WO2023000403A1 - 一种新型冠状病毒亚蛋白纳米疫苗及其制备方法和应用 - Google Patents

一种新型冠状病毒亚蛋白纳米疫苗及其制备方法和应用 Download PDF

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WO2023000403A1
WO2023000403A1 PCT/CN2021/111511 CN2021111511W WO2023000403A1 WO 2023000403 A1 WO2023000403 A1 WO 2023000403A1 CN 2021111511 W CN2021111511 W CN 2021111511W WO 2023000403 A1 WO2023000403 A1 WO 2023000403A1
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nano
vaccine
novel coronavirus
porphyrin
subprotein
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PCT/CN2021/111511
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English (en)
French (fr)
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郑俊武
金红林
黄浩
洪磊
卢利森
贺乾元
郭申元
郑成武
卫路
刘群
金秀妍
林明清
郭志坤
刘渝
郑永刚
侯兆水
王佶
田守云
陈成
康伟
彭瀚祺
高磊
马佳伟
谢逾豪
王翔宇
陈柱
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武汉圣润生物科技有限公司
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Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to a novel coronavirus subprotein nano-vaccine and its preparation method and application.
  • the novel coronavirus pneumonia (COVID-19) that broke out in the past two years is a respiratory disease caused by a new coronavirus (SARS-CoV-2), and its clinical manifestations range from mild flu-like symptoms to severe life-threatening pneumonia , with a very high infectivity and fatality rate.
  • SARS-CoV-2 coronavirus
  • Its main structure includes spike protein (S, spike), envelope protein (E, envelope), membrane protein/matrix protein (M, membrane/matrix) and nucleocapsid protein (N, uncleocapsid).
  • the S protein When the host is infected with the new coronavirus, the S protein will be cleaved by protease into S1 subunit and S2 subunit, and the receptor binding domain (RBD) of the S1 subunit will bind to angiotensin converting enzyme 2 (Angiotensin-converting enzyme 2) on the surface of the host cell. Converting enzyme 2, ACE2) binding, causing fever and pulmonary infection and other clinical manifestations. Neutralizing antibodies against the S protein can block the virus from invading host cells. Therefore, in view of the current situation of the global spread of pneumonia caused by the new coronavirus, inoculation of the new coronavirus vaccine developed against the S protein is an effective way to improve the spread of the epidemic.
  • RBD receptor binding domain
  • ACE2 angiotensin converting enzyme 2
  • Neutralizing antibodies against the S protein can block the virus from invading host cells. Therefore, in view of the current situation of the global spread of pneumonia caused by the new coronavirus, ino
  • Lipids Body refers to amphiphilic phospholipid molecules as the basic material, adding cholesterol and other auxiliary materials, in water, because the hydrophobic segments of amphiphilic phospholipids are gathered together, and the hydrophilic ends are exposed to water, thus self-assembling to form a hydrophobic middle and hydrophilic ends.
  • Bilayer structure vesicles albumin is the most abundant protein in plasma, using albumin as a carrier for insoluble drugs to prepare drug delivery nanoparticles will make it have low biotoxicity, low immune response, biodegradability and increase half-life and other advantages, the binding force between albumin and hydrophobic small molecule drugs is mainly van der Waals force and hydrogen bond force, hydrophobic small molecule drugs are mainly loaded with the IIA region of bovine serum albumin, which can significantly improve the solubility of drugs in plasma ; Amphiphilic block copolymers also mainly rely on their own hydrophobic domains to load hydrophobic small molecule drugs through hydrophilic and hydrophobic forces, such as PLGA, due to their excellent biocompatibility, low biotoxicity and good in vivo The degradability makes it certified by the US Food and Drug Administration and officially included in the US Pharmacopoeia as a pharmaceutical excipient, and has been approved for the in vivo delivery system of various drugs, but it is difficult for the above-mentioned carriers
  • the object of the present invention is to provide a novel coronavirus subprotein nano-vaccine and its preparation method and application, by coupling polylactic acid with porphyrins, and chelating Co 2+ ions that can be linked to histidine tags , forming an organic compound self-assembled by polylactic acid, porphyrin or porphyrin derivatives, and Co 2+ ion conjugates.
  • the core of the organic compound is coated with adjuvant, the core-shell surface is coated with lipid, and the surface is also efficiently loaded with 2019-nCoV antigenic protein, thereby realizing a nano-vaccine system co-delivered with 2019-nCoV antigenic protein and vaccine adjuvant, which can not only maximize the immunogenicity of 2019-nCoV recombinant subprotein, but also display Track its distribution in the organism; further, it is designed and loaded with polypeptides that can specifically target antigen-presenting cells, so as to more effectively promote the body's antiviral response.
  • the present invention provides a novel coronavirus subprotein nano-vaccine, said nano-vaccine comprising: organic compounds self-assembled by polylactic acid, porphyrin or porphyrin derivatives and Co 2+ ion conjugates; novel coronavirus antigenic protein; Vaccine adjuvants and lipids.
  • the organic compound self-assembled from polylactic acid, porphyrin or porphyrin derivatives and Co 2+ ion conjugates in the nano-vaccine has a core-shell structure, the core is a vaccine adjuvant, and the core-shell is wrapped with a lipid , the surface of the nucleocapsid is loaded with novel coronavirus antigenic protein.
  • the surface of the core-shell is also loaded with a targeting polypeptide targeting antigen-presenting cells, wherein the amino acid sequence of the targeting polypeptide is shown in SEQ ID NO:1.
  • the antigen-presenting cells include: dendritic cells and macrophages, and the targeting polypeptide is obtained by phage display technology.
  • novel coronavirus antigenic protein and the targeting polypeptide are respectively linked to organic compounds through histidine tags.
  • novel coronavirus antigenic protein is the RBD protein of the novel coronavirus.
  • porphyrin derivative is a porphyrin derivative containing only one carboxyl group.
  • porphyrin derivative is pyropheophorbide- ⁇ .
  • the vaccine adjuvant can be selected from: TLR7 agonist, TLR8 agonist, TLR9 agonist, QS-21 adjuvant.
  • the molecular weight of the polylactic acid is preferably 1500-3000.
  • the lipid is PEG-phospholipid, preferably PEG-DSPE, more preferably DSPE-PEG2000.
