EXAMPLE 1

[0081] Materials and Methods used Mice and Immunizations C57BL/6 and BALB/c 6- to 8-week-old mice were purchased from the Walter and Eliza Hall. Mice were immunized with 100 μl of antigen conjugated beads intradermally (ID) in the hind footpads.

[0082] Reagents:

[0083] All reagents including the antigen Ovalbumin (OVA, Grade III) and 1-Ethyl-3-(3-DimethylAminopropyl)Carbodiamide (EDAC) were purchased from Sigma unless otherwise stated. Monoclonal antibodies for FACScan and confocal studies were either purified in house from hybridoma lines on a Protein G column (Pharmacia) or purchased from Pharmigen. FITC conjugated and carboxylated fluospheres 0.02-2 μwere purchased from Molecular Probes and non-fluorescent carboxylated microspheres from Polysciences. Abs to the following markers were used: MHC II, MHC I, CD11c, CD11b, F4/80, NLD-145, CD8alpha, CD40, CD80 and CD86. The anti-Rab4 monoclonal antibody used in confocal studies was the kind gift of Dr. Russel (Peter McCallum Research Institute).

[0084] Antigen Presenting Cells:

[0085] 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 1×106 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 μM β-mercaptoethanol. and GM-CSF at 1000 units/ml and IL-4 at 0.2 ng/ ml were added. The 10 ml cultures were grown for 5-6 days in petri-dishes of a 100mm diameter at 37C in a humid CO2 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].

[0086] 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.

[0087] Cytotoxicity Assays:

[0088] These were performed as described [3]. Briefly, effector cells for cytotoxicity assays were generated by culturing spleen cells for 7 days at 2.5×106/ml in 2 ml well plates at 37° C. in a humid CO2 incubator with 10 μg/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 μM β mercaptoethanol. Interleukin 2 (10 U/ml, recombinant human IL2, Lymphocult HT, Biotest, UK) was added on day 3. Targets were 51Cr loaded EL4 cells, alone (background) or pre-pulsed for 1h at 37 ° C. with 10 μg/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.

[0089] Cytotoxic T cell Precursor( CTLp) Assays:

[0090] 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 μM 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 μl 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.

[0091] ELISPOT IFNγ Assays:

[0092] These were performed as described [3]. Briefly, 100 μl of 5×106/ml freshly isolated spleen cells were incubated with the stated stimuli for 18 hours on mixed acetate plates (MAHA Millipore) pre-coated with an anti-murine IFNγ mAb (R4), (EACC). Duplicate wells were set up for each condition. The media used was RPMI 1640 (CSL) supplemented as described above. After overnight incubation cells were washed off and the plates incubated with a second biotin conjugated mAb to murine IFNγ (XMG.21-biotin, Pharmigen, CA, USA), followed by an extravidin alkaline phosphatase (A-AP) conjugate at 1 μg/ml (Sigma). Spots of alkaline phosphatase activity were detected using a calorimetric AP detection kit (Biorad, Hercules, Calif., USA) and counted utilising a dissection microscope. The data are presented as spot forming units (sfu) per million cells. The SIINFEKL peptide was utilised at 2.5 μg/ml. Statistical analysis in protection studies the number of mice protected in each group was compared using a x2 test in the Statcalc program in the Epilnfo Version 5.0 package. In 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.

[0093] Bead uptake by dendritic cells and macrophages Three day old macrophage cultures grown on microscope slides and five day old dendritic cell cultures were fed with different size fluorescent microbeads for periods of 5 minutes to 24 hours. The cultures were then washed to remove free beads and prepared for FACScan or confocal analysis.

[0094] Analysis of Cells Taking Up Beads In Vivo:

[0095] 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.

