WO2019106427A2 - Procédé de criblage d'agents d'inversion de latence du vih-1 - Google Patents

Procédé de criblage d'agents d'inversion de latence du vih-1 Download PDF

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WO2019106427A2
WO2019106427A2 PCT/IB2018/001456 IB2018001456W WO2019106427A2 WO 2019106427 A2 WO2019106427 A2 WO 2019106427A2 IB 2018001456 W IB2018001456 W IB 2018001456W WO 2019106427 A2 WO2019106427 A2 WO 2019106427A2
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hiv
cells
inhibitor
lra
fraction
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PCT/IB2018/001456
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WO2019106427A3 (fr
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Privada Institut De Recerca De La Sida-Caixa Fundacio
Julia GARCIA PRADO
Alba RUIZ DE ANDRES
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Fundacio Privada Inst De Recerca De La Sida Caixa
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70514—CD4

Definitions

  • the present invention relates to a method for screening HIV-l latency reversing agents (LRA).
  • LRA HIV-l latency reversing agents
  • HIV infection is one of the major threats to global human health. It is estimated that more than 78 million people worldwide have been infected by the human immunodeficiency virus since 1981. Nearly half of these infected individuals have died of the resultant Acquired Immunodeficiency Syndrome (AIDS) during the same time frame. See UNAIDS, htp://www.unaids.ofg/, November 2017.
  • AIDS Acquired Immunodeficiency Syndrome
  • ART antiretroviral therapy
  • HDACi histone deacetylases inhibitors
  • PLCm protein kinase C
  • cytotoxic T-lymphocytes have been put forward as candidates to elicit this function due to their in vitro activity in eliminating reactivated cells and their key role in the control of the reservoir in natural HIV-l elite controllers. See Shan L, et al, Immunity 2012; 36(3):49l-50l, Descours B, et al, Clin Infect Dis 20l2;54(l0): 1495-503 and Noel N, et al, J Virol 2016; 90(13):6148-6158. Nonetheless, the direct effect of HDACi and PKCm in CTL activity should be taken into account to identify the therapeutic window of activity between latency-reversing agents and CTL clearance of reactivated cells.
  • the present invention is directed to a method for screening HIV-l latency reversing agents (LRA).
  • the method comprises the steps of (a) culturing in vitro a fraction of CD4+ T cells obtained from an HIV-infected subject with at least one latency reversing agent (LRA) for at least 48 hours, (b) co-culturing in vitro the fraction of CD4+ T cells with cytotoxic T-lymphocytes (CTL) obtained from the HIV-infected subject and (c) counting the killed CD4+ T cells in the fraction.
  • Said screening could be useful in the treatment and prevention of HIV-l infection and AIDS, such as by assisting in the optimization of ART and HAART protocols for HIV-infected subjects.
  • a method for screening latency reversing agents which comprises:
  • the LRA comprises a protein kinase C (PKC) modulator, a histone deacetylase (HDAC) inhibitor or a combination thereof.
  • PLC protein kinase C
  • HDAC histone deacetylase
  • HDAC inhibitor comprises panobinostat, romidepsin, trichostatin A and vorinostat.
  • the present invention refers to a kit comprising at least one of the LRA of the invention.
  • Fig. 1 Representation of the shock and kill RELI model. Briefly, the HIV-l permissive U937 cell line, transfected with the HLA-B* 27:05 gene, was infected for three days with a NL43 strain carrying a reporter GFP gene. The GFP negative population was then sorted and cultured in the presence of the protease inhibitor RTV to prevent viral spread and favor the establishment of an enriched latently infected state. RELI cells were reactivated with LRA in the presence of the integrase inhibitor RAL before being extensively washed and cultured in the presence or absence of HLA-class I matched HIV-specific CTL and RAL. 20 hours after coculture, cells were stained and analyzed by flow cytometry to evaluate HIV- 1 reactivation (shock) and CTL-mediated elimination of HIV-reactivated RELI cells (kill).
  • Fig. 2 Representative dot plots showing intracellular p24 levels from live RELI cells reactivated with HDACi combined or not with the PKC agonist bryostatin.
  • Fig. 3 Representative dot plots showing intracellular p24 and GFP levels from live RELI cells reactivated with LRA.
