WO2020143634A1 - 一种SamRNA疫苗及其制备方法 - Google Patents

一种SamRNA疫苗及其制备方法 Download PDF

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WO2020143634A1
WO2020143634A1 PCT/CN2020/070734 CN2020070734W WO2020143634A1 WO 2020143634 A1 WO2020143634 A1 WO 2020143634A1 CN 2020070734 W CN2020070734 W CN 2020070734W WO 2020143634 A1 WO2020143634 A1 WO 2020143634A1
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antigen
virus
samrna
adenovirus
plasmid
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French (fr)
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朱涛
李军强
巢守柏
莘春林
苗伟
路希山
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CanSino Biologics Inc
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CanSino Biologics Inc
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Priority to CA3125776A priority Critical patent/CA3125776A1/en
Priority to US17/420,608 priority patent/US20220088186A1/en
Priority to EP20739028.7A priority patent/EP3900741A4/en
Publication of WO2020143634A1 publication Critical patent/WO2020143634A1/zh
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Definitions

  • the invention belongs to the technical field of vaccines, and in particular relates to a modified adenovirus vector to produce mRNA vaccine and its preparation method, and the mRNA vaccine produced by the adenovirus vector can use RNA as a template for continuous amplification to form more mRNA , Referred to as samRNA (self-amplifying mRNA).
  • the mRNA vaccine is a genetic vaccine with immunity, safety and flexibility.
  • the mRNA vaccine can stimulate the immune system to produce balanced and long-term protection.
  • Some mRNA vaccines themselves have the characteristics of vaccine adjuvants, which stimulate the immune system by producing a variety of cytokines and other methods, enhance the immune body's ability to respond, shorten the immune response time, and increase antibody synthesis and release capabilities.
  • mRNA messenger RNA
  • This method of directly injecting mRNA can produce an immune response by expressing specific proteins, which is the prototype of mRNA therapy.
  • mRNA Compared with traditional vaccines, the safety of mRNA is more advantageous, it will not insert gene mutations, it can be degraded by normal cells, and its half-life can be changed by modifying sequence modification and delivery vectors. More importantly, traditional vaccines are weak against many new viruses, not to mention diseases like cancer that seriously threaten human health.
  • the mechanism of action of mRNA makes it like a recipe. As long as the RNA sequence is compiled, the cell can be turned into a small drug factory. The mRNA guides the cell to produce specific proteins to exert the systemic drug effect.
  • mRNA participates in the intermediate steps of DNA transcription and protein production.
  • RNA There are two types of RNA currently used to make vaccines, non-replicating mRNA and self-amplifying mRNA (SamRNA).
  • the antigens encoded by traditional mRNA vaccines contain 5′ and 3′ untranslated regions (UTRs), and self-amplifying RNA not only encodes antigens, but also has a sequence similar to the virus replication process, allowing it to replicate in cells and improve protein The amount of expression.
  • mRNA can be transcribed from an in vitro cDNA template, and an open reading frame (ORF) of protein encoding is added to the mRNA at the late stage of transcription, so that the synthesized mRNA has the function of encoding a protein.
  • ORF open reading frame
  • the open reading frame is composed of at least two important elements: a “hat” structure at the 5’ end and a “tail” of poly A.
  • UTRs untranslated regions
  • other complexes helps the stable transcription of mRNA.
  • adenovirus vector systems have been widely used in the development of gene therapy drugs and vaccines. There are also a large number of reports on adenovirus vector systems in clinic. Their safety and reliability as gene delivery vectors have been fully confirmed. . According to their ability to replicate, adenovirus vectors are classified into replication-type and replication-defective types. At present, replication-defective adenovirus vectors are more commonly used. Compared with replication-type adenovirus vectors, the former are more secure and have lower immunogenicity.
  • Adenovirus vector vaccine refers to the use of adenovirus as a carrier to recombine the protective antigen gene into the viral genome.
  • the antigen gene in the adenovirus vector is protected by the viral capsid protein, which can avoid the degradation of the carrier gene by various proteases in the host. It effectively solves the risk of mRNA degradation from the injection site to the host cell. After the adenovirus vector infects the host cell, the antigen gene or target gene carried can express the protein.
  • mRNA vaccines In addition to easy degradation and low bioavailability, mRNA vaccines also have a problem with copy number. Whether it is direct injection or the commonly used protamine-wrapped mRNA, an mRNA can only be effectively translated once in the organism, which greatly limits the availability of mRNA as a vaccine. Therefore, in order to solve this phenomenon, it is necessary to design a reproducible system to ensure the continuous production of mRNA in the host cell. After the adenovirus vector infects the host cell, the carrier genes are injected into the host cell, and these genes can use each base pair of the host cell to synthesize mRNA.
  • the adenovirus is a DNA virus, and the gene amplification ability of the DNA virus in the host body is much higher than that of the RNA virus. Therefore, using adenovirus as a vector will greatly increase the copy number of mRNA.
  • the copy number of the target gene is determined by the strength of the promoter of the adenovirus vector itself. Especially for replication-deficient adenovirus vectors, its replication function is still relatively weak.
  • the inventors modified the adenovirus gene to realize that the promoter of the adenovirus vector itself cooperates with the modified gene, which greatly improves the expression rate of the target antigen. It is also possible to add adjuvant DNA to the adenovirus vector carrying the mRNA gene.
  • the adenovirus vector system constructed in this way can not only improve the immunogenicity of the body, but also produce the effect of adjuvant.
  • the present invention provides a SamRNA vaccine.
  • the vaccine includes a recombinant viral vector.
  • the recombinant viral vector includes: i) a viral gene replication complex, and the viral gene replication complex includes an encoding viral gene replication-related protein nsP1.
  • the SamRNA vaccine further includes iii) a promoter for transcription of the antigen gene.
  • the recombinant viral vector is recombinant adenovirus, chimpanzee-type adenovirus, recombinant vesicular stomatitis virus, recombinant poxvirus, recombinant dengue virus, recombinant Kunjin virus, recombinant Sendai virus or recombinant canine distemper virus.
  • the recombinant virus vector is a recombinant adenovirus and chimpanzee-type adenovirus vector.
  • the adenovirus may be any one of Ad1-Ad52.
  • the antigens described in the present invention elicit an immune response against bacteria, viruses, fungi or parasites.
  • the antigen is human herpes zoster virus gE protein, rotavirus VP4 or VP7, HPV-L1 protein or Ebola virus gP protein.
  • the antigen of the present invention may also be a tumor-specific antigen, for example, the tumor-specific antigen is selected from NY-ESO-1, SSX2, SCP1, RAGE, BAGE, GAGE, MAGE family polypeptides, p53, p21/Ras, CDK4, MUM1 , Caspase-8, CIA0205, HLA-A2-R1701, ⁇ -catenin, TCR, BCR-abl, phosphoglyceraldehyde isomerase, KIA0205, CDC-27, LDLR-FUT, Galectin 4, Galectin 9, protease 3, WT 1.
  • the tumor-specific antigen is selected from NY-ESO-1, SSX2, SCP1, RAGE, BAGE, GAGE, MAGE family polypeptides, p53, p21/Ras, CDK4, MUM1 , Caspase-8, CIA0205, HLA-A2-R1701, ⁇ -catenin, TCR,
  • the recombinant viral vector is obtained by co-transfection of a modified adenovirus backbone plasmid and a shuttle plasmid containing an antigen gene.
  • the modified adenovirus skeleton plasmid includes a viral gene replication complex, the viral gene replication complex is a related protein derived from RNA virus encoding viral gene replication, and more preferably, the viral gene replication complex is derived from The nucleotide sequences of nsP1, nsP2, nsP3, and nsP4 of alphavirus.
  • the adenovirus backbone plasmid is selected from: pAdEasy-1, pAdEasy-2, pBHG11, pBHG-fiber5 or pBHG-fiber35.
  • the recombinant viral vector further includes: iv) a nucleotide sequence encoding at least one adjuvant, for example, the nucleotide sequence encoding at least one adjuvant is selected from Encoding GM-CSF, IL-17, IFNg, IL-15, IL-21, anti-PD1/2, lactoferrin, protamine, IL-1, IL-2, IL-3, IL-4, IL- 5.
