WO2022134487A1 - Vaccin recombinant sous-unitaire protéique contre le nouveau coronavirus - Google Patents

Vaccin recombinant sous-unitaire protéique contre le nouveau coronavirus Download PDF

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WO2022134487A1
WO2022134487A1 PCT/CN2021/098951 CN2021098951W WO2022134487A1 WO 2022134487 A1 WO2022134487 A1 WO 2022134487A1 CN 2021098951 W CN2021098951 W CN 2021098951W WO 2022134487 A1 WO2022134487 A1 WO 2022134487A1
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protein
ferritin
seq
fusion protein
amino acid
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宋春雨
徐伟伟
董若贝
兰青
陈小娟
司欢欢
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浙江鼎持生物制品有限公司
北京鼎持生物技术有限公司
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Definitions

  • the invention belongs to the field of genetic engineering, and particularly relates to a recombinant protein subunit vaccine of a novel coronavirus (SARS-CoV-2) and a preparation method thereof.
  • SARS-CoV-2 novel coronavirus
  • Coronaviruses are non-segmented single-stranded positive-stranded RNA viruses belonging to the subfamily Orthocoronavirinae of Nidovirales, Coronaviridae, and Coronavirinae. , ⁇ , ⁇ and ⁇ genera. To date, a total of 7 coronaviruses can infect humans: including 229E and NL63 of the alpha genus, OC43 and HKU1 of the beta genus, Middle East respiratory syndrome-related coronavirus (MERSr-CoV), severe acute respiratory syndrome-related coronavirus (SARSr -CoV) and novel coronavirus (SARS-CoV-2). At present, it is of great significance to provide a vaccine against the new coronavirus that can effectively induce the body's immune response, block the spread of the virus in the population, and prevent the virus from escaping in the body.
  • MERSr-CoV Middle East respiratory syndrome-related coronavirus
  • SARSr -CoV severe acute respiratory syndrome-related
  • the disease caused by the new coronavirus is the 2019 coronavirus disease COVID-19 (CoronaVirus Disease2019).
  • the virus of the genus Coronavirus is a positive-stranded single-stranded RNA virus with an outer envelope, with a diameter of about 80-120 nm and a genome of 27-32 Kb. Its genetic material is the largest among all RNA viruses.
  • the genome of the coronavirus encodes the spike protein, envelope protein, membrane protein and nucleocapsid protein in turn, and its human-to-human characteristics are determined by its capsid surface spike protein-S protein (spike glycoprotein) and host cell surface receptors.
  • S protein is produced as a single polypeptide and cleaved to form the S1 and S2 subunits, which are responsible for receptor binding (S1) and fusion with the host cell membrane (S2).
  • ACE2 angiotensin-converting enzyme 2
  • S1 subunit the receptor bound by the S1 subunit is angiotensin-converting enzyme 2 (ACE2).
  • ACE2 is an important cell surface receptor in humans and is widely distributed in the human heart, kidney, testis, gastrointestinal tract, brain and lung. , mainly involved in the regulation of cardiac function, blood pressure regulation, vascular protection and partial renal function.
  • COVID-19 patients have been shown to elicit robust neutralizing antibody responses against the 2019-nCoV spike protein, suggesting that this antigen may be promising in the context of protective vaccines. Therefore, the spike protein (S1) of coronaviruses is a key target for the development of novel vaccines, therapeutic antibodies and diagnostic technologies.
  • the total length of the S1 protein is 75kd, which is not easy to express and the yield is very low. Modifying the S1 protein to make it easier to express while retaining its receptor-binding activity, thereby increasing its yield has become the focus of research.
  • the popular new coronavirus began to mutate.
  • the new coronavirus mutant strain (B.1.1.7) was the first to break out in the UK, with 9 mutations in the S protein and a single point mutation of N501Y in the receptor binding domain, increasing the infectivity by 56%.
  • South African mutants (B.1.351), Brazilian mutants (P.1), California mutants (B.1.429), Finnish mutants (Fin-796H), and Indian mutants (B.1.617) appeared.
  • the infectivity of the South African mutant strain was confirmed to be enhanced by more than 50%, which directly led to a decrease in the protection rate of existing vaccines.
  • Moderna's mRNA vaccine mRNA-1273 still has a protective effect on B.1.1.7 and B.1.351 mutant strains, but the neutralizing titer of the convalescent serum samples used in this study was significantly lower for B.1.351 mutant strains ; while the mid-term protection result suddenly fell below 50%.
  • the mutation of the new coronavirus has severely impacted the effectiveness and protection of the existing vaccines based on the new coronavirus (2019-nCoV), and it is urgent to develop vaccines covering these new mutants.
  • the Helicobacter pylori ferritin multimerization platform has been used to display antigens such as influenza, HIV-1, and Epstein-Barr virus.
  • the principle is that Helicobacter pylori ferritin self-assembles to form 24-subunit particles with eight triple axes of symmetry. Assembly of protein nanoparticles displaying 8 copies of trimeric antigens at the surface 3-fold axis was facilitated by fusing a single antigen of the viral glycoprotein to the N-terminal region of the H. pylori ferritin subunit. Displaying the antigen on ferritin generally elicits a stronger neutralizing antibody response against the target pathogen than immunization with the antigen alone.
  • two influenza-functionalized ferritin vaccines have been shown to be safe and immunogenic in clinical trials (NCT03186781 and NCT03814720), and a stable basis has been established for large-scale manufacturing of ferritin-based vaccines .
  • the inventors removed the first 4 amino acids of the sequence from the original ferritin amino acid sequence, mutated the 19th N amino acid to Q amino acid, and carried out codon optimization according to the CHO expression system, An improved Helicobacter pylori ferritin multimerization platform was established.
