WO2021139147A1 - Vaccin à adénovirus bivalent - Google Patents

Vaccin à adénovirus bivalent Download PDF

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WO2021139147A1
WO2021139147A1 PCT/CN2020/106400 CN2020106400W WO2021139147A1 WO 2021139147 A1 WO2021139147 A1 WO 2021139147A1 CN 2020106400 W CN2020106400 W CN 2020106400W WO 2021139147 A1 WO2021139147 A1 WO 2021139147A1
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adenovirus
gene
replication
plasmid
orf2
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陈凌
杨臣臣
刘晓琳
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广州恩宝生物医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention belongs to the technical field of virus immunology, and specifically relates to an adenovirus bivalent vaccine.
  • Adenovirus is a double-stranded DNA virus with a genome length of about 35-40 kb. It is known that human adenoviruses are divided into 7 subgroups (A ⁇ G), including more than 50 serotypes (more than 90 genotypes). After infection, patients mainly cause acute respiratory diseases (adenovirus B and C subgroups) and conjunctivitis (Adenovirus B and D subgroups) and gastroenteritis (Adenovirus F subgroups 41 and 42, G subgroup 52). Respiratory tract infections caused by adenovirus are mostly caused by adenovirus types 3, 4 and 7. Ad4 and Ad7 broke out mainly in places where young people and teenagers gather such as troops and schools, and even led to the death of patients. However, there is no specific medicine for the treatment of adenovirus infection, and only supportive treatment can be taken clinically.
  • the vaccine is an oral live virus vaccine in the form of enteric-coated capsules, which are passed on human embryonic kidney diploid fibroblasts and are produced by freeze-dehydration and mixing with cellulose lactose.
  • the use of the vaccine effectively controlled the outbreak of adenovirus infections in the US military.
  • the Ad4 and Ad7 vaccines used by the U.S. military are extremely risky.
  • the vaccine is mainly low-dose wild-type adenovirus.
  • There is a risk that the residual live virus will be discharged from the intestine to pollute the living environment, and it is very easy to cause secondary pollution of the virus.
  • the safety is poor, so it cannot be widely used in the general population. Therefore, it is very necessary to develop a replication-deficient adenovirus vaccine that is highly safe and can prevent the strong strains of Ad4 and Ad7.
  • Replication-deficient adenovirus vectors have been widely used in vaccine development, gene therapy and other fields. They are not only safe, but also have a strong immune response in the organism. Studies have shown that the E1 gene of adenovirus is an essential gene for its replication and proliferation, and the E3 gene plays a key role in resisting the host's immune system. After knocking out the E1 and E3 genes, the adenovirus loses the ability to replicate in normal humans and has an attenuated phenotype in this respect. At the same time, the main surface antigens Hexon and Fiber of Ad4 and Ad7 are not affected and will not affect the immunogenicity of the vaccine.
  • replication-defective adenovirus as a vaccine can effectively increase the completeness and scope of the vaccine.
  • Replication-deficient adenovirus can be produced in complementary cell lines, such as 293 cells and PerC6 cells expressing the Ad5E1 gene.
  • complementary cell lines such as 293 cells and PerC6 cells expressing the Ad5E1 gene.
  • studies have found that many adenoviruses, especially non-C subgroup adenoviruses, have lower yields in these production cell lines after knocking out the E1 and E3 genes.
  • Ad5E1B 55K cannot interact with the B subtype adenovirus E4Orf6 protein. It cannot effectively inhibit the nucleation of host cell mRNA, and cannot increase the expression of late viral proteins.
  • adenoviruses require 293 cell lines or other cell lines expressing the corresponding E1 gene to be produced. Therefore, replication-defective Ad3, Ad4 and Ad7, which only knock out the E1 and E3 genes, are difficult to produce in the vaccine production cell line 293 or PerC6. Improving its production capacity in these cell lines is currently a bottleneck technical problem that needs to be resolved.
  • the purpose of the present invention is to overcome the defects of the prior art and provide a preparation method and application of replication-defective recombinant Ad4 and Ad7 that can be amplified on a large scale in vaccine production cell strains, and the replication-defective recombinant Ad4 and Ad7 can be used to a certain extent.
  • Ad4 and Ad7 bivalent vaccines are prepared by mixing Ad4 and Ad7 in a ratio of 5%.
