Composition for reactivating latent HIV

Abstract

The present disclosure provides a composition for reactivating latent HIV, the composition comprising xanthohumol, urolithin A, or deferiprone. Also provided is a method for screening substances capable of reactivating latent HIV.

Description

Composition for reactivating latent HIV infection

[0001] This patent application claims priority to Japanese Patent Application No. 2024-029993, the entire contents of which are incorporated herein by reference. The present disclosure relates to compositions for reactivating latently infected HIV.

[0002] Human immunodeficiency virus (HIV) infection causes severe systemic immunodeficiency, but the development of antiviral drugs has made it a controllable chronic disease. However, existing antiviral drugs cannot completely cure HIV infection, and latently infected HIV cells persist in the body for long periods of time. If a patient discontinues oral administration of antiviral drugs, viral particles are released from latently infected HIV cells within one month, resulting in a relapse of HIV-related bloodstream infection. Therefore, HIV-infected individuals must take oral antiviral drugs for the rest of their lives, and novel therapies that target latently infected cells are desperately needed.

[0003] A "shock and kill" approach has been proposed, which aims to achieve a radical cure by using a drug that reactivates HIV in latently infected HIV cells to express viral proteins and induce immune-mediated cell death (Non-Patent Document 1). Clinical trials of latency reversing agents (LRAs) have reported drugs that promote HIV transcription in infected individuals, but no drugs have yet been reported that can reduce the number of latently infected cells and prevent the recurrence of viremia. Existing candidate drugs include the HDAC inhibitors SAHA (vorinostat) (Non-Patent Document 2) and romidepsin (Non-Patent Document 3), the PKC agonist bryostatin-1 (Non-Patent Document 4), and the ALDH inhibitor disulfiram (Non-Patent Document 5). In recent years, immunomodulatory LRAs such as TLR agonists have also been reported, and it has been reported that the TLR7 agonist GS9620 delays the rebound of viremia in a monkey SIV model (Non-Patent Document 6).

[0004] Xanthohumol, a flavonoid found in hops, has been suggested to inhibit HIV replication and reverse transcription (Non-Patent Document 7). Urolithin A is an intestinal bacterial metabolite of ellagic acid and has been suggested to have inhibitory activity against HIV-1 integrase (Non-Patent Document 8). Deferiprone is an iron chelator approved by the FDA for the treatment of iron overload in thalassemia. It has been suggested that deferiprone inhibits hypusination of eIF5A to suppress HIV transcription (Non-Patent Document 9), and that it inhibits HIV transcription and replication and reduces the number of HIV RNA copies in peripheral blood in untreated HIV patients (Non-Patent Document 10). However, it is not known that xanthohumol, urolithin A, or deferiprone reactivate latent HIV infection.

[0005] Deeks SG. HIV: Shock and kill. Nature. 2012 Jul 25;487(7408):439-40.Archin NM, et al., Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. Nature. 2012 Jul 25;487(7408):482-5.Wei DG, et al., Histone deacetylase inhibitor romidepsin induces HIV expression in CD4 T cells from patients on suppressive antiretroviral therapy at concentrations achieved by clinical dosing. PLoS Pathog. 2014 Apr 10;10(4):e1004071.Gutierrez C, et al., Bryostatin-1 for latent virus reactivation in HIV-infected patients on antiretroviral therapy. AIDS. 2016 Jun 1;30(9):1385-92.Xing S, et al., Disulfiram reactivates latent HIV-1 in a Bcl-2-transduced primary CD4+ T cell model without inducing global T cell activation. J Virol. 2011 Jun;85(12):6060-4.Borducchi EN, et al., Antibody and TLR7 agonist delay viral rebound in SHIV-infected monkeys. Nature. 2018 Nov;563(7731):360-364.Wang Q, et al., Xanthohumol, a novel anti-HIV-1 agent purified from Hops Humulus lupulus.Antiviral Res. 2004 Dec;64(3):189-94.Rotich W, et al, HIV-1 Integrase Inhibitory Effects of Major Compounds Present in CareVidTM: An Anti-HIV Multi-Herbal Remedy. Life (Basel). 2022 Mar 12;12(3):417.Hoque M, et al., Inhibition of HIV-1 gene expression by Ciclopirox and Deferiprone, drugs that prevent hypusination of eukaryotic initiation factor 5A. Retrovirology. 2009 Oct 13;6:90.Saxena D, et al., Drug-Based Lead Discovery: The Novel Ablative Antiretroviral Profile of Deferiprone in HIV-1-Infected Cells and in HIV-Infected Treatment-Naive Subjects of a Double-Blind, Placebo-Controlled, Randomized Exploratory Trial. PLoS One. 2016 May 18;11(5):e0154842.

