US20040014708A1 - Composition comprising immunogenic microparticles - Google Patents

Composition comprising immunogenic microparticles Download PDF

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US20040014708A1
US20040014708A1 US10/380,588 US38058803A US2004014708A1 US 20040014708 A1 US20040014708 A1 US 20040014708A1 US 38058803 A US38058803 A US 38058803A US 2004014708 A1 US2004014708 A1 US 2004014708A1
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antigen
microparticles
cells
protein
composition
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Magdalena Plebanski
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PX Biosolutions Pty Ltd
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Austin Research Institute
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Priority claimed from AUPR4888A external-priority patent/AUPR488801A0/en
Priority claimed from AUPR4962A external-priority patent/AUPR496201A0/en
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Publication of US20040014708A1 publication Critical patent/US20040014708A1/en
Priority to US12/603,439 priority Critical patent/US8287877B2/en
Priority to US13/283,294 priority patent/US8846026B2/en
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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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to immunogenic compositions, vaccine compositions, methods of eliciting immune responses in a subject and methods of producing the compositions.
  • the mechanisms by which the immune system controls disease include the induction of neutralising antibodies (a humoral immune response), and the generation of cellular or T-cell responses.
  • the latter include T-helper cells (T H ) and cytotoxic T-lymphocytes (CTL).
  • T H T-helper cells
  • CTL cytotoxic T-lymphocytes
  • antibodies provide protection by preventing the virus from infecting cells.
  • Antibodies can also protect against bacteria e.g. pneumococci and staphylococci, by use of bactericidal mechanisms and by neutralising bacterial toxins.
  • T-cells can be stimulated when peptide fragments from an antigen are bound to molecules known as MHC I or MHC II (major histocompatability complex, class I or class II) and are displayed on the surface of professional APCs (antigen presenting cells) such as DCs (dendritic cells) or macrophages.
  • APCs antigen presenting cells
  • DCs dendritic cells
  • T-cells contain antigen receptors which they employ to monitor the surface of cells for the presence of the peptide fragments from the antigen.
  • the antigen receptors on T H cells recognise antigenic peptides bound to MHC II molecules.
  • the receptors on CTLs react with antigens displayed on class I molecules.
  • the stimulated T-cells amplify the immune response in that when a T-cell recognises a target cell which is infected with the pathogen, or that contain an epitope which it recognises, a chain of events is triggered and these eventually result in death of the infected cells.
  • some T-cells can stimulate secretion of cytokines or lymphokines, which in turn can exert effects that ultimately lead to inactivation or eradication of pathogens.
  • IgE production is induced along with other desired immunoglobulins such as IgA, IgG and IgM.
  • Vaccines that induce IgE are not desirable, as the immunoglobulin is involved in allergic responses.
  • Stimulation of IgA production is a “first line” defence for pathogens that infect via entry through a mucosal site or surface.
  • vaccines that can generate a high IgA secretory immune response without enhancing IgE production would also be valuable.
  • a vaccine results in stimulation of APCs
  • the degree of immune stimulation is sub-optimal.
  • dendritic cells or DCs are characterised by a series of subset of cells that can be distinguished from each other by surface molecules some of which are specific ligands that bind receptors on T cells. Accordingly, it would be desirable to produce a vaccine which would selectively target a subset of DCs, eg a subset capable of efficient CD8 T-cell priming since these T-cells play a vital role in protective immunity against many intracellular pathogens and cancer, but are notoriously difficult to induce.
  • VLP Non-infective viral like particles
  • VLPs composed of the surface Hepatitis B protein or yeast retro-transposon protein particles have been shown to be efficiently processed for MHC I presentation by macrophages to induce CD8 CTL responses in vitro and in vivo [2, 3].
  • VLPs are multimeric, lipid-containing protein particles the lipid content of which comprises more than 50% of the dry weight.
  • VLP are immunogenic not by virtue of size, but by biochemical composition. This would be consistent with the proposal that when antigen is presented in formulations containing lipid or detergent, they are able to fuse with the APC, possibly by damaging the cell membrane, and thus gain entry into the cytoplasm.
  • microspheres within which are entrapped antigens have been explored as a possible vaccine composition.
  • the microspheres are made from biodegradable polyesters of lactic and glycolic acids (PLA and PLGA).
  • PLA and PLGA biodegradable polyesters of lactic and glycolic acids
  • the microspheres are constructed such that their size and polymer composition control the rate at which they degrade. As the microspheres degrade, the entrapped antigen is released therefrom, and provides for a controlled release of antigen for stimulating the immune response. It is unlikely that these molecules would interface and react with immune cells in the same way as protein particles the make-up of which are biologically compatible with cellular membranes.
  • composition includes the antigen and microparticles. It may also include other components.
  • the antigen may be a peptide, protein, lipid, carbohydrate, nucleic acid or other type of molecule or a combination of any of these.
  • the antigen may be derived from a pathogen, tissue, cell, organ or molecule depending on the intended purpose of the composition, and may be a purified antigen, or be of recombinant origin produced in suitable vectors such as bacteria, yeast or cell cultures.
  • the pathogen for example may be any pathogen, intra or extracellular, antigenic portions or parts thereof, viral, bacterial or protozoal in origin such as HIV, influenza viruses, hepatitis viruses, malaria.
  • examples of the antigens envisaged by the present invention are as follows: pollens, hepatitis C virus, (HIV) core, E1, E2 and NS2 proteins, antigens from Plasmodium species such as P. vivax and other Plasmodium species including P.
  • falciparum circumsporozoite protein and human Plasmodium - falciparum , - vivax , - ovalae and malariae , TRAP, MSP-1, MSP-2, MSP-3, MSP4, MSP-5, AMA-1, RESA, SALSA, STARP, LSA1 and LSA3, HIV-gp120/160 envelope glycoprotein, streptococcus surface protein Ag, influenza nucleoprotein, haemagglutinin-neuraminidase surface infection, TcpA pilin. subunit, VP1 protein.
