WO2021196808A1 - Nouveau vaccin thérapeutique contre le nouveau coronavirus, son procédé de préparation et son utilisation - Google Patents

Nouveau vaccin thérapeutique contre le nouveau coronavirus, son procédé de préparation et son utilisation 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
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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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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention concerne un nouveau vaccin thérapeutique contre le nouveau coronavirus, comprenant : (1) un vaccin à particules de type viral composé d'un nouveau liposome conjugué à une protéine S antigénique de coronavirus de recombinaison et d'un immunostimulant, l'immunostimulant étant encapsulé dans le nouveau liposome conjugué à une protéine S antigénique de coronavirus de recombinaison ; ou (2) un vaccin à particules de type viral composé d'une nouvelle protéine S antigénique de coronavirus, d'un vecteur adénoviral recombiné génétiquement et d'un immunostimulant encapsulé par un liposome conjugué à un peptide transmembranaire, l'immunostimulant encapsulé par un liposome conjugué à un peptide transmembranaire étant préparé par encapsulation de l'immunostimulant dans le liposome conjugué à un peptide transmembranaire. L'invention concerne en outre un procédé de préparation du nouveau vaccin thérapeutique et une utilisation du nouveau vaccin thérapeutique dans la préparation d'un médicament contre le coronavirus. Le vaccin contre le nouveau coronavirus peut induire de manière significative une immunité humorale et une immunité cellulaire protectrice, déclencher l'immunité muqueuse, réguler l'état stable de l'immunité corporelle, améliorer les fonctions immunitaires et antivirales dans le corps, et induire significativement une fonction immunitaire spécifique contre le nouveau coronavirus, ce qui permet d'inhiber efficacement la pneumonie à coronavirus.
PCT/CN2020/142585 2020-03-30 2020-12-31 Nouveau vaccin thérapeutique contre le nouveau coronavirus, son procédé de préparation et son utilisation WO2021196808A1 (fr)

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