WO2021196808A1 - Novel therapeutic vaccine against novel coronavirus, preparation method therefor, and use thereof - Google Patents

Novel therapeutic vaccine against novel coronavirus, preparation method therefor, and use thereof Download PDF

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WO2021196808A1
WO2021196808A1 PCT/CN2020/142585 CN2020142585W WO2021196808A1 WO 2021196808 A1 WO2021196808 A1 WO 2021196808A1 CN 2020142585 W CN2020142585 W CN 2020142585W WO 2021196808 A1 WO2021196808 A1 WO 2021196808A1
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protein
coronavirus
antigen
recombinant
virus
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谭瀛轩
裴建武
向道凤
谭相石
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杭州星鳌生物科技有限公司
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Definitions

  • the invention belongs to the technical field of biomedicine, and specifically relates to a novel anti-coronavirus therapeutic vaccine, a preparation method thereof, and application in the preparation of anticoronavirus drugs.
  • Coronavirus is a large family of viruses, known to cause more serious diseases such as colds, Middle East respiratory syndrome and severe acute respiratory syndrome.
  • the new coronavirus is a new strain of coronavirus that has never been found in the human body before. Common signs of people infected with coronavirus include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, lung and kidney failure, and even death. There is currently no specific treatment for the disease caused by the new coronavirus. Since the outbreak of the new coronary pneumonia, from medical staff, experts and scholar to the general public, one of the focuses of the most attention has been the possible effective treatments and drugs for the new coronary pneumonia.
  • coronavirus pneumonia COVID-19
  • no preventive drug with a clear curative effect has been found.
  • Clinical trials of a variety of potentially effective drugs are being carried out rapidly.
  • the new coronavirus has 85% homology with SARS and MERS, considering that different viruses may have a common target, therefore, in the absence of specific drugs, exploring new uses of old drugs has become a relatively fast strategy.
  • Natural bacteria or viral infections or their vaccines can cause a wide range of immunity. In addition to inducing humoral immunity, it also induces immunity to resident memory T cells (TRM cells) in the lungs. There must be a delicate balance between the safety and immunogenicity of "replicative" vaccines, and it is only suitable for certain populations. In contrast, "non-replicating" pneumovirus vaccines induce poor T cell immunity in the respiratory tract and require effective mucosal adjuvants to overcome the immune regulation mechanism of the respiratory mucosa. However, despite decades of research, there is still a lack of effective mucosal adjuvants.
  • Type I interferons are the main immune mediators of protective immunity against viral infections, and can be strongly induced by pneumonia virus infection of alveolar epithelial cells (AECs) and immune cells. Therefore, the stimulating factor of interferon gene (STING) in these two cell types may be activated by the immune response induced by virus infection or replication vaccine.
  • STING interferon gene
  • the lung epithelial cells form a strong barrier to prevent the entry of nanoparticles and hydrophilic molecules, the STING agonist is delivered to the cytosol of AEC without destroying the integrity of the lung surface active (PS) layer. China remains a huge challenge.
  • cGAMP is a cytoplasmic DNA sensor, which acts as a second messenger to stimulate the induction of INF-I through STING, mediate the activation of TBK1 and IRF-3, and then initiate the transcription of INF- ⁇ genes.
  • STING is a transmembrane protein of the endoplasmic reticulum, which has an ENPP1 hydrolase.
  • ENPP1 hydrolase has a wide substrate specificity, including ATP and NAD + .
  • 2'3'-cGAMP is also a substrate of ENPP1. How to increase the effective metabolic time of immune agonists and quickly reach lung cells is a difficult challenge for scientists.
  • the main role of vaccines is to prevent diseases.
  • the role of vaccines is to enhance the body’s ability to prevent and resist diseases, and to prevent diseases; for humans and animals suffering from certain diseases or diseases, it is therapeutic
  • the role of the vaccine is to induce the body to produce a response to specific pathogenic factors to achieve the goal of eliminating the focus and treating the disease or patient. Therefore, it is urgent to develop new anti-coronavirus therapeutic vaccines, which can prevent and/or treat the inflammation of the neocoronavirus by affecting the body's immune system.
  • the invention provides a novel anti-new coronavirus therapeutic vaccine and a preparation method thereof.
  • the anti-new coronavirus therapeutic vaccine can significantly induce the specific immune function against the new coronavirus, effectively suppress viral inflammation, stimulate mucosal immunity, regulate the body's immune homeostasis, and enhance the immune and antiviral function of the body.
  • a new type of anti-new coronavirus therapeutic vaccine is provided.
  • a virus-like particle vaccine composed of recombinant neocoronavirus antigen S protein-coupled liposomes and an immune agonist encapsulated in recombinant neocoronavirus antigen S protein-coupled liposomes; or
  • a virus-like particle vaccine consisting of a recombinant adenovirus vector of the new coronavirus antigen S protein gene and a transmembrane peptide coupled liposome encapsulating immunostimulant.
  • the transmembrane peptide coupled liposome encapsulating immunostimulant The agent is prepared by encapsulating the immune agonist in a transmembrane peptide-coupled liposome;
  • the immune agonist is an agonist of the innate immune pathway (cGAS-STING-cGAMP-IRF3 pathway) STING or its transition metal complex, and the agonist of STING is the cyclic dinucleotide 2'3'-cGAMP or its derivatives;
  • the recombinant new coronavirus antigen S protein is a domain derivative of the COVID-19 virus S protein or the COVID-19 virus S protein;
  • the transmembrane peptide is a membrane targeting peptide or a targeted membrane vesicle-associated protein
  • the recombinant adenovirus vector of the new coronavirus antigen S protein gene is: a recombinant adenovirus with the COVID-19 virus S protein gene or the COVID-19 virus S protein domain gene, and the E1 and E3 regions of the early expression gene sequence of the adenovirus are deleted. Carrier.
  • the domain derivatives of the S protein of the COVID-19 virus include but are not limited to RBD, RBD-SD1 or RBD-SD1SD2.
  • the membrane targeting peptide is a transmembrane peptide gH625 of herpes simplex virus glycoprotein, and the amino acid sequence is HGLASTLTRWAHYNALIRAFGGG, SEQ ID NO:1;
  • the nanobody targeting membrane vesicle-associated protein is anti-PV1Nb, and the amino acid sequence is QVQLQQSGAELVKPGASVKLSCKASGYTFTDYYMYWVKQPPGQGLELIGEINPTNGDVNFNEMFKSKATLTVDTSSRTAYMQLSSLTSEDSAVYYCTSIHYWGQGTLVTVSAG:SG2, SEQ ID NO.
  • the preparation method of the transition metal complex of the STING agonist is: the transition metal ion metal salt and the STING agonist are heated under reflux and stirred in a water/alcohol mixed solvent, left to stand overnight, filtered, and the product is purified by an ion exchange column. have to.
  • the preparation method of the above-mentioned novel anti-new coronavirus therapeutic vaccine includes the following steps:
  • the thiolated recombinant new coronavirus antigen S protein is chemically bonded to the liposome and encapsulates the immune agonist.
  • the preparation method of the above-mentioned novel anti-new coronavirus therapeutic vaccine includes the following steps:
  • the sulfhydryl transmembrane peptide is chemically bonded to the liposome to encapsulate the immune agonist to obtain the transmembrane peptide-coupled liposome encapsulated immune agonist;
  • transmembrane peptide-coupled liposome encapsulated immune agonist is mixed with the recombinant adenovirus vector of the new coronavirus antigen S protein gene.
  • coronavirus infection diseases include, but are not limited to, viral pneumonia, viral nephritis, viral encephalitis, viral enteritis, or viral hepatitis caused by human or animal infection with coronavirus.
  • the vaccine can be separately prepared into unit preparations of different specifications or prepared into a pharmaceutical preparation through a pharmaceutically acceptable carrier.
  • drugs for the prevention and/or treatment of coronavirus infection diseases include intravenous injection preparations, nasal drip preparations, intravenous drip preparations, intramuscular injection preparations, subcutaneous injection preparations or oral preparations; oral preparations include but are not limited to capsules, tablets or Granules.
  • the present invention comprehensively studies and optimizes the functions and advantages of natural immune agonists, transmembrane peptides, and liposomes, and optimizes the composition of a new type of complex, which can avoid the rapid degradation of immune agonists in the body and can quickly target lung immune cells.
  • Lung epithelial cells inhibit viral inflammation.
  • the research of the present invention shows that the novel anti-new coronavirus therapeutic vaccine has potential application prospects in the prevention and treatment of coronavirus drugs, and can be used to prevent and treat a variety of coronavirus infection diseases, including viral inflammations such as new coronavirus pneumonia.
  • Example 1 Preparation of a novel anti-coronavirus therapeutic vaccine
  • the immunoagonist cyclic dinucleotide 2'3'-cGAMP is synthesized by the cyclized cGMP-AMP dinucleotide synthetase under the activation conditions after binding DNA according to the literature method, and the purity is above 98%.
  • the cyclic dinucleotide 2'3'-cGAMP metal complex has been verified by metal content analysis and elemental analysis.
  • MncGAMP chemical formula MnC 20 H 22 N 10 O 13 P 2 , molecular weight 727, elemental analysis percentage (theoretical value) (%): C, 32.65 (33.01); H, 2.98 (3.03); N, 18.86 (19.26) ; Mn, 7.21 (7.56).
  • ZncGAMP chemical formula ZnC 20 H 22 N 10 O 13 P 2 , molecular weight 737, elemental analysis percentage (theoretical value) (%): C, 32.28 (32.56); H, 2.69 (2.98); N, 18.68 (18.99) ; Zn, 8.46 (8.82).
  • COVID-19 virus S protein Spike protein
  • RBD domain S protein 319-541 amino acid sequence domain
  • the COVID-19 virus S protein or its RBD-SD1 domain (the 319-591 amino acid sequence domain of the S protein) or the RBD-SD1SD2 domain (the 319-732 amino acid sequence domain of the S protein) was prepared according to the above method, using S The protein gene or RBD-SD1 domain gene or RBD-SD1SD2 domain gene replaces the RBD domain gene, and the expression and purification method is the same.
  • the transmembrane peptide gH625 contains 23 amino acid residues (H 2 N-HGLASTLTRWAHYNALIRAFGGG-CONH 2 ), with a molecular weight of 2461 Da, and was synthesized by a solid-phase biotechnology company.
  • Anti-PV1Nb gene expression vector plasmid targeting membrane vesicle-associated protein was synthesized and prepared by Shanghai Biogenomics Company.
  • the expression vector plasmid adopts pET-22b(+), carries Amp+ resistance, and the protein sequence end is labeled 6His-tag
  • protein purification method uses affinity column NiNTA purification, the purity is 98%.
  • the freeze-dried powder is stored in an ultra-low temperature refrigerator for later use.
  • the Ellman method was used to determine the sulfhydryl group on the RBD-SD1 protein to verify the successful sulfhydrylation of RBD-SD1.
