AU2006204620B2 - Composition comprising immunogenic nanoparticles - Google Patents

Description

AUSTRALIA

Patents Act 1990 (Cth) Complete Specification (Divisional) The Austin Research Institute Invention Title Composition Comprising Immunogenic Nanoparticles The invention is described in the following statement: Blake Dawson Waldron Patent Services Level 39, 101 Collins Street Melbourne VIC 3000 Telephone: 61 3 9679 3065 Fax: +61 396793111 Ref: WJP GHE 03 1401 1402 200994512_1 COMPOSITION COMPRISING IMMUNOGENIC NANOPARTICLES The present invention relates to immunogenic compositions, vaccine compositions, methods of eliciting immune responses in a subject and methods of producing the compositions.

BACKGROUND OF THE INVENTION Manipulation of the immune systems of humans and animals is a recognised manner of avoiding or treating certain diseases or conditions.

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 Tcell responses. The latter include T-helper cells (TH) and cytotoxic T-lymphocytes (CTL).

In instances of viral infection e.g. polio or hepatitis, 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. The 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 TH cells recognise antigenic peptides bound to MHC II molecules.

By contrast, 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. In addition, 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.

Although there are many vaccines on the market there is a need to produce more effective and broad ranging vaccines for a number of diseases or conditions. There also remains a need for protection against infective agents or pathogens against which vaccines are 2009945121 currently unavailable or ineffective. In addition, there is a need for effective, single-dose vaccines, which are particularly desirable for economic reasons, for ease of delivery, and for patient or subject compliance.

Most vaccines suffer from the disadvantage that they are not able to induce an optimal combination of the various types of humoral and cellular responses so as to be immunologically effective. For instance, some vaccines only stimulate antibody responses when both antibody and cellular responses would be more efficacious. In other instances, multiple doses of the vaccines eg booster shots are required in order to attain protection against the relevant infective agent.

In some other instances, 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. Thus, vaccines that can generate a high IgA secretory immune response without enhancing IgE production would also be valuable.

In yet other instances, although a vaccine results in stimulation of APCs, the degree of immune stimulation is sub-optimal. For example, 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.

Further, with regard to vaccines extracellular antigens traditionally do not enter the MHC-I processing pathway in most cells. In general, the production of CTL immunity using nonliving vaccines is unlikely although alternative routes of processing and presentation for class 1 have been proposed in APC through the uptake of apoptotic cells, immune complexes and particles Non-infective viral like particles (VLP) composed of the surface Hepatitis B protein or yeast retro-transposon protein particles have been shown to be 200994512_1 cefficiently processed for MHC I presentation by macrophages to induce CD8 CTL Sresponses in vitro and in vivo VLPs are multimeric, lipid-containing protein particles the lipid content of which comprises more than 50% of the dry weight.

However, since Hepatitis B core protein particles fail to be immunogenic, and have a lower O 5 lipid content, it has been proposed that VLP are immunogenic not by virtue of size, but by INO 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, INO possibly by damaging the cell membrane, and thus gain entry into the cytoplasm.

C Over the past 15 years, much focus has been on DNA vaccines as a simple method to induce both humoral and cellular immune responses as well as protection from challenge in animal models, while they are considered to be inexpensive, extremely stable and considerably safer than attenuated viral vaccines [30-33]. Their ability to induce CD8 T cell responses makes DNA vaccines very attractive for anti-viral as well as anti-cancer immunisation strategies However, despite their potency in small animal models, considerably lower efficacy of naked plasmid DNA (pDNA) vaccines has been observed in human clinical trials, where substantially higher doses of DNA are required to induce protective immune responses [35-38]. Therefore, safe and effective DNA delivery strategies are needed to overcome these limitations and enhance the potency of DNA vaccines. Besides other optimisation strategies (reviewed in the use of particles as DNA delivery vehicles has emerged as a promising new approach to DNA vaccination Particularly DNA adsorption to the surface of cationic particles, such as polylactides and other polymers, through electrostatic interactions has shown to improve DNA vaccine efficacy [40-43].

The use of 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). 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.

200994512_ I However, the difficulties with this form of vaccine composition include antigen stability, the size of the spheres and the antigen-release kinetics, all of which still need to be resolved so as to produce a vaccine with good antigenicity and lasting immunogenicity, and to produce a vaccine that can be manufactured and administered economically In US patent no. 4,225,581, a composition comprising a mixture of heterogenous particles ranging in size is described as being useful for delivering antigens that are adsorbed onto the surface of the polymeric particles to the body. However, the successful delivery, antigenicity and immunogenicity of such a vaccine was not illustrated or shown.

Specifically, there was no reference to the induction of CD8 T cell responses, or even processing into the MHC class I presentation pathway. The polymeric material of the particles would be expected to have similar characteristics as PLA or PLGA nanoparticles discussed above.

Thus, it was not known prior to the present invention if the size per se of particles in the viral range administered as part of vaccines could induce immunogenic responses.

In work leading up to the present invention, the inventor has surprisingly found that nanoparticles about the same size as viruses associated with an antigen provide strong cellular and humoral antibody responses in subjects.

SUMMARY OF THE INVENTION In a first embodiment the invention provides an immunogenic composition comprising at least one nucleic acid molecule in association with nanoparticles, wherein the nucleic acid molecule encodes at least one antigen, and wherein the nanoparticles are about 0.02pm to about 0. 1pm in diameter. In a preferred embodiment the nanoparticles are about 0.04pm to about 0.05pm in diameter. In yet a further preferred embodiment the nanoparticles are about 0.05pm in diameter.

The term "comprising" used in relation to the immunogenic composition means that the composition includes the antigen and nanoparticles. It may also include other components.

200994512 1 N The term "antigen" refers to any molecule, moiety or entity capable of eliciting an immune t response. This includes cellular and/or humoral immune responses. Depending on the intended function of the composition one or more antigens may be included.

The antigen may be a peptide, protein, lipid, carbohydrate, nucleic acid or other type of molecule or a combination of any of these.

(N

z The antigen may be derived from a pathogen, tissue, cell, organ or molecule depending on CNi the intended purpose of the composition, and may be a purified antigen, or be of \0 Srecombinant origin produced in suitable vectors such as bacteria, yeast or cell cultures. The CN 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. Specifically, examples of the antigens envisaged by the present invention are as follows: pollens, hepatitis C virus, (HIV) core, El, E2 and NS2 proteins, antigens from Plasmodium species such as P. vivax and other Plasmodium species including P.

falciparum circumsporozoite protein (CS) and human Plasmodium-falciparum, -vivax, ovalae, -yoelli and malariae, TRAP, MSP-1, MSP-2, MSP-3, MSP-4, MSP-5, AMA-1, RESA, SALSA, STARP, LSA1 and LSA3, HIV-gpl20/160 envelope glycoprotein, streptococcus surface protein Ag, influenza nucleoprotein, haemagglutinin-neuraminidase surface infection, TcpA pilin subunit, VP1 protein. LMCV nucleoprotein, Leishmania major surface glycoprotein (gp63), Bordetella pertussis surface protein, rabies virus G protein, Streptococcus M protein, Staphylococcal proteins or Helicobacter pylori proteins, Syncyticial virus (RSV) F or G proteins, Epstein Barr virus (EBV) gp340 or nucleoantigen 3A, haemagglutinin, Borrelia burgdorferi outer surface protein (Osp) A, Mycobacterium tuberculosis 38kD lipoprotein or 30kD protein (Ag85), lOkD or 65kD proteins, Neisseria meningitidis class 1 outer protein, Varicella zoster virus IE62 and gpl, Rubella virus capsid protein, Hepatitis B virus pre S1 ag, Herpes simplex virus type I glycoprotein G or gp D or CP27, Murray valley encephalitis virus E glycoprotein, Hepatitis A virus VP1, polio virus capsid protein VP1, VP2, VP3 and VP6, chlamydia trachomatis surface protein, Hepatitis B virus envelope Ag pre S2, Human rhinovirus (HRV) capsid, papillomavirus peptides from oncogene E6 and E7, Listeria surface protein, Varicella virus envelope protein, Vaccinia virus envelope protein, Brucella surface protein, Rotavirus VP-3, VP-4, VP-5, VP-7 and VP-8, a combination of one or more of said antigens, an amino acid subunit of said antigens 200994512 1 comprising five or more amino acids in length or combinations of one or more of said subunits.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 associated with the nanoparticles.

The antigen may also be derived from any tumour type or malignancy. Examples of cancer types from which the antigens may be derived are breast, lung, pancreas and colon cancer and melanoma. Some further examples of 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.

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.

The term "in association with" refers to an association between the nanoparticle and the antigen. This may be by adsorption, conjugation or covalent coupling, or by electrostatic interaction. Preferably the antigen associates with the nanoparticles through electrostatic interaction between the antigen and poly-L-lysine (PLL) coated on the surface of the nanoparticles.

