CN113521274B - COVID-19 inactivated vaccine composition and application thereof - Google Patents
COVID-19 inactivated vaccine composition and application thereof Download PDFInfo
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- CN113521274B CN113521274B CN202010308114.8A CN202010308114A CN113521274B CN 113521274 B CN113521274 B CN 113521274B CN 202010308114 A CN202010308114 A CN 202010308114A CN 113521274 B CN113521274 B CN 113521274B
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Abstract
The invention belongs to the technical field of biology, in particular to the technical field of epidemic prevention and vaccine production, and particularly discloses COVID-19 inactivated vaccine composition and application thereof. The COVID-19 inactivated vaccine prepared by using the CpG oligodeoxynucleotide adjuvant can generate a strong immune response effect, can greatly prolong the residence time of the OVID-19 inactivated vaccine in vivo, and reduces the hydrolysis of various hydrolases. The preparation method of the inactivated vaccine is simple, the quality is easy to control, and the mass production is easy; the medicine has good safety and low toxic and side effects, and is particularly suitable for preventing and treating COVID-19 in various crowds including the crowds with low immune functions such as middle-aged and elderly people.
Description
Technical Field
The invention belongs to the technical field of biology, in particular to the technical field of epidemic prevention and vaccine production, and particularly relates to a COVID-19 inactivated vaccine composition and application thereof.
Background
The novel coronavirus COVID-19 is a novel coronavirus which is newly appeared, belongs to the coronavirus beta family, can be transmitted through the paths of respiratory tract spray transmission, contact, faeces-oral transmission and the like, and is extremely easy to infect in people.
In the development of vaccines, the use of adjuvants must be considered in order to prepare a safe and effective vaccine. The adjuvant is compatible with vaccine components, and can form stable, safe and immunogenic vaccine composition. Thus, research into adjuvants for use in vaccines has been an important step in the research process of vaccines. Adjuvants as nonspecific immunopotentiators play a critical role in inducing an effective immune response after vaccination.
Aluminum salt adjuvants are conventionally commonly employed, including aluminum hydroxide and aluminum phosphate and combinations thereof, are most widely used. Although the above-mentioned adjuvants are widely approved for use in vaccine preparation, in practical applications, the use of low-dose aluminum salt adjuvants alone has limited immunopotentiation effect on vaccines, while increased doses of aluminum salt adjuvants often have side effects such as swelling at injection site, granuloma, fever, pain, allergy, etc. Therefore, there is an urgent need to develop an ideal vaccine that is broader, safer, more efficient, and at the same time, easier to produce and use.
Disclosure of Invention
In order to solve the technical problems, the invention provides the following technical scheme.
In a first aspect, the present invention provides a COVID-19 inactivated vaccine composition comprising a CpG oligodeoxynucleotide as an adjuvant.
Further, as a preferred embodiment of the present invention, the COVID-19 inactivated vaccine composition further comprises COVID-19 inactivated strain.
As a preferred embodiment of the present invention, all nucleotides of said CpG oligodeoxynucleotide are thio-modified and said CpG oligodeoxynucleotide comprises at least 2 CpG units with a chain length of at least 20bp; further preferably, the CpG oligodeoxynucleotide sequence is:
5'-TGACTGTGAACGTTCGAGATGA-3'; or (b)
5'-TCGACGTTCGTCGTTCGTCGTTC-3'; or (b)
5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’。
As a preferred embodiment of the present invention, the COVID-19 inactivated strain has a fiber protein, an envelope protein, and a membrane protein. The COVID-19 inactivated virus strain is prepared by inactivating COVID-19 virus strain by using formalin or beta propiolactone.
As a preferred embodiment of the invention, the COVID-19 inactivated strain is cultured in vitro with Vero cells prior to inactivation of the COVID-19 strain.
As a preferred embodiment of the invention, the content of COVID-19 inactivated virus strains in the inactivated vaccine composition is 0.5-20 mug/dose; further preferred, the content of said COVID-19 inactivated strain in said inactivated vaccine composition is 1 μg/dose, 2 μg/dose, 3 μg/dose or 10 μg/dose.
