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EP4138910A1 - Novel combination for the treatment of aids - Google Patents

Novel combination for the treatment of aids

Info

Publication number
EP4138910A1
EP4138910A1 EP21721080.6A EP21721080A EP4138910A1 EP 4138910 A1 EP4138910 A1 EP 4138910A1 EP 21721080 A EP21721080 A EP 21721080A EP 4138910 A1 EP4138910 A1 EP 4138910A1
Authority
EP
European Patent Office
Prior art keywords
ifn
agent
cells
interferon
vaccine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21721080.6A
Other languages
German (de)
French (fr)
Inventor
Daniel Zagury
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
21C Bio SAS
Original Assignee
21C Bio SAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US16/857,715 external-priority patent/US12024558B2/en
Application filed by 21C Bio SAS filed Critical 21C Bio SAS
Publication of EP4138910A1 publication Critical patent/EP4138910A1/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/249—Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to a novel method for treating acquired immune deficiency syndrome (AIDS) in a subject in need thereof.
  • said method comprises the administration of a combination, a kit-of-parts, a composition or a pharmaceutical composition comprising a type III interferon blocking agent, an interferon-alpha (IFN-a) blocking agent, an antiretroviral (ART) agent and, optionally, an interferon-beta (IFN-b) blocking agent and/or a latency-reversing agent (LRA).
  • a type III interferon blocking agent an interferon-alpha (IFN-a) blocking agent, an antiretroviral (ART) agent and, optionally, an interferon-beta (IFN-b) blocking agent and/or a latency-reversing agent (LRA).
  • IFN-a interferon-alpha
  • ART antiretroviral
  • LRA latency-reversing agent
  • Antiretroviral therapy is thus far, a very efficient therapy in the treatment of patients infected with human immunodeficiency viruses (HIV). Indeed, patients treated with ART have a plasma viremia below detectable levels and thus can have normal life expectancy. However, these treatments are very onerous and can lead to uncertain long term cytotoxicity for HIV patients (Chun et ah, “Durable Control of HIV Infection in the Absence of Antiretroviral Therapy: Opportunities and Obstacles” JAMA, 2019 Jul 2;322(l):27-28).
  • CD8 cytotoxic T cells have less access to B-cell follicles and thus these cells are protected from the treatment, while in the gastrointestinal tract, many Thl7 cells expressing CCR5 are present, cells targeted preferentially by the vims, which can explain the high level of HIV DNA and RNA.
  • Macrophages from different tissues can also be infected and thus persist for example in the brain and this population was demonstrated as being the viral source of the viral rebound after ART arrest in a unique model of humanized myeloid-only mice (Honeycutt et ah, “HIV persistence in tissue macrophages of humanized myeloid-only mice during antiretroviral therapy” Nat Med, 2017; 23: 638-43).
  • the HIV-1 latent reservoir is a major hurdle to achieve a cure for HIV-1.
  • a new strategy consists in targeting the latent cells which escape the ART and can thus create a viral rebound after ART arrest.
  • One hypothesis was that the HIV integrated mostly into heterochromatin-repressed transcriptional regions into these latent cells, but it was surprisingly found that HIV can be latent while integrated in euchromatin- active transcriptional regions, suggesting that the epigenetic silencing occurs via cis- or trans- acting elements. So far, the therapies were focused on epigenetic mechanisms of latency and mostly histone deacetylase inhibitors (HDAC) inhibitors, but no clear reservoir reduction has been demonstrated.
  • HDAC histone deacetylase inhibitors
  • T-cell activating agents were also a new wave of therapy using for example protein kinase C (PKC) agonists but despite promising results in vitro , no reduction in the latent reservoir was reported in vivo.
  • PLC protein kinase C
  • One strategy could also be a “shock and kill” strategy wherein the “shock” phase will be to target the immune response towards the reservoir and to reverse latency and then to kill the cells, but this strategy requires to know which antigen will be presented by the re-activated cell.
  • the “shock and kill” strategy is based on inducing viral transcription of latent HIV-1 provirus followed by the selective killing of reactivated cells.
  • latent HIV reservoir represents the main obstacle to achieving sustained virologic remission in ART-treated HIV-infected individuals following ART treatment interruption. And the reservoir cell compartment still needs to be better understood in order to improve the cure strategy against HIV infection.
  • AIDS acquired immune deficiency syndrome
  • Interferons are a group of cytokines of different types. Type I interferons are systemic, while type III interferons are mainly mucosal (i.e. produced at the mucosal epithelial).
  • type I IFN-a and type III IFN-l locally is induced by viral replication occurring in the peripheral and mucosal reservoir cells (which contain latent integrated HIV proviruses), and is involved in the incomplete virus clearance.
  • the inventors have surprising shown the presence of type III interferons in the serum of patients after antiretroviral (cART) treatment, while the serum level of type I IFN had totally decreased after treatment.
  • This antiviral cytokine is locally produced by mucosal cells, which are still infected and replicating the virus. By their antiviral effects, these interferons locally reduce the ongoing viral replication occurring in these reservoir cells. This limits the local propagation of the virus to nearby cells, but at the time maintains the reservoir of infected cells.
  • the inventors have thus surprisingly shown that reactivation of the HIV-1 latent reservoirs is inhibited by type III interferons which are naturally produced by HIV 1 infected patients, especially when under antiretroviral treatment.
  • the inventors thus provide a new therapeutic approach based on the use of a type III interferon blocking agent to reverse the natural or therapeutically-induced mechanisms that inhibit HIV-1 reactivation in the latent reservoirs.
  • the inventors thus propose to block the type III interferon-l, the type I interferon-a, and optionally type I IFN interferon-b antiviral action in resistant cells containing proviral HIV-1 DNA in the HIV reservoirs, by repeated administration of specific agents blocking the production or of the activity of these cytokines, to reverse proviral latency, while administering ART agents to the patient until total viral clearance.
  • HIV-1 proviral DNA present in these reservoir cells may fully replicate viruses.
  • cART treatment which controls viral replication, may reduce and progressively eliminate these peripheral and mucosal cellular reservoirs.
  • the applicant thus provides a novel method for treating AIDS in a subject in need thereof, comprising administering to the subject a combination comprising: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • a type III interferon blocking agent ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • the invention relates to a combination comprising: i) a type III interferon blocking agent, ii) an interferon-alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) optionally, an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • a type III interferon blocking agent ii) an interferon-alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) optionally, an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • IFN-a interferon-alpha
  • IFN-b interferon-beta
  • ART antiretroviral
  • LRA latency-reversing agent
  • the type III interferon blocking agent is:
  • an agent neutralizing circulating IFN-l selected from the group comprising an active anti-IFN-l vaccine, such as an IFN-k-kinoid, IFN-l DNA-based or IFN-l RNA-based vaccine, and a passive anti-IFN-l vaccine, such as an anti-IFN-l antibody or an anti-IFN- l hyper-immune serum, or
  • an agent blocking type III interferon signaling selected from the group consisting of an active anti-IFNLRl vaccine, such as an IFNLR1 DNA-based or IFNLR1 RNA-based vaccine, and a passive anti-IFNLRl vaccine, such as an antibody that binds to the IFNLRl receptor.
  • an active anti-IFNLRl vaccine such as an IFNLR1 DNA-based or IFNLR1 RNA-based vaccine
  • a passive anti-IFNLRl vaccine such as an antibody that binds to the IFNLRl receptor.
  • the type III interferon blocking agent is an anti-IFN-l antibody, or an agent blocking type III interferon signaling selected from the group consisting of an active anti-IFNLRl vaccine, such as an IFNLRl DNA-based or IFNLRl RNA-based vaccine, and a passive anti-IFNLRl vaccine, such as an antibody that binds to the IFNLRl receptor.
  • an active anti-IFNLRl vaccine such as an IFNLRl DNA-based or IFNLRl RNA-based vaccine
  • a passive anti-IFNLRl vaccine such as an antibody that binds to the IFNLRl receptor.
  • the IFN-a blocking agent is selected from the group consisting of an agent neutralizing circulating IFN-a, an agent blocking IFN-a signaling, an agent depleting IFN-a producing cells, and an agent blocking IFN-a production; wherein the agent neutralizing circulating IFN-a is selected from the group comprising an active anti-IFN-a vaccine, such as an IFN-a-kinoid, IFN-a DNA-based or IFN-a RNA-based vaccine, and a passive anti-IFN-a vaccine, such as an anti-IFN-a antibody or anti-IFN-a hyper-immune serum; wherein the blocking agent of IFN-oc signaling is selected from the group consisting of an active anti-IFNARl or anti-IFNAR2 vaccine, such as an IFNAR1 or IFNAR2 DNA-based or an IFNAR1 or IFNAR2 RNA-based vaccine, a passive anti- IFNARl or anti-IFNAR2 vaccine, such as an anti-type I interfer
  • the IFN-b blocking agent is an agent neutralizing circulating IFN- b selected from the group comprising an active anti-IFN-b vaccine, such as an IFN-b- kinoid, IFN-b DNA-based or IFN-b RNA-based vaccine, and a passive anti-IFN-b vaccine, such as an anti-IFN-b antibody or an anti-IFN-b hyper-immune serum.
  • an active anti-IFN-b vaccine such as an IFN-b- kinoid, IFN-b DNA-based or IFN-b RNA-based vaccine
  • a passive anti-IFN-b vaccine such as an anti-IFN-b antibody or an anti-IFN-b hyper-immune serum.
  • the antiretroviral (ART) agent is selected from the group consisting of Nucleoside reverse transcriptase inhibitors (NRTIs), Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Protease inhibitors (Pis), Integrase inhibitors (INSTIs), Fusion inhibitors (FIs), Chemokine receptor antagonists (CCR5 antagonists) and Entry inhibitors (CD4-directed post-attachment inhibitors).
  • NRTIs Nucleoside reverse transcriptase inhibitors
  • NRTIs Non-nucleoside reverse transcriptase inhibitors
  • Protease inhibitors Protease inhibitors
  • INSTIs Integrase inhibitors
  • Fusion inhibitors FIs
  • CCR5 antagonists Chemokine receptor antagonists
  • Entry inhibitors CD4-directed post-attachment inhibitors
  • the latency-reversing agent is selected from the group consisting of PKC agonists, MAPK agonists, CCR5 antagonists, Tat vaccines, SMAC mimetics, inducers of P-TEFb release, activators of Akt pathway, benzotriazole derivatives, epigenetic modifiers and immunomodulatory LRAs.
  • Another aspect of the invention relates to a combination as described herein for use in the treatment of acquired immune deficiency syndrome (AIDS) in a subject in need thereof.
  • AIDS acquired immune deficiency syndrome
  • the presence of cells containing replication-competent proviral HIV DNA is assessed in a blood sample from the subject.
  • the type III interferon blocking agent, the IFN-a blocking agent, optionally the interferon-beta (IFN-b) blocking agent, the antiretroviral (ART) agent, and optionally the latency-reversing agent are for simultaneous, separate or sequential administration.
  • the type III interferon blocking agent, the IFN-a blocking agent, and optionally the interferon-beta (IFN-b) blocking agent are to be administered every week, every 2 weeks or every 3 weeks.
  • the antiretroviral (ART) agent, and optionally the latency-reversing agent are to be administered daily.
  • the type III interferon blocking agent, the IFN-a blocking agent, and optionally the interferon-beta (IFN-b) blocking agent are to be administered parenterally or intravenously.
  • one or more doses of the type III interferon blocking agent, the IFN-a blocking agent, and optionally the interferon-beta (IFN-b) blocking agent are to be administered to the subject before receiving said combination.
  • one or more doses of the antiretroviral (ART) agent, and optionally the latency-reversing agent, are to be administered to the subject before receiving said combination.
  • said combination is to be administered to the subject until no cell containing replication-competent proviral HIV DNA is detected in a blood sample from the subject.
  • the term “adjuvant” refers to a compound or combination of compounds that helps and enhances the pharmacological effect of a drug or a vaccine, or increases an immunogenic response.
  • administering means either directly administering a compound or composition of the present invention, or administering a prodrug, derivative or analog which will form an equivalent amount of the active compound or substance within the body.
  • routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
  • antigen refers to a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal.
  • an antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens.
  • the term "antigen” includes all related antigenic epitopes.
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.
  • Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.
  • a therapy decreases the infectious load or titer of a pathogen such as HIV, or one or more symptoms associated with infection.
  • fragment refers to a portion of a polypeptide that exhibits at least one useful epitope.
  • functional fragment(s) of a polypeptide refers to all fragments of a polypeptide that retain an activity, or a measurable portion of an activity, of the polypeptide from which the fragment is derived. Fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell.
  • An epitope is a region of a polypeptide capable of binding an immunoglobulin generated in response to contact with an antigen.
  • immunogenic peptide refers to a peptide which comprises an allele- specific motif or other sequence, such as an N-terminal repeat, such that the peptide will bind an MHC molecule and induce a cytotoxic T lymphocyte ("CTL") response, or a B cell response (for example antibody production) against the antigen from which the immunogenic peptide is derived.
  • CTL cytotoxic T lymphocyte
  • B cell response for example antibody production
  • immunogenic peptides are identified using sequence motifs or other methods, such as neural net or polynomial determinations known in the art.
  • algorithms are used to determine the "binding threshold" of peptides to select those with scores that give them a high probability of binding at a certain affinity and will be immunogenic.
  • the algorithms are based either on the effects on MHC binding of a particular amino acid at a particular position, the effects on antibody binding of a particular amino acid at a particular position, or the effects on binding of a particular substitution in a motif-containing peptide.
  • a "conserved residue" is one which appears in a significantly higher frequency than would be expected by random distribution at a particular position in a peptide.
  • immunogenic refers to the state of being able to mount a protective response upon exposure to an immunogenic agent.
  • Protective responses can be antibody-mediated or immune cell-mediated, and can be directed toward a particular pathogen or tumor antigen. Immunity can be acquired actively (such as by exposure to an immunogenic agent, either naturally or in a pharmaceutical composition) or passively (such as by administration of antibodies or in vitro stimulated and expanded T cells).
  • immunogenic agent either naturally or in a pharmaceutical composition
  • IFN-a alpha interferon
  • interferon-alpha refers to a family of more than 20 related but distinct members encoded by a cluster on chromosome 9 and all bind to the same IFN receptor.
  • the IFN-a2 have 3 recombinant variants (a2a, a2b, a2c) depending upon the cells of origin and the IFN-a2b is the predominant variant in human genome.
  • each subtype has a different binding capacity to the IFNAR, modulating the signaling transduction events and the biological effects in the target cells.
  • type III interferon also called interferon-lambda (IFN-l) refers to naturally occurring and/or recombinant cytokines of the type III interferon-lambda family. There are four IFN-l members in humans, IFN-/J/IL-29, IFN-k2/IL-28A, IFN-k3/IL-28B, IFN-/,4. -
  • IFN-b interferon-beta interferon
  • interferon-beta refers to a family of two related but distinct members IFN-bI and IRN-b3.
  • IFNAR IFN-a receptor
  • IFNAR1 IFNAR 1 /IFN AR2 heterodimer
  • isolated or “non-naturally occurring” with reference to a biological component (such as a nucleic acid molecule, protein organelle or cells), refers to a biological component altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated”.
  • An isolated nucleic acid or peptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • a preparation of isolated nucleic acid or peptide contains the nucleic acid or peptide at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, greater than about 96% pure, greater than about 97% pure, greater than about 98% pure, or greater than about 99% pure.
  • Nucleic acids and proteins that are "non-naturally occurring" or have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • An "isolated polypeptide" is one that has been identified and separated and/or recovered from a component of its natural environment.
  • subject refers to an animal, for example a mammal, primate or human, and include all mammals, such as e.g. non-human primate, (particularly higher primates), sheep, dog, rodent, (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, horses.
  • non-human primate particularly higher primates
  • sheep e.g. sheep
  • rodent e.g. mouse or rat
  • guinea pig e.g. goat
  • pig e.g. mouse or rat
  • rabbits cows, horses.
  • these terms refer to a human, to whom treatment, including prophylactic treatment, with the combination according to the present invention, is provided.
  • mutant refers to any difference in a nucleic acid or polypeptide sequence from a normal, consensus or “wild type” sequence.
  • a mutant is any protein or nucleic acid sequence comprising a mutation.
  • a cell or an organism with a mutation may also be referred to as a mutant.
  • Some types of coding sequence mutations include point mutations (differences in individual nucleotides or amino acids); silent mutations (differences in nucleotides that do not result in an amino acid changes); deletions (differences in which one or more nucleotides or amino acids are missing, up to and including a deletion of the entire coding sequence of a gene); frameshift mutations (differences in which deletion of a number of nucleotides indivisible by 3 results in an alteration of the amino acid sequence.
  • a mutation that results in a difference in an amino acid may also be called an amino acid substitution mutation.
  • Amino acid substitution mutations may be described by the amino acid change relative to wild type at a particular position in the amino acid sequence.
  • prevent refers to preventative measures, wherein the object is to reduce the chances that a subject will develop the pathologic condition or disorder over a given period of time. Such a reduction may be reflected, e.g., in a delayed onset of at least one symptom of the pathologic condition or disorder in the subject.
  • prophylactic refers to a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
  • a prophylactic treatment of a HIV or SIV infection in a subject refers to a treatment that allows the subject to become an elite controller (EC) i.e. to have a relatively high CD4 + T cell count (such as e.g. superior to 500 CD4 + T cells per microliter) and/or to maintain clinically undetectable plasma HIV-1 RNA level (such as e.g. HIV RNA ⁇ 50 copies/mL) during a prolonged period of time in the absence of any antiretroviral treatment (ART).
  • ART antiretroviral treatment
  • curative refers to a treatment administered to a subject suffering from a disease for the purpose of curing the disease, i.e. of making any sign of the disease disappear or becoming undetectable.
  • protein a polypeptide
  • amino acid sequence amino acid sequence
  • the terms “protein”, “peptide”, “polypeptide”, and “amino acid sequence” are used interchangeably herein to refer to polymers of amino acid residues of any length.
  • the polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
  • sample refers to a biological specimen obtained from a subject, such as a cell, fluid of tissue sample.
  • biological samples contain genomic DNA, RNA (including mRNA and microRNA), protein, or combinations thereof.
  • samples include, but are not limited to, saliva, blood, serum, urine, spinal fluid, tissue biopsy, surgical specimen, cells (such as PBMCs, white blood cells, lymphocytes, or other cells of the immune system) and autopsy material.
  • treatment refers to an intervention that ameliorates a sign or symptom of a disease or pathological condition.
  • HIV RNA viral load
  • CD4 T lymphocyte CD4 T lymphocyte
  • the efficacy of the treatment may be evaluated by the plasma viral RNA load of a "treated" human before and after the treatment, if it is reduced by at least about 10%, 20%, 30%, 40%, 50%, more preferably by at least about 70%, yet more preferably by at least about 75% or 80% or 85% or 90% or 95% or 98% or 99%, or even more (99.5%, 99.8%, 99.9%, 100%) the treatment is considered as effective, and/or by the monitoring of CD4 cell count before and after the treatment, if the absolute count of CD4 cell is increased by at least about 5%, 10%, 15%, 20%, 25% , more preferably by at least about 30%, yet more preferably by at least about 35% or 40% or 45% or 50% or 55% or 60% or 65%,
  • treatment also refers to any observable beneficial effect of the treatment.
  • the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease.
  • a therapeutic treatment is a treatment administered to a subject after signs and symptoms of the disease have developed.
  • a prophylactic treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs, for the purpose of decreasing the risk of developing pathology.
  • a prophylactic treatment of a HIV or SIV infection in a subject refers to a treatment that allows the subject to become an elite controller (EC) i.e. to have a relatively high CD4 + T cell count (such as e.g. superior to 500 CD4 + T cells per microliter) and/or to maintain clinically undetectable plasma HIV-1 RNA level (such as e.g. HIV RNA ⁇ 50 copies/mL) during a prolonged period of time in the absence of any antiretroviral treatment (ART).
  • a prophylactic treatment is a treatment administered to a subject suffering from a disease for the purpose of curing the disease, i.e. of making any sign of the disease disappear or becoming undetectable.
  • the term "vaccine” refers to an immunogenic product or composition that can be administered to a mammal, such as a human, to confer immunity, such as passive or active immunity, to a disease or other pathological condition.
  • Vaccines can be used preventively or therapeutically, either prophylactically or curatively.
  • vaccines can be used to reduce the likelihood of developing a disease (such as infection) or to reduce the severity of symptoms of a disease or condition, limit the progression of the disease or condition (such as infection), or limit the recurrence of a disease or condition.
  • virus refers to microscopic infectious organism that reproduces inside living cells.
  • a virus consists essentially of a core of nucleic acid (the viral genome) surrounded by a protein coat (capsid), and has the ability to replicate only inside a living cell.
  • “Viral replication” is the production of additional virus particles by the occurrence of at least one viral life cycle.
  • a virus may subvert the host cells' normal functions, causing the cell to behave in a manner determined by the virus. For example, a viral infection may result in a cell producing a cytokine, or responding to a cytokine, when the uninfected cell does not normally do so.
  • Particular viral species can alternatively enter into a "lysogenic" or “latent” infection. In the establishment of latency, the viral genome is replicated, but capsid proteins are not produced and assembled into viral particles.
  • the invention relates to a method for treating acquired immune deficiency syndrome (AIDS) in a subject in need thereof, comprising administering to the subject a combination comprising: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • the method comprises administering to the subject i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, and iv) an antiretroviral (ART) agent.
  • the method comprises administering to the subject i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, and iv) an antiretroviral (ART) agent.
  • a type III interferon blocking agent ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, and iv) an antiretroviral (ART) agent.
  • the method comprises administering to the subject i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • a type III interferon blocking agent ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • the method is a method of prophylactic treatment.
  • the method is a method of curative treatment.
  • the present invention further relates to a combination comprising: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) optionally, an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • a type III interferon blocking agent ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) optionally, an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • IFN-a interferon- alpha
  • IFN-b interferon-beta
  • ART antiretroviral
  • LRA latency-reversing agent
  • the present invention also relates to a combination for use as a medicament, wherein said combination comprises: i) a type III interferon blocking agent, ii) an interferon-alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • a type III interferon blocking agent ii) an interferon-alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • IFN-a interferon-alpha
  • IFN-b interferon-beta
  • ART antiretroviral
  • LRA latency-reversing agent
  • the present invention also relates to a combination for use in the treatment of acquired immune deficiency syndrome (AIDS) in a subject in need thereof, wherein said combination comprises: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • a type III interferon blocking agent ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • IFN-a interferon- alpha
  • IFN-b interferon-beta
  • ART anti
  • the combination for use comprises i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, and iv) an antiretroviral (ART) agent.
  • a type III interferon blocking agent ii) an interferon- alpha (IFN-a) blocking agent, and iv) an antiretroviral (ART) agent.
  • IFN-a interferon- alpha
  • ART antiretroviral
  • the combination for use comprises i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, and iv) an antiretroviral (ART) agent.
  • a type III interferon blocking agent ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, and iv) an antiretroviral (ART) agent.
  • the combination for use comprises i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • a type III interferon blocking agent ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • IFN-a interferon- alpha
  • IFN-b interferon-beta
  • ART antiretroviral
  • LRA latency-reversing agent
  • the present invention further relates to a kit-of-parts comprising: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) optionally, an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • a type III interferon blocking agent ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) optionally, an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • IFN-a interferon- alpha
  • IFN-b interferon-beta
  • ART antiretroviral
  • LRA latency-reversing agent
  • kits-of-parts for use as a medicament, wherein said kit-of-parts comprises: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) optionally, an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • kit-of-parts comprises: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) optionally, an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • IFN-a interferon- alpha
  • IFN-b interferon-beta
  • kits-of-parts for use in the treatment of acquired immune deficiency syndrome (AIDS) in a subject in need thereof, wherein said kit-of-parts comprises: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • a type III interferon blocking agent ii) an interferon- alpha (IFN-a) blocking agent
  • IFN-b interferon-beta
  • ART antiretroviral
  • LRA latency-reversing agent
  • the kit-of-parts for use comprises i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, and iv) an antiretroviral (ART) agent.
  • a type III interferon blocking agent ii) an interferon- alpha (IFN-a) blocking agent
  • ART antiretroviral
  • the kit-of-parts for use comprises i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, and iv) an antiretroviral (ART) agent.
  • a type III interferon blocking agent ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, and iv) an antiretroviral (ART) agent.
  • the kit-of-parts for use comprises i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • a type III interferon blocking agent ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
  • the combination or kit-of-parts as described herein is for use in the treatment of acquired immune deficiency syndrome (AIDS) in a subject in need thereof.
  • AIDS acquired immune deficiency syndrome
  • the subject is infected with a human immunodeficiency virus (HIV), or a simian immunodeficiency virus (SIV).
  • HAV human immunodeficiency virus
  • SIV simian immunodeficiency virus
  • kits-of-parts as described herein is also for use in the treatment of a human immunodeficiency vims (HIV) infection or a simian immunodeficiency virus (SIV) infection.
  • HIV human immunodeficiency vims
  • SIV simian immunodeficiency virus
  • HIV Due to the great variability in the HIV genome, which results from mutation, recombination, insertion and/or deletion, HIV has been classified in groups, subgroups, types, subtypes and genotypes. There are two major HIV groups (HIV-1 and HIV-2) and many subgroups because the HIV genome mutates constantly. The major difference between the groups and subgroups is associated with the viral envelope. HIV-1 is classified into a main group (M), said group M being divided into least nine genetically distinct subtypes. These are subtypes A, B, C, D, F, G, H, J and K. Many other subtypes resulting from in vivo recombination of the previous ones also exist (e.g., CRF).
  • the HIV antigen is related to a specific HIV group, subgroup, type, subtype or to a combination of several subtypes.
  • the HIV vims is HIV-1 or HIV-2, preferably HIV-1.
  • the subject is infected with an HIV-1 strain or an HIV-2 strain.
  • kits-of-parts as described herein is also for use in the treatment of an HIV-1 strain or an HIV-2 strain.
  • the HIV-1 vims is from group M and preferably subtype B (HXB2).
  • the subject is a mammal, a primate, preferably a human.
  • the combination or kit-of-parts as described herein is for use in the preventive or the prophylactic treatment of acquired immune deficiency syndrome (AIDS) in a subject in need thereof.
  • AIDS acquired immune deficiency syndrome
  • the combination or kit-of-parts as described herein is for use in the curative treatment of acquired immune deficiency syndrome (AIDS) in a subject in need thereof.
  • AIDS acquired immune deficiency syndrome
  • the subject has already received at least one dose of at least one antiretroviral (ART) agent, or is under antiretroviral therapy or under combined antiretroviral therapy (cART) comprising at least one antiretroviral (ART) agent, before being administered the combination of the invention.
  • ART antiretroviral
  • cART combined antiretroviral therapy
  • alpha interferon refers to a family of more than 20 related but distinct members encoded by a cluster on chromosome 9 and all bind to the same IFN receptor.
  • the IFN-a2 have 3 recombinant variants (a2a, a2b, a2c) depending upon the cells of origin and the IFN-a2b is the predominant variant in human genome.
  • each subtype has a different binding capacity to the IFNAR, modulating the signaling transduction events and the biological effects in the target cells.
  • the interferon- alpha blocking agent described herein is an agent neutralizing circulating IFN-a and/or an agent blocking IFN-a signaling, and/or an agent depleting IFN-a producing cells, and/or an agent blocking IFN-a production.
  • the interferon- alpha blocking agent described herein comprises at least one agent selected from: an agent neutralizing circulating IFN-a and/or an agent blocking IFN-a signaling, and/or an agent depleting IFN-a producing cells, and/or an agent blocking IFN-a production.
  • the agent neutralizing circulating IFN-a and/or the agent blocking IFN-a signaling, and/or the agent depleting IFN-a producing cells, and/or the agent blocking IFN-a production is/are an IFN-a antagonist.
  • the interferon-alpha blocking agent is selected from the group consisting of : an agent neutralizing circulating alpha interferon, an agent blocking interferon- alpha signaling, an agent depleting IFN-oc producing cells, and/or an agent blocking IFN-oc production, wherein the agent neutralizing circulating alpha interferon is selected from the group comprising active anti-IFN-oc vaccine including IFN-a-kinoid, IFN-a DNA-based or IFN-a RNA-based vaccine, or passive anti-IFN-oc vaccine including anti-IFN-oc antibodies or anti-IFN-oc hyper-immune serum, wherein the blocking agent of interferon- alpha signaling is selected from the group consisting of an active anti-IFNARl or anti-IFNAR2 vaccine, such as an IFNAR1 or IFNAR2 DNA-based or an IFNAR1 or IFNAR2 RNA-based vaccine, a passive anti-IFNARl or anti-IFNAR2 vaccine, such
  • the interferon- alpha blocking agent is an agent neutralizing circulating alpha interferon selected from the group consisting of active anti-IFN-a vaccine including IFN-a-kinoid, IFN-a DNA-based or IFN-a RNA-based vaccine, or passive anti-IFN-a vaccine including anti-IFN-a antibodies or anti-IFN-a hyper-immune serum
  • the blocking agent of interferon- alpha signaling is selected from the group consisting of an active anti-IFNARl or anti-IFNAR2 vaccine, such as an IFNARl or IFNAR2 DNA-based or an IFNAR1 or IFNAR2 RNA-based vaccine, a passive anti-IFNARl or anti-IFNAR2 vaccine, such as an anti-type I interferon R1 or R2 antibody, and an IFN-a endogenous regulator, such as SOSC1 or an aryl hydrocarbon receptor.
  • alpha interferon antagonist refers to a substance which interferes with or inhibits the IFN-a biological activity.
  • IFN-a biological activity refers to any activity occurring as a result of IFN-a binding to its receptor IFNAR (IFNARl /IFNAR2 heterodimer). Such binding can, for example, activate the JAK-STAT signaling cascade, and trigger tyrosine phosphorylation of a number of proteins including JAKs, TYK2, STAT proteins.
  • the blocking agent of interferon- alpha signaling can neutralize the fixation of the INF-oc to its receptor and/or block the signaling cascade induced by the binding of IFN-oc to its receptor.
  • the IFN-oc antagonist is selected from the group of active anti-IFN-oc vaccine (e.g., IFN- a-kinoid, IFN-a DNA-based or IFN-a RNA-based vaccine) or passive anti-IFN-oc vaccine
  • active anti-IFN-oc vaccine e.g., IFN- a-kinoid, IFN-a DNA-based or IFN-a RNA-based vaccine
  • passive anti-IFN-oc vaccine e.g., passive anti-IFN-oc vaccine
  • the agent neutralizing circulating IFN-a is a passive anti-IFN-a vaccine, such as an anti-IFN-a antibody or an anti-IFN-a hyper-immune serum.
  • the agent neutralizing circulating IFN-a is an anti-IFN-a antibody, preferably a neutralizing antibody.
  • the anti-IFN-a antibody may be a monoclonal or a polyclonal antibody, and is preferably a monoclonal antibody.
  • anti-IFN-a antibodies include, without limitation, Sifalimumab, Rontalizumab, MMHA-1 clone, MMHA-2 clone, MMHA-6 clone, MMHA-8 clone, MMHA-9 clone, MMHA-11 clone, MMHA-13 clone and MMHA-17 clone.
  • the agent neutralizing circulating IFN-a is an anti-IFN-a hyper-immune serum.
  • the agent neutralizing circulating IFN-a described herein is an IFN-a ligand inhibitor. In one embodiment, the agent neutralizing circulating IFN-a is a soluble receptor that binds IFN-a.
  • the agent neutralizing circulating IFN-a is an active anti-IFN-a vaccine.
  • an “active anti-IFN-a vaccine” designates a compound or composition that is capable of inducing the production of anti-IFN-a antibodies.
  • an “active anti-IFN-a vaccine” designates a compound or composition which, upon administration to a subject, is capable of inducing the production of anti-IFN-a auto-antibodies by said subject.
  • the active ingredient of the active anti-IFN-a vaccine may be a polypeptide, a protein, a DNA or an RNA molecule.
  • the active ingredient of the active anti-IFN-a vaccine is an IFN-a-kinoid.
  • a kinoid is an inactivated and/or non-toxic IFN derivative with immunogenic properties. Usually, it is in the form of a heterocomplex obtained by chemical binding of the IFN derivative to a carrier.
  • the kinoid may be used as an immunogen capable of inducing high affinity auto- antibodies against a given IFN. Immunization with kinoids thus induce high titers of neutralizing antibodies directed against the corresponding IFN.
  • the agent neutralizing circulating IFN- a described herein is an IFN- a-kinoid, such as, for example, Antiferon®.
  • the active ingredient of the active anti-IFN-a vaccine may also be a DNA molecule, for instance part(s) of or full-length IFN-a DNA, or an RNA molecule, for instance part(s) of or full-length IFN-a RNA.
  • the active anti-IFN-a vaccine may induce the production of antibodies that binds to one IFN-a subtype or to several IFN-a subtypes.
  • the RNA molecule used in the IFN-a RNA-based vaccine may be specific of one IFN-a subtype.
  • the active anti-IFN-a vaccine may comprise at least two RNA molecules corresponding to at least two IFN-a subtypes.
  • the interferon- alpha blocking agent is an agent blocking IFN- a signaling.
  • the agent blocking IFN-a signaling is an agent that antagonizes the type I IFN signaling pathway.
  • the agent blocking IFN-oc signaling described herein is an IFNAR antagonist.
  • the agent blocking IFN-oc signaling is an IFNAR1 antagonist. In another embodiment, the agent blocking IFN-oc signaling is an IFNAR2 antagonist. In one embodiment, the agent blocking IFN-oc signaling is a passive anti-IFNARl or anti-IFNAR2 vaccine, such as an anti-type I interferon R1 or R2 antibody.
  • the agent blocking IFN-oc signaling is an antibody that binds to IFNAR 1 or IFNAR2.
  • the agent blocking IFN-oc signaling is an active anti-IFNARl or anti-IFNAR2 vaccine, such as an IFNAR1 or IFNAR2 DNA-based or an IFNAR1 or IFNAR2 RNA-based vaccine.
  • an “active anti-IFNARl or anti-IFNAR2 vaccine” designates a compound or composition that is capable of inducing the production of anti-IFNARl or anti-IFNAR2 antibodies.
  • an “active anti-IFNARl or anti-IFNAR2 vaccine” designates a compound or composition which, upon administration to a subject, is capable of inducing the production of anti-IFNARl or anti-IFNAR2 auto-antibodies by said subject.
  • the active ingredient of the active anti-IFNARl or anti-IFNAR2 vaccine may be a DNA molecule, for instance part(s) of or full-length IFNAR1 or IFNAR2 DNA, or an RNA molecule, for instance part(s) of or full-length IFNAR 1 or IFNAR2 RNA.
