A novel corona covid-19 inactivated vaccine using a complex adjuvant containing a cpg oligodeoxynucleotide

Abstract

The application discloses a composite adjuvant containing CpG oligodeoxynucleotide and aluminum adjuvant, and a new coronavirus COVID-19 inactivated vaccine using the composite adjuvant. The CpG oligodeoxynucleotide is used in combination with the aluminum adjuvant, and a strong immune enhancement effect can be generated. When the composite adjuvant is applied to the new coronavirus COVID-19 inactivated vaccine, the residence time of the antigen in the body can be greatly prolonged, and the hydrolysis of the antigen by various hydrolytic enzymes is reduced. The preparation method of the new coronavirus COVID-19 inactivated vaccine provided by the application is simple, the quality is easy to control, the vaccine is easy to mass-produce, has good safety, low toxic and side effects, and is particularly suitable for being used for the prevention and treatment of the new coronavirus COVID-19 in various populations, including the low-immunity population such as the middle-aged and the old.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of epidemic prevention and vaccine production, in particular to a composite adjuvant containing CpG oligodeoxynucleotide and aluminum adjuvant and a new coronavirus COVID-19 inactivated vaccine using the composite adjuvant. BACKGROUND

[0002] The novel coronavirus COVID-19 discovered in 2019 belongs to the beta family of coronaviruses and can be transmitted through respiratory droplets, contact and fecal-oral transmission, and is highly infectious in the human population.

[0003] In the process of vaccine development, in order to prepare a safe and effective vaccine, the use of adjuvants must be considered. The compatibility of adjuvants with vaccine components allows the two to form a stable, safe, and immunogenic vaccine complex. Therefore, the study of adjuvants used in vaccines has always been an important part of the vaccine research process. Adjuvants, as non-specific immune enhancers, play a crucial role in inducing effective immune responses after vaccination.

[0004] Traditional aluminum salt adjuvants, including aluminum hydroxide and aluminum phosphate and their combinations, are the most widely used. Although the above adjuvants are widely approved for use in vaccine preparation, in actual use, the immunopotentiating effect of low-dose aluminum salt adjuvants alone on vaccines is limited, and increasing the dose of aluminum salt adjuvants often causes swelling at the injection site, granuloma, fever, pain, and allergic reactions. Therefore, it is urgent to develop an ideal vaccine adjuvant that is more broad-spectrum, safe, efficient, and easy to produce and use. SUMMARY

[0005] The first object of the present application is to provide a composite adjuvant. The composite adjuvant provided by the present application contains CpG oligodeoxynucleotide and aluminum adjuvant. Preferably, the composite adjuvant consists of CpG oligodeoxynucleotide and aluminum adjuvant.

[0006] CpG refers to a dinucleotide formed by cytosine (C) and guanine (G) connected by phosphodiester bonds (p). The CpG dinucleotide and each of the two bases at the 5' end and the 3' end of the CpG dinucleotide form a CpG motif. The CpG motif is also known as an immunostimulatory sequence (ISS), and CpG ODN refers to an oligodeoxynucleotide containing an unmethylated CpG motif. The immune system of vertebrates recognizes CpG DNA in bacterial genomes as a dangerous stimulus signal through pattern recognition receptors (PRRs), and then stimulates the body to produce an immune protective response.

[0007] The aluminum adjuvant described in the present application is selected from aluminum hydroxide and aluminum phosphate, and is preferably aluminum hydroxide. From the mechanism of immune response, the immune response of the aluminum adjuvant is biased towards a T helper cell type 2 (Th2) response, which is characterized by the secretion of Th2-type cytokines such as IL-4 and IL-5 and the production of IgG1 and IgE-type antibodies. Studies have shown that the aluminum hydroxide adjuvant can activate Th2 cells to secrete IL-4 and induce the expression of MHC-II molecules and CD83, CD86, etc., and ultimately induce a humoral immune response, but the cytotoxic T lymphocyte (CTL) response is weak or non-existent. CpG ODN can promote the maturation and activation of dendritic cells, macrophages and B cells, up-regulate the expression of CD80, CD86, CD40 and MHC-II molecules, promote the secretion of Thl-type cytokines such as IL-6, IL-12, IFN-γ, etc., and induce the body to produce a Thl-type immune response. In addition, after being endocytosed into immune cells, CpG ODN is recognized and combined with TLR9 on the endosome / lysosome of the cell, resulting in TLR9 dimerization, and ultimately promoting the secretion of a series of cytokines and chemokines by the related immune cells, triggering the intracellular bactericidal mechanism or inducing an inflammatory response, and producing a strong CTL induction effect.

[0008] The present application combines the traditional aluminum adjuvant with CpG oligodeoxynucleotides, which can complement each other in immune mechanisms, stimulate both cellular immunity and humoral immunity, and produce a stronger immune effect. By balancing Th1 and Th2 immune responses, the risk of ADE can be reduced.

