JP7731565B2 - Methods and reagents for detecting genetic factors in immune response to hepatitis B vaccines - Google Patents

Description

The present invention relates to methods and reagents for detecting genetic factors in immune responsiveness to hepatitis B vaccines.

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

The inventors have demonstrated that two haplotypes of HLA class II genes, HLA-DRB1 ^* 04:05-DQB1 ^* 04:01 and HLA-DRB1 ^* 14:06-DQB1 ^* 03:01, strongly contribute to low vaccine response (HBs antibody titer of 10 mIU/mL or less) in Japanese adults receiving Bimugen vaccination. On the other hand, two haplotypes of HLA class II genes, HLA-DRB1 ^* 08:03-DQB1 ^* 06:01 and HLA-DRB1 ^* 15:01-DQB1 ^* 06:02, and the BTNL2 gene, strongly contribute to Bimugen responsiveness (HBs antibody titer of more than 10 mIU/mL) (see, for example, 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.

However, much remains unknown about the genetic factors that contribute to immune responsiveness to HB vaccines, and no method for detecting them has been established.

The present invention was made in consideration of the above circumstances and provides a method and reagent for detecting genetic factors that contribute to immune responsiveness to a new hepatitis B vaccine.

As a result of extensive research to achieve the above-mentioned objective, the inventors conducted an analysis of the relationship between HLA class II gene haplotypes and the efficacy of hepatitis B vaccines using the HLA (Human Leukocyte Antigen) imputation method. They found that in individuals with HLA-DRB1*04:05-DQB*04:01, vaccination with either the Bimugen or Heptavax-II hepatitis B vaccines is less effective (immune response remains low), while in individuals with HLA-DRB1*13:02-DQB1*06:04, vaccination with Heptavax-II is less effective (immune response remains low), but vaccination with Bimugen is effective (immune response increases), leading to the completion of the present invention.

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 the haplotype of HLA class II genes in a DNA-containing sample from a subject;
determining that the subject has immune responsiveness to the Beamgen and no or low immune responsiveness to the Heptavax-II when HLA-DRB1*13:02-DQB1*06:04 is detected as the haplotype;
A method comprising:
(2) The method according to (1), further comprising determining that the subject does not have immune responsiveness to the Beamgen and the Heptavax-II when 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, 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 (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.

The methods and reagents described above make it possible to easily and reliably detect genetic factors that contribute to immune responsiveness to hepatitis B vaccine in a subject.

Details of a method and reagent for detecting genetic factors contributing to immune responsiveness to a hepatitis B vaccine according to one embodiment of the present invention (hereinafter sometimes abbreviated as "the method of this embodiment" and "the reagent of this embodiment") are provided below.

<Hepatitis B vaccine (HB vaccine)>
As used herein, hepatitis B vaccines are used to prevent infection or reactivation of hepatitis B virus, and examples include 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; and vaccines derived from genotype C (HBV/C) such as Bimugen.
Among these, the HB vaccines targeted by the method of this embodiment are Bimugen and Heptavax-II, which have a proven track record of use as HB vaccines in Japan.

<Method for detecting genetic factors in immune responsiveness to hepatitis B vaccine>
The method of this embodiment is a method for detecting genetic factors of immune responsiveness to a hepatitis B vaccine, wherein the hepatitis B vaccine is Bimugen and Heptavax-II;
Detecting the haplotype of HLA class II genes in a DNA-containing sample from a subject;
determining that the subject has immune responsiveness to the Beamgen and no or low immune responsiveness to the Heptavax-II when HLA-DRB1*13:02-DQB1*06:04 is detected as the haplotype;
Includes.

The inventors conducted a genome-wide association study (GWAS) on 1,193 specimens from Japanese adults who received the Bimugen vaccine and 555 specimens 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.

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

The method of this embodiment makes it possible to easily and reliably detect genetic factors that contribute to immune responsiveness to hepatitis B vaccine in a subject.

HLA is a human major histocompatibility complex (MHC) and is 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). The designations for alleles and polymorphisms in this specification are based on the HLA nomenclature.

