CN109069424A

CN109069424A - Liposome bacterin

Info

Publication number: CN109069424A
Application number: CN201680074917.3A
Authority: CN
Inventors: 迈克尔·戈德; 麦福思·扎曼
Original assignee: Griffith University
Current assignee: Griffith University
Priority date: 2015-10-27
Filing date: 2016-10-26
Publication date: 2018-12-21
Anticipated expiration: 2036-10-26
Also published as: WO2017070735A1; CN109069424B; CN106606775A

Abstract

It is suitable for the immunogenic agents for preventing, treating or being immunized for one or more different pathogens, it includes the carrier proteins of protein, its segment, variant or derivative and such as diphtheria toxoid positioned at intravesicular space derived from the one or more pathogen shown on lipid vesicle.Immunogenic agents are suitably adapted for intranasal administration and can cause mucosal immune response.Immunogenic agents can further include the activator of congenital immunity, such as the bile salt of 6,6 '-two behenate (TDB) of trehalose-and/or such as NaTDC.One or more pathogen can be A group streptococcus, virus or hookworm.

Description

Liposome bacterin

Technical field

The present invention relates to the prevention and treatment of communicable disease.More particularly it relates to pass through mucosa immunity-inducing Response treats or prevents the liposome bacterin of communicable disease and illness.

Background technique

It has been proved that general immunity is being prevented by the serum immune globulin (Ig) of systemic sites by a variety of different diseases Effective in disease caused by substance, but prevention mucosal sites colonize thus prevent interpersonal propagation in terms of be Invalid.Therefore, for some diseases, whole body vaccine inoculation is not optimal approach for inducing immune.In contrast, intranasal Administration resists the mucosal vaccine of a variety of organisms to be to have in terms of the antigen-specific immune response of inducing systemic and mucous membrane compartment Effect.Since this bilayer protective cap might is immune, mucosal vaccine inoculation is ideal strategy for resisting systemic and mucosal infections, The additional benefit that its pre- antiadhesion barrier having colonizes can also inhibit the propagation by droplet and aerosol from the upper respiratory tract.It is viscous Film vaccine inoculation also has advantage economically, this is an important considerations of vaccine development.Due to passing through nose approach The convenience of vaccine is given, it can be to avoid using needle set.Painless delivering facilitates better patient compliance.

Summary of the invention

The object of the present invention is to provide the immunogenic agents and delivery system that cause to the mucosal immune response of pathogen.? Broadly, the present invention relates to by the lipid vesicle for also including such as carrier protein of diphtheria toxoid (DT), pass through delivering Immunogenic protein, segment or variant promote or induce mucosal immunity.Suitably, carrier protein is located at intravesicular space.? In specific form, single immunogenic agents include a variety of different immunogenic proteins, piece from a variety of different pathogens Section or variant.

An aspect of of the present present invention provides the immunogenic agents for being suitable for being applied to mammal, and the immunogenic agents include One or more immunogenic proteins, its segment, variant or derivative, lipid vesicle and carrier protein or its segment or change Body.

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

In one embodiment, immunogenic agents be include panimmunity immunogenic peptide from different pathogens, its The lipid vesicle of segment, variant or derivative.

Another aspect provides the compositions comprising foregoing aspects of immunogenic agents.

In one embodiment, composition includes immunogenic agents, and the immunogenic agents include lipid vesicle, described Lipid vesicle includes identical or single pathogen or from identical or single pathogen panimmunity immunogenic peptide, its piece Section, variant or derivative.

In one embodiment, composition includes a variety of different immunogenic agents, and the immunogenic agents respectively wrap Containing different pathogen or from different pathogen one or more immunogenic proteins, its segment, variant or derivative Object.

In one embodiment, composition includes the single immunogenic agents of lipid vesicle, the lipid vesicle Comprising different pathogen or from different pathogen panimmunity immunogenic peptide, its segment, variant or derivative.

In one embodiment, composition includes a variety of different immunogenic agents, and the immunogenic agents respectively wrap Containing different pathogens or from different pathogens one or more immunogenic proteins, its segment, variant or derivative.

Another aspect provides the immune responses caused in mammals for one or more pathogen Method, the method includes the following steps: to mammal application comprising one or more immunogenic proteins, its segment, The immunogenic agents of variant or derivative, lipid vesicle and diphtheria toxoid (DT) or its segment or variant, or the group comprising it Object is closed, to cause the immune response for one or more pathogen in mammals.

Another aspect provides the method for being directed to one or more pathogen immunising mammals, the methods Include the following steps: to apply to mammal comprising one or more immunogenic proteins, its segment, variant or derivative, rouge The immunogenic agents of matter vesica and diphtheria toxoid (DT) or its segment or variant, or comprising its composition, to be directed to institute State one or more pathogen immunising mammals.

Of the invention still on the other hand providing is treated or prevented in mammals by one or more pathogenic infections Method, the method includes the following steps: to mammal application comprising one or more immunogenic proteins, its segment, The immunogenic agents of variant or derivative, lipid vesicle and diphtheria toxoid (DT) or its segment or variant, or the group comprising it Object is closed, to treated or prevented in mammals by one or more pathogenic infections.

Suitably, immunogenic agents cause mucosal immune response.

Typically, mucosal immune response includes generating IgA.

In a preferred form, immunogenic agents are through intranasal administration.

Suitably, immunogenic protein, its segment or variant exhibits are on the surface of lipid vesicle.Suitably, diphtheria class Toxin (DT) or its segment or variant are located at the intravesicular space in vesica.In preferred embodiments, the lipid vesicle It is liposome.

Suitably, immunogenic protein fragment or variant are esterified.In certain embodiments, in immunogenic protein Lysine (K) residue of the end N- of segment or variant is esterified.In a preferred form, lysine (K) residue of the end N- It is esterified by α and ε amine groups.In some embodiments, each lipid is all C₁₆Fatty acid, such as palmitinic acid.Preferably, Lysine (K) residue of the end N- is located in the introns amino acid sequence of the end N- of immunogenic protein fragment or variant.

In one particular embodiment, the immunogenic protein, segment or variant are A group streptococcus M albumen, its piece Section, variant or derivative.In other or optional embodiment, immunogenic protein is to promote neutrophil activity The reagent for restoring or enhancing.

In a particular embodiment, M protein fragments are the conservative region of M albumen or the conservative region comprising M albumen.? In one embodiment, the segment is immunogenic fragments, it includes p145 peptide or is contained in p145 peptide.Specific In embodiment, the immunogenic fragments are located in J8 peptide or its variant, or include J8 peptide or its variant.

Preferably, J8 peptide includes following amino acid sequence or is made of substantially following amino acid sequence: QAEDKVKQSREAKKQVEKALKQLEDKVQ(SEQ ID NO:1)。

It is widely implemented in scheme at one, the reagent for promoting neutrophil activity to restore or enhance is Protein S pyCEP Or its segment.

In preferred embodiments, SpyCEP segment is comprising following amino acid sequence or substantially by following amino acid sequence Composition: NSDNIKENQFEDFDEDWENF (SEQ ID NO:2).

In a specific embodiment, SpyCEP segment and M protein fragments can be merged to form single chimeric peptide.

In one embodiment, the chimeric peptide is following amino acid sequence or its variant, may include following amino acid Sequence or its variant, or be made of substantially following amino acid sequence or its variant: NSDNIKENQFEDFDEDWENFQAEDKVKQSREAKKQVEKALKQLEDKVQ(SEQ ID NO:3)。

In a specific embodiment, immunogenic protein, segment or variant are influenza viruses.Influenza virus can To be influenza A virus.Non-limitative example be following amino acid sequence, comprising following amino acid sequence or substantially by with Lower amino acid sequence composition: amino acid sequence MSLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO:4).Influenza virus can be with It is influenza B virus.Non-limitative example be following amino acid sequence or comprising following amino acid sequence or substantially by with Lower amino acid sequence composition: amino acid sequence PAKLLKERGFFGAIAGFLE (SEQ ID NO:5).

In a specific embodiment, immunogenic protein, segment or variant are rhinovirus.Non-limitative example It is following amino acid sequence, comprising following amino acid sequence or is substantially made of following amino acid sequence: amino acid sequence GAQVSTQKSGSHENQNILTNGSNQTFTVINY(SEQ ID NO:6).Another non-limitative example is following amino acid sequence Column comprising following amino acid sequence or are substantially made of following amino acid sequence: amino acid sequence GAQVSRQNVGTHSTQNMVSNGSSL(SEQ ID NO:7)。

In a specific embodiment, immunogenic protein, segment or variant are worms, such as hookworm.It is non- Limitative examples are following amino acid sequences, comprising following amino acid sequence or are substantially made of following amino acid sequence: ammonia Base acid sequence TSLIAGLKAQVEAIQKYIGAEL (SEQ ID NO:8).

In some embodiments, immunogenic agents can further include the activator of congenital immunity.It is described congenital to exempt from Epidemic disease activator can target c-type agglutinin, such as the derivable Ca of macrophage²⁺Dependence (c-type) agglutinin (" Mincle "). The activator of congenital immunity can be glycolipid class.Non-limiting example includes glycolipid, such as mycobacteria cord factor seaweed Two behenate (TDB) of analog trehalose -6,6'- or lipid A of two mycolate of sugar -6,6'- (TDM) and/or its synthesis Sugared lipid adjuvant.

In some embodiments, immunogenic agents can further include bile salt, such as NaTDC.

Unless the context requires otherwise, term " include (comprise) ", " including (comprises) " and " include (comprising) " or similar term means that nonexcludability includes, so that the element or feature list of narration not only wrap Containing illustrating or those of list, and it may include other elements or feature unlisted or illustrate.

Under the background of amino acid sequence, the amino acid sequence of narration is meant by "consisting essentially of" and is located at Other 1,2 of the end N- or C- or 3 amino acid.

As it is used herein, herein using indefinite article ' one (a) ' and ' one (an) ' to refer to or comprising odd number or again Number element or feature, and be not construed as meaning or limiting " one/one (one) " or " single " element or feature.

Detailed description of the invention

The idealized structure of Fig. 1 .J8-Lipo-DT.Liposome encapsulates DT, while being connected in N-terminal with introns KSS J8 and two palmitic acid molecule covalent coupling promotes J8 to be inserted into liposome membrane.

The J8 specific antibody response of mono- BALB/c mouse of Fig. 2.Mean antibody titers are indicated with bar.A) saliva IgA Titre.B) excrement IgA titre.C) serum IgG titers.It is for statistical analysis using single factor test ANOVA, then carry out Tukey thing (ns, p > 0.05 are examined afterwards；*, p < 0.05；*, p < 0.01；* *, p < 0.001).

Bacterial load of Fig. 3 in BALB/C mice, after carrying out intranasally attack with M1GAS bacterial strain.A) nasal cavity falls off Object.B) brush,throat.C) NALT is colonized.As a result it is indicated with following: for brush,throat, nasal cavity cast, in 1-3 It, 10 mouse/group average CFU+SEM；For NALT, on day 3.It is examined using graceful-Whitney U of nonparametric, non-matching It tests for statistical analysis, test group is compared to (ns, p > 0.05 with PBS control group；*, p < 0.05；*, p < 0.01；* *, p < 0.001).

The J8 specific antibody response (every group of n=5) of mono- B10.BR mouse of Fig. 4.Mean antibody titers are indicated with bar. A) saliva IgA titre.B) excrement IgA titre.C) serum IgG titers.It is for statistical analysis using single factor test ANOVA, then into Row Tukey post-hoc tests (ns, p > 0.05；*, p < 0.05；*, p < 0.01；* *, p < 0.001).

Fig. 5 assessment carries out the URT GAS challenge model of the bacterial load after intranasally attacking with M1 bacterial strain.A)B10.BR The nasal cavity cast of mouse.B) the brush,throat of B10.BR mouse.As a result with the 1-3 days, 5 mouse/group average CFU+SEM It indicates.Mann Whitney U test using nonparametric, non-matching is for statistical analysis, and test group and PBS control group are carried out Compare (ns, p > 0.05；*, p < 0.05).

The J8 specific antibody response of mono- BALB/c mouse of Fig. 6.Mean antibody titers are indicated with bar.A) saliva IgA Titre.B) serum IgG titers.It is for statistical analysis using single factor test ANOVA, then carry out Tukey post-hoc tests (ns, p > 0.05；*, p < 0.05；*, p < 0.01；* *, p < 0.001).

The chemotactic factor (CF) and cell factor of antigentic specificity secretion in Fig. 7 immune mouse.Splenocyte is taken out, and such as institute Show the following stimulations of addition: LPS (2 μ g/mL), J8 (10 μ g/mL) or individual culture medium.72 hours after stimulation, supernatant is separated Liquid, and the chemotactic factor (CF) of secretion or the level of cell factor are analyzed (referring to material and side using cytometric bead array Method).(ns, p > 0.05 for statistical analysis are examined using student t；*, p < 0.05；*, p < 0.01).

