WO2022000845A1 - Recombinant protein vaccine for preventing sars-cov-2 and preparation method therefor - Google Patents

Abstract

A target amino acid sequence of a SARS-CoV-2 recombinant protein vaccine, a target gene sequence of the recombinant protein vaccine, a eukaryotic cell expression vector containing the target gene sequence, a cell expressing the target amino acid sequence, and the recombinant protein vaccine having the target amino acid sequence as an antigen. The recombinant protein vaccine can effectively induce a body to produce cellular immunity and humoral immunity, can be quickly prepared, and is suitable for the prevention of novel coronavirus.

Description

A recombinant protein vaccine for preventing SARS-CoV-2 and preparation method thereof technical field

The invention relates to the field of epidemic prevention and vaccine production, in particular to a recombinant protein vaccine for preventing SARS-CoV-2 and a preparation method thereof.

Background technique

The novel coronavirus SARS-CoV-2 (WHO designation) is a novel coronavirus that has not been previously identified in humans and is capable of causing severe acute respiratory infection (SARI). Because the population lacks immunity to the new coronavirus SARS-CoV-2, and it is transmitted through respiratory droplets and can also be transmitted through contact, the population is generally susceptible, and there is currently no specific treatment for the disease caused by the new coronavirus. As of June 13, 2020, Johns Hopkins University in the United States has announced a total of 7,652,949 confirmed cases and 425,888 deaths worldwide.

Coronaviruses belong to the order of Taroviruses, Coronaviridae, and the genus Coronaviridae. They are viruses with an envelope and a linear single-stranded positive-stranded RNA genome. They are a large class of viruses that widely exist in nature. For different clinical symptoms ranging from the common cold to severe lung infections such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). SARS-CoV-2 and SARS-CoV belong to the beta family of coronaviruses. In view of the continuous human-to-human infection of the new coronavirus SARS-CoV-2 worldwide, it is urgent to develop a vaccine that can effectively prevent the infection of the new coronavirus SARS-CoV-2, which can effectively prevent the new coronavirus SARS-CoV-2 Pneumonia caused by infection, and even severe respiratory infection is spreading worldwide.

Vaccines currently used in the human body to prevent viral infection mainly include live attenuated vaccines prepared from attenuated viruses, such as live attenuated varicella vaccine, live attenuated measles vaccine, etc.; and inactivated whole virus particle vaccines, such as intestinal Virus EV71 inactivated vaccine, hepatitis A vaccine, etc. However, attenuated vaccines take a long time to reproduce the virus for many generations, and then gradually reduce most of the activity of the virus, which is difficult to meet the prevention needs of acute infectious diseases. Inactivated vaccines have many difficult problems in immune pathology. For example, some inactivated viruses enter the human body and will aggravate the disease when the virus invades.

The recombinant protein vaccine is a vaccine prepared by constructing the target antigen gene of the virus on an expression vector, and then transforming it into bacteria, yeast, mammalian or insect cells, and expressing a large amount of antigen protein under certain induction conditions. . The recombinant protein vaccine can induce the body to produce humoral immunity and cellular immunity, and can be quickly prepared, which is suitable for the prevention of new coronavirus.

SUMMARY OF THE INVENTION

The proteins encoded by SARS-CoV-2 include RNA polymerase proteins, spike proteins, envelope proteins, membrane proteins, and nucleocapsid proteins. Among them, the spike protein (Spike protein, hereinafter referred to as S protein) is a glycoprotein. This protein is both a determinant of the induction of protective immune responses and a site for binding to the host cell angiotensin-converting enzyme 2 (ACE2) receptor. And in mice and African green monkeys, S protein has been shown to induce serum neutralizing antibodies, which have the effect of neutralizing SARS-CoV. In addition, the S protein induces CD4+ and CD8+ T cell responses. Therefore, the present invention selects the S protein as the target for the research and development of the SARS-CoV-2 recombinant protein vaccine.

The first object of the present invention is to provide a target amino acid sequence of a recombinant protein vaccine for the prevention of SARS-CoV-2, including one or more amino acid sequences in SEQ1, SEQ2, and SEQ3, or with a similarity of more than 90%. amino acid sequence.

The above amino acid sequence is shown in the sequence listing of the present invention, specifically shown in Table 1.