As a preferred embodiment of the present invention, the content of the CpG oligodeoxynucleotide adjuvant in the inactivated vaccine composition is 0.25-5 mg/dose; further preferred, the content of CpG oligodeoxynucleotide adjuvant in the inactivated vaccine composition is 0.25 mg/dose, 1 mg/dose, 2 mg/dose or 5 mg/dose.
As a preferred embodiment of the present invention, the inactivated vaccine composition is a liquid vaccine, and more preferably an intramuscular liquid injection, a nasal liquid injection, an intradermal liquid injection or a subcutaneous liquid injection.
In a second aspect, the invention provides the use of an inactivated vaccine composition as described in the manufacture of a medicament for the prophylaxis and/or treatment of a disease caused by COVID-19 infection. Further preferred, the disease is pneumonia and syndrome, severe acute respiratory infection, intestinal disease, heart failure, renal failure or severe acute respiratory syndrome.
In a third aspect, the present invention provides the use of a CpG oligodeoxynucleotide as an adjuvant for a COVID-19 inactivated vaccine composition, all of the nucleotides of the CpG oligodeoxynucleotide being thio-modified and the CpG oligodeoxynucleotide comprising at least 2 CpG units, the chain length of which is at least 20bp; preferably, the CpG oligodeoxynucleotide sequence is:
5'-TGACTGTGAACGTTCGAGATGA-3'; or (b)
5'-TCGACGTTCGTCGTTCGTCGTTC-3'; or (b)
5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’。
CpG refers to dinucleotides composed of cytosine (C) and guanine (G) linked via phosphodiester bonds (phosphodiesterbonds, p), and CpG dinucleotides and two bases at the 5 'and 3' ends thereof form a CpG motif (CpG motifs). CpG motifs are also known as immunostimulatory sequences (immunostimulatory sequence, ISS), whereas CpG ODNs refer to oligodeoxynucleotides containing unmethylated CpG motifs. Because CpG dinucleotides occur less frequently and are mostly methylated in the genome of vertebrates, whereas CpG dinucleotides occur more frequently and are mostly unmethylated in the genome of bacteria, the immune system of vertebrates recognizes CpG DNA in the genome of bacteria as a dangerous stimulus signal through pattern recognition receptors (pattern recognition receptors, PRRs), thereby stimulating the organism to generate immune protection reaction.
The CpG oligodeoxynucleotide is used as an adjuvant of COVID-19 inactivated vaccines, so that the effects of enhancing immunity, prolonging the immunization time and reducing the antigen consumption can be achieved.
Preferably all nucleotides of the CpG oligodeoxynucleotide are thio-modified and the CpG oligodeoxynucleotide contains at least 2 CpG units and has a chain length of at least 20bp; further preferably, the CpG oligodeoxynucleotide sequence is:
5'-TGACTGTGAACGTTCGAGATGA-3'; or (b)
5'-TCGACGTTCGTCGTTCGTCGTTC-3'; or (b)
5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’。
In terms of immune response mechanism, cpG ODN can promote maturation and activation of dendritic cells, macrophages and B cells, up-regulate expression of CD80, CD86, CD40 and MHC-II molecules, promote secretion of IL-6, IL-12, IFN-gamma and other Thl type cytokines, and induce organisms to generate Thl type immune response. In addition, cpG ODN is recognized by and bound to TLR9 on endosomes/lysosomes in cells after uptake into immune cells via endocytosis, resulting in dimerization of TLR9, ultimately causing related immune cells to secrete a range of cytokines and chemokines, triggering intracellular bactericidal mechanisms or inducing inflammatory responses, producing strong CTL induction. Furthermore, th 2-type immunostimulation can also be reduced by avoiding the use of aluminium salt adjuvants, thereby reducing the risk of ADE.
