CN101180076B - Use of an influenza virus an oil-in-water emulsion adjuvant to induce CD4 T-cell and/or improved B-memory cell response - Google Patents

Abstract

The present invention relates to influenza vaccine formulations and vaccination regimes for immunising against influenza disease, in particular, the invention relates to a vaccine formulation containing an oil-in-water emulsion adjuvant and optional 3D-MPL, their use in medicine, in particular their use in augmenting immune responses to various antigens, and to methods of preparation, wherein the oil-in-water emulsion includes sterol, metabolizable oil and emulsifier.

Description

Influenza virus and oil in water emulsion adjuvant are induced the purposes in the immunogenic composition that the B-memory cell of CD-4 T cell and/or improvement replys in preparation

Technical field

The present invention relates to influenza vaccine formulation and the vaccination regimen that is used for immunity antagonism influenza disease.Specifically; The present invention relates to contain bacterin preparation, its purposes in medicine of oil in water emulsion adjuvant and the 3D-MPL that chooses wantonly; Especially it is increasing the purposes aspect the immunne response of influenza antigens; And relate to method for preparing, but wherein said O/w emulsion comprises sterol metabolism oil and emulsifying agent.

Technical background

Influenza virus is one of the most ubiquitous virus in the world, both can infect the mankind, also can infect domestic animal.Influenza produces great financial burden, sickness rate and even mortality rate.

Influenza virus is the RNA enveloped virus, and particulate diameter is about 125nm.Basic composition is of influenza virus: inner is and the bonded ribonucleic acid of nucleoprotein (RNA) nucleocapsid or core that external packets is around the viral tunicle of double-layer of lipoid structure and outer glycoprotein.Virus mainly is made up of stromatin by film inner layer, and skin mainly is the lipid material that the host originates.Influenza virus comprises two kinds of surface antigens: glycoprotein neuraminidase (NA) and hemagglutinin (HA), they are emerging on the particle surface with the long furcella of 10-12nm.Be exactly these surface proteins, especially hemagglutinin, determined the antigenic specificity of influenza subtype.

These surface antigens experience the change that some causes the variation of influenza antigen property progressively, sometimes apace.These antigenic changes are called ' drift ' and ' conversion '; Be unpredictalbe, see to have remarkable influence, because they finally cause occurring new influenza strain by the immunology viewpoint; Can make the viral escape immune system, almost all cause well-known epidemic diseases every year.

The strains of influenza viruses that joins each season in the influenza vaccines is confirmed by collaborative national health management board of WHO and production of vaccine merchant jointly.

HA is a most important antigen of confirming the serological specificity of different influenza strains.The albumen of this 75-80kD comprises numerous antigenic determinants, wherein several being located in the zone that the experience sequence changes in the different strains (strain specific determinant), and remaining is arranged in the total zone of many HA molecules (common determinant).

Influenza virus all causes almost annual winter is very popular, the infection rate of first (A) type or second (B) type virus in the time period in 6 weeks up to 40%.Influenza infection produces various diseases, by subclinical infection to slight upper respiratory tract infection to serious viral pneumonia.Typical flu outbreak causes that the sickness rate of pneumonia and lower respiratory illness increases, and evidence is that admission rate or mortality rate increase.Severity of disease is mainly determined by host's age, its immune state and infection site.

65 years old with above old people especially vulnerable, account for the 80-90% of all influenza associated deaths of developed country.The individuality of suffering from basic chronic disease also very might experience this complication.The child also possibly suffer from serious disease.Therefore, these colonies especially need protected.Except these ' risks ' the colony, the health adult's inoculation to contacting with the old people is also recommended by health authority.

Inoculation plays a crucial role to controlling annual flu outbreak.Present available influenza vaccines are inactivation influenza vaccines or attenuated live influenza vaccines.The inactivation influenza vaccines by 3 kinds maybe forms the antigen preparation thing forms: the inactivation totivirus, wherein the virion of purification by detergent or other reagent destruction with the subvirus body (so-called " split vaccine ") of lipin dissolving peplos or the HA or the NA (subunit vaccine) of purification.These inactivated vaccines intramusculars (i.m.) or intranasal (i.n.) give.

The influenza vaccines of all kinds all are trivalent vaccine usually.In general, their antigen of containing derives from two kinds of influenza A virus strains and a kind of Influenza B virus strain.According to single radiation immunity diffusion (SRD) (J.M.Wood etc.: An improved single radialimmunodiffusion technique for the assay of influenza haemaggiutininantigen:adaptation for potency determination of inactivated whole virusand subunit vaccines.J.Biol.Stand.5 (1977) 237-247; J.M.Wood etc.; International collaborative study of single radial diffusion and Immunoelectrophoresis techniques for the assay of haemaggiutininantigen of influenza virus.J.Biol.Stand.9 (1981) 317-330) detection; In most of the cases, standard 0.5 ml ID contains 15 μ g hemagglutinin antigen components of every kind of strain.

Present available influenza vaccines be considered to all age group all be safe (De Donato etc., 1999, Vaccine, 17,3094-3101).But, evidence suggests that seldom current influenza vaccines have effect in the child below 2 years old.And prevention has typically confirmed that the report ratio of the vaccine potency of influenza disease is 23-72% to old people, and this significantly is lower than effective percentage (Govaert, 1994, J.Am.Med.Assoc, 21, the 166-1665 of the 60-90% that younger adult is reported; Gross, 1995, Ann Intern.Med.123,523-527).Showed already; The effectiveness of influenza vaccines is relevant with the serum titer that the hemagglutination that is directed against Strain suppresses (HI) antibody; Several researchs have found that the HI titre that more old adult shows is lower than younger adult (Murasko, 2002, Experimental gerontology after the influenza immunity; 37,427-439).

The immunogenic novel vaccine that therefore, still need have improvement.Vaccine antigen is a kind of method that might strengthen the immunne response of subvirus isoantigen with the preparation of effective adjuvant.

Subunit influenza vaccines commercialization with the adjuvant MF59 adjuvantization of O/w emulsion form; Ability (De Donato etc., 1999, Vaccine that it induces the antibody titer that is higher than antibody titer that no adjuvant subunit vaccine obtains have been shown; 17,3094-3101).But, in publication afterwards, identical vaccine do not show its compare pattern that no adjuvant split vaccine improves (Puig-Barbera etc., 2004, Vaccine 23,283-289).

The influenza vaccines that still need improve are especially for elderly population.

The invention statement

Of the present invention aspect first; (a) influenza virus or its antigenicity prepared product and (b) the oil in water emulsion adjuvant purposes in producing immunogenic composition are provided; Said immunogenic composition be used for philtrum induce anti-said virus or antigenic composition below at least a: the CD4 T-cellullar immunologic response that i) improves; The B-memory cell that ii) improves is replied, but wherein said O/w emulsion comprises metabolism oil, sterol and emulsifying agent.

Aptly, said sterol is an alpha-tocopherol.In a specific embodiments, but said oil in water emulsion adjuvant comprises at least a metabolism oil, and its amount is the 0.5%-20% of cumulative volume, and has oil droplet, and at least 70% (with intensitometer) of oil droplet has the diameter that is lower than 1 μ m.

In a specific embodiments, to reply than what obtain with no adjuvant antigen or antigenic composition, said immunogenic composition can induce the CD4 T-cellullar immunologic response of improvement and the B-memory cell of improvement to reply.

Aspect second of the present invention; (a) influenza virus or its antigenicity prepared product and (b) the oil in water emulsion adjuvant purposes in producing immunogenic composition are provided; Said immunogenic composition is used to inoculate human immune injured individual or colony; For example excessive risk adult or old people, with to influenza, but wherein said O/w emulsion comprises metabolism oil, sterol and emulsifying agent.

In a preferred embodiment, but said oil in water emulsion adjuvant comprises at least a metabolism oil, and its amount is the 0.5%-20% of cumulative volume, and has oil droplet, and at least 70% (with intensitometer) of oil droplet has the diameter that is lower than 1 μ m.

Aspect the 3rd of the present invention; Influenza virus or its antigenicity prepared product purposes in producing immunogenic composition is provided; Said immunogenic composition is used to inoculate the people who had before inoculated with influenza virus or its antigenicity prepared product; Said influenza virus or its antigenicity prepared product are prepared with oil in water emulsion adjuvant, but said oil in water emulsion adjuvant comprises metabolism oil, sterol (being suitably alpha-tocopherol) and emulsifying agent.Preferably, inoculate and in last season, inoculation was to the experimenter of influenza, carrying out.Usually, inoculate after inoculation for the first time and to carry out at least 6 months, carry out after preferred 8-14 month, more preferably after about 10-12 is individual month, carry out.

Preferably; (a) influenza virus or its antigenicity prepared product and (b) the oil in water emulsion adjuvant purposes in producing immunogenic composition are provided; But said oil in water emulsion adjuvant comprises metabolism oil, sterol (for example alpha-tocopherol) and emulsifying agent; Said immunogenic composition is used to inoculate previous people with influenza virus or its antigenicity prepared product and oil in water emulsion adjuvant inoculation, but wherein said oil in water emulsion adjuvant comprises at least a metabolism oil, and its amount is the 0.5%-20% of cumulative volume; And having an oil droplet, at least 70% (with intensitometer) of oil droplet has and is lower than 1 μ m footpath.

In a further preferred embodiment; The immunogenic composition that is used to inoculate comprises influenza virus or its lytic virus antigenicity prepared product, they be used for inoculating for the first time susceptible poison or the total common CD4 T-cell epitope of its virus antigenicity prepared product or B cell epitope or both altogether.

Aspect the 4th of the present invention; Influenza virus or its anti-prepared product of (a) first influenza strain and (b) purposes of oil in water emulsion adjuvant in producing immunogenic composition are provided; But O/w emulsion comprises metabolism oil, sterol and emulsifying agent; Said immunogenicity is combined in the protective effect of the influenza infection that strain causes to influenza, and said influenza strain is the variant of said influenza strain.In a preferred embodiment, but at least a metabolism of said oil in water emulsion adjuvant oil, its amount is the 0.5%-20% of cumulative volume, and has oil droplet, at least 70% (with intensitometer) have the diameter that is lower than 1 μ m.

On the other hand; A kind of method of replying spoken sarcastically basis or colony (for example excessive risk adult or old people) with the immunogenic composition immunoprophylaxis is provided; The combination of said immunogenicity contain influenza virus or its antigenicity prepared product and as the O/w emulsion of preceding text definition help in another embodiment again; The present invention provides a kind of method that inoculates the previous people who inoculates with sick its virus antigenicity prepared product of influenza and oil in water emulsion adjuvant; Wherein but oil in water emulsion adjuvant comprises metabolism oil, sterol and emulsifying agent, and said method comprises that said people contains adjuvant or do not have the immunogenic composition of adjuvant influenza virus.Aptly, sterol is an alpha-tocopherol.

In a further embodiment; A kind of method is provided: inoculation crowd or individual antagonism one susceptible strain; Then inoculate said people or colony antagonism variant strains of influenza viruses; Includedly give said people: (i) first kind of compositions, it comprises virus or its antigenicity prepared product and oil in water emulsion adjuvant of first strains of influenza viruses, but said O/w emulsion assistant metabolism oil, sterol and emulsifying agent; (ii) second kind of immunogenic composition, the strains of influenza viruses variant of its said first strains of influenza viruses.Aptly, said sterol is educated phenol.

In another embodiment, the present invention provides the purposes of influenza virus or its antigenicity prepared product and oil in water emulsion adjuvant, but said oil in water emulsion adjuvant comprises metabolism oil, sterol and emulsifying agent.Aptly, said sterol is an alpha-tocopherol.

more on the other hand, the present invention provides a kind of method of design flow influenza vaccine, comprising:

1) select to contain the CD4+ epi-position influenza antigens and

2) the said influenza antigens of combination and the as above O/w emulsion of definition can induce enhanced CD4 to reply in said mammal when wherein said vaccine gives in mammal.

Others of the present invention and advantage further describe in following description of Preferred Embodiments.

Description of drawings

Fig. 1: the oil droplet particle size distribution of the SB62 O/w emulsion that detects according to PCS.Figure 1A has shown the granulometry result of the SB62 lot number 1023 that detects with Malvern Zetasizer 3000HS: A=dilution factor 1/10000 (Rec22 to Rec24) (analyzing with Contin and the optical model 1.5/0.01 that is fit to); B=dilution factor 1/20000 (Rec28 to Rec30) (analyzing) with Contin and the optical model 1.5/0.01 that is fit to.Figure 1B has shown the sketch map according to the record 22 (top) of intensity and record 23 (bottoms).

Fig. 2: the sketch map of MPL stock solution preparation.

Fig. 3: the sketch map of AS03+MPL adjuvant preparation.

Fig. 4: Explo Flu-001 clinical experiment.CD4 t cell response (Q1=first quartile, Q3=the 3rd quartile) to the cracking influenza antigens.

Fig. 5: Explo Flu-001 clinical experiment.CD8 t cell response (Q1=first quartile, Q3=the 3rd quartile) to the cracking influenza antigens.

Fig. 6: Explo Flu-001 clinical experiment.At the cross reactivity CD4 T-cell response that is directed against the cracking influenza antigen with Fluarix+AS03 inoculation back.

Fig. 7: Explo Flu-001 clinical experiment.Postvaccinal B cell memory is replied.

Fig. 8: Explo Flu-002 clinical experiment.Inoculating the cd4 t cell that the back cracking resistance separates influenza antigens replys.

Fig. 9: Explo Flu-002 clinical experiment.Anti-HI titre after inoculating.

Figure 10: ferret research I.Temperature monitoring (just exempt from and attack).Figure 10 A just exempts from, and Figure 10 B attacks.

Figure 11: ferret research I.Virus shedding.

Figure 12: ferret research II.Temperature monitoring (just exempt from and attack).Figure 12 A just exempts from, and Figure 12 B attacks.

Figure 13: ferret research II.Virus shedding.

Figure 14: ferret research II.HI titre to H3N2 A/ Panama (vaccine strain) (Figure 14 A) and H3N2A/ Wyoming (attack strain) (Figure 14 B).

Figure 15: mice study.Use full inactivation virus just to exempt from the frequency of CD4 T cell in the mice as C57BI/6 under the situation of stimulator antigen (back 7 days of immunity) again.

Figure 16: mice study.Use full inactivation virus just to exempt from the frequency of CD8 T cell in the mice as C57BI/6 under the situation of stimulator antigen (back 7 days of immunity) again.

Figure 17: mice study.Use the frequency of full inactivation virus as CD4 T cell (top) and cd8 t cell (bottom) in the C57BI/6 mice of just exempting from the allos strain under the situation of stimulator antigen (back 7 days of immunity) again.

Figure 18: people's clinical experiment.The B cell memory that the old people inoculates behind Fluarix, Fluarix+AS03, the Fluarix+AS03+MPL is replied (inoculating the difference between preceding and the inoculation back).

Figure 19: ferret research III.Before attacking with attack after temperature monitoring.

Figure 20: ferret research III.Before attacking with attack after virus shedding.

Figure 21: ferret research III.HI titre to H3N2A/ Wyoming (vaccine strain).

Figure 22: ferret research III.HI titre to H3N2A/ Panama (attack strain).

Figure 23: people's clinical experiment.HI titre (GMT) (persistence) when inoculation back 21 days, 90 days and 1 80 days.

Figure 24: people's clinical experiment.CD4 replys-hybrid antigen (persistence) of all double (producing the cells of at least two kinds of cytokines) check-when inoculation back 21 days, 90 days and 180 days.

Figure 25: people's clinical experiment.Compare with Fluarix and to use the HI titre in the clinical experiment that inoculates of AS03+MPL.

Figure 26: people's clinical experiment.The hybrid antigen in the time of 0 and 21 day of CMI-all double that CD4 replys check-after inoculation.

Figure 27: adopt people's clinical experiment of the AS03+MPL of two concentration.HI titre at 0 and 21 day.

Figure 28: adopt people's clinical experiment of the AS03+MPL of two concentration.Reactionogenicity.

Detailed Description Of The Invention

The inventor has been found that; Reply than what obtain with no adjuvant virus or its antigenicity prepared product; Contain influenza virus and antigenicity prepared product thereof and can reply at the CD4 T-cellullar immunologic response and/or the B cell memory of anti-said antigen of philtrum improvement or antigenic composition, but said oil in water emulsion adjuvant contains metabolism oil, sterol such as alpha-tocopherol and emulsifying agent together with the influenza preparation of oil in water emulsion adjuvant.Require the preparation of protection to be advantageously used in the CD4-T cell response of inducing influenza, this is replied and can detect the influenza epi-position of being offered by MHC II quasi-molecule.The applicant finds now, and the immune system that the said preparation efficient targeting is cell-mediated is so that increase the reactivity (when inoculation and infection) to the influenza strain of homology and drift.

Of the present invention have adjuvant influenza compositions to have several advantages:

1) immunogenicity of improving: they allow the weak immunne response among the old people (more than 50 years old, common over-65s) is returned to visible level in youngster (antibody and/or t cell response);

2) the cross protection pattern of improving: increase the cross-protection of resistance different (drift) influenza strain;

3) they also allow to use the antigen dose of minimizing to obtain similar replying, and have therefore guaranteed the effectiveness that in emergency circumstances (for example is very popular) and increases.

Specifically, according to people's number of subjects purpose evaluation of satisfying the protective effect of influenza dependency, compositions of the present invention can provide better anti-influenza serum protective effect after the immunity again.And the compositions that the present invention uses can also be induced following trend: than no adjunvant composition, inoculate B cell memory behind the people experimenter first time and reply higherly, and the humoral response after inoculating is higher.

The inventor also can show; Than the individuality that obtains the protection antibody level with no adjunvant composition, require the adjunvant composition that has of protection can in more individuality, induce and keep resisting the protection antibody level (for example referring to table 43) of the whole 3 kinds of strains that exist in the vaccine.

Therefore, in another embodiment again, require the compositions of protection can guarantee the persistence immunne response of influenza relevant disease.Specifically, said persistence refer to that the HI antibody mediated immunity is replied can be after inoculation at least 3 months, preferably satisfy administrative standard after at least 6 months.Specifically; Require the compositions of protection to induce protection antibody level in the individuality＞70% after at least 3 months, aptly in the individuality＞80% or aptly in the individuality＞90% at least a influenza strain that exists in the vaccine, preferred institute toxic strain.One concrete aspect, after inoculation, obtained at least 6 months to exist in the anti-vaccine combination at least a, aptly two kinds or all strains＞90% protection antibody level.

Strains of influenza viruses and antigen

Influenza virus or its antigenicity prepared product can be cracking influenza virus or its lytic virus antigenicity prepared product used according to the present invention.In an alternate embodiment, the influenza virus prepared product can comprise the inactivation influenza antigens of another type, the for example HA of inactivation totivirus or purification and NA (subunit vaccine), or influenza virus particles.In another embodiment again, influenza virus can be attenuated live influenza virus prepared product.

Aptly, cracking influenza virus or its lytic virus antigenicity prepared product are inactivation virus prepared product used according to the present invention, and wherein virion destroys with detergent or other reagent of lipin dissolving peplos.Aptly; Lytic virus or its lytic virus antigenicity prepared product are prepared as follows: with the full influenza virus of organic solvent or the detergent fragmentation infectivity or the inactivation of concentration of ordinary dissolution, remove whole or most of lytic agents and some or most of viral lipid material subsequently.Said its lytic virus antigenicity prepared product refers to possibly stand purification to a certain degree with respect to lytic virus and has kept the lytic virus prepared product of most of antigenic characteristic of lytic virus component.For example, when in ovum, producing, lytic virus can be by removing in the ovum foreign protein, and when perhaps in cell culture, producing, lytic virus can be by removing in the host cell impurity.Lytic virus antigenicity prepared product can comprise the lytic virus antigenicity component of more than one Strain.The vaccine that contains the vaccine (being called ' cracking influenza vaccines ') of lytic virus or contain lytic virus antigenicity prepared product generally comprises remaining stromatin and nucleoprotein, comprises lipid and membrane envelope albumen sometimes.This split-virus vaccine comprises major part or whole virus structural proteins usually, but the ratio that not necessarily in totivirus, exists with them is identical.

Perhaps, influenza virus can be the whole virus vaccine form.For pandemicity, it can show the advantage that surmounts split-virus vaccine, because it has avoided the relevant uncertainty that whether can successfully produce split-virus vaccine to new strains of influenza viruses.For some strain, the conventional detergent that is used to produce lytic virus can damage virus, and makes it useless.Although often might use different detergents and/or develop different split vaccine production methods, this can be consuming time, maybe be unavailable under pandemicity.The totivirus method also has the production of vaccine ability bigger than lytic virus except largely the definitiveness, because in the essential additional purification step of the suitable split vaccine of preparation, quite a large amount of antigen losses is arranged.

In another embodiment, the influenza virus prepared product is the subunit influenza vaccines form of purification.The subunit influenza vaccines generally comprise two kinds of main envelope protein HA and NA, possibly have the additional advantage that surmounts totivirus body vaccine, because their general reactionless originality, especially in the inoculator of youth.But the subunit vaccine recombinant production perhaps can be by disruptive virion purification.

In another embodiment, the influenza virus prepared product is the virion form.Virion is spheric unilamellar vesicle, and it has kept the functional viral envelope glycoprotein HA and the NA of true conformation, inserts in the phospholipid bilayer film of virion.

That said influenza virus or its antigenicity prepared product can be the ovum source or the tissue culture source.

For example, influenza antigen of the present invention or its antigenicity prepared product can derive from conventional embryonated egg method, and this method is cultivated influenza virus in ovum, and the allantoic fluid of purification collection.Ovum can be accumulated at short notice in a large number.Perhaps, they can derive from the new production process of any using-system culture, to cultivate virus or express recombinant influenza virus surface antigen.Be suitable for cultivating viral cellular matrix and comprise for example MDCK; For example MDCK or MDCK clone cell, MDCK like cell; MK cells, for example the AGMK cell comprises the Vero cell; Suitable pig cell system, or be suitable for producing any other mammalian cell types of the influenza virus of vaccine use.Suitable cellular matrix also comprises people's cell, for example the MRC-5 cell.Suitable cellular matrix is not limited to cell line; For example naive cell like little CEF, also comprises avian cell lines.

Influenza antigen or its antigenicity prepared product can be through any productions in numerous business methods, for example in the middle cracking influenza virus method of describing of patent No. DD 300833 and DD 211444 (being attached among this paper by reference).The cracking influenza virus uses solvent/detergent treatment to produce traditionally, for example TRI N BUTYL PHOSPHATE, perhaps diethyl ether combination Tween ^TM(being called " Tween-ether " cracking), this method is still used in some manufacturer.Other decomposition agent that uses at present comprises detergent or proteolytic enzyme or bile salts, the NaTDC of for example in patent No. DD155875 (being attached among this paper by reference), describing.The detergent that can be used as decomposition agent comprises cationic detergent, for example cetrimide (CTAB); Other ion detergent, for example lauryl sulfate, taurodeoxycholate; Or nonionic detergent, as above-mentioned a kind of, comprise Triton X-100 (for example at Lina etc., 2000, Biologicals 28, in the method that 95-103 describes) and Triton N-101; The perhaps combination of any two or more detergents.

The method for preparing of split vaccine can comprise numerous different filtrations and/or other separating step; Ultra centrifugal, ultrafiltration, band centrifugation and chromatography (for example ion exchange) step like various combinations; And optional for example employing heat, formaldehyde or β-propanoic acid lactone or ultraviolet deactivation step, it can carry out before or after cracking.Cracking process can be in batches, continuously or semi-continuous process carry out.The preferred cracking and the purification process that are used for the cracking immunogenic composition are described in WO02/097072.

Preferred cracking influenza vaccines antigen preparation thing of the present invention comprises the Tween 80 and/or the Triton X-100 of the remaining residual volume of production process, but these materials can add or adjust its concentration after the cracking antigen preparation.Preferably, Tween 80 exists with Triton X-100.The preferable range of the final concentration of these nonionic surfactant is in the vaccine dose: Tween 80:0.01-1%, more preferably from about 0.1% (volume)

Triton X-100:0.001-0.1% (weight/volume), more preferably 0.005-0.02% (weight/volume).

In a specific embodiments, the final concentration of Tween 80 is in the scope of 0.045%-0.09% (weight/volume).In another embodiment; Antigen provides as 2 times of spissated mixture; Its Tween that has 80 concentration must be used 2 times of adjuvant (or in control formulation, using buffer) dilutions when final preparation in the scope of 0.045%-0.2% (weight/volume).

In another embodiment, the final concentration of Triton X-100 is in the scope of 0.005%-0.017% (weight/volume).In another embodiment; Antigen provides as 2 times of spissated mixture; The Triton X-100 concentration that it has must be used 2 times of adjuvant (or in control formulation, using buffer) dilutions when final preparation in the scope of 0.005%-0.034% (weight/volume).

Preferably, the influenza virus prepared product prepares in the presence of low-level thimerosal, or preferably prepares under the situation of thimerosal not having.Preferably, the influenza prepared product of acquisition is stable under the situation that does not have the organic mercury antiseptic, and specifically, said prepared product does not contain remaining thimerosal.Specifically, the influenza virus prepared product is included under the situation that does not have thimerosal or stable hemagglutinin antigen under the situation of low-level thimerosal (being generally 5 μ g/ml or following).Specifically, the stable alpha tocopherol derivant such as the alpha tocopherol succinate (being also referred to as vitamin e succinate, i.e. VES) of utilizing of influenza B strain implemented.This prepared product and preparation method thereof is disclosed in WO 02/097072.

Preferred compositions contains the lytic virus isoantigen of 3 kinds of inactivations, and they recommend the strain preparation by the WHO in suitable influenza season.

Preferably, influenza virus or its antigenicity prepared product and oil in water emulsion adjuvant are included in the same container.This is called as ' single bottle of method '.Preferably, said bottle is a pre-filled syringe.In an alternate embodiment, influenza virus or its antigenicity prepared product and oil in water emulsion adjuvant are included in independent container or the bottle, and give to before the experimenter soon or mix when giving to the experimenter.This is called as ' two bottles of methods '.As an example; When vaccine is 2 component vaccines of 0.7ml accumulated dose volume; Spissated antigen (for example spissated trivalent inactivation lytic virus isoantigen) is present in 1 bottle (335 μ l) (antigen container), and pre-filled syringe contains adjuvant (360 μ l) (adjuvant container).In when injection, the content that uses the syringe that contains adjuvant will contain in the bottle of spissated trivalent inactivation lytic virus isoantigen takes out, and follows slight mixing syringe.Before injection, the pin of use is with the replacement of intramuscular pin, and volume is adjusted to 530 μ l.The potion reprovision has adjuvant influenza vaccines material standed for to be equivalent to 530 μ l.

Oil in water emulsion adjuvant

Adjunvant composition of the present invention comprises oil in water emulsion adjuvant, but preferably said emulsion comprises metabolism oil, and its amount is the 0.5%-20% of cumulative volume, and has oil droplet, and at least 70% (with intensitometer) of oil droplet has the diameter that is lower than 1 μ m.

Give in order to make any oil-in-water compositions be suitable for the people, but the oil phase of emulsion system must comprise metabolism oil.But the implication of term metabolism oil is well-known in this area.But metabolism may be defined as ' can be transformed through metabolism ' (Dorland ' s Illustrated Medical Dictionary, W.B.Sanders Company, the 25th edition (1974)).Said oil can be any vegetable oil, fish oil, animal oil or artificial oil, and they are nontoxic to the receiver, can be transformed through metabolism.Nut, seed and corn are the vegetable oil sources of using always.Artificial oil also is a part of the present invention; Can comprise the commodity carburetion, for example NEOBEE

etc.But specially suitable metabolism oil is zamene.Zamene (2,6,10,15,19; 23-hexamethyl-2,6,10,14,18; 22-tetracosa carbon six alkene) being a kind of unsaturated oils, being present in a large number in the shark liver oil, be present on a small quantity in olive oil, wheat germ oil, Testa oryzae oil and the yeast, is to be used for preferred especially oil of the present invention.But zamene is a metabolism oil because of the following fact: it is the biosynthetic intermediate of cholesterol (Merck index, the 10th edition, an entry number 8619).

O/w emulsion is from well-known in this area, shown to can be used as adjunvant composition (EP 399843; WO 95/17210).

Aptly, but metabolism oil exist with the amount of 0.5% to 20% (final concentration) of immunogenic composition cumulative volume, preferably 1.0% to 10% amount with cumulative volume exists, preferably 2.0% to 6.0% amount with cumulative volume exists.

In a specific embodiments, but metabolism oil exists with about final quantity of 0.5%, 1%, 3.5% or 5% of immunogenic composition cumulative volume.In another embodiment, but metabolism oil exist with about final quantity of 0.5%, 1%, 3.57% or 5% of immunogenic composition cumulative volume.