  • the present invention also provides a preparation method of the above-mentioned novel coronavirus subprotein nano-vaccine, comprising:
  • Step 1 reacting polylactic acid with porphyrin or porphyrin derivatives, making the hydroxyl at the end of polylactic acid condense with the carboxyl groups of porphyrin or porphyrin derivatives, so that porphyrin or porphyrin derivatives are bonded to polylactic acid for polymerization the end of the chain of things;
  • Step 2 adding Co 2+ ions to the product of step 1, so that Co 2+ ions are embedded in the porphyrin ring, to obtain organic compounds self-assembled by polylactic acid, porphyrin or porphyrin derivatives and Co 2+ ion conjugates ;
  • Step 3 mixing and dissolving the organic compound, lipid and vaccine adjuvant prepared in step 2, dripping in normal saline, evaporating the organic solvent to obtain a nanocarrier solution containing cobalt ion porphyrin rings evenly distributed in normal saline;
  • Step 4 Incubate the novel coronavirus antigenic protein linked with the histidine tag with the nanocarrier solution prepared in step 3 overnight, and ultracentrifuge to obtain a nanovaccine bound to the novel coronavirus antigenic protein.
  • the preparation method also includes: simultaneously incubating the novel coronavirus antigen protein containing histidine tag and the specific targeting polypeptide with the nanocarrier solution overnight, and then ultracentrifuging to obtain the nanovaccine.
  • step 1 polylactic acid is added to pyropheophorbide- ⁇ , and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4 -Dimethylaminopyridine, reacting in an ice bath until the disappearance of the starting alcohol is detected, then the reaction solution is extracted, washed and dried, and the solvent is removed to obtain the product PLA-pyropheophorbide.
  • the present invention also provides the application of the above novel coronavirus subprotein nano-vaccine in the preparation of immunogenic compositions for diseases related to novel coronavirus infection.
  • the present invention also provides the application of the above novel coronavirus subprotein nano-vaccine in the preparation of nano-tracers.
  • the beneficial effect of the present invention is: the present invention is formed by polylactic acid, porphyrin or porphyrin derivative and An organic compound self-assembled by a Co 2+ ion conjugate, the organic compound is connected to a histidine tag through a Co 2+ ion to achieve efficient loading of the new coronavirus antigen protein; and the chemically coupled porphyrin itself can produce The function of fluorescence traces the distribution of nanoparticles in the body; at the same time, the core of the vaccine is wrapped with an adjuvant, and the surface of the core-shell is wrapped with lipids, thus realizing the co-delivery of the new coronavirus antigen protein and vaccine adjuvant nano-vaccine system.
  • the present invention also designs and loads polypeptides that can target antigen-presenting cells on the surface of the compound, thereby promoting the massive uptake of nano-vaccine by DC cells and more effectively promoting the body's antiviral response. That is, the present invention provides a new type of coronavirus subprotein nano-vaccine with the functions of efficiently loading antigen, simultaneously carrying adjuvant and antigen, tracing the distribution of nano-vaccine, and specifically and efficiently targeting DC cells, and its preparation method is simple and efficient , providing a new and efficient approach and option for the effective prevention of novel coronavirus infection and the preparation of novel coronavirus subprotein nano-vaccine.
  • Fig. 1 is the nano vaccine NPQ-RBD structural representation prepared in the embodiment of the present invention 1;
  • Fig. 2 is the acquisition method of the polypeptide specifically targeting antigen-presenting cells in Example 1 of the present invention
  • Fig. 3 is the nano vaccine NPQ-RBD-AP structural representation prepared in the embodiment of the present invention 1;
  • Fig. 4 is the stability detection result of nano vaccine in the embodiment of the present invention 1;
  • Fig. 5 is the in vitro targeting detection result of the nano-vaccine in Example 2 of the present invention to mouse bone marrow-derived dendritic cells;
  • Fig. 6 is the detection result of the activation effect of nano-vaccine on mouse bone marrow-derived dendritic cells in Example 2 of the present invention.
  • Figure 7 is the detection result of the nano-vaccine targeting lymph nodes and antigen-presenting cells in the lymph nodes in Example 2 of the present invention.
  • Fig. 8 is the detection result of the antibody titer of the anti-RBD fragment produced by the nano-vaccine-induced animal serum in Example 2 of the present invention.
  • Fig. 9 is the detection result of the antibody titer of the anti-RBD fragment produced by the nano-vaccine NPQ-RBD in Example 2 of the present invention induced by animal serum over time;
  • Figure 10 is the detection result of the cellular immune effect of the nano-vaccine inducing the body to produce RBD fragments in Example 2 of the present invention.
  • Fig. 11 is the detection result of the nano-vaccine in Example 2 of the present invention on the resistance of hACE2 human-derived mice to lung infection caused by the new coronavirus COVID-19.
  • PLA was synthesized by ring-opening polymerization of L-lactide (L-LA) and D-lactide (D-LA) with n-hexanol as the initiator and stannous octoate (Sn(Oct) 2 ) as the catalyst. Its synthesis path is as follows:
  • DMF N,N-dimethylformamide
  • the currently known molecular weight of PLA can range from hundreds to tens of thousands. The larger its own molecular weight, the stronger its hydrophobicity, and the larger the difference between its molecular weight and that of excipients, the weaker its loading capacity. That is, PLA with different molecular weights can be prepared by adjusting the feed ratio, and the nanovaccine prepared based on PLA with different molecular weights has different loading capabilities for the target protein.
  • the present invention selects PLA with a molecular weight of 1500-3000 to maximize the loading of the novel coronavirus antigenic protein RBD protein.
  • the PLA prepared above was reacted with pyropheophorbide- ⁇ , and the hydroxyl group at the end of PLA reacted with pyropheophorbide- ⁇ .
  • the condensation reaction between the carboxyl groups of ⁇ makes pyropheophorbide- ⁇ bond to the end of the PLA polymer chain, and the synthesis route is as follows:
  • UV-vis ultraviolet-visible absorption spectrum
  • the present invention preferably finds that among all divalent metal ions that can bind to histidine tag, divalent Co
  • the binding efficiency of 2+ ions is the highest, so Co 2+ is selected to be chimerized into the porphyrin ring for binding to antigens containing histidine tags.