[0096] Cell Surface Marker Staining and Flow Cytometry: For surface staining, 5×105 cells were incubated with PE-labelled MoAb to surface markers F4/80 and NLD-145, CD. In cases where the antibody was not directly labelled after two washing steps the cells were incubated with a second PE-labelled antibody specific for the first antibody. Naive serum of the species where the second antibody was raised was used in a blocking step before incubation with the second antibody. After two washes, the cells were washed with PBS/0.2% paraformaldehyde and analysed with a FACScan flow cytometer. (Becton-Dickinson) and CellQuest software. Light scatter gates were set to exclude dead cells and nonlymphoid cells. Cells from bead immunized unstained for any surface marker and cells from naive mice stained with a PE-labelled antibody to a surface marker were used to determine compensation for overlap between the FITC and PE emission spectra.

[0097] Confocal Microscopy of Phagocytosed FITC-Labelled Beads:

[0098] An Olympus scanning confocal microscope was used with a Krypton-Argon laser source equipped with dual fluorescence and transmission detection to determine whether fluoresceinated beads of different sizes were phagocytosed by macrophages or dendritic cells. Serial sections through the samples were acquired at step sizes between 0.5-1.0 microns to determine phagocytosis and analysed on Optiscan Analyzer. Cells were excited at 488 nm and 568 nm for fluorescein and Alexa 594 respectively and detected through 530 nm and 610 nm band pass filters respectively. Throughout acquisition, laser power was kept below saturation levels and gain and offset parameters maintained within individual experiments.

[0099] ELISA Assays:

[0100] Antibody responses to OVA were measured using ELISA. Polyvinyl chloride microtitre plates were coated with OVA (10 μg/ 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.

EXAMPLE 2

Preferential Uptake of VSSP by Antigen Presenting Cells In Vitro and In Vivo

[0101] A number of studies using cells from the macrophage/monocyte lineage have shown particle size dependent phagocytosis, with optimal uptake at a 1 micron diameter [10, 11]. Uptake has been observed in dendritic cells, however, a comprehensive range of particle sizes has not been tested. The inventor was specifically interested to establish whether. protein coated particles of viral size (0.03-0.1 μm), would be efficiently taken up by dendritic cells or macrophages. FIG. 1 a shows that thioglycollate elicited peritoneal exudate macrophages intemalised both 1 m and 0.1 μm fluorescein-labelled particles (fluo-beads). Immature bone marrow derived dendritic cells, by contrast, were found to take in preferentially 0.1 μm 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 μm) 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. Particles of the 0.04-0.1 μm size were taken up preferentially by lymph node cells (FIG. 1b). 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. 1b). Bone marrow derived dendritic cells in vitro also took up VSSPs. As expected, these cells were of a predominantly myeloid phenotype [12].

EXAMPLE 3

VSSP Prime High Precursor Frequencies of Cytotoxic and IFNγ Secreting T Cells as well as Antibodies

[0102] 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 μm, boosted after 15 days and serum or spleen cells collected 10 days later. FIG. 2a shows optimal induction of IFNY secreting CD8 T cell induction to the MHC class I restricted SIINFEKL epitope achieved using 0.04 μm sized particles. CD4 T cells responding to OVA were found at similar precursor frequencies with OVA conjugated particles ranging from 0.04-1 micron. Cytotoxic T cells to SIINFEKL were also induced with VSSPs and correlated with the precursor frequency of IFNγ secreting T cells (R2=0.92)(FIG. 2b). Surprisingly, OVA-specific IgG was also found at the highest precursor frequencies in mice immunised with 0.04-1 μm particles, followed by those immunised with 1 μm particles (FIG. 2c). Similar immunogenicity results were found with using VSSPs without fluorescein. Thus, in contrast to many immunogens and adjuvants promoting preferentially a cellular or a humoral response, VSSPs were capable of inducing high levels of both.

