WO2023073116A1 - Méthode - Google Patents

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Publication number
WO2023073116A1
WO2023073116A1 PCT/EP2022/080106 EP2022080106W WO2023073116A1 WO 2023073116 A1 WO2023073116 A1 WO 2023073116A1 EP 2022080106 W EP2022080106 W EP 2022080106W WO 2023073116 A1 WO2023073116 A1 WO 2023073116A1
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Prior art keywords
cells
bacteria
subject
bacterial infection
photosensitizing agent
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PCT/EP2022/080106
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English (en)
Inventor
Anders HØGSET
Pål JOHANSEN
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Pci Biotech As
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Publication of WO2023073116A1 publication Critical patent/WO2023073116A1/fr

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    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0023—Agression treatment or altering
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • 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
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • 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
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells 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
    • 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

Definitions

  • the present invention relates to a method of generating an immune response to bacteria in a subject, e.g. for treatment or prevention, such as by vaccination or immunisation, involving the use of a photosensitizing agent and live bacteria, and irradiation with light of a wavelength effective to activate the photosensitizing agent.
  • the invention also relates to pharmaceutical compositions, useful in such a method and uses of those compositions or their components for generating an immune response.
  • the invention also provides therapeutic uses in which a population of cells are prepared ex vivo and administered to the subject in vivo to elicit an immune response, e.g. to achieve vaccination.
  • the invention is particularly concerned with the treatment or prevention of bacterial infection, e.g. tuberculosis.
  • Vaccines are very efficient in the prevention of infections caused by extracellular pathogens due to effective stimulation of pathogen-specific IgG antibodies. In contrast, intracellular antibody surveillance is not possible, for which reason vaccines are typically not effective in prevention and treatment of infections caused by intracellular pathogens such as Mycobacterium tuberculosis.
  • Intracellular survival of bacterial pathogens e.g. Mycobacterium tuberculosis, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus
  • pathogens e.g. Mycobacterium tuberculosis, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus
  • Mycobacterium tuberculosis is the cause of the devastating and widespread disease of tuberculosis.
  • the only approved vaccine against Mycobacterial tuberculosis (Mtb) is Mycobacterial bovis Bacille Calmette-Guerin (BCG), which has variable protective efficacy.
  • WHO recommends BCG in HIV-uninfected infants and juveniles as it provides protection against severe extrapulmonary tuberculosis forms, e.g. miliary and meningeal tuberculosis (WHO. (https://www.who.int/news- room/fact-sheets/detail/immunization-coveraqe, 2020).
  • WHO. https://www.who.int/news- room/fact-sheets/detail/immunization-coveraqe, 2020.
  • bovis BCG has been taken off many national immunization programs, due to low protective efficacy in pulmonary TB in adults (Fine, 2005, BMJ (Clinical Research Ed.), 331 , p647-648). After almost one century of M. bovis BCG vaccination, tuberculosis (TB) still causes millions of fatalities each year.
  • the conventional BCG vaccine has shown poor protection efficacy, especially in pulmonary TB in adults (Mangtani et al., 2014, Clin. Infect. Dis., 58, p470-480; Dockrell and Smith, 2017, Frontiers in Immunology, 8, p1134; and Schrager et al., 2020, The Lancet. Infectious Diseases. 20, e28-e37).
  • BCG typically fails to protect immune compromised or suppressed persons (Gonzalez et al., 1989, The Pediatric Infectious Disease Journal, 8, p201-206; Marciano et al., 2014, Journal of Allergy and Clinical Immunology, 133, p1134-1141 ; Al-Hammadi et al., 2017, BMC Research Notes, 10, p177; and Ong, et al., 2020, Int. J. Infect. Dis., 97, p117-125).
  • complementary healthcare interventions including new and effective prophylactic and therapeutic TB vaccines are urgently required.
  • M. bovis BCG The reason for the suboptimal protective efficacy of the M. bovis BCG vaccine is still not clear. It has been hypothesized that the attenuation process of M. bovis BCG may cause the lost of some important /Wtb-associated genes such as a 10-kDa culture filtrate protein (CFP-10) and a 6-kDa early-secreted antigenic target (ESTAT-6), which affects its immunogenicity. Moreover, some proteins in M. bovis BCG may inhibit the capacity of processing and presentation of mycobacterial antigens by antigen-presenting cells (APCs), including macrophages and dendritic cells (DCs), thus, shielding the subsequent immune responses (Tian et al., 2005, J.
  • APCs antigen-presenting cells
  • DCs dendritic cells
  • M. bovis BCG are phagocytosed by macrophages.
  • both Mtb and M. bovis BCG can arrest phagosome maturation (Fratti et al., 2003, PNAS USA, 100, p5437-5442; Vergne et al., 2004, Molecular Biology of the Cell, 15, p751-760; Vazquez et al., 2017, Infect. Immun. 85, e00720-16; and Panas et al., 2014, Infect. Immun., 82, p5317-5326).
  • PCI photochemical internalization
  • APCs antigen-presenting cells
  • the immunogenicity of M. bovis BCG was greatly improved using PCI treatment and provides a suitable method for generating an immune response to bacteria which may be used for therapeutic or prophylactic purposes.
  • PCI improves delivery of molecules into the cytosol and methods of vaccination which employ PCI are known.
  • PCI is a technique which uses a photosensitizing agent, in combination with an irradiation step to activate that agent, and is known to achieve release of molecules co-administered to a cell into the cell's cytosol. This technique allows molecules that are taken up by the cell into organelles, such as endosomes, to be released from these organelles into the cytosol, following irradiation.
  • PCI provides a mechanism for introducing otherwise membrane-impermeable (or poorly permeable) molecules into the cytosol of a cell in a manner which does not result in widespread cell destruction or cell death.
  • PCI photochemical internalisation
  • the molecule(s) to be internalised which in the present invention would be the live bacteria
  • a photosensitizing agent are brought into contact with a cell.
  • the photosensitizing agent and the molecule to be internalised are taken up into a cellular membrane-bound subcompartment within the cell, i.e. they are endocytosed into an intracellular vesicle (e.g. a lysosome or endosome).
  • the photosensitizing agent is activated which directly or indirectly generates reactive species which disrupt the intracellular vesicle's membranes. This allows the internalized molecule to be released into the cytosol.
  • photochemical internalisation was proposed for transporting a variety of different molecules, including therapeutic agents, into the cytosol i.e. into the interior of a cell.
  • WO 00/54802 utilises such a general method to present or express transfer molecules on a cell surface.
  • a molecule into the cell cytosol, it (or a part of that molecule) may be transported to the surface of the cell where it may be presented on the outside of the cell i.e. on the cell surface.
  • the method finds use in vaccination.
  • the first clinical trial testing PCIbased immunization has recently published (Otterhaug et al., 2021 , Front. Immunol., 11, article 576756).
  • PCI has surprisingly allowed considerable improvement of the immune responses generated to those bacteria, e.g. increased production of antigen-specific T cells and increased cytokine production by those cells.
  • PCI has not previously been used to internalize live bacteria and it was not previously recognized that improved access to the cell’s cytosol would improve the immune response which explains the alternative improvements being sought by others to improve vaccination.
  • the methods described herein improve immune responses and allow for treatment and prophylactic use of PCI to treat or prevent bacterial infections.
  • the present invention provides a method of generating an immune response to bacteria in a subject, comprising administering to cells of said subject live bacteria and a photosensitizing agent and irradiating cells of said subject with light of a wavelength effective to activate said photosensitizing agent, wherein an immune response is generated to said bacteria.
  • these methods employ only the above described two active ingredients (agents) in said methods and uses and the agents are present at appropriate levels (e.g. at the minimum levels described below) in the methods such that they affect the efficacy of the method (i.e. have an active role in enhancing PCI vaccination/antigen presentation/immune response stimulation).
  • the agents are present in buffers with no other active ingredients. Additional agents may, however, be added.
  • the photosensitizing agent and at least one live bacterium are each taken up into an intracellular vesicle in a cell of the subject; and when the cell is irradiated the membrane of the intracellular vesicle is disrupted releasing the molecules into the cytosol of the cell.
  • the different components may be taken up into the same or a different intracellular vesicle relative to each other. It has been found that active species produced by photosensitizers may extend beyond the vesicle in which they are contained and/or that vesicles may coalesce allowing the contents of a vesicle to be released by coalescing with a disrupted vesicle. As referred to herein "taken up” signifies that the molecule taken up is wholly contained within the vesicle.
  • the intracellular vesicle is bounded by membranes and may be any such vesicle resulting after endocytosis/phagocytosis, e.g. an endosome, lysosome or phagosome.
  • a "disrupted" compartment refers to destruction of the integrity of the membrane of that compartment either permanently or temporarily, sufficient to allow release of the molecules contained within it.
  • an “immune response” which may be generated may be humoral and cell- mediated immunity, for example the stimulation of antibody production, or the stimulation of cytotoxic or killer cells, which may recognise and destroy (or otherwise eliminate) cells expressing "foreign" antigens on their surface.
  • the term “stimulating an immune response” thus includes all types of immune responses and mechanisms for stimulating them and encompasses stimulating CTLs which forms a preferred aspect of the invention.
  • the immune response which is stimulated is cytotoxic CD8 T cells.
  • the extent of an immune response may be assessed by markers of an immune response, e.g. secreted molecules such as TNF-a or IFNy or the production of antigen specific T cells (e.g. assessed as described in the Examples).
  • the stimulation of cytotoxic cells or antibody-producing cells requires antigens to be presented to the cell to be stimulated in a particular manner by the antigen-presenting cells, for example MHC Class I presentation (e.g. activation of CD8 + cytotoxic T-cells requires MHC-I antigen presentation).
  • MHC Class I presentation e.g. activation of CD8 + cytotoxic T-cells requires MHC-I antigen presentation.
  • the immune response is stimulated via MHC-I presentation.
  • MHC-II presentation and stimulation of CD4 + T-cells is also a preferred feature of the methods and uses of the invention.
