A nebulized inhaled coronavirus vaccine and a method of making the same

Abstract

The application provides an atomized inhalation new crown vaccine and a preparation method thereof, the new crown vaccine is a recombinant protein, and a nucleotide sequence encoding the recombinant protein is obtained by connecting the following modules in series: a SP signal peptide nucleic acid artificial sequence, a S protein nucleic acid artificial sequence, a Tri T4 phage fibroin folding region nucleic acid artificial sequence, a Linker nucleic acid artificial sequence, a nucleic acid artificial sequence of a Fc antibody constant region, and a N nucleocapsid protein nucleic acid artificial sequence. In the selection process of the immunization antigen, in addition to the S protein, the relatively conservative nucleocapsid protein is also selected as the immunization antigen, so that the effective protection of the vaccine on the body can be maintained in the case of mutation of the new crown virus. The vaccine prepared by the application belongs to an inhalation new crown vaccine, can target the respiratory mucosa immunity through FcRn, and can enhance the immune protection efficiency of the vaccine.

Description

Technical Field

[0001] This invention provides a nebulized inhaled COVID-19 vaccine and its preparation method, belonging to the field of recombinant protein technology. Background Technology

[0002] Currently available COVID-19 vaccines include mRNA vaccines, adenovirus vector vaccines, inactivated vaccines, and recombinant protein vaccines. Inactivated vaccines have the most mature technology, but their production process is complex, quality control is stringent, and costs are high. Relatively speaking, recombinant protein vaccines are lower in cost and have fewer limitations on large-scale production capacity, but their immunogenicity is weaker and requires adjuvants to enhance it. The mRNA vaccines produced by Pfizer and Moderna are liposomal nanoparticle-packaged mRNA vaccines. These vaccines encode the viral spike glycoprotein. After intramuscular injection, the liposomal system allows the mRNA to enter the host cell, where it is translated into the S protein. The cell presents the S protein antigen on its membrane surface, thereby generating antibodies and cellular immunity. However, this type of vaccine is prone to causing autoimmune diseases and has side effects such as tumorigenesis. Adenovirus vector vaccines do not require manipulation of the infectious virus and have high safety, but they carry the risk of gene mutation and potential tumorigenesis. The AstraZeneca-Oxford University COVID-19 vaccine (AZD1222), developed in collaboration with Oxford University, and Johnson & Johnson's adenovirus vector-based COVID-19 vaccine (Ad26.COV2.S) have both reported cases of severe thrombosis after vaccination, indicating that adenovirus vector vaccines carry potential risks.

[0003] Since the novel coronavirus (Omega SARS-CoV-2) primarily enters the body through the respiratory epithelial mucosa cells, mucosal immunity is the body's first line of defense against the virus. Currently, most vaccines against respiratory infections are designed to provide protection through intramuscular or subcutaneous injection. While vaccination can stimulate a strong immune response, it is relatively weak against respiratory infections, partly because vaccines are unlikely to induce strong mucosal antibodies and T-cell or B-cell-mediated cellular immunity in the lungs.

[0004] Inhaled vaccines have the following advantages over injectable vaccines: First, they are convenient to administer, as nebulized inhalation can reduce the fear and pain associated with injections; second, they are highly safe, as the dosage of nebulized inhaled drugs is several times, or even ten or dozens of times, less than that of injected drugs, so the drugs are unlikely to cause adverse reactions in the body.

[0005] The inhaled COVID-19 vaccine developed by CanSino Biologics is an adenovirus vector recombinant COVID-19 vaccine, which has potential risks such as gene mutation and tumor formation.

