WO2023105247A1 - Methods and compositions to potentiate the immune response with lysine deacetylase inhibitors - Google Patents

Methods and compositions to potentiate the immune response with lysine deacetylase inhibitors Download PDF

Info

Publication number
WO2023105247A1
WO2023105247A1 PCT/GB2022/053173 GB2022053173W WO2023105247A1 WO 2023105247 A1 WO2023105247 A1 WO 2023105247A1 GB 2022053173 W GB2022053173 W GB 2022053173W WO 2023105247 A1 WO2023105247 A1 WO 2023105247A1
Authority
WO
WIPO (PCT)
Prior art keywords
kdaci
vaccine
dose
cells
administered
Prior art date
Application number
PCT/GB2022/053173
Other languages
French (fr)
Inventor
Eoin F. Mckinney
Kenneth G. C. Smith
John Sowerby
Prasanti KOTAGIRI
Original Assignee
Cambridge Enterprise Limited
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
Application filed by Cambridge Enterprise Limited filed Critical Cambridge Enterprise Limited
Publication of WO2023105247A1 publication Critical patent/WO2023105247A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4406—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 3, e.g. zimeldine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • 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
    • A61K39/12—Viral antigens
    • 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
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16311—Influenzavirus C, i.e. influenza C virus
    • C12N2760/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to methods and compositions to boost immune responses to both infection and vaccination against and infection.
  • Lasting protection after vaccination or infection depends on the induction of durable memory responses. Identifying means of expanding immune memory could increase vaccine efficacy, with enhanced cellular responses associated with reduced disease severity and maintenance of protective antibody levels in infectious diseases such as SARS-CoV-2 and influenza.
  • Lysine acetylation is a reversible post-translational modification controlled by the opposing activity of acetyltransferases and deacetylase enzymes (Drummond, D. C.et al. Clinical development of histone deacetylase inhibitors as anticancer agents. Annu Rev Pharmacol Toxicol 45, 495-528, 574 (2005)).
  • the known human KDAC enzymes fall into 4 main classes (l-IV), and effects of KDACi differ widely by target KDAC specificity, functional impact and target cell type (Choudhary, C. et al. Lysine acetylation targets protein complexes and co- regulates major cellular functions. Science 325, 834-840, (2009); Scholz, C.
  • the present invention is aimed at providing a method to boost immune responses to both infection with an infectious agent and vaccination against an infection with an infectious agent. It is also aimed at providing a treatment of infectious disease and vaccine adjuvant for the prevention of infection with an infectious agent.
  • KDACi lysine deacetylase inhibitors
  • MEC early memory precursor effector cell
  • KDACi induce durable memory responses to infectious disease antigens.
  • KDACi have been shown herein to enhance the immune response when used at a low dose of up to 50% of the clinically approved or standard (published) dose.
  • KDACi may be used to enhance the immune response when administering a vaccine, in particular a vaccine for the prevention of an infection with an infectious disease agent. Furthermore, KDACi may be used to boost immune responses to infection with an infectious disease agent and provide a treatment of infectious disease.
  • KDACi has surprisingly been shown herein to promote the memory precursor effector cell phenotype in CD8+ T cells, and as such can be used to enhance the immune response by promoting memory cell differentiation which leads to a strong and durable immune response.
  • an aspect of the invention relates to a method for enhancing an immune response in a subject comprising administering a lysine deacetylase inhibitor (KDACi) simultaneously, sequentially, or separately with a vaccine, wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
  • KDACi lysine deacetylase inhibitor
  • Another aspect relates to a method for the prevention of a disease comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine, and wherein the KDACi is wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
  • An aspect of the invention relates to a KDACi for use in the prevention of a disease, wherein the KDACi is administered simultaneously, sequentially, or separately with a vaccine, and wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 .
  • An aspect of the invention relates to an in vitro or in vivo method of promoting a T cell memory response comprising exposing cells to a KDACi capable of enhancing glutaminolysis.
  • An aspect of the invention relates to an in vitro or in vivo method of promoting differentiation of CD8+ T cells into memory precursor effector cells (MPEC), comprising exposing CD8+ T cells to a KDACi capable of enhancing glutaminolysis.
  • An aspect of the invention also relates to an in vitro or in vivo method of increasing IL7R expression in CD8+ T cells, comprising exposing CD8+ T cells to a KDACi capable of enhancing glutaminolysis.
  • a further aspect of the invention relates to an in vitro or in vivo method of enhancing glutaminolysis, comprising exposing CD8+ T cells to a KDACi.
  • An aspect of the invention also relates to a method of immunising a subject against a disease, comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine, and wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
  • Another aspect relates to a combination therapy comprising administration of administering a KDACi simultaneously, sequentially, or separately with a vaccine, and wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
  • An aspect of the invention relates to the use of a KDACi as an adjuvant for enhancing the efficacy of a vaccine wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 .
  • An aspect of the invention relates to a composition comprising a KDACi and a vaccine wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 .
  • An aspect of the invention relates to a kit comprising a KDACi and a vaccine and optionally instructions for use, wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
  • the invention relates to a method for treating a disease comprising administering a KDACi wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 .
  • the invention relates to a method for expanding cells for use in adoptive cell therapy comprising: providing a population of CD8+ T cells, contacting the population of CD8+ T cells to a KDACi capable of enhancing glutaminolysis to produce an expanded population of cells.
  • Transcriptomic screening identifies a KDACi subclass promoting memory differentiation in primary human CD8 T cells.
  • A Schematic illustration of transcriptomic approach to small molecule repurposing.
  • C Schematic illustration of in vitro phenotypic screen. (D-F).
  • KDACi-induced glutaminolysis promotes CD8 memory differentiation and a switch towards oxidative metabolism.
  • C Bubble plot showing KEGG pathway enrichment of 1115 unique proteins differentially acetylated by KDACi. mem and KDACi. eff compounds
  • A Metabolic pathways (blue) and other pathways (orange).
  • KDACi treatment induces transcriptomic and epigenetic modification in CD8 + T cells consistent with memory differentiation.
  • C Circular barplot showing association of each of 9 latent factors describing maximal variance in the transcriptome of valproate and vehicle treated CD8 + T cells with treatment group (valproate v valp+R162).
  • (E) Circular barplot showing fgsea enrichment of 50 consensus ‘hallmark’ gene signatures (radial axis) in ranked feature weights for LF7. Circular barplots radial axis -logwFDR.
  • Peak distribution (middle bar, % by region) and frequency of peaks/gene (lower bar) also indicated where multiple peaks were identified. Pathway names in red (left) indicate those known to influence T cell memory differentiation.
  • CNNB1 beta catenin
  • LEF1 Lymphoid enhancer binding factor 1
  • TCF7 Transcription factor 7
  • FIG. 4 KDACi treatment in vivo expands immune memory on vaccination and infection.
  • C Scatterplot (mean+/-sem) of MPEC (IL7R hi KLRG1 10 ) and SLEC (IL7R l0 KLRG1 hi ), CD44 hi and IL7R/PD1 expression (MFI) on CD3 + CD8 + splenocytes d7 post Nitrophenylacetyl[NP]-OVA immunisation.
  • D Schematic illustration of heterotypic influenza rechallenge model.
  • V Lung resident T memory (d30pi) pre-rechallenge.
  • F Mediastinal lymph node T cell populations, memory markers (G) and cell populations (H) post rechallenge.
  • V valproate treated
  • V/R valproate+R162 treated
  • O Scatterplot showing RBD-specific B cell and T cell ELISpot titres (y-axis, spot-forming units (SFU) per 10 5 splenocytes) d5 post RBD rechallenge by treatment regimen (valproate or vehicle).
  • SFU spot-forming units
  • NT50 SARS-CoV-2 neutralising antibody titre
  • PB plasmablasts
  • A, B Schematic illustration of the MEMRI study design (A) and experimental protocol (B).
  • C, D Line and scatterplots showing baseline-normalized influenza-specific (Dextramer*) CD8 + T cell expansion after seasonal influenza vaccination. C line plot valproate is shown in red (top line) and control in blue (bottom line).
  • E-G Baseline-normalized seasonal influenza specific IgG (E), IgA (F) valproate shown in red (top line and control shown in blue (bottom line). IgG and IgA titre using the Influenza A Hong Kong H3N2 vaccine strain. Significance assessed using Imm with cubic spline.
  • FIG. 1 Representative BCR network plots illustrating global BCR repertoire clonality and diversity, confirmed influenza-specific clones indicated in yellow.
  • H Scatter and boxplot showing somatic hypermutation rates (CDR-replacement per unique ‘flu-specific BCR sequence) in class-switched (right) and naive (lgM + lgD + , left) BCR clusters with confirmed ‘flu specificity.
  • I-J Representative examples (I) and summary scatterplots (J) of cluster breadth/expansion in naive (J, left: lgM + lgD + ) and class-switched (J, right) influenza-specific BCR clusters.
  • K-L Representative examples (K) and summary scatterplots (L) of cluster breadth in naive (L, left: lgM + lgD + ) and class-switched (L, right) influenza-specific BCR clusters.
  • BCR cluster sequences with 85% amino acid homology, identical CDR3 length and identical V/J gene.
  • Figure 7 Line and scatterplots showing effect (fold change vs vehicle control) of (A) the cell- permeable aKG donor dimethyl-ketoglutarate (DMKG) and (B-D) the glutaminolysis pathway inhibitors R162 (B), BPTES (C) and C968 (D).
  • CD8+ T cell phenotype was measured d6 after polyclonal stimulation with anti-CD3/28 bead.
  • Green cell division
  • red IL7R:PD1 ratio
  • blue CD25
  • purple live CD8+ T cell % of total viable events.
  • FIG. 8 (A, B) Line plot (A, mean +/- sem) and scatterplot (B, mean+/-sem) showing baseline- normalized influenza-specific CD8+ T cell number (Dextramer+) up to 30 days post vaccination with the 2020/21 seasonal influenza vaccine and treatment with either 100mg, 200mg valproate or no drug (control). (C-E) Line plots (mean +/- sem) for influenza-specific IgG (C), IgA (D) and RSV (E) by treatment group.
  • P values one tailed Mann Whitney test.
  • the present invention is based on the surprising finding that various KDAC inhibitors can enhance glutaminolysis - an anaplerotic pathway, feeding the TCA cycle through generation of a-ketoglutarate. It has been shown herein that the KDACi listed in Table 1 are capable of enhancing glutaminolysis. Through this mechanism KDACi promote the formation of memory precursor effector cells, as such this enhances the memory response and leads to a strong and durable immune response. The effects of KDACi expansion of both cellular and neutralising antibody memory responses in response to infection with an infectious agent can be used to boost immune responses to both infection and vaccination. Therefore, the invention provides a method for treating for an infectious disease. The invention also provides a method for preventing infection with an infectious disease agent.
  • the promotion of memory precursor cells elicited by KDACi can also be used to prime cells for use in therapies such as adoptive cell transfer. Therefore, the invention also provides methods of preparing memory precursor effector cells and methods of expanding or culturing cells for use in adoptive cell therapy. Methods for Enhancing an Immune Response
  • KDACi can be used to enhance the immune response elicited by a vaccine.
  • the invention relates to a method for enhancing an immune response in a subject comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine.
  • the invention relates to a method for enhancing an immune response in a subject comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine, wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
  • the invention in another aspect, relates to a method for preventing a disease comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine, wherein the KDACi is capable of enhancing glutaminolysis.
  • the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 .
  • the invention relates to a method for treating a disease comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine, wherein the KDACi is capable of enhancing glutaminolysis.
  • the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 .
  • KDAC Lysine deacetylase
  • HDAC histone deacetylase
  • KDAC and HDAC are a class of enzymes that catalyse the removal of acetyl groups from the NH2 terminal tail of histones. This removal of acetyl groups results in a closed chromatin structure and repression of gene expression.
  • KDAC and HDAC are used interchangeably herein.
  • Class I comprises KDAC1 , KDAC2, KDAC3 and KDAC8.
  • Class HA comprises KDAC4, KDAC5, KDAC7, and KDAC9.
  • Class IIB comprises KDAC6 and KDAC10.
  • Class 3 comprises sirtuins SIRT1 , SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7.
  • Class IV comprises KDAC11
  • KDACi Lysine deacetylase inhibitors
  • KDACi Lysine deacetylase inhibitors
  • KDACi can selectively alter gene transcription, in part, by chromatin remodelling and by changes in the structure of proteins in transcription factor complexes.
  • KDACi may be specific for certain KDAC classes.
  • a KDACi may target class I KDACs and inhibit KDAC1 , KDAC2, KDAC3 and KDAC8.
  • a class I KDACi refers to a compound which inhibits any of KDAC1 , KDAC2, KDAC3 and KDAC8.
  • a class I KDACi may also demonstrate inhibitory activity against other KDACi provided that it also targets one or more of KDAC1 , KDAC2, KDAC3 and KDAC8.
  • the class I KDACi only inhibits one or more of KDAC1 , KDAC2, KDAC3 and KDAC8.
  • Glutaminolysis is an anaplerotic pathway, feeding the TCA cycle through generation of a- ketoglutarate (aKG) while also creating energy and biosynthetic precursors such as nucleic acids and modulating cellular redox balance. It makes an important energetic contribution to T cell activation and can replenish TCA cycle intermediates as they are used for biosynthesis. Glutaminolysis can fuel stem cell-like oxidative metabolism in cancer cells, sustaining chronic proliferation akin to what is required of immune memory.
  • KDACi have demonstrated an effect in enhancing glutaminolysis in particular KDACi capable of inhibiting one or more of KDAC1 , KDAC2, KDAC3, KDAC4, KDAC5, KDAC7, KDAC9 or KDAC10 have been shown to enhance glutaminolysis.
  • KDACi listed in Table 1 have been shown to enhance glutaminolysis, however it will be apparent to the skilled person that other KDACi compounds may demonstrate the ability to enhance glutaminolysis.
  • Glutaminolysis is the process by which cells convert glutamine into TCA cycle metabolites through the activity of multiple enzymes including glutaminase, glutamate dehydrogenase, glutamate-oxaloacetate transaminase, glutamate pyruvate transaminase, phosphoserine transaminase.
  • enhanced glutaminolysis may be determined by upregulation of one or more of the enzymes involved in the glutaminolysis pathway or by identifying upregulated metabolites and protein acetylation patterns indicative of glutaminolysis. Metabolites and acetylation patterns may be identified via metabolomic analysis or via functional assessment of glutamine breakdown.
  • the KDACi capable of enhancing glutaminolysis is selected from abexinostat, belinostat, panobinostat, sodium butyrate, apicidin, mocetinostat, vorinostat, MS275, CI994 and/or valproate.
  • the valproate may be sodium valproate and/or valproic acid.
  • the KDACi is valproate and/or apicidin.
  • the KDACi is selected from one of the KDACi listed in Table 1 and the low dose is up to 45%, up to 40%, up to 35%, up to 30%, up to 25%, up to 20%, up to 15%, up to 10%, up to 5% of the dose listed in Table 1 for the selected KDACi.
  • the KDACi is selected from one of the KDACi listed in Table 1 and the low dose is from 1 to 50%, from 1 to 45%, from 1 to 40%, from 1 to 35%, from 1 to 30%, from 1 to 25%, from 1 to 20%, from 1 to 15%, from 1 to 10%, from 1 to 5% of the dose listed in Table 1 for the selected KDACi.
  • the KDACi is selected from one of the KDACi listed in Table 1 and the low dose is 1 , 2,3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39.
  • the KDACi is used at a dose of 1 to 25%, 1 to 20%, 1 to 15%, 1 to 10%, 1 to 5%, 5 to 25%, 5 to 20%, 5 to 15%, or 5 to 10% of the dose listed in Table 1 for the selected KDACi.
  • a vaccine is a substance used to stimulate an immune response and provide immunity against one or several diseases.
  • a vaccine is generally prepared from the causative agent of a disease, e.g. an infectious disease agent, its products, or a synthetic substitute, treated to act as an antigen without inducing the disease.
  • the term “vaccine” encompasses any vaccine which provides immunity against one or several diseases is used herein to refer to a biological preparation that induces an immunogenic response to a target antigen. Examples of vaccines include viral, bacterial, protein and nucleic acid vaccines.
  • the term "viral vaccine” refers to a virus that induces an immunogenic response to a target antigen.
  • Suitable antigens include tumour antigens, viral antigens, and in particular, antigens derived from viral pathogenic organisms such as HIV, HepC, FIV, LCMV, Ebola virus, as well as bacterial pathogens such as mycobacterium tuberculosis.
  • the vaccine is an infectious disease vaccine.
  • infectious disease vaccine refers to a vaccine that provides a level of immunity against an infectious disease cause by an infectious disease agent. Infectious diseases are generally caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi.
  • infectious disease agent refers to a pathogenic microorganism, such as bacteria, viruses, parasites or fungi. Also known as “communicable diseases”, infectious diseases can be spread directly or indirectly from one person to another.
  • infectious disease vaccine or “viral respiratory disease vaccine” does not extend to cancer vaccines. Thus, the infectious disease vaccine is not a cancer vaccine.
  • the infectious disease agent is a virus, for example and without limitation, a pox virus (e.g., vaccinia virus), zika virus, smallpox virus, marburg virus, flaviviruses (e.g.
  • influenza virus or antigens, such as F and G proteins or derivatives thereof), e.g., influenza A; or purified or recombinant proteins thereof, such as HA, NP, NA, or M proteins, or combinations thereof
  • parainfluenza virus e.g., sendai virus
  • respiratory syncytial virus rubeola virus
  • human immunodeficiency virus or antigens, e.g., such as tat, nef, gpl20 or gpl60
  • human papillomavirus or antigens, such as HPV6, 11 , 16, 18
  • varicella-zoster virus or antigens such as gpl, II and IE63
  • herpes simplex virus e.g., herpes simplex virus I, herpes simplex virus II; or antigens, e.g., such as gD
  • the infectious agent is a bacterium.
  • suitable bacteria or bacterially derived products for use in the vaccines and/or methods of the invention include Neisseria species, including N. gonorrhea and N. meningitidis (or antigens, such as, for example, capsular polysaccharides and conjugates thereof, transferrin-binding proteins, lactoferrin binding proteins, PilC, adhesins); Haemophilus species, e.g., H. influenzae-, S. pyogenes (or antigens, such as, for example, M proteins or fragments thereof, C5A protease, lipoteichoic acids), S. agalactiae, S.
  • Neisseria species including N. gonorrhea and N. meningitidis
  • antigens such as, for example, capsular polysaccharides and conjugates thereof, transferrin-binding proteins, lactoferrin binding proteins, PilC, adhesin
  • M catarrhalis also known as Branhamella catarrhalis (or antigens, such as, for example, high and low molecular weight adhesins and invasins); Bordetella spp, including B. pertussis (or antigens, such as, for example, pertactin, pertussis toxin or derivatives thereof, filamenteous hemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B. bronchiseptica; Mycobacterium species, including M.
  • tuberculosis or antigens, such as, for example, ESAT6, Antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L. pneumophila; Escherichia spp, including enterotoxic E. coli (or antigens, such as, for example, colonization factors, heat-labile toxin or derivatives thereof, heatstable toxin or derivatives thereof), enterohemorragic E. coli, enteropathogenic E.
  • antigens such as, for example, ESAT6, Antigen 85A, -B or -C
  • M. bovis M. leprae
  • M. avium M. paratuberculosis
  • M. smegmatis M. smegmatis
  • Legionella spp including L. pneumophila
  • Escherichia spp including entero
  • Vibrio spp including V. cholera (or antigens, such as, for example, cholera toxin or derivatives thereof); Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y enterocolitica (or antigens, such as, for example, a Yop protein), Y pestis, Y. pseudotuberculosis; Campylobacter spp, including C. jejuni (or antigens, such as, for example, toxins, adhesins and invasins) and C.
  • V. cholera or antigens, such as, for example, cholera toxin or derivatives thereof
  • Shigella spp including S. sonnei, S. dysenteriae, S. flexnerii
  • Yersinia spp including Y enterocolitica (or antigens, such as, for example, a Yop protein), Y
  • Salmonella spp including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis, S. typhimurium, and S. dysenteriae
  • Listeria species including L. monocytogenes
  • Helicobacter spp including H. pylori (for example urease, catalase, vacuolating toxin); Pseudomonas spp, including P. aeruginosa; Staphylococcus species, including S. aureus, S. epidermidis; Proteus species, e.g., P. mirabilis; Enterococcus species, including E. faecalis, E.
  • Clostridium species including C. tetani (or antigens, such as, for example, tetanus toxin and derivative thereof), C. botulinum (or antigens, such as, for example, botulinum toxin and derivative thereof), C. difficile (or antigens, such as, for example, Clostridium toxins A or B and derivatives thereof), and C. perfringens; Bacillus species, including B. anthracis (or antigens, such as, for example, botulinum toxin and derivatives thereof), B. cereus, B. circulans and B. megaterium; Corynebacterium species, including C.
  • diphtheriae or antigens, such as, for example, diphtheria toxin and derivatives thereof
  • Borrelia species including B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii (or antigens, such as, for example, OspA, OspC, DbpA, DbpB), B. afzelii (for example OspA, OspC, DbpA, DbpB), B. andersonii (or antigens, such as, for example, OspA, OspC, DbpA, DbpB), B. hermsii; Ehrlichia species, including E.
  • the infectious agent is a parasite, or a parasite derived product.
  • suitable parasite (or parasite derived products) for use in the vaccines and/or methods of the invention include Plasmodium species, including P. falciparum; Toxoplasma species, including T. gondii (or antigens, such as, for example SAG2, SAG3, Tg34); Entamoeba species, including E. histolytica; Babesia species, including B. microti; Trypanosoma species, including T cruzi; Giardia species, including G. lamblia; Leshmania species, including L. major, Pneumocystis species, including P. carinii; Trichomonas species, including T. vaginalis; and Schisostoma species, including S. mansoni.
  • the infectious agent is a fungus, or a fungal derived product.
  • the infectious agent is a protozoan, or a protozoan derived product.
  • Suitable protozoans (or protozoan derived products) for use in the vaccines and/or methods of the invention include, without limitation, protests (unicellular or multicellular), e.g., Plasmodium falciparum, and helminths, e.g., cestodes, nematodes, and trematodes.
  • the infectious disease vaccine is a respiratory disease vaccine.
  • the respiratory disease vaccine may be selected from a vaccine against Haemophilus influenza, a coronavirus, such as SARS-CoV-2, Haemophilus influenzae type B, measles virus, poliovirus, tetanus, tuberculosis, cholera, typhoid, dengue, diphtheria, hepatitis, Japanese encephalitis, meningococcal meningitis, mumps virus, pertussis, pneumococcal disease, rabies, rotavius, rubella, coronavirus, such as SARS-CoV-2, SARS-CoV, or MERS-CoV,
  • the infectious disease vaccine is a viral respiratory disease vaccine.
  • the viral respiratory disease vaccine may be selected from a vaccine against Haemophilus influenza, Haemophilus influenzae type B, measles virus, poliovirus, mumps virus, rubella, coronavirus, such as SARS-CoV-2, SARS-CoV, or MERS-CoV.
  • influenza hemagglutinin of influenza is one of the two main glycoproteins on the viral surface and a major target of neutralizing antibodies. Based on structure and antigenicity, there are eighteen defined subtypes (H1-H18) of IAV HAs belonging to two broad groups.
  • Influenza HA consists of an antigenically variable globular head domain containing the receptor-binding site (RBS) for viral attachment and a more conserved stem domain that mediates fusion of viral and cell membranes in the endosome.
  • the HA head domain is the immunodominant domain of the protein and is the target of most antibody responses induced by IAV vaccine or infection.
  • Influenza as used herein may refer to either influenza A, B, C or D.
  • Influenza A includes any of the influenza A subtypes including but not limited to H1 N1 , H3N2.
  • the vaccine is directed against influenza A or B.
  • Zoonotic influenza virus with H1 , H3, H5, H6, H7, H9 and H10 Has are also included.
  • the vaccine is directed against a coronavirus.
  • Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses composed of several proteins including the Spike (S), Envelope (E), Membrane (M) and Nucleocapsid (N) proteins.
  • S Spike
  • E Envelope
  • M Membrane
  • N Nucleocapsid
  • S glycoprotein is highly immunogenic with its receptor-binding domain (RBD) being a major target of the humoral response.
  • RBD receptor-binding domain
  • 2019 novel coronavirus 2019-nCoV
  • SARS-CoV-2 currently known as SARS-CoV-2, emerged in Wuhan city of the Chinese province of Hubei in December 2019.
  • SARS-CoV-2 causes a respiratory disease designated CoVID-19, with a median incubation period of 7 days (min: 3 days, max: 14 days).
  • Common symptoms include fever, cough, shortness of breath, muscle pain and fatigue while, in severe cases, the infection can lead to pneumonia, extensive lung damage and death, particularly in aged patients and in those with various underlying conditions.
  • the coronavirus is selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1.
  • the coronavirus is SARS-CoV-2.
  • SARS-CoV-2 includes any variant of SARS-CoV-2, including but not limited to D614G variant, “Cluster 5” variant, N501Y variant.
  • the infectious disease vaccine is selected from a whole pathogen vaccine, live attenuated vaccine, inactivated vaccine, whole killed vaccine, recombinant protein vaccine, toxoid vaccine, conjugate vaccine, virus-like particle (VLP) vaccine, outer membrane vesicle (OMV) vaccine, DNA vaccine, RNA vaccine, peptide vaccine or viral vectored vaccine.
  • the vector is an adenoviral vector.