Table 1 Amino acid sequence encoded by the target gene

The present invention aligns the amino acid sequences of the respective S proteins of 2003 Sars-CoV and SARS-CoV-2, and selects a sequence with higher conservation as the target amino acid sequence of the recombinant protein vaccine. Although these sequences vary in length, they cover the binding site of the Spike protein to the ACE2 receptor and the major protein surface epitopes.

The second object of the present invention is to provide the target gene sequence of the recombinant protein vaccine, which includes one or more amino acid sequences encoding SEQ1, SEQ2 and SEQ3 or amino acid sequences with a similarity of more than 90%.

As a preferred solution of the present invention, the target gene sequence is a codon-optimized DNA sequence.

The third object of the present invention is to provide a eukaryotic cell expression vector comprising the recombinant protein vaccine target gene sequence. It is constructed by inserting the target gene sequence into a eukaryotic cell expression vector.

The eukaryotic expression vector is preferably an expression vector that can be used in humans.

As a preferred solution of the present invention, the eukaryotic cell expression vector is pCHO1.0 vector. A schematic diagram of the pCHO1.0 vector is shown in FIG. 2 .

The fourth object of the present invention is to provide a cell expressing the recombinant protein vaccine antigen, into which the eukaryotic cell expression vector containing the target gene sequence is exogenously transferred.

As a preferred embodiment of the present invention, the cells are CHO cells. CHO cells (Chinese hamster ovary cells) were obtained from Chinese hamster ovary cells in 1957. The cells are epithelioid cells, usually grown in adherent but also in suspension, and are widely used to express proteins from recombinant DNA.

In the case of selecting CHO cells for antigen expression, the present invention preferably clones the target gene sequence into the eukaryotic cell expression vector after codon optimization of CHO cells, and then transfers it into CHO cells.

The fifth object of the present invention is to provide a recombinant protein vaccine for preventing SARS-CoV-2, the antigen of which comprises one or more amino acid sequences in SEQ1, SEQ2 and SEQ3, or amino acid sequences with a similarity of more than 90%.

As a preferred solution of the present invention, the content of the antigenic active ingredient in the recombinant protein vaccine is 5-200 μg/dose. Specifically, the content of the antigenic active ingredient in the recombinant protein vaccine may be 5 μg/dose, 20 μg/dose, 40 μg/dose or 200 μg/dose. In some specific embodiments of the present invention, the content of the antigenic active ingredient refers to the content of the vaccine stock solution in the vaccine product.

The vaccine provided by the present invention may also contain an immune adjuvant. The immune adjuvant can be selected from inorganic adjuvants, such as aluminum hydroxide adjuvant and the like. The immune adjuvant can also be an organic adjuvant, such as lipopolysaccharide, cytokine and the like.

As a preferred solution of the present invention, the recombinant protein vaccine contains 50-200 μg/dose of aluminum hydroxide adjuvant. Specifically, the content of the adjuvant in the recombinant protein vaccine can be 50 μg/dose, 100 μg/dose or 200 μg/dose.

The vaccine provided by the present invention is preferably a liquid vaccine, which can be in various dosage forms. Specifically, the recombinant protein vaccine can be intramuscular liquid injection, intravenous liquid injection, intranasal liquid injection, intradermal liquid injection or subcutaneous liquid injection. In practical application, it can be adjusted and selected according to clinical needs such as transfection efficiency and local immune monitoring, such as selecting a single dosage form for injection immunization, or selecting multiple mixed dosage forms for injection immunization.

The sixth object of the present invention is to provide a method for preparing the recombinant protein vaccine, which includes the following steps: transferring the eukaryotic cell expression vector containing the target gene sequence into cells for expression, and purifying the supernatant to obtain the vaccine stock solution.

As a preferred embodiment of the present invention, the cells are CHO cells. In the case of selecting CHO cells for antigen expression, the present invention preferably clones the target gene sequence into the eukaryotic cell expression vector after codon optimization of CHO cells, and then transfers it into CHO cells.

The purification can be carried out using conventional methods for purification of vaccine antigens in the art.

As a preferred solution of the present invention, the purification includes: after ultrafiltration and concentration of the supernatant, two-step chromatography of molecular sieve and anion exchange is performed, and the obtained chromatographic solution is filtered by a filter membrane, which is the vaccine stock solution . The filter membrane is preferably a 0.22 μm filter membrane.