  • Lysates or culture filtrates from the pathogens exemplified above may also be used as the antigen. Such fractions may be in purified, concentrated or diluted form, so long as they provide antigenicity and/or immunogenicity. Thus it makes it possible to “tailor-make” an immunogenic composition for a patient in accordance with the invention by using patient tumor lysates or a specific set of tumor proteins conjugated to the microparticles.
  • the antigen may also be derived from any tumour type or malignancy.
  • cancer types from which the antigens may be derived are breast, lung, pancreas and colon cancer and melanoma.
  • specific antigens obtained from tumours are melanoma specific antigen (for example, the MAGE series antigen), carcino embryonic antigen (CEA) from colon, nm23 cancer antigen and other cancer antigens or indeed antigens extracted from any tumour, e.g. mucin such as MUC-1 to MUC-7 antigens.
  • mucin such as MUC-1 to MUC-7 antigens.
  • Recombinant peptides or proteins alone or in combination may also be used.
  • the antigen may also be a synthetic epitope such as a mimic or peptidomimetic based on one or more of the antigens referred to above.
  • microparticle refers to a small particle. This may be in the form of a bead or sphere or any other suitable shape.
  • VSP virus sized particles
  • the microparticle is in the same size range as known viruses. This means that the microparticle is preferably less than about 0.50 ⁇ m. Preferably the microparticle is of such a size that it is adapted to elicit an immune response. In particular it is adapted to be taken up by antigen presenting cells within a human subject or an animal. More preferably the microparticles are between about 0.03 and 0.50 ⁇ m, preferably about 0.03 and 0.15 ⁇ m, still more preferably between about 0.03 and 0.10 ⁇ m.
  • the microparticles are about 0.03 to 0.05 ⁇ m, more preferably the microparticles are between about 0.03 ⁇ m and 0.049 ⁇ m, still more preferably the microparticles are between about 0.03 and about 0.04 ⁇ m or about 0.04 and 0.049 ⁇ m.
  • a population of microparticles to be used in accordance with the invention is of a uniform size. This means that the majority of the particles in a given population are of the stated size.
  • the immunogenic composition of the invention may be used in treatment, prophylaxis or prevention of the disease or condition caused by, or associated with contact with the antigen.
  • the composition may be used in the treatment or prophylaxis of certain cancers.
  • the invention provides a vaccine composition comprising microparticles associated with at least one antigen wherein the microparticles are in the same size range as viruses.
  • the composition of the invention is particularly useful and advantageous as it is an effective single-dose vaccine but may also be used in multiple dose regimes.
  • the invention provides a single-dose vaccine composition comprising microparticles associated with at least one antigen, wherein the microparticles are of the same size range as viruses.
  • single-dose it is meant that a humoral and/or cellular immune response is stimulated or enhanced to maximal levels ( “maximal” means that the levels are not capable of being further increased by repeated vaccination), or affords protection to the recipient of the composition, following one administration of the composition or. vaccine.
  • the administration may be by any suitable means eg. by injection i.m., i.p., i.v., orally, by inhalation, or by administration through a mucosal surface or site.
  • the antigen is conjugated to the surface of the microparticles.
  • the size of the microparticles is between about 0.03 and 0.5 ⁇ m, preferably about 0.03 to 0.15 ⁇ m, more preferably about 0.03 to 0.10 ⁇ m even more preferably about 0.03 to 0.05 ⁇ m in diameter, even more preferably about 0.03 to 0.049 ⁇ m. Still more preferably 0.03 to 0.04 ⁇ m or 0.04 to 0.049 ⁇ m
  • the microparticles are most preferably made of polystyrene, PLG, glass, calcium phosphate or gold.
  • each antigen for use in accordance with the invention is conjugated to microparticles of a uniform size.
  • microparticle has the meaning given above.
  • the microparticle is adapted to be taken up by antigen presenting cells in an animal.
  • the microparticles are between 0.03 and 0.5 ⁇ m, preferably between 0.03 and 0.15 ⁇ m. More preferably the microparticles are between about 0.03 and 0.10 ⁇ m, more preferably the microparticles are between about 0.03 ⁇ m and about 0.05 ⁇ m. Still more preferably the microparticles are about 0.03 to 0.049 or 0.04 and 0.049 ⁇ m.
  • the amount of vaccine composition of the invention delivered to a patient is not critical or limiting.
  • An effective amount of the vaccine composition is that which will stimulate an immune response against the antigen component, preferably after a single dose or administration and desirably, will result in strong cellular and humoral responses.
  • the amount of compositions delivered may vary according to the immune status of the patient (depending on whether the patient is immunosuppressed or immunostimulated), the judgement of attending physician or veterinarian, whether the composition is used as a vaccine to prevent or treat a disease state, or as a vaccine to prevent tumour formation, or whether the vaccine is used in the treatment of an existing tumour.
  • patients may receive from 1 ⁇ g to 10,000 ⁇ g of the composition of the invention, more preferably 50 ⁇ g to 5,000 ⁇ g, still more preferably 100 ⁇ g to 1,000 ⁇ g, and even more preferably 100 ⁇ g to 500 ⁇ g of the composition of the invention.
  • Adjuvants are not generally required. However, adjuvants may be used for immunization. Suitable adjuvants include alum, as well as any other adjuvant or adjuvants well known in the vaccine art for administration to humans.
  • the vaccine of the invention may be administered by injection, by administration via the oral route, by inhalation or by administration via a mucosal surface or site.
  • the vaccine is administered by means of a gene gun.
  • Ferrous microparticles and gold microparticles if used in accordance with the invention are especially suitable for administration by gene gun, However, other types of microparticles with antigens may be administered in this manner.
  • antigens derived from malaria libraries, DNA or plasmids have been shown to be effectively administered by gene gun in accordance with the procedure described in Smooker PM et al, “Expression library immunisation protects mice against a challenge with virulent malaria.”
