LIPOSOMAL VACCINE TECHNICAL FIELD THIS INVENTION relates to prevention and treatment of infectious diseases. More particularly, this invention relates to a liposomal vaccine for treating or preventing infectious diseases and conditions by inducing a mucosal immune response.

BACKGROUND

Systemic immunity has proven effective in preventing diseases caused by a variety of different pathogens through serum immunoglobulin (Ig) at systemic sites, but not in preventing colonisation of mucosal sites and thus person-to- person transmission.. Therefore, for some diseases, systemic vaccination is not the optimal approach for inducing immunity. In contrast, mucosal vaccines against various organisms administered nasally are effective in inducing antigen-specific immune responses in both systemic and mucosal compartments. Due to this two- layered protective immunity, mucosal vaccination is an ideal strategy for combating both systemic and mucosal infections with the added benefit that prevention of mucosal colonization will also inhibit transmission by droplets and aerosols from the upper respiratory tract. Mucosal vaccination is also economically advantageous, an important consideration for vaccine development. Owing to the ease of vaccine administration by the nasal route, use of needles can be avoided. Pain-free, delivery will aid in greater patient compliance.

SUMMARY

An objective of the present invention is to provide an immunogenic agent and delivery system that elicits a mucosal immune response to a pathogen. In a broad form, the invention relates to facilitating or inducing mucosal immunity to a by delivery of an immunogenic protein, fragment or variant, by way of a lipid vesicle that further comprises a carrier protein such as diptheria toxoid (DT). Suitably, the carrier protein is located in an intravesicular space. In a particular form, a single immunogenic agent comprises a plurality of different immunogenic proteins, fragments or variants from a plurality of different pathogens.

An aspect of the invention provides an immunogenic agent suitable for administration to a mammal, said immunogenic agent comprising one or a plurality of immunogenic proteins, fragmentd, variants or derivatives thereof, a lipid vesicle and a carrier protein or a fragment or variant thereof.

In one embodiment, the carrier protein is diptheria toxoid (DT).

In an embodiment, the immunogenic agent is a lipid vesicle comprising a plurality of immunogenic proteins, fragments, variants or derivatives thereof from different pathogens.

Another aspect of the invention provides a composition comprising the immunogenic agent of the aforementioned aspect.

In an embodiment, the composition comprises an immunogenic agent that includes a lipid vesicle comprising a plurality of immunogenic proteins, fragments, variants or derivatives thereof, of or from the same or single pathogen.

In an embodiment, the composition comprises a plurality of different immunogenic agents that respectively comprise one or plurality of immunogenic proteins, fragments, variants or derivatives thereof, of or from different pathogens.

In an embodiment, the composition comprises a single immunogenic agent that includes a lipid vesicle comprising a plurality of immunogenic proteins, fragments, variants or derivatives thereof, of or from different pathogens.

In an embodiment, the composition comprises a plurality of different immunogenic agents that respectively comprise one or plurality of immunogenic proteins, fragments, variants or derivatives thereof, of or from different pathogens.

Another aspect of the invention provides a method of eliciting an immune response to one or a plurality of pathogens in a mammal, said method including the step of administering to the mammal an immunogenic agent comprising one or a plurality of immunogenic proteins, fragments, variants or derivatives thereof, a lipid vesicle and diptheria toxoid (DT) or a fragment or variant thereof, or a composition comprising same, to thereby elicit an immune response to the one or plurality of pathogens in the mammal.

Another aspect of the invention provides a method of immunizing a mammal against one or a plurality of pathogens, said method including the step of administering to the mammal an immunogenic agent comprising one or a plurality of immunogenic proteins, fragments, variants or derivatives thereof, a lipid vesicle and diptheria toxoid (DT) or a fragment or variant thereof, or a composition comprising same, to thereby immunize the mammal against the one or plurality of pathogens. Yet another aspect of the invention provides a method of treating or preventing infection by one or a plurality of pathogens in a mammal, said method including the step of administering to the mammal an immunogenic agent comprising one or a plurality of immunogenic proteins, fragments, variants or derivatives thereof, a lipid vesicle and diptheria toxoid (DT) or a fragment or variant thereof, or a composition comprising same, to thereby treat or prevent the infection by the one or plurality of pathogens in the mammal.

Suitably, the immunogenic agent elicits a mucosal immune response.

Typically, the mucosal immune response includes the production of IgA. In a preferred form, the immunogenic agent is administered intranasally.

Suitably, the immunogenic protein, fragment or variant is displayed on the surface of the lipid vesicle. Suitably, the diptheria toxoid (DT) or a fragment or variant thereof is located in an intravesicular space within the vesicle. In a preferred embodiment, the lipid vesicle is a liposome.

Suitably, the immunogenic protein fragment or variant is lipidated. In certain embodiments, a lysine (K) residue at the N-terminus of the immunogenic protein fragment or variant is lipidated. In a preferred form an N-terminal lysine (K) residue is lipidated via a and ε amino groups. In some embodiments, the or each lipid is a C ₁₆ fatty acid such as palmitic acid. Preferably, the N-terminal lysine (K) residue is in a spacer amino acid sequence at the N-terminus of the immunogenic protein fragment or variant.

In one particular embodiment, the immunogenic protein, fragment or variant is a group A streptococcus M protein fragment, variant or derivative thereof. In an additional or alternative embodiment, the immunogenic protein is an agent that facilitates restoring or enhancing neutrophil activity.

In a particular embodiment, the M protein fragment is or comprises a conserved region of the M protein. In one embodiment, the fragment is an immunogenic fragment that comprises, or is contained within a pi 45 peptide. In a particular embodiment, the immunogenic fragment is within, or comprises, a J8 peptide or variant thereof.

Preferably, the J8 peptide comprises, or consists essentially of, the amino acid sequence Q AEDKVKQ SRE AKKQ VEK ALKQLEDKVQ (SEQ ID NO: l). In one broad embodiment, the agent that facilitates restoring or enhancing neutrophil activity is the protein is SpyCEP, or a fragment thereof.

In a preferred embodiment, the SpyCEP fragment comprises, or consists essentially of, the amino acid sequence NSDNIKENQFEDFDEDWENF (SEQ ID NO:2).

In one particular embodiment, the SpyCEP fragment and the M protein fragment may be fused to form a single, chimeric peptide.

In one embodiment, the chimeric peptide is, may comprise, or consist essentially of, the amino acid sequence NSDNIKENQFEDFDEDWE FQAEDKVKQSREAKKQVEKALKQLEDKVQ

(SEQ ID NO:3), or a variant thereof.

In one particular embodiment, the immunogenic protein, fragment or variant is of an influenza virus. The influenza virus may be influenza A. A non- limiting example is, comprises, or consists essentially of, the amino acid sequence M SLLTE VETPIRNEWGCRC D S SD (SEQ ID NO:4). The influenza virus may be influenza B. A non-limiting example is, or comprises, or consists essentially of, the amino acid sequence PAKLLKERGFFGAIAGFLE (SEQ ID NO: 5).

In one particular embodiment, the immunogenic protein, fragment or variant is of a rhinovirus. A non-limiting example is, comprises, or consists essentially of, the amino acid sequence

GAQVSTQKSGSHENQNILTNGSNQTFTVINY (SEQ ID NO: 6). Another non- limiting example is, comprises, or consists essentially of, the amino acid sequence GAQVSRQNVGTHSTQNMVSNGSSL (SEQ ID NO: 7).

In one particular embodiment, the immunogenic protein, fragment or variant is of a worm, such as a hookworm. A non-limiting example is, comprises, or consists essentially of, the amino acid sequence

T SLI AGLK AQ VE AIQK YIG AEL (SEQ ID NO:8).

In some embodiments, the immunogenic agent may further comprise an activator of innate immunity. The activator of innate immunity may target a C- type lectin such as macrophage inducible Ca ²⁺ -dependent (C-type) lectin ("Mincle"). The activatopr of innate imunity may be a glycolipid. Non-limiting examples include a glycolipid such as the mycobacterial cord factor trehalose- 6,6'-dimycolate (TDM) and/or its synthetic analogue trehalose-6,6'-dibehenate (TDB) or a lipid A glycolipid adjuvant. In some embodiments, the immunogenic agent may further comprise a bile salt such as sodium deoxycholate.

Unless the context requires otherwise, the terms "comprise", "comprises ^' " and "comprising", or similar terms are intended to mean a non-exclusive inclusion, such that a recited list of elements or features does not include those stated or listed elements solely, but may include other elements or features that are not listed or stated.

By "consisting essentially of in the context of an amino acid sequence is meant the recited amino acid sequence together with an additional one, two or three amino acids at the N- or C-terminus.

As used herein, the indefinite articles and 'an' are used here to refer to or encompass singular or plural elements or features and should not be taken as meaning or defining "one" or a "single" element or feature. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Idealized structure of J8-Lipo-DT. Liposome encapsulates DT while J8 attached to the spacer KSS at the N-terminus is covalently coupled to two palmitic acid molecules, facilitating the insertion of J8 into the liposome membrane.

Figure 2. J8-specific antibody response for individual BALB/c mice. Mean antibody titer are represented as a bar. A) Salivary IgA titer. B) Fecal IgA titer. C) Serum IgG titer. Statistical analysis was performed using a one-way ANOVA followed by the Tukey post hoc test (ns, p> 0.05; *, p< 0.05; **, p< 0.01; ***, p< 0.001).

Figure 3. Bacterial burden after intranasal challenge with Ml GAS strain in BALB/C mice. A) Nasal shedding. B) Throat swabs. C) Colonization of NALT. Results are represented as the mean CFU + SEM for 10 mice/group on days 1-3 for throat swabs, nasal shedding and day 3 for NALT. Statistical analysis was performed using a nonparametric, unpaired Mann- Whitney U test to compare test groups to the PBS control group (ns, p> 0.05; *, p< 0.05; **, p< 0.01; ***, p< 0.001).

Figure 4. J8-specific antibody response for individual B 10.BR mice (n=5 per group). Mean antibody titer are represented as a bar. A) Salivary IgA titer. B) Fecal IgA titer. C) Serum IgG titer. Statistical analysis was performed using a one-way ANOVA followed by the Tukey post hoc test (ns, p> 0.05; *, p< 0.05; **, / 0.01; ***, p< 0.001).

Figure 5. URT GAS challenge model assessing bacterial burden after intranasal challenge with Ml strain. A) Nasal shedding of B10.BR mice. B) Throat swabs of B10. BR mice. Results are represented as the mean CFU + SEM for 5 mice/group on days 1-3. Statistical analysis was performed using a nonparametric, unpaired Mann- Whitney U test to compare test groups to the PBS control group (ns, p> 0.05; *, p< 0.05).

