WO2023050484A1 - 一种表达载体、重组腺相关病毒及其在制备2019新型冠状病毒疫苗中的应用 - Google Patents

一种表达载体、重组腺相关病毒及其在制备2019新型冠状病毒疫苗中的应用 Download PDF

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WO2023050484A1
WO2023050484A1 PCT/CN2021/123797 CN2021123797W WO2023050484A1 WO 2023050484 A1 WO2023050484 A1 WO 2023050484A1 CN 2021123797 W CN2021123797 W CN 2021123797W WO 2023050484 A1 WO2023050484 A1 WO 2023050484A1
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coronavirus
associated virus
rbd
expression vector
nucleotide sequence
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French (fr)
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潘杏
何晓斌
方文晶
贾宁
余可
黄黎
张钰
杜亮
王梦蝶
刘宏均
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武汉枢密脑科学技术有限公司
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Definitions

  • the invention belongs to the technical field of bioengineering, and more specifically relates to an expression vector, a recombinant adeno-associated virus and their application in preparing a 2019 novel coronavirus vaccine.
  • Coronavirus is a class of enveloped single-stranded positive-strand RNA viruses, and it is the virus with the largest genome among known RNA viruses.
  • a total of 7 kinds of coronaviruses that can infect humans have been found, namely human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), Hong Kong type I human coronavirus (HCoV-HKU1), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and 2019 novel coronavirus (SARS-CoV-2).
  • SARS-CoV, MERS-CoV and SARS-CoV-2 are highly pathogenic human coronaviruses discovered so far, which have brought great harm to human health.
  • the 2019 Novel Coronavirus is an RNA virus that broke out rapidly around the world at the end of December 2019.
  • SARS-CoV-2 The 2019 Novel Coronavirus
  • the virus is highly contagious and highly concealed. People infected with the virus will have symptoms of varying degrees, including mild fever or mild cough, or severe pneumonia, and more severe cases may even cause death.
  • the prevention and control of the new crown epidemic in the world mainly lies in the control of the spread to uninfected people through measures such as isolation and disinfection, and at the same time, preventive vaccination against the new coronavirus.
  • the new coronavirus preventive vaccines currently approved for emergency use around the world include: (1) New coronavirus inactivated vaccine (Vero cells) (manufacturers: Sinopharm Beijing Bio, Sinopharm Wuhan Bio, Beijing Kexing Bio, Kangtai Bio); (2) ) Adenoviral vector new crown vaccine (manufacturer: CanSino Ad5, Johnson & Johnson Ad26, AstraZeneca Ad26, Gamaleya Ad5+Ad26); (3) mRNA new crown vaccine (manufacturer: Pfizer, Moderna); (4) recombinant protein new crown vaccine (manufacturer : Zhifeilong Koma, Novavax).
  • the inactivated vaccine uses the directly inactivated new coronavirus as an antigen to immunize the human body, thereby producing antibodies. It is the most widely used vaccine in my country. Kexing and Sinopharm vaccines are all inactivated vaccines. This type of vaccine technology is very mature and developed earlier. Its advantages lie in: extremely high safety, low risk of adverse reactions, and convenient storage conditions; disadvantages: low efficiency, long production cycle, and sometimes cause antibody dependence Antibody dependent enhancement (ADE), which aggravates viral infection.
  • ADE antibody dependence Antibody dependent enhancement
  • Adenovirus vector vaccines Currently, there are AstraZeneca vaccines, Russian vaccines, Johnson & Johnson vaccines, and CanSino vaccines as adenovirus vector vaccines.
  • the advantages of the vaccine are: fast production speed, suitable for sudden major epidemics, and high efficiency; the disadvantages are: poor safety and high adverse reaction rate.
  • This adverse reaction is mainly derived from the strong immune response of the human body to adenovirus, and there have been several deaths caused by this adverse reaction.
  • mRNA vaccines Both Pfizer and Moderna vaccines are mRNA vaccines, and they are also the mainstream vaccines currently used in the United States.
  • the mechanism is to modify the encoding mRNA of the antigenic determinant so that it does not have a strong ability to activate inflammation. After being absorbed by the cells, the antigenic determinants are expressed in the host cells, so that the immune system recognizes the antigens expressed by the host cells and produces cells. Antibodies that kill the virus.
  • the RNA vaccine carrier itself has certain immunogenicity. When exogenous nucleic acid infects cells, it will cause inflammation and activation of the latter, thereby degrading a large amount of RNA.
  • the advantages of this vaccine are: high effective rate, long duration of immunity, and fast production speed; the disadvantages are: strict storage conditions, high technical threshold, most countries cannot produce on a large scale, and the safety is lower than that of inactivated vaccines.
  • Recombinant protein new crown vaccine Zhifeilong Koma and Novavax vaccines are this type of vaccine.
  • Recombinant protein vaccines require the use of adjuvants to activate the immune response of the human body, and the choice of adjuvants has a great impact on the side effects of vaccines.
  • due to protein stability problems organisms cannot continuously produce antibodies for a long time.
  • the advantages are: the safety is second only to inactivated vaccines, the production speed is fast, and the efficiency is high; the disadvantages are: multiple immunizations are required, for example, Zhifeilong Koma's recombinant vaccine requires three injections.
  • Adeno-associated virus (adeno-associated virus, AAV) is a small, replication-deficient, non-enveloped virus that belongs to the family Parvoviridae. Due to the good safety of AAV (so far, wild-type AAV has never been found to be pathogenic to humans) ), low immunogenicity, ability to infect dividing cells and non-dividing cells, and recombinant AAV does not integrate into the host cell genome. In recent years, the use of AAV as a gene therapy vector has become a hot spot in gene therapy research. At present, clinical drugs for adeno-associated virus have been approved for marketing in the United States and Europe, and a large number of clinical cases have assisted in confirming its safety.
  • the object of the present invention is to provide an expression vector, a recombinant adeno-associated virus and its application in the preparation of 2019 novel coronavirus vaccine, by inserting TPA secretion signal peptide coding into the expression vector of double-stranded AAV
  • the sequence-linked RBD gene is used to realize the efficient, stable and long-term secretory expression of the new coronavirus RBD protein in the animal body.
  • the present invention provides an expression vector expressing the receptor binding domain of the coronavirus spike protein, which includes the expression cassette of the gene of interest and the AAV inverted terminal repeat sequence located at both ends of the coding region, the object
  • the gene expression cassette comprises an operably linked promoter from 5' to 3', a nucleotide sequence encoding a TPA secretion signal peptide and a nucleotide sequence encoding a coronavirus RBD, and the expression vector is used to prepare a recombinant double-stranded Adeno-associated virus.
  • the promoter is a CBh promoter, a CMV promoter, a CAG promoter or a CBA promoter.
  • the promoter is a CBh promoter.
  • the nucleotide sequence encoding the coronavirus RBD is optimized by human codons.
  • the coronavirus is the 2019 novel coronavirus, and the nucleotide sequence encoding the coronavirus RBD is shown in SEQ ID No.2.
  • the 3' end of the nucleotide sequence encoding the coronavirus RBD is operably linked to a nucleotide sequence encoding a targeting peptide, and the targeting peptide is an MHC2 receptor binding domain.
  • a recombinant adeno-associated virus expressing the receptor-binding domain of the coronavirus spike protein which is prepared by transfecting host cells with the above-mentioned expression vector.
  • the recombinant adeno-associated virus is double-stranded AAV.
  • the capsid protein of the recombinant adeno-associated virus is AAV2, AAV5, AAV6, AAV7, AAV8 or AAV9 serotype capsid protein.
  • the capsid protein of the recombinant adeno-associated virus is AAV6 or AAV9 serotype capsid protein.
  • a method for preparing recombinant adeno-associated virus includes the following steps: co-incubating the above-mentioned expression vector, helper plasmid pHelper and serotype plasmid pRepCap, in the presence of transfection reagent polyethyleneimine
  • the host cells are transfected under certain conditions, and after the cells are cultivated, the cells are collected by centrifugation, lysed and purified to obtain a purified liquid containing recombinant adeno-associated virus.
  • the rep gene in the serotype plasmid pRepCap is derived from AAV serotype 2, and the cap gene is derived from AAV serotype 6.
  • the coronavirus is the original strain of the 2019 novel coronavirus or a variant thereof.
  • the coronavirus is the 2019 novel coronavirus Delta variant or the 2019 novel coronavirus D614G variant.
  • the vaccine is prepared as an injection for intramuscular injection or nasal drip.
  • the vaccine further comprises pharmaceutically acceptable diluents and/or excipients.
  • the present invention induces the production of antibodies by expressing coronavirus RBD antigen molecules. Compared with the full-length S protein, the antibody titer is higher; on the RBD molecule, the extracellular expression of antigen fragments is improved by adding the secretory signal peptide TPA; at the same time, The selection of double-chain AAV (scAAV) greatly improves the expression efficiency of antigen molecules.
  • scAAV double-chain AAV
  • the expression vector of the present invention adopts the CBh promoter, which has better long-term expression effect and stronger stability; under the same expression intensity, selecting the CBh promoter can greatly reduce the injection dosage of the AAV vaccine, In order to reduce the adverse reactions of the body caused by high-dose AAV injection, it also reduces the cost.
  • the present invention designs three different targeting peptides on the antigen molecule expression carrier, screens out the MHC2 receptor binding domain targeting immune cells through experiments, and utilizes the expression carrier
  • the produced rAAV can achieve one-shot immunization and stably maintain a high level of neutralizing antibodies and total antibodies in the body. Compared with the existing vaccines that have been marketed, its persistence and stability are better.
  • AAV9 not only highly expresses antigen molecules in target tissues and muscles, but also infects other tissues and organs in the body such as the liver and nervous system. , which may cause potential vaccine safety issues; and AAV6 is recognized to target fewer tissues and has better muscle tropism.
