CN105412919A - Influenza vaccine, composition and use method - Google Patents

Abstract

The invention relates to a composition and a vaccine which contain mutational bordetella strains used for treating or preventing infection of mammal influenza viruses. In addition, the invention further provides a method for protecting mammals against influenza virus infection through the composition or the vaccine and/or inducing the immune response for the influenza viruses in mammals.

Description

Influenza vaccines, compositions and using method

The application is application number is " 200980160953.1 ", and the applying date is on June 15th, 2009, and denomination of invention is the divisional application of the application of " influenza vaccines, compositions and using method ".

Technical field

The present invention relates to microbiology and field of virology.

Background technology

There is the influenza virus of three types: A type, B-mode and influenza virus C, their difference in their epidemic diseases pattern are very large.Influenza A virus is best, also the most serious to public health threat by sign, and it can bring out large-scale epidemic disease or epidemic diseases.This virus also has the antigenicity of highly divergent isolate, makes to produce effective vaccine very difficult.

Influenza vaccines should be effective, safe, the nontoxic and the most most economical weapons preventing disease and control pathophoresis.Vaccinated main target activates the specific immune response adapted to, and mainly will produce B and the T lymphocyte for the antigen relevant to disease or cause of disease.

Can obtain some influenza vaccines at present, it is mainly containing inactivated vaccine.These vaccines can comprise two kinds of A type antigens (such as, H1N1 and H3N2) and the B-mode antigen of one.Available vaccine is generally containing whole virion, products of separated and subunit vaccine.Generally, if the homogeneity of these vaccines to newborn infectious disease is mated very much, they will have effect to the individuality of inoculation up to 90%.But they need to carry out every year upgrading to keep synchronous to change with the antigenicity of influenza virus.

In addition, a kind of attenuated live intranasal vaccine (LAIV) of the cold adaptation for seasonal influenza is recently reported.Resist about the Cotton Gossypii rat infecting H1N1 bacterial strain in a research of H3N2 virus and describe cross-protective immunity.But, be gene upset and the probability with wild type influenza virus miscegenation about one of LAIV main misgivings, its can produce a kind of newly, potential infectious bacterial strain.

The present invention is devoted to these and other problem solving influenza vaccines, provide vaccine and compositions, it uses bordetella (Bordetella) bacterial strain of sudden change as effective ingredient reagent, brings out the immunne response for one or more influenza strain.

In addition, description of Related Art uses bordetella bacterial strain to induce polytype vaccine for the immunne response that such as can cause the pertussal bordetella of people and compositions (WO2007104451 and WO2003102170); But these technology could not be disclosed in the method for the application of the invention in mammal, compositions and/or vaccine brings out method for the immunne response of influenza virus or compositions.

Therefore, demand is existed for the new type influenza vaccine extensively can resisting the disease caused by influenza infection.

Summary of the invention

The present invention relates to compositions and vaccine, it comprises the bordetella bacterial strain of the sudden change being used for the treatment of or preventing mammalian influenza virus to infect.In addition, invention further provides and use said composition or vaccine to protect mammal from influenza infection and/or the method for bringing out the immunne response for influenza virus in mammal.

In one aspect, the invention provides a kind of method of bringing out immunne response for influenza virus in mammal, comprise: to the bordetella bacterial strain of administration sudden change, wherein this bacterial strain comprises pertussis toxin, PT (ptx) gene of sudden change, cutaneous necrosis (dnt) gene of disappearance or sudden change and heterologous ampG gene.In some respects, this bordetella bacterial strain comprises bordetella pertussis bacterial strain.In some respects, wild type bordetella ampG gene is replaced by E. coli ampG gene.In other side, the sudden change of ptx gene comprises replaces the aminoacid of the aminoacid and/or participation catalysis that participate in Binding Capacity.In some respects, the amino acid whose replacement participating in Binding Capacity comprises K9R, and the amino acid whose replacement participating in catalysis comprises E129G.In some respects, this bordetella bacterial strain comprises the bacterial strain of three sudden changes.In some respects, this bordetella bacterial strain comprises BPZE1 bacterial strain.In other side, this bordetella bacterial strain is attenuation.In some respects, this bordetella bacterial strain comprises live strain.In other side, this bordetella bacterial strain does not comprise other heterologous gene except heterologous ampG gene.In some respects, this bordetella bacterial strain does not comprise and carries heterologous antigen and breathe the heterogenous expression platform of mucosa to mammal.In other side, the method comprises prevention further or treatment mammalian influenza virus infects.In some respects, this bordetella bacterial strain administration before influenza infection.In some respects, this bordetella bacterial strain is at influenza infection precontract 6 weeks or more time administration.In other side, this bordetella bacterial strain is at influenza infection precontract 12 weeks or more time administration.In some respects, this influenza virus comprises H3 or H1.In other side, this influenza virus comprises N2 or N1.

In some respects, this influenza virus comprises H3 and N2.In other side, this influenza virus comprises H1 and N1.In some respects, immunne response comprises Th1 immunne response.In some respects, this bacterial strain passes through subcutaneous (s.c.), Intradermal (i.d.), intramuscular (i.m.), intravenous (i.v.), oral or intranasal administration to mammal; Or by injecting or sucking.In other side, this bacterial strain intranasal administration.In some other sides, this bacterial strain is administered to the mammal needed for the protective immunity of influenza infection.This mammal is child in some respects.In some respects, this bacterial strain is administered once with single dose.In some respects, this bacterial strain administration is more than once.In some respects, this bacterial strain is with two dosed administrations twice.In other side, these two dosage are about every administration in 3 weeks.In some respects, the level of protection of influenza infection is resisted higher than about 60%.In other side, the level of protection of opposing influenza infection is higher than about 50%.In some respects, this mammal is people.

On the other hand; the invention provides the method for human body being brought out to the protective immune response for H3N2 influenza virus; comprise: to the BPZE1 bacterial strain of human body intranasal administration live body and attenuation before human infection H3N2 influenza virus, wherein this bacterial strain does not comprise and carries the heterogenous expression platform of heterologous antigen to human body respiration mucosa.

On the other hand, the invention provides the method for bringing out the immunne response for influenza virus at human body, comprising: to live bordetella bacterial strain to human administration, wherein this bacterial strain does not comprise and carries the heterogenous expression platform of heterologous antigen to human body respiration mucosa.

On the other hand; the invention provides the method for the disease that protection mammal causes from influenza infection; comprise: to the bordetella bacterial strain of mammal administration sudden change, it comprises the ptx gene of sudden change, the dnt gene of disappearance or sudden change and heterologous ampG gene.

On the other hand, the invention provides a kind of protective immunity for influenza infection, comprising: to the bordetella bacterial strain of mammal administration sudden change, it comprises the ptx gene of sudden change, the dnt gene of disappearance or sudden change and heterologous ampG gene.

On the other hand, the invention provides a kind of compositions being used for the treatment of or preventing mammalian influenza virus to infect, comprise: the bordetella bacterial strain of sudden change, wherein this bacterial strain comprises pertussis toxin, PT (ptx) gene of sudden change, cutaneous necrosis (dnt) gene of disappearance or sudden change and heterologous ampG gene.In some respects, this bordetella bacterial strain comprises bordetella pertussis bacterial strain.In other side, the ampG gene of wild type bordetella bacterial strain is replaced by E. coli ampG gene.In some respects, the sudden change of ptx gene comprises the replacement carried out the aminoacid of the aminoacid and/or participation catalysis that participate in Binding Capacity.In other side, the amino acid whose replacement participating in Binding Capacity comprises K9R, and the amino acid whose replacement participating in catalysis comprises E129G.In some respects, this bordetella bacterial strain comprises the bacterial strain of three sudden changes.

In some respects, this bordetella bacterial strain can comprise BPZE1 bacterial strain.In some respects, this bordetella bacterial strain can be attenuation.In some respects, this bordetella bacterial strain can comprise live strain.

In some respects, this bordetella bacterial strain can not comprise other heterologous gene except heterologous ampG gene.In some respects, this bordetella bacterial strain can not comprise and carries heterologous antigen and breathe the heterogenous expression platform of mucosa to mammal.

In some respects, said composition also can comprise pharmaceutically suitable excipient, carrier and/or carrier.In some respects, said composition also can comprise adjuvant.In other, said composition also can comprise the micromolecule that can affect influenza infection.

On the other hand, the invention provides a kind of vaccine, it comprises the compositions of the present invention being used for the treatment of or preventing mammalian influenza virus to infect.In some respects, this vaccine comprises bordetella strain composition described here.In some respects, this vaccine is configured to for nasal-cavity administration.

On the other hand, the invention provides a kind of bordetella bacterial strain, its preserving number is CNCMI-3585.

On the other hand, the invention provides a kind of bordetella bacterial strain, its preserving number is V09/009169.

In some respects, the invention provides a kind of compositions, it comprises the bordetella bacterial strain that preserving number is CNCMI-3585.On the other hand, present invention also offers a kind of vaccine, it comprises this and comprises the compositions that preserving number is the bordetella bacterial strain of CNCMI-3585, is used for the treatment of or prevents mammalian influenza virus to infect.In some respects, this vaccine can be configured to for nasal-cavity administration.

In some respects, the invention provides a kind of compositions, it comprises the bordetella bacterial strain that preserving number is V09/009169.On the other hand, present invention also offers a kind of vaccine, it comprises this and comprises the compositions that preserving number is the bordetella bacterial strain of V09/009169, is used for the treatment of or prevents mammalian influenza virus to infect.In some respects, this vaccine can be configured to for nasal-cavity administration.

Accompanying drawing explanation

By following description and accompanying drawing these and other feature, aspect and the advantage that the present invention may be better understood, wherein:

Fig. 1 shows with the mice of BPZE1 process the protective rate of the lethal hit of anti-mouse adaptability H3N2 virus.Grow up Balb/c mice by nose administration 5x10 ⁶the BPZE1 antibacterial of cfu, and use the mice adaptability H3N2 virus of fatal dose (2LD50) to attack afterwards at 3 weeks (closed squares) or 6 weeks (triangles).Every day monitors body weight change, carries out euthanasia when Mouse Weight loss exceedes 20% of original body weight to it.Survival rate and undressed mice (solid diamond) are compared.Often organize evaluation 10 animals.Result represents three independent experiments.

Fig. 2 shows with the mice of dead BPZE1 antibacterial and the BPZE1 bacterial treatment of living the protective rate of the lethal hit of anti-mouse adaptability H3N2 virus.Grow up Balb/c mice by nose administration 5 × 10 ⁶the BPZE1 antibacterial (triangles) alive of cfu or dead (closed square) BPZE1 antibacterial, and 6 weeks rear fatal dose (2LD50) mice adaptability H3N2 virus attack.Every day monitors body weight change, carries out euthanasia when Mouse Weight loss exceedes 20% of original body weight to it.Survival rate and undressed mice (solid diamond) are compared.Often organize evaluation 10 animals.Result represents two independent experiments.

Fig. 3 shows with the mice of BPZE1 viable bacteria process twice protective rate of the lethal hit of anti-mouse adaptability H3N2 virus.Adult Balb/c mice twice nose administration 5 × 10 in the interval of 4 weeks ⁶the BPZE1 antibacterial (closed square) of cfu, and use the mice adaptability H3N2 virus of fatal dose (2LD50) to attack after 4 weeks.Every day monitors body weight change, carries out euthanasia when Mouse Weight loss exceedes 20% of original body weight to it.Survival rate and undressed mice (solid diamond) are compared.Often organize evaluation 10 animals.Result represents two independent experiments.

Fig. 4 shows the protective rate of the lethal hit of the mice antagonism H1N1 influenza A virus with BPZE1 process.To grow up Balb/c mice three nose administrations 5 × 10 in the interval of 4 weeks and 3 weeks (triangles) ⁶the BPZE1 viable bacteria of cfu.A/PR/8/34 (H1N1) influenza A virus of animal with fatal dose (4LD50) was attacked in 2 weeks after BPZE1 process the last time.Every day monitors body weight change, carries out euthanasia when Mouse Weight loss exceedes 20% of original body weight to it.Survival rate and undressed mice (solid diamond) are compared.Often organize evaluation 10 animals.Result represents two independent experiments.

Fig. 5 shows the virus load of shielded mouse lung and not protected mouse lung.Adult Balb/c mice 2 nose administrations 5 × 10 in the interval of 4 weeks ⁶the BPZE1 viable bacteria of cfu, and use the mice adaptability H3N2 virus of fatal dose (2LD50) to attack after 4 weeks.Virus attack after 3 days often group kill five animals, get its pulmonary and carry out the TCID of mdck cell infection separately ₅₀vitro detection.Virus load and undressed mice are compared.Result represents three independent experiments.

