-Exo，从OVA免疫小鼠脾脏细胞提取出来的CD4 +T Exo简称为OVA-Exo，从乙肝疫苗免疫小鼠脾脏细胞提取出来的CD4 +T Exo简称为V-exo，把从正常小鼠脾脏细胞提取出来的CD4 +T exo简称为N-Exo。
(3)用BCA蛋白定量试剂盒测定CD4 +T exo的蛋白浓度：CD4 +T Exo悬液与等体积的裂解液(RIPA:PMSF＝250：1)混匀后，冰上放置30min，每10min震荡一次；4℃、12000g离心15min，收集上清。按照BCA蛋白定量试剂盒说明书操作步骤测定exosomes裂解上清中的蛋白浓度。
实施例3：CD4 +T Exo的鉴定
(1)透射电镜观察CD4 +T Exo形态：取20μL CD4 +T Exo悬液滴加于直径3mm的载样铜网上，室温静置2min；用滤纸轻轻吸干液体，滴加pH 6.8的2％磷钨酸溶液于铜网上，负染l min；滤纸吸干染液，白炽灯下烤干，透射电镜下观察。结果如图2所示，在透射电子显微镜下，可观察到CD4 +T Exo为圆形或椭圆形的双凹圆盘形微囊结构，有完整包膜，腔内为低电子密度成分，粒径主要分布在30-110nm。
(2)流式检测exosomes表面分子CD4以及CD25：80μg exosomes加入5μL乳胶免疫微球，室温孵育15min；加入1mL PBS，继续在室温下孵育2h，间断混匀；加入110μL 1M甘氨酸轻柔混匀，室温孵育30min；室温4000rpm离心3min，弃上清；1mL0.5％BSA PBS重悬，室温4000rpm离心3min，弃上清，重复洗涤两次；0.5mL 0.5％BSA  PBS重悬，加入10μL抗小鼠荧光抗体，4℃孵育30min；200μL 0.5％BSA PBS洗涤两次；200μL 0.5％BSA PBS重悬，上机检测。结果如图3所示，CD4 +T Exo表面表达CD4以及CD25分子。
(3)Western blot检测exosomes包含的蛋白分子CD63、Calnexin以及β-actin：配制5％的浓缩胶和12％分离胶，将变性的CD4 +T Exo按20μg蛋白总量上样，经100V恒压电泳后；再经350mA恒流电转90min后，5％脱脂牛奶封闭PVDF膜lh；一抗4℃孵育过夜；取出后用TBST洗膜，10min×3次，用辣根过氧化物酶标记的二抗，室温孵育90min；取出后用TBST洗膜，10min×3次；ImageQuant LAS 4000凝胶成像系统曝光显色，结果如图4所示，结果显示，CD4 +T Exo表达CD63及β-actin分子，不表达Calnexin，符合外泌体常规鉴定标准。
实施例4：CD4 +T Exo体外促进B细胞激活和增殖
(1)流式检测B细胞表面活化分子CD86、CD80、CD40以及MHCII分子的变化：磁珠分选OVA免疫小鼠脾脏CD19 +B细胞：分离小鼠脾脏细胞如同实例1步骤(2)，500μL重悬，按预得到10 7个CD19 +B细胞加入5μL anti-CD19biotin抗体；冰上孵育30min，每10min混匀一次；加入10mL PBE重悬，500g离心5min，弃上清，留100μL，混匀；加入10μL anti-biotin microbeads，置于冰上，孵育30min，每10min混匀1次；加10mLPBE，混匀，4℃、500g离心5min，弃上清，加500μL PBE，制得细胞悬液；将分选柱置于VarioMACS分选器上，用3mL的PBE润洗；加入细胞悬液，待最后1滴悬液流出后用3mL PBE洗涤分选柱3次；将分选柱撤移出分选器，加入5mLPBE液，推动柱栓，冲柱两次，收集流出的细胞悬液，即为CD19 +B细胞。按照5×10 5个B细胞/孔接种96孔板，加入不同浓度的OVA-CD4 +T Exo和N-CD4 +T Exo，同时加入PBS以及anti-IgM和anti-CD40功能性抗体作为阴阳性对照，每孔总体积为200μL，用含10％胎牛血清、pH 7.2的RPMI-1640培养液，37℃、5％CO2培养48h；500g离心5min，弃上清；100μLPBS重悬，加入抗小鼠荧光抗体和同型对照；4℃孵育30min，1mL PBS重悬，500g离心5min，弃上清；200μLPBS重悬，上机检测。如图5，与PBS对照组相比，10μg OVA-Exo或者N-Exo即可显著增强OVA免疫小鼠脾脏B细胞活化分子CD86和MHC II类分子的表达(P<0.05,P<0.001),且具有剂量依赖性，OVA-Exo相较于N-Exo功能更强，但对CD80和CD40的表达没有影响，其中OVA-Exo活化B细胞的效果更明显。
