The present invention relates to a method of vaccination or immunisation involving the use of a photosensitizing agent, an antigenic molecule, e.g. a vaccine component, and an agent which enhances the effect of photochemical

internalization (PCI) - mediated vaccination, wherein the agent is a virosome or a virus-like particle (VLP) as defined herein which comprises said antigenic molecule, and irradiation with light of a wavelength effective to activate the photosensitizing agent. The invention also relates to antigenic, e.g. vaccine compositions, useful in such a method. The invention also provides a method of generating antigen presenting cells which may be used to generate an immune response, e.g. for vaccination, which involves using the same components as above to introduce antigenic molecules, e.g. vaccine components, into cells to achieve antigen presentation, and to antigenic compositions useful in such a method. The invention also provides use of cells generated in vitro by such methods for administration to a patient in vivo to elicit an immune response, e.g. to achieve vaccination. A method of internalising an antigenic molecule into a cell is also provided.

Vaccination involves administration of antigenic molecules to provoke the immune system to stimulate development of an adaptive immunity to a pathogen. Vaccines can prevent or improve morbidity from infection. Vaccination is the most effective method of preventing infectious diseases, and widespread immunity due to vaccination is largely responsible for the worldwide eradication of smallpox and the restriction of diseases such as polio, measles, and tetanus from much of the world.

The active agent of a vaccine may be intact but inactivated (non-infective) or attenuated (with reduced infectivity) forms of the causative pathogens, or purified components of the pathogen that have been found to be immunogenic (e.g., outer coat proteins of a virus). Toxoids are produced for immunization against toxin- based diseases, such as the modification of tetanospasmin toxin of tetanus to remove its toxic effect but retain its immunogenic effect.

Since most vaccines are taken up by antigen presenting cells through endocytosis and transported via endosomes to lysosomes for antigen digestion and presentation via the MHC class-ll pathway, vaccination primarily activates CD4 T- helper cells and B cells. To combat disorders or diseases such as cancer, as well as intracellular infections, the stimulation of cytotoxic CD8 T-cell responses is important. However, the induction of cytotoxic CD8 T cells usually fails due to the difficulty in delivering antigen to the cytosol and to the MHC class-l pathway of antigen presentation. Photochemical internalisation (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.

The basic method of photochemical internalisation (PCI), is described in WO 96/07432 and WO 00/54802, which are incorporated herein by reference. In such methods, the molecule to be internalised (which in the present invention would be the antigenic molecule), and 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). On exposure of the cell to light of the appropriate wavelength, 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.

It was found that in such a method the functionality or the viability of the majority of the cells was not deleteriously affected. Thus, the utility of such a method, termed "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. Thus, following transport and release of 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. Such a method has particular utility in the field of vaccination, where vaccine components i.e. antigens or immunogens, may be introduced to a cell for presentation on the surface of that cell, in order to induce, facilitate or augment an immune response.

Whilst vaccination has achieved some noteworthy successes, there remains a need for alternative and improved vaccination methods. The present invention addresses this need. The present inventors have surprisingly found that, advantageously, a method involving the use of a photosensitizing agent and a virosome or a virus-like particle (VLP) as defined herein which comprises an antigenic molecule, e.g. a vaccine component, and irradiation with light of a wavelength effective to activate the photosensitizing agent results in improved vaccination or an improved immune response.

As will be described in more detail in the Examples below, it has been demonstrated that the method of the invention provides improved vaccination or an improved immune response, e.g. production of an increased amount of antigen- specific T cells. It is expected that synergistic effects are achieved.

Whilst not wishing to be bound by theory, it is believed that the methods of the invention result in increased antigen presentation on MHC Class I molecules leading to an increased CD8+ T cell responses and hence improved vaccination methods. As disclosed in the present Examples, a model system employing OT-1 cells can be used for assessing MHC class I presentation (see e.g. Delamarre et al. J. Exp. Med. 198:1 1 1 -122, 2003). In this model system MHC class I presentation of the antigen SIINFEKL leads to activation of the OT-1 T-cells, and the activation can be measured as an increase in proliferation of the antigen-specific T-cells or increased production of IFNy or IL-2.

Thus, in a first aspect the present invention provides a method of expressing an antigenic molecule or a part thereof on the surface of a cell, comprising contacting said cell with a photosensitizing agent, and a virosome or a virus-like particle (VLP) as defined herein which comprises said antigenic molecule, and irradiating the cell with light of a wavelength effective to activate the

photosensitising agent, wherein said antigenic molecule is released into the cytosol of the cell and the antigenic molecule or a part thereof is subsequently presented on the cell's surface.

Preferably this method (and subsequently described methods) employ only the above described two active ingredients (agents) in said method 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). Thus preferably the agents are present in buffers with no other active ingredients. In such methods said photosensitizing agent, and said virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule, are each taken up into an intracellular vesicle; and when the cell is irradiated the membrane of the intracellular vesicle is disrupted releasing the antigenic molecule into the cytosol of the cell.

The agents 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, e.g. an endosome or lysosome.

As used herein, 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 antigenic molecule contained within it.

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. Conveniently such a photosensitizing agent may be one which localises to intracellular compartments, particularly endosomes or lysosomes.

Photosensitisers may exert their effects by a variety of mechanisms, directly or indirectly. Thus for example, certain photosensitisers 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.

A range of such 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 method of the invention. There are many known photosensitising agents, including porphyrins, phthalocyanines, purpurins, chlorins, benzoporphyrins, lysomotropic weak bases, naphthalocyanines, cationic dyes and tetracyclines or derivatives thereof (Berg et al., (1997), J. Photochemistry and Photobiology, 65, 403-409). Other photosensitising agents include texaphyrins, pheophorbides, porphycenes, bacteriochlorins, ketochlorins, hematoporphyrin derivatives, and endogenous photosensitizers induced by 5-aminolevulinic acid and derivatives thereof, Photofrin, dimers or other conjugates between photosensitizers. Porphyrins are the most extensively studied photosensitising 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 skilled man will appreciate which photosensitisers are suitable for use in the present invention. Particularly preferred are photosensitizing agents which locate to endosome or lysosomes of cells. Thus, the photosensitizing agent is preferably an agent which is taken up into the internal compartments of lysosomes or endosomes. Preferably the photosensitizing agent is taken up into intracellular compartments by endocytosis. Preferred photosensitisers are di- and

tetrasulfonated aluminium phthalocyanine (e.g. AIPcS _2a ), sulfonated

tetraphenylporphines (TPPS _n ), sulfonated tetraphenyl bacteriochlorins (e.g.

TPBS _2a ), nile blue, chlorin e ₆ derivatives, uroporphyrin I, phylloerythrin,

hematoporphyrin and methylene blue. Further appropriate photosensitizers for use in the invention are described in WO03/020309, which is also incorporated herein by reference, namely sulfonated meso-tetraphenyl chlorins, preferably TPCS _2a . Preferred photosensitizing agents are amphiphilic photosensitizers (e.g.

disulfonated phosphosensitizers) such as amphiphilic phthalocyanines, porphyrins chlorins and/or bacteriochlorins, and in particular include TPPS _2a

(tetraphenylporphine disulfonate), AIPcS _2a (aluminium phthalocyanine disulfonate), TPCS _2a (tetraphenyl chlorin disulfonate) and TPBS _2a (tetraphenyl bacteriochlorin disulfonate), or pharmaceutically acceptable salts thereof. Also preferred are hydrophilic photosensitizing agents, for example TPPS ₄ (meso-tetraphenylporphine tetrasulfonate). Particularly preferred photosensitizing agents are sulfonated aluminium phthalocyanines, sulfonated tetraphenylporphines, sulfonated tetraphenylchlorins and sulfonated tetraphenylbacteriochlorins, preferably TPCS _2a, AIPcS _2a, TPPS ₄ and TPBS _2a . In a particularly preferred embodiment of the present invention the photosensitizing agent is the chlorin TPCS _2a (Disulfonated tetraphenyl chlorin, e.g. Amphinex ®).