  • the present invention designs an AAV6 expressing the coronavirus RBD antigen molecule, and selecting a better CBh promoter can realize that the expression level of the AAV6 antigen molecule is equivalent to that of AAV9.
  • the rAAV vaccine of the present invention is not only highly effective in immunity, but also maintains a high level of neutralizing antibodies for more than one year after one dose of immunization, especially for the more infectious and higher viral load of the new crown Delta mutation
  • the virus strain has stronger protective efficacy and stability, and the immune effect is obviously better than that of existing vaccines.
  • Fig. 1 is a fluorescence electron microscope image of cells infected with single-chain AAV (ssAAV) and double-chain AAV (scAAV) vectors for 48 hours and 72 hours in Example 1 of the present invention.
  • ssAAV single-chain AAV
  • scAAV double-chain AAV
  • Fig. 2 is a graph showing the detection of expression levels of mCherry expressed by different vectors after 72 hours of infection of cells in Example 1 of the present invention.
  • Fig. 3 is a schematic diagram of the construction of vector GT-0172 in Example 2 of the present invention.
  • Fig. 4 is a detection graph of total antibodies induced by mice infected with rAAV prepared by vector 0168 and rAAV prepared by vector 0173 respectively in Example 4 of the present invention.
  • Fig. 5 is a detection chart of neutralizing antibodies induced by mice infected with rAAV prepared by vector 0168 and rAAV prepared by vector 0173 in Example 4 of the present invention.
  • Figure 6 is the total antibody detection graph (A) and neutralizing antibody detection graph (B) induced by different doses of rAAV prepared by vector 0172 and rAAV prepared by vector 0173 in Example 5 of the present invention, respectively.
  • Fig. 7 is the detection diagram of total antibodies induced by serum type 6 rAAV prepared by vector 0172, serum type 9 rAAV and vector 0173 prepared by serum type 6 rAAV and serum type 9 rAAV respectively infecting mice in Example 5 of the present invention
  • A Detection chart of neutralizing antibody
  • B Detection chart of neutralizing antibody
  • Fig. 8 is a total antibody detection chart (A) and a neutralizing antibody detection chart (B) induced by rAAV prepared by vectors 0173, 0174, 0175, and 0176 in Example 6 of the present invention respectively infecting mice.
  • Figure 9 is a graph showing the protective efficacy of neutralizing antibodies induced by rAAV prepared by vectors 0173 and 0176 infecting mice for 90 days in Example 7 of the present invention against the original strain of the new coronavirus, the Delta variant strain, and the D614G variant strain.
  • Figure 10 is a graph showing the protective efficacy of neutralizing antibodies induced by rAAV prepared by vectors 0173 and 0176 in Example 7 of the present invention to mice infected with mice for 360 days, against the original strain of the new coronavirus, the Delta variant strain, and the D614G variant strain.
  • operably linked refers to the linkage of polynucleotide (or polypeptide) sequences in a functional relationship. Two nucleotide sequences are “operably linked” when they are placed into a functional relationship.
  • a transcriptional regulatory sequence eg, a promoter
  • a gene coding sequence if it affects the transcription of the gene coding sequence.
  • expression cassette refers to a nucleic acid construct comprising operably linked coding and regulatory sequences that when introduced into a host cell result in the transcription and/or translation of RNA or polypeptide, respectively.
  • An expression cassette is understood to include a promoter allowing the initiation of transcription, the open reading frame of the gene of interest and a transcription terminator. Typically, the promoter sequence is placed upstream of the gene of interest at a distance compatible with expression control.
  • Promoter is a DNA sequence that RNA polymerase recognizes, binds and initiates transcription. It contains the conserved sequence required for RNA polymerase specific binding and transcription initiation. Most of them are located upstream of the transcription initiation point of structural genes. The promoter itself is not transcription.
  • vector refers to a nucleic acid molecule designed to transport, transfer and/or store genetic material, as well as express genetic material and/or integrate genetic material into the chromosomal DNA of a host cell, such as plasmid vectors, cosmid vectors, artificial chromosomes , phage vectors and other viral vectors.
  • a vector usually consists of at least three basic units, namely a replication source, a selectable marker and a multiple cloning site.
  • recombinant adeno-associated virus vector refers to a recombinant non-replicating adeno-associated virus.
  • the recombinant adeno-associated virus (rAAV) vector includes a serotype protein capsid and wraps a recombinant genome, which includes functional 5' and 3' 'Inverted terminal repeats (inverted terminal repeats, ITR), the exogenous gene expression cassette is connected between the ITRs at both ends to replace the rep gene expression cassette and/or cap gene expression cassette of wild-type AAV. ITR sequences provide functional rescue, replication, and packaging of rAAV.
  • the ITR sequence is from AAV2.
  • the exogenous gene expression cassette usually consists of a series of expression control elements and coding regions.
  • AAV serotype plasmid pRepCap includes AAV rep gene expression cassette and cap gene expression cassette, which are used to express AAV Rep replication protein and VP capsid protein respectively.
  • the VP protein includes three subunits of VP1, VP2 and VP3, and AAV of different serotypes have different capsid protein coding sequences. Different AAV serotypes have certain differences in infection efficiency and tissue specificity.
  • the rep gene is derived from AAV serotype 2.
  • the AAV helper plasmid pHelper usually includes adenovirus VA, E4, E2A and other coding regions to provide the necessary functions for AAV replication to assist in the production of infectious AAV virus particles.
  • coronavirus there are 4-5 kinds of structural proteins encoded by the genome, which are spike protein (Spike protein, S protein), nucleocapsid protein (Nucleocapsid protein, N protein), membrane protein (Membrane protein, M protein). protein) and envelope protein (Envelope protein, E protein), a few species also have hemagglutinin glycoprotein (Haemaglutinin-esterase, HE protein).
  • spike protein spike protein
  • nucleocapsid protein Nucleocapsid protein, N protein
  • membrane protein Membrane protein, M protein
  • protein envelope protein
  • E protein envelope protein
  • the S protein has two subunits: S1 and S2, and the receptor binding domain (RBD) is located on the S1 subunit.
  • the S protein forms the spikes on the surface of the outer membrane of the virus particle in the form of a trimer, and its main function is to recognize the surface receptors of the host cell and mediate the fusion of the viral envelope with the cell membrane of the host cell.
  • Current studies have shown that both SARS-CoV and 2019-nCoV bind to human cell angiotensin-converting enzyme 2 (ACE2) receptors through the receptor-binding domain (RBD) on the spike protein, thereby triggering viral response to Invasion of human cells.
  • ACE2 angiotensin-converting enzyme 2
  • the 2019 novel coronavirus is currently mutating rapidly, and the main variants discovered include Alpha variant, Beta variant, Gamma variant, Delta variant and Lambda variant.
  • the mutation of the Alpha variant makes it easier to bind to target cell surface receptors, thereby greatly enhancing the infectivity; the outstanding feature of the Beta and Gamma strains is the ability to escape from the immune system; the Delta and Lambda strains have both high infectivity and immunity The ability to escape, and the short incubation period and high viral load indicate that its replication speed is greatly increased.
  • the Delta mutant strain is currently the most important epidemic strain in the world and in China.
  • L452R and E484Q mutations occur in the region where the spike protein binds to human cell angiotensin-converting enzyme 2 (ACE2) receptors, that is, the RBD region.
  • L452R improves the ability of the virus to invade cells, and E484Q helps to enhance the immune escape of the virus;
  • the present invention provides an expression vector expressing the receptor binding domain of the coronavirus spike protein, which comprises a target gene expression cassette and an adeno-associated virus inverted terminal repeat sequence located at both ends of the target gene expression cassette, the target gene
  • the expression cassette comprises an operably linked promoter from 5' to 3', a nucleotide sequence encoding a TPA secretion signal peptide and a nucleotide sequence encoding a coronavirus RBD, and the expression vector is used to prepare a recombinant double-stranded adenocarcinoma related virus.
  • TPA tissue-type plamnipen activator
  • TPA signal peptide can effectively promote the secretion of foreign proteins and improve their ability to induce antibody production.
  • the nucleotide sequence encoding the TPA secretion signal peptide is shown in SEQ ID No.4.
  • the promoters for promoting the expression of RBD antigen molecules can adopt various promoters that can normally promote expression in AAV expression vectors, including but not limited to CBh promoters, CAG promoters, CMV promoters, CBA promoters, etc. , preferably a CBh promoter, the nucleotide sequence of the CBh promoter is shown in SEQ ID No.6. Compared with the CAG promoter (1721bp), the fragment of the CBh promoter (794bp) is smaller.
  • the CBh promoter Compared with the CMV promoter, the CBh promoter has better long-term expression effect and higher stability; using the CBh promoter can greatly reduce The injection dose of AAV-RBD vaccine reduces the adverse reaction of the body caused by high-dose AAV injection, and also reduces the cost.
  • the nucleotide sequence encoding the coronavirus RBD is optimized by human codons, so that the RBD coding sequence matches the codon usage frequency of human cells, thereby increasing the protein expression level.
  • a human codon-optimized RBD coding sequence is designed for the 2019 novel coronavirus, and its nucleotide sequence is shown in SEQ ID No.2.
  • the C-terminus of the amino acid sequence of the RBD antigen polypeptide can be extended, so that the antigen molecule is richer in the cysteine of the original sequence, which is beneficial to the polymerization of the immune antigen protein, improves stability, and reduces Its degradation speed can induce the organism to produce sufficient immune response stably for a long time.
  • extended sequence please refer to Chinese patent documents CN111518175A and CN111996216A.
  • the 3' end of the nucleotide sequence encoding the coronavirus RBD is operably linked to a nucleotide sequence encoding a targeting peptide, and the targeting peptide is an MHC2 receptor binding domain to increase antigenicity.
  • Molecules target the targeting of immune cells, and at the same time, vaccine molecules expressing targeting peptides can stably maintain high levels of neutralizing antibodies and total antibodies in the body.
  • the nucleotide sequence encoding the MHC2 receptor binding domain is shown in SEQ ID No.5.
