CN113456810A - Novel anti-neocoronavirus therapeutic vaccine and preparation method and application thereof - Google Patents
Novel anti-neocoronavirus therapeutic vaccine and preparation method and application thereof Download PDFInfo
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Abstract
A novel therapeutic vaccine against new coronavirus, and its preparation method and application are provided. The invention belongs to the technical field of biological medicines, and particularly discloses a novel anti-new coronavirus therapeutic vaccine which comprises the following components in parts by weight: (1) the viroid-like particle vaccine consists of recombinant new coronavirus antigen S protein coupled liposome and an immune agonist, wherein the immune agonist is encapsulated in the recombinant new coronavirus antigen S protein coupled liposome; or (2) a virus-like particle vaccine consisting of a new coronavirus antigen S protein gene recombinant adenovirus vector and a transmembrane peptide coupled liposome encapsulated immune agonist, wherein the transmembrane peptide coupled liposome encapsulated immune agonist is prepared by encapsulating the immune agonist in the transmembrane peptide coupled liposome; and discloses a preparation method and application thereof in preparing anti-coronavirus medicines. The vaccine for resisting the new coronavirus can remarkably induce humoral immunity and protective cellular immunity, stimulate mucosal immunity, regulate the immune homeostasis of an organism, enhance the in-vivo immune antiviral function, remarkably induce the specific immune function for resisting the new coronavirus, and effectively inhibit the pneumonia of the coronavirus. Therefore, the novel vaccine has wide application prospect in preparing clinical medicines for preventing and/or treating the new coronavirus pneumonia.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a novel therapeutic vaccine for resisting novel coronavirus, a preparation method thereof and application thereof in preparing an anti-coronavirus medicament.
Background
Coronaviruses are a large family of viruses known to cause more severe diseases such as the common cold, middle east respiratory syndrome and severe acute respiratory syndrome. The novel coronavirus is a new strain of coronavirus that has not been previously discovered in humans. After people are infected with coronavirus, the common signs of the person are respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. 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 diseases caused by the novel coronavirus. Since the occurrence of new coronary pneumonia, one of the most concerned focuses from medical care personnel, experts and scholars to the general public is the treatment and medicine which can be effective on new coronary pneumonia. At present, no specific therapeutic drug for coronavirus pneumonia (COVID-19) exists, no preventive drug with definite curative effect is found, and clinical tests of various possible effective drugs are rapidly carried out. The new coronaviruses have 85 percent of homology with SARS and MERS, and different viruses can have common target spots, so that the exploration of new application of old medicines becomes a relatively rapid strategy under the condition of no specific medicines.
The development of vaccines against new coronaviruses has become a hot world area, and there are many types of vaccines currently developed, including recombinant DNA vaccines, RNA vaccines, protein vaccines, and the like. The vaccine clinical experiments are carried out in China, America and Germany, the new coronavirus vaccine which is firstly clinically applied in China is a recombinant DNA vaccine, the RNA vaccine which is clinically researched in the America is a prophylactic antiviral vaccine which is applied to healthy people. The vaccine is still in the process of development and approval.
Natural pathogen or viral infection or vaccines thereof can elicit broad immunity, in addition to inducing humoral immunity, also resident memory T cells (TRM cells) in the lung. A delicate balance between safety and immunogenicity of "replicative" vaccines must be achieved and they are only suitable for certain populations. In contrast, "non-replicating" pneumovirus vaccines induce poor T cell immunity in the respiratory tract and require potent mucosal adjuvants to overcome the immunoregulatory mechanisms 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 major immune mediators of protective immunity against viral infections and can be strongly induced by pneumoviral infection of Alveolar Epithelial Cells (AECs) as well as immune cells. Therefore, the interferon gene stimulating factor (STING) in both cell types may be activated by viral infection or by the immune response induced by a replicating vaccine. However, delivery of STING agonists into the AEC cytosol remains a significant challenge without disrupting the integrity of the lung surface active (PS) layer, since a strong barrier is formed outside the lung epithelium to prevent the entry of nanoparticles and hydrophilic molecules.
