WO2023165435A1

WO2023165435A1 - Recombinant spike protein, method for preparing same and use thereof

Info

Publication number: WO2023165435A1
Application number: PCT/CN2023/078329
Authority: WO
Inventors: 安娇; 王紫琰; 周晨亮; 江元翔; 胡冬冬; 刘革; 史立康; 曹夏尧
Original assignee: 上海泽润生物科技有限公司
Priority date: 2022-03-01
Filing date: 2023-02-27
Publication date: 2023-09-07
Also published as: CN116731192A

Abstract

Provided are a recombinant spike protein, an encoding nucleic acid thereof, a method for preparing same, a vaccine composition comprising the recombinant spike protein and use thereof. The recombinant spike protein can be used for preventing infection caused by SARS-CoV-2 or a variant thereof or diseases caused by the infection.

Description

Recombinant spike protein and its preparation method and use

technical field

The present invention belongs to the field of biomedicine, specifically, the present invention relates to a recombinant Spike protein, its encoding nucleic acid, its preparation method, a vaccine composition comprising the recombinant Spike protein and its use.

Background technique

New Coronary Pneumonia (COVID-19) is a respiratory disease caused by novel coronavirus (SARS-CoV-2) infection. As of December 31, 2021, more than 280 million people have been infected worldwide, and more than 5 million people have died of SARS-CoV-2 infection. SARS-CoV-2 belongs to subgroup B of the genus Beta and is an enveloped single-stranded positive-sense RNA virus. The virus genus also includes SARS-CoV (also known as SARS-CoV-1) and MERS-CoV. Invertase 2 (ACE2) binds to infect the host. As the only protein on the surface of virus particles, S protein is most vulnerable to the attack of specific neutralizing antibodies produced by the immune system. Current studies have shown that after the human body is infected with SARS-CoV-2, a large number of neutralizing antibodies against the S protein can be induced, and in vitro experiments have proved that these neutralizing antibodies can prevent the virus from infecting host cells. This is also the main reason why the S protein was chosen as the antigen in the previous vaccine development against SARS-CoV and MERS-CoV.

In the natural state, the S protein on the surface of the virus is in a trimer state. According to the structure and function of the protein, the S protein can be divided into two functional units: S1 and S2 protein subunits. The main function of the S1 subunit is to mediate the interaction with the host cell. Surface receptor binding, the function of the S2 subunit is to mediate the fusion of the virus and the host cell, and the main neutralizing epitope is concentrated on the receptor binding domain (RBD) of the S1 subunit. Therefore, the integrity and structural correctness of the S protein ensure the effectiveness of the vaccine. However, during the process of virus infection of cells, the natural S protein in the pre-fusion conformation (pre-fusion) is first hydrolyzed into S1 subunit and S2 subunit by protease, the S1 subunit dissociates, and the protease hydrolysis site on the S2 subunit point Then it is exposed, causing it to be further hydrolyzed by protease, so that the fusion peptide located inside the molecule is exposed, and the protein conformation is transformed into a post-fusion conformation (post-fusion), thereby mediating the fusion of the virus and the cell membrane. If the original amino acid sequence of the S protein is maintained for the recombinant expression of the S protein, due to the presence of proteases in the expression cells, the S protein is likely to be hydrolyzed and it is difficult to maintain the pre-fusion conformation, resulting in a decrease in the expression level of the S protein, and it is not conducive to its structure. correctness. Therefore, in the SARS-CoV-2 virus vaccine using the S protein as the antigen, the S protein needs to be modified by genetic engineering. On the one hand, the expression level of the S protein can be maintained to ensure the feasibility of vaccine industrialization; The correctness of the structure of the S protein ensures the effectiveness of the vaccine; in addition, the S protein expressed and prepared by in vitro recombination does not contain the genetic material of the SARS-CoV-2 virus, which ensures the safety of the vaccine.

With the spread of the new crown epidemic, the virus continues to evolve adaptively during the process of human-to-human transmission. Some of the mutant strains have significantly improved transmission ability and pathogenicity, and some strains have antigen escape. These mutant strains have caused The concern of the public health department and the public has been listed as Variants of Concern (VOC) by the World Health Organization (WHO). The key targets for these VOCs to attach and infect human cells, that is, the mutation of multiple key amino acid residues on the spike protein (S protein for short) is the main factor leading to its enhanced infectivity and immune escape, such as K417N, E484K , N501Y and D614G etc. Representative variants include Alpha (B.1.1.7, United Kingdom), Beta (B.1.351, South Africa), Gamma (P.1, Brazil), Delta (B.1.617.2, India) and Omicron ( B.1.1.529, South Africa).