The CpG oligodeoxynucleotide adjuvant and COVID-19 inactivated vaccine are combined according to the formula, so that a strong immune response effect can be generated, the retention time of the OVID-19 inactivated vaccine in the body can be prolonged to a great extent, and the hydrolysis effect of various hydrolases on the OVID-19 inactivated vaccine is reduced. The preparation method is simple, the quality is easy to control, and the mass production is easy to realize; the medicine has good safety and low toxic and side effects, and is particularly suitable for preventing and treating COVID-19 in various crowds including the crowds with low immune functions such as middle-aged and elderly people.
The preparation process of the COVID-19 inactivated virus strain in the COVID-19 inactivated vaccine composition provided by the invention comprises the following steps: the COVID-19 virus strain separated from clinical patients is subjected to industrial large-scale culture to obtain high-concentration COVID-19 virus solution, and an inactivation method and a purification method for effectively inactivating COVID-19 are successfully explored to prepare COVID-19 inactivated virus strain.
Wherein the inactivation method comprises inactivation with formalin or beta propiolactone. Preferably, the high concentration COVID-19 virus solution obtained is inactivated.
Inactivating by adopting formalin, wherein the concentration of the formalin is that the volume ratio of the formalin to virus liquid is 1:1000-1:4000, the inactivation time is 3-13 hours. Preferably, for safety reasons, the inactivation time is 9-39 hours or more to achieve inactivation of all viruses. Preferably, inactivation is carried out for four days with stirring at 37 ℃.
Beta propiolactone is adopted for inactivation, and the concentration of the beta propiolactone is 1 volume ratio of the beta propiolactone to the virus liquid: 4000-1:6000, the inactivation time is 16-24 hours. Preferably, for safety reasons, the inactivation time is 48-72 hours or more to achieve inactivation of all viruses. Preferably, the inactivation is carried out for 20 hours at 4℃with stirring. Then, the mixture was left at room temperature for 2 days or allowed to act at 37℃for 2 hours to decompose beta propiolactone.
The purification method of the inactivating liquid comprises the following steps:
(1) Clarifying the virus inactivating liquid;
(2) Concentrating the virus clarified liquid obtained in the step (1); and
(3) And (3) carrying out nuclease digestion treatment on the virus concentrate obtained in the step (2) to digest host DNA, thereby obtaining purified virus stock.
Preferably, in the step (1), the virus-inactivated solution is centrifuged for 30min under the centrifugal force of 2000-4000 g, sediment is removed, and the supernatant is obtained as the virus clarified solution. Preferably, centrifugation is carried out at 2-8 ℃. Or in the step (1), filtering and clarifying the virus inactivated solution through a deep membrane stack, and then filtering the solution through a 0.2-micrometer filter to obtain the virus clarified solution. Preferably, the deep layer membrane stack may be a diatomite deep layer membrane stack.
Preferably, in step (2), the virus supernatant is concentrated by ultrafiltration concentration. Preferably, the virus supernatant is concentrated by two-stage ultrafiltration, the first stage ultrafiltration preferably using a 100Kda pore size membrane pack, concentrating about 20-40 fold, and the second stage ultrafiltration preferably using a 300Kda pore size membrane pack, dialyzing 5-8 times against PBS, concentrating about 4-8 fold. Preferably, the concentration of PBS is 0.01 mole and the pH is 7.4.
Preferably, the step (2) is followed by a density gradient centrifugation of the purified virus. Preferably, sucrose density gradient centrifugation is used. The high sucrose concentration is 50-60% wv, the low sucrose concentration is 25-35% wv, and the ultra-chaotropic liquid is obtained by centrifugation at 28000-40000rpm for 16-20 hours at 2-8 ℃.
Preferably, chromatography is performed after density gradient centrifugation, preferably by using Sepharose four fast flow to perform chromatography on the chaotropic liquid, eluting with 0.01M PBS, and collecting the first flow through peak as virus refined solution.
Preferably, after concentrating the virus concentrate about 10-fold by ultrafiltration, the host DNA is digested by adding Benzonase nuclease. In the enzyme digestion, the concentration of the Benzonase is 25-50U/ml, the mixture is stirred for 2-4 hours, the mixture is kept stand for 14-20 hours, and the superfluous Benzonase nuclease is removed by ultrafiltration.
