KR100455902B1 - Surface protein preS1 peptide of HBV inducing immune activity of T-lymphocytes - Google Patents

Abstract

The present invention relates to a novel peptide derived from prees-1 (preS1) of the hepatitis B virus surface protein, and more particularly, to a peptide having an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 9 . The peptide of the present invention can be usefully used as an effective hepatitis B vaccine because of its high adhesion to human MHC II molecules and its high ability to induce T lymphocytes.

Description

Surface protein preS1 peptide of HBV inducing immune activity of T-lymphocytes

The present invention relates to a novel peptide derived from prees-1 (preS1) of the hepatitis B virus surface protein, and more particularly, to a peptide having an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 9 .

Hepatitis B virus (abbreviated as “HBV”) is an encapsulated DNA virus that affects about 300 million people worldwide. HBV is a major pathogen of hepatitis, which invades humans and causes acute or chronic hepatitis, which can worsen cirrhosis or liver cancer (Tiollais, P. and MA Buenda, Sci. Am ., 1991 , 264, 48-). 54).

This HBV coating consists of three surface antigen proteins, specifically, a major protein containing S antigen, a middle protein including S antigen and preS2 antigen, and an S antigen, preS2 antigen and preS1 antigen. Consisting of large proteins (Neurath, AR and SBH Kent, Adv. Vir. Res ., 1988 , 34, 65-142). All three surface antigens have epitopes that can neutralize the infectivity of the virus, especially the sites where epitopes and hepatitis B viruses bind to hepatocyte receptors, which have 56 amino acid actions at the amino terminus of the preS antigen. Are reported to exist together (Iwarson, S., et al., J. Med. Virol ., 1985 , 16, 89-96; Itoh, Y., et al., PNAS , 1986 , 84, 9174-). 9178; Neurath, AR, et al., Vaccine , 1989 , 7, 234-236; Neurath, AR, et al., Cell , 1986 , 46, 436-439; Pontisso, P., et al., Virology , 1989 , 173, 522-530; Petit, MA, et al., Mol. Immunol ., 1989 , 28, 517-530).

Until now, vaccines that prevent HBV infection mainly used virus antigens purely isolated and purified from human plasma, or antigens recombined by genetic engineering methods. However, most of them contain only the S antigen protein of HBV, so they respond to S antigens. For those who do not have difficulty using.

In addition to neutralizing HBV and inducing antibodies, HBV preS antigen protein is involved in virus clearance and recovery from acute HBV infection. Therefore, when preS antigen protein is used as a vaccine against HBV, It can be useful for overcoming non-responsiveness. Therefore, it is desirable to develop an HBV vaccine that generates an immune response not only to the S antigen protein but also to the preS antigen protein by linking the preS antigen site before the S antigen protein by genetic engineering method.

Until now, several fusion proteins including preS1 or preS2 antigen sites have been developed as vaccines using preS antigen proteins, but the studies have been insufficient in the actual expression and purification of preS antigen proteins (Lin, Y., et al. , J. Med. Virol ., 1991 , 33, 181-187; Kim, HS and Hong, HJ, Biotech. Lett ., 1995 , 17, 871-876; Rhyum, SB, et al., Biotech ., 1994 , 36, 221-230; Kumar, V., et al., Gene , 1992 , 110, 137-144).

In order to develop a vaccine against hepatitis B virus having excellent immunogenicity, the present inventors have a motif that can be well attached to the binding groove of HLA (Human Leukocyte Antigen) -DR in the sequence of the preS antigen protein. by synthesizing peptide sequences with (motif) and revealing that the synthesized peptides are well attached to MHC (major histocompatibility complex) -II type molecules and at the same time can significantly promote the differentiation of T lymphocytes essential for neutralizing antibody formation. The present invention has been completed.

An object of the present invention is to adhere to MHC (major histocompatibility complex) -II type molecules and at the same time pres-1 (preS1) of hepatitis B virus surface protein which can significantly promote the differentiation of T lymphocytes essential for neutralizing antibody formation. It is to provide a novel peptide derived.

1 is a graph showing the results of measurement using flow cytometry (FACS) that the expression of HLA-DR4 is increased in L929 cell line transformed with HLA-DR4,

Figure 2 is a graph showing the results of measuring the adhesion activity to the HLA-DR molecules HLA-DR4 expressing HLA-DR4 using FACS,

FIG. 3 is a graph showing the degree of T lymphocyte differentiation promotion of each candidate peptide derived from the preS portion of HBV to lymphocytes extracted from blood of a hepatitis B patient.