Fig. 6 show BPZE1 process after fatal dose virus attack with lung tissue in undressed mouse lung and cellular infiltration and CD3 ⁺t cell group.Grow up Balb/c mice by nose administration 5x10 ⁶the BPZE1 viable bacteria of cfu, and 6 weeks rear fatal dose (2LD50) mice adaptability H3N2 virus attack.Virus attack, after 3 days, carries out euthanasia to mice, to the bronchoalveolar lavage (B) that its pulmonary carries out histologic analysis (A) respectively or analyzes for cellular infiltration.Legend A: the control mice of infection shows severe inflammation, pulmonary edema (black arrow) and the severe necrotizing bronchitis (hollow arrow) with necrotic cell debris.The mice of BPZE1 process infected only show minimal inflammation and respiratory tract injured, and slight bronchioles peri lesion.Show representational region.Result is equivalent to every group analysis more than 40 regions (>5 region/cross section, 2 cross sections/mice and 4 mices/group).Legend B:a, non-immune mice is not under fire; B, the H3N2 virus attack of non-immune mice; The mice of c, BPZE1 process is not under fire; The mice H3N2 virus attack of d, BPZE1 process.Four animals often organizing each time point are evaluated respectively.**，p≤0.01；***，p≤0.001。Result represents two independent experiments.(C) CD3 of mouse lung ⁺the facs analysis of T cell group.Lethal H3N2 influenza viruse attack is after 3 days or 5 days, and the mice of untreated control mice and BPZE1 process twice carries out euthanasia, by the CD3 of its pulmonary of flow cytometry analysis ⁺t cell group.Four animals often organizing each time point are evaluated respectively.Result is expressed as CD3 in full pneumonocyte group ⁺the percentage ratio of T cell.Meansigma methods ± SD.Legend: a, non-immune mice is not under fire; The mice of b, BPZE1 process is not under fire; C, the H3N2 virus attack of non-immune mice is also put to death after 3 days; The mice of d, BPZE1 process is also put to death after 3 days with H3N2 virus attack; E, the H3N2 virus attack of non-immune mice is also put to death after 5 days; The mice of f, BPZE1 process is also put to death after 5 days with H3N2 virus attack.***，p≤0.001。

Fig. 7 shows mice and undressed mice the urging-composing with anti-inflammatory cytokine and chemotactic factor after lethal H3N2 virus attack of BPZE1 process.Grow up Balb/c mice twice nose administration 5x10 in the interval of 4 weeks ⁶the BPZE1 viable bacteria of cfu and the last time administration 4 weeks rear fatal dose (2LD50) mice adaptability H3N2 virus attack.Virus attack is after 1 day and 3 days, and often group is put to death 5 mices at each time point and collected bronchoalveolar lavage fluid (BALF).The cytokine relevant for each BALF sample detection 14 kinds of inflammatories and chemotactic factor.Legend: 1, untreated not under fire mice; The not under fire mice of 2, BPZE1 process; 3, untreated under fire mice and attack execution in latter 1 day; The under fire mice of 4, BPZE1 process and attack execution in latter 1 day; 5, untreated under fire mice and attack execution in latter 3 days; The under fire mice of 6, BPZE1 process and attack execution in latter 3 days.*，p≤0.05；**，p≤0.01；***，p≤0.001。

Fig. 8 shows the effect of bordetella pertussis (B.pertussis) specific immunity in cross protection.(A). before carrying out a day of lethal hit (2LD50) by mice adaptability H3N2 virus, the immune serum of non-immune serum (open triangles) or anti-H3N2 (filled circles) or anti-BPZE1 (open circles) is injected to adult non-immune Balb/c mice through abdominal cavity (ip.).Every day monitors body weight change, carries out euthanasia when Mouse Weight loss exceedes 20% of original body weight to it.Survival rate and undressed mice are compared (triangles).Often organize evaluation 10 animals.(B) & (C). the Balb/c mice that grows up is by nose administration 5x10 ⁶the BPZE1 viable bacteria of cfu, carries out euthanasia and collects its spleen after 6 weeks.Mixing (B) from 6 animals or individual (C) spleen cell is stimulated with BPZE1 lysate or heat inactivation H3N2 virion.Carry out according to following description ³h-thymus pyrimidine mixes (B) and IFN-γ ELISPOT analyzes (C).The result of the representative value of two groups of different experiments is similar; Meansigma methods ± SD; *, p≤0.05, * * *, p≤0.001.

Detailed description of the invention

Introduce and summary

The present invention relates to the compositions for treatment or flu-prevention viral infection in mammal and vaccine, they comprise the bordetella bacterial strain of sudden change.In addition, invention further provides use said composition or vaccine protection mammal from influenza infection and/or the method for bringing out the immunne response for influenza virus in mammal.

Unless otherwise stated, term in claims and description is according to give a definition.

Abbreviation " PTX " refers to pertussis toxin, PT as used herein, and it synthesizes and secretes a kind of ADP-ribosylating toxins.PTX is made up of five different subunits (being called S1-S5), and each complex contains the S4 of two copies.Each subunit is according to A-B structural arrangement.Part A has enzymatic activity, is formed at S1 subunit, and part B is receptor binding moiety, is made up of S2-S5 subunit.

Abridge as used herein " DNT " refer to pertussis dermotoxin, it is a kind of heat-labile toxin, can when intradermal injection inducing mouse and other laboratory animal generation local damage.

Abbreviation " TCT " refers to tracheal cell toxin as used herein, and it is the virulence factor of a kind of bordetella synthesis.TCT is a kind of Peptidoglycan fragment, can induce the production of il-1 and nitric oxide production synthesis.It can cause cilium to smoulder (stasis), and has lethal effect for airway epithelial cell.

Term " attenuation " refers to that weaken, hypotoxic bordetella bacterial strain, and it can be replied and produce protective immunity by immune stimulatory, but generally can not cause sick.

Term " rapid protective immunity " refers to the immunity had in the short time after sudden change bordetella bacterial strain of the present invention administration for bordetella.

Term " bordetella bacterial strain " or " bacterial strain " comprise the bacterial strain from bordetella pertussis (Bordetellapertussis), bordetella parapertussis (Bordetellaparapertussis) and bronchitis bordetella (Bordetellabronchiseptica).

Term " child " refers to 0 month to 18 years old large people or mammal.

" treatment " refers to any index successfully processing disease, disease or disorder or improve or prevent, and comprises any objective or subjective parameters, such as, alleviates; Alleviate; Transference cure or disease symptoms is more easily stood patient; Slow down and degenerate or weak speed; Or make deterioration so not weak for latter stage.The treatment of symptom or slow down can based on objective or subjective parameters; Comprise the inspection of doctor.Thus term " treatment " comprises uses compound of the present invention or reagent with prevention or the development postponing, slow down or stops or suppress the state of an illness relevant with disease described here, disease or disorder.Term " therapeutic effect " refers in experimenter disease, the minimizing of disease or side reaction, elimination or prevention.Use method of the present invention to carry out " treatment " or " process " but comprising prevention may cause increasing with the risk of disease described here, disease or disorderly relevant disease or disorder the symptom that the state of an illness does not also occur or shows and show effect in experimenter; Suppress the symptom (slow down or stop it to develop) of disease or disorder; The symptom palliated a disease or side reaction (comprising palliative treatment); With alleviate (causing degenerating) disease symptoms.Treatment can be preventative (prevention or postpone seizure of disease, or prevent its clinical or inferior clinical symptom) disease or the patient's condition performance after treatment suppress or mitigation symptoms.

Compositions of the present invention and known drug (or other compound) being carried out " drug combination " refers to by compositions and this medicine (or other compound) together administration certain time, thus the two all has treatment or diagnosis effect.This drug combination can comprise and walking abreast (that is, simultaneously), in front or administration in succession to this medicine (or other compound) relative to using for compositions of the present invention.Persons skilled in the art can be easy to suitable administration time, order and the dosage that judge certain drug (or other compound) and compositions of the present invention.

Term " protection " and " prevention " commutative use, mean prevention influenza infection as used herein.

" preventative vaccine " refers to that this vaccine prevention will be exposed to influenza virus postoperative infection future.

Term " immunogenic composition " or " compositions " refer to can induce immune response and thus there is antigenic compositions." immunne response " refers to immune any reaction.These reactions comprise the activity change of organic immune system for antigen, and it can comprise, such as, and the immunity of antibody producing, inducing cell mediation, the formation of complement activation or immunologic tolerance.

Term " disease " has this area generally known implication with understanding as used herein, comprises the function of host individual or any abnormal condition of health.Medical care and health personnel can by directly checking and/or carrying out analysis to draw to the result of one or more diagnostic detection to the diagnosis of specified disease.

Term " live vaccine composition ", " live vaccine ", " bacterial vaccine of living " and term similar represent a kind of compositions, and it includes and is provided to the alive bordetella of small part for the protective immunity of influenza.

Term " oral ", " enteral ", " through intestinal ", " per os ", " non-intestinal outer ", " non-is without intestinal " etc., represent, via gastral approach or mode, compound or compositions be administered to individuality.Compositions includes but not limited to through the example of " oral " administration, direct oral cavity swallows the vaccine combination of liquid or solid form, per nasal jejunum or the vaccine combination administration of gastrostomy tube, the duodenal administration of vaccine combination and rectally, such as, the suppository of release live bacterial vaccines bacterial strain described here is used.

Term " local application " refers to outer surface (comprising the outer surface of nose, lung and mouth) the drug administration reagent to skin or mucosa, thus this medicament through skin or mucosa outer surface and enter under tissue.Local application can cause medicament in the limited distribution of skin and surrounding tissue, or, after medicament is removed from treatment region with blood flow, the systemic distribution of medicament can be caused.In a preferred form, medicament is sent by percutaneous dosing, such as, uses skin plaster.Percutaneous dosing refers to that medicament passes skin (horny layer and epithelial layer) and spreads, and cutaneous manifestations is that a kind of obstacle that little medicament can be penetrated exists.On the contrary, corium is permeable, can absorb multiple solute and medicine, thus the easier skin through being scratched or otherwise peeling off epidermis and exposing corium of local application.Absorption through intact skin can obtain enhancing (such as by the combination carrying out active agents and oiliness carrier before dermatologic (embrocating process), cream, lubricant, penetration enhancers etc., be described in such as, Remington'sPharmaceuticalSciences, current edition, the people such as Gennaro edit).

Term " nasal-cavity administration " refers to push the nasal passage of experimenter or otherwise introduces active component thus its contact nose breathing epithelium any type of administration from this absorption to systemic circulation.Nasal-cavity administration also can comprise the olfactory epithelium of contact between nasal cavity top nasal septum and each main nasal passage sidewall.Nasal region directly around olfactory epithelium does not contact air-flow.Thus, generally need to adopt specific process to reach remarkable absorption through olfactory epithelium.

Term " aerosol " uses by its conventional sense, refers to that very meticulous liquid or solid granule is carried into the site for the treatment of use under stress by gaseous propellant.Medical aerosol of the present invention contains the therapeutical active compound of in fluid carrier and propellant mixture solubilized, dispersion or emulsifying.The form of aerosol can have solution, suspension, emulsion, powder or semi-solid preparation.Aerosol of the present invention is used for the respiratory tract administration through patient as granule that is meticulous, solid or liquid mist.Various types of propellant can be used, include but not limited to, hydro carbons or other suitable gas.Aerosol of the present invention is also sent by aerosol apparatus, and it produces the very meticulous liquid particles of evenly size substantially in gas.Preferably, the liquid containing reactive compound is broken up into drop, and it can be carried out aerosol apparatus by air-flow, enters patients with respiratory tract.

Term " improvement " refers to any useful therapeutic outcome obtained in the morbid state for the treatment of such as influenza associatcd disease state, comprises prevention, alleviates the order of severity or process, alleviation or healing.

Generally speaking, term " well-tolerated " refers to not exist affects the negative changes of the health status that treatment determines by treating the meeting caused.

" synergism " refers to a kind of interaction, and wherein the combined effect of two or more medicament is greater than the algebraical sum of its effect separately.

Term " external " refer to living organism outgrowth, be such as grown in tissue culture living cells in the process that occurs.

Term " in body " refers to the process occurred in living organism.

Term " mammal " comprises people and inhuman as used herein, and includes but not limited to people, non-human primates, Canidae, cat family, Muridae, Bovidae, equine and porcine animals.

Term " enough dosage " represents the dosage being enough to produce target effect, such as, be enough to the dosage of protein aggregation in regulating cell.

Term " in treatment effective dose " is the dosage effectively alleviating disease symptoms.In treatment, effective dose can be " preventative effective dose ", because prevention can be considered to treatment.

As used herein term " comprise ", " containing ", " comprising ", " having " or its other change any, mean and cover the containing of a kind of nonexcludability.Such as, comprise the process of series of elements, method, goods or equipment and need not be limited to only those elements, but element intrinsic in that other is not clearly listed or this process or method can be comprised.In addition, unless expressly stated to the contrary, "or" refers to the "or" of comprising property, and does not refer to the "or" of exclusiveness.Such as, be that genuine (or existence) and B are false (or not existing) by following arbitrary A or B:A that all can satisfy condition, A be false (or not existing) and B be genuine (or existence) and A and B is really (or existence).