  • the invention provides a preparation method of SamRNA vaccine, which includes: construction of modified adenovirus skeleton plasmid; cloning of antigen gene fragment; construction of shuttle plasmid; co-transfection and packaging of shuttle plasmid and adenovirus skeleton plasmid.
  • the construction of the modified adenovirus skeleton plasmid includes the cloning of the viral gene replication complex and the promoter, and the connection of the viral gene replication complex and the promoter to the adenovirus skeleton plasmid.
  • the adenovirus skeleton plasmid includes a viral gene replication complex
  • the viral gene replication complex is a gene sequence encoding viral replication-related proteins nsP1, nsP2, nsP3, and nsP4.
  • the amino acid sequences of the virus replication-related proteins nsP1, nsP2, nsP3 and nsP4 are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.
  • the adenovirus backbone plasmid further includes a promoter, and the promoter is a promoter that transcribes an antigen gene, and the sequence of the promoter is different due to a change in the nucleotide sequence encoding at least one antigen, preferably,
  • the gene sequence of the promoter is as SEQ ID NO.5.
  • the adenovirus backbone plasmid is selected from: pAdEasy-1, pAdEasy-2, pBHG11, pBHG-fiber5, pBHG-fiber35 and the like.
  • the construction method of the shuttle plasmid containing the antigen gene adopts conventional experimental methods in the industry, and this method is used in the technical field, for example, the synthesis of a nucleotide sequence encoding at least one antigen, and the synthesis of at least one antigen
  • the two ends of the nucleotide sequence are designed to introduce the same enzymes as the shuttle plasmid.
  • the target antigen and the shuttle plasmid are double digested respectively.
  • the nucleotide sequence encoding at least one antigen and the plasmid are connected by T4 DNA ligase. can.
  • T4 DNA ligase can.
  • the method for preparing the SamRNA vaccine provided by the present invention as long as any antigen gene (ie, a nucleotide sequence encoding at least one antigen) is inserted into the shuttle plasmid, a SamRNA vaccine that can prevent the disease will be developed.
  • the vaccine is obtained by co-transfection of the adenovirus skeleton plasmid inserted with the viral gene replication complex and the promoter gene and the shuttle plasmid containing the antigen gene.
  • the vaccine includes a recombinant viral vector, and the recombinant viral vector includes: i ) A viral gene replication complex, which includes nucleotide sequences encoding viral gene replication-related proteins nsP1, nsP2, nsP3, and nsP4, and ii) a nucleotide sequence encoding at least one antigen.
  • the antigen gene is a DNA sequence that can encode any antigen.
  • Any antigen gene that has been discovered or publicly reported can be recombined into an adenovirus vector through a shuttle plasmid. After the co-infection of the backbone plasmid and the shuttle plasmid, it can be obtained SamRNA vaccine according to the present invention.
  • the antigen gene is selected from the following: DNA sequence encoding human zoster virus gE protein, rotavirus VP4 or VP7 DNA sequence, HPV-L1 protein DNA sequence, Ebola virus gP protein DNA sequence Wait.
  • the shuttle vector is selected from: pDC311, pDC312, pDC315, pDC316, p-Shuttle, p-Shuttle-CMV, pAdTrack, pAdTrack-CMV, etc.
  • the present invention provides a pharmaceutical composition, which includes a SamRNA vaccine and pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable auxiliary materials include: diluents, solubilizers, binders, lubricants, suspending agents and the like.
  • the dosage form of the pharmaceutical composition includes but is not limited to: lyophilized preparation, liquid preparation, emulsion, etc.;
  • the pharmaceutical composition is a lyophilized preparation.
  • Excipients of the lyophilized preparation include mannitol, sucrose, human albumin, and PB buffer (for maintaining the pH of the preparation).
  • the mannitol concentration in the pharmaceutical composition is 10-500 mg/ml
  • the sucrose concentration is 10-500 mg/ml
  • the human albumin concentration is 25-100 mg/ml
  • the PB buffer concentration is 1-100mM.
  • the SamRNA vaccine is a liquid injection
  • the excipients added to the liquid preparation include human albumin and PB buffer.
  • the human albumin concentration is 25-100 mg/ml
  • the PB buffer concentration is 1-100 mM.
  • the present invention provides a modified adenovirus skeletal plasmid in which a viral gene replication complex gene is inserted, and the viral gene replication complex is a gene encoding viral gene replication-related proteins nsP1, nsP2, nsP3, and nsP4 sequence.
  • the gene sequence is inserted into the adenovirus skeleton plasmid in a nsP1---nsP2--nsP3--nsP4 connection manner, and one or more linkers are added between two adjacent protein genes.
  • the linker includes but is not limited to G4S, LE linker 1 (sequence TTAGAA), LE linker 2 (sequence CTCGAA) or DEL (sequence GATGAACTG).
  • the amino acid sequences encoding viral gene replication-related proteins nsP1, nsP2, nsP3, and nsP4 are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.
  • the adenovirus backbone plasmid further includes a promoter, and the promoter is a promoter that transcribes an antigen gene, and the sequence of the promoter is different due to changes in the antigen gene; the promoter may be derived from RNA virus non-structure
  • the promoter during protein replication can also be derived from common promoters on various expression vectors.
  • the gene sequence of the promoter is as SEQ ID NO.5.
  • the promoter and the replicase genes on the adenovirus backbone plasmid are codon-optimized.
  • the purpose of codon optimization is to increase the expression of the replicator in the host cell, thereby promoting transcription to form more copies of mRNA.
  • the amino acid sequences of nsP1, nsP2, nsP3 and nsP4 are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively. See SEQ ID NO.5 for the gene sequence of the promoter.
  • nsP1, nsP2, nsP3 and nsP4 and the promoter adopt a gene synthesis method, and those skilled in the art who have general knowledge know this synthesis method.
  • nsP1, nsP2, nsP3 and nsP4 are directly synthesized into one segment, and one or more linkers are added between adjacent genes.
  • the two ends of the synthesized sequence are added with enzyme cleavage sites, so the gene fragment can be easily inserted into the adenovirus backbone plasmid through T4DNA ligase.
  • the adenovirus backbone plasmid is selected from: pAdEasy-1, pAdEasy-2, pBHG11, pBHG-fiber5, pBHG-fiber35.
  • the invention provides a method for preparing a modified adenovirus skeleton plasmid, including the following steps:
  • amino acid sequences of nsP1, nsP2, nsP3, and nsP4 are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. See SEQ ID NO.5 for the gene sequence of the promoter.
  • the amino acid sequence of the viral gene replication complex nsP1, nsP2, nsP3 and nsP4 is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4.
  • the four protein genes are synthesized in the order of nsP1-linker--nsP2-linker--nsP3-linker--nsP4, and one or more linkers are added between the two protein genes.
  • the linker includes but is not limited to G4S, LE linker 1 (nucleotide sequence TTAGAA), LE linker 2 (nucleotide sequence CTCGAA) or DEL (nucleotide sequence GATGAACTG).
  • a restriction site is added to both sides of the gene fragment.
  • the gene fragments of the viral gene replication complex and the adenoviral skeleton plasmid are connected.
  • the specific operation is to perform double digestion of the adenoviral skeleton plasmid and the viral gene replication complex respectively, and the digestion is performed at 37°C;
  • the target gene fragments are recovered by post-glue.
  • the recovery of the target gene fragments adopts a gel recovery kit. Specific operations can be performed according to the instructions of the kit.
  • use the DNA ligation kit/T4 DNA ligase to ligate the double-digested viral gene replication complex gene fragment and adenovirus backbone plasmid, and ligate overnight.
  • the ligation product is then transformed into DH5a competent cells.
  • the clones identified by PCR were inoculated into LB medium and incubated at 37-2200rpm overnight. The following day, the bacterial cells were recovered, and the plasmid extraction kit was used to recover the plasmid.
  • the promoter gene fragment is inserted into the adenovirus backbone plasmid that has been linked to the viral gene replication complex fragment.
  • the insertion of the promoter gene fragment into the adenovirus backbone plasmid is similar to the above method.
  • the double-digestion of the adenoviral skeleton plasmid and the promoter, which have been connected to the viral gene replication complex, is performed at 37°C; after the digestion, the target gene fragments are recovered by gel, and the target gene fragments are recovered by using a gel recovery kit. The specific operation can be carried out according to the instructions of the kit.
