WO2021243974A1 - 一种SARS-CoV-2的融合蛋白及其疫苗组合物 - Google Patents

一种SARS-CoV-2的融合蛋白及其疫苗组合物 Download PDF

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WO2021243974A1
WO2021243974A1 PCT/CN2020/130222 CN2020130222W WO2021243974A1 WO 2021243974 A1 WO2021243974 A1 WO 2021243974A1 CN 2020130222 W CN2020130222 W CN 2020130222W WO 2021243974 A1 WO2021243974 A1 WO 2021243974A1
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fusion protein
rbd
mfc
cov
sars
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French (fr)
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刘中秋
廖国超
柯昌文
戚笑笑
柯碧霞
杨德盈
吴鹏
卢琳琳
冯潜
王彩艳
李自波
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广州中医药大学(广州中医药研究院)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a SARS-CoV-2 fusion protein and a vaccine composition thereof.
  • Novel Coronavirus Pneumonia (Corona Virus Disease 2019, COVID-19) is a viral infectious disease of human pneumonia caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
  • Common signs of people infected with coronavirus include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing; severe cases can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death.
  • This newly emerging infectious disease is spreading rapidly around the world.
  • As of April 2020 there have been more than 3 million cases worldwide and more than 200,000 deaths.
  • COVID-19 has become a serious public health problem.
  • SARS-CoV-2 seriously endangers human health, there are no specific drugs for the treatment and prevention of COVID-19. Therefore, there is an urgent need to develop a safe and effective SARS-CoV-2 vaccine to prevent COVID-19.
  • SARS-CoV-2 vaccines have entered the first phase of clinical trials, such as mRNA-1273 vaccine (Moderna), Ad5- nCoV vaccine (CanSino Biologicals), INO-4800 vaccine (Inovio), LV-SMENP-DC vaccine (Shenzhen Institute of Immunogene Therapy) and pathogen-specific aAPC vaccine (Shenzhen Institute of Immunogene Therapy), etc.
  • mRNA vaccines have high-efficiency, rapid development capabilities and low-cost production potential, and have broad application prospects in the prevention of infectious diseases, but the instability and low efficiency of in vivo administration often affect and restrict the further development of mRNA vaccines .
  • the viral vector vaccine is widely used because of its advantages of transient expression and high transduction efficiency, but there are serious problems in the production process.
  • DNA vaccines can induce a wider range of immune responses, but the introduction of exogenous DNA may cause changes in the host's genome or induce immune tolerance.
  • MNA microneedle array
  • Eun Kim et al. to deliver the SARS-CoV-2 subunit vaccine has been shown to cause an effective specific antibody response.
  • each vaccine strategy has its advantages and disadvantages. All new vaccines for SARS-CoV-2 prevention need to be further comprehensively evaluated for the effectiveness and safety of their immune enhancement.
  • RBD receptor-binding domain
  • ACE2 angiotensin-converting enzyme 2
  • the purpose of the present invention is to overcome the shortcomings of the prior art, and provide a fusion protein, the fusion protein includes the severe acute respiratory syndrome coronavirus 2 antigenic protein and immunoglobulin Fc fragment, the fusion protein is configured with an adjuvant.
  • the vaccine composition can achieve the purpose of inhibiting the replication of SARS-CoV-2, inhibiting the transmission of SARS-CoV-2 or preventing the common strain and variant strain of SARS-CoV-2 from colonizing in its host, thereby being able to effectively prevent and/or Treatment of new coronavirus pneumonia.
  • the technical solution adopted by the present invention is: a fusion protein, the fusion protein includes the Severe Acute Respiratory Syndrome Coronavirus 2 antigenic protein and the immunoglobulin Fc fragment.
  • the SARS-CoV-2 antigenic protein includes the RBD neutralizing epitope in the S protein fragment.
  • the amino acid sequence of the RBD neutralizing epitope is shown in SEQ ID No. 1.
  • the immunoglobulin Fc fragment includes an immunoglobulin Fc fragment of at least one of murine IgG, IgA, IgD, IgE, and IgM.
  • the murine IgG is selected from at least one of murine IgG1, IgG2a, IgG2b, and IgG3.
  • the amino acid sequence of the immunoglobulin Fc fragment is shown in SEQ ID No.2.
  • the amino acid sequence of the fusion protein is shown in SEQ ID No.3.
  • the present invention also provides a vaccine composition, which includes the fusion protein, and also includes an immunologically and pharmaceutically acceptable carrier or adjuvant.
  • the adjuvant includes aluminum adjuvant, Freund's adjuvant, monophosphoryl lipid A, RIBI adjuvant system, and ⁇ -galactose ceramide analog adjuvant At least one of them.
  • the present invention also provides the application of the vaccine composition in the preparation of drugs for the prevention and/or treatment of novel coronavirus pneumonia.
