WO2015136304A1 - Composés de polyamine et leurs utilisations - Google Patents

Composés de polyamine et leurs utilisations Download PDF

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Publication number
WO2015136304A1
WO2015136304A1 PCT/GB2015/050743 GB2015050743W WO2015136304A1 WO 2015136304 A1 WO2015136304 A1 WO 2015136304A1 GB 2015050743 W GB2015050743 W GB 2015050743W WO 2015136304 A1 WO2015136304 A1 WO 2015136304A1
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polyamine
cells
cell
optionally
administered
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PCT/GB2015/050743
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English (en)
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Daniel PULESTON
Anna Katherina SIMON
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Isis Innovation Limited
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Priority claimed from GB201404438A external-priority patent/GB201404438D0/en
Priority claimed from GB201420001A external-priority patent/GB201420001D0/en
Application filed by Isis Innovation Limited filed Critical Isis Innovation Limited
Publication of WO2015136304A1 publication Critical patent/WO2015136304A1/fr

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    • 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
    • A61K31/13—Amines
    • A61K31/132—Amines having two or more amino groups, e.g. spermidine, putrescine
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • 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
    • A61K2039/543—Mucosal route intranasal
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • 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
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic 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

Definitions

  • the present invention relates to polyamine compounds and their uses.
  • the present invention relates to the effect of polyamines on the immune response of a subject to an immunogen.
  • An effective and responsive immune system is essential to the maintenance of health, such that the numerous potential pathogens, toxins, aberrant neoplastic cells etc. encountered by a body do not result in pathology or disease. Similarly, such an effective and responsive immune system is required for therapeutic interventions such as vaccines to confer any benefit.
  • immune system If the immune system is suppressed, degraded or degenerate in any way, this can leave an individual susceptible to infection or disease.
  • a poorly functioning or ineffective immune system may be caused any of a number of factors, including infection (e.g. HIV), genetic defects (e.g. ADA-SCID), environmental factors (e.g. malnutrition), and aging (immunosenescence).
  • immunosenescence One particular, rarely addressed, cause of a poorly functioning immune system is immunosenescence. As individuals age, the cells of the immune system become less responsive to immunogens, with both primary and secondary immune responses demonstrating diminished or aberrant immune responses. In particular the adaptive immune response is decreased with reduced T cell memory formation or/and lower antibody titres. This leads to a two-fold problem: as a result of immunosenescence, aged individuals are more susceptible to infection, and at the same time are less responsive to prophylactic or therapeutic vaccination.
  • adjuvants work largely through stimulating other aspects of the immune system, often the innate immune system, in order to amplify the adaptive immune response (e.g. CpG or LPS binding to Toll-like receptors (TLRs)).
  • TLRs Toll-like receptors
  • polyamines such as spermidine has been shown to prolong the lifespan of yeast, flies, nematodes and mice, as well as the life-span of human peripheral blood mononuclear cells (PBMCs) by inhibition of necrotic cell death (Eisenberg et al., Nature Cell. Biol., 1 1 ; 1305-1314 (2009)).
  • PBMCs peripheral blood mononuclear cells
  • polyamines which occur naturally in healthy cells and are required for cell growth, can enhance the immune response of an individual to an immunogen such as a vaccine.
  • the use of polyamines is able to restore the immune response to vaccine of aged individuals to a level at least equivalent to healthy young controls.
  • Use of polyamines for this purpose has the added advantage of enhancing the immune response with minimal adverse side-effects.
  • the invention provides a polyamine for use in the treatment of immunosenescence in a subject.
  • a polyamine for use in the treatment of immunosenescence in a subject.
  • immunosenescence in a subject is characterised by a refractory immune response to an immunogen, for example a refractory adaptive immune response to an immunogen, such as a refractory T cell response and/or a refractory B cell response.
  • a refractory immune response to an immunogen for example a refractory adaptive immune response to an immunogen, such as a refractory T cell response and/or a refractory B cell response.
  • the polyamine may be administered following the administration to the subject of an immunogen.
  • the polyamine may be administered concurrently with the administration to the subject of an immunogen.
  • the polyamine may be administered according to any of these embodiments, alone or in any combination. That is, a polyamine for use in the treatment of
  • immunosenescence in a subject may be administered prior to, and/or
  • the immunogen to which the subject exhibits a refractory immune response and the immunogen administered to the subject is the same immunogen.
  • the invention provides a polyamine for use in enhancing the immune response of a subject to an immunogen.
  • the immune response is an adaptive immune response. That is, the immune response is a T cell (cellular) immune response and/or a B cell (humoral) immune response.
  • the immune response is a T cell immune response, for example a memory T cell response.
  • the immune response is a CD4 T cell immune response, for example a Th1 and/or a Th2 response.
  • the immune response is a CD8 T cell immune response, for example a CD8 memory T cell response.
  • the immune response is a B cell response.
  • the polyamine for use in enhancing the immune response of a subject is administered to the subject prior to the administration to the subject of an immunogen.
  • the polyamine may be administered to the subject following the administration to the subject of an immunogen.
  • the polyamine may be administered to the subject concurrently with the administration to the subject of an immunogen.
  • the polyamine may be administered according to any of these embodiments, alone or in any combination. That is, a polyamine for use in enhancing the immune response of a subject may be administered prior to, and/or concurrently with, and/or following the administration to the subject of an immunogen.
  • the immunogen is a vaccine, preferably a T cell vaccine.
  • the polyamine is a naturally-occurring polyamine.
  • the polyamine is putrescine, cadaverine, spermidine, or spermine (or an analogue thereof).
  • the polyamine is putrescine, cadaverine, spermidine, or spermine (or an analogue thereof).
  • the polyamine is putrescine, cadaverine, spermidine, or spermine (or an analogue thereof).
  • the polyamine is
  • the polyamine is administered to the subject in solution, preferably aqueous solution. In certain embodiments the polyamine is
  • the polyamine is administered to the subject at a concentration of 1 -10 mM, preferably 1 -5 mM, preferably 5 mM. In certain embodiments, the polyamine is administered to the subject at least once a day for 1 to 200 days, optionally 1 to 1 10 days, optionally 1 to 90 days, optionally 1 to 52 days, optionally 1 to 21 days, optionally 1 day (that is, wherein the polyamine is administered once). In an alternative embodiment the polyamine is
  • the invention provides a method of enhancing the immune response of a subject to an immunogen, the method comprising (i) administering a polyamine to the subject; and (ii) administering the immunogen to the subject.
  • step (i) is performed prior to and/or concurrently with and/or following step (ii).
  • the method enhances the adaptive immune response, preferably the T cell immune response. In preferred such embodiments, the method enhances the memory T cell immune response. In certain embodiments, the method enhances the CD8 T cell response, for example the memory CD8 T cell response, and/or the CD4 T cell response, for example the CD4 Th1 response and/or the CD4 Th2 response.
  • the immunogen is a vaccine, preferably a T cell vaccine.
  • the polyamine is a naturally-occurring polyamine.
  • the polyamine is putrescine, cadaverine, spermidine, or spermine.
  • the polyamine is spermidine.
  • the polyamine is administered to the subject in solution, preferably aqueous solution. In certain embodiments the polyamine is
  • the polyamine is administered to the subject at a concentration of 1 -10 mM, preferably 1 -5 mM, preferably 5 mM. In certain embodiments, the polyamine is administered to the subject at least once a day for 1 to 200 days, optionally 1 to 1 10 days, optionally 1 to 90 days, optionally 1 to 52 days, optionally 1 to 21 days, optionally 1 day (that is, wherein the polyamine is administered once). In an alternative embodiment the polyamine is
  • the invention provides a nutraceutical preparation comprising a polyamine, wherein said preparation is suitable for the treatment of
  • the nutraceutical preparation is suitable for oral administration, preferably ingestion. Accordingly, the invention provides an ingestible nutraceutical preparation comprising a polyamine, wherein said preparation is suitable for the treatment of immunosenescence.
  • the nutraceutical preparation is a beverage.
  • the nutraceutical preparation is a dairy-based preparation, for example a yoghurt.
  • the preparation comprises the polyamine in a concentration of 1 -10 mM, preferably 1 -5 mM, preferably 5 mM.
  • the invention provides a nutraceutical preparation according to the fourth aspect for use in the treatment of immunosenescence.
  • the preparation is administered at least once a day for 1 to 200 days, optionally 1 to 1 10 days, optionally 1 to 90 days, optionally 1 to 52 days, optionally 1 to 21 days, optionally 1 day, optionally wherein the preparation is administered once.
  • the preparation is administered weekly for 1 to 30 weeks, optionally 1 to 16 weeks, optionally 1 to 13 weeks, optionally 1 to 8 weeks, optionally 1 to 3 weeks, optionally 1 week.
  • FIG. 1 Poly amine treatment enhances the CD8 T cell response to vaccination in aged mice.