Figure 6. J8-specific antibody response for individual BALB/c mice. Mean antibody titer are represented as a bar. A) Salivary IgA titer. B) Serum IgG titer. Statistical analysis was performed using a one-way ANOVA followed by the Tukey post hoc test (ns, p> 0.05; *, p< 0.05; **, /?< 0.01; ***, /?< 0.001).

Figure 7. Antigen specific secreted chemokines and cytokines in immunized mice. Splenocytes were plated out and the following stimuli were added as indicated: LPS (2 μg/mL), J8 (10 μg/mL) or media alone. 72 hours post stimulation, supematants were isolated and levels of secreted chemokines or cytokines were assayed using a cytometric bead array (see Materials and Methods). Statistical analysis was performed using a Student's t test (ns, p> 0.05; *, p< 0.05; **, /?< 0.01).

Figure 8. Levels of surface markers on human DC subsets with and without treatment with reagents. The following stimuli were added as indicated: Polyinosinic:polycytidylic acid (pIC, 10 μg/mL), J8-Lipo-DT (150 μg/mL) or media alone. Cell surface markers were measured by a flow cytometer 24 hours post stimulation. A) CD123+ plasmacytoid DCs. B) CD141+ classical type 1 DCs. C) CDlc+ classical type 2 DCs. Values are expressed as the median fluorescence intensities (MFI) for pooled data from three individual donors ± SEM. Statistical analysis was performed using a nonparametric, unpaired Mann- Whitney U test to compare test groups to the media control group (ns, p> 0.05; *, p< 0.05; **, p< 0.01; ***, < 0.001).

Figure 9. J8-Lipo-DT induced secreted chemokines and cytokines in human dendritic cells. Dendritic cells were plated out and the following stimuli were added as indicated: pIC (10 μg/mL), J8-Lipo-DT (150 μg/mL) or media alone. Supematants were isolated 24 hours post stimulation and levels of secreted chemokines or cytokines were assayed using a cytometric bead array (see Materials and Methods). Statistical analysis was performed using a Student's t test (ns, p> 0.05; *, p< 0.05; **, p< 0.01; ***, p< 0.001).

Figure 10. SpyCEP peptide (S2; SEQ ID NO:2) liposomal delivery agent elicits a mucosal IgA response. Liposomes displaying palmitic acid lipidated S2 peptide or an S2-J8 chimera (SEQ ID NO: 3), together with intravesicular DT were administered intranasally to mice and S2-specific IgA titers measured.

Figure 11. J8+S2-Lipo-DT immunogenic agents induce an antigen-specific IgA, IgG response in mice.

Figure 12. J8-Lipo-DT immunogenic agents can be extruded to form nano- to micro-sized particles. Liposome size measurement was by Nanosizer (Dynamic light scattering or DLS).

Figure 13. The size of J8-Lipo-DT immunogenic agents size does not influence systemic IgG response.

Figure 14. Larger sized J8-Lipo-DT immunogenic agents induced a J8- specific mucosal response.

Figure 15. Size distribution of freed-dried J8-Lipo-DT powder reconstituted in PBS. Liposome size measurement was by Nanosizer (Dynamic light scattering or DLS).

Figure 16. Reconstituted, freeze-dried, J8-Lipo-DT liposomal immunogenic agents induced J8-specific systemic response without additional adjuvant.

Figure 17. Reconstituted, freeze-dried J8-Lipo-DT liposomal immunogenic agents induced a J8-specific mucosal response

Figure 18. Schematic depiction of a liposomal J8-Lipo-DT immunogenic agent comprising glycolipid adjuvants trehalose 6,6'-dibehenate (TDB).

Figure 19. TDB enhances mucosal IgA response induced by J8-Lipo-DT. Mice (n = 5 per group) were immunized intranasally with 30 μg of J8-Lipo- Dt+TDB. Mice were administered primary (Day 0) plus two boosts (days 21 & 42). Incorporation of TDB results in significantly higher mucosal IgA responses in both saliva & fecal samples in comparison to J8-Lipo-DT . The results are from a lyophilized powder version of J8-Lipo-DT+TDB which negates the stability issues of liposome

Figure 20. Schematic depiction of a liposomal immunogenic agent comprising the bile salt sodium deoxycholate. Figure 21. (A) Schematic depiction of a single immunogenic agent comprising a lipid vesicle, intravesicular DT and immunogenic proteins of, or from, a plurality of different pathogens, namely influenza A, influenza B and group A streptococcus; (B) Schematic depiction of a single immunogenic agent comprising a lipid vesicle, intravesicular DT and immunogenic proteins of, or from, a plurality of different pathogens, namely influenza A, influenza B and group A streptococcus and glycolipid adjuvants trehalose 6,6'-dibehenate (TDB) and Monophosphoryl 3-Deacyl Lipid A (3D-PHAD®).

Figure 22. Immunogenicity of a single immunogenic agent ("Multivax") against influenza A, influenza B and group A streptococcus compared to individual vaccination with separate immunogenic agenst against each of influenza A, influenza B and group A streptococcus ("Single Antigen-vax"), as measured by antigen-specific salivary IgA titre.

DETAILED DESCRIPTION

The present invention is at least partly predicated on the discovery that intranasal vaccination of mice with a liposomal immunogenic agent comprising an immunogenic peptide displayed on the liposome surface together with an intravesicular carrier protein such as diptheria toxoid (DT) resulted in mucosal and systemic antibody responses which were comparable to those induced by the established non- human compatible adjuvant, CTB. In the specific context of group A streptococcus and J8 peptide, the level of protective immunity induced by the liposomal formulation significantly exceeded that induced by J8/CTB. Furthermore, the cytokine response by purified human dendritic cell (DC) subsets suggests that such liposomes will be effective in inducing a mucosal J8-specific IgA and systemic IgG response in humans. In some embodiments, the liposomal immunogenic agent may comprise a SpyCEP peptide or other fragment thereof, alone or together with the J8 peptide. In a particular form, the liposomal immunogenic agent may be suitable for treating or preventing infections by particularly virulent strains or isolates of Group A streptococci that are resistant to the typical antibiotic treatments used for group A streptococcal infections. These strains or isolates typically cause serious infections of the skin (e.g necrotizing fasciitis) and in some cases may harbour a CovR/SCovR/S mutation. This may be generalizable to other pathogens and their associated diseases, disorders and conditions including, but not limited to, infuenza, rhinovirus and worms such as hookworms. Accordingly, an embodiment of the invention provides a single immunogenic agent that includes one or a plurality of immunogenic proteins, fragments, variants or derivatives of, or from, a plurality of different pathogens.

For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.

By "protein" is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L-amino acids as are well understood in the art.

The term "protein" includes and encompasses "peptide", which is typically used to describe a protein having no more than fifty (50) amino acids and "polypeptide", which is typically used to describe a protein having more than fifty (50) amino acids.

As generally used herein, when a protein is referred to as being "of or "from" a pathogen is meant that the protein comprises an amino acid sequence which is at least partly, or entirely, present in a protein of the pathogen.

A "fragment" is a segment, domain, portion or region of a protein, which constitutes less than 100% of the amino acid sequence of the protein. It will be appreciated that the fragment may be a single fragment or may be repeated alone or with other fragments.

In general, fragments may comprise, consist essentially of or consist of up to 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 100, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or 1600 amino acids of the full length protein.

As used herein, a protein "variant" shares a definable nucleotide or amino acid sequence relationship with a reference amino acid sequence. The "variant" protein may have one or a plurality of amino acids of the reference amino acid sequence deleted or substituted by different amino acids. It is well understood in the art that some amino acids may be substituted or deleted without changing the activity of the immunogenic fragment and/or protein (conservative substitutions). Preferably, protein variants share at least 70% or 75%, preferably at least 80%> or 85% or more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%) or 99% sequence identity with a reference amino acid sequence.

Terms used generally herein to describe sequence relationships between respective proteins and nucleic acids include "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". Because respective nucleic acids/proteins may each comprise (1) only one or more portions of a complete nucleic acid/protein sequence that are shared by the nucleic acids/proteins, and (2) one or more portions which are divergent between the nucleic acids/proteins, sequence comparisons are typically performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FAST A, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

The term "sequence identity" is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base {e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison {i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, "sequence identity ^' " may be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).

As used herein, "derivatives ^' " are molecules such as proteins, fragments or variants thereof that have been altered, for example by conjugation or complexing with other chemical moieties, by post-translational modification {e.g. phosphorylation, acetylation and the like), modification of glycosylation {e.g. adding, removing or altering glycosylation), lipidation and/or inclusion of additional amino acid sequences as would be understood in the art. In one particular embodiment, an additional amino acid sequence may comprise one or a plurality of lysine residues at an N and/or C-terminus thereof. The plurality of lysine residues {e.g polylysine) may be a linear sequence of lysine residues or may be branched chain sequences of lysine residues. These additional lysine residues may facilitate increased peptide solubility.

Additional amino acid sequences may include fusion partner amino acid sequences which create a fusion protein. By way of example, fusion partner amino acid sequences may assist in detection and/or purification of the isolated fusion protein. Non-limiting examples include metal-binding {e.g. polyhistidine) fusion partners, maltose binding protein (MBP), Protein A, glutathione S-transferase (GST), fluorescent protein sequences {e.g. GFP), epitope tags such as myc, FLAG and haemagglutinin tags. Other additional amino acid sequences include spacer sequences. One example of a spacer sequence is an amino acid sequence at the N- or C-terminus of an amino acid sequence of an immunogenic protein fragment or variant that includes a lysine (K) residue suitable for lipidation. Typically, the spacer amino acid sequence comprises two (2) to ten (10) amino acids, such as the three (3) amino acid sequence KSS. Other derivatives contemplated by the invention include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the immunogenic proteins, fragments and variants of the invention.

In this regard, the skilled person is referred to Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE, Eds. Coligan et al. (John Wiley & Sons NY 1995-2008) for more extensive methodology relating to chemical modification of proteins.

It will be appreciated that the immunogenic proteins, fragments and variants disclosed herein may be displayed on the surface of a lipid vesicle individually or as a chimera or fusion protein comprising multiple copies of the same peptide or multiple different peptides. A non-limiting example is the chimeric peptide of SEQ ID NO:3, as will be described in more detail hereinafter.

In the context of the present invention, the term "immunogenic" as used herein indicates the ability or potential to generate or elicit an immune response, such as to a pathogen or molecular components thereof, upon administration of the immunogenic agent to a mammal or other animal.