  • the inventors analyzed the S1 and S2 regions of the natural S protein nucleotide sequence of SARS-CoV-2, and obtained the S1 protein amino acid fragment according to the data. Furthermore, by mutating one base in the S1 nucleotide sequence, the S1 protein sequence of the present invention was obtained, and from the S1 protein sequence, SSO, SS1, SS2, and SS3 proteins were obtained by truncation. It should be noted that these proteins also have the above-mentioned mutations. Subsequently, the fusion protein was expressed using the above-mentioned modified Helicobacter pylori ferritin multimerization platform.
  • the term "S protein” is sometimes used hereinafter to collectively refer to the S1 protein and fragments thereof (SSO, SS1, SS2 or SS3 protein) of the present invention.
  • the inventors searched and analyzed the mutation sites of the new coronavirus South African mutant strain, Brazilian mutant strain, and California mutant strain in the S1 region of the natural S protein. , and finally identified a mutant sequence SS1t with artificially selected mutations that could provide protection against a variety of mutant strains. Compared with the SS1 protein of the present invention, it contains amino acid substitutions of K417N/E484K/N501Y/L452R/S477N/N439K. The inventors connected more than one mutant fragment SS1t through a linker, and also used the Helicobacter pylori ferritin multimerization platform to express the fusion protein.
  • the present invention produces functionalized nanoparticles by transfecting a single plasmid encoding the S protein or a fragment thereof (including the mutant fragment SS1t) with a fusion protein of a ferritin subunit into mammalian cells, which is incompatible with the need to conjugate the antigen to the ferritin after purification.
  • the nanoparticle platform on the carrier or scaffold is different.
  • the present invention immunizes Balb/c mice with a subunit vaccine prepared by S protein or its fragment-Ferritin fusion protein, which confirms that the generated antibody has the ability to strongly block the invasion of SARS-CoV-2 pseudovirus into target cells. It is verified that the fusion protein of the present invention can produce high titer neutralizing antibodies in vivo.
  • mice after immunizing Balb/c mice with a subunit vaccine prepared from the mutant fragment SS1t-Ferritin fusion protein, SS1t-SS1t-Ferritin fusion protein, the mice were successfully induced to produce high titers of neutralizing antibodies. And antibody can strongly prevent the pseudovirus of South African mutant strain (B.1.351) from invading target cells, and can evoke specific immune responses against a variety of mutant strains.
  • South African mutant strain B.1.351
  • the present invention provides the following technical solutions.
  • a fusion protein which is a polypeptide-linker-Helicobacter pylori ferritin (Ferritin), comprising:
  • the optimized fragments of the novel coronavirus S protein are one or more (preferably 2) S1 proteins or fragments thereof (SSO protein, SS1 protein, SS2 protein, SS3 protein, or a mutant fragment SS1t of SS1 protein),
  • amino acid sequence of the S1 protein is shown in SEQ ID NO: 1;
  • amino acid sequence of the SSO protein is shown in SEQ ID NO: 2;
  • amino acid sequence of the SS1 protein is shown in SEQ ID No: 3;
  • amino acid sequence of the SS2 protein is shown in SEQ ID No: 4.
  • amino acid sequence of the SS3 protein is shown in SEQ ID No: 5;
  • mutant fragment SS1t The amino acid sequence of the mutant fragment SS1t is shown in SEQ ID No: 19, and preferably, two or more mutant fragments SS1t are connected by linker 2.
  • the nucleotide sequence of the S1 protein is shown in SEQ ID NO: 8;
  • the nucleotide sequence of the SSO protein is shown in SEQ ID NO: 9;
  • the nucleotide sequence of the SS1 protein is shown in SEQ ID No: 10;
  • the nucleotide sequence of the SS2 protein is shown in SEQ ID No: 11;
  • the nucleotide sequence of the SS3 protein is shown in SEQ ID No: 12;
  • the nucleotide sequence of the mutant fragment SS1t is shown in SEQ ID No:21.
  • linker 1 and linker 2 are optionally the same or different, and are independently selected from: [A(EAAAK)nA], (GGGGS)3, (G)n, (XP)n, (GGGS)n, preferably, the amino acid sequence of at least one of linker 1 and linker 2 is the sequence shown in SEQ ID NO: 6, more preferably linker 1 and linker 2 are at the same time The sequence shown in SEQ ID NO:6.
  • fusion protein of any one of items 1 to 4 further comprising a purification tag, such as a His, Fc, HA, GST, Flag, MBP or FLAG tag.
  • a purification tag such as a His, Fc, HA, GST, Flag, MBP or FLAG tag.
  • a vector comprising the polynucleotide of item 6.
  • a host cell expressing the fusion protein according to any one of items 1 to 5, and/or including the polynucleotide according to item 6, and/or including the vector according to item 7.
  • a vaccine composition comprising the fusion protein according to any one of items 1 to 5, and optionally an immunologically and pharmaceutically acceptable carrier or adjuvant, wherein the adjuvant is, for example, an aluminum adjuvant, ISCOM, CpG, preferably aluminium adjuvant.
  • the adjuvant is, for example, an aluminum adjuvant, ISCOM, CpG, preferably aluminium adjuvant.
