Method for determining vaccine-induced immune response

Abstract

The purpose of the present invention is to provide a new marker that indicates the relevance between a vaccine and the immunity of a subject inoculated with the vaccine. More specifically, the purpose of the present invention is to provide, for example, a method for determining the waning of vaccine effect using a new marker correlated with a tendency of vaccine effect to wane. The problem is solved by providing a method for determining the waning of vaccine effect, the method comprising a step for detecting a mutation in an immunoglobulin heavy-chain region in DNA contained in a sample from a subject, wherein, when the mutation is detected in the immunoglobulin heavy-chain region, the vaccine effect in the subject is determined to have a high tendency to wane.

Description

Method for determining immune response to a vaccine

[0001] The present invention relates to a method for determining the attenuation of vaccine efficacy. The present invention also relates to a method for determining the immune responsiveness of a vaccine. The present invention further relates to a reagent for determining the immune responsiveness of a vaccine, and a reagent for determining the attenuation of vaccine efficacy.

[0002] The effectiveness of vaccines varies from person to person. Therefore, booster vaccinations may be necessary for people whose antibody titers do not increase easily after vaccination, or whose antibody titers tend to decrease after they increase.

[0003] Traditionally, antibody tests such as EIA-IgG, PA, NT, and HI have been used to measure antibody titers in the blood at regular intervals, and the decline in antibody titers has been continuously monitored to determine the timing of additional vaccination.

[0004] However, this method has the problem that it takes time to determine when to administer the booster vaccine, and it is not easy to carry out.

[0005] Meanwhile, there is a known technology for predicting vaccine efficacy using genomic mutations in specific regions as an indicator. For example, Patent Document 1 describes a method for detecting genetic factors affecting immune responsiveness to hepatitis B vaccines, in which a haplotype of HLA class II genes is detected in a DNA-containing sample derived from a subject, and if the haplotype detected is HLA-DRB1*13:02-DQB1*06:04, the subject is determined to have an immune responsiveness to Bimugen and no or low immune responsiveness to Heptavax-II.

[0006] Japanese Patent Application Laid-Open No. 2022-129868

[0007] However, to date, no markers other than HLA haplotypes that can determine the effects of vaccines have been reported, and there is a need for new markers.

[0008] Therefore, an object of the present invention is to provide a new marker that indicates the association between a vaccine and the immunity of a subject to be vaccinated with the vaccine. More specifically, the present invention provides a method for determining the attenuation of vaccine efficacy using a new marker that correlates with the tendency of vaccine efficacy to attenuate.

[0009] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have found for the first time that mutations in the immunoglobulin heavy chain region of chromosome 14 correlate with the tendency for vaccine efficacy to be attenuated. Further detailed studies have revealed that, among the immunoglobulin heavy chain regions of chromosome 14, mutations in the IGHG1 gene in particular correlate with the tendency for vaccine efficacy to be attenuated, and that mutations in rs1043109 and / or rs193160354 of the IGHG1 gene correlate with the tendency for vaccine efficacy to be attenuated. Furthermore, the present inventors have found that by combining mutations in the HLA region (e.g., mutations in rs4959098), which have previously been shown to be associated with immune responsiveness to HB vaccines, with mutations in rs1043109 and / or rs193160354 of the IGHG1 gene, the tendency for vaccine efficacy to be attenuated can be more accurately determined. Based on these findings, the present invention was completed.

[0010] That is, one aspect of the present invention relates to the following: [1] A method for determining attenuation of vaccine efficacy, comprising the step of detecting mutations in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and determining that the subject has a high tendency for vaccine efficacy to be attenuated if a mutation is detected in the immunoglobulin heavy chain region. [2] A method for determining immune responsiveness to a vaccine, comprising the step of detecting mutations in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and determining that the subject has a low immune responsiveness to the vaccine if a mutation is detected in the immunoglobulin heavy chain region. [3] The method of [1] or [2], further comprising the step of detecting mutations in an HLA region in the DNA in a DNA-containing sample derived from a subject. [4] The method of any of [1] to [3], wherein the mutation in the immunoglobulin heavy chain region is a mutation in the IGHG1 gene. [5] The method of any of [1] to [4], wherein the mutation in the immunoglobulin heavy chain region is a mutation in rs1043109 and / or rs193160354. [6] The method of any of [1] to [5], wherein the mutation in the immunoglobulin heavy chain region is a CC or CG mutation in rs1043109. [7] The method of any of [1] to [6], wherein the vaccine is a vaccine against a virus that causes a viral respiratory infection. [8] The method of any of [1] to [7], wherein the vaccine is a vaccine against at least one virus selected from the group consisting of coronavirus, influenza virus, and respiratory syncytial virus. [9] The method of any of [3] to [8], wherein the mutation in the HLA region is a mutation in rs4959098.

[10] The method of any of [3] to [9], wherein the mutation in the HLA region is an AA or AC mutation in rs4959098.

[11] The method according to any one of [3] to

[10] , wherein the mutation in the immunoglobulin heavy chain region is a CC or CG mutation in rs1043109, and the mutation in the HLA region is an AA or AC mutation in rs4959098.