[0006] The purpose of the present disclosure is to provide a new means for reactivating latently infected HIV.

[0007] The present inventors have found that knockout of PHB2, HSPA9, PAICS, or TIMM23 in latently infected cells results in HIV reactivation, and that treatment of latently infected cells with xanthohumol, urolithin A, or deferiprone results in HIV reactivation.

[0008] Accordingly, in some aspects, the present disclosure provides a composition for reactivating a latently infected human immunodeficiency virus (HIV), comprising xanthohumol, urolithin A, or deferiprone.

[0009] In some aspects, the present disclosure provides a composition for treating an HIV-infected individual comprising xanthohumol, urolithin A, or deferiprone.

[0010] In some aspects, the present disclosure provides a composition for preventing the onset of acquired immune deficiency syndrome (AIDS), comprising xanthohumol, urolithin A, or deferiprone.

[0011] In one aspect, the present disclosure provides a method for screening for a substance that reactivates latently infected HIV, the method comprising: (1) culturing T cells latently infected with HIV in the presence of a candidate substance, wherein the candidate substance is a substance that binds to a protein selected from PHB2, HSPA9, PAICS, and TIMM23 or a nucleic acid encoding the same, or an inhibitor of a protein selected from PHB2, HSPA9, PAICS, and TIMM23; (2) confirming whether HIV has been reactivated; and (3) if HIV has been reactivated, selecting the candidate substance as a substance that reactivates latently infected HIV.

[0012] The present disclosure provides a composition for reactivating latently infected HIV, or a method for screening for a substance that reactivates latently infected HIV.

[0013] HIV ＧＫＯAn overview of CRISPR screening using the method is shown. Results of CRISPR screening are shown. The percentage of HIV-reactivated cells is shown for JGL cells in which PHB2, HSPA9, PAICS, or TIMM23 have been knocked out. Western blots of each protein and viral protein p55 are shown for JGL cells in which PHB2, HSPA9, PAICS, or TIMM23 have been knocked out. The percentage of HIV-reactivated cells is shown for J-Lat5A8 in which PHB2, HSPA9, PAICS, or TIMM23 have been knocked out. The percentage of HIV-reactivated cells is shown for J-Lat6.3 in which PHB2, HSPA9, PAICS, or TIMM23 have been knocked out. The percentage of HIV-reactivated cells is shown for J-Lat8.4 in which PHB2, HSPA9, PAICS, or TIMM23 have been knocked out. Figure 1 shows the percentage of cells in which HIV was reactivated for J-Lat9.2 cells in which PHB2, HSPA9, PAICS, or TIMM23 was knocked out. Figure 2 shows the percentage of cells in which HIV was reactivated for J-Lat11.1 cells or JGL cells cultured in the presence of xanthohumol, urolithin A, or deferiprone.

[0014] Unless otherwise specified, terms used herein have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Definitions of some terms used herein are provided below, but these definitions take precedence over common understandings in this specification.

[0015] As used herein, when a numerical value is accompanied by the term "about," it is intended to encompass a range of ±10% of that value. For example, "about 20" is intended to include "18 to 22." A range of numerical values ​​includes all values ​​between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20 to 30" is intended to include "18 to 33."

[0016] Human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS). HIV belongs to the genus Lentivirus in the family Retroviridae and is genetically classified into HIV-1 and HIV-2. HIV-1 is the most prevalent type and is more infectious and pathogenic than HIV-2. As used herein, the terms "human immunodeficiency virus" and "HIV" include, but are not limited to, HIV-1 and HIV-2. In certain embodiments, the HIV is HIV-1 or HIV-2, preferably HIV-1.

