HK1128077B

HK1128077B - Intranasal influenza vaccine based on virosomes

Info

Publication number: HK1128077B
Application number: HK09105522.7A
Authority: HK
Inventors: A．J．克斯坦; L．杰勒兹; P．J．舍恩; J．J．P．瑙塔; D．H．范蕾内科莱希思
Original assignee: 雅培生物学有限责任公司
Priority date: 2006-03-22
Filing date: 2007-03-21
Publication date: 2013-05-31

Description

Intranasal influenza vaccine based on virus particles

Technical Field

The present invention relates to compositions and modes of administration, e.g., for inactivated influenza vaccines, wherein a systemic immune response positively correlated to clinical protection is obtained by a single intranasal or inhalational administration.

Background

Various proposals for immunization against influenza by the intranasal or oropharyngeal routes and using inactivated influenza antigens have been investigated as non-injectable (needle-less) alternatives to subcutaneous or intramuscular immunization. Experimental data supporting non-injection approaches have been generated in animal models. The concept of using inactivated influenza antigens (e.g. chemically inactivated whole virions, or further processed viral components such as split viruses, or purified surface antigens Hemagglutinin (HA) and/or Neuraminidase (NA)) for immunization via the intranasal route, supported by animal data, includes: the use of an adjuvant or immunostimulator (immunostimulator) in combination with an inactivated influenza antigen, or multiple immunizations are required. An adjuvant is any substance that enhances the immunogenicity of the antigen with which it is mixed. In humans, successful vaccination against influenza by the intranasal route has only been reported: (a) live (cold-adapted strain) influenza vaccine (FluMist)^TMMedImmune Vaccines Inc) (references 1, 2, 3), (b) viral particle-type influenza vaccine supplemented with heat labile toxin of e.coli (e.coli) (NasalFlu, Berna Biotech Ltd) (reference 4) or (c) use of high dose of antigen and repeat vaccination (references 5, 10, 11). Although live vaccines can induce a satisfactory immune response, the specific nature of becoming a live virus poses additional safety concerns and may induce side effects due to the need for viral replication in the vicinity of the upper respiratory tract. Furthermore, the required storage conditions also limit the commercialization of these products. The strong correlation between intranasal influenza vaccines using e.coli HLT as adjuvant and facial paralysis (Bell's Palsy) led to the withdrawal from the market of virion vaccines adjuvanted with HLT (reference 6).

The efficacy of an influenza vaccine in a given population can be assessed by: an immunogenicity parameter associated with the amount of anti-influenza antibody produced after vaccination is assessed. These immunogenicity parameters are commonly referred to as CHMP standards, which are used for annual re-licensing certification of inactivated influenza vaccines (reference 7). To date, successful immunization of humans against influenza, meeting these immunological requirements or CHMP criteria (reference 7), by using a single intranasal administration of an inactivated vaccine, without the addition of adjuvants, which are additional components of the vaccine, which are not derived from the infectious agent to be prevented by the vaccine, and which are added to the vaccine formulation for the purpose of enhancing the immune response to the antigen, has not been described. Thus, it is recognized that there remains a need in the art for an inactivated influenza vaccine composition that induces a satisfactory systemic immune response after a single intranasal administration, that is free of adjuvant, and that meets CHMP criteria with the single administration (reference 7).

The "CHMP standard" is defined as follows. In the Note for Guidance on harmony of requisitions for Influenza Vaccines of chmp (committee for medical Products for Human use), the following serological parameters were defined to assess the immunogenicity of inactivated Influenza Vaccines:

a seroprotection rate, wherein seroprotection is defined as a Haemoglobin Inhibition (HI) titer ≥ 40,

seroconversion, wherein seroconversion is defined as pre-inoculation HI titre <10, post-inoculation HI titre >40, or pre-inoculation HI titre > 10 and at least a 4-fold increase in HI titre,

mean fold increase value, which is the geometric mean of the increase in subjects (i.e., post-vaccination/pre-vaccination HI titer).