Preferably, O/w emulsion of the present invention system has the little oil droplet granularity of sub-micrometer range.Aptly, the diameter of oil droplet is in the scope of 120-750nm, and more preferably diameter is in the scope of 120-600nm.Most preferably, O/w emulsion comprises oil droplet, and the diameter of its at least 70% (with intensitometer) is lower than 500nm, and more preferably the diameter of at least 80% (with intensitometer) is lower than 300nm, and more preferably the diameter of at least 90% (with intensitometer) is in the scope of 120-200nm.

Oil droplet granularity of the present invention, promptly diameter provides with intensity.There is several method to measure droplet diameter size with intensitometer.Intensity uses particle size measuring instrument to detect, and detects through the dynamic light scattering such as Malvern Zetasizer 4000 or preferred Malvern Zetasizer 3000HS aptly.Detailed method provides at example II .2.First probability is to measure z average diameter ZAD through dynamic light scattering (PCS-photon correlation spectroscopy method); The extra polydispersity index (PDI) that provided of this method, ZAD and PDI this two all use the cumulant algorithm computation.These values do not need the information of particle refractive index.Second method is to measure full particle size distribution through another kind of algorithm Contin or NNLS or automatization " Malvern " method (default algorithm that is provided by particle size measuring instrument) to calculate droplet diameter.Mostly the particle refractive index owing to the complex combination thing is unknown under the situation, thus can only consider intensity distributions, if needs are arranged, by this acquisition average strength that distributes.

O/w emulsion comprises sterol.Sterol is well-known in this area, and for example cholesterol is known, for example is disclosed in Merck Index, the 11st edition, 341 pages, is naturally occurring sterol in the Animal fat.Other suitable sterol comprises cupreol, stigmasterol, ergosterol, alpha-tocopherol and ergocalciferol.Said sterol exists with the amount of 0.01% to 20% (weight/volume) of immunogenic composition cumulative volume aptly, and preferably the amount with 0.1% to 5% (weight/volume) exists.Preferably; When sterol is cholesterol; Its amount with 0.02% to 0.2% (weight/volume) of immunogenic composition cumulative volume exists; More preferably the amount with 0.02% (weight/volume) of 0.5ml vaccine dose volume exists, and perhaps the amount with 0.07% (weight/volume) of 0.5ml vaccine dose volume exists, and perhaps the amount with 0.1% (weight/volume) of 0.7ml vaccine dose volume exists.

Aptly, sterol is the alpha-tocopherol or derivatives thereof, for example the alpha-tocofecol succinic acid ester.Preferably; Alpha-tocopherol exists with the amount of 0.2% to 5.0% (volume) of immunogenic composition cumulative volume; More preferably the amount with 2.5% (volume) of 0.5ml vaccine dose volume exists; Perhaps the amount with 0.5% (volume) of 0.5ml vaccine dose volume exists, and perhaps exists with the 1.7-1.9% (volume) of 0.7ml vaccine dose volume, preferred 1.8% amount.For the purpose of distinct, the concentration that provides with volume can change the concentration of representing with weight/volume into through using following conversion coefficient: 5% (volume) alpha-tocopherol concentration equals 4.8% (weight/volume) alpha-tocopherol concentration.

O/w emulsion also can contain emulsifying agent.Amount that can 0.01-5.0% with immunogenicity composition weight meter (w/w) emulsifying agent exists, and (w/w) exists with the amount of 0.1-2.0% preferably by weight.Weight preferred concentrations in total compsn is 0.5-1.5% (w/w).

Emulsifying agent can be suitably Tween-81 (Tween 80).In a specific embodiments, 0.5ml vaccine dose volume contains 1% (w/w) Tween80, and 0.7ml vaccine dose volume contains 0.7% (w/w) Tween 80.In another embodiment, the concentration of Tween 80 is 0.2% (w/w).

Oil in water emulsion adjuvant can be used for other adjuvant or immunostimulant, but therefore important embodiment of the present invention is the oil-in-water preparation that contains zamene or another kind metabolism oil, alpha-tocopherol and tween 80.O/w emulsion also can comprise span 85 and/or lecithin.Typically, oil-in-water comprises the zamene of the 2-10% of immunogenic composition cumulative volume, the alpha-tocopherol of 2-10% and the Tween of 0.3-3% 80, and can be according to the method production of describing among the WO 95/17210.Preferably, zamene: the ratio of alpha-tocopherol is equal to or less than 1, because it provides more stable emulsion.Span 85 (Tween-85) also can be for example exists with 1% level.

The immunogenicity characteristic that is used for the immunogenic composition that the present invention inoculates for the first time

In the present invention; Than the CD4 T-cellullar immunologic response that the corresponding compositions (this paper is called ' common compositions ') with no adjuvant (promptly not containing any external source adjuvant) obtains, the influenza compositions can be induced the CD4 T-cellullar immunologic response of the improvement of anti-at least a composition antigen or antigenic composition.

Said ' the CD4 T-cellullar immunologic response of improvement ' refers to that the CD4 that after adjuvant immunity originality compositions is arranged, in human patients, obtains replys and is higher than the immunne response that obtains behind the same combination that is not having adjuvant.For example; With do not have after immunogenic composition adjuvant, that contain influenza virus or its antigenicity prepared product inductive replying and compare; When containing influenza virus or its antigenicity prepared product, in human patients, obtain higher CD4 T-cell response, but said oil in water emulsion adjuvant comprises metabolism oil, sterol such as alpha-tocopherol and emulsifying agent together with the immunogenic composition of oil in water emulsion adjuvant.This preparation will be advantageously used in the CD4-T cell response of inducing resisiting influenza virus, and this is replied and can detect the influenza epi-position of being offered by MHC II quasi-molecule.

Preferably, by being used for of the present inventionly having the inductive said immunne response of adjuvant cracking influenza virus compositions to be higher than with the inductive immunne response of any other no adjuvant normal flow influenza vaccine (for example subunit influenza vaccines or full influenza virus vaccine).

Particularly but not exclusively, acquisition among said ' the CD4 T-cellullar immunologic response of improvement ' patient's (being seronegative patient promptly) of on immunology, just exempting to said influenza virus or antigen.This seronegativity possibly be never in the face of this virus or antigenic said patient's (so-called ' originally ' patient) result, is the said patient's that when meeting with, can not reply said antigen result perhaps substitutingly.Preferably; The CD4 T-cellullar immunologic response of said improvement obtains in the impaired experimenter of immunne response; The impaired experimenter of said immunne response for example is the old people; Be generally 65 years old or more than, or have the excessive risk medical conditions less than 65 years old adult (' excessive risk adult '), or the child below 2 years old.

The CD4 T-cellullar immunologic response that improves can be estimated through detecting the cell number that produces any following cytokine:

Produce the cell of at least two kinds of different cytokines (CD40L, IL-2, IFN γ, TNF α)

Produce the cell of CD40L and another kind of cytokine (IL-2, TNF α, IFN γ) at least

Produce the cell of IL-2 and another kind of cytokine (CD40L, TNF α, IFN γ) at least

Produce the cell of IFN γ and another kind of cytokine (IL-2, TNF α, CD40L) at least

Produce the cell of TNF α and another kind of cytokine (IL-2, CD40L, IFN γ) at least

When with do not have adjunvant composition and compare the cell concentration that produces any above cytokine after adjunvant composition is being arranged when higher, with the CD4 T-cellullar immunologic response that has improvement.Usually, at least a, preferred two kinds in 5 kinds of situations mentioning of preceding text will be implemented.In a specific embodiments, compare with no adjuvant group, there is in the adjuvant group cell of producing whole 4 kinds of cytokines to exist with a large amount more.

In fact can give the back at single by the CD4 T-cellullar immunologic response that the improvement that adjuvant influenza compositions gives arranged of the present invention obtains.For example earthshaking meaning will be arranged at the outburst situation of the fast development agent method that places an order.In some cases, especially for elderly population, perhaps for the child (below 9 years old) of the inoculation of influenza first, give two doses identical this in season compositions possibly be useful.Second dose of said same combination (still being counted as ' compositions that is used for inoculating first ') can give in the process that initial immunne response is developing, and has time enough at interval.Usually, second dose of compositions gives several weeks or about 1 month (for example 2 weeks, 3 weeks, 4 weeks, 5 week or 6 weeks) after first dose, to help to trigger the immune system in no response or the low responder.

In a specific embodiments; Reply with inductive B memory cell in no adjunvant composition immune body and to compare, give said immunogenic composition and in the patient that adjuvant immunity originality compositions is arranged, alternatively or extraly induce the B-memory cell of improvement to reply.The B-memory cell that improves is replied to mean and when meeting with antigen, can be divided into the lymphocytic frequency of the plasmacytic peripheral blood B of antibody-secreting property and increase, and this stimulates and detect (referring to the embodiment part, for example the method for Elispot B cell memory) through vitro differentiation.

In another specific embodiments again, with the compositions inoculation CD8 is replied no detectable influence with the inoculation first that adjuvant is arranged.

The applicant is surprisingly found out that; Contain with the influenza virus of oil in water emulsion adjuvant preparation or compositions effective promotion t cell response in the immunocompromised host crowd of its antigenicity prepared product, but said oil in water emulsion adjuvant comprises metabolism oil, sterol such as alpha tocopherol and emulsifying agent.The applicant shows; According in elderly population, carrying out the relatedness evaluation that influenza inoculates back influenza vaccines protective effect; Compare with the serum protection that provides with no adjuvant influenza vaccinations, give single agent such as of the present invention the inoculation first can provide better serum protection with immunogenic composition.Than the immunne response that no adjuvant formulation obtains, require the adjuvant formulation that has of protection can also induce the CD4 T-cellullar immunologic response of the improvement of resisiting influenza virus.The responsiveness that this observed result increases in the time of maybe be with inoculation or in the face of the infection of influenza antigens property contact is relevant.And it also maybe be relevant with cross reactivity, and the ability that promptly strain is replied to the variant influenza is higher.Replying of this improvement maybe be useful especially in the crowd of immunocompromised host such as elderly population (65 years old with more than) and especially excessive risk elderly population.This can cause reducing whole M & M, and prevents promptly being admitted to hospital of pneumonia and other influenza appearance disease.This also can be useful to infant population (below 5 years old, preferred below 2 years old).And, and to compare with inductive the replying of no adjuvant formulation, it allows the more persistent CD4 t cell response of induction time, for example still has 1 year after the inoculation first.

Preferably, CD4 T-cellullar immunologic response for example at the CD4 T-cellullar immunologic response of the improvement of just not exempting to obtain among the experimenter, comprises that inducing cross reactivity CD4 T to assist replys.Specifically, the amount of cross reactivity CD4 T cell increases.Said ' cross reactivity ' CD4 replys the total epi-position that refers between the strain of CD4 T-cell-targeting influenza.

Usually, available influenza vaccines are only effectively resisted the influenza infection strain of the hemagglutinin with similar antigenicity characteristic.When infecting minor variations (the for example accumulation of point mutation or point mutation that (circulation) property influenza virus has experienced surface glycoprotein (hemagglutinin specifically); Cause amino acid change) time (antigenic drift variant Strain); Vaccine still can provide some protective effects; But it possibly only provide limited protective effect, because the new variant that produces possibly escaped previous influenza infection or inoculate inductive immunity.Antigenic drift be the annual popular reason that takes place during being very popular for twice (Wiley&Skehel, 1987, Ann.Rev.Biochem.56,365-394).Inducing to the present composition of cross reactivity CD4 T cell provides extra advantage; Because it also can provide cross-protection; In other words be the protective effect that anti-allos infects; Said allos infects the infection that is promptly caused by the circulation strains of influenza viruses, and this circulation strains of influenza viruses is the variant (variant for example drifts about) of the strains of influenza viruses that comprises in the immunogenic composition.This possibly be favourable when the circulation strain is difficult in ovum, breed or is difficult in tissue culture, produce, and makes the drift strain as alternative work strain.This accepts for the first time with some months or 1 year the experimenter at interval also maybe be favourable when inoculating for the second time, is used for the influenza strain of the immunogenic composition of immunity for the second time and is the drift variant strain of the strain that the compositions that is used for inoculating first uses.

Detection with cross reactivity CD4 T-cell after the influenza vaccinations

After giving classical trivalent influenza vaccines (3 week); The frequency of the peripheral blood CD4 T-cell that antigenicity strain prepared product (totivirus or cracking antigen) is replied significantly increases, a kind of antigen that exists in said antigenicity strain prepared product and the vaccine (H3N2:A/ Panama/2007/99; H1N1:A/ New Caledonia/20/99; B:B/ Shandong/7/97) (referring to EXAMPLE III) homology.If the influenza strain with being categorized as drift strain (H3N2:A/ Sydney/5/97, H1N1:A/ Beijing/262/95, B:B/ Pyrusussuriensis/166/98) stimulates peripheral blood CD4 T-cell again, then can see sizable frequency increases.

On the contrary, if be categorized as drift strain (H2N2:A/ Singapore/1/57 with the expert of the art; H9N2:A/ Hong Kong/1073/99) influenza strain stimulates peripheral blood CD4 T-cell again, then after inoculation, does not have observable increase.

The CD4 T-cell of influenza strain that can discern homology and drift simultaneously called after " cross reactivity " in presents.Require the adjuvant influenza compositions that has of protection purposes in the present invention can demonstrate the cross reactivity of different hypotype, because there is the observable cross reactivity of anti-drift influenza strain.

Consistent with above observed result, identify different influenza strains total CD4T-cell epitope (Gelder C etc., 1998, Int Immunol.10 (2): 211-22 at philtrum; Gelder CM etc., 1996 J Virol.70 (7): 4787-90; Gelder CM etc., 1995 J Virol.199569 (12): 7497-506).

In a specific embodiments; There is adjunvant composition that additional benefit can be provided; These interests provide the better protective effect of anti-circulation strain; Said circulation strain has experienced the about-face (for example gene recombinaton, the for example gene recombinaton between two different plant species) (antigenic drift) of hemagglutinin, and present available vaccine is invalid to it.

Other adjuvant

Compositions can comprise other adjuvant, specifically is TRL-4 part adjuvant, is suitably the non-toxic derivant of lipid A.Suitable TRL-4 part is 3 to take off-O-acidylate monophosphoryl lipid A (3D-MPL).Other suitable TLR-4 part is the F albumen of lipopolysaccharide (LPS) and derivant, MDP (muramyldipeptide) and RSV.

In one embodiment, said compositions can comprise Toll appearance receptor (TLR) 4 parts in addition, the non-toxic derivant of lipid A for example, monophosphoryl lipid A specifically, the 3-deacylated tRNA monophosphoryl lipid A (3D-MPL) of perhaps more specifically saying so.

3D-MPL sells (this paper is called MPL) by Corixa company with trade mark MPL , the CD4+T cell response that main promotion has IFN γ (Th1) phenotype.It can be according to disclosed method production among GB 2 220211 A.Chemically, it is the mixture with 3-deacylated tRNA monophosphoryl lipid A of 3,4,5 or 6 acidylate chains.Preferably, in the present composition, use granule 3D-MPL.The granular size that granule 3D-MPL has makes it can pass through 0.22 μ m filter aseptic filtration.This preparation is described in WO94/21292 and example II.

The 3D-MPL for example amount of each composition dosage 1-100 μ g (weight/volume) uses, and preferably uses with the amount of each composition dosage 10-50 μ g (weight/volume).The 3D-MPL of appropriate amount for example is any among each composition dosage 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 or the 50 μ g (weight/volume).More preferably, the amount of 3D-MPL is in the scope of each composition dosage 25-75 μ g (weight/volume).Usually, composition dosage will be at about 0.5ml to the scope of about 1ml.Typical vaccine dose is 0.5ml, 0.6ml, 0.7ml, 0.8ml, 0.9ml or 1ml.In a preferred embodiment, every ml vaccine combination comprises the final concentration of 50 μ g3D-MPL, or the final concentration of every 0.5ml vaccine dose 25 μ g 3D-MPL.In other preferred embodiment, every ml vaccine combination comprises the final concentration of 35.7 μ g or 71.4 μ g3D-MPL.Specifically, 0.5ml vaccine dose volume contains 25 μ g or 50 μ g 3D-MPL for every dose.

The dosage of MPL can strengthen to antigenic immunne response at philtrum aptly.Specifically, than no adjunvant composition, or than the compositions of making adjuvant with another kind of MPL amount, suitable MPL amount is improved the immune efficacy of said compositions, and the reactionogenicity pattern can be accepted simultaneously.

Synthetic lipid A derivant is known, and some of them are described to the TLR-4 agonist, and it includes but not limited to:

OM174 (2-deoxidation-6-o-[2-deoxidation-2-[(R)-3-dodecane acyl-oxygen base four-capryl is amino]-4-o-phosphono-β-D-glycopyranosyl]-2-[(R)-3-hydroxyl four capryl are amino]-α-D-glycopyranosyl dihydrogen phosphoric acid ester), (WO 95/14026)

OM 294 DP (3S, 9R)-3-[(R)-dodecane acyl-oxygen base four capryl are amino]-4-oxo-5-azepine-9 (R)-[(R)-3-hydroxyl four capryl are amino] last of the ten Heavenly stems-1,10-glycol, 1, two (dihydrogen phosphoric acid ester) (WO99/64301 and the WO 00/0462) of 10-

OM 197 MP-Ac DP (3S-, 9R)-3-[(R)-dodecane acyl-oxygen four capryl are amino]-4-oxo-5-azepine-9-[(R)-3-hydroxyl four capryl are amino] last of the ten Heavenly stems-1,10-glycol, 1-dihydrogen phosphoric acid ester 10-(6-aminocaprolc acid) (WO 01/46127)

Other suitable TLR-4 part for example is the F albumen of lipopolysaccharide and derivant thereof, MDP (muramyldipeptide) and respiratory syncytial virus.

Suitable immunostimulant is Quil A and derivant thereof to be used for another kind of the present invention.Quil A is the saponin prepared product that separates the Kui Laya genus Quillaia saponaria (Quilaja Saponaria Molina) from South America; At first (" Saponin adjuvants " described in 1974 by Dalsgaard etc.; Archiv.f ü r die gesamte Virusforschung, 44 volumes, Springer Verlag; Berlin, 243-254 page or leaf) has adjuvanticity.Separated the purification fragment of Quil A through HPLC, it has kept adjuvanticity, not the toxicity (EP 0 362 278) relevant with Quil A, for example QS7 and QS21 (being also referred to as QA7 and QA21).QS-21 is the natural saponin that a kind of Kui Laya of deriving from belongs to Quillaia saponaria (Quilaja Saponaria Molina) bark, and it induces CD8+ cytotoxic T cell (CTL), Th1 cell and main IgG2a antibody response, is preferred saponin within the scope of the present invention.

Described the concrete preparation of QS21 already, they are preferred especially, and these preparations also comprise sterol (WO96/33739).The saponin that constitutes the present invention's part can be O/w emulsion form (WO 95/17210).

The compositions that inoculates and be used to inoculate (reinforcement compositions)

One aspect of the present invention provides the purposes of influenza antigens in influenza immunogen property composition production; Said influenza immunogen property compositions is used to inoculate previous in order to the influenza virus of oil in water emulsion adjuvant preparation or the people of its antigenicity prepared product or the inoculation of its variant, but said oil in water emulsion adjuvant comprises metabolism oil, sterol such as α-Vitamin E and emulsifying agent.

Usually, be inoculated in first after the inoculation and carried out at least 6 months, carry out after preferred 8-14 month, more preferably from about carry out after 10-12 month.

The immunogenic composition that is used to inoculate (reinforcement compositions) can comprise the antigen preparation thing of any kind, itself or inactivation, or attenuation is active.It can comprise the antigen preparation thing of same type, i.e. HA and NA (subunit) vaccine or the virion of cracking influenza virus or its cracking influenza antigen property prepared product, totivirus body, purification are as the immunogenic composition of inoculating usefulness first.Perhaps; Strengthen compositions and can comprise the influenza antigen of the inoculation another kind of type outside the influenza antigens type of using, i.e. HA and NA (subunit) vaccine or the virion of cracking influenza virus or its cracking influenza antigen property prepared product, totivirus body, purification first.Strengthening compositions can have adjuvant or not have adjuvant.The reinforcement compositions of no adjuvant can be the Fluarix that intramuscular gives ^TM/ α-Rix

/ Influsplit

Said preparation comprises the inactivation lytic virus isoantigen that 3 kinds of suitable influenza season strains of being recommended by WHO prepare.

Therefore; In a preferred embodiment; The present invention provides (a) influenza virus or its antigenicity prepared product and (b) purposes of oil in water emulsion adjuvant in producing immunogenic composition; Said immunogenic composition is used to inoculate previous people with influenza virus or its antigenicity prepared product and oil in water emulsion adjuvant inoculation, but said oil in water emulsion adjuvant comprises metabolism oil, sterol and emulsifying agent.But said oil in water emulsion adjuvant preferably comprises at least a metabolism oil, and its amount is the 0.5%-20% of cumulative volume, and has oil droplet, and at least 70% (with intensitometer) of oil droplet has the diameter that is lower than 1 μ m.Aptly, said sterol is an alpha-tocopherol.

In a specific embodiments; The immunogenic composition that is used to inoculate (hereinafter is also referred to as ' reinforcement compositions ') comprises influenza virus or its antigenicity prepared product, they and influenza virus that is used for inoculating first or the total common CD4 T-cell epitope of its antigenicity prepared product.Common CD4 T-cell epitope means can be by the different antigenic peptide/sequence/epi-position of identical cd4 cell identification (referring to the epi-position of describing below for example: Gelder C etc., 1998, IntImmunol.10 (2): 211-22; Gelder CM etc., 1996 J Virol.70 (7): 4787-90; Gelder CM etc., 1995 J Virol.1995 69 (12): 7497-506).

In a preferred embodiment, the influenza strain can be relevant with the outburst of being very popular, or have and the relevant potentiality of outburst that are very popular.Specifically, when vaccine was polyvalent vaccine such as bivalent vaccine or trivalent vaccine, at least a strain was relevant with the outburst of being very popular, and perhaps had and the relevant potentiality of outburst that are very popular.Suitable strain is but is not limited to: H5N1, H9N2, H7N7, H2N2 and H1N1.

In another aspect of the present invention; Influenza virus or its antigenicity prepared product of (c) first influenza strain and (d) purposes of oil in water emulsion adjuvant in producing immunogenic composition are provided; But said oil in water emulsion adjuvant comprises metabolism oil, sterol and emulsifying agent; Said immunogenic composition is used for resisting the protective effect of the influenza infection that is caused by the influenza strain, and said influenza strain is the variant of the said first influenza strain.But said oil in water emulsion adjuvant preferably comprises at least a metabolism oil, and its amount is the 0.5%-20% of cumulative volume, and has oil droplet, and at least 70% (with intensitometer) of oil droplet has the diameter that is lower than 1 μ m.Aptly, said sterol is an alpha-tocopherol.

Usually, strengthen compositions and give in next influenza season in use, for example after first immunogenic composition, gave in about 1 year.Strengthen compositions and also give (the 4th time for the third time,, the 5th time inoculation etc.) subsequently every year.Strengthening compositions can be identical with the compositions that is used for inoculating first.Aptly, strengthen compositions and comprise influenza virus or its antigenicity prepared product, it is the variant strain of the influenza virus that is used for inoculating first.Specifically, strains of influenza viruses or its antigenicity prepared product are selected according to the reference material of World Health Organization's issue, make them be suitable for inoculating a year circulation influenza strain.

Influenza antigens that is used to inoculate or antigenic composition preferably comprise aforesaid aptly adjuvant or O/w emulsion.Said adjuvant can be oil in water emulsion adjuvant as indicated above, and it is preferred, comprises extra adjuvant alternatively, and for example the TLR-4 part like 3D-MPL or saponin, perhaps can be another kind of suitable adjuvant, for example aluminum or aluminum substitute, for example polyphosphazene.

Preferably; Reply than inoculate the inductive equity in back for the first time with no adjuvant influenza virus or its antigenicity prepared product; Inoculate and induce following any, preferred two or all: (i) CD4 of the improvement of resisiting influenza virus or its antigenicity prepared product replys, or the B cell memory that (ii) the improves humoral response of replying or (iii) improving.Preferably, have adjuvant influenza virus or its antigenicity prepared product to inoculate the inductive immunne response in back to be higher than and to inoculate back inductive correspondence with no adjunvant composition and reply with this paper definition.Preferably; In with the crowd who has adjuvant influenza virus compositions, preferred cracking influenza virus compositions to inoculate first, inoculate the inductive immunne response in back with no adjuvant influenza virus, preferred cracked influenza virus and be higher than with no adjuvant influenza virus compositions, preferably the correspondence among the crowd that inoculates first of cracking influenza virus compositions is replied.

The applicant shows; With contain influenza virus and as the reinforcement compositions of the oil in water emulsion adjuvant that defines of this paper inoculate the experimenter; The antibody titer that demonstrates is higher than with no adjunvant composition to be inoculated and with the respective value among the crowd of no adjunvant composition reinforcement first, but wherein said oil in water emulsion adjuvant comprises that metabolism is oily, sterol such as alpha tocopherol and emulsifying agent.Adjuvant strengthens to the effectiveness of the antibody response that inoculates known to the influenza virus inoculation or infect to have among the low old people who replys and be even more important.With regard to the CD4 T-cell response after improvement inoculates, it also is significant that the relevant interests of adjunvant composition are arranged.

Than the protective effect that control vaccine is given, the better cross response property that has adjunvant composition can induce anti-drift strain (the influenza strain in next influenza season) of the present invention.The persistency that said cross response sex expression goes out is higher than the persistency that obtains with no adjuvant formulation.

In embodiment 3, provide clinical before data for example shown the anti-special-shaped influenza infection of the present composition and the protective capability of the disease confirmed according to the body temperature reading.The clinical experiment data that in inoculating research, obtain are suitable for identical conclusion.

Strains of influenza viruses and antigen thereof

Said influenza virus or its antigenicity prepared product are suitably monovalent or polyvalent, for example bivalence or trivalent or tetravalence.Preferably, influenza virus or its antigenicity prepared product are tervalent or quaternary, have the antigen from 3 kinds of different influenza strains.

Perhaps, at least a strain is relevant with the outburst of being very popular, and perhaps has and the relevant potentiality of outburst that are very popular.

As a setting, in the time period between being very popular, with the relevant transmission of influenza virus of pandemic influenza virus before.Virus is propagated between the people with the immunity level different with the life early infection.In the time period of common 2-3, this propagation promotes changing to such an extent that be enough in general groups, cause the selection of epiphytotics new strain again; This process is called ' antigenic drift '.' drift variant ' all had Different Effects in different communities, area, country or continent in arbitrary year, although its general impacts often are similar in several years.In other words, when the crowd occurring it is not had the new influenza virus of immunity, flu outbreak takes place.Typical flu outbreak causes that the sickness rate of pneumonia and lower respiratory illness increases, and evidence is that admission rate or mortality rate increase.Old people or the people who suffers from basic chronic disease most possibly experience this complication, and the child also possibly suffer from serious disease.

New influenza virus in unpredictalbe interval, occurs, it has the critical surfaces antigenemia cell agglutinin with the complete different subtype of strain that seasonal current is capable before.At this moment, the antigen of generation can have 20% to 50% variation with previous corresponding albumen in philtrum circulation strain.This can cause viral escape ' herd immunity ', and generation is very popular.This phenomenon is called ' antigen conversion '.It is generally acknowledged that taken place at least in the past to be very popular, the influenza virus of different plant species such as fowl or swine influenza virus had passed species barrier at that time.If these viruses have the potentiality of interpersonal propagation, they can whole world diffusion in several months to one year, causes being very popular.For example, nineteen fifty-seven (Asia influenza is very popular), the H2N2 subtype virus has replaced H1N1 virus, and H1N1 virus just circulates among the crowds from the beginnings in 1918 that at first are separated to this virus at least.H2HA and N2 NA have experienced antigenic drift between nineteen fifty-seven to nineteen sixty-eight; The H3N2 influenza virus sub-strain that is occurred in nineteen sixty-eight (Mao flu is very popular) until HA substitutes, and after this N2 NA continues drift (Nakajima etc., 1991 together with H3 HA; Epidemiol.Infect.106,383-395).

Giving its strains of influenza viruses characteristic that causes the outbreak potential that is very popular is: compare it with the hemagglutinin in the current circulation strain and comprise new hemagglutinin, its can with or without the change of neuraminidase hypotype; It can be in the crowd horizontal transmission; It is morbific to the people.New hemagglutinin can be (possible many decades) also unconspicuous hemagglutinin, for example H2 in the crowd in long-time section.It does not also have circulation hemagglutinin in the crowd, the H5 that for example in bird, finds, H9, H7 or H6 before perhaps can be.Under any situation, most of or at least vast scale so that whole colony all do not run into antigen in the past, on immunology, be former initial state to antigen.

In general, under the environment that is very popular, some colony is increased by the risk of influenza infection.Old people, chronic disease and child be susceptible especially, but many youngsters are also risky with surperficial healthy people.For the H2 influenza, the risk that the partial mass of being born after nineteen sixty-eight bears increases.These colonies are importantly protected as soon as possible and with simple mode effectively.

