Method for preparing hepatitis A virus and hepatitis A virus prepared according to the method

Abstract

The present invention relates to a method for producing hepatitis A virus and the hepatitis A virus produced according to this method, and more particularly to: a method for producing hepatitis A virus and the hepatitis A virus produced according to this method, the method comprising the steps of: infecting a host cell with a virus obtained by transforming a host cell with a vector for preparing hepatitis A virus containing an expression cassette, the expression cassette containing a hepatitis A virus gene, and subculturing the host cell.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2019-0171279, filed on December 19, 2019, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a method for producing hepatitis A virus and hepatitis A virus produced according to the method, and more particularly to a method for preparing hepatitis A virus and hepatitis A virus prepared according to the method, the method comprising infecting a host cell with a virus and subculturing the host cell, the virus being obtained by transfecting a host cell with a vector for preparing hepatitis A virus into which an expression cassette containing a hepatitis A virus gene is inserted. Background Art

[0003] Hepatitis A is the most important cause of more than 70% of acute viral hepatitis in Korea. The hepatitis A virus (HAV) belongs to the family Picornaviridae, is about 27 nm in size, has no envelope, and its nucleic acid is single-stranded RNA. After an average incubation period of 28 days, HAV causes acute liver disease, characterized by clinical symptoms such as fever, anorexia, nausea and vomiting, abdominal pain, dark urine color, and jaundice (SM Lemon et al. J. Hepatol, 68(1):167-184(2018); Totsuka and Moritsugu, Intervirology, 42:63-68(1999)). According to a survey over the past 10 years, the number of hepatitis A patients is increasing in the western United States, the Middle East, and some Asian regions, raising concerns about the global spread of hepatitis A disease. Even in Korea, the number of people infected with hepatitis A is rapidly increasing among young people in their teens and twenties who have no immunity (Nwachuku and Gerba, Rev. Environ. Contam. Toxicol. 186:1-56(2006); Kim and Lee, Intervirology, 53(1):10-14(2010); Korea Centers for Disease Control and Prevention (KCDC) Infectious Diseases Portal Legal Infectious Disease Statistics by Disease - Hepatitis A). Especially in the case of adolescents of school age or test subjects, hepatitis A virus infection can cause personal and economic losses due to hospitalization and treatment. In the United States, it is well known that the direct and indirect costs per hepatitis A patient are $2,500 for adults and about $1,500 for those under 18 years old, and the annual medical costs caused by hepatitis A exceed $300 million (World Health Organization, 1999).

[0004] The main route of transmission of hepatitis A is the fecal-oral route, and hepatitis A is transmitted through contaminated food or drinking water. The Advisory Committee on Immunization Practices (ACIP) recommends vaccination for travelers leaving for areas where hepatitis A virus is endemic, workers in corresponding areas, men who have sex with men (MSM), patients with hepatitis B, patients with chronic liver disease and chronic renal failure, and children living in areas with a high incidence of hepatitis A (Nelson NP, Weng MK, Hofmeister MG, et al. Prevention of Hepatitis A Virus Infection in the United States: Recommendations of the Advisory Committee on Immunization Practices, 2020. MMWR Recomm Rep 2020;69(No. RR-5):1–38. DOI: http: / / dx.doi.org / 10.15585 / mmwr.rr6905a1).

[0005] To prepare hepatitis A vaccine, the preparation of hepatitis A virus (HAV) is necessarily accompanied, but the replication rate of hepatitis A virus is very slow (Cromeans et al. J. Gen. Virol. 70:2051-2062 (1989)). For viruses that usually infect humans, they can be isolated and replicated within a short incubation period of 2 to 3 days or as long as 7 days, but it takes about a month to obtain the virus for hepatitis A virus. A month is only equivalent to the situation where the virus adapts well to the cultured cells. Cell lines highly sensitive to hepatitis A virus (such as primary AGMK cells, FRhK-4, BS-C-1) used for amplifying hepatitis A virus are not suitable as cells for vaccine production. These cell lines have not been verified for cell line characterization and stability suitable for use as human vaccine production cell lines. In particular, the cells for which examples have been established are cell lines that can be cultured in vitro in culture flasks, etc., but are classified as materials from rhesus monkeys (Macaca mulatta) and African green monkeys (Cercopithecus aethiops) belonging to the Cercopithecidae family, which is an internationally endangered species restricted by CITES. Therefore, it is difficult to import them from ATCC (USA), which distributes and commercially sells cell lines, to Korea.

[0006] For isolation from human fecal samples and high - yield production in virus - producing cell lines, approximately 50 virus - infection passages are required to adapt HAV to the virus - producing cell line. For example, in the case of the master - seed virus (HAV 4380 or MRC5 / 9, master - seed) of the commercial vaccine Havrix, human - derived wt HM - 175 (human fecal suspension) was passaged 32 times in primary AGMK (African green monkey) cells to confirm the virus adapted to cell culture. Then, the corresponding virus (P - 32 AGMK - cell - adapted) was passaged 37 times in the MRC - 5 cell line and isolated as a virus clone (clone 25 - 4 - 21), and the clone was incubated again in MRC - 5 (38th passage). Thereafter, the 41st - passage virus, which was passaged 3 more times by virus infection in MRC - 5 cells, was used as the master - seed stock and was designated HAV 4380 (US6423318B1).

[0007] Hepatitis A vaccines are subject to mandatory vaccination designated by the government, but are often in short supply during HAV epidemics. The world's most commonly used hepatitis A vaccines, such as Havrix from GlaxoSmithKline (GSK) and Vaqta from Merck & Co., were sold out in South Korea during the 2019 hepatitis A epidemic and then suffered a heavy blow to the supply - demand difficulties, such as running out of supply at the end of 2019. These vaccines also had supply - demand imbalance problems in 2017, but the supply - demand instability was not resolved even in 2019 when there was a sudden outbreak of hepatitis A transmission. Currently, hepatitis A vaccines have been included in the national vaccination reserve plan for 2020 by the Ministry of Health and Welfare to stabilize the supply - demand of vaccines and efforts are being made to solve this problem. The reason for this supply - demand instability is that there is no commercially available vaccine developed with domestic technology for hepatitis A vaccines. Summary of the Invention

[0008] Technical Problem

[0009] In order to solve the above - mentioned technical problems and develop a method for stably and rapidly producing hepatitis A virus, the inventors of the present invention repeatedly and painstakingly studied. As a result, a method was found in which the number of passage - culture steps of about 50 times was shortened to only 6 times to obtain the virus, and an optimal combination of gene expression cassettes with higher yield expression in virus - producing cell lines. Then, the inventors found that the virus obtained by infecting host cells with an expression vector containing the genetic and functional sites of hepatitis A virus replicates at an astonishing speed through repeated passage - culture, to rapidly and stably produce hepatitis A virus, and then completed the present invention.

[0010] Therefore, an object of the present invention is to provide a hepatitis A virus gene defined by SEQ ID NO:1.

[0011] Another object of the present invention is to provide an expression cassette for preparing hepatitis A virus, which comprises the hepatitis A virus gene of SEQ ID NO:1.

[0012] Another object of the present invention is to provide a vector for preparing hepatitis A virus, which contains the said expression cassette.

[0013] Another object of the present invention is to provide hepatitis A virus prepared with the said vector.

[0014] Another object of the present invention is to provide a method for preparing hepatitis A virus for use in a vaccine, comprising the steps of: (a) transfecting a host cell with a vector for preparing hepatitis A virus into which an expression cassette is inserted, the expression cassette comprising the hepatitis A virus gene of SEQ ID NO:1; (b) obtaining virus from the host cell; (c) infecting a host cell with the obtained virus and subculturing the infected host cell; and (d) obtaining virus from the host cell.

[0015] Another object of the present invention is to provide hepatitis virus prepared according to the method.

[0016] Another object of the present invention is to provide a hepatitis A vaccine composition comprising hepatitis virus as an active ingredient.

[0017] Another object of the present invention is to provide a hepatitis A vaccine composition consisting of hepatitis virus.

[0018] Another object of the present invention is to provide a hepatitis A vaccine composition mainly consisting of hepatitis virus.

[0019] Another object of the present invention is to provide a kit comprising the said vaccine composition.

[0020] Another object of the present invention is to provide a pre-filled syringe filled with the said vaccine composition.

[0021] Another object of the present invention is to provide the use of the said hepatitis virus in the preparation of a hepatitis A vaccine.

[0022] Another object of the present invention is to provide the use of the said vaccine composition comprising hepatitis virus as an active ingredient in the prevention of hepatitis A.

[0023] Another object of the present invention is to provide a method for preventing hepatitis A, comprising administering an effective dose of the said vaccine composition comprising hepatitis virus as an active ingredient to a subject in need thereof.

[0024] Technical solution

[0025] To achieve the object of the present invention, the present invention provides a hepatitis A virus gene defined by SEQ ID NO:1.

[0026] To achieve another object of the present invention, the present invention provides an expression cassette for preparing hepatitis A virus, which comprises the hepatitis A virus gene of SEQ ID NO:1.

[0027] To achieve another object of the present invention, the present invention provides an expression vector for preparing hepatitis A virus, which comprises the expression cassette.

[0028] To achieve another object of the present invention, the present invention provides a hepatitis A virus prepared with the said vector.

[0029] To achieve another object of the present invention, the present invention provides a preparation method of hepatitis A virus for preparing a vaccine, comprising the following steps: (a) transfecting a host cell with a vector for preparing hepatitis A virus inserted with an expression cassette, the expression cassette comprising the hepatitis A virus gene of SEQ ID NO:1; (b) obtaining a virus from the host cell; (c) infecting the host cell with the obtained virus and subculturing the infected host cell; and (d) obtaining a virus from the host cell.

[0030] To achieve another object of the present invention, the present invention provides a hepatitis virus prepared according to the said method.

[0031] To achieve another object of the present invention, the present invention provides a hepatitis A vaccine composition comprising a hepatitis virus as an active ingredient.

[0032] In addition, the present invention provides a hepatitis A vaccine composition composed of a hepatitis virus.

[0033] In addition, the present invention provides a hepatitis A vaccine composition mainly composed of a hepatitis virus.

[0034] To achieve another object of the present invention, the present invention provides a kit comprising the said vaccine composition.

[0035] To achieve another object of the present invention, the present invention provides a prefilled syringe filled with the said vaccine composition.

[0036] To achieve another object of the present invention, the present invention provides the application of the said hepatitis virus in preparing a hepatitis A vaccine.

[0037] To achieve another object of the present invention, the present invention provides the application of a vaccine composition comprising a hepatitis virus as an active ingredient in preventing hepatitis A.

[0038] To achieve another object of the present invention, the present invention provides a method for preventing hepatitis A, which includes administering an effective dose of a vaccine composition containing hepatitis virus as an active ingredient to a subject in need.

[0039] The present invention will be described in detail below.

[0040] The present invention provides a hepatitis A virus gene defined by SEQ ID NO:1.

[0041] In addition, the present invention provides an expression cassette for preparing hepatitis A virus, which contains the hepatitis A virus gene of SEQ ID NO:1.

[0042] Compared with the gene of a commercial hepatitis A virus strain (ATCC VR-1402), the hepatitis A virus gene of SEQ ID NO:1 provided by the present invention includes A2876T and A3891T point mutations and has the characteristics suitable for high-yield and rapid subculture.

[0043] In particular, position 3891 is the main mutation where the amino acid changes from MET to LEU. The recombinant nucleotide sequence of the present invention is completed by mutating the main sites that can promote subculture and increase the yield.

[0044] According to one aspect of the present invention, it is confirmed that, as Figure 8 and 11 shown, compared with the commercially available wild-type hepatitis A virus, the productivity of the hepatitis A virus gene defined by SEQ ID NO:1 according to the present invention is more excellent.

[0045] In the related art, to obtain hepatitis A virus, at least 47 subcultures are required ( Figure 4 ), and there are disadvantages of being cumbersome, time-consuming, and costly (T. Cromeans et al., J Medical Virology 22:45-56, 1987). Generally, one subculture of HAV takes about one month, and it takes dozens of months to obtain hepatitis A virus for vaccine preparation from human feces according to the related art. In the present invention, the technical feature is to reduce the production cycle of hepatitis A virus to 4 to 5 months by using the hepatitis A virus gene of SEQ ID NO:1 with genetic mutations and the expression cassette containing this gene.

[0046] In the present invention, an "expression cassette" refers to a unit cassette that can express a target protein operably linked downstream of a signal peptide to be secreted and produced by including a promoter, a nucleotide sequence encoding a signal peptide, and a gene encoding a target protein. Various factors that can help effectively produce the target protein can be included inside or outside such an expression cassette.

[0047] In the present invention, "gene" generally refers to any segment of polynucleotide related to biological functions. Thus, a gene or polynucleotide includes introns and exons as in genomic sequences, or only coding sequences as in cDNA, such as an open reading frame starting from the start codon (methionine codon) and ending at the termination signal (stop codon). A gene or polynucleotide may also include sites that regulate its expression, such as transcription start sites, translation sites, and transcription termination sites. Therefore, a gene or polynucleotide includes a promoter and a ribosome binding site (usually, these regulatory elements are generally located about 60 to 250 nucleotides upstream of the start codon of the coding sequence or gene) and a transcription terminator (usually, the terminator is located within about 50 nucleotides downstream of the stop codon of the coding sequence or gene). A gene or polynucleotide also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein and contains regulatory sequences.

[0048] It will be apparent to those skilled in the art that the expression cassette provided by the present invention may include a nucleotide sequence encoding a polypeptide having substantially the same biological properties as the antigenicity or immunogenicity of a virus, while having at least 80% or more, preferably 90% or more, more preferably 95% or more sequence homology with the nucleotide sequence of the hepatitis A virus gene of SEQ ID NO: 1.

[0049] According to one aspect of the present invention, the expression cassette may include a promoter, a hammerhead (HH) ribozyme, and a hepatitis delta virus (HDV) ribozyme.