Fig. 8 with or unused reagent treated in the case where, the surface marker in people's DC subgroup is horizontal.As shown Add following stimulations: polyinosinic acid: poly- cytidylic acid (pIC, 10 μ g/mL), J8-Lipo-DT (150 μ g/mL) or independent Culture medium.24 hours after stimulation, pass through measured by flow cytometry cell surface marker.A) CD123+ Plasmacytoid DC.B) 1 type DC of CD141+ classics.C) 2 type DC of CD1c+ classics.It is worth glimmering with the middle position of the blended data from three independent donors Luminous intensity (MFI) ± SEM is indicated.Mann Whitney U test using nonparametric, non-matching is for statistical analysis, by test group (ns, p > 0.05 are compared with vehicle control group；*, p < 0.05；*, p < 0.01；* *, p < 0.001).

By the chemotactic factor (CF) and cell factor of J8-Lipo-DT secretion inducing in Fig. 9 human dendritic cell.Take out dendron shape Cell, and following stimulations: pIC (10 μ g/mL), J8-Lipo-DT (150 μ g/mL) or individual medium are added as shown.Thorn 24 hours separation supernatants after swashing, and the chemotactic factor (CF) of secretion or the horizontal of cell factor are carried out using cytometric bead array It analyzes (referring to material and method).(ns, p > 0.05 for statistical analysis are examined using student t；*, p < 0.05；*, p < 0.01；* *, p < 0.001).

Figure 10 .SpyCEP peptide (S2；SEQ ID NO:2) the initiation mucous membrane IgA response of liposome delivery reagent.It is intranasal to mouse Application shows the S2 peptide of palmitinic acid esterification or the liposome and intracapsular DT of S2-J8 chimera (SEQ ID NO:3), and measures S2 Specific IgA titre.

Inducing antigen-specific IgA, IgG response in mouse of Figure 11 .J8+S2-Lipo-DT immunogenic agents.

Figure 12 can squeeze J8-Lipo-DT immunogenic agents to form the particle of nanometer or micron-scale.Pass through Nanosizer (dynamic light scattering or DLS) carries out liposome size measurement.

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

The larger sized J8-Lipo-DT immunogenic agents of Figure 14 induce the response of J8 specific mucosal.

The particle diameter distribution of the J8-Lipo-DT powder for the freeze-drying restored in Figure 15 .PBS.Pass through Nanosizer (dynamic Light scattering or DLS) carry out liposome size measurement.

Figure 16 is without other adjuvants, the J8-Lipo-DT liposome immunization originality agent of the freeze-drying of recovery Induce J8 specificity more systemic response.

The J8-Lipo-DT liposome immunization originality agent for the freeze-drying that Figure 17 restores induces the response of J8 specific mucosal.

Figure 18 includes the liposome J8-Lipo-DT immunogene of 6,6 '-two behenate of sugared lipid adjuvant trehalose (TDB) Property agent schematic diagram description.

Figure 19 .TDB increases the mucous membrane IgA response of J8-Lipo-DT induction.J8-Lipo-Dt+TDB with 30 μ g is intranasal Immune mouse (every group of n=5).(the 0th day) is applied to mouse plus reinforcing (the 21st day and the 42nd day) twice for the first time.In saliva and In fecal specimens, compared with J8-Lipo-DT, being incorporated to TDB leads to significant higher mucous membrane IgA response.As a result freeze-dried powder is come from The J8-Lipo-DT+TDB of the stability problem for eliminating liposome of form.

Figure 20 includes the schematic diagram description of the liposome immunization originality agent of bile salt NaTDC.

Figure 21 (A) schematically depicts single immunogenic agents comprising lipid vesicle, intracapsular DT and a variety of Bu Tong diseases The immunogenic protein of substance or immunogenic protein from a variety of different pathogens, a variety of different pathogens, that is, A types Influenza virus, influenza B virus and A group streptococcus；(B) single immunogenic agents are schematically depicted comprising lipid capsule Bubble, the immunogenic protein of intracapsular DT and a variety of different pathogens or the immunogenic protein from a variety of different pathogens, with And 6,6 '-two behenate of sugared lipid adjuvant trehalose (TDB) and single phosphoryl 3- deacylation base lipid AInstitute State a variety of different pathogen i.e. influenza A virus, influenza B virus and A group streptococcus.

Figure 22 compares the separated immunogenic agents with each anti-influenza A virus, influenza B virus and A group streptococcus The list of individually inoculation (" Single Antigen-vax "), anti-influenza A virus, influenza B virus and A group streptococcus is immune The immunogenicity of originality agent (" Multivax "), as measured by antigentic specificity saliva IgA titre.

Specific embodiment

The present invention at least partly according to following discoveries based on: with the immunogenic peptide comprising being showed in surface of liposome with The liposome immunization originality agent of intracapsular carrier protein such as diphtheria toxoid (DT) carries out mouse intranasal vaccination, causes to glue Film and systemic antibody response, the response can be with those of the compatible adjuvant CTB inductions of non-human by establishing quite.In A Under the specific background of group streptococcus and J8 peptide, the level of the protective immunity induced by Liposomal formulation is significantly more than by J8/CTB The level induced.In addition, the cytokine response of human dendritic cell (DC) subgroup of purifying shows that this lipoid plastid is luring It will be effective for leading in people's mucous membrane J8 specificity IgA and systemic IgG response.In some embodiments, liposomal immunogen Property agent can comprising SpyCEP peptide or its other segment or also include individually J8 peptide.In concrete form, liposomal immunogen Property agent can be adapted for treating or preventing by treating the common antibiotics infected for A group streptococcus on resistant A group's chain Infection caused by the special velogen strain of coccus or separation bacterium.These bacterial strains or separation bacterium usually cause skin severe infections (example Such as necrotizing fasciitis), and in some cases, can be mutated containing CovR/SCovR/S.This may summarize to other cause of diseases Body disease relevant with them, illness and the patient's condition, including but not limited to influenza, rhinovirus and worm, such as hookworm.So this The embodiment of invention provides single immunogenic agents comprising a variety of different pathogen or from a variety of different diseases One or more immunogenic proteins, segment, variant or the derivative of substance.

For the object of the invention, " separation " means the material removed from its native state, or has received manually to grasp The material of work.Isolated material can be contained in substantially or essentially under its native state usually with its component, or can To be in artificial state together with component usually adjoint under its native state by operation.Isolated material can be day So, chemical synthesis or recombinant forms.

" protein " means amino acid polymer.As well known in the art, amino acid can be natural amino acid or non-day Right amino acid, D- amino acid or l-amino acid.

Term " protein " includes and covers " peptide " and " polypeptide ", and peptide has a no more than 50 (50) commonly used in description The protein of amino acid, polypeptide have the protein more than a amino acid in 50 (50) commonly used in description.

As on generally herein use, when protein is referred to as " pathogen " or means the egg " from pathogen " The white amino acid sequence including being at least partially or fully present in the albumen of pathogen.

" segment " is section, structural domain, part or the region of protein, constitutes the ammonia less than the protein 100% Base acid sequence.It will be recognized that segment can be individual segment, or can individually repeat, or can be repeated with other segments.

In general, segment may include full length protein at most 5,6,7,8,9,10,12,15,20,25,30,40,50,60, 70、80、90、100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、 950,100,1050,1100,1150,1200,1250,1300,1350,1400,1450,1500,1550 or 1600 amino acid, It is substantially made of the amino acid or is made of the amino acid.

As used herein, protein " variant " with referring to amino acid sequence share confirmable nucleotide or amino acid sequence Relationship." variant " protein can be lacked referring to one or more amino acid of amino acid sequence or the amino acid by difference Amino acid substitution.Known in field, some amino acid can be replaced or lack, without change immunogenic fragments and/or The activity (conservative replacement) of protein.Preferably, protein mutant with referring to amino acid sequence shared at least 70% or 75%, preferably at least 80% or 85%, or more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

Herein the term of the sequence relation between each protein of usually used description and nucleic acid include " comparison window ", " sequence identity ", " Percentage of sequence identity " and " substantially the same ".Since each nucleic acid/protein can respectively contain (1) Complete Nucleotide/protein sequence only one or multiple portions shared by nucleic acid/protein, and (2) nucleic acid/protein Between different one or more parts, usually compared by comparing sequence in " comparison window " to execute sequence, to identify and compare Compared with the sequence similarity of regional area." comparison window " refers to the general of usual 6,9 or 12 consecutive residues compared with canonical sequence Section in thought.Comparison window may include compared with canonical sequence about 20% or less and add or delete (i.e. vacancy), be used for The optimal comparison of each sequence.The optimal comparison of sequence for being aligned comparison window can be run by the computer of algorithm (the Geneworks program of Intelligenetics；7.0 (Genetics of Wisconsin science of heredity software package release Software Package Release 7.0) in GAP, BESTFIT, FASTA and TFASTA, Genetics Computer Group, 575Science Drive Madison, WI, USA are incorporated into herein by reference) or implemented by estimating, Pass through any generation optimal comparison (that is, generating the highest homology percentage in comparison window) of selected various methods. Such as blast program family disclosed in Altschul et al, 1997, Nucl.Acids Res.25:3389 can also be used as ginseng It examines, is incorporated into herein by reference.Being discussed in detail for sequence analysis can see the molecular biology that Ausubel et al. writes General handbook (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (John Wiley&Sons Inc NY, 1995- 1999) Unit 19.3.

The term as used herein " sequence identity " is used with its broadest meaning, to include about using canonical algorithm Suitable comparison, about the degree in the identical sequence of comparison window, the quantity of accurate nucleotide or amino acid match.Therefore, " Percentage of sequence identity " is calculated by following: being compared the sequence of two optimal comparisons in comparison window, is determined identical The number for the position that nucleic acid base (such as A, T, C, G, I) occurs in two sequences will be matched with obtaining matched positional number Positional number divided by positional number (that is, window size) total in comparison window, and by obtained result multiplied by 100 to obtain sequence identity Percentage.Such as, it is possible to understand that " sequence identity " mean by DNASIS computer program (windows editions, version 2 .5；It can From Hitachi Software engineering Co., Ltd., South San Francisco, California, USA are obtained ) calculate " match-percentage ".

As used herein, " derivative " is such as albumen being changed or the molecule of its segment or variant, such as this field Understood, such as by connecting or being complexed with other chemical parts, passes through posttranslational modification (such as phosphorylation, acetylation Deng), glycosylation modification (such as addition, removal or change glycosylation), esterification and/or include additional amino acid sequence. In one embodiment, additional amino acid sequence may include its N-terminal and/or C-terminal one or more lysines it is residual Base.Multiple lysine residues (for example, polylysine) can be the linear order of lysine residue or the branch of lysine residue Sequence.These additional lysine residues can promote increased peptide dissolubility.

Additional amino acid sequence may include the fusion partner amino acid sequence for generating fusion protein.For example, fusion partner Amino acid sequence can assist the detection and/or purifying of isolated fusion protein.Non-limiting example include metal bonding (for example, Polyhistidine) fusion partner, maltose binding protein (MBP), a-protein, glutathione s-transferase (GST), fluorescin Matter sequence (for example, GFP), Epitope tag such as myc, FLAG and hemagglutinin label.Other additional amino acid sequence packets Include spacer sequence.One example of spacer sequence is the N in immunogenic protein segment or the amino acid sequence of variant The amino acid sequence of end or C-terminal, it includes lysine (K) residues for being suitable for esterification.In general, introns amino acid sequence includes two A (2) are to ten (10) amino acid, such as three (3) amino acid sequence KSS.

Other derivatives that the present invention covers include but is not limited to the modification of side chain, during the synthesis of peptide or protein matter simultaneously Enter unnatural amino acid and/or its derivative, and use crosslinking agent, and to immunogenic protein of the invention, segment and Variant applies the other methods of conformation constraint.

In this regard, those skilled in the art can refer to the general handbook of protein science that Coligan et al. writes 15th chapter of (CURRENT PROTOCOLS IN PROTEIN SCIENCE) (John Wiley&Sons NY 1995-2008), The wider methodology of chemical modification about protein.

It should be understood that immunogenic protein disclosed herein, segment and variant can individually be presented on lipid vesicle Surface, or presented as the chimeric protein or fused protein of the identical peptide comprising multiple copies or multiple and different peptides.Non- limit Property example processed is the chimeric peptide of SEQ ID NO:3, be will be described in greater detail below.

In context of the present invention, the term as used herein " immunogenicity " refers to be used to mammal or other animals The ability or effect of immune response are generated or caused after immunogenic agents, such as are answered for pathogen or the immune of its molecular components It answers.

" causing immune response " means the generation or activity for the one or more elements for generating or stimulating immune system, including Cell immune system, antibody and/or natural immune system.Suitably, one or more elements of immune system include that B lymph is thin Born of the same parents, antibody, neutrophil cell, the dendritic cells including plasmacytoid dendritic cells, cell factor and/or chemotactic because Son.The non-limiting example of cell factor includes pro-inflammatory cytokine such as TNF-α, IL-6 and IL-1 (such as IL-1 β).Become The non-limiting example for changing the factor is neutrophil cell chemical inhibitor IL-8.Suitably, immune response is or including mucous membrane Immune response is generated for example including IgA.It preferably, is protective by the immune response that immunogenic agents cause.