[0009] As a preferred embodiment of the present application, the CpG oligodeoxynucleotide contains at least 2 CpG units, and the chain length is at least 20 bp, and all the nucleotides in the CpG oligodeoxynucleotide are sulfur-modified.

[0010] As a preferred scheme of the present application, the sequence of the CpG oligodeoxynucleotide is selected from the following sequences:

[0011] SEQ ID NO. 1: 5'-TGACTGTGAACGTTCGAGATGA-3'; or

[0012] SEQ ID NO. 2: 5'-TCGACGTTCGTCGTTCGTCGTTC-3'; or

[0013] SEQ ID NO. 3: 5' TCGTCGTTTTGTCGTTTTGTCGTT-3'.

[0014] As a preferred scheme of the present application, the mass ratio of the CpG oligodeoxynucleotide to the aluminum adjuvant is 1: (0.01-1). Further preferably, the mass ratio of the CpG oligodeoxynucleotide to the aluminum adjuvant is 1: (0.06-0.09), 1: (0.1-0.15), 1: (0.2-0.5), or 1: (0.6-0.9). Specifically, the mass ratio of the CpG oligodeoxynucleotide to the aluminum adjuvant can be 1:0.06, 1:0.09, 1:0.1, 1:0.15, 1:0.2, 1:0.5, 1:0.6, or 1:0.9. The present application determines the above most efficient complex adjuvant ratio scheme by studying the combination ratio scheme of the CpG oligodeoxynucleotide and the aluminum adjuvant, so as to ensure that the complex adjuvant can produce strong immune enhancement effect.

[0015] The second object of the present application is to provide the use of the complex adjuvant as an immune enhancer or immune adjuvant of a vaccine, or in improving the antigenicity of a vaccine or the immunogenicity of a vaccine.

[0016] As a preferred scheme of the present application, the vaccine is a COVID-19 inactivated vaccine. The use of the complex adjuvant as an adjuvant for the COVID-19 inactivated vaccine can better stimulate cellular immunity and produce stronger immune effect. This scheme has simple preparation method, easy quality control, and easy scale-up production. It can be used for effectively preventing and treating pneumonia caused by COVID-19 infection, and even severe respiratory infection. It provides technical support for disease prevention and control in China and even the whole world.

[0017] The third object of the present application is to provide a vaccine using the complex adjuvant. The use of the complex adjuvant in the vaccine provided by the present application can achieve the effects of enhancing immune effect, prolonging immune time, and reducing antigen dosage.

[0018] As a preferred scheme of the present application, in the vaccine, the dosage of the complex adjuvant is 0.3 mg / dose-6 mg / dose.

[0019] A fourth object of the present application is to provide a new coronavirus COVID-19 inactivated vaccine using the composite adjuvant. The new coronavirus COVID-19 inactivated vaccine provided by the present application can enhance the immune effect, prolong the immune time, and reduce the antigen dosage due to the use of the composite adjuvant.

[0020] As a preferred scheme of the present application, the dose of the composite adjuvant in the new coronavirus COVID-19 inactivated vaccine is 0.3 mg / dose to 6 mg / dose. Specifically, the content of the composite adjuvant in the inactivated vaccine can be 0.8 mg / dose, 0.95 mg / dose, 3.3 mg / dose, 3.45 mg / dose, 5.3 mg / dose, or 5.45 mg / dose.

[0021] As a preferred scheme of the present application, the new coronavirus COVID-19 inactivated vaccine contains 0.25 mg / dose to 5 mg / dose of CpG oligodeoxynucleotide adjuvant. Specifically, the content of the CpG oligodeoxynucleotide adjuvant in the inactivated vaccine can be 0.25 mg / dose, 0.5 mg / dose, 1 mg / dose, 3 mg / dose, 2 mg / dose, or 5 mg / dose.

[0022] As a preferred scheme of the present application, the new coronavirus COVID-19 inactivated vaccine contains 0.10 mg / dose to 0.45 mg / dose of aluminum hydroxide adjuvant. Specifically, the content of the aluminum hydroxide adjuvant in the inactivated vaccine can be 0.15 mg / dose, 0.3 mg / dose, or 0.45 mg / dose.

[0023] The new coronavirus COVID-19 inactivated vaccine provided by the present application contains a COVID-19 inactivated virus strain. The COVID-19 inactivated virus strain has a spike protein, an envelope protein, and a membrane protein.

[0024] As a preferred scheme of the present application, the COVID-19 inactivated virus strain is prepared by inactivating a COVID-19 virus strain using formalin or beta propiolactone.

[0025] As a preferred scheme of the present application, the COVID-19 inactivated virus strain is cultured in vitro using Vero cells before inactivation.