[12] The method of any of [1] to

[11] , wherein the step of detecting a mutation in the immunoglobulin heavy chain region in the DNA in a DNA-containing sample derived from a subject is performed before or after vaccination.

[13] A reagent for determining attenuation of vaccine efficacy, comprising one or more nucleic acid probes or primers for detecting a mutation in the immunoglobulin heavy chain region in the DNA in a DNA-containing sample derived from a subject.

[14] A reagent for determining immune responsiveness to a vaccine, comprising one or more nucleic acid probes or primers for detecting a mutation in the immunoglobulin heavy chain region in the DNA in a DNA-containing sample derived from a subject.

[15] The reagent of

[13] or

[14] , further comprising one or more nucleic acid probes or primers for detecting a mutation in the HLA region in the DNA in a DNA-containing sample derived from a subject.

[16] The reagent of any of

[13] to

[15] , wherein the mutation in the immunoglobulin heavy chain region is a mutation in the IGHG1 gene.

[17] The reagent according to any of

[13] to

[16] , wherein the mutation in the immunoglobulin heavy chain region is a mutation in rs1043109 and / or rs193160354.

[18] The reagent according to any of

[13] to

[17] , wherein the mutation in the immunoglobulin heavy chain region is a CC or CG mutation in rs1043109.

[19] The reagent according to any of

[13] to

[18] , wherein the vaccine is a vaccine against a virus that causes a viral respiratory infection.

[20] The reagent according to any of

[13] to

[19] , wherein the vaccine is a vaccine against at least one virus selected from the group consisting of coronavirus, influenza virus, and respiratory syncytial virus.

[21] The reagent according to any of

[15] to

[20] , wherein the mutation in the HLA region is a mutation in rs4959098.

[22] The reagent according to any of

[15] to

[21] , wherein the mutation in the HLA region is an AA or AC mutation in rs4959098.

[23] The reagent according to any one of

[15] to

[22] , wherein the mutation in the immunoglobulin heavy chain region is a CC or CG mutation in rs1043109, and the mutation in the HLA region is an AA or AC mutation in rs4959098.

[24] A kit comprising the reagent according to any one of

[13] to

[23] .

[0011] According to the present invention, it is possible to determine the attenuation of vaccine efficacy in a subject using a marker other than HLA. Also, according to the present invention, it is possible to determine the immune responsiveness of a subject to a vaccine using a marker other than HLA. Furthermore, according to the present invention, by using a marker other than HLA in combination with HLA, it is possible to more accurately determine the vaccine efficacy and responsiveness of a subject. Therefore, the present invention is extremely useful in fields where vaccines are applied.

[0012] Figure 1 shows the results of a genome-wide association study on antibody titers after two doses of SARS-CoV-2 vaccine. Figure 2 shows the results of a genome-wide association study (HLA and IHG1) on antibody titers after two doses of SARS-CoV-2 vaccine. Figure 3 shows the results of calculating the odds ratio and P value for antibody positivity after two doses of SARS-CoV-2 vaccine. Figure 4 shows the results of calculating the odds ratio and P value for antibody positivity after two doses of SARS-CoV-2 vaccine, limited to mCA. Figure 5 shows the results of calculating the odds ratio and P value for antibody positivity after two doses of SARS-CoV-2 vaccine, limited to mCA including two loci (MHC, IGH) detected in the SARS-CoV-2 vaccine antibody GWAS. Figure 6 shows the results of a genome-wide association study on antibody titers after influenza vaccination. In the figure, A-H1: A / Victoria / 1 / 2020 (H1N1), A-H3: A / Darwin / 9 / 2021 (H3N2), B-Vi: B / Austria / 1359417 / 2021 (Victoria lineage), and B-Ya: B / Phuket / 3073 / 2013 (Yamagata lineage) are shown.

[0013] Hereinafter, an example of an embodiment of the present invention will be described in detail, but the present invention is not limited to this.

[0014] (1. Definitions) As used herein, "assessing the attenuation of vaccine efficacy" means assessing (predicting) the rate, period, etc. at which the vaccine efficacy will attenuate in a subject to be vaccinated or in a subject who has been vaccinated.

[0015] As used herein, "determining the immune responsiveness of a vaccine" means evaluating (predicting) the amount of antibody titer produced by a vaccine in a subject who will be vaccinated or who has been vaccinated.

[0016] As used herein, "mutation in an immunoglobulin heavy chain region" refers to a DNA single nucleotide polymorphism (SNP) in the immunoglobulin heavy chain region on chromosome 14, in the case of humans, in the region encompassing IGHG1.

[0017] As used herein, the term "subject" refers to a subject to whom the method of the present invention for determining the attenuation of vaccine effect or the method of the present invention for determining the immune responsiveness of a vaccine is provided.

[0018] As used herein, the term "DNA-containing sample derived from a subject" refers to a sample collected from the living body of a subject and containing DNA as a part thereof.

[0019] As used herein, the term "viral respiratory infection" refers to a respiratory disease caused by a virus infecting the respiratory tract. A "viral respiratory infection" is also sometimes referred to as a "viral respiratory tract disease."

[0020] As used herein, the term "vaccine" has the meaning generally used in the art, and refers to a pharmaceutical product used to prevent infectious diseases.