A photosensitiser may be linked to a carrier to provide the photosensitising agent. Thus, in a preferred aspect of this embodiment of the invention the photosensitising agent is a conjugate of a photosensitiser and chitosan as defined in formula (I):

wherein n is an integer greater than or equal to 3;

R appears n times in said compound, and in 0.1 %-99.9% (preferably 0.5%-99.5%) of said total Rn groups, each R is a group A selected from: wherein each R-i, which may be the same or different, is selected from H, CH ₃ and -(CH ₂ ) _b -CH ₃ ; a is 1 , 2, 3, 4 or 5; and b is 0, 1 , 2, 3, 4 or 5 (in which the counter-ion may be, for example, CI ^" ); preferably Ri, is CH ₃ and b is 1 _, and

wherein Y is O; S; S0 _2; -NCH _3; or -N(CH ₂ )dCH _3; c=1 , 2, 3, 4 or 5; and d=1 , 2, 3, 4 or 5, preferably Y is NCH ₃ and c is 1 , wherein each R group may be the same or different, and in 0.1 %-99.9% (preferably 0.5%-99.5%) of said total Rn groups, each R is a group B selected from:

wherein

e is 0, 1 , 2, 3, 4 or 5; and f is 1 , 2, 3, 4 or 5; preferably e and f =1 ,

R ₂ is a group selected from:

W is a group selected from O, S, NH or N(CH ₃ ); preferably NH,

R ₃ is a group selected from:

and

V is a group selected from CO, S0 ₂ , PO, P0 ₂ H or CH ₂ ; preferably CO, and

R ₄ is a group (substituted in the o, m or p position), which may be the same or different, selected from H, -OH, -OCH ₃ , -CH ₃ , -COCH3, C(CH ₃ ) ₄ , -NH ₂ , -NHCH3, -N(CH ₃ ) ₂ and -NCOCH3, preferably H, wherein each R group may be the same or different.

The chitosan polymer has at least 3 units (n=3). However, preferably n is at least 10, 20, 50, 100, 500, 1000 e.g. from 10 to 100 or 10 to 50.

In a preferred embodiment R ₂ is selected from

TPCa ₂ In a fu

TPCc ₂

Preferably R ₂ or R ₃ is TPP _a , TPC _ai or TPC _c i .

Group A may provide 70 to 95% of the total Rn groups and group B may provide 5 to 30% of the total Rn groups.

In a most preferred embodiment the conjugate of a photosensitiser and chitosan is selected from (see numbering in Schemes 1 -5B in Figure 4):

19: B:25%, A:75%

In the above structures, the A/B % values provided refer to the proportion of Rn groups which are group A or B. The asterisks denote the remainder of the chitosan polymer.

These compounds may be made by synthesis methods which utilise procedures standard in the art, which will be familiar to the skilled man. By way of example, synthesis of the preferred conjugates discussed below, numbers 17, 19, 33 and 37, is shown in reaction schemes 1 -5B in Figure 1 (and see also Figure 1 legend).

An "antigenic" molecule as referred to herein is a molecule which itself, or a part thereof, is capable of stimulating an immune response, when presented to the immune system or immune cells in an appropriate manner. Advantageously, therefore the antigenic molecule will be a vaccine antigen or vaccine component, such as a polypeptide containing entity.

Many such antigens or antigenic vaccine components are known in the art and include all manner of bacterial or viral antigens or indeed antigens or antigenic components of any pathogenic species including protozoa or higher organisms. Whilst traditionally the antigenic components of vaccines have comprised whole organisms (whether live, dead or attenuated) i.e. whole cell vaccines, in addition sub-unit vaccines, i.e. vaccines based on particular antigenic components of organisms e.g. proteins or peptides, or even carbohydrates, have been widely investigated and reported in the literature. Any such "sub-unit"-based vaccine component may be used as the antigenic molecule of the present invention.

However, the invention finds particular utility in the field of peptide vaccines. Thus, a preferred antigenic molecule according to the invention is a peptide (which is defined herein to include peptides of both shorter and longer lengths i.e.

peptides, oligopeptides or polypeptides, and also protein molecules or fragments thereof e.g. peptides of 5-500 e.g. 10 to 250 such as 15 to 75, or 8 to 25 amino acids).

A vast number of peptide vaccine candidates have been proposed in the literature, for example in the treatment of viral diseases and infections such as AIDS/ HIV infection or influenza, canine parvovirus, bovine leukaemia virus, hepatitis, etc. (see e.g. Phanuphak et al., Asian Pac. J. Allergy. Immunol. 1997, 15(1 ), 41 -8; Naruse, Hokkaido Igaku Zasshi 1994, 69(4), 81 1 -20; Casal et al., J. Virol., 1995, 69(1 1 ), 7274-7; Belyakov et al., Proc. Natl. Acad. Sci. USA, 1998, 95(4), 1709-14; Naruse et al., Proc. Natl. Sci. USA, 1994 91 (20), 9588-92; Kabeya et ai, Vaccine 1996, 14(12), 1 1 18-22; Itoh et al., Proc. Natl. Acad. Sci. USA, 1986, 83(23) 9174-8. Similarly bacterial peptides may be used, as indeed may peptide antigens derived from other organisms or species.

In addition to antigens derived from pathogenic organisms, peptides have also been proposed for use as vaccines against cancer or other diseases such as multiple sclerosis. For example, mutant oncogene peptides hold great promise as cancer vaccines acting as antigens in the stimulation of cytotoxic T-lymphocytes. (Schirrmacher, Journal of Cancer Research and Clinical Oncology 1995, 121 , 443- 451 ; Curtis Cancer Chemotherapy and Biological Response Modifiers, 1997, 17, 316-327). Thus a melanoma antigen may be used as the antigenic molecule of the invention. In alternative embodiments, an antigenic molecule which is not a melanoma antigen may be used. A "melanoma antigen" as referred to herein is a molecule derived from a melanoma cell which itself, or a part thereof, is capable of stimulating an immune response, when presented to the immune system or immune cells in an appropriate manner. A molecule "derived" from a melanoma is a molecule which may appear in the melanoma cell or which is modified relative to the native molecule in the melanoma, e.g. by truncation, post-expression modification and/or sequence modification providing the modified molecule retains one or more epitopes from the native molecule which allows the modified molecule to generate an immune response which would recognise the native molecule. The melanoma antigen may be obtained by isolation from appropriate sources e.g. the subject's melanoma or may be synthesised e.g. by chemical synthesis or peptide/protein expression.

A synthetic peptide vaccine has also been evaluated for the treatment of metastatic melanoma (Rosenberg et al., Nat. Med. 1998, 4(3), 321 -7). A T-cell receptor peptide vaccine for the treatment of multiple sclerosis is described in Wilson et al., J. Neuroimmunol. 1997, 76(1 -2), 15-28. Any such peptide vaccine component may be used as the antigenic molecule of the invention, as indeed may any of the peptides described or proposed as peptide vaccines in the literature. The peptide may thus be synthetic or isolated or otherwise derived from an organism.

Once released in the cell cytosol by the photochemical internalisation process, the antigenic molecule may be processed by the antigen-processing machinery of the cell. Thus, the antigenic molecule expressed or presented on the surface of the cell may be a part or fragment of the antigenic molecule which is internalised (endocytosed). A "part" of an antigenic molecule which is presented or expressed preferably comprises a part which is generated by antigen-processing machinery within the cell. Parts may, however, be generated by other means which may be achieved through appropriate antigen design (e.g. pH sensitive bonds) or through other cell processing means. Conveniently such parts are of sufficient size to generate an immune response, e.g. in the case of peptides greater than 5, e.g. greater than 10 or 20 amino acids in size.

As discussed herein, the agent which enhances PCI-mediated vaccination is a virosome or a virus-like particle (VLP) which comprises said antigenic molecule. Thus the agent and the antigenic molecule together form the virosome or VLP.