Sequence data relating to the haplotypes may be data registered in databases such as GenBank (NIH genetic sequence data base) 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).
Of 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 as MHC class II antigen HLA-DRBI, partial (SEQ ID NO: 5, Genbank accession number AAD50971.1) with 89 amino acid residues. Of HLA-DRB1*13:02-DQB1*06:04, 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 as an MHC class II antigen, partial (SEQ ID NO: 6, Genbank accession number CBF35736.1) with 183 amino acid residues.
Hereinafter, the 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 the present embodiment." Furthermore, the term "alleles" may be referred to as "polymorphisms" or "SNPs."

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. Note that, as used herein, the term "polynucleotide" refers to a molecule containing two or more nucleotides linked together, and includes those of a length generally referred to as an oligonucleotide. Furthermore, the term "polynucleotide" as used herein may be either DNA or RNA.

These base sequences can be obtained from cDNA libraries, genomic libraries, etc. by preparing probes using appropriate fragments by methods known to those skilled in the art, and then using these probes to perform known hybridization methods such as colony hybridization, plaque hybridization, and Southern blotting.

The DNA-containing sample derived from a subject is not particularly limited as long as it is collected from the subject's living body, but 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, stool, and cells or tissues collected directly from the living body. It is preferable to extract DNA from these samples and use them as samples to be used for the detection described below.

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 can be detected in the genomic DNA or mRNA 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.

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

The DNA may also 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 also be used to extract mRNA. 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 random primers or poly-T primers.

HLA-DRB1*04:05-DQB*04:01 and HLA-DRB1*13:02-DQB1*06:04 can be detected in genomic DNA or mRNA prepared as described above 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.

Direct sequencing is performed by cloning the region containing the target HLA-DRB1*04:05-DQB*04:01 and HLA-DRB1*13:02-DQB1*06:04 in the DNA-containing sample from the 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, the region can be amplified by PCR using appropriate primers and then cloned by ligating it into an appropriate vector. Furthermore, subcloning into another vector is also possible, but is not limited to these methods. 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. Nucleotide sequences can be determined using known methods, including, but not limited to, manual sequencing using radioactive marker nucleotides and automated sequencing using dye terminators. Based on the nucleotide sequences thus obtained, it is determined whether the sample contains sequences corresponding to HLA-DRB1*04:05-DQB*04:01 and HLA-DRB1*13:02-DQB1*06:04.

The PCR method is performed using oligonucleotide primers (hereinafter sometimes referred to as "allele detection primers according to this embodiment") that hybridize only to sequences having the allele according to this embodiment or sequences having other alleles. As described above, the HLA-DRB1 gene has multiple SNPs. The allele detection primers 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. These primers are 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 primers for other alleles generate a PCR product, this indicates that the sample does not contain the allele according to this embodiment.

In the RFLP method, first, a region containing the allele of this embodiment to be detected is amplified by PCR. Next, this PCR product is cleaved with a restriction enzyme appropriate for the region containing the allele of this embodiment. The PCR products digested with the restriction enzyme are separated by gel electrophoresis and visualized by ethidium bromide staining. The fragment lengths are compared with molecular weight markers and, as a control, the PCR product described above that has not been treated with the restriction enzyme, to detect the presence of the allele of this embodiment in the sample.

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

The TaqMan PCR method uses an allele-specific TaqMan probe and Taq polymerase according to this embodiment to simultaneously detect SNPs and amplify regions containing the 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' to 3' nuclease activity. When the region containing the SNP site is amplified in the presence of these TaqMan probes and Taq polymerase using PCR primers designed to amplify the region containing the SNP site of interest, the TaqMan probe hybridizes to the target SNP site in the template DNA in parallel with the amplification. When the extension reaction from the forward primer reaches the TaqMan probe hybridized to the template, the 5' to 3' nuclease activity of Taq polymerase cleaves the fluorescent substance attached to the 5' end of the TaqMan probe. 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.