[0103] The results shown in FIG. 2 are based on injecting the same total amount of antigen after conjugation without eliminating residual soluble antigen. FIG. 3a 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. 3b 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. It could be argued that a higher amount of conjugated protein in smaller compared to larger particles could by itself result in higher VSSP immunogenicity. However, this was not so, since: 1) Immunogenicity peaked at 0.04 μm and 0.02 μm sized beads induced little reactivity (FIG. 2), 2) 0.04-0.1 micron VSSPs were consistently more immunogenic than 1 μm particles independently of the level of antigen conjugation, (3) Increasing the immunising concentration of 1 μm particles up to 100 fold (up to 1 mg/mouse) over that used for VSSPs failed to enhance immunogenicity to the levels seen with a range concentrations of antigen conjugated to VSSPs, 4) Immunising with equivalent amounts of bound protein on beads of different sizes, or with the same number of different sized beads, consistently showed VSSPs to be more immunogenic than larger particles across a range of concentrations (0.5-1000 ug total OVA, 0.5-50 ug conjugated OVA and 103-108 beads per animal).

EXAMPLE 4

A novel Pathway for the Uptake and Processing of Particles

[0104] It has been suggested that peptides derived from digestion of exogenous antigen through the class II MHC processing pathway may be regurgitated and subsequently bind to empty class I MHC molecules at the cell surface [1, 10] . This alternative mechanism of class I presentation is independent of transport into the endoplasmic reticulum (ER) mediated by TAP (transporter associated with antigen processing). Hepatitis B surface protein VLPs are processed for class I presentation in macrophages by a TAP independent mechanism [2] . The inventor immunised TAP knockout C57/BL mice with OVA-conjugated VSSPs. No T cell responses above background levels could be detected to SIINFEKL or OVA in TAP-KO animals suggesting VSSP processing for class I presentation was by contrast, TAP dependent. A TAP dependent mechanism of class I presentation of exogenous antigen has been described for proteins adsorbed onto 1 μm particles based on ‘leakiness’ of endocytic vesicles and accidental release of antigen into the cytoplasm [1, 10] . Processing of such large particles taken up by phagocytosis involves an early conjugation step with lysosomes expressing the Rab4 adaptor protein [13]. The inventor used confocal microscopy to determine whether VSSPs would be routed via this pathway. FIG. 4 shows that Rab4 and 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.

EXAMPLE 5

VSSP Induce Expansion of Antigen Presenting Cells In Vivo and In Vitro

[0105] C57BL/6 mice were left untreated (naïve) or immunized with fluorescent 0.1 μm 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.

[0106] The results (FIG. 5) show that 0.04, 0.05 and 0.1 μm polystyrene beads alone or conjugated to OVA were able to increase up to 4 fold the total number of cells recovered from the draining popliteal lymph node after intradermal immunisation. Moreover, they caused 1.5 fold increase in the proportion of NLDC145+ (dendritic cell marker) but not F4/80+ (monocyte/macrophage marker) cells. They also enhanced >1.5 fold the proportion of lymph node cells expressing the activation molecules CD40 and CD86. 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 μm beads, in this case we used 0.04 μm beads with a fluorescent green core.

[0107] 0.04, 0.05 μm 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).

[0108] These data together suggest that particles of 0.04-0.1 μm in size (VSSP) have the unsuspected and previously unknown ability to stimulate antigen presenting cells, including dendritic cells, and specifically cells expressing potent co-stimulatory molecules like CD40 and CD86 to proliferate and expand. This could further explain why they are so potent. Moreover, it suggests a mechanism by which VSSP may have an adjuvant effect (see Example 14 below) even for responses to antigen when it is not chemically conjugated to them.

EXAMPLE 6

Extended Phenotype of Cells Taking Up VSSP Rapidly After Injection

[0109] To assess further which cells take up VSSP rapidly after intradermal footpad injection (and thus may be responsible for subsequent activation of immunity), the draining popliteal draining lymph node was dissected. The phenotype of cells that had taken up 0.04 μm VSSP-OVA with a fluorescent core was then analysed by FACScan and compared to identical particles but which were 1 μm in size. FIG. 7 shows the proportion of 0.04 μm bead + or 1 μm bead+ cells expressing each phenotypic marker. Cells taking up 0.04 μm beads were mostly NLDC145+, CD40+ and CD86+. In addition more 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 μm beads may further explain why the immune responses we observed are so potent, particularly CD8 T cell responses.