[0026] The preparation process of the COVID-19 inactivated virus strain in the COVID-19 inactivated vaccine composition provided by the application preferably comprises: obtaining high-concentration COVID-19 virus liquid from the COVID-19 virus strain isolated from a clinical patient through industrial large-scale culture, and successfully exploring an effective inactivation method and a purification method for inactivating the COVID-19, thereby preparing the COVID-19 inactivated virus strain. The inactivation method comprises inactivation by using formaldehyde or beta propiolactone. Preferably, the obtained high-concentration COVID-19 virus liquid is inactivated.

[0027] In the inactivation by using formaldehyde, the concentration of the formaldehyde is that the volume ratio of formaldehyde to virus liquid is 1:1000-1:4000, and the inactivation time is 3-13 hours. Preferably, for safety, the inactivation time is 9-39 hours or more to inactivate all viruses. Preferably, the inactivation is stirred at 37°C for four days.

[0028] In the inactivation by using beta propiolactone, the concentration of the beta propiolactone is that the volume ratio of beta propiolactone to virus liquid is 1:4000-1:6000, and the inactivation time is 16-24 hours. Preferably, for safety, the inactivation time is 48-72 hours or more to inactivate all viruses. Preferably, the inactivation is stirred at 4°C for 20 hours. Then, the inactivation liquid is placed at room temperature for 2 days, or is treated at 37°C for 2 hours to decompose the beta propiolactone.

[0029] Specifically, the purification method of the inactivated liquid comprises the following steps:

[0030] (1) clarifying the virus inactivated liquid;

[0031] (2) concentrating the virus clarified liquid obtained in step (1); and

[0032] (3) performing nuclease digestion treatment on the virus concentrated liquid obtained in step (2) to digest host DNA, thereby obtaining a purified virus stock solution.

[0033] Preferably, in step (1), the virus inactivated liquid is centrifuged at a centrifugal force of 2000-4000g for 30 min, the precipitate is removed, and the supernatant is collected as the virus clarified liquid. Preferably, the centrifugation is performed at 2-8°C. Alternatively, in step (1), the virus inactivated liquid is clarified by filtration through a deep layer membrane stack, and then filtered through a 0.2-micron filter, thereby obtaining the virus clarified liquid. Preferably, the deep layer membrane stack can be a deep layer membrane stack of diatomite.

[0034] Preferably, the virus clarified liquid is concentrated by ultrafiltration in step (2). Preferably, the virus clarified liquid is concentrated by two-stage ultrafiltration, the first stage of ultrafiltration is preferably concentrated by about 20-40 times using a membrane package with a pore size of 100 Kda, and the second stage of ultrafiltration is preferably concentrated by about 4-8 times using a membrane package with a pore size of 300 Kda, and PBS dialysis is performed 5-8 times. Preferably, the concentration of PBS is 0.01 mol, and the pH is 7.4.

[0035] Preferably, density gradient centrifugation is performed after step (2) to purify the 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 centrifugation is performed at 28000-40000 rpm for 16-20 hours at 2-8℃ to obtain an ultra-centrifugation liquid.

[0036] Preferably, chromatography is performed after density gradient centrifugation. Preferably, Sepharose four fast flow is used for chromatography of the ultra-centrifugation liquid, and the eluate is 0.01M PBS, and the first flow-through peak is collected as the virus purified liquid.

[0037] Preferably, after the virus purified liquid is concentrated by ultrafiltration by about 10 times, Benzonase nuclease is added to digest the host DNA. In the enzyme digestion, the concentration of Benzonase is 25-50 U / ml, stirring is performed for 2-4 hours, and standing is performed for 14-20 hours, and the excess Benzonase nuclease is removed by ultrafiltration.

[0038] Preferably, the virus stock solution is obtained by sterilization filtration through a 0.22 μm membrane or by irradiation sterilization. The irradiation sterilization uses a dose of 4-10 kilograys of cobalt 60 to irradiate the sample for 60 minutes.

[0039] On the one hand, the COVID-19 virus strain has the spike protein (S protein, hereinafter referred to as S protein), envelope protein and membrane protein of the COVID-19 virus, and can cause the body fluid immune response to produce neutralizing antibodies. On the other hand, the COVID-19 virus strain is an inactivated strain. After inoculation of Vero-E6 cells or Vero cells, no pathological changes of Vero-E6 cells or Vero cells are caused. Using Vero cells as a matrix to culture COVID-19 virus in vitro, planar culture can be selected, such as using cell factories for culture, or suspension culture can be selected, such as using microcarriers for suspension culture in a cell fermentation tank.

[0040] The preparation method of the inactivated vaccine provided by the present application preferably comprises:

[0041] (1) a high-concentration COVID-19 virus liquid is obtained by industrialized large-scale culture of a COVID-19 virus strain isolated from a clinical patient;

[0042] (2) inactivating the COVID-19 virus liquid to obtain an inactivated COVID-19 virus inactivation liquid;

[0043] (3) purifying the inactivation liquid to obtain a purified virus stock solution; and

[0044] (4) mixing the virus stock solution with CpG oligodeoxynucleotide adjuvant to obtain an inactivated vaccine.

