Method for producing hepatitis A virus and hepatitis A virus produced by said method

Abstract

The present invention relates to a method for producing hepatitis A virus and the hepatitis A virus produced by this method. More specifically, the present invention relates to a method for producing hepatitis A virus, which comprises transforming host cells with a vector containing an expression cassette for producing hepatitis A virus containing a hepatitis A virus gene, infecting the host cells with the resulting virus, and subculturing the virus, and the hepatitis A virus produced by this method.

Description

[Technical field]

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

[0002] The present invention relates to a method for producing hepatitis A virus and the hepatitis A virus produced by this method, and more particularly to a method for producing hepatitis A virus, which comprises the steps of transforming host cells with a vector into which an expression cassette for producing hepatitis A virus containing a hepatitis A virus gene is inserted, infecting the host cells with the virus obtained, and subculturing the virus, and the hepatitis A virus produced by this method. [Background technology]

[0003] Hepatitis A is the most important cause of acute viral hepatitis in adults in Korea, accounting for more than 70% of cases. Hepatitis A virus (HAV) belongs to the picornavirus family, is non-enveloped, is approximately 27 nm in size, and has single-stranded RNA as its nucleic acid. After an average incubation period of 28 days, it causes acute liver disease characterized by clinical symptoms such as fever, loss of appetite, nausea and vomiting, abdominal pain, dark urine, 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 conducted in the last 10 years, the number of hepatitis A patients has been increasing in the western United States, the Middle East, and some Asian regions, raising concerns about the spread of hepatitis A disease worldwide. In South Korea, the number of people infected with Hepatitis A has recently been increasing rapidly among young people in their teens and twenties who have low 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 Disease Portal Statistics by Disease Type under Legal Infectious Diseases - Hepatitis A). In particular, for young people of school age and exam candidates, hospitalization and treatment due to hepatitis A infection can cause personal and economic harm. In the United States, the direct and indirect costs per hepatitis A patient are approximately $2,500 for adults and $1,500 for those under 18, and it is known that medical costs due to hepatitis A are approximately $300 million or more per year (World Health Organization, 1999).

[0004] The primary route of transmission of Hepatitis A is via the fecal-oral route, transmitted through contaminated food and water. The Advisory Committee on Immunization Practices (ACIP) recommends vaccination for outbound travelers and workers in areas with endemicity of hepatitis A virus, men who have sex with men (MSM), people with hepatitis B disease, people 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] In order to produce a hepatitis A vaccine, it is essential to produce the hepatitis A virus (HAV), which has a very slow replication rate (Cromeans et al. J. Gen. Virol. 70:2051-2062(1989)). Usually, viruses that infect humans can be replicated and isolated after 2-3 days or 7 days of culture, but the hepatitis A virus can be harvested after as little as one month of culture. This period of one month is limited only if the virus can adapt well to the cultured cells. Cell lines that are highly sensitive to the hepatitis A virus (e.g., Primary AGMK cell, FRhK-4, BS-C-1) used for amplifying the hepatitis A virus are not suitable as cells for producing vaccines. The characteristics and stability of these cell lines suitable for use as cell lines for producing vaccines for humans have not been verified. In particular, although the cells in question have been established as a cell line that can be cultured in vitro in flasks, etc., they are classified as substances derived from the Himalayan monkey (Macaca mulatta) and the Savannah monkey (Cercopithecus aethi), which belong to the family Long-tailed Monkeys and are internationally endangered species that are subject to restrictions under the CITES Convention, and it is difficult to import them into Japan from companies such as ATCC (US) that commercially sell cell lines.

[0006] To isolate HAV from human fecal samples and produce a high yield of virus in a producer cell line, approximately 50 virus infection passages are required to adapt the HAV to the producer cell line. As an 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 stool suspension) was passaged 32 times in primary AGMK (primary African green monkey) cells to confirm that the virus had adapted to cell culture. The virus (P-32 AGMK cell-adapted) was then cultured in MRC-5 cell lines up to passage 37, after which a virus clone (clone 25-4-21) was isolated. This clone was then cultured once more in MRC-5 cells (passage 38), and the virus was further passaged three times in MRC-5 cells to obtain the virus at passage 41, which was designated as the master seed stock, and this was designated as HAV 4380 (US6423318B1).

[0007] Hepatitis A vaccine is a mandatory vaccination designated by the country, but it is often in short supply during HAV epidemics. Havrix (GSK) by GlaxoSmithKline and Vaqta (Merck & Co., Inc.) by Merck, which are the most frequently used hepatitis A vaccines worldwide, sold out in Korea in 2019 when hepatitis A was prevalent, and supplies were suspended by the end of 2019, resulting in major supply and demand difficulties. This vaccine supply and demand imbalance problem occurred in 2017 as well, but the supply instability did not resolve in 2019 when a sudden hepatitis A epidemic occurred. Currently, the Ministry of Health and Welfare has included hepatitis A vaccine in the 2020 National Immunization Vaccine Stockpiling Plan to stabilize vaccine supply and demand, and efforts are underway to resolve this. This supply and demand instability provides the reason why there is currently no commercial hepatitis A vaccine developed using domestic technology. Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors have conducted intensive research to solve the above-mentioned technical problems and develop a method for stable and rapid production of Hepatitis A virus. As a result, they have found a method for obtaining the virus by shortening the number of subculture steps from about 50 times, which was previously required to obtain the virus, to just six times, and a combination of an optimal gene expression cassette that expresses the virus in a higher yield in a virus-producing cell line. They then infected host cells with an expression vector containing the Hepatitis A virus gene and functional region, and produced a virus. The inventors have found that the more the virus obtained is passaged, the more rapidly it replicates, enabling rapid and stable production of Hepatitis A virus, thereby completing the present invention.

[0009] Therefore, an object of the present invention is to provide a Hepatitis A virus gene represented by SEQ ID NO:1.

[0010] Another object of the present invention is to provide an expression cassette for producing Hepatitis A virus, comprising the Hepatitis A virus gene of SEQ ID NO:1.

[0011] Another object of the present invention is to provide a vector for producing Hepatitis A virus, which comprises the expression cassette.

[0012] Another object of the present invention is to provide a Hepatitis A virus produced by said vector.

[0013] Another object of the present invention is to provide a method for producing hepatitis A virus for vaccine production, comprising the steps of: (a) transforming a host cell with a vector into which an expression cassette for preparing hepatitis A virus, which includes the hepatitis A virus gene of SEQ ID NO: 1, is inserted; (b) acquiring the virus from the host cell; (c) infecting a host cell with the acquired virus and subculturing it; and (d) acquiring the virus from the host cell.

[0014] Another object of the present invention is to provide a hepatitis virus produced by said method.

[0015] Another object of the present invention is to provide a hepatitis A vaccine composition containing the hepatitis virus as an active ingredient.

[0016] Another object of the present invention is to provide a Hepatitis A vaccine composition comprising said hepatitis virus.

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

[0018] Another object of the present invention is to provide a kit comprising said vaccine composition.

[0019] Another object of the present invention is to provide a prefilled syringe containing the vaccine composition.

[0020] Another object of the present invention is to provide a use of said hepatitis virus for producing a hepatitis A vaccine.

[0021] Another object of the present invention is to provide a use of a vaccine composition containing the hepatitis virus as an active ingredient for preventing hepatitis A.

[0022] Another object of the present invention is to provide a method for preventing hepatitis A, which comprises administering an effective amount of a vaccine composition containing the hepatitis virus as an active ingredient to an individual in need thereof. [Means for solving the problem]

[0023] To achieve the above object of the present invention, the present invention provides a Hepatitis A virus gene represented by SEQ ID NO:1.

[0024] In order to achieve another object of the present invention, the present invention provides a method for the preparation of a hepatitis A virus gene of SEQ ID NO: 1. The present invention provides an expression cassette for producing hepatitis A virus, comprising the

[0025] In order to achieve another object of the present invention, there is provided an expression vector for producing Hepatitis A virus, comprising the above expression cassette.

[0026] In order to achieve another object of the present invention, the present invention provides a Hepatitis A virus produced by the above vector.

[0027] In order to achieve another object of the present invention, the present invention provides a method for producing Hepatitis A virus for producing a vaccine, comprising: (a) transforming a host cell with a vector into which an expression cassette for producing Hepatitis A virus, which includes the Hepatitis A virus gene of SEQ ID NO: 1, is inserted; (b) obtaining the virus from the host cell; (c) infecting a host cell with the obtained virus and subculturing the virus; and (d) obtaining the virus when cytopathic effect (CPE) appears in the host cell.

[0028] In order to achieve another object of the present invention, the present invention provides a hepatitis virus produced by the above method.

[0029] In order to achieve another object of the present invention, the present invention provides a hepatitis A vaccine composition comprising the hepatitis virus as an active ingredient.

[0030] The present invention also provides a hepatitis A vaccine composition comprising the hepatitis virus.

[0031] The present invention also provides a hepatitis A vaccine composition essentially consisting of the hepatitis virus.

[0032] In order to achieve another object of the present invention, the present invention provides a kit comprising the vaccine composition.

[0033] In order to achieve another object of the present invention, the present invention provides a prefilled syringe filled with the vaccine composition.

[0034] In order to achieve another object of the present invention, there is provided a use of said hepatitis virus for producing a hepatitis A vaccine.

[0035] In order to achieve another object of the present invention, there is provided a use of a vaccine composition containing the hepatitis virus as an active ingredient for preventing hepatitis A.

[0036] In order to achieve another object of the present invention, there is provided a method for preventing hepatitis A, which comprises administering an effective amount of a vaccine composition containing the hepatitis virus as an active ingredient to an individual in need thereof.

[0037] The present invention will be described in detail below.

[0038] The present invention provides a Hepatitis A virus gene represented by SEQ ID NO:1.

[0039] The present invention also provides an expression cassette for producing hepatitis A virus, comprising the hepatitis A virus gene of SEQ ID NO:1.

[0040] The Hepatitis A virus gene of SEQ ID NO: 1 provided by the present invention is a commercial Hepatitis A virus strain ( It contains A2876T and A3891T point mutations compared to the gene of ATCC VR-1402, which makes it suitable for high yield and rapid subculture.