  • DNA ligation kit/T4 DNA ligase to ligate the double-digested viral gene replication complex gene fragment and adenovirus backbone plasmid, and ligate overnight.
  • the ligation product is then transformed into DH5a competent cells.
  • the clones identified by PCR were inoculated into LB medium and incubated at 37-2200rpm overnight. The following day, the bacterial cells were recovered, and the plasmid extraction kit was used to recover the plasmid.
  • the constructed adenovirus backbone plasmid is identified by double enzyme digestion and PCR.
  • the invention solves the risk that the mRNA vaccine is degraded in a large amount in the body.
  • the existing packaging technology can avoid the massive degradation of the mRNA vaccine, it cannot solve the defect that the mRNA is difficult to directly enter the cell (low bioavailability).
  • the samRNA vaccine of the present invention is an adenovirus vector carrying an antigen, and the adenovirus vector can directly infect human and animal cells, thereby injecting the target antigen gene into the host cell.
  • the conventional mRNA vaccine can only be translated once after entering the host cell, and the samRNA vaccine of the present invention can use RNA as a template to synthesize a large amount of mRNA, thereby greatly increasing the expression amount of antigen.
  • ordinary replication-defective adenovirus vectors can only replicate in cells like HEK293. For humans and animals, they can only infect and cannot replicate, so their replication capacity in organisms is limited.
  • the nucleic acid is injected into the host cell. A series of dynamic reactions will then take place in the host cell, firstly the transcription of the viral gene replication complex gene and the translation of the protein. At the same time antigen gene is transcribed into mRNA. With the translation of the nsP1, nsP2, nsP3, and nsP4 proteins, the four proteins will form a viral genome replication complex of the viral genome.
  • mRNA is synthesized using the antigen gene RNA as a template.
  • mRNA utilizes various proteases from the host cell to synthesize the target antigen.
  • the synthesis of mRNA by the viral gene replication complex using RNA as a template is a continuous process, and the antigen expression lasts longer, so the effect of the modified adenovirus vector SamRNA vaccine is more durable.
  • viruses are used as carriers of SamRNA, but the shortcomings are also obvious.
  • the first viruses used for samRNA are all RNA viruses, and are sense RNA viruses. These viruses have limited infection and replication capabilities. Host restriction makes it very difficult to produce SamRNA with virus as a vector. Strict host cell restrictions make it difficult for conventional production processes to meet the production of this type of virus. In addition, the limited amplification capacity seriously affects the virus production.
  • SamRNA which uses virus as a carrier, is replaced by the gene of antigen during the process of genome recombination and construction. The advantage of deleting the outer membrane structural genes of the virus is that the virus will not assemble to form a virus after infecting animal and human host cells, reducing the risk of infection or disease.
  • the shortcomings are obvious, and the lack of structural genes makes it impossible for the virus vector vaccine to complete the assembly of virus particles under normal conditions, and the technical difficulty of commercial production is relatively high.
  • FIG. 1 Identification results of viral gene replication complex protein gene inserted into backbone plasmid
  • Figure 2 The identification results of the promoter sequence inserted into the backbone plasmid
  • FIG. 8 Immunogenicity of different vectors, the immunogenicity of the first immune on the left, and the immunogenicity of the second immune on the right;
  • Figure 10 Molecular adjuvants enhance the immunogenicity of Ad-SamRNA-gE, with the immunogenicity of the first immune on the left and the immunogenicity of the second immune on the right.
  • the gene sequences of viral gene replication complex nsP1, nsP2, nsP3 and nsP4 are SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, according to the above sequence Synthetic viral gene replication complex gene fragments, four antigen protein genes are synthesized into one large gene fragment.
  • the promoter synthesizes the promoter gene fragment according to SEQ ID NO.5, and adds enzyme cleavage sites at both ends of the promoter gene fragment.
  • the gene fragment of the viral gene replication complex was inserted into the pAdEasy-1 adenovirus skeleton plasmid, and the long fragments of the synthesized four viral gene replication complex protein genes contained pacI cleavage sites on both sides.
  • the pAdEasy-1 and 4 viral gene replication complex protein genes were digested with pac I enzyme, respectively. After the gel is recovered, it is ligated with T4 DNA ligase.
  • T4 DNA ligase there are two ways to connect the viral gene replication complex protein genes (insertion of the backbone plasmid backwards and insertion of the backbone plasmids backwards).
  • the promoter gene and the skeletal plasmid that has been inserted into the viral gene replication complex gene are digested with ClaI, and after the gel is recovered, they are ligated with T4 DNA ligase.
  • T4 DNA ligase there are two ways to connect the promoter protein genes (insertion of the backbone plasmid backwards and insertion of the backbone plasmids backwards).
  • After transforming DH5a pick a single gram and identify the vector inserted into the framework plasmid as expected by PCR.
  • the PCR identification results are shown in Figure 2. In this step, clones 3, 4, and 5 were correctly inserted into the promoter sequence.
  • the skeleton plasmids pAdEasy-13, 4 and 5 added to the viral gene replication complex and promoter sequence will be obtained.
  • the position of the promoter and viral gene replication complex in the pAdEasy-1 skeleton plasmid is shown in Figure 3. .
  • the promoter and viral gene replication complex sequences can be inserted into other backbone plasmids.
  • Ad adenovirus
  • the specific enzyme-cutting site is determined according to the selected recombination system and shuttle plasmid. Those skilled in the art will master this method.
  • the Adeasy vector system is taken as an example to show the construction process of Ad-SamRNA vaccine.
  • the gE antigen gene is ligated into the shuttle plasmid pS-C to form pS-C-gE, and the ligation method adopts an experimental method commonly used in the industry.
  • the shuttle plasmids pS-C and gE antigen genes were simultaneously double-digested with Kpn I and Xho I (see Table 1 below for the digestion reaction system). After the digestion reaction was completed, the target fragments were recovered with a gel recovery kit (see the instructions for the recovery method). Then the linearized antigen fragments and plasmids were ligated with T4 DNA ligase (see Table 2 below for the reaction system).
  • the construction method for other adenovirus vector systems is similar to the above.
  • the ligation reaction was carried out at 4°C, and the ligation was performed overnight.
  • the shuttle plasmid pS-C-gE containing the antigen gene is co-transfected with the adenovirus skeleton plasmid (such as pAdEasy-1) inserted into the viral gene replication complex and promoter, and the two plasmids will undergo homologous recombination and the Ad-Sa mRNA will be isolated -gE, the flow chart of the Ad-Sa mRNA construction is shown in Figure 4.
  • the adenovirus skeleton plasmid such as pAdEasy-1
  • the double-digestion method is used to verify the constructed Ad-SamRNA-gE.
  • the successfully constructed vector will be used for vaccine production and immunogenicity evaluation.
  • the results of the double-digestion are shown in Figure 5: Lane M is marker, 1 Double digestion for adenovirus vector, 2 for Ad-SamRNA-gE double digestion. The results show that the gE antigen gene has been correctly integrated into the adenovirus vector system.
  • Adenovirus vectors can multiply in 293 cells.
  • Admax adenovirus vector systems mostly use HEK293 cells, while Adeasy recombinant adenovirus vector cells use AD293 cells.
  • Ad-SamRNA-gE In addition to cell debris in the cell lysate, various contaminated proteins are Ad-SamRNA-gE, which can be removed by centrifugation. Compared with contaminated protein, Ad-SamRNA-gE molecular weight is much larger than contaminated protein. Therefore, pure virus can be obtained by centrifuging supernatant through CL-4B one-step process.
  • Ad-SamRNA-gE has the largest molecular weight, so it elutes first. Peak 1 in Figure 6 is Ad-SamRNA-gE.
  • the purity of the harvested Ad-SamRNA-gE was analyzed by HPLC.
  • the column of TSK5000 was selected for the experiment.
  • the HPLC result is shown in Figure 7. It can be seen that the harvested virus can not see the impurity peak, and the purity of the virus solution is very high.
  • the immunogenicity of Ad-Sa mRNA vaccine was displayed.
