WO2004072274A1 - Vaccin a base de pseudoparticules virales ayant comme support un replicon de recombinaison du virus de la dengue - Google Patents
Vaccin a base de pseudoparticules virales ayant comme support un replicon de recombinaison du virus de la dengue Download PDFInfo
- Publication number
- WO2004072274A1 WO2004072274A1 PCT/CN2004/000088 CN2004000088W WO2004072274A1 WO 2004072274 A1 WO2004072274 A1 WO 2004072274A1 CN 2004000088 W CN2004000088 W CN 2004000088W WO 2004072274 A1 WO2004072274 A1 WO 2004072274A1
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- dengue virus
- replicon
- virus
- structural protein
- dengue
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- C12N2810/00—Vectors comprising a targeting moiety
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Definitions
- the present invention relates to a dengue virus, specifically a recombinant vaccine in which a structural protein gene of the dengue virus is replaced with a specific antigen, and a preparation method and use thereof.
- HPV human papilloma virus
- the HPV infection rate and infection level increase gradually with the severity of cervical lesions, which can be briefly described as: chronic cervicitis-pseudocondyloma-wart-like lesions-genital warts-cervical intraepithelial neoplasia-cervical cancer.
- HPV has a wide range of infected people.
- the infection rate of Chinese women is 10-40%, and the infection rate of European women is as high as 40-60%.
- the incidence of cervical cancer is second only to breast cancer in women.
- cervical cancer patients According to statistics from the World Health Organization, there are about 1.7 million cervical cancer patients worldwide, about 500,000 new cases each year, and more than 200,000 deaths. There are about 140,000 patients with cervical cancer in China and about 40,000 new cases each year. More than 75% of cervical cancer cases are associated with two types of HPV infection (types 16 and 18). In clinical practice, radiotherapy, surgery and chemotherapy are often used to treat cervical cancer, and a few are combined with traditional Chinese medicine and Chinese medicine, but the effect is not satisfactory. The 5-year survival rate for patients with cervical cancer is 65%. Cervical cancer has a higher recurrence rate, and the prognosis of relapse cases is poor. The 2-year survival rate is only 5%. The cost of cervical cancer screening and treatment in the United States is approximately $ 570 million per year.
- the three traditional therapies most commonly used in the treatment of malignant tumors are surgery, chemotherapy, and radiotherapy, which have defects such as high recurrence rates, large side effects, and drug resistance.
- the biological treatment of tumors, especially tumor immunotherapy or gene therapy has theoretically established its strategic position as a new model of tumor treatment due to its theoretically high specificity and low side effects.
- the clinical application effect and people's expectations are still so far. There is a big gap.
- Dead vaccine This is a tumor vaccine tested in early humans. It is relatively safe, but because inducing cellular immunity requires the expression and presentation of antigens in living cells, it cannot cause an effective cellular immunity.
- the DNA virus vector retains a complete DNA replication system and is a live vaccine. It can effectively replicate and express oncogene fragments, can continuously stimulate the cellular immune system, and generate cytotoxic T cells (CTLs) against tumors.
- CTLs cytotoxic T cells
- cancer gene fragments are generally used as the antigen, so that the antigenicity is reduced. But this DNA virus vaccine is low in safety.
- this W00153467 discloses a recombinant flavivirus comprising an exogenous nucleotide sequence encoding an exogenous amino acid sequence. Infecting a host cell with a recombinant flavivirus can provide exogenous nucleic acid in the host cell and produce an antigenic polypeptide encoded by the exogenous nucleic acid, thereby triggering an immune response to the exogenous polypeptide.
- its disadvantage is that it uses the yellow fever virus that can autonomously replicate and proliferate, retains the entire yellow fever virus genome sequence, and has no deletion of structural protein genes. Although no packaging system is required to construct a vaccine, it is less safe.
- W09928487 discloses a method for delivering and expressing a foreign fragment using a flavivirus kunj in virus (KUN) as a vector, in which a part of a structural gene is excised first, and then the foreign fragment is inserted to obtain a replicon. After the replicon infects the host cell, the helper virus provides the structural protein, which is then packaged to form a pseudoviral particle.
- KUN flavivirus kunj in virus
- the helper virus provides the structural protein, which is then packaged to form a pseudoviral particle.
- the potency of such KUN pseudoviral particles is still not satisfactory, especially the effect of repeated immunization is poor.
- the KUN virus genome is relatively stable in the plasmid, and can be cloned and recombined using traditional cloning methods.
- KUN the genome of Dengzhang virus.
- W09928487 it is not possible to directly construct the vector by the method of W09928487. This may be because the KUN virus belongs to the Japanese encephalitis virus group and the dengue virus belongs to the Dengue virus group. The homology between the two is only about 45%.
- W002072803 discloses a method for constructing a subgenomic replicon of dengue virus, and its use as a dengue vaccine.
- the disclosure in this application is very inadequate and erroneous, and it is difficult to implement the technical solution of the invention based on its disclosure only.
