WO2006012695A1 - An immunogenic composition - Google Patents

An immunogenic composition Download PDF

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Publication number
WO2006012695A1
WO2006012695A1 PCT/AU2005/001163 AU2005001163W WO2006012695A1 WO 2006012695 A1 WO2006012695 A1 WO 2006012695A1 AU 2005001163 W AU2005001163 W AU 2005001163W WO 2006012695 A1 WO2006012695 A1 WO 2006012695A1
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microparticles
beads
population
composition
antigen
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PCT/AU2005/001163
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French (fr)
Inventor
Magdalena Plebanski
Anita Gamvrellis
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Panvax Limited
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Priority to AU2004904356 priority Critical
Priority to AU2004904356A priority patent/AU2004904356A0/en
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Publication of WO2006012695A1 publication Critical patent/WO2006012695A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/39Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6933Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained by reactions only involving carbon to carbon, e.g. poly(meth)acrylate, polystyrene, polyvinylpyrrolidone or polyvinylalcohol
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55555Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Immunogenic compositions comprising an antigen and two populations of microparticles, one population having an average size of less than 0.07 microns, the other having an average size of greater than 0.07 microns, where the smaller microparticles enhance cellular uptake of the larger microparticles and the cellular and humor immune responses to the antigen. Methods of obtaining immune responses using such compositions.

Description

AN IMMUNOGENIC COMPOSITION
FIELD OF THE INVENTION
The present invention relates to an immunogenic composition comprising micro- and nano-sized particles, as well as uses of the composition in, particularly, methods of eliciting an immune response in a subject.
BACKGROUND OF THE INVENTION
Manipulation of the immune systems of humans and animals is a recognised manner of treating or preventing certain diseases or conditions.
The mechanisms by which the immune system controls disease include the induction of neutralising antibodies (ie a humoral immune response), and the generation of cellular or T-cell responses. The latter include T-helper cells (TH) and cytotoxic T-lymphocytes (CTL).
In instances of viral infection (eg polio or hepatitis), antibodies provide protection by preventing the virus from infecting cells. Antibodies can also protect against bacteria (eg 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 the major histocompatability complex, class I or class II (ie MHC I or MHC II) and are displayed on the surface of antigen presenting cells (APCs) such as dendritic cells (DCs) 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. In contrast, the receptors on CTLs react with antigens displayed on MHC 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 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 immune 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 by 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.
On the other hand, stimulation of IgA production is a "first line" of 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 are desirable.
In yet other instances, a vaccine might result in stimulation of APCs, but the degree of immune stimulation is sub-optimal. For example, DCs are characterised by a series of subsets 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 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 non- living or sub-unit vaccines is unlikely, although alternative routes of processing and presentation for MHC I have been proposed in APCs through the uptake of apoptoptic cells, immune complexes and particles (Reimann J et al, 1999). Non-infective, viral-like particles (VLPs) composed of the Hepatitis B surface protein or yeast retro-transposon protein, have been shown to be efficiently processed for MHC I presentation by macrophages to induce CD8 CTL responses in vitro and in vivo (Schirmbeck R et al, 1995 and Plebanski M et al, 1998). 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 lipid content, it has been proposed that VLPs are immunogenic not by virtue of their size, but by virtue of their biochemical composition. This is consistent with the proposal that when antigen is presented in formulations containing lipid or detergent, they are able to fuse with APCs, possibly by damaging the cell membrane, and thus gain entry into the APC cytoplasm.
In International patent application no. PCT/AU01/01160 (WO 2002/22164), it has been shown that when micro- or nano-sized particles covalently conjugated to antigen are administered as part of an immunogenic composition to a subject, the size of the particles per se has a bearing on the type of immune response, or the relative strength of the types of immune response, that is/are elicited in the subject. That is, particles having an average diameter of, for example, 0.1 μm to 1.0 μm, which were covalently conjugated to antigen were able to elicit a strong humoral immune response, whereas smaller sized particles (eg having an average diameter of 0.03 μm to 0.05 μm) covalently conjugated to antigen were able to elicit a strong humoral immune response and a moderate to strong cellular immune response. The entire disclosure of WO 2002/22164 is to be regarded as incorporated herein by reference.
The present applicant has now extended the work described in WO 2002/22164 so as to examine the immunogenicity of compositions comprising a mixture of particle sizes. It is postulated that smaller sized particles (eg having an average diameter around 0.04-0.05 μm) are able to enhance the uptake by APCs of larger sized particles (eg having an average diameter around 0.5-1.0 μm) when the two sizes are co-injected. The result of this is that, at least, an enhanced cellular immune response may be elicited in a subject with larger sized beads covalently conjugated to antigen. Also, for a composition wherein both the larger and smaller sized particles are covalently conjugated to antigen, there is the potential to elicit a strong humoral immune response and a strong cellular immune response to the antigen. SUMMARY OF THE INVENTION
In a first aspect, the present invention provides an immunogenic composition comprising microparticles and at least one antigen, wherein said microparticles comprise a mixture of first and second microparticle populations, said first population having an average particle diameter size of greater than about 0.07 μm and said second population having an average particle diameter size of less than about 0.07 μm.
In a second aspect, there is provided an immunogenic composition according to the first aspect that is capable of eliciting a cellular immune response and/or a humoral immune response to the at least one antigen in said subject.
In a third aspect, the present invention provides a method of eliciting an immune response in a subject, said method comprising administering to said subject an immunologically- effective amount of a composition according to the first or second aspects of the invention.
In a fourth aspect, the present invention provides a method of enhancing the uptake of a first population of microparticles by antigen presenting cells (APCs) in a subject, said first population having an average particle diameter size of greater than about 0.07 μm, comprising administering to said subject said first population of microparticles with a second population of microparticles, said second population having an average particle diameter size of less than about 0.07 μm, wherein said first and second populations of microparticles are administered concurrently or consecutively in either order.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows that smaller (ie 0.04 μm) beads increases the uptake of larger (ie 1 μm) beads (n=2 experiments). Fluorescence of beads in murine LN after 24 hrs post injection with (A) 1 μm green beads, (B) 0.04 μm red beads, (C) 1 μm + 0.04 μm beads co-injected into the same site. Numbers indicate the percentage of cells that internalised microparticles, gated on large forward and side scatter cells. This gate was confirmed by back-gating to contain the CDlIc and CDlIb populations (APCs of dendritic and monocytoid lineage).