[0050] Preferably, the expression cassette sequentially includes a CMV promoter, a T7 promoter, a multiple cloning site (MCS), and an HH ribozyme site at the 5' end of the hepatitis A virus gene, and may sequentially include a hepatitis delta virus ribozyme, an MCS, and a polyA tail in the 3' end direction of the hepatitis A virus gene.

[0051] According to a preferred embodiment of the present invention, the CMV / T7 promoter can be defined by SEQ ID NO:2, the MCS sequence can be defined by SEQ ID NO:3 or 6, the HH ribozyme can be defined by SEQ ID NO:4, the HDV ribozyme can be defined by SEQ ID NO:5, and the polyA tail can be defined by SEQ ID NO:7. In an embodiment of the present invention, the CMV promoter site and the T7 promoter site capable of being transcribed in vitro by T7 RNA polymerase are inserted into the expression cassette (SEQ ID NO:2) for gene expression. The MCS is arranged after the promoter (SEQ ID NO:3) at the 5' end of the gene and before the polyA tail sequence (SEQ ID NO:6) for using restriction enzymes. The expression cassette is designed as a catalytic RNA cleavage structure with self-cleavage function, and the HH ribozyme (SEQ ID NO:4) and HDV ribozyme (SEQ ID NO:5) sites are located on both sides of the UTR-HAV polyprotein-UTR site, which is the HAV sequence site and can be separated (processed) only from the HAV mRNA structure to be targeted.

[0052] According to a preferred embodiment of the present invention, the expression cassette may comprise the nucleotide sequence of SEQ ID NO:8.

[0053] Furthermore, the present invention provides a vector for preparing hepatitis A virus comprising the expression cassette.

[0054] In the present invention, a "vector" is a vector capable of expressing a target protein in a suitable host cell and refers to a gene construct comprising necessary regulatory elements, which are operably linked to express the inserted gene. In the present invention, "operably linked" means that the nucleic acid sequence encoding the target protein is functionally linked to the nucleic acid expression regulatory sequence to perform a general function. The operable linkage with the vector can be prepared using genetic recombination techniques well-known in the art to which the present invention pertains, and site-specific DNA cleavage and ligation can be easily performed using enzymes well-known in the art to which the present invention pertains.

[0055] Suitable vectors for use in the present invention may not only comprise expression regulatory elements such as promoters, start codons, stop codons, polyadenylation signals, ribozymes, and enhancers, but also signal sequences for membrane targeting or secretion in addition to the hepatitis A gene of SEQ ID NO:1.

[0056] The start codon and the stop codon are generally considered to be part of the nucleotide sequence encoding an immunogenic target protein and are required to function in a host cell when the gene construct is administered, and need to be in frame with the coding sequence. The promoter can be constitutive or inducible. The promoters include, but are not limited to, the human elongation factor-1α (EF-1α), simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, cytomegalovirus (CMV), β-actin promoter, T7 promoter, and T3 promoter.

[0057] When the vector is a replicable expression vector, the vector can include an origin of replication, which is a specific nucleic acid sequence from which replication begins. As a recombinant expression vector, various types of vectors such as plasmids, viruses, and cosmids can be used. The type of the recombinant vector is not particularly limited as long as it can function to express the desired gene and produce the desired protein in various host cells of eukaryotic cells, but a vector that can produce a large amount of a promoter with strong activity and an exogenous protein in a form similar to the natural state but maintaining strong expression ability is preferred.

[0058] Eukaryotic expression vectors into which an expression cassette containing the hepatitis A virus gene of SEQ ID NO:1 can be inserted are known in the art. Non-limiting examples thereof include the pUC57 vector, the pcDNA3.1 vector, the pVAX1 vector (Life Technology, Cergy-Pontoise, France), and pBudCE4.1 (Life Technology), and the expression vectors of the present invention can be prepared using the vectors disclosed or mentioned herein or known to those skilled in the art. Preferably, the pUC57 vector can be used.

[0059] According to another preferred embodiment of the present invention, the vector can show Figure 2 the cleavage map of the HAV viral gene shown and the factors involved in gene expression to produce the virus.

[0060] The present invention provides a method for preparing hepatitis A virus for use in a vaccine, comprising the steps of: (a) transfecting a host cell with a vector for preparing hepatitis A virus into which an expression cassette is inserted, the expression cassette including the hepatitis A virus gene of SEQ ID NO:1; (b) obtaining a virus from the host cell; (c) infecting a host cell with the obtained virus and subculturing the infected host cell; and (d) obtaining a virus from the host cell.

[0061] (a) Transfecting a host cell with a vector into which an expression cassette is inserted, the expression cassette including the hepatitis A virus gene of SEQ ID NO:1 for preparing hepatitis A virus;

[0062] The "expression cassette" and "vector" can be applied in the same manner as described above.

[0063] In the present invention, a "host cell" refers to a eukaryotic cell that has been genetically altered or can be genetically altered by administering an exogenous polynucleotide such as a recombinant plasmid or vector. When referring to a genetically altered cell, the term includes the initially altered cell and its progeny. A polynucleotide or expression cassette containing the hepatitis A virus gene sequence of SEQ ID NO:1 can be inserted into a cloning vector and an expression vector, and then the vector can be injected into a suitable host cell for replication and amplification.

[0064] In step (a) of the present invention, there is no particular limitation on the type of host cell into which the vector is injected, but it can be a cell derived from a natural host of HAV (such as chimpanzee, monkey, human, etc.) or a cell used for vaccine production. The "cell used for vaccine production" can also be expressed as a cell substrate, which can be defined as having the ability to produce a drug as a raw material for preparing a biopharmaceutical or a cell culture drug in a cell line derived from a human or an animal.

[0065] In particular, in the present invention, the host cell is preferably used for producing biopharmaceuticals in the art because its safety as a cell for human vaccine production has been proven. For a detailed description of cells whose safety as cells for human vaccine production has been proven, reference can be made to Jordan and Sandig, Viruses, 6:1672 - 1700 (2014); WHO Technical Report Series, No. 978, Annex 3, and the entire content of these documents can be a reference for the present invention.

[0066] According to one aspect of the present invention, the cells for vaccine production can be selected from Vero, MA104, WI - 38, CHO, MDCK, Hi5, CEF, S9, human embryonic lung fibroblasts (such as MRC - 5, etc.), PER.C6, BHK - 21, CHO - K1 and their serum - free adapted cells. Preferably, the cells for vaccine production can be selected from MA104, Vero or their serum - free adapted cells. More preferably, the cells can be SF - Vero cells.

[0067] According to one aspect of the present invention, the cultivation of the host cell can be carried out as follows: The MA104 and Vero cell lines use EMEM (2% FBS - EMEM) medium containing 2% FBS, and the SF - Vero cell line is replaced with 2 mL of serum - free EMEM medium (SF - EMEM), and can be incubated in a CO2 incubator at 30 to 40 °C (preferably at 35 °C) with 3 to 7% (preferably 5%) CO2 for 2 to 4 weeks (preferably 3 weeks).

[0068] When the corresponding temperature range, carbon dioxide concentration range, and incubation days are insufficient or exceeded, an appropriate number of cell lines may not be generated, and appropriate transfection conditions for the HAV gene construct may not be formed either.

[0069] Those skilled in the art can gradually passage cells in a medium with reduced serum content until the cells can successfully survive and proliferate in a serum-free medium, thereby easily achieving cell adaptation for incubation in a serum-free medium, so that those skilled in the art can easily obtain serum-free adapted cell lines for each cell line.

[0070] In the present invention, the vector can be injected into host cells by any method known in the art. The vector for preparing hepatitis A virus into which an expression cassette including the hepatitis A virus gene of SEQ ID NO:1 is inserted can be injected into host cells in a variety of suitable ways, including endocytosis, transfection, electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other components; particle bombardment; lipofection; and viral vector injection (such as retroviral vector).

[0071] (b) Obtain the virus from the host cell;

[0072] After step (a), the host cells transfected with the vector are incubated in a nutrient medium to obtain hepatitis A virus as a seed, and the tolerated medium and incubation conditions can be appropriately selected and used according to the host cells at this time. During incubation, conditions such as temperature, pH of the medium, incubation time, etc. can be appropriately adjusted to suit cell growth and virus production. Thus, the virus produced or secreted in the host cells can be recovered from the supernatant of the medium or cell lysates, and can be separated and purified by conventional protein and virus separation techniques.

[0073] Then the virus obtained in step (b) is used to infect host cells and used as a seed for amplification. In one embodiment of the present invention, this virus is named the passage 0 (P0) virus.

[0074] (c) Infect host cells with the obtained virus and passage the infected host cells;

[0075] In step (c) of the present invention, the host cells are infected with the P0 virus obtained in step (b) and passaged repeatedly to produce a virus that can adapt to the host cells and undergo high-speed amplification.

[0076] According to one embodiment of the present invention, when performing infection passage, the host cells MA104 and Vero can be prepared at 1×10 5 to 1×10 7 cells / 5 mL, and the SF-Vero cells at 5×10 5to 5×10 7 cells / 5m preparation. When the host cells are prepared at a lower concentration than the corresponding concentration, it may be difficult to obtain a satisfactory titer, while when the host cells are prepared at a higher concentration than the corresponding concentration, uniform host cell infection may not occur, so it may not be economical.

[0077] According to one embodiment of the present invention, the host cells can be incubated in a 3 to 7% (preferably 5%) CO2 incubator at 30 to 40 °C (preferably 35 °C) for 2 to 4 weeks (preferably 3 weeks). When the corresponding temperature range, carbon dioxide concentration range, and culture days are insufficient or exceeded, an appropriate number of cell lines may not be produced, and appropriate subculture conditions may not be formed either.

[0078] According to one embodiment of the present invention, during the P1 infection passage, M104 and VERO cells are prepared at 5×105 to 5×10 7 cells / 30mL, and SF-Vero cells are prepared at 8×10 5 to 8×10 7 cells / 30mL. The medium can be removed from the prepared cells immediately before infection and washed 1 to 3 times with 30 mL of DPBS. When the corresponding conditions are insufficient or exceeded, the virus may not be obtained with the optimal yield.

[0079] According to one embodiment of the present invention, 1 to 10 mL of infection medium can be used for P1 to P4 infection passages, and 20 to 50 mL of infection medium can be used for P5 to P6 infection passages. When the corresponding conditions are insufficient or exceeded, the virus may not be obtained with the optimal yield.

[0080] In the present invention, passage refers to continuously maintaining the passage of the virus infecting the host cells even if, after the virus infects the host cells, the experimental confirmation of virus proliferation in the culture, i.e., the cytopathic effect or virus detection, is not confirmed.

[0081] The host cells infected with the virus in step (c) are preferably the same host cells as those used for preparing the P0 virus in step (a).

[0082] According to one embodiment of the present invention, the subculture can be specifically carried out as follows. The host cells are treated and infected with the P0 virus and then incubated. After culturing, the host cells are crushed and centrifuged to remove cell debris, and only the supernatant is obtained. The obtained supernatant is used for infecting the host cells for passage 1 (P1).

[0083] The process of infecting the host cells with the virus - culturing the host cells - crushing the host cells - obtaining the supernatant is repeated in one cycle for passage.

[0084] During the subculture process of one cycle of the present invention, the host cells are preferably cultured for 5 days to 30 days, more preferably 10 days to 25 days, even more preferably 17 days to 21 days, and most preferably 19 days to 21 days.

[0085] During each subculture process, the virus proliferation is carried out in the culture medium composition in which the host cells are usually incubated. The host cells are incubated in a standard commercial medium such as a medium supplemented with serum (e.g., 10% fetal bovine serum) or a serum-free medium under conditions of a CO2 concentration suitable for maintaining a neutral buffered pH (e.g., pH between 7.0 and 7.2) and controlled humidity. Optionally, the medium may contain additional nutrients such as L-glutamine, vitamins, sugars, amino acids, peptides, trace elements, sodium pyruvate, peptone, vitamins, sugars (e.g., glucose) and non-essential amino acids, as well as additional supplements that promote the desired growth properties (e.g., trypsin, β-mercaptoethanol, insulin, growth factors, amino acid complexes, etc.).

[0086] In some cases, for example, for the preparation of the virus, it is preferred to grow the host cells under serum-free conditions. The cells can be incubated adherently on a small scale, e.g., in a medium, culture tube or culture flask of less than 25 mL, or in a large culture flask (e.g., Cell Factory System), and can be incubated in microcarriers (e.g., Cytodex, GE Healthcare) in a stirred large culture flask, spinner flask and reactor culture solution. The microcarrier beads are small spheres (with a diameter range of 50 to 100 μm) that can provide a large surface area for adherent cell growth per unit volume of cell culture. For example, in the case of commercial virus production such as vaccine production, it is generally preferred to culture the cells in a bioreactor or fermenter. The volume of the bioreactor that can be used ranges from 1 L or less to more than 100 L. For example, an NBS bioreactor (New Brunswick Scientific, Edison, N.J.); Sartorius Stedim Biotech, Göttingen, Germany) or a Scale-X bioreactor (Scale-X single-use bioreactor system; Univercells Technologies, Belgium) to a commercial-scale bioreactor can also be used.

[0087] In the present invention, regardless of the culture volume, it is important to keep the temperature of the culture solution at 35 °C or below to ensure the effective preparation of hepatitis A virus. Generally, it is preferred to use a controller, such as a thermostat, or other device to sense and maintain the temperature of the cell culture system so that the temperature during virus replication does not exceed 35 °C.

[0088] Methods for maintaining cells in culture have been widely reported and are known in the art. General protocols are known in the art, and changes in conditions during cell culture can be readily determined by routine experimentation.

[0089] On the other hand, in the present invention, the above-mentioned subculture of one cycle per generation can be repeated 2 to 30 times, preferably 2 to 20 times, more preferably 4 to 20 times, even more preferably 4 to 15 times, and most preferably 4 to 10 times.

[0090] (d) Obtaining the virus from the host cell

[0091] Step (d) of the present invention is a step of repeating step (c) to obtain the host cell and its culture medium and recovering the virus for vaccine production.