Example 7

Uptake and Processing of Microparticles.

[0110] To address the mechanism of 0.04 μm bead-OVA uptake by dendritic cells (DC), bone-marrow derived DC were incubated with inhibitors of phagocytosis (cytochalasin D, CDD); clathrin pit (amiloride, AML) or caveole mediated internalisation (phorbol myristate acetate, PMA) [14, 15]. 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. OVA-fluorescent 0.04 μm beads were added for a further 3 hours and uptake was quantified by FACScan.

[0111] PMA and AML both decreased 0.04 μm beads-OVA uptake by DC, whereas CCD failed to cause any inhibition (FIG. 8a). Inhibition by PMA and AML was additive (FIG. 8a). This suggested clathrin pits and caveole could both be involved in VSSP uptake.

[0112] Amiloride acts by inhibiting Na+-H+ exchange necessary for receptor mediated endocytosis [14]. The inventor tested additionally ammonium chloride which inhibits the assembly of clathrin pits by interfering with cytosol acidification [14]. Ammonium chloride inhibited uptake of 0.04 but not of 1 μm size beads (FIG. 8b), 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. 8a). To confirm this, the inventor used another inhibitor of caveole, filipin. Filipin acts through cholesterol sequestration, whereas PMA affects the phosphorylation events regulating caveole internalisation [14, 15, 16, 17]. Similarly to PMA, filipin blocked 0.04 μm but not 1 μm bead uptake, confirming the inventor's hypothesis (FIG. 8b).

[0113] Caveole and clathrin pits can convey molecules to endosormal and lysosomal compartments [14, 16]. Alternatively, 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].

[0114] These results, together with those shown in Example 4, indicate a novel pathway for processing of antigens presented in accordance with the present invention. The immunogenic composition of the invention appears to be taken up by antigen presenting cells via caveole and/or clathrin pits, after which the antigens are processed by Rab4 independent and TAP dependent pathways for MHC class I presentation. This observation is novel in that the ability of caveole to induce the TAP-dependent antigen processing pathway and CD8 cells has not been reported before.

EXAMPLE 8

Comparison of Protective Efficacy

[0115] 50 nm (i.e. 0.05 μm) (VSSP) OVA conjugated particles were compared directly with OVA alone or 1000 nm (i.e. 1 μm) OVA conjugated particles, for ability to protect mice against subsequent subcutaneous challenge with 100,000 tumour cells (EL4) expressing OVA. All mice were immunised with 100 μg of either of the above (or nothing=naïve) intra-dermally once and then challenged with tumour 30 days later. FIG. 9a shows that VSSP-OVA prevented completely the growth of OVA expressing tumours, whereas OVA alone or with 1000 nm beads had a non-significant effect on protection.

EXAMPLE 9

A single VSSP Immunization Induces High Levels of Immunity and Protects Against Tumor Challenge

[0116] Intradermal immunisation with OVA-conjugated VSSPs induced IFNγ producing and cytotoxic SIINFEKL specific T cells (FIG. 10a and 1a and b). Additional immunizations did not further increase reactivity (FIG. 10b and 10c). T cells could be maintained at high precursor frequencies 82 days after immunization (FIG. 10d). Antibody responses were similarly maintained (FIG. 16). The CD8 T cell precursor frequency levels achieved by a single VSSP immunization were higher than those observed for single, or even multiple doses of VLP particles and are only comparable to the highly efficient heterologous prime/boost regimes [1, 2, 3, 19, 20]. High IFNγ 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. In addition, antibody levels were also increased following a single administration of antigen conjugated VSSP, similarly to those observed in FIG. 2c.

Further Work on VSSP Vaccines

[0117] 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.

