CN113521267A - COVID-19 recombinant protein vaccine composition and application - Google Patents
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Abstract
The invention discloses a COVID-19 recombinant protein vaccine composition which contains novel CpG oligodeoxynucleotide as an adjuvant. After the novel CpG oligodeoxynucleotide adjuvant used by the invention is combined with the COVID-19 recombinant protein vaccine according to a formula, a strong immune effect can be generated, the retention time of the COVID-19 recombinant protein vaccine in vivo can be prolonged to a great extent, and the hydrolysis effect of various hydrolytic enzymes on the vaccine can be reduced; the preparation method of the recombinant protein vaccine composition is simple, the quality is easy to control, and the large-scale production is easy to realize; and the composition has good safety and low toxic and side effects, and is particularly suitable for preventing and treating the novel coronavirus COVID-19 in various crowds, including middle-aged and old people with low immune function.
Description
Technical Field
The invention relates to the field of epidemic disease prevention and vaccine production, and particularly relates to a COVID-19 recombinant protein vaccine composition and application thereof.
Background
The coronavirus COVID-19 is a novel coronavirus, belongs to a coronavirus beta family, can be transmitted through respiratory droplet transmission, contact, excrement-mouth transmission and other paths, and is very susceptible to infection in people. There is currently no specific treatment for diseases caused by the novel coronavirus. By 4, 16 days in 2020, more than 200 million cases of global diagnosis are determined, and more than 13 million cases of death are determined, so that the development of a new coronavirus vaccine is urgently needed to block virus transmission and protect the global health of people.
The existing vaccines for preventing virus infection applied to human bodies mainly comprise attenuated live vaccines prepared by attenuating viruses, such as varicella attenuated live vaccines, measles attenuated live vaccines and the like; and inactivated whole virus particle vaccines, such as enterovirus EV71 type inactivated vaccine, hepatitis A vaccine and the like. However, it takes a long time for an attenuated vaccine to propagate the virus for many generations, and then gradually attenuates most of the activity of the virus, which is difficult to satisfy the prevention needs of acute infectious diseases. Inactivated vaccines present many problematic immunopathologies, for example, some inactivated viruses enter the human body to exacerbate the disease during viral invasion.
The recombinant protein vaccine is prepared by constructing target antigen genes of viruses on an expression vector, then transforming the target antigen genes into bacteria, yeast, mammals or insect cells, expressing a large amount of antigen proteins under certain induction conditions, and purifying the antigen proteins. The recombinant protein vaccine can induce the organism to generate humoral immunity and cellular immunity, and can be rapidly prepared.
In the vaccine development process, the use of adjuvants must be considered in order to prepare a safe and effective vaccine. The adjuvant and the vaccine components are compatible, so that the adjuvant and the vaccine components form a stable, safe and immunogenic vaccine compound. Therefore, the research on the adjuvant used by the vaccine is always an important link in the vaccine research process. Adjuvants, as non-specific immunopotentiators, play a crucial role in inducing effective immune responses after vaccination.
Traditional aluminum salt adjuvants, including aluminum hydroxide and aluminum phosphate and combinations thereof, are most widely used. Although the adjuvant is widely approved to be applied to vaccine preparation, in practical application, the single use of low dosage of aluminum salt adjuvant has limited immune enhancement effect on vaccine, and the use of the aluminum salt adjuvant with increased dosage often causes side reactions such as injection site swelling, granuloma, fever, pain, allergy and the like. Therefore, the development of ideal vaccine adjuvants which are more broad-spectrum, safe and efficient and are convenient to produce and use is urgent.
Disclosure of Invention
In order to solve the problems, the invention provides a COVID-19 recombinant protein vaccine composition and application thereof.
The first object of the present invention is to provide a COVID-19 recombinant protein vaccine composition comprising CpG oligodeoxynucleotide as an adjuvant.