  • CD8 + T cell kinetics in 14 week old and 25 month old wildtype and CD4-Atg7 _/" mice were tracked in the blood by tetramer following influenza vaccination. Mice were vaccinated on day 0 and day 22. Spermidine was administered in the drinking water 21 days prior to the first vaccination through to the experimental endpoint.
  • Figure 2 Poly amine treatment improves the CD8 + T cell response in the blood to influenza challenge in aged mice. Eight week old, 25 month old wildtype, and 25 month old CD4-Atg7 _/" mice were vaccinated on day 0 and day 22.
  • FIG. 3 Poly amine treatment improves the CD8 + T cell response in the lungs to influenza challenge in aged mice. Eight week old, 25 month old wildtype, and 25 month old CD4-Atg7 _/" mice were vaccinated on day 0 and day 22. Thirty days later they were challenged with 32 HAU wildtype X31 Influenza virus intranasally. The frequency (A) and absolute counts (B) of CD8 + T cells specific for NP 366 _374 was assessed in the lungs by tetramer on day 9 post-influenza challenge. Spermidine was administered in the drinking water 21 days prior to the first vaccination through to the experimental endpoint.
  • FIG. 4 Polyamine treatment improves the CD8 + T cell response to influenza infection in young mice. Eight week old mice were treated with spermidine in the drinking water at a concentration of 5mM for 8 weeks then infected with 0.001 X31 (H3N2) influenza. Data shown is the frequency CD8 + T cells specific for NP 366 . 3 74 in the lungs by tetramer on day 7 post infection. Figure 5. 8 week old mice were immunised intranasally with the influenza vaccine S-Flu. On day 7 the vaccine specific CD8 + T cell response was assessed in the lungs. From day 0 to day 7 spermidine was administered in the drinking water at a concentration of 5mM. Shown is the frequency of CD8 + T cells that are specific for the Influenza epitope NP 366 _37 4 .
  • Figure 6 8 week old mice were immunised intranasally with the influenza vaccine S-Flu. On day 14 the vaccine specific CD8 + T cell response was assessed in the lungs. In one group of mice, 100 ⁇ g of spermidine was administered intraperitoneally every other day from day 0 to day 14, with the first administration performed concurrently with vaccination (Figure 6A). For another group of mice, 500 ⁇ g of spermidine was administered intraperitoneally concurrently with the vaccination. Shown is the frequency of CD8 + T cells that are specific for the Influenza epitope NP 366 . 3 74.
  • Lymphopenia induces homeostatic proliferation and an activated CD8 + T cell phenotype in T-Atg7' ⁇ mice.
  • c) Absolute counts of CD4 + and CD8 + T cells in blood of J-AtgZ 1' and WT mice over time (n 4).
  • d) Percentage of CD44 hl cells in the splenic CD8 + T cell compartment of -Atg 1' and WT mice. Bar graphs depict the frequency of gated cells (representative of seven independent experiments), * p ⁇ 0.05 (n 4).
  • CD45.1 hosts reconstituted with a 1 :1 mix of -Atg 1' or WT BM (both CD45.2) with CD45.1 wild type BM.
  • AtgT gene expression in AtgT /+ and AtgT' ' ! cells T cells were isolated from the spleens of WT and J-Atg ⁇ mice by flow cytometry. mRNA was extracted and AtgT gene expression measured by q-PCR. Bar graph shows relative Atg7 expression in T cells from J-AtgT' ' m ⁇ ce compared to T cells from WT mice.
  • MCMV MCMV.
  • CD8 + T cell response to epitopes m38 (left panel) and IE3 (right panel) were tracked over time in blood by tetramer in WT and J-AtgZ 1' mice. Y-axis indicates the percentage of CD8 + T cells that are m38-specific.
  • CD8 + T cells from spleen were stained with CytolD at day 9 post-infection and assessed by flow cytometry. Histograms show examples of CD44'° CD8 + T cells from unimmunized mice (filled grey line) and in NP-specific CD8 + T cells from immunized mice (open black line). Quantification is by mean fluorescence intensity (MFI) of CytolD on gated indicated cell population and representative of two independent experiments.
  • MFI mean fluorescence intensity
  • Dot plots depict example of PD-1 and TIM-3 staining on gated CD45.2 + m45-tetramer + CD8 + T cells. Bar graph quantifies the percentage of (donor) CD45.2 + m45-tetramer + CD8 + T cells that are PD- ⁇ TIM-3 + at day 10 postinfection. * p ⁇ 0.05, by Mann-Whitney U-test (n 4-7). d) CD127 expression on AtgT' ' MCMV-specific CD8 + T cells in MCMV challenged BM chimeras.
  • IRF4 expression in AtgT' ' and AtgT' + splenic m45-specific CD8 + T cells on day 9, 15 and 22 post-infection. As a control, IRF4 was also measured in CD44'° CD8 + T cells from unimmunized mice (na ' ive). Quantification shows IRF4 mean fluorescence intensity from gated m45-tetramer + CD8 + T cells and CD44'° CD8 + T cells (na ' ive). Statistics - Student's t-test (n 4-5). b) EOMES expression in AtgT' ' and AtgT' + antigen-specific CD8 + T cells.
  • WT and J-AtgT' ' m ce were immunized with MCMV and EOMES expression was measured in m45-specific CD8 + T cells on day 9, 15, and 22.
  • AtgT ' memory CD8 + T cells show increased mitochondrial content, reactive oxygen species, apoptosis and fail to down-regulate GLUT-1 .
  • MCMV-immunized WT and J-Atg ⁇ mice were assessed for Bcl-2 expression in splenic m45-specific CD8 + T cells on day 9 and 22 post-infection.
  • AtgT 1' memory CD8 + T cells mount a significantly reduced recall response to secondary immunization.
  • mice were immunized with 0.00032 HAU PR8 influenza, followed by 0.32 HAU X31 on day 24, and 30 days later immunized for a third time with 32 HAU PR8.
  • Quantification shows frequency of CD8 + T cells that are NP-specific and are representative of two independent experiments. * p ⁇ 0.05, ** p ⁇ 0.01 , by Mann-Whitney U-test.
  • the AtgT' ' CD8 + T cell response to influenza vaccination was assessed for the third group.
  • mice WT and J-AtgT 1' mice were vaccinated twice, 22 days apart with 32 HAU of the live attenuated H1 N1 vaccine, S-Flu. Thirty days after the last vaccination, mice were challenged with 32 HAU X31 influenza. The CD8 + T cell response to NP was measured in lungs on day 23 post-challenge by tetramer. As a control, unvaccinated mice were challenged with 32 HAU X31 and culled on day 4 due to weight loss and morbidity. Quantification indicates the percentage of CD8 + T cells in the lung that are specific for NP on day 23 post- challenge. Example dot plots are shown. Data are representative of two independent experiments.
  • CD44 10 and CD44 hi CD8 + T cells were purified from 6 week old and 2 year old mice using fluorescent activated cell sorting. mRNA was extracted and the expression of essential autophagy genes was measured by q-PCR. Shown is the fold change in expression in CD8 + T cells from old mice relative to expression in young mice (normalized to gapdh and hprt).
  • mice Twenty-one days prior to the first vaccination, aged WT and T- Atg7' ⁇ mice were administered spermidine in the drinking water at a concentration of 5mM through to the experimental endpoint. As a control, twenty-three month old WT mice were administered water alone.
  • Y-axis depicts the frequency of CD8 + T cells that are specific for NP. ** p ⁇ 0.01 by Mann-Whitney Litest, h) CD8 + T cell kinetics to influenza vaccination and challenge in aged mice in the presence of spermidine.
  • NP-specific CD8 + T cell response to influenza challenge was measured by tetramer.
  • polyamine is an aliphatic organic compound having at least two primary amine groups. Polyamines may be cyclic, branched or single chained. Polyamines may further comprise at least one secondary amine group, for example 2 secondary amines. Polyamines may comprise 2-20 carbon atoms, preferably 4-10 carbon atoms. Polyamines may be synthetic polyamines, naturally-occurring polyamines or their analogues. Naturally-occurring
  • polyamines occur naturally in cells as physiologically active mediators in cellular processes.
  • exemplary naturally-occurring polyamines are putrescine, cadaverine, spermidine and spermine.
  • immunosenescence is a condition in which the functioning of a subject's immune system is deteriorating/has deteriorated in correlation with increasing age of that subject.
  • immunosenescence in a subject may be characterised by a refractory immune response to an immunogen.
  • the immune response to an immunogen of an older subject suffering from immunosenescence would be weaker in magnitude and/or effectiveness than the immune response of a healthy younger subject.
  • Treatment of immunosenescence may be prophylactic and/or therapeutic, preferably therapeutic.
  • Prophylactic treatment retards the progression of immunosenescence, or prevents it entirely.