[0118] The effectiveness of VSSP for therapy is shown in an additional two models. 1) 100% clearance of established tumours (see Example 10) and 2) protection against lethal malaria after a single administration of the vaccine.(see Example 11). This further confirms VSSP as an unusually potent and flexible vaccination protocol to develop single dose vaccines against a variety of diseases. Moreover, on the basis of these findings the inventor believes that VSSP may be used for therapy as well as prevention of cancer

EXAMPLE 10

Clearance of Established Tumours

[0119] The inventor has observed that a single immunisation with beads-OVA protects completely against subsequent challenge with tumours expressing OVA.

[0120] C57/B6 mice were immunised once with 100 μg bead(0.05 μm)-OVA (ID) or left untreated (naïve). After 30 days, mice were challenged with 5×10 6EG7 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).

[0121] The inventor now shows that already established 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).

[0122] 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).

EXAMPLE 11

Protection Against Malaria

[0123] The inventor demonstrates that polysterene beads of 0.05 μm 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.

[0124] Blood was collected from C57BU6mice infected with P. yoelii 17XL at 50% parasitaemia. Red blood cells (RBC) recovered after centrifugation 800 g for 15 min were freeze/thawed three times and sonicated (lysate). Lysate was conjugated to 0.05 μm particles as described above. Beads-lysate, beads-OVA or lysate alone were injected ID. Immunised or naive C57BL/6 mice were challenged two weeks later intra-peritoneally with 1,000,000 P. yoelii 17XL infected RBC.

[0125] All the animals survived the challenge, whereas 60% of the animals immunised with the lysate alone (without bead conjugation) failed to control the infection and died (FIG. 12). This is the first demonstration of a single dose vaccine being able to confer protection against blood-stage malaria. A single dose vaccine particularly attractive for malaria and other diseases present extensively in the Third world, since it simplifies administration and distribution of the vaccine, ensuring wide population coverage.

EXAMPLE 12

Cellular Immunity

[0126] 1) Polysterene beads of 0.05 μm in diameter conjugated to antigens other than OVA, such as the cancer antigen nm23 also induce strong cellular immunity as evidenced by the induction of high levels of IFN gamma secreting T cells (FIG. 13).

[0127] 2) As well as inducing cellular immunity, beads-OVA or beads-nm23 induced high levels of IL4 (FIG. 14). This lymphokine promotes the production of antibodies, which may explain why we observe good antibody induction as well as cellular immunity (which requires IFN gamma) was observed.

EXAMPLE 13

Antibody Immunity

[0128] 1) Polysterene bead of 0.05 μm in diameter conjugated to OVA induced, after a single intradermal (ID) injection induced high titres of IgG antibody (FIG. 15A).

[0129] 2) Despite high IgG levels, there are no detectable IgE levels. Therefore, this vaccine is demonstrated formally to have no risk of inducing potentially damaging (since they are involved in allergy) IgE responses (FIG. 15B).

EXAMPLE 14

Administration Via Different Routes and Induction of IgG and IgA Antibodies

[0130] With a view to identifying useful routes of administration of the vaccine in humans, as well as intra-dermally as described above, VSSP conjugated to OVA (100 μg/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). Surprisingly, in contrast to the initial observations using intradermal injection which induced high levels of IgG but little or no IgA, 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). 1

Unusually High IgG Responses After Two Doses

[0131] Immunisation twice with VSSP-OVA (100 μg/mouse 14 days apart) led to the generation of surprisingly high serum Ig and IgG antibody titres of >1/500,000 as assessed by ELISA. Similar results were obtained for specific IgG antibodies after two immunisations with the breast cancer antigen nm23 and the malaria antigen MSP4/5, when conjugated to VSSP.

EXAMPLE 15

Long-Lasting Responses

[0132] Responses to VSSP-OVA were surprisingly long-lasting. 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 μg/mouse). Panel B shows in addition that antigen has to be covalently conjugated to the solid particle for optimal immunogenicity.

EXAMPLE 16

Heterologous Prime-Boost

[0133] 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 μm 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.