Need be appreciated that and the invention is not restricted to ad hoc approach, reagent, compound, compositions or biosystem, it can change certainly.Will also be understood that and use the object of term to be only to describe particular aspects at this, and do not mean that restriction.As in this description and claim afterwards use, singulative " ", " one " comprise the indicant of plural number, clearly indicate in addition except in non-content.Thus such as, the meaning of " a kind of vaccine " contains the combination of two or more vaccine, etc.

" about " is when indicating the such as measurable magnitude such as dosage, time period as used herein, mean cover this particular value ± 20% or ± 10%, be more preferably ± 5%, be more preferably ± 1% and be even more preferably ± change of 0.1% because these changes are suitable for carrying out disclosed method.

Influenza virus type

The present invention is used for the treatment of at large or prevents mammalian influenza virus to infect.The present invention can the influenza virus of targeting three types: A type, B-mode and influenza virus C.Influenza A virus is divided into some hypotypes according to two of virus surface kinds of protein.These protein are called as hemagglutinin (H) and neuraminidase (N).Influenza A virus is divided into some hypotypes according to these two kinds of protein.There are 16 kinds of different hemagglutinin hypotypes: H1, H2, H3, H5, H4, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16; The neuraminidase hypotype different with 9 kinds: N1, N2, N3, N4, N5, N6, N7, N8 or N9, it all finds to some extent in wild type A avian influenza virus.Influenza A virus comprises A type (H1N1) and A type (H3N2), and it is all the example that the present invention could treat or prevent the influenza virus of institute's targeting in mammal.The general Symptom and disease caused by influenza infection can comprise: fever, cough, sneeze, pain, fatigue, headache, stream eye water, nasal congestion and stomachache.The present invention can be used for treatment or prevents these diseases.

Compositions

bordetella bacterial strain

The invention provides the bordetella bacterial strain of sudden change, it can be used as immunogenic composition or vaccine to bring out immunne response in mammal.In one aspect, the bordetella bacterial strain of this sudden change comprises the ptx gene of sudden change, the dnt gene of disappearance or sudden change and heterologous ampG gene.Heterologous ampG gene product can reduce the tracheal cell toxin quantity of generation in a large number.In one aspect, this bacterial strain is BPZE1.The initial strains carrying out suddenling change can be any bordetella bacterial strain, comprises bordetella pertussis, bordetella parapertussis and bronchitis bordetella.In one aspect, the initial strains for the bordetella bacterial strain obtaining this sudden change is bordetella pertussis.On the other hand, this bacterial strain is the bordetella bacterial strain of three sudden changes.On the other hand, the preserving number of this bordetella bacterial strain is CNCMI-3585.On the other hand, the preserving number of this bordetella bacterial strain is V09/009169.

The present invention is not limited only to said mutation body.It also can be other other sudden change, such as adenyl cyclase (AC) deficient mutants, lipopolysaccharide (LPS) deficient mutants, fibrous hemagglutinin (FHA) and any one bvg regulating element.

The structure of sudden change bordetella bacterial strain of the present invention can start from and the bordetella ampG gene in this bacterial strain is replaced with heterologous ampG gene.Any heterologous ampG gene known in the art all can be used to the present invention.Its example can comprise all gram negative bacteria that per generation discharges very small amount of Peptidoglycan fragment in culture medium.The example of gram negative bacteria includes but not limited to: colon bacillus (Escherichiacoli), Salmonella (Salmonella), enterobacteriaceae lactobacteriaceae (Enterobacteriaceae), pseudomonas (Pseudomonas), Moraxella (Moraxella), Helicobacter pylori (Helicobacter), long and narrow flat born of the same parents bacterium (Stenotrophomonas), Legionnella (Legionella) etc.Generally, by bordetella ampG gene is replaced with heterologous ampG gene, the scale producing tracheal cell toxin (TCT) in the bacterial strain obtained reveals the TCT residual activity lower than 1%.On the other hand, the amount expressing TCT in the bacterial strain obtained is the TCT residual activity of the TCT residual activity of about 0.6% to 1% or the TCT residual activity of about 0.4% to 3% or about 0.3% to 5%.

PTX is the main virulence factor causing general bordetella pertussis to infect, and is also a kind of main protective antigen.Because these attributes, natural ptx gene can be replaced by the version of sudden change, thus the toxin of enzymatic activity part S1 codase inactivation, but the immunogenicity of its pertussis toxin, PT is unaffected.This can realize by the lysine (Lys) of sequence the 9th is replaced with arginine (Arg) (K9R).In addition, the glutamic acid (Glu) of the 129th can be replaced by glycine (Gly) (E129G).These amino acid sites generally participate in Binding Capacity and catalysis respectively.In other side, other sudden change can also be carried out, such as, be described in U.S. Patent number 6,713,072, be hereby incorporated by, and any known or other can reduce the sudden change of neurotoxin active.In one aspect, first can apply allele and exchange version to delete ptx operon then insertion mutation.

In another aspect of this invention, allele can be applied exchange so that dnt gene is removed from this bordetella bacterial strain.Except all removing, enzymatic activity also can be suppressed by point mutation.Because DNT comprises the receptor binding domains of N end regions and the catalytic domain of C end, therefore point mutation is carried out to dnt gene, Cys-1305 is replaced with Ala-1305 and can suppress DNT activity (KashimotoT., KatahiraJ, CornejoWR, MasudaM, FukuohA, MatsuzawaT, OhnishiT, HoriguchiY. (1999) IdentificationoffunctionaldomainsofBordetelladermonecrot izingtoxin.Infect.Immun.67:3727-32.).

Except allele exchanges the ptx gene of insertion mutation and the dnt gene of suppressed or disappearance, the open reading frame of gene can by inserting gene order or plasmid interrupt.The method is also included within the present invention.Other method producing mutants which had is generally well known in the art.

In one aspect of the invention, the bacterial strain of this sudden change is called as BPZE1 bacterial strain, it is preserved in the national Culture Collection (CNCM) of Paris, FRA on March 9th, 2006 according to budapest treaty, and the preserving number of distribution is CNCMI-3585.The sudden change introducing BPZE1 generally causes weakening, but still allows bacterial clump breed and maintain.Thus on the other hand, the invention provides BPZE1, it can induce mucosa-immune and general immunity when being administered to the mammal of demand.In another aspect of this invention, the BPZE1 recombinant bacterial strain that expression three copies M2e polypeptide is constructed.This bacterial strain is preserved in the national measurement Research institute (NationalMeasurementInstitute of Australian 3207 Port Melbournes, Victoria on April 27th, 2009 according to budapest treaty, AGAL before), the preserving number of distribution is V09/009169.M2e is the outer part of born of the same parents of influenza m 2 protein.Its all high conservative in all influenza A viruss, has found that it can the opposing to influenza A virus of induction of antibodies mediation.The BPZE1 bacterial strain that M2e is produced in restructuring can bring out (such as; by viable bacteria nasal-cavity administration) a large amount of anti-M2e antibody response (local and whole body), thus significance protection can be carried out to H1N1 and H3N2 attack for independent BPZE1 antibacterial.

Sudden change bordetella bacterial strain of the present invention can be used in and to be used for the treatment of or in flu-prevention viral infection immunogenic composition.This immunogenic composition can for generation of immunne response, or the antibody response in mammal, or t cell response.Such as, t cell response can be that protection mammal is from influenza infection or from its impact/disease/symptom.

Sudden change bordetella bacterial strain of the present invention can use as live strain in vaccine or immunogenic composition.In one aspect, live strain is used to nasal-cavity administration, and the bacterial strain that chemistry or full-boiled process kill can be used for whole body or mucosal drug delivery.Attenuation at this bacterial strain of other side.

In other side of the present invention, this bacterial strain does not comprise other any heterologous genes except above-mentioned heterologous ampG gene.In other side, this bacterial strain does not comprise the expression platform (such as, see, WO2007104451) of allos.Generally, heterogenous expression platform carries heterologous antigen.In one aspect, heterogenous expression platform can be used for sending heterologous antigen to mammiferous respiratory mucosa.

adjuvant

Compositions of the present invention can with administration together with other immunomodulator comprising adjuvant.Term " adjuvant " refers to a kind of compound or the mixture that strengthen immunne response as used herein.Especially, compositions can comprise adjuvant.Following one or more can be included but not limited to for adjuvant of the present invention:

containing mineral adjunvant composition

Can be used as adjuvant and comprise mineral salt, such as aluminum salt and calcium salt for of the present invention containing mineral composition.The present invention includes such as that hydroxylate is (such as, oxyhydroxide), phosphate (such as, hydroxyl phosphate, orthophosphate), the mineral salt of sulfate etc. (such as, see VaccineDesign... (1995), Powell & Newman edits, ISBN:030644867X.Plenum. the 8th and 9 chapters), or the mixture of different minerals compound (such as, the mixture of phosphate and hydroxide adjuvant and optional excess phosphoric acid salt), its compound has any suitable form (such as, gel, crystal, amorphous, etc.), and be adsorbed to preferred salt.Also the granule (WO/0023105) of slaine is can be made into containing mineral composition.

Aluminum salt can be comprised, wherein Al in compositions of the present invention ₃ ⁺amount be 0.2 to 1.0mg/ agent.

oil-newborn adjuvant

Can be used as adjuvant and can comprise zamene-aqueous emulsion for oil-emulsion compositions of the present invention, such as MF59 (5% zamene, 0.5% Tween 80 and 0.5%Span85 use homogenizer to make submicron particles).See, such as, WO90/14837.Also see, Podda, " Theadjuvantedinfluenzavaccineswithnoveladjuvants:experie ncewiththeMF59-adjuvantedvaccine ", Vaccine19:2673-2680,2001.

In other related fields, the adjuvant for compositions is submicron O/w emulsion.The example of submicron O/w emulsion comprises zamene/aqueous emulsion as used herein, it comprises the MTP-PE of variable alternatively, such as comprise 4-5%w/v zamene, 0.25-1.0%w/v Tween 80 (polyoxyethylenesorbitan sorbitan monooleate), and/or 0.25-1.0%Span85 (sorbitol three oleic acid acid esters), and the submicron O/w emulsion of the optional different paddy ammonia-ALANINE-2-of N-acetylmuramyl-L alanyl-D-(1'-2'-bis-palmityl-s-n-glyceric acid-3-hydroxyl phosphoryl oxygen base)-ethamine (MTP-PE), such as, be called as submicron O/w emulsion (the international publication number WO90/14837 of " MF59 ", U.S. Patent number 6,299,884 and 6,451,325, be incorporated herein by reference in this entirety, with people such as Ott, " MF59--DesignandEvaluationofaSafeandPotentAdjuvantforHuma nVaccines ", be published in VaccineDesign:TheSubunitandAdjuvantApproach (Powell, and Newman M.F., M.J. edit) PlenumPress, NewYork, 1995, pp.277-296).MF59 can containing 4-5%w/v zamene (such as, 4.3%), 0.25-0.5%w/v Tween 80 and 0.5%w/vSpan85 and optionally comprise not commensurability MTP-PE, use such as Model110Y homogenizer (Microfluidics, Newton, MA) make submicron particles.Such as, the content of MTP-PE can be about 0-500 μ g/ agent or 0-250 μ g/ agent or 0-100 μ g/ agent.

Details for the preparation method of the submicron O/w emulsion of compositions, this emulsion and immunopotentiating agent such as muramyl peptide is described in international publication number WO90/14837 and U.S. Patent number 6,299,884 and 6,451,325, is incorporated herein by reference in this entirety.

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) also can be used as adjuvant of the present invention.

saponin adjuvant formulation

Saponin preparation also can be used as adjuvant of the present invention.Saponin is the heterocomplex of sterioside and triterpene glycosides, is found in the skin of various plants kind, leaf, stem, root and even flower.Saponin from Quillaia saponaria (QuillaiasaponariaMolina) skin is extensively studied by as adjuvant.Saponin also can be purchased from sarsaparilla (Smilaxornata), Caulis et folium pavettae hongkongensis (Gypsophillapaniculata) and Saponaria officinalis (Saponariaofficianalis) (Radix saponariae).Saponin adjuvant formulation can comprise pure preparations, such as QS21, and lipid formulations, such as immunostimulating complex (ISCOM; See below).

High performance thin layer chromatography (HPLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) has been used to carry out purification to Saponin compositions.Use the specific purified components of these technology to be differentiated, comprise QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C.The method of producing QS21 is disclosed in U.S. Patent number 5,057,540.Saponin preparation also can contain steroidal, such as cholesterol (see WO96/33739).

The combination of Saponin and cholesterol can be used for forming unique granule, is called ISCOM.ISCOM generally also comprises phosphoric acid lipid, such as PHOSPHATIDYL ETHANOLAMINE or phosphatidylcholine.Any known Saponin all can be used to ISCOM.Such as, ISCOM can comprise one or more QuilA, QHA and QHC.ISCOM is further described in EP0109942, WO96/11711 and WO96/33739.Alternatively, ISCOM can not containing extra detergent.See WO00/07621.