  • Therapeutic treatment restores the function of the immune system at least partially, preferably entirely.
  • therapeutic treatment of immunosenescence may partially or wholly restore the magnitude and/or efficacy of a subject's immune response to an immunogen to the level of a healthy younger subject.
  • the immune response may be any (measurable) immune response as defined herein, for example a T cell immune response.
  • an "adaptive immune response" to an immunogen is a cell- mediated and/or humoral immunogen-specific immune response mediated by at least T cells and B cells, respectively.
  • T cells are lymphocytes expressing a T cell receptor (TCR), for example an alpha/beta TCR or a gamma/delta TCR.
  • TCR T cell receptor
  • a cellular immune response, mediated by T cells, is typically characterised by cytokine production and direct cytolytic activity.
  • T cells can be broadly categorised as CD8 or CD4 T cells.
  • CD8 T cells, or cytotoxic T cells express CD8 glycoprotein and are MHC class I restricted.
  • CD4 T cells express CD4 glycoprotein and are MHC class II restricted.
  • CD4 cells may be sub-categorised as Th1 , Th2, Th 17 or regulatory T cells (Treg) according to the cytokines they express.
  • T cells may be categorised as effector or memory T cells, based on their function and phenotype.
  • immunogenic challenge may be characterised into two phases - the effector phase and the memory phase.
  • Effector T cells are highly active but short-lived, peaking approximately 5-1 5 days post-challenge. Memory T cells have greater persistence, often continuing to circulate for a considerable time post-challenge. Persistent memory T cells are then able to respond rapidly to any further challenges with the same immunogen - a so-called recall response.
  • Effector T cells in mice and humans are typically characterised by the expression of CD44 and the absence of CD62L, CCR7 and CD127.
  • Memory T cells are characterised by the expression of CD44 and may be further subcategorised into 'effector' and 'central' memory T cells depending on the expression or absence of CD62L, CCR7 and CD127.
  • Effector memory T cells are long-lived cells that reside at the biological site of a previous infection and lack CD62L, CCR7 and CD1 27 expression.
  • Central memory T cells are long-lived cells that circulate the blood and lymphatic system and express CD62L, CCR7 and CD127.
  • B cells or B lymphocytes, are lymphocytes expressing a B cell receptor (BCR).
  • BCR B cell receptor
  • B cells activated by an immunogenic challenge develop into two broad classes of B cells - plasma cells and memory B cells.
  • Plasma cells are large, short-lived B cells that produce large numbers of immunogen-specific antibodies during the effector phase of the B cell response.
  • Memory B cells produce far fewer antibodies during the effector phase but continue to circulate for a considerable time post-challenge. Persistent memory B cells are then able to respond rapidly to any further challenges with the same immunogen.
  • immunogen is meant a substance to which it is possible to elicit an immunogen-specific immune response, preferably an adaptive immune response i.e. a humoral (antibody) and/or cell-mediated (T-cell) response.
  • An immunogen may be an antigen.
  • An immunogen may be a protein, a lipid, a polysaccharide, a glycoprotein, a lipoprotein, a lipopolysaccharide, a nucleic acid, for example DNA or RNA, a virus, a bacterium, or any fragment of those.
  • An immunogen may be derived from a pathogen.
  • An immunogen may be a vaccine.
  • vacun is meant an immunogen administered to a subject for the purpose of preventing or treating a disease (i.e. a vaccine may be prophylactic or therapeutic).
  • a vaccine may comprise or consist of a nucleic acid or protein from a pathogen, wherein administration of the vaccine to a subject induces an immune response that prevents disease being caused by that pathogen.
  • T cell vaccine an immunogen administered to a subject for the purpose of preventing or treating a disease.
  • a vaccine may be prophylactic or therapeutic.
  • a vaccine may comprise or consist of a nucleic acid or protein from a pathogen, wherein administration of the vaccine to a subject induces an immune response that prevents disease being caused by that pathogen.
  • T cell vaccine is a vaccine in which the prophylactic or therapeutic effect of the vaccine is mediated through T cells.
  • Most vaccines in general use are antibody or B cell vaccines, in which the prophylactic or therapeutic effect is mediated by antibodies.
  • antibodies in which the prophylactic or therapeutic effect is mediated by antibodies.
  • diseases for example
  • TB Mycobacterium tuberculosis infection
  • HIV/AIDS HIV/AIDS
  • malaria HIV/AIDS
  • many cancers humoral immune responses have at most partial or sometimes negligible effect and it is therefore desirable to induce vaccine-specific T cells.
  • T cell vaccines are intended to primarily induce T cell immune responses.
  • T cell vaccines may also induce a humoral immune response.
  • subject is an animal, preferably a mammal, preferably a human. Enhancing an immune response
  • a "nutraceutical preparation” is a preparation derived from a food source, which is supplemented or fortified with at least one active ingredient in order to provide extra health benefits, in addition to the basic nutritional value found in the food source.
  • Polyamines are organic aliphatic compounds having at least two primary amine groups, optionally also having at least one, for example two secondary amines. Certain polyamines occur naturally in mammalian cells. These naturally-occurring polyamines have been shown to be active in a number of cellular processes. Examples of such naturally-occurring polyamines are putrescine, cadaverine, spermidine and spermine.
  • the present inventors have identified that supplementation of a subject with exogenous polyamine results in improved immune responses to immunogens.
  • One example of such an improved immune response is the effect of polyamine administration on immunosenescence.
  • mice old mice (-25 months old) have a weak adaptive immune response to immunogens such as vaccines. Moreover, if old mice are a weak adaptive immune response to immunogens such as vaccines. Moreover, if old mice are a weak adaptive immune response to immunogens such as vaccines. Moreover, if old mice are a weak adaptive immune response to immunogens such as vaccines. Moreover, if old mice are a weak adaptive immune response to immunogens such as vaccines. Moreover, if old mice are a weak adaptive immune response to immunogens such as vaccines. Moreover, if old mice are a weak adaptive immune response to immunogens such as vaccines. Moreover, if old mice are a weak adaptive immune response to immunogens such as vaccines. Moreover, if old mice are a weak adaptive immune response to immunogens such as vaccines. Moreover, if old mice are a weak adaptive immune response to immunogens such as vaccines. Moreover, if old mice are a weak adaptive immune response to immunogens such as vaccines.
  • the memory recall immune response to the boost is notably diminished in comparison to younger, healthy adult mice (6-8 weeks old) (Example 1 ). However, if old,
  • immunosenescent mice are provided with polyamine supplementation (e.g. exogenous spermidine), both the peak immune response and the memory recall response are restored to at least equivalent to that of healthy, younger adult mice (Example 1 ).
  • the memory immune response in treated mice following the boost is frequently better than that observed for control younger adult mice.
  • polyamine supplementation e.g. exogenous spermidine
  • the peak immune response to influenza infection is greatly increased compared to untreated control mice of equivalent age (Example 2). It is well-known that the protection afforded by a vaccine against a disease depends largely on the strength and persistence of an adaptive memory immune response.
  • Enhancing of the adaptive immune response to an immunogen is often achieved through the use of adjuvants or through multiple immunisations, or a combination of the two.
  • Adjuvants in general work through activating the innate immune system in order to provide additional stimulus signals to the adaptive immune system, for example through TLR signalling.
  • TLR signalling Such signals are general and non-specific and often lead to unwanted inflammatory side-effects such as swelling, redness/erythema, pain, fever and scarring.
  • use of multiple immunisations or higher doses of vaccines is often undesirable as it increases manufacturing expense, reduces compliance and high doses can lead to greater unwanted side-effects.
  • a further advantage of administering supplemental polyamines to enhance the immune response and/or treat immunosenescence is that such unwanted side-effects will be reduced.
  • Many polyamines are naturally-occurring polyamines which, unlike many adjuvants, do not activate the innate immune system. As such, there will be minimal unwanted inflammatory side-effects as a result of using polyamines to enhance a subject's immune response.
  • the present invention provides a polyamine for use in the treatment of immunosenescence in a subject.
  • immunosenescence in a subject is characterised by a refractory immune response to an immunogen.
  • a refractory immune response to an immunogen is an immune response that is weaker in magnitude and/or efficacy than the immune response of a healthy control subject.
  • a "healthy control” is a young, adult subject exhibiting the expected "immune age" and having no underlying conditions. An appropriate healthy control subject would be clearly apparent to the skilled person.
  • Treatment of immunosenescence results in an improvement in an immunosenescent subject's immune response to an immunogen compared to the equivalent immune response to the same immunogen without administration of a polyamine. This is, of course, a theoretical consideration because the same individual cannot be both treated and not treated.
  • the response following administration of the polyamine may be compared against the expected response of a healthy control and/or a control subject suffering from immunosenescence (and not receiving a polyamine).
  • treatment of immunosenescence results in an immune response to an immunogen equal to or greater than that observed for a healthy control.