  • Fig. 4 Correlation between p24 expression and GFP expression in RELI cells after LRA treatment.
  • the line indicates the fit of the data to a linear regression.
  • the Spearman correlation coefficient (rho) and the two-tailed p-value are shown.
  • the graph represents the mean ⁇ SEM of five independent experiments performed in duplicates.
  • Fig. 5 HIV-l reactivation measured as percentage of intracellular p24 levels and GFP expression.
  • the graph represents the mean ⁇ SD of two replicates from one representative experiment.
  • Fig. 6 HIV-l fold induction measured as the relative p24 expression level compared to untreated condition.
  • the graph represents the mean ⁇ SEM of five independent experiments performed in duplicates. The p-values were calculated using the one-sample t test. Only significant values are shown in the figure (p ⁇ 0.05 *, p ⁇ 0.005 **).
  • administering includes any method of delivering to a subject a cellular composition according to the present invention.
  • AIDS refers to the symptomatic phase of HIV infection, and includes both Acquired Immune Deficiency Syndrome (commonly known as AIDS) and “ARC,” or AIDS-Related Complex. See Adler M, et al., Brit. Med. J. 1987; 294: 1145-1147.
  • the immunological and clinical manifestations of AIDS are well known in the art and include, for example, opportunistic infections and cancers resulting from immune deficiency.
  • antiretroviral therapy refers to the administration of one or more antiretroviral drugs (i.e. HIV antiretrovirals) to inhibit the replication of HIV.
  • ART involves the administration of at least one antiretroviral agent (or, commonly, a cocktail of antiretrovirals) such as nucleoside reverse transcriptase inhibitor (e.g. zidovudine (AZT, lamivudine (3TC) and abacavir), non-nucleoside reverse transcriptase inhibitor (e.g. nevirapine and efavirenz) and protease inhibitor (e.g. indinavir, ritonavir and lopinavir).
  • nucleoside reverse transcriptase inhibitor e.g. zidovudine (AZT, lamivudine (3TC) and abacavir
  • non-nucleoside reverse transcriptase inhibitor e.g. nevirapine and efavirenz
  • protease inhibitor
  • HAART Highly Active Antiretroviral Therapy
  • HAART refers to treatment regimens designed to suppress aggressively HIV replication and disease progression.
  • HAART usually consists of three or more different drugs, such as, for example, two nucleoside reverse transcriptase inhibitors and a protease inhibitor.
  • histone deacetylase inhibitor refers to a compound that inhibits histone deacetylase enzymes.
  • Histone deacetylases remove acetyl groups from lysine residues on histones during this process, thus allowing histones to wrap DNA more tightly.
  • HDAC inhibitors prevent deacetylation and, therefore, affect gene expression. Examples of HDAC inhibitors include, but are not limited to, apicidin, abexinostat (i.e. PCI-24781), belinostat (i.e. Beleodaq®), BRD 4354, dacinostat (i.e.
  • LAQ- 824 depudecin, droxinostat, entinostat (i.e. SNDX-275, MS-275), givinostat (i.e. gavinostat, ITF 2357), KD 5170, LMK 235, M 344, MC 1568, MC 1742, MI 192, mocetinostat (i.e. MGCD-0103), NCH 51 (i.e. PTACH), NSC 3852 (i.e. 5-nitroso-8-quinolinol), niltubacin (i.e. MAZ-1391), oxamflatin (i.e. metacept 3), panobinostat (i.e.
  • Farydak® PCI 34051, pracinostat (i.e. SB 939), pyroxamide, quisinostat (i.e. JNJ-26481585), resminostat (i.e. 4SC- 201, RAS2410), RG2833 (i.e. RGFP109), RGFP966, Ricinus communis toxin (i.e. RC- Toxin), rocilinostat (i.e. ACY-1215), romidepsin (i.e. Istodax®, FK 228), scriptaid, sodium butyrate, sodium 4-phenylbutyrate, suberohydroxamic acid (i.e.
  • SBHA N,N'- dihydroxyoctanediamide
  • C-H 106 tacedinaline (i.e. N-acetyldinaline, Cl 994), TCS HDAC6 20b, TMP269, trichostatin A, tubacin, UF 010 (i.e. 4-bromo-N'- butylbenzohydrazide), valproic acid, and vorinostat (i.e. Zolinza®, SAHA).