As a preferred solution of the present invention, the method further comprises: formulating the vaccine stock solution into a vaccine. The dosage of the vaccine stock solution is preferably 20-200 μg/dose. Specifically, the content of the vaccine stock solution in the recombinant protein vaccine may be 25 μg/dose, 50 μg/dose, 100 μg/dose or 200 μg/dose.

The seventh object of the present invention is to provide the amino acid sequence, the gene sequence, the eukaryotic cell expression vector, the cell, the recombinant protein vaccine or the recombinant protein vaccine prepared by the method in the preparation of Use in medicines for preventing diseases caused by novel coronavirus SARS-CoV-2 infection.

Preferably, the disease is pneumonia caused by novel coronavirus SARS-CoV-2 infection, severe acute respiratory infection, intestinal disease, or severe acute respiratory syndrome.

The amino acid sequences provided by the present invention are relatively conservative sequences of the coronavirus beta family, and these sequences can be combined with ACE2 and are important antigenic determinants that cause immune responses, which can prevent both 2003 Sars-CoV and SARS-CoV-2, Can effectively induce the body to produce cellular immunity and humoral immunity. In addition, the recombinant protein vaccine provided by the present invention can be rapidly prepared, and is suitable for the prevention of novel coronavirus SARS-CoV-2.

The eighth object of the present invention is to provide a method for stimulating the production of novel coronavirus SARS-CoV-2 antibodies in vivo, the method comprising administering the aforementioned amino acid sequence, the aforementioned gene sequence, the aforementioned The eukaryotic cell expression vector, the cell described above, or the recombinant protein vaccine described above.

The ninth object of the present invention is to provide a method for preventing or treating infection by a novel coronavirus SARS-CoV-2, the method comprising administering the aforementioned amino acid sequence, the aforementioned gene sequence, the aforementioned The eukaryotic cell expression vector, the cell described above, or the recombinant protein vaccine described above.

The tenth object of the present invention is to provide a method for preventing or treating diseases caused by novel coronavirus SARS-CoV-2 infection, the method comprising administering the aforementioned amino acid sequence, the aforementioned gene sequence to a person in need , the aforementioned eukaryotic cell expression vector, the aforementioned cell or the aforementioned recombinant protein vaccine.

Further, the disease includes novel coronavirus pneumonia.

Description of drawings

Figure 1 is a schematic diagram of the results of the specific expression of S protein cytokines in serum.

detailed description

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention.

Example 1

This embodiment provides a method for preparing a SARS-CoV-2 recombinant protein vaccine stock solution, specifically:

(1) after the gene sequences encoding SEQ1, SEQ2 and SEQ3 are respectively subjected to CHO cell codon optimization, inserted into the expression vector of CHO, transfected into CHO cells, and the recombinant expression of the protein is carried out;

(2) Take the supernatant of the cell culture, carry out ultrafiltration and concentration, and filter the obtained chromatographic solution through a 0.22 μm filter membrane through affinity chromatography, ion exchange and molecular sieve to obtain a vaccine stock solution.

Example 2

This example provides a SARS-CoV-2 recombinant protein vaccine, which is a liquid vaccine, which contains 10 μg/dose of the vaccine stock solution provided in Example 1 and 50 μg/dose of aluminum hydroxide adjuvant. The vaccine number obtained in this example is recorded as 20200501.

Example 3

This example provides a SARS-CoV-2 recombinant protein vaccine, which is a liquid vaccine, which contains 20 μg/dose of the vaccine stock solution provided in Example 1 and 50 μg/dose of aluminum hydroxide adjuvant. The vaccine number obtained in this example is recorded as 20200502.

Example 4

This example provides a SARS-CoV-2 recombinant protein vaccine, which is a liquid vaccine, which contains 40 μg/dose of the vaccine stock solution provided in Example 1 and 50 μg/dose of aluminum hydroxide adjuvant. The vaccine number obtained in this example is recorded as 20200503.

Example 5

This example provides a SARS-CoV-2 recombinant protein vaccine, which is a liquid vaccine, which contains 80 μg/dose of the vaccine stock solution provided in Example 1 and 50 μg/dose of aluminum hydroxide adjuvant. The vaccine number obtained in this example is recorded as 20200504.

Example 6

This example provides a SARS-CoV-2 recombinant protein vaccine, which is a liquid vaccine, which contains 10 μg/dose of the vaccine stock solution provided in Example 1 and 200 μg/dose of aluminum hydroxide adjuvant. The vaccine number obtained in this example is recorded as 20200505.