Microbial and viral DNA in infected mammalian cells can induce endogenous potent immune responses by stimulating interferon secretion. The immune response of the Endoplasmic Reticulum (ER) receptor protein (STING) to cytoplasmic DNA is an essential factor. Recent studies have shown that cyclic cGMP-AMP dinucleotide synthetase (cGAS) endogenously catalyzes cGAMP synthesis under activated conditions upon DNA binding. cGAMP is a cytosolic DNA sensor that acts as a second messenger to stimulate the induction of INF-I by STING, mediating the activation of TBK1 and IRF-3, which in turn initiates transcription of the INF- β gene. STING is a transmembrane protein of the endoplasmic reticulum, which has a hydrolase enzyme ENPP 1. ENPP1 hydrolase has a rather broad substrate specificity, including ATP and NAD+Experiments have shown that 2'3' -cGAMP is also a substrate for ENPP 1. How to increase the effective metabolic time of an immune agonist and reach lung cells quickly is a difficult challenge for scientists.
The primary role of vaccines is to prevent disease. Aiming at normal human bodies and animal bodies, the vaccine has the function of enhancing the disease prevention and resistance of organisms and achieving the purpose of preventing diseases; in the case of human and animal subjects suffering from a disease or condition, the therapeutic vaccine is intended to induce the subject to respond to a particular pathogenic factor, thereby eliminating the disease or condition. Therefore, the development of novel therapeutic vaccines against the new coronavirus is urgently needed, which can prevent and/or treat the inflammation of the new coronavirus by influencing the immune system of the body.
Disclosure of Invention
The invention provides a novel therapeutic vaccine against new coronavirus and a preparation method thereof. The therapeutic vaccine for resisting the new coronavirus can remarkably induce the specific immune function for resisting the new coronavirus, effectively inhibit viral inflammation, stimulate mucosal immunity, regulate the immune steady state of an organism and enhance the immune and antiviral functions in vivo.
A novel therapeutic vaccine against neocoronavirus is disclosed
(1) A viroid-like particle vaccine consisting of recombinant neocoronavirus antigen S protein-coupled liposomes and an immune agonist, said immune agonist being encapsulated in the recombinant neocoronavirus antigen S protein-coupled liposomes; or
(2) A viroid-like particle vaccine consisting of a recombinant adenovirus vector of a new coronavirus antigen S protein gene and a transmembrane peptide-coupled liposome-encapsulated immune agonist, wherein the transmembrane peptide-coupled liposome-encapsulated immune agonist is prepared by encapsulating the immune agonist in a transmembrane peptide-coupled liposome;
the immune agonist is an agonist of a natural immune pathway (cGAS-STING-cGAMP-IRF3 pathway) STING or a transition metal complex thereof, and the agonist of STING is cyclic dinucleotide 2'3' -cGAMP or a derivative thereof;
the recombinant new coronavirus antigen S protein is a COVID-19 virus S protein or a structural domain derivative of the COVID-19 virus S protein;
the transmembrane peptide is a membrane targeting peptide or a targeting membrane vesicle associated protein;
the new coronavirus antigen S protein gene recombinant adenovirus vector is as follows: recombinant adenovirus vector with recombinant COVID-19 virus S protein gene or structural domain gene of COVID-19 virus S protein and deletion of early expression gene sequence E1 and E3 region of adenovirus.
Preferably, domain derivatives of the COVID-19 virus S protein include, but are not limited to, RBD-SD1 or RBD-SD1SD 2.
Preferably, the membrane targeting peptide is a section of transmembrane peptide gH625 of herpes simplex virus glycoprotein, and the amino acid sequence is HGLASTLTRWAHYNALIRAFGGG, SEQ ID NO 1;
the nano antibody of the targeting membrane vesicle associated protein is anti-PV1Nb, and the amino acid sequence is QVQLQQSGAE LVKPGASVKLSCKASGYTFTDYYMYWVKQPPGQGLELIGEINPTNGDVNFNEMFKSK ATLTVDTSSRTAYMQLSSLTSEDSAVYYCTSIHYWGQGTLVTVSAGSG, SEQ ID NO: 2.
Preferably, the transition metal complex of the agonist of STING is prepared by: heating, refluxing and stirring the transition metal ion metal salt and the agonist of STING in a water/alcohol mixed solvent, standing overnight, filtering, and purifying the product by an ion exchange column.
The preparation method of the novel anti-new coronavirus therapeutic vaccine comprises the following steps:
(1) carrying out sulfhydrylation on the recombinant new coronavirus antigen S protein;
(2) the thiolated recombinant neocoronavirus antigen S protein is fused to a liposomal chemical bond and encapsulates an immune agonist.