  • Co 2+ ions can be bound to the N atom in the center of the molecular skeleton in a coordinated manner , the synthesis path is as follows:
  • the RBD fragment is the key domain for the new coronavirus to bind to the ACE2 receptor of human lung epithelial cells, and it is also the most effective region currently routinely used to prepare vaccines. Construct the extracellularly secreted and expressed in cells RBD fragment lentiviral plasmids containing histidine tags, obtain puromycin-resistant lentiviruses capable of infecting cells, transfect CHO cells, and use puromycin to screen for expression CHO cell lines of RBD protein fragments. Expand the cell line to obtain supernatant or cell suspension; if it is cell suspension, add PBS solution combined with ultrasound to obtain PBS supernatant containing whole cell protein. A purification column containing a nickel ion column was used to obtain the RBD protein expressing only the histidine tag, followed by dialysis in saline solution to obtain the final desired RBD protein.
  • saponin adjuvants are considered to be the most promising adjuvants because they can induce both humoral immunity and cellular immunity at a low dose, and have been used in relevant clinical trials of tumor vaccines and virus vaccines Research and development, and due to the hydrophilic and hydrophobic properties of the adjuvant, it can be perfectly combined with the nano system, so this example uses QS-21 as the vaccine adjuvant, and DSPE-PEG2000 phospholipid as the nano system.
  • the present invention found that by adjusting the content ratio of DSPE-PEG2000 and PLA, the ability of the nanosystem to bind to proteins expressing histidine tags can be changed, and the system with the best loading effect is as follows:
  • the 10KD ultrafiltration centrifuge tube was centrifuged at 3000rpm to obtain a high-concentration saline solution containing nanocarriers; the excess RBD fragments were incubated with a solution containing nanocarrier NPQ at 4°C overnight, and then obtained by ultracentrifugation combined with RBD fragments and
  • the nano-vaccine of QS-21 (NPQ-RBD), its structural schematic diagram is shown in Figure 1.
  • Lymph node dendritic cells are important antigen-presenting cells in the body.
  • a polypeptide that can specifically target antigen-presenting cells was further screened and synthesized by phage technology.
  • the amino acid sequence is: LDLFRELPFEWLEALKQKLK (shown in SEQ ID NO.1), which is connected to the RBD protein with an organic compound through a histidine tag and loaded on the surface of the nano-vaccine.
  • the specific operation steps are:
  • nanocarrier NPQ solution Obtain the nanocarrier NPQ solution according to the above method, centrifuge the 10KD ultrafiltration centrifuge tube at 3000rpm to obtain the physiological saline solution containing the nanocarrier at a high concentration; The solution was incubated overnight at 4°C, and then ultracentrifuged to obtain a nanovaccine (NPQ-RBD-AP) combined with RBD fragments, targeting polypeptides and QS-21, the schematic diagram of which is shown in Figure 3 .
  • NPQ-RBD-AP nanovaccine
  • proteins with different molecular weights were selected respectively: His-IL-2 (interleukin-2 containing 6 histidine tags), His-IL-2 -RBD (RBD protein containing 6 histidine tags), His-BSA (bovine serum albumin containing 6 histidine tags), His-spike protein (new coronavirus Spike protein containing 6 histidine tags protein) as the research object; in the nano-carriers of equal concentration, add the above-mentioned protein of the same concentration (50nmol), after reacting at room temperature for 30 minutes, carry out HPLC identification, analyze the content of free protein, and according to the formula: the total protein added Content-free protein content/total protein content*100%, calculate the binding efficiency of different molecular weight proteins and NPQ carrier, the results are shown in Table 1:
  • nanocarrier NPQ prepared by the present invention has the highest loading efficiency for RBD protein, and its binding efficiency with NPQ is as high as 92%.
  • Nano-vaccine NPQ-RBD and NPQ-RBD-AP were respectively prepared by the method described in Example 1, and the particle size was detected to be between 100-200nm, and the nanoparticles in this range can effectively penetrate lymphatic vessels and be drained to the lymph nodes.
  • the amino acid fragments on the nano-NPQ-RBD were labeled with FITC molecules; the nano-particles were dialyzed in saline at 4°C, and the fractions were collected every day Solution, using a microplate reader to detect the relative FITC fluorescence intensity in the nanometer, and set the nanoparticle NP-RBD control group without adjuvant, and the group injected with only RBD antigen.
  • the detection results are shown in Figure 4.
  • nano-vaccine NPQ-RBD and NPQ-RBD-AP prepared by the present invention have good stability, can be stable for more than one week, and still carry more than 70% of RBD fragments;
  • Nanovaccine targets and activates mouse bone marrow-derived dendritic cells (BMDC) in vitro
  • BMDC BMDC to a six-well plate, 5 ⁇ 10 5 /well, and add 100 ⁇ L of NPQ-RBD and NPQ-RBD-AP suspension to each well.
  • the expression of CD80 and CD86, the markers of DC cell activation were detected by flow cytometry, and the expression of untreated DC cells, organic nanoparticles NP without adjuvant and RBD antigen, adjuvant DC cells were treated with QS21 and bacterial lipopolysaccharide LPS that non-specifically activates DC cells as controls, and the results are shown in FIG. 6 .
  • Nanovaccine targets lymph nodes and antigen-presenting cells in lymph nodes
  • NPQ-RBD and NPQ-RBD-AP carrying RBD fragments were injected subcutaneously at the base of the tail of C57 mice for two immunizations, and the mouse serum was detected on the 8th and 18th days after the second immunization Antibody titers against RBD fragments, and injection of PBS, adjuvant QS-21, RBD fragments without carrier, and nanoparticle NP-RBD without adjuvant were used as controls.
  • the method of taking blood from the orbital venous plexus is used to obtain 200-300 ⁇ L of mouse blood each time, coagulate at room temperature for half an hour, and then centrifuge at 4000rpm for 30 minutes to obtain mouse serum; use the ELisa reagent for detecting RBD antibodies
  • the box detects the titer of the IgG antibody against the RBD fragment contained in the mouse serum, wherein the detection results of different treatment groups are shown in Figure 8, and the detection results of the antibody titer of the nano-vaccine NPQ-RBD changing over time are shown in Figure 9 Show.
