CN106606775A

CN106606775A - Liposomal group A streptococcus vaccine

Info

Publication number: CN106606775A
Application number: CN201610222100.8A
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: 2016-04-11
Publication date: 2017-05-03
Also published as: WO2017070735A1; CN109069424B; CN109069424A

Abstract

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

Description

Liposome A group streptococcus vaccines

Invention field

The present invention relates to by caused by A streptococci bacterias or relevant with A streptococci bacterias Disease and disease prevention and treatment.More particularly it relates to be exempted from by inducing mucosal Epidemic disease response is treating or prevent the liposome epidemic disease by disease caused by A streptococci bacterias and disease Seedling.

Background of invention

Upper respiratory tract (URT) mucosa of A group streptococcus (GAS) the main infection mankind and skin, Cause extensive disease.Infection can cause toxic shock syndrome, necrotizing fasciitis with And myositis.The sickness rate of necrotizing fasciitis is ten a ten thousandths, and fatality rate is up to 70% (1). Disease-rheumatic fever (RF) and rheumatic heart disease (RHD) after streptococcal infection is also highly closed Note.According to estimates, there are the popular case and about 400,000 deaths of 1.56 thousand ten thousand RHD every year (2).Modal disease is pharyngitis after URT is colonized, and RF and RHD with it is untreated Constitutional is pharyngeal to infect closely related (3).GAS infection and correlation disease torrid areas, send out It is popular in the aboriginal communities of country and developed country in exhibition, cause annual 500000 death (4), this protrusion shows the urgent needss to vaccine.

GAS vaccine candidate objects can be divided into the vaccine (5) based on M albumen and non-M albumen.Carefully The coiled-coil protein that cellular surface M albumen is made up of 3 main domains, it is main Virulence determinants (6).The albumen is by the high amino terminal for becoming and for epidemiology molecule parting The A- repetitive structure domains of (emm or M typings), B- repetitive structure domains and conservative C- repeat to tie Structure domain constitutes (6).Main subunit vaccine in clinical research is based on amino terminal M eggs White polyvalent vaccine and conservative C- repeats M albumen peptide vaccines (5).Based on it in inducing systemic The success of immunology, these GAS vaccine candidate objects come into clinical trial (7). Prove, general immunity is sent out by the serum immune globulin (Ig) of systemic sites in prevention GAS It is effective in deep tissues and prevention disease, but colonizing thus in prevention mucosal sites Interpersonal propagation aspect is prevented to be invalid (8).Therefore, whole body vaccination is for luring The immunity of impedance GAS is not optimal approach.By contrast, anti-various biologies of nose administration The mucosal vaccine of body is have in terms of the antigen-specific immune response of inducing systemic and mucosa compartment (9-11) of effect.Due to the immunity of this bilayer protective cap might, mucosal vaccine inoculation is for opposing whole body Property and mucosa GAS infection are preferable strategies, and the benefit that its pre- antiadhesion barrier having is colonized can also Suppress the propagation (7) by the spittle from URT and aerosol.Mucosal vaccine inoculation also has Advantage economically, this is an important considerations of vaccine development.Due to by nose way Footpath gives the convenience of vaccine, can avoid using needle set (7).Painless delivering contributes to preferably Patient compliance.

Summary of the invention

It is an object of the invention to provide causing exempting from for the mucosal immune response to A streptococci bacterias Epidemic focus agent and delivery system.In a broad sense, the present invention relates to by also comprising such as diphtheria class The lipid vesicle of the carrier protein of toxin (DT), by delivering immunogenic protein, fragment or change Body, promotes or induces the Mucosal immunity to A streptococci bacterias.Suitably, carrier protein Positioned at vesicle internal pore.

An aspect of of the present present invention provides and is suitable to be applied to the immunogenic agents of mammal, described Immunogenic agents include immunogenicity A streptococci bacteria albumen, its fragment, variant or derivative Thing, lipid vesicle and carrier protein.

Another aspect provides the drug regimen comprising foregoing aspects of immunogenic agents Thing.

Preferably, pharmaceutical composition comprising immunogenic agents and pharmaceutically acceptable carrier, Diluent or excipient.

In embodiments, drug regimen does not include adjuvant.

Another aspect provides is used for according to foregoing aspects of immunogenic agents in preparation Cause the purposes in the medicine for the immunne response of A group streptococcus in mammal.

Another aspect provides is used for according to foregoing aspects of immunogenic agents in preparation Mammal is made to the purposes in the medicine of A group streptococcus immunity.

Another aspect provides is used for according to foregoing aspects of immunogenic agents in preparation Purposes in treatment or prevention mammal in the medicine of A group streptococcus infection.

Another aspect provides is used for according to foregoing aspects of immunogenic agents in preparation Treatment or prevent mammal in by A group streptococcus infection caused by or with A group streptococcus senses Purposes in the medicine of the related disease of dye.

In embodiments, immunogenic agents can be applied in the case where adjuvant is lacked.

Suitably, according to aforementioned aspect, immunogenic agents can cause mucosal immune response.

Typically, mucosal immune response is protected including IgA.

In a preferred form, apply in immunogenic agents per nasal.

Suitably, immunogenic protein, its fragment or variant exhibits are on the surface of lipid vesicle. Suitably, carrier protein is located at the vesicle internal pore in vesicle.In preferred embodiments, Lipid vesicle is liposome.

Suitably, immunogenic protein fragment or variant are lipidization.In some embodiments In, lysine (K) residue in immunogenic protein fragment or the N- ends of variant is lipidization 's.In a preferred form, lysine (K) residue of N- ends passes through<It is lipidization with ε amine groups. In some embodiments, each lipid is C₁₆Fatty acid, such as Palmic acid.Preferably, Lysine (K) residue of N- ends is located between the N- ends of immunogenic protein fragment or variant In sub- aminoacid sequence.

In one particular embodiment, immunogenic protein, its fragment or variant are A group's chains Coccus M albumen, its fragment, variant or derivant.In other or optional embodiment, Immunogenic protein is to promote neutrophil activity to recover or enhanced reagent.

In a particular embodiment, M protein fragments are for the conservative region of M albumen or comprising M eggs White conservative region.In one embodiment, the fragment is immunogenic fragments, and it is included P145 peptides are contained in p145 peptides.In a particular embodiment, the immunogenic fragments section It is internal in J8 peptides or its change, or comprising J8 peptides or its variant.

Preferably, J8 peptides are comprising following aminoacid sequence or are made up of following aminoacid sequence substantially： QAEDKVKQSREAKKQVEKALKQLEDKVQ(SEQ ID NO:1)。

It is widely implemented in scheme at one, promotes the reagent for recovering or strengthening neutrophil activity It is Protein S pyCEP or its fragment.

In preferred embodiments, SpyCEP fragments comprising following aminoacid sequence or it is basic by with Lower aminoacid sequence composition：NSDNIKENQFEDFDEDWENF(SEQ ID NO:2).

In a specific embodiment, SpyCEP fragments and M protein fragments can merge with shape Into single chimeric peptide.

In one embodiment, the chimeric peptide is following aminoacid sequence or its variant, can be wrapped Containing following aminoacid sequence or its variant, or it is made up of following aminoacid sequence or its variant substantially： NSDNIKENQFEDFDEDWENFQAEDKVKQSREAKKQVEKALKQLEDK VQ(SEQ ID NO:3)。

In some embodiments, immunogenic agents can also include the activator of innate immunity.Institute Stating innate immunity activator can be with targeting c-type agglutinin, the such as derivable Ca of macrophage²⁺Rely on Property (c-type) agglutinin (" Mincle ").Non-limiting examples include glycolipid, such as mycobacteria core The mycolate of '-two (TDM) of factor trehalose -6,6 and/or its synthesis analog trehalose -6,6 ' - Two behenates (TDB).

In some embodiments, immunogenic agents can also include cholate, such as NaTDC.

Unless the context requires otherwise, term " including (comprise) ", " including (comprises) " And " including (comprising) " or similar term mean that nonexcludability is included, so as to chat The element stated or feature list individually comprising it is illustrating or listing those, and can be comprising not arranging Other elements for going out or illustrating or feature.

Under the background of aminoacid sequence, the amino of narration is meant by " substantially by ... constitute " Acid sequence and other 1 positioned at N- or C- ends, 2 or 3 aminoacid.

As it is used herein, here uses indefinite article '/one kind (a) ' and ' one/one kind (an) ' to refer to or comprising odd number or plural elements or feature, and it is not construed as meaning or limits Fixed " one/a kind of (one) " or " single " element or feature.

Brief description

The idealized structure of Fig. 1 .J8-Lipo-DT.Liposome encapsulates DT, at the same N-terminal with J8 and two introns KSS connected palmitic acid molecule covalent coupling, promotes J8 insertion liposomees Film.

Fig. 2. the J8 specific antibody responses of single BALB/c mouse.Mean antibody titers are with bar Represent.A) saliva IgA titres.B) feces IgA titres.C) serum IgG titers.Using unidirectional ANOVA carries out statistical analysiss, then carries out Tukey post-hoc tests (ns, p>0.05；*, p< 0.05；*, p<0.01；* *, p<0.001).

Fig. 3. in BALB/C mice, with M1GAS bacterial strains the antibacterial after intranasal is excited is carried out Load.A) nasal cavity cast.B) brush,throat.C) NALT is colonized.As a result represented with following： For brush,throat, nasal cavity cast, at the 1-3 days, 10 mices/group average CFU +SEM；For NALT, at the 3rd day, 10 mices/group average CFU+SEM.Utilize Nonparametric, the Mann-Whitney U test of non-matching carry out statistical analysiss, by test group and PBS Matched group is compared (ns, p>0.05；*, p<0.05；*, p<0.01；* *, p<0.001).

Fig. 4. the J8 specific antibody responses (every group of n=5) of single B10.BR mice.Average antibody Titre is represented with bar.A) saliva IgA titres.B) feces IgA titres.C) serum IgG titers. Statistical analysiss are carried out using unidirectional ANOVA, Tukey post-hoc tests (ns, p is then carried out>0.05； *, p<0.05；*, p<0.01；* *, p<0.001).

Fig. 5. assessment carries out the URT GAS of the bacterial load after intranasal is excited with M1 bacterial strains and swashs Send out model.A) the nasal cavity cast of B10.BR mices.B) the brush,throat of B10.BR mices.Knot Fruit with the 1-3 days, 5 mices/group average CFU+SEM represent.Using nonparametric, non-match somebody with somebody To Mann-Whitney U test carry out statistical analysiss, test group is compared with PBS control group (ns, p>0.05；*, p<0.05).

Fig. 6. the J8 specific antibody responses of single BALB/c mouse.Mean antibody titers are with bar Represent.A) saliva IgA titres.B) serum IgG titers.Counted using unidirectional ANOVA Analysis, then carries out Tukey post-hoc tests (ns, p>0.05；*, p<0.05；*, p<0.01； * *, p<0.001).

Fig. 7. the chemotactic factor and cytokine of antigenic specificity secretion in the mice of immunity.Take out spleen Cell, and add following stimulations as shown：LPS (2 μ g/mL), J8 (10 μ g/mL) or single Culture medium.72 hours after stimulation, supernatant is separated, and using cytometric bead array to secretion The level of chemotactic factor or cytokine is analyzed (referring to materials and methods).Checked using student t Carry out statistical analysiss (ns, p>0.05；*, p<0.05；*, p<0.01).