[0021] As used herein, "HLA" refers to the human major histocompatibility complex (MHC), located on human chromosome 6. HLA is a membrane protein that binds to foreign antigen peptides from grafts, bacteria, viruses, etc., and presents them to T cells.

[0022] As used herein, "IGHG1 (gene)" refers to a gene encoding immunoglobulin heavy constant gamma 1 (immunoglobulin heavy chain constant γ1), located in the immunoglobulin heavy chain region of human chromosome 14 (NCBI Gene ID: 3500). The function of the IGHG1 gene is predicted to be involved in antigen-binding activity and immunoglobulin receptor-binding activity.

[0023] As used herein, the term "kit" refers to a package that includes containers (e.g., vials, plates, tubes, dishes, etc.) that contain specific materials.

[0024] (2. Method for Determining Decay of Vaccine Efficacy) The method for determining the decline of vaccine efficacy of the present invention (hereinafter sometimes referred to as "the present method for determining vaccine efficacy") comprises the step of detecting mutations in the immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and if a mutation is detected in the immunoglobulin heavy chain region, the subject is determined to have a high tendency for the vaccine efficacy to decline. In the present method for determining vaccine efficacy, DNA is extracted from a DNA-containing sample derived from a subject (hereinafter sometimes simply referred to as "sample"), and mutations in the immunoglobulin heavy chain region in the DNA are detected.

[0025] The vaccine used in this method for determining attenuation is preferably a vaccine against a virus that causes a viral respiratory infection. The type of vaccine is not particularly limited, and may be, for example, a live vaccine, an inactivated vaccine, a virus-like particle (VLP), a recombinant protein vaccine, an RNA vaccine, a DNA vaccine, a viral vector vaccine, or a toxoid. In one embodiment of the present invention, the vaccine used in this method for determining attenuation may be a vaccine against a bacterial respiratory infection (e.g., a pneumococcal vaccine).

[0026] Examples of viruses that cause viral respiratory infections include coronaviruses (e.g., alphacoronaviruses (e.g., human coronavirus 229E, human coronavirus NL63, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus), betacoronaviruses (e.g., human coronavirus HKU1, human coronavirus OC43, SARS-CoV, MERS-CoV, SARS-CoV 2 (SARS-CoV-2), porcine hemagglutinating encephalomyelitis virus, bovine coronavirus), gammacoronavirus, deltacoronavirus), influenza viruses (e.g., influenza A virus, influenza B virus, influenza C virus, influenza D virus), respiratory syncytial virus, adenovirus, human metapneumovirus, cytomegalovirus, etc. Among these, coronaviruses and influenza viruses, which are highly infectious, are preferred.

[0027] The subject is not particularly limited, but may be, for example, an animal, such as a human or a non-human mammal. Non-human mammals include mice, rats, cows, monkeys, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, and hamsters. The subject is preferably a human.

[0028] Examples of DNA-containing samples derived from subjects 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 directly collected from a living body, etc. DNA can be extracted from these samples and used as a sample in the detection described below.

[0029] The mutation in the immunoglobulin heavy chain region is preferably a mutation in the IGHG1 gene. Furthermore, the mutation in the immunoglobulin heavy chain region is more preferably a mutation in rs1043109 and / or rs193160354. When the mutation in the immunoglobulin heavy chain region is a mutation in rs1043109, it is even more preferably a CC or CG mutation. rs1043109 and rs193160354 are SNPs in the IGHG1 gene locus, and each notation indicates the registration number in the NCBI SNP Database.

[0030] The method for extracting DNA from a sample is not particularly limited, and any method known in the art can be used. Examples include the phenol / chloroform method and the cetyltrimethylammonium bromide (CTAB) method. Commercially available kits may also be used for DNA extraction. Examples of such kits include the Wizard® Genomic DNA Purification Kit (Promega).

[0031] In one embodiment of the present invention, the DNA may be cDNA. cDNA can be produced by extracting mRNA from a sample and synthesizing it using the mRNA as a template. The method for extracting RNA from a sample is not particularly limited, and any method known in the art can be used. For example, the guanidine isothiocyanate method can be used. Commercially available kits can be used to extract mRNA. Examples of such kits include the Nucleotrap (registered trademark) mRNA Kit (manufactured by Clontech). The method for synthesizing cDNA is also not particularly limited, and any method known in the art can be used. For example, cDNA can be synthesized by RT-PCR using random primers or polyT primers.

[0032] Mutations in the immunoglobulin heavy chain region of a DNA-containing sample derived from a subject can be detected by any method known in the art, including direct sequencing, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), hybridization, TaqMan® PCR, and mass spectrometry.

[0033] Direct sequencing is performed by cloning a region (e.g., an immunoglobulin heavy chain region) containing the target gene, rs1043109, rs193160354, or the like, in a DNA-containing sample derived from a subject into a vector or amplifying it by PCR, and then determining the nucleotide sequence of the region. Cloning, PCR, and nucleotide sequence determination can be performed using any method known in the art. Based on the nucleotide sequence thus obtained, it is determined whether the sample contains a mutation in rs1043109 or rs193160354.

