Methods and reagents for detecting genetic factors in immune responsiveness to hepatitis B vaccines

Abstract

To provide methods for detecting genetic factors of immune responsiveness to new hepatitis B vaccines.SOLUTION: The method for detecting genetic factors of immune responsiveness to hepatitis B vaccines Bimmugen (R) and Heptavax (R)-II comprises: detecting the haplotype of HLA class II gene in a DNA-containing sample derived from a subject; and when HLA-DRB1*13:02-DQB1*06:04 is detected as the haplotype, determining that the subject has immune responsiveness to Bimmugen but has no or low responsiveness to Heptavax II.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods and reagents for detecting genetic factors in immune responsiveness to hepatitis B vaccines. [Background technology]

[0002] Currently, hepatitis B vaccines (HB vaccines) are administered to protect against hepatitis B virus (HBV) in over 180 countries worldwide. There are multiple HBV genotypes, with genotype C (HBV / C) being the most common in Japan. Therefore, two vaccines are used: Bimugen (registered trademark) (hereafter, the term "registered trademark" will be omitted), a recombinant adsorbed HB vaccine (yeast-derived) for HBV / C, and Heptavax (registered trademark)-II, a recombinant adsorbed HB vaccine (yeast-derived) for genotype A (HBV / A). However, approximately 10% of recipients of Bimugen and Heptavax-II vaccines fail to develop the neutralizing antibody HBs antibody, the cause of which remains unknown.

[0003] The inventors have found that the HLA class II gene HLA-DRB1 is responsible for the low vaccine response (HBs antibody titer of 10mIU / mL or less) in Japanese adults who received Bimugen. * 04:05-DQB1 * 04:01 and HLA-DRB1 * 14:06-DQB1 * On the other hand, the HLA class II gene HLA-DRB1 is strongly associated with HBs antibody response (HBs antibody titer >10mIU / mL). * 08:03-DQB1 * 06:01 and HLA-DRB1 * 15:01-DQB1 * It has been revealed that two haplotypes of 06:02 and the BTNL2 gene strongly contribute to this mutation (see, for example, Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Nishida N et al., “Key HLA-DRB1-DQB1 Haplotypes and Role of the BTNL2 Gene for Response to a Hepatitis B Vaccine.”, Hepatology, Vol. 68, No. 3, pp. 848-858, 2018. Summary of the Invention [Problem to be solved by the invention]

[0005] However, much remains unknown about the genetic factors that influence immune response to HB vaccines, and no method has been established to detect them.

[0006] The present invention has been made in view of the above circumstances, and provides a method and reagent for detecting genetic factors in immune responsiveness to a novel hepatitis B vaccine. [Means for solving the problem]

[0007] As a result of extensive research to achieve the above-mentioned object, the inventors conducted an analysis of the relationship between HLA class II gene haplotypes and the effectiveness of hepatitis B vaccines using the HLA (Human Leukocyte Antigen) imputation method.The results showed that if a person has HLA-DRB1*04:05-DQB*04:01, vaccination with either the Bimugen or Heptavax-II hepatitis B vaccine is less effective (immune response remains low), while if a person has HLA-DRB1*13:02-DQB1*06:04, vaccination with Heptavax-II is unlikely to be effective (immune response remains low), but vaccination with Bimugen is effective (immune response increases), leading to the completion of the present invention.

[0008] That is, the present invention includes the following aspects. (1) A method for detecting genetic factors in immune responsiveness to hepatitis B vaccine, comprising: the hepatitis B vaccine is Bimugen and Heptavax-II; detecting a haplotype of an HLA class II gene in a DNA-containing sample from a subject; When HLA-DRB1*13:02-DQB1*06:04 is detected as the haplotype, determining that the subject has immune responsiveness to the Beamgen and has no or low immune responsiveness to the Heptavax-II; A method comprising: (2) The method described in (1), further comprising determining that the subject does not have immune responsiveness to the Beamgen and Heptavax-II if HLA-DRB1*04:05-DQB*04:01 is detected as the haplotype. (3) A reagent for detecting genetic factors in immune responsiveness to hepatitis B vaccine, A reagent comprising one or more nucleic acid probes or primers for detecting HLA-DRB1*13:02-DQB1*06:04, a haplotype of the HLA class II gene. (4) The reagent according to (3), further comprising one or more nucleic acid probes or primers for detecting HLA-DRB1*04:05-DQB*04:01, which is a haplotype of the HLA class II gene. [Effects of the Invention]

[0009] According to the method and reagent of the above embodiment, genetic factors in immune responsiveness to hepatitis B vaccine in a subject can be detected simply and reliably. DETAILED DESCRIPTION OF THE INVENTION

[0010] A method and reagent for detecting genetic factors related to the immune response to hepatitis B vaccine according to an embodiment of the present invention (hereinafter may be abbreviated as "the method of this embodiment" and "the reagent of this embodiment" respectively) will be described in detail below.

[0011] <Hepatitis B vaccine (HB vaccine)> In this specification, the hepatitis B vaccine is used to prevent hepatitis B virus infection and reactivation. For example, vaccines derived from Genotype A (HBV / A) such as Heptavax-II; vaccines derived from Genotype A2 (HBV / A2) such as Engerix-B and Recombivax HB; vaccines derived from Genotype C (HBV / C) such as BeamGen, etc. can be mentioned. Among them, the HB vaccines targeted by the method of this embodiment are BeamGen and Heptavax-II, which have a track record of use as HB vaccines in Japan.