- Antigen-presenting cells to prepare tumor vaccine The basic method is to first obtain dendritic cells from patients, perform primary cell culture and expansion, and then treat autologous dendritic cells with tumor-associated or specific antigens in vitro. Inoculated into the host, it activates T lymphocytes in the body, playing a therapeutic and protective role.
- This method is time-consuming, labor-intensive, and has high cost.
- the dendritic cell has low power for culturing, expanding, and activating, and it is difficult to achieve quality control. Therefore, this completely individualized and customized tumor vaccine method cannot be commercialized, and it is difficult to popularize and apply it.
- the purpose of the present invention is to provide a new vaccine with good safety, high immune titer, good repetitive immune effect in county, and suitable for industrial production, especially vaccines against tumors and tumors.
- a dengue virus recombinant replicon is provided, and the replicon is deleted from the entire coding sequence of the dengue virus preM protein and includes the following elements:
- the 3 'end of the exogenous nucleic acid sequence has a 3' end release element, or the coding region of the NS1 protein signal peptide has a 5 'release element, and the release element is selected from the group consisting of: SEQ ID
- the nucleotide sequence encoding the foot-and-mouth disease virus self-hydrolase shown in NO: 3 the nucleotide sequence encoding the signal peptide hydrolase substrate shown in SEQ ID NO: 44, and combinations thereof.
- the dengue virus is dengue virus type I, ⁇ , III or IV, or a combination thereof.
- the coding region of the NS1 protein signal peptide is the last 72 nucleotides of the E protein gene.
- the exogenous gene encodes an HPV antigen protein, an immunomodulatory factor or a complex of an HPV antigen and an immunomodulatory factor.
- a pseudoviral particle is provided, which is composed of the dengue virus recombinant replicon and the dengue virus structural protein package described above.
- a pharmaceutical composition comprising the above-mentioned dengue virus recombinant replicon or pseudoviral particle, and a pharmaceutically acceptable carrier.
- the use of the dengue virus recombinant replicon or pseudoviral particle of the present invention is provided for the preparation of a medicament for the treatment and prevention of the following diseases: tumors, viral diseases.
- a packaging cell for packaging the above-mentioned dengue virus recombinant replicon is provided, and the packaging cell is selected from the following group:
- a method for preparing a pseudoviral particle including the following steps:
- the packaging cell contains a dengue virus structural protein expression vector selected from the group consisting of: an expression vector containing a dengue genome sequence lacking NS3, and a gene expression vector regulated by tetracycline.
- FIG. 1 is a schematic diagram showing the preparation process of Method 1 in Example 4.
- FIG. 1 is a schematic diagram showing the preparation process of Method 1 in Example 4.
- FIG. 2 is a schematic diagram showing the preparation process of method 4 in Example 4.
- FIG. detailed description is a schematic diagram showing the preparation process of method 4 in Example 4.
- nt is a nucleotide.
- release element refers to a nucleic acid sequence located at the 3 'end of an antigen polypeptide coding sequence element or at the 5 end of an NS1 signal peptide coding region, and is used to release a complete antigenic polypeptide without an unrelated sequence when translated into a protein.
- a preferred release element is selected from the group consisting of a nucleotide sequence encoding a foot-and-mouth disease virus autohydrolase represented by SEQ ID NO: 3, a nucleotide sequence encoding a signal peptide hydrolase substrate represented by SEQ ID NO: 44, and a combination thereof .
- the release element is usually one, but it may be plural.
- immunological activity refers to the ability of natural, recombinant or synthetic peptides to induce specific humoral and / or cellular immune responses in mammals.
- antigenic polypeptide or “antigenic peptide” as used herein refers to an amino acid sequence that can elicit an immune response in a mammal, either alone or in combination with a helper molecule (such as a class I or II major histocompatibility antigen molecule).
- immune response includes cellular and / or humoral immune responses that are sufficient to inhibit or prevent infection; or to prevent or suppress the onset of diseases caused by microorganisms, especially pathogenic microorganisms.
- the term "subject” or “individual” or “patient” refers to any target that requires diagnosis or treatment, especially mammalian subjects, especially humans. Other subjects include cattle, dogs, cats, guinea pigs, rabbits, large animals Rat, mouse, horse, etc.
- the inventors confirmed for the first time after extensive experiments that the construction of the recombinant dengue virus replicon must meet the following conditions: 1.
- the complete nonstructural protein gene sequence must be retained. Any excision of the structural protein gene must ensure that it does not affect the expression of the non-structural protein of the original virus, nor can it cause frame-shift mutation of the non-structural protein and its signal peptide.
- the first 60 nt of the C protein gene (the coding sequence of the first 20 amino acids of the gp C protein) must be retained, because this sequence is complementary to the 5'-UTR and 3'-UTR to form a circle. Required structure. When it is less than 60nt, the efficiency of forming pseudoviral particles will be significantly reduced. Although it can still form pseudo particles when it is larger than 60nt (such as 81, 99, 120, 150, 180nt), the length of the foreign gene contained will decrease accordingly. Therefore, the preferred range is to retain the first 60-150nt of the protein C gene. First 60- 120nt of good land.
