WO2022057540A1 - Vaccin contre le sars-cov-2 à base de modification de revêtement lentiviral et de distribution d'arnm et procédé de préparation s'y rapportant - Google Patents

Vaccin contre le sars-cov-2 à base de modification de revêtement lentiviral et de distribution d'arnm et procédé de préparation s'y rapportant Download PDF

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WO2022057540A1
WO2022057540A1 PCT/CN2021/112661 CN2021112661W WO2022057540A1 WO 2022057540 A1 WO2022057540 A1 WO 2022057540A1 CN 2021112661 W CN2021112661 W CN 2021112661W WO 2022057540 A1 WO2022057540 A1 WO 2022057540A1
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spike
protein
sars
cov
mrna
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蔡宇伽
尹笛
凌思凯
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上海交通大学
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    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a method for preparing a SARS-CoV-2 mRNA vaccine, in particular to a method for preparing a coronavirus SARS-CoV-2 mRNA vaccine by a lentiviral vector.
  • Vaccines are an effective way to prevent infectious diseases caused by viruses and bacteria.
  • Common vaccine platforms include attenuated vaccines, inactivated viruses, recombinant proteins, polypeptides, genes, DNA and mRNA delivered by viral vectors.
  • Traditional vaccines such as attenuated and inactivated vaccines have complex production processes, slow production speed, and high safety level requirements, making it impossible to quickly respond to emergencies.
  • Viral vectors and DNA vaccines run the risk of integrating into the genome. Therefore, there is an urgent need to develop a vaccine preparation method that is safe, effective, and can be rapidly produced.
  • mRNA vaccine As a new generation vaccine, mRNA vaccine has gradually become a research hotspot. mRNA vaccines deliver the antigen's messenger RNA into the body and express the antigen in the body. Binding of antigens to receptors in the body triggers a specific immune response and produces antibodies. mRNA vaccines can be applied to both infectious and non-infectious diseases. Some companies have launched Phase I/II clinical trials of mRNA vaccines targeting tumors. mRNA vaccines have the following advantages: (1) mRNA vaccines are simple in design and can rapidly produce antigenic mRNAs of emerging viruses. (2) mRNA vaccines are relatively safe. In contrast to DNA vaccines, mRNA vaccines have no risk of integrating into the genome.
  • mRNA needs a carrier to enter the cell and function.
  • the most commonly used mRNA vaccine delivery method is liposome delivery. After the outbreak of the new coronavirus SARS-CoV-2 in 2019, the vaccine mRNA-1273 developed by Moderna is the world's first mRNA vaccine to enter clinical trials. mRNA-1273 is made from liposome-encapsulated Spike protein mRNA. However, the liposome delivery method is less efficient in infecting cells, and the infection efficiency plays a key role in the success of the vaccine.
  • the Chinese invention patent application with the application number of the previous patent document No. 201810533437.X discloses a lentiviral vector and a method for delivering exogenous RNA; using the principle of mutual binding between RNA binding proteins and the RNA sequences recognized by them, the RNA is combined with The protein is integrated into the backbone of the lentiviral GagPol long-chain protein, and the recognized RNA sequence is linked to the exogenous target RNA, so that the target RNA is packaged into the lentiviral particle during the assembly process of the lentiviral particle.
  • this method only delivers RNA, and it is difficult to induce neutralizing antibodies against conformational epitopes.
  • the purpose of the present invention is to provide a method for preparing a coronavirus SARS-CoV-2 mRNA vaccine by a lentiviral vector. Production of a SARS-CoV-2 vaccine that is both effective and safe.
  • the present invention is not only an mRNA vaccine, but also a protein vaccine.
  • the present invention wraps the mRNA of the spike protein Spike of SARS-CoV-2 through lentiviral particles. On this basis, by decorating the surface of lentiviral particles with Spike protein to simulate the appearance of the new coronavirus, the Spike protein is presented in the form of a three-dimensional structure to induce the body to produce neutralizing antibodies against conformational epitopes.