An example of a mass spectrometry-based SNP typing method is a method that combines MALDI-TOF/MS with primer extension. This method allows for high-throughput analysis and involves the following steps: 1) PCR, 2) PCR product purification, 3) primer extension reaction, 4) extension product purification, 5) mass spectrometry, and 6) genotype determination. First, a region containing the target SNP site is amplified from genomic DNA using PCR. PCR primers are designed so that they do not overlap with the bases at the SNP site. Purification is then performed using an enzymatic removal method using exonuclease and shrimp alkaline phosphatase, or by ethanol precipitation. Next, a primer extension reaction is performed using a genotyping primer designed so that its 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. When ddNTPs and DNA polymerase are added to the reaction system and subjected to a thermal cycle reaction, an oligomer one base longer than the genotyping primer is produced. The one-base longer oligomer produced in this extension reaction differs depending on the allele, depending on the design of the genotyping primer. The purified extension reaction product is subjected to mass spectrometry and analyzed from the mass spectrum.

Other detection methods include those that apply single-molecule fluorescence analysis as a high-throughput SNP typing method. 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).

DNA chip-based typing is also a type of typing method that allows for high throughput. A DNA chip has a variety of DNA probes aligned and fixed on a substrate. Labeled DNA samples are hybridized on the chip, and fluorescent signals from the probes are detected.

The Snipper method is an example of an SNP typing method that utilizes a gene amplification method other than PCR. This method utilizes rolling circle amplification (RCA), a DNA amplification method in which DNA polymerase synthesizes a complementary strand of DNA 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. It is designed to anneal to the target DNA and become circular. The 3' end of the probe is also designed to have a sequence complementary to the target SNP site. If the 3' end of the probe is perfectly complementary to the target SNP site, the probe will be circularized; however, if the 3' end of the probe is mismatched, the probe will not be circularized. The probe also has a backbone sequence between 40 and 50 bases long, and contains sequences complementary to two types of RCA amplification primers.

Other examples of SNP typing methods that use gene amplification methods other than PCR include typing methods that use the UCAN method or the LAMP method.

The UCAN method is an application 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 has modified DNA at the 3' end to prevent replication of the template DNA by DNA polymerase, and is designed so that the RNA portion binds 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 the DNA polymerase to proceed with the extension reaction 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.

The LAMP method, developed by Eiken Chemical, is an isothermal gene amplification method that defines six regions of a target gene (F3c, F2c, and F1c from the 3' end, and B3, B2, and B1 from the 5' end) and amplifies these six regions using four primers (FIP primer, F3 primer, BIP primer, and B3 primer). For typing purposes, only the target SNP site (one 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. In the absence of an SNP, a DNA synthesis reaction occurs from the dumbbell structure, which is the starting structure for the LAMP method, and the amplification reaction proceeds continuously. In the presence of an SNP, no DNA synthesis reaction occurs from the dumbbell structure, and the amplification reaction does not proceed.

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 to 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 to the 5' end, and the base corresponding to the SNP site can be any base. The FRET probe has a sequence complementary to the flap sequence at the 3' end. The 5' end of the FRET probe is labeled with a fluorescent dye and a quencher, but the FRET probe is designed to form a double strand within the molecule and is normally quenched. When these are reacted with template DNA, the allele probe forms a double strand with the template DNA, and the 3' end (any base portion) of the invader probe invades the SNP site. 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 is separated 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.

When using primers to detect SNPs, they are designed to be appropriate for the region to be amplified and the typing method. For example, it is preferable that the region can be completely amplified, and the sequence 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, such as having 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, there are no restrictions on the location of this site within the amplified region and it 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 to suit the detection method. 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 required for a primer to function 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, setting this temperature too low is undesirable because it can cause nonspecific reactions. Known primer design software can be used to confirm the Tm.

When using a probe to detect SNPs, the probe is designed to recognize the SNP site. In designing the probe, the SNP site may be recognized anywhere within the probe, depending on the typing method; depending on the typing method, it may be recognized at the end of the probe. When a polynucleotide for SNP detection is used as the probe, the length of the base sequence complementary to genomic DNA is typically 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.

In the method of this embodiment, if the haplotype detected is HLA-DRB1*13:02-DQB1*06:04, the subject is determined to have immune responsiveness to the Beamugen and no or low immune responsiveness to the Heptavax-II. In other words, if the subject has been vaccinated with Beamugen, 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 Bimugen and Heptavax-II.

The positive rate of immune reactivity to Bimugen can also be analyzed by using this to calculate diagnostic utility. 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%.