Fig. 8. with or in the case that unused reagent is treated, the surface marker in people's DC subgroups Thing level.Add following stimulations as shown：Polyinosinic acid：Poly- cytidylic acid (pIC, 10 μ g/mL), J8-Lipo-DT (150 μ g/mL) or single culture medium.24 hours after stimulation, By measured by flow cytometry cell surface marker.A) CD123+ Plasmacytoids DC.B) 1 CD141+ classical type DC.C) 2 CD1c+ classical types DC.Value is with from three individually confessions Average fluorescent strength (the MFI) ± SEM of the blended data of body is represented.Using nonparametric, non-matching Mann-Whitney U test carries out statistical analysiss, and test group is compared with vehicle control group (ns, p>0.05；*, p<0.05；*, p<0.01；* *, p<0.001).

Fig. 9. in human dendritic cell by J8-Lipo-DT secretion inducings chemotactic factor and cell because Son.Dendritic cell is taken out, and adds following stimulations as shown：pIC(10μg/mL)、J8-Lipo-DT (150 μ g/mL) or individually vehicle.Supernatant is separated, and utilizes streaming within 24 hours after stimulation Micropearl array is analyzed (referring to material and side to the chemotactic factor of secretion or the level of cytokine Method).Statistical analysiss (ns, p are carried out using student t inspections>0.05；*, p<0.05；*, p<0.01； * *, p<0.001).

Figure 10 .SpyCEP peptide (S2；SEQ ID NO:2) liposome delivery reagent causes mucosa IgA to answer Answer.Show the esterified S2 peptides of Palmic acid or S2-J8 chimeras (SEQ ID to mice intranasal administration NO:3) liposome and intracapsular DT, and measure S2 specificity IgA titres.

Figure 11 .J8+S2-Lipo-DT immunogenic agents inducing antigen-specific IgA, IgG in mice Response.

Figure 12. J8-Lipo-DT immunogenic agents can be extruded with formed nanometer or micron-scale Grain.Liposome size measurement is carried out by Nanosizer (dynamic light scattering or DLS).

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

Figure 14. the J8 specific mucosals of larger sized J8-Lipo-DT immunogenic agents induction should Answer.

The particle diameter distribution of the cryodesiccated J8-Lipo-DT powder restored in Figure 15 .PBS.Pass through Nanosizer (dynamic light scattering or DLS) carries out liposome size measurement.

Figure 16. in the case of without other adjuvants, the cryodesiccated J8-Lipo-DT lipids of recovery The J8 specificity more systemic responses of body immunogenic agents induction.

Figure 17. the J8 of the cryodesiccated J8-Lipo-DT liposome immunizations originality agent induction of recovery is special Different in nature mucosa response.

Figure 18. liposome immunization originality agent comprising the behenate of trehalose 6,6 '-two (TDB) is shown It is intended to description.

Figure 19. the schematic diagram description of the liposome immunization originality agent comprising cholate NaTDC.

Describe in detail

The present invention is at least partly according to following discoveries：With comprising the immunogen for being showed in surface of liposome Property peptide carries out mice nose with the liposome immunization originality agent of intracapsular carrier protein such as diphtheria toxoid (DT) Intradermal vaccine is inoculated with, and causes mucosa and systemic antibody response, and the response can be compatible with by non-human Adjuvant CTB induction those should compare.In A group streptococcus and the specific background of J8 peptides Under, the level of the protective immunity induced by Liposomal formulation is significantly more than being induced by J8/CTB Level.Additionally, the cytokine response induced by human dendritic cell (DC) subgroup of purification shows, This lipoid plastid will be effective in induction people's mucosa J8 specificitys IgA and systemic IgG response. In some embodiments, liposome immunization originality agent can individually comprising SpyCEP peptides or its its Its fragment also includes J8 peptides.In concrete form, liposome immunization originality agent goes for Treatment prevents by especially strong strain or to for the common antibiotics treatment of A group streptococcus infection Infection caused by A group streptococcus isolatess with resistance institute.These bacterial strains or isolatess are generally drawn Skin severe infections (such as necrotizing fasciitis) are played, and in some cases, can be accommodated CovR/SCovR/S is mutated.

For the object of the invention, " detached " means the material for having removed from its native state, or Manually-operated material is received.Detached material can substantially or essentially without usual With the component of its native state, or can be operated so as to usual adjoint its native state Component is in artificial state.Detached material can be natural, chemosynthesis or recombinant forms.

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

Term " protein " includes and covers " peptide " and " polypeptide " that peptide is generally used for description to be had not More than the protein of 50 (50) individual aminoacid, polypeptide is generally used for description to be had more than 50 (50) The protein of individual aminoacid.

" fragment " is section, domain, part or the region of protein, and it is constituted less than described The aminoacid sequence of protein 100%.It will be recognized that, fragment can be single fragment, or can Individually to repeat, or can repeat with other fragments.

Generally, fragment can 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、 1450th, 1500,1550 or 1600 aminoacid, it is consisting essentially of or be made from it.

As used herein, protein " variant " with have confirmable nucleoside with reference to aminoacid sequence Acid or aminoacid sequence relation." variant " protein can lack with reference to aminoacid sequence Or multiple aminoacid sequences, or replaced by different aminoacids.It is known in field, some aminoacid Can be replaced or lack, and the activity for not changing immunogenic fragments and/or protein is (conservative Type is replaced).Preferably, protein mutant with reference to aminoacid sequence total at least 70% or 75%, preferably at least 80% or 85%, or more preferably at least 90%, 91%, 92%, 93%, 94%th, 95%, 96%, 97%, 98% or 99% sequence iden.

What is be usually used herein describes the term bag of the sequence relation between each protein and nucleic acid Include " comparing window ", " sequence iden ", " Percentage of sequence identity " and " substantially the same ".By In each nucleic acid/protein can each self-contained (1) by nucleic acid/protein have Complete Nucleotide/albumen One or more different between the only one or some of matter sequence, and (2) nucleic acid/protein Part, generally by comparing come Perform sequence in " comparing window " comparative sequences, to identify and compare The sequence similarity of regional area." comparing window " refer to compare with canonical sequence usual 6,9 or Notional section of 12 consecutive residues.Relatively window can be included and be used for each sequence most The canonical sequence of ratio of greater inequality pair compares about 20% or less addition or deletes (i.e. room).For than Optimum comparison to comparing the sequence of window can be by the computer of algorithm operation (Wisconsin something lost Pass and learn (the Geneworks programs of Intelligenetics of software kit release 7.0；Genetics Software Package Release 7.0) in GAP, BESTFIT, FASTA and TFASTA, Genetics Computer Group, 575Science Drive Madison, WI, USA, by drawing With being incorporated into herein) or pass through range estimation and any one of the various methods that select is produced most Good comparison (that is, producing the highest homology percentage ratio for entirely comparing window) is implementing.For example Altschul et al,1997,Nucl.Acids Res.25:Blast program family disclosed in 3389 Can be incorporated into herein by quoting as reference.Discussing in detail for sequence analysis can be shown in In the general handbook (CURRENT of molecular biology that Eds.Ausubel et al. writes PROTOCOLS IN MOLECULAR BIOLOGY)(John Wiley&Sons Inc NY, Unit 19.3 1995-1999).

Term " sequence iden " used herein is used with its broadest implication, with including pass In the suitable comparison using canonical algorithm, with regard to the degree in relatively window identical sequence, accurately Nucleotide or amino acid match quantity.Therefore, " Percentage of sequence identity " is by following To calculate：The relatively two optimum sequences for comparing in relatively window, determine identical nucleic acid base (such as A, T, C, G, I) in two sequences occur position number with obtain match Positional number, by the positional number of matching divided by comparing total positional number (that is, window size) in window, and will The result for obtaining is multiplied by 100 to obtain Percentage of sequence identity.For example, it is possible to understand that " sequence Homogeneity " means by DNASIS computer programs (windows versions, version 2 .5；Can be from Hitachi Software engineering Co.,Ltd.,South San Francisco,California, USA obtain) calculate " match-percentage ".

As used herein, " derivant " is the such as albumen or its fragment or variant being changed Molecule, as understood in the art, such as, by being connected with other chemical parts or complexation, leads to Cross post translational modification (such as phosphorylation, acetylation etc.), glycosylation modification (for example addition, Remove or change glycosylation), it is lipidization and/or comprising extra aminoacid sequence.It is concrete one In embodiment, extra aminoacid sequence may include at one of its N-terminal and/or C-terminal or Multiple lysine residues.Multiple lysine residues (for example, PL200) can be residual for lysine The linear order of base or a chain-ordering of lysine residue.These extra lysine residues can promote Enter the peptide solubility of increase.

Extra aminoacid sequence may include the fusion partner aminoacid sequence for producing fusion protein. For example, fusion partner aminoacid sequence can aid in detection and/or the purification of detached fusion protein. Non-limiting examples include that metal combines (for example, polyhistidine) fusion partner, maltose and combines Protein (MBP), a-protein, glutathione s-transferase (GST), fluorescence protein sequence (for example, GFP), Epitope tag such as myc, FLAG and hemagglutinin labelling.Other volumes Outer aminoacid sequence includes spacer sequence.One example of spacer sequence is in immunogen Property protein fragments or variant aminoacid sequence N-terminal or the aminoacid sequence of C-terminal, its bag Containing being suitable to lipidization lysine (K) residue.Generally, introns aminoacid sequence includes two (2) To ten (10) aminoacid, such as three (3) aminoacid sequence KSS.

Other derivants that the present invention covers are included but is not limited to, the modification of side chain, in peptide or egg White matter is incorporated to alpha-non-natural amino acid and/or its derivant during synthesizing, and uses cross-linking agent, and Immunogenic protein, fragment and variant to the present invention applies the additive method of conformation constraint.

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

It should be understood that immunogenic protein disclosed herein, fragment and variant can be in individually The surface of present lipid vesicle, or as the identical peptide or multiple different peptides for including multiple copies Chimeric protein or fused protein are presented.Non-limiting examples are SEQ ID NO:3 it is chimeric Peptide, will be described in greater detail below.

In linguistic context of the present invention, term " immunogenicity " used herein is referred to mammal Or other animals use the ability or effect that immunne response is produced or caused after immunogenic agents, example Such as pathogen or the immunne response of its molecular components.

" initiation immunne response " means the product of one or more element of generation or stimulating immune system Raw or activity, including cell immune system, antibody and/or natural immune system.Suitably, exempt from One or more element of epidemic disease system includes bone-marrow-derived lymphocyte, antibody, neutrophilic leukocyte, includes Plasmacytoid dendritic cells are in interior dendritic cell, cytokine and/or chemotactic factor.Cell because The non-limiting examples of son include pro-inflammatory cytokine such as TNF-α, IL-6 and IL-1 (examples Such as IL-1 β).The non-limiting examples of chemotactic factor are neutrophilic leukocyte chemical inhibitor IL-8. Suitably, immunne response is or including mucosal immune response, for example, produces including IgA.It is preferred that Ground, the immunne response caused by immunogenic agents is protectiveness.

As this paper is general, term " immunity ", " vaccination " and " vaccine " refers to initiation for disease The protective immune response of substance, so as to the further infection of the pathogen is by least part of pre- The method and/or preparation of anti-or reduction.