Thus, in one embodiment the agent which comprises the antigenic molecule is a virosome. As referred to herein a "virosome" is a reconstituted empty virus envelope (preferably from influenza, but optionally from other viruses such as Sendai virus, Semliki Forest virus, vesicular stomatitis virus and Sindbis virus), which is devoid of the nucleocapsid including the genetic material of the source virus. The structure of a virosome is based on a liposome, i.e. they are tiny spherical vesicles with a typical mean diameter of 150 nm. They have a unilamellar phospholipid membrane (either a mono- or bi-layer) incorporating virus-derived proteins to allow the virosomes to fuse with target cells and deliver their contents to the cell. Virosomes contain the functional viral envelope glycoproteins such as influenza virus hemagglutinin (HA) and neuraminidase (NA) intercalated in the phospholipid bilayer membrane. Neuraminidase is an enzyme involved in the release of progeny virus from infected cells, by cleaving sugars that bind the mature viral particles. Hemagglutinin is a lectin that mediates binding of the virus to target cells and entry of the viral genome into the target cell. The HA and NA proteins provide the virosomes with properties which facilitate efficient vesicle uptake by and subsequent activation of cells of the immune system. HA confers structural stability and homogeneity to virosome-based formulations. The remainder of the virosome is made up of a mixture of synthetic phospholipids (that may be added during the reconstitution process) and natural phospholipids.

Virosomes carry antigenic molecules from the virus from which they are generated e.g. HA and NA. However, virosomes are versatile in that proteins or peptides derived from pathogens other than the source virus (e.g. influenza) can be incorporated into the vesicles for delivery to cells. Virosomes are not able to replicate but are pure fusion-active vesicles. Physical association of the virosome and antigen of interest can be achieved by a variety of methods, depending on the properties of the antigen. Antigens can be incorporated into virosomes, adsorbed to the virosome surface, or integrated into the lipid membrane, either via hydrophobic domains or lipid moieties cross-linked to the antigen. As noted above, the virosomes (or virus-like particles) comprise the antigenic molecule (also referred to as antigens herein). This extends to association by any means. As referred to herein said "association" includes incorporation, adsorption, integration, conjugation or binding as described herein. As discussed hereinafter, the virosome or VLP may additionally comprises the photosensitising agent. In such instances, as with the antigenic molecule, this extends to association by any means.

Virosomes are used in commercial influenza and hepatitis vaccines, for example as available from Crucell (Leiden, The Netherlands) e.g. the lnflexal®V, Epaxal® and Hepavax-Gene® vaccines from Crucell. These can also be utilized according to the methods of the present invention depending on the intended treatment, e.g. to vaccinate against influenza or hepatitis. Virosomes can be manufactured or produced according to methods known in the art, see for example Glijck and Metcalfe (Vaccine, 2003; 21 :61 1 -615) and Cusi et al. (Human Vaccines, 2006; 2(1 ): 1 -7). Commercially available virosomes as discussed herein may be used in the present invention.

Preferably an influenza virus, or its component parts, such as its envelope glycoproteins, and/or capsid proteins, is used to prepare the virosome to yield an influenza virosome.

Influenza viruses belong to the Orthomyxoviruses family of RNA viruses. The Orthomyxoviruses family comprises six genera: Influenzavirus A, Influenzavirus B, Influenzavirus C, Isavirus, Thogotovirus and a presently undescribed genus. By way of example, the influenza A virus particle or virion is 80-120 nm in diameter and usually roughly spherical, although filamentous forms can occur. The total genome length is 12000-15000 nucleotides. The influenza A genome contains eight pieces of segmented negative-sense RNA which encode 1 1 proteins (HA, NA, NP, M1 , M2, NS1 , NEP, PA, PB1 , PB1 -F2, PB2). The best-characterized of these viral proteins are hemagglutinin and neuraminidase, as employed in the production of virosomes. The virosome according to the present invention may comprise any of these proteins, or fragments thereof, preferably as the antigenic molecule.

As discussed above, virosomes can comprise proteins or peptides, i.e. antigens, from a wide variety of pathogens, for example, from a virus, bacterium, prion, parasite, fungus or diseased cell, particularly antigenic molecules as defined herein. The antigenic molecule may be derived from the virus used to generate the virosome or may be added to the virosome during its production, or a combination of these two options. If the antigenic molecule is viral in origin it may be from the virus from which the virosome has been produced or may be added to the virosome during its production.

Thus in one embodiment of the present invention the virosome comprises one or more proteins or peptides from a virus, preferably an influenza virus, preferably as the antigenic molecule. Conveniently this is achieved by using such a virus to derive the virosome, i.e. the virosome is prepared from that virus.

In an alternative, preferred embodiment, the virosome comprises one or more protein or peptide (antigen) which have been added during the production of to the virosome, i.e. are foreign antigens relative to the virus used to make the virosome.

The antigen may be derived from numerous sources as discussed herein, for example from pathogens, parasites or from diseased cells, for example tumour or cancer antigens, antigens from viruses, including hepatitis, HIV and HPV, antigens from bacteria such as Tuberculosis bacteria, antigens from parasites such as malarial antigens, and antigens from other intracellular bacteria or parasites. In a particularly preferred embodiment the antigen is from an influenza or hepatitis virus or a cancer cell.

The viral antigen may be from one of the following: Adenoviridae:

Adenoviruses; Arenaviruses: Guanarito virus, Junin virus, Lassa fever virus, Lujo virus, Machupo virus and Sabia virus; Bunyaviruses: Crimean-Congo hemorrhagic fever virus, Dobrava virus, Hantaan virus, Puumala virus, Rift Valley fever virus, Seoul virus and SFTS virus; Coronaviridae: Severe acute respiratory syndrome virus; Erythrovirus: Parvovirus B19; Flaviviruses: Akhurma virus, Dengue, Hepatitis C, Kyasanur Forest disease virus, Omsk hemorrhagic fever virus, Yellow fever; Filoviruses: Ebola virus and Marburg virus; Hepadnaviridae: Hepatitis B;

Hepeviridae: Hepatitis E; Herpesviruses: Cytomegalovirus, Epstein Barr virus, Varicella zoster virus, Human herpesvirus 6, Human herpesvirus 7 and Human herpesvirus 8; Orthomyxoviruses: Influenza; Parvoviridae: Parvovirus B19;

Picornaviruses: Echovirus, Hepatitis A; Reovirus: Colorado tick fever virus, Reovirus 3; or Hepatitis D. Alternatively, the antigen may be from Hepatitis B, C or E. The virosome of the present invention may comprise one or more protein or peptide, or fragment thereof, i.e. an antigen, from any of the above viruses, preferably as the antigenic molecule.

In an alternative embodiment the agent is a virus-like particle (VLP). Whilst virosomes typically are generated from viruses that have a lipid membrane envelope (such as the influenza virus), VLPs can also be generated from non- enveloped viruses (such as human papilloma virus (HPV)).

As referred to herein a "virus-like particle" comprises structural viral proteins, has a size in the order of 50-120nm and does not contain viral genetic material. The VLP may have a lipid membrane, although in some cases no lipid membrane is necessary. The presence of a lipid membrane generally depends on whether the corresponding virus has a lipid membrane, for example an influenza VLP may have a lipid membrane, whereas an HPV VLP may not. In cases where no lipid membrane is present the viral proteins may self-assemble into the VLP, for example under production in cell culture or in some cases the proteins may self- assemble in vitro following separate production.

These particulate structures are typically produced in cell culture and allow the insertion (e.g. into the lipid membrane) or fusion (genetic) of foreign antigenic sequences (e.g. non-viral), resulting in chimeric particles delivering foreign antigens on their surface. As mentioned above, the VLPs can be assembled automatically in cell culture, but alternatively the proteins to be included in the VLP may be produced separately and assembled (via self assembly) afterwards in a cell-free system. (Buonaguro et al., Expert Rev. Vaccines, 2009; 8(10):1379-98 and Kang et a/., Virus Res., 2009; 143(2):140-146 describes methods of producing VLPs.) For example, in the Gardasil vaccine from Merck & Co. the recombinant proteins are produced separately by fermentation in yeast and self-assembled afterwards.