[0041] In particular, the 3891st position is a major mutation that changes the amino acid from MET to LEU. Furthermore, the recombinant base sequence of the present invention was completed by mutating the major sites that facilitate subculture and increase yield.

[0042] According to one embodiment of the present invention, it was confirmed that the productivity of the hepatitis A virus of the gene represented by sequence number 1 of the present invention is superior to that of the wild-type hepatitis A virus that can be obtained locally, as shown in Figures 8 and 11.

[0043] In the conventional technology, at least 47 subcultures must be performed to obtain the hepatitis A virus (Figure 4), which is troublesome, time-consuming, and expensive (T. Cromeans et al., J Medical Virology 22:45-56, 1987). Normally, one subculture of HAV requires about one month, and conventional techniques require several tens of months to obtain hepatitis A virus for vaccine production from human feces. However, the present invention is characterized in that the production period of hepatitis A virus is shortened to 4 to 5 months by using the hepatitis A virus gene of SEQ ID NO: 1 having the above-mentioned gene mutation and an expression cassette containing the same.

[0044] In the present invention, the term "expression cassette" refers to a unit cassette that includes a base sequence encoding a promoter and a signal peptide, and a gene encoding a target protein, and that can be expressed for secretion of the target protein operably linked downstream of the signal peptide. Various factors that can supplement efficient production of the target protein may be included inside or outside such an expression cassette.

[0045] The term "gene" in the present invention is broadly used to refer to any segment of a polynucleotide associated with a biological function. Thus, a gene or polynucleotide includes introns and exons, such as a genomic sequence, or a coding sequence, such as a cDNA, for example, an open reading frame beginning with a start codon (methionine codon) and ending with a termination signal (stop codon). A gene and polynucleotide may also include regions that regulate its expression, such as transcription initiation, translation, and transcription termination. Thus, it also includes promoters and ribosome binding regions (these regulatory elements are generally located approximately 60-250 nucleotides upstream of the start codon of a coding sequence or gene), and transcription terminators (the terminators are generally located within approximately 50 nucleotides downstream of the stop codon of a 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 includes regulatory sequences.

[0046] It will be clearly understood by those skilled in the art that the expression cassette provided by the present invention can contain a base sequence encoding a polypeptide that has at least 80% or more, preferably 90% or more, and more preferably 95% or more sequence homology to the base sequence of the Hepatitis A virus gene of SEQ ID NO:1, while having substantially identical biological properties such as viral antigenicity or immunogenicity.

[0047] According to one embodiment of the present invention, the expression cassette can include a promoter, a hammerhead (HH) ribozyme and a hepatitis delta virus (HDV) ribozyme.

[0048] Preferably, the expression cassette comprises a CMV It may comprise a promoter, a T7 promoter, a multicloning region (MCS), and an HH ribozyme region, and may comprise, in the 3'-terminal direction of the hepatitis A virus gene, a hepatitis delta virus ribozyme, an MCS, and a polyA tail, in that order. According to a preferred embodiment of the present invention, the CMV / T7 promoter can be represented by SEQ ID NO: 2, the MCS sequence by SEQ ID NO: 3 or 6, the HH ribozyme by SEQ ID NO: 4, the HDV ribozyme by SEQ ID NO: 5, and the polyA tail by SEQ ID NO: 7. In one embodiment of the present invention, a CMV promoter region and a T7 promoter region capable of in-vitro transcription by T7 RNA polymerase were inserted into the expression cassette for gene expression (SEQ ID NO: 2). In order to allow the use of restriction enzymes, the MCS was placed behind the promoter at the 5' end of the gene (SEQ ID NO: 3) and in front of the polyA tail sequence (SEQ ID NO: 6). The HH ribozyme (SEQ ID NO: 4) and HDV ribozyme (SEQ ID NO: 5) regions, which serve as catalytic RNA cleavage structures with self-cleavage function, were located on both sides of the UTR-HAV polyprotein-UTR region, which is the HAV sequence region, and the expression cassette was designed so that only the targeted HAV mRNA structure could be isolated.

[0049] According to a preferred embodiment of the present invention, the expression cassette may comprise the base sequence of SEQ ID NO:8.

[0050] The present invention also provides a vector for preparing Hepatitis A virus, into which the expression cassette is inserted.

[0051] In the present invention, the term "vector" refers to a gene construct capable of expressing a target protein in an appropriate host cell, comprising essential regulatory elements operably linked to express a gene insert. In the present invention, "operably linked" refers to a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein being functionally linked to each other to perform a common function. The operably linked vector can be produced using recombinant gene technology well known in the technical field to which the present invention belongs, and site-specific DNA cleavage and ligation can be easily performed using enzymes generally known in the technical field to which the present invention belongs.

[0052] A vector suitable for use in the present invention may contain, in addition to the Hepatitis A gene of SEQ ID NO:1, expression regulatory elements such as a promoter, an initiation codon, a stop codon, a polyadenylation signal, a ribozyme and an enhancer, as well as a signal sequence for membrane targeting or secretion.

[0053] The initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein and must be functional in the host cell when the genetic construct is administered and in frame with the coding sequence. Common promoters may be constitutive or inducible. Promoters include, but are not limited to, 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.

[0054] When the vector is a replicable expression vector, it may contain a replication origin, which is a specific nucleic acid sequence from which replication is initiated. Various types of vectors, such as plasmids, viruses, and cosmids, may be used as recombinant expression vectors. The type of recombinant vector is not particularly limited, so long as it functions to express a desired gene in various eukaryotic host cells and produce a desired protein, but it is also preferred that the recombinant vector be capable of expressing a foreign protein in a form similar to its natural state while maintaining a promoter that exhibits strong activity and strong expression power. Vectors that can be produced in large quantities are preferred.

[0055] Eukaryotic expression vectors into which an expression cassette for producing hepatitis A virus containing the hepatitis A gene of SEQ ID NO: 1 can be inserted are known in the art. Non-limiting examples include pUC57 vector, pcDNA3.1 vector, pVAX1 vector (Life Technology, Cergy-Pontoise, France), and pBudCE4.1 (Life Technology), and vectors disclosed or mentioned in the present specification or known to those skilled in the relevant technical field can be used to construct the expression vector of the present invention. Preferably, pUC57 vector can be used.

[0056] According to another preferred embodiment of the invention, said vector may display the cleavage map of HAV viral genes and factors involved in gene expression for the generation of the virus as disclosed in FIG.

[0057] The present invention provides a method for producing Hepatitis A virus for producing a vaccine, comprising the steps of: (a) transforming a host cell with a vector into which an expression cassette for producing Hepatitis A virus, which includes the Hepatitis A virus gene of SEQ ID NO: 1, is inserted; (B) obtaining the virus from the host cell; (C) infecting a host cell with the obtained virus and subculturing the virus; and (D) obtaining the virus when a cytopathic effect (CPE) appears in the host cell.

[0058] (a) A step of transforming a host cell with a vector into which an expression cassette for producing Hepatitis A virus, which contains the Hepatitis A virus gene of SEQ ID NO: 1, has been inserted; the "expression cassette" and "vector" can be similarly applied as described above.

[0059] In the present invention, the "host cell" refers to a eukaryotic cell that has been genetically modified or can be genetically modified by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector. When referring to a genetically modified cell, the term includes the originally modified cell or all of its progeny. The 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 host cell suitable for replication and amplification.

[0060] The host cells into which the vector is injected in step (a) of the present invention are not particularly limited in type, and may be cells derived from the natural host of HAV (e.g., chinese monkeys, monkeys, humans, etc.) or vaccine-producing cells. The "vaccine-producing cells" may also be expressed as a cell substrate, and may be defined as a cell line derived from humans or animals that has the ability to produce pharmaceuticals as a raw material for producing biopharmaceuticals or cell culture pharmaceuticals.

[0061] In particular, in the present invention, it is preferable that the host cell has been proven safe as a cell for producing a human vaccine and is used in the industry for producing biological medicines. For a detailed description of cells proven safe as a cell for producing a human vaccine, Jordan and Sandig, Viruses, 6:1672-1700 (2014); WHO Technical Report Series, No. 978, Annex 3 may be referred to, and the entire contents of these documents may be used as a reference for the contents of the present invention.

[0062] According to one embodiment of the present invention, the vaccine production cells are selected from the group consisting of Vero, MA104, WI-38, CHO, MDCK, Hi5, CEF, S9, human embryonic lung fibroblasts (e.g., MRC-5, etc.), PER.C6, BHK-21, CHO-K1 and serum-free adapted cells thereof. Preferably, the vaccine producing cells may be selected from the group consisting of MA104, Vero or serum-free adapted cells thereof, and more preferably, SF-Vero cells.

[0063] According to one embodiment of the present invention, host cells can be cultured as follows: MA104 and Vero cell lines can be cultured in EMEM medium containing 2% FBS (2% FBS-EMEM), and SF-Vero cell line can be cultured in 2 mL of serum-free EMEM medium (SF-EMEM) at 30 to 40°C (preferably 35°C) in a 3 to 7% (preferably 5%) CO2 incubator for 2 to 4 weeks (preferably 3 weeks).

[0064] If the temperature ranges, carbon dioxide concentration ranges, and culture days are not met or exceeded, an appropriate number of cell lines may not be generated, and appropriate conditions for transforming the HAV gene construct may not be created.

[0065] Adaptation of cells to culture in serum-free medium can be easily achieved by one of ordinary skill in the relevant art by gradually subculturing the cells in medium with decreasing amounts of serum until the cells can successfully survive and grow in serum-free medium, and serum-free adapted cell lines of each of the above cell lines can be easily obtained by one of ordinary skill in the art.

[0066] The vector of the present invention may be injected into a host cell by any method known in the art. The vector containing the expression cassette for producing Hepatitis A virus, which contains the Hepatitis A virus gene of SEQ ID NO: 1, may be injected into a host cell by a number of suitable means, including intracellular transfection, transformation, electroporation, transformation using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other components; microparticle projection; lipofection; and viral vector injection (e.g., retroviral vector).