[0045] As a preferred scheme of the present application, the content of the COVID-19 inactivated virus strain (as an effective antigen component) in the inactivated vaccine composition is 0.5-10 μg / dose, preferably 2-4 μg / dose. Specifically, the content of the COVID-19 inactivated virus strain in the inactivated vaccine can be 1 μg / dose, 2 μg / dose, 3 μg / dose, 4 μg / dose or 10 μg / dose.

[0046] As a preferred scheme of the present application, the new coronavirus COVID-19 inactivated vaccine is a liquid preparation, which can adopt various dosage forms. Specifically, the inactivated vaccine can be an intramuscular liquid injection, an intravenous liquid injection, an intranasal liquid injection, an intradermal liquid injection or a subcutaneous liquid injection. In actual application, adjustment and selection can be made according to the clinical needs such as transfection efficiency, local immune surveillance, etc., such as selecting a single dosage form for injection immunization, or selecting a plurality of mixed dosage forms for injection immunization.

[0047] A fifth object of the present application is to provide the use of the new coronavirus COVID-19 inactivated vaccine using the composite adjuvant in the preparation of a medicament for preventing and / or treating diseases caused by COVID-19 infection. The diseases are preferably pneumonia and syndrome, severe acute respiratory infection, intestinal disease, heart failure, kidney failure or severe acute respiratory syndrome.

[0048] The new coronavirus COVID-19 inactivated vaccine using the composite adjuvant provided by the present application can greatly prolong the retention time of the vaccine antigen in the body and reduce the hydrolysis of various hydrolytic enzymes. The preparation method of the new coronavirus COVID-19 inactivated vaccine provided by the present application is simple, the quality is easy to control, easy to scale up production, and has good safety and low toxicity side effects, and is particularly suitable for the prevention and treatment of new coronavirus COVID-19 in various populations, including the elderly and other populations with low immune function. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 Schematic diagram of serum IgG titer of BALB / c mice immunized with COVID-19 inactivated vaccine with different CpG adjuvant and aluminum hydroxide adjuvant ratio scheme.

[0050] Figure 2 A schematic diagram showing the results of specific expression of S protein cytokines in serum. DETAILED DESCRIPTION

[0051] The following examples are intended to illustrate the present application but not to limit the scope of the present application.

[0052] In the following examples, the sequence of CpG oligodeoxynucleotide (adjuvant) is SEQ ID NO. 1: 5'-TGACTGTGAACGTTCGAGATGA-3'.

[0053] Example 1

[0054] The present example provides a composite adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of CpG oligodeoxynucleotide to aluminum hydroxide is 1:0.6.

[0055] The present example further provides a combination of the composite adjuvant and the inactivated vaccine of COVID-1 novel coronavirus.

[0056] Specifically, the CpG oligodeoxynucleotide adjuvant is added to the vaccine stock solution filtered by a 0.22 μm filter, so that the concentration of CpG adjuvant is 0.5 mg / dose, the content of aluminum hydroxide adjuvant is 0.3 mg / dose, and the content of vaccine antigen (i.e. the content of COVID-19 inactivated virus strain) is 2 μg / dose. The vaccine obtained in the present example is numbered as 20200401.

[0057] Example 2

[0058] The present example provides a composite adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of CpG oligodeoxynucleotide to aluminum hydroxide is 1:0.6.

[0059] The present example further provides a combination of the composite adjuvant and the inactivated vaccine of COVID-1 novel coronavirus.

[0060] Specifically, the CpG oligodeoxynucleotide adjuvant is added to the vaccine stock solution filtered by a 0.22 μm filter, so that the concentration of CpG adjuvant is 0.5 mg / dose, the content of aluminum hydroxide adjuvant is 0.3 mg / dose, and the content of vaccine antigen (i.e. the content of COVID-19 inactivated virus strain) is 4 μg / dose. The vaccine obtained in the present example is numbered as 20200402.

[0061] Example 3

[0062] The present example provides a composite adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of CpG oligodeoxynucleotide to aluminum hydroxide is 1:0.9.

[0063] The embodiment further provides a combination of the composite adjuvant and the inactivated vaccine of the novel coronavirus COVID-1.

[0064] Specifically, the CpG oligodeoxynucleotide adjuvant is added into the vaccine stock solution filtered by a 0.22 μm filter, so that the concentration of the CpG adjuvant is 0.5 mg / dose, the content of the aluminum hydroxide adjuvant is 0.45 mg / dose, and the content of the vaccine antigen (i.e. the content of the COVID-19 inactivated virus strain) is 2 μg / dose. The vaccine obtained in the embodiment is numbered as 20200403.

[0065] Embodiment 4

[0066] The embodiment provides a composite adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of the CpG oligodeoxynucleotide to the aluminum hydroxide is 1:0.9.