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 those infected with HBV. For example, it provides important information regarding the selection of treatment methods and medications for hepatitis B, as well as the prevention and onset of hepatitis B. In particular, for HBV-infected individuals, this information can be important for preventing HBV reactivation due to a weakened immune system, which can lead to severe hepatitis.

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. By detecting these other SNPs or haplotypes in combination with the detection of the above haplotypes, the presence or absence of immune responsiveness to hepatitis B vaccines can be determined with greater accuracy, further increasing the reliability of the diagnosis.

SNPs associated with the presence or absence of immune responsiveness to other hepatitis B vaccines include, for example, SNPs in the BTNL2 locus (NCBI SNP Database accession number rs4248166, etc.), SNPs in the IL1RL1 locus (NCBI SNP Database accession number rs9646944, etc.), SNPs in the STOX2 locus (NCBI SNP Database accession number rs2871385, etc.), SNPs in the MCPH1 locus (NCBI SNP Database accession number rs2732977, etc.), SNPs in the OXA1L locus (NCBI SNP Database accession number rs3132969, etc.), and SNPs in the BCL11B locus (NCBI (e.g., registration number rs1951122 in the SNP Database). 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 an immune response to the HB vaccine.

Examples of haplotypes associated with the presence or absence of immune responsiveness to hepatitis B vaccines include haplotypes of HLA class II genes (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.). One of these haplotypes may be detected alone, or two or more may be detected in combination. When at least one of the haplotypes HLA-DRB1 ^* 08:03-DQB1 ^* 06:01 and HLA-DRB1 ^* 15:01-DQB1 ^* 06:02 is detected in a sample, the sample can be determined to have immune responsiveness to the HB vaccine. On the other hand, when at least one of the haplotypes HLA-DRB1 ^* 04:05-DQB1 ^* 04:01 and HLA-DRB1 ^* 14:06-DQB1 ^* 03:01 is detected in a sample, the sample can be determined to have no immune responsiveness or a low immune responsiveness to the HB vaccine.
Furthermore, when at least one of the haplotypes HLA-DRB1 ^* 08:03-DQB1 ^* 06:01 and HLA-DRB1 ^* 15:01-DQB1 ^* 06:02 and at least one of the haplotypes HLA-DRB1 ^* 04:05-DQB1 ^* 04:01 and HLA-DRB1 ^* 14:06-DQB1 ^* 03:01 are detected in a sample, high reactivity to the vaccine is significant, and therefore the sample can be determined to have immune responsiveness to the HB vaccine.

Methods for detecting these other SNPs and haplotypes include methods similar to the haplotype detection methods described above.

<Reagent for detecting genetic factors in immune response to hepatitis B vaccine>
The reagent of this embodiment includes a nucleic acid probe or primers for detecting HLA-DRB1*13:02-DQB1*06:04, which is a haplotype of the HLA class II gene. The reagent of this embodiment is useful as a reagent for testing the presence or absence of immune responsiveness to a hepatitis B vaccine. Examples of HLA-DRB1*13:02-DQB1*06:04 include those exemplified in the above-mentioned "Method for detecting genetic factors in immune responsiveness to a hepatitis B vaccine." Furthermore, a person skilled in the art can appropriately prepare the nucleic acid probe or primers using the method described as a haplotype detection method in the above-mentioned "Method for detecting genetic factors in immune responsiveness to a hepatitis B vaccine."

The reagent of this embodiment preferably further comprises one or more nucleic acid probes or primers that detect HLA-DRB1*04:05-DQB*04:01, a haplotype of the HLA class II gene. By further including such nucleic acid probes or primers, it is possible to more efficiently detect subjects who have no or low immune responsiveness to Beamgen and Heptavax-II.

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, etc.

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 also be labeled with a fluorescent substance, a binding affinity substance such as biotin or digoxin, an enzyme, a radioactive isotope, 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, and α-galactosidase. Examples of radioactive isotopes include ¹²⁵ I, ¹³¹ I, ³ H, and ¹⁴ C. Examples of luminescent substances include luciferin, lucigenin, luminol, and luminol derivatives.

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 creating a calibration curve, a detector, etc. Examples of detectors include spectrometers, radiation detectors, light scattering detectors, and other devices capable of detecting the label on the nucleic acid probe or primer.