  • the fusion protein according to any one of items 1 to 5, the polynucleotide according to item 6, the vector according to item 7, the host cell according to item 8, or the vaccine composition according to item 9 Treatment and/or prevention of infection by novel coronavirus SARS-CoV-2 (preferably wild strain WH01, South African mutant strain B.1.351, Brazilian mutant strain P.1, California mutant strain B.1.429, Indian strain B.1.617), or Use of novel coronavirus disease COVID-19, or fusion protein according to any one of claims 1 to 5, polynucleotide according to claim 6, vector according to claim 7, and claim 8
  • the host cell, or the vaccine composition of claim 9 is prepared to treat and/or prevent the novel coronavirus SARS-CoV-2 (preferably wild strain WH01, South African mutant strain B.1.351, Brazilian mutant strain P.1, California mutant strain B.1.429, Indian strain B.1.617) infection, or use in the drug of the novel coronavirus disease COVID-19.
  • the preparation method of novel coronavirus SARS-CoV-2 vaccine is characterized in that, comprises the steps:
  • a method for inducing a specific immune response in an organism comprising administering to an individual the fusion protein of any one of items 1 to 5, the polynucleotide of item 6, the vector of item 7, and the item 8 The host cell, or the vaccine composition of item 9.
  • One embodiment of the present invention includes a method for efficiently expressing an optimized novel coronavirus S protein (S1 protein or fragments thereof S0, SS1, SS2, SS3, or a mutant fragment SS1t of one or more SS1 proteins), comprising adding the The nucleotide coding sequence for the polypeptide or fusion protein is introduced into CHO cells to express the polypeptide.
  • S1 protein or fragments thereof S0, SS1, SS2, SS3, or a mutant fragment SS1t of one or more SS1 proteins
  • the nucleotide coding sequence of the polypeptide or fusion protein is present in a recombinant plasmid, preferably by electroporation.
  • One embodiment of the present invention includes the use of the polypeptide or fusion protein in the preparation of an S protein (S1 protein or fragments thereof SSO, SS1, SS2, SS3, or one or more mutant fragments SS1t of SS1 proteins) antigen preparations.
  • S protein S1 protein or fragments thereof SSO, SS1, SS2, SS3, or one or more mutant fragments SS1t of SS1 proteins
  • One embodiment of the present invention includes the use of the polypeptide or fusion protein in the preparation of a preparation for diagnosing novel coronavirus.
  • One embodiment of the present invention includes the application of the polypeptide or fusion protein in the preparation of a novel coronavirus subunit vaccine.
  • CHO cells in addition to CHO cells, 293, Vero, etc. host cells can be used.
  • the purification is carried out by molecular sieve chromatography, and can also be carried out by affinity chromatography, for example, including filtering the cell supernatant expressing the antigen to remove cell debris, passing through a 10K ultrafiltration tube ( Millipore) for preliminary purification and concentration, and finally purified by molecular sieve chromatography using Siperose6 Increase10/300GL column (GE) to obtain high-purity target protein.
  • affinity chromatography for example, including filtering the cell supernatant expressing the antigen to remove cell debris, passing through a 10K ultrafiltration tube ( Millipore) for preliminary purification and concentration, and finally purified by molecular sieve chromatography using Siperose6 Increase10/300GL column (GE) to obtain high-purity target protein.
  • the present invention provides a kind of high-efficiency expression optimization method of novel coronavirus antigen and ferritin fusion expression, it comprises the aforementioned S protein (S1 protein or its fragment S0, SS1, SS2, SS3, or one or more SS1 proteins of the
  • S protein S1 protein or its fragment S0, SS1, SS2, SS3, or one or more SS1 proteins of the
  • the nucleotide coding sequence of the mutant fragment SS1t)-ferritin was introduced into CHO cells, and the host CHO cell strain was transfected with the above-mentioned recombinant plasmid based on electroporation, and the transfected host CHO cells were cloned, cultured and screened.
  • a CHO cell line was constructed.
  • the truncated novel coronavirus S1 or its fragment reduces the non-specific non-receptor binding region of the full sequence. Risk of heterosexual immunity.
  • the above-mentioned optimized novel coronavirus S protein (S1, SSO, SS1, SS2, SS3, or its artificially mutated fragment SS1t) and its fusion protein not only retain the S protein binding to the ACE2 receptor on the surface of human cells, but also make it easier to perform in CHO cells.
  • the obtained product is easy to purify, and the fusion protein appears as nanoparticle, which is very easy to separate from a single antigen small molecule, and appears as the first single peak on the SEC column, with higher purity and better separation. And while ensuring the neutralizing activity of the antibody, the yield is greatly improved (for example, from 14.23 mg/L to 346.16 mg/L).
  • the novel coronavirus antigen mutation fragment SS1t of the present invention is selected through combination and test based on the S1 protein mutation of South Africa, Brazil, California, and India mutant strains. Therefore, compared with the vaccine prepared against the classic strain 2019-nCoV, it can further cover the population in the environment where the mutant strain exists, provide more comprehensive protection, and is expected to have a higher protection rate to the population.
  • the repeated arrangement of antigens (the antigens are arranged on the surface of the spherical structure formed by ferritin) is realized.
  • the repeated arrangement of antigens can drive stronger humoral immune responses than single antigens, and can cause stronger B cell activation through antigen-driven B cell receptor (BCR) cross-linking, which may also potentially affect antigen transport and localization. effect.
  • BCR B cell receptor
  • the present invention uses ferritin nanoparticles to design and successfully display the nanoparticles of the novel coronavirus spike protein.
  • the ferritin-based antigen S protein-Ferritin
  • ferritin is a 24-sided sphere composed of 8 trimers, and the S protein is also a trimer, so the antigen in mammalian cells is Expression is similar to the spike trimer (S), which ensures that the fusion of the spike protein or its truncated and mutated fragment SS1t used in the present invention with ferritin does not negatively affect the production of the protein.