As general herein, term " immune ", " vaccine inoculation " and " vaccine " refers to that causing the protectiveness for being directed to pathogen exempts from Epidemic disease response, so that the further infection of the pathogen is at least partly prevented or reduced method and/or composition.

As used herein, term " pathogen " refers to cause disease, illness in animal, such as birds or mammal Or any living body or non-living body entity of the patient's condition.Pathogen can be virus, bacterium, protozoan or worm, but not limited to this. The specific non-limitative example of pathogen includes A streptococci bacteria, Respirovirus, such as influenza virus and rhinovirus and line Worm, such as hookworm.

As understood from the disclosure, the present invention provides lipid vesicle preparation, and it includes prepare to exempt from lipid vesicle Epidemic focus protein fragments, variant or derivative and carrier protein.As used herein widely, lipid vesicle can be rouge Plastid, microcell, multi-layer vesicles, protomere (micelle), vacuole or other imitated vesicle structures comprising lipid bilayer are suitably exempted from Epidemic focus protein fragments, variant or derivative are anchored into one or more lipids of lipid bilayer comprising promotion through deriving, from And immunogenic protein segment, variant or derivative are presented on the surface of lipid vesicle.In a preferred form, by α and/or ε amino esterification lysine (K) residue.To promote esterification, immunogenic protein segment, variant or derivative be can further include N-terminal introns, the N-terminal introns include by lysine (K) residue of esterification.Introns can usually include 2,3,4,5,6, 7,8,9 or 10 continuous amino acids.One embodiment is the introns KSS of three (3) amino acid.Lipid may include saturation or Unsaturated fatty acid (such as single insatiable hunger and/or how unsaturated).In some embodiments, the lipid or each lipid For C₁₆Fatty acid, such as palmitinic acid.However, it will also be understood that such as with C₁₂、C₁₃、C₁₄、C₁₅、C₁₇、C₁₈、C₁₉、C₂₀、C₂₁Or C₂₂Other lipids of saturation or unsaturation (for example, monounsaturated or how unsaturated) fatty acid of carbochain can be used for this hair It is bright.

Suitably, lipid vesicle includes the mixture for being capable of forming any lipid or lipid of Lipid Bilayer Structure.These packets Phosphatide is included, sterols, fatty acid and/or triglyceride including cholesterol, cholesteryl ester and phytosterol.Phosphatide Non-limiting example includes phosphatidyl choline (PC) (lecithin), phosphatidic acid, phosphatidyl-ethanolamine (PE) (cephalin), phosphatidyl Glycerol (PG), phosphatidylserine (PS), phosphatidylinositols (PI) and sphingomyelins (SM) or its natural or synthetic derivative. Natural derivative includes egg PC, egg PG, Soybean PC, hydrogenated soybean PC, soybean PG, brain PS, sheath ester (sphingolipids), brain SM, galactocerebroside, gangliosides, cerebroside, cephalin, cuorin and double hexadecyl acid ester.The derivative of synthesis Object include dipalmitoylphosphatidylcholine (DPPC), dinonanoylphosphatidylcholine (DDPC), two mustard phosphatidyl cholines (DEPC), Dimyristoyl phosphatidyl choline (DMPC), Distearoyl Phosphatidylcholine (DSPC), Dilauroyl Phosphatidylcholine (DLPC), Palmitoyl Phosphatidylcholine (POPC), palmityl dimyristoylphosphatidycholine (PMPC), palmitoylstearoylphosphatidyl Choline (PSPC), Dioleoyl Phosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylglycerol (DLPG), distearoylphosphatidylglycerol (DSPG), GLYCEROL,DIMYRISTOYL PHOSPHATIDYL (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), palmitoyl phosphatidylglycerol (POPG), di-myristoyl phosphatidic acid (DMPA), dipalmitophosphatidic acid (DPPA), G 12S3P (DSPA), two meat Myristoyl phosphatidyl-ethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), two myristoyl phosphatidylserines (DMPS), two palmityl phosphatidylserines (DPPS), Distearoyl Phosphatidylethanolamine (DSPE), dioleoyl phospholipid acyl ethyl alcohol Amine (DOPE), dioleoylphosphatidylserine (DOPS), two palmitoyl sphingomyelins (DPSM) and distearyl sphingomyelins (DSSM). Phosphatide is also possible to the derivative or the like of any above-mentioned phosphatide.

Suitably, the mixture of lipid may include desired molar ratio or it is expected each phosphatide of wt% ratio.Respective lipid it Between ratio can be in 20:1 between 1:1, including 15:1,12:1,10:1,7:1,5:1,4:1,3:1 and 2:1 or these narrations Any ratio between value.It is 7 two palmityl-sn- glycerol-3-phosphocholines (DPPC): 2 gallbladders it is, for example, possible to use molar ratio Sterol (CHOL): 1L- α-phosphatidyl glycerol (PG) forms liposome.

Suitably, lipid vesicle further includes carrier protein.Suitably, carrier protein is immunogenicity, or At least partly promote or enhance the immunogenicity of the immunogenic agents.In general, by carrier protein together with lipid vesicle It prepares so that carrier protein is located inside lipid vesicle in internal aqueous space.In some embodiments, carrier protein Matter is separated with its immunogenic protein fragment, variant or derivative.According in the embodiment, immunogenic protein fragment, Variant or derivative can be shown on capsule surface.In some embodiments, carrier protein can be with its described immunogenicity Protein fragments, variant or derivative fusion, combination or complexing.This may include recombinant protein fusion, chemical crosslinking and molecule Between be complexed, such as by biotin-avidin or other intermolecular bonding agents, while not limited to this.In this kind of implementation In scheme, described its immunogenic protein segment, variant or derivative are located inside lipid vesicle in internal aqueous space, It is merged with the carrier protein, combination or complexing.The embodiment can be particularly useful in oral delivery immunogenic agents, example In such as liposome comprising bile salt, as being described more particularly below herein.The non-limiting example of carrier protein includes Diphtheria toxoid (DT), tetanus toxoid (TT), such as CRM197 CRM protein and pertussis toxin mutants, although It is without being limited thereto.Also cover the segment and variant of carrier protein.In one embodiment, carrier protein is diphtheria class poison Plain (DT) or its segment.

In some embodiments, lipid vesicle further includes the activator of congenital immunity.Congenital immunity swashs Agent living can target the C- type agglutinin expressed by one or more cells relevant to congenital immunity.Preferred C- type agglutinin For the Ca of macrophage induction²⁺Dependence (C- type) agglutinin (" Mincle ").Non-limiting example includes glycolipid, such as is divided - 6,6 '-two mountain of analog trehalose of -6,6 '-two mycolate (TDM) of branch bacillus cord factor trehalose and/or its synthesis Yu acid esters (TDB) and/or the such as single phosphoryl 3- deacylation base lipid A of lipid A sugared lipid adjuvant can be 3D-With 3D (6- acyl group)Form.Although being not intended to be limited by any specific theory, thus it is speculated that congenital Property immune activator (such as above-mentioned) can enhance or improve the mucosal immunity caused by immunogenic agents.Preferably, glycolipid class May include in lipid vesicle, thus its account for total lipid in lipid vesicle no more than about 25%, 20%, 15%, 10% or 5%.

In some embodiments, lipid vesicle can further include bile acid or bile salt.Bile acid is usually two The steroids (in some embodiments including 24 carbon) of hydroxylating or trihydroxy, including cholic acid, deoxycholic aicd, goose deoxidation Cholic acid and ursodesoxycholic acid.Preferably, lipid vesicle include bile salt, as cholate, deoxycholate, goose deoxycholate or Ursodeoxycholic hydrochlorate.Preferably, bile salt is deoxysodium cholate.

In other embodiments, the liposome comprising immunogenic agents can be prepared as specific, selection or desired Particle size or size range.In some embodiments, the liposome of larger particle size can cause stronger mucosal immunity Response.

In other embodiments, the liposome comprising immunogenic agents can be lyophilized or be lyophilized to promote long-term storage. The liposome immunization originality agent of the freeze-drying of recovery causes and the comparable immune response of " fresh " liposome immunization originality agent.

In some embodiments, pathogen is A group streptococcus.

As used herein, term " A group streptococcus (streptococcus, Streptococci, Streptococcal, Strep) " and referred to as " GAS " refers to the streptococcus bacterium of Lan Shi A sero-group, is micrococcus scarlatinae (Streptococcus Pyogenes) the gram-positive β hemolytic bacteria planted.The important virulence factor of GAS is M protein, thin for strong anti-phagocytosis Born of the same parents' and in conjunction with serum factor H, it destroys C3- invertase and prevents the opsonification of C3b.These further include that toxicity is " prominent Variant ", for example, CovR/S the or CovRS mutant of Graham et al., 2002, PNAS USA 99 13855 description, although not It is limited to this.

Disease caused by A group streptococcus and the patient's condition include cellulitis, erysipelas, impetigo, scarlet fever, larynx infection it is for example anxious Property pharyngitis (" Strep throat "), bacteremia, toxic shock syndrome, necrotizing fasciitis, acute rheumatic fever and acute Glomerulonephritis, although not limited to this.

As used herein, " neutrophil cell " or neutrophil leucocyte are together with basophilic granulocyte and eosinophil Form a part of polymorphonuclear cell family (PMN).Neutrophil cell is relatively short-lived gulping down of being formed by stem cell Phagocyte and typically comprise 40% to 75% leucocyte in mammal.In addition to phagocytic, neutrophil cell release Soluble antimicrobial (such as granule protein) and generate that neutrophil cell is extracellular catches net.Neutrophil cell is to such as white The molecule of interleukin -8 (IL-8), C5a, fMLP and leukotriene B4 has a response, the molecule promote neutrophil cell to damage and/ Or the chemotaxis at the position of acute inflammation.

In one embodiment, immunogenic protein can be M albumen, its segment or variant.

As used herein, " M protein fragments " are that can be combined with immunogenicity and/or by antibody or antibody fragment Any segment of GAS M albumen.In general, segment is following, includes following or included in following interior: the C- of GAS M albumen is repeated The amino acid sequence in area or its segment.Non-limiting example includes p145, is 20mer, has amino acid sequence LRRDLDASREAKKQVEKALE(SEQ ID NO:4).In this respect, the segment of p145 amino acid sequence can reside in J8 peptide In.

As used herein, " J8 peptide " is comprising at least partly deriving from or corresponding to GAS M PROTEIN C-area's peptide amino acid The peptide of sequence.J8 peptide suitably includes conformation B- cell epitope and is free of potentially harmful T- cell auto epitope.Preferably J8 peptide amino acid sequence is QAEDKVKQKQLEDKVQ (SEQ ID NO:1) or its segment or change Body, wherein the residue of the overstriking corresponds to the residue 344 to 355 of GAS M albumen.In this embodiment, J8 is further The chimeric peptide of GCN4DNA- binding protein sequence including flank, the sequence help to maintain the correct helical fold and structure of J8 peptide Image structures.

In another embodiment, the examination that immunogenic protein can restore or enhance for promotion neutrophil activity Agent.

As used herein, " promote neutrophil activity restore or the reagent of enhancing " is directly or indirectly at least portion Point increase, enhancing or restore neutrophil cell generation, migration and/or chemotaxis and/or neutrophil cell one kind or The active molecule of panimmunity.In one embodiment, neutrophil cell inhibitor is immunized in the reagent initiation Response.In another embodiment, the reagent combination neutrophil cell inhibitor and at least partly make its inactivation.It is thermophilic Neutrophil leucocyte inhibitor can be to be derived from or the molecule from A group streptococcus.In a particular form, neutrophil(e) granule is thin Born of the same parents' inhibitor is the serine protease or its segment that proteolysis cuts interleukin 8.In one particular embodiment, in thermophilic Property granulocyte inhibitor be SpyCEP or its segment.SpyCEP is in human pathogen micrococcus scarlatinae (Streptococcus Pyogenes the serine protease of the 170-kDa Multidomain of surface expression), in thermophilic by catalytic pyrolysis and inactivation Property granulocyte chemoattractant interleukin-8 and play an important role in infection.The non-limiting reality of SpyCEP amino acid sequence Example can see accession number YP597949.1 and (micrococcus scarlatinae MGAS10270) and YP596076.1 (micrococcus scarlatinae MGAS9429).Therefore, in one particular embodiment, SpyCEP segment is or comprising SEQ ID NO:2 (NSDNIKENQFEDFDEDWENF) amino acid sequence shown in.It is proposed that SEQ ID NO:2 is or comprising excellent on SpyCEP Gesture epitope, can be with inducing function antibody.