- the signal peptide of the intact non-structural protein NS1 the last 24 amino acids of the SPE protein, must be retained.
- the corresponding coding sequence is the last 72 nt of the E protein gene. When it is less than 72nt, the efficiency of forming pseudoviral particles will be significantly reduced, and even pseudoparticles cannot be formed. Although more than 72 nt (such as 81, 99, 120, 150 nt) can still form false particles, but the length of the foreign genes contained will be reduced accordingly, so the preferred range is to retain 72-150 nt, more preferably 60-120 nt.
- Dengue virus structural protein coding region includes CpreME. Usually, a part of the coding sequence of the CpreME protein can be deleted (usually about 100-2000bp, preferably 500-2000bp). Of course, it is preferable to delete the coding region of CpreME as much as possible. Another preferred way is to completely remove preM to improve safety.
- the dengue virus that can be used in the present invention is not particularly limited, and may be any subtype of dengue virus. The genomic sequences of the four subtypes can be found in the following accession numbers:
- Dengue virus has the characteristics of dendritic cells, which can more effectively improve the curative effect of the vaccine.
- the specific ADE (antibody dependent enhancement of infection) phenomenon of dengue virus that is, after the first immunization with dengue virus, and the immunization with different subtypes of dengue virus again, the antibody-dependent infection is enhanced, thus Repeated immunization is very effective.
- the ADE phenomenon is used in a foreign protein expression system, which not only avoids the body's neutralizing effect on the carrier during repeated immunization, but also improves the efficiency of the vector entering the cell and the delivery efficiency of the foreign protein.
- immunity can be enhanced by inoculating recombinant dengue virus replicons of different subtypes.
- the exogenous gene applicable to the present invention is not particularly limited, and may be any tumor antigen gene, virus antigen gene, and immunoregulatory factor gene.
- antigens include (but are not limited to): human HPV Antigen (such as E6-E7 fragments of subtypes 16 and 18), HIV antigen, HBV antigen, HCV antigen, EBV antigen, HTLV-1 antigen, MAGE, BAGE, CAGE, etc.
- the length of the exogenous gene is not particularly limited, and is usually about 100-2000 bp, preferably 150-1200 bp.
- a release element (such as a 2A sequence) may be flanked at the 3 ′ end of the foreign gene.
- the vaccine of the present invention can prevent and treat tumors and viral diseases.
- the present invention provides preventive vaccines and therapeutic vaccines against diseases caused by HPV infection.
- the vaccines include fake dengue virus replicons and dengue virus structural protein packaging.
- Viral particles, the exogenous nucleic acid sequence of the dengue virus recombinant replicon encodes one or more HPV subtype antigen proteins, an immunomodulatory factor or a complex of HPV antigen and immunomodulatory factors, and the antigen of said HPV subtype
- the protein can be HPV 16 subtype or HPV 18 subtype E6, E7, E7-E6 oncoprotein, or HPV main capsid protein L1, secondary capsid protein L2, and the immunoregulatory factor can be IL2, IL12, IL18, GM- CSF, etc.
- the vaccine provided by the present invention particularly shows the advantages of a therapeutic vaccine.
- Vaccine against human papilloma virus HPV
- HPV human papilloma virus
- diseases caused by papilloma virus infection such as chronic cervicitis, pseudocondyloma, wart-like lesions, condyloma acuminatum, cervical intraepithelial neoplasia, and cervix Cancer, etc.
- diseases caused by papilloma virus infection such as chronic cervicitis, pseudocondyloma, wart-like lesions, condyloma acuminatum, cervical intraepithelial neoplasia, and cervix Cancer, etc.
- the following steps can be generally performed:
- a particularly preferred method is deletion of homologous recombination in yeast.
- the preferred method is still to perform homologous recombination in yeast: add homology to the ends of the foreign gene that are homologous to the sequences on both sides of the site to be inserted Arm, then introduce the foreign gene and the dengue virus genomic DNA with the deleted structural gene into yeast cells at the same time, and obtain the replicon inserted into the foreign gene by homologous recombination.
- the recombination rate of this method is close to 100%, and the construction process is relatively simple, controllable, and stable.
- the replicon After obtaining the replicon, it can be introduced into packaging cells to produce pseudoviral particles.
- One conventional method is to introduce a dengue virus replicon into a cell and then introduce a helper virus that expresses a structural protein.
- Another preferred method is to use an inducible expression plasmid or a constitutively expressed plasmid as a vector for the dengue virus structural protein gene, construct a packaging cell that induces expression, and then directly introduce the replicon (or pseudoviral particle) into the cell To produce pseudoviral particles.
- packaging cells that do not express the constitutive expression of the NS3 gene are used.
- the NS3 gene has a special packaging signal sequence.
- the dengue genome without the NS3 gene is packaged into pseudoviral particles.