CpG refers to a dinucleotide composed of cytosine (C) and guanine (G) linked via a phosphodiester bond (p), and CpG dinucleotides and two bases at the 5 'end and the 3' end of the dinucleotide constitute a CpG motif (CpG motif). The specific oligodeoxynucleotide serving as an adjuvant of the COVID-19 recombinant vaccine composition can achieve the purposes of enhancing the immune effect, prolonging the immune time and reducing the antigen dosage.
Preferably, the recombinant protein vaccine comprises (1) the receptor binding domain of the S protein of COVID-19 and (2) the Fc fragment of IgG.
Preferably, the recombinant protein vaccine comprises the ACE2 receptor binding domain and/or surface epitope of the S protein; the S protein is S protein of SARS or S protein of COVID-19.
Preferably, the recombinant protein vaccine comprises one or more amino acid sequences of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, or amino acid sequences with more than 90% of identity.
The amino acid sequence is shown in a sequence table of the invention, and is specifically shown in the following table 1.
Table 1: amino acid sequence
As a preferred embodiment of the invention, the content of the effective antigen components in the recombinant protein vaccine is 1-80 mug/dose; preferably, the content of the antigen effective component in the recombinant protein vaccine is 2.5 mu g/dose, 10 mu g/dose, 40 mu g/dose or 80 mu g/dose.
As a preferred embodiment of the invention, the content of the CpG oligodeoxynucleotide adjuvant in the recombinant protein vaccine is 0.25-5 mg/dose; preferably, the content of the CpG oligodeoxynucleotide adjuvant in the recombinant protein vaccine is 0.25 mg/dose, 1 mg/dose, 2 mg/dose or 5 mg/dose.
The invention researches the combination proportioning scheme of the novel CpG oligodeoxynucleotide adjuvant and the COVID-19 recombinant vaccine and determines the most efficient proportioning scheme. The COVID-19 recombinant vaccine using the scheme can generate strong immune response benefit.
As a preferred embodiment of the present invention, the recombinant protein vaccine composition is a liquid vaccine, preferably an intramuscular liquid injection, an 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 immune monitoring and the like, for example, a single preparation is selected for injection immunization, or a plurality of mixed preparations are selected for injection immunization.
The second purpose of the invention is to provide the application of the COVID-19 recombinant protein vaccine composition in preparing a medicament for preventing and/or treating diseases caused by COVID-19 infection, preferably, the diseases are pneumonia and syndromes, severe acute respiratory tract infection, intestinal diseases, heart failure, renal failure or severe acute respiratory tract syndrome.
The third purpose of the invention is to provide the application of CpG oligodeoxynucleotide as an immunopotentiator or immunologic adjuvant of the COVID-19 recombinant protein vaccine, or the application in improving the immunogenicity of the antigen or the vaccine of the COVID-19 recombinant protein vaccine.
As a preferred embodiment of the present invention, all nucleotides of the CpG oligodeoxynucleotide are thio-modified, and the CpG oligodeoxynucleotide contains at least 2 CpG units with a chain length of at least 20 bp; preferably, the CpG oligodeoxynucleotide sequence is:
5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 7); or
5'-TCGACGTTCGTCGTTCGTCGTTC-3' (SEQ ID NO: 8); or
5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’(SEQ ID NO:9)。
The recombinant protein vaccine provided by the invention can effectively induce an organism to generate cellular immunity and humoral immunity. The recombinant protein vaccine provided by the invention can be prepared quickly and is suitable for controlling the epidemic situation of the novel coronavirus COVID-19.
The preparation method of the recombinant protein vaccine composition is simple, the quality is easy to control, and the large-scale production is easy to realize. Can be used for effectively preventing and treating pneumonia and syndrome caused by novel coronavirus COVID-19 infection, and even serious respiratory tract infection. Provides technical support for the prevention and control of diseases in China and even all over the world.
Drawings
FIG. 1 is a schematic diagram of the serum IgG titer of BALB/c mice immunized with COVID-19 recombinant vaccines with different CpG adjuvants; the left bar represents the titer for 7 days and the right bar represents the titer for 14 days.
FIG. 2 is a diagram showing the results of specific expression of S protein cytokines in serum.