  • a whole pathogen virus uses the whole disease-causing pathogen to produce an immune response similar to that seen during natural infection.
  • a live attenuated vaccine utilises whole bacteria or viruses which have been weakened/attenuated so that they create a protective immune response but do not cause disease in healthy people. Attenuation may be achieved through genetic modification of the pathogen either as a naturally occurring phenomenon or as a modification specifically introduced.
  • Inactivated vaccines comprise whole bacteria or viruses which have been killed or have been altered, so that they cannot replicate.
  • a recombinant protein vaccine comprises one or more specific antigens from the surface of the pathogen and a generally acellular.
  • a toxoid vaccine comprises one or more specific antigens from the surface of a toxin produced by a pathogen.
  • a conjugate vaccine comprises a polysaccharide conjugated to an antigen from the cell surface of a pathogen or from a toxin produced by a pathogen.
  • a VLP comprises molecules that closely resemble viruses, but are non-infectious because they contain no viral genetic material. VLPs may be naturally occurring or synthesized through the individual expression of viral structural proteins, which can then self-assemble into the virus-like structure. In some cases, the antigens in a VLP vaccine are the viral structural proteins themselves. Alternatively, the VLPs can be manufactured to present antigens from another pathogen on the surface, or even multiple pathogens at once. As each VLP has multiple copies of an antigen on its surface it is more effective at stimulating an immune response that a single copy.
  • An OMV are vesicles which are naturally produced from the outer membrane of the bacterial outer cell wall and comprise many of the antigens found on the cell membrane but is a non-infectious particle.
  • An OMV may be modified so that toxic antigens are removed and antigens suitable for stimulating an immune response can be kept.
  • Nucleic acid vaccines comprise genetic material to stimulate a cell to produce the desired antigen.
  • RNA vaccines comprise mRNA inside a lipid membrane, wherein the mRNA encodes the desired antigen.
  • DNA vaccine comprise DNA wherein the DNA encodes the desired vaccine.
  • Viral vectored vaccines utilise viruses to deliver the genetic code of target vaccine antigens to cells of the body, in general the virus is harmless or has been modified to reduce virulence.
  • Viral vectored vaccine may be replicating or non-replicating.
  • Replicating viral vectors retain the ability to make new viral particles alongside delivering the vaccine antigen when used as a vaccine delivery platform.
  • Non-replicating viral vectors do not retain the ability to make new viral particles during the process of delivering the vaccine antigen to the cell.
  • the virus In non-replicating viral vectored vaccines, the virus has been modified to remove key viral genes that enable the virus to replicate.
  • the KDACi may be administered in combination with any commercially available infectious disease vaccine.
  • the infectious disease vaccine is a viral respiratory disease vaccine.
  • the infectious disease vaccine is against SARS-CoV-2 or influenza.
  • the vaccine is against coronavirus and may be selected from the following non-limiting list: Comirnaty (BNT162b2; Pfizer/BioNTech), mRNA-1273 (Moderna), AZD1222; (Vaxzevria, Covishield; AstraZeneca), Sputnik V (Gamaleya Research Institute, Acellena Contract Drug Research and Development), Sputnik Light (Gamaleya Research Institute, Acellena Contract Drug Research and Development), JNJ- 78436735 (Ad26.COV2.S; Johnson & Johnson), CoronaVac (Sinovac), BBlBP-CorV (Beijing Institute of Biological Products; China National Pharmaceutical Group (Sinopharm), EpiVacCorona (Federal Budgetary Research Institution State Research Center of Virology and Biotechnology) and Convidicea (PakVac, Ad5-nCoV; CanSino Biologies).
  • Comirnaty BNT162b2; Pfizer/BioN
  • the vaccine is against influenza and may be an inactivated influenza vaccines (I IV) and live attenuated influenza vaccines (LAIV).
  • the vaccine is a trivalent vaccine.
  • the vaccine is sleeted from Afluria, Fluarix, Flublok, Flulaval, Fluvirin or Fluzone.
  • non-vaccine strains refers to strains which the vaccine was not originally developed against.
  • the present invention provides methods of enhancing an immune response in a subject comprising administering a KDACi simultaneously, sequentially, or separately with an infectious disease vaccine, wherein the immune response enhances protection against non-vaccine strains of the infectious disease.
  • the inventors have also surprisingly shown that the effects of the KDACi, in particular valproate, on expanding memory are achieved when a low dose is administered. This dose is lower than the dosages used for KDACi, in particular valproate, for the treatment of cancer.
  • the KDACi preferably valproate
  • the KDACi may be administered at a dose of 10 to 950 mg/day, 15 to 850 mg/day, 20 to 750 mg/day, 25 to 650 mg/day, 30 to 550 mg/day, 35 to 450 mg/day, 40 to 350 mg/ day, 50 to 250 mg/day, 75 to 225 mg/day, 80 to 220 mg/day, 85 to 215mg/day, 90 to 210 mg/day, 95 to 205 mg/day, preferably at a dose of 100 to 200 mg/day.
  • the KDACi may be administered at a dose of 50 mg/day, 75 mg/day, 100 mg/day, 125 mg/day, 150 mg/day, 175 mg/day, 200 mg/day, 225 mg/day, 250 mg/day.
  • the KDACi may be administered at a dose of approximately 100 mg/day, approximately 100 mg/day may encompass a dose between 95 to 105 mg/day.
  • the KDACi may be administered at a dose of approximately 200 mg/day, approximately 200 mg/day may encompass a dose between 195 to 205 mg/day.
  • the dose administered per day is referred to as the total dose per day, as such the total dose may be split into multiple doses throughout the day, for example where a dose of 200mg/day is administered this may be split into two separate 100mg doses.
  • the total dose per day may be split into multiple administrations, for example the total dose may be split into 2, 3, 4, 5, or 6 administrations per day.
  • the KDACi preferably valproate
  • the KDACi, preferably valproate is administered at a dose of 200 mg/day.
  • the KDACi, preferably valproate is administered at a dose of 200 mg/day and the dose is split into two 100 mg doses taken at two separate time point during the day.
  • the correct dosage of the KDACi may vary according to the particular formulation, the mode of administration, and its particular site, host. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities may be taken into account.
  • abexinostat may be administered at a dose of 2 to 10 nM, 2.5 to 9.5 nM, 3 to 9 nM, 3.5 to 8.5 nM, 4 to 8 nM. 4.5 to 7.5 nM, 2.5 to 7.5 nM, 3.5 to 6.5 nM, 4.5 to 5.5 nM.
  • Abexinostat may be administered at a dose of 2nM, 5nM or 10nM.
  • Abexinostat may be administered at the clinically approved dose range of 0.1 pM.
  • a method for enhancing an immune response in a subject comprising administering abexinostat simultaneously, sequentially, or separately with a vaccine.
  • the abexinostat is administered at up to 50% of the dose recited in Table 1 . In an embodiment the abexinostat is administered in a dose of 2 to 10 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering abexinostat simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the abexinostat is administered in a dose of 2 to 10 nM.
  • belinostat may be administered at a dose of 5 to 800 nM, 5 to 700 nM, 5 to 600 nM, 5 to 500 nM, 5 to 400 nM, 5 to 300 nM, 5 to 200 nM, 5 to 100 nM, 5 to 50 nM, 5 to 40 nM, 5 to 30 nM, 5 to 20 nM, 5 to 15 nM., 10 to 800 nM, 10 to 700 nM, 10 to 600 nM, 10 to 500 nM, 10 to 400 nM, 10 to 300 nM, 10 to 200 nM, 10 to 100 nM, 10 to 50 nM, 10 to 40 nM, 10 to 30 nM, 10 to 20 nM, 10 to 15 nM.
  • Belinostat may be administered at a dose of 5 nM, 10 nM, or 15 nM. Belinostat may be administered at the clinically approved dose of 1 pM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering belinostat simultaneously, sequentially, or separately with a vaccine. In an embodiment the bellinostat is administered at up to 50% of the dose recited in Table 1. In an embodiment the belinostat is administered in a dose of 5 to 800 nM.
  • an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering belinostat simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the belinostat is administered in a dose of 5 to 800 nM.
  • panobinostat may be administered at a dose of 0.5 to 500 nM, 0.5 to 400 nM, 0.5 to 300 nM, 0.5 to 200 nM, 0.5 to 100 nM, 0.5 to 50 nM, 0.5 to 40 nM, 0.5 to 30 nM, 0.5 to 20 nM, 0.5 to 10 nM, 0.5 to 9 nM, 0.5 to 8 nM, 0.5 to 7 nM, 0.5 to 6 nM, 0.5 to 5 nM, 0.5 to 4 nM, 0.5 to 3 nM, 0.5 to 2 nM, 1 to 500 nM, 1 to 400 nM, 1 to 300 nM, 1 to 200 nM, 1 to 100 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, 1 to 10 nM, 1 to 9 nM, 1 to 8 nM
  • Panobinostat may be administered at a dose of 0.5 nM, 1 nM, or 2 nM. Panobinostat may be administered at the clinically approved dose of 500 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering panobinostat simultaneously, sequentially, or separately with a vaccine. In an embodiment the panobinostat is administered at up to 50% of the dose recited in Table 1 . In an embodiment the panobinostat is administered in a dose of 0.5 to 500 nM.
  • panobinostat in an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering panobinostat simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the panobinostat is administered in a dose of 0.5 to 500 nM.
  • butyrate may be administered at a dose of 0.1 x10 3 to 500x10 3 nM, 0.1x10 3 to 400x10 3 nM, 0.1x10 3 to 300x10 3 nM, 0.1x10 3 to 200x10 3 nM, 0.1x10 3 to 100x10 3 nM, 0.1x10 3 to 50x10 3 nM, 0.1x10 3 to 10x10 3 nM, 0.1x10 3 to 5x10 3 nM, 0.1 x10 3 to 1x10 3 nM, 0.1 x10 3 to 0.05x10 3 nM, 0.5 x10 3 to 500x10 3 nM, 0.5 x10 3 to 400x10 3 nM, 0.5 x10 3 to 300x10 3 nM, 0.5 x10 3 to 200x10 3 nM, 0.5 x10 3 to 100x10 3 nM, 0.5 x10 3 to 50x10 3 .
  • Butyrate may be administered at a dose of 0.5x10 3 nM, 1x10 3 nM, 2x10 3 nM. Butyrate may be administered at the clinically approved dose of 200 to 800pM.
  • a method for enhancing an immune response in a subject comprising administering butyrate simultaneously, sequentially, or separately with a vaccine.
  • the butyrate is administered at up to 50% of the dose recited in Table 1.
  • the butyrate is administered in a dose of 0.1 to 500x10 3 nM.
  • a method for enhancing an immune response in a subject comprising administering butyrate simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the butyrate is administered in a dose of 0.1 x10 3 to 500x10 3 nM.
  • apicidin may be administered at a dose of 0.01 to 100 nM, 0.01 to 90 nM, 0.01 to 80 nM, 0.01 to 70 nM, 0.01 to 60 nM, 0.01 to 50 nM, 0.01 to 40 nM, 0.01 to 30 nM, 0.01 to 20 nM, 0.01 to 10 nM, 0.01 to 5 nM, 0.01 to 1 nM, 0.01 to 0.9 nM, 0.01 to 0.8 nM, 0.01 to 0.7 nM, 0.01 to 0.6 nM, 0.01 to 0.5 nM , 0.01 to 0.4 nM, 0.01 to 0.3 nM, 0.01 to 0.2 nM, 0.01 to 0.1 nM, 0.01 to 0.09 nM, 0.01 to 0.08 nM, 0.01 to 0.07 nM, 0.01 to 0.06 nM, 0.01 to 0.05 nM, 0.01 to 0.04
  • Apicidin may be administered at a dose of 0.01 nM, 0.05 nM, or 0.1 nM. Apicidin may be administered at the clinically approved dose of 160 nM.
  • a method for enhancing an immune response in a subject comprising administering apicidin simultaneously, sequentially, or separately with a vaccine.
  • the apicidin is administered at up to 50% of the dose recited in Table 1.
  • the apicidin is administered in a dose of 0.01 to 100 nM.
  • a method for enhancing an immune response in a subject comprising administering apicidin simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the apicidin is administered in a dose of 0.01 to 100 nM.
  • mocetinostat may be administered at a dose of 0.01 x10 3 to 1x10 3 nM, 0.01 x10 3 to 0.9x10 3 nM, 0.01 x10 3 to 0.8x10 3 nM, 0.01 x10 3 to 0.7x10 3 nM, 0.01 x10 3 to 0.6x10 3 nM, 0.01 x10 3 to 0.5x10 3 nM, 0.01 x10 3 to 0.4x10 3 nM, 0.01 x10 3 to 0.3x10 3 nM, 0.01 x10 3 to 0.2x10 3 nM, 0.01 x10 3 to 0.1x10 3 nM, 0.1 x10 3 to 1x10 3 nM, 0.1 x10 3 to 0.9x10 3 nM, 0.1 x10 3 to 0.8x10 3 nM, 0.1 x10 3 to 0.7x10 3 nM, 0.1 x10
  • Mocetinostat may be administered at a dose of 0.1 x10 3 nM, 0.5 x10 3 nM, 1 x10 3 nM. Mocetinostat may be administered at the clinically approved dose of 100 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering mocetinostat simultaneously, sequentially, or separately with a vaccine. In an embodiment the mocetinostat is administered at up to 50% of the dose recited in Table 1 . In an embodiment the mocetinostat is administered in a dose of 0.01 to 1x10 3 nM.
  • a method for enhancing an immune response in a subject comprising administering mocetinostat simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the mocetinostat is administered in a dose of 0.01 x10 3 to 1x10 3 nM.
  • vorinostat may be administered at a dose of 0.2 x10 3 to 5x10 3 nM, 0.2 x10 3 to 4x10 3 nM, 0.2 x10 3 to 3x10 3 nM, 0.2 x10 3 to 2x10 3 nM, 0.2 x10 3 to 1x10 3 nM, 0.2 x10 3 to 0.5x10 3 nM, 0.2 x10 3 to 0.4x10 3 nM, 0.2 x10 3 to 0.3x10 3 nM, 1 x10 3 to 5x10 3 nM, 1 x10 3 to 4.5x10 3 nM, 1 x10 3 to 4x10 3 nM, 1 x10 3 to 3.5x10 3 nM, 1 x10 3 to 3x10 3 nM, 1 x10 3 to 2.5x10 3 nM, 1 x10 3 to 2x10 3 nM, 1 x10 3 to
  • Vorinostat may be administered at a dose of 1 .25 x10 3 nM, 2.5 x10 3 nM, 5 x10 3 nM. Vorinostat may be administered at the clinically approved dose of 500 nM.
  • a method for enhancing an immune response in a subject comprising administering vorinostat simultaneously, sequentially, or separately with a vaccine.
  • the vorinostat is administered at up to 50% of the dose recited in Table 1.
  • the vorinostat is administered in a dose of 0.2 to 5x10 3 nM.
  • a method for enhancing an immune response in a subject comprising administering vorinostat simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the vorinostat is administered in a dose of 0.2 x10 3 to 5x10 3 nM.
  • MS275 may be administered at a dose of 0.01x10 6 to 1x10® nM, 0.01x10 B to 0.9x10 6 nM, 0.01x10 6 to 0.8x10 6 nM, 0.01x10 6 to 0.7x10 6 nM, 0.01x10® to 0.6x10® nM, 0.01x10® to 0.5x10® nM, 0.01x10® to 0.4x10® nM, 0.01x10® to 0.3x10® nM, 0.01x10®to 0.2x10® nM, 0.01x10®to 0.1x10® nM, 0.01x10®to 0.09x10® nM, 0.01x10® to 0.08x10® nM, 0.01x10® to 0.07x10® nM, 0.01x10® to 0.06x10® nM, 0.01x10® to 0.05x10® nM, 0.01x10® to 0.04x
  • MS275 may be administered at a dose of 0.25x10® nM, 0.5x10® nM, 1x10® nM. MS275 may be administered at the clinically approved dose of 13 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering MS275 simultaneously, sequentially, or separately with a vaccine. In an embodiment the MS275 is administered at up to 50% of the dose recited in Table 1.
  • an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering MS275 simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the MS275 is administered in a dose of 0.01x10® to 1x10® nM,
  • CI994 may be administered at a dose of 0.01x10® to 2x10® nM, 0.01x10® to 1x10® nM, 0.01x10® to 0.9x10® nM, 0.01x10® to 0.8x10® nM, 0.01x10® to 0.7x10® nM, 0.01x10® to 0.6x10® nM, 0.01x10® to 0.5x10® nM, 0.01x10® to 0.4x10® nM, 0.01x10® to 0.3x10® nM, 0.01x10® to 0.2x10® nM, 0.01x10® to 0.1x10® nM, 0.01x10® to 0.09x10® nM, 0.01x10® to 0.08x10® nM, 0.01x10® to 0.07x10® nM, 0.01x10® to 0.06x10® nM, 0.01x10® to
  • CI994 may be administered at a dose of 0.1x10® nM, 0.5x10® nM, 1x10® nM. CI994 may be administered at the clinically approved dose of 2pM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering CI994 simultaneously, sequentially, or separately with a vaccine. In an embodiment the CI994 is administered at up to 50% of the dose recited in Table 1 .
  • the CI994 is administered in a dose of 0.01x10 B to 2x10 B nM
  • a method for enhancing an immune response in a subject comprising administering CI994 simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the CI994 is administered in a dose of 0.01x10 B to 2x10 B nM.
  • the KDACi can be administered simultaneously, sequentially or separately with a vaccine. It is not necessary that the KDACi and vaccine are packed together (but this is one embodiment of the invention). It is also not necessary that they are administered at the same time.
  • "separate" administration means that the drugs are administered as part of the same overall dosage regimen (which could comprise a number of days), but preferably on the same day.
  • "simultaneously” means that the drugs are to be taken together or administered together, or formulated as a single composition.
  • the KDACi and the vaccine may be formulated separately but administered together, via the same or different administration route. For example, the vaccine may be administered subcutaneously or intravenously and the KDACi may be administered orally.
  • “sequentially” means that the drugs are administered at about the same time one after another, and preferably within about 1 hour of each other.
  • the dosage regimen of the KDACi and the vaccine may comprise day 0 for example when the KDACi and vaccine are administered simultaneously.
  • the dosage regimen may comprise additional days wherein further doses of the KDACi and/or the vaccine are administered.
  • the further dose of KDACi may be administered in the absence of the vaccine.
  • the KDACi and the vaccine are administered simultaneously, sequentially or separately on day 0, and a further dose of the KDACi is administered on any or all of days 1 to 14, 1 to 13, 1 to 12, 1 to 11 , 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or day 1 following the administration of the KDACi and the vaccine.
  • Further doses of the KDACi may be administered on any or all of days 1 to 2, days 1 to 3, days 1 to 4, days 1 to 5, days 1 to 6 or days 1 to 7.
  • the KDACi and the vaccine are administered simultaneously, sequentially or separately on day 0, and a further dose of the KDACi is administered on any or all of days 1 to 7 following the administration of the KDACi and the vaccine.
  • subjects are administered the SARS-CoV-2 vaccination on day 0 along with a 100mg dose of sodium valproate.
  • the sodium valproate will then be administered for 7 days following the vaccination at a daily dose of 100mg.
  • the same protocol can used be when the SARS-CoV-2 booster vaccination is administered.
  • subjects are administered the influenza vaccination on day 0 along with a 10Omg or 200mg dose of sodium valproate.
  • the sodium valproate will then be administered for 7 days following the vaccination at a daily dose of 100mg or 200mg.
  • the same protocol can be used when the booster vaccination is administered.
  • valproate may refer to valproic acid. Where valproate or valproic acid are is used in the methods of the present invention the compound may be administered as valproic acid or the sodium salt form of valproic acid i.e. sodium valproate, other salt forms may be suitable. Where a salt form is administered the salt form converts to the active form of valproate ion within the blood.
  • the KDACi is administered to enhance an immune response to an infectious agent.
  • administration of the KDACi either alone orwith a vaccine can increase the intensity, rate, and duration of immune response, and/or shorten onset time of antibody responses.
  • the immune response to the vaccine is enhanced compared to the immune response generated by administration of the vaccine alone to the subject.
  • the immune system encompasses cellular immunity and humoral immunity.
  • Cellular immunity includes a network of cells and events. Memory T cells are key components of the immune response, are antigen-specific, are developed after exposure and recognition of a particular antigen.
  • Humoral immunity involves B cells and antibodies. When B cells become transformed to plasma cells, the plasma cells express and secrete antibodies. The secreted antibodies can subsequently bind to antigens. Expansion of germinal centre (GCB) and expansion of T cell follicular helper cells (TFH) are critical for promoting a high affinity antibody response.
  • GCB germinal centre
  • T cell follicular helper cells T cell follicular helper cells
  • immune response is defined as any response of the immune system, for example, of either a cell-mediated or humoral (i.e. antibody mediated) nature. In particular, the response is humoral.
  • immune responses can be assessed by a number of in vivo or in vitro assays well known to one skilled in the art including, but not limited to, antibody assays (for example ELISA assays) antigen specific cytotoxicity assays, production of cytokines (for example ELISPOT assays), etc. Suitable assays are also shown in the examples.
  • the enhanced immune response includes one or more of the following: expansion of germinal centre (GCB), expansion of T cell follicular helper cells (TFH), and/or differentiation of stimulated primary human CD8+ T cells into MPEC and expansion of MPEC.
  • GCB germinal centre
  • T cell follicular helper cells T cell follicular helper cells
  • differentiation of stimulated primary human CD8+ T cells into MPEC is promoted.
  • using a vaccine together with a KDACi at the dose described herein can stimulate greater protection against the infectious disease immunogenic targets expressing the antigen in the vaccine.
  • using a vaccine together with a KDACi results in a immune response with a greater breath thereby providing protection against both the vaccine strain and non-vaccine strain of the infectious disease.
  • using a vaccine together with a KDACi could lead to enhanced clonal expansion and memory and a quicker/faster, more intense, and more prolonged humoral response upon re-challenge to the antigen in question.
  • the combination of vaccine and a KDACi can produce an additive or synergistic effect and achieve greater benefit than using vaccine alone; and the combination also allows for possible use of smaller doses of the vaccine to achieve protective antibody titers.
  • a vaccine administration comprises a prime boost dosage regimen
  • the KDACi may be administered, as described herein, with both the priming dose of the vaccine and the boost dose of the vaccine or only one of these doses.
  • the “prime boost dosage regimen” refers to a regimen of immunization with the same immunogen during the prime and booster doses or a regimen of priming the immune system with an immunogen and then boosting with a different immunogen.
  • the prime and boost doses may be administered one or more weeks apart, one or more months apart or one or more years apart.
  • the KDACi is also administered separately, sequentially or simultaneously.
  • KDACi On subsequent days further doses of the KDACi may be administered, for example on any or all of days 1 to 14, 1 to 13, 1 to 12, 1 to 11 , 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or day 1 following the administration of the KDACi and the vaccine.
  • a prime boost dosage regimen is common with vaccines directed against SARS-CoV-2 and so this administration regimen, wherein the KDACi is administered with both the priming and boost doses of the SARS-CoV-2 vaccine, may be adopted.
  • the KDACi may be administered either with the priming dose of the vaccine or with the boost dose of the vaccine.
  • Administration of the KDACi and/or the vaccine may be via any reasonable route, for example any parenteral or enteral route.
  • any convenient route includes but is not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation.
  • Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration.
  • the KDACi or the infectious disease vaccine can take the form of one or more dosage units.
  • Administration of the KDACi may be performed by the oral, sublingual, buccal, intravenous, intramuscular, or subcutaneous route. Where more than one administration of the KDACi is used, the same or different routes of administration may be used.
  • Various oral dosage forms can be used, including such solid forms as tablets, capsules, liquids, granules and bulk powders. Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple- compressed, containing suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents.
  • Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents.
  • administration of the vaccine may be performed by intravenous, intramuscular, intradermal, intranasal, or subcutaneous route.
  • the vaccine and/or the KDACi can be in the form of a liquid, e.g., a solution, emulsion or suspension.
  • the liquid compositions can also include one or more of the following: sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides, polyethylene glycols, glycerin, or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the composition can be enclosed in an ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material.
  • An intravenous formulation of the vaccine or the KDACi may be in the form of a sterile injectable aqueous or non-aqueous (e.g. oleaginous) solution or suspension.
  • the sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example, a solution in 1 ,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, phosphate buffer solution, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils may be employed as a solvent or suspending medium.
  • any bland fixed oil may be employed, including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid may be used in the preparation of the intravenous formulation of the invention.
  • the vaccine or the KDACi can be prepared using methodology well known in the pharmaceutical art.
  • a composition intended to be administered by injection can be prepared by combining with water so as to form a solution.
  • a surfactant can be added to facilitate the formation of a homogeneous solution or suspension.
  • the subject is a mammal, preferably a human. It has been shown herein that the administration of the KDACi valproate in conjunction with a vaccine was able to enhance both the naive T cell response and the memory response.
  • the methods of the invention are particularly suitable for use in the elderly, children or immunocompromised subjects.
  • the subject is over the age of 65.
  • the subject is between the age of 1 year and 18 years old.
  • the subject is immunocompromised or immunosuppressed. Immunocompromised or immunosuppressed subject may include; transplant patients, subjects with weakened immune system, subjects taking immunosuppressive drugs, subjects with HIV/AIDS, cancer, diabetes or genetic disorders.
  • the invention relates to a KDACi for use in a method of enhancing an immune response in a subject comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine.
  • the invention also relates to a KDACi for use in the prevention or treatment of a disease, wherein the KDACi is administered simultaneously, sequentially, or separately with a vaccine, and wherein the KDACi is capable of enhancing glutaminolysis.
  • the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
  • the invention also relates to a KDACi for use in the treatment of a disease, wherein KDACi is capable of enhancing glutaminolysis.
  • KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 .
  • the invention also relates to a KDACi for use in the prevention of SARS-CoV-2, wherein the KDACi is administered simultaneously, sequentially, or separately with a SARS-CoV-2 vaccine.
  • the KDACi is administered at a dose of 10 to 950 mg/day.
  • the KDACi is valproate.
  • the invention also relates to a KDACi for use in the treatment of SARS-CoV-2, preferably wherein the KDACi is administered at a dose of 10 to 950 mg/day.
  • the KDACi is valproate.
  • the invention also relates to a KDACi for use in the prevention of influenza, wherein the KDACi is administered simultaneously, sequentially, or separately with an influenza vaccine, preferably wherein the KDACi is administered at a dose of 10 to 950 mg/day.
  • the KDACi is valproate.
  • the invention also relates to a KDACi for use in the treatment of influenza, preferably wherein the KDACi is administered at a dose of 10 to 950 mg/day.
  • the KDACi is valproate.