Example 7

This example provides a SARS-CoV-2 recombinant protein vaccine, which is a liquid vaccine, which contains 20 μg/dose of the vaccine stock solution provided in Example 1 and 200 μg/dose of aluminum hydroxide adjuvant. The vaccine number obtained in this example is recorded as 20200506.

Example 8

This example provides a SARS-CoV-2 recombinant protein vaccine, which is a liquid vaccine, which contains 40 μg/dose of the vaccine stock solution provided in Example 1 and 200 μg/dose of aluminum hydroxide adjuvant. The vaccine number obtained in this example is recorded as 20200507.

Example 9

This example provides a SARS-CoV-2 recombinant protein vaccine, which is a liquid vaccine, which contains 80 μg/dose of the vaccine stock solution provided in Example 1 and 200 μg/dose of aluminum hydroxide adjuvant. The vaccine number obtained in this example is recorded as 20200508.

Example 10 Effect of vaccine immunization in mice

The 8 groups of vaccines provided in Examples 2 to 9 were diluted at 1:4, 1:16, and 1:64 respectively, and BALB/c mice were inoculated with 10 mice in each group. They were immunized according to 0 and 7 days, and each mouse was intraperitoneally injected with one. Dosage, blood was collected 4 weeks after the first immunization, and neutralizing antibodies were detected. Neutralizing antibody titers with a GMT greater than 8 were considered positive and had a protective effect. The results are shown in Table 2.

Table 2 Neutralizing titers of BALB/c mice immunized with SARS-CoV-2 vaccine

The results show that the above SARS-CoV-2 vaccines with different active ingredients and adjuvant contents can produce sufficiently high neutralizing antibody titers when immunizing BALB/c mice. The vaccines provided by the present invention can induce BALB/c mice to produce neutralizing antibodies with protective ability.

Example 11 Effect of vaccine immunization in guinea pigs

The 4 groups of vaccines provided in Examples 2, 5, 6, and 9 were inoculated with guinea pigs, 4 in each group, and 1.0 ml (2 doses) was intramuscularly injected into each individual, according to the immunization procedure of immunization on 0, 7 days, and blood collection on 28 days, Serum neutralizing antibody titers were detected. The results are shown in Table 3.

Table 3 Neutralizing titers of SARS-CoV-2 vaccine immunized guinea pigs

Vaccine number	Neutralizing antibody titer (GMT)
20200501	32
20200504	64
20200505	128
20200508	128

The results showed that immunization of guinea pigs with different batches of SARS-CoV-2 vaccines could produce different levels of neutralizing antibody titers. All the vaccines provided by the invention can induce guinea pigs to produce good neutralizing antibodies.

Example 12 Effect of vaccine immunization in rabbits

The 4 groups of vaccines provided in Examples 2, 3, 4, and 5 were inoculated with New Zealand white rabbits respectively, with 4 rabbits in each group, and 1.0 ml (2 doses) was intramuscularly injected into each animal, and the immunization was performed according to the immunization of 0, 7 days, and 28 days of blood collection. procedure to detect serum neutralizing antibody titers. The results are shown in Table 4.

Table 4 Neutralizing titers of SARS-CoV-2 vaccine immunized rabbits

Vaccine number	Neutralizing antibody titer (GMT)
20200501	16
20200504	32
20200505	32
20200508	40

The results showed that different batches of SARS-CoV-2 vaccine immunized rabbits could produce different levels of neutralizing antibody titers. All the vaccines provided by the present invention can induce rabbits to produce good neutralizing antibodies.

Example 13 Cytokine Detection

BALB/c mice were immunized with vaccines numbered 20200501, 20200502, 20200503 and 20200504 according to the procedures of 0 and 7 days of immunization and blood collection on 28 days, respectively, and cell supernatants were collected for cellular immune detection. Intracellular cytokine staining and flow cytometry were used to detect CD4 ⁺ T cells specifically expressing S protein cytokines in serum. The results are shown in Figure 1.

The results showed that 20200501, 20200502, 20200503 and 20200504 could induce cellular immunity. Although vaccines with high content of active ingredients can produce higher levels of cellular immunity, vaccines within the antigen content range provided by the present invention can all cause good cellular immunity.