The preparation method of the novel anti-new coronavirus therapeutic vaccine comprises the following steps:
(1) carrying out sulfhydrylation on the membrane targeting peptide or the nano antibody of the targeting membrane vesicle associated protein to obtain sulfhydrylation transmembrane peptide;
(2) fusing thiolated transmembrane peptide with a liposome chemical bond to encapsulate the immune agonist, thereby obtaining the transmembrane peptide coupled liposome encapsulated immune agonist;
(3) the transmembrane peptide coupling liposome encapsulated immune agonist is mixed with a new coronavirus antigen S protein gene recombinant adenovirus vector.
The novel anti-new coronavirus therapeutic vaccine is applied to the preparation of the drugs for preventing and/or treating coronavirus infection diseases.
Preferably, the disease caused by coronavirus infection includes, but is not limited to, viral pneumonia, viral nephritis, viral encephalitis, viral enteritis, or viral hepatitis caused by human or animal infection with coronavirus.
Preferably, the vaccine can be prepared into unit preparations with different specifications or pharmaceutical preparations by pharmaceutically acceptable carriers.
Preferably, the medicament for preventing and/or treating the coronavirus infection disease comprises an intravenous injection preparation, a nasal cavity drip preparation, an intravenous drip preparation, an intramuscular injection preparation, a subcutaneous injection preparation or an oral preparation; oral formulations include, but are not limited to, capsules, tablets, or granules.
The invention comprehensively researches and optimizes the functions and advantages of the natural immune agonist, the transmembrane peptide and the liposome, optimizes and forms a novel compound, can avoid the too fast degradation of the immune agonist in vivo, can quickly target immune cells and epithelial cells of the lung, and inhibits viral inflammation.
The research of the invention shows that the novel therapeutic vaccine for resisting the new coronavirus has potential application prospect in the drugs for preventing and treating the coronavirus, and can be used for preventing and treating various coronavirus infection diseases including viral inflammations such as new coronavirus pneumonia and the like.
Detailed Description
The present invention will be described in detail with reference to examples. In the present invention, the following examples are given to better illustrate the present invention and are not intended to limit the scope of the present invention.
Example 1: preparation of novel therapeutic vaccines against neocoronavirus
(1) Preparation of immune agonists
The 2'3' -cGMAP serving as the cyclic-G-MP dinucleotide serving as the immune stimulant is synthesized by catalyzing cyclic-G-MP-AMP dinucleotide synthetase under the activation condition after DNA is combined according to a literature method, and the purity is over 98 percent.
Cyclic dinucleotide 2'3' -cGAMP Metal Complex ([ M (cGAMP))]M ═ Zn, Mn, etc. transition metal ions) from transition metal ion metal salts (MnCl)2·4H2O/or ZnCl21mmol) and cyclic dinucleotide 2'3' -cGAMP (1mmol) are heated, refluxed and stirred in a water/alcohol mixed solvent to react for 6 hours, and then the mixture is stood overnight and filtered, and then the product is purified by an ion exchange column to obtain the pure immune agonist metal complex. The cyclic dinucleotide 2'3' -cGAMP metal complex is verified by metal content analysis and element analysis.
MncGAMP: chemical formula MnC20H22N10O13P2Molecular 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 ZnC20H22N10O13P2Molecular weight 737, elemental analysis percentage (theoretical) (percent): c,32.28 (32.56); h,2.69 (2.98); n,18.68 (18.99); zn,8.46 (8.82).
(2) Preparation of recombinant new coronavirus antigen S protein (or membrane targeting peptide, or targeting membrane vesicle associated protein nano-antibody)
The RBD domain of the S protein (Spike protein) of the COVID-19 virus (S protein, amino acid sequence domain 319-541) is determined in accordance with the literature (Jun Lan et al, Crystal structure, from 2019-nCoV Spike receiver-binding domain bound with the ACE2 receiver, BioRxiu, doi:https:// orig/10.1101/2020.02.19.956235).
The COVID-19 virus S protein or the RBD-SD1 structural domain (the 319-591 amino acid sequence structural domain of the S protein) or the RBD-SD1SD2 structural domain (the 319-732 amino acid sequence structural domain of the S protein) is prepared by the method, the S protein gene or the RBD-SD1 structural domain gene or the RBD-SD1SD2 structural domain gene is used for replacing the RBD structural domain gene, and the expression and purification methods are the same.
The transmembrane peptide gH625 contains 23 amino acid residues (H)2N-HGLASTLTRWAHYNALIRAFGGG-CONH2) The molecular weight was 2461Da and was synthesized by Biotech on a solid phase.