[0017] Latent HIV infection refers to a reversible virus non-production state of HIV-infected cells. In memory T cells latently infected with HIV, viral transcription stops and viral proteins are not produced, but the ability to produce infectious virus particles is maintained. HIV-producing cells undergo cell death, but latently infected memory T cells survive for a long period of time. In patients with latently infected HIV, HIV particles or HIV RNA are usually not detected in the peripheral blood.

[0018] I. Compositions for Reactivating Latently Infected HIV As demonstrated in the Examples below, xanthohumol, urolithin A, and deferiprone reactivate latently infected HIV. HIV reactivation refers to the production of viral proteins by latently infected HIV. Therefore, administering a composition containing xanthohumol, urolithin A, or deferiprone to a subject can reactivate latently infected HIV in the subject's cells. Without being limited by theory, reactivation of HIV can induce cell death in infected cells by viral proteins, or can eliminate infected cells through immunity.

[0019] Xanthohumol (CAS number: 6754-58-1, 1-(2,4-dihydroxy-6-methoxy-3-[3-methylbut-2-en-1-yl]phenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one) is a compound represented by the following structural formula.

[0020] Urolithin A (CAS number: 1143-70-0, 3,8-dihydroxy-6H-benzo[c]chromen-6-one) is a compound represented by the following structural formula:

[0021] Deferiprone (CAS number: 30652-11-0, 3-hydroxy-1,2-dimethyl-4(1H)-pyridone) is a compound represented by the following structural formula.

[0022] In some embodiments, xanthohumol or deferiprone is used.

[0023] Pharmaceutically acceptable salts, solvates, or prodrugs of xanthohumol, urolithin A, or deferiprone may be used. References in this disclosure to xanthohumol, urolithin A, or deferiprone are intended to encompass pharmaceutically acceptable salts and solvates, and prodrugs thereof, unless inappropriate in the context.

[0024] A "pharmaceutically acceptable salt" may be a salt with an inorganic or organic acid. Preferred salts are salts with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid, or organic carboxylic or sulfonic acids such as acetic acid, trifluoroacetic acid, propionic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, or methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid or naphthalenedisulfonic acid.

[0025] Pharmaceutically acceptable salts may also be salts with conventional bases, for example alkali metal salts (e.g. sodium or potassium salts), alkaline earth metal salts (e.g. calcium or magnesium salts), or ammonium salts, especially sodium salts, derived from ammonia or organic amines (e.g. diethylamine, triethylamine, ethyldiisopropylamine, procaine, dibenzylamine, N-methylmorpholine, dihydroabietylamine, methylpiperidine, L-arginine, creatine, choline, L-lysine, ethylenediamine, benzathine, ethanolamine, meglumine or tromethamine).

[0026] "Solvate" means a compound that, in the solid or liquid state, forms a complex by coordination with solvent molecules. Preferred solvates are hydrates.

[0027] "Prodrug" means a prodrug of a compound that is suitable, within the scope of sound medical judgment, for use in contact with human or animal tissues without undue toxicity, irritation, allergic response, etc., commensurate with a reasonable benefit / risk ratio, and that is effective for the intended use. Prodrugs include compounds that are rapidly converted in vivo, for example by hydrolysis in the blood, to yield the parent compound of the above formula, as well as chemically modified versions of the parent compound that have enhanced bioavailability. A general description is found in T. Higuchi and V. Stella, "Prodrugs as Novel Delivery Systems," Vol. 14 of the ACS Symposium Series, and Edward B. Roche, ed., "Bioreversible Carriers in Drug Design," American Pharmaceutical Association and Pergamon Press, 1987 (both of which are incorporated herein by reference).

[0028] The subject to which the composition is administered is typically a patient latently infected with HIV. A patient latently infected with HIV refers to a patient who has a history of HIV infection and in whom HIV particles or HIV RNA are not detected in peripheral blood. For example, a patient latently infected with HIV is a patient who is receiving treatment with an anti-HIV drug and in whom HIV particles or HIV RNA are not detected in peripheral blood, but after discontinuing the treatment, HIV particles or HIV RNA are detected in peripheral blood. In some embodiments, the subject is a patient receiving treatment with an anti-HIV drug. In some embodiments, the subject is a patient receiving antiretroviral therapy (ART). In some embodiments, the subject is a patient receiving highly active antiretroviral therapy (HAART). The presence or absence of latently infected HIV can be determined, for example, by immunoPET imaging diagnosis.

