WO2008069598A1 - Method for preparing antigen of hepatitis a virus using transformed insect cells - Google Patents

Method for preparing antigen of hepatitis a virus using transformed insect cells Download PDF

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WO2008069598A1
WO2008069598A1 PCT/KR2007/006322 KR2007006322W WO2008069598A1 WO 2008069598 A1 WO2008069598 A1 WO 2008069598A1 KR 2007006322 W KR2007006322 W KR 2007006322W WO 2008069598 A1 WO2008069598 A1 WO 2008069598A1
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vpl
hepatitis
virus
cells
recombinant
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PCT/KR2007/006322
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French (fr)
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In Sik Chung
Wonyong Kim
Heeyoung Lee
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Medikan Inc.
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • A61K2039/541—Mucosal route
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    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • A—HUMAN NECESSITIES
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    • C12N2710/14011—Baculoviridae
    • C12N2710/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
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    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
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    • C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
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    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
    • C12N2770/32434—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present inventors have made many efforts to develop a hepatitis A virus vaccine, which is more stable and, at the same time, is easily administered orally.
  • the present inventors have found that, when trans formants, obtained by transforming insect cells with a recombinant vector, constructed by cloning VPl and VP3 genes, which are hepatitis A virus antigen genes, into an insect cell expression vector, are cultured, the hepatitis A virus antigens can be prepared, thereby completing the present invention.
  • the present invention provides a method for preparing a recombinant hepatitis A vims antigen, the method comprising culturing Drosophila melanogaster S2 transformed with an insect cell expression vector, in which a gene of hepatitis A virus antigen selected from the group consisting of VPl-S of SEQ ID NO: 1, VPl-L of SEQ ID NO: 2, and VP3 of
  • SEQ ID NO: 3 is inserted into an expression vector (pMT/BiP/V5-His) which contains a metallothionein promoter (P M ⁇ ), a BiP signal sequence (BiP SS), a V5 epitope (V5), polyhistidine (His6 tag) and restriction enzyme digestion sites and has a cleavage map of FIG. 1.
  • FIG. 1 is a schematic diagram of expression vector pMT/BiP/V5-His which is used to transform Drosophila melanogaster S2 cells such that the cells can express a hepatitis A virus antigen.
  • FIG. 2 shows schematic diagrams of pMT/BiP/VP3-V5-His, pMT/BiP/VPl-S- V5-His and pMT/BiP/VPl-L-V5-His according to the present invention.
  • FIG. 3 shows the results of SDS-PAGE (A) and Western blot analysis (B) of a His-tagged hepatitis A virus antigen obtained by culturing the transformed Drosophila melanogaster S2 cells.
  • M molecular weight marker
  • lane 1 cell fraction of non-transformed cell line
  • lanes 2, 3 and 4 a medium in which transformed Drosophila melanogaster S2 cells are expressing recombinant VP3, VPl-S and VPl-L
  • arrows recombinant VP3, VPl-S and VPl-L proteins.
  • FIG. 4 shows the production of an anti-VPl antibody induced by the intraperitoneal administration of recombinant VPl according to the present invention.
  • FIG. 5 shows the production of anti-VPl IgA induced by the oral administration of recombinant VPl according to the present invention.
  • FIG. 6 shows T-cell proliferation activity induced by the oral administration of recombinant VPl according to the present invention.
  • the present invention relates to a method for preparing a recombinant hepatitis A virus antigen by culturing Drosophila melanogaster S2 transformed with a recombinant vector expressing a hepatitis A virus antigen (VPl or VP3) gene.
  • a hepatitis A virus antigen VPl or VP3
  • the restriction enzyme digestion sites of the recombinant vector include Bg ⁇ ll and Apal, and the gene of hepatitis A virus antigen is inserted between BgHl and Apal.
  • the hepatitis A virus antigen was produced by cloning a VPl-S, VPl-L or VP3 gene, which are hepatitis A virus antigen genes, into the restriction enzyme digestion sites Bglll-Apal of an expression vector pMT/BiP/V5-His, introducing the vector into insect cells, Drosophila melanogaster S2 cells to transform the cells, and then culturing the transformed cells.
  • the VPl genes are genes containing a base sequence of base Nos. 1480- 2298 or a base sequence of base Nos. 1480-2379, which correspond to total lengths of 819 bp and 900 bp, respectively.
  • the VPl genes are named "VPl-S" of SEQ ID NO: 1, and "VPl-L” of SEQ ID NO: 2, respectively.
  • the VP3 gene is a gene containing a base sequence of base Nos. 742-1479 corresponding to a total length of 738 bp.
  • an insect cell expression vector pMT/BiP/V5-His is preferably used to express the hepatitis A virus antigen gene VPl-S, VPl-L or VP3, but the scope of the present invention is not necessarily limited thereto.
  • the pMT/BiP/V5-His vector (3.6 kb) is easily obtained from Invitrogen Co., CA, USA, and the vector contains a methallothionein promoter, a BiP signal sequence, a V5 epitope tag and a polyhistidine domain.
  • Drosophila melanogaster Schneider 2 cells (Invitrogen, U.S.A.) were used as host cells to express the hepatitis A virus antigen, but the scope of the present invention is not limited thereto.
  • Drosophila insect cells Spodoptera sp. cells, Trichoplusia sp. cells, silkworm cells and other insect cells may be used, as long as they can effectively express the hepatitis A virus antigen at high yield.