  • the present invention uses lentiviral particles as delivery carriers, (1) lentiviral particles have envelope proteins, and mRNA can be encapsulated in the enveloped particles to prevent mRNA from being degraded by nucleases; (2) lentiviral particles have high-efficiency infecting cells. (3) After the lentiviral vector enters the cell, the mRNA is released into the cytoplasm, and the viral antigen is efficiently expressed; (4) The lentiviral particle of the present invention is decorated with Spike protein, which can Spike proteins are presented to antigen-presenting cells in stable three-dimensional structures.
  • the present invention utilizes the interaction between the RNA binding protein and the RNA stem-loop structure it recognizes to package the Spike protein mRNA containing the recognizable RNA sequence into lentiviral particles.
  • the Spike protein can be assembled into the envelope structure of the lentivirus as a spike protein.
  • the present invention provides a SARS-CoV-2 vaccine based on lentiviral shell modification and mRNA delivery.
  • the SARS-CoV-2 vaccine is obtained by lentiviral particles as the shell wrapping the mRNA of the spike protein Spike of SARS-CoV-2. .
  • the envelope of the lentiviral particle is decorated with the spike protein Spike of SARS-CoV-2;
  • the gene of the spike protein Spike is a gene after codon optimization and amino acid mutation, and the sequence is as shown in SEQ ID NO. .9 shown.
  • the envelope of the lentiviral particle is also decorated with an envelope protein VSV-G, and the sequence of the plasmid expressing the envelope protein VSV-G is shown in SEQ ID NO.1.
  • the mRNA of the spike protein Spike is obtained by transcribing the spike protein Spike gene of SARS-CoV-2; the gene of the spike protein Spike is a gene after codon optimization and amino acid mutation, and the sequence is such as SEQ ID NO.9 is shown.
  • the invention also provides a preparation method of SARS-CoV-2 vaccine, which is prepared by transfecting the vaccine production plasmid into virus production cells; collecting supernatant, concentrating and preparing.
  • the specific steps of the preparation method are as follows:
  • the plasmid of the spike protein Spike mRNA is co-transfected into virus production cells; the stem-loop structure is placed in the expression frame of the spike protein Spike mRNA; the spike protein Spike mRNA is composed of the spike protein Spike gene of SARS-CoV-2 transcribed;
  • the plasmid expressing the RNA-binding protein-containing lentiviral GagPol long-chain protein is prepared by integrating the RNA-binding protein into the N-terminus of the third-generation lentiviral GagPol long-chain protein, and the RNA-binding protein is MS2 capsid protein;
  • the gene sequence of the MS2 capsid protein is shown in SEQ ID NO.5, and the plasmid sequence for expressing the RNA-binding protein-containing lentiviral GagPol long-chain protein is shown in SEQ ID NO.4.
  • the plasmid expressing the spike protein Spike mRNA containing the stem-loop structure recognized by the RNA-binding protein is obtained by fusing the RNA sequence of the stem-loop structure recognized by the RNA-binding protein with the mRNA sequence of the spike protein Spike. , the sequence of the resulting plasmid is shown in SEQ ID NO.11.
  • the stem-loop structure recognized by the RNA-containing protein is in the expression frame of Spike mRNA, and the RNA sequence of the stem-loop structure is shown in SEQ ID NO.8.
  • the virus producing cells are selected from 293T cells, 293FTX cells, and HEK293 cells;
  • the plasmid sequence for expressing lentivirus-containing GagPol long-chain protein is shown in SEQ ID NO.2.
  • the SARS-CoV-2 vaccine described in the present invention is a lentiviral vector that delivers the mRNA and protein of the spike protein Spike.
  • the lentiviral vector prepared in this way can also prepare the corresponding vaccine by delivering the mRNA of antigenic proteins of other viruses such as SARS, MERS, HIV, etc.
  • the SARS-CoV-2 vaccine using lentiviral particles as a delivery carrier of the present invention can be used to achieve the purpose of preventing the infection of the new coronavirus SARS-CoV-2.