By "elicit an immune response" is meant generate or stimulate the production or activity of one or more elements of the immune system inclusive of the cellular immune system, antibodies and/or the native immune system. Suitably, the one or more elements of the immune system include B lymphocytes, antibodies, neutrophils, dendritic cells inclusive of plasmacytoid dendritic cells, cytokines and/or chemokines. Non-limiting examples of cytokines include pro- inflammatory cytokines such as TNF-a, IL-6 and IL-1 (e.g IL-1 β). A non- limiting example of a chemokine is the neutrophil chemo-attractant IL-8. Suitably, the immune response is, or comprises, a mucosal immune response, such as including IgA production. Preferably, the immune response that is elicited by the immunogenic agent is protective.

As generally used herein the terms "immunize", "vaccinate" and "vaccine" refer to methods and/or compositions that elicit a protective immune response against a pathogen, whereby subsequent infection by the pathogen is at least partly prevented or minimized. The term "pathogen" as used herein relates to any living or non-living entity that is capable of causing a disease, disorder or condition in an animal, such as an avian or mammal. The pathogen may be a virus, bacterium, protozoan or worm, although without limitation thereto. Particular non-limiting examples of pathogens include group A streptococcus bacteria, respiratory viruses such as influenza virus and rhinovirus and nematodes such as hookworms.

As will be understood from the disclosure herein, the invention provides a lipid vesicle formulation that comprises an immunogenic protein fragment, variant or derivative and a carrier protein formulated in a lipid vesicle. As broadly used herein, the lipid vesicle may be a liposome, minicell, multilamellar vesicle, micelle, vacuole or other vesicular structure comprising a lipid bilayer, Suitably, the immunogenic protein fragment, variant or derivative is derivatized to comprise one or more lipids that facilitate anchoring to the lipid bilayer so that the immunogenic protein fragment, variant or derivative is displayed on the surface of the lipid vesicle. In a preferred form, a lysine (K) residue is lipidated via a and/or ε amino groups. To facilitate lipidation, the immunogenic protein fragment, variant or derivative may further comprise an N-terminal spacer comprising a lysine (K) residue that is lipidated. The spacer may typically comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids. An embodiment is the three (3) amino acid spacer KSS. The lipid may comprise a saturated or unsaturated fatty acid (e.g mono-unsaturated or polyunsaturated). In some embodiments, the or each lipid is a Ci6 fatty acid such as palmitic acid. However, it will also be appreciated that other lipids such as saturated or unsaturated (e.g mono-unsaturated or polyunsaturated) fatty acids having C ₁₂ , C ₁₃ C ₁₄ , C ₁₅ C ₁₇ , C _18; C19, C20, C ₂₁ or C22 carbon chains may be useful according to the invention.

The lipid vesicle suitably comprises any lipid or mixture of lipids capable of forming a lipid bilayer structure. These include a phospholipids, sterols inclusive of cholesterol, cholesterol-esters and phytosterols, fatty acids and/or triglycerides. Non-limiting examples of phospholipids include phosphatidylcholine (PC) (lecithin), phosphatidic acid, phosphatidylethanolamine (PE) (cephalin), phosphatidylglycerol (PG), phosphatidyl serine (PS), phosphatidylinositol (PI) and sphingomyelin (SM) or natural or synthetic derivatives thereof. Natural derivatives include egg PC, egg PG, soy bean PC, hydrogenated soy bean PC, soy bean PG, brain PS, sphingolipids, brain SM, galactocerebroside, gangliosides, cerebrosides, cephalin, cardiolipin, and dicetylphosphate. Synthetic derivatives include dipalmitoylphosphatidylcholine (DPPC), didecanoylphosphatidylcholine (DDPC), dierucoylphosphatidylcholine (DEPC), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dilaurylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoylmyristoylphosphatidylcholine (PMPC), palmitoylstearoylphosphatidylcholine (PSPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylglycerol (DLPG), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG), dimyristoylphosphatidic acid (DMPA), dipalmitoylphosphatidic acid (DPP A), distearoylphosphatidic acid (DSPA), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE) dioleoylphosphatidylserine (DOPS), dipalmitoylsphingomyelin (DPSM) and distearoylsphingomyelin (DSSM). The phospholipid can also be a derivative or analogue of any of the above phospholipids.

Suitably, a mixture of lipids may comprise each lipid at a desired molar or wt% ratio. The ratio between respective lipids may be between 20: 1 to 1 : 1, including 15: 1 12: 1, 10: 1, 7: 1, 5: 1, 4: 1, 3 : 1 and 2: 1 or any ratio between these stated values. By way of example, a liposome may be formed using a molar ratio of 7 dipalmitoyl-sn-glycero-3-phosphocholine (DPPC): 2 Cholesterol (CHOL): 1 L-a-phosphatidylglycerol (PG).

Suitably, the lipid vesicle further comprises a carrier protein. Suitably, the carrier protein is immunogenic, or at least partly facilitates or enhances the immunogenicity of the immungenic agent. Typically, the carrier protein is formulated with the lipid vesicle so that the carrier protein is located inside the lipid vesicle within the internal aqueous space. In some embodiments, the carrier protein is separate to the immunogenic protein fragment, variant or derivative thereof. According this embodiment, the immunogenic protein fragment, variant or derivative thereof may be displayed on the vesicle surface. In some embodiments, the carrier protein may be fused, conjugated or complexed with said immunogenic protein fragment, variant or derivative thereof. This includes recombinant protein fusions, chemical cross-linking and intermolecular complexing such as by way of biotin-avidin or other intermolecular binding agents, although without limitation thereto. In such embodiments said immunogenic protein fragment, variant or derivative thereof is located inside the lipid vesicle within the internal aqueous space, fused, conjugated or complexed with the carrier protein. This embodiment may be particularly suitable for oral delivery of the immunogenic agent, such as in liposomes comprising a bile salt, as will be described in more detail hereinafter. Non-limiting examples of carrier proteins include diptheria toxoid (DT), tetanus toxoid (TT), CRM proteins such as CRM197 and Pertussis toxin mutant, although without limitation thereto. Also contemplated are fragments and variants of carrier proteins. In one particular embodiment, the carrier protein is diptheria toxoid (DT), or a fragment thereof.

In some embodiments, the lipid vesicle further comprise an activator of innate immunity. The activator of innate immunity may target a C-type lectin expressed by one or more cells that are associated with innate immunity. A preferred C-type lectin is macrophage inducible Ca ²⁺ -dependent (C-type) lectin ("Mincle"). Non-limiting examples include a glycolipid such as the mycobacterial cord factor trehalose-6,6'-dimycolate (TDM) and/or its synthetic analogue trehalose-6,6'-dibehenate (TDB) and/or lipid A glycolipid adjuvants such as monophosphoryl 3-deacyl lipid A which mnay be in the form of PHAD®, 3D- PHAD® and 3D (6-acyl) PHAD®. Although not wishing to be bound by any particular theory, it is proposed that activators of innate immunity such as described above may enhance or improve mucosal immunity elicited by the immunogenic agent. Preferably, the glycolipid(s) may be included in the lipid vesicle so that it constitutes no more than about 25%, 20%, 15%, 10% or 5% of total lipid in the lipid vesicle.

In some embodiments, the lipid vesicle may further comprise a bile acid or bile salt. Bile acids are typically dihydroxylated or trihydroxylated steroids (in some embodiments comprising 24 carbons), including cholic acid, deoxycholic acid, chenodeoxycholic acid and ursodeoxycholic acid. Preferably, the lipid vesicle comprises a bile salt such as a cholate, deoxycholate, chenodeoxycholate or ursodeoxycholate salt. A preferred bile salt is sodium deoxycholate.

In other embodiments, immunogenic agents comprising liposomes may be produced at a particular, selected or desired particle size or size range. In some embodiments, larger particle size liposomes may elicit a stronger mucosal immune response.

In other embodiments, immunogenic agents comprising liposomes may be freeze-dried or lyophilized to facilitate longer term storage. Reconstituted freeze- dried liposomal immunogenic agents elicited an immune response comparable to that of "fresh" liposomal immunogenic agents

In some embodiments, the pathogen is group A streptococcus.

As used herein the terms "group A streptococcus", "Group A Streptococci", "Group A "Group A Strep" and the abbreviation "GAS" refer to streptococcal bacteria of Lancefield serogroup A which are gram positive β-hemolytic bacteria of the species Streptococcus pyogenes. An important virulence factor of GAS is M protein, which is strongly anti-phagocytic and binds to serum factor H, destroying C3-convertase and preventing opsonization by C3b. These also include virulent "mutants" such as CovR/S or CovRS mutants such as described in Graham et al., 2002, PNAS USA 99 13855, although without limitation thereto.

Diseases and conditions caused by group A streptococci include cellulitis, erysipelas, impetigo, scarlet fever, throat infections such as acute pharyngitis ("strep throat"), bacteremia, toxic shock syndrome, necrotizing fasciitis, acute rheumatic fever and acute glomerulonephritis, although without limitation thereto.

As used herein "neutrophils" or neutrophil granulocytes are cells that form part of the polymorphonuclear cell family (PMNs) together with basophils and eosinophils. Neutrophils are relatively short-lived phagocytic cells formed from bone marrow stem cells and typically constitute 40% to 75% of white blood cells in mammals. As well as being phagocytic neutrophils release soluble anti- microbials (e.g granule proteins) and generate neutrophil extracellular traps. Neutrophils are responsive to molecules such as interleukin-8 (IL-8), C5a, fMLP and leukotriene B4 which promote neutrophil chemotaxis to sites of injury and/or acute inflammation. In one embodiment, the immunogenic protein may be an M protein, fragment or variant thereof.

As used herein an " protein fragment" is any fragment of a GAS M protein that is immunogenic and/or is capable of being bound by an antibody or antibody fragment. Typically, the fragment is, comprises, or is contained within an amino acid sequence of a C-repeat region of a GAS M protein, or a fragment thereof. Non-limiting examples include pi 45 which is a 20mer with the amino acid sequence with the amino acid sequence LRRDLDASREAKKQVEKALE (SEQ ID NO:4). In this regard, fragments of the pl45 amino acid sequence may be present in a J8 peptide.

As used herein a "J8 peptide" is a peptide which comprises an amino acid sequence at least partly derived from, or corresponding to, a GAS M protein C- region peptide. J8 peptide suitably comprises a conformational B-cell epitope and lacks potentially deleterious T-cell autoepitopes. A preferred J8 peptide amino acid sequence is QAEDKVKQSREAKKQVEKALKQLEDKVQ (SEQ ID NO: 1) or a fragment or variant thereof, wherein the bolded residues correspond to residues 344 to 355 of the GAS M protein. In this embodiment, J8 is a chimeric peptide that further comprises flanking GCN4 DNA-binding protein sequences which assist maintaining the correct helical folding and conformational structure of the J8 peptide.

In another embodiment, the immunogenic protein may be an agent that facilitates restoring or enhancing neutrophil activity.