Another crowd who bears the risk of increase is the traveller.Today, whenever people than travelling more often in the past, and area China and Southeast Asia that maximum new virus occur have become welcome Reiseziel in recent years.This change of communication mode can make new virus in about several weeks rather than in several months or several years, arrive the whole world.

Therefore, for these crowds, need the inoculation protection to come under pandemicity or under potential pandemic situation especially to influenza.Suitable strain is but is not limited to: H5N1, H9N2, H7N7, H2N2 and H1N1.

Perhaps, said compositions can comprise more than 3 valencys, and for example two kinds of non-strains that are very popular add a kind of strain that is very popular.Perhaps, said compositions can comprise 3 kinds of strains that are very popular.

In a further embodiment; The present invention relates to a kind of vaccination regimen; Wherein inoculation first uses the cracking influenza compositions that contains at least a influenza strain to carry out; Wherein said influenza strain can cause the outburst of being very popular potentially, inoculates to use perhaps perhaps to carry out for the circulation strain of classical strain as epidemic strain.

CD4 epi-position among the HA

This antigenic drift mainly is present in the epi-position district of virus surface proteins hemagglutinin (HA) and neuraminidase (NA).Knownly be used for escaping that any difference of CD4 and B cell epitope all will play an important role in the influenza inoculation between the different influenza strains that the adaptability of host immune system replys by virus, and like this really.

Identify the total CD4 T-cell epitope of different influenza strains (referring to for example: Gelder C etc., 1998, Int Immunol.10 (2): 211-22 at philtrum; Gelder CM etc., 1996 JVirol.70 (7): 4787-90; With Gelder CM etc., 1995 J Viroi.1995 69 (12): 7497-506).

In a concrete embodiment; Inoculate and use the reinforcement compositions that contains influenza virus or its antigenicity prepared product to carry out, said influenza virus or its antigenicity prepared product and influenza antigen that is used for inoculation for the first time or the total common CD4 T-cell epitope of its antigenicity prepared product.Therefore; The immunogenic composition that the present invention relates to contain influenza virus or its antigenicity prepared product and oil in water emulsion adjuvant is inoculated the purposes in the component first what produce the multiple dose vaccine; But said oil in water emulsion adjuvant contains metabolism oil, sterol such as alpha-tocopherol and emulsifying agent; Said multiple dose vaccine also comprises as the influenza virus of booster dose or its antigenicity prepared product, the total common CD4 T-cell epitope of the influenza antigen of the dosage that gives or its virus antigenicity prepared product when they were inoculated with the first time.

Inoculation method

Compositions of the present invention can give through any suitable pipeline, for example Intradermal, mucosa such as intranasal, oral, intramuscular or subcutaneous.Other pipeline is well-known in this area.

The intramuscular pipeline is preferred to adjuvant influenza compositions is arranged.

Intradermal delivery is another suitable approach.Any suitable device all can be used for intradermal delivery, for example at US 4,886, and 499, US 5,190,521, US 5; 328,483, US5,527,288, US 4,270,537, US 5; 015,235, US 5,141, and 496, the hour hand device described among the US 5,417,662.Intradermal vaccine also can give through the device that the restriction pin gets into effective penetration length of skin, for example is described in device and the function equivalent thereof of WO99/34850 and EP1092444, and these two documents are attached among this paper by reference.Rapid injection device also suits, its through liquid fast injection device or through the effusive pin transmit fluid vaccine that pierces through horny layer and produce to arrive corium to corium.Rapid injection device is described in for example US5, and 480,381, US 5,599,302, US 5,334,144, US 5,993; 412, US 5,649, and 912, US 5,569,189, US 5,704,911, US 5,383; 851, US 5,893, and 397, US5,466,220, US 5,339,163, US 5,312; 335, US 5,503, and 627, US 5,064,413, US 5,520,639, US 4,596; 556, US 4,790, and 824, US 4,941,880, US4,940,460, WO 97/37705 and WO 97/13537.Pulverized powder/granule transfer device also suits, and it uses the vaccine of Compressed Gas powder quick form to pass through skin outer layer to corium.In addition, conventional syringe can be used for the classical Mantoux test method that Intradermal gives.

The approach that gives that another is suitable is a subcutaneous route.Any suitable device all can be used for subcutaneous transmission, for example classical pin.Preferably, use for example disclosed needleless fast injection device service in WO 01/05453, WO01/05452, WO 01/05451, WO 01/32243, WO 01/41840, WO 01/41839, WO 01/47585, WO 01/56637, WO 01/58512, WO 01/64269, WO01/78810, WO 01/91835, WO 01/97884, WO 02/09796, WO 02/34317.More preferably, said device is filled with liquid vaccine preparation in advance.

Perhaps, the vaccine intranasal gives.Typically, the vaccine topical administration preferably is not drawn in the lung in nasopharynx part.It is desirable to use bacterin preparation is transmitted into nasopharynx part and does not have or do not make it get into the intranasal transfer device in lung basically.

It is sprayer unit that the preferred intranasal of vaccine of the present invention gives device.Suitable commercial nose sprayer unit comprises Accuspray ^TM(Becton Dickinson).Nebulizer produces very tiny droplet, and it can be drawn in the lung easily, therefore can not effectively arrive nasal mucosa.Therefore not preferred nebulizer.

The preferred spray devices that is used for the intranasal purposes is the device of the pressure independent that provides of performance and user.These devices are called pressure territory device.Only, critical pressure just discharges liquid when being provided by nozzle.These devices obtain the spraying of drop size rule more easily.Be applicable to that pressure of the present invention territory device is known in this area, be described in for example WO 91/13281 and EP 311 863 B and EP 516 636, these documents are attached among this paper by reference.This device can be bought by Pfeiffer GmbH, also is described in Bommer, R.PharmaceuticalTechnology Europe, in JIUYUE, 1999.

Drop (water is detected as the liquid) scope that preferred intranasal device produces is 1-200 μ m, preferred 10-120 μ m.Have the suction risk less than 10 μ m, the drop that therefore it is desirable to below the 10 μ m is no more than about 5%.The above drop of 120 μ m spread not as less drop good, be no more than about 5% so it is desirable to the above drop of 120 μ m.

The dose double transmission is the more preferably characteristic that is used for the intranasal transmission system of vaccine of the present invention.The dose double device comprises two sub-doses of single vaccinating agent, and each nostril gives a sub-doses.In general, two sub-doses are present in the container, and the structure of device allows each single sub-doses that effectively transmits.Perhaps, can use single dose device to give vaccine of the present invention.

Perhaps, the present invention has also imagined epidermis or transdermal route of inoculation.

In a specific embodiments of the present invention, but the adjuvant immunity originality compositions intramuscular that has that gives first give, the reinforcement compositions that adjuvant is arranged or do not have an adjuvant can give through different approaches, for example Intradermal, subcutaneous or intranasal.In another embodiment, the compositions that gives first can comprise the standard HA content of each influenza strain 15 μ g, strengthens the HA that compositions can comprise low dosage, promptly is lower than 15 μ g, and can smaller size smaller gives according to the approach of giving.

Inoculation colony

The target group of inoculation can be the people of immunocompromised host.Compare with health adult, the people of immunocompromised host generally can not reply antigen, especially influenza antigens well.

Preferably, target group are the colonies that influenza just do not exempted from, and (for example with respect to the strain that is very popular) of they or former initial state perhaps before can not be replied influenza infection or inoculation.Preferably, target group are old peoples, be suitably 65 years old with more than; Younger excessive risk adult (be 18-64 year) is for example the people of health institution's work; The Young Adults that perhaps has high risk factor such as cardiovascular diseases and pneumonopathy or diabetes.Another target group are all 6 months children with above child, an especially 6-23 monthly age, and they have experienced the high relatively influenza admission rate of being correlated with.Preferably, target group are old peoples of over-65s.

Vaccination regimen, administration and additional effect standard

Aptly, immunogenic composition of the present invention is standard 0.5ml ID under most of situation, according to spreading (SRD) (J.M.Wood etc.: J.Biol.Stand.5 (1977) 237-247 according to single radiation immunity; J.M.Wood etc., J.Biol.Stand.9 (1981) 317-330) detect, contain 15 μ g hemagglutinin antigen components of each influenza strain.Aptly, the vaccine dose volume between 0.5ml to 1ml, specifically standard 0.5ml or 0.7ml vaccine dose volume.According to the HA concentration in the stock solution sample, can slightly adjust dose volume routinely.

Aptly, said immunogenic composition contains HA antigen-for example every influenza strain 1,2,3,4,5,6,7,8,9,10,11,12,13 of low dosage or any among the 14 μ gHA.Suitable low dosage HA between every influenza strain 1-7.5 μ gHA, aptly between every influenza strain 3.5-5 μ g HA, for example every influenza strain 3.75 μ g HA, typically every approximately influenza strain 5 μ gHA.

Advantageously, vaccine dose of the present invention, especially low dosage vaccine can be to provide than the little volume of routine injection cracking influenza vaccines that is generally every dose about 0.5,0.7 or 1ml.Small size dosage of the present invention preferred every dose less than 500 μ l, more preferably every dose less than 300 μ l, most preferably every dose is no more than about 200 μ l or following.

Therefore, be the dosage that in small size, has low antigen dose according to the preferred small size vaccine dose of one aspect of the invention, 15 μ g or about 7.5 μ gHA or about 3.0 μ g HA (every strain) for example have an appointment in about 200 μ l volumes.

Influenza medicine of the present invention preferably satisfies some international standard of vaccine.

Proposed to detect the standard that influenza vaccines are renderd a service in the world.The European Union's official standard that in following table 1, has shown effective influenza vaccine.In theory, for satisfying European Union's requirement, all influenza strains that in vaccine, comprise, influenza vaccines only need be satisfied a standard in the table.Compositions of the present invention satisfies at least one in these standards aptly.

Yet in practice, all Strain must satisfy at least two or whole three in the said standard, particularly for new generation vaccine (new generation vaccine that for example transmits through different approaches).In some cases, two standards maybe be enough.For example, can accept all Strain and satisfy two in three standards, and part but be not that whole Strain satisfy the 3rd standard (for example two kinds in three kinds of Strain).Requirement to adult colony (18-60 year) and old people colony (greater than 60 years old) is different.

Table 1

^*Serological conversion rate is defined as the inoculation back reaches at least 4 times inoculator to serum hemagglutinin inhibition (HI) titre increase of every kind of vaccine strain percentage rate.

^*Conversion factor definition inoculation back is to the increase multiple of the serum HI geometric mean titer (GMT) of every kind of vaccine strain.

^{* *}Protective rate is defined as inoculation back (to every kind of vaccine strain) serum HI titre and is equal to or higher than 1: 40 inoculator's percentage rate, can be counted as the indication protective effect usually.

Again on the one hand, it is a kind of to the known method that will pass through the disease design vaccine of healing of CD4+T cell activation or treatment that the present invention provides, and it comprises:

1) select to contain the CD4+ epi-position antigen and

2) oil in water emulsion adjuvant of said antigen of combination and preceding text definition can induce enhanced cd4 t cell to reply in said mammal when wherein said vaccine gives in said mammal.

The instruction of all lists of references of the application (comprise patent application and patented) all is attached among this paper by reference fully.

For avoiding feeling uncertain, inventor's intention be the term of this paper ' comprise ' in each case all randomly can by term " by ... form " substitute.

Optional embodiment

In an optional embodiment, particularly but not exclusively can when using cracking influenza antigens or its antigenicity prepared product, use any oil in water emulsion adjuvant.Therefore, under background of the present invention, also imagined following specific embodiments:

1.-(a) cracking influenza virus or its lytic virus antigenicity prepared product and (b) purposes of oil in water emulsion adjuvant in producing immunogenic composition; Said immunogenic composition philtrum induce anti-said antigen or its lytic virus antigenicity prepared product below at least a: the CD4 T-cell response that i) improves, the B cell memory that ii) improves is replied.

2.-(c) cracking influenza virus or its lytic virus antigenicity prepared product and (a) purposes of oil in water emulsion adjuvant in producing immunogenic composition; Said immunogenic composition is used to inoculate people's immunocompromised individuals or colony; For example excessive risk adult or old people are with to influenza.

3.-adjuvant is arranged or do not have influenza virus or its antigenicity prepared product purposes in producing immunogenic composition of adjuvant, said immunogenic composition is used to inoculate the previous people who inoculates with cracking influenza virus or its lytic virus antigenicity prepared product and oil in water emulsion adjuvant.Preferably, inoculate in having carried out the experimenter of influenza inoculation last season and carry out.Typically, inoculate and carrying out at least 6 months after the inoculation first, preferably carried out in 8-14 month the inoculation back first, more preferably carried out in 10-12 month in inoculation back first.

4.-preferably; (a) cracking influenza virus or its lytic virus antigenicity prepared product and (b) the oil in water emulsion adjuvant purposes in producing immunogenic composition are provided, and said immunogenic composition is used to inoculate the previous people who inoculates with cracking influenza virus or its lytic virus antigenicity prepared product and oil in water emulsion adjuvant.

5.-(e) from the cracking influenza virus of the first influenza strain or its lytic virus antigenicity prepared product and (f) purposes of oil in water emulsion adjuvant in producing immunogenic composition; Said immunogenic composition is used for resisting the protective effect of the influenza infection that is caused by the influenza strain, and said influenza strain is the variant of the said first influenza strain.

6.-in another embodiment; The immunogenic composition that is used to inoculate comprises cracking influenza virus or its lytic virus antigenicity prepared product, they and cracking influenza virus that is used for inoculating first or the total common CD4 T-cell epitope of its lytic virus antigenicity prepared product.

7.-the method with impaired human individual of immunogenic composition immunoprophylaxis or colony (for example excessive risk adult or old people), said immunogenic composition comprise cracking influenza virus or its lytic virus antigenicity prepared product and as the oil in water emulsion adjuvant that defines of preceding text.

8.-a method that inoculates previous people with cracking influenza virus or its lytic virus antigenicity prepared product and oil in water emulsion adjuvant inoculation comprises giving the immunogenic composition that contains influenza virus that said people has adjuvant or do not have adjuvant.

9.-one kind to human colony or individual resist a kind of strains of influenza viruses inoculation, follow the method that inoculates of said people or colony being carried out resistance body strains of influenza viruses; Said method comprises and gives cracking influenza virus that said people (i) contains first strains of influenza viruses or first compositions of its cracking influenza antigen property prepared product and oil in water emulsion adjuvant; (ii) second immunogenic composition, it comprises the strains of influenza viruses variant of said first strains of influenza viruses.

10.-the method for a design flow influenza vaccine comprises

1) select to contain the CD4+ epi-position influenza antigens and

2) the said influenza antigens of combination and the as above O/w emulsion of definition can induce enhanced CD4 to reply in said mammal when wherein said vaccine gives in mammal.

In a specific embodiments, to compare with replying of obtaining of no adjuvant antigen or antigenic composition, immunogenic composition can also induce the B-memory cell of CD4 T-cell response and the improvement of improvement to reply this two.

In all these embodiments, but said oil in water emulsion adjuvant comprises at least a metabolism oil aptly, and its amount is 0.5% to 20% of cumulative volume, and has oil droplet, and at least 70% (with intensitometer) of oil droplet has the diameter less than 1 μ m.

The present invention will further describe with reference to following non-limiting example:

Example I has been described the immune deciphering method that in mice, ferret and people's research, uses.

Example II has been described the O/w emulsion that is used for exemplary research and the preparation and the sign of adjuvant formulation.

EXAMPLE III has been described the clinical experiment of in the over-65s elderly population, carrying out with the vaccine that contains cracking influenza antigens prepared product and AS03 adjuvant.

EXAMPLE IV has been described second clinical experiment-inoculate experiment-in the over-65s elderly population, carry out with the vaccine that contains cracking influenza antigens prepared product and AS03 adjuvant.

EXAMPLE V has shown clinical preceding estimate (research I and research II) of influenza vaccines in ferret that adjuvant and no adjuvant are arranged.Detected temperatures monitoring, virus shedding and CD4 T-cell response.

Example VI has shown that influenza vaccines that adjuvant and no adjuvant are arranged are C57BI/6 originally mice and the clinical preceding evaluation of just exempting from the mice.

Example VII A has shown cracking and clinical preceding estimate of subunit influenza vaccines in the C57BI/6 mice of just exempting from the allos strain that adjuvant and no adjuvant are arranged.

Example VII A I has described the clinical experiment of in the over-65s elderly population, carrying out with the vaccine that contains cracking influenza antigens prepared product, and said prepared product contains AS03 adjuvant, AS03+MPL adjuvant or do not have the external source adjuvant.

Example I X has shown clinical preceding estimate (research III) of influenza vaccines in ferret that adjuvant and no adjuvant are arranged.Detected temperatures monitoring, virus shedding and HI titre.

Embodiment X has shown in the over-65s elderly population clinical experiment of carrying out with the vaccine that contains cracking influenza antigens prepared product: the immunogenicity persistent data when 90 days and 180 days, said prepared product contains AS03, is with or without the MPL adjuvant.

Embodiment XI has shown the clinical experiment of in the over-65s elderly population, carrying out with the vaccine that contains cracking influenza antigens prepared product, and said prepared product contains AS03 and MPL adjuvant.

Embodiment XII has shown the clinical experiment of in the over-65s elderly population, carrying out with the vaccine that contains cracking influenza antigens prepared product, and said prepared product contains the MPL adjuvant of AS03 and two concentration.

Example I-immune deciphering method

I.1. mice method

I.1.1. hemagglutination suppresses test

Test program

Use hemagglutination to suppress test (HI) and measure anti-hemagglutinin antibody titer to 3 kinds of strains of influenza viruses.The HI testing principle is based on the specific anti influenza antibodies and suppresses the ability through little chicken red blood cell (RBC) hemagglutination of influenza virus haemagglutinin (HA).Heat inactivation serum is handled with Kaolin and chicken RBC in advance, to remove nonspecific inhibitor.After pretreatment, with every kind of influenza strain incubation of 2 times of dilution serum and 4 HAUs.Add little chicken red blood cell then, and the record coagulation suppresses.Titre is expressed as the inverse of the high dilution of serum that suppresses hemagglutination fully.Because first dilution factor of serum is 1: 20, equal 10 titre so can not detection level be recorded as.

Statistical analysis

Use UNISTAT that postvaccinal HI titre is carried out statistical analysis.The method that is used for analytic variance can be sketched as follows:

The ■ data to number conversion

The Shapiro-Wilk check that ■ carries out each colony (group) is so that the normality of checking component cloth

■ Cochran check is so that confirm the variance homogeneity between the different groups (group)

The two-way analysis of variance that ■ carries out each group

■ is used for the Tukey HSD check of multiple comparisons

I.1.2 intracellular cytokine dyeing

This technology allows according to the quantitative antigen specific T lymphocyte of cytokine production: the effector-memory T cell of effector T cell and/or production IFN-γ and/or the maincenter memory t cell of production IL-2.Collected PBMC in back 7 days in immunity.

The external lymphoid cell that stimulates again in the presence of secreted inhibitor (Brefelldine).

Use fluorescent antibody (CD4, CD8, IFN-γ and IL-2) to handle these cells then through the routine immunization fluorescence method.The result is expressed as the frequency of cytokine positive cell in the CD4/CD8 T cell.PBMC was carried out in back 7 days the intracellular cytokine dyeing of T cell in immunity for the second time.Collect blood by mice, and be incorporated in the heparinization culture medium RPMI+ additive.For blood, the PBL suspension of RPMI+ additive dilution according to the method for recommending (in room temperature with centrifugal 20 minutes of 2500rpm) at Lympholyte-Mammal gradient higher slice.Remove the mononuclear cell at separating surface place, clean 2 times, the PBMC suspension is adjusted to 2 * 10 with RPMI 5% hyclone with the RPMI+ additive ⁶Individual cell/ml.

With Whole FI (1 μ g HA/ strain) with 1 * 10 ⁷The final concentration of individual cell/ml (tubular type FACS) carries out exo-antigen to PBMC to stimulate, then under the situation that adds anti--CD28 and anti--CD49d (being 1 μ g/ml) in 37 ℃ of incubations 2 hours.

At antigen again after the stimulation step, in the presence of Brefeldin (1 μ g/ml) in 37 ℃ of incubation PBMC that spend the night, to suppress cytokine secretion.IFN-γ/IL-2/CD4/CD8 dyeing is carried out as follows: clean cell suspending liquid, be resuspended in and contain 2%Fc closed reagent (1/50; 2.4G2) 50 μ l PBS 1%FCS in.In 4 ℃ of incubations after 10 minutes, add anti--CD4-PE (2/50) and resist-the 50 μ l mixture of CD8 perCp (3/50), in 4 ℃ of incubations 30 minutes.After in PBS 1%FCS, cleaning, made cell permeabilization in 20 minutes through being resuspended among the 200 μ l Cytofix-Cytoperm (Kit BD) and in 4 ℃ of incubations.Use Perm Wash (Kit BD) to clean cell then, and in order to the 50 μ l mixture resuspensions of anti--IFN-γ APC (1/50)+anti--IL-2 FITC (1/50) of Perm Wash dilution.In minimum 2 hours of 4 ℃ of incubations are the longest spend the night after, clean cell and be resuspended in the PBS 1%FCS+1% paraformaldehyde with PermWash.Carry out sample analysis through FACS.Gate (FSC/SSC) living cells is surveyed about 20,000 incidents (lymphocyte) on the CD4+T cell or 35,000 incidents.Calculate IFN-γ+or percentage rate of IL2+ to the crowd of CD4+ and CD8+ gate.

I.2. ferret method

I.2.1. hemagglutination suppresses test (HI)

Test program

Use hemagglutination to suppress test (HI) and measure anti-hemagglutinin antibody titer to 3 kinds of strains of influenza viruses.The HI testing principle is based on the specific anti influenza antibodies and suppresses the ability through little chicken red blood cell (RBC) hemagglutination of influenza virus haemagglutinin (HA).Serum at first uses 25% neuraminic acid enzymatic solution (RDE) to handle, and heat inactivation, to remove nonspecific inhibitor.After pretreatment, with every kind of influenza strain incubation of 2 times of dilution serum and 4 HAUs.Add little chicken red blood cell then, and the record coagulation suppresses.Titre is expressed as the inverse of the high dilution of serum that suppresses hemagglutination fully.Because first dilution factor of serum is 1: 10, equal 5 titre so can not detection level be recorded as.

Statistical analysis

Use UNISTAT that HI titre (41 days, before attacking) is carried out statistical analysis.The method that is used for analytic variance can be sketched as follows:

The ■ data to number conversion

The Shapiro-Wilk check that ■ carries out each colony (group) is so that the normality of checking component cloth

■ Cochran check is so that confirm the variance homogeneity between the different groups (group)

■ is to the interactive check of single factor ANOVA

■ is used for the Tukey HSD check of multiple comparisons

I.2.2. temperature monitoring

During attacking, monitor a temperature with pick off and through the telemetry record.Check and rebuild all implants, before putting into the abdominal cavity, newly calibrate through DSI (Data Sciences International, Centaurusweg 123,5015 TC Tilburg, The Netherlands).All animals all are housed in the independent cage independently between these detection periods.

Preceding 4 days of attack until attacking back 7 days per 15 minutes record temperature.

I.2.3. nose cleans

Carry out the nose cleaning through in two nostrils of clear-headed animal, giving 5ml PBS.Inoculum is collected in the culture dish, and places the shuttle on the dry ice.

The titration of virus of nose cleanout fluid

All nose samples are all at first through Spin X filter (Costar) aseptic filtration, to remove any bacterial pollutant.The continuous 10 times of dilution nose cleanout fluid of 50 μ l are transferred in the microtitration plate that contains 50 μ l culture medium (10 holes/dilution factor).Then with 100 μ l mdck cells (2.4 * 10 ⁵Individual cell/ml) adds each hole, and in 35 ℃ of incubation 5-7 days.

Behind 5-7 days incubations, take out culture medium carefully, add the culture medium that 100 μ l contain 1/20 WST-1, incubation is 18 hours again.

The survivaling cell number that the intensity of yellow first

dyestuff that when survivaling cell reduction WST-1, produces and titration of virus experiment are present in the hole when finishing is proportional, and quantitative through the absorbance that detects each hole at suitable wavelength (450nm).The intercepting value defined is not for infecting OD meansigma methods-0.3 OD (0.3 OD is equivalent to not infect control cells OD ± 3 standard deviation) of control cells.Be defined as negative score during OD <be defined as positive score during the intercepting value, on the contrary, OD>intercepting value.The virus shedding titre is measured through " Reed and Muench ", and is expressed as Log TCID50/ml.

I.3. the mensuration of immunne response among the appraiser

I.3.1. hemagglutination suppresses to measure

Use WHO influenza cooperation center, Center for Disease Control (CDC), Atlanta, the method that USA (1991) describes is through detecting HI TPPA immunne response.

Use 4 hemagglutination to suppress the suitable antigen and the 0.5% fowl red blood cell suspension of units (4 HIU), the freezing blood serum sample that melts is carried out the antibody titer detection with the micromethod of crossing with comprehensive verification of standard.Remove non-specific serum inhibitor through heat treatment and receptor destroying enzyme.

Estimate the HI antibody horizontal of the serum that obtains.Begin to prepare serial dilution degree (multiply by factor 2) with 1: 10 initial dilution factor, until final dilution factor 1: 20480.Showing that the high dilution step that suppresses (100%) hemagglutination fully is regarded as titration end-point.All experiments are all carried out with double.

I.3.2. neuraminic acid EIA

In encapsulating the microtitration plate of myosin, measure.The antiserum of 2 times of serial dilution degree of preparation mixes with influenza A H3N2, H1N1 or the Influenza B virus of normalized quantity.Test is based on the neuraminic acid enzyme bioactivity that is discharged neuraminic acid by the myosin enzymolysis.After downcutting terminal neuraminic acid, β-D-galactose-N-acetyl group-aminogalactose comes out.Add the peanut agglatinin from horseradish peroxidase (HRP) labelling of Semen arachidis hypogaeae to each hole, its specificity combines the galactose structure.Can be in substrate reactions detect and the quantitative amount of binding lectin with tetramethyl benzidine (TMB).Show and still suppress viral neuraminic acid enzymatic activity to reach at least 50% the highest antibody dilution be the NI titre.

I.3.3. neutralizing antibody is measured

The freezing blood serum sample that melts is carried out neutralizing antibody to be detected.The virus neutralization that is included in the antibody in the serum is tested with microneutralization and is measured.Serum need not further processing and can use in experiment.Each serum is tested with triplicate.The virus of normalized quantity is mixed with the serum of serial dilution and incubation, so that antibodies is viral.The cell suspending liquid that will contain the mdck cell of limited amount then adds in virus and the sero-fast mixture, in 33 ℃ of incubations.After incubation period, manifest virus replication through the erythrocytic agglutination of chicken.Utilize the method for Reed and Muench calculate serum 50% in and titre.

I.3.4. estimate cell-mediated immunity through cytokine flow cytometer (CFC)

Peripheral blood antigenic specificity CD4 and CD8 T cell can stimulate at external quilt again, to produce IL-2, CD40L, TNF-α and IFN, if corresponding antigen incubation with it.Therefore, antigenic specificity CD4 and CD8 T cell can be through the flow cytometer countings after routine immunization fluorescence labeled cell phenotype and intracellular cytokine production.In this research, influenza vaccines antigen and derive from the proteic peptide of specific influenza as the antigen that stimulates the influenza specific T-cells again.The result is expressed as the frequency of male CD4 of cytokine in CD4 or the CD8 T cell subsets or cd8 t cell.

I.3.5. statistical method

I.3.5.1. main terminal point

The part of demand and percentage rate, intensity and same inoculate related of whole body sign and symptom in postvaccinal 7 days follow-up period (i.e. inoculation day with 6 days subsequently) and whole process.

In postvaccinal 21 days follow-up period (i.e. inoculation day with 20 days subsequently) and whole process the percentage rate of the part of positive appeal and whole body sign and symptom, intensity and same with inoculate related.

The generation of serious adverse events in the whole research process.

I.3.5.2. secondary endpoints

For HI:

Observational variable:

At 0 day and 21 days: the serum hemagglutination suppressed (HI) and NI antibody titer, respectively to each tests of 3 kinds of strains of influenza viruses providing in the vaccine (anti--H1N1, anti--H3N2 and resist-B-antibody).

At 0 day and 21 days: NAT, respectively to each tests of 3 kinds of strains of influenza viruses providing in the vaccine.

The variable (having 95% confidence interval) of deriving:

The geometric mean titer (GMT) that has the serum HI antibody of 95% confidence interval (95%CI) before the inoculation and after the inoculation

The serological conversion rate that has 95%CI in the time of 21 days ^*

The conversion factor that has 95%CI in the time of 21 days ^*

The serum protective rate that has 95%CI in the time of 21 days ^{* *}

At the serum N I of all time points antibody GMT (having 95% confidence interval)

^*Serological conversion rate is defined as to increase during to the serum HI titre of every kind of vaccine strain the 21st day the time than the 0th day and reaches inoculator's percentage rate of at least 4 times.