[0067] The embodiment further provides a combination of the composite adjuvant and the inactivated vaccine of the novel coronavirus COVID-1.

[0068] Specifically, the CpG oligodeoxynucleotide adjuvant is added into the vaccine stock solution filtered by a 0.22 μm filter, so that the concentration of the CpG adjuvant is 0.5 mg / dose, the content of the aluminum hydroxide adjuvant is 0.45 mg / dose, and the content of the vaccine antigen (i.e. the content of the COVID-19 inactivated virus strain) is 4 μg / dose. The vaccine obtained in the embodiment is numbered as 20200404.

[0069] Embodiment 5

[0070] The embodiment provides a composite adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of the CpG oligodeoxynucleotide to the aluminum hydroxide is 1:0.1.

[0071] The embodiment further provides a combination of the composite adjuvant and the inactivated vaccine of the novel coronavirus COVID-1.

[0072] Specifically, the CpG oligodeoxynucleotide adjuvant is added into the vaccine stock solution filtered by a 0.22 μm filter, so that the concentration of the CpG adjuvant is 3 mg / dose, the content of the aluminum hydroxide adjuvant is 0.3 mg / dose, and the content of the vaccine antigen (i.e. the content of the COVID-19 inactivated virus strain) is 2 μg / dose. The vaccine obtained in the embodiment is numbered as 20200405.

[0073] Embodiment 6

[0074] The embodiment provides a composite adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of the CpG oligodeoxynucleotide to the aluminum hydroxide is 1:0.1.

[0075] The embodiment further provides a combination of the composite adjuvant and the inactivated vaccine of the novel coronavirus COVID-1.

[0076] Specifically, the CpG oligodeoxynucleotide adjuvant is added into the vaccine stock solution filtered by a 0.22 μm filter, so that the concentration of the CpG adjuvant is 3 mg / dose, the content of the aluminum hydroxide adjuvant is 0.3 mg / dose, and the content of the vaccine antigen (i.e. the content of the COVID-19 inactivated virus strain) is 4 μg / dose. The vaccine obtained in the embodiment is numbered as 20200406.

[0077] Embodiment 7

[0078] The embodiment provides a composite adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide, wherein the mass ratio of the CpG oligodeoxynucleotide to the aluminum hydroxide is 1:0.15.

[0079] The embodiment further provides a combination of the composite adjuvant and the inactivated vaccine of the novel coronavirus COVID-1.

[0080] Specifically, the CpG oligodeoxynucleotide adjuvant is added into the vaccine stock solution filtered by a 0.22 μm filter, so that the concentration of the CpG adjuvant is 3 mg / dose, the content of the aluminum hydroxide adjuvant is 0.45 mg / dose, and the content of the vaccine antigen (i.e. the content of the COVID-19 inactivated virus strain) is 2 μg / dose. The vaccine obtained in the embodiment is numbered as 20200407.

[0081] Embodiment 8

[0082] The embodiment provides a composite adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide, wherein the mass ratio of the CpG oligodeoxynucleotide to the aluminum hydroxide is 1:0.15.

[0083] The embodiment further provides a combination of the composite adjuvant and the inactivated vaccine of the novel coronavirus COVID-1.

[0084] Specifically, the CpG oligodeoxynucleotide adjuvant is added into the vaccine stock solution filtered by a 0.22 μm filter, so that the concentration of the CpG adjuvant is 3 mg / dose, the content of the aluminum hydroxide adjuvant is 0.45 mg / dose, and the content of the vaccine antigen (i.e. the content of the COVID-19 inactivated virus strain) is 4 μg / dose. The vaccine obtained in the embodiment is numbered as 20200408.

[0085] Embodiment 9

[0086] The embodiment provides a composite adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of the CpG oligodeoxynucleotide to the aluminum hydroxide is 1:0.06.

[0087] The embodiment further provides a combination mode of the composite adjuvant and a novel coronavirus COVID-1 inactivated vaccine.

[0088] Specifically, the CpG oligodeoxynucleotide adjuvant is added into a vaccine stock solution filtered through a 0.22 mu m filter, so that the CpG adjuvant concentration is 5 mg / dose, the aluminum hydroxide adjuvant content is 0.3 mg / dose, and the vaccine antigen content (that is, the COVID-19 inactivated virus strain content) is 2 ug / dose. The vaccine obtained in the embodiment is numbered as 20200409.

[0089] Embodiment 10

[0090] The embodiment provides a composite adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of the CpG oligodeoxynucleotide to the aluminum hydroxide is 1:0.06.

[0091] The embodiment further provides a combination mode of the composite adjuvant and a novel coronavirus COVID-1 inactivated vaccine.

[0092] Specifically, the CpG oligodeoxynucleotide adjuvant is added into a vaccine stock solution filtered through a 0.22 mu m filter, so that the CpG adjuvant concentration is 5 mg / dose, the aluminum hydroxide adjuvant content is 0.3 mg / dose, and the vaccine antigen content (that is, the COVID-19 inactivated virus strain content) is 2 ug / dose. The vaccine obtained in the embodiment is numbered as 20200409.