Detailed Description
The nucleotide sequence of SEQ ID NO 7 was used for CpG oligodeoxynucleotides in the following examples:
SEQ ID NO:7
5’-TGACTGTGAACGTTCGAGATGA-3’。
the antigen of the recombinant protein vaccine in each of the following examples has the amino acid sequence of SEQ ID NO 6:
SEQ ID NO:6
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAASVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWP。
the following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine.
The method specifically comprises the following steps: the CpG oligodeoxynucleotide adjuvant is added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the adjuvant concentration is 0.5 mg/dose, and the vaccine antigen content is 5 mu g/dose. The vaccine obtained in this example was numbered 20200401.
Example 2
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of an adjuvant is 0.5 mg/dose, and the content of a vaccine antigen is 10 mu g/dose. The vaccine obtained in this example was numbered 20200402.
Example 3
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of the adjuvant is 0.5 mg/dose, and the content of the vaccine antigen is 20 mu g/dose. The vaccine obtained in this example was numbered 20200403.
Example 4
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of the adjuvant is 0.5 mg/dose, and the content of the vaccine antigen is 40 mu g/dose. The vaccine obtained in this example was numbered 20200404.
Example 5
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of an adjuvant is 3 mg/dose, and the content of a vaccine antigen is 5 mu g/dose. The vaccine obtained in this example was numbered 20200405.
Example 6
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of an adjuvant is 3 mg/dose, and the content of a vaccine antigen is 10 mu g/dose. The vaccine obtained in this example was numbered 20200406.
Example 7
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of an adjuvant is 3 mg/dose, and the content of the vaccine antigen is 20 mu g/dose. The vaccine obtained in this example was numbered 20200407.
Example 8
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of an adjuvant is 3 mg/dose, and the content of a vaccine antigen is 40 mu g/dose. The vaccine obtained in this example was numbered 20200408.
Example 9
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of an adjuvant is 1 mg/dose, and the content of a vaccine antigen is 5 mu g/dose. The vaccine obtained in this example was numbered 20200409.
Example 10
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of an adjuvant is 1 mg/dose, and the content of a vaccine antigen is 10 mu g/dose. The vaccine obtained in this example was numbered 20200410.
Example 11
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of the adjuvant is 1 mg/dose, and the content of the vaccine antigen is 20 mu g/dose. The vaccine obtained in this example was numbered 20200411.
Example 12
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of the adjuvant is 1 mg/dose, and the content of the vaccine antigen is 40 mu g/dose. The vaccine obtained in this example was numbered 20200412.
Example 13
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of an adjuvant is 5 mg/dose, and the content of a vaccine antigen is 5 mu g/dose. The vaccine obtained in this example was numbered 20200413.
Example 14
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of an adjuvant is 5 mg/dose, and the content of a vaccine antigen is 10 mu g/dose. The vaccine obtained in this example was numbered 20200414.
Example 15
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of an adjuvant is 5 mg/dose, and the content of the vaccine antigen is 20 mu g/dose. The vaccine obtained in this example was numbered 20200415.
Example 16
This example provides a combination of the use of CpG oligodeoxynucleotide adjuvants and a novel coronavirus COVID-19 recombinant vaccine. The vaccine is a liquid vaccine, wherein the concentration of an adjuvant is 5 mg/dose, and the content of a vaccine antigen is 40 mu g/dose. The vaccine obtained in this example was numbered 20200416.
Experimental example 1: immunological evaluation of adjuvant Effect in mouse models
(1) Neutralizing antibody titer determination
As shown in Table 2, 8 groups of vaccines provided in the examples were diluted 1:4, 1:16, and 1:64 with physiological saline, and BALB/c mice were inoculated using a 1mL syringe, 10 mice in each group were immunized on days 0 and 7, one dose was injected into each abdominal cavity, blood was collected 4 weeks after the 1 st immunization, and neutralizing antibodies were detected. A neutralizing antibody titer GMT greater than 8 is considered positive and is considered protective. The results are shown in Table 3.