  • the present invention is expressed in mammalian cells (CHO), indicating that the present invention produces a spike protein with the new coronavirus (SARS-CoV-2) by natural-like glycosylation
  • SARS-CoV-2 new coronavirus
  • Antigen fragments with similar antigenicity are more conducive to eliciting the immune response of mammals such as humans to generate neutralizing antibodies against them.
  • Fig. 1 The pseudovirus inhibition rate-dilution factor curve of serum after intramuscular injection of S1 protein fragment-Ferritin fusion protein in mice (low dose group, 2 weeks after immunization for 3 times).
  • Example 1 Construction of expression vector and screening of S protein-Ferritin fusion gene
  • the S1 and S2 regions were analyzed (https://zhanglab.ccmb.med.umich.edu/C-I-TASSER/2019-nCov/) to obtain the original S1 protein amino acid fragment.
  • S1 sequence of the present invention amino acid sequence: as shown in SEQ ID No: 1, nucleotide sequence: as shown in SEQ ID No: 8
  • SSO, SS1, SS2, SS3 protein sequences were designed in a truncated manner.
  • the amino acid sequences of S0, SS1, SS2, and SS3 proteins are shown in SEQ ID Nos: 2 to 5, respectively, and the nucleotide sequences are shown in SEQ ID Nos: 9 to 12.
  • the original ferritin amino acid sequence was obtained from NCBI (https://www.ncbi.nlm.nih.gov/protein/WP_000949190.1?), the first 4 amino acids of the sequence were removed from the original ferritin amino acid sequence, and the 19th amino acid sequence was removed. Position N amino acid is mutated to Q amino acid to obtain the Helicobacter pylori Ferritin amino acid sequence shown in SEQ ID No: 7. Further codon optimization was carried out according to the CHO expression system to obtain the corresponding Ferritin nucleotide sequence (as shown in SEQ ID No: 18).
  • the nucleotide sequences after S1, SSO, SS1, SS2 or SS3 and ferritin are linked by the GGGSGGGS hinge sequence are respectively: SEQ ID No: 13: S1-Ferritin nucleotide sequence ; SEQ ID No: 14: SSO-Ferritin nucleotide sequence; SEQ ID No: 15: SS1-Ferritin nucleotide sequence; SEQ ID No: 16: SS2-Ferritin nucleotide sequence; SEQ ID No: 17: SS3 - Ferritin nucleotide sequence.
  • the nucleotide sequences SEQ.ID.NO.13-17 designed in Example 1.1 were synthesized by GenScript, and were respectively connected to the PMV universal vector by the company to obtain the corresponding PMV-S protein Ferritin plasmid. Then, using the pcDNA3.1 vector (purchased from Addgene), the corresponding five pcDNA3.1-S protein-ferritin particles were recombined and constructed respectively. The specific steps are described in detail below.
  • Enzymes XhoI and EcoRI were purchased from NEB Company. Add and mix samples into a 1.5mL EP tube according to the following table: The double-enzyme digestion reaction system is 10 ⁇ L, and the sample is added as shown in Table 1 below:
  • the above-mentioned double-enzyme digestion product was taken out and subjected to agarose gel electrophoresis to recover the DNA fragments therein.
  • the gel recovery kit was purchased from Tiangen Company.
  • step (3) Add an equal volume of PC buffer to the 1.5mL centrifuge tube in step (2), place it in a 50°C water bath for about 10 minutes, and gently turn the centrifuge tube up and down to ensure that the glue block is fully dissolved.
  • step (3) Add the solution obtained in step (3) into the adsorption column CB2, let stand for 2 min, 12,000 rpm, centrifuge for 1 min, pour off the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.
  • step (9) Store the DNA sample in step (8) at 4° C., prepare an agarose gel electrophoresis identification gel, and recover the DNA fragments for SEQ.ID.NO.13-17 respectively.
  • the DNA fragment recovered in 1.2.2 was inserted into pcDNA3.1 vector (purchased from Addgene).
  • ligation reaction product which contains the pcDNA3.1 vector to which the DNAs of SEQ.ID.NO.13-17 are respectively connected, which is called pcDNA3.1- S1 plasmid, pcDNA3.1-SS0 plasmid, pcDNA3.1-SS1 plasmid, pcDNA3.1-SS2 plasmid, pcDNA3.1-SS3 plasmid.
  • step (4) The ligation reaction product of step (4) can be directly subjected to transformation experiments, or can be stored at -20°C, thawed and transformed when needed. 1.2.4 Transformation reaction
  • step (1) After the completion of step (1), take out the sample tube, place it in a water bath at 42° C. for 45s, and then immediately take an ice bath for 90s to carry out transformation.
  • step (2) After step (2) is completed, take out the sample tube, add 200 ⁇ L of LB liquid medium to the sample tube in the ultra-clean workbench, and then place the sample tube on a constant temperature shaker at 37°C, 220rpm, and cultivate for 45min.
  • step (6) Invert the plate in step (5) in a biochemical constant temperature incubator, and cultivate at 37° C. for 15 hours.
  • the small plasmid kit was purchased from Tiangen Biochemical Technology Co., Ltd. The specific operations are as follows:
  • step (4) Add 250 ⁇ L of P2 buffer to the solution in step (4), and immediately and gently invert the centrifuge tube 6-8 times to mix. Let stand at room temperature for 2-4min.
  • step (6) Add 350 ⁇ L of P3 buffer to the solution in step (5), immediately and gently invert the centrifuge tube 6-8 times, and mix thoroughly. At this time, a white flocculent precipitate appears. Centrifuge at 12,000 rpm for 10 min.
  • step (7) Move the supernatant solution in step (6) to the center of the adsorption column CP3, centrifuge at 12,000 rpm for 1 min at room temperature, pour off the liquid in the collection tube, and put the adsorption column CP3 into the collection tube.
