TW201726164A - Liposomal vaccine - Google Patents

Abstract

An immunogenic agent suitable for preventing, treating or immunizing against one or a plurality of different pathogen comprises an immunogenic agent which comprises one or a plurality of pathogen-derived proteins, fragments, variants or derivatives thereof displayed on a lipid vesicle and a carrier protein such as diptheria toxoid located in an intravesicular space. The immunogenic agent may be suitable for intranasal administration and may be capable of eliciting a mucosal immune response. The immunogenic agent may further comprise an activator of innate immunity such as trehalose-6,6'-dibehenate and/or a bile salt such as sodium deoxycholate. The one or plurality of pathogens may be group A streptococcus, viruses or hookworms.

Description

Liposomal vaccine

The present invention relates to the prevention and treatment of infectious diseases. More specifically, the present invention relates to a liposome vaccine for treating or preventing infectious diseases and symptoms by inducing a mucosal immune response.

Systemic immunity has been shown to effectively prevent disease caused by a variety of different pathogens by serum immunoglobulin (Ig) in the whole body, but it cannot prevent colonization of the mucosa and thus prevent human infection. Therefore, for some diseases, systemic vaccination is not the best way to induce immunity. Conversely, mucosal vaccines against various organisms by nasal administration can effectively induce antigen-specific immune responses in both systemic and mucosal compartments. Because of this two-layer protective immunity, mucosal vaccines are ideal for combating both systemic and mucosal infections, and their additional benefits of preventing mucosal colonization will also inhibit the transmission of droplets and aerosols from the upper respiratory tract. Mucosal vaccination is also economically advantageous and is an important consideration for vaccine development. Since it is easier to administer the vaccine by the nasal route, the use of the needle can be avoided. Because of its lack of pain, patients will have greater compliance when delivered.

It is an object of the present invention to provide an immunogenic agent and a delivery system that induces a mucosal immune response to a pathogen. In a broad form, the invention relates to assisting or inducing a pathogen by delivering an immunogenic protein, fragment or variant in a manner that further comprises a lipid vesicle of a carrier protein such as diptheria toxoid (DT). Mucosal immunity. Suitably, the carrier protein is located in the intravesicular space. In a specific form, a single immunogenic agent comprises 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, carrier proteins or Fragment or variant.

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.

In another aspect of the invention, a composition comprising the immunogenic reagent of the above aspect is provided.

In one embodiment, the composition comprises an immunogenic agent comprising a plurality of immunogenic proteins, fragments, variants or derivatives thereof comprising the same or a single pathogen, or comprising a plurality of immunogens from the same or a single pathogen Lipid vesicles of a sex protein, fragment, variant or derivative thereof.

In one embodiment, the composition comprises one or more immunogenic proteins, fragments, variants or derivatives thereof, or one or more immunogenic proteins, fragments thereof, from different pathogens, A plurality of different immunogenic agents of a variant or derivative.

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

In one embodiment, the composition comprises one or more immunogenic proteins, fragments, variants or derivatives thereof, or one or more immunogenic proteins, fragments thereof, from different pathogens, A plurality of different immunogenic agents of a variant or derivative.

In another aspect of the invention, there is provided the use of an immunogenic agent for the manufacture of a medicament for inducing an immune response to one or more pathogens in a mammal, the immunogenic agent comprising one or more immunogenicities A protein, fragment, variant or derivative thereof, a lipid vesicle, and a diphtheria toxoid (DT), or a fragment or variant thereof, or a composition thereof.

In another aspect of the invention, there is provided the use of an immunogenic agent for the manufacture of a medicament for immunizing a mammal against one or more pathogens comprising one or more immunogenic proteins, A fragment, variant or derivative, lipid vesicle and diphtheria toxoid (DT) or a fragment or variant thereof, or a composition thereof.

In still another aspect of the present invention, there is provided the use of an immunogenic agent for the manufacture of a medicament for treating or preventing an infection caused by one or more pathogens in a mammal, the immunogenic reagent comprising one or more An immunogenic protein, a fragment, variant or derivative thereof, a lipid vesicle, and a diphtheria toxoid (DT), or a fragment or variant thereof, or a composition thereof.

Properly, the immunogenic agent induces a mucosal immune response

Typically, mucosal immune responses involve the production of IgA.

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

Suitably, the immunogenic protein, fragment, variant or derivative thereof is presented on the surface of the lipid vesicle. Suitably, the diphtheria toxoid (DT) or a fragment or variant thereof is located within the intracapsular space within the vesicle. In a preferred embodiment, the lipid vesicle is a liposome.

Suitably, the immunogenic protein fragment or variant is lipidated. In some 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 amino acid (K) residue is lipidated via alpha and epsilon amine groups. In some embodiments, each of the lipid or lipid-based, for example, palmitic acid C ₁₆ fatty acid. Preferably, the N-terminal cleavage of the amino acid (K) residue is in the spacer amino acid sequence at the N-terminus of the immunogenic protein fragment or variant.

In a specific embodiment, the immunogenic protein, fragment or variant system is a Group A Streptococcal M protein fragment, a variant or derivative thereof. In other or alternative embodiments, the immunogenic protein is an agent that facilitates recovery or enhancement of neutrophil activity.

In a specific embodiment, the M protein fragment is or comprises a reserved region of the M protein. In one embodiment, the fragment is an immunogenic fragment comprising or comprising a p145 peptide. In a particular embodiment, the immunogenic fragment is one in which or comprises a J8 peptide or a 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 facilitates recovery or enhancement of neutrophil activity is a protein of 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 particular embodiment, the SpyCEP fragment can be fused to the M protein fragment to form a single chimeric peptide.

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

In a specific embodiment, the immunogenic protein, fragment or variant system is an immunogenic protein, fragment or variant thereof of an influenza virus. The influenza virus can be a type A influenza virus. A non-limiting example is or comprises the amino acid sequence MSLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO: 11) or consists essentially of the amino acid sequence MSLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO: 11). The influenza virus can be type B influenza. A non-limiting example consists of or comprises the amino acid sequence PAKLLKERGFFGAIAGFLE (SEQ ID NO: 12), or substantially consists of PAKLLKERGFFGAIAGFLE (SEQ ID NO: 12).

In a specific embodiment, the immunogenic protein, fragment or variant system is an immunogenic protein, fragment or variant thereof of rhinovirus. A non-limiting example is or comprises the amino acid sequence GAQVSTQKSGSHENQNILTNGSNQTFTVINY (SEQ ID NO: 13) or consists essentially of the amino acid sequence GAQVSTQKSGSHENQNILTNGSNQTFTVINY (SEQ ID NO: 13). Another non-limiting example is or comprises the amino acid sequence GAQVSRQNVGTHSTQNMVSNGSSL (SEQ ID NO: 14) or consists essentially of the amino acid sequence GAQVSRQNVGTHSTQNMVSNGSSL (SEQ ID NO: 14).

In a particular embodiment, the immunogenic protein, fragment or variant system is a worm, such as an immunogenic protein, fragment or variant thereof of a hook worm. A non-limiting example is or comprises the amino acid sequence TSLIAGLKAQVEAIQKYIGAEL (SEQ ID NO: 15) or consists essentially of the amino acid sequence TSLIAGLKAQVEAIQKYIGAEL (SEQ ID NO: 15).

In some embodiments, the immunogenic agent can further comprise an innate immune activator. Activators of innate immunity can target C-type agins, such as macrophages ("Mincle"), which induce Ca ²⁺ -dependent (C-type) agglutination. The innate immune activator can be a glycolipid. Non-limiting examples include glycolipids, such as mycobacterial cord factor trehalose-6, 6'-dimycolate (TDM) and/or synthetic analogs thereof (analogue) Trehalose-6,6'-dibehenate (TDB) or lipid A glycolipid adjuvant.

In some embodiments, the immunogenic agent can further comprise a bile salt, such as sodium deoxycholate.

The terms "comprise", "comprises" and "comprising" or similar terms are intended to mean a non-exclusive inclusion, such that the recited elements or features include not only The elements are recited or recited, and may include other elements or features not listed or stated.

The amino acid sequence herein, by "consisting essentially of ", means that the amino acid sequence is an amine with one, two or three amino acids at the N-terminus or C-terminus. Base acid sequence.

As used herein, the indefinite article is used herein, "a (a)" and "a (AN)" means comprise a single or several or a plurality of elements or features, and should not be defined or represents "a (one)" or " A "single" element or feature.

The present invention is at least in part affirmed by the inclusion of an immunogenic peptide present on the surface of a liposome compared to a mouse induced by the established non-human compatible adjuvant CTB. For example, mice that are intranasally immunized with liposome immunogenicity of the intracapsular carrier protein of diphtheria toxoid (DT) cause mucosal and systemic antibody responses. In the specific full text of Group A Streptococcus and J8 peptides, the amount of protective immunity induced by the liposome preparation was significantly higher than that induced by J8/CTB. In addition, the interleukin reaction of purified human dendritic cell (DC) subsets proposed by the liposome will effectively induce mucosal J8-specific IgA and systemic IgG responses in humans. In some embodiments, the liposome immunogenic agent can comprise a separate SpyCEP peptide or other fragment thereof or together with a J8 peptide. In a particular form, the liposome immunogenic agent can be adapted to treat or prevent infection by isolates of particular virulent strains or group A streptococci, which can be tolerated for A Typical antibiotic treatment for streptococcal infections. This strain or isolate usually causes severe infection of the skin (for example, necrotizing fasciitis) and in some cases may harbor CovR/SCovR/S mutations. It can be analogized to include, but is not limited to, other pathogens of influenza, rhinovirus, and parasites (such as hookworms) or their associated diseases, abnormalities, and symptoms. Accordingly, embodiments of the invention provide a single immunogenic agent comprising one or more immunogenic proteins, fragments, variants or derivatives thereof, or one or more of a plurality of different pathogens An immunogenic protein, fragment, variant or derivative thereof.

For the purposes of the present invention, " isolated " means a material that has been removed from its natural state or that has been artificially manipulated. The separated material may be substantially or substantially freed from the composition normally associated with it in its natural state, or may be manipulated to facilitate its association with the composition normally associated with it in its natural state. The isolated material can be in a native, chemically synthesized or recombinant form.

By "protein (protein)" represents the amino acid-based polymer. The amino acid can be a natural or unnatural amino acid, D- or L-amino acid as understood in the art.

The term "protein (Protein)" and contains include "peptide (Peptide)", which is generally used to describe having less than fifty (50) amino acids of the protein, and "polypeptide (Polypeptide), which is generally used to describe having A protein of more than fifty (50) amino acids.

The commonly used herein, when the protein is attributable to "as the ...... (of)" or "from (from)" pathogen, which generally represents the protein comprises an amino acid sequence which at least a portion or the entire system is present in the Protein in the pathogen.

"Fragment (the fragment)" is based protein fragment, domain, portion or region, which consists of the amino acid sequence of the protein is less than 100%. It will be understood that the segments may be a single segment or may be repeated individually or with other segments.

Typically a segment may comprise, substantially consist of 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, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or 1600 amino acid full length proteins, or 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, 1000, 1050, 1100, 1150, 1200, 1250, 1300, It consists of a full length protein of 1350, 1400, 1450, 1500, 1550 or 1600 amino acids.

As used herein, a protein " variant " shares a definable nucleotide or amino acid sequence relationship with a reference amino acid sequence. A "variant" protein can have one or more amino acids of a reference amino acid sequence deleted or replaced by a different amino acid. It is understood in the art that a portion of the amino acid can be replaced or deleted without altering the activity of the immunogenic fragment and/or protein (conservative substitution). Preferably, the protein variant shares at least 70% or 75%, preferably at least 80% or 85% or more preferably at least 90%, 91%, 92%, 93%, 94%, with the reference amino acid sequence, 95%, 96%, 97%, 98% or 99% identical sequences.

Generally used herein to describe the sequence relationship between proteins and nucleic acids includes "comparison window", "sequence identity", "percentage of sequence identity" and "Substantial identity". Since the respective nucleic acids/proteins may each comprise (1) one or more proteins of only the complete nucleic acid/protein sequence shared by the nucleic acid/protein, and (2) one or more proteins that divergence between the nucleic acids/proteins Sequence comparisons are typically performed by comparing the sequences in a "comparison window" to identify and compare sequence similarities in local regions. "Comparative window" refers to a conceptual fragment of a 6, 9 or 12 contiguous residue that is typically compared to a reference sequence. The comparison window may contain about 20% or less of additionals or deletions (i.e., gaps) as compared to the optimal alignment of the respective sequences. The optimal alignment sequence for comparing the comparison windows can be processed by computerized execution of the algorithm (by Intelligenetics' Geneworks program; Wisconsin Genetics Software Package Release 7.0), GAP, BESTFIT , FASTA and TFASTA, Genetics Computer Group, 575 Scientific Drive Madison (575 Science Drive Madison), WI, USA incorporated herein by reference) or by inspection and best by any of a variety of methods selected Alignment (ie, producing the highest percentage of homology through the comparison window). Reference may also be made to the program of the BLAST family (BLAST family) as disclosed in, for example, Altschul et al., 1997, Nucl. Acids Res. 25 3389. A detailed discussion of sequence analysis can be based on Unit 19.3 of the CURRENT PROTOCOLS IN MOLECULAR BIOLOGY by Eds. Ausubel et al. (John Wiley and Sons Inc NY, 1995-1999) (Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al . (John Wiley & Sons Inc NY, 1995-1999)).