(2)CFSE染色检测B细胞的增殖:预热的0.1％BSA PBS重悬CD19 +B细胞，使细胞终浓度为1×10 6个细胞/mL；加入2.5mM的CFSE，置37℃孵育10min，持续震 荡混匀；用5倍体积的10％FBS RPMI 1640预冷培养液终止反应；置冰上孵育5min，500g离心5min，培养液洗2遍；用10％FBS RPMI 1640培养液重悬细胞，按照5×10 5个B细胞/孔接种96孔板，加入不同你浓度的OVA-Exo和N-Exo，同时加入PBS以及anti-IgM和anti-CD40功能性抗体作为阴阳性对照，每孔总体积为200μL；37℃、5％CO 2培养96h；300g离心10min，弃上清，200μL PBS重悬，流式检测各孔CFSE的增殖峰。如图6A、6B，4天后光镜下观察可见OVA-Exo和N-Exo处理组有明显的增殖团，且OVA-Exo处理组增值团更大更多。CFSE染色法也证实了CD4 +T Exo能够体外促进B细胞的增殖，与对照组PBS的增殖峰(13.1％)相比，OVA-Exo和N-Exo分别为33.7％以及27.5％，且OVA-Exo作用更强(本实验重复3次)，差异具有统计学意义(P<0.05)。
实施例5：CD4 +T Exo体外促进B细胞产生抗体
(1)CD4 +T Exo与B细胞共培养：OVA免疫小鼠脾脏CD19 +B细胞的磁珠分选：步骤同实施例3步骤(1)。按照5×10 5个B细胞/孔接种96孔板；加入不同浓度的OVA-Exo和N-Exo，同时加入PBS以及anti-IgM和anti-CD40功能性抗体作为阴阳性对照，每孔总体积为200μL，用含10％胎牛血清、pH 7.2的RPMI-1640培养液，37℃、5％CO2培养96h；收集上清，-20℃保存。
(2)共培养体系上清总IgG ELISA检测：按照小鼠IgG抗体ELISA检测试剂盒(联科生物)说明书操作步骤测定培养上清中总IgG的含量。如图7，不同来源CD4 +T exo与B细胞共培养后，在无任何B细胞激活剂存在的情况下，10μg的OVA-Exo和N-Exo即可显著加强B细胞体外产生IgG抗体的水平(P<0.001)，剂量增加到50μg时，这种作用更加明显(P<0.05)。与N-Exo处理组相比，OVA-Exo体外促进OVA免疫小鼠脾脏B细胞产生IgG抗体的能力更强(P<0.05)。
(3)共培养体系上清中OVA特异性IgG抗体ELISA检测：OVA抗原10μg/mL 4℃包被过夜；300μL洗涤液洗涤3遍,每遍3min；加入300μL封闭液37℃封闭2h；洗涤液洗涤3遍,每遍3min；用样品稀释液将待检样品稀释致所需浓度，每孔加入100μL，同时做两个复孔，并设阴性对照及阳性对照，37℃孵育60min；洗涤液洗涤3遍；每孔加入100μL酶标抗体，37℃孵育50min；洗涤液洗涤3遍；每孔加入100μL新配制的TMB底物溶液，室温避光反应15min；每孔中加入50μL的终止液，15min内酶标仪测OD值。如图8，不同来源CD4 +T Exo与B细胞共培养后，在无任何B细胞激活剂存在的情况下，10μg的OVA-Exo和N-Exo并不能显著加强OVA免疫小鼠脾脏B细胞体外产生OVA特异性IgG抗体的水平，而当剂量增加到50μg时，不同来源CD4 +T Exo便可显著加强B 细胞产生OVA特异性IgG抗体的水平(P<0.01)。与N-Exo处理组相比，OVA-Exo处理组对B细胞体外产生OVA IgG抗体的能力更强。