  • the immune body can effectively stimulate the body to produce humoral immunity and cellular immune response, and produce specific Ad4 and Ad7 neutralizing antibodies, which are used to prevent the infection of Ad4 and Ad7 pathogens.
  • a composition comprising replication-deficient human type 4 adenovirus and type 7 adenovirus.
  • the E1 and E3 genes of the replication-deficient type 4 adenovirus are deleted, and part of the coding frame of the E4 gene is replaced with the corresponding coding frame of the human type 5 adenovirus E4 gene.
  • the E1 and E3 genes of the replication-deficient human adenovirus type 7 are deleted, and part of the coding frame of the E4 gene is replaced with the corresponding coding frame of the human type 5 adenovirus E4 gene.
  • At least one of the E1 gene regions of the replication-deficient adenovirus integrates the foreign gene expression cassette.
  • the exogenous gene expression cassette contains a nucleotide sequence that can induce an immune response in the human body or generate a biological reporter molecule or a tracking molecule for detection, or a nucleotide sequence that can regulate gene function or a therapeutic molecule.
  • the mixing ratio of the number of replication-defective human adenovirus type 4 and replication-defective human adenovirus type 7 virus particles is between 1:10 and 10:1.
  • composition in preparing vaccines, detecting reagents, regulating gene function or medicines.
  • any of the above-mentioned compositions further includes a pharmaceutically acceptable adjuvant, carrier, diluent or excipient.
  • the present invention replaces the Orf2, Orf3, Orf4, and Orf6 coding frames of the E4 gene of Ad4 and Ad7 with the corresponding coding frames of the Ad5 E4 gene, which greatly improves the replication-defective Ad4 and The safety of Ad7 and its ability to replicate in production cell lines.
  • the Ad4 and Ad7 bivalent vaccine prepared by the present invention can effectively induce Ad4 and Ad7 specific humoral immunity and cellular immunity in experimental animals after primary immunization and booster immunization, and produce specific Ad4 and Ad7 neutralizing antibodies , Used to prevent the infection of Ad4 and Ad7 pathogens.
  • the bivalent vaccine of the present invention greatly improves the safety and scope of use of the vaccine while retaining its immunogenicity.
  • Figure 1 is a flow chart of the construction of pAd4 plasmid.
  • Figure 2 is a flow chart of the construction of pAd4 ⁇ E3 plasmid.
  • Figure 3 is a flow chart of the construction of pAd4 ⁇ E1 ⁇ E3 plasmid.
  • Figure 4 is a flow chart of the construction of pAd4 ⁇ E1 ⁇ E3 (Orf2-6) plasmid.
  • Figure 5 is a flow chart of the construction of pAd4 ⁇ E1 ⁇ E3(Orf2-6)-EGFP plasmid.
  • Figure 6 shows the enzyme digestion and identification results of pAd4 plasmid, pAd4 ⁇ E3 plasmid, pAd4 ⁇ E1 ⁇ E3 plasmid, pAd4 ⁇ E1 ⁇ E3 (Orf2-6) and pAd4 ⁇ E1 ⁇ E3 (Orf2-6)-EGFP plasmid.
  • Figure 7 shows the results of the production and purification of the replication-defective Ad4 vector.
  • Figure 8 shows the results of the plaque formation experiment of the replication-deficient Ad4 vector in HEK293 and A549 cells.
  • Figure 9 shows the construction flow chart (A) of pAd7 plasmid and the result of restriction digestion (B).
  • Figure 10 is the construction flow chart (A) and restriction diagram (B) of pAd7 ⁇ E3 plasmid.
  • Figure 11 is the construction flow chart (A) and restriction diagram (B) of pAd7 ⁇ E1 ⁇ E3 plasmid.
  • Figure 12 is the construction flow chart (A) and restriction diagram (B) of pAd7 ⁇ E1 ⁇ E3 (Orf2-6) plasmid.
  • Figure 13 is the construction flow chart (A) and restriction diagram (B) of pAd7 ⁇ E1 ⁇ E3(Orf2-6)-EGFP plasmid.
  • Figure 14 shows the results of the production and purification of the replication-defective Ad7 vector.
  • Figure 15 shows the results of the plaque formation experiment of the replication-deficient Ad7 vector in HEK293 and A549 cells.
  • Figure 16 shows the results of determination of the levels of Ad4 and Ad7 neutralizing antibodies in the serum of rhesus monkeys.
