Patent Application
20080063643
The peptide derived from hepatitis C virus of the invention is an HCV peptide, which contains the HLA-binding motif in its sequence and is recognized by an antibody present in the blood of a patient infected with hepatitis C virus.
In addition, the HCV peptide according to the invention contains an HLA-binding motif, i.e., an HLA-A2- or HLA-A24-binding motif in its sequence. This HCV peptide is also characterized by having a high immunogenicity as well as being recognized by cellular and humoral immunity.
Examples of the HCV peptide include those peptides listed in Tables 1 and 3. One of the particularly preferred HCV peptides of the invention is a peptide having the HLA-A2-binding motif with the following amino acid sequence:
IgG antibody reactive with this peptide is detected with significant proportion in patients infected with HCV with the detection ratio of 93% and the specificity for HCV infection of 100%. Further, particularly preferred HCV peptides of the invention are peptides having the HLA-A24-binding motif with any of the following amino acid sequences:
The HCV peptide according to the invention may be a peptide having a homology of at least 70%, preferably 80% or more, more preferably 90% or more, with the amino acid sequence of any of the peptides listed above.
The HCV peptide according to the invention may further comprise a peptide having a property of being recognized by an HLA-A2- or HLA-A24-restricted cytotoxic T cell (CTL). This type of antigenic peptide capable of binding to HLA is generated by intracellular degradation of an antigenic protein produced in a cell, and has a sequence motif for each HLA type. A cytotoxic T cell (CTL) recognizes a complex of the antigenic peptide and HLA and causes damage in an HCV-infected cell.
The polypeptide according to the invention comprises in its sequence the amino acid sequence of the peptide of the invention as described above, but the total number of amino acids is not particularly limited. In addition, in the polypeptide of the invention, the amino acid sequence other than that of the peptide are located at the N-terminal side and/or the C-terminal side or both sides of the amino acids of the peptide. Therefore, such a polypeptide has substantially the same function and effect as those of the above-mentioned peptide. When the term “peptide” is used herein, it should be construed that “polypeptide” is also included in the term unless otherwise specified.
The peptide having an amino acid sequence as described above can be obtained by a standard chemical synthesis, by an enzymatic degradation of a protein molecule, or by a recombinant DNA technique using a host transformed so as to express a nucleotide sequence encoding a desired amino acid sequence.
In the case where the desired peptide is produced by a chemical synthetic method, it can be produced by a technique which is well known per se and is commonly used in standard peptide chemistry. For example, the peptide can be synthesized by a solid phase synthetic method with a peptide synthesizer. A crude peptide obtained in this way can be purified by any of the purification method commonly used in protein chemistry, such as salting out, ultrafiltration, reverse-phase chromatography, ion-exchange chromatography and affinity chromatography.
In the case where the desired peptide is produced by a recombinant DNA technique, the desired peptide can be obtained by, for example, integrating a DNA fragment encoding a desired amino acid sequence synthesized as described above into an appropriate expression vector, transforming a microorganism or an animal cell using this expression vector and then culturing the thus obtained transformant. The expression vector that may be used in the invention includes a plasmid, a viral vector, etc.; which are well known in the art.
To transform a host cell with an expression vector in the peptide-producing technique described above, any of well known methods may be appropriately selected and used, such as a calcium chloride method, a calcium phosphate co-precipitation method, a DEAE-dextran method, a Lipofectin method or an electroporation method. The peptide can be purified by any of the above-mentioned purification methods from a cell extract or a culture supernatant collected from the culture medium.
A nucleotide, which is one of the other aspects of the invention, includes an oligonucleotide or a polynucleotide encoding the above-mentioned peptide derived from hepatitis C virus or the above-mentioned polypeptide. The nucleotide may include a ribonucleotide and a deoxynucleotide. The nucleotide may be modified by a method known in the art. Examples of the modification of the nucleotide include labeling, methylation, “caps”, substitution of one or more of naturally occurring nucleotides with an analog, and intranucleotide modification known in the art. Examples of the intranucleotide modification include nonionic modification (e.g., a methyl phosphonate, a phosphotriester, a phosphoramidate, etc.), ionic modification (e.g., a phosphorothioate, a phosphorodithioate etc.), modification of incorporating a pendant moiety such as a protein (e.g., a nuclease, a toxin, an antibody, a signal peptide etc.), modification with a chelating agent (e.g., a metal, boron etc.).
The above-mentioned nucleotide sequence allows for provision of a peptide or a polypeptide sequence of an HCV antigen encoded by the genome of HCV, as well as a peptide useful for a diagnostic test or as a component of a vaccine.
Once the nucleotide of the invention is obtained, it becomes possible to construct a nucleotide probe and a peptide useful for diagnosing a disease associated with HCV infection or for screening HCV infection in the blood or a blood preparation. Based on this nucleotide sequence, a DNA oligomer having, for example, 24 to 30 nucleotides or even more may be synthesized. The nucleotide can also be used as a probe for detecting HCV RNA in the serum of a subject or for screening the presence of HCV in the blood or blood products.
Furthermore, the nucleotide sequence allows for design and production of an HCV-specific peptide useful as a reagent for testing the presence of an antibody against HCV. An antibody raised against a purified peptide having this sequence may be used for detecting an HCV antigen in a subject infected with HCV and in blood products which might have been prepared from the blood of a person infected with HCV.