[0029] The method of administration of the composition is not particularly limited, and can be via common administration routes such as oral administration, parenteral administration, injection, infusion, and the like, and the composition can be in a dosage form suitable for each administration route. In some embodiments, the composition is administered orally. In some embodiments, the composition is administered intravenously.

[0030] Oral dosage forms include granules, fine granules, powders, coated tablets, tablets, powders, capsules, microcapsules, chewable tablets, solutions, suspensions, emulsions, and the like. Injectable dosage forms include common pharmaceutical dosage forms, such as intravenous injections, infusions, and formulations that extend the release of active substances. Intravenous or infusion dosage forms include aqueous and non-aqueous injection solutions (which may contain antioxidants, buffers, bacteriostats, isotonic agents, and the like) and aqueous and non-aqueous injection suspensions (which may contain suspending agents, thickeners, and the like). Injectable dosage forms may be provided in sealed ampoules or vials, or as lyophilized products that require only the addition of a sterile liquid (e.g., water for injection) immediately before use. Injectable solutions or suspensions may be prepared from powders, granules, or tablets.

[0031] These dosage forms are prepared by conventional formulation methods. Furthermore, various pharmaceutically acceptable pharmaceutical substances can be blended as needed for the formulation. The pharmaceutical substances can be appropriately selected depending on the dosage form of the preparation, and examples thereof include buffering agents, surfactants, stabilizers, preservatives, excipients, diluents, additives, disintegrants, binders, coating agents, lubricants, glidants, flavoring agents, sweeteners, solubilizers, etc.

[0032] The dosage and frequency of administration of the composition can be appropriately determined by one skilled in the art based on the subject's health condition, age, body weight, administration route, administration form, etc., so that an effective amount of the compound is administered to the subject. The effective amount in a given situation can be easily determined by routine experimentation and is within the skill and judgment of an ordinary clinician. The composition may be administered once, multiple times, or continuously. When administered multiple times, the composition may be administered, for example, once to several times a day, e.g., once, twice, or three times a day, every day or every few days, e.g., every 1, 2, 3, or 7 days. The administration period may be, for example, several weeks, several months, or several years. The timing and duration of administration are not limited, and a drug-free period may be included.

[0033] These compounds may be used alone or in combination with one or more additional active ingredients, particularly active ingredients for HIV reactivation, treatment of HIV infection, or prevention of AIDS. For example, xanthohumol, urolithin A, or deferiprone may be used in any combination, and xanthohumol, urolithin A, deferiprone, or combinations thereof may be used in combination with one or more additional active ingredients.

[0034] "Combining" ingredients refers not only to the use of dosage forms containing all ingredients and the use of combinations of dosage forms containing each ingredient separately, but also to the simultaneous administration of each ingredient or the delayed administration of any ingredient, so long as they are used for the treatment of HIV reactivation, HIV infection, or the prevention of AIDS. Two or more additional active ingredients can also be used in combination. Active ingredients suitable for combination include, for example, HDAC inhibitors such as SAHA (vorinostat) and romidepsin, PKC agonists such as bryostatin-1, ALDH inhibitors such as disulfiram, and TLR7 agonists such as GS9620.

[0035] In some embodiments, a composition for reactivating latently infected HIV is provided, the composition comprising xanthohumol, urolithin A, or deferiprone. In some embodiments, a method for reactivating latently infected HIV is provided, the method comprising administering to a subject an effective amount of xanthohumol, urolithin A, or deferiprone. In some embodiments, xanthohumol, urolithin A, or deferiprone are provided for reactivating latently infected HIV. In some embodiments, a use of xanthohumol, urolithin A, or deferiprone for reactivating latently infected HIV is provided. In some embodiments, a use of xanthohumol, urolithin A, or deferiprone for reactivating latently infected HIV is provided. In some embodiments, a use of xanthohumol, urolithin A, or deferiprone in the manufacture of a composition for reactivating latently infected HIV is provided.

[0036] The compositions of the present disclosure may be used to treat HIV-infected individuals, particularly those with a latent HIV infection. To treat HIV-infected individuals, the compositions of the present disclosure may be used in combination with one or more additional active ingredients.