  • the present invention Compared with the existing SARS-CoV-2 mRNA vaccine using liposome as a delivery medium, the present invention has the following beneficial effects:
  • the SARS-CoV-2 mRNA vaccine of the present invention is carried by lentiviral particles with Spike protein mRNA, and the efficiency of infecting cells is increased.
  • the envelope structure of the lentiviral particles of the SARS-CoV-2 mRNA vaccine of the present invention contains the Spike protein, thereby simulating the appearance of the new coronavirus, presenting the Spike protein in the form of a three-dimensional structure, and inducing the body to produce more effective neutralization antibody.
  • the present invention uses lentiviral particles as delivery carriers.
  • the lentiviral particles have envelope proteins, and mRNA can also be wrapped in the envelope particles to prevent mRNA from being degraded by nucleases; after the lentiviral vector enters the cell, the mRNA is released into the cytoplasm , viral antigens can be efficiently expressed.
  • Figure 1 shows: the structure of the Spike mRNA expression plasmid of the SARS-CoV-2 vaccine
  • Figure 2 Packaging strategy of SARS-CoV 2 mRNA vaccine
  • FIG. 3 shows: the situation of the Spike protein packaged in the SARS-CoV-2 vaccine
  • Figure 4 shows the Spike protein expressed by the mRNA carried by the SARS-CoV-2 vaccine infected 293T cells
  • FIG. 5 SARS-CoV-2 vaccinated mice produced neutralizing antibodies.
  • Lentiviral vector is a virus vector that cannot self-replicate which is transformed from HIV-1. Lentiviral vectors can efficiently infect dividing and non-dividing cells, and are commonly used in biological research and gene therapy. At present, the lentiviral vector has developed to the third generation, and the safety is getting higher and higher.
  • the Spike protein is the spike protein of the coronavirus that binds to cell surface receptors to infect cells. Spike protein can cause the body to immunize and produce corresponding antibodies, and designing vaccines based on this principle is a common method.
  • the purpose of the present invention is to package the Spike protein mRNA of SARS-CoV-2 into a lentiviral vector.
  • the principle of the present invention is: using the interaction between the RNA binding protein and the RNA stem-loop structure it recognizes, the Spike protein mRNA fused with the recognizable RNA sequence is packaged into lentiviral particles.
  • the lentiviral vector delivers the Spike protein mRNA of SARS-CoV-2 into cells to express the Spike protein; as a spike protein, the Spike protein can be assembled into the envelope structure of the lentivirus, and can be used as an antigen and a cell receptor. combine.
  • Embodiment 1 Plasmid construction method and packaging strategy of SARS-CoV-2 mRNA vaccine
  • This example relates to the preparation method of SARS-CoV-2 mRNA vaccine; the structure of Spike mRNA expression plasmid is shown in Figure 1, and the packaging strategy of SARS-CoV-2 mRNA vaccine is shown in Figure 2. Specific steps include:
  • Step 1 Plasmids expressing membrane proteins (such as VSV-G), plasmids expressing lentiviral GagPol long-chain proteins, plasmids expressing lentiviral GagPol long-chain proteins containing RNA-binding proteins, and stems recognized by the RNA-binding proteins Virus producing cells (eg 293T) are co-transfected with plasmids of the loop structure and the spike protein Spike mRNA. in,
  • the plasmid expressing membrane protein VSV-G is the well-known plasmid pMD2G, and its plasmid sequence is shown in SEQ ID NO.1.
  • the plasmid expressing the long-chain protein containing lentivirus GagPol is the integrase-deficient type of the well-known plasmid pMDlg/PRRE, and its plasmid sequence is shown in SEQ ID NO.2.
  • the helper plasmid is the well-known plasmid pRSV-REV.
  • the plasmid sequence is shown in SEQ ID NO.3.
  • the helper plasmid pRSV-REV was transfected into virus-producing cells together with the integrase-deficient plasmid of pMDlg/PRRE for vaccine production.