  • the recombinant protein VPl prepared according to the above method, was administered orally or intraperitoneally to mice, and then the amounts of anti-VPl IgG or anti-VPl IgA produced in serum and the small intestine were analyzed. As a result, the group administered with the VPl showed a high level of anti-VPl IgG or anti-VPl IgA.
  • the VPl can induce an immune response, when it is administered orally or intraperitoneally.
  • an MTT assay was performed. Specifically, the VPl and CT were administered orally to mice, a VPl -containing medium was added to T- cells isolated from the mice, and the proliferation of the T-cells was observed. As a result, the T-cells showed high absorbance due to cell division. However, the case of addition of OM (ovomucoid) showed an absorbance similar to that of the case where only the medium was added. This is because the recombinant VPl stimulated the T cells to proliferate.
  • the hepatitis A virus antigen produced according to the above method, is useful as a vaccine for preventing or treating hepatitis, because it can induce the production of specific antibodies.
  • the term "vaccine” refers to a composition which is used to prevent or treat epidemic diseases.
  • the vaccine either contains an antigen or enables the expression of the antigen, so that it can induce an immune response to the antigen.
  • the vaccine containing the recombinant vector of the present invention can be used in a desired form.
  • the term "vaccination” means administering a vaccine to induce active immunity (humoral immunity, cellular immunity, or both) in the body or culture system of organisms. This can suppress the infection, proliferation, contagion and/or spread of pathogens.
  • an epitope means a molecule containing one or more epitopes that will stimulate a host's immune system to induce a humoral and/or cellular antigen-specific immune response.
  • an epitope may consist of about three to several amino acids, an epitope in a protein generally contains about 7-15 amino acids, for example, 8, 9, 10, 12 or 14 amino acids.
  • antigen is also called “immunogen”. Also, if an antigen is expressed using a polynucleotide or vector encoding the antigen protein, this polynucleotide or vector are also called an “antigen” in the present invention and can be used as a vaccine component.
  • Example 1 Construction of recombinant vector 1-1 : Amplification of hepatitis A virus antigen gene
  • hepatitis A virus RNA was extracted using TRI reagent (Molecular Research Center, Cincinnati, USA). Using the extracted RNA, the Pl gene of hepatitis A virus was obtained through a series of nested RT-PCR processes.
  • cDNA was synthesized from 20 ng of the hepatitis A virus RNA using a random hexamer (Promega, USA), and then, the cDNA flanking region containing the hepatitis A virus Pl gene was amplified using primers of SEQ ID NO: 4 and SEQ ID NO: 5. From the flanking region, the hepatitis A virus Pl gene was amplified using primers of SEQ ID NO: 6 and SEQ ID NO: 7.
  • SEQ ID NO: 4 S'-AGTGGCCTTGACCACATTCTGT-S' SEQ ID NO: 5: 5'-ACTCCAAGTCTCCAGCTGCAATT-S'
  • SEQ ID NO: 6 5'-GCTCTAGAATGAATATGTCCAAACAAGG-S'
  • SEQ ID NO: 7 5'-GACTAGTCTCAAATCTTTTATCTTCCTC-S'
  • the PCR reactions were performed in the following conditions: predenaturation at 94 ° C for 3 min, and then 30 cycles of denaturation at 94 "C for 1 min, annealing at 55 ° C for 1 min, and extension at 72 ° C for 1 min, followed by final extension at 72 ° C for 7 min.
  • the amplified PCR product was ligated into a pCR2.1-TOPO vector (Invitrogen, USA), which was then introduced into E. coli TOPlO.
  • the E. coli strain containing the hepatitis A virus Pl gene was deposited in KCTC (KCTC 10987BP: E. coli TOP 10/pCR2.1 -TOPO(HAV Pl)), and the base sequence of the Pl gene is the same as SEQ ID NO: 8.
  • a base sequence of base Nos. 1480-2298 was designated as VPl-S of SEQ ID NO: 1; a base sequence of base Nos. 1480-2379, VPl-L of SEQ ID NO: 2; and a base sequence of base Nos. 742- 1479, VP3 of SEQ ID NO: 3.
  • the pMT/BiP/V5-His (3.6 kb, Invitrogen Co., CA) vector was treated with restriction enzymes BgIW and Apal at 37 ° C for 2 hours (FIG. 1). Also, HAV- VP3 and HAV-VPl, prepared in Example 1-1, were treated with restriction enzymes B gill and Apal at 37 ° C for 2 hours to obtain Bgl ⁇ l-Apal fragments containing the hepatitis A virus antigen gene.
  • the above-obtained BgIW- Apal fragments of HAV-VP3 and HAV-VPl were ligated into the BgIW and Apal digestion sites of the pMT/BiP/V5-His vector with a T4 DNA ligase at 25 °C for 2 hours, thus obtaining the desired expression vectors pMT/BiP/VPl-V5-His and pMT/BiP/VP3-V5-His for expressing the hepatitis A virus antigen (FIG. 2).
  • the VPl gene was a gene containing a base sequence of base Nos. 1480- 2298 or 1480-2379, corresponding to a total length of 819 bp or 900 bp, it was cloned into two types, HAV-Vl-S and HAV-Vl-L, as shown in FIG. 2.
  • Drosophila melanogaster S2 cells as host cells for expressing the hepatitis A virus antigen were cultured in 10% IMS (insect medium supplement, Sigma)- containing Shields and Sang M3 insect medium (Sigma) in a T-25 flask (Nunc, Denmark) at 27 ° C .