Research and development for the adjuvant based on Saponin can see the description of the people such as Barr, " ISCOMsandothersaponinbasedadjuvants ", AdvancedDrugDeliveryReviews32:247-27,1998.Also see people such as Sjolander, " UptakeandadjuvantactivityoforallydeliveredsaponinandISCO Mvaccines ", AdvancedDrugDeliveryReviews32:321-338,1998.

virion and virus-like particle (VLP)

Virion and virus-like particle (VLP) also can be used as adjuvant of the present invention.These structures are generally containing one or more protein from virus, and optional and phosphoric acid lipid carries out combining or preparation.They are generally non-pathogenic, not replicated and general not containing any protovirus genome.Virus protein can recombinant production or be separated from whole virus.These virus proteins being applicable to virion or VLP comprise derived from influenza virus (such as HA or NA), hepatitis B virus (such as core or capsid protein matter), Hepatitis E virus, Measles virus, Sindbis virus, Rota virus, foot and mouth disease disease virus, retrovirus, Norwalk virus, human papillomavirus, HIV, RNA-phage, QB-phage (such as coating protein matter), GA-phage, fr-phage, AP205 phage, with the protein of Ty (such as retrotransposon Ty protein matter pl).

antibacterial or mycobacterial derivatives

Can be used as adjuvant of the present invention and comprise antibacterial or mycobacterial derivatives, such as:

(1) non-toxic derivatives of enterobacterial lipopolysaccharide (LPS)

This derivant comprises LA (MPL) and 3-O-removes acyl group MPL (3dMPL).3dMPL is the 3 de--O-acidylate LA mixture with 4,5 or 6 acidylate chains.An example of 3 de--O-acidylate LA " granule " forms is disclosed in EP0689454." granule " of this 3dMPL is enough little, can carry out aseptic filtration (see EP0689454) by 0.22 micron membranes.Other avirulence LPS derivant comprises LA analogies, such as aminoalkyl glucosaminide phosphoric acid derivatives, such as, and RC-529.See people such as Johnson, BioorgMedChemLett9:2273-2278,1999.

(2) lipid A derivative

Lipid A derivative can comprise such as, from colibacillary lipid A derivative, OM-174.OM-174 is described in the people such as such as Meraldi, " OM-174; aNewAdjuvantwithaPotentialforHumanUse; InducesaProtectiveResponsewithAdministeredwiththeSynthet icC-TerminalFragment242-310fromthecircumsporozoiteprotei nofPlasmodiumberghei ", Vaccine21:2485-2491,2003; With people such as Pajak, " TheAdjuvantOM-174inducesboththemigrationandmaturationofm urinedendriticcellsinvivo ", Vaccine21:836-842,2003.

(3) immunostimulatory oligonucleotide

The immunostimulatory oligonucleotide that can be used as adjuvant of the present invention can comprise the nucleotide sequence (sequence containing non-methylated cytosine is connected through phosphate bond with guanosine) containing CpG motif.Find that antibacterial double-stranded RNA or the oligonucleotide containing the palindrome or poly-(dG) sequence have immunostimulating.

CpG can comprise nucleotide modification/analog such as D2EHDTPA and modify, and it can be double-strand or strand.Alternatively, guanosine can be replaced by analog such as 2'-deoxidation-7-denitrification guanosine.See people such as Kandimalla, " Divergentsyntheticnucleotidemotifrecognitionpattern:desi gnanddevelopmentofpotentimmunomodulatoryoligodeoxyribonu cleotideagentswithdistinctcytokineinductionprofiles ", NucleicAcidsResearch31:2393-2400,2003; The example that in WO02/26757 and WO99/62923, analog replaces.CpG ODN adjuvant effect is discussed further in the following documents to some extent: Krieg, " CpGmotifs:theactiveingredientinbacterialextracts? ", NatureMedicine (2003) 9 (7): 831-835; The people such as McCluskie, " Parenteralandmucosalprime-boostimmunizationstrategiesinm icewithhepatitisBsurfaceantigenandCpGDNA ", FEMSImmunologyandMedicalMicrobiology (2002) 32:179-185; W098/40100; U.S. Patent number 6,207,646; U.S. Patent number 6,239,116 and U.S. Patent number 6,429,199.

CpG sequence can such as, for Toll-sample receptor (TLR9), motif GTCGTT or TTCGTT.See people such as Kandimalla, " Toll-likereceptor9:modulationofrecognitionandcytokineind uctionbynovelsyntheticCpGDNAs ", BiochemicalSocietyTransactions (2003) 31 (part3): 654-658.CpG sequence can induce Th1 immunne response specifically, such as CpG-AODN, or it can be replied by elicit B cell more specifically, such as CpG-BODN.CpG-A and CpG-BODN is see people such as Blackwell, " CpG-A-InducedMonocyteIFN-gamma-InducibleProtein-10Produc tionisRegulatedbyPlasmacytoidDendriticCellDerivedIFN-alp ha ", J.Immunol.170:4061-4068,2003; Krieg, " FromAtoZonCpG ", TRENDSinImmunology23:64-65,2002, and WOO1/95935.

In some respects, CpG ODN can be built as its 5' end can by Receptor recognition.Alternatively, two kinds of CpG ODN sequences can be connected to its 3' end to form " immune body (immunomer) ".See, such as, the people such as Kandimalla, " SecondarystructuresinCpGoligonucleotidesaffectimmunostim ulatoryactivity ", BBRC306:948-95,2003; The people such as Kandimalla, " Toll-likereceptor9:modulationofrecognitionandcytokineind uctionbynovelsyntheticGpGDNAs ", BiochemicalSocietyTransactions31:664-658,2003; The people such as Bhagat, " CpGpenta-andhexadeoxyribonucleotidesaspotentimmunomodula toryagents " BBRC300:853-861,2003, and WO03/035836.

(4) ADP-ribosylating toxins and detoxified derivaties thereof thereof.

Bacterial ADPribosylating toxin and detoxified derivaties thereof thereof can be used as adjuvant of the present invention.Such as, toxin can derived from escherichia coli (that is, E.coli LT (LT)), cholera (CT) or pertussis (PTX).Detoxification ADP-ribosylating toxins is described in WO95/17211 as the application of mucosal adjuvant, is described in WO98/42375 as the outer adjuvant of intestinal.In some respects, adjuvant can be detoxification 192-GLY LT such as LT-K63, LT-R72 and LTR192G.ADP-ribosylating toxins and detoxified derivaties thereof thereof, particularly LT-K63 and LT-R72, application as adjuvant can find in the following documents, which is hereby incorporated by reference: the people such as Beignon respectively for it, " TheLTR72MutantofHeat-LabileEnterotoxinofEscherichiacoliE nahncestheAbilityofPeptideAntigenstoElicitCD4+TCellsandS ecreteGammaInterferonafterCoapplicationontoBareSkin ", InfectionandImmunity70:3012-3019,2002; The people such as Pizza, " Mucosalvaccines:nontoxicderivativesofLTandCTasmucosaladj uvants ", Vaccine19:2534-2541,2001; The people such as Pizza, " LTK63andLTR72, twomucosaladjuvantsreadyforclinicaltrials " Int.J.Med.Microbiol290:455-461,2003; The people such as Scharton-Kersten, " TranscutaneousImrnunizationwithBacterialADP-Ribosylating Exotoxins; SubunitsandUnrelatedAdjuvants ", InfectionandImmunity68:5306-5313,2000; The people such as Ryan, " MutantsofEscherichiacoliHeat-LabileToxinActasEffectiveMu cosalAdjuvantsforNasalDeliveryofanAcellularPertussisVacc ine:DifferentialEffectsoftheNontoxicABComplexandEnzymeAc tivityonTh1andTh2Cells " InfectionandImmunity67:6270-6280,2003; The people such as Partidos, " Heat-labileenterotoxinofEscherichiacolianditssite-direct edmutantLTK63enhancetheproliferativeandcytotoxicT-cellre sponsestointranasallyco-immunizedsyntheticpeptides ", Immunol.Lett.67:09-216,1999; The people such as Peppoloni, " MutantsoftheEscherichiacoliheat-labileenterotoxinassafea ndstrongadjuvantsforintranasaldeliveryofvaccines ", Vaccines2:285-293,2003; And people (2002) " Intranasalimmunizationwithinfluenzavaccineandadetoxified mutantofheatlabileenterotoxinfromEscherichiacoli (the LTK63) " J.ControlRelease85:263-270 such as Pine, 2002.The numerical reference of aminoacid replacement preferably based on the comparison of ADP-ribosylating toxins A and B subunit, see people such as Domenighini, Mol.Microbiol15:1165-1167,1995, it is incorporated herein by reference in this entirety.

bioadhesive polymer and mucoadhesive agent

Bioadhesive polymer and mucoadhesive agent also can be used as adjuvant of the present invention.Suitable bioadhesive polymer can comprise the hyaluronate microspheres (people such as Singh of esterification, J.Cont.Rele.70:267-276,2001) or the cross-linked derivant of mucoadhesive agent such as polyacrylic acid, polyvinyl alcohol, polyvidon polysaccharide and carboxymethyl cellulose.Chitosan-phospholipid complex also can be used as adjuvant of the present invention.See, such as, WO99/27960.

adjuvant microparticle

Microparticle also can be used as adjuvant of the present invention.It can consider to be formed from biodegradable and/or non-toxic material (such as, poly-'alpha '-hydroxy acids, poly butyric, poe, polyanhydride, polycaprolactone etc.), there is the microparticle of poly-(lactide coglycolide) (namely, particle diameter is about 100nm to about 150 μm, or diameter 200nm is to about 30 μm, or diameter is about 500nm to about 10 μm), be treated to alternatively and there is negative charged surface (such as, with SDS process) or positive charged surface is (such as, with cationic detergent, such as CTAB process).

adjuvant liposome

Be suitable as the example of the Liposomal formulation of adjuvant see U.S. Patent number 6,090,406, U.S. Patent number 5,916,588 and EP0626169.

I. polyoxyethylene ether and polyoxyethylene ester formulations

Be suitable as adjuvant of the present invention and also comprise polyoxyethylene ether and polyoxyethylene ester.WO99/52549。Said preparation may further include the combination (WO01/21207) of polyoxyethylene sorbitan ester surfactant and octyl phenol polyethers and polyoxyethylene alkyl ether or ester surfactant and at least one non-ionic surfactants as the combination (WO01/21152) of octyl phenol polyethers.

In some respects, polyoxyethylene ether can comprise: laureth9 (Laurel ether 9), polyoxyethylene-9-stearyl ether, polyoxyethylene-8-stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether or polyoxyethylene-23-lauryl ether.

polyphosphazene (PCPP)

PCPP preparation as adjuvant is described in, such as, the people such as Andrianov, " Preparationofhydrogelmicrospheresbycoacervationofaqueous polyphophazenesolutions ", Biomaterials19:109-115,1998, with people such as Payne, " ProteinReleasefromPolyphosphazeneMatrices ", Adv.Drug.DeliveryReview31:185-196,1998.

muramyl peptide

The example being suitable as the muramyl peptide of adjuvant of the present invention can comprise N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-fall muramyl-1-alanyl-d-isoglutamine (nor-MDP) and the different paddy acyl of N-N-acetylmuramyl-1-alanyl-d--1-alanine-2-(1'-2'-bis-palmityl-s-n-glycerol-3-di oxygen)-ethamine (MTP-PE).

imidazoquinolie compounds

The example being suitable as the imidazoquinolie compounds of adjuvant of the present invention can comprise imiquimod and homologue thereof, it is further described in Stanley, " Imiquimodandtheimidazoquinolones:mechanismofactionandthe rapeuticpotential " ClinExpDermatol27:571-577,2002 and Jones, " Resiquimod3M ", CurrOpinInvestigDrugs4:214-218,2003.

people's immunomodulator

The people's immunomodulator being suitable as adjuvant of the present invention can comprise cytokine, such as interleukin (such as, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferon (such as, interferon-γ), M-CSF and tumor necrosis factor.

adjuvant combination

The present invention also can contain the combination of one or more adjuvants above-mentioned.Such as, adjunvant composition can comprise:

(1) Saponin and O/w emulsion (WO99/11241);

(2) Saponin (such as, QS21)+avirulence LPS derivant (such as, 3dMPL) (see WO94/00153);

(3) Saponin (such as, QS21)+avirulence LPS derivant (such as, 3dMPL)+a kind of cholesterol;

(4) Saponin (such as, QS21)+3dMPL+IL-12 (optional+sterol) (WO98/57659);

(5) 3dMPL and such as QS21 and/or O/w emulsion are carried out combining (see european patent application 0835318,0735898 and 0761231);

(6) SAF, containing 10% squalane, 0.4% Tween 80,5%Pluronic block copolymer L121 and thr-MDP, itself or carry out miniflow and be emulsified into sub-micron emulsion, or vortex oscillation produces the emulsion of larger particles.

(7) Ribi adjuvant system (RAS) (RibiImmunochem), containing 2% zamene, 0.2% Tween 80 and one or more bacterial cell wall component being selected from monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferred MPL+CWS (Detox); With

(8) one or more mineral salts (such as aluminum salt)+LPS non-toxic derivatives (such as 3dPML).