  • Vaccination may be by single or multiple dose administration or via prime-boosting.
  • the invention provides a method of eliciting an immune response in a subject said method comprising administering to a subject an immunologically effective amount of a composition comprising at least one antigen associated with microparticles, wherein the microparticles are in the same size range as viruses.
  • the subject may be any human or animal in which it is desired to elicit an immune response.
  • This includes domestic animals, livestock (such as cattle, sheep, horses, cows, pigs, goats, llamas, poultry, ostriches, emus) and native and exotic animals, wild animals and feral animals.
  • An immunologically effective amount refers to an amount sufficient to generate an immune response in the subject, preferably after a single administration. This will vary depending on a number of factors including those discussed above, and may depend on whether the subject is a human or animal, its age, weight and so on.
  • immune response refers to the cellular and humoral responses as described above, and also to the response by cells that assist in mounting or amplifying the immune response as described above.
  • the immune response may be provided by the proliferation and/or expansion of dendritic cells, particularly DEC205+, CD40+ and CD86+ cells.
  • microparticle has the same meaning as given above.
  • the microparticle is between 0.03 and 0.5 ⁇ m, more preferably between about 0.03 and 0.15 ⁇ m, still more preferably between 0.03 and 0.1 ⁇ m.
  • the microparticle is between about 0.03 ⁇ m and 0.05 ⁇ m, even more preferably between about 0.03 and 0.04 or between about 0.19 and 0.049 ⁇ m.
  • the antigen/microparticle composition is particularly adapted to elicit a strong cellular and/or humoral immune response.
  • the invention provides a method of eliciting an immune response in a subject said method comprising administering to a subject an immunologically effective amount of a composition comprising at least one antigen associated with microparticles, wherein the microparticles are in the same size range as viruses and the immune response comprises the stimulation and/or proliferation of dendritic cells.
  • the microparticles are about 40 nm to 50 nm, most preferably 40 to 49 nm in size.
  • the invention provides a method of eliciting a protective immune response to an antigen via a single dose said method comprising administering, once only to a subject, an immunologically effective amount of a composition comprising at least one antigen associated with microparticles, wherein the microparticles are in the same size range as viruses and the immune response comprises the stimulation and/or proliferation of dendritic cells.
  • the microparticles are about 40 nm to 50 nm, most preferably about 40 to 49 nm in size.
  • FIG. 1 Panel A—Differential uptake of particles of different sizes by macrophages compared to dendritic cells. 1000 fluorescent beads of 0.02, 0.1 or 1 micron size per cell were incubated overnight with cultured peritoneal exudate macrophages or bone marrow derived dendritic cells from C57BL/B6 mice and the percentage of fluorescent cells assessed by FACSCan. One of three similar experiments is shown. Similar differences in uptake of different bead sizes were obtained using 10 fold higher bead concentrations, a 3 hour pulse with beads or Balb/c derived antigen presenting cells. Panel B—Virus sized particles are preferentially found in lymph node cells in vivo.
  • 0.1 micron bead uptake was significantly higher than 1 micron in lymph nodes collected also at days 3, 6 or 9 after inoculation;
  • Panels C & D Virus sized particles are taken up preferentially by lymph node NLDC145+ (also known as DEC205+) (panel C) and F4/80+ (panel D) cells.
  • Lymph node cells that have taken up fluorescent particles were assessed by FACScan analysis for co-expression of the dendritic cell marker NLDC145/DEC205 or the macrophage/monocyte marker F4/80. The data shows the percentage of NLDC145+ or F4/80+ cells that have become fluorescent due to bead uptake.
  • FIG. 2 Panel A—Induction of IFN ⁇ producing CD8 and CD4 T cells by immunization with OVA conjugated to beads of different sizes.
  • C57BUB6 mice were immunised intradermally twice (10 days interval) with 100 ⁇ g of OVA conjugated to 0.02,0.04,0.1,0.2,0.5,1 or 2 micron size beads and spleen T cell activity assessed 10 days after the booster immunisation by IFN ⁇ ELISPOT.
  • Responses were measured to the H-2 Kb restricted CD8 T cell epitope SIINFEKL or to whole OVA.
  • Panel B Correlation between T cells with cytotoxic activity and IFN ⁇ secreting T cells by ELISPOT in response to SIINFEKL C57BL/B6 mice were immunised with beads-OVA of different sizes and reactivity to SIINFEKL assessed by IFN ⁇ ELISPOT as described above.
  • PANEL C Antibody production induced by immunisation with OVA conjugated to beads of different sizes. Serum was collected from the mice described in Panel A and serum dilutions tested for OVA specific IgG reactivity by ELISA. Individual mice receiving 0.02, 0.04, 0.1,0.2, 0.5, 1 or 2 micron size OVA-bead immunisation are plotted. One of two similar experiments is shown.
  • FIG. 3 Covalent conjugation of antigen to beads necessary to induce optimal T cell responses.
  • Panel A-Bead-conjugated OVA alone accounts for MHC class I restricted T cell responses
  • C57BL/B6 mice were immunized with OVA conjugated covalently to 0.04 micron beads without prior dialysis (Control) or following dialysis against PBS through a 300 Kd exclusion membrane (Dialysed).
  • the induction of IFN ⁇ producing splenic SIINFEKL specific CD8 T cells was assessed 10 days after one intradermal immunization by ELISPOT.
  • the mean +/ ⁇ SE for 4 mice per group assessed by ELISPOT in duplicate wells is shown.
  • FIG. 4 A single immunization with viral sized beads-OVA is sufficient to induce long lasting high levels of MHC class I restricted T cells.
  • Panel A C57BL/B6 mice were immunized intradermally once, two or three times with beads-OVA (0.1 micron), each time 14 days apart and their IFN 7 response to SIINFEKL examined in each case 10 days after the last immunization by ELISPOT. Three mice were immunised per group and the data shows the mean of duplicate assays on each mouse. One of two similar experiments is shown.