Preferably, the virus stock is obtained by sterilizing with a 0.22 μm membrane, filtering, or sterilizing with irradiation. Irradiation sterilization the samples were irradiated with a dose of cobalt 60 of 4-10 kgy for 60 minutes.
In one aspect, the COVID-19 strain has the Spike protein (hereinafter abbreviated as S protein), envelope protein and membrane protein of COVID-19 virus, and is capable of causing the humoral immune response of the body to produce neutralizing antibodies.
In another aspect, the COVID-19 strain is an inactivated strain. The Vero-E6 cells or Vero cells after inoculation do not cause lesions in Vero-E6 cells or Vero cells. The use of Vero cells as a substrate for in vitro culture COVID-19 viruses may be selected for planar culture, e.g., using cell factories, or for suspension culture, e.g., using microcarriers in a cell fermenter.
The preparation method of the inactivated vaccine provided by the invention comprises the following steps:
(1) The COVID-19 virus strain separated from clinical patients is subjected to industrial large-scale culture to obtain high-concentration COVID-19 virus liquid;
(2) Inactivating COVID-19 virus liquid to obtain an inactivated COVID-19 virus inactivated liquid;
(3) Purifying the inactivated solution to obtain purified virus stock solution; and
(4) And mixing the virus stock solution with the CpG oligodeoxynucleotide adjuvant to obtain the inactivated vaccine.
The COVID-19 inactivated vaccine composition provided by the invention can be used for preparing medicines for preventing or treating diseases caused by COVID-19 infection by combining the CpG oligodeoxynucleotide adjuvant and the antigen active ingredient COVID-19 inactivated virus strain, and can effectively induce organisms to generate cellular immunity and humoral immunity. Such as pneumonia and syndromes caused by COVID-19 infections, severe acute respiratory infections, intestinal diseases, heart failure, renal failure or severe acute respiratory syndromes.
The inactivated vaccine provided by the invention can be rapidly prepared, and is suitable for controlling COVID-19 epidemic situations.
The vaccine provided by the invention is preferably a liquid vaccine, and can be in various dosage forms. In particular, the inactivated vaccine may be an intramuscular liquid injection, a intranasal liquid spray, an intradermal liquid injection or a subcutaneous liquid injection. In practical application, the preparation can be adjusted and selected according to clinical requirements such as transfection efficiency, local immunity monitoring and the like, for example, single dosage form is selected for injection immunization, or multiple mixed dosage forms are selected for injection immunization.
The invention determines the most efficient proportioning scheme by researching the combination proportion of the CpG oligodeoxynucleotide adjuvant and COVID-19 inactivated virus strains. The COVID-19 inactivated vaccine provided by the invention can generate strong immune response benefit. In addition, the inactivated vaccine has the advantages of simple preparation method, easy quality control and easy mass production. The inactivated vaccine composition provided by the invention can be used for effectively preventing and treating pneumonia caused by COVID-19 infection, even serious respiratory tract infection, and can provide technical support for prevention and control of diseases in China or even worldwide.
Drawings
FIG. 1 is a schematic representation of serum IgG titers of COVID-19 inactivated vaccine immunized BALB/c mice with different CpG adjuvants;
FIG. 2 is a schematic diagram showing the results of specific expression of S protein cytokines in serum.
Detailed Description
The invention is further illustrated by the following examples, it being understood that the examples are given solely for the purpose of illustration and are not intended to be limiting, as the invention is susceptible of simple modification within the spirit of the invention and is intended to be within the scope of the invention.
Example 1
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination.
The method comprises the following steps: the CpG oligodeoxynucleotide adjuvant is added into the inactivated and purified vaccine stock solution (i.e. virus stock solution) filtered by a 0.22 mu m filter membrane, so that the concentration of the adjuvant is 1 mg/dose, and the content of vaccine antigen (i.e. COVID-19 inactivated virus strain content) is 0.5 mu g/dose. The vaccine number obtained in this example is designated 20200401.