  • the agent blocking IFN-oc signaling can be an inhibitor of type I IFN signaling pathway.
  • Type I IFN signaling pathway inhibitors are well known in the art and include, without limitation, JAK1/2/3 inhibitors and STAT inhibitors. Accordingly, in one embodiment, the agent blocking IFN-oc signaling is selected from JAK1/2/3 inhibitors, STAT inhibitors, and Tyrosine Kinase 2 (TYK2) inhibitors.
  • Non-limiting examples of JAK1/2/3 inhibitors include Ruxolitinib, Tofacitinib and Baricitinib.
  • Non-limiting examples of TYK2 inhibitors include the BMS-986165 inhibitor.
  • the agent blocking IFN-oc signaling can be an endogenous negative regulator of type I IFN signaling pathway. Endogenous negative regulators are well known in the art and include, without limitation, SOCSl/3, F0X03, Aryl hydrocarbon Receptor (AhR) or other negative regulators. Accordingly, in one embodiment, the agent blocking interferon signaling is selected from SOCSl/3, F0X03 or Aryl hydrocarbon Receptor (AhR).
  • the agent blocking IFN-oc signaling is a PASylated antagonist.
  • PASylated antagonist of type I IFN are known in the art, see for example Nganou-Makamdop et al. (2016). PLoS Pathog 14(8): el007246.
  • the IFN- a antagonist described herein is an agent depleting IFN-oc producing cells.
  • IFN-OC producing cells refers to any cell that produce IFN-oc.
  • pDCs plasmacytoid dendritic cells
  • the agent depleting IFN- a producing cells depletes pDCs.
  • the agent depleting IFN- a producing cells is an antibody.
  • the antibody depletes pDCs, such as, for example, an anti-CD 123 antibody (i.e., anti-IL-3RA).
  • the IFN- a antagonist described herein is an agent that blocks the production of IFN- a.
  • the agent that blocks the production of the IFN-oc is an antibody.
  • the antibody blocks the production of IFN-oc by pDCs.
  • Said antibody can be, for example, an anti-BDCA2 (Blood DC Antigen 2) antibody.
  • the interferon-alpha blocking agent is selected from the group consisting of: an anti-IFN-oc antibody, preferably Sifalimumab, Rontalizumab, MMHA-1 clone, MMHA-2 clone, MMHA-6 clone, MMHA-8 clone, MMHA-9 clone, MMHA-1 1 clone, MMHA-13 clone or MMHA-17 clone, an anti-IFN-oc hyper-immune serum, an IFN-a-kinoid, such as e.g.
  • Antiferon® an IFN-a DNA-based or an IFN-a RNA-based vaccine, a soluble receptor that binds IFN-a, - an IFNAR1 or IFNAR2 antagonist, preferably an antibody that binds to IFNAR1 or
  • IFNAR2 an IFNAR1 or IFNAR2 DNA-based or an IFNAR1 or IFNAR2 RNA-based vaccine, a type I IFN signaling pathway inhibitors selected from a STAT inhibitor, a JAK1/2/3 inhibitor, such as e.g. Ruxolitinib, Tofacitinib or Baricitinib, and a TYK2 inhibitor, such as e.g. BMS-986165, an endogenous negative regulator of type I IFN signaling pathway selected from SOCSl/3, F0X03, Aryl hydrocarbon Receptor (AhR) or another negative regulator, a PASylated antagonist, an antibody depleting pDCs, preferably an anti-CD 123 (i.e. anti-IL-3RA) antibody, - an antibody blocking the production of IFN-a by pDCs, preferably an anti-BDCA2
  • a type I IFN signaling pathway inhibitors selected from a STAT inhibitor, a JAK1/2/3 inhibitor, such as
  • the interferon-alpha blocking agent is an anti-IFN-a antibody, preferably a monoclonal antibody, more preferably a neutralizing antibody.
  • type III interferon also called interferon-lambda (IFN-l) refers to naturally occurring and/or recombinant cytokines of the type III interferon-lambda family.
  • IFN-l interferon-lambda
  • the type III interferon is IFN-l.
  • the IFN-l refers to at least one IFN-l subtype, i.e. IFN-lI, IFN-k2 IRN-l3, IFN- 4.
  • the human IFN-lI has the following accession number NP_742152.1. In one embodiment, the human IFN-k2 has the following accession number NP_742150.1. In one embodiment, the human IFN-/3 has the following accession numbers NP_001333866.1 (isoform 1) or NP_742151.2 (isoform 2). In one embodiment, the human IFN-k4 has the following accession number NP_001263183.2.
  • the type III interferon to be blocked is a mucosal type III interferon or a mucosal IFN-l.
  • the interferon-lambda blocking agent described herein is an agent neutralizing circulating IFN-l and/or an agent blocking IFN-l signaling.
  • the interferon-lambda blocking agent is an agent neutralizing mucosal IFN- l and/or an agent blocking mucosal IFN-l signaling.
  • the agent neutralizing circulating IFN-l and/or the agent blocking IFN-l signaling is/are an IFN-l antagonist.
  • the at least one type III interferon blocking agent is:
  • an agent neutralizing circulating IFN-l selected from the group comprising an active anti-IFN-l vaccine, such as an IFN-k-kinoid, IFN-l DNA-based or IFN-l RNA-based vaccine, and a passive anti-IFN-l vaccine, such as an anti-IFN-l antibody or an anti-IFN- l hyper-immune serum, or
  • an agent blocking type III interferon signaling selected from the group consisting of an active anti-IFNLRl vaccine, such as an IFNLR1 DNA-based or IFNLR1 RNA-based vaccine, and a passive anti-IFNLRl vaccine, such as an antibody that binds to the IFNLR1 receptor.
  • an active anti-IFNLRl vaccine such as an IFNLR1 DNA-based or IFNLR1 RNA-based vaccine
  • a passive anti-IFNLRl vaccine such as an antibody that binds to the IFNLR1 receptor.
  • the term “lambda interferon antagonist” refers to a substance which interferes with or inhibits the IFN-l biological activity.
  • IFN-l biological activity refers to any activity occurring as a result of IFN-l binding to its receptor IFNLR (IFNLR1/IL10R2 heterodimer).
  • the signaling blocking agent of interferon can neutralize the fixation of the INF-l to its receptor and/or block the signaling cascade induced by the binding of IFN-l to its receptor.
  • the IFN-l antagonist is selected from the group of active anti-IFN-l vaccine (e.g ., IFN-k-kinoid, IFN-l DNA-based or IFN-l RNA-based vaccine) or passive anti-
  • IFN-l vaccine e.g., anti-IFN-l antibody or anti-IFN-l hyper- immune serum.
  • the agent neutralizing circulating IFN-l is a passive anti-IFN-l vaccine, such as an anti-IFN-l antibody or an anti-IFN-l hyper- immune serum.
  • the agent neutralizing circulating IFN-l is an anti-IFN-l antibody, preferably a neutralizing antibody.
  • the anti-interferon-lambda antibody may be a monoclonal or a polyclonal antibody, and is preferably a monoclonal antibody.
  • Non limiting examples of neutralizing anti-interferon-lambda antibodies include: the monoclonal anti-IL-29 (IFN-lI) antibody clone 6A11 (Invivogen), - the monoclonal anti-human IL-29 (IFN-lI) antibody clone #247801 (R&D systems), the monoclonal anti-IL-28A (IFN-k2) antibody clone 21C3 (Invivogen), the monoclonal anti-IL-28 A (IFN-k2) antibody clone MMHL-2 (PBL assay sciences), - the monoclonal anti-human IL-28A (IFN-k2) antibody Clone #248526
  • the agent neutralizing circulating IFN-l is an anti-IFN-l hyper-immune serum. In one embodiment, the agent neutralizing circulating IFN-l described herein is an IFN-l ligand inhibitor.
  • the agent neutralizing circulating IFN-l is a soluble receptor that binds IFN-l. In another embodiment, the agent neutralizing circulating IFN-l is an active anti-IFN-l vaccine.
  • an “active anti-IFN-l vaccine” designates a compound or composition that is capable of inducing the production of anti-IFN-l antibodies.
  • an “active anti-IFN-l vaccine” designates a compound or composition which, upon administration to a subject, is capable of inducing the production of anti-IFN-l auto-antibodies by said subject.
  • the active ingredient of the active anti-IFN-l vaccine may be a polypeptide, a protein, a DNA or an RNA molecule.
  • the active ingredient of the active anti-IFN-l vaccine is an IFN-k-kinoid.
  • a kinoid is an inactivated and/or non-toxic IFN derivative with immunogenic properties. Usually, it is in the form of a heterocomplex obtained by chemical binding of the IFN derivative to a carrier.
  • the kinoid may be used as an immunogen capable of inducing high affinity auto antibodies against a given IFN. Immunization with kinoids thus induce high titers of neutralizing antibodies directed against the corresponding IFN.
  • the active ingredient of the active anti-IFN-l vaccine may also be a DNA molecule, for instance part(s) of or full-length IFN-l DNA, or an RNA molecule, for instance part(s) of or full-length IFN-l RNA.
  • the active anti-IFN-l vaccine may induce the production of antibodies that binds to one IFN-l subtype or to several IFN-l subtypes selected from the group comprising IFN-lI, IFN-k2, IFN-/3 and IFN-k4.
  • the RNA molecule used in the IFN-l RNA- based vaccine may be specific of one IFN-l subtype, i.e. specific of IFN-lI, of IFN-k2, of IFN-/J or of IFN- 4.
  • the active anti-IFN-l vaccine may comprise at least two RNA molecules corresponding to at least two IFN-l subtypes selected from the group comprising IFN-lI, IRN-l2, IFN-/J and IFN- 4.
  • the type III interferon blocking agent is an agent blocking type III interferon signaling.
  • the agent blocking IFN-l signaling is an agent that antagonizes the type III IFN signaling pathway.
  • the agent blocking IFN-l signaling described herein is an IFNLR antagonist. In one embodiment, the agent blocking IFN-l signaling is an IFNLR 1 antagonist. In another embodiment, the agent blocking IFN-l signaling is an IL10R2 antagonist.
  • the agent blocking type III interferon signaling is a passive anti- IFNLR1 vaccine, such as an antibody that binds to the IFNLR 1 receptor.
  • the agent blocking type III interferon signaling is an antibody.
  • Such antibody can block or inhibit the biological effects of type III interferon and/or block or inhibit the type III interferon signaling pathway.
  • such antibody may bind to an epitope on the interferon-lambda receptor, impeding the binding of interferon- lambda to its receptor and thus the receptor signaling subsequent activation.
  • the heterodimeric receptor complex of interferon-lambda comprises IFNLR1 (IFNLRA, IL-28RA), and IL10R2 (IL-10RB).
  • IFNLR 1 confers ligand specificity and enables receptor assembly, while IL10R2 is shared with IL-10 family members and is required for signaling.
  • the agent blocking type III interferon signaling is an antibody that binds to IFNLR 1 or to IL10R2.
  • the agent blocking type III interferon signaling is an antibody that binds to the IFNLR 1 receptor.
  • Non- limiting examples of antibodies that bind to the IFNLR1 receptor include the clone MMHLR-1 (Pbl assay sciences) and the MHLICR2A1 antibody (Creative Biolabs).
  • the agent blocking type III interferon signaling is an active anti- IFNLRl vaccine, such as an IFNLR1 DNA-based or IFNLR1 RNA-based vaccine.
  • An “active anti-IFNLRl vaccine” designates a compound or composition that is capable of inducing the production of anti-IFNLRl antibodies.
  • an “active anti- IFNLRl vaccine” designates a compound or composition which, upon administration to a subject, is capable of inducing the production of anti-IFNLRl auto-antibodies by said subject.
  • the active ingredient of the active anti-IFNLRl vaccine may be a DNA molecule, for instance part(s) of or full-length IFNLR1 DNA, or an RNA molecule, for instance part(s) of or full-length IFNLR1 RNA.
  • the type III interferon blocking agent is a small chemical molecule entity (such as, for example, a chemical entity with a molecular weight less than 900 Daltons).
  • a chemical library may be tested in a ligand-receptor binding assay.
  • the small chemical molecule may bind and block the IFNLR1 receptor.
  • the type III interferon blocking agent is selected from the group consisting of: an anti-IFN-l antibody, preferably clone 6A11, clone #247801, clone 21C3, clone MMHL-2, clone #248526, clone 18F4, or clone MMHL-3.
  • an anti-IFN-l hyper-immune serum an IFN- -kinoid, - an IFN-l DNA-based or an IFN-l RNA-based vaccine, a soluble receptor that binds IFN-l, an IFNLR1 or IL10R2 antagonist, preferably an antibody that binds to IFNLR1 or IL10R2, more preferably an antibody that binds to IFNLR1 such as clone MMHLR- 1 or the MHLICR2A1 antibody, an IFNLR1 DNA-based or IFNLR1 RNA-based vaccine, and - a small chemical molecule that binds to IFNLR1.
  • beta interferon or “interferon-beta” refers to a family of two related but distinct members IFN-bI and IRN-b3. They both bind to the same IFN receptor, the IFN-a receptor (IFNAR), which is a cell surface receptor complex consisting of 2 chains: IFNAR1 and IFNAR2 (IFNAR 1 /IFN AR2 heterodimer). Binding of IFN-b to the IFNAR receptor triggers signaling transduction events and biological effects in the target cell.
  • IFN-a receptor IFN-a receptor
  • the interferon-beta blocking agent described herein is an agent neutralizing circulating IFN-b and/or an agent blocking IFN-b signaling.
  • the interferon-beta blocking agent described herein comprises at least one agent selected from an agent neutralizing circulating IFN-b and an agent blocking IFN-b signaling.
  • the agent neutralizing circulating IFN-b and/or the agent blocking IFN-b signaling is/are an IFN-b antagonist.
  • beta interferon antagonist refers to a substance which interferes with or inhibits the IFN-b biological activity.
  • IFN-b biological activity refers to any activity occurring as a result of IFN-b binding to its receptor
  • IFNAR IFNAR 1 /IFN AR2 heterodimer
  • Such binding can, for example, activate the JAK-STAT signaling cascade, and trigger tyrosine phosphorylation of a number of proteins including JAKs, TYK2, STAT proteins.
  • the signaling blocking agent of interferon can neutralize the fixation of the IFN-b to its receptor and/or block the signaling cascade induced by the binding of IFN-b to its receptor.
  • the IFN-b antagonist is selected from the group of active anti-IFN-b vaccine (e.g ., IFN-P-kinoid, IFN-b DNA-based or IFN-b RNA-based vaccine) or passive anti- IFN-b vaccine (e.g., anti-IFN-b antibody or anti-IFN-b hyper-immune serum).
  • active anti-IFN-b vaccine e.g ., IFN-P-kinoid, IFN-b DNA-based or IFN-b RNA-based vaccine
  • passive anti- IFN-b vaccine e.g., anti-IFN-b antibody or anti-IFN-b hyper-immune serum.
  • the agent neutralizing circulating IFN-b is a passive anti-IFN-b vaccine, such as an anti-IFN-b antibody or an anti-IFN-b hyper-immune serum.
  • the interferon-beta blocking agent is an agent neutralizing circulating IFN-b, wherein the agent neutralizing circulating IFN-b is an anti-IFN-b antibody or anti-IFN-b hyper-immune serum.
  • the agent neutralizing circulating IFN-b is an anti-IFN-b antibody, preferably a neutralizing antibody.
  • the anti-IFN-b antibody may be a monoclonal or a polyclonal antibody, and is preferably a monoclonal antibody.
  • Non-limiting examples of anti-IFN-b antibodies include: the neutralizing monoclonal antibody against human IFN-beta, clone 10B10 (Invivogen) the polyclonal anti-human IFN-beta antibodies (R&D systems) - the monoclonal anti-human IFN-beta antibodies clone #76703, clone #MMHB-3 and clone #937912 (R&D systems) the neutralizing polyclonal anti-human IFN-beta goat IgG (PBL assay sciences).
  • the agent neutralizing circulating IFN-b is an anti-IFN-b hyper-immune serum. In one embodiment, the agent neutralizing circulating IFN-b described herein is an IFN-b ligand inhibitor.
  • the agent neutralizing circulating IFN-b is a soluble receptor that binds IFN-b.
  • the interferon-beta blocking agent is an active anti-IFN-b vaccine.
  • An “active anti-IFN-b vaccine” designates a compound or composition that is capable of inducing the production of anti-IFN-b antibodies.
  • an “active anti-IFN-b vaccine” designates a compound or composition which, upon administration to a subject, is capable of inducing the production of anti-IFN-b auto-antibodies by said subject.
  • the active ingredient of the active anti-IFN-b vaccine may be a polypeptide, a protein, a DNA or an RNA molecule.
  • the active ingredient of the active anti-IFN-b vaccine is an IFN ⁇ -kinoid.
  • a kinoid is an inactivated and/or non-toxic IFN derivative with immunogenic properties. Usually, it is in the form of a heterocomplex obtained by chemical binding of the IFN derivative to a carrier.
  • the kinoid may be used as an immunogen capable of inducing high affinity auto antibodies against a given IFN. Immunization with kinoids thus induce high titers of neutralizing antibodies directed against the corresponding IFN.
  • the active ingredient of the active anti-IFN-b vaccine may also be a DNA molecule, for instance part(s) of or full-length IFN-b DNA, or an RNA molecule, for instance part(s) of or full-length IFN-b RNA.
  • the interferon-beta blocking agent is an agent blocking IFN-b signaling, wherein the blocking agent of IFN-b signaling is selected from the group consisting of anti-type I interferon R1 or R2 antibodies, SOSC1 and aryl hydrocarbon receptors.
  • the agent blocking IFN-b signaling described herein is an IFNAR antagonist. In one embodiment, the agent blocking IFN-b signaling is an IFNAR1 antagonist. In another embodiment, the agent blocking IFN-b signaling is an IFNAR2 antagonist. In one embodiment, the agent blocking IFN-b signaling is an antibody that binds to IFNAR 1 or IFNAR2. In one embodiment, the agent blocking IFN-b signaling is an agent that antagonizes the type I IFN signaling pathway.
  • the agent blocking IFN-b signaling can be an inhibitor of type I IFN signaling pathway.
  • Type I IFN signaling pathway inhibitors are well known in the art and include, without limitation, JAK1/2/3 inhibitors and STAT inhibitors. Accordingly, in one embodiment, the agent blocking IFN-b signaling is selected from JAK1/2/3 inhibitors, STAT inhibitors, and Tyrosine Kinase 2 (TYK2) inhibitors.
  • JAK1/2/3 inhibitors include Ruxolitinib, Tofacitinib and Baricitinib.
  • TYK2 inhibitors include the BMS-986165 inhibitor.
  • the agent blocking IFN-b signaling can be an endogenous negative regulator of type I IFN signaling pathway.
  • Endogenous negative regulators are well known in the art and include, without limitation, SOCSl/3, F0X03, Aryl hydrocarbon Receptor (AhR) or other negative regulators. Accordingly, in one embodiment, the agent blocking interferon signaling is selected from SOCSl/3, F0X03 or Aryl hydrocarbon Receptor (AhR).
  • the agent blocking IFN-b signaling is a PASylated antagonist.
  • PASylated antagonist of type I IFN are known in the art, see for example Nganou-Makamdop et al. (2016). PLoS Pathog 14(8): el007246.
  • antiretroviral therapy or “ART” or “highly active antiretroviral therapy” or “HAART” refers to any combination of antiretroviral (ARV) drugs to maximally suppress the HIV virus (e.g reduce viral load reduce HIV multiplication%), and stop the progression of HIV disease.
  • ARV antiretroviral
  • NRTIs non-nucleoside reverse transcriptase inhibitors
  • NRTIs nucleoside reverse transcriptase inhibitors
  • Pis protease inhibitors
  • CCR5 antagonists or CCR5 antagonists
  • INSTIs integrase strand
  • the antiretroviral (ART) agent is selected from the group consisting of Nucleoside reverse transcriptase inhibitors (NRTIs), Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Protease inhibitors (Pis), Integrase inhibitors (INSTIs), Fusion inhibitors (FIs), Chemokine receptor antagonists (CCR5 antagonists) and Entry inhibitors (CD4-directed post-attachment inhibitors).
  • NRTIs Nucleoside reverse transcriptase inhibitors
  • NRTIs Non-nucleoside reverse transcriptase inhibitors
  • Protease inhibitors Protease inhibitors
  • INSTIs Integrase inhibitors
  • Fusion inhibitors FIs
  • CCR5 antagonists Chemokine receptor antagonists
  • Entry inhibitors CD4-directed post-attachment inhibitors
  • Non-limiting examples of antiretroviral (ART) agents include:
  • Nucleoside reverse transcriptase inhibitors such as e.g.: o Abacavir (Ziagen®) o Didanosine (Videx®, Videx® EC) o Emtricitabine (Emtriva) o Lamivudine (Epivir®) o Stavudine (Zerit®) o Tenofovir disoproxil fumarate DF (Viread®) o Tenofovir alafenamide AF o Zidovudine (Retrovir®)
  • NRTIs Non-nucleoside reverse transcriptase inhibitors
  • NRTIs Non-nucleoside reverse transcriptase inhibitors
  • o Delavirdine Rescriptor®
  • Efavirenz Sustiva®
  • Estiva® Etravirine
  • Nevirapine Nevirapine
  • Veramune®, Viramune® XR Rilpivirine
  • Erfidelity® Doravirine
  • Protease inhibitors such as e.g.: o Atazanavir (Reyataz®) o Darunavir (Prezista®) o Fosamprenavir (Lexiva®) o Indinavir (Crixivan®) o Lopinavir/ritonavir (Kaletra®) o Nelfinavir (Viracept®) o Ritonavir (Norvir®) o Saquinavir (Invirase®) o Tipranavir (Aptivus®)
  • Protease inhibitors such as e.g.: o Atazanavir (Reyataz®) o Darunavir (Prezista®) o Fosamprenavir (Lexiva®) o Indinavir (Crixivan®) o Lopinavir/ritonavir (Kaletra®) o Nelfinavir (Viracept®) o Ritonavir (Norvir®)
  • Integrase inhibitors such as e.g.: o Raltegravir (Isentress®, Isentress® HD) o Dolutegravir (Tivicay®) o Elvitegravir (Vitekta®)
  • Chemokine receptor antagonist such as e.g. o Maraviroc (Selzentry®) - Fusion inhibitor (FI) such as e.g. o Enfuvirtide (Fuzeon®)
  • the ART agent according to the invention comprises several ART agents, or is a combination of several ART agents. These several ART agents are for instance chosen among the ART agents listed hereabove.
  • initial treatment regimens usually include two NTRIs combined with a third active antiretroviral drug, which may be in the INSTI, NNRTI, or PI class. They may sometimes include a booster, which may be cobicistat (Tybost®) or ritonavir (Norvir®).
  • a booster which may be cobicistat (Tybost®) or ritonavir (Norvir®).
  • the ART agent according to the invention comprises a combination of at least two ART agents, preferably chosen among the ART agents listed hereabove.
  • the ART agent according to the invention comprises a combination of two, three, four, five or six ART agents, preferably chosen among the ART agents listed hereabove.
  • Non-limiting examples of antiretroviral (ART) agents which are combination ARTs or combined ARTs (cARTs) include the following ART combinations: - Elvitegravir + cobicistat + emtricitabine + tenofovir DF (Stribild®)
  • Rilpivirine + emtricitabine + tenofovir AF (Odefsey®) Rilpivirine + emtricitabine + tenofovir DF (Complera®) Bictegravir + emtricitabine + tenofovir AF (Biktarvy®) Dolutegravir + abacavir + lamivudine (Triumeq®) Dolutegravir + rilpivirine (Juluca®)
  • Efavirenz + emtricitabine + tenofovir DF (Atripla®) Efavirenz + lamivudine + tenofovir DF (Symfi®) Doravirine + lamivudine + tenofovir DF (Delstrigo®) Emtricitabine + tenofovir AF (Descovy®)
  • Emtricitabine + tenofovir DF (Truvada®)
  • Lamivudine + tenofovir DF (Cimduo®)
  • the subject has already received at least one dose of at least one antiretroviral (ART) agent, or is under antiretroviral therapy or under combined antiretroviral therapy (cART) comprising at least one antiretroviral (ART) agent, before being administered the combination of the invention.
  • Said at least one antiretroviral (ART) agent may be the same antiretroviral (ART) agent as the one comprised in the combination of the invention, or it may be different from the antiretroviral (ART) agent comprised in the combination of the invention.
  • said at least one antiretroviral (ART) agent is the same antiretroviral (ART) agent as the one comprised in the combination of the invention.
  • the subject when being treated according to the method of the invention, the subject goes on receiving the at least one antiretroviral (ART) agent he/she has already been given before, while being further administered a type III interferon blocking agent, an interferon- alpha (IFN-a) blocking agent, optionally an interferon-beta (IFN-b) blocking agent, and optionally a latency-reversing agent (LRA).
  • IFN-a interferon- alpha
  • IFN-b interferon-beta
  • LRA latency-reversing agent
  • the subject has already received at least one dose of a combined antiretroviral therapy (cART) before being administered the combination of the invention, and said cART comprises Nucleoside reverse transcriptase inhibitors (NRTIs), Non- nucleoside reverse transcriptase inhibitors (NNRTIs) and Protease inhibitors (Pis).
  • cART combined antiretroviral therapy
  • NRTIs Nucleoside reverse transcriptase inhibitors
  • NRTIs Non- nucleoside reverse transcriptase inhibitors
  • Protease inhibitors Protease inhibitors
  • LRAs latency reversing agents
  • Non-limiting examples of latency reversing agent (LRA) include:
  • PKC agonists which for instance act on NF-KB activation, such as e.g. Prostratin Bryostatin-1 Ingenols: Ingenol-B, Ingenol 3,20-dibenzoate (Ingenol-db), ingenol-3-angelate (ingenol mebutate, PEP005);
  • MAPK agonist which for instance act on MAP Kinase activation, such as e.g. Procyanidin trimer Cl;
  • CCR5 antagonist which for instance act on NF-KB activation, such as e.g. Maraviroc;
  • Tat vaccine which for instance act on Activation of HIV-1 LTR, such as e.g. Tat Oyi vaccine, Tat-R5M4 protein;
  • SMAC mimetics which for instance act on Induction of non-canonical NF-KB pathways, such as e.g. SBI-0637142, Birinapant;
  • Inducers of P-TEFb release which for instance act on Release of P-TEFb, such as e.g. BETis: JQ1, I-BET, I-BET151, OTX015, UMB-136, MMQO, CPI-203, RVX-208, PFI-1, BI-2536 and BI-6727; HMBA;
  • Akt pathway which for instance act on Upregulation of Akt signaling pathway, such as e.g. Disulfiram;
  • Benzotriazole derivatives which for instance act on STAT5 activation, such as e.g. 1-hydroxybenzotriazol (HOBt);
  • Epigenetic modifiers which for instance act on HDAC inhibition, such as e.g. HDACis: TSA, trapoxin, SAHA, romidepsin, panobinostat, entinostat, givinostat, valproic acid, MRK-1/11, AR-42, fimepinostat, chidamide;
  • Epigenetic modifiers which for instance act on Suv39Hl, G9a, SMYD2, such as e.g. HMTis: chaetocin, EPZ-6438, GSK-343, DZNEP, BIX-01294, UNC-0638; Epigenetic modifiers, which for instance act on DNMT1, 3a, 3b, such as e.g. DNMTis: 5-AzaC, 5-AzadC; and
  • Immunomodulatory LRAs such as e.g. TLR agonists: TLR2 (Pam3CSK4), TLR7 (GS-9620), TLR8, TLR9 (MGN 1703) agonists; IL-15 agonist (ALT-803); Immune checkpoint inhibitors: anti-PD-1 (nivolumab, pembrolizumab), anti-CTLA-4 (ipilimumab).
  • the latency reversing agent is selected from the group consisting of PKC agonists, MAPK agonists, CCR5 antagonists, Tat vaccines, SMAC mimetics, inducers of P-TEFb release, activators of Akt pathway, benzotriazole derivatives, epigenetic modifiers and immunomodulatory LRAs.
  • the latency reversing agent is a Prostratin Bryostatin-1 Ingenol, such as Ingenol-B, Ingenol 3,20-dibenzoate (Ingenol-db), ingenol-3-angelate (ingenol mebutate, PEP005); Procyanidin trimer Cl; Maraviroc; Tat Oyi vaccine, Tat-R5M4 protein; SBI-0637142, Birinapant; a BETi such as JQ1, I-BET, I-BET151, OTX015, UMB-136, MMQO, CPI-203, RVX-208, PFI-1, BI-2536 and BI-6727 HMBA;
  • Prostratin Bryostatin-1 Ingenol such as Ingenol-B, Ingenol 3,20-dibenzoate (Ingenol-db), ingenol-3-angelate (ingenol mebutate, PEP005)
  • HDACi such as TSA, trapoxin, SAHA, romidepsin, panobinostat, entinostat, givinostat, valproic acid, MRK-1/11, AR-42, fimepinostat, chidamide
  • HMTi such as chaetocin, EPZ-6438, GSK-343, DZNEP, BIX-01294, UNC-0638
  • DNMTi such as 5-AzaC, 5-AzadC
  • TLR agonist such as a TLR2 agonist (Pam3CSK4), a TLR7 agonist (GS-9620), a TLR8 agonist, a TLR9 agonist (MGN 1703) agonist
  • an IL-15 agonist ALT- 803
  • an immune checkpoint inhibitor such as anti-PD-1 (nivolumab, pembrolizumab), or anti-CTLA-4 (i)
  • the latency reversing agent according to the invention comprises several LRAs, or is a combination of several LRAs, which are for instance chosen among the LRAs listed hereabove. In one embodiment, the latency reversing agent according to the invention comprises a combination of at least two LRAs, preferably chosen among the LRAs listed hereabove.
  • the latency reversing agent according to the invention comprises a combination of two, three, four, five or six LRAs, preferably chosen among the LRAs listed hereabove.
  • At least one agent comprised in the combination is comprised in a composition.
  • one, two or three agents selected from: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, are comprised in a single composition.
  • one or two agents selected from: iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA). are comprised in a single composition.
  • all agents comprised in the combination are comprised in a composition.
  • said composition consists essentially of the at least one agent of the combination according to the invention.
  • composition means that the at least one agent is the only therapeutic agent or agent with a biologic activity within said composition.
  • said composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable excipient.
  • excipient refers to any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA.
  • the excipient is an adjuvant, a stabilizer, an emulsifier, a thickener, a preservative, an antibiotic, an organic or inorganic acid or its salt, a sugar, an alcohol, an antioxidant, a diluent, a solvent, a filler, a binder, a sorbent, a buffering agent, a chelating agent, a lubricant, a coloring agent, or any other component
  • pharmaceutically acceptable is meant that the ingredients of a pharmaceutical composition are compatible with each other and not deleterious to the subject to which it is administered.
  • Examples of pharmaceutically acceptable excipient include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like or combinations thereof.
  • Pharmaceutically acceptable excipients that may be used in the pharmaceutical combination of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene- polyoxypropylene-
  • Another object of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising a combination of: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, and at least one pharmaceutically acceptable excipient, for use in the treatment of AIDS in a subject in need thereof.
  • a type III interferon blocking agent ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, and at least one pharmaceutically acceptable excipient, for use in the treatment of AIDS in a subject in need thereof.
  • IFN-a interferon- alpha
  • IFN-b interferon-beta
  • Another object of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising a combination of: iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA). and at least one pharmaceutically acceptable excipient, for use in the treatment of AIDS in a subject in need thereof.
  • the different agents of the combination or kit-of-parts according to the invention i.e. parts i), ii), iii), iv) and v) of the combination) are to be administered either simultaneously, separately or sequentially with respect to each other.
  • the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is formulated for administration to the subject.
  • the expression “combined preparation” or “combination” refers to any preparation comprising at least two components, such as e.g. parts i), ii), iii), iv) and/or v) of the combination of the invention.
  • the different components of the combined preparation, or of the combination may be used simultaneously, semi-simultaneously, separately, sequentially or spaced out over a period of time so as to obtain the maximum efficacy of the combination.
  • the components may be administered concurrently, i.e. simultaneously in time, or sequentially, i.e. one component is administered after the other one(s).
  • the other component(s) can be administered substantially immediately thereafter or after an effective time period.
  • the effective time period is the amount of time given for realization of maximum benefit from the administration of the components.
  • the combined preparations or combinations are not limited to those which are obtained by physical association of the constituents, but may also be in the form of separate products permitting a separate administration, which can be simultaneous or spaced out over a period of time.
  • the different components may be co -formulated.
  • the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention may be administered orally, intragastrically, parenterally, topically, by inhalation spray, rectally, nasally, buccally, preputially, vaginally or via an implanted reservoir.
  • the administration of each part of the combination of the invention can be done by the same route of administration or by a different route of administration.
  • the administration of i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, and iii) optionally, an interferon-beta (IFN-b) blocking agent is done by a route of administration, and the administration of iv) an antiretroviral (ART) agent and v) optionally, a latency-reversing agent (LRA) is done by another route of administration.
  • ART antiretroviral
  • LRA latency-reversing agent
  • the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is in an adapted form for an oral or an intragastric administration.
  • the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered orally or intragastrically to the subject, for example as a powder, a tablet, a capsule, and the like or as a tablet formulated for extended or sustained release or as an oral solution.
  • the antiretroviral (ART) agent and, optionally, the latency-reversing agent (LRA), are to be administered orally or intragastrically to the subject, for example as a powder, a tablet, a capsule, and the like or as a tablet formulated for extended or sustained release or as an oral solution.
  • Examples of forms adapted for oral or intragastric administration include, without being limited to, liquid, paste or solid compositions, and more particularly tablets, tablets formulated for extended or sustained release, capsules, pills, dragees, liquids, gels, syrups, slurries, suspensions, and the like.
  • the antiretroviral (ART) agent and, optionally, the latency-reversing agent (LRA) are in an adapted form for an oral or intragastric administration.
  • the antiretroviral (ART) agent and, optionally, the latency-reversing agent (LRA) are to be administered orally or intragastrically to the subject, for example as a capsule or as a tablet or as an oral solution.
  • the type III interferon blocking agent, the interferon-alpha (IFN- a) blocking agent and, optionally, the interferon-beta (IFN-b) blocking agent are in an adapted form for an oral or intragastric administration.
  • the type III interferon blocking agent, the interferon- alpha (IFN-a) blocking agent and, optionally, the interferon-beta (IFN-b) blocking agent are to be administered orally or intragastrically to the subject, for example as a capsule or as a tablet or as an oral solution.