1: PHA (phytohemagglutin), 2: control, 3: HA (hemagglutinin A) 308-318,

4: PreS1 82-92, 5: PreS1 82-92Y83, 6: PreS1 119-129,

7: PreS1 76-86, 8: PreS2 121-131, 9: PreS2 133-140,

10: PreS2 150-160, 11: PreS1 23-33, 12: PreS1 62-72

□: 3 µg / ml treatment ▒: 30 µg / ml treatment

In order to achieve the above object, the present invention provides a peptide having an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 9 .

Preferably, the peptide of the present invention may be acetylated, Fmoc, biotinylated amino group of the amino terminal, and the carboxyl group of the carboxyl terminal may be amidated.

The present inventors synthesized peptides described in SEQ ID NO: 1 to SEQ ID NO: 9 derived from the preS site of HBV by various kinds of small amount synthesis methods, and compared to SEQ ID NO: 10 and sequence derived from hemagglutinin A The peptide set forth in No. 11 was synthesized (see Table 1 ).

In addition, the present inventors constructed a cell line expressing HLA-DR at all times through transformation and analyzed the expression pattern of HLA-DR using flow cytometry, and as a result, compared to the control cell line in the transformed cell line. It was confirmed to express significant HLA-DR (see FIG. 1 ).

In addition, the inventors of the present invention use agretope to which peptides are attached to HLA-DR molecules using the synthesized peptides of the present invention described in SEQ ID NOS: 1 to SEQ ID NO: 11 and a transgenic cell line expressing HLA-DR. Activity was measured using FACS. As a result, the 23-33 peptide of preS1 described in SEQ ID NO: 1 and the 62-72 peptide of preS1 described in SEQ ID NO: 2 showed markedly increased attachment activity (see FIG. 2 ). In particular, it showed significantly increased adhesion than hemagglutinin 307-319 described in SEQ ID NO: 10 , which is reported to adhere well to HLA-DR.

In addition, the present inventors investigated the degree of differentiation of T lymphocytes by treating candidate lymphocytes derived from the preS portion of HBV to lymphocytes extracted from blood of hepatitis B patients exposed to hepatitis B virus. As a result, 23-33 peptides of preS1 and 62-72 peptides of preS1 described in SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention showed an activity that promotes T lymphocyte differentiation by an average of 5 times or more compared to the control group (see FIG. 3 ). ). In particular, it was confirmed that it exhibited more than twice the activity of HA (307-319) described in SEQ ID NO: 10 , which is already known as an epitope that promotes the differentiation of T lymphocytes.

From the above results, it was confirmed that the peptide of the present invention can be usefully used as an efficient hepatitis B vaccine because of its high adhesion ability to human MHCII molecules and high immune inducing ability of T lymphocytes.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples.

Example 1 Synthesis of Peptide Derived from the preS Portion

The present inventors synthesized various peptides derived from the preS site of HBV by various kinds of small amount synthesis methods, and synthesized peptides derived from hemagglutinin as a comparative group. Specifically, peptides were synthesized by a solid phase method using Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group of N _α -amino acid. The extension reaction of the peptide was carried out according to the N-hydroxybenzotriazole-dicyclohexylcarbodi-imide, ie HOBt-DCC method. After the synthesis was completed to the N terminal amino acid and washed several times with N-methylpyrrolidone and dichloromethane (dichloromethane) and dried with nitrogen gas. Trifluoroacetic acid: phenol: thianisole: water: triisoacetic acid (phenol: thioanisole: water: triisopropylsilane) in a 85: 5: 5: 3: 2 v / v solution for 2-3 hours The reaction was removed to remove the protecting group, the peptide was separated from the resin, and the peptide was precipitated with diethylether. The crude peptide thus obtained was acetonitrile gradient containing 0.05% trifluoroacetic acid and purified reverse phase high performance liquid chromatography column, Delta Pak, C ₁₈ 300 Å, Peptides were synthesized by purification using 1.9 × 30 cm, Waters).

As a result, as shown in Table 1 , the peptides described in SEQ ID NO: 1 to SEQ ID NO: 11 were synthesized.

Name of Peptide order preS1 (23-33) SEQ ID NO: 1 preS1 (62-72) SEQ ID NO: 2 preS1 (76-86) SEQ ID NO: 3 preS1 (82-92) SEQ ID NO: 4 preS1 (82-92) SEQ ID NO: 5 preS1 (119-129) SEQ ID NO: 6 preS2 (121-131) SEQ ID NO: 7 preS2 (139-149) SEQ ID NO: 8 preS2 (150-160) SEQ ID NO: 9 HA (307-319) SEQ ID NO: 10 HA (23-33) SEQ ID NO: 11

The present inventors used the following experiment after purifying the peptide synthesized above by 95% or more using high-performance liquid chromatography (HPLC).