[0067] (b) harvesting the virus from the host cell; After step (a), the host cells transformed with the vector are cultured in a nutrient medium to obtain the Hepatitis A virus to be used as a seed. In this case, the medium and culture conditions to be used can be appropriately selected and used as long as they are tolerated by the host cells. Conditions such as temperature, medium pH, and culture time can be appropriately adjusted so as to be suitable for cell growth and virus production during culture. As described above, the virus that can be produced or secreted in the host cells can be recovered from the supernatant of the culture medium or cell lysate, and can be separated and purified by conventional protein and virus separation techniques.

[0068] The virus obtained in step (b) is used as a seed for subsequent infection and amplification into host cells, and in one embodiment of the present invention, this is named Passage 0 (P0) virus.

[0069] (c) infecting a host cell with the obtained virus and subculturing the virus; Step (c) of the present invention is a step of producing a virus that can adapt to host cells and amplify at a rapid rate by infecting host cells with the P0 virus obtained in step (b) and repeatedly subculturing the virus.

[0070] According to one embodiment of the present invention, in the course of infection passage, the host cells MA104 and Vero are cultured at 1×10 5 ~1×10 7 cells / 5 mL, SF-Vero cells were 5 x 10 5 ~5×10 7 If the host cells are prepared at a concentration lower than this, it may be difficult to obtain a satisfactory titer. When prepared, infection of host cells may not be uniform and may be uneconomical.

[0071] According to one embodiment of the present invention, the host cells may be cultured in an incubator at 30 to 40° C. (preferably 35° C.) and 3 to 7% (preferably 5%) CO2 for 2 to 4 weeks (preferably 3 weeks). If the temperature range, carbon dioxide concentration range, and number of days of culture are not met or exceeded, an appropriate number of cell lines may not be generated, and appropriate conditions for subculture may not be formed.

[0072] According to one embodiment of the present invention, in the course of the P1 infection passage, M104 and VERO cells were cultured at 5×10 5 ~5×10 7 At cells / 30 mL, SF-Vero cells were 8 × 10 5 ~8×10 7 Preparation may be performed at 30 mL / cells, and the medium may be removed from the prepared cells in the same manner just before infection, and the cells may be washed 1 to 3 times with 30 mL of DPBS. If these conditions are not met or exceeded, the virus may not be obtained at the optimal yield.

[0073] According to one embodiment of the present invention, 1 to 10 mL of infection medium may be used in the progress of infection passages P1 to P4, and 20 to 50 mL of infection medium may be used in infection passages P5 to P6. If these conditions are not met or exceeded, the virus may not be obtained in an optimal yield.

[0074] In the present invention, the passaging refers to the continuous passage of host cells infected with a virus, even if there is no experimental confirmation of viral proliferation in culture after infecting host cells with the virus, i.e., no cytopathic effect or detection of the virus is confirmed.

[0075] In step (c), the host cells to be infected with the virus are preferably the same as the host cells used to produce the P0 virus in step (a).

[0076] According to one embodiment of the present invention, the subculture may specifically proceed according to the following method: The P0 virus is treated and infected into host cells, which are then cultured. After the culture, the host cells are disrupted and centrifuged to remove cell debris, and only the supernatant is collected. The collected supernatant passage 1 (P1) is used to infect host cells again.

[0077] The above-mentioned process of infecting host cells, culturing host cells, disrupting host cells and obtaining supernatant constitutes one cycle, which is then repeated for passage.

[0078] In the one cycle of passaging process of the present invention, the host cells can be cultured for preferably 5 to 30 days, more preferably 10 to 25 days, even more preferably 17 to 21 days, and most preferably 19 to 21 days.

[0079] During each of the above subculture steps, the virus is propagated in a medium composition in which the host cells are typically cultured. The host cells are cultured in a standard commercially available culture medium, such as medium supplemented with serum (e.g., 10% fetal bovine serum) or medium free of serum, under a CO2 concentration appropriate to maintain a neutral buffered pH (e.g., a 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, peptones, vitamins, sugars (e.g., glucose), non-essential amino acids, and additional supplements (e.g., trypsin, β-mercaptoethanol, insulin, growth factors, amino acid complexes, etc.) that promote desirable growth characteristics.

[0080] In some cases, e.g., for virus production, it is preferable to grow the host cells under serum-free conditions. The cells may be grown on a small scale, e.g., in less than 25 mL of medium, culture tubes, or the like. Adherent culture can be achieved in small or large flasks (e.g., Cell Factory System) and on microcarriers (e.g., Cytodex, GE Healthcare) in stirred large flasks, rotator bottles, or reactor cultures. Microcarrier beads are small spheres (diameters ranging from 50 to 100 μm) that provide a large surface area for adherent cell growth per volume of cell culture. For example, for commercial virus production, such as vaccine production, it is often preferred to culture cells in bioreactors or fermenters. Bioreactors are available in volumes from under 1 L to over 100 L, and laboratory-scale and commercial-scale bioreactors may be used, for example, NBS bioreactors (New Brunswick Scientific, Edison, NJ); Sartorius Stedim Biotech, Göttingen, Germany) or scale-X bioreactors (scale-X single-use bioreactor system; Univercells Technologies, Belgium).

[0081] In the present invention, regardless of culture volume, to ensure effective production of Hepatitis A virus, it is important to maintain the culture at a temperature below 35° C. Generally, it is preferred to utilize a modulator, such as an incubator or other device for sensing and maintaining the temperature of the cell culture system, to ensure that the temperature does not exceed 35° C. during the period of viral replication.

[0082] The process of maintaining cells in culture has been widely reported and is known in the art. General protocols are known in the art and changes in conditions during cell culture processes can be readily determined through routine experimentation.

[0083] On the other hand, in the present invention, the subculture may be performed by repeating one passage of the above-mentioned one cycle 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.

[0084] (d) harvesting the virus from the host cell; The step (d) of the present invention is a step of repeatedly harvesting the host cells and their culture medium in the step (c) and recovering the virus for vaccine production.

[0085] Preferably, step (d) allows harvesting of the virus when a cytopathic effect is exhibited in the host cells.

[0086] In the present invention, the cytopathic effect refers to all effects in cells caused by Hepatitis A virus infection, including, but not limited to, plaque formation, cell granulation and fragmentation, cell detachment from the support (e.g., cell-virus culture flask), cell shrinkage, cell aggregation, cell lysis, cell rounding degeneration, edema formation, induction of apoptosis, etc. The cytopathic effect can generally be confirmed by observation with a microscope or the naked eye.

[0087] Meanwhile, in step (d) of the present invention, step (d) may be performed by harvesting the virus from the cell culture medium and cells after the appearance of cytopathic effect in the host cells, and then performing additional subculture to harvest a desired amount of virus at each subculture, and then repeating the subculture.

[0088] In the present invention, the amount of the virus can be measured by viral titer (specifically, the content of Hepatitis A antigen), plaque size or morphology, particle density, or other means known in the art. This refers to the amount of virus measured by

[0089] According to one embodiment of the present invention, it was confirmed that in host cells that were repeatedly passaged according to the above-mentioned method, cytopathic effect (CPE) began to be observed from the third passage onwards, and the amount of virus released as the culture medium and intracellular virus significantly increased from the fourth or fifth passage onwards. It was also confirmed that the amount of virus released as the culture medium and intracellular virus was maintained without decreasing even with continued passage. As a representative example, virus particles were confirmed by electron microscopy (TEM) at the third passage of infection in the SF-Vero cell line, and electron microscope photographs of the produced virus particles are shown in Figures 5a to 5c.

[0090] Thus, in the present invention, the step (d) may be performed after the subculture in the step (c) has been performed at least twice, preferably three times, more preferably four times, but is not limited thereto. In the case where the amount of virus is to be further amplified even after the cytopathic effect in the host cells is observed, the virus can be obtained at the point where the desired level of the amount of virus is obtained by repeatedly performing subculture.

[0091] The method of the present invention may further comprise a step of purifying and inactivating the virus, so that the virus obtained after step (d) can be used as a vaccine.

[0092] In the present invention, the purification may be performed through a primary purification method or step, such as chromatography or density gradient ultracentrifugation purification. The chromatography may be resin ion exchange chromatography, hydrophobic interaction chromatography, mixed mode chromatography, membrane chromatography, etc. Also, ion exchange chromatography and size exclusion chromatography may be performed simultaneously, or ion exchange chromatography and multimodal chromatography may be performed simultaneously.

[0093] In the present invention, the inactivation step is performed to completely remove virus infectivity, and generally, this may be performed by chemical or physical means. Chemical inactivation may be performed by inactivating the virus with an inactivation reagent solution containing, for example, formaldehyde or beta-propiolactone at an appropriate concentration. If necessary, the remaining inactivated material may be neutralized afterwards. The material inactivated with formaldehyde may be neutralized with a formaldehyde neutralizer containing, for example, sodium sulfite or sodium bisulfite, and may be exchanged with a phosphate buffer, physiological saline, or a buffer that maintains the safety of the virus through diafiltration.

[0094] In the present invention, the order of the purification and inactivation steps is not particularly limited, and the purification step may be preceded by the inactivation step, or the inactivation step may be preceded by the purification step. Preferably, the purification step may be preceded by the inactivation step.

[0095] The present invention also provides a Hepatitis A virus produced by the method comprising the steps (a) to (d).

[0096] The hepatitis A virus produced by the method of the present invention has a very fast amplification rate in cell lines that are industrially used as vaccine production cell lines with their safety ensured or proven, or that are approved or certified as cell substrates for the production of biological medicines by health authorities of various countries, including WHO. Furthermore, the virus can be stably amplified even after repeated subculture for a long period of time in host cells, and is therefore very useful for the production of hepatitis A vaccines.

[0097] The present invention also provides a vaccine composition containing the virus as an active ingredient.

[0098] In the present invention, the vaccine may be a live vaccine, an attenuated vaccine or an inactivated vaccine.

