Method for detecting infection with aids virus

Abstract

The purpose of the present invention is to provide a method for detecting whether a subject is infected with AIDS virus (HIV) or is in a latent HIV infection state, or whether the subject is suspected of being infected with HIV or being in a latent HIV infection state.　The present invention provides a method for determining whether a mammalian subject is infected with HIV or is in a latent HIV infection state, or whether the mammalian subject is suspected of being infected with HIV or being in a latent HIV infection state, the method involving: a step for detecting the amount of 3-methylcytidine (m3C) in a sample derived from the subject; and a step for providing information on the detected amount for the determination.

Description

Methods for detecting HIV infection

[0001] The present invention relates to a method for detecting a subject who is infected with the AIDS virus (HIV), is in a latent HIV infection state, or is suspected of being infected with HIV. More specifically, the present invention relates to a method for detecting a subject who is infected with HIV, is in a latent HIV infection state, or is suspected of being infected with HIV, by using a modified nucleoside as a target.

[0002] According to a WHO report, in 2023, 1.3 million people worldwide were infected with HIV, 630,000 died from AIDS, and nearly 39 million people lived with HIV on a daily basis. The WHO has proposed the global elimination of HIV by 2030 as one of the Sustainable Development Goals (SDGs) targets for the entire world.

[0003] Currently, various HIV screening tests are used to detect HIV infection. In Japan, fourth-generation HIV screening tests are widely used. For example, Dynascreen TM One example is HIV Combo (https: / / www.globalpointofcare.abbott / jp / ja / product-details / alere-hiv-corobo.html). Fourth-generation tests simultaneously test for HIV antibodies and HIV antigens, significantly shortening the time from HIV infection to a positive test result, allowing detection in as little as 17 days. In addition, the PCR method, an assay method that extracts HIV RNA from the virus in plasma and quantifies the HIV genome copy number by RT-PCR, is widely used.

[0004] Once a diagnosis of HIV infection is made through testing, the patient is given antiviral therapy. However, existing antiviral drugs inhibit the replication of the HIV virus, meaning that infected individuals must continue taking antiviral medication for life.

[0005] Furthermore, while antiviral therapy reduces HIV RNA copy numbers to below the detection limit in many cases, this does not mean that HIV is eliminated from the body. Because HIV can remain latent within the human body by being integrated into the human genome, establishing a complete cure is extremely difficult. In such cases, an assay system is used in which genomic DNA is prepared from white blood cells in the blood, and the proviral genome integrated into the human genome is amplified by PCR. However, this assay method is time-consuming, costly, and labor-intensive, so a simpler method is desired. For these reasons, there is a strong need for biomarkers to monitor the pathogenesis of HIV infection while taking antiviral drugs, and markers that reflect the state of latent HIV infection.

[0006] The inventors have previously clarified and reported the physiological significance of chemical modifications in ribonucleic acid (RNA) in mammals. In the process, they discovered that modified nucleic acids are broken down into nucleosides and excreted outside the cell, and established a method for comprehensively analyzing modified nucleosides in blood and urine (Non-Patent Literature 1). It is known that some of these modified nucleosides are common across species, while others are species-specific.

[0007] The inventors also investigated modified nucleosides, 6-threonylcarbamoyladenosine (t) in blood or urine. ６ A) and / or 2-thiomethyl,6-threonylcarbamoyladenosine (ms ２ t ６ A method has been reported for determining whether a subject from which blood or urine is likely to be infected with COVID-19 or is infected with it, by measuring the amount of A).

[0008] WO2022 / 191244

[0009] Ogawa et al., Molecular Cell 81, 659-674, 2021. Nagayoshi Y, et al., Biomolecules. 2022 Sep 3;12(9):1233. doi: 10.3390 / biom12091233.

[0010] The object of the present invention is to provide a method for detecting a subject who is infected with HIV, is in a latent HIV infection state, or is suspected of being infected with HIV.