- packaging cells do not have the NS3 gene of the dengue virus, they cannot self-package to form pseudoviral particles.
- Pseudoviral particles obtained from such packaging cells contain only dengue virus recombinant replicons and do not require screening.
- the preferred method includes
- the packaging cell contains a gene expression cassette that is regulated by tetracycline (or other regulated expression method) to express the structural protein of dengue virus
- the recombinant dengue virus RNA replicon is introduced into the packaging cell, thereby generating dengue virus replication Daughter of pseudoviral particles.
- the invention also provides various compositions, including pharmaceutical compositions, especially vaccine compositions, comprising the recombinant dengue virus replicons and / or pseudoviral particles of the invention.
- compositions containing the recombinant dengue virus of the present invention may contain a buffering agent selected according to the actual use; it may also contain other substances suitable for the intended use. Those skilled in the art are good at selecting buffers, and a variety of buffers are known in the art to be suitable for the intended use.
- the composition may contain pharmaceutically acceptable excipients, many of which are known in the art and need not be discussed in detail herein. Various pharmacologically acceptable excipients have been detailed in various publications, including, for example, "Remington: Pharmacy and Pharmaceutical Practice", 19th Edition (1995) Mack Publishing Co ..
- the pharmaceutical composition can be prepared into various dosage forms, such as injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays and the like.
- Pharmaceutical grade organic or inorganic carriers and / or diluents suitable for oral or topical use can be used to formulate various compositions containing therapeutically active compounds.
- Diluents known in the art include aqueous media, vegetable and animal oils and fats. Stabilizers, wetting agents and emulsifiers, salts that change the osmotic pressure or various buffers to maintain a suitable pH value, and skin penetration enhancers can also be used as auxiliary materials.
- the recombinant dengue virus of the present invention can be formulated in a variety of ways.
- the vaccines of the present invention are formulated in a variety of methods well known in the art using suitable pharmaceutical carriers and / or vehicles.
- a suitable carrier is sterile saline.
- Other aqueous and non-aqueous isotonic aseptics can be used for this purpose Injectable solutions as well as aqueous and non-aqueous sterile suspensions (both known as pharmaceutically acceptable carriers and well known to those skilled in the art).
- the formulation of the vaccine composition of the present invention may also contain other ingredients, including, for example, adjuvants, stabilizers, pH adjusters, preservatives, and the like. These ingredients are well known to those skilled in the vaccine art.
- Adjuvants include (but are not limited to) aluminum salt adjuvants; saponin adjuvants; Ribi adjuvants (Ribi ImmunoChem Research In., Hamilton, MT); Montanide ISA Zuo Qi [J (Seppic, Paris, France); Hunter ' s TiterMax Zouqi! J (CytRx Corp., Norcross, GA); Gerbu Zouqi tj (Gerbu Biotechnik GmbH, Gaiberg, Germany), etc.
- other ingredients that regulate the immune response may be included in the formulation. Route and dosage
- the recombinant dengue virus replicon and / or pseudoviral particles of the present invention can be administered to an individual by known methods. These vaccines are usually administered using the same route of administration as conventional vaccines and / or mimicking pathogen infection routes.
- the vaccine composition may include a pharmaceutically acceptable carrier in addition to the recombinant Dengue virus.
- such a composition may include an adjuvant, a flavoring agent, or a stabilizer.
- Conventional and pharmaceutically acceptable routes of administration include: intranasal, intramuscular, intratracheal, subcutaneous, intradermal, intravaginal, intrapulmonary, intravenous, nasal, oral, or other parenteral routes of administration. If desired, the route of administration can be combined, or adjusted according to the antigenic peptide or disease condition.
- the vaccine composition may be administered in a single dose or multiple doses, and may include the administration of booster doses to elicit and / or maintain immunity.
- Dengue virus recombinant replicons and / or pseudoviral particles should be administered in an "effective amount" in an amount sufficient to elicit an immune response in the chosen route of administration and effectively promote protection of the host against symptoms such as viral infections and tumors.
- the amount of dengue virus replicon and / or pseudoviral particles selected for each vaccine dose is based on the amount that can elicit an immunoprotective response without significant side effects.
- parts of vaccine doses sufficient to produce from about 1- 1000 ⁇ ⁇ , preferably a 1-200 ⁇ ⁇ , more preferably 10-10 ( ⁇ g protein antigen.
- the recombinant dengue virus nucleic acid is calculated based on vaccine effective dose generally comprises administration of about 1 - lOOO g nucleic acid
- the general range of effective dose of the vaccine is about 102 - forming units (PFU 10 5 plaque --109, 103--107 or 10 '1 Standard research methods including antibody titers and other responses in the subject can be used to determine the optimal dosage for a particular vaccine.
- the need for booster doses can be determined by monitoring the level of immunity provided by the vaccine.
- Antibody titration in serum was evaluated After that, an enhanced dose of immunization may be required.