As understood from the disclosure, the present invention provides lipid vesicle preparation, and it includes and is formulated in Immunogenic protein fragment, variant or derivant in lipid vesicle, and carrier protein. As used herein widely, lipid vesicle can be liposome, microcell, multilamellar vesicle, micro- glue Grain, cavity or other imitated vesicle structures comprising lipid bilayer, suitably, immunogenic protein piece Derivative one or more lipid to be anchored into lipid bilayer comprising promotion of section, variant or derivant Jing, So as to immunogenic protein fragment, variant or derivant are presented on the surface of lipid vesicle. In preferred form, by α and/or ε amino lipids lysine (K) residues.To promote lipid Change, immunogenic protein fragment, variant or derivant can further include N-terminal introns, The N-terminal introns are included by lipidization lysine (K) residue.Introns generally can be wrapped Containing 2-10 continuous amino acid, such as the introns KSS of three (3) aminoacid.In some enforcements In scheme, the lipid or each lipid are C₁₆Fatty acid, such as Palmic acid.However, will also Understand, such as with C₁₂-C₂₂Saturation or unsaturation (for example, the single unsaturated) fatty acid of carbochain Other lipids can be used for the present invention.

Suitably, lipid vesicle includes that any lipid of Lipid Bilayer Structure, or lipid can be formed Mixture.These include phospholipid, including including cholesterol, cholesteryl ester and phytosterol Sterols, fatty acid and/or triglyceride.The non-limiting examples of phospholipid include phosphatidyl gallbladder Alkali (PC) (lecithin), phosphatidic acid, PHOSPHATIDYL ETHANOLAMINE (PE) (cephalin), phosphatidyl glycerol (PG), Phosphatidylserine (PS), phosphatidylinositols (PI) and sphingomyelins (SM), or its is naturally occurring or synthetic Derivant.Natural derivative includes egg PC, egg PG, SPC, hydrogenated soybean PC, big Bean PG, brain PS, sphingol ester (sphingolipids), brain SM, galactocerebroside, god Warp knuckle glycosides fat, cerebroside, cephalin, cardiolipin and double hexadecyl acid ester.Synthesis Derivant include dipalmitoyl phosphatidyl choline (DPPC), DDPC (DDPC), Two mustard phosphatidyl cholines (DEPC), dimyristoyl phosphatidyl choline (DMPC), distearyl Phosphatidyl choline (DSPC), DLPC (DLPC), palmitoyl-oleyl phospholipid Phatidylcholine (POPC), palmityl dimyristoylphosphatidycholine (PMPC), palmitostearate phosphorus Phosphatidylcholine (PSPC), DOPC (DOPC), DOPE (DOPE), PE (DLPG), DSPG (DSPG), It is GLYCEROL,DIMYRISTOYL PHOSPHATIDYL (DMPG), DPPG (DPPG), two hard Acyl phosphatidyl glycerol (DSPG), DOPG (DOPG), palmitoyl-oleyl phospholipid Acyl glycerol (POPG), two myristoyl phosphatidic acid (DMPA), DPPA (DPPA), G 12S3P (DSPA), DMPEA (DMPE), two palmityls PHOSPHATIDYL ETHANOLAMINE (DPPE), two myristoyl Phosphatidylserine (DMPS), two palmityl phosphorus Acyl serine (DPPS), DSPE (DSPE), dioleoyl phospholipid acyl second Hydramine (DOPE), DOPS (DOPS), two palmitoyl sphingomyelins (DPSM) With distearyl sphingomyelins (DSSM).Phospholipid can also be the derivant or class of arbitrary above-mentioned phospholipid Like thing.

Suitably, the mixture of lipid can include expectation mol ratio or expect the various of wt% ratios Phospholipid.It is, for example possible to use mol ratio is 7 two palmityls-sn- glycerol-3-phosphocholines (DPPC): 2 cholesterol (CHOL):1L- α-phosphatidyl glycerol (PG) forms liposome.

Suitably, lipid vesicle also includes carrier protein.Suitably, carrier protein is to exempt from Epidemic disease genic protein, or promote or strengthen the immunogenicity of the immunogenic agents at least in part. Generally, carrier protein is prepared so that carrier protein is located at lipid capsule together with lipid vesicle In the internal aqueouss space internally of bubble.In some embodiments, carrier protein can be with institute State immunogenic protein fragment or its variant or derivant fusion, combine or complexation.This can be with Including recombiant protein fusion, chemical crosslinking and intermolecular complexation, such as by biotin-antibiont Fibroin or other intermolecular bonding agent, while not limited to this.In this kind of embodiment, institute State immunogenic protein fragment or its variant or derivant is located inside lipid vesicle water internally Property space in, with the carrier protein fusion, combine or complexation.The embodiment can be with spy Do not have for oral delivery immune-active agent, such as liposome comprising bile salt, such as herein It is described more particularly below.The non-limiting examples of carrier protein include diphtheria toxoid (DT), tetanus toxoid (TT), the CRM protein of such as CRM197 and pertussis Toxin mutants, while not limited to this.It is also contemplated by the fragment and variant of carrier protein.One In specific embodiment, carrier protein is diphtheria toxoid (DT) or its fragment.

In some embodiments, the lipid vesicle also activator comprising innate immunity.It is congenital Property immunity activator can targeting be related to innate immunity is expressed by one or more cell C- type agglutinins.Preferred C- types agglutinin is the Ca of macrophage induction²⁺- dependency (C- types) Agglutinin (" Mincle ").Non-limiting examples include glycolipid, such as mycobacteria cord factor sea The behenyl of analog trehalose -6,6 '-two of algae -6,6 '-two mycolates of sugar (TDM) and/or its synthesis Acid esters (TDB).Although being not intended to be limited by any particular theory, thus it is speculated that innate immunity swash Dynamic agent such as TDB can strengthen or improve by the mucosal immunity of immunogenic agents initiation.

In some embodiments, lipid vesicle can also comprising bile acid or bile salt.Gallbladder The steroid of dihydroxy or trihydroxy of the juice acid usually with 24 carbon, including cholic acid, Deoxycholic acid, chenodeoxycholic acid and ursodesoxycholic acid.Preferably, lipid vesicle includes bile acid Salt, such as cholate, deoxycholate, CDC or ursodeoxycholic hydrochlorate.Preferably, Bile salt is sodium deoxycholate.

In other embodiments, the liposome comprising immunogenic agents can be prepared as it is specific, Select or desired particle size or size range.In some embodiments, larger particles The liposome of size can cause stronger mucosal immune response.

In other embodiments, the liposome comprising immunogenic agents can be lyophilized or low pressure is frozen Do to promote longer-term storage.The freeze-dried lipidosome immunogenic agents of recovery cause and " fresh " lipid The suitable immunne response of the immunne response of body immunogenic agents.

As used herein, term " A group streptococcus ", " A group streptococcus " and abbreviation " GAS " are referred to The streptococcus bacterium of Lan Shi A sero-groups, it is micrococcus scarlatinae (Streptococcus Pyogenes) the gram-positive β hemolytic bacterias planted.The important virulence factor of GAS is M eggs White matter, it is strongly anti-cytophagous and is combined with serum factor H, destruction C3- conversions The opsonification of enzyme and prevention C3b.These also include toxicity " mutant ", for example, Graham Et al., CovR/S the or CovRS mutants of the descriptions of 2002, PNAS USA 99 13855, to the greatest extent Pipe not limited to this.

Disease that A group streptococcus cause and the patient's condition include cellulitis, erysipelas, impetigo, scarlet Heat, larynx infection as acute pharyngitises (" Strep throat "), bacteremia, toxic shock syndrome, Necrotizing fasciitis, acute rheumatic fever and acute glomerulonephritiss, although not limited to this.

As used herein, " neutrophilic leukocyte " or neutrophilic granulocyte are forming part polymorphonuclear cell man Race (PMN) and the cell of basophilic granulocyte and eosinophilic granulocyte.Neutrophilic leukocyte is by bone marrow Stem cell formed relatively short-lived phagocyte and typically comprise in mammal 40% to 75% leukocyte.Phagocytic neutrophilic leukocyte not only discharges solvable anti-microbial (such as granule Albumen), and produce neutrophil cell extracellular bacteria net.Neutrophilic leukocyte is to such as interleukin-8 (IL-8), the molecule of C5a, fMLP and leukotriene B4 has response, and the molecule promotes neutral grain thin Born of the same parents are to damage and/or the chemotaxiss at acutely inflamed position.

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

As used herein, " M protein fragments " be with immunogenicity and/or can binding antibody or Any fragment of the GAS M albumen of antibody fragment.Generally, fragment for it is following, comprising it is following or bag It is contained in following：The aminoacid sequence of the C- duplicate blocks of GAS M albumen or its fragment.It is non-limiting Example includes p145, and it is 20mer, with aminoacid sequence LRRDLDASREAKKQVEKALE(SEQ ID NO:4).In this respect, p145 aminoacid The fragment of sequence may reside in J8 peptides.

As used herein, " J8 peptides " is to derive from or corresponding to GAS M albumen comprising at least part of The peptide of the aminoacid sequence of C- areas peptide.J8 peptides are suitably comprising conformation B- cell epitope and without can The T- cell auto epi-positions that can be harmful to.Preferred J8 peptide amino acid sequences are QAEDKVKQKQLEDKVQ(SEQ ID NO:1) or its fragment or Variant, wherein residue 344 to 355 of the residue of the overstriking corresponding to GAS M albumen.At this In embodiment, J8 be also including flank GCN4DNA- associated proteins sequences chimeric peptide, institute State sequence to help maintain the correct helical fold and conformational structure of J8 peptides.

In another embodiment, immunogenic protein can be promotion neutrophilic granulocyte activation recovering Or enhanced reagent.

As used herein, " promote neutrophilic granulocyte activation recovering or enhanced reagent " for directly or At least partly increase, strengthen or recover generation, migration and/or the chemotaxiss of neutrophilic leukocyte indirectly And/or the molecule of one or more immunologic competence of neutrophilic leukocyte.In one embodiment, Reagent causes the immunne response of centering granulocyte inhibitor.In another embodiment, reagent knot Close neutrophilic granulocyte inhibitor and at least in part inactivate it.Neutrophilic granulocyte inhibitor can be with Be from or from the molecule of A group streptococcus.In a particular form, neutrophilic granulocyte suppression Preparation is the serine protease or its fragment that Proteolytic enzyme cuts interleukin 8.In a specific reality In applying scheme, neutrophilic granulocyte inhibitor is SpyCEP or its fragment.The suppurative hammers of SpyCEP Bacterium is the surface expression in human pathogen micrococcus scarlatinae (Streptococcus pyogenes) The serine protease of 170-kDa Multidomains, it is in the infection by catalytic pyrolysiss and neutral grain Play an important role in the inactivation of cell chemoattractant interleukin-8.SpyCEP aminoacid sequences Non-limiting examples can see accession number YP597949.1 and (micrococcus scarlatinae ) and YP596076.1 (micrococcus scarlatinae MGAS9429) MGAS10270.Therefore, at one In particular, SpyCEP fragments are or comprising SEQ ID NO:2 (NSDNIKENQFEDFDEDWENF) aminoacid sequence shown in.Propose SEQ ID NO:2 For or comprising the Dominant Epitopes on SpyCEP, it can be with inducing function antibody.