[0011] The inventors comprehensively analyzed modified nucleosides in the blood of HIV-infected individuals and healthy individuals using a mass spectrometer. As a result, compared to healthy individuals, 3-methylcytidine (m) was found to be more prevalent in HIV-infected individuals and HIV-infected individuals taking antiviral drugs. ３ The present invention was completed by finding that the amount of C) was significantly low. The present invention includes the following: [1] A method for determining whether a mammalian subject is infected with the AIDS virus (HIV), is in a latent HIV infection state, or is suspected to be so, wherein the amount of 3-methylcytidine (m) in a sample derived from the subject is ３ A method comprising the steps of detecting an amount of C) and providing information of the detected amount for the determination. [2] The method according to [1], wherein the information of the detected amount is the amount of 3-methylcytidine detected in the sample, corrected using an internal standard (e.g., cytidine). [3] The method according to [1] or [2], wherein the information of the detected amount is information that can be used to diagnose whether a subject is infected with the AIDS virus (HIV), is in a latent HIV infection state, or is suspected to be infected, by comparing the amount of 3-methylcytidine detected in the sample with a predetermined reference value. [4] The method according to any one of [1] to [3], wherein the sample derived from the subject is plasma, serum, or urine. [5] The method according to any one of [1] to [4], wherein the subject is a human. [6] The method according to any one of [1] to [5], wherein the detection of the amount of 3-methylcytidine is performed by mass spectrometry (preferably tandem mass spectrometry (MS / MS)). [7] The sample is the sample that has undergone deproteinization and desalting treatment as described in [6] above. [8] The amount of 3-methylcytidine is detected by the ELISA method as described in any one of [1] to [5] above.

[0012] [9] A method for evaluating the therapeutic effect in a subject that is a mammal infected with the AIDS virus (HIV), wherein 3-methylcytidine (m) is present in a sample derived from the subject. ３ A method comprising the steps of detecting an amount of C) and providing information of the detected amount for the evaluation.

[10] The method according to [9], wherein the information of the detected amount is the amount of 3-methylcytidine detected in the sample, corrected using an internal standard (e.g., cytidine).

[11] The method according to [9] or

[10] , further comprising providing information of the amount of 3-methylcytidine previously detected in the same subject, together with the information of the detected amount.

[12] The method according to any one of [9] to

[11] , wherein the sample derived from the subject is plasma, serum, or urine.

[13] The method according to any one of [9] to

[12] , wherein the subject is human.

[14] The method according to any one of [9] to

[13] , wherein the detection of the amount of 3-methylcytidine is performed by mass spectrometry (preferably tandem mass spectrometry (MS / MS)).

[15] The method according to

[14] , wherein the sample is a deproteinized and desalted sample.

[16] The amount of 3-methylcytidine is detected by any one of the methods described in [9] to

[13] above, using the ELISA method.

[0013] The present invention also relates to a method for diagnosing whether a mammalian subject is infected with the AIDS virus (HIV), is in a latent HIV infection state, or is suspected to be so, comprising any of the following embodiments:

[17] A method for diagnosing whether a mammalian subject is infected with the AIDS virus (HIV), is in a latent HIV infection state, or is suspected to be so, comprising: 3-methylcytidine (m ３A method for diagnosing whether a subject is infected with HIV, in a latent HIV infection state, or suspected thereof, comprising the step of detecting the amount of (C), and diagnosing based on the information of the detected amount.

[18] The method for diagnosing according to

[17] above, wherein the information on the detected amount is the amount of 3-methylcytidine detected in a sample corrected using an internal standard substance (for example, cytidine).

[19] The method for diagnosing according to

[17] or

[18] above, wherein the diagnosing step is a step of diagnosing whether a subject is infected with HIV, in a latent HIV infection state, or suspected thereof by comparing the amount of the 3-methylcytidine in the detected sample with a predetermined reference value.

[20] The method for diagnosing according to any one of

[17] to

[19] above, wherein the sample derived from the subject is plasma, serum, or urine.

[21] The method according to any one of

[17] to

[20] above, wherein the subject is a human.

[22] The method for diagnosing according to any one of

[17] to

[21] above, wherein the detection of the amount of 3-methylcytidine is performed by mass spectrometry (preferably, tandem mass spectrometry (MS / MS) method).

[23] The method for diagnosing according to

[22] above, wherein the sample is a sample that has been subjected to protein removal treatment and desalting treatment.

[24] The method for diagnosing according to any one of

[17] to

[21] above, wherein the detection of the amount of 3-methylcytidine is performed by ELISA method.

[25] A method for evaluating the treatment effect and determining the treatment policy in a subject which is a mammal infected with the AIDS virus (HIV), comprising, in a sample derived from the subject, 3-methylcytidine (m ３A diagnostic method comprising a step of detecting the amount of (C), and a step of evaluating a treatment effect based on the detected amount and determining a treatment policy.

[26] The diagnostic method according to

[25] above, wherein the information on the detected amount is the amount of 3-methylcytidine detected in a sample corrected using an internal standard substance (for example, cytidine).