  • the hepatitis A virus antigen expression vector pMT/BiP/HAV (VP3, VPl-S, VPl-L)-V5-His, constructed in Example 1, and selection plasmid pCoHygro, at a ratio of 1 : 1, were introduced into the exponentially growing Drosophila melanogaster S2 cells using lipofectin according to the method described in
  • hepatitis A virus antigen was stably expressed in the transformed Drosophila melanogaster S2 cells was examined by Western blot analysis using a mouse anti-V5 polyclonal antibody (Invitrogen) (FIG. 3).
  • the Western blot analysis was performed using SDS-PAGE according to the method of Laemmli (Laemmli, U.K., Nature, 227:680, 1970). Specifically, the cells electrophoresed on gel were transferred onto nitrocellulose and allowed to react with the mouse anti-V5 polyclonal antibody. Then, the cells were labeled with a rabbit anti-mouse IgG alkaline phosphatase conjugate (1 : 1000, v/v). After the labeled cells were washed, BCIP/NBT solution (Amnesco Co., OH) was added thereto to develop a color, and distilled water was then added to stop the color development reaction.
  • BCIP/NBT solution Amnesco Co., OH
  • the VPl and VP3 antigens were expressed in the transformed Drosophila melanogaster S2 cells.
  • the recombinant VP3 had a molecular weight of about 33-34 kDa, which was 7 kDa larger than an expected molecular weight of 27 kDa.
  • the recombinant VPl-S had a molecular weight of about 38-39 kDa, which was 8 kDa larger than an expected molecular weight of 30 kDa.
  • the recombinant VPl-L had a molecular weight of about 39-40 kDa, which was 6 kDa larger than an expected molecular weight of 33 kDa.
  • the recombinant hepatitis A virus antigens were present in the transformed Drosophila melanogaster S2 cells and the culture medium. Densitometric scanning showed that the secreted hepatitis A virus antigens (i.e., the hepatitis A virus antigens present in the culture medium) accounted for about 85% of the total production of the hepatitis A virus antigens. That is, the transformed S2 cells effectively secreted the expressed hepatitis A virus antigens into the culture medium. In the non-transformed S2 cells, no recombinant hepatitis A virus antigen was detected.
  • 4-week-old female BALB/c mice and 5-week-old female C3H/He mice were divided into four groups and administered orally and intraperitoneally with the recombinant protein VPl, prepared in Example 3.
  • 50 ⁇ g of the VPl and an adjuvant were administered to the 5-week-old C3H/He mice three times at an interval of 2 weeks
  • 50 ⁇ g of the VPl and an adjuvant were administered to the 5-week-old BALB/c mice five times at an interval of 1 week.
  • the adjuvant used in the intraperitoneal administration the Freund's complete adjuvant (FCA) was used in the first immunization, and the Freund's incomplete
  • FIA adjuvant
  • CT choleta toxin
  • tissue solution was homogenized with a homogenizer for 1 minute, and then centrifuged at 4 ° C at 10,000 xg for 10 minutes to separate the supernatant.
  • the recombinant protein VPl was diluted in coating buffer (0.05 M Tris buffer, pH 9.0), and 100 ⁇ i of the dilution was dispensed into each well of a 96-well plate and left to stand at 4 °C overnight. Then, each well of the well plate was washed three times with 150 ⁇ of PBST (phosphate buffered saline (pH 7.4), 0.02% Tween 20). The above-isolated mouse serum was diluted 1000-fold in PBST, and 100 ⁇ i of the dilution was added to each well of the plate and left to stand at room temperature for 1 hour. After each well of the plate was washed again, 100 ⁇ i of an anti- mouse IgG-HRP conjugate, diluted 30,000-fold in PBST, was added to each well of the well plate and allowed to react at room temperature for 1 hour. After the following the PBST was diluted 1000-fold in PBST, and 100 ⁇ i of an anti- mouse IgG-
  • anti-VPl IgA in serum was conducted in the same manner as in the analysis of anti-VPl IgG.
  • 0.5 ⁇ g/m£ of an anti-mouse IgA-biotin conjugate, diluted in PBST was added to each well and left to stand at room temperature for 1 hour.
  • 50 pg/m£ of avidin-peroxidase was added to each well and left to stand at room temperature for 1 hour, and a color development reaction was performed.
  • the ELISA absorbance for anti-VPl IgG was as high as about 1.336 in the group treated with the VPl , but was 0.015 in the control group.
  • the ELISA absorbance for anti-VPl IgA in the group treated with the VPl was very low, which is similar to that for the control group. Accordingly, it can be seen that, when the VPl is administered intraperitoneally several times together with the Freund's adjuvant, IgG-like specific antibodies to VPl are produced in large amounts in serum, thus increasing immunity.
  • the analysis of anti-VPl IgA in the small intestines was performed in the same manner as in the analysis of anti-VP 1 IgA, except that the above-obtained small intestine extract, diluted 200-fold in 10% FBS-containing PBST, was used instead of serum as the test sample.
  • the absorbance at 450 nm was 0.322 in the group administered with the VPl, but was 0.045 in the control group.
