CN109069424B - Liposome vaccine - Google Patents

Abstract

An immunogenic agent suitable for prophylaxis, treatment or immunisation against one or more different pathogens comprising one or more pathogen-derived proteins, fragments, variants or derivatives thereof displayed on a lipid vesicle and a carrier protein such as diphtheria toxoid located within the intravesicular space. The immunogenic agent may be suitable for intranasal administration and is capable of eliciting a mucosal immune response. The immunogenic agent may further comprise an activator of innate immunity, for example trehalose-6, 6' -dibehenate (TDB) and/or bile salts such as sodium deoxycholate. The one or more pathogens may be group a streptococci, a virus, or hookworm.

Description

Liposome vaccine

Technical Field

The present invention relates to the prevention and treatment of infectious diseases. More particularly, the present invention relates to liposomal vaccines for treating or preventing infectious diseases and disorders by inducing mucosal immune responses.

Background

Systemic immunity through serum immunoglobulins (Ig) at systemic sites has been shown to be effective in preventing diseases caused by a variety of different pathogens, but ineffective in preventing colonization at mucosal sites and thus preventing human-to-human transmission. Thus, for some diseases, systemic vaccination is not the optimal route for inducing immunity. In contrast, nasal mucosal vaccines against a variety of organisms are effective in inducing antigen-specific immune responses in the systemic and mucosal compartments. Because of this double-layer protective immunity, mucosal vaccination is an ideal strategy for combating systemic and mucosal infections, with the additional benefit of pre-mucosal colonization that also inhibits transmission through droplets and aerosols from the upper respiratory tract. Mucosal vaccination also has economic advantages, which is an important consideration for vaccine development. Due to the convenience of administering vaccines via the nasal route, the use of needles can be avoided. Painless delivery contributes to better patient compliance.

Disclosure of Invention

It is an object of the present invention to provide immunogenic agents and delivery systems that elicit mucosal immune responses to pathogens. In a broad sense, the present invention relates to the promotion or induction of mucosal immunity by delivery of immunogenic proteins, fragments or variants by means of lipid vesicles further comprising a carrier protein such as Diphtheria Toxoid (DT). Suitably, the carrier protein is located in the intravesicular space. In particular forms, a single immunogenic agent includes a plurality of different immunogenic proteins, fragments or variants from a plurality of different pathogens.

One aspect of the invention provides an immunogenic agent suitable for administration to a mammal, the immunogenic agent comprising one or more immunogenic proteins, fragments, variants or derivatives thereof, lipid vesicles and a carrier protein or fragment or variant thereof.

In one embodiment, the carrier protein is Diphtheria Toxoid (DT).

In one 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 an immunogenic agent of the preceding aspect.

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

In one embodiment, the composition comprises a plurality of different immunogenic agents each comprising one or more immunogenic proteins, fragments, variants or derivatives thereof, of or from a different pathogen.

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

In one embodiment, the composition comprises a plurality of different immunogenic agents each comprising one or more immunogenic proteins, fragments, variants or derivatives thereof of or from a different pathogen.

Another aspect of the invention provides a method of eliciting an immune response against one or more pathogens in a mammal, the method comprising the steps of: administering to the mammal an immunogenic agent comprising one or more immunogenic proteins, fragments, variants or derivatives thereof, lipid vesicles and Diphtheria Toxoid (DT) or fragments or variants thereof, or a composition comprising the same, thereby eliciting an immune response in the mammal against the one or more pathogens.

Another aspect of the invention provides a method of immunizing a mammal against one or more pathogens, the method comprising the steps of: administering to the mammal an immunogenic agent comprising one or more immunogenic proteins, fragments, variants or derivatives thereof, lipid vesicles and Diphtheria Toxoid (DT) or fragments or variants thereof, or a composition comprising the same, thereby immunizing the mammal against the one or more pathogens.

Yet another aspect of the present invention provides a method of treating or preventing infection by one or more pathogens in a mammal, the method comprising the steps of: administering to a mammal an immunogenic agent comprising one or more immunogenic proteins, fragments, variants or derivatives thereof, lipid vesicles and Diphtheria Toxoid (DT) or fragments or variants thereof, or a composition comprising the same, thereby treating or preventing infection by the one or more pathogens in the mammal.

Suitably, the immunogenic agent elicits a mucosal immune response.

Typically, the mucosal immune response involves the production of IgA.

In a preferred form, the immunogenic agent is administered intranasally.

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

Suitably, the immunogenic protein fragment or variant is lipidated. In certain embodiments, the lysine (K) residue at the N-terminus of the immunogenic protein fragment or variant is lipidated. In a preferred form, the N-terminal lysine (K) residues are lipidated through the alpha and epsilon amino groups. In some embodiments, each lipid is C₁₆Fatty acids, such as palmitic acid. Preferably, the N-terminal lysine (K) residue is located in the spacer amino acid sequence at the N-terminus of the immunogenic protein fragment or variant.

In a particular embodiment, the immunogenic protein, fragment or variant is a group a streptococcus M protein, fragment, variant or derivative thereof. In additional or alternative embodiments, the immunogenic protein is an agent that promotes the recovery or enhancement of neutrophil activity.

In particular embodiments, the M protein fragment is or comprises a conserved region of the M protein. In one embodiment, the fragment is an immunogenic fragment comprising or comprised in a p145 peptide. In particular embodiments, the immunogenic fragment is located within the J8 peptide or variant thereof, or comprises the J8 peptide or variant thereof.

Preferably, the J8 peptide comprises or consists essentially of the amino acid sequence: QAEDKVKQSREAKKQVEKALKQLEDKVQ (SEQ ID NO: 1).

In a broad embodiment, the agent that promotes recovery or enhancement of neutrophil activity is the protein 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 a specific embodiment, the SpyCEP fragment and the M protein fragment can be fused to form a single chimeric peptide.

In one embodiment, the chimeric peptide is, may comprise or consist essentially of the following amino acid sequence or variant thereof: NSDNIKENQFEDFDEDWENFQAEDKVKQSREAKKQVEKALKQLEDKVQ (SEQ ID NO: 3).

In a particular embodiment, the immunogenic protein, fragment or variant is of an influenza virus. The influenza virus may be an influenza a virus. Non-limiting examples are, comprise or consist essentially of the amino acid sequence: amino acid sequence MSLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO: 4). The influenza virus may be influenza b virus. Non-limiting examples are the following amino acid sequences, or comprise or consist essentially of the amino acid sequence: amino acid sequence PAKLLKERGFFGAIAGFLE (SEQ ID NO: 5).

In a particular embodiment, the immunogenic protein, fragment or variant is rhinovirus. Non-limiting examples are, comprise or consist essentially of the amino acid sequence: amino acid sequence GAQVSTQKSGSHENQNILTNGSNQTFTVINY (SEQ ID NO: 6). Another non-limiting example is, comprises or consists essentially of the amino acid sequence of: amino acid sequence GAQVSRQNVGTHSTQNMVSNGSSL (SEQ ID NO: 7).

In a particular embodiment, the immunogenic protein, fragment or variant is helminth, such as hookworm. Non-limiting examples are, comprise or consist essentially of the amino acid sequence: amino acid sequence TSLIAGLKAQVEAIQKYIGAEL (SEQ ID NO: 8).

In some embodiments, the immunogenic agent may further comprise an activator of innate immunity. The innate immune activator may target C-type lectins, such as macrophage-inducible Ca²⁺Dependent (type C) lectins ("Mincle"). Activators of innate immunity can be glycolipids. Non-limiting examples include glycolipids such as mycosphase-6, 6 '-dimycolate (TDM) and/or its synthetic analogue trehalose-6, 6' -dibehenate (TDB) or lipid a glycolipid adjuvants.

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 only those elements or features that are set forth or listed, but may include other elements or features not listed or set forth.

By "consisting essentially of … …" in the context of an amino acid sequence is meant the recited amino acid sequence as well as1, 2, or 3 additional amino acids at the N-or C-terminus.

As used herein, the indefinite articles 'a' and 'an' are used herein to mean or include a singular or plural element or feature, and should not be taken as meaning or limiting "one" or "a single" element or feature.

Drawings

FIG. 1 is an idealized structure of J8-Lipo-DT. Liposomes encapsulate DT, while J8, which is linked to the spacer KSS at the N-terminus, is covalently coupled to two palmitic acid molecules, facilitating insertion of J8 into the liposome membrane.

FIG. 2J 8 specific antibody response of a single BALB/c mouse. The mean antibody titers are expressed in bar. A) Salivary IgA titer. B) Fecal IgA titer. C) Serum IgG titers. Statistical analysis was performed using one-way ANOVA followed by Tukey post-test (ns, p > 0.05;. p, p < 0.01;. p, p < 0.001).

FIG. 3 bacterial load following intranasal challenge with M1GAS strain in BALB/C mice. A) Nasal drops. B) A throat swab. C) Colonization of NALT. The results are expressed as follows: mean CFU + SEM of 10 mice/group on days 1-3 for throat swabs, nasal casts; for NALT, on day 3. Statistical analysis was performed using the non-parametric, unpaired mann-whitney U test to compare the test groups to the PBS control group (ns, p > 0.05;, p < 0.01;, p < 0.001).

Figure 4J 8-specific antibody responses of a single b10.br mouse (n-5 per group). The mean antibody titers are expressed in bar. A) Salivary IgA titer. B) Fecal IgA titer. C) Serum IgG titers. Statistical analysis was performed using one-way ANOVA followed by Tukey post-test (ns, p > 0.05;. p, p < 0.01;. p, p < 0.001).

FIG. 5 URT GAS challenge model to evaluate bacterial load after intranasal challenge with M1 strain. A) Nasal drops of br mice. B) Throat swab of br mice. Results are expressed as mean CFU + SEM of 5 mice/group on days 1-3. Statistical analysis was performed using the nonparametric, unpaired mann-whitney U test to compare the test groups to the PBS control group (ns, p > 0.05;. p < 0.05).

FIG. 6J 8 specific antibody response of a single BALB/c mouse. The mean antibody titers are expressed in bar. A) Salivary IgA titer. B) Serum IgG titers. Statistical analysis was performed using one-way ANOVA followed by Tukey post-test (ns, p > 0.05;. p, p < 0.01;. p, p < 0.001).

FIG. 7 antigen-specific secreted chemokines and cytokines in immunized mice. Splenocytes were removed and the following stimuli were added as indicated: LPS (2. mu.g/mL), J8 (10. mu.g/mL) or medium alone. 72 hours after stimulation, supernatants were separated and analyzed for levels of secreted chemokines or cytokines using flow bead arrays (see materials and methods). Statistical analysis was performed using student's t-test (ns, p > 0.05;. p < 0.01).

FIG. 8 surface marker levels on human DC subsets with and without treatment with agents. The following stimuli were added as indicated: polyinosinic acid: polycytosine nucleotides (pIC, 10. mu.g/mL), J8-Lipo-DT (150. mu.g/mL) or medium alone. Cell surface markers were measured by flow cytometry 24 hours after stimulation. A) CD123+ plasma-like cell DC. B) CD141+ classical type 1 DC. C) CD1c + classical type 2 DC. Values are expressed as Median Fluorescence Intensity (MFI) ± SEM of mixed data from three individual donors. Statistical analysis was performed using a non-parametric, unpaired mann-whitney U test to compare the test groups to the vehicle control group (ns, p > 0.05;, p < 0.01;, p < 0.001).

FIG. 9 chemokines and cytokines secreted by J8-Lipo-DT induced in human dendritic cells. Dendritic cells were removed and the following stimuli were added as indicated: pIC (10. mu.g/mL), J8-Lipo-DT (150. mu.g/mL) or vehicle alone. Supernatants were isolated 24 hours after stimulation and analyzed for levels of secreted chemokines or cytokines using flow bead arrays (see materials and methods). Statistical analysis was performed using student's t-test (ns, p > 0.05;. p < 0.05;. p, p < 0.01;. p < 0.001).

FIG. 10 Liposomal delivery of reagents for SpyCEP peptide (S2; SEQ ID NO:2) elicits mucosal IgA responses. Liposomes displaying palmitated S2 peptide or S2-J8 chimera (SEQ ID NO:3) and intravesicular DT were administered intranasally to mice and S2 specific IgA titers were measured.