  • step (2) The 1.5mL EP tube in step (1) was placed in a constant temperature water bath at 37°C, and the enzyme was digested overnight.
  • Example 2 Transfer of pcDNA3.1-S protein plasmids into CHO cells.
  • Example 1 The five pcDNA3.1-S1 protein Ferritin plasmids finally identified in Example 1 were extracted using a kit purchased from Tiangen Biochemical Technology Co., Ltd.
  • A. Preparation of medium dialyze FBS (purchased from Gibco, USA) with a 3500 dialysis bag, and then configure 1L of CSC-03 medium containing 10% dFBS. After the configuration, preheat in the incubator. The temperature was set to 37°C.
  • B. Host cell preparation inoculate a CHO cell line with an initial cell concentration of 0.5 ⁇ 10 6 cells/ml (introduced from ATCC by Beijing Dingchi Biotechnology Co., Ltd., introduction time: May 1, 2018, ATCC number: CCL61. After the cells were expanded and cultured in Beijing Dingchi Biotechnology Co., Ltd., a cell bank was established. The number of the cell bank is: BJDC-201800010) in a 125ml Erlenmeyer flask, and cultured in suspension for 3 days; the host was recorded with a Countstar automatic cell counter.
  • Cell CHO viable cell density and cell viability cells were taken according to the total amount of 1.0 ⁇ 10 7 cells, centrifuged to remove the supernatant, centrifuged at 800 r/m for 5 min; then washed twice with 5 mL of CD-Pro medium to remove the supernatant After the second wash, use 600 ⁇ L CD-Pro medium to resuspend the cells, and wait for use;
  • G Blow and suck the mixed electroporated cell solution, spread the cell solution into a 96-well plate at a volume ratio of 100 ⁇ L/well, and place the 96-well plate in an incubator at 37°C with 5% CO 2 .
  • MSX methionine imidosulfone
  • the addition amount is 30-50 ⁇ m; after culturing for about 20 days, the cell line is transferred to a 24-well plate for Culture, the medium is the monoclonal medium CSC-03 containing 5% dFBS (purchased from One Life Science (Shenzhen) Co., Ltd.);
  • a pair of 24-well plate cells were obtained by expansion. After standing at 37°C and 5% CO 2 for 7 days, the supernatant was taken for detection, and the positive cell lines were screened by ELISA method, that is, the transfected cells were successfully transfected.
  • Cell lines, the screened cell lines were transferred to 6-well plates for culture, and the medium was a monoclonal medium without dFBS; after 3 days of culture, they were transferred to a shake flask for culture, and the medium was CHO-K1+15um MSX without dFBS.
  • the CHO-K1 medium was purchased from One Life Science (Shenzhen) Co., Ltd.; the selection of the cell line and the adaptation of the cell line to the serum-free medium were completed during the culture process. The process is shown in the table 4 shown.
  • the screened cell lines were expanded, and NF604 medium (purchased from Shenzhen Eternal Life Branch) was added on the 4th day. When the cell viability was about 60%, centrifuge at 12,000 rmp for 15 min, collect the cell supernatant, and take 1 ml of the medium. The serum was detected, and the positive cell lines with high yield were screened by ELISA detection method (the primary antibody was anti-S-RBD, Yiqiao Shenzhou; the secondary antibody was goat anti-rabbit IgG-HRP, Soleibo), that is, the transfected cells were successfully transfected. cell line. Two more rounds of monoclonal screening were performed on the cell lines with high expression levels. A number of CHO cell lines stably expressing S1-Ferritin, SSO-Ferritin, SS1-Ferritin, SS2-Ferritin and SS3-Ferritin were obtained respectively.
  • Example 2 The cell supernatants containing five groups of optimized S protein-Ferritin obtained in Example 2 were purified by chromatography with GE's core 400. The specific steps are as follows: centrifuge the five groups of corresponding cell supernatants obtained in Example 2 at 10,000 ⁇ g for 30 min, collect the supernatants, filter them at 0.22um, and concentrate the filtered supernatants by 30kDa membrane-packed ultrafiltration 10-20 times. as a sample solution.
  • S protein and ferritin are fused to form particles with a diameter of about 30 nm, they have more advantages in separation on SEC columns.
  • the particles have basically no adsorption on the column and exist in the first chromatographic separation peak, with high purity and recovery The effect is good, but a single antigen will enter the pores on the column for many times when it passes through the column, and the separation is achieved according to the molecular weight.
  • the SS1-Ferritin antigen obtained in Example 3 was added with 1/10,000 thimerosal by weight, and then mixed with aluminum adjuvant (purchased from Thermo) 1:1 according to the experimental concentration to prepare the SS1-Ferritin subunit vaccine.
  • Example 5 Study on the immune response of SS1-Ferritin subunit vaccine
  • BALB/c mice were used, and the SS1-Ferritin subunit vaccine of the present invention was administered by intramuscular injection and subcutaneous injection.
  • the effective limiting dilution of the antibody produced in the peripheral blood of the mice was determined.
  • the dilution factor at the half inhibition rate was taken as EC50 (referring to the 50% inhibition dilution factor, inhibitory dilution, EC50), and the comparison among each group was carried out by a statistical method. From this, the SS1-Ferritin subunit vaccine immune response and immune response were evaluated.
  • mice 36 BALB/c mice (6-8 weeks, female) were divided into 6 groups of 6 mice as shown in Table 6 below.
  • the administration site of intramuscular injection was the quadriceps femoris muscle of mice, and the administration site of subcutaneous injection was subcutaneous on the back of the neck, and the volume of each injection was 200 ⁇ l/mice.