  • the present invention also provides a recombinant adeno-associated virus expressing the receptor binding domain of the coronavirus spike protein, which is prepared by transfecting host cells with the above expression vector.
  • the recombinant adeno-associated virus can be of various serotypes, correspondingly having different capsid proteins, and the recombinant adeno-associated virus of the present invention includes but not limited to AAV serotype 6 or 9.
  • the present invention also provides a preparation method of recombinant adeno-associated virus, comprising the following steps: co-incubating the above-mentioned expression vector, helper plasmid pHelper and serotype plasmid pRepCap, and transfecting host cells in the presence of transfection reagent polyethyleneimine After the cells are cultivated, the cells are collected by centrifugation, lysed and purified to obtain a purified solution containing recombinant adeno-associated virus.
  • the serotype plasmid pRepCap is pRep2Cap6, wherein the nucleotide sequence of the cap gene is shown in SEQ ID No.7.
  • HEK293T-based AAV helper-free system (AAVHelper-FreeSystem) is used for production, that is, the three-plasmid co-transfection method.
  • the AAV helper-free system contains expression vectors containing AAVITRs and rep/cap genes
  • the plasmid pRepCap and the helper plasmid pHelper are three plasmids.
  • the helper plasmid pHelper provides the adenoviral gene products (such as E2A, E4 and VARNA genes) required for the production of infectious AAV virus particles, and the remaining adenoviral gene products are produced by stably expressed adenoviral genes.
  • AAV-293T host cells of the viral E1 gene were provided. With the help of the helper plasmid, only the ITRs at both ends can package the carried target gene fragment into the adeno-associated virus particle.
  • There is no homologous sequence between the viral vector, pRepCap plasmid and helper plasmid so the recombinant AAV has no replication ability in theory.
  • the ITRs sequence and rep/cap gene of adeno-associated virus are expressed by independent plasmids, which have high safety.
  • Those skilled in the art can also construct expression plasmid and serotype plasmid production based on the insect system of SF9 cell line, such as Chinese patent application CN108699567A; or use a recombinant bacmid capable of more stable expression, which contains the rep gene of AAV , cap gene and core expression element ITR-GOI with exogenous target gene, such as Chinese patent application CN112553257A.
  • virus packaging systems based on herpes virus-assisted or adenovirus-assisted.
  • the recombinant adeno-associated virus prepared by the invention can be used to prepare a vaccine for preventing coronavirus.
  • the vaccine contains the recombinant adeno-associated virus, and the serum can still maintain high levels of total antibodies and neutralizing antibodies 360 days after the vaccine is injected.
  • the recombinant AAV is suitable for vaccine preparation of various coronaviruses such as SARS-CoV, MERS-CoV or SARS-CoV-2.
  • the coronavirus is the original strain of the 2019 novel coronavirus or its mutant strain, and the antibodies induced by the vaccine of the present invention have excellent immune effects on the original strain and the mutant strain of the 2019 novel coronavirus, especially against the more infectious Delta Mutant strain, the effect is obviously better than other existing vaccines.
  • the vaccine is made into an injection for intramuscular injection or nasal drip.
  • the vaccine further comprises pharmaceutically acceptable diluents and/or excipients.
  • Single-stranded AAV is used in the prior art to express the new crown vaccine molecule. This example verifies the difference in the expression levels of single- and double-stranded viruses in cells.
  • rAAV-CMV-mCHERRY-BGHpA ID: PB2-0948
  • pFD-scAAV-CMV-mCHERRY-BGHpA ID: GT-0118
  • Embodiment 2 Preparation of different antigen molecule carriers
  • the full-length S protein coding sequence of SARS-CoV-2 (2019-nCoV-WIV04) used in this example is the nucleotide sequence shown in SEQ ID No.1 commissioned by GenScript Company to synthesize, the nucleotide The sequence was optimized with human codons and named 2019-HnCOV-S. Select the main antigen recognition epitope RBD region of the S protein, as shown in SEQ ID No.2, and name it RBD; the amino acid sequence corresponding to this sequence is shown in SEQ ID No.3. On the basis of this sequence, the following signal peptide TPA and three targeting peptide sequences were designed, as shown in Table 1.
  • TPA is a secretion signal peptide, added to the N-terminal of the RBD sequence when the vector is constructed, and targeting peptides are respectively added to the C-terminal of the RBD sequence, and the targeting peptides used in the present invention are respectively the G protein receptor binding domain RVG of rabies virus Fragment, glycosylation site 2NNPLPQR peptide segment and MHC2 receptor binding domain targeting immune cells, wherein the nucleotide sequences encoding TPA secretion signal peptide and MHC2 receptor binding domain are respectively as SEQ ID No.4 and SEQ ID No.4 As shown in ID No.5, different plasmid vectors as shown in Table 2 were constructed.
  • Plasmid ID Plasmid name Length (bp) GT-0168 pFD-rAAV-ITR-CBh-2019-HnCOV-S-BGHpA 5184 GT-0172 pFD-scAAV-ITR-CMV+intron-TPA-RBD-BGHpA 2148 GT-0173 pFD-scAAV-ITR-CBh-TPA-RBD-BGHpA 2107 GT-0174 pFD-scAAV-ITR-CBh-TPA-RBD-RVG-BGHpA 2203 GT-0175 pFD-scAAV-ITR-CBh-TPA-RBD-2NNPLPQR-BGHpA 2161 GT-0176 pFD-scAAV-ITR-CBh-TPA-RBD-MHC2-BGHpA 2209
  • GT-0173 ⁇ GT-0176 and GT-0168 all use the CBh promoter, and their nucleotide sequences are shown in SEQ ID No.6; GT-0172 uses the CMV promoter.
  • GT-0168 is a single-chain AAV package, while the other molecules are all double-chain AAV packages.
  • Figure 3 taking GT-0172 as an example, the specific construction process of the carrier is as follows:
  • GenScript_MC_0101081_7695 human source optimized 2019-HnCoV-S as a template, design primers 0172-RBD-F and 0172-RBD-R, and clone the target fragment 0172-RBD with a size of 737bp.
  • the TPA fragment was synthesized and linked with 0172-RBD to obtain the 0172-TPA-RBD fragment with a size of 791bp.
  • PEI polyethyleneimine solution
  • the adeno-associated virus was further purified into finished products by iodixanol centrifugation-dialysis-ultrafiltration harvesting-sterile filtration, and the rAAV viruses numbered GT-0168, 0172, 0173, 0174, 0175 and 0176 in the above Example 2 were produced.
  • the physical titer of the virus was detected by SYBRGreen Q-PCR, and the purity was detected by SDS-PAGE silver staining.
  • the produced virus was subpackaged and stored in a -80°C refrigerator.
  • Orbital blood collection on day14, day30, day60, day90, day120, day240 of virus injection, negative control PBS group also requires blood collection. Transfer the collected serum to a clean EP tube and store in a -80°C refrigerator.
  • the antigen used for coating is the recombinant spike protein RBD-His, which is produced by our company by using the insect baculovirus system.
  • Dissolve RBD protein with 0.2M carbonate buffer (pH 9.6) prepare 1.0 ⁇ g/mL solution, add 100 ⁇ L per well to 96-well plate, and incubate overnight at 4°C; incubate at 37°C incubator the next day 1 h; wash the plate 3 times with PBST (PBS+0.1% Tween-20) (300 ⁇ L/well/time). After washing the plate, add 5% skimmed milk powder; add 300 ⁇ L to each well; incubate at 37°C for 1 h, and wash the plate 3 times with PBST.
  • PBST PBS+0.1% Tween-20
  • mice serum samples of the same group (GT-0168 virus injection group or GT-0173 virus injection group or PBS group) at each time point were serially diluted with PBS; Make three duplicate wells in parallel with each dilution gradient; place in a 37°C incubator and incubate for 2 hours, and wash the plate 3 times.
  • TMB solution 200 ⁇ L per well, and develop color at room temperature for 30 min in the dark.
  • Add 0.5M H 2 SO 4 solution 100 ⁇ L per well.
  • the OD average value + 3 times SD value of the PBS group test results is defined as the cut-off value, when the OD value of a certain dilution of the blood sample to be tested is greater than the cut-off value of the same dilution PBS group, and the next dilution If the OD value is less than or equal to the cut-off value of the PBS group with the same dilution, the dilution is defined as the IgG total antibody titer of the blood sample.
  • the result is a line graph with the antibody titer value as the ordinate and different time points as the abscissa.
  • the solid line represents GT-0173, the full-length S protein, and the dotted line represents GT-0168, the RBD protein, as shown in FIG. 4 .
  • VSV- ⁇ G-mCherry-SARS-CoV-2-S (abbreviated as VSV- ⁇ GS) pseudovirus is a vesicular stomatitis virus whose outer envelope is the new coronavirus S, which is developed and produced by the company, and expresses red fluorescent mCherry.
  • the TCID50 method to measure the infectious titer of the pseudovirus.
  • the blood sample to be tested was inactivated at 56°C for 30 minutes.
  • the inactivated mouse immune serum was serially diluted, the pseudovirus was diluted to 200 TCID50/well, and co-incubated with the blood sample to be tested for 1 hour at 37°C in a 5% CO 2 incubator.
  • 293T-ACE2-EGFP cells in culture were then added to the wells. No blood sample to be tested was added to the positive control well.
  • the back-titer plate add 8 replicate wells for each dilution virus of 1000 TCID50, 100 TCID50, 10 TCID50, 1 TCID50, and 0.1 TCID50 for detection, and calibrate the titer of the pseudovirus used.
  • the cells added with virus of 0.1TCID50 are negative quality control wells.
  • the number of virus-infected red fluorescent cells was recorded on day 3 after infection. On the third day, lesions appeared in the positive quality control wells, but no lesions appeared in the negative quality control wells, and the applicability condition of the experiment was established. The number of red fluorescent cells in each experimental group was observed and calculated, and the EC50 value was calculated by the Reed-Muench method as the neutralization titer of the serum.