M- protein amino acid sequence and SpyCEP ammonia comprising forming single, continuous amino acid sequence is also provided herein The chimeric peptide of base acid sequence.M- protein amino acid sequence can be located at the end C- of SpyCEP amino acid sequence, or vice versa. In one embodiment, chimeric peptide may include amino acid sequence NSDNIKENQFEDFDEDWENFQAEDKVKQSREAKKQ VEKALKQLEDKVQ (SEQ ID NO:3) or its variant.

In an alternate embodiment, it can produce each comprising M- protein amino acid sequence and SpyCEP amino acid sequence Liposome is used for " mixture " application.

In one particular embodiment, variant M albumen or peptide can include one or more in itself end N and/or C- Lysine residue.Multiple lysine residues (such as polylysine) can be the linear order of lysine residue or can be bad The branch chain-ordering of histidine residue.These other lysine residues can promote the dissolubility of increased peptide.

The non-limiting example of J8 peptide variant includes:

S R E A K K Q S R E A K K Q V E K A L K Q V E K A L C (SEQ ID NO:5)

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

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

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

Other variants can be based on such as Cooper et al., heptapeptide described in 1997.

For example, if it is known that epitope is located in alpha-helix protein structure conformation, the structure is folded into then can synthesize The mode peptide of elephant.Mode alpha-helix coiled coil peptide have been based on GCN4 leucine zipper structure (O ' Shea et al., 1991).First heptapeptide contains sequence MKQLEDK (SEQ ID NO:9), and it includes stablize coiled coil heptapeptide to repeat motif (a- B-c-d-e-f-g) several features (Cohen&Parry, 1990) present in n.These include in the big non-pole of position a and d Property residue, in position the acid/base of e and g to (Glu/Lys) (typically favor inter-chain ionic interaction) and position b, c, The polar group (consistent with the prediction of Lupas et al. (1991)) of f.The GCN4 peptide also a in position contains shared figured silk fabrics ammonia Acid.Also it is mentioned that when position a and d is occupied by V and L, coiled coil dimer be it is preferred (Harburyet al., 1994).Mode heptad repeat region derives from these common characteristics of GCN4 leucine zipper peptide: (VKQLEDK；SEQ ID NO: 10), there are the potentiality for forming alpha-helix coiled coil.The peptide becomes frame peptide.The overlapping fragments of comformational epitope in research are embedding Enter in mode coiled coil peptide to generate chimeric peptide.Whenever finding identical residue in helicon mode peptide and epitope sequences, It will be designed to ensure that in amino acid replacement (Cohen&Parry, 1990) the incorporation chimeric peptide of correct helical coil-coil conformation. Usually using following displacements: position a, V to I；B, K are to R；C, Q are to N；D, L are to A；E, E are to Q；, f:D to E；G, K are to R.It is all this A little replacement residues are commonly found in their own position in coiled-coil protein (Lupas et al., 1991).

It is described in Olive et al., the specific J8 peptide derivant of one in 2002, Infect&Immun.70 2734 For " lipid core peptide ".In one embodiment, lipid core peptide may be embodied in the poly- of the branch for being coupled to lipophilicity anchor The multiple J8 peptides (such as four J8 peptides) directly synthesized on two amino of each lysine of lysine core.

M protein fragments or variant and/or SpyCEP segment or variant can be derivatized comprising promoting to be anchored to above One or more lipids of the double-layer of lipoid.It in another embodiment, include M- protein amino acid sequence and SpyCEP The chimeric peptide (such as SEQ ID NO:3) of amino acid sequence can include introns amino acid sequence in its end N-.Therefore, exist SpyCEP segment or variant include in the embodiment of lipid vesicle, it can be divided together with M protein fragments or variant Esterification or can with esterification chimeric peptide exist.

In some embodiments, pathogen is influenza virus.In a specific embodiment, immunogenicity egg White, segment or variant are influenza A virus.Immunogenic protein or segment can be stromatin 2 or its segment.Non- limit Property example processed is or including amino acid sequence MSLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO:4).It is specific at one In embodiment, immunogenic protein, segment or variant are influenza B virus.Immunogenic protein or segment can be blood Solidifying fibroin or its segment.Non-limitative example is or including amino acid sequence PAKLLKERGFFGAIAGFLE (SEQ ID NO:5)。

In some embodiments, pathogen is rhinovirus.In a specific embodiment, immunogenic protein, Segment or variant are rhinovirus B albumen, such as capsid protein.Non-limitative example is or including amino acid sequence GAQVSTQKSGSHENQNILTNGSNQTFTVINY(SEQ ID NO:6).In another specific embodiment, immunogenicity Albumen, segment or variant are rhinovirus A albumen, such as capsid protein.Another non-limitative example is or including amino acid sequence It arranges GAQVSRQNVGTHSTQNMVSNGSSL (SEQ ID NO:7).

In some embodiments, pathogen is worm, such as hookworm.In a specific embodiment, immunogene Property albumen, segment or variant are American hookworm (Necator americanus).Non-limitative example is or including amino acid sequence It arranges TSLIAGLKAQVEAIQKYIGAEL (SEQ ID NO:8).

Isolated immunogenic protein, segment and/or derivative of the invention can pass through any hand known in the art Section generates, and the means include but is not limited to chemical synthesis, recombinant DNA technology and proteolytic cleavage to generate peptide fragment.

Chemical synthesis includes solid phase and liquid phase synthesis.Although with reference to SYNTHETIC VACCINES Ed.Nicholson 9th chapter and CURRENT PROTOCOLS IN PROTEIN of (Blackwell Scientific Publications) SCIENCE Eds.Coligan et al. is mentioned in the 15th chapter of (John Wiley&Sons, Inc.NY USA 1995-2008) The example of the chemical synthesising technology of confession, but such method is well known in the art.In this respect, it reference is also made to international publication WO 99/02550 and international publication WO 97/45444.

Recombinant protein can be used standard scheme and easily be prepared by those skilled in the art, the standard scheme It is described in such as Sambrook et al., MOLECULAR CLONING.A Laboratory Manual (Cold Spring Harbor Press, 1989), especially the 16th part and the 17th part；CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds.Ausubel et al., (John Wiley&Sons, Inc.NY USA 1995-2015), the especially the 10th Chapter and the 16th chapter；And CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds.Coligan et al., (John Wiley&Sons, Inc.NY USA 1995-2015), especially the 1st chapter, the 5th chapter and the 6th chapter.In general, recombinant protein preparation packet Include the nucleic acid for expressing coding protein in a suitable host cell.

As described above, the present invention provides immunogenic agents and/or they for prevent or treat mammal or its The purposes of the relevant disease of pathogen, illness or the patient's condition in its animal.

As used herein, " treatment (treating/treat/treatment) " refers to after initially forming at least partly Ground improves, eliminates or reduces the symptom of the relevant disease of pathogen, illness or the patient's condition or the therapeutic intervention of pathological signs.It controls Treatment is not necessarily absolutely beneficial to subject.Any method known to persons of ordinary skill in the art or mark can be used in beneficial effect Standard measures.

As used herein, it prevents (preventing/prevent/prevention) to refer to and is infecting or be exposed to cause of disease The effect started before body or its molecular components and/or before disease, the symptom of illness or the patient's condition or pathological signs breaking-out Process, to prevent from infecting and/or reduce symptom or pathological signs.It should be understood that such prevent from being not necessarily absolutely beneficial to tested Person." preventative " treatment is applied with reducing the purpose for developing the symptom of disease, illness or the patient's condition or the risk of pathological signs For do not show disease, illness or the patient's condition sign or only show early indication subject treatment.

In one embodiment, disease, illness or the patient's condition can be the relevant disease of A group streptococcus, illness or the patient's condition.

In the context of the present invention, " the relevant disease of A group streptococcus, illness or the patient's condition " means to be infected by A group streptococcus Caused any clinicopathologia and including cellulitis, erysipelas, impetigo, scarlet fever, larynx infect such as acpuei pharyngitis (" Strep throat "), bacteremia, toxic shock syndrome, necrotizing fasciitis, acute rheumatic fever and acute renal glomerulus Ephritis, although not limited to this.

As described above, the purposes disclosed herein for treating and/or being immunized includes including M egg to mammal application White tiles section, variant or derivative, lipid vesicle, carrier protein and/or SpyCEP peptide promote neutrophil activity extensive The immunogenic agents of multiple or enhancing other segments.

As disclosed herein, it treats and/or immune can also comprise administration of antibodies or antibody fragment and controlled with therapeutic GAS infection is treated, such as by targeting SpyCEP, and/or combination M albumen, its segment or variant at infection site (such as skin) Antibody or antibody fragment.

Antibody and antibody fragment can be polyclonal or monoclonal, natural or recombination.Antibody fragment include Fc, Fab or 2 segment of F (ab) and/or may include single-chain Fv antibody (scFv).Such scFv can for example basis be described in down Prepared by the method stated: U.S. Patent No. 5,091,513, European Patent No. 239,400 or Winter&Milstein, The article of 1991, Nature 349:293.Antibody can also include the polyvalent recombinant antibody fragment containing multiple scFv, such as double-strand Antibody, three chain antibodies and/or four chain antibodies and half chain antibody (demibodies) (such as the WO/ of dimerization activation 2007/062466).For example, such antibody can be according to being described in Holliger et al., 1993Proc Natl Acad Sci USA 90 6444；Or method in Kipriyanov, 2009Methods MolBiol 562 177 is made It is standby.The known scheme that can be applied to antibody generation, purifying and purposes can be seen, such as Coligan et al., CURRENT The 2nd chapter and Harlow, E.&Lane of PROTOCOLS IN IMMUNOLOGY (John Wiley&Sons NY, 1991-1994), D.Antibodies:A Laboratory Manual,Cold Spring Harbor,Cold Spring Harbor Laboratory,1988。

Method for generating polyclonal antibody is well known to those skilled in the art.What be can be used is exemplary Scheme is described in such as Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, sees above and Harlow& Lane, 1988, it sees above.In specific embodiments, anti-SpyCEP polyclonal antibody can obtained from or be purified from and come from It is exposed to or infects the human serum of the individual of A group streptococcus.It is alternatively possible to generate in the production species of such as horse for pure The polyclonal antibody of SpyCEP change or recombination or its immunogenic fragments, then then purifying before administration.

Standard method can be used to prepare in monoclonal antibody, for example, it is initially described inMilstein, 1975, Nature 256, the standard method in 495 article, or for example, by being described in Coligan et al., Prepared by its more recent improvement of CURRENT PROTOCOLS IN IMMUNOLOGY (seeing above), come from by immortalizing Inoculated one or more isolated albumen of the invention, segment, variant or derivative generation species splenocyte or its It generates the cell of antibody.Therefore, for M protein fragments, variant or derivative and/or neutrophil activity can be promoted extensive Multiple or enhancing reagent (such as SpyCEP) generates monoclonal antibody, in accordance with the purpose of the invention.In certain embodiments In, monoclonal antibody or its segment can be recombinant forms.If monoclonal antibody is initially thin by the spleen of non-human mammal Born of the same parents generate, then this may be particularly advantageous for " humanization " monoclonal antibody or segment.

For embodiment relevant to therapeutic antibodies, preferred M protein fragments can be p145 peptide.

The preferred segment of the SpyCEP generated for antibody may include following amino acid sequences or by following amino acid Sequence composition: NSDNIKENQFEDFDEDWENF (SEQ ID NO:2).

In some embodiments, disease, illness or the patient's condition can be the relevant disease of influenza virus, illness or the patient's condition. Influenza virus can cause disease that is propagated or otherwise infecting, referred to as " influenza ".Typical symptom includes fever, head Pain, cough, drowsiness, respiratory tract and nasopharyngeal mucus accumulation and secretion, myalgia, nausea and vomiting.Symptom can last for days Or continued for several weeks.In some cases, it can be possible to secondary respiratory tract bacterial infection occur, serious disease is caused in some cases Condition, such as pneumonia.Therefore, immunogenic agents of the invention and/or method can treat or prevent the relevant disease of influenza virus, disease Disease or the patient's condition are than as described above.

In some embodiments, disease, illness or the patient's condition can be the relevant disease of rhinovirus, illness or the patient's condition.Nose Viral (for example, rhinovirus A and rhinovirus B) is the kind that virus micro ribonucleic-acid Viraceae enterovirus belongs to.Rhinovirus is usually The virulence factor of common cold, symptom is similar to influenza, but general seriousness is lower and Secondary bacterial infections, such as The probability of pneumonia is lower.

Therefore, immunogenic agents of the invention and/or method can treat or prevent the relevant disease of rhinovirus, illness or disease Condition is as described above.

In some embodiments, disease, illness or the patient's condition can be the relevant disease of hookworm, illness or the patient's condition.Hookworm It is Nemata worm, colonizes in many different animals.The hookworm of the usually infection mankind may include American hookworm and 12 Duodenum 12 hook worm (Ancylostoma duodenalis).Hookworm has hook-shaped mouthpart, and hookworm is attached to intestinal wall, pierces through Blood vessel and blood is sucked, leads to serious anaemia in some cases.The hookworm infection in pregnancy period can cause the growth of fetus slow Stagnant, premature labor and low birth weight.Hookworm in children can cause intelligence, cognition and growth question.