[0099] The term "live vaccine" refers to a vaccine containing a live virus active component. The term "attenuated" refers to a vaccine in which the pathogenicity of a live virus has been weakened by artificial or natural factors, such that the virus is unable to cause disease in the body and only stimulates the immune system to induce immunity. Virus attenuation can be achieved through heat treatment of virus particles, exposure to ultraviolet light, and performing multiple serial passages in vitro or serial passages of the virus in cultured cells in an in vitro culture vessel such as a culture flask. Attenuation can also be achieved by making a defined genetic change, for example, by deleting a specific viral sequence known to provide toxicity or by inserting a sequence into the viral genome. The term "inactivated vaccine", also called an inactive vaccine, refers to a vaccine containing a virus from which infectivity has been removed. Examples include whole virus vaccines and split virus vaccines, which can be easily produced by known methods.

[0100] The vaccine composition of the present invention may further comprise one, two, or more adjuvants in addition to the aforementioned virus. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant may act primarily as a delivery system, act primarily as an immunomodulator, or have strong characteristics of both. Suitable adjuvants include those suitable for use in mammals, including humans.

[0101] Suitable immunoadjuvants for enhancing the effectiveness of the vaccine composition of the present invention include, but are not limited to, the following:

[0102] (1) Aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.).

[0103] (2) oil-in-water emulsion formulations (with or without muramyl peptides or other specific immunostimulants such as components of bacterial cell walls);

[0104] (3) Quil A or Stimulon )TM Particles using or produced from saponin adjuvants such as QS-21 [Antigenics, Framingham, MA] (U.S. Patent No. 5,057,540);

[0105] (4) synthetic polynucleotides;

[0106] (5) Cytokines, such as interleukins, interferons, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, tumor necrosis factor, etc.

[0107] In certain embodiments, an aluminum-based adjuvant may be used. An aluminum salt adjuvant may be an alum precipitated vaccine or an alum-adsorbed vaccine. Aluminium salts include alumina hydrate, aluminium oxide, etc. Aluminum, Aluminum Trihydrate, Aluminum Phosphate Gel, Superfos, Ampogel, Aluminum Hydroxide ( 2 These include, but are not limited to, aluminum hydroxyphosphate sulfate (aluminum phosphate adjuvant, APA), and amorphous alumina. Aluminum salts form antigen depots that slowly release antigens for 2-3 weeks and nonspecifically activate macrophages, complement, and innate immune mechanisms.

[0108] In yet another specific embodiment, the vaccine composition disclosed in the present invention may comprise a CpG oligonucleotide as an adjuvant. CpG oligonucleotide refers to an immunostimulatory CpG oligodeoxynucleotide (CpG ODN), and thus the terms are used interchangeably unless otherwise indicated.

[0109] The immunoadjuvant is appropriately selected depending on the amount and valence of the conjugate in the composition, and when an aluminum-based immunoadjuvant is used, it may be added so that the aluminum element is contained in the composition at 0.01 mg / mL to 1.0 mg / mL based on the aluminum element. Preferably, the aluminum element may be contained in the composition at 0.1 mg / mL to 0.6 mg / mL, or 0.1 mg / mL to 0.4 mg / mL, and more preferably, the aluminum element may be contained in the composition at 0.15 mg / mL to 0.35 mg / mL.

[0110] The vaccine composition of the present invention is not particularly limited in the formulation in which the composition is provided, so long as it achieves its intended effect.

[0111] The vaccine composition of the present invention can be formulated in a liquid form (i.e., a solution or suspension) or a lyophilized form. In one embodiment, the vaccine composition of the present invention is in a liquid form, preferably an aqueous liquid form. When provided as a liquid form, the vaccine composition of the present invention is provided in a packaged form in a container (preferably a syringe), and can be administered immediately without a separate vaccine composition reproduction process such as redispersion, and therefore the liquid form is also ideal for reproducing injections and certain effects, unlike the composition of a lyophilized formulation that requires resuspension in an aqueous medium.

[0112] The vaccine composition of the present invention can be formulated by various methods known in the art.For example, the hepatitis A virus can be formulated as a physiologically acceptable carrier to produce the composition.Examples of such carriers include, but are not limited to, water, buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), polysorbate 20 and dextrose solution.

[0113] The present specification provides a vaccine composition comprising the Hepatitis A virus disclosed in the present invention and a pharma- ceutically acceptable excipient, carrier, isotonicity agent, or diluent. The types of these excipients, carriers, or diluents to be used depending on the administration route of the pharmaceutical composition described below are known in the art.

[0114] As an example, pharma- ceutically acceptable carriers used in 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, saline, and buffer solutions. Pharmaceutical compositions may be isotonic, hypotonic, or hypertonic. However, pharmaceutical compositions administered by injection are preferably essentially isotonic. Thus, isotonicity or hypertonicity may be advantageous for storage of the composition. If the pharmaceutical composition is hypertonic, it may be diluted to be isotonic before administration. The isotonicity agent may be an ionic or non-ionic isotonicity agent. Iono Ionic tonicity agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, magnesium chloride, etc. Nonionic tonicity agents include, but are not limited to, sorbitol, glycerol, etc. Preferably, the pharmaceutical composition contains at least one pharma- ceutically acceptable buffer. For example, when the pharmaceutical composition is an injection, it is preferably composed of a buffer having a buffering capacity at pH 5.0 to pH 9.0, for example, pH 6.0 to pH 8.0, pH 6.8 to pH 7.5. The buffer may be selected from buffers composed of potassium phosphate, sodium monohydrogen phosphate, glutamate, carbonate, borate, lactate, citrate, histidine, glycine, triethanolamine, etc.

[0115] The vaccine compositions herein may further comprise one or more selected from the group consisting of buffers, salts, divalent cations, surfactants (particularly non-ionic detergents), cryoprotectants (e.g., sugars), antioxidants (e.g., chelating agents, preservatives) and antimicrobial agents.

[0116] The buffer may be any of those known in the art for use in pharmaceutical compositions, particularly vaccine compositions, and may be any of histidine, citric acid, phosphate, succinate, or Hepes (4-(2)-hydroxyethyl)-1-piperazineethanesulfonic acid). These may be used in any compound form, and for example, phosphate may be used in the form of sodium phosphate or potassium phosphate.

[0117] In one embodiment, the vaccine composition of the present invention comprises a salt. The type of the salt is not particularly limited as long as it is known in the art to be used in pharmaceutical compositions, particularly vaccine compositions, and may be selected from the group consisting of magnesium chloride, potassium chloride, sodium chloride, borochloride, and combinations thereof.

[0118] In one embodiment, the vaccine composition of the invention comprises a surfactant, hi a preferred embodiment, a non-ionic detergent is used. In one embodiment, the surfactant is polysorbate 20 (Tween™ 20), polysorbate 40 (Tween™ 40), polysorbate 60 (Tween™ 60), polysorbate 65 (Tween™ 65), polysorbate 80 (Tween™ 80), polysorbate 85 (Tween™ 85), Triton™ N-101, Triton™ X-100, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine-polypeptide oleate, polyoxyethylene-660-hydroxystearate (PEG-15, Solutol H 15), octylthioglucoside (OTG), octylglucoside (OG), nonylmaltoside (NM), lauryldimethylamine oxide (LDAO), dodecyl dimethylamine oxide (DDM), or the like. beta-D-maltoside and poloxamer.

[0119] In the present invention, the term "single dose" is used interchangeably with "unit dose" or "single dose", and the terms may be used interchangeably herein. Preferably, the unit dose may refer to a unit suitable for single administration to an animal, preferably a mammal, particularly a human, and each unit contains an antigenic substance calculated by a person skilled in the art to produce a preventive or immunizing effect against a disease of interest (particularly, without the risk of serious side effects at the same time). In a preferred embodiment, the vaccine composition provided by the present invention may be provided in a unit dose form.

[0120] The dosage may be determined appropriately by a person skilled in the art according to the technical common knowledge in the field of pharmaceutical formulations, such as the administration means or route. For example, a single dose for injection is 0.5 mL. The volume may be, but is not limited to, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, or 1.0 mL.

[0121] As a more convenient means for directly providing the above-mentioned vaccine compositions of the present invention to a user, the present invention provides a container filled with any one of the above-mentioned vaccine compositions.

[0122] The container may be of any type known in the art to be used for providing a pharmaceutical or vaccine composition. In one embodiment, the container is a prefilled syringe, a vial, a syringe, a sterile injection needle, or the like. The pharmaceutical composition is selected from the group consisting of a microneedle patch, a microneedle needle, an ampoule, and an administration cartridge.

[0123] A typical injection dose of the vaccine composition of the invention may be provided in a volume of 0.5 mL or 1.0 mL, more preferably 0.5 mL or 1.0 mL depending on the dose group.

[0124] Thus, the container or syringe as defined above may be provided filled with the vaccine composition of the present invention in a volume equivalent to the single injection dose. In one embodiment, the container or syringe may be provided 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.

[0125] The present invention also provides a kit comprising the vaccine composition of the present invention. The specific components of the kit may refer to the form of provision of the composition known in the art, depending on the form of provision of the composition. It is clear that any container filled with the vaccine composition described above is included in the kit.

[0126] As an example, a kit may be provided that includes one or more vials with or without a vaccine composition of the invention (whether in liquid or lyophilized form), one or more syringes with or without a vaccine composition of the invention, or a kit that includes one or more of each.

[0127] In addition, when the vaccine composition of the present invention is provided in a liquid dosage form, the vaccine substance is contained in a prefilled syringe, and in this case, the substance is a substance for administration to a patient. A sterile injection needle may be attached to the inlet of the prefilled syringe and the substance may be administered to a patient.

[0128] The kit may also include instructions for use that are provided to the user.

[0129] In the present invention, the kit may provide the vaccine composition in doses for a single vaccination schedule or in doses for a multiple (split) vaccination schedule.

[0130] In one embodiment, the vaccine composition disclosed in the present invention is for use as a medicament (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 bacterial infection, disease or condition in a subject. In particular, the vaccine composition disclosed in the present invention may be used for preventing, treating or improving Hepatitis A virus infection, disease or condition in a subject.