[0034] The polymerase chain reaction (PCR) method is carried out using oligonucleotide primers that hybridize only to a region containing rs1043109, rs193160354, etc. (for example, an immunoglobulin heavy chain region) or to a region other than those mentioned above.

[0035] The restriction fragment length polymorphism (RFLP) method involves PCR amplification of a region containing rs1043109, rs193160354, etc. (e.g., the immunoglobulin heavy chain region), and then cleaving the resulting PCR product with a restriction enzyme appropriate for the region. The restriction enzyme-digested PCR products are separated by gel electrophoresis and visualized by staining with ethidium bromide. The fragment lengths can be compared with molecular weight markers and with the PCR products not treated with restriction enzymes as controls to detect the presence of mutations in rs1043109 and rs193160354 in a sample.

[0036] The hybridization method is a method for determining the presence or absence of rs1043109 and rs193160354 mutations in a sample based on the ability of DNA in the sample to hybridize with complementary DNA molecules (e.g., oligonucleotide probes). This hybridization method can be performed using various hybridization and detection techniques, such as colony hybridization, plaque hybridization, and Southern blotting.

[0037] The TaqMan PCR method is a method in which, for example, detection of an SNP and amplification of a region containing the SNP are carried out simultaneously using TaqMan probes specific to rs1043109 and rs193160354 of the present invention and Taq polymerase.

[0038] An example of a method using mass spectrometry is a SNP typing method that applies MALDI-TOF / MS in combination with primer extension, which involves the following steps: 1) PCR, 2) purification of the PCR product, 3) primer extension reaction, 4) purification of the extension product, 5) mass spectrometry, and 6) genotype determination.

[0039] Other detection methods include methods that apply single molecule fluorescence analysis (for example, MF20 / 10S (Olympus)) and methods that use DNA chips.

[0040] An example of an SNP typing method that uses a gene amplification method other than PCR is the Snipper method, which is an application of the RCA method, a DNA amplification method in which DNA polymerase synthesizes a complementary strand of DNA while moving along a circular single-stranded DNA template.

[0041] 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.

[0042] The UCAN method is an application of the ICNA (isothermal gene amplification) method. In the UCAN method, a DNA-RNA-DNA chimeric oligonucleotide (DRD) is used as a primer precursor. When this DRD primer precursor is incubated with a template, an extension reaction by DNA polymerase proceeds, and the template DNA is amplified only if the DRD primer and template are perfectly matched.

[0043] The LAMP method involves defining six regions of a target gene (F3c, F2c, and F1c from the 3' end, and B3, B2, and B1 from the 5' end) and amplifying them using four primers (FIP primer, F3 primer, BIP primer, and 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. In the absence of an SNP, the amplification reaction proceeds continuously; in the presence of an SNP, the amplification reaction does not proceed.

[0044] The Invader method 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.

[0045] In this method for determining attenuation, if a mutation is detected in the immunoglobulin heavy chain region in the detection step, the subject is determined to have a high tendency for the vaccine effect to attenuate.

[0046] In one embodiment of the present invention, the tendency of the vaccine's effectiveness to decay can be relatively determined based on any criterion, including, but not limited to, the degree of decay (decay rate) of the vaccine's effectiveness in a subject in which no mutation is detected in the immunoglobulin heavy chain region and / or HLA region.

[0047] In one embodiment of the present invention, the method for assessing attenuation preferably further comprises the step of detecting mutations in the HLA region of DNA in a sample containing DNA derived from a subject. That is, it is preferable to combine the detection of mutations in the immunoglobulin heavy chain region with the detection of mutations in the HLA region. By including this step, the tendency for vaccine efficacy to decline can be assessed more accurately.

[0048] The mutation in the HLA region is preferably a mutation in rs4959098. When the mutation in the HLA region is a mutation in rs4959098, it is more preferably an AA or AC mutation. rs4959098 is an SNP in the HLA locus, and each notation indicates the registration number in the NCBI SNP Database.

[0049] The combination of a mutation in the immunoglobulin heavy chain region and a mutation in the HLA region may be any combination of rs1043109, rs193160354, and rs4959098. The combination of a mutation in the immunoglobulin heavy chain region and a mutation in the HLA region may be a combination of known SNPs other than the above SNPs.

[0050] When the mutation in the immunoglobulin heavy chain region is a mutation in rs1043109 and the mutation in the HLA region is a mutation in rs4959098, the antibody titer is more likely to decrease (the tendency to attenuate) in the order shown below.・rs1043109 is CC rs4959098 is AA (antibody titer is most likely to decrease) ・rs1043109 is CC rs4959098 is AC ・rs1043109 is CC rs4959098 is CC ・rs1043109 is CG rs4959098 is AA ・rs1043109 is CG rs4959098 is AC ・rs1043109 is CG rs4959098 is CC ・rs1043109 is GG rs4959098 is AA ・rs1043109 is GG rs4959098 is AC ・rs1043109 is GG rs4959098 is CC (antibody titer is least likely to decrease)

[0051] In one embodiment of the present invention, the mutation in the immunoglobulin heavy chain region may be a CC or CG mutation in rs1043109, and the mutation in the HLA region may be an AA or AC mutation in rs4959098.