  • the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is in a form adapted for parenteral administration.
  • the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is in an adapted form for an injection such as, for example, for intravenous, subcutaneous, intramuscular, intraperitoneal intradermal, transdermal injection or infusion.
  • the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be injected to the subject, by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion.
  • a sterile injectable form may be a solution or an aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic pharmaceutically acceptable diluent or solvent.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • oils such as olive oil or castor oil
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • a long-chain alcohol diluent or dispersant such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • surfactants such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • the type III interferon blocking agent, the interferon- alpha (IFN-a) blocking agent and, optionally, the interferon-beta (IFN-b) blocking agent are in an adapted form for a parenteral administration and/or injection.
  • the type III interferon blocking agent, the interferon- alpha (IFN-a) blocking agent and, optionally, the interferon-beta (IFN-b) blocking agent are to be administered parenterally and/or injected to the subject, by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion, preferably by intravenous injection.
  • the antiretroviral (ART) agent and, optionally, the latency-reversing agent (LRA) are in an adapted form for a parenteral administration and/or injection.
  • the antiretroviral (ART) agent and, optionally, the latency-reversing agent (LRA) are to be administered parenterally and/or injected to the subject, by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion, preferably by intravenous injection.
  • the type III interferon blocking agent, the interferon- alpha (IFN-a) blocking agent and, optionally, the interferon-beta (IFN-b) blocking agent are in an adapted form for a parenteral administration and/or injection, and are to be administered parenterally and/or injected to the subject, by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion, preferably by intravenous injection.
  • the antiretroviral (ART) agent and, optionally, the latency-reversing agent (LRA) are in an adapted form for an oral or intragastric administration, and are to be administered orally or intragastrically to the subject, for example as a capsule, a tablet or an oral solution.
  • the different agents of the combination or kit-of-parts according to the invention are to be administered either simultaneously, separately or sequentially with respect to each other.
  • Parts i), ii), iii), iv) and v) of the combination may be administered concurrently, i.e. simultaneously in time, or sequentially, i.e. administration of certain components of the combination followed by administration of other components of the combination.
  • the other component(s) can be administered substantially immediately thereafter or after an effective time period.
  • the effective time period is the amount of time given for realization of maximum benefit from the administration of the components.
  • the type III interferon blocking agent, the interferon- alpha (IFN-a) blocking agent, optionally the interferon-beta (IFN-b) blocking agent, the antiretroviral (ART) agent and, optionally, the latency-reversing agent (LRA) are all administered at the same time.
  • the type III interferon blocking agent, the IFN-a blocking agent, and optionally the interferon-beta (IFN-b) blocking agent are administered concurrently or simultaneously.
  • the antiretroviral (ART) agent, and optionally the latency-reversing agent are administered concurrently or simultaneously.
  • the type III interferon blocking agent, the interferon- alpha (IFN-a) blocking agent, and/or the interferon-beta (IFN-b) blocking agent are to be administered prior to the antiretroviral (ART) agent and/or the latency-reversing agent (LRA).
  • ART antiretroviral
  • LRA latency-reversing agent
  • the antiretroviral (ART) agent and/or the latency-reversing agent (LRA) are to be administered prior to the type III interferon blocking agent, the interferon- alpha (IFN-a) blocking agent, and/or the interferon-beta (IFN-b) blocking agent.
  • the subject receives one or more doses (one or more takes) of the type III interferon blocking agent, the IFN-a blocking agent, and/or the interferon-beta (IFN-b) blocking agent before starting receiving all parts i), ii), iii), iv) and v) of the combination.
  • the subject receives one or more doses (one or more takes) of the type III interferon blocking agent, the IFN-a blocking agent, and optionally the interferon-beta (IFN-b) blocking agent, during one or several weeks (e.g. 1, 2, 3, 4, 5, 6, 7, 8 or 10 weeks) in the absence of ART agent and LRA administration.
  • the subject continues receiving administrations of the type III interferon blocking agent, the IFN-a blocking agent, and/or the interferon-beta (IFN-b) blocking agent, and also receives administrations of the antiretroviral (ART) agent, and optionally the latency-reversing agent.
  • ART antiretroviral
  • the subject receives one or more doses (one or more takes) of the antiretroviral (ART) agent, and optionally the latency-reversing agent, during one or several weeks or months (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 10 or 12 weeks or months) in the absence of administration of the type III interferon blocking agent, the IFN-a blocking agent, and/or the interferon-beta (IFN-b) blocking agent.
  • ART antiretroviral
  • latency-reversing agent during one or several weeks or months (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 10 or 12 weeks or months) in the absence of administration of the type III interferon blocking agent, the IFN-a blocking agent, and/or the interferon-beta (IFN-b) blocking agent.
  • the subject continues receiving administrations of the antiretroviral (ART) agent, and optionally the latency-reversing agent, and also receives administrations of the type III interferon blocking agent, the IFN-a blocking agent, and optionally the interferon-beta (IFN-b) blocking agent.
  • ART antiretroviral
  • IFN-b interferon-beta
  • the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered to the subject in need thereof in a therapeutically effective amount.
  • terapéuticaally effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired preventive and/or therapeutic result.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disease being treated and the severity of the disease; activity of the specific agent(s), the combination, the kit- of-parts, the pharmaceutical composition or medicament employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific agent(s), the combination, the kit-of-parts, the pharmaceutical composition or medicament employed; the duration of the treatment; drugs used in combination or coincidental with the specific agent(s), the combination, the kit-of-parts, the pharmaceutical composition or medicament employed; and like factors well known in the medical arts.
  • the compound it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • the total dose required for each treatment may be administered by multiple doses or in a single dose.
  • a therapeutically effective amount of one of the agents i), ii), iii), iv) or v) of the combination of the invention ranges from about 0.1 mg/kg to about 10 mg/kg, from about 0.2 mg/kg to about 9 mg/kg, from about 0.3 mg/kg to about 8 mg/kg, from about 0.4 mg/kg to about 7.5 mg/kg, from about 0.5 mg/kg to about 7 mg/kg.
  • a therapeutically effective amount of the type III interferon blocking agent, the interferon-alpha (IFN-a) blocking agent, or the interferon-beta (IFN-b) blocking agent ranges from about 1 mg/kg to about 10 mg/kg, from about 2 mg/kg to about 9 mg/kg, from about 3 mg/kg to about 8 mg/kg, from about 4 mg/kg to about 7 mg/kg, from about 4 mg/kg to about 6 mg/kg.
  • a therapeutically effective amount of one of the agent i), ii), iii), iv) or v) of the combination of the invention ranges from about 1 mg to about 4000 mg, from about 10 mg to about 3000 mg, from about 25 mg to about 2000 mg, from about 50 mg to about 2000 mg, from about 100 mg to about 1500 mg, from about 200 mg to about 1000 mg.
  • a therapeutically effective amount of the antiretroviral (ART) agent or of the latency-reversing agent (LRA) ranges from about 1 mg to about 4000 mg, from about 10 mg to about 3000 mg, from about 25 mg to about 2000 mg, from about 50 mg to about 2000 mg, from about 100 mg to about 1500 mg, from about 200 mg to about 1000 mg.
  • the administration regimes typically have from 1 to 20 administrations of the different parts of the combination of the invention, but may have as few as one or two or four or eight or ten. In another embodiment, the administration regime is annual, biannual or other long interval (5-10 years).
  • a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered once a day, twice a day, three times a day or more.
  • a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered every day, every two days, every three days, every four days, every five days, every six days.
  • a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered every week, every two weeks, every three weeks, every four weeks, every five weeks.
  • a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered every month, every two months, every three months, every four months, every five months, every six months.
  • a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered every 12 hours, every 24 hours, every 36 hours, every 48 hours, every 60 hours, every 72 hours, every 96 hours.
  • a therapeutically effective amount of the type III interferon blocking agent, the interferon-alpha (IFN-a) blocking agent, or the interferon-beta (IFN- b) blocking agent (i.e. of one of the agents i), ii), iii)) is to be administered every week, every two weeks, every three weeks, every four weeks, every five weeks, preferably every two weeks.
  • a therapeutically effective amount of the antiretroviral (ART) agent or of the latency-reversing agent (LRA) is to be administered daily, for instance once a day, twice a day, or three times a day.
  • a therapeutically effective amount of the antiretroviral (ART) agent or of the latency-reversing agent (LRA) is a daily dose to be administered in one, two, three or more takes or in one, two, three or more injections.
  • the antiretroviral (ART) agent and optionally the latency- reversing agent are administered daily, and the IFN-a blocking agent, the type III interferon blocking agent, and optionally the interferon-beta (IFN-b) blocking agent, are administered every three weeks.
  • the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is for acute administration.
  • the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is for chronic administration.
  • a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered for a period of time ranging from about two weeks to about twenty-four weeks, from about two weeks to about twelve weeks, from about two weeks to about six weeks.
  • a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered for about 1 month, 2 months, 3 months, 6 months, 1 year or more.
  • a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered to the subject until no cell containing replication-competent proviral HIV DNA is detected in a blood sample from the subject.
  • the method of treatment of the invention further comprises assessing the presence of cells containing replication-competent proviral HIV DNA in a blood sample from the subject.
  • the presence of cells containing replication-competent proviral HIV DNA is assessed in a blood sample from the subject.
  • a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered to the subject until no cell containing replication-competent proviral HIV DNA is detected in a blood sample from the subject.
  • Cells containing replication-competent proviral HIV DNA also called “resistant cells” or “reservoir cells” may be detected in a sample, or their quantity, level or frequency in a sample may be measured by various assays known by the person skilled in the art.
  • cells containing replication-competent proviral HIV DNA may be detected in a sample, or their quantity, level or frequency in a sample may be measured by ex vivo viral outgrowth assay.
  • plasma HIV-1 RNA levels, size of the HIV reservoir and/or CD4+ T cell count may be typically monitored for instance every 2 weeks, after collection of blood samples, 1 day before administration of the interferon-blocking agents (i.e. parts i), ii) and iii) of the combination described herein).
  • Plasma HIV-1 RNA levels may for instance be determined using the Roche COB AS AmpliPrep/COBAS TaqMan HIV-1 Assay (version 2.0) or the Roche cobas HIV-1 quantitative nucleic acid test (cobas 6800), which quantitate HIV-1 RNA over a range of 2x1o 1 to lxlO 7 copies/ml. Size of the HIV reservoir may for instance be assessed with Quantitative Viral Outgrowth Assay (QVOA).
  • QVOA Quantitative Viral Outgrowth Assay
  • QVOA may typically be performed as previously described in Huang SH et al, 2018 J Clin Invest 128:876-889.
  • isolated CD4+ T cells may typically be plated out in serial dilutions (e.g. 2, 1, 0.5, 0.2, and 0.1 million per well), for instance into 12 wells in 24-well plates with added phytohemagglutinin (PHA; e.g. 2 pg/ml) and irradiated HIV-negative donor PBMC (e.g. 2 x 10 6 cells/well) to reactivate the infected cells.
  • PHA phytohemagglutinin
  • PBMC e.g. 2 x 10 6 cells/well
  • MOLT-4 CCR5 cells e.g. 2 x 10 6 cells/well
  • the p24 antigen in the culture supernatant may typically be quantified after 2 weeks of culture, for instance using an HIV p24 antigen enzyme-linked immunosorbent assay (ELISA) kit (Perkin-Elmer, Hopkinton, MA).
  • ELISA enzyme-linked immunosorbent assay
  • Estimated frequencies of cells with replication-competent HIV-1 may typically be calculated using limiting dilution analysis.
  • CD4+ T-cell counts may for instance be determined by a clinical flow cytometry assay.
  • administration of the interferon-blocking agents i.e. parts i), ii) and iii) of the combination described herein
  • VOA limiting dilution virus outgrowth assays
  • administration of the antiretroviral (ART) agent or/and the latency-reversing agent (LRA) (i.e. parts iv) and/or v) of the combination described herein) may be stopped when no cells with replication-competent HIV- 1 are detected with 3-4 consecutive limiting dilution virus outgrowth assays (VOA).
  • ART antiretroviral
  • LRA latency-reversing agent
  • timing in the treatment interruption is flexible.
  • AIDS symptoms or a detectable viral load
  • a certain time after interruption of the treatment such as e.g. after a few months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, etc. after interruption of the treatment.
  • treatment with the combination of the invention may then be resumed after a certain time of treatment interruption.
  • the combination of the invention is administered to the subject for a first period of time, then said treatment is interrupted during a period of time of a few months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 8 years or 10 years, and then the combination of the invention is administered anew to the subject for a second period of time, for instance until no cell containing replication-competent proviral HIV DNA is detected in a blood sample from the subject.
  • the subject may thus alternate between periods of time during which he receives administrations of the combination described herein and periods of time of treatment interruption.
  • kits-of-parts the composition or the pharmaceutical composition as described herein may be used alone.
  • the combination, the kit-of-parts, the composition or the pharmaceutical composition as described herein is used alone and comprises a therapeutically effective dose of each part each part of the combination (i.e. agent i), ii), iii), iv) or v)).
  • the combination, the kit-of-parts, the composition or the pharmaceutical composition as described herein is used in combination with at least one further therapeutic agent.
  • Such administration may be simultaneous, separate or sequential.
  • the agents may be administered as one composition or as separate compositions, as appropriate.
  • the further therapeutic agent is typically relevant for disorders to be treated.
  • FIG. 1 Antiviral activity of type I and type III interferons.
  • A Expression of ISGs in HepG2. HepG2 cells were treated with IFNa2a or IFN/J -4 (lO ng/ml). After 4 h of stimulation, qRT-PCR were used to examine the mRNA levels of the interferon-induced genes, IFIT1, MX1 and OASF and fold-changes was calculated by 2 DDa method as compared with non-treated cell control and using endogenous S14 mRNA level for normalization.
  • B Antiviral activity of type I and III IFNs against EMCV.
  • IFNa2a or IFNkl/2/3/4 (10 ng/ml) were added to HepG2 cells 24 h prior to challenge with EMCV. Forty-eight after infection with EMCV, cells were assayed for viability with a bioassay. A570 values were directly proportional to cell viability and therefore antiviral activity of the respective IFNs. IFN-a treatment without viral challenge was used as a baseline of the viability of the cells.
  • FIG. 1 Anti-proliferative activity of type I and type III interferons against CD4 + T cells.
  • CFSE-stained CD4 + T cells (10xl0 4 /well) were stimulated for 5 days in 96 round- bottomed microwells with allogeneic poly I:C matured DC in absence (control) or presence of 10 ng/ml of IFN-a2a, or IFN/J or IFNk2 or IFN/J or IFN/J.
  • anti-interferon type I receptor antibody was added. The percentage of CFSE dilution was evaluated by flow cytometry.
  • IFN-a2a but not IFN-type III induces the expression of ISGs in CD4 + T cells.
  • CD4 + T cells were treated with IFNa2a or IFN/J/2/3/4 (10 ng/ml). After 4 h of stimulation, qRT-PCR were used to examine the mRNA levels of the interferon-induced genes, IFIT1, MX1 and OASF and fold-changes was calculated by 2 DDa method as compared with non-treated cell control and using endogenous S14 mRNA level for normalization.
  • IFN-a2a but not IFN-type III stimulates the phosphorylation of Statl in CD4 + T cells.
  • CD4 + T cells were stimulated with 10 ng ml -1 of IFN-lI, IFN-/ , IFN-/J, IFN-k4, or IFN-a2a for 20 min, or were left unstimulated (control).
  • IFN-a2a but not IFN-type III increases CD38 expression in CD3/CD28 stimulated CD4 + T cells.
  • CD4 + T cells (4xl0 4 /well) were cultured in 96 round-bottomed microwells in the presence of ACD3-feeder (4xl0 4 /well) and plate-bound anti-CD3 mAb (2 pg/ml), soluble anti-CD28 mAb (2 pg/ml) with increasing dose of IFN-a2a or IFN type III.
  • CD38 Median Fluorescence Intensity (MFI) was measured by flow cytometry in CD3 + 7-AAD-CFSE + stimulated CD4 + T cells at the end of the culture.
  • FIG. 6 Comparison of CM CD8+ T cell distributions and serum IFN-a levels in HIV- 1-infected subjects and critical pathogenic role of IFN-a in human HIV-1 infection.
  • A Comparison of CM CD8+ T cell distributions in HIV- 1 -infected subjects;
  • FIG. 7 Serum levels of IFN-a and IFN-l, before and after cART treatment in HIV-1 patients originating from two distinct institutes ((A) Institute 1; (B) Institute 2) and control healthy donors.
  • FIG. 1 Schematic diagram of possible administration schedule for the combination of the invention.
  • Figure 9 HIV infection of NCG humanized mice induces IFN-I and IFN III production.
  • NCG humanized mice were intraperitoneally injected with HIV-NL4.3. Blood and spleen were removed from infected and non-infected humanized NCG mice at day 2 post infection, when the virus load has reached a plateau at day 10 and at day 30.
  • Graphs show IFN-a, IFN- 3 and IFN-lI mRNA levels in isolated PBMCS (A, B, C) and splenocytes (D, E, F) respectively.
  • Figure 10 Activated CD4 + T cells express IFN-lambda Receptor.
  • CD4+ T cells were cultured in presence of IL-2 without (black bars) or with anti-CD3 and anti-CD28 antibodies (1 pg/ml each) (grey bars) for 3 days.
  • IFNLR1 (IL28RA) and IL10RB expression was determined by RT-qPCR (A, B) and the detection of fixation of IFN- 3 on cultured CD4+ T cells (C) was assessed by IFN- 3 MFI measured by Flow cytometry.
  • IFIH1 (D) and IFI27 (E) mRNA induction in IFN- 3 -treated CD4+ T cells cultured as described above was determined by RT-qPCR.
  • Figure 12 Sequential treatment with two latency-reversing agents, DNA methylation inhibitor 5-AzadC and HDACI MS -275, induce IFN-1 Receptor expression on CD4 + T cells. 3-day-CD3/CD28/IL2-stimulated CD4+ T cells and J-Lat 10.6 cells were mock- treated or treated with 5-AzadC for 72 h with MS-275 for the last 24 h. At 72 h post treatment, mock (black bars) and 5-AzadC/MS-275 (grey bars) CD4 + T (A, B, C, D) and J-Lat 10.6 cells (E, F, G, H) were harvested and first analyzed for their capacity to express IFNLRl (IL28RA) by RT-qPCR (A and E).
  • IFN- 3 binding on T cells was assessed by IFN- 3 MFI measured by Flow cytometry (B and F).
  • the T cells’ sensitivity to IFN- l3 was evaluated by the identification of IFN- stimulated gene (ISG) proteins IFIH1 (C and G) and IFI27 (D and H) by RT-qPCR.
  • ISG IFN- stimulated gene
  • FIG. 13 The NtRTI TDF enhances the production of IFN- 3 by HT-29 cells.
  • Colon cancer HT-29 cells were incubated with dimethyl sulfoxide (DMSO) or tenofovir disoproxil fumarate (TDF, 25 mM). After 48 hours, IFN- 3 levels in treated HT-29 cells were evaluated by ELISA.
  • DMSO dimethyl sulfoxide
  • TDF tenofovir disoproxil fumarate
  • FIG 14 Experimental scheme for assessing inhibition of HIV infection in stimulated CD4+ T cells by IFN- 3 released by TDF treated HT-29 cells.
  • HT-29 cells were cultured in the bottom chamber of Transwell plates. When the culture was subconfluent, Tenofovir disoproxil fumarate (TDF) was added. After 24 hours of stimulation, activated CD4 T cells were deposited in the insert which is then placed in the culture well so as to be immersed in the culture medium of the HT-29 cells. When indicated, the CD4+ T cells were pre-treated with an IFNLRl (IL28RA) neutralizing antibody. After 24 hours of co- culture, the cells in the insert were harvested, infected and then placed back into the co culture for an additional 5 days of culture. The frequency of infected cells was determined by intracellular HIV p24 by FACS.
  • TDF Tenofovir disoproxil fumarate
  • FIG. 15 IFN- 3 secreted by the NtRTI TDF-treated HT-29 cells impairs CD4 + T cell infection.
  • Experiments were performed using 12-transwell chambers with a polycarbonate filter (0.2 pm pore size).
  • HT-29 were grown until subconfluence in the lower chamber.
  • HT-29 cells were treated with tenofovir disoproxil fumarate (TDF).
  • TDF tenofovir disoproxil fumarate
  • 3-day-CD3/CD28/IL2-stimulated CD4 + T cells were seeded in the upper chamber in presence of IL-2.
  • anti-IFNLRl (IL28RA) neutralizing Ab was added to the culture.
  • CD4 + T cells were isolated, infected with HIV NL4.3 and added again to the upper chamber.
  • FIG. 16 Experimental scheme for assessing the inhibition of HIV latency reversal in latently infected J-Lat cells by IFN- 3 released by TDF treated HT-29 cells.
  • HT-29 cells were cultured in the bottom chamber of Transwell plates. When the culture was subconfluent, Tenofovir disoproxil fumarate (TDF) was added. After 24 hours of stimulation, J-Lat 10.6 cells pretreated with 5-AzadC and MS-275 are deposited in the insert which was then placed in the culture well. When indicated, the J-Lat cells were pre- incubated with an IFNLR1 (IL28RA) neutralizing antibody. After 48 hours of co-culture, HIV reactivation was monitored as the percentage of living GFP-positive cells by Flow Cytometry analysis.
  • TDF Tenofovir disoproxil fumarate
  • FIG 17 IFN- 3 released by TDF-treated HT-29 cells inhibits in vitro HIV reactivation in latently infected J-Lat clone 10.6.
  • Experiments were performed using 12-transwell chambers with a polycarbonate filter (0.2 pm pore size).
  • HT-29 were grown until subconfluence in the lower chamber. Then HT-29 cells were treated with tenofovir disoproxil fumarate (TDF).
  • TDF tenofovir disoproxil fumarate
  • J-Lat clone 10.6 cells which had been pretreated for 72 hours with 5-AzadC and MS -275 as described above were seeded in the upper chamber.
  • FIG. 1 Schematic diagram of pre-clinical protocol for HIV-1 reservoirs elimination in HIV-1 infected humanized mice.
  • Example 1 Effects of type and type III interferons on innate and adaptative immune responses
  • HCC HepG2 and normal kidney epithelial Vero cell lines were obtained from ATCC. Cells were grown in Dulbecco’s Modified Eagle Medium supplemented with 10% heat- inactivated Fetal Bovine Serum, 2 mM L-glutamine, 1% penicillin and streptomycin solution in hypoxia 2%. Cancer cell lines were grown to 70-100% confluency, subsequently passaged for a maximum of 5 times and freshly thawed thereafter. Cells were detached by means of accutase, resuspended in FBS -containing medium and collected by means of centrifugation (300g, 3min). Cell numbers were determined by means of trypan blue.
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs are isolated by density gradient centrifugation on Ficoll-Hypaque (Pharmacia). PBMCs are used either as fresh cells or stored frozen in liquid nitrogen.
  • T-cell subsets and T cell-depleted accessory cells are isolated from either fresh or frozen PBMCs.
  • T cell-depleted accessory cells are isolated by negative selection from PBMCs by incubation with anti- CD3-coated Dynabeads (Dynal Biotech) and are irradiated at 3000 rad (referred to as ACD3-feeder).
  • CD4 + T cells are negatively selected from PBMCs with a CD4 + T-cell isolation kit (Miltenyi Biotec), yielding CD4 + T-cell populations at a purity of 96-99%.
  • T cell subsets are cultured either in IMDM supplemented with 5% SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES (IMDM-5 media) in hypoxia 2%.
  • Freezing and thawing of cells Cells were frozen in FBS containing 10% DMSO. Cryotubes were placed in CoolCell (Biocision) freezing containers and incubated at -80°C. After 2 days tubes were transferred to liquid nitrogen and stored until required. Thawing of cells was performed by placing cryotubes in a 37°C water bath for approximately 30 seconds. Next, cell suspension was mixed with equivalent volume of pre-warmed media and subsequently transferred to falcon tubes containing the same medium. Cells were pelleted by centrifugation (300g, 3min) to remove DMSO. The cell pellet was resuspended in cell culture medium
  • HepG2 cells were seeded at a density of 2 x 10 5 cells per well in 12-well plates and incubated for 24 h. Then, fresh media was added with the indicated interferons. The cells were incubated for 4 h and then lysed, and RNA was purified using an extraction kit (Qiagen), according to the manufacturer's instructions. Synthesis of cDNA was performed using the PrimeScript RT Reagent kit (TAKARA). Quantitative PCR was carried out using the Power SYBR Green PCR Master Mix (Applied Biosystems) on a LightCycler 480 instrument (Roche). Each reaction was carried out in duplicate in a total volume of 100 pL.
  • TAKARA PrimeScript RT Reagent kit
  • Virus production The virus used EMCV (FA strain) was grown on monolayers of Vero cells to complete cytopathic effect or until all cells were affected by the infection as determined by microscopy and prepared by two cycles of freezing and thawing, followed by centrifugation for 30 min at 5,000 x g for removal of cellular debris.
  • Antiviral assays were done on HepG2 cells, which were seeded in DMEM supplemented with 10% FCS at a density of 1.5 x 10 4 in 96-well plates and left to settle. The cells were incubated with indicated doses of IFNs for 24 h before challenge with EMCV. The cells were incubated with virus for 48 h. The medium was removed between each step. The viability of the cells was analyzed by a bioassay based on the dehydrogenase system; this system in intact cells will convert the substrate, MTT, into formazan (blue), which in turn can be measured spectrophotometrically. Briefly, the cells were given MTT and incubated for 2 h.
  • An extraction buffer (containing 6 to 11% sodium dodecyl sulfate and 45% N, N-dimethylformamide) was added to the cells, and the cells were then incubated overnight at 37°C. Subsequently, the absorbance at 570 nm was determined employing the extraction buffer as the blank probe. A570 was directly proportional to antiviral activity.
  • CD3 + T cells staining anti-CD4 (SK3)-APC, anti-CD3 (UCHTl)-FITC, anti-CD8 (RPA-T8)-BV421 are from Becton Dickinson. Cells are stained for surface markers (at 4°C in the dark for 30 min) using mixtures of Ab diluted in PBS containing 3% FBS, 2mM EDTA (FACS buffer).
  • STAT1 signaling analysis Flow cytometry analysis of STAT1 phosphorylation (pSTATl) was conducted in CD4 + T cells by using BD Phosflow technology according to the manufacturer’s instructions (BD Bio-sciences, San Jose, CA). CD4 + T cells were stimulated by incubation with interferon type I and Type III at 37°C for 20 min or left untreated. Activation was stopped by fixation using BD Phosflow Lyse/Fix Buffer (BD Biosciences) and cells were permeabilized with BD Perm Buffer III (BD Biosciences). Cells were stained with antibody recognizing specific phosphorylated STAT tyrosines: p-STATl (Y701)-PE.
  • CFSE staining CD4 + T cells were stained with 1 mM CFSE (CellTrace cell proliferation kit; Molecular Probes/Invitrogen) in PBS for 8 min at 37°C at a concentration of 1.107 cells/ml. The labeling reaction was stopped by washing twice the cell with RPMI-1640 culture medium containing 10% FBS. The cells were then re-suspended at the desired concentration and subsequently used for proliferation assays.
  • 7-AAD staining Apoptosis of stimulated CFSE-labeled CD4 + T was determined using the 7-AAD assay. Briefly, cultured cells were stained with 20 pg/mL nuclear dye 7-amino-actinomycin D (7-AAD; Sigma-Aldrich, St-Quentin Fallavier, France) for 30 minutes at 4°C. FSC/7-AAD dot plots distinguish living (FSC hlgl 77-AAD ) from apoptotic (FSC hlgh /7-AAD + ) cells and apoptotic bodies (FSC low /7-AAD + ) and debris ((FSC low /7-AAD ). Living cells were identified as CD3 + 7-AAD-FSC + cells.
  • T cell proliferation was assessed with CFSE-dilution assays.
  • CFSE-dilution assay at coculture completion, stimulated CFSE-labeled CD4 + T cells were harvested, co-stained with anti-CD3 mAh and 7-AAD, and the percentage of proliferating cells (defined as CFSE low fraction) in gated CD3 + 7-AAD cells was determined by flow cytometry.
  • CD38 Median Fluorescence Intensity (MFI) of CD38 expression was measured by flow cytometry in CD3 + 7-AAD-CFSE + stimulated CD4 + T cells at the end of the culture.
  • CD4 + T cell polyclonal stimulation CFSE-stained CD4 + T cells (5xl0 4 /well) were cultured in 96 round-bottomed microwells in the presence of ACD3 -feeder (lxl0 5 /well) and plate -bound anti-CD3 Ab (2 pg/ml), soluble anti-CD28 mAb (2 pg/ml).
  • CD4 + T cell proliferation was evaluated with CFSE dilution assays as described above by flow cytometry. Cells were stimulated in presence of different amounts of recombinant cytokines.
  • Allogeneic mixed lymphocyte reaction CFSE-stained CD4 + T cells (5xl0 4 /well) were cultured in 96 round-bottomed microwells in the presence of allogeneic mature DC. Proliferation of allo-activated CD4 + T cells with CFSE dilution assays as described above by flow cytometry. Cells were stimulated in presence of different amounts of recombinant cytokines.
  • CD4 + T cells were stimulated with IFN-lI, IFN-k2, IFN-k3, IFN-k4, or IFN-a2a (10 ng/ml) for 20 min, or were left unstimulated (control). Phosphorylated Statl levels was assessed by flow cytometry as described above.
  • Type I interferons IFN-a/b
  • type III IFNs IFN-l
  • Type III IFNs were originally identified as a novel ligand-receptor system acting in parallel with type I IFNs, but subsequent studies have provided increasing evidence for distinct roles for each IFN family.
  • the inventors aimed to evaluate the effects of type I and type III interferons on both innate (antiviral) and adaptive immune response (CD4 + T cell proliferation). Antiviral activities of types I and III
  • IFN type I and III interferon-stimulated genes
  • ISGs interferon- stimulated genes
  • the inventors further evaluate the capacity of both IFN to protect HepG2 cells from EMCV-induced cytopathogenic effect.
  • IFN type III and IFN- a2a have intrinsic cellular antiviral activity and are able to fully protect HepG2 cells challenged with EMCV.
  • CFSE labelled CD4 + T cells were first stimulated with poly I:C matured allogeneic dendritic cells in presence of different dose of IFNs. At 5 days post activation, the CFSE fluorescence dilution was analyzed. As shown in Figure 2, IFN-a2a inhibits the proliferation of stimulated CD4 + T cells, while IFN type III exhibits no ability to suppress their proliferation. Of note, when the MLR was performed in the presence of anti-interferon type I receptor antibody, CD4 + T cells exhibit a greater proliferation. Thus, IFN-type I but not IFN type III inhibit the proliferation of allo-activated CD4 + T cells.
  • ISGs are induced only in CD4 + T cells stimulated with IFN-a2a.
  • IFN-a2a but not IFN-type III induce the expression of ISGs in CD4 + T cells.
  • the Jak-STAT 1/2 pathway being the major regulators of the transcription of ISG, the inventors have analyzed the phosphorylation levels of Statl proteins in response to IFN-type I, or interferon type III within CD4 + T cells.
  • IFN-a2a was able to stimulate the phosphorylation of Statl within CD4 + T cells. Therefore, IFN-a2a but not IFN-type III induces tyrosine phosphorylation of STAT1 in CD4 + T cells.
  • PBMCs were thawed in RPMI 1640 with 10% fetal bovine serum (FBS) and washed in FACS buffer. Phenotypic staining was performed on 10 6 cells by incubation with a viability marker (AmCyan live-dead kit from Invitrogen) and with antibodies conjugated to CD3, CD4, CD8, CD45RA, CCR7. Subsequently, cells were washed, fixed with 4% paraformaldehyde for 5 min, washed, and acquired with an AURORA cytometer (Cytek).
  • FBS fetal bovine serum
  • Peripheral Blood samples were obtained either from healthy donors through Etableau Francais du Sang (EFS, Paris, France) or from Elite controller HIV-1 patients and chronically-HIV-infected patients pre and post combined ART treatment.
  • IFN-a and IFN-l (IL-28A) serum concentrations were measured using the high sensitivity Single-Molecule Array (Simoa®) technology (Digital ELISA technology) (Quanterix) according to the manufacturer’s instructions. Results
  • the relative frequencies of the CM populations within the CD8+ T cell compartments were evaluated in each of the subject groups.
  • the gating strategy to define this subset is the following. Briefly, singlet cells were defined, followed by gating on lymphocytes and live cells. Among the live cells, CD3+ T lymphocytes were identified, followed by the definition of CD8+ subpopulations. Subsequently, the expression of CD45RA and CCR7 was analyzed in the CD8+ T lymphocytes. Central memory T cells (TCM) are CD45RA- CCR7+.
  • Figure 6A shows that the level of CM CD8+ cells was significantly lower in non-controllers before cART than in other groups. Moreover, combined antiretroviral therapy (cART) results in increase of CM CD8+ cells.
  • Serum levels of IFN-a was measured in the 4 groups.
  • Figure 6B shows that the non-controller patients have significantly increased serum IFN-a levels before treatment compared with after treatment.
  • IFN-a inversely correlates with the percentage of CM CD8 + cells in HIV -infected patients without treatment
  • Serum from healthy control donors and HIV-1 infected patients, before and after cART treatment, originating from two distinct institutes were assayed by Simoa® for IFN-a and IFN- 2 proteins.
  • Latent HIV-1 reservoir represents the main obstacle in achieving sustained virologic remission in cART treated HIV-1 infected patients following ART treatment interruption. This is due to two opposing biologic processes occurring in parallel in patients under cART:
  • cART promotes an inhibition of the viral replication triggered by the HIV-1 proviral DNA present in activated peripheral and mucosal reservoir cells. It results from this process a minimal expression of viral particles in the body fluid ( ⁇ 40 copies/ml).
  • the second process is due to the production of type I IFN-a and type III IFN-l locally, induced by viral replication occurring in these still infected peripheral and mucosal cells, which contain latent integrated HIV proviruses.
  • these interferons locally reduce the ongoing viral replication occurring in these reservoir cells, which limits the local propagation of the virus to nearby cells but still maintains the reservoir of infected cells.
  • the local production of IFN-a and IFN-l by peripheral and mucosal cells, which are still infected and replicating the virus, is confirmed by the presence in the serum of post-cART patients of substantial concentrations of these antiviral cytokines (Figure 7).
  • HIV- 1 proviral DNA present in these reservoir cells may fully replicate viruses.