Example 2
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 1 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 1 mu g/dose. The vaccine number obtained in this example is designated 20200402.
Example 3
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 1 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 2 mu g/dose. The vaccine number obtained in this example is designated 20200403.
Example 4
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 1 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 4 mu g/dose. The vaccine number obtained in this example is designated 20200404.
Example 5
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 3 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 0.5 mu g/dose. The vaccine number obtained in this example is designated 20200405.
Example 6
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 3 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 1 mu g/dose. The vaccine number obtained in this example is designated 20200406.
Example 7
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 3 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 2 mu g/dose. The vaccine number obtained in this example is designated 20200407.
Example 8
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 3 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 4 mu g/dose. The vaccine number obtained in this example is designated 20200408.
Example 9
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 0.5 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 0.5 mu g/dose. The vaccine number obtained in this example is designated 20200409.
Example 10
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 0.5 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 1 mu g/dose. The vaccine number obtained in this example is designated 20200410.
Example 11
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 0.5 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 2 mu g/dose. The vaccine number obtained in this example is designated 20200411.
Example 12
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 0.5 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 4 mu g/dose. The vaccine number obtained in this example is designated 20200412.
Example 13
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 5 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 0.5 mu g/dose. The vaccine number obtained in this example is designated 20200413.
Example 14
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 5 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 1 mu g/dose. The vaccine number obtained in this example is designated 20200414.
Example 15
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 5 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 2 mu g/dose. The vaccine number obtained in this example is designated 20200415.
Example 16
The embodiment provides an inactivated vaccine, which uses CpG oligodeoxynucleotide as an adjuvant and COVID-19 inactivated virus strain combination. The vaccine is a liquid vaccine, cpG oligodeoxynucleotide adjuvant is added into vaccine stock solution filtered by a 0.22 mu m filter membrane, the concentration of the adjuvant is 5 mg/dose, and the content of vaccine antigen (namely COVID-19 inactivated virus strain content) is 4 mu g/dose. The vaccine number obtained in this example is designated 20200416.
The adjuvant CpG oligodeoxynucleotide sequences used in the above examples were:
5’-TGACTGTGAACGTTCGAGATGA-3’。
Example 17: immunological evaluation of adjuvant Effect on mouse model
1. Neutralizing antibody titer determination
The 8 groups of vaccines provided in examples 1-8 were diluted 1:4, 1:16, 1:64 with physiological saline, respectively, and BALB/c mice were vaccinated using 1mL syringe, 10 mice per group were immunized on day 0,7, one dose per intraperitoneal injection, 4 weeks after the 1 st immunization, and the neutralizing antibodies were detected. Neutralizing antibody titers GMT greater than 8 were considered positive and were considered protective. The measurement results are shown in Table 2.
Table 1 shows the experimental design of the immunological evaluation of the adjuvant effect on the mouse model.
TABLE 1
Table 2 shows the neutralization titers of COVID-19 inactivated vaccine immunized BALB/c mice with different CpG adjuvants.
TABLE 2
As can be seen from the results in Table 2, COVID-19 vaccine immunization of BALB/c mice with the different effective components and adjuvant contents resulted in sufficiently high neutralizing antibody titers. Vaccines with high adjuvant content are relatively more immunogenic than vaccines with low content of the same active ingredient. The invention provides a vaccine which can induce BALB/c mice to generate neutralizing antibodies with protective capability.