[0052] In another embodiment of the present invention, the step of detecting mutations in the immunoglobulin heavy chain region in DNA from a DNA-containing sample derived from a subject can be performed before or after vaccination. Whether performed before or after vaccination, a booster vaccination can be recommended for subjects determined to have a high tendency to attenuate.

[0053] In another embodiment of the present invention, the following methods are provided: [1-1] A method for determining attenuation of vaccine efficacy, comprising a step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and determining that the subject has a higher tendency for vaccine efficacy to attenuate when a mutation is detected in the immunoglobulin heavy chain region, compared to a subject in which no mutation is detected in the immunoglobulin heavy chain region. [1-2] A method for determining attenuation of vaccine efficacy, comprising a step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and determining that the subject has a faster tendency for vaccine efficacy to attenuate when a mutation is detected in the immunoglobulin heavy chain region. [1-3] A method for determining attenuation of vaccine efficacy, comprising a step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and determining that the subject has a faster tendency for vaccine efficacy to attenuate when a mutation is detected in the immunoglobulin heavy chain region, compared to a subject in which no mutation is detected in the immunoglobulin heavy chain region. [1-4] A method for determining the duration of vaccine effect, comprising the step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a sample containing DNA derived from a subject, and determining that the subject has a low duration of vaccine effect if a mutation is detected in the immunoglobulin heavy chain region. [1-5] A method for determining the duration of vaccine effect, comprising the step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a sample containing DNA derived from a subject, and determining that the subject has a low duration of vaccine effect if a mutation is detected in the immunoglobulin heavy chain region, compared to a subject in which no mutation is detected in the immunoglobulin heavy chain region.

[0054] In another embodiment of the present invention, the following methods are provided: [1-6] A method for assisting in the determination of attenuation of vaccine efficacy, comprising the step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and predicting that the subject will have a high tendency for the vaccine efficacy to attenuate if a mutation is detected in the immunoglobulin heavy chain region. [1-7] A method for assisting in the determination of attenuation of vaccine efficacy, comprising the step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and predicting that the subject will have a high tendency for the vaccine efficacy to attenuate compared to a subject in which no mutation is detected in the immunoglobulin heavy chain region. [1-8] A method for assisting in the determination of attenuation of vaccine efficacy, comprising the step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and predicting that the subject will have a faster tendency for the vaccine efficacy to attenuate if a mutation is detected in the immunoglobulin heavy chain region. [1-9] A method for assisting in determining the attenuation of vaccine effectiveness, comprising the step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, wherein if a mutation is detected in the immunoglobulin heavy chain region, the subject predicts that the vaccine effectiveness will attenuate more quickly compared to a subject in whom no mutation is detected in the immunoglobulin heavy chain region. [1-10] A method for assisting in determining the duration of vaccine effectiveness, comprising the step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, wherein if a mutation is detected in the immunoglobulin heavy chain region, the subject predicts that the vaccine effectiveness will last longer. [1-11] A method for assisting in determining the duration of vaccine effectiveness, comprising the step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, wherein if a mutation is detected in the immunoglobulin heavy chain region, the subject predicts that the vaccine effectiveness will last longer compared to a subject in whom no mutation is detected in the immunoglobulin heavy chain region.

[0055] (3. Method for determining immune responsiveness of a vaccine) The method for determining immune responsiveness of a vaccine of the present invention (hereinafter sometimes referred to as the "method for determining immune responsiveness") comprises the step of detecting mutations in the immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and determining that the subject has a low immune responsiveness to the vaccine if a mutation is detected in the immunoglobulin heavy chain region. It is generally assumed that a high tendency for vaccine efficacy to decline and low immune production (low immune responsiveness) are correlated (see, for example, Menegale et al., JAMA Network Open. 2023;6(5):e2310650. doi:10.1001 / jamanetworkopen.2023.10650).

[0056] In this method for determining immune responsiveness, the same description as above (2. Method for determining attenuation of vaccine effect) is applied except for the determination of immune responsiveness.

[0057] In the present method for determining immune responsiveness, if a mutation is detected in the immunoglobulin heavy chain region in the detection step, the subject is determined to have a low immune responsiveness to the vaccine.

[0058] In one embodiment of the present invention, the immune responsiveness of a vaccine can be relatively determined based on any criteria, including, but not limited to, the immune responsiveness of a vaccine to a subject in which no mutations are detected in the immunoglobulin heavy chain region and / or HLA region.

[0059] In another embodiment of the present invention, the following method is provided: [2-1] A method for determining immune responsiveness to a vaccine, comprising a step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and when a mutation is detected in the immunoglobulin heavy chain region, the subject is determined to have a lower immune responsiveness to a vaccine compared to a subject in which no mutation is detected in the immunoglobulin heavy chain region.

[0060] In another embodiment of the present invention, the following methods are provided: [2-2] A method for assisting in determining immune responsiveness to a vaccine, comprising the step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and predicting that the subject will have a low immune responsiveness to a vaccine if a mutation is detected in the immunoglobulin heavy chain region. [2-3] A method for assisting in determining immune responsiveness to a vaccine, comprising the step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and predicting that the subject will have a low immune responsiveness to a vaccine compared to a subject in which no mutation is detected in the immunoglobulin heavy chain region.