As used herein, an "agent that facilitates restoring or enhancing neutrophil activity" is a molecule that directly or indirectly at least partly increases, enhances or restores the production, migration and/or chemotaxis of neutrophils and/or one or more immunological activities of neutrophils. In one embodiment, the agent elicits an immune response to a neutrophil inhibitor. In another embodiment, the agent binds and at least partly inactivates the neutrophil inhibitor. The neutrophil inhibitor may be a molecule derived or originating from Group A Streptococcal bacteria. In one particular form the neutrophil inhibitor is a serine protease, or a fragment thereof, that proteolytically cleaves interleukin 8. In one particular embodiment, the neutrophil inhibitor is SpyCEP or a fragment thereof. SpyCEP is a 170-kDa multidomain serine protease expressed on the surface of the human pathogen Streptococcus pyogenes, which plays an important role in infection by catalyzing cleavage and inactivation of the neutrophil chemoattractant interleukin-8. Non-limiting examples of SpyCEP amino acid sequences may be found under accession numbers YP597949.1 and (S. pyogenes MGAS10270) and YP596076.1 (S. pyogenes MGAS9429). Accordingly, in one particular embodiment the SpyCEP fragment is, or comprises, the amino acid sequence set forth in SEQ ID NO:2 (NSDNIKENQFEDFDED WENF) . It is proposed that SEQ ID NO: 2 is, or comprises, the dominant epitope on SpyCEP that can induce functional antibodies.

Also provided herein is a chimeric peptide comprising an M-protein amino acid sequence and a SpyCEP amino acid sequence that form a single, contiguous amino acid sequence. The M-protein amino acid sequence may be located C- terminal to the SpyCEP amino acid sequence, or vice versa. In one embodiment, the chimeric peptide may comprise the amino acid sequence NSDNIKENQFEDFDED WENFQAEDKVKQSREAKKQVEKALKQLEDKVQ

(SEQ ID NO:3) or a variant thereof.

In an alternative embodiment, respective liposomes comprising an M- protein amino acid sequence and a SpyCEP amino acid sequence may be produced for administration as an "admixture".

In one particular embodiment, a variant M protein or peptide may comprise one or a plurality of lysine residues at an N and/or C-terminus thereof. The plurality of lysine residues (e.g polylysine) may be a linear sequence of lysine residues or may be branched chain sequences of lysine residues. These additional lysine residues may facilitate increased peptide solubility.

Non-limiting examples of J8 peptide variants include:

S R E A K K Q S R E A K K Q V E K A L K Q V E K A L C (SEQ ID NO:5) SREAKKQ SRE AKKQ VEKALKQ SRE AK C (SEQ ID NO:6) SREAKKQVE ALKQSRE A KKQVEKAL C (SEQ ID NO:7) SRE AKKQ VEK A LD A SREAKKQ VEKALC (SEQ ID NO:8)

Other variants may be based on heptads such as described in Cooper el al, 1997.

By way of example, if an epitope is known to reside within an a-helix protein structural conformation, then a model peptide can be synthesised to fold to this conformation. A model a-helical coiled coil peptide has been based on the structure of the GCN4 leucine zipper (O'Shea et al., 1991). The first heptad contains the sequence MKQLEDK (SEQ ID NO:9), which includes several of the features found in a stable coiled coil heptad repeat motif (a-b-c-d-e-f-g)n (Cohen & Parry, 1990). These include large apolar residues in the a and d positions, an acid/base pair (Glu/Lys) at positions e and g (usually favouring interchain ionic interactions), and polar groups in positions b, c, f (consistent with the prediction of Lupas etal. (1991)). The GCN4 peptide also contains a consensus valine in the a position. It has also been noted that when positions a and d are occupied by V and L a coiled coil dimer is favoured (Harbury et al, 1994). A model heptad repeat was derived from these consensus features of the GCN4 leucine zipper peptide: (VKQLEDK; SEQ ID NO: 10) with the potential to form a a-helical coiled coil. This peptide became the framework peptide. Overlapping fragments of a conformational epitope under study were embedded within the model coiled coil peptide to give a chimeric peptide. Amino acid substitutions, designed to ensure correct helical coiled coil conformations (Cohen & Parry, 1990) were incorporated into the chimeric peptides whenever an identical residue was found in both the helical model peptide and the epitope sequence. The following substitutions were typically used: position a, V to I; b, K to R; c, Q to N; d, L to A; e, E to Q; ,f: D to E; g, K to R. All of these replacement residues are commonly found at their respective position in coiled coil proteins (Lupas et al., 1991).

One particular J8 peptide derivative described in Olive et al., 2002, Infect & Immun. 70 2734 is a "lipid core peptide". In one embodiment, a lipid core peptide may comprise a plurality of J8 peptides (e.g four J8 peptides) synthesized directly onto two amino groups of each lysine of a branched polylysine core coupled to a lipophilic anchor.

The M protein fragment or variant and/or the SpyCEP fragment or variant may be derivatized to comprise one or more lipids that facilitate anchoring to the lipid bilayer as hereinbefore described. In yet another embodiment, a chimeric peptide comprising an M-protein amino acid sequence and a SpyCEP amino acid sequence (e.g. SEQ ID NO:3) may comprise a spacer amino acid sequence at the N-terminus thereof. Accordingly, in embodiments where a SpyCEP fragment or variant is included in the lipid vesicle, it may be separately lipidated along with the M protein fragment or variant or may be present as a chimeric peptide which is lipidated.

In some embodiments, the pathogen is influenza virus. In one particular embodiment, the immunogenic protein, fragment or variant is of influenza A virus. The immunogenic protein or fragment may be matrix protein 2, or a fragment thereof. A non-limiting example is, or comprises, the amino acid sequence M SLLTE VETPIRNEWGCRCND S SD (SEQ ID NO:4). In one particular embodiment, the immunogenic protein, fragment or variant is of influenza B virus. The immunogenic protein or fragment may be a haemagglutinin protein, or a fragment thereof. A non-limiting example is, or comprises, the amino acid sequence PAKLLKERGFFGAIAGFLE (SEQ II) NO.5).

In some embodiments, the pathogen is rhinovirus. In one particular embodiment, the immunogenic protein, fragment or variant is of a rhinovirus B protein, such as a capsid protein. A non-limiting example is, or comprises, the amino acid sequence GAQVSTQKSGSHENQNILTNGSNQTFTVINY (SEQ ID NO: 6). In another particular embodiment, the immunogenic protein, fragment or variant is of a rhinovirus A protein, such as a capsid protein. Another non- limiting example is, or comprises, the amino acid sequence GAQVSRQNVGTHSTQNMVSNGSSL (SEQ ID NO: 7).

In some embodiments, the pathogen is a worm, such as a hookworm. In one particular embodiment, the immunogenic protein, fragment or variant is of Necator americanus. A non-limiting example is, or comprises, the amino acid sequence T SLI AGLK AQ VE AIQKYIGAEL (SEQ ID NO:8).

The isolated immunogenic proteins, fragments and/or derivatives of the present invention may be produced by any means known in the art, including but not limited to, chemical synthesis, recombinant DNA technology and proteolytic cleavage to produce peptide fragments.

Chemical synthesis is inclusive of solid phase and solution phase synthesis. Such methods are well known in the art, although reference is made to examples of chemical synthesis techniques as provided in Chapter 9 of SYNTHETIC VACCINES Ed. Nicholson (Blackwell Scientific Publications) and Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. NY USA 1995-2008). In this regard, reference is also made to International Publication WO 99/02550 and International Publication WO 97/45444.

Recombinant proteins may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al, MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. NY USA 1995-2015), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. NY USA 1995-2015), in particular Chapters 1, 5 and 6. Typically, recombinant protein preparation includes expression of a nucleic acid encoding the protein in a suitable host cell.

As hereinbefore described, the invention provides immunogenic agents and/or use thereof for preventing or treating a pathogen-associated disease, disorder or condition in a mammal or other animal.

As used herein, "treating", "treat" or "treatment" refers to a therapeutic intervention that at least partly ameliorates, eliminates or reduces a symptom or pathological sign of a pathogen-associated disease, disorder or condition after it has begun to develop. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

As used herein, "preventing", "prevent" or "prevention" refers to a course of action initiated prior to infection by, or exposure to, a pathogen or molecular components thereof and/or before the onset of a symptom or pathological sign of the disease, disorder or condition, so as to prevent infection and/or reduce the symptom or pathological sign. It is to be understood that such preventing need not be absolute to be beneficial to a subject. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of the disease, disorder or condition, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom or pathological sign of the disease, disorder or condition.

In an embodiment, the disease, disorder or condition may be a group A- strep-associated disease, disorder or condition. In the context of the present invention, by "group A-strep-associated disease, disorder or condition " is meant any clinical pathology resulting from infection by group A strep and includes cellulitis, erysipelas, impetigo, scarlet fever, throat infections such as acute pharyngitis ("strep throat"), bacteremia, toxic shock syndrome, necrotizing fasciitis, acute rheumatic fever and acute glomerulonephritis, although without limitation thereto.

As hereinbefore described, the uses for treatment and/or immunization disclosed herein include administration of the immunogenic agent comprising an M protein fragment, variant or derivative, a lipid vesicle, carrier protein and/or a SpyCEP peptide or other fragment that facilitates restoring or enhancing neutrophil activity to a mammal.

As disclosed herein, treatment and/or immunization may include, in addition, administration of antibodies or antibody fragments to therapeutically treat GAS infections, such as by targeting SpyCEP at the site of infection {e.g. the skin) and/or antibodies or antibody fragments that bind an M protein, fragment or variant thereof.

Antibodies and antibody fragments may be polyclonal or monoclonal, native or recombinant. Antibody fragments include Fc, Fab or F(ab)2 fragments and/or may comprise single chain Fv antibodies (scFvs). Such scFvs may be prepared, for example, in accordance with the methods described respectively in United States Patent No 5,091,513, European Patent No 239,400 or the article by Winter & Milstein, 1991, Nature 349:293. Antibodies may also include multivalent recombinant antibody fragments, such as diabodies, triabodies and/or tetrabodies, comprising a plurality of scFvs, as well as dimerisation-activated demibodies {e.g. WO/2007/062466). By way of example, such antibodies may be prepared in accordance with the methods described in Holliger et al, 1993 Proc Natl Acad Sci USA 90 6444; or in Kipriyanov, 2009 Methods Mol Biol 562 177. Well-known protocols applicable to antibody production, purification and use may be found, for example, in Chapter 2 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991-1994) and Harlow, E. & Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988.

Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, supra, and in Harlow & Lane, 1988, supra. In a particular embodiment, anti-SpyCEP polyclonal antibodies may be obtained or purified from human sera from individuals exposed to, or infected by, Group A strep. Alternatively, polyclonal antibodies may be raised against purified or recombinant SpyCEP, or an immunogenic fragment thereof, in production species such as horses and then subsequently purified prior to administration.