CHMP requirements for influenza vaccine immunogenicity are: for each of the three virus strains in the vaccine, at least one of the following criteria is met:

standard of merit	Adults	Old people
			Protective rate of serum	>70％	>60％
Conversion rate of serum	>40％	>30％
			Mean multiple increment value	>2.5	>2.0

The invention is also applicable to children, from whom it has been shown that they mount an immune response in a manner comparable to that of adults (reference 8). The invention is also suitable for elderly subjects. Elderly people are over 60 years old.

Description of the invention

Surprisingly, contrary to preclinical rodent data and literature on human clinical experience, we found that the immune response of humans after a single intranasal vaccination with an inactivated influenza vaccine comprising reconstituted influenza virus envelopes met all three CHMP criteria for influenza vaccine efficacy for the age group of 18-60 years. One single intranasal administration was: to meet the CHMP criteria described above for immunogenicity of inactivated influenza vaccines, vaccination with the vaccine formulation is performed through one or both nostrils without repeated administration of the vaccine formulation. One single vaccine administration (by nasal, inhalation, oral, subcutaneous or intramuscular route) is typically the following vaccination schedule, which does not include: multiple administrations of vaccines at intervals of days or weeks are known in the art as priming and boosting. Formulations designed for intranasal or inhalational administration comprise a mixture of one or more active ingredients and excipients, and are prepared in a manner that permits intranasal or inhalational administration. The present invention provides a method for inducing a systemic immune response (circulating immunoglobulins or antibody-producing B cells) that meets the CHMP criteria, advantageously with a single intranasal or inhalational administration of a virosome influenza vaccine. The invention also provides a method for inducing a local or mucosal immune response comprising an increase in secreted immunoglobulins known as IgA at the mucosal membrane surface, advantageously with a single intranasal or inhalational administration of a virosome influenza vaccine. Induction of specific IgG and IgA responses following intranasal administration is related to the activity of lymphoid tissue in the nasal cavity (reference 12). Such tissues are known as nasal-associated lymphoid tissues (NALT), which have also been shown as mucosal induction sites for cellular immune responses (reference 13). Since viral particles are known to have the potential to induce an intracellular immune response (references 14, 15), the present invention also provides methods of inducing specific cytotoxic lymphocytes (CTLs).

Viral particles (virosomes) are lipid bilayers containing viral glycoproteins. Viral particles are typically produced by extracting membrane proteins and lipids from enveloped viruses with a detergent, followed by reconstitution of the characteristic bilayer by removal of the detergent. The invention also provides compositions of influenza virosomes comprising reconstituted influenza viral envelopes (in particular reconstituted without the addition of lipids and without the addition of immunomodulators of immunostimulants (commonly referred to as adjuvants)) for vaccination by aerosol administered to the mucosa of the naso-or oropharynx through one or both nostrils to achieve systemic and local immunity against influenza. A single administration by inhalation is also possible. Single oral mucosal administration is also possible.

Reconstituted influenza virus particles can be prepared from inactivated viruses, which can be solubilized with an opaque detergent that is removed by adsorption onto hydrophobic beads. The preparation may comprise a purified suspension of one or more influenza antigens selected from the group consisting of Hemagglutinin (HA), Neuraminidase (NA), derivatives of hemagglutinin and derivatives of neuraminidase. The viral membrane proteins haemagglutinin and neuraminidase can be reconstituted in a membrane composed of viral lipids (containing low levels of endotoxin and ovalbumin) (see reference 9). Derivatives of serum lectins and/or neuraminidases are hemagglutinin and/or neuraminidase molecules with modified amino acid sequences and/or structures. Amino acids may, for example, be deleted, substituted or added to the sequence. In addition, the glycosylation pattern can be altered. The derivatives retain the ability to induce an immune response when introduced into a host.

Influenza viruses used to prepare reconstituted viral particles can be cultured, for example, in eggs containing embryos, or in cell cultures of cells in adherent cells or suspensions. The virus may be, for example, a wild-type or a reassortant (reassortant) or genetically modified strain. The virus type may for example be any influenza a or B subtype, including epidemic strains.