FIG. 11J 8+ S2-Lipo-DT immunogenic agents induce antigen specific IgA, IgG responses in mice.

Figure 12J 8-Lipo-DT immunogenic agents can be extruded to form nano or micro sized particles. Liposome size measurements were performed by a Nanosizer (dynamic light scattering or DLS).

Figure 13 size of J8-Lipo-DT immunogenic agent did not affect systemic IgG response.

Figure 14, larger size J8-Lipo-DT immunogenic agents induced J8 specific mucosal responses.

Figure 15 particle size distribution of reconstituted freeze-dried J8-Lipo-DT powder in PBS. Liposome size measurements were performed by a Nanosizer (dynamic light scattering or DLS).

Figure 16 reconstituted lyophilized J8-Lipo-DT liposomal immunogenic agent induced a J8 specific systemic response in the absence of other adjuvants.

Figure 17 reconstituted lyophilized J8-Lipo-DT liposome immunogenic agent induced a J8 specific mucosal response.

FIG. 18 is a schematic depiction of a liposomal J8-Lipo-DT immunogenic agent comprising the glycolipid adjuvant trehalose 6,6' -dibehenate (TDB).

TDB increases the mucosal IgA response induced by J8-Lipo-DT. Mice were immunized intranasally with 30 μ g of J8-Lipo-Dt + TDB (n-5 per group). Mice were administered for the first time (day 0) plus two boosts (days 21 and 42). In saliva and stool samples, incorporation of TDB resulted in significantly higher mucosal IgA responses compared to J8-Lipo-DT. Results J8-Lipo-DT + TDB from lyophilized powder form that eliminated the stability problem of liposomes.

Figure 20 is a schematic depiction of a liposomal immunogenic agent comprising the bile salt sodium deoxycholate.

Fig. 21 (a) schematically depicts a single immunogenic agent comprising lipid vesicles, intravesicular DTs and immunogenic proteins of or from a plurality of different pathogens, i.e. influenza a virus, influenza b virus and group a streptococcus; (B) schematically depicted is a single immunogenic agent comprising lipid vesicles, intravesicular DT and immunogenic proteins from a variety of different pathogens or immunogenic proteins from a variety of different pathogens, together with the glycolipid adjuvants trehalose 6,6' -dibehenate (TDB) and monophosphoryl 3-deacylated lipid A

The multiple different pathogens are influenza a virus, influenza b virus and group a streptococcus.

Figure 22 immunogenicity of Single immunogenic agents ("Multivax") against influenza a, influenza b, and group a streptococci, as measured by Antigen-specific salivary IgA titers, compared to vaccination alone ("Single Antigen-vax") with each separate immunogenic agent against influenza a, influenza b, and group a streptococci.

Detailed Description

The present invention is based, at least in part, on the following findings: intranasal vaccination of mice with a liposomal immunogenic agent comprising an immunogenic peptide displayed on the surface of the liposome and an intravesicular carrier protein such as Diphtheria Toxoid (DT) elicited mucosal and systemic antibody responses comparable to those induced by the established non-human compatible adjuvant CTB. In the specific context of group A streptococci and the J8 peptide, the level of protective immunity induced by the liposomal formulation significantly exceeded that induced by J8/CTB. Furthermore, the cytokine response of purified subpopulations of human Dendritic Cells (DCs) suggests that such liposomes will be effective in inducing human mucosal J8-specific IgA and systemic IgG responses. In some embodiments, the liposomal immunogenic agent may comprise the SpyCEP peptide or other fragment thereof alone or in addition to the J8 peptide. In particular forms, the liposomal immunogenic agent may be suitable for use in the treatment or prevention of infection caused by a particularly virulent strain or isolate of group a streptococcus that is resistant to common antibiotic treatment for group a streptococcus infection. These strains or isolates often cause severe skin infections (e.g., necrotizing fasciitis) and, in some cases, may contain CovR/SCovR/S mutations. This may be generalized to other pathogens and their associated diseases, disorders and conditions, including but not limited to influenza, rhinoviruses and helminths, such as hookworms. Thus, embodiments of the invention provide a single immunogenic agent comprising one or more immunogenic proteins, fragments, variants or derivatives of or from a plurality of different pathogens.

For the purposes of the present invention, "isolated" means a material that has been removed from its natural state, or a material that has been subjected to manual manipulation. An isolated material may be substantially or essentially free of components that normally accompany it in its natural state, or may be manipulated to be in an artificial state along with components that normally accompany it in its natural state. The isolated material may be in natural, chemically synthesized, or recombinant form.

"protein" means an amino acid polymer. The amino acids may be natural or unnatural, D-or L-amino acids, as is well known in the art.

The term "protein" includes and encompasses "peptides", which are commonly used to describe proteins having no more than fifty (50) amino acids, and "polypeptides", which are commonly used to describe proteins having more than fifty (50) amino acids.

As used generally herein, when a protein is referred to as "pathogenic" or "from a pathogen" it is meant that the protein includes at least part or all of the amino acid sequence present in the protein of the pathogen.

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

Typically, a fragment 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 a full-length protein.

As used herein, a protein "variant" shares a determinable nucleotide or amino acid sequence relationship with a reference amino acid sequence. A "variant" protein may have one or more amino acids of the reference amino acid sequence deleted, or the amino acids replaced by different amino acids. It is well known in the art that some amino acids may be substituted or deleted without altering the activity of the immunogenic fragment and/or the protein (conservative substitutions). Preferably, the protein mutant shares 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 the reference amino acid sequence.

Terms commonly used herein to describe the sequence relationship between individual proteins and nucleic acids include "comparison window", "sequence identity", "percentage of sequence identity", and "substantially identical". Since each nucleic acid/protein may each comprise (1) only one or more portions of the complete nucleic acid/protein sequence shared by the nucleic acids/proteins, and (2) one or more portions that differ 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. "comparison window" refers to a conceptual segment of typically 6, 9, or 12 contiguous residues 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 the comparison windows can be performed by GAP, BESTFIT, FASTA and TFASTA in a Computer run of algorithms (Geneworks program of Intelligenetics; Wisconsin Genetics Software Package, Release 7.0(Genetics Software Package Release 7.0), Genetics Computer Group,575Science Drive Madison, Wis, USA, which is incorporated herein by reference) or by visual inspection, with the optimal alignment being generated by any of the various methods chosen (i.e., generating the highest percentage of homology in the comparison window). The BLAST program family disclosed, for example, in Altschul et al,1997, Nucl. acids Res.25:3389, which is incorporated herein by reference, may also be used as a reference. A detailed discussion of sequence analysis can be found IN element 19.3 of the general handbook of MOLECULAR BIOLOGY (Current PROTOCOLS IN MOLECULAR BIOLOGY) authored by Ausubel et al (John Wiley & Sons Inc NY,1995 1999).

The term "sequence identity" as used herein is used in its broadest sense to include the number of exact nucleotide or amino acid matches with respect to the degree of sequence identity over a comparison window, with respect to a suitable alignment using standard algorithms. Thus, "percent sequence identity" is calculated by: comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the identical nucleobase (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 comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, it is understood that "sequence identity" means a "percent match" calculated by a DNASIS computer program (windows edition, version 2.5; available from Hitachi Software engineering Co., Ltd., South San Francisco, Calif., USA).

As used herein, a "derivative" is a molecule, such as a protein or a fragment or variant thereof, that has been altered, e.g., by linkage or complexation with other chemical moieties, by post-translational modification (e.g., phosphorylation, acetylation, etc.), by glycosylation modification (e.g., addition, removal, or alteration of glycosylation), by lipidation, and/or comprises additional amino acid sequences, as understood in the art. In a particular embodiment, the additional amino acid sequence may include one or more lysine residues at its N-terminus and/or C-terminus. The plurality of lysine residues (e.g., polylysines) may be a linear sequence of lysine residues or a branched sequence of lysine residues. These additional lysine residues may promote increased peptide solubility.

The additional amino acid sequence may include the fusion partner amino acid sequence that produces the fusion protein. For example, the fusion partner amino acid sequence may aid in the 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 hemagglutinin tags. Other additional amino acid sequences include spacer sequences. One example of a spacer sequence is an amino acid sequence at the N-terminus or C-terminus of the amino acid sequence of an immunogenic protein fragment or variant, which comprises lysine (K) residues suitable for lipidation. Typically, the spacer amino acid sequence comprises two (2) to ten (10) amino acids, for example three (3) amino acid sequences KSS.

Other derivatives contemplated by the present invention include, but are not limited to, modification of side chains, incorporation of unnatural amino acids and/or derivatives thereof during peptide or protein synthesis, as well as the use of cross-linking agents, and other methods of imposing conformational constraints on the immunogenic proteins, fragments, and variants of the invention.

IN this regard, the person skilled IN the art can refer to Chapter 15 of the general handbook for PROTEIN SCIENCE (Current promoters IN PROTEIN SCIENCEs) authored by Coligan et al (John Wiley & Sons NY 1995-2008) for a more extensive methodology for the chemical modification of PROTEINs.

It is to be understood that the immunogenic proteins, fragments, and variants disclosed herein may be presented on the surface of lipid vesicles alone, or as a chimeric protein 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, described in more detail below.

In the context of the present invention, the term "immunogenic" as used herein refers to the ability or potency of an immunogenic agent to generate or elicit an immune response, e.g., against a pathogen or molecular component thereof, upon administration to a mammal or other animal.

By "eliciting an immune response" is meant producing or stimulating the production or activity of one or more elements of the immune system, including the cellular immune system, antibodies, and/or the innate immune system. Suitably, the one or more elements of the immune system comprise B lymphocytes, antibodies, neutrophils, dendritic cells including plasmacytoid dendritic cells, cytokines and/or chemokines. Non-limiting examples of cytokines include proinflammatory cytokines such as TNF- α, IL-6, and IL-1 (e.g., IL-1 β). A non-limiting example of a chemokine is neutrophil chemoattractant IL-8. Suitably, the immune response is or comprises a mucosal immune response, for example comprising IgA production. Preferably, the immune response elicited by the immunogenic agent is protective.

As used herein, the terms "immunize," "vaccinate," and "vaccine" refer to methods and/or compositions that elicit a protective immune response against a pathogen, whereby further infection by the pathogen is at least partially prevented or reduced.

As used herein, the term "pathogen" refers to any living or non-living entity capable of causing a disease, disorder, or condition in an animal, such as a bird or mammal. The pathogen may be a virus, bacteria, protozoa, or helminth, but is not limited thereto. Specific non-limiting examples of pathogens include group a streptococcal bacteria, respiratory viruses, such as influenza viruses and rhinoviruses, and nematodes, such as hookworms.

As will be appreciated from the present disclosure, the present invention provides lipid vesicle formulations comprising immunogenic protein fragments, variants or derivatives formulated in lipid vesicles, and a carrier protein. As used broadly herein, a lipid vesicle may be a liposome, minicell, multilamellar vesicle, micelle (micell), vacuole or other vesicle structure comprising a lipid bilayer, suitably an immunogenic protein fragment, variant or derivative is derivatized to comprise one or more lipids that facilitate anchoring to the lipid bilayer, such that the immunogenic protein fragment, variant or derivative is presented at the surface of the lipid vesicle. In a preferred form, lysine (K) residues are lipidated by alpha and/or epsilon 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 has been lipidated. The spacer may typically comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids. One embodiment is a three (3) amino acid spacer KSS. The lipids may include saturated or unsaturated fatty acids (e.g., mono-or polyunsaturated). In some embodiments, the or each lipid is C₁₆Fatty acids, such as palmitic acid. However, it will also be understood that such as having C₁₂、C₁₃、C₁₄、C₁₅、C₁₇、C₁₈、C₁₉、C₂₀、C₂₁Or C₂₂Other lipids of saturated or unsaturated (e.g., monounsaturated or polyunsaturated) fatty acids of the carbon chain can be used in the present invention.