Usually, when protein alone is used as the antigenic component of the vaccine, its immunogenicity is weak, and an adjuvant needs to be added to enhance the effect of the vaccine. Phase I clinical data of the first-generation candidate recombinant novel coronavirus vaccine against SARS-CoV-2 virus developed by the present inventors shows that it can induce good humoral and cellular immune responses in the body, and the vaccine has been proven to be effective in humans. good security. At present, the candidate vaccine has successfully entered Phase II clinical research. Therefore, during the development of the second-generation recombinant novel coronavirus vaccine, the adjuvant system of the first-generation vaccine was followed.

Aluminum adjuvant is a safe traditional adjuvant. According to the existing research results, the main content of aluminum adjuvant is Its main function is to store and release antigens, enhance the presentation of antigens, enhance the adaptive immune response mediated by type 2 helper cells (T helper cells 2, Th2), enhance the body's innate immune response and activate complement, etc. For the SARS-CoV-2 vaccine, inducing the body to produce a Th2-oriented immune response has the potential risk of causing vaccine-induced exacerbation of the disease (Vaccine-associated enhanced respiratory disease, VAERD). As an agonist of Toll-like receptor 9 (TLR9), CpG ODN can enhance humoral and cellular immune responses by activating downstream innate immune response pathways and inducing the expression of type I interferon and inflammatory factors. Studies have shown that CpG ODN mainly induces the body to produce an immune response that is inclined to Th1, so the combined use of CpG ODN in vaccines may help reduce the potential risk of VAERD. The safety of CpG ODN as a vaccine adjuvant has been clinically verified. CpG 1018 is used in Dynavax's hepatitis B vaccine (Heplisav-B), which was approved for marketing in the United States in 2017.

Contents of the invention

In the first aspect of the present invention, a recombinant S protein (hereinafter referred to as "rS protein") modified by genetic engineering means is provided. In order to restore the trimer structure of the S protein on the surface of the virus under natural conditions and increase the immunogenicity of the antigen through trimerization, the present invention removes the transmembrane region of the S protein and the inner segment of the C-terminal capsule through genetic engineering. , and at the same time, a domain that helps protein trimerization was added to the C-terminus of the protein to obtain the SΔTM protein.

Next, introduce hotspot amino acid mutations on the SΔTM protein to enhance its neutralizing immunogenicity and broad-spectrum against mutant strains; replace the S1/S2 enzyme cleavage site to prevent protease cleavage; introduce two consecutive A proline (Pro, P) mutation prevents the molecule from changing from a pre-fusion conformation to a post-fusion conformation; in order to further improve the stability and expression of the S protein mutant, the amino acid located in the flexible region of the S2 subunit is replaced by proline acid, thereby increasing its structural rigidity. The rS protein expressed and prepared by in vitro recombination does not contain the genetic material of the SARS-CoV-2 virus, which ensures the safety of the vaccine.

In a preferred embodiment, the rS protein has the amino acid sequence shown in SEQ ID NO: 1 or 3. In a further preferred embodiment, the rS protein is encoded by the nucleotide sequence shown in SEQ ID NO: 2 or 4, respectively.

In the second aspect of the present invention, a nucleic acid encoding the rS protein of the first aspect is provided. In a preferred embodiment, the nucleic acid has the nucleotide sequence shown in SEQ ID NO: 2 or 4.

In a third aspect of the present invention, an engineered cell is provided. In a further preferred embodiment, the cell genome is integrated with the nucleotide sequence shown in SEQ ID NO: 2 or 4. In a further preferred embodiment, said cells are capable of secreting and expressing rS protein. In a preferred embodiment, the engineered cells are CHO cells. In a preferred embodiment, the average expression level of the rS protein in the culture supernatant of the CHO cells is above 2000 mg/L, which is 8-10 times that of the first-generation vaccine antigen.

In a fourth aspect of the present invention, there is provided a method for secreting, expressing and isolating the rS protein of the first aspect by CHO cells, comprising the following steps:

(1) Cloning the nucleotide sequence shown in SEQ ID NO: 2 into the expression vector;

(2) transfecting the expression vector obtained in step (1) into CHO cells;

(3) Obtain a cell line stably expressing the rS protein through cell population screening and monoclonal screening;

(4) using the cell line obtained in step (3) for expression to obtain the culture supernatant containing the rS protein; and

(5) Purify the culture supernatant containing the rS protein obtained in step (4) to obtain purified rS protein, and the purification yield is more than 10 times that of the first-generation vaccine antigen.