  • the result is a line graph with the pseudovirus neutralizing antibody potency value EC50 as the ordinate and different time points as the abscissa.
  • the solid line represents GT-0173, the full-length S protein, and the dotted line represents GT-0168, the RBD protein, as shown in Figure 5 .
  • the neutralizing antibody titers of the full-length S protein were lower than those of the RBD protein, ranging from 2.81 to 7.99 times. It further shows that the molecular construction of the RBD antigen is better than that of the full-length S antigen.
  • the RBD molecules screened above were further evaluated for the expression intensity and persistence of different promoters CMV and CBh, and the efficiency of different serotypes AAV6 and AAV9 infecting muscle cells.
  • Virus injection Eight BALB/c mice aged 6-8 weeks were randomly selected in each group for intramuscular injection of GT-0172 or GT-0173, different serotypes (AAV6, AAV9) and different doses of virus, and the virus was diluted to 50 ⁇ L for injection. See Table 4 for virus injection grouping information. Among them, 5E+10vg/only is the low-dose group, 1E+11vg/only is the middle-dose group, and 2E+11vg/only is the high-dose group. In addition, 8 BALB/c mice aged 6-8 weeks were selected for intramuscular injection of PBS (50 ⁇ L) as a negative control.
  • PBS 50 ⁇ L
  • Orbital blood collection was performed on days 14, day 30, day 60, day 90, day 120, day 150, day 210, day 240, day 300, and day 360 of virus injection, and the negative control PBS group also required blood collection. Transfer the collected serum to a clean EP tube and store in a -80°C refrigerator.
  • the samples are retro-orbital venous plexus serum collected on day14, day30, day60, day90, day120, day150, day210, day240, day300, day360, and the detection method refers to the detection operation steps of Example 4.
  • the results of the promoter comparison are shown in Figure 6.
  • the solid circle line and dotted line represent the low and middle doses of GT-0173, respectively, and the solid and dotted triangle lines represent the high and middle doses of GT-0172, respectively.
  • the total antibody titers of the low-dose group of GT-0173 molecules at different time points are basically 1.22-1.93 times higher than those of the high-dose group of GT-0172 molecules, and the titers of neutralizing antibodies are basically 1.18-7.59 times higher.
  • the CBh promoter is far superior to the CMV promoter in the sustained and strong expression of antigen molecules, and the expression intensity of the low-dose group is higher than that of the CMV high-dose group. Selecting the CBh promoter can greatly reduce AAV- Injection dose of RBD vaccine, cost reduction and adverse reactions caused by high-dose AAV injection.
  • the comparison results of the two different serotypes are shown in Figure 7.
  • the solid circles and hollow circles represent the AAV6 and AAV9 types of the GT-0173 molecule, respectively; the solid triangles and hollow triangles represent the AAV6 and AAV9 types of the GT-0172 molecule, respectively.
  • the antibody titer of AAV9-GT-0172 molecule is significantly higher than that of AAV6, but on GT-0173 molecule, the total antibody level of AAV9 is only slightly higher than that of AAV6, and the neutralizing antibody level of AAV6 is reversed after 120 days Will be slightly higher than AAV9.
  • the antibody titer of AAV6-GT-0173 is higher than that of AAV9-GT-0172, further indicating that a well-selected promoter can make up for and span the expression difference caused by AAV serotype infection.
  • the RBD molecule was further modified, that is, the GT-0173 molecule was used as the basis for transformation. Including the nucleotide coding sequence of the MHC2 receptor binding domain targeting immune cells, the nucleotide coding sequence of the RVG fragment of the G protein receptor binding domain of rabies virus, and the nucleosides of the glycosylation site 2NNPLPQR peptide acid coding sequence.
  • Virus injection Eight BALB/c mice aged 6-8 weeks were randomly selected in each group for intramuscular injection of GT-0173 ⁇ GT-0176, the serotype was AAV6, the injection dose was 1E+11vg/mouse, and the virus was diluted to 50 ⁇ L for injection. See Table 6 for virus injection grouping information.
  • Orbital blood collection was performed on days 14, day 30, day 60, day 90, day 120, day 150, day 210, day 240, day 300, and day 360 of virus injection, and the negative control PBS group also required blood collection. Transfer the collected serum to a clean EP tube and store in a -80°C refrigerator.
  • the samples are retro-orbital venous plexus serum collected on day14, day30, day60, day90, day120, day150, day210, day240, day300, day360, and the detection method refers to the detection operation steps of Example 4.