  • the liposome materials including lecithin, cholesterol, 1,2-distearoyl-SN-glycerol-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000, mole ratio 57: 38:4:1 mixing), dissolved in methanol:chloroform (1:9(v/v)) solvent, spin-dried in a 40°C water bath to form a film; add 250mM(NH 4 ) 2 SO in a 65°C water bath 4 Hydrate into blank liposomes.
  • methanol:chloroform (1:9(v/v) methanol:chloroform (1:9(v/v)
  • the obtained complex has double-layer circular vesicles with good morphology.
  • the liposome diameter is about ⁇ 185nm, and the Zeta potential is ⁇ -23mV.
  • the immune agonist encapsulation rate is 75%, and it is stable under 4°C refrigeration conditions, and a 2.5% trehalose solution is used to make a freeze-dried powder for storage under refrigeration.
  • the above method is suitable for the replacement of various combinations of other immune agonists and other recombinant new coronavirus antigen S proteins.
  • the Ellman method was used to determine the sulfhydryl group on the transmembrane peptide to verify the successful sulfhydrylation of the transmembrane peptide.
  • Dissolve liposome materials including lecithin, cholesterol, 1,2-distearoyl-SN-glycerol-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000
  • methanol/ In a mixed solvent of chloroform vacuum rotary evaporation in a water bath to dry to form a film, and then add (NH 4 ) 2 SO 4 for hydration to make a blank liposome.
  • a liposome extruder a uniform single-compartment blank liposome was prepared by extruding through a 200nm polycarbonate microporous filter membrane.
  • the obtained complex (gH625 coupled liposome encapsulating MncGAMP, immunoagonist MncGAMP, transmembrane peptide gH625) was detected by TEM electron microscope.
  • the double-layer circular vesicles are in good shape and the liposome diameter is about ⁇ 175nm, Zeta potential ⁇ -22mV.
  • the immune agonist encapsulation rate is 78%, and it is stable under 4°C refrigeration conditions.
  • a 3% trehalose solution is used to make a freeze-dried powder for storage under refrigeration.
  • the recombinant adenovirus vector of the new coronavirus antigen S protein gene is completed by the outsourcing service of a biomedical technology company.
  • the recombinant adenovirus vector is constructed with the E1 and E3 regions of the early expression gene sequence of the adenovirus deleted:
  • adenovirus shuttle plasmid recombinant adenovirus vector plasmid with deletion of the early expression gene sequence E1 and E3 regions of adenovirus
  • the confirmed correct recombinant adenovirus shuttle plasmid and the backbone plasmid were co-transfected, packaged in 293A cells, and then amplified by adenovirus and purified by CsCl.
  • the packaged recombinant RBD-SD1 adenovirus vector is subjected to quality inspection. Quality inspection includes PCR and WB on the final product virus gene to confirm the existence of the target gene.
  • the gH625 coupled liposome encapsulated MncGAMP and the recombinant RBD-SD1 adenovirus vector were mixed to form a composition (200 ⁇ g MncGAMP: recombinant RBD-SD1 adenovirus vector 1 ⁇ 10 8 adenovirus particles). Finally, a 3% trehalose solution will be used to make this mixture into a freeze-dried powder for storage under refrigeration.
  • the preparation method of the new vaccine III is the same as the preparation of the new vaccine I (VFI), except that MncGAMP is replaced by ZncGAMP, and the other method steps are the same.
  • the obtained VFIII has double-layer circular vesicles with good morphology.
  • the liposome diameter is about ⁇ 187nm, and the Zeta potential is ⁇ -24mV.
  • the encapsulation rate of the immune agonist is 76%, and it is stable under refrigerated conditions at 4°C.
  • a 2.5% trehalose solution is used to make a freeze-dried powder for storage under refrigeration.
  • VFIV new vaccine IV
  • VFI new vaccine I
  • cGAMP is used to replace MncGAMP
  • antigen S protein domain RBD is replaced with antigen S protein domain RBD-SD1.
  • the other method steps are the same.
  • the obtained VFIV was detected by TEM electron microscope.
  • the double-layered circular vesicles were in good shape.
  • the liposome diameter was about ⁇ 184nm, and the Zeta potential was ⁇ -23mV.
  • the immune agonist encapsulation rate is 73%, and it is stable under 4°C refrigeration conditions. It is made into a lyophilized powder with a 2.5% trehalose solution and stored under refrigeration.
  • New vaccine I [antigen S protein domain RBD-SD1 coupled with liposome encapsulating MncGAMP]
  • VFII New vaccine II
  • New vaccine III [Antigen S protein domain RBD-SD1 coupled with liposome encapsulating ZncGAMP]
  • New vaccine III [antigen S protein domain RBD coupled liposome encapsulating cGAMP]
  • Immune agonist complex I [gH625 coupled liposome encapsulating cGAMP] (prepared according to step (4))
  • Immune agonist complex II [gH625 coupled liposome encapsulating MncGAMP] (prepared according to step (4))
  • mice C57BL/6 mice, male, weight 20-22g, 6-8 weeks old, purchased from Shanghai Slack Experimental Animal Co., Ltd. [Experimental animal quality certificate number: SCXK (Shanghai) 2007-0005]. All mice were free to forage and drink, and were raised at room temperature (23 ⁇ 2)°C. Feed and water are autoclaved, and all experimental feeding processes are SPF grade.
  • mice each group of 10 mice, a total of 9 groups, respectively, A: VFI; B: VFII; C: VFIII; D: VFIV; E: FI; F: FII; G: RBD-SD1 ; H: RBD; I: PBS blank.
  • Administration method nasal drip.
  • New vaccine I (10mg/kg MncGAMP, 100 ⁇ g RBD-SD1)
  • VFII New vaccines II (10mg / kg MncGAMP + recombinant adenoviral vector RBD-SD1 108)
  • New vaccine III (10mg/kg ZncGAMP, 100 ⁇ g RBD-SD1)
  • VFIV New Vaccine IV
  • Antigen S protein domain RBD (100 ⁇ g)
  • mice were put under anesthesia, the mice were fixed in a dorsal position, and the drug solution suspension of each group was slowly dripped through the inner wall of the mouse nostrils, and the drip volume was 60 ⁇ L (30 ⁇ L for each nostril).
  • the mouse was gently taken off the workbench, and the head and chest were raised slightly with folded paper towels to ensure smooth breathing of the mouse.
  • mice wake up they are put back into the squirrel cage. They were administered once on the 1, 7, and 14 days respectively, and the mouse lung lavage fluid and blood samples were obtained on the 21st day.
  • the ELISA method was used to determine the titers of immune agonist complexes and vaccine complexes induced to produce antibodies.
  • the experimental results are shown in Table 1.
  • the test results show that new vaccines (VFI, VFII, VFIII, VFIV) and immunoagonist liposome complexes (FI, FII) can significantly enhance or induce immune responses.
  • the new vaccines (VFI, VFII, VFIII, VFIV)
  • the effect is significantly higher than the immune agonist complex (FI, FII) and the single recombinant S protein domain RBD-SD1/RBD.
  • mice See Example 2 for the breeding and administration of mice.
  • Isotype control flow cytometry antibodies were purchased from eBiosciences, antibody magnets were purchased from Militeny Biotech, and flow cytometry was purchased from BD. After 21 days of immunization with three administrations, the spleen and lung tissues of the mice were taken, ground and crushed, and the cells were removed through 40 micron holes, and centrifuged at 1000 rpm for 10 minutes.
  • Example 4 The inhibitory effect of a novel anti-new coronavirus therapeutic vaccine on mouse coronavirus pneumonia
  • mice C57BL/6 mice, male, weighing 20-22g, 6-8 weeks old, SPF grade, from American Animals Inc., all mice foraging and drinking freely, at room temperature (23 ⁇ 2)°C Feeding under. Feed and water are autoclaved, and all experimental feeding processes are SPF grade.
  • Virus strain A virus strain suitable for laboratory use was purchased from the American ATCC Company: Coronavirus (ATCC VR-841). In this study, the virus experiment operation was commissioned by the American Animals Inc. Virus Laboratory.
  • Intranasal drip Keep the mouse in a sufficiently deep anesthesia state, fix the mouse in a dorsal position, and slowly drip the VR-841 virus suspension through the inner wall of the mouse nostril to ensure the largest lung Infection efficiency, the instillation volume is 60 ⁇ L (30 ⁇ L per nostril).
  • the mouse was gently taken off the workbench, and the head and chest were raised slightly with folded paper towels to ensure smooth breathing of the mouse. After the mice wake up, they are put back into the squirrel cage.
  • the mice in groups C, D, E, F, G, H, I, and J were administered once on the 2, 8 and 15 days respectively, and the drug solution suspension of each group was slowly passed through the inner wall of the mouse nostril. Instillation, the mouse lung lavage fluid and blood samples were obtained on the 21st day.
  • the immune agonist complex and vaccine complex induced protective cellular immunity titers were measured by ELISA.
  • Administration method nasal drip;
  • New vaccine I (10mg/kg MncGAMP, 100 ⁇ g RBD-SD1)
  • VFII New vaccines II (10mg / kg MncGAMP + recombinant adenoviral vector RBD-SD1 108)
  • New vaccine III (10mg/kg ZncGAMP, 100 ⁇ g RBD-SD1)
  • VFIV New Vaccine IV
  • Antigen S protein domain RBD (100 ⁇ g)
  • Method for obtaining mouse alveolar lavage fluid take an equal volume of PBS and inject it along the mouse trachea and aspirate it, repeat several times to obtain alveolar lavage fluid.
  • the collected serum is stored at -80°C.
  • ELISA method was used to detect the concentration of TNF-alpha and IL-1beta according to the kit instructions. After terminating the reaction, put the microtiter plate into the microplate reader slot, select the 450nm wavelength for detection, determine the standard and blank control area, detect the corresponding optical density value, and then draw the standard curve and calculate the corresponding concentration.
  • ICR mice were intraperitoneally injected with 1g/kg of new anti-coronavirus therapeutic vaccines (VFI, VFII, VFIII, VFIV) (prepared in PBS buffer) according to their body weight, and the toxicity and death of the mice within 14 days after administration were observed. It was found that after intraperitoneal injection of the mice, the mice moved normally. Within 14 days after the administration, the mice did not die. On the 15th day, all the mice were sacrificed, dissected, and visually inspected the various organs, and no obvious lesions were seen.