[0006] CN202210146014.9 discloses a method for preparing an inhalable nano-COVID-19 vaccine. The method uses Poly(I:C), a mimicking viral genetic material, as an adjuvant, negatively charged liposomes that efficiently enter lung macrophages as the viral capsid structure, and receptor-binding domains (RBDs) of SARS-CoV-2 to mimic the coronavirus structure. A catalyst and antigenic protein RBD are added to the liposome solution to attach the antigenic protein to the liposome surface. After purification and freeze-drying, the biomimetic viral nano-vaccine is obtained. A drawback of this vaccine is that if the antigenic protein RBD mutates, the protective effect of the vaccine will decrease or become ineffective. This is one of the main reasons why the effectiveness of many vaccines against the Omeprone variant is reduced. Furthermore, the immunogenicity of this vaccine is not ideal. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the present invention provides a nebulized inhalation COVID-19 vaccine and its preparation method, and achieves the following objectives: to improve the immune effect of the vaccine and maintain the effective protection of the body by the vaccine in the event of mutation of the COVID-19 virus.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] A nebulized COVID-19 vaccine, wherein the COVID-19 vaccine is a recombinant protein, and the nucleotide sequence encoding the recombinant protein is obtained by sequentially linking the following modules in series: SP signal peptide nucleic acid artificial sequence, S protein nucleic acid artificial sequence, Tri T4 phage fibrin folding region nucleic acid artificial sequence, Linker nucleic acid artificial sequence, Fc antibody constant region nucleic acid artificial sequence, and N nucleocapsid protein nucleic acid artificial sequence.

[0010] The artificial nucleic acid sequence of the SP signal peptide is shown in SEQ ID NO.1 of the sequence listing; the artificial nucleic acid sequence of the S protein is shown in SEQ ID NO.2 of the sequence listing; the artificial nucleic acid sequence of the Tri T4 phage fibrin folding region is shown in SEQ ID NO.3 of the sequence listing; the artificial nucleic acid sequence of the Linker is shown in SEQ ID NO.4 of the sequence listing; the artificial nucleic acid sequence of the Fc antibody constant region is shown in SEQ ID NO.5 of the sequence listing; and the artificial nucleic acid sequence of the N nucleocapsid protein is shown in SEQ ID NO.6 of the sequence listing.

[0011] The preparation method includes constructing recombinant plasmids, cell transfection, protein concentration, and purification.

[0012] The recombinant plasmid was constructed by synthesizing the entire expression cassette of the artificial sequences of SP signal peptide, S protein, Tri T4 phage fibrin folding region, Linker, Fc antibody constant region, and N nucleocapsid protein, and inserting them into the eukaryotic expression vector pcDNA3.0. The recombinant plasmid was then obtained through transformation and extraction.

[0013] The cell transfection involved transfecting Vero cells with the recombinant plasmid, collecting the cell supernatant, and obtaining the recombinant protein SN-Fc solution.

[0014] The protein concentration and purification process involves clarifying and filtering the recombinant protein SN-Fc solution, performing ultrafiltration concentration through a 250kD ultrafiltration membrane, anion exchange chromatography, cation exchange chromatography, sterile filtration, and concentration adjustment to obtain the COVID-19 vaccine.

[0015] The concentration adjustment involves using PBS buffer to adjust the concentration of the sterile filtered recombinant protein to 7.8-8.2 μg / mL.

[0016] This invention targets respiratory mucosal immunity with FcRn: the ligand Fc (constant region fragment of the heavy chain of immune antibodies) of the FcRn receptor, which is widely present on the surface of respiratory mucosal epithelial cells and alveolar cells, is linked to the antigen protein sequence, and some amino acid fragments of Fc are replaced, which can enhance the protective efficiency of the vaccine.

[0017] This invention optimizes the structure of the Fc (immune antibody heavy chain constant region fragment) protein to improve the effector function of Fc and enhance the protective efficiency of vaccines.

[0018] The artificial nucleic acid sequence of the constant region of the Fc antibody before optimization is shown in NCBI accession number AJ294730.1, and its corresponding amino acid sequence is abbreviated as AJ294730.1 Pro.

[0019] The amino acid sequence of the constant region of the Fc antibody of this invention is based on AJ294730.1 Pro, with 9 amino acid sites changed. Specifically, S replaces C at position 11, S replaces C at position 14, D replaces H at position 70, Q replaces T at position 92, D replaces L at position 94, H replaces Q at position 96, A replaces K at position 107, V replaces A at position 163, and S replaces N at position 219.

