CN113842455B - Adjuvant of novel coronavirus vaccine, application of adjuvant and novel coronavirus bivalent recombinant vaccine - Google Patents

Adjuvant of novel coronavirus vaccine, application of adjuvant and novel coronavirus bivalent recombinant vaccine Download PDF

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CN113842455B
CN113842455B CN202110995286.1A CN202110995286A CN113842455B CN 113842455 B CN113842455 B CN 113842455B CN 202110995286 A CN202110995286 A CN 202110995286A CN 113842455 B CN113842455 B CN 113842455B
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刘永江
张海江
贠炳岭
杨秀芬
伍树明
高文双
银飞
姜绪林
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Abstract

The invention discloses an adjuvant for a novel coronavirus COVID-19 vaccine and a bivalent vaccine developed by an antigen containing a novel coronavirus epidemic HuB strain and a south Africa mutant strain B.1.351. The adjuvant of the invention has the advantages of being better suitable for a sodium acetate buffer solution system with stable COVID-19 vaccine protein, and having high neutralizing antibody and cellular immunity level. The bivalent COVID-19 vaccine disclosed by the invention has a good protective effect on the infection of novel coronaviruses, particularly HuB strains, south Africa mutant strains and Delta mutant strains.

Description

Adjuvant of novel coronavirus vaccine, application of adjuvant and novel coronavirus bivalent recombinant vaccine
Technical Field
The invention belongs to the technical field of biology and medicine, and particularly relates to an adjuvant for a novel recombinant subunit vaccine of coronavirus and a novel bivalent vaccine of coronavirus COVID 19.
Background
The novel coronavirus belongs to a novel coronavirus of beta genus, and has envelope, round or elliptical particle, usually polymorphism, and diameter of 60-140 nm. The structural protein of the new coronavirus SARS-CoV-2 includes S protein, N protein, E protein and M protein. In SARS-CoV-2, only the neutralizing antibody directed against S protein can neutralize virus virulence and prevent virus infection to organism, and the Spike protein (S protein) on the mantle of coronavirus is a key protein for recognizing host cell Receptor in virus infection process, and the S protein is composed of two subunits of S1 and S2, wherein the S1 subunit contains Receptor Binding Domain (RBD) to mediate adsorption, and the S2 subunit mainly shows fusion activity.
The mutant strain in south Africa is a popular call, and refers to a mutant lineage (linkage) of a novel coronavirus detected in south Africa at 12/18 th day earlier than 2020. This mutation is then spread across the world at its very rapid rate, originally referred to as 501y.v2, to distinguish it from the N501Y mutation in the british mutant. They all belong to the B lineage mutations. The south African mutant lineage was named according to the New crown lineage nomenclature (B.1.351). This variant has become the local leader since then. B.1.351 contained multiple spike mutations in addition to D614G, including a cluster of mutations in NTD (e.g., 242-244del and R246I), 3 mutations in RBD (K417N, E484K and N501Y) and one mutation near the furin cleavage site (A701V). Wangcongfei et al published a research paper in Nature journal (antibody resistance of SARS-Cov-2 variantsB.1.351and B.1.1.7, Nature,2021) and revealed the protective efficiency of partially neutralizing antibodies and vaccine immune sera against mutants in south Africa. By comparison of the neutralizing activities, it was found that the neutralizing activity against the British mutant B.1.1.7 was substantially unchanged, but the neutralizing activity against the south African mutant B.1.351 was significantly reduced (12.4-fold, Moderna; 10.3-fold, feverer)
The novel coronavirus COVID-19 seriously harms the life health of people and is mainly used for prevention, and the vaccine can effectively prevent virus infection and severe pneumonia caused by the virus infection. For the novel coronavirus COVID-19, some researches have been carried out to obtain related vaccines, and the vaccines which are on the market at present are developed for the HuB strain (also called Hubei strain) novel coronavirus, so that the protection power on the south Africa mutant strain B.1.351 is poor, and the development of effective vaccines for the novel coronavirus of the south Africa mutant strain B.1.351 is urgently needed.