- the administration of adjuvants and / or immune stimulants can improve the immune response to the protein of the present invention. Compared with the prior art, the present invention has the following main advantages:
- Dendritic cells are the most effective antigen-presenting cells.
- the dengue virus has the characteristics of dendritic cells, and the dengue virus recombinant replicon is used as a vector to efficiently express and present an antigen in the infected cell, thereby stimulating effective immunity against the antigen.
- Recombinant dengue virus replicons of genes E6 and E7 which are both tumor antigens and viral antigens, can express E6 and E7 antigens in cells for a long time, effectively stimulating the immune system's immune response to the antigens.
- Dengue virus replicons are recombined from RNA viruses. Replicon replication and expression take place entirely in the cytoplasm, making it difficult to recombine oncogenes into the chromosomes of human cells.
- the recombinant dengue virus replicon is a recombinant dengue virus RNA from which the structural protein gene is removed. It can only be replicated and expressed in human cells, cannot form infectious virus particles, and will not cause secondary infection of surrounding cells.
- the immune system has the ability to recognize and remove recombinant replicons. Replicons do not exist in the body for a long time and have good safety.
- the frequency of administration is small (2 to 3 times), which reduces the pain caused to the patient during the treatment process.
- the recombinant replicon of dengue virus is used to transfect packaging cells that do not express the NS3 gene to produce pseudoviral particles.
- the NS3 gene has a special packaging signal sequence.
- the dengue genome lacking the NS3 gene cannot be packed into pseudoviral particles. Therefore, when the packaging cells cannot self-package into pseudoviral particles.
- the pseudoviral particles obtained by using such packaging cells all contain dengue virus recombinant replicons, which does not need to be screened, thereby improving the preparation efficiency of pseudoviral particles.
- RT-PCR was used to convert dengue virus RNA into three segments of dengue virus cDNA fragments (5 'end cDNA, 3' end cDNA and middle cDNA), and Xba I was added to the 5 'end of the 5' end cDNA fragment.
- Restriction site and Sp6 enhancer sequence Add Sac l enzyme site to the 3 'end of the 3' end fragment; Both ends of the middle fragment include the 3 'end of the 5' end cDNA fragment and the 3 'end of the cDNA fragment. 5 'end identical gene sequence.
- the sequence of the DNA primers used is as follows:
- Three dengue virus cDNA fragments were obtained with lengths of 5484 bp, 2530 bp, and 2922 bp, respectively.
- a) Use the BamH I endonuclease to position 1696 or 2203 of the dengue virus cDNA sequence (Gene Bank accession number: AF038403) (the BamH I restriction site inherent to the dengue virus gene) in c) of step 1 above.
- the resulting full-length dengue virus cDNA clone pRS / FLD2 was cut linear.
- a) Using the Fusing PCR method, a 96-base DNA fragment was prepared.
- the 5 'end of this fragment contained 45 base pairs derived from the 6th to 20th codons of the structural protein; the 3' end of the fragment contained The 45 base pairs are derived from codons 751 to 766 of the structural protein; the middle of this fragment includes a BamH I restriction site.
- the sequence of the DNA fragment is shown in SEQ ID NO: 5.
- Example 2 The dengue virus RNA replicon from which the sequence of the dengue virus structural protein has been removed is the same as the recombinant HPV dengue virus RNA replicon produced in Example 3. All four packaging methods in Example 4 can be used to prepare the dengue virus RNA replicon. Viral particles are used as controls in animal experiments.)
- Example 2 The dengue virus RNA replicon from which the sequence of the dengue virus structural protein has been removed is the same as the recombinant HPV dengue virus RNA replicon produced in Example 3. All four packaging methods in Example 4 can be used to prepare the dengue virus RNA replicon. Viral particles are used as controls in animal experiments.)
- Example 2 Example 2:
- E7-E6 sequence is the oncogene of HPV (Gene Bank accession number: AF486352;
- the 2A is a gene fragment of foot-and-mouth disease virus, which is composed of sixty bases, and its gene sequence is shown in SEQ ID NO: 3 in the sequence listing.
- HPV-16 prills (ATCC #: 45113D) are used as templates, respectively.
- E6 and E7 cDNA fragment were prepared.
- the partial sequence at the 5 'end of the E6 cDNA fragment is the same as the partial sequence at the 3' end of the E7 cDNA fragment.
- the partial sequence at the 3 'end of the E6 cDNA fragment is the same as the partial sequence at the 5' end of the 2A-containing fragment. Identical to nt 108-124 of dengue type II virus cDNA.
- -ll- CCAGGGTTGGACTCGAC (SEQ ID NO: 16) was used as a primer, and a llbp 2A-containing fragment was prepared by a fusion PCR method.
- the sequence at the 3 ′ end of the fragment was the same as that of the dengue type II virus cDNA nt 2275-2325.
- E6 cDNA fragment, E7 cDNA fragment and lA lbp fragment containing 2A at the same time in the PCR reaction tube as a template, and use GCGAGAAATACGCCTTTCAATATGCTGAAACGCGAGAGAAACATGCATGGAGAT ACACCTACA CSEQ ID NO: 17) as the 5 'end primer of the E7 cDNA fragment.