[0037] In some embodiments, a composition for treating an HIV-infected individual is provided, the composition comprising xanthohumol, urolithin A, or deferiprone. In some embodiments, a method for treating an HIV-infected individual is provided, the method comprising administering to a subject an effective amount of xanthohumol, urolithin A, or deferiprone. In some embodiments, xanthohumol, urolithin A, or deferiprone are provided for treating an HIV-infected individual. In some embodiments, the use of xanthohumol, urolithin A, or deferiprone for treating an HIV-infected individual is provided. In some embodiments, the use of xanthohumol, urolithin A, or deferiprone in the manufacture of a composition for treating an HIV-infected individual is provided.

[0038] The compositions of the present disclosure may be used to prevent the onset of AIDS. In the present disclosure, "preventing the onset of AIDS" includes preventing the onset of AIDS in HIV-infected individuals who have not yet developed AIDS, and preventing the recurrence of AIDS in HIV-infected individuals who have recovered from AIDS. To prevent the onset of AIDS, the compositions of the present disclosure may be used in combination with one or more additional active ingredients.

[0039] In some embodiments, a composition for preventing the onset of AIDS is provided, the composition comprising xanthohumol, urolithin A, or deferiprone. In some embodiments, a method for preventing the onset of AIDS is provided, the method comprising administering an effective amount of xanthohumol, urolithin A, or deferiprone to a subject. In some embodiments, xanthohumol, urolithin A, or deferiprone is provided for preventing the onset of AIDS. In some embodiments, a use of xanthohumol, urolithin A, or deferiprone for preventing the onset of AIDS is provided. In some embodiments, a use of xanthohumol, urolithin A, or deferiprone in the manufacture of a composition for preventing the onset of AIDS is provided.

[0040] II. Screening method for substances that reactivate latent HIV infection

[0041] As demonstrated in the Examples below, knockout of PHB2, HSPA9, PAICS, or TIMM23 in cells latently infected with HIV reactivates HIV. Therefore, these proteins or nucleic acids encoding them can be used to screen for substances that reactivate latently infected HIV.

[0042] The screening method involves (1) culturing T cells latently infected with HIV in the presence of a candidate substance.

[0043] The HIV used in the screening method may be modified to facilitate confirmation of reactivation, for example, an HIV that expresses a marker such as a fluorescent protein under the control of a viral promoter may be used.

[0044] The T cells may be any T cells that can be infected with HIV, and are typically CD4-positive T cells. The T cells may be derived from any tissue, and T cells induced to differentiate from pluripotent stem cells such as iPS cells or ES cells may be used. T cells established as a cell line may also be used. Typically, T cells latently infected with HIV express at least one of PHB2, HSPA9, PAICS, and TIMM23, preferably PHB2, HSPA9, PAICS, and TIMM23.

[0045] The candidate substance is a substance that binds to a protein selected from PHB2, HSPA9, PAICS and TIMM23 or a nucleic acid encoding the same, or an inhibitor of a protein selected from PHB2, HSPA9, PAICS and TIMM23.

[0046] PHB2, also known as prohibitin 2, is involved in several processes, such as defense responses against viruses, positive control of cell cycle phase transitions, and control of DNA-templated transcription. A representative amino acid sequence of human PHB2 is registered under GenBank accession number NP_001138303.1 (SEQ ID NO: 1), and the nucleotide sequence is registered under GenBank accession number NM_001144831.2 (SEQ ID NO: 2). In the present disclosure, PHB2 includes the products of all alleles.

[0047] HSPA9 is a member of the heat shock protein 70 family and is primarily localized in mitochondria. It plays a role in cell proliferation, stress response, and mitochondrial maintenance. A representative amino acid sequence of human HSPA9 is registered under GenBank accession number NP_004125.3 (SEQ ID NO: 3), and the nucleotide sequence is registered under GenBank accession number NM_004134.7 (SEQ ID NO: 4). In the present disclosure, HSPA9 includes all allelic products.

[0048] PAICS has phosphoribosylaminoimidazole carboxylase activity and phosphoribosylaminoimidazole succinocarboxamide synthase activity and catalyzes the biosynthesis of purines. A representative amino acid sequence of human PAICS is registered under GenBank Accession No. NP_001072992.1 (SEQ ID NO: 5), and the nucleotide sequence is registered under GenBank Accession No. NM_001079524.2 (SEQ ID NO: 6). In the present disclosure, PAICS includes the products of all alleles.