[0061] (4. Reagents) In one embodiment of the present invention, a reagent for determining the attenuation of vaccine efficacy is provided, which comprises one or more nucleic acid probes or primers for detecting mutations in the immunoglobulin heavy chain region of DNA in a DNA-containing sample derived from a subject (hereinafter, this may be referred to as "Reagent 1"). In another embodiment of the present invention, a reagent for determining the immune responsiveness of a vaccine is provided, which comprises one or more nucleic acid probes or primers for detecting mutations in the immunoglobulin heavy chain region of DNA in a DNA-containing sample derived from a subject (hereinafter, this may be referred to as "Reagent 2"). Furthermore, "Reagent 1" and "Reagent 2" will be collectively referred to as "Reagent" unless there is a need to distinguish between them.

[0062] The present reagent comprises one or more nucleic acid probes or primers for detecting mutations in the immunoglobulin heavy chain region in DNA in a sample containing the DNA derived from a subject.

[0063] (Nucleic Acid Probe) The nucleic acid probe contained in the present reagent is not particularly limited as long as it is a probe composed of a nucleic acid that can detect a mutation in the immunoglobulin heavy chain region in DNA in a sample containing DNA derived from a subject.

[0064] The nucleic acid probe contained in this reagent is designed so that the probe recognizes an 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 an SNP detection polynucleotide 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.

[0065] In one embodiment of the present invention, the nucleic acid probe contained in the present reagent is preferably a nucleic acid probe that can detect a mutation in a region containing the IGHG1 gene in the immunoglobulin heavy chain region of DNA in a DNA-containing sample derived from a subject.

[0066] In one embodiment of the present invention, the nucleic acid probe contained in the reagent is particularly preferably a nucleic acid probe capable of detecting, in a DNA-containing sample derived from a subject, mutations at rs1043109 and / or rs193160354 in the IGHG1 gene in the immunoglobulin heavy chain region of the DNA. In this embodiment, the nucleic acid probe may be a nucleic acid probe capable of detecting mutations at rs1043109 and rs193160354 individually, or a nucleic acid probe capable of simultaneously detecting mutations at rs1043109 and rs193160354.

[0067] In one embodiment of the present invention, the reagent preferably further comprises one or more nucleic acid probes for detecting a mutation in the HLA region in DNA in a DNA-containing sample derived from a subject. In this embodiment, the nucleic acid probe is particularly preferably a probe capable of detecting a mutation in HLA rs4959098 in the DNA in a DNA-containing sample derived from a subject.

[0068] (Primers) The primers contained in the present reagent are not particularly limited as long as they are primers that can amplify an immunoglobulin heavy chain region in DNA in a sample containing DNA derived from a subject and detect mutations in said region.

[0069] The primers contained in this reagent are designed to be suitable 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 usable 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.

[0070] In one embodiment of the present invention, the primers contained in the present reagent are preferably primers that can amplify a region containing the IGHG1 gene in the immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and detect mutations in the gene.

[0071] In one embodiment of the present invention, the primers contained in the reagent are particularly preferably primers that can amplify a region containing rs1043109 and / or rs193160354 in the IGHG1 gene in the immunoglobulin heavy chain region in a DNA-containing sample derived from a subject, and detect mutations in rs1043109 and / or rs193160354. In this embodiment, the primers may be primers that can amplify the regions containing rs1043109 and rs193160354, respectively, or may be primers that can simultaneously amplify the regions containing rs1043109 and rs193160354.

[0072] In one embodiment of the present invention, the reagent preferably further comprises one or more primers for detecting a mutation in the HLA region in DNA in a DNA-containing sample derived from a subject. Furthermore, in one embodiment of the present invention, the primers are particularly preferably primers capable of amplifying a region containing HLA rs4959098 in the DNA in a DNA-containing sample derived from a subject and detecting a mutation in rs4959098.

[0073] (5. Kit) In one embodiment of the present invention, a kit is provided that includes the present reagent 1. In another embodiment of the present invention, a kit is provided that includes the present reagent 2. These two kits are hereinafter collectively referred to as "the present kit."

[0074] The kit may contain components commonly used in this type of kit, such as distilled water, salts, proteins, surfactants, reaction buffer, MgSO, deoxynucleotide triphosphates (dNTPs), DNA polymerase, etc. The kit may further contain an instruction manual, instructions for synthesizing the nucleic acid of the present invention, or other package inserts.

[0075] This kit is widely used in the vaccine field to determine the attenuation of vaccine efficacy, the immune response of vaccines, etc.

[0076] The present invention will be described in more detail below using examples, but these are not intended to limit the scope of the present invention. Note that all references cited throughout this specification are incorporated herein by reference in their entirety.

[0077] (1. Genome-wide association analysis) The following QC was performed on data obtained by genotyping the genomic DNA of 2,271 vaccine recipients at Keio University using the Asian Screening Array chip.

[0078] <Sample QC> Based on the information from high-quality SNPs obtained through genotyping, the genome analysis tool PLINK was used to estimate the gender of each sample and the kinship relationships between samples. Consistency between the gender in clinical information and the gender estimated from the genotype was confirmed. Furthermore, samples estimated to be from outside the mainland in principal component analysis were excluded from the analysis to prevent bias due to population structure. As a result of sample QC, 2,137 samples remained.