Suitably, the lipid vesicle comprises any lipid, or mixture of lipids, capable of forming a lipid bilayer structure. These include phospholipids, sterols including cholesterol, cholesterol esters and plant sterols, fatty acids and/or triglycerides. Non-limiting examples of phospholipids include Phosphatidylcholine (PC) (lecithin), phosphatidic acid, Phosphatidylethanolamine (PE) (cephalin), Phosphatidylglycerol (PG), Phosphatidylserine (PS), Phosphatidylinositol (PI), and Sphingomyelin (SM), or natural or synthetic derivatives thereof. Natural derivatives include egg PC, egg PG, soybean PC, hydrogenated soybean PC, soybean PG, brain PS, sphingolipids (sphingolipids), brain SM, galactocerebroside, ganglioside, cerebroside, cephalin, cardiolipin and dicetyl phosphate. Synthetic derivatives include Dipalmitoylphosphatidylcholine (DPPC), didecanoyl phosphatidylcholine (DDPC), Dipalmitoylphosphatidylcholine (DEPC), Dimyristoylphosphatidylcholine (DMPC), Distearoylphosphatidylcholine (DSPC), dilauroyl phosphatidylcholine (DLPC), Palmitoyl Oleoyl Phosphatidylcholine (POPC), Palmitoyl Myristoyl Phosphatidylcholine (PMPC), Palmitoyl Stearoyl Phosphatidylcholine (PSPC), dioleoyl phosphatidylcholine (DOPC), dioleoyl phosphatidylethanolamine (DOPE), dilauroyl phosphatidylglycerol (DLPG), distearoyl phosphatidylglycerol (DSPG), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearoyl glycerol (DSPG), dioleoyl phosphatidylglycerol (DOPG), Palmitoyl Oleoyl Phosphatidylglycerol (POPG), dimyristoyl phosphatidic acid (DMPA), Dipalmitoylphosphatidic acid (DPPA), distearoylphosphatidic acid (DSPA), Dimyristoylphosphatidylethanolamine (DMPE), Dipalmitoylphosphatidylethanolamine (DPPE), Dimyristoylphosphatidylserine (DMPS), Dipalmitoylphosphatidylserine (DPPS), Distearoylphosphatidylethanolamine (DSPE), Dioleoylphosphatidylethanolamine (DOPE), Dioleoylphosphatidylserine (DOPS), Dipalmitoylphosphatidylcholine (DPSM) and Distearoylsphingomyelin (DSSM). The phospholipid may also be a derivative or analog of any of the above phospholipids.

Suitably, the mixture of lipids may comprise the respective phospholipids in a desired molar ratio or in a desired wt% ratio. The ratio between the respective lipids may be between 20:1 and 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 recited values. For example, liposomes can be formed using a molar ratio of 7 dipalmitoyl-sn-glycero-3-phosphocholine (DPPC):2 Cholesterol (CHOL):1L- α -Phosphatidylglycerol (PG).

Suitably, the lipid vesicle further comprises a carrier protein. Suitably, the carrier protein is immunogenic, or at least partially contributes to or enhances the immunogenicity of said immunogenic agent. Typically, the carrier protein is formulated with the lipid vesicle such that the carrier protein is located within the interior aqueous space of the lipid vesicle. In some embodiments, the carrier protein is separate from the immunogenic protein fragment, variant, or derivative thereof. According to this embodiment, the immunogenic protein fragment, variant or derivative thereof may be displayed on the surface of the vesicle. In some embodiments, the carrier protein may be fused, conjugated or complexed to said immunogenic protein fragment, variant or derivative thereof. This may include recombinant protein fusion, chemical cross-linking, and intermolecular complexation, for example, by biotin-avidin or other intermolecular binding agents, although not limited thereto. In such embodiments, the immunogenic protein fragment, variant or derivative thereof is located within the lipid vesicle in the internal aqueous space, fused, bound or complexed to the carrier protein. This embodiment may be particularly useful for oral delivery of immunogenic agents, such as liposomes comprising bile salts, as described in more detail herein below. Non-limiting examples of carrier proteins include Diphtheria Toxoid (DT), Tetanus Toxoid (TT), CRM proteins such as CRM197, and pertussis toxin mutants, although not limited thereto. Fragments and variants of the carrier protein are also contemplated. In a particular embodiment, the carrier protein is Diphtheria Toxoid (DT) or a fragment thereof.

In some embodiments, the lipid vesicle further comprises an activator of innate immunity. Activators of innate immunity may target C-type lectins associated with innate immunity that are expressed by one or more cells. Preferred C-type lectins are macrophage-induced Ca²⁺-dependent (C-type) lectins ("Mincle"). Non-limiting examples include glycolipids, e.g., mycose-6, 6 '-dimycolate (TDM) and/or its synthetic analog trehalose-6, 6' -dibehenate (TDB) and/or lipid A glycolipid adjuvants such as monophosphoryl 3-deacylated lipid A, which may be monophosphoryl 3-deacylated lipid A

3D-

And 3D (6-acyl)

In the form of (1). While not wishing to be bound by any particular theory, it is speculated that an activator of innate immunity (such as those described above) may enhance or improve mucosal immunity elicited by an immunogenic agent. Preferably, the glycolipid may be included in the lipid vesicle such that it comprises no more than about 25%, 20%, 15%, 10% or 5% of the total lipid in the lipid vesicle.

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

In other embodiments, liposomes containing an immunogenic agent can be prepared to a particular, selected, or desired particle size or size range. In some embodiments, liposomes of larger particle size can elicit a stronger mucosal immune response.

In other embodiments, liposomes comprising an immunogenic agent can be lyophilized or lyophilized to facilitate long-term storage. The reconstituted lyophilized liposomal immunogenic agent elicits an immune response comparable to that of the "fresh" liposomal immunogenic agent.

In some embodiments, the pathogen is group a streptococcus.

As used herein, the terms "group a Streptococcus (Streptococcus, Strep)" and simply "GAS" refer to Streptococcus bacteria of the lan's serogroup a, which are gram-positive β -hemolytic bacteria of the species Streptococcus pyogenes (Streptococcus pyogenes). An important virulence factor of GAS is the M protein, which is strongly anti-phagocytic and binds to serum factor H, destroying the C3-convertase and preventing the opsonin action of C3 b. These also include toxic "mutants," such as, for example, the CovR/S or CovRS mutants described by Graham et al, 2002, PNAS USA 9913855, although not limited thereto.

Diseases and conditions caused by group a streptococci include, but are not limited to, cellulitis, erysipelas, impetigo, scarlet fever, throat infections such as acute pharyngitis ("streptococcal laryngitis"), bacteremia, toxic shock syndrome, necrotizing fasciitis, acute rheumatic fever, and acute glomerulonephritis.

As used herein, a "neutrophil" or neutrophil forms part of a polymorphonuclear cell family (PMN) together with basophils and eosinophils. Neutrophils are relatively short-lived phagocytic cells formed by bone marrow stem cells and typically constitute 40% to 75% of the leukocytes in mammals. In addition to phagocytosis, neutrophils release soluble antimicrobials (e.g., granulins) and produce a neutrophil extracellular trap. Neutrophils respond to molecules such as interleukin-8 (IL-8), C5a, fMLP, and leukotriene B4, which promote chemotaxis of neutrophils to sites of injury and/or acute inflammation.

In one embodiment, the immunogenic protein may be an M protein, a fragment or variant thereof.

As used herein, an "M protein fragment" is any fragment of a GAS M protein that is immunogenic and/or capable of being bound by an antibody or antibody fragment. Typically, fragments are, include, or are within the following: an amino acid sequence of the C-repeat region of GAS M protein or a fragment thereof. Non-limiting examples include p145, which is a 20mer having the amino acid sequence LRRDLDASREAKKQVEKALE (SEQ ID NO: 4). In this regard, fragments of the p145 amino acid sequence may be present in the J8 peptide.

As used herein, a "J8 peptide" is a peptide comprising an amino acid sequence at least partially derived from or corresponding to a peptide of the C-region of a GAS M protein. The J8 peptide suitably comprises conformational B-cell epitopes and is free of potentially harmful T-cell self epitopes. A preferred amino acid sequence of the J8 peptide isQAEDKVKQ

KQLEDKVQ (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 further comprising flanking GCN4 DNA-binding protein sequences that help maintain the correct helical fold and conformational structure of the J8 peptide.

In another embodiment, the immunogenic protein may be an agent that promotes the recovery or enhancement of neutrophil activity.

As used herein, an "agent that promotes recovery or enhancement of neutrophil activity" is a molecule that directly or indirectly at least partially increases, enhances or restores 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 to and at least partially inactivates a neutrophil inhibitor. The neutrophil inhibitor may be a molecule derived or derived from group a streptococcus. In a particular form, the neutrophil inhibitor is a serine protease or fragment thereof that proteolytically cleaves interleukin 8. In a 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 (Streptococcus pyogenes) which plays an important role in infection by catalytic cleavage and inactivation of the neutrophil chemoattractant interleukin-8. Non-limiting examples of SpyCEP amino acid sequences can be found in accession numbers YP597949.1 and (streptococcus pyogenes MGAS10270) and YP596076.1 (streptococcus pyogenes MGAS 9429). Thus, in a particular embodiment, the SpyCEP fragment is or comprises the amino acid sequence set forth in SEQ ID NO 2 (NSDNIKENQFEDFDEDWENF). It is proposed that SEQ ID NO 2 is or comprises a dominant epitope on SpyCEP, which can induce functional antibodies.

Also provided herein are chimeric peptides comprising an M-protein amino acid sequence and a SpyCEP amino acid sequence forming 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 amino acid sequence NSDNIKENQFEDFDEDWENFQAEDKVKQSREAKKQVEKALKQLEDKVQ (SEQ ID NO:3) or a variant thereof.

In an alternative embodiment, individual liposomes comprising the M-protein amino acid sequence and the SpyCEP amino acid sequence can be produced for administration in a "cocktail".

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

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)

S R E A K K Q S R E A K K Q V E K A L K Q S R E A K C(SEQ ID NO：6)

S R E A K K Q V E K A L K Q S R E A K K Q V E K A L C(SEQ ID NO：7)

S R E A K K Q V E K A L D A S R E A K K Q V E K A L C(SEQ ID NO：8)

other variants may be based on heptapeptides such as described in Cooper et al, 1997.

For example, if an epitope is known to be located within the structural conformation of an α -helical protein, a model peptide can be synthesized that folds into that conformation. The model α -helical coiled-coil peptide has been based on the structure of the leucine zipper of GCN4 (O' Shea et al, 1991). The first heptad contains the sequence MKQLEDK (SEQ ID NO:9), which contains several features present in the stable coiled-coil heptad repeat motif (a-b-c-d-e-f-g) n (Cohen & Parry, 1990). These include large non-polar residues at positions a and d, acid/base pairs (Glu/Lys) at positions e and g (generally favouring interchain ionic interactions) and polar groups at positions b, c, f (consistent with predictions of Lupas et al (1991)). The GCN4 peptide also contains a common valine at position a. It has also been noted that coiled-coil dimers are preferred when positions a and d are occupied by V and L (Harburyet, 1994). The pattern heptad repeat region is derived from these common features of the GCN4 leucine zipper peptide: (VKQLEDK; SEQ ID NO:10) has the potential to form alpha-helical coiled coils. The peptide becomes a framework peptide. Overlapping fragments of the conformational epitope under study were embedded in a model coiled-coil peptide to produce chimeric peptides. Whenever identical residues are found in the helical pattern peptide and epitope sequence, amino acid substitutions designed to ensure the correct helical coiled-coil conformation (Cohen & Parry,1990) are incorporated into the chimeric peptide. The following substitutions are generally used: position a, V to I; b, K to R; c, Q to N; d, L to A; e, E to Q; f is D to E; g, K to R. All these replacement residues are usually found at their respective positions in coiled coil proteins (Lupas et al, 1991).

One particular J8 peptide derivative described in Olive et al, 2002, Infect & Immun.702734 is the "lipid core peptide". In one embodiment, the lipid core peptide may comprise a plurality of J8 peptides (e.g., four J8 peptides) synthesized directly on the two amino groups of each lysine of a branched polylysine core coupled to a lipophilic anchor.