  • the solid circle line represents GT-0176
  • the solid triangle line represents the control molecule GT-0173
  • the solid square line represents GT-0174
  • the dotted line represents GT-0175.
  • scAAV6-TPA-RBD and scAAV6-TPA-RBD-MHC2 vaccine molecules can achieve one-shot immunization, and stably maintain high levels of neutralizing antibodies and total antibodies in the body, compared with existing The marketed vaccines have better persistence and stability.
  • the protective power of the vaccines that have been on the market to the Delta strain that is currently more contagious and more virulent is 2.5-7.4 times lower than that of the B1 strain on the 28th day to 90 days, while the vaccine of the present invention The vaccine only decreased by 1.68-2.09 times at 90 days. It can be seen that although the new crown vaccine of the present invention is aimed at the original strain, it has stronger protective effect on the mutant strain, better stability, and is better than the existing vaccine. .

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Abstract

一种表达载体、重组腺相关病毒及其在制备2019新型冠状病毒疫苗中的应用，该表达载体包括目的基因表达盒和位于所述目的基因表达盒两端的腺相关病毒反向末端重复序列，所述目的基因表达盒包含从5'至3'的、可操作连接的启动子、编码TPA分泌信号肽的核苷酸序列及编码冠状病毒刺突蛋白受体结合结构域（RBD）的核苷酸序列，所述表达载体用于制备重组双链腺相关病毒。利用所述表达载体制备的重组腺相关病毒能够在体内高效、稳定、长期地分泌表达冠状病毒RBD蛋白，诱导产生血清中和抗体，对包括变异株在内的2019新型冠状病毒具有中和效果，并持续表达，具有很好的应用前景。

Description

一种表达载体、重组腺相关病毒及其在制备2019新型冠状病毒疫苗中的应用 【技术领域】
本发明属于生物工程技术领域，更具体地，涉及一种表达载体、重组腺相关病毒及其在制备2019新型冠状病毒疫苗中的应用。
【背景技术】
冠状病毒(Coronavirus)是一类有包膜的单股正链RNA病毒，是目前已知RNA病毒中基因组最大的病毒。目前共发现7种可以感染人的冠状病毒，分别是人冠状病毒229E(HCoV-229E)、人冠状病毒OC43(HCoV-OC43)、人冠状病毒NL63(HCoV-NL63)、香港I型人冠状病毒(HCoV-HKU1)、严重急性呼吸道综合征冠状病毒(SARS-CoV)、中东呼吸综合征冠状病毒(MERS-CoV)及2019新型冠状病毒(SARS-CoV-2)。其中，SARS-CoV、MERS-CoV和SARS-CoV-2是目前发现的人高致病性冠状病毒，给人类健康带来了极大危害。
2019新型冠状病毒(SARS-CoV-2)为2019年12月底在全球范围内迅速爆发的RNA病毒，截止2021年9月2日，全球新型冠状病毒感染累计病例2.16亿人，累计死亡病例450万人。该病毒具有高传染性和高隐蔽性，感染病毒的人会出现不同程度的症状，包括轻症样的发烧或轻微咳嗽，或是重症样的肺炎，而更为严重的甚至造成患者死亡。目前全世界对于新冠疫情的防控主要在于通过隔离消毒等措施，控制向未感染人群的传播，同时接种新型冠状病毒预防性疫苗。目前全球获批紧急使用的新型冠状病毒预防性疫苗包括：(1)新型冠状病毒灭活疫苗(Vero细胞)(厂家：国药北京生物、国药武汉生物、北京科兴生物、康泰生物)；(2)腺病毒载体新冠疫苗(厂家：康希诺Ad5、强生Ad26、阿斯利康Ad26、Gamaleya Ad5+Ad26)；(3)mRNA新冠疫苗(厂家：辉瑞、Moderna)；(4)重组蛋白新冠疫苗(厂家：智飞龙科马、Novavax)。
目前获批紧急使用的新型冠状病毒疫苗各有其优劣。
灭活疫苗是使用直接灭活的新型冠状病毒作为抗原对人体进行免疫，从而产生抗体，是我国使用最广泛的疫苗，科兴、国药疫苗均为灭活疫苗。这类疫苗技术十分成熟，发展较早，其优势在于：极高的安全性，很低的不良反应几率，方便的储存条件；劣势为：有效率较低，生产周期长，有时会引起抗体依赖性增强效应(antibody dependent enhancement，ADE)，加重病毒感染。
腺病毒载体疫苗：目前有阿斯利康疫苗、俄罗斯疫苗、强生疫苗、康希诺疫苗为腺病毒载体疫苗。该疫苗优势在于：生产速度快，适用于突发重大流行病，有效率高；劣势在于：安全性差，不良反应率极高。这种不良反应主要是源自人体对腺病毒的强烈免疫反应，目前已出现几例由该不良反应引发的死亡案例。
mRNA疫苗：辉瑞、Moderna疫苗均为mRNA疫苗，也是目前美国所采用的主流疫苗。其机理为通过修饰抗原决定簇的编码mRNA使其不具备较强的炎症激活能力，在被细胞吸收后，在宿主细胞内表达抗原决定簇，使得免疫系统识别该宿主细胞表达的抗原后产生细胞免疫抗体，从而杀灭病毒。但RNA疫苗载体本身具有一定的免疫原性，外源性核酸感染细胞时会引起后者炎症活化，从而使得大量RNA被降解。该疫苗优势为：有效率高，免疫持续时间长，生产速度快；缺点为：储存条件严苛，技术门槛高，多数国家无法大规模生产，安全性低于灭活疫苗。
重组蛋白新冠疫苗：智飞龙科马和Novavax疫苗为此类型疫苗。重组蛋白疫苗需要辅助使用佐剂激活人体的免疫应答，而佐剂的选择对疫苗副作用的产生具有较大影响，同时由于蛋白质稳定性的问题，生物体无法长期持续性产生抗体。优势为：安全性仅次于灭活疫苗，生产速度较快，有效率高；缺点为：需要多次免疫，如智飞龙科马的重组疫苗需要免疫三针。
腺相关病毒(adeno-associated virus，AAV)是一类小的、复制缺陷的、无包膜的病毒，属于细小病毒科，由于AAV存在安全性好(迄今从未发现野生型AAV对人体致病)、免疫原性低、能感染分裂细胞和非分裂细胞、重组AAV不整合到宿主细胞基因组等优点，近年来，用AAV作基因治疗载体已成为基因治疗研究的热点。目前美国以及欧洲均已批准腺相关病毒的临床药物上市，有大量临床案例辅助证实了其安全性。
目前基于重组AAV的新冠疫苗相较于其他种类疫苗研究较少，缺乏AAV新冠疫苗的长效性研究，迫切需要开发一种针对预防新型冠状病毒SARS-CoV-2具有长效性、稳定性强的AAV载体疫苗。
【发明内容】
针对现有技术的缺陷，本发明的目的在于提供一种表达载体、重组腺相关病毒及其在制备2019新型冠状病毒疫苗中的应用，通过在双链AAV的表达载体中插入TPA分泌信号肽编码序列连接的RBD基因，以实现新型冠状病毒RBD蛋白在动物机体内的高效、稳定地、长期地的分泌表达。
为实现上述目的，本发明提供了一种表达冠状病毒刺突蛋白受体结合结构域的表达载 体，其包括目的基因表达盒和位于所述编码区两端的AAV反向末端重复序列，所述目的基因表达盒包含从5’至3’的、可操作连接的启动子、编码TPA分泌信号肽的核苷酸序列及编码冠状病毒RBD的核苷酸序列，所述表达载体用于制备重组双链腺相关病毒。
优选地，所述启动子为CBh启动子、CMV启动子、CAG启动子或CBA启动子。
优选地，所述启动子为CBh启动子。
优选地，所述编码冠状病毒RBD的核苷酸序列经过人源密码子优化。
优选地，所述冠状病毒为2019新型冠状病毒，所述编码冠状病毒RBD的核苷酸序列如SEQ ID No.2所示。
优选地，所述编码冠状病毒RBD的核苷酸序列的3’端可操作连接有编码靶向肽的核苷酸序列，所述靶向肽为MHC2受体结合结构域。
按照本发明的另一方面，提供了一种表达冠状病毒刺突蛋白受体结合结构域的重组腺相关病毒，其由上述表达载体参与转染宿主细胞而制得。
优选地，所述重组腺相关病毒为双链AAV。
优选地，所述重组腺相关病毒的衣壳蛋白为AAV2、AAV5、AAV6、AAV7、AAV8或AAV9血清型衣壳蛋白。
优选地，所述重组腺相关病毒的衣壳蛋白为AAV6或AAV9血清型衣壳蛋白。
按照本发明的另一方面，提供了一种重组腺相关病毒的制备方法，其包括如下步骤：将上述表达载体、辅助质粒pHelper和血清型质粒pRepCap共孵育，在转染试剂聚乙烯亚胺存在的条件下转染宿主细胞，培育细胞后，离心收集细胞，经过裂解和纯化，得到含有重组腺相关病毒的纯化液。
优选地，所述血清型质粒pRepCap中rep基因来源于AAV血清2型，cap基因来源于AAV血清6型。
按照本发明的另一方面，提供了上述重组腺相关病毒在制备预防冠状病毒的疫苗中的应用。
优选地，所述冠状病毒为2019新型冠状病毒原始株或其变异株。
优选地，所述冠状病毒为2019新型冠状病毒Delta变异株或2019新型冠状病毒D614G变异株。
优选地，所述疫苗制成注射剂，用于肌肉注射或鼻腔滴注。
优选地，所述疫苗还包含药学上可接受的稀释剂和/或赋形剂。
总体而言，通过本发明所构思的以上技术方案与现有技术相比，具有以下有益效果：
(1)本发明通过表达冠状病毒RBD抗原分子诱导产生抗体，相比全长S蛋白，抗体效价更高；在RBD分子上通过添加分泌信号肽TPA，提高抗原片段的胞外表达；同时，选择双链AAV(scAAV)，极大地提高了抗原分子的表达效率。
(2)相较于CMV启动子，本发明表达载体采用CBh启动子，长期表达效果更好，稳定性更强；在同样的表达强度下，选择CBh启动子可大大降低AAV疫苗的注射计量，以减小高剂量AAV注射后引起的机体不良反应，同时这样也降低了成本。
(3)本发明为了提高抗原分子的靶向性，在抗原分子表达载体上设计了三种不同靶向肽，经过实验筛选出了靶向免疫细胞的MHC2受体结合结构域，利用该表达载体生产的rAAV能够实现一针免疫，且在机体中稳定保持较高的中和抗体和总抗体水平，相较于现有的已上市的疫苗，其持续性、稳定性更好。