Therefore, immunogenic agents of the invention and/or method can treat or prevent the relevant disease of worm, illness or the patient's condition Than as described above.

In specific embodiments, preceding method can carry out as follows:

(i) immunogenic agents are applied, the immunogenic agents include one or more differences of single or identical pathogen Protein, segment, variant or derivative；

(ii) apply a variety of different immunogenic agents, the immunogenic agents respectively include different pathogens one kind or A variety of different protein, segment, variant or derivative；Or

(iii) immunogenic agents are applied, the immunogenic agents include one or more different eggs of different pathogens White matter, segment, variant or derivative；

To:

Cause the immune response for being directed to the pathogen；

It is immune for the pathogen；Or

Prevent or treat one or more diseases, illness or the patient's condition caused by one or more pathogen.

In some aspects and in embodiment, immunogenic agents can be applied with composition forms.

In specific embodiments, the composition may include:

Immunogenic agents, the immunogenic agents include one or more different albumen of identical or single pathogen Matter, segment, variant or derivative；

A variety of different immunogenic agents, the immunogenic agents respectively include one or more albumen of different pathogens Matter, segment, variant or derivative；Or

Immunogenic agents, the immunogenic agents include one or more different protein of different pathogens, segment, Variant or derivative；

In a preferred form, the composition includes acceptable carrier, diluent or excipient.

" acceptable carrier, diluent or excipient " means the solid that can be used safely in systemic administration or liquid filling Agent, diluent or encapsulated substance.Depending on specific administration method, variety carrier well known in the art, diluent can be used And excipient.These can be selected from including sugar, starch, cellulose and its derivates, malt, gelatin, talcum, calcium sulfate, plant Oil, synthetic oil, polyalcohol, alginic acid, phosphate buffer, emulsifier, isotonic saline solution and salt (such as inorganic acid salt, including salt Hydrochlorate, bromate and sulfate；Acylate, such as acetate, propionate and malonate), the group of water and apirogen water.

The available reference for describing acceptable carrier, diluent and excipient is Remington ' s Pharmaceutical Sciences (Mack Publishing Co.N.J.USA, 1991), is incorporated herein by reference.

Preferably, in order to cause the purpose of immune response, certain immunizing agents can be with immunogenic agents group disclosed herein It shares in preparation.

Term " immunizing agent " includes carrier well known in the art, delivery agents, immunostimulant and/or assistant within its scope Agent.This field will be understood that, immunostimulant and adjuvant refer to or the immunogenicities including one or more enhancing preparations and/or The substance of effect.Suitable immunostimulant and the non-limiting example of adjuvant include: saualane and squalene (or plant or dynamic Other oil in object source)；Block copolymer；Detergent such as tweenMineral oil such as Drakeol or Marcol, vegetable oil such as peanut oil；The adjuvant in corynebacteria source such as corynebacterium (Corynebacterium parvum)；The adjuvant in Propionibacterium source such as propionibacterium acnes (Propionibacterium acne)；Ox type branch bar Bacterium (Mycobacterium bovis) (BCG vaccine or BCG)；Pertussis bordetella (Bordetella pertussis) Antigen；Tetanus toxoid；Diphtheria toxoid；Surface reactive material such as cetylamine, octadecylamine, octadecyl amino-acid ester, molten Blood lecithin, dimethyl double dioctadecylammonium bromides, N, N- bis- octadecyl-N ', N ' two (2- ethoxy-malonamide), Methoxyl group hexadecane glycerol and pluronic polyalcohol；Polyamine such as pyrans, dextran sulfate, poly- IC card pop；Peptide such as born of the same parents Wall acyl dipeptides and derivative, dimethylglycine, stimulin；Oil emu；And mineral coagulant such as aluminum phosphate, aluminium hydroxide Or alum；Interleukin such as interleukin-22 and interleukin 12；Monokine such as interleukin-11；Tumor necrosis factor；Interferon example Such as interferon；Immunostimulating DNA such as CpG DNA, composition such as saponin(e-aluminium hydroxide or Quil-A aluminium hydroxide； Liposome；WithAdjuvant；Mycobacterial cell wall extract；The glycopeptide of synthesis such as cell wall Acyl dipeptides or other derivatives；Avridine；Lipid A derivative；Dextran sulfate；Individual deae dextran or and aluminum phosphate Deae dextran together；Carboxypolymethylene such as Carbopol ' EMA；Acrylic copolymer emulsion such as Neocryl A640 (example Such as U.S. Patent number 5,047,238)；Water-in-oil emulsifier such as Montanide ISA 720；Poliovirus, cowpox Or animal poxvirus protein；Or their mixture.

Immunizing agent may include: carrier protein such as thyroglobulin；Albumin such as human serum albumins；Poison Element, toxoid come from tetanus, diphtheria, pertussis, pseudomonad (Pseudomonas), Escherichia coli (E.coli), grape Any mutant cross reactivity substance of the toxin of coccus (Staphylococcus) and streptococcus (Streptococcus) (CRM)；Polyaminoacid is for example poly- (lysine: glutamic acid)；Influenza；Rotavirus vp 6, parvovirus VP1 and VP2；B-mode liver Scorching viral core protein；Hepatitis type B virus recombinant vaccine etc..It is alternatively possible to using carrier protein segment or epitope or The other immunogenic proteins of person.It is, for example, possible to use the t cell epitopes of bacteriotoxin, toxoid or CRM.It, can be in this aspect With reference to U.S. Patent number 5,785,973, it is incorporated by reference herein.

It is expected that any suitable scheme is for generating vaccine preparation.Exemplary arrangement includes, such as New Generation Vaccines(1997,Levine et al.,Marcel Dekker,Inc.New York,Basel,Hong Kong it those of described in), is incorporated by reference herein.

Any safe administration route can be used, comprising: intranasal administration, oral administration, rectally, parenteral are given Medicine, sublingual administration, Buccal administration, intravenous administration, intra-articular administration, intramuscular adminstration, intradermal administration, subcutaneous administration, suction Enter administration, eye drops, Intraperitoneal medication, intracerebroventricular administration, local administration, mucosa delivery and percutaneous dosing, although unlimited In this.

Dosage form include tablet, dispersion liquid, suspension, injection, solution, syrup, pastille, capsule, nasal spray, suppository, Aerosol, dermal patch etc..These dosage forms can also include the injection specially designed for the purpose or implantation control release device Or the implantation material of improved other forms, to additionally be played a role in the form of this kind.Controlled release may be by coating hydrophobic polymeric Object realizes that the hydrophobic polymer includes acrylic resin, wax, higher aliphatic, polylactic acid and polyglycolic acid and certain fibres Tie up plain derivative such as hydroxypropyl methyl cellulose.

Composition can be in isolated unit, such as capsule, wafer, functional food/feed or tablet, respectively include pre- The one or more therapeutic agents of the invention for first determining amount in powder or particle capsule or are in waterborne liquid, non-aqueous liquid, water Packet fat liquor or solution or suspension in water-in-oil liquid lotion.Such preparation can be prepared by any method of pharmacy, but It is that all methods all include the following steps: one or more reagents as described above and constitute one or more essential components Carrier in combination.In general, preparing the preparation by following: by reagent of the invention and liquid-carrier or fine crushing consolidating Body carrier or the two mix uniformly and nearly, then, if it is desired, product is moulded into desired form.

Above-mentioned preparation can be used in the mode compatible with the dosage form with effective amount.In the context of the present invention, it applies Dosage for patient should be enough to generate beneficial response in patients after a suitable period of time.The amount of medicament to be administered May depend on subject to be treated, including its age, gender, weight and general health, depending on doctor judgement because Element.

In a specific embodiment, the composition is suitable for through being intranasally applied to subject.

As conventionally used herein, term " patient ", " individual " and " subject " is in treatment disclosed herein or times of preparation What used under the background of mammalian subject.Therefore, method disclosed herein and preparation can be used for medical treatment and/or animal doctor answers With.In a preferred form, the mammal is behaved.

To be well understood the present invention can and generate actual effect, referring to following non-limiting embodiment.

Embodiment

Embodiment 1

Foreword

The upper respiratory tract (URT) mucous membrane and skin of A group streptococcus (GAS) main infection people, leads to numerous diseases.Infection It can lead to toxic shock syndrome, necrotizing fasciitis and muscle inflammation.Necrotizing fasciitis disease incidence is 10 a ten thousandths, and The death rate is up to 70% (1).Streptococcosis-rheumatic fever (RF) and rheumatic heart disease (RHD)-is equally concerned afterwards.Estimate There are about 15,600,000 RHD to show illness example, and annual almost 400,000 death (2) for meter.Bacterial colonization most common disease after URT It is pharyngitis, and RF and RHD and the pharyngeal infection closely related (3) of untreated primary.GAS infection and its related disease are in heat It is generally existing in Local resident with area, developing country and developed country, lead to 500,000 death (4) every year, highlights Vaccine needs.

GAS vaccine candidate object can be divided into the vaccine (5) based on M albumen and non-M albumen.The M albumen of cell surface is (by 3 The coiled-coil protein of main domains composition) it is main virulence determinants (6).The amino terminal that the protein is become by height With the A- repetitive structure domain for being used for epidemiology molecule parting (emm or M parting)；B- repetitive structure domain and conservative C- repeat to tie Structure domain (6) composition.Major subunit vaccine in clinical research is polyvaccine based on amino terminal M albumen and conservative C- repeats M albumen peptide vaccine (5).Based on the success of its inducing systemic immunity, these GAS vaccine candidate objects have entered clinical examination Test (7).Being proved general immunity effectively prevents GAS from disseminating to deep tissues, and passes through sero-immunity ball egg in whole body site White (Ig) prevents disease, but without prevent mucosal site colonize and thus interpersonal propagation (8).Therefore, entirely The best approach of body vaccine and the non-induced immunity for GAS.On the contrary, the mucous membrane epidemic disease for various organisms of nasal administration Seedling is in whole body and mucous membrane compartment effectively induction of antigen-specific immune response (9-11).Since such bilayer protective cap might is exempted from Epidemic disease power, mucosal vaccine are the ideal strategies for coping with whole body and mucous membrane GAS infection, and increased benefit is the resistance colonized to mucous membrane It only can also inhibit the propagation (7) by drop and aerosol from UTR.Mucosal vaccine be economically also it is beneficial, this is The important consideration of vaccine development.It, can be from using needle set (7) due to being easy to nasal administration vaccine.Painless delivering facilitates bigger Patient compliance.

Based on the minimum B cell epitope of the conservative C3- repetitive structure domain from M albumen, we have determined a kind of epidemic disease before this Seedling candidate peptide J8 (12).The J8 peptide (QAEDKVKQKQLEDKVQ；SEQ ID NO:1) it is embedding Peptide is closed, 12 amino acid from the area C- (being shown as boldface type) are contained, flank is GCN4DNA- binding protein sequence, with Maintain correct helical conformation structure (13).When being connected to carrier protein diphtheria toxoid (DT) and applied together with alum When, J8 protects mouse to prevent whole body and the skin attack (4,13) of a variety of GAS bacterial strains induction of IgG antibody.Moreover, when with The mucosal adjuvants CTB (14,15) for being limited to animal is applied together, or when applying as proteasome (16), the M egg based on GAS The vaccine candidate object of white conserved region, which is effectively protected, prevents GAS intranasal infection.When fixed induction of mucosal immunity and reduced URT When growing, it is determined that the correlation generated with IgA.After understanding this point, our target is to develop people's compatibility based on J8 to glue Film vaccine.

However, exploitation for people mucosal vaccine limitation first is that, lack safely and effectively mucosal adjuvants (17,18).

The spherical vesica that liposome is made of biocompatible phospholipids bilayer can load and deliver hydrophilic and hydrophobic point Sub (19).Liposome has passed through intra-nasal route and has safely been delivered to human body (20,21).However, presenting the liposome of peptide antigen simultaneously The ideal platform of non-induced peptide-specific antibody response.Peptide only includes limited can activate for needed for B cell antibody reaction T helper cell epitope.They are needed to be conjugated to " carrier " protein so that it generates immunity in outbreeding group, because This its presented by liposome it is not most suitable.But, the immunogenicity enhancing assigned by particle such as liposome is not odd It is strange, because natural pathogen is also particle, and (22) are identified by immune system well.Liposome is mutual with antigen presenting cell The natural of effect is inclined to as presenting cardinal principle (23) of the antigen to immune system with liposome.The purpose of the research is The Liposomal formulation (when lacking auxiliary agent) based on J8 is developed, wherein lipophilicity J8 construct is incorporated into double-layer of lipoid, and parent Aqueous carrier protein (DT) is encapsulated in internal contain in core water.