EXAMPLE 17

Material Composition of the Solid Core for VSSP

[0134] The inventor's hypothesis that the 0.04-0.05 μm size of solid core is the principal determinant of VSSP immunogenicity predicts that particles made of material other than polystyrene would be highly immunogenic within this size range. Thus, she compared immunogenicity in mice after a single immunisation intradermally with 0.05 μm particles made of polystyrene (PS) or of glass (G1 or G2) and conjugated to OVA using the same chemical procedure (G1) or binding using. glutaraldehyde (G2). FIG. 18 shows that VSSP made of either polystyrene or glass were similarly highly immunogenic inducing a high precursor frequency of IFNg producing T cells to SIINFEKL by ELISPOT. Therefore, the solid core of VSSP for protein conjugation can be provided by glass as well as polystyrene, and it is anticipated that other materials for the solid core will also be functional, for example PLG.

EXAMPLE 18

Conjugation of Antigen to VSSP

[0135] The results show that mixing antigen with 0.05 μm particles makes it more immunogenic than antigen alone, but that covalent linkage is necessary for optimal immunogenicity. The chemical procedure used to conjugate antigen to VSSP could therefore theoretically be a determinant of immunogenicity. Specifically the overall charge of the particle could promote interaction with specific serum or other endogenous proteins. These in turn could theoretically promote uptake by dendritic cells, and cause high immunogenicity. To test for this, mice were immunised with 50nm (i.e. 0.05 μm) particles having different charges on the surface. 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). By quenching the reaction with ethanolamine charges can be neutralised except for the net charge of OVA after conjugation (Alcohol, FIG. 19). By quenching with ethylenediamine a positive charge is introduced (Amine, FIG. 19). By quenching with aminoacetaldehyde dimethyl acetal (Aldehyde, FIG. 19) potentially useful aldehyde groups can be introduced. All three modifications of the conjugation protocol resulted in highly immunogenic particles, with amine and alcohol modifications being comparable to glycine, and aldehyde slightly less immunogenic. Therefore it is highly unlikely immunogenicity results from non-specific adsorption of serum proteins as the introduction of opposite charges to the particle results in similar immunogenicity. Moreover, some alternative modifications to the conjugation procedure, and changes in charges can be introduced with no decrease in immunogenicity.

DISCUSSION

[0136] In view of the results above, the 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.

[0137] The optimal size of the VSSP coincides with that of most known viruses (30-150 nm). Hence, it is tempting to speculate 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. Indeed, antigens targeted through this pathway elicited surprisingly broad (comprising both humoral and cellular arms of the immune response) and strong responses (inducing rapidly long lasting high effector T cell precursor frequencies) suggesting the immune system may be geared to react fully to particles of viral size. Previous studies utilising VLPs (i.e. pure antigen not linked to a particle) comprised of HepB surface proteins, or the yeast retrotransposon protein (Ty) have also shown broad and long lasting immunity induced by a single immunizing dose, although responses were still 10-100 fold lower than with VSSPs [1, 2, 3, 19, 20]. However it has been assumed that characteristics other than size alone, such as their lipid or mannan content, or membrane biding proteins are responsible for the ability of VLPs to induce class I restricted T cell responses [1]. Not wishing to be bound by theory, the inventor considers that the combination of efficient targeting to antigen presenting cells such as dendritic cells in vivo by VSSPs, followed by potential slow antigen release by proteolysis from VSSPs, may have generated particularly powerful immunogens.

[0138] 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). 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.

[0139] At present, priming of DCs for effective stimulation of CTLs is by ex vivo pulsing of DCs but this is expensive and logistically difficult. 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 μm 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.

[0140] Other advantages of the 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.

[0141] Further studies will involve the use of the VSSPs to determine the physiological mechanisms that make them elicit the unique immune response obtained.

[0142] It is to be understood that various other embodiments of the invention that is described herein may be made by those skilled in the art without departing from the central concept underlying the invention.

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