In another aspects of the invention, the antibody includes a chimeric antibody, a modified antibody, a monovalent antibody, Fab, F(ab′)2, Fab′, a single chain Fv (scFV) protein and a single domain antibody, which are reactive to the peptide derived from hepatitis C virus or the polypeptide. These antibodies can immunologically recognize the peptide or the polypeptide.
In another aspects of the invention, a vector is a construct comprising the nucleotide and can transform a selected host cell to express heterologous coding sequences in the host. The expression vector may be either a cloning vector or an integration-type vector.
The cloning vector may include, for example, a plasmid and a virus (such as an adenovirus vector). The cloning vector is a replicon that can transform a host cell and has an ability to replicate in the cell (e.g., a plasmid, a chromosome, a virus, a cosmid and the like).
An integration vector is a vector that does not serve as a replicon in a host cell, but has an ability to integrate its contents into a replicon (typically a chromosome) in the host to stably transform the host.
Another aspect of the invention provides a method of inducing a cytotoxic T cell targeted to an HCV-infected cell using the peptide. The induction method of the invention may comprise, for example, contacting the peptide with a cell expressing HLA-A2 to present the peptide on the HLA-A2 molecule, stimulating a T cell with the cell presenting the peptide with HLA-A2, and inducing the T cell into CTL. The HLA-A2-expressing cell to be used in this method may be collected from the blood of an HCV patient, however, it can also be prepared by introducing a gene encoding HLA-A2 into a cell that does not express HLA-A2. Thus, the peptide according to the invention is useful for detection and diagnosis of HCV infection and is also useful as a pharmaceutical composition, such as a vaccine for the treatment of a disease associated with hepatitis C virus.
CTL as induced in this way will target and attack a cell infected with HCV, since a CTL may be also as a pharmaceutical composition for a cell therapy.
In another aspect of the invention, the peptide derived from hepatitis C virus, polypeptide, nucleotide and/or antibody or substance with an antibody-like activity is useful for detecting or diagnosing HCV infection.
The detection of HCV and the diagnosis of a disease associated with HCV can be carried out by, for example, detecting the presence of the peptide, detecting the presence or amount of the corresponding nucleotide sequence, determining the biological distribution of the peptide in an individual and/or determining the amount of the peptide present in a specimen derived from an individual. Such a method may utilize the interaction and/or reactivity with the nucleotide sequence encoding the peptide. In other words, the detection of HCV and the diagnosis of a disease associated with HCV may be effected by assaying the peptide as a marker. The assay can be carried out by a well known method by, for example, an antigen-antibody reaction system, an enzymatic reaction system, and a PCR reaction system.
Monitoring of the effect of the treatment and the prediction the prognosis of a disease associated with HCV can be carried out by monitoring the concentration of an antibody reactive to the peptide in the blood of a subject having a disease associated with HCV. It is particularly preferred to measure the level of anti-C-35 IgG or anti-NS5A-2132 IgG in the blood of a subject.
In still another aspect of the invention, the pharmaceutical composition includes a vaccine. The vaccine comprises the peptide derived from hepatitis c virus, the polypeptide and/or the nucleotide, and may suitably contain a pharmaceutically acceptable adjuvant and/or carrier. An adjuvant can enhance the immune response, and may include incomplete Freund's adjuvant or aluminum hydroxide gel. The carrier may include, for example, a diluent such as PBS or distilled water, physiological saline.
The pharmaceutical composition of the invention can be administered orally, parenterally or transdermally depending on the administration form, for example, via intravenous administration or subcutaneous administration. Examples of the dosage form include tablets, granules, soft capsules, hard capsules, liquids, oils, and emulsions. The dose of such a pharmaceutical composition can suitably selected depending on the symptoms of the patient to be treated. In general, the amount of the peptide may range from 0.1 to 10 mg per day for an adult, and the composition may be administered once every several days or several months.
The entire contents of all patents and reference documents explicitly cited in the present specification are incorporated herein by reference. In addition, the entire contents described in the specification and drawings of Japanese Patent Application No. 2003-330258 to which this application claims priority are also incorporated herein by reference.
The invention will be described in more detail with reference to Examples below. These Examples are provided to illustrate the invention more specifically, but does not limit the scope of the invention.
The abbreviations for amino acids used in the following Examples are as follows.
A denotes alanine, C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; P, proline; R, arginine; S, serine; T, threonine; V, valine; and Y, tyrosine.
Synthetic peptides having an HLA-A2-binding motif or an HLA-A24-binding motif from a conserved region of HCV genotype 1b protein were used in this study (Table 1). A peptide derived from HIV having an HLA-A2-binding motif was used as a negative control. All of these peptides were purchased from a commercially supplier. The purity was analyzed by reverse-phase high-pressure liquid chromatography and found to be 90% or higher.
The level of the peptide-specific IgG in the serum was measured by ELISA. First, each peptide was dissolved in dimethyl sulfoxide (DMSO) and stored at −80° C. The peptide was diluted with 0.1 M sodium carbonate/sodium bicarbonate solution containing disuccinimidyl suberate (DSS) as a crosslinker. An ELISA plate was coated with 20 μg/well of peptide at 4° C. overnight. The wells were washed 3 times with 0.05% Tween 20/PBS (PBST), and the plate was blocked with Block Ace (registered trademark) and left overnight at 4° C. Plasma or serum samples were diluted in 1:100, 1:200 and 1:400 with 0.05% Tween 20/Block Ace, and 100 μl of each of the samples was added to each well. After the samples were incubated at 37° C. for 2 hours, the plate was washed 9 times with PBST, 100 μl of a 1:1000 diluted rabbit anti-human IgG (γ-chain specific) was added to each well, and incubated at 37° C. for 2 hours. The plate was washed 9 times with PBST, and 100 μl of 1:100 diluted mouse anti-rabbit IgG conjugated to a horseradish peroxidase-dextran polymer was added to each well, and then the plate was incubated at room temperature for 40 minutes. After the plate was washed, 100 μl of a tetramethylbenzidine substrate solution was added to each well. The reaction was stopped by the addition of 2.0 M phosphoric acid. The cutoff value was calculated as the mean±3 SD of the optical density values of the control obtained from the healthy donors.