  • the method of generating an immune response may be used to treat or prevent a bacterial infection.
  • treatment refers to reducing, alleviating or eliminating one or more symptoms of the bacterial infection which is being treated, relative to the symptoms prior to treatment. Such symptoms may be correlated with the abundance of bacteria present in the infected cells of the subject.
  • symptoms may be bacterial load (e.g. as assessed in blood, skin, sputum or larynx, trachea, bronchi or lung samples, swabs or washings) or infiltrates, consolidations, cavities and/or lesions in the lung (e.g. as assessed by X-ray or CT scan), by way of example.
  • Prevention refers to delaying or preventing the onset of the symptoms of the bacterial infection. Prevention may be absolute (such that no bacterial infection occurs) or may be effective only in some individuals, or cells, or for a limited amount of time, e.g. a reduced set or level of symptoms.
  • vaccination is the use of an antigen to elicit an immune response which is prophylactic or therapeutic against the development (or further development) of a disease, disorder or infection, wherein that disease, disorder or infection is associated with abnormal expression or presence of that antigen (in this case a bacterial antigen).
  • a disease, disorder or infection is associated with abnormal expression or presence of that antigen (in this case a bacterial antigen).
  • the methods or uses may be used as prophylactic or therapeutic vaccination against a bacterial infection. In therapeutic vaccination treatment of an existing bacterial infection is achieved whereas in prophylactic vaccination, a bacterial infection is prevented or its severity or spread reduced.
  • the live bacteria are used to generate an immune response to that live bacteria.
  • the live bacteria which are administered correspond to the bacteria responsible for the bacterial infection.
  • the bacteria responsible for the bacterial infection may be the same as, or different to, the bacteria used to generate an immune response.
  • Mycobacterium tuberculosis may be used as the live bacteria and may also be the bacteria responsible for the bacterial infection.
  • the live bacteria that may be used may be different to the bacteria responsible for the bacterial infection.
  • the bacterial infection may be Mycobacterial tuberculosis (in humans) caused by Mycobacterium tuberculosis, but the live bacteria may be Mycobacterium bovis (as is currently used for vaccination in humans).
  • the live bacteria “correspond” to the bacteria responsible for the bacterial infection.
  • the live bacteria are sufficiently similar to the bacteria responsible for the bacterial infection that they generate an immune response that recognizes the bacteria responsible for the bacterial infection to effect treatment/prevention. This will generally mean that they have common epitopes.
  • live bacteria are bacteria which exhibit one or more indicators of viability, such as an intact cell membrane, metabolic activity and/or reproducibility. Metabolic activity and reproducibility may only be evident once the bacteria are placed in the correct environment.
  • the bacteria are able to reproduce, i.e. grow and divide. Generally, the bacteria are unable to reproduce sufficiently to cause disease but are able to generate an immune response before clearance by the immune system. The low levels of bacteria that may result from such reproduction are not considered a bacterial infection according to the invention in which continued and extensive proliferation occurs.
  • the live bacteria may be pathogenic bacteria which cause infectious diseases, or bacteria corresponding to pathogenic bacteria. However, the bacteria do not cause disease in a subject to which they are administered.
  • Bacteria “corresponding” to pathogenic bacteria are bacteria which are sufficiently structurally (and optionally functionally) related to the pathogenic bacteria that an immune response to the corresponding bacteria would also target the pathogenic bacteria when present.
  • Corresponding bacteria include bacteria which are derived from the pathogenic bacteria. Thus, for example, where necessary the pathogenic bacteria are modified or altered, e.g. attenuated, to avoid disease by reducing their virulence, but if used in unmodified form or in appropriate amounts would be pathogenic.
  • the bacteria are modified to reduce their virulence, i.e. they are attenuated.
  • the live bacteria are attenuated, for example Mycobacterium bovis BCG or an attenuated Salmonella enterica serovar Typhimurium bacteria.
  • the live bacteria may be Pasteur BCG strain M. bovis BCG 1721, optionally in which the zinc metalloprotease 1 (amp1) gene has been deleted, as described in the examples.
  • Preferred live bacteria are those which result in intracellular bacterial infection, (or correspond to such pathogenic bacteria able to cause such an infection) namely bacteria which are taken up into the cell and are able to survive and replicate in that cell.
  • the bacteria may additionally exist and replicate outside the cell.
  • Such intracellular bacteria may be present at any location within the cell, e.g. within the cytosol or in a membrane-contained subcompartment such as a lysosome, endosome or phagosome.
  • intracellular bacteria include Mycobacterium (e.g. M. tuberculosis or M. bovis), Pseudomonas (e.g. P. aeruginosa), Escherichia (e.g. E.
  • coli coli
  • Staphylococcus e.g. S. aureus or S. epidermidis
  • Salmonella Salmonella enterica serovar Typhimurium
  • Other intracellular bacteria include Listeria (e.g. Listeria monocytogenes), Francisella (e.g. Francisella tularensis), Coxiella (e.g. C. burnetii) and Rickettsia.
  • live bacteria are Mycobacterium bovis or Mycobacterium tuberculosis bacteria.
  • references herein to bacteria in the plural include the singular.
  • use of live bacteria results in the internalization of one or more bacterium per cell, and the presentation of an antigenic portion(s) of one or more bacterium on the cell surface of each cell.
  • a “bacterial infection” is invasion of a cell(s) or bodily tissue by bacteria that proliferate at that site and which may result in injury to that cell or tissue.
  • a cell which is “infected” contains one or more bacterium capable of survival and potentially replication in that cell.
  • the bacterial infection is caused by the bacteria described hereinbefore, preferably by intracellular bacteria selected from the genera Mycobacterium, Pseudomonas, Escherichia, Staphylococcus and Salmonella.
  • intracellular bacterial infections by bacteria selected from the genera Coxiella, Listeria, Francisella and Rickettsia.
  • the infection is present in cells of, or associated with, bones, blood, the heart, urinary tract, lung, skin or mucosal surfaces.
  • Preferred bacterial infections to be treated include osteomyelitis, bacteremia, tuberculosis, Q-fever and endocarditis and (sub)cutaneous skin or mucosal infections/damages such as chronic wounds, ulcers, abscesses and diabetic foot infection as well as oral and nasal infections such as chronic rhinosinusitis and periodontitis.
  • Particularly preferred is the treatment of tuberculosis.
  • a “photosensitizing agent” as referred to herein is a compound that is capable of translating the energy of absorbed light into chemical reactions when the agent is activated on illumination at an appropriate wavelength and intensity to generate an activated species.
  • the highly reactive end products of these processes can result in cyto- and vascular toxicity.
  • a photosensitizing agent may be one which localises to intracellular compartments, particularly endosomes, phagosomes or lysosomes.
  • Photosensitizing agents may exert their effects by a variety of mechanisms, directly or indirectly. Thus for example, certain photosensitizing agents become directly toxic when activated by light, whereas others act to generate toxic species, e.g. oxidising agents such as singlet oxygen or other reactive oxygen species, which are extremely destructive to cellular material and biomolecules such as lipids, proteins and nucleic acids.
  • oxidising agents such as singlet oxygen or other reactive oxygen species
  • photosensitizing agents are known in the art and are described in the literature, including in WO96/07432, which is incorporated herein by reference, and may be used in methods and uses of the invention.
  • photosensitizing agents including porphyrins, phthalocyanines and chlorins, (Berg et al., 1997, J. Photochemistry and Photobiology, 65, p403-409, incorporated herein by reference).
  • Other photosensitizing agents include bacteriochlorins.
  • Porphyrins are the most extensively studied photosensitizing agents. Their molecular structure includes four pyrrole rings linked together via methine bridges. They are natural compounds which are often capable of forming metal-complexes. For example in the case of the oxygen transport protein hemoglobin, an iron atom is introduced into the porphyrin core of heme B.
  • Chlorins are large heterocyclic aromatic rings consisting, at the core, of three pyrroles and one pyrroline coupled through four methine linkages. Unlike porphyrin, a chlorin is therefore largely aromatic, but not aromatic through the entire circumference of the ring.
  • the photosensitizing agent is preferably an agent which is taken up into the internal compartments of lysosomes, phagosomes or endosomes.
  • the photosensitizing agent is taken up into intracellular compartments by endocytosis or phagocytosis.
  • Preferred photosensitizing agents are amphiphilic photosensitizers (e.g.
  • disulphonated photosensitizing agents such as amphiphilic phthalocyanines, porphyrins, chlorins, and/or bacteriochlorins, and in particular include sulfonated (preferably disulfonated) meso-tetraphenyl chlorins, porphyrins, phthalocyanines and bacteriochlorins.
  • the photosensitizing agent is selected from a porphyrin, phthalocyanine, purpurin, chlorin, benzoporphyrin, lysomotropic weak base, naphthalocyanine, cationic dye, tetracycline, 5-aminolevulinic acid and/or esters thereof, or a derivative of any of said agents, preferably TPPS4, TPPS2a, AIPcS2a, TPCS2a, 5-aminolevulinic acid or esters of 5-aminolevulinic acids, or pharmaceutically acceptable salts thereof.
  • Particularly preferred photosensitizing agents are sulfonated aluminium phthalocyanines, sulfonated tetraphenylporphines, sulfonated tetraphenylchlorins and sulfonated tetraphenylbacteriochlorins.
  • TPPS2a tetraphenylporphine disulfonate
  • AIPcS2a aluminium phthalocyanine disulfonate
  • TPPS4 meso-tetraphenylporphine tetrasulfonate
  • TPCS2a tetraphenyl chlorin disulfonate
  • TPBS2a tetraphenyl bacteriochlorin disulfonate
  • the photosensitizing agent is TPCS2a (Disulfonated tetraphenyl chlorin, e.g. Amphinex ®).
  • TPCS2a Disulfonated tetraphenyl chlorin, e.g. Amphinex ®
  • the arrow indicates the structural difference between the two molecules.