[0092] Preferably, in step (d), the virus can be obtained when a cytopathic effect appears in the host cell.

[0093] In the present invention, the cytopathic effect refers to all effects caused by hepatitis A virus infection in cells. The cytopathic effect includes plaque formation, cell granulation and fragmentation, detachment of cells from the support (such as a cell-virus culture flask), cell shrinkage, cell aggregation, cell lysis, cell rounding and degeneration, cell shedding, apoptosis induction, etc., but is not limited thereto. The cytopathic effect can usually also be confirmed by microscopic or naked-eye observation.

[0094] Meanwhile, in step (d) of the present invention, after the cytopathic effect appears in the host cell and the virus is obtained from the cell culture solution and the cells, additional subculture is performed to obtain the expected amount of virus per passage, and step (d) can also be carried out by repeating the passage again.

[0095] In the present invention, the amount of virus refers to the amount of virus measured by virus titer (specifically, the content of hepatitis A antigen), the size or shape of plaques, particle density, or other methods known in the art.

[0096] According to one embodiment of the present invention, the cytopathic effect (CPE) was observed starting from the 3rd passage in the host cells subcultured according to the above method, and a significant increase in the amount of virus released into the culture medium and the amount of virus in the cells was observed after the 4th or 5th passage. In addition, it was confirmed that even when the passage was continued, the amount of virus released into the culture medium or the amount of virus in the cells did not decrease but remained unchanged. As a representative example, virus particles were confirmed in the 3rd passage of infection subculture in the SF-Vero cell line by transmission electron microscopy (TEM), and electron micrographs of the prepared virus particles are shown in FIGS. 5A to 5C.

[0097] Therefore, in the present invention, step (d) can be carried out after subculture in step (c) is performed at least twice, preferably three or more times, more preferably four or more times, but the present invention is not limited thereto. Even after the cytopathic effect is observed in the host cell, when it is desired to further amplify the amount of virus, the virus can be obtained when the desired level of virus is obtained by repeating subculture.

[0098] The method of the present invention may further include a virus purification step and a virus inactivation step in order to use the virus obtained after step (d) as a vaccine.

[0099] In the present invention, purification can be carried out by a main purification method or a main purification step, such as chromatography or density gradient ultracentrifugation purification method. Chromatography may include resin ion exchange chromatography, hydrophobic interaction chromatography, mixed chromatography, membrane chromatography, etc. In addition, ion exchange chromatography and size exclusion chromatography can be carried out simultaneously, and ion exchange chromatography and multi-column chromatography can be carried out simultaneously.

[0100] In the present invention, the inactivation step is carried out to completely remove virus infectivity. Generally, the inactivation step can be carried out chemically or physically. For chemical inactivation, the virus can be inactivated with an inactivation solution containing, for example, an appropriate concentration of formaldehyde or β-propiolactone. If necessary, the residual inactivation material can be neutralized later. The material inactivated with formaldehyde can be neutralized with a formaldehyde neutralizing agent containing, for example, sodium sulfite or sodium bisulfite, and can be replaced with phosphate buffer, physiological saline or buffer solution to maintain virus safety by diafiltration.

[0101] In the present invention, the order of the purification and inactivation steps is not particularly limited. The inactivation step can be carried out after purification, or the purification step can be carried out after inactivation. Preferably, the purification step can be carried out after inactivation.

[0102] The present invention provides hepatitis A virus prepared according to the method including steps (a) to (d).

[0103] The hepatitis A virus prepared according to the method of the present invention has a very fast amplification rate in cell lines that have been industrially used, and the safety of the cell lines as vaccine production cell lines has been guaranteed or confirmed by national health departments including the WHO, or has been approved or qualified as a cell substrate for the production of biopharmaceuticals. In addition, even after long-term subculture is repeated in the host cell, the virus can be stably amplified, so the virus can be very usefully used for the production of hepatitis A vaccine.

[0104] The present invention also provides a vaccine composition containing the virus as an active ingredient.

[0105] In the present invention, the vaccine can be a live vaccine, an attenuated vaccine or an inactivated vaccine.

[0106] A "live vaccine" refers to a vaccine containing a live virus active ingredient. "Attenuation" means weakening the pathogenicity of a live virus through artificial or natural factors, inducing immunity by only stimulating the immune system without causing disease in the body. Attenuation of a virus can be achieved by performing heat treatment of virus particles, ultraviolet irradiation of the virus, in vitro high-order continuous passage culture, or multiple consecutive virus passages in cells incubated in an in vitro culture container such as a culture flask. Attenuation can also be achieved by performing different genetic modifications, for example, by specifically deleting virus sequences known to provide toxicity or inserting sequences into the virus genome. An "inactivated vaccine", also known as a killed vaccine, is a vaccine containing a virus from which infectivity has been removed. Examples thereof include whole virus vaccines and split virus vaccines that can be easily prepared by known methods.

[0107] In addition to the above viruses, the vaccine composition of the present invention may further include one, two or three or more adjuvants. The term "adjuvant" refers to a compound or mixture that enhances the immune response against an antigen. Adjuvants can mainly be used as a delivery system, mainly as an immunomodulator, or have the strong characteristics of both. Suitable adjuvants include those suitable for mammals including humans.

[0108] Adjuvants suitable for enhancing the efficacy of the vaccine composition of the present invention include but are not limited to the following materials:

[0109] (1) Aluminum salts (Alum) (such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.);

[0110] (2) Oil-in-water emulsion preparations (with or without other specific immune stimulants such as muramyl peptides or bacterial cell wall components);

[0111] (3) Particles used in combination with saponin adjuvants such as Quil A or STIMULON TM QS-21 [Antigenics, Framingham, MA] (U.S. Patent No. 5,057,540) or particles produced therefrom;

[0112] (4) Synthetic polynucleotides; and

[0113] (5) Cytokines such as interleukin, interferon, granulocyte macrophage colony-stimulating factor, macrophage colony-stimulating factor, tumor necrosis factor, etc.

[0114] In a specific embodiment, an aluminum-based adjuvant can be used. Aluminum salt adjuvants can be aluminum-precipitated vaccines or aluminum-adsorbed vaccines. Aluminum salts include hydrated aluminum oxide, aluminum oxide, aluminum trihydrate, aluminum phosphate gel, Superfos, amphogel, aluminum hydroxide (III), aluminum phosphate adjuvant (APA), amorphous aluminum oxide, etc., but are not limited thereto. Aluminum salts form an antigen depot that slowly releases antigens within 2 to 3 weeks to non-specifically activate macrophages, complement, and innate immune mechanisms.

[0115] In another specific embodiment, the vaccine composition disclosed in the present invention can include CpG oligonucleotides as adjuvants. CpG oligonucleotides refer to immunostimulatory CpG oligodeoxynucleotides (CpG ODNs), and thus these terms can be used interchangeably unless otherwise specified.

[0116] The adjuvant is appropriately selected according to the amount and valence of the conjugate in the composition. However, when an aluminum-based adjuvant is used, aluminum element can be added, and the amount thereof included in the composition is 0.01 mg / mL to 1.0 mg / mL based on the aluminum element. Preferably, the content of aluminum element in the composition can be 0.1 mg / mL to 0.6 mg / mL, or 0.1 mg / mL to 0.4 mg / mL, and more preferably the content of aluminum element in the composition can be 0.15 mg / mL to 0.35 mg / mL.

[0117] As long as the vaccine composition of the present invention achieves its effect, there is no particular limitation on the formulation providing the composition.

[0118] The vaccine composition of the present invention can be formulated in a liquid form (i.e., solution or suspension) or in a lyophilized form. In one embodiment, the vaccine composition of the present invention is in a liquid form, preferably in an aqueous liquid form. When provided as a liquid preparation, the vaccine composition of the present invention is provided in a form in which the liquid preparation is packaged in a container (preferably a syringe) for direct administration without a separate vaccine composition reproduction process such as redispersion. Therefore, different from the composition of a lyophilized preparation that needs to be resuspended in an aqueous medium, the liquid preparation can be ideal for injection and for reproducing a certain effect.

[0119] The formulation of the vaccine composition of the present invention can be carried out using various methods known in the art. For example, the composition can be prepared by formulating hepatitis A virus in a physiologically acceptable carrier. Examples of the carrier include water, buffered saline, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), polysorbate 20, and glucose solution, but are not limited thereto.

[0120] The present invention provides a vaccine composition, which comprises hepatitis A virus disclosed by the present invention and a pharmaceutically acceptable excipient, carrier, isotonic agent or diluent. The types of the excipient, carrier or diluent are known in the art and can be used according to the administration route of the pharmaceutical composition described below.

[0121] In one example, pharmaceutically acceptable carriers for liquid formulations include aqueous or non-aqueous solvents, suspensions, emulsions and oils. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol and ethyl oleate. Aqueous solvents include water, aqueous solutions, emulsions or suspensions, physiological saline and buffer solutions. The pharmaceutical composition can be isotonic, hypotonic or hypertonic. However, the pharmaceutical composition administered by injection is preferably substantially isotonic. Therefore, isotonicity or hypertonicity can be advantageous for the storage of the composition. When the pharmaceutical composition is hypertonic, the pharmaceutical composition can be diluted to isotonicity before administration. The isotonic agent can be an ionic isotonic agent or a non-ionic isotonic agent. Ionic isotonic agents include sodium chloride, calcium chloride, potassium chloride, magnesium chloride, etc., but are not limited thereto. Non-ionic isotonic agents include sorbitol, glycerol, etc., but are not limited thereto. Preferably, at least one pharmaceutically acceptable buffer is included. For example, when the pharmaceutical composition is an injection, the pharmaceutical composition preferably contains a buffer having buffering capacity at pH 5.0 to pH 9.0, such as pH 6.0 to pH 8.0 and pH 6.8 to pH 7.5. The buffer can be selected from buffers composed of potassium phosphate, monosodium phosphate, glutamate, carbonate, borate, lactate, citrate, histidine, glycine, triethanolamine, etc.

[0122] The vaccine composition of the present invention may additionally include at least one selected from buffers, salts, divalent cations, surfactants (especially non-ionic detergents), cryoprotectants (such as sugars), antioxidants (such as chelating agents), preservatives and antifungal agents.

[0123] The type of buffer is not particularly limited as long as the buffer is known in the art for use in pharmaceutical compositions, especially vaccine compositions, but histidine, citrate, phosphate, succinate or 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes) can be used. These buffers can be used in the form of any compound. For example, phosphate can be used in the form of sodium phosphate and potassium phosphate.

[0124] In one embodiment, the vaccine composition of the present invention contains a salt. The type of the salt is not particularly limited as long as the salt is known in the art for use in pharmaceutical compositions, especially vaccine compositions, but can be selected from magnesium chloride, potassium chloride, sodium chloride, borate chloride and combinations thereof.

[0125] In one embodiment, the vaccine composition of the present invention comprises a surfactant. In a preferred embodiment, a non-ionic detergent is used. In one embodiment, the surfactant is selected from polysorbate 20 (Tween TM 20), polysorbate 40 (Tween TM 40), polysorbate 60 (Tween TM 60), polysorbate 65 (Twin TM 65), polysorbate 80 (Twin TM 80), polysorbate 85 (Twin TM 85), Triton TM N-101, Triton TM X-100, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H 15), octyl thioglucoside (OTG), octyl glucoside (OG), nonyl maltoside (NM), lauryldimethylamine oxide (LDAO), dodecyl β-D-maltoside (DDM), and poloxamer.

[0126] In the present invention, "per dose" has the same meaning as "unit dose" or "one dose" and can be used interchangeably herein. Preferably, the unit dose may refer to a unit suitable as a unit dose for animals, preferably mammals, especially humans, and each unit contains an antigenic material calculated by those skilled in the art to have a preventive or immunizing effect on the expected disease (especially without the risk of serious side effects at the same time). In a preferred embodiment, the vaccine composition provided by the present invention can be provided in the form of unit doses.

[0127] The dose can be a suitable amount set by those skilled in the art according to the common knowledge in the field of drug preparation, for example, set according to its administration mode or administration route, etc. For example, a single injection dose can be 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, or 1.0 mL, but is not limited thereto.

[0128] As a more convenient way to directly provide the above vaccine composition of the present invention to users, the present invention provides a container filled with any of the above vaccine compositions.

[0129] The type of the container is not particularly limited as long as the container is known in the art for providing a pharmaceutical composition or a vaccine composition. In one embodiment, the container is selected from pre-filled syringes, vials, syringes, sterile disposable needles, microneedle patches, ampoules, and graduated cylinders.

[0130] Depending on the administration group, a typical single-dose injection of the vaccine composition of the present invention can be provided in a volume of 0.5 mL or 1.0 mL, more preferably 0.5 mL or 1.0 mL.

[0131] Thus, a container or syringe as defined above can be provided, wherein the vaccine composition of the present invention is filled therein in a single-dose injection volume. In one embodiment, the container or syringe can be provided as filled with any one of the vaccine compositions defined in the present invention in a volume of, for example, 0.5 mL or 1.0 mL.

[0132] The present invention also provides a kit comprising the above-mentioned vaccine composition of the present invention. The specific components of the kit can be provided with reference to the known forms in the art according to the provided form of the composition. Obviously, any container containing the above vaccine composition is included in the kit.

[0133] In one example, the kit can be provided with one or more vials containing or not containing the vaccine composition (liquid preparation or lyophilized preparation) of the present invention, one or more syringes containing or not containing the vaccine composition of the present invention, or a kit comprising all of the vials or syringes.

[0134] In addition, when the vaccine composition of the present invention is provided as a liquid preparation, the vaccine material is contained in a pre-filled syringe, and at this time, the material becomes the material for patient administration. The vaccine composition can be administered to a patient by connecting a sterile injection needle to the inlet of the pre-filled syringe.

[0135] The kit can also include a package insert provided to the user.

[0136] In the present invention, the kit can provide the vaccine composition in doses of a single vaccination schedule or can provide the doses in a multiple (split) vaccination schedule.