  • Figure 17 shows the results of determining the levels of Ad3, Ad11 and Ad14 cross-neutralizing antibodies in the serum of rhesus monkeys.
  • Figure 18 shows the results of the PMBC ELISPOT experiment in rhesus monkeys.
  • human type 4 adenovirus, human type 7 adenovirus refers to the type 4 adenovirus and type 7 adenovirus known to those of ordinary skill in the art.
  • the adenovirus genome used in the examples is also derived from these known adenoviruses.
  • Human adenovirus The replication-deficient human type 4 adenovirus vector and human type 7 adenovirus vector of the present invention are not limited to the specific clinical isolates used in the examples.
  • Ad4 genome as a template for PCR amplification, recombinant arms Ad4-L and Ad4-R were obtained.
  • Ad4-L primer sequence
  • Ad4-L Fw ATAGAATTCGGGGTGGAGTGTTTTTGCAAG (SEQ ID NO.1);
  • Ad4-L Rw TTTACTAGTGTTTAAACGTAATCGAAACCTCCACGTAATGG (SEQ ID NO. 2).
  • PCR program 95°C, 30 seconds; 62°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • Ad4-R primer sequence
  • Ad4-R Fw ACTAGTAGCTGGATCCAAGCCTCGAGGCACTACAATG (SEQ ID NO.3);
  • Ad4-R Rw CCTGCCGTTCGACGATGCGATCGCCATCATCAATAATATACCTTATAGATGG (SEQ ID NO. 4).
  • PCR program 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 80 seconds; 25 cycles.
  • Homologous recombinase was used to connect to pSIMPLE 19 (EcoRV) vector (TaKaRa) to obtain Ad4 genome circularized shuttle plasmid pT-Ad4 (L+R).
  • pT-Ad4(L+R) was linearized with SpeI and BamHI, it was co-transformed with Ad4 genome into BJ5183 competent cells; after screening for ampicillin resistance, the plasmid was manually extracted and further transformed into XL-Blue competent cells (Beijing Sibai Hui Biotechnology Co., Ltd.); manually extract the plasmid to obtain pAd4, the technical process is shown in Figure 1, and the restriction diagram is shown in Figure 6.
  • L-delE3 (or called delE3-4L) primer sequence:
  • L-delE3 F GACATTGATTATTGACTAGTTTCAACACCTGGACCACTGCC (SEQ ID NO.5);
  • L-delE3 R ATTTAAATTGGAATTCAAGGTCAGAGACTGGTTGAAGGATG (SEQ ID NO. 6).
  • PCR program 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • R-delE3 F GAATTCCAATTTAAATAGCAGTCTGGCGATACCAAGG (SEQ ID NO. 7);
  • R-delE3 R GTTTAAACGGGCCCTCTAGACATTCTTGGTGGTGACAGGGTC (SEQ ID NO. 8).
  • PCR program 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • the shuttle plasmid pVax-delE3(L+R) which knocked out the E3 gene was obtained by ligating the homologous recombinase to pVax vector.
  • L-delE1 (or L-delK) primer sequence:
  • L-delE1 F CCAGATATACGCGTGTATACCATCATCAATAATATACCTTATAGATGG (SEQ ID NO.9);
  • L-delE1 R GATATCAAGTTAATTAAAATCGAAACCTCCACGTAAAC (SEQ ID NO. 10).
  • PCR program 95°C, 30 seconds; 50°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • R-delE1 (or R-delK) primer sequence:
  • R-delE1 F TTAATTAACTTGATATCGTGTGGATGTGACGGAGGAC (SEQ ID NO.11);
  • R-delE1 R GCCCAGTAGAAGCGCCGGTGCGGGATTATTAGTGGAACTTGAG (SEQ ID NO. 12).
  • PCR program 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • Homologous recombinase was used to connect to the pVax vector (Invitrogen) to obtain the shuttle plasmid pVax-delE1(L+R) which knocked out the E1 gene.
  • pVax-delE1(L+R) was linearized with BstZ17I+SgrAI double enzyme digestion, it was co-transformed with pAd4 ⁇ E3 linearized by PsiI to transform BJ5183 competent cells (Stratagene); after screening for ampicillin resistance, the plasmid was manually extracted, and further Transform XL-Blue competent cells (Beijing Sibaihui Biotechnology Co., Ltd.); manually extract the plasmid to obtain the pAd4 ⁇ E1 ⁇ E3 plasmid.