[0131] The present invention also relates to a vaccine composition comprising a vaccine composition for use in a patient in need thereof, the ... for use in a patient in need thereof, the vaccine composition for use in a patient in need thereof, the vaccine composition for use in a patient in need thereof, the vaccine composition for use in a patient in need thereof, the vaccine composition for use in a patient in The present invention provides a vaccination method for preventing hepatitis A, which comprises:

[0132] In the present invention, the "effective amount" refers to an amount that, when administered to an individual, exhibits an effect of killing the Hepatitis A virus or improving, treating or preventing infection, disease or condition associated with the Hepatitis A virus.

[0133] In one embodiment, the effective amount is an immunologically effective amount. The immunologically effective amount refers to an amount of antigen or vaccine sufficient to induce any one of cellular (T cell) or humoral (B cell or antibody) immune responses, as measured by standard assays known to those skilled in the art. It may be measured by measuring the level of antigen-specific antiserum or antigen-neutralizing serum induced by an antigen as an immunogen, such as a viral antigen or its vaccine, or by detecting cytokines secreted by T cells stimulated by a viral antigen. The protective level of immune response may also be measured by confirming the reduction of viral infection caused by the antigen or the prevention of disease caused by infection in immunized individuals.

[0134] In one embodiment, the effective amount is a prophylactically effective amount. The term "prevention" in the present invention means suppressing the occurrence of a disease or illness in individuals who have not been diagnosed with the disease or illness but are prone to such disease or illness. Thus, the term "prophylactically effective amount" in the present specification means an amount sufficient to achieve the pharmacological effect.

[0135] In a specific embodiment, the vaccine composition disclosed in the present invention may be used to prevent Hepatitis A in a subject. Thus, the present invention provides a method for preventing Hepatitis A, comprising administering an effective amount of the aforementioned vaccine composition of the present invention to an individual in need thereof.

[0136] In the present invention, the term "individual", which may be used interchangeably with "subject", refers to an animal, preferably a mammal, particularly a human, and may be a mammal such as a cat, sheep, pig, horse, cow or dog, or a cell, tissue, organ, etc. derived from an animal. The individual may be a patient in need of the effect.

[0137] In one embodiment of the present invention, the immunocompromised individual disclosed in the present invention is any male or female human.

[0138] The vaccine composition of the present invention may be used to protect or treat humans susceptible to Hepatitis A virus infection by administering the vaccine composition via intramuscular, intraperitoneal, intradermal, subcutaneous, rectal, systemic or mucosal routes, without any particular limitation on the route of administration, so long as the vaccine composition achieves its intended effect in the body.

[0139] The vaccine composition of the present invention may be provided in a single dose or multiple doses. In some cases, as little as a single dose of the vaccine composition of the present invention is required, but in some situations, such as under conditions of greater immunodeficiency, a second, third or fourth dose may be provided. Following initial vaccination, the subject may be further immunized once or several times (multiple times) at appropriate intervals.

[0140] The present invention also provides a kit comprising the vaccine composition. The present invention also provides a prefilled syringe containing the vaccine composition.

[0141] The kit and the pre-filled syringe may include the HAV vaccine composition and a pharma- ceutically acceptable carrier, and may further include guidelines on basic matters to be observed in order to prevent HAV infection (such as precautions for administration, administration cycle, storage temperature, and expiration period).

[0142] The invention provides the use of said virus for producing a Hepatitis A vaccine.

[0143] The present invention provides use of a vaccine composition containing the hepatitis virus as an active ingredient for preventing hepatitis A.

[0144] The present invention provides a method for preventing hepatitis A, which comprises administering an effective amount of a vaccine composition containing the hepatitis virus as an active ingredient to an individual in need thereof.

[0145] As used herein, the term "comprising" is used interchangeably with "containing" or "characterized by" and does not exclude additional components or method steps not specifically mentioned in a composition or method according to the invention. Additionally, the term "consisting of" means excluding additional elements, steps, ingredients, etc. that are not otherwise specified. The term "essentially consisting of" means that the scope of a composition or method may include, in addition to the materials or steps specified, substances or steps that do not substantially affect its basic properties. Effect of the Invention

[0146] According to the method for producing the hepatitis A virus provided by the present invention, it is possible to produce a hepatitis A virus that stably amplifies within a short period of time, and the method can be very usefully used as a raw material for the hepatitis A vaccine virus. [Brief description of the drawings]

[0147] [Figure 1] 1 is a diagram showing a method for producing Hepatitis A virus (HAV) for vaccine production according to the method of the present invention. A seed virus is obtained from a host cell transformed with a vector containing an HAV gene, and the seed virus is infected into the same host cell and subcultured, whereby HAV can be rapidly and stably amplified. [Diagram 2] FIG. 2 shows a cleavage map of the vector used to transform host cells to produce a seed virus in the present invention. [Figure 3a] , [Figure 3b]Figures 3a and 3b are diagrams showing the steps of a method for producing HAV in MA104 cells and Vero cells according to the method of the present invention (Figure 3a) and a method for producing HAV in serum-free adapted Vero cells (SF-Vero) (Figure 3b). [Figure 4] FIG. 4 is a step-by-step diagram of a prior art method for producing a HAV. [Figure 5a] , [Figure 5b] , [Figure 5c] Figures 5a to 5c show transmission electron microscope (TEM) images of viruses isolated at passage 3 (P3) after infection of MA104 (Figure 5a), Vero (Figure 5b), and SF-Vero (Figure 5c) with seed virus produced by the method of the present invention. [Figure 6] Figure 6 shows the results of confirming the virus content (titer) at passage 1 (P1), which is a blind passage step for virus rescue after P0, and passage 5 (P5) in the process of Figures 3a and 3b of the present invention. The virus titer in the cell lysate and culture supernatant of each passage P1 to P5 was measured by absorbance value. [Figure 7a] , [Figure 7b] Figures 7a and 7b show the quantitative analysis results of the virus content (titer) confirmed in the cell lysate sample isolated at step p6 of the procedure of Figures 3a and 3b of the present invention (Figure 7a), and also show the results of detecting the infected virus in the cells by immunofluorescence assay (IFA) after infecting MA104, Vero, and SF-Vero cells with the seed virus produced by P6 subculture (Figure 7b). [Figure 8]Fig. 8 shows the results of measuring the virus content after infection of host cells (MA104, Vero, SF-Vero) with commercially available HAV by a conventional method ("A" in Fig. 8) or infection of the same host cells with seed viruses produced by the methods of Fig. 3a and Fig. 3b ("B" in Fig. 8) from serial virus infection passages 1 to 6. The virus titer was measured by absorbance from the infected host cell lysates and culture supernatants collected at each of the six passages by ELISA, and the results show a relative comparison of virus growth in host cells "A" and "B". [Figure 9a] , [Figure 9b] Figures 9a and 9b show the results of virus titers being confirmed ten times in the supernatant and cells after infecting SF-Vero and MRC-5 cell lines with the seed virus produced by the method of Figure 3b. [Figure 10] Figure 10 shows the virus titer (antigen content) as a function of days of virus culture in a T-flask (175 cm2). To confirm the state of virus growth after infection with the seed virus of the invention, infected samples were collected at intervals of 3 to 4 days, and the virus titer was measured from the supernatant and cell lysate. [Figure 11] Figure 11 shows the results of infecting host cells in a Cell Factory 10 (CF10) culture vessel with commercially available HAV by conventional methods (A), or infecting host cells (MA104, Vero or SF-Vero) with seed virus produced by the method of the present invention (B), and confirming the virus titer in the cell lysate at passage P11. [Figure 12]12 is a diagram showing the titers of anti-HAV serum analyzed in blood of experimental animals before (Day-5), after the second vaccination at two-week intervals (Day28), and after the third vaccination (Day42) with the inactivated HAV antigen produced by the method of the present invention or a commercial HAV vaccine (Havrix, GSK). SK144 and SK72 are groups in which the virus of the present invention was purified and inactivated, and administered at 144 EL.U (3.0 IU) and 72 EL.U (1.5 IU), respectively, while HVR144 and HVR72 are groups in which the commercial product Havrix was administered at 144 EL.U and 72 EL.U. As negative controls for the test, the normal group was administered saline instead of the viral antigen, and the alum group was administered only alum adjuvant without the antigen. [Figure 13] FIG. 13 shows the average body weight of each animal group and the occurrence of abnormal reactions due to antigen inoculation during the course of the experiment shown in FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0148] The present invention will now be described in detail with reference to the following examples, although it should be noted that the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0149] Experimental Method The experimental methods used in the present invention are summarized in FIG.

[0150] 1. Gene synthesis and vector construction In the present invention, the base sequence of the HAV gene used for gene synthesis is shown in SEQ ID NO: 1. The HAV gene is functionally composed of the base sequence of the 5'-terminal UTR (untranslated region), polyprotein gene, and 3'-terminal UTR in that order. The HAV gene contains the base sequences of the CMV promoter-T7 promoter (SEQ ID NO: 2), multicloning site (MCS, SEQ ID NO: 3), and HH (hammerhead) ribozyme (SEQ ID NO: 4) in the 5'-terminal direction, and the hepatitis delta virus ( The base sequences of the HDV ribozyme (SEQ ID NO: 5), MCS (SEQ ID NO: 6), and bGH poly A terminator (SEQ ID NO: 7) are located in this region. The base sequence including the HAV gene consisting of the base sequence of SEQ ID NO: 1 and all other functional regions such as the promoter is shown in SEQ ID NO: 8.

[0151] The synthesized hepatitis A virus gene of SEQ ID NO: 8 was cloned into pUC57 vector using KpnI (GGTACC) and SalI (GTCGAC) restriction enzymes. The completed plasmid construction map is shown in Figure 2, and was named HAV expression vector.

[0152] 2. Transformation with HAV expression vector MA104 (ECACC, 85102918) and Vero (WHO) (ECACC, 88020401) cell lines were cultured at 2 × 10 5 The cells were prepared in EMEM (Lonza) medium containing 10% and 5% FBS at a cell / well / 2 mL, respectively. Serum Free Vero cells (serum-free adapted cells derived from Vero (WHO), SF-Vero) were prepared at a cell / well / 2 mL. 5 Prepare serum-free EMEM medium at 2 mL cells / well and incubate at 5% CO. 2、 The cells were cultured in an incubator at 37° C. The medium used for the cell preparation was used as the culture medium for each cell.