  • Control group 1 gE protein mRNA injection group, referred to as mRNA-gE for short;
  • Control group 2 gE protein antigen gene vaccine with common adenovirus as carrier, referred to as Ad-gE for short;
  • Negative control group normal saline immunization group
  • mice Take NIH mice, 10 in each group, weighing 12-14g,
  • Immunization method subcutaneous injection, the experimental group, the control group 1, the control group 2 single injection dose of 1 ⁇ 10 8 IFU, each mouse is immunized with two needles, two needles are separated by 4 weeks, and the second needle is collected from the anterior orbit Blood-free, the eyeballs were collected 28 days after the last immunization to collect the second-free serum, and the antibody titer in the serum was measured by ELISA. The results are shown in Figure 8.
  • Fig. 9 shows that compared with the control group Ad-gE, the expression level of gE protein was significantly higher after the test sample Ad-SamRNA-gE infected the cells.
  • the immunogenicity of Ad-SamRNA-gE in Example 4 is much higher than that of Ad-gE.
  • the immunogenicity of Ad-Sa mRNA vaccine was displayed.
  • Negative control group normal saline immunization group
  • mice Take NIH mice, 10 in each group, weighing 12-14g,
  • Immunization method subcutaneous injection, the experimental group, the control group 1, the control group 2 single injection dose of 1 ⁇ 10 8 IFU, each mouse is immunized with two needles, two needles are separated by 4 weeks, and the second needle is collected from the anterior orbit Blood-free, the eyeballs were collected 28 days after the last immunization to collect the second serum, and the antibody titer in the serum was measured by ELISA. The results are shown in Figure 10.

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Abstract

本发明公开了一种SamRNA疫苗，所述疫苗包括重组病毒载体，所述的重组病毒载体包括：i)病毒基因复制复合体，所述的病毒基因复制复合体包括编码病毒基因复制相关蛋白nsP1、nsP2、nsP3和nsP4的核苷酸序列，和ii)编码至少一种抗原的核苷酸序列。

Description

一种SamRNA疫苗及其制备方法 技术领域
本发明属于疫苗技术领域，具体涉及修饰化腺病毒载体生产mRNA疫苗的工艺技术及其制备方法，而且该腺病毒载体所生产的mRNA疫苗，可以RNA为模板进行持续的扩增形成更多的mRNA，简称samRNA(self-amplifying mRNA)。
背景技术
mRNA疫苗是具有免疫性、安全性、灵活性的基因疫苗，mRNA疫苗能够刺激免疫系统产生平衡、长效的保护。部分mRNA疫苗本身具有疫苗佐剂特性，通过产生多种细胞因子等不同方式刺激免疫系统，增强免疫机体反应能力，缩短免疫应答时间，加大抗体合成释放能力。
1990年，科学家将体外转录的信使RNA(mRNA)注射入小鼠体内，通过检测发现其可在小鼠体内表达活性，产生相关蛋白且具有剂量依赖性。这种直接注射mRNA的方法能够通过表达特定蛋白，产生免疫反应，这就是mRNA疗法的雏形。
相比传统疫苗，mRNA的安全性更有优势，不会插入基因突变，可以被正常细胞降解，通过调节序列修饰和递送载体可以改变其半衰期等。更重要的是，传统疫苗对很多新型病毒有心无力，更别提像癌症这种严重威胁人类健康的疾病了。而mRNA的作用机理使它就像一本餐谱一样，只要编好RNA序列，就可以将细胞变成小型的药物工厂，mRNA引导细胞自己产生特定蛋白发挥系统药效。
mRNA疫苗作用机理：mRNA参与DNA转录和蛋白质生成的中间步骤。目前用于制造疫苗的有两种RNA，非复制型(non-replicating)mRNA和自我扩增型(self-amplifying)mRNA(SamRNA)。传统mRNA疫苗编码的抗原包含5′和3′的未翻译区(UTRs)，而自我扩增型RNA不仅能编码抗原，还有类似病毒复制过程的序列，使其可以在细胞内复制，提高蛋白表达量。
mRNA可以通过体外cDNA模板转录而成，转录后期在mRNA增加蛋白质编码的开放阅读框(open reading frame，ORF)，使合成的mRNA具有编码蛋白质的功能。开放 阅读框至少由两个重要元素组成：5’端的“帽子”结构和一条多聚A(poly A)的“尾巴”。此外，增加不转录区域(untranslated regions，UTRs)及其他复合物协助mRNA的稳定转录。
但是，裸露的mRNA直接进入体内会被降解，而高效的mRNA递送是疫苗药效的保证，因此开发高效的mRNA递送载体是确保疫苗有效的关键因素。目前mRNA的给药方式多以皮内或者节内注射为主，但是这种注射方式由于体内mRNA的降解，因此需要大量给药才能激发机体产生有效的免疫应答。有文献报道可以用鱼精蛋白，以及其他的高分子，比如脂质体来包裹mRNA，从而避免机体内蛋白酶对mRNA的大量破坏。虽然这种包裹方式相比较直接注射可以有效增加机体对抗原的吸收量，但是仍然存在一些缺陷需要克服，比如有效的包裹量，包裹后的mRNA在体内的释放时间，以及mRNA在体内的释放和转移等。
目前，腺病毒(adenovirus，Ad)载体系统已经大量的被应用于基因治疗药物以及疫苗的开发，临床中也有腺病毒载体系统的大量报道，其安全性和作为基因递送载体的可靠性已经充分证实。根据其能否复制，将腺病毒载体分为复制型和复制缺陷型。目前复制缺陷型腺病毒载体更为常用，与复制型腺病毒载体相比，前者的安全性更好，载体自身的免疫原性更低。腺病毒载体疫苗是指以腺病毒作为载体，将保护性抗原基因重组到病毒基因组中，腺病毒载体中的抗原基因有病毒衣壳蛋白保护，可以避免宿主体内各种蛋白酶对携带基因的降解，有效解决了mRNA从注射部位到宿主细胞被降解风险，腺病毒载体在感染宿主细胞后，所携带的抗原基因或者目标基因就可以表达蛋白。
mRNA疫苗除了易于降解、生物利用度低之外，还有一个问题就是拷贝数。无论是直接注射，还是常用的鱼精蛋白包裹的mRNA，都是一个mRNA只能在生物体内有效的翻译一次，这极大的限制了mRNA作为疫苗的利用度。因此，为解决此现象，需要设计一个可复制的系统，保证在宿主细胞内能不断的产生mRNA。该腺病毒载体感染宿主细胞后，将携带基因注入宿主细胞，这些基因可以利用宿主细胞的各碱基对合成mRNA。更为关键的是腺病毒为DNA病毒，DNA病毒在宿主体内的基因扩增能力远高于RNA病毒。因此 以腺病毒作为载体将极大提高mRNA的拷贝数。