Figure 2 shows the competitive immunogenicity of mice immunised with 1 μm beads conjugated to OVA and unconjugated 0.04 μm beads. ELISPOT assay at day 9 for IFNγ production from mice immunised with 1 μm beads conjugated to OVA alone, co- immunisation of 1 μm beads conjugated to OVA plus 0.04 μm naked beads at the same site and at different sites (C57BL/6 mice immunised once with lOOμg OVA, n=3/group).
Figure 3 shows the localisation of beads to draining lymph nodes. C57BL/6 mice were immunised ID in the footpad with OVA conjugated to 0.02, 0.04 or 1 μm fluorescent (FITC) beads and the draining popliteal lymph nodes (LN) dissected at 48 and 14 days after immunisation. The data is presented as the mean FITC % positive cells +/- standard error (SE).
Figure 4 shows the phenotypic characterisation of microparticle-positive cells at 48 hr after injection of 0.02, 0.04 or 1 μm beads: the mean +/- SE for 3-14 mice tested for each marker is shown.
Figure 5 shows enhanced cell trafficking to the draining lymph node and consequent immunogenicity of larger beads by co-injection with naked 0.04 μm beads. Green fluorescent large 1 μm beads were injected alone (20 μl of 1% solids) or together with 0.04 μm red beads (10 μl of 1% solids each) intradermally in the footpad of C57BL/6 mice and draining popliteal lymph nodes (LN) dissected 24 hr later for FACScan analysis. Red fluorescent 0.04 μm beads alone were injected as controls and these LN cells used to set up the red-green FACScan compensation. 20-100,000 events were collected for each sample type. The data shows the mean percentage +/- SE of green fluorescent cells (positive for 1 μm beads) from the total LN cells from 3 animals per group. Enhanced numbers of 1 μm microparticle-positive cells (green only) in the LN were found upon 0.04 μm beads co- injection (p<0.0005). There was no increase in the uptake of the red 0.04 μm beads (not shown). One of two similar experiments is shown. Similar enhanced lμm bead uptake was seen with unconjugated or OVA-conjugated beads.
Figure 6 shows spleen cells from mice immunised ID with 1 μm OVA-conjugated beads alone (1 μm-OVA beads) or mixed with unconjugated 0.04 μm beads (1 μm-OVA beads plus 0.04 μm beads) as above were tested after 9 days for the induction of IFN-γ producing CD8 T cells by re-stimulation with the peptide SIINFEKL (SEQ ID NO: 1) in ELISPOT assays. Enhanced induction of IFN-γ was found after co-immunisation (p<0.005). Figure 7 shows cellular immune responses as measured by IFN-γ production in splenocytes from mice immunised with OVA alone or OVA conjugated to 0.04 μm or 0.5 μm beads. Data is expressed as "spots/million cells + SEM (n=4).
Figure 8 shows specific anti-OVA antibody activities in serum from mice immunised with OVA conjugated to 0.04 μm or 0.5 μm beads. The data is expressed as "average OD at 450 nm + STDEV" (n=4). Pre= pre-bleed data.
Figure 9 shows cellular immune responses as measured by IFN-γ production in splenocytes from mice immunised with 0.5 μm beads conjugated to OVA and naked 0.04 μm beads either together (C), sequentially (18 hours apart; D) or at different sites (0.04 μm beads were injected in the left foot pad and 0.5 μm beads conjugated to OVA were injected into the right foot pad; E). Data is expressed as "spots/million cells + SEM" (n=4).
Figure 10 shows specific anti-OVA antibody activities in serum from mice immunised with OVA conjugated 0.5 μm beads and naked 0.04 μm beads administered either together, sequentially or at different sites. Data is expressed as "average OD at 450 nm + STDEV" (n=4).
Figure 11 shows the uptake of beads by CDlIc positive dendritic cells in the lymph nodes and measured by flow cytometry. (A) % of 0.04 μm bead positive CDl Ic cells in the left vs right draining lymph node, (B) % of 0.5 μm bead positive CDlIc cells in the left vs right draining lymph nodes.
Figure 12 shows cellular immune responses as measured by IFN-γ production in splenocytes from mice immunised with two different sizes of smaller beads (0.043 μm or 0.067 μm beads) together with OVA conjugated 1.0 μm beads. Data is expressed in "spots/million cells ± SEM" (n=4).
Figure 13 shows specific anti-OVA antibody activities in serum from mice immunised with two different sizes of smaller beads (0.043 μm or 0.067 μm beads) together with OVA conjugated 1.0 μm beads. Data is expressed as "average OD at 450 nm + STDEV" (n=4). Figure 14 shows cellular immune responses as measured by IFN-γ production in splenocytes from mice immunised with OVA absorbed onto 0.5 μm beads and naked 0.04 μm beads. Data is expressed in "spots/million cells + SEM" (n=4).
Figure 15 shows specific anti-OVA antibody activities in serum from mice immunised with OVA absorbed onto 0.5 μm beads and naked 0.04 μm beads. Data is expressed as "average OD at 450 nm ± STDEV" (n=4).
Figure 16 shows the uptake of OVA absorbed 0.5 μm beads by CDlIc positive dendritic cells in the lymph nodes and measured by flow cytometry.
DETAILED DESCRIPTION OF THE INVENTION
In a first aspect, the present invention provides an immunogenic composition comprising microparticles and at least one antigen, wherein said microparticles comprise a mixture of first and second microparticle populations, said first population having an average particle diameter size of greater than about 0.07 μm and said second population having an average particle diameter size of less than 0.07 μm.