These VLPs may contain viral antigens from the virus from which they are derived, but optionally or alternatively, they can be used as carriers for foreign antigens, including non-protein antigens, via chemical conjugation. Particulate structures, represent a very efficient system for delivering antigens to antigen presenting cells (APC) which, in turn, trigger and amplify the adaptive immune response. HPV VLPs are commercially available as Gardasil or Cervarix

(GlaxoSmith Kline) vaccines.

The VLP according to the present invention may comprise any of the antigens discussed herein. In a preferred embodiment of the present invention the VLP comprises one or more protein or peptide, or fragment thereof, i.e. an antigen from a virus from the papillomavirus family (optionally the VLP may be prepared from such a virus or by expressing genes from said virus), preferably as the antigenic molecule. A VLP derived or prepared from a papillomavirus family virus or its component parts (e.g. envelope and/or capsid proteins) is referred to herein as a papillomavirus VLP.

Papillomaviridae is an ancient taxonomic family of non-enveloped DNA viruses known as papillomaviruses. Several hundred species of papillomaviruses, have been identified. Infection by most papillomavirus types, depending on the type, is either asymptomatic (e.g. most Beta-PVs) or causes small benign tumors, known as papillomas or warts (e.g. human papillomavirusl , HPV6 or HPV1 1 ). Papillomaviruses are usually considered as highly host- and tissue-tropic, and are thought to rarely be transmitted between species.

Papillomaviruses replicate exclusively in the basal layer of the body surface tissues. All known papillomavirus types infect a particular body surface, typically the skin or mucosal epithelium of the genitals, anus, mouth, or airways. For example, human papillomavirus (HPV) type 1 tends to infect the soles of the feet, and HPV type 2 the palms of the hands, where they may cause warts. Additionally, there are descriptions of the presence papillomavirus DNA in the blood and in the peripheral blood mononuclear cells.

Papillomaviruses are non-enveloped, i.e. the outer shell or capsid of the virus is not covered by a lipid membrane. A single viral protein, L1 , is necessary and sufficient for formation of a 55-60 nanometer capsid composed of 72 star- shaped capsomers. As for the majority of non-enveloped viruses, the capsid is geometrically regular and presents icosahedral symmetry. Self-assembled virus-like particles composed of L1 are the basis of a successful group of prophylactic HPV vaccines designed to elicit virus-neutralizing antibodies that protect against initial HPV infection. The papillomavirus genome is a double-stranded circular DNA molecule -8,000 base pairs in length. It is packaged within the L1 shell along with cellular histone proteins, which serve to wrap and condense DNA.

The papillomavirus capsid also contains a viral protein known as L2, which is less abundant. Although not clear how L2 is arranged within the virion, it is known to perform several important functions, including facilitating the packaging of the viral genome into nascent virions as well as the infectious entry of the virus into new host cells. L2 is of interest as a possible target for more broadly protective HPV vaccines.

The papillomavirus genome is divided into an early region (E), encoding six (E1 , E2, E4, E5, E6, and E7) open reading frames (ORF) that are expressed immediately after initial infection of a host cell, and a late region (L) encoding a major capsid protein L1 and a minor capsid protein L2. All viral ORFs are encoded on one DNA strand.

In a particularly preferred embodiment the VLP comprises a protein or peptide, or fragment thereof, i.e. an antigen from a Human papillomavirus (HPV) (e.g. is derived from said virus) which is preferably a protein or part thereof of one of the early or late proteins referred to herein, preferably as the antigenic molecule. As used 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.

The term "cell" is used herein to include all eukaryotic cells (including insect cells and fungal cells). Representative "cells" thus include all types of mammalian and non-mammalian animal cells (such as fish cells), plant cells, insect cells, fungal cells and protozoa. Preferably, however, the cells are mammalian, for example cells from cats, dogs, horses, donkeys, sheep, pigs, goats, cows, mice, rats, rabbits, guinea pigs, but most preferably from humans. The cell which is subjected to the methods, uses etc. of the invention may be any cell which is capable of expressing, or presenting on its surface a molecule which is administered or transported into its cytosol. The cell is conveniently an immune cell i.e. a cell involved in the immune response. However, other cells may also present antigen to the immune system and these also fall within the scope of the invention. The cells according to the present invention are thus advantageously antigen-presenting cells as described hereinafter. The antigen-presenting cell may be involved in any aspect or "arm" of the immune response as defined herein.

The stimulation of cytotoxic 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-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. Alternatively, 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. 52: 1 -123). If the peptide is of foreign antigen origin, the peptide-MHC class I complex will be recognised by CD8+ cytotoxic T-cells (CTLs). 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. Immunity against the foreign antigen may be established if a sufficient amount of the antigen can be introduced into the cytosol (Yewdell and Bennink, 1992, supra; Rock, 1996, Immunology Today 17: 131 -137). This is the basis for development of inter alia cancer vaccines. One of the largest practical problems is to introduce sufficient amounts of antigens (or parts of the antigen) into the cytosol. This may be solved according to the present invention.

As mentioned previously, once released in the cell cytosol by the

photochemical internalisation process, the antigenic molecule may be processed by the antigen-processing machinery of the cell and presented on the cell surface in an appropriate manner e.g. by Class I MHC. This processing may involve degradation of the antigen, e.g. degradation of a protein or polypeptide antigen into peptides, which peptides are then complexed with molecules of the MHC for presentation. Thus, the antigenic molecule expressed or presented on the surface of the cell according to the present invention may be a part or fragment of the antigenic molecule which is internalised (endocytosed).

A variety of different cell types can present antigen on their surface, including for example, 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. Preferably the cell is a professional antigen-presenting cell.

For antigen presentation by an antigen-presenting cell to a cytotoxic T-cell (CTL) the antigenic molecule needs to enter the cytosol of the antigen-presenting cell (Germain, Cell, 1994, 76, 287-299).

In embodiments of the invention, for example involving an in vitro or ex vivo method, or alternatively an in vivo method, the cell is a dendritic cell. Dendritic cells are immune cells forming part of the mammalian immune system. Their main function is to process antigenic material and present it on the surface to other cells of the immune system. Once activated, they migrate to the lymph nodes where they interact with T cells and B cells to initiate the adaptive immune response.

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.

The dendritic cells may be derived from any appropriate source of dendritic cells, such as from the skin, inner lining of the nose, lungs, stomach and intestines or the blood. In a particularly preferred embodiment of the present invention the dendritic cells are derived from bone marrow.

Dendritic cells may be isolated from natural sources for use in the in vitro methods of the invention or may be generated in vitro. Dendritic cells arise from monocytes, i.e. white blood cells which circulate in the body and, depending on the right signal, can differentiate into either dendritic cells or macrophages. The monocytes in turn are formed from stem cells in the bone marrow. Monocyte- derived dendritic cells can be generated in vitro from peripheral blood mononuclear cells (PBMCs). Plating of PBMCs in a tissue culture flask permits adherence of monocytes. Treatment of these monocytes with interleukin 4 (IL-4) and granulocyte- macrophage colony stimulating factor (GM-CSF) leads to differentiation to immature dendritic cells (iDCs) in about a week. Subsequent treatment with tumor necrosis factor (TNF) further differentiates the iDCs into mature dendritic cells.

As used herein "contacting" refers to bringing the cells and the

photosensitizing agent and/or the virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule 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 or in vivo at body temperature, i.e. 36-38°C.

The cell may be contacted with the photosensitizing agent and the virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule sequentially or simultaneously. Preferably, and conveniently the components are contacted with the cell simultaneously. These agents may be taken up by the cell into the same or different intracellular compartments (e.g. they may be co-translocated). In a preferred embodiment the virosome or VLP may comprise both the antigenic molecule and the photosensitizing agent such that both components contact the cell simultaneously and are taken up into the same intracellular compartment. This is particularly appropriate in the case of

photosensitiser:chitosan conjugates described herein.

The cells are then exposed to light of suitable wavelengths to activate the photosensitizing compound 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 antigenic molecule) 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.