Monoclonal antibodies may be produced using the standard method as for example, originally described in an article by Kohler & Milstein, 1975, Nature 256, 495, or by more recent modifications thereof as for example, described in Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, supra by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of the isolated proteins, fragments, variants or derivatives of the invention. Accordingly, monoclonal antibodies may be raised against an M protein fragment, variant or derivative and/or the agent that facilitates restoring or enhancing neutrophil activity (e.g SpyCEP) for use according to the invention. In certain embodiments, the monoclonal antibody or fragment thereof may be in recombinant form. This may be particularly advantageous for "humanizing" the monoclonal antibody or fragment if the monoclonal antibody is initially produced by spleen cells of a non- human mammal.

For embodiments relating to therapeutic antibodies, a preferred M protein fragment may be a p 145 peptide.

A preferred fragment of SpyCEP for antibody production may comprise or consist of the amino acid sequence NSDNIKENQFEDFDEDWENF (SEQ ID NO:2).

In some embodiments, the disease, disorder or condition may be an influenza virus-associated disease, disorder or condition. Influenza virus may cause a transmissable or otherwise infectious disease known as the "flu". Typical symptoms include fever, headache, coughing, lethargy, respiratory and nasopharyngeal mucous build-up and secretionm muscular pain, nausea and vomiting. Symptoms may last for a few days or persist for several weeks. In some cases, secondary respiratory bacterial infections may arise, in some cases causing severe conditions such as pneumonia. Accordingly, the immunogenic agents and/or methods of the invention may treat or prevent influenza virus-associated disease, disorder or conditions such as described above.

In some embodiments, the disease, disorder or condition may be a rhinovirus-associated disease, disorder or condition. Rhinoviruses (e.g. Rhinovirus A and Rhinovirus B) are species in the genus Enterovirus of the Picornaviridae family of viruses. Rhinoviruses are typically the causative agents of the common cold, the symptoms of which are similar to influenza but generally less severe and with a lower probability of secondary bacterial infections such as pneumonia.

Accordingly, the immunogenic agents and/or methods of the invention may treat or prevent rhinovirus-associated disease, disorder or conditions such as described above.

In some embodiments, the disease, disorder or condition may be a hookworm-associated disease, disorder or condition. Hookworms are nematode worms that infest a variety of different animals. Hookworms that typically infect humans may include Necator americanus and Ancylostoma duodenalis. Hookworms have hook-like mouthparts with attach the hookworm to the wall of the gut, puncturing the blood vessels and feeding on blood, leading in some cases to severe anaemia. Hookworm infection in pregnancy can cause retarded growth of the fetus, premature birth and a low birth weight. Hookworms in children can cause intellectual, cognitive and growth problems.

Accordingly, the immunogenic agents and/or methods of the invention may treat or prevent worm-associated disease, disorder or conditions such as described above.

In particular embodiments, the aforementioned methods may be performed as follows:

(i) administering an immunogenic agent that comprises one or a plurality of different proteins, fragments, variants or derivatives of a single or same pathogen;

(ii) administering a plurality of different immunogenic agents that respectively comprise one or a plurality of different proteins, fragments, variants or derivatives of different pathogens; or (iii) administering an immunogenic agent that comprises one or a plurality of different proteins, fragments, variants or derivatives of different pathogens;

to thereby:

(a) elicit an immune response to the pathogen(s);

(b) immunize against the pathogen(s); or

(c) prevent or treat one or a plurality of diseases, disorders or conditions caused by the one or plurality of pathogens.

In certain aspects and embodiments, the immunogenic agent may be administered in the form of a composition.

In particular embodiments, the composition may comprise:

(I) an immunogenic agent comprising one or a plurality of different proteins, fragments, variants or derivatives of a same or single pathogen;

(II) a plurality of different immunogenic agents respectively comprising one or a plurality of proteins, fragments, variants or derivatives of different pathogens; or

(III) an immunogenic agent comprising one or a plurality of different proteins, fragments, variants or derivatives of different pathogens; In a preferred form, the composition comprises an acceptable carrier, diluent or excipient.

By "acceptable carrier, diluent or excipient" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, diluent and excipients well known in the art may be used. These may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates, water and pyrogen- free water.

A useful reference describing acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991) which is incorporated herein by reference. Preferably, for the purposes of eliciting an immune response, certain immunological agents may be used in the formulation in combination with the immunogenic agent disclosed herein.

The term "immunological agent" includes within its scope carriers, delivery agents, immunostimulants and/or adjuvants as are well known in the art. As will be understood in the art, immunostimulants and adjuvants refer to or include one or more substances that enhance the immunogenicity and/or efficacy of a formulation. Non-limiting examples of suitable immunostimulants and adjuvants include squalane and squalene (or other oils of plant or animal origin); block copolymers; detergents such as Tween®-80; Quil® A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium-derived adjuvants such as Corynebacterium parvum Propionibacterium-denwed adjuvants such as Propionibacterium acne] Mycobacterium bovis (Bacille Calmette and Guerin or BCG); Bordetella pertussis antigens; tetanus toxoid; diphtheria toxoid; surface active substances such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dicoctadecyl-N', N'bis(2- hydroxyethyl-propanediamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran, dextransulfate, poly IC carbopol; peptides such as muramyl dipeptide and derivatives, dimethylglycine, tuftsin; oil emulsions; and mineral gels such as aluminium phosphate, aluminium hydroxide or alum; interleukins such as interleukin 2 and interleukin 12; monokines such as interleukin 1 ; tumour necrosis factor; interferons such as gamma interferon; immunostimulatory DNA such as CpG DNA, combinations such as saponin-aluminium hydroxide or Quil-A aluminium hydroxide; liposomes; ISCOM® and ISCOMATRIX® adjuvant; mycobacterial cell wall extract; synthetic glycopeptides such as muramyl dipeptides or other derivatives; Avridine; Lipid A derivatives; dextran sulfate; DEAE-Dextran alone or with aluminium phosphate; carboxypolymethylene such as Carbopol' EMA; acrylic copolymer emulsions such as Neocryl A640 (e.g. U.S. Pat. No. 5,047,238); water in oil emulsifiers such as Montanide ISA 720; poliovirus, vaccinia or animal poxvirus proteins; or mixtures thereof. Immunological agents may include carrier proteins such as thyroglobulin; albumins such as human serum albumin; toxins, toxoids or any mutant crossreactive material (CRM) of the toxin from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus; polyamino acids such as poly(lysine: glutamic acid); influenza; Rotavirus VP6, Parvovirus VP1 and VP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine and the like. Alternatively, a fragment or epitope of a carrier protein or other immunogenic protein may be used. For example, a T cell epitope of a bacterial toxin, toxoid or CRM may be used. In this regard, reference may be made to U.S. Patent No 5,785,973 which is incorporated herein by reference.

Any suitable procedure is contemplated for producing vaccine formulations. Exemplary procedures include, for example, those described in New Generation Vaccines (1997, Levine et al, Marcel Dekker, Inc. New York, Basel, Hong Kong), which is incorporated herein by reference.

Any safe route of administration may be employed, including intranasal, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intramuscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, topical, mucosal and transdermal administration, although without limitation thereto.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, nasal sprays, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release may be effected by coating with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose.

Compositions may be presented as discrete units such as capsules, sachets, functional foods/feeds or tablets each containing a pre-determined amount of one or more therapeutic agents of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in- water emulsion or a water-in-oil liquid emulsion. Such formulations may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the formulations are prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

The above formulations may be administered in a manner compatible with the dosage formulation, and in such amount as effective. The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial response in a patient over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.

In a particular embodiment, the composition is suitable for intranasal administration to a subject.

As generally used herein, the terms "patient", "individuaF and "subject" are used in the context of any mammalian recipient of a treatment or formulation disclosed herein. Accordingly, the methods and formulations disclosed herein may have medical and/or veterinary applications. In a preferred form, the mammal is a human.

So that the invention may be fully understood and put into practical effect, reference is made to the following non-limiting Examples.

EXAMPLES EXAMPLE 1

INTRODUCTION

Group A streptococcus (GAS) infects primarily the upper respiratory tract

(URT) mucosa but also the skin of humans, resulting in a broad spectrum of diseases. Infections can lead to toxic shock syndrome, necrotising fasciitis, and myositis. Necrotising fasciitis has an incidence of 1 in 100,000 and a mortality rate of up to 70% (1). The post streptococcal diseases - rheumatic fever (RF) and rheumatic heart disease (RHD) - are also of great concern. There is estimated to be 15.6 million prevalent cases of RHD and almost 400,000 deaths per year (2). The most common disease following colonization of the URT is pharyngitis and RF and RHD are closely linked to untreated primary pharyngeal infections (3). GAS infection and associated diseases are prevalent in the tropics, in developing countries, and in Indigenous populations of developed countries resulting in 500,000 deaths each year (4) highlighting the urgent need for a vaccine.

GAS vaccine candidates can be divided into M protein and non M protein- based vaccines (5). The cell surface M protein, a coiled-coil protein consisting of 3 major domains, is a major virulence determinant (6). This protein consists of a hypervariable amino-terminal and A-repeat domains used for epidemiologic molecular typing (emm or M typing); a B-repeat domain and a conserved C-repeat domain (6). The leading subunit vaccines under clinical investigations are the amino-terminal M protein-based multivalent vaccines and conserved C-repeat M protein peptide vaccines (5). These GAS vaccine candidates have entered into clinical trials based on their success in inducing systemic immunity (7). Systemic immunity has proven effective in preventing GAS dissemination to deep tissues and preventing disease through serum immunoglobulin (Ig) at systemic sites, but not in preventing colonisation of mucosal sites and thus person-to-person transmission (8). Therefore, systemic vaccination is not the optimal approach for inducing immunity against GAS. In contrast, mucosal vaccines against various organisms administered nasally are effective in inducing antigen-specific immune responses in both systemic and mucosal compartments (9-11). Due to this two- layered protective immunity, mucosal vaccination is an ideal strategy for combating both systemic and mucosal GAS infection with the added benefit that prevention of mucosal colonization will also inhibit transmission by droplets and aerosols from the URT (7). Mucosal vaccination is also economically advantageous, an important consideration for vaccine development. Owing to the ease of vaccine administration by the nasal route, use of needles can be avoided (7). Pain-free, delivery will aid in greater patient compliance.