  • VFI new anti-coronavirus therapeutic vaccines

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Abstract

Disclosed is a novel therapeutic vaccine against novel coronavirus, comprising: (1) a virus-like particle vaccine composed of a recombinant novel coronavirus antigen S protein-conjugated liposome and an immunostimulant, wherein the immunostimulant is encapsulated in the recombinant novel coronavirus antigen S protein-conjugated liposome; or (2) a virus-like particle vaccine composed of novel coronavirus antigen S protein, a genetically recombinant adenoviral vector, and an immunostimulant encapsulated by a transmembrane peptide-conjugated liposome, wherein the immunostimulant encapsulated by a transmembrane peptide-conjugated liposome is prepared by encapsulating the immunostimulant in the transmembrane peptide-conjugated liposome. Further disclosed are a preparation method for the novel therapeutic vaccine and a use of the novel therapeutic vaccine in the preparation of a drug against coronavirus. The vaccine against novel coronavirus can significantly induce humoral immunity and protective cellular immunity, trigger mucosal immunity, regulate the stable state of body immunity, enhance immune and anti-viral functions in the body, and significantly induce an immune function specific to novel coronavirus, thereby effectively inhibiting coronavirus pneumonia.

Description

一种新型抗新冠病毒治疗性疫苗及其制备方法和应用A novel anti-new coronavirus therapeutic vaccine and its preparation method and application 技术领域Technical field
本发明属于生物医药技术领域，具体涉及一种新型抗新型冠状病毒治疗性疫苗及其制备方法和在制备抗冠状病毒药物中的应用。The invention belongs to the technical field of biomedicine, and specifically relates to a novel anti-coronavirus therapeutic vaccine, a preparation method thereof, and application in the preparation of anticoronavirus drugs.
背景技术Background technique
冠状病毒是一个大型病毒家族，已知可引起感冒、中东呼吸综合征和严重急性呼吸综合征等较严重疾病。新型冠状病毒是以前从未在人体中发现的冠状病毒新毒株。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。在较严重病例中，感染可导致肺炎、严重急性呼吸综合征、肺肾衰竭，甚至死亡。目前对于新型冠状病毒所致疾病并没有特异治疗方法。新冠肺炎疫情发生以来，从医护人员、专家学者到普罗大众，最关注的焦点之一，就是可能对新冠肺炎有效的疗法与药物。目前，尚无新型冠状病毒肺炎(COVID-19)的特效治疗药物，也未发现明确疗效的预防性药物，多种可能有效药物的临床试验正在迅速开展。新冠病毒与SARS和MERS同源性达到85％，考虑不同病毒可能有共同的靶点，因此，在没有特效药的情况下，探索老药新用成为相对较快速的策略。Coronavirus is a large family of viruses, known to cause more serious diseases such as colds, Middle East respiratory syndrome and severe acute respiratory syndrome. The new coronavirus is a new strain of coronavirus that has never been found in the human body before. Common signs of people infected with coronavirus include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, lung and kidney failure, and even death. There is currently no specific treatment for the disease caused by the new coronavirus. Since the outbreak of the new coronary pneumonia, from medical staff, experts and scholars to the general public, one of the focuses of the most attention has been the possible effective treatments and drugs for the new coronary pneumonia. At present, there is no specific treatment drug for the new type of coronavirus pneumonia (COVID-19), and no preventive drug with a clear curative effect has been found. Clinical trials of a variety of potentially effective drugs are being carried out rapidly. The new coronavirus has 85% homology with SARS and MERS, considering that different viruses may have a common target, therefore, in the absence of specific drugs, exploring new uses of old drugs has become a relatively fast strategy.
抗新冠病毒的疫苗研发已经成为世界范围热点领域，目前研发的疫苗种类有多种，包括重组DNA疫苗、RNA疫苗、蛋白疫苗等。中国、美国、德国领先开展了疫苗临床实验，中国最先在临床应用的新冠病毒疫苗是重组DNA疫苗，美国临床研究的是RNA疫苗，全部都是应用于健康人的预防性抗病毒疫苗。上述疫苗仍处于研发、审批程序中。The research and development of vaccines against the new coronavirus has become a worldwide hot spot. There are many types of vaccines currently being developed, including recombinant DNA vaccines, RNA vaccines, and protein vaccines. China, the United States, and Germany have pioneered clinical trials of vaccines. China's first clinical application of the new coronavirus vaccine is a recombinant DNA vaccine, and the United States clinical research is an RNA vaccine, all of which are preventive antiviral vaccines for healthy people. The above-mentioned vaccines are still in the development and approval process.
天然病菌或病毒感染或其疫苗都可引起广泛的免疫，除了诱发体液免疫外，还会诱导肺部驻留记忆T细胞(TRM细胞)免疫。“复制性”疫苗的安全性和免疫原性之间必须达到微妙的平衡，并且其仅适用于某些人群。相比之下，“非复制性”肺炎病毒疫苗在呼吸道中诱导较差的T细胞免疫，并且需要有效的粘膜佐剂来克服呼吸道粘膜的免疫调节机制。然而，尽管进行了数十年的研究，仍然缺乏有效的粘膜佐剂。I型干扰素(IFN-Is)是针对病毒感染的保护性免疫的主要免疫介质，并且可以通过肺泡上皮细胞(AECs)以及免疫细胞的肺炎病毒感染而强烈诱导。因此，这两种细胞类型中的干扰素基因刺激因子(STING)可能会由病毒感染或复制疫苗诱导的免疫应答而被激活。然而，由于肺上皮细胞外形成了强大的屏障来阻 止纳米颗粒和亲水性分子进入，在不破坏肺表面活性(PS)层完整性的情况下，将STING激动剂递送到AEC的胞质溶胶中仍然是一个巨大的挑战。Natural bacteria or viral infections or their vaccines can cause a wide range of immunity. In addition to inducing humoral immunity, it also induces immunity to resident memory T cells (TRM cells) in the lungs. There must be a delicate balance between the safety and immunogenicity of "replicative" vaccines, and it is only suitable for certain populations. In contrast, "non-replicating" pneumovirus vaccines induce poor T cell immunity in the respiratory tract and require effective mucosal adjuvants to overcome the immune regulation mechanism of the respiratory mucosa. However, despite decades of research, there is still a lack of effective mucosal adjuvants. Type I interferons (IFN-Is) are the main immune mediators of protective immunity against viral infections, and can be strongly induced by pneumonia virus infection of alveolar epithelial cells (AECs) and immune cells. Therefore, the stimulating factor of interferon gene (STING) in these two cell types may be activated by the immune response induced by virus infection or replication vaccine. However, because the lung epithelial cells form a strong barrier to prevent the entry of nanoparticles and hydrophilic molecules, the STING agonist is delivered to the cytosol of AEC without destroying the integrity of the lung surface active (PS) layer. China remains a huge challenge.
在感染的哺乳动物细胞中微生物和病毒DNA能通过刺激干扰素分泌诱导内源强有力的免疫应答。内质网(ER)受体蛋白(STING)对胞质DNA的免疫应答是必需的因素。最近的研究表明，环化cGMP-AMP二核苷酸合成酶(cGAS)在结合DNA后的活化条件下，内源性地催化cGAMP的合成。cGAMP是一种胞质DNA传感器，它作为第二信使通过STING刺激INF-I的感应，介导TBK1和IRF-3的活化，进而启动INF-β基因的转录。STING是内质网的跨膜蛋白，内质网上具有一种ENPP1的水解酶。ENPP1水解酶具有相当宽的底物特异性，包括ATP和NAD +，实验显示2'3'-cGAMP也是ENPP1的底物。如何增加免疫激动剂的有效代谢时间，快速抵达肺部细胞是对科学家的艰巨挑战。 Microbial and viral DNA in infected mammalian cells can induce a strong endogenous immune response by stimulating the secretion of interferon. The endoplasmic reticulum (ER) receptor protein (STING) is an essential factor for the immune response to cytoplasmic DNA. Recent studies have shown that circularized cGMP-AMP dinucleotide synthase (cGAS) endogenously catalyzes the synthesis of cGAMP under the activation conditions after binding to DNA. cGAMP is a cytoplasmic DNA sensor, which acts as a second messenger to stimulate the induction of INF-I through STING, mediate the activation of TBK1 and IRF-3, and then initiate the transcription of INF-β genes. STING is a transmembrane protein of the endoplasmic reticulum, which has an ENPP1 hydrolase. ENPP1 hydrolase has a wide substrate specificity, including ATP and NAD + . Experiments show that 2'3'-cGAMP is also a substrate of ENPP1. How to increase the effective metabolic time of immune agonists and quickly reach lung cells is a difficult challenge for scientists.
疫苗的主要作用在于预防疾病。针对正常的人体和动物体而言，疫苗的作用在于增强机体的防病和抗病能力，起到预防疾病的目的；对于患有某种疾病或者病患的人体和动物体而言，治疗性疫苗的作用要诱导机体产生针对特定致病因素的反应，达到消除病灶，治疗疾病或者病患的目的。因此，亟待开发新型抗新冠病毒治疗性疫苗，通过影响机体的免疫系统达到预防和/或治疗新冠病毒炎症的目的。The main role of vaccines is to prevent diseases. For normal humans and animals, the role of vaccines is to enhance the body’s ability to prevent and resist diseases, and to prevent diseases; for humans and animals suffering from certain diseases or diseases, it is therapeutic The role of the vaccine is to induce the body to produce a response to specific pathogenic factors to achieve the goal of eliminating the focus and treating the disease or patient. Therefore, it is urgent to develop new anti-coronavirus therapeutic vaccines, which can prevent and/or treat the inflammation of the neocoronavirus by affecting the body's immune system.
发明内容Summary of the invention
本发明提供了一种新型抗新冠病毒治疗性疫苗及其制备方法。该抗新冠病毒治疗性疫苗可显著诱发抗新冠病毒的特异免疫功能，有效抑制病毒性炎症，激发黏膜免疫、调节机体免疫稳态，增强体内免疫抗病毒功能。The invention provides a novel anti-new coronavirus therapeutic vaccine and a preparation method thereof. The anti-new coronavirus therapeutic vaccine can significantly induce the specific immune function against the new coronavirus, effectively suppress viral inflammation, stimulate mucosal immunity, regulate the body's immune homeostasis, and enhance the immune and antiviral function of the body.