One hundred microliters of a plasma or serum sample diluted with 0.05% Tween 20/Block Ace (registered trademark) was added to each well and allowed to be absorbed to the peptide (20 μg/well) coated on the well of the plate at 37° C. for 2 hours. After this procedure was repeated 3 times, the peptide-specific IgG level in the supernatant was measured by ELISA. The antibody bound to the peptide-coated plate in the initial adsorption step was eluted with 30 μl/well of 5 M NaCl/50 mM citrate buffer (pH 3.0) (neutralized with 0.05% Tween 20/Block Ace containing 1.0 M Tris-HCl (pH 7.5)), and the peptide-specific IgG level in the eluted fraction was measured by ELISA.
A peptide-specific CTL was detected by a method commonly used in this technical field. PBMCs were added to each well of a round-bottomed 96-well microculture plate at 1×105 cells per well, and the cells were incubated in 200 μl of a medium with 10 μM peptide. The composition of this medium is as follows: 45% RPMI-1640, 45% AIM-V (registered trademark), 10% fetal calf serum (FCS), 100 U/ml interleukin 2 (IL-2), and 1 mM MEM non-essential amino acid solution. On days 3, 6 and 9 of culture, one half of each culture solution was removed and replaced with a fresh medium containing a corresponding peptide (20 μg/ml). On day 12 of culture, the cultured cells were collected and assayed for the interferon gamma (IFN-γ) production in response to CIR-A2402 cell or T2 previously pulsed with the corresponding peptide or an HIV peptide as a negative control. Four wells were used for each peptide and the assay was carried out twice. The background IFN-γ production in response to the HIV peptide was subtracted from the obtained data value. For the inhibition assay, a peptide-reactive CTL was purified using a CD8 isolation kit, and assayed for the peptide-specific IFN-γ production in the presence of 20 μg/ml of any of an anti-HLA class I (W6/32, IgG2a) monoclonal antibody, an anti-CD8 (Nu-Ts/c, IgG2a) monoclonal antibody and an anti-HLA class II (H-DR-1, IgG2a) monoclonal antibody. The background IFN-γ production was subtracted from the obtained data value.
The values are expressed as the mean±SD. A statistical analysis was carried out using the Mann-Whitney U-test and the chi-square test, and a P value less than 0.05 was considered statistically significant.
The level of IgG that reacts with each of the 62 types of peptides was measured in the sera from 12 patients infected with HCV and 10 healthy donors (HDs) (Table 1). The prevalence of the antibody against each peptide is shown in Table 1. A plurality of peptides were found that highly reacted with IgG in the blood from the patients infected with HCV and rarely react with the sera from the healthy donors.
The peptides include No. 3: NS5A-2132 (corresponding to the peptide at positions 2132-2140 of HCV NS5A protein, hereinafter abbreviated in a similar fashion), No. 11: NS3-1266, No. 12: E2-488, No. 13: E1-213, No. 22: NS2-947, No. 25: NS3-1081, No. 32: C-176, No. 38: C-173, No. 40: C-35, and No. 62: NS5B-2990. Representative results of measuring IgG level in the blood from the patients infected with HCV by the ELISA method are shown in
In the serum from the patient shown in
In the sera from 4 patients shown in
Among the antibodies listed in Table 1, the antibody reactive to C-35 peptide was detected at 100% in the patients infected with HCV and at 0% in the healthy donors, indicating that it has high specificity to HCV. The sequence of C-35 peptide of HCV has a partial homology with peptides of various viruses, such as tet protein (the amino acid sequence at positions 54-58) or env protein (amino acid sequences at positions 5-9, 158-162, and 717-727) of HIV-1. It also shows a homology with various proteins of HTLV-I (such as pol 724-755, rex 5-9, rex 310-313 and tax 70-74). Thus, the antibody reactive to C-35 peptide was further examined in a double-blind study of 156 cases in total, including 60 cases of diseases associated with HCV infection (22 cases of chronic hepatitis C, 20 cases of liver cirrhosis and 18 cases of liver cancer), 24 cases of subjects infected with hepatitis B virus, 10 cases of hepatitis B virus vaccine recipients, 27 cases of healthy donors, 22 cases of patients with an autoimmune disease, 10 cases of subjects infected with HTLV-I and 3 cases of subjects infected with HIV. The results are shown in Table 2. The HCV specificity and the detection rate of the antibody reactive to C-35 peptide were 88.5% and 93.3%, respectively.