[27] Further, comparing the amount of 3-methylcytidine previously detected in the same subject with the detected amount, evaluating the treatment effect in the subject, and determining a treatment policy, the diagnostic method according to

[25] or

[26] above.

[28] The diagnostic method according to any one of

[25] to

[27] above, wherein the sample derived from the subject is plasma, serum, or urine.

[29] The diagnostic method according to any one of

[25] to

[28] above, wherein the subject is a human.

[30] The diagnostic method according to any one of

[25] to

[29] above, wherein the detection of the amount of 3-methylcytidine is performed by a mass spectrometry method (preferably, tandem mass spectrometry (MS / MS) method).

[31] The diagnostic method according to

[30] above, wherein the sample is a sample that has been subjected to protein removal treatment and desalting treatment.

[32] The diagnostic method according to any one of

[26] to

[29] above, wherein the detection of the amount of 3-methylcytidine is performed by an ELISA method.

[0014] By the method of the present invention, it is possible to determine whether a subject is infected with the AIDS virus (HIV) or in a state of latent HIV infection, or is suspected thereof. By the method of the present invention, it is also possible to evaluate the treatment effect in a subject.

[0015] Results of comprehensively analyzing modified nucleic acids in plasma for healthy individuals, untreated HIV-positive individuals, and HIV-positive individuals during ART (taking antiviral agents). The average value of healthy individuals is set to 1. For healthy individuals, untreated HIV-positive individuals, and HIV-positive individuals during ART (taking antiviral agents), m in plasma ３ Results of measuring the amount of C. The data represents the average value ± SEM, and the points indicate the measurement results of individual subjects (the same applies in the following drawings unless otherwise specified). m in cultured cells infected with HIV-1 ３ Results of measuring the amount of C. For healthy Japanese individuals and HIV-positive individuals during ART, m in plasma ３This is the result of comparing the amount of mC. Taking the average value of the mC amount in healthy individuals as 1, the mC amount in each sample (subject) is shown as a relative value (log). ３ This is the measurement result (nM) of the mC amount in the plasma and urine of healthy Japanese individuals and HIV-positive individuals during ART. ３ ２ This is the result of comparing the amount of mC. Taking the average value of the mC amount in healthy individuals as 1, the mC amount in each sample (subject) is shown as a relative value (log). ３ This is the measurement result (nM) of the mC amount in the plasma and urine of healthy Japanese individuals and HIV-positive individuals during ART.

[0016] Hereinafter, the present invention will be described with exemplary embodiments as examples, along with preferred methods and materials that can be used in the implementation of the present invention. However, the present invention is not limited to the embodiments described below. Unless otherwise specified in the text, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains. Also, any materials and methods equivalent or similar to those described in this specification can be similarly used in the implementation of the present invention. Additionally, all publications and patents cited in this specification in relation to the present invention are incorporated herein by reference and constitute a part of this specification, for example, indicating methods, materials, etc. that can be used in the present invention.

[0017] In this specification, the description "A to B" indicating a numerical range means a numerical range including the endpoints A and B. Also, in this specification, "about" is used in the sense of allowing ±10%. When intending the lower limit, it is used in the sense of allowing minus 10%, and when intending the upper limit, it is used in the sense of allowing plus 10%.

[0018] In this specification, ART or antiretroviral therapy means a drug therapy for the treatment of HIV infection, which means administering various antiretroviral agents to a patient. Although ART can suppress the growth of HIV, it is difficult to completely eliminate HIV from the body. By appropriately continuing ART, HIV-infected individuals can keep the viral load below the detection limit and prevent the progression of the infection.

[0019] ​In this specification, "latent HIV infection" means a state in which HIV has not completely disappeared and remains in the body, but the amount of HIV in the blood is suppressed to below the detection limit of conventional detection methods by ART, indicating a state in which HIV infection is managed and its progression is suppressed by ART. Conventional detection methods include, for example, HIV RNA quantification methods that detect HIV RNA in the blood by PCR, and "below the detection limit" means the detection limit value of the measurement method used, for example, 100 to 200 copies / mL or less.

[0020] In this invention, "subject" or "patient" refers to, but is not limited to, any mammal, including, humans; non-human primates including chimpanzees, other apes and monkey species; domestic animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; and small animals or laboratory animals including rodents such as mice, rats and guinea pigs, and is preferably human. Furthermore, "subject" or "patient" also includes adults, infants and newborns.