一种新型抗新冠病毒治疗性疫苗，所述疫苗为A new type of anti-new coronavirus therapeutic vaccine, the vaccine is
(1).由重组新冠病毒抗原S蛋白偶联脂质体与免疫激动剂构成的仿病毒颗粒疫苗，所述免疫激动剂包封于重组新冠病毒抗原S蛋白偶联脂质体中；或(1) A virus-like particle vaccine composed of recombinant neocoronavirus antigen S protein-coupled liposomes and an immune agonist encapsulated in recombinant neocoronavirus antigen S protein-coupled liposomes; or
(2).由新冠病毒抗原S蛋白基因重组腺病毒载体与跨膜肽偶联脂质体包封免疫激动剂构成的仿病毒颗粒疫苗，所述跨膜肽偶联脂质体包封免疫激动剂为将所述免疫激动剂包封于跨膜肽偶联脂质体中制得；(2). A virus-like particle vaccine consisting of a recombinant adenovirus vector of the new coronavirus antigen S protein gene and a transmembrane peptide coupled liposome encapsulating immunostimulant. The transmembrane peptide coupled liposome encapsulating immunostimulant The agent is prepared by encapsulating the immune agonist in a transmembrane peptide-coupled liposome;
免疫激动剂为天然免疫通路(cGAS-STING-cGAMP-IRF3通路)STING的激动剂或 其过渡金属配合物，STING的激动剂为环二核苷酸2’3’-cGAMP或其衍生物；The immune agonist is an agonist of the innate immune pathway (cGAS-STING-cGAMP-IRF3 pathway) STING or its transition metal complex, and the agonist of STING is the cyclic dinucleotide 2'3'-cGAMP or its derivatives;
重组新冠病毒抗原S蛋白为COVID-19病毒S蛋白或COVID-19病毒S蛋白的结构域衍生物；The recombinant new coronavirus antigen S protein is a domain derivative of the COVID-19 virus S protein or the COVID-19 virus S protein;
跨膜肽为膜靶向肽或靶向膜囊泡关联蛋白；The transmembrane peptide is a membrane targeting peptide or a targeted membrane vesicle-associated protein;
新冠病毒抗原S蛋白基因重组腺病毒载体为：重组有COVID-19病毒S蛋白基因或COVID-19病毒S蛋白的结构域基因，并且缺失腺病毒的早期表达基因序列E1和E3区的重组腺病毒载体。The recombinant adenovirus vector of the new coronavirus antigen S protein gene is: a recombinant adenovirus with the COVID-19 virus S protein gene or the COVID-19 virus S protein domain gene, and the E1 and E3 regions of the early expression gene sequence of the adenovirus are deleted. Carrier.
优选地，COVID-19病毒S蛋白的结构域衍生物包括但不限于RBD、RBD-SD1或RBD-SD1SD2。Preferably, the domain derivatives of the S protein of the COVID-19 virus include but are not limited to RBD, RBD-SD1 or RBD-SD1SD2.
优选地，膜靶向肽为单纯疱疹病毒糖蛋白的一段跨膜肽gH625，氨基酸序列为HGLASTLTRWAHYNALIRAFGGG，SEQ ID NO:1；Preferably, the membrane targeting peptide is a transmembrane peptide gH625 of herpes simplex virus glycoprotein, and the amino acid sequence is HGLASTLTRWAHYNALIRAFGGG, SEQ ID NO:1;
靶向膜囊泡关联蛋白的纳米抗体为anti-PV1Nb,氨基酸序列为QVQLQQSGAELVKPGASVKLSCKASGYTFTDYYMYWVKQPPGQGLELIGEINPTNGDVNFNEMFKSKATLTVDTSSRTAYMQLSSLTSEDSAVYYCTSIHYWGQGTLVTVSAGSG，SEQ ID NO:2。The nanobody targeting membrane vesicle-associated protein is anti-PV1Nb, and the amino acid sequence is QVQLQQSGAELVKPGASVKLSCKASGYTFTDYYMYWVKQPPGQGLELIGEINPTNGDVNFNEMFKSKATLTVDTSSRTAYMQLSSLTSEDSAVYYCTSIHYWGQGTLVTVSAG:SG2, SEQ ID NO.
优选地，STING的激动剂的过渡金属配合物制备方法为：过渡金属离子金属盐与STING的激动剂在水/醇混合溶剂中加热回流搅拌，静置过夜，过滤，产物经离子交换柱纯化制得。Preferably, the preparation method of the transition metal complex of the STING agonist is: the transition metal ion metal salt and the STING agonist are heated under reflux and stirred in a water/alcohol mixed solvent, left to stand overnight, filtered, and the product is purified by an ion exchange column. have to.
上述新型抗新冠病毒治疗性疫苗的制备方法，包括如下步骤：The preparation method of the above-mentioned novel anti-new coronavirus therapeutic vaccine includes the following steps:
(1)对重组新冠病毒抗原S蛋白进行巯基化；(1) Sulfhydrylation of recombinant new coronavirus antigen S protein;
(2)巯基化重组新冠病毒抗原S蛋白与脂质体化学键融合，并且包封免疫激动剂。(2) The thiolated recombinant new coronavirus antigen S protein is chemically bonded to the liposome and encapsulates the immune agonist.
上述新型抗新冠病毒治疗性疫苗的制备方法，包括如下步骤：The preparation method of the above-mentioned novel anti-new coronavirus therapeutic vaccine includes the following steps:
(1)对膜靶向肽或靶向膜囊泡关联蛋白的纳米抗体进行巯基化，得到巯基化跨膜肽；(1) Sulfhydrylation of membrane targeting peptides or nanobodies targeting membrane vesicle-associated proteins to obtain thiolated transmembrane peptides;
(2)巯基化跨膜肽与脂质体化学键融合，包封免疫激动剂，得到跨膜肽偶联脂质体包封免疫激动剂；(2) The sulfhydryl transmembrane peptide is chemically bonded to the liposome to encapsulate the immune agonist to obtain the transmembrane peptide-coupled liposome encapsulated immune agonist;
(3)跨膜肽偶联脂质体包封免疫激动剂与新冠病毒抗原S蛋白基因重组腺病毒载 体混合。(3) The transmembrane peptide-coupled liposome encapsulated immune agonist is mixed with the recombinant adenovirus vector of the new coronavirus antigen S protein gene.
上述新型抗新冠病毒治疗性疫苗在制备预防和/或治疗冠状病毒感染疾病药物中的应用。The application of the above-mentioned novel anti-new coronavirus therapeutic vaccine in the preparation of drugs for the prevention and/or treatment of coronavirus infections.
优选地，冠状病毒感染疾病包括但不限于人或动物感染冠状病毒引起的病毒性肺炎、病毒性肾炎、病毒性脑炎、病毒性肠炎或病毒性肝炎。Preferably, coronavirus infection diseases include, but are not limited to, viral pneumonia, viral nephritis, viral encephalitis, viral enteritis, or viral hepatitis caused by human or animal infection with coronavirus.
优选地，疫苗可单独制备成不同规格的单位制剂或通过药学上可接受的载体制备成药物制剂。Preferably, the vaccine can be separately prepared into unit preparations of different specifications or prepared into a pharmaceutical preparation through a pharmaceutically acceptable carrier.
优选地，预防和/或治疗冠状病毒感染疾病药物包括静脉注射制剂、鼻腔滴注制剂、静脉滴注制剂、肌肉注射制剂、皮下注射制剂或口服制剂；口服制剂包括但不限于胶囊、片剂或颗粒剂。Preferably, drugs for the prevention and/or treatment of coronavirus infection diseases include intravenous injection preparations, nasal drip preparations, intravenous drip preparations, intramuscular injection preparations, subcutaneous injection preparations or oral preparations; oral preparations include but are not limited to capsules, tablets or Granules.
本发明综合研究优化天然免疫激动剂、跨膜肽、脂质体的作用和优点，优化组成一种新型复合物，其能避免免疫激动剂体内过快降解，能快速靶向肺部免疫细胞，肺部上皮细胞，抑制病毒性炎症。The present invention comprehensively studies and optimizes the functions and advantages of natural immune agonists, transmembrane peptides, and liposomes, and optimizes the composition of a new type of complex, which can avoid the rapid degradation of immune agonists in the body and can quickly target lung immune cells. Lung epithelial cells, inhibit viral inflammation.
本发明研究表明，新型抗新冠病毒治疗性疫苗在预防和治疗冠状病毒药物中有潜在应用前景，可用于预防和治疗多种冠状病毒感染疾病包括新冠病毒性肺炎等病毒性炎症。The research of the present invention shows that the novel anti-new coronavirus therapeutic vaccine has potential application prospects in the prevention and treatment of coronavirus drugs, and can be used to prevent and treat a variety of coronavirus infection diseases, including viral inflammations such as new coronavirus pneumonia.
具体实施方式Detailed ways
下面通过实施例具体说明本发明的内容。在本发明中，以下所述的实施例是为了更好地阐述本发明，并不是用来限制本发明的范围。The content of the present invention will be described in detail through the following examples. In the present invention, the following embodiments are used to better illustrate the present invention, and are not used to limit the scope of the present invention.
实施例1：新型抗新冠病毒治疗性疫苗的制备Example 1: Preparation of a novel anti-coronavirus therapeutic vaccine
(1)免疫激动剂的制备(1) Preparation of immune agonists
免疫激动剂环二核苷酸2’3’-cGAMP，按文献方法在结合DNA后的活化条件下，由环化cGMP-AMP二核苷酸合成酶催化合成，纯度在98％以上。The immunoagonist cyclic dinucleotide 2'3'-cGAMP is synthesized by the cyclized cGMP-AMP dinucleotide synthetase under the activation conditions after binding DNA according to the literature method, and the purity is above 98%.
环二核苷酸2’3’-cGAMP金属配合物([M(cGAMP)],M＝Zn,Mn,等过渡金属离子)由过渡金属离子金属盐(MnCl 2·4H 2O/或ZnCl 2,1mmol)与环二核苷酸2’3’-cGAMP(1mmol)在水/醇混合溶剂中加热回流搅拌反应6小时条件下生成，静置过夜，过滤，然后产物经离子交换柱纯化，得到纯的免疫激动剂金属配合物。环二核苷酸2’3’-cGAMP金属配合物经过金 属含量分析和元素分析验证。 Cyclic dinucleotide 2'3'-cGAMP metal complex ([M(cGAMP)], M=Zn, Mn, and other transition metal ions) is composed of transition metal ion metal salt (MnCl 2 ·4H 2 O/or ZnCl 2 ,1mmol) and cyclic dinucleotide 2'3'-cGAMP (1mmol) in a water/alcohol mixed solvent, heated under reflux and stirred for 6 hours to generate, let stand overnight, filter, and then the product is purified by ion exchange column to obtain Pure immune agonist metal complex. The cyclic dinucleotide 2'3'-cGAMP metal complex has been verified by metal content analysis and elemental analysis.
MncGAMP：化学式MnC 20H 22N 10O 13P 2,分子量727，元素分析百分含量(理论值)(％)：C,32.65(33.01)；H,2.98(3.03)；N,18.86(19.26)；Mn,7.21(7.56)。 MncGAMP: chemical formula MnC 20 H 22 N 10 O 13 P 2 , molecular weight 727, elemental analysis percentage (theoretical value) (%): C, 32.65 (33.01); H, 2.98 (3.03); N, 18.86 (19.26) ; Mn, 7.21 (7.56).
ZncGAMP：化学式ZnC 20H 22N 10O 13P 2，分子量737，元素分析百分含量(理论值)(％)：C,32.28(32.56)；H,2.69(2.98)；N,18.68(18.99)；Zn,8.46(8.82)。 ZncGAMP: chemical formula ZnC 20 H 22 N 10 O 13 P 2 , molecular weight 737, elemental analysis percentage (theoretical value) (%): C, 32.28 (32.56); H, 2.69 (2.98); N, 18.68 (18.99) ; Zn, 8.46 (8.82).