Six types of HLA-A24 binding peptides (Nos. 3, 12, 13, 25, 32 and 38) and 18 types of HLA-A2 binding peptides (Nos. 40 to 57) were incubated with peripheral blood mononuclear cells (PBMCs) obtained from HLA-A24+ or HLA-A2+ HCV-infected patients (5 patients in each group). As a control, the PBMCs were incubated with an HIV peptide. Then, the incubated PBMCs were tested for their ability to produce IFN-γ in response to CIR-A2402 cell or T2 cell pulsed with a corresponding peptide. Six types of HLA-A24 binding peptides (Nos. 3, 12, 13, 25, 32 and 38) could induce CTL from the PBMC obtained from 10 HLA-A24+ HCV patients (
The cytotoxic activity of peptide-stimulated PBMC was measured by the 51Cr release assay. The representative results are shown in
These PBMCs showed a significantly higher level of cytotoxic activity against the C1R-A2402 cell and the T2 cell preloaded with the corresponding peptide (except for an HIV peptide as a negative control).
The HLA class I restriction of the cytotoxicity was tested by the inhibition assay using an anti-CD8 monoclonal antibody (
The detection of IgG reactive to the HCV peptide was carried out more extensively in HLA-A24+ HCV-infected patients. Sixty HCV-1b+ patients were found to be seropositive for an anti-HCV antibody (Ab) indicated by the second generation or the third generation immunoassay test. The genotype of HCV contained in the sera of the patients was determined by RT-PCR. The patients were diagnosed at the time of serum sampling by biochemical analysis, echography, computed tomography and histological finding. The results of diagnosis are as follows: chronic hepatitis (CH, n=24), liver cirrhosis (LC, n=18) and hepatic cell carcinoma (HCC, n=18). A negative control was serum samples from 20 healthy donors (HDs) who had normal hepatic function and no medical history of hepatitis virus.
Forty-four types of synthetic peptides that were derived from a highly conserved region of HCV-1b protein and selected based on a high binding score to HLA-A24 molecule (Bioinformatics and Molecular Analysis Section, NIH, Bethesda, Md.) were used. A peptide derived from HIV that has an HLA-A24 binding motif (RYLRDQQLLGI (SEQ ID NO: 63)) served as a negative control. The binding scores and sequences of the peptides are shown in Table 3. For the screening of the reactivity of peptide to serum IgG, desalted grade of the peptides (>70%) were purchased from BioSynthesis (Lewisville, Tex.) or SynPep (Dublin, Calif.). A purified peptide (>90%) was used in the subsequent experiments. The plasma level of peptide-specific IgG was measured by an enzyme-linked immunosorbent assay (ELISA). The specificity of anti-peptide IgG in a plasma sample was tested by the absorption and elution of peptide-reactive IgG.
Peptide-specific CTL precursor cells were detected by the method as described in Hida et al. (Cancer Immunol Immunother 2002; 51: 219-228). For an inhibition assay, 20 μg/ml of anti-HLA class I (W6/32, IgG2a), anti-CD8 (Nu-Ts/c, IgG2a), anti-CD4 (Nu-Th/i, IgG1b) and anti-HLA class II (H-DR-1, IgG) monoclonal antibodies were used.
The cDNAs of NS5A, HLA-A2402 and HLA-A2601 were obtained by RT-PCR from Huh7 (NNU-50-1) cell, C1R-A2402 cell and KE4-CTL cell, respectively, and cloned into an expression vector pCR3.1 (Invitrogen, San Diego, Calif.). The Huh7 (NNU50-1) cell line is a replicon cell derived from a cell line infected with HCV-1b and expresses proteins from NS3 to NS5B (Kishine et al., Biochem Biophys Res Commun 2002; 293: 993-999). Then, 100 ng of NS5A, HLA-A2402 or NS5A and HLA-A2601 cDNA (as a negative control) were mixed in 100 μl of Opti-MEM (Invitrogen) containing 0.6 μl of Fugene6 (Roche Molecular Biochemicals, Indianapolis, Ind.) and incubated for 30 minutes. Then, 100 μl of the mixture was added to COS-7 cells (1×104 cells) and incubated for 6 hours. One hundred microliters of RPMI-1640 medium containing 20% FCS was added to the mixture and COS-7 cells were cultured for 2 days, followed by the addition of NS5A 2132-2140-stimulated PBMCs (2×105 cells/well). After incubation for 18 hours, 100 μl of the supernatant was removed and IFN-γ concentration was assayed by ELISA in triplicate. In order to prepare a stable transfectant cell line, either pCR3.1-NS5A or pCR3.1-HLA-A3101 (negative control) was transfected to C1R-A2402 cell by electroporation using a Gene Pulser (BioRAD, Richmond, Calif.) These cells were cultured for 30 to 40 days in the presence of geneticin (G418), and then geneticin resistant cells were collected. The expression of NS5A protein in the transfectant was assayed by Western blot analysis using an anti-NS5 polyclonal antibody (Abcam Limited, Cambridge, UK).
Huh7 cell line was incubated in a well of a flat-bottomed 96-well microculture plate in the presence of various sera for 24, 48 and 72 hours. After the incubation, Huh7 cells were counted with a Cell Count kit-8 (10 μl/well) (Dojindo, Japan). For ADCC assay, PBMCs newly isolated from a HLA-A24+ HD were pre-incubated for 1.5 hours in 10% FCS and RPMI-1640 medium containing inactivated serum (56° C., 30 minutes) from any of various patients and HD (as a negative control) C1R-A2402 cells or C1R cells (as a negative control) were incubated for 2 hours with NS5A 2132-2140 peptide (10 μM) and radiolabeled with Na251CrO4 for 1.5 hours, washed and used as target cells. The peptide-stimulated PBMCs were incubated in a well of a round-bottomed 96-well microculture plate with the target cells at an effecter to the target cell (E/T) ratio of 40, 20 or 10 to 1. After incubation at 37° C. for 6 hours, cell-free supernatant was collected and the ADCC activity was assayed (Shomura, et al., Br J Cancer 2004; 90: 1563-1571).