  • the photosensitizing agent may be attached to or associated with or conjugated to one or more carrier molecules or targeting molecules which can act to facilitate or increase the uptake of the photosensitizing agent.
  • the photosensitizing agent may be linked to a carrier.
  • the photosensitizing agent may be provided in the form of a conjugate, e.g. a chitosan- based conjugate, for example a conjugate disclosed in WO2013/189663, which is hereby incorporated by reference.
  • the “subject” as used herein refers to a mammal, reptile, bird, insect or fish.
  • the subject is a mammal, particularly a primate (preferably a human), domestic or companion animal, livestock or laboratory animal.
  • preferred animals include humans, cats, dogs, horses, donkeys, sheep, pigs, goats, cows, monkeys, mice, rats, rabbits or guinea pigs.
  • the live bacteria and photosensitizing agent are administered to cells in the subject and those cells are irradiated.
  • the cells for administration may be selected depending on whether the method is for treatment or prevention and the preferred mode of administration.
  • the cells to which the agents are administered may be cells which are infected or local to those which are infected.
  • non-infected, non-local cells may be targeted which provide an immune response with systemic effects.
  • cells are selected which are readily accessible for irradiation.
  • administration is preferably intradermal and thus preferred cells are skin cells.
  • cells actively involved in generating an immune response may be targeted, e.g. lymphoid tissues.
  • the cells are those which readily present antigen on their surface, as discussed below in relation to the generation of cells ex vivo.
  • the subject may be treated by generating a population of cells in which each cell expresses an antigenic portion of a live bacterium on its surface and then administering those cells to the subject.
  • the invention provides a method of generating an immune response to bacteria in a subject, preferably for treating or preventing a bacterial infection in said subject, comprising preparing a population of cells in which each cell expresses an antigenic portion of a live bacterium on its surface by contacting said cells with said live bacteria, as defined hereinbefore and said photosensitizing agent as defined hereinbefore, wherein when treating or preventing a bacterial infection said live bacteria correspond to the bacteria responsible for the bacterial infection, and irradiating the cells with light of a wavelength effective to activate the photosensitizing agent and release said live bacteria into the cytosol of the cells to present an antigenic portion thereof on each of the cells’ surfaces, and subsequently administering said cells to said subject.
  • Cells suitable for this purpose are those which can present antigen on their surface, i.e. any cell which is capable of expressing, or presenting on its surface a molecule which is administered or transported into its cytosol. Conveniently it is an immune cell i.e. a cell involved in the immune response. However, other cells may also present antigen to the immune system and may also be used. Such antigen- presenting cells may be involved in any aspect or "arm" of the immune response as defined herein.
  • expressing or “presenting” refers to the presence of the antigenic molecule or a part thereof on the surface of said cell such that at least a portion of that molecule is exposed and accessible to the environment surrounding that cell, preferably such that an immune response may be generated to the presented molecule or part thereof.
  • Expression on the "surface” may be achieved in which the molecule to be expressed is in contact with the cell membrane and/or components which may be present or caused to be present in that membrane.
  • cytotoxic cells require antigens to be presented to the cell to be stimulated in a particular manner by the antigen-presenting cells, for example MHC Class I presentation (e.g. activation of CD8 + cytotoxic T-cells requires MHC-1 antigen presentation).
  • Antibody-producing cells may also be stimulated by presentation of antigen by the antigen-presenting cells.
  • Antigens may be taken up by antigen-presenting cells by endocytosis and degraded in the endocytic vesicles to peptides. These peptides may bind to MHC class II molecules in the endosomes and be transported to the cell surface where the peptide-MHC class II complex may be recognised by CD4+ T helper cells and induce an immune response.
  • proteins in the cytosol may be degraded, e.g. by proteasomes and transported into endoplasmic reticulum by means of TAP (transporter associated with antigen presentation) where the peptides may bind to MHC class I molecules and be transported to the cell surface (Yewdell and Bennink, 1992, Adv. Immunol.
  • the peptide-MHC class I complex will be recognised by CD8+ cytotoxic T-cells (CTLs).
  • CTLs cytotoxic T-cells
  • the CTLs will bind to the peptide-MHC (HLA) class I complex and thereby be activated, start to proliferate and form a clone of CTLs.
  • the target cell and other target cells with the same peptide-MHC class I complex on the cells surface may be killed by the CTL clone.
  • the live bacteria may be processed by the antigen-processing machinery of the cell and an antigenic portion thereof (i.e. an antigenic portion of a formerly live bacterium) presented on the cell surface in an appropriate manner e.g. by Class I MHC.
  • This processing will require degradation of the bacteria, e.g. into peptides, which peptides are then complexed with molecules of the MHC for presentation.
  • a part or fragment of the live bacterium (the antigenic portion) is expressed or presented on the surface of the cell.
  • lymphocytes both T and B cells
  • dendritic cells macrophages etc.
  • others include for example cancer cells e.g. melanoma cells. These cells are referred to herein as "antigen-presenting cells”.
  • “Professional antigen-presenting cells” which are cells of the immune system principally involved in the presentation of antigen to effector cells of the immune system are known in the art and described in the literature and include B lymphocytes, dendritic cells and macrophages.
  • the cell is a professional antigen-presenting cell.
  • Dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells which are characterized by high endocytic activity and low T-cell activation potential. Once they have come into contact with a presentable antigen, they become activated into mature dendritic cells and begin to migrate to the lymph node. Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces and upon maturation present those fragments at their cell surface using MHC molecules. In the alternative macrophages may be used.
  • the dendritic cells or macrophages may be derived from any appropriate source, such as from the skin, inner lining of the nose, lungs, stomach and intestines or the blood.
  • the dendritic cells are derived from bone marrow.
  • the cells may be isolated from natural sources for use in the ex vivo methods of the invention or may be generated ex vivo, e.g. by culture of peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • a “population” of cells is a plurality of cells of essentially identical form, e.g. a single cell type prepared by the same method.
  • the population of cells may be comprised in a larger collection of cells. For example a collection of cells may be treated but only some of those cells may express an antigenic portion of a live bacterium on their surface. Only those modified cells provide the population of cells but they may be present with other cells which are not so modified.
  • Each cell in the cell population expresses at least one antigenic portion of a live bacterium on its surface.
  • An "antigenic portion" of the live bacterium is a portion of the bacterium which is capable of stimulating an immune response, when presented to the immune system or immune cells in an appropriate manner. Processing of live bacteria by cells through the MHC I pathway results in destruction of the bacteria and presentation (or expression) of one or more antigenic portion on the cell surface.
  • the antigenic portion is a peptide it is of sufficient size to generate an immune response, e.g. greater than 5, e.g. greater than 10 or 20 amino acids in size.
  • the cells are contacted with the live bacteria.
  • contacting refers to bringing the cell(s) and the various agents used in the method into physical contact with one another under conditions appropriate for internalization into the cells, e.g. preferably at 37°C in an appropriate nutritional medium, e.g. from 25-39°C. In the in vivo methods a similar contact is achieved in the subject’s cells.
  • the cells may be contacted with the photosensitizing agent and the live bacteria used in the method (or use) as defined herein sequentially, separately or simultaneously.
  • the agents are contacted with the cell(s) simultaneously and preferably are applied to the cell(s) together as described in more detail hereinafter.
  • the different agents may be taken up by the cell(s) into the same or different intracellular compartments (e.g. they may be co-translocated).
  • the agents are not applied together, i.e. are applied at a different time and/or by a different administration route.
  • the cells are then exposed to light of suitable wavelengths to activate the photosensitizing agents which in turn leads to the disruption of the intracellular compartment membranes.
  • WO 02/44396 (which is incorporated herein by reference) describes a method in which the order of the steps in the method may be arranged such that for example the photosensitizing agent is contacted with the cells and activated by irradiation before the molecule to be internalised (in this case the live bacteria) is brought into contact with the cells.
  • This method takes advantage of the fact that it is not necessary for the molecule to be internalised to be present in the same cellular subcompartment as the photosensitizing agent at the time of irradiation.
  • said photosensitizing agent and said live bacteria as defined herein are applied (or administered) to the cells together, or separately relative to one another. Irradiation is then performed at a time when the photosensitizing agent and the live bacteria appear in the same intracellular compartment. This is referred to as a "light after" method and is a preferred aspect of the invention.
  • the method can be performed by contacting the cells with the photosensitizing agent first, followed by contact with the live bacteria to be used as defined herein, and irradiation is performed after uptake of the photosensitizing agent into an intracellular compartment, but prior to the cellular uptake of the live bacteria into an intracellular compartment containing the photosensitizing agent (e.g. they may be present in a different intracellular compartment at the time of light exposure), preferably prior to cellular uptake into any intracellular compartment, e.g. prior to any cellular uptake.
  • the photosensitizing agent may be administered followed by irradiation and then administration of the live bacteria. This is the so-called "light before" method.
  • Internalisation refers to the intracellular, e.g. cytosolic, delivery of molecules.
  • internalisation may include the step of release of molecules from intracellular/membrane bound compartments into the cytosol of the cells.
  • cellular uptake or “translocation” refers to one of the steps of internalisation in which molecules external to the cell membrane are taken into the cell such that they are found interior to the outer lying cell membrane, e.g. by endocytosis, phagocytosis or other appropriate uptake mechanisms, for example into or associated with intracellular membrane-restricted compartments, for example the endoplasmic reticulum, Golgi body, lysosomes, endosomes, phagosomes etc.
  • the methods or uses described herein may be performed on a cell(s) that is already infected with bacteria, i.e. as a treatment. However, the method (or use) may also be performed on a cell(s) that is not, or not yet, infected with bacteria. The latter is appropriate as a prevention method or to generate a cell ex vivo for in vivo uses, or as a treatment method as treatment does not require performing the method on affected cells.
  • the cell(s) may be a cell(s) which is identified to be at risk of infection (e.g. at a site where infection is likely) or cells which are preferred for antigen presentation.
  • the step of contacting the cells with the different agents may be carried out in any convenient or desired way.