The M protein fragment or variant and/or SpyCEP fragment or variant may be derivatized to comprise one or more lipids that facilitate anchoring to the lipid bilayer described above. In 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 its N-terminus. Thus, in embodiments where the SpyCEP fragment or variant is included in a lipid vesicle, it may be lipidated separately along with the M protein fragment or variant or may exist as a lipidated chimeric peptide.

In some embodiments, the pathogen is an influenza virus. In a 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 includes, amino acid sequence MSLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO: 4). In a particular embodiment, the immunogenic protein, fragment or variant is of influenza b virus. The immunogenic protein or fragment can be a hemagglutinin protein, or a fragment thereof. A non-limiting example is or includes the amino acid sequence PAKLLKERGFFGAIAGFLE (SEQ ID NO: 5).

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

In some embodiments, the pathogen is a helminth, such as hookworm. In a particular embodiment, the immunogenic protein, fragment or variant is hookworm americanus (cateor americanus). A non-limiting example is or includes the amino acid sequence TSLIAGLKAQVEAIQKYIGAEL (SEQ ID NO: 8).

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

Chemical synthesis includes solid phase and liquid phase synthesis. Although reference is made to the example of the chemical synthesis technique provided IN chapter 9 of SYNTHETIC VACCINES Ed. Nicholson (Black well Scientific Publications) and chapter 15 of Current PROTOCOLS IN PROTEIN SCIENCE eds. Coligan et al, (John Wiley & Sons, Inc. NY USA 1995-2008), such methods are well known IN the art. In this respect, reference is also made to international publication WO 99/02550 and international publication WO 97/45444.

Recombinant proteins can be conveniently prepared by those skilled in the art using standard protocols described, for example, in Sambrook et al, MOLECULAR clone.a Laboratory Manual (Cold Spring Harbor Press,1989), particularly sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY eds. ausubel et al, (John Wiley & Sons, inc. ny USA 1995-2015), particularly chapters 10 and 16; and CURRENT promoters IN process SCIENCE eds. colligan et al, (John Wiley & Sons, inc. ny USA 1995-. Generally, recombinant protein production involves expression of a nucleic acid encoding the protein in a suitable host cell.

As described above, the present invention provides immunogenic agents and/or their use for the prevention or treatment of pathogen-associated diseases, disorders or conditions in mammals or other animals.

As used herein, "treatment" refers to a therapeutic intervention that at least partially ameliorates, eliminates, or reduces symptoms or pathological signs of a pathogen-associated disease, disorder, or condition after onset of formation. Treatment is not necessarily an absolute benefit to the subject. The beneficial effect can be determined using any method or standard known to one of ordinary skill in the art.

As used herein, prevention (prevention/prevention) refers to a course of action that begins before infection or exposure to a pathogen or molecular component thereof and/or before onset of symptoms or pathological signs of a disease, disorder, or condition, in order to prevent infection and/or reduce symptoms or pathological signs. It is to be understood that such prevention is not necessarily an absolute benefit to the subject. "prophylactic" treatment is treatment administered to a subject who does not show signs of a disease, disorder or condition, or shows only early signs, with the purpose of reducing the risk of developing symptoms or pathological signs of the disease, disorder or condition.

In one embodiment, the disease, disorder, or condition may be a group a streptococcus-associated disease, disorder, or condition.

In the context of the present invention, "group a streptococcus-associated disease, disorder or condition" means any clinical pathology resulting from group a streptococcus infection and includes, although not limited to, cellulitis, erysipelas, impetigo, scarlet fever, throat infections such as acute pharyngitis ("streptococcal laryngitis"), bacteremia, toxic shock syndrome, necrotizing fasciitis, acute rheumatic fever and acute glomerulonephritis.

As described above, the uses disclosed herein for treatment and/or immunization include administering to a mammal an immunogenic agent comprising a fragment, variant or derivative of M protein, a lipid vesicle, a carrier protein and/or a SpyCEP peptide or other fragment that promotes recovery or enhancement of neutrophil activity.

As disclosed herein, treatment and/or immunization may additionally include administration of an antibody or antibody fragment to therapeutically treat GAS infection, e.g., by targeting SpyCEP at the site of infection (e.g., skin), and/or an antibody or antibody fragment that binds M protein, a fragment or variant thereof.

Antibodies and antibody fragments may be polyclonal or monoclonal, natural or recombinant. Antibody fragments include Fc, Fab or f (ab)2 fragments and/or may include single chain Fv antibodies (scFv). Such scfvs may be prepared, for example, according to the methods described below, respectively: U.S. Pat. No. 5,091,513, European patent No. 239,400 or Winter & Milstein,1991, Nature 349: 293. Antibodies may also include multivalent recombinant antibody fragments comprising multiple scfvs, such as diabodies, triabodies and/or tetrabodies, as well as dimerization-activated half-chain antibodies (demibodies) (e.g., WO/2007/062466). For example, such antibodies can be prepared according to the methods described in Holliger et al, 1993Proc Natl Acad Sci USA 906444; or Kipriyanov,2009Methods MolBiol 562177. Well-known PROTOCOLS that can be applied to antibody production, purification and use can be found, for example, IN Coligan et al, Current PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991-.

Methods for producing polyclonal antibodies are well known to those skilled in the art. Exemplary PROTOCOLS that can be used are described, for example, IN Coligan et al, Current PROTOCOLS IN IMMUNOLOGY, supra and Harlow & Lane,1988, supra. In particular embodiments, the anti-SpyCEP polyclonal antibody may be obtained or purified from human serum from an individual exposed to or infected with group a streptococcus. Alternatively, polyclonal antibodies to purified or recombinant SpyCEP or immunogenic fragments thereof can be produced in a producer species, such as horses, and then subsequently purified prior to administration.

Monoclonal antibodies can be prepared using standard methods, such as those described initially in

Standard methods IN the article of Milstein,1975, Nature 256,495, or by more recent modifications thereof, such as described IN Coligan et al, Current PROTOCOLS IN IMMUNOLOGY (supra), by immortalizing spleen cells or other antibody-producing cells from an organism species that has been inoculated with one or more of the isolated proteins, fragments, variants or derivatives of the invention. Thus, monoclonal antibodies may be generated against fragments, variants or derivatives of the M protein and/or agents that promote the restoration or enhancement of 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 if the monoclonal antibody is originally produced by splenocytes from a non-human mammal, which may be a "humanized" monoclonal antibody or fragment.

For embodiments related to therapeutic antibodies, a preferred M protein fragment may be a p145 peptide.

Preferred fragments 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 viruses can cause transmitted or otherwise transmitted disease, referred to as "flu". Typical symptoms include fever, headache, cough, lethargy, accumulation and secretion of respiratory and nasopharyngeal mucus, muscle pain, nausea and vomiting. Symptoms may last days or weeks. In some cases, secondary respiratory bacterial infections may occur, in some cases causing serious conditions such as pneumonia. Thus, the immunogenic agents and/or methods of the invention can treat or prevent influenza virus-associated diseases, disorders, or conditions such as those described above.

In some embodiments, the disease, disorder, or condition can be a rhinovirus-related disease, disorder, or condition. Rhinoviruses (e.g., rhinovirus a and rhinovirus B) are species of the enterovirus genus of the virus family picornaviridae. Rhinoviruses are often the causative agent of the common cold, with symptoms similar to influenza, but generally less severe and with a lower probability of secondary bacterial infections, such as pneumonia.

Thus, the immunogenic agents and/or methods of the invention can treat or prevent rhinovirus-related diseases, disorders, or conditions, such as those described above.

In some embodiments, the disease, disorder, or condition can be a hookworm-related disease, disorder, or condition. Hookworms are nematode worms that are parasitic in many different animals. Hookworms that normally infect humans may include hookworms americanus and Ancylostoma duodenale (Ancylostoma duodenalis). Hookworms have hooked mouthpieces that attach the hookworm to the intestinal wall, puncture the blood vessel and suck blood, in some cases resulting in severe anemia. Hookworm infection during pregnancy can cause growth retardation, preterm birth, and low birth weight in the fetus. Hookworms in children can cause intelligence, cognition, and growth problems.

Thus, the immunogenic agents and/or methods of the invention can treat or prevent helminth-associated diseases, disorders or conditions such as those described above.

In a specific embodiment, the foregoing process may be carried out as follows:

(i) administering an immunogenic agent comprising one or more different proteins, fragments, variants or derivatives of a single or the same pathogen;

(ii) administering a plurality of different immunogenic agents, each comprising one or more different proteins, fragments, variants or derivatives of a different pathogen; or

(iii) Administering an immunogenic agent comprising one or more different proteins, fragments, variants or derivatives of different pathogens;

so that:

eliciting an immune response against the pathogen;

(ii) immunity against the pathogen; or

Preventing or treating one or more diseases, disorders or conditions caused by the one or more pathogens.

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

In particular embodiments, the composition may comprise:

an immunogenic agent comprising one or more different proteins, fragments, variants or derivatives of the same or a single pathogen;

a plurality of different immunogenic agents each comprising one or more proteins, fragments, variants or derivatives of a different pathogen; or

An immunogenic agent comprising one or more 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 safe for systemic administration. Depending on the particular route of administration, a variety of carriers, diluents, and excipients well known in the art may be used. These may be selected from the group comprising sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers, emulsifiers, isotonic saline and salts (e.g. inorganic acid salts including hydrochlorides, bromates and sulfates; organic acid salts such as acetates, propionates and malonates), water and pyrogen-free water.

A useful reference to describe acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing co.n.j.usa,1991), which is incorporated herein by reference.

Preferably, certain immunogenic agents may be used in combination with the immunogenic agents disclosed herein in a formulation for the purpose of eliciting an immune response.

The term "immunizing agent" includes within its scope carriers, delivery agents, immunostimulants and/or adjuvants well known in the art. It will be understood in the art that immunostimulants and adjuvants refer to or include one or more substances that enhance the immunogenicity and/or efficacy of the formulation. Non-limiting examples of suitable immunostimulants and adjuvants include: squalane and squalene (or other oils of plant or animal origin); a block copolymer; detergents such as tweens

Mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil; corynebacterium-derived adjuvants such as Corynebacterium parvum (Corynebacterium parvum); propionibacterium-derived adjuvants such as Propionibacterium acnes (Propionibacterium acnes); mycobacterium bovis (Mycobacterium Bovis) (BCG); bordetella pertussis (Bordetella pertussis) antigen; tetanus toxoid; diphtheria toxoid; surface-active substances such as hexadecylamine, octadecylamine, octadecylamino acid ester, lysolecithin, dimethyldioctadecylammonium bromide, N-dioctadecyl-N ', N' -di (2-hydroxyethyl-malonamide), methoxyhexadecane glycerol and pluronic polyol; polyamines such as pyrans, dextran sulfate, poly IC carbopol; peptides such as muramyl dipeptide and derivatives, dimethylglycine, deglutition promoters; an oil emulsion; and mineral gels such as aluminum phosphate, aluminum hydroxide or alum; interleukins such as interleukin 2 and interleukin 12; monokines such as interleukin 1; tumor necrosis factor; interferons such as gamma interferon; immunostimulatory DNA such as CpG DNA, compositions such as saponin-aluminum hydroxide or Quil-a aluminum hydroxide; a liposome;

and

an adjuvant; a mycobacterial cell wall extract; synthetic glycopeptides such asMuramyl dipeptide or other derivative; alfvudine; a lipid a derivative; dextran sulfate; DEAE dextran alone or with aluminum 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.

The immunizing agent may comprise: carrier proteins such as thyroglobulin; albumins such as human serum albumin; toxins, toxoids or any mutant cross-reactive material (CRM) from the toxins of tetanus, diphtheria, pertussis, Pseudomonas (Pseudomonas), escherichia coli (e.coli), Staphylococcus (Staphylococcus), and Streptococcus (Streptococcus); polyamino acids such as poly (lysine: glutamic acid); influenza; rotavirus VP6, parvovirus VP1 and VP 2; hepatitis b virus core protein; hepatitis B virus recombinant vaccine, and the like. Alternatively, fragments or epitopes of the carrier protein or other immunogenic proteins may be used. For example, T cell epitopes of bacterial toxins, toxoids or CRM can 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 protocol is contemplated for generating the vaccine formulation. Exemplary protocols 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 administration, oral administration, rectal administration, parenteral administration, sublingual administration, buccal administration, intravenous administration, intra-articular administration, intramuscular administration, intradermal administration, subcutaneous administration, inhalation administration, intraocular administration, intraperitoneal administration, intracerebroventricular administration, topical administration, mucosal administration, and transdermal administration, although not limited thereto.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, lozenges, capsules, nasal sprays, suppositories, aerosols, dermal patches and the like. These dosage forms may also include injections or implants designed specifically for this purpose for the purpose of implanting a controlled release device or other form of implant modified to additionally function in that form. Controlled release may be achieved by coating hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose.