^*Conversion factor is defined as than the 0th day increase multiple to the serum HI GMT of every kind of vaccine strain the 21st day the time.

^{* *}Protective rate is defined as inoculation back (to every kind of vaccine strain) inoculator's percentage rate that serum HI titre equals 40, regards this percentage rate as the indication protective effect usually.

(CMI) replys for cell-mediated immunity

Observational variable

The 0th day with 21 days: in the different tests per 10 ⁶The frequency of the central male CD4/CD8 cell of cytokine.The all quantitative CD4/CD8 T cell of each test replying to following material:

Peptide influenza (pf) antigen (these antigenic precise nature need not provide/explain with the source)

Cracking influenza (sf) antigen

Full influenza (wf) antigen

The variable of deriving:

Produce the cell of at least two kinds of different cytokines (CD40L, IL-2, IFN γ, TNF α)

Produce the cell of CD40L and another kind of cytokine (IL-2, TNF α, IFN γ) at least

Produce the cell of IL-2 and another kind of cytokine (CD40L, TNF α, IFN γ) at least

Produce the cell of IFN γ and another kind of cytokine (IL-2, TNF α, CD40L) at least

Produce the cell of TNF α and another kind of cytokine (IL-2, CD40L, IFN γ) at least

I.3.5.3. immunogenicity analysis

Immunogenicity is analyzed based on whole inoculation crowd.For each treatment group, calculate following parameter (having 95% confidence interval):

The geometric mean titer (GMT) of HI and NI antibody titer when the 0th day and 21 days

The geometric mean titer (GMT) of NAT when the 0th day and 21 days

Conversion factor in the time of 21 days

Serological conversion rate in the time of 21 days (SC), be defined as than the 0th day the 21st day the time increase of serum HI titre reach inoculator's percentage rate of at least 4 times.

Protective rate in the time of 21 days is defined as inoculator's percentage rate of serum HI titre=1: 40.

Sum up the frequency (descriptive statistics) that the CD4/CD8T-lymphocytic emiocytosis is replied at each time point (the 0th day, the 21st day) to each inoculation group with to every kind of antigen (peptide influenza (pf) antigen, cracking influenza (sf) antigen and full influenza (wf) antigen).

For each inoculation group and every kind of antigen (pf, sf and wf), individuality is replied the descriptive statistic of difference between time point (before the inoculation back-inoculation) in each 5 kinds different tests.

Use the relatively local difference between 3 groups of nonparametric test (Kruskall-Wallis check), in each 5 kinds different tests, calculate every kind of antigenic statistical p-value.All statistical test all are two tails.Being less than or equal to 0.05 P-value, to be counted as statistics significant.The preparation of example II-O/w emulsion and adjuvant formulation and sign

Except as otherwise noted, otherwise oil/aqueous emulsion of in embodiment subsequently, using comprise organic facies that 2 kinds of oil (alpha-tocopherol and zamene) form and contain the PBS water of Tween 80 as emulsifying agent.Except as otherwise noted; Otherwise the oil in water emulsion adjuvant preparation that in embodiment subsequently, uses is all processed and is contained following O/w emulsion component (providing final concentration): 2.5% zamene (volume), 2.5% alpha-tocopherol (volume), 0.9% polyoxyethylene sorbitan monoleate (volume) (Tween 80), and referring to WO 95/17210.This emulsion is called AS03 in embodiment subsequently, being prepared as follows is 2 times of concentrate.

II.1. the preparation of emulsion SB62

II.1.1. laboratory scale preparation

Tween 80 is dissolved in the PBS (PBS), obtains 2% PBS solution.For 2 times of concentrated emulsions of 100ml are provided, vortex 5g DL alpha tocopherol and 5ml zamene are with thorough mixing.Add 90ml PBS/Tween solution, thoroughly mix.Then the emulsion that obtains is passed through syringe, use M110S microfluidic device microjet at last.The oil droplet that obtains has the size (being expressed as the Z average that detects through PCS) of about 120-180nm.Other adjuvant/antigen component is incorporated as in the emulsion of single mixture.

II.1.2. amplify the preparation of scale

Through under strong mixing, mixing oil phase of forming by hydrophobic components (alpha-tocopherol and zamene) and the water that contains water-soluble component (Tween 80 and PBS mod (modified form), pH 6.8), preparation SB62 emulsion preparation thing.When stirring, oil phase (1/10 cumulative volume) is transferred to water (9/10 cumulative volume), in stirring at room mixture 15 minutes.In the interaction chamber of micro-fluidic device, the mixture that obtains is applied shearing force, impulsive force and cavitation force (15000 PSI, 8 circulations) then, to produce submicron oil droplet (being distributed between the 100-200nm).The pH that obtains is between 6.8 ± 0.1.Make the SB62 emulsion through 0.22 μ m membrane filtration degerming then, with the emulsifiable concentrate stored frozen of degerming in 2-8 ℃ Cupac container.Dead volume with aseptic noble gas (nitrogen or argon) feeding SB62 emulsifying semi-finished product container continues at least 15 seconds.

The final composition of SB62 emulsion is following:

Tween 80:1.8% (volume) 19.4mg/ml; Zamene: 5% (volume) 42.8mg/ml; Alpha-tocopherol: 5% (volume) 47.5mg/ml; PBS-mod:NaCl 121mM, KCl 2.38mM, Na2HPO4 7.14mM, KH2PO4 1.3mM; PH 6.8 ± 0.1.

II.2. the dynamic light scattering of oil droplet granularity detects

II.2.1. brief introduction

The size of droplet diameter is measured according to following method and under following experiment condition.The oil droplet granularity Detection provides with intensity detection, and is expressed as the z average that detects through PCS.

II.2.2. sample preparation

Oil in water emulsion adjuvant is carried out granularity Detection, and said oil in water emulsion adjuvant is: according to SB62, AS03 and the AS03+MPL (50 μ g/ml) of multiplying gauge modeling method preparation, back two kinds are faced and use preceding preparation.The composition of sample provides (referring to chapters and sections II.2.4) hereinafter.Sample is with 4000 times to 8000 times of PBS7.4 dilutions.

As contrast, with 10mM NaCl dilution PL-Nanocal granulometry article 100nm (catalog number (Cat.No.) 6011-1015).

II.2.3.Malvern Zetasizer 3000HS granularity Detection

All granularity Detection all use two Malvern Zetasizer 3000HS to detect.Sample detects at the plastics cuvette that is used for the Malvern analysis with suitable dilution factor (be generally 4000 times to 20000 times dilution factor, depend on sample concentration), and adopts two kinds of optical modes:

-or real particle refractive index 0 and imaginary refractive index 0.

-or real particle refractive index 1.5 and imaginary refractive index 0.01 (according to visible value in document, emulsion being adopted the optical mode that is fit to).

Technical conditions are:

-optical maser wavelength: 532nm (Zeta3000HS).

-laser power: 50mW (Zeta3000HS).

-in the scattered light (Zeta3000HS) of 90 ° of detections.

-temperature: 25 ℃,

-the persistent period: confirm automatically by software,

-number of times: 3 continuous detecting,

-z average diameter: through the cumulant analysis

-particle size distribution: through Contin or automatic mode.

The Malvern of automatization algorithm uses the combination of cumulant, Contin and non-negative least square (NNLS) algorithm.

Because Mie is theoretical, intensity distributions can change volume distributed median into.

II.2.4. result's (referring to table 2)

Cumulant is analyzed (Z average diameter):

Table 2

( ^*) being prepared as follows: water for injection, PBS 10x concentrated solution, 250 μ l SB62 emulsions and 25 μ g MPL mix, to reach 280 μ l final volume.

Z average diameter (ZAD) size is measured with the amount of scattered light of various granularities in the sample.This value is relevant with the unimorph analysis of sample, is mainly used in the reproduction purpose.

Counting rate (CR) is the testing result of scattered light: it is corresponding to the thousands of photon of per second.

Polydispersity (Poly) index is the width that distributes.It is the dimensionless testing result of distribution popularity.

Contin and automated analysis:

According to above elaboration and have the method preparation of minor alteration and estimate two kinds of other SB62 prepared products (2 times of spissated AS03):

For the best counting rate value that obtains to be used for Zetasizer 3000HS definite two dilution factor: 10000x and 20000x, be used for the plastics cuvette test sample that Malvern analyzes.

The result is shown in table 3.

Table 3

The value that obtain "-" this moment is inconsistent.

These results illustrate the preparation 1023 in Fig. 1.Visible by it, most of particle (for example at least 80%) has the diameter less than 300nm with intensitometer.

II.2.5. whole conclusion

Detect different dilution SB62 preparations with Malvern Zetasizer 3000HS with two kinds of optical modes.The granularity ZAD of the preparation of more than estimating (average strength that promptly obtains through the cumulant analysis) is about 150-155nm.

When using the cumulant algorithm, we observe dilution factor to ZAD and not influence of polydispersity.

II.3. contain the preparation of the AS03 of MPL

The preparation of II.3.1.MPL liquid suspension

MPL (in whole file, use, it is that 3D-MPL is the abbreviation of 3-O-deacylated tRNA monophosphoryl lipid A) stock solution is prepared by MPL lyophilized powder.MPL stock solution is stable raw material dense (about 1mg/ml) aqueous dispersion, and it can be used for vaccine or adjuvant preparation at any time.The sketch map of preparation process provides in Fig. 2.

For maximum batch 12g, MPL stock solution goods are stored in the aseptic glass container.The dispersion of MPL is made up of following steps:

-with the MPL powder suspension in water for injection

-through adding any big aggregation of thermal depolymerization (heat treatment)

-through microjet with particle size reduction to 100nm to 200nm

-with Sartoclean pre-filtrating equipment 0.8/0.65 μ m pre-filtering goods

-in room temperature aseptic filtration goods (Sartobran P device, 0.22 μ m)

The MPL powder produces stable colloid aqueous dispersion (the MPL granularity is less than 200nm) through the microjet lyophilizing.The MPL freeze-dried powder is dispersed in the water for injection, so that obtain rough 10mg/ml suspension.Under agitation suspension is heat-treated then.After being cooled to room temperature, the beginning microjet is handled, so that reduce granularity.Use micro fluidic device M110EH, reach minimum through the microjet interaction chamber dispersion liquid that circulates continuously through amount (cycle-index: n to limit pressure _Min) carry out microjet.Represent the microjet persistent period of cycle-index to calculate based on flow velocity that detects and discrete volume.For the locking equipment of giving under the setting pressure, the flow velocity of acquisition can change to the whole activity cycle between another interaction chamber and specific interaction chamber at one to some extent.In the present embodiment, the interaction chamber of use is the micro-fluidic device of F20Y type.Since microjet render a service with pressure-flow velocity to relevant, so the processing time can be a collection of to variation between another batch.The time that 1 circulation needs calculates based on flow velocity.The flow velocity of being assert is the flow velocity that just before with the MPL gatherer, detects with water for injection.1 circulation is defined as the MPL cumulative volume through 1 the needed time of device (with a minute expression).The following calculating time that n circulation needs that obtains:

Amount (ml)/flow velocity (ml/ minute) of the MPL of n * handle

Correspondingly revise cycle-index thus.Minimal circulation amount (the n that will implement used preferred equipment and interaction chamber has been described _Min).According to n _MinThe definite global cycle amount that will implement of the granularity Detection result who carries out after the circulation.Confirm particle size restrictions (d based on historical data _Lim).Detect through photon correlation spectroscopy method (PCS) technology and realize d _LimBe expressed as monotype result (Z _Average).Under this restriction, can be at n _MinThe circulation back stops microjet.On this restriction, continue microjet, until obtaining satisfied particle size reduction, carry out 50 circulations at most again.

If behind microjet, do not begin immediately to filter, then dispersive MPL is stored in+and 2 to+8 ℃, wait for being transferred to filtration zone.

Behind microjet, dilute dispersion liquid with water for injection, through 0.22 μ m filter aseptic filtration under laminar flow.Last MPL concentration is 1mg/ml (0.80-1.20mg/ml).

The preparation of II.3.2.AS03+MPL adjuvant vaccine: single bottle of method

In the AS03 adjuvant formulation, add MPL, final concentration is between every vaccinating agent 10-50 μ g.

PBS10 times of concentrated solution when concentrated (1 times be pH7.4) and the SB62 mixture that contains Tween, TritonX-100 and VES (vitamin e succinate) are added in the water for injection.Investigate the amount of the detergent that exists in the influenza strain, so that reach the target final concentration: 750 μ g/mlTween, 80,110 μ g/ml Triton X-100 and 100 μ g/ml VES.After stirring in 5 minutes, add every kind of target influenza strain (for example strain H1N1, H3N2 and the B in classical 3 valency vaccines) of 15 μ g.After stirring in 15 minutes, add 250 μ l SB62 emulsions, add 25 μ g or 50 μ gMPL then.

The sketch map of preparation process provides in Fig. 3.Table 4 has provided the final composition that contains the AS03 of MPL in each human dose.

Table 4

^*PBS mod 10x concentrated solution pH 6.8=KH ₂PO ₄, Na ₂HPO ₄, NaCl, KCl-HCl

^*MPL is every dose 25 μ g or 50 μ g

The preparation of II.3.3.AS03+MPL adjuvant vaccine: two bottles of methods

Can be equipped with same preparation by two bottles of legal systems through mixing 2 times of spissated antigens or antigen preparation thing and AS03 (SB62 250 μ l) or AS03+MPL (SB62 250 μ l+25 μ g or 50 μ g MPL) adjuvant.In the case, it carries out as follows.The production of AS25-adjuvant influenza vaccines is made up of 3 key steps:

1) the trivalent semi-finished product of the no adjuvant of preparation (2x concentrates) also divide in the antigen container of packing into

2) preparation AS03+MPL adjuvant

3) the instant reprovision of AS03+MPL adjuvant split-virus vaccine.

1) the trivalent semi-finished product of the no adjuvant of preparation (2x concentrates) also divide in the antigen container of packing into

The HA content that the volume of 3 kinds of unit price stock solutions detects in each unit price stock solution before based on preparation, and based on the target volume of 1100ml.The premix of spissated PBS and Tween 80, Triton X-100 and alpha-tocopherol hydrogen succinate ester is diluted in the water for injection.Then with 3 kinds of spissated unit price stock solutions (A/ New Caledonia, A/ New York, B/ Jiangsu) serial dilution go into to obtain PBS/(pH 7.4 for Tween 80-Triton X-100-alpha-tocopherol hydrogen succinate ester solution; 1 37mM NaCl; 2.7mM KCl, 8.1mM Na ₂HPO ₄, 1.47mM KH ₂PO ₄990 μ g/ml Tween 80; 150 μ g/ml Triton X-100 and 130 μ g/ml alpha-tocopherol hydrogen succinate esters) in, so that have the final concentration of HA (17.5 μ g HA/B strains/380 μ l trivalent semi-finished product) of HA/ml trivalent semi-finished product (15 μ g HA/A strains/380 μ l trivalent semi-finished product) and the 46 μ g B strains of 39.47 μ g A strains (H1N1, H3N2).Adding between every kind of unit price stock solution, reach 10-30 minute in the stirring at room mixture.After adding last a kind of unit price stock solution, stirred 15-30 minute, check pH, and with HCl or NaOH with pH regulator to 7.2 ± 0.2.

Antigenic trivalent semi-finished product are aseptic subpackaged goes in the aseptic I type of 3-ml (European Pharmacopoeia) vial.Every bottle contains 470 μ l volumes (380 μ l+90 μ l overcharge).

2) preparation AS03/MPL adjuvant stock solution is also divided in the adjuvant container of packing into.

Prepare adjuvant AS03/MPL:SB62 emulsion (method among the chapters and sections II.1.2) and MPL (method among the chapters and sections II.3.1) through mixing two kinds of components.The MPL stock solution of 1 times of spissated PBS mod (preparing) and SB62 stock solution and 1mg/ml through the spissated PBS mod of 10x is diluted in the water for injection.Measure MPL concentration,, be suitably the about 25 μ g of each final vaccine for man dosage so that reach the final content of 10-50 μ g.Mixture is in stirring at room 5-30 minute, with NaOH (0.05 or 0.5 M)/HCl (0.03 M or 0.3 M) with pH regulator to 6.8 ± 0.1.After 5-30 minute, mixture is passed through 0.22 μ m membrane filtration degerming in the room temperature restir.Implement aseptic noble gas (nitrogen) and feed, in minimum 1 minute process, in minute packaging container, to produce inertia head space gas.Aseptic AS03+MPL adjuvant is stored in+and 2-8 ℃, go in the aseptic I type of 1.25-ml (European Pharmacopoeia) glass syringe until aseptic subpackaged.Each syringe contains 80 μ l excess volumes (320 μ l+80 μ l overcharge).

When injection, the content that will contain the pre-filled syringe of adjuvant injects the bottle that contains spissated trivalent inactivation lytic virus isoantigen.After mixing, content is drawn in the syringe, pin is replaced with the intramuscular pin.The AS25 adjuvant influenza candidate vaccine of 1 dose of reprovision is equivalent to 0.7mL.

II.4. in oil in water emulsion formulation, contain the preparation of the immunogenic composition of influenza antigens and optional MPL

In the SB62 of II.1 emulsion, add isopyknic 2 times of spissated cracking influenza antigens (Fluarix ^TM) (HA of every kind of strain of 15 μ g) and mixing.When suitable it is mixed with 50 μ g/ml MPL, obtain final preparation.

The clinical experiment (Explo-Flu-001) that EXAMPLE III-in the old people of over-65s is carried out with the vaccine that contains cracking influenza antigens prepared product and AS03 adjuvant

In the elderly population of over-65s, carried out open random research of I phase in 2003, so that estimate the reactionogenicity and the immunogenicity of the GlaxoSmithKline Biologicals influenza candidate vaccine that contains adjuvant AS03., intramuscular detected HI (promptly anti-hemagglutinin antibody titer, NAT and anti-neuraminidase antibody titer) and cell-mediated immune responses (CD4 and/or CD8 t cell response) after giving 1 dose of AS03 adjuvant vaccine or WV vaccine in 21 days.Fluarix ^TMAs reference.

III.1. research design

3 groups of following vaccines of experimenter's intramuscular acceptance abreast:

1 group of 50 experimenter of ■ accept 1 dose of reprovision and the SV influenza vaccines (FluAS03) that adjuvant is arranged

1 group of 50 experimenter of ■ accept 1 dose of totivirus influenza vaccine (FIuWVV)

1 group of 50 experimenter of ■ accept 1 dose of Fluarix ^TM(Fluarix)=and the contrast vaccine program: 1 influenza vaccine of injection in the 0th day, collect blood sample, carried out interpretive analysis (HI TPPA, NI TPPA, neutralizing antibody are measured and CMI analyzes) at 21 days and draw research conclusion.

The standard 3 valency cracking influenza vaccines-Fluarix that in this research, use ^TMBe a kind of at the commercialized vaccine by GlaxoSmithKline Biologicals exploitation and production in 2003.

III.2. vaccine is formed and is given (table 5)

III.2.1. vaccine production

AS03 adjuvant influenza vaccines

The influenza vaccines material standed for of AS03 adjuvantization is a kind of 2 component vaccines, is made up of with the preparatory filling I type glass syringe (335 μ l) (adjuvant container) that contains the SB62 emulsion the 3 valency inactivation lytic virus isoantigens that concentrate that in I type vial (335 μ l) (antigen container), provide.When injection, the content of antigen container is removed by means of SB62 emulsion pre-filled syringe, follows slight mixing syringe.SB62 emulsion and vaccine antigen mixing reprovision AS03 adjuvant.Before injection, the pin of use is replaced by the intramuscular pin, and volume is adjusted to 500 μ l.

The AS03 adjuvant influenza vaccines of 1 dose of reprovision are equivalent to 0.5ml, are contained in registered Fluarix ^TM/ α-Rix 15 μ g HA of every kind of strains of influenza viruses in the vaccine, and comprise 10.68mg zamene, 11.86mg DL-alpha tocopherol and 4.85mg polysorbate80 (Tween 80).

Preparation

The production of the influenza vaccines of AS03 adjuvantization is made up of 3 key steps:

1) 3 valency semi-finished product of the no adjuvant of preparation also divide in the antigen container of packing into

The HA content that the volume of 3 kinds of unit price stock solutions detects in each unit price stock solution before based on preparation, and based on the target volume of 800ml.The premix of spissated PBS and Tween 80, Triton X-100 and alpha-tocopherol hydrogen succinate ester is diluted in the water for injection.Then with 3 kinds of spissated unit price stock solutions (strain A/ New Caledonia-, strain A/ Panama-, strain B/ Shandong-) serial dilution go into to obtain PBS/(pH 7.4 for Tween 80-Triton X-100-alpha-tocopherol hydrogen succinate ester solution; 137mM NaCl; 2.7mM KCl, 8.1mM Na ₂HPO ₄, 1.47mM KH ₂PO ₄1500 μ g/ml Tween 80; 220 μ g/mlTriton X-100 and 200 μ g/ml alpha-tocopherol hydrogen succinate esters) in, so that have the final concentration of HA (17.5 μ g HA/B strains/250 μ l trivalent semi-finished product) of HA/ml trivalent semi-finished product (15 μ g HA/A strains/250 μ l trivalent semi-finished product) and the 70 μ g B strains of 60 μ g A strains.Adding between every kind of unit price stock solution, reach 10 minutes in the stirring at room mixture.After adding last a kind of unit price stock solution, stirred 15 minutes, check pH, and with HCl or NaOH with pH regulator to 7.2 ± 0.1.

Go in the aseptic vial of 3-ml I type antigenic 3 valency semi-finished product are aseptic subpackaged.Every bottle contains 34% volume plussage (335 μ l cumulative volume).

2) the aseptic stock solution of preparation SB62 emulsion is also divided in the adjuvant container of packing into

Water: when stirring, 902ml Tween 80 is mixed (regulating back pH=6.8) with HCl with 44105ml PBS-mod buffer.

Oil phase: when stirring, the 2550ml zamene is added in the 2550ml alpha-tocopherol.

Mix water and oil phase: when stirring, 5000ml oil phase (1/10 cumulative volume) is transferred to 45007ml water (9/10 cumulative volume).Mixture was in stirring at room 15 minutes.

Emulsifying: in the interaction chamber of micro-fluidic device, the mixture that obtains is applied shearing force, impulsive force and cavitation force (15000 PSI, 8 circulations), to produce submicron droplets (being distributed between the 100-200nm).The pH that obtains is between 6.8 ± 0.1.

Aseptic filtration: make the SB62 emulsion through 0.22 μ m membrane filtration degerming, with aseptic emulsifiable concentrate stored frozen in 2-8 ℃ Cupac container.The dead volume that aseptic noble gas (nitrogen or argon) is fed SB62 emulsifying semi-finished product container reaches at least 15 seconds.

The amount of all the components that provides all is used to prepare 50 L emulsions, and provides with volume.In fact, amount is with the densitometry of composition.The density of PBS is regarded as and equals 1.

The final composition of SB62 emulsion is following:

Table 5

Go in the aseptic I type of the 1.25-ml glass syringe aseptic SB62 emulsifiable concentrate is aseptic subpackaged then.Each syringe contains 34% volume plussage (335 μ l cumulative volume).

3) the instant reprovision of AS03 adjuvant split-virus vaccine.

When injection, the content that contains the bottle that concentrates 3 valency inactivation lytic virus isoantigens is removed by means of the syringe that contains the SB62 emulsion, follows slight mixing syringe.SB62 emulsion and vaccine antigen mixing reprovision AS03 adjuvant.

III.2.2. vaccine is formed (table 6) and is given

Table 6

The vaccine intramuscular gives the deltoid region in non-habitual arm.Close observation inoculator at least 30 minutes implements the suitable therapeutic treatment of utilization easily for rare anaphylactic reaction after giving vaccine.

III.3. study the result of colony

148 experimenters have been recruited in this research altogether: 49 experimenters organize at FluAS03, and 49 experimenters organize at Fluarix, and 50 experimenters organize at FIuWVV.Total inoculation crowd's mean age is 71.8 years old when inoculation, standard deviation 6.0 years old.It is similar that experimenter's mean age and sex are distributed between 3 vaccine group.

III.4. safety conclusion

It is safe in research colony (being the old people of over-65s), using the influenza vaccines of AS03 adjuvantization, is well tolerable clinically.

III.5. immunogenicity result

Immunogenicity to total inoculation crowd is analyzed.

III.5.1. HI

In order to estimate, calculate the following parameter (having 95% confidence interval) of each treatment group by the vaccine-induced HI of AS03 adjuvantization:

The geometric mean titer (GMT) of HI and NI antibody titer when the 0th day and 21 days

The geometric mean titer (GMT) of NAT when the 0th day and 21 days

Serological conversion rate in the time of 21 days (SC) is defined as than the serum HI titre increase the 21st day time the in the 0th day and reaches inoculator's percentage rate of at least 4 times.

Conversion factor in the time of 21 days is defined as than the 0th day increase multiple to the serum HI GMT of every kind of vaccine strain the 21st day the time.

Protective rate in the time of 21 days is defined as inoculator's percentage rate of serum HI titre=1: 40.

The anti-hemagglutinin antibody response of III.5.1.1

A) HI geometric mean titer (GMT)

GMT with HI antibody of 95%CI is shown in table 7 (GMT of anti-HI antibody).

Be in the same range as to antibody GMT before the inoculation of all vaccine strains in 3 groups.After inoculation, anti-hemagglutinin antibody horizontal significantly increases.A kind of trend is being arranged after the inoculation: the GMT to the HI antibody of whole 3 kinds of vaccine strains in FluAS03 and Fluarix group is higher, although the 95%CI between Fluarix group and the FIuWVV group has some overlapping.

Table 7

The available experimenter's number of N=result;

The 95%CI=95% confidence interval; The LL=lower limit; The UL=upper limit;

MIN/MAX=minimum/maximum;

Preceding the 0th day of PRE=inoculation;

Back the 21st day of PI (D21)=inoculation

B) conversion factor of anti-HI antibody titer, serum protective rate and serological conversion rate (relevant) with the protective effect of philtrum

The result is shown in table 8.

Conversion factor representative was than the 0th day increase multiple to the serum HI GMT of each vaccine strain the 21st day the time.Conversion factor changes by 6.1 to 13.6 according to Strain and vaccine.This conversion factor is superior to 2.0 times of GMT increases that European official requires widely.

Experimenter's ratio of serum protective rate representative serum HI titre >=40 in the time of the 21st day.When the research beginning, half experimenter (scope is between 34.0%-69.4%) of all groups has the protection antibody level to institute's toxic strain.In the time of the 21st day, in 3 groups to the serum protective rate of different virus strain in 88.0% to 100% scope.With regard to protective effect, this means that surpassing 88% experimenter has after inoculation >=40 serum HI titre, be considered to have the protective effect of anti-3 kinds of strains.This ratio is superior to widely that European official requires >=60 years old elderly population in 60% serum protective rate.

Serological conversion rate representative than the 0th day the 21st day the time serum HI titre have the experimenter's ratio that reaches at least 4 times of increases.Whole response rate to 3 kinds of strains equates in 3 groups basically.In order to be considered to effectively and to meet European Union's standard, vaccine should be induced in=60 years old elderly population and surpassed 30% positive rate of rotation.In this research, the positive rate of rotation of 3 groups surpasses 50%.

Table 8

N=experimenter's sum

Sum up:

A kind of trend is being arranged after the inoculation: the GMT to the HI antibody of whole 3 kinds of vaccine strains in FluAS03 and Fluarix group is higher, although the 95%CI between Fluarix group and the FIuWVV group has some overlapping.

Conversion factor changes by 6.1 to 13.6 according to Strain and vaccine.This conversion factor is superior to 2.0 times of GMT increases that European official requires greatly.

In the time of the 21st day, in 3 groups to the serum protective rate of different virus strain in 88.0% to 100% scope.This ratio is superior to greatly that European official requires >=60 years old elderly population in 60% serum protective rate.

In this research, the serological conversion rate of 3 groups surpasses 50%.Whole response rate to 3 kinds of strains equates in 3 groups basically.

The III.5.1.2 NAT

In order to characterize the immunne response of inoculating to influenza among the old people better, estimate to the antigenic serum antibody response of neutralization.The result is shown in table 9 (geometric mean titer of serum protective rate and anti-NAT (GMT)) and table 10 (at back 21 days anti-neutral positive rates of rotation (increasing multiple=4) of inoculation).

Detect the NAT of anti-3 kinds of influenza strains in the serum of the preceding and immune back of immunity.Measure following parameter:

The geometric mean titer (GMT) that has the serum neutralizing antibody of 95% confidence interval (95%CI) before the inoculation and after the inoculation

The positive rate of rotation that in the time of 21 days, has 95%CI, be defined as than the 0th day the 21st day the time HI titre have the inoculator's percentage rate that reaches at least 4 times of increases.