  • the invention also relates the use of a KDACi in the manufacture of a medicament for the prevention and/or treatment of a disease wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
  • the KDACi is administered together with an infectious disease vaccine.
  • the terms “treat”, “treatment” or “treating,” as used herein refer to administering a compound to a subject for prophylactic and/or therapeutic purposes.
  • the KDACi is for use as part of an immunization regimen
  • the KDACi is for use in the prevention of an infectious disease by enhancing the production of memory precursor effector cells.
  • the KDACi can also be used to treat disease, in particular at the early onset of the disease.
  • the KDACi is being used to prevent the onset of an infectious disease and is administered prophylactically together with a vaccine.
  • prevention refers to preventing the onset of an infectious disease or preventing an infection.
  • prevention can refer to a reduction in the risk of contracting a disease.
  • prophylactic or preventative treatment refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition.
  • therapeutic treatment refers to administering treatment to a subject already suffering from a disease or condition.
  • the invention also relates to an in vivo, in vitro or ex vivo method of promoting a T cell memory response, comprising exposing cells to a KDACi.
  • the KDACi is administered at a dose as set out herein.
  • the KDACi may enhance the T cell memory response.
  • the enhanced T cell memory response may be characterised by an expanded population of memory precursor effector cells. Memory precursor effector cells can be characterized by the presences of specific markers and cell surface markers. Methods to identify and quantify these markers are well known in the art.
  • Memory precursor effector cells may be characterised by a number of markers, examples include but are not limited to; ILR7 hi PD1 10 .
  • the term " hi ” and “ l0 ” refer to relative expression of these markers on the cells.
  • Io may referto cells wherein there is no expression of the markers, it may also refer to cells wherein there is low expression of the markers relative to other cells in the sample.
  • hi may refer to cells wherein there is high expression of the markers, for example where there is high expression of the markers relative to other cells in the sample.
  • the invention also relates to an in vivo, in vitro or ex vivo method of promoting differentiation of CD8+ T cells into memory precursor effector cells, comprising exposing CD8+ T cells to a class I KDACi.
  • CD8+ T cells as used herein relates to human CD8+ T cells.
  • differentiation refers to cell differentiation and has is usual meaning in the art and refers to the process wherein a cell transitions from one cell type to another. Cell differentiation can be characterised by the presence of cell surface markers.
  • CD8+ T cells may be identified by the following cell surface markers CD3+CD8+.
  • the transition of CD8+ T cells into memory precursor effector cells may be characterised by an increase in cell population expressing ILR7 hi PD1 10 .
  • CD8+ T cells may be obtained from a subject and then exposed in vitro/ex vivo in order to stimulate differentiation of the CD8+ T cells into memory precursor effector cells.
  • the expanded population of memory precursor effector cells may be reintroduced to a subject.
  • the KDACi is administered at a dose as set out herein.
  • KDACi stimulates expression of IL7R (interleukin-7 receptor).
  • An aspect of the invention related to an in vivo, in vitro or ex vivo method of enhancing IL7R expression in CD8+ T cells, comprising exposing CD8+ T cells to a KDACi.
  • IL7R plays a key role in the development of lymphocytes and VDJ recombination.
  • Enhanced expression of IL7R may be detected using standard techniques including but not limited to include but are not limited to include but are not limited to affinity-based separation methods, magnetic cell sorting techniques, fluorescence-based cell sorting techniques such as FACS (fluorescence activated cell sorting).
  • FACS fluorescence activated cell sorting
  • Adoptive cell therapy is a type of immunotherapy wherein T cells are provided to a subject in order to enhance the subjects immune response to diseases such as cancer.
  • Types of adoptive cell therapy include chimeric antigen receptor T-cell (CAR T-cell) therapy and tumor-infiltrating lymphocyte (TIL) therapy.
  • An aspect of the invention relates to a method for expanding cells for use in adoptive cell therapy comprising: providing a population of CD8+ T cells, contacting the population of CD8+ T cells to a KDACi capable of enhancing glutaminolysis to produce an expanded population of cells.
  • the term “expanding cells” as used herein refers to culturing cells under conditions such that the number of cells increases.
  • the resultant expanded population of cells comprises memory precursor effector cells.
  • the expanded population of cells may be characteristed by specific cell surface markers for example the memory precursor effector cells are characterised by the markers IL7Rhi, PD1 lo.
  • the expanded population of memory precursor effector cells of are further characterised by the markers IL7Rhi, PD1 lo, CX3CR1 , CCR7, CD27 and CD62L.
  • the invention also provides a method of providing a population of cells enriched for memory precursor effector cells comprising: providing a population of CD8+ T cells, contacting the population of CD8+ T cells to a KDACi capable of enhancing glutaminolysis to produce an expanded population of cells.
  • the invention relates to a method of treatment comprising administering a population of cells expanded by the methods described herein.
  • An embodiment relates to an expanded population of cells for use in therapy wherein the expanded population of cells are obtained by the following method: providing a population of CD8+ T cells, contacting the population of CD8+ T cells to a KDACi capable of enhancing glutaminolysis to produce an expanded population of cells.
  • the cells for use in therapy or methods of treatment may be characterised as memory precursor effector cells as identified by one or more of the cell surface markers IL7Rhi, PD1 lo, CX3CR1 , CCR7, CD27 and CD62L, in particular IL7Rhi, PD1 lo.
  • the cells may be for use in adoptive cell therapy
  • glutaminolysis refers to the process which feeds the TCA cycle through the generation of a- ketoglutarate.
  • the process of glutaminolysis comprises a series of biochemical reactions by which the amino acid glutamine is lysed to glutamate, aspartate, CO2, pyruvate, lactate, alanine and citrate.
  • an aspect of the invention relates to an in vivo, in vitro or ex vivo method of enhancing glutaminolysis, comprising exposing CD8+ T cells to a KDACi.
  • the KDACi is administered at one of the doses as set out above.
  • valproate is used as the KDACi
  • valproate is administered at a dose of 10 to 950 mg/day.
  • cells are exposed to the KDACi in vivo, in vitro or ex vivo.
  • the cells may be exposed to the KDACi by administering the KDACi directly to the subject.
  • cells may be obtained from a subject and exposed to the KDACi. Once the cells have been exposed to the KDACi the cells may be returned to the subject.
  • the cells may also be exposed to a viral respiratory disease vaccine.
  • the viral respiratory disease vaccine may be any vaccine as described herein.
  • the cells may be exposed to the viral respiratory disease vaccine in vivo, in vitro or ex vivo. As such the cells may be exposed to the viral respiratory disease vaccine by administering the viral respiratory disease vaccine directly to the subject. Alternatively, cells may be obtained from a subject and exposed to the viral respiratory disease vaccine. Once the cells have been exposed to the viral respiratory disease vaccine, the cells may be returned to the subject.
  • the invention relates to a method of immunising a subject against a disease, comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine, and wherein the KDACi is capable of enhancing glutaminolysis.
  • the invention relates to the use of a KDACi as an adjuvant for enhancing the efficacy of a vaccine.
  • adjuvant refers to a substance that enhances the immune system's response to the presence of an antigen.
  • An adjuvant is commonly used to improve the effectiveness of a vaccine.
  • the use of the KDACi as an adjuvant may be performed by administering the KDACi in combination with a vaccine, preferably an infectious disease vaccine, the KDACi and the vaccine may be administered separately, sequentially or simultaneously. Where the KDACi is used as an adjuvant for administration to a subject, it is provided in a dose as set out herein.
  • the KDACi is one of the listed in Table 1 and used at a dose of up to 50% of the dose set out in Table 1 for said KDACi.
  • valproate is used as the KDACi, valproate is administered at a dose of 10 to 950 mg/day.
  • the invention also relates to a composition comprising a KDACi and a vaccine wherein the KDACi is capable of enhancing glutaminolysis.
  • the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
  • the composition may comprise the KDACi a vaccine formulated together, preferably an infectious disease vaccine.
  • the composition may comprise one or more additional active ingredients, pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • the compositions may optionally include other drug actives.
  • the invention also relates to a kit comprising a KDACi and a vaccine and optionally instructions for use, wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
  • the kit may comprise the KDACi and a vaccine formulated together, preferably an infectious disease vaccine.
  • the kit may comprise the KDACi and i vaccine formulated separately.
  • the kit may also comprise one or more additional active ingredients, pharmaceutically acceptable carrier, diluent, excipient or adjuvant, and optionally instructions for use.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antiviral, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • substances which can serve as pharmaceutically-acceptable carriers or components thereof, are sugars, such as lactose, dextrose, glucose and sucrose; starches, such as com starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the TWEENS; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives
  • Example 1 Genomic drug repurposing expands immune memory following immunisation and infection Drug repurposing allows the rapid use of existing therapeutics for new disease indications and can be directed by matching disease-associated transcriptional changes to those induced by candidate small molecules.
  • KDACi lysine deacetylase inhibitory
  • MPEC memory precursor effector cell
  • KDACi treatment reproduced - and glutaminolysis inhibition reversed - the transcriptional memory signatures used for initial screening with concurrent epigenetic modification of Wnt pathway genes controlling glutamine breakdown.
  • KDACi sodium valproate
  • the high- throughput assay tested each compound’s ability to modulate differentiation of stimulated primary human CD8+ T cells into memory precursor effector cells (MPEC), characterised by increased expression of IL7R on dividing cells (Fig1 C).
  • MPEC memory precursor effector cells
  • Fig1 C memory precursor effector cells
  • 68 metabolites were found to be differentially expressed (Fig2D, 103) amongst which the ‘alanine, aspartate and glutamate’ pathway demonstrated strongest enrichment, greatest predicted pathway impact while showing similar enrichment amongst differentially-acetylated proteins also (Fig2E, Fig 6). Mapping differential metabolites onto this pathway revealed a specific and significant (FDR ⁇ 5%,) increase in both metabolites and acetylation of enzymes regulating glutamine breakdown and the TCA cycle (Fig2F).
  • Glutaminolysis feeds the TCA cycle through generation of a-ketoglutarate (aKG), creating energy and biosynthetic precursors such as nucleic acids and modulating cellular redox balance. It makes an important energetic contribution to T cell activation and can replenish TCA cycle intermediates as they are used for biosynthesis. Glutaminolysis is known to promote B cell memory formation and can fuel stem cell-like oxidative metabolism in cancer cells, sustaining chronic proliferation. This process is controlled by Wnt/B-catenin signalling, a pathway also known to arrest effector T cell differentiation and promote memory formation. However, the specific contribution of glutaminolysis to T cell memory formation remains unclear. We therefore sought to determine whether KDACi.
  • mem-induced glutaminolysis had a functional impact on T cell ‘metabolic switching’ - a relative increase in oxidative over glycolytic metabolism on activation that is a critical determinant of memory differentiation.
  • Low-dose valproate treatment enhanced oxidative metabolism and reduced the glycolytic rate (Fig2G-l) without changing cell activation, proliferation orglucose uptake (Fig2J, K).
  • Fig2G-l valproate-induced metabolic switching
  • Fig2L-M MPEC differentiation
  • MPEC differentiation could also be induced by bypassing glutaminolysis through direct provision of the cell-permeable aKG analogue dimethyl ketoglutarate (DMKG: Fig 7).
  • DMKG cell-permeable aKG analogue dimethyl ketoglutarate
  • RNAseq analysis showed marked changes in stimulated CD8+ T cell transcriptomes induced by valproate treatment (Fig3A, compared to vehicle-treated, stimulated controls) that could be reversed by concurrent inhibition of glutaminolysis (Fig3B).
  • Fig3A stimulated CD8+ T cell transcriptomes induced by valproate treatment
  • Fig3B glutaminolysis
  • Valproate treatment during primary infection gMHV, Fig4A
  • immunisation Nitrophenylacetyl(NP)-OVA, Fig4B
  • Fig4C primary intranasal infection with H3N2 influenza (A/HKx31) is followed by intranasal rechallenge with a recombinant H1 N1 strain (A/PR8/34).
  • the PR8 strain shares all internal proteins with HKx31 but is serologically distinct, allowing detection of secondary T cell responses without confounding effects of antibody mediated viral clearance.
  • Secondary influenza responses are characterised by expansion of antigen-specific T cells in mediastinal lymph nodes (MLN) followed by migration to lung with severe infection characterised by diffuse lymphocytic infiltration and virus-induced lung immunopathology.
  • MN mediastinal lymph nodes
  • Fig4D d30pi
  • Fig4E, F expanded secondary lymphoid responses on rechallenge characterised by enhanced expression of a memory phenotype (IL7RhiPD1 Io, Fig 4G) and reduced pulmonary lymphocytic infiltration (Fig4H).
  • a memory phenotype Io, Fig 4G
  • Fig4H reduced pulmonary lymphocytic infiltration
  • influenza model was specifically designed to test secondary T cell responses (with no sharing of B cell epitopes between primary infection and rechallenge), we also observed an expansion of germinal centre B (GCB) and T follicular helper cells (TFH), critical for promoting a high-affinity antibody response (Fig4H).
  • GCB germinal centre B
  • T follicular helper cells T follicular helper cells
  • Fig 4L Treated animals again showed expansion of germinal centre and plasmablast responses (Fig 4L) alongside a >2-fold expansion in the magnitude of RBD-specific B and T cell memory responses following peptide rechallenge (Fig 4L, M) along with a similar expansion in SARS-CoV-2 neutralising antibody titre (Fig 4N).
  • Example 2 Experimental medicine study in healthy volunteers receiving immune challenge by seasonal influenza vaccination with/without concurrent administration of low-dose sodium valproate
  • CD8 A*02:01
  • Samples will be processed in batches to minimize batch artefact structure once all have been collected. Serum samples will be used to quantitate influenza specific humoral responses (ELISA, HAI titre) at each timepoint while frozen, viable cell aliquots will be used to identify and sort influenza specific CD8 and CD4 T cells for quantitation and sequencing.
  • ELISA ELISA
  • HAI titre ELISA, HAI titre
  • viable cell aliquots will be used to identify and sort influenza specific CD8 and CD4 T cells for quantitation and sequencing.
  • the immune traits being measured as outcomes in this study design have been developed taking into account previous evidence of immunological correlates of protection following influenza vaccination:
  • Influenza responses to seasonal vaccination are demonstrably lower in aged individuals compared to younger vaccines.
  • influenza-specific IgA titre in plasma was expanded > 10-fold in the treatment group on day 7 post-vaccination although levels had fallen in both groups by day 30 (Influenza A/Hong Kong H3, Fig 5F).
  • influenza-specific HAI titre was increased >1 .5 fold in the treatment group (Fig 5G) with much of the observed humoral response due to the lowest dose treatment group .
  • the impact on the CD8+ T cell response was observed to be greater with the higher dose treatment.
  • KDACi can have direct effects on histone acetylation and, while the reversal of treatment effects by inhibition of glutaminolysis (both in vitro and in vivo) suggests that epigenetic modulation is secondary to immunometabolic modulation, our experiments do not definitively differentiate these two possibilities.
  • KDACi can have immunomodulatory effects, and in particular that cell survival, proliferation and differentiation can be altered by certain compounds. Our data are consistent with this - we see a biphasic dose response relationship with limitations in cell cycle and proliferation at higher doses and promotion of cellular memory only at lower doses. Only through screening multiple KDACi compounds was the subclass effect apparent.
  • the MEMRI study described here indicates that low dose KDACi (valproate) treatment can significantly expand the size, breadth and durability of vaccine-induced cellular and humoral immunity (Fig 5).
  • the study does not confirm clinical efficacy of the observed expansion but uses validated correlates of vaccine-induced protection from infection as key endpoints.
  • Data from both murine and human studies are consistent with a model in which enhanced cellular and humoral responses are secondary to expanded germinal centre and TFH cell differentiation, rather than direct expansion of T cell memory alone.
  • Example 3 Experimental medicine study in healthy volunteers receiving immune challenge by SARS-CoV-2 vaccination with/without concurrent administration of low- dose sodium valproate
  • Subjects will be administered the SARS-CoV-2 vaccination on day 0 along with a 10Omg dose of sodium valproate. The sodium valproate will then be administered for 7 days following the vaccination. The same protocol will be used when the SARS-CoV-2 booster vaccination is administered.
  • Control subjects will also be included in the study who will not receive sodium valproate. Blood samples will be taken on the day of the first and second vaccinations as well as 6 months and 12 months after the first vaccination.
  • Purified T cells (>95%) werelabelled with 2.5pM CFSE (Invitrogen) and resuspended in complete RPMI 1640 (Sigma Aldrich) in the presence of 10% FCS and then stimulated in sterile, U-bottomed 96 well culture plates (Greiner) using MACS iBead particles (1 :2 bead:cell ratio, Miltenyi) conjugated to anti-CD2/CD3/CD28 in the presence of non-limiting IL2 (10ng/ml, Gibco Life technologies) for 6 days. Selected compounds were added at the time of stimulation (dO), or 2 (d2) or 4 (d4) days after as indicated.
  • T cell memory subsets were isolated using flow cytometric sorting (FACSArialll cell sorter, BD Biosciences) following MACS enrichment of CD8+ T cells as above, into naive (Tn, CD45RA+CD62L+), effector memory (Tern, CD45RA-CD62L-), central memory (CD45RA- CD62L+) and Temra (CD45RA+CD62L+) populations before co-culture with drugs as indicated.
  • T cells were stimulated in the presence of treatments as indicated for 6 days before undergoing high-throughput immunophenotypic screening (BD LSR Fortessa HTS) with quantification of IL7R.
  • BD LSR Fortessa HTS high-throughput immunophenotypic screening
  • IL7R BD Biosciences, Clone HIL-7R- M21
  • CD25 CD25
  • live/dead discrimination AquaFluorescent amine-reactive dye, Invitrogen.
  • Data was analysed using FlowJo software (Tree Star), extracting immunophenotypic traits (IL7Rmfi, division (CFSEdil), CD25mfi, % live cells) with ratios calculated against pooled vehicle treated controls in triplicate.
  • Compounds were selected from the in silico screen on the basis of consistent enrichment against multiple signatures taking into consideration likely impact on cellular viability, toxicity and availability. Additional ligands and cytokines with a demonstrated association with T cell memory formation were also included with Fc-Chimaeric ligands hybridised to individual wells of a 96- well U-bottomed culture plate (Greiner) for 2h at 37C before washing in sterile complete RPMI- 1640 and addition of cell suspensions for stimulation. Dose ranges for inclusion were selected on the basis of demonstrated in vitro IC50 for target effects with 3 doses included per treatment. Effects were considered by dose and also by peak effect per treatment with each value compared to the median of triplicate repeat vehicle stimulation.
  • SILAC Stable isotope labelling with amino acids in cell culture
  • MS mass spectroscopic
  • OCR oxygen consumption rate
  • ECAR XF Glycolysis Stress test
  • NBDG ThermoFisher N13195
  • live/dead AquaFluorescent amine-reactive dye, Invitrogen
  • IL7R BD Biosciences, Clone HIL-7R-M21
  • CD8+ T cells were stimulated and co-cultured in the presence of either a dose range of sodium valproate (Sigma) or of the specific glutaminolysis inhibitors GDH1 (Focus Biomolecules), C968 (Sigma) or BPTES (Sigma), or with the cell-permeable a- ketoglutarate analogue dimethyl-2-ketoglutarate (DMKG, Sigma) as indicated.
  • Raw sequence read QC was undertaken using fastqc followed by adapter removal (TrimGalore), extraction of ribosomal reads (BBsplit) and sequence mapping (HISAT2) to the reference genome (GRCh38).
  • Merged bam file QC was undertaken using the QoRTs package in R with assimilation using multiQC.
  • Merged bam files were read into R using featurecounts (Rsubread) with feature filtration to >cpm1 in >6 samples and annotation using BioMart followed by pairwise differential expression using DGEList (edgeR).
  • Matrix factorisation analysis was undertaken using the MOFA package in R with feature set enrichment performed using the Bioconductor package fgsea in R with gene sets extracted from MSigDB using the msigdbr package as indicated.
  • ATACseq was undertaken following the protocol established by Buenrostro et al 201545 with modifications. Following CD8+ T cell culture as above, 50,000 cells were lysed (1 M Tris-HCI) and the nuclear pellet transposed using the Tn5 transposase (TDE1 , Illumina) at 37C for 30min before DNA isolation (Qiagen 386 minElute) and PCR amplification (NEBnext) guided by qPCR (KAPA-SYBR) estimation of reporter signal v cycle number and PCR cycle length tailored to 1/3 maximal amplification. Libraries were purified (AMPure XP) with quality assessed using an Agilent BioAnalyser 2100 and RNA quantification performed using a NanoDrop ND-1000 spectrophotometer.
  • TDE1 Tn5 transposase
  • NEBnext PCR amplification
  • KAPA-SYBR qPCR
  • Primary CD8+ T cells were isolated, stimulated and cultured as above for 6 days in the presence of either sodium valproate (175pM, Sigma) or vehicle control ( ⁇ 0.001 %DMSO) followed by flow cytometric sorting (FACSArialll cell sorter, BD Biosciences) into divided CFSEIolL7Rhi and CFSEIolL7Rlo subpopulations. These were rested for 5 days in complete RPMI-1640 (Sigma), with normalization of live cell numbers (1x10 6 /ml) and CFSE-relabelling before polyclonal restimulation repeated as for the primary stimulation (anti- CD2/3/28, Miltenyi).
  • C57BL/6 mice were infected i/n with gMHV virus or immunised with 200pl NP-412 OVA in alum with spleens harvested d7 (NP-OVA) or d9pi (yMHV).
  • NP-OVA spleens harvested d7
  • yMHV d9pi
  • C57BL/6 mice were given a primary i/n challenge with 500pfu H3N2 influenza (A/HKx31) and a secondary challenge with 105pfu 415 i/n H1 N1 (A/PR8/34) influenza d31 post-primary challenge with tissues (lung, spleen, MLN, blood) harvested on d30pi and d38pi for stable memory and 417 rechallenge assessment respectively (Fig4C).
  • mice were administered sodium valproate 0.1 % w/v in drinking water, sodium valproate plus R162 (90% corn oil, 10% DMSO) 20mg/kg/day intraperitoneally or matched vehicle control as indicated (Fig4).
  • R162 90% corn oil, 10% DMSO
  • mice were administered receptor binding domain (RBD) peptide (gift, YM) 50pg s/c in QuilA adjuvant either once (R1) orthree times (R2) as indicated (Fig4K).
  • RBD receptor binding domain
  • Recombinant SARS-CoV-2 Spike RBD glycoprotein carrying a C-terminal hexahistidine purification (His6) tag was expressed in the Pichia pastoris X-33 yeast strain using the pPICZa A expression vector (ThermoFisher). Cultures were grown on the 10-liter scale in an Eppendorf BioFlo 510 fermenter with active control of temperature, pH, dissolved oxygen concentration and methanol feed (as the carbon source) to maximize protein yield based on a protocol developed previously to purify SARS-CoV-1 RBD46.47.
  • RBD glycoprotein was secreted into the cell culture supernatant and purified by nickel-affinity and size-exclusion chromatography. To produce untagged RBD, the His6 tag was removed using Carboxypeptidase A (Sigma), followed by nickel-affinity chromatography to remove uncleaved protein and size-exclusion chromatography.
  • Tissue processing Splenocytes and draining lymph nodes were weighed and then homogenised through a 70pm cell strainer (BD bioscience) using complete tissue culture media [IMDM (Thermo), 10% Fetal Bovine Serum (Sigma), 1 mM Sodium Pyruvate (Thermo), 1 X Non-essential Amino Acid Solution (Sigma), 2mM 444 Glutamax (Thermo), 100U/ml Penicillin-Streptomycin (Thermo) and 50pM b- 445 Mercaptoethanol (Sigma)].
  • IMDM Thermo
  • 10% Fetal Bovine Serum Sigma
  • 1 mM Sodium Pyruvate Thermo
  • 1 X Non-essential Amino Acid Solution Sigma
  • 2mM 444 Glutamax Thermo
  • 100U/ml Penicillin-Streptomycin Thermo
  • 50pM b- 445 Mercaptoethanol Sigma
  • MultiScreen-IP Filter PDVF, 0.45 pm ELISPOT plates (Merck Millipore) were first activated in 70% ethanol for 2 minutes before being washed twice with sterile PBS. Plates were then coated overnight at 4°C with 50pl of purified RBP protein in PBS (5pg/ml). The next day, plates were washed twice with sterile PBS and blocked with 200pl complete tissue culture media for 2 hours at 37°C. After this time, blocking media was aspirated and 10OpI of splenocyte suspensions were added in two concentrations (10 6 /mL and 10 6 /mL) to appropriate triplicate wells. Plates were then incubated at 37°C overnight.
  • IFNg ELISPOTS plates were prepared with ethanol and washed as above before being coated with 50pl of anti-mouse IFNg (AN18, Thermo) at 2ug/ml in PBS. Plates were incubated overnight at 4°C before being washed and blocked as described above. First, 50pl of culture media containing 60U/ml IL-2 (Peprotech) and 20pg/ml purified RBP protein were added to the appropriate wells, followed by 50pl of cell suspension corresponding to 104 or 105 cells per well. Plates were then incubated at 37°C for 48 hours before cells were aspirated and washed as above.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Pain & Pain Management (AREA)
  • Pulmonology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The invention relates to methods and compositions to boost immune responses to both infection and vaccination against and infection. In particular, the present invention is aimed at providing a method to boost immune responses to both infection with an infectious agent and vaccination against an infection with an infectious agent. It is also aimed at providing a treatment of infectious disease and vaccine adjuvant for the prevention of infection with an infectious agent.