  • the technical process is shown in Figure 3, and the restriction map is shown in Figure 6.
  • a PacI restriction site was introduced into the pro-E1 region of the adenovirus genome in the resulting pAd4 ⁇ E1 ⁇ E3 plasmid to facilitate subsequent cloning.
  • 4E4R F CATTGATTATTGACTAGAGTATACCATGCTGGCGCGGCTGACCTAGCT (SEQ ID NO.13);
  • PCR program 95°C, 30 seconds; 60°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • PCR program 95°C, 30 seconds; 60°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • the Ad5 genome was used as a template for PCR amplification to obtain Orf2-6 of the Ad5 adenovirus E4 gene.
  • Orf2-6 primer sequence :
  • Orf2-6 F TCCTCGGTGGTTGGAATCACAGCTACATGGGGGTAGAGTCATAATCG (SEQ ID NO.17);
  • Orf2-6 R CCAAAAACACTAACCATGCTGGAATGCAGAAACCCGCAGACATGTTTGAG (SEQ ID NO.18).
  • PCR program 95°C, 30 seconds; 65°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • Homologous recombinase was used to connect to the pGK143-(L+R) vector linearized by BamHI to obtain the shuttle plasmid pGK143-Orf2-6 modified by the Ad4 E4 gene.
  • pGK143-Orf2-6 was linearized with BstZ17I+SgrAI double enzyme digestion, it was co-transformed with SwaI linearized pAd4 ⁇ E1 ⁇ E3 to transform BJ5183 competent cells (Stratagene); after screening for ampicillin resistance, the plasmid was manually extracted and further transformed into XL- Blue Competent Cells (Beijing Sibaihui Biotechnology Co., Ltd.); the plasmid was manually extracted to obtain the pAd4 ⁇ E1 ⁇ E3 (Orf2-6) plasmid.
  • the technical process is shown in Figure 4, and the restriction map is shown in Figure 6.
  • PCR program 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • PCR program 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 1 minute, 30 seconds; 25 cycles.
  • the homologous recombination arms 4SE1L and 4SE1R of the E1 region were connected to the pVax vector by using the homologous recombinase (Vazyme) ligation to obtain the shuttle plasmid pGK41-(L+R) carrying the recombination arms.
  • PCR program 95°C, 30 seconds; 66°C, 30 seconds; 72°C, 30s; 25 cycles.
  • Homologous recombinase (Vazyme) was used to connect the CMV-EGFP-BGH expression cassette to the pGK41-(L+R) vector to obtain the shuttle plasmid pGK41-EGFP carrying the recombination arm.
  • pGK41-EGFP plasmid was cut with BstZ17I+SgrAI and recovered by ethanol precipitation; pAd4 ⁇ E1 ⁇ E3 (Orf2-6) was linearized with PacI and recovered by ethanol precipitation; co-transformed with BJ5183, homologous recombination yielded pAd4 ⁇ E1 ⁇ E3 (Orf2-6) carrying the foreign gene expression cassette -EGFP plasmid, the technical process is shown in Figure 5. Refer to Figure 6 for the results of double restriction digestion.
  • pAd4 ⁇ E1 ⁇ E3(Orf2-6) and pAd4 ⁇ E1 ⁇ E3(Orf2-6)-EGFP were linearized with AsiSI, recovered by ethanol precipitation, and transfected with cationic liposome transfection method into 293 cells. 8 hours after transfection, 2 ml Incubate in DMEM medium with 5% fetal bovine serum for 7-10 days to observe the cytopathic changes; after the virus has emerged, collect the cells and culture supernatant, freeze and thaw them in a 37-degree water bath and liquid nitrogen three times and centrifuge to remove cell debris.
  • plaque formation experiments were used to identify the growth ability of replication-deficient Ad4 virus in HEK293 helper cells and A549 non-helper cells.
  • 293 or A549 cells in the six-well plate grow to 90% full, they are infected with Ad4 ⁇ E1 ⁇ E3(Orf2-6)-EGFP, and the infection titer is 1X10 7 Vp/well.
  • the medium was aspirated, and a 1% agarose gel (containing 1% agarose, 1% BSA, 1 ⁇ MEM medium) was spread. After being placed in a 37°C incubator for 9-12 days, observe the formation of virus clones under a fluorescence microscope, and take pictures and record. The result is shown in Figure 8.