  • cART treatment which controls viral replication, may reduce and progressively eliminate these peripheral and mucosal cellular reservoirs.
  • Example 3 Clinical protocol for AIDS treatment in human patients Study design 1
  • Study eligibility criteria includes patients having ongoing ART (>three drugs) with plasma HIV-1 RNA levels ⁇ 40 copies/ml as well as a CD4+ T cell count > 500 cells/pl (and optionally ⁇ 500 cells/pl). Patients receive infusions of neutralizing monoclonal antibodies anti-IFN type I and type III receptors at 2-week intervals in parallel with their ongoing daily cART therapy (Figure 8). Plasma HIV-1 RNA levels, size of the reservoir and CD4+ T cell counts are monitored every 2 weeks, after collection of blood samples, 1 day before each mAbs infusion.
  • Study eligibility criteria includes patients having ongoing ART (>three drugs) with plasma HIV-1 RNA levels ⁇ 40 copies/ml as well as a CD4+ T cell count > 500 cells/pl (and optionally ⁇ 500 cells/pl). Patients receive infusions of anti-IFN-a, anti-IFN-l monoclonal antibodies and optionally anti-IFN-b monoclonal antibodies at 2-week intervals in parallel with their ongoing daily cART therapy (Figure 8). Plasma HIV-1 RNA levels, size of the reservoir and CD4+ T cell counts are monitored every 2 weeks, after collection of blood samples, 1 day before each mAbs infusion. Plasma HIV-1 RNA Levels
  • HIV-1 RNA levels are determined using the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Assay (version 2.0) or the Roche cobas HIV-1 quantitative nucleic acid test (cobas 6800), which quantitate HIV-1 RNA over a range of 2x101 to 1x107 copies/ml.
  • QVOA Quantitative Viral Outgrowth Assay
  • p24 antigen is quantified in the culture supernatant, using an HIV p24 antigen enzyme-linked immunosorbent assay (ELISA) kit (Perkin-Elmer, Hopkinton, MA), and estimated frequencies of cells with replication-competent HIV-1 are calculated using limiting dilution analysis.
  • ELISA enzyme-linked immunosorbent assay
  • CD4+ T-cell counts are determined by a clinical flow cytometry assay.
  • mice Animal experiments were carried out with female NOD/Shi-scid/IL-2RYnull (NOG) immunodeficient mouse strain. Mice were humanized using hematopoietic stem cells (CD34+) isolated from human cord blood.
  • NOG NOD/Shi-scid/IL-2RYnull
  • Humanized mice were intraperitoneally injected with HIV-NL4.3, and mock-infected mice were subsequently injected with an equal volume of the vehicle. Blood and spleen were removed from infected and non-infected humanized NCG mice at day 2 post infection, when the vims load has reached a plateau at day 10 and at day 30.
  • IFN-a IFN-lI and IFN-/.3 mRNA isolation and real-time PCR analysis.
  • the conditions for PCR were as follows: 10 min at 95°C, then 40 cycles of amplification at 95°C for 15 s and 60 s at 60°C, and finally 15 s at 95°C, 30 s at 60°C, and 15 s at 95°C.
  • Statistical analyses were performed by the 2-AACT method.
  • mice were sacrificed on the day of infection and 10 and 35 days after infection.
  • PBMCs and splenocytes were recovered and the presence of IFN-oc, IFN- 3, and IFN-lI mRNA was assessed by RT-qPCR.
  • Figure 9 shows that both types of IFN were produced after infection. IFN-oc was most notably present in PBMCs, whereas IFN- 3 and IFN-lI were present in splenocytes.
  • Example 5 Activated CD4 + T cells express IFN-l Receptor.
  • CD4 + T cells were purified from peripheral blood mononuclear cells (PBMCs) obtained from anonymous healthy blood donors (EFS). Ficoll (Ficoll Hystopaque; Sigma) density centrifugation was performed as per the manufacturer’ s instructions, and CD4 + cells were negatively selected using magnetic beads (CD4 + T-cell isolation kit I; Miltenyi Biotec). Purity was assessed following cell isolation by staining with anti-CD4-Alexa Fluor 700 (RPA-T4) and all samples were >97% CD4 + by flow cytometry.
  • CD4+ T cells were cultured in RPMI 1640 supplemented with 10% FBS (Gibco), 100 IU penicillin, 100 pg/mL streptomycin, 0.1 M Hepes, 2 mM L-glutamine, and/or recombinant human IL-2. Cells were maintained at 37 °C in a 5% C02 humidified incubator.
  • CD4 + T cells were co-stimulated through CD3 and CD28 using plate-bound antibodies for 3 days or no simulation.
  • Anti-CD3 OKT3
  • CD28.2 anti-CD28
  • IL-28RA IL-10RB
  • IFIH1 IFI27 mRNA isolation and real-time PCR analysis.
  • the conditions for PCR were as follows: 10 min at 95°C, then 40 cycles of amplification at 95°C for 15 s and 60 s at 60°C, and finally 15 s at 95°C, 30 s at 60°C, and 15 s at 95°C.
  • Statistical analyses were performed by the 2-AACT method.
  • IFN-/J binding assay to quantify the presence of IL-28RA on CD4+ T cells
  • cells were treated with or without His-tagged IFN-/J (R&D Systems) diluted in PBS containing 1% BSA on ice for 60 min at indicated doses. Cells were then washed and stained with anti-His PE (Miltenyi Biotec) for 40 min in the dark and a viability dye.
  • the MFIs of IFN-/J binding on CD4 + T cells were determined by flow cytometry in living cells as follows: MFI (His-tagged IFN-/J + anti-His PE) - MFI (anti-His PE).
  • IFN type I receptors IFN AR 1/2
  • IFNLR1 IFN receptor 1
  • epithelial cells neutrophils and dendritic cells.
  • CD4 + T cells We assessed the ability of CD4 + T cells to express IFNLR according to their level of activation.
  • Figure 10 shows that while purified CD4+ T cells cultured in the presence of IL-2 alone expressed basal levels of IL-28RA mRNA and IFNLR, the 3-day-CD3/CD28/IL- 2-stimulated cells displayed high level of IFNLR, at mRNA and protein levels.
  • Example 6 IFN- 3 inhibits HIV-1 infection of activated CD4 + T cells.
  • CD4 + T cell purification and culture CD4 + T cells were purified and cultured as described in Example 5.
  • IFN-a, IFN- 3 and anti-IFNAR2 neutralizing antibody were purchased from Bio-Techne and anti-IL28RA (IFNLR1) neutralizing antibody from PBL assays science.
  • Viral stocks were generated by transfection of HEK 293T with polyethylenimine (Polysciences). Two days after transfection, culture supernatants were collected, clarified at 441 x g for 5 min, and filtered (0.45 pm).
  • CD4 + T cells were co-stimulated through CD3 and CD28 using plate-bound antibodies for 3 days or no simulation.
  • Anti-CD3 OKT3
  • CD28.2 anti-CD28
  • Stimulated CD4 + T cell HIV-1 infections occurred after 3 days of CD3/CD28 stimulation.
  • Infections with HIV-NL4.3 were performed overnight in the presence of polybrene (2 pg/mL), and fresh media were replaced. The frequency of infected cells was determined by intracellular HIV p24 by FACS. When indicated, IFN-a, IFN-/J, anti- IFNAR2 and anti-IL-28RA neutralizing antibodies were added 24 hours before infection and for an additional 5 days in culture after infection.
  • CD4 + T cell purification and culture CD4 + T cells were purified, cultured and co-stimulated as described in Example 5.
  • J-Lat clone 10.6 The human cell line Jurkat (J-Lat) clone 10.6, was obtained from AIDS Reagent Program, National Institutes of Health (Germantown, MD). This cell line was grown in complete culture medium, as the primary cells, and maintained at 37 °C in a 5% C02 incubator. J- Lat clone 10.6 harbors a HIV provirus containing the GFP open reading frame (ORF) instead of nef and a frameshift mutation in env.
  • ORF GFP open reading frame
  • 5-AzadC was purchased from Sigma Aldrich, MS-275 from Enzo Life Sciences and FN- l3 from Bio-Techne. LRA treatment of stimulated CD4 + T cells
  • Stimulated CD4 + T cells were submitted to a 48-h 5-AzadC pretreatment followed by a 24-h MS -275 induction, corresponding to a total 72-h 5-AzadC treatment.
  • HIV reactivation in vitro in latently infected J-Lat clone 10.6 HIV reactivation was performed by a 48-h 5-AzadC pretreatment followed by a 24-h MS- 275 induction, corresponding to a total 72-h 5-AzadC treatment. Reactivation of HIV was monitored as the percentage of living GFP-positive cells, according to forward and side laser light scatter flow cytometry analysis in a FACS ARIA3 flow cytometer (BD Biosciences). The data were analyzed using FlowJo software. IL-28RA, IFIH1 and IFI27 mRNA isolation and real-time PCR analysis.
  • the conditions for PCR were as follows: 10 min at 95°C, then 40 cycles of amplification at 95°C for 15 s and 60 s at 60°C, and finally 15 s at 95°C, 30 s at 60°C, and 15 s at 95°C.
  • Statistical analyses were performed by the 2-AACT method.
  • IFN-/J binding assay to quantify the presence of IL-28RA on CD4+ T cells
  • cells were treated with or without His-tagged IFN-/J (R&D Systems) diluted in PBS containing 1% BSA on ice for 60 min at indicated doses. Cells were then washed and stained with anti-His PE (Miltenyi Biotec) for 40 min in the dark and a viability dye.
  • the MFIs of IFN-/J binding on CD4 + T cells were determined by flow cytometry in living cells as follows: MFI (His-tagged IFN-/J + anti-His PE) - MFI (anti-His PE). Results
  • CD4 + T cells were treated sequentially with two latency- reversing agents, DNA methylation inhibitor 5-AzadC and HDACI MS-275, a combination of molecule known to reactivate HIV.
  • GFP Green Fluorescent Protein
  • Example 8 The NtRTI TDF enhances the production of IFN-L3 by HT-29 cells.
  • HT-29 cells Human colon adenocarcinoma HT-29 cells (ATCC, HTB-38) were cultured in 25 cm 2 culture flasks in McCoy's 5 A medium supplemented with 10% heat-inactivated FCS (Gibco), penicillin (100 U/mL) and streptomycin (100 pg/mL). TDF treatment of HT -29 cells
  • HT-29 cells were incubated with dimethyl sulfoxide (DMSO) or tenofovir disoproxil fumarate (TDF, 25 mM) for 48 hours.
  • DMSO dimethyl sulfoxide
  • TDF tenofovir disoproxil fumarate
  • IFN- /. 3 analysis of supernatants The levels of IFN-k3 were assayed using an ELISA kit according to the manufacturer's instructions.
  • TDF Tenofovir disoproxil fumarate
  • NRTI nucleotide reverse transcriptase inhibitor
  • CD4 + T cells were purified and cultured as described in Example 5.
  • Human colon adenocarcinoma HT-29 cells (ATCC, HTB-38) were cultured as described in Example 8.
  • Anti-IL28RA neutralizing antibody was purchased from PBL assays science. Production of Viral Stocks
  • HIV NL4.3 was obtained from the AIDS Research and Reference Reagent Program. Viral stocks were generated by transfection of HEK 293T with polyethylenimine (Polysciences). Two days after transfection, culture supernatants were collected, clarified at 441 x g for 5 min, and filtered (0.45 pm).
  • Transwell co-culture model for assessing inhibition of HIV infection in stimulated CD4+ T cells by IFN-/.3 released by TDF-treated HT-29 cells.
  • HT-29 were grown until subconfluence in the lower chamber. Then HT-29 cells were treated with tenofovir disoproxil fumarate (TDF). After 24h of TDF treatment, 3-day-CD3/CD28/IL2-stimulated CD4 + T cells were seeded in presence of IL- 2 in the upper chamber, which is then placed in the culture well so as to be immersed in the culture medium of the HT-29 cells. When indicated, the CD4+ T cells were pre-treated with an IFNLR1 (IL28RA) neutralizing antibody. After 24h of co-culture, CD4 + T cells were isolated, infected with HIV NL4.3 and then placed back into the co-culture for an additional 5 days of culture. The frequency of infected cells was determined by intracellular HIV p24 by FACS.
  • TDF tenofovir disoproxil fumarate
  • Transwell culture assay to evaluate the IFN- 3 antiviral activity.
  • the test was performed in 12-well Transwell plates with stimulated CD4 + T cells at sufficient concentrations (Figure 14). Briefly, HT-29 cells were cultured in the bottom chamber of Transwell plates. When the culture was subconfluent, Tenofovir disoproxil fumarate (TDF) was added. After 24 hours of stimulation, activated CD4 T cells were deposited in the insert which was then placed in the culture well. When indicated, the CD4+ T cells were pre-treated with an IFNLR1 (IL28RA) neutralizing antibody.
  • IFNLR1 IFNLR1
  • FIG. 15 shows that the IFN- 3 released by TDF-treated HT-29 cells was able to impair HIV-1 CD4 + infection.
  • Example 10 IFN-L3 released by TDF-treated HT-29 cells inhibit in vitro HIV reactivation in latently infected J-Lat clone 10.6.
  • 5-AzadC was purchased from Sigma Aldrich; MS-275 from Enzo Life Sciences; and IFN- l3, from Bio-Techne.
  • Anti-IL28RA neutralizing antibody was purchased from PBL assays science.
  • HT-29 cell culture Human colon adenocarcinoma HT-29 cells (ATCC, HTB-38) were cultured as described in Example 8.
  • the human cell line Jurkat (J-Lat) clone 10.6 was cultured as described in Example 8.
  • Transwell co-culture model for assessing the inhibition of HIV latency reversal in latently infected J-Lat cells by IFN- /. 3 released by TDF-treated HT-29 cells.
  • Transwell cultures (12 wells) with confluent HT-29 monolayers were used for co-culture with J-LAT 10.6 cells.
  • HT-29 were grown until subconfluence on Transwell bottom.
  • HT-29 cells were treated with Tenofovir disoproxil fumarate (TDF).
  • TDF Tenofovir disoproxil fumarate
  • J-LAT 10.6 cells pretreated with 5-AzadC and MS-275 for 72h were seeded on Transwell filters.
  • anti-IFNLRl (IL28RA) neutralizing Abs were added to the culture 2 hours before the J-Lat 10.6 cells seeding.
  • HIV reactivation was monitored as the percentage of living GFP-positive cells, according to forward and side laser light scatter flow cytometry analysis in a FACS ARIA3 flow cytometer (BD Biosciences). The data were analyzed using FlowJo software. Results
  • Example 11 Pre- Clinical protocol for HIV-1 reservoirs elimination in HIV-1 infected humanized mice.
  • mice Two humanized mouse models are used, BLT mice and NCG mice.
  • Humanized BLT mice with > 50% of circulating human CD45+ cells at 24 weeks post-transplantation and humanized NCG mice with > 20% of circulating human CD45+ cells at 24 weeks post transplantation are infected with HIV-1 through retro-orbital (BLT mice) or intraperitoneal injection (NCG mice).
  • Hu-mice infected with HIV-1 for 7-9 weeks are treated with cART.
  • the study is structured into two arms ( Figure 18). The mice are injected with a-IFNARl mAh and a-IFNLRl mAh (experimental group) or with corresponding isotype mAbs (control group) twice a week starting from week 4 after cART.
  • cART is maintained for an additional 2.5 weeks after the last antibody treatment. At the end of the cART treatment, animals are sacrificed. Some animals are kept after cART has been discontinued to measure time to viral detectability after treatment interruption. These groups of “viral rebound” are monitored for another three to six weeks or longer if they remain undetectable in plasma viral loads. Monitoring of viral load upon HIV-1 infection and under cART treatment is performed every two weeks. Plasma HIV-1 RNA Levels
  • HIV-1 RNA levels are determined using the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Assay (version 2.0) or the Roche cobas HIV-1 quantitative nucleic acid test (cobas 6800), which quantitate HIV-1 RNA over a range of 2x1o 1 to lxlO 7 copies/ml. Cell-associated HIV-1 DNA detection.
  • HIV-1 DNA is quantified by real time PCR. DNA from serial dilutions of ACH2 cells, which contain 1 copy of HIV genome in each cell, is used to generate a standard curve. Cell-associated HIV-1 RNA detection.
  • HIV-1 RNA is extracted from spleen or bone marrow cells using the RNeasy plus mini kit (Qiagen). HIV-1 RNA is detected as described above. The HIV-1 RNA expression levels are normalized to human CD4 mRNA controls, and the result is calculated as fold change in gene expression. Quantitative Viral Outgrowth Assay
  • QVOA Quantitative Viral Outgrowth Assay
  • p24 antigen is quantified in the culture supernatant, using an HIV p24 antigen enzyme-linked immunosorbent assay (ELISA) kit (Perkin-Elmer, Hopkinton, MA), and estimated frequencies of cells with replication-competent HIV-1 are calculated using limiting dilution analysis.
  • ELISA enzyme-linked immunosorbent assay
  • lymphoid formations called reservoirs. These formations are found anatomically on the intestinal or vaginal mucous membranes initially (during the primary infection), and then within the peripheral lymph nodes or spleens. These lymphoid formations contain virions in their circulating lymph and host three categories of CD4+ T cells:
  • infected CD4+ T cells that are immune-activated by specific antigenic stimulation 2) infected CD4+ T cells that are immune-resting. These cells are resting although they carry the proviral DNA integrated into their genome. These cells do not synthesize viral proteins until they are immune-stimulated.
  • CD4+ T cells that are not carrying the proviral DNA and are resting while awaiting their specific antigenic stimulation. These formations actually maintain virus synthesis thanks to infected CD4+ T cells undergoing specific immune activation induced by their antigen. These cells produce stromal lymph virions and circulating blood virions and may spread the viral infection to other lymphoid formations. These latter formations in turn become reservoir formations.
  • interferon- stimulated genes maintaining viral latency: these are a set of molecules that inhibit the transcription of HIV genes when they are integrated into their DNA.
  • Mucosal type III and systemic type I interferons represent the first natural anti viral defense blocking the integration of proviral DNA in target CD4+ T cells that carry HIV receptors.
  • IFN messengers present in the stromal lymph of the lymphoid formations, act in a paracrine and dose-dependent manner in neighboring target CD4+ T cells that carry their specific type I and type III receptors. Their action induces the expression of ISGs which inhibit the synthesis of viral proteins, such as ISG15.
  • CD4+ T cells constitutively express type I IFN receptors and inducibly express type III IFN receptors. Activation of these receptors by IFNs prevents the expression of virions within CD4+ T cells by blocking viral protein synthesis and virion production.
  • the infected resting cells in the lymphoid reservoir formations that have the proviral DNA integrated into their genome, as well as the healthy uninfected resting cells are protected, do not synthesize viral proteins and do not produce viruses.
  • infected cells of these lymphoid formations which are immune- activated by their antigenic stimulation and which already produce the virions, are not sensitive to the IFNs action, but they produce the infectious virions.
  • HIV-infected and immune-activated CD4+ T cells maintain their viral production and die by apoptosis. However, before disappearing, the virions they produce induce the production of mucosal type III antiviral IFNs in the mucous membranes and of systemic type I IFNs which protect the infected or non-inf ected resting CD4+ T cells (category 2 and 3) in a paracrine and dose-dependent manner.
  • Infected CD4+ T cells which carry the integrated proviral DNA, but are not immune-activated by their specific stimulation within the lymphoid foci, are protected from viral production by the production of ISGs induced by type III and type I IFNs. Indeed, the activation of type I and III IFN receptors that they express activate the ISGMLs responsible for the latency induced by the IFNs.
  • IFNs are mainly produced by APCs (pDC, mDC and macrophages).
  • Stopping cART affects all three categories of CD4+ T cells of the reservoir lymphoid formations. It follows that:
  • the infected and non-immune- stimulated cells wait for their antigenic stimulation to be activated and release the ISGMLs of their latency. These cells will induce the synthesis of viral proteins and the production of virions circulating in the stromal lymph of the lymphoid formations and then in the peripheral blood and will disseminate the infection to other so far spared lymphoid formations.
  • the invention proposes to combine these two treatments with agents blocking mucosal type III IFNs produced on the mucous membranes, such as neutralizing antibodies. This combination appears necessary to clear out the lymphoid reservoir formations of their virus-producing cells that maintain the infection.

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Abstract

The present invention relates to a novel method for treating acquired immune deficiency syndrome (AIDS) in a subject in need thereof. In particular, said method comprises the administration of a combination, a kit-of-parts, a composition or a pharmaceutical composition comprising a type III interferon blocking agent, an interferon-alpha (IFN-α) blocking agent, an antiretroviral (ART) agent and, optionally, an interferon-beta (IFN-β) blocking agent and/or a latency-reversing agent (LRA).

Description

NOVEL COMBINATION FOR THE TREATMENT OF AIDS
FIELD OF INVENTION
The present invention relates to a novel method for treating acquired immune deficiency syndrome (AIDS) in a subject in need thereof. In particular, said method comprises the administration of a combination, a kit-of-parts, a composition or a pharmaceutical composition comprising a type III interferon blocking agent, an interferon-alpha (IFN-a) blocking agent, an antiretroviral (ART) agent and, optionally, an interferon-beta (IFN-b) blocking agent and/or a latency-reversing agent (LRA).
BACKGROUND OF INVENTION
Antiretroviral therapy (ART) is thus far, a very efficient therapy in the treatment of patients infected with human immunodeficiency viruses (HIV). Indeed, patients treated with ART have a plasma viremia below detectable levels and thus can have normal life expectancy. However, these treatments are very onerous and can lead to uncertain long term cytotoxicity for HIV patients (Chun et ah, “Durable Control of HIV Infection in the Absence of Antiretroviral Therapy: Opportunities and Obstacles” JAMA, 2019 Jul 2;322(l):27-28).
In addition, the most considerable issue with ART is that when patients stopped their treatment and are thus in an analytic treatment interruption (ATI), a rapid rebound of plasma viremia is observed. This phenomenon suggests that not all HIV-infected cells are eliminated by ART and that some infected cells remain in the organism. The scientific community is thus wondering how and where HIV is able to persist in patient during ART. Two different types of cells were identified: latent and productive infected cells. In patients treated with ART, latent cells were identified in many T-cells types (including stem cell memory, central memory, transitional memory, effector memory and naive CD4 T cells), suggesting that these cells can contribute to viral rebound after ART arrest, but this mechanism still remains poorly understood (Pitman et ah, “Barriers and strategies to achieve a cure for HIV” Lancet HIV, 2018 Jun;5(6):e317-e328). Under ART treatment, lymph nodes and gastrointestinal tract have much higher concentration of HIV DNA and RNA per CD4 T cells than other tissues ( e.g . blood). Indeed, in lymph nodes, CD8 cytotoxic T cells have less access to B-cell follicles and thus these cells are protected from the treatment, while in the gastrointestinal tract, many Thl7 cells expressing CCR5 are present, cells targeted preferentially by the vims, which can explain the high level of HIV DNA and RNA. Macrophages from different tissues can also be infected and thus persist for example in the brain and this population was demonstrated as being the viral source of the viral rebound after ART arrest in a unique model of humanized myeloid-only mice (Honeycutt et ah, “HIV persistence in tissue macrophages of humanized myeloid-only mice during antiretroviral therapy” Nat Med, 2017; 23: 638-43).
Furthermore, the study of Rabezanahary et ah, found that in the spleen mesenteric and peripheral lymph nodes of SIVmac251-infected rhesus macaques, effector memory and follicular helper T CD4 have the highest frequency of viral DNA and RNA. Interestingly, two weeks after ART arrest, a viral rebound was observed due to a rapid seeding of SIV from visceral lymphoid tissues producing viral RNA, highlighting the importance of these anatomical sites, reservoirs of HIV, in the viral rebound (Rabezanahary et ah, “Despite early antiretroviral therapy effector memory and follicular helper CD4 T cells are major reservoirs in visceral lymphoid tissues of SIV-infected macaques” Mucosal Immunology, 2020 13:149-160). Many other clinical strategies were investigated including targeting the viral replication cycle (BM transplantation of patients with hematopoietic stem cells from donors homozygous for CCD5A32 or WT CCR5, gene therapy by editing CCR5, latency reversal agents), HIV-specific immune enhancement strategies (T-cell vaccines, bNAbs and CAR T cells) or immune modulation strategies (immune-checkpoint blockers, vedolizumab, IL15 superagonist or sirolimus) (Pitman et ah, “Barriers and strategies to achieve a cure for HIV” Lancet HIV, 2018 Jun;5(6):e317-e328).
The HIV-1 latent reservoir is a major hurdle to achieve a cure for HIV-1. A new strategy consists in targeting the latent cells which escape the ART and can thus create a viral rebound after ART arrest. One hypothesis was that the HIV integrated mostly into heterochromatin-repressed transcriptional regions into these latent cells, but it was surprisingly found that HIV can be latent while integrated in euchromatin- active transcriptional regions, suggesting that the epigenetic silencing occurs via cis- or trans- acting elements. So far, the therapies were focused on epigenetic mechanisms of latency and mostly histone deacetylase inhibitors (HDAC) inhibitors, but no clear reservoir reduction has been demonstrated. T-cell activating agents were also a new wave of therapy using for example protein kinase C (PKC) agonists but despite promising results in vitro , no reduction in the latent reservoir was reported in vivo. One strategy could also be a “shock and kill” strategy wherein the “shock” phase will be to target the immune response towards the reservoir and to reverse latency and then to kill the cells, but this strategy requires to know which antigen will be presented by the re-activated cell.
Many others strategies are currently under investigation (Sengupta et ah, “Targeting the Latent Reservoir for HIV-1” Immunity, 2018 15;48(5):872-895 and Ait-Ammar el ah, “Current Status of Latency Reversing Agents Facing the Heterogeneity of HIV- 1 Cellular and Tissue Reservoirs” Frontiers in Microbiology, 202024; 10:3060). Although several latency-reversing agents (LRAs) have been identified and tested, none of them has been able to efficiently eradicate the HIV-1 latent reservoir.
Alternative therapeutic approaches aimed at reversing HIV latency in presence of antiretroviral therapy have been tested. One of them was aimed at controlling the effects of type I interferons, cytokines known to have a crucial role in the immunopathogenesis of AIDS during chronic HIV-1 infection. IFNAR blockade in the presence of cART was found to reduce the size of HIV-1 reservoirs in lymphoid tissues in humanized mice persistently infected with HIV-1, but was insufficient to efficiently eradicate these reservoirs.
The “shock and kill” strategy is based on inducing viral transcription of latent HIV-1 provirus followed by the selective killing of reactivated cells.
Inefficient eradication of the HIV-1 latent reservoir is due to the persistence of resting cells containing latent HIV-1 provirus, since only reactivated cells are killed by antiretroviral (ART) agents in the “kill” phase of the “shock and kill” strategy. In summary, latent HIV reservoir represents the main obstacle to achieving sustained virologic remission in ART-treated HIV-infected individuals following ART treatment interruption. And the reservoir cell compartment still needs to be better understood in order to improve the cure strategy against HIV infection. Thus, there is still an important and urgent need for a novel treatment of HIV infection or acquired immune deficiency syndrome (AIDS) which allows sustained or complete virologic remission in ART-treated HIV patients.
This could be achieved by finding new strategies that induce reactivation of cells constituting the HIV-1 latent reservoirs and viral transcription of latent HIV-1 provims in these cells, or for therapeutic approaches that reverse mechanisms that inhibit such HIV-1 reactivation in the latent reservoirs.
Interferons are a group of cytokines of different types. Type I interferons are systemic, while type III interferons are mainly mucosal (i.e. produced at the mucosal epithelial).
Based on their discovery of new biological properties of type I and type III interferons, the inventors propose to improve existing therapeutic strategies for treating AIDS caused by HIV.
In particular, they have shown that the production of type I IFN-a and type III IFN-l locally is induced by viral replication occurring in the peripheral and mucosal reservoir cells (which contain latent integrated HIV proviruses), and is involved in the incomplete virus clearance.
Indeed, the inventors have surprising shown the presence of type III interferons in the serum of patients after antiretroviral (cART) treatment, while the serum level of type I IFN had totally decreased after treatment. This antiviral cytokine is locally produced by mucosal cells, which are still infected and replicating the virus. By their antiviral effects, these interferons locally reduce the ongoing viral replication occurring in these reservoir cells. This limits the local propagation of the virus to nearby cells, but at the time maintains the reservoir of infected cells. The inventors have thus surprisingly shown that reactivation of the HIV-1 latent reservoirs is inhibited by type III interferons which are naturally produced by HIV 1 infected patients, especially when under antiretroviral treatment.
Moreover, the inventors have surprisingly shown that this inhibition of HIV reactivation in the latently infected cells may be reversed by type III interferon blocking agents.
The inventors thus provide a new therapeutic approach based on the use of a type III interferon blocking agent to reverse the natural or therapeutically-induced mechanisms that inhibit HIV-1 reactivation in the latent reservoirs.
This new therapeutic approach, combined with the “kill” phase of the “shock and kill” strategy performed by antiretroviral therapy (ART), will allow complete viral clearance from the latent reservoirs.
The inventors thus propose to block the type III interferon-l, the type I interferon-a, and optionally type I IFN interferon-b antiviral action in resistant cells containing proviral HIV-1 DNA in the HIV reservoirs, by repeated administration of specific agents blocking the production or of the activity of these cytokines, to reverse proviral latency, while administering ART agents to the patient until total viral clearance.
Indeed, following the blocking of the peripheral and mucosal antiviral interferons, which hinder expression of viral replication in reservoir cells, the HIV-1 proviral DNA present in these reservoir cells may fully replicate viruses. In turn, cART treatment, which controls viral replication, may reduce and progressively eliminate these peripheral and mucosal cellular reservoirs.
The applicant thus provides a novel method for treating AIDS in a subject in need thereof, comprising administering to the subject a combination comprising: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA). SUMMARY
The invention relates to a combination comprising: i) a type III interferon blocking agent, ii) an interferon-alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) optionally, an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
In some embodiments, the type III interferon blocking agent is:
- an agent neutralizing circulating IFN-l selected from the group comprising an active anti-IFN-l vaccine, such as an IFN-k-kinoid, IFN-l DNA-based or IFN-l RNA-based vaccine, and a passive anti-IFN-l vaccine, such as an anti-IFN-l antibody or an anti-IFN- l hyper-immune serum, or
- an agent blocking type III interferon signaling selected from the group consisting of an active anti-IFNLRl vaccine, such as an IFNLR1 DNA-based or IFNLR1 RNA-based vaccine, and a passive anti-IFNLRl vaccine, such as an antibody that binds to the IFNLRl receptor.
In some embodiments, the type III interferon blocking agent is an anti-IFN-l antibody, or an agent blocking type III interferon signaling selected from the group consisting of an active anti-IFNLRl vaccine, such as an IFNLRl DNA-based or IFNLRl RNA-based vaccine, and a passive anti-IFNLRl vaccine, such as an antibody that binds to the IFNLRl receptor.
In some embodiments, the IFN-a blocking agent is selected from the group consisting of an agent neutralizing circulating IFN-a, an agent blocking IFN-a signaling, an agent depleting IFN-a producing cells, and an agent blocking IFN-a production; wherein the agent neutralizing circulating IFN-a is selected from the group comprising an active anti-IFN-a vaccine, such as an IFN-a-kinoid, IFN-a DNA-based or IFN-a RNA-based vaccine, and a passive anti-IFN-a vaccine, such as an anti-IFN-a antibody or anti-IFN-a hyper-immune serum; wherein the blocking agent of IFN-oc signaling is selected from the group consisting of an active anti-IFNARl or anti-IFNAR2 vaccine, such as an IFNAR1 or IFNAR2 DNA-based or an IFNAR1 or IFNAR2 RNA-based vaccine, a passive anti- IFNARl or anti-IFNAR2 vaccine, such as an anti-type I interferon R1 or R2 antibody, and an IFN-oc endogenous regulator, such as SOSC1 or an aryl hydrocarbon receptor; wherein the agent depleting IFN-oc producing cells is an agent depleting plasmacytoid dendritic cells (pDCs); and wherein the agent blocking IFN-oc production is an agent blocking the production of IFN- oc by pDCs. In some embodiments, the IFN-b blocking agent is an agent neutralizing circulating IFN- b selected from the group comprising an active anti-IFN-b vaccine, such as an IFN-b- kinoid, IFN-b DNA-based or IFN-b RNA-based vaccine, and a passive anti-IFN-b vaccine, such as an anti-IFN-b antibody or an anti-IFN-b hyper-immune serum.
In some embodiments, the antiretroviral (ART) agent is selected from the group consisting of Nucleoside reverse transcriptase inhibitors (NRTIs), Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Protease inhibitors (Pis), Integrase inhibitors (INSTIs), Fusion inhibitors (FIs), Chemokine receptor antagonists (CCR5 antagonists) and Entry inhibitors (CD4-directed post-attachment inhibitors).
In some embodiments, the latency-reversing agent (LRA) is selected from the group consisting of PKC agonists, MAPK agonists, CCR5 antagonists, Tat vaccines, SMAC mimetics, inducers of P-TEFb release, activators of Akt pathway, benzotriazole derivatives, epigenetic modifiers and immunomodulatory LRAs.
Another aspect of the invention relates to a combination as described herein for use in the treatment of acquired immune deficiency syndrome (AIDS) in a subject in need thereof. In some embodiments, the presence of cells containing replication-competent proviral HIV DNA is assessed in a blood sample from the subject.
In some embodiments, the type III interferon blocking agent, the IFN-a blocking agent, optionally the interferon-beta (IFN-b) blocking agent, the antiretroviral (ART) agent, and optionally the latency-reversing agent are for simultaneous, separate or sequential administration.
In some embodiments, the type III interferon blocking agent, the IFN-a blocking agent, and optionally the interferon-beta (IFN-b) blocking agent are to be administered every week, every 2 weeks or every 3 weeks.
In some embodiments, the antiretroviral (ART) agent, and optionally the latency-reversing agent, are to be administered daily.
In some embodiments, the type III interferon blocking agent, the IFN-a blocking agent, and optionally the interferon-beta (IFN-b) blocking agent are to be administered parenterally or intravenously.
In some embodiments, one or more doses of the type III interferon blocking agent, the IFN-a blocking agent, and optionally the interferon-beta (IFN-b) blocking agent are to be administered to the subject before receiving said combination.
In some embodiments, one or more doses of the antiretroviral (ART) agent, and optionally the latency-reversing agent, are to be administered to the subject before receiving said combination.
In some embodiments, said combination is to be administered to the subject until no cell containing replication-competent proviral HIV DNA is detected in a blood sample from the subject.