We have previously defined a vaccine candidate peptide, J8, based on a minimal B cell epitope from the conserved C3 -repeat domain of the M protein (12). The J8 peptide (QAEDKVKQSREAKKQVEKALKQLEDKVQ; SEQ ID NO: l) is a chimeric peptide that contains 12 amino acids from the C-region (shown in bold) flanked by GCN4 DNA-binding protein sequences to maintain the correct helical conformational structure (13). When linked to the carrier protein diphtheria toxoid (DT) and administered with Alum, J8 induces IgG antibodies that protect mice from systemic and skin challenge with multiple GAS strains (4, 13). Furthermore, vaccine candidates based on the conserved region of the M protein of GAS were efficacious in protection against intranasal infection with GAS when administered with the animal-restricted mucosal adjuvant, CTB (14, 15) or when administered as proteasomes (16). Where mucosal immunity and reduced URT colonization were induced, a correlation with the production of IgA was identified. With this in mind, our aim was to develop a J8-based, human compatible, mucosal vaccine.

However, one of the limitations in the development of mucosal vaccines for use in humans is the lack of safe and effective mucosal adjuvants (17, 18).

Liposomes are spherical vesicles composed of biocompatible phospholipid bilayers and can be loaded with and deliver both hydrophilic and hydrophobic molecules (19). Liposomes have been administered safely to humans via the intranasal route (20, 21). However, liposomes that present peptide antigens are not ideal platforms to induce peptide-specific antibody responses. Peptides can only contain limited antigenic epitopes capable of activating helper T cells necessary for an antibody response by B cells. They require conjugation to 'carrier' proteins to render them immunogenic in an outbred population and as such are not ideally suited for presentation by liposomes. Nevertheless, the immunogenicity enhancements conferred by particulates such as liposome are unsurprising, as natural pathogens are also particulates and are well-recognised by the immune system (22). The natural tendency of liposomes to interact with antigen presenting cells has served as the primary rationale for using liposomes to present antigens to the immune system (23). The aim of this study was to develop a J8-based liposomal formulation (in the absence of an adjuvant) where a lipophilic J8 construct was incorporated within the lipid bilayers and a hydrophilic carrier protein (DT) was encapsulated in the inner aqueous core.

MATERIALS AND METHODS

Mice. All animal protocols used were approved by the Griffith University Research Ethics Review Board for Animal-Based Work, GU Ref No: GLY/09/14/AEC. This study was carried out in strict accordance with the National Health and Medical Research Council (NHMRC) of Australia guidelines for laboratory animals. Methods were chosen to minimize pain and distress to the mice and animals were observed daily by trained animal care staff. Mice were terminated using a C0 ₂ inhalation chamber. Human blood. With written informed consent, blood was obtained from donors by a phlebotomist at the Griffith university health centre. Approval of the study was granted by the Griffith University Human Research Ethics Committee (GU HREC, Protocol # GLY/03/14/HREC). Samples were de-identified prior to handling by laboratory personnel.

J8-Lipo-DT formulation. To promote noncovalent complexing of J8 to liposome bilayer, a hydrophobic anchor consisting of two palmitic acid (CI 6) was added to the epsilon and primary amine group of the lysine in a tripeptide spacer (consisting of Lys Ser Ser) present in J8 amino-terminus (C16-C16-KSSJ8). This construct was manufactured by Chinapeptides Co., Ltd. (Shanghai, China). The expected molecular weight of the construct (MW 4061.97 g/mol) was confirmed by ESI-MS, and the product obtained at greater than 95% purity (by analytical RP-HPLC area under the curve analysis). Liposomes were prepared using the thin film hydration method (42). Lipids from Avanti Polar Lipids, Inc. (Alabaster, United States) were used at a molar ratio of 7 dipalmitoyl-sn-glycero-3- phosphocholine (DPPC): 2 Cholesterol (CHOL): 1 L-a-phosphatidylglycerol (PG). Lipids in chloroform (CHC1 ₃ ) solution were coated onto round-bottom flasks using a rotary evaporator along with predetermined amount of C16-C16- KSSJ8. The volumes used were 0.7 ml of DPPC (10 mg/ml) in CHC1 ₃ , 0.2 ml of CHOL (5 mg/ml) in CHC1 ₃ , and 0.1 ml of PG (10 mg/ml) in CHC1 ₃ . The lipid thin film was then hydrated and dispersed in 1 mL of phosphate buffer saline (PBS) containing a predetermined amount of DT by vigorous mixing at room temperature. The resultant liposomal suspension was centrifuged at 16, 162 g for 10 min, the supernatant removed and the liposome pellet resuspended in an appropriate volume of PBS to be administered in mice. To determine DT encapsulation efficiency, the supernatant was collected and the amount of unentrapped DT in supernatant was determined using a NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific, Massachusetts, United States). Subtraction of supernatant DT concentration from starting DT concentration in PBS used for rehydration of lipids to produce liposomes allowed quantification of encapsulation efficiency. The average particle size (nm) of liposome was measured at 25°C using a Nanosizer (Zetasizer Nano Series ZS, Malvern Instruments, United Kingdom) with disposable capillary cuvettes. Size was analysed using a noninvasive backscatter system and measurements taken with a 173° scattering angle. Correlation times were based on 10 seconds per run and at least five consecutive runs were made per measurement. The results are the average of triplicate independent measurements analysed using Dispersion Technology Software (Malvern Instruments, United Kingdom). Homogenous size distribution as determined by a low polydispersity index (PDI) of 0.238 was shown for J8-Lipo- DT. The PDI is an indication of how narrow the sample size distribution is and values greater than 0.7 indicate samples with a broad size distribution.

Intranasal immunization of mice. B10.BR and BALB/c mice (Animal Resources Centre, Western Australia, Australia) to be immunized were anesthetized by use of a mixture of xylazine and ketamine (1 : 1 : 10 mixture of xylazine: ketamine: H ₂ 0). Mice were administered 30 μg of J8-Lipo-DT alone in a total volume of 20 μΙ_, PBS (10 μΐνηατβ) whilst control mice were administered 20 μΙ_, of PBS (10 μΐνηατβ). Positive control mice received 30 μg of J8 conjugated to DT, co-administered with 10 μg of CTB (Sigma Aldrich, St. Louis, United States) in a total volume of 20 μΙ_, PBS. The mice received 2 booster immunizations 21 days apart in the same fashion as the primary immunization. Other control groups received equivalent amounts of J8, DT or liposome alone as described above.

Serum, saliva and fecal sample collection. Serum was collected on days 20, 40, and 60 after primary immunization to determine the level of J8-specific systemic antibodies. Blood was collected from mice via the tail artery and allowed to clot for at least 30 min at 37 °C. Serum was collected after centrifugation for 10 min at

1000 g, heat inactivated for 10 min at 56 °C and stored at -20 °C.

Mice were administered 50 μΐ _^ of a 0.1% solution of pilocarpine to induce salivation. Saliva was then collected in eppendorf tubes containing 2 μΙ_, of 50 mmol/L phenylmethylsulfonyl fluoride (PMSF) protease inhibitor (Sigma

Aldrich). Particulate matter was separated by centrifugation for 10 min at 13,000 g and samples were stored at -80°C.

Six to 10 freshly voided fecal pellets were collected from individual mice, frozen and then lyophilized. The dry weight of fecal solids was determined before they were resuspended by vortexing in 5% nonfat dry milk, 50 mmol/L EDTA (Sigma Aldrich), 0.1 mg/mL soyabean trypsin inhibitor (Sigma Aldrich), and 2 mmol/L PMSF (20 μΙ7η¾ of dry weight). Solid matter was separated by centrifugation for 10 min at 15,000 g. The supernatants were stored at -80°C.

Determination of antibody titers by enzyme linked immunosorbent assay (ELISA). ELISA was used to measure J8-specific serum IgG and mucosal IgA as described elsewhere (43). J8 peptides were diluted to 0.5 mg/ml in carbonate coating buffer, pH 9.6, and coated onto polycarbonate plates in a volume of 100 μΐ/well overnight at 4°C. Unbound peptide was removed and the wells blocked with 150 μΐ of 5% skim milk PBS-Tween 20 for 2 h at 37°C. The plates were then washed 3 times with PBS-Tween 20 buffer. Samples were serially diluted down the plate in 0.5% skim milk PBS-Tween 20 buffer, starting at an initial dilution of 1 : 100 to a final dilution of 1 : 12,800 for sera and 1 :2 to 1 :256 for saliva/ fecal samples. Each sample was diluted to a final volume of 100 μΐ and incubated for 1.5 h at 37°C. The plates were washed 5 times and peroxidase conjugated goat anti-mouse IgG or IgA (Invivogen, San Diego, United States) were added at a dilution of 1 :3000 or 1 : 1000 respectively in 0.5% skim milk PBS-Tween20 for 1.5 h at 37°C. After washing, 100 μΐ of OPD substrate (Sigma Aldrich) was added according to the manufacturer's instructions and incubated at room temperature for 30 min in the dark. The absorbance was measured at 450 nm in a Victor ³ 1420 multilabel counter (Perkin Elmer Life and Analytical Sciences, Shelton, United States). The titer was described as the lowest dilution that gave an absorbance of >3 standard deviation (SD) above the mean absorbance of negative control wells (containing normal mouse serum immunized with PBS). Statistical significance (p< 0.05) was determined using a one-way analysis of variance (ANOVA) with Tukey post hoc test using GraphPad Prism 5 software (GraphPad, California, United States). Procedure for GAS challenge. Immunized and control mice were challenged intranasally with a predetermined dose of the GAS strain Ml on day 63 after primary immunization. The GAS strain Ml had been serially passaged in mouse spleen to enhance virulence, and made streptomycin-resistant to enable GAS to be distinguished in throat swabs from normal murine bacterial flora (44). To determine GAS colonization, throat swabs were obtained from mice on days 1-3 after challenge. The throat swabs were streaked out on Todd-Hewitt agar plates containing 2% defibrinated horse blood and incubated overnight at 37°C. Bacterial burden in nasal shedding was determined by pressing the nares of each mouse onto the surface of Columbia blood agar (CBA) plates ten times (triplicate CBA plates/mouse/day) and exhaled particles were streaked out. On day 3 mice were culled, organ samples were homogenized in PBS and samples were plated in triplicate using the pour plate method. For nasal shedding and throat swabs, results are represented as the mean colony forming units (CFU) + standard errors of the means (SEM) for 10 mice/group on days 1, 2 and 3. For organ samples, results are represented as the mean CFU + SEM for 10 mice/group on day 3. Differences were analysed with GraphPad Prism 5 using a nonparametric, unpaired Mann- Whitney U test to compare test groups to the PBS control group (p < 0.05 was considered significant).