  • the nano-vaccine prepared by the present invention can effectively induce the production of antibodies in the RBD region, and the antibody titer produced is more than 3000 times that of the control group, which proves that the vaccine has the potential to prevent new coronaviruses.
  • the titer can exist in the mouse serum for at least 10 days, which proves the persistence of the antibody produced by the nanovaccine.
  • Nano-vaccine induces the body to produce cellular immunity against RBD fragments
  • Nanoparticle NP-RBD served as a control. A total of two immunizations were performed. One month after the second immunization, the mice were sacrificed by vertebral dislocation, the mouse spleen and lymph node cells were extracted, and the erythrocytes were lysed by ACK to make a single cell suspension.
  • NPQ-RBD and NPQ-RBD-AP carrying RBD fragments were injected subcutaneously at the base of the tail of the mice, and injected with PBS, adjuvant QS-21, and carrier-free respectively.
  • RBD fragment, nanoparticle NP-RBD without adjuvant served as control.
  • the nano-vaccine NPQ-RBD and NPQ-RBD-AP prepared by the present invention can effectively prevent hACE2 human-derived mice from being infected with the new coronavirus.

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Abstract

一种新型冠状病毒亚蛋白纳米疫苗及其制备方法和应用，所述纳米疫苗包括：由聚乳酸、卟啉或卟啉衍生物以及Co 2+离子结合物自组装的有机化合物；新型冠状病毒抗原蛋白；疫苗佐剂和脂质。所述合成的有机化合物，其核心包裹有佐剂，表面高效装载有新型冠状病毒抗原蛋白，实现了新型冠状病毒抗原蛋白和疫苗佐剂共同递送的纳米疫苗体系，可以最大限度发挥新型冠状病毒重组亚蛋白的免疫原性，还可通过荧光分子示踪其在生物体内分布情况。此外，所述纳米疫苗还连接了可特异性靶向抗原提呈细胞的多肽，促进纳米疫苗被DC细胞大量摄取，促进抗病毒反应。所述制备方法简单高效，为有效预防新型冠状病毒感染提供了途径。

Description

一种新型冠状病毒亚蛋白纳米疫苗及其制备方法和应用 技术领域
本发明属于生物医药领域，具体涉及一种新型冠状病毒亚蛋白纳米疫苗及其制备方法和应用。
背景技术
近两年爆发的新型冠状病毒肺炎(COVID-19)是由新型冠状病毒(SARS-CoV-2)引起的一种呼吸***疾病，其临床表现从轻症的流感类症状到重症危机生命的肺炎，具备非常高的传染性和致死率。其主要结构包括刺突蛋白(S，spike)、包膜蛋白(E，envelope)、膜蛋白/基质蛋白(M，membrane/matrix)和核衣壳蛋白(N，uncleocapsid)。当宿主感染新型冠状病毒后，S蛋白会被蛋白酶切割分成S1亚基和S2亚基，S1亚基的受体结合域(RBD)会通过与宿主细胞表面的血管紧张素转化酶2(Angiotensin-converting enzyme 2，ACE2)结合，引起发烧以及肺部感染等临床表现。针对S蛋白的中和抗体可以阻断病毒侵入宿主细胞。因此，鉴于目前新型冠状病毒引起的肺炎在全球蔓延的局势，接种针对S蛋白开发的新型冠状病毒疫苗的是改善疫情蔓延的有效方法。
近年来，致力于实现抗原和佐剂共同递送的纳米疫苗平台开发迅速，脂质体，白蛋白以及一些通过FDA的两亲性嵌段共聚物是近些年用于递送平台的纳米载体，脂质体是指由两亲型磷脂分子为基础材料，加入胆固醇等其他辅料，在水中由于两亲性磷脂疏水段聚集在一起，亲水端暴露在水中原因从而自组装形成中间疏水两端亲水的双分子层结构囊泡；白蛋白是血浆中最丰富的蛋白，使用白蛋白作为难溶性药物的载体制备药物递送纳米粒子，会使其具有低生物毒性，低免疫反应，可生物降解和提升半衰期等优点，白蛋白与疏水小分子药物之间的结合力主要为范德华力和氢键作用力，疏水类小分子药物主要装载牛血清白蛋白的IIA区域，能够显著的提升药物在血浆中的溶解度；两亲性嵌段共聚物也是主要依靠自身的疏水结构域通过亲疏水作用力装载疏水性小分子药 物，例如PLGA，凭借着其优异的生物相容性，低生物毒性和在生物体内良好的降解性使其通过美国食品药品管理局的认证并被正式作为药用辅料收录进美国药典，并被批准用于多种药物的体内递送***，但是上述载体均很难实现新型冠状病毒S蛋白的装载，这是由于新冠S蛋白的分子量较大(140KD)，具有较大的空间位阻效应，无法有效大容量装载于纳米体系中；并且S蛋白的抗原的空间效应使之只能通过吸附作用或者油包水装载到纳米粒子内核，上述两种装载方式不仅效率低下，并且更容易释放，难以稳定保存。