(4)目前已报道的AAV疫苗大多选择靶向肌肉较好的AAV血清9型，但有文献报告AAV9除了在靶组织肌肉中高表达抗原分子以外，还会感染肝脏、神经系统等全身其他组织器官，这可能会引起潜在的疫苗安全性问题；而AAV6公认靶向的组织会更少，且肌肉嗜性较好。本发明设计一种表达冠状病毒RBD抗原分子的AAV6，选择较优的CBh启动子能够实现AAV6抗原分子的表达水平与AAV9相当。
(5)本发明rAAV疫苗不仅免疫性高效，且在一针剂量的免疫后，较高的中和抗体水平能够维持一年以上，尤其针对传染性更强、病毒载量更高的新冠Delta变异毒株，具有更强的保护效力和稳定性，免疫效果明显优于现有疫苗。
【附图说明】
图1为本发明实施例1中单链AAV(ssAAV)和双链AAV(scAAV)载体感染细胞48h和72h后的荧光电镜图。
图2为本发明实施例1中感染细胞72h后不同载体表达mCherry的表达量检测图。
图3为本发明实施例2中载体GT-0172的构建示意图。
图4为本发明实施例4中载体0168制备的rAAV和载体0173制备的rAAV分别感染小鼠诱导产生的总抗体检测图。
图5为本发明实施例4中载体0168制备的rAAV和载体0173制备的rAAV分别感染小鼠诱导产生的中和抗体检测图。
图6为本发明实施例5中载体0172制备的rAAV和载体0173制备的rAAV以不同剂量分别感染小鼠诱导产生的总抗体检测图(A)和中和抗体检测图(B)。
图7为本发明实施例5中载体0172制备的血清6型rAAV、血清9型rAAV和载体 0173制备的血清6型rAAV、血清9型rAAV分别感染小鼠诱导产生的总抗体检测图(A)和中和抗体检测图(B)。
图8为本发明实施例6中载体0173、0174、0175、0176制备的rAAV分别感染小鼠诱导产生的总抗体检测图(A)和中和抗体检测图(B)。
图9为本发明实施例7中载体0173、0176制备的rAAV分别感染小鼠90天诱导产生的中和抗体对新冠病毒原始株、Delta变异株、D614G变异株的保护效力检测图。
图10为本发明实施例7中载体0173、0176制备的rAAV分别感染小鼠360天诱导产生的中和抗体对新冠病毒原始株、Delta变异株、D614G变异株的保护效力检测图。
【具体实施方式】
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
术语解释
正如本文所用的，术语“可操作连接”是指多核苷酸(或多肽)序列以功能性关系的连接。当两段核苷酸序列置于功能性关系时，这两段核苷酸序列是“可操作连接”的。例如，转录调控序列(例如启动子)如果影响某基因编码序列的转录，则其与该基因编码序列可操作连接。
术语“表达盒”是指包含引入宿主细胞时可操作连接的编码序列和调控序列的核酸构建体，分别导致RNA或多肽的转录和/或翻译。表达盒应理解为包括允许转录开始的启动子、目的基因开放阅读框和转录终止子。通常，启动子序列置于目的基因上游，与目的基因的距离与表达控制相容。启动子是RNA聚合酶识别、结合和开始转录的一段DNA序列，它含有RNA聚合酶特异性结合和转录起始所需的保守序列，多数位于结构基因转录起始点的上游，启动子本身不被转录。
术语“载体”是指设计用于转运、转移和/或存储遗传物质，以及表达遗传物质和/或将遗传物质整合到宿主细胞染色体DNA中的核酸分子，例如质粒载体、粘粒载体、人工染色体、噬菌体载体和其他病毒载体。载体通常由至少三个基本单元组成，即复制源、选择标记和多克隆位点。
术语“重组腺相关病毒载体”是指重组非复制型的腺相关病毒，重组腺相关病毒(rAAV)载体包括血清型蛋白衣壳，并包裹着重组的基因组，基因组包括功能性的5’和3’反向末端重复序列(inverted terminal repeats，ITR)，两端ITR之间连接外源基因表达盒替代野生 型AAV的rep基因表达盒和/或cap基因表达盒。ITR序列提供对rAAV功能性拯救、复制、包装。如果突变去除AAV两端ITR中的任一D序列，导致无法正确地剪切ssDNA，就会使得基因组二聚体显著增加，在复制时形成双链反向重复序列，最终包装进入AAV衣壳形成双链AAV(scAAV)。一些实施例中，ITR序列来自AAV2。外源基因表达盒通常由一系列表达调控元件和编码区组成。
AAV血清型质粒pRepCap包括AAV的rep基因表达盒和cap基因表达盒，分别用于表达AAV的Rep复制蛋白和VP衣壳蛋白。本领域技术人员已知的，VP蛋白包括VP1、VP2、VP3三种亚基，不同血清型的AAV具有不同的衣壳蛋白编码序列。不同的AAV血清型在感染效率和组织特异性方面存在一定的差异。一些实施例中，rep基因来源于AAV血清2型。
AAV辅助质粒pHelper通常包括腺病毒VA、E4、E2A等编码区域提供AAV复制必须的功能，以辅助生产出具有感染性的AAV病毒颗粒。
冠状病毒根据种类的不同，其基因组编码的结构蛋白有4-5种，分别为刺突蛋白(Spike protein，S蛋白)，核衣壳蛋白(Nucleocapsidprotein，N蛋白)，膜蛋白(Membrane protein，M蛋白)和包膜蛋白(Envelope protein，E蛋白)，少数种类还有血凝素糖蛋白(Haemaglutinin-esterase，HE蛋白)。其中，S蛋白有两个亚基：S1和S2，受体结合结构域(RBD)位于S1亚基上。S蛋白以三聚体的形式组成病毒粒子外膜表面的刺突，其主要功能是识别宿主细胞表面受体，并介导病毒包膜与宿主细胞的细胞膜融合。目前有研究表明，SARS-CoV病毒与2019新型冠状病毒均通过其刺突蛋白上的受体结合结构域(RBD)与人体细胞血管紧张素转化酶2(ACE2)受体结合，从而引发病毒对人体细胞的入侵。
2019新型冠状病毒目前突变迅速，发现的主要变异株包括Alpha变异株、Beta变异株、Gamma变异株、Delta变异株和Lambda变异株。Alpha变异株的突变让它与靶细胞表面受体更容易结合，从而传染性大大增强；Beta和Gamma株的突出特点是具有免疫逃逸的本领；Delta和Lambda株则同时具有了高传染性和免疫逃逸的能力，并且潜伏期短、病毒载量高，说明其复制速度大大增加。其中，Delta变异毒株是目前全球和国内最重要的流行株，其相较于原始毒株一共有15处突变，有6处发生在刺突蛋白上，其中又有3处突变比较关键：L452R和E484Q突变发生在刺突蛋白与人体细胞血管紧张素转化酶2(ACE2)受体结合的区域，即RBD区，L452R提高了病毒侵入细胞的能力，E484Q有助于增强病毒的免疫逃逸；第三处突变P681R也能使病毒更有效地进入细胞。这些突变的综合效应使Delta变异毒株能部分避开一些中和抗体，传染性增强。
本发明提供的一种表达冠状病毒刺突蛋白受体结合结构域的表达载体，其包括目的基因表达盒和位于所述目的基因表达盒两端的腺相关病毒反向末端重复序列，所述目的基因表达盒包含从5’至3’的、可操作连接的启动子、编码TPA分泌信号肽的核苷酸序列及编码冠状病毒RBD的核苷酸序列，所述表达载体用于制备重组双链腺相关病毒。
组织纤溶酶原激活剂(tissue-type plamnipen activator，TPA)又称组织型纤维蛋白溶酶原激活剂。TPA信号肽可有效促进外源蛋白的分泌，提高其诱导抗体产生的能力。一些实施例中，编码TPA分泌信号肽的核苷酸序列如SEQ ID No.4所示。
一些实施例中，启动RBD抗原分子表达的启动子可采用在AAV表达载体中能够正常启动表达的各种启动子，包括但不限于CBh启动子、CAG启动子、CMV启动子、CBA启动子等，优选为CBh启动子，CBh启动子的核苷酸序列如SEQ ID No.6所示。相较于CAG启动子(1721bp)，CBh启动子(794bp)的片段更小，相较于CMV启动子，CBh启动子的长期表达效果更好，稳定性更高；采用CBh启动子可以大大降低AAV-RBD疫苗的注射剂量，从而减小了高剂量AAV注射后引起的机体不良反应，同时还降低了成本。
一些实施例中，所述编码冠状病毒RBD的核苷酸序列经过人源密码子优化，使得RBD编码序列与人体细胞的密码子使用频率相匹配，从而提高蛋白表达水平。优选地，针对2019新型冠状病毒设计人源密码子优化的RBD编码序列，其核苷酸序列如SEQ ID No.2所示。
一些实施例中，可以对RBD抗原多肽的氨基酸序列C端进行延伸，使得抗原分子更富含有原序列自有的半胱氨酸，有利于免疫抗原蛋白的聚体化，提高稳定性，降低其降解速度，从而能长期稳定地诱导生物体产生足够的免疫应答，具体延伸序列可参见中国专利文献CN111518175A和CN111996216A。
一些实施例中，所述编码冠状病毒RBD的核苷酸序列的3’端可操作连接有编码靶向肽的核苷酸序列，所述靶向肽为MHC2受体结合结构域，以提高抗原分子靶向免疫细胞的靶向性，同时表达有靶向肽的疫苗分子在机体中能稳定保持较高的中和抗体和总抗体水平。编码MHC2受体结合结构域的核苷酸序列如SEQ ID No.5所示。
本发明还提供一种表达冠状病毒刺突蛋白受体结合结构域的重组腺相关病毒，其由以上表达载体参与转染宿主细胞而制得。
一些实施例中，该重组腺相关病毒可以为各种血清型，对应具有不同的衣壳蛋白，本发明的重组腺相关病毒包括但不限于AAV血清6型或9型。
本发明还提供一种重组腺相关病毒的制备方法，包括如下步骤：将上述表达载体、辅助质粒pHelper和血清型质粒pRepCap共孵育，在转染试剂聚乙烯亚胺存在的条件下转染 宿主细胞，培育细胞后，离心收集细胞，经过裂解和纯化，得到含有重组腺相关病毒的纯化液。
一些实施例中，所述血清型质粒pRepCap为pRep2Cap6，其中cap基因的核苷酸序列如SEQ ID No.7所示。
重组腺相关病毒的制备可采用本领域技术人员已知的工艺方法。本发明实施例中采用的是基于HEK293T的AAV无辅助病毒系统(AAVHelper-FreeSystem)进行生产，即三质粒共转染法，AAV无辅助病毒系统中含有包含AAVITRs的表达载体、包含rep/cap基因的质粒pRepCap和辅助质粒pHelper这三种质粒，辅助质粒pHelper提供生产具感染性AAV病毒颗粒所需的腺病毒基因产物(如E2A、E4和VARNA基因)，其余的腺病毒基因产物由稳定表达腺病毒E1基因的AAV-293T宿主细胞提供。在辅助质粒的帮助下，仅需两端的ITR就能将携带的目的基因片段包装进入腺相关病毒颗粒。病毒载体、pRepCap质粒和辅助质粒之间不具有同源性序列，因此重组AAV在理论上不具有复制能力。腺相关病毒的ITRs序列和rep/cap基因分别由独立的质粒表达，具有高度安全性。
本领域技术人员还可以基于SF9细胞系的昆虫系统构建表达质粒和血清型质粒生产，例如中国专利申请CN108699567A；或者利用能够更稳定传代表达的重组杆粒，该重组杆粒包含AAV的rep基因、cap基因和带有外源目的基因的核心表达元件ITR-GOI，例如中国专利申请CN112553257A。此外，还有基于疱疹病毒辅助或者腺病毒辅助的病毒包装系统等。
本发明制备的重组腺相关病毒可用于制备预防冠状病毒的疫苗，该疫苗包含所述重组腺相关病毒，疫苗注射360天后血清中仍能保持较高的总抗体和中和抗体水平。