Although the present invention has been described in detail above with general description, specific embodiments and tests, some modifications or improvements can be made on the basis of the present invention, which is obvious to those skilled in the art . Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (21)

1. A target amino acid sequence of a SARS-CoV-2 recombinant protein vaccine, characterized in that it includes one or more amino acid sequences in SEQ1, SEQ2 and SEQ3, or an amino acid sequence with a similarity of more than 90%.
2. A target gene sequence of a SARS-CoV-2 recombinant protein vaccine is characterized in that it comprises a DNA sequence encoding one or more amino acid sequences in SEQ1, SEQ2 and SEQ3 or an amino acid sequence with a similarity of more than 90%.
3. The target gene sequence according to claim 2, wherein the target gene sequence is a codon-optimized DNA sequence.
4. A eukaryotic cell expression vector comprising the target gene sequence of claim 2 or 3.
5. The eukaryotic cell expression vector according to claim 4, wherein the eukaryotic cell expression vector is an expression vector that can be used in humans.
6. The eukaryotic cell expression vector according to claim 4, wherein the eukaryotic cell expression vector is a pCHO1.0 vector.
7. A cell expressing a SARS-CoV-2 recombinant protein vaccine antigen, into which the eukaryotic cell expression vector described in any one of claims 4 to 6 is exogenously transferred.
8. The cell according to claim 7, wherein the cell is a CHO cell.
9. A recombinant protein vaccine for preventing SARS-CoV-2, the antigen of which comprises one or more amino acid sequences of SEQ1, SEQ2 and SEQ3, or amino acid sequences whose similarity is more than 90%.
10. The recombinant protein vaccine according to claim 9, wherein the content of the antigenic active ingredient in the recombinant protein vaccine is 5-200 μg/dose.
11. The recombinant protein vaccine according to claim 10, wherein the vaccine is a liquid vaccine.
12. The recombinant protein vaccine according to claim 11, wherein the vaccine is intramuscular liquid injection, intranasal liquid injection, intradermal liquid injection or subcutaneous liquid injection.
13. A method for preparing a recombinant protein vaccine for preventing coronavirus, comprising the steps of: transferring the eukaryotic cell expression vector described in any one of claims 4 to 6 into cells for expression, and purifying the supernatant to obtain Vaccine stock solution.
14. The preparation method according to claim 13, wherein the cells are CHO cells.
15. The preparation method according to claim 13, wherein the purification comprises: after the supernatant is concentrated by ultrafiltration, two-step chromatography of molecular sieve and anion exchange is performed, and the obtained chromatographic solution is filtered through a filter membrane.
16. The preparation method according to claim 13, wherein the method further comprises: formulating the vaccine stock solution into a vaccine; preferably, formulating the vaccine stock solution into a vaccine at a dosage of 20-200 μg/dose.
17. The amino acid sequence according to claim 1, the gene sequence according to claim 2 or 3, the eukaryotic cell expression vector according to any one of claims 4 to 6, the cell according to claim 7 or 8, and any one of claims 9 to 15 The application of the recombinant protein vaccine described in item 1 or the recombinant protein vaccine prepared by the method of any one of claims 15 to 17 in the preparation of a medicine for preventing diseases caused by novel coronavirus SARS-CoV-2 infection.
18. The application according to claim 17, wherein the disease is pneumonia caused by novel coronavirus SARS-CoV-2 infection, severe acute respiratory infection, intestinal disease, or severe acute respiratory syndrome.
19. A method for stimulating the production of novel coronavirus SARS-CoV-2 antibodies in vivo, characterized in that the method comprises administering the amino acid sequence according to claim 1, the gene sequence according to claim 2 or 3, the claim The eukaryotic cell expression vector of any one of 4 to 6, the cell of claim 7 or 8, or the recombinant protein vaccine of any one of claims 9 to 15.
20. A method for preventing or treating infection by novel coronavirus SARS-CoV-2, characterized in that the method comprises administering the amino acid sequence of claim 1, the gene sequence of claim 2 or 3, the The eukaryotic cell expression vector of any one of 4 to 6, the cell of claim 7 or 8, or the recombinant protein vaccine of any one of claims 9 to 15.
21. A method for preventing or treating diseases caused by novel coronavirus SARS-CoV-2 infection, characterized in that the method comprises administering the amino acid sequence of claim 1 and the gene sequence of claim 2 or 3 to those in need . The eukaryotic cell expression vector of any one of claims 4 to 6, the cell of claim 7 or 8, or the recombinant protein vaccine of any one of claims 9 to 15.

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