Thus in one embodiment, said photosensitizing agent and/or said virosome or virus-like particle (VLP) as defined herein which comprises said antigenic molecule are applied to the cell together, or separately relative to one another. Irradiation is then performed at a time when these components appear in the same intracellular compartment. This is referred to as a "light after" method. In an alternative embodiment, said method can be performed by contacting said cell with the photosensitizing agent first, followed by contact with the virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule, and irradiation is performed after uptake of the photosensitizing agent into an intracellular compartment, but prior to the cellular uptake of the virosome or virus-like particle (VLP) into an intracellular compartment containing said photosensitizing agent (e.g. it 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. Thus for example the photosensitizing agent may be administered followed by irradiation and then administration of the remaining agent. This is the so-called "light before" method.

"Internalisation" as used herein, refers to the intracellular, e.g. cytosolic, delivery of molecules. In the present case "internalisation" may include the step of release of molecules from intracellular/membrane bound compartments into the cytosol of the cells.

As used herein, "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 or other appropriate uptake mechanisms, for example into or associated with intracellular membrane-restricted compartments, for example the endoplasmic reticulum, Golgi body, lysosomes, endosomes etc.

The step of contacting the cells with the various agents may be carried out in any convenient or desired way. Thus, if the contacting step is to be carried out in vitro 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 agents can simply be added to the medium under appropriate conditions, for example at an appropriate concentration and for an appropriate length of time. For example, the cells may be contacted with the agents in the presence of serum-free medium, or with serum-containing medium.

The comments below discuss the application of the agents to the cells separately. As discussed above however, these agents may be applied to cells together, separately, simultaneously or sequentially. As referred to herein, the application of the agents used in the methods of the invention may be to cells in vitro or in vivo. In the latter case, the application may be via direct (i.e. localized) or indirect (i.e. systemically 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 and the target cell type and location. The 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. For example photosensitizing agents as described herein may be used at a concentration of for example 10 to 50 μg ml. For in vitro use the range can be much broader, e.g. 0.0005-500 μg ml. For in vivo human treatments the photosensitizing agent may be used in the range 0.05-20 mg/kg body weight when administered systemically. If administered locally, for example by intradermal, subcutaneous or intratumoural administration, the dose may be in the region of 1 - 5000 pg, for example 10-2500, 25-1000, 50-500, 10-300 or 100-300μg. Preferably the dose is selected from 100μg, 150μg, 200μg and 250μg. Preferably the dose is 75-125 μg, e.g. 100μg. The doses provided are for a human of average weight (i.e. 70kg). For intradermal injection the photosensitiser dose may be dissolved in 100 μΙ-1 ml, i.e. the concentration may be in the range of 1 -50000 μg 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 concentration of antigen to be used will depend on the antigen which is to be used. Conveniently a concentration of 0.001-500 g/ml (e.g. 20-500, 20-300, 20-100 Mg/ml, 20-50, 10-50, 5-50, 1 -50, 0.01 -50, or 0.001 -50 Mg/ml) antigen may be used in vitro presented in the virosome/VLP. For a peptide antigen a lower concentration e.g. of 0.001 -500, e.g. 0.001 -1 , 5, 25, 50 or 100 μg/ml may be used. For a protein antigen a higher concentration of e.g. 0.5-500 μg/ml may be used. For in vivo use the protein antigen dose may be in the range 0.5-500 μg, for example 10-100 μg. For peptide antigens an in vivo dose of 0.1 -4000μg, e.g. 0.1 - 2000μg, 0.1 -1000 μg or 0.1 -500 pg, for example 10-200μg, may be employed. Such doses are appropriate for local administration. An appropriate concentration can be determined depending on the efficiency of uptake of the agent in question into the cells in question and the final concentration it is desired to achieve in the cells.

The concentration of the virosome or virus-like particle (VLP) will also depend on the particular virosome or VLP which is to be used. Conveniently a concentration of 1 -100 μς ηιΙ (e.g. 20-100 μςΛτιΙ, or 20-50 μς/ηιΙ) may be used in vitro. In vivo, for local administration, typically virosomes or VLPs will contain 10- 200 μg of total antigen protein or peptide. The amount required varies little between different animals.

In most cases the photosensitizing agent and the virosome or virus-like particle (VLP) which comprises the antigenic molecule are administered together, but this may be varied. Thus different times or modes or sites of administration (or contact with the cell) are contemplated for the different components and such methods are encompassed within the scope of the invention. In a preferred embodiment of the products, methods and uses of invention the virosome or VLP may comprise both the antigenic molecule and the photosensitizing agent such that simultaneous administration may be achieved. This is particularly appropriate in the case of photosensitiser:chitosan conjugates described herein.

In one embodiment the virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule is administered separately from the photosensitiser, for example in a separate formulation. Thus, in one embodiment the virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule may be administered prior to administration of the

photosensitiser, for example 24 hours before the photosensitizer or, e.g.

approximately 2 hours prior to illumination.

The contact between the cell and the photosensitizing agent and/or the virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule is conveniently from 15 minutes to 24 hours, e.g. 30 minutes to four hours, preferably from 1 .5 to 2.5 hours.

In a preferred embodiment the initial incubation of the cell is with the photosensitising agent. In one embodiment the time between the administration of the photosensitizing agent and the virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule is a matter of hours. For example, the photosensitizing agent may be applied 16 to 20 hours, e.g. 18 hours, before illumination, and the virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule may be applied 1 to 3 hours, e.g. 2 hours before illumination. Thus, the time between the administration of the photosensitizing agent and the virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule may be in the range of 15 to 23 hours.

Thus, the cell is then incubated with the virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule after the incubation with the photosensitiser. Conveniently the cells may be placed into photosensitizer/antigen-free medium after the contact with the photosensitizer/antigen 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

photosensitiser and the virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule.

In vivo 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 tumour, tissue or organ which is to be treated/irradiated, the cells near the injection 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 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 hours may be used.

In addition, agents not administered directly to the site of interest 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.

Nevertheless, although the situation in vivo is more complicated than in vitro, the underlying concept of the present invention is still the same, i.e. the time at which the molecules 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 other agent 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 other agent is in contact with the cells for a period of time sufficient to allow its uptake into the cells.

For administration of agents described herein in vivo, any local mode of administration common or standard in the art may be used, e.g. injection, infusion, topical administration, transdermal administration, both to internal and external body surfaces etc. For in vivo use, the invention can be used in relation to any tissue which contains cells to which the photosensitising agent containing compound or the antigenic molecule to be internalized is localized, including body fluid locations, as well as solid tissues. All tissues can be treated as long as the photosensitiser is taken up by the target cells, and the light can be properly delivered. Preferred modes of administration are intradermal, subcutaneous, topical or intratumoural administration or injection. Preferably administration is by intradermal injection.

To achieve the desired outcome, e.g. antigen presentation, generation of an immune response or vaccination, the methods or parts thereof may be repeated, e.g. "re-vaccination" may take place. Thus, the method in its entirety may be performed multiple times (e.g. 2, 3 or more times) after an appropriate interval or parts of the method may be repeated, e.g. further administration of the virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule or additional irradiation steps. For example, the method or part of the method 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. 7 to 20 days, preferably 14 days, or weeks, e.g. between 1 and 5 weeks (for example, 1 , 2, 3 or 4 weeks) after it was first performed. All or part of the method may be repeated multiple times at appropriate intervals of time, e.g. every two weeks or 14 days. In a preferred embodiment the method is repeated at least once. In another embodiment the method is repeated twice.

In one embodiment, parts of the method of the invention may be carried out prior to the method of the invention being carried out. For example, the method may be carried out one or more times, for example twice, in the absence of

photosensitiser and illumination before the method of the invention is carried out. Part of the method may be carried out a matter of days, e.g. 7 or 14 days, or weeks, e.g. 1 , 3 or 4 weeks before the method of the invention is carried out. Part of the method may be repeated one or more times at these time intervals before the method of the invention is carried out. Thus, in a preferred aspect, the antigenic molecule is administered (e.g. to the subject) equal to or greater than 2 times (e.g. at the time intervals discussed above), wherein at least one administration of said antigenic molecule is performed in accordance with the method of the invention.