腺病毒载体疫苗感染宿主细胞后，目的基因的拷贝数通过腺病毒载体本身的启动子强弱来决定，尤其对复制缺陷型腺病毒载体而言，其复制功能还是比较弱。为了解决这个问题，在本发明中，发明人对腺病毒基因进行修饰，实现腺病毒载体本身的启动子与修饰基因共同作用，极大提高了目标抗原的表达率。也可以在携带mRNA基因的腺病毒载体中加入佐剂的DNA，如此构建的腺病毒载体系统不仅可以提高机体免疫原性，也可以产生佐剂的效果。
发明内容
本发明提供一种SamRNA疫苗，所述的疫苗包括重组病毒载体，所述的重组病毒载体包括：i)病毒基因复制复合体，所述的病毒基因复制复合体包括编码病毒基因复制相关蛋白nsP1、nsP2、nsP3和nsP4的核苷酸序列和ii)编码至少一种抗原的核苷酸序列。
优选的，所述SamRNA疫苗还包括iii)转录抗原基因的启动子。
优选的，所述的重组病毒载体为重组腺病毒、黑猩猩型腺病毒、重组水疱性口炎病毒、重组痘病毒、重组登革热病毒、重组昆津病毒、重组仙台病毒或重组犬瘟热病毒。更优选的，所述的重组病毒载体为重组腺病毒和黑猩猩型腺病毒载体，更优选的，所述的腺病毒可以为Ad1-Ad52中任意一种。
本发明所述的抗原引起针对细菌、病毒、真菌或寄生虫的免疫应答。
在本发明的一个实施方式中，所述的抗原为人带状疱疹病毒gE蛋白、轮状病毒VP4或VP7、HPV-L1蛋白或埃博拉病毒的gP蛋白。
本发明所述的抗原还可以为肿瘤特异抗原，例如所述肿瘤特异抗原选自NY-ESO-1、SSX2、SCP1、RAGE、BAGE、GAGE、MAGE家族多肽、p53、p21/Ras、CDK4、MUM1、caspase-8、CIA0205、HLA-A2-R1701、β-联蛋白、TCR、BCR-abl、磷酸甘油醛异构酶、KIA0205、CDC-27、LDLR-FUT、Galectin 4、Galectin 9、蛋白酶3、WT 1、碳酸酐酶、醛缩酶A、PRAME、HER-2/neu、乳腺珠蛋白、甲胎蛋白、KSA、胃泌素、端粒酶催化蛋白、MUC-1、G-250、p53、癌胚抗原、黑素瘤-黑素细胞分化抗原、PAP、PSA、PSMA、PSH-P1、 PSM-P1、PSM-P2、p15、Hom/Mel-40、H-Ras、E2A-PRL、H4-RET、IGH-IGK、MYL-RAR、EB病毒抗原、EBNA、人乳头瘤病毒抗原、乙型和丙型肝炎病毒抗原、人类嗜T淋巴细胞病毒抗原、TSP-180、p185erbB2、p180erbB-3、c-met、mn-23H1、TAG-72-4、CA 19-9、CA 72-4、CAM 17.1、NuMa、K-ras、p16、TAGE、PSCA、CT7、43-9F、5T4、791Tgp72、β-HCG、BCA225、BTAA、CA 125、CA 15-3(CA 27.29\BCAA)、CA 195、CA 242、CA-50、CAM43、CD68\KP1、CO-029、FGF-5、Ga733(EpCAM)、HTgp-175、M344、MA-50、MG7-Ag、MOV18、NB/70K、NY-CO-1、RCAS1、SDCCAG16、TAAL6、TAG72、TLP、TPS。
优选的，所述的重组病毒载体由修饰化腺病毒骨架质粒与含有抗原基因的穿梭质粒共转染得到。所述修饰化腺病毒骨架质粒上包括病毒基因复制复合体，所述病毒基因复制复合体为来自于RNA病毒编码病毒基因复制的相关蛋白，更优先的，所述病毒基因复制复合体为来自于alphavirus的nsP1、nsP2、nsP3和nsP4的核苷酸序列。所述腺病毒骨架质粒选自：pAdEasy-1、pAdEasy-2、pBHG11、pBHG-fiber5或pBHG-fiber35。
在本发明的一个实施方式中，所述的重组病毒载体还包括：iv)编码至少一种佐剂的核苷酸序列，例如，所述的编码至少一种佐剂的核苷酸序列选自编码GM-CSF、IL-17、IFNg、IL-15、IL-21、抗PD1/2、乳铁蛋白、鱼精蛋白、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-12、INF-α、INF-γ、Lymphotoxin-α、hGH的核苷酸序列。
本发明提供一种SamRNA疫苗的制备方法，包括：修饰化腺病毒骨架质粒的构建；抗原基因片段的克隆；穿梭质粒的构建；穿梭质粒与腺病毒骨架质粒共转染与包装。
优选的，所述修饰化腺病毒骨架质粒的构建包括所述病毒基因复制复合体和启动子的克隆，以及病毒基因复制复合体和启动子与腺病毒骨架质粒的连接。
优选的，所述腺病毒骨架质粒上包括病毒基因复制复合体，所述病毒基因复制复合体为编码病毒复制相关蛋白nsP1、nsP2、nsP3和nsP4的基因序列。所述的病毒复制相关蛋白nsP1、nsP2、nsP3和nsP4的氨基酸序列见SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4。
优选的，所述腺病毒骨架质粒上还包括启动子，所述启动子为转录抗原基因的启动子， 启动子的序列由于编码至少一种抗原的核苷酸序列的改变而不同，优选的，在本发明中，启动子的基因序列如SEQ ID NO.5。
所述腺病毒骨架质粒选自：pAdEasy-1、pAdEasy-2、pBHG11、pBHG-fiber5、pBHG-fiber35等。
优选的，含有抗原基因的穿梭质粒的构建方法采用行业内常规的实验方法，本技术领域都采用该方法，例如合成编码至少一种抗原的核苷酸序列，所合成的编码至少一种抗原的核苷酸序列两端设计引入和穿梭质粒相同的酶，分别双酶切目标抗原和穿梭质粒，回收核苷酸片段后，以T4DNA连接酶连接编码至少一种抗原的核苷酸序列和质粒即可。具体可以参考骨架质粒中引入病毒基因复制复合体和启动子的方法。
本发明提供的SamRNA疫苗的制备方法，只要在穿梭质粒中插入任何抗原基因(即编码至少一种抗原的核苷酸序列)，就会开发出一个可预防该疾病的SamRNA疫苗。所述疫苗上述插入病毒基因复制复合体和启动子基因的腺病毒骨架质粒与含有抗原基因的穿梭质粒共转染重组得到，所述的疫苗包括重组病毒载体，所述的重组病毒载体包括：i)病毒基因复制复合体，所述的病毒基因复制复合体包括编码病毒基因复制相关蛋白nsP1、nsP2、nsP3和nsP4的核苷酸序列，和ii)编码至少一种抗原的核苷酸序列。
优选的，所述抗原基因为可编码任意抗原的DNA序列，任何已经发现或者公开报道的抗原基因都可以通过穿梭质粒，重组到腺病毒载体中，骨架质粒和穿梭质粒共感染后，即可得到本发明所述的SamRNA疫苗。
优选的，所述抗原基因选自：编码人带状疱疹病毒gE蛋白的DNA序列、轮状病毒VP4或VP7的DNA序列、HPV-L1蛋白的DNA序列，埃博拉病毒的gP蛋白的DNA序列等。
所述穿梭载体选自：pDC311、pDC312、pDC315、pDC316、p-Shuttle、p-Shuttle-CMV、pAdTrack、pAdTrack-CMV等。
本发明提供一种药物组合物，所述药物组合物包括SamRNA疫苗和药学上可接受的辅料。
所述药学上可接受的辅料包括：稀释剂、增溶剂、粘合剂、润滑剂、助悬剂等。
所述药物组合物的剂型包括但不限于：冻干制剂、液体制剂、乳剂等；
优选的，所述的药物组合物为冻干制剂，冻干制剂的辅料例如包括甘露醇、蔗糖、人白蛋白、PB缓冲液(用于维持制剂的pH)。在本发明的实施方式中，药物组合物中所述的甘露醇浓度为10-500mg/ml，蔗糖浓度为10-500mg/ml，人白蛋白浓度为25-100mg/ml，PB缓冲液浓度为1-100mM。
优选的，SamRNA疫苗为液体针剂，液体制剂中加入的辅料包括人白蛋白，和PB缓冲液。所述人白蛋白浓度为25-100mg/ml，PB缓冲液浓度为1-100mM。
本发明提供一种修饰化腺病毒骨架质粒，所述腺病毒骨架质粒上插入病毒基因复制复合体基因，所述病毒基因复制复合体为编码病毒基因复制相关蛋白nsP1、nsP2、nsP3和nsP4的基因序列。所述基因序列以nsP1--nsP2--nsP3--nsP4连接的方式插入腺病毒骨架质粒中，相邻两个蛋白基因之间加入一个或多个linker。所述linker包括但不限于G4S、LE linker 1(序列TTAGAA)、LE linker 2(序列CTCGAA)或DEL(序列GATGAACTG)。编码病毒基因复制相关蛋白nsP1、nsP2、nsP3和nsP4的氨基酸序列分别为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4。
优选的，所述腺病毒骨架质粒上还包括启动子，所述启动子为转录抗原基因的启动子，启动子的序列由于抗原基因的改变而不同；所述启动子可以来自于RNA病毒非结构蛋白复制时的启动子，也可以来自于各种表达载体上常见的启动子。优选的，在本发明中，启动子的基因序列如SEQ ID NO.5。