  • treatment of immunosenescence results in an immune response to an immunogen greater than that observed for a control subject suffering from immunosenescence (and not receiving a polyamine).
  • the refractory immune response restored and/or enhanced by the treatment of immunosenescence is an adaptive immune response.
  • the refractory immune response is a T cell response, preferably a memory T cell response.
  • the T cell response is a CD8 T cell response.
  • the T cell response is a CD4 T cell response, for example a Th 1 CD4 response and/or a Th2 CD4 response and/or a Th 17 CD4 response.
  • the refractory immune response restored by the treatment of immunosenescence is a CD8 memory T cell response.
  • the refractory immune response restored by the treatment of immunosenescence is a B cell response, for example a memory B cell response.
  • the polyamine is administered at least one day (24 hours) prior to the administration of an immunogen.
  • the polyamine is administered from 1 to 200 days prior to the administration of the immunogen, optionally 1 to 1 10 days, optionally 1 to 90 days, , optionally 1 to 52 days, preferably 1 to 21 days prior to administration of the immunogen.
  • immunosenescence in a subject is administered to the subject following the administration of an immunogen.
  • the polyamine is administered at least one day (24 hours) following the administration of an immunogen.
  • the polyamine is administered from 1 to
  • the polyamine is administered within 24 hours of the administration of an immunogen. In certain embodiments, the polyamine is administered within 1 2 hours, optionally within 6 hours, preferably simultaneously with the administration of the immunogen.
  • the polyamine and immunogen may be co-administered in the form of a single composition.
  • a polyamine may be used to treat immunosenescence in a subject according to any one or more of these embodiments in combination. That is, in certain embodiments, the polyamine is administered to the subject prior to and/or concurrently with and/or following the administration of an immunogen.
  • the polyamine is administered at least once a day. In certain such embodiments, the polyamine is administered once a day for 1 to 200 days, optionally for 1 to 1 1 0 days, optionally for 1 to 90 days, optionally for 1 to 52 days, optionally for 1 to 21 days, optionally for 1 day.
  • the polyamine is administered at least once a week.
  • the polyamine may be administered for 1 to 30 weeks, optionally 1 to 1 6 weeks, optionally 1 to 13 weeks, optionally for 1 to 8 weeks, optionally for 1 to 3 weeks, optionally for 1 week. Any one or more of these embodiments may be used in combination with those embodiments wherein the polyamine is administered prior to administration of an immunogen. That is, for example, in certain embodiments wherein the polyamine is administered prior to the administration of an immunogen, the polyamine is administered once 200 days prior to the administration of an immunogen.
  • the polyamine is administered at least once a day for 200 days prior to administration of an immunogen. Equally, in certain embodiments, the polyamine is administered at least once a day for 21 days beginning 200 days, optionally beginning 52 days, preferably beginning 21 days prior to the administration of an immunogen.
  • the polyamine is administered prior to administration of an immunogen
  • the polyamine is administered at least once a week beginning 200 days prior to administration of an immunogen.
  • the polyamine is administered once a week for 1 -3 weeks beginning 200 days, optionally beginning 52 days, preferably beginning 21 days prior to the administration of an immunogen.
  • the polyamine is administered to a subject at least once a day for 1 to 200 days, preferably 1 to 52 days, preferably 1 to 21 days, more preferably for 21 days prior to administration of an immunogen to the subject.
  • any one or more of these embodiments may be used in combination with those embodiments wherein the polyamine is administered following administration of an immunogen. That is, for example, in certain embodiments wherein the polyamine is administered following the administration of an immunogen, the polyamine is administered once 200 days following the administration of an immunogen. Equally, in certain embodiments the polyamine is administered at least once a day for 200 days following administration of an immunogen. Equally, in certain embodiments, the polyamine is administered at least once a day for 21 days ending 200 days, optionally ending 52 days, preferably ending 21 days following the administration of an immunogen.
  • the polyamine is administered following administration of an immunogen
  • the polyamine is administered at least once a week ending 200 days following administration of an immunogen.
  • the polyamine is administered once a week for 1 -3 weeks ending 200 days, optionally ending 52 days, preferably ending 21 days prior to the administration of an immunogen.
  • the polyamine is administered to a subject at least once a day for 1 to 200 days, preferably 1 to 100 days, preferably 1 to 52 days, for example 1 to 21 days following administration of an immunogen to the subject. In certain more preferred embodiments, the polyamine is administered to a subject at least once a day for 52 days following administration of an immunogen to the subject.
  • any one or more of these embodiments may be used in combination with those embodiments wherein the polyamine is administered concurrently with administration of an immunogen. That is, in certain embodiments, wherein the polyamine is administered at least once a day for a number of days, or at least once a week for a number of weeks, the specified number of days or weeks begins, ends or spans the day on which an immunogen is administered. In certain preferred embodiments, the polyamine is administered at least once a day for 21 days prior to administration of an immunogen, is administered concurrently with administration of the immunogen, and is administered for 52 days following administration - that is, the polyamine is administered at least once a day for between 1 to 1 10 days (74 days) spanning the administration of the immunogen.
  • the immunogen may be administered more than once, for example, where the immunogen is a vaccine, as a boost. In such
  • the above-described schedules of administration refer to the first administration of the immunogen.
  • the polyamine may also be administered prior to and/or concurrently with and/or following any subsequent administration of the immunogen.
  • the above schedules of administration of the polyamine may also apply to subsequent administration of the immunogen.
  • the present invention provides a polyamine for use in enhancing an immune response of a subject to an immunogen.
  • enhancing an immune response of a subject to an immunogen is increasing the magnitude and/or efficacy of a subject's immune response to a level greater than that observed for an untreated control subject.
  • the immune response enhanced by use of a polyamine is an adaptive immune response.
  • the immune response is a T cell response, preferably a memory T cell response.
  • the T cell response is a CD8 T cell response.
  • the T cell response is a CD4 T cell response, for example a Th1 CD4 response and/or a Th2 CD4 response and/or a Th17 CD4 response.
  • the enhanced immune response is a CD8 memory T cell response.
  • the enhanced immune response is a B cell response, for example a memory B cell response.
  • the polyamine for use in enhancing the immune response of a subject to an immunogen is administered to the subject prior to the administration of an immunogen.
  • the polyamine is administered at least one day (24 hours) prior to the administration of an immunogen.
  • the polyamine is administered from 1 to 200 days prior to the administration of the immunogen, optionally 1 to 1 10 days, optionally 1 to 90 days, optionally 1 to 52 days, preferably 1 to 21 days prior to administration of the immunogen.
  • the polyamine for use in enhancing the immune response of a subject is administered to the subject following the administration of an immunogen.
  • the polyamine is administered at least one day (24 hours) following the administration of an immunogen.
  • the polyamine is administered from 1 to 200 days following the administration of the immunogen, optionally 1 to 1 10 days, optionally 1 to 90 days, optionally 1 to 52 days, preferably 1 to 21 days following administration of an immunogen.
  • the polyamine for use in enhancing the immune response of a subject is administered to the subject concurrently with the administration of an immunogen.
  • the polyamine is administered within 24 hours of the administration of an immunogen.
  • the polyamine is administered within 12 hours, optionally within 6 hours, preferably simultaneously with the administration of the immunogen.
  • a polyamine may be used to enhance the immune response of a subject according to any one or more of these embodiments in combination. That is, in certain embodiments, the polyamine is administered to the subject prior to and/or concurrently with and/or following the administration of an immunogen.
  • the polyamine is administered at least once a day. In certain such embodiments, the polyamine is administered once a day for 1 to 200 days, optionally for 1 to 52 days, optionally for 1 to 21 days, optionally for 1 day.
  • the polyamine is administered at least once a week for 1 to 30 weeks, optionally 1 to 16 weeks, optionally 1 to 13 weeks, optionally for 1 to 8 weeks, optionally for 1 to 3 weeks, optionally for 1 week.
  • any one or more of these embodiments may be used in combination with those embodiments wherein the polyamine is administered prior to administration of an immunogen. That is, for example, in certain embodiments wherein the polyamine is administered prior to the administration of an immunogen, the polyamine is administered once 200 days prior to the administration of an immunogen.
  • the polyamine is administered at least once a day for 200 days prior to administration of an immunogen. Equally, in certain embodiments, the polyamine is administered at least once a day for 21 days beginning 200 days, optionally beginning 52 days, preferably beginning 21 days prior to the administration of an immunogen.
  • the polyamine is administered prior to administration of an immunogen
  • the polyamine is administered at least once a week beginning 200 days prior to administration of an immunogen.
  • the polyamine is administered once a week for 1 -3 weeks beginning 200 days, optionally beginning 52 days, preferably beginning 21 days prior to the administration of an immunogen.