The term nanoparticle refers to a small particle. This may be in the form of a bead or sphere or any other suitable shape.

The term "virus sized particles" (VSP) is used in this document to describe certain embodiments of the immunogenic composition of the invention. It should be understood that the term VSP has only been adopted for convenience and does not limit the invention to the size of known viruses. For Example, particles of the same size as unknown viruses are also contemplated by the invention.

Preferably the nanoparticle has a solid core providing stability to the associated antigens as distinct from the microspheres of the prior art which are hollow or encapsulate molecules.

2009945121 For convenience these are referred to herein as virus sized solid particles (VSSP) where the antigen is present on the outside of the particle. The particles used to make VSSP are available from the manufacturer and are substantially of uniform size within ±10% of the stated size).

The term "solid core" means substantially solid the particles are not hollow). The nanoparticle may be composed of any suitable material so long as it does not detract from the function of the immunogenic composition. Thus the nanoparticles may be made from materials such as latex, ferrous molecules, gold (such as gold nanoparticles), glass, calcium phosphate, polystyrene or biodegradable and biocompatible polymers such as PLG (Polylysine Preferably, the nanoparticles are composed of polystyrene, PLG or gold.

Most preferably, the nanoparticles are made from polystyrene.

The nanoparticle is in the same size range as known viruses. This means that the nanoparticle is preferably about 0.020g.m to 0.10 tm in diameter, more preferably about 0.04pm to about 0.05pm in diameter, and even more preferably about 0.05pm in diameter.

It is also preferred that the nanoparticle 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.

In a preferred embodiment, a population of nanoparticles to be used in accordance with the invention, eg in one dose of vaccination, is of a uniform size. This means that the majority of the particles in a given population are of the stated size.

Preferably the nanoparticle/antigen composition is particularly adapted to elicit a cellular and/or humoral immune response. The cellular response is preferably selected from the group consisting of activation, maturation or proliferation of TH cells, in particular IFN and IL4 producing T cells, CTLs, particularly CD8 CTL and B cells. Preferably the nanoparticle/antigen composition elicits mechanisms for MHC class I presentation of antigens which are taken up by a hitherto unknown mechanism involving caveole and/or clathrin pits for further processing by Rab 4 independent and TAP dependent processes as explained in Examples 4 and 7 herein. The humoral response is preferably selected from the group consisting of IgA, IgD, IgG, IgM and subclasses thereof.

200994512_1 Cells which assist in mounting or amplifying an immune response may also be stimulated by the composition. These included but are not limited to APCs such as DCs of both myeloid or lymphoid origin, and macrophages. The maturation, activation or proliferation of such cells are contemplated, as are the co-stimulatory ligands or molecules on such cells that interact with T-cells, eg CD 40, CD 80 and CD 86.

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. For example, the composition may be used in the treatment or prophylaxis of certain cancers.

In another embodiment the invention provides a vaccine composition comprising at least one nucleic acid molecule in association with nanoparticles, wherein the nucleic acid molecule encodes at least one antigen, and wherein the nanoparticles 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.

Thus, in one embodiment, the invention provides a single-dose vaccine composition comprising at least one nucleic acid molecule in association with nanoparticles, wherein the nucleic acid molecule encodes at least one antigen, and wherein the nanoparticles are about 0.02pm to about 0.10pm in diameter. Preferably the nanoparticles are about 0.04pm to about 0.05pm in diameter, and even more preferably about 0.05P m in diameter.

By "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 orally, by inhalation, or by administration through a mucosal surface or site.

Preferably, the antigen associates with the surface of the nanoparticles. The nanoparticles are most preferably made of polystyrene, PLG, glass, calcium phosphate or gold. In a preferred embodiment, each antigen for use in accordance with the invention associates with nanoparticles of a uniform size.

200994512 1 In a further embodiment, the invention provides a single-dose vaccine composition that is capable of mounting a humoral and a cellular immune response, the composition comprisingat least one nucleic acid molecule in association with nanoparticles, wherein the nucleic acid molecule encodes at least one antigen, and wherein the nanoparticles are about 0.04pm to 0. 10pm in diameter, preferably 0.04p m to 0.05p m in diameter, and even more preferably 0.05pm in diameter.

The cellular response is preferably selected from the group consisting of stimulation, maturation or proliferation of TH cells, CTLs and B cells. The humoral response is preferably selected from the group consisting of IgA, IgG, IgM and subclasses thereof.

Preferably, IgG, IgA and/or IgM responses are stimulated.

Cells which assist in mounting or amplifying an immune response may also be stimulated by the composition. These included but are not limited to APCs such as DCs of both myeloid or lymphoid origin, and macrophages. The maturation, activation or proliferation of such cells are contemplated.

The term "comprising" has the same meaning given above.

The term nanoparticle has the meaning given above. Preferably the nanoparticle is adapted to be taken up by antigen presenting cells in an animal.

The terms "antigen" and "associated with" have the meanings given above. Any suitable antigen may be used depending on which condition/disease it is intended to vaccinate against.

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 prevert 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. By way of example, 2009945121 patients may receive from 1 gLg to 10,000 gg of the composition of the invention, more preferably 50 pg 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. In one embodiment, the vaccine is administered by means of a gene gun. Ferrous nanoparticles and gold nanoparticles if used in accordance with the invention are especially suitable for administration by gene gun, However, other types of nanoparticles with antigens may be administered in this manner. For example, 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." Vaccine, 18(22): 2533-2540, 2000, incorporated herein in its entirety by this reference. Vaccination may be by single or multiple dose administration or via prime-boosting.

In a further embodiment 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 nucleic acid molecule in association with nanoparticles, wherein the nucleic acid molecule encodes at least one antigen, and wherein the nanoparticles are about 0.02Vm to about 0. 10pm in diameter, preferably about 0.04P m to about 0.05pm in diameter, and even more preferably about 0.05pm in diameter.

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.

The term "comprising" has the same meaning as given above.

2009945121 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.

The terms "antigen" and "associated with" have the same meanings as given above.

The term "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. In particular the immune response may be provided by the proliferation and/or expansion of dendritic cells, particularly DEC205+, CD40+ and CD86+ cells.

The term nanoparticle has the same meaning as given above. Preferably the antigen/nanoparticler composition is particularly adapted to elicit a strong cellular and/or humoral immune response.

In a preferred embodiment 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 nucleic acid molecule in association with nanoparticles, wherein the nucleic acid molecule encodes at least one antigen, and wherein the nanoparticles are about 0.04p m to 0.05V m in diameter, nanoparticlesand the immune response comprises the stimulation and/or proliferation of dendritic cells.

In another embodiment 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 nucleic acid molecule in association with nanoparticles, wherein the nucleic acid molecule encodes at least one antigen, and wherein the nanoparticles are about 0.02pm to about 0.10pm in diameter, preferably about 0.04pm to about 0.05pm in diameter, and even more preferably about 0.05pm in diameter, and wherein the immune response comprises the stimulation and/or proliferation of dendritic cells.

As previously stated, the immunogenic composition of the invention may be used in treatment, prophylaxis or prevention of the disease or condition caused by, or associated 200994512_ I with contact with the antigen. For example, the composition may be used in the treatment or prophylaxis of certain cancers.

In another embodiment the invention provides a method of in vivo delivery of an antigen to dendritic cells in order to elicit an immune response said method comprising administering to a subject an immunologically effective amount of a composition comprising at least one nucleic acid molecule in association with nanoparticles, wherein the nucleic acid molecule encodes at least one antigen, and wherein the nanoparticles are about 0.02pm to about 0.04pm in diameter, preferably about 0.04pm to about 0.05pm in diameter, and even more preferably about 0.05pm in diameter, and the immune response comprises the stimulation and/or proliferation of dendritic cells.

The invention also extends to a method of producing an immunogenic nanoparticle/antigen composition comprising contacting with one or more antigens such that the nanoparticles and antigens become associated. Those skilled in the art will be familiar with the techniques used to produce such a composition.

DETAILED DESCRIPTION OF THE INVENTION The invention will now be described with reference to the following non-limiting examples and figures.

Figure 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 C57BLB6 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. LEFT C57BL/B6 mice were inoculated intradermally in the footpad with 50p.l of 0.1% solution of fluorescent beads of different sizes (0.02, 0.04, 0.1, 0.2, 0.5, 1 and 2 micron) and the draining popliteal lymph nodes removed 10 days later to assess the percentage of cells that have taken up the beads by FACScan. The data is shown as mean percentage of fluorescent cells Standard error 200994512 1 S(SE) of triplicate samples. The 0.04 and 0.1 micron particle sizes had significantly higher

J

uptake to any other sized particles (p>0.001). In similar experiments using comparatively only 0.1 or 1 micron beads, 0.1 micron bead uptake was significantly higher than 1 micron Sin 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+) O (panel C) and F4/80+ (panel D) cells. Lymph node cells that have taken up fluorescent Sparticles 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 N percentage of NLDC 145+ or F4/80+ cells that have become fluorescent due to bead uptake.