  • Example 5 Measurement of effect of recombinant VPl on T-cell proliferation activity
  • an MTT assay was performed. Specifically, the spleen of the BALB/c mouse, in which an immune response has been induced by the oral administration of VPl, was extracted and separated into cells. 50 ⁇ i of the cells were placed in each of a 96-well plate at a cell density of I x IO 7 cells/ mi and cultured in a CO 2 incubator at 37 ° C in the presence of a suitable concentration of antigen. In the culture, an RPMI medium, containing 10% FBS, mercaptoethanol and an antibiotic, was used.
  • MTT 3-(4,5-dimethylthiazolyl-2)-2,5-di ⁇ henyltetrazolium bromide
  • the absorbance was about 1.74-fold higher than that in the case where only the medium was added. Accordingly, it could be observed that, due to the oral administration of VPl, the IgA-specific antibody was produced in the small intestines, and in addition, antigen stimulation at the cell level occurred.
  • the recombinant hepatitis A virus VPl according to the present invention induces the production of a specific antibody in serum when it is administered intraperitoneally, and in addition, it induces the production of a specific antibody in the small intestine when it is administered orally. This is believed to also be associated with the mitogen activity of VPl .
  • the infection route of hepatitis A virus is the oral cavity
  • the IgA- specific antibody which is produced in the small intestine upon the oral administration of VPl, will be useful to prevent hepatitis A virus from entering the body.
  • a recombinant hepatitis A virus antigen can be obtained by culturing Drosophila melanogaster S2 transformed with a recombinant vector containing hepatitis A virus antigen gene VPl or VP3 which cause hepatitis A in the human body. Also, it is possible to prepare a vaccine containing this virus antigen as an active ingredient.
  • the present invention has advantages in that, in the production of hepatitis A

Abstract

The present invention relates to a method for stably preparing the VPl or VP2 antigen protein of hepatitis A virus using insect cells transformed with a recombinant vector containing the antigen gene of hepatitis A virus. When the transformed insect cells expressing the antigen gene of hepatitis A virus is used, a recombinant hepatitis A virus antigen can be stably prepared, and it can be used as a vaccine.

Description

was insignificant. Thus, the precise expression level of the antigen in the expression system has not yet been reported, and there has been no patent application relating to the preparation of the antigen.
Accordingly, the present inventors have made many efforts to develop a hepatitis A virus vaccine, which is more stable and, at the same time, is easily administered orally. As a result, the present inventors have found that, when trans formants, obtained by transforming insect cells with a recombinant vector, constructed by cloning VPl and VP3 genes, which are hepatitis A virus antigen genes, into an insect cell expression vector, are cultured, the hepatitis A virus antigens can be prepared, thereby completing the present invention.
SUMMARY OF THE INVENTION
Therefore, it is a main object of the present invention to provide a method for preparing a recombinant hepatitis A virus antigen using insect cells transformed with a recombinant vector containing a hepatitis A virus antigen gene.
To achieve the above object, the present invention provides a method for preparing a recombinant hepatitis A vims antigen, the method comprising culturing Drosophila melanogaster S2 transformed with an insect cell expression vector, in which a gene of hepatitis A virus antigen selected from the group consisting of VPl-S of SEQ ID NO: 1, VPl-L of SEQ ID NO: 2, and VP3 of
SEQ ID NO: 3, is inserted into an expression vector (pMT/BiP/V5-His) which contains a metallothionein promoter (PMτ), a BiP signal sequence (BiP SS), a V5 epitope (V5), polyhistidine (His6 tag) and restriction enzyme digestion sites and has a cleavage map of FIG. 1.
Other features and aspects of the present invention will be apparent from the following detailed description and the appended claims. BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a schematic diagram of expression vector pMT/BiP/V5-His which is used to transform Drosophila melanogaster S2 cells such that the cells can express a hepatitis A virus antigen.
FIG. 2 shows schematic diagrams of pMT/BiP/VP3-V5-His, pMT/BiP/VPl-S- V5-His and pMT/BiP/VPl-L-V5-His according to the present invention.
FIG. 3 shows the results of SDS-PAGE (A) and Western blot analysis (B) of a His-tagged hepatitis A virus antigen obtained by culturing the transformed Drosophila melanogaster S2 cells. In FIG. 3, M: molecular weight marker; lane 1: cell fraction of non-transformed cell line; lanes 2, 3 and 4: a medium in which transformed Drosophila melanogaster S2 cells are expressing recombinant VP3, VPl-S and VPl-L; and arrows: recombinant VP3, VPl-S and VPl-L proteins.
FIG. 4 shows the production of an anti-VPl antibody induced by the intraperitoneal administration of recombinant VPl according to the present invention.
FIG. 5 shows the production of anti-VPl IgA induced by the oral administration of recombinant VPl according to the present invention.
FIG. 6 shows T-cell proliferation activity induced by the oral administration of recombinant VPl according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS In one aspect, the present invention relates to a method for preparing a recombinant hepatitis A virus antigen by culturing Drosophila melanogaster S2 transformed with a recombinant vector expressing a hepatitis A virus antigen (VPl or VP3) gene.
In the present invention, preferably, the restriction enzyme digestion sites of the recombinant vector include Bgϊll and Apal, and the gene of hepatitis A virus antigen is inserted between BgHl and Apal.
In an example of the present invention, the hepatitis A virus antigen was produced by cloning a VPl-S, VPl-L or VP3 gene, which are hepatitis A virus antigen genes, into the restriction enzyme digestion sites Bglll-Apal of an expression vector pMT/BiP/V5-His, introducing the vector into insect cells, Drosophila melanogaster S2 cells to transform the cells, and then culturing the transformed cells.