Preferably, the first population of microparticles has an average particle diameter size of greater than 0.08 μm and, more preferably, greater than 0.09 μm. More preferably, the first population of microparticles has an average particle diameter size in the range of from about 0.09 to 5.0 μm, still more preferably in the range of from about 0.1 to 2.0 μm, even more preferably in the range of from about 0.45 to 1.2 μm, most preferably in the range of from about 0.5 to 1.0 μm.
Preferably, the second population of microparticles has an average particle diameter size of less than 0.067 μm and, more preferably, less than 0.06 μm or less than about 0.05 μm. More preferably, the second population of microparticles has an average particle diameter size in the range of from about 0.02 μm to 0.05 μm. Most preferably, the average particle diameter size is about 0.035-0.049 μm.
Preferably, the composition is intended to be administered as a vaccine.
The "antigen" for use in the immunogenic composition of the present invention can be any molecule, moiety or entity capable of eliciting an immune response in a subject. This includes both humoral (ie antibody mediated) and/or cellular (ie T-cell mediated) immune responses.
Depending upon the intended function of the composition, one or more antigens may be included in the composition.
Various types of antigens may be used in the composition of the invention including peptide (eg an epitope), protein (or an antigenic fragment thereof), lipid, carbohydrate, nucleic acid or other type of molecule or a combination of any of these.
Antigens may be derived from a pathogen, tissue, cell, organ or molecule or other substance depending upon the intended purpose of the composition, and may be a purified antigen, cell lysate or culture filtrate, or be of recombinant origin produced in suitable hosts such as bacteria, yeast or mammalian or insect cell lines.
Antigens derived from pathogens may be antigens derived from, for example, intra or extracellular pathogens or antigenic fragments thereof including viral (eg human immunodeficiency viruses, influenza viruses and hepatitis viruses), bacterial (eg Streptococci, Leishmania etc) or protozoal (eg malaria) pathogens or antigenic fragments thereof.
Specific examples of antigens derived from pathogens and envisaged for use in the present invention are as follows: human immunodeficiency virus (HIV) core, Gag, Pol, Tat, Rev, Nef (and fragments thereof), El, E2 and NS2 proteins and gpl20/160 envelope glycoprotein; Hepatitis B virus (HBV) pre Sl Ag; Hepatitis C virus (HCV) proteins;
Influenza nucleoprotein and haemagglutinin-neuraminidase; Respiratory syncyticial virus (RSV) F and G proteins; Epstein Barr virus (EBV) gp340 and nucleoantigen 3A; Foot and mouth disease virus (FMDV) protein; Cytomegalovirus (CMV) protein; Human papilloma virus (HPV) strain 16 E7 protein-derived peptide (ie E7.1; RAHYNIVTF (SEQ ID NO: I)); Dengue surface or core capsid protein; Streptococcal surface and M proteins; Leishmania major surface glycoprotein (gp63); Bordetella pertussis surface protein; Staphylococcal proteins; Helicobacter pylori proteins; Mycobacterium tuberculosis 38kD lipoprotein and 3OkD protein (Ag85), 1OkD and 65kD proteins; Neisseria meningitidis class 1 outer protein; antigens from Plasmodium species such as P vivax, P falciparum and other Plasmodium species including circumsporozite protein (CS) and TRAP, MSP-I, MSP-2, MSP-3, MSP-4, MSP-5, AMA-I, RESA, SALSA, STARP, LSAl and LSA3; or a combination of one or more of said antigens or antigenic fragments thereof.
Antigens derived from a tissue, cell or organ may be antigens associated with a cancer, such as a cancer of the skin, colon, breast, lung or pancreas.
Specific examples of antigens associated with a cancer and envisaged for use in the present invention are as follows: melanoma specific antigen (eg the MAGE series antigen); carcinoembryonic antigen (CEA) from colon; nm23 cancer antigen; prostatic specific antigen (PSA); mucin antigens (eg the MUC-I to MUC-7 antigens); or a combination of one or more of said antigens or antigenic fragments thereof. Other examples of suitable cancer-associated antigens are mentioned in Berzofsky JA et al., 2004, the entire disclosure of which is to be regarded as incorporated herein by reference.
Other antigens derived from a tissue, cell or organ may be antigens associated with a degenerative disease such as Alzheimer's disease (AD).
Antigens derived from a molecule, may be antigens derived from pollen.
The present invention contemplates the use of free antigen (eg antigen that is in soluble form) and/or antigen that is covalently or non-covalently bound (eg adsorbed to the surface) to the surface of the microparticles.
Accordingly, in one embodiment of the first aspect, the invention provides an immunogenic composition comprising said first and second populations of microparticles and said at least one antigen, wherein said at least one antigen is in a free, soluble form.
In a further embodiment of the first aspect, the invention provides an immunogenic composition comprising said first and second populations of microparticles and said at least one antigen, wherein said at least one antigen is covalently or non-covalently bound to microparticles of the said first population.
In a still further embodiment of the first aspect, the invention provides an immunogenic composition comprising said first and second populations of microparticles and said at least one antigen, wherein said at least one antigen is covalently or non-covalently bound to microparticles of the said first population and said second population of microparticles. The at least one antigen may be bound to microparticles of the first and/or second populations via a linker molecule (eg a flexible peptide sequence such as those containing a short string of Glycines (G)).
The amount of the at least one antigen utilised in the immunogenic composition of the present invention may be in the range of 2 to 2000 μg per single injection, more preferably, 10 to 1000 μg per single injection, still more preferably, 20to 500 μg per single injection, and even more preferably, 20 to 200 μg per single injection.
The term "microparticles" as used herein refers to small, micro- or nano-sized particles. Such microparticles may be in the form of a bead or sphere or any other suitable shape able to be taken up by APCs.