  • the polyamine is administered to a subject at least once a day for 1 to 200 days, preferably 1 to 52 days, preferably 1 to 21 days, more preferably for 21 days prior to administration of an immunogen to the subject.
  • any one or more of these embodiments may be used in combination with those embodiments wherein the polyamine is administered following administration of an immunogen. That is, for example, in certain embodiments wherein the polyamine is administered following the administration of an immunogen, the polyamine is administered once 200 days following the administration of an immunogen. Equally, in certain embodiments the polyamine is administered at least once a day for 200 days following administration of an immunogen. Equally, in certain embodiments, the polyamine is administered at least once a day for 21 days ending 200 days, optionally ending 52 days, preferably ending 21 days following the administration of an immunogen.
  • the polyamine is administered at least once a week ending 200 days following administration of an immunogen.
  • the polyamine is administered once a week for 1 -3 weeks ending 200 days, optionally ending 52 days, preferably ending 21 days prior to the administration of an immunogen.
  • the polyamine is administered to a subject at least once a day for 1 to 200 days, preferably 1 to 100 days, preferably 1 to 52 days, for example 1 to 21 days.
  • the polyamine is administered to a subject at least once a day for 52 days following administration of an immunogen to the subject.
  • any one or more of these embodiments may be used in combination with those embodiments wherein the polyamine is administered concurrently with administration of an immunogen. That is, in certain embodiments, wherein the polyamine is administered at least once a day for a number of days, or at least once a week for a number of weeks, the specified number of days or weeks begins, ends or spans the day on which an immunogen is administered. In certain preferred embodiments, the polyamine is administered at least once a day for 21 days prior to administration of an immunogen, is administered concurrently with administration of the immunogen, and is administered for 52 days following administration - that is, the polyamine is administered at least once a day for between 1 to 1 10 days (74 days) spanning the administration of the immunogen.
  • the immunogen may be administered more than once, for example, where the immunogen is a vaccine, as a boost. In such
  • the above-described schedules of administration refer to the first administration of the immunogen.
  • the polyamine may also be administered prior to and/or concurrently with and/or following any subsequent administration of the immunogen.
  • the above schedules of administration of the polyamine may thus also apply to subsequent administration of the immunogen.
  • the invention provides a method of enhancing the immune response of a subject to an immunogen, the method comprising:
  • enhancing an immune response of a subject to an immunogen is increasing the magnitude and/or efficacy of a subject's immune response to a level greater than that observed for an untreated control subject.
  • the immune response enhanced by the method is an adaptive immune response.
  • the immune response is a T cell response, preferably a memory T cell response.
  • the T cell response is a CD8 T cell response.
  • the T cell response is a CD4 T cell response, for example a Th1 CD4 response and/or a Th2 CD4 response and/or a Th17 CD4 response.
  • the enhanced immune response is a CD8 memory T cell response.
  • the enhanced immune response is a B cell response, for example a memory B cell response.
  • administration of a polyamine to a subject according to step (i) is prior to the administration of an immunogen to the subject in step (ii).
  • the polyamine is administered at least one day (24 hours) prior to the administration of an immunogen.
  • the polyamine is administered from 1 to 200 days prior to the administration of the immunogen, optionally between 1 to 100 days, between 1 to 52 days, preferably between 1 to 21 days prior to administration of the immunogen.
  • administration of a polyamine to a subject according to step (i) is following the administration of an immunogen to the subject in step (ii).
  • the polyamine is administered at least one day (24 hours) following the administration of an immunogen.
  • the polyamine is administered from 1 to 200 days following the administration of the immunogen, optionally 1 to 1 10 days, optionally 1 to 90 days,, optionally 1 to 52 days, preferably 1 to 21 days following administration of an immunogen.
  • administration of a polyamine to a subject according to step (i) is concurrent with the administration of an immunogen to the subject in step (ii).
  • the polyamine is administered within 24 hours of the administration of an immunogen.
  • the polyamine is administered within 12 hours, optionally within 6 hours, preferably simultaneously with the administration of the immunogen.
  • a polyamine may be used to enhance the immune response of a subject according to any one or more of these embodiments in combination. That is, in certain embodiments, the polyamine is administered to the subject prior to and/or concurrently with and/or following the administration of an immunogen.
  • the polyamine is administered at least once a day. In certain such embodiments, the polyamine is administered once a day for 1 to 200 days, optionally for 1 to 52 days, optionally for 1 to 21 days, optionally for 1 day.
  • the polyamine is administered at least once a week for 1 to 30 weeks, optionally 1 to 16 weeks, optionally 1 to 13 weeks, optionally for 1 to 8 weeks, optionally for 1 to 3 weeks, optionally for 1 week.
  • any one or more of these embodiments may be used in combination with those embodiments wherein the polyamine is administered prior to administration of an immunogen. That is, for example, in certain embodiments wherein the polyamine is administered prior to the administration of an immunogen, the polyamine is administered once 200 days prior to the administration of an immunogen.
  • the polyamine is administered at least once a day for 200 days prior to administration of an immunogen. Equally, in certain embodiments, the polyamine is administered at least once a day for 21 days beginning 200 days, optionally beginning 52 days, preferably beginning 21 days prior to the administration of an immunogen.
  • the polyamine is administered prior to administration of an immunogen
  • the polyamine is administered at least once a week beginning 200 days prior to administration of an immunogen.
  • the polyamine is administered once a week for 1 -3 weeks beginning 200 days, optionally beginning 52 days, preferably beginning 21 days prior to the administration of an immunogen.
  • the polyamine is administered to a subject at least once a day for 1 to 200 days, preferably 1 to 52 days, preferably 1 to 21 days, more preferably for 21 days prior to administration of an immunogen to the subject.
  • any one or more of these embodiments may be used in combination with those embodiments wherein the polyamine is administered following administration of an immunogen. That is, for example, in certain embodiments wherein the polyamine is administered following the administration of an immunogen, the polyamine is administered once 200 days following the administration of an immunogen. Equally, in certain embodiments the polyamine is administered at least once a day for 200 days following administration of an immunogen. Equally, in certain embodiments, the polyamine is administered at least once a day for 21 days ending 200 days, optionally ending 52 days, preferably ending 21 days following the administration of an immunogen.
  • the polyamine is administered following administration of an immunogen
  • the polyamine is administered at least once a week ending 200 days following administration of an immunogen.
  • the polyamine is administered once a week for 1 -3 weeks ending 200 days, optionally ending 52 days, preferably ending 21 days prior to the administration of an immunogen.
  • the polyamine is administered to a subject at least once a day for 1 to 200 days, preferably 1 to 100 days, preferably 1 to 52 days, for example 1 to 21 days. In certain more preferred embodiments, the polyamine is administered to a subject at least once a day for 52 days following administration of an immunogen to the subject.
  • any one or more of these embodiments may be used in combination with those embodiments wherein the polyamine is administered concurrently with administration of an immunogen. That is, in certain embodiments, wherein the polyamine is administered at least once a day for a number of days, or at least once a week for a number of weeks, the specified number of days or weeks begins, ends or spans the day on which an immunogen is administered. In certain preferred embodiments, the polyamine is administered at least once a day for 21 days prior to administration of an immunogen, is administered concurrently with administration of the immunogen, and is administered for 52 days following administration - that is, the polyamine is administered at least once a day for between 1 to 1 10 days (74 days) spanning the administration of the immunogen.
  • the immunogen may be administered more than once, for example, where the immunogen is a vaccine, as a boost. In such
  • the above-described schedules of administration refer to the first administration of the immunogen.
  • the polyamine may also be administered prior to and/or concurrently with and/or following any subsequent administration of the immunogen.
  • the above referenced schedules of administration of the polyamine may also be applied to subsequent
  • the polyamine administered to the subject is a naturally- occurring polyamine, a synthetic polyamine, or an analogue thereof.
  • the polyamine is putrescine, cadaverine, spermidine, or spermine, or an analogue thereof.
  • the polyamine is spermidine.
  • the polyamine is administered in solution, preferably in aqueous solution. In certain embodiments the polyamine is administered at a concentration 1 -10 mM, such as 1 -5 mM, optionally 5 mM.
  • the polyamine is administered orally. In certain such embodiments, the polyamine is ingested. In certain preferred embodiments the polyamine is administered as an ingestible composition, for example in a beverage or foodstuff, preferably a beverage. In a preferred embodiment, the polyamine is administered dissolved in water.
  • the polyamine is administered by injection.
  • the polyamine is administered intravenously, intramuscularly, intraperitoneally, intradermal ⁇ , or subcutaneously.
  • the polyamine is administered by gavage, by bronchoscope, by nebuliser, or by suppository.
  • an immunogen is a substance which stimulates an immune response when administered to a subject with a healthy immune system.
  • the immunogen may be an antigen.
  • the immunogen is derived from a pathogen.
  • Pathogens from which an immunogen may be derived include viruses, bacteria, fungi, protozoans and helminths.