发明内容
本发明的目的在于提供一种新型冠状病毒亚蛋白纳米疫苗及其制备方法和应用，通过将聚乳酸与卟啉类物质偶联，并螯合有可与组氨酸标签连接的Co 2+离子，形成由聚乳酸、卟啉或卟啉衍生物以及Co 2+离子结合物自组装的有机化合物，该有机化合物的核心中包裹有佐剂，核壳表面包裹有脂质，表面还高效装载有新型冠状病毒抗原蛋白，从而实现了新型冠状病毒抗原蛋白和疫苗佐剂共同递送的纳米疫苗体系，该体系不仅可最大限度发挥新型冠状病毒重组亚蛋白的免疫原性，同时还可通过荧光分子示踪其在生物体内分布情况；进一步还通过设计并装载有可特异性靶向抗原提呈细胞的多肽，从而更有效促进机体的抗病毒反应。
为实现上述目的，本发明采用的技术方案是：
本发明提供了一种新型冠状病毒亚蛋白纳米疫苗，所述纳米疫苗包括：由聚乳酸、卟啉或卟啉衍生物以及Co 2+离子结合物自组装的有机化合物；新型冠状病毒抗原蛋白；疫苗佐剂和脂质。
进一步地，所述纳米疫苗中由聚乳酸、卟啉或卟啉衍生物以及Co 2+离子结合物自组装的有机化合物为核壳结构，核内为疫苗佐剂，核壳上包裹有脂质，核壳表面负载有新型冠状病毒抗原蛋白。
进一步地，所述核壳表面还负载有靶向抗原提呈细胞的靶向多肽，其中所 述靶向多肽的氨基酸序列如SEQ ID NO:1所示。其中所述抗原提呈细胞包括：树突状细胞和巨噬细胞，并且该靶向多肽通过噬菌体展示技术获得。
进一步地，所述新型冠状病毒抗原蛋白和靶向多肽分别通过组氨酸标签与有机化合物连接。
进一步地，所述新型冠状病毒抗原蛋白为新型冠状病毒的RBD蛋白。
进一步地，所述卟啉衍生物为只含有一个羧基基团的卟啉衍生物。
进一步地，所述卟啉衍生物为焦脱镁叶绿酸-α。
进一步地，所述疫苗佐剂可选自：TLR7激动剂、TLR8激动剂、TLR9激动剂、QS-21佐剂。
进一步地，所述聚乳酸的分子量大小优选为1500-3000。
进一步地，所述脂质为PEG-磷脂，优选为PEG-DSPE，更优选为DSPE-PEG2000。
进一步地，所述有机化合物的结构式为：
Figure PCTCN2021111511-appb-000001
本发明还提供了上述新型冠状病毒亚蛋白纳米疫苗的制备方法，包括：
步骤1、将聚乳酸与卟啉或卟啉衍生物反应，使聚乳酸末端的羟基与卟啉或卟啉衍生物的羧基进行缩合反应，使卟啉或卟啉衍生物键合到聚乳酸聚合物链的末端；
步骤2、向步骤1的产物中加入Co 2+离子，使Co 2+离子嵌入卟啉环中，得到由聚乳酸、卟啉或卟啉衍生物以及Co 2+离子结合物自组装的有机化合物；
步骤3、将步骤2制得的有机化合物、脂质和疫苗佐剂混合溶解，滴入生理 盐水中，蒸发有机溶剂获得含有钴离子卟啉环的均匀分布于生理盐水中的纳米载体溶液；
步骤4、将连接有组氨酸标签的新型冠状病毒抗原蛋白，与步骤3制得的纳米载体溶液孵育过夜，超速离心获得结合有新型冠状病毒抗原蛋白的纳米疫苗。
进一步的，所述制备方法中还包括：将含有组氨酸标签的新型冠状病毒抗原蛋白以及特异性靶向多肽同时与纳米载体溶液孵育过夜，然后超速离心获得纳米疫苗。
进一步的，所述步骤1中，向焦脱镁叶绿酸-α中加入聚乳酸，然后加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和4-二甲氨基吡啶，冰浴反应至检测到起始醇消失，然后将反应液经萃取、洗涤干燥，并除去溶剂后得到产物PLA-焦脱镁叶绿酸。
本发明还提供了上述新型冠状病毒亚蛋白纳米疫苗在制备与新型冠状病毒感染相关疾病的免疫原性组合物中的应用。
本发明还提供了上述新型冠状病毒亚蛋白纳米疫苗在制备纳米示踪剂中的应用。
与现有技术相比，本发明的有益效果是：本发明通过将聚乳酸与卟啉类物质偶联并螯合有Co 2+离子，形成了由聚乳酸、卟啉或卟啉衍生物以及Co 2+离子结合物自组装的有机化合物，该有机化合物通过Co 2+离子与组氨酸标签连接，实现了高效装载新型冠状病毒抗原蛋白；并利用化学偶联的卟啉类物质本身可以产生荧光的功能，示踪纳米颗粒的在体分布；同时还在核心包裹疫苗有佐剂，核壳表面包裹有脂质，从而实现了新型冠状病毒抗原蛋白和疫苗佐剂共同递送的纳米疫苗体系。进一步地，本发明还设计并在化合物表面装载有可靶向抗原提呈细胞的多肽，从而促进纳米疫苗被DC细胞大量摄取，更有效促进机体的抗病毒反应。即本发明提供了一种兼具高效装载抗原、同时携载佐剂和抗原、示踪纳米疫苗分布并特异性高效靶向DC细胞功能的新型冠状病毒亚蛋白纳米疫 苗，并且其制备方法简单高效，为有效预防新型冠状病毒感染、新型冠状病毒亚蛋白纳米疫苗的制备提供了一个全新高效的途径与选择。
附图说明
图1为本发明实施例1中制备的纳米疫苗NPQ-RBD结构示意图；
图2为本发明实施例1中特异性靶向抗原提呈细胞的多肽的获取方式；
图3为本发明实施例1中制备的纳米疫苗NPQ-RBD-AP结构示意图；
图4为本发明实施例1中纳米疫苗的稳定性检测结果；
图5为本发明实施例2中纳米疫苗对小鼠骨髓来源树突状细胞的体外靶向性检测结果；
图6为本发明实施例2中纳米疫苗对小鼠骨髓来源树突状细胞的激活效应检测结果；