Figure 2: Panel A Induction of IFN y 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|lg 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 IFNy ELISPOT. Responses were measured to the H-2Kb restricted CD8 T cell epitope SIINFEKL or to whole OVA. In the case of assessing reactivity to OVA spleen cells were depleted from CD8 T cells before the assay with magnetic beads (Dynabeads) to quantify OVA reactive CD4 T cells. SIINFEKL was used at plg/ml and OVA at 25 gg/ml. One of three similar experiments is shown. Two mice per group were immunized for each bead size. ELISPOT cultures were done in duplicates and average values of spot forming units (SFU) per million cells tested are shown. The standard deviation (SD) was always less than 20% of the mean. Panel B Correlation between T cells with cytotoxic activity and IFNy 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 IFNy ELISPOT as described above. In addition, the number of SIINFEKL specific T cells with cytotoxic activity was determined in parallel by limiting dilution analysis. Chromium loaded EL4 cells alone or pre-pulsed with 2.5 gig/ml of SIINFEKL were used as targets. The data shown illustrates the strong correlation (R square= 0.9254) found between the two assays. One of two similar experiments is shown.

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, 200994512_1 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.

Figure 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 C57BLB6 mice were immunized with OVA conjugated covalently to 0.04 micron beads without prior dialysis (Control) or following dialysis against PBS through a 300Kd exclusion membrane (Dialysed). The induction of IFNy 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. PANEL B Co-administration of beads and soluble OVA is not sufficient to induce optimal MHC class I restricted T cells responses. C57BUB6 mice were immunized with OVA conjugated covalently to 0.1 micron beads (Beads conjugated-OVA) or mixed with OVA prior to injection (Beads/OVA mixed). The induction of IFNy producing splenic SIINFEKL specific CD8 T cells was assessed 10 days after one intradermal immunization by ELISPOT. The mean T cell precursor frequency for 2 mice per group assessed by ELISPOT in duplicate wells is shown.

Figure 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 IFNy 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 IFNy responses to SIINFEKL or OVA tested by ELISPOT 12 or 82 days later. Antibody levels to OVA measured as in Figure 2 were maintained at day 82.

Figure 5: Assessment of CD40 expression by cells that have taken up beads in the draining lymph node after intra-dermal immunization Popliteal LN cells from naive C57BU6 mice (left) or mice immunized with fluorescent 0.04gim fluo-beads intradermally in the footpad (right) were dissected 48 hours after injection and analysed for expression of CD40 by staining with PE conjugated antibodies specific to these markers. FL-1 FITC positive cells 2009945121 (bead+) and FL-2= PE positive cells (marker+). Background staining was negligible The left panel represents popliteal LN cells from non-immunised animals, the right panel represents the same type of cells from VSP-OVA immunised animals.

Figure 6: Induction of immature and mature murine DC proliferation in response to 0.1gm OVA-beads. DCs were cultured from bone marrow cells extracted from the tibia and femur of the hind legs of C57BL/6 mice, with the addition of GM-CSF and IL-4. After 5 days in culture, the cells were separated into the experimental conditions at 1.25 x 10 6 cells/1.25ml and pulsed with conjugated beads to OVA 1pm) at 1000 beads/cell. After 4 hours of pulsing, LPS and TNFa were added to appropriate cultures. The cells continued to incubate overnight, and proliferation thymidine assay set up the next day and incubated overnight.

Each value represents triplicate averages SD (*p<0.00001 between unpulsed DCs and experimental groups, unpaired t-test).

Figure 7: Phenotypic characterisation of APC taking up 0.04 compared to 1gm particles in vivo C57/B6 mice were injected in the footpad with 50.l of 0.04 or 1 im 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 l.m fluobead+ cells had significantly different expression of DEC205, F4/80, CD40, CD80 and CD86 (p<0.05).

Figure 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 gM (grey); amiloride (AML) at 0 (black),l (white), 3 mM (grey) (white), or cytochalasin D (CDD) at 0 (black), 0.25 (white) or 0.5 gg/ml (grey) for 30 min and 0.04 gm-OVA-fluorescent particles added a further 3 hours. Selective inhibition of caveolae, clathrin coated pit formation, or phagocytosis has been reported for 10 jM PMA, 3 mM amiloride and 0.5 gg/ml CCD, respectively 14, 15. When used together PMA was kept constant at 10 jlM and AML was added at 1 (white) or 3 mM (grey). The number of fluorescent cells was assessed by FACScan.

Data is presented as the mean SD of triplicate cultures.

Figure 8B: Confirmation of the mechanism of uptake DC were incubated with nothing, CDD 1 lg/ml, filipin (FIL) at 1 Lg/ml or ammonium chloride (AC) at 40 mM for 30 min and 2009945121 0.04 pm or 1 nm fluorescent beads added a further 3 hours. Selective inhibition of caveolae or clathrin coated pit formation has been reported for 1 .tg/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.

Figure 9: Soluble OVA and 1 um-OVA beads fail to induce comparable protection to 0.05 um-OVA beads. C57/B6 mice were immunised ID with OVA conjugated to 0.05 um or 1 um beads, soluble OVA or left untreated and then challenged as above. Data is presented as the individual tumour sizes at day 10 for 8 animals in each group. One of two similar experiments is shown. The difference in the frequency of tumours between the 0.05 um- OVA bead group and each one of the other groups was significant: P=0.0001 vs. naive; p=0.0007 vs. soluble and p=0.0035 vs. 1 um bead-OVA.

Figure 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. 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.

Figure 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 5x10 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 200994512 1 animals left untreated (Naive) and 6 immunised ID with OVA-VSSP (Immunised).

Individual growth curves are shown day 3-13 after immunisation.

Figure 12: Survival of mice to lethal malaria challenge after VSSP immunisation. C57/B6 mice immunised intradermally once with 100 gig of VSSP-OVA, VSSP-lysate or lysate alone were challenged with 500,000 lethal Plasmodium yoelii 17XL infected C57/B6 redblood cells. Survival was monitored daily. 5 animals were challenged per group and one of six representative experiments is shown. In similar experiments naive mice had survival after 2 weeks (8/20 mice). The lysate was generated by repeated freeze-thaw of P.

yoelii 17XL infected red-cells and ultra centrifugation and conjugated to VSP using the standard protocol.

Figure 13: The antigen nm23 was conjugated to 0.05 Lp bead (VSP) as described before for the antigen OVA, injected intradermally into mice at 100.tg/mouse and 10 days later IFN gamma reactivity assessed in the spleens of immunised animals by ELISPOT. The data id presented as the precursor frequency of cells responding to nm23 per million spleen cells as spot forming units (SFU/million) the standard devation of the mean The individual responses of three mice (ml-m3) are shown.

Figure 14: The cancer antigen nm23 or OVA were conjugated to VSP per standard protocol and 100 Gig/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.

Figure 15A: Antibody reactivity to OVA in the sera of mice immunised once intradermally with 0.05im beads conjugated to OVA (VSP-OVA) and assessed 90 days later by ELISA (B group) in comparison to non-immunised controls (A group).

Figure 15B: The same sera from mice in Figure 15A was tested for the presence of OVA specific IgE antibodies by ELISA, in two naive mice (A2 and A3) and three VSP-OVA mice (B2, 3 and Figure 16: PANEL A Induction of long lasting antibody responses by a single immunisation. C57/B6 mice were immunised once with OVA conjugated to 0.04pm beads 200994512_1 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. Naive 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). OVA alone failed to induce IgG responses over PBS immunised animals and OVA in Complete Freunds Adjuvant (CFA) induced IgG responses a log higher than single dose 0.05p.m beads-OVA (not shown). PANEL B Induction of long lasting high levels of IFNy producing T-cells by a single immunisation with 0.0.4 lim beads OVA C57/B6 mice were immunised ID once with OVA conjugated to 0.04[n beads (black or chequered bar), soluble OVA in PBS (white bar) or with OVA mixed in with 0.041gm beads (grey bar). Precursor frequency of SIINFEKL reactive spleen T-cells was assessed 10 days later (back, white and grey bars) or 12 months later (chequered bar) by IFNy ELISPOT. Four mice were tested per group and one of two similar experiments is shown. Average values of spot forming units (SFU) per million cells standard deviation (SE) are shown for each group. In similar experiments using 10 times less antigen (10 Rtg VSP-OVA) a single immunisation induced 102 56 SIINFEKL specific spleen cells per million Cytotoxic T-cell responses in standard Chromium release assays were also observed 10 days after a single immunisation Specific lysis for 3/3 animals at E:T ratio 20:1; not shown).