The term "sequence identical" is used in the broadest sense herein to encompass the number of precise nucleotide or amino acid matches that have the same degree of alignment with respect to the sequence through the comparison window, taking into account the appropriate alignment using standard algorithms. Therefore, the "sequence of the same sequence" is generated by comparing the two optimal alignment sequences through the comparison window and defining the number of positions of the same nucleic acid base (eg, A, T, C, G, I) in the two sequences. The number of matching locations, the number of matching locations is divided by the total number of comparison window locations (ie, window size) and the result is multiplied by 100 to produce the same percentage of the sequence. For example, "same sequence" can be understood as using the DNASIS computer program (version 2.5 for windows); purchased from Hitachi Software engineering Co., Ltd., South San Francisco (South San) Francisco), California, USA)) The meaning of the "match percentage" calculated.

As used herein, "derivatives (Derivatives)" system in the art, for example by understood with other chemical moieties conjugated (Conjugation) or composite, by post-translational modifications (post-translational modification) (e.g., phosphoric acid Molecules, such as proteins, fragments or variants thereof, modified by glycosylation (eg, addition, removal or alteration of glycation), lipidation, and/or incorporation of additional amino acid sequences body. In a particular embodiment, the additional amino acid sequence can comprise one or more lysine residues at its N-terminus and/or C-terminus. The plurality of lysine residues (eg, polyaminic acid) can be a linear sequence of an lysine residue or a branched sequence that can be an amide residue. This additional lytic acid residue can be beneficial to increase the solubility of the peptide.

The additional amino acid sequence can comprise a fusion partner amino acid sequence which produces a fusion protein. For example, the fusion partner amino acid sequence can aid in the detection and/or purification of the isolated fusion protein. Non-limiting examples include metal binding (eg, polyhistidine) fusion partner, maltose binding protein (MBP), protein A, glutathione S-transferase (GST) )), fluorescent protein sequences (eg, GFP), epitope tags such as myc, FLAG, and haemagglutinin tags. Other additional amino acid sequences comprise spacer sequences. An example of a spacer sequence is an amino acid sequence at the N-terminus or C-terminus of the amino acid sequence of the immunogenic protein fragment or a variant comprising an lysine (K) residue suitable for lipidation. Typically, the spacer amino acid sequence comprises two (2) to ten (10) amino acids, such as three (3) amino acid sequences KSS.

Other derivatives contemplated by the present invention include, but are not limited to, modifications to the side chain, incorporation of their derivatives in the unnatural amino acid and/or peptide, or protein synthesis and use of crosslinkers, and Other methods of enhancing conformational constraints on immunogenic proteins, fragments and variants of the invention.

In this regard, those of ordinary skill in the art are referred to in Chapter 15 of the CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley and Sons NY, 1995-2008) for more A wide range of methods for chemical modification.

The immunogenic proteins, fragments and variants disclosed herein will be understood to be each present on the surface of a lipid vesicle or as a chimeric or fusion protein comprising multiple copies or multiple different peptides of the same peptide. A non-limiting example is the chimeric peptide of SEQ ID NO: 3, which will be described in more detail below.

In the context of the present invention, such as "immunogenic (immunogenic)" as used herein, are diagrams of mammals or other animals after administration of an immunogenic agent, for example, pathogen or a molecule or composition to induce an immune response to produce power or potential.

By " elicit an immune response " is meant the production or activity of one or more elements that produce or stimulate an immune system, an antibody, and/or a native immune system comprising a cellular immune system. Suitably, one or more elements of the immune system comprise B lymphocytes, antibodies, neutrophils, dendritic cells comprising plasmacytoid dendritic cells, interleukins and/or chemical activins. Non-limiting examples of interleukins include pro-inflammatory interleukins such as TNF-[alpha], IL-6 and IL-1 (e.g., IL- l[beta]). A non-limiting example of a chemical activin is the chemo-attractant 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 term generally "immunization (immunize)", "vaccination (vaccinate)" and "vaccine (Vaccine)" means to induce a protective immune response against the pathogen of the method, and / or formulation, thereby at least partially avoided Or minimize subsequent infection with the pathogen.

As used herein, a "pathogen (pathogen)" system for the animals have the ability to cause any disease, disorder or condition which has no individual life or the life of the animals such as birds or mammals, human beings are also included. The pathogen can be, but is not limited to, a virus, a bacterium, a protozoa or a parasite. Specific non-limiting examples of pathogens include bacteria, such as group A streptococci, respiratory viruses, such as influenza viruses, and rhinoviruses and parasites, such as nematodes, which also contain hookworms.

As understood from the disclosure herein, the invention provides a lipid vesicle formulation comprising an immunogenic protein fragment, variant or derivative and a carrier protein formulated in a lipid vesicle. As widely used herein, a lipid vesicle can be a liposome, a minicell, a multilamellar vesicle, a micelle, a vacuole, or other vesicular structure comprising a lipid bilayer ( Vesicular structure), suitably, an immunogenic protein fragment, variant or derivative is derivatized to comprise one or more lipids that are useful for immobilization in a lipid bilayer such that the immunogenic protein fragment, variant or derivative is present On the surface of lipid vesicles. In a preferred form, the lysine (K) residue is lipidated via an alpha and/or epsilon amine group. To facilitate lipidation, the immunogenic protein fragment, variant or derivative may further comprise an N-terminal spacer comprising a lipidated lysine (K) residue. The spacing may typically comprise from 2 to 10 linked amino acids, such as three (3) amino acid spacer KSS. In some embodiments, each of the lipid or lipid is C ₁₆ fatty acids such as palmitic acid. However, it will also be appreciated that other lipids, for example, a C ₁₂ -C ₂₂ saturated or unsaturated carbon chain (e.g., units of unsaturated (mono-unsaturated)) according to the present invention, fatty acid can be used.

The lipid vesicles suitably comprise any lipid or mixed lipid that can form a lipid bilayer structure. It comprises phospholipids, sterols, fatty acids and/or triglycerides comprising cholesterol (cholesterol), cholesterol esters (cholesterol-esters) and phytosterols. Non-limiting examples of phospholipids include phosphatidylcholine (PC) (lecithin), phosphatidic acid, phosphatidylethanolamine (PE), cephalin, and phospholipids. Phosphatidylglycerol (PG), phospholipidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM) or natural or synthetic derivatives thereof. Natural derivatives include egg PC (egg PC), egg PG, soybean PC, hydrogenated oil soybean PC, soybean PG, brain PS, sphingolipid, brain SM, galactocerebroside, ganglioside (gangliosides), cerebrosides, cephalin, cardiolipin, and dicecityl phosphate. The synthetic derivative comprises dipalmitoylphosphatidylcholine (DPPC), didecanoylphosphatidylcholine (DDPC), dierucoylphosphatidylcholine (DEPC), and two flesh. Dimyristoylphosphatidylcholine (DMPC), disearoylphosphatidylcholine (DSPC), dilaurylphosphatidylcholine (DLPC), palmitoyl phosphatidylcholine choline (palmitoyloleoylphosphatidylcholine (POPC)), palmitosylphosphatidylcholine (PMPC), palmitoylstearoylphosphatidylcholine (PSPC), dioleoylphosphatidylcholine (DOPC) )), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylglycerol (DLPG), disearoylphosphatidylglycerol (DSPG), dimylopectin phospholipid (dimyristoylphosphatidylglycerol (DMPG)), Dipalmitoylphosphatidylglycerol (DPPG), disearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG) )), dimyristoylphosphatidic acid (DMPA), dipalmitoylphosphatidic acid (DPPA), disearoylphosphatidic acid (DSPA), dimylopectin Dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS) ), disearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylserine (DOPS), dipalmitoside sphingomyelin Dipalmitoylsph Ingomylin (DPSM)) and distearoylsphingomyelin (DSSM). The phospholipid may also be a derivative or analog of any of the above phospholipids.

Suitably, the mixed lipids may comprise a desired molar or wt% ratio between the individual lipids. For example, liposomes can utilize 7 7 dipalmitoyl-sn-glycero-3-phosphocholine (DPPC): 2 cholesterol (CHOL): 1 L-α-phospholipid The molar ratio of glycerol (L-α-phosphatidylglycerol (PG)) is formed.

Suitably, the lipid vesicle further comprises a carrier protein. Suitably, the carrier protein is an immunogenic protein, or at least partially facilitates or enhances the immunogenicity of the immunogenic agent. Typically, the carrier protein is prepared as a lipid vesicle such that the carrier protein is located in the internal aqueous space inside the lipid vesicle. In some embodiments, the carrier protein can be fused, conjugated or complexed with the immunogenic protein fragment, variant or derivative thereof. It includes recombinant protein fusion, chemical cross-linking, and intermolecular complexing, for example, by biotin-avidin or other intermolecular binding agents, but not Limited to this. In this embodiment, the immunogenic protein fragment, variant or derivative thereof is fused, conjugated or complexed with the carrier protein site in the internal aqueous space inside the lipid vesicle. This embodiment may be particularly useful for orally administered immunogenic agents, such as liposomes comprising bile salts, which are described in more detail below. Non-limiting examples of carrier proteins include diphtheria toxoid (DT), tetanus toxoid (TT), CRM protein such as CRM197, and Pertussis toxin mutant, but are not limited thereto. Fragments and variants of carrier proteins are also contemplated. In a specific 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. An activator of innate immunity can target a type C agglutinin expressed by one or more cells associated with innate immunity. Preferably, the C-type agglutinin is a macrophage ("Mincle") that induces a Ca ²⁺ -dependent (C-type) agglutinin. Non-limiting examples include glycolipids such as mycobacterium cord-like factor trehalose-6,6'-dimyristate (TDM) and/or its synthetic analog trehalose-6,6'-dibehenate ( TDB) and/or a lipid A glycolipid adjuvant, such as monophosphoryl 3-dethiol-lipid A, which may be in the form of PHAD®, 3D-PHAD® and 3D (6-acyl) PHAD®. While not wishing to be bound by any particular theory, the activators of innate immunity as suggested above may enhance or ameliorate mucosal immunity induced by immunogenic agents. Preferably, one or more glycolipids may be included in the lipid vesicles such that they do not occupy more than about 25%, about 20%, about 15%, about 10% of the total lipid in the lipid vesicles.

In some embodiments, the lipid vesicles may further comprise bile acid or bile salts. The bile acid is usually a dihydroxylated or trihydroxylated sterol having 24 carbons, which comprises cholic acid, deoxycholic acid, chenodeoxycholic acid. (chenodeoxycholic acid) and ursodeoxycholic acid. Preferably, the lipid vesicles comprise a bile salt such as cholate, deoxycholate, chenodeoxycholate or ursodeoxycholate salt. Preferably the bile salt is sodium deoxycholate.

In other embodiments, the immunogenic agent comprising the liposome can be made to a particular size, size or range of specific, selected or desired. In some embodiments, larger particle size liposomes can induce a stronger mucosal immune response.

In other embodiments, the immunogenic agent comprising the liposome can be freeze dried or lyophilized to facilitate long term storage. The immune response elicited by the recombinant freeze-dried liposome immunogenic agent can be compared to a "fresh" liposome immunogenic reagent.

In some embodiments, the pathogen is group A streptococci.

As used herein, the terms " group A streptococcus ", " Group A Streptococci ", " Group A Streptococcal ", " Group A Streptococcus " ( Group A) "Strep " and the abbreviation "GAS" refer to the Streptococcus of Lancefield serogroup A, which is a Gram-positive β-hemolytic bacteria of Streptococcus pyogenes . ). An important causative agent of GAS is the M protein, which is strongly anti-phagocytic and linked to serum factor H, destroys C3-convertase and avoids opsonization by C3b. As described by Graham et al ., PNAS USA 99 13855 (Graham et al ., 2002, PNAS USA 99 13855), it also encompasses pathogenic "mutations" such as CovR/S or CovRS mutations, but is not limited thereto.

Diseases and symptoms caused by group A streptococci include cellulitis, erysipelas, impetigo, scarlet fever, laryngeal infection such as acute pharyngitis ( Strept throat, bacteremia, toxic shock syndrome, necrotizing fasciitis, acute rheumatic fever, and acute glomerulonephritis, but are not limited thereto.

As used herein, "neutrophils (neutrophils)" or neutrophil cell line to form granules with basophil granulocyte (basophils) and eosinophil (eosinophil) family polymorphonuclear cells (polymorphonuclear cell family (PMNs ))part. Neutrophils produce relatively short-lived phagocytic cells from bone marrow stem cells and typically constitute 40% to 75% of white blood cells in mammals. The phagocytic neutrophils release soluble anti-microbials (eg, granule proteins) and produce neutrophil extracellular traps. The neutrophil reacts with molecules such as interleukin 8 (IL-8), C5a, fMLP, and leukotriene B4, which promote chemotaxis of the neutrophils to the site of injury and/or acute inflammation.