[0012] <Method for detecting genetic factors related to the immune response to hepatitis B vaccine> The method of this embodiment is a method for detecting genetic factors related to the immune response to hepatitis B vaccine, wherein the hepatitis B vaccine is BeamGen and Heptavax-II, detecting the haplotype of the HLA class II gene in a DNA-containing sample derived from a subject, when the haplotype HLA-DRB1*13:02-DQB1*06:04 is detected, it is determined that the subject has an immune response to BeamGen and does not have or has a low immune response to Heptavax-II, and includes.

[0013] The inventors conducted genome-wide association studies (GWAS) on 1,193 samples from Japanese adults who received the Bimugen vaccine and 555 samples from Japanese adults who received the Heptavax-II vaccine, and identified HLA-DRB1*04:05-DQB*04:01 and HLA-DRB1*13:02-DQB1*06:04 as haplotypes of the HLA class II gene associated with immune responsiveness to hepatitis B vaccine.

[0014] Therefore, it is preferable that the method of this embodiment further includes determining that the subject does not have immune responsiveness to the Bimmugen and the Heptavax-II if HLA-DRB1*04:05-DQB*04:01 is detected as the haplotype.

[0015] According to the method of this embodiment, genetic factors in immune responsiveness to hepatitis B vaccine in a subject can be detected simply and reliably.

[0016] HLA is a human major histocompatibility complex (MHC), a membrane protein that binds to foreign antigen peptides from transplants, bacteria, viruses, etc. and presents them to T cells. Many alleles are known to exist in HLA, and information on these is available in the HLA nomenclature (http: / / hla.alleles.org / announcement.html) and elsewhere. The designations for alleles and polymorphisms in this specification are based on the HLA nomenclature designation.

[0017] Sequence data for the above haplotypes may be data registered in databases such as GenBank (NIH genetic sequence database) and DDBJ (DNA Data Bank of Japan). For example, in HLA-DRB1*04:05-DQB1*04:01, the gDNA of the HLA-DRB1 gene is 11,074 bp and is registered in GenBank as HLA-DRB1 (SEQ ID NO: 1, GenBank accession number NM_002124.4), and as a 266-amino acid MHC class II antigen (SEQ ID NO: 2, GenBank accession number AAB42072.1). Of HLA-DRB1*04:05-DQB1*04:01, the gDNA of the HLA-DQB1 gene is 7191 bp and is registered in GenBank as HLA-DQB1 (SEQ ID NO: 3, GenBank accession number NM_002123.5), and has 229 amino acid residues and is registered as MHC class II HLA-DQ-beta-1 (SEQ ID NO: 4, GenBank accession number AAC41967.1). Among HLA-DRB1*13:02-DQB1*06:04, the gDNA of the HLA-DRB1 gene is 11,704 bp and is registered in GenBank as HLA-DRB1 (SEQ ID NO: 1, GenBank accession number NM_002124.4), and the 89 amino acid residues are MHC class II antigen HLA-DRBI, partial (SEQ ID NO: 5, GenBank accession number AAD50971.1). Among HLA-DRB1*13:02-DQB1*06:04, the gDNA of the HLA-DQB1 gene is 7,191 bp and is registered in GenBank as HLA-DQB1 (SEQ ID NO: 3, GenBank accession number NM_002123.5), and the 183 amino acid residues are MHC class II antigen, partial (SEQ ID NO: 6, GenBank accession number CBF35736.1). Hereinafter, the above haplotypes HLA-DRB1*04:05-DQB*04:01 and HLA-DRB1*13:02-DQB1*06:04 may be referred to as "alleles associated with immune responsiveness to hepatitis B vaccine" or "alleles related to this embodiment." Furthermore, "alleles" may be referred to as "polymorphisms" or "SNPs."

[0018] Alleles include single-stranded and double-stranded DNA as well as their RNA complements, and may be naturally occurring or artificially produced. Examples of DNA include, but are not limited to, genomic DNA, cDNA corresponding to the genomic DNA, chemically synthesized DNA, DNA amplified by PCR, combinations thereof, and DNA-RNA hybrids. As used herein, the term "polynucleotide" refers to a molecule containing two or more nucleotides, including those of a length generally referred to as an oligonucleotide. The term "polynucleotide" as used herein may be either DNA or RNA.

[0019] These base sequences can be obtained from cDNA libraries, genomic libraries, etc. by preparing a probe using an appropriate fragment by a method known to those skilled in the art, and then using this probe by known hybridization methods such as colony hybridization, plaque hybridization, Southern blotting, etc.

[0020] The DNA-containing sample derived from a subject is not particularly limited as long as it is collected from the subject's living body, and examples include, but are not limited to, blood, serum, plasma, urine, puffy coat, saliva, semen, thoracic exudate, cerebrospinal fluid, tears, sputum, mucus, lymph, ascites, pleural effusion, amniotic fluid, bladder washings, bronchoalveolar lavage fluid, hair, feces, cells or tissues collected directly from the living body, etc. It is preferable to extract DNA from these samples and use them as samples to be used for detection, as described below.

[0021] Detection of haplotypes of HLA class II genes can be performed at the gene level or protein level. For example, genomic DNA or mRNA can be prepared from a sample, and alleles associated with immune responsiveness to hepatitis B vaccines in the genomic DNA or mRNA can be detected based on the base sequence. Alternatively, human HLA-DP molecule protein can be prepared from a sample, and HLA-DRB1*04:05-DQB*04:01 and HLA-DRB1*13:02-DQB1*06:04 in the DP molecule can be detected using, for example, antibodies. These methods are briefly described below.