The nano antibody (Anti-PV1Nb) gene expression vector plasmid of the targeting membrane vesicle associated protein is prepared by synthesis of Shanghai biological gene company, the expression vector plasmid adopts pET-22b (+) and carries Amp + resistance, the tail end of a protein sequence is marked with 6His-tag to assist in purification, an escherichia coli high-efficiency expression system is used, and a protein purification method is used for purification by using an affinity column NiNTA, wherein the purity is up to 98%. And storing the freeze-dried powder in an ultra-low temperature refrigerator for later use.
(3) Preparation of novel vaccine I (VFI)
First, the COVID-19 virus S protein RBD-SD1 domain was subjected to terminal thiolation (EDTA solution was added to make the final concentration of EDTA 5mM, and a thiolation reagent (Traut' S reagent, available from Sigma) was added dropwise with stirring), and after stirring in a water bath, the mixture was incubated for 1 hour with exclusion of light, and excess thiolation reagent was removed with a desalting column).
The Ellman method is used for determining the sulfydryl on the RBD-SD1 protein, and the success of sulfhydrylation of the RBD-SD1 is verified.
Dissolving a liposome material (comprising lecithin, cholesterol, 1, 2-distearoyl-SN-glycerol-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000, mixed according to a molar ratio of 57:38:4: 1) in methanol: in chloroform (1:9(v/v)) solvent, forming a film by vacuum spin-drying in a water bath at 40 ℃; 250mM (NH) was added to a 65 ℃ water bath4)2SO4Hydrating to blank liposome. And extruding the mixture by a liposome extruder through a 200nm polycarbonate microporous filter membrane to prepare the uniform single-chamber blank liposome.
MncGAMP solution was added to the single-chamber blank liposome, incubated at 60 ℃ for 1h, added with terminally thiolated RBD-SD1 (1mg blank liposome: 20. mu.gBD-SD 1), and incubated overnight at room temperature in the absence of light. Unencapsulated immune agonist and unattached RBD-SD1 were removed using a 30kD concentrator tube at 4000rpm and 4 ℃.
The obtained compound is detected by a TEM electron microscope, the double-layer round vesicle has good shape, the diameter of the liposome is about 185nm, and the Zeta potential is minus 23 mV. The immune agonist has an entrapment rate of 75%, is stable under a refrigeration condition at 4 ℃, and is prepared into freeze-dried powder by using 2.5% trehalose solution for refrigeration and preservation.
The method is suitable for replacing other immune agonists and other recombinant new coronavirus antigen S proteins in various combinations.
(4) Preparation of transmembrane peptide (gH625 or Anti-PV1Nb) coupled Liposomal Encapsulated immune agonists
According to the preparation method of the above (3), the RBD-SD1 protein is replaced by the transmembrane peptide gH625 or Anti-PV1Nb, and the transmembrane peptide is first subjected to terminal thiolation (EDTA solution is added to the transmembrane peptide solution so that the final concentration of EDTA is 5mM, a thiolation reagent Trut's reagent is added dropwise under stirring, and after stirring in a water bath, incubation is carried out for 1 hour in the absence of light, and excess thiolation reagent is removed by a desalting column).
The success of thiol-ylation of the transmembrane peptide was verified by measuring thiol groups on the transmembrane peptide by the Ellman method.
Dissolving liposome material (including lecithin, cholesterol, 1, 2-distearoyl-SN-glycerol-3-phosphorylethanolamine-N-maleimide-polyethylene glycol 2000) in methanol/chloroform mixed solvent, and adding waterVacuum rotary evaporation in bath to dry and form film, then adding (NH)4)2SO4Hydrating to obtain blank liposome. And extruding the mixture by a liposome extruder through a 200nm polycarbonate microporous filter membrane to prepare the uniform single-chamber blank liposome.
To a single-compartment blank liposome was added the immune agonist solution, incubated at 60 ℃ for 1h, added the terminal thiolated transmembrane peptide (1mg blank liposome: 20. mu.g gH625 or Anti-PV1Nb), and incubated overnight at room temperature in the absence of light. Unencapsulated immune agonist drug and unattached transmembrane peptide gH625 or Anti-PV1Nb were removed using a 30kD concentrator tube at 4000rpm at 4 ℃.
The obtained complex (gH625 coupled liposome encapsulated MncGAMP, immune agonist MncGAMP and transmembrane peptide gH625) is detected by a TEM (Transmission Electron microscope), and the double-layer round vesicle has good shape, the diameter of the liposome is about 175nm, and the Zeta potential is minus 22 mV. The immune agonist has an entrapment rate of 78%, is stable under a refrigeration condition at 4 ℃, and is prepared into freeze-dried powder by using a 3% trehalose solution for refrigeration and preservation.