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

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

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

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

The agent that facilitates restoring or enhancing neutrophil activity , as used herein, directly or indirectly increases, enhances or restores the production, movement and/ or continuation of neutrophils . / or chemotactic, and / or one or more immunologically active molecules of the neutrophil. In one embodiment, the agent induces an immune response to a neutrophil inhibitor. In another embodiment, the agent binds and at least partially does not activate the neutrophil inhibitor. The neutrophil inhibitor can be a molecule derived or derived from Group A Streptococcus. In a particular form, the neutrophil inhibitor is a serine protease or a fragment thereof that proteolytically cleaves interleukin 8. In a specific embodiment, the neutrophil inhibitor is SpyCEP or a fragment thereof. SpyCEP is a 170-kDa multidomain serine protease expressed on the surface of human pathogenic Streptococcus pyogenes, which plays an important role in infection by catalyzing shear and not activating neutrophil chemoattractant protein-8. character of. Non-limiting examples of SpyCEP amino acid sequences can be based on SEQ ID NO: YP597949.1 and ( S. pyogenes MGAS 10270) and YP 596076.1 ( S. pyogenes MGAS 9429). Thus, in a particular embodiment, the SpyCEP fragment is or comprises the SEQ ID NO: 2 (NSDNIKENQFEDFDEDWENF) amino acid sequence. Its proposed SEQ ID NO: 2 is or is a dominant epitope on SpyCEP that induces functional antibodies.

Furthermore, provided herein is a chimeric peptide comprising an M protein amino acid sequence and a SpyCEP amino acid sequence which form a single, contiguous amino acid sequence. The M protein amino acid sequence can be located at the C-terminus of the SpyCEP amino acid sequence or vice versa. In one embodiment, the chimeric peptide may comprise the amino acid sequence NSDNIKENQFEDFDEDWENFQAEDKVKQSREAKKQVEKALKQLEDKVQ (SEQ ID NO: 3) or a variant thereof.

In an alternate embodiment, the respective liposomes comprising the M protein amino acid sequence and the SpyCEP amino acid sequence can be produced for administration as an "admixture."

In a particular embodiment, the variant M protein or peptide may comprise one or more lysine residues at its N-terminus and/or C-terminus. The plurality of lysine residues (eg, polyaminic acid) can be a linear sequence of an lysine residue or a branched sequence that can be an amide residue. This additional lytic acid residue can be beneficial to increase the solubility of the peptide.

Non-limiting examples of J8 peptide variants include: SREAKKQSREAKKQVEKALKQ VEKALC (SEQ ID NO: 5) SREAKKQSREAKKQVEKALKQ SREAKC (SEQ ID NO: 6) SREAKKQVEKALKQSREAKKQ VEKALC (SEQ ID NO: 7) SREAKKQVEK ALDASREAKKQVEKALC (SEQ ID NO: 8) Other variants It may be based on, for example, the heptapeptide described by Cooper et al., 1997.

For example, if the epitope is known to be in the alpha-helical protein structural conformation, then the model peptide can be synthesized to fold into this configuration. It is based on the structure of the GCN4 leucine zipper (O'Shea et al., 1991) to design a model of the alpha-helical coiled-coil peptide. The first heptapeptide contains the sequence MKQLEDK (SEQ ID NO: 9), which contains several features found in the stable helically coiled heptad repeat motif (abcdefg) n (Cohen and Parry, 1990). . It contains large non-polar residues at the a and d positions, acid/base pairs at the e and g positions (Glu/Lys) (usually favoring ionic interactions between chains) and at positions b , c and f Polar group (consistent with Lupas et al.'s prediction (1991)). The GCN4 peptide also contains a consensus valine in the a position. It should also be noted that a coiled coil dimer is preferred when occupying the a and d positions by V and L (Harbury et al., 1994). A heptad repeat model derived from the consensus feature of GCN4 leucine zipper peptide (VKQLEDK; SEQ ID NO: 10): has the potential to form an alpha-helical coiled-coil. This peptide becomes a framework peptide. Overlapping fragments based on the studied conformational epitope are embedded in the model coiled coil peptide to form a chimeric peptide. Whenever the same residue is found in both the helical model peptide and the epitope sequence, it will be designed to ensure that the correct helical coiled helical configuration (Cohen and Parry, 1990) is incorporated into the chimeric acid substitution Peptide. The following permutations are commonly used: at position a, V is changed to I; b is position, K is replaced by R; c is position, Q is replaced by N; at position d , L is replaced by A; at position e, E is replaced by Q; f position, D is replaced by E; at the g position, K is replaced by R. All such replacement residues are common in their respective positions of the coiled-coil protein (Lupas et al., 1991).

A specific J8 peptide derivative described in Olive et al., 2002, Infect & Immun. 70 2734 is a "lipid core peptide". In one embodiment, the lipid core peptide may comprise a plurality of amino acids synthesized directly by coupling two amino acids in each of the lysines of the branched polyaminic acid core to a lipophilic anchor. A J8 peptide (for example, four J8 peptides).

M protein fragments or variants and/or SpyCEP fragments or variants can be derivatized to comprise one or more lipids that facilitate immobilization to the lipid bilayer described above. In yet another embodiment, a chimeric peptide comprising an M protein amino acid sequence and a SpyCEP amino acid sequence (eg, SEQ ID NO: 3) can comprise a spacer amino acid sequence at its N-terminus. Thus, the SpyCEP fragment or variant in the examples is contained in a lipid vesicle, which can be presented as a chimeric peptide that is lipidated separately or can be lipidated with the M protein fragment or variant.

In some embodiments, the pathogen is an influenza virus, and in a particular embodiment, the immunogenic protein, fragment or variant is an immunogenic protein, fragment or variant thereof of influenza A virus. The immunogenic protein or fragment can be matrix protein 2 or a fragment thereof. Non-limiting examples are or include the amino acid sequence MSLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO: 11). In a specific embodiment, the immunogenic protein, fragment or variant is an immunogenic protein, fragment or variant thereof of influenza B virus. The immunogenic protein or fragment may be a haemagglutinin protein or a fragment thereof. Non-limiting examples are or include the amino acid sequence PAKLLKERGFFGAIAGFLE (SEQ ID NO: 12).

In some embodiments, the pathogen is a rhinovirus. In a specific embodiment, the immunogenic protein, fragment or variant is a type B rhinovirus protein, such as a capsid protein. Non-limiting examples are or include the amino acid sequence GAQVSTQKSGSHENQNILTNGSNQTFTVINY (SEQ ID NO: 13). In another specific embodiment, the immunogenic protein, fragment or variant is an immunogenic protein, fragment or variant of a rhinovirus type A, such as a capsid protein. Another non-limiting example is or comprises the amino acid sequence GAQVSRQNVGTHSTQNMVSNGSSL (SEQ ID NO: 14).

In some embodiments, the pathogen is a parasite, such as a hookworm. In a specific embodiment, the immunogenic protein, fragment or variant is an immunogenic protein, fragment or variant of Necator americanus . Non-limiting examples are or include the amino acid sequence TSLIAGLKAQVEAIQKYIGAEL (SEQ ID NO: 15).

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

Chemical synthesis consists of solid and liquid synthesis. Although reference is made to Ed. Nicholson (Blackwell Scientific Publications) Synthetic Vaccine (SYNTHETIC VACCINES) Chapter 9 and Eds. Coligan et al. Protein Science Laboratory Manual, Chapter 15 (John Wiley and Sons, Inc. NY) An example of a chemical synthesis technique provided by USA 1995-2008) is made, but the method is a method known in the art. In this respect, it is also produced in accordance with International Publication No. WO 99/02550 and International Publication No. WO 97/45444.

Recombinant proteins are utilized by those of ordinary skill in the art using, for example, the molecular laboratory of Sambrook et al. (MOLECULAR CLONING. A Laboratory Manual) (Cold Spring Harbor Press, 1989), in particular In paragraphs 16 and 17; Eds. Ausubel et al. Molecular Biology Laboratory Manual (John Wiley and Sons, Inc. NY USA 1995-2008), particularly in Chapters 10 and 16; and Eds. The Coligan et al. Protein Science Laboratory Manual (John Wiley and Sons, Inc. NY USA 1995-2008), especially in the standard experimental procedures described in Chapters 1, 5, and 6, can be conveniently prepared. Generally, recombinant protein preparation encompasses the expression of a nucleic acid encoding a protein in a suitable host cell.

As described above, the present invention provides an immunogenic agent and/or use thereof for preventing or treating a disease, abnormality or symptom associated with a pathogen in a mammal or other animal.

Such as "treatment (treating)", as used herein, "treatment (treat)" or "treatment (treatment)" means at least part of its development after the improvement (ameliorates), elimination (eliminates) or mitigate a disease associated with the pathogen Therapeutic intervention of the symptoms or pathological signs of abnormalities or symptoms. This treatment does not need to be absolutely beneficial to the individual. Advantageous effects can be defined using any method or standard known to those of ordinary skill in the art.

As used herein, "prevention (preventin g)", "prevention (prevent)" or "prophylaxis (prevention)" means before or by exposure to group A streptococcal infection and / or associated with group A streptococcal The course of action initiated prior to the onset of a disease, abnormality or symptom of the pathology or pathology to avoid infection and/or reduce signs or pathological signs. It will be understood that this prevention need not be absolutely beneficial to the individual. "Preventive (prophylactic)" treatment is the individual signs of the disease, disorder or condition which has not yet appeared, or only early signs of administered treatment to reduce the risk of developing a disease, disorder or pathological symptoms or signs of symptoms of purpose .

The disease, abnormality or symptom may be a disease, abnormality or symptom associated with group A streptococcus.

In the entire text of the present invention, " group A-strep-associated disease (disorder or condition )" means any clinical pathology caused by group A streptococcal infection. And includes cellulitis, erysipelas, pustular fever, scarlet fever, laryngeal infections such as acute pharyngitis (streptotoxic laryngitis), bacteremia, toxic shock syndrome, necrotizing fasciitis, acute rheumatic fever and acute Renal glomerulonephritis, but not limited to this.

As described herein above, the use of the invention for treatment and/or immunization comprises administering to a mammal an M protein fragment, variant or derivative, lipid vesicle, comprising a protein fragment that is useful for rejuvenating or enhancing neutrophil activity, An immunogenic agent for carrier proteins and/or SpyCEP peptides or other fragments.

As disclosed herein, in addition, the treatment and/or immunization may comprise the therapeutic treatment of a GAS infection by an antibody or antibody fragment, for example by targeting SpyCEP and/or binding M protein at the site of infection (eg, skin), An antibody or antibody fragment of a fragment or variant.

The antibody and antibody fragment may be polyclonal or monoclonal, natural or recombinant. Antibody fragments comprise Fc, Fab or F(ab)2 fragments and/or may comprise single chain Fv antibodies (scFvs). Such scFvs can be prepared, for example, according to the methods described in U.S. Patent No. 5,091,513, European Patent No. 239,400, or by the respectives of Winter and Milstein, 1991, Nature 349:293 (Winter & Milstein, 1991, Nature 349:293). The antibody may also comprise multivalent recombinant antibody fragments, such as diabodies, triabodies, and/or tetrabodies, comprising a plurality of scFvs and dimerisation-activated demibodies ( For example, WO/2007/062466). For example, such antibodies can be prepared according to the method described by Holliger et al, 1993 Proc Natl Acad Sci USA 90 6444; or Kipriyanov, 2009 Methods Mol Biol 562 177. Known experimental procedures can be based, for example, on the antibodies of Corigan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, Chapter 2 (John Wiley and Sons NY, 1991-1994) and Harlow, E. and Lane, D. : Laboratory Manual , Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988 (Harlow, E. & Lane, D. Antibodies: A Laboratory Manual , Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988) for antibody production, purification, and use.

Methods for producing polyclonal antibodies are known to those of ordinary skill in the art. Exemplary experimental procedures can be performed, for example, in the immunological experimental manipulation manual of Coligan et al., supra, and Harlow and Lane, 1988, supra. In a particular embodiment, the anti-SpyCEP polyclonal antibody can be obtained or purified from an individual exposed to Group A Streptococcus or human serum infected with Group A Streptococcus. Alternatively, multiple strains of antibodies can be produced against purified or recombinant SpyCEP or immunogenic fragments thereof in, for example, a horse producing species, and then purified prior to administration.

Monoclonal antibodies can be utilized, for example, by standard methods originally described in the article by Köhler and Milstein, 1975, Nature 256 , 495 or by, for example, the manual of the immunology experiment in Coligan et al., supra, the more recent modifications described. The method is produced by immortalizing a spleen of an isolated protein, fragment, variant or derivative of one or more of the invention or other antibody producing cells derived from the producing species. Thus, a monoclonal antibody can be produced against an M protein fragment, variant or derivative and/or an agent that facilitates recovery or enhancement of neutrophil activity (eg, SpyCEP) in accordance with the use of the invention. In some embodiments, the monoclonal antibodies or fragments thereof can be in recombinant form. If a monoclonal antibody is originally produced by a non-human mammalian spleen cell, it may be particularly advantageous for "humanizing" monoclonal antibodies or fragments.

In an embodiment associated with a therapeutic antibody, a preferred M protein fragment can be a p145 peptide.

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

In some embodiments, the disease, abnormality, and symptoms can be influenza virus related diseases, abnormalities, or symptoms. Influenza viruses can cause transmissions known as "flu" or other infectious diseases. Typical symptoms include fever, headache, cough, lethargy, mucus production in the respiratory and nasopharynx, and secretory muscle pain, nausea and vomiting. This symptom can last for days or weeks. In some cases, secondary respiratory bacterial infections may occur, and in some cases, severe symptoms such as pneumonia may occur. Accordingly, the immunogenic agent of the present invention and/or its use can treat or prevent a disease, abnormality or symptom associated with the influenza virus as described above.