Description

METHODS AND COMPOSITIONS TO POTENTIATE THE IMMUNE RESPONSE WITH LYSINE DEACETYLASE INHIBITORS
Field of Invention
The invention relates to methods and compositions to boost immune responses to both infection and vaccination against and infection.
Introduction
Lasting protection after vaccination or infection depends on the induction of durable memory responses. Identifying means of expanding immune memory could increase vaccine efficacy, with enhanced cellular responses associated with reduced disease severity and maintenance of protective antibody levels in infectious diseases such as SARS-CoV-2 and influenza.
Lysine acetylation is a reversible post-translational modification controlled by the opposing activity of acetyltransferases and deacetylase enzymes (Drummond, D. C.et al. Clinical development of histone deacetylase inhibitors as anticancer agents. Annu Rev Pharmacol Toxicol 45, 495-528, 574 (2005)). The known human KDAC enzymes fall into 4 main classes (l-IV), and effects of KDACi differ widely by target KDAC specificity, functional impact and target cell type (Choudhary, C. et al. Lysine acetylation targets protein complexes and co- regulates major cellular functions. Science 325, 834-840, (2009); Scholz, C. et al. Acetylation site specificities of lysine deacetylase inhibitors in human cells. Nat Biotechnol 33, 415-423, (2015); Bantscheff, M. et al. Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes. Nat Biotechnol 29, 255- 265, (2011)). Promotion of growth arrest or death of transformed cells has resulted in the development of some KDACi as cancer therapeutics, and some have also been reported to modulate immune cell differentiation and function but not immune memory (Dinarello et al Histone Deacetylase Inhibitors for Treating a Spectrum of Diseases Not Related to Cancer. Mol Med 17(5-6)333-352, 2011 ; Li, Y. & Seto, E. HDACs and HDAC Inhibitors in Cancer Development and Therapy. Cold Spring Harb Perspect Med 6, (2016); Moreira, J. M., Scheipers, P. & Sorensen, P. The histone deacetylase inhibitor Trichostatin A modulates CD4+ T cell responses. BMC Cancer 3, 30, (2003); Tao, R. et al. Deacetylase inhibition promotes the generation and function of regulatory T cells. Nat Med 13, 1299-1307, (2007); Licciardi, P. V. & Karagiannis, T. C. Regulation of immune responses by histone deacetylase inhibitors. ISRN Hematol 2012, 690901 , (2012)).
The present invention is aimed at providing a method to boost immune responses to both infection with an infectious agent and vaccination against an infection with an infectious agent. It is also aimed at providing a treatment of infectious disease and vaccine adjuvant for the prevention of infection with an infectious agent.
Summary of the Invention The present inventors have surprisingly identified that lysine deacetylase inhibitors (KDACi) have the ability to modulate T-cell memory responses by expanding immune memory cells. In particular, KDACi have ability to promote both an early memory precursor effector cell (MEC) phenotype and expanded stable memory populations and an enhanced secondary recall response. This is critical for promoting a high-affinity antibody response to infectious disease antigens. Thus, KDACi induce durable memory responses to infectious disease antigens. Surprisingly KDACi have been shown herein to enhance the immune response when used at a low dose of up to 50% of the clinically approved or standard (published) dose. Therefore, KDACi may be used to enhance the immune response when administering a vaccine, in particular a vaccine for the prevention of an infection with an infectious disease agent. Furthermore, KDACi may be used to boost immune responses to infection with an infectious disease agent and provide a treatment of infectious disease.
Thus, administration of KDACi has surprisingly been shown herein to promote the memory precursor effector cell phenotype in CD8+ T cells, and as such can be used to enhance the immune response by promoting memory cell differentiation which leads to a strong and durable immune response.
As such, an aspect of the invention relates to a method for enhancing an immune response in a subject comprising administering a lysine deacetylase inhibitor (KDACi) simultaneously, sequentially, or separately with a vaccine, wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
Another aspect relates to a method for the prevention of a disease comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine, and wherein the KDACi is wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
An aspect of the invention relates to a KDACi for use in the prevention of a disease, wherein the KDACi is administered simultaneously, sequentially, or separately with a vaccine, and wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 .
An aspect of the invention relates to an in vitro or in vivo method of promoting a T cell memory response comprising exposing cells to a KDACi capable of enhancing glutaminolysis.
An aspect of the invention relates to an in vitro or in vivo method of promoting differentiation of CD8+ T cells into memory precursor effector cells (MPEC), comprising exposing CD8+ T cells to a KDACi capable of enhancing glutaminolysis. An aspect of the invention also relates to an in vitro or in vivo method of increasing IL7R expression in CD8+ T cells, comprising exposing CD8+ T cells to a KDACi capable of enhancing glutaminolysis.
A further aspect of the invention relates to an in vitro or in vivo method of enhancing glutaminolysis, comprising exposing CD8+ T cells to a KDACi.
An aspect of the invention also relates to a method of immunising a subject against a disease, comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine, and wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
Another aspect relates to a combination therapy comprising administration of administering a KDACi simultaneously, sequentially, or separately with a vaccine, and wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
An aspect of the invention relates to the use of a KDACi as an adjuvant for enhancing the efficacy of a vaccine wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 .
An aspect of the invention relates to a composition comprising a KDACi and a vaccine wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 .
An aspect of the invention relates to a kit comprising a KDACi and a vaccine and optionally instructions for use, wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
In yet another aspect, the invention relates to a method for treating a disease comprising administering a KDACi wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 .
In a further aspect, the invention relates to a method for expanding cells for use in adoptive cell therapy comprising: providing a population of CD8+ T cells, contacting the population of CD8+ T cells to a KDACi capable of enhancing glutaminolysis to produce an expanded population of cells. Figures
Figure 1. Transcriptomic screening identifies a KDACi subclass promoting memory differentiation in primary human CD8 T cells.
(A) Schematic illustration of transcriptomic approach to small molecule repurposing. (B) Bubble plots showing enrichment score (x-axis) against significance (y-axis) for matching of three CD8+ T cell memory signatures against drug response signatures collated from GEO. Red symbols indicate compounds with KDACi activity, n=700 drug signatures screened against each memory signature. Plots illustrate those with overlap of at least n=5. (C) Schematic illustration of in vitro phenotypic screen. (D-F). Scatter plots showing maximum change in IL7Rhi MPEC% (y-axis, fold change v vehicle) against % cell division (x-axis, fc v vehicle) for (D) each of 42 selected treatments (n=844, mean +/-sem), (E) 13 selected treatments given 4 days after stimulation or (F) 6 KDACi given to sorted T cell memory subsets on dO. (G, H) Representative (G) and summary (H) scatterplots illustrating dose-dependent induction of an MPEC phenotype (IL7RhiPD110) on valproate treatment (mean +/-sem). Inset numbers (G) indicate % population indicated, inset histograms indicate proliferation (cfse dilution). (I, J) Line and scatterplots showing MPEC phenotype on treatment with low dose valproate (I, n=8) and apicidin (J, n=5). y-axes, ratio v vehicle treatment: red, IL7R:PD1 ratio, green, cell division, blue, activation (CD25+) and purple, cell survival by low dose of sodium valproate (H) and apicidin (J). *<0.05, **<0.01 , ***<0.001 . MPEC = memory precursor effector cell, SLEC = short lived effector cell, fc = fold change.
Figure 2. KDACi-induced glutaminolysis promotes CD8 memory differentiation and a switch towards oxidative metabolism.
(A) Unrooted tree plots illustrating unsupervised clustering of KDACi compounds by site-specific acetylomic changes induced on treatment of HEK cells. Leaf diameter indicates peak MPEC expansion (A, IL7R fold change v vehicle, red up/blue down) and induced total acetylation (B, panAcK fold change v vehicle, green) on treatment of stimulated primary CD8* T cells. Internal pie charts (A) indicate the target KDAC enzyme class(es) of each compound. Class l= target enzyme 1 , 2, 3, 8, class ll= target enzyme 4, 5, 7, 9, 6, 8, class IV= target enzyme 11 , class lll= sirts 1 , 2, 3, 4, 5, 6, 7). (C) Bubble plot showing KEGG pathway enrichment of 1115 unique proteins differentially acetylated by KDACi. mem and KDACi. eff compounds (A). Metabolic pathways (blue) and other pathways (orange). (D) Volcano plot showing significance (y-axis, - log FDR) and magnitude (logFC, x-axis) of differential metabolites (red/blue) identified in metabolomic analysis of CD8+T cells, d6 post after stimulation with KDACi (valproate/apicidin, n=16) or vehicle (n=8). (E) Bubble plot showing metabolic pathway enrichment analysis (pathway impact, x-axis v significance, y-axis) for differential metabolites (n=68, FDR<0.05) identified in (D). Pathways coloured blue were significantly (FDR<0.05) KDACi-induced in both metabolomic and acetylomic analyses. (F) KDACi (valproate/apicidin) induced metabolites (green, from D) and acetylated proteins (red, from A) mapped onto the glutaminolysis pathway (HMDB SMP02298) (G) Scatter/line plots and (H) summary scatterplot showing baseline- normalised oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) against time for CD8+T cells stimulated with valproate (red), vehicle (black) or valproate + R162 (green), n=5, mean+Z-sem. (I, J) Representative scatterplots (I) and summary bar plot (J, mean +/- sem, n=4) showing fluoroglucose uptake (NBDG, inset histograms) by MPEC phenotype (IL7RhiPD110) and valproate treatment. (K, L) Line and scatter plots (mean+/-sem) showing MPEC differentiation (IL7RhiPD1101 fold change (fc) vs vehicle control, y-axis) on treatment (dO) with valproate (K, x-axis, n=8) or with 125|j.M sodium valproate + increasing dose of R162 (L, x-axis, n=5). P = 2 way ANOVA.
Figure 3. KDACi treatment induces transcriptomic and epigenetic modification in CD8+ T cells consistent with memory differentiation.
(A, B) Volcano plots showing differentially expressed genes (DEG, red/blue, FDR<0.05) induced by treatment of stimulated primary CD8+T cells (n=8) for 6d with 175pM valproate (A) or valproate+25pM R162 (B). DEG in (B) are colored as in (A) to illustrate impact of R162 inhibition. (C) Circular barplot showing association of each of 9 latent factors describing maximal variance in the transcriptome of valproate and vehicle treated CD8+ T cells with treatment group (valproate v valp+R162). (D) Circular barplot showing fgsea enrichment (-logwFDR, radial axis) of LF7 gene weights for selected T cell activation and memory signatures. (E) Circular barplot showing fgsea enrichment of 50 consensus ‘hallmark’ gene signatures (radial axis) in ranked feature weights for LF7. Circular barplots radial axis = -logwFDR. (F) Annotated line plot showing weighted contribution (y-axis) of all genes (x-axis, ranked by weight) to LF7 with annotation for genes upregulated by memory (red) and exhausted (blue) CD8 T cells (from the Icmv chronic infection model, gse9650). (G) Annotated line plot showing weighted contribution (y-axis) of all genes (x-axis, ranked by weight) to LF7 (correlated with valp/R162 treatment, Fig3C) with annotation for selected genes (red) from the hallmark oxidative phosphorylation pathway, fgsea enrichment performed on whole pathway (n=200, M5936) with top 50 genes illustrated. (H) Waterfall plot showing the top enriched pathways (y-axis heatmap, -logwFDR) amongst 3320 ranked genes (x-axis) with differential ATACseq peaks identified on valproate treatment of primary, stimulated CD8+ T cells (n=6 per group). Peak distribution (middle bar, % by region) and frequency of peaks/gene (lower bar) also indicated where multiple peaks were identified. Pathway names in red (left) indicate those known to influence T cell memory differentiation. (I) Representative track plots illustrating increased open chromatin peaks in Wnt pathway genes beta catenin (CTNNB1), Lymphoid enhancer binding factor 1 (LEF1) and Transcription factor 7 (TCF7). (J) Line and scatterplot showing beta-catenin induction in stimulated CD8+ Tcells (n=6) with increasing dose of sodium valproate (x-axis) .*<0.05, **<0.01 , ***<0.001 .
Figure 4. KDACi treatment in vivo expands immune memory on vaccination and infection. (A) Schematic illustration of in vivo testing of KDACi-induced MPEC differentiation. X-axis= time (days), y-axis= T cell response. (B) Scatterplot showing MPEC ratio (IL7Rhi l°) in CD44+CD8+ splenocytes d9 post yMHV infection in KDACi (valproate/apicidin) or vehicle treated mice (treatment dO to d9, n=5 per group). (C) Scatterplot (mean+/-sem) of MPEC (IL7RhiKLRG110) and SLEC (IL7Rl0KLRG1hi), CD44hi and IL7R/PD1 expression (MFI) on CD3+CD8+ splenocytes d7 post Nitrophenylacetyl[NP]-OVA immunisation. (D) Schematic illustration of heterotypic influenza rechallenge model. (E-H) Scatterplots (Iog2 ratio vs vehicle, mean+/-sem, n=7-9 per group) illustrating immune cell populations following valproate (V) or combined valproate/R162 (V/R) treatment in the heterotypic ‘flu rechallenge model (D). (E) Lung resident T memory (d30pi) pre-rechallenge. (F) Mediastinal lymph node T cell populations, memory markers (G) and cell populations (H) post rechallenge. V = valproate treated, V/R = valproate+R162 treated, NP = influenza A nucleoprotein. Red dashed line = vehicle control level. (I-L) Representative immunofluorescence microscopy images (I, J, K) and summary scatterplot (L, mean +/- sem, n=3) showing peribronchial distribution of proliferating cells (I, Ki67+), T cells (J, K CD3+8+), Treg cells (K, CD3+Foxp3+) and B cells (K, CD19+) on valproate treatment compared to combined valproate/R162 treatment, br = bronchiole, 2-way ANOVA P = 0.01 , mean +/-sem of Ki67 fluorescence assessed in n=16 100 .M segments in each of n=3 biological replicates. (M) Schematic illustration of SARS-CoV-2 receptor binding domain (RBD) immunisation regimen with valproate treatment or vehicle dO-7. (N) Scatterplot (mean+/-sem, n=8-10 per group) showing splenic B cell populations d5 post RBD challenge. (O) Scatterplot showing RBD-specific B cell and T cell ELISpot titres (y-axis, spot-forming units (SFU) per 105 splenocytes) d5 post RBD rechallenge by treatment regimen (valproate or vehicle). (P) Scatterplot (mean+/-sem) showing SARS-CoV-2 neutralising antibody titre (NT50) by treatment group (valproate/vehicle). Red = below detection threshold, plotted at NT=10 for illustrative purposes. PB = plasmablasts, GL7 = germinal centre marker. * = P<0.05, **<0.01 , ***<0.001 .
Figure 5. Modulating Emerging Memory after Immunisation (the MEMRI study)
(A, B) Schematic illustration of the MEMRI study design (A) and experimental protocol (B). (C, D) Line and scatterplots showing baseline-normalized influenza-specific (Dextramer*) CD8+ T cell expansion after seasonal influenza vaccination. C line plot valproate is shown in red (top line) and control in blue (bottom line). (E-G) Baseline-normalized seasonal influenza specific IgG (E), IgA (F) valproate shown in red (top line and control shown in blue (bottom line). IgG and IgA titre using the Influenza A Hong Kong H3N2 vaccine strain. Significance assessed using Imm with cubic spline. (G) Representative BCR network plots illustrating global BCR repertoire clonality and diversity, confirmed influenza-specific clones indicated in yellow. (H) Scatter and boxplot showing somatic hypermutation rates (CDR-replacement per unique ‘flu-specific BCR sequence) in class-switched (right) and naive (lgM+lgD+, left) BCR clusters with confirmed ‘flu specificity. (I-J) Representative examples (I) and summary scatterplots (J) of cluster breadth/expansion in naive (J, left: lgM+lgD+) and class-switched (J, right) influenza-specific BCR clusters. (K-L) Representative examples (K) and summary scatterplots (L) of cluster breadth in naive (L, left: lgM+lgD+) and class-switched (L, right) influenza-specific BCR clusters. BCR cluster = sequences with 85% amino acid homology, identical CDR3 length and identical V/J gene. Figure 6. (A) Line and scatterplots showing dose dependent impact of KDACi treatment (x-axis) on CD8+ T cell phenotype (y axis). Red = KDACi. mem (apicidin, pano, CI994, vorino, mocet, belino, MS275, abexino, butyrate), Blue = KDACi. eff (sirtinol, bufex, nicotin, tenovin, PCI34051 , tubacin). Open circle = reported IC50 KDACi effect, closed circle = clinically approved dose where available.
Figure 7. Line and scatterplots showing effect (fold change vs vehicle control) of (A) the cell- permeable aKG donor dimethyl-ketoglutarate (DMKG) and (B-D) the glutaminolysis pathway inhibitors R162 (B), BPTES (C) and C968 (D). CD8+ T cell phenotype was measured d6 after polyclonal stimulation with anti-CD3/28 bead. Green = cell division, red = IL7R:PD1 ratio, blue = CD25, purple = live CD8+ T cell % of total viable events.
Figure 8. (A, B) Line plot (A, mean +/- sem) and scatterplot (B, mean+/-sem) showing baseline- normalized influenza-specific CD8+ T cell number (Dextramer+) up to 30 days post vaccination with the 2020/21 seasonal influenza vaccine and treatment with either 100mg, 200mg valproate or no drug (control). (C-E) Line plots (mean +/- sem) for influenza-specific IgG (C), IgA (D) and RSV (E) by treatment group. C-E: X-axis=days post vaccine, y-axis= ratio v baseline, E: X- axis=days post vaccine, y-axis= ratio v baseline tire. P values = one tailed Mann Whitney test.
Detailed Description
The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology, chemistry, biochemistry and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature, see, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012).
The present invention is based on the surprising finding that various KDAC inhibitors can enhance glutaminolysis - an anaplerotic pathway, feeding the TCA cycle through generation of a-ketoglutarate. It has been shown herein that the KDACi listed in Table 1 are capable of enhancing glutaminolysis. Through this mechanism KDACi promote the formation of memory precursor effector cells, as such this enhances the memory response and leads to a strong and durable immune response. The effects of KDACi expansion of both cellular and neutralising antibody memory responses in response to infection with an infectious agent can be used to boost immune responses to both infection and vaccination. Therefore, the invention provides a method for treating for an infectious disease. The invention also provides a method for preventing infection with an infectious disease agent. The promotion of memory precursor cells elicited by KDACi can also be used to prime cells for use in therapies such as adoptive cell transfer. Therefore, the invention also provides methods of preparing memory precursor effector cells and methods of expanding or culturing cells for use in adoptive cell therapy. Methods for Enhancing an Immune Response
The present inventors have shown that KDACi can be used to enhance the immune response elicited by a vaccine. As such the invention relates to a method for enhancing an immune response in a subject comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine.
In an aspect the invention relates to a method for enhancing an immune response in a subject comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine, wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
Table 1
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000011_0001
In another aspect, the invention relates to a method for preventing a disease comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine, wherein the KDACi is capable of enhancing glutaminolysis. In an embodiment the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 .
In yet another aspect, the invention relates to a method for treating a disease comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine, wherein the KDACi is capable of enhancing glutaminolysis. In an embodiment the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 .
Lysine deacetylase (KDAC), also known as histone deacetylase (HDAC), are a class of enzymes that catalyse the removal of acetyl groups from the NH2 terminal tail of histones. This removal of acetyl groups results in a closed chromatin structure and repression of gene expression. The term KDAC and HDAC are used interchangeably herein. There are 18 known KDACs which are split into five classifications based on their sequence homology to the originally identified yeast enzymes. The classifications are as follows, class I, class HA, class IIB, class III and class IV. Class I comprises KDAC1 , KDAC2, KDAC3 and KDAC8. Class HA comprises KDAC4, KDAC5, KDAC7, and KDAC9. Class IIB comprises KDAC6 and KDAC10. Class 3 comprises sirtuins SIRT1 , SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7. Class IV comprises KDAC11 .
Lysine deacetylase inhibitors (KDACi) are compounds that inhibit the action of KDAC. As such KDACi can selectively alter gene transcription, in part, by chromatin remodelling and by changes in the structure of proteins in transcription factor complexes. KDACi may be specific for certain KDAC classes. For example, a KDACi may target class I KDACs and inhibit KDAC1 , KDAC2, KDAC3 and KDAC8. A class I KDACi refers to a compound which inhibits any of KDAC1 , KDAC2, KDAC3 and KDAC8. A class I KDACi may also demonstrate inhibitory activity against other KDACi provided that it also targets one or more of KDAC1 , KDAC2, KDAC3 and KDAC8. Alternatively, the class I KDACi only inhibits one or more of KDAC1 , KDAC2, KDAC3 and KDAC8.
It has been shown herein that certain KDACi are capable of enhancing glutaminolysis. Glutaminolysis is an anaplerotic pathway, feeding the TCA cycle through generation of a- ketoglutarate (aKG) while also creating energy and biosynthetic precursors such as nucleic acids and modulating cellular redox balance. It makes an important energetic contribution to T cell activation and can replenish TCA cycle intermediates as they are used for biosynthesis. Glutaminolysis can fuel stem cell-like oxidative metabolism in cancer cells, sustaining chronic proliferation akin to what is required of immune memory. Specific KDACi have demonstrated an effect in enhancing glutaminolysis in particular KDACi capable of inhibiting one or more of KDAC1 , KDAC2, KDAC3, KDAC4, KDAC5, KDAC7, KDAC9 or KDAC10 have been shown to enhance glutaminolysis. As shown herein the KDACi listed in Table 1 have been shown to enhance glutaminolysis, however it will be apparent to the skilled person that other KDACi compounds may demonstrate the ability to enhance glutaminolysis. Glutaminolysis is the process by which cells convert glutamine into TCA cycle metabolites through the activity of multiple enzymes including glutaminase, glutamate dehydrogenase, glutamate-oxaloacetate transaminase, glutamate pyruvate transaminase, phosphoserine transaminase. As such, enhanced glutaminolysis may be determined by upregulation of one or more of the enzymes involved in the glutaminolysis pathway or by identifying upregulated metabolites and protein acetylation patterns indicative of glutaminolysis. Metabolites and acetylation patterns may be identified via metabolomic analysis or via functional assessment of glutamine breakdown.
In an embodiment the KDACi capable of enhancing glutaminolysis is selected from abexinostat, belinostat, panobinostat, sodium butyrate, apicidin, mocetinostat, vorinostat, MS275, CI994 and/or valproate. The valproate may be sodium valproate and/or valproic acid. In one embodiment the KDACi is valproate and/or apicidin.
It has been shown herein that is it advantageous to use the KDACi at a low dose, for example a dose that is up to 50% of the dose listed in Table 1. In an embodiment the KDACi is selected from one of the KDACi listed in Table 1 and the low dose is up to 45%, up to 40%, up to 35%, up to 30%, up to 25%, up to 20%, up to 15%, up to 10%, up to 5% of the dose listed in Table 1 for the selected KDACi. In an embodiment the KDACi is selected from one of the KDACi listed in Table 1 and the low dose is from 1 to 50%, from 1 to 45%, from 1 to 40%, from 1 to 35%, from 1 to 30%, from 1 to 25%, from 1 to 20%, from 1 to 15%, from 1 to 10%, from 1 to 5% of the dose listed in Table 1 for the selected KDACi. In an embodiment the the KDACi is selected from one of the KDACi listed in Table 1 and the low dose is 1 , 2,3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39. 40 , 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50% of the dose listed in Table 1 for the selected KDACi. Preferably the KDACi is used at a dose of 1 to 25%, 1 to 20%, 1 to 15%, 1 to 10%, 1 to 5%, 5 to 25%, 5 to 20%, 5 to 15%, or 5 to 10% of the dose listed in Table 1 for the selected KDACi.
As used herein, a vaccine is a substance used to stimulate an immune response and provide immunity against one or several diseases. A vaccine is generally prepared from the causative agent of a disease, e.g. an infectious disease agent, its products, or a synthetic substitute, treated to act as an antigen without inducing the disease. The term “vaccine” encompasses any vaccine which provides immunity against one or several diseases is used herein to refer to a biological preparation that induces an immunogenic response to a target antigen. Examples of vaccines include viral, bacterial, protein and nucleic acid vaccines. The term "viral vaccine" refers to a virus that induces an immunogenic response to a target antigen. Suitable antigens include tumour antigens, viral antigens, and in particular, antigens derived from viral pathogenic organisms such as HIV, HepC, FIV, LCMV, Ebola virus, as well as bacterial pathogens such as mycobacterium tuberculosis.
In an embodiment the vaccine is an infectious disease vaccine. The term “infectious disease vaccine” as used herein refers to a vaccine that provides a level of immunity against an infectious disease cause by an infectious disease agent. Infectious diseases are generally caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi. Thus, the term infectious disease agent as used herein refers to a pathogenic microorganism, such as bacteria, viruses, parasites or fungi. Also known as “communicable diseases”, infectious diseases can be spread directly or indirectly from one person to another. As used herein the terms “infectious disease vaccine” or “viral respiratory disease vaccine” does not extend to cancer vaccines. Thus, the infectious disease vaccine is not a cancer vaccine.
In one embodiment, the infectious disease agent (or infectious agent product) is a virus, for example and without limitation, a pox virus (e.g., vaccinia virus), zika virus, smallpox virus, marburg virus, flaviviruses (e.g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus, Japanese Encephalitis Virus), influenza virus (or antigens, such as F and G proteins or derivatives thereof), e.g., influenza A; or purified or recombinant proteins thereof, such as HA, NP, NA, or M proteins, or combinations thereof), parainfluenza virus (e.g., sendai virus), respiratory syncytial virus, rubeola virus, human immunodeficiency virus (or antigens, e.g., such as tat, nef, gpl20 or gpl60), human papillomavirus (or antigens, such as HPV6, 11 , 16, 18), varicella-zoster virus (or antigens such as gpl, II and IE63), herpes simplex virus (e.g., herpes simplex virus I, herpes simplex virus II; or antigens, e.g., such as gD or derivatives thereof or Immediate Early protein such as ICP27 from HSV1 or HSV2), cytomegalovirus (or antigens such as gB or derivatives thereof), Epstein-Barr virus (or antigens, such as gp350 or derivatives thereof), JC virus, rhabdovirus, rotavirus, rhinovirus, adenovirus, papillomavirus, parvovirus, picomavirus, poliovirus, virus that causes mumps, virus that causes rabies, reovirus, rubella virus, togavirus, orthomyxovirus, retrovirus, hepadnavirus, hantavirus, junin virion, filovirus (e.g., ebola virus), coxsackievirus, equine encephalitis virus, Rift Valley fever virus, alphavirus (e.g., Chikungunyavirus, sindbis virus), hepatitis A virus, hepatitis B virus (or antigens thereof, for example Hepatitis B Surface antigen or a derivative thereof), hepatitis C virus, hepatitis D virus, or hepatitis E virus.
In one embodiment, the infectious agent is a bacterium. Non-limiting examples of suitable bacteria (or bacterially derived products) for use in the vaccines and/or methods of the invention include Neisseria species, including N. gonorrhea and N. meningitidis (or antigens, such as, for example, capsular polysaccharides and conjugates thereof, transferrin-binding proteins, lactoferrin binding proteins, PilC, adhesins); Haemophilus species, e.g., H. influenzae-, S. pyogenes (or antigens, such as, for example, M proteins or fragments thereof, C5A protease, lipoteichoic acids), S. agalactiae, S. mutans; H. ducreyi; Moraxella spp, including M catarrhalis, also known as Branhamella catarrhalis (or antigens, such as, for example, high and low molecular weight adhesins and invasins); Bordetella spp, including B. pertussis (or antigens, such as, for example, pertactin, pertussis toxin or derivatives thereof, filamenteous hemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B. bronchiseptica; Mycobacterium species, including M. tuberculosis (or antigens, such as, for example, ESAT6, Antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L. pneumophila; Escherichia spp, including enterotoxic E. coli (or antigens, such as, for example, colonization factors, heat-labile toxin or derivatives thereof, heatstable toxin or derivatives thereof), enterohemorragic E. coli, enteropathogenic E. coli (or antigens, such as, for example, shiga toxin-like toxin orderivatives thereof); Vibrio spp, including V. cholera (or antigens, such as, for example, cholera toxin or derivatives thereof); Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y enterocolitica (or antigens, such as, for example, a Yop protein), Y pestis, Y. pseudotuberculosis; Campylobacter spp, including C. jejuni (or antigens, such as, for example, toxins, adhesins and invasins) and C. coli; Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis, S. typhimurium, and S. dysenteriae; Listeria species, including L. monocytogenes; Helicobacter spp, including H. pylori (for example urease, catalase, vacuolating toxin); Pseudomonas spp, including P. aeruginosa; Staphylococcus species, including S. aureus, S. epidermidis; Proteus species, e.g., P. mirabilis; Enterococcus species, including E. faecalis, E. faecium; Clostridium species, including C. tetani (or antigens, such as, for example, tetanus toxin and derivative thereof), C. botulinum (or antigens, such as, for example, botulinum toxin and derivative thereof), C. difficile (or antigens, such as, for example, Clostridium toxins A or B and derivatives thereof), and C. perfringens; Bacillus species, including B. anthracis (or antigens, such as, for example, botulinum toxin and derivatives thereof), B. cereus, B. circulans and B. megaterium; Corynebacterium species, including C. diphtheriae (or antigens, such as, for example, diphtheria toxin and derivatives thereof); Borrelia species, including B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii (or antigens, such as, for example, OspA, OspC, DbpA, DbpB), B. afzelii (for example OspA, OspC, DbpA, DbpB), B. andersonii (or antigens, such as, for example, OspA, OspC, DbpA, DbpB), B. hermsii; Ehrlichia species, including E. equi and the agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii; Chlamydia species, including C. trachomatis (or antigens, such as, for example, MOMP, heparin-binding proteins), C. pneumoniae (for example MOMP, heparin-binding proteins), C. psittaci; Leptospira species, including L. interrogans; Streptococcus species, such as S. pyogenes, S. agalactiae, S. pneumonia Treponema species, including T. pallidum (or antigens, such as, for example, the rare outer membrane proteins), T denticola, and T. hyodysenteriae.