  • the cells may conveniently be maintained in an aqueous medium, such as for example appropriate cell culture medium, and at the appropriate time point the various agents can simply be added to the medium under appropriate conditions, for example at an appropriate concentration and for an appropriate length of time.
  • the cells may be contacted with the agents in the presence of serum- free medium, or with serum-containing medium.
  • the application of the agents used in the methods and uses of the invention may be to cells ex vivo or in vivo. In the former case the cells generated ex vivo are to be administered to the subject. In the latter case, the application may be via direct (i.e. localized, such as topically or by injection) or indirect (i.e. systemic or non-localized) administration as described in more detail hereinbelow.
  • the photosensitizing agent is brought into contact with the cells at an appropriate concentration and for an appropriate length of time which can easily be determined by a skilled person using routine techniques, and will depend on such factors as the particular photosensitizing agent used, the mode of administration, the course of treatment, the age and weight of the patient/subject, the medical indication, the body or body area to be treated and may be varied or adjusted according to choice.
  • concentration of the photosensitizing agent is conveniently such that once taken up into the cell, e.g. into, or associated with, one or more of its intracellular compartments and activated by irradiation, one or more cell structures are disrupted e.g. one or more intracellular compartments are lysed or disrupted.
  • photosensitizing agents as described herein may be used at a concentration of for example 0.1 to 50 pg/ml.
  • the range can be much broader, e.g. 0.0005-500 pg/ml.
  • the photosensitizing agent may be used in the range 0.05-20 mg/kg body weight when administered systemically.
  • a range of 0.005-20mg/kg body weight may be used for systemic administration.
  • the total dose provided may be in the order of 1-5000 pg, for example 10-2500, 25-1000, 50-500, 10-300, 25-200, or 100-300pg.
  • the dose is selected from 100pg, 150pg, 200pg and 250pg.
  • the dose is 75-125 pg, e.g. 100 pg.
  • Local administration is preferred, for example by topical or intradermal administration, and in this case the dose may be in the region of 0.001 - 500 pg or 0.1-500 pg, for example 0.001-0.1 , 0.0025-1 , 0.01-50, 0.0025-250, 1-250, preferably between 5 and 150pg, preferably between 25 and 100pg, for example 50pg.
  • the dose is selected from 25g, 40pg, 60pg and 75pg.
  • the dose is 25-75 pg.
  • the doses provided are for a human of average weight (i.e. 70kg).
  • the photosensitizer dose may be dissolved in 100 pl-1 ml, i.e. the concentration may be in the range of 0.01-50000 pg/ml or 1-50000 pg/ml. In smaller animals the concentration range may be different and can be adjusted accordingly though when administered locally, little variation in dosing is necessary for different animals.
  • the amount of the live bacteria as defined herein to be used will also depend on the particular bacteria which is to be used, the mode of administration, the course of treatment, the age and weight of the patient/subject, the medical indication, the body or body area to be treated and may be varied or adjusted according to choice.
  • the live bacteria may be used in an amount of 1 x 10 2 to 1 x 1O 10 CFU (colony forming units), preferably between 1 x 10 4 to 1 x 10 8 CFU, preferably 1 x 10 5 to 1 x 10 7 CFU.
  • a similar amount may be used for ex vivo and in vivo use. Similar amounts may be used regardless of administration route and, for example, the above amounts may be used for local delivery, e.g. intradermal delivery.
  • the doses provided are for a human of average weight (i.e. 70kg). In most cases the photosensitizing agent and the live bacteria are administered together (in the in vivo and ex vivo methods), but this may be varied. Thus different times or modes or sites of administration (or contact with the cell) are contemplated for the components and such methods and uses are encompassed within the scope of the invention.
  • the live bacteria as defined herein are administered separately from the photosensitizing agent, for example in a separate formulation, e.g. systemically, e.g. via oral administration.
  • the separate administration may be simultaneous (e.g. via different administration routes in vivo but at the same time) or may be consecutive.
  • the live bacteria or the photosensitizing agent may be administered prior to administration of the photosensitizer or live bacteria, respectively, for example up to 24 or 48 hours before.
  • the separate administrations are separated by less than 48, 24, 12, 8, 4 or 2 hours.
  • the photosensitizer may be administered in one location (e.g. locally to avoid skin photosensitivity) and the live bacteria may then be administered in a second location (e.g. systemically or at a different site). In an alternative they may be administered at the same location but sequentially/consecutively.
  • the contact between the cell(s) and the photosensitizing agent and/or live bacteria as defined herein is conveniently from 15 minutes to 24 (or 48) hours (e.g. 15 or 30 minutes to 4 hours, e.g. 1-2 hours).
  • the contacting may be simultaneous or sequential or the timing of contacting with the separate components may overlap, as discussed above.
  • the contacting step refers to the total contact time of the cell(s) with the agent in question and that contacting time may be made up of a number of discrete separate contacting steps.
  • the agent may be removed from contact with the cell(s) for a period of time before the irradiation step. In ex vivo methods, in a preferred aspect, the contacting step for each agent may be 15 (or 30) minutes to 24 hours.
  • a similar contact time may be used, but the cells may not be contacted with the agents immediately after administration (e.g. where systemic administration is used) and in such cases the agents will need to be administered sufficiently before illumination so that the agents reach the target cells and have contact with those cells for the required contact time, as discussed hereinbelow in more detail. This is mitigated by local delivery, which is preferred, allowing contact to be achieved shortly after administration.
  • the initial incubation of the cell is with the photosensitizing agent.
  • the time between the administration of the photosensitizing agent and the live bacteria is a matter of hours.
  • the photosensitizing agent may be applied 16 to 20 (or 40 to 44) hours, e.g. 18 hours, before illumination
  • the live bacteria may be applied 1 to 3 hours, e.g. 2 hours before illumination.
  • the time between the administration of the photosensitizing agent and the live bacteria may be in the range of 15 to 23 (or 47) hours. This timing applies regardless of which agent is administered first.
  • both the photosensitizing agent and the live bacteria are applied at the same time (which may be applied separately or together).
  • live bacteria and the photosensitizing agent are mixed together (e.g. for 1 to 6 hours, e.g. for 2-4 hours) to form a complex prior to application to the cell or administration to the subject, as described in the examples.
  • the cell(s) may be placed into photosensitizer/bacteria-free medium after the contact with the photosensitizer/live bacteria and before irradiation, e.g. for 30 minutes to 4 hours, e.g. from 1.5 to 2.5 hours, depending on the timing of the incubation with the photosensitizer and the live bacterial.
  • an appropriate method and time of incubation by which the various agents are brought into contact with the target cells will be dependent on factors such as the mode of administration and the type of agents which are used. For example, if the agents are injected into a tissue or organ which is to be treated/irradiated, or applied topically, the cells near the injection or application point will come into contact with and hence tend to take up the agents more rapidly than the cells located at a greater distance from the injection or application point, which are likely to come into contact with the agents at a later time point and lower concentration. Conveniently a time of 6-24 (or 6-48) hours may be used.
  • agents administered remote to the site may take some time to arrive at the target cells and it may thus take longer post-administration e.g. several days, in order for a sufficient or optimal amount of the agents to accumulate in a target cell or tissue.
  • the time of administration required for individual cells in vivo is thus likely to vary depending on these and other parameters.
  • the time at which the agents come into contact with the target cells must be such that before irradiation occurs an appropriate amount of the photosensitizing agent has been taken up by the target cells and either: (i) before or during irradiation the live bacteria has either been taken up, or will be taken up after sufficient contact with the target cells, into the cell, for example into the same or different intracellular compartments relative to the photosensitizing agent or (ii) after irradiation the live bacteria are in contact with the cells for a period of time sufficient to allow its uptake into the cells.
  • any mode of administration common or standard in the art may be used, e.g. oral, parenteral (e.g. intramuscular, transdermal, subcutaneous, percutaneous, intraperitoneal, intrathecal or intravenous), intestinal, buccal, rectal or topical, both to internal and external body surfaces etc.
  • parenteral e.g. intramuscular, transdermal, subcutaneous, percutaneous, intraperitoneal, intrathecal or intravenous
  • intestinal, buccal, rectal or topical both to internal and external body surfaces etc.
  • the invention can be used in relation to any tissue which contains cells to which the photosensitizing agent containing compound or the live bacterial is localized, including body fluid locations, as well as solid tissues. All tissues can be treated as long as the photosensitizer is taken up by the target cells, and the light can be properly delivered.
  • Preferred modes of administration are intradermal, topical administration or injection.
  • administration is local, e.g. topical (such as via a cream or gel, e.g. on the skin surface or by other means, e.g. intradermal administration such as by injection.
  • topical such as via a cream or gel, e.g. on the skin surface or by other means, e.g. intradermal administration such as by injection.
  • intradermal administration such as by injection.
  • the photosensitizing agent is administered locally.
  • Modes of administration may be selected depending on the subject and the effect to be achieved, i.e. prevention or treatment. Where treatment is contemplated administration may be close to or at the site of infection. However, as the methods and uses of the invention generate an immune response which would have systemic effects, sites distant to the site of infection may be used.
  • Administration may be by application of the required agents to the cell(s) at the time of the treatment/prevention method (e.g. administration to the patient/subject) or one or more of the required agents may be provided in a formulation which allows slow or controlled (on demand) release, which may then be followed with the treatment/prevention step.
  • the live bacteria and/or the photosensitizing agent may be provided on or within (e.g. embedded within or impregnated in) a biomaterial to be used on or in a patient/subject.
  • Such agents may be released slowly with time or released on demand and the treatment /prevention step initiated by irradiation (optionally with administration of one of the required agents if not present in/on the biomaterial). Conveniently in assessing timings and doses such administration is considered local administration.
  • any mode of administration of the cell population which is common or standard in the art may be used, e.g. injection or infusion, by an appropriate route. Both systemic and local forms of administration are contemplated.
  • the cells are administered by intralymphatic injection.