[0022] [Preparation of genomic DNA or mRNA from DNA-containing samples] The DNA extraction method is not particularly limited, and can be performed using known methods. Examples include the phenol / chloroform method and the cetyltrimethylammonium bromide (CTAB) method. DNA extraction can also be performed using commercially available kits. Examples of such kits include the Wizard Genomic DNA Purification Kit (Promega).

[0023] Alternatively, the DNA may be cDNA synthesized by extracting mRNA and using the mRNA as a template. There are no particular limitations on the method for extracting mRNA, and it can be extracted using known methods. Examples include the guanidine isothiocyanate method. A commercially available kit may be used for mRNA extraction. Examples of such kits include the NucleoTrap (registered trademark) mRNA Kit (manufactured by Clontech). There are also no particular limitations on the method for synthesizing cDNA, and it can be synthesized using known methods. For example, cDNA can be synthesized from RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) using a random primer or a polyT primer.

[0024] HLA-DRB1*04:05-DQB*04:01 and HLA-DRB1*13:02-DQB1*06:04 in the genomic DNA or mRNA prepared as described above can be detected using genetic polymorphism detection methods known in the art. Examples include direct sequencing, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), hybridization, TaqMan® PCR (hereinafter, the term "registered trademark" will be omitted), and mass spectrometry. However, because the HLA-DRB1 gene contains many polymorphic sites, particularly within the second exon, detecting these alleles requires distinguishing between multiple polymorphisms. These methods are described below.

[0025] Direct sequencing is performed by cloning the target region containing HLA-DRB1*04:05-DQB*04:01 and HLA-DRB1*13:02-DQB1*06:04 in a DNA-containing sample from a subject into a vector or amplifying it by PCR, and then determining the nucleotide sequence of the region. Cloning can be performed by screening a cDNA library using an appropriate probe. Alternatively, cloning can be performed by amplifying the target region by PCR using appropriate primers and ligating it into an appropriate vector. Subcloning into another vector is also possible, but is not limited to these. Examples of vectors that can be used include commercially available plasmid vectors, such as pBlue-Script SK(+) (Stratagene), pGEM-T (Promega), pAmp (Gibco-BRL), p-Direct (Clontech), and pCR2.1-TOPO (Invitrogene), as well as viral vectors, artificial chromosome vectors, and cosmid vectors. The base sequence can be determined by known methods, including, but not limited to, manual sequencing using radioactive marker nucleotides and automated sequencing using dye terminators. Based on the base sequence thus obtained, it is determined whether the sample has sequences corresponding to HLA-DRB1*04:05-DQB*04:01 and HLA-DRB1*13:02-DQB1*06:04.

[0026] The PCR method is performed using an oligonucleotide primer (hereinafter sometimes referred to as the "allele detection primer according to this embodiment") that hybridizes only to a sequence having the allele according to this embodiment or a sequence having another allele. As described above, there are multiple SNPs in the HLA-DRB1 gene. The allele detection primer according to this embodiment may be a single primer capable of detecting all SNPs, or a combination of two or more primers capable of detecting each SNP. The primer is used to amplify the DNA of the sample. If only the allele detection primer according to this embodiment generates a PCR product, the sample contains the allele according to this embodiment. If only the primer for the other allele generates a PCR product, it indicates that the sample does not contain the allele according to this embodiment.

[0027] In the RFLP method, first, a region containing the allele of the present embodiment to be detected is amplified by PCR. The PCR product is then cleaved with a restriction enzyme appropriate for the region containing the allele of the present embodiment. The PCR product digested with the restriction enzyme is separated by gel electrophoresis and visualized by ethidium bromide staining. The fragment lengths can be compared with molecular weight markers and, as a control, with the PCR product not treated with the restriction enzyme, to detect the presence of the allele of the present embodiment in a sample.

[0028] The hybridization method determines the presence or absence of the allele of this embodiment in a sample based on the ability of sample-derived DNA to hybridize with complementary DNA molecules (e.g., oligonucleotide probes). This hybridization method can be performed using various hybridization and detection techniques, including colony hybridization, plaque hybridization, and Southern blotting. For detailed procedures for the hybridization method, see "Molecular Cloning, A Laboratory Manual 3rd ed." (Cold Spring Harbor Press (2001); particularly Sections 6-7), "Current Protocols in Molecular Biology" (John Wiley & Sons (1987-1997); particularly Sections 6.3-6.4), and "DNA Cloning 1: Core Techniques, A Practical Approach 2nd ed." (Oxford University (1995); particularly Section 2.10 for hybridization conditions). Furthermore, hybridization can also be detected using a DNA chip. In this method, an oligonucleotide probe specific to the allele according to this embodiment is designed and attached to a solid support, and then a DNA sample derived from a specimen is contacted with the DNA chip to detect hybridization.