(5) Preparation of new coronavirus antigen S protein gene recombinant adenovirus vector
The new coronavirus antigen S protein gene recombinant adenovirus vector is completed by outsourcing service of biological medicine science and technology company, and is constructed by adopting a recombinant adenovirus vector which lacks early expression gene sequences E1 and E3 of adenovirus:
inserting a target gene (COVID-19 virus S protein gene or structural domain gene of COVID-19 virus S protein) into an adenovirus shuttle plasmid (recombinant adenovirus vector plasmid deleting adenovirus early expression gene sequences E1 and E3 regions), cotransfecting the correctly determined recombinant adenovirus shuttle plasmid and a skeleton plasmid (deleting adenovirus early expression gene sequences E1 and E3 regions), packaging in 293A cells, performing adenovirus amplification and CsCl purification, and then performing quality inspection on the packaged recombinant RBD-SD1 adenovirus vector. Quality control involves PCR and WB of the final product viral genes to confirm the presence of the gene of interest.
(6) Novel vaccine II (VFII)
The gH 625-coupled liposome-encapsulated MncGAMP was mixed with a recombinant RBD-SD1 adenovirus vector (200. mu.g MncGAMP: heavyGroup RBD-SD1 adenovirus vector 1X 108Adenovirus particles). Finally, the mixture is prepared into freeze-dried powder by using a 3% trehalose solution for refrigeration and preservation.
(7) Novel vaccine III (VFIII)
The preparation of novel vaccine III (VFIII) is carried out in the same manner as novel vaccine I (VFI) except that ZncGAMP is used instead of MncGAMP, and the other steps are the same. The obtained VFIII is detected by a TEM electron microscope, the double-layer round vesicle has good shape, the diameter of the liposome is about 187nm, and the Zeta potential is minus 24 mV. The immune agonist has an entrapment rate of 76%, is stable under a refrigeration condition at 4 ℃, and is prepared into freeze-dried powder by using a 2.5% trehalose solution for refrigeration and preservation.
(8) Novel vaccine IV (VFIV)
The preparation method of the novel vaccine IV (VFIV) is the same as that of the novel vaccine I (VFI), except that MncGAMP is replaced by cGAMP, and the antigen S protein domain RBD is replaced by the antigen S protein domain RBD-SD1, and other steps are the same.
The VFIV obtained by TEM electron microscope detection has double-layer round vesicles with good shape, liposome diameter of about 184nm and Zeta potential of-23 mV. The immune agonist has an entrapment rate of 73%, is stable under a refrigeration condition at 4 ℃, and is prepared into freeze-dried powder for refrigeration and storage by using a 2.5% trehalose solution.
(9) The obtained novel vaccine for resisting new coronavirus
Novel vaccine I (VFI), [ antigen S protein domain RBD-SD1 coupled liposome Encapsulated MncGAMP ]
Novel vaccine II (VFII), [ (gH 625-coupled liposome-encapsulated MncGAMP) recombinant RBD-SD 1-containing adenovirus vector ]
Novel vaccine III (VFIII) [ antigen S protein Domain RBD-SD1 coupled liposome Encapsulated ZncGAMP ]
Novel vaccine III (VFIV) [ antigen S protein Domain RBD conjugated Liposome Encapsulated cGAMP ]
Immune agonist complex I (FI), [ gH625 coupled liposome-encapsulated cGAMP ] (prepared as in step (4))
Immunoagonist Complex II (FII), [ gH625 coupled Liposome Encapsulated MncGAMP ] (prepared as in step (4))
Example 2 study of the specific immune function of a novel therapeutic vaccine against the New coronavirus
Experimental animals: c57BL/6 mice, male, 20-22 g in weight, 6-8 weeks old, purchased from shanghai slaike laboratory animals llc [ laboratory animal quality certification no: SCXK (Shanghai) 2007 + 0005 ]. All mice were left free to feed and drink water and were kept at room temperature (23 + -2) ° c. The feed and water are sterilized by high pressure, and the whole experimental feeding process is SPF grade.
Mouse immunization: grouping mice: every 10 groups, 9 groups in total, are respectively, A: VFI; b is VFII; c is VFIII; d: VFIV; e, FI; f: FII; g: RBD-SD 1; h, RBD; and I, PBS blank.
The administration mode comprises the following steps: and (4) nasal cavity instillation.