  • Panel B Mice were immunized once with beads—OVA (0.1 micron) and IFN ⁇ responses to SIINFEKL or OVA tested by ELISPOT 12 or 82 days later. Antibody levels to OVA measured as in FIG. 2 were maintained at day 82 .
  • FIG. 7 Phenotypic characterisation of APC taking up 0.04 compared to 1 ⁇ m particles in vivo C57/B6 mice were injected in the footpad with 50 ⁇ l of 0.04 or 1 ⁇ m fluobeads-OVA. Draining popliteal LN were analysed 48 hours later for co-staining of bead positive cells with cell markers of activation and antigen presenting cell lineage, the mean +/ ⁇ SE for 3-14 mice/marker is shown. 0.04 and 1 ⁇ m fluobead+ cells had significantly different expression of DEC205, F4/80, CD40, CD80 and CD86 (p ⁇ 0.05).
  • FIG. 8A Mechanism of viral size particle uptake by DC. Bone marrow derived cultured DC were incubated with phrobol myristate acetate (PMA) at 0 (black),5 (white),10 ⁇ M (grey); amiloride (AML) at 0 (black),1 (white), 3 mM (grey) (white), or cytochalasin D (CDD) at 0 (black), 0.25 (white) or 0.5 ⁇ g/ml (grey) for 30 min and 0.04 ⁇ m-0VA-fluorescent particles added a further 3 hours.
  • PMA phrobol myristate acetate
  • AML amiloride
  • CDD cytochalasin D
  • FIG. 8B Confirmation of the mechanism of uptake DC were incubated with nothing, CDD 1 ⁇ g/ml, filipin (FIL) at 1 ⁇ g/ml or ammonium chloride (AC) at 40 mM for 30 min and 0.04 ⁇ m or 1 ⁇ m fluorescent beads added a further 3 hours. Selective inhibition of caveolae or clathrin coated pit formation has been reported for 1 ⁇ g/ml filipin and 40 mM ammonium chloride, respectively 14-17, 29. The number of fluorescent cells was assessed by FACScan. Data is presented as the mean +/ ⁇ SD of triplicate cultures.
  • FIG. 9 Soluble OVA and 1 um-OVA beads fail to induce comparable protection to 0.05 um-OVA beads.
  • FIG. 10 Viral sized particles do not co-localize with early endosomes (LEFT). Bone marrow derived DC were incubated overnight with 0.1 micron beads-OVA (500 beads/cell), washed gently to remove free beads and prepared for confocal microscopy by spinning onto glass slides. Cells were then fixed in paraformaldehyde, permeabilised with triton and stained the presence of the early endosomal marker Rab4 using a biotin conjugated monoclonal antibody followed by streptavidin-Alexa. Similar results were observed with unconjugated 0.04 and 0.1 micron beads and one of three experiments is shown.
  • LFT early endosomes
  • Fluorescent 0.1 micron beads similarly failed to co-localise with Rab4 staining using DC incubated with beads for 30 minutes or for 3 hours.
  • (RIGHT) Mice were injected intradermally in the hind footpad with 0.1 micron beads-OVA and the draining popliteal lymph nodes dissected 48 hours later for confocal analysis as described above. No co-localization was observed for the Rab4 marker and OVA conjugated or unconjugated 0.1 micron or 0.04 micron beads. One of three experiments is shown. By contrast co-localization was confirmed for the positive control mice immunized with 1 micron sized fluorescent beads.
  • FIG. 11 Protection against tumour PANEL A C57/B6 mice immunised intradermally (ID) once with OVA-VSSP (immunised) or left untreated (naive) were challenged 30 days later subcutaneously with 5 ⁇ 10 6 EG7 (tumour cells). Tumours were measured using calipers. Individual tumour growth curves for 10 animals per group are shown. PANEL B Tumours were induced as above and at day 8 of tumour growth (day 0 of immunisation) 6 animals left untreated (Na ⁇ fve) and 6 immunised ID with OVA-VSSP (Immunised). Individual growth curves are shown day 3-13 after immunisation.
  • FIG. 12 Survival of mice to lethal malaria challenge after VSSP immunisation.
  • FIG. 13 The antigen nm23 was conjugated to 0.05 ⁇ bead (VSP) as described before for the antigen OVA, injected intradermally into mice at 100 ⁇ g/mouse and 10 days later IFN gamma reactivity assessed in the spleens of immunised animals by ELISPOT.
  • VSP 0.05 ⁇ bead
  • SD spot forming units
  • FIG. 14 The cancer antigen nm23 or OVA were conjugated to VSP per standard protocol and 100 ⁇ g/mouse injected intradermally. 10 days later the induction of IL4 secreting cells was assessed by ELISPOT. Data is presented as SFU/million +/ ⁇ SD for three individual mice immunised with each immunogen.
  • FIG. 15A Antibody reactivity to OVA in the sera of mice immunised once intradermally with 0.05 ⁇ m beads conjugated to OVA (VSP-OVA) and assessed 90 days later by ELISA (B group) in comparison to non-immunised controls (A group).
  • FIG. 15B The same sera from mice in FIG. 15A was tested for the presence of OVA specific IgE antibodies by ELISA, in two na ⁇ ve mice (A2 and A3) and three VSP-OVA mice (B2, 3 and 5).
  • FIG. 16 PANEL A Induction of long lasting antibody responses by a single immunisation.
  • C57/B6 mice were immunised once with OVA conjugated to 0.04 ⁇ m beads and sera collected at different time-points.
  • the mean optical density at 405nm +/ ⁇ SE for each group of four animals in OVA specific IgG ELISA is shown. Na ⁇ ve sera is shown as negative control.
  • One of two similar experiments is shown. Similar ELISA results were obtained for total Ig and no IgM or IgA was detected (not shown).