  • 1x10 4 to 1x10 8 cells are administered per kg of subject (e.g. 1.4x10 4 to 2.8x10 6 per kg in human).
  • a dose of 0.1- 20x10 7 cells may be administered in a dose, i.e. per dose, for example as a vaccination dose. The dose can be repeated at later times if necessary.
  • the methods (or uses described hereinafter) or parts thereof may be repeated, e.g. “re-vaccination” may take place.
  • the method (or use) in its entirety may be performed multiple times (e.g. 2, 3 or more times) after an appropriate interval or parts of the method (or use) may be repeated, e.g. further administration of the live bacteria and/or photosensitizing agent as defined herein or additional irradiation steps.
  • the method (or use) or part of the method (or use) may be performed again a matter of days, e.g. between 5 and 60 days (for example 7, 14, 15, 21, 22, 42 or 51 days), e.g.
  • the method (or use) may be repeated multiple times at appropriate intervals of time, e.g. every two weeks or 14 days. In a preferred embodiment the method (or use) is repeated at least once. In another embodiment the method (or use) is repeated twice.
  • “Irradiation" to activate the photosensitizing agent refers to the administration of light directly or indirectly as described hereinafter.
  • the cell(s) which may be ex vivo or present in a subject
  • Irradiation of the cell or subject may occur approximately 15 minutes to 24 (or 48) hours after administration of the various agents for use in the methods and uses as defined herein. In methods involving the ex vivo production of treated cells, irradiation may occur from, for example 15 to 120 minutes after administration.
  • a longer time after administration may be required if contact time with the target cells within the subject is not immediate (depending on the route of administration), e.g. from 15 minutes to 24 (or 48) hours after administration.
  • irradiation is performed 6 to 24 hours after administration of both the photosensitizing agent and the live bacteria, preferably 14 to 22 hours, e.g. 18 hours after administration.
  • the light irradiation step to activate the photosensitizing agent may take place according to techniques and procedures well known in the art.
  • the dose, wavelength and duration of the illumination must be sufficient to activate the photosensitizing agent, i.e. to generate reactive species.
  • the wavelength of light to be used is selected according to the photosensitizing agent to be used. Suitable artificial light sources are well known in the art, e.g. using blue (400-475nm) or red (620-750nm) wavelength light. For TPCS2a, both blue and red light. For example, a wavelength of between 400 and 500nm, more preferably between 400 and 450nm, e.g. from 430-440nm, and even more preferably approximately 435nm, or 435nm may be used. In the alternative light with a wavelength of between 630 and 675nm may be used, e.g. from 645- 660nm, e.g. 652nm. Where appropriate the photosensitizer, e.g. a porphyrin or chlorin, may be activated by green light, for example the KillerRed (Evrogen, Moscow, Russia) photosensitizer may be activated by green light.
  • the photosensitizer e.g. a porphy
  • Suitable light sources are well known in the art, for example the LumiSource® lamp of PCI Biotech AS.
  • an LED-based illumination device which has an adjustable output power of up to 60mW and an emission spectra of 430-435nm may be used.
  • a suitable source of illumination is the PCI Biotech AS 652nm laser system SN576003 diode laser, although any suitable red light source may be used.
  • the time for which the cells are exposed to light in the methods and uses of the present invention may vary.
  • the efficiency of the internalisation of a molecule into the cytosol increases with increased exposure to light to a maximum beyond which cell damage and hence cell death increases.
  • a preferred length of time for the irradiation step depends on factors such as the target cell, the photosensitizer, the amount of the photosensitizer accumulated in the target cells or tissue and the overlap between the absorption spectrum of the photosensitizer and the emission spectrum of the light source.
  • the length of time for the irradiation step is in the order of seconds to minutes or up to several hours (even up to 12 hours), e.g. preferably up to 60 minutes e.g. from 10 seconds to 60 minutes, preferably 1 to 30 minutes, preferably for 4 to 10 minutes, preferably for 6 minutes.
  • Shorter irradiation times may also be used, for example 1 to 60 seconds, e.g. 10-50, 20-40 or 25-35 seconds, e.g. when higher doses of photosensitizing agent are used or when ex vivo irradiation methods are used.
  • Appropriate light doses can be selected by a person skilled in the art and again will depend on the photosensitizer used and the amount of photosensitizer accumulated in the target cell(s) or tissues.
  • the light doses are usually lower when photosensitizers with higher extinction coefficients (e.g. in the red area, or blue area if blue light is used, depending on the photosensitizer used) of the visible spectrum are used.
  • a light dose in the range of 0.24 - 7.2J/cm 2 at a fluence range of 0.05-20 mW/cm 2 e.g.
  • 2.0 mW/cm 2 may be used when an LED-based illumination device which has an adjustable output power of up to 60mW and an emission spectra of 430-435nm is employed.
  • the LumiSource® lamp a light dose in the range of 0.1-6J/cm 2 at a fluence range of 0.1- 20 (e.g. 13 as provided by Lumisource®) mW/cm 2 is appropriate.
  • a light dose of 0.03-1 J/cm 2 e.g. 0.3J/cm 2
  • at a fluence range of 0.1-5 mW/cm 2 e.g. 0.81 mW/cm 2
  • 0.03-1 J/cm 2 e.g. 0.3J/cm 2
  • a fluence range of 0.1-5 mW/cm 2 e.g. 0.81 mW/cm 2
  • the methods and uses of the invention may inevitably give rise to some cell damage by virtue of the photochemical treatment i.e. by photodynamic therapy effects through the generation of toxic species on activation of the photosensitizing agent. Depending on the proposed use, this cell death may not be of consequence and may indeed be advantageous to remove some bacteria-infected cells. In most embodiments, however, cell death is avoided, e.g. to allow immune responses to be generated.
  • the methods and uses of the invention may be modified such that the fraction or proportion of the surviving cells is regulated by selecting the light dose in relation to the concentration or dose of the photosensitizing agent. Again, such techniques are known in the art.
  • substantially all of the cells, or a significant majority are not killed (of those subject to the treatment).
  • Ex vivo cell viability following PCI treatment can be measured by standard techniques known in the art such as the MTS test.
  • In vivo cell death of one or more cell types may be assessed within a 1cm radius of the point of administration (or at a certain depth of tissue), e.g. by microscopy. As cell death may not occur instantly, the % cell death refers to the percent of cells which remain viable within a few hours of irradiation (e.g. up to 4 hours after irradiation) but preferably refers to the % viable cells 4 or more hours after irradiation.
  • the various agents used in the methods or uses of the invention may be administered to the subject (or cells) separately, sequentially or simultaneously.
  • the photosensitizing agent and the live bacteria may be provided in a composition.
  • they may be in separate solutions or compositions allowing different mechanisms or timings for administration or application.
  • co-administration and “coapplication” refers to use of both components in the same method or use rather than simultaneous use (either in terms of timing or in the same composition).
  • the present invention provides a composition comprising live bacteria as described herein and a photosensitizing agent as described herein.
  • the composition may be in the form of a pharmaceutical composition comprising in addition one or more pharmaceutically acceptable diluents, carriers or excipients.
  • compositions may be formulated in any convenient manner according to techniques and procedures known in the pharmaceutical art, e.g. using one or more pharmaceutically acceptable diluents, carriers or excipients.
  • the compositions may be formulated as slow or delayed release compositions.
  • “Pharmaceutically acceptable” as referred to herein refers to ingredients that are compatible with other ingredients of the compositions (or products) as well as physiologically acceptable to the recipient.
  • the nature of the composition and carriers or excipient materials, dosages etc. may be selected in routine manner according to choice and the desired route of administration, purpose of treatment etc.
  • Dosages may likewise be determined in routine manner and may depend upon the nature of the molecule (or components of the composition or product), purpose of treatment, age of patient/subject, mode of administration etc. In connection with the photosensitizing agent, the potency/ability to disrupt membranes on irradiation, should also be taken into account.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a population of cells in which each cell expresses an antigenic portion of a live bacterium on its surface, wherein said cells are obtainable by a method comprising contacting said cells with said live bacteria as described herein and said photosensitizing agent as described herein, and irradiating the cells with light of a wavelength effective to activate the photosensitizing agent and release said live bacteria into the cytosol of the cells to present an antigenic portion thereof on each of the cells’ surfaces, and one or more pharmaceutically acceptable diluents, carriers or excipients, wherein preferably the cells are macrophages.
  • the method for generating the cells ex vivo is as described hereinbefore for the generation of cells for delivery in vivo for the therapeutic and prophylactic methods and uses described herein.
  • compositions as defined hereinbefore for use in prophylaxis or therapy may be used as described hereinbefore, particularly for use in generating an immune response to bacteria in a subject, preferably for treating or preventing a bacterial infection in said subject wherein said live bacteria correspond to the bacteria responsible for the bacterial infection.
  • the use may comprise a method of generating an immune response as described hereinbefore, e.g. the use is intended for that specific purpose, or the use is directed to a preferred aspect of that method, e.g. the intended mode or dose of administration or irradiation.
  • the present invention provides use of a composition as defined herein in the manufacture of a medicament for generating an immune response to bacteria in a subject, preferably for treating or preventing a bacterial infection in the subject wherein the live bacteria correspond to the bacteria responsible for the bacterial infection.
  • the invention also provides live bacteria as defined hereinbefore and a photosensitizing agent as defined hereinbefore for use in prophylaxis or therapy.
  • Such use may be to generate an immune response to bacteria in a subject, preferably for treating or preventing a bacterial infection in the subject wherein the live bacteria correspond to the bacteria responsible for the bacterial infection.
  • the use is intended to generate an immune response by using a method, or aspects of that method, as defined hereinbefore.
  • the use may comprise preparing a population of cells in which each cell expresses an antigenic portion of a live bacterium on its surface by contacting said cells with live bacteria as defined herein and a photosensitizing agent as defined herein and irradiating the cells with light of a wavelength effective to activate the photosensitizing agent and release the live bacteria into the cytosol of the cells to present an antigenic portion thereof on each of the cells’ surfaces.