  • HIV include HIV-l and HIV-2, SHIV and SIV.“HIV- 1” means the human immunodeficiency virus type-l. HIV-l includes, but is not limited to, extracellular virus particles and the forms of HIV-l associated with HIV-l infected cells. The HIV-l virus may represent any of the known major subtypes (Classes A, B, C, D E, F, G and H) or outlying subtype (Group O) including laboratory strains and primary isolates.“HIV-2” means the human immunodeficiency virus type-2. HIV-2 includes, but is not limited to, extracellular virus particles and the forms of HIV-2 associated with HIV-2 infected cells.
  • SIV refers to simian immunodeficiency virus which is an HIV-like virus that infects monkeys, chimpanzees, and other nonhuman primates. SIV includes, but is not limited to, extracellular virus particles and the forms of SIV associated with SIV infected cells.
  • HIV infection refers to indications of the presence of the HIV virus in an individual including asymptomatic seropositivity, AIDS-related complex (ARC), and acquired immunodeficiency syndrome (AIDS).
  • ARC AIDS-related complex
  • AIDS acquired immunodeficiency syndrome
  • Integrase inhibitor refers to a compound that can block the action of integrase, a viral enzyme that inserts the viral genome into the DNA of the host cell. Integrase inhibitors include integrase strand transfer inhibitors (INSTIs) and integrase binding inhibitors (INBIs).
  • An“HIV integrase inhibitor” is an integrase inhibitor used in the treatment and prevention of HIV infection and AIDS. Examples of HIV integrase inhibitors include, but are not limited to, aurintricarboxylic acid, bictegravir (i.e. GS-9883), cabotegravir (i.e.
  • GSK1265744, GSK744) caffeic acid phenethyl ester, curcumin, chicoric acid, 3,5- dicaffeoylquinic acid, 4-(2,3-dihydropyrano[4,3,2-de]quinolin-7-yl)-a-(l, 1 -dimethyl ethoxy)- 2-methyl-3-quinolineacetic acid (i.e. BI 224436), dolutegravir (i.e. Tivicay®), elvitegravir (i.e. Vitekta®), globoidnan A, MK-2048, quercetin, raltegravir (i.e. Isentress®), tyrphostin, and their derivatives.
  • dolutegravir i.e. Tivicay®
  • elvitegravir i.e. Vitekta®
  • globoidnan A MK-2048
  • quercetin quercetin
  • latency reversing agents include, but not are not limited to, protein kinase C modulators (PKCm), bromo and external (BET) bromodomain inhibitors, histone deacetylase inhibitors (HDACi), acetaldehyde dehydrogenase inhibitors (e.g. disulfiram), activators of nuclear factor kappa-light chain-enhancer of activated B cells (NF- KB), and activators of the AKT pathway.
  • PICm protein kinase C modulators
  • BET bromo and external bromodomain inhibitors
  • HDACi histone deacetylase inhibitors
  • acetaldehyde dehydrogenase inhibitors e.g. disulfiram
  • activators of nuclear factor kappa-light chain-enhancer of activated B cells NF- KB
  • activators of the AKT pathway activators of the AKT pathway.
  • BET inhibitors include, but are not limited to, CPI 203,
  • HIV protease inhibitor refers to a compound that can prevent viral replication by binding selectively to viral proteases and, therefore, blocks the cleavage of protein precursors that are necessary for making infectious viral particles.
  • An“HIV protease inhibitor” is a protease inhibitor used in the treatment and prevention of HIV infection and AIDS.
  • HIV protease inhibitors include, but are not limited to, amprenavir (i.e. Agenerase®), atazanavir (i.e. Reyataz®), darunavir (i.e. Prezista®), fosamprenavir (i.e. Lexiva®, Telzir®), indinavir (i.e.
  • Crixivan® lopinavir (i.e. Kaletra®), nelfmavir (i.e. Viracept®), ritonavir (i.e. Norvir®), saquinavir (i.e. Invirase®, Fortovase®), and tipranavir (Aptivus®).
  • PKC modulator refers to a compound that affects the activity of a protein kinase C (“PKC”) enzyme.