Microparticles suitable for use in the present invention may be composed of any suitable material and may, therefore, be microparticles composed from protein (eg virus-like particles (VLPs)) or saponins (eg iscoms). However, preferably, the microparticles are composed of a material that is substantially immunologically inert (ie the material itself does not provoke any substantial immune response). Thus, the microparticles may be composed from materials such as gold, glass, silica, and polystyrene. However, more preferably, the microparticles are composed of a material that is substantially immunologically inert and which is biodegradable and biocompatible, such as ferrous molecules, calcium phosphate, and carbohydrate-based polymers including polylysine G (PLG) and poly(glycolide) (PGA). Particularly preferred, are microparticles composed of a biodegradable and biocompatible polyacetal such as those described in United States patent no. 5,863,990, the entire disclosure of which is to be regarded as incorporated herein by reference. The microparticles are preferably solid (ie having a solid rather than hollow core) and may, therefore, be of a substantially homogenous composition throughout.
The immunogenic composition of the present invention may comprise any ratio of first population microparticles: second population microparticles which, preferably, results in enhanced uptake of microparticles of the first population by APCs. However, preferred ratios range from about 10:1 to about 1:10. More preferably, the ratio of first population microparticles:second population microparticles is about 1:1.
The immunogenic composition of the present invention is adapted to elicit at least a humoral immune response to the at least one antigen, however the immunogenic composition may be adapted to elicit both a humoral immune response and a cellular immune response to the at least one antigen. The humoral response elicited by the immunogenic composition is preferably selected from the group consisting of IgA, IgD, IgG, IgM and subclasses thereof. The cellular immune response elicited by the immunogenic composition is preferably selected from the group consisting of activation, maturation or proliferation of TH cells, in particular IFN- and IL4-producing T-cells, and CTLs, particularly CD8 CTLs and B-cells.
The immunogenic composition of the present invention may also cause the activation, maturation and/or proliferation of cells which assist in mounting or amplifying an immune response. These include but are not limited to APCs (especially dendritic cells of myeloid or lymphoid origin) and/or macrophages.
The immunogenic composition of the present invention may be used in the treatment, prophylaxis or prevention of a disease or condition caused by, or associated with contact with, the at least one antigen. For example, the immunogenic composition may be used in the treatment or prophylaxis of certain viral diseases, cancers or degenerative diseases such as AD.
The immunogenic composition of the present invention offers the possibility of use as an effective single-dose vaccine, but may also be used in multiple vaccine dose regimes.
By the term "single dose", it is meant that a humoral immune response and/or cellular immune response is elicited at a maximal level (ie a level that is substantially incapable of being further increased by further vaccination), or is immunoprotective, following one administration of the immunogenic composition.
Administration of the immunogenic composition may be by any suitable means, for example, by intramuscular (i.m.), intraperitoneal (i.p.), intravenous (i.v.), intradermal (i.d.) or subcutaneous (s.c.) injection, oral administration, by inhalation, or by administration through a mucosal surface or site.
The amount of the immunogenic composition of the present invention that may be delivered to a subject will be an "immunologically-effective amount", that is an amount which is effective to elicit an immune response against the at least one antigen, and may vary according to, for example, the immune status of the subject (ie depending upon whether the subject is immunosuppressed or immunostimulated), and the judgement of attending physician or veterinarian. However, by way of example, the subject may receive from 1 μg to 10,000 μg, more preferably, 50 μg to 5,000 μg, still more preferably, 100 μg to 1,000 μg, and even more preferably, 100 μg to 500 μg of the immunogenic composition of the present invention per administration.
The immunogenic composition may further comprise one or more suitable adjuvants.
Suitable adjuvants include alum, as well as any other adjuvant or adjuvants well known in the vaccine art for administration to humans. Preferred adjuvants include growth factors and cytokines such as chemokines and cytokines that bias immune reactivity to ThI or Th2. Such growth factors and cytokines may be included in the immunogenic composition of the present invention at a concentration in the range of up to 33%, preferably up to 10% and more preferably up to 5%, by weight of the composition.
In a second aspect, there is provided an immunogenic composition according to the first aspect that is capable of eliciting a cellular immune response and/or a humoral immune response in said subject.
In a third aspect, the present invention provides a method of eliciting an immune response in a subject, said method comprising administering to said subject an immunologically- effective amount of a composition according to the first or second aspects of the invention.
The subject may be a human or any other animal in which it is desired to elicit an immune response. This includes companion animals (such as dogs and cats), livestock (such as cattle, sheep, horses, cows, pigs, goats, llamas, poultry, ostriches, emus) and native and exotic animals, wild animals and feral animals.
Preferably, the immune response elicited by the method of the third aspect involves activation, maturation and/or proliferation of APCs (especially dendritic cells of myeloid or lymphoid origin) and/or macrophages. More preferably, the immune response elicited by the method of the third aspect involves activation, maturation and/or proliferation of DEC205+, CD40+ and CD86+ cells.
In a fourth aspect, the present invention provides a method of enhancing uptake of a first population of microparticles by antigen presenting cells (APCs) in a subject, said first population having an average particle diameter size of greater than about 0.07 μm, comprising administering to said subject said first population of microparticles with a second population of microparticles, said second population having an average particle diameter size of less than about 0.07 μm, wherein said first and second populations of microparticles are administered concurrently or consecutively in either order.
Advantages achieved by the immunogenic composition of the present invention may also be achieved by separately administering the first and second populations of microparticles to a subject, either concurrently or consecutively. Preferably, the separate administrations are made by the same route of administration. More preferably, the separate administrations are made by i.m., i.p., Lv., i.d. or s.c. injection at the same, or adjacent sites.
By "adjacent sites" it is meant that the sites are separated by no more than about 20.0 cm, and are generally within about 0.02 to 10.0 cm of each other, more preferably, within about 0.05 to 1.0 cm of each other.
Where the separate administrations are made consecutively, the period of time between the administrations may generally be no more than about 12 hours, but may be between 5 and 120 minutes in duration. However, most preferably, for consecutive administrations, the administrations will be separated by a period of 5 to 120 seconds.