[0029] The TaqMan PCR method uses an allele-specific TaqMan probe according to the present embodiment and Taq polymerase to simultaneously detect SNPs and amplify regions containing SNPs. The TaqMan probe is an oligonucleotide of approximately 20 bases, labeled with a fluorescent substance at the 5' end and a quencher at the 3' end, and is designed to hybridize to the target SNP site. Taq polymerase has 5'→3' nuclease activity. When a region containing the SNP site is amplified using PCR primers designed to amplify the region containing the target SNP site in the presence of the TaqMan probe and Taq polymerase, the TaqMan probe hybridizes to the target SNP site in template DNA in parallel with the amplification. When the extension reaction from the forward primer reaches the TaqMan probe hybridized to the template, the fluorescent substance attached to the 5' end of the TaqMan probe is cleaved by the 5'→3' nuclease activity of the Taq polymerase. As a result, the released fluorescent substance is no longer affected by the quencher and emits fluorescence. SNP detection becomes possible by measuring the fluorescence intensity.

[0030] Mass spectrometry-based SNP typing, for example, can be performed using MALDI-TOF / MS in combination with primer extension. This method allows for high-throughput analysis and involves the following steps: 1) PCR, 2) PCR product purification, 3) primer extension, 4) extension product purification, 5) mass spectrometry, and 6) genotyping. First, a region containing the target SNP site is amplified from genomic DNA by PCR. PCR primers are designed so that they do not overlap with the SNP site base. The primers are then purified by enzymatic removal using exonuclease and shrimp alkaline phosphatase or by ethanol precipitation. Next, a primer extension reaction is performed using a genotyping primer whose 3' end is directly adjacent to the SNP site. The PCR product is denatured at high temperature, and excess genotyping primer is added and annealed. ddNTPs and DNA polymerase are added to the reaction system, followed by thermal cycling to generate oligomers one base longer than the genotyping primer. The oligomers generated by this extension reaction are different for each allele depending on the design of the genotyping primer. The purified extension reaction products are subjected to mass spectrometry and analyzed from the mass spectrum.

[0031] Other detection methods include a high-throughput SNP typing method that applies single-molecule fluorescence analysis. For example, the MF20 / 10S (manufactured by Olympus) is a system that employs this method. Specifically, it uses a confocal laser optical system and a highly sensitive photodetector to measure and analyze the translational diffusion time at the single-molecule level of fluorescently labeled primers amplified by PCR using complementary and non-complementary primers in an ultra-small area of ​​approximately 1 femtoliter (1 / 1000 trillionth of a liter).

[0032] Another high-throughput typing method is the use of DNA chips, which have a variety of DNA probes arrayed and fixed on a substrate. Labeled DNA samples are hybridized on the chip, and fluorescent signals from the probes are detected.

[0033] The Snipper method is an example of an SNP typing method that uses gene amplification methods other than PCR. This method utilizes rolling circle amplification (RCA), a DNA amplification method in which DNA polymerase synthesizes complementary DNA strands while moving along a circular single-stranded DNA template. The probe is an oligo DNA between 80 and 90 bases long, containing sequences of 10 and 20 bases at both ends that are complementary to the 5' and 3' ends of the target SNP site, respectively, and is designed to anneal to the target DNA and form a circular form. The 3' end of the probe is also designed to be complementary to the target SNP site. If the 3' end of the probe is completely complementary to the target SNP site, the probe will circularize; however, if the 3' end of the probe is mismatched, the probe will not circularize. The probe also has a backbone sequence between 40 and 50 bases long and contains sequences complementary to two RCA amplification primers.

[0034] Other examples of SNP typing methods that utilize gene amplification methods other than PCR include typing methods that utilize the UCAN method and the LAMP method.

[0035] The UCAN method is an adaptation of the ICAN method, an isothermal gene amplification method developed by Takara Bio. The UCAN method uses a DNA-RNA-DNA chimeric oligonucleotide (DRD) as a primer precursor. This DRD primer precursor is modified at the 3' end to prevent DNA polymerase from replicating the template DNA, and is designed to allow the RNA portion to bind to the SNP site. When this DRD primer precursor is incubated with the template, the coexisting RNase H cleaves the RNA portion of the paired DRD primer only if the DRD primer and template are perfectly matched. This removes the modified DNA from the 3' end of the primer, creating a new one, allowing DNA polymerase to elongate the primer and amplify the template DNA. On the other hand, if the DRD primer and template DNA do not match, RNase H does not cleave the DRD primer, and DNA amplification does not occur. After the perfectly matched DRD primer precursor is cleaved by RNase H, the amplification reaction proceeds via the ICAN reaction mechanism.

[0036] The LAMP method is a gene isothermal amplification method developed by Eiken Chemical. It defines six regions of the target gene (F3c, F2c, F1c from the 3' end, and B3, B2, B1 from the 5' end) and amplifies them using four primers (FIP primer, F3 primer, BIP primer, B3 primer) for these six regions. For typing purposes, only the target SNP site (single base) is required between F1 and B1, and the FIP and BIP primers are designed so that the single base of the SNP is located at their 5' ends. If there is no SNP, DNA synthesis occurs from the dumbbell structure, which is the starting structure for the LAMP method, and the amplification reaction proceeds continuously. If there is an SNP, DNA synthesis from the dumbbell structure does not occur, and the amplification reaction does not proceed.