  • CFA Complete Freunds Adjuvant
  • OVA C57/B6 mice were immunised ID once with OVA conjugated to 0.04 ⁇ m beads (black or chequered bar), soluble OVA in PBS (white bar) or with OVA mixed in with 0.04 ⁇ m beads (grey bar).
  • FIG. 17 Prime/boost C57/B6 animals were left untreated (Nothing) or primed intradermally with 100ug of peptide cp 13-32 from MUC1 conjugated to 700ug of KLH in Complete Freunds Adjuvant (cp13), mannan conjugated recombinant MUC1-GST fusion protein (MFP) or 0.1 um VSP conjugated to MUC1-GST fusion protein (VSP). 14 days later animals were boosted intradermally with a million infectious vaccinia virus expressing the MUC1 protein, and reactivity to the epitopes in cp13-32 assessed by IFNg ELISPOT 10 days later. The data shown is the mean number of IFNg producing cells +/ ⁇ SE per million spleen cells averaged for 2-3 animals/group.
  • FIG. 18 Comparison of polystyrene and glass 0.05 um VSP-OVA particles.
  • Polystyrene 0.05 ⁇ m beads were conjugated to OVA as before (PS) and compared to OVA conjugated glass beads in the same way (G1).
  • a different chemical procedure was compared for the glass beads. Briefly, glass beads were weighed and suspended to 2.5%solids in PBS and washed twice. PBS was removed by 5 minute centrifugation in a microfuge. The bead pellet was resuspended in 8% gluteraldehyde in PBS ph 7.4 and mixed gently at room temperature overnight.
  • the beads were then washed 3 ⁇ with PBS resuspended in PBS and 500 ⁇ g of protein per ml was added and mixed gently for 5 hours. The beads were then pelleted and the reaction was stopped by resuspending the pellet in o.5 M ethanolamine and mixing for 30 minutes. The beads were then washed in PBS and used for immunization (G2). Polystyrene (PS) or glass (G1 or G2) VSP-OVA were immunised intradermally at 100 ug/mouise and SIINFEKL specific IFNg secerting T cells quantified 10 days later from spleens by ELISPOT. The data shows individual mean+/ ⁇ SE for three animals per group.
  • FIG. 19 Mode of bead conjugation and immunogenicity.
  • Ovalbumin at 2 mg/ml in 50 mM MES buffer (ph 6.0) was mixed with the polystyrene carboxy modified 0.05 ⁇ m beads (2% solids) for 15 minutes.
  • 1-ethyl-3-(3-dimethylaminopropyl)-carbodiiamide was added to each preparation at 4 mg/ml (pH 6.5) and incubated at room temperature for 2 hours.
  • the standard (Glycine) was quenched with 7 mg/ml of glycine or 20 ⁇ l of 1 M ethanolamine (amine) pH 7.4, or 20 ⁇ l of 1 M aminoacetaldehyde dimethyl acetal (aldehyde) pH 8.0, or 20 ⁇ l of 1 M ethylenediammne (alcohol) pH 7.4.
  • the preparations were incubated at room temperature for approximately 16 hours. All the preparations were dialysed overnight in PBS at 4° C.
  • the aldehyde preparation was quenched further with 20 ⁇ l of 1 M HCL and incubated for 4 hours, and dialysed overnight in PBS at 4° C.
  • mice were immunised with 100 ug intradermally of each one of these VSSP-OVA particles and immunogenicity assessed in spleens. by IFNg ELISPOT to the CD8 T cell epitope SIINFEKL. Results are shown as mean+/ ⁇ SE of SFU/million spleen cells for each animal.
  • mice C57BL/6 and BALB/c 6- to 8-week-old mice were purchased from the Walter and Eliza Hall. Mice were immunized with 100 ⁇ l of antigen conjugated beads intradermally (ID) in the hind footpads.
  • ID intradermally
  • Ovalbumin Ovalbumin
  • EDAC 1-Ethyl-3-(3-DimethylAminopropyl)Carbodiamide
  • Denditric cells were prepared from bone marrow monocytes with minor modifications of previously published methods [5]. Briefly cells were harvested from tibia and long bones of the hind limbs by flushing out the cells from the bone cavities with media, following by red cells lysis. The cells were plated out at 1 ⁇ 10 6 cells/ml in RPMI ((CSL, AUST) supplemented with 10% heat inactivated foetal calf serum (FCS), 4mM L-glutamine, 100 U/ml penicillin, 100mg/ml streptomycin sulphate and 100 ⁇ M ⁇ -mercaptoethanol. and GM-CSF at 1000 units/ml and IL-4 at 0.2 ng/ ml were added.
  • RPMI ((CSL, AUST) supplemented with 10% heat inactivated foetal calf serum (FCS), 4mM L-glutamine, 100 U/ml penicillin, 100mg/ml streptomycin sulphate and 100 ⁇ M
  • the 10 ml cultures were grown for 5-6 days in petri-dishes of a 100mm diameter at 37C in a humid CO 2 incubator. Macrophages were recovered from the intraperitoneal (IP) cavity of mice three days after IP injection of thioglycollate, and cultured for 3 days to enrich for adherent cell fractions as described [6].
  • IP intraperitoneal
  • Bead-antigen conjugation was performed following the manufacturers instructions. Briefly, OVA was diluted to 2.0 mg/ml in 0.05M MES buffer pH 6.0 mixed in a volume ratio of 1:1 with beads of 2% solids/volume. The mixture was rocked gently for 15 minutes and then 4 mg/ml EDAC was added. The pH of the mixture was adjusted to 6.5 with dilute NaOH and the mixture was rocked gently for two to three hours. The reaction was stopped with glycine to a final concentration of 100 mM. After 30 minutes of mixing the preparation was dialysed overnight in the cold in PBS. The preparation was either used immediately or stored at 4 ° C. with 0.01% azide for later use.