- TCGAC (SEQ ID NO: 18) was used as the 3 'end primer of the 2A-containing fragment, and the full-length E7-E6-2A cDNA fragment was obtained by PCR. The two ends of this fragment contained the same genes as the dengue virus cDNA, respectively. sequence.
- Example 3
- the cDNA clone of HP V-dengue virus RNA replicon produced in step 2 was purified from yeast using a Qiagen column (QIAGEN Inc.).
- HPV-dengue virus RNA replicon cDNA clone cloned from yeast was used to transform Stabl2 TM bacteria (purchased from Invitrogen, USA). A large number of HPV-dengue virus RNA replicon cDNA clones can be prepared using this system.
- the recombinant HPV dengue virus RNA replicon was transfected into host cell BHK-21 (Baby hamster kidney) (ATCC #: CCL-10) by electroporation.
- the recombinant HPV dengue virus RNA replicon can be expressed in HPV E7-E6 protein is replicated and expressed in host cells.
- Method 1 Using a tetracycline-regulated gene expression system to prepare a vaccine
- a tetracycline-regulated gene expression system was used to transform BHK-21 (Baby hamster kidney) (ATCC #: CCL-10) to develop a cell line capable of regulating the expression of dengue virus structural proteins.
- Tetracycline-regulated gene expression systems Tet-Off Gene Expression systems purchased from Clontech, USA. This plasmid is capable of expressing a regulatory protein called "tetracycline-controlled inverse activation protein", which is involved in the regulation of gene expression plasmid transcription.
- This gene expression system includes pTet- Off regulator vectors, pTRE2 response vectors, and pTK-Hyg selection vectors.
- GGCG SEQ ID NO: 19
- ATATATCTAGACTAGGCCTGCACC ATAACTCCCAA SEQ ID NO: 20
- pTRE2 Clontech
- the digested product was purified using a Qiagen spin column (QIAGEN Inc.).
- the two DNA fragments were ligated with T4 ligase (New England Biolab), and transformed into E. coli, thereby cloning and generating the dengue virus structural protein recombinant pTRE2 plasmid.
- the pTet-Off regulator vector was transformed into BHK-21 cells by electric shock, and then the BHK-21 Tet-off cell line was selected.
- the tetracycline-regulated gene expression system inserted into the dengue virus structural protein gene generated in step 1 was transformed into BHK-21 Tet-off (Baby hamster kidney) by electric shock to obtain a regulated and highly expressed dengue virus structural protein. ⁇ -21 cell line. 4.
- the recombinant HPV dengue virus RNA replicon produced in Example 3 was transformed into the above-mentioned cell line by an electric shock method.
- One day later, the tetracycline-regulated gene expression system induced high expression of dengue virus structural proteins in this cell line.
- culture supernatants were collected, to give 104 per ml packed off recombinant dengue virus RNA replicon HPV pseudovirions.
- Method 2 Use Sindbis RNA replicon to prepare vaccine
- ID NO: 22 was used as a primer to prepare a DNA fragment of the C coding region of the dengue virus structural protein gene by PCR.
- TAACTCCCAA (SEQ ID NO: 24) was used as a primer to prepare a DNA fragment of the preM-E coding region of the dengue virus structural protein gene by PCR.
- Sindbis RNA replicon cDNA clone (STRATAGENE) was used as a template, ATTACACCTGTCCTACTGGCA (SEQ ID NO: 25) and CATGGTAAG CTCTAGCTAGAG (SEQ ID NO: 26) were used as primers, and a Sindbis 26S DNA fragment was prepared by PCR.
- the 21nt sequence at the 3 'end of the DNA fragment of the preM-E coding region is identical to the partial gene sequence at the 5' end of the Sindbis 26S DNA fragment.
- the 3 'end 22nt sequence of the Sindbis 26S DNA fragment is the same as the partial gene sequence of the 5' end of the C coding region DNA fragment.
- preM-E coding region DNA fragment and Sindbis 26S DNA fragment as templates, GCGCTCTAGAATGACTGCAGGCATGATCA TTATG (SEQ ID NO: 27) and GTATAGAGCTCTTATCTGCGTCT CCTGTTCAAGAT (SEQ ID NO: 28) as primers, and fusing PCR Method to prepare the preME-26S-C fragment.
- GCGCTCTAGAATGACTGCAGGCATGATCA TTATG SEQ ID NO: 27
- GTATAGAGCTCTTATCTGCGTCT CCTGTTCAAGAT SEQ ID NO: 28
- STRATAGENE SindbisRNA replicon cDNA clone
- STRATAGENE SindbisRNA replicon cDNA clone
- the two DNA fragments were ligated with T4 ligase (New England Bio lab) and transformed into E. coli, and cloned to produce dengue virus structural protein gene recombination Sindbis RNA replicon cDNA clone, and used Qiagen spin column (QIAGEN Inc.) Perform purification.