[0153] After 18 to 24 hours, the culture medium was removed from the culture plate, washed twice with 2 mL of DPBS, and 2 mL of culture medium was added to each well. 1.0 μg of HAV expression vector (plasmid), 35 μL of Lipofectamine LTX-Plus (ThermoFisher), and 960 μL of Opti-MEM (ThermoFisher) were mixed in a conical tube and left at room temperature for 15 minutes. 200 μL of the mixture was added to each plate well and incubated in 5% CO. 2、 The cells were cultured in a 37°C incubator. After 24 hours, the supernatants of all the cells were removed, and the MA104 and Vero cell lines were replaced with EMEM containing 2% FBS (2%FBS-EMEM), and the SF-Vero cell line was replaced with 2mL of serum-free EMEM medium (SF-EMEM), and the cells were cultured in a 35°C, 5% CO2 incubator for 3 weeks. The cultured MA104, Vero, and SF-Vero cells were collected and suspended in 500μL of EMEM medium, and freeze / thawed three times. To remove the cell debris remaining after the crushing, the cells were centrifuged at 10,000g for 1 minute, and only the supernatant was collected again and used as virus P0 (seed virus).

[0154] 3. Blind passage for virus rescue For infection passage progression, first blind passage with P1 virus, place 1 x 10 MA104 and Vero in a T25 flask. 6 cells / 5mL, SF-Vero 5×10 6 At the time of P1-P4 infection passage, MA104 and Vero cells were prepared in a T25 flask at 7 × 10 5 At a concentration of cells / 5 mL, SF-Vero cells were 1 × 10 6 The cells were cultured at 37°C and 5% CO2 in culture medium at 5 mL / cell. After 24 h, the culture medium was removed from the prepared cells, and 5 mL of DPBS was added to each flask to wash the cells twice just before infection. For the P5 and P6 infection passages, 5 × 10 MA104 and Vero cells were added to a T175 flask one day before the infection passage from P4 was started. 6 cells / 30 mL, SF-Vero cells were 8 × 106 The medium was similarly removed from the cells prepared at 30 mL / cells just before infection, and the cells were washed twice with 30 mL of DPBS.

[0155] EMEM medium containing 2% FBS was used as the virus infection medium for MA104 and Vero cell lines, and EMEM without FBS was used as the infection medium for the SF-Vero cell line. 5 mL of infection medium for each cell type was used for the P1-P4 infection passages, and 35 mL of infection medium was used for the P5-P6 infection passages. In the P6 passage, five T17 Infection passage was performed using 5 flasks. The prepared P0 sample was added to the prepared cells, and the mixture was left at 35°C and 5% CO2 for 1 hour, after which 5 mL (35 mL) of infection medium was added.

[0156] For 21 days after infection of the P0 sample, the culture medium was changed at 7-day intervals to maintain the infected cells. On the 21st day after infection, the cell lysate was collected, and freezing / thawing (-70℃ / 37℃) was performed three times in the same manner as when collecting P0, cell debris was removed, and the cells were centrifuged. The supernatant was collected and used as the virus culture medium, which was designated as passage 1 (P1). When progressing from P0 to P1, the medium was changed every week, but when passing from P1 to P6, the medium was changed only on the 7th day after infection. A total of six passages were performed consecutively, with one passage period being 20-21 days. The above-mentioned cell infection and passage progression process are shown in Figure 3a and Figure 3b.

[0157] After one passage, the culture supernatant and cell lysate were collected from the virus-infected flasks. When collecting the cell lysate, 2 mL of trypsin-Versene (Lonza) solution was added to the flask from which the supernatant had been removed, and the flask was washed and then removed. Another 2 mL of trypsin-Versene solution was added and the flask was left in a 37°C incubator for 5 minutes, after which the cells were detached. The 2 mL of trypsin-Versene suspension with the cells suspended was transferred to a conical tube and centrifuged to collect the cells in a cell pellet state.

[0158] 1 mL (T25, P1-P4) or 5 mL (T175, P5) of EMEM was added to the cell lysate (pellet), which was then frozen / thawed three times and centrifuged, after which the cell debris was removed and the supernatant (cell lysate sample) was prepared. After centrifugation with the culture supernatant collected from each infection passage, 200 μL of the cell lysate sample was transferred to a microcentrifuge tube for virus titer analysis and frozen until analysis. All the remaining cell lysate samples, excluding those for analysis, were used for the next infection.

[0159] P1-P5 were titrated by ELISA qualitative analysis. In passage 6, the culture supernatant was removed, all the infected cells were collected, and suspended in 5 ml of serum-free EMEM. After five freeze-thaw cycles, the cells were centrifuged to remove cell debris, and only the supernatant was collected and stored as the seed virus. The titer of P6 virus was measured by ELISA qualitative analysis.

[0160] 4. Confirmation of the cytopathic effect of the blind passage process Cytopathic effect of the host cells due to virus infection (CPE) was examined by microscopy. In MA104 and Vero cell lines, no cytopathic effect was observed until the P4 passage, but CPE due to the virus, such as cell debris and detachment, was observed from the P5 passage, and mild CPE was observed after the P3 passage in the SF-Vero cell line.

[0161] 5. HAV antigen analysis ELISA HAV antigen measurements in samples collected during the blind passage of the virus were performed by qualitative and quantitative analysis. Qualitative analysis confirmed the antigen by absorbance (450 nm) using HAV-specific ELISA, and quantitative analysis used a standard product (Inactivated HAV BRP, 1350 IU / mL, Y0001192, EDQM) to create a standard curve and measure the HAV virus titer (antigen content) in the samples. The analytical kit used was the HAV-Antigen ELISA kit (Mediagnost, E12), and the virus titer unit was expressed in IU / mL according to the standard product. The commercial hepatitis A virus strain used (ATCC VR-1402) was also quantified in a similar manner.

[0162] 6. Transmission Electron Microscopy (TEM) A portion of the cell lysate collected after the P6 subculture in Figures 3a and 3b was used for transmission electron microscopy. Negative staining was performed with uranyl acetate for 15 seconds, and the samples were observed under a transmission electron microscope (JEM-1011, JEOL), and viral particles were confirmed using a Camera-Megaview III Imaging device.

[0163] 7. Immunofluorescence Assay (IFA) 5 x 10 cells in a 24-well culture plate 3 MA104 and Vero cells were cultured at 8 x 10 cells / well / 0.5 mL. 3SF-Vero cells were prepared by suspending them in each culture medium at a concentration of cells / well / 0.5 mL, and cultured for 24 hours at 37°C and 5% CO2 conditions. The medium was removed from the cultured cells, and 300 μL / well of DPBS was added and washed. The seed virus produced in Figure 3 was diluted with the infection medium of each cell and treated with the cells at a concentration of 0.1 IU / well, and 0.5 mL of infection medium was added to each well and cultured at 35°C in a 5% CO2 incubator. On the 7th day after the infection, all the supernatant of the cells was removed, and the cells were washed twice with 0.5 mL of DPBS. All the DPBS remaining in the cells was removed, and 0.2 mL of 3.7% formaldehyde solution was added, and the cells were left at room temperature for 30 minutes. The formaldehyde solution was removed, and the cells were washed three times with DPBS as described above. 0.2% Triton X-100 buffer solution was added at 250 μL / well, left at room temperature for 5 minutes, and then washed three times with 0.5 mL of DPBS. The primary antibody (Anti-HAV Surface Ag, Raybiotech) was diluted 1 / 500 in PBS, added at 250 μL / well, and reacted at room temperature for 1 hour. Then, the secondary antibody (Goat anti-Mouse IgG Alexa488, ThermoFisher) was diluted 1 / 4000 and added to the cells in the same volume as the primary antibody, left at room temperature for 1 hour, and then washed five times with DPBS to remove it. DPBS was added to the cells at 250 μL / well, and the cells were observed under a fluorescent microscope (Magnification Photographed with 100x, Eclipse Ts2-FL, Nikon.

[0164] 8. Confirmation of seed virus infection (Infection test) The infection patterns of the seed viruses prepared in Figures 3a and 3b were compared with those 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 the ELISA described in this patent. 24 hours before virus infection, 2 × 10 6 The SF-Vero cell line was 3.5 × 10 6The cells were prepared in 12 mL of medium. 2.0 IU of each of the seed virus and commercial virus stock was suspended in 1 mL of infection medium and added to each cell in a T75 flask that had been washed with 10 mL of DPBS. After 1 hour of reaction in a 35°C, 5% CO2 incubator, 11 mL of infection medium was added and cultured. 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 virus infection passages were performed during the 21-day culture, and the collection of samples for virus content analysis and successive infection passages were the same as in the blind passage, and the cell lysate samples were finally suspended in 5 mL of serum-free EMEM at the time of collection and used as samples for the next infection passage.

[0165] In addition, a total cell count of 8 × 10 6 MRC-5 (ECACC, 05011802) cell line and SF-Vero were prepared at 3.5 × 10 cells / 12 mL. 6 The seed virus prepared in Figure 3b was prepared at a cell concentration of 12 cells / mL and subjected to a total of six consecutive infection passages under the same conditions as the T75 flask infection described above. The virus titers (contents) in some of the supernatants and some of the cell lysates collected at each virus infection passage were measured and compared. The culture medium for the MRC-5 cell line was EMEM medium containing 10% FBS, and the infection medium was used at a concentration of 2%. The method for collecting the supernatant and cells after virus infection was the same as that described in the blind passage execution method, which involves repeated freezing and thawing.

[0166] 9. Seed virus growth confirmation test One day before infection, culture 2 x 10 SF-Vero cells in a T175 flask. 7 Seed cells / 35mL After seeding, the cells were cultured at 37℃ and 5% CO2. Nine T175 flasks with the same cell density were prepared, eight of which were infected with the virus and the remaining were normal cell controls. On the day of infection, the culture medium was removed from all flasks, and the cells were washed with 30 mL of DPBS in each flask. A virus infection solution containing 15 IU of SF-Vero-derived seed virus in 35 mL of culture medium was prepared and added to the washed T175 flask. A total of eight flasks were infected with 15 IU in the same manner. The infected cells were cultured in a 35℃, 5% CO2 incubator, and the supernatant and cell lysate were collected on days 3, 7, 10, 14, 17, 21, 24, and 28 post-infection (dpi) to measure the virus titer (content).