Aluminum salt and MF59 are the examples for injectable influenza vaccines adjuvant.Bacteriotoxin and bioadhesive polymer are the examples of the adjuvant used together with the mucosal delivery vaccine of such as nasal cavity vaccine.All above-mentioned adjuvants and persons skilled in the art other adjuvant known can use technology well known in the art to carry out preparing for intranasal administration.

preparation and carrier

Be used for the treatment of or flu-prevention viral infection relevant disease (details sees below) method also contain by the present invention.Described method of the present invention comprises the compositions of the present invention of effective dose on drug treatment.Compositions of the present invention can be formulated as pharmaceutical composition.These compositionss can also comprise pharmaceutically acceptable excipient, carrier, buffer, stabilizing agent except one or more this bacterial strain, or other material well known to those skilled in the art.These materials should be generally avirulent, and generally should not clash with the effect of active component.The exact nature of carrier or other material can judge according to route of administration, such as, and oral, intravenous, skin or subcutaneous, nasal cavity, intramuscular or intraperitoneal routes.

The pharmaceutical composition of oral administration can be tablet, capsule, powder or liquid form.Tablet can comprise solid carrier such as gelatin or adjuvant.Composition of liquid medicine generally comprises liquid-carrier such as water, oil, animal or plant oil, mineral oil or artificial oil.Normal saline solution, glucose or other polysaccharide solution or glycol can also be contained, such as ethylene glycol, propylene glycol or Polyethylene Glycol.

For intravenous, skin or subcutaneous injection, or in afflicted area injection, active component exists with the form of the outer acceptable aqueous solution of intestinal, and it is not containing pyrogen, and it has suitable pH, isotonicity and stability.Relevant technical staff in the field has the ability to use such as isotonic vehicle to prepare suitable solution, such as chloride injection, the injection of woods grignard or lactated Ringer injection.Also antiseptic, stabilizing agent, buffer, antioxidant and/or other additive can be contained as required.

Dosage is " in treatment effective dose " or " preventative effective dose " (according to possible situation, although prevention can be considered to treatment) preferably, and it is enough to demonstrate benefit to individuality.Actual dosage and medicine-feeding rate and time will depend on character and the order of severity of the disease for the treatment of.Treatment prescription, such as the decision of dosage etc., is the responsibility of general practitioner and other doctor, and generally will consider the other factors that the situation of disease to be treated, individual patients, administering position, medication and doctor know.The example of above-mentioned technology and scheme can at the Remington ' sPharmaceuticalScience of latest edition, and MackPublishingCompany, Easton, PA find in (" Remington ' s ").

Generally, compositions can be individually dosed, or with other therapeutic combination administration, its according to the state of an illness to be treated can simultaneously administration also can administration in succession.

Method

route of administration

Compositions of the present invention is generally administered directly to mammal.Directly send and can be undertaken (such as by parenteral, subcutaneous, intraperitoneal, Intradermal, intravenous, intramuscular or be administered to interstice), or mucosal drug delivery, such as by rectum, oral (such as, tablet, spraying), vagina, locally, percutaneous is (see such as, WO99/27961) or through skin (see such as, WO02/074244 and WO02/064162), suck, intranasal (see such as, WO03/028760), eyes, ear, pulmonary or other mucosal drug delivery.Compositions also can carry out local application by being transferred directly to skin surface.Local application can carry out by any equipment, or by use the equipment of binder or similar binder directly contact exposed skin to carry out (see, such as, U.S. Patent number 6,348,450).

In some respects, the mode of administration be that intestinal is outer, the combination of mucosa or mucosa and Parenteral immunization.In other side, the mode of administration is that intestinal is outer, mucosa or the mucosa of 1-2 time and the combination of Parenteral immunization altogether of interval 1-3 week.At related aspect, route of administration includes but not limited to intranasal administration.

administration flow process and dosage

The present invention can comprise to mammal administration sudden change bordetella bacterial strain to bring out the immunne response (such as, TH1 immunne response) of the influenza virus can attacking such as H3N2.The example of sudden change bordetella bacterial strain of the present invention is described above.Generally, the administration of the bordetella bacterial strain of this sudden change is by the protective immunity for influenza virus, is used for the treatment of or prevents the influenza infection of mammal such as people.In some respects, the bordetella of this sudden change before influenza infection administration with flu-prevention viral infection.Generally, the bordetella strain bacterial strain of sudden change be less than 1 in influenza infection precontract, 1,2,3,4,5,6,7,8,9,10,11,12, or more week be administered to mammal.

In one aspect, the method for the treatment of or flu-prevention viral infection comprises the compositions of the present invention to there being the experimenter of demand to use single dose, such as, and BPZE1.In related fields, dosing step is undertaken by mucosa, such as, and intranasal.

In other side, compositions administration of the present invention more than once, such as, twice.The number of times of medication can change as required, such as to the number of times of mammal administration can be 1,2,3,4,5,6,7,8,9,10, or more time.In one aspect, the method for the treatment of or flu-prevention viral infection comprises and (containing such as there being the experimenter of demand to use the first immunogenic composition of the present invention, BPZE1), the second immunogenic composition (containing such as, BPZE1) is then used.Generally, the time range between the compositions of each dosage can be about 1-6 days, or about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,20,30,40,50,60,70,80,90, or more week.In related fields, the time range between each dosage is about 3 weeks.In other side, can use immune strengthening method, wherein compositions of the present invention first for " bringing out (priming) " step, then can be used for " strengthening (boosting) " step by compositions of the present invention.

Compositions generally can be used to bring out whole body and/or mucosa-immune, such as, bring out whole body and/or the mucosa-immune of enhancement.Such as, induced serum IgG can be passed through and/or intestinal IgA immunne response characterizes immunne response.Generally, the level of protection that infected by influenza infects can more than 50%, such as, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.In one aspect, level of protection can reach 100%.In other side, level of protection lower than 50%, such as, 20%.In other side, the bacterial number in each dosage is regulated in mammal, obtain effective immunne response.Bacterial number in each dosage or cfu can be about 1,10,100,1000,10000,100000,1000000,5x10 ⁶, or more or described each dosage between any dosage.

The co-administered of compositions and other reagent also can be comprised in other side the present invention.General various compositions/reagent can be sent according to any order.Thus sending the various aspects of multiple different compositions or reagent, the bordetella bacterial strain of this sudden change carries out administration before not needing the reagent such as the micromolecule that all maybe can affect influenza infection at such as medicine, siRNA, miRNA, immunogenic peptide.The example of other reagent comprises neuraminidase inhibitor and M2 inhibitor (diamantane (obsolete)).Such as, bring out step and can comprise and send one or more reagent, strengthen step and can comprise the bordetella bacterial strain sending one or more sudden changes.In other side, the multiple dosing of the bordetella bacterial strain of sudden change can carry out after the multiple dosing of reagent.Administration can be carried out according to any order.Thus bordetella bacterial strain and one or more reagent of one or more sudden changes described here can carry out co-administered, such as, to bring out immunoreation according to any order and any route of administration known in the art.

In the present invention, dosage treatment can according to single dose schedule or multiple dose drug regimen.Such as, multiple dose can be used to bring out Immunization programme and/or potential booster immunization strategy.In multiple dose medication in the works, various medicament can carry out administration by identical or different approach, such as, brings out and bring out strengthen outward with intestinal with mucosa reinforcement, mucosa outside intestinal, etc.In other side, dosage control can strengthen the activity of antibody response, causes the antibody with character in having.External neutralization analysis can be used for detect neutralizing antibody (see people such as such as Asanaka, JVirology102:10327,2005; The people such as Wobus, PLOSBiology2; E432; With people such as Dubekti, JMedicalVirology66:400).

for measuring the test of immunne response effect or existence

A kind of method evaluating the therapeutic effect after compositions administration of the present invention relates to be monitored infection.A kind of method evaluating the preventive effect after compositions administration of the present invention relates to be monitored the immunne response of the antigen in compositions.The another kind of immunogenic method evaluating compositions of the present invention is isolated protein or protein mixture with by immunoblotting screening patients serum or mucosal secretions.Occur between protein and patients serum to create immunne response to compositions before positive reaction then shows patient.

Check the another kind of method of the therapeutic effect after compositions administration of the present invention to relate to monitor infection.A kind of method of the preventive effect after compositions administration of the present invention is checked to relate to general to the antigen in compositions (such as monitoring the level of production of IgG1 and IgG2a) and the immunne response of mucosal (such as monitoring the level of production of IgA) is monitored.Generally, detect serum specific antibody response before attacking after immunity, and detect the response of mucosa specific antibody after immunity and after attacking.The immunogenicity of the present composition can be evaluated in animal model before to host's administration of such as people in vitro and in vivo.

Effect of compositions of the present invention also can be undertaken by the infected animal model attacking such as mice by compositions detecting in body.Compositions can derived from attack the identical bacterial strain of bacterial strain, or can not derived from the bacterial strain identical with attacking bacterial strain.In body, efficacy models can include but not limited to: the Infection in Rodents model of (i) end user bacterial strain; (ii) mouse disorders model, it is the Murine models using mice Adaptive strain, such as, mice is had to the bacterial strain of specific toxicity; (iii) primate model of end user's separator.

The present invention induction immunne response can be Th1 immunne response and TH2 response one or both.Immunne response can be immunne response that is that improve or strengthen or that change.Immunne response can be one or both of general and mucosal immunne response.Such as, immunne response can be the whole body and/or mucosa response that strengthen.The whole body strengthened and/or mucosa-immune are reflected in TH1 and/or the TH2 immunne response of enhancing.Such as, the immunne response of enhancing can comprise the increase of the output of IgG1 and/or IgG2a and/or IgA.Mucosal immune response can be TH2 immunne response on the other hand.Such as, mucosal immune response can comprise the output increase of IgA.

Generally, the TH2 cell of activation strengthens the generation of antibody, thus is valuable in the outer infection of response born of the same parents.The TH2 cell of activation generally can secrete one or more IL-4, IL-5, IL-6 and IL-10.TH2 immunne response also can cause the generation of IgG1, IgE, IgA and/or memory B cell to provide following protection.In a word, TH2 immunne response can comprise the increase of one or more one or more cytokines (such as IL-4, IL-5, IL-6 and IL-10) relevant to TH2 immunne response, or the increase of the output of IgG1, IgE, IgA and memory B cell.Such as, the TH2 immunne response of enhancing can comprise the increase of IgG1 output.

Th1 immunne response can comprise the increase of the increase of one or more CTL, the increase of one or more cytokines relevant to Th1 immunne response (such as IL-2, IFN-γ and TNF-α), the increase of activated macrophage, the increase of NK activity or IgG2a output.Such as, the TH1 immunne response of enhancing can comprise the increase of IgG2a output.

Compositions of the present invention, particularly immunogenic composition containing one or more bacterial strains of the present invention, can be used alone or use with other agent combination, and the optional immunomodulatory agents with Th1 and/or Th2 can be induced to reply uses.

Compositions of the present invention can inducing cell mediation immunne response and humoral immunoresponse(HI) effectively to resist influenza infection.Preferably, this immune response inducing (such as, neutralizes) antibody for a long time and can respond the cell-mediated immunity be exposed to future in one or more infectious antigens fast.

experimenter and mammal

Compositions of the present invention is generally used for the Influenza virus strain of the mammalian subject of preventing or treating such as people.In some respects, experimenter can comprise old man's (such as, >65 year), child's (such as, <5 year), inpatient, working healthily person, arms service personal and soldier, Food processing personnel, anemia of pregnant woman, chronic and overseas trip crowd.Compositions is generally applicable to these crowds and population, or doctor think need crowd.

Test kit

The present invention also provides the test kit of the container containing one or more compositionss of the present invention.Compositions can be liquid form or lyophilizing.Suitable composition container comprises, such as, and bottle, medicine bottle, injection tube and test tube.Container can adopt multiple material, comprises glass or plastics.Container can have aseptic entrance point (such as, container can be parenteral solutions bag or the medicine bottle with the stopper that can be punctured by hypodermic needle).

Test kit may further include the second container containing pharmaceutically acceptable buffer (saline of such as phosphoric acid-buffering, ringer's solution or glucose solution).It also can contain other material useful to terminal use, comprises other pharmaceutically acceptable formulation soln such as buffer, diluent, filter, pin and syringe or other delivery device.Test kit may further include the Part III containing adjuvant.

Test kit also can comprise the package insert of the description with the method infected for immune induction, prevention infection or treatment.This package insert can be not approved rough draft package insert, the package insert also can ratified by Food and Drug Administration (FDA) or other supervision department.

The present invention also provides a kind of pre-fill to be filled with the delivery device of compositions of the present invention.

Pharmaceutical composition is made generally in aseptic and substantially isotonic, and specifies in accordance with the GMP (GMP) of FDA completely.

Those skilled in the art can carry out different modifications and change according to description before to compositions and method.All these are modified in the scope of the claim all belonging to additional, are also included in the lump at this.Each scope mentioned comprises whole combinations and time combination of this scope, and at the optional network specific digit symbol that this comprises.