According to a specific embodiment of the present invention, the CHO cells used in the step (2) are CHO-K1Q cells.

In the fifth aspect of the present invention, a recombinant novel coronavirus vaccine composition is provided, which comprises the rS protein of the first aspect and pharmaceutically acceptable excipients.

In some preferred embodiments, the excipient is aluminum adjuvant in combination with CpG ODN adjuvant. In a further embodiment, the aluminum adjuvant is aluminum hydroxide. In a further embodiment, the CpG ODN adjuvant is CpG 7909. In a further embodiment, the excipient is aluminum hydroxide in combination with CpG 7909.

In some preferred embodiments, the vaccine composition of the present invention contains 10μg-100μg/0.5ml rS protein. In a preferred embodiment, the content of rS protein is 25μg-50μg/0.5ml.

In some preferred embodiments, the content of the aluminum adjuvant is between 100 μg-1000 μg/0.5 ml. In a preferred embodiment, the content of aluminum adjuvant is between 250μg-500μg/0.5ml. In a preferred embodiment, the vaccine composition of the invention contains 500 μg/0.5 ml of aluminum hydroxide.

In some preferred embodiments, the content of CpG ODN adjuvant is between 100 μg-1000 μg/0.5ml. In a preferred embodiment, the content of CpG ODN adjuvant is between 250 μg-500 μg/0.5ml. In a preferred embodiment, the vaccine composition of the invention contains 500 μg/0.5 ml of CpG 7909.

In the sixth aspect of the present invention, there is provided the use of the rS protein of the first aspect in the preparation of a vaccine composition, which is used to prevent infection caused by the new coronavirus or its mutant strain or caused by the infection disease. In a preferred embodiment, the vaccine composition has strong immunogenicity. In a preferred embodiment, the novel coronavirus is a prototype strain or a variant strain or a combination thereof. In a preferred embodiment, the mutant strain is Alpha strain, Beta strain, Gamma strain, Delta strain, Omicron strain or a combination thereof, or a new mutant strain containing a combination of mutation sites of these mutant strains. In a preferred embodiment, the mutant strain is a Beta strain, a Delta strain, an Omicron strain or a combination thereof.

In a preferred embodiment, the pharmaceutical vaccine composition is used to prevent infection caused by prototype strain, Beta strain, Delta strain, Omicron strain or a combination thereof or disease caused by the infection.

The rS protein provided by the present invention has a pre-fusion conformation and is in a trimer state, has good immunogenicity in animal models, and can be used to prepare vaccine compositions for the prevention of novel coronaviruses. In addition, the present invention also provides a method for efficiently expressing rS protein in CHO cells, the method is high in expression, quick and easy, and can realize large-scale production.

The recombinant new coronavirus vaccine composition provided by the present invention can induce high titer high levels of pseudoviruses against different mutant strains in animal models, such as BALB/c mouse models. Flat cross-neutralizing antibodies and high levels of cellular immune response. The immune response induced by the vaccine composition combined with the adjuvant was significantly better than that induced by the rS protein without adjuvant and the vaccine composition with only aluminum adjuvant or only CpG7909 adjuvant. In addition, the vaccine composition also demonstrated its good immunogenicity and cellular immune response in the rhesus monkey animal model, as well as high-level cross-neutralizing antibody activity against a broad spectrum of VOCs and prototype strains of pseudoviruses and live viruses. In addition, the vaccine composition can significantly reduce the viral load in throat swabs and anal swabs, lungs and trachea-bronchus of rhesus monkeys after challenge, and alleviate the pathological damage of the lungs, indicating that the vaccine The composition may have better protective efficacy in non-human primates as well as humans.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings:

Figure 1. The plasmid map of the expression vector SNT70-S.

Figure 2. Denaturing and reducing SDS-PAGE analysis of purified rS protein.

Figure 3. SE-HPLC analysis of purified rS protein.

Figure 4. Activity and stability analysis of rS protein at 25°C.

Figure 5. Results of pseudovirus neutralizing antibody titer in serum of BALB/c mice after second immunization.

Figure 6. The results of the binding antibody titer in the serum of BALB/c mice after the second immunization in the dose ratio experiment.