A statistical analysis was carried out using Student's t-test and the Mann-Whitney U-test. Values of P<0.05 were considered statistically significant.
In order to screen for a humoral response to the peptides, the level of IgG reactive to each of the 44 types of peptides was assayed in the plasma of 12 patients infected with HCV-1b. The plasma of 10 HDs was used as a negative control. The level of peptide-reactive IgG was measured by ELISA for serially diluted plasma samples and reported by the values of absorbance (OD) of each sample. The cut-off value of these peptides at a plasma dilution of 1:100 was set at 0.18 OD (mean (0.08)+2SD (0.05×2) of 10 HDs). A significant level (>0.18 OD at a plasma dilution of 1:100) of IgG reactive to NS5A-2132, C-176, E2-488, E1-213, C-173 and NS3-1081 peptides were detectable in the plasma of 12, 11, 10, 9, 8 and 5 patients among the 12 patients, respectively (
a)The level of peptide-specific IgG in the sera of 12 HCV-1b+ patients was measured by ELISA. The cut-off value was determined to be 0.18.
b)The binging score of the peptide to HLA-A24 molecule was searched using Bioinformatics and Molecular Anaylsis Section, NIH, Bethesda, MD (http://bimas.dcrt.nih.gov/molbio/hla_bind/).
Each of the 6 types of the above peptides having a purity of 90% or more and the negative control peptide were tested for reactivity to each of the serum samples from 60 HCV-1b+ patients (CH, n=24; LC, n=18; and HCC, n=18) and 20 HDs (
The values for each of these samples at a dilution of 100:1 were plotted. The mean±SD of IgG (OD value) was as follows: NS5A-2132 (0.48±0.36), E2-488 (0.18±0.19), E1-213 (0.10±0.21), NS3-1081 (0.036±0.14) C-176 (0.09±0.14) and C-173 (0.04±0.13). The asterisks indicate P<0.05 in the Mann-Whitney U-test. The average level of IgG reactive to NS5A-2132, E2-488, E1-213 or C-173 was statistically significantly higher than that of HDs, however, it was not the case with NS3-1081 or C-176. IgG showing a significant level of reactivity (>0.101 OD at a serum dilution of 1:100) to NS5A-2132, E2-488, E1-213, C-173, NS3-1081 and C-176 peptides was detected in the plasma of 57 (95%) , 44 (73%) 28 (47%), 23 (38%), 21 (35%) and 17 (28%) patients of the 60 patients tested, respectively. On the contrary, a significant level of IgG could not be detected in any of the sera of 20 HDs (
The peptide specificity of IgG reactive to each of these peptides was examined by absorption and elution tests. Each serum sample was absorbed by either a corresponding peptide or an irrelevant peptide (HIV; used as a negative control) at 37° C. for 3 times and the level of peptide-specific IgG was measured by ELISA (
As expected, each IgG to the 6 types of peptides was all absorbed by the corresponding peptide, but not absorbed by the irrelevant peptide (HIV peptide). Furthermore, this IgG was eluted from a binging fraction by the corresponding peptide, but not eluted by the irrelevant peptide. Taken together, these results suggested that IgG reactive to each peptide be specific to the peptide derived from HCV-1b.
PBMCs from HLA-A24+ patients infected with HCV-1b (n=12, 6 CH patients, 3 LC patients, and 3 HCC patients) and HLA-A24+ HDs (n=5) without HCV infection were cultured with each of the 6 types of peptides (purity >90%) or a control HIV peptide for 13 days, and examined for IFN production in response to C1R-A2402 cell pulsed with a corresponding peptide. PBMCs from HLA-A24+ HCV-1b+ patients (n=12) and HDs (n=5) were stimulated with any of the 6 types of peptides in 4 wells (15×104/well). On day 14 of the culture, the peptide-stimulated PBMCs (80 to 120×104/well) were separately collected from each well and divided into quarters. Two portions of them were tested separately for the ability to produce IFN-γ in response to C1R-A2402 cells pulsed with the corresponding peptide. The remaining two portions were tested with cells using the negative control peptide (HIV). The level of IFN-γ produced in response to the HIV peptide (<50 pg/ml) was subtracted as background. The asterisks indicate p<0.05 in the two-sided Student's t-test.