Disclosure of Invention
The present inventors developed vaccines against the HuB strain and the south african mutant strain b.1.351 of the novel coronavirus, respectively (application nos. 202011454175.1 and 202110659137.8). Through optimization of the adjuvant and optimization research of the vaccine proportion, the invention successfully develops the bivalent vaccine, and can have good protection effect on the infection of the two new coronavirus and diseases caused by the infection.
The invention provides a novel adjuvant for coronavirus vaccines, which is characterized by comprising one of the following components:
a. squalene, tween-80, span-85, sodium citrate, citric acid and water;
b. squalene, tween-80, span-85, and acetic acid-sodium acetate;
c. squalene, span-85, SDS, SolutolHS-15, acetic acid-sodium acetate.
Preferably, it is constituted by one of the following components, calculated on a 5ml solution basis:
a. 200mg of squalene 190-5, 20-30mg of tween-8020, 8535-40 mg of span-8535, 12-14mg of sodium citrate, 0.8-0.9mg of citric acid and 5ml of water;
b. 230mg of squalene 200-;
c. 230mg of squalene 200-.
More preferably, it consists of the following components, calculated as 5ml of solution: 215mg of squalene, 8025 mg of tween-8025 mg of span-8525 mg and 5ml of acetic acid-sodium acetate aqueous solution.
In one embodiment, the adjuvant is prepared as follows:
weighing squalene, tween-80 and span-85, or/and SDS, stirring and mixing uniformly to obtain an oil phase; adding acetic acid-sodium acetate aqueous solution water phase, mixing oil phase and water phase, and stirring with a dispersion machine to form primary emulsion; homogenizing with ultrasonic or high pressure homogenizer or microfluidizer to obtain refined milk.
The invention further provides the application of the adjuvant in the preparation of novel coronavirus vaccine preparations.
Preferably, the vaccine is the following fusion protein of the novel coronavirus COVID-19 epitope: the vaccine consists of a novel coronavirus COVID-19 epitope fragment and an immunoglobulin Fc fragment, wherein the epitope fragment of the novel coronavirus COVID-19 vaccine is an RBD fragment in an S1 subunit and an SD1 fragment. More preferably, the novel coronavirus vaccine is a HuB strain vaccine and/or a south african mutant strain b.1.351 vaccine of the novel coronavirus.
The invention further provides a bivalent recombinant vaccine against the novel coronavirus, which is characterized by comprising a combination of recombinant vaccines against the HuB strain and the south Africa mutant strain, wherein the HuB strain vaccine against the novel coronavirus is composed of an epitope fragment and an immunoglobulin Fc fragment of the HuB strain of the novel coronavirus, and the epitope fragment of the HuB strain of the novel coronavirus is an RBD fragment and an SD1 fragment in a S1 subunit; the south Africa mutant strain B.1.351 vaccine for the novel coronavirus is composed of an epitope fragment and an immunoglobulin Fc fragment of the novel coronavirus south Africa mutant strain B.1.351, wherein the epitope fragment of the novel coronavirus south Africa mutant strain B.1.351 is an RBD fragment in a S1 subunit and an SD1 fragment. ..
Preferably, the adjuvant adopts the adjuvant described above; preferably, the vaccine dose ratio of the HuB strain vaccine against the novel coronavirus and the south african mutant strain b.1.351 is 2:1, preferably 60 mug of the two, 40 mug of the two, 20 mug of the two and 15 mug of the 30 mug of the two, more preferably 40 mug of the two and 20 mug of the two.
More preferably, the fusion protein of the HuB strain vaccine against the novel coronavirus has an amino acid sequence shown as SEQ ID NO. 1; the fusion protein of south Africa mutant strain B.1.351 vaccine for the novel coronavirus has an amino acid sequence shown as SEQ ID NO. 2.