  • the plasmid expressing the RNA-binding protein-containing lentiviral GagPol long-chain protein is obtained by fusing the MS2 capsid protein with the lentiviral GagPol long-chain protein.
  • the plasmid sequence for expressing the RNA-binding protein-containing lentiviral GagPol long-chain protein is shown in SEQ ID NO.4.
  • the gene sequence of the MS2 capsid protein is shown in SEQ ID NO.5, and the encoded protein can be connected to the GagPol long-chain protein and placed at the N-terminus of the GagPol long-chain protein.
  • the primer sequence is F: 5'-tagcaccggtcgccaccatggcctctaattttactca-3' (SEQ ID NO.13), R: 5'-tagctccggactgcacaa tcggatagttctggctgtatatcccgctgttggcgg-3' (SEQ ID NO.14);
  • the plasmid (pCMV-nCoV-S-6XMS2-mut-flag) containing the stem-loop structure recognized by the RNA binding protein and the Spike mRNA of the spike protein is expressed by the RNA of the stem-loop structure recognized by the MS2 capsid protein
  • the sequence was obtained by fusion with the mRNA sequence of the SARS-CoV-2 Spike protein.
  • the plasmid structure is shown in Figure 1, and the sequence of pCMV-nCoV-S-6XMS2-mut-flag is shown in SEQ ID NO.11.
  • RNA sequence recognized by the MS2 capsid protein is shown in SEQ ID NO.7, and the sequence can be connected with the exogenous target RNA in a single or multiple repeating manner to form an exogenous structure containing single or multiple stem-loop structures.
  • source RNA sequence of interest The sequence of 6 repetitions used in this example (there are connecting sequences at both ends of each RNA sequence) is obtained by gene synthesis, and the sequence is shown in SEQ ID NO.8.
  • the mRNA sequence of the Spike protein is obtained by transcribing the spike protein Spike gene of SARS-CoV-2; the SARS-CoV-2 Spike protein gene is obtained by gene synthesis after codon optimization, and amino acid mutations (K1003P and V1004P). The resulting gene sequence is shown in SEQ ID NO.9.
  • the plasmid preparation scheme for expressing the mRNA containing the stem-loop structure recognized by the RNA-binding protein and the spike protein Spike is as follows:
  • the gene can transcribe the spike protein Spike mRNA;
  • the primer sequences are F: 5'-tagcccgcggacgcgtgccaccatggactggacctggatcct-3' (SEQ ID NO.16), R: 5'-ttacttatcgtcatcgtctt-3' (SEQ ID NO.17);
  • the gene sequence of the stem-loop structure RNA was amplified by PCR, and the primers were F: 5'-aagacgatgacgataagtaaaccggtcttgcagcacatacatgag-3' (SEQ ID NO.18) and R: 5'-tagcccgcgggccaagcttaaggtgcacac-3' (SEQ ID NO.19).
  • Fusion PCR fuse the two genes amplified in step 2 into one, the primers are: F: 5'-tagcacgcgtgccaccatggactggacctggatcct-3' (SEQ ID NO.20), R: 5'-tagcccgcgggccaagcttaaggtgcacac-3' (SEQ ID NO.21).
  • T4 ligase to ligate the vector plasmid and the insert fragment of step 4 and then transform, extract the plasmid, and specifically transform, the steps of extracting the plasmid are: the ligation system is transformed with competent XL1-Blue cells, single clones are picked and shaken bacteria, Finally, the plasmid was extracted with a plasmid extraction kit.
  • pCMV-nCoV-S-6XMS2-mut-flag containing the stem-loop structure recognized by the RNA-binding protein and the Spike mRNA of the spike protein
  • Step 2 Collect the supernatant containing virus particles and concentrate by high-speed centrifugation or HPLC to obtain high titer mRNA-containing lentivirus particles, namely SARS-CoV-2 mRNA vaccine.
  • the mRNA vaccine preparation method introduced in the present invention can be used to prepare SARS-CoV-2 mRNA vaccines, and can also prepare vaccines of other viruses.