The compositions may be in discrete units, such as capsules, sachets, functional food/feed or tablets, each of which contains a predetermined amount of one or more therapeutic agents of the invention, in powder or granular capsules or as a solution or suspension in an aqueous liquid, non-aqueous liquid, oil-in-water emulsion or water-in-oil liquid emulsion. Such formulations may be prepared by any pharmaceutical method, but all methods include the steps of: one or more reagents as described above are combined with the carrier which constitutes one or more of the necessary ingredients. In general, the formulations are prepared by: the agents of the invention are mixed homogeneously and intimately with liquid carriers or finely divided solid carriers or both, and the product is then, if desired, shaped into the desired form.

The above formulations can be used in effective amounts in a manner compatible with the dosage form. In the context of the present invention, the dose administered to a patient should be sufficient to produce a beneficial response in the patient after an appropriate period of time. The amount of agent to be administered may depend on the subject to be treated, including its age, sex, weight and general health, depending on factors at the discretion of a physician.

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

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

In order 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 to the word

Group A Streptococci (GAS) primarily infect the Upper Respiratory Tract (URT) mucosa and skin in humans, causing numerous diseases. Infection can lead to toxic shock syndrome, necrotizing fasciitis and myositis. The incidence of necrotizing fasciitis is 10 ten-thousandth, and the mortality rate is as high as 70% (1). Streptococcus mutans, Rheumatic Fever (RF) and Rheumatic Heart Disease (RHD), are also of great interest. An estimated approximately 1560 million RHD present cases and almost 40 million deaths per year (2). The most common disease after bacterial colonization of the URT is pharyngitis, and RF and RHD are closely related to untreated primary pharyngeal infections (3). GAS infection and its associated diseases are prevalent among local populations in tropical regions, developing and developed countries, resulting in 50 million deaths each year (4), highlighting the urgent need for vaccines.

GAS vaccine candidates can be divided into M protein-based and non-M protein-based vaccines (5). The M protein on the cell surface (coiled coil protein consisting of 3 major domains) is the major virulence determinant (6). The protein consists of a hypervariable amino terminus and an a-repeat domain for epidemiological molecular typing (emm or M typing); b-repeat domain and conserved C-repeat domain (6). The major subunit vaccines in clinical studies are amino-terminal M protein based multivalent vaccines and conserved C-repeat M protein peptide vaccines (5). Based on their success in inducing systemic immunity, these GAS vaccine candidates have entered clinical trials (7). Systemic immunity has been shown to effectively prevent GAS dissemination to deep tissues and disease prevention by serum immunoglobulins (Ig) at systemic sites, but not to prevent colonization at mucosal sites and thus interpersonal transmission (8). Therefore, systemic vaccines are not the best way to induce immunity against GAS. In contrast, mucosal vaccines administered nasally against a variety of organisms effectively induce antigen-specific immune responses in the systemic and mucosal compartments (9-11). Due to such a double-layer protective immunity, mucosal vaccines are an ideal strategy to cope with systemic and mucosal GAS infections with the added benefit that the prevention of mucosal colonization will also inhibit transmission through droplets and aerosols from the UTR (7). Mucosal vaccines are also economically beneficial, which is an important consideration for the development of vaccines. The use of needles (7) is avoided due to the ease of nasal administration of the vaccine. Painless delivery contributes to greater patient compliance.

Based on the smallest B cell epitope from the conserved C3-repeat domain of the M protein, we previously identified a vaccine candidate peptide J8 (12). The J8 peptide (QAEDKVKQ)

KQLEDKVQ; SEQ ID NO:1) is a chimeric peptide containing 12 amino acids from the C-region (shown in bold type) flanked by GCN4 DNA-binding protein sequences to maintain the correct helical conformation (13). When linked to the carrier protein Diphtheria Toxoid (DT) and administered with alum, J8 induced IgG antibodies that protected mice against systemic and skin challenge by various GAS strains (4, 13). Moreover, vaccine candidates based on conserved regions of the M protein of GAS are effectively protected against GAS intranasal infection when administered with animal-restricted mucosal adjuvants CTB (14,15), or when administered as proteasomes (16). When mucosal immunity and reduced URT colonization were induced, a correlation with IgA production was determined. After this is clear, we aimed to develop human compatible mucosal vaccines based on J8.

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

Liposomes are spherical vesicles consisting of a biocompatible phospholipid bilayer, which can be loaded and deliver hydrophilic and hydrophobic molecules (19). Liposomes have been safely delivered to the human body by intranasal route (20, 21). However, liposomes presenting peptide antigens are not an ideal platform for inducing peptide-specific antibody responses. The peptides contain only a limited number of epitopes capable of activating helper T cells required for a B cell antibody response. They need to be conjugated to a "carrier" protein to immunize them in the outcrossing population, and so their presentation by liposomes is not very suitable. However, the enhanced immunogenicity conferred by particles such as liposomes is not surprising, since the natural pathogen is also a particle and is well recognized by the immune system (22). The natural tendency of liposomes to interact with antigen presenting cells has been the primary principle of using liposomes to present antigen to the immune system (23). The objective of this study was to develop a liposome formulation based on J8 (in the absence of adjuvant) in which the lipophilic J8 construct was incorporated into the lipid bilayer and the hydrophilic carrier protein (DT) was encapsulated in the inner aqueous core.

Materials and methods

A mouse. All Animal protocols used were approved by the University of Griffith for Animal Work-Based Research Ethics Review Board for Animal-Based Work, GU Ref No: GLY/09/14/AEC. The study was conducted strictly according to the guidelines for experimental animals of the australian National Health and Medical Research Council (NHMRC). The selected method minimizes pain and affliction to the mice, and animals are observed daily by trained animal caregivers. By using CO₂The mice were sacrificed by aspiration.

Human blood. Consented by written consent, blood was obtained from donors by a blood drawing technician at the university of griffis health center. The study was approved by the university of Greefield human research ethics Committee (GUHREC, Protocol # GLY/03/14/HREC). The samples were de-identified before the experimenter handled them.

J8-lipid-DT formulation. To facilitate non-covalent coordination of J8 to the lipid bilayer, a hydrophobic anchor consisting of two palmitic acids (C16) was added to the epsilon and primary amino groups of lysine of a tripeptide spacer (consisting of Lys Ser) present at the amino terminus of J8 (C16-C16-KSSJ 8). This construct was produced by Shanghai hayao biotech ltd (China peptides co., ltd., Shanghai, China). The predicted molecular weight of this construct (MW 4061.97g/mol) was verified by ESI-MS to give a product purity of greater than 95% (by analytical RP-HPLC area under curve analysis). Liposomes are prepared using membrane hydration (42). Lipids from Avanti Polar Lipids were used in a molar ratio of 7 dipalmitoyl-sn-glycero-3-phosphocholine (DPPC):2 cholesterol cholestol (chol):1L- α -Phosphatidylglycerol (PG). Chloroform is evaporated by a rotary evaporator(CHCl₃) Liposomes in solution were coated into round bottom flasks with a predetermined amount of C16-C16-KSSJ 8. The volumes used were: 0.7ml DPPC (10mg/ml) in CHCl₃In 0.2ml CHOL (5mg/ml) in CHCl₃And 0.1ml PG (10mg/ml) in CHCl₃In (1). The lipid film was then hydrated and dispersed by vigorous stirring in 1mL of phosphate buffer containing a predetermined amount of DT at room temperature. The resulting liposome suspension was centrifuged at 16,162g for 10min, the supernatant was removed, and the liposome pellets were resuspended in an appropriate amount of PBS to be administered to mice. To determine the encapsulation efficiency of DT, the supernatant was collected and the amount of unencapsulated DT in the supernatant was determined using a NanoDrop 2000 uv-vis spectrophotometer (Thermo Scientific, Massachusetts, United States). The DT concentration of the supernatant was subtracted from the initial DT concentration of PBS used to rehydrate the lipids to produce liposomes, allowing quantification of encapsulation efficiency. The mean particle size (nm) of the liposomes was measured at 25 ℃ using a nanometer particle sizer (Zetasizer Nano Series ZS, Malvern Instruments, United Kingdom) with disposable capillary cuvettes. Dimensions were analyzed with a non-invasive backscatter system and measured at a 173 ° scatter angle. The relevant time is based on 10 seconds per run, with at least 5 consecutive runs per measurement. The results are the average of triplicate independent measurements analyzed using the scatter technique software. A homogeneous size distribution as determined by a low polydispersity index (PDI) of 0.238 is shown for J8-Lipo-DT. PDI is an indication of how narrow the sample size distribution is, with values greater than 0.7 indicating that the sample has a broad size distribution.

Intranasal immunization of mice. With xylazine and ketamine (xylazine: ketamine: H)₂O ═ 1:1:10 mixture), and anesthetizing the b10.br and BALB/c mice to be immunized (Animal Resources Centre, Western Australia, Australia). Mice were administered 30 μ g of J8-Lipo-DT alone in a total volume of 20 μ L PBS (10 μ L/nostril), while control mice were administered 20 μ L PBS (10 μ L/nostril). Positive control mice received 30 μ g of J8 conjugated to DT, while 10 μ g of CTB (Sigma Aldrich, st. louis, United States) was administered in a total volume of 20 μ L PBS. In the same manner as the primary immunization, mice received two boosts at 21-day intervals. Other control groups received equal amounts of J8, DT alone as described aboveOr liposomes.

Serum, saliva and fecal samples were collected. After primary immunization, sera were collected at days 20, 40 and 60 to determine the level of J8-specific systemic antibodies. Blood from the mice was collected via the tail artery and allowed to clot at 37 ℃ for at least 30 min. Serum was collected after centrifugation at 1000g for 10min, heat inactivated at 56 ℃ for 10min and stored at-20 ℃.

Mice were administered 50 μ L of 0.1% pilocarpine solution to induce salivation. Saliva was collected into a microcentrifuge tube containing 2. mu.L of 50mmol/L phenylmethylsulfonyl fluoride (PMSF) protease inhibitor (Sigma Aldrich). The particulate material was separated by centrifugation at 13,000g for 10min and the samples were stored at-80 ℃.

6-10 parts of fresh fecal pellets were collected from a single mouse, frozen and lyophilized. The dry weight of the fecal solids was determined and then resuspended by vortexing in 5% skim milk powder, 50mmol/L EDTA (Sigma Aldrich), 0.1mg/mL soybean trypsin inhibitor (Sigma Aldrich) and 2mmol/L PMSF (20. mu.L/mg dry weight). The solid material was separated by centrifugation at 15,000g for 10min and the supernatant was stored at-80 ℃.