In one embodiment, the infectious agent is a parasite, or a parasite derived product. Non-limiting examples of suitable parasite (or parasite derived products) for use in the vaccines and/or methods of the invention include Plasmodium species, including P. falciparum; Toxoplasma species, including T. gondii (or antigens, such as, for example SAG2, SAG3, Tg34); Entamoeba species, including E. histolytica; Babesia species, including B. microti; Trypanosoma species, including T cruzi; Giardia species, including G. lamblia; Leshmania species, including L. major, Pneumocystis species, including P. carinii; Trichomonas species, including T. vaginalis; and Schisostoma species, including S. mansoni.
In another embodiment, the infectious agent is a fungus, or a fungal derived product. In another embodiment, the infectious agent is a protozoan, or a protozoan derived product. Suitable protozoans (or protozoan derived products) for use in the vaccines and/or methods of the invention include, without limitation, protests (unicellular or multicellular), e.g., Plasmodium falciparum, and helminths, e.g., cestodes, nematodes, and trematodes.
Thus, in one embodiment, the infectious disease vaccine is a respiratory disease vaccine. The respiratory disease vaccine may be selected from a vaccine against Haemophilus influenza, a coronavirus, such as SARS-CoV-2, Haemophilus influenzae type B, measles virus, poliovirus, tetanus, tuberculosis, cholera, typhoid, dengue, diphtheria, hepatitis, Japanese encephalitis, meningococcal meningitis, mumps virus, pertussis, pneumococcal disease, rabies, rotavius, rubella, coronavirus, such as SARS-CoV-2, SARS-CoV, or MERS-CoV,
In one embodiment, the infectious disease vaccine is a viral respiratory disease vaccine. The viral respiratory disease vaccine may be selected from a vaccine against Haemophilus influenza, Haemophilus influenzae type B, measles virus, poliovirus, mumps virus, rubella, coronavirus, such as SARS-CoV-2, SARS-CoV, or MERS-CoV.
The hemagglutinin of influenza is one of the two main glycoproteins on the viral surface and a major target of neutralizing antibodies. Based on structure and antigenicity, there are eighteen defined subtypes (H1-H18) of IAV HAs belonging to two broad groups. Influenza HA consists of an antigenically variable globular head domain containing the receptor-binding site (RBS) for viral attachment and a more conserved stem domain that mediates fusion of viral and cell membranes in the endosome. The HA head domain is the immunodominant domain of the protein and is the target of most antibody responses induced by IAV vaccine or infection. However, due to the high level of sequence and antigenic diversity occurring in the HA head domain and the incorporation of large number of glycans in this region to evade immune recognition, most head domain specific antibodies exhibit a very narrow breadth of protection. Influenza as used herein may refer to either influenza A, B, C or D. Influenza A includes any of the influenza A subtypes including but not limited to H1 N1 , H3N2. In one embodiment, the vaccine is directed against influenza A or B. Zoonotic influenza virus with H1 , H3, H5, H6, H7, H9 and H10 Has are also included.
In an embodiment, the vaccine is directed against a coronavirus. Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses composed of several proteins including the Spike (S), Envelope (E), Membrane (M) and Nucleocapsid (N) proteins. The S glycoprotein is highly immunogenic with its receptor-binding domain (RBD) being a major target of the humoral response. The 2019 novel coronavirus (2019-nCoV), currently known as SARS-CoV-2, emerged in Wuhan city of the Chinese province of Hubei in December 2019.
Infection by SARS-CoV-2 causes a respiratory disease designated CoVID-19, with a median incubation period of 7 days (min: 3 days, max: 14 days). Common symptoms include fever, cough, shortness of breath, muscle pain and fatigue while, in severe cases, the infection can lead to pneumonia, extensive lung damage and death, particularly in aged patients and in those with various underlying conditions.
In an embodiment the coronavirus is selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1. In one embodiment, the coronavirus is SARS-CoV-2. SARS-CoV-2 includes any variant of SARS-CoV-2, including but not limited to D614G variant, “Cluster 5” variant, N501Y variant.
Various types of vaccine may be used, for example the infectious disease vaccine is selected from a whole pathogen vaccine, live attenuated vaccine, inactivated vaccine, whole killed vaccine, recombinant protein vaccine, toxoid vaccine, conjugate vaccine, virus-like particle (VLP) vaccine, outer membrane vesicle (OMV) vaccine, DNA vaccine, RNA vaccine, peptide vaccine or viral vectored vaccine. In one embodiment, the vector is an adenoviral vector.
A whole pathogen virus uses the whole disease-causing pathogen to produce an immune response similar to that seen during natural infection. A live attenuated vaccine utilises whole bacteria or viruses which have been weakened/attenuated so that they create a protective immune response but do not cause disease in healthy people. Attenuation may be achieved through genetic modification of the pathogen either as a naturally occurring phenomenon or as a modification specifically introduced. Inactivated vaccines comprise whole bacteria or viruses which have been killed or have been altered, so that they cannot replicate. A recombinant protein vaccine comprises one or more specific antigens from the surface of the pathogen and a generally acellular. A toxoid vaccine comprises one or more specific antigens from the surface of a toxin produced by a pathogen. A conjugate vaccine comprises a polysaccharide conjugated to an antigen from the cell surface of a pathogen or from a toxin produced by a pathogen. A VLP comprises molecules that closely resemble viruses, but are non-infectious because they contain no viral genetic material. VLPs may be naturally occurring or synthesized through the individual expression of viral structural proteins, which can then self-assemble into the virus-like structure. In some cases, the antigens in a VLP vaccine are the viral structural proteins themselves. Alternatively, the VLPs can be manufactured to present antigens from another pathogen on the surface, or even multiple pathogens at once. As each VLP has multiple copies of an antigen on its surface it is more effective at stimulating an immune response that a single copy. An OMV are vesicles which are naturally produced from the outer membrane of the bacterial outer cell wall and comprise many of the antigens found on the cell membrane but is a non-infectious particle. An OMV may be modified so that toxic antigens are removed and antigens suitable for stimulating an immune response can be kept. Nucleic acid vaccines comprise genetic material to stimulate a cell to produce the desired antigen. RNA vaccines comprise mRNA inside a lipid membrane, wherein the mRNA encodes the desired antigen. DNA vaccine comprise DNA wherein the DNA encodes the desired vaccine. Viral vectored vaccines utilise viruses to deliver the genetic code of target vaccine antigens to cells of the body, in general the virus is harmless or has been modified to reduce virulence. Viral vectored vaccine may be replicating or non-replicating. Replicating viral vectors retain the ability to make new viral particles alongside delivering the vaccine antigen when used as a vaccine delivery platform. Non-replicating viral vectors do not retain the ability to make new viral particles during the process of delivering the vaccine antigen to the cell. In non-replicating viral vectored vaccines, the virus has been modified to remove key viral genes that enable the virus to replicate.
The KDACi may be administered in combination with any commercially available infectious disease vaccine. In a preferred embodiment, the infectious disease vaccine is a viral respiratory disease vaccine. In an embodiment the infectious disease vaccine is against SARS-CoV-2 or influenza.
In one example of the various aspects of the invention, the vaccine is against coronavirus and may be selected from the following non-limiting list: Comirnaty (BNT162b2; Pfizer/BioNTech), mRNA-1273 (Moderna), AZD1222; (Vaxzevria, Covishield; AstraZeneca), Sputnik V (Gamaleya Research Institute, Acellena Contract Drug Research and Development), Sputnik Light (Gamaleya Research Institute, Acellena Contract Drug Research and Development), JNJ- 78436735 (Ad26.COV2.S; Johnson & Johnson), CoronaVac (Sinovac), BBlBP-CorV (Beijing Institute of Biological Products; China National Pharmaceutical Group (Sinopharm), EpiVacCorona (Federal Budgetary Research Institution State Research Center of Virology and Biotechnology) and Convidicea (PakVac, Ad5-nCoV; CanSino Biologies).
In one example of the various aspects of the invention, the vaccine is against influenza and may be an inactivated influenza vaccines (I IV) and live attenuated influenza vaccines (LAIV). In one embodiment, the vaccine is a trivalent vaccine. In one embodiment, the vaccine is sleeted from Afluria, Fluarix, Flublok, Flulaval, Fluvirin or Fluzone.
The inventors have shown herein that treatment with KDACi is capable of enhancing both the naive and memory immune responses as such co administration of KDACi with a vaccine has the ability to increase the breadth of the immune response and thereby improving immune protection against non-vaccine strains. The term “non-vaccine strains” refers to strains which the vaccine was not originally developed against. As such the present invention provides methods of enhancing an immune response in a subject comprising administering a KDACi simultaneously, sequentially, or separately with an infectious disease vaccine, wherein the immune response enhances protection against non-vaccine strains of the infectious disease.
The inventors have also surprisingly shown that the effects of the KDACi, in particular valproate, on expanding memory are achieved when a low dose is administered. This dose is lower than the dosages used for KDACi, in particular valproate, for the treatment of cancer. The KDACi, preferably valproate, may be administered at a dose of 10 to 950 mg/day, 15 to 850 mg/day, 20 to 750 mg/day, 25 to 650 mg/day, 30 to 550 mg/day, 35 to 450 mg/day, 40 to 350 mg/ day, 50 to 250 mg/day, 75 to 225 mg/day, 80 to 220 mg/day, 85 to 215mg/day, 90 to 210 mg/day, 95 to 205 mg/day, preferably at a dose of 100 to 200 mg/day. The KDACi may be administered at a dose of 50 mg/day, 75 mg/day, 100 mg/day, 125 mg/day, 150 mg/day, 175 mg/day, 200 mg/day, 225 mg/day, 250 mg/day. The KDACi may be administered at a dose of approximately 100 mg/day, approximately 100 mg/day may encompass a dose between 95 to 105 mg/day. The KDACi may be administered at a dose of approximately 200 mg/day, approximately 200 mg/day may encompass a dose between 195 to 205 mg/day. The dose administered per day is referred to as the total dose per day, as such the total dose may be split into multiple doses throughout the day, for example where a dose of 200mg/day is administered this may be split into two separate 100mg doses. The total dose per day may be split into multiple administrations, for example the total dose may be split into 2, 3, 4, 5, or 6 administrations per day.
In an embodiment the KDACi, preferably valproate, is administered at a dose of 100 mg/day. In an embodiment the KDACi, preferably valproate, is administered at a dose of 200 mg/day. In an embodiment the KDACi, preferably valproate, is administered at a dose of 200 mg/day and the dose is split into two 100 mg doses taken at two separate time point during the day. The correct dosage of the KDACi may vary according to the particular formulation, the mode of administration, and its particular site, host. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities may be taken into account.
Where abexinostat is used in the methods of the invention, abexinostat may be administered at a dose of 2 to 10 nM, 2.5 to 9.5 nM, 3 to 9 nM, 3.5 to 8.5 nM, 4 to 8 nM. 4.5 to 7.5 nM, 2.5 to 7.5 nM, 3.5 to 6.5 nM, 4.5 to 5.5 nM. Abexinostat may be administered at a dose of 2nM, 5nM or 10nM. Abexinostat may be administered at the clinically approved dose range of 0.1 pM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering abexinostat simultaneously, sequentially, or separately with a vaccine. In an embodiment the abexinostat is administered at up to 50% of the dose recited in Table 1 . In an embodiment the abexinostat is administered in a dose of 2 to 10 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering abexinostat simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the abexinostat is administered in a dose of 2 to 10 nM.
Where belinostat is used in the methods of the invention, belinostat may be administered at a dose of 5 to 800 nM, 5 to 700 nM, 5 to 600 nM, 5 to 500 nM, 5 to 400 nM, 5 to 300 nM, 5 to 200 nM, 5 to 100 nM, 5 to 50 nM, 5 to 40 nM, 5 to 30 nM, 5 to 20 nM, 5 to 15 nM., 10 to 800 nM, 10 to 700 nM, 10 to 600 nM, 10 to 500 nM, 10 to 400 nM, 10 to 300 nM, 10 to 200 nM, 10 to 100 nM, 10 to 50 nM, 10 to 40 nM, 10 to 30 nM, 10 to 20 nM, 10 to 15 nM. Belinostat may be administered at a dose of 5 nM, 10 nM, or 15 nM. Belinostat may be administered at the clinically approved dose of 1 pM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering belinostat simultaneously, sequentially, or separately with a vaccine. In an embodiment the bellinostat is administered at up to 50% of the dose recited in Table 1. In an embodiment the belinostat is administered in a dose of 5 to 800 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering belinostat simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the belinostat is administered in a dose of 5 to 800 nM.
Where panobinostat is used in the methods of the invention, panobinostat may be administered at a dose of 0.5 to 500 nM, 0.5 to 400 nM, 0.5 to 300 nM, 0.5 to 200 nM, 0.5 to 100 nM, 0.5 to 50 nM, 0.5 to 40 nM, 0.5 to 30 nM, 0.5 to 20 nM, 0.5 to 10 nM, 0.5 to 9 nM, 0.5 to 8 nM, 0.5 to 7 nM, 0.5 to 6 nM, 0.5 to 5 nM, 0.5 to 4 nM, 0.5 to 3 nM, 0.5 to 2 nM, 1 to 500 nM, 1 to 400 nM, 1 to 300 nM, 1 to 200 nM, 1 to 100 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, 1 to 10 nM, 1 to 9 nM, 1 to 8 nM, 1 to 7 nM, 1 to 6 nM, 1 to 5 nM, 1 to 4 nM, 1 to 3 nM, 1 to 2 nM. Panobinostat may be administered at a dose of 0.5 nM, 1 nM, or 2 nM. Panobinostat may be administered at the clinically approved dose of 500 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering panobinostat simultaneously, sequentially, or separately with a vaccine. In an embodiment the panobinostat is administered at up to 50% of the dose recited in Table 1 . In an embodiment the panobinostat is administered in a dose of 0.5 to 500 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering panobinostat simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the panobinostat is administered in a dose of 0.5 to 500 nM.
Where butyrate is used in the methods of the invention, butyrate may be administered at a dose of 0.1 x103 to 500x103 nM, 0.1x103 to 400x103 nM, 0.1x103 to 300x103 nM, 0.1x103 to 200x103 nM, 0.1x103 to 100x103 nM, 0.1x103 to 50x103 nM, 0.1x103 to 10x103 nM, 0.1x103 to 5x103 nM, 0.1 x103 to 1x103 nM, 0.1 x103 to 0.05x103 nM, 0.5 x103 to 500x103 nM, 0.5 x103 to 400x103 nM, 0.5 x103 to 300x103 nM, 0.5 x103 to 200x103 nM, 0.5 x103 to 100x103 nM, 0.5 x103 to 50x103 nM, 0.5 x103 to 10x103 nM, 0.5 x103 to 5x103 nM, 0.5 x103 to 0.1x103 nM, 0.5 x103 to 0.1x103 nM. Butyrate may be administered at a dose of 0.5x103 nM, 1x103 nM, 2x103 nM. Butyrate may be administered at the clinically approved dose of 200 to 800pM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering butyrate simultaneously, sequentially, or separately with a vaccine. In an embodiment the butyrate is administered at up to 50% of the dose recited in Table 1. In an embodiment the butyrate is administered in a dose of 0.1 to 500x103 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering butyrate simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the butyrate is administered in a dose of 0.1 x103 to 500x103 nM.
Where apicidin is used in the methods of the invention, apicidin may be administered at a dose of 0.01 to 100 nM, 0.01 to 90 nM, 0.01 to 80 nM, 0.01 to 70 nM, 0.01 to 60 nM, 0.01 to 50 nM, 0.01 to 40 nM, 0.01 to 30 nM, 0.01 to 20 nM, 0.01 to 10 nM, 0.01 to 5 nM, 0.01 to 1 nM, 0.01 to 0.9 nM, 0.01 to 0.8 nM, 0.01 to 0.7 nM, 0.01 to 0.6 nM, 0.01 to 0.5 nM , 0.01 to 0.4 nM, 0.01 to 0.3 nM, 0.01 to 0.2 nM, 0.01 to 0.1 nM, 0.01 to 0.09 nM, 0.01 to 0.08 nM, 0.01 to 0.07 nM, 0.01 to 0.06 nM, 0.01 to 0.05 nM, 0.01 to 0.04 nM, 0.01 to 0.03 nM, 0.01 to 0.02 nM, 0.05 to 100 nM, 0.05 to 90 nM, 0.05 to 80 nM, 0.05 to 70 nM, 0.05 to 60 nM, 0.05 to 50 nM, 0.05 to 40 nM, 0.05 to 30 nM, 0.05 to 20 nM, 0.05 to 10 nM, 0.05 to 5 nM, 0.05 to 1 nM, 0.05 to 0.9 nM, 0.05 to 0.8 nM, 0.05 to 0.7 nM, 0.05 to 0.6 nM, 0.05 to 0.5 nM , 0.05 to 0.4 nM, 0.05 to 0.3 nM, 0.05 to 0.2 nM, 0.05 to 0.1 nM, 0.05 to 0.09 nM, 0.05 to 0.08 nM, 0.05 to 0.07 nM, 0.05 to 0.06 nM. Apicidin may be administered at a dose of 0.01 nM, 0.05 nM, or 0.1 nM. Apicidin may be administered at the clinically approved dose of 160 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering apicidin simultaneously, sequentially, or separately with a vaccine. In an embodiment the apicidin is administered at up to 50% of the dose recited in Table 1. In an embodiment the apicidin is administered in a dose of 0.01 to 100 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering apicidin simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the apicidin is administered in a dose of 0.01 to 100 nM.
Where mocetinostat is used in the methods of the invention, mocetinostat may be administered at a dose of 0.01 x103 to 1x103 nM, 0.01 x103 to 0.9x103 nM, 0.01 x103 to 0.8x103 nM, 0.01 x103 to 0.7x103 nM, 0.01 x103 to 0.6x103 nM, 0.01 x103 to 0.5x103 nM, 0.01 x103 to 0.4x103 nM, 0.01 x103 to 0.3x103 nM, 0.01 x103 to 0.2x103 nM, 0.01 x103 to 0.1x103 nM, 0.1 x103 to 1x103 nM, 0.1 x103 to 0.9x103 nM, 0.1 x103 to 0.8x103 nM, 0.1 x103 to 0.7x103 nM, 0.1 x103 to 0.6x103 nM, 0.1 x103 to 0.5x103 nM, 0.1 x103 to 0.4x103 nM, 0.1 x103 to 0.3x103 nM, 0.1 x103 to 0.2x103 nM. Mocetinostat may be administered at a dose of 0.1 x103 nM, 0.5 x103 nM, 1 x103 nM. Mocetinostat may be administered at the clinically approved dose of 100 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering mocetinostat simultaneously, sequentially, or separately with a vaccine. In an embodiment the mocetinostat is administered at up to 50% of the dose recited in Table 1 . In an embodiment the mocetinostat is administered in a dose of 0.01 to 1x103 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering mocetinostat simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the mocetinostat is administered in a dose of 0.01 x103 to 1x103 nM.
Where vorinostat is used in the methods of the invention, vorinostat may be administered at a dose of 0.2 x103 to 5x103 nM, 0.2 x103 to 4x103 nM, 0.2 x103 to 3x103 nM, 0.2 x103 to 2x103 nM, 0.2 x103 to 1x103 nM, 0.2 x103 to 0.5x103 nM, 0.2 x103 to 0.4x103 nM, 0.2 x103 to 0.3x103 nM, 1 x103 to 5x103 nM, 1 x103 to 4.5x103 nM, 1 x103 to 4x103 nM, 1 x103 to 3.5x103 nM, 1 x103 to 3x103 nM, 1 x103 to 2.5x103 nM, 1 x103 to 2x103 nM, 1 x103 to 1 .5x103 nM. Vorinostat may be administered at a dose of 1 .25 x103 nM, 2.5 x103 nM, 5 x103 nM. Vorinostat may be administered at the clinically approved dose of 500 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering vorinostat simultaneously, sequentially, or separately with a vaccine. In an embodiment the vorinostat is administered at up to 50% of the dose recited in Table 1. In an embodiment the vorinostat is administered in a dose of 0.2 to 5x103 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering vorinostat simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the vorinostat is administered in a dose of 0.2 x103to 5x103 nM.
Where MS275 is used in the methods of the invention, MS275 may be administered at a dose of 0.01x106 to 1x10® nM, 0.01x10B to 0.9x106 nM, 0.01x106 to 0.8x106 nM, 0.01x106 to 0.7x106 nM, 0.01x10® to 0.6x10® nM, 0.01x10® to 0.5x10® nM, 0.01x10® to 0.4x10® nM, 0.01x10® to 0.3x10® nM, 0.01x10®to 0.2x10® nM, 0.01x10®to 0.1x10® nM, 0.01x10®to 0.09x10® nM, 0.01x10® to 0.08x10® nM, 0.01x10® to 0.07x10® nM, 0.01x10® to 0.06x10® nM, 0.01x10® to 0.05x10® nM, 0.01x10® to 0.04x10® nM, 0.01x10® to 0.03x10® nM, 0.01x10® to 0.02x10® nM 0.25x10® to 1x10® nM, 0.25x10® to 0.9x10® nM, 0.25x10® to 0.8x10® nM, 0.25x10® to 0.7x10® nM, 0.25x10® to 0.6x10® nM, 0.25x10® to 0.5x10® nM, 0.25x10® to 0.4x10® nM, 0.25x10® to 0.3x10® nM. MS275 may be administered at a dose of 0.25x10® nM, 0.5x10® nM, 1x10® nM. MS275 may be administered at the clinically approved dose of 13 nM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering MS275 simultaneously, sequentially, or separately with a vaccine. In an embodiment the MS275 is administered at up to 50% of the dose recited in Table 1. In an embodiment the MS275 is administered in a dose of 0.01x10® to 1x10® nM,ln an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering MS275 simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the MS275 is administered in a dose of 0.01x10® to 1x10® nM,
Where CI994 is used in the methods of the invention, CI994 may be administered at a dose of 0.01x10® to 2x10® nM, 0.01x10® to 1x10® nM, 0.01x10® to 0.9x10® nM, 0.01x10® to 0.8x10® nM, 0.01x10® to 0.7x10® nM, 0.01x10® to 0.6x10® nM, 0.01x10® to 0.5x10® nM, 0.01x10® to 0.4x10® nM, 0.01x10® to 0.3x10® nM, 0.01x10® to 0.2x10® nM, 0.01x10® to 0.1x10® nM, 0.01x10® to 0.09x10® nM, 0.01x10® to 0.08x10® nM, 0.01x10® to 0.07x10® nM, 0.01x10® to 0.06x10® nM, 0.01x10® to 0.05x10® nM, 0.01x10® to 0.04x10® nM, 0.01x10® to 0.03x10® nM, 0.01x10® to 0.02x10® nM 0.25x10® to 1x10® nM, 0.25x10® to 0.9x10® nM, 0.25x10® to 0.8x10® nM, 0.25x10® to 0.7x10® nM, 0.25x10® to 0.6x10® nM, 0.25x10® to 0.5x10® nM, 0.25x10® to 0.4x10® nM, 0.25x10®to 0.3x10® nM. CI994 may be administered at a dose of 0.1x10® nM, 0.5x10® nM, 1x10® nM. CI994 may be administered at the clinically approved dose of 2pM. In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering CI994 simultaneously, sequentially, or separately with a vaccine. In an embodiment the CI994 is administered at up to 50% of the dose recited in Table 1 . In an embodiment the CI994 is administered in a dose of 0.01x10Bto 2x10B nM, In an embodiment of the invention relates to a method for enhancing an immune response in a subject comprising administering CI994 simultaneously, sequentially, or separately with a viral respiratory disease vaccine, wherein the CI994 is administered in a dose of 0.01x10B to 2x10B nM.
According to the invention the KDACi can be administered simultaneously, sequentially or separately with a vaccine. It is not necessary that the KDACi and vaccine are packed together (but this is one embodiment of the invention). It is also not necessary that they are administered at the same time. As used herein, "separate" administration means that the drugs are administered as part of the same overall dosage regimen (which could comprise a number of days), but preferably on the same day. As used herein "simultaneously" means that the drugs are to be taken together or administered together, or formulated as a single composition. The KDACi and the vaccine may be formulated separately but administered together, via the same or different administration route. For example, the vaccine may be administered subcutaneously or intravenously and the KDACi may be administered orally. As used herein, "sequentially" means that the drugs are administered at about the same time one after another, and preferably within about 1 hour of each other.
The dosage regimen of the KDACi and the vaccine may comprise day 0 for example when the KDACi and vaccine are administered simultaneously. The dosage regimen may comprise additional days wherein further doses of the KDACi and/or the vaccine are administered. The further dose of KDACi may be administered in the absence of the vaccine. For example, the KDACi and the vaccine are administered simultaneously, sequentially or separately on day 0, and a further dose of the KDACi is administered on any or all of days 1 to 14, 1 to 13, 1 to 12, 1 to 11 , 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or day 1 following the administration of the KDACi and the vaccine. Further doses of the KDACi may be administered on any or all of days 1 to 2, days 1 to 3, days 1 to 4, days 1 to 5, days 1 to 6 or days 1 to 7. In an embodiment the KDACi and the vaccine are administered simultaneously, sequentially or separately on day 0, and a further dose of the KDACi is administered on any or all of days 1 to 7 following the administration of the KDACi and the vaccine.
For example, in embodiments relating to administration with a SARS-CoV-2 vaccine, subjects are administered the SARS-CoV-2 vaccination on day 0 along with a 100mg dose of sodium valproate. The sodium valproate will then be administered for 7 days following the vaccination at a daily dose of 100mg. The same protocol can used be when the SARS-CoV-2 booster vaccination is administered.
For example, in embodiments relating to administration with an influenza, subjects are administered the influenza vaccination on day 0 along with a 10Omg or 200mg dose of sodium valproate. The sodium valproate will then be administered for 7 days following the vaccination at a daily dose of 100mg or 200mg. The same protocol can be used when the booster vaccination is administered.
As used herein the term “valproate” may refer to valproic acid. Where valproate or valproic acid are is used in the methods of the present invention the compound may be administered as valproic acid or the sodium salt form of valproic acid i.e. sodium valproate, other salt forms may be suitable. Where a salt form is administered the salt form converts to the active form of valproate ion within the blood.
The KDACi is administered to enhance an immune response to an infectious agent. In particular, administration of the KDACi either alone orwith a vaccine can increase the intensity, rate, and duration of immune response, and/or shorten onset time of antibody responses. When administered with a vaccine, the immune response to the vaccine is enhanced compared to the immune response generated by administration of the vaccine alone to the subject.
The immune system encompasses cellular immunity and humoral immunity. Cellular immunity includes a network of cells and events. Memory T cells are key components of the immune response, are antigen-specific, are developed after exposure and recognition of a particular antigen. Humoral immunity involves B cells and antibodies. When B cells become transformed to plasma cells, the plasma cells express and secrete antibodies. The secreted antibodies can subsequently bind to antigens. Expansion of germinal centre (GCB) and expansion of T cell follicular helper cells (TFH) are critical for promoting a high affinity antibody response.
Thus, the term “inducing and/or enhancing an immune response” means that the method evokes and/or enhances any response of the subject’s immune system. “Immune response” is defined as any response of the immune system, for example, of either a cell-mediated or humoral (i.e. antibody mediated) nature. In particular, the response is humoral. These immune responses can be assessed by a number of in vivo or in vitro assays well known to one skilled in the art including, but not limited to, antibody assays (for example ELISA assays) antigen specific cytotoxicity assays, production of cytokines (for example ELISPOT assays), etc. Suitable assays are also shown in the examples.
In particular, as used herein, the enhanced immune response includes one or more of the following: expansion of germinal centre (GCB), expansion of T cell follicular helper cells (TFH), and/or differentiation of stimulated primary human CD8+ T cells into MPEC and expansion of MPEC. Thus, in one embodiment, the production of memory T cells produced during infectious disease challenge simulated by administration of the vaccine. In one embodiment, differentiation of stimulated primary human CD8+ T cells into MPEC is promoted.
Therefore, using a vaccine together with a KDACi at the dose described herein can stimulate greater protection against the infectious disease immunogenic targets expressing the antigen in the vaccine. In particular, as discussed above, using a vaccine together with a KDACi results in a immune response with a greater breath thereby providing protection against both the vaccine strain and non-vaccine strain of the infectious disease. In addition, using a vaccine together with a KDACi could lead to enhanced clonal expansion and memory and a quicker/faster, more intense, and more prolonged humoral response upon re-challenge to the antigen in question. The combination of vaccine and a KDACi can produce an additive or synergistic effect and achieve greater benefit than using vaccine alone; and the combination also allows for possible use of smaller doses of the vaccine to achieve protective antibody titers.