  • PKCs are enzymes that regulate the function of other proteins through the phosphorylation of hydroxyl groups located in the serine and threonine residues of said proteins.
  • PKC modulators can be antagonists (i.e. inhibitors), agonists (i.e. activators) or both. Examples of PKC antagonists include, but are not limited to, bisindolylmaleimide 1 (i.e.
  • BIM-l, GF 109203X and its related compounds BIM-2, BIM-3 and BIM-8, [Ala 107 J- MBP (104-118), [Ala 113 ]-MBP (104-118), C-l, calphostin C (i.e. UCN 1028C, PKF 115584), CGP 53353, chelerythrine, D-erythro-sphingosine, dihydrosphingosine (i.e. sphinganine), enzastaurin (i.e. LY317615), [Glu 27 ]-PKC (19-36), Go 6976, Go 6983, gossypol, melittin, myricitrin (i.e.
  • PKC agonists include, but are not limited to, bryostatin A (i.e. bryostatin 1, NSC 339555) and its analogs bryostatin 2 and bryostatin 3, diacyl glycerol (DAG) mimics, FR 236924 (i.e. DCP- LA), ingenol mebutate (i.e. Picato®, ingenol-3-angelate, PEP005) and its analogs, phorbol 12, 13 -dibutyrate (i.e. PDbu), phorbol l2-myristate l3-acetate (i.e. PMA), prostratin, pseudo RACK1, SC-10, and SC-9.
  • bryostatin A i.e. bryostatin 1, NSC 339555
  • DAG diacyl glycerol
  • FR 236924 i.e. DCP- LA
  • ingenol mebutate i.e. Picato®, ingenol-3
  • subject refers to an individual or animal, such as a human, a nonhuman primate (e.g. chimpanzees and other apes and monkey species); farm animals, such as birds, fish, cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs.
  • a nonhuman primate e.g. chimpanzees and other apes and monkey species
  • farm animals such as birds, fish, cattle, sheep, pigs, goats and horses
  • domestic mammals such as dogs and cats
  • laboratory animals including rodents, such as mice, rats and guinea pigs.
  • rodents such as mice, rats and guinea pigs.
  • the term“subject” encompasses an embryo and a fetus. In a preferred embodiment, the subject is a human.
  • control of the disease progression refers to mean the beneficial or desired clinical results that include, but are not limited to, reduction of the symptoms, reduction of the duration of the disease, stabilization of pathological states (specifically to avoid additional deterioration), delaying the progression of the disease, improving the pathological state and remission (both partial and total).
  • the control of progression of the disease also involves an extension of survival compared with the expected survival if treatment was not applied.
  • the terms “treat” and“treatment” refer specifically to stopping or slowing the infection and destruction of healthy CD4+ T cells in an HIV-infected subject. It also refers to the stopping and slowing of the onset of symptoms of the acquired immunodeficiency disease such as extreme low CD4+ T cell count and repeated infections by opportunistic pathogens.
  • Beneficial or desired clinical results include, but are not limited to, an increase in absolute naive CD4+ T cell count (range 10-3520), an increase in the percentage of CD4+ T cell over total circulating immune cells (range 1-50%), or an increase in CD4+ T cell count as a percentage of normal CD4+ T cell count in an uninfected subject (range 1-161%).“Treatment” can also mean prolonging survival of the infected subject as compared to expected survival if the subject does not receive any HIV targeted treatment.
  • the present invention refers to a method for screening latency reversing agents (LRA) which comprises the steps of (a) culturing in vitro a fraction of CD4+ T cells obtained from a HIV-infected subject with at least one latency reversing agent (LRA) for at least 48 hours, (b) co-culturing in vitro the fraction of CD4+ T cells with cytotoxic T- lymphocytes (CTL) obtained from the HIV-infected subject and (c) counting the killed CD4+ T cells in the fraction.
  • LRA latency reversing agents
  • the LRA comprises a protein kinase C (PKC) modulator, a histone deacetylase (HD AC) inhibitor or a combination thereof.
  • PLC protein kinase C
  • HD AC histone deacetylase
  • the PKC modulator is a PKC agonist.