[0079] <Variant QC> To remove SNPs that could not be successfully genotyped using the ASA chip, SNPs that deviated significantly from Hardy-Weinberg equilibrium were excluded. Furthermore, all called samples showed a call rate of 98% or higher. SNPs with a call rate of >99% and SNPs with minor allele counts of less than 5 in the entire dataset were excluded. SNPs were compared with existing allele frequency information from the Japanese population and SNPs showing a difference of 5% or more in allele frequency were excluded. As a result of variant QC, 537,027 SNPs on the autosomes and X chromosome were obtained at a level suitable for analysis.

[0080] <Genotype imputation> Variants were imputed using the haplotype inference tool Shapeit4 and the genotype imputation tool Minimac4 with a reference panel (n = 4,561) derived from Japanese whole-genome sequencing data. Imputed variants were selected using cutoffs of Rsq ≥ 0.3 and minor allele frequency ≥ 0.005, resulting in 9,113,947 variants in the autosomes and X chromosome.

[0081] <GWAS for quantitative traits> A genome-wide association study (GWAS) was conducted treating antibody titers and T cell immunity (CD4, CD4 / 8) as quantitative traits. The test was performed using Regenie, which can perform association analyses taking into account the kinship between samples. Covariates included vaccine type, age, sex, age 2, age x sex, age 2 x sex, and the top 10 principal components of genome-wide genotypes.

[0082] Antibody titers: Antibody titers were measured in n = 2,137 individuals. The class II HLA region on chromosome 6 (rs4959098, P = 3.1 × 10-12) and the immunoglobulin heavy chain region on chromosome 14 (rs1043109 and rs193160354, P = 4.8 × 10-14) showed genome-wide significant associations (Figures 1 and 2). The two lead variants in the immunoglobulin heavy chain region on chromosome 14, rs1043109 and rs193160354, showed identical statistical significance and were completely linked in the imputed reference panel used in this study. Both of these SNPs were located in exons of the IGHG1 gene; rs1043109 was a missense mutation, and rs193160354 was a synonymous substitution. Furthermore, when referring to the 1000 Genomes Project and gnomAD, it showed population specificity, being a rare variant with a frequency of less than 0.005 in populations other than East Asians.

[0083] (2. Association Analysis with Mosaic Chromosome Alterations (mCA)) Next, we analyzed the association with mCA on autosomes. For each chromosome, mCA carriers were defined separately for the short and long arms for loss and CN-LOH, and for gain, regardless of whether they were mCA carriers. A definition (Any) was also prepared that combined the presence or absence of mCA on both the short and long arms regardless of type. For analysis, odds ratios and p-values ​​for antibody positivity after two doses of vaccine were calculated using the Cochran-Mantel-Haenszel test stratified by sex and 5-year age bins according to (Loh PR 2020). The data consisted of gain = 2,388, loss = 3,718, copy-neutral LOH = 8,184, and unknown = 5,339. Results showed a negative association with mCA on 6p, particularly CN-LOH on 6p. An association was also observed with mCA on 14q (Figure 3).

[0084] Next, because the mCA cell ratio is also considered important in terms of its impact on antibody production, we limited the analysis to those with a cell fraction >10% (the cutoff for expanded mCA in Zekavat SM 2021). When we performed a similar analysis limited to expanded mCA, mCA at 14q showed the strongest association (Figure 4).

[0085] When limited to expanded mCA, the chromosomal regions containing 6p and 14q, two loci (MHC, IGH) detected in the vaccine antibody GWAS, showed the strongest association. Therefore, we performed an analysis limited to mCA containing these regions and confirmed significant associations in both regions (Figure 5).

[0086] (3. Evaluation of SNP combinations of IGHG1 and HLA) IGHG1 and HLA were evaluated for SNP and allele combinations that are likely to reduce antibody titers after vaccination. Based on the data obtained above (2. Association analysis with mosaic chromosome mutation (mCA)), the pattern rs1043109 GG, rs4959098 CC, which is the pattern least likely to reduce antibody titers, was used as the standard, and the SD (standard deviation) of the effect on antibody titer reduction was calculated for other combinations. The results are shown in Table 1.

[0087]

[0088] As is clear from Table 1, it was found that the antibody titer is most likely to decrease with the SNP (rs1043109) in the IGHG1 region in the order CC, CG, and GG. Furthermore, it has been previously reported that the antibody titer is most likely to decrease with the SNP (rs4959098) in the HLA region in the order AA, AC, and CC. When the SNP (rs1043109) in the IGHG1 region and the SNP (rs4959098) in the HLA region were evaluated in combination, it was found that the antibody titer was most likely to decrease with the SNP (rs1043109) in the IGHG1 region in CC and the SNP (rs4959098) in the HLA region in AA.