[0137] In one embodiment, the vaccine composition disclosed herein is used as a drug (pharmaceutical composition). The vaccine composition disclosed in the present invention can be used as a pharmaceutical composition in various therapeutic or prophylactic methods for preventing, treating or improving a bacterial infection, disease or disorder in a subject. In particular, the vaccine composition disclosed in the present invention can be used for preventing, treating or improving an infection, disease or disorder caused by hepatitis A virus in a subject.

[0138] In addition, the present invention provides a method for vaccinating against hepatitis A, which is characterized by administering an effective amount of the above-mentioned vaccine composition of the present invention to an individual in need thereof.

[0139] In the present invention, an "effective amount" refers to an amount that exhibits an effect of killing or improving hepatitis A virus, treating or preventing an infection, disease or disorder associated with hepatitis A virus when administered to an individual.

[0140] In one embodiment, the effective amount is an immunologically effective amount. An immunologically effective amount is the amount of an antigen or vaccine that is sufficient to elicit a cellular (T cell) or humoral (B cell or antibody) immune response as measured by standard assays known to those of skill in the art. The level of an antigen, such as a viral antigen or antigen-specific antiserum or neutralizing antibody induced by a vaccine thereof, as an immunogen can be measured by detecting cytokines secreted by T cells stimulated by the viral antigen or can be measured. In addition, the protective level of the immune response can be measured by identifying a reduction in antigen-derived viral infection in the immunized individual or a disease caused by preventing infection.

[0141] In one embodiment, the effective amount is a prophylactically effective amount. In the present invention, the term "prophylaxis" refers to inhibiting the occurrence of a disorder or disease in an individual who has never been diagnosed with the disorder or disease but is predisposed to having the disorder or disease. Thus, as used herein, the term "prophylactically effective amount" refers to an amount sufficient to achieve the pharmacological effect.

[0142] In a specific embodiment, the vaccine composition disclosed herein can be used to prevent hepatitis A in a subject. Accordingly, the present invention provides a method for preventing hepatitis A, comprising administering an effective amount of the vaccine composition of the present invention to an individual in need thereof.

[0143] In the present invention, the term "individual" can be used interchangeably with "subject" and can be an animal, preferably a mammal, particularly including a human, such as a cat, sheep, pig, horse, cow or dog, etc., and can also be cells, tissues, organs, etc. derived from an animal. The individual can be a patient in need of the effect.

[0144] In one embodiment of the present invention, the immunosuppressed individuals disclosed herein are any human male or human female.

[0145] In the vaccine composition of the present invention, the route of administration is not particularly limited as long as the desired effect is achieved in vivo, but the vaccine composition is administered by intramuscular, intraperitoneal, intradermal, subcutaneous, rectal, systemic or mucosal routes for protecting or treating humans susceptible to hepatitis A virus.

[0146] The vaccine composition of the present invention can be provided in a single dose or multiple doses. In some cases, a single dose of the vaccine composition of the present invention is sufficient, but in some cases, such as in greater immunodeficiency conditions, a second, third or fourth dose can also be provided. After the initial vaccination, the subject can be given one or several (multiple) additional immunizations at appropriate time intervals.

[0147] The present invention also provides a kit comprising the vaccine composition.

[0148] The present invention also provides a pre-filled syringe filled with the vaccine composition.

[0149] The kit and the pre-filled syringe may include an HAV vaccine composition and a pharmaceutically acceptable carrier. In addition, the kit and the pre-filled syringe may further include instructions on the basic matters to be observed for preventing HAV infection (matters to be noted during administration, dosing cycle, storage temperature, expiration date, etc.).

[0150] The present invention provides the use of the virus for preparing a hepatitis A vaccine.

[0151] The present invention provides the use of a vaccine composition comprising a hepatitis virus as an active ingredient in the prevention of hepatitis A.

[0152] The present invention provides a method for preventing hepatitis A, comprising administering an effective dose of the vaccine composition comprising a hepatitis virus as an active ingredient to an individual in need.

[0153] As used herein, the term "comprising" has the same meaning as "including" or "characterized by", and does not exclude other ingredients or method steps not specifically mentioned in the composition or method according to the present invention. Unless otherwise specified, the term "consisting of" means excluding additional elements, steps or ingredients, etc. The term "consisting essentially of" means that within the scope of the composition or method, in addition to the materials or steps described, it also includes materials or steps that do not substantially affect its basic properties.

[0154] Advantageous Effects

[0155] According to the method for preparing hepatitis A virus provided by the present invention, hepatitis A virus that can be stably amplified in a short time can be prepared, which is very useful as a raw material for hepatitis A vaccine virus. Brief Description of the Drawings

[0156] Figure 1 It is a diagram illustrating a method for producing hepatitis A virus (HAV) for vaccine preparation according to the method of the present invention. By obtaining a seed virus from a host cell transfected with a vector containing the HAV gene, infecting the same host cell with the seed virus and performing passage culture thereon, HAV can be rapidly and stably amplified.

[0157] Figure 2 It is a diagram illustrating a cleavage map of the vector for transfecting a host cell for preparing a seed virus in the present invention.

[0158] Figures 3A and 3B are schematic diagrams exemplifying the method for stepwise preparation of HAV from MA104 cells and Vero cells according to the method of the present invention (Figure 3A), and the method for preparation of HAV from serum-free adapted Vero cells (SF-Vero) (Figure 3B).

[0159] Figure 4 is a schematic diagram of the method for stepwise preparation of HAV in the related art.

[0160] Figures 5A to 5C are diagrams of the virus isolated at passage 3 (P3) after the seed virus prepared according to the method of the present invention infects MA104 (Figure 5A), Vero (Figure 5B), and SF-Vero (Figure 5C) as observed by transmission electron microscopy (TEM).

[0161] Figure 6 is the result of confirming the virus content (titer) identified in the 5th passage (P5) in the 1st passage (P1), which is the blind passage step for virus rescue after P0 during the methods of Figures 3A and 3B of the present invention. The virus titers in the cell lysates and culture supernatants at each passage from P1 to P5 were measured as absorbance values.

[0162] Figures 7A and 7B exemplify the quantitative analysis results of the virus content (titer) identified in the cell lysate samples isolated from p6 in the methods of Figures 3A and 3B of the present invention (Figure 7A), and the results of detecting the virus infected in cells by immunofluorescence assay (IFA) after the seed virus prepared in the 6th passage culture infects MA104, Vero, and SF-Vero cells (Figure 7B).

[0163] Figure 8 is after commercially available HAV infects host cells MA104, Vero, and SF-Vero ( Figure 8 A ) or after the seed virus prepared according to the methods of Figures 3A and 3B infects the same host cells ( Figure 8 B ), the figure of measuring the virus content after continuously performing virus infection passages 1 to 6 times. In addition, Figure 8 exemplifies the absorbance of the virus titers in the infected host cell lysates and culture supernatants harvested at each of the 6 passages measured by ELISA, and the result of relatively comparing the virus proliferation in host cells A and B.

[0164] Figures 9A and 9B are the results of confirming the virus titers in the supernatant and cells after the seed virus prepared according to the method of Figure 3B infects the SF-Vero cell line and the MRC-5 cell line 10 times.

[0165] Figure 10The example illustrates the confirmation of virus titer (antigen content) according to the number of days of incubation of the virus in a T-flask (175 cm 2 ). To confirm the virus growth pattern after infection with the seed virus of the present invention, infected samples were harvested every 3 to 4 days, and the virus titer was measured and described from the supernatant and cell lysate.

[0166] Figure 11 The example illustrates the results of confirming the virus titer in the cell lysate of passage P11 by infecting host cells ( Figure 11 A) in a cell factory 10 (CF10) culture container with a commercially available HAV or infecting host cells MA104, Vero, or SF-Vero ( Figure 11 B) with the seed virus prepared according to the method of the present invention.

[0167] Figure 12 The example illustrates a graph of the anti-HAV serum titers analyzed in the blood of experimental animals before inoculation with the inactivated antigen of HAV prepared according to the method of the present invention or a commercial HAV vaccine (Havrix, GSK) (day -5), after two inoculations at 2-week intervals (day 28), and after three inoculations (day 42). SK144 and SK72 are groups administered the purified and inactivated virus of the present invention at 144 EL.U (3.0 IU) and 72 EL.U (1.5 IU) respectively, and HVR144 and HVR72 are groups administered commercial Havrix at 144 EL.U and 72 EL.U. As a negative control for the experiment, the normal group was administered physiological saline instead of the virus antigen, and the aluminum group was administered only the aluminum adjuvant without the antigen.

[0168] Figure 13 is an example showing that during the Figure 12 experiment, by observing the average body weight of each animal group, a graph of whether there are adverse reactions due to antigen inoculation was observed. Detailed Description of the Invention

[0169] Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrative purposes of the present invention, and the content of the present invention is not limited to the following examples.

[0170] Experimental Methods

[0171] In Figure 1 the experimental methods conducted in the present invention are summarized.

[0172] 1. Gene Synthesis and Vector Preparation

[0173] In the present invention, the nucleotide sequence of the HAV gene for gene synthesis is shown in SEQ ID NO:1. The HAV gene contains a nucleotide sequence, and its functional order is 5' untranslated region (UTR), polyprotein gene, and 3' UTR. The nucleotide sequences of CMV promoter - T7 promoter (SEQ ID NO:2), multiple cloning site (MCS, SEQ ID NO:3), and hammerhead (HH) ribozyme (SEQ ID NO:4) are included in the 5'-end direction of the HAV gene, and the nucleotide sequences of hepatitis delta virus (HDV) ribozyme (SEQ ID NO:5), MCS (SEQ ID NO:6), and bGH polyA terminator (SEQ ID NO:7) are located in the 3'-end direction. The nucleotide sequence consisting of all other functional regions such as the HAV gene, promoter, etc. and composed of the nucleotide sequence of SEQ ID NO:1 is defined by SEQ ID NO:8.

[0174] The synthesized hepatitis A virus gene of SEQ ID NO:8 was cloned into the pUC57 vector using the KpnI (GGTACC) and SalI (GTCGAC) restriction enzymes. The complete plasmid construction diagram is as Figure 2 shown, and it is called the HAV expression vector.

[0175] 2. Transfection using the HAV expression vector

[0176] MA104 (ECACC, 85102918) and Vero (WHO) (ECACC, 88020401) cell lines were prepared in 6-well culture plates in EMEM (Lonza) medium containing 10% and 5% FBS respectively at 2×10 5 cells / well / 2 mL. Serum-free Vero cells (serum-free adapted cells from Vero (WHO), SF-Vero) were prepared in serum-free EMEM medium at 4×10 5 cells / well / 2 mL and incubated in a 5% CO2 incubator at 37°C. The medium used for cell preparation became the medium for each cell.

[0177] After 18 to 24 hours, remove the medium from the culture plates, wash twice with 2 mL of DPBS, and add 2 mL of medium to each well. Mix 1.0 μg of the HAV expression vector (plasmid), 35 μL of Lipofectamine LTX-Plus (ThermoFisher), and 960 μL of Opti-MEM (ThermoFisher) in a conical tube and incubate at room temperature for 15 minutes. Add 200 μL of this mixture to each well of the plate and incubate in a 5% CO2, 37 °C incubator. After 24 hours, remove the supernatant from all cells, change the medium of the MA104 and Vero cell lines to EMEM containing 2% FBS (2% FBS-EMEM), change the medium of the SF-Vero cell line to 2 mL of serum-free EMEM medium (SF-EMEM), and incubate the cell lines in a 35 °C, 5% CO2 incubator for 3 weeks. Harvest the incubated MA104, Vero, and SF-Vero cells in 500 μL of EMEM medium, suspend, and freeze / thaw 3 times. To remove the remaining fragmented cell debris, after centrifugation at 10,000 g for 1 minute, only harvest the supernatant again and designate it as virus P0 (seed virus).

[0178] 3. Blind passage for virus rescue

[0179] When performing an infective passage as the first blind passage P1 using the P0 virus, 1×10 6 cells / 5 mL of MA104 and Vero and 5×10 6 cells / 5 mL of SF-Vero are prepared in a T25 culture flask 24 hours before infection. For the P1 to P4 infective passages, in a T25 culture flask, MA104 and Vero cells at a concentration of 7×10 5 cells / 5 mL and SF-Vero cells at a concentration of 1×10 6 cells / 5 mL are incubated in the medium at 37 °C and 5% CO2. After 24 hours, remove the medium from the prepared cells, add 5 mL of DPBS just before infection, and wash twice. In the P5 and P6 infective passages, 1 day before the P4 infective passage, 5×10 6 cells / 30 mL of MA104 and Vero cells and 8×10 6 cells / 30 mL of SF-Vero cells are prepared in a T175 culture flask, and the medium is removed from the prepared cells in the same manner just before infection and washed twice with 30 mL of DPBS.

[0180] EMEM medium containing 2% FBS is the virus infection medium for MA104 and Vero cell lines, and EMEM medium without FBS is used as the infection medium for SF-Vero cell line. 5 mL of each cell infection medium is used for P1 to P4 infection passages, and 35 mL of infection medium is used for P5 and P6 infection passages. In the P6 passage, five T175 culture flasks are used for infection passage. The prepared P0 sample is added to the prepared cells, placed at 35 °C and 5% CO2 for 1 hour, and then 5 mL (35 mL) of infection medium is added.

[0181] After infection with the P0 sample, the medium is changed every 7 days for 21 days, and the infected cells are maintained. On the 21st day after infection, the cell lysate is harvested and subjected to 3 freeze-thaw cycles (-70 °C / 37 °C) as in the P0 harvest, then the cell debris is removed and centrifuged, and only the supernatant is harvested and used as the virus medium, designated as the first passage (P1). During the passage from P0 to P1, the medium is changed once a week, but during the passage from P1 to P6, the medium is changed only on the 7th day after infection. With a passage cycle of 20 to 21 days, 6 passages are carried out continuously. The above processes of cell infection and passage are shown in Figures 3A and 3B.