- step b) linearizing the Sindbis RNA replicon cDNA clone of the dengue virus structural protein gene generated in step a) with Xho I endonuclease;
- Sindbis that recombines linearized dengue virus structural protein gene
- the RNA replicon cDNA clone is transcribed into RNA to obtain the Sindbis RNA replicon of the dengue virus structural protein gene recombination.
- BHK-21 cells (ATCC #: CCL-10) were transfected with the recombinant HPV dengue virus RNA replicon produced in Example 3, and after 24 hours, the dengue virus structural protein gene generated in step 2 c) above was used.
- the recombinant Sindbis RNA replicon was transfected into the cell again. Both transfection were performed by electric shock, 0.4cm Gene Pulser Cuvette, 200 V / 950uF. After one to two days, culture supernatants were collected, to obtain 104-105 ml per recombinant dengue virus RNA replicon HPV pseudovirions.
- Method 3 Preparation of vaccine using pseudoviral particles of Sindbis RNA replicon
- Method 4 Establishment of a BHK-21 packaging cell line transformed with NS3 deleted dengue virus cDNA
- CMV cytomegalovirus, cytomegalovirus
- DEN3' end was prepared by PCR.
- a DNA fragment (hepat it is delta virus ant i genomi c ribozyme (HDVr)) was prepared by PCR using 3 and 9 r as primers (referred to as HDVr, SEQ ID NO: 30).
- pcDNA3 (purchased from IWITROGEN, USA) as a template and ⁇ and ⁇ 3 ⁇ 44 as primers
- a gangster fragment of bovine growth hormone pol y A (bovine growth hormone poly A, BGH pA) (abbreviated as pA) was prepared by PCR.
- a DNA fragment of DEN3 'end-HDVr- pA was prepared by using the above three fragments as templates and 5' / ⁇ ⁇ 0 2 ⁇ port ⁇ 4 as primers by fusing PCR method.
- the sequence of the DNA primers used is as follows:
- the pRS / CMV-DEN5 'end-DEN3' end-HDVr- pA clone generated in step 4 above was digested with Apal, and the pRS / FLD2 prepared in the first step c) of Example 1 was Xbal and Sac I perform digestion.
- the above digested product was purified, and the purified product was transformed into yeast, and the same sequence in yeast was used to recombine A to generate a full-length eDNA clone of dengue virus RMA replicon (referred to as pRS / CMV / D2) .
- AACAACCAAATC (SEQ ID NO: 41) and CTCCACATTTTCCAAGATTTGGTTG
- TTCTTAATTCC (SEQ ID NO: 42) was used as a primer, and a 75 bp long DNA fragment was generated by PCR with the sequence CAGACTGAAAAAAGTATTGAAGACAATCCAGAGATCGAAGGAATTAAG AACAACCAAATCTTGGAAAATGTGGAG (SEQ ID NO: 43).
- the linearized full-length The cDNA clone of the dengue virus RNA replicon was transformed into yeast together with a 75bp long DNA fragment.
- the linearized gene sequence of the partial NS3 coding region of the dengue virus RNA replicon ( nt 5059-6215) was excised, and a cDNA clone of NS3 deleted dengue virus was obtained.
- Example 5 pcDNA3 (purchased from INVITROGEN, USA) was also transfected with BHK-21 cell (ATCC #: CCL-10). After the cells were cultured for one day, they were transformed into a cell culture solution containing G418 (purchased from SIGMA, USA). The recombinant HPV dengue virus RNA replicon produced in Example 3 was used to transform an anti-G418 cell line and a packaging cell line was selected. The production cell line may reach 105 recombinant dengue virus RNA replicon HPV pseudovirions.
- Example 5 Example 5
- JHU-1 HPV tumor cells contained the HPV virus E6 / E7 oncogene. 1 ⁇ 10 5 JHU-1 HPV tumor cells in 500 ⁇ l of PBS buffer were injected into the umbilical abdomen (FLANK) of eight-week-old C57BL / 6 mice. JHU-1 HPV tumor cells can induce 100% of solid tumors. Seven days after injection of JHU-1 HPV tumor cells, the tumor could be touched quickly. And fourteen days later, the induced tumors can be more than 6mm.
- HPV virus vaccine Treatment of HPV virus vaccine: The entire experiment was divided into four groups of eight C57BL / 6 mice.
- PFU Infectious Particle of HPV replion-containing VLP.
- Each mouse in the tumor control group and vaccine treatment group was injected with 1X10 5 JHU-1CELLS. Fourteen days after injection of JHU-1 cells, the vaccine treatment group and mice vaccine control group were injected subcutaneously into the particles 107 of an effective vaccine. After seven days and, again, an effective vaccine is injected subcutaneously 10 7 PFU of the particles as an additional treatment.
- Example 4 the following recombinant dengue virus type II replicons were constructed in the same manner as described in Examples 1, 2, and 3, except that the 5 'sequence of the protein C gene and the length of the signal peptide of the NS1 protein were different.