Where a vaccine administration comprises a prime boost dosage regimen, the KDACi may be administered, as described herein, with both the priming dose of the vaccine and the boost dose of the vaccine or only one of these doses. As used herein the “prime boost dosage regimen” refers to a regimen of immunization with the same immunogen during the prime and booster doses or a regimen of priming the immune system with an immunogen and then boosting with a different immunogen. During a prime boost dosage regimen, the prime and boost doses may be administered one or more weeks apart, one or more months apart or one or more years apart. When the prime dose of the vaccine is administered on day 0 the KDACi is also administered separately, sequentially or simultaneously. On subsequent days further doses of the KDACi may be administered, for example on any or all of days 1 to 14, 1 to 13, 1 to 12, 1 to 11 , 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or day 1 following the administration of the KDACi and the vaccine. A prime boost dosage regimen is common with vaccines directed against SARS-CoV-2 and so this administration regimen, wherein the KDACi is administered with both the priming and boost doses of the SARS-CoV-2 vaccine, may be adopted. Alternatively, the KDACi may be administered either with the priming dose of the vaccine or with the boost dose of the vaccine.
Administration of the KDACi and/or the vaccine may be via any reasonable route, for example any parenteral or enteral route. For example, any convenient route, includes but is not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. As discussed above, the KDACi or the infectious disease vaccine can take the form of one or more dosage units. Administration of the KDACi may be performed by the oral, sublingual, buccal, intravenous, intramuscular, or subcutaneous route. Where more than one administration of the KDACi is used, the same or different routes of administration may be used. Various oral dosage forms can be used, including such solid forms as tablets, capsules, liquids, granules and bulk powders. Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple- compressed, containing suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents.
In one embodiment, administration of the vaccine may be performed by intravenous, intramuscular, intradermal, intranasal, or subcutaneous route.
The vaccine and/or the KDACi can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid compositions, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides, polyethylene glycols, glycerin, or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The composition can be enclosed in an ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material.
An intravenous formulation of the vaccine or the KDACi may be in the form of a sterile injectable aqueous or non-aqueous (e.g. oleaginous) solution or suspension. The sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example, a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, phosphate buffer solution, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of the intravenous formulation of the invention.
The vaccine or the KDACi can be prepared using methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining with water so as to form a solution. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension. In an embodiment the subject is a mammal, preferably a human. It has been shown herein that the administration of the KDACi valproate in conjunction with a vaccine was able to enhance both the naive T cell response and the memory response. As such, the methods of the invention are particularly suitable for use in the elderly, children or immunocompromised subjects. In an embodiment the subject is over the age of 65. In an embodiment the subject is between the age of 1 year and 18 years old. In an embodiment the subject is immunocompromised or immunosuppressed. Immunocompromised or immunosuppressed subject may include; transplant patients, subjects with weakened immune system, subjects taking immunosuppressive drugs, subjects with HIV/AIDS, cancer, diabetes or genetic disorders.
The invention relates to a KDACi for use in a method of enhancing an immune response in a subject comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine.
The invention also relates to a KDACi for use in the prevention or treatment of a disease, wherein the KDACi is administered simultaneously, sequentially, or separately with a vaccine, and wherein the KDACi is capable of enhancing glutaminolysis. In an embodiment the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1.
The invention also relates to a KDACi for use in the treatment of a disease, wherein KDACi is capable of enhancing glutaminolysis. In an embodiment the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 .
The invention also relates to a KDACi for use in the prevention of SARS-CoV-2, wherein the KDACi is administered simultaneously, sequentially, or separately with a SARS-CoV-2 vaccine. Preferably wherein the KDACi is administered at a dose of 10 to 950 mg/day. In an embodiment the KDACi is valproate.
The invention also relates to a KDACi for use in the treatment of SARS-CoV-2, preferably wherein the KDACi is administered at a dose of 10 to 950 mg/day. In an embodiment the KDACi is valproate.
The invention also relates to a KDACi for use in the prevention of influenza, wherein the KDACi is administered simultaneously, sequentially, or separately with an influenza vaccine, preferably wherein the KDACi is administered at a dose of 10 to 950 mg/day. In an embodiment the KDACi is valproate. The invention also relates to a KDACi for use in the treatment of influenza, preferably wherein the KDACi is administered at a dose of 10 to 950 mg/day. In an embodiment the KDACi is valproate.
The invention also relates the use of a KDACi in the manufacture of a medicament for the prevention and/or treatment of a disease wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1. In one embodiment, the KDACi is administered together with an infectious disease vaccine.
The terms "treat”, "treatment” or "treating,” as used herein refer to administering a compound to a subject for prophylactic and/or therapeutic purposes. Where the KDACi is for use as part of an immunization regimen, the KDACi is for use in the prevention of an infectious disease by enhancing the production of memory precursor effector cells. As explained herein, the KDACi can also be used to treat disease, in particular at the early onset of the disease. Thus in one embodiment, the KDACi is being used to prevent the onset of an infectious disease and is administered prophylactically together with a vaccine. As such the term “prevention” refers to preventing the onset of an infectious disease or preventing an infection. The term “prevention” can refer to a reduction in the risk of contracting a disease. The term "prophylactic or preventative treatment” refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. The term “therapeutic treatment” refers to administering treatment to a subject already suffering from a disease or condition.
Methods of Enhancing Memory T cell Response
It has been shown herein that exposure to KDACi results in expanded memory cell populations and improved recall responses. As such the invention also relates to an in vivo, in vitro or ex vivo method of promoting a T cell memory response, comprising exposing cells to a KDACi. According to the method, for administration to a human subject, the KDACi is administered at a dose as set out herein. In particular the KDACi may enhance the T cell memory response. The enhanced T cell memory response may be characterised by an expanded population of memory precursor effector cells. Memory precursor effector cells can be characterized by the presences of specific markers and cell surface markers. Methods to identify and quantify these markers are well known in the art. Examples of suitable methods include but are not limited to affinity- based separation methods, magnetic cell sorting techniques, fluorescence-based cell sorting techniques such as FACS (fluorescence activated cell sorting). Memory precursor effector cells may be characterised by a number of markers, examples include but are not limited to; ILR7hiPD110. The term "hi” and “l0” refer to relative expression of these markers on the cells. “Io” may referto cells wherein there is no expression of the markers, it may also refer to cells wherein there is low expression of the markers relative to other cells in the sample. “hi” may refer to cells wherein there is high expression of the markers, for example where there is high expression of the markers relative to other cells in the sample.
The invention also relates to an in vivo, in vitro or ex vivo method of promoting differentiation of CD8+ T cells into memory precursor effector cells, comprising exposing CD8+ T cells to a class I KDACi. Unless otherwise specified, the term CD8+ T cells as used herein relates to human CD8+ T cells. As used herein, the term “differentiation” refers to cell differentiation and has is usual meaning in the art and refers to the process wherein a cell transitions from one cell type to another. Cell differentiation can be characterised by the presence of cell surface markers. CD8+ T cells may be identified by the following cell surface markers CD3+CD8+. The transition of CD8+ T cells into memory precursor effector cells may be characterised by an increase in cell population expressing ILR7hiPD110. CD8+ T cells may be obtained from a subject and then exposed in vitro/ex vivo in order to stimulate differentiation of the CD8+ T cells into memory precursor effector cells. The expanded population of memory precursor effector cells may be reintroduced to a subject. According to the method, for administration to a human subject, the KDACi is administered at a dose as set out herein.
It has been shown herein that KDACi stimulates expression of IL7R (interleukin-7 receptor). An aspect of the invention related to an in vivo, in vitro or ex vivo method of enhancing IL7R expression in CD8+ T cells, comprising exposing CD8+ T cells to a KDACi. IL7R plays a key role in the development of lymphocytes and VDJ recombination. Enhanced expression of IL7R may be detected using standard techniques including but not limited to include but are not limited to affinity-based separation methods, magnetic cell sorting techniques, fluorescence-based cell sorting techniques such as FACS (fluorescence activated cell sorting). According to the method, for administration to a human subject, the KDACi is administered at a dose as set out herein.
A shown herein exposing cells to KDACi results in a cell population with strong and durable immune responses. Therefore these cell populations are particularly suitable for use in therapies such as adoptive cell therapy. Adoptive cell therapy is a type of immunotherapy wherein T cells are provided to a subject in order to enhance the subjects immune response to diseases such as cancer. Types of adoptive cell therapy include chimeric antigen receptor T-cell (CAR T-cell) therapy and tumor-infiltrating lymphocyte (TIL) therapy. An aspect of the invention relates to a method for expanding cells for use in adoptive cell therapy comprising: providing a population of CD8+ T cells, contacting the population of CD8+ T cells to a KDACi capable of enhancing glutaminolysis to produce an expanded population of cells. The term “expanding cells” as used herein refers to culturing cells under conditions such that the number of cells increases. In an embodiment the resultant expanded population of cells comprises memory precursor effector cells. The expanded population of cells may be characteristed by specific cell surface markers for example the memory precursor effector cells are characterised by the markers IL7Rhi, PD1 lo. In an embodiment the expanded population of memory precursor effector cells of are further characterised by the markers IL7Rhi, PD1 lo, CX3CR1 , CCR7, CD27 and CD62L.
The invention also provides a method of providing a population of cells enriched for memory precursor effector cells comprising: providing a population of CD8+ T cells, contacting the population of CD8+ T cells to a KDACi capable of enhancing glutaminolysis to produce an expanded population of cells.
In an aspect the invention relates to a method of treatment comprising administering a population of cells expanded by the methods described herein. An embodiment relates to an expanded population of cells for use in therapy wherein the expanded population of cells are obtained by the following method: providing a population of CD8+ T cells, contacting the population of CD8+ T cells to a KDACi capable of enhancing glutaminolysis to produce an expanded population of cells.
The cells for use in therapy or methods of treatment may be characterised as memory precursor effector cells as identified by one or more of the cell surface markers IL7Rhi, PD1 lo, CX3CR1 , CCR7, CD27 and CD62L, in particular IL7Rhi, PD1 lo. The cells may be for use in adoptive cell therapy
Further it has been shown herein that KDACi enhance glutaminolysis in cells. As used herein “glutaminolysis” refers to the process which feeds the TCA cycle through the generation of a- ketoglutarate. The process of glutaminolysis comprises a series of biochemical reactions by which the amino acid glutamine is lysed to glutamate, aspartate, CO2, pyruvate, lactate, alanine and citrate. As such, an aspect of the invention relates to an in vivo, in vitro or ex vivo method of enhancing glutaminolysis, comprising exposing CD8+ T cells to a KDACi. According to the method, for administration to a human subject, the KDACi is administered at one of the doses as set out above. In an embodiment where valproate is used as the KDACi, valproate is administered at a dose of 10 to 950 mg/day.
In the methods described herein cells are exposed to the KDACi in vivo, in vitro or ex vivo. As such the cells may be exposed to the KDACi by administering the KDACi directly to the subject. Alternatively, cells may be obtained from a subject and exposed to the KDACi. Once the cells have been exposed to the KDACi the cells may be returned to the subject.
In the methods described herein the cells may also be exposed to a viral respiratory disease vaccine. The viral respiratory disease vaccine may be any vaccine as described herein. The cells may be exposed to the viral respiratory disease vaccine in vivo, in vitro or ex vivo. As such the cells may be exposed to the viral respiratory disease vaccine by administering the viral respiratory disease vaccine directly to the subject. Alternatively, cells may be obtained from a subject and exposed to the viral respiratory disease vaccine. Once the cells have been exposed to the viral respiratory disease vaccine, the cells may be returned to the subject.
Methods of Immunising
The invention relates to a method of immunising a subject against a disease, comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine, and wherein the KDACi is capable of enhancing glutaminolysis.
The invention relates to the use of a KDACi as an adjuvant for enhancing the efficacy of a vaccine. As used herein the term "adjuvant” refers to a substance that enhances the immune system's response to the presence of an antigen. An adjuvant is commonly used to improve the effectiveness of a vaccine. The use of the KDACi as an adjuvant may be performed by administering the KDACi in combination with a vaccine, preferably an infectious disease vaccine, the KDACi and the vaccine may be administered separately, sequentially or simultaneously. Where the KDACi is used as an adjuvant for administration to a subject, it is provided in a dose as set out herein. In an embodiment the KDACi is one of the listed in Table 1 and used at a dose of up to 50% of the dose set out in Table 1 for said KDACi. In an embodiment where valproate is used as the KDACi, valproate is administered at a dose of 10 to 950 mg/day.
The invention also relates to a composition comprising a KDACi and a vaccine wherein the KDACi is capable of enhancing glutaminolysis. In an embodiment the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1. The composition may comprise the KDACi a vaccine formulated together, preferably an infectious disease vaccine. The composition may comprise one or more additional active ingredients, pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The compositions may optionally include other drug actives.
The invention also relates to a kit comprising a KDACi and a vaccine and optionally instructions for use, wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1. The kit may comprise the KDACi and a vaccine formulated together, preferably an infectious disease vaccine. The kit may comprise the KDACi and i vaccine formulated separately. The kit may also comprise one or more additional active ingredients, pharmaceutically acceptable carrier, diluent, excipient or adjuvant, and optionally instructions for use.
The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antiviral, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Some examples of substances, which can serve as pharmaceutically-acceptable carriers or components thereof, are sugars, such as lactose, dextrose, glucose and sucrose; starches, such as com starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the TWEENS; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.
All documents mentioned in this specification are incorporated herein by reference in their entirety.
The invention is further described in the non-limiting examples.
Examples
Example 1 - Genomic drug repurposing expands immune memory following immunisation and infection Drug repurposing allows the rapid use of existing therapeutics for new disease indications and can be directed by matching disease-associated transcriptional changes to those induced by candidate small molecules. Here we screen multiple transcriptional signatures of T cell memory against libraries of drug response signatures, identifying a subgroup of compounds with lysine deacetylase inhibitory (KDACi) activity with the ability to promote, at low doses, a memory precursor effector cell (MPEC) phenotype in primary human CD8+ T cells. Using combined proteomics, metabolomics, transcriptomics and epigenomics on in vitro treated cells, we identify enhanced glutaminolysis and oxidative metabolism during T cell activation as the immunometabolic mechanism promoting memory cell differentiation. KDACi treatment reproduced - and glutaminolysis inhibition reversed - the transcriptional memory signatures used for initial screening with concurrent epigenetic modification of Wnt pathway genes controlling glutamine breakdown. We demonstrate in vivo efficacy of a repurposed, clinically-approved KDACi (sodium valproate) in four murine models of immunisation and infection, including SARS- CoV-2 spike protein immunisation, showing expanded memory populations and improved recall responses. Memory expansion in vivo is also reversed by inhibition of glutaminolysis, highlighting its contribution to T cell memory formation. The observed expansion of both cellular and neutralising antibody memory responses to SARS-CoV-2 immunisation suggest that rapid translation of selected KDACi may enhance the efficacy of vaccination strategies.
Results
To identify existing small molecules with the potential to modulate T cell memory responses we undertook an in silica repurposing screen. We matched three independent transcriptional signatures of CD8+ T cell memory (Fig1A, B) against three libraries of small molecule transcriptional response signatures comprising over 30,000 compounds (Methods). Amongst predicted matches we observed multiple instances of compounds with lysine (K) deacetylase inhibitory (KDACi) activity (Fig1 B), with strongest enrichment from drug libraries containing signatures from human immune and primary cells. We therefore included 16 KDACi compounds amongst selected treatments in an in vitro phenotypic screen comprising 844 assays across 3 doses per compound. The doses of each compound are set out in the table below. The high- throughput assay tested each compound’s ability to modulate differentiation of stimulated primary human CD8+ T cells into memory precursor effector cells (MPEC), characterised by increased expression of IL7R on dividing cells (Fig1 C). We observed significant expansion of MPEC following treatment with multiple KDACi with the effect diminishing if treatment was delayed following stimulation (Fig1 E) and arising predominantly from differentiation of naive T cells, ratherthan from restimulation of those in a pre-existing memory pool (Fig1 F).
Table 2. KDACi dose range selection
Figure imgf000033_0001
Figure imgf000034_0001
Following KDACi treatment of primary human CD8+ T cells we observed a biphasic dose response, with MPEC expansion occurring only at low doses at which no impact on cell survival, proliferation or activation was seen (Fig1 G, H). Higher doses limited cell proliferation and survival consistent with their clinical use as anti-proliferative agents in the context of cancer (Fig 6).
Dose-dependent MPEC differentiation was characterised by expansion of a population of IL7RhiPD1 Io cells (Figi l-J) and was apparent with only a subset (9/16) of KDACi (Figi H-J, Fig6). We therefore sought to investigate the mechanism of enhanced memory differentiation, systematically comparing genomic analyses of compounds promoting MPEC differentiation (KDACi. mem) with compounds favouring short lived effector differentiation (KDACi.eff, Fig. 2A 84 and Fig 6). We reasoned that KDACi-induced differences in site-specific protein acetylation may be responsible and compared acetylated protein data quantified by mass spectrometry in KDACi-treated HEK cells. Consistent with this, unsupervised clustering of proteomic acetylation signatures derived from 88 segregated compounds by their effect on MPEC differentiation (Fig2A), but not by target KDAC enzyme class (Fig2A) or by the magnitude of total induced acetylation (quantified in primary stimulated CD8+ T cells, Fig2B, C). This indicated that proteinspecific acetylation - ratherthan simply increased total acetylation - was important so we further investigated protein specific acetylation differences folowing KDACi. mem or KDACi.eff exposure. Analysis of peptide acetylomic data identified 1560 acetylation sites from 1116 proteins differentially induced by KDACi. mem and KDACi.eff compounds, with pathway enrichment analysis indicating over-representation of multiple metabolic pathways amongst them . Given this result, we undertook targeted mass spectrometric metabolomic analysis of primary human CD8+ T cells stimulated in the presence of selected KDACi.mem, sodium valproate and apicidin (compounds selected for largest effect sizes at lowest concentrations, Fig 6Fig1 H-K). Compared to vehicle-treated stimulated control cells, 68 metabolites were found to be differentially expressed (Fig2D, 103) amongst which the ‘alanine, aspartate and glutamate’ pathway demonstrated strongest enrichment, greatest predicted pathway impact while showing similar enrichment amongst differentially-acetylated proteins also (Fig2E, Fig 6). Mapping differential metabolites onto this pathway revealed a specific and significant (FDR<5%,) increase in both metabolites and acetylation of enzymes regulating glutamine breakdown and the TCA cycle (Fig2F).
Glutaminolysis feeds the TCA cycle through generation of a-ketoglutarate (aKG), creating energy and biosynthetic precursors such as nucleic acids and modulating cellular redox balance. It makes an important energetic contribution to T cell activation and can replenish TCA cycle intermediates as they are used for biosynthesis. Glutaminolysis is known to promote B cell memory formation and can fuel stem cell-like oxidative metabolism in cancer cells, sustaining chronic proliferation. This process is controlled by Wnt/B-catenin signalling, a pathway also known to arrest effector T cell differentiation and promote memory formation. However, the specific contribution of glutaminolysis to T cell memory formation remains unclear. We therefore sought to determine whether KDACi. mem-induced glutaminolysis had a functional impact on T cell ‘metabolic switching’ - a relative increase in oxidative over glycolytic metabolism on activation that is a critical determinant of memory differentiation. Low-dose valproate treatment enhanced oxidative metabolism and reduced the glycolytic rate (Fig2G-l) without changing cell activation, proliferation orglucose uptake (Fig2J, K). Both valproate-induced metabolic switching (Fig2G-l) and MPEC differentiation (Fig2L-M) could be reversed by administration ofthe specific glutaminolysis inhibitor R162 (which targets GLUD1 : Fig2F Fig 7). MPEC differentiation could also be induced by bypassing glutaminolysis through direct provision of the cell-permeable aKG analogue dimethyl ketoglutarate (DMKG: Fig 7). Together, these data indicate that increased glutaminolysis induced by a subgroup of KDACi in activated primary human CD8+ T cells promotes a switch to oxidative metabolism, promoting their differentiation towards a memory precursor phenotype.
We next sought to test whether valproate treatment of stimulated CD8+ T cells would induce global transcriptional changes consistent with T cell memory differentiation. RNAseq analysis showed marked changes in stimulated CD8+ T cell transcriptomes induced by valproate treatment (Fig3A, compared to vehicle-treated, stimulated controls) that could be reversed by concurrent inhibition of glutaminolysis (Fig3B). To better understand these changes we performed a matrix factorisation analysis (methods), identifying 9 latent factors of which one (LF7) showed strong, specific association with valproate treatment 144 (Fig3C, FDR<1 %). Systematic enrichment analysis of T cell differentiation signatures amongst LF7 gene weights identified specific polarisation of T cell memory and exhaustion signatures used for initial compound screening (Fig3D, E). Enrichment analysis using consensus ‘hallmark’ signalling pathways (Fig3F) identified specific induction of two signatures, reflecting oxidative phosphorylation and also Myc signalling. Myc is a known upstream regulator of Wnt signaling and memory formation and also glutaminolysis-driven functional metabolic switching. Other latent factors reflected inter-individual differences in the extent of T cell activation and proliferation but were not associated with response to valproate treatment.
The generation of durable cellular memory requires epigenetic modification as cells undergo functional differentiation. We therefore used ATACseq analysis of valproate or vehicle-treated and stimulated CD8+ T cells to identify differentially open chromatin regions (Fig3G, H). Amongst differential peaks in 3220 genes associated with 92 pathways we observed strongest enrichment for the Wnt/B-catenin pathway (Fig3G), known to regulate glutaminolysis and a stem-like T cell memory phenotype. In addition, 6 of the top 9 valproate-induced pathways have established roles in T cell memory formation including HIF1 a-33, Hippo and glutamate signalling (Fig3G). We observed significant expansion of peaks in both promoter and regulatory 3’UTR regions in key Wnt pathway genes including CTNNB1 , LEF1 and TCF726 165 (Fig3H) and confirmed dose dependent beta catenin (CTNNB1) induction by valproate treatment of CD8+ T cells in vitro (Fig31) . We next sought to investigate whether the memory phenotype induced on primary stimulation was preserved on subsequent restimulation in vitro. We sorted IL7Rhi/lo MPEC/SLECs after either KDACi (valproate/apicidin) or vehicle treatment and, after resting and normalisation of cell numbers, restimulated them in vitro (using polyclonal anti-CD2/3/28 microbeads). In both cases, but especially after KDACi pre-treatment, the initial MPEG phenotype observed on primary stimulation was preserved on restimulation (Fig3J-L).
Epigenetic modification and preservation of MPEC phenotype indicated that durable expansion of memory may occur following KDACi. mem treatment. We therefore next tested whether low dose valproate treatment during a primary challenge could enhance immune memory in vivo. This was undertaken in four different murine models of infection or immunisation, testing early MPEC differentiation (d7post-immunisation/infection (pi)), the stable T cell memory pool (>d30pi) and recall responses on rechallenge (d5 post-rechallenge (pr)). Valproate treatment during primary infection (gMHV, Fig4A) or immunisation (Nitrophenylacetyl(NP)-OVA, Fig4B) promoted MPEC differentiation as with treatment of stimulated CD8+ T cells in vitro. Consequently, to allow assessment of functional in vivo memory responses, we used a heterosubtypic influenza rechallenge model. In this model (Fig4C) primary intranasal infection with H3N2 influenza (A/HKx31) is followed by intranasal rechallenge with a recombinant H1 N1 strain (A/PR8/34). The PR8 strain shares all internal proteins with HKx31 but is serologically distinct, allowing detection of secondary T cell responses without confounding effects of antibody mediated viral clearance. Secondary influenza responses are characterised by expansion of antigen-specific T cells in mediastinal lymph nodes (MLN) followed by migration to lung with severe infection characterised by diffuse lymphocytic infiltration and virus-induced lung immunopathology. Following low dose valproate treatment for 7 days during the primary infection (~10% clinically approved dose,), we observed significant expansion of lung-resident T cell memory by d30pi (Fig4D). This was followed by expanded secondary lymphoid responses on rechallenge (Fig4E, F) characterised by enhanced expression of a memory phenotype (IL7RhiPD1 Io, Fig 4G) and reduced pulmonary lymphocytic infiltration (Fig4H). In each case, concurrent administration of the glutaminolysis inhibitor (R162) reversed the valproate-induced expansion of T cell memory (Fig4D-H, VR). Although valproate treatment resulted in an overall reduction in lung T cell infiltration on rechallenge, we observed marked clustering of proliferating cells around the peribronchiolar site of infection (Fig 4I, J). This is consistent with the enhanced antigen-specific memory seen at d30pi compared to a more diffuse parenchymal T cell infiltrate characteristic of immunopathology seen in the controls (Fig 4I-J). Epigenetic (ATACseq) analysis of CD44+CD8+ T cells from spleens of valproate-treated animals again showed enrichment of Wnt, Hippo and HIF pathways amongst differentially open chromatin regions, consistent with in vitro observations on similarly treated primary human CD8+ T cells (Fig 3G). Although the influenza model was specifically designed to test secondary T cell responses (with no sharing of B cell epitopes between primary infection and rechallenge), we also observed an expansion of germinal centre B (GCB) and T follicular helper cells (TFH), critical for promoting a high-affinity antibody response (Fig4H). We therefore tested the impact of valproate treatment on both cellular and humoral responses in a murine model of SARS-CoV2 immunisation. Mice were immunised with SARS-CoV2 receptor binding domain protein (RBD) 221 alongside low dose oral valproate treatment (Fig 4K). Treated animals again showed expansion of germinal centre and plasmablast responses (Fig 4L) alongside a >2-fold expansion in the magnitude of RBD-specific B and T cell memory responses following peptide rechallenge (Fig 4L, M) along with a similar expansion in SARS-CoV-2 neutralising antibody titre (Fig 4N).