[0182] After completing one passage, the culture supernatant and cell lysate are harvested separately from the virus-infected culture flasks. When harvesting the cell lysate, 2 mL of trypsin-Versene (Lonza) solution is added to the culture flask from which the supernatant has been removed, washed and removed, then 2 mL of trypsin-Versene solution is added again, placed in a 37 °C incubator for 5 minutes, and the cells are separated. The 2 mL trypsin-Versene suspension containing the cells is transferred to a conical tube and centrifuged to harvest the cell pellet.

[0183] The cell lysate (pellet) is added to 1 mL of EMEM (T25, P1 - P4) or 5 mL of EMEM (T175, P5), centrifuged after 3 freeze-thaw cycles, and then the supernatant from which the cell debris has been removed (cell lysate sample) is prepared. After centrifuging the harvested culture supernatant from each infection passage, 200 μL of the cell lysate sample is transferred to a microcentrifuge tube for virus titer analysis and frozen until analysis. Except for analysis, all the remaining cell lysate samples are used for the next infection.

[0184] The titers of P1 to P5 are measured by ELISA qualitative analysis. At the 6th passage (P6), the culture supernatant is removed, all the infected cells are harvested, suspended in 5 mL of serum-free EMEM, centrifuged after 5 freeze-thaw cycles to remove the cell debris, and then only the supernatant is harvested and stored as the seed virus. The titer of the P6 virus is measured by ELISA qualitative analysis.

[0185] 4. Confirm the cytopathic effect in the blind passage process

[0186] Examine the cytopathic effect (CPE) of host cells due to virus infection by microscopy. In the MA104 and Vero cell lines, virus-induced cytopathic effects were not shown until passage P4, but CPEs such as cell lysis and cell detachment caused by the virus were confirmed starting from passage P5. The SF-Vero cell line showed mild CPE after passage P3.

[0187] 5. HAV antigen analysis ELISA

[0188] Determine the HAV antigen in the samples harvested during the blind passage of the virus by qualitative and quantitative analysis. For qualitative analysis, perform HAV-specific ELISA to confirm the antigen by absorbance (optical density, 450 nm). For quantitative analysis, draw a standard curve by applying a standard (inactivated HAV BRP, 1350 IU / mL, Y0001192, EDQM) and measure the HAV virus titer (antigen content) in the samples. Use the detection kit HAV-Antigen ELISA kit (Mediagnost, E12), and express the virus titer unit as IU / mL according to the standard. The commercially available hepatitis A virus strain (ATCC VR-1402) used was also quantified by the same method.

[0189] 6. Transmission electron microscopy (TEM)

[0190] As shown in Figures 3A and 3B, observe the transmission electron microscopy images using a portion of the cell lysate harvested after passage culture at P6. Use an EM grid coated with a carbon support film (formvar-carbon), perform negative staining with 2% uranyl acetate for 15 seconds, observe with a transmission electron microscope (JEM-1011, JEOL), and identify virus particles using a Camera-Megaview III imaging device.

[0191] 7. Immunofluorescence assay (IFA)

[0192] MA104 and Vero cells under the condition of 5×10 3 cells / well / 0.5 mL and 8×10 3SF-Vero cells at a concentration of [number of cells] / well / 0.5 mL were suspended and prepared in each medium in a 24-well culture plate and incubated at 37 °C and 5% CO2 for 24 hours. The medium was removed from the incubated cells and the cells were washed by adding 300 μL / well of DPBS. The seed virus prepared as shown in Figure 3 was diluted with the infection medium per cell, treated at a concentration of 0.1 IU / well in the cells, and 0.5 mL of the infection medium was added to each well and incubated in a 35 °C and 5% CO2 incubator. On the 7th day from the day of infection, the cell supernatant was completely removed and washed twice with 0.5 mL of DPB. All the remaining DPBS in the cells was removed, 0.2 mL of 3.7% formaldehyde solution was added, and then left at room temperature for 30 minutes. The formaldehyde solution was removed in the same manner as above and washed 3 times with DPBS. 0.2% Triton X-100 buffer solution was added at 250 μL / well, left at room temperature for 5 minutes, and then washed 3 times with 0.5 mL of DPBS. The primary antibody (anti-HAV surface Ag, Raybiotech) was diluted to 1 / 500 in PBS, added at 250 μL / well, and reacted at room temperature for 1 hour. Thereafter, the secondary antibody (goat anti-mouse IgG Alexa488, ThermoFisher) was diluted to 1 / 4000 and added to the cells at the same dose as the treatment dose of the primary antibody, left at room temperature for 1 hour, then washed 5 times with DPBS and removed. DPBS was added to the cells at 250 μL / well, and the cells were photographed with a fluorescence microscope (magnification 100×, Eclipse Ts2-FL, Nikon).

[0193] 8. Confirmation of seed virus infection (infection test)

[0194] The seed virus prepared as shown in Figures 3A and 3B was compared with the infection pattern of a commercial hepatitis A virus strain (ATCC VR-1402). For this purpose, the virus titer (content) of the commercial virus strain was quantified by ELISA described in this patent. 24 hours before virus infection, MA104 and Vero cell lines were prepared at [number of cells] in 12 mL of medium in a T75 culture flask, and the SF-Vero cell line was prepared at [number of cells] in 12 mL of medium 6 and the SF-Vero cell line was prepared at 3.5×10 6Cell preparation. The seed virus and the commercial virus strain were suspended at 2.0 IU in 1 mL of infection medium, washed with 10 mL of DPBS and added to each cell prepared in a T75 culture flask. After reacting for 1 hour in a 35 °C, 5% CO2 incubator, 11 mL of infection medium was added and incubated. The cells were cultured for 21 days, and the medium was replaced with fresh infection medium on the 7th day after infection. A total of 10 passages of virus infection of the 21-day incubation were carried out, and the continuous infection passages and blind passages for sample harvesting and virus content analysis were the same. When harvesting the cell lysate sample, the cells were finally suspended in 5 mL of serum-free EMEM and used as a sample for the next infection passage.

[0195] In addition, the MRC-5 (ECACC, 05011802) cell line was prepared in a T75 culture flask, with a total of 8×10 6 cells / 12 mL, and SF-Vero was prepared at a cell concentration of 3.5×10 6 cells / 12 mL. The seed virus prepared as shown in Figure 3B was subjected to a total of 6 consecutive infection passages under the same conditions as the T75 culture flask infection described above. The virus titers (contents) in some supernatant and some cell lysate samples harvested at each virus infection passage were measured and compared. The medium used for the MRC-5 cell line contained 10% FBS and 2% concentration of infection medium in the EMEM medium. The methods for harvesting the supernatant and cells after virus infection were the same as the method of repeated freeze-thawing, and the specific methods were the same as those described in the method for performing blind passages.

[0196] 9. Growth confirmation test of seed virus

[0197] One day before infection, SF-Vero cells were seeded at 2×10 7 cells / 35 mL in a T175 culture flask and incubated at 37 °C and 5% CO2 conditions. A total of 9 T175 culture flasks with the same cell density were prepared, 8 of which were infected with the virus and the rest served as normal cell controls. After removing the medium from all culture flasks on the day of infection, the cells in each flask were washed with 30 mL of DPBS. A virus infection solution containing 15 IU of SF-Vero-derived seed virus in 35 mL of medium was prepared and added to the washed T175 culture flasks. A total of 8 flasks were equally infected with 15 IU of SF-Vero-derived seed virus. When the infected cells were incubated in a 35 °C, 5% CO2 incubator, the supernatant and cell lysate were harvested on the 3rd, 7th, 10th, 14th, 17th, 21st, 24th, and 28th days post-infection (dpi), respectively, and the virus titers (contents) were measured.

[0198] 10. Incubation of seed virus cell factory

[0199] The superinfection passage of each cell-derived seed virus was carried out in T175 culture flasks in the same manner and then progressed to the 11th passage (MA104, Vero) and the 12th passage (SF-Vero) to increase the cell adaptability of the virus and harvest the infected cells during the corresponding process. In the method of superinfection passage, the first seed virus harvested from P6 was infected and incubated twice in a T175 culture flask to obtain the virus of the P8 generation. When infecting P9, the virus sample obtained from P8 was quantitatively infected at a concentration of 15 IU / T175. Similarly, the virus was passaged to P11 and P12 by repeated infection-harvest. The P11 (MA104, Vero) and P12 (SF-Vero) passage viruses of the seed virus were quantified and the virus corresponding to 500 IU was aliquoted into separate cryotubes. Using Figure 6 the commercial virus sample obtained as shown after 6 passages, the commercial virus was infected and passaged to P11 (MA104, Vero) and P12 (SF-Vero) in the same manner as the superinfection passage to obtain the virus. In the superinfection passage, the supernatant was not collected, only the infected cells were harvested, the cells were disrupted with a Sonifier for 40 seconds, centrifuged, and then only the supernatant was collected and used. The seed virus and the commercial virus were each used at an equal amount of 500 IU for host cell infection of CF10 (6320 cm2, ThermoFisher). The infection method in CF10 was as follows. MA104, Vero, and SF-Vero cells were prepared in a medium of 2.0×10 8 cells / 1.5 L. After 16 to 18 hours, the medium was removed, and the cells were washed once with 500 mL of DPBS and removed. A virus infection solution was prepared by adding 500 IU of the prepared virus to 200 mL of EMEM medium and added to the washed CF10. The virus infection solution was incubated at 35 °C and 5% CO2 for 60 minutes, and the culture flask was tilted every 15 minutes to evenly adsorb the virus diluent onto the cells. After virus adsorption, 1.5 L of infection medium (MA104, Vero: 2% FBS-EMEM; SF-Vero: SF-EMEM) was added to each cell and incubated at 35 °C and 5% CO2 for 21 days. On the 7th day of virus incubation, the medium was replaced with fresh infection medium.

[0200] After infection, the harvesting process of the virus-infected culture in CF10 is as follows. After infection, the supernatant is removed from the CF10 container. CF10 is washed with 500 mL of DPBS and removed, 200 mL of TrypLE Express (ThermoFisher) is added and reacted in a 37 °C incubator for 3 to 5 minutes. 200 mL of serum-free EMEM is added, and approximately 400 mL of the infected cell suspension is harvested in a 2 L square flask. The harvested suspension is centrifuged at 5000 g for 10 minutes, the supernatant is removed, and only the cell pellet is recovered. 100 mL of phosphate buffer (50 mM, pH 7.0) is added to the cell pellet, suspended, and then sonicated (amplitude 40%, 2 minutes) with a Sonifier (SFX550, Branson) for cell lysis. Approximately 100 mL of the cell lysate is centrifuged (5000 g, AllegraX-15R, SX4750A), and the supernatant is transferred to a new sterile 1 L square flask. After centrifugation, approximately 100 μL of the 100 mL supernatant is stored for ELSIA analysis. 400 mL of phosphate buffer (50 mM, pH 7.0) is added and used for the purification process. The cell lysates derived from MA104 and Vero are stored separately, and the lysate of the virus-infected cells incubated in SF-Vero is used for antigen purification for animal experiment administration.

[0201] 11. Virus purification and inactivation

[0202] The recovered cell lysate is purified successively using a capsule filter (Sartopure PP3, 5 μm, Sartorius Stedim) and a depth filter (Supra50, 050PDH4, PALL). The purified harvest is buffer-exchanged with phosphate buffer (50 mM, pH 7.0) using a 100 kDa ultrafiltration / diafiltration (UF / DF) filter ( 2Mini, P2B100A01, Merck, Millipore), filtered and concentrated 10-fold, and then treated with benzonase (1 unit). DEAE Fast Flow (GEHealthcare) column equilibrated with phosphate buffer (50 mM, pH 7.0) is used for ion exchange chromatography (IEC) at a rate of 10 mL / min. Approximately 200 mL fractions are collected, and a 10 kDa UF / DF filter ( The 2 Mini, P2B010A01, Merck Millipore) was buffer-exchanged with phosphate buffer (50 mM, pH 7.0) and concentrated 5-fold. Size-exclusion chromatography (SEC) was performed on the concentrate using HiPrep 26 / 60 Sephacryl S-200 HR (GE Healthcare) at a rate of 1 mL / min.

[0203] After SEC, approximately 40 mL of the antigen fraction was collected and concentrated 80-fold using a 10 kDa filter ( 2 Mini, P2B010A01, Merck Millipore) to obtain purified HAV antigen. Formaldehyde was added to the obtained antigen at a concentration of 270 to 370 μg / mL for virus inactivation and reacted at 37 °C for 5 days. Thereafter, the antigen was diafiltered using a 10 kDa filter and sterile filtered using a 0.22 μm filter ( Gold). Twenty-four hours before each administration in animal experiments, aluminum hydroxide was suspended in adsorption buffer (pH 7.1 - 8.0), mixed with the antigen, and stirred at 4 °C for 16 hours or longer.

[0204] 12. Animal experiments

[0205] The aluminum-adsorbed antigen was administered to mice (BALB / c, 4 weeks old, 10 subjects per group). As a control for the animal experiments, a commercial HAV vaccine (Havrix, GSK) was used, and the antigen (3.0 IU and 1.5 IU) and control doses (144 EL.U and 72 EL.U, ELISA units) administered to the mice were set to 1 / 10 of the human (adult and infant) doses of the commercial vaccine. After separating only the antigen by dissociating the aluminum salt bound to the control (commercial vaccine), the dose setting of the administered antigen was adjusted to the same level by measuring and comparing the antigen of the present invention and its amount. In the animal experiments, the antigen was administered 3 times at 2-week intervals by intramuscular injection (IM). After administration, sera were separated from the whole blood of the mice to measure the anti-total HAV antibody titers. 97 / 646 (NIBSC, International Standard for Anti-Hepatitis A, Immunoglobulin) was set as the standard control material, and anti-HAV ELISA (E10, Mediagnost) or anti-hepatitis A virus IgG ELISA (4660, ALPHA Diagnostic International) was used during the measurement.