Figure 17: Prime/boost C57/B6 animals were left untreated (Nothing) or primed intradermally with 100ug of peptide cp13-32 from MUC1 conjugated to 700ug of KLH in Complete Freunds Adjuvant (cpl3), mannan conjugated recombinant MUC1-GST fusion protein (MFP) or 0.lum VSP conjugated to MUC1-GST fusion protein (VSP). 14 days later animals were boosted intradermally with a million infectious vaccinia virus expressing the MUC protein, and reactivity to the epitopes in cpl3-32 assessed by IFNg ELISPOT days later. The data shown is the mean number of IFNg producing cells SE per million spleen cells averaged for 2-3 animals/group.

Figure 18: Comparison of polystyrene and glass 0.05 um VSP-OVA particles. Polystyrene 0.05pm beads were conjugated to OVA as before (PS) and compared to OVA conjugated glass beads in the same way In addition, a different chemical procedure was compared for the glass beads. Briefly, glass beads were weighed and suspended to in PBS and washed twice. PBS was removed by 5 minute centrifugation in a 200994512 1 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 3x with PBS resuspended in PBS and 500pg 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 M ethanolamine and mixing for 30 minutes. The beads were then washed in PBS and used for immunization Polystyrene (PS) or glass (G1 or G2) VSP-OVA were immunised intradermally at 100 ug/mouise and SIINFEKL specific IFNg secerting T cells quantified days later from spleens by ELISPOT. The data shows individual mean+/- SE for three animals per group.

Figure 19: Mode of bead conjugation and immunogenicity. Ovalbumin at 2 mg/ml in mM MES buffer (ph 6.0) was mixed with the polystyrene carboxy modified 0.05 pm beads 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 pl of I M ethanolamine (amine) pH 7.4, or 20 gl of 1 M aminoacetaldehyde dimethyl acetal (aldehyde) pH 8.0, or pl 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 0 C. The aldehyde preparation was quenched further with 20 pl of 1 M HCL and incubated for 4 hours, and dialysed overnight in PBS at 4 0 C. 2-3 C57/B6 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.

Figure 20: Cell numbers in popliteal lymph nodes upon injection with nanoparticles alone.

C57BU6 mice were injected ID with OVA conjugated or unconjugated 0.04 im, 0.1 p.m or 1.0 pm nanoparticles. Popliteal lymph nodes (LN) were removed after 2 days and teased into single cell suspensions with supplemented RPMI. Cells were diluted in trypan blue to identify dead cells and counted using a haemocytometer and bright field microscopy. The mean cell count SD is shown with the number of mice tested indicated by n=x.

Figure 21: Division of activated lymph node cells expressing CD11 upon injection with nanoparticles alone. C57BL/6 mice were injected with OVA conjugated or unconjugated 200994512_1 0.04 imr, 0.1 pm and 1.0 pm nanoparticles or soluble OVA. Popliteal LN were removed after 2 days, pulsed with CSFE and cultured for 3 days at 37 0 C. Cells were labelled with or CD86 antibodies and acquired on FACScan (10-20,000 counts collected). Cells were analysed using the CellQuest software. >750 counts were used for analysis. Cells were gated on the expression of CD40 or CD86 that had divided. The initial CFSE loading of the cells was used to determine the proportion of cells that had divided. The percentages of CD40 or CD86 cells that had divided and expressed CD1 Ic (A and B) or CD1 lb (C and D) are shown as fold increase above naive division levels.

Figure 22: Proliferation of lymph node cells with variously sized nanoparticles.

Proliferation of LN cells 48 hours after injection with 0.02 jim, 0.04 im and 1.0 [im nanoparticles alone. Proliferation was measured by tritiated thymidine incorporation assay.

Count per minute (cpm) meand standard deviation of triplicate samples for each condition.

Figure 23: Injection of mice with nanoparticles in admixture with soluble antigen and effects on T cell immunogenicity. C57BU6 mice were immunised intradermally once with soluble OVA OVA mixed with beads (0.04/OVA, n=2) or conjugated to beads (0.04- OVA, n=4) at 100 lig of OVA at 1% carrier solids and spleen T cell responses to SIINFEKL assessed 10 days later by IFN-y ELISPOT assay. Precursor frequency shown as mean spot forming units (SFU) per million cells SE.

Figure 24: Induction of DCs to proliferate upon culture with nanoparticles. 5 day cultured IL4/GM-CSF grown DCs were pulsed with 0.04 pm-OVA particles (1 x 104, 1 x 105 and 1 x 106 nanoparticles/cell) overnight and proliferation quantified the next day by tritiated thymidine incorporation overnight. Each value represents triplicate averages SD between un-pulsed cells and experimental groups, unpaired T-test).

Figure 25: Injection of mice with nanoparticles in admixture with a malarial antigen. Mice were injected IP with 500,000 P yoelii-parasitised red cells and either left untreated (naive), or injected with 1% solids of 0.05 nim nanoparticles alone or MSP4/5 recombinant protein alone at 20Lg or the conjugate of 0.05 j.m nanoparticles and MSP4/5, and monitored for 2009945121 parisitemia daily by Giemsa stain of thin and thick blood smears. P values compared to naive control are shown below each graph.

Figure 26: DNA band retardation analysis. Binding of DNA to PLL-nanoparticles masks the negative charges in the DNA backbone which facilitate its migration during gel electrophoresis. Gel migration of sOVA-Cl complexed with cationic polystyrene 0.05 pm nanoparticles was completely abolished whereas naked sOVA-C simply mixed with polystyrene nanoparticles shows similar migration to sOVA-Cl alone. Equal amounts of DNA (200 ng) were loaded on a 1% agarose gel. Lambda EcoRI/HindIII marker was used to assess plasmid size.

Figure 27: Immunogenicity of sOVA-C1/PLL-nanoparticle complexes after two immunisatons. C57BU6 mice were immunised twice, 14 days apart, withl0 pg sOVA either on its own (naked sOVA-C1), complexed with PLL-coated nanoparticles (sOVA- CL/PLL-0.05 pm) or simply mixed with nanoparticles (sOVA-CI 0.05pm). Control mice received no immunisation. 14 days after boost immunisation splenocytes were assessed for IFNy secretion by ex vivo ELISpot assay. Bars represent mean spot forming units SD (n 2 mice/group). OVA-specific serum antibody responses were measured by ELISA. Bars represent total Ig titres of 7 individual mice 14 days after boost immunisations. Isotyping of OVA-specific antibodies. Bars represent total Ig titres of 7 individual mice 14 days after boost immunisations. All antibody titres were calculated as lowest dilution with A450nm above mean A450nm of 7 naive mice 3 SD. Results are expressed as the mean of the IgG 1, IgG2a and IgG2b titres, respectively SEM from 7 mice in each group. indicates p<0.05.

Figure 28: Comparison of DNA band retardation after complexation with PLL-coated polystyrene particles of 0.02, 0.05 or 1.0 pm in diameter. All particles successfully complexed the pDNA and retarded its migration through the gel. Equal amounts of DNA (200 ng) were loaded on a 1% agarose gel. Lambda EcoRI/HindIII marker was used to assess plasmid size.

Figure 29: Immunogenicity after 4 immunisations with sOVA-C /PLL-polystyrene particles of different sizes. C57BU6 mice received four immunisation in 14 day intervals with 10 pg sOVA either on its own or complexed with PLL-coated polystyrene particles of 200994512 1 0.05 or 0.02 pm in diameter. Control mice received no immunisation. 14 days after the last immunisation splenocytes were assessed for IFNy secretion by ex vivo ELISpot assay. Bars represent mean spot forming units SD (n 2 mice/group). indicates p<0.05. OVA-specific serum antibody responses were measured 14 days after the last immunisation. Bars represent the mean of the total Ig titres SEM from 5 mice in each group. Antibody titres were calculated as lowest dilution with A450nm above mean A450nm of 5 naive mice 3 SD. indicates p<0.05; indicates antibody titre of mice immunised with sOVA/PLL-0.05 pm particles significantly higher than naked sOVA group (p<0.05).

Figure 30: Tumour challenge after 2 immunisations with sOVA-C l/PLL-nanoparticles.

C57BU6 mice were immunised twice in a 14 day interval withl0 pg sOVA either on its own or complexed with PLL-coated nanoparticles. Control mice received no immunisation. 21 days after boost immunisation mice were challenged s.c. with 107 EG7 cells. Individual tumour sizes are shown for each mouse (n 5 mice/group). Average tumour size per group on day 8 after tumour challenge. Mice immunised with sOVA/PLL- 0.05 pm nanoparticles displayed significantly reduced to absent tumours (p<0.0001).

Example 1: 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 100l of antigen conjugated beads intradermally (ID) in the hind footpads or into the base of the tail. All immunizations were given 14 days apart.

Reagents: 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 0.0 2 2 4 were purchased from Molecular Probes and non-fluorescent carboxylated microspheres from Polysciences.

Abs to the following markers were used: MHC II, MHC I, CD1 Ic, CD1 lb, 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).