  • the vaccine composition of the present invention can recombinantly express a large amount of the components of the vaccine composition, that is, the fusion protein, by genetic engineering means, which is not only time-consuming and short, but also convenient for large-scale production.
  • Figure 1 is an evaluation diagram of the fusion protein vaccine RBD-mFc of Example 1-2 of the present invention and the vaccine composition containing the fusion protein RBD-mFc/Al and RBD-mFc/FA to induce the immune activity of the antibody IgG produced in mice.
  • Figure 2 is an evaluation diagram of the immunological activity of the fusion protein vaccine RBD-mFc of Example 1-2 of the present invention and the vaccine composition RBD-mFc/Al and RBD-mFc/FA induced by the fusion protein of the IgG subtype in mice .
  • Figure 3 is a diagram showing the immune activity evaluation of the fusion protein vaccine RBD-mFc of Example 1-2 of the present invention and the vaccine composition RBD-mFc/Al and RBD-mFc/FA containing the fusion protein in the antibody neutralization experiment induced by mice .
  • Figure 4 is an evaluation diagram of the cytokine IFN- ⁇ produced by mice induced by the fusion protein vaccine RBD-mFc of Example 1-2 and the vaccine composition RBD-mFc/Al and RBD-mFc/FA containing the fusion protein.
  • FIG. 5 is an evaluation diagram of the fusion protein vaccine RBD-mFc of Example 1-2 of the present invention and the vaccine composition RBD-mFc/Al and RBD-mFc/FA containing the fusion protein to induce the production of cytokine IL-4 in mice.
  • Figure 6 is the fusion protein vaccine RBD-mFc of Example 1-2 of the present invention and the vaccine composition containing the fusion protein RBD-mFc/Al and RBD-mFc/FA induced mice to specifically recognize and transfect the antibody serum Flow cytometric evaluation diagram of ACE2 carrier human renal epithelial cell line 293T.
  • fusion protein has two different meanings, one is the expression product of two gene recombination obtained through DNA recombination technology, which connects two different proteins into one macromolecule; the other It is connected chemically.
  • SEQ ID No. 1 The amino acid sequence of the RBD neutralizing epitope in the S protein fragment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2);
  • SEQ ID No. 2 The amino acid sequence of the Fc protein in mouse immunoglobulin IgG;
  • SEQ ID No. 3 the amino acid sequence of the fusion protein RBD-mFc
  • SEQ ID No. 4 The nucleotide sequence of the fusion protein RBD-mFc.
  • NCBI https://www.ncbi.nlm.nih.gov
  • the gene sequence of the RBD neutralizing epitope in the S protein fragment of the COVID-19 virus (GeneI D:43740578) reported by NCBI (https://www.ncbi.nlm.nih.gov) (the amino acid sequence is as SEQ I D No 1)
  • the gene sequence of the Fc protein in mouse immunoglobulin IgG amino acid sequence is shown in S EQ ID No. 2
  • the fusion protein is prepared by genetic engineering.
  • the nucleotide sequence of fully synthesized RBD-mFc (nucleotide sequence is shown in SEQ ID No. 4, amino acid sequence is shown in SEQ ID No. 3), and introduced at the 5'-end and 3'-end of the gene Specific enzyme cut sites BamHI and XhoI, the mammalian cell expression vector PCDNA3.1 and RBD-mFc genes were simultaneously digested with BamHI and XhoI, and then subjected to 10g/L agarose gel electrophoresis after digestion and coagulation Gel recovery purification kit to purify the digested product, mix the two digested and purified products at a molar ratio of 3:1, ligate with T4 DNA ligase for 1h, and use low-temperature CaCl 2 to prepare E.coli DH5 ⁇ competent bacteria Transform, and then spread in LB solid medium containing ampicillin.
  • step (4) Take 50 ⁇ L of the cell culture medium cultured for 72h after adding the transfection enhancer in step (4), centrifuge at 4°C, 12000r/min for 5 minutes, prepare the sample with the protein loading buffer containing the reducing agent and electrophoresis; after the electrophoresis is completed, use The protein was transferred to the PVDF membrane activated by methanol by wet transfer method; after transfer, the PVDF membrane was sealed with 5% skim milk on a room temperature shaker for 1 hour, and anti-Gc rabbit polyclonal antibody (1:5000) and StrepII-tag were used respectively.
  • HRP horse anti-rabbit secondary antibody
  • Vaccine composition complete Freund’s adjuvant RBD-mFc/FA (RBD-mFc/CFA); use PBS buffer salt solution to dilute the RBD-mFc fusion protein prepared in Example 1, and add incomplete Freund’s adjuvant, and stir Make it into a white emulsion to obtain a vaccine composition incomplete Freund's adjuvant RBD-mFc/FA (RBD-mFc/IFA) with a fusion protein content of 10 ⁇ g/mL.