DEFINITIONS
In the present invention, the following terms have the following meanings:
"About" preceding a figure encompasses plus or minus 10%, or less, of the value of said figure. It is to be understood that the value to which the term "about” refers is itself also specifically, and preferably, disclosed. As used herein, the term "adjuvant" refers to a compound or combination of compounds that helps and enhances the pharmacological effect of a drug or a vaccine, or increases an immunogenic response.
The term "administering" means either directly administering a compound or composition of the present invention, or administering a prodrug, derivative or analog which will form an equivalent amount of the active compound or substance within the body. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes. - The term "antigen" refers to a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term "antigen" includes all related antigenic epitopes. "Epitope" or "antigenic determinant" refers to a site on an antigen to which B and/or T cells respond. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.
The term "decrease" refers to reducing the quality, amount, or strength of something. For example, a therapy (such as the methods provided herein) decreases the infectious load or titer of a pathogen such as HIV, or one or more symptoms associated with infection.
The term "fragment" refers to a portion of a polypeptide that exhibits at least one useful epitope. The phrase "functional fragment(s) of a polypeptide" refers to all fragments of a polypeptide that retain an activity, or a measurable portion of an activity, of the polypeptide from which the fragment is derived. Fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell. An epitope is a region of a polypeptide capable of binding an immunoglobulin generated in response to contact with an antigen.
The term "immunogenic peptide" (or "antigenic peptide") refers to a peptide which comprises an allele- specific motif or other sequence, such as an N-terminal repeat, such that the peptide will bind an MHC molecule and induce a cytotoxic T lymphocyte ("CTL") response, or a B cell response (for example antibody production) against the antigen from which the immunogenic peptide is derived. In one embodiment, immunogenic peptides are identified using sequence motifs or other methods, such as neural net or polynomial determinations known in the art. Typically, algorithms are used to determine the "binding threshold" of peptides to select those with scores that give them a high probability of binding at a certain affinity and will be immunogenic. The algorithms are based either on the effects on MHC binding of a particular amino acid at a particular position, the effects on antibody binding of a particular amino acid at a particular position, or the effects on binding of a particular substitution in a motif-containing peptide. Within the context of an immunogenic peptide, a "conserved residue" is one which appears in a significantly higher frequency than would be expected by random distribution at a particular position in a peptide.
The term "immunity" refers to the state of being able to mount a protective response upon exposure to an immunogenic agent. Protective responses can be antibody-mediated or immune cell-mediated, and can be directed toward a particular pathogen or tumor antigen. Immunity can be acquired actively (such as by exposure to an immunogenic agent, either naturally or in a pharmaceutical composition) or passively (such as by administration of antibodies or in vitro stimulated and expanded T cells). - As used herein, the term "alpha interferon" (IFN-a) or “interferon-alpha” refers to a family of more than 20 related but distinct members encoded by a cluster on chromosome 9 and all bind to the same IFN receptor. Among these, the IFN-a2 have 3 recombinant variants (a2a, a2b, a2c) depending upon the cells of origin and the IFN-a2b is the predominant variant in human genome. There is evidence though that each subtype has a different binding capacity to the IFNAR, modulating the signaling transduction events and the biological effects in the target cells.
The term "type III interferon", also called interferon-lambda (IFN-l), refers to naturally occurring and/or recombinant cytokines of the type III interferon-lambda family. There are four IFN-l members in humans, IFN-/J/IL-29, IFN-k2/IL-28A, IFN-k3/IL-28B, IFN-/,4. - The term "beta interferon" (IFN-b) or “interferon-beta” refers to a family of two related but distinct members IFN-bI and IRN-b3. They both bind to the same IFN receptor, the IFN-a receptor (IFNAR), which is a cell surface receptor complex consisting of 2 chains: IFNAR1 and IFNAR2 (IFNAR 1 /IFN AR2 heterodimer). Binding of IFN-b to the IFNAR receptor triggers signaling transduction events and biological effects in the target cell.
The term "isolated" or "non-naturally occurring" with reference to a biological component (such as a nucleic acid molecule, protein organelle or cells), refers to a biological component altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated”. An isolated nucleic acid or peptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. Typically, a preparation of isolated nucleic acid or peptide contains the nucleic acid or peptide at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, greater than about 96% pure, greater than about 97% pure, greater than about 98% pure, or greater than about 99% pure. Nucleic acids and proteins that are "non-naturally occurring" or have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. An "isolated polypeptide" is one that has been identified and separated and/or recovered from a component of its natural environment.
The terms "subject", "individual," and "patient" are used interchangeably herein, and refer to an animal, for example a mammal, primate or human, and include all mammals, such as e.g. non-human primate, (particularly higher primates), sheep, dog, rodent, (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, horses. In particular, these terms refer to a human, to whom treatment, including prophylactic treatment, with the combination according to the present invention, is provided.
The term "mutation" refers to any difference in a nucleic acid or polypeptide sequence from a normal, consensus or "wild type" sequence. A mutant is any protein or nucleic acid sequence comprising a mutation. In addition, a cell or an organism with a mutation may also be referred to as a mutant. Some types of coding sequence mutations include point mutations (differences in individual nucleotides or amino acids); silent mutations (differences in nucleotides that do not result in an amino acid changes); deletions (differences in which one or more nucleotides or amino acids are missing, up to and including a deletion of the entire coding sequence of a gene); frameshift mutations (differences in which deletion of a number of nucleotides indivisible by 3 results in an alteration of the amino acid sequence. A mutation that results in a difference in an amino acid may also be called an amino acid substitution mutation. Amino acid substitution mutations may be described by the amino acid change relative to wild type at a particular position in the amino acid sequence.
As used herein, the terms "prevent", "preventing" and "prevention" refer to preventative measures, wherein the object is to reduce the chances that a subject will develop the pathologic condition or disorder over a given period of time. Such a reduction may be reflected, e.g., in a delayed onset of at least one symptom of the pathologic condition or disorder in the subject.
The term "prophylactic" refers to a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. In particular, a prophylactic treatment of a HIV or SIV infection in a subject refers to a treatment that allows the subject to become an elite controller (EC) i.e. to have a relatively high CD4+ T cell count (such as e.g. superior to 500 CD4+ T cells per microliter) and/or to maintain clinically undetectable plasma HIV-1 RNA level (such as e.g. HIV RNA <50 copies/mL) during a prolonged period of time in the absence of any antiretroviral treatment (ART). - The term "therapeutic" refers to a treatment administered to a subject who exhibit early or established signs of a disease.
The term "curative" refers to a treatment administered to a subject suffering from a disease for the purpose of curing the disease, i.e. of making any sign of the disease disappear or becoming undetectable. - The terms "protein", "peptide", "polypeptide", and "amino acid sequence" are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
The term "sample" or "biological sample" refers to a biological specimen obtained from a subject, such as a cell, fluid of tissue sample. In some cases, biological samples contain genomic DNA, RNA (including mRNA and microRNA), protein, or combinations thereof. Examples of samples include, but are not limited to, saliva, blood, serum, urine, spinal fluid, tissue biopsy, surgical specimen, cells (such as PBMCs, white blood cells, lymphocytes, or other cells of the immune system) and autopsy material. - As used herein, the term "treatment" refers to an intervention that ameliorates a sign or symptom of a disease or pathological condition. For example, in case of HIV infection, HIV RNA (viral load) and CD4 T lymphocyte (CD4) cell count are the two surrogate markers of antiretroviral treatment (ART) responses and HIV disease progression that have been used for decades to manage and monitor HIV infection. Thus, the efficacy of the treatment may be evaluated by the plasma viral RNA load of a "treated" human before and after the treatment, if it is reduced by at least about 10%, 20%, 30%, 40%, 50%, more preferably by at least about 70%, yet more preferably by at least about 75% or 80% or 85% or 90% or 95% or 98% or 99%, or even more (99.5%, 99.8%, 99.9%, 100%) the treatment is considered as effective, and/or by the monitoring of CD4 cell count before and after the treatment, if the absolute count of CD4 cell is increased by at least about 5%, 10%, 15%, 20%, 25% , more preferably by at least about 30%, yet more preferably by at least about 35% or 40% or 45% or 50% or 55% or 60% or 65%, or even more the treatment is considered as effective. As used herein, the terms "treatment", "treat" and "treating," with reference to a disease, pathological condition or symptom, also refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. A therapeutic treatment is a treatment administered to a subject after signs and symptoms of the disease have developed. A prophylactic treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs, for the purpose of decreasing the risk of developing pathology. Also, a prophylactic treatment of a HIV or SIV infection in a subject refers to a treatment that allows the subject to become an elite controller (EC) i.e. to have a relatively high CD4+ T cell count (such as e.g. superior to 500 CD4+ T cells per microliter) and/or to maintain clinically undetectable plasma HIV-1 RNA level (such as e.g. HIV RNA <50 copies/mL) during a prolonged period of time in the absence of any antiretroviral treatment (ART). A prophylactic treatment is a treatment administered to a subject suffering from a disease for the purpose of curing the disease, i.e. of making any sign of the disease disappear or becoming undetectable.
As used herein, the term "vaccine" refers to an immunogenic product or composition that can be administered to a mammal, such as a human, to confer immunity, such as passive or active immunity, to a disease or other pathological condition. Vaccines can be used preventively or therapeutically, either prophylactically or curatively. Thus, vaccines can be used to reduce the likelihood of developing a disease (such as infection) or to reduce the severity of symptoms of a disease or condition, limit the progression of the disease or condition (such as infection), or limit the recurrence of a disease or condition.
The term "virus" refers to microscopic infectious organism that reproduces inside living cells. A virus consists essentially of a core of nucleic acid (the viral genome) surrounded by a protein coat (capsid), and has the ability to replicate only inside a living cell. "Viral replication" is the production of additional virus particles by the occurrence of at least one viral life cycle. A virus may subvert the host cells' normal functions, causing the cell to behave in a manner determined by the virus. For example, a viral infection may result in a cell producing a cytokine, or responding to a cytokine, when the uninfected cell does not normally do so. Particular viral species can alternatively enter into a "lysogenic" or "latent" infection. In the establishment of latency, the viral genome is replicated, but capsid proteins are not produced and assembled into viral particles.
DETAILED DESCRIPTION
The invention relates to a method for treating acquired immune deficiency syndrome (AIDS) in a subject in need thereof, comprising administering to the subject a combination comprising: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA). In one embodiment, the method comprises administering to the subject i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, and iv) an antiretroviral (ART) agent.
In another embodiment, the method comprises administering to the subject i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, and iv) an antiretroviral (ART) agent.
In another embodiment, the method comprises administering to the subject i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA). In some embodiments, the method is a method of prophylactic treatment.
In some embodiments, the method is a method of curative treatment.
The present invention further relates to a combination comprising: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) optionally, an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA). The present invention also relates to a combination for use as a medicament, wherein said combination comprises: i) a type III interferon blocking agent, ii) an interferon-alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
The present invention also relates to a combination for use in the treatment of acquired immune deficiency syndrome (AIDS) in a subject in need thereof, wherein said combination comprises: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
In one embodiment, the combination for use comprises i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, and iv) an antiretroviral (ART) agent.
In one embodiment, the combination for use comprises i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, and iv) an antiretroviral (ART) agent.
In one embodiment, the combination for use comprises i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
The present invention further relates to a kit-of-parts comprising: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) optionally, an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
The present invention further relates to a kit-of-parts for use as a medicament, wherein said kit-of-parts comprises: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) optionally, an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA). The present invention also relates to a kit-of-parts for use in the treatment of acquired immune deficiency syndrome (AIDS) in a subject in need thereof, wherein said kit-of-parts comprises: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA).
In one embodiment, the kit-of-parts for use comprises i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, and iv) an antiretroviral (ART) agent.
In one embodiment, the kit-of-parts for use comprises i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, and iv) an antiretroviral (ART) agent.
In one embodiment, the kit-of-parts for use comprises i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) an interferon-beta (IFN-b) blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA). According to the present invention, the combination or kit-of-parts as described herein is for use in the treatment of acquired immune deficiency syndrome (AIDS) in a subject in need thereof.
In one embodiment, the subject is infected with a human immunodeficiency virus (HIV), or a simian immunodeficiency virus (SIV).
Thus, the combination or kit-of-parts as described herein is also for use in the treatment of a human immunodeficiency vims (HIV) infection or a simian immunodeficiency virus (SIV) infection.
Due to the great variability in the HIV genome, which results from mutation, recombination, insertion and/or deletion, HIV has been classified in groups, subgroups, types, subtypes and genotypes. There are two major HIV groups (HIV-1 and HIV-2) and many subgroups because the HIV genome mutates constantly. The major difference between the groups and subgroups is associated with the viral envelope. HIV-1 is classified into a main group (M), said group M being divided into least nine genetically distinct subtypes. These are subtypes A, B, C, D, F, G, H, J and K. Many other subtypes resulting from in vivo recombination of the previous ones also exist (e.g., CRF). In one embodiment, the HIV antigen is related to a specific HIV group, subgroup, type, subtype or to a combination of several subtypes.
In one embodiment, the HIV vims is HIV-1 or HIV-2, preferably HIV-1. In one embodiment, the subject is infected with an HIV-1 strain or an HIV-2 strain.
Thus, the combination or kit-of-parts as described herein is also for use in the treatment of an HIV-1 strain or an HIV-2 strain.
In one embodiment, the HIV-1 vims is from group M and preferably subtype B (HXB2).
In one embodiment, the subject is a mammal, a primate, preferably a human. In an embodiment, the combination or kit-of-parts as described herein is for use in the preventive or the prophylactic treatment of acquired immune deficiency syndrome (AIDS) in a subject in need thereof.
In another embodiment, the combination or kit-of-parts as described herein is for use in the curative treatment of acquired immune deficiency syndrome (AIDS) in a subject in need thereof.
In some embodiments, the subject has already received at least one dose of at least one antiretroviral (ART) agent, or is under antiretroviral therapy or under combined antiretroviral therapy (cART) comprising at least one antiretroviral (ART) agent, before being administered the combination of the invention.
As used herein, the term "alpha interferon" (IFN-a) or “interferon-alpha” refers to a family of more than 20 related but distinct members encoded by a cluster on chromosome 9 and all bind to the same IFN receptor. Among these, the IFN-a2 have 3 recombinant variants (a2a, a2b, a2c) depending upon the cells of origin and the IFN-a2b is the predominant variant in human genome. There is evidence though that each subtype has a different binding capacity to the IFNAR, modulating the signaling transduction events and the biological effects in the target cells.
In one embodiment, the interferon- alpha blocking agent described herein is an agent neutralizing circulating IFN-a and/or an agent blocking IFN-a signaling, and/or an agent depleting IFN-a producing cells, and/or an agent blocking IFN-a production.
In one embodiment, the interferon- alpha blocking agent described herein comprises at least one agent selected from: an agent neutralizing circulating IFN-a and/or an agent blocking IFN-a signaling, and/or an agent depleting IFN-a producing cells, and/or an agent blocking IFN-a production. In one embodiment, the agent neutralizing circulating IFN-a and/or the agent blocking IFN-a signaling, and/or the agent depleting IFN-a producing cells, and/or the agent blocking IFN-a production is/are an IFN-a antagonist. In some embodiments, the interferon-alpha blocking agent is selected from the group consisting of : an agent neutralizing circulating alpha interferon, an agent blocking interferon- alpha signaling, an agent depleting IFN-oc producing cells, and/or an agent blocking IFN-oc production, wherein the agent neutralizing circulating alpha interferon is selected from the group comprising active anti-IFN-oc vaccine including IFN-a-kinoid, IFN-a DNA-based or IFN-a RNA-based vaccine, or passive anti-IFN-oc vaccine including anti-IFN-oc antibodies or anti-IFN-oc hyper-immune serum, wherein the blocking agent of interferon- alpha signaling is selected from the group consisting of an active anti-IFNARl or anti-IFNAR2 vaccine, such as an IFNAR1 or IFNAR2 DNA-based or an IFNAR1 or IFNAR2 RNA-based vaccine, a passive anti-IFNARl or anti-IFNAR2 vaccine, such as an anti-type I interferon R1 or R2 antibody, and an IFN-a endogenous regulator, such as SOSC1 or an aryl hydrocarbon receptor, wherein the agent depleting IFN-a producing cells is an agent depleting plasmacytoid dendritic cells (pDCs), and wherein the agent blocking IFN-a production is an agent blocking the production of IFN-a by pDCs. In some embodiments, the interferon- alpha blocking agent is an agent neutralizing circulating alpha interferon selected from the group consisting of active anti-IFN-a vaccine including IFN-a-kinoid, IFN-a DNA-based or IFN-a RNA-based vaccine, or passive anti-IFN-a vaccine including anti-IFN-a antibodies or anti-IFN-a hyper-immune serum, and the blocking agent of interferon- alpha signaling is selected from the group consisting of an active anti-IFNARl or anti-IFNAR2 vaccine, such as an IFNARl or IFNAR2 DNA-based or an IFNAR1 or IFNAR2 RNA-based vaccine, a passive anti-IFNARl or anti-IFNAR2 vaccine, such as an anti-type I interferon R1 or R2 antibody, and an IFN-a endogenous regulator, such as SOSC1 or an aryl hydrocarbon receptor. As used herein, the term "alpha interferon antagonist" refers to a substance which interferes with or inhibits the IFN-a biological activity. "IFN-a biological activity" as used herein refers to any activity occurring as a result of IFN-a binding to its receptor IFNAR (IFNARl /IFNAR2 heterodimer). Such binding can, for example, activate the JAK-STAT signaling cascade, and trigger tyrosine phosphorylation of a number of proteins including JAKs, TYK2, STAT proteins. Thus, the blocking agent of interferon- alpha signaling can neutralize the fixation of the INF-oc to its receptor and/or block the signaling cascade induced by the binding of IFN-oc to its receptor. In some embodiments, the IFN-oc antagonist is selected from the group of active anti-IFN-oc vaccine (e.g., IFN- a-kinoid, IFN-a DNA-based or IFN-a RNA-based vaccine) or passive anti-IFN-oc vaccine
(e.g., anti-IFN-oc antibody or anti-IFN-oc hyper-immune serum). See for example Noel el al. (2018). Cytokine Growth Factor Rev 40:99-112.
In one embodiment, the agent neutralizing circulating IFN-a is a passive anti-IFN-a vaccine, such as an anti-IFN-a antibody or an anti-IFN-a hyper-immune serum. In one embodiment, the agent neutralizing circulating IFN-a is an anti-IFN-a antibody, preferably a neutralizing antibody. The anti-IFN-a antibody may be a monoclonal or a polyclonal antibody, and is preferably a monoclonal antibody.
Examples of anti-IFN-a antibodies include, without limitation, Sifalimumab, Rontalizumab, MMHA-1 clone, MMHA-2 clone, MMHA-6 clone, MMHA-8 clone, MMHA-9 clone, MMHA-11 clone, MMHA-13 clone and MMHA-17 clone.
In one embodiment, the agent neutralizing circulating IFN-a is an anti-IFN-a hyper-immune serum.
In one embodiment, the agent neutralizing circulating IFN-a described herein is an IFN-a ligand inhibitor. In one embodiment, the agent neutralizing circulating IFN-a is a soluble receptor that binds IFN-a.
In another embodiment, the agent neutralizing circulating IFN-a is an active anti-IFN-a vaccine.
An “active anti-IFN-a vaccine” designates a compound or composition that is capable of inducing the production of anti-IFN-a antibodies. For instance, an “active anti-IFN-a vaccine” designates a compound or composition which, upon administration to a subject, is capable of inducing the production of anti-IFN-a auto-antibodies by said subject.
The active ingredient of the active anti-IFN-a vaccine may be a polypeptide, a protein, a DNA or an RNA molecule. For instance, the active ingredient of the active anti-IFN-a vaccine is an IFN-a-kinoid. A kinoid is an inactivated and/or non-toxic IFN derivative with immunogenic properties. Usually, it is in the form of a heterocomplex obtained by chemical binding of the IFN derivative to a carrier.
The kinoid may be used as an immunogen capable of inducing high affinity auto- antibodies against a given IFN. Immunization with kinoids thus induce high titers of neutralizing antibodies directed against the corresponding IFN.
In one embodiment, the agent neutralizing circulating IFN- a described herein is an IFN- a-kinoid, such as, for example, Antiferon®.
The active ingredient of the active anti-IFN-a vaccine may also be a DNA molecule, for instance part(s) of or full-length IFN-a DNA, or an RNA molecule, for instance part(s) of or full-length IFN-a RNA.
The active anti-IFN-a vaccine may induce the production of antibodies that binds to one IFN-a subtype or to several IFN-a subtypes. For instance, the RNA molecule used in the IFN-a RNA-based vaccine may be specific of one IFN-a subtype. Alternatively, the active anti-IFN-a vaccine may comprise at least two RNA molecules corresponding to at least two IFN-a subtypes.
In another embodiment, the interferon- alpha blocking agent is an agent blocking IFN- a signaling.
In one embodiment, the agent blocking IFN-a signaling is an agent that antagonizes the type I IFN signaling pathway. In one embodiment, the agent blocking IFN-oc signaling described herein is an IFNAR antagonist.
In one embodiment, the agent blocking IFN-oc signaling is an IFNAR1 antagonist. In another embodiment, the agent blocking IFN-oc signaling is an IFNAR2 antagonist. In one embodiment, the agent blocking IFN-oc signaling is a passive anti-IFNARl or anti-IFNAR2 vaccine, such as an anti-type I interferon R1 or R2 antibody.
In one embodiment, the agent blocking IFN-oc signaling is an antibody that binds to IFNAR 1 or IFNAR2.
In another embodiment, the agent blocking IFN-oc signaling is an active anti-IFNARl or anti-IFNAR2 vaccine, such as an IFNAR1 or IFNAR2 DNA-based or an IFNAR1 or IFNAR2 RNA-based vaccine.
An “active anti-IFNARl or anti-IFNAR2 vaccine” designates a compound or composition that is capable of inducing the production of anti-IFNARl or anti-IFNAR2 antibodies. For instance, an “active anti-IFNARl or anti-IFNAR2 vaccine” designates a compound or composition which, upon administration to a subject, is capable of inducing the production of anti-IFNARl or anti-IFNAR2 auto-antibodies by said subject.
The active ingredient of the active anti-IFNARl or anti-IFNAR2 vaccine may be a DNA molecule, for instance part(s) of or full-length IFNAR1 or IFNAR2 DNA, or an RNA molecule, for instance part(s) of or full-length IFNAR 1 or IFNAR2 RNA. In one embodiment, the agent blocking IFN-oc signaling can be an inhibitor of type I IFN signaling pathway. Type I IFN signaling pathway inhibitors are well known in the art and include, without limitation, JAK1/2/3 inhibitors and STAT inhibitors. Accordingly, in one embodiment, the agent blocking IFN-oc signaling is selected from JAK1/2/3 inhibitors, STAT inhibitors, and Tyrosine Kinase 2 (TYK2) inhibitors. Non-limiting examples of JAK1/2/3 inhibitors include Ruxolitinib, Tofacitinib and Baricitinib. Non-limiting examples of TYK2 inhibitors include the BMS-986165 inhibitor. In one embodiment, the agent blocking IFN-oc signaling can be an endogenous negative regulator of type I IFN signaling pathway. Endogenous negative regulators are well known in the art and include, without limitation, SOCSl/3, F0X03, Aryl hydrocarbon Receptor (AhR) or other negative regulators. Accordingly, in one embodiment, the agent blocking interferon signaling is selected from SOCSl/3, F0X03 or Aryl hydrocarbon Receptor (AhR).
In one embodiment, the agent blocking IFN-oc signaling is a PASylated antagonist. PASylated antagonist of type I IFN are known in the art, see for example Nganou-Makamdop et al. (2018). PLoS Pathog 14(8): el007246. In one embodiment, the IFN- a antagonist described herein is an agent depleting IFN-oc producing cells.
As used herein, the term “IFN-OC producing cells” refers to any cell that produce IFN-oc. In particular, it is well known in the art that the plasmacytoid dendritic cells (pDCs) are the main producer of IFN-oc. Thus, in one embodiment, the agent depleting IFN- a producing cells depletes pDCs.
In one embodiment, the agent depleting IFN- a producing cells is an antibody. In one embodiment, the antibody depletes pDCs, such as, for example, an anti-CD 123 antibody (i.e., anti-IL-3RA).
In one embodiment, the IFN- a antagonist described herein is an agent that blocks the production of IFN- a.
In one embodiment, the agent that blocks the production of the IFN-oc is an antibody. In one embodiment, the antibody blocks the production of IFN-oc by pDCs. Said antibody can be, for example, an anti-BDCA2 (Blood DC Antigen 2) antibody. In some embodiments, the interferon-alpha blocking agent is selected from the group consisting of: an anti-IFN-oc antibody, preferably Sifalimumab, Rontalizumab, MMHA-1 clone, MMHA-2 clone, MMHA-6 clone, MMHA-8 clone, MMHA-9 clone, MMHA-1 1 clone, MMHA-13 clone or MMHA-17 clone, an anti-IFN-oc hyper-immune serum, an IFN-a-kinoid, such as e.g. Antiferon®, an IFN-a DNA-based or an IFN-a RNA-based vaccine, a soluble receptor that binds IFN-a, - an IFNAR1 or IFNAR2 antagonist, preferably an antibody that binds to IFNAR1 or
IFNAR2, an IFNAR1 or IFNAR2 DNA-based or an IFNAR1 or IFNAR2 RNA-based vaccine, a type I IFN signaling pathway inhibitors selected from a STAT inhibitor, a JAK1/2/3 inhibitor, such as e.g. Ruxolitinib, Tofacitinib or Baricitinib, and a TYK2 inhibitor, such as e.g. BMS-986165, an endogenous negative regulator of type I IFN signaling pathway selected from SOCSl/3, F0X03, Aryl hydrocarbon Receptor (AhR) or another negative regulator, a PASylated antagonist, an antibody depleting pDCs, preferably an anti-CD 123 (i.e. anti-IL-3RA) antibody, - an antibody blocking the production of IFN-a by pDCs, preferably an anti-BDCA2
(Blood DC Antigen 2) antibody.
In one embodiment, the interferon-alpha blocking agent is an anti-IFN-a antibody, preferably a monoclonal antibody, more preferably a neutralizing antibody.
As used herein, the term "type III interferon", also called interferon-lambda (IFN-l), refers to naturally occurring and/or recombinant cytokines of the type III interferon-lambda family. There are four IFN-l members in humans, IFN-/J/IL-29, IFN-k2/IL-28A, IFN-k3/IL-28B, IFN-/,4.
In one embodiment, the type III interferon is IFN-l. In one embodiment, the IFN-l refers to at least one IFN-l subtype, i.e. IFN-lI, IFN-k2 IRN-l3, IFN- 4.
In one embodiment, the human IFN-lI has the following accession number NP_742152.1. In one embodiment, the human IFN-k2 has the following accession number NP_742150.1. In one embodiment, the human IFN-/3 has the following accession numbers NP_001333866.1 (isoform 1) or NP_742151.2 (isoform 2). In one embodiment, the human IFN-k4 has the following accession number NP_001263183.2.
In some embodiments, the type III interferon to be blocked is a mucosal type III interferon or a mucosal IFN-l. In one embodiment, the interferon-lambda blocking agent described herein is an agent neutralizing circulating IFN-l and/or an agent blocking IFN-l signaling.
Preferably, the interferon-lambda blocking agent is an agent neutralizing mucosal IFN- l and/or an agent blocking mucosal IFN-l signaling.
In one embodiment, the agent neutralizing circulating IFN-l and/or the agent blocking IFN-l signaling is/are an IFN-l antagonist.
In some embodiments, the at least one type III interferon blocking agent is:
- an agent neutralizing circulating IFN-l selected from the group comprising an active anti-IFN-l vaccine, such as an IFN-k-kinoid, IFN-l DNA-based or IFN-l RNA-based vaccine, and a passive anti-IFN-l vaccine, such as an anti-IFN-l antibody or an anti-IFN- l hyper-immune serum, or
- an agent blocking type III interferon signaling selected from the group consisting of an active anti-IFNLRl vaccine, such as an IFNLR1 DNA-based or IFNLR1 RNA-based vaccine, and a passive anti-IFNLRl vaccine, such as an antibody that binds to the IFNLR1 receptor. As used herein, the term "lambda interferon antagonist" refers to a substance which interferes with or inhibits the IFN-l biological activity. "IFN-l biological activity" as used herein refers to any activity occurring as a result of IFN-l binding to its receptor IFNLR (IFNLR1/IL10R2 heterodimer). Thus, the signaling blocking agent of interferon can neutralize the fixation of the INF-l to its receptor and/or block the signaling cascade induced by the binding of IFN-l to its receptor. In some embodiments, the IFN-l antagonist is selected from the group of active anti-IFN-l vaccine ( e.g ., IFN-k-kinoid, IFN-l DNA-based or IFN-l RNA-based vaccine) or passive anti-
IFN-l vaccine (e.g., anti-IFN-l antibody or anti-IFN-l hyper- immune serum).
In one embodiment, the agent neutralizing circulating IFN-l is a passive anti-IFN-l vaccine, such as an anti-IFN-l antibody or an anti-IFN-l hyper- immune serum.
In one embodiment, the agent neutralizing circulating IFN-l is an anti-IFN-l antibody, preferably a neutralizing antibody.
The anti-interferon-lambda antibody may be a monoclonal or a polyclonal antibody, and is preferably a monoclonal antibody.
Non limiting examples of neutralizing anti-interferon-lambda antibodies include: the monoclonal anti-IL-29 (IFN-lI) antibody clone 6A11 (Invivogen), - the monoclonal anti-human IL-29 (IFN-lI) antibody clone #247801 (R&D systems), the monoclonal anti-IL-28A (IFN-k2) antibody clone 21C3 (Invivogen), the monoclonal anti-IL-28 A (IFN-k2) antibody clone MMHL-2 (PBL assay sciences), - the monoclonal anti-human IL-28A (IFN-k2) antibody Clone #248526
(R&D systems), the polyclonal anti-human IL-28A (IFN-k2) antibody (R&D systems), the monoclonal anti IL-28B (IFN-k3) antibody clone 18F4 (Invivogen), and the monoclonal anti-IL-28B (IFN-k3) antibody clone MMHL-3 (PBL assay sciences).
In one embodiment, the agent neutralizing circulating IFN-l is an anti-IFN-l hyper-immune serum. In one embodiment, the agent neutralizing circulating IFN-l described herein is an IFN-l ligand inhibitor.
In one embodiment, the agent neutralizing circulating IFN-l is a soluble receptor that binds IFN-l. In another embodiment, the agent neutralizing circulating IFN-l is an active anti-IFN-l vaccine.
An “active anti-IFN-l vaccine” designates a compound or composition that is capable of inducing the production of anti-IFN-l antibodies. For instance, an “active anti-IFN-l vaccine” designates a compound or composition which, upon administration to a subject, is capable of inducing the production of anti-IFN-l auto-antibodies by said subject.
The active ingredient of the active anti-IFN-l vaccine may be a polypeptide, a protein, a DNA or an RNA molecule.
For instance, the active ingredient of the active anti-IFN-l vaccine is an IFN-k-kinoid. A kinoid is an inactivated and/or non-toxic IFN derivative with immunogenic properties. Usually, it is in the form of a heterocomplex obtained by chemical binding of the IFN derivative to a carrier.
The kinoid may be used as an immunogen capable of inducing high affinity auto antibodies against a given IFN. Immunization with kinoids thus induce high titers of neutralizing antibodies directed against the corresponding IFN. The active ingredient of the active anti-IFN-l vaccine may also be a DNA molecule, for instance part(s) of or full-length IFN-l DNA, or an RNA molecule, for instance part(s) of or full-length IFN-l RNA.
The active anti-IFN-l vaccine may induce the production of antibodies that binds to one IFN-l subtype or to several IFN-l subtypes selected from the group comprising IFN-lI, IFN-k2, IFN-/3 and IFN-k4. For instance, the RNA molecule used in the IFN-l RNA- based vaccine may be specific of one IFN-l subtype, i.e. specific of IFN-lI, of IFN-k2, of IFN-/J or of IFN- 4. Alternatively, the active anti-IFN-l vaccine may comprise at least two RNA molecules corresponding to at least two IFN-l subtypes selected from the group comprising IFN-lI, IRN-l2, IFN-/J and IFN- 4.
In another embodiment, the type III interferon blocking agent is an agent blocking type III interferon signaling.
In one embodiment, the agent blocking IFN-l signaling is an agent that antagonizes the type III IFN signaling pathway.
In one embodiment, the agent blocking IFN-l signaling described herein is an IFNLR antagonist. In one embodiment, the agent blocking IFN-l signaling is an IFNLR 1 antagonist. In another embodiment, the agent blocking IFN-l signaling is an IL10R2 antagonist.
In one embodiment, the agent blocking type III interferon signaling is a passive anti- IFNLR1 vaccine, such as an antibody that binds to the IFNLR 1 receptor.
In one embodiment, the agent blocking type III interferon signaling is an antibody. Such antibody can block or inhibit the biological effects of type III interferon and/or block or inhibit the type III interferon signaling pathway. For example, such antibody may bind to an epitope on the interferon-lambda receptor, impeding the binding of interferon- lambda to its receptor and thus the receptor signaling subsequent activation. The heterodimeric receptor complex of interferon-lambda (IFNLR) comprises IFNLR1 (IFNLRA, IL-28RA), and IL10R2 (IL-10RB). IFNLR 1 confers ligand specificity and enables receptor assembly, while IL10R2 is shared with IL-10 family members and is required for signaling.
Thus, in one embodiment, the agent blocking type III interferon signaling is an antibody that binds to IFNLR 1 or to IL10R2. In one embodiment, the agent blocking type III interferon signaling is an antibody that binds to the IFNLR 1 receptor. Non- limiting examples of antibodies that bind to the IFNLR1 receptor include the clone MMHLR-1 (Pbl assay sciences) and the MHLICR2A1 antibody (Creative Biolabs).
In another embodiment, the agent blocking type III interferon signaling is an active anti- IFNLRl vaccine, such as an IFNLR1 DNA-based or IFNLR1 RNA-based vaccine. An “active anti-IFNLRl vaccine” designates a compound or composition that is capable of inducing the production of anti-IFNLRl antibodies. For instance, an “active anti- IFNLRl vaccine” designates a compound or composition which, upon administration to a subject, is capable of inducing the production of anti-IFNLRl auto-antibodies by said subject. The active ingredient of the active anti-IFNLRl vaccine may be a DNA molecule, for instance part(s) of or full-length IFNLR1 DNA, or an RNA molecule, for instance part(s) of or full-length IFNLR1 RNA.