(2)重组新冠病毒抗原S蛋白(或膜靶向肽，或靶向膜囊泡关联蛋白纳米抗体)的制备(2) Preparation of recombinant new coronavirus antigen S protein (or membrane targeting peptide, or targeted membrane vesicle-associated protein nanobody)
COVID-19病毒S蛋白(Spike protein，刺突蛋白)RBD结构域(S蛋白第319-541氨基酸序列结构域)按文献(Jun Lan et al.,Crystal structure of the 2019-nCoV spike receptor-binding domain bound with the ACE2 receptor,BioRxiu,doi: https://doi.org/10.1101/2020.02.19.956235) 方法制备。 COVID-19 virus S protein (Spike protein) RBD domain (S protein 319-541 amino acid sequence domain) according to the literature (Jun Lan et al., Crystal structure of the 2019-nCoV spike receptor-binding domain) bound with the ACE2 receptor, BioRxiu, doi: https://doi.org/10.1101/2020.02.19.956235) method.
COVID-19病毒S蛋白或其RBD－SD1结构域(S蛋白第319-591氨基酸序列结构域)或RBD-SD1SD2结构域(S蛋白第319-732氨基酸序列结构域)按以上方法制备，用S蛋白基因或RBD－SD1结构域基因或RBD-SD1SD2结构域基因替换RBD结构域基因，表达纯化方法相同。The COVID-19 virus S protein or its RBD-SD1 domain (the 319-591 amino acid sequence domain of the S protein) or the RBD-SD1SD2 domain (the 319-732 amino acid sequence domain of the S protein) was prepared according to the above method, using S The protein gene or RBD-SD1 domain gene or RBD-SD1SD2 domain gene replaces the RBD domain gene, and the expression and purification method is the same.
跨膜肽gH625含有23个氨基酸残基(H 2N-HGLASTLTRWAHYNALIRAFGGG-CONH 2)，分子量是2461Da，由生物技术公司固相合成。 The transmembrane peptide gH625 contains 23 amino acid residues (H 2 N-HGLASTLTRWAHYNALIRAFGGG-CONH 2 ), with a molecular weight of 2461 Da, and was synthesized by a solid-phase biotechnology company.
靶向膜囊泡关联蛋白的纳米抗体(Anti-PV1Nb)基因表达载体质粒由上海生物基因公司合成制备，表达载体质粒采用pET-22b(+)，携带Amp+抗性，蛋白序列末端标记6His-tag帮助纯化，用大肠杆菌高效表达体系，蛋白纯化方法用亲和柱NiNTA纯化，纯度达98％。冻干粉于超低温冰箱保存备用。Anti-PV1Nb gene expression vector plasmid targeting membrane vesicle-associated protein was synthesized and prepared by Shanghai Biogenomics Company. The expression vector plasmid adopts pET-22b(+), carries Amp+ resistance, and the protein sequence end is labeled 6His-tag To help with purification, use E. coli high-efficiency expression system, protein purification method uses affinity column NiNTA purification, the purity is 98%. The freeze-dried powder is stored in an ultra-low temperature refrigerator for later use.
(3)新型疫苗I(VFI)的制备(3) Preparation of new vaccine I (VFI)
首先，对COVID-19病毒S蛋白RBD-SD1结构域进行末端巯基化(加入EDTA溶液使EDTA终浓度为5mM，搅动下滴加巯基化试剂(Traut’s reagent，购于Sigma公司)，水浴中搅动后，避光孵育1小时，用脱盐柱除去过量巯基化试剂)。First, perform terminal sulfhydrylation on the RBD-SD1 domain of COVID-19 virus S protein (add EDTA solution to make the final concentration of EDTA 5mM, add sulfhydryl reagent (Traut's reagent, purchased from Sigma) under agitation, after stirring in a water bath , Incubate for 1 hour in the dark, and use a desalting column to remove excess sulfhydryl reagent).
用Ellman方法测定RBD-SD1蛋白上的巯基，验证RBD-SD1巯基化成功。The Ellman method was used to determine the sulfhydryl group on the RBD-SD1 protein to verify the successful sulfhydrylation of RBD-SD1.
将脂质体材料(包括卵磷脂、胆固醇、1,2-二硬脂酰-SN-甘油-3-磷酰乙醇胺-N-马来酰亚胺-聚乙二醇2000，按摩尔比例57:38:4:1混合)，溶解于甲醇：氯仿(1:9(v/v))溶剂中，在40℃水浴中真空旋干成膜；在65℃水浴中加入250mM(NH 4) 2SO 4水化成空白脂质体。用脂 质体挤出器，经过200nm聚碳酸酯微孔滤膜挤出制备成均匀单室空白脂质体。 The liposome materials (including lecithin, cholesterol, 1,2-distearoyl-SN-glycerol-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000, mole ratio 57: 38:4:1 mixing), dissolved in methanol:chloroform (1:9(v/v)) solvent, spin-dried in a 40℃ water bath to form a film; add 250mM(NH 4 ) 2 SO in a 65℃ water bath 4 Hydrate into blank liposomes. Using a liposome extruder, a uniform single-compartment blank liposome was prepared by extruding through a 200nm polycarbonate microporous filter membrane.
向单室空白脂质体中加入MncGAMP溶液，60℃孵育1h，加入末端巯基化的RBD-SD1(1mg空白脂质体:20μg RBD-SD1)，室温避光孵育过夜。用30kD的浓缩管，4000rpm，4℃除去未包封免疫激动剂和未连接RBD-SD1。Add MncGAMP solution to single-chamber blank liposomes, incubate at 60°C for 1h, add terminal sulfhydryl RBD-SD1 (1mg blank liposome: 20μg RBD-SD1), and incubate overnight at room temperature in the dark. Use 30kD concentrated tube, 4000rpm, 4℃ to remove unencapsulated immunoagonist and unconnected RBD-SD1.
用TEM电镜检测所得到的复合物，双层圆形囊泡，形态良好，脂质体直径约为～185nm，Zeta电位～－23mV。免疫激动剂包封率为75％，4℃冷藏条件下稳定，使用2.5％海藻糖溶液制成冻干粉冷藏保存。Detected by TEM electron microscope, the obtained complex has double-layer circular vesicles with good morphology. The liposome diameter is about ~185nm, and the Zeta potential is ~-23mV. The immune agonist encapsulation rate is 75%, and it is stable under 4°C refrigeration conditions, and a 2.5% trehalose solution is used to make a freeze-dried powder for storage under refrigeration.
上述方法适用于其他免疫激动剂、其他重组新冠病毒抗原S蛋白各种组合的替换使用。The above method is suitable for the replacement of various combinations of other immune agonists and other recombinant new coronavirus antigen S proteins.
(4)跨膜肽(gH625或Anti-PV1Nb)偶联脂质体包封免疫激动剂的制备(4) Preparation of transmembrane peptide (gH625 or Anti-PV1Nb) coupled liposome encapsulated immunoagonist
按以上(3)的制备方法，用跨膜肽gH625或Anti-PV1Nb替换RBD-SD1蛋白，首先，对跨膜肽进行末端巯基化(在跨膜肽溶液中加入EDTA溶液使EDTA终浓度为5mM，搅动下滴加巯基化试剂Traut’s reagent，水浴中搅动后，避光孵育1小时；用脱盐柱除去过量巯基化试剂)。According to the preparation method of (3) above, replace the RBD-SD1 protein with the transmembrane peptide gH625 or Anti-PV1Nb. First, carry out terminal sulfhydrylation of the transmembrane peptide (add EDTA solution to the transmembrane peptide solution to make the final concentration of EDTA 5mM , Add sulfhydryl reagent Traut's reagent dropwise under agitation. After stirring in a water bath, incubate in the dark for 1 hour; use a desalting column to remove excess sulfhydryl reagent).
用Ellman方法测定跨膜肽上的巯基，验证跨膜肽巯基化成功。The Ellman method was used to determine the sulfhydryl group on the transmembrane peptide to verify the successful sulfhydrylation of the transmembrane peptide.
将脂质体材料(包括卵磷脂、胆固醇、1,2-二硬脂酰-SN-甘油-3-磷酰乙醇胺-N-马来酰亚胺-聚乙二醇2000)，溶解于甲醇/氯仿混合溶剂中，在水浴中真空旋转蒸发干成膜，然后加入(NH 4) 2SO 4水化制成空白脂质体。用脂质体挤出器，经过200nm聚碳酸酯微孔滤膜挤出制备成均匀单室空白脂质体。 Dissolve liposome materials (including lecithin, cholesterol, 1,2-distearoyl-SN-glycerol-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000) in methanol/ In a mixed solvent of chloroform, vacuum rotary evaporation in a water bath to dry to form a film, and then add (NH 4 ) 2 SO 4 for hydration to make a blank liposome. Using a liposome extruder, a uniform single-compartment blank liposome was prepared by extruding through a 200nm polycarbonate microporous filter membrane.
向单室空白脂质体中加入免疫激动剂溶液，60℃孵育1h，加入末端巯基化的跨膜肽(1mg空白脂质体:20μg gH625或Anti-PV1Nb)，室温避光孵育过夜。用30kD的浓缩管，4000rpm，4℃除去未包封免疫激动剂药物和未连接跨膜肽gH625或Anti-PV1Nb。Add immunoagonist solution to the single-compartment blank liposome, incubate at 60°C for 1h, add terminal sulfhydryl transmembrane peptide (1mg blank liposome: 20μg gH625 or Anti-PV1Nb), and incubate overnight at room temperature in the dark. Use a 30kD concentrated tube, 4000rpm, 4℃ to remove unencapsulated immunoagonist drugs and unattached transmembrane peptide gH625 or Anti-PV1Nb.
用TEM电镜检测所得到的复合物(gH625偶联脂质体包封MncGAMP，免疫激动剂为MncGAMP，跨膜肽为gH625)，双层圆形囊泡，形态良好，脂质体直径约为～175nm，Zeta电位～－22mV。免疫激动剂包封率为78％，4℃冷藏条件下稳定，使用3％海藻糖溶液制成冻干粉冷藏保存。The obtained complex (gH625 coupled liposome encapsulating MncGAMP, immunoagonist MncGAMP, transmembrane peptide gH625) was detected by TEM electron microscope. The double-layer circular vesicles are in good shape and the liposome diameter is about ~ 175nm, Zeta potential ~ -22mV. The immune agonist encapsulation rate is 78%, and it is stable under 4°C refrigeration conditions. A 3% trehalose solution is used to make a freeze-dried powder for storage under refrigeration.