  • the PKC agonist comprises bryostatin A, bryostatin 2, bryostatin 3, DAG mimics, FR 236924, ingenol mebutate, phorbol 12, 13 -dibutyrate, phorbol l2-myristate l3-acetate, prostratin, pseudo RACK1, SC-10, and SC-9 or a combination thereof. More preferably, the PKC agonist comprises bryostatin A.
  • the HDAC inhibitor comprises apicidin, abexinostat, belinostat, BRD 4354, dacinostat, depudecin, droxinostat, entinostat, givinostat, KD 5170, LMK 235, M 344, MC 1568, MC 1742, MI 192, mocetinostat, NCH 51, 5-nitroso-8- quinolinol, niltubacin, oxamflatin, panobinostat, PCI 34051, pracinostat, pyroxamide, quisinostat, resminostat, RG2833, RGFP966, Ricinus communis toxin, rocilinostat, romidepsin, scriptaid, sodium butyrate, sodium 4-phenylbutyrate, suberohydroxamic acid, C- H 106, tacedinaline, TCS HDAC6 20b, TMP269, trichostatin A, tub
  • a combination of a PKC modulator and a HDAC inhibitor is employed as LRA.
  • a combination of a PKC agonist and a HDAC inhibitor is used.
  • a combination of a PKC antagonist and a HDAC inhibitor is employed.
  • a combination of: a PKC agonist and panobinostat, a PKC agonist and romidepsin, a PKC agonist and trichostatin A, or a PKC agonist and vorinostat is employed.
  • a combination of: bryostatin A and panobinostat, bryostatin A and romidepsin, bryostatin A and trichostatin A, or bryostatin A and vorinostat is used.
  • an HIV protease inhibitor is added to the fraction of CD4+ T cells of step (a) before culturing said fraction with the LRA.
  • amprenavir, atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, nelfmavir, ritonavir, saquinavir, and tipranavir or a combination thereof is used as protease inhibitor. More preferably, ritonavir is employed as protease inhibitor.
  • the LRA and HIV protease inhibitor of the method of the invention comprise, respectively, (a) a combination of a PKC agonist and panobinostat and (b) ritonavir, (a) a combination of a PKC agonist and romidepsin and (b) ritonavir, (a) a combination of a PKC agonist and trichostatin A and (b) ritonavir, or (a) a combination of a PKC agonist and vorinostat and (b) ritonavir.
  • the LRA and HIV protease inhibitor of the method comprise, respectively, (a) a combination of bryostatin A and panobinostat and
  • ritonavir (a) a combination of bryostatin A and romidepsin and (b) ritonavir, (a) a combination of bryostatin A and trichostatin A and (b) ritonavir, or (a) a combination of bryostatin A and vorinostat and (b) ritonavir.
  • an HIV integrase inhibitor is added to the fraction of CD4+ T cells of step (a) when culturing said fraction with the LRA.
  • cabotegravir, dolutegravir, elvitegravir, and raltegravir or a combination thereof is used as integrase inhibitor. More preferably, raltegravir is employed as integrase inhibitor.
  • the LRA and HIV integrase inhibitor of the method of the invention comprise, respectively, (a) a combination of a PKC agonist and panobinostat and
  • raltegravir (a) a combination of a PKC agonist and romidepsin and (c) raltegravir, (a) a combination of a PKC agonist and trichostatin A and (c) raltegravir, or (a) a combination of a PKC agonist and vorinostat and (c) raltegravir.
  • the LRA and HIV integrase inhibitor of the method comprise, respectively, (a) a combination of bryostatin A and panobinostat and (c) raltegravir, (a) a combination of bryostatin A and romidepsin and (c) raltegravir, (a) a combination of bryostatin A and trichostatin A and (c) raltegravir, or (a) a combination of bryostatin A and vorinostat and (c) raltegravir.
  • the LRA, HIV protease inhibitor and HIV integrase inhibitor of the method of the invention comprise, respectively, (a) a combination of a PKC agonist and panobinostat, (b) ritonavir, and (c) raltegravir, (a) a combination of a PKC agonist and romidepsin, (b) ritonavir, and (c) raltegravir, (a) a combination of a PKC agonist and trichostatin A, (b) ritonavir, and (c) raltegravir, or (a) a combination of a PKC agonist and vorinostat, (b) ritonavir, and (c) raltegravir.