  • the immunogen is a protein, a polysaccharide, a nucleotide, a lipid, a lipoprotein, a lipopolysaccharide, or a glycoprotein.
  • the immunogen is a toxin.
  • the immunogen is a vaccine.
  • a vaccine may be a protein vaccine, a DNA vaccine, a bacterial vaccine or a viral vaccine.
  • a bacterial vaccine may be a live, live-attenuated or killed bacterial vaccine.
  • a viral vaccine may be a live, live-attenuated or killed viral vaccine.
  • the immunogen is a viral vector vaccine.
  • the viral vector vaccine comprises an exogenous nucleotide comprising and/or encoding an antigen from a pathogen.
  • the immunogen is a T cell vaccine.
  • the immunogen is a viral T cell vaccine. In certain embodiments the immunogen is an influenza vaccine. In certain embodiments the immunogen is a live-attenuated influenza vaccine.
  • the subject is a mammal, preferably a human.
  • immunosenescence is most commonly exhibited in elderly individuals. Therefore, in certain embodiments of the first, second and third aspects of the invention, the subject is an elderly subject.
  • the elderly subject is a human subject, the subject may be 60 years old or older, 65 years old or older, 70 years old or older.
  • Treatment of immunosenescence in a subject may be characterised by the subject exhibiting an improved immune response to an immunogen compared to the response exhibited by an untreated
  • the response in a treated subject may be at least equivalent to the immune response exhibited by a subject not exhibiting immunosenescence, such as a healthy adult subject.
  • a healthy adult may be a subject from 20-60 years old, optionally 25-50 years old, optionally 25-40 years old, and who is not exhibiting immunosenescence.
  • the invention provides a nutraceutical preparation comprising a polyamine, wherein said preparation is suitable for the treatment of
  • the nutraceutical preparation is suitable for oral administration, preferably ingestion. Accordingly, the invention provides an ingestible nutraceutical preparation comprising a polyamine, wherein said preparation is suitable for the treatment of immunosenescence.
  • the nutraceutical preparation is a beverage.
  • the nutraceutical preparation is a dairy-based preparation, for example a yoghurt.
  • the nutraceutical preparation may additionally contain suitable flavouring and/or colouring agents to improve the appearance and/or taste of the preparation.
  • the nutraceutical preparation may be a liquid or may be a powder such as a freeze-dried or spray-dried composition to permit
  • the nutraceutical preparation comprises a polyamine in solution, preferably in aqueous solution. In certain embodiments the nutraceutical preparation comprises a polyamine in a concentration of 1 -10 mM, preferably 1 -5 mM, preferably 5 mM.
  • the nutraceutical preparation may be packaged to deliver a single dosage unit of the polyamine. This may be a suitable volume of liquid or for powdered forms the packaging may be provided with a suitable measure together with instructions for reconstituting the preparation.
  • the nutraceutical preparation is for use in the treatment of immunosenescence.
  • the nutraceutical preparation is administered at least once a day for 1 to 200 days, optionally 1 to 1 10 days, optionally 1 to 90 days, optionally 1 to 52 days, optionally 1 to 21 days, optionally 1 day, optionally wherein the nutraceutical is administered once.
  • the nutraceutical preparation for use in the treatment of immunosenescence is administered weekly for 1 to 30 weeks, optionally 1 to 1 6 weeks, optionally 1 to 1 3 weeks, optionally 1 to 8 weeks, optionally 1 to 3 weeks, optionally 1 week.
  • Example 1 The use of a polyamine in the treatment of immunosenescence was examined in an influenza vaccination and challenge model system.
  • CD4-Atg7 _/ lack the autophagy gene Atg7 ⁇ r CD4 expressing cells - primarily T cells. Because all T cells go through a CD4 expressing stage in the thymus, Atg7 s excised in CD8 + T cells as well as CD4 + T cells. The loss of Atg7 renders these mice autophagy-deficient in the T cell lineage.
  • a polyamine (spermidine) was applied to the drinking water at a concentration of 5 mM from a 1 M aqueous stock solution. Fresh spermidine-containing drinking water was supplied every 3-5 days.
  • mice were immunised intranasally with an influenza vaccine at a concentration of 32 HAU in 50 ⁇ _.
  • the vaccine in question is derived from a H1 H1 influenza strain and is attenuated due to deletion of the haemagglutinin signal sequence, meaning it can efficiently infect cells but cannot replicate and spread cell to cell.
  • mice were vaccinated on day 0 and day 22 before being challenged with 32 HAU X31 H3N2 influenza.
  • the CD8 + T cell response to influenza vaccination and infection in the blood and lungs was determined with a MHC class I tetramer specific for the
  • nucleoprotein present in both the influenza vaccine and X31 influenza. Following tetramer staining, samples were stained with anti-CD8a and assessed on a flow cytometer.
  • the initial CD8 + T cell response to primary vaccination is largely normal, a second 'booster' vaccination is unable to improve the frequency of circulating CD8 + T cells specific for Influenza Nucleoprotein (NP) (herein referred to as vaccine-specific CD8 + T cells) in old mice as it does in young mice.
  • NP Influenza Nucleoprotein
  • the frequency of vaccine-specific CD8 + T cells is 4-fold lower in aged mice compared to young.
  • mice are autophagy deficient in the T cell lineage due to a T cell-specific deletion of the essential autophagy gene Atg7, suggesting spermidine may mediate its effect through autophagy.
  • the CD8 + T cell response in the blood following influenza challenge of vaccinated mice was assessed by tetramer.
  • a polyamine spermidine
  • spermidine a polyamine
  • the CD8 + T cell response in the lungs following influenza challenge in vaccinated mice was assessed by tetramer. Influenza challenge induces a large CD8 + T cell response in the lungs of young mice following vaccination (at least 60% tetramer+) (A). In comparison, the frequency of NP-specific CD8 + T cells in the lungs of old mice was significantly reduced - a response characteristic of immunosenescence. Oral intake of a polyamine (spermidine) in old mice increased the frequency of NP-specific CD8 + T cells following influenza challenge. Similarly, polyamine treatment was also able to substantially enhance the absolute number of NP-specific CD8 + T cells in elderly mice, beyond even what was observed in the lungs of young mice (B).
  • polyamine treatment can act to treat immunosenescence in old mice by enhancing the immune response to both vaccination and infection.
  • a polyamine (spermidine) was applied to the drinking water at a concentration of 5 mM from a 1 M aqueous stock solution. Fresh spermidine-containing drinking water was supplied every 3-5 days.
  • mice were immunised with 0.001 HAU X31 Influenza intranasally in 50 ⁇ _.
  • the CD8 + T cell response to influenza infection in the lungs was determined with a MHC class I tetramer specific for the immunodominant epitope in C57BL/6 mice, NP 366 -374 (amino acid sequence: ASNENMETM). This epitope is found within the conserved Influenza nucleoprotein (NP) present in both the influenza vaccine and X31 influenza. Following tetramer staining, samples were stained with anti-CD8a and assessed on a flow cytometer.
  • mice Following eight weeks of oral polyamine treatment, eight week old mice were infected with influenza and the CD8 + T cells response in the lungs was assessed by tetramer on day 7. Polyamine-treated mice exhibited a five-fold increase in the frequency of NP-specific CD8 + T cells (5%) in the lungs, compared to untreated controls (1 %). This demonstrates that polyamine treatment can act to enhance the immune response to infection in young, healthy mice.
  • Figure 5 clearly shows that continuous oral administration of spermidine following vaccination increases the immune response to a vaccine antigen.
  • mice 8 week old mice were immunised intranasally with the influenza vaccine S-Flu. On day 14 the vaccine specific CD8 + T cell response was assessed in the lungs.
  • 100 ⁇ g of spermidine was administered intraperitoneally every other day from day 0 to day 14, with the first administration performed concurrently with vaccination (Figure 6A).
  • 500 ⁇ g of spermidine was administered intraperitoneally concurrently with the vaccination.
  • Figure 6 shows the frequency of CD8 + T cells that are specific for the Influenza epitope NP 366 . 3 74.
  • Figure 6A shows that parenteral administration of spermidine following
  • CD8 + T cells During infection CD8 + T cells initially expand then contract, leaving a small memory pool providing long lasting immunity. While it has been described that CD8 + T cell memory formation becomes defective in old age, the cellular mechanism is largely unknown. Autophagy is a major cellular lysosomal degradation pathway of bulk material and levels are known to fall with age. Here, we describe a novel role for autophagy in CD8 + T cell memory formation. Mice lacking the autophagy gene Atg7 n T cells failed to establish CD8 + T cell memory to influenza and MCMV infection. Interestingly, autophagy levels were diminished in CD8 + T cells from aged mice. We could rejuvenate CD8 + T cell responses in elderly mice in an autophagy-dependent manner using the compound spermidine. This study suggests novel immune modulators to enhance T cell responses in the aged.