In some embodiments, the disease, disorder, or condition can be a rhinovirus-associated disease, disorder, or condition. Rhinovirus (eg, type A rhinovirus and type B rhinovirus) species of the genus Enterovirus of the Picornaviridae family of viruses. Rhinoviruses are usually the cause of the common cold, with symptoms similar to those of influenza but less severe and less likely to cause secondary bacterial infections such as pneumonia.

Accordingly, the immunogenic agent of the present invention and/or its use can treat or prevent rhinovirus-related diseases, abnormalities and symptoms as described above.

In some embodiments, the disease, abnormality, or condition can be a hookworm-related disease, disorder, or symptom. Hookworm is a nematode that invades many different animals. Hookworms that normally infect humans can include Necator americanus and Ancylostoma duodenalis . Hookworms have a hook-like mouth to allow hookworms to attach to the intestinal wall, pierce blood vessels and feed on blood, and in some cases cause severe anemia. Infected hookworms in pregnant women can cause fetal growth retardation, premature birth and low birth weight. Hookworms that infect children can cause mental, cognitive and growth disorders.

Accordingly, the immunogenic agent of the present invention and/or its use can treat or prevent a parasitic-related disease, abnormality or symptom as described above.

In some embodiments, the foregoing uses may comprise: (i) an immunogenic agent comprising one or a plurality of different proteins, fragments, variants or derivatives; (ii) comprising different pathogens, respectively a plurality of different immunogenic agents of one or more different proteins, fragments, variants or derivatives; or (iii) immunization comprising one or a plurality of different proteins, fragments, variants or derivatives An original reagent; it is used to: (i) induce an immune response to one or more pathogens; (ii) immunize against one or more pathogens; or (iii) prevent or treat one or more of the pathogens Or multiple diseases, abnormalities or symptoms.

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

In a particular embodiment, the composition can comprise: (i) an immunogenic agent comprising one or a plurality of different proteins, fragments, variants or derivatives; (ii) comprising different pathogens, respectively a plurality of different immunogenic agents of one or more different proteins, fragments, variants or derivatives; or (iii) immunization comprising one or a plurality of different proteins, fragments, variants or derivatives In situ reagent; In a preferred form, the composition comprises an acceptable carrier, diluent or excipient.

By "acceptable carrier, diluent or excipient (acceptable carrier, diluent or excipient)" line represents safely used in systemic administration of solid or liquid filler, diluent or encapsulating substance. Various carriers, diluents and excipients known in the art can be used depending on the particular route of administration. It may be selected from the group consisting of sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols. , alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and, for example, hydrochlorides, bromides, and sulfates Mineral acid salts, salts of organic acids such as acetates, propionates, and malonates, water, and pyrogen-free water Group

A useful reference for the description of acceptable carriers, diluents and excipients is incorporated herein by reference to Remington's Pharmaceutical Sciences (Mack Publishing Co. N. J. USA, 1991).

Preferably, for the purpose of eliciting an immune response, a portion of the immunological reagent can be used in combination with the immunogenic reagents disclosed herein for formulation.

The term " immunological agent " encompasses vectors, delivery agents, immunostimulants, and/or adjuvants within the scope thereof as known in the art. As is understood in the art, immunostimulants and adjuvants refer to or comprise one or more substances that enhance the effectiveness of the immunogenicity and/or formulation. Non-limiting examples of suitable immunostimulants and adjuvants include squalane and squalene (or other oils of plant or animal origin); block copolymers; for example, Tween ®-80 detergent; Quil® a, for example mineral oil Drakeol or Marcol, vegetable oils such as peanut oil; for example, small Corynebacterium (Corynebacterium parvum) derived adjuvant, Corynebacterium; e.g. P. acnes (Propionibacterium acne) Propionibacterium-derived adjuvant; Mycobacterium bovis (Bacille Calmette and Guerin or BCG); Bordetella pertussis antigen; tetanus toxoid; diphtheria toxoid; eg hexadecylamine (hexadecylamine), octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide , N,N -dioctadecyl-N¢, N¢ N- dicoctadecyl-N¢, N¢bis(2-hydroxyethyl-propanediamine) Methoxy hexadecyl A surface active substance of methoxyhexadecylglycerol and pluronic polyols; for example, a polyamine of pyran, dextransulfate, poly IC carbopol ( Polyamines; for example, muramyl dipeptides and derivatives, dimethylglycine, tuftsin peptides; oil emulsifiers; and, for example, aluminum phosphate, a mineral hydroxide of aluminium hydroxide or alum; a medium such as interleukin 2 and interleukin 12; a mononuclear factor such as interleukin-1; tumor necrosis factor; For example, an interferon of gamma interferon; an immunostimulatory DNA such as CpG DNA, a composition such as saponin-aluminium hydroxide or Quil-A aluminium hydroxide; Liposomes; ISCOM® and ISCOMATRIX® adjuvants; mycobacterial cell wall extracts; synthetic glycopeptides such as cell wall dipeptides or other derivatives; Avridine; lipid A derivatives Dextran sulfate; DEAE-Dextran alone or in combination with aluminum phosphate; carboxypolymethylene such as Carbopol'EMA; for example, Neocryl A640 (for example, US Patent No. 5,047,238) of acrylic copolymer emulsions; for example, water in oil emulsifiers of Montanide ISA 720; poliovirus, vaccine or animal pox virus Animal poxvirus proteins; or mixtures thereof.

Immunizing agent may include, for example, thyroglobulin (Thyroglobulin) a carrier protein; e.g. (human serum albumin) human albumin serum albumin; from tetanus, diphtheria, pertussis, Pseudomonas (of Pseudomonas), Escherichia coli (E. coli ), Staphylococcus (Staphylococcus) and streptococci (Streptococcus) toxin of the toxin, toxoid or mutants of any cross-reactive material (CRM); such as poly (lysine: glutamic acid) (poly (lysine: glutamic acid ) Polyamino acid; influenza; Rotavirus VP6, Parvovirus VP1 and VP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine. Alternatively, fragments or epitopes of carrier proteins or other immunogenic proteins can be used. For example, bacterial cell toxins, toxoids or T cell epitopes of CRM can be used. In this regard, it can be made by reference to U.S. Patent No. 5,785,973, which is incorporated herein by reference.

Any suitable step is considered for the production of a vaccine formulation. Exemplary steps include, for example, New Generation Vaccines (1997, Levine et al, Marcel Dekker, Inc. New York, Basel, Hong Kong), incorporated herein by reference. The steps.

Any safe route of administration may be employed, including intranasal, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular (intra- Articular), intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, local, mucosa And transdermal administration, but is not limited thereto.

Dosage forms include tablets, dispersing agents, suspending agents, injections, solutions, syrups, troches, capsules, nasal sprays, suppositories, aerosols, transdermal patches, and the like. . The dosage form may also comprise injection or implantation of a controlled release device or modification designed specifically for this purpose to additionally be implanted in this manner. Controlled release may include the inclusion of acrylic resins, waxes, higher aliphatic alcohols, polylactic acid and polyglycolic acids, and, for example, hydroxypropyl methylcellulose (for example) The hydrophobic polymer coating of a part of the cellulose derivative of hydroxypropylmethyl cellulose) is affected.

The compositions may each comprise a predetermined amount of one or more therapeutic agents of the invention, such as capsules, sachets, functional foods/feeds or tablets, in powders or granules or in aqueous liquids. Suspensions, non-aqueous liquids, oil-in-water emulsions or water-in-oil liquid emulsions are present. The formulation may be prepared by any of the methods of pharmacy, but all methods comprise the step of combining one or more of the above formulations with a carrier comprised of one or more of the desired ingredients. In general, the formulations are prepared by uniformly and intimately admixing the formulation of the present invention with a liquid carrier or a finely divided solid carrier or both, and, if desired, shaping the product into the desired form.

The above formulations may be administered in a manner compatible with the dosage form and may be administered in an effective dosage. In the context of the present invention, the dose administered to the patient should be sufficient to provide the patient with a beneficial response at an appropriate time. The amount of the agent can be administered depending on factors such as the age, sex, weight, and general health of the patient being treated, according to the judgment of the physician.

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

As used herein, the term commonly used "patients (patient)", "individual (individual)" and "individual (subjec t)" as used throughout the text to accept disclosed herein or any mammalian therapeutic agents. Thus, the methods and formulations disclosed herein can be used in medical and/or veterinary applications. In a preferred form, the mammal is a human.

Therefore, the present invention can be fully understood and achieved with reference to the following non-limiting examples.

Example

Example 1 Introduction

Group A Streptococcus (GAS) mainly infects the upper respiratory tract (URT) mucosa, but it also infects human skin, causing disease prevalence. Infection can lead to toxic shock syndrome, necrotizing fasciitis and myositis. Necrotizing fasciitis has an incidence of 100,000 and a mortality rate of up to 70% (1). Rheumatoid fever (RF) and rheumatic heart disease (RHD) after streptococcal disease are also highly relevant. It is estimated to have 15.6 million RHD epidemics and approximately 400,000 deaths per year (2). The most common diseases after URT colonization are pharyngitis and RF and RHD, which are closely related to untreated pharyngeal infections (3). GAS infections and related diseases are prevalent in tropical regions of developing countries and highlight the urgent need for vaccines in the indigenous populations of developing countries (4), which cause 500,000 deaths per year.

GAS vaccine candidates can be divided into M protein and non-M protein vaccines (5). The cell surface M protein of coiled-coil protein consisting of three major domains is the major causative factor (6). This protein consists of a highly variable amine terminus and an A repeat domain for epidemiological molecular types ( emm or M); a B repeat domain and a C repeat domain (6). According to the clinical investigation, the leading subunit vaccine is an amino-based M-protein multivalent vaccine and a C-repetitive M-protein peptide vaccine (5). The GAS vaccine candidate has entered clinical trials based on its successful induction of systemic immunity (7). Systemic immunity has been shown to effectively prevent GAS from spreading to deep tissues and prevent disease through serum immunoglobulin (Ig) throughout the body, but it does not prevent colonization of the mucosal site and thus prevents human-to-human infection (8). Therefore, systemic vaccination is not the best way to induce immunity against GAS. Conversely, mucosal vaccines that administer nasal mucosa against various microorganisms effectively induce antigen-specific immune responses in both systemic and mucosal compartments (9-11). Because of the two-layer protective immunity, it is an ideal strategy for combating both systemic and mucosal GAS infections, and its additional benefit of preventing mucosal colonization will also inhibit droplets and aerosol infection from URT (7). Mucosal vaccination is also economically advantageous and is an important consideration for vaccine development. Since the administration of the vaccine by the nasal route is easier, the use of the needle can be avoided (7). Because of its lack of pain, patients will have greater compliance when delivered.

The vaccine candidate peptide J8 (12) was previously defined as a minimal B cell epitope based on the retained C3 repeat domain from the M protein. The J8 peptide (QAEDKVKQ SREAKKQVEKAL KQLEDKVQ; SEQ ID NO: 1) is a chimeric peptide containing 12 amino acids from the C region (shown in bold) located on the side of the GCN4 DNA binding protein sequence to maintain the correct helix Configuration structure (13). When conjugated to the carrier protein diphtheria toxoid (DT) and administered with alum, J8 induced protection of mice from systemic and skin-stimulated IgG antibodies due to multiple GAS strains (4, 13). Furthermore, when administered with an animal-restricted mucosal adjuvant CTB (14, 15) or when administered as a proteasome (16), a vaccine candidate based on the reserved region of the GAS-based M protein is effectively Protect against intranasal infections due to GAS. When mucosal immunity was induced and URT colonization was reduced, it was identified that it was associated with IgA production. In view of this, the purpose is to develop a J8-type, human compatible mucosal vaccine.

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 composed of biocompatible phospholipid bilayers and are capable of loading and transporting both hydrophilic and hydrophobic molecules (19). Phospholipids are safely administered to humans via the intranasal route (20, 21). However, liposomes that exhibit peptide antigens are not an ideal platform for inducing peptide specific antibody responses. The peptide may only contain a restricted epitope of helper T cells required to activate an antibody response to B cells. It needs to be conjugated to a "vector" protein to present its immunogenicity in the outbred population and render it undesirably suitable for presentation by liposomes. However, since natural pathogens are also particulate and have been recognized by the immune system, there is no doubt that immunogenicity is enhanced by particulate matter such as liposomes (22). The natural tendency to interact liposomes with antigen presenting cells is the primary theory of presenting antigens to the immune system using liposomes (23). The goal of this study was to develop a J8 liposome formulation (in the absence of an adjuvant) in which the lipophilic J8 construct was incorporated into the lipid bilayer and the hydrophilic carrier protein (DT) was encapsulated in the internal aqueous core. Materials and Methods

Mouse. All animal operations were approved for use by the Griffith University Research Ethics Review Board for Animal-Based Work, GU Ref No: GLY/09/14/AEC. This study was conducted in strict accordance with the National Health and Medical Research Council (NHMRC) of Australia guidelines. Methods for minimizing pain and pain in mice are selected and animals are observed daily by trained animal caregivers. The mice were terminated using a CO ₂ inhalation chamber.