  • Blood collection method 2 weeks after each immunization during the animal survival period, before the injection on the same day, blood was collected from the intraocular canthal vein after isoflurane anesthesia, and 120 ⁇ l of blood was collected from each mouse; 2 weeks after the last immunization, anesthesia and The animals were dissected, blood was collected from the large abdominal vein, and the serum was separated for neutralizing antibody detection. During the whole experiment, the feeding situation of animals and so on were observed.
  • SARS-CoV-2 pseudovirus SARS-CoV-2 pseudovirus, batch number: 20200424, titer: 1.4 ⁇ 10 5 TCID50/ml; batch number: 20200609, titer: 1.35 ⁇ 10 5 TCID50/ml, purchased from National Center for Drug Safety Evaluation and Monitoring AIDS room.
  • Huh-7 cells were purchased from the AIDS Department of the National Center for Drug Safety Evaluation and Monitoring.
  • DMEM Cat. No. 12430054, by thermo Fisher.
  • Serum negative control healthy BALB/c mouse serum. Among them, the serum obtained in the above-mentioned example is referred to as a serum sample.
  • VICTOR X5 chemiluminescence instrument manufacturer: Perkin Elmer.
  • Cell control column medium, cells.
  • Virus control column medium, cells, virus.
  • Serum sample column culture medium, cells, serum samples, virus.
  • Serum Negative Control Column Medium, Cells, Serum Negative Control, Virus.
  • the prepared Huh7 cells with a confluence rate of 80% to 90% were digested with trypsin, and then added with DMEM complete medium to mix well, and the cell concentration was adjusted to 2 ⁇ 10 5 /ml.
  • the ratio of the RLU of the virus control column to the RLU of the cell control column is greater than or equal to 1000;
  • the EC50 value of the negative serum control is less than 30 when calculated according to the following formula, and the test is judged to be established; or the RLU of the negative serum control at 30-fold dilution is used to calculate the luminescence intensity as follows, and its mean +-2SD is used as the negative serum control. Reference range, when the luminescence intensity of the negative serum control is within the reference range, the test is determined to be established;
  • Neutralization inhibition rate [1-(luminescence intensity of serum sample group-luminescence intensity of cell control)/(luminescence intensity of serum negative control group-luminescence intensity of cell control)] ⁇ 100%.
  • the results showed that in the intramuscular injection adjuvant group and the subcutaneous injection adjuvant group, the 50% inhibition dilution factor EC50 was less than 30, which was the same level as the EC50 of the negative blank control. No inhibitory effect was shown, i.e., there were no medium antibodies against SARS-CoV-2. High titers of neutralizing antibodies were produced in all mice injected with the subunit vaccine. In addition, in the intramuscular injection low-dose group, the subcutaneous injection adjuvant group, and the subcutaneous injection booster group, the inhibitory effect of each mouse against pseudovirus was enhanced with the increase of the number of immunizations (Table 7, Figure 1).
  • the geometric mean value of EC50 of all groups injected with subunit vaccine showed a trend that the inhibitory effect on pseudovirus increased with the increase of the number of immunizations (Table 8).
  • the intramuscular low-dose group After three immunizations, the intramuscular low-dose group finally reached an extremely high 50% inhibitory dilution of 24143 (individual mice reached 55358, 65610), and the subcutaneous injection booster group finally reached an extremely high 50% inhibitory dilution of 23346 multiple.
  • the immunization effect did not increase with increasing dose in the intramuscular high-dose group compared with the intramuscular low-dose group. For the reason, we noticed that there was a trend of first increase and then decrease in some of these mice, especially in mice (F15), although the EC50 after the third immunization was only 3132.99, it experienced 12593 such a respectable high value. It is suggested that by increasing the immunization dose, the effect is more rapid in some mice receiving intramuscular injection, so that the induced neutralizing antibodies reach the peak earlier. Compared with the mice in the high-dose intramuscular injection group, the first dose increase in the subcutaneous injection booster group delayed the arrival of the peak EC50, and the EC50 was still as high as 23346 2 weeks after the third immunization.
  • the tolerance of the animals under the immunization dose of 25 ⁇ g/animal was observed.
  • the activity, body temperature, and feeding behavior of the mice in the subcutaneous injection high-dose group were normal, and no death occurred.
  • the geometric mean of EC50 after the second immunization was 5313, which was also obtained. good immune effect.
  • the excellent safety of the subunit vaccine of the present invention is suggested.
  • S1-Ferritin, SSO-Ferritin, SS2-Ferritin, SS3-Ferritin were prepared as subunit vaccines by the same method as in Example 4, and neutralizing antibody detection experiments were carried out in the same scheme as in Example 5, and the vaccines were immunized. Response efficacy and immune response were evaluated.
  • mice 36 BALB/c mice (6-8 weeks, female) were divided into 6 groups as shown in Table 12 below, with 6 mice in each group.
  • the intramuscular injection was used, the administration site was the quadriceps femoris muscle of the mice, and the number of immunizations was twice, except that it was the same as in Example 5, and the injection volume for each time was the same as 200 ⁇ l/mice.
  • the test schedule and administration dose are shown in Table 12.
  • the immunized animals did not die, the basic indicators such as body weight and body temperature were stable, and there was no redness and swelling at the inoculation site.
  • the mixture of serum collected from 6 mice in the same group was used as a sample, and the neutralization inhibition rate of each group after the initial dilution of 30 times was measured by the same method, and converted into EC50. It was confirmed that the EC50 and other indicators of the above fusion proteins all had the same effect as that of SS1-Ferritin.
  • Table 13 shows the detection results of serum at the time point 2 weeks after the second immunization.
  • the EC50 of the samples in the SS0-Ferritin group was 7194.