优选的，腺病毒骨架质粒上的启动子以及复制体基因经过密码子优化，密码子优化的目的是提高复制体在宿主细胞内的表达，从而促进转录形成更多拷贝的mRNA。nsP1、nsP2、nsP3和nsP4的氨基酸序列分别为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4。启动子的基因序列见SEQ ID NO.5。
所述nsP1、nsP2、nsP3和nsP4和启动子的序列采用基因合成的方法，本领域掌握一般知识的技术人员都知道该合成方法。其中nsP1、nsP2、nsP3和nsP4直接合成为一个片 段，并且相邻基因之间加入一个或多个linker。所合成的序列两端加入酶切位点，因此该基因片段通过T4DNA连接酶就可以很简单的插入腺病毒骨架质粒。
所述腺病毒骨架质粒选自：pAdEasy-1、pAdEasy-2、pBHG11、pBHG-fiber5、pBHG-fiber35。
本发明提供一种制备修饰化腺病毒骨架质粒的方法，包括如下步骤：
病毒基因复制复合体和启动子基因的克隆，所述nsP1、nsP2、nsP3和nsP4的氨基酸序列分别为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4。启动子的基因序列见SEQ ID NO.5。
本领域掌握常规技术都可以提供该基因的合成业务；基因合成采用本领域常规的实验技术。合成的启动子基因片段，两端加入酶切位点。
优选的，病毒基因复制复合体nsP1、nsP2、nsP3和nsP4的氨基酸序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4。四个蛋白基因采用nsP1—linker--nsP2—linker--nsP3—linker--nsP4顺序进行合成一个基因片段，两个蛋白基因之间加入一个或多个linker。所述linker包括但不限于G4S、LE linker 1(核苷酸序列TTAGAA)、LE linker 2(核苷酸序列CTCGAA)或DEL(核苷酸序列GATGAACTG)。该基因片段的两侧加入酶切位点。
病毒基因复制复合体和启动子基因片段插入腺病毒骨架质粒：病毒基因复制复合体和启动子基因片段和腺病毒骨架质粒的连接采用行业内常规的实验方法，掌握本行业分子生物学一般知识的人都可以进行该操作。
优选的，将病毒基因复制复合体基因片段和腺病毒骨架质粒相连接，具体的操作为分别将腺病毒骨架质粒与病毒基因复制复合体进行双酶切，酶切在37℃进行；酶切结束后胶回收目的基因片段，目的基因片段的回收采用胶回收试剂盒，具体的操作可依据试剂盒的说明书进行即可。然后用DNA连接试剂盒/T4DNA连接酶将双酶切后的病毒基因复制复合体基因片段以及腺病毒骨架质粒进行连接，连接过夜。之后将连接产物转化DH5a感受态细胞。PCR鉴定阳性的克隆接种LB培养基，在37℃200-2250rpm条件下过夜。第二天回 收菌体，同时用质粒抽提试剂盒回收质粒。
优选的，将启动子基因片段插入已经连接病毒基因复制复合体片段的腺病毒骨架质粒。启动子基因片段插入腺病毒骨架质粒和上述方法类似。分别将已经连接病毒基因复制复合体的腺病毒骨架质粒与启动子进行双酶切，酶切在37℃进行；酶切结束后胶回收目的基因片段，目的基因片段的回收采用胶回收试剂盒，具体的操作可依据试剂盒的说明书进行即可。然后用DNA连接试剂盒/T4DNA连接酶将双酶切后的病毒基因复制复合体基因片段以及腺病毒骨架质粒进行连接，连接过夜。之后将连接产物转化DH5a感受态细胞。PCR鉴定阳性的克隆接种LB培养基，在37℃200-2250rpm条件下过夜。第二天回收菌体，同时用质粒抽提试剂盒回收质粒。
优选的，构建好腺病毒骨架质粒以双酶切，以及PCR的方法进行鉴定。
本发明解决了mRNA疫苗在体内被大量降解的风险，现有的包裹技术尽管可以避免mRNA疫苗的大量降解，但是解决不了mRNA难以直接进入细胞的缺陷(生物利用度低)。本发明所述的samRNA疫苗为携带有抗原的腺病毒载体，腺病毒载体可以直接感染人和动物细胞，从而将目标抗原基因注入宿主细胞。常规的mRNA疫苗进入宿主细胞后只能翻译一次，而本发明所述的samRNA疫苗可以以RNA为模板，合成大量的mRNA，从而极大的提高抗原的表达量。
普通复制缺陷型腺病毒载体，只能在类似于HEK293这样的细胞中进行复制，对于人和动物而言，只能感染，不能复制，因此在生物体内的复制能力有限。但是本发明所提供的插入病毒基因复制复合体和启动子的腺病毒载体疫苗感染宿主细胞后，将核酸注入宿主细胞内。随后在宿主细胞内将发生一系列动态反应，首先是病毒基因复制复合体基因的转录以及蛋白质的翻译。同时抗原基因被转录为mRNA。随着nsP1，nsP2，nsP3，nsP4蛋白的翻译，四个蛋白将形成病毒基因组病毒基因复制复合体，随后在该病毒基因复制复合体的作用下，以抗原基因RNA为模板，大量合成mRNA，这些mRNA利用宿主细胞的各种蛋白酶，合成目标抗原。所述的病毒基因复制复合体以RNA为模板合成mRNA是一个持续的过程，抗原表达持续的时间更长，因此修饰化腺病毒载体SamRNA疫苗的作用更持久。
文献报道以病毒作为SamRNA的载体，但是缺点也很明显，首先用于samRNA的病毒均为RNA病毒，而且为正义RNA病毒，这些病毒的感染和复制能力都有限。宿主限制性导致以病毒为载体的SamRNA生产非常困难，严格的宿主细胞限制导致常规的生产工艺很难满足这类病毒的生产，此外扩增能力有限更是严重影响病毒的产量。此外以病毒为载体的SamRNA，在基因组重组构建的过程中，RNA病毒的外膜结构基因被抗原的基因替代。病毒外膜结构基因的删除的优点是病毒感染动物和人宿主细胞后不会组装形成病毒，降低在感染或者致病的风险。但是缺点显而易见，结构基因的缺失，导致该病毒载体疫苗常规条件下没有办法完成病毒颗粒的组装，商业化生产的技术难度较高。
附图说明
图1病毒基因复制复合体蛋白基因插入骨架质粒鉴定结果；
图2启动子序列插入骨架质粒鉴定结果；
图3修饰化的骨架质粒；
图4 Ad-SamRNA-gE构建流程图；
图5 Ad-SamRNA-gE双酶切检定；
图6 CL-4B纯化Ad-SamRNA-gE图谱；
图7 Ad-SamRNA-gE纯度分析；
图8不同载体的免疫原性，左侧为一免的免疫原性，右侧为二免的免疫原性；
图 9不同载体gE基因的表达量；
图10分子佐剂增强Ad-SamRNA-gE的免疫原性，左侧为一免的免疫原性，右侧为二免的免疫原性。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1病毒基因复制复合体和启动子基因片段插入骨架质粒
病毒基因复制复合体和启动子基因片段的合成：
在具体实施过程中，病毒基因复制复合体nsP1、nsP2、nsP3和nsP4的基因序列分别为SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9，依据上述序列合成病毒基因复制复合体基因片段，四个抗原蛋白基因合成为一个大的基因片段。
启动子依据SEQ ID NO.5合成启动子基因片段，同时在启动子基因片段两端加入酶切位点。
以pAdEasy-1腺病毒骨架质粒为例，呈现病毒基因复制复合体和启动子插入pAdEasy-1的过程。
首先将病毒基因复制复合体基因片段插入pAdEasy-1腺病毒骨架质粒，合成的4个病毒基因复制复合体蛋白基因长片段两侧都含有pacI酶切位点。分别以pac I酶酶切pAdEasy-1和4个病毒基因复制复合体蛋白基因。胶回收后，用T4DNA连接酶进行连接。理论上病毒基因复制复合体蛋白基因有两种连接方式(倒着插入骨架质粒和顺着插入骨架质粒)，转化DH5a后，分别挑单克，通过PCR的方法来鉴别按照预期插入骨架质粒的载体。PCR鉴定结果见图1，1号和2号克隆都正确插入病毒基因复制复合体蛋白基因片段，选择1号克隆进行启动子基因片段插入的操作。
将启动子基因和已经插入病毒基因复制复合体基因的骨架质粒，以ClaI进行酶切，胶回收后，用T4DNA连接酶进行连接。理论上启动子蛋白基因有两种连接方式(倒着插入骨架质粒和顺着插入骨架质粒)，转化DH5a后，分别挑单克，通过PCR的方法来鉴别按照预期插入骨架质粒的载体。PCR鉴定结果见图2，该步操作中，3号、4号、5号克隆正确插入启动子序列。
经过以上两步，将得到加入病毒基因复制复合体和启动子序列的骨架质粒pAdEasy-13号，4号和5号，pAdEasy-1骨架质粒中启动子和病毒基因复制复合体的位置见图3。
根据同样的方法可以在其他骨架质粒上完成启动子和病毒基因复制复合体序列的插入。
实施例2：Ad-SamRNA-gE的构建