200994512_1 Plasmid and plasmid purification: The sOVA-C 1 plasmid, encoding for soluble chicken ovalbumin under the CMV promoter has been described Plasmid DNA was maintained and purified from Escherichia coli DHIO3 using Qiagen Plasmid Maxi Kit (Qiagen, Germany) according to the manufacturer's instructions.

Nanoparticles: Nanoparticles were sourced from three different manufacturers. The 0.05 pm nanoparticles were obtained from Polysciences (catalog no. 15913), and while sold as 0.05 pm diameter nanoparticles, these have been generally found to have a mean diameter of either 0.047 pm or 0.049 pm and usually a deviation of 0.0015 pim or 0.007 jim respectively. The 0.04 jm nanoparticles, on the other hand, were obtained from Molecular Probes (catalog no. F-8795) and actually have a mean diameter of 0.043 im with a deviation of 0.006 LJm. These 0.04 pm nanoparticles comprise a fluorescent yellow-green core. Alternatively, 0.04 pm nanoparticles were sourced from Sigma (catalog no. L-5030) and have a mean diameter of 0.046 pm 19.1%. These nanoparticles comprise a fluorescent yellow-green core.

PPL-coating of polystyrene particles: Poly-L-Lysine was conjugated to carboxylated polystyrene particles as previously described [45] with minor modifications. Briefly, carboxylated polystyrene microspheres with (Molecular Probes) or without fluorescent labels (Polysciences, Germany.) of 0.02, 0.05 or 1.0 pm in diameter, were mixed with 1 mg/ml poly-L-lysine (mol wt 15,000-30,000, Sigma), in 0.05 M MES (2morpholinoethanesulfonic acid) buffer pH 6.2 for 10 min. The 0.05 pm nanoparticles were used at 0.2% solid, whereas the amounts of 0.02 and 1.0 pm particles were adjusted to match the surface area of the 0.05 pm nanoparticles. For covalent conjugation, N-Ethyl-N'- (3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) (Sigma, USA) was added at 4 mg/ml, the pH was adjusted to 6.5 with NaOH and the mixture placed on a rocker for 2 hours at room temperature Glycine was added to a final concentration of 100 mM and the mixture was rocked for further 30 min. The preparations were dialysed overnight against cold PBS using a 14 kD membrane cut off.

DNA band retardation analysis: pDNA/PLL-particle complexes were analysed on 1% agarose (Promega, USA) gels. The volumes loaded contained 200 ng DNA. DNA complexation was considered successful when the migration of the DNA band was retarded 200994512_1 in the gelAntigen presenting cells: Denditric cells were prepared from bone marrow monocytes with minor modifications of previously published methods 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 lx10 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 pM P-mercaptoethanol. and GM-CSF at 1000 units/ml and IL-4 at 0.2 ng/ ml were added. The 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 Bead-antigen conjugation: 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 minutes of mixing the preparation was dialysed overnight in the cold in PBS. The preparation was either used immediately or stored at 4 0 C with 0.01% azide for later use.

Antigens: The following antigens were used for ELISA or in vitro re-stimulation: chicken egg ovalbumin (Sigma, Australia), the minimum OVA CD8 T cell epitope SIINFEKL

(OVA

257 -264) and the CD4 T cell epitope ISQAVHAAHAEINEAGR (OVA 323 339 All peptides were synthesised by Auspep, (Melbourne, Australia).

Cytotoxicity assays: These were performed as described Briefly, effector cells for cytotoxicity assays were generated by culturing spleen cells for 7 days at 2.5 x 10 6 /ml in 2 ml well plates at 37oC 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 PM p mercaptoethanol. Interleukin 2 (10 U/ml, recombinant human IL2, Lymphocult HT, Biotest, UK) was added on day 3. Targets were SCr loaded EL4 cells, alone (background) or pre- 200994512 1 pulsed for lh at 37 0 C with 10g/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 triton) were set up for all targets in quadruplicate. Supematants were harvested after 4h.

%Lysis was calculated as 100x ((Experimental release -Spontaneous release)/ (Maximum release-Spontaneous release). Specific Lysis was %Lysis with peptide- %Lysis with no peptide.

Cytotoxic T cell precursor( CTLp) assays: CTLp assays were performed as described previously CTLp frequencies were determined from a minimum of 32 replicates, for at least 6 effector cell numbers (1x103 1.28x105). Cells were cultured in U-bottomed microtitre trays, with 5x105 mitomycin C treated syngeneic spleen cells, in DMEM supplemented with 10% foetal calf serum, 5 1 M of SIINFEKL or OVA and 10 U/ml rhIL-2.

Seven days later, each microculture was assayed for cytotoxicity by replacing 100 ml of culture medium with 100 pl target cell suspension containing 104 51Cr-labelled EL4 OR EG7 as targets. Cytotoxic activity was considered to be present if in each well 51Cr 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 rIL2 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, CTLp frequency assays were performed three times and the individual frequencies did not differ by more than 20% from the mean value.

ELISPOT IFNyassays: These were performed as described Briefly, 100 p1 of 5 x 106 /ml freshly isolated spleen cells were incubated with the stated stimuli for 16 or 18 hours on mixed acetate plates (MAHA Millipore) pre-coated with an anti-murine IFNy mAb (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 IFNy (XMG.21biotin,Pharmigen, CA, USA), followed by an extravidin alkaline phosphatase (A-AP) conjugate at 1 l.g/ml (Sigma). Spots of alkaline phosphatase activity were detected using a colorimetric AP detection kit (Biorad, Hercules, CA, USA) and counted utilising a dissection 200994512 1 microscope. The data are presented as spot forming units (sfu) per million cells. Alternatively, the number of spot forming units (SFU) per well was scored using an AID ELISPOT reader (Autoimmun Diagnastika GmbH, Germany) and the AID Elispot software version 2.9 (Autoimmun Diagnastika GmbH, Germany). Data are presented as mean SFU standard deviation. The SIINFEKL peptide (CD8 epitope) was utilised at 2.5 gg/ ml, and the ISQAVHAAHAEINEAGR peptide (CD4 epitope) utilised at 25 g/ ml.

Statistical analysis: Statistical analysis in protection studies the number of mice protected in each group was compared using a X 2 test in the Statcalc program in the Epilnfo Version 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 package. Linear regression analysis was used to assess correlation between immunogenicity and protection using the SPSS for Windows statistical program package.

Values ofp for differences between mean SFU by ELISPOT or mean inverse titre by ELISA for diversely immunized animal groups were determined using unpaired two-tailed equal variance Student-t test 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.

Analysis of cells taking up beads in vivo: 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.

Cell surface marker staining andflow cytometry: For surface staining, 5x10 5 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- 200994512_1 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.

Confocal microscopy of phagocytosed FITC-labelled beads: 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 488nm and 568nm for fluorescein and Alexa 594 respectively and detected through 530nm and 610nm band pass filters respectively. Throughout acquisition, laser power was kept below saturation levels and gain and offset parameters maintained within individual experiments.

ELISA assays: Antibody responses to OVA were measured using ELISA. Polyvinyl chloride microtitre plates were coated with OVA (10 glg/ ml in 0.2 M NaHCO3 buffer, pH 9,6) overnight at 4 0 C. The plates were washed 4x with PBS/0.05% Tween20 and 4x 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. Alternatively, for IgG isotyping, sera were incubated with rat-anti-mouse IgG1-HRP, rat-anti-mouse IgG2a-HRP or with rat-anti-mouse IgG2b-biotin followed by Steptavidin-HRP (all Pharmingen, USA). The assay was developed with TMB solution (Zymed, USA), stopped with 1M HCI and read at A 4 50 nm. Antibody titres were defined as the lowest serum dilution with A450nm above the mean A450nm 3 standard deviations of naive control sera values in the same assay.

200994512 1 Tumour challenge: EG7 cells are H-2 b EL4 thymoma cells that stably express OVA [461.

Cells were cultured in RPMI-1640 supplemented with 10% heat inactivated FCS, 2 mM Lglutamine, 100 units/ml penicillin, 100 pg/ml streptomycin, 100 mM p-mercaptoethanol and 0.2 M HEPES buffer under standard tissue culture conditions. 21 days after the last immunisation mice were injected subcutaneously with 10 7 OVA expressing EG7 cells in 100 il volumes of sterile PBS. Tumours were measured every second day using a vernier calliper.

Example 2: Preferential uptake of VSSP by antigen presenting cells in vitro and in vivo 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 would be efficiently taken up by dendritic cells or macrophages. Figure la shows that thioglycollate elicited peritoneal exudate macrophages internalised 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 2pm) 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 im size were taken up preferentially by lymph node cells (Figure Ib). 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 (Figure Ib). Bone marrow derived dendritic cells in vitro also took up VSSPs. As expected, these cells were of a predominantly myeloid phenotype [12].

200994512 1 Example 3: VSSP prime high precursor frequencies of cytotoxic and IFNy secreting T cells as well as antibodies.