[0206] Experimental results

[0207] In the present invention, a gene expression cassette was designed to express the HAV gene, and the corresponding cassette was synthesized to obtain a cassette expression vector. The synthesized HAV expression vector was transfected into three cell lines, MA104, Vero, and SF-Vero. The gene-transfected cells (transfectants) were lysed to infect the same cells, and blind passage or virus-infected passage was performed until virus particles were confirmed to isolate the virus. Thus, a predetermined amount of virus was confirmed to be prepared as a seed virus that can be used for future vaccine production and research. The seed virus was prepared by only 6 passages of culture, and it was confirmed that after preparation, the seed virus was prepared in a sterile state by performing mycoplasma and sterility tests.

[0208] In the case of existing commercial vaccines, to prepare the virus (master seed lot) for vaccine production, the virus underwent a multiple cell culture adaptation process by passage culture in MRC-5 to establish the virus, which required primary AGMK cultures and serum. Among the seed viruses prepared by this method, the seed virus (926 IU / mL) prepared from the serum-free vaccine production cell line (SF-Vero) was a virus derived from the vaccine cell line and had high applicability for commercial vaccine development. After the corresponding seed virus was amplified, purified, inactivated to remove infectivity, adsorbed to an adjuvant, and then administered to mice, it was confirmed that the antiserum titer of the antigen administered in this patent was similar to that of the commercial vaccine.

[0209] The method of producing a virus using the above vector and the HAV expression cassette for virus production is schematically illustrated and described in Figure 1 and 2 respectively. In Figure 1 two incubation processes, transfection and blind passage, performed in the MA104 and Vero cell lines and in the SF-Vero cell line are listed, and the processes together with the required time are exemplified in FIGS. 3A and 3B. After the infection passage from P1 to P5, as Figure 6As shown, the virus titers (contents) in the supernatant and cell lysates harvested and measured at each passage were confirmed. Even after multiple passages after transfection, the virus was confirmed by ELISA, demonstrating that the virus stably amplified in the supernatant and cell lysate samples. It was confirmed that the relative detection amount of the virus in the cell lysate was higher than that in the supernatant, which may reflect the characteristics of non-lytic HAV. After the next passage of P6, virus particles were immediately confirmed by electron microscopy from the cell lysate (Figures 5A to 5C), and the virus content was quantified and confirmed to be 2371 IU / mL (MA104), 586 IU / mL (Vero), and 926 IU / mL (SF-Vero) (Figure 7A). Referring to the fact that the MA104 cell line is used for the study of the infection of enteroviruses such as hepatitis A virus that proliferate in the gastrointestinal tract (JH Lee et al., 2013), the seed virus used for the study was amplified from the cell line and used for securing. The Vero and SF-Vero cell lines have the same origin, but their virus culture conditions vary depending on the presence or absence of serum. In particular, SF-Vero cells were selected together to confirm that the virus of the present invention can be successfully amplified even under serum-free conditions. As the content of the seed virus, it was confirmed that the seed virus derived from SF-Vero incubated and isolated under serum-free culture conditions had a relatively higher content compared to the seed virus derived from Vero cells. The prepared seed virus was infected in a 24-well plate at a content of 0.1 IU / well, and the infectivity of the virus was confirmed by immunofluorescence on the 7th day (Figure 7B).

[0210] The three types of prepared seed viruses shown in Figure 7A and a commercial virus (ATCC stock, 227 IU / mL) were infected with 2.0 IU as described in the method for confirming the infection of the seed virus in Experimental Method 8, and the amplification degrees were compared when the seed virus of the present invention and the commercial virus infected the same cell line. The virus was detected from the supernatant and cell lysates of the infection cultures that underwent 6 consecutive infection passages, and compared with the commercial virus strain ( Figure 8 A), it was confirmed that the seed virus of the present invention ( Figure 8 B) had a stable and relatively high virus titer ( Figure 8 ).

[0211] In addition, the MRC-5 cell line, which has been used as a production cell for existing hepatitis A vaccines and requires serum during the incubation process, and the serum-free culture vaccine production cell SF-Vero cell line used for virus production and infection in the present invention were set as infected cells, and a comparison was made by infecting with 2.0 IU of the seed virus prepared in SF-Vero in Figure 3B. During a total of 10 passages, the supernatant and cell lysates were analyzed by ELISA, and the virus titers (contents) were relatively compared using absorbance values. As a result, virus production in SF-Vero cells had a pattern where the virus was detected starting from the first generation after infection, and starting from the second generation, the virus titers measured in the cell lysates were consistently detected in the ELISA reaction (Figure 9A). In the MRC-5 passages, there was one passage where the virus detection level measured in the harvested cell lysate samples was similar, but no constant increasing pattern of virus titers was shown. In particular, the virus detected in the supernatant was lower, contrary to the infection passage results for vaccine production in SF-Vero (Figure 9B). Thus, it was confirmed that the seed virus prepared by the method of the present invention was successfully amplified in SF-Vero cells, and according to the cultures after infection passages, the cell adaptability of the virus was higher in the SF-Vero cell line than in the MRC-5 cells. The measured absorbance result values are reported together with the graphs (Figure 9A and 9B).

[0212] Before performing CF10 culture to obtain the virus for animal experiments, the SF-Vero-derived seed virus prepared in the present invention was infected in a T175 culture flask, and the virus titers (contents) were confirmed at intervals of 3 to 4 days as the number of days changed ( Figure 10 ). This operation was preferentially carried out to confirm the culture time of the virus of the present invention and at the same time set the virus content per culture unit area of the infected cells. As a result of the measurement after virus infection, starting from the 10th day after infection, the amount of virus in the cells increased, as shown in Figures ( Figure 6 , 7A and 7B, 8 and 9), and it was confirmed that the virus amplified in the cells had a higher titer compared to the supernatant. In particular, the maximum virus content was confirmed around the 20th to 21st day after infection. It was confirmed that the virus amplification (3537 IU, 21 dpi) was approximately 230 times higher than the initial infection amount (15 IU) ( Figure 10 ).

[0213] Reference Figure 11, as the measurement result of the virus titer obtained by incubating the seed virus and the commercial virus with the same passage number of infection under the same conditions in CF10, the virus titer B prepared and passaged according to the method of the present invention is significantly higher than the virus titer A obtained by incubating the commercial virus obtained by the general method A for a long time as needed in the same manner. The significant level means that on the premise of the same virus culture area and the same culture method, the content of titer B according to the method of the present invention is increased by 150% (MA104, 1.53 times), 470% (Vero, 4.70 times) and 251% (SF-Vero, 2.51 times) compared with titer A. When subculturing under the same conditions, the fact that the virus titer of the method of the present invention is higher than that of the conventional method means that the HAV virus can be prepared faster and more stably by the method of the present invention. It is speculated that the difference in virus titer between cell lines is due to the different susceptibilities of cells to hepatitis A virus infection, but it should be confirmed later through the characteristic research of the seed virus of the present invention. Importantly, in the present invention, it is meaningful to develop a new virus strain and apply it to the vaccine production strain, and its culture potential has been confirmed from the commercial production of the existing MRC-5-based hepatitis A vaccine.

[0214] Meanwhile, Figure 12 It shows the antibodies against the inactivated antigen produced by the serum separated from the collected whole blood of mice after incubating, purifying and inactivating the seed virus prepared by the present invention and then administering the seed virus to the experimental animals of mice 3 times at intervals of 2 weeks. Figure 13 It is a graph showing the average body weight of each group of animals during the animal experiment. SK144 and SK72 are the groups administered with the virus of the present invention at 3 IU (144 EL.U) and 1.5 IU (72 EL.U) respectively after purification and inactivation, while HVR144 and HVR72 are the groups administered with the commercial product Havrix at 144 EL.U and 72 EL.U.

[0215] Refer to Figure 12 , Day - 5 refers to the mouse serum 5 days before the first administration, Day 28 refers to the serum 14 days after the second administration, and Day 42 refers to the serum 14 days after the third administration. The bar graph of each injection group represents the concentration of anti-HAV antibodies (total IgG anti-HAV serum) present in the mouse serum harvested every day. The serum concentration was measured by the antibody titration analysis method described in 12. Animal Experiment in the experimental method.

[0216] In the sera of each administration group on the -5th day before administration, no increase in anti-HAV serum titer was observed in any group. In the serum analysis on the 28th day, it was confirmed that the antibody titers between the SK144 group (average antibody 5.742 mIU / mL) and the HVR144 group (average antibody 5.783 mIU / mL) (p>0.9999) were similar to those between the SK72 group (4.377 mIU / mL) and the HVR72 group (4.875 mIU / mL) (p = 0.3895). In the serum analysis on the 42nd day, it was confirmed that the antibody titers between the SK144 group (average antibody 6.002 mIU / mL) and the HVR144 group (average antibody 6.223 mIU / mL) (p = 0.8825) were similar to those between the SK72 group (5.432 mIU / mL) and the HVR72 group (5.446 mIU / mL) (p>0.9999).

[0217] Figure 12 It is shown that there is no significant difference in immunological efficacy between the HAV antigen prepared by the method according to the present invention and the commercial hepatitis A vaccine (Havrix). For reference, since the human dose cannot be administered to mice, the 1 / 10 human dose is used and the doses are calculated based on the adult dose of 1440 EL.U / injection dose and the child dose of 720 EL.U / injection dose of the commercial hepatitis A vaccine.

[0218] Reference Figure 13 , from the start date to the end date of the animal experiment, no adverse reactions of unknown cause, mouse stress, immediate adverse reactions after antigen administration, and weight loss that may affect the immunogenicity assay were observed. In addition, even in visual observations, no adverse reactions were confirmed by administering the purified inactivated antigen and control material of the present invention.