Preparation and maturation of DCs. Peripheral blood was collected from healthy volunteers and fractionated over Ficoll-Paque (Amersham Pharmacia, Uppsala, Sweden) by a standard procedure. PBMCs were harvested by centrifugation over Ficoll Paque, washed and resuspended at a final density of 1 x 10 ⁸ per 0.35 mL MACS buffer (Miltenyi Biotec S.L., Germany). DCs were isolated using the Pan DC isolation kit (Miltenyi Biotec) according to the manufacturer's instructions. The resultant DC population was resuspended in RPMI 1640 (Gibco, Gaithersburg, United States) complete media (with 2 mM 1- glutamine, 1% non-essential amino acids, 1 % Pen-strep, 10 mM HEPES) supplemented with 10% FCS (Gibco). DCs (2 X 10 ⁶ ) in a total volume of 0.2 mL was plated out and the following stimuli were added as indicated: pIC (Invivogen, San Diego, United States) at 10 μg/mL, J8-Lipo-DT (150 μg/mL) or complete media alone for 24 hours. Supernatants were collected after 24 h and stored at - 20°C.

Immunophenotype analysis by flow cytometry. For analysis of surface expression of various markers, treated DCs were stained with one or more of the following fluorophore-labeled mAb and analysed by flow cytometry using an LSR Fortessa cytometer (Becton Dickinson, California, United States) and FlowJo software (Treestar, Inc., California, United States). The resultant populations were assessed by flow cytometric analysis using the following antibodies (Becton Dickinson): anti-HL A-DR- V450, -CDlc-APC, -CD80-PE- Cy7, -CD83-PE-TexasRed, -CD86-PE, -CD123-Percp-5.5, -CD141-APC-Cy7. After staining with the appropriate antibodies in the dark at 4°C for 30 min, cells were washed twice in PBS, and fixed in 1% paraformaldehyde. Gating was on large granular cells, and 2000-5000 gated events were collected from each sample. Briefly, ULA-DR positive cells were gated to define human DCs and further subdivided into the CD141+ conventional DCs type 1, CDlc+ DCs conventional (myeloid) DCs type 2 and CD123+ plasmacytoid DCs according to previously established methods (45). Mean fluorescence intensity (MFI) values were determined on the gated population. The data are reported as means + SEM, and differences were analysed with GraphPad Prism 5 software (GraphPad, California, United States) using Student's t test. P values under 0.05 were considered as significant.

In vitro stimulation of splenocytes with antigen. The cytometric bead array (CBA) assay and flow cytometry analysis were used to quantify the pro- inflammatory response produced by splenocytes after stimulation with the J8 peptide. Briefly, single-cell suspensions of spleens from J8-Lipo-DT immunized mice free of erythrocytes were prepared in RPMI 1640 media. Splenocytes (4 x 10 ⁵ )in a total volume of 0.1 mL was plated out and the following stimuli were added as indicated: LPS (Sigma Aldrich) at 2 μg/mL, J8 (10 μg/mL) or RPMI 1640 media alone for 72 h. Supernatants were isolated After 72 h and stored at - 80°C for CBA flow cytometric analysis.

Quantification of secreted chemokines and cytokines by CBA. Levels of accumulated inflammatory cytokines were quantified according to the manufacturer's instructions. For the Mouse Inflammation Kit CBA, volumes of samples and standards were scaled down to 10 μΐ, and 2 μΐ of each capture bead was used. Supernatants from human DC wells were used for the Human Inflammation Kit CBA (Becton Dickinson) according to the manufacturer's recommendations. Samples were run on an LSR Fortessa cytometer and data analysed with FCAP array (vl .01 for Windows) software (Becton Dickinson). The data are reported as means + standard errors of the means (SEM), and differences were analysed with GraphPad Prism 5 software using Student's t test. P values under 0.05 were considered significant.

RESULTS

Liposomes with surface-associated J8 peptide and containing diphtheria toxoid (Fig. 1) were constructed as described in Materials and Methods. The administered formulation contained 30 μg of J8 per dose linked to the liposome surface using a palmitic acid-based moiety. Internally, liposomes contained 30 μg of DT per dose. The average diameter of the liposomes was 1.8 μπι (standard deviation = 100.3 nm) measured by dynamic light scattering (see Materials and Methods).

Using a primary and 2-boost regimen, BALB/c mice (10 per group) were immunized intranasally with J8-Lipo-DT and various controls: liposomes alone (Lipo); liposomes encapsulating DT (Lipo-DT); J8 embedded on the surface of liposomes but without encapsulated DT (J8-Lipo); J8-DT+CTB; PBS+CTB; and PBS.

To assess the efficacy of J8-Lipo-DT in comparison to other constructs, mice vaccinated intranasally were then challenged intra-nasally with the pharyngeal isolate Ml GAS strain obtained from a patient with scarlet fever (16). Prior to challenge, we observed that J8-Lipo-DT induced higher J8-specific IgA (fecal and salivary) and serum IgG titers than J8-Lipo. Although the difference between J8-Lipo-DT and J8-Lipo was not statistically significant for any one group, J8-Lipo-DT was observed to be superior for the salivary IgA response, the fecal IgA response and the serum IgG response (Fig. 2A-C). Post challenge with GAS, the bacterial burden in nasal discharge was significantly lower in J8-Lipo- DT immunized mice in comparison to the PBS group and comparable to mice immunized with J8-DT+CTB (Day 3, Fig. 3 A).

Surprisingly however, J8-DT+CTB-immunized mice were not protected from colonization of the throat or NALT whereas J8-Lipo-DT-immunized mice showed significant protection against colonization in both compartments (Fig. 3 B and C). Protection due to J8-Lipo-DT was significantly better than that induced by J8-Lipo. Murine NALT is a portal of entry for persistent GAS infection (24), and is a functional homologue of human tonsils (25). Thus, these results highlight the efficacy of J8-Lipo-DT in reducing bio-burden in the preferential site of mucosal GAS infection.

We then asked whether J8-Lipo-DT would similarly protect mice of a different strain. J8-DT/CTB and PBS served as control immunogens. Immunization of BIO.BR mice (n= 5) with J8-Lipo-DT induced significant J8- specific antibody titers (Fig. 4). Mucosal antibody titers in saliva and fecal samples were comparable to those in mice immunized with J8-DT+CTB (Fig. 4A- B). To test whether J8-Lipo-DT would protect from GAS infection in B IO.BR mice a further cohort of mice (n=5) were immunized and challenged with GAS Ml strain. Nasal shedding and throat swabs were monitored over a 3-day observation period. By day 2, J8-Lipo-DT and J8-DT+CTB immunized mice had undetectable bio-burden in nasal shedding (Fig. 5A). Similarly to BALB/c mice, the data also demonstrated an absence of bacteria in J8-Lipo-DT immunized mice by day 2 post-challenge for throat swabs, whereas J8-DT+CTB immunised mice still had detectable level of GAS in throat swabs on day 3 (Fig. 5β).

Previous studies demonstrated that while peptides encapsulated within liposomes did not induce an immunoglobulin response, liposomes plus lipid A (a component of lipopolysaccharides) were able to induce an antibody response following intra-peritoneal immunization (26), thus suggesting that the lipid tail of lipid A may act as an adjuvant. To ask whether the double C16 lipid tail anchoring J8 to the liposome surface was responsible for induction of the antibody response a further cohort of mice were immunized with C16-C16-KSSJ8, J8- Lipo-DT, J8-DT+CTB or PBS. We observed that J8-Lipo-DT and J8-DT+CTB were immunogenic, whereas C16-C16-KSSJ8 was not (Fig. 6A-B).

The cytokine response of spleen cells from intranasally immunized mice was measured to determine whether intranasal immunization induced a systemic cellular immune response that may explain the self adjuvanticity of J8-Lipo-DT and the switch of the isotype of antibodies to IgA. Pro-inflammatory cytokines (gamma interferon [IFN-γ], interleukin 1 [IL-1], IL-6, IL-12p70, monocyte chemotactic protein 1 [MCP-1], and tumor necrosis factor alpha [TNF-a]) were analysed. B IO.BR mice immunized with J8-Lipo-DT were sacrificed and splenocytes were stimulated with J8, LPS or media. We observed significant IFN- γ, MCP-1 and IL-6 production in response to J8 and LPS (Fig. 7). Other cytokines assessed were not detected. The results demonstrated that vaccination with J8-Lipo-DT induced a pro-inflammatory response, providing a potential mechanism for the self-adjuvanticity of J8-Lipo-DT. In particular, IL-6 is known to be responsible for the switch of the antibody response to IgA (27). Furthermore, the chemo-attractant MCP-1 is known to play a major role in GAS defence mechanisms (28).

To evaluate the potential of J8-Lipo-DT to induce an effective immune response with self-adjuvanting activity in humans, dendritic cell subsets were isolated from the blood of three healthy volunteers and stimulated with J8-Lipo- DT. Mature DCs are potent antigen presenting cells, expressing high levels of cell surface molecules involved in antigen presentation and co-stimulation that facilitate antigen recognition and cell-cell interactions. To characterize human DC maturation, the modulation of various cell surface molecules in response to J8- Lipo-DT was examined by flow cytometry (Fig. 8A-C). The synthetic double- stranded RNA adjuvant, polyriboinosinic-polyribocytidylic acid (pIC) was used as a control (29). Levels of the costimulatory molecules CD80, CD83 and CD86 were significantly higher on CD123+ plasmacytoid DCs (pDCs) cultured with J8- Lipo-DT (Fig. 8A). Expression of CD80 was also increased in the two subsets of classical DCs (cDC), CD 141+ classical type 1 DCs and CDlc+ classical type 2 DCs (Fig. 8fi-C). In addition, CD86 expression was also increased for CD141+ DCs (Fig. 8fi).

To further define the interaction with human DCs, levels of pro- inflammatory cytokines post stimulation were evaluated using the cytometric bead array. We observed increased expression of the pro-inflammatory cytokines (T F-a, IL-6 and IL-1 beta (IL-1 β)) as well as the neutrophil chemo-attractant, IL-8 (Fig. 9). Neutrophils are known to be crucial to IgA control of GAS infections (30). Elevated levels of anti-inflammatory cytokine, IL-10, were also observed (Fig. 9). This is possibly due to the regulatory effects of IL-10 in DC maturation steps and counterbalancing host pro-inflammatory responses (31, 32). However, the IL-6 response, in particular, suggests that J8-lipo-DT would lead to IgA switching in humans and, together with a neutrophil response (via IL-8), would well position the host to control a GAS infection. As shown in FIG. 10, SpyCEP peptide (S2; SEQ ID N0:2) displayed by liposomes together with intravesicular DT, elicited a mucosal IgA response, both when displayed as alone and as an S2-J8 chimera (SEQ ID NO:3).

Referring to FIG. 11, J8+S2-Lipo-DT induced antigen specific IgA, IgG responses. Comparable immune responses were observed in response to J8-Lipo- DTand S2-Lipo-DT. Different formulation strategies employing either (i) both epitopes in a liposome (J8+S2-Lipo-DT) or (ii) an admixture of J8/S2S2-Lipo-DT liposomes.