  • the replication-defective Ad4 ⁇ E1 ⁇ E3(Orf2-6)-EGFP can only form plaques in HEK293 cells, but not in A549 cells. This indicates that the replication-deficient Ad4 vector can effectively proliferate in HEK293 cells with E1 gene complementation, but it has no replication ability in non-helper cells such as A549 cells and has an attenuated phenotype. At the same time, the results also show that the replication-defective human type 4 adenovirus vector can carry the reporter gene into the target cell, so it can be used in the reporter tracer system.
  • the left arm (L-Ad7) and the right arm (R-Ad7) of the Ad7 genome were obtained by PCR.
  • L-Ad7-F ACTGCGATCGCCTCTCTATTTAATATACCTTATAGATGG (SEQ ID NO.25);
  • L-Ad7-R ACATGGATCCTCACTGAAGATAATCTCCTGTGG (SEQ ID NO. 26).
  • PCR conditions 95°C, 3min; 95°C, 30s; 56°C for 30s; 72°C, 40s; cycles 30; 72°C, 5min; 12°C storage.
  • R-Ad7-F AGCTGGATCCGAACCACCAGTAATATCATCAAAG (SEQ ID NO.27);
  • R-Ad7-R TGAGCGATCGCCTCTCTATATAATATACCTTATAGATGGAA (SEQ ID NO. 28).
  • the PCR product and the T vector were ligated with three fragments using Exnase recombinase to obtain pT-Ad7(L+R).
  • pT-Ad7(L+R) was digested and linearized with BamHI, and then co-transformed with Ad7 genome into BJ5183 competent cells for recombination, and the ampicillin resistance plate was used for resistance screening, and the selected single clones were amplified and extracted
  • the plasmid was transformed into XL-Blue chemically competent cells, and the plasmid was extracted to obtain pAd7, which was identified by different enzyme digestion methods.
  • pAd7 introduced two AsisI digestion sites on both sides of the genome to facilitate subsequent linearization of the modified Ad7 genome Rescue the virus. The specific construction process is shown in Figure 9.
  • L- ⁇ E3-F CATACTAGTCTGTCTACTTCAACCCCTTCTCCG (SEQ ID NO.29);
  • L- ⁇ E3-R GCAGAATTCATTTAAATGGAGGAAGGGTCTGGGTCTTCTG (SEQ ID NO. 30).
  • PCR conditions 95°C, 3min; 95°C, 30s; 63°C 30s; 72°C, 30s; cycles 30; 72°C, 5min; 12°C storage.
  • R- ⁇ E3-F GCAGATATCATTTAAATAGACCCTATGCGGCCTAAGAGAC (SEQ ID NO.31);
  • R- ⁇ E3-R ACATCTAGAGACAGTTGGCTCTGGTGGGGT (SEQ ID NO.32).
  • PCR conditions 95°C, 3min; 95°C, 30s; 61°C 30s; 72°C, 40s; cycles 30; 72°C, 5min; 12°C storage.
  • L- ⁇ E3 was digested with SpeI+EcoRI, it was ligated to the pVax vector digested with the same restriction to obtain pVax-L- ⁇ E3.
  • R- ⁇ E3 was digested with EcoRV+XbaI and connected to the pVax-L- ⁇ E3 backbone of the same digestion to obtain pVax- ⁇ E3(L+R).
  • pVax- ⁇ E3(L+R) was linearized with SpeI+XbaI
  • pAd7 was linearized with EcoRI, recovered by ethanol precipitation, and then co-transformed into BJ5183 competent cells, spread to ampicillin resistant plates, hand-extracted plasmids, and continued to transform XL-Blue Competent cells were hand-extracted and the plasmids were digested for identification.
  • the genomic plasmid pAd7 ⁇ E3 with the E3 gene knocked out and the only single restriction site SwaI introduced in the E3 region was obtained.
  • the insertion of the SwaI restriction site facilitates linearization in the E3 gene region.
  • the schematic diagram of the construction of the shuttle plasmid and pAd7 ⁇ E3 plasmid and the restriction enzyme digestion results of the large plasmid are shown in Figure 10.