"Irradiation" to activate the photosensitising agent refers to the

administration of light directly or indirectly as described hereinafter. Thus subjects or cells may be illuminated with a light source for example directly (e.g. on single cells in vitro) or indirectly, e.g. in vivo when the cells are below the surface of the skin or are in the form of a layer of cells not all of which are directly illuminated, i.e. without the screen of other cells. Illumination of the cell or subject may occur approximately 18-24 hours after administration of the photosensitizing agent and the virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule.

The light irradiation step to activate the photosensitising agent may take place according to techniques and procedures well known in the art. The wavelength of light to be used is selected according to the photosensitising 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 TPCS _2a 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. Where appropriate the photosensitiser, e.g. a porphyrin or chlorin, may be activated by green light (e.g. around 514nm), for example the KillerRed (Evrogen, Moscow, Russia) photosensitiser may be activated by green light.

Suitable light sources are well known in the art, for example the

LumiSource® lamp of PCI Biotech AS. Alternatively, 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. For red light, 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 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, 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. Generally, 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 0.25 or 1 to 30 minutes, e.g. from 0.5 to 3 minutes or from 1 to 5 minutes or from 1 to 10 minutes e.g. from 3 to 7 minutes, and preferably approximately 3 minutes, e.g. 2.5 to 3.5 minutes. Shorter irradiation times may also be used, for example 1 to 60 seconds, e.g. 10-50, 20-40 or 25-35 seconds.

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 cells 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 photosensitiser used) of the visible spectrum are used. For example, 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. Alternatively, e.g. if the LumiSource® lamp is employed a light dose in the range of 0.1 -6 J/cm ² at a fluence range of 0.1 - 20 (e.g. 13 as provided by Lumisource®) mW/cm ² is appropriate. For red light, 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 , may be used. Furthermore, if cell viability is to be maintained, the generation of excessive levels of toxic species is to be avoided and the relevant parameters may be adjusted accordingly.

The methods 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 for some applications (e.g. cancer treatment). In most embodiments, however, cell death is avoided to allow the generation of an immune response from the presenting cell. The methods 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 of the photosensitizing agent. Again, such techniques are known in the art.

Preferably, substantially all of the cells, or a significant majority (e.g. at least 75%, more preferably at least 80, 85, 90 or 95% of the cells) are not killed. In vitro 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 1 cm 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 method may be performed in vivo, in vitro or ex vivo. Preferably the method is used in vitro or ex vivo to generate cells for administration in vivo or the method is used in vivo. Thus in a preferred feature, the method may be used to generate an immune response in a subject.

Thus, in a further aspect the present invention provides a method of generating an immune response in a subject, comprising administering to said subject a photosensitizing agent and a virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule, and irradiating said subject with light of a wavelength effective to activate said photosensitizing agent, wherein an immune response is generated.

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. Preferably 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 IL-2 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). Preferably the immune response is stimulated via MHC-I presentation.

Preferably the immune response is used to treat or prevent a disease, disorder or infection, e.g. a viral infection, preferably influenza or papillomavirus, or cancer. In methods and uses described herein the cancer may be melanoma. In alternative embodiments, the cancer may be a cancer which is not melanoma.

Preferably the method is used for vaccination. As referred to herein, "vaccination" is the use of an antigen (or a molecule containing 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. Preferably the disease is cancer (and the vaccination is therapeutic) and the infection is a viral infection, preferably influenza or papillamovirus (and the vaccination is prophylactic).

In a preferred embodiment of the present invention, the subject of the method, e.g. vaccination, is a non-mammalian animal (e.g. a fish) or a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit or guinea pig, but most preferably the subject is a human.

Preferably the methods described herein achieve synergy, i.e. the extent of cell surface presentation or the immune response generated is enhanced more than the combined enhancement observed by (i) performing the method with the antigenic molecule in the absence of the virosome or virus-like particle (VLP) and (ii) performing the method with the virosome or virus-like particle (VLP) comprising the antigenic molecule in the absence of the photosensitizing agent and the irradiation step, i.e. synergy between the methods is observed. The level of cell surface presentation or immune response generation may be assessed by appropriate means, e.g. number of antigen-specific CD8+ cells or levels of markers of immune response activation, e.g. IFNy or IL-2.

"Synergy" as used to herein refers to a quantitative improvement over merely additive effects.

The various agents used in the methods of the invention may be

administered to the subject separately, sequentially or simultaneously.

Aspects and features discussed above in relation to the method of expressing an antigenic molecule or a part thereof on the surface of a cell of the present invention, where appropriate, are also applicable to the method of generating an immune response above.

The invention also provides a method for introducing an antigenic molecule into the cytosol of a cell, comprising contacting said cell with a photosensitising agent and a virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule to be introduced, and irradiating the cell with light of a wavelength effective to activate the photosensitising agent. Once activated, intracellular compartments within said cell containing said compound release the molecule contained in these compartments into the cytosol.

The methods of the invention above may be used in vitro or in vivo, for example either for in situ treatment or for ex vivo treatment followed by the administration of the treated cells to the body.

The invention further provides a cell expressing an antigenic molecule, or a part thereof, on its surface, or a population thereof, which cell is obtainable (or obtained) by any of the methods as defined herein. Also provided is the cell or cell population for use in prophylaxis, or therapy, as described hereinafter.

The cell population may be provided in a pharmaceutical composition comprising in addition one or more pharmaceutically acceptable diluents, carriers or excipients.

The present invention also provides a pharmaceutical composition comprising a photosensitizing agent, and a virosome or virus-like particle (VLP) as defined herein which comprises an antigenic molecule and one or more

pharmaceutically acceptable diluents, carriers or excipients. These compositions (and products of the invention) 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. "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, 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 cells, for example antigen presenting cells, may be prepared in vitro. In treatment methods, these cells may be administered to a body in vivo or a body tissue ex vivo such that those cells may stimulate an immune response, e.g. for prophylactic or therapeutic purposes.

Thus the invention further provides a cell population (or composition containing the same) as defined herein, or a photosensitizing agent and a virosome or virus-like particle (VLP) as defined herein which comprises an antigenic molecule, for use in prophylaxis or therapy or for use in stimulating an immune response, for example for vaccination purposes, e.g. for stimulating CTLs, in a subject, preferably for treating or preventing a disease, disorder or infection in said subject, particularly for treating or preventing cancer or a viral infection, preferably influenza or papillomavirus. Alternatively defined the present invention provides use of (i) a cell population, (ii) a composition as defined herein, or (iii) a

photosensitizing agent and a virosome or virus-like particle (VLP) as defined herein which comprises an antigenic molecule, for the preparation of a medicament for use in stimulating an immune response (e.g. for stimulating CTLs) in a subject, preferably for treating or preventing a disease, disorder or infection in said subject, preferably for vaccination and/or for treating or preventing cancer or a viral infection, preferably influenza or papillomavirus, wherein preferably said immune response is stimulated by a method as defined herein.

Said stimulation, treatment or prevention preferably comprises administering said medicament to said subject. The photosensitizing agent and the virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule may be combined and presented in a composition. (In a preferred aspect, and as described hereinbefore, the virosome or VLP may comprise both the antigenic molecule and the

photosensitizing agent.) Alternatively expressed, the invention provides use of a photosensitizing agent and a virosome or virus-like particle (VLP) as defined herein which comprises an antigenic molecule in the manufacture of a medicament for stimulating an immune response (e.g. for stimulating CTLs in a subject), preferably to treat or prevent a disease, disorder or infection in said subject, particularly for vaccination purposes, wherein said medicament comprises a population of cells expressing an antigenic molecule or a part thereof on the surface of said cells obtainable by a method as defined herein, for administration to said subject.

Preferably the cell population is obtained by such methods. The population is for administration to the subject.