The inventor researches an optimized adjuvant aiming at a vaccine developed by a HuB strain and a south Africa mutant strain B.1.351 of a novel coronavirus, and the optimized adjuvant has the advantages of containing an acetic acid-sodium acetate buffer solution system which is better suitable for stabilizing the protein of the novel coronavirus, and having high neutralizing antibody and cellular immunity levels. On the basis, the bivalent recombinant vaccine disclosed by the invention is obtained through research, has broad spectrum, and experiments prove that the vaccine can provide protection for HuB strains, south Africa mutant strains and Delta mutant strains. The bivalent recombinant vaccines of the present invention also have synergistic effects, and in one experiment, it was shown that the neutralizing activity increased from 5337 to 16188 when combined at 2:1, the neutralizing antibody level of the HuB strain increased by about 3-fold compared to HuB strain vaccine alone, and the neutralizing antibody level of the south african strain increased from 629 to 3658, by about 5-fold. In addition, experiments also show that the Delta mutant strain also has very good immune protection effect, which is unexpected, and further shows that the bivalent vaccine has broad spectrum, so that the cost for developing the vaccine can be reduced greatly. Thus, it is reasonable to believe that the bivalent vaccine of the present invention has synergistic protection as well for other types of mutants.
Detailed Description
The invention will be further described with reference to specific embodiments for better understanding, but the invention is not to be construed as being limited thereto.
The experimental protocol and procedures were carried out according to the usual procedures, if not otherwise specified.
Example one, adjuvant configuration and optimization
1. Adjuvant formulations
As the main components of the referenced positive control MF59 adjuvant are squalene, Tween 80 and span 85, and the buffer solution is a citric acid system, in addition, the recombinant expressed new coronin is shown to be more stable in a sodium acetate buffer system with the pH of about 5.6 through preliminary research, so that the adjuvant optimization of the citric acid buffer system and the sodium acetate buffer system is respectively carried out, and simultaneously, the research of SDS, SolutolHS-15 and RH40 which have similar protein protection functions with the Tween 80 is added.
Formulation of adjuvants the adjuvant test formulations are as follows:
Figure GDA0003574072660000041
weighing squalene, tween-80 and span-85 or/and SDS, stirring and mixing uniformly to obtain oil phase. Adding acetic acid-sodium acetate solution into water phase. And (3) uniformly mixing the oil phase and the water phase, stirring the mixture by a dispersion machine respectively for 5 to 20 minutes at 1000 to 10000 rpm to form primary emulsion, and treating the primary emulsion by a microfluidizer at 500 to 3000bar for 1 to 5 cycles for homogenization to obtain refined emulsion.
2. Mouse immunization and neutralizing antibody detection
The HuB strain recombinant proteins (see 202110659137.8 for a preparation method thereof) are respectively mixed with the adjuvant combinations, and mice are immunized. 100 female BALB/c mice were purchased for 6-8 weeks and randomly divided into 2 groups (experimental and negative control) of 10 mice each. Each mouse was immunized muscle with 1ug of protein. The booster immunization was performed twice every two weeks for a total of two immunizations. From the first immunization, blood was collected from the mice at two weeks intervals, and the supernatant was centrifuged to carry out the detection of neutralizing antibody titer.
The titer of the neutralizing antibody is detected by adopting a novel coronavirus pseudovirus neutralizing method, and the experimental method comprises the following steps:
(1) cell preparation: the day before the experiment, will be about 1x104Inoculum size of individual cells/well cells to be infected were seeded in 96-well cell culture plates.
(2) Pseudovirus infection: taking out the frozen pseudovirus, thawing, sucking the pseudovirus with required amount, adding the pseudovirus into the cell culture system to infect the target cell after complete thawing, and replacing fresh culture medium for continuous culture after 6-8H after virus infection.
(3) Infection detection: after the cells are infected with pseudovirus 48-72H, the infection efficiency is judged by observing the activity of the detected luciferase.
(4) The maximum dilution of serum at which 50% of infection was inhibited was taken as the neutralizing antibody titer. The results are given in the table below.