(5)新冠病毒抗原S蛋白基因重组腺病毒载体的制备(5) Preparation of recombinant adenovirus vector of new coronavirus antigen S protein gene
新冠病毒抗原S蛋白基因重组腺病毒载体由生物医药科技公司外包服务完成，采用缺失了腺病毒的早期表达基因序列E1和E3区的重组腺病毒载体进行构建:The recombinant adenovirus vector of the new coronavirus antigen S protein gene is completed by the outsourcing service of a biomedical technology company. The recombinant adenovirus vector is constructed with the E1 and E3 regions of the early expression gene sequence of the adenovirus deleted:
将目的基因(COVID-19病毒S蛋白基因或COVID-19病毒S蛋白的结构域基因)插入腺病毒穿梭质粒(缺失了腺病毒的早期表达基因序列E1和E3区的重组腺病毒载体质粒)中，将确定正确的重组腺病毒穿梭质粒和骨架质粒(缺失了腺病毒的早期表达基因序列E1和E3区)共转染，在293A细胞中进行包装，经过腺病毒扩增和CsCl纯化，然后对包装出的重组RBD－SD1腺病毒载体进行质检。质检包括对终产品病毒基因进行PCR和WB以确认目的基因的存在。Insert the target gene (COVID-19 virus S protein gene or COVID-19 virus S protein domain gene) into the adenovirus shuttle plasmid (recombinant adenovirus vector plasmid with deletion of the early expression gene sequence E1 and E3 regions of adenovirus) , The confirmed correct recombinant adenovirus shuttle plasmid and the backbone plasmid (with the deletion of the early expression gene sequence E1 and E3 regions of adenovirus) were co-transfected, packaged in 293A cells, and then amplified by adenovirus and purified by CsCl. The packaged recombinant RBD-SD1 adenovirus vector is subjected to quality inspection. Quality inspection includes PCR and WB on the final product virus gene to confirm the existence of the target gene.
(6)新型疫苗II(VFII)(6) New vaccine II (VFII)
将gH625偶联脂质体包封MncGAMP与重组RBD－SD1腺病毒载体混合组成(200μg MncGAMP：重组RBD－SD1腺病毒载体1×10 8腺病毒粒子)。最后将使用3％海藻糖溶液将此混合物制成冻干粉冷藏保存。 The gH625 coupled liposome encapsulated MncGAMP and the recombinant RBD-SD1 adenovirus vector were mixed to form a composition (200μg MncGAMP: recombinant RBD-SD1 adenovirus vector 1×10 8 adenovirus particles). Finally, a 3% trehalose solution will be used to make this mixture into a freeze-dried powder for storage under refrigeration.
(7)新型疫苗III(VFIII)(7) New vaccine III (VFIII)
新型疫苗III(VFIII)的制备方法同新型疫苗I(VFI)的制备，只是用ZncGAMP替换MncGAMP，其它方法步骤相同。用TEM电镜检测所得到的VFIII，双层圆形囊泡，形态良好，脂质体直径约为～187nm，Zeta电位～－24mV。免疫激动剂包封率为76％，4℃冷藏条件下稳定，使用2.5％海藻糖溶液制成冻干粉冷藏保存。The preparation method of the new vaccine III (VFIII) is the same as the preparation of the new vaccine I (VFI), except that MncGAMP is replaced by ZncGAMP, and the other method steps are the same. Detected by TEM electron microscope, the obtained VFIII has double-layer circular vesicles with good morphology. The liposome diameter is about ~187nm, and the Zeta potential is ~-24mV. The encapsulation rate of the immune agonist is 76%, and it is stable under refrigerated conditions at 4°C. A 2.5% trehalose solution is used to make a freeze-dried powder for storage under refrigeration.
(8)新型疫苗IV(VFIV)(8) New vaccine IV (VFIV)
新型疫苗IV(VFIV)的制备方法同新型疫苗I(VFI)的制备，只是用cGAMP替换MncGAMP,抗原S蛋白结构域RBD替换抗原S蛋白结构域RBD-SD1，其它方法步骤相同。The preparation method of the new vaccine IV (VFIV) is the same as the preparation of the new vaccine I (VFI), except that cGAMP is used to replace MncGAMP, and the antigen S protein domain RBD is replaced with antigen S protein domain RBD-SD1. The other method steps are the same.
用TEM电镜检测所得到的VFIV，双层圆形囊泡，形态良好，脂质体直径约为～184nm，Zeta电位～－23mV。免疫激动剂包封率为73％，4℃冷藏条件下稳定，使用2.5％海藻糖溶液制成冻干粉冷藏保存。The obtained VFIV was detected by TEM electron microscope. The double-layered circular vesicles were in good shape. The liposome diameter was about ~184nm, and the Zeta potential was ~-23mV. The immune agonist encapsulation rate is 73%, and it is stable under 4°C refrigeration conditions. It is made into a lyophilized powder with a 2.5% trehalose solution and stored under refrigeration.
(9)制得的新型抗新冠病毒治疗性疫苗(9) New anti-coronavirus therapeutic vaccine prepared
新型疫苗I(VFI)，[抗原S蛋白结构域RBD-SD1偶联脂质体包封MncGAMP]New vaccine I (VFI), [antigen S protein domain RBD-SD1 coupled with liposome encapsulating MncGAMP]
新型疫苗II(VFII)，[(gH625偶联脂质体包封MncGAMP)含重组RBD－SD1腺病毒载体]New vaccine II (VFII), [(gH625 coupled liposome encapsulating MncGAMP) containing recombinant RBD-SD1 adenovirus vector]
新疫苗III(VFIII)   [抗原S蛋白结构域RBD-SD1偶联脂质体包封ZncGAMP]New vaccine III (VFIII) [Antigen S protein domain RBD-SD1 coupled with liposome encapsulating ZncGAMP]
新疫苗III(VFIV)   [抗原S蛋白结构域RBD偶联脂质体包封cGAMP]New vaccine III (VFIV) [antigen S protein domain RBD coupled liposome encapsulating cGAMP]
免疫激动剂复合物I(FI)，[gH625偶联脂质体包封cGAMP](按步骤(4)制备)Immune agonist complex I (FI), [gH625 coupled liposome encapsulating cGAMP] (prepared according to step (4))
免疫激动剂复合物II(FII)，[gH625偶联脂质体包封MncGAMP](按步骤(4)制备)Immune agonist complex II (FII), [gH625 coupled liposome encapsulating MncGAMP] (prepared according to step (4))
实施例2.新型抗新冠病毒治疗性疫苗特异免疫功能研究Example 2. Research on specific immune function of new anti-coronavirus therapeutic vaccine
实验动物：C57BL/6小鼠，雄性，体重20－22g，6-8周龄，购于上海斯莱克实验动物有限责任公司[实验动物质量合格证号：SCXK(沪)2007-0005]。所有小鼠均自由觅食和饮水，在室温(23±2)℃下饲养。饲料及水均经高压灭菌处理，全部实验饲养过程为SPF级。Experimental animals: C57BL/6 mice, male, weight 20-22g, 6-8 weeks old, purchased from Shanghai Slack Experimental Animal Co., Ltd. [Experimental animal quality certificate number: SCXK (Shanghai) 2007-0005]. All mice were free to forage and drink, and were raised at room temperature (23±2)°C. Feed and water are autoclaved, and all experimental feeding processes are SPF grade.
小鼠免疫：小鼠分组：每10只一组，共9组，分别为，A：VFI；B:VFII；C:VFIII；D：VFIV；E:FI；F：FII；G：RBD-SD1；H:RBD；I:PBS空白。Mouse Immunization: Grouping of mice: each group of 10 mice, a total of 9 groups, respectively, A: VFI; B: VFII; C: VFIII; D: VFIV; E: FI; F: FII; G: RBD-SD1 ; H: RBD; I: PBS blank.
给药方式：鼻腔滴注。Administration method: nasal drip.
给药剂量：Dosage:
新型疫苗I(VFI)，(10mg/kg MncGAMP，100μg RBD-SD1)New vaccine I (VFI), (10mg/kg MncGAMP, 100μg RBD-SD1)
新型疫苗II(VFII)，(10mg/kg MncGAMP+重组RBD－SD1腺病毒载体10 8) New vaccines II (VFII), (10mg / kg MncGAMP + recombinant adenoviral vector RBD-SD1 108)
新型疫苗III(VFIII)，(10mg/kg ZncGAMP，100μg RBD-SD1)New vaccine III (VFIII), (10mg/kg ZncGAMP, 100μg RBD-SD1)
新型疫苗IV(VFIV)，(10mg/kg cGAMP，100μg RBD)New Vaccine IV (VFIV), (10mg/kg cGAMP, 100μg RBD)
免疫激动剂复合物I(FI)，(10mg/kg cGAMP)Immune Agonist Complex I (FI), (10mg/kg cGAMP)
免疫激动剂复合物II(FII)，(10mg/kg MncGAMP)Immune Agonist Complex II (FII), (10mg/kg MncGAMP)
抗原S蛋白结构域RBD-SD1 (100μg)Antigen S protein domain RBD-SD1 (100μg)
抗原S蛋白结构域RBD    (100μg)Antigen S protein domain RBD (100μg)
使小鼠处于麻醉状态，将小鼠以背卧姿势固定，并慢慢地分别将各组药溶液悬液通过小鼠鼻孔内壁逐滴滴入，滴入体积为60μL(每个鼻孔30μL)。将小鼠轻轻从工作台拿下，并将头部和胸部用折叠的纸巾小幅度垫高，以保证小鼠顺畅的呼吸。待小鼠苏醒后，放回鼠笼。分别在第1，7，14天各给药一次，在第21天获得小鼠肺灌洗液和取血样。用ELISA法测定免疫激动剂复合物及疫苗复合物诱导产生抗体的效价。The mice were put under anesthesia, the mice were fixed in a dorsal position, and the drug solution suspension of each group was slowly dripped through the inner wall of the mouse nostrils, and the drip volume was 60 μL (30 μL for each nostril). The mouse was gently taken off the workbench, and the head and chest were raised slightly with folded paper towels to ensure smooth breathing of the mouse. After the mice wake up, they are put back into the squirrel cage. They were administered once on the 1, 7, and 14 days respectively, and the mouse lung lavage fluid and blood samples were obtained on the 21st day. The ELISA method was used to determine the titers of immune agonist complexes and vaccine complexes induced to produce antibodies.
小鼠肺泡灌洗液获得方法：Method of obtaining mouse alveolar lavage fluid:
取等体积PBS沿小鼠气管注射后吸出，反复几次，获得肺泡灌洗液。收集的血清，于－80℃保存。Take an equal volume of PBS and inject it along the mouse trachea and aspirate it, repeat it several times to obtain alveolar lavage fluid. The collected serum is stored at -80°C.
实验结果见表1。测定结果显示，新型疫苗(VFI、VFII、VFIII、VFIV)及免疫激动剂脂质体复合物(FI、FII)均能显著剂或诱发免疫应答，新型疫苗(VFI、VFII、VFIII、VFIV)的效果显著高于免疫激动剂复合物(FI、FII)及单独重组S蛋白结构域RBD-SD1/RBD。The experimental results are shown in Table 1. The test results show that new vaccines (VFI, VFII, VFIII, VFIV) and immunoagonist liposome complexes (FI, FII) can significantly enhance or induce immune responses. The new vaccines (VFI, VFII, VFIII, VFIV) The effect is significantly higher than the immune agonist complex (FI, FII) and the single recombinant S protein domain RBD-SD1/RBD.