[0049] TIMM23 is present in the inner mitochondrial membrane and is a component of a complex that mediates the transmembrane transport of proteins containing transit peptides. A representative amino acid sequence of human TIMM23 is registered under GenBank Accession No. NP_006318.1 (SEQ ID NO: 7), and the nucleotide sequence is registered under GenBank Accession No. NM_006327.4 (SEQ ID NO: 8). In the present disclosure, TIMM23 includes the products of all alleles.

[0050] The term "test substance" encompasses any substance, such as peptides, proteins, amino acids, nucleic acids, lipids, carbohydrates, and small molecules. Test substances are typically purified or isolated, but may also be unpurified or unisolated crude preparations or live cells that produce the test substance. For example, a candidate substance is an antibody or ligand that binds to PHB2, HSPA9, PAICS, or TIMM23, or an inhibitor of PHB2, HSPA9, PAICS, or TIMM23. For example, a candidate substance is an oligonucleotide that binds to a nucleic acid encoding PHB2, HSPA9, PAICS, or TIMM23.

[0051] The screening method includes (2) confirming whether HIV has been reactivated. HIV reactivation can be confirmed by detecting or measuring viral proteins produced by HIV.

[0052] In steps (1) and (2), for example, HIV ＧＫＯA dual-color HIV reporter (DuoFluol) virus model, such as the HIV-1 virus model (see U.S. Pat. No. 10,106,818; V. Calvanese et al., "Dual-color HIV reporters trace a population of latent infected cells and enable their purification", Virology 446 (2013) 283-292; Emilie Battivelli, et al., (2018) "Distinct chromatin functional states correlate with HIV latency reactivation in infected primary CD4+ T cells", eLife 7:e34655) may be used.

[0053] The screening method includes (3) selecting a candidate substance as a substance that reactivates latently infected HIV when HIV is reactivated. The selected candidate substance can be used in the same manner as xanthohumol, urolithin A, and deferiprone described above.

[0054] For example, the following embodiments are provided: [1] A composition for reactivating latently infected human immunodeficiency virus (HIV), comprising xanthohumol, urolithin A, or deferiprone. [2] The composition described in 1 above, comprising xanthohumol or deferiprone. [3] The composition described in 1 or 2 above, for administration to a subject undergoing treatment with an anti-HIV drug. [4] The composition described in any one of 1 to 3 above, for administration to a subject undergoing highly active antiretroviral therapy (HAART). [5] A composition for treating HIV patients, comprising xanthohumol, urolithin A, or deferiprone. [6] A composition for preventing the onset of AIDS, comprising xanthohumol, urolithin A, or deferiprone. [7] A method for screening for a substance that reactivates latently infected HIV, comprising: (1) culturing T cells latently infected with HIV in the presence of a candidate substance, wherein the candidate substance is a substance that binds to a protein selected from PHB2, HSPA9, PAICS, and TIMM23, or a nucleic acid encoding the protein, or an inhibitor of PHB2, HSPA9, PAICS, or TIMM23; (2) confirming whether HIV has been reactivated; and (3) if HIV has been reactivated, selecting the candidate substance as a substance that reactivates latently infected HIV.

[0055] Experiment 1: CRISPR screening of HIV transcriptional reactivators using a latently infected cell model HIV ＧＫＯUsing a latently infected cell model (Emilie Battivelli, et al., (2018) "Distinct chromatin functional states correlate with HIV latency reactivation in infected primary CD4+ T cells" eLife 7:e34655) and CRISPR screening (Tzelepis et al., 2016, "A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia" Cell Reports 17, 1193-1205), we screened for genes that reactivate latently infected cells. [Method] HIV was transfected into Jurkat T cells. ＧＫＯ The JGL cells were infected with HIV-1, and the fraction of latently infected cells that were mKO2-single positive was separated using a cell sorter (JGL cells). Next, Cas9 was introduced into the JGL cells using a lentiviral vector (JGL-Cas9 cells). The JGL-Cas9 cells were then infected with the gRNA library virus to obtain HIV-1. ＧＫＯ The GFP-positive fraction, which indicates reactivation of HIV, was separated using a cell sorter, and the count of the introduced gRNA was compared with that of the cell group before separation. ＧＫＯ An overview of the CRISPR screening experiment using the gene is shown in Figure 1. [Results] The results of the CRISPR screening are shown in Figure 2. Four genes, PHB2, HSPA9, TIMM23, and PAICS, were identified.