Table 2: experimental design for immunological evaluation of adjuvant effect on mouse model
| Examples | Vaccine numbering | Antigen content (μ g/dose) | CpG adjuvant content (mg/dose) |
| 1 | 20200401 | 5 | 0.5 |
| 2 | 20200402 | 10 | 0.5 |
| 3 | 20200403 | 20 | 0.5 |
| 4 | 20200404 | 40 | 0.5 |
| 5 | 20200405 | 5 | 3 |
| 6 | 20200406 | 10 | 3 |
| 7 | 20200407 | 20 | 3 |
| 8 | 20200408 | 40 | 3 |
Table 3: neutralization potency of COVID-19 recombinant vaccine immune BALB/c mice with different CpG adjuvants
The results show that the COVID-19 vaccines with different effective components and adjuvant contents can generate enough high neutralizing antibody titer to immunize BALB/c mice. Vaccines with high adjuvant content are relatively more immunogenic than vaccines with the same active ingredient in lower amounts. The vaccines provided by the invention can induce BALB/c mice to generate protective neutralizing antibodies.
(2) IgG antibody titer determination
The 8 groups of vaccines provided in the examples were subjected to IgG antibody titer determination as shown in table 2. The IgG antibody titer detection method comprises the following steps:
coating the new coronavirus on an enzyme label plate according to the protein concentration of 1 mu g/ml and 100 mu l/hole, coating overnight at the temperature of 2-8 ℃ or coating for more than 2 hours at the temperature of 37 ℃, washing the plate, patting the plate to be dry, sealing by adopting 0.01M PBS containing 1% BSA or 10% calf serum, sealing at the temperature of 200 mu l/hole, sealing for 1-2 hours at the temperature of 37 ℃, throwing off liquid in the hole, and draining for later use. Diluting a sample to be detected and negative serum by adopting the sealing liquid series, adding a sealed enzyme label plate into each hole of 100 mu l, incubating for 60-70 minutes at 37 ℃, washing the plate and drying; adding HRP enzyme-labeled antibody of the corresponding anti species, incubating for 45-60 minutes at 37 ℃, washing the plate and patting dry; adding 50 μ l of each developing solution A/B, developing at 37 deg.C for 10-15 min, adding 2M H2SO4And (6) terminating. And (4) analyzing results: and 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 OD value of the negative control is less than 0.05, the value is calculated as 0.05.
The results are shown in FIG. 1.
Experimental example 2: cytokine detection
The vaccines in the table 2 are used for immunizing BALB/c mice according to the procedures of 0 day immunization, 7 days immunization and 28 days blood collection respectively, and cell supernatants are collected for cell immune detection. Method for detecting CD4 of specific expression S protein cytokine in serum by adopting intracellular cytokine staining method and flow cytometry method+T cells.
(1) Isolation of splenic lymphocytes
Mice were sacrificed by cervical dislocation and soaked in 70% alcohol for about 3 min. The spleens were aseptically removed from the mice in a biosafety cabinet and placed on a 200 mesh cell sieve in a sterile plate. 10mL of RPMI1640 complete medium was added, the spleen gently ground into single cells with a syringe plunger, and the cell screen was rinsed with 10mL of RPMI1640 complete medium to obtain more splenocytes. The spleen cell suspension was transferred to a 50mL centrifuge tube and centrifuged horizontally at 500g for 5 min. The supernatant was discarded, the cells were resuspended in 3m |1 × erythrocyte lysate, lysed at room temperature for 5min, 27mL of RPMI1640 complete medium was added, and centrifuged horizontally at 500g for 5 min. Discarding the supernatant, washing the cells once with 20mL of RPMI1640 complete medium, resuspending the cells with an appropriate amount of medium, filtering the cells through a 200-mesh cell sieve into a 10mL test tube, diluting 50 μ L of the cells by 20 times, and counting the cells for later use.