[0021] In this invention, "sample" means a sample derived from any subject that may contain modified nucleosides. While not particularly limited, a bodily fluid sample derived from the subject is preferably used as the sample. "Body fluid sample" means, and is not limited to, any liquid sample that can be isolated from the body of an individual, but examples include blood, plasma, serum, saliva, urine, tears, sweat, etc. Preferably, the body fluid sample is plasma, serum, or urine. In one embodiment, the sample used in this invention is of human origin.

[0022] In this invention, being infected with the AIDS virus (HIV) means that the subject can be determined to be infected with HIV, the causative virus. In this invention, being in a latent HIV infection state means that the subject can be determined to be in a state in which HIV has not completely disappeared and remains in the body, but the amount of HIV in the blood is suppressed to below the detection limit by ART. In this invention, being suspected of being infected means that the subject is suspected to be infected with HIV or to be in a latent HIV infection state.

[0023] A subject determined to be infected with or suspected of being infected with HIV by the method of the present invention can be diagnosed as being infected with HIV by combining it with other methods for detecting HIV infection. Other detection methods are not limited to those described above, but include PCR testing, antibody testing, and antigen testing, with PCR testing being preferred. For example, the testing methods described in the "2020 Guidelines for the Diagnosis of HIV-1 / 2 Infection in Clinical Practice" can be cited.

[0024] A subject determined to be in a latent HIV infection state or suspected to be so by the method of the present invention can be diagnosed as being in a latent HIV infection state by combining this information with the patient's medical history and a detection method capable of detecting latent HIV infection. While not limited to this, a detection method capable of detecting latent HIV infection could include detecting HIV-derived DNA integrated into the host cell genome by PCR.

[0025] If, by the method of the present invention, or in combination with the method of the present invention, a subject is determined to be HIV-infected or in a latent HIV infection state, the subject is considered to be a candidate for treatment of HIV infection. In this specification, a subject being a candidate for treatment of HIV infection means that it can be determined that the subject is infected with HIV, the causative virus, and requires treatment.

[0026] In this specification, evaluating the therapeutic effect on a subject infected with HIV means evaluating the therapeutic effect on a subject who is infected with HIV, the causative virus, and who is currently being treated or has been treated in the past, for example, by administering an antiviral agent, and that this can be done by the method of the present invention.

[0027] For the diagnosis and testing of HIV infection, you can refer to, for example, the "Diagnostic Guidelines for HIV-1 / 2 Infection in Clinical Practice, 2020 Edition," which was jointly formulated by the Japanese AIDS Society and the Japanese Society of Clinical Laboratory Medicine. For the treatment of HIV infection, you can refer to, for example, the "Guidelines for Anti-HIV Treatment (March 2024)," which was created under the Ministry of Health, Labour and Welfare's Administrative Promotion Research Project Grant for AIDS Countermeasures Policy Research. In addition, you can refer to other guidelines such as the WHO's clinical practice guidelines, the U.S. National Institutes of Health, and guidelines published by various countries.

[0028] In one embodiment, the present invention includes the step of detecting the amount of 3-methylcytidine (m3C) in a sample derived from a subject in order to determine whether the subject is infected with HIV, is in a latent HIV infection state, or is suspected to be infected with HIV.

[0029] In one embodiment, the present invention further includes the step of providing information on the amount of detected 3-methylcytidine (m3C) in order to determine whether a subject is infected with HIV, is in a latent HIV infection state, or is suspected to be infected with HIV.

[0030] In one embodiment, the present invention includes a step of detecting the amount of 3-methylcytidine (m3C) in a sample derived from an HIV-infected subject in order to evaluate the therapeutic effect in that subject.

[0031] In one embodiment, the present invention further includes a step of providing information on the amount of detected 3-methylcytidine (m3C) in order to evaluate the therapeutic effect in a subject.

[0032] The modified nucleoside to be detected in this invention is 3-methylcytidine (m ３ C) is 3-methylcytidine (m ３ C) has a structure in which a methyl group is bonded to the carbon at the 3rd position of cytidine. 3-methylcytidine, along with other modified nucleic acids, is one of the modified nucleosides targeted in RNA epigenetics research, and is represented by the following structure.

[0033]

[0034] In the present invention, m ３ The detection of C is not particularly limited as long as it can be detected by any method, but it is preferably detected by mass spectrometry or ELISA.