Table 9

The spissated Flu vaccine of group 1:Flu vaccine mixing adjuvant 2x;

Group 2:Flu vaccine Flu vaccine;

Group 3:Flu vaccine FluWVV vaccine;

The available experimenter's number of N=result;

Number/percentage rate of the experimenter of n/%=titre in particular range;

The 95%CI=95% confidence interval; The LL=lower limit; The UL=upper limit;

Preceding the 0th day of PRE=inoculation; Back the 21st day of PI (D21)=inoculation

Table 10

Group 1:Flu vaccine (DFLU58A16) mixes the spissated Flu vaccine of adjuvant (D621024A8) 2x;

Group 2:Flu vaccine (1885489) Flu vaccine;

Group 3:Flu vaccine (DFLU59A2) Flu WVV vaccine;

Preceding and the available experimenter's number of inoculation back result of N=inoculation;

N=respondent number;

%=respondent percentage rate (n/N * 100);

Accurate 95% confidence interval of 95%CI=; The LL=lower limit; The UL=upper limit;

Main discovery is:

For 3 kinds of vaccines, in the time of 21 days, to the serum protective rate of two kinds of A strains acquisitions 100%.For the B strain, the serum protective rate in 3 groups is in 92% to 100% scope.

After the inoculation, the GMT to institute's toxic strain in 3 groups has remarkable increase.Yet, a kind of trend is arranged: in FluAS03 and Fluarix group, be directed against GMT ratio height in FIuWVV of the neutralizing antibody of whole 3 vaccine strains, although the 95%CI between Fluarix group and the FIuWVV group has some overlapping.

For serological conversion rate, equal basically in 3 groups to the overall response rate of 3 kinds of strains.

In all groups, the result is with to analyze the result who is obtained consistent to anti-hemagglutinin antibody.

III.5.1.3 neuraminidase (NA) antibody titer

In order to be characterized in the immunne response of in the elderly population influenza being inoculated better, estimate the antigenic serum antibody response of neuraminidase.Similar with the HI antibody titer, measure following terminal point:

GMT (adopting the logarithm titre to transform the antilogarithm of meansigma methods)

Serological conversion rate, be defined as than the 0th day 21 days the time HI titre to every kind of vaccine strain have the inoculator's percentage rate that reaches at least 4 times of increases.

The GMT and the serological conversion rate that in table 11 (anti-NA antibody GMT) and table 12 (postvaccinal NA serological conversion rate (the 21st day) (4 times of increases)), have shown NI antibody with 95%CI.

Table 11

FluAS03: with the blended Flu vaccine of AS03 adjuvant (D621024A8) (DFLU58A16)

Fluarix:Flu vaccine (18854B9)

FluWVV:Flu WVV vaccine (DFLU59A2)

Before the PRE=inoculation, back the 21st day of PI (D21)=inoculation

95%CI, LL and UL=95% confidence interval, lower limit and the upper limit

Table 12

FluAS03: with the blended Flu vaccine of AS03 adjuvant (D621024A8) (DFLU58A16), Fluarix:Flu vaccine (18854B9), FluWVV:Flu WVV vaccine (DFLU59A2)

Preceding and the available experimenter's number of inoculation back result of N=inoculation;

N=respondent number;

%=respondent ratio (n/N * 100);

Accurate 95% confidence interval of 95%CI=; The LL=lower limit; The UL=upper limit

The main discovery is:

Value to observed GMT of hemagglutinin and serological conversion rate is higher than the observed value of neural ammonia Suan enzyme.

Antibody GMT is in same range as before in 3 groups, being directed against the inoculation of all vaccine strains.After inoculation, anti-neuraminidase antibody horizontal significantly increases.As for the HI antibody titer, a kind of trend is being arranged after the inoculation: the GMT to the HI antibody of whole 3 kinds of vaccine strains in FluAS03 and Fluarix group is higher, although the 95%CI between Fluarix group and the FIuWVV group has some overlapping.

About serological conversion rate, in 3 groups and concerning 3 kinds of strains, equate basically to the overall response rate of 3 kinds of strains.

Our result shows, the healthy elderly development of influenza inoculation goes out the good antibody response to neuraminidase in this research, no matter which kind of influenza vaccine.But, to neuraminidase antigenic reply to be lower than to hemagglutinin is antigenic reply.

III.5.2. cellullar immunologic response

Can be at external peripheral blood antigenic specificity CD4 and the CD8 T cell of stimulating again, to produce IL-2, CD40L, TNF-α and IFN γ, if corresponding antigen incubation with it.Therefore, antigenic specificity CD4 and CD8 T cell can be through the flow cytometer countings after routine immunization fluorescence labeled cell phenotype and intracellular cytokine production.In this research, influenza vaccines antigen and derive from the proteic peptide of specific influenza and come to stimulate again the influenza specific T-cells as antigen.The result of CD4 and CD8 t cell response is provided in table 13-18.

The antigenic specificity CD4T-cell response that table 13 is represented with the cell that produces two kinds of different cytokines at least: to inoculating descriptive statistic (always inoculating the crowd) preceding and postvaccinal CD40L/IL2/TNF-α/IFN-γ

[0528]?

Group 1:FluAS03:Flu vaccine Fluarix ^TM, mix with the AS03 adjuvant

Group 2:Fluarix:Flu vaccine Fluarix ^TM

Group 3:FIuWVV:Flu WVV vaccine

The SD=standard deviation; Min, Max=minimum, maximum

The Q1=first quartile; Q3=the 3rd quartile

The available experimenter's number of N=result

P-value=Kruskall-Wallis checks (nonparametric technique), tests the local difference (Wilcoxon rank test) in the time of the 21st day between 3 groups

The antigenic specificity CD4T-cell response that table 14 is represented with the cell that produces two kinds of different cytokines at least: to before inoculating and after the inoculation DifferenceDescriptive statistic (always inoculating the crowd)

[0539]Table 15 is to produce the antigenic specificity CD4 T-cell response that the cell of CD40L and another kind of cytokine is at least represented: to before inoculating with inoculation after DifferenceDescriptive statistics (always inoculating the crowd)

[0542]Table 16 has produced the antigenic specificity CD4 T-cell response that the cell of IFN γ and another kind of cytokine is at least represented: to before inoculating with inoculation after DifferenceDescriptive statistics (always inoculating the crowd)

[0545]Table 17 is to produce the antigenic specificity CD4 T-cell response that the cell of IL2 and another kind of cytokine is at least represented: to before inoculating with inoculation after DifferenceDescriptive statistics (always inoculating the crowd)

[0548]Table 18 is to produce the antigenic specificity CD4 T-cell response that the cell of TNF α and another kind of cytokine is at least represented: to before inoculating with inoculation after DifferenceDescriptive statistics (always inoculating the crowd)

[0551]The result also is expressed as the frequency of male CD4 of cytokine in CD4 or the CD8 T cell subsets or CD8 T cell, and is shown in Fig. 4 and Fig. 5.

In similar analysis, use the influenza antigens of drift strain (A/H1N1/ Beijing/262/95 (H1N1d), A/H3N2/ Sydney/5/97 (H3N2d), B/ Pyrusussuriensis/166/98 (Bd)) or conversion strain (A/ Singapore/1/57 (H2N2), A/ Hong Kong/1073/99 (H9N2)) to estimate cross reactivity CD4 T-cell response.The result is expressed as the frequency of the male CD4 T of cytokine cell, is shown in Fig. 6.

The main discovery is:

With Fluarix and totivirus inoculation the reinforcement a little CD4 T-cell response.Induce strong CD4 T-cell response (Fig. 4) with Flu AS03 inoculation, it is significant on statistics.After stimulated in vitro, drawing identical conclusion with cracking antigen or totivirus, also is like this to all cells factor of studying (IL-2, IFN γ, TNF α and CD40L).

The major part individuality has the CD8 T-cell response of anti-full influenza virus, but inoculation does not have detectable influence (promptly inoculating preceding=inoculation back), whichsoever seminar (Fig. 5) to the CD-8 t cell response.

Only induce low-level cross reactivity CD4 T-cell response (Fig. 6) with the Fluarix inoculation.Induce the strong CD4 T-cell response that resists the strain of drift influenza with the FluAS03 inoculation, it is significant (Fig. 6) on statistics.Basically do not detect replying of anti-conversion strain.

III.5.3.B-cell ELISPOT memory

The III.5.3.1 target

Reply in order to characterize better by the inductive CMI of AS03 adjuvant influenza vaccines, evaluation use influenza vaccines strain or anti-human normal immunoglobulin are external evoked to be divided into plasmacytic B-cell Elispot memory response, so that the plasma cell of counting influenza or IgG secretion.The result is described in table 19 and table 20 and Fig. 7.

Select the part among 22 experimenters that accept 1 dose of FluAS03 vaccine first and 21 experimenters that accept 1 dose of Fluarix vaccine first, to use the influence of B-cell memory Elispot technology assessment inoculation to influenza specificity memory B cell.Measure following terminal point:

At the 0th day and the 21st day: detected the influenza specificity memory B cell among all experimenters through B-cell Elispot.The result is expressed as per 1,000,000 (10 ⁶) the influenza specific antibody forms the frequency of cell among the antibody forming cell.

Difference before inoculation back (the 21st day) and the inoculation between (the 0th day) also is expressed as per 1,000,000 (10 ⁶) the influenza specific antibody forms the frequency of cell among the antibody forming cell.

III.5.3.2. statistical method

Each inoculation group is expressed as per 1,000,000 (10 at the descriptive statistics of the 0th day and the 21st day ⁶) the influenza specific antibody forms the frequency of cell among the individual antibody forming cell.The descriptive statistics of (before the inoculation back-inoculation) individual variation is per 1,000,000 (10 between the 21st day and the 0th day ⁶) the influenza specific antibody forms the frequency of cell among the individual antibody forming cell.

The Wilcoxon check is used to contrast the local difference between two groups, calculates to statistical p-value of every kind in 3 kinds of strains (A/ New Caledonia, A/ Panama and B/ Shandong).

III.5.3.3 result

Than the Fluarix group, there is the trend of a kind of AS03 of helping adjuvant influenza vaccines.For the strain of A/ New Caledonia, have the statistically-significant difference (p-value=0.021) that is beneficial to FluAS03 than Fluarix.Do not observe two significant differences between the group for A/ Panama and the strain of B/ Shandong.

Table 19 B-cell memory: to the descriptive statistics and 10 of (the 0th day) before inoculating and inoculation back (the 21st day) ⁶The inferential statistics of (the 21st day) frequency after antigen in the individual product IgG plasma cell (a part of experimenter)-plasmacytic inoculation

[0571]Group 1:Flu vaccine Fluarix+AS03 oil in water emulsion adjuvant

Group 2:Flu vaccine Fluarix ^TM

The SD=standard deviation

Min, Max=minimum, maximum

The Q1=first quartile

Q3=the 3rd quartile

The available experimenter's number of N=result

P-value=Wilcoxon checks (nonparametric technique), the local difference when testing the 21st day between 2 groups (Wilcoxon rank test).

Table 20 B-cell memory: to 10 ⁶In the individual product IgG plasma cell (a part of experimenter) antigenic specificity plasma cell frequency after inoculation (the 21st day) with inoculate before the descriptive statistics and the inferential statistics of difference between (the 0th day)

Group 1:Flu vaccine Fluarix+AS03 oil in water emulsion adjuvant

Group 2:Flu vaccine Fluarix ^TM

The SD=standard deviation

Min, Max=minimum, maximum

The Q1=first quartile

Q3=the 3rd quartile

The available experimenter's number of N=result

P-value=Wilcoxon checks (nonparametric technique), the local difference when testing the 21st day between 2 groups (Wilcoxon rank test).

III.6. whole conclusion

III.6.1. reactionogenicity and safety results

Although the influenza immunity has significantly reduced the risk of pneumonia and associated death, old people's inoculation only provides the protective effect of the influenza disease of 23-72%.Vaccine antigen is the attractive method of a kind of enhancing to the immunne response of subunit antigen with the preparation of effective adjuvant.This research design be used for estimating (1) with O/w emulsion be the influenza vaccines of AS03 adjuvantization at safety of healthy geriatric philtrum and reactionogenicity, (2) antibody and cell-mediated immune responses.The reactionogenicity data show, bring out more part and General Symptoms with the influenza vaccines of AS03 adjuvantization than other two kinds of vaccines.But,, do not observe 3 kinds of differences between the vaccine for the adverse events of positive appeal.Can be reached a conclusion by these results, the reactionogenicity of candidate vaccine and safety are gratifying, are acceptable clinically.

III.6.2. immunogenicity result

With regard to immunne response, 3 kinds of vaccines exceeded European official to the requirements of the lytic virus body influenza vaccines of registration in every year (" the guide explanation that requires about the coordinated flow influenza vaccine ", be used for immunological evaluation year strain change-CPMP/BWP/214/96).In this research the test 3 kinds of influenza vaccines be immunogenic at the healthy geriatric philtrum, these old peoples to the influenza hemagglutinin with in antigen development good antibody response (table 21).

Table 21

(CMI) replys for cell-mediated immunity, replys (comprise drift strain) with the inductive CD4 of the influenza vaccines of AS03 adjuvantization and significantly is better than other two kinds of vaccines (Fluarix and full influenza virus vaccine).But inoculation is replied CD8 does not have detectable influence.

Reply about the B cell memory,, have the trend that is beneficial to the adjuvant influenza vaccines than no Adjuvanted vaccines.

EXAMPLE IV-usefulness contains the clinical experiment-Explo-Flu-002 of vaccine in the elderly population of over-65s of cracking influenza antigens prepared product and AS03 adjuvant

The GlaxoSmithKline Biologicals influenza candidate vaccine that contains adjuvant AS03 for evaluation is reactionogenicity and the immunogenicity in the over-65s elderly population of inoculation candidate vaccine in the Explo-Flu-001 clinical experiment in 2003 formerly, has carried out opening, controlled I/II phase and has studied.For immunogenicity and safety evaluatio, Fluarix ^TMVaccine (is called α-rix in Belgium ^TM) as reference.

IV.1. target

After intramuscular gives the vaccine of 1 dose of AS03 adjuvantization 21 days, detect HI (promptly anti-hemagglutinin antibody titer) and cell-mediated immune responses (CD4 and/or CD8 t cell response) and B memory cell and reply.Fluarix ^TMAs reference.

Target is:

Whether the Flu (40 experimenters) that 1) confirms the AS03 adjuvantization has confirmed its strongest immunostimulatory activity to the individual immunity of influenza antigens inoculation of CD4-and/or CD8-mediation with respect to Fluarix (18 experimenters);

2) use vertical analysis research AS03 to do the influence of adjuvant to the preceding immunne response (inoculating back 1 year replying just in 2003 first) of inoculation in 2004.

IV.2. research design, vaccine are formed and terminal point

40＞65 years old experimenter of ■, they had before accepted the influenza vaccines (FluAS03) of 1 dose of AS03 adjuvantization in Explo-Flu-001 clinical experiment in 2003.

1 about 20 over-65s experimenter's of ■ matched group, they had before accepted 1 dose of Fluarix in Explo-Flu-001 clinical experiment in 2003 ^TM(Fluarix).

IV.2.1. vaccine is formed

The vaccine composition is similar to the vaccine composition that is used to study Explo-Flu-001, and the influenza strain that only is included in the vaccine (vaccine in 2004) is different.Strain is following:

A/ New Caledonia/20/99 (IVR-116) (H1N1)=A/ New Caledonia/(H1N1)-similar strain

A/ Wyoming/3/2003 (X-147) (H3N2)=A/ Fujian (H3N2)-similar strain

B/ Jiangsu/10/2003=B/ Shanghai-similar strain

IV.2.2. immunogenicity (HI) terminal point

GMT (adopting the logarithm titre to transform the antilogarithm of meansigma methods)

Conversion factor (than the increase multiple of the 0th day serum HI GMT the 21st day the time)

Serological conversion rate (than the 0th day the 21st day the time HI titre to every kind of vaccine strain have the inoculator's percentage rate that reaches at least 4 times of increases)

Protective rate (serum HI titre in the time of the 21st day >=1: 40 inoculator's percentage rate)

The IV.2.3.CMI-terminal point

Observational variable:

At the 0th day and the 21st day: per 10 ⁶Be directed against the frequency of the positive CD4/CD8 cell of cytokine of 4 kinds of different cytokines in the individual cell.Each test is all quantitatively to following CD4/CD8 t cell response:

3 kinds of antigenic mixture below the ■

■ New Caledonia antigen

■ Wyoming antigen

■ Jiangsu antigen

The variable of deriving:

In 5 kinds of different tests (cytokine) by the antigenic specificity CD4 T cell and the CD8 t cell response of following expression:

1. produce the cell of at least two kinds of different cytokines (CD40L, IL-2, IFN γ, TNF α)

2. produce the cell of CD40L and another kind of cytokine (IL-2, TNF α, IFN γ) at least

3. produce the cell of IL-2 and another kind of cytokine (CD40L, TNF α, IFN γ) at least

4. produce the cell of IFN γ and another kind of cytokine (IL-2, TNF α, CD40L) at least

5. produce the cell of TNF α and another kind of cytokine (IL-2, CD40L, IFN γ) at least

IV.2.4.CMI analyzes

First CMI analyzes based on total inoculation crowd (FluAS03 group N=40 name experimenter, Fluarix group N=18 name experimenter).

Vertical analysis is based on the dynamic crowd of Explo-Flu-001 (crack protein) and Explo-Flu-002 (blended flu antigen) research:

Before the ■ inoculation: FluAS03 group N=36 name experimenter, Fluarix group N=15 name experimenter.

Before the ■ inoculation back-inoculation: FluAS03 group N=34 name experimenter, Fluarix group N=15 name experimenter.

(a) through the descriptive statistics of every kind of antigen, every kind of cytokine, each vaccine group and each time point (before the inoculation and inoculation back) is summed up the frequency that CD4/CD8 T-lymphocytic emiocytosis is replied.

The descriptive statistics of (b) every kind of antigen, every kind of cytokine and each vaccine group individuality between time point (inoculation before with inoculation afterwards) being replied difference is tabulated.

(c) for inoculation back time point with (inoculation back-preceding), use the relatively local difference between two vaccine group of nonparametric Wilcoxon check, and the statistical p-values below calculating with regard to 4 kinds of different cytokines:

-to the CD4 T-cell response of New Caledonia, Wyoming, Jiangsu and 3 kinds of strain mixture

-to the CD8 T-cell response of New Caledonia, Wyoming, Jiangsu and 3 kinds of strain mixture

(d) non parametric tests (Wilcoxon-check) also is used for:

-according in each vaccine group Between Explo-Flu-001 and the Explo-Flu-002The frequency of specific C D4, the kinetics of (the 0th day) immunne response before the research inoculation

-according to study at Explo-Flu-001 and Explo-Flu-002 each in Between 2 vaccine groupThe frequency of specific C D4, the kinetics of (the 0th day) immunne response before the research inoculation

-according in each vaccine group Between Explo-Flu-001 and the Explo-Flu-002The frequency difference of specific C D4 (before the inoculation back-inoculation), the kinetics of research immunne response

-according to study at Explo-Flu-001 and Explo-Flu-002 each in Between 2 vaccine groupThe frequency difference (inoculation back-inoculation preceding) of specific C D4, study the kinetics of immunne response

All significance test all are two tails.Being less than or equal to 0.05 P-value, to be considered to statistics significant.

IV.3. result

The result is expressed as the frequency of positive CD4 of cytokine in CD4 or the CD8 T cell subsets or cd8 t cell.

IV.3.1. antigenic specificity CD4 T-lymphocyte

Through the descriptive statistics of every kind of antigen, every kind of cytokine, each vaccine group and each time point (before the inoculation and inoculation back) is summed up the frequency that antigenic specificity CD4 T-lymphocytic emiocytosis is replied.

In table 22, shown descriptive statistics for every kind of antigen, each 5 kinds of different cells factor and each vaccine group CD4 T-lymphocyte responses individual variation between time point (before the inoculation back-inoculation).

The descriptive statistics (always inoculating the crowd) of antigenic specificity CD4 T-lymphocyte responses difference between (at the 0th day) before table 22 inoculation back (at the 21st day) and the inoculation

[0656]The SD=standard deviation

Min, Max=minimum, maximum

The Q1=first quartile

Q3=the 3rd quartile

The number of the available test subject of N=result

Vaccine-induced CD4 T cell demonstrates and can continue at least 1 year, because with comparing between the individuality with the Fluarix/AS03 inoculation individuality and the previous year of Fluarix inoculation, there is observable difference in CD4 T-cell response level before the inoculation.The result also is shown in Fig. 8, has shown the CD4 T-cell response that is directed against the cracking influenza antigen before inoculating and afterwards.Therefore D0 indicates persistence corresponding to back 12 months of inoculation in first year.

Than the lymphocytic frequency difference of antigenic specificity CD4 T-between back 2 groups of inoculation (according to the Wilcoxon check), nearly all p-value is considered to significant (referring to table 23) all less than 0.05 on statistics, and this helps the FluAS03 group.

Table 23 inferential statistics: The 21st dayFrom antigenic specificity between 2 vaccine group of Wilcoxon rank test CD4The p value of T-lymphocyte responses (always inoculating the crowd)

P-value: Wilcoxon check (nonparametric technique) test is the local difference (Wilcoxon rank test) between 2 groups in the time of the 21st day.

Individual variation (before the inoculation back-inoculation) through antigenic specificity CD4-T lymphocyte responses frequency between 2 groups of Wilcoxon check contrast; Following antigen-combination of cytokines occurs less than 0.05 and is considered to the significant p-value of statistics: blended influenza antigen-alldouble, blended influenza antigen-IFN γ and Jiangsu-IFN γ, this helps FluAS03 group (table 24).

Table 24 inferential statistics: through the Wilcoxon rank test calculate not on the same group between antigenic specificity CD4The T-lymphocyte responses Difference between (the 0th day) before (the 21st day) and the inoculation after inoculationP value (always inoculating the crowd)

Local difference (Wilcoxon rank test) between 2 groups of P-value: Wilcoxon check (nonparametric technique) test.

IV.3.2. antigenic specificity CD8 T-lymphocyte

Through the descriptive statistics of every kind of antigen, every kind of cytokine, each vaccine group and each time point (before the inoculation and inoculation back) is summed up the frequency that antigenic specificity CD8 T-lymphocytic emiocytosis is replied, be similar to the method that the CD4 t cell response is adopted.

Through the lymphocytic frequency difference of antigenic specificity CD8 T-between back 2 groups of Wilcoxon check contrast inoculation, all p-values all are higher than 0.05, are considered to not be that statistics is significant.Through the individual variation (before the inoculation back-inoculation) of antigenic specificity CD8 T-lymphocyte responses frequency between 2 groups of Wilcoxon check contrast, all p-values all are higher than 0.05, are considered to not be that statistics is significant.

IV.3.3. dynamic analysis: the immunne response of (inoculating back 1 year first) before the inoculation in 2003

Through each descriptive statistics, the descriptive statistics of each and each vaccine group of two researchs in the his-and-hers watches 27 of every kind of cytokine in the his-and-hers watches 25 and each vaccine group and two researchs, sum up the frequency that inoculation pro-antigen specific C D4 T-lymphocytic emiocytosis is replied.Inferential statistic provides in table 16 and table 28.

The descriptive statistics (kinetics) of (the 0th day) specific C D4 T lymphocyte responses inoculation before the table 25 pair inoculation

The SD=standard deviation

Min, Max=minimum, maximum

The Q1=first quartile

Q3=the 3rd quartile

The available experimenter's number of N=result

Through the lymphocytic frequency difference of antigenic specificity CD4 T-of each vaccine group between 2 researchs of Wilcoxon check contrast, only for the FluAS03 group with adopt the situation of TNF α cytokine to occur and be considered to the significant p-value of statistics (helping Explo-Flu-002) (referring to table 26) less than 0.05.

Table 26 inferential statistic: between the difference research from the Wilcoxon rank test The 0th dayAntigenic specificity CD4The p-value (kinetics) of T-lymphocyte responses

Between 2 groups of P-value: Wilcoxon check (nonparametric technique) test the 21st day local difference (Wilcoxon rank test).

The descriptive statistics (kinetics) of (the 0th day) specific C D4 T lymphocyte responses inoculation before the table 27 pair inoculation

The SD=standard deviation

Min, Max=minimum, maximum

The Q1=first quartile

Q3=the 3rd quartile

The available experimenter's number of N=result

Through the difference of antigenic specificity CD4 T-lymphocyte frequency between 2 vaccine group of Wilcoxon check contrast of each research, all p values of Explo-Flu-002 are considered to significant (helping FluAS03) (referring to table 28) of statistics all less than 0.05.

Table 28 inferential statistic: from the Wilcoxon rank test not on the same group between The 21st dayAntigenic specificity CD4The p-value (kinetics) of T-lymphocyte responses

Between 2 groups of P-value: Wilcoxon check (nonparametric technique) test the 21st day local difference (Wilcoxon rank test).

IV.3.4. dynamic analysis: deduct the preceding immunne response of inoculation after the inoculation

Through every kind of cytokine in the his-and-hers watches 29 and the descriptive statistics of each vaccine group and each research, each research and the descriptive statistics of each vaccine group in the his-and-hers watches 31, be summarised in the frequency that the antigenic specificity CD4 T-lymphocytic emiocytosis of (inoculation back-inoculation is preceding) time point is replied.Inferential statistic provides in table 30 and table 32.

The descriptive statistics (kinetics) of specific C D4 T lymphocyte responses inoculation between (the 0th day) before table 29 inoculation back (the 21st day) and the inoculation

The SD=standard deviation

Min, Max=minimum, maximum

The Q1=first quartile

Q3=the 3rd quartile

The available experimenter's number of N=result

Difference through antigenic specificity CD4 T-lymphocyte frequency between 2 researchs of Wilcoxon check contrast of each vaccine group; All p-values of FluAS03 group are considered to significant (helping Explo-Flu-001) (referring to table 30) of statistics all less than 0.05.

The inferential statistic of difference between (the 21st day) and the inoculation preceding (the 0th day) after the table 30 pair inoculation: between the difference research from the Wilcoxon rank test The 21st dayAntigenic specificity CD4The p-value (kinetics) of T-lymphocyte responses

The 21st day local difference (Wilcoxon rank test) between 2 groups of P-value: Wilcoxon check (nonparametric technique) test.

The descriptive statistics (kinetics) of specific C D4 T lymphocyte responses inoculation difference between (the 21st day) and the inoculation preceding (the 0th day) after the table 31 pair inoculation

The SD=standard deviation

Min, Max=minimum, maximum

The Q1=first quartile

Q3=the 3rd quartile

The available experimenter's number of N=result

Through the difference of antigenic specificity CD4 T-lymphocyte frequency between 2 vaccine group of Wilcoxon check contrast of each research, only the p value of Explo-Flu-001 is considered to significant (helping FluAS03) (referring to table 32) of statistics less than 0.05.

Table 32 inferential statistic: from the Wilcoxon rank test not on the same group between The 21st dayAntigenic specificity CD4The p-value (kinetics) of T-lymphocyte responses

Between 2 groups of P-value: Wilcoxon check (nonparametric technique) test the 21st day local difference (Wilcoxon rank test).

The IV.4.HI titre

The result is shown in Fig. 9 and Biao 33-36.

Table 33: geometric mean titer of anti-HI titre (GMT) and seroprevalence (GMT calculates to the experimenter of inoculation)

Before the PRE=inoculation

Back 21 days of PI (D21)=inoculation

95%CI, LL and UL=95% confidence interval, lower limit and the upper limit

S+=seropositivity experimenter's number

Table 34: the conversion factor of anti-HI titre (experimenters of all inoculations)

N=experimenter's sum

GMR=geometric average ratio (the average logarithmic antilogarithm of the 21st day/the 0th day titre ratio)

The 95%CI=95% confidence interval

Table 35: the serum protective rate of anti-HI titre (experimenters of all inoculations)

Before the PRE=inoculation

Back 21 days of PI (D21)=inoculation

The available experimenter's number of N=result

The experimenter number of n=titre in particular range

The experimenter percentage rate of %=titre in particular range

Table 36: the 21st day serological conversion rate of PI (increasing multiple=4) (all experimenters of inoculation)

Preceding and the available experimenter's number of inoculation back result of N=inoculation

N=respondent number

%=respondent ratio (n/N * 100)

Accurate 95% confidence interval of 95%CI=; The LL=lower limit, the UL=upper limit

IV.5. whole conclusion

Clinical research confirmation thus; With regard to the frequency of influenza specific C D4 T cell; Also just to inoculating research D0 (Explo Flu 002; Promptly ± 1 year) till the persistency of the immunne response that excites of Flu-AS03 inoculation first (in Explo Flu 001 first exempt from inoculation), have Adjuvanted vaccines Flu-AS03 to be superior to the no Adjuvanted vaccines Fluarix of equity.And this is replied and can discern the drift influenza strain that exists in the novel vaccine, and discerns the strain of influenza vaccines in 2004.