2. IgG antibody titer determination
The 8 groups of vaccines provided in examples 1-8 were subjected to IgG antibody titer determinations as shown in table 1. The method for detecting the titer of the IgG antibody is as follows:
COVID-19 virus is coated on an ELISA plate according to the protein concentration of 1 mug/ml and 100 mug/Kong Baobei at the temperature of 2-8 ℃ overnight or 37 ℃ for more than 2 hours, washed and patted dry, blocked by 0.01M PBS containing 1% BSA or 10% calf serum, blocked by 200 mug/hole, blocked for 1-2 hours at 37 ℃, and the liquid is thrown off and drained for standby. Serial dilution is carried out on the sample to be detected and negative serum control by adopting the sealing liquid, 100 mu l of each hole is added with a sealed ELISA plate, incubation is carried out for 60-70 minutes at 37 ℃, and the plate is washed and dried; adding HRP enzyme-labeled antibody corresponding to the anti-species, incubating for 45-60 minutes at 37 ℃, washing the plate and beating; after 50. Mu.l of each of the color-developing solutions A/B was added and the color development was performed at 37℃for 10-15 minutes, the addition of 2MH2SO4 was stopped. Analysis of results: the highest dilution factor when the OD value of the sample is more than or equal to 2.1 times of the OD value of the negative serum under the same dilution factor is the IgG antibody titer of the sample. If the negative control OD value is less than 0.05, it is calculated as 0.05.
The results are shown in FIG. 1, and FIG. 1 is a graph of serum IgG titers of mice immunized with BALB/c by COVID-19 inactivated vaccine with different CpG adjuvants.
Example 18: cytokine detection
The vaccines in Table 1 were immunized according to the procedures of 0 and 7 days, and 28 days of blood collection, BALB/c mice were immunized respectively, and cell supernatants were collected for cell immunodetection. And detecting CD4+T cells which specifically express the S protein cytokines in serum by adopting an intracellular cytokine staining method and a flow cytometry method. Specifically, the following is described.
(1) Separation of spleen lymphocytes
Mice were sacrificed by cervical dislocation and immersed in 70% alcohol for about 3min. The mice were aseptically removed from the spleens in a biosafety cabinet and placed on a 200 mesh cell sieve placed in a sterile dish. 10mL of RPMI1640 complete medium was added, the spleen was gently ground into single cells with a syringe plunger, and the cell sieve was rinsed with 10mL of RPMI1640 complete medium to obtain more spleen cells. The spleen cell suspension was transferred to a 50mL centrifuge tube and centrifuged at 500g level for 5min. The supernatant was discarded, the cells resuspended in 3m1×erythrocyte lysate, lysed at room temperature for 5min, added 27mL of RPM 1640 complete medium and centrifuged at 500g level for 5min. The supernatant was discarded, the cells were washed once with 20mL of complete medium of RPM 1640, resuspended in appropriate amount of medium, filtered through a 200 mesh cell sieve into 10mL tubes, and 50. Mu.L diluted 20-fold were counted for use.
(2) In vitro stimulation of mouse spleen cells
The above isolated mouse spleen cells were diluted to 4X 10 6 cells/mL in appropriate amounts and added to a 24-well plate at 0.5mL per well. Each mouse was provided with a specific CTL epitope stimulation well and no stimulation well, respectively. Specific epitope concentration was 2 μg/mL per peptide, and equivalent amount of DMSO was added without stimulation. As a positive control, wells were stimulated with PMA and ionomycin, where the PMA concentration was 100ng/mL and ionomycin concentration was 1. Mu.g/mL. While 1. Mu. L Brilliant Violet. Mu.421. TM. Anti-mouse CD107a was added to each well. After culturing the cells in a 5% CO 2 cell incubator at 37℃for 1 hour, an appropriate amount of GolgiStop and/or GolgiPlug was added to each well as a blocker of cytokine secretion. Staining of the relevant antigen was performed after a total of 6 hours of incubation for flow cytometry detection of intracellular cytokines.