Antibody titers were identified by enzyme-linked immunosorbent assay (ELISA). J8-specific serum IgG and mucosal IgA were measured using ELISA as described elsewhere (43). The J8 peptide was diluted to 0.5mg/ml with carbonate coating buffer (pH 9.6) and coated on polycarbonate plates in a volume of 100. mu.l/well and incubated overnight at 4 ℃. Unbound peptides were removed and the wells were blocked with 150 μ l of 5% skim milk PBS-tween 20 for 2 hours at 37 ℃. The plates were then washed 3 times with PBS-tween 20 buffer. Samples were serially diluted in plates 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 serum), and 1:2 to 1:256 (for saliva/stool samples). Each sample was diluted to a final volume of 100. mu.l and incubated at 37 ℃ for 1.5 hours. The plates were washed 5 times and peroxidase-labeled goat anti-mouse IgG or IgA (Invivogen, San Diego, United States) was added at a dilution of 1:3000 or 1:1000, respectively, in 0.5% skim milk PBS-Tween and incubated at 37 ℃ for 1.5 hours. After washing, according to the manufacturer's instructionsThe book was added 100 μ l of OPD substrate (Sigma Aldrich) and incubated in the dark at room temperature for 30 minutes. In Victor³1420 multiple-marker counter (Perkin Elmer Life and Analytical Sciences, Shelton, United States) measures absorbance at 450 nm. Titers are described as being above the average absorbance obtained in negative control wells (containing normal mouse serum immunized with PBS)>The lowest dilution of absorbance at 3 Standard Deviation (SD). Statistical significance (p) was determined using one-way analysis of variance (ANOVA) and Tukey post hoc tests using GraphPad Prism 5 software (GraphPad, California, United States)<0.05)。

And (5) the process of the GAS attack. On day 63 after the primary immunization, the immunized and control mice were challenged intranasally with a predetermined dose of GAS strain M1. GAS strain M1 has been serially passaged in mouse spleens to increase virulence and acquired streptomycin resistance to differentiate GAS in pharyngeal swabs from normal murine bacterial flora (44). To determine the colonization of GAS, pharyngeal swabs were obtained from mice 1-3 days after challenge. The pharyngeal swabs were streaked onto Todd-Hewitt agar plates containing 2% defibrinated horse blood and incubated overnight at 37 ℃. Bacterial load in nasal drops was determined by pressing the nostrils of each mouse ten times (triplicate CBA plates/mouse/day) against the surface of a Columbian Blood Agar (CBA) plate, and expired particles were streaked. On day 3, mice were sorted, organ samples were homogenized in PBS, and samples were inoculated in triplicate using cast-plating. For nasal drops and pharyngeal swabs, results are expressed as mean Colony Forming Units (CFU) + Standard Error of Mean (SEM) for 10 mice/group on days 1, 2 and 3. For organ samples, results are expressed as mean CFU + SEM for 10 mice/group on day 3. The test group was compared to the PBS control group using GraphPad Prism 5, using nonparametric, unpaired Mann-Whitney U-test for variability (p <0.05 was considered significant).

And (4) preparing and maturing the DCs. Peripheral blood was collected from healthy volunteers and fractionated by standard procedures based on Ficoll-Paque (Amersham Pharmacia, Uppsala, Sweden). PBMC were harvested by centrifugation based on Ficoll Paque, washed and buffered at MACS per 0.35mL(Miltenyi Biotec S.L.，Germany)1x 10⁸The final density of individual cells was resuspended. DCs were isolated using a Pan DC isolation kit (Miltenyi Biotec) according to the manufacturer's instructions. The resulting DC population was resuspended in RPMI 1640(Gibco, Gaithersburg, United States) complete medium (with 2mM l-glutamine, 1% non-essential amino acids, 1% Pen-strep, 10mM HEPES) supplemented with 10% FCS (Gibco). Inoculate a Total volume of 0.2mL of DCs (2X 10⁶) And the following stimuli were added as indicated: mu.g/mL pIC (Invivogen, San Diego, United States), J8-Lipo-DT (150. mu.g/mL) or complete medium only, incubated for 24 hours. After 24 hours the supernatant was collected and stored at-20 ℃.

Immunophenotypic analysis was performed by flow cytometry. To analyze the surface expression of various markers, the treated DCs were stained with one or more fluorophore-labeled mabs described below and analyzed by flow cytometry using LSR fortesta cytometers (Becton Dickinson, California, United States) and FlowJo software (Treestar, inc., California, United States). The resulting populations were evaluated by flow cytometry analysis using the following antibodies (Becton Dickinson): anti-HLA-DR-V450, -CD1c-APC, -CD80-PE-Cy7, -CD83-PE-TexasRed, -CD86-PE, -CD123-Percp-5.5, -CD141-APC-Cy 7. After dark staining with the appropriate antibody at 4 ℃ for 30min, the cells were washed twice with PBS and fixed with 1% paraformaldehyde. Gating was performed based on large granular cells and 2000-. Briefly, HLA-DR positive cells were gated to define human DCs, and further subdivided into CD141+ conventional DC type 1, CD1c + DC conventional (myeloid) DC type 2, and CD123+ plasmacytoid DCs according to previously established methods (45). Mean Fluorescence Intensity (MFI) values were determined based on gated populations. The data are reported as mean + SEM and the variability analyzed using student's t-test with GraphPad Prism 5 software (GraphPad, California, United States). P values less than 0.05 were considered significant.

Splenocytes were stimulated in vitro with antigen. Quantification of pro-inflammatory responses produced by splenocytes stimulated with J8 peptide using flow microsphere array (CBA) analysis and flow cytometry analysisAnd (6) answering. Briefly, single cell suspensions of spleens from J8-Lipo-DT immunized mice without erythrocytes were prepared in RPMI 1640 medium. Splenocytes were seeded in a total volume of 0.1mL (4X 10)⁵) And the following stimuli were added as indicated: 2 μ g/mL LPS (Sigma Aldrich), J8(10 μ g/mL) or RPMI 1640 medium only, incubated for 72 hours. After 72 hours the supernatant was separated and stored at-80 ℃ for CBA flow cytometry analysis.

Secreted chemokines and cytokines were quantified by CBA. The level of accumulated inflammatory cytokines was quantified according to the manufacturer's instructions. For the mouse inflammation kit CBA, the volume of the sample and standard was reduced to 10 μ Ι, and 2 μ Ι of each capture bead was used. Supernatants from human DC wells were used with the human inflammation kit cba (becton dickinson) according to the manufacturer's recommendations. Samples were run on an LSR Fortessa cytometer and data analyzed using FCAP array (v1.01for Windows) software (Becton Dickinson). The data are reported as mean + Standard Error of Mean (SEM) and differences were analyzed using student's t-test with GraphPad Prism 5 software. P values less than 0.05 were considered significant.

Results

Liposomes with surface-associated J8 peptide and containing diphtheria toxoid were constructed as described in materials and methods (figure 1). The administered formulation contained 30 μ g J8 attached to the surface of the liposomes using a palmitic acid based moiety per dose. Liposomes contained 30 μ g DT per dose inside. The mean diameter of the liposomes as measured by dynamic light scattering was 1.8 μm (standard deviation 100.3nm) (see materials and methods).

BALB/c mice were immunized nasally with J8-Lipo-DT and various controls using primary and 2 boost protocols (10 per group): liposomes alone (Lipo); liposomes encapsulating DT (Lipo-DT); j8 DT embedded in the liposome surface but without encapsulation (J8-Lipo); J8-DT + CTB; PBS + CTB; and a PBS.

To evaluate the efficacy of J8-Lipo-DT in comparison to other constructs, the nasally immunized mice were then challenged intranasally with the pharyngeal isolate M1GAS strain obtained from scarlet fever patients (16). Before challenge, we observed that J8-Lipo-DT induced higher J8-specific IgA (stool and saliva) and serum IgG titers compared to J8-Lipo. Although the difference between J8-Lipo-DT and J8-Lipo was not statistically significant for any of the groups, J8-Lipo-DT was observed to be superior for salivary IgA response, fecal IgA response and serum IgG response (FIGS. 2A-C). After challenge with GAS, the bacterial burden in the nasal discharge was significantly lower in J8-Lipo-DT immunized mice compared to the PBS group and comparable to J8-DT + CTB immunized mice (day 3, fig. 3A).

However, unexpectedly, J8-DT + CTB immunized mice did not protect against colonization of the throat or NALT, whereas J8-Lipo-DT immunized mice showed significant protection against colonization in both compartments (fig. 3B and C). The protection of J8-Lipo-DT is significantly better than that induced by J8-Lipo. Murine NALT is an entry portal for persistent GAS infection (24) and is a functional homolog of the human tonsil (25). Thus, these results underscore the efficacy of J8-Lipo-DT in reducing bioburden in the preferred site of mucosal GAS infection.

We then asked whether J8-Lipo-DT similarly protected mice of different strains. J8-DT/CTB and PBS served as control immunogens. Immunization of b10.br mice with J8-Lipo-DT (n ═ 5) induced significant J8-specific antibody titers (fig. 4). Mucosal antibody titers in saliva and stool samples were comparable to those in J8-DT + CTB immunized mice (FIGS. 4A-B). To test whether J8-Lipo-DT will protect against GAS infection in b10.br mice, another population of mice (n ═ 5) was immunized and challenged with GAS M1 strain. Nasal drops and pharyngeal swabs were monitored over a 3 day observation period. By day 2, J8-Lipo-DT and J8-DT + CTB immunized mice had undetectable bioburden in nasal drops (FIG. 5A). Similar to BALB/c mice, the data also demonstrated that by day 2 post challenge, pharyngeal swabs from J8-Lipo-DT immunized mice were free of bacteria, while J8-DT + CTB immunized mice still had detectable levels of GAS in the pharyngeal swabs at day 3 (fig. 5B).

Previous studies demonstrated that although the peptide encapsulated within liposomes did not induce an immunoglobulin response, liposomes plus lipid a (a component of lipopolysaccharide) induced an antibody response following intraperitoneal immunization (26), thus suggesting that the lipid tail of lipid a may act as an adjuvant. To answer whether the double C16 lipid tail anchoring J8 to the liposome surface was responsible for induction of antibody responses, another mouse population was 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, while C16-C16-KSSJ8 were not (FIGS. 6A-B).

Cytokine responses from splenocytes from nasally immunized mice were measured to determine whether the nasal immunization induced a systemic cellular immune response that could explain the self-adjuvanticity and antibody isotype to IgA conversion of J8-Lipo-DT. Proinflammatory cytokines (interferon-gamma, IFN-gamma, interleukin 1 IL-1, IL-6, IL-12p70, monocyte chemotactic protein 1 MCP-1, and tumor necrosis factor alpha TNF-alpha) were analyzed. J8-Lipo-DT immunized B10.BR mice were sacrificed and splenocytes stimulated with J8, LPS or vehicle. We observed significant IFN-. gamma., MCP-1 and IL-6 production in response to J8 and LPS (FIG. 7). No other cytokines evaluated were detected. This result demonstrates that immunization with J8-Lipo-DT induces a pro-inflammatory response, providing a potential mechanism for self-adjuvanticity of J8-Lipo-DT. In particular, IL-6 is known to be responsible for the conversion of antibody responses to IgA (27). Furthermore, the chemoattractant MCP-1 is known to play a major role in GAS defense mechanisms (28).

To evaluate the potential of J8-Lipo-DT to induce an effective immune response with self-adjuvant activity in humans, a subset of dendritic cells was isolated from 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 promote antigen recognition and cell-cell interactions. To characterize human DC maturation, various cell surface molecules were examined for modulation in response to J8-Lipo-DT by flow cytometry (fig. 8A-C). Synthetic double-stranded RNA adjuvant, poly-inosine cytosine nucleotides (pIC) was used as a control (29). The levels of co-stimulatory molecules CD80, CD83 and CD86 on CD123+ plasma-like cell dc (pdc) cultured with J8-Lipo-DT were significantly higher (fig. 8A). Expression of CD80 was also increased in two classical DC subsets (cdcs), CD141+ classical 1-type DC and CD1C + classical 2-type DC (fig. 8B-C). Furthermore, CD86 expression of CD141+ DC was also increased (fig. 8B).

To further clarify the interaction with human DCs, the level of proinflammatory cytokines after stimulation was assessed using a flow microbead array. We observed increased expression of pro-inflammatory cytokines (TNF- α, IL-6 and IL-1 β (IL-1 β)) and neutrophil chemoattractant IL-8 (figure 9). Neutrophils are known to be critical for IgA control of GAS infection (30). Elevated levels of the anti-inflammatory cytokine IL-10 were also observed (FIG. 9). This may be due to the regulatory role of IL-10 in the DC maturation step and in balancing the pro-inflammatory response of the host (31, 32). However, in particular, the IL-6 response suggests that J8-lipo-DT will cause IgA conversion in humans, and that neutrophil responses (via IL-8) will give good host control of GAS infection. As shown in FIG. 10, the SpyCEP peptide displayed by liposomes (S2; SEQ ID NO:2) together with the intravesicular DT elicited mucosal IgA responses when displayed alone and as the S2-J8 chimeric (SEQ ID NO: 3).