It is also provided herein comprising the M- Argine Monohydrochloride sequences for forming single, continuous aminoacid sequence The chimeric peptide of row and SpyCEP aminoacid sequences.M- protein amino acid sequences may be located at SpyCEP The C- ends of aminoacid sequence, or vice versa it is as the same.In one embodiment, chimeric peptide can be wrapped Containing aminoacid sequence NSDNIKENQFEDFDEDWENFQAEDKVKQSREAKKQVEKALKQLEDK VQ(SEQ ID NO:Or its variant 3).

In an alternate embodiment, can produce comprising M- protein amino acid sequences and SpyCEP ammonia Each liposome of base acid sequence is used for " mixture " administration.

In one particular embodiment, variant M albumen or peptide can be in itself N and/or C- ends Comprising one or more lysine residues.Multiple lysine residues (such as polylysine) can be bad ammonia The linear order of sour residue can be a chain-ordering of lysine residue.These other bad ammonia Sour residue can promote the solubility of increased peptide.

The non-limiting examples of J8 peptide variants include：

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

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

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

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

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

For example, if it is known that epi-position is located in alpha-helix protein structure conformation, then Ke Yihe Into the pattern peptide for being folded into the conformation.We are based on the structure design of GCN4 leucine zippers pattern Alpha-helix coiled coil peptide (O ' Shea et al., 1991).First heptapeptide contains sequence MKQLEDK (SEQ ID NO:9), it includes to stablize and exists in coiled coil heptapeptide repetition motif (a-b-c-d-e-f-g) n Some features (Cohen＆Parry, 1990).These are included in the big nonpolar residual of position a and d Base, in position the acid/base of e and g to (Glu/Lys) (typically favor inter-chain ionic interact) and The polar group (consistent with the prediction of Lupasetal. (1991)) of b, c, f in position.GCN4 peptides are also In position, a contains total L-Valine.Also it is mentioned that when position a and d is occupied by V and L, rolling up Bent spiral dimer for it is preferred (Harburyet al., 1994).Pattern heptad repeat region derives from These common characteristics of GCN4 leucine zipper peptides：(VKQLEDK；SEQ ID NO:10), it can Alpha-helix coiled coil can be formed.The peptide is changed into framework peptide.The superimposed sheets of the comformational epitope in research Producing chimeric peptide in section embedded model coiled coil peptide.Whenever in pattern peptide and epitope sequences send out During existing identical residue, the amino acid replacement of correct helical coil-coil conformation will be designed to ensure that (1990) Cohen＆Parry is mixed in chimeric peptide.Following displacements are usually used：Position a, V to I； B, K to R；C, Q to N；D, L to A；E, E to Q；,f:D to E；G, K to R.It is all this It is a little replace residues be commonly found in respective position in coiled-coil protein (Lupasetal., 1991).

It is described in Olive et al., the specific J8 of in 2002, Infect＆Immun.70 2734 Peptide derivant is " lipid core peptide ".In one embodiment, lipid core peptide can be included Two amino of each lysine of the direct polylysine core in the branch for being coupled to lipotropy anchor Multiple J8 peptides (such as four J8 peptides) of upper synthesis.

M protein fragments or variant and/or SpyCEP fragments or variant can be derivatized with comprising promoting Enter one or more lipids on the lipid upper strata for being anchored to mentioned above.In another embodiment, Chimeric peptide comprising M- protein amino acid sequences and SpyCEP aminoacid sequences (such as SEQ ID NO:3) introns aminoacid sequence can be included in its N- end.Therefore, in SpyCEP fragments or Variant is included in the embodiment of lipid vesicle, can be divided together with M protein fragments or variant It is not lipidization or can exist with lipidization chimeric peptide.

The detached immunogenic protein of the present invention, fragment and/or derivant can be by this areas Producing, the means include but is not limited to chemosynthesis, recombinant DNA technology to any means known With proteolytic cleavage producing fragments of peptides.

Chemosynthesis include solid phase and liquid phase synthesis.Although with reference to SYNTHETIC VACCINES 9th chapter and CURRENT of Ed.Nicholson (Blackwell Scientific Publications) PROTOCOLS INPROTEIN SCIENCE Eds.Coliganet al.,(John Wiley& Sons, Inc.NY USA 1995-2008) the 15th chapter in provide chemical synthesising technology example, But what such method was well known in the art.In this respect, reference is also made to international publication WO 99/02550 With international publication WO 97/45444.

Recombiant protein can easily be prepared using standard scheme by those skilled in the art, The standard scheme is described in such as Sambrooket al., MOLECULAR CLONING.A Laboratory Manual (Cold Spring Harbor Press, 1989), particularly the 16th part and 17th part；CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubelet al., (John Wiley＆Sons, Inc.NY USA 1995-2008), the particularly the 10th Chapter and the 16th chapter；And CURRENT PROTOCOLS INPROTEIN SCIENCE Eds. Coliganet al., (John Wiley＆Sons, Inc.NY USA 1995-2008), the particularly the 1st Chapter, the 5th chapter and the 6th chapter.Generally, recombiant protein is prepared and is included in table in suitable host cell Up to the nucleic acid of encoding proteins.

As described above, the invention provides immunogenic agents and/or they be used for prevent or treat the food in one's mouth The purposes of pathogen related disease, disease or the patient's condition in newborn animal or other animals.

As used herein, " treat (treating/treat/treatment) " and refer to after initially forming Improve at least in part, eliminate or reduce the related disease of pathogen, the symptom of disease or the patient's condition or The therapeutic intervention of pathological signs.Treatment is not necessarily definitely beneficial to object.Beneficial effect can be with Determined using any method known to persons of ordinary skill in the art or standard.

As used herein, prevent from (preventing/prevent/prevention) to refer to infecting or be exposed to Before A group streptococcus and/or the related disease, disease or the patient's condition of A group streptococcus symptom or pathology Learn sign outbreak before start to prevent from infecting and/or reducing the effect of symptom or pathological signs Process.It is also contemplated that such prevent from being not necessarily definitely beneficial to object.It is " preventative " treatment be for Reduce the purpose with the symptom of disease, disease or the patient's condition or the risk of pathological signs, apply In controlling for the sign for not showing disease, disease or the patient's condition or the object for only showing early indication Treat.

Disease, disease or the patient's condition can be the related disease of A group streptococcus, disease or the patient's condition.

In the context of the present invention, " the related disease of A group streptococcus, disease or the patient's condition " mean by A group streptococcus any clinical pathology for causing of infection and including cellulitis, erysipelas, pustule Disease, scarlet fever, larynx infection such as acute pharyngitises (" Strep throat "), bacteremia, toxic shock Syndrome, necrotizing fasciitis, acute rheumatic fever and acute glomerulonephritiss, although not limited to This.

As described above, the purposes for treatment and/or immunity disclosed herein is included to mammal Administration includes M protein fragments, variant or derivant, lipid vesicle, carrier protein and/or SpyCEP The immunogenic agents of peptide or promotion neutrophilic granulocyte activation recovering or enhanced other fragments.

As disclosed herein, treatment and/or immunity can be comprised additionally in as by targeting SpyCEP Infection site (such as skin) apply treatment on treat GAS infection antibody or antibody fragment and/or With reference to the antibody or antibody fragment of M albumen, its fragment or variant.

Antibody and antibody fragment can be polyclone or monoclonal, natural or restructuring.It is anti- Body fragment includes the fragment of Fc, Fab or F (ab) 2 and/or can include single-chain Fv antibody (scFv).This Class scFv can be prepared for example according to following methods are described in：U.S. Patent No. No. 5,091,513, European Patent No. No. 239,400 or Winter＆Milstein, 1991, Nature 349:293 article.Antibody can also include the polyvalent recombinant antibody fragment containing multiple scFv, Such as double-chain antibody, three chain antibodies and/or four chain antibodies, and half chain antibody of dimerization activation (demibodies) (such as WO/2007/062466).For example, this antibody-like can be according to description In Holliger et al., 1993ProcNatlAcadSci USA 90 6444；Or Kipriyanov, Method in 2009Methods MolBiol562 177 is preparing.Can be applicable to antibody produce, it is pure Changing can see with scheme known to purposes, such as Coliganet al., CURRENT PROTOCOLS IN IMMUNOLOGY's (John Wiley＆Sons NY, 1991-1994) 2nd chapter and Harlow, E.＆Lane, D.Antibodies:A Laboratory Manual,Cold Spring Harbor,Cold Spring Harbor Laboratory,1988。

Method for producing polyclonal antibody is to those skilled in the art well known.Can be with The exemplary arrangement for using is described in such as Coliganet al., CURRENT PROTOCOLS IN IMMUNOLOGY, sees above and Harlow＆Lane, 1988, in seeing above.In particular implementation In scheme, anti-SpyCEP polyclonal antibodies can be derived from or be purified from from being exposed to or infect A The individual human serum of group streptococcus.Alternatively, for purification or restructuring SpyCEP or its Immunogenic fragments, polyclonal antibody can be raised in the generation species of such as horse, then applied With subsequent purification before.

Monoclonal antibody can pass through one or more detached egg from the inoculated present invention In vain, the cell of the immortalization spleen of the generation species of fragment, variant or derivant or other generation antibody Use standard methods to prepare, such as it is initially described in&Milstein,1975,Nature 256, Article in 495 or for example, by being described in Coliganet al., CURRENT PROTOCOLS The article of the recent modification of IN IMMUNOLOGY (seeing above) is producing.Therefore, for basis M protein fragments, variant or derivant and/or promote neutrophilic granulocyte activation recovering that the present invention is used Or enhanced reagent (such as SpyCEP), monoclonal antibody can be raised.In certain embodiments, Monoclonal antibody or its fragment can be recombinant forms.If monoclonal antibody is initially fed by non-human The splenocyte of newborn animal is produced, then this may be particularly advantageous for " humanization " monoclonal antibody or piece Section.

For the embodiment related to therapeutic antibodies, preferred M protein fragments can be p145 Peptide.

For antibody produce SpyCEP preferred fragment can include following aminoacid sequences or It is made up of following aminoacid sequences：NSDNIKENQFEDFDEDWENF(SEQ ID NO:2).

In some aspects with embodiment in, immunogenic agents can be applied with dosage form.

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

" acceptable carrier, diluent or excipient " means that systemic administration can be used safely in Solid or liquid filler, diluent or encapsulated substance.Depending on specific route of administration, can be with Using variety carrier well known in the art, diluent and excipient.These can selected from sugar, starch, It is cellulose and its derivates, Fructus Hordei Germinatus, gelatin, Talcum, calcium sulfate, vegetable oil, artificial oil, many First alcohol, alginic acid, phosphate buffer, emulsifying agent, isotonic saline solution and salt are (such as inorganic acid salt, bag Include hydrochlorate, bromate and sulfate；Acylate, such as acetate, propionate and malonate), Water and apirogen water.

Description acceptable carrier, the free reference material of diluent and excipient are Remington’s Pharmaceutical Sciences(Mack Publishing Co.N.J.USA, 1991), it is incorporated herein by.

Preferably, in order to cause the purpose of immunne response, some immunological reagents can with it is public herein The immunogenic agents opened are combined in preparation.