Fig. 7. The results of serum neutralizing antibody titers against pseudoviruses after the second immunization of BALB/c mice in the dose ratio experiment. A: prototype strain; B: Beta strain; and C: Delta strain.

Figure 8. ELISPOT (A) and ICS (B) results of BALB/c mice after the second immunization in the dose ratio experiment.

Figure 9. Titer results of binding antibody (A), live virus neutralizing antibody (B) and pseudovirus neutralizing antibody (C) in rhesus monkeys after the second immunization.

Figure 10. Results of ICS (A and B) and ELISPOT (C) in rhesus monkeys after the second immunization.

Figure 11. Viral load results in swabs (A and B) and tissues (C and D) in rhesus monkeys post-challenge.

Figure 12. Pathological results of the lungs of the vaccine group and the adjuvant control group after challenge in rhesus monkeys.

Detailed ways

The following examples are only for the purpose of illustrating the technical solutions of the present invention, rather than limiting the scope of protection of the present invention.

Example 1: Cloning construction, expression and purification of the S protein extracellular segment of SARS-CoV-2 virus

1. Selection of S protein sequence and gene synthesis

Based on the inventor's antigenic design for the first-generation recombinant novel coronavirus vaccine, the S protein extracellular segment amino acid sequence (1-1213) of the SARS-CoV-2 virus prototype strain (genome sequence accession number: MN908947) was still selected as the protein basis for optimization. The same as the antigen design of the first-generation vaccine, in order to better realize the secretory expression of the S protein, the signal peptide MFVFLVLLPLVSS of the S protein itself was replaced with a strong secretory signal peptide MEFGLSWLFLVAILKGVQC; in order to maintain the stability of the protein and lock it before fusion ( pre-fusion) conformation, on the one hand, the S protein S1/S2 protease cleavage site ⁶⁸² RRAR ⁶⁸⁵ was replaced by GGSG, and the key site amino acid ⁹⁸⁶ KV ⁹⁸⁷ was mutated into two consecutive prolines (Proline, P); A T4 fibritin motif (GYIPEAPRDGQAYVRKDGEWVLLSTFL) was introduced at its C-terminus to further stabilize the trimerization state of the S protein. In addition, four hotspot amino acid mutations, K417N, E484K, N501Y, and D614G, were introduced into the S protein to enhance its neutralizing immunogenicity and broad-spectrum against mutant strains; it will be located at positions 817 and 892 of the S2 subunit , 899th and 942nd amino acids were replaced by prolines (F817P, A892P, A899P and A942P), thereby increasing its structural rigidity. The amino acid sequence of the designed rS protein is shown in SEQ ID NO: 1, which contains 1248 amino acids, of which the N-terminal 19 amino acids are signal peptides, which will be excised during the secretory expression process, so the obtained target S protein The antigen is 1229 amino acids (SEQ ID NO: 3).

In order to facilitate the high-efficiency expression of rS protein in CHO cells, the codons preferred by CHO cells were selected to optimize the coding gene. The optimized nucleotide sequence is shown in SEQ ID NO: 2 and artificially synthesized. It should be noted that the optimization principle is not simply to choose CHO The codon with the highest frequency in the cell, but a more complex optimization scheme. There are three principles for overall optimization: first, according to the degeneracy of codons, replace the original codons with the high-frequency codons corresponding to each amino acid in CHO cells; second, to avoid excessive codons in the transcribed mRNA GC content affects its secondary structure, which in turn affects translation efficiency. During the optimization process, try to control the GC% of the gene at 40-60%; third, avoid some commonly used restriction enzyme sites.

2. Cloning construction of rS protein expression vector

The 5' and 3' ends of the above-mentioned synthesized genes contained Hind III and EcoR I restriction endonuclease sites respectively, and the fragments were obtained by double digestion and cloned into the expression vector SNT70 (Figure 1). Vector SNT70 carries an ampicillin resistance gene and a glutamine synthetase selection marker. Cytomegalovirus (CMV) promoter/enhancer sequences were used for expression of the gene of interest. The CMV promoter is a strong promoter commonly used to promote the expression of eukaryotic genes. The corresponding expression vector SNT70-S was obtained by cloning and construction, and the sequence was confirmed to be correct by enzyme digestion and sequencing.