[0093] Embodiment 11

[0094] The embodiment provides a composite adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of the CpG oligodeoxynucleotide to the aluminum hydroxide is 1:0.09.

[0095] The embodiment further provides a combination mode of the composite adjuvant and a novel coronavirus COVID-1 inactivated vaccine.

[0096] Specifically, the CpG oligodeoxynucleotide adjuvant is added into a vaccine stock solution filtered through a 0.22 mu m filter, so that the CpG adjuvant concentration is 5 mg / dose, the aluminum hydroxide adjuvant content is 0.3 mg / dose, and the vaccine antigen content (that is, the COVID-19 inactivated virus strain content) is 2 ug / dose. The vaccine obtained in the embodiment is numbered as 20200409.

[0097] Embodiment 12

[0098] The present embodiment provides a composite adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of CpG oligodeoxynucleotide to aluminum hydroxide is 1:0.09.

[0099] The present embodiment further provides a combination of the composite adjuvant and the inactivated vaccine of COVID-1 novel coronavirus.

[0100] Specifically, the CpG oligodeoxynucleotide adjuvant is added to the vaccine stock solution filtered by a 0.22 μm filter, so that the concentration of the CpG adjuvant is 5 mg / dose, the content of the aluminum hydroxide adjuvant is 0.45 mg / dose, and the content of the vaccine antigen (i.e. the content of the COVID-19 inactivated virus strain) is 4 μg / dose. The vaccine obtained in the present embodiment is numbered as 20200412.

[0101] Comparative Example 1

[0102] The present comparative example provides a combination of the aluminum hydroxide adjuvant and the inactivated vaccine of COVID-19 novel coronavirus.

[0103] Specifically, the aluminum hydroxide adjuvant is added to the vaccine stock solution filtered by a 0.22 μm filter, so that the content of the aluminum hydroxide adjuvant is 0.45 mg / dose, and the content of the vaccine antigen (i.e. the content of the COVID-19 inactivated virus strain) is 2 μg / dose. The vaccine obtained in the present comparative example is numbered as 20200413.

[0104] Comparative Example 2

[0105] The present comparative example provides a combination of the CpG oligodeoxynucleotide adjuvant and the inactivated vaccine of COVID-19 novel coronavirus.

[0106] Specifically, the CpG oligodeoxynucleotide adjuvant is added to the vaccine stock solution filtered by a 0.22 μm filter, so that the concentration of the CpG adjuvant is 5 mg / dose, and the content of the vaccine antigen (i.e. the content of the COVID-19 inactivated virus strain) is 2 μg / dose. The vaccine obtained in the present comparative example is numbered as 20200414.

[0107] Experimental Example 1: Immunological evaluation of adjuvant effect on mouse model

[0108] 1. Measurement of neutralizing antibody titer

[0109] The 14 groups of vaccines provided by the examples and comparative examples are respectively diluted with physiological saline at 1:4, 1:16, and 1:64. BALB / c mice are inoculated with 1 mL syringes, 10 mice per group, immunized at 0, 7 days, and each mouse is injected intraperitoneally with one dose. Blood is collected 4 weeks after the first immunization, and the neutralizing antibody is detected. The neutralizing antibody titer GMT greater than 8 is considered positive, indicating a protective effect. The results are shown in Table 1.

[0110] Table 1: Neutralizing titer of BALB / c mice immunized with COVID-19 inactivated vaccine

[0111]

[0112] The results showed that immunization of BALB / c mice with COVID-19 vaccines of different active ingredients and adjuvant contents all produced sufficiently high neutralizing antibody titers. Vaccines with higher adjuvant contents exhibited relatively higher immunogenicity than vaccines with lower adjuvant contents of the same active ingredient. The vaccines provided by this invention can all induce protective neutralizing antibodies in BALB / c mice.

[0113] 2. IgG antibody titer determination

[0114] The IgG antibody titers of a total of 14 groups of vaccines provided in the various examples and comparative examples were determined. The method for detecting IgG antibody titers is as follows:

[0115] 100 μl of SARS-CoV-2 protein at a concentration of 1 μg / ml was coated onto an ELISA plate and incubated overnight at 2-8°C or for at least 2 hours at 37°C. After washing and drying, the plate was blocked with 200 μl of 0.01M PBS containing 1% BSA or 10% fetal bovine serum at 37°C for 1-2 hours. The liquid was then discarded and the plate was drained. The test samples and negative serum controls were serially diluted with the above blocking buffer and added to each well (100 μl). The plates were incubated at 37°C for 60-70 minutes, washed, and dried. The corresponding species-specific HRP-labeled antibody was added and incubated at 37°C for 45-60 minutes. The plates were then washed and dried. 50 μl each of chromogenic solutions A and B were added and incubated at 37°C for 10-15 minutes. Finally, 2M H₂SO₄ was added to stop the reaction. Results analysis: The highest dilution where the OD value of the sample is ≥ 2.1 times the OD value of the negative serum at the same dilution is the IgG antibody titer of the sample. If the negative control OD value is less than 0.05, it shall be calculated as 0.05.