Administration dose:
novel vaccine I (VFI), (10mg/kg MncGAMP, 100. mu.g RBD-SD1)
Novel vaccine II (VFII), (10mg/kg MncGAMP + recombinant RBD-SD1 adenovirus vector 108)
Novel vaccine III (VFIII), (10mg/kg ZncGAMP, 100. mu. gRBD-SD1)
Novel vaccine IV (VFIV), (10mg/kg cGAMP, 100. mu. gRGD)
Immune agonist complex I (FI), (10mg/kg cGAMP)
Immune agonist complex II (FII), (10mg/kg MncGAMP)
Antigen S protein Domain RBD-SD1 (100. mu.g)
Antigen S protein Domain RBD (100. mu.g)
The mice were anesthetized, fixed in a dorsal decubitus position, and each group of the drug solution suspensions was slowly dropped through the inner walls of the nostrils of the mice in a volume of 60 μ L (30 μ L per nostril), respectively. The mouse was gently removed from the table and the head and chest were raised with a small amount of folded paper towels to ensure smooth breathing of the mouse. After the mice recovered, the mice were returned to their cages. The administration was performed once on days 1, 7 and 14, respectively, and mouse lung lavage fluid and blood samples were obtained on day 21. The titer of the antibody induced by the immune agonist compound and the vaccine compound is determined by ELISA method.
The mouse alveolar lavage fluid obtaining method comprises the following steps:
the same volume of PBS was injected along the mouse trachea and then aspirated out for several times to obtain alveolar lavage fluid. The collected serum was stored at-80 ℃.
The results are shown in Table 1. The determination result shows that the novel vaccines (VFI, VFII, VFIII and VFIV) and the immune agonist liposome complex (FI and FII) can obviously take effect or induce immune response, and the effect of the novel vaccines (VFI, VFII, VFIII and VFIV) is obviously higher than that of the immune agonist complex (FI and FII) and the single recombinant S protein domain RBD-SD 1/RBD.
TABLE 1 specific immune function potency of novel vaccines against neocoronavirus
Example 3 novel therapeutic vaccine against New coronavirus induces protective specific cellular Immunity Studies
Mice were bred, dosed, etc. as in example 2. Isotype control flow antibodies were purchased from eBiosciences, antibody magnetic strains from milteny biotech, and flow cytometers from BD. 21 days after three times of administration immunization, spleen and lung tissues of mice are taken, respectively ground and mashed, cells are removed through 40-micron holes, the cells are centrifuged at 1000rpm for 10 minutes, immune cells which are not cracked are separated, DC (CD40\ CD80\ CD86\ MHCII) and T (CD8+) cells are separated through antibody magnetic strains, corresponding FAC antibodies (diluted by FACS buffer solution) are added, isotype control antibodies serve as negative controls, the cells are incubated for 1 hour after the addition of the antibodies, centrifuged, washed by PBS, samples are analyzed by a flow cytometer, proper cells are sorted, and the fluorescence intensity (MFI) of the selected cells is measured, and the flow results are shown in Table 2. Flow cytometry results show that the novel vaccines (VFI, VFII, VFIII and VFIV) and immune agonist complexes (FI and FII) can remarkably activate dendritic cell DC and T cells, and the effect of the novel vaccines (VFI, VFII, VFIII and VFIV) is remarkably higher than that of the immune agonist complexes (FI and FII) and antigen S protein domain RBD-SD 1/RBD.
TABLE 2 novel vaccines against neocoronavirus to induce protective cellular immune effects
Example 4 inhibition of mouse coronavirus pneumonia by novel therapeutic vaccines against the New coronavirus
Experimental animals: c57BL/6 mice, male, 20-22 g in weight, 6-8 weeks old, SPF grade, from American Animals Inc., all mice were left to eat and drink water freely and were bred at room temperature (23 + -2) ° C. The feed and water are sterilized by high pressure, and the whole experimental feeding process is SPF grade.
Animal grouping: the 60 mice were randomly divided into 10 groups (n ═ 6), specifically: group a, normal control group; group B, pneumonia model group; group C, immune agonist complex, group FI; group D, immune agonist complex, FII; group E, novel vaccine VFI group; group F, novel vaccine VFII; group G, novel vaccine VFIII group; group H, novel vaccine VFIV; group I, antigen S protein domain RBD-SD 1; group J, antigen S protein domain RBD group.
Building a pneumovirus model mouse:
virus strain: strains suitable for laboratory use are available from ATCC companies in the united states: coronavirus (ATCC VR-841), and the virus experimental procedures in this study were carried out by American Animals Inc. Virus laboratories, USA.