Example 2 Preparation of Modified Peptides (Biotinylated Peptides)

20% piperidine / N-methylpyrrolidone solution was added to the peptide coupled to the N-terminal amino acid by the above method to remove the Fmoc group, and N-methylpyrrolidone and After washing with dichloromethane (NH) -Biotin was added to the biotin was coupled. After the synthesis, the mixture was washed several times with N-methylpyrrolidone and dichloromethane and dried with nitrogen gas. Trifluoroacetic acid: phenol: thioanisole: water: triisoacetic acid (phenol: thiothiosole: water: triisopropylsilane) in a 85: 5: 5: 3: 2 v / v solution for 2-3 hours The protecting group was removed, the peptide was separated from the resin, and the peptide was precipitated with diethylether. The crude peptide thus obtained was synthesized by acetonitrile gradient containing 0.05% trifluoroacetic acid and purified using a purified reverse phase high performance liquid chromatography column to synthesize biotinylated peptides.

Experimental Example 1 Expression of HLA-DR in a Cell Line Transformed with HLA-DR

The present inventors cloned HLA-DRA1 * 0101 and HLA-DRB1 * 0405 into pCDNA3.1 / Neo and pCDNA3.1 / Hygro (Invitrogen) to transform L929 cell line to construct a cell line expressing HLA-DR at all times. Expression of HLA-DR was analyzed using flow cytometry. Since the L929 cell line does not generally express class II molecules, all class II molecules that interact with a given peptide can be considered to be transformed HLA-DR4 molecules. PCDNA3.1 from Invitrogen using the CMV promoter was used as a transformation vector, and the vector containing the whole cDNA of DRB1 * 0405 (pUB) and DRA0101 (pUA) was treated with EcoRI and HindIII, respectively. Only DNA was purified on 1.2% TAE-agarose gel and then cloned into pcDNA 3.1 vector. The vectors thus prepared were named pcDNA-DRB and pcDNA-DRA, respectively, and the vectors were purified with high purity using Qiagen's plasmid preparation kit for transformation. Transformation was performed using L929 cells in log growth phase. Introduction of the plasmid into the cells was performed using LipofectAMINE (Life Technology Inc., Gaithersburg, MD). To do this, L929 cells were mixed well with 1 μg of plasmid DNA in 100 μl DMEM medium at 1 × 10 ⁵ cells / ml, and 6 μl of PLUS reagent (Life Technology Inc.) was added thereto and left at room temperature for 15 minutes. Meanwhile, 1 μl of LipofectAMINE was mixed well in 100 μl DMEM, and after 15 minutes, the PLUS / DNA solution was mixed with LipofectAMINE and left for another 15 minutes. Transformation was performed by mixing the DNA / LipofectAMINE mixture (200 μl) well in each well and then standing at 37 ° C. for 3 hours. Selection for neomycin (neomycin G418) was performed to prepare transformants, cell lines in which the HLA-DR4 protein is still expressed. Since MHC-class II molecules can form stable dimers at the cell surface only when both α and β chains are expressed simultaneously, double-transfection was used to introduce both plasmids in one transformation. . For successful expression, G418 was selected by adding 500 μg / ml to the cell culture medium. After 3 weeks, surviving transformants were limited to dilution by single colony to class II molecules with high yield. Expressing clones were selected by the MACS method. Selected cell line clones were identified by the flow cytometry using RT-PCR method and DR2 specific fluorescent antibody L243 (DAKO Corp.) to confirm the expression of HLA-DR4 on the cell surface.

As a result, it was confirmed that the expression of HLA-DR is significantly higher in the transformed cell line than the control L929 cell line ( FIG. 1 ).

Experimental Example 2 Measurement of Adhesion Ability to HLA-DR of Peptides Derived from preS Part

The present inventors FACS agritope (agretope) activity that peptides are attached to the HLA-DR molecule using the peptide of the present invention synthesized in Example 1 and L929 cell line expressing HLA-DRB1 ^* 0405 DRA1 ^* 0101 Measured using. Specifically, in order to verify that the T-cell epitope site in the vaccine candidate antigen peptide binds with the actual HLA-DR4, a biotylated antigen using a technique of biotilizing the N-terminus during chemical synthesis of the peptide Peptides were prepared. The biotylated peptide was dissolved in HBSS, and cultured with L929 cell line transformed with HLA-DR4 or cell line expressing HLA-DR4, respectively, to measure the binding activity. Specifically, peptide antigens are prepared at concentrations of 0.001 mg / ml, 0.01 mg / ml, and 0.1 mg / ml, and the cells are suspended in RPMI-1640 medium containing 0.1% sodium azide. The mixture was incubated for 1 hour at 37 ℃. Cells were then washed with ice-cold HBSS and incubated at 4 ° C. for 30 minutes with streptoavidin bound to FITC. Specific peptide / MHC attachment reactions were analyzed using flow cytometer. To this end, cells incubated with peptide were suspended in 250 μl PBS to analyze cells with FACScalibur (Becton Dickinson, Mountain View, CA), a flow cytometer, and final statistics were obtained using software (Cell Quest).