表1.新型抗新冠病毒疫苗特异免疫功能效价Table 1. Specific immune function titers of new anti-coronavirus vaccines
Figure PCTCN2020142585-appb-000001
Figure PCTCN2020142585-appb-000001
实施例3新型抗新冠病毒治疗性疫苗诱发保护性特异细胞免疫研究Example 3 Research on the protective specific cellular immunity induced by a novel anti-new coronavirus therapeutic vaccine
小鼠饲养、给药等见实施例2。同型对照流式抗体购自eBiosciences,抗体磁株购于Militeny Biotech,流式细胞仪购于BD公司。三次给药免疫21天后取小鼠脾脏、肺组织，分别研磨捣碎，过40微米孔脱过滤细胞，1000rpm离心10分钟，分离未被裂解的免疫细胞，用抗体磁株分离DC(CD40\CD80\CD86\MHCII)、T(CD8+)细胞，加入对应的FAC抗体(用FACS缓冲液稀释)，同型对照抗体作为阴性对照，抗体加入后孵育1小时后离心，用PBS清洗，用流式细胞仪分析样品，分选合适的细胞，测定选定细胞的荧光强度(MFI)，流式结果见表2。流式细胞测定结果显示，新型疫苗(VFI、VFII、VFIII、VFIV)及免疫激动剂复合物(FI、FII)均能显著活化树突状细胞DC和T细胞，新型疫苗(VFI、VFII、VFIII、VFIV)的效果显著高于免疫激动剂复合物(FI、FII)及抗原S蛋白结构域RBD-SD1/RBD。See Example 2 for the breeding and administration of mice. Isotype control flow cytometry antibodies were purchased from eBiosciences, antibody magnets were purchased from Militeny Biotech, and flow cytometry was purchased from BD. After 21 days of immunization with three administrations, the spleen and lung tissues of the mice were taken, ground and crushed, and the cells were removed through 40 micron holes, and centrifuged at 1000 rpm for 10 minutes. \CD86\MHCII), T(CD8+) cells, add the corresponding FAC antibody (diluted with FACS buffer), the isotype control antibody as a negative control, after the antibody is added, incubate for 1 hour, centrifuge, wash with PBS, and use flow cytometry Analyze the samples, sort the appropriate cells, and measure the fluorescence intensity (MFI) of the selected cells. The flow cytometry results are shown in Table 2. The results of flow cytometry showed that the new vaccines (VFI, VFII, VFIII, VFIV) and immune agonist complexes (FI, FII) can significantly activate dendritic cells DC and T cells, new vaccines (VFI, VFII, VFIII) , VFIV) is significantly higher than the immune agonist complex (FI, FII) and antigen S protein domain RBD-SD1/RBD.
表2.新型抗新冠病毒疫苗诱发保护性细胞免疫效果Table 2. Protective cellular immunity induced by the new anti-coronavirus vaccine
Figure PCTCN2020142585-appb-000002
Figure PCTCN2020142585-appb-000002
实施例4.新型抗新冠病毒治疗性疫苗对小鼠冠状病毒性肺炎的抑制作用Example 4. The inhibitory effect of a novel anti-new coronavirus therapeutic vaccine on mouse coronavirus pneumonia
实验动物：C57BL/6小鼠，雄性，体重20－22g，6-8周龄，SPF级，来源于American Animals Inc.，所有小鼠均自由觅食和饮水，在室温(23±2)℃下饲养。饲料及水均经高压灭菌处理，全部实验饲养过程为SPF级。Experimental animals: C57BL/6 mice, male, weighing 20-22g, 6-8 weeks old, SPF grade, from American Animals Inc., all mice foraging and drinking freely, at room temperature (23±2)℃ Feeding under. Feed and water are autoclaved, and all experimental feeding processes are SPF grade.
动物分组：将60只小鼠随机分为10组(n＝6)，具体分组为：A组，正常对照组；B组，肺炎模型组；C组，免疫激动剂复合物，FI组；D组，免疫激动剂复合物，FII组；E组，新型疫苗VFI组；F组，新型疫苗VFII组；G组，新型疫苗VFIII组；H组，新型疫苗VFIV组；I组，抗原S蛋白结构域RBD－SD1组；J组，抗原S蛋白结构域RBD组。Animal grouping: 60 mice were randomly divided into 10 groups (n=6), specifically grouped into: group A, normal control group; group B, pneumonia model group; group C, immune agonist complex, FI group; D Group, immune agonist complex, FII group; E group, new vaccine VFI group; F group, new vaccine VFII group; G group, new vaccine VFIII group; H group, new vaccine VFIV group; I group, antigen S protein structure Domain RBD-SD1 group; J group, antigen S protein domain RBD group.
肺炎病毒模型小鼠建立：Establishment of pneumonia virus model mice:
病毒株：适合在实验室使用的病毒株购于美国ATCC公司：冠状病毒(ATCC VR－841)，该研究中病毒实验操作委托美国American Animals Inc.病毒实验室完成。Virus strain: A virus strain suitable for laboratory use was purchased from the American ATCC Company: Coronavirus (ATCC VR-841). In this study, the virus experiment operation was commissioned by the American Animals Inc. Virus Laboratory.
鼻内滴注：使小鼠处于足够深的麻醉状态，将小鼠以背卧姿势固定，并慢慢地将VR－841病毒悬液通过小鼠鼻孔内壁逐滴滴入，为保证最大的肺部感染效率，滴入体积为60μL(每个鼻孔30μL)。将小鼠轻轻从工作台拿下，并将头部和胸部用折叠的纸巾小幅度垫高，以保证小鼠顺畅的呼吸。待小鼠苏醒后，放回鼠笼。分别对C、D、E、F、G、H、I、J组小 鼠在第2，8，15天各给药一次，慢慢地分别将各组药溶液悬液通过小鼠鼻孔内壁逐滴滴入，在第21天获得小鼠肺灌洗液和取血样。用ELISA法测定免疫激动剂复合物及疫苗复合物诱导产生保护性细胞免疫效价。Intranasal drip: Keep the mouse in a sufficiently deep anesthesia state, fix the mouse in a dorsal position, and slowly drip the VR-841 virus suspension through the inner wall of the mouse nostril to ensure the largest lung Infection efficiency, the instillation volume is 60 μL (30 μL per nostril). The mouse was gently taken off the workbench, and the head and chest were raised slightly with folded paper towels to ensure smooth breathing of the mouse. After the mice wake up, they are put back into the squirrel cage. The mice in groups C, D, E, F, G, H, I, and J were administered once on the 2, 8 and 15 days respectively, and the drug solution suspension of each group was slowly passed through the inner wall of the mouse nostril. Instillation, the mouse lung lavage fluid and blood samples were obtained on the 21st day. The immune agonist complex and vaccine complex induced protective cellular immunity titers were measured by ELISA.
给药方式：鼻腔滴注；Administration method: nasal drip;
给药剂量：Dosage:
新型疫苗I(VFI)，(10mg/kg MncGAMP，100μg RBD-SD1)New vaccine I (VFI), (10mg/kg MncGAMP, 100μg RBD-SD1)
新型疫苗II(VFII)，(10mg/kg MncGAMP+重组RBD－SD1腺病毒载体10 8) New vaccines II (VFII), (10mg / kg MncGAMP + recombinant adenoviral vector RBD-SD1 108)
新型疫苗III(VFIII)，(10mg/kg ZncGAMP，100μg RBD-SD1)New vaccine III (VFIII), (10mg/kg ZncGAMP, 100μg RBD-SD1)
新型疫苗IV(VFIV)，(10mg/kg cGAMP，100μg RBD)New Vaccine IV (VFIV), (10mg/kg cGAMP, 100μg RBD)
免疫激动剂复合物I(FI)，(10mg/kg cGAMP)Immune Agonist Complex I (FI), (10mg/kg cGAMP)
免疫激动剂复合物II(FII)，(10mg/kg MncGAMP)Immune Agonist Complex II (FII), (10mg/kg MncGAMP)
抗原S蛋白结构域RBD-SD1 (100μg)Antigen S protein domain RBD-SD1 (100μg)
抗原S蛋白结构域RBD     (100μg)Antigen S protein domain RBD (100μg)
小鼠肺泡灌洗液获得方法：取等体积PBS沿小鼠气管注射后吸出，反复几次，获得肺泡灌洗液。收集的血清，于－80℃保存。采用ELISA法，按试剂盒说明书检测TNF-alpha、IL-1beta浓度。终止反应后，将酶标板放入酶标仪槽内，选择450nm波长检测，确定标准品和空白对照区域，检测相应的光密度值，然后绘制标准曲线并计算相应的浓度。在小鼠肺炎模型中，促炎性细胞因子IL-1beta和TNF-alpha在血清和肺泡灌洗液中的含量都显著上升，新型疫苗和免疫激动剂复合物给药后均不同程度地降低了两者的含量。新型疫苗(VFI、VFII、VFIII、VFIV)的效果显著高于免疫激动剂复合物(FI、FII)及抗原S蛋白结构域RBD－SD1或RBD。不同药物显示抑制小鼠肺炎作用的结果见表3。Method for obtaining mouse alveolar lavage fluid: take an equal volume of PBS and inject it along the mouse trachea and aspirate it, repeat several times to obtain alveolar lavage fluid. The collected serum is stored at -80°C. ELISA method was used to detect the concentration of TNF-alpha and IL-1beta according to the kit instructions. After terminating the reaction, put the microtiter plate into the microplate reader slot, select the 450nm wavelength for detection, determine the standard and blank control area, detect the corresponding optical density value, and then draw the standard curve and calculate the corresponding concentration. In the mouse pneumonia model, the levels of pro-inflammatory cytokines IL-1beta and TNF-alpha in serum and alveolar lavage fluid were significantly increased, and the new vaccines and immune agonist complexes were all reduced to varying degrees after administration. The content of both. The effect of new vaccines (VFI, VFII, VFIII, VFIV) is significantly higher than that of immune agonist complexes (FI, FII) and antigen S protein domain RBD-SD1 or RBD. Table 3 shows the results of different drugs inhibiting pneumonia in mice.