Material and method

Mouse.All animal protocols used are directed to the research ethics to be worked based on animal through Griffith University and examined The committee (Griffith University Research Ethics Review Board for Animal-Based Work, GU Ref No:GLY/09/14/AEC) approval.Strictly according to Australian National Health and Med Res Co (NHMRC) the guide about experimental animal implements the research.Selected method reduces the pain and torment of mouse to greatest extent, Animal is observed daily by trained animal caregivers.Using CO₂Suction chamber puts to death mouse.

The blood of people.Written informed consent is obtained by blood drawing techniques personnel from donor in health center of Griffith University Obtain blood.The research is by human research Ethics Committee of Griffith University (GUHREC, Protocol#GLY/03/14/HREC) Approval.Before experimenter handles sample, sample is gone into identificationization.

J8- lipid-DT preparation.For the non-covalent coordination for promoting J8 and double-layer of lipoid, will be made of two palmitinic acids (C16) Hydrophobic anchor be added to the ε and primary for being present in the lysine of tripeptides introns (being made of Lys Ser Ser) of J8 amino terminal Amino (C16-C16-KSSJ8).The construct by Shanghai Qiangyao Biotechnology Co., Ltd. (Chinapeptides Co., Ltd., Shanghai, China) production.The estimated molecular weight (MW 4061.97g/mol) of the construct is verified by ESI-MS, The product purity of acquisition is greater than 95% (passing through the analytical RP-HPLC area under tracing analysis).Using film aquation method (42) liposome is prepared.Using the rouge for coming from Avanti Polar Lipids company (Alabaster, United States) Matter, molar ratio are 7 two palmityl-sn- glycerol-3-phosphocholines (DPPC): 2 cholesterol Cholesterol (CHOL): 1L- α-phosphatidyl glycerol (PG).Using rotary evaporator by chloroform (CHCl₃) liposome and predetermined amount in solution C16- C16-KSSJ8 is applied to round-bottomed flask.Volume used are as follows: the DPPC (10mg/ml) of 0.7ml is in CHCl₃In, 0.2ml's CHOL (5mg/ml) is in CHCl₃In and 0.1ml PG (10mg/ml) in CHCl₃In.Then by lipid membrane aquation, and Room temperature is scattered in the 1mL phosphate buffer containing predetermined amount DT by being vigorously stirred.By obtained liposome suspension with 16, 162g is centrifuged 10min, removes supernatant, liposome bead is resuspended in proper amount of to be administered in the PBS of mouse.For determination The packaging efficiency of DT is collected supernatant, and is measured in supernatant using 2000 ultraviolet-uisible spectrophotometer of NanoDrop and do not wrapped The amount (Thermo Scientific, Massachusetts, United States) of the DT of envelope.From for rehydration lipid with The PBS starting DT concentration for generating liposome subtracts the DT concentration of supernatant, allows quantitative encapsulation efficiency.Once in 25 DEG C of apparatus Property capillary pipe cuvette nano particle size instrument (Zetasizer Nano Series ZS, Malvern Instruments, United Kingdom) measurement liposome average grain diameter (nm).With Noninvasive backscatter system analyze size, and with 173 ° of angle of scatterings measure.The relevant time is based on each run 10 seconds, each measurement at least continuous operation 5 times.The result is that Using scattering technology software analysis it is triplicate independently measure be averaged.It shows for J8-Lipo-DT and is referred to by low polydispersion The homogeneous size distribution that number (PDI) 0.238 is determined.PDI is that sample size is distributed how narrow instruction, and value is greater than 0.7 instruction sample Product have wide size distribution.

The intranasal immunisations of mouse.With xylazine and ketamine (xylazine: ketamine: H₂The mixing of O=1:1:10 Object) mixture, anaesthetize B10.BR and BALB/c mouse (Animal Resources Centre, Western to be immunized Australia,Australia).To the 30 independent J8-Lipo- of μ g in mouse application 20 μ L PBS of total volume (10 nostril μ L/) DT, and control mice applies the PBS (10 nostril μ L/) of 20 μ L.Positive control mice receives the J8 for being conjugated to DT of 30 μ g, simultaneously Apply the 10 μ g CTB (Sigma Aldrich, St.Louis, United States) in 20 μ L PBS of total volume.With with it is first Identical mode is immunized, mouse interval receives booster immunization twice in 21 days.Other control groups receive the independent of equivalent as described above J8, DT or liposome.

Serum, saliva and faecal samples are collected.After initial immunity, in the 20th, 40 and 60 day collection serum, with measurement The level of J8- specificity whole body antibody.The blood that mouse is collected by arteria caudalis allows to be aggregated at least 30min in 37 DEG C.With Serum is collected after 1000g centrifugation 10min, is stored in 56 DEG C of heat inactivation 10min and at -20 DEG C.

The 0.1% Pilocarpus jaborandi aqueous slkali of 50 μ L is applied with secretion inducing saliva to mouse.By saliva collection to containing 2 μ L 50mmol/L phenylmethylsulfonyl fluoride (PMSF) protease inhibitors (Sigma Aldrich) microcentrifugal tube in.With 13, 000g is centrifuged 10min and separates particle matter, and sample is stored at -80 DEG C.

6-10 parts of fresh excreta excrement balls are collected from single mouse, freezes and is lyophilized.The dry weight of excrement solid is measured, then 5% skimmed milk power, 50mmol/L EDTA (Sigma Aldrich), 0.1mg/mL Soybean Trypsin are resuspended in by being vortexed In enzyme inhibitor (Sigma Aldrich) and 2mmol/L PMSF (20 μ L/mg dry weight).It is solid with 15,000g centrifugation 10min separation Body substance stores supernatant at -80 DEG C.

Antibody titer is identified by Enzyme Linked Immunoadsorbent Assay (ELISA).As described at other (43) use ELISA measures J8- specific serum IgG and mucous membrane IgA.J8 peptide is diluted to carbonate coating buffer (pH 9.6) 0.5mg/ml, and be coated on polycarbonate plate with the volume in 100 holes μ l/, it is incubated overnight in 4 DEG C.Removal is unbonded Peptide, and closed in the hole 2 hours in 37 DEG C with the 5% skimmed milk PBS- polysorbas20 of 150 μ l.Then slow with PBS- polysorbas20 Fliud flushing washs the plate 3 times.In 0.5% skimmed milk PBS- polysorbas20 buffer, sample is serially diluted in plate, It starts from the initial of 1:100 and is diluted to 1:12, and 800 final dilution (for serum) and 1:2 to 1:256 are (for saliva Liquid/fecal specimens).Each sample is diluted to the final volume of 100 μ l, and is incubated for 1.5 hours in 37 DEG C.The plate is washed 5 It is secondary, and by peroxidase labelling goat anti-mouse IgG or IgA (Invivogen, San Diego, United States) It is respectively added in 0.5% skimmed milk PBS- tween with the dilution of 1:3000 or 1:1000, is incubated for 1.5 hours in 37 DEG C.It washes After washing, 100 μ l OPD substrates (Sigma Aldrich) are added according to the manufacturer's instructions, and dark incubation 30 at room temperature Minute.In Victor³1420 multiple labeling calculating instruments (Perkin Elmer Life and Analytical Sciences, Shelton, United States) in, absorbance is measured under 450nm.Titre is described as obtaining (to be contained in negative control hole Useful PBS immune Normal Mouse Serum) mean light absorbency on > the minimum dilution of the absorbances of 3 standard deviations (SD) Degree.Using one-way analysis of variance (ANOVA) and Tukey post-hoc tests, 5 software of GraphPad Prism is utilized (GraphPad, California, United States) measures significance,statistical (p < 0.05).

The process of GAS attack.The 63rd day after initial immunity, it is immunized with the intranasal attack of GAS bacterial strain M1 warp of predetermined close small Mouse and control mice.GAS bacterial strain M1 is continuously passed in mouse spleen to improve virulence, and is obtained streptomycin resistance and made The GAS obtained in throat swab is different from normal mouse bacteria flora (44).In order to measure colonizing for GAS, 1-3 days after attack, from small Mouse obtains throat swab.Throat swab scribing line is incubated at the Todd-Hewitt agar plate for defibrinating horse blood containing 2% On, and be incubated overnight in 37 DEG C.By the surface ten times that the nostril of each mouse is pressed in Columbia Blood Agar (CBA) plate (triplicate CBA plate/mouse/day) measures the bacterial load in nose cast, and the particle of exhalation is crossed and is cultivated.The 3 days, mouse is sorted, so that organ samples is homogenized in PBS, and use pouring plate method, by three parts of sample Inoculation.For nose cast and throat swab, it is poor to be as a result represented as average clonogenic unit (CFU)+standard error of the mean (SEM), for the 1st, 2 and 3 day 10 mouse/groups.For organ samples, it is as a result represented as average CFU+SEM, for the 3 days 10 mouse/groups.With GraphPad Prism 5, nonparametric, non-paired Mann-Whitney U check analysis are utilized Otherness compares test group and PBS control group (p < 0.05 is considered significant).

The preparation and maturation of DC.Peripheral blood is collected from healthy volunteer, and is based on Ficoll-Paque (Amersham Pharmacia, Uppsala, Sweden), it is classified by standard procedure.Based on Ficoll Paque, it is harvested by centrifugation PBMC is washed and with every 0.35mL MACS buffer (Miltenyi Biotec S.L., Germany) 1x 10⁸A cell Final densities are resuspended.Using Pan DC separating kit (Miltenyi Biotec), DC is separated according to the manufacturer's instructions. Obtained DC group is resuspended in RPMI 1640 (Gibco, Gaithersburg, the United for being supplemented with 10%FCS (Gibco) States) complete medium (has 2mM l- glutamine, 1% nonessential amino acid, 1%Pen-strep, 10mM HEPES).It is inoculated with the DC (2X 10 of total volume 0.2mL⁶), and by the shown following stimulants of addition: the pIC of 10 μ g/mL (Invivogen, San Diego, United States), J8-Lipo-DT (150 μ g/mL) or only complete medium are incubated for 24 Hour.Supernatant is collected after 24 hours, and is stored in -20 DEG C.

Immunophenotype analysis is carried out by flow cytometry.In order to analyze the surface expression of various markers, with a kind of or The mAb of a variety of following fluorogen labels dyes processed DC, and utilizes LSR Fortessa cell counter (Becton Dickinson, California, United States) and FlowJo software (Treestar, Inc., California, United States), it is analyzed by flow cytometry.Utilize following antibody (Becton Dickinson), the group assessed by flow cytometry: anti-HLA-DR-V450 ,-CD1c-APC ,-CD80- PE-Cy7,-CD83-PE-TexasRed,-CD86-PE,-CD123-Percp-5.5,-CD141-APC-Cy7.Resisted with suitable Body twice by cells rinsed with PBS, and is fixed after 4 DEG C secretly dyeing 30 minutes with 1% paraformaldehyde.Based on big granular Cell carries out gating, and from 2000-5000 gating events of each sample collection.In short, HLA-DR positive cell is set Door is further subdivided into 1 type of CD141+ routine DC, CD1c+ according to method (45) established before to limit people DC Conventional 2 type of (marrow sample) DC of DC and CD123+ Plasmacytoid DC.Based on gating group, average fluorescent strength (MFI) value is measured.Institute State data and be reported as average value+SEM, and examined using student t, with 5 software of GraphPad Prism (GraphPad, California, United States) analysis otherness.P value less than 0.05 is considered significant.

Splenocyte is stimulated with antigen in vitro.It is quantified using flow microsphere array (CBA) analysis and flow cytometry The proinflammatory response generated after the stimulation of J8 peptide by splenocyte.In short, preparation comes from J8- in 1640 culture medium of RPMI The single cell suspension without red blood cell of the spleen of mouse is immunized in Lipo-DT.It is inoculated with the splenocyte (4x of total volume 0.1mL 10⁵), and following stimulants: 2 μ g/mL LPS (Sigma Aldrich), J8 (10 μ g/mL) or only RPMI are added in accordance with the instructions 1640 culture mediums are incubated for 72 hours.Supernatant is separated after 72 hours, and is stored in -80 DEG C, is used for CBA flow cytometry.

Pass through the CBA chemotactic factor (CF) quantitatively secreted and cell factor.The inflammation quantitatively accumulated according to the manufacturer's instructions The level of property cell factor.For mouse anti-inflammatory agent box CBA, the volume of sample and reference substance is reduced to 10 μ l, and makes With every kind of capture pearl of 2 μ l.According to the recommendation of manufacturer, supernatant user's anti-inflammatory agent box CBA from the hole people DC (Becton Dickinson).The Run sample on LSR Fortessa cell counter, and with FCAP array (v1.01for Windows) software (Becton Dickinson) analyzes data.The data are reported as average value+mean value standard error (SEM), it and using student t examines, analyzes difference with 5 software of GraphPad Prism.P value less than 0.05 is considered as Significantly.