[0020] The detailed comparison results between the amino acid sequence of the constant region of the Fc antibody of this invention (SEQ ID NO.7) and the amino acid sequence of the pre-optimized AJ294730.1 Pro are attached. Figure 1 .

[0021] Among them, the cysteine ​​[11, 14] mutation keeps Fc monomers to avoid the formation of dimers, the lysine

[107] mutation can avoid CDC (complement-mediated cytotoxicity) caused by Fc binding to C1q, and other mutations can increase Fc effector function.

[0022] In addition to selecting the S protein, this invention also selects the relatively conserved nucleocapsid protein as an immune antigen, which can maintain the vaccine's effective protection of the body in the event of mutation of the novel coronavirus.

[0023] This invention optimizes the amino acid sequence of the S protein to ensure the full-length fusion state of the S protein and enhance the protective efficiency of the vaccine.

[0024] The amino acid sequence of the S protein before optimization is the sequence shown in NCBI accession number UOT54456.1. The amino acid sequence of the S protein of this invention is obtained by changing four amino acid sites in the amino acid sequence shown in UOT54456.1. Specifically, A replaces R at position 680, A replaces R at position 812, P replaces K at position 983, and P replaces V at position 984. The specific comparison results of the amino acid sequences are attached. Figure 2 .

[0025] Replace R with A in bit 680, replace R with A in bit 812, replace K with P in bit 983, and replace V with P in bit 984.

[0026] The amino acid sequence of the S protein before optimization (accession number: UOT54456.1) corresponds to the nucleic acid sequence shown in NCBI accession number OW791050.1.

[0027] This invention incorporates the T4 phage trimer region into the vaccine: maintaining the trimer state before S protein fusion, thereby maximally inducing local and systemic immune responses to protect the body from viral infection.

[0028] Compared with the prior art, the present invention achieves the following beneficial effects:

[0029] (1) In the selection of immunogenic antigens, this invention, in addition to using the S protein, also selects the relatively conserved nucleocapsid protein as an immunogenic antigen. This ensures that the vaccine can effectively protect the body even when the novel coronavirus mutates. Furthermore, the vaccine prepared by this invention is a recombinant protein vaccine, which avoids the serious side effects caused by mRNA vaccines and adenovirus vector vaccines.

[0030] (2) The present invention has optimized the amino acid and nucleotide sequences of the S protein and the amino acid and nucleotide sequences of the Fc. The prepared vaccine can increase the content of IgG in mouse serum and improve the immune effect of recombinant protein, thereby enhancing the protective efficiency of the vaccine. The vaccine prepared in this invention, after immunizing mice twice, showed an OD value of 1.84 in mouse serum at a wavelength of 450 nm after 2 weeks, which was significantly higher than that of control group 1 and control group 2. Attached Figure Description

[0031] Figure 1 The comparison results are as follows: the amino acid sequence of the constant region of the Fc antibody of the present invention (SEQ ID NO.7) is compared with the amino acid sequence of AJ294730.1Pro before optimization;

[0032] Figure 2 The comparison results are shown for amino acid sequences of the S protein of the present invention (SEQ ID NO.8) and amino acid sequences of the S protein before optimization (accession number: UOT54456.1), specifically amino acid segments 631-990.

[0033] Figure 3 This is a diagram illustrating the immune response of the COVID-19 vaccine of this invention. Detailed Implementation

[0034] Example 1: Construction of recombinant plasmid: pcDNA3.0-SN

[0035] This invention selects the S protein and nucleocapsid protein as immune antigens, which bind to the ligand Fc (the constant region fragment of the heavy chain of immune antibodies) of the FcRn receptor expressed by respiratory mucosal epithelial cells to target respiratory mucosal immunity, and designs an aerosolized inhaled COVID-19 (Omeprone) vaccine.

[0036] The gene sequences of the S protein and nucleocapsid protein were cloned into the eukaryotic expression vector pcDNA3.0 to generate the enveloped recombinant plasmid pcDNA3.0-SN.