Together these data indicate that short duration, low dose sodium valproate treatment following primary immunisation or infection results in a significant expansion of both early MPEC and expanded stable memory populations and an enhanced secondary recall response. Combined genomic analyses indicate that this expansion is directed by increased glutaminolysis, oxidative metabolism and Wnt/Myc signalling accompanied by epigenetic modification of Wnt and other memory associated pathway genes. These effects of valproate treatment may be reversed both in vitro and in vivo by specifically blocking glutaminolysis, indicating an immunometabolic mechanism underpinning treatment-induced memory differentiation. With demonstrated in vivo efficacy at low dose, selected KDACi could be rapidly translated to boost immune memory responses to both infection and vaccination, including to influenza and SARS-CoV2 vaccines. Such enhanced cellular memory may be of particular benefit in the elderly, where vaccine responses may be suboptimal, or to limit the ability of new viral variants to evade vaccine- induced serological protection. Example 2 - Experimental medicine study in healthy volunteers receiving immune challenge by seasonal influenza vaccination with/without concurrent administration of low-dose sodium valproate
This is an experimental medicine study into the development of immunological memory following seasonal influenza vaccine aiming to investigate the extent and mechanism by which a repurposed drug alters that process.
The population we aimed to study were healthy adults in two broad age groups (18-65 years and >65 years) who are positive for specific HLA alleles that will facilitate identification of antigen specific CD4 (HLA-DRB*07:01 , HLA-DRB*11 :01 , HLA-DRB*04:01) and CD8 (A*02:01) T cells. Individualswill be identified and invited to participate through the NIHR Cambridge BioResource, to facilitate identification by HLA haplotype.
We aimed to recruit up to 180 volunteers, to achieve full datasets from 50 volunteers per treatment group (1 .vaccine ,+drug.dose#1 (100mg/day), 2.vaccine+drug.dose#2 (200mg/day) of the study (allowing for 20% dropout). Group 3 received seasonal influenza immunization alone, groups 1 and 2 received oral low dose sodium valproate from day 0 to day 7 inclusive. Each group was further stratified by age into those 18-65 years old and those >65 years old. Peripheral blood samples will be collected before and after vaccination (dayO and day7) and will be processed to retain serum and peripheral blood mononuclear cells (PBMC) for storage as viable aliquots of 107 cells. Samples will be processed in batches to minimize batch artefact structure once all have been collected. Serum samples will be used to quantitate influenza specific humoral responses (ELISA, HAI titre) at each timepoint while frozen, viable cell aliquots will be used to identify and sort influenza specific CD8 and CD4 T cells for quantitation and sequencing. The immune traits being measured as outcomes in this study design have been developed taking into account previous evidence of immunological correlates of protection following influenza vaccination:
• The cellular response following influenza vaccine shows a significant correlation with protection from severe infection.
• Increased titre of influenza specific antibody is a correlate of protection from influenza infection following seasonal vaccination
• Influenza responses to seasonal vaccination are demonstrably lower in aged individuals compared to younger vaccines.
• There is a dose-dependent effect of sodium valproate on memory differentiation during polyclonal stimulation of T lymphocytes in vitro culture.
• There is a differential effect of sodium valproate on memory and naive populations during polyclonal stimulation of T lymphocytes in vitro culture.
Results - Modulating Emerging Memory Responses to Immunisation (The MEMRI study) Neutralising antibody responses have been established as robust correlates of protection with multiple vaccines, while enhanced cellular responses associate with reduced disease severity and maintenance of protective antibody levels in both SARS-CoV-2 and influenza. In human influenza infection the antibody response has been established as a robust correlate of protection from infection while the magnitude of the heterotypic T cell response was associated with reduced severity of influenza infection during the 2009-2001 H1 N1 pandemic. We therefore sought to test whether repurposed low dose sodium valproate treatment could act as an immunometabolic adjuvant, promoting expanded cellular or humoral correlates of protection after seasonal influenza vaccination. We designed an interventional experimental medicine study (the MEMRI study, Fig 5A, + Protocol, methods) with stratified randomisation of HLA - genotyped (A*-0201) healthy volunteers to receive either sodium valproate (at one of two low doses (100 or 200mg/day) equivalent to 5-10% of its approved dose as an anti-convulsant,) or no additional treatment daily for seven days starting on the day of seasonal influenza (2020/21) vaccination (Fig 5B). Blood samples taken both before (day 0) and at days 7 and 30 following vaccination (Fig 5B) were used to quantify both the CD8+ T cell response to the immunodominant HLA-A*0201 restricted influenza protein MP1 and the humoral response using both vaccine-specific influenza antibody titres and functional haemagglutination inhibition (HAI) assays. We observed treatment-induced expansion of baseline-normalized influenza-specific cellular and humoral responses post vaccination with low dose sodium valproate treatment resulting in both a >10-fold expansion and maintenance of ‘flu-specific CD8+ T cells (Fig 5C, D) and a >2-fold expansion and maintenance of vaccine strain-specific influenza IgG titre (Fig 5E). Mean influenza-specific IgA titre in plasma was expanded > 10-fold in the treatment group on day 7 post-vaccination although levels had fallen in both groups by day 30 (Influenza A/Hong Kong H3, Fig 5F). Overall, influenza-specific HAI titre was increased >1 .5 fold in the treatment group (Fig 5G) with much of the observed humoral response due to the lowest dose treatment group . Conversely, the impact on the CD8+ T cell response was observed to be greater with the higher dose treatment.
We also observed post-vaccination expansion of influenza-specific IgG and IgA titres to viral serotypes that were not included in the seasonal vaccine used, including some which were increased by sodium valproate treatment. This implied that treatment may promote a broader response, as well as expansion and maintenance. Such a hypothesis is also supported by our observations of increased TFH and germinal centre B cells (Fig4B, M) in the murine influenza (Fig 4B) and SARS-CoV-2 immunisation models (Fig 4M) as these changes would be expected to result in increased affinity maturation and somatic hypermutation leading to a broader, more effective vaccine-induced antibody response. To test this hypothesis further, we undertook targeted B cell receptor sequencing and repertoire analysis of whole blood samples from the MEMRI study. In the global BCR repertoire we observed longitudinal changes in isotype usage, somatic hypermutation and clonal diversity peaking at day 7 post-vaccination. However, no global repertoire differences were observed between treatment groups (Fig 5H). To investigate changes in the influenza-specific BCR repertoire, we matched the observed BCR sequences to those of over 27,000 known influenza-specific clones (methods), identifying 5274 BCR clusters with confirmed influenza specificity. Amongst these sequences, valproate treatment was associated with significant increases in somatic hypermutation (Fig 51), clonal expansion (Fig 5J, K) and diversity (Fig 5L, M). Measures of expansion and diversity were increased by treatment in both class-switched and naive (lgM+lgD+) sequences (Fig5K, M) suggesting a treatment effect on both de novo primary and also secondary influenza-specific responses following vaccination.
Discussion
Prior to this study, it was known that metabolic changes could be causal drivers of immune cell differentiation as well as important consequences of that pro cess (McKinney and Smith, 2018). It was also known that metabolic intermediates - such as acetate(Qiu et al., 2019) - could serve as signalling events to modify critical cellular processes(Choudhary et al., 2009) including chromatin accessibility, such as are necessary to mediate long-lived changes in cellular phenotype(Choudhary et al., 2014). Consequently, what were initially known as ‘histone deacetylases’ have expanded to become lysine (K) deacetylases, capable of modulating many protein groups and with many targets in the mitochondria(Scholz et al., 2015). Our data are consistent with a model in which a subclass of KDACi compounds are capable of altering protein acetylation resulting in increased glutaminolysis during initial antigen encounter and cellular activation. The resulting switch towards oxidative phosphorylation promotes immune cell differentiation towards a memory phenotype and enhanced secondary responses. T cell exposure to certain KDACi at the time of activation results in acquisition of a memory phenotype along with functional, transcriptional and epigenetic features of memory. However, it is less clear whether other immune cell types (such as CD4+ T cells and B cells) undergo similar cell-intrinsic epigenetic modulation, or whether altering the phenotype of one cell type may have secondary consequences on the entire response. In addition, KDACi can have direct effects on histone acetylation and, while the reversal of treatment effects by inhibition of glutaminolysis (both in vitro and in vivo) suggests that epigenetic modulation is secondary to immunometabolic modulation, our experiments do not definitively differentiate these two possibilities.
It was also previously known that KDACi can have immunomodulatory effects, and in particular that cell survival, proliferation and differentiation can be altered by certain compounds. Our data are consistent with this - we see a biphasic dose response relationship with limitations in cell cycle and proliferation at higher doses and promotion of cellular memory only at lower doses. Only through screening multiple KDACi compounds was the subclass effect apparent.
Our experimental approach demonstrates that, by combining multiple genomic analyses with both in silico and in vitro phenotypic screening, it is possible to identify repurposable small molecules capable of rapid clinical translation. Similar approaches to the identification of repurposable small molecules have been tried before, although with only rare examples of validation in human studies(Pushpakom et al., 2019). However, the inclusion of multi-omic analysis also allows us to also consider the drugs screened to be molecular probes, using genomic data to interpret their mechanism of action and inform our biological understanding of immune memory in the process. A stepwise progression from in silico screen, through in vitro mechanistic evaluation into preclinical and ultimately human experimental medicine studies offers the potential to understand why as well as which compounds should be repurposed.
The MEMRI study described here indicates that low dose KDACi (valproate) treatment can significantly expand the size, breadth and durability of vaccine-induced cellular and humoral immunity (Fig 5). The study does not confirm clinical efficacy of the observed expansion but uses validated correlates of vaccine-induced protection from infection as key endpoints. Data from both murine and human studies are consistent with a model in which enhanced cellular and humoral responses are secondary to expanded germinal centre and TFH cell differentiation, rather than direct expansion of T cell memory alone. Our observation that both antigen naive (lgM+lgD+) and secondary (d7 lgG+) humoral responses show increased expansion, breadth and SHM on treatment suggests that susceptible groups - such as the elderly - who are more reliant on heterologous secondary responses for vaccine-induced protection could gain particular benefit from KDACi adjuvanted vaccination.
Together these data indicate that short duration, low dose sodium valproate treatment following primary immunisation or infection results in a significant expansion of immune memory in mice and humans. Multi-'omic analyses indicate that this expansion is directed by increased glutaminolysis, oxidative metabolism and Wnt/Myc signalling accompanied by epigenetic modification of Wnt and other memory associated pathway genes. These effects of valproate treatment may be reversed both in vitro and in vivo by specifically blocking glutaminolysis, indicating an immunometabolic mechanism underpinning treatment-induced memory differentiation. With demonstrated in vivo efficacy at low dose, selected KDACi could be rapidly translated to boost immune memory responses to both infection and vaccination, including to influenza and SARS-CoV2 vaccines. Such enhanced cellular memory may be of particular benefit in the elderly, where vaccine responses may be suboptimal, or to limit the ability of new viral variants to evade vaccine-induced serological protection.
Example 3 - Experimental medicine study in healthy volunteers receiving immune challenge by SARS-CoV-2 vaccination with/without concurrent administration of low- dose sodium valproate
This is an experimental medicine study into the development of immunological memory following seasonal influenza vaccine aiming to investigate the extent and mechanism by which a repurposed drug alters that process.
Subjects will be administered the SARS-CoV-2 vaccination on day 0 along with a 10Omg dose of sodium valproate. The sodium valproate will then be administered for 7 days following the vaccination. The same protocol will be used when the SARS-CoV-2 booster vaccination is administered.
Control subjects will also be included in the study who will not receive sodium valproate. Blood samples will be taken on the day of the first and second vaccinations as well as 6 months and 12 months after the first vaccination.
Methods
In silico screen
Target CD8+ T cell memory signatures defining CD8 SLEC v MPEC (ImmuneSigDB M3027, M3028), CD8+ exhaustion v memory (GSE41867, ImmuneSigDB, M9480.9482), CD2 memory (E-MTAB-3470) were obtained from MSigDB or GEO. Signatures were screened against characteristic direction drug response signature libraries curated from GEO (Drug_perturbations_from_GEO_2014), LINCS (L100 chem perturbation) and the connectivity map (CMAP) using the EnrichR platform. Compounds with significant overlap (overlap >=5 and adjusted P95%) were considered for inclusion for in vitro phenotypic screening. Combined enrichment scores are 11 255 computed as modified Fisher’s exact test multiplied by deviation from an expected rank value.
In vitro phenotypic screen
Primary human CD8+ T cells were separated from leucocyte cones obtained from NHS Blood and Transplant (Addenbrooke’s Hospital, Cambridge, UK) by centrifugation over ficoll and positive selection using magnetic beads as previously described. The purity of separated cell subsets was determined by three-colour flow cytometry. Purified T cells (>95%) werelabelled with 2.5pM CFSE (Invitrogen) and resuspended in complete RPMI 1640 (Sigma Aldrich) in the presence of 10% FCS and then stimulated in sterile, U-bottomed 96 well culture plates (Greiner) using MACS iBead particles (1 :2 bead:cell ratio, Miltenyi) conjugated to anti-CD2/CD3/CD28 in the presence of non-limiting IL2 (10ng/ml, Gibco Life technologies) for 6 days. Selected compounds were added at the time of stimulation (dO), or 2 (d2) or 4 (d4) days after as indicated. T cell memory subsets were isolated using flow cytometric sorting (FACSArialll cell sorter, BD Biosciences) following MACS enrichment of CD8+ T cells as above, into naive (Tn, CD45RA+CD62L+), effector memory (Tern, CD45RA-CD62L-), central memory (CD45RA- CD62L+) and Temra (CD45RA+CD62L+) populations before co-culture with drugs as indicated. T cells were stimulated in the presence of treatments as indicated for 6 days before undergoing high-throughput immunophenotypic screening (BD LSR Fortessa HTS) with quantification of IL7R. Reactions were standardised with multicolour calibration particles (BD Biosciences) with saturating concentrations of the following antibodies: IL7R (BD Biosciences, Clone HIL-7R- M21), CD25 (Clone M-A251), live/dead discrimination (AquaFluorescent amine-reactive dye, Invitrogen). Data was analysed using FlowJo software (Tree Star), extracting immunophenotypic traits (IL7Rmfi, division (CFSEdil), CD25mfi, % live cells) with ratios calculated against pooled vehicle treated controls in triplicate.
Compound selection and titration
Compounds were selected from the in silico screen on the basis of consistent enrichment against multiple signatures taking into consideration likely impact on cellular viability, toxicity and availability. Additional ligands and cytokines with a demonstrated association with T cell memory formation were also included with Fc-Chimaeric ligands hybridised to individual wells of a 96- well U-bottomed culture plate (Greiner) for 2h at 37C before washing in sterile complete RPMI- 1640 and addition of cell suspensions for stimulation. Dose ranges for inclusion were selected on the basis of demonstrated in vitro IC50 for target effects with 3 doses included per treatment. Effects were considered by dose and also by peak effect per treatment with each value compared to the median of triplicate repeat vehicle stimulation. In vitro KDACi efficacy was confirmed by measuring impact on total cellular protein acetylation by flow cytometry (acetylated lysine multiMab mix, CST: Fig2B) and for selected compounds (apicidin and sodium valproate) taken into genomic, functional and in vivo assays standard concentrations were generated using targeted LC-MS/MS quantitation (PK/B Core facility, CRUK, Cambridge).
Lysine Acetylation proteomic data
Stable isotope labelling with amino acids in cell culture (SILAC) mass spectroscopic (MS) quantitation of enriched acetylated peptides was obtained from. We extracted SILAC H/L ratios from KDACi-treated HeLa cells, verifying comparability between and then combining data across technical replicates. Median H/L ratios were converted to a pairwise distance matrix using the ape and phylobase packages in R before visualisation as an annotated, unrooted tree using iTOL (https://itol.embl.de/). KDACi compounds were classified on the basis of MPEG phenotype induction (KDACi. mem/eff) and differentially acetylated peptides were determined by comparison between these groups (threshold fdr 5%). Peptides were summarised to protein level and pathway enrichment analysis of differentially acetylated proteins was undertaken against the KEGG pathway database using EnrichR.
Metabolomic analysis
Primary human CD8+ T cells were snap frozen in liquid nitrogen following 20 isolation stimulation and culture as described above in the presence of either apicidin (1.4nM), valproate (87.5pM) or vehicle control (<0.001 % DMSO n=8 per group). Cell pellets were subjected to LC-MS/MS analysis (Metabolon Ltd) with identified compounds normalised to dsDNA concentration, log transformed and missing values imputed as the lowest value identified per compound. A Mixed model ANOVA with biological replicate as a random effect was used to identify differential compounds between experimental conditions (FDRq) Pathway enrichment of differential metabolites was undertaken using MetaboAnalyst42 and metabolite set enrichment analysis, taking pathways from HMDB43 as indicated.
Seahorse metabolic flux analysis
Measurement of oxygen consumption rate (OCR, XF MitoStress test, Agilent) and extracellular acidification rate (ECAR, XF Glycolysis Stress test, Agilent) was undertaken using the Seahorse XFp energy phenotype system (Agilent) according to the manufacturer’s instructions. Primary human CD8+ T cells were isolated, stimulated and cultured as above for 2 days in the presence of either 87.5pM sodium valproate before being transferred to Seahorse-XF base medium (Agilent, Cat No. 103335-100), immobilised onto a Seahorse XF96 cell culture microplate (Agilent) using Cell-Tak (Corning) with concurrent aliquots taken for immunophenotypic assessment including live/dead (AquaFluorescent amine-reactive dye, Invitrogen) and cell proliferation (CFSE dilution) quantification. Raw data was extracted using Wave software (Agilent) and normalised to baseline values with comparative flux estimates obtained from summarized peak values across biological replicates.
Glucose uptake assay
Primary human CD8+ T cells were isolated, stimluated and cultured as above for 6 days after which cells were incubated in glucose-free media for 60min at 37C in the presence of 50pM NBDG (ThermoFisher N13195) before staining with live/dead (AquaFluorescent amine-reactive dye, Invitrogen) and IL7R (BD Biosciences, Clone HIL-7R-M21) and acquisition of NBDG fluorescence on a BD LSR Fortessa instrument.
Glutaminolysis inhibition
Following isolation as above, CD8+ T cells were stimulated and co-cultured in the presence of either a dose range of sodium valproate (Sigma) or of the specific glutaminolysis inhibitors GDH1 (Focus Biomolecules), C968 (Sigma) or BPTES (Sigma), or with the cell-permeable a- ketoglutarate analogue dimethyl-2-ketoglutarate (DMKG, Sigma) as indicated.
RNA sequencing
Primary CD8+ T cells were cultured and stimulated as above for 6 days in the presence of either sodium valproate (175pM, Sigma) or sodium valproate plus R162 (175pM/25pM, Focus Biomolecules) before total RNA was extracted using an RNeasy mini kit (Qiagen) with quality assessed using an Agilent BioAnalyser 2100 and RNA quantification performed using a NanoDrop ND-1000 367 spectrophotometer. Sequencing library preparation was performed using the SMARTer stranded Total RNA-Seq kit v2 pico input (Takara Bio) and 75bp paired end sequencing performed on a NextSeq500 instrument with titrated 1 % PhiX. Raw sequence read QC was undertaken using fastqc followed by adapter removal (TrimGalore), extraction of ribosomal reads (BBsplit) and sequence mapping (HISAT2) to the reference genome (GRCh38). Merged bam file QC was undertaken using the QoRTs package in R with assimilation using multiQC. Merged bam files were read into R using featurecounts (Rsubread) with feature filtration to >cpm1 in >6 samples and annotation using BioMart followed by pairwise differential expression using DGEList (edgeR). Matrix factorisation analysis was undertaken using the MOFA package in R with feature set enrichment performed using the Bioconductor package fgsea in R with gene sets extracted from MSigDB using the msigdbr package as indicated.
ATAC sequencing
ATACseq was undertaken following the protocol established by Buenrostro et al 201545 with modifications. Following CD8+ T cell culture as above, 50,000 cells were lysed (1 M Tris-HCI) and the nuclear pellet transposed using the Tn5 transposase (TDE1 , Illumina) at 37C for 30min before DNA isolation (Qiagen 386 minElute) and PCR amplification (NEBnext) guided by qPCR (KAPA-SYBR) estimation of reporter signal v cycle number and PCR cycle length tailored to 1/3 maximal amplification. Libraries were purified (AMPure XP) with quality assessed using an Agilent BioAnalyser 2100 and RNA quantification performed using a NanoDrop ND-1000 spectrophotometer. 50bp paired-end sequencing was performed on a NextSeq500 instrument with downstream QC matching against ENCODE standards and peak calling and differential peak identification undertaken using the esATAC BioConductor package in R. KEGG pathway enrichment and visualisation of treatment-specific differential peaks was undertaken using the BioConductor packages clusterProfiler, GenVisR and rtracklayer in R. in vitro restimulation
Primary CD8+ T cells were isolated, stimulated and cultured as above for 6 days in the presence of either sodium valproate (175pM, Sigma) or vehicle control (<0.001 %DMSO) followed by flow cytometric sorting (FACSArialll cell sorter, BD Biosciences) into divided CFSEIolL7Rhi and CFSEIolL7Rlo subpopulations. These were rested for 5 days in complete RPMI-1640 (Sigma), with normalization of live cell numbers (1x106/ml) and CFSE-relabelling before polyclonal restimulation repeated as for the primary stimulation (anti- CD2/3/28, Miltenyi). After 5 days restimulation cells were analysed by flow cytometry (BD LSR Fortessa) with staining for live/dead (AquaFluorescent amine-reactive dye, Invitrogen) and IL7R (BD Biosciences, Clone HIL-7R-M21)
Infection and immunisation models
C57BL/6 mice were infected i/n with gMHV virus or immunised with 200pl NP-412 OVA in alum with spleens harvested d7 (NP-OVA) or d9pi (yMHV). For the heterotypic influenza model C57BL/6 mice were given a primary i/n challenge with 500pfu H3N2 influenza (A/HKx31) and a secondary challenge with 105pfu 415 i/n H1 N1 (A/PR8/34) influenza d31 post-primary challenge with tissues (lung, spleen, MLN, blood) harvested on d30pi and d38pi for stable memory and 417 rechallenge assessment respectively (Fig4C). For each model mice were administered sodium valproate 0.1 % w/v in drinking water, sodium valproate plus R162 (90% corn oil, 10% DMSO) 20mg/kg/day intraperitoneally or matched vehicle control as indicated (Fig4). For SARS- CoV-2 immunisation, mice were administered receptor binding domain (RBD) peptide (gift, YM) 50pg s/c in QuilA adjuvant either once (R1) orthree times (R2) as indicated (Fig4K). Tail bleeds were taken (d58pi) prior to RBD re-immunisation (d60) with tissues harvested on d65pi.
Expression and purification of SARS-CoV-2 RBD
Recombinant SARS-CoV-2 Spike RBD glycoprotein carrying a C-terminal hexahistidine purification (His6) tag was expressed in the Pichia pastoris X-33 yeast strain using the pPICZa A expression vector (ThermoFisher). Cultures were grown on the 10-liter scale in an Eppendorf BioFlo 510 fermenter with active control of temperature, pH, dissolved oxygen concentration and methanol feed (as the carbon source) to maximize protein yield based on a protocol developed previously to purify SARS-CoV-1 RBD46.47. RBD glycoprotein was secreted into the cell culture supernatant and purified by nickel-affinity and size-exclusion chromatography. To produce untagged RBD, the His6 tag was removed using Carboxypeptidase A (Sigma), followed by nickel-affinity chromatography to remove uncleaved protein and size-exclusion chromatography.
Tissue processing Splenocytes and draining lymph nodes were weighed and then homogenised through a 70pm cell strainer (BD bioscience) using complete tissue culture media [IMDM (Thermo), 10% Fetal Bovine Serum (Sigma), 1 mM Sodium Pyruvate (Thermo), 1 X Non-essential Amino Acid Solution (Sigma), 2mM 444 Glutamax (Thermo), 100U/ml Penicillin-Streptomycin (Thermo) and 50pM b- 445 Mercaptoethanol (Sigma)]. Splenocyte solutions were red blood cell lysed, resuspended in culture media and counted before use.
ELISPOTs
For B cell ELISPOTS, MultiScreen-IP Filter PDVF, 0.45 pm ELISPOT plates (Merck Millipore) were first activated in 70% ethanol for 2 minutes before being washed twice with sterile PBS. Plates were then coated overnight at 4°C with 50pl of purified RBP protein in PBS (5pg/ml). The next day, plates were washed twice with sterile PBS and blocked with 200pl complete tissue culture media for 2 hours at 37°C. After this time, blocking media was aspirated and 10OpI of splenocyte suspensions were added in two concentrations (106/mL and 106/mL) to appropriate triplicate wells. Plates were then incubated at 37°C overnight. After 18hrs cells were aspirated from the wells and plates were washed 3 times with PBS/0.05% Tween-20 (Sigma) [PBST] followed by 3 washes in PBS. 50pl of goat anti-mouse IgG-HRP conjugate (Southern Biotech) in PBS at 1 :2000 dilution was then added to the plates and incubated at room temperature for 1 hour. Plates were then washed as previous, before being developed with 3-Amino-9- ethylcarbazole tablets (Sigma) using the manufacturer’s instructions. After stopping development, plates were left to dry for 48 hours before being read and analysed using an ELISPOT plate scanner (ELISPOT Reader System, AID) and spots enumerated using Imaged (National Institutes of Health).
For IFNg ELISPOTS, plates were prepared with ethanol and washed as above before being coated with 50pl of anti-mouse IFNg (AN18, Thermo) at 2ug/ml in PBS. Plates were incubated overnight at 4°C before being washed and blocked as described above. First, 50pl of culture media containing 60U/ml IL-2 (Peprotech) and 20pg/ml purified RBP protein were added to the appropriate wells, followed by 50pl of cell suspension corresponding to 104 or 105 cells per well. Plates were then incubated at 37°C for 48 hours before cells were aspirated and washed as above. 50pl of biotinylated anti-mouse IFNg (R4-6A2, Thermo) in PBS/0.5% BSA (Fischer) was then added to each well and plates were incubated for 2 hours at room temperature before being washed. 50pl of Streptavidin-horseradish peroxidase (Thermo) diluted 1 :5000 in PBS/0.5% BSA was then added to each 20 well and plates were incubated for 1 hour at room temperature, washed and then developed as above.
Immunophenotyping
After harvesting cell suspensions were normalised to 1x106 /ml and stained with non-limiting concentrations of IL7R (CloneA7R34, Biolegend), PD1 (Clone RMP1- 30, Biolegend), KLRG1 0, CD44 (ClonelM7, Biolegend), NP-dextramer (Immudex), GL7 (Clone GL7, Biolegend), CD8 (Clone 53-6.7, Biolegend), BCL6 4(ClonelG191 E/A8, Biolegend), FOXP3 (Clone FJK-16S, ThermoFisher), B220 (Clone RA3-6B2, Biolegend) before data acquisition by flow cytometry (BD LSR Fortessa) and analysis in FlowJo (BD Biosciences) and prism 6.0 (Graphpad).
Multiplex IHC
Lung and spleen samples were fixed and processed into FFPE blocks. 4.5um paraffin sections were collected on Super Frost Plus slides and dried overnight. For immunostaining slides were deparaffinized in xylene and rehydrated with series of graded ethanol. Antigen Retrieval was performed by gentle simmering in 700ml of Tris-EDTA, pH9 buffer, following by endogenous peroxidases blocking and incubation with 2.5% Horse Serum. Ki67 antibody (1 :500) were incubated in a humidified chamber overnight at +4C, detected using ImmPRESS anti-Rabbit- HRP Polymer detection system (VectorLab, MP-7401) and visualized using TSA-Plus Opal 620 (Akoya, FP1495001 KT). Slides were then placed in Tris-EDTA, ph9 buffer and simmered for 15 min. anti-CD3 antibodies (1 :50) were applied and incubated in a humidified chamber overnight at +4C. As before primary anti-CD3 antibodies were detected with ImmPRESS anti-Rabbit-HRP 21 Polymer detection system and visualized with TSA-Plus Opal 520 (Akoya, FP1495001 KT). Slides were mounted with Dapi-supplemented ProlongGold and imaged using Zeiss Apotome 2 microscope and Hamamatsu monochrome camera.