  • effector cells for cytotoxicity assays were generated by culturing spleen cells for 7 days at 2.5 ⁇ 10 6 /ml in 2 ml well plates at 37° C. in a humid CO 2 incubator with 10 ⁇ g/ml of the peptide antigen in RPMI medium (CSL, AUST) supplemented with 10% heat inactivated foetal calf serum (FCS), 4mM L-glutamine, 100 U/ml penicillin, 100mg/ml streptomycin sulphate and 100 ⁇ M ⁇ mercaptoethanol.
  • CSL, AUST RPMI medium
  • FCS heat inactivated foetal calf serum
  • 4mM L-glutamine 100 U/ml penicillin
  • 100mg/ml streptomycin sulphate 100 ⁇ M ⁇ mercaptoethanol.
  • Interleukin 2 (10 U/ml, recombinant human IL2, Lymphocult HT, Biotest, UK) was added on day 3.
  • Targets were 51 Cr loaded EL4 cells, alone (background) or pre-pulsed for 1h at 37 ° C. with 10 ⁇ g/ml of the SIINFEKL peptide or EG7 an ovalbumin transformed EL4 cell line. Unless otherwise stated assays were performed in duplicate at an effector:target ratio of 20:1. Spontaneous lysis (with media alone) and maximum lysis (with 5% triton) were set up for all targets in quadruplicate. Supernatants were harvested after 4 h.
  • % Lysis was calculated as 100 ⁇ ((Experimental release-Spontaneous release)/(Maximum release-Spontaneous release). % Specific Lysis (% SL) was % Lysis with peptide- % Lysis with no peptide.
  • CTLp assays were performed as described previously [7 ] CTLp frequencies were determined from a minimum of 32 replicates, for at least 6 effector cell numbers (1 ⁇ 103 ⁇ 1.28 ⁇ 105).
  • Cells were cultured in U-bottomed microtitre trays, with 5 ⁇ 105 mitomycin C treated syngeneic spleen cells, in DMEM supplemented with 10% foetal calf serum, 5 ⁇ M of SIINFEKL or OVA and 10 U/ml rhlL-2. Seven days later, each microculture was assayed for cytotoxicity by replacing 100 ml of culture medium with 100 ⁇ l target cell suspension containing 104 51 Cr-labelled EL4 OR EG7 as targets.
  • Cytotoxic activity was considered to be present if in each well 51 Cr release was found three standard deviations above the mean isotope release from 104 effectors cultured with stimulators only or from stimulator cells with peptide only or rlL2 only.
  • a linear relationship (0.987 ⁇ r2 ⁇ 1) existed between the number of responder cells, represented on a linear scale, and the frequency of negative wells on a logarithmic scale.
  • CTLp frequencies were determined as the inverse of responder cell dose required to generate 37% negative wells[8, 9].
  • CTLp frequency assays were performed three times and the individual frequencies did not differ by more than 20% from the mean value.
  • mice protected in each group was compared using a x 2 test in the Statcalc program in the Epilnfo Version 5.0 package.
  • immunogenicity studies the ELISPOT and Chromium release responses were compared between groups using the Student's t test with the Microsof Excel Version 5.0a package. Linear regression analysis was used to assess correlation between immunogenicity and protection using the SPSS for Windows statistical program package.
  • Draining lymph nodes and spleens were collected from bead immuninuzed mice at various time intervals from 12 hrs post immunization and up to 12 days. Cells were collected and after red blood cell lysis and washing they were prepared for FACscan or confocal analysis.
  • Antibody responses to OVA were measured using ELISA.
  • Polyvinyl chloride microtitre plates were coated with OVA (10 ⁇ g/ ml in 0.2 M NaHCO3 buffer, pH 9,6) overnight at 4° C. The plates were washed 4 ⁇ with PBS/0.05% Tween20 and 4 ⁇ with PBS and then blocked for non-specific binding with 2% bovine serum albumin for 1 h at room temperature. After washing as above serial dilutions of the mouse sera were added and incubated for a further 1 h at room temperature. Non immune mouse serum was used as the negative control.
  • the plates were washed and the bound antibody detected using horseradish-peroxidase-conjugated sheep anti-mouse Ig (Selinus, AUS) and the chromogenic substrate 2,2′′-azino-di(3-ethylbenzthiazoline) sulphonate (Amersham, UK).
  • the absorption at 405 nm was recorded using an EL 312e microplate reader.
  • FIG. 1 a shows that thioglycollate elicited peritoneal exudate macrophages intemalised both 1 m and 0.1 ⁇ m fluorescein-labelled particles (fluo-beads).
  • Immature bone marrow derived dendritic cells by contrast, were found to take in preferentially 0.1 ⁇ m sized fluo-beads. Confocal microscopy was used to confirm the particles were inside of the cells (not shown). This in vitro data suggested viral sized solid particles (VSSP) could also be preferentially taken up by antigen presenting cells in vivo. Fluorescent polysterene protein conjugated particles in a range of sizes (0.02, 0.04, 0.1, 0.2, 0.5, 1 and 2 ⁇ m) were injected intradermally (ID) into the footpad of C57BL/B6 mice and cells from the draining popliteal lymph nodes collected 10 days later for FACScan analysis.
  • ID intradermally
  • FIG. 1 b Particles of the 0.04-0.1 ⁇ m size were taken up preferentially by lymph node cells. Similar results were obtained analysing lymph node cells on days 1, 3, 6 and 10 after particle injection, and with unconjugated particles.
  • VSSPs were efficiently taken up by antigen presenting cell expressing both macrophage and dendritic cell surface markers (FIG. 1 b ). Bone marrow derived dendritic cells in vitro also took up VSSPs. As expected, these cells were of a predominantly myeloid phenotype [12].
  • Efficient VSSP uptake by dendritic cells in vivo suggested their use for targeted antigen delivery and potential as novel vaccines.
  • C57/BL mice were immunised with ovalbumin (OVA) coated particles of 0.02, 0.04,0.1,0.2,0.5,1 or 2 ⁇ m, boosted after 15 days and serum or spleen cells collected 10 days later.