Figure GDA0003574072660000042
Figure GDA0003574072660000051
The results of the neutralizing antibody experiments show that the neutralizing antibody levels of the serum of the mice after two weeks of the second immunization and the adjuvant formulas 5, 8 and 9 are the highest, the titer can reach about 6000, and the neutralizing antibody levels of the adjuvant formulas 3, 6, 7 and 10 and the positive control MF59 are equivalent to each other and are about 3500. The neutralizing antibody levels for adjuvant formulations 1, 2, 4 were significantly lower than those for the other formulations.
3. Detection of mouse cellular immunity level
(1) ELISPOT coating: add 20. mu.l of 70% ethanol per well to pre-wet PVDF membrane and spin dry. Wash with sterile PBS, add 100. mu.l per well, wash 2 times to remove ethanol. The coated antibody was diluted with sterile PBS and 100. mu.l of diluted coated antibody (final concentration 10. mu.g/ml) was added to each well. Coating was carried out overnight at 4 ℃ or for 2 hours at room temperature.
(2) And (3) sealing: the coating solution was poured and washed 3 times with sterile PBS. And finally, drying the sterilized absorbent paper in a buckling manner. Add 200. mu.l of cell culture medium to each well and block, incubate for 1 hour at room temperature.
(3) Cell culture: the blocking solution was poured, washed and the prepared cells were added directly. (initial assay, stimulus concentration gradient, and/or cell count gradient pre-assay) wells of the experimental group were filled with cells and stimulus, positive control group was filled with 10. mu.l PHA per well (final PHA concentration 1-4ug/mL), and negative control group was filled with cells per well. Culturing in a carbon dioxide incubator at 37 deg.C for 16-24 h.
(4) And (3) detection: the plate was washed 3 times with PBS/0.05% Tween-20. Spin-dry, dilute the detection antibody with antibody diluent (PBS + 1% BSA). Add 1: 100. mu.l of the diluted biotin-labeled detection antibody (1000 ml) was incubated at room temperature for 2 hours. The plate was washed 3 times with PBS/0.05% Tween-20. Spin-dry, dilute the enzyme avidin with antibody diluent (PBS + 1% BSA), add 1: 1000 diluted avidin was incubated at room temperature for 1 h.
(5) Color development: the plate was washed 3 times with PBS/0.05% Tween-20 and 1 time with PBS. Patting to dry. 50. mu.l of BCIP. NBT developing solution was added. Standing at room temperature for 15-45min, and keeping out of the sun.
(6) The development was stopped, the plates were rinsed with tap water, dried at room temperature and observed under an inverted microscope for spot counting, the results of which are given in the table below.
Figure GDA0003574072660000052
Figure GDA0003574072660000061
The ELISPOT method detects the cytokines IFN-r and IL-2, and the results show that the three adjuvant formulas of 5, 8 and 9 and the positive control MF59 adjuvant have obvious positive, which indicates that the cell immunity exists. While the other prescriptions were negative, indicating no cellular immunity.
Through the research of the ten adjuvant formulas, since the sodium acetate buffer system for the adjuvant No. 8 is better suitable for the stabilization of the new crown vaccine protein and the combination of the neutralizing antibody and the cellular immunity level is highest, the adjuvant No. 8 of the adjuvant is selected as the adjuvant of the new crown vaccine and is named as KL02 adjuvant.
Example two, Effect of different adjuvants on neutralizing antibody levels in New crown vaccines
The HuB strain recombinant protein (the preparation method is shown in 202110659137.8) is respectively mixed with different adjuvant combinations, and the mice are immunized. Each group containing 10 animals. Each mouse was immunized muscle with 1ug of protein. The booster immunization was performed two weeks apart. Blood of the mice is collected three weeks after the first immunization, and the supernatant is centrifuged to carry out the detection of the neutralizing antibody titer of the pseudovirus method and the detection of the cell factors of the ELISPOT method. Two weeks after the second immunization, mouse sera were collected and tested for pseudovirus neutralizing antibodies.