  • mice Twenty C57BL/6 mice aged 6-8 weeks were randomly divided into 4 groups with 5 mice in each group.
  • the vaccines prepared were injected on the 0th and 28th days, each with 5 mice in each group. Only 0.1mL per injection.
  • RBD-mFc group intramuscular injection of the vaccine RBD-mFc prepared in Example 2 on day 0 and 28;
  • RBD-mFc/Al group The vaccine composition RBD-mFc/Al prepared in Example 2 was injected intramuscularly on the 0th and 28th days;
  • RBD-mFc/FA group On day 0, the vaccine composition prepared in Example 2 was injected intramuscularly with complete Freund’s adjuvant RBD-mFc/FA (RBD-mFc/CFA); on day 28, the vaccine composition prepared in Example 2 was injected intramuscularly
  • the vaccine composition is incomplete Freund's adjuvant RBD-mFc/FA (RBD-mFc/IFA).
  • Control group as the blank control group, the same dose of PBS solution with pH 7.4 was injected.
  • mice Blood was collected on the 28th and 42nd days. Each mouse was taken from 0.1mL to 0.2mL, placed at 0°C for 60 minutes, centrifuged at 4000 rpm for 15 minutes, and the upper clear serum was used for ELISA detection and analysis. The mice were euthanized for 42 days, and the spleens were taken to make a single cell suspension for subsequent ELISPOT experiments.
  • mice serum of the primary or secondary immunization (1:300) to each well, add 100 ⁇ L to each well, and incubate in a 37°C incubator for 1 hour; wash the plate 3 times; after the first immunization or Add HRP (horseradish peroxidase) labeled IgG to the secondary immunized mouse serogroup, and add HRP (horseradish peroxidase) labeled IgG1, IgG2b, IgG2c, and IgG3 to the secondary immunized mouse serogroup. , Add 100 ⁇ L to each well, incubate at room temperature for 1 hour; wash the plate 3 times.
  • TMB solution add 100 ⁇ L to each well, and develop color in the dark at room temperature for 20 minutes.
  • Add 0.5M H 2 SO 4 solution to stop the color development add 100 ⁇ L to each well, and detect the absorbance with a microplate reader.
  • the detection wavelength is 450 nm, and 570 nm is used as the background wavelength.
  • the results are shown in Figure 1.
  • the absorbance (OD value) is plotted against different subunit vaccines.
  • the subunit fusion protein vaccine RBD-mFc and its vaccine composition RBD-mFc/Al and RBD- in Example 2 of the present invention mFc/FA can induce the production of high titer IgG antibodies in mice, and the RBD-mFc/FA group with Freund's adjuvant has the best effect.
  • Vero E6 cells (2 ⁇ 10 4 cells/well) were seeded into a 96-well plate and cultured overnight at 37° C. and 5% CO 2 until a monolayer was formed.
  • the 100TCID50 of SARS-CoV-2 was mixed with the mouse antiserum prepared in step (1) of Example 3 that was serially diluted 4-fold, incubated at 37°C for one hour, and the serum sample was heated at 56°C for 30 minutes, and then added Mix with Vero E6 cells.
  • cells infected with 100TCID50 SARS-CoV-2 were used as a positive control, while cells without virus were used as a negative control, and then CPE (cytopathic effect) was recorded on the 3rd day after infection.
  • the neutralization titer of serum inoculated with RBD-mFc fusion protein that completely inhibited 50% of the CPE of the well was calculated by the Reed-Muench method.
  • NA neutralizing antibody
  • the ELISPOT kit was used to detect cytokines.
  • the 96-well plate is pre-coated with anti-mouse IFN- ⁇ or IL-4.
  • Each group made 3 sub-wells and cultured at 37°C and 5% CO 2 for 24 hours.
  • mice vaccinated with RBD-mFc/Al caused INF- ⁇ and IL-4 points on day 42 to be 3.33 times and 7.35 times that of the blank group, respectively. It shows that under the action of aluminum adjuvant, RBD-mFc can induce Th1 and Th2 immune responses at the same time; in addition, RBD-mFc/FA and RBD-mFc induce more INF- ⁇ than IL-4, indicating different adjuvants
  • the drug has a significant effect on the immune effect induced by RBD-mFc.
  • the subunit vaccine RBD-mFc prepared by binding protein RBD to the Fc domain of immunoglobulin to obtain a fusion protein, and a vaccine composition prepared by adding an adjuvant to the fusion protein; it can promote selective absorption and induce The specific immune response against SARS-CoV-2 can achieve the purpose of inhibiting the replication of SARS-CoV-2, inhibiting the transmission of SARS-CoV-2, or preventing common and variant SARS-CoV-2 from colonizing its host.