HCV peptides of C-173, C-176, E1-213, E2-488, NS3-1081 and NS5A-2132 induced IFN-γ production at a significant level (p<0.05) in 1, 3, 4, 6, 2 and 7 of 12 patients (
The cytotoxicity of these peptide-stimulated PBMC was examined by a 6-hour 51Cr release assay (
The peptide-stimulated PBMC used in the experiment shown in
Cytotoxicity against C1R-A2402 cells pulsed with each of 3 types of peptides was inhibited by the anti-HLA-class I (W6/32) or CD8 monoclonal antibody (mAb), but not by any of the other mAbs tested, indicating that the peptide-specific cytotoxicity is mediated mainly by CD8+T cells in an HLA-class I restricted manner (
Then, it was examined whether NS5A-2132 peptide-stimulated PBMCs recognize a naturally processed peptide using COS-7 cell that was transiently co-transfected with NS5A and HLA-A2401 genes as a target cell. As a negative control, COS-7 cell that was transiently co-transfected with NS5A and HLA-A2601 genes was used. The NS5A-2132 peptide-stimulated PBMCs from patients #1 and #2 were tested for their ability to recognize COS-7 cell transiently co-transfected with NS5A and HLA-A2401 genes as a target cell to produce IFN-γ. As a negative control, COS-7 cell that was transiently co-transfected with NS5A and HLA-A2601 genes was used. The values are expressed as the mean±SD of percentage of IFN-γ production in triplicate assays at an E/T ratio of 20:1 (
As expected, the peptide-stimulated PBMCs from patients #1 and #2 showing a CTL activity (
Next, cytotoxicity was tested against C1R-A2402 cell that was stably transfected with NS5A gene and used as a target cell. The expression of HLA-A24 molecule in C1R-A2402 cell that was stably transfected with NS5A gene and the expression of HLA-A31 molecule in C1R-A2402 cell that was stably transfected with HLA-A31 gene (negative control) were analyzed by a flow cytometry using a FACScan (
Then, cytotoxicity was tested by a 6-hour 51Cr release assay using C1R-A2402 cell that was stably transfected with NS5A gene as a target cell. C1R-A2402 cell that was stably transfected with a negative control gene (HLA-A3101) was used as a negative control, and C1R-A2402 cell that was pulsed with NS5A-2132 peptide was used as a positive control. A 6-hour 51Cr release assay was carried out at three different E/T ratios. The values are expressed as the mean±SD of percentage of specific lysis. The asterisks indicate p<0.05 in the two-sided Student's t-test. The PBMC stimulated with NS5A-2132 peptide showed a higher cytotoxicity level against both C1R-A2402 cell that was transfected with NS5A gene and non-transfected C1R-A2402 cell that was previously pulsed with a corresponding peptide, compared with the cytotoxicity against C1R-A2402 cell that was transfected with the negative control gene (
In order to understand more about the possibility of biological role of anti-peptide IgG, it was examined whether anti-NS5A-2132 IgG recognizes the whole NS5A protein in absorption and elution assays. NS5A-2132 and an HIV peptide were used as a positive control and a negative control, respectively (see the description above for the details of the absorption and elution tests). Anti-peptide IgG was neither absorbed nor eluted with the whole NS5 protein (
Then, Huh7 cells were incubated up to three days in the presence of sera from patients (sera from 2 HCV-1b+ patients whose sera showed a high level of anti-NS5A-2132 activity). As a negative control, sera from 2 HDs and FCS were used. The number of Huh7 (NNU50-1) cells that could survive was counted with a Cell Count kit-8 (10 μl/well) and the mean value of three assays are shown. None of the tested sera inhibited the growth of Huh7 (NNU50-1) cells under these culture conditions (
Sera were obtained from 33 patients with a disease associated with HCV in the cohort study conducted between 1995 and 2002. The results of the follow-up survey on the 33 patients are shown in Table 4. The sera obtained from patients with chronic hepatitis (CH, n=68), liver cirrhosis (LC, n=43) and hepatic cell carcinoma (HCC, n=52) were also used in the analysis. These patients were diagnosed at the time of the first serum sampling by biochemical and histological findings, echography and computed tomography. An anti-HCV antibody was assayed using a chemiluminescence enzyme immunoassay (CLEIA) kit (Lumipulse II HCV, Fujirebio Inc., Tokyo, Japan) or the second generation or the third generation enzyme immunoassay test (SRL, Tokyo, Japan). HCV-RNA in the sera was detected using RT-PCR (SRL, Tokyo, Japan). The HCV genotype was determined by directly sequencing HCV in the sera of the patients by RT-PCR (SRL, Tokyo, Japan). The sera were also obtained from subjects who were not infected with HCV, which include 24 subjects with an autoimmune disease (6 patients with systematic lupus erythematosus, one with Behcet's disease and 17 with atopic dermatitis), 17 cases of hepatitis B virus (HBV) infection (positive for HBV surface antigen), 3 patients having human immunodeficiency virus (HIV) and 10 cases with human T cell leukemia virus type I (HTLV-1) infection. As a negative control, the sera obtained from 37 persons who did not have the history of a viral hepatitis or vaccination of HBV and had normal hepatic function were tested.
Two peptides with a purity of 90% or more were purchased from BIOSYNTHESIS (Lewisville, Tex.): HCV-1b core protein 35-44 (YLLPRRGPRL (SEQ ID NO: 1) capable of inducing an HLA-A2-restricted CTL activity) and HCV-1b NS5A protein 2132-2140 (RYAPACKPL (SEQ ID NO: 2) capable of inducing an HLA-A24-restricted CTL activity). As a negative control, peptides derived from HIV with an HLA-A2-binding motif (SLYNTVATL (SEQ ID NO: 64)) and an HLA-A24-binding motif (RYLRDQQLLGI (SEQ ID NO: 63)) were used.
The level of peptide-specific IgG was measured by an enzyme-linked immunosorbent assay (ELISA). Briefly, each peptide was dissolved in dimethyl sulfoxide (DMSO) and stored at −20° C. The peptide (20 μg/well) diluted with 0.1 M carbonate buffer containing disuccinimidyl suberate (DSS) (PIERCE, Rockford, Ill.) as a chemical crosslinker was bound to an ELISA plate. The wells were washed 3 times with 0.05% Tween 20/PBS (PBST). Then, the plate was blocked overnight at 4° C. with Block Ace (Yukijirushi, Tokyo, Japan). A serum sample was diluted 1:100, 1:200 or 1:400 with 0.05% Tween 20/Block Ace, and 100 μl/well of the sample was added to each well. After the sample was incubated at 37° C. for 2 hours, the plate was washed 9 times with PBST, and further incubated at 37° C. for 2 hours with 100 μl/well of a 1:1000 diluted rabbit anti-human IgG (γ-chain specific) (DAKO Glostrup, Denmark). After the plate was washed 9 times, 100 μl/well of a 1:100 diluted anti-rabbit IgG-conjugated horseradish peroxidase-dextran polymer (En Vision, DAKO) was added to each well, and then the plate was incubated at room temperature for 40 minutes. After the plate was washed, 100 μl/well of a tetramethylbenzidine substrate solution (KPL, Guildford, UK) was added, and the reaction was stopped by the addition of 1.0 M phosphoric acid.