一些实施例中，所述重组AAV适用于SARS-CoV、MERS-CoV或SARS-CoV-2等各种冠状病毒的疫苗制备。优选地，该冠状病毒为2019新型冠状病毒原始株或其变异株，本发明疫苗诱导产生的抗体对2019新型冠状病毒原始株和变异株均有优异的免疫效果，尤其针对传染性更强的Delta变异株，效果明显优于其它现有疫苗。
一些实施例中，所述疫苗制成注射剂，用于肌肉注射或鼻腔滴注。
一些实施例中，所述疫苗还包含药学上可接受的稀释剂和/或赋形剂。
以下结合具体实施例，对上述技术方案详细说明。
以下实施例中未注明具体条件的实验方法，通常按照常规条件，例如分子克隆实验指南(Sambrook等,New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商提供的说明书条件。
以下实施例中所采用的材料如无特殊说明，均可从商业途径得到。
实施例1
现有技术中使用的均是单链AAV表达新冠疫苗分子，本实施例对单、双链病毒在细胞中表达水平的差异进行了验证。
1、质粒构建
构建2种rAAV质粒，rAAV-CMV-mCHERRY-BGHpA(编号：PB2-0948)和pFD-scAAV-CMV-mCHERRY-BGHpA(编号：GT-0118)，分别用于制备单链AAV和双链AAV。
2、病毒包装和滴度检测
HEK293T细胞在转染前24h以每培养皿1.5×10 6个细胞的密度在100mm培养皿中接种，加入核心质粒pAAV-GOI(PB2-0948或GT-0118)：血清型质粒pRepCap：辅助质粒pHelper＝5μg：10μg：7.5μg和45μL转染试剂聚乙烯亚胺溶液(PEI，1mg/mL)孵育转染，转染72h后，离心收集细胞，经过裂解和纯化，收获病毒上清。用SYBRGreen Q-PCR检测2种病毒上清的物理滴度。
3、感染HepG2细胞并拍照
以MOI＝1E+05感染HepG2细胞，感染48h和72h后在荧光显微镜下进行红色荧光拍照。
由图1看出，感染48h和72h后，双链AAV的表达强度明显高于单链AAV。
4、RT-PCR。
感染72h后收获细胞，用RNA提取试剂盒分别提取RNA，进行RT-PCR检测mCherry的表达量，并用GraphPad Prism软件进行作图。
由图2看出，两者间目的基因的表达量存在着显著性的差异，说明双链AAV表达目的基因的水平远高于单链AAV。因此，本发明在后续实验中选择双链AAV进行新冠疫苗分子的表达。
实施例2不同抗原分子载体的制备
本实施例中使用的SARS-CoV-2(2019-nCoV-WIV04)全长S蛋白编码序列是委托金斯瑞公司合成的如SEQ ID No.1所示的核苷酸序列，该核苷酸序列经过人源密码子优化，命名为2019-HnCOV-S。选取S蛋白的主要抗原识别表位RBD区域，如SEQ ID No.2所示，命名为RBD；该序列对应的氨基酸序列如SEQ ID No.3所示。在该序列的基础上，设计以下信号肽TPA和3种靶向肽序列，如表1所示。
表1信号肽和靶向肽序列信息
Figure PCTCN2021123797-appb-000001
其中TPA为分泌信号肽，载体构建时加在RBD序列的N端，靶向肽分别加在RBD序列的C端，本发明采用的靶向肽分别为狂犬病毒的G蛋白受体结合结构域RVG片段、糖基化位点2NNPLPQR肽段和靶向免疫细胞的MHC2受体结合结构域，其中编码TPA分泌信号肽和MHC2受体结合结构域的核苷酸序列分别如SEQ ID No.4和SEQ ID No.5所示，构建如表2所示的不同质粒载体。
表2不同质粒载体组成信息
质粒编号 质粒名称 长度(bp)
GT-0168 pFD-rAAV-ITR-CBh-2019-HnCOV-S-BGHpA 5184
GT-0172 pFD-scAAV-ITR-CMV+intron-TPA-RBD-BGHpA 2148
GT-0173 pFD-scAAV-ITR-CBh-TPA-RBD-BGHpA 2107
GT-0174 pFD-scAAV-ITR-CBh-TPA-RBD-RVG-BGHpA 2203
GT-0175 pFD-scAAV-ITR-CBh-TPA-RBD-2NNPLPQR-BGHpA 2161
GT-0176 pFD-scAAV-ITR-CBh-TPA-RBD-MHC2-BGHpA 2209
其中GT-0173～GT-0176和GT-0168均采用CBh启动子，其核苷酸序列如SEQ ID No.6所示；GT-0172采用CMV启动子。GT-0168是单链AAV包装，而其他的分子均是双链AAV包装。如图3所示，以GT-0172为例，载体的具体构建过程如下：
(1)使用限制性内切酶AgeI和SacI双酶切PB2-894载体pFD-scAAV-ITR-CMV+intron-EGFP-BGHpA，得到大小分别为6779bp和758bp的酶切片段，回收大小6779bp片段，命名984-AS。
(2)以金斯瑞_MC_0101081_7695(人源优化)2019-HnCoV-S为模板，设计引物0172-RBD-F和0172-RBD-R，克隆目的片段0172-RBD，大小为737bp。合成TPA片段，并与0172-RBD进行酶连，得到0172-TPA-RBD片段，大小为791bp。
(3)0172-TPA-RBD片段同源重组，转化，用引物0172-TPA-F和0172-RBD-R进行菌落PCR鉴定，片段大小791bp。
(4)使用引物CX-BHGpA-R进行测序验证。本实施例所用引物信息见表3。
表3引物信息
引物名称 引物序列
0172-RBD-F gctgtgtgctgctgctgtgtggagcagtcttcgtttcgcccagggtgcagccaaccgagt
0172-TPA-F tgcctttctctccacagaccggtgccaccatggatgcaatgaagagagggctctgctgtgtgctgctgctgtgt
0172-RBD-R agtcgaggctgatcagcgagctctagtcgacttagaagttcacgcacttgttcttc
CX-BGHpA-R ggcaaacaacagatggctgg
其他载体的构建参照以上构建过程。
实施例3病毒的包装和生产
HEK293T细胞在转染前24h以每培养皿1.5×10 6个细胞的密度在100mm培养皿中接种，加入核心质粒pAAV-GOI：血清型质粒pRep2Cap6：辅助质粒pHelper＝5μg：10μg：7.5μg和45μL转染试剂聚乙烯亚胺溶液(PEI，1mg/mL)孵育转染，转染72h后，将收获收集的细胞混液加入1/10体积的氯仿37℃剧烈振摇1h，加NaCl至终浓度1mol/L振摇溶解后，4℃、12000rpm/min离心15min，取出上层水相，弃氯仿和沉淀，加PEG8000至终浓度10％(w/v)，振摇溶解后，冰浴放置1h。11000rpm/min离心15min，弃去上清，用1/10原细胞混液体积的PBS吹打洗脱并重悬，加入核酸酶至终浓度1μg/mL，室温消化30min，加入等体积的氯仿抽提，4℃、12000rpm/min离心5min，取出水相，即完成纯化至中间产品。采用碘克沙醇离心-透析-超滤收获-除菌过滤进行腺相关病毒进一步纯化成成品，生产得到以上实施例2中编号GT-0168、0172、0173、0174、0175、0176rAAV病毒。病毒的物理滴度采用SYBRGreen Q-PCR检测，纯度采用SDS-PAGE银染的方法。生产得到的病毒分装保存于-80℃冰箱。
实施例4全长S(GT-0168)和RBD(GT-0173)免疫BALB/c小鼠
1、病毒注射
每组随机选取6只6-8周龄BALB/c小鼠进行肌肉注射GT-0168(注射剂量1E+11vg/只，注射体积为50μL)或GT-0173(注射剂量1E+11vg/只，注射体积为50μL)。另外选取3只6-8周龄BALB/c小鼠进行肌肉注射PBS(50μL)作为阴性对照。注射病毒day14，day30，day60，day90，day120，day240眼眶采血，阴性对照PBS组也需要采血。将收集的血清转移到干净的EP管中，保存在-80℃冰箱。
2、总抗体检测
包被用的抗原为重组刺突蛋白RBD-His，由本公司自行生产，采用昆虫杆状病毒系统生产。用0.2M碳酸盐缓冲液(pH 9.6)溶解RBD蛋白，配制成1.0μg/mL溶液，以每孔 100μL的量加入96孔板，放入4℃孵育过夜；第二天37℃培养箱孵育1h；用PBST(PBS+0.1％吐温-20)洗板3次(300μL/孔/次)。洗板后，加入5％脱脂奶粉；每孔加入300μL；37℃孵育1h，用PBST洗板3次。每个时间点的同组(GT-0168病毒注射组或GT-0173病毒注射组或PBS组)小鼠血清样品分别用PBS梯度稀释；将稀释好的血清以每孔100μL加入96孔板，每个稀释梯度平行做三个复孔；放置在37℃培养箱孵育2h，洗板3次。分别将HRP(辣根过氧化物酶)标记的羊抗鼠IgG二抗稀释10000倍，每孔加入100μL，室温孵育1h；洗板3次。加入TMB溶液，每孔加入200μL，室温避光显色30min。加入0.5M H 2SO 4溶液，每孔加100μL。立即用酶标仪检测吸光度，检测波长为450nm。PBS组检测结果的OD平均值+3倍SD值定义为cut-off值，当待测血样的某一个稀释度的OD值大于同稀释度PBS组的cut-off值，且下一个稀释度的OD值小于或等于同稀释度PBS组的cut-off值，则定义该稀释度为该血样的IgG总抗体效价。
结果以抗体效价值为纵坐标、不同时间点为横坐标作折线图，实线代表GT-0173即全长S蛋白，虚线代表GT-0168即RBD蛋白，如图4所示。从14天到240天，全长S蛋白的总抗体效价均低于RBD蛋白，基本上都是低8倍。说明RBD抗原要比全长S抗原的分子构建更优。
3、中和抗体检测
使用假病毒中和法检测待测血样。检测用293T-ACE2-EGFP细胞为本公司自行研发生产的单克隆细胞系，在293T细胞膜上稳定表达ACE2受体，为新冠病毒和新冠假病毒的敏感细胞系。VSV-△G-mCherry-SARS-CoV-2-S(简写为VSV-△G-S)假病毒为本公司自行研发生产的外包膜为新冠S的水泡性口炎病毒，表达红色荧光mCherry。正式实验前，使用TCID50法测定假病毒的感染滴度。检测时，将待测血样56℃灭活30min。取已灭活的小鼠免疫血清梯度稀释，假病毒稀释至200 TCID50/孔，与待测血样在37℃ 5％CO 2培养箱中共孵育1h。随后在孔中加入培养中的293T-ACE2-EGFP细胞。阳性对照孔不加待测血样。回滴板中，按照1000 TCID50、100 TCID50、10 TCID50、1 TCID50、0.1 TCID50每个稀释度病毒加8个复孔进行检测，校准所用的假病毒滴度。0.1TCID50的病毒加入的细胞为阴性质控孔。在感染后第3天记录感染病毒的红色荧光细胞数量。第三天阳性质控孔出现病变，阴性质控孔无病变，该实验的适用性条件成立。观察计算各实验组红色荧光细胞数，通过Reed-Muench法计算EC50值作为血清的中和效价。
结果以假病毒中和抗体效价值EC50为纵坐标、不同时间点为横坐标作折线图，实线代表GT-0173即全长S蛋白，虚线代表GT-0168即RBD蛋白，如图5所示。从30天到 240天，全长S蛋白的中和抗体效价均低于RBD蛋白，在2.81-7.99倍间。进一步说明RBD抗原要比全长S抗原的分子构建更优。