As a result

The building as described in material and method has the relevant J8 peptide in surface and the liposome containing diphtheria toxoid (Fig. 1).It is administered every dose of the preparation 30 μ g J8 comprising being connected to surface of liposome using the part based on palmitinic acid.Lipid Body contains every dose of 30 μ g DT in inside.The average diameter of the liposome as measured by dynamic light scattering is 1.8 μm of (standard deviations Difference=100.3nm) (referring to material and method).

Using first and 2 strengthened schemes, with J8-Lipo-DT and (every group 10 of control intranasal immunizations BALB/c mouse a variety of Only): individual liposome (Lipo)；Encapsulate the liposome (Lipo-DT) of DT；J8 is embedded in surface of liposome but the DT without encapsulating (J8-Lipo)；J8-DT+CTB；PBS+CTB；And PBS.

In order to compared with other constructs relatively to evaluate J8-Lipo-DT the effect of, then by intranasal immunizations be inoculated with mouse With M1GAS plants of pharynx isolate intranasal attacks (16) for being obtained from scarlet fever patient.Before attack, it is observed that comparing J8- Lipo, J8-Lipo-DT induce higher J8- specificity IgA (excrement and saliva) and serum IgG titers.Although for any One group, difference between J8-Lipo-DT and J8-Lipo is not statistically significant, it has been observed that J8-Lipo-DT for Saliva IgA response, excrement IgA response and serum IgG response are preferably (Fig. 2A-C).After being excited with GAS, with PBS group phase Than, in the mouse that J8-Lipo-DT is immunized, bacterium in nasal mucus burden is significantly lower, and be immunized with J8-DT+CTB Mouse is quite (the 3rd day, Fig. 3 A).

However, it was unexpectedly determined that J8-DT+CTB immune mouse is not protected from throat or the field planting of NALT, And J8-Lipo-DT immune mouse then shows and is significantly protected from two every indoor field planting (Fig. 3 B and C).J8- The protection of Lipo-DT is significantly better than the protection that J8-Lipo is induced.Mouse NALT is the portal of entry (24) for continuing GAS infection, and It and is the functional homologue (25) of human tonsil.Therefore, these results, which highlight J8-Lipo-DT, is reducing mucous membrane GAS infection In preferred sites the effect of biological load.

Then, whether our query J8-Lipo-DT can similarly protect the mouse of different lines.J8-DT/CTB and PBS Served as control immunogene.B10.BR mouse (n=5) is immunized induction of significant J8- Specific antibody titre with J8-Lipo-DT (Fig. 4).The titre in mouse is immunized quite (Fig. 4 A-B) in mucosal antibody titers and J8-DT+CTB in saliva and fecal specimens. In order to test whether J8-Lipo-DT will be protected from GAS infection in B10.BR mouse, another mouse population (n=5) is exempted from Epidemic disease is simultaneously attacked with M1 plants of GAS.Nose cast and throat swab were monitored within 3 days observation periods.By the 2nd day, J8-Lipo-DT and J8-DT+CTB immune mouse has undetectable biological load (Fig. 5 A) in nose cast.It is similar with BALB/c mouse, Data also demonstrate that when to after exciting the 2nd day, the throat swab of J8-Lipo-DT immune mouse does not have bacterium, and J8-DT+CTB exempts from The mouse of epidemic disease on day 3 when still have the GAS (Fig. 5 B) of detectable level in throat swab.

Research before confirms, although the peptide encapsulated in liposome does not induce immunoglobulin response, liposome Add lipid A (ingredient of lipopolysaccharides) to can induce after Intraperitoneal immunization antibody response (26), thus prompts the broadtail of lipid A can To work as adjuvant.In order to answer whether the J8 double C16 broadtails for being anchored to surface of liposome are responsible for luring for antibody response It leads, another mouse population is immune with C16-C16-KSSJ8, J8-Lipo-DT, J8-DT+CTB or PBS.It is observed that J8- Lipo-DT and J8-DT+CTB is immunogenicity, and C16-C16-KSSJ8 is not (Fig. 6 A-B).

The cytokine response of the splenocyte from intranasal immunizations mouse is measured, to determine intranasal immunizations whether induction of can With explain J8-Lipo-DT self adjuvanticity and conversion from antibody isotype to IgA systemic cell immune response.It analyzes Pro-inflammatory cytokine (interferon [IFN-γ], interleukin 1 [IL-1], IL-6, IL-12p70, monocyte chemotactic Albumen 1 [MCP-1] and tumor necrosis factor α [TNF-α]).By J8-Lipo-DT be immunized B10.BR mouse put to death, and with J8, LPS or medium stimulate splenocyte.It is observed that significant response generates (figure in the IFN-γ of J8 and LPS, MCP-1 and IL-6 7).It is not detected the other cell factors assessed.It should be as a result, it was confirmed that with J8-Lipo-DT immunity inoculation induction of proinflammatory Property response, provides the potential mechanism of self adjuvanticity of J8-Lipo-DT.It is known that IL-6 be responsible for antibody response to The conversion (27) of IgA.Furthermore it is known that chemical inhibitor MCP-1 plays a major role in GAS defense mechanism (28).

There is the potential of effective immune response of self adjuvanticity in order to evaluate J8-Lipo-DT in people's Immune inducing in vivo, from The blood of three healthy volunteers separates dendritic cells subset, and is stimulated with J8-Lipo-DT.Mature DC is that effective antigen is in Delivery cell, the high-caliber antigen presentation for participating in promoting antigen recognizing and cell-ECM interaction of expression and costimulation Cell surface molecule.It is mature in order to characterize people DC, check the response of various kinds of cell surface molecular in J8- by flow cytometry The adjustment (Fig. 8 A-C) of Lipo-DT.The double-stranded RNA adjuvant of synthesis, Polyriboinosinic polyribocytidylic acid (pIC) are used as control (29).Costimulatory molecules CD80, CD83 and the CD86 on CD123+ Plasmacytoid DC (pDC) cultivated together with J8-Lipo-DT Horizontal significantly higher (Fig. 8 A).In two classics DC subsets (cDC), 12 type DC of type DC and CD1c+ classics of CD141+ classics, The expression of CD80 also increases (Fig. 8 B-C).In addition, the CD86 expression of CD141+DC is also increased (Fig. 8 B).

In order to further clarify the interaction with people DC, using cytometric bead array assessment stimulation after proinflammatory cytokine because The level of son.It is observed that pro-inflammatory cytokine (TNF-α, IL-6 and IL-1 β (IL-1 β)) and neutrophil cell The increased expression (Fig. 9) of chemical inhibitor IL-8.Known neutrophil cell is most important to the IgA control of GAS infection (30).Raised levels of anti-inflammatory cytokines IL-10 (Fig. 9) is also observed.This may be since IL-10 is in DC maturing step With the adjustment effect (31,32) in balance host pro-inflammatory response.However, specifically, IL-6 response prompt, J8-lipo-DT It will lead to IgA conversion and the neutrophil cell response (via IL-8) in human body, place will be made to control GAS infection well. As shown in Figure 10, when individually being shown and being shown as S2-J8 block polymer (SEQ ID NO:3), by lipid body display SpyCEP peptide (S2；SEQ ID NO:2) and intracapsular DT caused mucous membrane IgA response together.

Referring to Figure 11, J8+S2-Lipo-DT is induction of antigentic specificity IgA, IgG response.It observes to J8-Lipo-DT With the comparable immune response of S2-Lipo-DT.Two kind epitope (J8+S2-s of the different preparation strategies using (i) in liposome ) or the mixture of (ii) J8/S2S2-Lipo-DT liposome Lipo-DT.

The infection of A group streptococcus can cause a variety of Skin and soft tissue infections, and some of which is severe, is even threatened Life.Thus it is of interest that checking the skin infection whether J8-Lipo-DT can be protected from after 88/30 plant of attack.It adopts With significant IgG after the initial experiment display J8-Lipo-DT intranasal immunizations of the nasal administration of the liposome individually comprising DT and J8 Titre, but without protection in skin attack measurement (data are not shown).It is carrying out through different liposome preparation or is leading to Cross the enhancing with the subcutaneous administration J8-DT+Alum whole body IgG response being immunized.

It discusses

We have been developed for the mucosal activity subunit liposome bacterin candidate of A group streptococcus (GAS).By lipid The displaying of GAS specific b cells epitope on the immunostimulatory properties of body and the encapsulation and surface of liposome of protein carrier DT It combines.Peptide and carrier protein are all required for best immunity.The mucosal immunity that complex liposome is induced is better than The mucosal immunity that the peptide-protein conjugate applied together with CTB is induced.

Mucosal immunity, for the means of the protective immunity of infectious diseases, causes many concerns as excitation.It is big absolutely Mucomembranous surface occurs or originates in for part infection.Therefore, the application that can induce the vaccine of mucosa-protective immune response is reason It is thinking and with practicability.In practice, it often confirms to be difficult to stimulate out strong mucosal IgA immune response, and utilizes It is unsatisfactory that subunit peptide antigen carries out the process that mucosal vaccine inoculation is made great efforts.This is partly due to be difficult to stimulate out and routine The comparable strong immune response of method based on full organism.It adds adjuvant and is conjugated with protein carrier subunit antigen Source as T cell auxiliary has been found to be effective for systemic immune.But mucosa immunity-inducing power needs newly Strategy.

" landform " position of the associated antigen of liposome influences antigen processing and the presentation to B cell and T helper cell (33).It has been shown that the antigen of exposure is preferentially processed on surface of liposome, and presented by B cell, and the antigen of liposome encapsulation It is more effectively processed and is presented to by antigen presenting cell T cell (34).Vaccine candidate object J8-Lipo-DT represents conjunction as a result, The subunit liposome bacterin of reason designs, this ensures that B cell epitope is associated with liposome bilayer, is combined with being exposed The Ig receptor of B cell, while the encapsulation permission of DT effectively delivers, processes and is presented to T cell.

We demonstrate that GAS is removed in the URT tissue including NALT.The effective nasopharynx that vaccine candidate object provides is immune Power shows that the ideal potentiality for reducing RF and RHD, RF and RHD and primary pharyngeal infection are associated (7).In mankind URT, almond Gland is that the primary of GAS stores position, maintains the endemic illness (25) in global range.The tonsilar functional analogue of the mankind GAS is colonized in NALT reduction prompt can reduce that the mankind are tonsilar to be colonized and infect using the intranasal immunisations of J8-Lipo-DT, To reduce the propagation (25) of GAS.

Although the peptide that liposome previously has been reported for delivering encapsulation carrys out inducing cellular immune response, such liposome is not IgA or IgG response is induced, unless there are strong adjuvant, such as LipidA (26,35).It might be that the lipid tail on J8 Portion provides adjuvanticity, therefore makes contributions to the immunogenicity of J8-Lipo-DT；However, itself there is lipid tail J8 peptide does not have immunogenicity, this demonstrates the need for Liposomal formulation.The immunostimulatory activity of itself adjuvant of liposome is previously It has been reported that, and is proved to be due to the interaction with antigen presenting cell and promotees the induction (36) of inflammatory response.Ours grinds The vitro detection studied carefully discloses the induction of antigentic specificity Chemokines and cell factor in immune mouse.Especially it is worth It is to be noted that the secretion of antigentic specificity MCP-1 and IL-6.MCP-1 is lymphocyte, monocyte and antigen presenting cell Chemistry ingratiates with agent (37).Be prompted before participate in mediate mucosal inflammation, due to can increase significantly mucous membrane IgA secretion and by It is reported as strong mucosal adjuvants (37).

Although antigen can be presented to immune system by many cell types, the starting of nave T cell need it is mature, Antigen presentation and be only found in professional antigen in delivery cell (such as DC) costimulatory molecules engagement (38).DC is bridge joint To congenital and adaptive immune response the key element (39) of infection.Mature DC generates inflammatory cytokine, raises thorn altogether Sharp and antigen presenting molecules, and migrate to lymph node, in lymph node, their function is as the strong of naive Tlymphocyte Antigen presenting cell, to start adaptive immune response.We demonstrate that J8-Lipo-DT mediates mankind DC after external exposure The expression of upper cell surface activation and maturity symbol object, induction include the pro-inflammatory cytokine of IL-6 and IL-8.People and mouse IL- 6 play key effect in B cell terminal differentiation, and in mucosal sites, stimulate the proliferation and secretion of IgA in mucosal sites (27).The presence (30) of neutrophil cell is needed for the IgA specific immunity of GAS.IL-8 is in the dynamic of neutrophil cell Key effect is played in member and activation.In this regard, the SpyCEP S2 peptide (SEQ shown by liposome particles delivery system is administered alone ID NO:2) or be administered in combination J8 peptide cause peptide specific IgA.

Therefore, assigning in the mankind can be used liposome platform to the basic mechanism of the immunity of GAS infection to mediate, Based on research with the relevance of clinical application provide support.