In the method of the fourth aspect, one or both of the first and second populations of microparticles may be administered as an immunogenic composition further comprising at least one antigen. The at least one antigen may be present in the immunogenic composition as free antigen and/or be covalently or non-covalently bound to the surface of the microparticles.
Thus, the method of the fourth aspect contemplates, for example, the concurrent or consecutive (in either order) administration of a first immunogenic composition comprising said first population of microparticles and at least one antigen (in free, soluble form and/or covalently or non-covalently bound to the microparticles) and a second immunogenic composition comprising said second population of microparticles. In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non-limiting examples.
EXAMPLES
The examples show, in particular, that when "small" microparticles (ie microparticles having an average diameter of less than 0.07 μm) and "large" microparticles (ie microparticles having an average diameter of greater than 0.07 μm) are administered into a subject, uptake by lymph node APCs of the large microparticles is enhanced. Further, the examples show that consecutive administration of small "naked" microparticles (ie microparticles having an average diameter of less than 0.07 μm and having no covalently conjugated antigen) at the same site as large antigen-conjugated microparticles (ie microparticles having an average diameter of greater than 0.07 μm covalently conjugated to an antigen) enhances the immunogenicity of the large microparticles. Therefore, the examples indicate that the small microparticles act as an adjuvant to the large microparticles.
While the applicant is not to be bound by theory, it is believed that small microparticles are taken up by lymph node APCs (particularly, dendritic cells of subsets DEC205+, CD40+ and CD86+) by a mechanism inhibited by phorbol myristate acetate (PMA) and, therefore, likely involving caveole and/or clathrin pits (which are believed to be capable of taking up particles of no more than 0.07 μm in size) whereby they may cause activation, maturation and/or proliferation of APCs and enhance the transfer to and uptake of large microparticles by lymph node APCs. Specifically, the small microparticles may (i) activate DCs so as to become more phagocytic to the large microparticles, (ii) signal DCs that have taken up large microparticles to migrate to the lymph node, (iii) locally induce DCs to secrete cytokines thereby attracting more DCs to the site of administration of the microparticles and generally enhancing the numbers of DCs migrating to the lymph node, and/or (iv) causing proliferation of DCs thereby enhancing the numbers of DCs in the lymph node. Any antigen which may be covalently conjugated to the small microparticles and/or the large microparticles appear to be processed in a manner for MHC I presentation. In the case of antigen covalently conjugated to small microparticles, this presentation is by a mechanism that involves, it is believed, Rab 4-independent and TAP dependent processes. It may be appreciated from the applicant's theory that the small particles (ie the second population of microparticles) may have an average particle size that is 0.07 μm in size or slightly greater and, importantly, still uptaken by lymph node DCs by a mechanism inhibited by PMA. Accordingly, it is to be understood that the present invention extends to the use of first and second microparticle populations wherein the second population has an average particle size which allows their uptake by DCs through a mechanism inhibited by PMA. In this case, the first population will preferably have an average particle size that is substantially larger than that of the second population, such that the microparticles are still uptaken by APCs but through a mechanism other than one that is inhibited by PMA.
EXAMPLE 1: Materials and methods
Microparticles
The microparticles were obtained from three different manufacturers.
Carboxylated polystyrene microspheres were obtained from Polysciences of sizes: 0.02 μm, 0.04 μm, 1 μm and 2 μm. Actual average diameter size of the "0.04 μm" Polysciences beads is 0.047-0.049 μm.
Carboxylated 0.04 μm and 1 μm fluorescent red labelled beads were obtained by Molecular Probes. NH2-modified 0.043 μm and 0.067 μm fluorescent green labelled beads were obtained from IDC, Carboxylated 0.5 μm fluorescent yellow-green labelled beads were obtained from Molecular Probes..
Mice and immunisation
H-2Kb C57BL/6, 6-8 week old mice (6 mice per experimental group unless indicated otherwise) were immunised with 100 μg of antigen and lOOμl of 1% solids of polystyrene microspheres, by intradermal (i.d.) injection into the mouse footpad.
Bead-antigen conjugation
The microspheres/beads were adjusted to 2% solids, mixed 1:1 (vol:vol) with 2mg/ml of Ovalbumin (OVA, Grade III, Sigma), in 0.04M MES ([2-N-morpholino]ethanesulfonic acid) buffer pH 6.0 for 15 min. For covalent conjugation, 1-ethyl (3- dimethylaminopropyl) carbodiimide (Sigma) was added at 4mg/ml, pH adjusted to 6.5 with NaOH and preparations (mixed or conjugated) rocked for 3 hours at room temperature (RT). Glycine was added to 100 mM for 30 min before overnight dialysis in cold PBS using a 12kD membrane cut off. After dialysis, the volume was adjusted to maintain 1% solids and lmg/ml OVA. Endotoxin levels (<3 EU/ml in all final bead preparations tested) were assayed by standard methods. Efficiency of conjugation was determined in representative experiments by inclusion of trace amounts of 125I-labeled OVA. After 125I-labeled conjugation, lOOμm samples were taken and free OVA removed by repeated centrifugation. Solids and PBS washings were counted to determine percentage antigen conjugated. Conjugated beads were stored at 4° and sonicated for 15 minutes before use.
Dendritic cell preparation from lymph nodes with collagenase digest
Popliteal lymph nodes were harvested from mice by dissection. Excess fat and connective tissue were removed. The lymph nodes were then placed into a Petri-dish containing 2 ml of cold complete RPMI containing 10% foetal calf serum. The lymph nodes were macerated with the frosted edges of two microscope slides. The cell slurry was then collected into a small digestion tube and each petri dish was thoroughly washed with 2 ml of cold complete RPMI containing 10% foetal calf serum.