Specifically, the construction of expression plasmid SNT70-S can be carried out as follows:

a. Use Hind III and EcoR I to double digest the SNT70 plasmid and the pUC57-S plasmid (including the nucleotide sequence encoding rS protein) obtained by gene synthesis entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. (GENEWIZ), and agarose After gel separation, use a DNA gel recovery kit to recover, and purify the digested target fragment and digested carrier SNT70.

b. Ligation reaction: The purified target fragment (about 3.8kb) and the digested product of the vector SNT70 (about 9.4kb) were ligated with T4 ligase to construct the expression plasmid SNT70-S.

c. Transform the ligation reaction product into Top10 competent cells, smear LB medium plates, and obtain monoclonal transformed colonies.

d. Pick 10 monoclonal colonies for PCR amplification and sequencing verification. Subsequently, the correct clones were verified by sequencing, streaked twice on the LB medium plate, and the isolated single clones were transferred to LB liquid medium (containing 100 μg/mL ampicillin), 37 ℃, 220rpm shaking culture overnight, a large amount of plasmid DNA was extracted, and the finally obtained plasmid was named SNT70-S.

3. Transfection of SNT70-S plasmid, expression and purification of rS protein

Through the method in the above 2, a large number of expression plasmids were prepared, linearized by PvuI digestion, and then transfected into the host cell CHO-K1Q by electroporation. The electroporation transfection method is as follows: take 2.4× ¹⁰⁷ cells, centrifuge at 1000rpm for 5min, and discard the supernatant; resuspend the cells with 20ml of CHO CD medium, centrifuge at 1000rpm for 5min, discard the supernatant; mix 1520μl of CHOCD medium and 80μl of plasmid Place them in the cells after centrifugation, resuspend and mix them evenly; take 800 μl of the mixed solution in two electroporation cups, and place the electroporation cups in the electroporation apparatus for electric shock (electroporation program: 300V, 900μF, exponential pulse, resistance (∞ )); after electric shock, the cells in the two electroporation cups were merged and transferred to a square bottle containing 20ml of preheated CHO CD04 medium (containing a final concentration of 25mM L-methionine sulfoximine, MSX), and pressure recovery and filter. In this example, 4 sets of transfection experiments were carried out.

24h after electroporation, on the 7th day and every 2-4 days thereafter, the cell density and viability were detected, the cell density was adjusted to 0.5×10 ⁶ cells/ml, and placed in a 37°C, 10% CO2 incubator for static culture. When the cell density is greater than 1×10 ⁶ cells/ml and the cell viability is greater than 90%, the pressurized screening of the cell population is completed. Then, 14-day fed-batch culture was used to evaluate the expression level of the cell population obtained by pressurized screening. The expression level is evaluated by using Biolayer Interferometry (BLI for short), that is, the protein G sensor is first combined with the 2G4 antibody, and then the 2G4 antibody is combined with the target protein, and the protein level of the sample to be tested is calculated by establishing a standard curve. concentration.

Next, the automated cell plating and imaging system (VIPS) was used to perform monoclonalization and screening of the cell population obtained by pressurized screening by limiting dilution method, and the selected monoclonals were expanded during fed-batch culture. The supernatant of the above-mentioned cloned cells was collected, and samples were taken to detect the expression level of the target protein, and the best 3 clones were selected by comprehensively considering the growth of the clones, the density of viable cells, the viability, the final lactic acid content and related product quality parameters. , which is the dominant cell line. Cell culture supernatant containing rS protein can be obtained by culturing and expressing the cell line obtained above in a bioreactor, and the above supernatant is also sampled for BLI detection. The results show that the average expression level of rS protein in the culture supernatant of the above cell lines can reach 200-300 mg/L, which meets the requirements of vaccine production.

In order to obtain the rS protein antigen, the culture supernatant of the dominant cell line is processed by conventional methods, including virus inactivation, anion exchange chromatography, cation exchange chromatography, gel filtration chromatography and virus removal nanofiltration, and then S protein can be obtained as a trimer with SDS-PAGE purity exceeding 90% ( FIG. 2 ) ( FIG. 3 ).

Further, the stability of the rS protein stock solution and the finished vaccine was analyzed. The ELISA test results showed that even if incubated at 25°C for 7 days, the rS protein stock solution could still maintain more than 80% of the activity, and the candidate vaccines incubated at the same temperature for 30 days The finished vaccine can also maintain more than 80% of its activity (Figure 4).