Term " immunology agent (reagent) " includes carrier, the delivering examination in the range of it well known in the art Agent, immunostimulant and/or adjuvant.This area will be understood that, immunostimulant and adjuvant refer to or The material of the immunogenicity comprising one or more enhancing preparation and/or effect.Suitable immunostimulation The non-limiting examples of agent and adjuvant include：Squalane and Squalene (or other plants or animal origin Oil)；Block copolymer；Detergent such as tween Mineral oil such as Drakeol or Marcol, vegetable oil such as Oleum Arachidis hypogaeae semen；The adjuvant such as coryne bacterium parvum in corynebacterium source (Corynebacterium parvum)；The adjuvant such as propionibacterium acness in propionibacterium source (Propionibacterium acne)；Mycobacterium bovis BCG (Mycobacterium bovis) (bacillus calmette-guerin vaccine or BCG)；Bordetella pertussis (Bordetella pertussis) antigen；Tetanus toxoid；In vain Larynx toxoid；Surfactant such as cetylamine, 18-amine., octadecyl amino-acid ester, haemolysis ovum Phospholipid, dimethyl double dioctadecylammonium bromide, N, N-dicoctadecyl-N ', N ' two (2- hydroxyl second Base-Malondiamide), methoxyl group hexadecane glycerol and Pluronic polyolss；Polyamine such as pyrans, sulphuric acid Portugal gather Sugared, poly- IC carboxylics ethylene；Peptide such as muramyldipeptide and derivant, dimethylglycine, stimulin； Oil emulsion；And mineral coagulant such as aluminum phosphate, aluminium hydroxide or aluminum；Interleukin such as interleukin-22 and Interleukin 12；Monokine such as interleukin-11；Tumor necrosis factor；Interferon such as IFN-γ； Immunostimulating DNA such as CpG DNA, compositionss such as saponin aluminium hydroxide or Quil A hydroxides Aluminum；Liposome；WithAdjuvant；Mycobacterial cell wall extract； The glycopeptide of synthesis such as muramyldipeptide or other derivants；A Fuliding；Lipid A derivative；Sulfur Sour glucosan；Single deae dextran or the deae dextran together with aluminum phosphate；Carboxylic gathers Ethylene such as Carbopol ' EMA；(for example the U.S. is special for acrylic copolymer emulsion such as Neocryl A640 Profit number is 5,047,238)；Water-in-oil emulsifier such as Montanide ISA 720；Polioviruses, Cowpox or animal poxvirus protein；Or their mixture.

Immunological reagent can be included：Carrier protein such as Elityran；Albumin such as human serum Albumin；Toxin, toxoid or from tetanus, diphtheria, pertussis, pseudomonass, large intestine Any mutant cross reactivity material (CRM) of bacillus, staphylococcuses and streptococcic toxin； For example poly- (the lysine of polyamino acid：Glutamic acid)；Influenza；Rotavirus vp 6, parvovirus VP1 And VP2；Hepatitis B virus core protein；Hepatitis B viruss recombiant vaccine etc..Alternatively, The fragment or epi-position or other immunogenic proteins of carrier protein can be used.For example, can make With the t cell epitope of bacteriotoxin, toxoid or CRM.In this aspect, the U.S. is may be referred to special Profit number 5,785,973, is incorporated by reference herein.

Any suitable scheme is considered for producing bacterin preparation.Exemplary arrangement includes, for example New Generation Vaccines(1997,Levine et al.,Marcel Dekker,Inc.New York, Basel, Hong Kong) described in those, be incorporated by reference herein.

Any safe route of administration can be adopted, including：Intranasal administration, oral administration, rectum Administration, parenteral, sublingual administration, Buccal administration, intravenous administration, intra-articular administration, Intramuscular adminstration, intradermal administration, subcutaneous administration, inhalation, eye drops, intraperitoneal are given Medicine, intracerebroventricular administration, local administration, mucosa delivery and percutaneous dosing, while not limited to this.

Dosage form includes tablet, dispersion liquid, suspension, injection, solution, syrup, lozenge, glue Capsule, nasal spray, suppository, aerosol, transdermal patches etc..These dosage forms can also be included for this The heeling-in of the purpose release device that specially injection of design or implantation are controlled or improved other forms Agent, so as to additionally play a role in this kind of form.Controlled release may be affected by hydrophobic polymer is coated, The hydrophobic polymer includes acrylic resin, wax, high fatty alcohol, polylactic acid and polyglycolic acid And some cellulose derivatives such as hydroxypropyl methyl cellulose.

Preparation can be in detached unit, such as capsule, wafer, functional food/feedstuff or tablet, It respectively includes one or more therapeutic agent of the invention of predetermined volume；In powder or granule capsule or Person in the solution in waterborne liquid, non-aqueous liquid, oil-in-water emulsion or water-in-oil liquid emulsion or Suspension.Such preparation can be prepared by any method of pharmacy, but under all methods all include State step：By one or more required component of one or more reagent as described above and composition Carrier in combination.In general, preparing the preparation by following：By the reagent and liquid of the present invention Body carrier or the solid carrier for fully grinding or both is uniform and nearly mix, then, if Need, product is moulded into desired form.

Above-mentioned preparation can be used in the mode compatible with the dosage form, and its dosage used is effective 's.In the context of the present invention, being applied to the dosage of patient should be enough to after a suitable period of time Beneficial response is produced in patients.The amount of the medicament in patient to be administered may depend on to be treated Object, including its age, sex, body weight and general health, depending on doctor judge because Element.

In a specific embodiment, the preparation is suitable to be applied to object in per nasal.

As this paper is conventionally used, term " patient ", " individuality " and " object " is used in disclosed herein Treatment or any mammalian subject of preparation.Therefore, method disclosed herein and preparation are available In medical treatment and/or veterinary application.In a preferred form, the mammal is behaved.

To make the present invention to be well understood and produce actual effect, with reference to following non-limiting enforcement Example.

Embodiment

Embodiment 1

Foreword

Upper respiratory tract (URT) mucosa of A group streptococcus (GAS) main infection people and skin, lead Cause numerous diseases.Infection can cause toxic shock syndrome, necrotizing fasciitis and muscle inflammation. Necrotizing fasciitis sickness rate is 10 a ten thousandths, and mortality rate is up to 70% (1).Streptococcus afterwards Disease-rheumatic fever (RF) and rheumatic heart disease (RHD)-equally receive much concern.There are about 15,600,000 The existing illness examples of RHD, and annual almost 400,000 death (2).Bacterial colonization is most normal after URT The disease seen is pharyngitis, and RF and RHD is closely related (3) with untreated pharyngeal main infection. The Local resident of GAS infection and its relevant disease in torrid areas, developing country and developed country Middle generally existing, causes every year 500000 death (4), has highlighted needing badly for vaccine.

GAS vaccine candidate objects can be divided into the vaccine (5) based on M albumen and non-M albumen.Cell table The M albumen (FZ of the coiling being made up of 3 main domains) in face is that main toxicity is determined Determine factor (6).The albumen is by the high amino terminal for becoming and for epidemiology molecule parting (emm or M Typing) A- repetitive structure domains；B- repetitive structure domains and conservative C- repetitive structure domains (6) composition. It is amino terminal M albumen-base polyvalent vaccine and conservative based on the major subunit vaccine of clinical research C- repeat M albumen peptide vaccines (5).Based on the success of its inducing systemic immunity, these GAS Vaccine candidate object has been enter into clinical trial (7).It is proved general immunity and effectively prevents GAS to deep layer Tissue infectious disease, and disease is prevented by serum immune globulin (Ig) in whole body site, but without resistance Only its colonizing in mucosal site, so as to cause human-to-human transmission (8).Therefore, whole body epidemic disease The best approach of Seedling and non-induced for the immunity of GAS.Conversely, nasal administration for various The mucosal vaccine of organism is in whole body and mucosa compartment effectively induction of antigen-specific immune response (9-11).Due to such bilayer protective cap might immunity, mucosal vaccine is reply whole body and mucosa GAS The ideal strategy of infection, its increased benefit is that the prevention colonized to mucosa also can be by from UTR Drop and aerosol suppress infect (7).In view of the development of vaccine, mucosal vaccine is economically It is beneficial.Due to being easy to nasal administration vaccine, can avoid using needle set (7).Painless delivering is helped In the very big cooperation for obtaining patient.

Based on the minimum B cell epi-position of the conservative C3- repetitive structure domains from M albumen, we this Before determine a kind of vaccine candidate object peptide J8 (12).The J8 peptides (QAEDKVKQKQLEDKVQ；SEQ ID NO:1) it is chimeric peptide, It contains 12 aminoacid from C- areas (being shown as boldface type), and flank has GCN4 DNA- to tie Hop protein sequence, to maintain correct helical conformation structure (13).When being connected to carrier protein diphtheria class Toxin (DT) and when applying together with Alumen, induction of IgG antibody, its protection mice resists many to J8 The whole body and skin for planting GAS bacterial strains is excited (4,13).And, when with the mucosal adjuvants for being limited to animal CTB (14,15) apply together, or the M eggs when applying as proteasome (16), based on GAS The vaccine candidate object of white conserved region is effectively protected resists GAS intranasal infections.When induction of mucosa When immunity and the URT for reducing are colonized, it is determined that the dependency produced with IgA.Understand this point with Afterwards, our target is people compatibility mucosal vaccine of the exploitation based on J8.

However, it is to lack safely and effectively mucosa to be developed for one of restriction of mucosal vaccine of people Auxiliary agent (17,18).

The spherical vesicle that liposome is made up of biocompatible phospholipids bilayer, it can load and deliver Hydrophilic and hydrophobic molecule (19).Liposome is safely delivered to human body (20,21) by intra-nasal route. However, presenting the ideal platform of the liposome not induction peptide specific antibody response of peptide antigen.Peptide Only comprising the limited epitope that can activate the helper T cell needed for B cell antibody reaction. They need to be conjugated to " carrier " albumen so that it produces immunity in outbreeding population, therefore it is by fat Plastid is not most suitable to present.But, the immunogenicity for being given by granule such as liposome strengthens No wonder, because natural pathogen is also granule, and is recognized well (22) by immune system.Fat Conduct presents antigen with liposome extremely to the natural tendency that plastid interacts with antigen-presenting cell Immune cardinal principle (23).The purpose of the research be exploitation based on J8 Liposomal formulation ( When lacking auxiliary agent), wherein lipotropy J8 constructs are merged in double-layer of lipoid, and hydrophilic carrier albumen (DT) be encapsulated in inside containing in core water.

Material and method

Mice.

Using the equal Jing Griffith Universities of all animal protocols based on animal work Ethic review committee Member meeting (Griffith University Research Ethics Review Board for Animal-Based Work,GU Ref No:GLY/09/14/AEC) ratify.Strict implement Australia is implemented in the research National Health and the guide with regard to laboratory animal of Med Res Co (NHMRC).Selected side Method reduces to greatest extent the pain of mice and torments, and by trained experimenter, daily observation is dynamic Thing.Using CO₂Suction chamber puts to death mice.

The blood of people.

Jing Informed Consent Forms, in health center of Griffith University by blood drawing techniques personnel from donor Obtain blood.The research by human research Ethics Committee of Griffith University (GU HREC, Protocol#GLY/03/14/HREC) ratify.Experimenter is processed before sample, is gone identificationization.

J8- lipid-DT preparations.