A statistical analysis was carried out using the χ2-test. Values of P<0.05 were considered statistically significant.
The cross reactivity of two antibodies reactive to the peptides derived from HCV-1b was examined. The sera obtained from 60 HCV patients and patients who are not included in the cohort study (CH, n=22, LC, n=21 and HCC, n=17) were examined for reactivity to the peptides of the core 35-44 (C-35) and NS5A 2132-2140 (NS5A-2132). The sera were also obtained from 24 subjects with an autoimmune disease, 17 cases of HBV infection and 10 cases of HTLV-1 infection. The sera obtained from 37 HDs were used as a negative control. The level of peptide-reactive IgG contained in the serially diluted serum samples was measured by ELISA. The results are reported by the absorbance (OD) of each sample. The representative results of OD at a serum dilution of 100:1 are shown. The cut-off value was set at 0.093 (mean+2 SD of OD from HDs). The statistical analysis was carried out using the χ2-test. Values of P<0.05 were considered statistically significant.
As a result, a significant level of anti-C-35 IgG was detected in 56 out of 60 HCV-positive patients (93.3%) and there was no significant difference in the IgG level among the 3 groups (CH, LC and HCC patients) (
Then, the correlation between the HLA-class IA phenotype and the level of anti-C-35 IgG or anti-NS5A-2132 IgG was examined in 29 patients with a disease associated with HCV. The HLA-class IA phenotype was determined by a standard serological method. Nine patients were HLA-A2+, 13 patients were A24+, and the remaining 7 patients were A2−-A24−. The levels of Anti-C-35 and anti-NS5A-2132 were measured by a standard ELISA and the OD value of each patient at a serum dilution of 100:1 is shown in the figure. Regardless of difference in the HLA-class IA phenotype, both antibodies were detected in the majority of the patients infected with HCV (
Also the correlation between the HCV genotype and the level of anti-C-35 IgG or anti-NS5A-2132 IgG was examined in a double blind study in patients infected with HCV-1b (n=29), HCV-2a (n=16) and HCV-2b (n=3). The HCV genotype was determined by directly sequencing HCV in the serum of the patient. The results of all subjects at a dilution of 100:1 are shown in
The anti-C-35 antibody was detected in 26 out of 30 patients infected with HCV-1b, 15 out of 15 patients infected with HCV-2a and 3 out of 3 patients infected with HCV-2b, respectively. Similarly, the anti-NS5A-2132 antibody was detected in the sera of 23 out of 30 patients infected with HCV-1b, 10 out of 16 patients infected with HCV-2a and 3 out of 3 patients infected with HCV-2b, respectively (
The above results suggest that the level of the anti-NS5A-2132 antibody correlates with the prognosis of an individual infected with HCV but not the case with the anti-C-35 antibody.
Then, the sera of CH (n=24), LC (n=22), HCC (n=26) and HDs (n=9) were newly prepared and the level of anti-NS5A-2132 IgG was measured (
In order to further examine the correlation between the anti-NS5A-2132 antibody and the prognosis of an HCV-positive patient at an individual level, the serum levels of the two antibodies were examined in a sample from the cohort study, in which habitants infected with HCV were annually screened from 1990 to 2002. This study was done with 66 serum samples in total, which were obtained from 33 patients and collected twice in 1995 and 2002 from the same individuals. The diagnoses of these 33 patients were CH (n=17), LC (n=1), asymptomatic carrier (ASC) (n=4), and a past medical history of HCV infection (an individual spontaneously recovered) (n=11) at the time of 1995, and CH (n=13), LC (n=4), HCC (n=2), asymptomatic carrier (ASC) (n=1), and a past medical history of HCV infection (n=13) at the time of 2002. In other words, in the period of seven years, the progress of disease was not observed in 26 patients, while the progress was observed in the remaining 7 patients (Table 4). The OD values of the anti-C-35 antibody and the anti-NS5A-2132 IgG in each patient measured at a serum dilution of 100:1 in 1995 and 2002 are shown in
Then, the 33 subjects were subdivided into 5 groups (CH, LC, HCC, a past medical history of HCV infection (a recovered individual), and ASC) and the levels of the two antibodies at a serum dilution of 100:1 in 2002 were plotted (
The level of IgG reactive to HCV peptide in the sera of HLA-A24+ HCV-infected patients was measured in the same manner as in Example 1 using the Luminex assay, which is considered to be more sensitive than the ELISA method.