Opposite with inoculation in 1 year, previous when inoculating with adjuvant Fluarix is arranged ^TMThe inoculation individual the time demonstrate than no adjuvant Fluarix ^TMThe HI titre response that the individuality of inoculation increases.Can be observed a kind of trend: the HI titre of anti-H1N1 and H3N2 strain increases to 1.5-2 doubly, and the HI titre of anti-B strain has the statistics that has shown to be increased.

Evaluation before the influenza vaccines of EXAMPLE V-have adjuvant and no adjuvant clinical in ferret

The effectiveness of first research-novel formulation AS03 and AS03+MPI

V.1. ultimate principle and target

With regard to the sensitivity that infects and clinical response this two, the influenza infection in the ferret model exactly likes human influenza.

Formerly do not carry out under the situation of Strain immunity, ferret is to the infection of influenza A and influenza B this two sensitivity very.Therefore, its protective effect of being given by the influenza vaccines that give for research provides splendid model system.

The various 3 valency split vaccines that adjuvant arranged or do not have the adjuvant effectiveness to virus shedding in the minimizing symptom (body temperature) of the ferret attacked with the homology strain and the nasal discharge has been studied in this research.

The target of this experiment is to show the effectiveness of adjuvant influenza vaccines than common (no adjuvant) vaccine is arranged.

Terminal point is:

1) main terminal point: reduce homology and attack the virus shedding in the nose cleanout fluid of back;

2) secondary endpoints: reply and monitor the temperature of just exempting from and attacking front and back through the IHA analysing body fluid.

V.2. experimental design

V.2.1. treatment/group (table 37)

(Hampshire UK) obtains the 14-20 female ferret (Mediterranean ferret (Mustela putorius furo)) (6 ferret/groups) in age in week by MISAY Consultancy.Ferret at the 0th day with xenogenesis hypotype strain H1N1 A/ Stockholm/24/90 (4 Log TCID ₅₀/ ml) just exempt from.At the 21st day, with the combination intramuscular injection ferret of H1N1 A/ New Caledonia/20/99 of total man's dosage (500 μ g vaccine doses, 15 μ g HA/ strains), H3N2 A/ Panama/2007/99 and B/ Shandong/7/97.Passed through the intranasal approach with homotype strain H3N2 A/ Panama/2007/99 (4.51 Log TCID at the 41st day then ₅₀/ ml) attack ferret.

Table 37

V.2.2. the preparation of bacterin preparation

1: three minute common (no adjuvant) preparation (500 μ l) of preparation:

10 times of concentrated solutions of PBS when concentrated (1 times be pH7.4) and the mixture (amount counts the detergent that exists in the strain) that contains Tween 80, TritonX-100 and VES are added in the water for injection.The detergent amount that reaches is following: every 1ml has 750 μ g Tween, 80,110 μ g TritonX-100 and 100 μ g VES.Stir after 5 minutes, be sequentially added into every kind of strain H1N1 of 15 μ g, H3N2 and 17.5 μ g B strains, stirred 10 minutes between each the adding.Preparation is in stirring at room 15 minutes, if do not give immediately then be stored in 4 ℃.

Preparation 2: with the trivalent cracking influenza preparation (500 μ l) of AS03 adjuvantization:

10 times of concentrated solutions of PBS when concentrated (1 times be pH7.4) and the mixture (amount counts the detergent that exists in the strain) that contains Tween 80, TritonX-100 and VES are added in the water for injection.The detergent amount that reaches is following: every 1ml has 750 μ g Tween, 80,110 μ g TritonX-100 and 100 μ g VES.Stir after 5 minutes, add every kind of strain H1N1 of 15 μ g, H3N2 and 17.5 μ g B strains, stirred 10 minutes between each the adding.Stir after 15 minutes, add 250 μ lSB62 emulsions (like the preparation of the instruction in example II .1).In stirring at room preparation 15 minutes, if do not give immediately then be stored in 4 ℃.

Preparation 3: with the trivalent cracking influenza preparation of AS03+MPL adjuvantization

10 times of concentrated solutions of PBS when concentrated (1 times be pH 7.4) and the mixture (amount counts the detergent that exists in the strain) that contains Tween 80, TritonX-100 and VES are added in the water for injection.The detergent amount that reaches is following: every 1ml has 750 μ g Tween, 80,110 μ g TritonX-100 and 100 μ g VES.Stir after 5 minutes, add every kind of strain H1N1 of 15 μ g, H3N2 and 17.5 μ gB strains, stirred 10 minutes between each the adding.Stir after 15 minutes, add 250 μ l SB62 emulsions (like the preparation of the instruction in example II .1).Restir mixture 15 minutes, the suspension that is prepared by the instruction like embodiment II.3.1 afterwards adds 25 μ g MPL immediately.In stirring at room preparation 15 minutes, if do not give immediately then be stored in 4 ℃.

Remarks: in every kind of preparation, adding 10 times of concentrated solutions of PBS and ooze to waiting, is 1 times of concentrated solution in final volume.Calculate H ₂The O volume is to reach target volume.

V.2.3. understand by (table 38)

Table 38

[0776]In=is independent/and Po=is blended

V.3. result

Result's sketch map provides in Figure 10 and Figure 11.

V.3.1. temperature monitoring

With pick off and through tele rcording (according to the method that in I.2.2, a details) monitoring temperature.Check and rebuild all implants, before putting into the abdominal cavity, newly calibrate through DSI.All animals all are housed in the independent cage independently between these detection periods.

Until attacking back 5 days per 15 minutes record temperature, calculate meansigma methods preceding 3 days of attack by Japan and China.The result of baseline to baseline body temperature is shown in Figure 10 A (having shown-1 to+3 days result) and 10B (having shown-2 to+3 days result).

After attack, only after with common vaccine of trivalent cracking or PBS immunity, observe the body temperature peak value.After trivalent split vaccine immunity, do not observe peak value with AS03 or AS03+MPL adjuvantization.

V.3.2. virus shedding (Figure 11)

Every group of 6 animals are carried out the titration of virus of nose cleanout fluid.

Carry out the nose cleaning through in two nostrils of clear-headed animal, giving 5ml PBS.Inoculum is collected in the culture dish, and places the shuttle of-80 ℃ (dry ice).

All nose samples are all at first through Spin X filter (Costar) aseptic filtration, to remove any germ contamination.The nose cleanout fluid of the continuous 10 times of dilutions of 50 μ l is transferred in the microtitration plate that contains 50 μ l culture medium (10 holes/dilution factor).Then with 100 μ l mdck cells (2.4 * 10 ⁵Individual cell/ml) adds each hole, and in 35 ℃ of incubations, reaches cell until control cells and converge, for example 5-7 days.Behind 6-7 days incubations, take out culture medium carefully, add the culture medium that 100 μ l contain 1/20WST-1, incubation is 18 hours again.

The survivaling cell number that the intensity of the cured dyestuff of yellow first that when survivaling cell reduction WST-1, produces and titration of virus experiment are present in the hole when finishing is proportional, and quantitative through the absorbance that detects each hole at suitable wavelength (450nm).The intercepting value defined is not for infecting OD meansigma methods-0.3 OD (0.3 OD is equivalent to not infect control cells OD ± 3 standard deviation) of control cells.Be defined as negative score during OD <be defined as positive score during the intercepting value, on the contrary, OD>intercepting value.The virus shedding titre is measured through " Reed and Muench ", and is expressed as Log TCID ₅₀/ ml.

Than common vaccine of trivalent cracking or PBS, after attacking, observe lower virus shedding with the trivalent split vaccine of AS03 or AS03+MPL adjuvantization.The protective effect of adopting AS03 is than AS03+MPL good slightly (referring to attacking the back the 2nd day).Because every group size of animal is few, so be not sure of significance,statistical.

V.3.3. experiment conclusion

Than the common vaccine of trivalent cracking, observe higher humoral response (HI titre) (2 kinds in 3 kinds of strains (being H3N2 and B strain) are at least 2 times) to whole 3 kinds of strains with the trivalent split vaccine of AS03 or AS03+MPL adjuvantization.

With regard to regard to the protection in the ferret is renderd a service, AS03 and AS03+MPL preparation demonstrate extra interests (lower virus shedding and temperature) (Figure 10 and 11).

After the attack, do not observe the reinforcement of humoral response after the trivalent split vaccine immunity with AS03 or AS03+MPL adjuvantization.

The special-shaped Attack Research of second research-in ferret: the effectiveness that shows the novel formulation of test

V.4. ultimate principle and target

This research has been studied the effectiveness that adjuvant is arranged or do not have the various trivalent split vaccines of adjuvant through ability that alleviates symptom (body temperature) and the effect that allos is attacked the virus shedding in the immune ferret nasal discharge in back thereof.

V.5. experimental design

(Hampshire UK) obtains the 14-20 female ferret (Mediterranean ferret (Mustela putorius furo)) (6 ferret/groups) in age in week by MISAY Consultancy.Test 4 groups:

^*Fluarix

^*Trivalent split vaccine AS03

^*Trivalent split vaccine AS03+MPL

^*PBS

Ferret at the 0th day with xenogenesis hypotype strain H1N1 A/ Stockholm/24/90 (4 LogTCID ₅₀/ ml) just exempt from.At the 21st day, with the combination intramuscular injection ferret of H1N1 A/ New Caledonia/20/99 of total man's dosage (500 μ g vaccine doses, 15 μ g HA/ strains), H3N2 A/ Panama/2007/99 and B/ Shandong/7/97 (17.5 μ g HA).Passed through the intranasal approach with xenogenesis hypotype strain H3N2 A/ Wyoming/3/2003 (4.51 Log TCID at the 43rd day then ₅₀/ ml) attack ferret.

V.6. result

Result's sketch map provides in Figure 12 and Figure 13.

V.6.1. temperature monitoring

During attacking, monitor a temperature with pick off and through tele rcording.The inspection and rebuild all implants, before putting into the abdominal cavity, newly calibrate through DSI.All animals all are housed in the independent cage independently between these detection periods.

Result (Figure 12) shows:

-in the high transmutability of just exempting between one group of front and back observation and another group.Baseline seems before just exempting from, to exempt from than just back high.

Although there is high transmutability in-body temperature, only in the ferret of PBS (6/6 ferret), the common vaccine of trivalent cracking (5/6 ferret) and AS03 adjuvant trivalent split vaccine (2/6 ferret) immunity, after attacking, observe peak value.After with the immunity of AS03+MPL adjuvant trivalent split vaccine, do not observe peak value (0/6 ferret).

-with regard to the heating prevention, it is effective that anti-allos strain AS03 seems to be not so good as AS03+MPL.We can not conclude that the adjuvant differences is the probability that comes from the preceding antibody horizontal of different attacks.

V.6.2. virus shedding (Figure 13)

Carry out the nose cleaning through in two nostrils of clear-headed animal, giving 5ml PBS.Inoculum is collected in the culture dish, and places the shuttle of-80 ℃ (dry ice).

All nose samples are all at first through Spin X filter (Costar) aseptic filtration, to remove any germ contamination.The nose cleanout fluid of the continuous 10 times of dilutions of 50 μ l is transferred in the microtitration plate that contains 50 μ l culture medium (10 holes/dilution factor).Then with 100 μ l mdck cells (2.4 * 10 ⁵Individual cell/ml) adds each hole, and in 35 ℃ of incubations, reaches cell until control cells and converge, and for example reaches 5-7 days.Behind 6-7 days incubations, take out culture medium carefully, add the culture medium that 100 μ l contain 1/20 WST-1, incubation is 18 hours again.

The survivaling cell number that the intensity of the cured dyestuff of yellow first that when survivaling cell reduction WST-1, produces and titration of virus experiment are present in the hole when finishing is proportional, and quantitative through the absorbance that detects each hole at suitable wavelength (450nm).The intercepting value defined is not for infecting OD meansigma methods-0.3 OD (0.3 OD is equivalent to not infect control cells OD ± 3 standard deviation) of control cells.Be defined as negative score during OD <be defined as positive score during the intercepting value, on the contrary, OD>intercepting value.The virus shedding titre is measured through " Reed and Muench ", and is expressed as Log TCID ₅₀/ ml.

Virus shedding after just exempting from

Detect and just exempt from preceding the 1st day to the virus shedding of just exempting from the 7th day 12 the blood ermines in back.The result representes with hybrid mode.

Virus sweep in the ferret is arranged just exempting from back 7 days observation stations.

Virus shedding after the attack

Detect 6 ferret/rents by attacking preceding 1 day to attacking back 7 days virus shedding.

Attacked back 2 days; Than ferret with common vaccine of trivalent cracking and PBS immunity; In ferret, observe significantly lower virus titer (compare with common vaccine, the difference of adjuvant group AS03/AS03+MPL is respectively 1.25/1.22 log and 1.67/1.64 log) of statistics with AS03 and the immunity of AS03+MPL adjuvant trivalent split vaccine.

At the 50th day, in the nose cleanout fluid, do not detect virus.

V.6.3. hemagglutination suppresses test (HI titre) (Figure 14 A and B)

Just exempt from preceding 1 day, just exempt from back 21 days, back 22 days of immunity and attack and collected blood serum sample in back 14 days.

Use hemagglutination inhibition test (HI) to measure anti-hemagglutinin antibody titer to H3N2 influenza virus (vaccine strain and attack strain).The HI testing principle is based on the specific anti influenza antibodies suppresses the hemagglutination of little chicken red blood cell (RBC) through influenza virus haemagglutinin (HA) ability.Serum at first uses 25% neuraminic acid enzymatic solution (RDE) to handle, and heat inactivation, to remove nonspecific inhibitor.After the pretreatment, with every kind of influenza strain incubation of 2 times of dilution serum and 4 HAUs.Add little chicken red blood cell then, and the record coagulation suppresses.Titre is expressed as the dilution inverse of the highest serum that suppresses hemagglutination fully.Because first serum dilution is 1: 10, so be to equal 5 titre with undetectable horizontal recording.

The result:

The result is shown in Figure 14 A and 14B.After with the immunity of H3N2 A/ Panama, the humoral response (HI titre) that in the ferret with AS03 or the immunity of AS03+MPL adjuvant trivalent split vaccine, observes is than using no adjuvant (common) trivalent split vaccine (Fluarix ^TM) humoral response that observes behind the immune ferret is high.

In the ferret immune, observe similar HI titre with H3N2 A/ Panama of AS03 or AS03+MPL adjuvantization.

Only behind the vaccine immunity that contains A/ Panama H3N2 strain, observe cross reactivity HI titre (after with the common vaccine immunity of trivalent cracking, not observing) to allos strain A/ Wyoming H3N2 with AS03 or AS03+MPL adjuvantization.

In with allos strain A/ Panama H3N2 immunity and ferret, observe the enhancing of A/ Wyoming specificity HI titre with A/ Wyoming H3N2 attack.As expect that attack on the contrary with homology, allos is attacked and caused with the Panamanian specificity HI of A/ titre increase in the ferret of A/ Panama H3N2 immunity of AS03 and AS03+MPL adjuvantization.

V.6.4. the conclusion that should test

As expect, compare with the situation (do not have and strengthen) after homology is attacked, after allos is attacked, observe the reinforcement of anti-H3N2 HI titre.

But, after allos and homology are attacked, observe similar protective effect (virus shedding).Example VI-have the influenza vaccines of adjuvant and no adjuvant just to exempt from clinical preceding evaluation the in the mice at C57BI/6

VI.1. experimental design and target

Common with Fluarix (no adjuvant) vaccine is compared, and in Explo-Flu-001 clinical research (referring to EXAMPLE III), observes to the CD4 t cell response of trivalent influenza split vaccine AS03 significantly higher.Between these two groups, do not observe the difference of CD8 T cell and humoral response this two.

Target is to select the deciphering mode, is similar to the observed CMI of philtrum and replys in mice, to induce.Specifically, target is to reply through using split vaccine AS03 or split vaccine AS03+MPL in mice, to demonstrate than the high CMI of the common vaccine of cracking.

VI.1.1. treatment/group

By Harlan Horst, Netherland obtains the 6-8 female C57BI/6 mice (15 mice/groups) in age in week.Testing group is:

The common vaccine of-trivalent cracking

-trivalent split vaccine AS03

-trivalent split vaccine AS03+MPL

-PBS

Mice was just exempted from xenogenesis hypotype strain (the full inactivation H1N1 of 5 μ g HA A/ Johannesburg/82/96, H3N2 A/ Sydney/5/97, B/ Harbin/7/94) at the 0th day.At the 28th day, with 1.5 μ g HA trivalent split vaccines (A/ New Caledonia/20/99, A/ Panama/2007/99, B/ Shandong/7/97) common vaccine or Adjuvanted vaccines (referring to following group) intramuscular injection mice is arranged.

VI.1.2. the preparation of bacterin preparation

In every kind of preparation, add 10 times of concentrated solutions of PBS and ooze to reach etc., in final volume 1 times of concentrated solution.Formic acid H ₂The O volume is to reach target volume.

Cracking trivalent common (no adjuvant) preparation:

Preparation 1 (reaching 500 μ l): 10 times of concentrated solutions of PBS when concentrated (1 times be pH 7.4) and the mixture (amount counts the detergent that exists in the strain) that contains Tween 80, Triton X-100 and VES are added in the water for injection.The detergent amount that reaches is following: every 1ml has 750 μ gTween, 80,110 μ g Triton X-100 and 100 μ g VES.After stirring in 5 minutes, add 15 μ g every kind of strain H1N1, H3N2 and B, stirred 10 minutes between each the adding.Preparation is in stirring at room 15 minutes, if do not give immediately then be stored in 4 ℃.

Cracking trivalent preparation with oil in water emulsion adjuvant AS03 adjuvantization:

10 times of concentrated solutions of PBS when concentrated (1 times be pH 7.4) and the mixture (amount counts the detergent that exists in the strain) that contains Tween 80, TritonX-100 and VES are added in the water for injection.The detergent amount that reaches is following: every 1ml has 750 μ g Tween, 80,110 μ g TritonX-100 and 100 μ g VES.Stir after 5 minutes, add 15 μ g every kind of strain H1N1, H3N2 and B, stirred 10 minutes between each the adding.Stir after 15 minutes, add 250 μ lSB62 emulsions (like the preparation of the instruction in example II .1).In stirring at room preparation 15 minutes, if do not give immediately then be stored in 4 ℃.

Trivalent cracking preparation with the AS03+MPL adjuvantization

10 times of concentrated solutions of PBS when concentrated (1 times be pH7.4) and the mixture (amount counts the detergent that exists in the strain) that contains Tween 80, TritonX-100 and VES are added in the water for injection.The detergent amount that reaches is following: every 1ml has 750 μ g Tween, 80,110 μ g TritonX-100 and 100 μ g VES.Stir after 5 minutes, add 15 μ g every kind of strain H1N1, H3N2 and B, stirred 10 minutes between each the adding.Stir after 15 minutes, add 250 μ lSB62 emulsions (like the preparation of the instruction in example II .1).Restir mixture 15 minutes adds 25 μ g MPL afterwards immediately.In stirring at room preparation 15 minutes, if do not give immediately then be stored in 4 ℃.

VI.1.3. understand

CMI analyzes (ICS:CD4/CD8, IL-2/IFNg dyeing)

Collected the PBMC that just exempts from mice in back 7 days in immunity.In mixture/group, test them.

VI.2. result

Through using C57BI/6 just to exempt from mice and, confirm to demonstrate higher CD4 with CD8+T cell frequency and the condition of hanging down background as the full inactivation virus 1 μ g/ml of stimulator antigen again.The result is shown in Figure 15 (CD4 T-cell response) and Figure 16 (CD8 T-cell response).

Under these conditions, might induce:

Than the higher CD4 t cell response of the common vaccine of cracking, as viewed at philtrum to cracking AS03 vaccine.

Than the higher CD4 t cell response of the common vaccine of cracking to cracking AS03+MPL vaccine.

Similar CD8 t cell response between common vaccine of cracking and cracking AS03 vaccine is as viewed at philtrum.

Compare to the higher trend of the CD8 t cell response of AS03+MPL with cracking AS03 vaccine or the common vaccine of cracking.

The clinical preceding evaluation of the cracking of example VII A-in the C57BI/6 mice of just exempting from, have adjuvant and no adjuvant and subunit influenza vaccines with the allos strain

VII.1. experimental design and purpose

Common with Fluarix (no adjuvant) vaccine is compared, and in Explo-Flu-001 clinical research (referring to EXAMPLE III), observes to the CD4 t cell response of trivalent influenza virus cracking AS03 vaccine significantly higher.Between these two groups, do not observe the difference of CD8 T cell and humoral response this two.

The C57BI/6 mice of using the allos strain just to exempt from is developed a kind of animal model, and this model has reappeared in the observed similar immune pattern of philtrum.For ICS (intracellular cytokine dyeing), stimulate again and carry out with the inactivation totivirus.

Purpose is the commercial split vaccine (Fluarix of comparison GlaxoSmithKline ^TM) with respect to subunit vaccine (the vaccine Fluad of Chiron ^TM) inductive CMI replys and reply with the CMI that these vaccines of AS03 or AS03+MPL or another kind of oil in water emulsion adjuvant (OW) adjuvantization obtain.

VII.1.1. treatment/group

By Harlan Horst, Netherland obtains the 6-8 female C57BI/6 mice (24 mice/groups) in age in week.Mice was just exempted from xenogenesis hypotype strain (the full formaldehyde inactivation of 5 μ g HA H1N1A/ Johannesburg/82/96, H3N2 A/ Sydney/5/97, B/ Harbin/7/94) intranasal at the 0th day.At the 29th day, with 1.5 μ g HA trivalent cracking (A/ New Caledonia/20/99, A/ Wyoming/3/2003, B/ Jiangsu/10/2003) common vaccines or Adjuvanted vaccines (referring to the group in the following table 39) intramuscular injection mice is arranged.

Table 39

^*Fluarix ^TM

^*As at the OW that produces with the elaboration in the lower part

VII.1.2. the preparation of bacterin preparation

The preparation of OW

The O/w emulsion that is called OW according to the prescription preparation of announcing in the description pamphlet that in Chiron Behring FIuAd vaccine, comprises.

Water for injection, 36.67mg citric acid and 627.4mg Trisodium citrate dihydrate are mixed, volume is adjusted to 200ml.This buffer of 470mg Tween 80 and 94.47ml is mixed, and this mixture is called " solution A ".Through under magnetic agitation, mixing 3.9g zamene and 470mg Span 85 preparation oily mixtures.Then solution A is added in the oily mixture, the final volume of acquisition is 100ml.Then, mixture at first through 18G * 11/2 pin, is put into the micro-fluidic device of M110S (deriving from Microfluidics) with two samples, to reduce the oil droplet size then.When each sample obtains the granularity of about 150nm, merge two samples, filter through 0.2 μ m filter.The sample that when T0, is combined obtains the z average of 143nm, and polydispersity is 0.10, and in the z average of 4 ℃ of storages acquisition 145nm after 4 months, polydispersity is 0.06.This granularity uses Zetasizer 3000HS (deriving from Malvern) under following technical conditions, to obtain:

-optical maser wavelength: 532nm (Zeta3000HS)

-laser power: 50mW (Zeta3000HS)

-in the scattered light (Zeta3000HS) of 90 ° of detections

-temperature: 25 ℃

-the persistent period: confirm automatically by software

-number of times: 3 continuous detecting

-z average diameter: utilize the cumulant analysis

The preparation (for 1ml) of group 1:

10 times of concentrated solutions of PBS when concentrated (1 times be pH 7.4) and the mixture (amount counts the detergent that exists in the strain) that contains Tween 80, TritonX-100 and VES are added in the water for injection, to reach final concentration: 375 μ g/ml Tween, 80,55 μ g/ml Triton X-100 and 50 μ g/mlVES.Stir after 5 minutes, add 15 μ g every kind of strain H1N1, H3N2 and B, stirred 10 minutes between each the adding.Preparation is in stirring at room 15 minutes, if do not give immediately then be stored in 4 ℃.

The preparation (for 1ml) of group 2:

10 times of concentrated solutions of PBS when concentrated (1 times be pH 7.4) and the mixture (amount counts the detergent that exists in the strain) that contains Tween 80, TritonX-100 and VES are added in the water for injection, to reach final concentration: 375 μ g/ml Tween, 80,55 μ g/ml Triton X-100 and 50 μ g/ml VES.Stir after 5 minutes, add 15 μ g every kind of strain H1N1, H3N2 and B, stirred 10 minutes between each the adding.Stir after 15 minutes, add 250 μ l OW emulsions.Stirred preparation 15 minutes, if do not give immediately then be stored in 4 ℃.

The preparation (for 1ml) of group 3:

10 times of concentrated solutions of PBS when concentrated (1 times be pH 7.4) and the mixture (amount counts the detergent that exists in the strain) that contains Tween 80, TritonX-100 and VES are added in the water for injection, to reach final concentration: 375 μ g/ml Tween, 80,55 μ g/ml Triton X-100 and 50 μ g/ml VES.Stir after 5 minutes, add 15 μ g every kind of strain H1N1, H3N2 and B, stirred 10 minutes between each the adding.Stir after 15 minutes, add 250 μ l SB62 emulsions.Stirred preparation 15 minutes, if do not give immediately then be stored in 4 ℃.

The preparation (for 1ml) of group 4:

10 times of concentrated solutions of PBS when concentrated (1 times be pH 7.4) and the mixture (amount counts the detergent that exists in the strain) that contains Tween 80, TritonX-100 and VES are added in the water for injection, to reach final concentration: 375 μ g/ml Tween, 80,55 μ g/ml Triton X-100 and 50 μ g/ml VES.Stir after 5 minutes, add 15 μ g every kind of strain H1N1, H3N2 and B, stirred 10 minutes between each the adding.Stir after 15 minutes, add 250 μ l SB62 emulsions.Restir mixture 15 minutes adds 25 μ g MPL afterwards immediately.Stirred preparation 15 minutes, if do not give immediately then be stored in 4 ℃.

The preparation (for 1ml) of group 5:

Mix isopyknic PBS and FluAdTM/Gripguard ^TMVaccine (commercial vaccine).Stirred preparation 15 minutes, if do not give immediately then be stored in 4 ℃.

The preparation (for 1ml) of group 6:

The Aggripal that 250 μ l PBS mod pH 7.4 is added 1 dose of 500 μ l ^TMIn (commercial vaccine).Stir after 15 minutes, add 250 μ l SB62 (according to the method preparation that large-scale production is detailed).Stirred preparation 15 minutes, if do not give immediately then be stored in 4 ℃.

The preparation (for 1ml) of group 7:

The Aggripal that PBS mod pH 7.4 is added 1 dose of 500 μ l ^TMIn (commercial vaccine) (to reach final volume 1ml).Stir after 15 minutes, add 250 μ l SB62 (according to the method preparation that large-scale production is detailed).Add 25 μ g MPL then.Stirred preparation 15 minutes, if do not give immediately then be stored in 4 ℃.

The preparation (for 1ml) of group 8:

250 μ l PBS mod pH 7.4 are added among the Aggripal of 1 dose of 500 μ l.Stir after 15 minutes, add 250 μ l, stirred preparation 15 minutes, if do not give immediately then be stored in 4 ℃ as organizing the OW of 2 preparations.

The preparation (for 1ml) of group 9:

Mix isopyknic PBS mod pH 7.4 and Aggripal.Stirred preparation 15 minutes, if do not give immediately then be stored in 4 ℃.

VII.1.3. understand by (table 40)

CMI (ICS): back 7 days of immunity.

Among the IHA/ and measure: back 21 days of immunity.

Table 40

In=is independent/and Po=is blended

CMI analyzes (ICS:CD4/CD8, IL-2/IFNg dyeing)

, test in mixture/group by 24 mices/group results PBMC back 7 days of immunity.

VII.2. result

VII.2.1. humoral immunity

The hemagglutination of just exempting to detect anti-3 kinds of vaccine strains in the serum of 24 animal/groups in back 16 days with immunity in back 14 days in intranasal allos suppresses activity.

For 3 kinds of strains and all groups, after immunity, observe the HI titre of reinforcement.

For identical adjuvant and 3 kinds of strains, HI titre like subunit vaccine and the split vaccine inductive phase.

For 3 kinds of strains, compare Fluad with Aggripal OW and observe similar HI titre.

For H1N1 and B strain, do not observe the difference between Fluarix and the Aggripal.

For 3 kinds of strains, compare with the normal stream influenza vaccine, with the AS03 adjuvant influenza vaccines (split vaccine or subunit vaccine) that are with or without MPL the time observe on the statistics significantly higher H I titre.

Compare with the normal stream influenza vaccine, only significantly higher on statistics with the HI titre of the influenza vaccines (split vaccine or subunit vaccine) of OW adjuvantization for A/ Wyoming strain.

VII.2.2. cell-mediated immune responses (at the back 7 days ICS of immunity)

CD4 t cell response-Figure 17 top

Collected the PBMC of 24 mice/groups in back 7 days in immunity, and test in 1 mixture/group.The trivalent totivirus of inactivation (1 μ g/ml) is used as stimulator antigen again.The result is shown in Figure 17 top.