To promote the non-covalent coordination of J8 and double-layer of lipoid, will be by dredging that two Palmic acids (C16) constitute Water anchor adds to the bad ammonia for being present in the aminoterminal tripeptides introns of J8 (being made up of Lys Ser Ser) The ε and primary amino radical (C16-C16-KSSJ8) of acid.The construct is shone by force the limited public affairs of biotechnology by Shanghai Department (Chinapeptides Co., Ltd., Shanghai, China) production.The estimated molecular weight of the construct (MW 4061.97g/mol) is verified by ESI-MS, and the product purity of acquisition (passes through more than 95% Analyze under the curve of analytical RP-HPLC areas).Lipid is prepared using film water cooperation usage (42) Body.Using the liposome from Avanti Polar Lipids companies (Alabaster, United States), Mol ratio is, 7 two palmityls-sn- glycerol-3-phosphocholines (DPPC):2 cholesterol Cholesterol(CHOL):1L- α-phosphatidyl glycerol (PG).Using rotary evaporator by chloroform (CHCl₃) liposome in solution is applied to round-bottomed flask with the C16-C16-KSSJ8 of scheduled volume.Institute Volume is：The DPPC (10mg/ml) of 0.7ml is in CHCl₃In, the CHOL (5 of 0.2ml Mg/ml) in CHCl₃In, and the PG (10mg/ml) of 0.1ml is in CHCl₃In.Then by fat Thin film aquation, and it is scattered in the 1mL phosphate containing scheduled volume DT by being stirred vigorously in room temperature Buffer.The liposome suspension for obtaining is centrifuged into 10min with 16,162g, supernatant is removed, Liposome bead is resuspended in proper amount of to be administered in the PBS of mice.To determine the envelope of DT Dress efficiency, collects supernatant, and is determined using the ultraviolet-uisible spectrophotometers of NanoDrop 2000 Amount (Thermo Scientific, Massachusetts, the United of non-encapsulated DT in supernatant States).Supernatant is deducted with the PBS startings DT concentration for producing liposome from for rehydration lipid DT concentration, it is allowed to quantitative encapsulation efficiency.In 25 DEG C of apparatus once property capillary pipe cuvette Nano-particle analyser (Zetasizer Nano Series ZS, Malvern Instruments, United Kingdom the mean diameter (nm) of liposome) is measured.With Noninvasive backscatter system analysis ruler It is very little, and measured with 173 ° of angle of scatterings.The related time is based on each run 10 seconds, each survey Amount is at least continuously run 5 times.Using scattering technology software (Dispersion Technology Software, Malvern Instruments, United Kingdom) analyze the meansigma methodss of independent three repeated measure Obtain a result.Determined by the low polydispersity index (PDI) 0.238 shown for J8-Lipo-DT equal Matter size distribution.PDI is that sample size is distributed how narrow instruction, and its value indicates sample tool more than 0.7 There is wide size distribution.

The intranasal immunisations of mice.

With xylazine and ketamine (thiazine:Ketamine:H₂O=1:1:10 mixture) mixture, The B10.BR and BALB/c mouse (Animal Resources Centre, Western of immunity is treated in anesthesia Australia,Australia).30 μ g in the μ L PBS of cumulative volume 20 (10 μ L/ nostrils) are applied to mice Independent J8-Lipo-DT, and control mice applies the PBS (10 μ L/ nostrils) of 20 μ L.Positive control Mice receives the J8 for being conjugated to DT of 30 μ g, and 10 μ g in the μ L PBS of cumulative volume 20 are administered simultaneously CTB(Sigma Aldrich,St.Louis,United States).With with initial immunity identical mode, Mice is spaced 21 days and receives booster immunization twice.Other control mices receive as mentioned above the list of equivalent Only J8, DT or liposome.

Serum, saliva and faecal samples are collected.

After initial immunity, serum was collected at the 20th, 40 and 60 days, to determine J8- specificity whole bodies The level of antibody.The blood of mice is collected by arteria caudalis, in 37 DEG C coagulation at least 30min is allowed. To collect serum after 1000g centrifugation 10min, in 56 DEG C of heat inactivation 10min and at -20 DEG C Storage.

The 0.1% Pilocarpus jaborandi aqueous slkali of 50 μ L is applied to mice with secretion inducing saliva.By saliva Collect to 50mmol/L Phenylmethanesulfonyl fluorides (PMSF) protease inhibitor (Sigma containing 2 μ L Aldrich in microcentrifugal tube).Particle matter is separated with 13,000g centrifugation 10min, by sample In -80 DEG C of storages.

The fresh Excreta excrement ball of 6-10 parts, freezing and lyophilizing are collected from single mice.Determine excrement ball The dry weight of solid, is then resuspended in 5% defatted milk powder, 50mmol/L EDTA by being vortexed (Sigma Aldrich), 0.1mg/mL soybean trypsin inhibitors (Sigma Aldrich) and 2 In mmol/L PMSF (20 μ L/mg dry weights).10min separating solid substances matter is centrifuged with 15,000g, By supernatant in -80 DEG C of storages.

Antibody titer is identified by Enzyme Linked Immunoadsorbent Assay (ELISA)

(43) as described by other, measured using ELISA J8- specific serums IgG and Mucosa IgA, 0.5mg/ml is diluted to carbonate coating buffer (pH 9.6) by J8 peptides, and And be coated on polycarbonate plate with the volume in 100 μ l/ holes, in 4 DEG C of overnight incubations, go Except uncombined peptide, and will be described in 37 DEG C with the 5% skimmed milk PBS- polysorbas20s of 150 μ l Hole is closed 2 hours, is then washed the plate 3 times with PBS- polysorbas20s buffer, 0.5% In skimmed milk PBS- polysorbas20 buffer, sample is serially diluted in plate, it starts In 1:100 are initially diluted to 1:12,800 final dilution (for serum), and 1:2 to 1:256 (for saliva/fecal specimens), by each diluted sample into 100 μ l cumulative volume, and in 37 DEG C are incubated 1.5 hours, and the plate is washed 5 times, and by peroxidase labelling goat Anti-mouse IgG or IgA (Invivogen, San Diego, United States) are with 1:3000 or 1:1000 dilution factor is respectively added in 0.5% skimmed milk PBS- tweens, and in 37 DEG C 1.5 are incubated Hour, after washing, 100 μ l OPD substrate (Sigma are added according to the description of manufacturer Aldrich), and secretly it is incubated 30 minutes at room temperature, in Victor³1420 multiple labeling calculating instruments In (Perkin Elmer Life and Analytical Sciences, Shelton, United States), Absorbance is measured under 450nm, titre is described as obtaining exceeding negative control hole (containing useful The Normal Mouse Serum of PBS immunity) mean light absorbency>The absorbance of 3 standard deviations (SD) Minimum dilution factor, using one factor analysis of variance (ANOVA) and Tukey post-hoc tests, profit Determined with the softwares of GraphPad Prism 5 (GraphPad, California, United States) and united Meter learns significance (p<0.05).

The process that GAS is excited

The 63rd day after initial immunity, exempted from exciting in the GAS bacterial strain M1 per nasal of predetermined close Epidemic disease mice and control mice.GAS bacterial strains M1 is continuously passed on to carry in mouse spleen High virulence, and streptomycin resistance is obtained so that the GAS in throat swab and normal Mus bacteria flora Different (44).In order to determine colonizing for GAS, excite latter 1-3 days, from mice throat swab obtained, By the streak culture Todd-Hewitt agar in horse blood of defibrinating containing 2% of the throat swab On plate, and in 37 DEG C of night incubation.By the way that the nostril of each mice is pressed in into Colombia's blood fine jade The surface ten times (the CBA plates/mice/day of three repetitions) of fat (CBA) plate is determined in nose cast Bacterial load, and by exhalation particle it is streak culture.3rd day, mice is chosen, Organ samples are made to homogenize in PBS, and using pouring plate method, by sample with three weights Multiple mode is inoculated with, and for nose cast and throat swab, is as a result represented as average Clone formation Unit (CFU)+standard error of the mean poor (SEM), for 10 mices of the 1st, 2 and 3 days / group.For organ samples, average CFU+SEM is as a result represented as, for the 3rd day 10 mice/groups.With GraphPad Prism 5, using nonparametric, non-paired Mann-Whitney U check analyses diversityes are comparing test group and PBS control group (p<0.05 It is considered as significant).

The preparation of DC and maturation

Peripheral blood is collected from healthy volunteer, and based on Ficoll-Paque (Amersham Pharmacia, Uppsala, Sweden), it is classified by standard procedure, based on Ficoll Paque, by the way that PBMC is harvested by centrifugation, washs and with every 0.35mL MACS buffer (Miltenyi Biotec S.L., Germany) 1x10⁸The final densities of individual cell are resuspended.Utilize Pan DC separating kits (Miltenyi Biotec), according to the description of manufacturer DC is separated, The DC colonies for obtaining are resuspended in the RPMI 1640 for being supplemented with 10%FCS (Gibco) (Gibco, Gaithersburg, United States) complete medium (has 2mM l- glutamy Amine, 1% non essential amino acid, 1%Pen-strep, 10mM HEPES).Inoculation cumulative volume 0.2 DC (the 2X10 of mL⁶), and by the following stimulus object of shown addition：Single 10 μ g/mL's PIC (Invivogen, San Diego, United States), single J8-Lipo-DT (150 μ g/mL) or single complete medium, it is incubated 24 hours.Supernatant is collected after 24 hours, And it is stored in -20 DEG C.

Immunophenotype analysis are carried out by flow cytometry

In order to analyze the surface expression of various marks, with one or more following fluorogen labelling MAb dye to processing DC, and using LSR Fortessa cell counter (Becton Dickinson, California, United States) and FlowJo softwares (Treestar, Inc., California, United States), it is analyzed by flow cytometry.Using following antibody (Becton Dickinson), the colony for obtaining is assessed by flow cytometry：It is anti- HLA-DR-V450、-CD1c-APC、-CD80-PE-Cy7、-CD83-PE-TexasRed、 -CD86-PE、-CD123-Percp-5.5、-CD141-APC-Cy7.With suitable antibody in 4 DEG C Dark dyeing, and is fixed afterwards for 30 minutes by cells rinsed with PBS twice with 1% paraformaldehyde. Gating is carried out based on big granular cell, and from 2000-5000 gatings of each sample collection Event, in short, HLA-DR positive cells gating is limited people DC, and according to it The method (45) of front establishment is further subdivided into the CD141+ routine types of DC 1, CD1c+DC Conventional (marrow sample) types of DC 2 and CD123+ Plasmacytoid DC.Based on gating colony, determine flat Equal fluorescence intensity (MFI) value.The data are registered as meansigma methodss+SEM, and utilize Student ' s t are checked, with GraphPad Prism 5 software (GraphPad, California, United States) diversity is analyzed.P values less than 0.05 are considered as significant.

Stimulate splenocyte with antigen in vitro

Come quantitative due to J8 using flow microsphere array (CBA) analysis and flow cytometry The proinflammatory response that the post-stimulatory splenocyte of peptide is produced.In short, in the culture medium of RPMI 1640 The middle single-cell suspension without erythrocyte for preparing the spleen from J8-Lipo-DT immune mouses Liquid.Splenocyte (the 4x10 of inoculation cumulative volume 0.1mL⁵), and add following stimulations in accordance with the instructions Thing：Single 2 μ g/mL LPS (Sigma Aldrich), J8 (10 μ g/mL) or RPMI 1640 Culture medium, is incubated 72 hours.Supernatant is separated within 72 hours afterwards, and is stored in -80 DEG C, use In CBA flow cytometries.