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 2pm, boosted after 15 days and serum or spleen cells collected 10 days later. Figure 2a shows optimal induction of IFNy secreting CD8 T cell induction to the MHC class I restricted SIINFEKL epitope achieved using 0.04pm 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 IFNy secreting T cells (R2=0.92)(Figure 2b). Surprisingly, OVA-specific IgG was also found at the highest precursor frequencies in mice immunised with 0.04gm particles, followed by those immunised with 1pm particles (Figure 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.

The results shown in Figure 2 are based on injecting the same total amount of antigen after conjugation without eliminating residual soluble antigen. Figure 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. Figure 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 0.041m and 0.02gm sized beads induced little reactivity (Figure 2) 0.04-0.1 micron VSSPs were consistently more immunogenic than lpm particles independently of the level of antigen conjugation, Increasing the immunising concentration of l.gm particles up to 100 fold (up to 1 mg/mouse) over that used for VSSPs failed to enhance immunogenicity to 200994512_1 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 10 3 -108 beads per animal).

Example 4: A novel pathway for the uptake and processing of particles 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 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 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 llm particles based on 'leakiness' of endocytic vesicles and accidental release of antigen into the cytoplasm 10] Processing of such large particles taken up by phagocytosis involves an early conjugation step with lysosomes expressing the Rab4 adaptor protein The inventor used confocal microscopy to determine whether VSSPs would be routed via this pathway. Figure 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 C57BU6 mice were left untreated (naive) or immunized with fluorescent 0. l1 m fluo-beads intradermally in the foot pad. Popliteal lymph node (LN) were dissected 48 hours after 200994512 1 injection and analysed for expression of CD40 by staining with PE conjugated antibodies specific for this marker.

The results (Figure 5) show that 0.04, 0.05 and 0. 1mrn 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. Figure 5 shows an example of the increase in CD40+ cells after immunisation observed by FACScan (33% to It also shows that many of these cells have taken up the 0.041m beads, in this case we used 0.04pm beads with a fluorescent green core.

0.04, 0.05tm 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 (Figure 6).

These data together suggest that particles of 0.04-0.1 m 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 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 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 0.04am VSSP-OVA with a fluorescent core was then analysed by FACScan and compared to identical particles but which were ilam in size. Figure 7 shows the proportion of 0.04am bead+ or ljam bead+ cells expressing each phenotypic marker. Cells taking up 0.04gm 200994512 1 beads were mostly NLDC145+, CD40+ and CD86+. In addition more CD1 CD4+ and CD8+ cells were 0.04 than 1 im bead+. This highly activated DC phenotype of cells that have taken up 0.04tm beads may further explain why the immune responses we observed are so potent, particularly CD8 T cell responses.

Example 7: Uptake and processing of nanoparticles To address the mechanism of 0.04pm bead-OVA uptake by dendritic cells bonemarrow 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. OVAfluorescent 0.04 pm beads were added for a further 3 hours and uptake was quantified by FACScan.

PMA and AML both decreased 0.04 pm 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.

Amiloride acts by inhibiting Na+-H+ exchange necessary for receptor mediated endocytosis The inventor tested additionally ammonium chloride which inhibits the assembly of clathrin pits by interfering with cytosol acidification Ammonium chloride inhibited uptake of 0.04 but not of 1lim 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 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 interalisation [14, 15, 16, 17].

Similarly to PMA, filipin blocked 0.04pm but not l.tm bead uptake, confirming the inventor's hypothesis (Fig 8b).

Caveole and clathrin pits can convey molecules to endosomal and lysosomal compartments [14, 16]. Alternatively, caveole may deliver antigen directly into the cytosol [17] leading to 200994512 1 cytoplasmic processing and TAP dependent transport into the endoplasmic reticulum for presentation with MHC class I 18].

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 nm 0.05gm) (VSSP) OVA conjugated particles were compared directly with OVA alone or 1000nm lgm) 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 gg of either of the above (or nothing=naive) intra-dermally once and then challenged with tumour 30 days later. Figure 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 Intradermal immunisation with OVA-conjugated VSSPs induced IFNy producing.and cytotoxic SIINFEKL specific T cells (Figure 10a and la and Additional immunizations did not further increase reactivity (Figure 10b and 10c). T cells could be maintained at high precursor frequencies 82 days after immunization (Figure 10d). Antibody responses were similarly maintained (Figure 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 2, 3, 19, 20]. High IFNy 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- 200994512 1 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 Figure 2c.

Further work on VSSP vaccines 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.

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

C57/B6 mice were immunised once with 100gg bead(0.05pgm)-OVA (ID) or left untreated (naive). After 30 days, mice were challenged with 5x10 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).

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 200994512_1 N within two weeks after a single injection. This therapeutic ability is highly unusual for any t cancer vaccine, and makes this vaccination vehicle highly promising for development of a therapeutic vaccine (Figure 11B).

Mucin-1 or Mud is a breast cancer associated antigen. Immunisation once with VSSP- Mud protein also inhibited tumour formation in mice challenged with tumour cell lines Sexpressing the breast cancer antigen (see Figure 11A).

CN- Example 11: Protection Against Malaria

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0 The inventor demonstrates that polysterene beads of 0.05 gm 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.

Blood was collected from C57BU6 mice infected with P. yoelii 17XL at 50% parasitaemia.

Red blood cells (RBC) recovered after centrifugation 800g for 15 min were freeze/thawed three times and sonicated (lysate). Lysate was conjugated to 0.05gpm particles as described above. Beads-lysate, beads-OVA or lysate alone were injected ID. Immunised or naive C57BU6 mice were challenged two weeks later intra-peritoneally with 1,000,000 P. yoelii 17XL infected RBC.

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 (Figure 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 1) Polysterene beads of 0.05 Lim 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 (Figure 13).

200994512 1 2) As well as inducing cellular immunity, beads-OVA or beads-nm23 induced high levels of IL4 (Figure 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 1) Polysterene bead of 0.05 pm in diameter conjugated to OVA induced, after a single intradermal (ID) injection induced high titres of IgG antibody (Figure 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 (Figure Example 14: Administration via different routes and induction of IgG and IgA antibodies 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 jig/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 intradermal, all routes induced T cells which secreted IFNg to SIINFEKL or to OVA by ELISPOT assay (1/50,000 tol/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 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).

Table 1 Route Serum IgG titre Serum IgA titre Intra-rectal <1/100, <1/100, <1/100 1/1280, 1/640, 1/1280 200994512_1 Intra-peritoneal 1/1640, 1/100, >1/5120, >1/5120, >1,5120 1/400 Intra-nasal <1/100, <1/100, 1/160, 1/320, <1/100, <1/100 1/320, 1/640 subcutaneous 1/800, >1/5120, >1/5120, >1/5120 1/200, 1/6560 Unusually high IgG responses after two doses: Immunisation twice with VSSP-OVA (100 gig/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 Responses to VSSP-OVA were surprisingly long-lasting. Figure 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.

Example 16: Heterologous prime-boost Vaccinia-MUC1 was used to boost responses of animals primed with nothing, peptide cpl3- 32 (cpl3) from MUC1 in complete Freund's Adjuvant (CFA), Mannan conjugated MUC1 (recombinant MUC 1-GST fusion protein)(M-FP) or 0.1 Lgm VSSP-MUC (recombinant MUC1-GST fusion protein)(VSSP). Figure 17 shows responses to the peptide 13-32 region of MUC 1 were enhanced in the VSSP-MUC primed, Vaccina-MUC 1 boosted group compared to animals that received Vaccinia MUC 1 alone (Nothing/V compared to 200994512 1 VSSP/V). Therefore VSSP-antigen would be suitable for use in heterologous Prime-boost protocols.

Example 17: Material composition of the solid core for VSSP The inventor's hypothesis that the 0.04-0.05 pm 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 0.05mn particles made of polystyrene (PS) or of glass (G I or G2) and conjugated to OVA using the same chemical procedure (G1) or binding using glutaraldehyde Figure 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 The results show that mixing antigen with 0.05 pm 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 0.051pm) 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 Figure 19). By quenching the reaction with ethanolamine charges can be neutralised except for the net charge of OVA after conjugation (Alcohol, Figure 19). By quenching with ethylenediamine a positive charge is introduced (Amine, Figure 19). By quenching with aminoacetaldehyde dimethyl acetal (Aldehyde, Figure 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 200994512 1 N comparable to glycine, and aldehyde slightly less immunogenic. Therefore it is highly tb unlikely immunogenicity results from non-specific adsorption of serum proteins as the introduction of opposite charges to the particle results in similar immunogenicity.

CC Moreover, some alternative modifications to the conjugation procedure, and changes in charges can be introduced with no decrease in immunogenicity.

SDISCUSSION

ri In view of the results above, the composition of the invention provides a way to further

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Simprove or optimise vaccines or vaccination strategy that could apply to a variety of C infections, cancer or other diseases.

The optimal size of the VSSP coincides with that of most known viruses (30-150nm).