With regard to the influenza all-virus specific C D4+T cell of expressing IL-2, IFN-γ or these two kinds of cytokines (Figure 17 top):

1.GSK adjuvant demonstrates and previous observation (example VI) identical trend: AS03+MPL is superior to AS03, and AS03 is superior to the result with common vaccine acquisition again.All observe this trend for split vaccine or subunit vaccine this two.

2. which kind of preparation (common vaccine, AS03 or AS03+MPL) no matter, the split vaccine CD4+T cell response that all the induction ratio subunit vaccine is high.

3.Fluad (subunit+O/w emulsion OW-is referring to the preparation part) seems to induce and the similar frequency of the common vaccine of Fluarix.

4. preparation trivalent split vaccine/AS03 or trivalent split vaccine/AS03+MPL induce the CD4+T cell response that is higher than preparation subunit/O/w emulsion OW.

CD8 t cell response-Figure 17 bottom

After immunity, collected PBMC by 24 mices/group on the 7th day, and in 1 mixture/group, test.The trivalent totivirus of inactivation (1 μ g/ml) is used as stimulator antigen again.

With regard to the influenza all-virus specific C D8+T cell of expressing IL-2, IFN-γ or two kinds of cytokines (Figure 17 bottom):

The intercepting value of this experiment is higher relatively, and reason is that the background that the PBS negative control group is observed is high.

But, compare with other bacterin preparation, for observe higher specific C D8 t cell response with trivalent split vaccine/AS03+MPL mice immunized.

VII.3. the result summarizes and conclusion

Obtain following result:

1) the influenza virus specific C D4+T cell that obtained through ICS in back 7 days in immunity shows:

1. compare Fluad with Fluarix and obtain similar replying.

2. compare with no Adjuvanted vaccines,, have adjuvant formulation all to induce higher immunne response for cracking influenza vaccines (like what observe) and subunit vaccine (Aggripal) (not estimating) this two at philtrum at philtrum.The oil in water emulsion adjuvant AS03 (group 4 and group 9) that adds MPL produces high the replying than oil in water emulsion adjuvant AS03 (group 3 and group 8).

3. compare the trend (Figure 17) that has the higher CD4 to split vaccine/AS03+MPL to reply with split vaccine/AS03.

4. by inductive reply (the contrast groups 1-4 and group 5-9) that is superior to the subunit vaccine acquisition that reply of split vaccine.

5. no matter split vaccine does adjuvant (group 3 and 4) with having still with the AS03 that does not have MPL, all demonstrates than the high CD4+T cell response of subunit vaccine (Fluad (group 5) or Aggripal+OW (group 7)).

2) the influenza virus specific C D8+T cell that obtained through ICS in back 7 days in immunity does not demonstrate the difference that between split vaccine/AS3 and common split vaccine (like what observe at philtrum), observes.Compare with common split vaccine with split vaccine/AS3, the CD8+T cell response that uses split vaccine/AS3+MPL to obtain has higher trend.

3) for identical adjuvant and 3 kinds of strains, HI titre like subunit vaccine and the split vaccine inductive phase.For 3 kinds of strains; Compare with the normal stream influenza vaccine, when influenza vaccines (subunit vaccine or split vaccine) observe titre significantly higher on the statistics (only for A/ Wyoming strain influenza vaccines OW>normal stream influenza vaccine) during with AS03 or AS03+MPL adjuvant.

Example VII A I-is with contain the clinical experiment that cracking influenza antigens prepared product and the vaccine that is with or without the AS03 of MPL adjuvant carry out in the over-65s elderly population

VIII.1. experimental design

In over-65s (>=65 years old) elderly population, open, at random, the controlled I phase studies, so that estimate and Fluarix ^TMVaccine (is called α-Rix in Belgium ^TM) compare the reactionogenicity and the immunogenicity of the GlaxoSmithKline Biologicals influenza candidate vaccine that contains adjuvant AS03 or AS03+MPL that intramuscular gives.

Estimate 3 parallel-group:

1 group of 50 experimenter accepts the SV influenza vaccines (Flu AS03) of 1 dose of reprovision and AS03 adjuvantization

1 group of 50 experimenter accepts the SV influenza vaccines (Flu AS03+MPL) of 1 dose of reprovision and Flu AS03+MPL adjuvantization

50 experimenters of 1 matched group accept 1 dose of Fluarix ^TM(Fluarix)

VIII.2. vaccine is formed and is given

The strain that in 3 kinds of vaccines, uses is to recommend to be used for the 2004-2005 Northern Hemisphere strain in influenza season, i.e. A/ New Caledonia/20/99 (H1N1), the new California of A//3/2003 (H3N2) and B/ Jiangsu/10/2003 by WHO.Same Fluarix ^TM/ α-Rix ^TMThe same, commercial vaccine has every dose of Adjuvanted vaccines (AS03 or AS03+MPL) to contain 15 μ g hemagglutinins (HA) of every kind of strains of influenza viruses as comparison.

It is a kind of 2 component vaccines that adjuvant influenza candidate vaccine is arranged, and is made up of with the preparatory filling I type glass syringe that contains adjuvant (AS03 or AS03+MPL) the 3 valency inactivation lytic virus isoantigens that concentrate that in I type vial, provide.They prepare like the detailed description in example II.Being used to has 3 kinds of inactivation lytic virus isoantigens (unit price stock solution) of adjuvant influenza candidate vaccine preparation and is used to prepare commercial Fluarix ^TMThe active component of/α-Rix is identical.

The vaccine of AS03 adjuvantization:

The influenza candidate vaccine of AS03 adjuvantization is a kind of 2 component vaccines, is made up of with the preparatory filling I type glass syringe (335 μ l) (adjuvant container) that contains the SB62 emulsion the 3 valency inactivation lytic virus isoantigens that concentrate that in I type vial (335 μ l) (antigen container), provide.The description and the composition of AS03 candidate vaccine are set forth in EXAMPLE III.

The vaccine of AS03+MPL adjuvantization:

In brief; The influenza vaccines material standed for of AS03+MPL adjuvantization is a kind of 2 component vaccines, is made up of with the preparatory filling I type glass syringe (360 μ l) (adjuvant container) that contains the AS03+MPL adjuvant the 3 valency inactivation lytic virus isoantigens that concentrate that in I type vial (335 μ l) (antigen container), provide.When injection, the content of antigen container contains the syringe of AS03+MPL adjuvant by taking out in the bottle through use, follow slight mixing syringe.Before injection, the pin of use is replaced by the intramuscular pin, and volume is adjusted to 530 μ l.The AS03+MPL adjuvant influenza candidate vaccine of 1 dose of reprovision is equivalent to 530 μ l.For obtaining 15 μ g HA of every kind of influenza strain when the AS03+MPL adjuvant vaccine reprovision, with Fluarix ^TM(i.e. 30 μ g HA/ml) compare, and the lytic virus isoantigen of inactivation is concentrated 2 times (i.e. 60 μ gHA/ml) in the antigen container.

The forming and middle report identical of table 45 (referring to embodiment XI), just influenza strain difference by the adjuvant influenza vaccines of 1 dose of reprovision.Two kinds of vaccines all intramuscular give.

VIII.3.CMI target, terminal point and result

The CMI target is the compositions of confirming with respect to no any adjuvant, with which kind of immunogenic composition between the preparation of AS03 or AS03+MPL adjuvantization the CD4 of influenza antigens inoculation individuality and the immunity of CD8 mediation is had the strongest immunostimulatory activity.

VIII.3.1.CMI terminal point and result

Observational variable

At the 0th day and the 21st day: per 10 ⁶Be directed against the frequency of the positive CD4/CD8 cell of cytokine of 5 kinds of different cytokines in the individual cell.Each test is quantitatively to following CD4/CD8 t cell response:

-following 3 kinds of antigenic mixture

-New Caledonia antigen

-Wyoming antigen

-Jiangsu antigen

The variable of deriving:

Antigenic specificity CD4 and the CD8-T-cell response in 5 kinds of different tests, represented with following cell:

(a) cell of at least two kinds of different cytokines of generation (CD40L, IL-2, IFN γ, TNF α)

(b) produce the cell of CD40L and another kind of cytokine (IL-2, TNF α, IFN γ) at least

(c) produce the cell of IL-2 and another kind of cytokine (CD40L, TNF α, IFN γ) at least

(d) produce the cell of IFN γ and another kind of cytokine (IL-2, TNF α, CD40L) at least

(e) produce the cell of TNF α and another kind of cytokine (IL-2, CD40L, IFN γ) at least

The CMI response analysis:

CMI analyzes based on total inoculation crowd.

(a) for each treatment group, to each inoculation group, each time point (the 0th day, the 21st day) and every kind of antigen: 3 kinds of different strains of New Caledonia, Wyoming and Jiangsu and merging are measured the frequencies that CD4/CD8 T-lymphocytic emiocytosis are replied.

(b) to each 5 kinds of different cells factor, between time point (before the inoculation back-inoculation) to the descriptive statistics of each inoculation group and various antigenic individual variations of replying.

(c) contrast 3 groups following aspect with regard to 5 kinds of different cytokines:

-to the CD4 T-cell response of 3 kinds of strains of New Caledonia, Wyoming, Jiangsu and merging

-to the CD8 T-cell response of 3 kinds of strains of New Caledonia, Wyoming, Jiangsu and merging

(d) non parametric tests (Kruskall-Wallis check) is used to contrast the local difference between 3 groups, calculates various antigenic statistical p-values to each 5 kinds of different cytokines.

(e) the Wilcoxon check is respectively applied for and checks Flu AS03+MPL that Fluarix, FluAS03+MPL are formed comparing 2 between the Fluarix Flu AS03 and Flu AS03.

(f) all significance test all are two tails.Being less than or equal to 0.05 P-value, to be considered to statistics significant.

VIII.3.2.CMI result

The result is expressed as the frequency of male CD4 of cytokine in CD4 or the CD8 T cell subsets or CD8 T cell.

The lymphocytic frequency of antigenic specificity CD4 T-

(a) with in EXAMPLE III, implemented similar, the frequency of replying to each inoculation group, each time point (the 0th day, the 21st day) and every kind of antigen (merging, New Caledonia, Wyoming and Jiangsu) mensuration antigenic specificity CD4 T-lymphocytic emiocytosis.

(b) through the lymphocytic frequency difference of antigenic specificity CD4 T-between 3 groups of Kruskall-Wallis check contrast, all p-values are all less than 0.05, and it is significant to be considered to statistics.

(c) through the lymphocytic frequency difference of antigenic specificity CD4 T-between Wilcoxon check contrast Flu AS03+MPL and the Fluarix group, all p-values are all less than 0.05, and it is significant to be considered to statistics.

(d) through the lymphocytic frequency difference of antigenic specificity CD4 T-between Wilcoxon check contrast Flu AS03 and the Fluarix group, all p-values are all less than 0.05, and it is significant to be considered to statistics.

(e) through the lymphocytic frequency difference of antigenic specificity CD4 T-between Wilcoxon check contrast Flu and the Flu AS03+MPL group, all p-values are all less than 0.05, and it is significant to be considered to statistics.

The lymphocytic individual variation of CD4T-between time point (before the inoculation back-inoculation)

(a) with in EXAMPLE III, implemented similar, do descriptive statistics for each 5 kinds of different cytokines to the individual variation of each inoculation group and every kind of antigen CD4 T-lymphocyte responses between time point (before the inoculation back-inoculation).

(b) after the inoculation through antigenic specificity CD4 T-lymphocyte responses between 3 groups of Kruskall-Wallis check contrast-inoculation before individual variation, all p-values are all less than 0.001, are considered to that highly statistics is significant.

(c) use after the inoculation of antigenic specificity CD4 T-lymphocyte responses between Wilcoxon check contrast Flu AS03+MPL and the Fluarix-inoculation before individual variation, all p-values are all less than 0.05, it is significant to be considered to statistics.

(d) use after the inoculation of antigenic specificity CD4 T-lymphocyte responses between Wilcoxon check contrast Flu AS03 and the Fluarix-inoculation before individual variation, all p-values are all less than 0.001, are considered to that highly statistics is significant.

(e) use after the inoculation of antigenic specificity CD4 T-lymphocyte responses between Wilcoxon check contrast Flu AS03+MPL and the Flu AS03-inoculation before individual variation, all p-values are considered to not be that all greater than 0.05 statistics is significant.

The VIII.4.B cell memory is replied target, terminal point and result

To be research compare with Fluarix in the elderly population target of research, when carrying out 1 intramuscular inoculation with the influenza candidate vaccine that contains adjuvant AS03+MPL or AS03, whether the frequency of the memory B cell of influenza virus antigenic specificity significantly induced.The frequency of memory B cell is through B cell Elispot evaluation of measuring.

The VIII.4.1.B cell memory is replied terminal point

Terminal point is:

(a) in the time of the 0th, 21 day: with regard to 1,000,000 (10 ⁶) individual specific anti protoplasmic cell frequency of producing in the IgG plasma cell, in all experimenters, all detect the cell that in the memory B cell of In vitro culture, produces through B cell ELISPOST.

(b) after inoculation before (the 21st day) and the inoculation difference between (the 0th day) also be expressed as per 1,000,000 (10 ⁶) the influenza specific antibody forms the frequency of cell among the individual antibody forming cell.

VIII.4.2.B cell memory response result

Measure per 1,000,000 (10 ⁶) the influenza specific antibody forms the frequency of cell among the individual antibody forming cell.The result shows; Between Flu AS03+MPL that obtains through Wilcoxon check and the Fluarix group frequency of influenza antigen specificity memory B cell as far as B/ Jiangsu strain significantly (p＜0.05) higher, then can be not like this to other two kinds of strains (A strain New Caledonia and Wyoming).

Also measured the individual variation of the specific memory B cell of influenza antigens between time point (before the inoculation back-inoculation).The result shows; The individual variation of influenza antigen specificity memory B cell frequency between time point (before the inoculation back-inoculation) is higher as far as B/ Jiangsu strain remarkable (p＜0.05) between Flu AS03+MPL that obtains through Kruskall-Wallis check and the Fluarix group, and can be not like this to other two kinds of strains (A strain New Caledonia and Wyoming).

The result is shown in Figure 18.

Example I X-has clinical preceding estimate (research III) of influenza vaccines in ferret of adjuvant and no adjuvant

IX.1. ultimate principle and target

(the Fluarix of the no adjuvant of this research contrast ^TM) or with the commercial trivalent of the GSK of AS03+MPL adjuvantization CrackingInfluenza vaccines and two kinds of other commercializations SubunitVaccine:

-Fluad ^TM, Chiron has adjuvant subunit vaccine (adjuvant is the MF59 adjuvant of Chiron),

-Agrippal ^TM, the commercial subunit vaccine of the no adjuvant of Chiron, it does adjuvant with the AS03 adjuvant in this research.

The target of this experiment is to estimate these vaccines alleviate symptom (body temperature and virus shedding) in the nasal discharge of the ferret of attacking with the allos strain ability.

Terminal point is:

1) main terminal point: after allos is attacked, alleviate the virus shedding in the nose cleanout fluid:

2) secondary endpoints: reply through the IHA analysing body fluid, and monitoring just exempts to attack with allos the temperature of front and back.

IX.2. experimental design

IX.2.1. treatment

(Hampshire UK) obtains the 14-20 female ferret (Mediterranean ferret (Mustela putorius furo)) in age in week by MISAY Consultancy.Ferret at the 0th day with xenogenesis hypotype strain H1N1 A/ Stockholm/24/90 (4 Log TCID ₅₀/ ml) intranasal is just exempted from.At the 21st day, with the combination intramuscular injection ferret of H1N1 A/ New Caledonia/20/99, H3N2 A/ Wyoming/3/2003 and B/ Jiangsu/10/2003 of total man's dosage (1ml vaccine dose, 15 μ g HA/ strains).Passed through the intranasal approach with special-shaped strain H3N2 A/ Panama/2007/99 (4.51 Log TCID at the 42nd day then ₅₀/ ml) attack ferret through the intranasal approach.Each group (6 ferret/groups) is described in table 41.The deciphering mode of implementing is specified in table 42.

Table 41

IX.2.2. the preparation of bacterin preparation

The trivalent cracking common (no adjuvant formulation: 1ml preparation:

10 times of concentrated solutions of PBS when concentrated (1 times be pH 7.4) and the mixture (amount counts the detergent that exists in the strain) that contains Tween 80, TritonX-100 and VES are added in the water for injection.The detergent amount that reaches is following: every 1ml has 375 μ g Tween, 80,55 μ g TritonX-100 and 50 μ g VES.Stir after 5 minutes, add every kind of strain H1N1 of 15 μ g, H3N2 and 17.5 μ g B strains, stirred 10 minutes between each the adding.Preparation is in stirring at room 15 minutes, if do not give immediately then be stored in 4 ℃.

Trivalent cracking preparation with the AS03+MPL adjuvantization: 1ml preparation

10 times of concentrated solutions of PBS when concentrated (1 times be pH 7.4) and the mixture (amount counts the detergent that exists in the strain) that contains Tween 80, TritonX-100 and VES are added in the water for injection.The detergent amount that reaches is following: every 1ml has 375 μ g Tween, 80,55 μ g TritonX-100 and 50 μ g VES.Stir after 5 minutes, add 15 μ g every kind of strain H1N1, H3N2 and B, stirred 10 minutes between each the adding.Stir after 15 minutes, add 250 μ l SB62 emulsions (like the preparation of the instruction in example II .1).Restir mixture 15 minutes adds 25 μ g MPL afterwards immediately.In stirring at room preparation 15 minutes, if do not give immediately then be stored in 4 ℃.

FluAd ^TMPreparation: 1ml preparation:

In PBS pH of buffer 7.4, prepare FluAd ^TM2 times of diluents of vaccine.Agrippal ^TMAS03 preparation: 1ml preparation:

250 μ l PBS pH of buffer 7.4 are added 1 dose of Aggripal ^TMIn.After the mixing, add 250 μ l SB62 emulsions (like the preparation of the detailed description in example II .1).Mixture is in stirring at room.

IX.2.2. understand

Table 42

In=is independent/and Po=is blended

IX.3. result (Figure 19-22)

IX.3.1. temperature monitoring

With pick off and with monitoring a temperature through tele rcording.Check and rebuild all implants, before putting into the abdominal cavity, newly calibrate through DSI.All animals all are housed in the independent cage independently between these detection periods.By attacking preceding 2 days, calculate mean temperature by Japan and China until attacking back 4 days per 15 minutes record temperature.The result is shown in Figure 19.

The result:

After the attack, with no adjuvant (common) trivalent split vaccine (Fluarix ^TM) or subunit vaccine Fluad ^TMObserve the body temperature peak value behind (it contains the MF59 O/w emulsion) immune ferret.With the trivalent split vaccine of AS03+MPL adjuvantization with the subunit A grippal of AS03 adjuvantization ^TMAll do not observe peak value behind the immunity ferret.In a word,, all demonstrate the vaccine that contains AS03 prevents fervescence after attack surcharge, after attacking, can not prevent that in ferret fervescence is opposite here with the vaccine that contains MF59 for cracking and subunit test vaccine.

IX.3.2. virus shedding

Every group of 6 animals are carried out the titration of virus that nose cleans thing.Carry out the nose cleaning through in two nostrils of clear-headed animal, giving 5ml PBS.Inoculum is collected in the culture dish, and places the shuttle on the dry ice (80 ℃).

All nose samples are all at first through Spin X filter (Costar) aseptic filtration, to remove any germ contamination.The nose cleanout fluid of the continuous 10 times of dilutions of 50 μ l is transferred in the microtitration plate that contains 50 μ l culture medium (10 holes/dilution factor).Then with 100 μ l mdck cells (2.4 * 10 ⁵Individual cell/ml) adds each hole, and in 35 ℃ of incubation 5-7 days.Behind 5-7 days incubations, take out culture medium carefully, add the culture medium that 100 μ l contain 1/20 WST-1, incubation is 18 hours again.

The survivaling cell number that the intensity of the cured dyestuff of yellow first that when survivaling cell reduction WST-1, produces and titration of virus experiment are present in the hole when finishing is proportional, and quantitative through the absorbance that detects each hole at suitable wavelength (450 nm).The intercepting value defined is not for infecting OD meansigma methods-0.3 OD (0.3 OD is equivalent to not infect control cells OD ± 3 standard deviation) of control cells.Be defined as negative score during OD <be defined as positive score during the intercepting value, on the contrary, OD>intercepting value.The virus shedding titre is measured through " Reed and Muench ", and is expressed as Log TCID ₅₀/ ml.

The result:

The result is shown in Figure 20.With usefulness no adjuvant (common) trivalent split vaccine (Fluarix ^TM) or Fluad ^TMObserved low-down virus shedding reduces and compares behind the subunit vaccine immunity ferret, with the trivalent split vaccine of AS03+MPL adjuvantization or with the Agrippal of AS03 adjuvantization ^TMSubunit vaccine is observed lower virus shedding after attacking.

With to fervescence discuss similar, than the vaccine observation that contains MF59 to the surcharge that contains the AS03 vaccine.

The IX.3.3.HI titre

Use hemagglutination to suppress test (HI) and measure anti-hemagglutinin antibody titer to the H3N2 strains of influenza viruses.The HI testing principle is based on the specific anti influenza antibodies suppresses the hemagglutination of little chicken red blood cell (RBC) through influenza virus haemagglutinin (HA) ability.Serum at first uses 25% neuraminic acid enzymatic solution (RDE) to handle and heat inactivation, to remove nonspecific inhibitor.After pretreatment, with the serum of 2 times of dilutions and every kind of influenza strain incubation of 4 HAUs.Add little chicken red blood cell then, and the record coagulation suppresses.Titre is expressed as the inverse of the high dilution of serum that suppresses hemagglutination fully.Because first dilution factor of serum is 1: 10, equal 5 titre so can not detection level be recorded as.

The result:

After with H3N2 A/ Wyoming immunity, with usefulness no adjuvant (common) trivalent split vaccine (Fluarix ^TM) or Fluad ^TMObserved humoral response is compared behind the subunit vaccine immunity ferret, with the trivalent split vaccine of AS03+MPL adjuvantization or the Agrippal of AS03 adjuvantization ^TMObserve higher humoral response (HI titre) (Figure 21) in the ferret of subunit vaccine immunity.

After with H3N2 A/ Wyoming immunity, and compare, with the trivalent split vaccine of AS03+MPL adjuvantization or the Agrippal of AS03 adjuvantization with the ferret of the common vaccine of trivalent cracking or Fluad immunity ^TMIn the ferret of immunity, also observe (Figure 22) to the Panamanian higher humoral response of drift strain H3N2 A/ (HI titre) that is used as the attack strain.

With our adjuvant (AS03 or AS03+MPL) observe to this cross reaction of allos strain with AS03+MPL adjuvant trivalent split vaccine or AS03 adjuvant Agrippal ^TMThe subunit vaccine immunity also uses the protective effect that observes in the ferret of this allos strain attack to be associated subsequently.Can't help the vaccine (FIuAd of MF59 adjuvantization by vaccine-induced this cross reactivity that contains AS03 to the allos strain ^TM) induce.

Embodiment X-is with containing cracking influenza antigens prepared product and have or do not have the clinical experiment that the vaccine of the AS03 of MPL adjuvant carries out in the over-65s elderly population: the immunogenicity persistent data the 90th day and 180 days

X.1. research design

In the elderly population of over-65s (>=65 years old), open, at random, the controlled I phase studies, and (is called α-Rix in Belgium so that estimate to compare with the FluarixTM vaccine ^TM), the reactionogenicity and the immunogenicity of the GlaxoSmithKline Biologicals influenza candidate vaccine that contains adjuvant AS03 or AS03+MPL that intramuscular gives.The research of in example VII A I, reporting is followed in this research.

Estimate three parallel-group:

1 group of 50 experimenter accepts the SV influenza vaccines (Flu AS03) of 1 dose of reprovision and AS03 adjuvantization

1 group of 50 experimenter accepts the SV influenza vaccines (Flu AS03+MPL) of 1 dose of reprovision and Flu AS03+MPL adjuvantization

50 experimenters of 1 matched group accept 1 dose of Fluarix ^TM(Fluarix)

X.2. immunogenicity result

X.2.1. HI terminal point and result

In order to estimate vaccine-induced HI and persistency thereof, to the following parameter of each treatment set of calculated by AS03 and AS03+MPL adjuvantization.

At the 0th, 21,90 and 180 day: each the serum hemagglutination of testing separately that is directed against 3 kinds of strains of influenza viruses that in vaccine, provide suppressed (HI) antibody titer (resist-H1N1, resist-H3N2 and anti--B-antibody).

The serum HI antibody GMT that had 95%CI at the 0th, 21,90 and 180 day

The serological conversion rate that had 95%CI at the 21st, 90 and 180 day

The conversion factor that had 95%CI at the 21st day

The serum protective rate that had 95%CI at the 0th, 21,90 and 180 day

The result

GMT with HI antibody of 95%CI is shown in Figure 23.Be in the same range as to GMT before the inoculation of the antibody of whole 3 kinds of vaccine strains in 3 groups.After inoculation, anti-hemagglutinin antibody horizontal significantly increases.But, inoculation back to the inoculation of the antibody of 3 kinds of vaccine strains after GMT as far as all vaccines all still in identical scope.At the 21st day; For A/ New Caledonia and B/ Jiangsu strain; Observe to compare and help 2 kinds of slight tendency that Adjuvanted vaccines is arranged, have in the Adjuvanted vaccines, observe higher GMT to A/ Wyoming and B/ Jiangsu strain with FLU AS03 at two kinds with Fluarix.

Observed identical trend at the 90th day.At the 180th day, concerning 3 kinds of vaccines, to the antibody GMT of 3 kinds of vaccine strains in same range as.

In the experimenter more than 60 years old, all influenza vaccines are all satisfied European official to the requirement of the influenza inactivated vaccines of every year registration [" about being used for the guide explanation that requires of the coordinated flow influenza vaccine that changes of strain of immunological evaluation year " (CPMP/BWP/214/96)].

In back 3 months of inoculation (90 days) and 6 months (180 days), the seminar that whichever is investigated, minimum 60% the protective rate that the serum protective rate is higher than still all that European official requires.At the 90th day, 30% the minimum serological conversion rate that in 3 vaccine group, all vaccine strains is reached still all that European official requires was except the A/ New Caledonia strain of Fluarix.At the 180th day, still can reach this serological conversion rate with 3 kinds of vaccines to A/ Wyoming and the strain of B/ Jiangsu, but to the strain of A/ New Caledonia not all right (table 43 and table 44).

Table 43 suppresses titre with the serum hemagglutination and is superior to or equals the serum protective rate (immunogenic ATP crowd) that inoculator's percentage rate of 1: 40 is represented

The available experimenter's number of N=result

Number/percentage rate of the experimenter of n/%=titre in particular range

Titre before the PRE=inoculation

PI (D21)=at the 21st day adopts inoculation back blood sample

PI (D90)=at the 90th day adopts inoculation back blood sample

PI (D180)=at the 180th day adopts inoculation back blood sample

The hemagglutination of representing table 44 suppresses the serological conversion rate of (HI) antibody titer, is defined as than the 0th day serum HI titre at each inoculation back time point to have the inoculator's percentage rate (immunogenic ATP crowd) that reaches 4 times of increases at least

Preceding and the available experimenter's number of inoculation back result of N=inoculation

N/%=has experimenter's number/percentage rate of at least 4 times of increases

Accurate 95% confidence interval of 95%CI=; The LL=lower limit, the UL=upper limit

X.2.2.CMI reply terminal point and result

In order to estimate, each is treated the following parameter of set of calculated by inductive cellullar immunologic response of Adjuvanted vaccines and persistency thereof are arranged:

In each time point (the 0th, 21,90 with 180 days): in different tests per 10 ⁶The frequency of the positive CD4/CD8 cell of cytokine in the individual cell is (at the 0th day and 21 days that investigate separately and merge New Caledonia, Wyoming and Jiangsu antigen; At the 90th day and 180 days that investigate separately and merge New Caledonia, Wyoming, Jiangsu and New York antigen).

All double: the cell (CD40L, IFN-γ, IL-2, TNF-α) that produces at least two kinds of different cytokines.

CD40L: produce the cell of CD40L and another kind of cytokine (IFN-γ, IL-2, TNF-α) at least.

IFN-γ: produce the cell of IFN-γ and another kind of cytokine (CD40L, IL-2, TNF-α) at least.

IL-2: produce the cell of IL-2 and another kind of cytokine (CD40L, IFN-γ, TNF-α) at least.

TNF-α: produce the cell of TNF-α and another kind of cytokine (CD40L, IFN-γ, IL-2) at least.

The result

The main discovery is (Figure 24):

(a) inoculation is back 21 days, than the Fluarix group, has the frequency of the male CD4 T of cytokine cell (IL-2, CD40L, TNF-α and IFN-γ) in the Adjuvanted vaccines group significantly higher at 2.But, between two kinds of adjuvants, do not have significant difference.