Although the details that invention has above been undertaken being convenient to understand by the mode of example describes, but to those skilled in the art, disclosed content obviously also contains some changes and modifies, it can be implemented within the scope of the appended claims and need not pay excessive experimental work, it is only descriptive, instead of restrictive.

embodiment

It is below the example implementing particular aspects of the present invention.These examples only for descriptive object, but not for limiting scope of the present invention by any way.Although made great efforts the accuracy ensureing the numeral (such as, quantity, temperature etc.) used, obviously should allow to there is some experimental erroies and deviation.

Operation of the present invention is unless otherwise stated, use the protein chemistry of this area routine, biochemistry, recombinant DNA technology and method of pharmacy.These technology have complete explanation in the literature.See, such as, T.E.Creighton, Proteins:StructuresandMolecularProperties (W.H.FreemanandCompany, 1993); A.L.Lehninger, Biochemistry (WorthPublishers, Inc., current edition); The people such as Sambrook, MolecularCloning:ALaboratoryManual (second edition, 1989); MethodsInEnzymology (S.Colowick and N.Kaplan edits, AcademicPress, Inc.); Remington's; The CareyandSundbergAdvancedOrganicChemistry third edition (PlenumPress), A and B rolls up, 1992).

Materials and methods

Bacterial isolates and growth conditions

Bacterial isolates in this research is bordetella pertussis BPZE1, a kind of TohamaI derivant of anti-streptomycin, lack cutaneous necrosis (DNT) encoding gene, the pertussis toxin, PT (PT) of inactivation and tracheal cell toxin (TCT) (22) of background level can have been produced.BPZE1 antibacterial is at 37 DEG C, be supplemented with 1% glycerol, 10% defibrinated Sanguis caprae seu ovis and 100 μ g/ml streptomycin (SigmaChemicalCO., StLouis, Mo.) Bordet-Gengou (BG) agar (Difco, Detroit, Mich.) middle growth 72h.Containing 1g/l seven-(2,6-bis--O-methyl)-beta-schardinger dextrin-(Sigma) Stainer-Scholte (SS) culture medium in carry out the liquid culture (people such as MenozziFD according to description before, " Identificationandpurificationoftransferring-andlactoferr in-bindingproteinsofBordetellapertussisandBordetellabron chiseptica ", Infect.Immun59:3982-3988,1991).95 DEG C of heat inactivations 1 hour.

Intranasal infection.Be placed in 6-8 week large female Balb/c mice not containing the independent ventilation cage tool of specific pathogen, all experiments all carries out according to the guidance of the zooscopy board of directors of NUS.About the process of BPZE1, to taking ataractic mice intranasal administration containing the 5x10 that has an appointment ⁶the aseptic PBS of the 20 μ l (PBST) being supplemented with 0.05% Tween 80 (Sigma) of the survival of colony-forming units (cfu) or the BPZE1 antibacterial of death once, twice or three times, (the people such as MielcarekN as previously mentioned, " IntranasalprimingwithrecombinantBordetellapertussisforth einductionofasystemicimmuneresponseagainstaheterologousa ntigen ", Infectimmune65:544-550,1997).For influenza infection, take ataractic mice by nose administration containing having an appointment 2 × 10 ⁶tCID ₅₀the aseptic PBS of the 20 μ l (people such as NarasarajuT being supplemented with penicillin and streptomycin of mice adaptability the 10th generation A/Aichi/2/68 (H3N2) virus, " AdaptationofhumaninfluenzaH3N2virusinamousepneumonitismo del:insightsintoviralvirulence, tissuetropismandhostpathogenesis ", MicrobesInfect11:2-11, 2009), or the aseptic PBS of 20 μ l being supplemented with penicillin and streptomycin of H1N1 (A/PR/8/34) influenza virus (ATCC#VR-95) of 4 times of lethal dosage (LD) 50.Use and often organize the survival rate of 10 mices detections based on body weight loss, when Mouse Weight loss exceedes 20% of original body weight, euthanasia is carried out to it.

Virus titer detects

Collect the lung of mice and use Mechanical Crushing (Omnihomogenizer) to homogenize, by using WHO (WHO, " WHOManualonAnimalsInfluenzaDiagnosisandSurveillance " (WorldHealthOrganization, Geneva), the method for modifying 2002) reported is to carry out TCID 50 (TCID ₅₀) analyze with the existence detecting life virus.In brief, the lung homogenate thing of 10 times of serial dilutions of 100 μ l is inculcated to 90% on 96-orifice plate and merge Madin-Darby dog kidney (MDCK) cell of state.By orifice plate at 35 DEG C of humidified incubator (5%CO ₂) in cultivate 3 days.Cytopathic effect (CPE) minimizing by 50% measures TCID ₅₀, draw logTCID ₅₀/ lung.Often group detects 5 mices respectively to each time point.

Histopathological examination

Often organize execution 4 mices, within 3 days after virus attack, collect its pulmonary.Pulmonary is removed and uses the PBS containing 10% formalin to be fixed.After fixing, enclosed paraffin, cut into slices and use H & E to dye.

Cellular infiltration in bronchoalveolar lavage fluid (BALF)

Aseptic PBS respectively by injection 1ml extremely puts to death the pulmonary of animal and carries out a lavation step to ensure that the infiltration of two panels lung in this process is to reclaim individual BALF.Then under 400g by centrifugal for BALF 10min, remove supernatant ,-80 DEG C store be used for cytokines measurement.Blood-counter system is used to detect full BALF cell number.Use cytospin (ThermoShandon) by cell blots to microscope slide, the Wright dyeing course modified is used to carry out dyeing (the people such as KimmanTG, " Developmentandantigenspecificityofthelymphophoproliferat ionresponseofpigstopseudorabiesvirus:dichotomybetweensec ondaryB-andT-cellresponses ", Immunology86:372-378,1995).The Morphologic Parameters of use standard identifies different cell types.Result is expressed as the percentage ratio that each cell type accounts for whole cell mass.Often open slice, thin piece and be considered to whole 500 cells.Often organize and detect 4 mices respectively.

Facs analysis.

Put to death mice, collect pulmonary, 2ml contain 0.5mg/ml digestive enzyme (Roche), 1%FCS and 2U/mlDNaseI (Qiagen) RPMI digestion buffer in 37 DEG C of digestion pulmonary 15min prepare single cell suspension, the then centrifugal 20min of room temperature under 600g in the Ficoll-PaqueTMPLUS (GE).Collecting cell, washs 2 times with aseptic FACS buffer (2%FCS, 5mMEDTA, PBS).By 10 ⁶the anti-mouse CD3 antibody (eBioscience) of individual cell FITC-labelling dyes, and analyzes on CyAnTMADP cell counter (Dako).Often group detects 5 mices respectively to each time point.

Cytokine and chemotactic factor analysis

The cytokine and chemotactic factor that produce in ProcartaCytokineProfiling test kit detection BALF supernatant is used according to the explanation of manufacturer (Panomics).After Ab and Streptavidin-PE complex are hatched and detected to the beadlet with Ab-coupling, Bio-Plex instrument (Bio-Rad) runs sample.Determine following somatomedin, cytokine and inflammatory mediator: GM-CSF, KC, IL-1 β, IL-6, IFN-γ and TNF-α, IFN-α, MCP-1RANTES, IL-10.In addition, the level of TGF-β is have detected according to explanation end user/mice TGF β 1ELISA test kit (eBioscience) of manufacturer (eBioscience).

Passive transfer test

In the interval of 4 weeks, twice 5x10 is infected to 10 adult Balb/c mice nasal cavities ⁶the BPZE1 viable bacteria of cfu produces the anti-bordetella pertussis immune serum of high titre.To injection (ip.) 10 in 10 non-immune Balb/c mouse peritoneums of growing up of another group ^5.5tCID ₅₀complete Freund's adjuvant in heat inactivation people/Aichi/2/68 (H3N2) virus (HI-H3N2), and to strengthen by the HI-H3N2 virus in mutually commensurability complete Freund's adjuvant after 2 weeks.Within 2 weeks after reinforcement, collect the immune serum from each mice group, merged, detect anti-pertussis and anti-influenza antibodies titre by ELISA.In addition, whether existed by the neutralizing antibody of neutralization analysis detection HI-H3N2 serum.Filtration sterilization is carried out to immune serum, 56 DEG C heating 30min carry out inactivation, and be stored in-80 DEG C stand-by.Collect from contrasting the serum of non-immune mice as negative control.

Non-immune, the anti-BPZE1 of 200 μ l or anti-H3N2 immune serum was injected in all large receptor Balb/c mouse peritoneums of 6-8 before 1 day with mice adaptability H3N2 virus attack.Monitoring body weight loss is to measure survival rate.Every group analysis 10 mices.

T cell proliferation assay

By mix tritium-labeled ( ³h) thymus pyrimidine detects lymphocytic propagation, as the description (people such as BaoZ of other document, " Glycogensynthasekinase-3betainhibitionattenuatesasthmain mice ", AmJRespirCritCareMed176:431-438,2007).In brief, aseptically collect mice (often group 6 mices) spleen from non-immune and BPZE1 process and mix.Prepare single cell suspension also with the centrifugal 20min of room temperature under Ficoll-PaqueTMPLUS (GE) 600g.With spleen cell inoculation 96-hole circle base plate (NUNC) be separated, density is 2 × 10 ⁵cells/well 100 μ l culture medium (is supplemented with 10%FCS, 5 × 10 ^-5the RPMI640 of M beta-mercaptoethanol, 2mML-glutamate, Glu, 10mMHEPES, 200U/ml penicillin, 200 μ g/ml streptomycins).Add in spleen cell containing 20 μ g/mlBPZE1 intact cells lysate or heat inactivations 10 ⁵tCID ₅₀the 100 μ l culture medium of mice adaptability H3N2 influenza virus (HI-H3N2) (test antigen).The ovum amniotic fluid using 100ul not infect respectively and the 100ul culture medium containing 5 μ g/ml concanavalin As (conA) are as simulation and active control.37 ^o5%CO under C ₂after hatching 3 days in environment, culture 20 μ lRPMI complete mediums of 0.4 μ Ci [3H] thymus pyrimidine are carried out pulse.After hatching 18 hours, collecting cell, washs and uses TopCountNXT ^tMmicroplateScintillation and LuminescenceCounter (PerkinElmer) detects the radioactivity mixed.Result be expressed as correspond to test antigen exist under take in [ ³h] thymus pyrimidine meansigma methods with containing taking under test antigen [ ³h] stimulation index (SI) of ratio of meansigma methods of thymus pyrimidine.SI>2 then thinks positive.Each sample analysis four is parallel.

IFN-α ELISPOT analyzes

Use BD mice ELISPOT series (BDPharMingen) to carry out ELISPOT according to the explanation of manufacturer and analyze the frequency (frequency) that mensuration antigen-specificity produces the spleen cell of IFN-γ.In brief, prepare the single cell suspension from each mouse spleen of non-immune and BPZE1 process and be placed in 4 DEG C of pre-coated 96-microwell plate (Millipore having 100 μ l [aseptic PBS contains the anti-IFN-gamma antibodies of 5 μ g/ml] that spend the night, Bedford, MA), wash three times and at room temperature close 2 hours with the RPMI1640 containing 10%FCS.Then by cell at 20 μ g/mlBPZE1 intact cells lysate or heat inactivations 10 ⁵tCID ₅₀mice adaptability H3N2 influenza virus (HI-H3N2) or 5 μ g/mlconA at 37 DEG C 5%CO ₂12-20hr is hatched in environment.Washing microwell plate also at room temperature adds the anti-mouse IFN-gamma antibodies 2 hours of biotin coupling.After washing, add Streptavidin-HRP conjugate, incubated at room 1 hour.Again wash each hole, and cultivate until speckle is visible with 3-amino-9-Ethy-Carbazole (AEC) substrate solution.After drying, use 4000 (Biosystem) calculates the cell number forming speckle.Often organize and analyze 6 animals respectively.

Statistical analysis

Unless otherwise stated, error bar represents mean ± SD, meansigma methods uses the not paired T inspection of the bilateral of 5% significance level to compare, wherein * p≤0.05, * * p≤0.01 and * * * p≤0.001.

embodiment 1: live body attenuation bordetella pertussis single nasal-cavity administration is protected from H3N2 influenza challenge.

The mice adaptability H3N2 influenza virus (people such as NarasarajuT is obtained to the Balb/c mice that grows up by the lung-lung continuous passage of A/Aichi/2/68 (H3N2) virus, " AdaptationofhumaninfluenzaH3N2virusinamousepneumonitismo del:insightsintoviralvirulence; tissuetropismandhostpathogenesis ", MicrobesInfect11:2-11,2009).10th generation (P10) strains expressed goes out high toxicity, causes gangrenosum acne and inflammatory damage to be transmitted to Different Organs outside the pulmonary of infected animals.Nasal-cavity administration 2x10 ⁶tCID ₅₀p10 viral suspension cause the (people such as NarasarajuT of animal dead in 4 days, " AdaptationofhumaninfluenzaH3N2virusinamousepneumonitismo del:insightsintoviralvirulence; tissuetropismandhostpathogenesis ", MicrobesInfect11:2-11,2009).