In another embodiment, the type III interferon blocking agent is a small chemical molecule entity (such as, for example, a chemical entity with a molecular weight less than 900 Daltons). Methods of screening chemical libraries to identify small chemical molecule entities which may be potential drug candidates are known in the art. For example, a chemical library may be tested in a ligand-receptor binding assay. For instance, the small chemical molecule may bind and block the IFNLR1 receptor.
In some embodiments, the type III interferon blocking agent is selected from the group consisting of: an anti-IFN-l antibody, preferably clone 6A11, clone #247801, clone 21C3, clone MMHL-2, clone #248526, clone 18F4, or clone MMHL-3. an anti-IFN-l hyper-immune serum, an IFN- -kinoid, - an IFN-l DNA-based or an IFN-l RNA-based vaccine, a soluble receptor that binds IFN-l, an IFNLR1 or IL10R2 antagonist, preferably an antibody that binds to IFNLR1 or IL10R2, more preferably an antibody that binds to IFNLR1 such as clone MMHLR- 1 or the MHLICR2A1 antibody, an IFNLR1 DNA-based or IFNLR1 RNA-based vaccine, and - a small chemical molecule that binds to IFNLR1.
As used herein, the term "beta interferon" (IFN-b) or “interferon-beta” refers to a family of two related but distinct members IFN-bI and IRN-b3. They both bind to the same IFN receptor, the IFN-a receptor (IFNAR), which is a cell surface receptor complex consisting of 2 chains: IFNAR1 and IFNAR2 (IFNAR 1 /IFN AR2 heterodimer). Binding of IFN-b to the IFNAR receptor triggers signaling transduction events and biological effects in the target cell.
In one embodiment, the interferon-beta blocking agent described herein is an agent neutralizing circulating IFN-b and/or an agent blocking IFN-b signaling.
In one embodiment, the interferon-beta blocking agent described herein comprises at least one agent selected from an agent neutralizing circulating IFN-b and an agent blocking IFN-b signaling.
In one embodiment, the agent neutralizing circulating IFN-b and/or the agent blocking IFN-b signaling is/are an IFN-b antagonist.
As used herein, the term "beta interferon antagonist" refers to a substance which interferes with or inhibits the IFN-b biological activity. " IFN-b biological activity" as used herein refers to any activity occurring as a result of IFN-b binding to its receptor
IFNAR (IFNAR 1 /IFN AR2 heterodimer). Such binding can, for example, activate the JAK-STAT signaling cascade, and trigger tyrosine phosphorylation of a number of proteins including JAKs, TYK2, STAT proteins. Thus, the signaling blocking agent of interferon can neutralize the fixation of the IFN-b to its receptor and/or block the signaling cascade induced by the binding of IFN-b to its receptor. In some embodiments, the IFN-b antagonist is selected from the group of active anti-IFN-b vaccine ( e.g ., IFN-P-kinoid, IFN-b DNA-based or IFN-b RNA-based vaccine) or passive anti- IFN-b vaccine (e.g., anti-IFN-b antibody or anti-IFN-b hyper-immune serum).
In one embodiment, the agent neutralizing circulating IFN-b is a passive anti-IFN-b vaccine, such as an anti-IFN-b antibody or an anti-IFN-b hyper-immune serum. In one embodiment, the interferon-beta blocking agent is an agent neutralizing circulating IFN-b, wherein the agent neutralizing circulating IFN-b is an anti-IFN-b antibody or anti-IFN-b hyper-immune serum.
In one embodiment, the agent neutralizing circulating IFN-b is an anti-IFN-b antibody, preferably a neutralizing antibody. The anti-IFN-b antibody may be a monoclonal or a polyclonal antibody, and is preferably a monoclonal antibody.
Non-limiting examples of anti-IFN-b antibodies include: the neutralizing monoclonal antibody against human IFN-beta, clone 10B10 (Invivogen) the polyclonal anti-human IFN-beta antibodies (R&D systems) - the monoclonal anti-human IFN-beta antibodies clone #76703, clone #MMHB-3 and clone #937912 (R&D systems) the neutralizing polyclonal anti-human IFN-beta goat IgG (PBL assay sciences).
In one embodiment, the agent neutralizing circulating IFN-b is an anti-IFN-b hyper-immune serum. In one embodiment, the agent neutralizing circulating IFN-b described herein is an IFN-b ligand inhibitor.
In one embodiment, the agent neutralizing circulating IFN-b is a soluble receptor that binds IFN-b.
In another embodiment, the interferon-beta blocking agent is an active anti-IFN-b vaccine. An “active anti-IFN-b vaccine” designates a compound or composition that is capable of inducing the production of anti-IFN-b antibodies. For instance, an “active anti-IFN-b vaccine” designates a compound or composition which, upon administration to a subject, is capable of inducing the production of anti-IFN-b auto-antibodies by said subject. The active ingredient of the active anti-IFN-b vaccine may be a polypeptide, a protein, a DNA or an RNA molecule.
For instance, the active ingredient of the active anti-IFN-b vaccine is an IFN^-kinoid. A kinoid is an inactivated and/or non-toxic IFN derivative with immunogenic properties. Usually, it is in the form of a heterocomplex obtained by chemical binding of the IFN derivative to a carrier.
The kinoid may be used as an immunogen capable of inducing high affinity auto antibodies against a given IFN. Immunization with kinoids thus induce high titers of neutralizing antibodies directed against the corresponding IFN.
The active ingredient of the active anti-IFN-b vaccine may also be a DNA molecule, for instance part(s) of or full-length IFN-b DNA, or an RNA molecule, for instance part(s) of or full-length IFN-b RNA.
In one embodiment, the interferon-beta blocking agent is an agent blocking IFN-b signaling, wherein the blocking agent of IFN-b signaling is selected from the group consisting of anti-type I interferon R1 or R2 antibodies, SOSC1 and aryl hydrocarbon receptors.
In one embodiment, the agent blocking IFN-b signaling described herein is an IFNAR antagonist. In one embodiment, the agent blocking IFN-b signaling is an IFNAR1 antagonist. In another embodiment, the agent blocking IFN-b signaling is an IFNAR2 antagonist. In one embodiment, the agent blocking IFN-b signaling is an antibody that binds to IFNAR 1 or IFNAR2. In one embodiment, the agent blocking IFN-b signaling is an agent that antagonizes the type I IFN signaling pathway.
In one embodiment, the agent blocking IFN-b signaling can be an inhibitor of type I IFN signaling pathway. Type I IFN signaling pathway inhibitors are well known in the art and include, without limitation, JAK1/2/3 inhibitors and STAT inhibitors. Accordingly, in one embodiment, the agent blocking IFN-b signaling is selected from JAK1/2/3 inhibitors, STAT inhibitors, and Tyrosine Kinase 2 (TYK2) inhibitors. Non-limiting examples of JAK1/2/3 inhibitors include Ruxolitinib, Tofacitinib and Baricitinib. Non-limiting examples of TYK2 inhibitors include the BMS-986165 inhibitor. In one embodiment, the agent blocking IFN-b signaling can be an endogenous negative regulator of type I IFN signaling pathway. Endogenous negative regulators are well known in the art and include, without limitation, SOCSl/3, F0X03, Aryl hydrocarbon Receptor (AhR) or other negative regulators. Accordingly, in one embodiment, the agent blocking interferon signaling is selected from SOCSl/3, F0X03 or Aryl hydrocarbon Receptor (AhR).
In one embodiment, the agent blocking IFN-b signaling is a PASylated antagonist. PASylated antagonist of type I IFN are known in the art, see for example Nganou-Makamdop et al. (2018). PLoS Pathog 14(8): el007246.
As used herein, the term "antiretroviral therapy" or "ART" or "highly active antiretroviral therapy" or "HAART" refers to any combination of antiretroviral (ARV) drugs to maximally suppress the HIV virus ( e.g reduce viral load reduce HIV multiplication...), and stop the progression of HIV disease. There are several classes of HIV drug, such as, for example, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), post- attachment inhibitors (or entry inhibitors), protease inhibitors (Pis), CCR5 antagonists, integrase strand transfer inhibitors (INSTIs), fusion inhibitors (FIs).
In one embodiment, the antiretroviral (ART) agent is selected from the group consisting of Nucleoside reverse transcriptase inhibitors (NRTIs), Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Protease inhibitors (Pis), Integrase inhibitors (INSTIs), Fusion inhibitors (FIs), Chemokine receptor antagonists (CCR5 antagonists) and Entry inhibitors (CD4-directed post-attachment inhibitors).
Non-limiting examples of antiretroviral (ART) agents include:
Nucleoside reverse transcriptase inhibitors (NRTIs) such as e.g.: o Abacavir (Ziagen®) o Didanosine (Videx®, Videx® EC) o Emtricitabine (Emtriva) o Lamivudine (Epivir®) o Stavudine (Zerit®) o Tenofovir disoproxil fumarate DF (Viread®) o Tenofovir alafenamide AF o Zidovudine (Retrovir®)
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) such as e.g. : o Delavirdine (Rescriptor®) o Efavirenz (Sustiva®) o Etravirine (Intelence®) o Nevirapine (Viramune®, Viramune® XR) o Rilpivirine (Edurant®) o Doravirine (Pifeltro®)
Protease inhibitors (Pis) such as e.g.: o Atazanavir (Reyataz®) o Darunavir (Prezista®) o Fosamprenavir (Lexiva®) o Indinavir (Crixivan®) o Lopinavir/ritonavir (Kaletra®) o Nelfinavir (Viracept®) o Ritonavir (Norvir®) o Saquinavir (Invirase®) o Tipranavir (Aptivus®)
Integrase inhibitors (INSTIs) such as e.g.: o Raltegravir (Isentress®, Isentress® HD) o Dolutegravir (Tivicay®) o Elvitegravir (Vitekta®)
Chemokine receptor antagonist (CCR5 antagonist) such as e.g. o Maraviroc (Selzentry®) - Fusion inhibitor (FI) such as e.g. o Enfuvirtide (Fuzeon®)
Entry inhibitor such as e.g. o Ibalizumab (Trogarzo®) and any combination thereof. In one embodiment, the ART agent according to the invention comprises several ART agents, or is a combination of several ART agents. These several ART agents are for instance chosen among the ART agents listed hereabove.
Generally, initial treatment regimens usually include two NTRIs combined with a third active antiretroviral drug, which may be in the INSTI, NNRTI, or PI class. They may sometimes include a booster, which may be cobicistat (Tybost®) or ritonavir (Norvir®).
In one embodiment, the ART agent according to the invention comprises a combination of at least two ART agents, preferably chosen among the ART agents listed hereabove.
In one embodiment, the ART agent according to the invention comprises a combination of two, three, four, five or six ART agents, preferably chosen among the ART agents listed hereabove.
ART combination products are known in the art and some of them are approved as complete daily regimens.
Non-limiting examples of antiretroviral (ART) agents which are combination ARTs or combined ARTs (cARTs) include the following ART combinations: - Elvitegravir + cobicistat + emtricitabine + tenofovir DF (Stribild®)
Elvitegravir + cobicistat + emtricitabine + tenofovir AF (Genvoya®)
Darunavir + cobicistat + emtricitabine + tenofovir AF (Symtuza®)
Rilpivirine + emtricitabine + tenofovir AF (Odefsey®) Rilpivirine + emtricitabine + tenofovir DF (Complera®) Bictegravir + emtricitabine + tenofovir AF (Biktarvy®) Dolutegravir + abacavir + lamivudine (Triumeq®) Dolutegravir + rilpivirine (Juluca®)
Dolutegravir + lamivudine (Dovato®)
Efavirenz + emtricitabine + tenofovir DF (Atripla®) Efavirenz + lamivudine + tenofovir DF (Symfi®) Doravirine + lamivudine + tenofovir DF (Delstrigo®) Emtricitabine + tenofovir AF (Descovy®)
Emtricitabine + tenofovir DF (Truvada®)
Abacavir + lamivudine (Epzicom®)
Lamivudine + tenofovir DF (Cimduo®)
Abacavir + lamivudine + zidovudine (Trizivir®) Zidovudine + lamivudine (Combivir®)
Atazanavir + cobicistat (Evotaz®)
Darunavir ethanolate + cobicistat (Prezcobix®).
In some embodiments, the subject has already received at least one dose of at least one antiretroviral (ART) agent, or is under antiretroviral therapy or under combined antiretroviral therapy (cART) comprising at least one antiretroviral (ART) agent, before being administered the combination of the invention. Said at least one antiretroviral (ART) agent may be the same antiretroviral (ART) agent as the one comprised in the combination of the invention, or it may be different from the antiretroviral (ART) agent comprised in the combination of the invention.
In one embodiment, said at least one antiretroviral (ART) agent is the same antiretroviral (ART) agent as the one comprised in the combination of the invention. In this case, when being treated according to the method of the invention, the subject goes on receiving the at least one antiretroviral (ART) agent he/she has already been given before, while being further administered a type III interferon blocking agent, an interferon- alpha (IFN-a) blocking agent, optionally an interferon-beta (IFN-b) blocking agent, and optionally a latency-reversing agent (LRA). In some embodiments, the subject has already received at least one dose of a combined antiretroviral therapy (cART) before being administered the combination of the invention, and said cART comprises Nucleoside reverse transcriptase inhibitors (NRTIs), Non- nucleoside reverse transcriptase inhibitors (NNRTIs) and Protease inhibitors (Pis).
One option for eradicating HIV-1 reservoirs is based on HIV-1 reactivation in latently- infected cells while maintaining antiretroviral therapy (ART) in order to prevent spreading of the infection by the neosynthesized vims. Several latency reversing agents (LRAs) with distinct mechanistic classes have been characterized to reactivate HIV-1 viral gene expression. Some LRAs have shown their potential to reverse HIV-1 latency in order to purge latent HIV-1.
Non-limiting examples of latency reversing agent (LRA) include:
PKC agonists, which for instance act on NF-KB activation, such as e.g. Prostratin Bryostatin-1 Ingenols: Ingenol-B, Ingenol 3,20-dibenzoate (Ingenol-db), ingenol-3-angelate (ingenol mebutate, PEP005);
MAPK agonist, which for instance act on MAP Kinase activation, such as e.g. Procyanidin trimer Cl;
CCR5 antagonist, which for instance act on NF-KB activation, such as e.g. Maraviroc;
Tat vaccine, which for instance act on Activation of HIV-1 LTR, such as e.g. Tat Oyi vaccine, Tat-R5M4 protein;
SMAC mimetics, which for instance act on Induction of non-canonical NF-KB pathways, such as e.g. SBI-0637142, Birinapant;
Inducers of P-TEFb release, which for instance act on Release of P-TEFb, such as e.g. BETis: JQ1, I-BET, I-BET151, OTX015, UMB-136, MMQO, CPI-203, RVX-208, PFI-1, BI-2536 and BI-6727; HMBA;
Activators of Akt pathway, which for instance act on Upregulation of Akt signaling pathway, such as e.g. Disulfiram;
Benzotriazole derivatives, which for instance act on STAT5 activation, such as e.g. 1-hydroxybenzotriazol (HOBt); Epigenetic modifiers, which for instance act on HDAC inhibition, such as e.g. HDACis: TSA, trapoxin, SAHA, romidepsin, panobinostat, entinostat, givinostat, valproic acid, MRK-1/11, AR-42, fimepinostat, chidamide;
Epigenetic modifiers, which for instance act on Suv39Hl, G9a, SMYD2, such as e.g. HMTis: chaetocin, EPZ-6438, GSK-343, DZNEP, BIX-01294, UNC-0638; Epigenetic modifiers, which for instance act on DNMT1, 3a, 3b, such as e.g. DNMTis: 5-AzaC, 5-AzadC; and
Immunomodulatory LRAs, such as e.g. TLR agonists: TLR2 (Pam3CSK4), TLR7 (GS-9620), TLR8, TLR9 (MGN 1703) agonists; IL-15 agonist (ALT-803); Immune checkpoint inhibitors: anti-PD-1 (nivolumab, pembrolizumab), anti-CTLA-4 (ipilimumab).
In some embodiments, the latency reversing agent (LRA) is selected from the group consisting of PKC agonists, MAPK agonists, CCR5 antagonists, Tat vaccines, SMAC mimetics, inducers of P-TEFb release, activators of Akt pathway, benzotriazole derivatives, epigenetic modifiers and immunomodulatory LRAs.
In some embodiments, the latency reversing agent (LRA) is a Prostratin Bryostatin-1 Ingenol, such as Ingenol-B, Ingenol 3,20-dibenzoate (Ingenol-db), ingenol-3-angelate (ingenol mebutate, PEP005); Procyanidin trimer Cl; Maraviroc; Tat Oyi vaccine, Tat-R5M4 protein; SBI-0637142, Birinapant; a BETi such as JQ1, I-BET, I-BET151, OTX015, UMB-136, MMQO, CPI-203, RVX-208, PFI-1, BI-2536 and BI-6727 HMBA;
Disulfiram; 1-hydroxybenzotriazol (HOBt); a HDACi such as TSA, trapoxin, SAHA, romidepsin, panobinostat, entinostat, givinostat, valproic acid, MRK-1/11, AR-42, fimepinostat, chidamide; a HMTi such as chaetocin, EPZ-6438, GSK-343, DZNEP, BIX-01294, UNC-0638; a DNMTi such as 5-AzaC, 5-AzadC; a TLR agonist such as a TLR2 agonist (Pam3CSK4), a TLR7 agonist (GS-9620), a TLR8 agonist, a TLR9 agonist (MGN 1703) agonist; an IL-15 agonist (ALT- 803); and/or an immune checkpoint inhibitor such as anti-PD-1 (nivolumab, pembrolizumab), or anti-CTLA-4 (ipilimumab).
In one embodiment, the latency reversing agent according to the invention comprises several LRAs, or is a combination of several LRAs, which are for instance chosen among the LRAs listed hereabove. In one embodiment, the latency reversing agent according to the invention comprises a combination of at least two LRAs, preferably chosen among the LRAs listed hereabove.
In one embodiment, the latency reversing agent according to the invention comprises a combination of two, three, four, five or six LRAs, preferably chosen among the LRAs listed hereabove.
In one embodiment, at least one agent comprised in the combination is comprised in a composition.
In one embodiment, one, two or three agents selected from: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, are comprised in a single composition.
In one embodiment, one or two agents selected from: iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA). are comprised in a single composition.
In one embodiment, all agents comprised in the combination (i.e. agents i), ii), iii), iv) and v)) are comprised in a composition.
In some embodiments, said composition consists essentially of the at least one agent of the combination according to the invention.
As used herein, "consisting essentially of", with reference to a composition, means that the at least one agent is the only therapeutic agent or agent with a biologic activity within said composition.
In one embodiment, said composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable excipient. As used herein, the term "excipient" refers to any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. In general, the nature of the excipient will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA. In one embodiment, the excipient is an adjuvant, a stabilizer, an emulsifier, a thickener, a preservative, an antibiotic, an organic or inorganic acid or its salt, a sugar, an alcohol, an antioxidant, a diluent, a solvent, a filler, a binder, a sorbent, a buffering agent, a chelating agent, a lubricant, a coloring agent, or any other component By "pharmaceutically acceptable" is meant that the ingredients of a pharmaceutical composition are compatible with each other and not deleterious to the subject to which it is administered. Examples of pharmaceutically acceptable excipient include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like or combinations thereof. Pharmaceutically acceptable excipients that may be used in the pharmaceutical combination of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat. In one embodiment, said composition is a vaccine composition. In one embodiment, said vaccine composition further comprises at least one adjuvant.
Another object of the invention is a pharmaceutical composition comprising a combination of: i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, iii) optionally, an interferon-beta (IFN-b) blocking agent, and at least one pharmaceutically acceptable excipient, for use in the treatment of AIDS in a subject in need thereof.
Another object of the invention is a pharmaceutical composition comprising a combination of: iv) an antiretroviral (ART) agent, and v) optionally, a latency-reversing agent (LRA). and at least one pharmaceutically acceptable excipient, for use in the treatment of AIDS in a subject in need thereof. The different agents of the combination or kit-of-parts according to the invention (i.e. parts i), ii), iii), iv) and v) of the combination) are to be administered either simultaneously, separately or sequentially with respect to each other.
According to one embodiment, the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is formulated for administration to the subject. The expression “combined preparation” or “combination” refers to any preparation comprising at least two components, such as e.g. parts i), ii), iii), iv) and/or v) of the combination of the invention. The different components of the combined preparation, or of the combination, may be used simultaneously, semi-simultaneously, separately, sequentially or spaced out over a period of time so as to obtain the maximum efficacy of the combination.
For instance, they may be administered concurrently, i.e. simultaneously in time, or sequentially, i.e. one component is administered after the other one(s). After administration of the first component, the other component(s) can be administered substantially immediately thereafter or after an effective time period. The effective time period is the amount of time given for realization of maximum benefit from the administration of the components.
As a result, for the purposes of the present invention, the combined preparations or combinations are not limited to those which are obtained by physical association of the constituents, but may also be in the form of separate products permitting a separate administration, which can be simultaneous or spaced out over a period of time.
Alternatively, the different components may be co -formulated.
In one embodiment, the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention may be administered orally, intragastrically, parenterally, topically, by inhalation spray, rectally, nasally, buccally, preputially, vaginally or via an implanted reservoir.
In one embodiment, the administration of each part of the combination of the invention (i.e. agent i), ii), iii), iv) or v) of the combination of the invention) can be done by the same route of administration or by a different route of administration. In one embodiment, the administration of i) a type III interferon blocking agent, ii) an interferon- alpha (IFN-a) blocking agent, and iii) optionally, an interferon-beta (IFN-b) blocking agent, is done by a route of administration, and the administration of iv) an antiretroviral (ART) agent and v) optionally, a latency-reversing agent (LRA) is done by another route of administration.
In one embodiment, the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is in an adapted form for an oral or an intragastric administration. Thus, in one embodiment, the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered orally or intragastrically to the subject, for example as a powder, a tablet, a capsule, and the like or as a tablet formulated for extended or sustained release or as an oral solution. For instance, the antiretroviral (ART) agent and, optionally, the latency-reversing agent (LRA), are to be administered orally or intragastrically to the subject, for example as a powder, a tablet, a capsule, and the like or as a tablet formulated for extended or sustained release or as an oral solution.
Examples of forms adapted for oral or intragastric administration include, without being limited to, liquid, paste or solid compositions, and more particularly tablets, tablets formulated for extended or sustained release, capsules, pills, dragees, liquids, gels, syrups, slurries, suspensions, and the like.
In one embodiment, the antiretroviral (ART) agent and, optionally, the latency-reversing agent (LRA) are in an adapted form for an oral or intragastric administration. Thus, in one embodiment, the antiretroviral (ART) agent and, optionally, the latency-reversing agent (LRA) are to be administered orally or intragastrically to the subject, for example as a capsule or as a tablet or as an oral solution.
In another embodiment, the type III interferon blocking agent, the interferon-alpha (IFN- a) blocking agent and, optionally, the interferon-beta (IFN-b) blocking agent, are in an adapted form for an oral or intragastric administration. Thus, in one embodiment, the type III interferon blocking agent, the interferon- alpha (IFN-a) blocking agent and, optionally, the interferon-beta (IFN-b) blocking agent, are to be administered orally or intragastrically to the subject, for example as a capsule or as a tablet or as an oral solution. In one embodiment, the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is in a form adapted for parenteral administration.
In another embodiment, the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is in an adapted form for an injection such as, for example, for intravenous, subcutaneous, intramuscular, intraperitoneal intradermal, transdermal injection or infusion. Thus, the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be injected to the subject, by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion.
A sterile injectable form may be a solution or an aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic pharmaceutically acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. In one embodiment, the type III interferon blocking agent, the interferon- alpha (IFN-a) blocking agent and, optionally, the interferon-beta (IFN-b) blocking agent, are in an adapted form for a parenteral administration and/or injection. Thus, in another embodiment, the type III interferon blocking agent, the interferon- alpha (IFN-a) blocking agent and, optionally, the interferon-beta (IFN-b) blocking agent, are to be administered parenterally and/or injected to the subject, by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion, preferably by intravenous injection.
In another embodiment, the antiretroviral (ART) agent and, optionally, the latency-reversing agent (LRA) are in an adapted form for a parenteral administration and/or injection. Thus, in another embodiment, the antiretroviral (ART) agent and, optionally, the latency-reversing agent (LRA) are to be administered parenterally and/or injected to the subject, by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion, preferably by intravenous injection. Preferably, the type III interferon blocking agent, the interferon- alpha (IFN-a) blocking agent and, optionally, the interferon-beta (IFN-b) blocking agent, are in an adapted form for a parenteral administration and/or injection, and are to be administered parenterally and/or injected to the subject, by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion, preferably by intravenous injection. Preferably, the antiretroviral (ART) agent and, optionally, the latency-reversing agent (LRA) are in an adapted form for an oral or intragastric administration, and are to be administered orally or intragastrically to the subject, for example as a capsule, a tablet or an oral solution.
The different agents of the combination or kit-of-parts according to the invention (i.e. parts i), ii), iii), iv) and v) of the combination) are to be administered either simultaneously, separately or sequentially with respect to each other.
Parts i), ii), iii), iv) and v) of the combination may be administered concurrently, i.e. simultaneously in time, or sequentially, i.e. administration of certain components of the combination followed by administration of other components of the combination. After administration of the first component(s), the other component(s) can be administered substantially immediately thereafter or after an effective time period. The effective time period is the amount of time given for realization of maximum benefit from the administration of the components. In one embodiment, the type III interferon blocking agent, the interferon- alpha (IFN-a) blocking agent, optionally the interferon-beta (IFN-b) blocking agent, the antiretroviral (ART) agent and, optionally, the latency-reversing agent (LRA) are all administered at the same time.
In one embodiment, the type III interferon blocking agent, the IFN-a blocking agent, and optionally the interferon-beta (IFN-b) blocking agent are administered concurrently or simultaneously.
In one embodiment, the antiretroviral (ART) agent, and optionally the latency-reversing agent are administered concurrently or simultaneously.
In one embodiment, the type III interferon blocking agent, the interferon- alpha (IFN-a) blocking agent, and/or the interferon-beta (IFN-b) blocking agent, are to be administered prior to the antiretroviral (ART) agent and/or the latency-reversing agent (LRA).
In one embodiment, the antiretroviral (ART) agent and/or the latency-reversing agent (LRA) are to be administered prior to the type III interferon blocking agent, the interferon- alpha (IFN-a) blocking agent, and/or the interferon-beta (IFN-b) blocking agent. In one embodiment, the subject receives one or more doses (one or more takes) of the type III interferon blocking agent, the IFN-a blocking agent, and/or the interferon-beta (IFN-b) blocking agent before starting receiving all parts i), ii), iii), iv) and v) of the combination.
For instance, in a first period of time, the subject receives one or more doses (one or more takes) of the type III interferon blocking agent, the IFN-a blocking agent, and optionally the interferon-beta (IFN-b) blocking agent, during one or several weeks (e.g. 1, 2, 3, 4, 5, 6, 7, 8 or 10 weeks) in the absence of ART agent and LRA administration. Then, in a second period of time, the subject continues receiving administrations of the type III interferon blocking agent, the IFN-a blocking agent, and/or the interferon-beta (IFN-b) blocking agent, and also receives administrations of the antiretroviral (ART) agent, and optionally the latency-reversing agent.
Alternatively, in a first period of time, the subject receives one or more doses (one or more takes) of the antiretroviral (ART) agent, and optionally the latency-reversing agent, during one or several weeks or months (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 10 or 12 weeks or months) in the absence of administration of the type III interferon blocking agent, the IFN-a blocking agent, and/or the interferon-beta (IFN-b) blocking agent. Then, in a second period of time, the subject continues receiving administrations of the antiretroviral (ART) agent, and optionally the latency-reversing agent, and also receives administrations of the type III interferon blocking agent, the IFN-a blocking agent, and optionally the interferon-beta (IFN-b) blocking agent.
In one embodiment, the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered to the subject in need thereof in a therapeutically effective amount.
The term “therapeutically effective amount”, as used herein, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired preventive and/or therapeutic result.
It will be however understood that the total daily usage of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disease being treated and the severity of the disease; activity of the specific agent(s), the combination, the kit- of-parts, the pharmaceutical composition or medicament employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific agent(s), the combination, the kit-of-parts, the pharmaceutical composition or medicament employed; the duration of the treatment; drugs used in combination or coincidental with the specific agent(s), the combination, the kit-of-parts, the pharmaceutical composition or medicament employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. The total dose required for each treatment may be administered by multiple doses or in a single dose.
In one embodiment, a therapeutically effective amount of one of the agents i), ii), iii), iv) or v) of the combination of the invention ranges from about 0.1 mg/kg to about 10 mg/kg, from about 0.2 mg/kg to about 9 mg/kg, from about 0.3 mg/kg to about 8 mg/kg, from about 0.4 mg/kg to about 7.5 mg/kg, from about 0.5 mg/kg to about 7 mg/kg.
In one embodiment, a therapeutically effective amount of the type III interferon blocking agent, the interferon-alpha (IFN-a) blocking agent, or the interferon-beta (IFN-b) blocking agent (i.e. of one of the agents i), ii), iii)) ranges from about 1 mg/kg to about 10 mg/kg, from about 2 mg/kg to about 9 mg/kg, from about 3 mg/kg to about 8 mg/kg, from about 4 mg/kg to about 7 mg/kg, from about 4 mg/kg to about 6 mg/kg.
In one embodiment, a therapeutically effective amount of one of the agent i), ii), iii), iv) or v) of the combination of the invention ranges from about 1 mg to about 4000 mg, from about 10 mg to about 3000 mg, from about 25 mg to about 2000 mg, from about 50 mg to about 2000 mg, from about 100 mg to about 1500 mg, from about 200 mg to about 1000 mg.
In one embodiment, a therapeutically effective amount of the antiretroviral (ART) agent or of the latency-reversing agent (LRA) (i.e. of one of the agents iv) or v)) ranges from about 1 mg to about 4000 mg, from about 10 mg to about 3000 mg, from about 25 mg to about 2000 mg, from about 50 mg to about 2000 mg, from about 100 mg to about 1500 mg, from about 200 mg to about 1000 mg.
Usually, there is a set time interval between separate administrations of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention. While this interval varies for every subject, typically it ranges from 1 days to several weeks, and is often 1, 2, 4, 6 or 8 days, or 1, 2, 4, 6 or 8 weeks. In one embodiment, the administration regimes typically have from 1 to 20 administrations of the different parts of the combination of the invention, but may have as few as one or two or four or eight or ten. In another embodiment, the administration regime is annual, biannual or other long interval (5-10 years). In one embodiment, a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered once a day, twice a day, three times a day or more.
In one embodiment, a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered every day, every two days, every three days, every four days, every five days, every six days.
In one embodiment, a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered every week, every two weeks, every three weeks, every four weeks, every five weeks.
In one embodiment, a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered every month, every two months, every three months, every four months, every five months, every six months.
In one embodiment, a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered every 12 hours, every 24 hours, every 36 hours, every 48 hours, every 60 hours, every 72 hours, every 96 hours. In a preferred embodiment, a therapeutically effective amount of the type III interferon blocking agent, the interferon-alpha (IFN-a) blocking agent, or the interferon-beta (IFN- b) blocking agent (i.e. of one of the agents i), ii), iii)) is to be administered every week, every two weeks, every three weeks, every four weeks, every five weeks, preferably every two weeks.
In a preferred embodiment, a therapeutically effective amount of the antiretroviral (ART) agent or of the latency-reversing agent (LRA) (i.e. of one of the agents iv) or v)) is to be administered daily, for instance once a day, twice a day, or three times a day.
In a preferred embodiment, a therapeutically effective amount of the antiretroviral (ART) agent or of the latency-reversing agent (LRA) (i.e. of one of the agents iv) or v)) is a daily dose to be administered in one, two, three or more takes or in one, two, three or more injections. In a preferred embodiment, the antiretroviral (ART) agent and optionally the latency- reversing agent are administered daily, and the IFN-a blocking agent, the type III interferon blocking agent, and optionally the interferon-beta (IFN-b) blocking agent, are administered every three weeks.
In one embodiment, the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is for acute administration. Preferably, the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is for chronic administration.
In one embodiment, a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered for a period of time ranging from about two weeks to about twenty-four weeks, from about two weeks to about twelve weeks, from about two weeks to about six weeks.
In one embodiment, a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered for about 1 month, 2 months, 3 months, 6 months, 1 year or more. Alternatively, a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered to the subject until no cell containing replication-competent proviral HIV DNA is detected in a blood sample from the subject. Indeed, in one embodiment, the method of treatment of the invention further comprises assessing the presence of cells containing replication-competent proviral HIV DNA in a blood sample from the subject.
In another embodiment, the presence of cells containing replication-competent proviral HIV DNA is assessed in a blood sample from the subject. In some embodiments, a therapeutically effective amount of the agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-parts, the composition or the pharmaceutical composition of the invention is to be administered to the subject until no cell containing replication-competent proviral HIV DNA is detected in a blood sample from the subject. Cells containing replication-competent proviral HIV DNA, also called “resistant cells” or “reservoir cells” may be detected in a sample, or their quantity, level or frequency in a sample may be measured by various assays known by the person skilled in the art.
For instance, cells containing replication-competent proviral HIV DNA may be detected in a sample, or their quantity, level or frequency in a sample may be measured by ex vivo viral outgrowth assay.
For example, plasma HIV-1 RNA levels, size of the HIV reservoir and/or CD4+ T cell count may be typically monitored for instance every 2 weeks, after collection of blood samples, 1 day before administration of the interferon-blocking agents (i.e. parts i), ii) and iii) of the combination described herein). Plasma HIV-1 RNA levels may for instance be determined using the Roche COB AS AmpliPrep/COBAS TaqMan HIV-1 Assay (version 2.0) or the Roche cobas HIV-1 quantitative nucleic acid test (cobas 6800), which quantitate HIV-1 RNA over a range of 2x1o1 to lxlO7 copies/ml. Size of the HIV reservoir may for instance be assessed with Quantitative Viral Outgrowth Assay (QVOA).