For collagenase digestion, 1 ml of DNAse and 7 mg of Collagenase was mixed in each tube by gentle vortexing on ice. 62.5 μl DNAse/collagenase per each lymph node was added to the digest tube. The digest tube(s) were then incubated at room temperature for 20-25 minutes on a rocker or rotation wheel. 37.5 μl EDTA (0.1M) per lymph node was then added to stop the collagenase digestion and the cell suspension incubated for 5 minutes at room temperature with mixing. The collagenase digest slurry was then collected with a glass pipette and put through a cell filter into a pre-labelled tube. The volume was then made up to 9 ml with cold complete RPMI containing 10% foetal calf serum. 1 ml of cold EDTA/FCS (1:11 of 0. IM EDTA in neat foetal calf serum) was then added by underlay and the tubes centrifuged at 1700 rpm for 7 minutes at 4°C. The supernatant was then removed and the cells resuspended in 10 ml of complete RPMI containing 10% foetal calf serum, counted and plated into a V bottom plate for flow cytometry analysis (FACS).
ELISPOT assays
As described previously (Plebanski, M., et al 1998), splenocytes were incubated with the class I epitope SIINFEKL (SEQ ID NO: 2)at 2.5μg/ml or OVA at 25μg/ml or OVA plus helper epitope [KISQAVHAAHAEINEAGREV (SEQ ID No 3)] at 25μg/ml for 18 hours on plates (MAHA Millipore) pre-coated with anti-IFNγ mAb (clone R4, ATCC). Cells were discarded and plates incubated with anti-IFNγ mAB-biotin (XMG.21-biotin, Pharmingen), followed by extravidin-alkaline phosphatase (AP) (Sigma) and spots detected with an AP kit (Biorad). Data are presented as mean spot forming units (sfu) per million cells +/- standard error of the mean (SEM) or standard deviation (STDEV).
ELISA for anti-OVA antibodies
Plates (96 well) coated with OVA (10 μg/ml in 0.2 M NaHCO3 buffer, pH9.6) were incubated with test or control mouse sera, followed by incubation with HRP-conjugated sheep anti-mouse Ig (Silenus, AUST) and detected with ABTS. Titre was defined as the serum dilution at which the 405 nm OD was above the mean + 3 standard deviations of naϊve sera values.
Phenotypic analysis
Draining popliteal lymph node cells, were isolated from mice treated by ID footpad injection with beads (antigen coupled, with or without red or green fluroescent labels). These cells, as a single cell suspension, were incubated on ice for 30 min with phycoerythrin (PE) conjugated mAb to CD40, CD80, CD86 (Pharmigen) or mAb hybridoma supernatants to DEC205, F4/80 followed PE-mouse anti-rat (Pharmigen) in cold PBS/10% FCS, washed 3X with the latter and analysed by FACScan (Becton & Dickinson).
Statistics
P values for difference between mean spot forming units (sfu) by ELISPOT were determined using two-tailed Student t-test.
EXAMPLE 2: Immunisation of mouse footpad with red and/or green beads, of the same or different size, at the same or different sites
A shown in Figure 1, fluorescent green labelled 1 μm beads (Polysciences) showed a low level of uptake in the draining lymph nodes with only 2.6% of the lymph node (LN) APCs (of dendritic and monocytoid lineage) positive for green beads (A). In contrast, the fluorescent red labelled 0.04 μm beads (Polysciences) were taken up by draining LN APCs (11.7%) as shown in (B). The mixed bead preparation (ie 1 μm beads + 0.04 μm beads) showed enhanced uptake of green beads (4.7%) by cells that also contained red beads, indicating that the presence of the smaller 0.04 μm beads in the preparation promoted greater uptake (ie nearly 2-fold) by APCs of the larger 1 μm beads. However, there still remained a proportion of red microparticle-positive cells (7.1%) with no green 1 μm beads, thereby suggesting preferential uptake of the smaller beads by APCs. The origin of the APCs was confirmed by back-gating to contain the DCl Ic and CDl Ib populations.
EXAMPLE 3: Examination of immunogenicity in mice immunised in the foot pad with a combination of 1 μm beads conjugated to OVA and naked 0.04 μm beads at the same or different sites
Figure 2 shows the results of competitive immunogenicity assays from mice immunised with either 1 μm beads (Polysciences) that were conjugated to OVA alone (1 μm beads/OVA), or with both 1 μm/OVA conjugated beads and naked 0.04 μm beads ((Polysciences); that is 0.04 μm beads with no conjugated antigen) either at the same site or at different sites. When 1 μm beads and 0.04 μm beads were injected at the same or separate sites, the 0.04 μm beads were preferentially internalised by APCs compared with the 1 μm beads (not shown). However, co-immunisation of naked 0.04 μm beads at the same site as OVA-conjugated 1 μm beads was found to enhance the immunogenicity of the 1 μm beads implying that the 0.04 μm beads act as "mix-in" adjuvants to the larger beads. Therefore, high numbers of MHC class I restricted T cells and T cells responsive to OVA were induced by immunisation of OVA-conjugated 1 μm beads together with naked 0.04 μm beads.
EXAMPLE 4: In vivo localisation of beads
The fate of different sizes of fluorescent OVA-conjugated beads after ID injection was followed. At various intervals from 48 hours to 14 days post immunisation, cells that had taken up beads were found in the draining lymph node. At both 48 hours and 14 days, the 0.04 μm beads (Polysciences) were found in more cells than other smaller (ie 0.02 μm) or larger (ie 1 μm) sized beads (Figure 3). Phenotypic analysis at 48 hrs of the lymph node cells showed preferential localisation of the 0.04 μm beads to cells which expressed the markers DEC205+, CD40+ and CD86+. These markers are characteristic of the mature/activated DC subset of subcutaneous origin (Figure 4). In contrast, at 48 hr 1 μm beads localised preferentially to F4/80+, CD80+ cells representing a macrophage-like subset of APC (Shortman K et ah, 2002) and 0.02 μm beads were found in CD40+ cells, but not DEC205+ or F4/80+ cells. After 14 days, although fewer bead-positive cells were detected, the same phenotypic profile was seen in these cells, with the 0.04 μm beads continuing to localise preferentially to DEC205+ and the 1 μm beads to F4/80+ cells, with co-stimulatory marker expression similar to that seen at 48 hrs (data not shown). The results indicate that 0.04 μm beads are efficient antigen carriers to the DC in the draining lymph node and that the beads persist within LN cells for more than 14 days (considering both total LN uptake and cell phenotype). EXAMPLE 5: Localisation studies
The finding that the 0.04 μm beads (Polysciences) preferentially localised to DCs in the draining LN suggests that beads of this size cause activation of DCs and migration from dermal sites. To investigate this, fluorescent red labelled 0.04 μm beads were mixed with fluorescent green labelled 1 μm beads (Polysciences) and injected ID to determine whether this would enhance the localisation of the larger beads to draining lymph nodes. Figures 5 and 6 show that there was enhanced transport of larger beads in mixtures compared with 1 μm beads injected alone (Figure 5, P<0.0005). OVA-conjugated 1 μm beads mixed with naked 0.04 μm beads also induced greater T cell responses compared with 1 μm OVA- conjugated beads alone as measured by induction of IFN-γ producing CD8 T cells by re- stimulation with SπNFEKL (SEQ ID NO: 2) in ELISPOT assays.