Figure GDA0003574072660000062
The results show that the neutralizing activity of the KL02 adjuvant and MF59 adjuvant group was significantly higher than that of the aluminum hydroxide adjuvant, aluminum phosphate adjuvant and CpG adjuvant group in the serum collected two weeks after the second immunization.
Figure GDA0003574072660000063
The results showed that in serum collected three weeks after one immunization, the cytokines were positive for the KL02 adjuvant and MF59 adjuvant groups, while negative for the aluminum hydroxide adjuvant, aluminum phosphate adjuvant and CpG adjuvant groups, indicating that the KL02 adjuvant and MF59 adjuvant groups had cellular immunity, while the other adjuvant groups had no cellular immunity.
EXAMPLE III study of antigen dose
The HuB strain recombinant protein (the preparation method is shown in 202110659137.8) is respectively mixed with KL02 adjuvant or aluminum hydroxide adjuvant, and the rhesus monkey is immunized. The KL02 adjuvant group was given two doses to immunize 4 monkeys separately, and the aluminum hydroxide adjuvant group was given one dose for a total of 12 rhesus monkeys. The booster immunizations were performed once every 4 weeks for a total of three times. Serum was collected after the second and third immunizations, respectively, and the neutralizing antibody was detected using live virus from the new corona HuB strain using the new corona HuB virus neutralizing antibody detection method.
The detection method of the neutralizing antibody of the new coronavirus comprises the following steps: the 50% virus neutralizing antibody titer was detected using cytopathic effect (cytopathic effect) assay. Serum samples were inactivated by incubation at 56 ℃ for 30 min. Vero E6 cells at 1X104Culturing for 18-24 h on a 96-well plate after inoculation. The next day, inactivated sera were diluted in 3-fold gradients per stage, with 6 replicates per dilution. 70 μ L of the serially diluted serum was mixed with 70 μ L of 140TCID50 neocoronavirus in a 96-well plate at 37 deg.C (5% CO)2) After 2h incubation, 100. mu.L of the mixture was added to 96-well plates filled with Vero E6 cells and incubated for 4 days. The cytotoxic effect of each well is recorded by observation under a microscope, and the maximum dilution factor of serum capable of inhibiting the lesion of 50 percent of cell samples is calculated and determined by a Reed-Muench method and is used as the neutralizing antibody titer.
Figure GDA0003574072660000071
(remark: the highest dilution factor of the serum in this experiment is 1:128, and the result shows that the actual neutralizing antibody titer level of the serum with 1:128 exceeds 1: 128.)
The result shows that the titer of the neutralizing antibody in the serum after the three times of immunization is higher than that of the neutralizing antibody after the second time of immunization, which shows that the vaccine has better effect than 2 times of immunization after 3 times of immunization; in serum after three times of immunization, the neutralizing antibody titer of the KL02 adjuvant group with low dose and high dose is obviously higher than that of the aluminum hydroxide adjuvant group, and the experimental result of 20 mu g antigen immunization of the KL02 adjuvant group shows that the antibody titer of 2 monkeys is 64, two monkeys is 128, while the antibody titer of 60 mu g antigen immunization group and 4 monkeys is 128, which indicates that the effect of the 60 mu g antigen immunization group is better; in addition, the rhesus monkey No. 5 had only 16 in two weeks of immunization, but reached 128 in two weeks of immunization, indicating that the 60 μ g dose was able to more eliminate individual differences. Therefore, selection of the dose, preferably 60 μ g, may provide better protection of neutralizing antibodies.
EXAMPLE four, antigen matching Studies of bivalent New crown vaccines
Recombinant proteins of HuB strain and south Africa strain with different ratios (the preparation methods are respectively shown in 202011454175.1 and 202110659137.8) are respectively mixed with KL02 adjuvant, and mice are immunized. Each group had 10. Each mouse was immunized intramuscularly with a total of 3. mu.g of protein. The booster immunizations were performed two weeks apart. Blood of the mice is collected two weeks after the third immunization, and the supernatant is centrifuged to carry out the detection of the neutralizing antibody titer by the pseudovirus method.