Embodiment 2: the preparation of vaccine composition

In order to study the technical effect of the vaccine composition provided by the present invention, the inventor has made the following multiple vaccine composition preparations (0.5ml/dose), each composition contains rS protein, aluminum hydroxide adjuvant and/or or CpG 7909. The specific preparation method is as follows: firstly, the CpG 7909 sample is adsorbed on the aluminum hydroxide adjuvant, and prepared into different ratios of CpG 7909/aluminum hydroxide adjuvant (w/w) adsorption samples (the content of the aluminum hydroxide adjuvant here is substantially The above is the content of aluminum element); then add different concentrations of rS protein stock solution to the CpG 7909/aluminum hydroxide adjuvant sample. After the above formulations are thoroughly mixed, if not administered immediately, store at 2-8°C. The specific ratio is shown in Table 1.

Table 1: Components of each vaccine composition

Example 3: Comparing the immunogenicity of the vaccine composition of rS protein combined with different adjuvants in BALB/c mice

1. Mouse grouping and animal immunization strategy

Fifty SPF grade female BALB/c mice (5-6 weeks old) were grouped. 10 mice in each group, 5 groups in total. For group information, see Table 2. The mice were immunized according to the grouping situation in Table 2. The immunization method was intramuscular injection into the inner thigh of the mice, and the injection volume was 50 μl/dose/mouse (1/10 the intended dose for humans). Each mouse was immunized twice with an interval of three weeks.

Table 2: Animal groups comparing the immunogenicity of vaccine compositions of rS protein combined with different adjuvants

2. Detection of neutralizing antibody response induced by vaccine composition

Two weeks after the second immunization, blood was collected from mice in each group, and serum was separated. Serum neutralizing antibody titers were detected by neutralization experiments against prototype strain, Beta strain and Delta strain pseudovirus (Fig. 5). Among them, * represents p<0.05; ** represents p<0.01; **** represents p<0.0001.

Compared with the adjuvant control group (AH+CpG7909), a certain level of neutralizing antibody titers against the rS protein pseudovirus could be detected in serum two weeks after the second immunization of mice in each group, and against the prototype strain , Beta strain and Delta strain pseudovirus strains, the antibody titers from high to low were double adjuvant group (rS+AH+CpG7909), single aluminum adjuvant group (rS+AH), single CpG 7909 group (rS+ CpG7909), no adjuvant group (rS).

Based on the above results, rS protein combined with double adjuvant as a component in the vaccine composition of the present invention is significantly better than rS protein combined with aluminum adjuvant alone or CpG 7909 alone or without any adjuvants.

Example 4: Studying the Dosing Ratio of Antigens and Adjuvants in Vaccine Compositions in BALB/c Mice

1. Mouse grouping and animal immunization strategy

100 SPF grade female BALB/c mice (6-8 weeks old) were grouped. 10 in each group, a total of 10 groups. For group information, see Table 3. The mice were immunized according to the grouping situation in Table 3. The immunization method was intramuscular injection into the inner thigh of the mice, and the injection volume was 50 μl/dose/mouse (1/10 the intended dose for humans). Each mouse was immunized twice with an interval of three weeks.

Table 3: Grouping of BALB/c mice in dose-matching experiments

2. Detection of Vaccine-Induced Antibody Response

Two weeks after the second immunization, blood was collected from mice in each group, and serum was separated. The IgG antibody titer against rS protein in the serum was detected by ELISA; the serum neutralizing antibody titer was detected by a pseudovirus-based neutralization experiment.

Antigen-specific binding antibodies were hardly detected in the double adjuvant control group; except for the combination of 5.0 μg rS protein/50 μg aluminum hydroxide adjuvant group and 1.0 μl rS protein/25 μg aluminum hydroxide adjuvant/50 μg CpG 7909 group Except for relatively low antibody levels, the other groups all induced high levels of binding antibody titers (≥10 ⁶ ) ( FIG. 6 ).

From the results of the neutralizing antibody titers in the serum of each group of mice detected based on the pseudovirus neutralization experiment, the antibody titers of the adjuvant control group for the prototype strain, the Beta strain and the Delta strain pseudovirus strains were not detected, Other groups induced higher levels of neutralizing antibody titers. Wherein, when the dose of rS protein increases, the titer against each pseudovirus strain increases with the increase of antigen dose. When the fixed dose of aluminum hydroxide adjuvant was 50 μg and the dose of rS protein was 5.0 μg, the titer against each pseudovirus strain increased with the increase of CpG7909 dose. When the fixed dose of CpG7909 was 50 μg, and the dose of rS protein was 1.0 μg, 2.5 μg and 5.0 μg, the titer against each pseudovirus strain increased with the increase of aluminum hydroxide dose ( FIG. 7 ).