  • the LRA, HIV protease inhibitor and HIV integrase inhibitor of the method comprise, respectively, (a) a combination of bryostatin A and panobinostat, (b) ritonavir, and (c) raltegravir, (a) a combination of bryostatin A and romidepsin, (b) ritonavir, and (c) raltegravir, (a) a combination of bryostatin A and trichostatin A, (b) ritonavir, and (c) raltegravir, or (a) a combination of bryostatin A and vorinostat, (b) ritonavir, and (c) raltegravir.
  • kits comprising at least one of the LRA, HIV protease inhibitors or HIV integrase inhibitors of the invention.
  • the kit comprises a combination of at least one PKC modulator and at least one HD AC inhibitor according to the invention. More preferably, the kit comprises a combination of at least one PKC agonist and at least one HDAC inhibitor.
  • the kit comprises (a) a combination of at least one PKC agonist and at least one HDAC inhibitor and (b) at least one HIV protease inhibitor.
  • the kit comprises (a) a combination of at least one PKC agonist and at least one HDAC inhibitor and (c) at least one HIV integrase inhibitor.
  • the kit comprises (a) a combination of at least one PKC agonist and at least one HDAC inhibitor, (b) at least one HIV protease inhibitor, and (c) at least one HIV integrase inhibitor.
  • the kit may include one or more PKC agonists, HDAC inhibitors, HIV protease inhibitors, and HIV integrase inhibitors or combinations thereof.
  • kits of the invention may be optionally packed in suitable containers and be labeled for screening LRA.
  • the components of the kits may be stored in unit or multi-dose containers as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution.
  • the containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (e.g. the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the kits may further comprise more containers comprising a pharmaceutically acceptable carrier.
  • kits may further include other materials desirable from a commercial and user standpoint, including, but not limited to, buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable cells or other active agents used in the method of the invention.
  • the kits can contain instructions customarily included in commercial packages of diagnostic and screening products that contain information, for example, about the usage, dosage, manufacture, administration, contraindications or warnings concerning the use of such diagnostic and screening products.
  • HLA-B*2705 expressing cells were obtained from the University of Oxford (Oxford, GB). HLA-B*2705 restricted cytotoxic T-lymphocytes (CTL) specific for the recognition of HIV-I epitopes KKlO (KRWIILGLNK) and KY9 (KRKGGIGGY) and KF11 (KAFSPEVIPMF) were prepared following protocols known in the art. See Payne R, el al ., J Virol 2010; 84(20): 10543-10557 and Kloverpris H, et al, J Virol 2013; 87(19): 10889- 10894.
  • CTL cytotoxic T-lymphocytes
  • R10 Prior to infection, the U937-HLA-B*B2705 expressing cells were cultured since day - 3 in a R10 + geneticin 0.5 mg/mL (G418, Cat. No. 10131027, Thermo Fisher Scientific Inc., Waltham, MA, USA) culture medium. A final culture concentration of 0.8-1 M/mL was reached.
  • R10 is composed of RPMI 1640 medium (Cat. No. 21875, Thermo Fisher Scientific Inc., Waltham, MA, USA), penicillin-streptomycin (5,000 UI/mL, Cat. No. 15070063, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA).
  • the magnetofection plate was then incubated. 0.8 M cells were added to 24-well plates in a volume of 300 pL R10. The virus was mixed with the VM (100 pL NL43-GFP: 4 pL VM). The mixture was incubated for 15 minutes at room temperature. Viral stocks from 1M TCID50/mL were preferably used. The magnetized virus was pipetted carefully into the target wells, making sure of not disturbing the cell layer at the bottom of the wells.
  • the 24-well plates were placed on the magnetofection plates making sure that the magnets were properly lined up. The plates were then incubated at 37°C for 40 minutes. Afterwards, the infected cells were harvested, re-suspended in PBS IX (Cat. No. 10010023, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) and transferred to a microcentrifuge tube. The plates were washed with 1 mL of PBS IX to make sure that all cells were harvested. The cells were subsequently washed twice with PBS IX and were centrifuged at 400 g for 5 minutes.