In an alternative embodiment the present invention provides a

photosensitizing agent and a virosome or virus-like particle (VLP) as defined herein which comprises an antigenic molecule for use in expressing said antigenic molecule or a part thereof on the surface of a cell to stimulate an immune response (e.g. for stimulating CTLs) in a subject, preferably to treat or prevent a disease, disorder or infection in said subject, wherein said use comprises a method as defined herein, preferably to prepare a population of cells, e.g. dendritic cells. These cells may then be administered to the subject.

The invention further provides a product comprising a photosensitizing agent and a virosome or virus-like particle (VLP) as defined herein which comprises an antigenic molecule as a combined preparation for simultaneous, separate or sequential use in stimulating an immune response in a subject (or for expressing an antigenic molecule or a part thereof on the surface of a cell or for internalising an antigenic molecule into the cytosol of a cell) in a method as defined herein, preferably to treat or prevent a disease, disorder or infection in a subject. In one embodiment, the combined preparation may be for simultaneous use in which the virosome or VLP comprises both the antigenic molecule and the photosensitizing agent.

The present invention also provides a kit for use in stimulating an immune response in a subject, preferably for treating or preventing a disease, disorder or infection in said subject, for example for use in vaccination or immunisation, or for expressing an antigenic molecule or a part thereof on the surface of a cell or for internalising an antigenic molecule into the cytosol of a cell in a method as defined herein, said kit comprising

a first container containing a photosensitizing agent as defined herein; and a second container containing a virosome or virus-like particle (VLP) as defined herein which comprises the antigenic molecule.

In another aspect of the invention, when the virosome or VLP comprises both the antigenic molecule and the photosensitising agent, the kit may comprise only a single container.

The products and kits of the invention may be used to achieve cell surface presentation (or therapeutic methods) as defined herein.

In a yet further embodiment the present invention provides a method of generating an immune response (e.g. for stimulating CTLs) in a subject, preferably to treat or prevent a disease, disorder or infection in said subject, comprising preparing a population of cells according to the method defined herein, and subsequently administering said cells to said subject.

The antigenic presentation achieved by the claimed invention may advantageously result in the stimulation of an immune response when the treated cells are administered in vivo. Preferably an immune response which confers protection against subsequent challenge by an entity comprising or containing said antigenic molecule or part thereof is generated, and consequently the invention finds particular utility as a method of vaccination.

The disease, disorder or infection is any disease, disorder or infection which may be treated or prevented by the generation of an immune response, e.g. by eliminating abnormal or foreign cells which may be identified on the basis of an antigen (or its level of expression) which allows discrimination (and elimination) relative to normal cells. Selection of the antigenic molecule to be used determines the disease, disorder or infection to be treated. Based on the antigenic molecules discussed above, the methods, uses, compositions, products, kits and so forth, described herein may be used to treat or prevent against, for example, infections (e.g. viral or bacterial as mentioned hereinbefore), cancers or multiple sclerosis. Prevention of such diseases, disorders or infection may constitute vaccination.

As defined herein "treatment" refers to reducing, alleviating or eliminating one or more symptoms of the disease, disorder or infection which is being treated, relative to the symptoms prior to treatment. "Prevention" (or prophylaxis) refers to delaying or preventing the onset of the symptoms of the disease, disorder or infection. Prevention may be absolute (such that no disease occurs) or may be effective only in some individuals or for a limited amount of time. For in vivo administration of the cells, 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. Conveniently, the cells are administered by intralymphatic injection. Preferably 1 x10 ⁴ to 1 x10 ⁸ cells are administered per kg of subject (e.g. 1 .4x10 ⁴ to 2.8x10 ⁶ per kg in human). Thus, for example, in a 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 invention will now be described in more detail in the following non- limiting Examples with reference to the following drawings in which:

Figure 1 shows Scheme 1 : synthetic route for synthesis of compound 5. Reagents and conditions: (a) propionic acid, reflux, 1 h (20%); (b) NaN0 ₂ (1 .8 eq), TFA, rt, 3min. 67%) ; (c) SnCI ₂ .2H ₂ 0, cone. HCI, 60° C, 1 h (88%) ; (d) Bromoacetyl bromide, Et ₃ N, CH ₂ CI ₂ , rt, 1 h (64%) (e) Piperazine, CH ₂ CI _2, rt, 1 h (94%).

Scheme 2. Synthesis of N-modified Chitosan derivatives (TPP-CS-TMA & TPP-CS- MP). Here A-represents 1 ^st batch compounds and B-presents 2 ^nd batch

compounds. Reagents and conditions: (a) MeS0 ₃ H/ H ₂ 0, 10°C-rt, 1 h, (90%); (b) TBDMSCI, imidazole, DMSO, rt, 24h (96 %) ; (c) Bromoacetyl bromide, Et ₃ N, CH ₂ CI ₂ , -20°C, 1 h (92%) ; (d) compound 5 i.e. TPP-NH-Pip (0.1 or 0.25 eq), Et ₃ N, CHCI ₃ , rt, 2h (92-90 %) (e) NMe ₃ or 1 -methyl piperazine, CHCI _3, rt, 24h (f) TBAF, NMP, 55°C, 24h or cone. HCI/ MeOH, rt, 24h.

Scheme 3 - Synthesis scheme for compounds 1 , 3 20 and 21 .

Reactions and conditions: ((a) Propionic acid, reflux, 1 h, (20%); (b) NaN0 ₂ (1 .8 eq.), TFA, rt, 3min.; (c) SnCI ₂ .2H ₂ 0, cone. HCI, 60 °C, 1 h, (54 %); (di) p- Toluenesulfonylhydrazide, K ₂ C0 _3, pyridine, reflux, 24h; (d ₂ ) o-Chloranil, CH ₂ CI ₂ , rt, (80%); (e) Chloroacetyl chloride, Et ₃ N, CH ₂ CI ₂ , rt, 2h, in s/ ^' fi/-(f) Piperazine, CH ₂ CI ₂ , rt, 12h, (61 %). All derivatives of compound 20 and 21 will contain the TPCai and the TPCa ₂ isomer. However only the TPCai structure is shown in schemes and in the structure drawings.

Scheme 4 - synthesis scheme for compounds 22-28. Reactions and conditions: (a) Acetyl chloride, MeOH, reflux, 24h, (87 %); (b) BF ₃ .Et ₂ 0, CHCI ₃ , rt, p-chloranil, 48h, (14%); (c) 2N KOH (in MeOH), THF:Pyridine (10:1 ), reflux, 24h (71 %); (di) p- Toluenesulfonylhydrazide, K ₂ C0 _3, Pyridine, reflux, 24h; (d ₂ ) o-chloranil, CH ₂ CI ₂ : MeOH (75:25), rt, (70 %); (e) EDCI.HCI, HOBT, Et ₃ N, N-Boc-piperazine 5, DMF, rt, 24h (54 %) (f) TFA, CH ₂ CI _2, rt, 1 h (89 %). All derivatives of compound 26-28 will contain the TPCci and the TPCc ₂ isomer. However, only the TPCci structure is shown in schemes and in the structure drawings.

Scheme 5A and 5B. Reagents and conditions (6A) : (a) compound 21 i.e. TPC-NH- Pip (0.1 eq), Et ₃ N, CHCI ₃ , rt, 2h (78%) (b) NMe ₃ or 1 -methyl piperazine, CHCI _3, rt, 24h. Reagents and conditions (6b) : a) compound 28 i.e. TPC-CO-Pip (0.1 eq), Et ₃ N, NMP, 75 °C, 12h (89 %) (b) NMe ₃ or 1 -methyl piperazine, CHCI _3, rt, 24h.

EXAMPLES

Materials and methods

Mice

C57BL/6 mice are purchased from Harlan (Horst, The Netherlands). OT-I mice transgenic for the T-cell receptor that recognises the MHC class-l restricted epitope OVA ₂ 57-264 from ovalbumin (OVA) are bred in facilities at the University of Zurich (originally purchased from Taconic Europe (Ry, Denmark)). All mice are kept under specified pathogen-free (SPF) conditions, and the procedures performed are approved by Swiss Veterinary authorities. In the OT-1 mice, the gene for the T-cell receptor has been engineered in such a way that nearly all of the CD8+ T-cells in these mice (called OT-1 cells) will specifically recognize the specific peptide epitope (SIINFEKL) from the ovalbumin (OVA) antigen.