The reagent of this embodiment may include, in addition to the nucleic acid probes or primers that detect the two types of haplotypes described above, nucleic acid probes or primers that detect other SNPs or haplotypes that are associated with the presence or absence of immune responsiveness to hepatitis B vaccines. Examples of such SNPs and haplotypes include those similar to 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 that detect these SNPs and 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."

The present invention will be explained below using examples, but the present invention is not limited to the following examples.

<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, manufactured by Chemo-Sero-Therapeutic Research Institute). Individuals who had received the Heptavax-II vaccine (manufactured by 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.

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 an anti-HBc II kit, respectively, and a fully automated chemiluminescent enzyme immunoassay system using an Architect i2000SR analyzer (Abbott Japan). Individuals with HBcAb positivity (>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 bengen. They were divided into the following three groups:
group_0, low response group, HBsAb ≦10 mIU/mL, n = 107.
group_1, intermediate response group, 10 mIU/mL < HBsAb ≤ 100 mIU/mL, n = 351.
group_2, high response group, 100 mIU/mL<HBsAb≦1000 mIU/mL, n=735.
The clinical information of the 1,193 individuals has been compiled by group at the following URL (http://onlinelibrary.wiley.com/doi/10.1002/hep.29876/suppinfo).
Similarly, 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 ≦10 mIU/mL, n = 66.
group_1, intermediate response group, 10 mIU/mL < HBsAb ≤ 100 mIU/mL, n = 124.
group_2, high response group, 100 mIU/mL<HBsAb, n=305.

[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.

[HLA imputation 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.

[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 carried out to compare the two groups in Status-1 and Status-2 shown below.

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

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 in a GWAS targeting Heptavax-II (P value < 0.05) showed a very high odds ratio (R2 = 0.8856).However, when comparing Heptavax-II vaccinated and Beamgen vaccinated patients in three groups, no SNPs were found to be genome-wide significant.
These findings indicate that GWAS was unable to detect significant differences in genes involved in immune responsiveness to the two types of hepatitis B vaccines.

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 ≦10 mIU/mL, n = 66.
group_1, intermediate response group, 10 mIU/mL < HBsAb ≤ 100 mIU/mL, n = 124.
group_2, high response group, 100 mIU/mL<HBsAb, n=305.

Next, the association between haplotypes and alleles in each response group was analyzed using the HLA imputation method.

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).

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. Since the odds ratio (OR) for each was greater than 1, it was suggested that having these haplotypes and alleles significantly increased the probability of being in group 0, meaning that individuals were more likely to be in the low response group.

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.

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 (OR) indicated that individuals with HLA-DRB1*04:05-DQB1*04:01 were significantly associated with a larger Heptavax-II group, meaning they were more likely to be in the high or intermediate-high response group in the Heptavax-II group. On the other hand, individuals with HLA-DRB1*13:02-DQB1*06:04 were significantly associated with a larger Beamgen group, meaning they were more likely to be in the high response group in the Beamgen 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 responder, intermediate responder, 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, respectively.

Next, we compared the relationship between HLA haplotypes and the high Bimugen and Heptavax-II responders, as well as the healthy control group. The results are shown in Table 5.

As shown in Table 5, HLA-DRB1*13:02-DQB1*06:04 showed a significant association when comparing the Heptavax-II high responder group with the healthy control group. On the other hand, no significant correlation was found for this haplotype between the healthy control group and the Bimugen high responder group. Furthermore, because the odds ratio (OR) when comparing the Heptavax-II high responder group with 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 group with HLA-DRB1*13:02-DQB1*06:04 was significantly more prevalent in the healthy control group than in the Heptavax-II high responder group, which indirectly suggests that groups with this haplotype are less likely to become high responders to Heptavax-II vaccination.

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

The results revealed that with HLA-DRB1*04:05, many antigen peptides from Heptavax-II showed stable binding. Furthermore, a search for antigen peptide sequences that were unstable with Heptavax-II but stable with Bimugen in binding to HLA-DRB1*13:02 revealed 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).

These 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; conversely, 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.

The method and reagent of this embodiment make it possible to easily and reliably detect genetic factors that contribute to immune responsiveness to hepatitis B vaccine in a subject.

Claims (4)

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) . 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. A reagent for detecting genetic factors of immune responsiveness to 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) , 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. 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.