  • FIG. 2 a shows optimal induction of IFNY secreting CD8 T cell induction to the MHC class I restricted SIINFEKL epitope achieved using 0.04 ⁇ m sized particles.
  • CD4 T cells responding to OVA were found at similar precursor frequencies with OVA conjugated particles ranging from 0.04-1 micron.
  • FIG. 2 The results shown in FIG. 2 are based on injecting the same total amount of antigen after conjugation without eliminating residual soluble antigen.
  • FIG. 3 a shows that similar T cell responses were obtained with 0.04 micron VSSPs after the soluble antigen was eliminated by dialysis or ultracentrifugation. Therefore, there was no significant contribution from the soluble antigen to the observed T cell responses. This was further supported by the comparison of conjugated and unconjugated OVA and VSSP mixes.
  • FIG. 3 b shows that only covalently conjugated VSSP induced high levels of SIINFEKL specific T cells. Thus, covalent attachment to the VSSPs was necessary to target OVA into the class I presentation pathway and induce class I restricted T cells in vivo.
  • VSSPs were consistently more immunogenic than 1 ⁇ m particles independently of the level of antigen conjugation
  • VSSP fluo-bead containing vesicles did not co-localise either in bone marrow derived dendritic cells in culture, or in vivo in lymph node cells 24 hours after intradermal VSSP administration.
  • VSSPs therefore may use a processing pathway which differs from that used by both VLPs or larger particles in that it is Rab4 independent and TAP dependent. The mechanism was further investigated in Example 7.
  • C57BL/6 mice were left untreated (na ⁇ ve) or immunized with fluorescent 0.1 ⁇ m fluo-beads intradermally in the foot pad.
  • Popliteal lymph node (LN) were dissected 48 hours after injection and analysed for expression of CD40 by staining with PE conjugated antibodies specific for this marker.
  • FIG. 5 shows an example of the increase in CD40+ cells after immunisation observed by FACScan (33% to 63%). It also shows that many of these cells have taken up the 0.04 ⁇ m beads, in this case we used 0.04 ⁇ m beads with a fluorescent green core.
  • 0.04, 0.05 ⁇ m and 0.1 polystyrene beads alone or conjugated to OVA were able to induce dendritic cells purified from mouse bone marrow to proliferation in vitro. Immature, but not mature (after activation with LPS and TFN-alpha) dendritic cells were susceptible to this activating effect of VSSP (FIG. 6).
  • VSSP particles of 0.04-0.1 ⁇ m in size
  • FIG. 7 shows the proportion of 0.04 ⁇ m bead + or 1 ⁇ m bead+ cells expressing each phenotypic marker.
  • Cells taking up 0.04 ⁇ m beads were mostly NLDC145+, CD40+ and CD86+.
  • CD11 c+, CD4+ and CD8+ cells were 0.04 than 1 m bead+. This highly activated DC phenotype of cells that have taken up 0.04 ⁇ m beads may further explain why the immune responses we observed are so potent, particularly CD8 T cell responses.
  • DC dendritic cells
  • cytochalasin D CDD
  • clathrin pit amiloride, AML
  • caveole mediated internalisation phorbol myristate acetate, PMA
  • DC were cultured in triplicate with PMA, AML, CDD, filipin (FIL) or ammonium chloride (AM) (all from Sigma) at the stated concentrations for 30 min.
  • FIL filipin
  • AM ammonium chloride
  • Amiloride acts by inhibiting Na+-H+ exchange necessary for receptor mediated endocytosis [14].
  • Ammonium chloride inhibited uptake of 0.04 but not of 1 ⁇ m size beads (FIG. 8 b ), confirming a role for clathrin pits in VSSP uptake.
  • Caveole have been suggested to mediate a novel mechanism for uptake of viral particles in DC [15].
  • the inventor results using PMA suggest that caveole were involved in VSSP uptake in DC (FIG. 8 a ). To confirm this, the inventor used another inhibitor of caveole, filipin.
  • Caveole and clathrin pits can convey molecules to endosormal and lysosomal compartments [14, 16].
  • caveole may deliver antigen directly into the cytosol [17] leading to cytoplasmic processing and TAP dependent transport into the endoplasmic reticulum for presentation with MHC class I [1, 18].
  • High IFN ⁇ producing and cytotoxic T cell precursor frequencies are associated with protection against many intracellular pathogens and cancer [21, 22, 23].
  • the inventor immunised C57/BL mice with a single intradermal dose of OVA-conjugated VSSPs and then challenged them with the EG7 tumor cell line, which expresses cytoplasmic OVA and is a target for cytotoxic SIINFEKL specific T cells in vitro.
  • the results show that VSSP immunised mice were completely protected against tumor challenge, whereas all the naive controls developed tumors.
  • antibody levels were also increased following a single administration of antigen conjugated VSSP, similarly to those observed in FIG. 2 c.
  • Examples 10 to 13 described below formally demonstrate that VSSP can be used with a variety of antigens and induce broad immunity comprising both IFN and IL4 producing T cells. High levels of IgG, but not the potentially allergenic IgE antibodies are also induced after a single dose.
  • mice were immunised once with 100 ⁇ g bead(0.05 ⁇ m)-OVA (ID) or left untreated (na ⁇ ve). After 30 days, mice were challenged with 5 ⁇ 10 6 EG7 tumour cell lines. Tumour size was measured using calipers on days 3-13 after immunisation. For regression studies, mice were given the EG7 cells and eight days later divided into groups of similar tumour size distribution. One group was left untreated and the other was immunised with bead-OVA after 3 days (ie day 11 after administration of tumour cell line).
  • tumours can be cured by a single immunisation into a tumour bearing mouse. Tumours were cleared from immunised mice within two weeks after a single injection. This therapeutic ability is highly unusual for any cancer vaccine, and makes this vaccination vehicle highly promising for development of a therapeutic vaccine (FIG. 11B).