EXAMPLE 6: Variation of bead composition
It is expected that the enhanced APC uptake and immunogenicity for the larger beads shown in the examples when co-administered with the smaller beads is independent of the material from which the beads are composed. Indeed, in other experimentation, 0.04 μm gold beads and 0.04 μm silica beads have demonstrated similar effects on APC proliferation as that seen with 0.04 μm polystyrene beads.
EXAMPLE 7: Effects on immunogenicity and antibody responses when larger and smaller beads are mixed
T cell responsiveness as measured by IFN-γ production was determined from mice immunised with OVA alone (soluble OVA) or OVA conjugated to 0.04 μm beads (Molecular Probes) or 0.5 μm beads (Molecular Probes). The results presented in Figure 7 show that 0.04 μm beads alone induced greater IFN-gamma responses compared with 0.5 μm beads alone. However, when 0.04 μm beads (whether naked or conjugated to OVA) were mixed with 0.5 μm OVA conjugated beads, immunogenicity of the 0.5 μm OVA conjugated beads was enhanced compared with 0.5 μm beads alone. T cells responsive to the SπNFEKL (SEQ ID NO: 2) epitope was greater when the 0.5 μm beads were conjugated to OVA compared with soluble OVA, beads alone or naϊve controls (P<0.05)
Anti-OVA antibody responses in serum from mice immunised with OVA conjugated to beads were measured by ELISA assay as shown in Figure 8. The results show that 0.04 μm beads conjugated to OVA induced a higher antibody response compared with 0.5 μm beads conjugated to OVA (see D vs E). Naked 0.04 μm beads mixed with 0.5 μm beads conjugated to OVA demonstrated an enhanced antibody response compared with 0.5 μm beads conjugated to OVA alone (see C vs E). AU immunisations with beads conjugated to OVA yielded higher antibody responses compared with OVA alone (see L). EXAMPLE 8: The effect of time between injections and site of injection on immunogenicity, antibody responses and bead uptake when smaller and larger beads are mixed
An immunogenicity assay was conducted as above to determine the cellular response in splenocytes from mice immunised with OVA conjugated to 0.5 μm beads (Molecular Probes) as shown in Figure 9. Consistent with earlier results, the inclusion of 0.04 μm naked beads (Molecular Probes) to the OVA conjugated 0.5 μm beads demonstrated an enhanced IFN-gamma response compared with the OVA conjugated 0.5 μm beads alone (see C vs B; p=0.03). This enhanced effect was not observed when the beads were administered sequentially (ie after 18 hours; see D vs B). Similarly, no enhanced effect was observed when the beads were administered at different sites (see E vs B).
Anti-OVA antibody responses in serum from mice immunised with OVA conjugated to beads were measured by ELISA assay as shown in Figure 10. 0.04 μm naked beads when mixed with 0.5 μm beads conjugated to OVA, induced antibody responses comparable to 0.04 μm beads conjugated to OVA alone (see E vs C). The addition of 0.04 μm naked beads to the 0.5 μm beads conjugated to OVA resulted in an enhanced antibody response compared with 0.5 μm beads conjugated to OVA alone (see C vs G). It was found that an improved antibody response was still observed when 0.04 μm naked beads were administered at the same site 18 hours after administration of 0.5 μm beads conjugated to OVA compared with 0.5 μm beads conjugated to OVA alone (see D vs G), although not at the level seen when the beads were co-administered (see C vs G). However, antibody responses to the 0.5 μm beads conjugated to OVA were not enhanced to the same extent when the 0.04 μm naked beads were administered at a different location (see J vs G).
Naked 0.04 μm beads or 0.04 μm beads mixed with 0.5 μm beads conjugated to OVA, were taken up equally well by dendritic cells in the draining lymph node. However,
Naked 0.04 μm beads injected at a distant site showed minimal uptake by dendritic cells in the distant lymph node (Figure HA). Similarly, 0.5 μm beads conjugated to OVA injected into the right foot pad where taken up by dendritic cells in the right draining lymph node and not the left lymph node (Figure HB).
Accordingly, the results demonstrate that enhanced immunogenicity of the larger bead formulations by smaller naked beads requires simultaneous co-administration of naked and antigen conjugated beads for the induction of optimum cellular responses. Nevertheless, antibody responses were still enhanced with the sequential administration of OVA conjugated 0.5 μm beads followed by naked 0.04 μm beads. However, administration of small beads at a distant site (ie opposite foot pads) to the OVA conjugated 0.5 μm beads did not improve immunogenicity (humoral or cellular). EXAMPLE 9: Comparison of the immunogenicity and antibody responses in mice injected with two different sizes of smaller beads (0.043 μm beads or 0.067 μm beads) mixed with larger beads
An immunogenicity assay was conducted as described above to determine the cellular response in splenocytes from mice immunised with OVA conjugated 1.0 μm beads (IDC) mixed with either 0.067 μm beads (IDC) or 0.043 μm beads (IDC). Induction of IFN- gamma responses in both groups was enhanced compared with naive controls. There was no significant difference in the response observed between the two sizes of smaller beads (Figure 12; p>0.05).