Immunisation protocol

On day 0 female C57BL/6 mice are injected with 1 .5 x 10 ⁶ splenocytes from

Rag2/OT-1 mice intravenously in the tail vein (for experiments using OVA as antigen). In this way the mice that are vaccinated have a "background" of CD8 T- cells that can respond to the SIINFEKL-epitope from OVA if, and only if, this is properly presented on MHC class I on antigen presenting cells. Thus, the transfer of OT-1 cells "amplifies" the detection system in the vaccinated mice making it possible to easily assay for the effect of in vivo vaccination by measuring antigen specific CD8+ T-cells and IFN-γ and IL-2 production.

4 hours later the animals are vaccinated by intradermal injection at the abdomen (2 x 50 μΙ of solutions containing the ingredients specified below). 6 groups of 4 animals received total doses of:

Group 1 : 250 g TPCS _2a (Amphinex) + 100 g ovalbumin (OVA, Grade V,

Sigma-Aldrich). Group 2: 250 μg TPCS _2a + influenza virosomes (e.g. lnflexal ^® V) containing 100 μg ovalbumin.

Group 3: 250 μg TPCS _2a + HPV VLPs (e.g. Gardasil).

Group 4: 100 μg ovalbumin.

Group 5: influenza virosomes (e.g. lnflexal ^® V) containing 100 μg ovalbumin. Group 6: HPV VLPs (e.g. Gardasil).

On day 1 the animals in groups 1 , 2 and 3 are anaesthetized and illuminated for 6 minutes with blue light using a LumiSource lamp (PCI Biotech AS). The animals are illuminated about 18 h after injection of the antigen solution, the fluence rate of the illumination is about 13 mW/cm ² On day 7 the mice are bled from the tail vein and the blood cells are stained with SIINFEKL pentamer (Prolmmune) or a relevant HPV antigen, and CD8 and CD44 antibodies for flow cytometry analysis (see protocols below). On day 14 the mice are euthanized and the spleens are collected. One aliquot of the splenocytes is restimulated with the SIINFEKL peptide (EMC microcollections, Tuebingen, Germany) or a relevant HPV antigen, stained for intracellular IFN-γ expression and analysed by Flow cytometry analysis (see below). Another aliquot of the splenocytes is resuspended in cell culture medium, kept in this medium overnight (purely for practical reasons) without restimulation stained by SIINFEKL-pentamer or a relevant HPV antigen as described above and analysed by flow cytometry (see protocol below).

SIINFEKL-pentamer-staining of spleen cells

SIINFEKL-pentamer staining and flow cytometry on spleen cells is performed on cells that have been resuspended in cell medium and kept in this medium overnight (purely for practical reasons) without restimulation. Similar conditions are used for staining for a HPV antigen.

SIINFEKL-pentamer staining and Flow cytometry

5-10 drops of whole tail blood are collected and 0.5 ml of Red Cell Lyse solution (Sigma) is added. After 5-6 minutes, cells are spun down and washed twice with 0.5 ml PBS. The cell pellet is resuspended in FACS buffer (2% FCS/PBS with 0.01 % Na-azide), transferred to a U-formed 96 well plate and incubated with FcR- blocking antibodies (1 .0 μΙ Anti-CD16/CD32 from Pharmingen) for 10 min on ice, (1 μΙ + 49 μΙ FACS buffer). Without washing, the SIINFEKL-pentamer-PE (Prolmmune; 5 ul per sample) (or equivalent HPV antigen) is added, mixed and incubated at 37°C for 15 min. Without washing, a fluorescence-labeled CD8 or CD44 is added to a final concentration of 1 :100, and incubated on ice for 25-45 min. Cells are washed in 100μΙ FACS buffer and suspended in 100μΙ FACS buffer. Cells are analysed with FACSCanto.

Splenocyte restimulation ex vivo

Splenocytes are isolated and prepared for intracellular staining by crushing the spleen and separating cells in 2% FCS/PBS, by agitation in lysis buffer (Sigma) for 1 -2 minutes and washing in 2% FCS/PBS. 1 ml of the cell suspension in complete medium is added per well of a 24-well plate (500,000 cells/ml) and

5 μg/ml SIINFEKL (or HPV antigen) is added to each well and incubated overnight at 37°C. Brefeldin A (1 -2 μg/ml) is added to each well and incubated for 4 hours at 37°C. Cells are transferred to U-formed 96 well plates, washed in 2% FCS/PBS and resuspended in 50μΙ FACS buffer with FcR-blocking antibodies (1.0μΙ anti- CD16/CD32 from Pharmingen), and incubated on ice for 10 minutes. Without washing, cells are incubated with surface antibodies CD8 or CD44 for 20-45 min on ice (dark), washed in FACS buffer and fixed by resuspending in 100 ul

paraformaldehyde (PFA) (1 % in PBS) for 10-20 minutes on ice. Cells are washed in FACS buffer, resuspended in 100μΙ NP40 (0.1 % in PBS) and incubated for 3 minutes on ice. After washing in FACS buffer, a fluorescence-labelled interferon- gamma antibody is added and incubated for 35 min on ice in the dark. After washing and suspension in FACS buffer, the cells are analysed with FACSCanto using FlowJo 8.5.2 software (Tree Star, Inc., Ashland, OR).

Flow cytometry

The frequency of OVA(or HPV)-specific T-cells is determined by flow cytometry (FACSCanto from BD Biosciences, San Jose, USA). Before the flow cytometry run a compensation is performed using beads stained with each antibody separately. Before antibody staining, the red blood cells are lysed using Red Cell Lyse solution (Sigma). 10 000 CD8 ⁺ events were recorded for each sample, and the percentage of SIINFEKL-pentamer (or HPV antigen) positive cells is calculated using FlowJo 8.5.2 software from Tree Star, Inc. (Ashland, OR) http://www.flowjo.com/.

ELISA

ELISA is performed using the Ready-set Go! kit (eBioscience) for the relevant molecules according to the manufacturer's instructions. Appropriate ELISAs for HPV antigens are known. Example 1 : Effect of virosomes VLPs on in vivo vaccination with OVA or an HPV antigen

Mice are vaccinated in vivo by the immunisation protocol described above. Blood is isolated after 7 days and spleen after 14 days. Blood is analysed for antigen- specific CD8+ T cells and spleen cells are either analysed directly for antigen- specific CD8+ T-cells or for IFN-γ or IL-2 production after restimulation in vitro.

Level of antigen-specific T-cells in blood and spleen

The level of antigen-specific T-cells is measured by flow cytometry, using a fluorescently labelled antigen-specific "pentamer" (or HPV antigen) that binds specifically to the antigen-specific T-cells. The number of antigen specific CD8+ T- cells in % of the total CD8+ T-cells in the animal is determined (see the staining and flow cytometry analysis described in the immunisation protocol and details of SIINFEKL staining).

In the case of the OVA experiments, the endogenous T-cells serve as an internal control for the antigen-specificity of the effect, since a general stimulation effect on T-cells will affect also the endogenous T-cells not leading to an increase in the % of the antigen-specific cells. Typically the % of OT-1 cells is measured before vaccination and at time point(s) after vaccination. The effect of the antigen alone ("conventional vaccination") is compared to the effect of antigen+PCI.

Level of IFN-γ production in spleen cells after ex vivo stimulation with antigen (flow cytometry)

Spleens removed on day 14 of vaccination are subject to splenocyte isolation and restimulation with SIINFEKL antigen peptide (or HPV antigen) and intracellular staining for IFN-γ production for analysis of CD8+ T cells by flow cytometry as described in the protocols above.

Level of IFN-γ and IL-2 production in spleen cells after ex vivo stimulation with antigen (ELISA)

Spleens removed on day 14 of vaccination are subject to splenocyte isolation and restimulation with SIINFEKL antigen peptide (or HPV antigen) and IFN-γ and IL-2 production analysis by ELISA as described in the protocols above.