(b) there are all statistical discrepancies between Adjuvanted vaccines and the Fluarix to be retained to the 90th day and the 180th day, except below the 180th day several:

For All double, CD40L, IFN-γ and IL2 (only Wyoming strain) with for Alldouble, CD40L and TNF-α (only New York strain), between FluAS03/MPL and Fluarix, there is not statistically-significant difference,

For IL2 (only Jiangsu strain), between FluAS03 and Fluarix, there is not statistically-significant difference

(c) to the 90th day and the 180th day, confirming does not have the significant difference of statistics between two kinds of vaccines that adjuvant arranged.

(d) for two kinds of vaccines that adjuvant is arranged, the significant difference that is directed against the CD4 T-lymphocyte responses of all cells factor of being studied (IL-2, CD40L, TNF-α and IFN-γ) before the inoculation and between the inoculation back (the 21st day) is higher than Fluarix ^TMBut, between two kinds of adjuvants, do not detect significant difference.

(e) inoculation is replied CD8 does not have detectable influence, treatment group whichsoever.The clinical experiment that embodiment XI-carries out in the over-65s elderly population with the vaccine that contains cracking influenza antigens prepared product and AS03 and MPL adjuvant

XI.1. research design and target

In over-65s (＞65 years old) elderly population, open, the controlled I/II phase studies; So that estimate the reactionogenicity and the immunogenicity of the GlaxoSmithKline Biologicals influenza candidate vaccine that contains the AS03+MPL adjuvant, said elderly population was before used same candidate vaccination in 2004.For carrying out immunogenicity and safety evaluatio, Fluarix ^TMVaccine (is called α-Rix in Belgium ^TM) as reference.

Estimate 2 parallel-group:

1 group of about 50 experimenter, what they had before accepted 1 dose of reprovision in clinical experiment in advance has adjuvant influenza vaccines

About 50 experimenters of 1 matched group (Fluarix) have accepted 1 dose of Fluarix in advance in the middle of their clinical experiment formerly

1 target of this research is to estimate the HI that inoculates (anti-hemagglutinin and anti--MPL titre) that after dispenser first, had adjuvant influenza vaccines Flu AS03+MPL to carry out in about 1 year.For comparing purpose, formerly accept Fluarix in the experiment ^TMThe experimenter accept 1 dose of commercial vaccine, and constitute the matched group of this experiment.

XI.2. vaccine is formed and is given

The strain that in 3 kinds of vaccines, uses is to recommend to be used for the 2005-2006 Northern Hemisphere strain in influenza season, i.e. A/ New Caledonia/20/99 (H1N1), the new California of A//7/2004 (H3N2) and B/ Jiangsu/10/2003 by WHO.Same Fluarix ^TM/ α-Rix ^TMThe same, commercial vaccine is as comparison, every dose of 15 μ g hemagglutinins (HA) that contain every kind of strains of influenza viruses of the vaccine of AS03+MPL adjuvantization (back literary composition abbreviates " Adjuvanted vaccines is arranged " as).

The influenza candidate vaccine that adjuvant is arranged is a kind of 2 component vaccines, is made up of with the preparatory filling I type glass syringe that contains the AS03+MPL adjuvant the 3 valency inactivation lytic virus isoantigens that concentrate that in I type vial, provide.They are according to the method preparation of detailing in the example II.

When injection, the content that will contain the pre-filled syringe of adjuvant injects and contains the bottle that concentrates trivalent inactivation lytic virus isoantigen.After mixing, content is drawn in the syringe, pin is replaced with the intramuscular pin.The adjuvant influenza candidate vaccine that has of 1 dose of reprovision is equivalent to 0.7ml.It is the vaccine of preservative free that adjuvant influenza candidate vaccine is arranged.

1 dose of reprovision by providing forming in table 45 of adjuvant influenza vaccines.Two kinds of vaccines all intramuscular give.

Table 45 reprovision has the composition of adjuvant (AS03+MPL) influenza candidate vaccine

XI.3. immunogenicity result

XI.3.1. resist-HA HI terminal point and result

Observational variable:

At 0 day and 21 days: the serum hemagglutination suppressed (HI) antibody titer, respectively to each tests of 3 kinds of strains of influenza viruses providing in the vaccine (anti--H1N1, anti--H3N2 and anti--B-antibody).

The variable (having 95% confidence interval) of deriving:

(f) has the geometric mean titer (GMT) of the serum HI antibody of 95% confidence interval (95%CI) before the inoculation with after the inoculation

(g) has the serological conversion rate of 95%CI 21 days the time ^*

(h) has the seroconversion factor of 95%CI 21 days the time ^*

(i) has the serum protective rate of 95%CI 21 days the time ^{* *}

^*Serological conversion rate is defined as inoculator's percentage rate of HI titre before the inoculation to every kind of vaccine strain＜1: 10 and inoculation back titre>=1: 40, or titre before the inoculation>=1: 10 and inoculation back titre increase to minimum 4 times inoculator's percentage rate.

^*The seroconversion factor is defined as than the 0th day increase multiple to the serum HI GMT of every kind of vaccine strain the 21st day the time.

^{* *}Protective rate is defined as inoculator's percentage rate of serum HI titre>=40, inoculation back (to every kind of vaccine strain), regards this percentage rate as the indication protective effect usually.

The result

As expect that the most subjects in preceding 2 groups of inoculation has been seropositive to 3 kinds of strains.Be in the same range as in 2 groups to GMT before the inoculation of whole 3 kinds of vaccine strains.Than the Fluarix group, in Flu AS03+MPL group, higher trend is arranged, although 95%CI is eclipsed (Figure 25) to GMT after the inoculation of whole 3 kinds of vaccine strains.

In the experimenter more than 60 years old, 2 kinds of influenza vaccines are satisfied European official to the requirement of the influenza inactivated vaccines of registration in every year [about being used for the guide explanation that requires of the coordinated flow influenza vaccine that changes of strain of immunological evaluation year " (CPMP/BWP/214/96)] (table 46).

Serum protective rate, serological conversion rate and the conversion factor (immunogenic ATP crowd) of table 46 in the time of the 21st day

N=experimenter's sum; %=titre in the time of the 21st day is in the experimenter's percentage rate in the particular range; The CI=confidence interval

The clinical experiment that embodiment XII-carries out in the over-65s elderly population with the vaccine that contains cracking influenza antigens prepared product, with the AS03 and the MPL of two variable concentrations adjuvant is arranged

XII.1. research design and target

With the Fluarix that in adult (18-40 year), gives ^TM(be called α-Rix in Belgium ^TM) compare, concerning the cell-mediated immune response of the GlaxoSmithKline Biologicals influenza candidate vaccine that contains various adjuvants that in elderly population (65 years old and more than), gives, open, the I/II phase is at random studied and shows non-bad effect property.

Estimate 4 parallel-group:

(a) adult of 75 in a matched group (18-40 year) accepts 1 dose of Fluarix ^TM(Fluarix group)

(b) 200 old experimenters (65 years old and more than) were divided into 3 groups at random according to 3: 3: 2:

-one group of 75 experimenter, they accept the influenza vaccines (concentration 1-25 μ g) with the AS03+MPL adjuvantization

-one group of 75 experimenter, they accept the influenza vaccines (concentration 2-50 μ g) with the AS03+MPL adjuvantization

-having 50 experimenters' reference Flu group, they accept 1 dose of Fluarix ^TM

Primary goal

Primary goal is with regard to the lymphocytic frequency of influenza specific C D4 T-that produces at least two kinds of different cytokines (CD40L, IL-2, TNF-α, IFN-γ), with the Fluarix that in adult (18-40 year), gives ^TMCompare, the adjuvant influenza vaccines that have that in old experimenter (65 years old or more than), give showed non-bad effect property in back 21 days in inoculation.

By-end

By-end is

(a) estimate safety and the reactionogenicity that usefulness in the middle of after intramuscular gives vaccine in old people (65 years old and more than) 21 days has adjuvant candidate influenza vaccinations.Fluarix ^TMAs reference.

(b) estimate with 21,90 and 180 days the HI (anti-hemagglutinin titre) in adjuvant influenza candidate vaccine inoculation back is arranged.Fluarix ^TMAs reference.

The 3rd target

The 3rd target is the cell-mediated immune responses of estimating with there being after the adjuvant influenza vaccinations 21,90 and 180 days (producing IFN-γ, IL-2, CD40L and TNF-α and memory B-cell response).Fluarix ^TMBe used as reference.

XII.2. vaccine is formed and is given

Also be used for the research of setting forth at embodiment XI with the influenza vaccines of AS03+MPL (every dose 25 μ g) system's adjuvantization.Influenza vaccines with AS03+MPL (every dose 50 μ g) system's adjuvantization have same composition, and just MPL concentration doubles.The method of describing to the influenza vaccines of AS03+MPL adjuvantization among method and the example VII A I is identical, and unique difference is that MPL concentration doubles.

Contrast: full dose F luarix ^TM, give through IM.

Every experimenter carries out predetermined following up a case by regular visits to 4 times: when 0,21,90 and 180 day at every turn follow up a case by regular visits to, collect blood sample, to estimate immunogenicity.

Vaccine program: carried out 1 influenza vaccines injection at the 0th day.

XII.3. immunogenicity result

XII.3.1.CMI terminal point and result

The main terminal point of estimating

At the 21st day: in the test that produces at least two kinds of different cytokines (IL-2, IFN-γ, TNF-α and CD40L), according to per 10 ⁶All experimenters' of influenza specific C D4 T-lymphocyte frequency CMI replys in the individual cell

Reply for estimating CMI, analyze the frequency of influenza specific C D4 as follows:

The ANCOVA model that use relates to the logarithmic transformation titre obtains the GM ratio according to influenza specific C D4 frequency between the group that Adjuvanted vaccines and Flu YNG inoculation is arranged.The ANCOVA model comprises as the vaccine group of fixed effect with as logarithmic transformation titre before the inoculation of regressor.GM ratio and 98.75%CI thereof derive from the exponential transform of correspondence group opposite in the model.The 98.75%CI of the GM of adjustment obtains through the exponential transform of the 98.75%CI of the group least square method of above ANCOVA model.

The result- Rational analysis (table 47)

After the external stimulation again with " antigen II of merging ", the lymphocytic adjustment back GM of influenza specific C D4 T-and the GM ratio (and 98.75%CI) that produced at least two kinds of cytokines (IL-2, IFN-γ, TNF-α and CD40L) in the 21st day are shown in table 47.The adjuvant influenza vaccines are arranged for every kind, the upper limit of the bilateral 98.75%CI of GM ratio is far below 2.0 clinical limit.This has shown the Fluarix that gives than in the 18-40 adult in year ^TMTwo kinds of giving to old experimenter of vaccine have the non-bad effect property of adjuvant influenza vaccines with regard to frequency after the inoculation of influenza specific C D40.

The GM ratio (immunogenic ATP crowd) of the adjustment of the influenza specific C D4 of at least two kinds of cytokines of table 47 generation in the 21st day

The GM=of adjustment is according to the geometric average antibody of baseline titre adjustment; Preceding and the available experimenter's number of inoculation back result of N=inoculation; 98.8% confidence interval (Ancova model :) of the GM ratio of 98.8%CI=adjustment to baseline adjustment; The LL=upper limit; The UL=lower limit.

The result- Descriptive analysis (Figure 26)

The main discovery is:

If CMI replys in youngster than in the old people high before the inoculation

After the inoculation,

Zero influenza vaccines have been replied booster action to the CMI in the Young Adults (18-40 year)

Zero CMI in the old people who accepts the adjuvant influenza vaccines reply with Young Adults in CMI reply quite.

When we contrast Fluarix (18-40 year) and FlU/AS03+MPL (concentration 1), except IFN γ, for all tests, with Fluarix ^TM(18-40 year) compares, and the difference of CD4 T lymphocyte responses that is directed against all cells factor of being studied (IL-2, CD40L, TNF-α and IFN-γ) before the inoculation and between the inoculation back is all significantly higher when employing has Adjuvanted vaccines.

Should be pointed out that stimulated in vitro with Flu strain (i) B/ Jiangsu, (ii) A/H3N2/ New York and (iii) the A/H3N2/ Wyoming carry out, rather than be included in the A/H1N1/ New Caledonia in the vaccine.But the preliminary data that comprises from a part of experimenter's A/H1N1/ New Caledonia vaccine strain shows that the result is similar.

The result- Estimate the 3rd terminal point (table 48)

In order to estimate the 3rd terminal point, detected the frequency of influenza specific C D4/CD8 T-lymphocyte and memory B cell at the 0th, 21,90 and 180 day.

Summed up for every kind of antigen, each vaccine group the influenza specific cell factor lymphocytic frequency of male CD4/CD8 T-(descriptive statistics) at the 0th and 21 day.

The local difference that non parametric tests (Wilcoxon check) is used to contrast between two groups (has the adjuvant influenza vaccines to Fluarix ^TM), calculate in each test to every kind of antigenic statistical p-value.In each test, calculate 21 days/0 day descriptive statistics of replying individual variation between (before the inoculation back/inoculation) to each inoculation group and every kind of antigen.

Non parametric tests (Wilcoxon check) is used to contrast individual variation (inoculation before the inoculation back/inoculation), calculates every kind of antigenic statistical p-value in each different check.

The p-value that is used to contrast the Wilcoxon check of influenza specific C D4 T-lymphocyte frequency difference is shown in table 48.

Table 48 inferential statistic: from the CD4 T cell of Kruskal-Wallis check the p-of each time point value (immunogenic ATP crowd)

Group 1: with the influenza vaccines (concentration 1) of AS03+MPL adjuvantization

Group 2: with the influenza vaccines (concentration 2) of AS03+MPL adjuvantization

Main Conclusions is:

(a) the GM frequency all is similar in all old subject group before the inoculation of influenza specific C D4, but more excellent in the adult in 18-40 year.

(b) in the old experimenter that the Adjuvanted vaccines inoculation is arranged and at Fluarix ^TMAmong the 18-40 year adult of inoculation, the lymphocytic inoculation of influenza specific C D4 T afterwards (the 21st day) frequency is similar.

(c) and Fluarix ^TMCompare, with after Adjuvanted vaccines inoculation is arranged, afterwards (the 21st day) frequency is significantly higher for the lymphocytic inoculation of influenza specific C D4 T among the old experimenter.

(d) all similar basically in all groups before the inoculation of influenza specific C D8 T cell with inoculation back GM frequency.

The result- Estimate the HI terminal point

Observational variable:

At the 0th, 21,90 and 180 day: the serum hemagglutination suppressed (HI) antibody titer, to each test separately (resist-H1N1, resist-H3N2 and anti--B antibody) of 3 kinds of strains of influenza viruses that provide in the vaccine.

The intercepting value of the HI antibody of anti-all vaccine antigens is confirmed (equaling 1: 10) by laboratory before analysis.Seronegativity experimenter is the experimenter that its antibody titer is lower than the intercepting value.Seropositivity experimenter is the experimenter of its antibody titer more than or equal to the intercepting value.Being lower than the antibody titer of measuring the intercepting value provides with any value of half intercepting value.

Based on the HI antibody titer, calculate following parameter:

(j) at the geometric mean titer (GMT) of the HI antibody of the 0th day and 21 days, adopt the antilogarithm of logarithm titre conversion meansigma methods to calculate

(k) the seroconversion factor (SF) the 21st day the time is defined as than the increase multiple of the 0th day serum HI GMT the 21st day the time.

(I) serological conversion rate (SC) the 21st day the time is defined as inoculator's percentage rate of HI titre before the inoculation＜1: 10 and inoculation back titre >=1: 40, or titre before the inoculation >=1: 10 and inoculation back titre have the minimum inoculator's percentage rate that reaches 4 times of increases.

(m) the serum protective rate (SPR) the 21st day the time is defined as the inoculator's of serum HI titre >=1: 40 percentage rate.

Obtain the 95%CI of the GM of each group respectively.Suppose the number conversion titre and at first to obtain 95%CI number conversion titre meansigma methods with the unknown variance normal distribution.Then through the index conversion of the 95%CI of number conversion titre meansigma methods being obtained the 95%CI of GM.

The serology result of the disappearance that antibody specific detects is not replaced.Therefore, put serum-free in preset time and learn the determination and analysis that result's experimenter does not influence this time point.

The HI result ( Figure 27 and table 49)

GMT is in the same range as in 4 treatment groups before the inoculation of the HI antibody of whole 3 kinds of vaccine strains.After inoculation, the standard Fluarix in the same community, 2 kinds of adjuvants that increase the humoral response among the old people have tangible effect.

GMT is

For AS03+MPL (concentration 2), significantly higher to the GMT of H1N1,

For two kinds of adjuvants, significantly higher to the GMT of H3N2 and B,

Inoculate back 21 days, Fluarix (18-40 year) experimenter replys higher to the HI of New Caledonia and the strain of B/ Jiangsu.

Shown in table 49; In the experimenter more than 60 years old, there are the adjuvant influenza vaccines to exceed European official to the requirement of the lytic virus body influenza vaccines of every year registration [" about being used for the guide explanation that requires of the coordinated flow influenza vaccine that changes of strain of immunological evaluation year " (CPMP/BWP/214/96)].

After inoculation, with regard to the serum protective rate of HI antibody, Fluarix (>=65 years old) group and following group have significant difference:

Flu AS03+MPL (concentration 2) is for the strain of A/ New Caledonia

For each vaccine strain, 2 kinds of serum protective rates that adjuvant influenza vaccines groups arranged are in the same range as than Fluarix (18-40 year) group.

With regard to the positive rate of rotation of HI antibody, Fluarix (>=65 years old) group and following group have significant difference:

Flu AS03+MPL (concentration 2) is for the strain of A/ New Caledonia

Flu AS03+MPL (concentration 1) is for the strain of B/ Jiangsu

For each vaccine strain, 2 kinds of positive rates of rotation that adjuvant influenza vaccines groups arranged are in the same range as than Fluarix (18-40 year) group, except the strain of New Caledonia.

Table 49 is the 21st day serum protective rate, positive rate of rotation and conversion factor (immunogenic ATP crowd)

N=experimenter's sum; %=titre in the time of 21 days is in the experimenter's percentage rate in the particular range; The CI=confidence interval

XII.3.2. immunogenicity conclusion

(a) than the adult in 18-40 year, frequency is obviously relatively poor before the inoculation of the influenza specific C D4 among the old people.Using Fluarix ^TMAfter the inoculation, than youngster, frequency after the inoculation among the old people (the 21st day) is still relatively poor.On the contrary, than in 18-40 year adult, using Fluarix ^TMInoculation, the non-bad effect property of frequency after with the inoculation that shows influenza specific C D4 after having Adjuvanted vaccines to inoculate old experimenter.

(b) about the HI according to the HI antibody titer, all influenza vaccines are all satisfied European official to the requirement of the influenza inactivated vaccines of every year registration [" about being used for the guide explanation that requires of the coordinated flow influenza vaccine that changes of strain of immunological evaluation year " (CPMP/BWP/214/96)].In the old people, have Adjuvanted vaccines to mediate at least a trend: the HI to the influenza hemagglutinin compares Fluarix ^TMHigh.Table 50 has been summed up than Fluarix ^TMSignificantly different by the difference that the Adjuvanted vaccines mediation is arranged in old experimenter between the HI of every kind of vaccine strain.Than using Fluarix ^TMThe 18-40 year adult of inoculation demonstrates a kind of trend with the old experimenter that the Adjuvanted vaccines inoculation is arranged: GMT and the seroconversion factor are higher after the inoculation of anti-A/ New York strain in the time of the 21st day.

Table 50 has the significant difference of HI between Adjuvanted vaccines and the Fluarix in old experimenter

XII.4. reactionogenicity result

XII.4.1. the record of adverse events (AE)

Be recorded in the demand symptom (referring to table 51) that occurs in the middle of 7 days follow-up period (inoculation day with subsequently 6 days).Also write down the positive appeal symptom that in the middle of 21 days follow-up period (inoculation day with subsequently 20+3 days), occurs.Like intensity at the following AE of evaluation described in the table 52.

The part of table 51 demand/whole body adverse events

Attention: temperature is record at night.In one day, should carry out temperature detection in addition At All Other Times, the record maximum temperature.

The strength grade of demand symptom among table 52 adult

^*Heating is defined as axillaty temperature>=37.5 ℃ (99.5

)

The local injection position is rubescent/the following record of maximum intensity of swelling:

The 0th, 0mm; The 1st,＞0 to≤20mm; The 2nd,＞20 to≤50mm; The 3rd,＞50 mm.

The maximum intensity of following record heating:

The 1st,＞37.5 to≤38.0 ℃; The 2nd,＞38.0 to≤39.0 ℃; The 3rd,＞39.0 ℃.

Researcher carries out intensity to all other AE and judges that other AE is the symptom of positive appeal, is included in the SAE that reports in the middle of the research.Evaluation is based on the clinical judgment of researcher.The intensity distribution of every kind of AE of record is a following kind:

1 (slightly)=experimenter is the AE of tolerance easily, causes minimum discomfort, and does not disturb daily activity;

2 (medium)=discomforts are enough to disturb the AE of normal activity every day;

The AE of 3 (seriously)=obstruction normal activity every day (in adult/teenager, this AE for example can hinder work/go to school, and should give correct treatment).

XII.4.2. write down adverse events (AE)

With regard to local and General Symptoms this two, find that employing has Adjuvanted vaccines observed reactionogenicity in old experimenter to use Fluarix in than same community ^TMObserved height.But, its demonstrate with the colony of being grown up in observed level similar.Than using Fluarix ^TMObserved reactionogenicity is compared in old experimenter, and the sickness rate of symptom and intensity are increasing (Figure 28) after the Adjuvanted vaccines inoculation is arranged.In all cases, symptom all disappears fast.

3 grades of symptoms show a kind of trend: have the group of Adjuvanted vaccines (wherein MPL is a low concentration) to compare with acceptance, accepting the highest MPL concentration has the group of vaccine of adjuvant higher.But in all cases, symptom all disappears fast.

Claims (39)

1. (a) influenza virus or its antigenicity prepared product and (b) purposes of oil in water emulsion adjuvant in producing immunogenic composition; Said immunogenic composition be used for philtrum induce anti-said virus or antigenic composition below at least a: the CD4T-cellullar immunologic response that i) improves; The B-memory cell that ii) improves is replied; Wherein said O/w emulsion comprises zamene, alpha-tocopherol and Tween 80, and said influenza virus or its antigenicity prepared product comprise CD4T-cell epitope or B cell epitope or both.

2. influenza virus or its antigenicity prepared product and oil in water emulsion adjuvant are in the purposes of preparation in the immunogenic composition; Said immunogenic composition is used to inoculate the old people to influenza; Wherein said O/w emulsion comprises zamene, alpha-tocopherol and Tween 80, and said influenza virus or its antigenicity prepared product comprise CD4T-cell epitope or B cell epitope or both.

3. the purposes of claim 2, wherein said compositions are induced the CD4T-cellullar immunologic response of the improvement of anti-said virus or antigenic composition in said old experimenter.

4. each purposes among the claim 1-3, wherein said O/w emulsion comprises oil droplet, and at least 70% of said oil droplet is lower than 1 μ m with the intensitometer diameter.

5. each purposes among the claim 1-3, wherein said O/w emulsion comprises oil droplet, and at least 70% of said oil droplet is lower than 500nm with the intensitometer diameter.

6. each purposes among the claim 1-3, wherein said O/w emulsion comprises oil droplet, and at least 80% of said oil droplet is lower than 300nm with the intensitometer diameter.

7. each purposes among the claim 1-3, wherein said O/w emulsion comprises oil droplet, said oil droplet at least 90% with the intensitometer diameter in the scope of 120-200nm.

8. each purposes among the claim 1-3, wherein said alpha-tocopherol exists with 1.0% to 20% amount of said immunogenic composition cumulative volume.

9. the purposes of claim 8, wherein said alpha-tocopherol exists with 1.0% to 5.0% amount of said immunogenic composition cumulative volume.

10. each purposes among the claim 1-3, wherein zamene exists with 0.5% to 20% amount of said immunogenic composition cumulative volume.

11. the purposes of claim 10, wherein zamene exists with 1.0% to 10% amount of said immunogenic composition cumulative volume.

12. the purposes of claim 11, wherein zamene exists with 2.0% to 6.0% amount of said immunogenic composition cumulative volume.

13. each purposes, wherein zamene among the claim 1-3: the ratio of alpha-tocopherol is equal to or less than 1.

14. each purposes among the claim 1-3, wherein Tween 80 exists with the amount of 0.01% to 5.0% weight (w/w) of said immunogenic composition.

15. the purposes of claim 14, wherein Tween 80 exists with the amount of 0.1% to 2.0% weight (w/w) of said immunogenic composition.

16. each purposes among the claim 1-3, wherein said oil in water emulsion adjuvant has following composition: the alpha-tocopherol of the zamene of 2-10% weight, 2-10% weight and the Tween 80 of 0.3-3% weight.

17. each purposes among the claim 1-3, wherein said immunogenic composition also comprises 3D-MPL.

18. the purposes of claim 17, wherein 3D-MPL exists with the amount of every dose of compositions 10-50 μ g.

19. the purposes of claim 18, wherein 3D-MPL exists with the amount of every dose of compositions 25 μ g.

20. each purposes among the claim 1-3, the giving of wherein said immunogenic composition induces the CD4T-cellullar immunologic response of improvement and the B-memory cell of improvement to reply the two extraly.

21. each purposes among the claim 1-3, wherein said CD4T-cellullar immunologic response comprise that inducing cross reactivity CD4T to assist replys.

22. each purposes among the claim 1-3, wherein said target group are more than 50 years old.

23. each purposes among the claim 1-3, wherein said target group are the old people of over-65s.

24. influenza virus or its antigenicity prepared product purposes in producing immunogenic composition; Said immunogenic composition is used to inoculate previous people with influenza virus or its antigenicity prepared product and oil in water emulsion adjuvant inoculation; Said oil in water emulsion adjuvant comprises zamene, alpha-tocopherol and Tween 80, and said influenza virus or its antigenicity prepared product comprise CD4T-cell epitope or B cell epitope or both.

25. the purposes of claim 24, the wherein said compositions that is used to inoculate comprises 3D-MPL.

26. each purposes among the claim 24-25, wherein said oil in water emulsion adjuvant such as claim 1,2 and 4-18 in each definition.

27. each purposes among the claim 24-25; The wherein said immunogenic composition that is used to inoculate comprises influenza virus or its antigenicity prepared product; Said influenza virus or its antigenicity prepared product are at least a below having with the cracking influenza virus that is used for inoculation for the first time or its lytic virus antigenicity prepared product: i) common CD4T-cell epitope, ii) common B cell epitope.

28. each purposes among the claim 24-25; Immunne response after wherein inoculating is following any or two kinds or all: the CD4 of the improvement of resisiting influenza virus or its antigenicity prepared product replys, or the humoral response that improves or the B cell memory of improvement are replied.

29. (a) influenza virus or its antigenicity prepared product and (b) purposes of oil in water emulsion adjuvant in the preparation immunogenic composition; Said immunogenic composition is used to resist the protective effect of the influenza infection that the influenza virus as said influenza virus drift variant causes; Wherein said O/w emulsion comprises zamene, alpha-tocopherol and Tween 80, and said influenza virus or its antigenicity prepared product comprise CD4T-cell epitope or B cell epitope or both.

30. each purposes in the claim 1,2,24 and 29, wherein said influenza virus or its antigenicity prepared product are monovalent, bivalence or tervalent.

31. the purposes of claim 30, wherein said influenza virus or its antigenicity prepared product are from three kinds of different influenza strains.

32. the purposes of claim 31, wherein at least a strain is relevant with the outburst of being very popular, and perhaps has and the relevant potentiality of outburst that are very popular.

33. the purposes of claim 32, the wherein said strain that is very popular is selected from: H5N1, H9N2, H7N7, H2N2 and H1N1.

34. the purposes of claim 24, wherein inoculation is for the first time carried out with the cracking influenza compositions that contains the influenza strain that can cause the outburst of being very popular potentially, and the said strain that is very popular with circulating that inoculates carries out.

35. each purposes in the claim 1,2,24 and 29, wherein said immunogenic composition comprises the HA antigen that is less than 15 μ g.

36. each purposes in the claim 1,2,24 and 29, wherein said influenza antigens or its antigenicity prepared product be the ovum source or the tissue culture source.

37. a method that designs vaccine, said vaccine will pass through the influenza disease that the CD4+T cell activation is cured or treated to known, and said method comprises:

1) select to contain the CD4+ epi-position influenza antigen and

2) oil in water emulsion adjuvant of the said antigen of combination and each definition in claim 1,2,4-16, wherein said vaccine can induce the enhanced CD4 of resisiting influenza virus or its antigenicity prepared product to reply when giving said mammal in said mammal.

38. each purposes in the claim 1,2,24,29 and 37, wherein said influenza virus is selected from: cracking influenza virus, full influenza virus, subunit influenza virus, influenza virus particles and antigenicity prepared product thereof.

39. the purposes of claim 38, wherein said influenza virus are cracking influenza antigen or its antigen preparation thing.

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