  • T cell population Upon successful clearance of a pathogen, the majority of short-lived effector T cells die and the remaining cells differentiate into a memory T cell population that provides long lasting immunity. While the cytokines, surface molecules and signaling components involved in T cell memory formation have been extensively studied, the molecular pathways supporting these cell fate decisions are poorly understood.
  • the T memory population is (1 ) quiescent, (2) long-lived and actively maintained and (3) relies on mitochondrial respiration 1 . Both reduced cell cycling and longevity of this stem-cell-like population demand rigorous maintenance of the cytoplasm as debris cannot be diluted to daughter cells, reminiscent of true stem cells 2 ' 3 ' 4 . Across mammalian cell types, clearance of debris and damaged organelles such as mitochondria is typically executed via autophagy 5 .
  • BM bone marrow
  • Atg7flox/flox mice were bred with CD4-Cre mice to generate mice with defective autophagy in both CD4 + and CD8 + T lymphocytes -AtgT').
  • Successful excision and thereby absence of Atg7 mRNA and Atg7 protein was confirmed in purified T cells ( Figure 8, a and b, respectively).
  • Using the imaging flow cytometer (ImageStream) to count LC3 puncta in CD4 + and CD8 + T cells 15 we demonstrated that functional autophagy was significantly diminished in Atg7' ⁇ CD8 + T cells ( Figure 8c,with examples of ImageStream images in right panel).
  • using a classical technique to detect lipidated LC3 we confirmed that basal autophagy was diminished in the presence and absence of the autophagy flux inhibitor Bafilomycin A (Figure 8d).
  • AtgT' ' CD8 + T cells exhibited an activated phenotype with increased CD44 expression (Fig 7d) and decreased CD62L expression (Fig 7e), resembling a "virtual memory” compartment 19 .
  • CD62L + CD44 hl a central effector memory
  • -AtgT 1' mice accumulated CD8 + T cells with an effector memory phenotype (CD62L ⁇ CD44 hl ) (Fig 8e).
  • the expression of the homeostatic proliferation marker CD24 20 was found to be significantly increased on AtgT' ' CD8 + T cells (Fig 7g).
  • BM bone marrow
  • Fig 9c mixed bone marrow
  • AtgT' ' CD8 + T cell phenotype was no longer detected in BM chimeras as measured by the frequency of donor CD45.2 + CD8 + T cells expressing CD62L + (Fig 7h) and CD44 hi (Fig T ⁇ ).
  • CD127 " KLRG1 + ) CD8 + T cells was in fact normal in J-AtgT' ' mice (Fig 12b). This suggests that autophagy is required to maintain the memory compartment rather than to establish it. We then tested whether the lack of memory response is due to exhaustion of autophagy-deficient antigen-specific CD8 + T cells. While in T- AtgT' ' mice, we found a sharp increase of antigen-specific CD8 + T cells co- expressing the exhaustion markers PD-1 + and TIM-3 + ; this was not found in the mixed BM chimeras.
  • AtgT' ' CD8 + T cells were undergoing more cell death than wild type cells. While this is well described for na ' ive autophagy deficient T cells 7 , it had not been analyzed in antigen-specific T cells. As expected, in wild type mice challenged with MCMV, we found highest levels of cell death among antigen-specific CD8 + T cells just after the peak of the effector phase on day 9, whereas in J-AtgT' ' mice, cell death was found to increase over time (Fig 14a). This could not be explained by a change in levels of the anti-apoptotic protein Bcl2 (Fig 15a).
  • mitochondrial quality and reactive oxygen species (ROS) via mitophagy the degradation of mitochondria, has been found to prevent cell death in T cells 18 .
  • mitochondrial content (Fig 14b) and mitochondrial ROS (Fig 14c) were significantly increased in AtgT' ' antigen-specific CD8 + T cells. While this likely explains the increased cell death, as shown for other hematopoietic cells 21 and T cells 18 , mitophagy is also thought to be essential for maintenance of healthy mitochondrial energy generation.
  • GLUT-1 is upregulated on CD8 + T eff (day 9) and then downregulated on CD8 + T mem (day 22) in wild type mice as CD8 + T cells switch to mitochondrial respiration (Fig 14d).
  • antigen-specific Atg7' ⁇ CD8 + T eff express more GLUT-1 and downregulation does not occur to the same extent at the later time points compared to WT cells (day 22) (Fig 14d). This suggests that autophagy supports the metabolic switch during the differentiation from T eff to T mem . This was confirmed using the GLUT-1 antibody (Fig 15b).
  • treatment with the anti-diabetic drug metformin that induces mitochondrial ⁇ - oxidation metabolism in T cells 26 did not rescue the memory T cell compartment in this model (data not shown).
  • J-Atg7 / ⁇ mice were able to mount a recall CD8 + T cell response to a secondary infection.
  • H3N2 heterologous virus strain expressing different surface antigens it is possible to significantly diminish the influence of antibodies in mediating immunity to secondary infection.
  • heterotypic immunity relies heavily on cross-reactive CD8 + T cell responses 3 31 , as opposed to homotypic immunity (for example PR8 primed, PR8 challenged) to which influenza-specific antibodies contribute 30 ' 32 .
  • H1 N1 vaccinated -AtgT ⁇ mice could mount secondary CD8 + T responses to heterotypic viral challenge.
  • Mice were vaccinated twice, 22 days apart then challenged with X31 influenza. Large numbers of influenza NP-specific CD8 + T cells were detected in the lungs of vaccinated wild type mice 5 days post-challenge, but not in -AtgT ⁇ mice (Fig 16b).
  • CD8 + T cells from old mice also showed significantly decreased autophagic flux detected by counting LC3 spots in NP-specific CD8 + T cells from young and old mice both in the presence and absence of an autophagy flux inhibitor (Fig 18b and 18c). This was confirmed by using two flow cytometry based autophagy detection, also in NP-specific CD8 + T cells ( Figure 19a and b).
  • spermidine induces autophagy in T cells in vitro in a dose and time-dependent manner shown by increased levels of LC3-II (Fig 18d). Spermidine levels in the blood are known to decrease with age 31 .
  • spermidine was administered to old and young mice via the drinking water at concentrations known to induce autophagy 14 during the influenza vaccination protocols as in Fig 16b-d.
  • old mice do not mount a robust CD8 + T cell response as compared to young mice.
  • Spermidine dramatically enhanced influenza-specific CD8 + T cell responses in old vaccinated wildtype mice but not in old vaccinated -AtgT ⁇ mice as tracked over time in blood (Fig 18g).
  • the response to live influenza challenge in the blood was improved 2-3 fold (back to levels similar to young mice) when spermidine was administered to old mice, but not in the absence of autophagy (Fig 18h).
  • spermidine is significantly more attractive than previously published T mem - boosting compounds such as metformin 26 and rapamycin 10 , that come with unwelcome side effects and toxicity in humans, as spermidine would not be expected to have these effects.
  • CD4-Cre mice 51 were from Adeline Hajjar (University of Washington) and crossed with Atg7 lox/ilox 52 to obtain -Atg '- mice (CD4-Cre + AtgT ,+ ) on a C57BL/6 background. Unless otherwise stated, all mice were 6-12 weeks of age at the start of each experiment and were age and sex matched. CD4-Cre AtgT /+ mice were used as wildtype controls and were littermates where possible. No phenotype was observed in CD4-Cre + AtgT' ⁇ mice. Old mice, and young control mice mice were purchased from Charles River, UK.
  • mice for bone marrow chimeras were purchased from Biomedical Services, Oxford. All mice were housed in Biomedical Services, Oxford and animal experiments were approved by the local ethical review committee and performed under UK project license (PPL 39/2809).
  • PE/FITC/APC PE/FITC/APC
  • TCRp H57-597
  • CD3 145-2C1 1
  • CD62L MEL-14
  • IM7 FITC/PE-Cy7
  • KLRG1 2F1 ) FITC/PE-Cy7;
  • IRF4 (3E4) FITC; EOMES (Dan1 1 mag) PE-Cy7; CD127 (A7R34) FITC/PE/eF450; Ki-67
  • Anti-CD16/32 (Fc Block, eBioscience) was generally added to antibody mix to minimize non-specific staining.
  • LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Life Technologies) was used prior to surface staining to exclude dead cells.
  • cytoplasmic intracellular staining (Bcl-2), cells were stained for surface antibodies then fixed in IC fixation buffer before permeabilisation with perm buffer (eBioscience). Cells were then suspended in perm buffer at RT for antibody staining.
  • nuclear targets IRF, EOMES, Ki-67
  • after surface staining cells were fixed and permeabilised with FoxP3 Fixation/Permeabilisation kit (ebioscience) before resuspension in perm buffer for intracellular staining at RT.