[0056] Experiment 2: Confirmation of CRISPR knockout of candidate genes and HIV transcriptional reactivation using a latently infected cell model (JGL) [Method] The gRNA that knocks out the identified gene was introduced alone into JGL-Cas9 cells, and HIV ＧＫＯThe percentage of GFP-positive fractions, which indicate reactivation of HIV-1, was confirmed by FACS, and the expression of the protein encoded by the target gene and the viral protein (HIV-1 gag p55) was confirmed by Western blot. [Results] The results of FACS are shown in Figure 3. Knocking out PHB2, HSPA9, TIMM23, or PAICS resulted in the reactivation of HIV-1. ＧＫＯ The number of latently infected cells in which the virus was reactivated increased compared to the control. The results of Western blot analysis are shown in Figure 4.

[0057] Experiment 3: CRISPR knockout of candidate genes and confirmation of HIV transcriptional reactivation using latently infected model cell lines J-Lat5A8, 6.3, 8.4, and 9.2 [Method] The gRNA that knocks out the identified gene was introduced alone into latently infected J-Lat5A8, 6.3, 8.4, and 9.2 cells, and the percentage of GFP-positive fractions, indicating HIV reactivation, was confirmed by FACS. J-Lat5A8 (Chan JK, et al., (2013) Calcium / Calcineurin Synergizes with Prostratin to Promote NF-kB Dependent Activation of Latent HIV. PLoS ONE 8(10): e77749. doi:10.1371 / journal.pone.0077749) and J-Lat6.3, 8.4, 9.2 (Albert Jordan, et al., HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. The EMBO Journal Vol.22 No.8 pp.1868-1877, 2003) are latently infected cells with a single HIV-1 integration site, and express GFP upon HIV reactivation. [Results] The results are shown in Figures 5 to 8. Knocking out PHB2, HSPA9, TIMM23, or PAICS increased latently infected cells in which HIV was reactivated compared to controls.

[0058] Experiment 4: Effect of drugs on HIV transcriptional reactivation in a latently infected cell model [Method] J-Lat11.1 cells or JGL cells, which are latently infected cell models, were cultured in the presence of a PHB2 inhibitor (xanthohumol) or a mitophagy inducer (urolithin A or deferiprone), and the percentage of GFP-positive fractions, indicating HIV reactivation, was determined by FACS. [Results] The results are shown in Figure 9. In the presence of xanthohumol, urolithin A, or deferiprone, the number of latently infected cells in which HIV was reactivated increased compared to the control.

[0059] The present disclosure provides a method for reactivating latent HIV infection, which can be used in the medical field.

Claims

1. A composition for reactivating latently infected human immunodeficiency virus (HIV), comprising xanthohumol, urolithin A, or deferiprone.

2. The composition of claim 1, comprising xanthohumol or deferiprone.

3. The composition of claim 1 for administration to a subject undergoing treatment with an anti-HIV drug.

4. The composition of claim 1 for administration to a subject receiving highly active antiretroviral therapy (HAART).

5. A composition for treating an HIV-infected individual, comprising xanthohumol, urolithin A, or deferiprone.

6. A composition for preventing the onset of acquired immune deficiency syndrome (AIDS), comprising xanthohumol, urolithin A, or deferiprone.

7. A method for screening for a substance that reactivates latently infected HIV, comprising: (1) culturing T cells latently infected with HIV in the presence of a candidate substance, wherein the candidate substance is a substance that binds to a protein selected from PHB2, HSPA9, PAICS, and TIMM23, or a nucleic acid encoding the same, or an inhibitor of a protein selected from PHB2, HSPA9, PAICS, and TIMM23; (2) confirming whether HIV has been reactivated; and (3) if HIV has been reactivated, selecting the candidate substance as a substance that reactivates latently infected HIV.

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