CN102580077A - Intranasal influenza vaccine based on virosomes - Google Patents
Intranasal influenza vaccine based on virosomes Download PDFInfo
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Abstract
The present invention provides a composition of influenza virosomes comprising reconstituted envelopes of said virus, wherein the viral envelopes are entirely derived from influenza viral particles, wherein no lipid is added from an external source to the reconstituted virosomes, wherein the virosomes comprise the influenza antigens haemagglutinin and/or neuraminidase or derivatives thereof, wherein no separate adjuvant and/or immune stimulator is added to the composition, and wherein the composition is for intranasal or inhalational administration, which composition is characterized in that a single intranasal or inhalational administration to a human being is sufficient for the induction of a systemic immune response and/or a local immune response and/or a cytotoxic lymphocytes response against said influenza antigens, which systemic response is in accordance with the CHMP criteria for influenza vaccine, and wherein the dose of haemagglutinin per viral strain per intranasal or inhalational administration is lower than or equal to 30 [mu]g. The invention also provides the use of reconstituted influenza virosomes for the manufacture of said composition, and accordingly manufactured vaccine formulations.
Description
The application be that March 21, application number in 2007 are 200780010217.9 the applying date, denomination of invention divides an application for the one Chinese patent application of " based on the intranasal influenza vaccine of virion ".
Invention field
The present invention relates to, for example, be used for the compositions and the administering mode of inactivated influenza vaccine, wherein, single intranasal or inhalation have obtained and the positively related systemic immune response of clinical protection.
Background of invention
Be studied as non-injection (needleless needle-less) alternative route subcutaneous or that intramuscular is immune through intranasal or oropharynx approach and the multiple imagination of using the deactivation influenza antigens to carry out immunity to influenza.In animal model, produced the experimental data of supporting non-injection means.Obtain that animal data is supported, use the deactivation influenza antigens (complete virus particle of chemical mode deactivation for example; Perhaps by the virus component of further processing; The virus that for example is cut open; Perhaps purified surface antigen hemagglutinin (HA) and/or neuraminidase (NA)) thinking of carrying out immunity through the intranasal approach comprises: uses adjuvant or immunostimulant (immune stimulator) and the combination of deactivation influenza antigens, perhaps needs repeatedly immunity.Adjuvant is immunogenic any material that can strengthen with its antigens mixed.In the mankind, only reported through the successful vaccination of intranasal approach opposing influenza: (strain of the cold adaptation property) influenza vaccines (FluMist that (a) lives
TMMedImmune Vaccines Inc) (list of references 1,2,3); (b) virion type influenza vaccines; Its assistant has the heat-labile toxin (NasalFlu, Berna Biotech Ltd) (list of references 4) of escherichia coli (E.coli) or (c) use high dose antigen and repetition vaccination (list of references 5,10,11).Though live vaccine can be induced satisfied immunne response, its special properties that can become live virus has brought extra safety worries, and because therefore near need be upper respiratory tract virus replication might induce side effect.In addition, required holding conditions has also limited the commercialization of these products.Use escherichia coli HLT to withdraw from (list of references 6) from market as the virus particle vaccine that the strong dependency between the intranasal influenza vaccine of adjuvant and the facial paralysis (Bell ' s Palsy) causes helping HLT.
The curative effect of influenza vaccines in given crowd can be assessed through following method: the relevant immunogenicity parameter of quantity of evaluating the influenza antibody that produces with the inoculation back.These immunogenicity parameters are commonly called the CHMP standard, and it is used for inactivated influenza vaccine is carried out permitting again every year authentication (list of references 7).Up to now; A single intranasal through using inactivated vaccine is bestowed; (it is the supplementary element in the vaccine not add adjuvant; It is from the infectious reagent that will prevent with vaccine, and is to be added in the bacterin preparation for the purpose that strengthens antigenic immunne response), just meet that these immunologys require or CHMP standard (list of references 7), that the mankind are resisted the successful immunity of influenza is not on the books as yet.Therefore; People recognize, still have the demand to following inactivated influenza vaccine compositions in the art, and said compositions can be induced satisfied general immunoreation after the single intranasal administration; And do not contain adjuvant, and can meet CHMP standard (list of references 7) through said single-dose.
Said " CHMP standard " is according to hereinafter described being defined.In the Note for Guidance on Harmonisation of Requirements for of CHMP (Committee for Medicinal Products for Human Use) Influenza Vaccines; Defined following serology parameter, to evaluate the immunogenicity of inactivated influenza vaccine:
-serum protective rate, wherein serum protection be defined as hemagglutination suppress (Haemagglutination Inhibition HI) tires >=40,
-seroconversion rate, wherein seroconversion be defined as that HI tires before the inoculation＜10, inoculation back HI tires >=40, perhaps, before the inoculation HI tire >=10 and tire at least 4 times increase of HI,
-average multiple value added, this is to the geometrical mean that increases in the experimenter (that is HI tired before, inoculation back HI tired/inoculates).
Require to the immunogenic CHMP of influenza vaccines to be: for every kind in three kinds of Strain in the vaccine, meet at least one in the criterion:
| Standard | The adult | The old people |
| The serum protective rate | ＞70％ | ＞60％ |
| Seroconversion rate | ＞40％ | ＞30％ |
| Average multiple value added | ＞2.5 | ＞2.0 |
The present invention also is applicable to the child, demonstrates from them, and they make immunne response (list of references 8) with the mode suitable with the adult.The present invention also is applicable to old experimenter.Old artificial surpass 60 years old.
Invention is described
The present invention relates to:
1. the compositions that comprises influenza virus particles; Said virion comprises the reconstruct peplos of said virus; Wherein said peplos obtains from influenza virus particles fully; Wherein do not add lipid to said reconstruct virion from external source; Wherein said virion comprises said influenza antigens hemagglutinin and/or neuraminidase or derivatives thereof; Wherein add independent adjuvant and/or immunostimulant to said compositions, and wherein said compositions designs as intranasal or inhalation preparation, said compositions is characterised in that said preparation can the said influenza antigens hemagglutinin of reactance and/or the systemic immune response and/or the local immune response of neuraminidase or derivatives thereof to the mankind's single intranasal or inhalation.
2. like project 1 described compositions, wherein the hemagglutinin dosage of every kind of each intranasal of Strain or inhalation is less than or equal to 30 μ g.
3. like any described compositions in project 1 or 2, the single intranasal of wherein said preparation or inhalation can also the inducing cytotoxic lymphocyte responses.
4. like any described compositions in the project 1 to 3, wherein said immunne response meets the CHMP standard to influenza vaccines.
5. like project 4 described compositionss; Wherein said immunne response provides one or more in following: as far as adult＞70% and/or as far as the serum protective rate of old people＞60%; To adult＞40% and/or to the seroconversion rate of old people＞30%, and increase to adult＞2.5 and/or to the average multiple of old people＞2.0.
6. like any described compositions in the project 2 to 5, wherein, the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 25 μ g.
7. like any described compositions in the project 2 to 5, wherein, the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 20 μ g.
8. like any described compositions in the project 2 to 5, wherein, the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 15 μ g.
9. like any described compositions in the project 2 to 5, wherein, the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 10 μ g.
10. like any described compositions in the project 2 to 5, wherein, the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 5 μ g.
11. like any described compositions in the project 1 to 10, wherein, said compositions is the bacterin preparation that comprises the pharmaceutical carrier that is used for intranasal or inhalation.
12. the influenza virus particles that comprises the reconstruct peplos of said virus is used to prepare the purposes of the compositions that is used for intranasal or inhalation; Wherein said peplos obtains from influenza virus particles fully; Wherein do not add lipid to said reconstruct virion from external source; Wherein said virion comprises influenza antigens hemagglutinin and/or neuraminidase or derivatives thereof; Wherein add independent adjuvant and/or immunostimulant to said compositions, said compositions is characterised in that systemic immune response and/or the local immune response that said compositions is enough to said influenza antigens hemagglutinin of reactance and/or neuraminidase or derivatives thereof to the mankind's single intranasal or inhalation.
13. like project 12 described purposes, wherein the hemagglutinin dosage of every kind of each intranasal of Strain or inhalation is equal to or less than 30 μ g.
14. like any described purposes in project 12 or 13, the single intranasal of wherein said preparation or inhalation be the inducing cytotoxic lymphocyte responses also.
15. like any described purposes in the project 12 to 14, wherein said immunne response meets the CHMP standard to influenza vaccines.
16. like project 15 described purposes; Wherein said immunne response provides one or more in following: as far as adult＞70% and/or as far as the serum protective rate of old people＞60%; To adult＞40% and/or to the seroconversion rate of old people＞30%, and increase to adult＞2.5 and/or to the average multiple of old people＞2.0.
17. like any described purposes in the project 13 to 16, wherein the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 25 μ g.
18. like any described purposes in the project 13 to 16, wherein the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 20 μ g.
19. like any described purposes in the project 13 to 16, wherein the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 15 μ g.
20. like any described purposes in the project 13 to 16, wherein the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 10 μ g.
21. like any described purposes in the project 13 to 16, wherein the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 5 μ g.
22. like any described purposes in the project 12 to 21, wherein the compositions of preparation is a bacterin preparation.
23. comprise the bacterin preparation of the compositions of influenza virus particles; Said virion comprises the reconstruct peplos of said virus; Wherein said peplos obtains from influenza virus particles fully; Wherein do not add lipid from external source to the reconstruct virion, wherein virion comprises influenza antigens hemagglutinin and/or neuraminidase or derivatives thereof, does not wherein add independent adjuvant and/or immunostimulant to said compositions; This vaccine is characterised in that to designing said vaccine to the mankind's single intranasal or inhalation, and wherein the hemagglutinin dosage of every kind of each intranasal of Strain or inhalation is equal to or less than 30 μ g.
24. like project 23 described bacterin preparations, wherein the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 25 μ g.
25. like project 23 described bacterin preparations, wherein the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 20 μ g.
26. like project 23 described bacterin preparations, wherein the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 15 μ g.
27. like project 23 described bacterin preparations, wherein the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 10 μ g.
28. like project 23 described bacterin preparations, wherein the dosage of the hemagglutinin of every kind of each intranasal of Strain or inhalation is less than or equal to 5 μ g.
29. like any described bacterin preparation in the project 23 to 28; The single intranasal of wherein said preparation or inhalation can be in the said mankind induce immune response; Said immunne response comprises: the systemic immune response of opposing influenza antigens hemagglutinin and/or neuraminidase or derivatives thereof; The local immune response of opposing influenza antigens hemagglutinin and/or neuraminidase or derivatives thereof, and in the immunne response of cytotoxic lymphocyte mediation one or multinomial.
30. like project 29 described bacterin preparations, wherein said immunne response meets the CHMP standard to influenza vaccines.
31. like project 30 described bacterin preparations; Wherein, Said immunne response provides one or more in following: as far as adult＞70% and/or as far as the serum protective rate of old people＞60%; To adult＞40% and/or to the seroconversion rate of old people＞30%, and increase to adult＞2.5 and/or to the average multiple of old people＞2.0.
32. be used for the device of intranasal or inhalation, said device comprises according to any described bacterin preparation in the project 23 to 31.
33. like project 32 described devices, wherein, said device comprises a certain amount of bacterin preparation that is used for single intranasal or inhalation.
34. like the device that project 32 or 33 is stated, wherein, said device is disposable.
Amazing ground; With clinical preceding Rodents data and opposite about the document of the clinical experience of the mankind; We find; For 18-60 year age group, carry out the single intranasal vaccination with the inactivated influenza vaccine that comprises reconstruct influenza virus peplos after, human immunne response meets whole three the CHMP standards to the influenza vaccines curative effect.One time the single intranasal is bestowed and is: for meeting to the immunogenic above-mentioned CHMP standard of inactivated influenza vaccine, carry out the inoculation of bacterin preparation through one or two nostril, and need not to repeat to bestow bacterin preparation.One time the single vaccine is bestowed (through nose, suction, oral, subcutaneous or intramuscular approach) normally following inoculation arrangement, and it does not comprise: in this area as first (priming) with reinforcement (boosting) and known, with interval repeatedly the bestowing of a couple of days or several weeks to vaccine.The preparation that is designed to intranasal or inhalation preparation comprises the mixture of one or more active components and excipient, is to prepare according to the mode that allows intranasal or inhalation.The invention provides a kind of method, be used to induce the systemic immune response (the B cell of circulation immunoglobulin or generation antibody) that meets the CHMP standard, advantageously, said method uses single intranasal or the inhalation to the virion influenza vaccines to carry out.The present invention also provides a kind of method; Be used to induce part or mucosal immune response; Said replying comprises on the mucosa film surface that as the increase of the known immunoglobulin,exocrine of IgA, advantageously, said method is used single intranasal of virion influenza vaccines or inhalation are carried out.After intranasal is bestowed inducing of replying of specific IgG and IgA related to adenoid activity in the nasal cavity (list of references 12).This type of tissue is known as nose associated lymphoid tissue (NALT), and it also is shown as the mucosa inducing site (list of references 13) that is used for cellullar immunologic response.Because the known viruse granule has the probability (list of references 14,15) of immunne response in the inducing cell, so the present invention also provides the method for inducing specific cytotoxic lymphocyte (CTL).
Virion (virosome) is the double-layer of lipoid that contains VGP.Usually through extracting memebrane protein and lipid from the band enveloped virus, then come reconstruct characteristic bilayer to produce virion through removing said detergent with detergent.The present invention also provides the compositions of influenza virus particles; Its influenza virus peplos that comprises reconstruct (particularly; No longer add lipid, and do not add immunomodulator and the reconstruct of immunostimulant (being commonly referred to adjuvant)), be used for inoculating through aerosol; Said aerosol is bestowed the mucosa of nasopharynx or oropharynx through one or two nostril, to obtain the general and the local immunity of opposing influenza.It also is feasible that the single that carries out through suction is bestowed.It also is feasible that the single oral mucosa is bestowed.
Can prepare the reconstruct influenza virus particles from inactivation of viruses, can use the detergent that to dialyse that inactivation of viruses is dissolved, remove said detergent on the hydrophobic beadlet through being adsorbed onto.Prepared product can comprise the purified suspension of one or more influenza antigens, and said influenza antigens is selected from hemagglutinin (HA), neuraminidase (NA), the derivant of hemagglutinin and the derivant of neuraminidase.Can in the film that viral lipid (containing low-level endotoxin and ovalbumin) constitutes, carry out reconstruct (document 9 sees reference) to virus membrane antigen hemagglutinin and neuraminidase.The derivant of serum agglutinin and/or neuraminidase is to have through the aminoacid sequence of modification and/or the hemagglutinin and/or the neuraminidase molecule of structure.Aminoacid can for example be deleted, replaced or added in the sequence.In addition, glycosylation mode can be changed.Derivant remains with the ability of induce immune response when importing the host.
Can be in the egg that for example contains the embryo, or cultivate the influenza virus that is used to prepare the reconstruct virion in the cell culture of the cell in cell that adheres to or suspension.Virus for example can be the strain of (reassortant) wild type or reprovision or process genetic modification.Virus Type can for example be any influenza A or B hypotype, comprises epidemic strain.
The present invention also provides vaccine.The term vaccine should be understood that to have immunocompetent pharmaceutical preparations.In some embodiments, vaccine can comprise the harmless variant or the derivant of pathogenic microbes, and for example, stimulating immune system produces the opposing to true pathogen.In some embodiments,, vaccine for example can induce adaptive immunity when bestowing the host.Vaccine can contain the death or the weakening form of pathogen or pathogen component, for example the antigenicity component of pathogen.The vaccine production thing can also contain pharmaceutical carrier, and said pharmaceutical carrier can design to the AD HOC that vaccine will be bestowed, and for example is directed against the pharmaceutical carrier of intranasal or inhalation design.Influenza vaccines can comprise one or more not degeneration influenza antigens, its one or more can induce the special immunne response of influenza.
The invention provides the compositions that contains influenza virus particles, said influenza virus particles comprises the reconstruct peplos of said virus, and wherein said compositions is designed to intranasal or inhalation preparation.The present invention also provides virion wherein to comprise the said compositions of influenza antigens hemagglutinin and/or neuraminidase or derivatives thereof.The said compositions that the present invention also provides peplos wherein to obtain from virion fully.The present invention also provides the said compositions that does not wherein have lipid to add to the reconstruct virion from external source.The present invention also provides and has not wherein added the independent adjuvant and/or the said compositions of immunostimulant to said compositions.The present invention also provides the said compositions that wherein can induce systemic immune response to experimenter's single intranasal or inhalation.The present invention also provides the said compositions that wherein can also induce local immune response to experimenter's single intranasal or inhalation.The present invention also provide can also the inducing cytotoxic lymphocyte responses to experimenter's single intranasal or inhalation said compositions.The present invention also provides and wherein in the mankind, has shown the said compositions of inducing the ability that systemic immune response and/or local immune response and/or cytotoxic lymphocyte reply.The present invention also provides wherein, and immunne response comprises the said compositions to the immunne response of influenza antigens hemagglutinin and/or neuraminidase or derivatives thereof.A kind of preferred embodiment in, the present invention also provides immunne response wherein to meet the said compositions of the CHMP standard that is used for influenza vaccines.The present invention also provides immunne response wherein that one or more the said compositions in following is provided: as far as adult＞70% and/or as far as the serum protective rate of old people＞60%; To adult＞40% and/or to the seroconversion rate of old people＞30%, and increase to adult＞2.5 and/or to the average multiple of old people＞2.0.A kind of especially preferred embodiment in, the present invention also provides wherein the hemagglutinin dosage of every kind of each intranasal of Strain or inhalation to be equal to or less than the said compositions of 30 μ g.At last, the present invention provides also wherein that compositions is the said compositions that comprises the bacterin preparation of the pharmaceutical carrier that is used for intranasal or inhalation.
The present invention also provides the influenza virus particles of the reconstruct peplos that comprises said virus to be used to prepare the purposes of the compositions that is used for intranasal or inhalation.The present invention also provides so said purposes: wherein, influenza virus particles comprises influenza antigens hemagglutinin and/or neuraminidase or derivatives thereof.The present invention also provides so said purposes: wherein peplos obtains from influenza virus particles fully.The present invention also provides so said purposes: wherein do not have lipid to join the reconstruct virion from external source.The present invention also provides so said purposes: wherein do not add independent adjuvant and/or immunostimulant to said compositions.The present invention also provides so said purposes: wherein single intranasal or the inhalation to the experimenter is enough to induce systemic immune response.The present invention also provides so said purposes: wherein also induce single intranasal from local immune response to experimenter or inhalation.The present invention also provides so said purposes: wherein to experimenter's single intranasal or inhalation inducing cytotoxic lymphocyte responses also.The present invention also provides so said purposes: the experimenter who wherein accepts administration is human.The present invention also provides so said purposes: inductive immunne response comprises the immunne response to influenza antigens hemagglutinin and/or neuraminidase or derivatives thereof.A kind of preferred embodiment in, the present invention also provides so said purposes, wherein compositions is induced the immunne response that meets to the CHMP standard of influenza vaccines.The present invention also provides so said purposes; Wherein immunne response provides one or more in following: as far as adult＞70% and/or as far as the serum protective rate of old people＞60%; To adult＞40% and/or to the seroconversion rate of old people＞30%, and increase to adult＞2.5 and/or to the average multiple of old people＞2.0.A kind of especially preferred embodiment in, the present invention also provides so said purposes, wherein the applied dose of the hemagglutinin of every kind of each intranasal of Strain or inhalation is equal to or less than 30 μ g.At last, the compositions of wherein preparation also is provided is the said purposes of bacterin preparation in the present invention.
Therefore; In one embodiment; The invention provides the compositions of the influenza virus particles of the reconstruct peplos that comprises said virus; Wherein said peplos obtains from influenza virus particles fully, does not wherein add lipid from external source to the reconstruct virion, and wherein virion comprises influenza antigens hemagglutinin and/or neuraminidase or derivatives thereof; Wherein do not add independent adjuvant and/or immunostimulant to said compositions; And wherein said compositions designs as intranasal or inhalation preparation, said compositions be characterised in that said preparation to the mankind's single intranasal or inhalation can the said influenza antigens of reactance systemic immune response and/or local immune response, said more systemic response can meet the CHMP standard that is used for influenza vaccines; And wherein, the hemagglutinin dosage of every kind of each intranasal of Strain or inhalation is equal to or less than 30 μ g.
According to another kind of embodiment; The influenza virus particles that the invention provides the reconstruct peplos that comprises said virus is used to prepare the purposes of the compositions that is used for intranasal or inhalation; Wherein said peplos obtains from influenza virus particles fully; Wherein do not add lipid to the reconstruct virion from external source; Wherein virion comprises influenza antigens hemagglutinin and/or neuraminidase or derivatives thereof; Wherein add independent adjuvant and/or immunostimulant to said compositions, the purposes that said influenza virus particles is used to prepare the compositions that is used for intranasal or inhalation is characterised in that, systemic immune response from the said influenza antigens of reactance to the mankind's single intranasal or inhalation and/or local immune response that said compositions is enough to; Saidly reply the CHMP standard that can meet, and wherein the hemagglutinin dosage of every kind of each intranasal of Strain or inhalation is equal to or less than 30 μ g to influenza vaccines.
According to another kind of embodiment; The invention provides a kind of bacterin preparation that comprises the compositions of influenza virus particles; Said virion comprises the reconstruct peplos of said virus, and wherein, said peplos obtains from influenza virus particles fully; Wherein do not add lipid to the reconstruct virion from external source; Wherein virion comprises influenza antigens hemagglutinin and/or neuraminidase or derivatives thereof, does not wherein add independent adjuvant and/or immunostimulant to said compositions, and wherein vaccine is characterised in that to single intranasal or inhalation to the mankind and designs vaccine; And wherein the hemagglutinin dosage of every kind of each intranasal of Strain or inhalation is equal to or less than 30 μ g.Advantageously, the said single intranasal of said preparation or inhalation can be induced general and/or local immune response in the said mankind.Also provide according to the present invention and to have comprised a certain amount of device that is used for the said bacterin preparation of single intranasal or inhalation.
The hemagglutinin of every kind of each intranasal of Strain or inhalation can also be less than or equal to 25 μ g, 20 μ g, 15 μ g, 10 μ g or 5 μ g according to application dose of the present invention.
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(8)Daubeney，P.，Taylor，C.J.，McGaw，J.，Brown，Brown，E.M.，Ghosal，S.，Keeton，B.R.，Palache，B.，Kerstens，R.Immunogenicity?and?tolerability?of?a?trivalent?influenza?subunit?vaccine(InfluvacR)in?high-risk?children?aged?6?months?to?4?years.BJCP?1997?March，51(2)：87-90
(9)Stegmann，T.，Morselt，H.W.M.，Booy，F.P.，Van?Breemen，J.F.L.，Scherphof，G.，Wilschut，J.Functional?reconstitution?of?influenza?viris?envelopes.EMBO?Journal?1987，6(9)：2651-2659
(10)Treanor?J，Nolan?C，O’Brien?D，Burt?D，Lowell?G，Linden?J，Fries?L.Intranasal?administration?of?a?proteosome-influenza?vaccine?is?well-tolerated?and?induces?serum?and?nasal?secretion?influenza?antibodies?in?healthy?human?subjects.Vaccine?2006；24(3)：254-62
(11)Read?R.C.，Naylor?S.C.Potter?C.W.，Bond?J.，Jabbal-Gill?I.，Fisher?A.，Illum?L.，Jennings?R.Effective?nasal?influenza?vaccine?delivery?using?chitosan.Vaccine?2005；23(35)：4367-74
(12)Kuper?CF，Koornstra?PJ，Hameleers?DM，Biewenga?J，Spit?BJ，Duijvestein?AM，van?Breda?Vriesman?PJ，Sminia?T.The?role?of?nasopharyngeal?lymphoid?tissue.Immunol.Today?199213：219-24
(13)Zuercher?AW，Coffin?SE，Thurnheer?MC，Fundova?P，Cebra?JJ.Nasal-associated?lymphoid?tissue?is?mucosal?inductive?site?for?virus-specific?humoral?and?cellular?immune?responses.J.Immunol.2002?168：1796-803
(14)Huckriede?A，Bungener?L，Stegmann?T，Daemen?T，Medema?J，Palache?AM，Wilschut?J.The?virosome?concept?for?influenza?vaccines.Vaccine?2005?23(Suppl?1)：S26-38
(15)Glück?R，Burri?KG，Metcalfe?I，Adjuvant?and?antigen?delivery?properties?of?virosomes.Curr.Drug?Deliv.20052：395-400
Embodiment
The LPP virus particle vaccine of embodiment 1 in the BALB/C mice in 8 ages in week; On the suboptimum HA dosage level, to the intranasal comparison of multiple HA/LPP ratio
The group (10 every group) that the negative female Balb/c mice of anti-influenza sera is formed is bestowed through intranasal and is obtained LPP (lipopeptid)-virus particle vaccine; Its HA/LPP ratio is 1: 1.5,1: 0.7,1: 0.4,1: 0 (promptly not having LPP), and every doses has the HA of 2 μ g.In addition, the matched group of 10 female mices is accepted 0 μ g HA/ dosage (intranasal of carrier is bestowed).
Preparation contains four kinds of prepared products of the virion of LPP.In brief, through centrifugal inactivating influenza virus in the 30-40% sucrose solution is precipitated.Again it is viral to suspend, and is dissolved in and contains in detergent eight Polyethylene Glycol monododecyl ethers (the Octaethylene glycol monododecyl ether) buffer (OEG).Subsequently, through the ultra centrifugal virus nucleocapsid body of removing.The supernatant that will contain OEG is divided into 4 aliquot volume; Add the not commensurability lipopeptid P3CSK4 (P3CSK4:N-palmityl-S-[2, two (Petiolus Trachycarpi acyl-oxygen)-(the 2RS)-propyl group of 3-]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine) in the OEG buffer that contains.With the buffer adjusted volume that contains OEG.Remove OEG on the hydrophobic resin through being adsorbed onto.This has caused containing the formation of the virion of LPP, a kind ofly in their films, contains HA and NA and (alternatively) contain the reconstruct of LPP in their films viral vesicle.After OEG removes, be the pvdf membrane filtration of 0.22 μ m with the virion via hole diameter.
Initiation material is the HA of 20mg, and it contains 252I.U. endotoxin/100 μ g HA from influenza A/Wyoming/3/2003X-147 (type A/Fujian/411/200 (H3N2) strain).After the dissolving, 4 batches of the preparations of summarizing according to table 1.
The preparation of table 1 virion
Carrier is made up of 5mM Hepes, 145mM NaCl, 1mM EDTA (pH 7.4).Concerning group E (seeing table 2), be the pvdf membrane filtration of 0.22 μ m with the carrier via hole diameter.Said according to table 2, to intranasal immunity, 4 batches of virions of preparation are diluted to the concentration of 200 μ g/ml, to the intramuscular immunity, be diluted to the concentration of 67 μ g/ml, divide the bottle (2 bottles every group) of putting into 1ml.Use these vaccine group according to what table 4 was summarized.
The preparation of table 2 vaccine
| Group number | Preparation |
| A | VIR-2004-11 |
| B | VIR-2004-12 |
| C | VIR-2004-13 |
| D | VIR-2004-14 |
| E | Carrier * |
*Carrier: via hole diameter is that 5mM Hepes, 145mM NaCl, the 1mM EDTA (pH 7.4) of the pvdf membrane of 0.22 μ m filters.
Analysis to preparation
The preparation that is used for this research to some kinds of variable analyses shown in the table 3.
Table 3 is for the analytical data of the virion that is used to prepare vaccine
aThe Lowry test, principle: after basic copper sulfate and the processing of Folin-ciocalteu phenol reagent, protein forms blueness.Use albumin BSA standard as a reference, measure protein content from the absorbance of 750nm.
Lowry，OH，NJ?Rosebrough，AL?Farr，and?RJ?Randall.J.Biol.Chem.193：265.1951.
Oostra，GM，NS?Mathewson，and?GN?Catravas.Anal.Biochem.89：31.1978.
Stoscheck，CM.Quantitation?of?Protein.Methods?in?Enzymology?182：50-69(1990).
Hartree，EF.Anal?Biochem?48：422-427(1972).
bPhEur: monograph 2053 and 2.7.1 joint
cPrinciple: each phospholipid contains single phosphorus atoms, and it can be used for phospholipid is carried out quantitatively.Destroy phospholipid through perchloric acid, through the phosphate that the molybdate complexation produces, molybdate is produced blue product by the ascorbic acid reduction.Measure color at the 812nm place with spectrophotometer.Quantitative in addition through comprising that the phosphoric acid caliberator comes the amount of phospholipid in the sample.
Ames?BN.Assay?of?inorganic?phosphate，total?phosphate?and?phosphatases.Meth.Enzymol.1966；8：115-118
CJF，van?Gent?CM?&?Pries?C.A?rapid?and?sensitive?sub-micro?phosphorus?determination.Anal.Chim.Acta?1961；24：203-204
dPh.Eur.2.6.14
eOvalbumin ELISA is direct sandwich style enzyme immunization method, and the fixed polyclone antiovalbumin antibody that wherein is used to catch and antiovalbumin-HRP conjugate are as detection system.Conjugate and sample are cultivated simultaneously.Remove unconjugated component through washing step.With substrate (TMB and H
2O
2) join in the hole.Come the existence of the bonded conjugate of specificity in the indicator hole through the colour developing of blueness.In substrate, add sulphuric acid and come cessation reaction, it causes, and change color is yellow in the product.Read absorbance (OD) at 450nm.For obtaining optimal result, use with reference to light filter at the 620nm place.The replying of ovalbumin standard (0.3-20.0ng/ml) that from test, comprises set up standard curve.The concentration of unknown sample is inserted in the standard curve and is read.
fAccording to monograph 0869 and 2053: the purity of checking (monovalent pooled) gleanings that unit price merges through PAGE.Electrophoresis: carry out according to Ph.Eur2.2.31.
Test macro
Test animal
Use seven treated animals, ten every group female Balb/c mices (BALB/cAnNCrl).
Handling at the beginning, mice is 8-9 age in week, and weight is 17-19g.
With unit price LPP virion influenza vaccines (A/Wyoming) animal is carried out intranasal vaccination the 0th day and the 14th day, carry out thanatopsy inoculating back 21 days for the second time.
Intranasal: slight isoflurane/O at the back
2/ N
2Intranasal vaccination test substances (10 μ l are divided on two nostrils and carry out) in the animal of O anesthesia.
Table 4 is handled and is arranged
| Group number | Bestow approach | Bacterin preparation | Female numbering | Number of animals |
| A | Intranasal | 2 μ g HA, the HA/LPP ratio is 1: 1.5 | 10 | 01-10 |
| B | Intranasal | 2 μ g HA, the HA/LPP ratio is 1: 0.7 | 10 | 11-20 |
| C | Intranasal | 2 μ g HA, the HA/LPP ratio is 1: 0.4 | 10 | 21-30 |
| D | Intranasal | 2 μ g HA, the HA/LPP ratio is 1: 0 | 10 | 31-40 |
| E | Intranasal | 2 μ g HA, the HA/LPP ratio is 0: 0 | 10 | 41-50 |
Before inoculation for the first time and after inoculating 14 days for the first time, at isoflurane/O
2/ N
2The eye socket blood sample is collected in O anesthesia down.The 35th day, put to death animal, collect blood sample (at O
2/ CO
2Anesthesia is down through abdominal aorta or cardiac puncture blood-letting).Collect from the serum of whole samples, cryogenic refrigeration is stored in the PA tube up to handling in being lower than-10 ℃.
Influenza virus makes red blood cell (RBCs) coagulation, and this can be prevented from when having enough special viral antibodies.This phenomenon provides the basis of hemagglutination inhibition (HI) test, this test be used to survey with quantitative serum in specific antiviral antibody.Serum is joined among influenza virus and the turkey RBCs.Some diluents are tested (analysis of tiring).HI tires and is defined as the inverse that still can suppress hemagglutinative high dilution.Come geometrical averages-were calculated tire (GMT) by hereinafter is said:
1) each log (tiring) is calculated, obtains multiple arithmetic mean of instantaneous value twice:
[log (tires
1)+log (tires
2)]/2
2) calculate the arithmetic mean of instantaneous value of each log (tiring)
3) GMT
(group)=10EXP (cell mean log (tiring))
Statistical analysis
Through inoculation group and natural law, use the geometric average titer to summarize HI and tire.The HI that analyzes the 35th day group that transforms through log through linear regression tires, with the amount of LPP and the dose response relationship between the GMT in the research vaccine.
The result
The HI analysis of tiring
GMT is shown in Table 5.
Table 5 geometric average titer
The 0th day, in mice, can not detect HA specific antibody (that is, all HI tire＜10).
The 14th day, in through the great majority in the mice of intranasal approach (i.n.) inoculation, can not detect the HA specific antibody.All tire≤and 10,80), (HI tires: 35) (HI's mice among the group C tires: 160) with organizing a mice among the D (HI's mice in group A tires:.
The 35th day, observed the aborning dose response of HA specific antibody, that is, in vaccine, add more LPP and cause higher antibody titer.
The HI that between group, relatively (changed through log) the 35th day through linear regression tires.The regression slope of match has highly significant difference (P＜0.0001).Therefore, LPP content in the observed vaccine and the dose response relationship between the GMT are though statistically significant.
Conclusion:
The reconstruct influenza virus particles of the no adjuvant of usefulness carries out the repetition intranasal vaccination to mice and does not induce the systemic immune response that can be measured to.With same HA dosage level (2 μ g HA/ dosage), the LPP dosage that adopt to rise has demonstrated LPP dose dependent immunne response with assistant with the repetition intranasal vaccination that the reconstruct influenza virus particles of LPP carries out.With the present invention fully opposite (embodiment 2 of face as follows); Be considered to support the generally acknowledged conclusion of this area before these data; Promptly; Using immunostimulant (LPP in this case) is necessary for carrying out intranasal vaccination with inactivated influenza vaccine, even influenza antigens (HA) is present in the reconstruct virion.
Embodiment 2 double blindings, at random, parallel group research, with the safety of research lipopeptid adjuvant and for the effect of virion subunit influenza vaccines curative effect (after intranasal transports in age>=18 and≤40 healthy young adult)
With contain every strain 150mcg HA/mL and 315mcg LPP/mL, help and to have the reconstruct influenza virus particles of LPP (lipopeptid) healthy human volunteer to be carried out intranasal vaccination with the dose volume (each nostril 0.1ml) of 0.2ml.Carry out intranasal vaccination with containing the dose volume (each nostril 0.1ml) of reconstruct influenza virus particles every strain 150mcg HA/mL, that do not have LPP to similar group with 0.2ml.The research purpose is the viewpoint of in the man, showing in the checking mice,, for after carrying out intranasal vaccination with inactivated influenza vaccine, obtaining satisfied systemic immune response, needs to use adjuvant (for example LPP) that is.
Research design:
This is double blinding, the parallel-group research of in the healthy young experimenter of age>=18 and≤40, carrying out at random.This research is carried out in a research center: Swiss Pharma Contract Ltd., Basel, SUI.Main researcher is doctor M.Seiberling.Research has two parts.In part I, the evaluation assistant has the safety of the virion subunit influenza vaccines of LPP in 12 experimenters.With the LPP-RVM (viromembrane of LPP reconstruct; Influenza vaccines-surface antigen, deactivation, virion-Zuo has LPP) nine experimenters of inoculation, with RVM (influenza vaccines-surface antigen, deactivation, virion-) inoculate three experimenters.In the part II of research, curative effect and the safety of (50 every group) evaluation LPP-RVM in 100 experimenters.
Research is carried out in the health volunteer.In addition, during research beginning the first three years, the experimenter who participates in the part II of research does not inoculate to influenza.This has increased study population's among the part II homogeneity through reduce to have the number to the experimenter of the antibody of influenza that is pre-existing in as far as possible.
Part I:
In preceding 14 days of inoculation (the 1st visit), after said experimenter has provided informed consent, screen he or she to comprising, and carry out physical examination he or she with exclusion standard.In this time gone to a doctor, the sample of gathering nasal epithelial cells was used for cytological analysis, tests with glucide and measures baseline cilium (cilia) activity.
Go to a doctor (the 1st day) at the 2nd time, get the 4-6mL blood sample, be used for the standard analysis of Hematology Changes, get the 6-10mL blood sample and be used for the standard biological chemical analysis, and the evaluation vital sign.Through behind the randomization, inoculate the experimenter with one of two kinds of bacterin preparations, the experimenter stopped first 24 hours on the spot after inoculation, to monitor part and systemic reaction and the adverse events that produces immediately.After inoculation the 4th with twenty four hours evaluation vital sign.In addition, after 24 hours, get two parts of blood samples and be used for standard hematology (4-6mL) and biochemistry (6-10mL) analysis; After the inoculation, the sample of gathering nasal epithelial cells is used for cytological analysis, inoculates back cilium activity analysis with the glucide test.Provide questionnaire (questionnaire I) to the experimenter, let them take home, evaluate part and the systemic reaction of next day (the 3rd day).
The experimenter must be released the back of going home at them and get back to the research point two weeks: the 3rd time go to a doctor (the 4th day).In this time gone to a doctor, evaluation part and systemic reaction, record was last time and this any spontaneous adverse events that takes place between going to a doctor.Two parts of blood samples of this treatment with external measures are used for standard hematology (4-6mL) and biochemistry (6-10mL) is analyzed, and the evaluation vital sign.
In two weeks of back of inoculation for the first time, at the 15th day, said experimenter got back to research point (the 4th visit).In this time gone to a doctor, the sample of collecting nasal epithelial cells was used for cytological analysis, and is active with glucide experimental measurement cilium, writes down the adverse events that takes place between the 3rd prescription on individual diagnosis and the 4th prescription on individual diagnosis.
Part II:
In preceding 14 days of blood sampling and nose washings (wash) sampling for the first time (the 1st visit), after said experimenter has provided informed consent, screen he or she to comprising with exclusion standard, detect his or her health through physical examination.
Went to a doctor (1 day at the 2nd time; This prescription on individual diagnosis can be carried out with the 1st merging of going to a doctor), get the 6-10mL blood sample and be used for baseline hemagglutination inhibition (HI) titration, and blood sampling carries out standard hematology (4-6mL) and standard biological chemistry (6-10mL) is analyzed.Collect nose washings sample and be used for establishment of base line nose IgA antibody titer.
Next day, the 3rd time go to a doctor (the 1st day), after evaluation, said experimenter is carried out randomization to vital sign, inoculate with the single dose of one of two kinds of preparations of nose influenza vaccines.In postvaccinal first hour, monitor any local response, systemic reaction and adverse events immediately on the spot.Afterwards, evaluate vital sign again, the experimenter obtains questionnaire and takes home, at postvaccinal first seven days, writes down part and the systemic reaction of every day.
Back (the 4th prescription on individual diagnosis of two weeks; The 15th day), get the 6-10mL blood sample and be used for the HI titration, get the outer blood sample of two shares and be used for standard hematology (4-6mL) and biochemistry (6-10mL) analysis, get nose washings sample and be used for the analysis of nose IgA antibody titer.
Effective evaluation
Be the evaluation effect, at the-1 day (baseline) and the 15th day collection blood sample and nose washings sample.
Blood sample
Collect 6-10mL blood, suppress (HI) antibody titer to measure hemagglutination.
1Blood collecting with solidify after (under the room temperature at least 30 minutes), separation of serum, and keep freezing (20 ℃) is up to the analysis of tiring.Repeat the antibody titer analysis with double.Tiring of sample is the geometrical mean of twice mensuration.The inoculation before with postvaccinal serum by the analysis of tiring simultaneously.
Nose washings sample
For collecting nose washings sample,, under rhinoscopy control, use the saline (37 ℃) of 6mL preheating in a nostril.Said experimenter is asked to make washing liquid to flow at the head 60 ° of angles of inclining.The washing liquid of collecting is applied to second nostril, and it is washed under identical condition.In sample, add listerine (sample volume 1/100).Listerine contains 10mg/ml and is dissolved in the bovine serum albumin in the 100mM Tris HCl buffer, and pH 8.Come directly clarification sample through low-speed centrifugal (800xg, 10 minutes), with its be divided into aliquot (with avoid from now on to sample repeat thaw), place it on the dry ice, advance-80 ℃ up to transfer.
Measure the IgA level in the nose washing sample through ELISA, test with Wilcoxon and carry out statistical analysis.Influenza vaccines are used as envelope antigen in 96 orifice plates.Through sealing nonspecific binding site with sealing buffer incubation.The nose washings with sealing buffer twice dilution (12 parts of diluents of every duplicate samples), is used for the influenza specific antibody is adsorbed onto the antigen of 96 orifice plates.With before the bonded anti-people's antibody of enzyme (being combined with horse horseradish peroxidase or the alkali phosphatase) incubation 96 orifice plates are being washed.Remove unconjugated anti-people's antibody through washing, measure the amount of influenza strain specific antibody through measuring light density after the substrate that is used for enzyme reaction in adding.
Bacterin preparation
Two kinds of different influenza vaccine formulations are used for this research.Two kinds of preparations all contain the virus antigen that WHO is Southern Hemisphere recommendation in 2005
2, its dosage level is the every strain 30mcg of the dosage of every 0.2ml.
——————————————
1 Palmer DF, Dowdle WR, Coleman MT, Schild GC. hemagglutination reaction suppresses experiment. be used for the advanced person's of influenza diagnosis laboratory technique .U.S.Dept.Hlth.Ed.Welfare, P.H.S.Atlanta; 1975:25-62
2 WHO recommend is used for the influenza virus vaccine compositions .Weekly Epidem Rec.2004 in influenza season in 2005; 79:369-376
The strain of-A/New Caledonia/20/99/ (H1N1) appearance
The strain of-A/Wellington/1/2004 (H3N2) appearance
The strain of-B/Shanghai/361/2002 appearance
In brief, through centrifugal inactivating influenza virus in the 30-40% sucrose solution is precipitated.Again it is viral to suspend, and is dissolved in and contains detergent, in the buffer of eight Polyethylene Glycol monododecyl ethers (OEG).Subsequently, through the ultra centrifugal virus nucleocapsid body of removing.Lipopeptid P3CSK4 with containing in the OEG buffer regulates the supernatant that contains OEG; Perhaps under the situation of the reconstruct viromembrane that does not contain LPP; Only regulate (P3CSK4:N-palmityl-S-[2, two (Petiolus Trachycarpi acyl-oxygen)-(the 2RS)-propyl group of 3-]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine) with the buffer that contains OEG.Remove OEG on the hydrophobic resin through being adsorbed onto.The formation of this viromembrane that has caused containing LPP or do not contain LPP (the reconstruct virus vesicle that in film, contains HA and NA and in film, contain LPP alternatively).After OEG removes, be the pvdf membrane filtration of 0.22 μ m with the virion via hole diameter.
For every kind of strain of virus, preparation has or does not have the independent prepared product (table 6) of LPP.The amount of the LPP that adds is corresponding to the HA/LPP ratio of 1: 0.7 (w/w).
The preparation of table 6 virion
| Batch | LPP | Strain |
| VIR-2005-09 | Exist | Influenza B/Jiangsu/10/2003 |
| VIR-2005-11 | Exist | Influenza A/New Caledonia/20/1999 IVR-116 reassortant |
| VIR-2005-13 | Exist | Influenza A/Wellington/1/2004IVR-139 reassortant |
| VIR-2005-10 | Do not exist | Influenza B/Jiangsu/10/2003 |
| VIR-2005-12 | Do not exist | Influenza A/New Caledonia/20/1999IVR-116 reassortant |
| VIR-2005-14 | Do not exist | Influenza A/Wellington/1/2004IVR-139 reassortant |
The analysis of table 7 pair preparation
aThe Lowry test, principle: after basic copper sulfate and the processing of Folin-ciocalteu phenol reagent, protein forms blueness.Use albumin BSA standard as a reference, measure protein content from the absorbance of 750nm.
Lowry，OH，NJ?Rosebrough，AL?Farr，and?RJ?Randall.J.Biol.Chem.193：265.1951.
Oostra，GM，NS?Mathewson，and?GN?Catravas.Anal.Biochem.89：31.1978.
Stoscheck，CM.Quantitation?of?Protein.Methods?in?Enzymology?182：50-69(1990).
Hartree，EF.Anal?Biochem?48：422-427(1972).
bPhEur: monograph 2053 and 2.7.1 joint
cPrinciple: each phospholipid contains single phosphorus atoms, and it can be used for phospholipid is carried out quantitatively.Destroy phospholipid through perchloric acid, through the phosphate that the molybdate complexation produces, molybdate is produced blue product by the ascorbic acid reduction.Measure color at the 812nm place with spectrophotometer.Quantitative in addition through comprising that the phosphoric acid caliberator comes the amount of phospholipid in the sample.
Ames?BN.Assay?of?inorganic?phosphate，total?phosphate?and?phosphatases.Meth.Enzymol.1966；8：115-118
CJF，van?Gent?CM?&?Pries?C.A?rapid?and?sensitive?sub-micro?phosphorus?determination.Anal.Chim.Acta?1961；24：203-204
dPh.Eur.2.6.14，
eOvalbumin ELISA is direct sandwich style enzyme immunization method, and the fixed polyclone antiovalbumin antibody that wherein is used to catch and antiovalbumin-HRP conjugate are as detection system.Conjugate and sample are cultivated simultaneously.Remove unconjugated component through washing step.With substrate (TMB and H
2O
2) join in the hole.Come the existence of the bonded conjugate of specificity in the indicator hole through the colour developing of blueness.In substrate, add sulphuric acid and come cessation reaction, it causes, and change color is yellow in the product.Read absorbance (OD) at 450nm.Be to obtain optimal result, use the 620nm place with reference to light filter.The ovalbumin standard (0.3-20.0ng/ml) that comprises replys the preparation standard curve from test.The concentration of unknown sample is inserted in the standard curve and is read.
fAccording to monograph 0869 and 2053: the purity of checking the gleanings that unit price merges through PAGE.Electrophoresis: carry out according to Ph.Eur 2.2.31.
Effect
Pass through Wilcoxon ' s rank summation and test bilateral significant level, comparing between two inoculation group the 15th day every kind of Strain through the HI antibody titer of log conversion and the 15th day nose IgA antibody titer with 0.05.
Also through every kind of Strain and each inoculation group are calculated the HI antibody titer that following three parameters are analyzed the 15th day.
-serum protective rate, wherein serum protection be defined as hemagglutination suppress (HI) tire >=40,
-seroconversion rate, wherein seroconversion be defined as that HI tires before the inoculation＜10, inoculation back HI tires >=40, perhaps, before the inoculation HI tire >=10 and tire at least 4 times increase of HI,
-average multiple increases, the promptly said HI geometrical mean that multiple increases of tiring.
Principle according to per-protocol and purpose treatment (intent-to-treat) is analyzed effect data.But in view of this is the research of so-called Proof-Of Principle type, it is basic a kind of that per-protocol analysis is considered to.The sample of purpose treatment is by being become by some inoculation back effect data sets of inoculation experimenter.The per-protocol sample is by having accomplished scheme and not taking place being formed by the inoculation experimenter of main scheme deviation.Main deviation is including but not limited to: comprise or the deviation of exclusion standard banning drugs use etc.In addition, the experimenter of the concurrent influenza infection of laboratory proofing and the experimenter that lost the basic effect data are also got rid of from the per-protocol sample.Research data base non-blind before, the decision whether the experimenter is got rid of from per-protocol.
The result
Whether table 8 decision is got rid of the experimenter from per-protocol.Carry out after the intranasal vaccination CHMP assessment with virion influenza vaccines (RVM) to HI
RVM: virion influenza vaccines
LPP-RVM: assistant has the virion influenza vaccines of lipopeptid
Table 8 carries out after the intranasal vaccination CHMP assessment (continuing) to HI with virion influenza vaccines (RVM)
RVM: virion influenza vaccines
LPP-RVM: assistant has the virion influenza vaccines of lipopeptid
Table 8 carries out after the intranasal vaccination CHMP assessment (continuing) to HI with virion influenza vaccines (RVM)
RVM: virion influenza vaccines
LPP-RVM: assistant has the virion influenza vaccines of lipopeptid
Table 9 nose washings IgA tire (GMT)
Conclusion
Unexpectedly and with same vaccine batch clinical before data (embodiment 1) and WO 04/110486 and described (the Gluck U of clinical data; Gebbers JO; Gluck R, Phase 1 evaluation of intranasal virosomal influenza vaccine with and without Escherichia coli heat-labile toxin in adult volunteers.J Virol.1999 Sep; 73 (9): 7780-6) opposite, in the human colony that the reconstruct virion influenza vaccines with no adjuvant only once inoculate, observed gratifying, meet systemic immune response to the CHMP standard of influenza vaccines.
Claims (11)
1. be used for single intranasal or inhalation and ability reactance influenza antigens hemagglutinin and/or the whole body of neuraminidase and/or the compositions of local immune response to the mankind; Said compositions comprises the influenza virus particles that the reconstruct peplos by said virus forms, wherein:
● said peplos obtains from influenza virus particles fully,
● do not add lipid to said reconstruct virion from external source,
● said virion comprises influenza hemagglutinin and/or neuraminidase,
● the dosage of the hemagglutinin of every kind of Strain of every single intranasal or inhalation is 5 μ g to 30 μ g,
It is characterized in that not in compositions, adding additional adjuvant and/or immunostimulant, and the single intranasal of wherein said preparation or inhalation can also the inducing cytotoxic lymphocyte responses.
2. according to the compositions of claim 1, wherein said immunne response meets the CHMP standard to influenza vaccines.
3. according to the compositions of claim 1 or 2; Wherein said immunne response provide following in one or more: for adult＞70% and/or for the serum protective rate of old people＞60%; Increase for adult＞40% and/or for the seroconversion rate of old people＞30% with for adult＞2.5 and/or for the average multiple of old people＞2.0.
4. the influenza virus particles that is formed by the reconstruct peplos of said virus is used for to the mankind's single intranasal or inhalation and can reactance influenza antigens hemagglutinin and/or the purposes of the compositions of the whole body of neuraminidase and/or local immune response in preparation; Said compositions comprises the influenza virus particles that the reconstruct peplos by said virus forms, wherein:
● said peplos obtains from influenza virus particles fully,
● do not add lipid to said reconstruct virion from external source,
● said virion comprises influenza hemagglutinin and/or neuraminidase,
● the dosage of the hemagglutinin of every kind of Strain of every single intranasal or inhalation is 5 μ g to 30 μ g,
It is characterized in that not in compositions, adding additional adjuvant and/or immunostimulant, and the single intranasal of wherein said preparation or inhalation can also the inducing cytotoxic lymphocyte responses.
5. according to the purposes of claim 4, wherein said immunne response meets the CHMP standard to influenza vaccines.
6. according to the purposes of claim 4 or 5; Wherein said immunne response provide following in one or more: for adult＞70% and/or for the serum protective rate of old people＞60%; Increase for adult＞40% and/or for the seroconversion rate of old people＞30% with for adult＞2.5 and/or for the average multiple of old people＞2.0.
7. according to purposes any in the claim 4 to 6, the compositions of wherein said preparation is a bacterin preparation.
8. comprise bacterin preparation according to compositions any in the claim 1 to 3.
9. the device that comprises said bacterin preparation according to Claim 8 and be used for the mechanism of said vaccine atomizing that is used for intranasal or inhalation.
10. according to the device of claim 9, wherein said device comprises a certain amount of bacterin preparation that is used for single intranasal or inhalation.
11. according to the device of claim 9 or 10, wherein said device is disposable.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP06111534 | 2006-03-22 | ||
| EP06111534.1 | 2006-03-22 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007800102179A Division CN101405027B (en) | 2006-03-22 | 2007-03-21 | Intranasal influenza vaccine based on virosomes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102580077A true CN102580077A (en) | 2012-07-18 |
Family
ID=36648714
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007800102179A Expired - Fee Related CN101405027B (en) | 2006-03-22 | 2007-03-21 | Intranasal influenza vaccine based on virosomes |
| CN2012100495363A Pending CN102580077A (en) | 2006-03-22 | 2007-03-21 | Intranasal influenza vaccine based on virosomes |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007800102179A Expired - Fee Related CN101405027B (en) | 2006-03-22 | 2007-03-21 | Intranasal influenza vaccine based on virosomes |
Country Status (6)
| Country | Link |
|---|---|
| CN (2) | CN101405027B (en) |
| HK (1) | HK1128077A1 (en) |
| MX (1) | MX2008012046A (en) |
| TW (1) | TWI397419B (en) |
| UA (1) | UA93236C2 (en) |
| ZA (1) | ZA200807880B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107375920A (en) * | 2017-06-23 | 2017-11-24 | 中农威特生物科技股份有限公司 | A kind of porcine reproductive and respiratory syndrome virus swine influenza virus reconstruct virosomes vaccine and its production and use |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0919243A1 (en) * | 1997-11-25 | 1999-06-02 | Duphar International Research B.V | Vaccine containing B subunits of heat-labile enterotoxin (LTB) of Escherichia coli as an adjuvant |
| TWI329130B (en) * | 2003-06-19 | 2010-08-21 | Bestewil Holding Bv | Functionally reconstituted viral membranes containing adjuvant |
-
2007
- 2007-03-19 TW TW096109319A patent/TWI397419B/en not_active IP Right Cessation
- 2007-03-21 CN CN2007800102179A patent/CN101405027B/en not_active Expired - Fee Related
- 2007-03-21 MX MX2008012046A patent/MX2008012046A/en active IP Right Grant
- 2007-03-21 UA UAA200812366A patent/UA93236C2/en unknown
- 2007-03-21 CN CN2012100495363A patent/CN102580077A/en active Pending
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2008
- 2008-09-12 ZA ZA200807880A patent/ZA200807880B/en unknown
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2009
- 2009-06-19 HK HK09105522.7A patent/HK1128077A1/en not_active IP Right Cessation
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107375920A (en) * | 2017-06-23 | 2017-11-24 | 中农威特生物科技股份有限公司 | A kind of porcine reproductive and respiratory syndrome virus swine influenza virus reconstruct virosomes vaccine and its production and use |
| CN107375920B (en) * | 2017-06-23 | 2020-08-11 | 中农威特生物科技股份有限公司 | Porcine reproductive and respiratory syndrome virus-swine influenza virus reconstituted virosome vaccine and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| TW200808345A (en) | 2008-02-16 |
| UA93236C2 (en) | 2011-01-25 |
| MX2008012046A (en) | 2009-01-22 |
| ZA200807880B (en) | 2009-09-30 |
| CN101405027A (en) | 2009-04-08 |
| CN101405027B (en) | 2013-01-02 |
| TWI397419B (en) | 2013-06-01 |
| HK1128077A1 (en) | 2009-10-16 |
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