Human blood. Blood was obtained from the donor at the Griffith university health centre with written consent. The study was approved by the Griffith University Human Research Ethics Committee (GU HREC, Protocol # GLY/03/14/HREC). Samples are de-identified before being processed by laboratory personnel.

J8-Lipo-DT preparation. To facilitate non-covalent complexation of J8 to the liposome bilayer, a hydrophobic anchor consisting of two palmitic acids (C16) is added to the tripeptide spacer presented at the amine end of J8 (consisting of Lys Ser Ser) The epsilon of the lysine and the primary amine (C16-C16-KSSJ8). This composition was manufactured by Chinapeptides Co., Ltd. (Shanghai, China). The expected molecular weight of this composition (MW 4061.97 g/mol) was confirmed by ESI-MS, and the obtained product had a purity of more than 95% (RP-HPLC area under analysis curve). Liposomes were prepared using a thin film hydration method (42). The lipid from Avanti Polar Lipids, Inc. (Alabaster, USA) is 7 dipalmitoyl-sn-glycerate-3-phosphocholine (DPPC): 2 cholesterol (CHOL): 1 L-α-phospholipid glycerol ( PG) is used by Mobi. The lipid in the chloroform (CHCl ₃ ) solution was coated with a predetermined amount of C16-C16-KSSJ8 in a round-bottom flask using a rotary evaporator. It is used in an amount of 0.7 ml of CHCl ₃ DPPC (10 mg / ml), in 0.2 ml CHCl ₃ in the CHOL (5 mg / ml), and the CHCl ₃ in 0.1 ml of PG (10 mg / ml). The lipid film was then hydrated by intimate mixing at room temperature and dispersed in 1 mL of phosphate buffered saline (PBS) containing a predetermined amount of DT. The resultant liposomal suspension was centrifuged at 16,162 g for 10 minutes, the supernatant was removed, and the liposome pellet was resuspended in an appropriate amount of PBS to be administered to the mice. To define the DT encapsulation rate, supernatants were collected and the amount of unpackaged DT in the supernatant was defined using a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific, Massachusetts, USA). The initial DT concentration in the rehydrated PBS for lipids was subtracted from the DT concentration of the supernatant to produce a predetermined quantitation encapsulation ratio for the liposomes. The average particle size (nm) of the liposomes was measured at 25 ° C using a disposable capillary cuvettes using a Nanosizer (Zetasizer Nano Series ZS, Malvern Instruments, United Kingdom). The dimensions were analyzed using a non-invasive backscatter system and taking measurements of the 173° scattering angle. The correlation time is based on a continuous operation of 10 seconds per operation and at least five measurements per measurement. The result was an average of three independent measurements repeated using Dispersion Technology Software (Malvern Instruments, UK). The Homogenous size distribution is defined by the low polydispersity index (PDI) of 0.238 as indicated by J8-Lipo-DT. PDI is a representation of how narrow the sample size distribution is, and a value greater than 0.7 indicates that the sample has a broad size distribution.

Intranasal immunization of mice. B10.BR and BALB/c (Animal Resources Centre, Western Australia, Australia) are mixtures of xylazine and ketamine (xylazine:ketamine:H) ₂ :1:1 mixture of ₂ O) anesthetize for immunization. Mice were administered 30 μg of J8-Lipo-DT alone in a total volume of 20 μL PBS (10 μL/nostril), while control mice were administered 20 μL of PBS (10 μL/nostril). Positive control mice received 30 μg of J8 conjugated to DT in total volume of 20 μL PBS and co-administered 10 μg of CTB (Sigma Aldrich, St. Louis, USA) ). Mice received two booster immunizations in the same manner as the primary immunization at 21 days apart. The other control groups received the above equivalent amounts of J8, DT or liposomes separately.

Collect serum, saliva and stool samples. Sera were collected 20 days, 40 days, and 60 days after the initial immunization to define the extent of J8-specific systemic antibodies. Blood is collected from the blood of the mouse via a tail artery and allowed to agglutinate for at least 30 minutes at 37 °C. Serum was collected after centrifugation at 1000 g for 10 minutes, heat inactivated at 56 °C for 10 minutes and stored at –20 °C.

Mice were administered 50 μL of a 0.1% solution of pilocarpine to induce salivation. Saliva was collected in a microcentrifuge tube containing 2 μL of 50 mmol/L phenylmethylsulfonyl fluoride (PMSF) protease inhibitor (Sigma Aldrich). The Particulate matter was separated by centrifugation at 13,000 g for 10 minutes and the sample was stored at −80 °C.

Six to ten fresh interstitial fecal pellets were collected from each mouse, frozen and then lyophilized. In feces by vortexing in 5% fat-free dry milk powder, 50 mmol/L EDTA (Sigma Aldrich), 0.1 mg/mL soybean trypsin inhibitor (Sigma Aldrich) and 2 mmol/L PMSF (20 μL) The dry weight of the fecal solids was determined before resuspending at /mg dry weight. The solid material was separated by centrifugation at 15,000 g for 10 minutes. Store the supernatant at −80 °C.

Antibody titers were defined by enzyme linked immunosorbent assay (ELISA) . As described above, ELISA is used to determine J8-specific serum IgG and mucosal IgA (43). The J8 peptide was diluted to 0.5 mg/ml in a carbonate coating buffer, pH 9.6, and coated on a polycarbonate plate at a volume of 100 μl/well at 4°. Place a night under C. Unbound peptides were removed and wells were blocked with 150 μl of 5% skim milk powder PBS-Tween 20 for 2 hours at 37 °C. The plates were then washed 3 times with PBS-Tween 20 buffer. Samples were serially diluted in plates with 0.5% skim milk powder PBS-Tween 20 buffer, starting with a 1:100 initial dilution of serum to a final dilution of 1:12,800, for saliva/fecal samples 1:2 to 1 :256. Each sample was diluted to a final volume of 100 μl and incubated at 37 ° C for 1.5 hours. The plate was washed 5 times and added to peroxidase conjugated goat anti-mouse IgG or IgA (Invivogen, San Diego, USA) diluted to 1:3000 or 1:1000 with 0.5% skim milk powder PBS-Tween20 at 37°. 1.5 hours under C. After washing, 100 μl of OPD matrix (Sigma Aldrich) was added according to the manufacturer's instructions and incubated for 30 minutes at room temperature in the dark. Absorbance at 450 nm was measured with a Victor ³ 1420 multilabel counter (Perkin Elmer Life and Analytical Sciences, Shelton, USA). The titer is described as the lowest dilution, which gives >3 standard deviation (SD) absorbance above the mean absorbance of negative control wells (containing normal mouse serum immunized with PBS). Significant differences ( p < 0.05) were defined using one-way analysis of variance (ANOVA) and Tukey post hoc detection using GraphPad Prism 5 software (GraphPad, California, USA).

Steps for GAS excitation. The immunized and control group mice were intranasally challenged with a predetermined dose of GAS strain M1 63 days after the primary immunization. GAS strain M1 has been serially passaged in the spleen of mice to enhance pathogenicity, and is resistant to streptomycin-resistant to distinguish GAS from normal murine bacterial flora in laryngeal wiping. (44). To define GAS colonization, a laryngeal wipe was obtained from the mice 1 to 3 days after challenge. The throat was wiped on a Todd-Hewitt agar plate containing 2% defibrinated horse blood and streaked and incubated at 37 ° C overnight. The bacterial load from the nose was obtained by pressing the nostrils of each mouse on the surface of a Columbia blood agar (CBA) disk ten times (three times CBA disk/mouse/day) and exhaled particles. Define by streaking. The mice were sacrificed on day 3, the organ samples were homogenized in PBS and the samples were plated three times using a pour plate method. For nasal outflow and throat swab, the results were expressed as mean colony forming units (CFU) + mean standard deviation (SEM) of 10 mice/group at 1 day, 2 days, and 3 days. For organ samples, the results are expressed as mean CFU + SEM on day 3 of 10 mice per group. The difference was analyzed by GraphPad Prism 5 using a parent-free, unpaired Mann-Whitney U assay comparing the test cohort with the PBS control cohort ( p < 0.05 was considered significant).

Preparation and maturation of DC . Peripheral blood was collected from healthy volunteers and fractionated by Ficoll-Paque (Amersham Pharmacia, Uppsala, Sweden (Sweden)) by standard procedures. A final density of PBMC of 1 x 10 ⁸ per 0.35 mL of MACS buffer (Miltenyi Biotec SL, Germany) was obtained by centrifugation, washing and resuspension by Ficoll Paque. The DC was separated using a Pan DC separation kit (Miltenyi Biotec) according to the manufacturer's instructions. Synthetic DC population resuspended in RPMI 1640 (Gibco, Gaithersburg, USA) complete medium (with 2 mM 1-l-glutamine, 1% non-essential amino acids, 1%) Pen-strep, 10 mM HEPES) and supplemented with 10% FCS (Gibco). Spread 0.2 mL of total volume of DCs (2 x 10 ⁶ ) and then add the following instructions for stimulation: 10 μg/mL of pIC (Invivogen, San Diego, USA), J8-Lipo-DT (150 μg/mL) or Stimulation of complete medium alone for 24 hours. The supernatant was collected after 24 hours and stored at -20oC.

Immune typing analysis by flow cytometry (Immunophenotype). For analysis of the surface representation of various markers, the treated DCs were stained with one or more of the following fluorophore-labeled mAbs and by using LSR Fortessa cytometer (Becton Dickinson, California, USA) and FlowJo software (Treestar, Inc., California, USA) Flow cytometry for analysis. Synthetic populations were determined by flow cytometry 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-Cy7. After staining with an appropriate antibody at 4 ° C for 30 minutes in the dark, the cells were washed twice with PBS and fixed with 1% paraformaldehyde. Gating is a large granular cell, and 2000–5000 framed events are collected from each sample. Briefly, HLA-DR positive cells were selected to define human DCs and further subdivided into CD141+1 conventional DCs (CDs), CD1c+ 2 DCs (bone marrow) DCs, and according to previously established methods. CD123+ plasma cell-like DC (45). The average fluorescence intensity (MFI) value is defined in the box selection group. Data were reported as mean + SEM and the difference was analyzed using Student's t-test using GraphPad Prism 5 software (GraphPad, California, USA). A P value of less than 0.05 was considered significant.

Splenocytes are stimulated with antigen in vitro. Flow microsphere array (CBA) analysis and flow cytometry analysis were used to quantify the pro-inflammatory response produced by splenocytes after stimulation with J8 peptide. Briefly, a single cell suspension of erythrocytes from the J8-Lipo-DT immunized mice was prepared in RPMI 1640 medium. Sprinkle a total volume of 0.1 mL of splenocytes (4 x 10 ⁵ ) and then stimulate with the following instructions: 2 μg/mL of LPS (Sigma Aldrich), J8 (10 μg/mL) or RPMI 1640 alone for 72 hours. . The supernatant was separated after 72 hours and stored at -80 °C for CBA flow cytometry analysis.

Chemical activin and interleukin secreted by CBA . The amount of accumulated inflammatory interleukins was quantified according to the manufacturer's instructions. For the mouse inflamed kit CBA, the amount of sample and standard was reduced to 10 μl, and 2 μl of capture bead was used for each. Supernatants from human DC cells were used in human inflammatory kit CBA (Becton Dickinson) according to the manufacturer's recommendations. The samples were run on an LSR Fortessa cytometer and the data was analyzed using FCAP array (v1.01 for Windows) software (Becton Dickinson). Data were reported as mean + mean standard deviation (SEM) and the differences were analyzed using the StudentPaint 5 software using Student's t-test. A P value of less than 0.05 was considered significant. result

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

Using primary and secondary-boost regimens, BALB/c mice (10 per group) were J8-Lipo-DT and each control group: liposome alone (Lipo); liposome encapsulated DT (Lipo-DT); J8 embedded Intranasal immunization was performed on the surface of liposomes but not in DT (J8-Lipo); J8-DT+CTB; PBS+CTB; and PBS.

To assess the efficacy of J8-Lipo-DT compared to other constructs, intranasal vaccinated mice were then challenged intranasally by the pharyngeal isolated M1 GAS strain obtained from patients with scarlet fever (16). Before challenge, we observed that J8-Lipo-DT induced higher J8-specific IgA (feces and saliva) and serum IgG titers than J8-Lipo. Although the difference between J8-Lipo-DT and J8-Lipo was not statistically significant in any of the groups, it was observed that J8-Lipo-DT has superior salivary IgA response, fecal IgA response, and serum IgG response (p. 2 Figure A to Part C). After the GAS challenge, the bacterial load in the nose, J8-Lipo-DT immunized mice were significantly lower than the PBS group, and comparable to the J8-DT+CTB immunized mice (Day 3, 3rd) Figure A)).

Surprisingly, J8-DT+CTB immunized mice did not protect the larynx or NALT colonization, whereas J8-Lipo-DT immunized mice showed significant prevention of colonization in both chambers (Fig. 3) Part B and Part C). The protection of J8-Lipo-DT is significantly better than that induced by J8-Lipo. Murine NALT is the entry point for persistent GAS infection (24) and is a functional homolog of the human tonsil (25). Therefore, this result highlights the efficacy of J8-Lipo-DT in reducing the bio-burden of the preferential site of mucosal GAS infection.