Intranasal instillation: the mice were placed under sufficient depth of anesthesia, held in a dorsal decubitus position, and the VR-841 viral suspension was slowly instilled through the inner walls of the mouse's nostrils in a volume of 60 μ L (30 μ L per nostril) to ensure maximum lung infection efficiency. The mouse was gently removed from the table and the head and chest were raised with a small amount of folded paper towels to ensure smooth breathing of the mouse. After the mice recovered, the mice were returned to their cages. C, D, E, F, G, H, I, J groups of mice were administered once each on days 2, 8, and 15, and each group of drug solution suspensions was slowly and separately instilled through the inner walls of the nostrils of the mice to obtain lung lavage fluid and blood samples on day 21. The ELISA method is used for measuring the protective cell immunity titer induced by the immune agonist compound and the vaccine compound.
The administration mode comprises the following steps: nasal instillation;
administration dose:
novel vaccine I (VFI), (10mg/kg MncGAMP, 100. mu.g RBD-SD1)
Novel vaccine II (VFII), (10mg/kg MncGAMP + recombinant RBD-SD1 adenovirus vector 108)
Novel vaccine III (VFIII), (10mg/kg ZncGAMP, 100. mu.g RBD-SD1)
Novel vaccine IV (VFIV), (10mg/kg cGAMP, 100. mu.g RBD)
Immune agonist complex I (FI), (10mg/kg cGAMP)
Immune agonist complex II (FII), (10mg/kg MncGAMP)
Antigen S protein Domain RBD-SD1 (100. mu.g)
Antigen S protein Domain RBD (100. mu.g)
The mouse alveolar lavage fluid obtaining method comprises the following steps: the same volume of PBS was injected along the mouse trachea and then aspirated out for several times to obtain alveolar lavage fluid. The collected serum was stored at-80 ℃. The concentrations of TNF-alpha and IL-1beta are detected by ELISA method according to the kit instruction. After the reaction is stopped, the ELISA plate is placed in a groove of an ELISA reader, the wavelength of 450nm is selected for detection, a standard substance and a blank control area are determined, the corresponding optical density value is detected, then a standard curve is drawn, and the corresponding concentration is calculated. In a mouse pneumonia model, the content of proinflammatory cytokines IL-1beta and TNF-alpha in serum and alveolar lavage fluid is obviously increased, and the content of the proinflammatory cytokines IL-1beta and TNF-alpha in the serum and alveolar lavage fluid is reduced to different degrees after the novel vaccine and immune agonist compound are administered. The effect of the novel vaccine (VFI, VFII, VFIII, VFIV) is obviously higher than that of immune agonist complex (FI, FII) and antigen S protein domain RBD-SD1 or RBD. The results of the various drugs showing inhibition of pneumonia in mice are shown in table 3.
TABLE 3 therapeutic effect of novel anti-neocoronavirus vaccines on pneumonia in mice
Experimental results show that the novel anti-new coronavirus vaccines (VFI, VFII, VFIII and VFIV) and the immune agonist liposome complex (FI and FII) can inhibit proinflammatory cytokines of mouse pneumonia to different degrees, and have obvious treatment effect on mouse viral pneumonia inflammation. The results show that the novel anti-new coronavirus vaccines (VFI, VFII, VFIII and VFIV) have the effect on resisting the mouse pneumonia which is remarkably superior to that of immune agonist liposome complexes (FI and FII) and virus antigen protein domains RBD-SD1 and RBD. The novel anti-new coronavirus vaccine has the effect of resisting mouse coronavirus pneumonia.
EXAMPLE 5 acute toxicity Studies of novel therapeutic vaccines against New coronavirus
Experimental Material
40 ICR mice (purchased from Shanghaisleke laboratory animal Limited liability company [ laboratory animal quality qualification No.: SCXK (Shanghai) 2007-0005]), half of male and female animals, with the weight of 20-22 g, and the animals are fed with pellet feed and can freely eat and drink water.
Experimental methods
ICR mice were injected intraperitoneally with 1g/kg of novel therapeutic vaccines (VFI, VFII, VFIII, VFIV) against neocoronaviruses (PBS buffer) per body weight, and the mice were observed for toxicity and death within 14 days after administration. As a result, the mice were found to be normally active after intraperitoneal injection. Within 14 days after administration, the mice did not die, and on day 15, all mice were sacrificed, dissected, and examined by naked eyes for each organ, and no obvious lesion was observed.