[0037] The Invader method does not use nucleic acid amplification, but instead uses two types of non-fluorescently labeled probes (allele probe and Invader probe), one type of fluorescently labeled probe (FRET probe), and the endonuclease cleavase. The allele probe has a sequence complementary to the template DNA from the SNP site at the 3' end, and a flap sequence unrelated to the template DNA at the 5' end of the probe. The Invader probe has a sequence complementary to the template DNA from the SNP site at the 5' end, with the base corresponding to the SNP site being an arbitrary base. The FRET probe has a sequence complementary to the flap sequence at the 3' end. The 5' end of the other probe is labeled with a fluorescent dye and a quencher, but the FRET probe is designed to form a double-stranded chain within the molecule and is usually quenched. When these probes are reacted with template DNA, the 3' end of the Invader probe (any base portion) invades the SNP site when the allele probe forms a double-stranded chain with the template DNA. Cleavase recognizes the structure where the base has invaded and cleaves the flap portion of the allele probe. Next, when this released flap binds to the complementary sequence of the FRET probe, the 3' end of the flap invades the intramolecular double-stranded portion of the FRET probe. As in the case of the allele probe and invader probe described above, cleavase recognizes the structure where the base of the flap has invaded the FRET probe and cleaves the fluorescent dye of the FRET probe. The fluorescent dye separates from the quencher, causing fluorescence. If the allele probe does not match the template DNA, the specific structure recognized by cleavase is not formed, and the flap is not cleaved.

[0038] When primers are used to detect SNPs, they are designed to be suitable for the region to be amplified and the typing method. For example, it is preferable that the primers be capable of completely amplifying the region, and the primer sequences can be designed based on the sequences near both ends of the region. Primer design techniques are well known in the art, and primers that can be used in the method of this embodiment are designed to satisfy conditions for specific annealing, for example, to have a length and base composition (melting temperature) that enable specific annealing. The length of the region to be amplified is not limited as long as it does not interfere with typing, and may be increased or decreased as appropriate depending on the detection method. Furthermore, while a portion of the amplified region contains an SNP site, the position of this site within the amplified region is not limited and may be positioned appropriately depending on the detection method (typing method). Therefore, when designing primers, the positional relationship between the primer and the SNP site can be freely designed according to the detection method, and primers can be designed taking into account the characteristics of the typing method as long as they hybridize to a region containing the SNP to be detected (e.g., a continuous region of 50 to 500 bases in length). The length of a primer that functions as a primer is preferably 10 to 100 bases, more preferably 15 to 50 bases, and even more preferably 15 to 30 bases. Furthermore, when designing a primer, it is preferable to confirm its melting temperature (Tm), which is the temperature at which 50% of any nucleic acid strand hybridizes with its complementary strand. In order for the template DNA and the primer to form a double strand and anneal, the annealing temperature must be optimized. However, a temperature that is too low is undesirable because it can cause nonspecific reactions. Known primer design software can be used to confirm the Tm.

[0039] When a probe is used to detect SNPs, it is designed so that the probe recognizes the SNP site. In designing the probe, the SNP site may be recognized at any location within the probe depending on the typing method, and may be recognized at the end of the probe depending on the typing method. When a polynucleotide for SNP detection is used as the probe, the length of the base sequence complementary to genomic DNA is usually 15 to 200 bases, preferably 15 to 100 bases, and more preferably 15 to 50 bases, but may be longer or shorter depending on the typing method.

[0040] In the method of this embodiment, if the haplotype HLA-DRB1*13:02-DQB1*06:04 is detected, the subject is determined to have immune responsiveness to the Beamgen and no or low immune responsiveness to Heptavax-II. In other words, if the subject has been vaccinated with Beamgen, this indicates that the subject may have HBs antibodies. Furthermore, in the method of this embodiment, if HLA-DRB1*04:05-DQB*04:01 is detected as the haplotype, the subject is determined to have no immune responsiveness to the Bimmugen and Heptavax-II.

[0041] It can also be used to calculate diagnostic utility and analyze the positive rate of immune reactivity to Bimugen. The positive rate here refers to the proportion of samples with the HLA-DRB1*13:02-DQB1*06:04 haplotype among all samples. For example, of the 1,193 Japanese adults described in the examples herein, 123 samples had HLA-DRB1*13:02-DQB1*06:04, and 1,070 samples did not have HLA-DRB1*13:02-DQB1, resulting in a positive rate of approximately 10.3%.

[0042] The presence or absence of immune responsiveness to the HB vaccine is important information not only for healthy individuals who are about to receive the HB vaccine or who have already received the HB vaccine, but also for HBV-infected individuals, and provides important information for, for example, selecting treatment methods and therapeutic drugs for hepatitis B, as well as for preventing and preventing the onset of hepatitis B. In particular, for HBV-infected individuals, this information can be important for preventing fulminant hepatitis caused by HBV reactivation due to a weakened immune system, etc.

[0043] In the method of this embodiment, in addition to detecting the above haplotypes, other SNPs or haplotypes associated with the presence or absence of immune responsiveness to hepatitis B vaccines may also be detected. Detecting these other SNPs or haplotypes in combination with the detection of the above haplotypes allows for more accurate determination of the presence or absence of immune responsiveness to hepatitis B vaccines, further increasing the reliability of the diagnosis.

[0044] Examples of SNPs associated with the presence or absence of immune responsiveness to other hepatitis B vaccines include SNPs in the BTNL2 locus (accession number rs4248166 in the NCBI SNP Database, etc.), SNPs in the IL1RL1 locus (accession number rs9646944 in the NCBI SNP Database, etc.), SNPs in the STOX2 locus (accession number rs2871385 in the NCBI SNP Database, etc.), SNPs in the MCPH1 locus (accession number rs2732977 in the NCBI SNP Database, etc.), SNPs in the OXA1L locus (accession number rs3132969 in the NCBI SNP Database, etc.), and SNPs in the BCL11B locus (accession number rs1951122 in the NCBI SNP Database, etc.). These SNPs may be detected singly or in combination of two or more. When these SNPs are detected in a sample, it can be determined that the sample has immune responsiveness to an HB vaccine.