QVOA may typically be performed as previously described in Huang SH et al, 2018 J Clin Invest 128:876-889. Briefly, isolated CD4+ T cells may typically be plated out in serial dilutions (e.g. 2, 1, 0.5, 0.2, and 0.1 million per well), for instance into 12 wells in 24-well plates with added phytohemagglutinin (PHA; e.g. 2 pg/ml) and irradiated HIV-negative donor PBMC (e.g. 2 x 106 cells/well) to reactivate the infected cells. MOLT-4 CCR5 cells (e.g. 2 x 106 cells/well) may typically be added after 2 days of culture, to amplify the HIV. The p24 antigen in the culture supernatant may typically be quantified after 2 weeks of culture, for instance using an HIV p24 antigen enzyme-linked immunosorbent assay (ELISA) kit (Perkin-Elmer, Hopkinton, MA). Estimated frequencies of cells with replication-competent HIV-1 may typically be calculated using limiting dilution analysis.
CD4+ T-cell counts may for instance be determined by a clinical flow cytometry assay. As a non-limiting example, administration of the interferon-blocking agents (i.e. parts i), ii) and iii) of the combination described herein) may be stopped when no cells with replication-competent HIV-1 are detected following 2-3 consecutive limiting dilution virus outgrowth assays (VOA).
As a non-limiting example, administration of the antiretroviral (ART) agent or/and the latency-reversing agent (LRA) (i.e. parts iv) and/or v) of the combination described herein) may be stopped when no cells with replication-competent HIV- 1 are detected with 3-4 consecutive limiting dilution virus outgrowth assays (VOA).
It will be apparent to the person skilled in the art that such timing in the treatment interruption is flexible. In some cases, if viral clearance was not complete, AIDS symptoms (or a detectable viral load) may appear anew after a certain time after interruption of the treatment, such as e.g. after a few months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, etc. after interruption of the treatment. In these cases, treatment with the combination of the invention may then be resumed after a certain time of treatment interruption.
Thus, in one embodiment, the combination of the invention is administered to the subject for a first period of time, then said treatment is interrupted during a period of time of a few months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 8 years or 10 years, and then the combination of the invention is administered anew to the subject for a second period of time, for instance until no cell containing replication-competent proviral HIV DNA is detected in a blood sample from the subject.
The subject may thus alternate between periods of time during which he receives administrations of the combination described herein and periods of time of treatment interruption.
The combination, the kit-of-parts, the composition or the pharmaceutical composition as described herein may be used alone.
Thus, in one embodiment, the combination, the kit-of-parts, the composition or the pharmaceutical composition as described herein is used alone and comprises a therapeutically effective dose of each part each part of the combination (i.e. agent i), ii), iii), iv) or v)).
In another embodiment, the combination, the kit-of-parts, the composition or the pharmaceutical composition as described herein is used in combination with at least one further therapeutic agent.
Such administration may be simultaneous, separate or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate. The further therapeutic agent is typically relevant for disorders to be treated. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Antiviral activity of type I and type III interferons. (A) Expression of ISGs in HepG2. HepG2 cells were treated with IFNa2a or IFN/J -4 (lO ng/ml). After 4 h of stimulation, qRT-PCR were used to examine the mRNA levels of the interferon-induced genes, IFIT1, MX1 and OASF and fold-changes was calculated by 2 DDa method as compared with non-treated cell control and using endogenous S14 mRNA level for normalization. (B) Antiviral activity of type I and III IFNs against EMCV. IFNa2a or IFNkl/2/3/4 (10 ng/ml) were added to HepG2 cells 24 h prior to challenge with EMCV. Forty-eight after infection with EMCV, cells were assayed for viability with a bioassay. A570 values were directly proportional to cell viability and therefore antiviral activity of the respective IFNs. IFN-a treatment without viral challenge was used as a baseline of the viability of the cells.
Figure 2. Anti-proliferative activity of type I and type III interferons against CD4+ T cells. CFSE-stained CD4+T cells (10xl04/well) were stimulated for 5 days in 96 round- bottomed microwells with allogeneic poly I:C matured DC in absence (control) or presence of 10 ng/ml of IFN-a2a, or IFN/J or IFNk2 or IFN/J or IFN/J. When indicated, anti-interferon type I receptor antibody was added. The percentage of CFSE dilution was evaluated by flow cytometry.
Figure 3. IFN-a2a but not IFN-type III induces the expression of ISGs in CD4+ T cells. CD4+ T cells were treated with IFNa2a or IFN/J/2/3/4 (10 ng/ml). After 4 h of stimulation, qRT-PCR were used to examine the mRNA levels of the interferon-induced genes, IFIT1, MX1 and OASF and fold-changes was calculated by 2 DDa method as compared with non-treated cell control and using endogenous S14 mRNA level for normalization.
Figure 4. IFN-a2a but not IFN-type III stimulates the phosphorylation of Statl in CD4+ T cells. CD4+ T cells were stimulated with 10 ng ml-1 of IFN-lI, IFN-/ , IFN-/J, IFN-k4, or IFN-a2a for 20 min, or were left unstimulated (control). Increases in pSTATl were evaluated as a ratio of induction over baseline levels (MFI fold change = MFI cytokine-stimulated/MFI untreated cells) Figure 5. IFN-a2a but not IFN-type III increases CD38 expression in CD3/CD28 stimulated CD4+ T cells. CFSE-stained CD4+ T cells (4xl04/well) were cultured in 96 round-bottomed microwells in the presence of ACD3-feeder (4xl04/well) and plate-bound anti-CD3 mAb (2 pg/ml), soluble anti-CD28 mAb (2 pg/ml) with increasing dose of IFN-a2a or IFN type III. CD38 Median Fluorescence Intensity (MFI) was measured by flow cytometry in CD3+ 7-AAD-CFSE+ stimulated CD4+ T cells at the end of the culture.
Figure 6. Comparison of CM CD8+ T cell distributions and serum IFN-a levels in HIV- 1-infected subjects and critical pathogenic role of IFN-a in human HIV-1 infection. (A) Comparison of CM CD8+ T cell distributions in HIV- 1 -infected subjects;
(B) Comparison of serum IFN-a levels in HIV- 1 -infected subjects; (C) Relationship between CD8+ CM frequency and serum IFN-a level in non-treated HIV patients (EC and pre-cART group).
Figure 7. Serum levels of IFN-a and IFN-l, before and after cART treatment in HIV-1 patients originating from two distinct institutes ((A) Institute 1; (B) Institute 2) and control healthy donors.
Figure 8. Schematic diagram of possible administration schedule for the combination of the invention.
Figure 9: HIV infection of NCG humanized mice induces IFN-I and IFN III production. NCG humanized mice were intraperitoneally injected with HIV-NL4.3. Blood and spleen were removed from infected and non-infected humanized NCG mice at day 2 post infection, when the virus load has reached a plateau at day 10 and at day 30. Graphs show IFN-a, IFN- 3 and IFN-lI mRNA levels in isolated PBMCS (A, B, C) and splenocytes (D, E, F) respectively. Figure 10: Activated CD4+ T cells express IFN-lambda Receptor. CD4+ T cells were cultured in presence of IL-2 without (black bars) or with anti-CD3 and anti-CD28 antibodies (1 pg/ml each) (grey bars) for 3 days. IFNLR1 (IL28RA) and IL10RB expression was determined by RT-qPCR (A, B) and the detection of fixation of IFN- 3 on cultured CD4+ T cells (C) was assessed by IFN- 3 MFI measured by Flow cytometry. IFIH1 (D) and IFI27 (E) mRNA induction in IFN- 3 -treated CD4+ T cells cultured as described above was determined by RT-qPCR.
Figure 11: IFN-k3 inhibits HIV-1 infection of activated CD4+ T cells. 3 day- CD3/CD28/IL2-stimulated CD4+ T cells were treated with the indicated reagents IFN- l3, IFN-a, anti-IFNLRl (IL28RA), anti-IFNAR2 neutralizing antibodies. 24 hours after, treated cells were infected with HIV NL4.3. Graphs show the frequency of infected cells 5 days post-infection using intracellular Gag p24 staining (n = 4 donors).
Figure 12: Sequential treatment with two latency-reversing agents, DNA methylation inhibitor 5-AzadC and HDACI MS -275, induce IFN-1 Receptor expression on CD4+ T cells. 3-day-CD3/CD28/IL2-stimulated CD4+ T cells and J-Lat 10.6 cells were mock- treated or treated with 5-AzadC for 72 h with MS-275 for the last 24 h. At 72 h post treatment, mock (black bars) and 5-AzadC/MS-275 (grey bars) CD4+ T (A, B, C, D) and J-Lat 10.6 cells (E, F, G, H) were harvested and first analyzed for their capacity to express IFNLRl (IL28RA) by RT-qPCR (A and E). Then samples were cultured with IFN- 3. The presence of IFN- 3 binding on T cells was assessed by IFN- 3 MFI measured by Flow cytometry (B and F). The T cells’ sensitivity to IFN- l3 was evaluated by the identification of IFN- stimulated gene (ISG) proteins IFIH1 (C and G) and IFI27 (D and H) by RT-qPCR.
Figure 13: The NtRTI TDF enhances the production of IFN- 3 by HT-29 cells. Colon cancer HT-29 cells were incubated with dimethyl sulfoxide (DMSO) or tenofovir disoproxil fumarate (TDF, 25 mM). After 48 hours, IFN- 3 levels in treated HT-29 cells were evaluated by ELISA.
Figure 14: Experimental scheme for assessing inhibition of HIV infection in stimulated CD4+ T cells by IFN- 3 released by TDF treated HT-29 cells. HT-29 cells were cultured in the bottom chamber of Transwell plates. When the culture was subconfluent, Tenofovir disoproxil fumarate (TDF) was added. After 24 hours of stimulation, activated CD4 T cells were deposited in the insert which is then placed in the culture well so as to be immersed in the culture medium of the HT-29 cells. When indicated, the CD4+ T cells were pre-treated with an IFNLRl (IL28RA) neutralizing antibody. After 24 hours of co- culture, the cells in the insert were harvested, infected and then placed back into the co culture for an additional 5 days of culture. The frequency of infected cells was determined by intracellular HIV p24 by FACS.
Figure 15: IFN- 3 secreted by the NtRTI TDF-treated HT-29 cells impairs CD4+ T cell infection. Experiments were performed using 12-transwell chambers with a polycarbonate filter (0.2 pm pore size). HT-29 were grown until subconfluence in the lower chamber. Then HT-29 cells were treated with tenofovir disoproxil fumarate (TDF). After 24h of TDF treatment, 3-day-CD3/CD28/IL2-stimulated CD4+ T cells were seeded in the upper chamber in presence of IL-2. When indicated, anti-IFNLRl (IL28RA) neutralizing Ab was added to the culture. After 24h of co-culture, CD4+ T cells were isolated, infected with HIV NL4.3 and added again to the upper chamber. Graphs show the frequency of infected cells 5 days post-infection using intracellular Gag p24 staining (n = 3 donors).
Figure 16: Experimental scheme for assessing the inhibition of HIV latency reversal in latently infected J-Lat cells by IFN- 3 released by TDF treated HT-29 cells. HT-29 cells were cultured in the bottom chamber of Transwell plates. When the culture was subconfluent, Tenofovir disoproxil fumarate (TDF) was added. After 24 hours of stimulation, J-Lat 10.6 cells pretreated with 5-AzadC and MS-275 are deposited in the insert which was then placed in the culture well. When indicated, the J-Lat cells were pre- incubated with an IFNLR1 (IL28RA) neutralizing antibody. After 48 hours of co-culture, HIV reactivation was monitored as the percentage of living GFP-positive cells by Flow Cytometry analysis.
Figure 17: IFN- 3 released by TDF-treated HT-29 cells inhibits in vitro HIV reactivation in latently infected J-Lat clone 10.6. Experiments were performed using 12-transwell chambers with a polycarbonate filter (0.2 pm pore size). HT-29 were grown until subconfluence in the lower chamber. Then HT-29 cells were treated with tenofovir disoproxil fumarate (TDF). After 24 h of TDF treatment, J-Lat clone 10.6 cells which had been pretreated for 72 hours with 5-AzadC and MS -275 as described above were seeded in the upper chamber. When indicated, anti-IFNLRl (IL28RA) Ab was added to the culture. After 48 hours of co-culture, HIV reactivation was measured by quantification of GFP expression by flow cytometry (n = 4 donors).
Figure 18. Schematic diagram of pre-clinical protocol for HIV-1 reservoirs elimination in HIV-1 infected humanized mice.
EXAMPLES
The present invention is further illustrated by the following examples.
Example 1: Effects of type and type III interferons on innate and adaptative immune responses
Material and Methods
Human cell lines
HCC HepG2 and normal kidney epithelial Vero cell lines were obtained from ATCC. Cells were grown in Dulbecco’s Modified Eagle Medium supplemented with 10% heat- inactivated Fetal Bovine Serum, 2 mM L-glutamine, 1% penicillin and streptomycin solution in hypoxia 2%. Cancer cell lines were grown to 70-100% confluency, subsequently passaged for a maximum of 5 times and freshly thawed thereafter. Cells were detached by means of accutase, resuspended in FBS -containing medium and collected by means of centrifugation (300g, 3min). Cell numbers were determined by means of trypan blue.
Human Blood Sample
Blood samples from healthy individuals originated from Etablissement Francais du Sang (EFS, Paris). Blood cells are collected using standard procedures.
Cell Purification and culture
Peripheral blood mononuclear cells (PBMCs) are isolated by density gradient centrifugation on Ficoll-Hypaque (Pharmacia). PBMCs are used either as fresh cells or stored frozen in liquid nitrogen. T-cell subsets and T cell-depleted accessory cells (ACD3 cells) are isolated from either fresh or frozen PBMCs. T cell-depleted accessory cells (ACD3 cells) are isolated by negative selection from PBMCs by incubation with anti- CD3-coated Dynabeads (Dynal Biotech) and are irradiated at 3000 rad (referred to as ACD3-feeder). CD4+ T cells are negatively selected from PBMCs with a CD4+ T-cell isolation kit (Miltenyi Biotec), yielding CD4+ T-cell populations at a purity of 96-99%. T cell subsets are cultured either in IMDM supplemented with 5% SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES (IMDM-5 media) in hypoxia 2%.
Freezing and thawing of cells Cells were frozen in FBS containing 10% DMSO. Cryotubes were placed in CoolCell (Biocision) freezing containers and incubated at -80°C. After 2 days tubes were transferred to liquid nitrogen and stored until required. Thawing of cells was performed by placing cryotubes in a 37°C water bath for approximately 30 seconds. Next, cell suspension was mixed with equivalent volume of pre-warmed media and subsequently transferred to falcon tubes containing the same medium. Cells were pelleted by centrifugation (300g, 3min) to remove DMSO. The cell pellet was resuspended in cell culture medium
Real-time PCRfor ISGs detection
HepG2 cells were seeded at a density of 2 x 105 cells per well in 12-well plates and incubated for 24 h. Then, fresh media was added with the indicated interferons. The cells were incubated for 4 h and then lysed, and RNA was purified using an extraction kit (Qiagen), according to the manufacturer's instructions. Synthesis of cDNA was performed using the PrimeScript RT Reagent kit (TAKARA). Quantitative PCR was carried out using the Power SYBR Green PCR Master Mix (Applied Biosystems) on a LightCycler 480 instrument (Roche). Each reaction was carried out in duplicate in a total volume of 100 pL. Primers were designed to be intron-spanning using Primer3 or Primer Express® v3.0 software (Applied Biosystems). To measure the cellular transcriptional response to IFN stimulation, 3 ISG targets, MXI, OASL and ISG15, were selected based on published results investigating the transcriptional response in IFN-stimulated PBMCs (see, for example, Waddell et al. (2010) PLoS One. 5(3):e97532). For gene induction assays, fold change values were calculated using the AACt method. The geometric mean of the Ct values of the reference genes, S14, was used as a reference value.
Virus production The virus used EMCV (FA strain) was grown on monolayers of Vero cells to complete cytopathic effect or until all cells were affected by the infection as determined by microscopy and prepared by two cycles of freezing and thawing, followed by centrifugation for 30 min at 5,000 x g for removal of cellular debris.
Antiviral assay Antiviral assays were done on HepG2 cells, which were seeded in DMEM supplemented with 10% FCS at a density of 1.5 x 104 in 96-well plates and left to settle. The cells were incubated with indicated doses of IFNs for 24 h before challenge with EMCV. The cells were incubated with virus for 48 h. The medium was removed between each step. The viability of the cells was analyzed by a bioassay based on the dehydrogenase system; this system in intact cells will convert the substrate, MTT, into formazan (blue), which in turn can be measured spectrophotometrically. Briefly, the cells were given MTT and incubated for 2 h. An extraction buffer (containing 6 to 11% sodium dodecyl sulfate and 45% N, N-dimethylformamide) was added to the cells, and the cells were then incubated overnight at 37°C. Subsequently, the absorbance at 570 nm was determined employing the extraction buffer as the blank probe. A570 was directly proportional to antiviral activity.
Flow cytometry analysis
CD3+ T cells staining: anti-CD4 (SK3)-APC, anti-CD3 (UCHTl)-FITC, anti-CD8 (RPA-T8)-BV421 are from Becton Dickinson. Cells are stained for surface markers (at 4°C in the dark for 30 min) using mixtures of Ab diluted in PBS containing 3% FBS, 2mM EDTA (FACS buffer).
STAT1 signaling analysis: Flow cytometry analysis of STAT1 phosphorylation (pSTATl) was conducted in CD4+ T cells by using BD Phosflow technology according to the manufacturer’s instructions (BD Bio-sciences, San Jose, CA). CD4+ T cells were stimulated by incubation with interferon type I and Type III at 37°C for 20 min or left untreated. Activation was stopped by fixation using BD Phosflow Lyse/Fix Buffer (BD Biosciences) and cells were permeabilized with BD Perm Buffer III (BD Biosciences). Cells were stained with antibody recognizing specific phosphorylated STAT tyrosines: p-STATl (Y701)-PE. In multiparametric immunophenotyping experiments, cells were simultaneously stained with anti-CD3-FITC and 7-AAD. Increases in pSTATl were assayed as a ratio of induction over baseline levels (MFI fold change = MFI cytokine-stimulated/MFI untreated cells) CFSE staining: CD4+ T cells were stained with 1 mM CFSE (CellTrace cell proliferation kit; Molecular Probes/Invitrogen) in PBS for 8 min at 37°C at a concentration of 1.107 cells/ml. The labeling reaction was stopped by washing twice the cell with RPMI-1640 culture medium containing 10% FBS. The cells were then re-suspended at the desired concentration and subsequently used for proliferation assays. 7-AAD staining: Apoptosis of stimulated CFSE-labeled CD4+ T was determined using the 7-AAD assay. Briefly, cultured cells were stained with 20 pg/mL nuclear dye 7-amino-actinomycin D (7-AAD; Sigma-Aldrich, St-Quentin Fallavier, France) for 30 minutes at 4°C. FSC/7-AAD dot plots distinguish living (FSChlgl77-AAD ) from apoptotic (FSChlgh/7-AAD+) cells and apoptotic bodies (FSClow/7-AAD+) and debris ((FSClow/7-AAD ). Living cells were identified as CD3+ 7-AAD-FSC+ cells.
Appropriate isotype control Abs are used for each staining combination. Samples are acquired on a BD LSR FORTESSA flow cytometer using BD FACSDIVA 8.0.1 software (Becton Dickinson). Results are expressed in percentage (%) or in mean fluorescence intensity (MFI). Functionnal assay
T cell proliferation: T cell proliferation was assessed with CFSE-dilution assays. For CFSE-dilution assay, at coculture completion, stimulated CFSE-labeled CD4+ T cells were harvested, co-stained with anti-CD3 mAh and 7-AAD, and the percentage of proliferating cells (defined as CFSE low fraction) in gated CD3+ 7-AAD cells was determined by flow cytometry.
T cell activation: CD38 Median Fluorescence Intensity (MFI) of CD38 expression was measured by flow cytometry in CD3+ 7-AAD-CFSE+ stimulated CD4+ T cells at the end of the culture. CD4+ T cell polyclonal stimulation: CFSE-stained CD4+ T cells (5xl04/well) were cultured in 96 round-bottomed microwells in the presence of ACD3 -feeder (lxl05/well) and plate -bound anti-CD3 Ab (2 pg/ml), soluble anti-CD28 mAb (2 pg/ml). CD4+ T cell proliferation was evaluated with CFSE dilution assays as described above by flow cytometry. Cells were stimulated in presence of different amounts of recombinant cytokines.
Allogeneic mixed lymphocyte reaction: CFSE-stained CD4+ T cells (5xl04/well) were cultured in 96 round-bottomed microwells in the presence of allogeneic mature DC. Proliferation of allo-activated CD4+ T cells with CFSE dilution assays as described above by flow cytometry. Cells were stimulated in presence of different amounts of recombinant cytokines.
Statl phosphorylation analysis: CD4+ T cells were stimulated with IFN-lI, IFN-k2, IFN-k3, IFN-k4, or IFN-a2a (10 ng/ml) for 20 min, or were left unstimulated (control). Phosphorylated Statl levels was assessed by flow cytometry as described above.
Results Type I interferons (IFN-a/b) and the more recently identified type III IFNs (IFN-l) function as the first line of defense against virus infection, and regulate the development of both innate and adaptive immune responses. Type III IFNs were originally identified as a novel ligand-receptor system acting in parallel with type I IFNs, but subsequent studies have provided increasing evidence for distinct roles for each IFN family. The inventors aimed to evaluate the effects of type I and type III interferons on both innate (antiviral) and adaptive immune response (CD4+T cell proliferation). Antiviral activities of types I and III
The ability of IFN type I and III to induce the expression of interferon-stimulated genes (ISGs) was analyzed by qPCR.
Briefly, the antiviral activity of type I and III was tested in HepG2 cells treated with IFN-a2a, IFN/J , IFNk2, IFN/J or IFN 4 for 4 hours. Then the induction of the well-known interferon- stimulated genes (ISGs) MX1, IFIT1 and OASL was monitored by qPCR.
As shown in Figure 1A, all five interferons clearly induced all three ISGs.
Since the investigated ISGs are functionally related to an antiviral defense, the inventors further evaluate the capacity of both IFN to protect HepG2 cells from EMCV-induced cytopathogenic effect.
Briefly, cells were seeded in a 96-well microtiter plate and treated with the indicated amount of IFNs for 24 h and then challenged with EMCV for 20 h. Cell survival was measured by an MTT coloring assay. As shown in Figure IB, IFN type III and IFN- a2a have intrinsic cellular antiviral activity and are able to fully protect HepG2 cells challenged with EMCV.
Anti-proliferative activity of type I and type III interferons against CD4+ T cells proliferation
The effect of IFN-type I and IFN type III on CD4+ T cells proliferation in response either to polyclonal or to allogeneic stimulation was evaluated in a mixed lymphocyte reaction (MLR) assay.
Briefly, CFSE labelled CD4+ T cells were first stimulated with poly I:C matured allogeneic dendritic cells in presence of different dose of IFNs. At 5 days post activation, the CFSE fluorescence dilution was analyzed. As shown in Figure 2, IFN-a2a inhibits the proliferation of stimulated CD4+ T cells, while IFN type III exhibits no ability to suppress their proliferation. Of note, when the MLR was performed in the presence of anti-interferon type I receptor antibody, CD4+ T cells exhibit a greater proliferation. Thus, IFN-type I but not IFN type III inhibit the proliferation of allo-activated CD4+ T cells.
Moreover, the analysis of mRNA levels of the interferon-induced genes (ISG), IFIT1, MX1 and OASL in IFNs treated CD4+ T cells confirmed the lack or minimal sensitivity of CD4+ T cells to interferon type III.
Indeed, as shown in Figure 3, ISGs are induced only in CD4+ T cells stimulated with IFN-a2a. Thus, IFN-a2a but not IFN-type III induce the expression of ISGs in CD4+ T cells. Because the Jak-STAT 1/2 pathway being the major regulators of the transcription of ISG, the inventors have analyzed the phosphorylation levels of Statl proteins in response to IFN-type I, or interferon type III within CD4+T cells.
As shown in Figure 4, only IFN-a2a was able to stimulate the phosphorylation of Statl within CD4+ T cells. Therefore, IFN-a2a but not IFN-type III induces tyrosine phosphorylation of STAT1 in CD4+ T cells.
Induction of chronic immune activation in presence of type I and III interferons.
Because chronic immune activation has been reasoned to be a significant contributor to disease progression in HIV- 1 -infected subjects, it is possible to monitor disease progression by measuring the expression of activation markers on CD4+ T cell surface. Thus, the inventors have evaluated, by flow cytometry, the capacity of both IFNs to increase the CD38 expression on stimulated CD4+ T cells.
As shown in Figure 5, only IFN-a2a was able to enhance the expression of CD38 on stimulated CD4+ T cells.
Collectively, these ex vivo experiments show that while exhibiting anti-viral activity, as does IFN-a, interferon type III, by contrast to the immunosuppressive IFN-a have no effect on CD4+ T cell activation and proliferation. Indeed, interferon type III do not inhibit the initiation of the adaptative immune reaction as do IFN- a2a. In conclusion, while interferon type I and type III are induced by the same viral stimulating factors and exhibit similar signature profiles, their biological activity appears not redundant but rather complementary. Indeed, following viral infection, during the innate phase of the immune response, interferons type III exert their antiviral effects in mucosal sites whereas IFN-a act more systemically in the whole organism. Furthermore, the subsequent adaptive immune reaction is inhibited at its initiation level by the immunosuppressive effect of the IFN-a on activated CD4+ T cells.
Example 2: Critical pathogenic role of IFN-a and IFN-l in human HIV-1 infection
Material and Methods Cryopreserved PBMCs were thawed in RPMI 1640 with 10% fetal bovine serum (FBS) and washed in FACS buffer. Phenotypic staining was performed on 106 cells by incubation with a viability marker (AmCyan live-dead kit from Invitrogen) and with antibodies conjugated to CD3, CD4, CD8, CD45RA, CCR7. Subsequently, cells were washed, fixed with 4% paraformaldehyde for 5 min, washed, and acquired with an AURORA cytometer (Cytek).
Peripheral Blood samples were obtained either from healthy donors through Etablissement Francais du Sang (EFS, Paris, France) or from Elite controller HIV-1 patients and chronically-HIV-infected patients pre and post combined ART treatment.
Blood cells were collected using standard procedures. The study was performed according to the Helsinki declaration, and the study protocol was reviewed and approved by the local Ethics Committee. All samples were de-identified prior to use in this study.
Frozen serums were thawed at 4°C and centrifuged at 4000 G for 10 min at 4°C. IFN-a and IFN-l (IL-28A) serum concentrations were measured using the high sensitivity Single-Molecule Array (Simoa®) technology (Digital ELISA technology) (Quanterix) according to the manufacturer’s instructions. Results
Comparison of central memory (CM) CDS + T cell distributions in HIV-1 -infected subjects
In study of chronically HIV- 1 -infected subjects, the following groups were studied:
(i) elite controllers (EC) who naturally suppress HIV-1 in the absence of combined antiretroviral therapy treatment (c-ART)
(ii) non-controllers before (pre-cART) and after cART (post-cART) treatment, and
(iii) a cohort of age-matched healthy donor (HD) subjects.
The relative frequencies of the CM populations within the CD8+ T cell compartments were evaluated in each of the subject groups.
The gating strategy to define this subset is the following. Briefly, singlet cells were defined, followed by gating on lymphocytes and live cells. Among the live cells, CD3+ T lymphocytes were identified, followed by the definition of CD8+ subpopulations. Subsequently, the expression of CD45RA and CCR7 was analyzed in the CD8+ T lymphocytes. Central memory T cells (TCM) are CD45RA- CCR7+.
Figure 6A shows that the level of CM CD8+ cells was significantly lower in non-controllers before cART than in other groups. Moreover, combined antiretroviral therapy (cART) results in increase of CM CD8+ cells.
Comparison of serum IFN-a levels in HIV- 1 -infected subjects
Serum levels of IFN-a was measured in the 4 groups. Figure 6B shows that the non-controller patients have significantly increased serum IFN-a levels before treatment compared with after treatment.
IFN-a inversely correlates with the percentage of CM CD8+ cells in HIV -infected patients without treatment
In the population of HIV infected patients (EC pre-cART patients), the inventors explored the potential correlations between the level of CM CD8+ cells and the serum IFN-a levels. In this study, there was a significant negative correlation between the frequency of CM CD8+ cells and serum IFN-a levels (spearman correlation r = -0,667; p < 0.005). This reflects the critical pathogenic effect of IFN-a on T cell proliferation in secondary organs (see Figure 6C). Comparison of serum IFN-l levels in HIV -1 -infected subjects
Serum from healthy control donors and HIV-1 infected patients, before and after cART treatment, originating from two distinct institutes were assayed by Simoa® for IFN-a and IFN- 2 proteins.
Before cART treatment, serum levels of both IFN-a and IFN-l were higher in patients versus controls. After cART treatment, the levels of IFN-a had decreased but the levels of IFN-l remained unchanged (Figure 7A) or increased slightly (Figure 7B).
Conclusion
Latent HIV-1 reservoir represents the main obstacle in achieving sustained virologic remission in cART treated HIV-1 infected patients following ART treatment interruption. This is due to two opposing biologic processes occurring in parallel in patients under cART:
1- On the one hand, cART promotes an inhibition of the viral replication triggered by the HIV-1 proviral DNA present in activated peripheral and mucosal reservoir cells. It results from this process a minimal expression of viral particles in the body fluid (<40 copies/ml).
2- On the other hand, the second process is due to the production of type I IFN-a and type III IFN-l locally, induced by viral replication occurring in these still infected peripheral and mucosal cells, which contain latent integrated HIV proviruses. By their antiviral effects, these interferons locally reduce the ongoing viral replication occurring in these reservoir cells, which limits the local propagation of the virus to nearby cells but still maintains the reservoir of infected cells. The local production of IFN-a and IFN-l by peripheral and mucosal cells, which are still infected and replicating the virus, is confirmed by the presence in the serum of post-cART patients of substantial concentrations of these antiviral cytokines (Figure 7).
Following the blocking of the peripheral and mucosal antiviral interferons, which hinder expression of viral replication in reservoir cells, the HIV- 1 proviral DNA present in these reservoir cells may fully replicate viruses. In turn, cART treatment, which controls viral replication, may reduce and progressively eliminate these peripheral and mucosal cellular reservoirs.
Example 3: Clinical protocol for AIDS treatment in human patients Study design 1
Study eligibility criteria includes patients having ongoing ART (>three drugs) with plasma HIV-1 RNA levels < 40 copies/ml as well as a CD4+ T cell count > 500 cells/pl (and optionally < 500 cells/pl). Patients receive infusions of neutralizing monoclonal antibodies anti-IFN type I and type III receptors at 2-week intervals in parallel with their ongoing daily cART therapy (Figure 8). Plasma HIV-1 RNA levels, size of the reservoir and CD4+ T cell counts are monitored every 2 weeks, after collection of blood samples, 1 day before each mAbs infusion.
Study design 2
Study eligibility criteria includes patients having ongoing ART (>three drugs) with plasma HIV-1 RNA levels < 40 copies/ml as well as a CD4+ T cell count > 500 cells/pl (and optionally < 500 cells/pl). Patients receive infusions of anti-IFN-a, anti-IFN-l monoclonal antibodies and optionally anti-IFN-b monoclonal antibodies at 2-week intervals in parallel with their ongoing daily cART therapy (Figure 8). Plasma HIV-1 RNA levels, size of the reservoir and CD4+ T cell counts are monitored every 2 weeks, after collection of blood samples, 1 day before each mAbs infusion. Plasma HIV-1 RNA Levels
HIV-1 RNA levels are determined using the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Assay (version 2.0) or the Roche cobas HIV-1 quantitative nucleic acid test (cobas 6800), which quantitate HIV-1 RNA over a range of 2x101 to 1x107 copies/ml.
Quantitative Viral Outgrowth Assay
Size of the HIV reservoir is assessed with Quantitative Viral Outgrowth Assay (QVOA) as previously described (Huang etal, 2018, J Clin Invest 128:876-889). Briefly, isolated CD4+ T cells are plated out in serial dilutions (2, 1, 0.5, 0.2, and 0.1 million per well) into 12 wells in a 24-well plate with phytohemagglutinin (PHA; 2 pg/ml) and irradiated HIV-negative donor PBMC (2xl06 cells/well) to reactivate the infected cells. After 2 days of culture, MOLT-4 CCR5 cells (2xl06 cells/well) are added to amplify the HIV. After 2 weeks of culture, p24 antigen is quantified in the culture supernatant, using an HIV p24 antigen enzyme-linked immunosorbent assay (ELISA) kit (Perkin-Elmer, Hopkinton, MA), and estimated frequencies of cells with replication-competent HIV-1 are calculated using limiting dilution analysis.
CD4+ T-cell counts
CD4+ T-cell counts are determined by a clinical flow cytometry assay.
Treatment interruption Monoclonal antibodies infusion is stopped when no cells with replication-competent HIV-1 are detected following 2-3 consecutive limiting dilution virus outgrowth assays (VOA). Then, ART treatment is stopped when no cells with replication-competent HIV-1 are detected with 3-4 consecutive limiting dilution virus outgrowth assays. Example 4: Humanized HIV-infected mice produce type and type III interferons.
Material and Methods
Animals
Animal experiments were carried out with female NOD/Shi-scid/IL-2RYnull (NOG) immunodeficient mouse strain. Mice were humanized using hematopoietic stem cells (CD34+) isolated from human cord blood.
Humanized animal model
Humanized mice were intraperitoneally injected with HIV-NL4.3, and mock-infected mice were subsequently injected with an equal volume of the vehicle. Blood and spleen were removed from infected and non-infected humanized NCG mice at day 2 post infection, when the vims load has reached a plateau at day 10 and at day 30.
IFN-a, IFN-lI and IFN-/.3 mRNA isolation and real-time PCR analysis.
RNA was extracted from cells. Samples were DNase treated and then converted into cDNA (reverse transcribed) using the Superscript III First-Strand Synthesis system (Invitrogen). The cDNA was used at 1:10, and expression of IFN-a, IFN-lI, and IFN- l3 were analyzed using the SYBR Green PCR-based protocol with the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Gene-specific forward and reverse primers were designed using Primer Express software 1.5 (Applied Biosystems). The housekeeping RPS14 gene was used to normalize cDNA across samples. The conditions for PCR were as follows: 10 min at 95°C, then 40 cycles of amplification at 95°C for 15 s and 60 s at 60°C, and finally 15 s at 95°C, 30 s at 60°C, and 15 s at 95°C. Ct was normalized to RPS 14 (ACt = Ct sample - Ct RPS 14). Statistical analyses were performed by the 2-AACT method.