  • the samples in the S1-Ferritin, SS1-Ferritin, SS2-Ferritin, and SS3-Ferritin groups had higher titers, so the inhibition rate was 7290 when the dilution factor was 7290. Still well above 50% reporting their EC50 as >7290.
  • the subunit vaccines prepared with S1-Ferritin, SS0-Ferritin, SS2-Ferritin, and SS3-Ferritin can play a better antibody neutralization effect on the new coronavirus pseudovirus, and can For subsequent novel coronavirus vaccine preparation and vaccine screening.
  • fusion proteins of two or three S protein fragments (S1, SSO, SS2, SS3) and Ferritin were prepared by the same operation as in the above example: 2S1-Ferritin, 2SSO-Ferritin, 2SS2-Ferritin, 2SS3-Ferritin, 3SS1-Ferritin, etc., can have a better antibody neutralization effect on pseudoviruses, reflecting the same effect as that of single-segment-Ferritin.
  • the amino acid sequence shown by SEQ ID NO: 19 was obtained by introducing selected mutated amino acid sites, namely K417N/E484K/N501Y/L452R/S477N/N439K, into the above SS1 fragment. This sequence was named SS1t and the corresponding nucleotide sequence is shown in SEQ ID NO:21.
  • linker 2 is the sequence shown in SEQ ID NO: 6, the amino acid sequence after connecting the two SS1t with the linker 2 is shown in SEQ ID NO: 20, hereinafter, also referred to as fragment 2SS1t, the corresponding core
  • SEQ ID NO:22 The nucleotide sequence is shown in SEQ ID NO:22.
  • linker 1 is the sequence shown in SEQ ID NO:6
  • amino acid sequence after linking SS1t and ferritin with linker 1 is shown in SEQ ID No:23
  • fragment 2SS1t and ferritin after linking with linker 1 The amino acid sequence is shown in SEQ ID No:24.
  • the coding sequence corresponding to the amino acid sequence shown in SEQ ID NO: 19 was synthesized by BGI, and the company was connected to the PMV universal carrier (BGI) to obtain the corresponding SS1t protein plasmid (PMV-SS1t plasmid).
  • Example 8 the cell supernatants containing SS1t-Ferritin and 2SS1t-Ferritin proteins collected in Example 8 were purified by chromatography with GE's core 400.
  • the purified SS1t-Ferritin and 2SS1t-Ferritin antigen proteins were obtained, and their amino acid sequences were SEQ ID NO.23 and SEQ ID NO.24, which can be used as subunit vaccine candidates for new coronavirus mutant strains.
  • SS1t-Ferritin and 2SS1t-Ferritin subunit vaccines were prepared using the SS1t-Ferritin and 2SS1t-Ferritin antigenic proteins obtained above, respectively.
  • BALB/c mice were used to administer the SS1t-Ferritin subunit vaccine of the present invention by intramuscular injection. Except for the following operations, the operations in this example and the 50% inhibition dilution ratio (the definition of EC50) were the same as In the same manner as in Example 5, the immune response and immune response induced by the SS1t-Ferritin subunit vaccine in vivo were evaluated.
  • mice Thirty BALB/c mice (6-8 weeks, female) were divided into 5 groups of 6 mice as shown in Table 7 below.
  • the injection site was the quadriceps femoris muscle of mice, and the drugs administered to each group were as follows.
  • Negative control aluminum adjuvant (purchased from Thermo), control product: Kelefu (novel coronavirus inactivated vaccine (Vero cells), SINOVAC).
  • Test product the SS1t-Ferritin subunit vaccine of the present invention (hereinafter also referred to as the subunit vaccine of the present invention).
  • the intramuscular injections were administered according to Table 9. Except for the test product group, the administration volume was 100 ⁇ l/piece. During the test, check the animal clinical indicators (see Table 10) and observe whether there is any abnormality such as redness and swelling at the injection site. During the survival of the animals, 2 weeks after each immunization, blood was collected from the intraocular canthal vein, and the blood was collected from each mouse ⁇ 120 ⁇ l/time, and the serum was separated by the usual method for neutralizing antibody detection.
  • Serum serial dilution Serum samples collected and isolated from immunized animals were inactivated at 56°C for 0.5 h, and 2-fold serial dilutions were performed with cell culture medium (DMEM, provided by thermo Fisher). Except for the control product group (diluted from 1:128 to 1:16384), the dilution ratios were all 1:4 to 1:512.
  • DMEM cell culture medium
  • the specific dilution process is as follows: label the wells of 96-well plate in sequence from 1:4 to 1:512; add 180 ⁇ l virus maintenance solution containing 0.5% penicillin-streptomycin double antibody to the first well, and add 120 ⁇ l containing 0.5% penicillin to other wells Streptomycin double-antibody virus maintenance solution; pipette 60 ⁇ l of serum into the first well, put the pipette tip below the liquid surface and gently pipette to make a 1:4 dilution serum sample; take 120 ⁇ l of 1 :4 dilution of the liquid to the second well, and mix gently; repeat the above dilution steps to 1:512, and aspirate 120 ⁇ l of the liquid from the last well.
  • Hazardous waste disposal The above operations are carried out with sterile filter tips. Every time a new tip is replaced, the old sterile filter tips absorb part of the disinfectant and put them in a disinfection container containing effective chlorine 5000mg/L84 disinfectant. After soaking for 30 minutes, discard it in a medical garbage bag for high-pressure treatment.
  • Neutralization The above virus suspension was added to the serially diluted serum (120 ⁇ l:120 ⁇ l) at a ratio of 1:1, mixed in a 96-well culture plate, and the plate was placed in a CO 2 incubator (37 ⁇ 1°C) for 2 hours. , to obtain a neutralized mixture.