  • Absolute cell counts were performed on peripheral blood taken from the lateral tail vein of live animals, collected in heparin-coated tubes (Microvette 300, Sarstedt (Numbrecht, Germany) to avoid coagulation. Cell counts were calculated with BD TruCount tubes (BD Bioscience, NJ, USA) according to the manufacturers instructions.
  • Tetramers were generated as previously described 53 and MHC-Class I monomers stored at -80 ° C. Biotinylated monomers were tetrammzed with Streptavidin PE or APC at a the right concentration to achieve 1 :1 ratio with biotin binding sites, and added in 1 /1 0 th volumes waiting 1 0 minutes between additions. Tetramerized complexes were stored at 4°C.
  • Peptide sequences for MCMV tetramers were as follows: m45 985 HGIRNASFI 993 ' H-2D b -restricted; m38 316 SSPPMFRV 323 , H-2K b -restricted; IE3 416 RALEYKNL 423 , H-2K b -restricted.
  • the peptide sequence for the influenza tetramer was as follows: NP 366 AS N E N M ET M 374 , H-2D b -restricted. Cells were always stained with tetramers prior to surface antibody staining in PBS 2% FCS at 37 ° C for 1 5 minutes.
  • splenocytes were stained with m45-tetramer and surface antibody as described above, then stained with LIVE/DEAD Fixable Violet Dead Cell Stain Kit. Cells were finally stained with Annexin V PE-Cy7 (eBioscience) in Annexin V binding buffer at room temperature. Apoptotic cells were determined as LIVE/DEAD cell dye negative (live cells), Annexin V positive.
  • GLUT-1 was measured through binding to its ligand, the receptor binding domain (RBD) of a recombinant glycoprotein from the human T lymphotrophic Virus (HTLV) fused to eGFP (H RBD -eGFP) 54 .
  • RBD receptor binding domain
  • H RBD -eGFP eGFP
  • MitoTracker Green Life Technologies, Carlsbad, CA, USA
  • MitoSOX Red Life Technologies
  • BM cells were extracted from a single 8-week-old wildtype, -AtgT ⁇ (both CD45.2 + ), and C57BL/6 SJL mouse expressing CD45.1 . After lysis erythrocytes, 3x10 6 wildtype or -AtgT ⁇ BM cells were added to 3x10 6 CD45.1 + BM cells (1 :1 CD45.2 + :CD45.1 + ) in a total volume of 200 ⁇ PBS.
  • the 1 :1 BM mix was injected i.v 2h after lethal irradiation (450cGy twice, 4 hours apart) to C57BL/6 SJL CD45.1 + recipients (total 6x10 6 BM cells per mouse in 200 ⁇ ).
  • mixed BM chimera were immunized with MCMV or PR8 influenza, or left unimmunized.
  • WT and J-Atg ⁇ mice were always used as controls. Immunizations. Mice were immunized i.v. with 1 x10 6 p.f.u MCMV in 100 ⁇ (Smith strain ATCC: VR194). Unimmunized controls were injected with 100 ⁇ PBS i.v.
  • mice were administered intranasally with either A/PR/8/34 (PR8, H1 N1 Cambridge) influenza or X31 (H3N2) influenza at the stated dose in 50 ⁇ viral dilution media (VDM; DMEM 0.1 % BSA, 10 mM HEPES, 100U/ml Penicillin 100 ⁇ g/mL streptomycin and 2mM Glutamine; all from Sigma, St Louis, MO, USA).
  • VDM viral dilution media
  • DMEM 0.1 % BSA 10 mM HEPES, 100U/ml Penicillin 100 ⁇ g/mL streptomycin and 2mM Glutamine; all from Sigma, St Louis, MO, USA.
  • Mice were anaesthetized with isofluorane and droplets of VDM-containing virus were applied to the nares until the total 50 ⁇ was inhaled. Unimmunized mice were always used as controls (50 ⁇ of VDM alone).
  • mice were immunized intranasally with 32 HAU pseudotyped H1 N1 influenza (S-Flu) 33 , using the influenza infection protocol described above, and challenged with 32 HAU X31 . Unvaccinated and fully na ' ive mice were used as controls in all influenza vaccine experiments. In unvaccinated mice that received X31 challenge, a combination of weight loss and clinical score was used as a humane endpoint.
  • q-PCR. Cells were purified with a MoFlo cell sorter (Beckman Coulter) by their surface markers. RNA was extracted using RNeasy Kit (Qiagen, Hilden, Germany) and quantified using a Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA).
  • Real-time quantitative PCR using comparative Ct method (AACt) was utilized to evaluate gene expression using validated Taqman probes (AB) on a 7500 Fast Real-time PCR machine (AB). Conditions: 1 . 50°C, 2 min; 2. 95°C, 10 min;3. 95°C 15 sec; 4. 60°C 1 minute; 40 cycles of 3-4.
  • the assay IDs for the primers of the analyzed genes are as follows: Mm00504340_m1 (Atg5), Mm00512209_m1 (Atg7), Mm01264428_m1 (Atg9), Mm00470550_m1 (AtgW), Mm00503201_m1 (Atg12), Mm0051717_m1 (beclin l), Mm00458724_m1 (Map 1lc3a), Mm01545399_m1 (hprt), Mm99999915_g1 (gapdh).
  • ImageStream. ImageStream (Amnis imaging flow cytometer, MA, USA) has previously been used to determine autophagic flux 15 .
  • To determine LC3 spot count we stained cells for LC3-II (following cell surface staining) using the FlowCellect Autophagy LC3 Antibody-based Assay Kit (FCCH100171 , Merk-Millipore) according to the manufacture's instructions.
  • FCCH100171 FlowCellect Autophagy LC3 Antibody-based Assay Kit
  • LC3 spot count was quantified using Ideas software (Amnis), which contains a specialised, objective spot counting feature. Images collected were all at x60 magnification.
  • the membrane was blotted using the following primary antibodies: LC3 (L8918, Sigma)(1 :1000), GAPDH (MAB374, Millipore)(1 :10000), pS6 (221 1 , Cell Signaling Technology, Danvers, MA, USA)(1 :5000).
  • LC3 L8918, Sigma
  • GAPDH MAB374, Millipore
  • pS6 221 1 , Cell Signaling Technology, Danvers, MA, USA
  • IRDye secondary antibodies were bought from LI-COR (Lincoln, NE, USA): (926-3221 1 , 926-68020)(1 :15000).
  • TCID 50 Influenza Viral Titers
  • TCID 50 Determination of the 50% tissue culture infective dose (TCID 50 ) was performed on MDCK-SIAT1 cells in a 96 well flat- bottom plate performed by serial dilution of lung homogenates onto 3x10 4 MDCK-SIAT1 cells followed by incubation for 72 hours at 37 °C.
  • Virus was then detected by hemagglutination where 50 ⁇ _ 1 % (vol/vol) human erythrocytes (adjusted so that a 1 :2 dilution gave an optical density at 600nm) was added to 50 ⁇ _ of the serially diluted lung homogenates/MDCK-SIAT1 supernatent in a 96 well V-bottom plate. Hemagglutination was analyzed by the loss of teardrop formation after tilting the plate. TCID 50 was calculated as described by Reed and Muench 55 . Statistical Analyses. All data were presented as mean ⁇ s.e.m.
  • lymphocytes recognize influenza haemagglutinin that lacks a signal sequence. Nature 324, 575-577, doi:10.1038/324575a0 (1986).

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Abstract

La présente invention concerne des composés de polyamine et leur utilisation dans l'amélioration d'une réponse immunitaire à un immunogène. L'invention concerne également l'utilisation de composés de polyamine pour le traitement de l'immunosénescence. En outre, l'invention concerne des composés nutraceutiques comprenant des composés de polyamine.
PCT/GB2015/050743 2014-03-13 2015-03-13 Composés de polyamine et leurs utilisations WO2015136304A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GR20180100105A (el) * 2018-03-15 2019-10-16 Αθανασιος Κωνσταντινου Αναγνωστοπουλος Ελληνικα γαλακτομικα προϊοντα ενισχυμενα/εμπλουτισμενα με σπερμιδινη και μεθοδος παρασκευης τους
WO2020241445A1 (fr) * 2019-05-24 2020-12-03 学校法人慈恵大学 Agent pharmaceutique, solution médicinale pour le nettoyage d'alvéoles pulmonaires et nébuliseur
CN114250198A (zh) * 2021-08-13 2022-03-29 北京肿瘤医院(北京大学肿瘤医院) 一种增强免疫细胞抗肿瘤效果的方法
CN114344287A (zh) * 2021-12-24 2022-04-15 苏州麦轮生物科技有限公司 一种含有亚精胺的营养组分及其应用
WO2023133342A3 (fr) * 2022-01-10 2023-09-07 The Children's Medical Center Corporation Méthodes et compositions se rapportant à l'immunisation de patients immunitaires distincts

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