图7为本发明实施例2中纳米疫苗靶向***和***中的抗原提呈细胞的检测结果；
图8为本发明实施例2中纳米疫苗诱导动物血清产生抗RBD片段的抗体效价检测结果；
图9为本发明实施例2中纳米疫苗NPQ-RBD诱导动物血清产生抗RBD片段的抗体效价随时间变化的检测结果；
图10为本发明实施例2中纳米疫苗诱导机体产生针对RBD片段的细胞免疫效应检测结果；
图11为本发明实施例2中纳米疫苗对hACE2人源性小鼠抵抗新型冠状病毒COVID-19导致的肺部感染的检测结果。
具体实施方式
下面将结合本发明中的实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动条件下所 获得的所有其它实施例，都属于本发明保护的范围。
实施例1新型冠状病毒亚蛋白纳米疫苗的制备
1、聚乳酸PLA的合成
以正己醇作为引发剂，辛酸亚锡(Sn(Oct) 2)作为催化剂，通过L-丙交酯(L-LA)和D-丙交酯(D-LA)开环聚合的方法合成PLA，其合成路径如下：
Figure PCTCN2021111511-appb-000002
具体操作步骤为：
于手套箱中称取0.2500g正己醇置于干燥的安瓿瓶中，加入L-LA，D-LA各2.1180g，加入0.31mL预先配置好的浓度为0.02g/mL的Sn(Oct) 2溶液,最后加入21.0mL干燥的甲苯。聚合反应在130℃、N 2环境中反应2天。溶液用200mL冰***沉降，得到的产物室温下干燥24h。然后产物用N,N-二甲基甲酰胺(DMF)溶解，通过透析袋(MWCO＝1000D)在DMF中透析48h除去少量未反应的单体，转移至去离子水中透析去除有机溶剂，经冷冻干燥得最终产物。
1H NMR各个峰的成功归属及峰面积的计算证明了该聚合物成功合成。目前已知的PLA分子量可从几百到几万，其自身分子量越大疏水性越强，与辅料的分子量相差越大，其装载能力越弱。即通过调节投料比可制备得到不同的分子量的PLA，而基于不同分子量的PLA制备的纳米疫苗对于目标蛋白的装载能力有所不同。经优选，本发明选用分子量1500-3000的PLA以最大程度实现新型冠状病毒抗原蛋白RBD蛋白的装载。
2、PLA与卟啉类物质共价结合
本实施例以卟啉类物质：焦脱镁叶绿酸-α为例，将上述制得的PLA与焦脱镁叶绿酸-α反应，通过PLA末端的羟基与焦脱镁叶绿酸-α的羧基之间的缩合反应，使焦脱镁叶绿酸-α键合到PLA聚合物链的末端，合成路径如下：
Figure PCTCN2021111511-appb-000003
具体步骤为：
向焦脱镁叶绿酸(53.4mg)的无水二氯甲烷溶液中加入162.3mg PLA，然后加入38.6mg的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、49.8mg的4-二甲氨基吡啶，于冰浴中反应24h，TCL点板检测起始醇消失后，将反应液倒入水中，用二氯甲烷多次萃取产物，合并二氯甲烷相，然后依次用4％NaHCO 3水溶液洗涤、饱和食盐水洗涤，无水硫酸镁干燥12h。抽滤得产物的二氯甲烷溶液，然后通过旋转蒸发仪除去溶剂得到粗产物,通过透析袋(MWCO＝1000D)透析48h除去少量杂质，然后冷冻干燥得到产物PLA-焦脱镁叶绿酸。所有反应过程避光进行。
1H NMR中Ppa及PDLLA的峰成功归属、反应物Ppa中羧基峰的消失证明该缩合反应成功进行。紫外-可见吸收光谱(UV-vis)显示该材料与Ppa有相近的吸收峰位置。
3、Co 2+离子的配位螯合
通过在卟啉环中嵌入金属离子，利用金属离子实现与带有组氨酸标签的目的抗原的结合，本发明经优选发现在所有能够结合组氨酸标签的二价金属离子中，二价Co 2+离子的结合效率最高，因此选用Co 2+嵌合到卟啉环中，用于结合含有组氨酸标签的抗原，Co 2+离子可以在分子骨架中心位置于N原子以配位方式结合，合成路径如下：
Figure PCTCN2021111511-appb-000004
具体步骤为：
向上述制得的PLA-焦脱镁叶绿酸(50mg)的二氯甲烷溶液中(10.0mL)加入氯化钴的饱和甲醇溶液(1.0mL)并搅拌12h。旋转蒸发仪除去溶剂，然后用2.0mL二氯甲烷溶解，依次用4％NaHCO 3水溶液洗涤、饱和食盐水洗涤，无水硫酸镁干燥，过滤，通过旋转蒸发仪除去溶剂得到产物，即结合钴离子的卟啉环的PLA有机化合物。UV-vis显示Co 2+配位后，吸收峰位置发生红移。
4、RBD蛋白的纯化
RBD片段是新型冠状病毒结合人源性肺上皮细胞ACE2受体的关键结构域，也是目前常规被用来制备疫苗的最有效的区域。分别构建胞外分泌的和在细胞中表达的含有组氨酸标签的RBD片段慢病毒质粒，获得能够感染细胞的含有嘌呤霉素抗性的慢病毒，转染CHO细胞，使用嘌呤霉素筛选获得表达RBD蛋白片段的CHO细胞系。扩大培养该细胞系，获得上清液或者细胞悬浮液；如果是细胞悬浮液，通过添加PBS溶液结合超声，获得含有全细胞蛋白的PBS上清液。使用含有镍离子柱的纯化柱子获得仅表达组氨酸标签的RBD蛋白，接着在生理盐水溶液中进行透析，获得最终所需的RBD蛋白。
5、RBD蛋白的装载
相比较于其它佐剂而言，皂苷佐剂因为低剂量便可以同时引发体液免疫和细胞免疫效应，被认为是目前最有前景的佐剂，已经被用于肿瘤疫苗和病毒疫苗的相关临床实验研发，并且由于佐剂的亲疏水特性，能够与纳米体系完美结合，因此本实施例选用了QS-21作为疫苗佐剂，并选用DSPE-PEG2000磷脂作为纳米体系。本发明研究发现通过调整DSPE-PEG2000和PLA的含量比例，可改变该纳米体系结合表达组氨酸标签的蛋白的能力，装载效果最优的体系如下：