[0116] The results are as follows Figure 1 As shown. Figure 1 The bar chart shown represents the IgG antibody titer results for each vaccine group, which consists of two columns: the left column represents 7 days (7D) and the right column represents 14 days (14D).

[0117] Experiment Example 2: Cytokine Detection

[0118] Fourteen groups of vaccines provided in the various examples and comparative examples were used to immunize BALB / c mice according to a schedule of 0 and 7 days, with blood collection on day 28. Cell supernatants were collected for cellular immunoassay. Intracellular cytokine staining and flow cytometry were used to detect CD4+ T cells in serum that specifically express S protein cytokines.

[0119] (1) Isolation of splenocytes

[0120] The mice were sacrificed at the site of cervical dislocation and the spleen was removed aseptically in a biological safety cabinet and placed on a 200-mesh cell strainer in a sterile petri dish. 10 mL of RPMI 1640 complete medium was added and the spleen was gently ground into single cells using a syringe plunger. The cell strainer was rinsed with 10 mL of RPMI 1640 complete medium to obtain more splenocytes. The splenocyte suspension was transferred to a 50-mL centrifuge tube and centrifuged at 500 g for 5 min. The supernatant was discarded and the cells were resuspended in 3 mL of lx red blood cell lysis solution, lysed at room temperature for 5 min, and then added to 27 mL of RPMI 1640 complete medium and centrifuged at 500 g for 5 min. The supernatant was discarded and the cells were washed once with 20 mL of RPMI 1640 complete medium, resuspended in an appropriate amount of medium, filtered through a 200-mesh cell strainer into a 10-mL test tube, and diluted 20-fold for 50 μL for counting, ready for use.

[0121] (2) In vitro stimulation of mouse splenocytes

[0122] The above isolated mouse splenocytes were diluted to 4 x 10 6 cells / mL, and 0.5 mL was added to each well of a 24-well plate. Each mouse was set up with a specific CTL epitope stimulation well and a non-stimulation well. The specific epitope concentration was 2 μg / mL per peptide, and an equal amount of DMSO was added to the non-stimulation well. As a positive control, PMA and ionomycin were added to the stimulation well, with a PMA concentration of 100 ng / mL and an ionomycin concentration of 1 μg / mL. 1 μL of Brilliant Violet 421™ anti-mouse CD 107a was also added to each well. After incubation at 37°C in a 5% CO2 cell incubator for 1 hour, an appropriate amount of GolgiStop and / or GolgiPlug was added to each well as a blocking agent for cytokine secretion. After a total incubation of 6 hours, the relevant antigen was stained for intracellular cytokine flow cytometry detection.

[0123] (3) Staining of cell surface antigens and intracellular cytokines

[0124] After 6 hours of in vitro stimulation, the spleen cells were transferred to flow tubes, centrifuged at 500g for 5 minutes at 4°C, and the supernatant was discarded. The fluorescently labeled antibodies PerCP / Cy5.5-conjugated anti-CD3 (clone 145-2c11) and FITC conjugated anti-CD8 (clone 53-6.7) were diluted in PBS+2% FBS according to the recommended usage, 50 μL was added to each tube, mixed gently, and incubated at 4°C for 30 minutes. After 30 minutes, 3 mL of PBS+2% FBS was added to each tube, centrifuged at 500g for 5 minutes at 4°C, and the supernatant was discarded. 200 μL of Cytofix / Cytoperm™ Fixation and Permeabilizaiton Solution was added to each tube, and the cells were fixed and perforated at 4°C for 20 minutes. After 20 minutes, 1 mL of 1x Perm / Wash™ Buffer was added to each tube, centrifuged at 600g for 5 minutes at 4°C, and the supernatant was discarded. The PE conjugated anti-IFN-γ (clone XMG1.2) antibody was diluted in 1x Perm / Wash™ Buffer according to the recommended usage, 50 μL was added to each tube, mixed gently, and incubated at 4°C for 30 minutes. Finally, each tube was washed once with 1 mL of 1x Perm / Wash™ Buffer and 3 mL of PBS, respectively, resuspended with 200 μL of PBS after the supernatant was discarded, and detected on the machine. In order to adjust the fluorescence compensation between dyes during detection, non-stained tubes, PerCP / Cy5.5-conjugated anti-CD3 single-stained tubes, FITC conjugated anti-CD8 single-stained tubes, and PE conjugated anti-IFN-γ single-stained tubes were set up, and the PE conjugated anti-IFN-γ single-stained tubes used positive stimulated cells.