(2) In vitro stimulation of mouse splenocytes
Diluting the separated mouse spleen cells to 4 × 106cells/mL, 0.5mL per well, was added to a 24-well plate. A specific CTL epitope stimulation hole and a stimulation hole are respectively arranged in each mouse. The specific epitope concentration was 2. mu.g/mL per peptide, without stimulation an equivalent amount of DMSO was added. As positive controls, PMA and ionomycin stimulated wells were added, with PMA concentration of 100ng/mL and ionomycin concentration of 1. mu.g/mL. At the same time, 1. mu.L of Brilliant Violet 421TM anti-mouse CD107a was added to each well. After the cells were cultured in a 5% CO2 cell culture chamber at 37 ℃ for 1 hour, a suitable amount of GolgiStop and/or GolgiPlug was added per well as a blocking agent for cytokine secretion. After a total of 6 hours of culture, staining of the relevant antigens was performed for flow cytometry detection of intracellular cytokines.
(3) Cell surface antigen and intracellular cytokine staining
After 6 hours of in vitro stimulation of splenocytes, they were transferred to a flow tube, centrifuged at 500g for 5 minutes at 4 ℃ and the supernatant discarded. Appropriate amounts of the fluorescently labeled antibodies PerCP/Cy5.5-conjugated anti-CD3(clone 145-2c11) and FITC conjugated anti-CD8(clone 53-6.7) were diluted with PBS + 2% FBS at the recommended usage amounts as the instructions, 50. mu.L of each tube was added, mixed gently and left at 4 ℃ for 30 minutes. After 30 minutes, 3mL PBS + 2% FBS was added to each tube, centrifuged at 500g for 5 minutes at 4 ℃ and the supernatant was discarded. Cells were fixed and perforated by adding 200. mu.L of Cytofix/CytopermTM Fixation and Permeabilizaton Solution per tube, standing 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 discarded. An appropriate amount of PE conjugated anti-IFN-. gamma. (clone XMG1.2) antibody was diluted with 1 XPerm/WashTM Buffer as recommended in the specification, 50. mu.L of the diluted antibody was added to each tube, and the mixture was 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, the supernatant was discarded, resuspended in 200. mu.L of PBS, and tested on the machine. To adjust the fluorescence compensation between the dyes in the assay, unstained tubes, singly-stained PerCP/Cy5.5-conjugated anti-CD3 tubes, singly-stained FITC conjugated anti-CD8 tubes, and singly-stained PEconjugated anti-IFN-. gamma.tubes were set, wherein the PE conjugated anti-IFN-. gamma.singly-stained tubes used positively-stimulated cells.
(4) Flow cytometry detection
Flow cytometry detection was performed using a BD FACS cantm. The appropriate voltage is first adjusted for each channel, the fluorescence compensation between each dye is adjusted using a single fluorescent dye sample, then the samples are loaded in sequence and the data is collected.
(5) Intracellular cytokine staining flow cytometry detection results
The detection result of intracellular cytokine staining flow cytometry shows that the splenocytes of the immunized mice are stimulated by the epitope, and then the CD4+T cells can secrete IFN gamma and IL-2 cytokines in a large amount, and the expression level is obviously higher than that of a control group. The results are shown in FIG. 2.