The Balb/c mice intranasal inoculation BPZE1 viable bacteria that grows up also was attacked by the mice adaptability H3N2 virus of lethal dosage then after 3 or 6 weeks.Based on the survival rate display nasal cavity BPZE1 process of body weight change after 3 weeks mice under fire be not subject to remarkable protection, and the protection of 60% is in BPZE1 process acquisition (Fig. 1) afterwards in 6 weeks.

embodiment 2: live body but not the BPZE1 antibacterial of death are protected from the attack of lethal H3N2.

The BPZE1 antibacterial nasal-cavity administration of adult Balb/c mice live body or death once, and is attacked in the mice adaptability H3N2 virus of BPZE1 process 6 weeks rear fatal dose.The antibacterial of result display death does not provide any significance for H3N2 protection (Fig. 2), shows that the bacterial clump of mouse lung is required for eliciting protective mechanism.

embodiment 3: stiffening effect.

Balb/c mice uses BPZE1 viable bacteria nasal-cavity administration twice in 4 weekly intervals, and BPZE1 administration was attacked by the mice adaptability H3N2 virus of lethal dosage after 4 weeks the last time.Animal for BPZE1 process obtains protective rate and the minimum body weight change (Fig. 3) of 100%.Within two weeks after reinforcement, carry out virus attack and have also been obtained similar protective rate.These data show that secondary nasal-cavity administration BPZE1 viable bacteria not only increases Vaccine effectiveness, and shorten the time required for triggering protective mechanism.

embodiment 4:BPZE1 antibacterial provides the protection of attacking for H1N1 virus

Further developed the protection potentiality of BPZE1 antibacterial for influenza A virus.It once can not be protected from the lethal hit (data do not show) of 6 weeks rear employment A/PR/8/34 (H1N1) influenza A viruss to mice BPZE1 viable bacteria nasal-cavity administration.But, three successive administrations of BPZE1 viable bacteria give for H1N1 virus 50% protection (Fig. 4).These phenomenons show that BPZE1 antibacterial can have the potentiality resisting all influenza A viruss, although its effect is change.

embodiment 5: virus load does not reduce in shielded mice.

In order to characterize the cross protection for influenza A virus further, for be undertaken quantitatively by the mice of BPZE1 bacterial treatment twice and the lung virus carrying capacity of control mice.Within 3 days, detect virus load after infection, it corresponds to by the virus titer peak value of the mice of the mice adaptability H3N2 viral infection (people such as NarasarajuT, " AdaptationofhumaninfluenzaH3N2virusinamousepneumonitismo del:insightsintoviralvirulence; tissuetropismandhostpathogenesis ", MicrobesInfect11:2-11,2009).Do not observe the significant difference (Fig. 5) of two treated animal virus loads.This result shows the intersecting protective mechanism that triggers in the animal of BPZE1 process directly targeting virion and/or infected cell.

the immunopathogenesis that embodiment 6:BPZE1 process protection mice is induced from influenza and lymphocyte loss.

By have detected lung immunopathogenesis to the Histological research of the infected animal of BPZE1 process and the Lung sections of control mice.As (the people such as NarasarajuT expected before and describe, " AdaptationofhumaninfluenzaH3N2virusinamousepneumonitismo del:insightsintoviralvirulence; tissuetropismandhostpathogenesis ", MicrobesInfect11:2-11,2009), the control mice infected shows with the extensive inflammation of inflammatory cell, serious bronchopneumonia and interstitial pneumonia, bronchus and alveolar are filled with downright bad fragment, and height emphysema and intermediate edema (Fig7A).Otherwise, the mouse lung of shielded BPZE1 process only observe minor irritation, minimal air flue and alveolar damage and relevant to minimal edema slight blood vessel week/damages in bronchioles week.

Also have detected the cell mass of the bronchoalveolar lavage fluid (BALF) be present in from protected and not protected animal.Although the total cellular score existed in the BALF of two treated animals suitable (11.8 × 10 ⁵with 16.1 × 10 ⁵), but in the mice of BPZE1 process, found obviously more macrophage quantity and lower neutrophilic granulocyte quantity (Fig. 6 B).

In addition, the lymphocyte populations of the pulmonary being present in protected and not protected mice is also analyzed.Lymphocyte depletion is infecting the mice (people such as KashJC of high pathogenicity H1N1 (1918) and H5N1 influenza virus, " Genomicanalysisofincreasedhostimmuneandcelldeathresponse sinducedby1918influenzavirus ", Nature443:578-581,2006, the people such as UiprasertkulM, " ApoptosisandPathogenesisofAvianInfluenzaA (H5N1) VirusinHumans ", EmergInfectDis13:708-712,2007, the people such as LuX, " Amousemodelfortheevaluationofpathogenesisandimmunitytoin fluenzaA (H5N1) virusesisolatedfromhumans ", JVirol73:5903 – 5911, 1999) and infect the mice (people such as NarasarajuT of mice adaptability H3N2 virus strains used in this work, " AdaptationofhumaninfluenzaH3N2virusinamousepneumonitismo del:insightsintoviralvirulence, tissuetropismandhostpathogenesis ", MicrobesInfect11:2-11, 2009) report was had in.At this, with BPZE1 bacterial treatment Balb/c mice twice, and attack by mice adaptability H3N2 virus after 4 weeks.Influenza challenge 3 and collect the facs analysis of pulmonary for T cell group of mice for 5 days afterwards.After virus attack 3 days, CD3 in the control mice of infection and the mice of BPZE1 process ⁺the lymphocytic percentage ratio of T and suitable (Fig. 6 C) in this animal before attack.But virus attack observed significant CD3 after 5 days in the control animal infected ⁺t cell loss (Fig. 6 C), (the people such as NarasarajuT as reported before, " AdaptationofhumaninfluenzaH3N2virusinamousepneumonitismo del:insightsintoviralvirulence; tissuetropismandhostpathogenesis ", MicrobesInfect11:2-11,2009).On the contrary, in the animal of shielded BPZE1 immunity, T cell group keeps constant (Fig. 6 C) before and after attack, shows the Lymphocyte depletion that BPZE1 process prevents influenza to induce.

embodiment 7: in the mice of shielded BPZE1 process pro-inflammatory cytokine and chemotactic because of the output of son reduces.

Lethal H3N2 virus attack 1 and after 3 days, major proinflammatory cytokine and chemotactic factor in the mice BALF from shielded BPZE1 process being detected, and compare with not protected mice.The pro-inflammatory cytokine of all detections in shielded animal groups BALF and chemotactic factor are all than the remarkable reduction (Fig. 7) detected in not protected mice.For contribute to influenza induction immunopathogenesis and main pro-inflammatory cytokine IL-1 β, IL-6, IFN-γ and the TNF-α relevant with disease severity for, all observed difference at the 1st and the 3rd day.(people such as deJongMD, " FataloutcomeofhumaninfluenzaA (H5N1) isassociatedwithhighviralloadandhypercytokinemia ", NatMed12:1203-1207,2006; The people such as BeigelJH, " AvianinfluenzaA (H5N1) infectioninhumans:, NEnglJMed353:1374-1385,2005; The people such as PeirisJS, " Re-emergenceoffatalhumaninfluenzaAsubtypeH5N1disease ", Lancet363:617 – 619,2004; The people such as SchmitzN, " Interleukin-1isresponsibleforacutelungimmunopatholoybuti ncreasessurvivalofrespiratoryinfluenzavirusinfection ", JVirol79:6441-6448,2005).Find that the level of IFN-α, MCP-1 and RANTES significantly reduces in virus attack for 1 day afterwards, and the generation of IL-12 (p70), GM-CSF and KC reduces after 3 days in attack.Significantly, find that the generation of IL-12 in shielded animal groups is totally constrained, it is consistent with the low-level of IFN-γ.

In addition; the level significant difference of protection and the anti-inflammatory cytokine IL-10 do not watched for animals and TGF-β do not detected, this eliminate 1 type regulatory T-cell (Tr1) and participate in (probability) (Fig. 7) in cross protection mechanism.

embodiment 8:bordetella pertussis the adaptive immunity of specificity does not participate in cross protection.

Have detected the existence of cross reaction (and protectiveness) antibody in the animal of BPZE1 process and/or T cell.First, BLAST retrieval does not obtain bordetella pertussis and has the epi-position (data do not show) of any coupling between influenza A H3N2 and H1N1 virus.Secondly, the immune serum of the mice of BPZE1 process can not react with whole H3N2 virion in elisa assay, can not neutralize virus (data do not show) in its neutralization analysis in vivo.3rd, high titre anti-BPZE1 immune serum does not provide any protection for H3N2 lethal hit in passive transfer test in vitro, and gives the protective rate (Fig. 8 A) of 100% for the immune serum of heat inactivation H3N2 virus.4th, propagation and IFN-γ ELISPOT analyze the Mouse spleen cells showing BPZE1 process respectively and do not breed when stimulating with H3N2 virion, also do not produce IFN-J (Fig. 8 B and C).Generally speaking, these data strongly support bordetella pertussis specific immunity and do not play any effect in for the cross protections of influenza A virus.

Discuss

Serious respiratory tract disease and immunopathogenesis and high case fatality rate have become the feature that the mankind and other mammal generation high pathogenic avian influenza virus infect.But, cause the potential mechanism of this sever immune pathological effects still not illustrated completely.Especially, the relation between the generation of virus load, immunopathogenesis and disease is still subtle.Several researchs before have reported and a kind ofly in influenza infection animal model, have reduced mortality rate and significantly do not reduce the immunopathogenesis of virus load; Such as, along with MIP-1 α gene disruption observes inflammatory cell infiltration and the pulmonary lesion of minimizing, but along with the virus sweep postponed (people such as CookDN, " RequirementofMIP-1alphaforaninflammatoryresponsetovirali nfection ", Science269:1583-1585,1995).Similar, CCR2 (major receptors of MCP-1) but-the deficient mice mortality rate showing minimizing and the lung cells reduced infiltrate the virus load (people such as DawsonTC of the remarkable increase relevant with tissue injury, " ContrastingeffectsofCCR5andCCR2deficiencyinthepulmonaryi nflammatoryresponsetoinfluenzaAvirus ", AmJPathol156:1951-1959,2000).On the contrary, the mice that IL-1R knocks out shows the mortality rate of the increase relevant to the virus sweep postponed, but the lighter pathology (people such as SchmitzN, " Interleukin-1isresponsibleforacutelungimmunopatholoybuti ncreasessurvivalofrespiratoryinfluenzavirusinfection ", JVirol79:6441-6448,2005).

At this, the cross protection for influenza A virus that we report bordetella pertussis mediation does not cause lower lung virus carrying capacity.Otherwise the mice of shielded BPZE1 process shows the reduction of the output of minimum pulmonary's immunopathogenesis and major proinflammatory cytokine and chemotactic factor.Our discovery is consistent with a widespread consensus, i.e. disease severity (the people such as LaGrutaNL very relevant to high cytokine/Chemokines Levels, " Aquestionofself-preservation:immunopathologyininfluenzav irusinfection ", ImmunolCellBiol85:85-922007).Be characterized as the serum and pulmonary's chemotactic factor that are caused by the uncontrolled activation of host immune system and the excessive cytokine storm (storm) of cytokine levels, really relevant (the people such as KashJC lethal to the laboratory animal of 1918H1N1 and the H5N1 influenza infection be reconstructed, " Genomicanalysisofincreasedhostimmuneandcelldeathresponse sinducedby1918influenzavirus ", Nature443:578-581,2006; The people such as SimonAK, " Tumornecrosisfactor-relatedapoptosis-inducingligandinTce lldevelopment:sensitivityofhumanthymocytes ", ProcNatlAcadSciUSA98:5158-5163,2001; The people such as KobasaD, " Aberrantinnateimmuneresponseinlethalinfectionofmacaquesw iththe1918influenzavirus ", Nature445:319-323,2007; The people such as TumpeyTM, " Characterizationofthereconstructed1918Spanishinfluenzapa ndemicvirus ", Science310:77-80,2005); And with the lethal relevant (people such as deJongMD of the mankind, " FataloutcomeofhumaninfluenzaA (H5N1) isassociatedwithhighviralloadandhypercytokinemia ", NatMed12:1203-1207,2006; The people such as BeigelJH, " AvianinfluenzaA (H5N1) infectioninhumans:, NEnglJMed353:1374-1385,2005; The people such as UiprasertkulM, " ApoptosisandPathogenesisofAvianInfluenzaA (H5N1) VirusinHumans ", EmergInfectDis13:708-712,2007; The people such as PeirisJS, " Re-emergenceoffatalhumaninfluenzaAsubtypeH5N1disease ", Lancet363:617 – 619,2004).In addition, tissue and pathological hallmarks show that excessive host response plays pivotal role in the extreme at least partly pathology that mediation is relevant to highly pathogenic influenza virus strongly.But the effect of the individual cells factor in influenza infection is unclear yet, it often has front and negative effect simultaneously; Although the virus sweep that its generation may be carried out for the immunoeffectors cell by supplementary and/or reactivate infection site is important, but its inflammation character may cause the tissue injury (people such as LaGrutaNL equally, " Aquestionofself-preservation:immunopathologyininfluenzav irusinfection ", ImmunolCellBiol85:85-922007).