Herein, the VPl genes are genes containing a base sequence of base Nos. 1480- 2298 or a base sequence of base Nos. 1480-2379, which correspond to total lengths of 819 bp and 900 bp, respectively. The VPl genes are named "VPl-S" of SEQ ID NO: 1, and "VPl-L" of SEQ ID NO: 2, respectively. Also, the VP3 gene is a gene containing a base sequence of base Nos. 742-1479 corresponding to a total length of 738 bp.
In the present invention, an insect cell expression vector pMT/BiP/V5-His is preferably used to express the hepatitis A virus antigen gene VPl-S, VPl-L or VP3, but the scope of the present invention is not necessarily limited thereto.
The pMT/BiP/V5-His vector (3.6 kb) is easily obtained from Invitrogen Co., CA, USA, and the vector contains a methallothionein promoter, a BiP signal sequence, a V5 epitope tag and a polyhistidine domain. Meanwhile, in the present invention, Drosophila melanogaster Schneider 2 cells (Invitrogen, U.S.A.) were used as host cells to express the hepatitis A virus antigen, but the scope of the present invention is not limited thereto. For example, in addition to Drosophila insect cells, Spodoptera sp. cells, Trichoplusia sp. cells, silkworm cells and other insect cells may be used, as long as they can effectively express the hepatitis A virus antigen at high yield.
In another example of the present invention, the recombinant protein VPl, prepared according to the above method, was administered orally or intraperitoneally to mice, and then the amounts of anti-VPl IgG or anti-VPl IgA produced in serum and the small intestine were analyzed. As a result, the group administered with the VPl showed a high level of anti-VPl IgG or anti-VPl IgA.
This suggests that the VPl can induce an immune response, when it is administered orally or intraperitoneally.
Also, in order to examine the effect of the recombinant protein VPl on T-cell proliferation activity, an MTT assay was performed. Specifically, the VPl and CT were administered orally to mice, a VPl -containing medium was added to T- cells isolated from the mice, and the proliferation of the T-cells was observed. As a result, the T-cells showed high absorbance due to cell division. However, the case of addition of OM (ovomucoid) showed an absorbance similar to that of the case where only the medium was added. This is because the recombinant VPl stimulated the T cells to proliferate.
From the above results, it can be seen that the hepatitis A virus antigen, produced according to the above method, is useful as a vaccine for preventing or treating hepatitis, because it can induce the production of specific antibodies.
As used herein, the term "vaccine" refers to a composition which is used to prevent or treat epidemic diseases. The vaccine either contains an antigen or enables the expression of the antigen, so that it can induce an immune response to the antigen. To prevent or treat the infection, contagion and spread of hepatitis A virus, the vaccine containing the recombinant vector of the present invention can be used in a desired form.
As used herein, the term "vaccination" means administering a vaccine to induce active immunity (humoral immunity, cellular immunity, or both) in the body or culture system of organisms. This can suppress the infection, proliferation, contagion and/or spread of pathogens.
As used herein, the term "antigen" means a molecule containing one or more epitopes that will stimulate a host's immune system to induce a humoral and/or cellular antigen-specific immune response. Although an epitope may consist of about three to several amino acids, an epitope in a protein generally contains about 7-15 amino acids, for example, 8, 9, 10, 12 or 14 amino acids.
The term "antigen" is also called "immunogen". Also, if an antigen is expressed using a polynucleotide or vector encoding the antigen protein, this polynucleotide or vector are also called an "antigen" in the present invention and can be used as a vaccine component.
Examples
Hereinafter, the present invention will be described in further detail with reference to examples. It will be apparent to one skilled in the art that these examples are for illustrative purpose only and are not construed to limit the scope of the present invention.
Example 1 : Construction of recombinant vector 1-1 : Amplification of hepatitis A virus antigen gene
From fecal samples of Korean male patients diagnosed as hepatitis A, hepatitis A virus RNA was extracted using TRI reagent (Molecular Research Center, Cincinnati, USA). Using the extracted RNA, the Pl gene of hepatitis A virus was obtained through a series of nested RT-PCR processes.
Specifically, cDNA was synthesized from 20 ng of the hepatitis A virus RNA using a random hexamer (Promega, USA), and then, the cDNA flanking region containing the hepatitis A virus Pl gene was amplified using primers of SEQ ID NO: 4 and SEQ ID NO: 5. From the flanking region, the hepatitis A virus Pl gene was amplified using primers of SEQ ID NO: 6 and SEQ ID NO: 7.
SEQ ID NO: 4: S'-AGTGGCCTTGACCACATTCTGT-S' SEQ ID NO: 5: 5'-ACTCCAAGTCTCCAGCTGCAATT-S'
SEQ ID NO: 6: 5'-GCTCTAGAATGAATATGTCCAAACAAGG-S' SEQ ID NO: 7: 5'-GACTAGTCTCAAATCTTTTATCTTCCTC-S'
The PCR reactions were performed in the following conditions: predenaturation at 94 °C for 3 min, and then 30 cycles of denaturation at 94 "C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min, followed by final extension at 72 °C for 7 min.