[0167] 10. Cultivation of seeded virus cell factories The seed virus derived from each cell was further infected and passaged in a T175 flask in the same manner, progressing to passage 11 (MA104, Vero) and passage 12 (SF-Vero) to increase the cell fitness of the virus, and the infected cells were collected during the process. The method of further infection passage was to obtain the virus of passage P8 by infecting and culturing the first seed virus harvested at P6 twice in a T175 flask, and at the time of infection at P9, the virus sample obtained at P8 was quantified and infected at a concentration of 15 IU / T175. Similarly, passages were repeated with infection and recovery up to P11 and P12. The seed virus of passages P11 (MA104, Vero) and P12 (SF-Vero) were quantified, and the virus equivalent to 500 IU was dispensed into a separate cryovial. The commercial virus was obtained by infection passage at P11 (MA104, Vero) and P12 (SF-Vero) in the same manner as the additional infection passage described above, using the commercial virus sample obtained after six passages in Figure 6. The virus was obtained by infection passage at P11 (MA104, Vero) and P12 (SF-Vero). The supernatant was not collected during the additional infection passage, and only the infected cells were collected. The cells were disrupted with a Sonifier for 40 seconds and centrifuged, after which the supernatant was collected and used. The seed virus and the commercial virus were each 500 IU in the same amount and incubated with CF10 (6320 cm 2The infection method for CF10 was as follows: MA104, Vero, and SF-Vero cells were infected with 2.0 × 10 8 The cells were prepared at 1.5L per cell. After 16-18 hours, the culture medium was removed, and the cells were washed once with 500mL of DPBS. 500 IU of the virus was added to 200mL of EMEM medium to prepare a virus infection solution, which was then added to the washed CF10. The cells were cultured for 60 minutes at 35°C and 5% CO2, and the flask was tilted every 15 minutes to allow the virus dilution to be uniformly adsorbed onto the cells. After virus adsorption, 1.5L of infection medium for each cell type (MA104, Vero: 2% FBS-EMEM, SF-Vero: SF-EMEM) was added, and the cells were cultured for 21 days at 35°C and 5% CO2. On the 7th day of virus culture, the medium was replaced with a new infection medium.

[0168] The procedure for harvesting virus-infected cultures in CF10 after infection is as follows. The upper layer culture medium was removed from the infected CF10 container. The CF10 was washed with 500 mL of DPBS and then removed, and 200 mL of TrypLE Express (ThermoFisher) was added and reacted in a 37°C incubator for 3 to 5 minutes. 200 mL of serum-free EMEM was added, and the infected cell suspension with a volume of approximately 400 mL was collected in a 2 L square bottle. The collected suspension was centrifuged at 5000 g for 10 minutes, the supernatant was discarded, and only the cell pellet was collected. 100 mL of phosphate buffer (50 mM, pH 7.0) was added to the cell pellet to suspend it, and then the cells were disrupted by sonication with a Sonifier (SFX550, Branson) (amplitude 40%, 2 minutes). Approximately 100 mL of cell lysate was centrifuged (5000 g, Alegra X-15R, SX4750A) and the supernatant was transferred to a new sterile 1 L square bottle. After centrifugation, 100 μL of the 100 mL of supernatant was stored and used for ELISA analysis. 400 mL of phosphate buffer (50 mM, pH 7.0) was added and used for the purification process. Cell lysates from MA104 and Vero were stored separately, and antigen purification for animal test administration was performed using lysates of virus-infected cells cultured in SF-Vero.

[0169] 11. Virus purification and inactivation The collected cell lysate was purified sequentially using a capsule filter (Sartopure PP3, 5 μm, Sartorius Stedim) and a depth filter (Supra 50, 050PDH4, PALL). The purified harvest was buffer exchanged into phosphate buffer (50 mM, pH 7.0) using a 100 kDa ultrafiltration / diafiltration (UF / DF) filter (Pellicon®2 Mini, P2B100A01, Merck Millipore) and treated with benzoin (1 unit) after filtration and 10-fold concentration. Ion exchange chromatography (IEC) was performed at a rate of 10 mL / min using a DEAE Sepharose® Fast Flow (GE Healthcare) column equilibrated with phosphate buffer (50 mM, pH 7.0). Fractions of approximately 200 mL were collected and buffer exchanged into phosphate buffer (50 mM, pH 7.0) using a 10 kDa UF / DF filter (Pellicon® 2 mini, P2B010A01, Merck Millipore) and concentrated 5-fold. The concentrate was subjected to size exclusion chromatography (SEC) using a HiPrep 26 / 60 Sephacryl S-200 HR (GE Healthcare) at a rate of 1 mL / min.

[0170] After SEC, about 40 mL of antigen fraction was collected and concentrated 80-fold using a 10 kDa (Pellicon (registered trademark) 2Mini, P2B010A01, Merck Millipore) filter to obtain purified HAV antigen. To inactivate the virus, formaldehyde was added to the collected antigen at a concentration of 270-370 μg / mL and reacted at 37 ° C for 5 days. After that, it was decomposed and diafiltered using a 10 kDa filter, and sterilized and filtered using a 0.22 μm filter (Millipak (registered trademark) Gold). 24 hours before administration of each dose in the animal test, alum hydroxide was suspended in an adsorption buffer (pH 7.1-8.0) and mixed with the antigen, and then stirred at 4 ° C for more than 16 hours.

[0171] 12. Animal testing Mice (BALB / c, 4 weeks old, 10 per group) were administered the antigen adsorbed to the Alum. A commercial HAV vaccine (Habrics, GSK) was used as a control group for the animal test, and the antigen (3.0IU and 1.5IU) administered to the mice and the dose of the control group (144EL.U and 72EL.U, ELISA Unit) were set to 1 / 10 of the human (adult and child) dose proposed by the commercial vaccine. The dose of the administered antigen was set by desorbing the alum salt bound to the control group (commercial vaccine) and separating only the antigen, and then measuring and comparing the amount of the patented antigen together to adjust the dose to the same level. In the animal test, the antigen was administered three times at 2-week intervals via muscle (IM, intramuscular injection). After administration, serum was separated from the whole blood of the mice, and the titer of anti-total HAV antibodies was measured. The standard control substance was 97 / 646 (NIBSC, International Standard for Anti-Hepatitis A, Immunoglobulin), and measurements were performed using Anti-HAV ELISA (E10, Mediagnost) or Anti-Hepatitis A Virus IgG ELISA (4660, ALPHA Diagnostic International).

[0172] Experimental Results In the present invention, a gene expression cassette was designed to express the HAV gene, and the cassette expression vector was obtained by synthesizing the cassette. The synthetic HAV expression vector was transformed into three types of cell lines (MA104, Vero, and SF-Vero), and the transformed cells (transfectants) were disrupted and infected into the same cells to obtain viral particles. The virus was isolated by passage (blind passage or virus-infected passage) until a certain amount of virus was confirmed. This confirmed a certain amount of virus, and produced a seed virus that can be used for future vaccine production and research. The seed virus was produced by only six subcultures, and after production, mycoplasma and sterility tests were performed to confirm that it was produced in a sterile condition.

[0173] In the case of conventional commercial vaccines, in order to produce a virus (master seed lot) used for vaccine production, a cell culture adaptation process is performed several times, in which the virus is established by primary AGMK culture and subculture in MRC-5, which requires serum. In contrast, in the present invention, a method of isolating the virus using a gene expression vector is devised and applied. Among the seed viruses produced by the devised method, the seed virus (926 IU / mL) produced in a serum-free cultured vaccine production cell line (SF-Vero) is a virus derived from a vaccine cell line, and is considered to have high applicability when developing a commercial vaccine. The seed virus was amplified, purified, and inactivated to remove infectivity, and then adsorbed with adjuvant and administered to mice, and it was confirmed that the antiserum titer against the administered antigen of the patent was similar to that of the commercial vaccine.

[0174] The virus production method using the above-mentioned vector and the HAV expression cassette used for virus production are shown in Figures 1 and 2, respectively. The process in Figure 1 lists two culture processes performed in MA104 and Vero cell lines, and SF-Vero cell lines, and the transformation and blind passage processes are shown in Figures 3a and 3b along with the required time. After infection passage from P1 to P5, the virus titer (content) in the supernatant and cell lysate collected and measured at each passage was confirmed as shown in Figure 6. Although only a few passages had passed after transformation, the virus was confirmed in the ELISA detection phase, and it was confirmed that the virus was stably amplified in the supernatant and cell lysate samples. The relative virus detection amount was confirmed to be higher in the cell lysate than in the supernatant, which can be judged to reflect the characteristics of non-clinical HAV. Immediately after the next P6 passage was completed, virus particles were confirmed from the cell lysate by electron microscopy (Figures 5a to 5c), and the virus content was quantified to obtain 2371 The virulence factors of the virus in the present invention were identified as 1.1 IU / mL (MA104), 586 IU / mL (Vero), and 926 IU / mL (SF-Vero) (Figure 7a). The MA104 cell line was used to amplify and secure seed viruses for research, in reference to its use in infection research of enteric viruses such as Hepatitis A virus (JH Lee et al., 2013). Vero and SF-Vero cell lines have the same origin, but the culture conditions for the virus differ 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 smoothly amplified even under serum-free conditions. It was confirmed that the seed virus content of SF-Vero-derived seed virus cultured and isolated under serum-free culture conditions was relatively high compared to the seed virus derived from Vero cells. The prepared seed virus was used to infect a 24-well plate at a content of 0.1 IU / well, and the infectivity of the virus was confirmed by immunofluorescence reaction on the 7th day (FIG. 7b).