We demonstrate that mankind pDC increases both mature and costimulation markers when being stimulated with J8-Lipo-DT. Mankind pDC easily swallows and processes the antigen (40) being packaged in particle delivery system, shows that particle delivery system can be used for Promote effective delivering of the antigen to pDC.According to it is believed that our result shows for the first time with the particle delivery body based on liposome It is the ability of stimulating human pDC.The mankind pDC initially identified in blood is subsequent in spleen, lymph node and including tonsil Mucosal sites in be detected (41).Therefore, the vaccine delivery based on liposome is potential is exploited for targeting the DC Subgroup is for desired mucosal immune response in the mankind.

Embodiment 2

Following experiment is carried out to study influence of the liposome size to immunogenicity.

Liposome extruding is completed with thermal modules, and the mini extruder of 1mL syringe (Avanti Polar is utilized Lipids).Make to pass through 50nm, 400nm, 1000mm filter (Avanti Polar Lipids) solution 11 times of rehydration, simultaneously Thermal modules are set as~40 DEG C.Liposome size measurement carries out (dynamic light scattering or DLS) by Nanosizer.

As shown in figure 12, J8-Lipo-DT can be extruded and be formed the particle of nanometer or micron-scale.Particle size It is most of that there is narrow molecular weight distribution (low polydispersity index < 0.3).The data of Figure 13 show, J8-Lipo-DT size Systemic IgG response is not influenced.But as shown in figure 14, the liposome-induced J8 specific mucosal response of larger size.

Embodiment 3

Following experiment is carried out to study influence of the freeze-drying to immunogenicity of liposome.With containing 10% trehalose MilliQ water makes liposome membrane rehydration, is then lyophilized.1,4 and 7 week after freeze-drying, J8-Lipo-DT powder is restored with PBS.

Figure 15 shows the size knot by Nanosizer (dynamic light scattering or DLS) the liposome size measurement carried out Fruit.The major part of particle size has narrow molecular weight distribution (low polydispersity index < 0.3).Figure 16 shows, the jelly of recovery The liposome-induced J8 specificity whole body response of dry J8-Lipo-DT, without additional adjuvant.This be with it is freshly prepared The comparable immune response of J8-Lipo-DT.Trehalose is important the immunogenicity of the J8-Lipo-DT of freeze-drying.Figure 17 displays, the liposome-induced J8 specific mucosal response of the J8-Lipo-DT of the freeze-drying of recovery.

Embodiment 4

Further experiment determines the glycolipid Class Activation agent comprising congenital immunity, such as 6,6 '-two behenate of trehalose (TDB) and 3D-Immunogenic liposome the effect of, for example schematically shown in Figure 18.TDB is based in liposome The percentage and liposome formulation of total phospholipids.Using the phosphatide of 9mg, the ratio that TDB is used is 20% (1.8mg) of the amount.It is logical It crosses immune rear antibody titer (IgA and IgG antibody) and measures effect using the skin attack experiment of GAS bacterial strain.Data are shown in In Figure 19.With the J8-Lipo-Dt+TDB intranasal vaccinated mice (n=5/ group) of 30ug.(the 0th day) is applied to mouse and adds for the first time Strengthen (the 21st day and the 42nd day) twice.In saliva and fecal specimens, compared with J8-Lipo-DT, addition TDB results in aobvious Write higher mucous membrane IgA response.As a result the J8-Lipo-DT+TDB from freezing powder type, which raises the stabilizations of liposome Property.

Further experiment can determine whether the effect of including the immunogenic liposome of bile salt (such as NaTDC).Packet The example of the liposome of the NaTDC containing bile salt is illustrated schematically in Figure 20.It will be noted that immunogenic agents are (in the situation Under, J8 peptide) it can merge or be conjugated to carrier protein (such as DT) or can show on the surface of liposome.When phosphatide is hydrated When, bile salt will be formulated in liposome to generate liposome (referred to as " the bile body of the liposome containing bile salt (bilosomes)").Preparing for bile body is as follows.

It is the sorbitol anhydride tristearate (150mmol) of 7:3:1, cholesterol and double by molar ratio in round-bottomed flask The J8 of hexadecanyl phosphate (DCP) and 150g modified with palmitic acid moieties is dissolved in 10mL chloroform.It is steamed by rotation It sends out instrument and removes solvent, to form film on the glass surface of round-bottomed flask.Then, with (the bile of NaTDC containing 100mg Salt) and the 3.5mL PBS (pH 7.4) of 150g diphtheria toxoid make film hydration.

Embodiment 5

Influenza A virus, influenza B virus are directed to by antigentic specificity saliva IgA titrimetry lipid vesicle With the immunogenicity of A group streptococcus.Figure 21 shows the schematic depiction of immunogenic agents, and the immunogenic agents include single Lipid vesicle, the lipid vesicle have respective immune from each influenza A virus, influenza B virus and A group streptococcus It is former.What is shown in Figure 22 includes respectively exempting from from each influenza A virus, influenza B virus and A group streptococcus as the result is shown The lipid vesicle (showing in such as Figure 21 A) of epidemic focus is induced in mouse for the immune of these each pathogen.Further work The immunogenicity of the immunogenic agents including sugared lipid adjuvant schematically shown in such as Figure 21 B will be studied.

In short, this research reports the mucosal activity GAS vaccine candidate object based on liposome for the first time.Our discovery for Overcome the problems, such as at present in exploitation for preventing to be important a step in the infection of mucosal sites and the GAS vaccine of Community Communication. This research provides for how liposome particles delivery system integrally induces ideal mucosal immune response to fight GAS infection Important mechanism enlightenment.The strategy reported herein is directed to subunit's mucosal vaccine phase of other pathogens organism with exploitation It closes.Non-limitative example includes influenza virus, rhinovirus and hookworm, as described previously.In some embodiments, single Lipid vesicle may include the immunogene for a variety of different pathogens.

In the present specification, in order to describe the preferred embodiments of the invention, and be not to limit the invention to In any one embodiment or the specific collection of feature.It, can in the case where not departing from wide in range spirit and scope of the invention To be made various changes and modifications to the embodiment for being described herein and showing.

By herein cited all computer programs, algorithm, patent and scientific literature by reference it is whole simultaneously Enter herein.

Bibliography

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Claims

1. being suitable for the immunogenic agents applied to mammal, the immunogenic agents include one or more immunogenicity eggs White, its segment, variant or derivative, lipid vesicle and carrier protein or its segment or variant.

2. immunogenic agents as described in claim 1, are suitable for intranasal administration.

3. the immunogenic agents as described in claim 1 or claim 2 can cause mucosal immunity to be answered in mammals It answers.

4. the immunogenic agents as described in any one of aforementioned claim, wherein the carrier protein is located at intravesicular space.

5. the immunogenic agents as described in any one of aforementioned claim, wherein the carrier protein is diphtheria toxoid (DT)。

6. the immunogenic agents as described in any one of aforementioned claim, wherein the immunogenic protein, its segment, variant or Derivative is shown on the surface of the lipid vesicle.

7. the immunogenic agents as described in any one of aforementioned claim, wherein the immunogenic protein, its segment, variant or Derivative is fused to or is conjugated to the carrier protein.

8. the immunogenic agents as described in any one of aforementioned claim, wherein the lipid vesicle is liposome.

9. the immunogenic agents as described in any one of aforementioned claim, it includes identical pathogen or come from identical pathogen One or more immunogenic proteins, its segment, variant or derivative.

10. it includes each in a variety of different pathogens such as immunogenic agents of any of claims 1-8 Or one or more immunogenic proteins, its segment, variant or the derivative of each in a variety of different pathogens.

11. such as claim 9 or immunogenic agents described in any one of claim 10, wherein the pathogen is bacterium, virus or compacted Worm.

12. the immunogenic agents as described in any one of aforementioned claim, wherein the immunogenic protein, segment or variant are A streptococci bacteria M albumen, its segment, variant or derivative.

13. immunogenic agents as claimed in claim 12, wherein the segment is located within J8 peptide or its variant, or comprising J8 peptide or its variant.

14. immunogenic agents as claimed in claim 13, wherein the J8 peptide is following amino acid sequence, comprising following amino It acid sequence or is made of substantially following amino acid sequence:

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

15. the immunogenic agents as described in any one of claim 1-14, wherein the immunogenic protein is to promote neutrality Granulocyte activation recovering or the reagent of enhancing, or the reagent comprising promoting neutrophil leucocyte activation recovering or enhancing.

16. immunogenic agents as claimed in claim 15, wherein the examination for promoting neutrophil leucocyte activation recovering or enhancing Agent is protein, is SpyCEP or its segment.

17. immunogenic agents as claimed in claim 16, wherein the SpyCEP segment be following amino acid sequence, comprising with Lower amino acid sequence is made of following amino acid sequence substantially:

Amino acid sequence NSDNIKENQFEDFDEDWENF (SEQ ID NO:2).

18. the immunogenic agents as described in any one of aforementioned claim, wherein the immunogenic protein, its segment or variant Include the SpyCEP segment and M protein fragments as single chimeric peptide.

19. immunogenic agents as claimed in claim 18, wherein the chimeric peptide is following amino acid sequence or its variant, packet Containing following amino acid sequence or its variant or substantially it is made of following amino acid sequence or its variant:

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

20. the immunogenic agents as described in any one of aforementioned claim, wherein the immunogenic protein, segment or variant are Immunogenic protein, segment or the variant of influenza virus.

21. immunogenic agents as claimed in claim 20, wherein the immunogenic protein, segment or variant are following amino Acid sequence including following amino acid sequence are substantially made of following amino acid sequence: amino acid sequence MSLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO:4) or amino acid sequence PAKLLKERGFFGAIAGFLE (SEQ ID NO:5)。

22. the immunogenic agents as described in any one of aforementioned claim, wherein the immunogenic protein, segment or variant are Immunogenic protein, segment or the variant of rhinovirus.

23. immunogenic agents as claimed in claim 22, wherein the immunogenic protein, segment or variant are following amino Acid sequence including following amino acid sequence are substantially made of following amino acid sequence: amino acid sequence GAQVSTQKSGSHENQNILTNGSNQTFTVINY (SEQ ID NO:6) or amino acid sequence GAQVSRQNVGTHSTQNMVSNGSSL(SEQ ID NO:7)。

24. the immunogenic agents as described in any one of aforementioned claim, wherein the immunogenic protein, segment or variant are Immunogenic protein, segment or the variant of hookworm.

25. immunogenic agents as claimed in claim 24, wherein the immunogenic protein, segment or variant are following amino Acid sequence including following amino acid sequence are substantially made of following amino acid sequence: amino acid sequence TSLIAGLKAQVEAIQKYIGAEL(SEQ ID NO:8)。

26. the immunogenic agents as described in any one of aforementioned claim, further include the activator of congenital immunity.

27. immunogenic agents as claimed in claim 26, wherein the activator of the congenital immunity is glycolipid or comprising glycolipid.

28. the immunogenic agents as described in claim 26 or 27, wherein the activator of the congenital immunity is trehalose -6, 6 '-two behenates (TDB) or lipid A sugared lipid adjuvant.

29. the immunogenic agents as described in any one of aforementioned claim, further include bile salt.

30. immunogenic agents as claimed in claim 29, wherein the bile salt is NaTDC.

31. the immunogenic agents as described in any one of aforementioned claim are freeze-drying or freeze-drying.

32. the immunogenic agents as described in any one of aforementioned claim, are come with selected size or selected size range Production.

33. a kind of composition, it includes the immunogenic agents described in any one of preceding claims.

34. composition as claimed in claim 33, it includes single immunogenic agents, the immunogenic agents include identical disease Substance or from identical pathogen one or more immunogenic proteins, its segment, variant or derivative.

35. composition as claimed in claim 33, it includes single immunogenic agents, the immunogenic agents include it is a variety of not With it is each in pathogen or in a variety of different pathogens each one or more immunogenic proteins, its Segment, variant or derivative.

36. composition as claimed in claim 33, it includes a variety of different immunogenic agents, the different immunogenicity Agent respectively contain different pathogens or from different pathogens one or more immunogenic proteins, its segment, variant or Derivative.

37. one kind causes the method for the immune response for one or more pathogen in mammals, the method includes Following step: to immunogenic agents or such as claim of the mammal application as described in any one of claim 1-32 Composition described in any one of 33-36, to cause in the mammal for one or more pathogen Immune response.

38. a kind of method for one or more pathogen immunising mammals, the method includes the following steps: to described Mammal immunogenic agents of the application as described in any one of claim 1-32 or such as any one of claim 33-36 institute The composition stated, so that the mammal be immunized for one or more pathogen.

39. a kind of method for treating or preventing the infection as caused by one or more pathogen in mammal, the method packet Include following step: to mammal immunogenic agents of the application as described in any one of claim 1-32 or as right is wanted Composition described in any one of 33-36 is sought, to treat or prevent in the mammal by one or more cause of diseases It is infected caused by body.

40. the method as described in any one of claim 33-39, wherein the immunogenic agents or composition are by intranasal administration To the mammal.

41. the method as described in any one of claim 33-40, wherein the immunogenic agents cause mucosal immune response.

42. the method as described in any one of claim 33-41, wherein the mammal is people.