以带状疱疹的gE蛋白抗原为例，进行Ad(腺病毒)-SamRNA疫苗构建工艺的展示。
合成gE抗原基因，同时在基因片段的两侧加入酶切位点，具体的酶切位点根据所选择的重组系统以及穿梭质粒来定，本领域专业技术人员都掌握该方法。下面以Adeasy载体系统为例进行Ad-SamRNA疫苗构建工艺的展示。
首先将gE抗原基因连接到穿梭质粒pS-C中形成pS-C-gE，所述连接方法采用业内常用的实验方法。穿梭质粒pS-C以及gE抗原基因以KpnⅠ、XhoⅠ同时进行双酶切(酶切反应体系见下表1)，酶切反应结束后，用胶回收试剂盒回收目的片段(回收方法见说明书)。然后以T4DNA连接酶连接线性化的抗原片段和质粒(反应体系见下表2)。
对于采用其他腺病毒载体系统的构建方法和上述类似。
表1：双酶切反应体系(50ul)
Figure PCTCN2020070734-appb-000001
双酶切反应在37℃酶切至少4h以上。
表2酶连接反应体系(10ul)
Figure PCTCN2020070734-appb-000002
连接反应在4℃进行，过夜连接。
含有抗原基因的穿梭质粒pS-C-gE和插入病毒基因复制复合体、启动子的腺病毒骨架质粒(如pAdEasy-1)共转染，两个质粒将发生同源重组，分离得到Ad-SamRNA-gE，所述Ad-SamRNA构建流程图见图4。
采用双酶切的方法对构建好Ad-SamRNA-gE进行检定，检定构建成功的载体将用于疫苗的生产和免疫原性的评价，双酶切的结果如下图5：泳道M为marker，1为腺病毒载体双酶切，2为Ad-SamRNA-gE双酶切。结果表明，gE抗原基因已经正确的整和到腺病毒载体系统中。
实施例3 Ad-SamRNA-gE疫苗的制备
以带状疱疹的gE蛋白抗原为例，进行Ad-SamRNA疫苗制备工艺的展示。
腺病毒载体可在293细胞中大量繁殖，Admax腺病毒载体系统多采用HEK293细胞，而采用Adeasy重组腺病毒载体细胞则采用AD293细胞。
Ad-SamRNA-gE以MOI＝5—10感染293细胞，感染至少40h后8000g离心10min收集细胞沉淀。细胞沉淀以PB或者裂解(2mM MgCl 2，50mM HEPES，PH7.5)缓冲液溶解后，-80℃反复冻融三次裂解细胞，离心去除细胞碎片，上清过CL-4B，经一步层析就可以得到目标病毒。
细胞裂解液中除了细胞碎片，各种杂蛋白就是Ad-SamRNA-gE，细胞碎片通过离心就可以去除。相比较杂蛋白，Ad-SamRNA-gE分子量远大于杂蛋白，因此，离心上清液通过CL-4B一步工艺就可以得到纯病毒。
CL-4B纯化Ad-SamRNA-gE结果如图6，Ad-SamRNA-gE分子量最大，因此最先洗脱，图6中的峰1即为Ad-SamRNA-gE。
以HPLC分析收获的Ad-SamRNA-gE的纯度，实验选择TSK5000的柱子，HPLC结果见图7，可以看出收获的病毒看不到杂质峰，病毒液的纯度很高。
实施例4 Ad-SamRNA-gE疫苗免疫原性研究
以带状疱疹的gE蛋白抗原为例，进行Ad-SamRNA疫苗免疫原性的展示。
实验组：实施例3制备的Ad-SamRNA-gE组；
对照组1：gE蛋白mRNA直接注射组，简称为mRNA-gE；
对照组2：普通腺病毒为载体的gE蛋白抗原基因疫苗，简称为Ad-gE；
阴性对照组：生理盐水免疫组
实验动物：取NIH小鼠，每组10只，体重为12-14g，
免疫方式：皮下注射，实验组、对照组1、对照组2药物单次注射剂量为1×10 8IFU，每只小鼠免疫两针，两针间隔4周，第二针免前眼眶采集一免血，最后一次免后28天摘眼球采集二免血清，ELISA法测定血清中的抗体滴度，结果如图8所示。
根据图8结果可以看出，Ad-SamRNA-gE的免疫原性优于Ad-gE，也明显优于mRNA-gE，差异显著。因此，证明本发明所制备的以修饰化腺病毒为载体的抗原基因疫苗，可极大提高mRNA疫苗的免疫原性。
实施例5 Ad-SamRNA-gE和Ad-gE表达量的测定
将构建好Ad-SamRNA-gE和Ad-gE以MOI＝10感染293细胞，37℃5％CO 2培养箱培养40h后离心收集细胞上清和沉淀，细胞沉淀以裂解缓冲液(2mM MgCl 2，50mM HEPES，PH7.5)裂解；通过SDS-PAGE分析两个载体中gE蛋白的表达量。结果见图9.
图9中说明相比较对照组Ad-gE，供试品Ad-SamRNA-gE感染细胞后gE蛋白的表达量明显高。和实施例4中Ad-SamRNA-gE的免疫原性远高于Ad-gE相对应。
实施例6分子佐剂对Ad-SamRNA-gE的免疫原性
以带状疱疹的gE蛋白抗原为例，进行Ad-SamRNA疫苗免疫原性的展示。
实验组：分别制备含有C3b和不含有C3b的Ad-SamRNA-gE；
阴性对照组：生理盐水免疫组
实验动物：取NIH小鼠，每组10只，体重为12-14g，
免疫方式：皮下注射，实验组、对照组1、对照组2药物单次注射剂量为1×10 8IFU，每只小鼠免疫两针，两针间隔4周，第二针免前眼眶采集一免血，最后一次免后28天摘眼球采集二免血清，ELISA法测定血清中的抗体滴度，结果如图10所示。
根据图10结果可以看出，C3b可增强Ad-SamRNA-gE的免疫原性。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Figure PCTCN2020070734-appb-000003
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Figure PCTCN2020070734-appb-000006
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Claims (14)

  1. 一种SamRNA疫苗，所述的疫苗包括重组病毒载体，所述的重组病毒载体包括：i)病毒基因复制复合体，所述的病毒基因复制复合体包括编码病毒基因复制相关蛋白nsP1、nsP2、nsP3和nsP4的核苷酸序列，和ii)编码至少一种抗原的核苷酸序列。
  2. 根据权利要求1所述的SamRNA疫苗，其特征在于，所述的重组病毒载体为重组腺病毒、黑猩猩型腺病毒、重组水疱性口炎病毒、重组痘病毒、重组登革热病毒、重组昆津病毒、重组仙台病毒或重组犬瘟热病毒。
  3. 根据权利要求1所述的SamRNA疫苗，其特征在于，所述的抗原引起针对细菌、病毒、真菌或寄生虫的免疫应答。
  4. 根据权利要求3所述的SamRNA疫苗，其特征在于，所述的抗原为人带状疱疹病毒gE蛋白、轮状病毒VP4或VP7、HPV-L1蛋白或埃博拉病毒的gP蛋白。
  5. 根据权利要求1所述的SamRNA疫苗，其特征在于，所述的抗原为肿瘤特异抗原，选自NY-ESO-1、SSX2、SCP1、RAGE、BAGE、GAGE、MAGE家族多肽、p53、p21/Ras、CDK4、MUM1、caspase-8、CIA0205、HLA-A2-R1701、β-联蛋白、TCR、BCR-abl、磷酸甘油醛异构酶、KIA0205、CDC-27、LDLR-FUT、Galectin 4、Galectin 9、蛋白酶3、WT1、碳酸酐酶、醛缩酶A、PRAME、HER-2/neu、乳腺珠蛋白、甲胎蛋白、KSA、胃泌素、端粒酶催化蛋白、MUC-1、G-250、p53、癌胚抗原、黑素瘤-黑素细胞分化抗原、PAP、PSA、PSMA、PSH-P1、PSM-P1、PSM-P2、p15、Hom/Mel-40、H-Ras、E2A-PRL、H4-RET、IGH-IGK、MYL-RAR、EB病毒抗原、EBNA、人乳头瘤病毒抗原、乙型和丙型肝炎病毒抗原、人类嗜T淋巴细胞病毒抗原、TSP-180、p185erbB2、p180erbB-3、c-met、mn-23H1、TAG-72-4、CA 19-9、CA 72-4、CAM 17.1、NuMa、K-ras、p16、TAGE、PSCA、CT7、43-9F、5T4、791Tgp72、β-HCG、BCA225、BTAA、CA 125、CA 15-3(CA 27.29\BCAA)、CA 195、CA 242、CA-50、CAM43、CD68\KP1、CO-029、FGF-5、Ga733(EpCAM)、HTgp-175、M344、MA-50、MG7-Ag、MOV18、NB/70K、NY-CO-1、RCAS1、SDCCAG16、TAAL6、TAG72、TLP、TPS。
  6. 根据权利要求1所述的SamRNA疫苗，其特征在于，所述的重组病毒载体由修饰 化腺病毒骨架质粒与含有编码至少一种抗原的核苷酸序列的穿梭质粒共转染得到，所述修饰化腺病毒骨架质粒上包括病毒基因复制复合体，所述病毒基因复制复合体为编码病毒基因复制相关蛋白nsP1、nsP2、nsP3和nsP4的基因序列。