[0035] In one embodiment of the method of the present invention, m in the sample ３ The amount of C can also be detected using mass spectrometry. "Mass spectrometry" or "MS" is an analytical technique for identifying compounds by their mass. It involves ionizing a sample by applying energy such as high voltage, and then filtering, detecting, and / or measuring the ions based on their mass-to-charge ratio (m / z). There are many types of mass spectrometers, depending on the ionization method and detection method used. ３ Any method that can be used for detecting C is acceptable and can be used without particular limitations. Various mass spectrometers, including MS and its improved versions such as TOF-MS and MALDI-TOF-MS, are commercially available and can be used as appropriate in this invention.

[0036] When using mass spectrometry, detection is possible with a single mass spectrometer, but preferably, a tandem mass spectrometer (tandem MS / MS) is used, in which two mass spectrometers are connected in tandem. A tandem MS / MS is a device in which two mass spectrometers (MS) are connected in series and have a collision activation chamber between them. First, the sample is ionized by the first MS, then only ions of a specific mass number are selected and guided into the collision activation chamber, where they are collided with an inert gas such as Xe (xenon). After that, secondary ions (product ions) generated from the ions selected by the first MS are detected by the second MS.

[0037] Ionization methods include electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), electron ionization (EI), fast electron beam ionization (FAB) / liquid secondary ionization (LSIMS), matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption, thermal spray / plasma spray ionization, and particle beam ionization. Those skilled in the art can appropriately select the ionization method based on the analyte to be measured, the type of sample, the type of detector, and the choice of positive or negative ion mode. ３ The above methods can be used as appropriate, and are not particularly limited as long as C can be detected.

[0038] Tandem MS / MS is typically performed using selective reaction monitoring (SRM). Selective reaction monitoring refers to operating the mass spectrometer in a multi-stage mass spectrometry with two or more stages to continuously detect only the signal levels of specific product ions produced from the analyte, instead of acquiring product ion spectra. In SRM, tandem mass spectrometry can be spatial or temporal.

[0039] When using mass spectrometry, it is preferable to connect liquid chromatography (LC) or gas chromatography (GC), more preferably LC, before the mass spectrometer (MS or MS / MS). After separating the sample with LC or GC, it is introduced into the mass spectrometer for analysis. By connecting LC or GC before the mass spectrometer, good analysis can be performed even when using blood or urine samples, for example.

[0040] Types of chromatography that can be used in LC include partition chromatography, normal-phase liquid chromatography (NPLC), substitution chromatography, reverse-phase liquid chromatography (RPLC), size exclusion chromatography, ion exchange chromatography, and affinity chromatography.

[0041] Generally, a mass spectrometer consists of a sample introduction unit, an ionization unit (ion source), a mass separation unit (analyzer), a detection unit (detector), a vacuum pump, and an instrument control / data processing unit (data system).

[0042] Examples of analyzers used in mass spectrometers include triple quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers, with triple quadrupole analyzers or quadrupole time-of-flight (QTOF) analyzers being preferred. From the viewpoint that commercially available instrument platforms advantageous for SRM assays often use triple quadrupole analyzers, it is more preferable to perform tandem MS / MS using a triple quadrupole analyzer in the detection method of the present invention. Here, "triple quadrupole" means not only quadrupoles but also cases where multipole or stacked electrode is used instead of quadrupoles, as is usually understood by those skilled in the art. Furthermore, in the detection of the present invention, mass spectrometry may be performed in negative ion mode or in positive ion mode.

[0043] In the method of the present invention, ３ For the detection of C, a triplicate quadrupole LC / MS / MS is preferably used, in which an LC is coupled before the mass spectrometer.

[0044] In the method of the present invention, using mass spectrometry, ３ When detecting C, it is preferable to pre-treat the sample with deproteinization and / or desalting. These pretreatments allow for sensitive and accurate detection of the target substance.

[0045] Methods for deproteinization generally include immobilization by protein denaturation (addition of acids such as perchloric acid, trichloroacetic acid, and metaphosphate; addition of water-miscible organic solvents such as acetone, acetonitrile, methanol, and ethanol; heating and cooling), and physical removal (ultrafiltration using membrane filters (centrifugal filtration devices, etc.); dialysis using dialysis tubes; and ultracentrifugation). Furthermore, deproteinization can also be performed by using penetration-limiting packing materials such as reverse-phase packing materials, hybrid packing materials, and hydrophilic polymer packing materials. ３While not limited to methods that do not interfere with the detection of C, a preferred deproteinization method is to deproteinize using a method of immobilization by protein denaturation with a water-miscible organic solvent, such as using methanol. Deproteinization methods are well known and can be carried out according to standard procedures. While not particularly limited, for example, deproteinization can be performed by adding 0.2 to 20 times, preferably 1 to 5 times, the amount of ethanol or methanol to a sample (preferably a body fluid sample), allowing it to react for a sufficient time for protein denaturation (e.g., 15 minutes), then centrifuging under conditions sufficient to precipitate the denatured protein (e.g., 12,000 × g for 15 minutes), and collecting the supernatant (organic solvent layer) to obtain a deproteinized sample. The deproteinized sample can be applied to an LC as is, or after drying using a centrifugal evaporator, etc., and dissolving it in a suitable solvent such as distilled water.