Results We studied the kinetics and localization of type I and type III IFN appearance in HIV- infected humanized mice. Mice were sacrificed on the day of infection and 10 and 35 days after infection. PBMCs and splenocytes were recovered and the presence of IFN-oc, IFN- 3, and IFN-lI mRNA was assessed by RT-qPCR. Figure 9 shows that both types of IFN were produced after infection. IFN-oc was most notably present in PBMCs, whereas IFN- 3 and IFN-lI were present in splenocytes. Example 5: Activated CD4+ T cells express IFN-l Receptor.
Material and Methods
CD4+ T cell purification and culture
CD4+ T cells were purified from peripheral blood mononuclear cells (PBMCs) obtained from anonymous healthy blood donors (EFS). Ficoll (Ficoll Hystopaque; Sigma) density centrifugation was performed as per the manufacturer’ s instructions, and CD4+ cells were negatively selected using magnetic beads (CD4+ T-cell isolation kit I; Miltenyi Biotec). Purity was assessed following cell isolation by staining with anti-CD4-Alexa Fluor 700 (RPA-T4) and all samples were >97% CD4+ by flow cytometry.
CD4+ T cells were cultured in RPMI 1640 supplemented with 10% FBS (Gibco), 100 IU penicillin, 100 pg/mL streptomycin, 0.1 M Hepes, 2 mM L-glutamine, and/or recombinant human IL-2. Cells were maintained at 37 °C in a 5% C02 humidified incubator.
CD4+ T cells were co-stimulated through CD3 and CD28 using plate-bound antibodies for 3 days or no simulation. Anti-CD3 (OKT3) was used at 1 pg/mL (BioLegend, San Diego, CA) and anti-CD28 (CD28.2) at 1 pg/mL (BioLegend, San Diego, CA).
IL-28RA, IL-10RB, IFIH1 and IFI27 mRNA isolation and real-time PCR analysis.
RNA was extracted from cells. Samples were DNase treated and then converted into cDNA (reverse transcribed) using the Superscript III First-Strand Synthesis system (Invitrogen). The cDNA was used at 1:10, and expression of IL-28RA, IL-10RB, IFIH1 and IFI27 were analyzed using the SYBR Green PCR-based protocol with the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Gene-specific forward and reverse primers were designed using Primer Express software 1.5 (Applied Biosystems). The housekeeping RPS14 gene was used to normalize cDNA across samples. The conditions for PCR were as follows: 10 min at 95°C, then 40 cycles of amplification at 95°C for 15 s and 60 s at 60°C, and finally 15 s at 95°C, 30 s at 60°C, and 15 s at 95°C. Ct was normalized to RPS14 (ACt = Ct sample - Ct RPS14). Statistical analyses were performed by the 2-AACT method.
Gene expression analysis of interferon stimulating genes IFIH1 and IFI27
Cells were treated with 100 IU/ml of type I IFN (IFN-a) or 10 ng/ml of type III IFN (IFN-k3). Total RNA was isolated at 16 hours post-treatment.
Flow Cytometry Analysis. For IFN-/J binding assay to quantify the presence of IL-28RA on CD4+ T cells, cells were treated with or without His-tagged IFN-/J (R&D Systems) diluted in PBS containing 1% BSA on ice for 60 min at indicated doses. Cells were then washed and stained with anti-His PE (Miltenyi Biotec) for 40 min in the dark and a viability dye. The MFIs of IFN-/J binding on CD4+ T cells were determined by flow cytometry in living cells as follows: MFI (His-tagged IFN-/J + anti-His PE) - MFI (anti-His PE).
Results
While IFN type I receptors (IFN AR 1/2) are known to be ubiquitous and expressed on almost all cell types, IFNLR1 is described to be more restricted and to be highly expressed on epithelial cells, neutrophils and dendritic cells. Some teams reported that CD4+ T cells were sensitive to type III IFN, others indicate that they were not. These discordant results are due to the different biological contexts when the analysis of the expression of the receptor was performed.
We assessed the ability of CD4+ T cells to express IFNLR according to their level of activation. We purified primary CD4+ T cells and stimulated them without or with anti- CD3 and anti-CD28 antibodies in the presence of IL-2. After three days of culture, we studied the presence of IL28RA and IL-10RB transcripts (these two molecules constituting IFNLR), the ability of the cells to bind IFN- 3 and to produce the IFIH1 and IFI27 mRNA interferon stimulated genes (ISG) after their culture in the presence of IFN- l3. Figure 10 shows that while purified CD4+ T cells cultured in the presence of IL-2 alone expressed basal levels of IL-28RA mRNA and IFNLR, the 3-day-CD3/CD28/IL- 2-stimulated cells displayed high level of IFNLR, at mRNA and protein levels. The IFIH1 and IFI27 mRNA detection in IFN- 3 -treated stimulated CD4+ T cells confirmed the presence of a functional IFNLR at their surface.
Example 6: IFN- 3 inhibits HIV-1 infection of activated CD4+ T cells.
Material and Methods
CD4+ T cell purification and culture CD4+ T cells were purified and cultured as described in Example 5.
Reagents
IFN-a, IFN- 3 and anti-IFNAR2 neutralizing antibody were purchased from Bio-Techne and anti-IL28RA (IFNLR1) neutralizing antibody from PBL assays science.
Production of Viral Stocks HIV NL4.3 was obtained from the AIDS Research and Reference Reagent Program. Viral stocks were generated by transfection of HEK 293T with polyethylenimine (Polysciences). Two days after transfection, culture supernatants were collected, clarified at 441 x g for 5 min, and filtered (0.45 pm).
HIV Infection Experiments CD4+ T cells were co-stimulated through CD3 and CD28 using plate-bound antibodies for 3 days or no simulation. Anti-CD3 (OKT3) was used at 1 pg/mL (BioLegend, San Diego, CA) and anti-CD28 (CD28.2) at 1 pg/mL (BioLegend, San Diego, CA). Stimulated CD4+ T cell HIV-1 infections occurred after 3 days of CD3/CD28 stimulation.
Infections with HIV-NL4.3 were performed overnight in the presence of polybrene (2 pg/mL), and fresh media were replaced. The frequency of infected cells was determined by intracellular HIV p24 by FACS. When indicated, IFN-a, IFN-/J, anti- IFNAR2 and anti-IL-28RA neutralizing antibodies were added 24 hours before infection and for an additional 5 days in culture after infection.
Results We then investigated the ability of type III IFNs to prevent HIV-1 infection of activated CD4+ T cells, using as control type I IFN known to strongly inhibits HIV-1 infection. Figure 11 shows that both types of IFN inhibited infection of activated CD4+ T cells, with type I IFN inhibition being slightly more important. Interestingly, stimulation of CD4+ T cells by both IFNs simultaneously increased the blockage of infection. Example 7: Sequential treatment with two latency-reversing agents, DNA methylation inhibitor 5-AzadC and HDACI MS-275, induce IFN-l Receptor expression on CD4+ T cells.
Material and Methods
CD4+ T cell purification and culture CD4+ T cells were purified, cultured and co-stimulated as described in Example 5.
Jurkat cell culture
The human cell line Jurkat (J-Lat) clone 10.6, was obtained from AIDS Reagent Program, National Institutes of Health (Germantown, MD). This cell line was grown in complete culture medium, as the primary cells, and maintained at 37 °C in a 5% C02 incubator. J- Lat clone 10.6 harbors a HIV provirus containing the GFP open reading frame (ORF) instead of nef and a frameshift mutation in env.
Reagents
5-AzadC was purchased from Sigma Aldrich, MS-275 from Enzo Life Sciences and FN- l3 from Bio-Techne. LRA treatment of stimulated CD4+ T cells
Stimulated CD4+ T cells were submitted to a 48-h 5-AzadC pretreatment followed by a 24-h MS -275 induction, corresponding to a total 72-h 5-AzadC treatment.
HIV reactivation in vitro in latently infected J-Lat clone 10.6. HIV reactivation was performed by a 48-h 5-AzadC pretreatment followed by a 24-h MS- 275 induction, corresponding to a total 72-h 5-AzadC treatment. Reactivation of HIV was monitored as the percentage of living GFP-positive cells, according to forward and side laser light scatter flow cytometry analysis in a FACS ARIA3 flow cytometer (BD Biosciences). The data were analyzed using FlowJo software. IL-28RA, IFIH1 and IFI27 mRNA isolation and real-time PCR analysis.
RNA was extracted from cells. Samples were DNase treated and then converted into cDNA (reverse transcribed) using the Superscript III First-Strand Synthesis system (Invitrogen). The cDNA was used at 1:10, and expression of IL-28RA, IFIH1 and IFI27 were analyzed using the SYBR Green PCR-based protocol with the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Gene-specific forward and reverse primers were designed using Primer Express software 1.5 (Applied Biosystems). The housekeeping RPS14 gene was used to normalize cDNA across samples. The conditions for PCR were as follows: 10 min at 95°C, then 40 cycles of amplification at 95°C for 15 s and 60 s at 60°C, and finally 15 s at 95°C, 30 s at 60°C, and 15 s at 95°C. Ct was normalized to RPS 14 (ACt = Ct sample - Ct RPS 14). Statistical analyses were performed by the 2-AACT method.
Flow Cytometry Analysis.
For IFN-/J binding assay to quantify the presence of IL-28RA on CD4+ T cells, cells were treated with or without His-tagged IFN-/J (R&D Systems) diluted in PBS containing 1% BSA on ice for 60 min at indicated doses. Cells were then washed and stained with anti-His PE (Miltenyi Biotec) for 40 min in the dark and a viability dye. The MFIs of IFN-/J binding on CD4+ T cells were determined by flow cytometry in living cells as follows: MFI (His-tagged IFN-/J + anti-His PE) - MFI (anti-His PE). Results
Reactivation of HIV gene expression in latently infected cells together with an efficient cART has been proposed as an adjuvant therapy aimed at eliminating/decreasing the reservoir size. So far, none of the LRAs that have been tested have been successful in eradicating HIV-1 latent reservoir.
We investigated whether these molecules could influence the IFNLR expression in CD4+ T cells. Briefly, stimulated CD4+ T cells and latently infected J-Lat 10.6 cells (cell lines that harbor an HIV provirus containing the Green Fluorescent Protein (GFP) ORF instead of nef and a frameshift mutation in env) were treated sequentially with two latency- reversing agents, DNA methylation inhibitor 5-AzadC and HDACI MS-275, a combination of molecule known to reactivate HIV. After a 72-h treatment with 5-AzadC and MS -275 (MS -275 having been added during the last 24 hours), we studied the presence of IL28RA (IFNLR1) transcript in both cells, their ability to bind IFN- 3 and to produce the IFIH1 and IFI27 mRNA interferon stimulated genes (ISG) after their culture in the presence of IFN- 3. Figure 12 shows that the sequential association of these two molecules increased IFNLR mRNA and protein levels in both T cells. In addition, their stimulation with IFN- 3 led to the induction of the IFIH1 and IFI27 ISG.
Collectively these data indicate that this LRA combination treatment could make the cells sensitive to type III interferon. Example 8: The NtRTI TDF enhances the production of IFN-L3 by HT-29 cells.
Material and Methods
HT-29 cell culture
Human colon adenocarcinoma HT-29 cells (ATCC, HTB-38) were cultured in 25 cm2 culture flasks in McCoy's 5 A medium supplemented with 10% heat-inactivated FCS (Gibco), penicillin (100 U/mL) and streptomycin (100 pg/mL). TDF treatment of HT -29 cells
HT-29 cells were incubated with dimethyl sulfoxide (DMSO) or tenofovir disoproxil fumarate (TDF, 25 mM) for 48 hours.
IFN-/.3 analysis of supernatants The levels of IFN-k3 were assayed using an ELISA kit according to the manufacturer's instructions.
Results
Since patients undergoing antiretroviral treatment have a persistent type 1/type III interferon signature, we looked at the effects of certain antiviral molecules on type III IFN production. Since HIV infection occurs at the gastrointestinal level and antiviral treatments are taken orally, we looked at the effect of Tenofovir disoproxil fumarate (TDF), a nucleotide reverse transcriptase inhibitor (NRTI) of HIV commonly used, on intestinal epithelial HT-29 cells. Figure 13 shows that IFN- 3 was detected in the culture supernatants of HT-29 cells treated with TDF. Example 9: IFN-k3 secreted by TDF-treated HT-29 cells impairs CD4+ T cells infection.
Material and Methods
CD4+ T cell purification and culture
CD4+ T cells were purified and cultured as described in Example 5. HT-29 cell culture
Human colon adenocarcinoma HT-29 cells (ATCC, HTB-38) were cultured as described in Example 8.
Reagents
Anti-IL28RA neutralizing antibody was purchased from PBL assays science. Production of Viral Stocks
HIV NL4.3 was obtained from the AIDS Research and Reference Reagent Program. Viral stocks were generated by transfection of HEK 293T with polyethylenimine (Polysciences). Two days after transfection, culture supernatants were collected, clarified at 441 x g for 5 min, and filtered (0.45 pm).
Transwell co-culture model for assessing inhibition of HIV infection in stimulated CD4+ T cells by IFN-/.3 released by TDF-treated HT-29 cells.
Experiments were performed using 12-transwell chambers with a polycarbonate filter (0.2 pm pore size). HT-29 were grown until subconfluence in the lower chamber. Then HT-29 cells were treated with tenofovir disoproxil fumarate (TDF). After 24h of TDF treatment, 3-day-CD3/CD28/IL2-stimulated CD4+ T cells were seeded in presence of IL- 2 in the upper chamber, which is then placed in the culture well so as to be immersed in the culture medium of the HT-29 cells. When indicated, the CD4+ T cells were pre-treated with an IFNLR1 (IL28RA) neutralizing antibody. After 24h of co-culture, CD4+ T cells were isolated, infected with HIV NL4.3 and then placed back into the co-culture for an additional 5 days of culture. The frequency of infected cells was determined by intracellular HIV p24 by FACS.
Flow Cytometry Analysis.
For the HIV p24 intracellular staining, cells were fixed and permeabilized with the BD fix and perm kit and stained with Kc-57-RDl (1:100; Beckam-Coulter).
Results
In order to get closer to an in vivo context, we developed a Transwell culture assay to evaluate the IFN- 3 antiviral activity. The test was performed in 12-well Transwell plates with stimulated CD4+ T cells at sufficient concentrations (Figure 14). Briefly, HT-29 cells were cultured in the bottom chamber of Transwell plates. When the culture was subconfluent, Tenofovir disoproxil fumarate (TDF) was added. After 24 hours of stimulation, activated CD4 T cells were deposited in the insert which was then placed in the culture well. When indicated, the CD4+ T cells were pre-treated with an IFNLR1 (IL28RA) neutralizing antibody. After 24 hours of co-culture, the cells in the insert were harvested, infected and then placed back into the co-culture for an additional 5 days of culture. The frequency of infected cells was determined by intracellular HIV p24 by FACS. In this context, Figure 15 shows that the IFN- 3 released by TDF-treated HT-29 cells was able to impair HIV-1 CD4+ infection.
Example 10: IFN-L3 released by TDF-treated HT-29 cells inhibit in vitro HIV reactivation in latently infected J-Lat clone 10.6.
Material and Methods Reagents
5-AzadC was purchased from Sigma Aldrich; MS-275 from Enzo Life Sciences; and IFN- l3, from Bio-Techne. Anti-IL28RA neutralizing antibody was purchased from PBL assays science.
HT-29 cell culture Human colon adenocarcinoma HT-29 cells (ATCC, HTB-38) were cultured as described in Example 8.
Jurkat cell culture
The human cell line Jurkat (J-Lat) clone 10.6 was cultured as described in Example 8.
Transwell co-culture model for assessing the inhibition of HIV latency reversal in latently infected J-Lat cells by IFN-/.3 released by TDF-treated HT-29 cells.
Transwell cultures (12 wells) with confluent HT-29 monolayers were used for co-culture with J-LAT 10.6 cells. HT-29 were grown until subconfluence on Transwell bottom. Then HT-29 cells were treated with Tenofovir disoproxil fumarate (TDF). After 24h of TDF treatment, J-LAT 10.6 cells pretreated with 5-AzadC and MS-275 for 72h were seeded on Transwell filters. When indicated, anti-IFNLRl (IL28RA) neutralizing Abs were added to the culture 2 hours before the J-Lat 10.6 cells seeding. After 48 h of co- culture, HIV reactivation was monitored as the percentage of living GFP-positive cells, according to forward and side laser light scatter flow cytometry analysis in a FACS ARIA3 flow cytometer (BD Biosciences). The data were analyzed using FlowJo software. Results
We finally determined whether type III interferon could impair HIV-1 latency reversal. We used the Transwell culture assay we have set up to evaluate the IFN- 3 antiviral activity. We placed the HT-29 cells in the bottom chamber of Transwell plates and the J- Lat cells pretreated with 5-AzadC and MS -275 in the insert (Figure 16). Figure 17 shows that IFN- 3 released by TDF-treated HT-29 cells inhibited HIV latency reversal.
Example 11: Pre- Clinical protocol for HIV-1 reservoirs elimination in HIV-1 infected humanized mice.
Study design Two humanized mouse models are used, BLT mice and NCG mice. Humanized BLT mice with > 50% of circulating human CD45+ cells at 24 weeks post-transplantation and humanized NCG mice with > 20% of circulating human CD45+ cells at 24 weeks post transplantation are infected with HIV-1 through retro-orbital (BLT mice) or intraperitoneal injection (NCG mice). Hu-mice infected with HIV-1 for 7-9 weeks are treated with cART. The study is structured into two arms (Figure 18). The mice are injected with a-IFNARl mAh and a-IFNLRl mAh (experimental group) or with corresponding isotype mAbs (control group) twice a week starting from week 4 after cART. cART is maintained for an additional 2.5 weeks after the last antibody treatment. At the end of the cART treatment, animals are sacrificed. Some animals are kept after cART has been discontinued to measure time to viral detectability after treatment interruption. These groups of “viral rebound” are monitored for another three to six weeks or longer if they remain undetectable in plasma viral loads. Monitoring of viral load upon HIV-1 infection and under cART treatment is performed every two weeks. Plasma HIV-1 RNA Levels
HIV-1 RNA levels are determined using the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Assay (version 2.0) or the Roche cobas HIV-1 quantitative nucleic acid test (cobas 6800), which quantitate HIV-1 RNA over a range of 2x1o1 to lxlO7 copies/ml. Cell-associated HIV-1 DNA detection.
To measure total cell-associated HIV-1 DNA, nucleic acid is extracted from spleen and bone marrow cells using the DNeasy mini kit (Qiagen). HIV-1 DNA is quantified by real time PCR. DNA from serial dilutions of ACH2 cells, which contain 1 copy of HIV genome in each cell, is used to generate a standard curve. Cell-associated HIV-1 RNA detection.
To measure total cell-associated HIV-1 RNA, nucleic acid is extracted from spleen or bone marrow cells using the RNeasy plus mini kit (Qiagen). HIV-1 RNA is detected as described above. The HIV-1 RNA expression levels are normalized to human CD4 mRNA controls, and the result is calculated as fold change in gene expression. Quantitative Viral Outgrowth Assay
Size of the HIV reservoir is assessed with Quantitative Viral Outgrowth Assay (QVOA) as previously described (Huang etal., 2018, J Clin Invest 128:876-889). Briefly, isolated CD4+ T cells are plated out in serial dilutions (2, 1, 0.5, 0.2, and 0.1 million per well) into 12 wells in a 24-well plate with phytohemagglutinin (PHA; 2 pg/ml) and irradiated HIV-negative donor PBMC (2xl06 cells/well) to reactivate the infected cells. After 2 days of culture, MOLT-4 CCR5 cells (2xl06 cells/well) are added to amplify the HIV. After 2 weeks of culture, p24 antigen is quantified in the culture supernatant, using an HIV p24 antigen enzyme-linked immunosorbent assay (ELISA) kit (Perkin-Elmer, Hopkinton, MA), and estimated frequencies of cells with replication-competent HIV-1 are calculated using limiting dilution analysis. Example 12: Discussion
Lymphoid reservoir formations present before cART in infected patients
HIV infection in patients is maintained by some lymphoid formations called reservoirs. These formations are found anatomically on the intestinal or vaginal mucous membranes initially (during the primary infection), and then within the peripheral lymph nodes or spleens. These lymphoid formations contain virions in their circulating lymph and host three categories of CD4+ T cells:
1) infected CD4+ T cells that are immune-activated by specific antigenic stimulation, 2) infected CD4+ T cells that are immune-resting. These cells are resting although they carry the proviral DNA integrated into their genome. These cells do not synthesize viral proteins until they are immune-stimulated.
3) healthy CD4+ T cells that are not carrying the proviral DNA and are resting while awaiting their specific antigenic stimulation. These formations actually maintain virus synthesis thanks to infected CD4+ T cells undergoing specific immune activation induced by their antigen. These cells produce stromal lymph virions and circulating blood virions and may spread the viral infection to other lymphoid formations. These latter formations in turn become reservoir formations.
What happens under cART within these reservoir formations which contain the three categories of CD4+ T cells?
The behavior of these three categories of CD4 T cells is different during cART treatment. Their behavior is determined by the combined, and sometimes antagonistic, biological effect of three factors:
1) the presence of anti-viral messengers, namely mucosal type III interferon and systemic type I interferon induced by viral particles. 2) pharmacological treatments (cART) which block any synthesis of viral proteins and virion production.
3) interferon- stimulated genes (ISGs) maintaining viral latency: these are a set of molecules that inhibit the transcription of HIV genes when they are integrated into their DNA.
Mucosal type III and systemic type I interferons (IFNs) represent the first natural anti viral defense blocking the integration of proviral DNA in target CD4+ T cells that carry HIV receptors. These IFN messengers, present in the stromal lymph of the lymphoid formations, act in a paracrine and dose-dependent manner in neighboring target CD4+ T cells that carry their specific type I and type III receptors. Their action induces the expression of ISGs which inhibit the synthesis of viral proteins, such as ISG15.
It should be noted that CD4+ T cells constitutively express type I IFN receptors and inducibly express type III IFN receptors. Activation of these receptors by IFNs prevents the expression of virions within CD4+ T cells by blocking viral protein synthesis and virion production. Thus, the infected resting cells in the lymphoid reservoir formations, that have the proviral DNA integrated into their genome, as well as the healthy uninfected resting cells are protected, do not synthesize viral proteins and do not produce viruses. On the other hand, infected cells of these lymphoid formations, which are immune- activated by their antigenic stimulation and which already produce the virions, are not sensitive to the IFNs action, but they produce the infectious virions.
How does each of these three categories ofCD4+ T cells evolve within the mucosal and peripheral lymphoid reservoir formations under cART?
1) HIV-infected and immune-activated CD4+ T cells (category 1) maintain their viral production and die by apoptosis. However, before disappearing, the virions they produce induce the production of mucosal type III antiviral IFNs in the mucous membranes and of systemic type I IFNs which protect the infected or non-inf ected resting CD4+ T cells (category 2 and 3) in a paracrine and dose-dependent manner. 2) Infected CD4+ T cells which carry the integrated proviral DNA, but are not immune-activated by their specific stimulation within the lymphoid foci, are protected from viral production by the production of ISGs induced by type III and type I IFNs. Indeed, the activation of type I and III IFN receptors that they express activate the ISGMLs responsible for the latency induced by the IFNs. IFNs are mainly produced by APCs (pDC, mDC and macrophages).
3) Uninfected cells not carrying proviral DNA (category 3) remain resting. These are healthy cells that are spared thanks to the paracrine action of IFNs during their specific antigenic stimulation, that behave like healthy and immune-activated CD4+ T cells. cART interruption
Stopping cART affects all three categories of CD4+ T cells of the reservoir lymphoid formations. It follows that:
1) The infected cells which were immune-activated during cART died after producing the virions which induce the secretion by APCs of type III and type I antiviral IFNs in a paracrine and dose-dependent manner.
2) The infected and non-immune- stimulated cells wait for their antigenic stimulation to be activated and release the ISGMLs of their latency. These cells will induce the synthesis of viral proteins and the production of virions circulating in the stromal lymph of the lymphoid formations and then in the peripheral blood and will disseminate the infection to other so far spared lymphoid formations.
3) The uninfected and not yet stimulated cells will no longer be protected by the IFNs and during their specific stimulation will also produce viruses circulating in the stromal lymph. After production of virions, these cells will disappear by apoptosis, which reduces the supply of CD4+ T cells in the body. How to clear out lymphoid reservoir formations in patients under cART optionally in combination with antibodies neutralizing IF N alpha?
The invention proposes to combine these two treatments with agents blocking mucosal type III IFNs produced on the mucous membranes, such as neutralizing antibodies. This combination appears necessary to clear out the lymphoid reservoir formations of their virus-producing cells that maintain the infection.
Indeed, our analysis of sera from patients under cART (still expressing a certain virion concentration) shows the presence of mucosal IFN-lambda acting on the mucous membranes, which is the initial site of infection. The gradual disappearance of infected CD4+ T cells by apoptosis, whether they are activated (category 1) or resting (categories 2 and 3), will allow eradication of the HIV infection. This elimination can be controlled by mucosal lambda IFNs in the mucous membranes and by systemic type I IFNs, which will prevent the decrease in CD4+ T cells in the body. This decrease in CD4+ T cells actually decreases adaptive immune capacity and facilitates progression to disease. The viral load (number of virions/ml) at the time of the set point after the primary infection will be a major indicator of the time required to clear out the lymphoid reservoir formations to eradicate the infection.

Claims

1. A combination for use in the treatment of acquired immune deficiency syndrome
(AIDS) in a subject in need thereof, wherein said subject is under antiretroviral therapy, said combination comprising: i) at least one type III interferon blocking agent, ii) at least one interferon- alpha (IFN-a) blocking agent, iii) optionally, at least one interferon-beta (IFN-b) blocking agent, iv) at least one antiretroviral (ART) agent, and v) optionally, at least one latency-reversing agent (LRA).
2. The combination for the use according to claim 1, wherein the at least one type III interferon blocking agent is:
- an agent neutralizing circulating IFN-l selected from the group consisting of an active anti-IFN-l vaccine, such as an IFN-k-kinoid, IFN-l DNA-based or IFN-l RNA-based vaccine, and a passive anti-IFN-l vaccine, such as an anti-IFN-l antibody or an anti-IFN-l hyper- immune serum, or
- an agent blocking type III interferon signaling selected from the group consisting of an active anti-IFNLRl vaccine, such as an IFNLR1 DNA-based or IFNLR1 RNA- based vaccine, and a passive anti-IFNLRl vaccine, such as an antibody that binds to the IFNLR1 receptor.
3. The combination for the use according to claim 1 or 2, wherein the at least one
IFN-a blocking agent is selected from the group consisting of an agent neutralizing circulating IFN-a, an agent blocking IFN-a signaling, an agent depleting IFN-a producing cells, and an agent blocking IFN-a production; wherein the agent neutralizing circulating IFN-a is selected from the group consisting of an active anti-IFN-a vaccine, such as an IFN-a-kinoid, IFN-a DNA-based or IFN-a RNA-based vaccine, and a passive anti-IFN-a vaccine, such as an anti-IFN-a antibody or an anti-IFN-a hyper-immune serum; wherein the blocking agent of IFN-oc signaling is selected from the group consisting of an active anti-IFNARl or anti-IFNAR2 vaccine, such as an IFNAR1 or IFNAR2 DNA-based vaccine or an IFNAR1 or IFNAR2 RNA-based vaccine, a passive anti- IFNARl or anti-IFNAR2 vaccine, such as an anti-type I interferon R1 or R2 antibody, and an IFN- a endogenous regulator, such as SOSC1 or an aryl hydrocarbon receptor; wherein the agent depleting IFN-oc producing cells is an agent depleting plasmacytoid dendritic cells (pDCs); and wherein the agent blocking IFN-oc production is an agent blocking the production of IFN-oc by pDCs.
4. The combination for the use according to any one of claims 1 to 3, wherein the at least one IFN-b blocking agent is an agent neutralizing circulating IFN-b selected from the group consisting of an active anti-IFN-b vaccine, such as an IFN^-kinoid, IFN-b DNA-based or IFN-b RNA-based vaccine, and a passive anti-IFN-b vaccine, such as an anti-IFN-b antibody or an anti-IFN-b hyper-immune serum.
5. The combination for the use according to any one of claims 1 to 4, wherein the at least one antiretroviral (ART) agent is selected from the group consisting of Nucleoside reverse transcriptase inhibitors (NRTIs), Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Protease inhibitors (Pis), Integrase inhibitors (INSTIs), Fusion inhibitors (FIs), Chemokine receptor antagonists (CCR5 antagonists) and Entry inhibitors (CD4-directed post- attachment inhibitors).
6. The combination for the use according to any one of claims 1 to 5, wherein the at least one latency-reversing agent (LRA) is selected from the group consisting of PKC agonists, MAPK agonists, CCR5 antagonists, Tat vaccines, SMAC mimetics, inducers of P-TEFb release, activators of Akt pathway, benzotriazole derivatives, epigenetic modifiers and immunomodulatory LRAs.
7. The combination for the use according to any one of claims 1 to 6, wherein the presence of cells containing replication-competent proviral HIV DNA is assessed in a blood sample from the subject.
8. The combination for the use according to any one of claims 1 to 7, wherein the at least one type III interferon blocking agent, the at least one IFN-a blocking agent, optionally the at least one interferon-beta (IFN-b) blocking agent, the at least one antiretroviral (ART) agent, and optionally the at least one latency-reversing agent are for simultaneous, separate or sequential administration.
9. The combination for the use according to any one of claims 1 to 8, wherein the at least one type III interferon blocking agent, the at least one IFN-a blocking agent, and optionally the at least one interferon-beta (IFN-b) blocking agent are to be administered every week, every 2 weeks or every 3 weeks.
10. The combination for the use according to any one of claims 1 to 9, wherein the at least one antiretroviral (ART) agent, and optionally the at least one latency-reversing agent, are to be administered daily.
11. The combination for the use according to any one of claims 1 to 10, wherein the at least one type III interferon blocking agent, the at least one IFN-a blocking agent, and optionally the at least one interferon-beta (IFN-b) blocking agent are to be administered parenterally or intravenously.
12. The combination for the use according to any one of claims 1 to 11, wherein one or more doses of the at least one type III interferon blocking agent, the at least one IFN-a blocking agent, and optionally the at least one interferon-beta (IFN-b) blocking agent are to be administered to the subject before receiving said combination.
13. The combination for the use according to any one of claims 1 to 12, wherein one or more doses of the at least one antiretroviral (ART) agent, and optionally the at least one latency-reversing agent, are to be administered to the subject before receiving said combination.
14. The combination for the use according to any one of claims 1 to 13, wherein said combination is to be administered to the subject until no cell containing replication-competent proviral HIV DNA is detected in a blood sample from the subject.
15. A combination comprising: i) at least one type III interferon blocking agent, ii) at least one interferon- alpha (IFN-a) blocking agent, iii) optionally, at least one interferon-beta (IFN-b) blocking agent, iv) optionally, at least one antiretroviral (ART) agent, and v) optionally, at least one latency-reversing agent (LRA).
16. The combination according to claim 15, wherein the at least one type III interferon blocking agent is:
- an agent neutralizing circulating IFN-l selected from the group consisting of an active anti-IFN-l vaccine, such as an IFN-k-kinoid, IFN-l DNA-based or IFN-l RNA-based vaccine, and a passive anti-IFN-l vaccine, such as an anti-IFN-l antibody or an anti-IFN-l hyper- immune serum, or - an agent blocking type III interferon signaling selected from the group consisting of an active anti-IFNLRl vaccine, such as an IFNLR1 DNA-based or IFNLR1 RNA- based vaccine, and a passive anti-IFNLRl vaccine, such as an antibody that binds to the IFNLR1 receptor.
17. The combination according to claim 15 or 16, wherein the at least one IFN-a blocking agent is selected from the group consisting of an agent neutralizing circulating
IFN-a, an agent blocking IFN-a signaling, an agent depleting IFN-a producing cells, and an agent blocking IFN-a production; wherein the agent neutralizing circulating IFN-a is selected from the group consisting of an active anti-IFN-a vaccine, such as an IFN-a-kinoid, IFN-a DNA- based or IFN-a RNA-based vaccine, and a passive anti-IFN-a vaccine, such as an anti-IFN-a antibody or an anti-IFN-a hyper-immune serum; wherein the blocking agent of IFN-a signaling is selected from the group consisting of an active anti-IFNARl or anti-IFNAR2 vaccine, such as an IFNAR1 or IFNAR2 DNA-based vaccine or an IFNAR1 or IFNAR2 RNA-based vaccine, a passive anti- IFNAR1 or anti-IFNAR2 vaccine, such as an anti-type I interferon R1 or R2 antibody, and an IFN- a endogenous regulator, such as SOSC1 or an aryl hydrocarbon receptor; wherein the agent depleting IFN-oc producing cells is an agent depleting plasmacytoid dendritic cells (pDCs); and wherein the agent blocking IFN-oc production is an agent blocking the production of IFN-oc by pDCs.
18. The combination according to any one of claims 15 to 17, wherein the at least one IFN-b blocking agent is an agent neutralizing circulating IFN-b selected from the group consisting of an active anti-IFN-b vaccine, such as an IFN^-kinoid, IFN-b
DNA-based or IFN-b RNA-based vaccine, and a passive anti-IFN-b vaccine, such as an anti-IFN-b antibody or an anti-IFN-b hyper-immune serum.
19. The combination according to any one of claims 15 to 18, wherein the at least one antiretroviral (ART) agent is selected from the group consisting of Nucleoside reverse transcriptase inhibitors (NRTIs), Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Protease inhibitors (Pis), Integrase inhibitors (INSTIs), Fusion inhibitors (FIs), Chemokine receptor antagonists (CCR5 antagonists) and Entry inhibitors (CD4-directed post- attachment inhibitors).
20. The combination according to any one of claims 15 to 19, wherein the at least one latency-reversing agent (LRA) is selected from the group consisting of PKC agonists,
MAPK agonists, CCR5 antagonists, Tat vaccines, SMAC mimetics, inducers of P-TEFb release, activators of Akt pathway, benzotriazole derivatives, epigenetic modifiers and immunomodulatory LRAs.
EP21721080.6A 2020-04-24 2021-04-23 Novel combination for the treatment of aids Pending EP4138910A1 (en)

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US20180169108A1 (en) * 2015-02-20 2018-06-21 The Johns Hopkins University Drug Combinations For the Treatment of HIV
US11136399B2 (en) * 2016-07-14 2021-10-05 Institute Of Biophysics, Chinese Academy Of Sciences Type I interferon receptor antibody and use thereof
US20190284286A1 (en) * 2016-10-27 2019-09-19 The University Of North Carolina At Chapel Hill Modulation of Type I Interferons to Reactivate HIV-1 Reservoir and Enhance HIV-1 Treatment
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