  • PBS IX Cat. No. 10010023, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA
  • the infected cells were placed in 24-well plates at a final concentration of 0.8 M/mL in 1 mL of R10 + geneticin 0.5 mg/mL for 72 hours.
  • the culture was checked at 24 and 48 hours after infection to monitor cellular growth.
  • Fresh new media i.e. around 1 mL was added to the wells if necessary.
  • the frequency of GFP positive cells was measured by flow cytometry after 72 hours of infection.
  • the cells were counted using microbeads.
  • a 10-20% range of GFP-expressing cells was employed to ensure that, after sorting the positive fraction, an appropriate level of proviral DNA was present for monitoring HIV-l reactivation in the resting-like GFP negative fraction.
  • the cells were centrifuged and re-suspended in R20 to a final concentration of 5-7 M/mL in a 15 mL tube. The cells were then placed on ice until sorting.
  • the GFP negative fraction was sorted using a BD FACS Aria II Cell Sorter (BD Biosciences, San Jose, CA, USA) or a similar device.
  • the cells were cultured in a T25 flask to a final concentration of 0.8 M/mL in R10+ geneticin 0.5 mg/mL medium in the presence of 1 mM of ritonavir (RTV, Cat. No. A2169, Sigma-Aldrich Corp., Saint Louis, MO, USA). The cells were cultured during 4 days and were monitored for cell growth. Fresh media + RTV were added if the culture turned yellow.
  • RTV ritonavir
  • the cell culture was enriched in HIV-l latently infected cells (or Resting-Like“RELI” cells). Under these experimental conditions, the RELI cells had 0.6-0.9 copies of HIV-l proviral DNA per million cells.
  • RELI cells were counted and added to round-bottom 96-well plates until a final concentration of lM/mL in R10 + raltegravir (RLT 100 nM, Cat. No. CDS023737, Sigma-Aldrich Corp., Saint Louis, MO, USA) medium per well was attained.
  • Latency reactivating agents were added for 48 hours to the RELI cells to assess viral reactivation. See Table 1. LRAs were also added to uninfected cells to assess cellular toxicity. All LRA-treated cells were assessed in both co-culture and non-co-culture conditions in duplicates.
  • the HIV-l reactivation "SHOCK" was analyzed by the percentage of intracellular viral protein p24 from non-dead RELI cells.
  • CTLs were counted and their viability was checked.
  • the CTLs were seeded at a final concentration of 2 M/mL in 24-well plates with a RPMI 1640 medium containing 50% of human AB serum (Cat. No. H3667, Sigma-Aldrich Corp., Saint Louis, MO, USA).
  • the CTLs were cultured overnight in order to reduce cytokine background production.
  • the RELI cells were washed at least twice with PBS IX for 5 minutes at 800 rpm. Afterwards, the cells were re-suspended in a LRA- free R10 + RLT (100 nM) medium. RMD-treated cells were washed 5 hours post- reactivation.
  • the RELI cells/CTL co-culture was set up in round-bottom 96-well plates. CTLs were counted and re-suspended in RPMI with 10% of human AB serum (Cat. No. H3667, Sigma-Aldrich Corp., Saint Louis, MO, USA) to a final concentration of 1 M/mL. A CDl07a-PE-Cy5 antibody (clone H4A3, Cat. No. 555802, BD Biosciences, San Jose, CA, USA) was added to the CTL culture. The CTLs were then mixed with the reactivated RELI cells in a 1 : 1 ratio (100,000 RELI cells: 100,000 CTLs). The CTLs and RELI cells were co cultured for 20 hours.
  • the cells were incubated at 37°C for 3 hours in the presence of brefeldin A at 2.5 pL/mL (Golgiplug solution, Cat. No. 555029, BD Biosciences, San Jose, CA, USA) and monensin at 1.6 pL/mL (GolgiStop solution, Cat. No. 554724, BD Biosciences, San Jose, CA, USA) diluted in PBS IX (50 pL/well).
  • brefeldin A 2.5 pL/mL
  • monensin at 1.6 pL/mL
  • PBS IX 50 pL/well
  • the culture supernatant was removed.
  • the culture supernatant was removed.