Next we explored whether J8-Lipo-DT also protects mice of different strains. J8-DT/CTB and PBS were used as control immunogens. B10.BR mice (n=5) induced significant J8-specific antibody titers with J8-Lipo-DT immunization (Fig. 4). The mucosal antibody titers in saliva and stool samples were comparable to those immunized with J8-DT+CTB (Fig. 4, Parts A and B). To test whether J8-Lipo-DT protected B10.BR mice from GAS infection, another group of mice (n=5) was immunized and challenged with the GAS M1 strain. Nasal outflow and throat swabs were monitored during 3 days of observation. By day 2, J8-Lipo-DT and J8-DT+CTB immunized mice had no bioburden detected in the nose (Fig. 5A). Similar to BALB/c mice, the data also showed that J8-Lipo-DT immunized mice had no bacteria in the throat wipe on the second day after challenge, whereas J8-DT+CTB immunized mice had a throat wipe on the third day. The amount of GAS can still be detected (Fig. 5, Part B).

Previous studies have shown that when a peptide encapsulated in a liposome cannot induce an immunoglobulin reaction, liposome plus lipid A (a component of lipopolysaccharides) can be induced after intra-peritoneal immunization. The antibody reacts (26), thus indicating that the lipid tail of lipid A can act as an adjuvant. To investigate whether the double C16 lipid tail that immobilizes J8 on the surface of liposomes is responsible for the induction of antibody responses, another group of mice were immunized with C16-C16-KSSJ8, J8-Lipo-DT, J8-DT+CTB or PBS. . We observed that J8-Lipo-DT and J8-DT+CTB were immunogenic, whereas C16-C16-KSSJ8 did not (Fig. 6 Part A and Part B).

The interleukin response of spleen cells from mice immunized intranasally was determined to define whether intranasal immunization induces a systemic cellular immune response, which may explain the self adjuvantity of J8-Lipo-DT and the IgA Switch of isotype antibody. Analysis of proinflammatory interferon (gamma interferon [IFN-γ]), interleukin 1 [IL-1], IL-6, IL-12p70, monocyte chemotactic protein 1 (monocyte chemotactic protein 1) ) [MCP-1] and tumor necrosis factor alpha [TNF-α]). B10.BR mice immunized with J8-Lipo-DT were sacrificed and splenocytes were stimulated with J8, LPS or medium. We observed that significant IFN-γ, MCP-1 and IL-6 were produced corresponding to J8 and LPS (Fig. 7). No other interleukins were assessed for evaluation. This result demonstrates that vaccination with J8-Lipo-DT induces a pro-inflammatory response, providing a potential mechanism for the autoadjuvant of J8-Lipo-DT. In particular, IL-6 is known to be responsible for the antibody response to switch IgA (27). In addition, the chemotactic MCP-1 is known to play a major role in the GAS defense mechanism (28).

To assess the possibility of J8-Lipo-DT having autologous adjuvant activity in humans to elicit an effective immune response, dendritic cell subsets were isolated from the blood of three healthy volunteers and stimulated with J8-Lipo-DT. Mature DCs are potent antigen-presenting cells that exhibit high levels of cell surface molecules involved in antigen presentation and co-stimulation that facilitates antigen recognition and cell-cell interactions. To characterize the maturation of human DCs, the regulation of various cell surface molecules corresponding to J8-Lipo-DT was determined by flow cytometry (Figure 8, Panels A to C). A synthetic double-stranded RNA adjuvant, polyriboadenosyl-polyribose cytidine (pIC) was used as the control group (29). The amount of costimulatory molecules CD80, CD83 and CD86 was significantly higher in CD123+ plasmacytoid DCs (pDCs) cultured in J8-Lipo-DT (Fig. 8 Panel A). The performance of CD80 was also increased in two subsets of CD141+typical type 1 DC and CD1c+typical type 2 DC typical DCs (cDC) (Fig. 8 Part B to Part C). In addition, CD86 performance also increased in CD141+ DC (Fig. 8B).

To further determine interaction with human DCs, the amount of proinflammatory cytokines after stimulation was assessed using a flow microsphere array. We observed an increase in the expression of proinflammatory interleukins (TNF-α, IL-6 and IL-1 beta (IL-1 β)) and neutrophil chemoattractant IL-8 (Fig. 9). Neutrophils are known to be the key to IgA control of GAS infection (30). An increase in the amount of anti-inflammatory interleukin IL10 was also observed (Fig. 9). It may be due to the regulation of IL-10 in the DC maturation step and to counteract the host's inflammatory response (31, 32). However, in particular the IL-6 response showed that J8-lipo-DT would cause a human IgA switch and, together with a neutrophil response (via IL-8), would help the host to control GAS infection. As shown in Figure 10, when both are presented alone or as S2-J8 chimeras (SEQ ID NO: 3), the SpyCEP peptide (S2; SEQ ID NO) presented by the liposome together with the intracapsular DT : 2) Inducing mucosal IgA response.

Referring to Figure 11, J8+S2-Lipo-DT induces antigen-specific IgA and IgG responses. A comparable immune response was observed corresponding to J8-Lipo-DT and S2-Lipo-DT. Different formulation strategies employ (i) a mixture of two epitopes in the liposome (J8+S2-Lipo-DT) or (ii) J8/S2S2-Lipo-DT liposomes.

Group A streptococcal infections can cause a variety of skin and soft tissue infections, some of which are severe or even life-threatening. It is therefore of interest to see if J8-Lipo-DT can prevent skin infections after challenge with the 88/30 strain. Initial experiments with intranasal administration of liposomes containing only DT and J8 showed significant IgG titers after intranasal immunization with J8-Lipo-DT, but showed no protection in skin challenge tests (data not shown) ). Systemic IgG responses are currently being enhanced by immunization with different liposomal formulations or by subcutaneous administration of J8-DT + alum. discuss

We have developed a mucosal activator cell liposome vaccine candidate for group A streptococci (GAS). The immunostimulatory properties of the liposome bind to the encapsulation of the protein carrier DT and present a GAS-specific B cell epitope on the surface of the liposome. Both the peptide and the carrier protein are required for optimal immunity. Mucosal immunity induced by complex liposomes is superior to induction by peptide-protein and CTB conjugate administration.

Mucosal immunity has attracted much attention as a way to induce protective immunity against infectious diseases. The vast majority of infections occur on the mucosal surface or from the mucosal surface. Therefore, vaccine applications that induce mucosal protective immune responses are desirable and practical. In particular, the use of subunit peptide antigens often proves to be difficult to stimulate a strong mucosal IgA immune response and the progress in mucosal vaccination results is disappointing. Part of it is difficult to stimulate an effective immune response compared to the traditional whole-organism basic method. The addition of an adjuvant and conjugation of the subunit antigen to the carrier protein as a source of T cell help proves to be effective for systemic immunity. However, the induction of mucosal immunity requires novel strategies.

The topographical position of the antigen-associated liposomes affects antigen processing and presentation of B cells and helper T cells (33). It has been demonstrated that antigens exposed on the surface of liposomes are preferentially treated and presented by B cells, while antigen-coated liposomes are more efficiently processed and presented to T cells by antigen presenting cells (34). Therefore, the vaccine candidate J8-Lipo-DT has a well-designed subunit liposome vaccine design, ensuring that the B cell epitope is associated with the liposome bilayer and the Ig receptor (receptor) bound to the B cell is exposed, while the DT coating Allow efficient delivery, processing and presentation to T cells.

We have demonstrated clearance of GAS in URT tissue, which includes NALT. Nasopharyngeal immunity effective by vaccine candidates has the potential to reduce the potential of RF and RHD associated with primary pharyngeal infection (7). In human URT, the tonsils are the main accumulates of GAS, which are persistent endemic diseases worldwide (25). Reduction of GAS colonization in functional homologous NALT in human tonsils suggests that intranasal immunization with J8-Lipo-DT will reduce colonization and infection of human tonsils, thus reducing GAS transmission (25).

Although liposomes have been previously reported to encapsulate peptides to induce cellular immune responses, in addition to the presence of effective adjuvants, such as lipid A, the liposomes do not induce IgA or IgG responses (26, 35). Since the lipid tail on J8 may provide adjuvant activity, it contributes to the immunogenicity of J8-Lipo-DT; however, all of the J8 peptides with lipid tails do not demonstrate immunogens for liposome preparations. Sexual needs. The autologous adjuvant immunostimulatory activity of liposomes has been previously reported and is shown to be due to cell interaction with the antigen and induce a pro-inflammatory response (36). In vitro assays in this study showed induction of antigen-specific inflammatory chemical activins and interleukins in immunized mice. Of concern is the secretion of antigen-specific MCP-1 and IL-6. MCP-1 is a chemoattractant that presents cells to lymphocytes, monocytes, and antigens (37). Previously associated mediator mucosal inflammation has been reported as a possible mucosal adjuvant due to a significant increase in mucosal IgA secretion (37).

Although antigens can be presented to the immune system by various cell types, the primary naive T cells require maturation, antigen presentation, and involvement of co-stimulatory molecules found only in specialized antigen-presenting cells, such as DCs ( 38). DC is a key element in bridging congenital and adaptive immune responses to infection (39). Mature DCs produce inflammatory interleukins, up-regulate co-stimulation and antigen-presenting molecules and move to lymph nodes, which function to present cells as effective antigens to naive T cells to initiate an adaptive immune response. Based on the induction of proinflammatory cytokines exposed in vitro and containing IL-6 and IL-8, we have demonstrated that J8-Lipo-DT mediates the expression of cell surface activation and maturation markers on human DCs. Human and murine IL-6 play a key role in B cell terminal differentiation and stimulate proliferation at the mucosal site and secrete IgA at the mucosal site (27). IgA-specific immunity against GAS requires the presence of neutrophils (30). IL-8 is critical in the recruitment and activation of neutrophils. In this regard, administration of the SpyCEP S2 peptide (SEQ ID NO: 2) is presented by a separate liposome particle delivery system or in combination with J8 peptide-inducing peptide-specific IgA.

Therefore, the underlying mechanisms that confer human immunity to GAS infection can be mediated through the liposome platform, which has important implications for the relevance of clinical basic research.

We demonstrate that human pDC increases both maturation and co-stimulatory markers after stimulation with J8-Lipo-DT. Human pDCs readily phagocytose and process antigens (40) trapped in the particle delivery system, which means that the particle delivery system can be used to facilitate efficient delivery of antigen to pDC. To the best of our knowledge, our results show for the first time that liposome-like particle delivery systems can stimulate human pDC. Human pDC is initially identified in the blood and subsequently detected in the spleen, lymph nodes, and mucosal sites containing the tonsils (41). Thus, liposome vaccine delivery can potentially be utilized in humans to target DC subsets for the desired mucosal immune response. Example 2

Experiments were conducted to investigate the effect of liposome size on immunogenicity.

Liposomal extrusion was performed with a heat block, 1 mL syringe-mini-extruder (Avanti Polar Lipids). The rehydrated solution was passed through a 50 nm, 400 nm, 1000 mm filter (Avanti Polar Lipids) 11 times while the thermal block was set at ~40 °C. Liposomal size measurements were performed with Nanosizer (Dynamic Light Scattering or DLS).

As shown in Figure 12, J8-Lipo-DT can be extruded to form nano-micron sized particles. The primary particle size has a narrow molecular-weight distribution (< low polydispersity index of 0.3). The data shown in Figure 13 shows that the J8-Lipo-DT size does not affect the systemic IgG response. However, as shown in Figure 14, larger size liposomes induce a J8-specific mucosal response. Example 3

Experiments were conducted to investigate the effect of freeze-dried liposomes on immunogenicity. The liposome film was rehydrated with milliQ water containing 10% trehalose and then lyophilized. The J8-Lipo-DT powder was reconstituted in PBS after freeze drying for 1 week, 4 weeks, and 7 weeks.

Figure 15 shows the size of the liposome size results as measured by Nanosizer (Dynamic Light Scattering or DLS). The main particle size has a narrow molecular-weight distribution (<low polydispersity index of < 0.3). Figure 16 shows that the reconstituted, freeze-dried J8-Lipo-DT liposomes induced a J8-specific systemic response without the addition of an adjuvant. It has a comparable immune response to freshly made J8-Lipo-DT. Trehalose is important for the immunogenicity of freeze-dried J8-Lipo-DT. Figure 17 demonstrates that the reconstituted, freeze-dried J8-Lipo-DT liposomes induce a J8-specific mucosal response. Example 4

Further experimental definitions, as shown in Figure 18, schematically illustrate the efficacy of immunogenic liposomes containing innate immune glycolipid activators such as trehalose-6,6'-dibehenate (TDB) and 3D PHAD® . TDB is formulated with liposomes based on % of total phospholipids in the liposomes. 9 mg of phospholipid was used and its 20% ratio of TDB (1.8 mg) was used. Efficacy was determined by antibody titer after immunization (IgA and IgG antibodies) and skin challenge experiments were performed with GAS strain. The data is shown in Figure 19. Mice (n=5 per group) were immunized intranasally via 30 μg of J8-Lipo-Dt+TDB. Mice were administered initially (Day 0) and boosted twice (Day 21 and Day 42). Compared with J8-Lipo-DT, the results of the combination with TDB were significantly higher in mucosal IgA responses in both saliva and fecal samples. This result is derived from the freeze-dried powder protocol of J8-Lipo-DT+TDB, which enhances the stability of the liposomes.