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Abstract

一种SARS-CoV-2的融合蛋白及其疫苗组合物。所述融合蛋白及含该融合蛋白制备的疫苗组合物能诱导针对SARS-CoV-2的特异性免疫反应，达到抑制SARS-CoV-2的复制、抑制SARS-CoV-2的转播或阻止SARS-CoV-2普通株和变异株在其宿主体内定居的目的，所述融合蛋白及含该融合蛋白的疫苗组合物，能有效预防和/或治疗新型冠状病毒肺炎（Corona Virus Disease 2019，COVID-19）。该融合蛋白可利用基因工程技术进行大量重组表达，耗时短且可便于大规模生产。

Description

一种SARS-CoV-2的融合蛋白及其疫苗组合物 技术领域
本发明属于生物技术领域，具体涉及一种SARS-CoV-2的融合蛋白及其疫苗组合物。
背景技术
新型冠状病毒肺炎(Corona Virus Disease 2019，COVID-19)是由严重急性呼吸综合征冠状病毒2(Severe Acute Respiratory Syndrome Coronavirus 2，SARS-CoV-2)引起人肺炎的病毒性传染病。人感染了冠状病毒后常见体征有呼吸道症状、发热、咳嗽、气促和呼吸困难等；严重者可导致肺炎、严重急性呼吸综合征、肾衰竭，甚至死亡。这种新出现的传染病在世界各地迅速传播，截至2020年4月，全世界已有300多万例，造成20多万人死亡，COVID-19已成为一个严峻的公共卫生问题。虽然SARS-CoV-2严重危害人类的健康，但是目前还没有治疗和预防COVID-19的具体药物。因此，迫切需要开发安全有效的SARS-CoV-2疫苗来预防COVID-19。
目前，全球范围内SARS-CoV-2疫苗的研发正快速进行中；世界范围内，有几种SARS-CoV-2疫苗已进入临床第一阶段试验，如mRNA-1273疫苗(Moderna)、Ad5-nCoV疫苗(CanSino Biologicals)、INO-4800疫苗(Inovio)、LV-SMENP-DC疫苗(深圳市免疫基因治疗研究院)和pathogen-specific aAPC疫苗(深圳市免疫基因治疗研究院)等。其中，mRNA疫苗具有高效、快速的开发能力和低成本的生产潜力，在预防传染病方面具有广阔的应用前景，但体内给药的不稳定性和低效率常常影响和制约着mRNA疫苗的进一步开发。作为第三代疫苗的病毒载体疫苗因其具有瞬时表达和高转导效率的优点而被广泛使用，但生产过程中存在严重问题。DNA疫苗可以诱导更广泛的免疫应答类型，但外源性DNA的引入可能导致宿主基因组的改变或诱导免疫耐受。此外，Eun Kim等人设计和生产的微针阵列(MNA)递送SARS-CoV-2亚单位疫苗，已被证明能够引起有效的特异性抗体反应。目前每种疫苗策略都有其优点和缺点， 所有用于SARS-CoV-2预防的新疫苗还待进一步综合评估其免疫增强的有效性和安全性。
研究人员发现SARS-CoV-2表面刺突糖蛋白受体结合区(receptor-binding domain,RBD)可直接与功能细胞受体血管紧张素转换酶2(ACE2)结合，在病毒进入功能细胞受体和发病过程中起着重要作用；因此，SARS-CoV-2的RBD蛋白可作为开发SARS-CoV-2病毒抑制剂和亚单位疫苗的一个有吸引力的靶点。但是小分子量的SARS-CoV-2 RBD(～21KDa)具有免疫原性差，不能诱导细胞毒性T淋巴细胞反应，诱导产生的免疫反应持续时间较短，需要多次接种等缺点，不适合单独制备成疫苗。因此，如何提高亚单位疫苗的有效性与安全性是急需解决的技术问题。
发明内容
本发明的目的在于克服现有技术的不足，提供一种融合蛋白，该融合蛋白包括严重急性呼吸综合征冠状病毒2抗原性蛋白和免疫球蛋白Fc片段，利用该融合蛋白与佐剂配置得到的疫苗组合物，可达到抑制SARS-CoV-2的复制、抑制SARS-CoV-2的转播或阻止SARS-CoV-2普通株和变异株在其宿主体内定居的目的，从而能有效预防和/或治疗新型冠状病毒肺炎。
为实现上述目的，本发明采取的技术方案为：一种融合蛋白，所述融合蛋白包括严重急性呼吸综合征冠状病毒2抗原性蛋白和免疫球蛋白Fc片段。