表3.新型抗新冠病毒疫苗对小鼠肺炎的治疗作用Table 3. Therapeutic effects of new anti-coronavirus vaccines on pneumonia in mice
Figure PCTCN2020142585-appb-000003
Figure PCTCN2020142585-appb-000003
实验结果表明，新型抗新冠病毒疫苗(VFI、VFII、VFIII、VFIV)和免疫激动剂脂质体复合物(FI、FII)均能不同程度地抑制小鼠肺炎促炎细胞因子，对小鼠病毒性肺炎炎症有明显的治疗作用。结果显示，新型抗新冠病毒疫苗(VFI、VFII、VFIII、VFIV)的抗小鼠肺炎的作用显著优于免疫激动剂脂质体复合物(FI、FII)及病毒抗原蛋白结构域RBD－SD1和RBD。该类新型抗新冠病毒疫苗具有抗小鼠冠状病毒肺炎的作用。Experimental results show that the new anti-coronavirus vaccines (VFI, VFII, VFIII, VFIV) and immune agonist liposome complexes (FI, FII) can inhibit mouse pneumonia pro-inflammatory cytokines to varying degrees, and have a positive effect on mouse viruses. Pneumonia inflammation has obvious therapeutic effect. The results show that the new anti-coronavirus vaccines (VFI, VFII, VFIII, VFIV) have significantly better anti-mouse pneumonia effects than immunoagonist liposome complexes (FI, FII) and viral antigen protein domains RBD-SD1 and RBD. This new type of anti-coronavirus vaccine has the effect of anti-coronavirus pneumonia in mice.
实施例5新型抗新冠病毒治疗性疫苗的急性毒性研究Example 5 Study on the acute toxicity of a new anti-coronavirus therapeutic vaccine
实验材料Experimental Materials
ICR小鼠40只(购于上海斯莱克实验动物有限责任公司[实验动物质量合格证号：SCXK(沪)2007-0005])，雌雄各半，体重20～22g，动物以颗粒饲料喂养，自由摄食和饮水。40 ICR mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd. [Experimental Animal Quality Certificate No.: SCXK (Shanghai) 2007-0005]), half male and half, weighing 20-22g, the animals are fed with pellet feed, free Eat and drink.
实验方法experimental method
ICR小鼠按体重分别腹腔注射1g/kg的新型抗新冠病毒治疗性疫苗(VFI、VFII、VFIII、VFIV)(PBS缓冲液配制)，观察给药后小鼠14天内的毒性反应及死亡情况。结果发现，小鼠腹腔注射给药后，小鼠活动正常。给药后14天内，小鼠未出现死亡，第15天，全部小鼠处死，解剖，肉眼检查各脏器，均未见明显病变。ICR mice were intraperitoneally injected with 1g/kg of new anti-coronavirus therapeutic vaccines (VFI, VFII, VFIII, VFIV) (prepared in PBS buffer) according to their body weight, and the toxicity and death of the mice within 14 days after administration were observed. It was found that after intraperitoneal injection of the mice, the mice moved normally. Within 14 days after the administration, the mice did not die. On the 15th day, all the mice were sacrificed, dissected, and visually inspected the various organs, and no obvious lesions were seen.
实验结果Experimental result
上述急性毒性实验结果表明，腹腔注射给药最大耐受量MTD不低于1g/Kg，说明两种新型抗新冠病毒治疗性疫苗的急性毒性低。The above-mentioned acute toxicity test results show that the maximum tolerated dose MTD for intraperitoneal injection is not less than 1g/Kg, indicating that the acute toxicity of the two new anti-coronavirus therapeutic vaccines is low.

Claims (9)

  1. 一种新型抗新冠病毒治疗性疫苗，其特征在于，所述疫苗为A novel anti-new coronavirus therapeutic vaccine, which is characterized in that the vaccine is
    (1).由重组新冠病毒抗原S蛋白偶联脂质体与免疫激动剂构成的仿病毒颗粒疫苗，所述免疫激动剂包封于重组新冠病毒抗原S蛋白偶联脂质体中；(1) A virus-like particle vaccine composed of recombinant neocoronavirus antigen S protein-coupled liposomes and an immune agonist encapsulated in recombinant neocoronavirus antigen S protein-coupled liposomes;
    或or
    (2).由新冠病毒抗原S蛋白基因重组腺病毒载体与跨膜肽偶联脂质体包封免疫激动剂构成的仿病毒颗粒疫苗，所述跨膜肽偶联脂质体包封免疫激动剂为将所述免疫激动剂包封于跨膜肽偶联脂质体中制得；(2). A virus-like particle vaccine consisting of a recombinant adenovirus vector of the new coronavirus antigen S protein gene and a transmembrane peptide coupled liposome encapsulating immunostimulant. The transmembrane peptide coupled liposome encapsulating immunostimulant The agent is prepared by encapsulating the immune agonist in a transmembrane peptide-coupled liposome;
    所述免疫激动剂为STING的激动剂或其过渡金属配合物，所述STING的激动剂为环二核苷酸2’3’-cGAMP或其衍生物；The immune agonist is an agonist of STING or a transition metal complex thereof, and the agonist of STING is a cyclic dinucleotide 2'3'-cGAMP or a derivative thereof;
    所述重组新冠病毒抗原S蛋白为COVID-19病毒S蛋白或COVID-19病毒S蛋白的结构域衍生物；The recombinant new coronavirus antigen S protein is the COVID-19 virus S protein or a domain derivative of the COVID-19 virus S protein;
    所述跨膜肽为膜靶向肽或靶向膜囊泡关联蛋白；The transmembrane peptide is a membrane targeting peptide or a targeted membrane vesicle-associated protein;
    所述新冠病毒抗原S蛋白基因重组腺病毒载体为：重组有COVID-19病毒S蛋白基因或COVID-19病毒S蛋白的结构域基因，并且缺失了腺病毒的早期表达基因序列E1和E3区的重组腺病毒载体。The recombinant adenovirus vector of the new coronavirus antigen S protein gene is: recombined with the COVID-19 virus S protein gene or the COVID-19 virus S protein domain gene, and has deleted the early expression gene sequence E1 and E3 regions of the adenovirus Recombinant adenovirus vector.
  2. 根据权利要求1所述的一种新型抗新冠病毒治疗性疫苗，其特征在于，The novel anti-coronavirus therapeutic vaccine according to claim 1, characterized in that:
    所述COVID-19病毒S蛋白的结构域衍生物包括但不限于RBD、RBD-SD1或RBD-SD1SD2。The domain derivatives of the COVID-19 virus S protein include but are not limited to RBD, RBD-SD1 or RBD-SD1SD2.
  3. 根据权利要求1所述的一种新型抗新冠病毒治疗性疫苗，其特征在于，The novel anti-coronavirus therapeutic vaccine according to claim 1, characterized in that:
    所述膜靶向肽为单纯疱疹病毒糖蛋白的一段跨膜肽gH625，氨基酸序列为HGLASTLTRWAHYNALIRAFGGG，SEQ ID NO:1；The membrane targeting peptide is a transmembrane peptide gH625 of herpes simplex virus glycoprotein, and the amino acid sequence is HGLASTLTRWAHYNALIRAFGGG, SEQ ID NO:1;
    所述靶向膜囊泡关联蛋白的纳米抗体为anti-PV1 Nb，氨基酸序列为QVQLQQSGAELVKPGASVKLSCKASGYTFTDYYMYWVKQPPGQGLELIGEINPTNGDVNFNEMFKSKATLTVDTSSRTAYMQLSSLTSEDSAVYYCTSIH YWGQGTLVTVSAGSG，SEQ ID NO:2。The nanobody targeting membrane vesicle-associated protein is anti-PV1 Nb, and the amino acid sequence is QVQLQQSGAELVKPGASVKLSCKASGYTFTDYYMYWVKQPPGQGLELIGEINPTNGDVNFNEMFKSKATLTVDTSSRTAYMQLSSLTSEDSAVYYCTSIH YWGQGTLVIDTVSAGNOSG2, SEQ.
  4. 根据权利要求1所述的一种新型抗新冠病毒治疗性疫苗的制备方法，其特征在于，包括如下步骤：The preparation method of a novel anti-new coronavirus therapeutic vaccine according to claim 1, characterized in that it comprises the following steps:
    (1)对重组新冠病毒抗原S蛋白进行巯基化，得到巯基化重组新冠病毒抗原S蛋白；(1) Carry out sulfhydrylation on the recombinant neocoronavirus antigen S protein to obtain the sulfhydryl recombinant neocoronavirus antigen S protein;
    (2)巯基化重组新冠病毒抗原S蛋白与脂质体化学键融合，并且包封免疫激动剂。(2) The thiolated recombinant new coronavirus antigen S protein is chemically bonded to the liposome and encapsulates the immune agonist.
  5. 根据权利要求1所述的一种新型抗新冠病毒治疗性疫苗的制备方法，其特征在于，包括如下步骤：The preparation method of a novel anti-new coronavirus therapeutic vaccine according to claim 1, characterized in that it comprises the following steps:
    (1)对膜靶向肽或靶向膜囊泡关联蛋白的纳米抗体进行巯基化，得到巯基化跨膜肽；(1) Sulfhydrylation of membrane targeting peptides or nanobodies targeting membrane vesicle-associated proteins to obtain thiolated transmembrane peptides;
    (2)巯基化跨膜肽与脂质体化学键融合，包封免疫激动剂，得到跨膜肽偶联脂质体包封免疫激动剂；(2) The sulfhydryl transmembrane peptide is chemically bonded to the liposome to encapsulate the immune agonist to obtain the transmembrane peptide-coupled liposome encapsulated immune agonist;
    (3)跨膜肽偶联脂质体包封免疫激动剂与新冠病毒抗原S蛋白基因重组腺病毒载体混合。(3) The transmembrane peptide-coupled liposome encapsulated immune agonist is mixed with the recombinant adenovirus vector of the new coronavirus antigen S protein gene.
  6. 根据权利要求1所述的新型抗新冠病毒治疗性疫苗在制备预防和/或治疗冠状病毒感染疾病药物中的应用。The use of the novel anti-new coronavirus therapeutic vaccine according to claim 1 in the preparation of drugs for the prevention and/or treatment of coronavirus infections.
  7. 根据权利要求6所述的应用，其特征在于，所述冠状病毒感染疾病包括但不限于人或动物感染冠状病毒引起的病毒性肺炎、病毒性肾炎、病毒性脑炎、病毒性肠炎或病毒性肝炎。The application according to claim 6, characterized in that the coronavirus infection disease includes, but is not limited to, viral pneumonia, viral nephritis, viral encephalitis, viral enteritis, or viral pneumonia caused by human or animal infection with coronavirus. hepatitis.
  8. 根据权利要求6所述的应用，其特征在于，所述疫苗可单独制备成不同规格的单位制剂或通过药学上可接受的载体制备成药物制剂。The application according to claim 6, wherein the vaccine can be separately prepared into unit preparations of different specifications or prepared into pharmaceutical preparations through a pharmaceutically acceptable carrier.
  9. 根据权利要求6所述的应用，其特征在于，所述预防和/或治疗冠状病毒感染疾病药物包括静脉注射制剂、鼻腔滴注制剂、静脉滴注制剂、肌肉注射制剂、皮下注射制剂或口服制剂；所述口服制剂包括但不限于胶囊、片剂或颗粒剂。The application according to claim 6, characterized in that the drugs for prevention and/or treatment of coronavirus infection diseases comprise intravenous injection preparations, nasal drip preparations, intravenous drip preparations, intramuscular injection preparations, subcutaneous injection preparations or oral preparations ; The oral preparations include, but are not limited to, capsules, tablets or granules.
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