Claims

47
1 . A method for enhancing an immune response in a subject comprising administering a lysine deacetylase inhibitor (KDACi) simultaneously, sequentially, or separately with a vaccine, wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 for the selected KDACi.
2. The method according to claim 1 , wherein the KDACi is provided at a dose of up to 25% of the dose listed in Table 1.
3. The method according to claim 1 or 2, wherein the vaccine is an infectious disease vaccine against a viral, bacterial, fungal or parasitic infectious agent.
4. The method according to a preceding claim, wherein the vaccine is a respiratory disease vaccine selected from a vaccine against Haemophilus influenza, a coronavirus, such as SARS-CoV-2, Haemophilus influenzae type B, measles virus, poliovirus, tetanus, tuberculosis, cholera, typhoid, dengue, diphtheria, hepatitis, Japanese encephalitis, meningococcal meningitis, mumps virus, pertussis, pneumococcal disease, rabies, rotavius, rubella, coronavirus, such as SARS-CoV-2, SARS-CoV, or MERS-CoV,
5. The method according to a preceding claim, wherein the vaccine is selected from a whole pathogen vaccine, live attenuated vaccine, inactivated vaccine, whole killed vaccine, recombinant protein vaccine, toxoid vaccine, conjugate vaccine, virus-like particle (VLP) vaccine, outer membrane vesicle (OMV) vaccine, DNA vaccine, RNA vaccine, or viral vectored vaccine.
6. The method according to a preceding claim, wherein the KDACi is administered at a dose of 10 to 950 mg/day, or 10 to 500 mg/day, or 50 to 250 mg/day, or 75 to 225 mg/day, preferably at a dose of 100 to 200 mg/day.
7. The method according to a claim 5, wherein the KDACi is valproate and administered at a dose of 100 mg/day.
8. The method according to claim 5, wherein the KDACi valproate and is administered at a dose of 200 mg/day.
9. The method according to a preceding claim, wherein the KDACi and the vaccine are administered on day 0, and wherein a further dose of the KDACi is administered on days 1 to 2, days 1 to 3, days 1 to 4, days 1 to 5, days 1 to 6 or days 1 to 7.
10. The method according to a preceding claim, wherein the KDACi is administered by the oral, sublingual, buccal, intravenous, intramuscular, or subcutaneous route.
11 . The method according to a preceding claim, wherein the KDACi is provided as a tablet, capsule or liquid.
12. The method according to a preceding claim, wherein the vaccine is administered by the intravenous, intramuscular, or subcutaneous route. 48 A method for the treatment of a disease comprising administering a lysine deacetylase inhibitor (KDACi) wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 for the selected KDACi. A KDACi for use in the prevention of a disease, wherein the KDACi is administered simultaneously, sequentially, or separately with a vaccine, wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 for the selected KDACi. A KDACi for use in the prevention of SARS-CoV-2, wherein the KDACi is administered simultaneously, sequentially, or separately with a SARS-CoV-2 vaccine. A KDACi for use in the prevention of influenza, wherein the KDACi is administered simultaneously, sequentially, or separately with an influenza vaccine. A method of promoting a T cell memory response comprising exposing cells to a KDACi capable of enhancing glutaminolysis. A method of promoting differentiation of CD8+ T cells into memory precursor effector cells, comprising exposing CD8+ T cells to a KDACi capable of enhancing glutaminolysis. A method of increasing IL7R expression in CD8+ T cells, comprising exposing CD8+ T cells to a KDACi capable of enhancing glutaminolysis. A method of enhancing glutaminolysis comprising exposing CD8+ T cells to a KDACi capable of inhibiting one or more of KDAC1 , KDAC2, KDAC3, KDAC4, KDAC5, KDAC7, KDAC9 or KDAC10. The method of any one of claims 17 to 20, wherein the cells are exposed to the KDACi in vivo, in vitro or ex vivo. The method of any one of claims 17 to 21 , wherein the cells are also exposed to a vaccine. A method of immunising a subject against a disease, comprising administering a KDACi simultaneously, sequentially, or separately with a vaccine, and wherein the KDACi is capable of enhancing glutaminolysis. Use of a class I KDACi as an adjuvant for enhancing the efficacy of a vaccine wherein the KDACi is capable of enhancing glutaminolysis. A composition comprising a KDACi and a vaccine wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1 . A kit comprising a KDACi and an infectious disease vaccine and optionally instructions for use, wherein the KDACi is selected from the KDACi listed in Table 1 and provided at up to 50% of the dose listed in Table 1. A method for enhancing an immune response in a subject comprising administering valproate simultaneously, sequentially, or separately with a vaccine, wherein the valproate is administered in a dose of 10 to 950 mg/day. 49
28. A method for enhancing an immune response in a subject comprising administering abexinostat simultaneously, sequentially, or separately with a vaccine, wherein the abexinostat is administered in a dose of 2 to 10 nM.
29. A method for enhancing an immune response in a subject comprising administering bellinostat simultaneously, sequentially, or separately with a vaccine, wherein the bellin ostat is administered in a dose of 5 to 800 nM.
30. A method for enhancing an immune response in a subject comprising administering panobinostat simultaneously, sequentially, or separately with a disease vaccine, wherein the panobinostat is administered in a dose of 0.5 to 500 nM,
31. A method for enhancing an immune response in a subject comprising administering butyrate simultaneously, sequentially, or separately with a vaccine, wherein the sodium butyrate is administered in a dose of 0.1 x103 to 500x103 nM.
32. A method for enhancing an immune response in a subject comprising administering apicidin simultaneously, sequentially, or separately with a vaccine, wherein the apicidin is administered in a dose of 0.01 to 100 nM.
33. A method for enhancing an immune response in a subject comprising administering mocetinostat simultaneously, sequentially, or separately with a vaccine, wherein the mocetinostat is administered in a dose of 0.01 x103 to 1x103 nM.
34. A method for enhancing an immune response in a subject comprising administering vorinostat simultaneously, sequentially, or separately with a vaccine, wherein the vorinostat is administered in a dose of 0.2 x103 to 5x103 nM.
35. A method for enhancing an immune response in a subject comprising administering MS275 simultaneously, sequentially, or separately with a vaccine, wherein the MS275 is administered in a dose of 0.01x10® to 1x10® nM,
36. A method for enhancing an immune response in a subject comprising administering CI994 simultaneously, sequentially, or separately with a vaccine, wherein the CI994 is administered in a dose of 0.01x10®to 2x10® nM.
37. A method for expanding cells for use in adoptive cell therapy comprising: providing a population of CD8+ T cells, contacting the population of CD8+ T cells to a KDACi capable of enhancing glutaminolysis to produce an expanded population of cells.
38. The method of claim 37, wherein the expanded population of cells comprises memory precursor effector cells
39. The method of claims 37 to 38, wherein the expanded population of cells are characterised by the markers IL7Rhi, PD110.
40. The method of claims 37 to 39, wherein the expanded population of cells are further characterised by the markers IL7Rhi, PD110, CX3CR1 +, CCR7+, CD27+ and CD62L+.
PCT/GB2022/053173 2021-12-10 2022-12-12 Methods and compositions to potentiate the immune response with lysine deacetylase inhibitors WO2023105247A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB2117903.1 2021-12-10
GBGB2117903.1A GB202117903D0 (en) 2021-12-10 2021-12-10 Methods and compositions to potentiate the immune response

Publications (1)

Publication Number Publication Date
WO2023105247A1 true WO2023105247A1 (en) 2023-06-15

Family

ID=80080087

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2022/053173 WO2023105247A1 (en) 2021-12-10 2022-12-12 Methods and compositions to potentiate the immune response with lysine deacetylase inhibitors

Country Status (2)

Country Link
GB (1) GB202117903D0 (en)
WO (1) WO2023105247A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012122629A1 (en) * 2011-03-11 2012-09-20 Mcmaster University A method of vaccination comprising a histone deacetylase inhibitor
WO2020030634A1 (en) * 2018-08-06 2020-02-13 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and compositions for treating cancers

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012122629A1 (en) * 2011-03-11 2012-09-20 Mcmaster University A method of vaccination comprising a histone deacetylase inhibitor
WO2020030634A1 (en) * 2018-08-06 2020-02-13 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and compositions for treating cancers

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
BANTSCHEFF, M. ET AL.: "Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes", NAT BIOTECHNOL, vol. 29, 2011, pages 255 - 265, XP002652670, DOI: 10.1038/nbt.1759
CHOUDHARY, C. ET AL.: "Lysine acetylation targets protein complexes and co- regulates major cellular functions", SCIENCE, vol. 325, 2009, pages 834 - 840, XP055151659, DOI: 10.1126/science.1175371
DINARELLO ET AL.: "Histone Deacetylase Inhibitors for Treating a Spectrum of Diseases Not Related to Cancer", MOL MED, vol. 17, no. 5-6, 2011, pages 333 - 352, XP002676921, DOI: 10.2119/MOLMED.2011.00116
DRUMMOND, D. C. ET AL.: "Clinical development of histone deacetylase inhibitors as anticancer agents", ANNU REV PHARMACOL TOXICOL, vol. 45, 2005, pages 495 - 528, XP055002358, DOI: 10.1146/annurev.pharmtox.45.120403.095825
GREENSAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR LABORATORY PRESS
LI, Y.SETO, E.: "HDACs and HDAC Inhibitors in Cancer Development and Therapy", COLD SPRING HARB PERSPECT MED, vol. 6, 2016, XP055899214, DOI: 10.1101/cshperspect.a026831
LICCIARDI, P. V.KARAGIANNIS, T. C.: "Regulation of immune responses by histone deacetylase inhibitors", ISRN HEMATOL, vol. 2012, 2012, pages 690901
MCCAW TYLER R ET AL: "Histone deacetylase inhibition promotes intratumoral CD8T-cell responses, sensitizing murine breast tumors to anti-PD1", CANCER IMMUNOLOGY IMMUNOTHERAPY, SPRINGER, BERLIN/HEIDELBERG, vol. 68, no. 12, 12 November 2019 (2019-11-12), pages 2081 - 2094, XP036952137, ISSN: 0340-7004, [retrieved on 20191112], DOI: 10.1007/S00262-019-02430-9 *
MOREIRA, J. MSCHEIPERS, P.SORENSEN, P.: "The histone deacetylase inhibitor Trichostatin A modulates CD4+ T cell responses", BMC CANCER, vol. 3, 2003, pages 30, XP021004596, DOI: 10.1186/1471-2407-3-30
SCHOLZ, C. ET AL.: "Acetylation site specificities of lysine deacetylase inhibitors in human cells.", NAT BIOTECHNOL, vol. 33, 2015, pages 415 - 423
TAO, R. ET AL.: "Deacetylase inhibition promotes the generation and function of regulatory T cells", NAT MED, vol. 13, 2007, pages 1299 - 1307, XP002538143, DOI: 10.1038/NM1652

Also Published As

Publication number Publication date
GB202117903D0 (en) 2022-01-26

Similar Documents

Publication Publication Date Title
RU2742993C2 (en) Prime-boost modes involving introduction of at least one mrna structure
BE1023915A9 (en) NOVEL METHODS FOR INDUCING AN IMMUNE RESPONSE
CN114096675A (en) Coronavirus immunogenic compositions and uses thereof
US20220105170A1 (en) African swine fever vaccine
JP7223989B2 (en) Immunomodulatory compositions and methods of use thereof
JP2023510542A (en) Universal vaccines against immunogens of pathogenic organisms promoting organism-specific and intergroup protection
WO2012131069A1 (en) Short chain fatty acids and their derivatives for use in treatment immunogenic disorders
WO2022197720A2 (en) Compositions and methods for treating coronavirus infection
JP2022545771A (en) Drug composition that produces a safe amount of nitric oxide in the body and its use
ES2748632T3 (en) Pharmaceutical composition comprising propionic acid for use in the treatment of viral infections
JP2007508319A5 (en)
WO2015136304A1 (en) Polyamine compounds and uses thereof
WO2023105247A1 (en) Methods and compositions to potentiate the immune response with lysine deacetylase inhibitors
Wan et al. As a genetic adjuvant, CTA improves the immunogenicity of DNA vaccines in an ADP-ribosyltransferase activity-and IL-6-dependent manner
Wang et al. Protective immunity against acute toxoplasmosis in BALB/c mice induced by a DNA vaccine encoding Toxoplasma gondii 10 kDa excretory–secretory antigen (TgESA10)
Dröge et al. [23] Thiols and the immune system: Effect of N-acetylcysteine on T cell system in human subjects
Ouaked et al. Non-clinical evaluation of local and systemic immunity induced by different vaccination strategies of the candidate tuberculosis vaccine M72/AS01
JP4526763B2 (en) Inactivation of MEP pathway genes
JP2005526729A (en) Strategies for retroviral immunotherapy
Wang et al. Oral immunisation of mice with a recombinant rabies virus vaccine incorporating the heat-labile enterotoxin B subunit of Escherichia coli in an attenuated Salmonella strain
Yang et al. Intranasal immunization with a Middle East respiratory syndrome-coronavirus antigen conjugated to the M-cell targeting ligand Co4B enhances antigen-specific mucosal and systemic immunity and protects against infection
WO2015077442A2 (en) Grass pollen immunogens and methods and uses for immune response modulation
WO2024084785A1 (en) Composition suitable for use as rs virus vaccine
US20240115693A1 (en) Sars-cov-2 antigen nanoparticles and uses there of
WO2011144360A1 (en) Marker vaccine for classical swine fever

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22829815

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE