CN101511385A - Making influenza virus vaccines without using eggs - Google Patents

Abstract

Currently, the steps performed prior to release of influenza strains to vaccine manufacturers involve passaging influenza virus through eggs. The invention aims to provide procedures useful in manufacturing influenza vaccines, in which the use of eggs is reduced, and preferably is avoided altogether. For instance, rather than use chicken eggs for influenza vaccine isolation, MDCK cells (Madin Darby canine kidney cells) may be used e.g. growing in suspension, growing in a serum-free medium, growing in a protein-free medium, being non-tumorigenic, grown in the absence of an overlay medium, etc.

Description

Do not use egg to prepare influenza virus vaccine

It is for referencial use that all documents that this paper is quoted are included this paper in full in.

Technical field

The invention belongs to the manufacturing field of the vaccine that is protected from influenza infection.

Background technology

The preparation method of the seasonal vaccine of antagonism human influenza virus infection may further comprise the steps [1,2] at present: the Strain of (a) separating circulation; (b) isolating virus is carried out antigen and genetic analysis; (c) be chosen in the Strain that is suitable in the season that to arrive; (d) by a reprovision or high growth kind of the strain of employing reverse genetics preparation; (e) will plant strain and be distributed to the production of vaccine merchant; (f) estimate the suitability of this kind strain in commercial production by the manufacturer; (g) cultivate kind of strain, make vaccine by this virus then to prepare virus.

Step in this process (a)-(e) is undertaken by the international influenza center of FDA and government permission, obtains the subsidy of World Health Organization (WHO) usually; Step (f) and (g) undertaken by manufacturer oneself.

Step (d) is converted into virus from the form of natural suitable infection human body can grow to the form that height is tired under industrial condition of culture.For influenza A virus, this step generally includes and produces the 6:2 reassortant, this reassortant comprises HA and the NA encoding gene group section from the strain of selecting in (c), and all the other 6 genome sections come effective strain of cultivating in the comfortable egg, and this strain is A/PR/8/34 normally.After the reprovision step, strain goes down to posterity in containing the embryo egg repeatedly to realize that egg adapts to and growth.For Influenza B virus, can directly obtain to have the prototype-strain of good growth characteristics usually and in containing the embryo egg, go down to posterity repeatedly and need not to produce reprovision virus.

Therefore, being distributed to the step of carrying out before the production of vaccine merchant is included in the egg influenza virus is gone down to posterity.Even cultivating virus on the cell substrate rather than on the egg by the manufacturer in step (g), some stage between virus will be accepted the manufacturer of the separation of step (a) and step (e) goes down to posterity by egg.

For example, step (a) comprises makes substrate contact with patient's sample, makes that the virus on any sample can infect substrate.Then, the virus that the substrate amplification exists, the virus after obtaining increasing is used for further research.This step is carried out in egg or mammalian cell.The known former generation isolated cells of can be used for comprises: MRC-5 cell [3], Vero cell [4,5], mdck cell [6], HepG2 cell [7], LLC MK2 cell [8] etc.But, usually still use egg to separate to be used to make the reference strain of influenza virus.The use of egg is most important in current operation, fourth quarter in 2003, FDA has vetoed the use (A/Fujian/411/2002) of most of suitable H3N2 strains, because they are not from egg isolating [29] at first, and does not also obtain antigenicity similarly from the isolating strain of egg.

Past attempts proposes not re-use egg in a plurality of stages of production of Influenza virus.

List of references 10 proposes, vaccine should adopt (i) through going down to posterity height growth clinical separation strain or (ii) from the deutero-reassortant of mammal strains of influenza viruses of at least a natural origin, cultivate in cell culture, prerequisite is that described separated strain or reassortant do not go down to posterity through fowl egg.Therefore, list of references 10 described methods are from being beneficial to the kind virus beginning of cultivating through selection or operation selected cell culture.

List of references 11 has compared virus that goes down to posterity by egg and the virus that goes down to posterity by mdck cell, but selects the former to be used for vaccine production especially.

List of references 12 suggestion, the kind virus of pandemic influenza vaccine can prepare in the following manner: the strain directly breeding rather than on mammalian cell cultures of being very popular by embryonated egg, but point out that it is the interval that is very popular to make necessary that egg goes down to posterity.The reason that this mandatory egg goes down to posterity is, it is believed that egg goes down to posterity can be used as " filter " of foreign substance: administrative organization accepts, carry out the initial clinical process that is separated to the final vaccine product that gives human body from human body, in the birds system, carry out a series of foreign substance and influenza virus that can prevent mammalian-type of going down to posterity and duplicate jointly.

The purpose of this invention is to provide the additional method that is applicable to that influenza vaccines produce, this method has reduced the use of egg, preferably avoids using egg.One concrete aspect, the purpose of this invention is to provide the useful extra and operation that improves in the influenza virus separation process.

Summary of the invention

Though past attempts proposes not re-use egg in a plurality of stages of production of Influenza virus, the present invention is different with these suggestions in many aspects.

Plant the preparation of virus

A first aspect of the present invention provides preparation to be used to make the method for the influenza kind virus of vaccine, and this method may further comprise the steps: (i) use directly obtain from the patient or from the influenza infection cell line of former generation separator; (ii) the virus with gained infected cell system in the step (i) at least once goes down to posterity; (iii) step infected cell is (ii) cultivated with the preparation influenza virus.From step (iii) the influenza virus of culture purification promptly can be used as kind of a virus.

Different with list of references 12, the influenza virus of using in the step (i) is Influenza B virus or non-being very popular property influenza A virus, promptly is influenza A virus H1N1 or H3N2 strain at present.

Step (i), (ii) or (iii) do not relate to and in egg, carry out Virus culture or go down to posterity.Preferably, at least two steps, all three steps are all carried out in identical cell type ideally, for example all carry out in mdck cell.

Step going down to posterity (ii) generally includes: influenza virus is duplicated in cell culture; For example collect the virus of duplicating from culture supernatants; With the replication-competent virus of collecting is transferred in the cell culture that does not infect.Can repeat this process.At least once after going down to posterity, (iii) make virus replication in step, collect virus, just virus is used as kind of virus rather than transfers to the culture that does not infect and further goes down to posterity.

The used cell line of first aspect preferably is not the human cell line.By avoiding end user's cell, even do not use egg also can keep " filtering " to external reagent.Because with people's close relationship, cell line preferably is not primate cell system yet, for example is not Vero cell line (monkey-kidney cells system).List of references 10 proposes to use the Vero cell during vaccine is made, but this cell can infect many Human virus, thereby any other Human virus who exists in the influenza virus sample that uses in the step (i) will parallel production with influenza virus, causes final pollution of planting virus.

The preferred cell system that is used for first aspect present invention is a canine cells system, as mdck cell system (Madin-Darby canine kidney cell line), is different from the concrete disclosure of list of references 10.The further details of mdck cell provides hereinafter.Have been found that mdck cell has " filtering " effect that is equivalent to the antagonism foreign substance that fowl egg can realize.Therefore, based on this discovery, mdck cell can be used for replacing egg and can not increase managing risk, goes down to posterity and can have growth advantage between the MDCK culture period though reported the influenza virus egg in the list of references 13, has avoided the cultivation in the egg.

List of references 14 is described, and from people patient's isolated influenza strain, need not to carry out any going down to posterity in egg or cell culture, can effectively cultivate in comprising the mdck cell culture of serum-free medium.Therefore, the infection of step (i) can be adopted the influenza virus from the clinical sample (for example by pharyngeal swab etc.) that the patient directly obtains, and perhaps can adopt and pass through isolating virus of former generation.In some cases, in step (i) former generation before, separates and can carry out in egg, is the separator that does not use egg to obtain but be used for preferred former generation of the present invention separator, the separator that for example obtains in mammalian cell.The known former generation isolated cells of can be used for includes but not limited to: MRC-5 cell [3], Vero cell [4,5], mdck cell [6], HepG2 cell [7], LLC MK2 cell [8] etc.

If the present invention adopts former generation separator in step (i), in former generation, separates and preferably carries out in the cell type identical with step (i)-(iii).Known MDCK is applicable to that former generation of influenza virus separates, goes down to posterity and cultivate, but below will describe the isolating improved form of MDCK.

In order to understand the history of influenza virus separator as much as possible, the preferred employing from clinical isolates directly obtains virus rather than uses former generation separator.Present influenza monitoring system comprises that hospital separates in former generation, delivers to country and international influenza center with strain interested.Except that using patient's sample to carry out the former generation separation, also need to reserve a part usually and store (for example by freezing), separate once more thereby can recover original material.If the sample that can obtain to store then can use this sample to replace former generation separator in step (i).

If the source of separator is not clear, especially use is not when directly the virus of acquisition begins from clinical sample, can use reverse genetics to produce new Strain in step (i) with (iii), this new Strain has at least one viral genome section from parental virus.Adopt in step (i) and (iii) reverse genetics can be from the viral product that step is used (iii) the viral product of use in the separating step (i), thereby can be used as the filter of the foreign substance that antagonism may introduce before at step (i).As " filter ", reverse genetic learns a skill and also can be used for other reasons except by this way, for example produces reassortant, handles coded sequence, replaces particular section etc.Further details will provide in second aspect present invention.

Unite and use learn a skill cell culture with influenza virus of reverse genetic

A second aspect of the present invention provides preparation to be used to make the method for the influenza kind virus of vaccine, and this method may further comprise the steps: (i) use directly obtain from the patient or from the influenza infection cell line of former generation separator; The cDNA that (ii) prepares at least one viral RNA section of the influenza virus that produces by gained infected cell system in the step (i), and in the reverse genetics method, adopting this cDNA to prepare new influenza virus, at least one viral RNA section is identical with the influenza virus of step (i) in this new influenza virus; (iii) use new influenza infection cell line, cultivate this cell line then to prepare new influenza virus.

The virus of using in the step (i) can be the influenza A virus of any hypotype, perhaps can be Influenza B virus.Preferred subtypes of influenza A virus is H1, H3 and H5.

Step (i), (ii) or (iii) do not relate to and in egg, carry out Virus culture or go down to posterity.Preferably, in the cell of same type, carry out in steps, for example they can carry out in mdck cell.

Other features of second aspect present invention are as mentioned described in the first aspect.Therefore, preferably use mdck cell, or the like.

Hereinafter describing reverse genetic in more detail learns a skill.The (ii) middle genome section that shifts of step can comprise the HA section, can comprise NA section and/or one or more other sections.

Plant virus

First and second aspects of the present invention provide plants virus.These kinds virus can be used in every way.

The sign (for example) of kind of virus comprises, nucleic acid and/or protein sequence are checked order, and checks the antigen dependency of itself and other strain (strain of for example circulating), checks its immunogenicity, or the like.

Plant virus and can be used for causing antiserum.

Plant virus and can distribute to the production of vaccine merchant.

Planting virus can store for future use.

Plant virus and can be used for the preparation work seed lot.This system can realize that the safety of original species virus stores, and can adopt the work seed lot to carry out daily use.Can plant work virus freezing standby.The preparation of work seed lot can be included in the step of carrying out Virus culture in the cell culture, preferably carries out on the cell type identical with making kind virus.Do not adopt and produce the preparation work seed lot in the egg.

Plant virus and can be used for infection cell system, cultured cell system is to be provided for the viral of production of vaccine or to be used to prepare diagnostic reagent.

The present invention plants virus and has many common characteristics with the deutero-kind virus of using at present of egg, but in the many aspects difference.

For example, the preferred influenza A virus kind of the present invention virus comprises the viral section from the PR/8/34 influenza virus less than 6 (promptly 0,1,2,3,4 or 5), and is following described in more detail.

Need not the production of vaccine of egg

A third aspect of the present invention provides preparation to be used to make the method for the influenza virus of vaccine, this method may further comprise the steps: (i) obtain influenza virus that circulates in colony or the influenza virus that comprises hemagglutinin, this hemagglutinin has the representative antigenicity of the influenza virus that circulates in colony; The influenza infection cell line of (ii) using step (i) to obtain; (iii) will by step (ii) the virus that obtains of infected cell system at least once go down to posterity, to obtain kind of a strain; (iv) incubation step kind strain (iii) is to produce influenza virus.

There is mutual difference the same (seeing above) with first and second aspects, a fourth aspect of the present invention provides preparation to be used to make the method for the influenza virus of vaccine, this method may further comprise the steps: (i) obtain influenza virus that circulates in colony or the influenza virus that comprises hemagglutinin, this hemagglutinin has the representative antigenicity of the influenza virus that circulates in colony; The influenza infection cell line of (ii) using step (i) to obtain; The cDNA that (iii) prepares at least one viral RNA section of the influenza virus that produces by gained infected cell system in the step (i), and in the reverse genetics method, adopting this cDNA to prepare influenza kind virus, at least one viral RNA section is identical with the influenza virus of step (i) in this influenza kind virus; (iv), cultivate then from step cell line (iii) to produce influenza virus through going down to posterity with influenza kind virus infected cell system.

The present invention also provides the method for preparing influenza virus vaccine, and this method comprises these steps (i)-(iv) of third and fourth aspect, be then step (v): treatment step (iv) gained virus to produce vaccine.(the technology details that adopt v) provide step hereinafter.

Step (i), (ii), (iii), (iv) or (v) do not relate in egg and to cultivate or the virus that goes down to posterity.

The virus of using in the step (i) can be the influenza A virus of any hypotype, perhaps can be Influenza B virus.Preferred subtypes of influenza A virus is H1, H3 and H5, for example H1N1 or H3N2.

The virus of using in the step (i) begins just never to breed on egg since initial separation, preferably from patient that virus is provided at first until any stage the process of step (i) beginning all never breed at egg.

In fact the term that uses in the influenza vaccines field " representative antigenicity " is used for describing and may circulate extensively in colony, but can cause the immunne response of the strain in the antagonism circulation, is convenient to the Strain of producing simultaneously.The serum (for example) that the strain of " representative antigenicity " causes can suppress the strain that circulates in hemagglutination inhibition test.

Step (i) can comprise from many different strains, selects the strain that further uses then.For example, step (i) can comprise from many different influenza A virus H1N1 strains, selects the strain that further uses then.Step (i) can comprise from many different influenza A virus H3N2 strains, selects the strain that further uses then.Step (i) can comprise from many different Influenza B virus strains, selects the strain that further uses then.Conventional immunity and the serology standard of using when selection will be based on the strain selecting to be included in the influenza virus for example selects to have representative antigenic strain of the most common strain in the circulation and/or pathogenic strain.

Step (iii) afterwards can be carried out following steps: prove that kind of virus has the representative antigenicity of step (i) gained strain.This verification step can carry out before step (iv) begins, and perhaps can carry out with step is (iv) parallel.

The present invention also provides the method for preparing influenza virus vaccine, and this method may further comprise the steps: the virus of the method preparation of the step (i)-(iv) of the step (i)-(iii) that comprises first aspect of (a) passing or second aspect; (b) handle this virus with the preparation vaccine.

Therefore, third and fourth aspect of the present invention can be from patient's sample (or former generation separator) preparation influenza vaccines, and the influenza virus that is used to prepare vaccine was not all gone down to posterity through egg in any stage.

The virus reprovision

A fifth aspect of the present invention provides the method for the influenza virus of preparation reprovision, this method may further comprise the steps: (i) with first strains of influenza viruses with first group of genome section and the second strains of influenza viruses infection cell system with second group of genome section, wherein said first strain has the HA section of the required hemagglutinin of coding; (ii) the infected cell of incubation step (i) is with the preparation influenza virus, at least one section is from first group of genome section in this influenza virus, at least one section is from second group of genome section, and prerequisite is that described at least one section from first group of genome section comprises the HA section from first strain.Therefore, this method at least can be with the HA sector transfer of first strain to second strain, has from the new reassortant of the different virus genome section set of first or second strain and do not need to use egg thereby produce.

From step (ii) the influenza virus of culture purification can be used for the described application of other parts of this paper.With respect to first strain, the growth characteristics of reassortant improves.

Reassortant also can comprise the NA section from the required neuraminidase of coding of first strain.

Usually, the section ratio from first strain and second strain that is comprised in the reassortant is 1:7,2:6,3:5,4:4,5:3,6:2 or 7:1.Usually, most of section is from second strain.

This method can be used for preparing the reassortant of influenza A virus and Influenza B virus.For influenza A virus, in some embodiments, second virus is PR/8/34, but also can adopt other strains, comprises having only 1,2,3,4 or 5 virus that section is identical with PR/8/34 in those NP, M, NS, PA, PB1 or the PB2 section.

The influenza virus that the present invention also provides the reallocating method by the 5th aspect to obtain.The present invention also provides should the application of virus in vaccine is made.

Step (i) or (ii) do not relate to and in egg, carry out Virus culture, reprovision or go down to posterity.The reprovision process can be easily with described in other parts of this paper separate and the used identical cell type (for example mdck cell) that goes down to posterity in carry out.

First strain should be directly to obtain or from the influenza virus of former generation separator from the patient.

Virus is separated

As mentioned above, exist the use egg to carry out the isolating strong tendency of influenza virus.On the contrary, a sixth aspect of the present invention adopts mdck cell.In some embodiments, suspension culture mdck cell.In other embodiments, mdck cell is cultivated at serum-free medium or in protein-free medium.In other embodiments, mdck cell is a non-tumorigenic.In other embodiments, mdck cell is not covering cultivation in the presence of the culture medium.

Therefore, the invention provides from the method for patient's sample separation influenza virus, this method may further comprise the steps: cultivate patient's sample with mdck cell, described mdck cell is cultivated in suspending nutrient solution.

The present invention also provides from the method for patient's sample separation influenza virus, and this method may further comprise the steps: cultivate patient's sample with mdck cell, described mdck cell is cultivated in serum-free medium.

The present invention also provides from the method for patient's sample separation influenza virus, and this method may further comprise the steps: cultivate patient's sample with mdck cell, described mdck cell is cultivated in protein-free medium.

The present invention also provides from the method for patient's sample separation influenza virus, and this method may further comprise the steps: cultivate patient's sample with the non-tumorigenic mdck cell.

The present invention also provides from the method for patient's sample separation influenza virus, and this method may further comprise the steps: cultivate patient's sample with mdck cell, do not provide the covering culture medium to described mdck cell.

The present invention also provides from the method for patient's sample separation influenza virus, and this method may further comprise the steps: cultivate patient's sample with mdck cell, described mdck cell is cultivated in the serum-free suspending nutrient solution.

The present invention also provides from the method for patient's sample separation influenza virus, and this method may further comprise the steps: cultivate patient's sample with mdck cell, described mdck cell is cultivated in no protein suspending culture fluid.

The present invention also provides from the method for patient's sample separation influenza virus, and this method may further comprise the steps: cultivate patient's sample with the non-tumorigenic mdck cell, described mdck cell is cultivated in suspending nutrient solution.

The present invention also provides from the method for patient's sample separation influenza virus, and this method may further comprise the steps: cultivate patient's sample with the non-tumorigenic mdck cell, described mdck cell is cultivated in the serum-free suspending nutrient solution.

The present invention also provides from the method for patient's sample separation influenza virus, and this method may further comprise the steps: cultivate patient's sample with the non-tumorigenic mdck cell, described mdck cell is cultivated in no protein suspending culture fluid.

The present invention also provides by one of said method isolated influenza virus.The present invention also provides should the application of virus in vaccine is made.

The cultivation of patient's sample and mdck cell causes mdck cell by influenza virus usually, and for example human influenza virus, especially influenza virus A hominis infect.Virus can be duplicated in cell, collects the cell that duplicates then.Randomly, can in downstream processes, adopt following steps, for example detect, characterize, analyze, prepare kind of a virus, operation etc.

After separating in mdck cell, virus also can go down to posterity in mdck cell and/or cultivate.Mode as an alternative perhaps also can be at non-mdck cell, and perhaps egg perhaps goes down to posterity in other substrates and/or cultivates.

A sixth aspect of the present invention relates to the use of mdck cell system.Original mdck cell is to obtain from ATCC, but a sixth aspect of the present invention adopts the derivant of this cell line.As follows, the separation in these derivants is better than the separation in original mdck cell is.Suitable mdck cell and feature thereof be more detailed description hereinafter.

For example, some embodiments of the 6th aspect adopt and can in suspending nutrient solution the spontaneous mdck cell that duplicates be.List of references 30 has been described the mdck cell system that is adapted at cultivating in the suspending nutrient solution, and this cell line (' MDCK 33016 ') is particularly useful for the method for the 6th aspect.MDCK 33016 can grow in serum-free medium, and need not to cover culture medium can grow.The another kind of mdck cell that can cultivate in suspending nutrient solution (comprising serum-free medium) is to be ' B-702 ' cell line [36; Vide infra].

The non-tumorigenic mdck cell system that is used for the 6th aspect comprises those that list of references 37 is described, for example ' MDCK-S ', ' MDCK-SF101 ', ' MDCK-SF102 ' and ' MDCK-SF103 ' (vide infra).

In some embodiments aspect the 6th, in serum-free medium and/or protein-free medium, cultivate mdck cell.

Different with the method for prior art, sixth aspect present invention can be avoided using between the influenza virus separation period and cover culture medium.

Before the preferred contact mdck cell as indicated above, in egg, do not cultivate virus.Preferred, virus of cultivating in mdck cell as indicated above is not cultivated in egg subsequently yet.

For the used patient's sample in the 6th aspect, the clinical sample that uses during influenza virus separates can be a various forms, but generally includes respiratory secretions, includes but not limited to: direct aspirate; Collutory; The nasal cavity cleaning mixture; The nasal cavity swab; The pharyngeal canal swab; Pharyngeal swab etc.These samples obtain from doubtful patient by influenza infection usually, comprise the patient who suffers from the new type influenza Strain.

Can be used for preparing the influenza kind virus that is used to make vaccine by the 6th aspect method isolated influenza virus.Therefore, a sixth aspect of the present invention also can comprise the step that the virus that is from infected mdck cell is at least once gone down to posterity.Then, this method can comprise the step of cultivation infected cell with the preparation influenza virus.The influenza virus of purification can be used as kind of a virus behind the incubation step, as described in other parts of this paper.

The source that also can be used as the reverse genetic technology according to the isolating virus in the 6th aspect.Therefore, can prepare cDNA from least one viral RNA section according to the 6th aspect isolated influenza virus.Then, this cDNA can be used for the reverse genetic process so that prepare the new influenza virus that has at least one identical viral RNA section with isolated influenza virus.Then, this new influenza virus can be used for infecting (for example) cell line with further cultivation.

A sixth aspect of the present invention can be used for separating any suitable influenza virus, comprises the human influenza virus.These viruses can be influenza A virus, Influenza B virus or influenza virus C.Common and useful subtypes of influenza A virus is H1, H3 and H5.

In present interval in the stage of being very popular, vaccine generally includes two kinds of influenza A strains (H1N1 and H3N2) and a kind of influenza B strain.The 6th aspect can be used for separating these strains, or separates the pandemic disease strain, as H2, H5, H7 or H9 hypotype strain.Usually, the 6th aspect can be used for separating the influenza A virus with one of HA hypotype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.

Can comprise with oligosaccharide according to sixth aspect present invention isolated influenza virus and to compare, more be partial to and the bonded hemagglutinin of oligosaccharide with the terminal disaccharide of Sia (α 2,6) Gal with the terminal disaccharide of Sia (α 2,3) Gal.Preferably, find that separation method of the present invention helps stable viral HA sequence and the oligosaccharide preference thereof of keeping.

Can be used for vaccine manufacturing and Therapeutic Method according to the isolating virus in the 6th aspect.Therefore, the present invention also provides the application that is used for improving the medicine of patient's immunne response according to the antigen of the isolating virus preparation in the 6th aspect in manufacturing.

Receptors bind

The human influenza virus is in conjunction with the receptor oligosaccharide with terminal disaccharide of Sia (α 2,6) Gal (α-2,6 is connected to the sialic acid of galactose), and egg has the receptor oligosaccharide of the terminal disaccharide of Sia (α 2,3) Gal.The growth of human influenza virus in egg provides with Sia (α 2,6) Gal combination and compared, and the selection that is more prone to Sia (α 2,3) the bonded hemagglutinin of Gal is pressed.

The same with egg, the Vero cell is mainly expressed Sia (α 2,3) Gal receptors [15].On the contrary, mdck cell and PER.C6 cell are expressed Sia (α 2,3) Gal and Sia (α 2,6) Gal simultaneously.List of references 16 report transfection mdck cells are beneficial to select Sia (α 2,6) Gal combination with overexpression α-2,6 sialyltransferase.But, even without this operation, also can on mdck cell, cultivate influenza virus, and not make their deflection Sia (α 2,3) Gal combinations.Therefore, the present invention can adopt and express Sia (α 2,3) Gal and Sia simultaneously (α 2,6) cell of Gal, the influenza virus that is produced have with the oligosaccharide that contains the terminal disaccharide of (α 2,3) Gal to be compared, more be partial to contain the terminal disaccharide of Sia (α 2,6) Gal oligosaccharide in conjunction with preference.

In the preferred implementation aspect the present invention first and second, the influenza virus that is used to infect in the step (i) has with respect to the oligosaccharide that contains the terminal disaccharide of Sia (α 2,3) Gal, more be partial to contain the terminal disaccharide of Sia (α 2,6) Gal oligosaccharide in conjunction with preference.(ii) keep during (iii) in step in conjunction with preference with step, the influenza virus that makes step (iii) be produced has that (α 2 with respect to containing Sia, 3) oligosaccharide of the terminal disaccharide of Gal, more be partial to contain the terminal disaccharide of Sia (α 2,6) Gal oligosaccharide in conjunction with preference.

In the preferred implementation aspect the present invention third and fourth, the influenza virus that step is used to infect in (ii) has with respect to the oligosaccharide that contains the terminal disaccharide of Sia (α 2,3) Gal, more be partial to contain the terminal disaccharide of Sia (α 2,6) Gal oligosaccharide in conjunction with preference.Thisly (iii) keep during (iv) in step with step in conjunction with preference, the influenza virus that makes step (iv) be produced has that (α 2 with respect to containing Sia, 3) oligosaccharide of the terminal disaccharide of Gal, more be partial to contain the terminal disaccharide of Sia (α 2,6) Gal oligosaccharide in conjunction with preference.

In order to determine whether virus has with respect to the oligosaccharide that contains the terminal disaccharide of Sia (α 2,3) Gal, more be partial to contain the terminal disaccharide of Sia (α 2,6) Gal oligosaccharide in conjunction with preference, can utilize various analytical tests.For example, list of references 17 has been described mensuration influenza virus receptor-test in conjunction with active insoluble enzyme joint-trial, and it is the quantitative assay affinity constant delicately.List of references 18 adopts assessment virus, and (ovomucoid has Sia (α 2,3) Gal determinant with two kinds of different sialoglycoproteins; With pig α ₂Macroglobulin has Sia (α 2,6) Gal determinant) the test of bonded solid phase, also described and estimated virus and two kinds of receptor analogs: free sialic acid (Neu5Ac) and 3 '-the bonded test of sialyl lactose (Neu5Ac α 2-3Gal β 1-4Glc).List of references 19 has been reported the test of adopting the polysaccharide array that can know the receptor preference of distinguishing α 2,3 or α 2,6 connections.List of references 20 has been reported a kind of based on the agglutinative test of human red blood cell that comprises Sia (α 2,6) Gal or Sia (α 2,3) Gal through enzyme modification.According to test type, can utilize virus itself directly to test, perhaps can utilize and test indirectly from the hemagglutinin of viral purification.

Reference material

The process of making at present influenza virus comprises the reference reagent that prepare every kind of strain, and promptly (i) resists-the HA serum and the (ii) whole virus particles of purification.Can utilize these calibration reagents to measure HA level in large quantities of antigens that manufacturer produces in the SRID test, thereby this batch antigen diluent can be obtained the vaccine that every dosage has required HA content.

The reference strain goes down to posterity by egg and produces strain and cultivate according to egg and be optimized in present method, and serum and antigen in this moment reference reagent mate well.Yet find that the antigen coupling of the serum that produces is relatively poor in cell culture, be likely because the selection in different system is pressed different.Reactivity difference between control serum and the antigen represents that the HA level is underestimated, cause the dosage of (i) given batch low and (ii) in the vaccine HA dosage too much.

In order to overcome the derive matching problem of the serum that antigen and egg derived material produce of cell culture, the invention provides based on the reference material that does not adapt to the virus that the egg base cultivates.

Therefore, the invention provides from animal and prepare sero-fast method, this method may further comprise the steps: the influenza virus hemagglutinin that (i) gives the animal purification; (ii) reclaim the animal serum that contains this hemagglutinin identification antibody then, it is characterized in that, the virus of cultivating in the next comfortable cell line of the hemagglutinin that uses in the step (i) from animal.

The hemagglutinin that uses in the step (i) is preferably from the virus of never cultivating in egg.For example, hemagglutinin can have with respect to the oligosaccharide that contains the terminal disaccharide of Sia (α 2,3) Gal, more be partial to contain the terminal disaccharide of Sia (α 2,6) Gal oligosaccharide in conjunction with preference.

Can utilize mammal cell line (cell line for example as herein described) as MDCK in the polysaccharide that obtains of growth carry out glycosylation modified to being used to produce sero-fast hemagglutinin.

Antiserum can be influenza A virus and Influenza B virus antiserum.

Animal is mammal preferably, as goat, and more preferably sheep.Antiserum is not difficult to prepare in sheep, promptly handles the HA that extracts purified virus, sedimentation purification on saccharose gradient then by bromelain.Give sheep with the hemagglutinin of the about 50 μ g of dosage with complete Freund's adjuvant (FCA) intramuscular.Give dosage 10 μ g after two weeks, give dosage again 2-4 time with weekly interval then.Then, collect serum.Before the use, can dilute (for example with containing the PBS buffer of Hydrazoic acid,sodium salt) this serum and be loaded in the container.This serum can be exposed to acid pH (for example pH5 continues 2 hours) to meet the foot and mouth disease regulation.

The present invention also provides from animal and has prepared sero-fast method, and this method may further comprise the steps: (i) cultivate influenza virus in cell line; The (ii) viral purification hemagglutinin antigen of cultivating from step (i); (iii) with step (ii) the hemagglutinin of purification give animal; (iv) reclaim the animal serum that contains hemagglutinin identification antibody then from animal.

The present invention also provides the antiserum that obtains by these methods.

The present invention also provides and has comprised this sero-fast gel.Therefore, above-mentionedly prepare sero-fast method from animal and comprise antiserum and the blended additional step of gel.Gel is applicable to and carries out the SRID test that for example, it is an agarose gel.

Except antiserum is provided, the present invention also provides the antigen reference material.Therefore, the invention provides the method for preparing the antigen reference material, this method may further comprise the steps: (i) cultivate influenza virus in cell line; The (ii) virus of cultivating in the purification step (i); (iii) inactivation of viruses is characterized in that, the influenza virus of test was never cultivated in egg in the step (i).This method can be further comprising the steps of: (iv) with the viral lyophilizing of deactivation.

The virus of test was never cultivated in egg in the step (i).For example, hemagglutinin can have with respect to the oligosaccharide that contains the terminal disaccharide of Sia (α 2,3) Gal, more be partial to contain the terminal disaccharide of Sia (α 2,6) Gal oligosaccharide in conjunction with preference.

Reference material does not contain egg derived material (for example, do not contain ovalbumin, do not contain ovomucoid, do not contain chicken DNA).Can utilize mammal cell line (cell line for example as herein described) as MDCK in the polysaccharide that obtains of growth carry out glycosylation modified to the glycoprotein in the reference material.Reference material can be the reference material of influenza A virus and Influenza B virus.

Reference material is generally to using, so the present invention also provides the kit that comprises following material: (i) antiserum that obtains from these methods and the antigen reference material that (ii) obtains from these methods.

The present invention also provides the method for preparing kit, and this method may further comprise the steps: (i) prepare antiserum as mentioned above; (ii) prepare the antigen reference material as mentioned above; (iii) step (i) is become kit with (ii) product mix.

Antigen and antiserum are suitable and can be used for the SRID test, the invention provides the unidirectional radioimmunodiffusion test of influenza virus hemagglutinin, it is characterized in that the antigen reference material that obtains antiserum and/or obtain from these methods from these methods is adopted in this test.SRID test may further comprise the steps: preparation comprises sero-fast gel, and antigen reference material (the use medium is rebuild when needing) is applied to gel (adding in the hand-hole usually), makes then that antigen is radial to be diffused in the gel.Available detergent such as zwitterionic detergent are handled antigen before using.

By technology preparation of the present invention or isolating virus (comprising kind of a virus).

The preferred influenza A virus of the present invention (comprise kind of a virus, with mdck cell from the virus of patient's sample separation, reprovision virus etc.) comprises the viral section from the PR/8/34 influenza virus less than 6 (promptly 0,1,2,3,4 or 5).Preferably do not contain the PR/8/34 section.If there are one or more sections of PR/8/34 arbitrarily, then they do not comprise the PR/8/34HA section and do not comprise the PR/8/34NA section usually.Therefore, preferred virus is at least one virus that does not derive from PR/8/34 among section NP, M, NS, PA, PB1 and/or the PB2.More preferably, at least one does not derive from PR/8/34 among section NP, M, PA, PB1 and/or the PB2.Therefore, the present invention has improved currently available vaccines, existing vaccines by the epitope antigen that contains that adds one or more representative circulation strains in normal HA and NA antigen.

Similarly, preferred influenza A virus comprises the viral section from AA/6/60 influenza virus (A/AnnArbor/6/60) less than 6 (promptly 0,1,2,3,4 or 5).Preferably do not contain the AA/6/60 section.If there are one or more sections of AA/6/60 arbitrarily, then they do not comprise the AA/6/60HA section and do not comprise the AA/6/60NA section usually.Therefore, preferred virus is at least one virus that does not derive from AA/6/60 among section NP, M, NS, PA, PB1 and/or the PB2.More preferably, at least one does not derive from AA/6/60 among section NP, M, PA, PB1 and/or the PB2.

Preferred Influenza B virus comprises the viral section from AA/1/66 influenza virus (A/Ann Arbor/1/66) less than 6 (promptly 0,1,2,3,4 or 5).Preferably do not contain the AA/1/66 section.If there are one or more sections of AA/1/66 arbitrarily, then they do not comprise the AA/1/66HA section and do not comprise the AA/1/66NA section usually.Therefore, preferred virus is at least one virus that does not derive from AA/1/66 among section NP, M, NS, PA, PB1 and/or the PB2.More preferably, at least one does not derive from AA/1/66 among section NP, M, PA, PB1 and/or the PB2.

The preferred influenza virus of the present invention (comprise kind of a virus, with mdck cell from the virus of patient's sample separation, reprovision virus etc.) comprises that (α 2 with respect to having Sia, 3) oligosaccharide of the terminal disaccharide of Gal, more be partial to hemagglutinin in conjunction with oligosaccharide with the terminal disaccharide of Sia (α 2,6) Gal.This described in more detail as mentioned in conjunction with preference.

The preferred influenza virus of the present invention (comprise kind of a virus, with mdck cell from the virus of patient's sample separation, reprovision virus etc.) comprises that glycosylation pattern is different from the glycoprotein of egg derived virus (comprising hemagglutinin).Therefore, glycoprotein will comprise the unexistent sugared shape of egg cultivation virus, for example can have non-birds sugar key, comprise mammal sugar key.

Cell line

The present invention relates to use the cell line of supporting that influenza virus is duplicated, and avoid using egg.Cell line is the mammal source normally.Suitable mammalian cell source includes but not limited to: hamster, cattle, primates (comprising people and monkey) and canine cells, but the use of primate cell is not preferred.Can use various cell types, as nephrocyte, fibroblast, retina cell, pneumonocyte etc.The example of suitable hamster cell is the cell line that is called BHK21 or HKCC.Suitable MC is (for example) cercopithecus aethiops cell, for example nephrocyte such as Vero cell line [21-23].Suitable canine cells is (for example) nephrocyte, as CLDK and mdck cell system.

Therefore, suitable cell line includes but not limited to: MDCK; CHO; CLDK; HKCC; 293T; BHK; Vero; MRC5; PER.C6[24]; FRhL2; WI-38; Deng.Suitable cell line can be obtained by various sources, for example American type culture collection (ATCC) [25], Ke Lier (Coriell) cell bank [26] or European cell culture preservation center (ECACC).For example, ATCC provides various different Vero cells, and catalog number (Cat.No.) is CCL81, CCL81.2, CRL-1586 and CRL-1587; And mdck cell, catalog number (Cat.No.) are provided is CCL34.PER.C6 can be available from ECACC, and preserving number is 96022940.Any of these cell type can be used for cultivation of the present invention, reprovision and/or goes down to posterity.

Most preferred cell line is to have mammalian type glycosylated cells system.As a kind of more not alternative of preferred mammal cell line, [for example can on avian cell line, cultivate virus, list of references 27-29], these avian cell lines comprise the cell line from duck (for example duck retina cell) or chicken (for example chick embryo fibroblast (CEF)) etc., but the use of mammalian cell means, vaccine can not contain the protein (as ovalbumin and ovomucoid) of birds DNA and egg, thereby reduces allergenicity.

The most preferred cell line that is used to cultivate influenza virus is the mdck cell system [30-33] from Madin-Darby canine kidney.Original mdck cell system can be available from ATCC (CCL34), but also can utilize the derived cell system of this cell line.For example, list of references 30 discloses the (' MDCK of mdck cell system of suitable suspension culture

33016 ' or ' 33016-PF ', preserving number DSM ACC 2219 also is found in list of references 34 and 35).Similarly, list of references 36 discloses the MDCK derived cell system (' B-702 ', preserving number FERM BP-7449) of suspension culture in serum-free medium.List of references 37 discloses the non-tumorigenic mdck cell, comprise ' MDCK-S ' (ATCC PTA-6500), ' MDCK-SF101 ' (ATCC PTA-6501), ' MDCK-SF102 ' (ATCC PTA-6502) and ' MDCK-SF103 ' (PTA-6503).List of references 38 discloses the mdck cell system with the infection of being very easy to, and comprises ' MDCK.5F1 ' cell (ATCC CRL12042).The present invention can adopt any of these mdck cell system.

Virus can be cultivated on adhere-wall culture or suspension cultured cells.Also can use microcarrier to cultivate.In some embodiments, cell may use suspension culture.Cell line is preferably cultivated in serum-free medium and/or protein-free medium.In content of the present invention, culture medium is meant the serum-free medium that does not have from the serum additive in human or animal source.The cell of growing in this culture itself comprises protein under natural situation, but protein-free medium should be understood to and is illustrated in the culture medium of carrying out cell proliferation under the condition that does not comprise (not adding in the culture medium) protein, somatomedin, other proteins additives and non-serum proteins, but can randomly comprise the necessary protein of (in culture medium) viral growth such as trypsin or other protease.

During virus replication, support cell line that influenza virus duplicates preferably to cultivate [39] (for example, 30-36 ℃, or about 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃) under 37 ℃ the temperature being lower than.For example, aspect the 6th, mdck cell can cultivated (before the separating step, during or afterwards) under these temperature, especially during the virus replication.

The method of propagative viruses (for example in cultured cells, in the mdck cell of cultivating, cultivate influenza virus according to the 6th aspect) generally include following steps: to cultured cell inoculation strain to be cultivated, with the infected required time of cell culture and virus breeding, for example the time of measuring by virus titer or antigen presentation (as, inoculation back 24-168 hour), collect the virus of breeding.Ratio with virus and cell is 1:500-1:1, preferred 1:100-1:5, and more preferably 1:50-1:10 is (by PFU or TCID ₅₀Detection) cell of inoculated and cultured.Virus can be added in the cell suspension, or put on cell monolayer, virus is in 25-40 ℃, and preferred 28-37 ℃, absorption is at least 60 minutes on cell, but is less than 300 minutes usually, preferably between 90-240 minute.Can remove the cell culture (for example, monolayer) of infection by freeze thawing or enzymatic catalysis, with the viral level in the culture supernatant that increases results.Make the liquid deactivation or the freezing preservation of collection then.With about 0.0001-10, preferred 0.002-5, more preferably the infection multiplicity of 0.001-2 (" m.o.i. ") infects cultured cells.More preferably with about 0.01 m.o.i. infection cell.Infect the back and collected the cell that infects in 30-60 hour.Preferred 34-48 hour collecting cell after infection.More preferably 38-40 hour collecting cell after infection.Usually during cell culture, add protease (normally trypsin) with releasing virus, any suitable stage that can be in the training period, for example before the inoculation, inoculation simultaneously or the inoculation back add protease [39].

In a preferred embodiment, mdck cell especially, cell line is no more than 40 times from the passage number in main working cell storehouse.

Virus inoculation thing and viral cultures preferably do not contain (that is, obtaining the negative result of pollution after tested) herpes simplex virus, respiratory syncytial virus, parainfluenza virus 3, sars coronavirus, adenovirus, rhinovirus, reovirus, polyoma virus, birnavirus, circovirus virus and/or parvovirus [40].Similarly, the preferred mdck cell that uses in the 6th aspect is not contain (that is, obtaining the negative result of pollution after tested) herpes simplex virus, respiratory syncytial virus, parainfluenza virus 3, sars coronavirus, adenovirus, rhinovirus, reovirus, polyoma virus, birnavirus, circovirus virus and/or parvovirus.Especially preferably there is not herpes simplex virus.

The mdck cell that uses among the present invention does not preferably contain G418 drug resistance labelling (list of references 16).Therefore, this cell line is responsive to the G418 treatment.

The cell line of using among the present invention does not preferably contain exogenous plasmid (list of references 16), unless the required any exogenous plasmid of reverse genetic technology.

The reverse genetic technology

As mentioned above, the present invention can directly use clinical isolates or former generation separator.Yet in addition, the present invention also can use recombined strain, comprises the strain (for example, list of references 41-45) that produces with the reverse genetic technology.The reverse genetic technology can utilize the manipulation in vitro of plasmid to produce the combination of viral section, is convenient to operate coded sequence or non-coding sequence in the viral section, to introduce sudden change etc.This technology can be used for first type and Influenza B virus.

The reverse genetic technology for example generally comprises expression (a), the dna molecular of the required viral RNA molecule of coding that starts by polI promoter, bacteria RNA polymerase promoter, phage polymerase promoter etc., (b) for example, the dna molecular of the coding virus protein that starts by the polII promoter, so that in cell, express two types DNA, cause being assembled into complete infectious viral particle.DNA preferably provides all viral RNAs and protein, but also may provide some RNA and protein with helper virus.The preferred plasmid method [46-48] that adopts different plasmids to produce each viral RNA, these methods also comprise uses plasmid expression all or some virus protein (for example only PB1, PB2, PA and NP albumen), uses 12 kinds of plasmids in the certain methods.

In order to reduce required plasmid quantity, in the recent period method [49] with a plurality of rna plymerase is transcribe box (being used for synthetic viral RNA) merge to same plasmid (as encode 1,2,3,4,5,6,7 or the sequence of all 8 influenza A vRNA sections), and with a plurality of protein-coding regions and rna plymerase ii promoter merge to another plasmid (as encode 1,2,3,4,5,6,7 or the sequence of all 8 kinds of influenza A mRNA transcripies) on.The preferred aspect of list of references 49 methods comprises: (a) PB1, PB2 and PA mRNA coding region are positioned on the same plasmid; (b) all 8 vRNA coding regions are positioned on the same plasmid.Comprise NA and HA sections on the plasmid and other six sections to be positioned at also may be favourable on another plasmid.

Because the species specificity of polI promoter can utilize dog polI promoter [50] when carrying out reverse genetic in mdck cell.As using the encode another kind of alternative of viral RNA section of polI promoter, can utilize phage polymerase promoter [51].For example, can use the promoter of SP6, T3 or T7 polymerase easily.Because the species specificity of polI promoter, for the more suitable use phage polymerase promoter of many cell types (for example MDCK), but necessary plasmid transfection cell with the encoding exogenous polymerase.

In other technology, can use polI and polII double-promoter] by a template encode simultaneously viral RNA and effable mRNA[52,53.

Though be used for generally including six RNA sections that are derived from the PR/8/34 influenza A virus (HA and N section from vaccine strain, i.e. 6:2 reassortant), avoid using egg to mean and to eliminate the PR/8/34 section at the strain that egg is cultivated.Therefore, influenza A virus can comprise the viral section from the PR/8/34 influenza virus less than 6 (promptly 0,1,2,3,4 or 5).Therefore, preferred virus is at least one virus that does not derive from PR/8/34 among section NP, M, NS, PA, PB1 and/or the PB2.Virus can be included in the NS section that originates from the bird flu virus.

When the present invention uses the reverse genetic technology, can make the viral RNA sector transfer that is derived from influenza virus in the genome of purpose influenza virus.Thereby will have at least one identical viral RNA section in these two kinds of viruses.The identical copies of whole section represented here in term " identical ", but the also extensible modification copy that is illustrated in this section that contains modification in coding region or the noncoding region.When in the coding region, modifying, can not change the immunogenicity and/or the activity of coded protein basically.Therefore, can near the HA1/HA2 cleavage site, operate the HA section and not change the ability of bringing out effective anti-HA antibody after it is giving the patient.Therefore, can use the reverse genetic technology to modify natural HA, for example remove and cause virus in birds, to produce highly pathogenic determinant (for example hyperalkaline zone around the HA1/HA2 cleavage site) according to the isolating virus in the 6th aspect.

Vaccine production

Can obtain various forms of influenza virus vaccines the 17th and 18 chapters of list of references 54 (for example referring to) at present.Vaccine is usually based on live virus or inactivation of viruses.Inactivated vaccine can be based on whole virus particles, " division " virion or based on the surface antigen of purification.Influenza antigens also can the virion form occur.All can use the present invention when making the vaccine of these types arbitrarily.

Live virus comprises the FLUMIST of Midi Miu Ni company (MedImmune) ^TMProduct (trivalent live virus).Prepare vaccine by the following method: on suitable substrate, cultivate virus, then from containing the fluid purification virion of virion.For example, described fluid can be by centrifugalize and stable with buffer (for example containing sucrose, potassium phosphate and monosodium glutamate).

When adopting inactivation of viruses, this vaccine can comprise the surface antigen (comprise hemagglutinin, also comprise neuraminidase usually) of complete virion, division virion or purification.The chemical mode that is used for inactivation of viruses comprises and uses one or more following reagent of effective dose to handle: detergent, formaldehyde, beta-propiolactone, methylene blue, psoralen, carboxyl fullerene (C60), diethylamine (binary ethylamine), acetyl group aziridine or their combination.The method non-chemically of inactivation of virus known in the art, for example UV ray or gamma-rays radiation.

Can be by the whole bag of tricks by containing viral liquid results virion.Purification process can comprise with linear Sucrose gradient solutions (containing detergent with the break virus granule) and carries out band centrifugation.After the optional dilution, can be by the diafiltration purifying antigen.

The virion of handling purification with detergent (as ether, polysorbate80, dexycholate, three normal-butyl phosphate, triton x-100, triton N101, cetab, Tergitol NP9 etc.) is to obtain the lytic virus granule, thereby produce the subviral particle preparation, comprise " tween-ether " cleavage method.The method of cracking influenza virus is well known in the art, for example referring to list of references 55-60 etc.Decomposition agent destruction or fragmentation intact virus that general use destroys concentration come lytic virus, and no matter this virus has or not infectivity.This destruction causes the dissolving wholly or in part of virus protein, changes the integrity of virus.Preferred decomposition agent is nonionic and ion-type (for example cationic) surfactant; as alkyl polyglucoside; the alkyl sulfide glycosides; acyl group sugar; sulfobetaines; betanin; polyoxyethylene alkyl ether; N; N-dialkyl group-glucamide; Hecameg; alkyl phenoxy-polyethoxy ethanol; NP9; quaternary ammonium compound; sarcosyl; CTAB (cetab); three normal-butyl phosphate esters; Sai Talong (Cetavlon); myristyl front three ammonium salt; lipofection reagent; Li Feitaming (lipofectamine) and DOT-MA; octyl group-or Nonylphenoxy polyoxy ethanol (as the triton surfactant, as triton x-100 or triton N101); polyoxyethylene sorbitan alcohol ester (tween surfactants); polyoxyethylene ether; polyoxyethylene ester etc.A kind of useful cleavage method utilizes the continuous action of NaTDC and formaldehyde, and (for example in sucrose density gradient solution) can take place during the initial purification of virion in cracking.Therefore, cracking process can comprise: clarification contains the material (to remove non-virion material) of virion, and the virion that concentrates results (for example uses adsorption method, as CaHPO ₄Absorption), from non-virion material separation whole virus particles,, filter (for example ultrafiltration) then to remove unwanted material with decomposition agent lytic virus granule (for example) in the density gradient centrifugation step with the saccharose gradient that contains decomposition agent such as NaTDC.Cracked virion can be resuspended in the isotonic sodium chlorrde solution of sodium phosphate buffer.BEGRIVAC ^TM, FLUARIX ^TM, FLUZONE ^TMAnd FLUSHIELD ^TMProduct is a split vaccine.

The SAV of purification comprises influenza surface antigen hemagglutinin, generally also comprises neuraminidase.These method of protein of preparation purified form are well known in the art.FLUVIRIN ^TM, AGRIPPAL ^TMAnd INFLUVAC ^TMProduct is the subunit vaccine.

The deactivation influenza antigens of another kind of form is virion [61] (the viral sample liposome particles that does not contain nucleic acid).Virion can be removed nucleocapsid and prepare with the film that reconstruction contains viral glycoprotein then by with detergent solubilising influenza virus.The another kind of method for preparing virion comprises: adding viromembrane glycoprotein is excessive to phospholipid, obtains having in the film liposome of virus protein.The present invention can be used for storing a large amount of virion, as INFLEXAL V ^TMAnd INVAVAC ^TMProduct.

Influenza virus can be attenuated.Influenza virus can be a responsive to temperature type.Influenza virus can be a cold adaptation sexually transmitted disease (STD) poison.These three kinds of features are particularly useful when using live virus as antigen.

HA is the main immunogens in the inactivated influenza vaccine, generally detects by SRID and makes the vaccine dose standardization with reference to the HA level.Vaccine contains the 15 μ g HA/ strains of having an appointment usually, though for example child, or also can use lower dosage under the pandemicity.With higher dosage (for example, 3 * or 9 * dosage [62,63]) the same, used fractional doses, 1/2 (that is, 7.5 μ g HA/ strains), 1/4 and 1/8[81 for example, 82].Therefore, vaccine can comprise 0.1-150 μ g, preferred 0.1-50 μ g, and for example 0.1-20 μ g, 0.1-15 μ g, 0.1-10 μ g, 0.1-7.5 μ g, 0.5-5 μ gHA/ influenza strain, or the like.Concrete dosage comprises, for example each strain is about 45, about 30, about 15, about 10, about 7.5, about 5, about 3.8, about 1.9, about 1.5, or the like.

For live vaccine, by (the TCID of intermediate value TCID ₅₀) rather than HA content detect administration, the TCID of every kind of strain ₅₀Generally 10 ⁶-10 ⁸Between (preferred 10 ^6.5-10 ^7.5).

The used strain of the present invention can contain the natural HA that finds in the wild-type virus, or modified HA.For example, the known HA of modification causes virus in the morbific determinant of birds camber (near for example, the hyperalkaline zone (hyper-basic region) HA1 and the HA2 cleavage site) to remove.

The influenza virus strain that is used for vaccine is with becoming season.Present being very popular interval, vaccine generally comprises two kinds of influenza A strains (H1N1 and H3N2) and a kind of influenza B strain, generally is trivalent vaccine.It (is that the vaccine receiver never crosses the strain that immunity contacts with it with the general population that the present invention also can adopt the strain that is very popular, influenza A virus particularly), as H2, H5, H7 or H9 hypotype strain, the influenza vaccines of strain of being very popular can be univalent vaccines, perhaps can be based on the normal trivalent vaccine that is supplemented with the strain that is very popular.Yet according to the antigenic characteristic that comprises in season and the vaccine, the present invention can be protected from the infection of one or more hypotypes among HA hypotype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or the H16.The present invention can be protected from the infection of one or more hypotypes among influenza A virus NA hypotype N1, N2, N3, N4, N5, N6, N7, N8 or the N9.

The present composition is not only applicable to the be very popular immunity of interval of immunity, is particularly useful for obtaining the immunity to the strain that is very popular.The feature of influenza strain that may cause the outburst of being very popular is: (a) compare with the hemagglutinin in people's strain of present circulation, it contains new hemagglutinin, promptly there is many decades in the crowd, not have the hemagglutinin of finding (as H2), the hemagglutinin of perhaps never finding in the crowd (as usually only appearing at H5, H6 or the H9 in the flock of birds body) is to such an extent as to crowd's hemagglutinin of immune contacted this strain not; (b) it can be in the crowd horizontal transmission; (c) it can make the people cause a disease.The virus that contains H5 hemagglutinin type is preferred for obtaining to pandemic influenza, as the immunity of H5N1 strain.Other possible strain comprises H5N3, H9N2, H2N2, H7N1 and H7N7, and any strain that is very popular that other may occur.In the H5 hypotype, virus is divided into that HA evolves 1, HA evolves 1 ', HA evolve 2 or a HA 3[64 that evolves], it is 1 especially relevant with 3 to evolve.

It is the strain that the antagonism viral therapy has resistance (for example oseltamivir [65] and/or zanamivir being had resistance) that its antigen can usefully be included in other strain in the present composition, comprises the resistance strain [66] that is very popular.

Therefore, the present composition can comprise one or more (as 1,2,3,4 or more kinds of) influenza virus strains, comprises the antigen of influenza A virus and/or Influenza B virus.When vaccine comprised more than one influenza strains, the different strains of general single culture after gathering in the crops virus and preparing antigen mixed them.Therefore, the inventive method can comprise the antigenic step of mixing more than one influenza strains.Preferably include antigenic trivalent vaccine from two kinds of influenza A virus strains and a kind of Influenza B virus strain.

In some embodiments of the present invention, compositions can comprise the antigen from a kind of influenza A strain.In some embodiments, compositions can comprise the antigen from two kinds of influenza A strains, as long as these two kinds of strains are not H1N1 and H3N2.In some embodiments, compositions can comprise the antigen from two or more influenza A strains.

The invention provides the method for the influenza antigen that uses in the preparation vaccine, this method may further comprise the steps: (i) accept influenza virus; (ii) use this influenza infection cell line; (iii) incubation step infection cell (ii) is to produce influenza virus.Step (iii) gained virus can be used for preparing vaccine, for example prepares vaccine by methods such as deactivation, preparations.The influenza virus that step (i) is accepted has one or more following characteristics: (a) never bred on the egg substrate; (b) from mdck cell, as separating in the mdck cell of cultivating in MDCK 33016 cells and/or the serum-free medium; (c) never on the substrate that contains the blood serum medium cultivation, breed; (d) produce with the reverse genetic technology; (e) it is a kind of influenza A virus, wherein less than 6 viral sections from the PR/8/34 influenza virus and/or less than 6 viral sections from the AA/6/60 influenza virus, perhaps it is a kind of Influenza B virus, wherein less than 6 viral sections from the AA/1/66 influenza virus; (f) it comprises hemagglutinin, and hemagglutinin has with respect to the oligosaccharide that contains the terminal disaccharide of Sia (α 2,3) Gal, more be partial to contain the terminal disaccharide of Sia (α 2,6) Gal oligosaccharide in conjunction with preference; And/or (g) it has the glycoprotein (comprising hemagglutinin) that glycosylation pattern is different from the egg derived virus.Therefore, may be as the influenza virus of accepting in the acquisition step (i) as described in other parts of this paper.

Host cell DNA

When cultivating virus with cell line, standard method is that to make residual cells in the final vaccine be that the amount of DNA minimizes, so that the carcinogenic activity of DNA is minimized.

Therefore, when cultivating virus with cell line, then every dose of compositions preferably contains and is less than the 10ng residual host cell DNA of (preferably being less than 1ng, more preferably less than 100pg), though can there be the host cell DNA of trace.

Host cell DNA content＜10ng in preferred per 15 μ g hemagglutinins (as＜1ng,＜100pg) vaccine, and host cell DNA content＜10ng in every 0.25ml volume (as＜1ng,＜100pg) vaccine.Host cell DNA content＜10ng in more preferably per 50 μ g hemagglutinins (as＜1ng,＜100pg) vaccine, and host cell DNA content＜10ng in every 0.5ml volume (as＜1ng,＜100pg) vaccine.

The average length of preferred any remaining host cell DNA is less than 500bp, for example less than 400bp, less than 300bp, less than 200bp, less than 100bp, or the like.

Can adopt the standard purification method, for example chromatography etc. is removed contaminative DNA during vaccine production.Handle by nuclease, for example can promote to remove residual host cell DNA with the DNA enzyme.List of references 67 and 68 has disclosed and has reduced the facilitated method that host cell DNA pollutes, it relates to the processing of two steps, at first can use the DNA enzyme (for example, Benzonase) during Virus culture, can during breaking, virion use cationic detegent (for example, CTAB) then.Use alkylating agent, for example beta-propiolactone is handled and also be can be used for removing host cell DNA, and this method also can be preferred for inactivation of viruses particle [69].

Now, the mensuration of residual host cell DNA is the Routine Management requirement of biological product, and it is in technical staff's general ability scope.The experiment that is used to measure DNA generally is the experiment of having verified [70,71].Can and can quantized condition describe the performance characteristic of confirmatory experiment by mathematics, and identify possible source of error.Usually test some feature of this experiment, as accuracy, accuracy, specificity.In case proofread and correct (for example proofreading and correct) and detected certain experiment, then can carry out quantitative DNA routinely and measure with the host cell DNA of known standard amount.Can adopt three kinds of quantitative basic fundamentals of DNA: hybridizing method, as Southern trace or slot blot [72]; Method of immunity is as Threshold ^TMSystem [73]; And quantitative PCR [74].Those skilled in the art are familiar with these methods, but the accurate feature of each method, and for example the selection of hybridization probe, selection of primers and/or primer of being used to increase etc. may depend on the host cell of being studied.Threshold from a minute subset company (Molecular Devices) ^TMSystem is the quantitative experiment of the total DNA of pik level, has been used for the level [73] of monitoring bio medicine contaminating dna.Model experiment comprises that biotinylation ssDNA is conjugated protein, form the reaction complex in non-sequence-specific mode between the link coupled anti-ssDNA antibody of urase and the DNA.Comprise all experiment components in total DNA mensuration test kit (Total DNA Assay Kit) available from the manufacturer.A plurality of commodity production merchants all provide in order to detect the quantitative PCR experiment of residual host cell DNA, for example AppTec ^TM(the AppTec of laboratory service company ^TMLaboratory Services), BioReliance ^TMAo Xi scientific ﹠ technical corporation of company (Althea Technologies) etc.Chemiluminescence hybrid experiment and total DNA Threshold that the host cell DNA of measuring Human virus's vaccine is polluted ^TMThe comparison of system is referring to list of references 75.

Pharmaceutical composition

Vaccine combination constructed in accordance is pharmaceutically acceptable.Except influenza antigens, they comprise one or more pharmaceutical carriers and/or excipient usually.As described below, also can comprise adjuvant.To comprehensive discussion of these components document 76 that sees reference.

Vaccine combination is aqueous form normally.

Vaccine combination can comprise antiseptic, for example thimerosal or 2-phenyl phenol.Yet these vaccines preferably are substantially free of (that is, being less than 5 μ g/ml) hydrargyrum material, for example do not contain thimerosal [59,77].More preferably not mercurous vaccine.Can mix the succedaneum [59] of succinic acid alpha-tocopherol as the hydrargyrum material.The vaccine that does not especially preferably contain antiseptic.

Preferably comprise physiology salt, for example sodium salt is with control tension force.Sodium chloride (NaCl) between the preferred 1-20 mg/ml.Other salt that can exist comprises potassium chloride, potassium dihydrogen phosphate, dehydration sodium hydrogen phosphate (disodium phosphate dehydrate), magnesium chloride, calcium chloride etc.

The osmolality of vaccine combination between 200mOsm/kg-400mOsm/kg, between the preferred 240-360mOsm/kg, more preferably is in the 290-310mOsm/kg scope usually.Had in the past and reported that osmolality did not influence [78] to the pain due to the vaccination, but preferably osmolality was maintained in this scope.

Vaccine combination can comprise one or more buffer agents.Typical buffer agent comprises: phosphate buffer; The Tris buffer agent; Borate buffer; The succinate buffer agent; Histidine buffer (histidine buffer that particularly contains aluminum hydroxide adjuvant); Or citrate buffer agent.Contained buffer agent scope generally is 5-20mM.

The pH of vaccine combination is usually between 5.0-8.1, and is more common between 6.0-8.0, for example between 6.5 and 7.5, or between the 7.0-7.8.Therefore, the inventive method can comprise the step that the pH of elder generation's adjusting bulk vaccine packs again.

Vaccine combination is preferably aseptic.The preferred apyrogeneity of compositions, for example every dosage contain＜1EU (endotoxin unit, gauge), preferred every dosage＜0.1EU.The preferred GF of compositions.

Vaccine combination of the present invention, particularly can comprise detergent for cracking or SAV, polyoxyethylene sorbitan esters surfactant (being called " tween ") for example, Octoxinol (for example, octoxynol 9 (triton x-100) or uncle's octylphenoxy polyethoxy ethanol), cetab (CTAB), or NaTDC.The detergent that can only have trace.Therefore, the content of Octoxinol (octoxynol)-10 and polysorbate80 is lower than 1 mg/ml separately in the vaccine.The residual component of other trace can be antibiotic (for example, neomycin, kanamycin, a polymyxin B).

Vaccine combination can comprise the once material of immunity, maybe can comprise the repeatedly material (that is " multiple dose " kit) of immunity.In the multiple dose configuration, preferably comprise antiseptic.Except in multi-dose compositions, comprising antiseptic, also compositions can be placed the container that the sterile adapter that is used for transfer of material is housed.

The influenza vaccines dose volume that generally gives is about 0.5ml, but can give the child with a half-value dose (that is about 0.25ml).

Compositions and kit are preferably kept between 2 ℃-8 ℃.Should be freezing they.Preferably avoid the light direct projection.

Adjuvant

The present composition should comprise adjuvant, and the effect of adjuvant is to strengthen the immunne response (humoral immunization and/or cellular immunization) that causes in accepting the patient of compositions.Once described adjuvant was used with influenza vaccines.Use aluminium hydroxide in the list of references 79 and 80, use the mixture of aluminium hydroxide and aluminum phosphate in the list of references 81.List of references 82 has also been described the application of aluminum salt adjuvant.The FLUAD of uncommon imperial vaccine company (ChironVaccines) ^TMProduct comprises O/w emulsion.

Can be used for adjuvant of the present invention includes but not limited to:

The mineral composition that contains that comprises calcium salt and aluminum salt (or its mixture).Calcium salt comprises calcium phosphate (as list of references 83 disclosed " CAP " granule).Aluminum salt comprises aluminium hydroxide, aluminum phosphate, aluminum sulfate etc., and described salt is taked any suitable form (as gel, crystal, amorphous state etc.).Preferably be adsorbed in these salt.Also the compositions that contains mineral can be made the granule [84] of slaine.Aluminum salt adjuvant is described in beginning in more detail below.

Cytokine induction agent (describing in detail as follows)

Saponin [the 22nd chapter of list of references 112] is at bark, leaf, stem, the root of numerous species plant even the steroline of finding in spending and the heterogeneous population of triterpene glucosides.Broad research as the saponin from Quillaia saponaria (Quillaia saponaria) Molina bark of adjuvant.But saponin also commercialization available from beautiful colored Rhizoma Smilacis Chinensis (Smilax ornata) (sarsaparilla), Caulis et folium pavettae hongkongensis (Gypsophilla paniculata) (wedding gauze kerchief flower) and Saponaria officinalis (Saponaria officianalis) (Radix saponariae).The saponin adjuvant preparation comprises purification preparation such as QS21 and lipid formulations such as ISCOM.QS21 is with trade mark Stimulon ^TMSell.HPLC and RP-HPLC purification astragalin composition have been adopted.Identified specific components, comprised QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C with these technology purification.The method for preparing QS21 is referring to list of references 85.The saponin preparation also can comprise sterol, as cholesterol [86].The combination of saponin and cholesterol can be used for forming the unique granule [the 23rd chapter of list of references 112] that is called immunostimulating complex (ISCOM).ISCOM also contains phospholipid such as PHOSPHATIDYL ETHANOLAMINE or phosphatidylcholine usually.Can adopt any known saponin among the ISCOM.ISCOM preferably comprises one or more among QuilA, QHA and the QHC.Further described ISCOM among the list of references 86-88.Randomly, ISCOM can not contain other detergent [89].Exploitation can be referring to list of references 90 and 91 based on the summary of the adjuvant of saponin.

Fat adjuvant (describing in detail as follows)

Antibacterial ADP-ribosylation toxin (for example E.coli LT " LT ", cholera toxin " CT " or pertussis toxin, PT " PT ") and their detoxifcation derivant, as be called the mutant toxin [92] of LT-K63 and LT-R72.Antidotal ADP-ribosylation toxin is described in list of references 93 as mucosal adjuvants, is described in list of references 94 as the parenteral adjuvant.

Bioadhesive polymer and mucoadhesive are as hyaluronic acid microsphere [95] or the chitosan and the derivant [96] thereof of esterification

The microgranule that is formed by biodegradable and avirulence material (is that diameter is that about 100nm is to about 150 μ m, more preferably from about 200nm is to about 30 μ m, perhaps about 500nm is to the granule of about 10 μ m), described material for example poly-(alpha-hydroxy acid), poly hydroxybutyric acid, poe, polyanhydride, polycaprolactone or the like, wherein preferred poly (glycolide-lactide) copolymer, randomly to its processing, make it have electronegative surface (for example handling) or positively charged surface (for example using catioic detergent, for example CTAB) processing with SDS.

Liposome (list of references 112 the 13rd and 14 chapters).The example that is suitable as the Liposomal formulation of the adjuvant document 97-99 that sees reference is described.

Polyoxyethylene ether and polyoxyethylene ester [100].This type of preparation also comprises the polyoxyethylene Sorbitan ester surfactant [101] with Octoxinol combination, and with the polyoxyethylene alkyl ether or the ester surfactant [102] of at least a other nonionic surfactant (for example Octoxinol) combination.Preferred polyoxyethylene ether is selected from: polyoxyethylene-9-Laurel ether (laureth 9), polyoxyethylene-9-stearyl (steoryl) ether, polyoxyethylene-8-stearyl ether, polyoxyethylene-4-Laurel ether, polyoxyethylene-35-Laurel ether and polyoxyethylene-23-Laurel ether.

Muramyl peptide; for example N-acetyl group-muramyl-L-Threonyl-D-isoglutamine (" thr-MDP "), N-acetyl group-nor-muramyl-L-alanyl-D-isoglutamine (" nor-MDP "), the different paddy-L-Ala-two palmityl propionic acid amide .s of N-acetyl glucosamine-N-acetyl group muramyl-L-A1-D-(N-acetylglucsaminyl-N-acetylmuramyl-L-A1-D-isoglu-L-Ala-dipalmitoxypropylamide) (" DTP-DPP ", or " Theramide ^TM") or N-acetyl group muramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1 '-2 '-two palmityls-sn-glyceryl-3-hydroxyl phosphorus acyloxy)-ethamine (" MTP-PE ").

From the outer membrane protein albuminous body goods of first kind of gram negative bacteria preparation and combination derived from lipopolysaccharide (LPS) goods of second kind of gram negative bacteria, wherein said outer membrane protein albuminous body and LPS goods form stable non-covalent adjuvant complex.This class complex comprises ＂ IVX-908 ＂, the complex that it is made up of Neisseria meningitidis adventitia and LPS.They have been used as the adjuvant [103] of influenza vaccines.

Methylinosine 5 '-phosplate (" MIMP ") [104].

Polyhydroxylated Pyrrolizidine (pyrrolizidine) chemical compound [105] or its pharmaceutically acceptable salt or the derivant that are shown below:

Wherein R is selected from down group: hydrogen, straight or branched, do not replace or replace, saturated or undersaturated acyl group, alkyl (for example, cycloalkyl), thiazolinyl, alkynyl and aryl.Example includes but not limited to: casurin (casuarine), casurin-6-α-D-glucopyranose, 3-table-casurin, 7-table-casurin, 3, and 7-two table-casurins, or the like.

γ inulin [106] or derivatives thereof is as algae inulin (algammulin).

The CD1d part is as the alpha-galactoside ceramide.

Polyethylene-the bridged piperazine derivatives of polyoxy ion (polyoxidonium) polymer [107,108] or other N-oxidation.

These adjuvants and the more detailed description of other adjuvanticity materials see reference document 112 and 113.

Compositions can comprise two or more described adjuvants.For example, compositions should comprise O/w emulsion and cytokine induction agent simultaneously, because this combination can improve the cytokine response that influenza vaccines cause, to reply as interferon gamma, this improvement effect is more far better with the situation of emulsion or cytokine induction agent than single.

Antigen in the compositions and adjuvant generally form mixture.

Oil in water emulsion adjuvant

Found that O/w emulsion is specially adapted in the influenza virus vaccine of adjuvant preparation.Known various such emulsion, they comprise at least a oil and at least a surfactant usually, and wherein one or more oil and one or more surfactants are biodegradables (but metabolism) and biocompatible.Oil droplet general diameter in the emulsion is lower than 5 μ m, even can have sub-micron diameter, can realize these small sizes with the Micro Fluid instrument, thereby stable emulsion can be provided.Preferred size is less than the oil droplet of 220nm, because can carry out filtration sterilization to them.

The present invention can use the oil such as animal origin (for example fish) or plant origin.The vegetable oil source comprises nut, seed and corn.The example of macadamia nut oil is the most normal Oleum Arachidis hypogaeae semen of buying, soybean oil, cocos nucifera oil and olive oil.For example, can utilize the Jojoba oil that obtains from flash Fructus Crotonis (jojoba bean).Seed oil comprises safflower oil, Oleum Gossypii semen, Oleum Helianthi, Semen Sesami wet goods.In corn oil, Semen Maydis oil is the easiest to be buied, but the oil of other corn such as Semen Tritici aestivi, Herba bromi japonici, rye (Secale cereale L.), rice, Herba Eragrostidis pilosae (teff), black Semen Tritici aestivi etc. also can utilize.Nut and the seed oil initial substance suitable by hydrolysis, separation and esterification can prepare natural non-existent glycerol and 1 in the seed oil, the 6-10 carbocyclic aliphatic acid esters of 2-propylene glycol.The fat of mammal milk and oils are metabolizable, therefore can be used for implementing the present invention.Well knownly obtain the required separation of pure oil, purification, saponification and other method from animal origin.But most of fishes contain the metabolism oil of being not difficult to reclaim.For example, the example of the available several fish oil of the present invention is cod liver oil, shark liver oil and whale oil, as spermaceti.Can 5-carbon isoprene unit synthesize many side chain oil by biochemical method, these side chain oil are commonly referred to terpenoid.Shark liver oil contains the unsaturated terpenoid of side chain that is called Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-24 hexenes (tetracosahexaene)), and this is that the present invention is particularly preferred.Squalane is the saturated analogues of Squalene, and it also is preferred oil.The fish oil that comprises Squalene and squalane is not difficult to buy from commercial source, maybe can obtain by methods known in the art.Other preferred oil is tocopherol (vide infra).Can utilize the mixture of oil.Can be according to " HLB " (hydrophilic) of surfactant with its classification.The HLB of preferred surfactant of the present invention is at least 10, and preferably at least 15, more preferably at least 16.The available surfactant of the present invention comprises but is not limited to: polyoxyethylene sorbitan esters surfactant (being commonly referred to tween), particularly polysorbate20 and polysorbate80; The copolymer of oxirane (EO), expoxy propane (PO) and/or epoxy butane (BO) is with DOWFAX ^TMFor trade name is sold for example linear EO/PO block copolymer; The Octoxinol that multiple ethyoxyl (oxygen-1,2-second two bases) group number possibility is different, wherein interested especially is octoxynol 9 (triton x-100 or uncle's octylphenoxy polyethoxy ethanol); (Octylphenoxy) polyethoxy ethanol (IGEPAL CA-630/NP-40); Phospholipid, for example poly-choline (lecithin) of phosphatidyl; Nonyl phenol ethoxylate, for example Tergitol ^TMNP series; Derived from the polyoxyethylene aliphatic ether (being called the Brij surfactant) of lauryl alcohol, spermol, stearyl alcohol and oleyl alcohol, trietbhlene glycol list lauryl ether (triethyleneglycol monolauryl ether) (Brij30) for example; And sorbitan esters (being commonly referred to span (SPAN)), for example anhydrosorbitol trioleate (sorbester p37) and sorbitan monolaurate.Preferred nonionic surfactant.Contained preferred surfactant is Tween 80 (polyoxyethylene sorbitan monoleate), sorbester p37 (anhydrosorbitol trioleate), lecithin and triton x-100 in the emulsion.

Can utilize surfactant mixtures, for example Tween 80/sorbester p37 mixture.Polyoxyethylene sorbitan esters, for example polyoxyethylene sorbitan monoleate (Tween 80) and Octoxinol, for example the mixture of uncle's octylphenoxy polyethoxy ethanol (triton x-100) also is suitable for.Another useful mixture comprises laureth 9 and adds polyoxyethylene sorbitan esters and/or Octoxinol.

The preferable amount of surfactant (weight %) is: polyoxyethylene sorbitan esters (for example, Tween 80) 0.01-1%, particularly about 0.1%; Octyl group-or Nonylphenoxy polyoxy ethanol (for example other detergent of triton x-100 or triton series) 0.001-0.1%, particularly 0.005-0.02%; Polyoxyethylene ether (for example laureth 9) 0.1-20%, preferred 0.1-10%, particularly 0.1-1% or about 0.5%.

The used concrete oil in water emulsion adjuvant of the present invention includes but not limited to:

The submicron emulsion of Squalene, Tween 80 and sorbester p37.By volume, the composition of described emulsion can be about 5% Squalenes, about 0.5% polysorbate80 and about 0.5% sorbester p37.By weight, these ratios can be 4.3% Squalenes, 0.5% polysorbate80 and 0.48% sorbester p37.This adjuvant is called " MF59 " [109-111], is described in detail as the 12nd chapter of the 10th Zhanghe list of references 112 of list of references 112.The MF59 emulsion preferably comprises citrate ions, for example the 10mM sodium citrate buffer solution.

The emulsion of Squalene, tocopherol and Tween 80.This emulsion can comprise phosphate-buffered saline.It also can comprise sorbester p37 (for example, 1%) and/or lecithin.These emulsions can have 2-10% Squalene, 2-10% tocopherol and 0.3-3% Tween 80, Squalene: the weight ratio of tocopherol is preferred≤and 1, because this can provide more stable emulsion.The volume ratio of Squalene and Tween 80 can be about 5:2.Tween 80 can be dissolved in and obtain 2% solution among the PBS, then the mixture of this solution of 90ml with (5g DL-alpha-tocopherol and 5ml Squalene) be mixed, this mixture of Micro Fluid prepares a kind of like this emulsion subsequently.The emulsion that obtains can have the submicron oil droplet, for example average diameter between 100-250nm, preferably about 180nm.

The emulsion of Squalene, tocopherol and triton detergent (for example, triton x-100).This emulsion also can comprise 3d-MPL (seeing below).This emulsion can comprise phosphate buffer.

The emulsion that contains polysorbate (for example, polysorbate80), triton detergent (for example, triton x-100) and tocopherol (for example, alpha-tocofecol succinic acid ester).The mass ratio of these three kinds of components that this emulsion comprised (for example is about 75:11:10,750 μ g/ml polysorbate80s, 110 μ g/ml triton x-100s and 100 μ g/ml alpha-tocofecol succinic acid esters), these concentration should comprise the influence from antigenic these components.This emulsion also can comprise Squalene.This emulsion also can comprise 3d-MPL (seeing below).Water can contain phosphate buffer.

Squalane, polysorbate80 and poloxamer 401 (the general sieve stream of ＂ Buddhist nun gram ^TML121 ＂) emulsion.Can use phosphate-buffered saline, pH7.4 prepares this emulsion.This emulsion is a kind of useful muramyldipeptide delivery vector, with the threonyl-MDP coupling [114] of preparing with ＂ SAF-I ＂ adjuvant (0.05-1%Thr-MDP, 5% Squalene alkane, 2.5% general sieve stream Buddhist nun restrain L121 and 0.2% polysorbate80).Can be not and the Thr-MDP coupling yet, for example use ＂ AF ＂ adjuvant (5% squalane, 1.25% general sieve stream Buddhist nun restrain L121 and 0.2% polysorbate80) preparation [115].Preferred microfluidization.

The emulsion that comprises Squalene, aqueous solvent, polyoxyethylene alkyl ether hydrophilic nonionic surfactant (for example polyoxyethylene (12) cetyl/stearyl ether) and hydrophobicity nonionic surfactant (for example sorbitan esters or mannide ester, as dehydrating sorbitol monooleate or ' sorbester p17 ').The size of preferably thermoreversible and/or at least 90% oil droplet (by volume) of emulsion is less than 200nm[116].Also can comprise in the emulsion following one or more: aldehyde alcohol; Cryoprotective agent (for example sugared) as dodecyl maltoside and/or sucrose; And/or alkyl poly glucoside.This emulsion can be by lyophilizing.

The emulsion [117] of Squalene, poloxamer 105 and Abil Care.The final concentration of these components (weight) is 5% Squalene, 4% poloxamer 105 (pluoronics polyhydric alcohol) and 2%AbilCare 85 (two PEG/PPG-16/16PEG/PPG-16/16 simethicone in the Adjuvanted vaccines; Caprylic/capric triglyceride).

Emulsion with oil, 0.1-10% phospholipid and 0.05-5% non-ionic surface active agent of 0.5-50%.As described in list of references 118, preferred phospholipid fraction is phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidylinositols, phosphatidyl glycerol, phosphatidic acid, sphingomyelins and cuorin.Preferred submicron droplets size.

The submicron O/w emulsion of nonmetabolizable oil (for example, light mineral oil) and at least a surfactant (for example, lecithin, Tween 80 or sorbester p17).Can comprise additive; for example QuilA saponin, cholesterol, saponin-the lipophilic conjugate (for example; list of references 119 described GPI-0100; prepare by the carboxyl of aliphatic amine by glucuronic acid added on the deacylated tRNA basis soap glycosides), dimethyl two (octadecyl) ammonium bromide and/or N; N-two (octadecyl)-N, N-two (2-ethoxy) propane diamine.

The emulsion [120] that comprises mineral oil, nonionic lipotropy ethoxylized fatty alcohol and nonionic hydrophilic surfactant active (for example, ethoxylized fatty alcohol and/or polyox-yethylene-polyoxypropylene block copolymer).

The emulsion [120] that comprises mineral oil, nonionic lipotropy ethoxylized fatty alcohol and nonionic lipophilic surfactant (for example, ethoxylized fatty alcohol and/or polyox-yethylene-polyoxypropylene block copolymer).

Wherein saponin (for example, QuilA or QS21) and sterin (for example, cholesterol) are combined into the micellar emulsion of helical form [121].

These emulsions are preferably mixed with antigen in sending temporarily.Therefore, in the vaccine of packing or distribution, adjuvant and antigen are separately deposited usually, at last preparation immediately in use.Antigen generally exists in aqueous form, makes finally to prepare vaccine by mixing two kinds of liquid.The volume ratio that is used for blended two kinds of liquid can different (for example, between 5:1 and 1:5), but normally about 1:1.

After antigen and adjuvant mixed, hemagglutinin antigen maintained in the aqueous solution usually, but himself may distribute near oil/water termination.Enter the hemagglutinin usually few (if any) of the oil phase of emulsion.

If compositions comprises tocopherol, α, β, γ, δ, ε or ξ tocopherol are all available, but preferred alpha-tocopherol.Tocopherol can take several forms, for example different salt and/or isomer.Salt comprises organic salt, for example succinate, acetate, nicotinate or the like.D-alpha-tocopherol and DL-alpha-tocopherol are all available.Preferably comprise tocopherol in the used vaccine of gerontal patient's (for example, 60 years old or bigger), because it is reported that vitamin E has positive influences [122] to immunne response in this patient's group.They also have anti-oxidation characteristics, thereby help stabilizing solution [123].Preferred alpha-tocopherol is the DL-alpha-tocopherol, and the preferred salt of this tocopherol is a succinate.Have now found that the part cooperation that succinate in vivo can be relevant with TNF-.In addition, known alpha-tocofecol succinic acid salt is compatible with influenza vaccines, is the useful antiseptic [59] of replacement for mercury chemical compound.

The cytokine induction agent

The cytokine induction agent that is included in when giving the patient in the present composition can cause immunne response, to discharge cytokine, comprises interferon and interleukin.Known cytokine response and early stage relevant with the decision stage [124] at the host defense of influenza infection.Preferred medicament can cause the release of following one or more materials: interferon gamma; Interleukin-11; Interleukin-22; Interleukin 12; TNF α; TNF β; With GM CSF.Preferred medicament can bring out the release with Th1-type immunne response related cytokine, interferon gamma for example, TNF α, interleukin-22.Preferred interferon gamma and the interleukin-22 of stimulating.

Therefore, the result who accepts the present composition is that when stimulating with influenza antigens, the patient has the T cell that discharges required cytokine in the antigenic specificity mode.For example, during external contact influenza virus hemagglutinin, will discharge IFN-from the T cell of blood purification.It is known in the art measuring this method of replying of peripheral blood lymphocytes (PBMC), and these methods comprise ELISA, ELISPOT, flow cytometry and PCR in real time.For example, list of references 125 has been reported the immunne response of monitoring at the T cells with antigenic specificity mediation of tetanus toxoid, the particularly IFN-research of replying, find that ELISPOT is inductive the replying and the spontaneous the highest method of sensitivity of replying of difference antigen specific T T-, but the Flow cytometry kytoplasm inner cell factor is to measure once more the most effectual way of effect of stimulation.

Suitable cytokine induction agent includes but not limited to:

Immunostimulatory oligonucleotide for example contains the oligonucleotide or the double-stranded RNA of CpG motif (containing the dinucleotide sequence that is connected in the cytosine that do not methylate of guanine by phosphate bond) or contains the oligonucleotide of palindrome or contain the oligonucleotide of poly-(dG) sequence.

(' 3dMPL ' is also referred to as ' MPL to 3-O-deacylated tRNA base monophosphoryl lipid A ^TM') [126-129].

Imidazoquinolie compounds, for example imiquimod (" R837 ") [130,131], resiquimod (" R-848 ") [132] and their analog; With their salt (for example, hydrochlorate).Other details of immunostimulating imidazoquinolie document 133-137 that sees reference.

Thiosemicarbazones chemical compound, for example those of list of references 138 disclosures.List of references 138 has also been described the method for preparation, manufacturing and screening reactive compound.Thiosemicarbazones is especially effective for stimulation human peripheral blood mononuclear cell generation cytokine such as TNF-α.

Couroupitine A chemical compound, for example those of list of references 139 disclosures.List of references 139 has also been described the method for preparation, manufacturing and screening reactive compound.Thiosemicarbazones is especially effective for stimulation human peripheral blood mononuclear cell generation cytokine such as TNF-α.

Nucleoside analog, for example: (a) Ai Shatuola shore (Isatorabine) (ANA-245; 7-thia-8-oxo guanosine) and prodrug:

(b) ANA975; (c) ANA-025-1; (d) ANA380; (e) the described chemical compound of list of references 140-142; (f) chemical compound shown in the following formula:

In the formula:

R ₁And R ₂Independent separately be H, halogen ,-NR _aR _b,-OH, C _1-6The C of alkoxyl, replacement _1-6The heterocyclic radical of alkoxyl, heterocyclic radical, replacement, C _6-10The C of aryl, replacement _6-10Aryl, C _1-6The C of alkyl or replacement _1-6Alkyl;

R ₃Do not exist, or H, C _1-6The C of alkyl, replacement _1-6Alkyl, C _6-10The C of aryl, replacement _6-10The heterocyclic radical of aryl, heterocyclic radical or replacement;

R ₄And R ₅Independent separately be H, halogen, heterocyclic radical, replacement heterocyclic radical ,-C (O)-R _d, C _1-6The C of alkyl, replacement _1-6Alkyl, or be combined together to form 5 yuan of rings, as R _4-5:

Shown in the key place realize combination.

X ₁And X ₂Independent separately is N, C, O or S;

R ₈Be H, halogen ,-OH, C _1-6Alkyl, C _2-6Thiazolinyl, C ₂- ₆Alkynyl ,-OH ,-NR _aR _b,-(CH ₂) _n-O-R _c,-O-(C _1-6Alkyl) ,-S (O) _pR _eOr-C (O)-R _d

R ₉Be H, C _1-6The C of alkyl, replacement _1-6The heterocyclic radical of alkyl, heterocyclic radical, replacement or R _9a, R wherein _9aBe:

Shown in the key place realize combination.

R ₁₀And R ₁₁Independent separately is H, halogen, C _1-6The C of alkoxyl, replacement _1-6Alkoxyl ,-NR _aR _bOr-OH;

R _aAnd R _bIndependent separately is H, C _1-6The C of alkyl, replacement _1-6Alkyl ,-C (O) R _d, C _6-10Aryl;

R _cIndependent separately is H, phosphate-based, diphosphonic acid ester group, triphosphoric acid ester group, C _1-6The C of alkyl or replacement _1-6Alkyl;

R _dIndependent separately is H, halogen, C _1-6The C of alkyl, replacement _1-6Alkyl, C _1-6The C of alkoxyl, replacement _1-6Alkoxyl ,-NH ₂,-NH (C _1-6Alkyl) ,-NH (C of replacement _1-6Alkyl) ,-N (C _1-6Alkyl) ₂,-N (the C of replacement _1-6Alkyl) ₂, C _6-10Aryl or heterocyclic radical;

R _eIndependent separately is H, C _1-6The C of alkyl, replacement _1-6Alkyl, C _6-10The C of aryl, replacement _6-10The heterocyclic radical of aryl, heterocyclic radical or replacement;

R _fBe H, C independently of one another _1-6The C of alkyl, replacement _1-6Alkyl ,-C (O) R _d, phosphate-based, diphosphonic acid ester group or triphosphoric acid ester group;

N independently is 0,1,2 or 3 separately;

P independently is 0,1 or 2 separately; Perhaps

(g) (a) each pharmaceutically acceptable salt-(f), (a)-(f) in each tautomer, or the pharmaceutically acceptable salt of this tautomer.

Loxoribine (Loxoribine) (7-pi-allyl-8-oxo guanosine) [143].

The chemical compound that list of references 144 discloses; comprise: the acyl piperazine chemical compound; indole dione (Indoledione) chemical compound; tetrahydroisoquinoline (THIQ) chemical compound; benzo ring diketone (Benzocyclodione) chemical compound; amino azepine vinyl (Aminoazavinyl) chemical compound; amino benzimidazole quinolinones (Aminobenzimidazole quinolinone) is chemical compound [145,146] (ABIQ); hydroxyl phthalic amide (Hydrapthalamide) chemical compound; benzophenone cpd isoxazole compound; sterol compounds; quinazolone (Quinazilinone) chemical compound; azole compounds [147]; anthraquinone compounds; quinoxaline compounds; triaizine compounds; pyrazoles pyrimidine (Pyrazalopyrimidine) chemical compound and benzazole (Benzazole) chemical compound [148].

The chemical compound that list of references 149 discloses.

The aminoalkyl glucosaminide phosphate derivant, as RC-529[150,151].

Phosphonitrile, poly-[two (carboxyl phenoxy group) phosphonitrile] for example described in the list of references 152 and 153 (" PCPP ", poly[di (carboxylatophenoxy) phosphazene]).

Micromolecule immunostimulant (SMIP), for example:

N2-methyl isophthalic acid-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;

N2, N2-dimethyl-1-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-2,4-diamidogen;

N2-ethyl-N2-methyl isophthalic acid-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;

N2-methyl isophthalic acid-(2-methyl-propyl)-N2-propyl group-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;

1-(2-methyl-propyl)-N2-propyl group-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;

N2-butyl-1-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;

N2-butyl-N2-methyl isophthalic acid-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;

N2-methyl isophthalic acid-(2-methyl-propyl)-N2-amyl group-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;

N2-methyl isophthalic acid-(2-methyl-propyl)-N2-third-2-thiazolinyl-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;

1-(2-methyl-propyl)-2-[(phenyl methyl) sulfur]-1H-imidazo [4,5-c] quinoline-4-amine;

1-(2-methyl-propyl)-2-(rosickyite base)-1H-imidazo [4,5-c] quinoline-4-amine;

2-[[4-amino-1-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-2-yl] (methyl) amino] ethanol;

2-[[4-amino-1-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-2-yl] (methyl) amino] ethylhexoate;

4-amino-1-(2-methyl-propyl)-1,3-dihydro-2H-imidazo [4,5-c] quinoline-2-one-;

N2-butyl-1-(2-methyl-propyl)-N4, two (phenyl methyl)-1H-imidazo [4, the 5-c] quinoline-2 of N4-, 4-diamidogen;

N2-butyl-N2-methyl isophthalic acid-(2-methyl-propyl)-N4, two (phenyl methyl)-1H-imidazo [4, the 5-c] quinoline-2 of N4-, 4-diamidogen;

N2-methyl isophthalic acid-(2-methyl-propyl)-N4, two (phenyl methyl)-1H-imidazo [4, the 5-c] quinoline-2 of N4-, 4-diamidogen;

N2, N2-dimethyl-1-(2-methyl-propyl)-N4, two (phenyl methyl)-1H-imidazo [4, the 5-c] quinoline-2 of N4-, 4-diamidogen;

1-{4-amino-2-[methyl (propyl group) amino]-1H-imidazo [4,5-c] quinoline-1-yl }-2-methyl propan-2-ol;

1-[4-amino-2-(propyl group amino)-1H-imidazo [4,5-c] quinoline-1-yl]-2-methyl propan-2-ol;

N4, N4-dibenzyl-1-(2-methoxyl group-2-methyl-propyl)-N2-propyl group-1H-imidazo [4,5-c] quinoline-2,4-diamidogen.

Being used for cytokine induction agent of the present invention can be Toll sample receptor (TLR) regulator and/or agonist.For example, they can be one or more the agonist in people TLR1, TLR2, TLR3, TLR4, TLR7, TLR8 and/or the TLR9 albumen.Preferred cytokine induction agent is the agonist (for example, imidazoquinolines) of TLR7 and/or the agonist (for example, CpG oligonucleotide) of TLR9.These immunostimulants can be used for activating the innate immunity approach.

Can add the cytokine induction agent in each stage of present composition production process.For example, it can be positioned at antigen composition, then this mixture is joined in the O/w emulsion.Another kind of optional mode is that the cytokine induction agent is arranged in O/w emulsion, in this case, reagent can be added emulsion components before emulsifying, perhaps can add in the emulsion after emulsifying.Similarly, reagent can condense upon in the emulsion droplet.The position of cytokine induction agent in final composition and distribution will be depended on its hydrophile/lipophile characteristic, and for example this medicament can be positioned on water, oil phase and/or the oil-water interfaces.

This cytokine induction agent can be coupled to another kind of medicament, as antigen (as CRM197).The summary of relevant micromolecule coupling technology is provided in the list of references 154.As the optional mode of another kind, adjuvant can non-covalent with other reagent (for example by hydrophobic interaction or ionic interaction) combine.

Two kinds of preferred cytokine induction agent are (a) immunostimulatory oligonucleotide and (b) 3dMPL.

Immunostimulatory oligonucleotide can comprise nucleotide modification/analog, modifies as thiophosphate, can be double-stranded or (except that RNA) strand.List of references 155,156 and 157 discloses possible similar replacement, for example uses 2 '-deoxidation-7-denitrogenation guanosine to replace guanosine.The adjuvant effect of CpG oligonucleotide further has been discussed among the list of references 158-163.CpG sequence may lead TLR9, for example motif GTCGTT or TTCGTT[164].But CpG sequence specificity is induced the Th1 immunne response, CpG-A ODN (oligodeoxyribonucleotide) for example, or induce B cell response, for example CpG-BODN more specifically.CpG-A and CpG-B ODN have been discussed among the list of references 165-167.The preferred CpG-AODN of CpG.Making its 5 ' end can be receptor when preferably making up the CpG oligonucleotide discerns.Choose wantonly 3 ' of two CpG oligonucleotide sequences are held the formation " immune aggressiveness " that is connected.Referring to for example, list of references 164 and 168-170.Useful CpG adjuvant is CpG7909, is also referred to as ProMune ^TM(thunder pharmacy group company of section (Coley Pharmaceutical Group, Inc.)).

Except that using the CpG sequence, also can use TpG sequence [171].These oligonucleotide can not contain unmethylated CpG motif.

Immunostimulatory oligonucleotide can be rich in pyrimidine.For example, it can comprise a plurality of successive thymidine nucleotide (for example, list of references 171 described TTTT), and/or its nucleotide is formed and can be contained〉25% thymidine (for example,〉35%, 40%, 50%, 60%, 80%, or the like).For example, it can comprise a plurality of successive cytidylic acids (for example, list of references 171 described CCCC), and/or its nucleotide is formed and can be contained〉25% cytosine (for example,〉35%, 40%, 50%, 60%, 80%, or the like).These oligonucleotide can not contain unmethylated CpG motif.

Immunostimulatory oligonucleotide comprises at least 20 nucleotide usually.They can comprise and be less than 100 nucleotide.

3dMPL (be also referred to as 3 take off-O-acidylate monophosphoryl lipid A or 3-O-deacylated tRNA base-4 '-monophosphoryl lipid A) be in the monophosphoryl lipid A 3 of reducing end glycosamine by the adjuvant of deacylation.3dMPL is from the preparation of the no heptose mutant (heptoselessmutant) of salmonella minnesota (Salmonella minnesota), and it chemically is being similar to lipid A but lacks sour unsettled phosphoryl and alkali labile acyl group.The cell of its activated monocyte/macrophage pedigree stimulates several release of cytokines, comprises IL-1, IL-12, TNF-α and GM-GSF (being also shown in list of references 172).List of references 173 has been described the preparation of 3dMPL at first.

3dMPL can take the form (for example, having 3,4,5 or 6 acyl chains that length may be different) of the different correlation molecule mixture of acidylate degree.By the N-acyl groupization, 3 ' also by the O-acyl groupization for the 2-position carbon (that is, 2 and 2 ') of two glycosamines (being also referred to as 2-deoxidation-2-amino-glucose) monosaccharide.The group that links to each other with carbon 2 is suc as formula-NH-CO-CH ₂-CR ¹R ^{1 '}Shown in.The group that links to each other with carbon 2 ' is suc as formula-NH-CO-CH ₂-CR ²R ^{2 '}Shown in.The group that links to each other with carbon 3 ' is suc as formula-O-CO-CH ₂-CR ³R ^{3 '}Shown in.Representational structure is:

Radicals R ¹, R ²And R ³Independently be-(CH separately ₂) _n-CH ₃The n value preferably between 8-16, more preferably between 9-12, most preferably 10.

Radicals R ^{1 '}, R ^{2 '}And R ^{3 '}Independently be separately: (a)-H; (b)-OH; Or (c)-O-CO-R ⁴, R wherein ⁴Be-H or-(CH ₂) _m-CH ₃, wherein the m value preferably between 8-16, more preferably 10,12 or 14.At 2, m preferred 14.In 2 ' position, m preferred 10.In 3 ' position, m preferred 12.So radicals R ^{1 '}, R ^{2 '}And R ^{3 '}Preferred dodecylic acid, tetradecanoic acid or hexadecanoic acid-the O-acyl group.

Work as R ^{1 '}, R ^{2 '}And R ^{3 '}All be-during H, 3d-MPL have only 3 acyl chains (2,2 ' and 3 ' position on respectively have one).Work as R ^{1 '}, R ^{2 '}And R ^{3 '}In have only two and be-during H, 3d-MPL can have 4 acyl chains.Work as R ^{1 '}, R ^{2 '}And R ^{3 '}In have only one and be-during H, 3d-MPL can have 5 acyl chains.Work as R ^{1 '}, R ^{2 '}And R ^{3 '}In none be-during H, 3d-MPL can have 6 acyl chains.The used 3d-MPL adjuvant of the present invention can be the mixture with these forms of 3-6 bar acyl chain; but preferably comprise 3d-MPL in the mixture with 6 acyl chains; to guarantee that particularly 6 acyl chain forms account for 10% of total 3d-MPL at least by weight, for example 〉=20%, 〉=30%, 〉=40%, 〉=50% or higher.The 3d-MPL that discovery has 6 acyl chains is the most activated adjuvant form.

Therefore, the most preferred form of the 3dMPL that comprises in the present composition has following general formula (IV):

General formula (IV)

When adopting the 3dMPL of form of mixtures, mention that the consumption of 3dMPL in the present composition or concentration refer to all kinds of blended 3dMPL in this mixture.

Under aqueous conditions, 3dMPL can form and vary in size, for example diameter＜150nm or micellar aggregates or the granule of 500nm.The present invention can use any or the two, can select preferable granule by routine test.Smaller particles is preferred for the present invention's (for example, thereby the enough little clarification aqueous suspension that can obtain 3dMPL) [174] because of its preferable activity.The average diameter of preferred particulates is less than 220nm, is more preferably less than 200nm or less than 150nm or less than 120nm, average diameter even can be less than 100nm.Yet in most applications, average diameter can be less than 50nm.These granules are enough little, thereby are suitable for filtration sterilization.Can be by the routine techniques assessment particle diameter of dynamic light scattering, this technology can disclose average particulate diameter.When mentioning particulate diameter and be x nm, distribution of particles generally near this meansigma methods, but in quantitative terms at least 50% the particulate diameter of (for example, 〉=60%, 〉=70%, 〉=80%, 〉=90% or more) in x ± 25% scope.

It is favourable that 3dMPL and O/w emulsion are used in combination.Preferably all 3dMPL are positioned at the aqueous phase of emulsion basically.

Can use 3dMPL separately, or with one or more other chemical compound couplings.For example, can be with 3dMPL and following substances coupling: QS21 saponin [175] (being included in [176] in the O/w emulsion), immunostimulatory oligonucleotide, QS21 and immunostimulatory oligonucleotide, aluminum phosphate [177], aluminium hydroxide [178] or aluminum phosphate and aluminium hydroxide.

The fat adjuvant

The fatty adjuvant that uses among the present invention comprises above-mentioned O/w emulsion, for example also comprises:

The compound or its salt of formula I, II or III:

As described in list of references 179, as ' ER 803058 ', ' ER 803732 ', ' ER 804053 ', ' ER804058 ', ' ER 804059 ', ' ER 804442 ', ' ER 804680 ', ' ER 804764 ', ' ER 803022 ' or ' ER 804057 ', as:

Escherichia coli (Escherichia coli) are as the derivant (as described in list of references 180 and 181) of the lipid A of OM174.

Cation lipid is total to lipid (co-lipid) with (being generally neutral), plants acyl phospholipids acyl-ethanolamine (＂ Vaxfectin as aminopropyl-dimethyl-macene acyloxy-bromination third ammonium-two ^TM＂) or the preparation of aminopropyl-dimethyl-two dodecyl oxygen base-bromination third ammoniums-dioleoyl phosphatidyl-ethanolamine (＂ GAP-DLRIE:DOPE ＂).Preferably contain-N-(3-aminopropyl)-N, N-dimethyl-2, two (suitable-9-tetradecene acyloxy)-1-third ammonium salts of 3-(2, the preparation [182] of 3-bis (syn-9-tetradeceneyloxy)-1-propanaminium).

3-O-deacylated tRNA base monophosphoryl lipid A (as mentioned above).

The chemical compound that contains the lipid that is connected in the acyclic main chain of phosphoric acid is as TLR4 antagonist E5564[183,184]:

Aluminum salt adjuvant

Can use the adjuvant that is called aluminium hydroxide and aluminum phosphate.These titles are conventional, but just use for convenient, accurately do not describe existing actual chemistry and constitute [the 9th chapter of the document 112 that for example, sees reference].The present invention can be with any " hydroxide " that is conventionally used as adjuvant or " phosphate " adjuvant.

The adjuvant that is called " aluminium hydroxide " generally is an aluminum oxyhydroxide salt, and it is partially crystallizable at least usually.Aluminum oxyhydroxide is with molecular formula AlO (OH) expression, itself and other aluminium compound, for example aluminium hydroxide Al (OH) ₃Difference be infrared (IR) spectrum, particularly at 1070cm ^-1There is absorption band in the place and at 3090-3100cm ^-1There is intensive acromion [the 9th chapter of list of references 112] in the place.The width (WHH) of half-peak eminence diffraction zone has reflected the crystallization degree of aluminum hydroxide adjuvant, and the not good granule of crystallization shows stronger spectral line broadening because of crystalline size is less.Surface area increases with the increase of WHH, and the adjuvant that the WHH value is bigger shows that the ability of adsorption antigen is stronger.Aluminum hydroxide adjuvant is typical fibre morphology (for example, transmission electron micrograph is being seen).The pI of aluminum hydroxide adjuvant is usually about 11, and promptly adjuvant itself has positive surface charge under physiological pH.It is reported that during pH7.4, the absorbability of aluminum hydroxide adjuvant is at every mg Al ⁺⁺⁺1.8-2.6mg between the protein.

The adjuvant that is called " aluminum phosphate " generally is an Adju-Phos, and these adjuvants also often contain a small amount of sulfate radical (that is hydroxyl phosphoric acid aluminum sulfate).Can obtain these adjuvants by precipitation, reaction condition during the precipitation and concentration affects phosphate radical replace the degree of hydroxyl in this salt.PO in the hydroxyl phosphate ₄/ Al mol ratio is usually between 0.3-1.2.Hydroxyl phosphate is different from strict AlPO because of there being hydroxyl ₄For example, 3164cm ^-1IR band (for example, when being heated to 200 ℃) show and have structural hydroxyl [the 9th chapter of list of references 112].

The PO of aluminum phosphate adjuvant ₄/ Al ³⁺Mol ratio usually between 0.3-1.2, preferably between 0.8-1.2, more preferably 0.95 ± 0.1.Aluminum phosphate is normally unbodied, particularly hydroxyl phosphate.Typical adjuvant is PO ₄/ Al ³⁺Mol ratio contains 0.6mg Al between 0.84-0.92 ³⁺The amorphous Adju-Phos of/ml.Aluminum phosphate normally granular (for example, the dull and stereotyped sample form seen in the transmission electron micrograph).The representative diameter of these granules behind any antigen of absorption is 0.5-20 μ m (for example, about 5-10 μ m).It is reported that during pH7.4, the absorbability of aluminum phosphate adjuvant is at every mg Al ⁺⁺⁺0.7-1.5mg between the protein.

The degree of the point of zero electric charge of aluminum phosphate (PZC) and phosphate radical substituted hydroxy is inversely proportional to, and should the replacement degree according to the used reaction condition of this salt of precipitation preparation with reactant concentration and different.Concentration that also can be by changing free phosphate anion in the solution (phosphate radical many more=PZC acidity is high more) or by for example adding histidine buffering liquid buffer such as (making PZC have more alkalescence) changes PZC.The PZC of the used aluminum phosphate of the present invention usually between 4.0-7.0, more preferably between 5.0-6.5, for example about 5.7.

The aluminum salt suspension that is used to prepare the present composition can contain buffer agent (for example, phosphate or histidine or Tris buffer agent), but not necessarily.These suspensions are preferably aseptic, apyrogeneity.Suspension can contain free aqueous phosphate anion, for example concentration between the 1.0-20mM, preferably between 5-15mM, 10mM more preferably from about.These suspensions also can contain sodium chloride.

The present invention can use adjuvant to comprise the mixture [81] of aluminium hydroxide and aluminum phosphate.In this case, aluminum phosphate may be more than aluminium hydroxide, and for example weight ratio is at least 2:1, for example 〉=5:1, 〉=6:1, 〉=7:1, 〉=8:1, 〉=9:1, or the like.

Give Al in patient's the compositions ⁺⁺⁺Concentration preferably be lower than 10 mg/ml, for example≤5 mg/ml ,≤4 mg/ml ,≤3 mg/ml ,≤2 mg/ml ,≤1 mg/ml, or the like.Preferred range is between the 0.3-1 mg/ml.Preferably be up to 0.85 milligram/agent.

Except that comprising one or more aluminum salt adjuvants, the adjuvant component also can comprise one or more other adjuvant or immunostimulant.Other this class component includes but not limited to: 3-O-deacylated tRNA base monophosphoryl lipid A adjuvant (' 3dMPL '); And/or O/w emulsion.

The packing of vaccine combination

The suitable vessel of the present composition (or kit component) comprises bottle, syringe (for example, disposable syringe), nasal atomizer etc.These containers should be aseptic.

If compositions/component is stored in the bottle, described bottle is preferably made by glass or plastic material.Preferred earlier bottle the sterilization adds compositions again.For avoiding the patient to latex problem hypersensitive, preferably with the plug seal bottle of no latex, all packaging material preferably all do not contain latex.Bottle can be equipped with single dose vaccine, multiple dose (" multiple dose " bottle) maybe can be housed, for example 10 doses.Preferably make bottle with flint glass.

Bottle can be equipped with lid (for example, Lushi (Luer) snap close), thereby the syringe of filling in advance can be inserted in the lid, the inclusions of syringe is pushed in the bottle (for example, rebuild freeze-dried material wherein), the inclusions of bottle is drawn back in the syringe again.After syringe taken out, load onto syringe needle from bottle, give the patient compositions.Lid is positioned at seals or covering, seal or covering contacts lid again thereby will remove earlier.Bottle can have lid, thereby can aseptic taking-up its inclusions, particularly multiple dose vials.

If certain component is packaged into syringe, syringe can be equipped with syringe needle.If be unkitted syringe needle, can provide independent syringe needle to be used for assembling and use with syringe.This syringe needle can have sheath.The preferred security syringe needle.Commonly No. 3,1 in2, No. 5,1 in2 and No. 5 syringe needles of 5/8 in2.Syringe can post peelable label, has printed the effect duration of lot number, influenza season and inclusions on it, thereby has helped scorekeeping.The piston of syringe preferably has restrain unit, thereby can prevent that plunger accident during aspirating from dropping out.Syringe can be equipped with latex rubber lid and/or plunger.Disposable syringe can contain single dose vaccine.Before loading onto syringe needle, syringe generally is equipped with the pin medicated cap with apical end, and described pin medicated cap is preferably made by butyl rubber.If syringe and syringe needle be packing separately, then preferably the butyl rubber cover is housed to syringe needle.Preferred syringe is with " Tip-Lok " ^TMThose that put goods on the market for trade name.

Can do the labelling that shows the half-value dose volume to container, for example to help to be delivered to the child.For example, the syringe that contains 0.5ml dosage can have the labelling that shows the 0.25ml volume.

If use glass container (for example, syringe or bottle), the then container that preferably uses pyrex rather than soda-lime glass to make.

(for example, being contained in the same box) inset can be provided with kit or compositions, and this inset comprises the details of vaccine, the operation instructions of administration for example, antigenic details etc. in the vaccine.Operation instructions also can comprise warning, for example prepare epinephrine solution in case the anaphylaxis after the vaccination, or the like.

The administration of Therapeutic Method and vaccine

The invention provides vaccine constructed in accordance.

Vaccine combination constructed in accordance is fit to give human patients, the invention provides the method that causes patient's immunne response, and this method comprises the step that gives patient's present composition.

The present invention also provides the present composition as medicine.

The present invention also provides influenza antigen prepared in accordance with the present invention to cause application in the medicine of patient's immunne response in manufacturing.

The immunne response that causes by these methods and applications generally includes antibody response, preferred protection antibody reaction.The method of the postvaccinal antibody response of well known assessment influenza virus vaccine, neutralising capacity and protective effect.Human research proof is at the antibody titer of human influenza virus's hemagglutinin relevant with protective effect (during the blood serum sample hemagglutination-about 30-40 of inhibition titre, the protective effect of homology viral infection being about 50%) [185].Generally detect antibody response by hemagglutination inhibition, microneutralization, unidirectional radioimmunodiffusion (SRID) and/or unidirectional radiation haemolysis (SRH).Well known these experimental techniques.

Can give the present composition by the whole bag of tricks.Most preferred immunization route is intramuscular injection (for example, being injected in arm or the lower limb), but other available approach comprises subcutaneous injection, intranasal [186-188], oral [189], intradermal [190,191], transdermal, percutaneous approach such as [192].

The vaccine of the present invention's preparation can be used for treating child and adult.Influenza vaccines recommend to be used for department of pediatrics and adult's immunity inoculation at present, and the age was from 6 months.Therefore, the patient can be less than 1 years old, 1-5 year, 5-15 year, 15-55 year or at least 55 years old.The preferred old people of patient who accepts vaccine (for example, 〉=50 years old, 〉=60 years old, preferred 〉=65 years old), youngster (for example, ≤ 5 years old), inpatient, health care personnel, army and army personnel, conceived women, chronic disease, immunodeficiency patient take antiviral compound (for example, oseltamivir or zanamivir chemical compound in preceding 7 days accepting vaccine; For example oseltamivir phosphate vide infra) the patient and the people who goes abroad.Yet these vaccines are not only applicable to these colonies, also can be applicable to widely among the crowd.For epidemic isolates, preferably give all age group.

1,2 or 3 of the CPMP standard that preferred composition of the present invention satisfy to be renderd a service.In adult's (18-60 year), these standards are: (1) 〉=70% serum protection; (2) 〉=40% seroconversion; And/or (3) GMT increase 〉=2.5-doubly.Old people's (〉 60 years old) in, these standards are: (1) 〉=60% serum protection; (2) 〉=30% seroconversion; And/or (3) GMT increase 〉=2-doubly.These standards are the open label research (open label study) according at least 50 patients.

Can treat by single dose scheme or multiple dose scheme.Multiple dose can be used for just exempting from scheme and/or booster immunization scheme.In the multiple dose scheme, can give various dosage by identical or different approach, for example just exempt to adopt approach outside the gastrointestinal tract and booster immunization employing mucosal route, perhaps just exempt to adopt mucosal route and the outer approach of booster immunization employing gastrointestinal tract etc.Give an above dosage (generally being two dosage) and be particularly useful for the not patient of immunity contact of immunity, for example the people of never received influenza vaccines perhaps is used for the new HA hypotype of immunity inoculation (as hypotype of the outburst of being very popular).Generally the interval with at least 1 week (for example about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks etc.) gives a plurality of dosage.

Can be basically simultaneously (for example with vaccine of the present invention and other vaccine, can be during the same medical consultation at health care expert or vaccination center or going to a doctor) give the patient, for example give simultaneously basically: Measles Vaccine with following vaccine, mumps Vaccine, rubella vaccine, the MMR vaccine, chickenpox vaccine, the MMRV vaccine, diphtheria vaccine, tetanus vaccine, pertussis vaccine, the DTP vaccine, link coupled influenza B haemophilus vaccine, the poliovirus vaccine of deactivation, hepatitis B virus vaccine, the meningococcal conjugates vaccine (for example, tetravalence A-C-W135-Y vaccine), respiratory syncytial virus vaccine, the streptococcus pneumoniae conjugate vaccines, or the like.Basically give in the gerontal patient particularly useful simultaneously with Pnu-Imune 23 and/or meningococcus vaccine.

Similarly, can be with vaccine of the present invention and antiviral compound, the antiviral compound (for example, oseltamivir and/or zanamivir) of particularly effectively resisting influenza sense poison (for example, can during health care expert's a same medical consultation or going to a doctor) basically simultaneously gives the patient.These antiviral (chemical compound) comprise neuraminidase inhibitor; (3R for example; 4R; 5S)-4-acetyl-amino-5-amino-3 (1-ethyl propoxyl group)-1-cyclohexene-1-carboxylic acid or 5-(acetyl-amino)-4-[(amino imino methyl)-amino]-2; 6-dehydration-3; 4, the 5-three deoxidations-D-glyceryl-D-galactose ninth of the ten Heavenly Stems (galactonon)-2-olefin(e) acid (enonic acid) comprise their ester (for example ethyl ester) and their salt (for example phosphate).Preferred antiviral compound is that (3R, 4R 5S)-4-acetyl-amino-5-amino-3 (1-ethyl propoxyl group)-1-cyclohexene-1-carboxylic acid, ethyl ester phosphate (1:1), are also referred to as oseltamivir phosphate (oseltamivir phosphate capsule (TAMIFLU) ^TM).

General introduction

Term " contain " comprise " comprising " and " by ... form ", the compositions that for example " contains " X can only be made up of maybe X can comprise other material, for example X+Y.

Word " basically " is not got rid of " fully ", and for example the compositions of " essentially no " Y can not have Y fully.This word " basically " can optionally save from the present invention's definition.

The term " about " relevant with numerical value x represent, for example x ± 10%.

Except as otherwise noted, comprise that the method for the step of mixing two or more components is without any need for specific order by merging.Therefore, can any order mix these components.When three kinds of components were arranged, two kinds of components can be mixed mutually, then blended component were mixed with the third component again etc.

When material was used for cultured cell, they should particularly not suffer from the source of mad cow disease (BSE) available from not suffering from Transmissible spongiform encephalopathy (TSE) with animal (specifically being cattle).Generally, preferred cultured cell in the material that does not contain animal origin fully.

When certain chemical compound gave human body as the part of compositions, this chemical compound can be replaced by suitable prodrug.

Brief Description Of Drawings

Fig. 1 has shown the flow process of separating influenza virus from clinical sample.

Fig. 2 has compared the HA titre of isolating 9 kinds of virus sample in the MDCK-33016 cell.In every kind of sample, the post of left-hand side represented for the 2nd generation, and dexter post represented for the 5th generation.

The HA titre of 10 influenza virus samples that Fig. 3 comparison is cultivated in three kinds of different mdck cell types.For each sample, three posts connotation separately is: 33016 of suspension culture is represented on the left side, 33016 of intermediate representation adhere-wall culture, and the CCL-34MDCK cell is represented on the right.

Fig. 4 has shown combining of three kinds of viruses and SNA or MAA agglutinin.Fig. 4 A has shown the combination of initially-separate thing, and 4B has shown the combination after the growth in MDCK 33016 cells, and 4C has shown the combination after the growth in egg.

Fig. 5 and 6 demonstration viruses combine with 3-SL or 6-SLN's.Have six groups in this two width of cloth figure, be respectively: three groups on the limit of keeping left is illustrated under the variable concentrations (1 μ M, 0.5 μ M, 0.25 μ M) and the combining of 3-SL; Keep right three groups on limit is illustrated under the variable concentrations (0.25 μ M, 0.125 μ M, 0.0625 μ M) and the combining of 6-SLN.In this each group of six groups, each square column is represented different virus.In Fig. 5, three square columns from left to right are: (i) virus of cell separation; The (ii) isolating virus of egg; (iii) birds virus.In Fig. 6, four square columns from left to right are: the virus after (i) carrying out going down to posterity for twice in egg; Virus after (ii) in MDCK, carrying out going down to posterity for twice; Virus after (iii) in egg, carrying out going down to posterity for five times; Virus after (iv) in MDCK, carrying out going down to posterity for five times.

Implement mode of the present invention

Isolated viral from patient's sample

Between the influenza seasonal period of the 2006-2007 Northern Hemisphere, obtain the clinical sample (nasal cavity or throat swab) that contains first type and/or Influenza B virus hypotype from child and adult.By determining hemagglutinin (HA) titre, polymerase chain reaction (PCR) and titration of virus, the susceptibility of MDCK 33016 cell lines (DSMACC2219) of serum-free suspension culture and reliability and MDCK CCL 34 cell lines (ATCC) and the egg set up are compared.

248 influenza positive have been differentiated by diagnostic polymerase chain reaction (PCR).Estimating influenza virus by the following method in MDCK33016 cell line and egg duplicates and isolating susceptibility and reliability: (i) hemagglutinin (HA) titre; (ii) be used to measure the real-time polymerase chain reaction (PCR) of viral load; (iii) titration of virus.By the HA gene of second filial generation separator in the original clinical sample and in mdck cell and the egg is checked order, estimate the accuracy that duplicates in the cell.The virus titer that will be obtained by the separator of MDCK 33016 cells of suspension culture and the MDCK 33016 cell gained results of adhere-wall culture compare.

The result shows that MDCKCCL 34 cell lines that the separating power of MDCK33016 suspension cell line is better than having set up significantly are better than egg gained result.After in MDCK 33016 cells, virus sample being gone down to posterity, all do not identify aminoacid replacement in the separator.On the contrary, nearly all egg virus that goes down to posterity all comprises one or more aminoacid replacement, mainly occurs in the HA1 gene.Sudden change after going down to posterity in the egg in the observed HA gene antibody binding site can cause the immunogenicity of influenza virus to change.