[0046] For desalting, known desalting methods used in analysis can be used as appropriate. Furthermore, the deproteinization method described above can also be used to perform desalting.

[0047] In one embodiment of the method of the present invention, based on the results measured using a mass spectrometer, the m in the sample is measured. ３ Detect the amount of C. ３ The detection of the amount of C may or may not be corrected using an internal standard, but detection can be performed with greater accuracy by using an internal standard. Examples of internal standards, though not limited to those listed, include cytidine in plasma or serum if the sample is a blood sample, and creatinine, urea nitrogen, uric acid, cytidine, adenosine, and 3-amino-3-carboxypropyluridine (acp) in urine if the sample is urine. ３ U) can be cited. Using the measured values ​​of these internal standards, the measured m ３ By correcting the amount of C, the target substance can be detected with greater accuracy. ３ The detection of C may also be performed using a pre-prepared calibration curve.

[0048] The detection method of the present invention involves m in subjects not infected with HIV (e.g., healthy individuals). ３ By pre-setting an average value for the amount of C (for example, as a reference value), if the amount in the sample from the target source is low compared to that value, it can be determined that the person is infected with HIV, is in a latent HIV infection state, or is suspected of being infected.

[0049] target m ３ If the amount of C is, for example, about 1 / 5 to about 1 / 100 compared to a healthy person, preferably about 1 / 5 to about 1 / 50, and more preferably about 1 / 10 to about 1 / 40, it can be determined that the person is infected with HIV or is suspected of being infected. ３ If the amount of C is, for example, about 1 / 2 to 1 / 10 of that of a healthy person, preferably about 1 / 3 to 1 / 10, and more preferably about 1 / 4 to 1 / 8, it can be determined that the person is in a latent HIV infection state or is suspected to be so. If both HIV infection and a latent HIV infection state are suspected, it can be determined whether or not the person is in a latent infection state by detecting the number of HIV viruses in the blood, for example, using an HIV RNA quantitative analysis method.

[0050] In one embodiment of the method of the present invention, m in the sample ３ The amount of C can also be detected using the ELISA method. The ELISA method is a method of detection and quantification that involves binding a specific antibody to a target antigen contained in a sample and using an enzymatic reaction. In the method of the present invention, m ３ Detection can be performed using an antibody against C. Antibodies against modified nucleosides can be those produced according to conventional methods, or antibodies produced by a third party or commercially available antibodies can be used. Either polyclonal or monoclonal antibodies can be used, but monoclonal antibodies are preferred.

[0051] In the method of the present invention, using the ELISA method, ３ When detecting the amount of C, this can be done according to conventional methods. For example, the direct method, indirect method, sandwich method, or competing method can be used, but the sandwich method is preferred.

[0052] In one embodiment of the present invention, the method of the present invention can be used to determine whether a subject is infected with HIV, is in a latent HIV infection state, or is suspected to be infected with HIV. In one embodiment of the method of the present invention, m is measured in a sample of the subject. ３ By comparing the amount of C with a predetermined reference value, it can be determined that the subject is infected with HIV, is in a latent HIV infection state, or is suspected of being infected. The predetermined reference value is not limited to this, but for example, it may be the amount of m detected in a healthy subject (healthy person). ３ The value of C, or a value obtained by adding a value to that number that can effectively eliminate false positives, can be given. The value of m in the subject relative to the cutoff value (reference value) is given. ３ If the amount of C is significantly reduced, it can be determined that the subject is infected with HIV or is in a latent HIV infection state. Whether the reduction is significant can be determined appropriately according to the sensitivity required for the detection method. For example, in determining HIV infection, it may be set to approximately 1 / 5 or less, or approximately 1 / 10 or less, of the average value for healthy individuals. For example, in determining a latent HIV infection state, it may be set to approximately 1 / 2 or less, approximately 1 / 3 or less, or approximately 1 / 4 or less, of the average value for healthy individuals, and approximately 1 / 10 or more.