[0045] The haplotypes associated with immune responsiveness to hepatitis B vaccine include the haplotype of HLA class II gene (HLA-DRB1 * 08:03-DQB1 * 06:01, HLA-DRB1 * 14:06-DQB1 * 03:01, HLA-DRB1 * 15:01-DQB1 * 06:02, HLA-DRB1 * 01:01-DQB1 * 05:01, etc.) These haplotypes may be detected singly or in combination of two or more. * 08:03-DQB1 * 06:01 and HLA-DRB1 * 15:01-DQB1 * When at least one of the haplotypes HLA-DRB1, 06, 02 is detected, the sample can be determined to have immune responsiveness to the HB vaccine. * 04:05-DQB1 * 04:01 and HLA-DRB1 * 14:06-DQB1 * When at least one of the haplotypes 03:01 is detected, it can be determined that the specimen does not have an immune response to the HB vaccine or has a low immune response. In addition, HLA-DRB1 * 08:03-DQB1 * 06:01 and HLA-DRB1 * 15:01-DQB1 * At least one of the haplotypes 06:02 and HLA-DRB1 * 04:05-DQB1 * 04:01 and HLA-DRB1 * 14:06-DQB1 *When both of at least one of the haplotypes at 03:01 are detected, since the high reactivity to the vaccine becomes significant, it can be determined that the specimen has an immune responsiveness to the HB vaccine.

[0046] As methods for detecting these other SNPs and haplotypes, the same methods as the above-described haplotype detection methods can be mentioned.

[0047] <Reagent for Detecting Genetic Factors of Immune Responsiveness to Hepatitis B Vaccine> The reagent of the present embodiment includes a nucleic acid probe or primer that detects HLA-DRB1*13:02-DQB1*06:04, which is a haplotype of the HLA class II gene. The reagent of the present embodiment is useful as a reagent for examining the presence or absence of immune responsiveness to the hepatitis B vaccine. Examples of HLA-DRB1*13:02-DQB1*06:04 include the same ones as those exemplified in the above-mentioned "Method for Detecting Genetic Factors of Immune Responsiveness to Hepatitis B Vaccine". Also, for the nucleic acid probe or primer, those skilled in the art can appropriately prepare it using the methods described as haplotype detection methods in the above-mentioned "Method for Detecting Genetic Factors of Immune Responsiveness to Hepatitis B Vaccine".

[0048] The reagent of the present embodiment preferably further includes one or more nucleic acid probes or primers that detect HLA-DRB1*04:05-DQB*04:01, which is a haplotype of the HLA class II gene. By further including the nucleic acid probe or primer, it is possible to more efficiently detect subjects who do not have or have low immune responsiveness to Beamgen and Heptavax-II.

[0049] In addition to the nucleic acid probe or primer, the reagent of this embodiment may further include reagents, positive controls, solvents, and solutes commonly used in SNP typing. Examples of such reagents include deoxynucleotide triphosphates (dNTPs) and DNA polymerase. Examples of solvents and solutes include distilled water, pH buffer reagents, salts, proteins, surfactants, and the like.

[0050] The nucleic acid probe or primer may contain a sequence unrelated to the two types of haplotypes described above. The nucleic acid probe or primer may also be a chimera of DNA and RNA. The nucleic acid probe or primer may be labeled with a fluorescent substance, a binding affinity substance such as biotin or digoxin, an enzyme, a radioisotope, a luminescent substance, or the like. Examples of fluorescent substances include fluorescamine and fluorescein isothiocyanate. Examples of enzymes include peroxidase, alkaline phosphatase, malate dehydratase, α-glucosidase, α-galactosidase, and the like. Examples of radioisotopes include 125 I, 131 I, 3 H, 14 Examples of luminescent substances include luciferin, lucigenin, luminol, and luminol derivatives.

[0051] In addition to the nucleic acid probe or primer, the reagent of this embodiment may also include a reference sample to serve as a comparison standard or for preparing a calibration curve, a detector, etc. Examples of detectors include those capable of detecting the label of the nucleic acid probe or primer, such as a spectrometer, a radiation detector, and a light scattering detector.

[0052] The reagent of this embodiment may contain, in addition to the nucleic acid probes or primers for detecting the two types of haplotypes described above, nucleic acid probes or primers for detecting other SNPs or haplotypes associated with the presence or absence of immune responsiveness to hepatitis B vaccines. Examples of such SNPs and haplotypes include those exemplified in the above-mentioned "Method for detecting genetic factors in immune responsiveness to hepatitis B vaccines." Furthermore, a person skilled in the art can appropriately prepare nucleic acid probes or primers for detecting these SNPs or haplotypes using the method described as a haplotype detection method in the above-mentioned "Method for detecting genetic factors in immune responsiveness to hepatitis B vaccines." [Example]

[0053] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0054] <Materials and measurement methods> [Samples and clinical data] The genomic DNA samples from 1,193 Japanese adults used in this study to detect genetic factors in immune responsiveness to Bimugen were all obtained from healthy adult volunteers (aged 18 years or older) who had received three 0.5 mL vaccinations at 0, 1, and 6 months with recombinant adsorbed HB vaccine (Bimugen, Chemo-Sero-Therapeutic Research Institute). Individuals who had received the Heptavax-II vaccine (MSD KK) were not included in the genomic DNA samples from these 1,193 individuals. Meanwhile, the genomic DNA samples from 555 Japanese adults used in this study to detect genetic factors in immune responsiveness to Heptavax-II were all obtained from healthy adult volunteers (aged 18 years or older) who had received three 0.5 mL vaccinations at 0, 1, and 6 months with recombinant adsorbed HB vaccine (Heptavax-II, manufactured by MSD KK). Individuals who had received the Bimugen vaccine (manufactured by the Chemo-Sero-Therapeutic Research Institute) were not included in the genomic DNA samples from these 555 individuals.