[0089] (4. Influenza Vaccine Antibody Titer Analysis) Influenza vaccine antibody titer analysis was performed using Sanger sequencing results. Specifically, the analysis was performed using antibody titer data from 2021 / 2022 influenza vaccine recipients from Keio University. The data shows measurement data for the following four types: Type A: A / Victoria / 1 / 2020 (H1N1), A / Darwin / 9 / 2021 (H3N2) Type B: B / Phuket / 3073 / 2013 (Yamagata lineage), B / Austria / 1359417 / 2021 (Victoria lineage) Of the above, A (H1N1) and B (Yamagata) correspond to the vaccinated strains for the 2021 / 2022 vaccine.

[0090] Associations among variants detected in SARS-CoV-2 vaccine GWAS: Previously performed association analyses were performed using genotypes obtained by Sanger sequencing on (1) log-transformed trait values, (2) int-transformed residuals obtained by adjusting the log-transformed values ​​for covariates, (3) the difference between log-transformed values ​​before and after influenza vaccination, and (4) int-transformed residuals obtained by adjusting the difference for covariates. The covariates used in the association analysis were age, sex, age 2, age × sex, age 2 × sex, and the top 10 principal components. A significant association was observed for the nominal variables, although it did not correspond to the vaccine strain. For 572 recipients of the Pfizer vaccine, information on influenza antibody titers before and after influenza vaccination was available, so we checked whether an effect beyond the vaccine type was observed. As a result, an overall negative association was observed for influenza vaccines (Figure 6).

[0091] The present invention is extremely useful, particularly in the field of vaccines, because it enables assessment of the attenuation of vaccine efficacy, assessment of vaccine immune responsiveness, etc. This application is based on Japanese Patent Application No. 2024-016714 (filing date: February 6, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A method for determining the attenuation of vaccine effectiveness, comprising the step of detecting a mutation in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and determining that the subject has a high tendency for the vaccine effectiveness to be attenuated if a mutation is detected in the immunoglobulin heavy chain region.

2. A method for determining immune responsiveness to a vaccine, comprising the step of detecting mutations in an immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject, and determining that the subject has a low immune responsiveness to a vaccine if a mutation is detected in the immunoglobulin heavy chain region.

3. The method of claim 1 or 2, further comprising the step of detecting mutations in the HLA region in DNA in a DNA-containing sample derived from a subject.

4. The method according to claim 1 or 2, wherein the mutation in the immunoglobulin heavy chain region is a mutation in the IGHG1 gene.

5. The method according to claim 1 or 2, wherein the mutation in the immunoglobulin heavy chain region is a mutation of rs1043109 and / or rs193160354.

6. The method of claim 1 or 2, wherein the mutation in the immunoglobulin heavy chain region is a CC or CG mutation of rs1043109.

7. The method according to claim 1 or 2, wherein the vaccine is a vaccine against a virus that causes a viral respiratory infection.

8. The method according to claim 1 or 2, wherein the vaccine is a vaccine against at least one virus selected from the group consisting of coronavirus, influenza virus, and respiratory syncytial virus.

9. The method according to claim 3, wherein the mutation in the HLA region is a mutation in rs4959098.

10. The method of claim 3, wherein the mutation in the HLA region is an AA or AC mutation in rs4959098.

11. The method of claim 3, wherein the mutation in the immunoglobulin heavy chain region is a CC or CG mutation in rs1043109, and the mutation in the HLA region is an AA or AC mutation in rs4959098.

12. The method of claim 1 or 2, wherein the step of detecting mutations in the immunoglobulin heavy chain region in the DNA of a DNA-containing sample derived from the subject is carried out before or after vaccination.

13. A reagent for determining the attenuation of vaccine efficacy, comprising one or more nucleic acid probes or primers that detect mutations in the immunoglobulin heavy chain region in a DNA-containing sample derived from a subject.

14. A reagent for determining immune responsiveness to a vaccine, comprising one or more nucleic acid probes or primers that detect mutations in the immunoglobulin heavy chain region in DNA in a DNA-containing sample derived from a subject.

15. The reagent according to claim 13 or 14, further comprising one or more nucleic acid probes or primers for detecting mutations in the HLA region in DNA in a DNA-containing sample derived from a subject.

16. The reagent according to claim 13 or 14, wherein the mutation in the immunoglobulin heavy chain region is a mutation in the IGHG1 gene.

17. The reagent according to claim 13 or 14, wherein the mutation in the immunoglobulin heavy chain region is a mutation of rs1043109 and / or rs193160354.

18. The reagent described in claim 13 or 14, wherein the mutation in the immunoglobulin heavy chain region is a CC or CG mutation of rs1043109.

19. The reagent according to claim 13 or 14, wherein the vaccine is a vaccine against a virus that causes a viral respiratory infection.

20. The reagent according to claim 13 or 14, wherein the vaccine is a vaccine against at least one virus selected from the group consisting of coronavirus, influenza virus, and respiratory syncytial virus.

21. The reagent according to claim 15, wherein the mutation in the HLA region is a mutation in rs4959098.

22. The reagent described in claim 15, wherein the mutation in the HLA region is the AA or AC mutation of rs4959098.

23. The reagent described in claim 15, wherein the mutation in the immunoglobulin heavy chain region is a CC or CG mutation in rs1043109, and the mutation in the HLA region is an AA or AC mutation in rs4959098.

24. A kit comprising the reagent according to claim 13 or 14.

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