Further experiments will define the efficacy of immunogenic liposomes containing a bile salt such as sodium deoxycholate. An example of a liposome containing sodium bile salt deoxycholate is schematically illustrated in Figure 20. It should be noted that the immunogenic agent (this example refers to the J8 peptide) can be fused or conjugated to a carrier protein (eg, DT) or can be presented on the surface of the liposome. When the phospholipid is dehydrated to produce a liposome, the bile salt preparation is referred to as a liposome (the bile salt containing the liposome is referred to as "bilosomes"). The preparation of bile salts is as follows.

A round bottom flask with 150 μg of J8 modified with palmitic acid, sorbitan tristearate (150 mmol), cholesterol and dihexadecyl phosphate (DCP) in molar ratio 7:3:1 was dissolved in 10 mL of chloroform. The solvent was removed by a rotary evaporator to form a film on the glass surface of the original flask. Next, the film was hydrated with 150 μg of diphtheria toxoid in 3.5 mL of PBS (pH 7.4) containing 100 mg of sodium deoxycholate (bile salt). Example 5

The immunogenicity of lipid vesicles for influenza A, influenza B, and group A streptococci is determined by antigen-specific salivary IgA titers. 21A and 21B are schematic diagrams showing an immunogenic reagent comprising a single lipid having respective immunogens from each of influenza A, influenza B, and group A streptococci. Vesicles. The results shown in Fig. 22 show that lipid vesicles (as shown in Fig. 21A) containing the respective immunogens from each of influenza A, influenza B, and group A streptococcus are induced against mice. Immunization of each of the above pathogens. As schematically illustrated in Figure 21B, further studies will explore the immunogenicity of immunogenic agents comprising glycolipid adjuvants.

In conclusion, this result is the first report of a mucosal activated GAS vaccine candidate for liposomes. Our findings in the development of GAS vaccines are important steps to overcome current barriers to prevent infection and community infection at mucosal sites. This study provides an important institutional perspective on how liposome microparticle delivery systems can collectively induce a desired mucosal immune response against GAS infection. The strategies contained herein are related to the development of subunit mucosal vaccines against other pathogenic organisms. Non-limiting examples include the aforementioned influenza virus, rhinovirus, and hookworm. In some embodiments, a single lipid vesicle can comprise an immunogen against a plurality of different pathogens.

Throughout the specification, the present invention is intended to describe the preferred embodiments of the invention, and not to limit the invention to any particular embodiment. Various changes and modifications can be made to the described embodiments and the embodiments described herein without departing from the scope of the invention.

All computer programs, algorithms, patents, and scientific literature referred to herein are hereby incorporated by reference. Reference material

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21. Childers NK; Tong G and Michalek SM (1997), nasal immunization of humans with liposomes containing S. mutans antigens. Oral Microbiology and Immunology, 12(6): 329-335. (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 and Toth I (2013), nano vaccine and its mode of action. Method, (San Diego, Calif.) , 60(3): 226-231. (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), a liposome as a carrier for antigens and adjuvants. Journal of Immunology Methods, 140(1): 1-13. (Alving CR (1991) Liposomes as carriers of antigens and adjuvants. Journal of immunological methods 140(1): 1-13.)

24, Cleary PP; Zhang Y and Park HS (2004), nasal related lymphoid tissue and M cells, a window against persistent streptococcal infection. Indian Journal of Medical Research, 119 Suppl: 57-60. (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 and Cleary PP (2005), active and passive intranasal immunization with the streptococcal surface protein C5a peptide to prevent the functional homology of the human tonsils and the infection of lymphoid tissues associated with murine nasal mucosa. Infection and Immunity, 73(12): 7878-7886. (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), Reaction of antibodies and cytotoxic T lymphocytes to a peptide antigen associated with a single liposome. Vaccine, 13(12): 1111-1122. (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), interleukin and IgA synthesis. Human and mouse interleukin-6 induce high IgA secretion in IgA committed B cells. Journal of Experimental Medicine, 169(6): 2133-2148. (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 and Medina E (2010), an abnormal inflammatory response to S. pyogenes in mice lacking myeloid differentiation factor 88. American Journal of Pathology, 176(2): 754-763. (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), Polyriboadenosine: Polyribonucleotide (poly(I:C)) induces functional activation of stable maturation of human dendritic cells. Journal of Immunology (Baltimore, Md.: 1950) 163(1): 57-61. (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 the specificity of IgA antibodies to the retained epitopes in the M protein of Group A Streptococcus in the original Australian endemic community. International Immunology, 11(4): 569-576. (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), the proinflammatory mediators of interleukin-1β (IL-1β), IL-6, IL- in children with severe falciparum malaria and matching complex malaria or health control groups. 8. Serum levels of IL-10, tumor necrosis factor alpha and IL-12 (p70). Infection and Immunity 72 (10): 5630-5637. (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 immunity 72(10):5630-5637.)

32. Samarasinghe R et al. (2006), induction of anti-inflammatory interleukin IL10 in dendritic cells following a toll-like receptor signal. Journal of Interferon and Interleukin Research: Official Journal of International Association of Interferon and Interleukin Studies, 26(12): 893-900. (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 and Szoka FC, Jr. (1989), Effects of liposome encapsulation on antigen presentation in vitro. A comparison presented by peritoneal macrophages and B cell tumors. Journal of Immunology (Baltimore, Md.: 1950) 142(5): 1437-1443. (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 and Unanue ER (1991), liposome-encapsulated antigens in lysosomes, treatment and expression of T cells. Cell, 64(2): 393-401. (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 and Kida H (2002), intranasal administration of a synthetic peptide vaccine encapsulated in liposomes with anti-CD40 antibodies in mice to induce anti-influenza A The protection of the virus is immune. Vaccine, 20 (25-26): 3123-3129. (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 a vaccine delivery system: a review of recent advances. Progress in the treatment of vaccines, 2 (6): 159-182. (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 and Ferrari MG (2005), mucosal adjuvant. Current medical design, 11 (6): 801-811. (Stevceva L & Ferrari MG (2005) Mucosal adjuvants. Current pharmaceutical design 11(6): 801-811.)

38. Gamvrellis A et al. (2004), a vaccine that promotes entry of antibodies into dendritic cells. Immunology and Cell Biology, 82(5): 506-516. (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. (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, treat and present exogenous particle antigens. Journal of Immunology (Baltimore, Md.: 1950) 184(8): 4276-4283. (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 and Ginhoux F (2014), comparing true dendritic cell populations across species. Cellular Immunology, 291 (1-2): 3-10. (Dutertre CA, Wang LF, & Ginhoux F (2014) Aligning bona fide dendritic cell populations across species. Cellular immunology 291(1-2): 3-10.)

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43. Zaman M et al. (2014), Group A Streptococcal Vaccine Candidates: Contribution of epitopes to size, antigen-presenting cell interaction, and immunogenicity. Nanomedicine (London, UK), 9(17): 2613-2624. (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 and Good MF (2002), protecting mice from A by intranasal immunization with a peptide vaccine containing a M protein B cell epitope and a T cell self epitope. Group of streptococcal infections. Vaccine, 20 (21-22): 2816-2825. (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 and Radford KJ (2010), Separation of human blood DC subtypes. Methods of Molecular Biology (Clifton, NJ) , 595:45-54. (Kassianos AJ, Jongbloed SL, Hart DN, & Radford KJ (2010) Isolation of human blood DC subtypes. Methods in molecular biology (Clifton, NJ) 595:45-54.)

no

Figure 1 is an idealized structure of J8-Lipo-DT. The liposome encapsulates DT, while J8 attached to the N-terminal spacer KSS is covalently coupled to two palmitic acid molecules to facilitate insertion of J8 into the liposome membrane.

Figure 2 is the response of J8-specific antibodies to individual BALB/c mice. The average antibody titer is represented by a histogram. A) IgA titer for saliva. B) is the IgA titer of feces. C) is the IgG titer of the serum. Statistical analysis was performed after Tukey post hoc test using one-way ANOVA (ns, p >0.05; *, p <0.05; **, p <0.01; ** *, p < 0.001).

Figure 3 is the Bacterial burden after intranasal challenge of BA1/C mice with M1 GAS strain. A) Nasal shedding. B) Wipe for the pharynx. C) is the colonization of NALT. The results of throat swabs, nasal outflows, and NALT on day 3 of day 10 to day 3 of 10 mice/population were expressed as mean CFU + SEM. Statistical analysis was performed using a nonparametric, unpaired Mann-Whitney U assay for comparison of test cohorts with PBS control cohorts (ns, p >0.05; *, p <0.05; **, p <0.01; ***, p < 0.001).

Figure 4 is the response of J8-specific antibodies to individual B10.BR mice (n=5 per group). The average antibody titer is represented by a histogram. A) IgA titer for saliva. B) is the IgA titer of feces. C) is the IgG titer of the serum. Statistical analysis was performed after Tukey's post hoc test using single factor variance analysis (ns, p >0.05; *, p <0.05; **, p <0.01; ***, p < 0.001).

Figure 5 is a URT GAS challenge mode to assess the bacterial load after intranasal challenge with the M1 strain. A) is the nasal outflow of B10.BR mice. B) Wipe the throat of B10.BR mice. The results of 5 mice/population on day 1 to day 3 were expressed as mean CFU + SEM. Statistical analysis was performed using a Mann-Whitney U assay with no parent number and no pairing to compare the test cohort with the PBS control cohort (ns, p >0.05; *, p < 0.05).

Figure 6 is the response of J8-specific antibodies to individual BALB/c mice. The average antibody titer is represented by a histogram. A) IgA titer for saliva. B) is the IgG titer of the serum. Statistical analysis was performed after Tukey's post hoc test using single factor variance analysis (ns, p >0.05; *, p <0.05; **, p <0.01; ***, p < 0.001).

Figure 7 is a chemical activin and interleukin secreted specifically by antigen in immunized mice. Splenocytes were plated and subsequently stimulated by the following instructions: LPS (2 μg/mL), J8 (10 μg/mL) or medium alone. After 72 hours of stimulation, the supernatant was separated and the amount of released chemical activin or interleukin was analyzed using a cytometric bead array (reference materials and methods). Statistical analysis was performed using Student's t test (ns, p >0.05; *, p <0.05; **, p < 0.01).

Figure 8 is the amount of surface markers in the human DC subsets with and without reagents. Stimulation was then added as follows: polyinosinic: polycytidylic acid (pIC, 10 μg/mL), J8-Lipo-DT (150 μg/mL) or medium alone. Cell surface markers were determined by flow cytometry 24 hours after stimulation. A) is CD123+ plasmacytoid DC. B) is CD141+ classic type 1 DC. C) is CD1c+classical type 2 DC. Values are expressed as the median fluorescence intensities (MFI) from the data of the three respective donors ± SEM. Statistical analysis was performed using a Mann-Whitney U assay with no parent number and no pairing to compare test groups with media control groups (ns, p >0.05; *, p <0.05; **, p <0.01; ***, p < 0.001).

Figure 9 shows J8-Lipo-DT inducing the secretion of chemical activin and interleukin in human dendritic cells. Dendritic cells were plated out and subsequently stimulated by the addition of the following instructions: pIC (10 μg/mL), J8-Lipo-DT (150 μg/mL) or medium alone. The supernatant was separated after 24 hours of stimulation and the amount of released chemical activin or interleukin was analyzed using a flow microsphere array (reference materials and methods). Statistical analysis was performed using Student's t test (ns, p >0.05; *, p <0.05; **, p <0.01; ***, p < 0.001).

Figure 10 is a mucosal IgA response induced by a lipid delivery reagent of SpyCEP peptide (S2; SEQ ID NO: 2). Liposomes showing palmitate lipidated S2 peptide or S2-J8 chimera (SEQ ID NO: 3) liposomes were administered intranasally to mice with intracapsular DT and assayed for S2 specific IgA efficacy price.

Figure 11 shows the antigen-specific IgA and IgG responses in mice induced by the J8+S2-Lipo-DT immunogenic reagent.

Figure 12 is a J8-Lipo-DT immunogenic reagent that can be extruded to form nano- to micro-sized particles. Liposomal size was determined by Nanosizer assay (Dynamic Light Scattering or DLS).

Figure 13 is the size of the J8-Lipo-DT immunogenic reagent does not affect the systemic IgG response.

Figure 14 is a larger size J8-Lipo-DT immunogenic reagent that induces a J8-specific mucosal response.

Figure 15 is the size distribution of the freeze-dried J8-Lipo-DT powder reconstituted in PBS. Liposomal size was determined by Nanosizer assay (Dynamic Light Scattering or DLS).

Figure 16 is a recombinant, freeze-dried J8-Lipo-DT liposome immunogenic agent that induces a J8-specific systemic response without additional adjuvant.

Figure 17 is a recombinant, freeze-dried J8-Lipo-DT liposome immunogenic reagent that induces a J8-specific mucosal response.

Figure 18 is a schematic representation of a liposome J8-Lipo-DT immunogenic reagent comprising the glycolipid adjuvant trehalose-6,6'-dibehenate (TDB).

Figure 19 shows TDB enhancing the mucosal IgA response induced by J8-Lipo-DT. Mice (n=5 per group) were immunized intranasally with 30 μg of J8-Lipo-DT+TDB. The mice were administered on the initial (Day 0) and two boosts (Day 21 and Day 42). The results in combination with TDB showed a significantly higher mucosal IgA response in both saliva and fecal samples than in the administration of J8-Lipo-DT. This result stems from the freeze-dried powder protocol of J8-Lipo-DT+TDB, which eliminates the problem of liposome stability.