The chemotactic factor quantitatively secreted by CBA

The level of the inflammatory cytokine quantitatively accumulated according to the description of manufacturer.For little The volume of Mus anti-inflammatory agent box CBA, sample and reference material is reduced to 10 μ l, and uses 2 Every kind of capture pearl of μ l.According to the recommendation of manufacturer, the supernatant from people DC holes uses people Anti-inflammatory agent box CBA (Becton Dickinson).Transport on LSR Fortessa cell counters Row sample, and with FCAP arrays (v1.01for Windows) software (Becton Dickinson) Analytical data, the data are registered as the standard error (SEM) of meansigma methodss+average, and profit Checked with student t, with the software analysis differences of GraphPad Prism 5.P values less than 0.05 It is considered as significant.

As a result

As material and the structure described in method have the related J8 peptides in surface and containing diphtheria class poison The liposome (Fig. 1) of element.Using the part based on Palmic acid by containing the administration per the μ g J8 of dosage 30 Preparation is connected to surface of liposome.Liposome is internally containing per the μ g DT of dosage 30.By dynamic Measured by light scattering, the average diameter of liposome is 1.8 μm (standard deviations=100.3nm) (ginseng See material and method).

Using first and 2 strengthened schemes, with J8-Lipo-DT and various control intranasal immunizations BALB/c mouse (10 per group)：Single liposome (Lipo)；The liposome of encapsulating DT (Lipo-DT)；But J8 is embedded in surface of liposome without the DT (J8-Lipo) of encapsulating；J8-DT+CTB； PBS+CTB；And PBS.

, relatively evaluating effect of J8-Lipo-DT, then per nasal is exempted from order to compared with other constructs The mice of epidemic disease inoculation is excited (16) with the pharynx separator M1GAS strain intranasal available from scarlet fever patient. Before exciting, it is observed that comparing J8-Lipo, it is special that J8-Lipo-DT induces higher J8- Property IgA (feces and saliva) and serum IgG titers.Although for any group of, J8-Lipo-DT and Difference between J8-Lipo is statistically notable, it has been observed that J8-Lipo-DT is for saliva Liquid IgA responses, feces IgA responses and serum IgG response are preferably (Fig. 2A-C).Use GAS After exciting, compared with PBS groups, in the mice of J8-Lipo-DT immunity, the antibacterial in nasal mucus Burden is significantly lower, and suitable (the 3rd day, Fig. 3 A) with the mice with J8-DT+CTB immunity.

However, it was unexpectedly determined that the mice of J8-DT+CTB immunity do not defend throat or The field planting of NALT, and the mice of J8-Lipo-DT immunity then shows and significantly defend in Liang Ge areas Field planting (Fig. 3 B and C) in domain.Lured because the protection of J8-Lipo-DT is significantly better than J8-Lipo The protection led.Mus NALT is the portal of entry (24) of lasting GAS infection, and is human tonsil Functional homologue (25).Therefore, these results highlight J8-Lipo-DT and are reducing mucosa GAS Effect in the biological load of the preferred sites of infection.

Then, whether our query J8-Lipo-DT can similarly protect the mice of different lines. J8-DT/CTB and PBS served as control immunogens.With J8-Lipo-DT immunity B10.BR mice (n= 5) induction of significant J8- Specific antibody titres (Fig. 4).Mucoantibody in saliva and fecal specimens Titre (Fig. 4 A-B) suitable with the titre in J8-DT+CTB immune mouses.In order to test Whether J8-Lipo-DT will defend the GAS infection in B10.BR mices, another mouse population (n=5) With GAS M1 strains immunity and excite.Nasal mucus and throat swab are monitored within the observation period of 3 days.To 2 days, the mice of J8-Lipo-DT and J8-DT+CTB immunity had undetectable life in nasal mucus Thing bears (Fig. 5 A).Similar with BALB/c mouse, data are also demonstrated that, to after exciting 2 days, The throat swab of the mice of J8-Lipo-DT immunity does not have antibacterial, and at the 3rd day, J8-DT+CTB exempted from Still there is the GAS (Fig. 5 B) of detectable level in the throat swab of the mice of epidemic disease.

Research before is confirmed, although the peptide of encapsulating does not induce immunoglobulin response in lipid body, But liposome adds lipid A (composition of lipopolysaccharide) energy induction of antibodies response after Intraperitoneal immunization (26), thus prompting lipid A broadtail can work as adjuvant.In order to answer J8 grapplings Whether it is responsible for the induction of antibody response in double C16 broadtails of surface of liposome, another mouse population is used C16-C16-KSSJ8, J8-Lipo-DT, J8-DT+CTB or PBS are immune.It is observed that J8-Lipo-DT and J8-DT+CTB are immunogenic, and C16-C16-KSSJ8 is not (figure 6A-B)。

The cytokine response of the splenocyte from intranasal immunizations mice is measured, to determine intranasal immunizations Whether induction of the cellullar immunologic response of whole body, it can explain self adjuvanticity of J8-Lipo-DT With conversion from antibody isotype to IgA.Analyze pro-inflammatory cytokine (IFN-γ [IFN-γ], Interleukin 1 [IL-1], IL-6, IL-12p70, MCP 1 [MCP-1] and Tumor necrosis factor α [TNF-α]).By the B10.BR sacrifices of J8-Lipo-DT immunity, it is used in combination J8, LPS or medium stimulate splenocyte.It is observed that significantly respond to the IFN-γ of J8 and LPS, MCP-1 and IL-6 produces (Fig. 7).It is not detected other cytokines assessed.The knot Fruit confirms, with J8-Lipo-DT immunity inoculations induction of proinflammatory response, there is provided J8-Lipo-DT The potential mechanism of self adjuvanticity.It is known that IL-6 is responsible for antibody response turning to IgA Change (27).Furthermore it is known that chemical inhibitor MCP-1 plays a major role (28) in GAS defense mechanisms.

Answer in effective immunity that people's Immune inducing in vivo has self adjuvanticity to evaluate J8-Lipo-DT The effect answered, from the blood separation dendritic cell subset of three healthy volunteers, and uses J8-Lipo-DT Stimulate.Ripe DC is effective antigen-presenting cell, expresses the high-caliber promotion antigen that participates in and knows Antigen presentation and the cell surface molecule of costimulation that other and cell-ECM interacts.For table Traveller on a long journey DC is ripe, checks that various kinds of cell surface molecular responds to J8-Lipo-DT by flow cytometry Adjustment (Fig. 8 A-C).The double-stranded RNA adjuvant of synthesis, polyriboinosinic acid-polyribocytidylic acid (pIC) with comparing (29).CD123+ Plasmacytoid DC (pDC) cultivated together with J8-Lipo-DT On costimulatory moleculeses CD80, CD83 and CD86 level significantly higher (Fig. 8 A).At two Classical DC subsets (cDC), in CD141+ 1 types DC of classics and CD1c+ 2 types DC of classics, The expression of CD80 also increases (Fig. 8 B-C).Additionally, the CD86 expression of CD141+DC is also to increase (Fig. 8 B).

In order to further clearly with the interaction of people DC, using Cytometric bead array The level of pro-inflammatory cytokine after assessment stimulation.It is observed that pro-inflammatory cytokine The increasing of (TNF-α, IL-6 and IL-1 β (IL-1 β)) and neutrophil cell chemical inhibitor IL-8 Plus expression (Fig. 9).Known neutrophil cell controls most important (30) to the IgA of GAS infection. It was additionally observed that anti-inflammatory cytokines IL-10 (Fig. 9) of elevated levels.This is likely due to IL-10 Adjustment effect (31,32) in DC maturing steps and balance host pro-inflammatory response.However, tool For body, IL-6 responses prompting, J8-lipo-DT changes the IgA caused in human body, and it is thermophilic in Property granulocyte response (via IL-8), will orientate host as control GAS infection well.As schemed Shown in 10, when individually being shown and as S2-J8 block polymers (SEQ ID NO:3) when showing, by SpyCEP peptide (the S2 that liposome and intracapsular DT are shown；SEQ ID NO:2) mucosa has been caused IgA responses.

With reference to Figure 11, J8+S2-Lipo-DT is induction of antigenic specificity IgA, IgG responses.Observation To the suitable immunne response for responding to J8-Lipo-DT and S2-Lipo-DT.Different preparation strategies Two kinds of epi-positions (J8+S2-Lipo-DT) in liposome or (ii) J8/S2S2-Lipo-DT fat using (i) The admixture of plastid.

The infection of A group streptococcus can cause various Skin and soft tissue infections, and some of which is severe , even life-threatening.Thus it is of interest that checking whether J8-Lipo-DT can defend 88/30 plant excite after skin infection.Applied using the single per nasal of the liposome comprising DT and J8 Initial experiment shows, significant IgG titres after J8-Lipo-DT intranasal immunizations, but in skin Skin is excited in measure unprotected (data do not show).It is working on by different liposome preparation or logical Cross the enhancing of the whole body IgG responses with the immunity of subcutaneous administration J8-DT+Alum.

Discuss

We have been developed for the mucosal activity subunit liposome epidemic disease of A group streptococcus (GAS) Seedling candidate.By the encapsulation of the mucosal irritation properties of liposome and protein carrier DT and liposome The displaying of the GAS specific b cells epi-positions on surface combines.Peptide and carrier protein are all Required for optimal immunity.The mucosal immunity that complex liposome is induced is better than and CTB mono- Act the mucosal immunity that the peptide-protein conjugate for giving is induced.

Mucosal immunity is caused as the means for exciting the protective immunity for infectious disease Strong concern.There is or originate in mucomembranous surface in overwhelming majority infection.Accordingly, it is capable to induce The application of the vaccine of mucosa-protective immunne response is preferable and feasible.In practice, Stimulate and strong mucosal IgA immune response and be usually proved to extremely difficult, and utilize subunit The process that peptide antigen carries out mucosal vaccine inoculation effort is unsatisfactory.This be to a certain extent by In being difficult to stimulate the strong immunne response suitable with the conventional method based on full organism.Add Plus adjuvant and using the conjugated source as helper T cell of subunit antigen and protein carrier by Confirm for systemic immune is effective.But, mucosa immunity-inducing power remains a need for new plan Slightly.

" landform " position of the associated antigen of liposome affect antigen processing and to B cell and The presentation (33) of helper T cell.It has been shown that exposed antigen is preferentially added on surface of liposome Work, and presented by B cell, and the antigen of liposome encapsulation is more effectively processed, and by resisting Original presents presented by cells and gives T cell (34).Thus, vaccine candidate object J8-Lipo-DT is represented Rational subunit liposome bacterin design, this guarantees that B cell epi-position is related to liposome bilayer Connection, the Ig receptors of B cell are combined to be exposed, while the encapsulation of DT is allowed effectively Delivering, process and be presented to T cell.

We demonstrate that GAS is eliminated in the URT tissues including NALT.Vaccine candidate object There is provided effective nasopharynx immunity show reduce RF and RHD preferable potentiality, RF and RHD is associated (7) with main pharyngeal infection.In mankind URT, tonsil is the master of GAS Position is stored, causes in global range property disease (25) where persistence.Mankind's tonsil Functional analogue NALT in GAS colonize reduction prompting using J8-Lipo-DT intranasal Immunity can reduce that the mankind are tonsilar to be colonized and infect, so as to reduce the propagation (25) of GAS.