  • CD8-V450 antibody pacific blue, clone RPA-T8, Cat. No. 558207, BD Biosciences, San Jose, CA, USA
  • PBS IX PBS IX
  • the cells were washed twice with 200 pL PBS IX at 800 g for 3 minutes. The culture supernatant was removed. Alternatively, the cells can be stored overnight in the Fixation Medium at 4°C. 11)
  • the following antibodies (i) p24-HIV (clone KC57- RD1, Cat. No. 6604667, Beckman Coulter, Inc., Brea, CA, USA), (ii) MIRIb- FITC (clone 24006, Cat. No. IC271F, R&D Systems, Inc. Minneapolis, MN, USA) and (iii) IFNg-PE-Cy7 (clone 4S.B3, Cat. No.
  • the CTL“KILL” was calculated taking into account the reduction of non-dead, CD8 negative, p24 positive cells from co-cultures of RELI cells and CTLs, i.e. 100- [(% p24 in non-dead CD8 negative cells co-cultured with CD8 positive cells / % p24 in non-dead CD8 negative cells in the absence of CD8 positive cells) x 100]
  • the CTL activation profile in response to viral reactivation was analyzed by the secretion of CD 107a, MIPlb and IFNg from live, CD8 positive CTLs.
  • Banerjee C, et al. BET bromodomain inhibition as a novel strategy for reactivation of HIV-l, J Leukoc Biol 2012; 92: 1147-1154.
  • HDACis Histone deacetylase inhibitors
  • Boehm D, et al. BET bromodomain-targeting compounds reactivate HIV from latency via a Tat-independent mechanism, Cell Cycle 2013: 12:452-462.
  • Bosque A et al. , Induction of HIV-l latency and reactivation in primary memory CD4+ T cells, Blood 2009; 113:58-65.
  • Contreras X, et al. Suberoylanilide hydroxamic acid reactivates HIV from latently infected cells, J Biol Chem 2009; 284:6782-6789.
  • Gallastegui E, et al. Combination of biological screening in a cellular model of viral latency and virtual screening identifies novel compounds that reactivate HIV-l, J Virol 2012; 86:3795-3808.
  • Hermankova M, et al. Analysis of human immunodeficiency virus type 1 gene expression in latently infected resting CD4+ T lymphocytes in vivo , J Virol 2003; 77:7383-7392.
  • Kauder S, et al. Epigenetic regulation of HIV- 1 latency by cytosine methylation, PLoS Pathog 2009; 5:el000495Korin Y, et al. , Effects of prostratin on T cell activation and human immunodeficiency virus latency, J Virol 2002; 76:8118-8123.
  • Keedy K, et al. A limited group of class I histone deacetylases acts to repress human immunodeficiency virus type 1 expression J Virol 2009; 83:4749-4756.
  • Rasmussen T, et al. Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial, The Lancet HIV 2014; l(l):el3-e2l.
  • Shan L, et al. Stimulation of HIV-l- specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation, Immunity 2012; 36:491- 501. 55. Shan L, et al. , A novel PCR assay for quantification of HIV-l RNA, J Virol 2013; 87(1 l):652l-6525.
  • 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; l0(4):el00407l.
  • Williams S, et al. Prostratin antagonizes HIV latency by activating NF-kB, J Biol Chem 2004; 279:42008-42017.
  • Williams S, et al. NF-kB p50 promotes HIV latency through HD AC recruitment and repression of transcriptional initiation, EMBO J 2006; 25: 139-149.
  • liver is a major organ for clearing simian immunodeficiency virus in rhesus monkeys, J Virol 2002; 76(l0):527l-5273.

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Abstract

La présente invention concerne un procédé avec des agents d'inversion de latence du VIH-1 (LRA). Le procédé comprend les étapes consistant à : (a) cultiver in vitro une fraction de lymphocytes T CD4+ obtenus à partir d'un sujet infecté par le VIH avec au moins un agent d'inversion de latence (LRA) pendant au moins 48 heures, (b) co-cultiver in vitro la fraction de lymphocytes T CD4+ avec des lymphocytes-T cytotoxiques (CTL) obtenus à partir du sujet infecté par le VIH et (c) compter les lymphocytes T CD4+ tués dans la fraction.
PCT/IB2018/001456 2017-11-22 2018-11-21 Procédé de criblage d'agents d'inversion de latence du vih-1 WO2019106427A2 (fr)

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