[0037] The construction method of pcDNA3.0-SN recombinant plasmid is as follows:

[0038] Sequences 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 were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and inserted into the eukaryotic expression vector pcDNA3.0 to obtain the recombinant plasmid pcDNA3.0-SN. The recombinant plasmid pcDNA3.0-SN was transformed into E. coli (DH5α), positive clones were picked, and PCR identification was performed. The plasmid was extracted and sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the sequencing confirmed its correctness, indicating successful construction of pcDNA3.0-SN. The pcDNA3.0-SN plasmid was extracted from the positive clones, diluted to 2 μg / μL, and stored at -80℃ for later use.

[0039] The nucleic acid sequences of each module in the recombinant plasmid were sequentially linked in the following order:

[0040] ① SP signal peptide nucleic acid artificial sequence (SEQ ID NO.1)

[0041] ②Artificial sequence of S protein nucleic acid (SEQ ID NO.2)

[0042] ③Artificial nucleic acid sequence of the fibrin folding region of Tri T4 bacteriophage (SEQ ID NO.3)

[0043] ④Linker nucleic acid artificial sequence (SEQ ID NO.4)

[0044] ⑤ Artificial nucleic acid sequence of the constant region of the Fc antibody (SEQ ID NO.5)

[0045] ⑥Artificial sequence of N nucleocapsid protein nucleic acid (SEQ ID NO.6);

[0046] The amino acid sequence corresponding to the artificial nucleic acid sequence SEQ ID NO.5 in the constant region of the Fc antibody is SEQ ID NO.7;

[0047] The amino acid sequence corresponding to the artificial nucleic acid sequence SEQ ID NO.2 of the S protein is SEQ ID NO.8.

[0048] This invention optimizes the gene sequences of the Fc antibody constant region and the S protein.

[0049] The artificial nucleic acid sequence of the constant region of the Fc antibody before optimization is shown in NCBI accession number AJ294730.1, and its corresponding amino acid sequence is abbreviated as AJ294730.1 Pro.

[0050] The amino acid sequence of the constant region of the Fc antibody of this invention is based on AJ294730.1 Pro, with 9 amino acid sites changed. Specifically, S replaces C at position 11, S replaces C at position 14, D replaces H at position 70, Q replaces T at position 92, D replaces L at position 94, H replaces Q at position 96, A replaces K at position 107, V replaces A at position 163, and S replaces N at position 219.

[0051] The detailed comparison results between the amino acid sequence of the constant region of the Fc antibody of this invention (SEQ ID NO.7) and the amino acid sequence of the pre-optimized AJ294730.1 Pro are attached. Figure 1 .

[0052] The amino acid sequence of the S protein before optimization is the sequence shown in NCBI accession number UOT54456.1. The amino acid sequence of the S protein of this invention is obtained by changing four amino acid sites in the amino acid sequence shown in UOT54456.1. Specifically, A replaces R at position 680, A replaces R at position 812, P replaces K at position 983, and P replaces V at position 984. The specific comparison results of the amino acid sequences are attached. Figure 2 .

[0053] The amino acid sequence of the S protein before optimization (accession number: UOT54456.1) corresponds to the nucleic acid sequence shown in NCBI accession number OW791050.1.

[0054] Example 2, Cell Transfection

[0055] Vero cells were seeded in 75 cm cells containing DMEM and 10% (vol) FBS. 2 In culture flasks (cell density at inoculation was 10-1) 5 / cm 2 Add 10 mL of culture medium to the culture flask and culture at 37°C with 5% CO2. When the cell adhesion rate reaches 70%, prepare for transfection. Take two sterile 1.5 mL EP tubes and prepare the reaction system according to the following components: ① Serum-free DMEM: 2 mL, pcDNA3.0-SN recombinant plasmid: 10 μg; ② Serum-free DMEM: 2 mL, liposome transfection reagent: 96 μL. After incubating at room temperature for 20 min, mix ① and ② and add them evenly to a Vero cell culture flask. Incubate in a CO2 incubator. 12 h after transfection, carefully aspirate the cell culture medium and add 10 mL of fresh DMEM medium containing 10% (vol) FBS for further culture. After changing the medium for 48 h, collect the cell supernatant to obtain the recombinant protein SN-Fc solution.