Although the peptide that liposome has been reported for delivering encapsulation carrys out inducing cellular immune response, this The liposome of sample will not induce IgA or IgG responses, unless there is strong adjuvant, such as Lipid A (26,35).It might be that the lipid tail on J8 provides adjuvanticity, therefore The immunogenicity of J8-Lipo-DT is made contributions；However, in the J8 itself with lipid tail Peptide does not have immunogenicity, and this demonstrates the need for Liposomal formulation.Itself adjuvant of liposome is exempted from Epidemic disease stimulating activity it has been reported that and be proved to be due to the interaction of antigen-presenting cell and Promote the induction (36) of inflammatory response.Vitro detection in our research is disclosed in immune mouse The induction of antigenic specificity Chemokines and cytokine.Particularly noteworthy is antigen The secretion of specificity MCP-1 and IL-6.MCP-1 is lymphocyte, mononuclear cell and antigen Chemistry in delivery cell ingratiates with agent (37).Be prompted before participate in mediation mucosal inflammation, its due to Mucosa IgA secretions can be significantly increased and strong mucosal adjuvants (37) have been reported as.

Although antigen can be presented to immune system, nave T cell by many cell types Preparation need the ripe of antigen and present, and be only found in professional antigen in delivery cell (for example The engagement (38) of costimulatory moleculeses DC).DC is that the congenital and adaptability bridged to infecting is exempted from The key element (39) of epidemic disease response.Ripe DC produces inflammatory cytokine, raises costimulation and resists Original presents molecule, and migrates to lymph node, and in lymph node, their function is as natural The strong antigen-presenting cell of T lymphocytes, to start adaptive immune response.We demonstrate that, The pro-inflammatory cytokine of IL-6 and IL-8, J8-Lipo-DT are included by external exposure and induction The expression of cell surface activation and maturity symbol thing on mediation mankind DC.People and Mus IL-6 are in B Pivotal role is played in cell terminal differentiation, and in mucosal sites, in its stimulating mucosal position The propagation of IgA and secretion (27).Need neutrophil(e) granule thin for the IgA specific immunitys of GAS The presence (30) of born of the same parents.IL-8 plays pivotal role in the mobilization and activation of neutrophil cell. In this regard, individually giving SpyCEP S2 peptides (the SEQ ID shown by liposome particles delivery system NO:2) or J8 peptides are given in combination inspire peptide specific IgA.

Therefore, the basic mechanism of the immunity to GAS infection is given in the mankind can use fat Plastid platform mediating, based on research provide with the relatedness of clinical practice and support.

We demonstrate that, when being stimulated with J8-Lipo-DT, mankind pDC increase it is ripe and Costimulation mark.Mankind pDC easily swallows and processes and is packaged in particle delivery system Antigen (40), shows that particle delivery system can be used to promote effective delivering of the antigen to pDC.According to It is believed that our result confirms to stimulate people with the particle delivery system based on liposome first The ability of class pDC.The mankind pDC for initially identifying in blood is subsequently in spleen, lymph Knot and including being detected (41) in tonsilar mucosal sites.Therefore, based on liposome The potential human mucosa being exploited for required for the DC subgroups are targeted of vaccine delivery is exempted from Epidemic disease response.

Embodiment 2

Carry out following experiment to study liposome size to immunogenic impact.

Liposome extruding is completed with thermal modules, using the mini extruder of 1mL syringes (Avanti Polar Lipids).The solution for making rehydration passes through 50nm, 400nm, 1000mm Filter 11 times (Avanti Polar Lipids), while thermal modules are set as into～40 DEG C.Liposome Dimensional measurement is carried out (dynamic light scattering or DLS) by Nanosizer.

As shown in figure 12, J8-Lipo-DT can be extruded and formed nanometer or micron-scale Grain.The major part of particle size has narrow molecular weight distribution (low polydispersity index<0.3). The data display of Figure 13, J8-Lipo-DT sizes do not affect systemic IgG responses.But, such as Shown in Figure 14, the liposome-induced J8 specific mucosals response of large-size.

Embodiment 3

Carry out following experiment to study the lyophilization of liposome to immunogenic impact.With containing The milliQ water of 10% trehalose makes liposome membrane rehydration, then lyophilizing.1,4 after lyophilizing With 7 weeks, J8-Lipo-DT powder is restored with PBS.

Figure 15 shows the liposome chi carried out by Nanosizer (dynamic light scattering or DLS) The size results of very little measurement.The major part of particle size has narrow molecular weight distribution (low many points Scattered sex index<0.3).Figure 16 shows, the liposome-induced J8 of J8-Lipo-DT of the lyophilizing of recovery Specific system response, without extra adjuvant.This is the J8-Lipo-DT with fresh preparation Suitable immunne response.Trehalose is weight for the immunogenicity of cryodesiccated J8-Lipo-DT Want.Figure 17 shows that the liposome-induced J8 specificitys of J8-Lipo-DT of the lyophilizing of recovery glue Film response.

Embodiment 4

Further experiment can determine whether the immunogen comprising the behenate of trehalose 6,6 '-two (TDB) Effect of property liposome.TDB and the percentage that liposome formulation is based on total phospholipidses in liposome Than.Using the phospholipid of 9mg, the ratio that TDB is used is 20% (1.8mg) of the amount.Pass through Surveyed using antibody titer (IgA and IgG antibody) after the immunity of GAS bacterial strains and skin excitation experiment Amount effect.The example of the liposome comprising the behenate of trehalose 6,6 '-two (TDB) is referring to Figure 18 Schematic diagram.

Further experiment can determine whether the immunogenicity lipid comprising cholate (such as NaTDC) Effect of body.Schematic diagram of the example of the liposome comprising cholate NaTDC referring to Figure 19. When phospholipid is hydrated, cholate can be formulated in liposome to produce the liposome (lipid containing cholate Body is referred to as " bile body (bilosomes) ").Preparing for bile body is as follows.

It is 7 by mol ratio in round-bottomed flask:3:1 sorbitan tristearate (150 Mmol), cholesterol and dicetyl phosphate ester (DCP) and 150g use palmitic acid moieties The J8 of modification is dissolved in 10mL chloroforms.Solvent is removed by Rotary Evaporators, to justify Thin film is formed on the glass surface of bottom flask.Then, (the bile of NaTDC containing 100mg is used Salt) and the 3.5mL PBS (pH 7.4) of 150g diphtheria toxoid make film hydration.

In a word, mucosal activity GAS vaccine candidate of this research reported first based on liposome Thing.Ours finds that direction is overcome in the infection and Community Communication for being developed for prevention mucosal sites GAS vaccines in the difficult problem that currently encounters stepped an important step.This research is liposome How grain delivery system integrally induces preferable mucosal immune response to resist GAS infection Enlighten there is provided important mechanism.

In this manual, in order to describe the preferred embodiments of the invention, and not It is the specific collection for limiting the invention to any one embodiment or feature.Without departing from this In the case of the wide in range spirit and scope of invention, embodiment that can be to being described herein and showing Make various changes and modifications.

By herein cited all of computer program, algorithm, patent and scientific literature by drawing Mode is integrally incorporated herein.

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Claims

1. the immunogenic agents applied to mammal are suitable for, and the immunogenic agents are included exempts from Epidemic focus A streptococci bacteria albumen, its fragment, variant or derivant, lipid vesicle and Carrier protein.

2. immunogenic agents as claimed in claim 1, it is suitable for intranasal administration.

3. immunogenic agents as claimed in claim 1 or 2, it can cause mammal Mucosal immune response.

4. immunogenic agents as described in aforementioned any one claim, wherein the carrier protein Positioned at vesicle internal pore.

5. immunogenic agents as described in aforementioned any one claim, wherein the carrier protein For diphtheria toxoid (DT) or its variant or fragment.

6. immunogenic agents as described in aforementioned any one claim, wherein the immunogenicity A streptococci bacteria protein fragments, its fragment, variant or derivant are illustrated in lipid vesicle table Face.

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

8. immunogenic agents as described in aforementioned any one claim, wherein the immunogenicity A streptococci bacteria albumen, its fragment or variant are A group streptococcus M protein fragments, variant Or derivatives thereof.

9. immunogenic agents as claimed in claim 8, wherein the fragment be located at J8 peptides or its Within variant, or comprising J8 peptides or its variant.

10. immunogenic agents as claimed in claim 9, wherein the J8 peptides are following amino Acid sequence, it is made up of following aminoacid sequence comprising following aminoacid sequence or substantially：

QAEDKVKQSREAKKQVEKALKQLEDKVQ(SEQ ID NO:1)。

11. immunogenic agents as any one of claim 1-10, wherein the immunity Immunogenic peptide is to promote neutrophilic granulocyte activation recovering or enhanced reagent.

12. immunogenic agents as claimed in claim 11, wherein the promotion neutrophilic granulocyte Activation recovering or enhanced reagent are albumen, are SpyCEP or its fragment.

13. immunogenic agents as claimed in claim 12, wherein the SpyCEP fragments are Following aminoacid sequence, it is comprising following aminoacid sequence or basic by following aminoacid sequence group Into：

NSDNIKENQFEDFDEDWENF(SEQ ID NO:2)。

14. immunogenic agents as described in aforementioned any one claim, wherein the immunogen Property albumen, its fragment or variant form the SpyCEP fragments and M of single chimeric peptide comprising fusion Protein fragments.

15. immunogenic agents as claimed in claim 14, wherein the chimeric peptide is following ammonia Base acid sequence or its variant, it is comprising following aminoacid sequence or its variant or basic by following ammonia Base acid sequence or its variant composition：

NSDNIKENQFEDFDEDWENFQAEDKVKQSREAKKQVEKALKQ LEDKVQ(SEQ ID NO:3)。

16. immunogenic agents as described in aforementioned any one claim, it also exempts from comprising congenital Epidemic disease activator.

17. immunogenic agents as claimed in claim 16, wherein the innate immunity activator For the behenate of trehalose -6,6 '-two (TDB).

18. immunogenic agents as described in aforementioned any one claim, it also includes cholate.

19. immunogenic agents as claimed in claim 18, wherein the cholate is deoxycholic acid Sodium.

20. immunogenic agents as described in aforementioned any one claim, it is cryodesiccated Or lyophilizing.

21. immunogenic agents as described in aforementioned any one claim, its with select size or Produce selecting in magnitude range.

22. pharmaceutical compositions, it includes the immunogenicity any one of claim 1-21 Agent and pharmaceutically acceptable carrier, diluent or excipient.

23. immunogenic agents as claimed in claim 22, it does not contain adjuvant.

Immunogenic agents any one of 24. claim 1-21 are being prepared for causing the food in one's mouth For the purposes in the medicine of A group streptococcus immunne response in newborn animal.

Immunogenic agents any one of 25. claim 1-21 are being prepared for making suckling Animal is to the purposes in the medicine of A group streptococcus immunity.

Immunogenic agents any one of 26. claim 1-21 prepare for treatment or Purposes in prevention mammal in the medicine of A group streptococcus infection.

Immunogenic agents any one of 27. claim 1-21 prepare for treatment or It is being caused by the infection of A group streptococcus in prevention mammal or related to the infection of A group streptococcus Purposes in the medicine of disease or the patient's condition.

28. purposes as any one of claim 24-27, wherein the immunogenicity Mammal is applied in agent per nasal.

29. purposes as any one of claim 24-28, wherein the immunogenicity Agent can cause mucosal immune response.

30. purposes as claimed in claim 29, wherein the immunne response is protectiveness.

31. purposes as any one of claim 24-30, wherein the mammal For people.