用1mL四氢呋喃溶解含有上述有机化合物、DSPE-PEG2000磷脂以及QS-21佐剂，其中所述有机化合物、DSPE-PEG2000磷脂以及QS-21佐剂的质量比为：1:10:10。溶解后的溶液逐滴滴入温度为70℃的生理盐水中，旋转蒸发有机溶剂，获得含有钴离子卟啉环的均匀分布于生理盐水中的纳米载体NPQ溶液；
10KD超滤离心管3000rpm离心获取高浓度含量的包含纳米载体的生理盐水溶液；将过量的RBD片段与含有纳米载体NPQ的溶液在4℃条件下孵育过夜，然后通过超速离心获得结合有RBD片段和QS-21的纳米疫苗(NPQ-RBD)，其结构示意图如图1所示。
6、RBD蛋白和靶向多肽的共同装载
***树突状细胞(DC)是机体重要的抗原提呈细胞，为提高纳米疫苗促进机体的抗病毒反应，进一步通过噬菌体技术筛选并且合成了一个可特异性靶向抗原提呈细胞的多肽，其氨基酸序列为：LDLFRELPFEWLEALKQKLK(SEQ ID NO.1所示)，将其与RBD蛋白分别通过组氨酸标签与有机化合物连接，并装载于纳米疫苗表面，具体操作步骤为：
按上述方法获得纳米载体NPQ溶液，10KD超滤离心管3000rpm离心获取高浓度含量的包含纳米载体的生理盐水溶液；将过量的RBD片段、含有组氨酸标签的靶向多肽与含有纳米载体的NPQ溶液在4℃条件下孵育过夜，然后通过超速离心获得结合有RBD片段、靶向多肽和QS-21的纳米疫苗(NPQ-RBD-AP)，其结构示意图如图3所示。
7、不同分子量蛋白与NPQ载体的结合效率分析
为验证本发明优选制得的NPQ载体对于RBD蛋白片段具有高效的结合效率，分别选取了不同分子量的蛋白质：His-IL-2(含有6个组氨酸标签的白细胞介素-2)、His-RBD(含有6个组氨酸标签的RBD蛋白)、His-BSA(含有6个组氨酸标签的牛血清白蛋白)、His-spike protein(含有6个组氨酸标签的新型冠状病毒Spike的蛋白)作为研究对象；在相等浓度的纳米载体中，加入相同浓度(50nmol)的上述蛋白，室温反应30分钟后，进行HPLC鉴定，分析游离的蛋白的含量，并根据公式：加入的总蛋白含量-游离蛋白含量/总蛋白含量*100％，计算不同分子量蛋白与NPQ载体的结合效率，结果如表1所示：
表1不同分子量与NPQ载体的结合效率
蛋白名称 分子量 摩尔质量 与NPQ结合效率
His-IL-2 15KD 50nmol 72％
His-RBD 26.1KD 50nmol 92％
His-BSA 66.5KD 50nmol 75％
His-spike protein 190KD 50nmol 62％
结果显示，本发明制备得到的纳米载体NPQ对于RBD蛋白具有最高的装载效率，其与NPQ的结合效率高达92％。
实施例2纳米疫苗NPQ-RBD和NPQ-RBD-AP的效果验证
1、纳米疫苗的特性表征
采用实施例1所述的方法分别制备得到了纳米疫苗NPQ-RBD和NPQ-RBD-AP，经检测其粒径为100-200nm之间，该区间大小的纳米颗粒可有效穿透***被引流至***。
为验证携载RBD片段的纳米疫苗NPQ-RBD和NPQ-RBD-AP的稳定性，使用FITC分子标记了纳米NPQ-RBD上的氨基酸片段；将该纳米至于4℃生理盐水中透析，每天收集部分溶液，使用酶标仪检测纳米中的相对FITC荧光强度，并设置不含佐剂的纳米颗粒NP-RBD对照组，以及仅注射RBD抗原的组别，检测结果如图4所示。
结果显示，本发明制备得到的纳米疫苗NPQ-RBD和NPQ-RBD-AP具有很好的稳定性，均可稳定一个星期以上，并仍然携带超过70％的RBD片段；
2、纳米疫苗体外靶向和激活小鼠骨髓来源树突状细胞(BMDC)
于无菌条件下提取雄性7-8周小鼠的骨髓，在体外用20ug/mL GM-CSF刺激7天后，即可获得小鼠骨髓来源树突状细胞BMDC，将BMDC置于共聚焦小皿中进行培养，FITC染色NPQ-RBD和NPQ-RBD-AP，使用20x物镜拍摄BMDC摄取纳米的情况，检测结果如图5所示。
结果显示，NPQ-RBD和NPQ-RBD-AP均可被树突状细胞摄取，其中携载有靶向抗原提呈细胞的多肽的纳米疫苗可被树突状细胞高效摄取。
将BMDC加到六孔板中，5×10 5/孔，向每孔中加入NPQ-RBD和 NPQ-RBD-AP悬液100μL。继续在培养箱中培养24h后，通过流式细胞仪检测DC细胞活化的标志物CD80、CD86的表达，并以未处理的DC细胞、不含佐剂和RBD抗原的有机纳米颗粒NP、佐剂QS21以及非特异性活化DC细胞的细菌脂多糖LPS分别处理DC细胞作为对照，结果如图6所示。
结果显示，相较于未处理的DC细胞，NPQ-RBD和NPQ-RBD-AP处理后的DC细胞中CD80、CD86的表达显著升高，即证明NPQ-RBD和NPQ-RBD-AP纳米疫苗可体外能显著激活小鼠骨髓来源树突状细胞(BMDC)；此外，该实验结果也证实了纳米载体NP本身也可以在一定程度上活化DC细胞。
3、纳米疫苗靶向***和***中的抗原提呈细胞
使用罗丹明标记纳米疫苗NPQ-RBD-AP上的RBD片段，然后向小鼠的尾根部注射100μL上述溶液；24h后使用小动物活体成像检测该纳米疫苗到达***的程度，检测结果如图7所示；接着将剥离的***进行冰冻切片以及免疫荧光操作，使用CD11c抗体标记DC细胞，使用F4-80抗体标记巨噬细胞，使用激光共聚焦显微镜观察***中纳米的定位，定位检测结果如图7所示。结果所示，所述纳米疫苗NPQ-RBD-AP可有效到达引流***部位，并且能够特异性富集于抗原提呈细胞区域，如DC细胞和巨噬细胞。
4、纳米疫苗诱导动物血清中产生抗RBD片段的抗体效价检测