The amplified PCR product was ligated into a pCR2.1-TOPO vector (Invitrogen, USA), which was then introduced into E. coli TOPlO. The E. coli strain containing the hepatitis A virus Pl gene was deposited in KCTC (KCTC 10987BP: E. coli TOP 10/pCR2.1 -TOPO(HAV Pl)), and the base sequence of the Pl gene is the same as SEQ ID NO: 8. Herein, a base sequence of base Nos. 1480-2298 was designated as VPl-S of SEQ ID NO: 1; a base sequence of base Nos. 1480-2379, VPl-L of SEQ ID NO: 2; and a base sequence of base Nos. 742- 1479, VP3 of SEQ ID NO: 3.
1-2: Construction of expression vector
The pMT/BiP/V5-His (3.6 kb, Invitrogen Co., CA) vector was treated with restriction enzymes BgIW and Apal at 37 °C for 2 hours (FIG. 1). Also, HAV- VP3 and HAV-VPl, prepared in Example 1-1, were treated with restriction enzymes B gill and Apal at 37 °C for 2 hours to obtain Bglϊl-Apal fragments containing the hepatitis A virus antigen gene.
The above-obtained BgIW- Apal fragments of HAV-VP3 and HAV-VPl were ligated into the BgIW and Apal digestion sites of the pMT/BiP/V5-His vector with a T4 DNA ligase at 25 °C for 2 hours, thus obtaining the desired expression vectors pMT/BiP/VPl-V5-His and pMT/BiP/VP3-V5-His for expressing the hepatitis A virus antigen (FIG. 2).
Because the VPl gene was a gene containing a base sequence of base Nos. 1480- 2298 or 1480-2379, corresponding to a total length of 819 bp or 900 bp, it was cloned into two types, HAV-Vl-S and HAV-Vl-L, as shown in FIG. 2.
Whether the insertion of the genes in the above-constructed recombinant expression vectors pMT/BiP/VPl-V5-His and ρMT/BiP/VP3-V5-His was achieved in the right direction, and reading frame, were analyzed by restriction enzyme mapping and DNA sequencing.
Example 2: Construction of transformants
Drosophila melanogaster S2 cells as host cells for expressing the hepatitis A virus antigen were cultured in 10% IMS (insect medium supplement, Sigma)- containing Shields and Sang M3 insect medium (Sigma) in a T-25 flask (Nunc, Denmark) at 27 °C .
The hepatitis A virus antigen expression vector pMT/BiP/HAV (VP3, VPl-S, VPl-L)-V5-His, constructed in Example 1, and selection plasmid pCoHygro, at a ratio of 1 : 1, were introduced into the exponentially growing Drosophila melanogaster S2 cells using lipofectin according to the method described in
Korean Patent Registration No. 10-0557343. Then, antibiotic hygromycin (300 βglmi) was added to the culture medium, and a process of selecting a transformed cell line was performed for about 4 weeks, thus establishing a stably transformed cell line.
Example 3: Examination of recombinant protein expression in transformants
Whether the recombinant hepatitis A virus antigen was stably expressed in the transformed Drosophila melanogaster S2 cells was examined by Western blot analysis using a mouse anti-V5 polyclonal antibody (Invitrogen) (FIG. 3). The Western blot analysis was performed using SDS-PAGE according to the method of Laemmli (Laemmli, U.K., Nature, 227:680, 1970). Specifically, the cells electrophoresed on gel were transferred onto nitrocellulose and allowed to react with the mouse anti-V5 polyclonal antibody. Then, the cells were labeled with a rabbit anti-mouse IgG alkaline phosphatase conjugate (1 : 1000, v/v). After the labeled cells were washed, BCIP/NBT solution (Amnesco Co., OH) was added thereto to develop a color, and distilled water was then added to stop the color development reaction.
As a result, as shown in FIG. 3, the VPl and VP3 antigens were expressed in the transformed Drosophila melanogaster S2 cells. The recombinant VP3 had a molecular weight of about 33-34 kDa, which was 7 kDa larger than an expected molecular weight of 27 kDa. The recombinant VPl-S had a molecular weight of about 38-39 kDa, which was 8 kDa larger than an expected molecular weight of 30 kDa. Also, the recombinant VPl-L had a molecular weight of about 39-40 kDa, which was 6 kDa larger than an expected molecular weight of 33 kDa.
It was observed that the recombinant hepatitis A virus antigens were present in the transformed Drosophila melanogaster S2 cells and the culture medium. Densitometric scanning showed that the secreted hepatitis A virus antigens (i.e., the hepatitis A virus antigens present in the culture medium) accounted for about 85% of the total production of the hepatitis A virus antigens. That is, the transformed S2 cells effectively secreted the expressed hepatitis A virus antigens into the culture medium. In the non-transformed S2 cells, no recombinant hepatitis A virus antigen was detected.
The above results suggest that the expression of the recombinant hepatitis A virus antigens in the transformed S2 cells was induced by the hepatitis A virus antigen gene-containing pMT/BiP/HAV(VP3, VPl-S, VPl-L)-V5-His expression vector introduced into the cells. Also, the amount of recombinant hepatitis A virus antigens produced in the transformed S2 cells was about 4 mg/£.