  • L- ⁇ E1-F ACTCACCGGCGGCGATCGCCTCTCTATTTAATATACCTTATAGATGG (SEQ ID NO.33);
  • L- ⁇ E1-R ATCACAATTGAATTCGTTTAAACGTAATCGAAACCTCCACGTAA (SEQ ID NO.34).
  • PCR conditions 95°C, 3min; 95°C, 30s; 54°C for 30s; 72°C, 30s; cycles 30; 72°C, 5min; 12°C storage.
  • R-SE1-F ATAGAATTC ACTAGTGAGGCCCGATCATTTGGTGCT (SEQ ID NO.35);
  • R-SE1-R ACGTATAC CTATCATTATGGATGAGTGCATGG (SEQ ID NO. 36).
  • PCR conditions 95°C, 3min; 95°C, 30s; 61°C 30s; 72°C, 1min 10s; cycles 30; 72°C, 5min; 12°C storage.
  • the PCR product and the T vector were ligated with three fragments using Exnase recombinase to obtain pT-Ad7(L+R).
  • pT-Ad7- ⁇ E1(L+R) was linearized with Bstz17I
  • pAd7 was linearized with AatII
  • the insertion of the PmeI restriction site facilitates linearization in the E1 gene region.
  • the construction diagram of the shuttle plasmid and pAd7 ⁇ E1 ⁇ E3 plasmid and the restriction enzyme digestion results of the large plasmid are shown in Figure 11.
  • L-SE4-F CGCGGATCTTCCAGAGATGTTTAAACAACCAGTTACTCCTAGAACAGTCAGC (SEQ ID NO.37);
  • R-SE4-R GCCTGCCGTTCGACGATGTTTAAAC CAGCTGGCACGACAGGTTTC (SEQ ID NO.40)
  • L-SE4 and R-SE4 fragments obtained by PCR and the blunt-ended T vector were ligated with three fragments to obtain p7SE4.
  • Ad5 Orf2-6-F TCACAGTCCAACTGCT CCTACATGGGGGTAGAGTCATAATCG (SEQ ID NO.41);
  • p7SE4-F CAATAGGTTACCGCGCTGCG (SEQ ID NO.43);
  • p7SE4 (Orf2-6) uses PmeI for restriction digestion and linearization
  • pAd7 ⁇ E1 ⁇ E3 uses SwaI for restriction digestion and linearization.
  • the above two digestion products are recovered by ethanol precipitation, and the BJ5183 competent cells are co-transformed for recombination, and the ampicillin plate is resistant.
  • the left arm SE1L and the right arm SE1R of the E1 gene region shuttle plasmid were obtained by PCR using the Ad7 genome as a template.
  • SE1L-F CCAGATATACGCGTGTATACTTAATTAACGGCATCAGAGCAGATTGTACTG (SEQ ID NO.45);
  • SE1L-R GTTTAAACAAGATTTAAATGTAATCGAAACCTCCACGTAAACG (SEQ ID NO. 46).
  • SE1R-F ATTTAAATCTTGTTTAAACGAATTCACTAGTGAGGCCCGATC (SEQ ID NO.47);
  • SE1R-R GCCCAGTAGAAGCGCCGGTGTTAATTAACAAGTAGCTTGTCCTCAGCCAGG (SEQ ID NO. 48).
  • the plasmid pVax was digested with Bstz17I+SgraI to recover the plasmid backbone, and then SE1L was obtained with the above PCR, and SE1R was ligated with three fragments using Exnase enzyme to obtain pSE1LR.
  • the primer sequence for amplifying CMV-EGFP-BGH is the primer sequence for amplifying CMV-EGFP-BGH
  • CMV-EGFP-BGH-F ACTAGTGAATTCGTTTACTAGTTATTAATAGTAATCAATTACGGG (SEQ ID NO.49);
  • pSE1LR was linearized by PmeI digestion, and then ligated with the CMV-EGFP-BGH expression box obtained by PCR amplification with Exnase enzyme to obtain pGK71-EGFP.
  • pGK71-EGFP is linearized with PacI
  • pAd7 ⁇ E1 ⁇ E3 (Orf2-6) is linearized with PmeI digestion
  • the above two digestion products are recovered by ethanol precipitation
  • the BJ5183 competent cells are co-transformed for recombination, and the ampicillin plate is resistant
  • Screening after screening the monoclonal amplification, the plasmid is extracted and transformed into XL-Blue competent cells, the plasmid is extracted to obtain pAd7 ⁇ E1 ⁇ E3(Orf2-6)-EGFP, and the plasmid is extracted and identified by restriction enzyme digestion.