Figure GDA0003574072660000081
The experimental results show that the HuB strain: the neutralizing antibody titer is highest when the antigen ratio of the south Africa strain is 2: 1. The results also show that after the bivalent vaccine is formed by the two protein components, the vaccine can protect both epidemic strains and south African strains, besides the broad spectrum of the vaccine is increased, the experimental results show that the neutralizing antibody levels of the two protein components also have a synergistic enhancement effect, when the two protein components are combined in a 2:1 mode, the titer of the neutralizing antibody of the epidemic strain pseudovirus is increased from 1:5337 to 1:16188, the neutralizing antibody level of the epidemic strain pseudovirus is increased by about 3 times compared with that of the epidemic strain vaccine alone, and the neutralizing antibody level of the south African strain pseudovirus is increased from 1:629 to 1:3658 and is increased by about 5 times.
In combination with a rhesus dosage experiment, in the bivalent vaccine determined by the invention, a HuB strain is adopted: the south Africa strain is 2:1, the proportion of 60 mug to 30 mug and the optimal dose proportion are respectively, and each dose of vaccine is 0.5 ml.
EXAMPLE V synergistic protective Effect of bivalent New crown vaccine on other types
The antigen with the determined antigen ratio in example four (HuB strain: south Africa strain antigen ratio of 2:1) was mixed with KL02 adjuvant, and 10 mice were immunized. Each mouse was immunized intramuscularly with a total of 3. mu.g of protein. The booster immunizations were performed two weeks apart. Blood was collected from the mice two weeks after the start of the third immunization, the supernatant was centrifuged and the neutralizing antibodies were detected by the new coronavirus neutralizing antibody assay, and the values listed are mean values.
Figure GDA0003574072660000082
From the above tabulated data, it can be seen that after the bivalent vaccine is used to immunize mice, in addition to the HuB strain, the south Africa mutant strain, the Delta mutant strain has very good immune protection effect, which is unexpected, and the bivalent vaccine has broad spectrum, so that the cost for developing the vaccine can be greatly reduced.
<110> Beijing Kangle guard Biotechnology Ltd
<120> adjuvant of novel coronavirus vaccine, application thereof and novel coronavirus bivalent recombinant vaccine
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<170> PatentIn Version 3.1
<210>1
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<213> novel coronavirus COVID-19 HuB strain RBDSD1-hFc
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MFVFLVLLPL VSSQCRVQPT ESIVRFPNIT NLCPFGEVFN ATRFASVYAW NRKRISNCVA 60
DYSVLYNSAS FSTFKCYGVS PTKLNDLCFT NVYADSFVIR GDEVRQIAPG QTGKIADYNY 120
KLPDDFTGCV IAWNSNNLDS KVGGNYNYLY RLFRKSNLKP FERDISTEIY QAGSTPCNGV 180
EGFNCYFPLQ SYGFQPTNGV GYQPYRVVVL SFELLHAPAT VCGPKKSTNL VKNKCVNFNF 240
NGLTGTGVLT ESNKKFLPFQ QFGRDIADTT DAVRDPQTLE ILDITPCSDK THTCPPCPAP 300
ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR 360
EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP 420
PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV 480
DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK 515
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DYSVLYNSAS FSTFKCYGVS PTKLNDLCFT NVYADSFVIR GDEVRQIAPG QTGNIADYNY 120
KLPDDFTGCV IAWNSNNLDS KVGGNYNYLY RLFRKSNLKP FERDISTEIY QAGSTPCNGV 180
KGFNCYFPLQ SYGFQPTYGV GYQPYRVVVL SFELLHAPAT VCGPKKSTNL VKNKCVNFNF 240
NGLTGTGVLT ESNKKFLPFQ QFGRDIADTT DAVRDPQTLE ILDITPCSDK THTCPPCPAP 300
ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR 360
EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP 420
PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV 480
DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK 515

Claims (12)

1. A novel coronavirus Covid-19 vaccine containing a fusion protein with an amino acid sequence shown as SEQ ID NO. 1and an adjuvant, which is characterized in that the adjuvant consists of the following components in 5ml of solution: 230mg of squalene 200-;
the novel coronavirus Covid-19 vaccine is a vaccine against a HuB strain.