Example 4: Measurement of immune activity of recombinant protein VPl
4-week-old female BALB/c mice and 5-week-old female C3H/He mice (Orientbio Inc., Korea) were divided into four groups and administered orally and intraperitoneally with the recombinant protein VPl, prepared in Example 3. In the intraperitoneal administration, 50 μg of the VPl and an adjuvant were administered to the 5-week-old C3H/He mice three times at an interval of 2 weeks, and in the oral administration, 50 μg of the VPl and an adjuvant were administered to the 5-week-old BALB/c mice five times at an interval of 1 week. As the adjuvant used in the intraperitoneal administration, the Freund's complete adjuvant (FCA) was used in the first immunization, and the Freund's incomplete
10 adjuvant (FIA) was used in additional administrations, and as the adjuvant in the oral administration, choleta toxin (CT) was used (Table 1).
Table 1 : Design of animal experiments
Figure imgf000011_0001
In order to analyze an antibody in blood, induced by the intraperitoneal administration, blood was collected from the tail vein at 1 week after the last administration and coagulated, and serum was then isolated by centrifugation.
In order to analyze an antibody in small intestines, induced by the oral administration, a 10 cm length of small intestine was cut from the experimental mice one week after the last administration, and a 2-fold volume (v/w) of PBS was added thereto. The tissue solution was homogenized with a homogenizer for 1 minute, and then centrifuged at 4°C at 10,000 xg for 10 minutes to separate the supernatant.
In order to analyze anti-VPl IgG present in serum, the recombinant protein VPl was diluted in coating buffer (0.05 M Tris buffer, pH 9.0), and 100 μi of the dilution was dispensed into each well of a 96-well plate and left to stand at 4 °C overnight. Then, each well of the well plate was washed three times with 150 ≠ of PBST (phosphate buffered saline (pH 7.4), 0.02% Tween 20). The above-isolated mouse serum was diluted 1000-fold in PBST, and 100 μi of the dilution was added to each well of the plate and left to stand at room temperature for 1 hour. After each well of the plate was washed again, 100 μi of an anti- mouse IgG-HRP conjugate, diluted 30,000-fold in PBST, was added to each well of the well plate and allowed to react at room temperature for 1 hour. After the
11 well plate was washed, 100 βi of a color-developing reagent (TMB and 3% H2O2) was added to each well of the plate and allowed to react for 30 minutes. Then, 100 μJL of 2 M H2SO4 was added to each well of the plate to stop the color development reaction, and the absorbance at 450 nm was measured with an ELISA reader.
The analysis of anti-VPl IgA in serum was conducted in the same manner as in the analysis of anti-VPl IgG. However, in place of the anti-mouse IgG-HRP conjugate, 0.5 μg/m£ of an anti-mouse IgA-biotin conjugate, diluted in PBST, was added to each well and left to stand at room temperature for 1 hour. After each well of the plate was washed, 50 pg/m£ of avidin-peroxidase was added to each well and left to stand at room temperature for 1 hour, and a color development reaction was performed.
As a result, as shown in FIG. 4, the ELISA absorbance for anti-VPl IgG was as high as about 1.336 in the group treated with the VPl , but was 0.015 in the control group. However, the ELISA absorbance for anti-VPl IgA in the group treated with the VPl was very low, which is similar to that for the control group. Accordingly, it can be seen that, when the VPl is administered intraperitoneally several times together with the Freund's adjuvant, IgG-like specific antibodies to VPl are produced in large amounts in serum, thus increasing immunity.
Also, the analysis of anti-VPl IgA in the small intestines was performed in the same manner as in the analysis of anti-VP 1 IgA, except that the above-obtained small intestine extract, diluted 200-fold in 10% FBS-containing PBST, was used instead of serum as the test sample. As a result, as shown in FIG. 5, the absorbance at 450 nm was 0.322 in the group administered with the VPl, but was 0.045 in the control group.
Meanwhile, the analysis of anti-VPl IgG in the small intestine was performed.
12 As a result, the absorbance for anti-VPl IgG in the test group was very low without a significant difference from the control group. Accordingly, it can be seen that, when VPl is administered orally several times together with CT, a specific antibody to VPl is produced in the small intestine. This suggests that the recombinant protein VPl has a good immunity induction, when it is administered orally.
Example 5: Measurement of effect of recombinant VPl on T-cell proliferation activity
In order to examine the effect of the recombinant protein VPl, prepared in Example 3, on T-cell proliferation activity, an MTT assay was performed. Specifically, the spleen of the BALB/c mouse, in which an immune response has been induced by the oral administration of VPl, was extracted and separated into cells. 50 μi of the cells were placed in each of a 96-well plate at a cell density of I x IO7 cells/ mi and cultured in a CO2 incubator at 37 °C in the presence of a suitable concentration of antigen. In the culture, an RPMI medium, containing 10% FBS, mercaptoethanol and an antibiotic, was used. After 3 days of the culture, 5 mgM of MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diρhenyltetrazolium bromide) solution was added to the well plate in an amount of 20 μi per each well and left to stand for 4 hours. Then, 100 μi of 0.01 M HCl containing 10% SDS was added to each well of the plate and incubated overnight, and then the absorbance at 595 nm was measured.
As a result, as shown in FIG. 6, in the case where the VPl -containing medium was added to the T-cells obtained from the mice administered orally with VPl and CT, the absorbance was about 1.74-fold higher than that in the case where only the medium was added. Accordingly, it could be observed that, due to the oral administration of VPl, the IgA-specific antibody was produced in the small intestines, and in addition, antigen stimulation at the cell level occurred.