  • Embodiment 2 The situation of Spike protein packaged by SARS-CoV-2 mRNA vaccine
  • the specific steps are the same as in Example 1, and the C-terminal of the SARS-CoV-2 Spike protein gene used contains 1 copy of the Flag protein gene.
  • the p24 content was detected with a lentiviral titer kit. Take 300ng of p24 for Western Blot (immunoblotting test).
  • the primary antibody showing the position of the Spike protein band was mouse-derived anti-Flag; the primary antibody showing the position of the p24 protein band was mouse-derived anti-p24, and the secondary antibody was anti-Mouse IgG. Deglycosylase with PNGase F.
  • the Flag is at the C-terminus of the Spike protein, so the S2 subunit is detected), and the content of the Spike protein is higher than that of the SARS-CoV-2 mRNA vaccine particles containing the unmutated Spike gene (VSP-S indicates , the preparation method is basically the same as that in Example 1, the only difference is: in the preparation of the plasmid expressing the stem-loop structure recognized by the RNA binding protein and the spike protein Spike mRNA, the SARS-CoV-2 Spike protein gene used was not carried out. amino acid mutations).
  • Figure 3 also compares the common lentiviruses (IDLV-S/IDLV-S-mut) containing the Spike protein gene (unmutated/mutated) in the nucleic acid vectors respectively.
  • the preparation method is basically the same as that in Example 1, except that the expression membrane Protein plasmid (such as VSV-G), plasmid expressing lentiviral GagPol long-chain protein, and lentiviral vector plasmid expressing Spike protein gene (unmutated/mutated) are co-transfected into cells to produce.
  • the expression membrane Protein plasmid such as VSV-G
  • plasmid expressing lentiviral GagPol long-chain protein lentiviral vector plasmid expressing Spike protein gene (unmutated/mutated
  • the expression Spike protein gene (amino acid mutation) lentiviral vector plasmid (which is obtained by replacing the GFP gene in the lentiviral vector plasmid pCCL-PGK-eGFP by the Spike protein gene (amino acid mutation), the gene sequence is shown in SEQ ID NO. 12) can not or very Carry less Spike protein.
  • the real S2 subunit (about 80kDa) is obtained after deglycosylation.
  • Common lentiviral particle IDLV is used as a negative control.
  • Example 3 SARS-CoV-2 vaccine infected 293T cells, and the Spike protein expressed by the carried mRNA condition
  • 293T cells were seeded in a 48-well plate at 4 ⁇ 10 4 cells/well. 24 hours later, p24 300ng SARS-CoV-2 mRNA vaccine (VSP-S-mut prepared in Example 2), SARS-CoV-2 mRNA vaccine containing unmutated Spike gene (VSP-S prepared in Example 2) and The nucleic acid vector contains a common lentivirus (IDLV-S, IDLV-S-mut) containing the Spike protein gene (unmutated/mutated). At the same time, 293T cells were transfected with 2ug of plasmids pCMV-nCoV-S-6XMS2-flag and pCMV-nCoV-S-6XMS2-mut-flag.

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Abstract

La présente invention concerne un vaccin contre le SARS-COV-2 et un procédé de préparation s'y rapportant. Le vaccin est obtenu en utilisant des particules lentivirales comme revêtement pour envelopper l'ARNm d'une protéine spike du SARS-CoV-2, le revêtement (particules lentivirales) étant modifié avec un antigène de protéine spike du SARS-CoV-2.
PCT/CN2021/112661 2020-09-21 2021-08-16 Vaccin contre le sars-cov-2 à base de modification de revêtement lentiviral et de distribution d'arnm et procédé de préparation s'y rapportant WO2022057540A1 (fr)

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CN202010995678.3 2020-09-21
CN202010995678.3A CN112168958B (zh) 2020-09-21 2020-09-21 基于慢病毒外壳修饰和mRNA递送的SARS-CoV-2疫苗及其制备方法

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WO2022057540A1 true WO2022057540A1 (fr) 2022-03-24

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