As a result, the 23-33 peptide of preS1 described in SEQ ID NO: 1 and the 62-72 peptide of preS1 described in SEQ ID NO: 2 showed markedly increased attachment activity ( FIG. 2 ). In particular, it showed significantly increased adhesion than hemagglutinin 307-319 described in SEQ ID NO: 10 , which is reported to adhere well to HLA-DR.

Experimental Example 3 Promoting Differentiation of T Lymphocytes by Peptides Derived from the preS Portion

The present inventors investigated the degree of differentiation of T lymphocytes by treating candidate lymphocytes derived from the preS portion of HBV to lymphocytes extracted from blood of hepatitis B patients exposed to hepatitis B virus. Specifically, PBMC (peripheral blood monocyte cells) were isolated from the blood of hepatitis B patients after treatment with histopac (Histopaque 1077, Sigma), a density gradient solution. Genomic DNA was isolated from the isolated PBMCs and HLA typing was performed using the DR SSP kit (Dynal Corp) to select only the DR4 type and then plated at 96-well microplates at PBMC 5 × 10 ⁶ cells / ml. The preS peptide synthesized on the plate was treated with 3 μg / ml, 10 μg / ml, 30 μg / ml, and cultured for 4 days, followed by 16 hours with 1 μCi of [ ³ H] -methyl thymidine (Dupont). Was treated and measured by liquid scintillation spectroscopy (Beckman Coulter). The ability to promote lymphocyte differentiation by peptide was expressed by SI (Stimulation Index), which is the ratio of treated group to untreated peptide.

As a result, the 23-33 peptides of preS1 and 62-72 peptides of preS1 described in SEQ ID NO: 1 and SEQ ID NO: 2 , respectively, had activity of promoting T lymphocyte differentiation by an average of 5 times or more at concentrations of 3 µg / ml and 30 µg / ml, respectively. ( FIG. 3 ). In particular, it was confirmed that the antibody exhibits more than twice the activity of the HA (307-319) peptide described in SEQ ID NO: 10 , which is known as an epitope that promotes the differentiation of T lymphocytes.

As described above, the peptide derived from the preS portion of the HBV of the present invention may be useful as an effective hepatitis B vaccine because of its high adhesion ability to human MHCII molecules and high T lymphocyte immune induction ability.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Surface protein preS1 peptide of HBV inducing immune activity of T-lymphocytes <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 11 <212> PRT <213> Hepatitis B virus <400> 1 Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala 1 5 10 <210> 2 <211> 11 <212> PRT <213> Hepatitis B virus <400> 2 Ala Phe Gly Pro Gly Phe Thr Pro Pro His Gly 1 5 10 <210> 3 <211> 11 <212> PRT <213> Hepatitis B virus <400> 3 Gly Trp Ser Pro Gln Ala Gln Gly Val Leu Thr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Hepatitis B virus <400> 4 Gln Gly Val Leu Thr Thr Val Pro Val Ala Pro 1 5 10 <210> 5 <211> 11 <212> PRT <213> Hepatitis B virus <400> 5 Gln Gly Val Leu Thr Thr Val Pro Val Ala Pro 1 5 10 <210> 6 <211> 11 <212> PRT <213> Hepatitis B virus <400> 6 Ala Met Gln Trp Asn Ser Thr Thr Phe His Gln 1 5 10 <210> 7 <211> 11 <212> PRT < 213> Hepatitis B virus <400> 7 Gln Trp Asn Ser Thr Thr Phe His Gln Ala Leu 1 5 10 <210> 8 <211> 11 <212> PRT <213> Hepatitis B virus <400> 8 Leu Tyr Phe Pro Ala Gly Gly Ser Ser Ser Gly 1 5 10 <210> 9 <211> 11 <212> PRT <213> Hepatitis B virus <400> 9 Thr Val Asn Pro Val Pro Thr Thr Ala Ser Pro 1 5 10 <210> 10 < 211> 13 <212> PRT <213> Hepatitis B virus <400> 10 Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr 1 5 10 <210> 11 <211> 11 <212> PRT <213> Hepatitis B virus <400> 11 Gly Thr Leu Val Lys Thr Ile Thr Asp Asp Gln 1 5 10

Claims (4)

An epitope of hepatitis B virus pris-1 antigen represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 9 . A vaccine composition for preventing or treating hepatitis B virus, comprising the epitope of claim 1 as an active ingredient. The epitope or vaccine composition according to claim 1 or 2, wherein the amino group at the amino terminus of the epitope is acetylated, Fmocated, or biotinylated. The epitope or vaccine composition according to claim 1 or 2, wherein the carboxyl group at the carboxyl terminus of the epitope is amidated.