  • the population of cells is to be administered to the subject.
  • live bacteria as defined herein or a photosensitizing agent as defined herein in the manufacture of a medicament for generating an immune response to bacteria in a subject, preferably for treating or preventing a bacterial infection in said subject wherein said live bacteria correspond to the bacteria responsible for the bacterial infection.
  • the use may comprise a method (or aspects thereof) as defined herein.
  • the medicament may comprise the live bacteria or the photosensitizing agent and the medicament is used to generate an immune response as described herein, in which the agent not present in the medicament, is also used (i.e. is co-administered or co-used with the medicament).
  • the medicament may comprise a population of cells in which each cell expresses an antigenic portion of a live bacterium on its surface, wherein the cells are obtainable by a method comprising contacting said cells with said live bacteria as defined herein and said photosensitizing agent as defined herein, and irradiating the cells with light of a wavelength effective to activate the photosensitizing agent and release said live bacteria into the cytosol of the cells to present an antigenic portion thereof on each of the cells’ surfaces, for administration to said subject.
  • Also provided by the invention is a product comprising live bacteria as defined herein and a photosensitizing agent as defined herein, as a combined preparation for simultaneous, separate or sequential use in generating an immune response to bacteria in a subject, preferably for treating or preventing a bacterial infection in said subject wherein said live bacteria correspond to the bacteria responsible for the bacterial infection, wherein preferably the use comprises a method (or aspects thereof) as defined hereinbefore.
  • the invention provides a kit for use in generating an immune response to bacteria in a subject, preferably for treating or preventing a bacterial infection in the subject wherein the live bacteria correspond to the bacteria responsible for the bacterial infection, and the kit comprising a first container containing a photosensitizing agent as defined herein; and a second container containing live bacteria as defined herein, wherein preferably the use comprises a method (or aspects thereof) as defined hereinbefore.
  • the products and kits of the invention may be used to generate an immune response, preferably to treat or prevent an infection as defined hereinbefore.
  • an immune response preferably to treat or prevent an infection as defined hereinbefore.
  • Figure 1 shows PCI-based BCG vaccination induced strong PPD-specific CD8 and CD4 and T-cell responses in mice.
  • A-E Groups of 8-10 C57B/6 mice were vaccinated i.d. with 4x10 6 CFU of Pasteur BCG (BCG), with a mixture of BCG and 50 pg TPCS2a (BCG+PCI) or left untreated (llntr). After 18 h, the mice were light treated. Two weeks later, mice were challenged i.d. with 10 pg of PPD, and a DTH reaction in the ears was measured as ear swelling at 0, 24, 48, and 72 hours after the challenge (A).
  • PPD specific T-cell responses were assessed in splenocytes by IFN-y ELISPOT assay for total T cells (B) or by flow cytometry for measurement of IFN-y and CD44 expression in CD4 and CD8 T cells (C).
  • Splenocytes were also cultured ex vivo with PPD and cytokine secretion measured by ELISA (D).
  • Pasteur (Past) BCG, Pasteur BCGAzmpI , and Denmark (DK) BCG were used for vaccination ⁇ PCI as above (E).
  • IFN-y-producing and CD44- expressing CD4 and CD8 T cells were monitored by flow cytometry (E).
  • Figure 2 shows light is indispensable for the adjuvant effect of PCI in M. bovis BCG vaccination.
  • Two weeks after vaccination, splenocytes were assessed for PPD-specific T-cell responses by IFN-y ELISPOT (A), or by intracellular IFN-y staining and flow cytometry of CD44-expressing CD4 and CD8 T cells (B).
  • C-D Comparison of T- cell responses in mice receiving combined BCG and PCI treatment or timely separated BCG and PCI.
  • FIG. 3 shows MHC class-l-restricted epitope-specific CD8 T-cell responses measured after PCI-based BCG::OVA vaccination.
  • Mice were spiked with 1x10 6 naive OT-I cells one day prior to vaccination.
  • A MHC class l-pentamers for IMYNYPAM
  • SIINFEKL SIINFEKL
  • Splenocytes were also re-stimulated ex vivo with each peptide for analysis of specific IFN-y and TNF-a cytokine production in supernatants (D and H). Experiments were repeated three times. The shown data are means and SD (A-C and E-F) or Box and whiskers with 5-95 percentile (D and H).
  • FIG 4 shows comparison of T-cell responses after PCI-based BCG vaccination and i.v. BCG vaccination.
  • FIG. 5 shows heterologous prime-boost vaccination with PPD and BCG and with PCI-facilitated priming.
  • bovis BCG bovis BCG.
  • PPD-specific CD8 and CD4 T-cell responses were analyzed in mouse splenocytes with IFN-y intracellular staining and flow cytometry (B).
  • Data shown means and SDs, and the treatment outcomes were analyzed by one-way ANOVA, correcting for multiple testing, the significance level was set to 95%. The experiments were repeated twice with comparable results.
  • Figure 6 shows PCI treatment increased MHC class l-restricted antigenpresentation in secondary lymphoid organs after BCG vaccination.
  • Two days after vaccination mice were euthanized and spleen, inguinal, mesenteric, and axillary LNs were isolated and single-cell suspension thereof were used as APCs (1 x10 5 ) to present SIINFEKL OT-I cells (2x10 4 ) in co-cultures. After 72 h, IFN-y concentrations in the supernatants were measured with ELISA. Means and SDs are shown.
  • Figure 7 shows PCI treatment induced macrophage activation ex vivo.
  • A Experimental scheme. BCG (1x10 7 CFU/ml) and TPCS2a (0.5 ug/ml) were mixed and co-incubated at 37 °C and for 2 h in PBS. Control samples contained BCG only. After washing thrice in PBS to remove non-bound photosensitizer, BCG- TPCS2a samples or BCG only were added to RAW 264.7 macrophage cultures (MOI 2:1). Negative control samples contained macrophages only. The samples were incubated overnight at 37 °C, washed thrice, and treated with 3 min of light.
  • Figure 8 shows strong inflammation induced directly by BCG-PCI bound macrophages was correlated with optimal antigen presentation in vivo.
  • Groups of five BALB/c mice were immunized with 1x10 6 RAW264.7 macrophages loaded with BCG, or BCG and TPCS2a as described in Figure 7.
  • the cellular vaccines were injected directly into inguinal LNs.
  • Six days later, the mice were euthanized and inflammatory responses were monitored in one inguinal LN from each animal with intracellular staining of TNF-a producing CD11b + cells (A).
  • the second inguinal LNs were analyzed for PPD-specific IFN-y and TNF-a production in CD4 (B) and CD8
  • mice 6-8 weeks of age were purchased from Envigo (Horst, the Netherlands) or Janvier (Genest-Saint-lsle, France) and used age-matched after 1-3 weeks of acclimatization in the animal facility Biêts University Hospital of Zurich.
  • Rag2-deficient transgenic OT-I mice B6.129S6-Rag2' m7Fwa Tg(TcraTcrb)1100Mjb) that recognize the MHC class-l- restricted H-2K b epitope SIINFEKL from ovalbumin (aa257-264) were purchased from Taconic Europe (Ry, Denmark) and further bred in the animal facility.
  • mice All animals were kept under specific pathogen free conditions, in individually ventilated cages, in groups of 4-5 mice, at 21°C, and with a 12h-12h light dark cycle. Only female mice were used, and the experiments took place during daytime and under laminar airflow in biosafety hoods. The animal experiments were reviewed by the local ethical review board, approved by the Veterinary authorities of Cet Zurich (ZH 52/2016 and ZH 170/2019), and performed in accordance with Swiss animal law and regulations.
  • PPD Tuberculin PPD
  • IMYNYPAM Mycobacterium tuberculosis TB10.44-11
  • SIINFEKL Ovalbumin aa257-264
  • TPCS2a The photosensitizer tetraphenyl chlorine disulfonate
  • PBS Phosphate-buffered saline
  • FCS Fetal Calf Serum
  • L- Glutamine L- Glutamine
  • Normocin Normocin
  • Mouse macrophage cell line RAW264.7 (ATCCOTIB-71 TM, BALB/c derived H-2d) were purchased from ATCC (Manassas, VA). Cells were cultured in RPMI-1640 medium supplemented with 10% FCS, 2 mM L-glutamine and 0.1 mg/ml Normocin (RPMI-C medium).
  • M. bovis BCG M. bovis BCG 1721 wild type BCG
  • Pasteur BCGAzmpl with depleted zinc metalloprotease 1 (zmp1) gene Johansen et al., 2011, Clin. Vaccine Immunol., 18, 907-913
  • Denmark M. bovis BCG strain DK BCG::OVA
  • Pasteur BCG::DsRed that contains fluorescent DsRed (provided by Dr Peter Sander).
  • All BCG strains were produced in house and grown in Middlebrook 7H9 broth supplemented with 0.05% Tween-80 and Middlebrook oleic acid-albumin-dextrose- catalase obtained from BD Biosciences (Becton Dickinson, Franklin Lakes, NJ).
  • TPCS2a was mixed with the antigen ⁇ M. bovis BCG or PPD) in PBS, kept light protected, and used for injection within 60 min. Based on initial TPCS2a dose-finding studies, the final dose of TPCS2a was set at 50 pg.
  • a total volume of 100 pl of the vaccine was injected intradermally (i.d.) in the shaved abdominal region using 500 pl syringes with 29G needles.
  • the vaccine was split into two injections 50 pl each to the left and right side of the abdominal midline.
  • the mice were anaesthetized with intraperitoneal injection of ketamine (25mg/kg body weight) and xylazine (4 mg/kg body weight) mixture in PBS.
  • the sedated mice were then placed belly-down on the LumiSource® (PCI Biotech) light source and illuminated with blue light (peak emission at 435 nm) for 6 minutes (4.86 J/cm 2 ) as described previously (Hakerud et al., 2014, J.