In the animal respiratory of shielded BPZE1 process, the output of pro-inflammatory cytokine and chemotactic factor reduces probably affects cellular infiltration and activated immune cell; Really in the BALF of shielded animal, significantly reduced neutrophil cell number is observed, it is consistent with the low-level of KC and TNF-α, these two kinds of cytokines participate in supplementing of neutrophil cell in infected tissue and activate (the people such as LaGrutaNL, " Aquestionofself-preservation:immunopathologyininfluenzav irusinfection ", ImmunolCellBiol85:85-922007; The people such as KipsJC, " Tumornecrosisfactorcausesbronchialhyperresponsivenessinr ats ", AmRevRespirDis145:332 – 336,1992; The people such as HeadleyAS, " Infectionsandtheinflammatoryresponseinacuterespiratorydi stresssyndrome ", Chest111:1306-1321,1997).In addition, the activation probably compromising some immunocytes, such as natural killer cell (NK) and cytotoxin CD8 are produced in the suppression of the IL-12 detected in shielded animal ⁺t cell.These two kinds of cell types have been described to potential harmful, and participate in the immunopathogenesis (people such as LaGrutaNL of inflammatory mediator release, " Aquestionofself-preservation:immunopathologyininfluenzav irusinfection ", ImmunolCellBiol85:85-922007).Consistent therewith, observe IFN-γ---NK and CD8 of shielded animal BALF ⁺the mark of T cell activation---output significantly reduces.In addition, the IFN-γ output reduced can affect the response of neutrophil cell for virus, comprise oxidative burst, antigen presentation induction and chemotactic factor and produce (EllisTN and BeamanBL, " Interferon-gammaactivationofpolymorphonuclearneutrophilf unction ", Immunology112:2 – 11,2004; FarrarMA and SchreiberRD, " Themolecularcellbiologyofinterferon-gammaanditsreceptor ", AnnuRevImmunol11:571,1993).

What is interesting is, observe the macrophage number significantly raised in the mice BALF of shielded BPZE1 process, although the level of itself MCP-1 and GM-CSF reduces, the latter relates separately to mononuclear cell and supplements and be divided into macrophage.But, it should be noted that and be pulmonary alveolar macrophage (AM) but not the main macrophage group (people such as JakubzickC organizing resident macrophage to constitute to reclaim in BALF, " Modulationofdendriticcellstraffickingtoandfromtheairways ", JImmunol176:3578-3584,2006).Thus, can expect that shielded mice can show the macrophage group of reduction for the control mice infected in its lung tissue.

AM has shown by the regulatory T-cell function (people such as StricklandDH, " RegulationofT-cellfunctioninlungtissuebypulmonaryalveola rmacrophages ", Immunology80:266-272,1993) and by suppressing the dendritic cell maturation (people such as HoltPG, " Down-regulationoftheantigenpresentingfunction (s) ofpulmonarydendriticcellsinvivobyresidentalveolarmacroph ages ", JExpMed177:397-407,1993; BilykN and HoltPG, " Inhibitionoftheimmunosuppressiveactivityofresidentpulmon aryalveolarmacrophagesbygranulocyte/macrophagecolonystim ulatingfactor ", JExpMed177:1773-1777,1993; The people such as StumblesPA, " Airwaydendriticcells:co-ordinatorsofimmunologicalhomeost asisandimmunityintherespiratorytract ", APMIS111:741-755,2003) and migrate to the mesenteric lymph node (people such as JakubzickC, " Modulationofdendriticcellstraffickingtoandfromtheairways ", JImmunol176:3578-3584,2006) inhibition for inflammatory reaction is shown.Therefore we can suppose that the inflammation that the pulmonary alveolar macrophage of the comparatively high amts of being induced by influenza challenge contributes in the animal of shielded BPZE1 process suppresses/controls.

In addition, we find CD3 ⁺t cell group remains unchanged when the animal of shielded BPZE1 process is subject to virus attack, and has found CD3 in the control mice infected ⁺the remarkable reduction of T cell ratio.(the people such as KashJC is had been reported before Lymphocyte depletion in highly pathogenic influenza infection process, " Genomicanalysisofincreasedhostimmuneandcelldeathresponse sinducedby1918influenzavirus ", Nature443:578-581,2006; The people such as UiprasertkulM, " ApoptosisandPathogenesisofAvianInfluenzaA (H5N1) VirusinHumans ", EmergInfectDis13:708-712,2007; The people such as NarasarajuT, " AdaptationofhumaninfluenzaH3N2virusinamousepneumonitismo del:insightsintoviralvirulence; tissuetropismandhostpathogenesis ", MicrobesInfect11:2-11,2009; The people such as LuX, " Amousemodelfortheevaluationofpathogenesisandimmunitytoin fluenzaA (H5N1) virusesisolatedfromhumans ", JVirol73:5903 – 5911,1999), and experimental evidence showed cell apoptosis be a kind of potential mechanism (people such as UiprasertkulM, " ApoptosisandPathogenesisofAvianInfluenzaA (H5N1) VirusinHumans ", EmergInfectDis13:708-712,2007; The people such as LuX, " Amousemodelfortheevaluationofpathogenesisandimmunitytoin fluenzaA (H5N1) virusesisolatedfromhumans ", JVirol73:5903 – 5911,1999).Due to protection and the difference not observing virus load in not watching for animals, therefore Lymphocyte Apoptosis can not be self direct molten cell effect of virus.Otherwise, our data are consistent with research before, it thinks in the mankind and mice of H5N1-infection, Lymphocyte Apoptosis may by cytokine dysregulation host immune response overactivity cause (the people such as UiprasertkulM, " ApoptosisandPathogenesisofAvianInfluenzaA (H5N1) VirusinHumans ", EmergInfectDis13:708-712,2007; The people such as MainesTR, " Pathogenesisofemergingavianinfluenzavirusesinmammalsandt hehostinnateimmuneresponse ", ImmunolRev225:68-84,2008).Especially, known TNF-α and the associated TNF-superfamily member (TRAIL) comprising the relevant apoptosis induction ligand of TNF can the induction of T cell apoptosis (people such as SimonAK, " Tumornecrosisfactor-relatedapoptosis-inducingligandinTce lldevelopment:sensitivityofhumanthymocytes ", ProcNatlAcadSciUSA98:5158-5163,2001; The people such as WangJ, " ThecriticalroleofLIGHT, aTNFfamilymember, inTcelldevelopment ", JImmunol167:5099 – 5105,2001).Consistent therewith, the TNF-α of reduced levels detected in the BALF when the mice of shielded BPZE1 process is subject to influenza challenge, thus be translated as lower t cell proliferation potentially.

Also do not identify the protectiveness mechanism that cross protection is relevant.But our adaptive immunity (comprising cross reacting antibody and T cell) of data display bordetella pertussis-specificity has nothing to do therewith.Viewedly be live body but not the BPZE1 antibacterial of death provides protection, this shows that pulmonary's bacterium colony of antibacterial is formed, that is, prolongation be exposed to host immune system under, be required for eliciting protective mechanism.In addition, the derived need of protectiveness mechanism more than 3 weeks because do not protected significantly by the mice of influenza H3N2 virus attack once and after 3 weeks with the process of BPZE1 viable bacteria.But second time BPZE1 process can be shortened the time needed for eliciting protective mechanism and increase protective rate, shows that some memory cells produce when bringing out, it can be responsively rapider when second time runs into BPZE1 antibacterial, and responsiveness is larger.Finally, observing three continuous BPZE1 viable bacteria nasal-cavity administrations provides the protection of 50% to be necessary for for people A/PR/8/34 (H1N1) influenza virus.The different protective rates obtained are attacked for H3N2 and H1N1 virus and illustrates that the molecule disease-mechanism of being induced by two-strain is different.But even if may be different for the Vaccine effectiveness of different subtype, bordetella pertussis remains a kind of promising influenza A virus broad-spectrum vaccine.

A lot of activity of bordetella pertussis virulence factor are all devoted to immunomodulating, thus suppress, destroy and escape the system of defense (CarbonettiNH of host, " ImmunomodulationinthepathogenesisofBordetellapertussisin fectionanddisease ", CurrOpinPharmacol7:1-7,2007).Toll-sample receptor (TLR)-4 intracellular signaling starts and controls the immunne response for bordetella pertussis, its generation (people such as HigginsSC by the arborescent cell (DC) of pathology in inflammatory response and restriction air flue can be suppressed to induce anti-inflammatory cytokine IL-10, " Toll-likereceptor4-mediatedinnateIL-10activatesantigen-s pecificregulatoryTcellsandconfersresistancetoBordetellap ertussisbyinhibitinginflammatorypathology ", JImmunol171:3119-3127, 2003).Show fiber hemagglutinin (FHA), the main adhesin that bordetella pertussis produces, IL-10 can be stimulated to produce and suppress the production of IL-12 in the macrophage of TLR-induction and DC, cause the development (people such as McGuirkP of the 1 type T regulating cell (Tr1) secreting IL-10, " Pathogen-specificTregulatory1cellsinducedintherespirator ytractbyabacterialmoleculethatstimulatesinterleukin10pro ductionbydendriticcells:anovelstrategyforevasionofprotec tiveThelpertype1responsesbyBordetellapertussis ", JExpMed195:221-231, 2002).What is interesting is, the Formulations for systemic administration of recent discovery FHA can reduce inflammatory bowel in the colitis model of a T cell mediation, this supports the anti-inflammatory effect (people such as BraatH of FHA, " Preventionofexperimentalcolitisbyparenteraladministratio nofapathogen-derivedimmunomodulatorymolecule ", Gut56:351-357,2007).But, do not find to protect and in unprotected mice, the production of IL-10 and TGF-has any difference at this, this eliminate bordetella pertussis give for the cross protection of influenza A virus in relate to FHA mediation the Tr1 Potential feasibility of inducing.

We report in the Balb/c mouse model in serious pneumonia at this; the nasal-cavity administration of the attenuated strain (BPZE1) of pertussis cause of disease agent---bordetella pertussis provides the protection of the effective and persistence of the lethal hit for mice adaptability H3N2 influenza A virus, and provides the protection compared with low degree to people H1N1 (A/PR/8/34) influenza A virus.Although the cell related in this cross protection and molecule still have to be identified, our data show that the downward of the adaptive immunity of bordetella pertussis specificity and Tr1 mediation may have nothing to do therewith.Importantly, we find that this cross protection does not cause the reduction of virus load.On the contrary, the mice of shielded BPZE1 process shows minimum pulmonary's immunopathogenesis, this with the neutrophil infiltrates reduced in its BALF and the various main pro-inflammatory cytokine of minimizing and the generation of chemotactic factor consistent.Thus our discovery shows that the protection for the fatal pneumonia of influenza A virus induction can by slowing down inflammation and T suppression cell factor storm obtains consumingly, and demonstrate BPZE1 antibacterial as the potential application of effective preventative means providing protection for viral influenza a virus infection.

All in order to various object, entirety is incorporated herein by reference at this for all publications of quoting in this description and patent application, as distinguishingly with indicate often kind of publication individually or patent application is incorporated herein by reference the same in order to various object.

Although aforementioned invention has carried out details description by the mode of explanation and embodiment, but its object is to understand conveniently, the various distortion that those of ordinary skill in the art obviously can make technical scheme of the present invention and improvement, and the spirit or scope of additional claim can not be departed from.

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Claims (10)

1. the bordetella bacterial strain of the sudden change of the attenuation of a work is for the preparation of bringing out for the purposes in the medicine of the protective immunity of influenza A virus; wherein said bacterial strain comprises pertussis toxin, PT (ptx) gene of sudden change, cutaneous necrosis (dnt) gene of disappearance or sudden change and heterologous ampG gene, but is retained in the ability that mammiferous pulmonary grows surely.

2. purposes according to claim 1, wherein wild type bordetella bacterial strain ampG gene is replaced by E. coli ampG gene.

3. purposes according to claim 2, wherein the described sudden change of ptx gene comprises participating in the aminoacid of Binding Capacity and/or participating in the aminoacid of catalysis and replace.

4. purposes according to claim 3, the amino acid whose replacement of wherein said participation Binding Capacity comprises K9R, and the amino acid whose replacement of described participation catalysis comprises E129G.

5. purposes according to claim 1, the administration before influenza infection of wherein said bordetella bacterial strain.

6. purposes according to claim 5, wherein said bordetella bacterial strain carries out administration in influenza infection precontract 6 weeks or longer time.

7. purposes according to claim 6, wherein said bordetella bacterial strain carries out administration in influenza infection precontract 12 weeks or longer time.

8. purposes according to claim 1, wherein said medicine is with more than one dosed administration.

9. purposes according to claim 8, wherein said medicine is with two dosed administrations twice.

10. purposes according to claim 9, wherein said two dosage are about every administration in 3 weeks.