实施例5不同启动子、不同血清型AAV-RBD病毒免疫BALB/c小鼠
以上筛选出的RBD分子进一步评估不同启动子CMV和CBh的表达强度和持续性，以及不同血清型AAV6和AAV9感染肌肉细胞的效率。
1、病毒注射。每组随机选取8只6-8周龄BALB/c小鼠进行肌肉注射GT-0172或GT-0173，不同血清型(AAV6、AAV9)和不同剂量病毒，病毒稀释至50μL进行注射。病毒注射分组信息见表4。其中5E+10vg/只为低剂量组，1E+11vg/只为中剂量组，2E+11vg/只为高剂量组。另外选取8只6-8周龄BALB/c小鼠进行肌肉注射PBS(50μL)作为阴性对照。注射病毒day14，day30，day60，day90，day120，day150，day210，day240，day300，day360后进行眼眶采血，阴性对照PBS组也需要采血。将收集的血清转移到干净的EP管中，保存在-80℃冰箱。
表4实施例5中病毒注射分组信息
Figure PCTCN2021123797-appb-000002
2、总抗体和中和抗体检测。样品为day14，day30，day60，day90，day120，day150，day210，day240，day300，day360采的眼眶后静脉丛血清，检测方法参考实施例4的检测操作步骤。
启动子比较结果见图6，圆实线和虚线分别代表GT-0173的低剂量和中剂量，三角形实线和虚线分别代表GT-0172的高剂量和中剂量。结合图6和表5可以明显看出，GT-0173分子的低剂量组不同时间点的总抗体效价基本上均比GT-0172分子高剂量组高1.22-1.93倍，中和抗体效价基本上高1.18-7.59倍。由此可见，CBh启动子在抗原分子的持续强启动表达上要远远优于CMV启动子，其低剂量组的表达强度比CMV的高剂量组还高，选择CBh启动子可以大大降低AAV-RBD疫苗的注射剂量，降低成本以及高剂量AAV注射后引 起的机体不良反应。
表5不同启动子CMV和CBh不同剂量抗体效价比值
Figure PCTCN2021123797-appb-000003
2种不同血清型比较结果见图7，实心圆形和空心圆形分别代表GT-0173分子的AAV6型和AAV9型；实心三角形和空心三角形分别代表GT-0172分子的AAV6型和AAV9型。从图中可以明显看出AAV9-GT-0172分子的抗体效价要明显高于AAV6，但是在GT-0173分子上，AAV9总抗体水平只是略高于AAV6，中和抗体水平AAV6在120天后反而会略高于AAV9。同时，AAV6-GT-0173的抗体效价要高于AAV9-GT-0172，进一步说明选择好的启动子，可以弥补和跨越AAV血清型感染带来的表达差异。
实施例6不同改造AAV-RBD病毒免疫BALB/c小鼠
在以上筛选出的CBh启动子的基础上进一步改造RBD分子，即以GT-0173分子为基础进行改造。包括添加靶向免疫细胞的MHC2受体结合结构域的核苷酸编码序列、狂犬病毒的G蛋白受体结合结构域RVG片段的核苷酸编码序列以及糖基化位点2NNPLPQR肽段的核苷酸编码序列。
1、病毒注射。每组随机选取8只6-8周龄BALB/c小鼠进行肌肉注射GT-0173～GT-0176，血清型为AAV6，注射剂量1E+11vg/只，病毒稀释至50μL进行注射。病毒注射分组信息见表6。另外选取8只6-8周龄BALB/c小鼠进行肌肉注射PBS(50μL)作为阴性对照。注射病毒day14，day30，day60，day90，day120，day150，day210，day240，day300，day360进行眼眶采血，阴性对照PBS组也需要采血。将收集的血清转移到干净的EP管中，保存在-80℃冰箱。
表6实施例6中病毒注射分组信息
编号 抗原名称 剂量(vg) 数量(n)
GT-0173 TPA-RBD 1E+11 8
GT-0174 TPA-RBD-RVG 1E+11 8
GT-0175 TPA-RBD-2NNPLPQR 1E+11 8
GT-0176 TPA-RBD-MHC2 1E+11 8
2、总抗体和中和抗体检测。样品为day14，day30，day60，day90，day120，day150，day210，day240，day300，day360采的眼眶后静脉丛血清，检测方法参考实施例4的检测操作步骤。
图8中圆实线代表GT-0176，三角形实线代表对照分子GT-0173，正方形实线代表GT-0174，正方形虚线代表GT-0175，从总抗体和中和抗体的检测结果看，添加RVG片段和糖基化位点2NNPLPQR肽段都使得RBD抗原分子的总抗体和中和抗体效价下降严重，而添加靶向免疫细胞的MHC2受体结合结构域在第30天至210天均提高了RBD抗原分子的总抗体水平，且在第150天时总抗体效价达到751209.7，而不改造的RBD分子最高只能达到375604.9。从中和抗体的检测结果看，在RBD上添加MHC2片段所激起的机体中和抗体水平和不改造的RBD相差不大，分析原因，MHC2片段是靶向免疫细胞的一个结构域，添加这个片段会增加RBD分子对免疫细胞的嗜性，从而更快的进入免疫细胞诱导B细胞的成熟，释放抗体。而中和抗体则不受这个过程的影响。因此，我们得出结论，scAAV6-TPA-RBD和scAAV6-TPA-RBD-MHC2疫苗分子能够实现一针免疫，且在机体中稳定保持较高的中和抗体和总抗体水平，相较于现有的已上市的疫苗，其持续性、稳定性更好。
实施例7AAV-RBD疫苗对新冠Delta毒株和B1株(D614G)的保护效力
将实施例6中GT-0173疫苗和GT-0176疫苗免疫的BALB/c小鼠血样：90天和360天，进行新冠Delta毒株和B1株(D614G)的中和抗体检测试验，评估这2种疫苗对新冠变异株的保护力。检测方法参见实施例4中的中和抗体检测方法。VSV-△G-mCherry-SARS-CoV-2-S假病毒中的S膜蛋白由原始株(野生株)的换成新冠Delta毒株和B1株(D614G)，该病毒由枢密自行生产。评估AAV-RBD疫苗对新冠Delta毒株和B1株(D614G)的保护效力，结果如图9。
从图9可以看出，疫苗血清样本对Delta毒株和B1株(D614G)的保护效力都略有下降，下降的倍数见表7。
表7本发明疫苗免疫后不同时间点对Delta毒株保护效力下降倍数
免疫后不同时间点下降倍数 0173下降倍数 0176下降倍数
  Day90 Day 360 Day 90 Day 360
与原始株相比 2.79 2.61 1.99 2.24
与B1株(D614G)相比 2.09 0.97 1.68 1.83
从表7可以看出，相较于原始株和B1株(D614G)，免疫后90天和360天的血样，对Delta毒株的保护效力基本上都下降了2倍左右，疫苗的稳定保护性较好。这比文献公开报道的其他上市疫苗对于Delta株的中和检测数据要更优，保护效力更强，稳定性更好。
表8其他上市疫苗对于Delta株的中和检测数据
Figure PCTCN2021123797-appb-000004
备注：
[1]Tada Takuya,ZhouHao,Samanovic Marie I et al.Comparison of Neutralizing Antibody Titers Elicited by mRNA and Adenoviral Vector Vaccine against SARS-CoV-2 Variants.[J].bioRxiv,2021,doi:10.1101/2021.07.19.452771.
[2]Wall Emma C,WuMary,Harvey Ruth et al.Neutralising antibody activity against SARS-CoV-2 VOCs B.1.617.2 and B.1.351 by BNT162b2 vaccination.[J].Lancet,2021,397:2331-2333.
[3]Wall Emma C,WuMary,Harvey Ruth et al.AZD1222-induced neutralising antibody activity against SARS-CoV-2 Delta VOC.[J].Lancet,2021,398:207-209.
[4]Sapkal G N,Yadav P D,Sahay R R,et al.Neutralization of Delta variant with sera of Covishield vaccinees and COVID-19-recovered vaccinated individuals[J].Journal of Travel Medicine,2021.
从表8可以看出，已上市的疫苗对目前传染性更强、毒性更猛的Delta毒株的保护力在第28天-90天时较B1株下降了2.5倍-7.4倍，而本发明的疫苗在90天时只下降了1.68倍-2.09倍，由此可见，本发明的新冠疫苗虽然针对的是原始株，但是对变异株的保护效力更强，稳定更好，比现有的疫苗更优。
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种表达冠状病毒刺突蛋白受体结合结构域的表达载体，其特征在于：包括目的基因表达盒和位于所述目的基因表达盒两端的腺相关病毒反向末端重复序列，所述目的基因表达盒包含从5’至3’的、可操作连接的启动子、编码TPA分泌信号肽的核苷酸序列及编码冠状病毒RBD的核苷酸序列，所述表达载体用于制备重组双链腺相关病毒。
  2. 根据权利要求1所述的表达载体，其特征在于：所述启动子为CBh启动子、CMV启动子、CAG启动子或CBA启动子。
  3. 根据权利要求1所述的表达载体，其特征在于：所述编码冠状病毒RBD的核苷酸序列经过人源密码子优化。
  4. 根据权利要求3所述的表达载体，其特征在于：所述冠状病毒为2019新型冠状病毒，所述编码冠状病毒RBD的核苷酸序列如SEQ ID No.2所示。
  5. 根据权利要求1所述的表达载体，其特征在于：所述编码冠状病毒RBD的核苷酸序列的3’端可操作连接有编码靶向肽的核苷酸序列，所述靶向肽为MHC2受体结合结构域。
  6. 一种表达冠状病毒刺突蛋白受体结合结构域的重组腺相关病毒，其特征在于：由权利要求1-5任一所述的表达载体参与转染宿主细胞而制得。
  7. 根据权利要求6所述的重组腺相关病毒，其特征在于：所述重组腺相关病毒的衣壳蛋白为AAV2、AAV5、AAV6、AAV7、AAV8或AAV9血清型衣壳蛋白。
  8. 权利要求6所述的重组腺相关病毒在制备预防冠状病毒的疫苗中的应用。
  9. 根据权利要求8所述的应用，其特征在于：所述冠状病毒为2019新型冠状病毒原始株或其变异株。
  10. 根据权利要求8所述的应用，其特征在于：所述疫苗制成注射剂，用于肌肉注射或鼻腔滴注。
PCT/CN2021/123797 2021-09-28 2021-10-14 一种表达载体、重组腺相关病毒及其在制备2019新型冠状病毒疫苗中的应用 WO2023050484A1 (zh)

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