实施例6：CD4 +T Exo体内加强SRBC免疫小鼠免疫效果
CD4 +T Exo对SRBC作为抗原免疫小鼠的佐剂效应：取6-8w雌性Babl/c小鼠构建SRBC免疫小鼠模型，分为PBS对照组、N-Exo处理组、S-Exo处理组，每组5只。在第0天和第9天腹腔注射5％的SRBC，同时尾静注射50μg相应CD4 +T Exo，PBS作为对照。于第16天、23天、30天、40天、50天尾静脉取血，检测小鼠外周血血清抗SRBC IgG的含量，用OD值表示(图9A)。结果发现，经S-Exo处理后的SRBC免疫小鼠，随着时间的延长，血清中SRBC抗体的水平逐渐增高，且均大于N-Exo处理组以及对照组，在初次免疫后50天差异最大，且具有统计学意义(P<0.05)。而经N-Exo处理后，与PBS对照组相比，SRBC免疫小鼠外周血血清中的SRBC特异性IgG抗体水平无明显差异，也就是说，只有抗原特异性的CD4 +T Exo才能够在体内促进SRBC免疫小鼠外周血中的特异性抗体水平(图9B)。
实施例7：CD4 +T Exo体内加强乙肝疫苗免疫小鼠免疫效果
CD4 +T Exo对乙肝疫苗免疫小鼠的佐剂效应：取6-8w雌性Babl/c小鼠构建乙肝疫苗免疫小鼠模型，分为PBS对照组、N-Exo处理组、V-Exo处理组，每组5只。在第0天和第14天于小鼠后大腿肌肉注射乙肝疫苗100μL(含2μg的HbsAg)，接种的同时尾静脉注射50μg相应外泌体，PBS作为对照。于第16天、23天、30天、40天、50天尾静脉取血，检测小鼠外周血血清HbsAb的含量，用OD值表示(图10A)。结果显示，只有经V-Exo处理后才能显著提高乙肝疫苗免疫小鼠外周血血清中的HbsAb抗体水平，而N-Exo或PBS处理对血清中的HbsAb水平并没有影响(图10B)，提示CD4 +T Exo体内发挥佐剂效应是抗原特异性的。除此之外，经不同CD4 +T Exo处理后，实验小鼠骨髓中CD19 -CD138 +浆细胞的比例均显著上升，与PBS对照组相比差异具有统计学意义(P<0.05)，但对小鼠脾脏中浆细胞的比例没有影响(图11)。同时检测不同CD4 +T Exo处理对乙肝疫苗免疫小鼠脾脏内T细胞亚群的影响，结果发现N-Exo和V-Exo处理后均能够提高Th2细胞以及CD8 +T细胞的比例，促进机体细胞免疫应答(图12、图13)。

Claims (10)

  1. 一种CD4 +T细胞来源的exosomes，其特征在于，所述exosomes是一种细胞间信息传递的亚细胞结构的复合信息体；为圆形或椭圆形的双凹圆盘形微囊结构，有完整包膜，腔内为低电子密度成分，粒径主要分布在30-110nm。
  2. 根据权利要求1所述的一种CD4 +T细胞来源的exosomes，其特征在于，所述exosomes来源于特异性抗原免疫刺激过的个体或正常个体。
  3. 权利要求1或2所述的一种CD4 +T细胞来源的exosomes在制备疫苗佐剂中的应用。
  4. 根据权利要求3所述的一种CD4 +T细胞来源的exosomes在制备疫苗佐剂中的应用，其特征在于，所述疫苗为传染病疫苗。
  5. 根据权利要求3所述的一种CD4 +T细胞来源的exosomes在制备疫苗佐剂中的应用，其特征在于，所述疫苗为传染病疫苗为乙肝疫苗。
  6. 权利要求1或2所述的一种CD4+T细胞来源的exosomes在制备疫苗中的应用。
  7. 权利要求6所述的一种CD4+T细胞来源的exosomes在制备传染病疫苗中的应用。
  8. 根据权利要求7所述的一种CD4+T细胞来源的exosomes在制备传染病疫苗中的应用，其特征在于，所述疫苗为乙肝疫苗。
  9. 一种疫苗佐剂，其特征在于，所述疫苗佐剂为乙肝疫苗佐剂，所述佐剂含有CD4 +T细胞来源的exosomes。
  10. 一种疫苗，其特征在于，所述疫苗为乙肝疫苗，所述疫苗含有CD4 +T细胞来源的exosomes。
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