作为本发明所述的融合蛋白的优选实施方式，所述严重急性呼吸综合征冠状病毒2抗原性蛋白包括S蛋白片段中RBD中和表位。
作为本发明所述的融合蛋白的优选实施方式，所述RBD中和表位的氨基酸序列如SEQ ID No.1所示。
作为本发明所述的融合蛋白的优选实施方式，所述的免疫球蛋白Fc片段包括鼠源IgG、IgA、IgD、IgE、IgM中至少一种的免疫球蛋白Fc片段。
作为本发明所述的融合蛋白的优选实施方式，所述的鼠源IgG选自鼠源IgG1、IgG2a、IgG2b、IgG3的至少一种。
作为本发明所述的融合蛋白的优选实施方式，所述免疫球蛋白Fc片段的氨基酸序列如SEQ ID No.2所示。
作为本发明所述的融合蛋白的优选实施方式，所述融合蛋白的氨基酸序列 如SEQ ID No.3所示。
本发明还提供了一种疫苗组合物，所述疫苗组合物包括所述的融合蛋白，还包括免疫学和药学上接受的载体或佐剂。
作为本发明所述的疫苗组合物的优选实施方式，所述佐剂包括铝佐剂、弗氏佐剂、单磷酰脂质A、RIBI佐剂系统、α-半乳糖神经酰胺类似物佐剂中的至少一种。
另外，本发明还提供所述的疫苗组合物在制备预防和/或治疗新型冠状病毒肺炎的药物中的应用。
本发明的有益效果：
(1)将SARS-CoV-2的RBD蛋白结合到鼠源免疫球蛋白Fc域得到融合蛋白RBD-mFc，并加入佐剂制成疫苗组合物，不仅可以增加疫苗的溶解度、稳定性，延长疫苗在体内半衰期；还促进选择性吸收并诱导针对SARS-CoV-2的特异性免疫反应，达到抑制SARS-CoV-2的复制、抑制SARS-CoV-2的转播或阻止SARS-CoV-2普通株和变异株在其宿主体内定居的目的，从而能有效预防和/或治疗新型冠状病毒肺炎；
(2)本发明的疫苗组合物可通过基因工程手段对疫苗组合物的组分即所述的融合蛋白进行大量重组表达，不仅耗时短，还可便于大规模生产。
附图说明
图1是本发明实施例1-2的融合蛋白疫苗RBD-mFc和含该融合蛋白的疫苗组合物RBD-mFc/Al与RBD-mFc/FA诱导小鼠产生的抗体IgG免疫活性评价图。
图2是本发明实施例1-2的融合蛋白疫苗RBD-mFc和含该融合蛋白的疫苗组合物RBD-mFc/Al与RBD-mFc/FA诱导小鼠产生的抗体IgG亚型免疫活性评价图。
图3是本发明实施例1-2的融合蛋白疫苗RBD-mFc和含该融合蛋白的疫苗组合物RBD-mFc/Al与RBD-mFc/FA诱导小鼠产生的抗体中和实验免疫活性评价图。
图4是本发明实施例1-2的融合蛋白疫苗RBD-mFc和含该融合蛋白的疫苗组合物RBD-mFc/Al与RBD-mFc/FA诱导小鼠产生的细胞因子IFN-γ评价图。
图5是本发明实施例1-2的融合蛋白疫苗RBD-mFc和含该融合蛋白的疫苗组合物RBD-mFc/Al与RBD-mFc/FA诱导小鼠产生的细胞因子IL-4评价图。
图6是本发明实施例1-2的融合蛋白疫苗RBD-mFc和含该融合蛋白的疫苗组合物RBD-mFc/Al与RBD-mFc/FA诱导小鼠产生的抗体血清特异性识别转染了ACE2载体的人肾上皮细胞系293T的流式细胞实验评价图。
具体实施方式
为更清楚地表述本发明的技术方案，下面结合具体实施例进一步说明，但不能用于限制本发明，此仅是本发明的部分实施例。
术语融合蛋白：融合蛋白(fusion protein)有两种不同的含义，一种是通过DNA重组技术得到的两个基因重组后的表达产物，将两个不同的蛋白质连成一个大分子；另一种是通过化学方法连接。
序列表：
(1)SEQ ID No.1：严重急性呼吸综合征冠状病毒2(SARS-CoV-2)中S蛋白片段中RBD中和表位的氨基酸序列；
(2)SEQ ID No.2：鼠免疫球蛋白IgG中Fc蛋白的氨基酸序列；
(3)SEQ ID No.3：融合蛋白RBD-mFc的氨基酸序列；
(4)SEQ ID No.4：融合蛋白RBD-mFc的核苷酸序列。
实施例1：融合蛋白RBD-mFc的制备、鉴定及含量测定
根据NCBI(https://www.ncbi.nlm.nih.gov)报道的COVID-19病毒(GeneI D:43740578)中S蛋白片段中RBD中和表位的基因序列(氨基酸序列如SEQ I D No.1所示)，以及鼠免疫球蛋白IgG中Fc蛋白的基因序列(氨基酸序列如S EQ ID No.2所示)，通过基因工程手段制备融合蛋白。