The results show that the vaccines in the antigen content range provided by the invention can cause good cellular immunity. Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Beijing Koxing vitamin technology Co., Ltd
<120> COVID-19 recombinant protein vaccine composition and application
<130> RYP2010643.6
<160> 9
<170> PatentIn version 3.5
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Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Arg Val Gln
1 5 10 15
Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro
20 25 30
Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp
35 40 45
Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr
50 55 60
Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr
65 70 75 80
Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val
85 90 95
Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys
100 105 110
Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val
115 120 125
Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr
130 135 140
Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu
145 150 155 160
Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn
165 170 175
Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe
180 185 190
Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu
195 200 205
Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys
210 215 220
Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly Gly Gly Gly
225 230 235 240
Ser Gly Gly Gly Gly Ser Ile Ser Arg Thr Pro Glu Val Thr Cys Val
245 250 255
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
260 265 270
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
275 280 285
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
290 295 300
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
305 310 315 320
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
325 330 335
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
340 345 350
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
355 360 365
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
370 375 380
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
385 390 395 400
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
405 410 415
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
420 425 430
Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440
<210> 2
<211> 198
<212> PRT
<213> Artificial sequence
<400> 2
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
1 5 10 15
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
20 25 30
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
35 40 45
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
50 55 60
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
65 70 75 80
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
85 90 95
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
100 105 110
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
115 120 125
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
130 135 140
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
145 150 155 160
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
165 170 175
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
180 185 190
Cys Gly Pro Lys Lys Ser
195
<210> 3
<211> 345
<212> PRT
<213> Artificial sequence
<400> 3
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
1 5 10 15
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
20 25 30
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
35 40 45
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
50 55 60
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
65 70 75 80
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
85 90 95
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
100 105 110
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
115 120 125
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
130 135 140
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
145 150 155 160
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
165 170 175
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
180 185 190
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
195 200 205
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
210 215 220
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
225 230 235 240
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
245 250 255
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
260 265 270
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
275 280 285
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
290 295 300
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
305 310 315 320
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
325 330 335
Gly Ile Cys Ala Ser Tyr Gln Thr Gln
340 345
<210> 4
<211> 197
<212> PRT
<213> Artificial sequence
<400> 4
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
1 5 10 15
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
20 25 30
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
35 40 45
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
50 55 60
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
65 70 75 80
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
85 90 95
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
100 105 110
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
115 120 125
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
130 135 140
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
145 150 155 160
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
165 170 175
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
180 185 190
Thr Val Cys Gly Pro
195
<210> 5
<211> 261
<212> PRT
<213> Artificial sequence
<400> 5
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
1 5 10 15
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
20 25 30
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
35 40 45
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
50 55 60
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
65 70 75 80
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
85 90 95
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
100 105 110
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
115 120 125
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
130 135 140
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
145 150 155 160
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
165 170 175
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
180 185 190
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
195 200 205
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
210 215 220
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
225 230 235 240
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
245 250 255
Pro Cys Ser Phe Gly
260
<210> 6
<211> 1213
<212> PRT
<213> Artificial sequence
<400> 6
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Ala Ser Val Ala
675 680 685
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
690 695 700
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
705 710 715 720
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
725 730 735
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
740 745 750
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
755 760 765
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
770 775 780
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
785 790 795 800
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
805 810 815
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
820 825 830
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
835 840 845
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
850 855 860
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
865 870 875 880
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
885 890 895
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
900 905 910
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
915 920 925
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
930 935 940
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
945 950 955 960
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
965 970 975
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
980 985 990
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
995 1000 1005
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn
1010 1015 1020
Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys
1025 1030 1035
Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
1040 1045 1050
Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val
1055 1060 1065
Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His
1070 1075 1080
Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn
1085 1090 1095
Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln
1100 1105 1110
Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val
1115 1120 1125
Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro
1130 1135 1140
Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn
1145 1150 1155
His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn
1160 1165 1170
Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu
1175 1180 1185
Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
1190 1195 1200
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro
1205 1210
<210> 7
<211> 22
<212> DNA
<213> Artificial sequence
<400> 7
tgactgtgaa cgttcgagat ga 22
<210> 8
<211> 23
<212> DNA
<213> Artificial sequence
<400> 8
tcgacgttcg tcgttcgtcg ttc 23
<210> 9
<211> 24
<212> DNA
<213> Artificial sequence
<400> 9
tcgtcgtttt gtcgttttgt cgtt 24
Claims (10)
1. A COVID-19 recombinant protein vaccine composition comprising CpG oligodeoxynucleotide as an adjuvant.
2. The COVID-19 recombinant protein vaccine composition of claim 1, which comprises (1) the receptor binding domain of the S protein of COVID-19 and (2) the Fc fragment of IgG.
3. The COVID-19 recombinant protein vaccine composition of claim 1, comprising the ACE2 receptor binding domain and/or surface epitope of the S protein; the S protein is S protein of SARS or S protein of COVID-19.
4. The COVID-19 recombinant protein vaccine composition of claim 1, comprising one or more of the amino acid sequences of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, or an amino acid sequence with more than 90% identity thereto.