The 55% clinical sample discriminating of obtaining from the acute respiratory disease patient is the influenza positive, has following Virus Type: 79%A/H3N2; 12.5%A/H1N1; 1.6%B; 0.4%H3/B and 6.5% is typing not.Can utilize MDCK 33016 cells from clinical sample isolated viral (Fig. 1).On the contrary, inject the virus that is derived from clinical sample of egg, all fail to realize successfully to separate.Obtain similar negative findings in the chick embryo fibroblast (CEF) with prepared fresh.Have only MDCK 33016 culture supernatants of utilizing positive HA titre could separate and set up influenza virus in the egg.

Further inoculate egg, be used for the reference purpose with the thing that obtains at first that is derived from each cell.Adopt the whole bag of tricks, the quantity of successful isolated viral has also shown the quantity of the different virus type of injecting shown in Fig. 1 square frame.All obtain rational HA titre (〉 32 from all three kinds of virus subtypes second pass egg of MDCK 33016 cell separation after generation) and virus titer (〉 1 x 10 ⁶), two kinds of titres increase (Fig. 2) with passage number.

For all three kinds of hypotypes, when separating influenza virus by clinical swab, the MDCK33016 cell of suspension growth is better than attached cell system (CCL-34).Suspension cell line is higher to the sensitivity of positive influenza swab material, shown in the response rate (table 1).HA sequence and original separator after will carrying out different passage numbers in MDCK 33016 cells and egg compare (table 2), isolating influenza A strain is not found sudden change yet after 5 times even go down to posterity in MDCK 33016 cells, and promptly there is sudden change in isolating influenza A strain at the proteic antibody combining site of HA after 2 times and go down to posterity in the egg.Isolating influenza B strain is not all found sudden change in MDCK 33016 or egg.

Viral yield after separator duplicates in MDCK 33016 suspension cells is than high at least one logarithm value of MDCK 33016 attached cells.

Therefore, MDCK 33016 suspension cell lines are the ideal systems that separate and duplicate wild type influenza strain, because it has the separating power higher than egg.And, because height duplicates accuracy, use the cell separation thing to prepare the human influenza vaccine and can produce more reliable vaccine.Coupling between the strain that comprises in the wild type strain of circulation and the vaccine improves, and causes vaccine to produce better influenza protective effect.

Conclusion: (a) with respect to egg, all virus stains separation of succeeding in MDCK 33016 cells; (b) from the virus stain of MDCK 33016 cell separation can be egg successful reproduction; (c) compare with attached cell, the response rate of all three kinds of influenza virus sub-strains is preferable in the MDCK of suspension growth 33016 cells; (d) compare, do not exist the HA gene to replace in any separator of in MDCK 33016 cells, cultivating, this replacement promptly occurs after twice and in egg, go down to posterity with hyle.Therefore, MDCK 33016 suspension cell lines are to be applicable to very much the substrate that separates and breed human influenza virus's hypotype, because it can go down to posterity to the wild type influenza virus from clinical isolates highly reliably, and can keep the real features of wild-type virus.

Receptors bind

The virus of original isolating virus, egg cultivation and the Virus receptors preference that MDCK cultivates have been studied.Research is adopted has 2; 3-sialic acid connecting key (MAA) or 2; the agglutinin of 6-sialic acid connecting key (SNA); or 2; 3-sialyl lactose (3SL; the analog of one hatching egg receptor) and 2,6-sialic acid-N-acetyllactosamine (6-SLN, a kind of people's receptor analogs) sialic acid glycopolymers (sialylglycopolymer) [193].

Fig. 4 illustrates the representative studies result.The peak of labelling shows and the combining of SNA or MAA agglutinin.Protovirus (4A) has different SNA and MAA peak with the virus (4B) of cultivating on MDCK 33016, and viral SNA that in egg, cultivates and MAA peak overlapping substantially (4C).

Further adopting 3-SL and 6-SLN to check binding specificity in the experiment.Example results is shown in Figure 5.The combination in figure left side shows birds receptor preference, and right side associative list person of good sense receptor preference.As shown in Figure 5, the virus of cell separation has strong preference to people's receptor.

Fig. 6 has shown the data of using the Stuttgart separator (A/H1N1) after carrying out in egg or in MDCK 33016 suspension cells going down to posterity for 2 times or 5 times to obtain.MDCK go down to posterity virus 6SLN is had strong preference.

Conclusion is, clinical people's first type or B virus that all MDCK cultivate are easier in 6SLN with respect to 3SL, but find some separators in this test to two kinds of not combinations of material.Different with original clinical isolates, egg adapts to virus can be in conjunction with 3SL, or to 3SL and not combinations of 6SLN.

Cultivate the change that causes in the egg

In mdck cell, separate each strain of first type and Influenza B virus, go down to posterity at the most 5 times by following substrate then: egg; Mdck cell CCL-34; Mdck cell 33016; Vero cell or HEK 293-T cell.Check order and measure the HA titre viral HA gene in the back of at every turn going down to posterity.

Though (for example, HA sequence A/H1N1/Bayern/7/95) is stable existence in egg and MDCK 33016 go down to posterity process, and other are not like this for some strains.For example, go down to posterity in egg after 2 times, the HA sequence of A/H1N1/Nordrhein Westfalen/1/05 sudden change D203N occurs at antibody combining site D, the R329K sudden change also occurs after repeated transmission generation 2 times.On the contrary, do not change by sequence in the MDCK 33016 parallel viruses that go down to posterity.

For the A/H1N1/NRW/1/05 strain, do not see growth during with the Vero cell culture.Other four kinds of substrates can be supported its growth, but HA titre difference.For example, observing titre in the egg is 32-256, but the titre lower (16-32) that the 293T cell produces, and the titre higher (32-512) that MDCK 33016 produces.

Should be understood that and only described the present invention by way of example, can in scope of the present invention and design, make amendment.

Table 1: the response rate after the influenza positive goes down to posterity in MDCK 33016 and ATCC (CCL-34) cell line for the first time

* comprise that available two kinds of mdck cells are isolating 1 double infection sample (H3/B)

Table 2: the comparison of the hemagglutinin sequence of hemagglutinin sequence after 2 times or 5 times of in MDCK 33016-PF cell or egg, going down to posterity and original separator

0=does not have detectable sudden change

* for original separator, only obtain the HA1 sequence

The comparison of * and P2 (MDCK 33016) separator

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Claims (51)

1. method for preparing the influenza kind virus that is used to make vaccine said method comprising the steps of: (i) use directly obtain from the patient or from the influenza infection cell line of former generation separator; (ii) the virus with gained infected cell system in the step (i) at least once goes down to posterity; (iii) step infected cell is (ii) cultivated with preparation and be used as kind of the influenza virus of virus, wherein, used influenza virus is the H1 strain or the H3 strain of Influenza B virus or influenza A virus in the step (i).

2. method for preparing the influenza kind virus that is used to make vaccine said method comprising the steps of: (i) use directly obtain from the patient or from the influenza infection cell line of former generation separator; The cDNA that (ii) prepares at least one viral RNA section of the influenza virus that produces by gained infected cell system in the step (i), and in the reverse genetics method, adopting this cDNA to prepare new influenza virus, at least one viral RNA section is identical with the influenza virus of step (i) in this new influenza virus; (iii) use described new influenza infection cell line, this cultured cell system is used as kind of the new influenza virus of virus with preparation then.

3. the method for claim 1 is characterized in that, step going down to posterity in (ii) is that the cell by identical with used cell type in the step (i) carries out.

4. each described method of claim as described above is characterized in that, step (i), (ii) or does not (iii) relate to influenza virus and cultivates in egg or go down to posterity.

5. each described method of claim as described above is characterized in that described cell line is inhuman mammal cell line.

6. each described method of claim as described above is characterized in that, described cell line is mdck cell system.

7. each described method of claim as described above is characterized in that, described kind virus is checked order.

8. each described method of claim as described above is characterized in that, causes antiserum with described kind virus.

9. each described method of claim as described above is characterized in that, with the viral preparation work seed lot of described kind.

10. each described method of claim as described above is characterized in that, makes vaccine with described kind virus.

11. each described method of claim is characterized in that as described above, described kind of viral genome do not have the PR/8/34 section.

12. each described method of claim is characterized in that as described above, described kind virus contains hemagglutinin, this hemagglutinin has and contains Sia (α 2,3) oligosaccharide of the terminal disaccharide of Gal is compared, more be partial in conjunction with the oligosaccharide that contains the terminal disaccharide of Sia (α 2,6) Gal in conjunction with preference.

13. method for preparing the influenza virus that is used to make vaccine, said method comprising the steps of: (i) obtain influenza virus that circulates in colony or the influenza virus that comprises hemagglutinin, this hemagglutinin has the representative antigenicity of the influenza virus that circulates in colony; The influenza infection cell line of (ii) using step (i) to obtain; (iii) will by step (ii) the virus that obtains of infected cell system at least once go down to posterity, to obtain kind of a strain; (iv) incubation step kind strain (iii) is to produce influenza virus.

14. method for preparing the influenza virus that is used to make vaccine, said method comprising the steps of: (i) obtain influenza virus that circulates in colony or the influenza virus that comprises hemagglutinin, this hemagglutinin has the representative antigenicity of the influenza virus that circulates in colony; The influenza infection cell line of (ii) using step (i) to obtain; The cDNA that (iii) prepares at least one viral RNA section of the influenza virus that produces by gained infected cell system in the step (i), and in the reverse genetics method, adopting this cDNA to prepare influenza kind virus, at least one viral RNA section is identical with the influenza virus of step (i) in this influenza kind virus; (iv) be to cultivate then from step cell line (iii) to produce influenza virus through going down to posterity with described influenza kind virus infected cell.

15. a method for preparing influenza virus vaccine, this method may further comprise the steps after as claim 13 or 14 described steps (i)-(iv): (v) treatment step (iv) gained virus with the preparation vaccine.

16. method as claimed in claim 15 is characterized in that, described step (v) relates to the described virus of deactivation.

17. method as claimed in claim 16 is characterized in that, described vaccine is the whole virus particles vaccine.

18. method as claimed in claim 16 is characterized in that, described vaccine is the division virus particle vaccine.

19. method as claimed in claim 16 is characterized in that, described vaccine is a SAV.

20. method as claimed in claim 16 is characterized in that, described vaccine is the virion vaccine.

21., it is characterized in that the every dosage of described vaccine comprises the residual host cell DNA less than 10ng as each described method among the claim 15-20.

Carry out making multiple independent strains of influenza viruses 22. a method of making the multivalence influenza vaccines, this method comprise, and described independent vaccine mixing is made the multivalence influenza vaccines as each described method among the claim 15-20.

23. method as claimed in claim 22 is characterized in that, described multivalence influenza vaccines have two kinds of influenza A virus strains and a kind of Influenza B virus strain.

24., it is characterized in that described vaccine is substantially free of hydrargyrum as each described method among the claim 15-22.

25., it is characterized in that described vaccine comprises adjuvant as each described method among the claim 15-24.

26. one kind prepares sero-fast method by animal, said method comprising the steps of: the influenza virus hemagglutinin that (i) gives described animal purification; (ii) reclaim the serum of the identification antibody that contains this hemagglutinin then, the virus of cultivating in the next comfortable cell line of the hemagglutinin that adopts in the described step (i) from described animal.

27. one kind prepares sero-fast method by animal, said method comprising the steps of: (i) cultivate influenza virus in cell line; The (ii) viral purification hemagglutinin antigen of from step (i), cultivating; (iii) give described animal from step purification hemagglutinin (ii); (iv) reclaim the serum of the identification antibody that contains this hemagglutinin then from described animal.

28., it is characterized in that described animal is a sheep as claim 26 or 27 described methods.

29., it is characterized in that described method also comprises described antiserum and the blended step of gel that is applicable to that SRID analyzes as each described method among the claim 26-28.

30. one kind can be by the antiserum as method obtains as described in each among the claim 26-28.

31. a method for preparing the antigen reference material said method comprising the steps of: (i) in cell line, cultivate influenza virus; The (ii) virus of cultivating in the purification step (i); (iii) deactivation should virus, and the influenza virus of adopting in the step (i) was never cultivated in egg; (iv) this inactivation of viruses of lyophilizing.

32. the method from patient's sample separation influenza virus, this method comprise the step of cultivating patient's sample with mdck cell, wherein, described mdck cell is grown in suspending nutrient solution.

33. the method from patient's sample separation influenza virus, this method comprise the step of cultivating patient's sample with mdck cell, wherein, described mdck cell is grown in serum-free medium.

34. the method from patient's sample separation influenza virus, this method comprise the step of cultivating patient's sample with mdck cell, wherein, described mdck cell is grown in protein-free medium.

35. the method from patient's sample separation influenza virus, this method comprise the step of cultivating patient's sample with the non-tumorigenic mdck cell.

36. the method from patient's sample separation influenza virus, this method comprise the step of cultivating patient's sample with mdck cell, wherein, do not provide the covering culture medium to described mdck cell.

37. the method from patient's sample separation influenza virus, this method comprise the step of cultivating patient's sample with mdck cell, wherein, described mdck cell is grown in the serum-free suspending nutrient solution.

38. the method from patient's sample separation influenza virus, this method comprise the step of cultivating patient's sample with mdck cell, wherein, described mdck cell is grown in no protein suspending culture fluid.

39. the method from patient's sample separation influenza virus, this method comprise the step of cultivating patient's sample with the non-tumorigenic mdck cell, wherein, described mdck cell is grown in suspending nutrient solution.

40. the method from patient's sample separation influenza virus, this method comprise the step of cultivating patient's sample with the non-tumorigenic mdck cell, wherein, described mdck cell is grown in the serum-free suspending nutrient solution.

41. the method from patient's sample separation influenza virus, this method comprise the step of cultivating patient's sample with the non-tumorigenic mdck cell, wherein, described mdck cell is grown in no protein suspending culture fluid.

42. one kind by each described method isolated influenza virus among the claim 32-41.

43. method for preparing the reprovision influenza virus, said method comprising the steps of: (i) with first strains of influenza viruses with first group of genome section and the second strains of influenza viruses infection cell system with second group of genome section, wherein said first strain has the HA section of the required hemagglutinin of coding; (ii) the infected cell of incubation step (i) is with the preparation influenza virus, at least one section is from first group of genome section in this influenza virus, at least one section is from second group of genome section, and prerequisite is that described at least one section from first group of genome section comprises the HA section from described first strain.

44. a method for preparing the influenza antigen that uses in vaccine said method comprising the steps of: (i) accept the influenza virus never on the egg substrate, bred; (ii) use this influenza infection cell line; (iii) incubation step infected cell (ii) is with the preparation influenza virus.

45. a method for preparing the influenza antigen that uses in vaccine said method comprising the steps of: (i) be received in isolated influenza virus in the MDCK33016 cell; (ii) use this influenza infection cell line; (iii) incubation step infected cell (ii) is with the preparation influenza virus.

46. a method for preparing the influenza antigen that uses in vaccine said method comprising the steps of: (i) accept never the influenza virus that bred on the substrate that blood serum medium cultivates containing; (ii) use this influenza infection cell line; (iii) incubation step infected cell (ii) is with the preparation influenza virus.

47. a method for preparing the influenza antigen that uses in vaccine said method comprising the steps of: (i) acceptance utilizes the influenza virus that the reverse genetic technology produces; (ii) use this influenza infection cell line; (iii) incubation step infected cell (ii) is with the preparation influenza virus.

48. a method for preparing the influenza antigen that uses in vaccine said method comprising the steps of: (i) accept to contain less than 6 influenza A viruss from the viral section of PR/8/34 influenza virus; (ii) use this influenza infection cell line; (iii) incubation step infected cell (ii) is with the preparation influenza virus.

49. a method for preparing the influenza antigen that uses in vaccine said method comprising the steps of: (i) accept to contain less than 6 influenza A viruss from the viral section of AA/6/60 influenza virus; (ii) use this influenza infection cell line; (iii) incubation step infected cell (ii) is with the preparation influenza virus.

50. method for preparing the influenza antigen that in vaccine, uses, said method comprising the steps of: (i) accept influenza virus, hemagglutinin in this influenza virus has that (α 2 with respect to containing Sia, 3) oligosaccharide of the terminal disaccharide of Gal, be more prone in conjunction with the oligosaccharide that contains the terminal disaccharide of Sia (α 2,6) Gal in conjunction with preference; (ii) use this influenza infection cell line; (iii) incubation step infected cell (ii) is with the preparation influenza virus.

51. a method for preparing the influenza antigen that uses in vaccine said method comprising the steps of: (i) accept to have the influenza virus of not observed hemagglutinin in the egg and/or neuraminidase sugar shape; (ii) use this influenza infection cell line; (iii) incubation step infected cell (ii) is with the preparation influenza virus.

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