  7. 根据权利要求6所述的SamRNA疫苗，其特征在于，所述腺病毒骨架质粒选自：pAdEasy-1、pAdEasy-2、pBHG11、pBHG-fiber5或pBHG-fiber35。
  8. 根据权利要求1所述的SamRNA疫苗，其特征在于，所述的重组病毒载体还包括：iii)转录抗原基因的启动子。
  9. 根据权利要求1所述的SamRNA疫苗，其特征在于，所述的重组病毒载体还包括：iv)编码至少一种佐剂的核苷酸序列。
  10. 根据权利要求8所述的SamRNA疫苗，其特征在于，所述的佐剂选自C3b、GM-CSF、IL-17、IFN、IL-15、IL-21、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-12、INF-α、INF-γ、CpG。
  11. 一种权利要求1所述的SamRNA疫苗的制备方法，包括：修饰化腺病毒骨架质粒的构建；抗原基因片段的克隆；穿梭质粒的构建；穿梭质粒与腺病毒骨架质粒共转染与包装，所述修饰化腺病毒骨架质粒上包括病毒基因复制复合体，所述病毒基因复制复合体为编码病毒基因复制相关蛋白nsP1、nsP2、nsP3和nsP4的基因序列。
  12. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1-10任一所述的SamRNA疫苗和药学上可接受的辅料。
  13. 权利要求1-10任一所述的SamRNA疫苗或权利要求12所述的药物组合物在制备用于哺乳动物的疫苗中的应用。
  14. 一种修饰化腺病毒骨架质粒，其特征在于，所述腺病毒骨架质粒上包括病毒基因复制复合体，所述病毒基因复制复合体为编码病毒基因复制相关蛋白nsP1、nsP2、nsP3和nsP4的基因序列，所述腺病毒骨架质粒选自：pAdEasy-1、pAdEasy-2、pBHG11、pBHG-fiber5或pBHG-fiber35。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11793843B2 (en) 2019-01-10 2023-10-24 Janssen Biotech, Inc. Prostate neoantigens and their uses
US12018289B2 (en) 2019-11-18 2024-06-25 Janssen Biotech, Inc. Vaccines based on mutant CALR and JAK2 and their uses
US12295997B2 (en) 2020-07-06 2025-05-13 Janssen Biotech, Inc. Prostate neoantigens and their uses

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109806390A (zh) * 2019-01-07 2019-05-28 康希诺生物股份公司 一种SamRNA疫苗及其制备方法
CN110448689A (zh) * 2019-08-06 2019-11-15 太仓美诺恒康生物技术有限公司 mRNA疫苗及其试剂盒、应用
CN111926042B (zh) * 2020-06-09 2021-12-07 深圳豪石生物科技有限公司 一种树突状细胞制剂及其应用
CN112625095B (zh) * 2021-01-13 2023-01-03 武汉科前生物股份有限公司 一种猪轮状病毒重组蛋白以及表达该蛋白的重组腺病毒和应用
CN116875520B (zh) * 2023-07-12 2024-06-14 吉林农业大学 表达核糖体失活蛋白的乳酸菌及其在抗轮状病毒中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101365484A (zh) * 2005-08-15 2009-02-11 瓦克辛公司 通过给药非复制型载体化疫苗而进行的对禽类的免疫
WO2018014008A1 (en) * 2016-07-15 2018-01-18 Etubics Corporation Compositions and methods for alphavirus vaccination
WO2018223103A1 (en) * 2017-06-02 2018-12-06 Etubics Corporation Compositions and methods for tumor vaccination and immunotherapy involving her antigens
CN109806390A (zh) * 2019-01-07 2019-05-28 康希诺生物股份公司 一种SamRNA疫苗及其制备方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MXPA03001680A (es) * 2000-08-29 2004-11-01 Wyeth Corp Empaque de particulas de un replicon del virus de acido ribonucleico (arn) de hebra positiva.
GR20060100496A (el) * 2006-09-01 2008-04-15 Παρθενιος Μπουλικας ΛΙΠΟΣΩΜΑΤΙΚΑ ΕΓΚΑΨΥΛΙΩΜΕΝΟΙ ΦΟΡΕΙΣ-ΥΒΡΙΔΙΟΥ ΑΔΕΝΟΙΟΥ - ΙΟΥ SEMLIKI FOREST (SFV), ΠΟΥ ΦΕΡΕΙ ΚΑΤΑΣΚΕΥΑΣΜΑΤΑ RNAi ΚΑΙ ΘΕΡΑΠΕΥΤΙΚΑ ΓΟΝΙΔΙΑ ΠΟΥ ΧΡΗΣΙΜΟΠΟΙΟΥΝΤΑΙ ΕΝΑΝΤΙΟΝ ΣΤΟΧΩΝ ΚΑΡΚΙΝΟΥ ΚΑΙ ΑΛΛΩΝ ΑΣΘΕΝΕΙΩΝ
CN106008667B (zh) * 2016-06-03 2019-11-26 南开大学 短肽、其作为疫苗佐剂的应用及疫苗
CN111956797B (zh) * 2020-07-10 2022-05-13 清华大学 新型疫苗佐剂及其在新冠肺炎疫苗和其他疫苗中的应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101365484A (zh) * 2005-08-15 2009-02-11 瓦克辛公司 通过给药非复制型载体化疫苗而进行的对禽类的免疫
WO2018014008A1 (en) * 2016-07-15 2018-01-18 Etubics Corporation Compositions and methods for alphavirus vaccination
WO2018223103A1 (en) * 2017-06-02 2018-12-06 Etubics Corporation Compositions and methods for tumor vaccination and immunotherapy involving her antigens
CN109806390A (zh) * 2019-01-07 2019-05-28 康希诺生物股份公司 一种SamRNA疫苗及其制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3900741A4 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11793843B2 (en) 2019-01-10 2023-10-24 Janssen Biotech, Inc. Prostate neoantigens and their uses
US12582683B2 (en) 2019-01-10 2026-03-24 Janssen Biotech, Inc. Prostate neoantigens and their uses
US12018289B2 (en) 2019-11-18 2024-06-25 Janssen Biotech, Inc. Vaccines based on mutant CALR and JAK2 and their uses
US12295997B2 (en) 2020-07-06 2025-05-13 Janssen Biotech, Inc. Prostate neoantigens and their uses

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