[0055] Serum anti-HBV surface antibody (HBsAb) and serum anti-HBV core antibody (HBcAb) were confirmed before vaccination and 1 month after the final vaccination using an anti-HBs kit and anti-HBc II kit, respectively, and a fully automated chemiluminescent enzyme immunoassay system using the Architect i2000SR analyzer (Abbott Japan). Individuals who were HBcAb-positive (>1.0 S / CO) were not included in this study. In this study, 1,193 Japanese adults were used to detect genetic factors in immune responsiveness to the above-mentioned bemgenins and were divided into the following three groups: group_0, low response group, HBsAb ≤ 10mIU / mL, n = 107. group_1, intermediate response group, 10mIU / mL <HBsAb≦100mIU / mL、n=351。 group_2, high response group, 100mIU / mL <HBsAb≦1000mIU / mL、n=735。 Clinical information for the 1,193 individuals is summarized by group at the following URL (http: / / onlinelibrary.wiley.com / doi / 10.1002 / hep.29876 / suppinfo). The 555 Japanese adult cases used to detect genetic factors in immune responsiveness to Heptavax-II were also classified into the following three groups: group_0, low response group, HBsAb ≤ 10mIU / mL, n = 66. group_1, intermediate response group, 10mIU / mL <HBsAb≦100mIU / mL、n=124。 group_2, high response group, 100mIU / mL <HBsAb、n=305。

[0056] [Genome-wide association study (GWAS)] Genome-wide SNP analysis was performed on the above genomic DNA samples using the Affymetrix Axiom Genome-Wide ASI 1 Array according to the manufacturer's instructions.

[0057] [HLA imuputation method] The SNP data for the 555 cases were extracted from the extended MHC (xMHC) region, ranging from 25,759,242 bp to 33,534,827 bp, based on the hg19 position. Using the same method as previously reported (Non-Patent Document 1), two-field HLA genotype imputation was performed for the three HLA class II genes using the HIBAG R package. For HLA-DRB1, DQB1, and DPB1, our own Japanese reference imputation was used for HLA genotype imputation. A call threshold (CT > 0.5) was used for quality control after imputation. A total of 515 samples, consisting of 63 cases in Group_0, 174 cases in Group_1, and 278 cases in Group_2, showed three predicted HLA genotypes, all of which met the threshold. In total, 22 HLA-DRB1, 14 HLA-DQB1, and 11 HLA-DPB1 genotypes were assigned to HLA class II genes.

[0058] [Example 1] The genome-wide SNP analysis was carried out using the genomic DNA samples from the above 555 cases, and a genome-wide association study (GWAS) was conducted to compare the two groups in Status-1 and Status-2 shown below.

[0059] Status-1: group_0 (low response group, HBsAb≦10mIU / mL, n=66) vs. group_1+group_2 (intermediate response group + high response group, HBsAb>10mIU / mL, n=489) Status-2: Group_0 (low response group, HBsAb≦10mIU / mL, n=66) vs. Group_2 (high response group, HBsAb>100mIU / mL, n=305)

[0060] In a previous study, among the SNPs that were significantly associated with Beamgen response in a GWAS targeting Beamgen (P value < 0.0001), 72 SNPs that were also significantly associated with response (P value < 0.05) in a GWAS targeting Heptavax-II showed a very high correlation odds ratio (R2 = 0.8856).However, when comparing Heptavax-II and Beamgen recipients in three groups, no SNPs were found to be genome-wide significant. These findings suggest that GWAS was unable to detect significant differences in genes involved in immune response to the two types of hepatitis B vaccines.

[0061] Subsequently, HLA-associated analysis was performed. Specifically, in the HLA-associated analysis, 555 Japanese adults used to detect genetic factors related to immune responsiveness to Heptavax-II were similarly classified into the following three groups: group_0, low response group, HBsAb ≤ 10mIU / mL, n = 66. group_1, intermediate response group, 10mIU / mL <HBsAb≦100mIU / mL、n=124。 group_2, high response group, 100mIU / mL <HBsAb、n=305。

[0062] Next, the association between haplotypes and alleles in each response group was analyzed using the HLA imuputation method.

[0063] First, we examined the relationship between each reaction group and HLA haplotype. The results are shown in Table 1 (comparison between group_0 and group_2) and Table 2 (comparison between group_0 and group_1 + group_2).

[0064] [Table 1]

[0065] [Table 2]

[0066] As shown in Tables 1 and 2, the haplotype (HLA-DRB1*04:05-DQB1*04:01) and allele (DPB*05:01) showed a significant association. The odds ratio (OR) for each was greater than 1, suggesting that having these haplotypes and alleles significantly increases the probability of being in group 0, meaning that individuals are more likely to be in the low response group.