[0219] Industrial Applicability

[0220] The method for preparing hepatitis A virus provided by the present invention can prepare stably amplified hepatitis A virus in a short time, which is very useful for preparing hepatitis A vaccine. In addition, the method can be used as a source technology for the development of hepatitis A vaccine technology, which has not been developed in South Korea. Sequence Listing <110> SK Biotech Co., Ltd. <120> Method for Preparing Hepatitis A Virus and Hepatitis A Virus Prepared According to the Method <130> OP22-0020 / PCT / CN <150> KR 10-2019-0171279 <151> 2019-12-19 <150> PCT / KR 2020 / 018703 <151> 2020-12-18 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 7487 <212> DNA <213> Artificial Sequence <220> <223> Hepatitis A virus polyprotein nucleotide sequence <400> 1 ttcaagaagg gtctccggga atttccggag tccctcttgg aagtccatgg tgaggggact 60 tgatacctca ccgccgtttg cctaggctat aggctaaatt ttccctttcc cttttccctt 120 tcctattccc tttgttttgc ttgtaaatat tgatttgtaa atattgattc ctgcaggttc 180 agggttctta aatctgtttc tctataagaa cactcatttc acgctttctg tcttctttct 240 tccagggctc tccccttgcc ctaggctctg gccgttgcgc ccggcggggt caactccatg 300 attagcatgg agctgtagga gtctaaattg gggacacaga tgtttggaac gtcaccttgc 360 agtgttaact tggctttcat gaatctcttt gatcttccac aaggggtagg ctacgggtga 420 aacctcttag gctaatactt ctatgaagag atgccttgga tagggtaaca gcggcggata 480 ttggtgagtt gttaagacaa aaaccattca acgccggagg actgactctc atccagtgga 540 tgcattgagt ggattgactg tcggggctgt ctttaggctt aattccagac ctctctgtgc 600 ttggggcaaa catcatttgg ccttaaatgg gattctgtga gaggggatcc ctccattgcc 660 agctggactg ttctttgggg ccttatgtgg tgtttgccgc tgaggtactc aggggcattt 720 aggtttttcc tcattcttaa ataataatga acatgtctag acaaggtatt ttccagactg 780 ttgggagtgg tcttgaccac atcctgtctt tggcagacat tgaggaagag caaatgattc 840 aatcagttga taggactgca gtgactggtg cttcttattt tacttctgtg gatcaatctt 900 cagttcatac agctgaggtt ggatcacacc aggttgaacc tttgagaacc tctgttgata 960 aacccggttc aaagaggact cagggagaga aatttttctt gattcattct gcagattggc 1020 ttactacaca tgctcttttc catgaagttg caaaattgga tgtggtgaaa ttattataca 1080 atgagcagtt tgctgttcaa gggttgttga gataccatac atatgcaaga tttggcattg 1140 aaattcaagt tcagataaac cctacacctt tccaacaggg gggattgatc tgtgctatgg 1200 ttcctggtga ccagagctat ggttctatag catcattgac tgtttatcct catggtttgt 1260 taaattgcaa tattaacaat gtggttagaa taaaggttcc atttatttac acaagaggtg 1320 cttaccactt taaagatcca caatacccag tttgggaatt gacaattaga gtttggtcag 1380 aattaaatat tgggacagga acttcagctt atacttcact caatgtttta gctagattta 1440 cagatttgga gttgcatgga ttaactcctc tttctacaca aatgatgaga aatgaattta 1500 gggtcagtac tactgagaat gtggtgaatc tgtcaaatta tgaagatgca agagcaaaga 1560 tgtcttttgc tttggatcag gaagattgga aatctgatcc gtcccagggt ggtgggatca 1620 aaattactca ttttactact tggacatcta ttccaacttt ggctgctcag tttccattta 1680 atgcttcaga ctcagttggt caacaaatta aagttattcc agttgaccca tattttttcc 1740 aaatgacaaa taaaaatcct gaccaaaaat gtataactgc tttggcttct atttgtcaga 1800 tgttttgttt ttggagagga gatcttgtct ttgattttca agtttttccc accaaatatc 1860 attcaggtag attactgttt tgttttgttc ctggcaatga gctaatagat gtttctggaa 1920 tcacattaaa gcaagcaact actgctcctt gtgcagtaat ggatattaca ggagtgcagt 1980 caactttgag atttcgtgtt ccctggattt ctgacactcc ttacagagtg aacaggtata 2040 caaagtcagc acatcagaaa ggtgagtaca ctgccattgg gaagcttatt gtgtattgtt 2100 ataacagatt gacctctcct tctaacgttg cttcccatgt cagagtgaat gtttatcttt 2160 cagcaattaa cttggaatgt tttgctcctc tttatcatgc tatggatgtt actacacaag 2220 ttggagatga ttctggaggt ttttcaacaa cagtttctac agaacagaat gttccagatc 2280 cccaagttgg tataacaacc atgaaagatt tgaaaggaaa agctaacaga gggaaaatgg 2340 atgtttcagg agtacaagca cctgtgggag ctatcacaac aattgaggat ccagttttag 2400 caaagaaagt acctgagaca tttcctgaat tgaaacctgg agaatccaga catacatcag 2460 atcatatgtc catctacaag tttatgggaa ggtctcattt cttgtgcact tttacattca 2520 attcaaataa taaagagtac acatttccta taaccttgtc ttcaacctct aatcctcctc 2580 atggtttgcc atcaacactg aggtggtttt tcaacttgtt tcagttgtat agagggcctt 2640 tagatctgac aattattatt acaggagcaa ctgatgtaga tggcatggcc tggtttactc 2700 cagtaggtct tgccgttgat actccttggg tagagaagga gtcagctttg tctattgact 2760 acaaaactgc tcttggagct gtcagattta acacaaggag aacagggaac attcagatta 2820 gattaccatg gtattcttat ttatatgctg tgtctggagc actggatggt ttgggtgaca 2880 agacagattc tacatttgga ttggtttcta ttcagattgc aaattacaat cattctgatg 2940 aatacttgtc ttttagttgt tatttgtctg tcacagaaca atcagagttt tattttccca 3000 gagctccatt gaactcaaat gccatgttac ccactgaatc aatgatgagc agaattgcag 3060 ctggagactt ggagtcatca gtggatgatc ctagatcaga ggaagataaa agatttgaga 3120 gtcatataga atgcaggaag ccatataaag aactgagatt agaagttggg aaacaaagac 3180 tcaagtatgc tcaggaagaa ttgtcaaatg aagtacttcc accccctagg aaaatgaagg 3240 gactgttttc acaagccaat atttctcttt tttatactga ggagcatgaa atgatgaagt 3300 tttcctggag aggtgtgact gctgatacta gagctttaag gaggtttgga ttctctttgg 3360 ccgcaggcag aagtgtgtgg actcttgaaa tggatgctgg ggttcttact gggagactga 3420 ttagattgaa tgatgagaaa tggacagaaa tgaaggatga caagattgtt tcattgattg 3480 aaaagtttac aagtaacaaa tattggtcca aagtgaattt cccacatggg atgttggatc 3540 ttgaagaaat tgctgccaat tctaaggatt ttcctaacat gtctgaaacg gatttgtgtt 3600 tcttgctgca ttggttaaat ccaaagaaaa ttaatttagc agatagaatg cttggattgt 3660 ctggagttca ggaaattaaa gaacaaggtg ttggattaat agcagagtgt agaactttct 3720 tacattctat tgctggaact ttaaaatcta tgatgtttgg atttcatcat tctgtgactg 3780 ttgaaattat aaacactgtg ctctgttttg ttaagagtgg aattttgctt tatgtaatac 3840 aacaattgaa tcaggatgaa cattctcaca taattggtct gttgagagtc ttgaattatg 3900 tagatattgg ttgttcagtt atttcatgtg gcaaagtttt ttccaaaatg ctggaaacag 3960 tctttaattg gcaaatggac tccagaatga tggagttaag gactcagagt ttttccaact 4020 ggttaagaga tatttgttct gggatcacca tttttaaaaa cttcaaggat ggaatttgtt 4080 ggctttatac aaaattaaag gacttttatg aagtgaatta tggcaagaag aaggacattt 4140 taaatattct taaagataac caacaaaaaa tagagaaagc cattgaggaa gccgataaat 4200 tttgcatttt gcaaatccaa gatgtggaaa aatctgaaca gtatcagaaa ggggttgact 4260 tgatacaaaa attgagaact gtttattcaa tggctcaggt tgatccaaat ttaatggttc 4320 atttgtcacc tttgagagat tgtatagcaa gagttcatca gaaacttaaa aaccttggat 4380 ttataaatca ggcaatggta acgagatgtg agccagttgt ttgttattta catggcaaaa 4440 gagggggagg aaagagctta acatcaattg cattggcaac caaaatttgt aaacattatg 4500 gtgttgagcc tgaaaagaat atctatacta aacctgtggc ttcagattac tgggatggat 4560 atagtggaca attagtttgc atcattgatg atattggcca aaacacaaca gatgaggact 4620 ggtcagattt ttgtcagtta gtgtcaggat gtccaatgag attaaacatg gcctctcttg 4680 aggagaaggg taggcatttt tcttctcctt ttataatagc aacttcaaat tggtcaaatc 4740 caagtccaaa aacagtttat gttaaggaag caattgaccg cagactccat ttcaaggttg 4800 aagttaaacc tgcttcattt ttcaaaaatc ctcacaatga tatgttgaat gttaatttag 4860 ctaaaacaaa tgatgcaatc aaagatatgt cttgtgttga tttgataatg gatggacata 4920 atgtttcatt gatggatttg ctcagttctt tagtcatgac agttgatatt agaaaacaaa 4980 acatgactga attcatggag ttgtggtctc agggaatttc agatgataat gatagtgcag 5040 tagctgagtt tttccagtct tttccatctg gtgaaccatc gaactctaaa ttatctggct 5100 ttttccaatc tgttactaat cacaagtggg ttgctgtggg agctgcagtt ggcattcttg 5160 gagtgctcgt tggaggatgg gttgtgtata agcatttctc ccacaaagag gaagaaccaa 5220 tcccagctga aggggtatat catggtgtaa ctaagcccaa gcatgtgatt aaattagatg 5280 cagatccagt agaatctcag tcaactttgg aaatagcagg actggttagg aagaacttgg 5340 ttcagtttgg agttggagag aagaatggat gtgtgagatg ggttatgaat gccttgggag 5400 tgaaagatga ttggctgctt gtgccttccc atgcttataa atttgagaaa gattatgaaa 5460 tgatggagtt ttattttaat agaggtggaa cttactattc aatttcagct ggtaatgttg 5520 ttattcaatc tttggatgtg ggattccagg atgttgttct gatgaaggtt cctacaattc 5580 ctaagtttag agatattact gagcatttta ttaagaaagg ggatgtgcct agagctttga 5640 atcgcctggc aacattagtg acaactgtaa atggaacccc tatgttaatt tctgagggcc 5700 cactaaagat ggaagagaaa gctacttatg ttcataagaa aaatgatggt acaacagttg 5760 atttaactgt ggatcaggca tggagaggaa aaggcgaagg tcttcctgga atgtgtggtg 5820 gggccttggt ttcatcgaat caatctatac agaatgcaat cttgggcatc catgttgctg 5880 gaggaaattc aattcttgtt gcaaaattgg ttactcaaga aatgttccaa aatattgata 5940 agaaaattga aagtcagaga attatgaaag tggagtttac tcagtgttca atgaatgtgg 6000 tctccaaaac gctttttaga aagagtccca tttatcatca cattgataaa accatgatta 6060 attttcctgc agctatgccc ttttctaaag ctgaaattga tccaatggct gtgatgttat 6120 ctaagtattc attacctatt gtagaagaac cagagggtta taaagaggct tcaatttttt 6180 atcaaaataa aatagtgggt aagactcagt tagttgatga ttttctagat cttgatatgg 6240 ccattacagg ggccccagga attgatgcta tcaacatgga ttcatctcct ggatttcctt 6300 atgtccagga gaagttgacc aaaagagatt taatttggtt ggatgaaaat ggtttattgc 6360 tgggagttca tccaagattg gctcagagaa tcttattcaa tactgtcatg atggaaaatt 6420 gttctgattt ggatgttgtt tttacaacct gtccaaaaga tgaattgagg ccattagaga 6480 aagtgttgga atcaaaaaca agagctattg atgcttgtcc tctggattac acaattttgt 6540 gccgaatgta ttggggtcca gctattagtt attttcattt gaatccaggt ttccatacag 6600 gtgttgctat tggcatagat cctgataaac agtgggatga actatttaaa acaatgataa 6660 gattcggaga tgttggtctt gatttagatt tctctgcttt tgatgctagt cttagtccat 6720 ttatgattag agaagcaggt agaatcatga gtgaactatc tggaactcca tcccattttg 6780 gcacagctct tatcaatact atcatttatt ccaagcattt gctgtataac tgttgttacc 6840 atgtctgtgg ttcaatgccc tctgggtctc cttgtacagc tttgctaaat tcaattatta 6900 ataatgtcaa tttgtattat gtgttttcta agatatttgg aaagtctcca gttttctttt 6960 gtcaggcttt gaagattctc tgttatggag atgatgtttt aatagttttc tctcgagatg 7020 ttcagattga taatcttgat ttgattggac aaaaaattgt agatgagttt aagaaacttg 7080 gcatgacagc tacttctgct gacaagaatg tacctcagct gaaaccagtt tcggaattga 7140 cttttctcaa aagatctttc aatttggtag aggatagaat tagacctgca atttcggaaa 7200 aaacaatttg gtctttaata gcatggcaga gaagtaacgc tgagtttgag cagaacttag 7260 aaaatgctca gtggtttgct tttatgcatg gctatgagtt ttatcagaaa ttctattatt 7320 ttgttcagtc ctgtttggag aaagagatga tagaatacag acttaaatct tatgattggt 7380 ggagaatgag attttatgac cagtgtttca tttgtgacct ttcatgattt gtttaaatga 7440 actttcttaa aatttctgag gtttgtttat ttcttttatc agtaaat 7487 <210> 2 <211> 646 <212> DNA <213> Artificial Sequence <220> <223> CMV Immediate Early Promoter - T7 Promoter <400> 2 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc 60 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 120 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 180 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 240 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 300 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 360 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 420 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 480 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 540 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga 600 acccactgct tactggctta tcgaaattaa tacgactcac tatagg 646 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> MCS Sequence <400> 3 gagctctcgc gaatgcatga tatcggatcc tcgag 35 <210> 4 <211> 51 <212> DNA <213> Artificial sequence <220> <223> HH ribozyme <400> 4 ccttcttgaa ctgatgaggc cgaaaggccg aaaacccggt atcccgggtt c 51 <210> 5 <211> 68 <212> DNA <213> Artificial sequence <220> <223> HDV ribozyme <400> 5 ggccggcatg gtcccagcct cctcgctggc gccggctggg caacatgctt cggcatggcg 60 aatgggac 68 <210> 6 <211> 28 <212> DNA <213> Artificial sequence <220> <223> MCS sequence 2 <400> 6 gaattctgca gaggcctgca tgcaagct 28 <210> 7 <211> 227 <212> DNA <213> Artificial sequence <220> <223> bGH polyA terminator <400> 7 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 60 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 120 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 180 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatg 227 <210> 8 <211> 8543 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of the HAV expression cassette <400> 8 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc 60 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 120 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 180 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 240 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 300 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 360 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 420 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 480 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 540 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga 600 acccactgct tactggctta tcgaaattaa tacgactcac tatagggagc tctcgcgaat 660 gcatgatatc ggatcctcga gccttcttga actgatgagg ccgaaaggcc gaaaacccgg 720 tatcccgggt tcttcaagaa gggtctccgg gaatttccgg agtccctctt ggaagtccat 780 ggtgagggga cttgatacct caccgccgtt tgcctaggct ataggctaaa ttttcccttt 840 cccttttccc tttcctattc cctttgtttt gcttgtaaat attgatttgt aaatattgat 900 tcctgcaggt tcagggttct taaatctgtt tctctataag aacactcatt tcacgctttc 960 tgtcttcttt cttccagggc tctccccttg ccctaggctc tggccgttgc gcccggcggg 1020 gtcaactcca tgattagcat ggagctgtag gagtctaaat tggggacaca gatgtttgga 1080 acgtcacctt gcagtgttaa cttggctttc atgaatctct ttgatcttcc acaaggggta 1140 ggctacgggt gaaacctctt aggctaatac ttctatgaag agatgccttg gatagggtaa 1200 cagcggcgga tattggtgag ttgttaagac aaaaaccatt caacgccgga ggactgactc 1260 tcatccagtg gatgcattga gtggattgac tgtcggggct gtctttaggc ttaattccag 1320 acctctctgt gcttggggca aacatcattt ggccttaaat gggattctgt gagaggggat 1380 ccctccattg ccagctggac tgttctttgg ggccttatgt ggtgtttgcc gctgaggtac 1440 tcaggggcat ttaggttttt cctcattctt aaataataat gaacatgtct agacaaggta 1500 ttttccagac tgttgggagt ggtcttgacc acatcctgtc tttggcagac attgaggaag 1560 agcaaatgat tcaatcagtt gataggactg cagtgactgg tgcttcttat tttacttctg 1620 tggatcaatc ttcagttcat acagctgagg ttggatcaca ccaggttgaa cctttgagaa 1680 cctctgttga taaacccggt tcaaagagga ctcagggaga gaaatttttc ttgattcatt 1740 ctgcagattg gcttactaca catgctcttt tccatgaagt tgcaaaattg gatgtggtga 1800 aattattata caatgagcag tttgctgttc aagggttgtt gagataccat acatatgcaa 1860 gatttggcat tgaaattcaa gttcagataa accctacacc tttccaacag gggggattga 1920 tctgtgctat ggttcctggt gaccagagct atggttctat agcatcattg actgtttatc 1980 ctcatggttt gttaaattgc aatattaaca atgtggttag aataaaggtt ccatttattt 2040 acacaagagg tgcttaccac tttaaagatc cacaataccc agtttgggaa ttgacaatta 2100 gagtttggtc agaattaaat attgggacag gaacttcagc ttatacttca ctcaatgttt 2160 tagctagatt tacagatttg gagttgcatg gattaactcc tctttctaca caaatgatga 2220 gaaatgaatt tagggtcagt actactgaga atgtggtgaa tctgtcaaat tatgaagatg 2280 caagagcaaa gatgtctttt gctttggatc aggaagattg gaaatctgat ccgtcccagg 2340 gtggtgggat caaaattact cattttacta cttggacatc tattccaact ttggctgctc 2400 agtttccatt taatgcttca gactcagttg gtcaacaaat taaagttatt ccagttgacc 2460 catatttttt ccaaatgaca aataaaaatc ctgaccaaaa atgtataact gctttggctt 2520 ctatttgtca gatgttttgt ttttggagag gagatcttgt ctttgatttt caagtttttc 2580 ccaccaaata tcattcaggt agattactgt tttgttttgt tcctggcaat gagctaatag 2640 atgtttctgg aatcacatta aagcaagcaa ctactgctcc ttgtgcagta atggatatta 2700 caggagtgca gtcaactttg agatttcgtg ttccctggat ttctgacact ccttacagag 2760 tgaacaggta tacaaagtca gcacatcaga aaggtgagta cactgccatt gggaagctta 2820 ttgtgtattg ttataacaga ttgacctctc cttctaacgt tgcttcccat gtcagagtga 2880 atgtttatct ttcagcaatt aacttggaat gttttgctcc tctttatcat gctatggatg 2940 ttactacaca agttggagat gattctggag gtttttcaac aacagtttct acagaacaga 3000 atgttccaga tccccaagtt ggtataacaa ccatgaaaga tttgaaagga aaagctaaca 3060 gagggaaaat ggatgtttca ggagtacaag cacctgtggg agctatcaca acaattgagg 3120 atccagtttt agcaaagaaa gtacctgaga catttcctga attgaaacct ggagaatcca 3180 gacatacatc agatcatatg tccatctaca agtttatggg aaggtctcat ttcttgtgca 3240 cttttacatt caattcaaat aataaagagt acacatttcc tataaccttg tcttcaacct 3300 ctaatcctcc tcatggtttg ccatcaacac tgaggtggtt tttcaacttg tttcagttgt 3360 atagagggcc tttagatctg acaattatta ttacaggagc aactgatgta gatggcatgg 3420 cctggtttac tccagtaggt cttgccgttg atactccttg ggtagagaag gagtcagctt 3480 tgtctattga ctacaaaact gctcttggag ctgtcagatt taacacaagg agaacaggga 3540 acattcagat tagattacca tggtattctt atttatatgc tgtgtctgga gcactggatg 3600 gtttgggtga caagacagat tctacatttg gattggtttc tattcagatt gcaaattaca 3660 atcattctga tgaatacttg tcttttagtt gttatttgtc tgtcacagaa caatcagagt 3720 tttattttcc cagagctcca ttgaactcaa atgccatgtt acccactgaa tcaatgatga 3780 gcagaattgc agctggagac ttggagtcat cagtggatga tcctagatca gaggaagata 3840 aaagatttga gagtcatata gaatgcagga agccatataa agaactgaga ttagaagttg 3900 ggaaacaaag actcaagtat gctcaggaag aattgtcaaa tgaagtactt ccacccccta 3960 ggaaaatgaa gggactgttt tcacaagcca atatttctct tttttatact gaggagcatg 4020 aaatgatgaa gttttcctgg agaggtgtga ctgctgatac tagagcttta aggaggtttg 4080 gattctcttt ggccgcaggc agaagtgtgt ggactcttga aatggatgct ggggttctta 4140 ctgggagact gattagattg aatgatgaga aatggacaga aatgaaggat gacaagattg 4200 tttcattgat tgaaaagttt acaagtaaca aatattggtc caaagtgaat ttcccacatg 4260 ggatgttgga tcttgaagaa attgctgcca attctaagga ttttcctaac atgtctgaaa 4320 cggatttgtg tttcttgctg cattggttaa atccaaagaa aattaattta gcagatagaa 4380 tgcttggatt gtctggagtt caggaaatta aagaacaagg tgttggatta atagcagagt 4440 gtagaacttt cttacattct attgctggaa ctttaaaatc tatgatgttt ggatttcatc 4500 attctgtgac tgttgaaatt ataaacactg tgctctgttt tgttaagagt ggaattttgc 4560 tttatgtaat acaacaattg aatcaggatg aacattctca cataattggt ctgttgagag 4620 tcttgaatta tgtagatatt ggttgttcag ttatttcatg tggcaaagtt ttttccaaaa 4680 tgctggaaac agtctttaat tggcaaatgg actccagaat gatggagtta aggactcaga 4740 gtttttccaa ctggttaaga gatatttgtt ctgggatcac catttttaaa aacttcaagg 4800 atggaatttg ttggctttat acaaaattaa aggactttta tgaagtgaat tatggcaaga 4860 agaaggacat tttaaatatt cttaaagata accaacaaaa aatagagaaa gccattgagg 4920 aagccgataa attttgcatt ttgcaaatcc aagatgtgga aaaatctgaa cagtatcaga 4980 aaggggttga cttgatacaa aaattgagaa ctgtttattc aatggctcag gttgatccaa 5040 atttaatggt tcatttgtca cctttgagag attgtatagc aagagttcat cagaaactta 5100 aaaaccttgg atttataaat caggcaatgg taacgagatg tgagccagtt gtttgttatt 5160 tacatggcaa aagaggggga ggaaagagct taacatcaat tgcattggca accaaaattt 5220 gtaaacatta tggtgttgag cctgaaaaga atatctatac taaacctgtg gcttcagatt 5280 actgggatgg atatagtgga caattagttt gcatcattga tgatattggc caaaacacaa 5340 cagatgagga ctggtcagat ttttgtcagt tagtgtcagg atgtccaatg agattaaaca 5400 tggcctctct tgaggagaag ggtaggcatt tttcttctcc ttttataata gcaacttcaa 5460 attggtcaaa tccaagtcca aaaacagttt atgttaagga agcaattgac cgcagactcc 5520 atttcaaggt tgaagttaaa cctgcttcat ttttcaaaaa tcctcacaat gatatgttga 5580 atgttaattt agctaaaaca aatgatgcaa tcaaagatat gtcttgtgtt gatttgataa 5640 tggatggaca taatgtttca ttgatggatt tgctcagttc tttagtcatg acagttgata 5700 ttagaaaaca aaacatgact gaattcatgg agttgtggtc tcagggaatt tcagatgata 5760 atgatagtgc agtagctgag tttttccagt cttttccatc tggtgaacca tcgaactcta 5820 aattatctgg ctttttccaa tctgttacta atcacaagtg ggttgctgtg ggagctgcag 5880 ttggcattct tggagtgctc gttggaggat gggttgtgta taagcatttc tcccacaaag 5940 aggaagaacc aatcccagct gaaggggtat atcatggtgt aactaagccc aagcatgtga 6000 ttaaattaga tgcagatcca gtagaatctc agtcaacttt ggaaatagca ggactggtta 6060 ggaagaactt ggttcagttt ggagttggag agaagaatgg atgtgtgaga tgggttatga 6120 atgccttggg agtgaaagat gattggctgc ttgtgccttc ccatgcttat aaatttgaga 6180 aagattatga aatgatggag ttttatttta atagaggtgg aacttactat tcaatttcag 6240 ctggtaatgt tgttattcaa tctttggatg tgggattcca ggatgttgtt ctgatgaagg 6300 ttcctacaat tcctaagttt agagatatta ctgagcattt tattaagaaa ggggatgtgc 6360 ctagagcttt gaatcgcctg gcaacattag tgacaactgt aaatggaacc cctatgttaa 6420 tttctgaggg cccactaaag atggaagaga aagctactta tgttcataag aaaaatgatg 6480 gtacaacagt tgatttaact gtggatcagg catggagagg aaaaggcgaa ggtcttcctg 6540 gaatgtgtgg tggggccttg gtttcatcga atcaatctat acagaatgca atcttgggca 6600 tccatgttgc tggaggaaat tcaattcttg ttgcaaaatt ggttactcaa gaaatgttcc 6660 aaaatattga taagaaaatt gaaagtcaga gaattatgaa agtggagttt actcagtgtt 6720 caatgaatgt ggtctccaaa acgcttttta gaaagagtcc catttatcat cacattgata 6780 aaaccatgat taattttcct gcagctatgc ccttttctaa agctgaaatt gatccaatgg 6840 ctgtgatgtt atctaagtat tcattaccta ttgtagaaga accagagggt tataaagagg 6900 cttcaatttt ttatcaaaat aaaatagtgg gtaagactca gttagttgat gattttctag 6960 atcttgatat ggccattaca ggggccccag gaattgatgc tatcaacatg gattcatctc 7020 ctggatttcc ttatgtccag gagaagttga ccaaaagaga tttaatttgg ttggatgaaa 7080 atggtttatt gctgggagtt catccaagat tggctcagag aatcttattc aatactgtca 7140 tgatggaaaa ttgttctgat ttggatgttg tttttacaac ctgtccaaaa gatgaattga 7200 ggccattaga gaaagtgttg gaatcaaaaa caagagctat tgatgcttgt cctctggatt 7260 acacaatttt gtgccgaatg tattggggtc cagctattag ttattttcat ttgaatccag 7320 gtttccatac aggtgttgct attggcatag atcctgataa acagtgggat gaactattta 7380 aaacaatgat aagattcgga gatgttggtc ttgatttaga tttctctgct tttgatgcta 7440 gtcttagtcc atttatgatt agagaagcag gtagaatcat gagtgaacta tctggaactc 7500 catcccattt tggcacagct cttatcaata ctatcattta ttccaagcat ttgctgtata 7560 actgttgtta ccatgtctgt ggttcaatgc cctctgggtc tccttgtaca gctttgctaa 7620 attcaattat taataatgtc aatttgtatt atgtgttttc taagatattt ggaaagtctc 7680 cagttttctt ttgtcaggct ttgaagattc tctgttatgg agatgatgtt ttaatagttt 7740 tctctcgaga tgttcagatt gataatcttg atttgattgg acaaaaaatt gtagatgagt 7800 ttaagaaact tggcatgaca gctacttctg ctgacaagaa tgtacctcag ctgaaaccag 7860 tttcggaatt gacttttctc aaaagatctt tcaatttggt agaggataga attagacctg 7920 caatttcgga aaaaacaatt tggtctttaa tagcatggca gagaagtaac gctgagtttg 7980 agcagaactt agaaaatgct cagtggtttg cttttatgca tggctatgag ttttatcaga 8040 aattctatta ttttgttcag tcctgtttgg agaaagagat gatagaatac agacttaaat 8100 cttatgattg gtggagaatg agattttatg accagtgttt catttgtgac ctttcatgat 8160 ttgtttaaat gaactttctt aaaatttctg aggtttgttt atttctttta tcagtaaatg 8220 gccggcatgg tcccagcctc ctcgctggcg ccggctgggc aacatgcttc ggcatggcga 8280 atgggacgaa ttctgcagag gcctgcatgc aagcttcgac tgtgccttct agttgccagc 8340 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 8400 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 8460 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 8520 ctggggatgc ggtgggctct atg 8543