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 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 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 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.

2009945121 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- I (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.

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 0.04-0.05gm 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 nanoparticles according to the invention.

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.

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

200994512 Example 19: Unconjugated nanoparticles induce proliferation of APCs To examine the differences in the dynamics of viral and bacterial-sized particles to induce local immune responses, OVA conjugated or unconjugated 0.04 im, 0.1 tpm and 1.0 pm nanoparticles or soluble OVA were injected ID in the mouse footpad, and popliteal lymph nodes (LN) were removed 2 days later. Soluble OVA was used as a non-particulate antigen to determine whether the free OVA in the particle preparation could augment cell recruitment. Macroscopically, it was generally observed that the appearance of the popliteal LN in the nanoparticle-injected mice was significantly larger than the naive LN. Also, generally, there was over a 4-fold increase in cells within the LN of mice injected with 0.04 pm nanoparticles, over a 3-fold increase after 0.1 pim and 1.0 pm nanoparticles and 0.04ptm, 0.1 lpm and 1.0 pm nanoparticle-OVA injection, and a 2-fold increase with soluble OVA injection as shown in Figure 20, wherein each replicate represents two popliteal LNs/mouse (denoted by Therefore, the nanoparticles per se stimulate recruitment of cells from the periphery, as well as their potential cellular division.

The popliteal LNs of mice injected ID in the footpad with OVA conjugated and unconjugated 0.04 ptm, 0.1 pm and 1.0pm nanoparticles were examined for T cell, DC and macrophage cell surface markers by FACS analysis (data not shown). There was minimal variation in CD4 and CD8 T cell numbers between the nanoparticle groups post injection, although their numbers were decreased compared to the naive animals. This may be attributable to the increased influx of APCs loaded with antigen. Similar levels of CD1 c, CD1 lb and CD205 were observed upon injection of all sizes of nanoparticles. The macrophage and minor DC population marker F4/80 did not decrease upon injection with pm and 1.0 pm-OVA nanoparticles. However, comparing the proportion of cells to the naive control, APC populations appeared to decrease, indicating migration through the efferent lymphatics to secondary lymphoid organs or a change in the proportion of DCs within the LN upon recruitment of T and B cells from the periphery.

To determine whether the nanoparticles on their own were capable of inducing proliferation of LN cells generally, C57BL/6 mice were injected ID in the footpad with OVA conjugated or unconjugated 40 pm nanoparticles or soluble OVA, and popliteal LNs removed after 2 days and a tritiated thymidine incorporation assay conducted (data not shown). The results 200994512_1 indicated that the induction of proliferation of LN cells could occur due to antigen or to the unconjugated nanoparticles. Moreover, it was found that the proliferative response correlated with the number of nanoparticles injected (data not shown). The results therefore indicated that the nanoparticles per se were inducing a non-specific stimulation and cell expansion in the LN.

To investigate whether APCs, in particular DCs, present in the popliteal LN were induced to proliferate in response to stimulation with the nanoparticles, the LN cells were loaded with pM of carboxy-fluorescein diacetate, succinimidyl ester (CFSE) and cultured for 3 days.

Division was primarily determined by examining the percentage of CFSE loaded cells that had divided upon injection with nanoparticles. Since the CFSE is retained in the cytoplasm, during each round of division, the relative intensity of the dye is decreased by half. The cells were then stained for CD1 Ic and CD1 lb markers and co-stimulatory molecules, and analysed for cell division. LN cells were gated on CD40 or CD86 expressing cells that had divided, and then examined for the co-expression of DC markers. Figure 21 and (b) show the percentage fold increase of CD40 and CD86 divided cells that express the CD1 c marker and the CD1 Ib marker respectively. The results indicated that of the CD40 divided cells, there was a similar increase in the percentage of CD1 lc+ cells upon injection of 0.04 pm, 0.04 gim-OVA, 0.1 pim, 0.1 jm-OVA and 1.0 pm-OVA nanoparticles compared to naive controls, whereas there was no increase upon injection of 1.0 p.m nanoparticles alone or OVA alone. Of the CD86 divided cells, there was a considerable increase in the CD1 lc+ cells upon injection of 0.04 pim-OVA nanoparticles only. Of the CD40 and CD86 divided cells, there was a 2-fold increase of CD40+ divided cells that express CD 1 b upon injection of viral size particles, but not bacterial size particles.

The results of this example indicate that upon ID injection of viral size nanoparticles, activated DC and not macrophages are stimulated to expand.

Example 20: Unconjugated nanoparticles cause LN cell proliferation that is dependent on the size of the nanoparticle C57BL/6 mice were injected with 50 jl of 1% solids of nanoparticles of 0.02 pm, 0.04jim and 1.0.m. LN cells were harvested 48 hr after injection and proliferation assessed in a standard overnight tritiated thymidine incorporation assay (Figure 22). Significant 200994512 1 proliferation above naive was observed to be induced by 0.02 pm and 0.04 pm but not pm nanoparticles.

Example 21: Unconjugated nanoparticles enhance T cell immunogenicity C57BL/6 mice were immunised ID with soluble OVA OVA mixed with 0.04 pm nanoparticles (0.04/OVA, n=2) or OVA conjugated to nanoparticles (0.04-OVA, n=4) at 100 pg of OVA at 1% carrier solids. Spleen T cell responses to the antigen, SIINFEKL gg/ml) were assessed 10 days later by IFN gamma ELISPOT assay. As shown in Figure 23, unconjugated nanoparticles mixed with OVA had a significantly enhanced response (P<0.05) over OVA alone. Thus, when 0.04 pm unconjugated nanoparticles are co-injected with OVA, enhanced immunogenicity is observed compared with immunisation with OVA alone.

Example 22: Expansion of antigen presenting cells in vitro Since it had been seen that LN cells expanded in vivo without further antigenic stimulus, it was considered that APCs might have the ability to expand in vitro upon nanoparticle contact. Previous in vitro experiments by others (see Hamilton and colleagues (2000) J.

Leukocyte Biology 67(2):226-232) found that certain particulate adjuvants could induce macrophages to proliferate. Indeed, particulate material such as Aluminium hydroxide, OxLDL and aggregated LDL, calcium phosphate and silica had been observed to induce BMD-macrophage survival and DNA synthesis. Thus, to address whether nanoparticles could induce APC proliferation, murine DCs grown in IL4/GM-CSF for 5 days were pulsed with 40 nm-OVA particles overnight, and their proliferation analysed using the thymidine incorporation assay. Figure 24 shows a representative of 5 similar experiments showing that in vitro grown DCs can be induced to proliferate in response to interaction with 0.04 pm nanoparticles. More particularly, it was shown that 5 day old IL4/GM-CSF BM-derived DCs can proliferate in vitro upon nanoparticle contact, and that the optimal stimulatory nanoparticle concentration ranged between 10,000 and 100,000 nanoparticles/cell.

Example 23: Protection against malaria by unconjugated nanoparticles 200994512_1 Eight Balb/c mice were given 500,000 Plasmodium yoelii (YM) parasitised red cells intraperitoneally and either left untreated (naive) or injected with 1% solids 0.05 tm nanoparticles alone or MSP4/5 recombinant protein alone at 20 gg (MSP4/5 alone) or the conjugate of 0.05 .tm-MSP4/5 and monitored for parasitemia daily. Animals given nanoparticles alone or 0.05 pm-MSP4/5 but not MSP4/5 alone had, at days 7, 8, and 9, significantly less parasites than untreated mice (Figure 25). Thus immunisation with unconjugated nanoparticles was able to decrease parasite load.

Example 24: Preparation of pDNA/PLL-polystyrene particle complexes The inventors investigated whether polystyrene nanoparticles could enhance the efficacy of DNA vaccines by using the sOVA-Cl plasmid, which contains the gene for OVA under the control of the CMV promoter To facilitate binding between the plasmid DNA (pDNA) and the nanoparticles we conjugated poly-L-lysine (PLL) to carboxylated polystyrene nanoparticles of 0.05 pm in diameter using the same method we described previously for the conjugation of protein antigen This covalently bound PLL provides a cationic linker that allows electrostatic binding to the negatively charged pDNA [47].

DNA binding to PLL-coated nanoparticles was performed by slowly adding the PLL-coated nanoparticles into pDNA solution of equal volume in the presence of 700 mM NaCl at a rate of 10 pl per minute under constant vortexing. Because the negatively charged phosphate groups of the DNA backbone are masked after electrostatic binding to the positively charged amino groups of the PLL the successfully complexed DNA loses its ability to migrate to the anode during agarose gel electrophoresis. Therefore DNA binding to the nanoparticles was considered successful when a retardation of the DNA band in a 1% agarose gel was observed. As illustrated in Figure 26, full retardation was seen for pDNA/PLL-nanoparticles but not for pDNA that had simply been mixed with 0.05 pm nanoparticles without the PLL linker. We therefore conclude that the presence of the cationic PLL linker is essential for the binding of the DNA to the particles.