(3) Cell surface antigen and intracellular cytokine staining
After 6 hours of in vitro stimulation, splenocytes were transferred to a flow tube and centrifuged at 500g for 5 minutes at 4℃and the supernatant discarded. A suitable amount of the fluorescent-labeled antibodies PerCP/Cy5.5-conjugated anti-CD3 (clone 145-2c 11) and FITC conjugated anti-CD8 (clone 53-6.7) was diluted with PBS+2% FBS in the amounts recommended in the instructions, 50. Mu.L of each tube was added, gently mixed, and left at 4℃for 30 minutes. After 30 minutes, 3mL PBS+2% FBS was added to each tube, and 500g was centrifuged for 5 minutes at 4℃and the supernatant was discarded. mu.L Cytofix/CytopermTM Fixation and Permeabilizaiton Solution was added to each tube, and the cells were fixed and perforated by placing at 4℃for 20 minutes. After 20 minutes, 1mL of 1 XPerm/WashTM Buffer was added to each tube, centrifuged at 600g for 5 minutes at 4℃and the supernatant was discarded. A suitable amount of PE conjugated anti-IFN-gamma (clone XMG 1.2) antibody was diluted with 1 XPerm/WashTM Buffer in accordance with the instructions recommended to use, 50. Mu.L of each tube was added, gently mixed and left at 4℃for 30 minutes. Finally, each tube was washed once with 1mL of 1 XPerm/WashTM Buffer and 3mL of PBS, respectively, the supernatant was discarded and resuspended in 200. Mu.L of PBS and checked on the machine. To adjust the fluorescence compensation between the dyes at the time of detection, a non-staining tube, a single-staining PerCP/Cy5.5-conjugated anti-CD3 tube, a single-staining FITC conjugated anti-CD8 tube and a single-staining PEconjugated anti-IFN-gamma tube were set, wherein the PE conjugated anti-IFN-gamma single-staining tube used positively stimulated cells.
(4) Flow cytometry detection
Flow cytometry detection was performed using BD FACS CantoTM. Firstly, regulating proper voltage of each channel, regulating fluorescence compensation among dyes by using a single fluorescence staining sample, then loading samples in sequence, and collecting data.
(5) Intracellular cytokine staining flow cytometry detection results
The detection result of intracellular cytokine staining flow cytometry shows that after the spleen cells of the immunized mice are stimulated by the epitope, CD4+ T cells can secrete a large amount of IFNgamma and IL-2 cytokines, and the expression level is obviously higher than that of a control group. The results are shown in FIG. 2.
The results in fig. 2 show that vaccines within the antigen content range provided by the present invention all elicit good cellular immunity.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Sequence listing
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Claims (8)
1. A COVID-19 inactivated vaccine composition comprising CpG oligodeoxynucleotides as an adjuvant and COVID-19 inactivated viral strain;
All the nucleotides of the CpG oligodeoxynucleotide are thio-modified, and the CpG oligodeoxynucleotide sequence is as follows: 5'-TGACTGTGAACGTTCGAGATGA-3';
The content of COVID-19 inactivated virus strains in the inactivated vaccine composition is 2-4 mug/dose, and the content of CpG oligodeoxynucleotide adjuvant is 1-3 mg/dose.
2. The inactivated vaccine composition according to claim 1, wherein the COVID-19 inactivated strain is obtained by inactivating COVID-19 strain with formalin or beta propiolactone.
3. The inactivated vaccine composition according to claim 1, wherein the COVID-19 inactivated strain is cultured in vitro using Vero cells for COVID-19 strain prior to inactivation.
4. An inactivated vaccine composition according to any one of claims 1 to 3, wherein the COVID-19 inactivated strain is present in the inactivated vaccine composition in an amount of 2 μg/dose, 3 μg/dose or 4 μg/dose.
5. An inactivated vaccine composition according to any one of claims 1 to 3, wherein the CpG oligodeoxynucleotide adjuvant content of the inactivated vaccine composition is 1 mg/dose, 2 mg/dose or 3 mg/dose.
6. An inactivated vaccine composition according to any one of claims 1 to 3, which is a liquid vaccine.
7. An inactivated vaccine composition according to any one of claims 1 to 3, wherein the vaccine is an intramuscular liquid injection, a intranasal liquid spray, an intradermal liquid injection or a subcutaneous liquid injection.
8. Use of an inactivated vaccine composition as claimed in any one of claims 1 to 7 in the manufacture of a medicament for the prophylaxis and/or treatment of a disease caused by COVID-19 infection.
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