[0053] The cutoff value can be appropriately set by those skilled in the art from the viewpoint of sensitivity, specificity, positive predictive value for disease (infection), and negative predictive value for infection. For example, it can be set based on ROC curve analysis. Alternatively, the positive or negative result of the target can be determined using another known HIV detection method (e.g., PCR method), and the cutoff value for the method of the present invention can be determined by comparing the results with data obtained when the same target is measured using the method of the present invention.

[0054] In another embodiment of the present invention, the detection method of the present invention can be used to evaluate the therapeutic effect in subjects infected with HIV. The evaluation of the therapeutic effect in subjects is, for example, measured in a sample of the subject. ３This can be done by comparing the amount of C before and after treatment or over time during treatment. In evaluating the therapeutic effect in the method of the present invention, the target m ３ If the amount of C increases after treatment compared to before treatment, it can be determined that the treatment is effective. Based on this result, the treatment plan and medication regimen for the patient can be determined or changed.

[0055] The present invention will be described in more detail by the following examples, but these examples are merely illustrative and do not limit the scope of the present invention in any way.

[0056] The present invention will be described in more detail by the following examples, but these examples are merely illustrative and do not limit the scope of the present invention in any way.

[0057] (Example 1) Comprehensive analysis of modified nucleosides in plasma from HIV-positive and HIV-negative volunteers Plasma was collected from HIV-positive and HIV-negative volunteers from Tanzanians and cryopreserved. Specifically, plasma was prepared from blood collected from 30 healthy individuals, 40 HIV-positive, untreated volunteers, and 30 HIV-positive volunteers undergoing ART (Antiretroviral Therapy), i.e., taking antiviral drugs, and then cryopreserved. Subsequently, the cryopreserved plasma samples were thawed at room temperature, and 100 μL was deproteinized and desalted using a column (Nanosep with 3K Omega). 2 μL of the sample was comprehensively analyzed for modified nucleosides using an ultrafast triple quadrupole mass spectrometer (Shimadzu LCMS-8050). The measurement results are shown in Figure 1. Compared to healthy individuals, 3-methylcytidine (m) was more effective in both untreated HIV-positive individuals and HIV-positive individuals undergoing ART. ３ C) was significantly lower.

[0058] Therefore, the relative abundance of 3-methylcytidine in plasma was compared among healthy individuals, untreated HIV-positive individuals, and HIV-positive individuals undergoing ART. The results are shown in Figure 2.

[0059] HIV-positive individuals should be aware of 3-methylcytidine (m ３It was found that the amount of C) was significantly lower compared to healthy individuals. Furthermore, in HIV-infected individuals whose plasma viral RNA was significantly reduced by taking antiviral drugs (including HIV-infected individuals whose RNA was reduced to below the detection limit), 3-methylcytidine (m ３ The amount of C) had recovered compared to untreated HIV-positive individuals, but remained low compared to healthy individuals. These results suggest that the amount of 3-methylcytidine (m) in plasma (blood) is low. ３ It was found that measuring the amount of C) could detect whether a subject was infected with HIV. Furthermore, even in HIV-positive individuals undergoing ART, 3-methylcytidine (m) ３ Since the amount of C) is significantly lower compared to healthy individuals, it was found that HIV itself is reduced by ART, and that even in HIV-positive individuals undergoing treatment who are difficult to detect by detection methods targeting HIV copy number or antigen, the method of the present invention can detect HIV. Furthermore, in HIV-positive individuals undergoing ART, 3-methylcytidine (m ３ Although the amount of C) is significantly increased compared to untreated HIV-positive individuals, it remains significantly lower compared to healthy individuals, therefore, in the subjects, m ３ It was also found that measuring the amount of C can be used to evaluate the therapeutic effect of ART in HIV-infected patients.

[0060] (Example 2) Changes in modified nucleosides of cultured cells associated with HIV-1 infection. The commonly used HIV-1 NL43 strain was used to infect Jurkat cells and peripheral mononuclear cells (PBMCs) derived from healthy individuals. ３ The amount of C was measured. In summary, Jurkat cells (2 x 10) ５ cells) or PBMC (2x10 ６50 μL of HIV-1 solution (Jurkat cells / RT activity: 100 mU, PBMC / RT activity: 10 mU) was added to cell culture medium containing cells and the cells were infected. After 6 days, the RNA of each cell was purified with Trizol. The RNA concentration was adjusted to 1,000 ng / μL using a spectrophotometer (DeNovix DS-11). Subsequently, the RNA was degraded to nucleosides using RNA-degrading enzymes, and 2 μL of the sample was comprehensively analyzed for modified nucleosides using an ultrafast triple quadrupole mass spectrometer (Shimadzu Corporation: LCMS-8050). The results are shown in Figure 3. As a result, with HIV-1 infection, m ３ A significant decrease in C was observed.