在C57小鼠尾根部皮下分别注射100μL携带有RBD片段的纳米疫苗NPQ-RBD和NPQ-RBD-AP，共免疫两次，于第二次免疫后的第8天和第18天检测小鼠血清中针对RBD片段的抗体效价，并分别以注射PBS、佐剂QS-21、不含载体的RBD片段、不含佐剂的纳米颗粒NP-RBD作为对照。
小鼠血清的获取：采用眼眶静脉丛取血的方法，每次获取小鼠血液200-300μL，常温下凝固半个小时，然后4000rpm离心30分钟，获取小鼠血清；使用检测RBD抗体的ELisa试剂盒检测小鼠血清中含有的抗RBD片段的IgG抗体的效价，其中不同处理组的检测结果如图8所示，纳米疫苗NPQ-RBD的抗体效 价随时间变化的检测结果如图9所示。
结果显示，采用本发明制备得到的纳米疫苗可有效诱导RBD区域的抗体产生，并且产生的抗体效价为对照组的3000倍以上，即证明了该疫苗具有预防新型冠状病毒的潜力，同时该抗体效价可以在小鼠血清中存在至少10天以上，证明了该纳米疫苗产生抗体的持久性。
5、纳米疫苗诱导机体产生针对RBD片段的细胞免疫效应
在C57小鼠尾根部皮下注射100μL携带有RBD片段的纳米疫苗NPQ-RBD和NPQ-RBD-AP，并分别以注射PBS、佐剂QS-21、不含载体的RBD片段、不含佐剂的纳米颗粒NP-RBD作为对照。共免疫两次，在免疫第二次的一个月后，劲椎脱臼处死小鼠，提取小鼠脾脏和***细胞，ACK裂解红细胞，制成单细胞悬液。加入RBD蛋白片段再次刺激T细胞，使用流式技术检测细胞悬液中的CD8阳性T细胞中，表达IL-2、TNF-α和IFN-γ等细胞因子的比例，同时检测CD4阳性T细胞中表达IFN-γ的细胞比例，用于判断小鼠适应性免疫的生成。检测结果如图10所示。
结果显示，本发明制备得到的纳米疫苗NPQ-RBD和NPQ-RBD-AP均具有很强的诱导机体产生针对RBD片段的细胞免疫的功效。
6、纳米疫苗抵抗新型冠状病毒COVID-19导致的肺部感染
使用hACE2人源性小鼠，在该小鼠尾根部皮下注射100μL携带有RBD片段的纳米疫苗NPQ-RBD和NPQ-RBD-AP，并分别以注射PBS、佐剂QS-21、不含载体的RBD片段、不含佐剂的纳米颗粒NP-RBD作为对照。共免疫两次；在第二次免疫后的两周，对人源性hACE2小鼠进行攻毒实验，即使用COVID-19病毒感染该小鼠，在感染小鼠后的一周，使用PBS灌洗小鼠肺部，收集肺灌洗液，检测小鼠肺部感染新型冠状病毒情况，并通过检测新型冠状病毒mRNA来判定小鼠感染情况；将小鼠劲椎处死，获取小鼠肺部，并进行HE染色，评判小鼠肺部炎症情况，检测结果如图11所示，其中图11-B为RNA拷贝数检测结果， 11-C为抗体效价检测结果，11-D为肺部炎症观测结果。
结果显示，小鼠肺部无明显病变，即本发明制备得到的纳米疫苗NPQ-RBD和NPQ-RBD-AP可有效预防hACE2人源性小鼠感染新型冠状病毒。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。

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  1. 一种新型冠状病毒亚蛋白纳米疫苗，其特征在于，所述纳米疫苗包括：由聚乳酸、卟啉或卟啉衍生物以及Co 2+离子结合物自组装的有机化合物；新型冠状病毒抗原蛋白；疫苗佐剂和脂质。
  2. 根据权利要求1所述的新型冠状病毒亚蛋白纳米疫苗，其特征在于，所述纳米疫苗中由聚乳酸、卟啉或卟啉衍生物以及Co 2+离子结合物自组装的有机化合物为核壳结构，核内为疫苗佐剂，核壳上包裹有脂质，核壳表面负载有新型冠状病毒抗原蛋白。
  3. 根据权利要求2所述的新型冠状病毒亚蛋白纳米疫苗，其特征在于，所述核壳表面还负载有靶向抗原提呈细胞的靶向多肽，其中所述靶向多肽的氨基酸序列如SEQ ID NO:1所示。
  4. 根据权利要求3所述的新型冠状病毒亚蛋白纳米疫苗，其特征在于，所述新型冠状病毒抗原蛋白和靶向多肽分别通过组氨酸标签与有机化合物连接。
  5. 根据权利要求1所述的新型冠状病毒亚蛋白纳米疫苗，其特征在于，所述新型冠状病毒抗原蛋白为新型冠状病毒的RBD蛋白。
  6. 根据权利要求1所述的新型冠状病毒亚蛋白纳米疫苗，其特征在于，所述卟啉衍生物为只含有一个羧基基团的卟啉衍生物。
  7. 根据权利要求6所述的新型冠状病毒亚蛋白纳米疫苗，其特征在于，所述有机化合物的结构式为：
    Figure PCTCN2021111511-appb-100001
  8. 如权利要求1-7任一项所述的新型冠状病毒亚蛋白纳米疫苗的制备方法，其特征在于，所述方法包括：
    步骤1、将聚乳酸与卟啉或卟啉衍生物反应，使聚乳酸末端的羟基与卟啉或卟啉衍生物的羧基进行缩合反应，使卟啉或卟啉衍生物键合到聚乳酸聚合物链的末端；
    步骤2、向步骤1的产物中加入Co 2+离子，使Co 2+离子嵌入卟啉环中，得到由聚乳酸、卟啉或卟啉衍生物以及Co 2+离子结合物自组装的有机化合物；
    步骤3、将步骤2制得的有机化合物、脂质和疫苗佐剂混合溶解，滴入生理盐水中，蒸发有机溶剂获得含有钴离子卟啉环的均匀分布于生理盐水中的纳米载体溶液；
    步骤4、将连接有组氨酸标签的新型冠状病毒抗原蛋白，与步骤3制得的纳米载体溶液孵育过夜，超速离心获得结合有新型冠状病毒抗原蛋白的纳米疫苗。
  9. 如权利要求1-7任一项所述的新型冠状病毒亚蛋白纳米疫苗在制备与新型冠状病毒感染相关疾病的免疫原性组合物中的应用。
  10. 如权利要求1-7任一项所述的新型冠状病毒亚蛋白纳米疫苗在制备纳米示踪剂中的应用。
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