- the method 4 in Example 4 was used to prepare pseudoviral particles. The results are as follows:
- Example 4 the following dengue virus recombinant replicons were constructed in the same manner as described in Examples 1, 2, and 3, except that the type I dengue virus in Examples 1-3 was replaced with the type I dengue virus Then, the method 4 in Example 4 was used to prepare pseudoviral particles. Then, the mice were administered according to the same protocol of the treatment group in Example 5. The experimental animals were 4 mice whose tumors did not disappear in Example 5.
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP04706596A EP1593738A4 (en) | 2003-01-30 | 2004-01-30 | VIRUSELY PARTICULATE VACCINE WITH RECOMBINANT DENGUEVIRUS REPLICANT AS ITS SUPPORT |
US11/192,923 US20060018928A1 (en) | 2003-01-30 | 2005-07-29 | Virus-like particle containing a dengue virus recombinant replicon |
US11/971,343 US20090197319A1 (en) | 2003-01-30 | 2008-01-09 | Virus-Like Particle Containing A Dengue Recombinant Replicon |
US12/129,908 US20090149405A1 (en) | 2003-01-30 | 2008-05-30 | Virus-like particle containing a Dengue virus recombinant replicon |
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CN03115273 | 2003-01-30 | ||
CN03115272 | 2003-01-30 | ||
CN03115272.4 | 2003-01-30 | ||
CN03115273.2 | 2003-01-30 |
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US19434205A Continuation-In-Part | 2003-01-30 | 2005-08-01 |
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PCT/CN2004/000088 WO2004072274A1 (fr) | 2003-01-30 | 2004-01-30 | Vaccin a base de pseudoparticules virales ayant comme support un replicon de recombinaison du virus de la dengue |
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EP1986686A4 (en) * | 2006-02-13 | 2010-08-11 | Integral Molecular Inc | VIRUS REPORTER OF DENGUE AND METHODS OF MAKING AND USING SAME |
EA016490B1 (ru) | 2006-02-27 | 2012-05-30 | Дзе Борд Оф Риджентс Оф Дзе Юниверсити Оф Техас Систем | Псевдоинфекционный флавивирус и его применение |
US8993744B2 (en) | 2010-05-21 | 2015-03-31 | University of Pittsburgh—of the Commonwealth System of Higher Education | Universal dengue virus sequences and methods of use |
US8685649B2 (en) | 2010-06-10 | 2014-04-01 | The United States Of America As Represented By The Secretary Of The Navy | RT-LAMP assay for the detection of pan-serotype dengue virus |
US11845939B2 (en) * | 2016-12-05 | 2023-12-19 | Janssen Pharmaceuticals, Inc. | Compositions and methods for enhancing gene expression |
CN113604483A (zh) * | 2021-07-13 | 2021-11-05 | 中山大学 | 一种登革病毒血清3型流行株gz14d3复制子及其应用 |
Citations (3)
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WO1999028487A1 (en) * | 1997-11-28 | 1999-06-10 | The Crown In The Right Of The Queensland Department Of Health | Flavivirus expression and delivery system |
WO2001053467A1 (en) * | 2000-01-21 | 2001-07-26 | The Regents Of The University Of California | Recombinant flaviviruses and methods of use thereof |
WO2002072803A2 (en) * | 2001-03-09 | 2002-09-19 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Subgenomic replicons of the flavivirus dengue |
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US6893866B1 (en) * | 1997-11-28 | 2005-05-17 | The Crown In The Right Of The Queensland Department Of Health | Flavivirus expression and delivery system |
US20030021805A1 (en) * | 2001-05-29 | 2003-01-30 | Barber Glen N. | Generation of HCV-like particles and chimeric HCV virus |
NZ533237A (en) * | 2001-11-26 | 2005-11-25 | Univ Queensland | Flavivirus vaccine delivery system |
-
2004
- 2004-01-30 WO PCT/CN2004/000088 patent/WO2004072274A1/zh active Application Filing
- 2004-01-30 EP EP04706596A patent/EP1593738A4/en not_active Withdrawn
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2005
- 2005-07-29 US US11/192,923 patent/US20060018928A1/en not_active Abandoned
-
2008
- 2008-01-09 US US11/971,343 patent/US20090197319A1/en not_active Abandoned
- 2008-05-30 US US12/129,908 patent/US20090149405A1/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999028487A1 (en) * | 1997-11-28 | 1999-06-10 | The Crown In The Right Of The Queensland Department Of Health | Flavivirus expression and delivery system |
WO2001053467A1 (en) * | 2000-01-21 | 2001-07-26 | The Regents Of The University Of California | Recombinant flaviviruses and methods of use thereof |
WO2002072803A2 (en) * | 2001-03-09 | 2002-09-19 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Subgenomic replicons of the flavivirus dengue |
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US20060018928A1 (en) | 2006-01-26 |
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US20090197319A1 (en) | 2009-08-06 |
US20090149405A1 (en) | 2009-06-11 |
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