[0061] (Example 3) m in a sample from a Japanese HIV-1 infected patient ３ Detection of C: Plasma and urine samples were collected from HIV-positive and HIV-negative Japanese volunteers, and 3-methylcytidine (m) was detected in the plasma and urine. ３ C) The quantity was measured. Specifically, plasma was prepared from blood collected from 10 healthy individuals and 30 volunteers who were HIV-positive and undergoing ART (i.e., taking antiviral drugs), and frozen for storage. In addition, urine samples were collected from the aforementioned 10 healthy individuals and 10 of the 30 HIV-positive individuals undergoing ART, and frozen for storage. The frozen plasma and urine samples were thawed at room temperature, and 100 μL was deproteinized and desalted using a column (Nanosep with 3K Omega). Then, 2 μL of the sample was analyzed using an ultrafast triple quadrupole mass spectrometer (Shimadzu LCMS-8050) ３ The amount of C was measured. The plasma levels of m³ were compared between healthy individuals and HIV-positive individuals (undergoing ART). ３ Comparison results of C quantity (relative value: log ２ Figure 4 shows the same result in Japanese individuals, particularly in HIV-positive individuals (those undergoing ART). ３ The amount of C was significantly reduced. Also, the amount of m in each sample (subject) ３ Figure 5 shows the measurement results (nM) of C levels. In both plasma and urine, m was observed in HIV-positive individuals (during ART). ３ The C levels were reduced, and it was shown that it could be detected in both blood and urine samples.

[0062] The above detailed description merely illustrates the object and subject matter of the present invention and does not limit the scope of the appended claims. Various modifications and substitutions to the embodiments described without departing from the scope of the appended claims will be apparent to those skilled in the art from the teachings described herein.

[0063] The present invention makes it possible to determine whether a subject is infected with the AIDS virus (HIV), is in a latent HIV infection state, or is suspected of being so. The method of the present invention also makes it possible to evaluate the therapeutic effect on the subject.

Claims

1. A method for determining whether a mammalian subject is infected with the AIDS virus (HIV), is in a latent HIV infection state, or is suspected to be infected, the method comprising the steps of detecting the amount of 3-methylcytidine (m3C) in a sample derived from the subject, and providing information on the detected amount for the purpose of the determination.

2. The method according to claim 1, wherein the information on the detected amount is the amount of 3-methylcytidine detected in the sample, corrected using an internal standard.

3. The method according to claim 1, wherein the information on the detected amount is information that can be used to diagnose whether a subject is infected with the AIDS virus (HIV), is in a latent HIV infection state, or is suspected of being infected, by comparing the amount of 3-methylcytidine in the detected sample with a predetermined reference value.

4. The method according to claim 1, wherein the sample derived from the subject is plasma, serum, or urine.

5. The method according to claim 4, wherein the subject is a human.

6. The method according to any one of claims 1 to 5, wherein the amount of 3-methylcytidine is detected by mass spectrometry.

7. The method according to claim 6, wherein the sample is a sample that has undergone deproteinization and desalting treatment.

8. The method according to any one of claims 1 to 5, wherein the amount of 3-methylcytidine is detected by ELISA.

9. A method for evaluating the therapeutic effect in a subject that is a mammal infected with the AIDS virus (HIV), comprising the steps of detecting the amount of 3-methylcytidine (m3C) in a sample derived from the subject, and providing information on the detected amount for the evaluation.

10. The method according to claim 9, wherein the information on the detected amount is the amount of 3-methylcytidine detected in the sample, corrected using an internal standard (e.g., cytidine).

11. The method according to claim 9, further comprising providing information on the amount of 3-methylcytidine previously detected in the same subject, together with the information on the detected amount.

12. The method according to claim 9, wherein the sample derived from the subject is plasma, serum, or urine.

13. The method according to claim 12, wherein the subject is a human.

14. The method according to any one of claims 9 to 13, wherein the amount of 3-methylcytidine is detected by mass spectrometry.

15. The method according to claim 14, wherein the sample is a sample that has undergone deproteinization and desalting treatment.

16. The method according to any one of claims 9 to 13, wherein the amount of 3-methylcytidine is detected by ELISA.

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