(1)COVID-19抗原性蛋白RBD和mFc融合蛋白的克隆构建及鉴定
全合成RBD-mFc的核苷酸序列(核苷酸序列如SEQ ID No.4所示，氨基酸序列如SEQ ID No.3所示)，并在该基因5'-端和3'-端引入特异性酶切位点BamHI和XhoI，将哺乳动物细胞表达载体PCDNA3.1和RBD-mFc基因，同时用BamHI和XhoI双酶切，酶切后进行10g/L琼脂糖凝胶电泳，并经过凝胶回收纯化试剂盒将酶切产物进行纯化，将两者酶切纯化的产物按照3∶1的摩尔比混合，用T4 DNA连接酶连接1h，用低温CaCl 2制备的E.coli DH5α感受态细菌转化，然后涂布于含氨苄青霉素的LB固体培养基中。次日，随机挑取10个单菌落，分别接种于0.5mL含氨苄青霉素的LB培养液中，37℃下150r/min振荡3h。取1μL作为模板以通用引物进行菌落PCR鉴定，如果结果经琼脂糖凝胶电泳显示大小正确则用质粒提取试剂盒提取质粒DNA。测序采用Sanger双脱氧链终止法，对引物所在序列的两端进行测定，测序工作由广州艾基生物工程公司完成，构建得到序列正确的重组表达质粒PCDNA3.1-RBD-mFc。
(2)RBD-mFc蛋白在Expi293F悬浮型细胞中的表达
转染前，将3×10 6Expi293F细胞接种于30mL Expi293F表达培养基中，于5％CO 2、37℃、120r/min条件下持续培养数小时，当细胞密度达3.5×10 6/mL时，将80μL ExpiFectamine293转染试剂加入1.5mL培养基中混匀，室温孵育5分钟。将30μg表达质粒加入1.5mL培养基中混匀，然后将孵育好的转染试剂与培养基的混合物加入质粒与培养基的混合物中轻轻混匀，室温孵育25分钟，最后缓慢加入细胞培养瓶中。培养16小时后，在上述细胞培养瓶中分别加入150μL转染增强剂1和1.5mL转染增强剂2(具体步骤参见赛默飞公司试剂盒Expi293 TM Expression System Kit(Cat.No.A14635))，培养72小时后收集细胞进行RBD-mFc表达量的鉴定。
(3)RBD-mFc蛋白表达量的鉴定
取步骤(4)中添加转染增强剂后培养72h的细胞培养液50μL，4℃、12000r/min离心5分钟，用含有还原剂的蛋白上样缓冲液制样并电泳；电泳结束后，用湿转方法将蛋白转移到经甲醇激活的PVDF膜上；转膜后，用5％脱脂牛奶对PVDF膜室温摇床封闭1小时，分别用抗Gc兔多抗(1∶5000)和StrepⅡ-tag(HRP)抗体(1∶30000)与封闭后的PVDF室温孵育1.5小时；用TBST清洗PVDF膜3次，每次10分钟；以1∶20000的比例加入羊抗兔二抗(HRP)室温孵育1h；用TBST清洗PVDF膜3次，每次10分钟；最后利用Western印迹显色液进行显色。对RBD-mFc蛋白在Expi293F细胞中表达量的鉴定。我们确定转染后72小时为收取细胞上清的最佳时间。
(4)RBD-mFc蛋白的纯化及鉴定
在确定最佳细胞上清收获时间后，利用MabSelect SuRe LX亲和柱对收获后 的细胞上清进行纯化，同时在纯化过程中收取目的蛋白的洗脱峰、柱前和柱后，进行SDS-PAGE分析，在收取的洗脱峰中获得了纯度较好的RBD-mFc蛋白。用BCA方法测定的纯化后RBD-mFc蛋白浓度为0.7mg/mL。最后纯化后的RBD-mFc蛋白进行了蛋白印迹鉴定。
实施例2：亚单位疫苗的制备
(1)使用PBS缓冲盐溶液将实施例1制备的RBD-mFc融合蛋白进行稀释，并充分混匀，得到重组的融合蛋白的含量为10μg/mL的疫苗RBD-mFc。
(2)使用PBS缓冲盐溶液将实施例1制备的RBD-mFc融合蛋白进行稀释，并加入铝佐剂充分混匀，得到重组的融合蛋白的含量为10μg/mL的疫苗组合物RBD-mFc/Al；
(3)使用PBS缓冲盐溶液将实施例1制备的RBD-mFc融合蛋白进行稀释，并加入完全弗氏佐剂，搅拌使其成为白色乳剂，即得到重组的融合蛋白的含量为10μg/mL的疫苗组合物完全福氏佐剂RBD-mFc/FA(RBD-mFc/CFA)；使用PBS缓冲盐溶液将实施例1制备的RBD-mFc融合蛋白进行稀释，并加入非完全弗氏佐剂，搅拌使其成为白色乳剂，即得到融合蛋白的含量为10μg/mL的疫苗组合物不完全福氏佐剂RBD-mFc/FA(RBD-mFc/IFA)。