When antibody responses were examined, there was no significant difference in antibody production induced by either OVA conjugated 1.0 μm beads mixed with 0.043 μm beads or OVA conjugated 1.0 μm beads mixed with 0.067 μm beads (I vs L in Figure 13). Responses were greater compared with naϊve controls (K).
EXAMPLE 10: 0.04 μm beads enhance the immunogenicity and uptake of OVA absorbed 0.5 μm beads
IFN-gamma responses to the SIINFEKL (SEQ ID NO: 2) epitope were measured for 0.5 μm beads (Molecular Probes) onto which the OVA antigen was absorbed (Figure 14). The addition of naked 0.04 μm beads (Molecular Probes) to the OVA absorbed 0.5 μm beads resulted in a significantly higher IFN-gamma response compared with OVA absorbed 0.5 μm beads alone (P<0.05).
Anti-OVA antibody responses were measured by ELISA assay as shown in Figure 15. The addition of naked 0.04 μm beads did not enhance the antibody response induced by the OVA absorbed 0.5 μm beads (compare J and K). However, the antibody response was still greater compared with soluble OVA suggesting that the beads were providing an adjuvant effect.
As shown in Figure 16, uptake of the larger OVA absorbed 0.5 μm beads was enhanced by the addition of naked 0.04 μm beads in the draining lymph nodes.
Thus, in summary, the cellular response and in vivo uptake of OVA-absorbed 0.5 μm beads was improved by the addition of naked 0.04 μm beads. Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
AU publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
REFERENCES
1. Berzofsky JA et al, Progress on new vaccine strategies for the immunotherapy and prevention of cancer, J Clin Invest, 113(11):1515-1525, 2004.
2. Plebanski M et al, Protection from Plasmodium berghei infection by priming and boosting T cells to a single class I-restricted epitope with recombinant carriers suitable for human use, Eur J Immunol, 28:4345-4355, 1998.
3. Reimann J and Schirmbeck R, Alternative pathways for processing exogenous and endogenous antigens that can generate peptides for MHC class I-restricted presentation, Immunol Rev, 172:131-152, 1999.
4. Schirmbeck R et al, Hepatitis B virus small surface antigen particles are processed in a novel endosomal pathway for major histocompatibility complex class I- restricted epitope presentation, Eur J Immunol, 25:1063-1070, 1995.
5. Shortman K and Liu Y, Mouse and human dendritic cell types, Nat Rev Immunol, 2:151-161, 2002.

Claims

1. An immunogenic composition comprising microparticles and at least one antigen, wherein said microparticles comprise a mixture of first and second microparticle populations, said first population having an average particle diameter size of greater than about 0.07 μm and said second population having an average particle diameter size of less than 0.07 μm.
2. A composition according to claim 1, wherein the first population of microparticles has an average diameter size of greater than 0.09 μm.
3. A composition according to claim 1 or 2, wherein the first population of microparticles has an average particle diameter size in the range of from about 0.5 to 1.0 μm.
4. A composition according to claim 1, wherein the second population of microparticles has an average particle diameter size of less than 0.067 μm.
5. A composition according to claim 1, wherein the second population of microparticles has an average particle diameter size of less than 0.05 μm.
6. A composition according to claim 4 or 5, wherein the second population of microparticles has an average particle diameter size in the range of from about 0.40-0.43 μm.
7. A composition according to any one of the preceding claims, wherein the composition is administered as a vaccine.
8. A composition according to any one of the preceding claims, wherein said at least one antigen is in a free, soluble form.
9. A composition according to any one of claims 1 to 6, wherein said at least one antigen is covalently or non-covalently bound to microparticles of the said first population.
10. A composition according to any one of claims 1 to 6, wherein said at least one antigen is covalently or non-covalently bound to microparticles of the said first population and said second population of microparticles.
11. A composition according to claim 9 or 10, wherein the antigen is covalently bound to microparticles of the said first population and/or said second population of microparticles.
12. A composition according to any one of the preceding claims, wherein the microparticles are composed of a material that is substantially immunologically inert.
13. A composition according to any one of the preceding claims, wherein the microparticles are composed of a material which is biodegradable and biocompatible.
14. A composition according to any one of the preceding claims, wherein the microparticles have a solid core.
15. A method of eliciting an immune response in a subject, said method comprising administering to said subject an immunologically-effective amount of a composition according to any one of the preceding claims.
16. A method of enhancing uptake of a first population of microparticles by antigen presenting cells (APCs) in a subject, said first population having an average particle diameter size of greater than about 0.07 μm, comprising administering to said subject said first population of microparticles with a second population of microparticles, said second population having an average particle diameter size of less than about 0.07 μm, wherein said first and second populations of microparticles are administered concurrently or consecutively in either order.
17. A method according to claim 16, wherein one or both of the first and second populations of microparticles is/are administered as an immunogenic composition further comprising at least one antigen.
18. A composition according to claim 16 or 17, wherein said at least one antigen is in a free, soluble form.
19. A composition according to any one of claims 16 to 18, wherein said at least one antigen is covalently or non-covalently bound to microparticles of the said first population.
20. A composition according to any one of claims 16 to 18, wherein said at least one antigen is covalently or non-covalently bound to microparticles of the said first population and said second population of microparticles.
21. A composition according to claim 19 or 20, wherein the antigen is covalently bound to microparticles of the said first population and/or said second population of microparticles.
22. A composition according to any one of claims 16 to 21, wherein the microparticles are composed of a material that is substantially immunologically inert.
23. A composition according to any one of the claims 16 to 22, wherein the microparticles are composed of a material which is biodegradable and biocompatible.
24. A composition according to any one of claims 16 to 23, wherein the microparticles have a solid core.
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