3. Vaccine-induced cellular immune response detection

Two weeks after the second immunization, 5 mice in each group were sacrificed, and spleens were taken, and the cellular immune response induced by the vaccine was detected by ELISpot and ICS. The results showed that there was a certain correlation between the secretion level of cytokines and the dose of antigen in the 1.0 μg, 2.5 μg and 5.0 μg rS groups, and the secretion level of cytokines could reach a relatively high level among groups with the same antigen dose and different adjuvant dose ratios. High levels (Fig. 8A). Vaccine compositions with different proportions of 2.5 μg and 5.0 μg rS protein can induce a higher ratio of CD4+ T positive cells. With the increase of antigen dose, IL-2+, IFN-γ+, TNF-α+ The proportion of CD4+ T cells increased (Fig. 8B).

Based on the above results, the vaccine compositions with different antigen doses and adjuvant dose ratios all showed good binding antibody levels and pseudovirus cross-neutralizing antibody levels in this experiment, and showed a certain dose effect. Therefore, the 5.0 μg rS protein/50 μg aluminum hydroxide/50 μg CpG7909 group will be used as a candidate vaccine composition to further explore and verify its immunogenicity and protective efficacy in rhesus monkeys.

Example 5: Studying the Immunogenicity and Protective Efficacy of Vaccine Compositions in the Rhesus Monkey Model

1. Rhesus monkey grouping and animal immunization strategy

Rhesus monkeys were randomly divided into 2 groups, 6 monkeys in each group (3 male and 3 monkeys). Two immunizations were given by intramuscular injection with an interval of three weeks between immunizations. The vaccine information of each group is shown in Table 4.

Table 4: Vaccine information of each group

2. Detection of Vaccine-Induced Antibody Response

Blood was collected before the first immunization, 1 week after the first immunization, 2 weeks after the first immunization, 1 week after the second immunization and 2 weeks after the second immunization, and the serum was separated. by ELISA Experimental method The binding antibody titer of the serum was detected, and the neutralizing antibody titer of the serum was detected by a pseudovirus neutralization experiment and a live virus neutralization experiment. The results showed that the vaccine composition exhibited a trend of higher binding antibody titer and the antibody titer reached the highest level after the second immunization ( FIG. 9A ). In the pseudovirus neutralization experiment, the high antibody titer level of the vaccine composition against the prototype strain pseudovirus and the 4 VOC pseudoviruses was fully demonstrated, which indicated that the vaccine composition had better cross-neutralizing activity ( FIG. 9C ). The live virus neutralization experiment further verified that the vaccine composition has a strong neutralizing ability for the prototype virus, Beta strain, Delta strain and Omicron strain virus on rhesus monkeys ( FIG. 9B ).

3. Detection of Vaccine-Induced Cellular Responses

Two weeks after the second immunization, blood was collected and PBMCs were isolated, and the cellular immune response induced by the vaccine was detected by ELISpot and ICS. From the results of cellular immunity testing, the vaccine group had a higher level of cellular immunity than the adjuvant control group, and the cellular immunogenicity of the vaccine group in the rhesus monkey model was mainly CD4+ T cell responses (Figure 10A and Figure 10B), and simultaneously exhibited higher levels of T cell immune responses in Th1 and Th2 pathways (Figure 10C).

4. Protective efficacy of vaccines

Six weeks after the completion of the second immunization, all animals were infected with tracheal intubation and inoculated with virus fluid at a dose of 1×10 ⁵ TCID50. Swabs were collected every day to detect changes in viral load in different samples. On the 7th day after the challenge, all experimental animals were dissected to observe the lung pathological changes, and the viral RNA load in the lung tissue samples was detected.