  • Culturing Vero cells with mixed liquid discard the cell culture medium in the 96-well plate cultured with Vero cells, and add 100 ⁇ l/well of virus maintenance solution containing 0.5% penicillin-streptomycin double antibody with a multi-channel pipette. Then, 100 ⁇ l per well of the neutralized mixture was added to the wells, and 2 replicate wells were set for each dilution. At the same time, the wells in which 200 ⁇ l/well of virus maintenance solution containing 0.5% penicillin-streptomycin double antibody were added were set as normal cell controls.
  • the 96-well plate was covered and fixed up and down with paper tape after inoculation, placed in a lunch box padded with non-woven fabric, and placed in a CO 2 incubator ( 37 ⁇ 1°C) for 96h.
  • Back drop test Dilute the above virus suspension as above, and dilute to a gradient of 10 -1 to 10 -3 in a 10-fold manner. Take a new 96-well plate plated with Vero cells, discard the cell culture liquid, add 150 ⁇ l/well of virus maintenance solution containing 0.5% penicillin-streptomycin double antibody with a multi-channel pipette, and add 50 ⁇ l/well of virus maintenance solution. 10 0 to 10 -3 of the virus dilution was inoculated into the above plate, and 8 replicate wells were set for each dilution, and cultured for 96 hours. A normal cell control was set as above.
  • CPE cytopathic effect
  • the changes of cell CPE were observed under an inverted microscope.
  • the normal cell control should have no cytopathic changes, and the positive control had CPE changes.
  • the reciprocal of the dilution is the neutralizing antibody titer of the sample;
  • the neutralizing antibody titer of the sample is the reciprocal of the average value of the two dilutions; when both adjacent dilutions have 1 well lesions, the The neutralizing antibody titer of a sample is the reciprocal of the mean of the two dilutions.
  • ⁇ Seronegative/positive judgment standard 1:12 is used as the positive/negative judgment standard, ⁇ 1:12 is negative, and ⁇ 1:12 is positive.
  • the inventors also conducted the above experiments using pseudoviruses of South African mutant 501Y.V2, California mutant B.1.429, and Indian mutant B.1.617 (for example, the pseudoviruses were purchased from the AIDS Department of the National Center for Drug Safety Evaluation and Monitoring).
  • the animals given the SS1t-Ferritin subunit vaccine of the present invention produced higher titers of neutralizing antibodies, and with the increase of the number of immunizations, the 50% inhibition dilution ratio increased, and the The inhibitory effect of pseudovirus was enhanced.
  • the geometric mean EC50s of the three dose groups of the test products appeared to increase with the number of immunizations.
  • the middle-dose group finally reached an extremely high 50% inhibitory dilution factor of 32836.18
  • the low-dose group finally reached an extremely high 50% inhibitory dilution factor of 17235.83, which was much higher than that of the control product group.
  • the tolerance of animals was observed by setting a high immunization dose of 10 ⁇ g/vaccine in this example.
  • the subunit vaccine prepared by the present invention can be used to prevent novel coronavirus (SARS-CoV-2) infection or novel coronavirus disease (COVID-19), including its various mutant strains (such as , Brazilian mutant strain) infection.
  • SARS-CoV-2 novel coronavirus
  • COVID-19 novel coronavirus disease
  • the fusion protein provided by the present invention comprising S1 protein, SSO, SS1, SS2, SS3 protein or a mutant fragment SS1t of more than one SS1 protein and Helicobacter pylori ferritin is easier to express in CHO cells, and can significantly improve the S protein (S1, SSO, SS1, SS2, SS3, or a mutant fragment SS1t of more than one SS1 protein)-Ferritin expression level, which can be used to prepare vaccines for the prevention of novel coronavirus (including South African mutants) disease (COVID-19 ), laying a solid foundation for the production of human subunit vaccines for novel coronavirus.
  • novel coronavirus including South African mutants
  • COVID-19 South African mutants
  • the present invention also relates to the S protein-Ferritin fusion gene, vector, cell, preparation method, treatment method or pharmaceutical use of the novel coronavirus (SARS-CoV-2), which can be applied to basic research on the principle of action of the virus, differential diagnosis, rapid Determination, epidemiological investigation, animal model preparation and other wide-ranging fields.
  • SARS-CoV-2 novel coronavirus

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Abstract

L'invention concerne une protéine de fusion d'un polypeptide et d'une ferritine Helicobacter Pylori, et un vaccin sous-unitaire pour la prévention d'une infection par le nouveau coronavirus (SARS-CoV-2) préparé à l'aide de la protéine de fusion. Le polypeptide comprend une ou plusieurs protéines S1 ou des fragments de celles-ci (SS0, SS1, SS2 ou SS3, ou un fragment mutant SS1t de la protéine SS1), et leurs séquences d'acides aminés sont dérivées des séquences de la protéine S du nouveau coronavirus et sont optimisées. Le polypeptide ou le fragment peut être fusionné avec la ferritine d'helicobacter pylori à codon optimisé au moyen d'un lieur pour former une protéine de fusion à exprimer. La protéine de fusion a l'avantage d'être facile à purifier tout en ayant un niveau d'expression élevé dans des cellules CHO, et peut produire un anticorps neutralisant à titre élevé contre le nouveau coronavirus (SARS-CoV-2) après immunisation d'un animal, et peut couvrir diverses souches de virus du nouveau coronavirus notamment des souches mutantes.
PCT/CN2021/098951 2020-12-22 2021-06-08 Vaccin recombinant sous-unitaire protéique contre le nouveau coronavirus WO2022134487A1 (fr)

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