Referring to FIG. 11, J8+ S2-Lipo-DT induced an antigen-specific IgA, IgG response. Comparable immune responses to J8-Lipo-DT and S2-Lipo-DT were observed. Different formulation strategies employ either (i) two epitopes in the liposome (J8+ S2-Lipo-DT) or (ii) a mixture 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 was of interest to see if J8-Lipo-DT could protect against skin infection after 88/30 strain challenge. Initial experiments with nasal administration of liposomes containing DT and J8 alone showed significant IgG titers after nasal immunization with J8-Lipo-DT, but no protection in the skin challenge assay (data not shown). Enhancement of systemic IgG responses by different liposomal formulations or by immunization with subcutaneous administration of J8-DT + Alum is ongoing.

Discussion of the related Art

We have developed mucosal active subunit liposome vaccine candidates for Group A Streptococci (GAS). The immunostimulatory properties of liposomes are combined with the encapsulation of the protein carrier DT and the display of GAS-specific B-cell epitopes on the liposome surface. Both peptides and carrier proteins are required for optimal immunity. The mucosal immunity induced by the complex liposomes was superior to that induced by the peptide-protein conjugate administered with CTB.

Mucosal immunity has attracted much attention as a means of eliciting protective immunity against infectious diseases. The vast majority of infections occur or originate at mucosal surfaces. Therefore, the use of vaccines that induce mucosal protective immune responses is desirable and has utility. In practice, it has often proven difficult to stimulate a strong mucosal IgA immune response, and the process of mucosal vaccination efforts with subunit peptide antigens is not ideal. This is due in part to the difficulty in stimulating a strong immune response comparable to conventional whole-organism based methods. The addition of adjuvants and conjugation of subunit antigens to protein carriers have proven effective for systemic immunity as a source of T cell help. However, new strategies are needed to induce mucosal immunity.

The "topographic" location of the liposome-associated antigen affects antigen processing and presentation to B cells and helper T cells (33). It has been shown that antigens exposed on the liposome surface are preferentially processed and presented by B cells, while liposome-encapsulated antigens are more efficiently processed and presented to T cells by antigen presenting cells (34). Thus, the vaccine candidate J8-Lipo-DT represents a reasonable subunit liposome vaccine design, which ensures that B cell epitopes are associated with the liposome bilayer to be exposed to bind the Ig receptor of B cells, while encapsulation of DT allows for efficient delivery, processing and presentation to T cells.

We demonstrated that GAS is cleared in URT tissues including NALT. The potent nasopharyngeal immunity provided by vaccine candidates has shown the ideal potential to reduce RF and RHD, which are associated with primary pharyngeal infections (7). In human URTs, the tonsils are the primary reservoir of GAS, maintaining endemic disease worldwide (25). Reduction of GAS colonization in the functional analog of human tonsils NALT suggests that intranasal immunization with J8-Lipo-DT reduces colonization and infection of human tonsils, thereby reducing GAS spread (25).

Although liposomes have previously been reported for the delivery of encapsulated peptides to induce cellular immune responses, such liposomes do not induce IgA or IgG responses unless a strong adjuvant, such as LipidA (26, 35), is present. It is likely that the lipid tail on J8 provides adjuvant activity and therefore contributes to the immunogenicity of J8-Lipo-DT; however, the J8 peptide, which has its own lipid tail, is not immunogenic, indicating the need for a liposome formulation. The immunostimulatory activity of liposomal self-adjuvants has been previously reported and demonstrated to be due to interaction with antigen presenting cells and induction of pro-inflammatory responses (36). In vitro assays in our studies revealed induction of antigen-specific inflammatory chemokines and cytokines in immunized mice. Of particular note 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 suggested to be involved in mediating mucosal inflammation, which has been reported as a strong mucosal adjuvant due to the ability to significantly increase mucosal IgA secretion (37).

While antigens can be presented to the immune system by many cell types, the priming of natural T cells requires maturation, antigen presentation, and engagement of costimulatory molecules found only on professional antigen presenting cells (e.g., DCs) (38). DCs are key elements that bridge 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 strong antigen-presenting cells of natural T lymphocytes to initiate an adaptive immune response. We demonstrate that, following in vitro exposure, J8-Lipo-DT mediates cell surface activation and expression of maturation markers on human DCs, inducing pro-inflammatory cytokines including IL-6 and IL-8. Human and murine IL-6 play a key role in B cell terminal differentiation and at mucosal sites, it stimulates IgA proliferation and secretion in mucosal sites (27). IgA-specific immunity against GAS requires the presence of neutrophils (30). IL-8 plays a key role in neutrophil mobilization and activation. In this regard, administration of SpyCEP S2 peptide (SEQ ID NO:2) displayed by the liposomal particle delivery system alone or in combination with J8 peptide resulted in peptide-specific IgA.

Thus, the underlying mechanism that confers immunity to GAS infection in humans can be mediated using a liposome platform, providing support for the relevance of basic research to clinical applications.

We demonstrate that human pDC increases both maturation and co-stimulatory markers when stimulated with J8-Lipo-DT. Human pdcs readily phagocytose and process antigens encapsulated in a particle delivery system (40), indicating that the particle delivery system can be used to facilitate efficient delivery of antigens to pdcs. Our results are believed to demonstrate for the first time the ability to stimulate human pDC with a liposome-based particle delivery system. The human pDC originally identified in the blood has subsequently been detected in the spleen, lymph nodes, and mucosal sites including the tonsils (41). Thus, liposome-based vaccine delivery has the potential to be developed to target this subset of DCs for the desired mucosal immune response in humans.

Example 2

The following experiments were performed to investigate the effect of liposome size on immunogenicity.

Liposome extrusion was accomplished using a thermal block using a 1mL syringe mini extruder (Avanti Polar Lipids). The rehydrated solution was passed 11 times through a 50nm, 400nm, 1000mm filter (Avanti Polar Lipids) while the thermal block was set to-40 ℃. Liposome size measurements were performed by a Nanosizer (dynamic light scattering or DLS).

As shown in fig. 12, J8-Lipo-DT can be extruded to form nano-or micro-sized particles. The majority of the particle size has a narrow molecular weight distribution (low polydispersity index < 0.3). The data in FIG. 13 show that J8-Lipo-DT size does not affect systemic IgG responses. However, as shown in figure 14, the larger size liposomes induced a J8 specific mucosal response.

Example 3

The following experiments were performed to investigate the effect of freeze-drying of liposomes on immunogenicity. The liposome films were rehydrated with milliQ water containing 10% trehalose and then lyophilized. 1, 4 and 7 weeks after lyophilization, J8-Lipo-DT powder was reconstituted with PBS.

Figure 15 shows the size results of liposome size measurements by Nanosizer (dynamic light scattering or DLS). The majority of the particle size has a narrow molecular weight distribution (low polydispersity index < 0.3). Figure 16 shows that reconstituted lyophilized J8-Lipo-DT liposomes induced a J8 specific systemic response without the need for additional adjuvant. This is an immune response comparable to freshly prepared J8-Lipo-DT. Trehalose is important for the immunogenicity of freeze-dried J8-Lipo-DT. Figure 17 shows that reconstituted lyophilized J8-Lipo-DT liposomes induced a J8 specific mucosal response.

Example 4

Further experiments confirmed that glycolipid activators containing innate immunity, such as trehalose 6,6' -dibehenate (TDB) and 3D-

The efficacy of the immunogenic liposomes, such as shown schematically in figure 18. TDB is formulated with liposomes based on the percentage of total phospholipids in the liposomes. 9mg of phospholipid were used, and the proportion of TDB used was 20% of this amount (1.8 mg). Efficacy was measured by post-immunization antibody titers (IgA and IgG antibodies) and skin challenge experiments with GAS strains. The data are shown in figure 19. Mice were immunized intranasally with 30ug of J8-Lipo-Dt + TDB (n-5/group). Mice were administered for the first time (day 0) plus two boosts (days 21 and 42). In both saliva and stool samples, addition of TDB resulted in significantly higher mucosal IgA responses compared to J8-Lipo-DT. The results are from J8-Lipo-DT + TDB in frozen powder form, which improves the stability of the liposomes.

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

In a round bottom flask, sorbitan tristearate (150mmol) in a 7:3:1 molar ratio, cholesterol and dicetyl phosphate (DCP) and 150g of J8 partially modified with palmitic acid were dissolved in 10mL of chloroform. The solvent was removed by rotary evaporator to form a thin film on the glass surface of the round bottom flask. Then, the membrane was hydrated with 3.5mL PBS (pH 7.4) containing 100mg sodium deoxycholate (bile salts) and 150g diphtheria toxoid.

Example 5

Immunogenicity of lipid vesicles against influenza a, influenza b and group a streptococci was measured by antigen-specific salivary IgA titration. Figure 21 shows a schematic depiction of an immunogenic agent comprising a single lipid vesicle with respective immunogens from each influenza a virus, influenza b virus and group a streptococcus. The results shown in figure 22 show that lipid vesicles comprising respective immunogens from each influenza a virus, influenza b virus and group a streptococcus (as shown in figure 21A) induced immunity against each of these pathogens in mice. Further work will investigate the immunogenicity of immunogenic agents including glycolipid-based adjuvants such as shown schematically in figure 21B.

In summary, this study reports for the first time liposome-based mucosal active GAS vaccine candidates. Our discovery is an important step towards overcoming the current challenges in developing GAS vaccines for pre-adhesion to membranous sites of infection and community transmission. This study provides an important mechanistic hint as to how liposomal particle delivery systems can globally induce the desired mucosal immune response to combat GAS infection. The strategy reported here is relevant for the development of subunit mucosal vaccines against other pathogenic organisms. Non-limiting examples include influenza virus, rhinovirus, and hookworm, as described above. In some embodiments, a single lipid vesicle can include immunogens against multiple different pathogens.

In the present 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 by reference in its entirety.

Reference to the literature

1.Baker A(2012)Group A streptococcal infections in primary care:a case report.The British journal of general practice:the journal of the Royal College of General Practitioners 62(600):388-389.

2.Zuhlke LJ&Steer AC(2013)Estimates of the global burden of rheumatic heart disease.Global heart 8(3):189-195.

3.Nkomo VT(2007)Epidemiology and prevention of valvular heart diseases and infective endocarditis in Africa.Heart(British Cardiac Society)93(12):1510-1519.

4.Pandey M,et al.(2015)A synthetic M protein peptide synergizes with a CXC chemokine protease to induce vaccine-mediated protection against virulent streptococcal pyoderma and bacteremia.Journal of immunology(Baltimore,Md.:1950)194(12):5915-5925.

5.Steer AC,Dale JB,&Carapetis JR(2013)Progress toward a global group a streptococcal vaccine.The Pediatric infectious disease journal 32(2):180-182.

6.Metzgar D&Zampolli A(2011)The M protein of group A Streptococcus is a key virulence factor and a clinically relevant strain identification marker.Virulence2(5):402-412.

7.Georgousakis MM,McMillan DJ,Batzloff MR,&Sriprakash KS(2009)Moving forward:a mucosal vaccine against group A streptococcus.Expert review of vaccines 8(6):747-760.

8.Gamba MA,et al.(1997)Familial transmission of a serious disease--producing group A streptococcus clone:case reports and review.Clinical infectious diseases:an official publication of the Infectious Diseases Society of America 24(6):1118-1121.

9.Langermann S,Palaszynski S,Sadziene A,Stover CK,&Koenig S(1994)Systemic and mucosal immunity induced by BCG vector expressing outer-surface protein A of Borrelia burgdorferi.Nature 372(6506):552-555.

10.Wu HY&Russell MW(1998)Induction of mucosal and systemic immune responses by intranasal immunization using recombinant cholera toxin B subunit as an adjuvant.Vaccine 16(2-3):286-292.

11.Baldridge JR,Yorgensen Y,Ward JR,&Ulrich JT(2000)Monophosphoryl lipid A enhances mucosal and systemic immunity to vaccine antigens following intranasal administration.Vaccine 18(22):2416-2425.