The results of swab viral load showed that viral RNA could be detected in the anal swab samples of 2 animals in the adjuvant group, but no RNA was detected in the vaccine group. The viral load of throat swabs in the adjuvant group changed with the time of infection, viral RNA remained at a high level in throat swabs, and the viral load of throat swabs reached its peak on the second day after challenge. The viral loads of throat swabs of most animals in the vaccine group were below the detection threshold or continued to decrease, and the vaccine had a certain protective effect on the upper respiratory tract (Figure 11A and Figure 11B). The results of lung and tracheal viral loads on the 7th day after challenge showed that the tracheal and bronchial viral loads of the vaccine group decreased by about 3 logs compared with the adjuvant control group (Fig. 11D). Compared with the adjuvant group, the range of viral load in the lung organs of the vaccine group decreased by >3 log10, indicating that the vaccine can have a good protective effect on the lungs and trachea (Fig. 11C). According to According to the extent and scope of pathological changes in the lungs of the control group, the pathological damage to the lungs of the animals in the control group caused by the virus was "severe viral pneumonia with pulmonary fibrosis". The lung pathological damage of the vaccine group was "moderate, mild-moderate and mild viral pneumonia with pulmonary fibrosis".

Based on the above results, the vaccine composition with a dose of 50 μg rS protein/500 μg aluminum hydroxide/500 μg CpG7909 had good immunogenicity and cellular immune response in rhesus macaques, as well as a high level of cross-neutralization against pseudoviruses and live viruses active. In addition, the vaccine can significantly reduce the viral load in swabs and tissues, and effectively reduce the pathological damage of the lungs, indicating that the vaccine composition also has a good protective effect.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Sequence information:

The rS protein amino acid sequence is shown in SEQ ID NO: 1, which contains 1248 amino acids, of which 19 amino acids at the N-terminal are signal peptides:

The nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO: 1:

The amino acid sequence of rS protein is shown as SEQ ID NO: 3, which contains 1229 amino acids:

The nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO: 3:

Claims

A recombinant spike protein, characterized in that the recombinant spike protein has the amino acid sequence shown in SEQ ID NO: 1 or 3.
A nucleic acid encoding a recombinant spike protein, characterized in that the nucleic acid has a nucleotide sequence shown in SEQ ID NO: 2 or 4.
An engineered cell, characterized in that the cell genome is integrated with the nucleotide sequence shown in SEQ ID NO: 2 or 4.
The cell according to claim 3, wherein the cell can secrete and express recombinant spike protein.
The cell according to claim 3, wherein said cell is a CHO cell.
A method for secreting and expressing by CHO cells and preparing a recombinant spike protein having the amino acid sequence shown in SEQ ID NO: 3, comprising the steps of:

(1) Cloning the nucleotide sequence shown in SEQ ID NO: 2 into the expression vector;

(2) transfecting the expression vector obtained in step (1) into CHO cells;

(3) Obtaining a cell line stably expressing the recombinant Spike protein through screening of cell populations and monoclonal screening;

(4) using the cell line obtained in step (3) for expression to obtain a culture supernatant containing the recombinant spike protein; and

(5) Purifying the culture supernatant containing the recombinant Spike protein obtained in step (4) to obtain a purified recombinant Spike protein.
A vaccine composition, characterized in that the vaccine composition comprises a recombinant Spike protein having the amino acid sequence shown in SEQ ID NO: 3 and a pharmaceutically acceptable excipient.
The vaccine composition according to claim 7, characterized in that the vaccine composition contains 10 μg-100 μg/0.5ml, preferably 25 μg-50 μg/0.5ml of the recombinant Spike protein.
The vaccine composition according to claim 7, wherein the excipient is adjuvant.
The vaccine composition according to claim 9, wherein the adjuvant is aluminum adjuvant combined with CpG ODN adjuvant.
The vaccine composition according to claim 10, wherein the aluminum adjuvant is aluminum hydroxide.
The vaccine composition according to claim 10, characterized in that the vaccine composition contains 100μg-1000μg/0.5ml, preferably 250μg-500μg/0.5ml of aluminum adjuvant.
The vaccine composition according to claim 10, wherein the vaccine composition contains 500 μg/0.5ml of aluminum hydroxide.
The vaccine composition according to claim 10, wherein the CpG ODN adjuvant is CpG 7909.
The vaccine composition according to claim 10, characterized in that, the vaccine composition contains 100 μg-1000 μg/0.5ml, preferably 250 μg-500 μg/0.5ml of CpG ODN adjuvant.
The vaccine composition of claim 10, wherein the vaccine composition contains 500 μg/0.5ml of CpG 7909.
The use of the recombinant spike protein according to claim 1 in the preparation of a vaccine composition, which is used to prevent infection caused by the novel coronavirus or its mutant strain or diseases caused by the infection.
The use according to claim 17, wherein the mutant strain is Alpha strain, Beta strain, Gamma strain, Delta strain, Omicron strain or a combination thereof, or a new mutant strain containing a combination of mutation sites of these mutant strains.