[0056] Example 3: Protein Concentration and Purification

[0057] The main steps for concentrating and purifying the recombinant protein SN-Fc solution are as follows:

[0058] ① Clarification and filtration: A clear liquid is obtained by low-speed centrifugation;

[0059] The recombinant protein SN-Fc solution prepared in Example 2 was centrifuged at low speed for 10 min at 300 g. The supernatant was filtered through a 0.45 μm filter to obtain a clear solution.

[0060] ② Ultrafiltration concentration: The clarified liquid is concentrated and washed using a 250kD ultrafiltration membrane to obtain a concentrated liquid;

[0061] ③ Anion exchange column chromatography: The concentrated solution was subjected to anion exchange column chromatography and eluted with PBS buffer (pH 6.5) to obtain the first step column purification solution;

[0062] ④ Cation column chromatography: The first step column purification solution is subjected to cation column chromatography and eluted with PBS buffer (pH 6.5) to obtain the second step column purification solution;

[0063] ⑤ Sterilization filtration: The second-step column purification solution was sterilized by passing it through a 0.2μm filter to obtain recombinant protein S-Fc.

[0064] The concentration of the concentrated and purified recombinant protein SN-Fc was adjusted to 8 μg / mL using PBS buffer at pH 7.4.

[0065] Example 4: Respiratory tract immune induction of specific antibody immune response in mice using recombinant protein

[0066] Comparative Group 1: Plasmids were constructed according to the method of Example 1, with the difference being that the artificial nucleic acid sequence before Fc optimization (accession number: AJ294730.1) was used instead of the artificial nucleic acid sequence after Fc optimization (SEQ ID NO.5) of this invention during plasmid construction. Recombinant protein was then prepared according to the methods of Examples 2 and 3, and named SN-Fc1.

[0067] Comparative Group 2: Plasmids were constructed according to the method of Example 1, with the difference that the artificial nucleic acid sequence of the S protein before optimization (accession number: OW791050.1) was used instead of the artificial nucleic acid sequence of the S protein after optimization (SEQ ID NO.2) in this invention during plasmid construction. Recombinant protein was prepared according to the methods of Examples 2 and 3, and named Comparative SN-Fc2.

[0068] The recombinant protein SN-Fc prepared in this invention, the recombinant protein SN-Fc1 prepared in control group 1, and the recombinant protein SN-Fc2 prepared in control group 2 were administered to mice via intranasal immunization to test the production of immune antibodies in the mice.

[0069] Healthy female BALB / c mice, 8 weeks old (19-21g), were selected to test the production of immune antibodies in the mice. The mice were randomly divided into 4 groups: control group, experimental group, control group 1, and control group 2, with 8 mice in each group.

[0070] The specific groupings are shown below:

[0071] Control group: PBS solution (8 μg) with pH 7.4 for immunization;

[0072] Experimental group: Recombinant immune protein SN-Fc (8 μg);

[0073] Control group 1: Recombinant immune protein SN-Fc1 (8 μg);

[0074] Control group 2: Recombinant immune protein SN-Fc2 (8 μg);

[0075] The immunization methods are as follows:

[0076] ①According to the above grouping, mice were immunized intranasally using a nebulizer:

[0077] ② A booster immunization with the same dose was administered two weeks after the initial immunization.

[0078] ③ Two immunizations were administered.

[0079] Two weeks after the booster immunization, intravenous blood was collected, incubated at 37°C for 30 min, centrifuged at 3000 rpm for 30 min, and serum was collected. The serum IgG titer of mice was determined using the SARS-CoV-2 S protein IgG antibody ELISA kit (Frdbio).

[0080] Specific methods:

[0081] First, 50 ng of S protein was used to coat ELISA plates overnight at 4°C. The next day, PBS buffer containing 10% (vol) FBS and 0.5% (vol) Tween 20 was added to each well, and the plates were blocked at 37°C for 1 hour. After blocking, the plates were washed with PBST solution; 100 μl of the test serum diluted 1:10 (v / v) was added to each well, and the plates were incubated at 37°C for 60 min, followed by thorough washing with washing buffer; 100 μl of HRP-labeled biotinylated anti-human IgG antibody working solution was added, and the plates were incubated at 37°C for 60 min, followed by thorough washing with washing buffer; 100 μL of HRP-streptavidin conjugate working solution was added to each well, and the plates were incubated at 37°C for 60 min, followed by thorough washing with washing buffer; substrate solution was added, and the plates were incubated at 37°C for 10 min; finally, 100 μl of stop solution was added, and the OD value was measured at 450 nm.