Example 25: sOVA-Cl/nanoparticle complexes induce strong OVA-specific CD8 T cell responses in vivo as well as specific antibodies To assess the immunogenicity of the pDNA/nanoparticle complexes, C57BL/6 mice were immunised twice, 2 weeks apart, into the base of the tail with 100 pl of either sOVA- 2009945121 C1/PLL-nanoparticle conjugate, sOVA-C1 pDNA simply mixed with 0.05 pm nanoparticles or with naked sOVA-Cl. The amount of pDNA in each of the formulations was 10 pg per mouse per injection. Age-matched mice were used as negative controls and received no immunisation. 14 days after the last immunisation, splenic T cell responses were assessed by ex vivo ELISpot assay The well characterized OVA epitope SIINFEKL [48] was used for the restimulation of CD8 T cell responses and the OVA helper epitope OVA 32 3 339 to measure CD4 T cell responses of isolated splenocytes. Figure 27A shows that sOVA- C1/PLL-nanoparticle complexes induced strong SIINFEKL-specific CD8 T cell responses (p<0.0 5 after two immunisations whereas neither naked sOVA-Cl nor sOVA-C1 mixed with nanoparticles elicited significant IFNy responses. In contrast to the strong CD8 T cells response, no reactivity to OVA 323 339 was observed indicating that no IFNy secreting CD4 T cells specific for this epitope had been induced. Furthermore no IL-4 secreting T cells were detected in either of the groups (data not shown). Serum was collected 14 days after the last immunisation and analysed for the presence of OVA-specific antibodies. As shown in Figure 27B, only mice immunised with sOVA-C1/PLL-nanoparticles had significant OVAspecific antibodies whereas no significant antibody titres could be detected in mice that received naked pDNA or sOVA-Cl mixed with 0.05 pm nanoparticles. IgG isotyping revealed that immunisation with sOVA-Cl/PLL-nanoparticles mainly induced IgG1 antibodies and to a lesser extent IgG2a and IgG2b (Figure 27C). This IgG1 dominance has previously been observed for DNA encoding for soluble OVA No significant IgM or IgE could be detected in any of the groups (data not shown).

Together these results demonstrate that pDNA complexed to PLL-coated nanoparticles induce both cellular and humoral immune responses in mice with an efficacy which by far exceeds the efficacy of pDNA alone. Furthermore, binding of the pDNA to the particles via the PLL linker is essential for the induction of immune responses. Therefore; the enhanced immunogenicity of sOVA-Cl/PLL-nanoparticle conjugates is not simply the result of an adjuvant effect of co-administration of nanoparticles.

Example 26: Induction of OVA specific immune responses in vivo is particle size dependent 200994512 1 The size of particles used as DNA carriers has been suggested to be of importance for the vaccine efficacy. Recent studies have shown that nanoparticles might be more successful at stimulating immune responses in vivo than nanoparticles [39, 49]. To assess which particles size would be most effective as a carrier for DNA vaccines, PLL-coated polystyrene nanoparticles of 1.0 pm in diameter were compared to PLL-coated nanoparticles of 0.05 or 0.02 pm in diameter for induction of immune responses. The amount of 0.02 and 1.0 pm particles used was calculated to represent a total surface area equal to the 0.05 pm nanoparticles to ensure the same reaction area for the binding of the PLL.

DNA band retardation analysis showed that all particles successfully complexed sOVA-C1 pDNA (Figure 28). 100 pl of each formulation containing 10 pg of sOVA-Cl pDNA was injected into C57BL/6 mice and the immune responses 14 days after the fourth immunisations measured.

Figure 29 shows that although all particle sizes were able to complex sOVA-C1 pDNA, only pDNA complexes to polystyrene particles of 0.05 pm in diameter induced OVAspecific CD8 T cell responses, as measured by ex vivo IFNy ELISpot assay (Figure 29A).

Interestingly, 4 immunisations of naked sOVA-C1 pDNA also induced small but significant IFNy responses above background, but these responses were significantly lower compared to the strong response induced by sOVA-C1 complexed to 0.05 pm nanoparticles (Figure 29B; p<0.05). No significant IL-4 secretion was detected in any of the groups (data not shown). Antibody responses were tested 14 days after the last immunisation. Again, sOVA-C1/PLL-0.05 pm particles induced significant anti-OVA antibodies but not pDNA complexed to particles of 1.0 or 0.02 pm in diameter (Figure 29B). While injection of naked sOVA-Cl resulted in moderate antibody responses, high anti-OVA antibody titres were observed in mice immunised with sOVA-CI/PLL-0.05 pm particles These data clearly show that the immunogenicity of DNA/PLL-particles complexes strongly depends on the size of the particle, with 0.05 pm nanoparticles being the only efficient carrier out of the sizes tested in this study. This size was further confirmed to be capable of promoting both strong CD8 T cell and antibody responses after 2 or 4 immunizations.

Example 27: Immunisation with sOVA-C1/0.05 pm nanoparticle complexes protects from EG7 tumour challenge: 200994512 1 The inventors showed that immunisation with nanoparticle-PLL/sOVA complexes induced high CD8 T cell responses as well as high antibody titres. To test whether these immune responses were protective against tumour challenge, mice were immunised twice with 10 pg sOVA-C1 alone or complexed to PLL-coated 0.05 pm polystyrene nanoparticles and challenged with 10 7 EG7 thymoma cells 21 days after boost immunisation. EG7 thymoma cells stably express OVA and represent a non-lethal tumour model. In the naive mice, the tumours grew to their maximum at day 7-9 and were rejected around day 14 (Figure In the group immunised with sOVA/PLL 0.05 pm polystyrene nanoparticles complexes 2/5 mice had no detectable tumours, 1/5 mice developed a small tumour which was cleared by day 8 and 2/5 mice showed delayed onset of tumour growth (Figure Of the mice immunised with naked sOVA-Cl only 1/5 mice cleared the tumour on day 8, the others show tumour growth similar to naive mice (Figure 30A). Comparison of the average tumour size per group on day 8 after challenge, which represents the peak day of tumour growth clearly shows a highly significant reduction of tumour size in mice immunised with sOVA-C1/PLL-0.05pm nanoparticles (Figure 30D; p<0.0001), but in none of the other groups.

200994512 1

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Claims (10)

1. 7-- 53 CLAIMS 1. An immunogenic composition comprising at least one nucleic acid molecule in association with nanoparticles, wherein the nucleic acid molecule encodes at least one antigen, and wherein the nanoparticles are about 0.02pm to about 0.1pm in diameter. 2. An immunogenic composition of claim 1 wherein the nanoparticles are about 0.04pm to about 0.5pm in diameter. 3. An immunogenic composition of claim 1 or claim 2 wherein the nanoparticles are about 0.5pm in diameter. 4. An immunogenic composition of any one of claims 1 to 3 wherein the nanoparticles are of a substantially uniform size. An immunogenic composition of any one of claims 1 to 4 wherein the nanoparticles comprise a solid core. 6. An immunogenic composition of any one of claims 1 to 5 wherein the nanoparticles are composed of a substantially inert material. 7. An immunogenic composition of claim 6 wherein the inert material is selected from the group consisting of latex, ferrous molecules, gold, glass, calcium phosphate, polystyrene, poly lysine G, biodegradable polymers, biocompatible polymers, and combinations thereof.
2. 8. A method of eliciting an immune response in a subject, the method comprising administering to the subject an immunologically effective amount of an immunogenic composition comprising at least one nucleic acid molecule in association with nanoparticles, wherein the nucleic acid molecule encodes at least one antigen, and wherein the nanoparticles are about 0.02pm to about 0.10pm in diameter.
3. 9. A method of claim 9 wherein the nanoparticles are about 0.04pm to about 0.05pm in diameter. 54 O N
4. 10. A method of claim 8 or claim 9 wherein the nanoparticles are about 0.05pm in o diameter
5. 11. A method of any one of claims 8 to 10 wherein the nanoparticles are of a substantially uniform size. S 5 12. A method of any one of claims 8 to 11 wherein the nanoparticles comprise a solid core.
6. 13. A method of any one of claims 8 to 12 wherein the nanoparticles are composed of a Ssubstantially inert material.
7. 14. A method of claim 13 wherein the inert material is selected from the group consisting of latex, ferrous molecules, gold, glass, calcium phosphate, polystyrene, poly lysine G, biodegradable polymers, biocompatible polymers, and combinations thereof. The method of any one of claims 8 to 14 wherein the immune response is at least a CD8 and/or B cell immune response.
8. 16. The method of claim 15 wherein the B cell immune response results in the production of antibodies specific to the antigen of the immunogenic composition.
9. 17. The method of claim 16 wherein the antibodies are selected from the group consisting of IgGI, IgG2a and IgG2b antibodies.
10. 18. The method of claim 17 wherein the antibodies are IgG 1 antibodies.