13 Meanwhile, in the case where the VP 1 -containing medium was added to the T- cells obtained from the mice administered orally with PBS and CT, the absorbance was about 1.29-fold higher than that in the case where only the medium was added. On the other hand, in the case where OM (ovomucoid) was added as a control group, the absorbance was almost equal to the case where only the medium was added. That is, when VPl was brought into contact with the spleen cells which did not undergo the antigen stimulation with VPl, significant cell proliferation appeared, unlike the case where the cells were treated with OM. This suggests that VPl has mitogen activity.
Putting together the above results relating to the immuno genicity of VP 1 , it can be seen that the recombinant hepatitis A virus VPl according to the present invention induces the production of a specific antibody in serum when it is administered intraperitoneally, and in addition, it induces the production of a specific antibody in the small intestine when it is administered orally. This is believed to also be associated with the mitogen activity of VPl . Particularly, considering that the infection route of hepatitis A virus is the oral cavity, the IgA- specific antibody, which is produced in the small intestine upon the oral administration of VPl, will be useful to prevent hepatitis A virus from entering the body.
INDUSTRIAL APPLICABILITY
As described in detail above, according to the present invention, a recombinant hepatitis A virus antigen can be obtained by culturing Drosophila melanogaster S2 transformed with a recombinant vector containing hepatitis A virus antigen gene VPl or VP3 which cause hepatitis A in the human body. Also, it is possible to prepare a vaccine containing this virus antigen as an active ingredient. The present invention has advantages in that, in the production of hepatitis A
14 virus envelope proteins VPl and VP3, a complicated process such as baculovirus infection is not required, the envelop proteins are expressed at high levels, and the production of the proteins can be performed using a continuous process.
Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
15

Claims

THE CLAIMS
What is Claimed is:
L A method for preparing a recombinant hepatitis A virus antigen, the method comprising culturing Drosophila melanogaster S2 transformed with an insect cell expression vector, in which a gene of hepatitis A virus antigen selected from the group consisting of VPl-S of SEQ ID NO: 1, VPl-L of SEQ ID NO: 2, and VP3 of SEQ ID NO: 3, is inserted into an expression vector (pMT/BiP/V5-His) which contains a metallothionein promoter (PMT)> a BiP signal sequence (BiP SS), a V5 epitope (V5), polyhistidine (His6 tag) and restriction enzyme digestion sites and has a cleavage map of FIG. 1.
2. The method for preparing a hepatitis A virus antigen according to claim 1, wherein said restriction enzyme digestion sites of the recombinant vector include BgIU and Apal, and the gene of hepatitis A virus antigen is inserted between BgIlI and Apaϊ.
16
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012122858A1 (en) * 2011-03-17 2012-09-20 中国科学院上海巴斯德研究所 Method for producing virus-like particle by using drosophila cell and applications thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101012830B1 (en) * 2008-04-25 2011-02-10 메디칸(주) Novel Gene Coding Antigen of Hepatitis A Virus and Plants Transformed by the Gene

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002014478A2 (en) * 2000-08-16 2002-02-21 Apovia, Inc. IMMUNOGENIC HBc CHIMER PARTICLES HAVING ENHANCED STABILITY
KR20020068148A (en) * 2001-02-20 2002-08-27 동화약품공업주식회사 Divalent vaccine against hepatitis b and hepatitis d and a preparing method thereof
KR20020078708A (en) * 2001-04-10 2002-10-19 부광약품 주식회사 The Novel Nucleotide Seguence of Hepatitis A Virus from Korean Patients

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7118745B1 (en) 1999-09-27 2006-10-10 The Regents Of The University Of California Engineering antibodies that bind irreversibly

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002014478A2 (en) * 2000-08-16 2002-02-21 Apovia, Inc. IMMUNOGENIC HBc CHIMER PARTICLES HAVING ENHANCED STABILITY
KR20020068148A (en) * 2001-02-20 2002-08-27 동화약품공업주식회사 Divalent vaccine against hepatitis b and hepatitis d and a preparing method thereof
KR20020078708A (en) * 2001-04-10 2002-10-19 부광약품 주식회사 The Novel Nucleotide Seguence of Hepatitis A Virus from Korean Patients

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HARMON S.A. ET AL.: "Expression of hepatitis A virus capsid sequences in insect cells", VIRUS RES., vol. 10, no. 2-3, May 1988 (1988-05-01), pages 273 - 280, XP023706630, DOI: doi:10.1016/0168-1702(88)90022-6 *
JEON H.K. ET AL.: "Functional expression of recombinant tumstatin in stably transformed by Drosophila melanogaster S2 cells", D5: BIOTECHNOLOGY LETTERS, vol. 25, no. 3, February 2003 (2003-02-01), pages 185 - 189 *
KARAYIANNIS P. ET AL.: "Hepatitis vaccines", BRITISH MEDICAL BULLETIN, vol. 70, no. 1, 2004, pages 29 - 49, XP008045299, DOI: doi:10.1093/bmb/ldh024 *
PARK J.H. ET AL.: "Production of recombinant endostatin from stably transformed Drosophila melanogaster S2 cells", BIOTECHNOLOGY LETTERS, vol. 21, no. 9, September 1999 (1999-09-01), pages 729 - 733 *
STANIC A.K. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 100, no. 4, 18 February 2003 (2003-02-18), pages 1849 - 1854 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012122858A1 (en) * 2011-03-17 2012-09-20 中国科学院上海巴斯德研究所 Method for producing virus-like particle by using drosophila cell and applications thereof

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