[0067] Next, the relationship between HLA haplotypes and Heptavax-II responsiveness was compared with the relationship between HLA haplotypes and Bimugen responsiveness in a previous study of 1,193 Japanese adults (see Non-Patent Document 1). Table 3 shows the proportions of HLA haplotypes in the high response group (Group_2) for both Heptavax-II and Bimugen. Table 4 shows the proportions of HLA haplotypes in the intermediate / high response groups (Group_1 + Group_2) for both Heptavax-II and Bimugen.

[0068] [Table 3]

[0069] [Table 4]

[0070] As shown in Tables 3 and 4, two haplotypes (HLA-DRB1*04:05-DQB1*04:01 and HLA-DRB1*13:02-DQB1*06:04) showed a significant association. The respective odds ratios (ORs) indicated that HLA-DRB1*04:05-DQB1*04:01 significantly increased the proportion of patients in the Heptavax-II group, meaning that patients were more likely to be in the high and intermediate-high response groups in the Heptavax-II group. On the other hand, HLA-DRB1*13:02-DQB1*06:04 significantly increased the proportion of patients in the Birmingham group, meaning that patients were more likely to be in the high response group in the Birmingham group. HLA-DRB1*04:05-DQB1*04:01 is a haplotype that has already been shown to make people who have this haplotype more likely to be in the low responder group for both Heptavax-II and Bimugen vaccines, based on comparisons with the low, intermediate, and high responder groups. On the other hand, a significant correlation was first observed for HLA-DRB1*13:02-DQB1*06:04 when compared with the medium to high response groups in Heptavax-II and Beamgen.

[0071] Therefore, we next compared the relationship between HLA haplotypes and the high responder groups of Bimugen and Heptavax-II, as well as the healthy control group. The results are shown in Table 5.

[0072] [Table 5]

[0073] As shown in Table 5, HLA-DRB1*13:02-DQB1*06:04 showed a significant association between the Heptavax-II high responder group and the healthy control group. On the other hand, no significant correlation was observed between the healthy control group and the Bimugen high responder group for this haplotype. Since the odds ratio (OR) for the comparison between the Heptavax-II high responder group and the healthy control group was less than 1, the probability of having these haplotypes was significantly higher in the healthy control group. In other words, the proportion of those with HLA-DRB1*13:02-DQB1*06:04 was significantly higher in the healthy control group than in the Heptavax-II high responder group, which indirectly suggests that those with this haplotype are less likely to become high responders to Heptavax-II vaccination.

[0074] In addition, the IEDB (Immune Epitope Database) was used to predict binding between HLA-DRB1*04:05 or HLA-DRB1*13:02 and antigen peptides of Bimmugen or Heptavax-II.

[0075] The results showed that the antigenic peptides of Heptavax-II showed more stable binding to HLA-DRB1*04:05. Furthermore, when searching for antigenic peptide sequences that were unstable with Heptavax-II but stable with Bimugen in binding to HLA-DRB1*13:02, a sequence unique to Heptavax-II, consisting of 15 amino acid residues from the 134th to 148th amino acid residues from the N-terminus (FPSCCCTKP[T / S]DGNCT: SEQ ID NO: 7), was found.

[0076] The above results suggest that if a sample has HLA-DRB1*04:05-DQB1*04:01, the sample tends to have no or low immune responsiveness to Bimugen and Heptavax-II, whereas if a sample has HLA-DRB1*13:02-DQB1*06:04, the sample tends to have immune responsiveness to Bimugen and no or low immune responsiveness to Heptavax-II. [Industrial Applicability]

[0077] According to the method and reagent of this embodiment, genetic factors in immune responsiveness to hepatitis B vaccine in a subject can be detected simply and reliably.

Claims

1. 1. A method for detecting genetic factors in immune responsiveness to hepatitis B vaccine, comprising: the hepatitis B vaccine is a yeast-derived recombinant adsorbed HB vaccine corresponding to genotype A (HBV / A) and a yeast-derived recombinant adsorbed HB vaccine corresponding to genotype C (HBV / C); Detecting the haplotype of HLA class II genes in a DNA-containing sample from a subject; and determining, when HLA-DRB1*13:02-DQB1*06:04 is detected as the haplotype, that the subject has immune responsiveness to the yeast-derived recombinant precipitated HB vaccine corresponding to genotype C (HBV / C) and has no or low immune responsiveness to the yeast-derived recombinant precipitated HB vaccine corresponding to genotype A (HBV / A).

2. The method of claim 1, further comprising determining that the subject does not have immune responsiveness to the yeast-derived recombinant adsorbed HB vaccine corresponding to genotype A (HBV / A) and the yeast-derived recombinant adsorbed HB vaccine corresponding to genotype C (HBV / C) when HLA-DRB1*04:05-DQB1*04:01 is detected as the haplotype.

3. A reagent for detecting genetic factors of immune responsiveness to a yeast-derived recombinant precipitated HB vaccine corresponding to genotype A (HBV / A) and a yeast-derived recombinant precipitated HB vaccine corresponding to genotype C (HBV / C), comprising: A reagent comprising one or more nucleic acid probes or primers for detecting HLA-DRB1*13:02-DQB1*06:04, which is a haplotype of the HLA class II gene.

4. The reagent according to claim 3, further comprising one or more nucleic acid probes or primers for detecting HLA-DRB1*04:05-DQB1*04:01, which is a haplotype of the HLA class II gene.

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