  • Mucin-1 or Muc1 is a breast cancer associated antigen. Immunisation once with VSSP-Muc1 protein also inhibited tumour formation in mice challenged with tumour cell lines expressing the breast cancer antigen (see FIG. 11A).
  • polysterene beads of 0.05 ⁇ m in diameter may be used as a vehicle to induce protection against malaria in mice.
  • a lysate from Plasmodium yoelii infected red cells was conjugated to beads and used to immunise mice which were then challenged with a lethal dose of the parasites.
  • VSSP conjugated to OVA 100 ⁇ g/mouse was administered to mice intra-peritoneally, sub-cutaneously, intra-nasally and intra-rectally. 3-4 mice per group were tested 30 days after a single immunisation. Similarly to the intra-dermal, all routes induced T cells which secreted IFNg to SIINFEKL or to OVA by ELISPOT assay (1/50,000 to1/2,000 spleen cells).
  • VSSP-OVA by these other routes induced serum IgA responses, and the intra-rectal and intra-nasal route did not induce detectable IgG (titre ⁇ 1/100) (Table 1).
  • VSSP by the intra-rectal, intra-peritoneal, intra-nasal and subcutaneous routes could therefore also be used to induce protective immunity to diseases where IgA plays a protective role, such as mucosal infections (eg. in lung, cervix or gut).
  • Serum IgG titre Intra-rectal ⁇ 1/100, ⁇ 1/100, 1/1280, 1/640, ⁇ 1/100 1/1280 Intra-peritoneal 1/1640, 1/100, >1/5120, >1/5120, 1/400 >1,5120
  • FIG. 16 shows that strong IgG OVA specific antibody by ELISA (Panel A) and CD8 T cell responses to SIINFEKL by IFNg ELISPOT (Panel B) present one year after a single intradermal immunisation (100 ⁇ g/mouse).
  • Panel B shows in addition that antigen has to be covalently conjugated to the solid particle for optimal immunogenicity.
  • Vaccinia-MUC1 was used to boost responses of animals primed with nothing, peptide cp13-32 (cp13) from MUC1 in complete Freund's Adjuvant (CFA), Mannan conjugated MUC1 (recombinant MUC1-GST fusion protein)(M-FP) or 0.1 ⁇ m VSSP-MUC1 (recombinant MUC1-GST fusion protein)(VSSP).
  • FIG. 17 shows responses to the peptide 13-32 region of MUC1 were enhanced in the VSSP-MUC1 primed, Vaccina-MUC1 boosted group compared to animals that received Vaccinia MUC1 alone (NothingN compared to VSSPN). Therefore VSSP-antigen would be suitable for use in heterologous Prime-boost protocols.
  • OVA-VSSP has an overall negative charge due to use of carboxylate modified nanoparticles and quenching the activated carboxylic acid groups after conjugation of OVA with glycine (Glycine FIG. 19).
  • Glycine FIG. 19 By quenching the reaction with ethanolamine charges can be neutralised except for the net charge of OVA after conjugation (Alcohol, FIG. 19).
  • aminoacetaldehyde dimethyl acetal Aldehyde, FIG. 19
  • composition of the invention provides a way to further improve or optimise vaccines or vaccination strategy that could apply to a variety of infections, cancer or other diseases.
  • the optimal size of the VSSP coincides with that of most known viruses (30-150 nm). Hence, it is believed that use of the VSSPs is biologically significant. From the above observations, the inventor believes that the immune system may be geared to react fully to particles of the size range of the VSSPs. Before the present invention, it was not known or understood that the stimulation of an immune response could depend to a great extent on the size of an immune stimulant that falls within the size range of viruses, especially when epitopes from other pathogens eg bacteria, fungi are considerably large.
  • VLPs i.e. pure antigen not linked to a particle
  • yeast retrotransposon protein Ty
  • VSSPs Use of VSSP as novel vaccines was demonstrated by the ability of a single immunising dose to protect against subsequent challenge with tumor cells in the OVA model.
  • the inventor has also observed broad and strong immunogenicity and protection to an antigen expressed in breast cancer, mucin-1 (MUC-1).
  • MUC-1 mucin-1
  • the intradermal route of administration utilised in their animal studies may be easily implemented in humans.
  • VSSPs may thus offer a particularly attractive and simple strategy for human vaccine development, in particular to diseases where both humoral and cellular immunity participate in generating protection, such as malaria, cancer and viral diseases, notably, AIDS and hepatitis [10, 12, 21, 25-28].
  • the targeting of recombinant antigen to class I presentation pathway also offers the possibility of inducing T cell responses to multiple epitopes, and thus would extend the use of such vaccines in a MHC diverse target human population.
  • the present invention provides an alternative to efficiently deliver antigens to DCs in vivo, leading to the subsequent induction of high numbers of antigen specific CD8 T-cells and immune protection.
  • the ability of the VSSPs within the narrow size range of 0.04-0.05 ⁇ m to induce singularly high CD8 T-cell levels could be the consequence of efficient uptake by APCs or by a potent subset, targeting to the MHC 1 processing pathway and/or direct stimulation of APC function.
  • Uptake of the VSSPs was found to be enhanced in the lymph node, compared to other sizes, and this enhancement was attributed to increased frequencies of particle positive DEC205+ cells, a marker of DCs.
  • DCs are powerful APC and expression of CD40 and CD86 further characterises a subset capable of efficient CD8 T-cell priming. These markers were found in a high proportion of VSSP+ cells. Thus, uptake and selective localisation of VSSP in this potent DC subset in vivo could explain the immunogenicity of the microparticles according to the invention.
  • VSSP of the invention include the ability to induce immune responses including IgA production following administration via a number of routes, and their suitability for prime-boost vaccination strategy.
  • Plebanski Preparation of lymphocytes and identification of lymphocyte subpopulations in Lymphocytes: a practical approach 1999; Edited by Rowland-Jones, S L and McMichael, A J:1-26.

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