12.Hayman WA,et al.(1997)Mapping the minimal murine T cell and B cell epitopes within a peptide vaccine candidate from the conserved region of the M protein of group A streptococcus.International immunology 9(11):1723-1733.

13.Batzloff MR,et al.(2003)Protection against group A streptococcus by immunization with J8-diphtheria toxoid:contribution of J8-and diphtheria toxoid-specific antibodies to protection.The Journal of infectious diseases 187(10):1598-1608.

14.Bessen D&Fischetti VA(1990)Synthetic peptide vaccine against mucosal colonization by group A streptococci.I.Protection against a heterologous M serotype with shared C repeat region epitopes.Journal of immunology(Baltimore,Md.:1950)145(4):1251-1256.

15.Bessen D&Fischetti VA(1988)Influence of intranasal immunization with synthetic peptides corresponding to conserved epitopes of M protein on mucosal colonization by group A streptococci.Infection and immunity 56(10):2666-2672.

16.Batzloff MR,et al.(2005)Toward the development of an antidisease,transmission-blocking intranasal vaccine for group a streptococcus.The Journal of infectious diseases 192(8):1450-1455.

17.Zeng L,et al.(2015)Compound 48/80 acts as a potent mucosal adjuvant for vaccination against Streptococcus pneumoniae infection in young mice.Vaccine 33(8):1008-1016.

18.Zaman M,Chandrudu S,&Toth I(2013)Strategies for intranasal delivery of vaccines.Drug delivery and translational research 3(1):100-109.

19.Giddam AK,Zaman M,Skwarczynski M,&Toth I(2012)Liposome-based delivery system for vaccine candidates:constructing an effective formulation.Nanomedicine(London,England)7(12):1877-1893.

20.Childers NK,et al.(1999)A controlled clinical study of the effect of nasal immunization with a Streptococcus mutans antigen alone or incorporated into liposomes on induction of immune responses.Infection and immunity 67(2):618-623.

21.Childers NK,Tong G,&Michalek SM(1997)Nasal immunization of humans with dehydrated liposomes containing Streptococcus mutans antigen.Oral microbiology and immunology 12(6):329-335.

22.Zaman M,Good MF,&Toth I(2013)Nanovaccines and their mode of action.Methods(San Diego,Calif.)60(3):226-231.

23.Alving CR(1991)Liposomes as carriers of antigens and adjuvants.Journal of immunological methods 140(1):1-13.

24.Cleary PP,Zhang Y,&Park HS(2004)Nasal associated lymphoid tissue&M cells,a window to persistent streptococcal infections.The Indian journal of medical research 119 Suppl:57-60.

25.Park HS&Cleary PP(2005)Active and passive intranasal immunizations with streptococcal surface protein C5a peptidase prevent infection of murine nasal mucosa-associated lymphoid tissue,a functional homologue of human tonsils.Infection and immunity 73(12):7878-7886.

26.White WI,et al.(1995)Antibody and cytotoxic T-lymphocyte responses to a single liposome-associated peptide antigen.Vaccine 13(12):1111-1122.

27.Beagley KW,et al.(1989)Interleukins and IgA synthesis.Human and murine interleukin 6 induce high rate IgA secretion in IgA-committed B cells.The Journal of experimental medicine 169(6):2133-2148.

28.Loof TG,Goldmann O,Gessner A,Herwald H,&Medina E(2010)Aberrant inflammatory response to Streptococcus pyogenes in mice lacking myeloid differentiation factor 88.The American journal of pathology 176(2):754-763.

29.Verdijk RM,et al.(1999)Polyriboinosinic polyribocytidylic acid(poly(I:C))induces stable maturation of functionally active human dendritic cells.Journal of immunology(Baltimore,Md.:1950)163(1):57-61.

30.Brandt ER,et al.(1999)Functional analysis of IgA antibodies specific for a conserved epitope within the M protein of group A streptococci from Australian Aboriginal endemic communities.International immunology 11(4):569-576.

31.Lyke KE,et al.(2004)Serum levels of the proinflammatory cytokines interleukin-1 beta(IL-1beta),IL-6,IL-8,IL-10,tumor necrosis factor alpha,and IL-12(p70)in Malian children with severe Plasmodium falciparum malaria and matched uncomplicated malaria or healthy controls.Infection and immunity72(10):5630-5637.

32.Samarasinghe R,et al.(2006)Induction of an anti-inflammatory cytokine,IL-10,in dendritic cells after toll-like receptor signaling.Journal of interferon&cytokine research:the official journal of the International Society for Interferon and Cytokine Research 26(12):893-900.

33.Dal Monte P&Szoka FC,Jr.(1989)Effect of liposome encapsulation on antigen presentation in vitro.Comparison of presentation by peritoneal macrophages and B cell tumors.Journal of immunology(Baltimore,Md.:1950)142(5):1437-1443.

34.Harding CV,Collins DS,Slot JW,Geuze HJ,&Unanue ER(1991)Liposome-encapsulated antigens are processed in lysosomes,recycled,and presented to T cells.Cell 64(2):393-401.

35.Ninomiya A,Ogasawara K,Kajino K,Takada A,&Kida H(2002)Intranasal administration of a synthetic peptide vaccine encapsulated in liposome together with an anti-CD40 antibody induces protective immunity against influenza A virus in mice.Vaccine 20(25-26):3123-3129.

36.Schwendener RA(2014)Liposomes as vaccine delivery systems:a review of the recent advances.Therapeutic advances in vaccines 2(6):159-182.

37.Stevceva L&Ferrari MG(2005)Mucosal adjuvants.Current pharmaceutical design 11(6):801-811.

38.Gamvrellis A,et al.(2004)Vaccines that facilitate antigen entry into dendritic cells.Immunology and cell biology 82(5):506-516.

39.Klechevsky E(2015)Functional Diversity of Human Dendritic Cells.Advances in experimental medicine and biology 850:43-54.

40.Tel J,et al.(2010)Human plasmacytoid dendritic cells phagocytose,process,and present exogenous particulate antigen.Journal of immunology(Baltimore,Md.:1950)184(8):4276-4283.

41.Dutertre CA,Wang LF,&Ginhoux F(2014)Aligning bona fide dendritic cell populations across species.Cellular immunology 291(1-2):3-10.

42.Szoka F,Jr.&Papahadjopoulos D(1980)Comparative properties and methods of preparation of lipid vesicles(liposomes).Annual review of biophysics and bioengineering 9:467-508.

43.Zaman M,et al.(2014)Group A Streptococcal vaccine candidate:contribution of epitope to size,antigen presenting cell interaction and immunogenicity.Nanomedicine(London,England)9(17):2613-2624.

44.Olive C,Clair T,Yarwood P,&Good MF(2002)Protection of mice from group A streptococcal infection by intranasal immunisation with a peptide vaccine that contains a conserved M protein B cell epitope and lacks a T cell autoepitope.Vaccine 20(21-22):2816-2825.

45.Kassianos AJ,Jongbloed SL,Hart DN,&Radford KJ(2010)Isolation of human blood DC subtypes.Methods in molecular biology(Clifton,N.J.)595:45-54.

Claims (24)

1. An immunogenic agent suitable for intranasal administration to a mammal, the immunogenic agent comprising one or more immunogenic proteins or fragments thereof, a lipid vesicle and a carrier protein, wherein the carrier protein is located within the lipid vesicle space and the immunogenic protein or fragment thereof is displayed on the surface of the lipid vesicle, wherein the immunogenic agent is capable of eliciting a mucosal immune response in a mammal, and wherein the carrier protein is a diphtheria toxoid, wherein the one or more immunogenic proteins are selected from the group a streptococcal bacterial M protein or fragment thereof, an immunogenic protein of influenza a virus or fragment thereof, and an immunogenic protein of influenza b virus or fragment thereof.

2. The immunogenic agent of claim 1, wherein the lipid vesicle is a liposome.

3. The immunogenic agent of claim 1 or 2 comprising one or more immunogenic proteins or fragments thereof of or from the same pathogen.

4. The immunogenic agent of claim 1 or 2 comprising one or more immunogenic proteins or fragments thereof of or from each of a plurality of different pathogens.

5. The immunogenic agent of claim 1 or 2, wherein the fragment of the group a streptococcal bacterial M protein is located within the J8 peptide or comprises the J8 peptide.

6. The immunogenic agent of claim 5, wherein the J8 peptide comprises or consists of the amino acid sequence:

amino acid sequence QAEDKVKQSREAKKQVEKALKQLEDKVQ (SEQ ID NO: 1).

7. The immunogenic agent of any one of claims 1-2 and 6, wherein the immunogenic protein or fragment thereof comprises a SpyCEP fragment and an M protein fragment as a single chimeric peptide or a mixture comprising a SpyCEP fragment and an M protein fragment, wherein the SpyCEP fragment promotes neutrophil activity recovery or enhancement.

8. The immunogenic agent of claim 7, wherein the SpyCEP fragment comprises or consists of the amino acid sequence of SEQ ID NO: amino acid sequence NSDNIKENQFEDFDEDWENF (SEQ ID NO: 2); or

Wherein the chimeric peptide comprises or consists of the amino acid sequence:

amino acid sequence NSDNIKENQFEDFDEDWENFQAEDKVKQSREAKKQVEKALKQLEDKVQ (SEQ ID NO: 3).

9. The immunogenic agent of claim 1, wherein the immunogenic protein or fragment thereof comprises or consists of the amino acid sequence of seq id no: amino acid sequence MSLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO:4) or amino acid sequence PAKLLKERGFFGAIAGFLE (SEQ ID NO: 5).

10. The immunogenic agent of any one of claims 1-2, 6, 8 and 9, further comprising an activator of innate immunity.

11. The immunogenic agent of claim 10, wherein the activator of innate immunity is or comprises a glycolipid.

12. The immunogenic agent of claim 10, wherein the activator of innate immunity is trehalose-6, 6' -dibehenate (TDB) or lipid a glycolipid adjuvant.

13. The immunogenic agent of any one of claims 1-2, 6, 8, 9, 11 and 12, further comprising a bile salt.

14. The immunogenic agent of claim 13, wherein the bile salt is sodium deoxycholate.

15. The immunogenic agent of any one of claims 1-2, 6, 8, 9, 11, 12 and 14 which is freeze-dried or lyophilized.

16. The immunogenic agent of any one of claims 1-2, 6, 8, 9, 11, 12 and 14 produced in a selected size or a selected size range.

17. A composition comprising the immunogenic agent of any one of claims 1-16.

18. The composition of claim 17, comprising a single immunogenic agent comprising one or more immunogenic proteins or fragments thereof of or from the same pathogen.

19. The composition of claim 17, comprising a single immunogenic agent comprising one or more immunogenic proteins or fragments thereof of or from each of a plurality of different pathogens.

20. The composition of claim 17, comprising a plurality of different immunogenic agents each comprising one or more immunogenic proteins or fragments thereof of or from a different pathogen.

21. Use of the immunogenic agent of any one of claims 1-16 or the composition of any one of claims 17-20 in the manufacture of a formulation for eliciting a mucosal immune response in a mammal against one or more pathogens, wherein the immunogenic agent or composition is prepared for intranasal administration to the mammal and the pathogen is selected from the group consisting of group a streptococcal bacteria, influenza a virus and influenza b virus.

22. Use of the immunogenic agent of any one of claims 1-16 or the composition of any one of claims 17-20 in the preparation of a formulation for immunizing a mammal against one or more pathogens, wherein the immunogenic agent or composition is prepared for intranasal administration to the mammal and the immunogenic agent elicits a mucosal immune response, and the pathogen is selected from the group consisting of group a streptococcal bacteria, influenza a virus, and influenza b virus.

23. Use of the immunogenic agent of any one of claims 1-16 or the composition of any one of claims 17-20 in the manufacture of a medicament for the treatment or prevention of an infection in a mammal caused by one or more pathogens, wherein the immunogenic agent or composition is prepared for intranasal administration to the mammal and the immunogenic agent elicits a mucosal immune response and the pathogen is selected from group a streptococcal bacteria, influenza a virus and influenza b virus.

24. The use of any one of claims 21-23, wherein the mammal is a human.

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