Results of the experiment
The results of the acute toxicity experiments show that the maximum tolerated dose MTD of intraperitoneal injection administration is not less than 1g/Kg, which indicates that the acute toxicity of the two novel anti-new coronavirus therapeutic vaccines is low.
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Claims (9)
1. A novel therapeutic vaccine against neocoronavirus, characterized in that said vaccine is
(1) A viroid-like particle vaccine consisting of recombinant neocoronavirus antigen S protein-coupled liposomes and an immune agonist, said immune agonist being encapsulated in the recombinant neocoronavirus antigen S protein-coupled liposomes;
or
(2) A viroid-like particle vaccine consisting of a recombinant adenovirus vector of a new coronavirus antigen S protein gene and a transmembrane peptide-coupled liposome-encapsulated immune agonist, wherein the transmembrane peptide-coupled liposome-encapsulated immune agonist is prepared by encapsulating the immune agonist in a transmembrane peptide-coupled liposome;
the immune agonist is an agonist of STING or a transition metal complex thereof, and the agonist of STING is cyclic dinucleotide 2'3' -cGAMP or a derivative thereof;
the recombinant new coronavirus antigen S protein is a COVID-19 virus S protein or a structural domain derivative of the COVID-19 virus S protein;
the transmembrane peptide is a membrane targeting peptide or a targeting membrane vesicle associated protein;
the new coronavirus antigen S protein gene recombinant adenovirus vector is as follows: recombinant adenovirus vector with recombinant COVID-19 virus S protein gene or structural domain gene of COVID-19 virus S protein and deleted early expression gene sequence E1 and E3 region of adenovirus.
2. The novel therapeutic vaccine against a neocoronavirus according to claim 1,
the structural domain derivatives of the COVID-19 virus S protein include but are not limited to RBD, RBD-SD1 or RBD-SD1SD 2.
3. The novel therapeutic vaccine against a neocoronavirus according to claim 1,
the membrane targeting peptide is a section of transmembrane peptide gH625 of herpes simplex virus glycoprotein, and the amino acid sequence is HGLASTLTRWAHYNALIRAFGGG, SEQ ID NO: 1;
the nano antibody of the targeting membrane vesicle associated protein is anti-PV1Nb, and the amino acid sequence is QVQLQQSGAELVKPGASVKLSCKASGYTFTDYYMYWVKQPPGQGLELIGEINPTNGDVNFNEMFKSKATLTVDTSSRTAYMQLSSLTSEDSAVYYCTSIHYWGQGTLVTVSAGSG, SEQ ID NO: 2.
4. The process for the preparation of a novel therapeutic vaccine against neocoronaviruses according to claim 1, characterized in that it comprises the following steps:
(1) carrying out sulfhydrylation on the recombinant new coronavirus antigen S protein to obtain sulfhydrylation recombinant new coronavirus antigen S protein;
(2) the thiolated recombinant neocoronavirus antigen S protein is fused to a liposomal chemical bond and encapsulates an immune agonist.
5. The process for the preparation of a novel therapeutic vaccine against neocoronaviruses according to claim 1, characterized in that it comprises the following steps:
(1) carrying out sulfhydrylation on the membrane targeting peptide or the nano antibody of the targeting membrane vesicle associated protein to obtain sulfhydrylation transmembrane peptide;
(2) fusing thiolated transmembrane peptide with a liposome chemical bond to encapsulate the immune agonist, thereby obtaining the transmembrane peptide coupled liposome encapsulated immune agonist;
(3) the transmembrane peptide coupling liposome encapsulated immune agonist is mixed with a new coronavirus antigen S protein gene recombinant adenovirus vector.
6. Use of the novel therapeutic vaccine against neocoronavirus according to claim 1 for the preparation of a medicament for the prevention and/or treatment of a disease caused by coronavirus infection.
7. The use of claim 6, wherein the disease caused by coronavirus infection includes, but is not limited to, viral pneumonia, viral nephritis, viral encephalitis, viral enteritis, or viral hepatitis caused by human or animal infection with coronavirus.
8. The use according to claim 6, wherein the vaccine can be prepared separately in unit preparations of different specifications or in pharmaceutical preparations with pharmaceutically acceptable carriers.
9. The use according to claim 6, wherein the medicament for preventing and/or treating a coronavirus infection disease comprises an intravenous injection preparation, a nasal drip preparation, an intravenous drip preparation, an intramuscular injection preparation, a subcutaneous injection preparation, or an oral preparation; the oral preparation includes but is not limited to capsules, tablets or granules.
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