Figure 20 is a schematic representation of a liposome immunogenic reagent comprising sodium bile salt deoxycholate.

Figure 21A is an immunogenic protein containing or containing immunolipids from lipid vesicles, intracapsular DT, and a plurality of different pathogens (ie, influenza A, influenza B, and group A streptococci). Schematic diagram of a single immunogenic reagent for a protein; Figure 21B shows an immunogen comprising a lipid vesicle, an intracapsular DT, and a plurality of different pathogens (ie, influenza A, influenza B, group A streptococci) Protein or immunogenic protein and glycolipid adjuvant (trehalose-6,6'-dibehenate (TDB) and monophosphoryl 3-deacyl Schematic representation of a single immunogenic reagent of Lipid A, 3D-PHAD®)).

Figure 22 compares the single immunogenic reagents for influenza A, influenza B, and group A streptococci by the measurement of antigen-specific salivary IgA titre ("Multivax The immunogenicity of ") is compared to individual vaccines ("Single Antigen-vax") for individual immunogenic agents for each of influenza A, influenza B, and group A streptococci.

<110> Griffith University <120> Liposomal Vaccine <130> 31639TW3 <150> AU2015904403 <151> 2015-10-27 <150> TW105111153 <151> 2016-04-08 <150> AU2016901026 <151> 2016-03-18 <160> 15 <170> PatentIn Version 3.5 <210> 1 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 1 Gln Ala Glu Asp Lys Val Lys Gln Ser Arg Glu Ala Lys Lys Gln Val 1 5 10 15 Glu Lys Ala Leu Lys Gln Leu Glu Asp Lys Val Gln 20 25 <210> 2 <211> 20 <212> PRT <213> Purulent chain Streptococcus pyogenes <400> 2 Asn Ser Asp Asn Ile Lys Glu Asn Gln Phe Glu Asp Phe Asp Glu Asp 1 5 10 15 Trp Glu Asn Phe 20 <210> 3 <211> 48 <212> PRT <213> Artificial sequence <220> <223> Synthetic chimeric peptide <400> 3 Asn Ser Asp Asn Ile Lys Glu Asn Gln Phe Glu Asp Phe Asp Glu Asp 1 5 10 15 Trp Glu Asn Phe Gln Ala Glu Asp Lys Val Lys Gln Ser Arg Glu Ala 20 25 30 Lys Lys Gln Val Glu Lys Ala Leu Lys Gln Leu Glu Asp Lys Val Gln 35 40 45 <210> 4 <211> 20 <212> PRT <213> Streptococcus pyogenes <400> 4 Leu Arg Arg Asp Leu Asp Ala Ser Arg Glu Ala Lys Lys Gln Val Glu 1 5 10 15 Lys Ala Leu Glu 20 <210> 5 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 5 Ser Arg Glu Ala Lys Lys Gln Ser Arg Glu Ala Lys Lys Gln Val Glu 1 5 10 15 Lys Ala Leu Lys Gln Val Glu Lys Ala Leu Cys 20 25 <210> 6 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide variant < 400> 6 Ser Arg Glu Ala Lys Lys Gln Ser Arg Glu Ala Lys Lys Gln Val Glu 1 5 10 15 Lys Ala Leu Lys Gln Ser Arg Glu Ala Lys Cys 20 25 <210> 7 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide variant <400> 7 Ser Arg Glu Ala Lys Lys Gln Val Glu Lys Ala Leu Lys Gln Ser Arg 1 5 10 15 Glu Ala Lys Lys Gln Val Glu Lys Ala Leu Cys 20 25 <210> 8 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide variant <400> 8 Ser Arg Glu Ala Lys Lys Gln Val Glu Lys Ala Leu Asp Ala Ser Arg 1 5 10 15 Glu Ala Lys Lys Gln Val Glu Lys Ala Leu Cys 20 25 <210> 9 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Modeled peptide <400> 9 Met Lys Gln Leu Glu Asp Lys 1 5 <210> 10 <211> 7 <212> PRT <213> Artificial sequence < 220> <223> Analog peptide <400> 10 Val Lys Gln Leu Glu Asp Lys 1 5 <210> 11 <211> 24 <212> PRT <213> Influenza A virus <400> 11 Met Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly 1 5 10 15 Cys Arg Cys Asn Asp Ser Ser Asp 20 <210> 12 <211> 19 <212> PRT <213> Type B Epidemic Cold Virus (Influenza B virus) <400> 12 Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala Gly 1 5 10 15 Phe Leu Glu <210> 13 <211> 31 <212> PRT <213> Rhinovirus <400> 13 Gly Ala Gln Val Ser Thr Gln Lys Ser Gly Ser His Glu Asn Gln Asn 1 5 10 15 Ile Leu Thr Asn Gly Ser Asn Gln Thr Phe Thr Val Ile Asn Tyr 20 25 30 <210> 14 <211> 24 <212> PRT <213> Rhinovirus <400> 14 Gly Ala Gln Val Ser Arg Gln Asn Val Gly Thr His Ser Thr Gln Asn 1 5 10 15 Met Val Ser Asn Gly Ser Ser Leu 20 < 210> 15 <211> 22 <212> PRT <213> Necator americanus <400> 15 Thr Ser Leu Ile Ala Gly Leu Lys Ala Gln Val Glu Ala Ile Gln Lys 1 5 10 15 Tyr Ile Gly Ala Glu Leu 20

no

Claims (48)

An immunogenic agent suitable for administration to a mammal comprising one or more immunogenic proteins, fragments thereof, variants or derivatives thereof, a lipid vesicle, a carrier protein or a fragment thereof or a variant thereof. The immunogenic reagent of claim 1, which is suitable for intranasal administration. The immunogenic agent according to claim 1, which is capable of inducing a mucosal immune response in the mammal. The immunogenic agent of claim 1, wherein the carrier protein is located in an intracapsular space. The immunogenic agent according to claim 1, wherein the carrier protein is diphtheria toxoid (DT). The immunogenic agent according to claim 1, wherein the immunogenic protein, a fragment thereof, a variant thereof or a derivative thereof is present on the surface of the lipid vesicle. The immunogenic agent according to claim 1, wherein the immunogenic protein, a fragment thereof, a variant thereof or a derivative thereof is fused or conjugated to the carrier protein. The immunogenic agent according to claim 1, wherein the lipid vesicle is a liposome. The immunogenic agent according to claim 1, which comprises one or a plurality of immunogenic protein fragments, variants or derivatives thereof, or one or a plurality of immunogenic proteins from the pathogen A fragment, a variant thereof or a derivative thereof. An immunogenic agent according to claim 1, which comprises one or a plurality of immunogenic protein fragments, variants or derivatives thereof, or a plurality of different pathogens from the plurality of different pathogens One of each or a plurality of immunogenic protein fragments, variants thereof or derivatives thereof. The immunogenic agent according to claim 9, wherein the pathogen is a bacterium, a virus or a parasite. The immunogenic agent according to claim 1, wherein the immunological protein, a fragment thereof or a variant thereof is a fragment, variant or derivative of the group A Streptococcus M protein. The immunogenic agent according to claim 12, wherein the fragment is in a J8 peptide or J8 peptide variant or comprises the J8 peptide or J8 peptide variant. The immunogenic reagent according to the invention of claim 13, wherein the J8 peptide is or comprises the amino acid sequence QAEDKVKQSREAKKQVEKALKQLEDKVQ (SEQ ID NO: 1) or mainly the amino acid sequence QAEDKVKQSREAKKQVEKALKQLEDKVQ (SEQ ID NO: 1) ) composed of. The immunogenic agent of claim 1, wherein the immunogenic protein is or comprises an agent that facilitates recovery or enhancement of neutrophil activity. The immunogenic agent according to claim 15, wherein the agent which is beneficial for restoring or enhancing neutrophil activity is a protein of SpyCEP or a fragment of SpyCEP. The immunogenic reagent according to claim 16, wherein the SpyCEP fragment is or comprises the amino acid sequence NSDNIKENQFEDFDEDWENF (SEQ ID NO: 2) or mainly consists of the amino acid sequence NSDNIKENQFEDFDEDWENF (SEQ ID NO: 2) Composed of. The immunogenic agent according to claim 1, wherein the immunogenic protein, a fragment thereof or a variant thereof comprises a SpyCEP fragment and an M protein fragment as a single chimeric peptide. The immunogenic reagent according to claim 18, wherein the chimeric peptide is or comprises an amino acid sequence NSDNIKENQFEDFDEDWENFQAEDKVKQSREAKKQVEKALKQLEDKVQ (SEQ ID NO: 3) or a variant thereof, or mainly consists of an amino acid sequence NSDNIKENQFEDFDEDWENFQAEDKVKQSREAKKQVEKALKQLEDKVQ (SEQ ID NO: 3) or a variant thereof. The immunogenic agent according to claim 1, wherein the immunogenic protein, a fragment thereof or a variant thereof is an immunogenic protein, fragment or variant of influenza virus. The immunogenic agent according to claim 20, wherein the immunogenic protein, a fragment thereof or a variant thereof thereof comprises or comprises an amino acid sequence MSLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO: 11) or PAKLLKERGFFGAIAGFLE (SEQ ID NO: 12) or consists essentially of the amino acid sequence MSLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO: 11) or PAKLLKERGFFGAIAGFLE (SEQ ID NO: 12). The immunogenic agent according to claim 1, wherein the immunogenic protein, a fragment thereof or a variant thereof is an immunogenic protein, fragment or variant of rhinovirus. The immunogenic agent according to claim 22, wherein the immunogenic protein, a fragment thereof or a variant thereof thereof comprises or comprises an amino acid sequence GAQVSTQKSGSHENQNILTNGSNQTFTVINY (SEQ ID NO: 13) or GAQVSRQNVGTHSTQNMVSNGSSL (SEQ ID NO: 14) or consists essentially of the amino acid sequence GAQVSTQKSGSHENQNILTNGSNQTFTVINY (SEQ ID NO: 13) or GAQVSRQNVGTHSTQNMVSNGSSL (SEQ ID NO: 14). The immunogenic agent according to claim 1, wherein the immunogenic protein, a fragment thereof or a variant thereof is an immunogenic protein, fragment or variant of hookworm. The immunogenic agent according to claim 24, wherein the immunogenic protein, a fragment thereof or a variant thereof thereof comprises or comprises the amino acid sequence TSLIAGLKAQVEAIQKYIGAEL (SEQ ID NO: 15) or is mainly composed of an amino acid sequence. Composition of TSLIAGLKAQVEAIQKYIGAEL (SEQ ID NO: 15). The immunogenic agent of claim 1, further comprising an innate immune activator. The immunogenic agent according to claim 26, wherein the innate immune activator is or comprises a glycolipid. The immunogenic agent according to claim 26, wherein the innate immune activator is trehalose-6,6'-dibehenate (TDB) or a lipid A glycolipid adjuvant. The immunogenic agent according to claim 1, which further comprises a bile salt. The immunogenic agent according to claim 29, wherein the bile salt is sodium deoxycholate. The immunogenic agent of claim 1, which is freeze-dried or lyophilized. The immunogenic agent of claim 1, which is made to a selected size or in a selected size range. A composition comprising the immunogenic agent according to item 1 of the patent application. The composition of claim 33, comprising a single immunogenic agent comprising one or a plurality of immunogenic protein fragments, variants or derivatives thereof, of the same pathogen, Or one or a plurality of immunogenic protein fragments, variants thereof or derivatives thereof from the same pathogen. The composition of claim 33, comprising a single immunogenic reagent comprising one or more immunogenic protein fragments of each of a plurality of different pathogens, variants thereof Or a derivative thereof, or one or more immunogenic protein fragments, variants or derivatives thereof, from each of the plurality of different pathogens. The composition of claim 33, comprising a plurality of complex immunogenic agents comprising one or a plurality of immunogenic protein fragments, variants thereof, or a derivative, or one or a plurality of immunogenic protein fragments, variants thereof or derivatives thereof from the different pathogens. An immunogenic agent according to any one of claims 1 to 32, or a composition according to any one of claims 33 to 36, for use in the manufacture of a mammal for induction Use of an agent for one or more pathogens for an immune response. An immunogenic agent according to any one of claims 1 to 32, or a composition according to any one of claims 33 to 36, for use in the manufacture of one or more The use of a medicament for pathogenic immune mammals. An immunogenic agent according to any one of claims 1 to 32, or a composition according to any one of claims 33 to 36, for use in the manufacture of a medicament for the treatment or prevention of mammals Use of an agent for infection caused by one or more pathogens. The use of claim 37, wherein the medicament is administered intranasally to the mammal. The use of claim 37, wherein the immunogenic agent induces a mucosal immune response. The use of claim 37, wherein the mammal is a human. The use of claim 38, wherein the agent is administered intranasally to the mammal. The use of claim 38, wherein the immunogenic agent induces a mucosal immune response. The use of claim 38, wherein the mammal is a human. The use of claim 39, wherein the medicament is administered intranasally to the mammal. The use of claim 39, wherein the immunogenic agent induces a mucosal immune response. The use of claim 39, wherein the mammal is a human.