  • naive SlINFEKL-specific CD8 T cells freshly prepared from lymph nodes (LNs) and spleen of transgenic OT-I mice were intravenously (/.v.) injected into the recipient C57BL/6 mice one day prior to vaccination with BCG::OVA and 50 pg TPCS2a. The mice were illuminated 18 hours later for 6 minutes.
  • mice were vaccinated with M. bovis BCG by i.v. injection. Briefly, mice were pre-warmed under the red warm lamp for 5 minutes, the local tail veins were disinfected with 70% ethanol, and 4x10 6 CFU of M. bovis BCG in 100 pl PBS were injected into the tail vein using 300 pl syringes with 30G needles.
  • mice with macrophages infected with BCG and treated with PCI ex vivo Vaccination of mice with macrophages infected with BCG and treated with PCI ex vivo
  • RAW264.7 macrophages H-2k d were cultured at 1 xio 6 cells per well in 24-well plates in a volume of 2 ml. The wells were then added 100 pl of 10 pg/ml of TPCS2a and 100 pl 2X10 7 CFU M. bovis BCG and cultured for 18 hours. The culture supernatants were then removed and replaced with fresh supplemented medium and cultured for another 2 hours. After that, the cells were then rinsed three times with cold PBS and collected by scraping into Eppendorf tubes.
  • mice were anaesthetized with ketamine and xylazine as described above, and small skin incisions in the left and right inguinal region were made aseptically. Using 500 pl syringes with 29G needles, 5X10 5 cells in 10 pl was injected directly into each LN. The skin incisions were closed using surgical thread. Delayed-type hypersensitivity test
  • DTH delayed-type hypersensitivity
  • the DTH reaction was monitored by measuring the ear thickness using a spring-loaded Mitutoyo (Kawasaki, Japan) precision digital thickness gauge (provided by Blois- Ruegger Corp., llrdorf, CH).
  • Mitutoyo Know, Japan
  • Precision digital thickness gauge provided by Blois- Ruegger Corp., llrdorf, CH.
  • 10 pl PBS was injected into the right ear, and the ear swelling was calculated by subtracting right-ear values (neg. control) form left-ear values (PPD samples).
  • Intracellular cytokine analysis in spleen and LN cells was performed using the protocols provided by the manufacturer (BD Biosciences). The cells were suspended in RPMI-C medium and re-stimulated with 5 pg/ml PPD overnight, or with 1 pM SIINFEKL or IMYNYPAM peptide at 37 °C, 5% CO 2 for 6 hours; splenocytes were treated with Red Blood Cell Lysis Buffer (Sigma-Aldrich) before incubation. During the last 4 hours, 5 pg/ml Brefeldin A (BFA, Sigma-Aldrich) was added.
  • BFA Red Blood Cell Lysis Buffer
  • the cells were then washed in cold PBS, fixed with Cytofix/CytopermTM solution, and permeabilized with Perm/WashTM solution (BD Biosciences, San Diego, CA).
  • the cells were incubated with anti-CD16/CD32 Ab for 15 minutes to block FcRs then stained with rat anti-mouse CD4 PE, CD8 PerCP-Cy5.5, CD44- FITC, IFN-y APC and TNF-a PE-Cy7 (all Invitrogen) for 45 minutes. All steps were intercepted by washing in cold PBS, and the incubations were performed protected from light and on ice.
  • venous blood was collected and erythrocytes were lysed with Red Blood Cell Lysis Buffer (Sigma-Aldrich) before FcRs blocking with anti-CD16/CD32 Ab.
  • the mouse peripheral blood mononuclear cells (PBMCs) were stained with PE-conjugated H- 2K b /Pro5 peptide-specific pentamers (Proimmune) for 15 minutes 37 °C and then with anti-CD8-PerCP-Cy5.5 and anti-CD44-FITC for 45 minutes.
  • Antigen-specific IFN-y production was also analyzed by mouse IFN-y ELISPOT assay according to the manufacturer’s protocol (eBioscience). Briefly, multiscreen 96-well PVDF plates (Millipore, Wohlen, Switzerland) were coated with 1 pg/ml antimouse IFN-y antibody overnight at 4 °C. The plates were then blocked with RPMI-C medium before freshly prepared splenocytes were seeded at 2x10 5 cells per well) and re-stimulated with indicated antigens (5 pg/ml of PPD, 1 pg/ml IMYNYPAM or SIINFEKL peptides) at 37 °C, 5 % CO2, and incubated for 18 hours. The ELISPOT plates were developed using the provided protocol and spots were read with AID EliSpot Reader System (Autoimmun Diagnostika, Strassberg, DE).
  • ELISA was performed to analyze cytokine secretion in supernatants of splenocytes after ex vivo antigen re-stimulation. Briefly, 5x10 5 splenocytes were re-stimulated in round-bottom 96-well plates with indicated antigens (5 pg/ml of PPD, 0.1 pg/ml IMYNYPAM or SIINFEKL peptides, respectively) and cultured at 37° C, 5% CO2.
  • the cell culture supernatants were collected at different time points and analyzed for secretion of IL-2, TNF-a (24 h), IFN-y and IL-17A (72 h) using Ready-Set-GO® ELISA Kits as described by the manufacturer (eBioscience).
  • the suspensions of BCG-TPCS2a or BCG was then added to RAW264.7 macrophages (1x10 6 BCG to 5x10 5 macrophages per ml) and incubated in RPMI-C medium in 6-well cell culture plates at 37 °C, 5% CO2 and protected from light. After overnight incubation, the RAW cells loaded with BCG-TPCS2a or BCG were collected, washed 3 times with PBS and illuminated with LumiSource® light source for 3 minutes. Afterwards, the BCG-PCI or BCG-loaded RAW264.7 macrophages were used freshly for testing macrophage activation ex vivo and for intralymphatic injection (see below and Results).
  • ICS intracellular cytokine staining
  • 5 pg/ml BFA was added to the RAW-BCG ⁇ PCI cell cultures, then cells were incubated for 4 hours at 37°C.
  • macrophages were collected, washed, fixed and permeabilized as described above for intracellular inflammatory cytokine staining thereafter.
  • the samples were acquired using FACSCanto flow cytometer (BD Biosciences) and results were analyzed using FlowJo v.10 software (Tree Star). Live macrophages were first gated based on forward and side scatter properties. Inflammatory macrophages were defined as the TNF-a or Pro I L-1 p positive cell population from total CD11b positive cells.
  • mice Six days after intralymphatic injection with RAW macrophages loaded either with BCG-PCI or BCG alone or untreated RAW264.7 cells (see above), mice were euthanized and the injected inguinal LNs were harvested. Intracellular TNF-a producing cells in CD11b positive population was measured with flow cytometry. Specific T cell responses in the inguinal LNs were also assessed with IFN-y ICS as described above.
  • C57BL/6 mice were injected intradermally in the belly region with BCG with or without TPCS2a as described above. After light treatment on day 1, skin samples were collected on days 2 and 8 post BCG administration, fixed in 4% formalin in PBS overnight, dehydrated, and embedded in paraffin. Sections of 3 pm were cut on a microtome and the sections stained with hematoxylin and eosin (H&E). Additional sections were subjected to immunohistochemistry for identification of CD11b-positive myeloid cells (monocytes, macrophages, neutrophils and granulocytes), CD11c-positive DCs, CD68-positive macrophages, as well as CD4- and CD8-positive T cells (Sophistolab AG, Muttenz, Switzerland).
  • CD11b-positive myeloid cells monocytes, macrophages, neutrophils and granulocytes
  • CD11c-positive DCs CD68-positive macrophages
  • CD4- and CD8-positive T cells Sophisto
  • the sections were evaluated by a board certified dermatopathologist, scanned on Aperio ScanScope CS, and the images prepared using Aperio ImageScope software v11.2.0.780. Inflammatory infiltrates in different regions were manually counted from the representative sections by two independent researchers and by using QuPath software (Bankhead et al., 2017, Scientific Reports, 7, 16878).
  • mice were vaccinated i.d. with a mixture of M. bovis BCG and photosensitizer TPCS2a and treated with light after 18 hours.
  • a cutaneous DTH reaction to mycobacterial proteins (PPD) was determined as a measure for cellular immunity.
  • PPD-specific ear swelling was observed 24-72 hours post challenge in all mice BCG-vaccinated mice (Fig. 1A).
  • At the peak of the DTH reaction (48 h) approx. 100 pm swelling was measured in BCG-vaccinated mice, while mice that also received PCI treatment showed a swelling of approx. 225 pm, suggesting that PCI increased the immunogenicity of BCG (p ⁇ 0.0001 by 2-way ANOVA).
  • mice No ear swelling was observed in nonvaccinated mice or after challenge with PBS.
  • the mice were then euthanized and the splenocytes re-stimulated ex vivo with PPD.
  • ELISPOT revealed that the frequency of PPD-specific IFN-y-producing T cells was increased after combined BCG vaccination and PCI treatment as compared to BCG vaccination alone (Fig.
  • the phenotype of T-cell responses was further characterized by ELISA in cultures of splenocytes re-stimulated ex vivo with PPD.
  • the secretion of IL-2, TNF- a, IFN-y, IL-17A, and IL-10 was strongly elevated in BCG-vaccinated mice as compared to non-vaccinated mice, and PCI additionally and significantly triggered the release of all cytokines but IL-10 (Fig. 1D).
  • the beneficial effect of PCI on T-cell responses upon M. bovis BCG vaccination was confirmed using three different strains of BCG (Fig. 1E), e.g. Pasteur M. bovis BCG (Past. BCG), Denmark M.
  • bovis BCG DK BCG
  • Pasteur M. bovis BCG lacking a Zink metalloprotease gene (Past. BCGAzmpI) (Johansen et al., 2011, supra). Liqht-activation of the photosensitizer was indispensable for the effect of PCI on BCG vaccination
  • Illumination is expected to play a key role in PCI-mediated delivery of antigens from the phagosomal compartment into the cytosol, where the antigen can be loaded on MHC class I molecules for subsequent cross presentation to T cells.