Claims

1. Hepatitis A virus gene, whose nucleic acid sequence is as shown in SEQ ID NO:

1.

2. Expression cassette for preparing hepatitis A virus, which contains the hepatitis A virus gene as described in claim 1.

3. The expression cassette as described in claim 2, wherein the expression cassette contains a promoter, a hammerhead (HH) ribozyme and a hepatitis delta virus (HDV) ribozyme.

4. The expression cassette as described in claim 2, wherein the expression cassette contains the nucleotide sequence of SEQ ID NO:

8.

5. Vector for preparing hepatitis A virus, which contains the expression cassette as described in any one of claims 2 to 4.

6. Preparation method of hepatitis A virus for preparing a vaccine, including the following steps: (a) Transfecting a host cell with a vector for preparing hepatitis A virus containing an expression cassette, the expression cassette containing the hepatitis A virus gene of SEQ ID NO:1; (b) Obtaining the virus from the host cell; (c) Infecting the host cell with the obtained virus and subculturing the infected host cell; and (d) Obtaining the virus from the host cell.

7. The preparation method as described in claim 6, wherein the expression cassette sequentially contains a CMV promoter, a T7 promoter, a multiple cloning site (MCS) and a hammerhead (HH) ribozyme site in the 5' end direction of the hepatitis A virus gene, and sequentially contains a hepatitis delta virus (HDV) ribozyme, an MCS and a poly-A tail in the 3' end direction.

8. The preparation method as described in claim 6, wherein the expression cassette contains the nucleotide sequence of SEQ ID NO:

8.

9. The preparation method as described in claim 6, wherein the host cell is selected from Vero, MA104, WI-38, BHK-21, CHO, MDCK, Hi5, CEF and Sf9.

10. The preparation method as described in claim 6, wherein the host cell is a cell adapted to serum-free medium.

11. The preparation method as described in claim 6, wherein the subculturing is carried out 2 to 30 times.

12. The preparation method as described in claim 6, wherein in step (d), the host cell shows a cytopathic effect in 3 or more subcultures.

13. The preparation method as described in claim 6, wherein the host cells in steps (a) and (c) are the same cells.

14. The preparation method as described in claim 6, further comprising: A purification step, an inactivation step or a purification and inactivation step for the virus after step (d).

15. Hepatitis A virus prepared according to the method as described in any one of claims 6 to 14.

16. Hepatitis A vaccine composition containing the virus as described in claim 15 as an active ingredient.

17. The hepatitis A vaccine composition as described in claim 16, wherein the vaccine is a live vaccine, an attenuated vaccine or an inactivated vaccine.

18. The hepatitis A vaccine composition as described in claim 17, further comprising an adjuvant.

19. Kit containing the vaccine composition as described in claim 16.

20. A pre-filled syringe filled with the vaccine composition as described in claim 16.

21. Use of the virus according to claim 15 for the preparation of a hepatitis A vaccine.

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