[0082] The test results are attached. Figure 3 See Table 1.

[0083]

[0084] like Figure 3 As shown in Table 1, compared to the control group, the recombinant proteins in the experimental group, control group 1, and control group 2 all induced BALB / c mice to produce neutralizing antibodies. However, compared to control group 1 and control group 2, the recombinant protein in the experimental group significantly increased the ability to induce mice to produce neutralizing antibody IgG, and the difference was statistically significant. This indicates that the recombinant protein SN-Fc prepared in this invention has the best effect in inducing specific antibody immune responses in mice. That is, optimizing the Fc nucleic acid sequence and the S protein nucleic acid sequence can improve the immunogenicity of the recombinant protein, thereby enhancing the protective efficiency of the vaccine.

Claims

1. An aerosolized inhalation COVID vaccine, characterized in that: The new coronavirus vaccine is a recombinant protein, and the nucleotide sequence encoding the recombinant protein is obtained by sequentially connecting the following modules: an SP signal peptide nucleic acid artificial sequence, an S protein nucleic acid artificial sequence, a T4 phage fibritin folding region nucleic acid artificial sequence, a Linker nucleic acid artificial sequence, an Fc antibody constant region nucleic acid artificial sequence, and an N nucleocapsid protein nucleic acid artificial sequence. The SP signal peptide nucleic acid artificial sequence is shown in SEQ ID NO. 1 in the sequence listing; the S protein nucleic acid artificial sequence is shown in SEQ ID NO. 2 in the sequence listing; the T4 phage fibritin folding region nucleic acid artificial sequence is shown in SEQ ID NO. 3 in the sequence listing; the Linker nucleic acid artificial sequence is shown in SEQ ID NO. 4 in the sequence listing; the Fc antibody constant region nucleic acid artificial sequence is shown in SEQ ID NO. 5 in the sequence listing; and the N nucleocapsid protein nucleic acid artificial sequence is shown in SEQ ID NO. 6 in the sequence listing.

2. A process for the preparation of the new coronavirus vaccine as claimed in claim 1, wherein: The preparation method comprises constructing a recombinant plasmid, cell transfection, protein concentration and purification.

3. The process for the preparation of the new coronavirus vaccine as claimed in claim 2, wherein: The recombinant plasmid is constructed by synthesizing the entire expression frame of the SP signal peptide nucleic acid artificial sequence, the S protein nucleic acid artificial sequence, the T4 phage fibritin folding region nucleic acid artificial sequence, the Linker nucleic acid artificial sequence, the Fc antibody constant region nucleic acid artificial sequence, and the N nucleocapsid protein nucleic acid artificial sequence, and inserting the entire expression frame into a eukaryotic expression vector pcDNA3.0 vector, and then transforming and extracting to obtain the recombinant plasmid.

4. The process for the preparation of the new coronavirus vaccine as claimed in claim 2, wherein: The cell transfection is performed by transfecting the recombinant plasmid into Vero cells, collecting the cell supernatant, and obtaining a recombinant protein SN-Fc solution.

5. The method of claim 4, wherein the SARS-CoV-2 vaccine is prepared by: The protein concentration and purification are performed by subjecting the recombinant protein SN-Fc solution to clarification filtration, 250kD ultrafiltration membrane ultrafiltration concentration, anion column chromatography, cation column chromatography, sterilization filtration, and concentration adjustment to obtain the new coronavirus vaccine.

6. The method of claim 5, wherein the SARS-CoV-2 vaccine is prepared by: The concentration adjustment is performed by adjusting the concentration of the recombinant protein after sterilization filtration to 7.8-8.2 μg / mL using a PBS buffer.

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