实施例3：ELISA免疫分析
(1)小鼠免疫
取6-8周龄的C57BL/6小鼠20只，随机分为4组，每组5只，采用一次初始免疫和二次增强免疫方案，分别在第0和28天注射制备的疫苗，每只每次注射量0.1mL。
RBD-mFc组：第0和28天肌肉注射实施例2所制备的疫苗RBD-mFc；
RBD-mFc/Al组：第0和28天肌肉注射实施例2所制备的疫苗组合物RBD-mFc/Al；
RBD-mFc/FA组：第0天肌肉注射实施例2所制备的疫苗组合物完全福氏佐剂RBD-mFc/FA(RBD-mFc/CFA)；第28天肌肉注射实施例2所制备的疫苗组合物不完全福氏佐剂RBD-mFc/FA(RBD-mFc/IFA)。
Control组：为空白对照组，注射相同剂量的pH7.4的PBS溶液。
第28天、42天取血，每只小鼠取血取0.1mL到0.2mL，在0℃放置60分钟，4000转/分钟离心15分钟，取上层清亮血清用于ELISA检测分析，并在第42天对小鼠进行安乐死，取脾脏制成单细胞混悬液用于后续ELISPOT实验。
(2)ELISA免疫分析
用0.1M碳酸盐缓冲液(pH 9.6)将RBD-His(广州环球基因工程有限公司采用人工合成的方法制备而成)配置成0.33μg/mL溶液，以每孔100μL的量加入96孔板，放入4℃孵育过夜；第二天放入37℃培养箱孵育1小时；用PBST(PBS+0.1％吐温20)洗板3次，每孔加入300μL洗液；洗板后加入2％脱脂奶粉，每孔加入250μL，37℃孵育1小时；用PBST洗板3次。分别在每个孔中加入一次免疫或二次免疫的小鼠血清(1:300)，每孔加入100μL，放置在37℃培养箱孵育1个小时；洗板3次后；分别在一次免疫或二次免疫的小鼠血清组加入HRP(辣根过氧化物酶)标记的IgG，在二次免疫的小鼠血清组加入HRP(辣根过氧化物酶)标记的IgG1、IgG2b、IgG2c与IgG3，每孔加入100μL，室温孵育1小时；洗板3次。加入TMB溶液，每孔加入100μL，室温避光显色20分钟。加入0.5M H 2SO 4溶液终止显色，每孔加100μL，酶标仪检测吸光度，检测波长为450nm，570nm作为背景波长。
结果如图1所示，将吸光度(OD值)相对于不同亚单位疫苗作图，本发明实施例2中的亚单位融合蛋白疫苗RBD-mFc及其疫苗组合物RBD-mFc/Al与RBD-mFc/FA，在小鼠体内均能诱导产生高滴度的IgG抗体，其中加弗氏佐剂组RBD-mFc/FA效果最好。
如图2所示，将吸光度(OD值)相对于不同亚型抗体作图，RBD-mFc/Al和RBD-mFc/FA免疫的小鼠显著增强了IgG1和IgG2b的分泌，表明RBD-mFc可以同时诱导Th2和Th1免疫反应并产生强效的抗体。
实施例4：抗体中和实验
将Vero E6细胞(2×10 4细胞/孔)接种到96孔板中，在37℃和5％CO 2下培养过夜，直到形成单层。将SARS-CoV-2的100TCID50与连续4倍稀释的实施例3步骤(1)中制备得到的小鼠抗血清混合，在37℃孵育一小时，将血清样品在56℃加热30分钟，随后加入与Vero E6细胞混合。在每种测定中，感染 了100TCID50 SARS-CoV-2的细胞为阳性对照，而无病毒的细胞为阴性对照，然后在感染后第3天记录CPE(细胞病变效应)。通过Reed-Muench法计算了完全抑制孔的50％CPE的用RBD-mFc融合蛋白接种的血清的中和效价。
中和抗体(NA)滴度越高，说明病毒复制水平越低，对病毒感染的防护能力越强。如图3所示，疫苗组合物RBD-mFc/FA诱导的抗体血清的中和抗体滴度显著高于其他疫苗，表明在弗氏佐剂的作用下，融合蛋白疫苗RBD-mFc可产生强效的体液免疫应答，其诱导产生的抗体血清具有较强的中和活性，并保护细胞免受SARS-CoV-2感染。
实施例5：ELISPOT实验