  • the specific construction of pAd7 ⁇ E1 ⁇ E3(Orf2-6)-EGFP Refer to Figure 13 for the process and the identification results of large plasmids.
  • pAd7 ⁇ E1 ⁇ E3(Orf2-6) and pAd7 ⁇ E1 ⁇ E3(Orf2-6)-EGFP were linearized with AsiSI, recovered by ethanol precipitation, and transfected into 293 cells by cationic liposome transfection method, 4-6 hours after transfection, added 2 ml of DMEM medium containing 5% fetal bovine serum, incubate for 7-10 days, observe the cell pathology; after the virus is found, collect the cells and culture supernatant, freeze and thaw them in a 37°C water bath and liquid nitrogen three times and remove them by centrifugation Cell debris, the supernatant infects 10 cm dishes; 2-3 days later, collect cells and culture supernatant, freeze-thaw three times and centrifuge to remove cell debris, supernatants infect 10-15 15 cm dishes; 2-3 days later, collect Cells were repeatedly frozen and thawed three times and centrifuged to remove cell debris.
  • virus concentration OD260 ⁇ dilution factor ⁇ 36/genome length (Kb); virus stock solution is frozen at -80°C.
  • the results of the production and purification of the replication-defective Ad7 vector are shown in Figure 14.
  • the plaque experiment was used to determine the replication ability of the replication-deficient Ad7 vector in the helper cell 293 and the non-helper cell A549. Inoculate 293 or A549 cells in a 6-well cell plate. When the cell density approaches 100%, dilute the harvested P1 generation Ad7 ⁇ E1 ⁇ E3(Orf2-6)-EGFP virus stock solution and infect 293 or A549 cells respectively, each virus Do two repetitions for the concentration. After the virus infects the cells for 2 hours, aspirate the medium, and spread about 2ml agarose gel on each well (containing 1ml 1.4% agarose, 1ml 1 ⁇ MEM medium, 200ul BSA, 1 ⁇ penicillin antibiotic).
  • the replication-defective Ad4 and Ad7 vaccines purified by cesium chloride density gradient force centrifugation were carried out until the concentration of Ad4 was 4 ⁇ 10 11 vp/ml and the concentration of Ad7 was 4 ⁇ 10 11 vp/ml, and stored at -80°C.
  • Ad4 and Ad7 tetravalent vaccines were designed. As shown in Table 1, the immunogenicity of Ad4 and Ad7 bivalent vaccines was evaluated according to the designed immunization protocol.
  • Ad4 and Ad7 bivalent vaccines Ad4: 2 ⁇ 10 10 vp/ml, Ad7: 2 ⁇ 10 10 vp/ml).

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Abstract

La présente invention concerne un vaccin à adénovirus bivalent comprenant un adénovirus de type 4 humain à réplication déficiente et un adénovirus de type 7 humain à réplication déficiente. Les gènes E1 et E3 de l'adénovirus de type 4 humain à réplication déficiente et l'adénovirus de type 7 humain à réplication déficiente sont délétés. Une partie de la cassette de codage du gène E4 est remplacée par la cassette de codage correspondante du gène E4 de l'adénovirus de type 5 humain. Le vaccin à adénovirus bivalent peut stimuler efficacement le corps pour produire une réponse immunitaire humorale et une réponse immunitaire cellulaire pour produire des anticorps neutralisants spécifiques à titre élevé destinés à prévenir une infection pathogène.
PCT/CN2020/106400 2020-01-08 2020-07-31 Vaccin à adénovirus bivalent WO2021139147A1 (fr)

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CN111166875A (zh) * 2020-01-08 2020-05-19 广州恩宝生物医药科技有限公司 一种腺病毒二价疫苗
CN113908265B (zh) * 2020-07-10 2024-03-15 上海市公共卫生临床中心 一种人4型、7型腺病毒减毒活疫苗及其应用
CN112156181A (zh) * 2020-09-29 2021-01-01 广州恩宝生物医药科技有限公司 一种腺病毒四价疫苗
CN114150005B (zh) * 2022-02-09 2022-04-22 广州恩宝生物医药科技有限公司 用于预防SARS-CoV-2奥密克戎株的腺病毒载体疫苗

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