Group A streptococcal infections, can cause a variety of skin and soft- tissue infections, some of which are severe and even life-threatening. Therefore it is of interest to see if J8-Lipo-DT can protect against skin infection post-challenge with 88/30 strain. Initial experiments employing Intranasal administration of liposomes comprising DT and J8 alone showed significant IgG titer post intranasal immunization with J8-Lipo-DT but there was no protection in a skin challenge assay (data not shown). Enhancement of systemic IgG responses through different liposome formulations or through immunization with J8- DT+Alum given subcutaneously is being undertaken

DISCUSSION

We have developed a mucosally active subunit liposomal vaccine candidate for group A streptococcus (GAS). The immunostimulatory properties of liposomes were combined with encapsulation of the protein carrier DT, and display of a GAS-specific B cell epitope on the liposome surface. Both the peptide and the carrier protein were required for optimal immunity. Mucosal immunity induced by the compound liposome was superior to that induced by the peptide- protein conjugate administered with CTB.

Mucosal immunization, as a means of eliciting protective immunity against infectious diseases, has attracted much interest. The vast majority of infections occur at, or begin from, mucosal surfaces. Therefore, the application of a vaccine that can induce a mucosal protective immune response is both desirable and practical. In practice it has often proven difficult to stimulate strong mucosal IgA immune responses and the progress in mucosal vaccination efforts using subunit peptide antigens has been disappointing. This is due in part to difficulty in stimulating potent immune responses in comparison to traditional whole-organism based approaches. Addition of an adjuvant and conjugation of subunit antigens to protein carriers as sources of T cell help have proven effective for systemic immunity. However, induction of mucosal immunity requires novel strategies.

The topographical position of liposome associated antigens affects antigen processing and presentation to B and helper T cells (33). It has been demonstrated that antigens exposed on the liposome surface are preferentially processed and presented by B cells whilst liposome encapsulated antigen are more effectively processed and presented to T cells by antigen presenting cells (34). As such, the vaccine candidate J8-Lipo-DT represents a rational subunit liposomal vaccine design, ensuring B cell epitopes are liposome bilayer-associated to be exposed to bind to the Ig receptor of a B cell whilst encapsulation of DT allows effective delivery, processing and presentation to T cells.

We demonstrated clearance of GAS in the URT tissue, including NALT. Effective nasopharyngeal immunity by the vaccine candidate shows promising potential to reduce RF and RHD, which are linked to primary pharyngeal infections (7). In the human URT, tonsils are the primary reservoirs for GAS, sustaining endemic disease across the globe (25). Reduction of GAS colonization in the functional homolog of human tonsils, the NALT, suggests that intranasal immunization with J8-Lipo-DT will reduce colonization and infection of human tonsils, thus reducing transmission of GAS (25).

While liposomes have previously been reported to deliver encapsulated peptides to induce a cellular immune response, such liposomes do not induce an IgA nor an IgG response except in the presence of a potent adjuvant such as Lipid A (26, 35). It is possible that the lipid tail on J8 provided adjuvant activity thus contributing to the immunogenicity of J8-Lipo-DT; however, the J8 peptide with the lipid tail on its own was not immunogenic demonstrating the need for the liposome formulation. Self-adjuvanting immunostimulatory activities of liposomes have been previously reported and shown to be due to interaction with antigen presenting cells and induction of a pro-inflammatory response (36). In vitro assays in our study revealed induction of antigen specific inflammatory chemokines and cytokines in immunized mice. Of particular interest is the secretion of antigen specific MCP-1 and IL-6. MCP-1 is a chemoattractant for lymphocytes, monocytes, and antigen presenting cells (37). Previously implicated in mediating mucosal inflammation, it has been reported as a potential mucosal adjuvant due to significantly increasing mucosal IgA secretion (37).

Although antigen can be presented to the immune system by many cell types, the priming of naive T cells requires maturation, presentation of antigen and engagement of costimulatory molecules only found on professional antigen presenting cells such as DCs (38). DCs are key elements bridging the innate and adaptive immune responses to infection (39). Mature DCs produce inflammatory cytokines, up-regulate costimulatory and antigen presenting molecules, and migrate to lymph nodes where they function as potent antigen presenting cells for naive T lymphocytes to initiate adaptive immune responses. We demonstrated J8- Lipo-DT mediated expression of cell -surface activation and maturation markers on human DCs upon in vitro exposure and induction of pro-inflammatory cytokines including IL-6 and IL-8. Human and murine IL-6 plays a critical role in B cell terminal differentiation, and in mucosal sites it stimulates proliferation and secretion of IgA in mucosal sites (27). IgA specific immunity against GAS requires the presence of neutrophils (30), IL-8 has a key role in the recruitment and activation of neutrophils. In this regard, administration of the SpyCEP S2 peptide (SEQ ID NO:2) displayed by the liposomal particulate delivery system alone or in conjunction with J8 peptide elicited peptide-specific IgA.

Therefore, mechanisms that are fundamental to conferring immunity in humans to GAS infection can be mediated using the liposome platform, leveraging the relevance of the basic research to the clinical setting.

We demonstrated that human pDCs increased both maturation and costimulatory markers upon stimulation with J8-Lipo-DT. Human pDCs readily phagocytose and process antigens entrapped in particulate delivery systems (40), indicating that particulate delivery systems can be used to facilitate efficient delivery of antigens to pDCs. To the best of our knowledge, our results show for the first time the ability to stimulate human pDCs with a liposome-based particulate delivery system. Human pDCs initially identified in the blood, have subsequently been detected in the spleen, lymph nodes and mucosal sites including tonsils (41). Therefore, liposome-based vaccine delivery could potentially be exploited to target this DC subset for desired mucosal immune responses in humans.

EXAMPLE 2 Experiments were undertaken to investigate the effect of liposome size on immunogenicity.

Liposome extrusion was done with a heat block, 1 mL syringe mini- extruder (Avanti Polar Lipids). The rehydrated solution was passed eleven times through a 50 nm, 400 nm, 1000 mm filter (Avanti Polar Lipids) while the heat block was set at ~40°C. Liposome size measurement was performed by Nanosizer (Dynamic light scattering or DLS).

As shown in Figure 12, J8-Lipo-DT can be extruded to form nano- to micro-sized particles The majority of particle sizes had a narrow molecular- weight distribution (low polydispersity index of < 0.3). The data shown in Figure 13 suggest that J8-Lipo-DT size does not influence systemic IgG response. However, as shown in Figure 14, larger sized liposomes induced a J8-specific mucosal response.

EXAMPLE 3

Experiments were undertaken to investigate the effects freeze-drying of liposomes on immunogenicity. Liposome thin films were rehydrated with milliQ water containing 10% Trehalose and then lyophilized. Post 1, 4 and 7 weeks lyophilization, J8-Lipo-DT powder was reconstituted in PBS.

Figure 15 shows the size results of liposome size measurement by Nanosizer (Dynamic light scattering or DLS). The majority of particle sizes had a narrow molecular-weight distribution (low polydispersity index of < 0.3). Figure 16 shows that reconstituted, freeze-dried, J8-Lipo-DT liposomes induced J8- specific systemic response without additional adjuvant This was a comparable immune response to freshly made J8-Lipo-DT. Trehalose was important for the immunogenicity of freeze-dryed J8-Lipo-DT. Figure 17 demonstrates that reconstituted, freeze-dried J8-Lipo-DT liposomes induced a J8-specific mucosal response

EXAMPLE 4

Further experiments determine the efficacy of immunogenic liposomes comprising glycolipid activators of innate immunity such as trehalose 6,6'- dibehenate (TDB) and 3D-PHAD® such as shown scehmatically in FIG. 18. TDB is formulated with liposomes based on % of total phospholipids in liposomes. 9 mg of phospholipids are used and TDB is used at radio of 20% of this (1.8 mg). Efficacy is measured by antibody titers post-immunization (IgA and IgG antibodies) and skin challenge experiment with a GAS strain. Data are shown in FIG. 19. Mice (n = 5/ group) were immunized intranasally with 30ug of J8-Lipo- Dt+TDB. Mice were administered primary (Day 0) plus two boosts (days 21 & 42). Incorporation of TDB results in significantly higher mucosal IgA responses in both saliva & fecal samples in comparison to J8-Lipo-DT. The results are from a lyophilized powder version of J8-Lipo-DT+TDB which improved the stability of the liposome

Further experiments will determine the efficacy of immunogenic liposomes comprising a bile salt such as sodium deoxycholate. An example of a liposome comprising the bile salt sodium deoxycholate is shown schematically in FIG. 20. It will be noted that the immunogenic agent (in this case J8 peptide) may be fused or conjugated to the carreier protein (e.g. DT) or may be displayed on the surface of the liposome. Bile salt will be formulated in liposomes when phospholipids are hydrated to produce liposomes (bile salt containing liposomes are referred to as "bilosomes"). Preparation of bilosomes is as follows.

Sorbitan tristearate (150 mmol), cholesterol and dicetyl phosphate (DCP) in a molar ratio of 7:3 : 1 is dissolved in 10 mL chloroform in a round-bottomed flaskalong with 150 μg of J8 modified with palmitic acid moiety. Solvent is removed by rotary evaporator to form a thin film on the glass surface of a round- bottomed flask. The film is then hydrated with 3.5 mL PBS (pH 7.4), containing 100 mg of sodium deoxycholate (bile salt) along with 150 μg of diphtheria toxoid.

EXAMPLE 5

The immunogenicity of a lipid vesicle against influenza A, influenza B and group A streptococcus was measured by antigen-specific salivary IgA titre. FIG. 21 shows a schematic depiction of the immunogenic agent comprising a single lipid vesicle having respective immunogens from each of influenza A, influenza B and group A streptococcus. The results shown in FIG. 22 show that a lipid vesicle comprising respective immunogens from each of influenza A, influenza B and group A streptococcus (as shown in FIG 21 A) induced immunity against each of these pathogens in mice. Further work will investigate the immunogenicity of an immunogenic agen comprising glycolipid adjuvants such as shown schematically in FIG. 2 IB.

In conclusion, this work is the first to report on a liposome-based, mucosally active GAS vaccine candidate. Our findings are an important step toward overcoming current obstacles in the development of a GAS vaccine to prevent infection at mucosal sites and community dissemination. The study provides important mechanistic insights into how liposomal particulate delivery systems can collectively induce the desired mucosal immune responses to combat GAS infection. The strategy reported here is relevant to the development of subunit mucosal vaccines against other pathogenic organisms. Non-limiting examples include influenza virus, rhinovirus and hookworms as hereinbefore described. In some embodiments, a single lipid vesicle may comprise immunogens against a plurality of different pathogens.

Throughout this specification, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated herein without departing from the broad spirit and scope of the invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference in their entirety.

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