[0175] As described in "Experimental Method 8. Method for confirming seed virus infection," 2.0 IU of the three types of seed viruses produced in Figure 7a and a commercial virus (ATCC stock, 227 IU / mL) were infected, and the amplification degree when the seed virus of the invention and the commercial virus were infected into the same cell line was compared. The virus was detected from the supernatant and cell lysate of the infected culture that had been serially passaged a total of six times, and it was confirmed that the virus titer of the seed virus of the invention ("B" in Figure 8) was measured to be stable and relatively high compared to the commercial virus stock ("A" in Figure 8) (Figure 8).

[0176] In addition, the MRC-5 cell line, which is used as a production cell for conventional Hepatitis A vaccine and requires serum during culture, and the SF-Vero cell line, which is a serum-free cultured vaccine production cell used for the production and infection of the virus in this patent, were used as infected cells, and 2.0 IU of the seed virus produced in SF-Vero in Figure 3b were infected and compared. As a result of performing ELISA analysis on the supernatant and cell lysate during the progress of a total of 10 passages and comparing the virus titer (content) relative to the absorbance value, the virus production in SF-Vero cells showed a pattern in which the virus began to be detected from the first passage after infection, and from the second passage, the virus titer measured in the cell lysate was consistently detected by ELISA reaction (Figure 9a). In the passage in MRC-5, the virus detection in the collected cell lysate sample was measured at a similar level at some passages, but did not show a constant pattern of increasing virus titer. In particular, in contrast to the results of infection passage in SF-Vero for vaccine production, the virus detection in the supernatant was low (Figure 9b). This confirmed that the seed virus produced by the method of the present invention was amplified smoothly in SF-Vero cells, and that the cellular adaptability of the virus after infection and passage was higher in the SF-Vero cell line than in MRC-5 cells. The measured absorbance results are reported along with graphs (Figures 9a and 9b).

[0177] Before CF10 culture to obtain the virus to be used in animal experiments, a T175 flask was infected with the seed virus derived from SF-Vero produced in the present invention, and the change in the virus titer (content) depending on the number of days of culture was confirmed at intervals of 3-4 days (Figure 10). This was carried out preferentially to set the virus content per unit area of ​​culture of the virus-infected cells and to confirm the culture period of the virus of the present invention. As a result of measurement after virus infection, the amount of virus in the cells increased starting from 10 days after infection, and it was confirmed that the virus amplified in the cells had a higher titer than the supernatant, as in the above figures (Figures 6, 7a and 7b, Figures 8 and 9). In particular, it was confirmed that the virus content was maximum around 20-21 days after infection. The virus amplification was confirmed to be about 230 times (3537 IU, 21 dpi) compared to the initial infected virus amount (15 IU) (Figure 10).

[0178] Referring to FIG. 11, the titers of the virus obtained by culturing seed virus and commercial virus having the same infection passage number in CF10 under the same conditions were measured. It was confirmed that the titer of the virus produced and passaged according to the method of the present invention (B) was significantly higher than the titer of the virus obtained by similarly culturing the commercial virus produced by the conventional method (A) that requires a long time. The significant level indicates that the titer (B) of the method of the present invention, which assumes the same virus culture area and the same culture method, was increased by 150% (MA104, 1.53 times), 470% (Vero, 4.70) times, and 251% (SF-Vero, 2.51 times) compared to (A). It was confirmed that the virus titer was higher than that of the conventional method when passage culture was carried out under the same conditions, which means that the method of the present invention can produce HAV virus more quickly and stably. The difference in virus titer between each cell line is due to the susceptibility to Hepatitis A virus infection (Ccell This is presumed to be due to differences in the susceptibility of the seed virus, but this will need to be confirmed through future studies of the characteristics of the seed virus of the present invention. In particular, the present invention is significant in that it goes beyond the commercial production of existing MRC-5-based Hepatitis A vaccines, develops a new virus strain, applies it to vaccine production strains, and confirms its culturability.

[0179] Meanwhile, Fig. 12 shows the results of culturing, purifying and inactivating the seed virus produced in the present invention, administering it to experimental mice three times at two-week intervals, and then confirming whether or not the mice produced antibodies against the inactivated antigen administered with serum isolated from the whole blood of the mice. Fig. 13 shows the average body weight of animals in each group during the animal experiment. SK144 and SK72 are groups in which the virus of the present invention was purified and inactivated, and administered 3 IU (144 EL.U) and 1.5 IU (72 EL.U), respectively, while HVR144 and HVR72 are groups in which the commercial product Havrix was administered at 144 EL.U. The groups were administered EL.U and 72 EL.U.

[0180] Referring to Figure 12, Day 5 (Day-5) refers to mouse serum 5 days before the first administration, Day 28 (Day28) refers to serum 14 days after the second administration, and Day 42 (Day42) refers to serum 14 days after the third administration. The bar graphs for each administration group indicate the concentration of anti-HAV antibodies (total IgG anti-HAV serum) present in the mouse serum collected on each date. The serum concentration was measured using the antibody titer analysis method described in the Experimental Method section "12. Animal Experiments."

[0181] No increase in anti-HAV serum was observed in any of the groups on day 5 before administration. Serum analysis on day 28 confirmed that the antibody titers of the SK144 group (mean antibody 5.742mIU / mL) and the HVR144 group (mean antibody 5.783mIU / mL) were similar (p>0.9999), and that the SK72 group (4.377mIU / mL) and the HVR72 group (4.875mIU / mL) were also at similar levels (p=0.3895). Serum analysis on day 42 confirmed that antibody titers between the SK144 group (mean antibody 6.002mIU / mL) and the HVR144 group (mean antibody 6.223mIU / mL) (p=0.8825), and antibodies between the SK72 group (5.432mIU / mL) and the HVR72 group (5.446mIU / mL) were at similar levels (p>0.9999).

[0182] 12 shows that there is no significant difference in the immunological efficacy of the HAV antigen prepared according to the method of the present invention compared to the commercialized Hepatitis A vaccine (Havrix). For reference, since mice cannot be administered with the human dose, 1 / 10 of the human volume was used, and the calculation was based on the adult dose of 1440 EL.U / infectious dose and the pediatric dose of 720 EL.U / infectious dose of the commercial Hepatitis A vaccine.

[0183] 13, from the start date to the end date of the animal test, no abnormal reactions of unknown causes were observed by the naked eye, no immediate abnormal reactions after antigen administration that may affect mouse stress and immunogenicity measurement, and no weight loss were found. In addition, no abnormal reactions due to the administered purified inactivated antigen of the present invention and the control substance were confirmed by visual observation. [Industrial Applicability]

[0184] According to the method for producing the hepatitis A virus provided by the present invention, it is possible to produce a hepatitis A virus that stably amplifies in a short period of time, and the method is highly useful for producing a hepatitis A vaccine and has high industrial applicability. In addition, the method can be used as a source technology for the development of hepatitis A vaccine technology that has not been developed domestically in Korea.

Claims

1. A Hepatitis A virus gene comprising the base sequence of SEQ ID NO:

1.

2. An expression cassette for producing hepatitis A virus, comprising the hepatitis A virus gene according to claim 1.

3. 3. The expression cassette of claim 2, wherein the expression cassette comprises a promoter, an HH (Hammerhead) ribozyme and a Hepatitis Delta Virus (HDV) ribozyme.

4. The expression cassette of claim 2 , wherein the expression cassette comprises the base sequence of SEQ ID NO:

8.

5. A vector for producing Hepatitis A virus, comprising the expression cassette according to any one of claims 2 to 4.

6. A Hepatitis A virus produced by the vector of claim 5.

7. (a) transforming a host cell in vitro with a vector comprising an expression cassette for producing a hepatitis A virus, the expression cassette comprising a hepatitis A virus gene comprising the base sequence of SEQ ID NO:1; (b) harvesting the virus from the host cell; (c) infecting a host cell with the obtained virus and subculturing it; (d) a method for producing a Hepatitis A virus for the production of a vaccine, comprising the step of obtaining the virus from said host cell.

8. The method for producing a vaccine according to claim 7, wherein the expression cassette comprises, in the order from the 5' end of the Hepatitis A virus gene, a CMV promoter, a T7 promoter, a multicloning region (MCS), and a Hammerhead (HH) ribozyme region, and, in the order from the 3' end of the Hepatitis A virus gene, a Hepatitis Delta Virus (HDV) ribozyme, an MCS, and a polyA chain. A method for producing a hepatitis A virus.

9. The method for producing a Hepatitis A virus for producing a vaccine according to claim 7, wherein the expression cassette comprises the base sequence of SEQ ID NO:

8.

10. The method for producing a Hepatitis A virus for producing a vaccine according to claim 7, wherein the host cell is selected from the group consisting of Vero, MA104, WI-38, BHK-21, CHO, MDCK, Hi5, CEF and Sf9.

11. The method for producing a Hepatitis A virus for producing a vaccine according to claim 7, wherein the cells are adapted to a serum-free medium.

12. The method for producing a hepatitis A virus for producing a vaccine according to claim 7, wherein the subculture is carried out 2 to 30 times.

13. The method for producing a Hepatitis A virus for producing a vaccine according to claim 7, wherein in step (d), a cytopathic effect appears in the host cells after three or more subcultures.

14. The method for producing a Hepatitis A virus for producing a vaccine according to claim 7, wherein the host cells in steps (a) and (c) are the same cells.

15. The method for producing a Hepatitis A virus for producing a vaccine according to claim 7, further comprising a step of purifying, inactivating, or purifying and inactivating the virus after step (d).

16. A Hepatitis A virus produced by the method according to any one of claims 7 to 15.

17. A Hepatitis A vaccine composition comprising the virus according to claim 16 as an active ingredient.

18. 18. The Hepatitis A vaccine composition of claim 17, wherein the vaccine is a live vaccine, an attenuated vaccine or an inactivated vaccine.

19. 20. The Hepatitis A vaccine composition of claim 18, wherein the composition further comprises an adjuvant.

20. A kit comprising the vaccine composition of claim 17.

21. A prefilled syringe containing the vaccine composition according to claim 17.

22. 17. Use of the virus according to claim 16 for the production of a Hepatitis A vaccine.

23. The use of the virus described in claim 22, wherein the Hepatitis A vaccine is a vaccine for the prevention of Hepatitis A.

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