2. The novel coronavirus Covid-19 vaccine of claim 1, wherein the adjuvant has a pH of 5.6.
3. The novel coronavirus Covid-19 vaccine of claim 2, wherein the adjuvant comprises the following components in a 5ml solution: 215mg of squalene, 8025 mg of tween-8025 mg, 8525 mg of span and 5ml of acetic acid-sodium acetate aqueous solution in total volume.
4. The novel coronavirus Covid-19 vaccine of any one of claims 1 to 3, wherein the adjuvant is prepared by the following method:
weighing squalene, tween-80 and span-85, and stirring and mixing to obtain oil phase; adding acetic acid-sodium acetate solution water phase; mixing oil phase and water phase, and stirring with a disperser to obtain primary emulsion; homogenizing with ultrasonic homogenizer or high pressure homogenizer to obtain refined milk.
5. A novel coronavirus Covid-19 vaccine containing a fusion protein with an amino acid sequence shown as SEQ ID NO.2 and an adjuvant, which is characterized in that the adjuvant consists of the following components in 5ml of solution: 230mg of squalene 200-;
the novel coronavirus Covid-19 vaccine is a vaccine against the south Africa mutant strain B.1.351.
6. The novel coronavirus Covid-19 vaccine of claim 5, wherein the adjuvant has a pH of 5.6.
7. The novel coronavirus Covid-19 vaccine of claim 6, wherein the adjuvant comprises the following components in a 5ml solution: 215mg of squalene, 8025 mg of tween-8025 mg, 8525 mg of span and 5ml of acetic acid-sodium acetate aqueous solution in total volume.
8. The novel coronavirus Covid-19 vaccine of any one of claims 5 to 7, wherein the adjuvant is prepared by the following method:
weighing squalene, tween-80 and span-85, and stirring and mixing to obtain oil phase; adding acetic acid-sodium acetate solution water phase; mixing oil phase and water phase, and stirring with a disperser to obtain primary emulsion; homogenizing with ultrasonic homogenizer or high pressure homogenizer to obtain refined milk.
9. A novel coronavirus Covid-19 vaccine containing a fusion protein with an amino acid sequence shown as SEQ ID NO.1, a fusion protein shown as SEQ ID NO.2 and an adjuvant is characterized in that the adjuvant consists of the following components in 5ml of solution: 230mg of squalene 200-;
the fusion protein with the amino acid sequence shown as SEQ ID NO.1 is a vaccine aiming at a HuB strain of the novel coronavirus Covid-19, and the fusion protein shown as SEQ ID NO.2 is a vaccine aiming at a south Africa mutant strain B.1.351 of the novel coronavirus Covid-19.
10. The novel coronavirus Covid-19 vaccine of claim 9, wherein the adjuvant has a pH of 5.6.
11. The novel coronavirus Covid-19 vaccine of claim 10, wherein the adjuvant comprises the following components in a 5ml solution: 215mg of squalene, 8025 mg of tween-8025 mg, 8525 mg of span and 5ml of acetic acid-sodium acetate aqueous solution in total volume.
12. The novel coronavirus Covid-19 vaccine of any one of claims 9 to 11, wherein the adjuvant is prepared by the following method:
weighing squalene, tween-80 and span-85, and stirring and mixing to obtain oil phase; adding acetic acid-sodium acetate solution water phase; mixing oil phase and water phase, and stirring with a disperser to obtain primary emulsion; homogenizing with ultrasonic homogenizer or high pressure homogenizer to obtain refined milk.
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