[0125] (4) Flow cytometry detection

[0126] Flow cytometry detection was performed using a BD FACS Canto™. First, the appropriate voltage was adjusted for each channel, the fluorescence compensation between dyes was adjusted using single-fluorescently stained samples, and then the samples were sequentially loaded and data was collected.

[0127] 5. Intracellular cytokine staining flow cytometry detection results

[0128] The detection results are as follows Figure 2The new coronavirus COVID-19 inactivated vaccine can induce the increase of serum antibody level. The new coronavirus vaccine using the composite adjuvant can produce high level of specific antibody, and the immune effect is obviously stronger than that of the control vaccine using aluminum hydroxide adjuvant and CpG oligodeoxynucleotide alone. The experiment shows the good effect of the composite adjuvant in the new coronavirus COVID-19 inactivated vaccine.

[0129] Although the present application has been described in detail with general description, specific embodiments and experiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, are within the scope of the present application claimed. SEQUENCE LISTING <110> Beijing Wantai Biological Pharmacy Co., Ltd. <120> A new coronavirus COVID-19 inactivated vaccine using composite adjuvant containing CpG oligodeoxynucleotide <130> RYP2010640.3 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <400> 1 tgactgtgaa cgttcgagat ga 22 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <400> 2 tcgacgttcg tcgttcgtcg ttc 23 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <400> 3 tcgtcgtttt gtcgttttgt cgtt 24

Claims

1. Use of a composite adjuvant in the preparation of a new coronavirus COVID-19 inactivated vaccine, characterized in that, the composite adjuvant is composed of CpG oligodeoxynucleotide and aluminum adjuvant; the mass ratio of CpG oligodeoxynucleotide to aluminum adjuvant is 1: (0.06-0.09); in the new coronavirus COVID-19 inactivated vaccine, the dose of CpG oligodeoxynucleotide is 0.25 mg-5 mg / dose, and the dose of aluminum adjuvant is 0.10 mg-0.45 mg / dose; the sequence of the CpG oligodeoxynucleotide is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that, all nucleotides in the CpG oligodeoxynucleotide are sulfur-modified.

3. The use according to any one of claims 1-2, characterized in that, the aluminum adjuvant is aluminum hydroxide.

4. A new coronavirus COVID-19 inactivated vaccine containing a composite adjuvant, characterized in that, the composite adjuvant is composed of CpG oligodeoxynucleotide and aluminum adjuvant; the mass ratio of CpG oligodeoxynucleotide to aluminum adjuvant is 1: (0.06-0.09); in the new coronavirus COVID-19 inactivated vaccine, the dose of CpG oligodeoxynucleotide is 0.25 mg-5 mg / dose, and the dose of aluminum adjuvant is 0.10 mg-0.45 mg / dose; the sequence of the CpG oligodeoxynucleotide is shown in SEQ ID NO.

1.

5. The new coronavirus COVID-19 inactivated vaccine according to claim 4, characterized in that, all nucleotides in the CpG oligodeoxynucleotide are sulfur-modified.

6. The new coronavirus COVID-19 inactivated vaccine according to claim 4, characterized in that, the aluminum adjuvant is aluminum hydroxide.

7. The new coronavirus COVID-19 inactivated vaccine according to any one of claims 4-6, characterized in that, in the new coronavirus COVID-19 inactivated vaccine, the dose of the composite adjuvant is 0.3 mg / dose-6 mg / dose.

8. The inactivated vaccine according to claim 7, characterized in that, the inactivated vaccine contains a COVID-19 inactivated virus strain.

9. The inactivated vaccine according to claim 8, characterized in that, The content of the COVID-19 inactivated virus strain in the vaccine is 0.5-10 μg / dose.

10. The inactivated vaccine according to claim 8, characterized in that, The COVID-19 inactivated virus strain is prepared by inactivating a COVID-19 virus strain using formalin or beta propiolactone.

11. The inactivated vaccine according to claim 10, characterized in that, the COVID-19 inactivated virus strain is cultured in vitro using Vero cells before inactivation.

12. The new coronavirus COVID-19 inactivated vaccine according to any one of claims 4-6, 8-11, characterized in that, the vaccine is a liquid preparation.

13. The new coronavirus COVID-19 inactivated vaccine according to claim 12, characterized in that, the vaccine is an intramuscular liquid injection, an intranasal liquid spray, an intradermal liquid injection, or a subcutaneous liquid injection.

14. Use of the new inactivated vaccine against COVID-19 according to any one of claims 4 to 13 for the preparation of a medicament for the prevention and / or treatment of a disease caused by a COVID-19 infection.

15. The inactivated vaccine according to claim 14, characterized in that, The disease is pneumonia and syndrome.

16. The inactivated vaccine of claim 14, characterized in that, The disease is severe acute respiratory infection.

17. The inactivated vaccine of claim 14, characterized in that, The disease is intestinal disease, heart failure, kidney failure or severe acute respiratory syndrome.

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