5. The COVID-19 recombinant protein vaccine composition according to any one of claims 1 to 4, wherein the content of the antigen effective component in the recombinant protein vaccine is 1 to 80 μ g/dose; preferably, the content of the antigen effective component in the recombinant protein vaccine is 2.5 mu g/dose, 10 mu g/dose, 40 mu g/dose or 80 mu g/dose.
6. The COVID-19 recombinant protein vaccine composition of any one of claims 1-4, wherein the content of CpG oligodeoxynucleotide adjuvants in the recombinant protein vaccine is 0.25-5 mg/dose; preferably, the content of the CpG oligodeoxynucleotide adjuvant in the recombinant protein vaccine is 0.25 mg/dose, 1 mg/dose, 2 mg/dose or 5 mg/dose.
7. The COVID-19 recombinant protein vaccine composition of any one of claims 1-6, wherein the recombinant protein vaccine composition is a liquid vaccine, preferably an intramuscular liquid injection, an intranasal liquid spray, an intradermal liquid injection, or a subcutaneous liquid injection.
8. Use of a COVID-19 recombinant protein vaccine composition according to any one of claims 1 to 7 in the manufacture of a medicament for the prevention and/or treatment of a disease caused by a COVID-19 infection; preferably, the disease is pneumonia and syndrome, severe acute respiratory infection, intestinal disease, heart failure, renal failure or severe acute respiratory syndrome.
Use of CpG oligodeoxynucleotides as immunopotentiators or immunoadjuvants for a COVID-19 recombinant protein vaccine, or for increasing the immunogenicity of a antigen or vaccine of a COVID-19 recombinant protein vaccine.
10. The use according to claim 9, wherein 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 20 bp; preferably, the CpG oligodeoxynucleotide sequence is:
5'-TGACTGTGAACGTTCGAGATGA-3', respectively; or
5'-TCGACGTTCGTCGTTCGTCGTTC-3', respectively; or
5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2014203624A1 (en) * | 2007-08-01 | 2014-07-24 | Idera Pharmaceuticals, Inc. | Novel synthetic agonists of TLR9 |
| CN108728444A (en) * | 2017-04-18 | 2018-11-02 | 长春华普生物技术股份有限公司 | Immunoregulation polynucleotide and its application |
| CN110951756A (en) * | 2020-02-23 | 2020-04-03 | 广州恩宝生物医药科技有限公司 | Nucleic acid sequence for expressing SARS-CoV-2 virus antigen peptide and its application |
| CN110974950A (en) * | 2020-03-05 | 2020-04-10 | 广州恩宝生物医药科技有限公司 | Adenovirus vector vaccine for preventing SARS-CoV-2 infection |
-
2020
- 2020-04-17 CN CN202010307478.4A patent/CN113521267A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2014203624A1 (en) * | 2007-08-01 | 2014-07-24 | Idera Pharmaceuticals, Inc. | Novel synthetic agonists of TLR9 |
| CN108728444A (en) * | 2017-04-18 | 2018-11-02 | 长春华普生物技术股份有限公司 | Immunoregulation polynucleotide and its application |
| CN110753756A (en) * | 2017-04-18 | 2020-02-04 | 长春华普生物技术股份有限公司 | Immunomodulatory polynucleotides and uses thereof |
| CN110951756A (en) * | 2020-02-23 | 2020-04-03 | 广州恩宝生物医药科技有限公司 | Nucleic acid sequence for expressing SARS-CoV-2 virus antigen peptide and its application |
| CN110974950A (en) * | 2020-03-05 | 2020-04-10 | 广州恩宝生物医药科技有限公司 | Adenovirus vector vaccine for preventing SARS-CoV-2 infection |
Non-Patent Citations (2)
| Title |
|---|
| BRIAN D. QUINLAN等: "The SARS-CoV-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement", BIORXIV, pages 5 - 8 * |
| TUNG THANH LE等: "The COVID-19 vaccine development landscape", NATURE REVIEWS DRUG DISCOVERY, pages 305 * |
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