Each peptide was coupled to microbeads (manufactured by Luminex Corporation, xMAP Multi-Analyte COOH Beads), which was encoded with a classification code for the content of each fluorescent dye (hereinafter referred to as color-coded) according to the manufacturer's instruction. One hundred microliters of unprepared color-coded microbeads were put in each well of a filter plate and aspirated, and then washed twice with a washing buffer (phosphate buffered saline (PBS) (pH 7.4±0.1), Tween (registered trademark) 20 (0.05% v/v)) and each well was aspirated at the same time. Then, 50 μl of 0.1 M MES (2-morpholinoethanesulfonic acid) buffer (pH 7.0) was added, and 10 μl of EDC (N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride) (1 mg/ml/0.1 M MES buffer (pH 7.0)) was added to each well. Several hundred microliters of the peptide (1 mg/ml, 0.1 MMES buffer, pH 7.0) was mixed with the washed microbeads in each well. After the peptide and the mixed microbeads were reacted in the dark for 20 minutes at room temperature, 10 μl of EDC (1 mg/ml/0.1 M MES buffer (pH 7.0)) was added to each well and the mixture was allowed to react in the dark for 20 minutes at room temperature. this process was repeated twice. After the excess solution was aspirated, 100 μl of 1 M Tris-HCl buffer (pH 7.0) was added to each well and the mixture was allowed to react in the dark for 15 minutes at room temperature. Then, the beads in each well were washed 3 times with the washing buffer as above and collected in a storage solution containing 0.05% sodium azide in Block Ace (registered trademark).
Microbeads for each well are prepared by placing a bead solution obtained by binding a peptide to the color-coded microbeads in such a manner that about 5,000 microbeads are contained in each well of a filter plate (each well contains about 1 μl of single type of bead). A bead mixture was prepared by mixing equal amount of ten types of microbeads prepared in this manner and diluting the mixture with the above-mentioned washing buffer (PBS, TWEEN (registered trademark) 20 (0.05% v/v)) so as to give a total amount of about 25 μl.
Serum was used in this study. One hundred microliters of each serum dilution was prepared by diluting the serum at 1:100 to 1:1000 with a reaction buffer (PBS (pH 7.4±0.1), Tween (registered trademark) 20 (0.05% v/v) , and bovine serum albumin (BSA) 10 mg/ml).
A biotinylated goat anti-human IgG (γ-chain specific) diluted with the reaction buffer was used as a secondary antibody. Streptavidin (1 mg/ml) labeled with PE (phycoerythrin) (SRPE) was diluted to 20 μl (1/50 dilution) with the above-mentioned reaction buffer and used as a fluorescent dye-labeled streptavidin.
One hundred microliters of the washing buffer was put in each well of a filter plate and then removed by aspiration. This washing step was carried out twice. Then, a 96-well filter plate containing 25 μl of the above-mentioned bead mixture in each well was washed with the washing buffer twice. After the washing, 100 μl of the sample was added to all wells. The filter plate was covered and shaken by a plate shaker (300 rpm) in the dark for 2 hours at room temperature and aspirated. Then, 100 μl of the washing buffer was put in each well and removed by aspiration. This washing step was carried out three times.
Then, 100 μl of the biotinylated secondary antibody, i.e., biotinylated goat anti-human IgG (γ-chain specific) , was added to each well and the filter plate was covered and shaken by a plate shaker (300 rpm) in the dark for 1 hour at room temperature. After aspiration, 100 μl of the washing buffer was put in each well and removed by aspiration. This washing step was carried out three times.
Subsequently, 100 μl of SRPE was added to each well and the filter plate was covered and shaken by a plate shaker (300 rpm) in the dark for 30 minutes at room temperature. After aspiration, 100 μl of the washing buffer was put in each well and removed by aspiration. This washing step was carried out three times. Thereafter, 100 μl of the washing buffer was added to each well and shaken by a plate shaker (300 rpm) for 2 to 3 minutes, and then 50 μl of the sample thus obtained was assayed by a fluorescence flowmetric system. The results are shown in
An anti-virus effect of a vaccine comprising a peptide derived from hepatitis C virus of the present invention was examined in HCV-infected HLA-A2+ or HLA-A24+ subjects. All the subjects were infected with HCV-1b and showed no response to a treatment with interferon and ribavirin. Peptides derived from hepatitis C virus, C-35, NS5A-2132, E2-488, E1-213 and NS3-1081 were synthesized, purified and stored as lyophilized powder under GMP compliance. The C-35, NS5A-2132, E2-488, and NS3-1081 peptides were dissolved in a small amount of DMSO (1 mg/10 to 25 μl) and E1-213 was dissolved in 7% sodium hydrogen carbonate solution for injection (Meylon) (1 mg/15 μl). Each solution was diluted with physiological saline for injection (1 to 2 mg/ml) and sterilized by filtration through 0.22 μm filter. Each of the resulting solutions was mixed with an equal amount of an adjuvant (Montanide ISA-51), to prepare emulsified injection solutions. C-35 injection solution was administered to 3 subjects of HLA-A2+ and NS5A-2132 injection solution was administered to 5 subjects of HLA-A24+. The peptide was administered every two weeks by injecting the emulsion containing 0.3 mg of the peptide per dose into the lateral region. The level of HCV RNA and the values of GOT, GPT, γ-GTP and AFP were monitored continuously.
The results are shown in
The peptide derived from hepatitis C virus according to the invention contains an HLA-binding motif in its sequence. it is recognized by an antibody reactive to hepatitis C virus, and also has a property of being recognized by an HLA-A2- or HLA-A24-restricted cytotoxic T cell.
Accordingly, the peptide derived from hepatitis C virus of the invention could be an effective vaccine for a disease attributable to HCV infection.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003-330258 | Sep 2003 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP04/14312 | 9/22/2004 | WO | 00 | 1/3/2007 |