采用ELISPOT试剂盒检测细胞因子。96孔板预包被anti-mouse IFN-γ或者IL-4。将不含FBS(胎牛血清)的RPMI-1640(200μL/孔)添加到每个孔中以激活单克隆抗体，室温下孵育2小时，弃掉培养基，将接种了疫苗的和对照组的脾细胞接种于板中(2.5×10 5细胞/孔)，同时加入10μg/mL的RBD-His，每组做3个副孔，在37℃和5％CO 2环境下培养24小时。在4℃温度下，用冰冷的双蒸水溶解细胞10分钟；1×洗涤缓冲液洗板6次；加入生物素化的anti-mouse IFN-γ或IL-4抗体(1:100)，在37℃下孵育一小时。洗板后加入链霉亲和素-辣根过氧化物酶(1:100)，孵育1h。洗板后，添加3-氨基-9-乙基咔唑(100μL/孔)。加入AEC可以在37℃的黑暗环境中形成斑点，持续30分钟，然后用dd H 2O停止反应并风干板。用显微镜计数分泌的IFN-γ和分泌IL-4的T细胞的斑点。
结果如图4与图5所示，与对照组相比，接种RBD-mFc/Al的小鼠在第42天导致INF-γ和IL-4点数量分别是空白组的3.33倍和7.35倍，说明在铝佐剂的作用下，RBD-mFc可以同时诱导产生Th1和Th2免疫反应；此外，RBD-mFc/FA和RBD-mFc诱导产生的INF-γ多于产生IL-4，表明不同的佐剂对RBD-mFc诱导的免疫作用有显著的影响。
实施例6：流式细胞实验
将人肾上皮细胞系293T用人ACE2载体转染48h，分离并用HBSS洗涤细胞3次，计数使293T/ACE2 1.0×10 6细胞/管，将小鼠血清(1:10稀释)加入到 293T/ACE2细胞中，然后加入1μg/mL RBD-His蛋白，在冰上孵育1h。用HBSS洗涤3次后，加入anti-His-FITC，在冰上避光孵育1h，用HBSS洗涤3次后通过流式细胞仪检测平均荧光强度。
结果如图6所示，与Positive control(阳性对照组)相比，亚单位疫苗RBD-mFc及疫苗组合物RBD-mFc/FA和RBD-mFc/Al的峰图明显向左移，接近于Negative control组(阴性对照组)，Control组(空白血清)峰图明显右移，与Positive control组相近，说明亚单位疫苗RBD-mFc及疫苗组合物RBD-mFc/FA和RBD-mFc/Al的小鼠血清在1：10稀释时有效阻断了RBD与ACE2/293T细胞的结合，而对照的空白血清在相同稀释度下没有抑制作用。
综上所述，蛋白RBD结合到免疫球蛋白Fc域得到融合蛋白制得的亚单位疫苗RBD-mFc，及在该融合蛋白中加入佐剂制成的疫苗组合物；可以促进选择性吸收并诱导针对SARS-CoV-2的特异性免疫反应，达到抑制SARS-CoV-2的复制、抑制SARS-CoV-2的转播或阻止SARS-CoV-2普通株和变异株在其宿主体内定居的目的。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (10)

  1. 一种融合蛋白，其特征在于，所述融合蛋白包括严重急性呼吸综合征冠状病毒2抗原性蛋白和免疫球蛋白Fc片段。
  2. 根据权利要求1所述融合蛋白，其特征在于，所述严重急性呼吸综合征冠状病毒2抗原性蛋白包括S蛋白片段中RBD中和表位。
  3. 根据权利要求2所述融合蛋白，其特征在于，所述RBD中和表位的氨基酸序列如SEQ ID No.1所示。
  4. 根据权利1所述的融合蛋白，其特征在于，所述的免疫球蛋白Fc片段包括鼠源IgG、IgA、IgD、IgE、IgM中至少一种的免疫球蛋白Fc片段。
  5. 根据权利要求4所述的融合蛋白，其特征在于，所述的鼠源IgG选自鼠源IgG1、IgG2a、IgG2b、IgG3的至少一种。
  6. 根据权利要求1所述的融合蛋白，其特征在于，所述免疫球蛋白Fc片段的氨基酸序列如SEQ ID No.2所示。
  7. 根据权利要求1所述的融合蛋白，其特征在于，所述融合蛋白的氨基酸序列如SEQ ID No.3所示。
  8. 一种疫苗组合物，其特征在于，所述疫苗组合物包括权利要求1-7任一所述的融合蛋白，还包括免疫学和药学上接受的载体或佐剂。
  9. 根据权利要求8所述的疫苗组合物，其特征在于，所述佐剂包括铝佐剂、弗氏佐剂、单磷酰脂质A、RIBI佐剂系统、α-半乳糖神经酰胺类似物佐剂中的至少一种。
  10. 权利要求8-9任一所述的疫苗组合物在制备预防和/或治疗新型冠状病毒肺炎的药物中的应用。
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