  • C57BL/6 mice were vaccinated with BCG and TPCS2a. Half of all animals received light treatment after 18 hours, while the other half was not treated with light. Mice vaccinated with BCG alone or left untreated were included as controls, but also received light treatment as for the PCI-treated group. Two weeks later, PPD specific T-cell activation was detected in all BCG-vaccinated mice (Figs. 2A-B).
  • mice were photochemically treated prior to the BCG vaccination: TPCS2a on day 0, light on day 1 , BCG on day 2.
  • Another group was vaccinated with the mixture of M. bovis BCG and TPCS2a on day 0 and received light treatment on day 1 as normal (Fig. 2C).
  • Inflammation is important as it allows for immune response directed against the antigen delivered in the vaccine, which protects from the development of the disease.
  • Local skin inflammation was detected in mice that received the BCG vaccine and consecutive PCI treatment, observed as inflammatory infiltrate (composed of neutrophils, eosinophils and histiocytes), thickening of epidermis (acanthosis) and edema. Additionally, activation of a photosensitizer by illumination resulted in ulceration, necrosis of epidermis and compact hyperkeratosis. Formation of fibrotic scar tissue was observed eight days after the BCG+PCI vaccination (data not shown).
  • CD8 T-cell responses are expected to be important for effective prevention or treatment of TB infection.
  • the intrinsic mycobacterial H-2k b MHC class-l-binding epitope IMYNYPAM was used.
  • the Denmark M. bovis BCG strain was genetically modified to express the H-2k b MHC class-l-binding epitope SIINFEKL from ovalbumin (provided by Dr Peter Sander).
  • lymphocytes from naive OT-I transgenic mice were adoptively transferred into recipient C57BL/6 mice one day prior to vaccination.
  • IMYNYPAM- Fig. 3A
  • SIINFEKL- Fig. 3E
  • IMYNYPAM- Fig. 3A
  • SIINFEKL- Fig. 3E
  • splenocyte analysis ex vivo also demonstrated more robust IMYNYPAM- and SHNFEKL-specific CD8 T- cell responses after PCI treatment and as measured by IFN-y producing CD8 T cells by ELISPOT (Figs. 3B and 3F) and intracellular cytokine staining and flow cytometry (Figs.
  • mice vaccinated with BCG and PCI secreted significantly more IFN-y and TNF-a cytokines into cultures of cells re-stimulated with IMYNYPAM or SIINFEKL (Figs. 3D and 3H).
  • i.v. BCG vaccination was proposed to improve efficacy of BCG vaccination (Dockrell and Butkeviciute, 2021, in press, Vaccine). Therefore, we compared T-cell responses induced by PCI-based and i.v. BCG vaccination.
  • the frequency of IMYNYPAM-specific CD8 T cells in blood of mice was significantly higher (p ⁇ 0.0001) higher after PCI-based BCG vaccination than after i.v. BCG administration, and the response was more long lasting (Fig. 4A).
  • Fig. 4A Nine weeks post vaccination, significantly higher frequency of IMYNYPAM-specific IFN-y producing CD8 T cells were detected in splenocytes (Fig.
  • PPD is a mixture of intracellular bacterial proteins obtained from filtrates of M. tuberculosis cultures, is used in TB diagnosis, but is weakly immunogenic in mice.
  • mice were injected i.d. with 10 or 100 pg PPD, combined with PCI treatment (50 pg of TPCS2a and 6 min light). Seven days later, PPD-specific IFN-y secretion was assessed in splenocytes by flow cytometry. As shown in Figure 5A, very low numbers of IFN-y producing CD4 and CD8 T cells were detected in mice that received 10 pg PPD ⁇ TPCS2a and in mice that received 100 pg PPD only.
  • mice that received 100 pg PPD with PCI showed slightly stronger CD8 and CD4 T-cell responses.
  • a small inoculum of M. bovis BCG (1x10 6 CFU) was injected intradermally in all PPD-primed mice.
  • BCG vaccination resulted in PPD-specific IFN-y production in CD4 and CD8 T cells of all tested mice (Fig. 5B).
  • T-cell responses were determined in mice primed with 100 pg PPD combined with PCI than in mice primed with 100 pg PPD only.
  • PCI-based BCG vaccination improved antigen presentation
  • mice were vaccinated with BCG::OVA and PCI or with BCG::OVA only. Two days later, LNs and spleens were isolated and used as MHC class I antigen-bearing APCs and co-cultured with naive H-2b OT-I CD8 T cells. Mesenteric and axillary LN cells from vaccinated mice did not trigger IFN-y production in the OT-I cultures (Fig. 6). However, IFN-y could be determined in supernatants of OT-I cells cultured with inguinal LN cells or with splenocytes from mice treated with BCG.
  • PCI further enhanced cross-presentation to CD8 T cells.
  • antigen cross-presentation may have been initiated in inguinal LNs and spleens.
  • PCI treatment elicited the activation of BCG-loaded macrophages ex vivo
  • M. bovis BCG was incubated with TPCS2a for two hours to allow binding of photosensitizer on BCG. After removal of free TPCS2a, the BCG- TPCS2a combination or BCG alone was incubated with RAW264.7 macrophages overnight to allow BCG uptake (Fig. 7A). The macrophage cultures were harvested, washed to remove any free BCG, and illuminated to trigger the activation of BCG- associated TPCS2a. The cells were split in two to (i) test the activation status and the secretion of pro-inflammatory cytokines after further 24 hours culturing or (ii) to use for intralymphatic immunization (see below).
  • TNF-a and pro-IL-1 p secreted by BCG-infected and PCI-treated RAW264.7 cells were analyzed by flow cytometry (Fig. 7B). Compared to untreated RAW264.7 cells, significantly higher frequencies of cytokine-producing cells were measured in BCG-infected RAW264.7 macrophages. Approx. 10% of all cells produced TNF-a and 5% produced pro I L-1 p. The corresponding frequencies for BCG-infected and PCI-treated macrophages were approx. 20% and 10%.
  • BCG- and PCI-treated RAW264.7 macrophages were injected directly into the inguinal LNs of syngeneic BALB/c mice. Six days later, the mice were euthanized and the inguinal LNs were removed and analyzed by flow cytometry. Interestingly, TNF-a-producing CD11b-positive cells were detected in all LN preparations, the strongest response observed in animals immunized with BCG- and PCI-treated RAW264.7 macrophages (Fig. 8A).
  • the macrophages treated with PCI ex vivo also induced strongest local inflammation in vivo.
  • the activation of CD8 (Fig. 8B) and CD4 (Fig. 8C) T cells in the injected LNs was determined. While BCG-infected macrophages only induced IFN-y production in inguinal CD8 T cells, PCI-treated and BCG-infected macrophages triggered both IFN-y- and TNF-a-production in inguinal CD8 T cells.
  • the analysis of CD4 cells from mice immunized with macrophages revealed the beneficial effect of PCI for IFN-y production but not for TNF-a- production.
  • results show that a live microbial vaccine can be combined with a photochemical compound and light for cross presentation of antigens to CD8 T cells.
  • results reveal that PCI treatment strongly improves the antigenspecific T-cell responses to M. bovis BCG. Both enhanced CD4 and CD8 T-cell responses were observed. It is believed that light activation of TPCS2a resulted in partial antigen release into the cytosol which accessed the MHC I pathway of antigen presentation to stimulate CD8 T cells, while non-released antigen remain in the phagolysosomes for subsequent presentation via MHC class II to achieve CD4 T cell stimulation. This relocation from default MHC class II to MHC class I antigen presentation was strictly dependent on light.
  • BCG-specific IFN-y-secreting cells could be measured, by ELISPOT, ELISA, and by flow cytometry, and the absolute number as well as frequency of such cells were significantly increasing in mice that received PCI treatment with the vaccination.
  • the T-cell responses induced by PCI-mediated BCG vaccination was typically multifunctional, the T cells expressing IFN-y, TNF-a, IL-2, and IL-17. The latter results aligns well with published reports suggesting that Th17 cells, which naturally traffic to the airways, can accelerate the recruitment of protective Th1 cells in pulmonary TB (Khader et al., 2007, Nat.
  • IFN-y is a key effector cytokine in the control of Mtb infection
  • IL-2 is a T-cell growth factor that also assures long-term survival of lymphocytes
  • IFN-y-producing cytotoxic CD8 T cells have also been proven vital in the elimination of intracellular bacterial infections (Wong and Pamer, 2003, Ann. Rev. Immunol., 21, p29-70) including Mtb (van Pinxteren et al., 2000, Eur. J. Immunol., 30, p3689-3698; Chen et al., 2009, PLOS Pathogens, 5, e1000392; and Axelsson-Robertson et al., 2015, Int. J. Infect. Dis., 32, p23-29), for which reason several Mtb vaccine developments aimed at stimulating also antigenspecific CD8 T cell (Behar et al., 2007, Expert Review of Vaccines, 6, p441-456;
  • BCG vaccines have been suggested to be important for vaccine efficacy (Waeckerle-Men et al., 2013, Vaccine, 31 , p1057-1064; and Moliva et al., 2015, Vaccine, 33, p5035-5041).
  • intravenous (/.v.) administration of BCG to non-human primates prevented TB infection (Darrah et al., 2020, supra).
  • Antigen-responsive CD4 and CD8 T-cell responses in secondary lymphoid organs and in lung tissues were substantially higher after i.v. BCG vaccination, compared to vaccination via the intradermal route or aerosol delivery.

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Abstract

La présente invention concerne une méthode de production d'une réponse immunitaire à des bactéries chez un sujet, consistant à administrer des bactéries vivantes (telles que le BCG) et un agent photosensibilisant à des cellules du sujet et à irradier les cellules avec une lumière d'une longueur d'onde efficace pour activer l'agent photosensibilisant en vue de générer une réponse immunitaire aux bactéries. L'invention concerne également des compositions pharmaceutiques, des produits et des kits à cet effet.
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