The invention also provides vaccines. The term vaccine is to be understood as a pharmaceutical preparation with immunological activity. In certain embodiments, the vaccine may comprise harmless variants or derivatives of pathogenic microorganisms, for example, to stimulate the immune system to develop resistance against the actual pathogen. In certain embodiments, the vaccine, for example, induces adaptive immunity when administered to a host. Vaccines can contain killed or weakened forms of a pathogen or a component of a pathogen, such as an antigenic component of a pathogen. The vaccine preparation may also contain a pharmaceutical carrier which may be designed for the particular mode in which the vaccine is to be administered, for example for intranasal or inhalation administration. Influenza vaccines can comprise one or more undenatured influenza antigens, one or more of which can induce an influenza-specific immune response.

The present invention provides a composition comprising influenza virosomes comprising reconstituted envelopes of said virus, wherein the composition is designed for intranasal or inhalational administration. The invention also provides said composition wherein the viral particles comprise the influenza antigens haemagglutinin and/or neuraminidase or derivatives thereof. The invention also provides said composition wherein the viral envelope is entirely obtained from a virion. The invention also provides said composition wherein no lipid is added from an external source to the reconstituted viral particles. The invention also provides the composition wherein no separate adjuvant and/or immunostimulant is added to the composition. The invention also provides said composition wherein a single intranasal or inhalational administration to a subject induces a systemic immune response. The invention also provides said composition wherein a single intranasal or inhalational administration to a subject is also capable of inducing a local immune response. The invention also provides said composition which is also capable of inducing a cytotoxic lymphocyte response to a single intranasal or inhalational administration to a subject. The invention also provides said composition wherein the ability to induce a systemic immune response and/or a local immune response and/or a cytotoxic lymphocyte response is shown in humans. The invention also provides said composition wherein the immune response comprises an immune response against the influenza antigens haemagglutinin and/or neuraminidase or derivatives thereof. In a preferred embodiment, the invention also provides said composition wherein the immune response meets the CHMP criteria for influenza vaccines. The invention also provides the composition wherein the immune response provides one or more of: a seroprotection rate of > 70% for adults and/or > 60% for elderly, a seroconversion rate of > 40% for adults and/or > 30% for elderly, and an average fold increase of >2.5 for adults and/or >2.0 for elderly. In a particularly preferred embodiment, the invention also provides said composition wherein the dose of haemagglutinin per viral strain per intranasal or inhalational administration is equal to or lower than 30 μ g. Finally, the invention also provides a composition wherein the composition is a vaccine formulation comprising a pharmaceutical carrier for intranasal or inhalational administration.

The invention also provides the use of influenza virosomes comprising reconstituted envelopes of said viruses for the preparation of a composition for intranasal or inhalational administration. The invention also provides such a use: wherein the influenza virus particles comprise influenza antigens haemagglutinin and/or neuraminidase or derivatives thereof. The invention also provides such a use: wherein the viral envelope is obtained entirely from influenza virions. The invention also provides such a use: wherein no lipid is exogenously added to the reconstituted viral particles. The invention also provides such a use: wherein no separate adjuvant and/or immunostimulant is added to the composition. The invention also provides such a use: wherein a single intranasal or inhalational administration to the subject is sufficient to induce a systemic immune response. The invention also provides such a use: wherein a single intranasal or inhalational administration to the subject also induces a local immune response. The invention also provides such a use: wherein a single intranasal or inhalational administration to the subject also induces a cytotoxic lymphocyte response. The invention also provides such a use: wherein the subject receiving the administration is a human. The invention also provides such a use: the induced immune response comprises an immune response against the influenza antigens haemagglutinin and/or neuraminidase or derivatives thereof. In a preferred embodiment, the invention also provides such use, wherein the composition induces an immune response that meets the CHMP criteria for influenza vaccines. The invention also provides such a use wherein the immune response provides one or more of: a seroprotection rate of > 70% for adults and/or > 60% for elderly, a seroconversion rate of > 40% for adults and/or > 30% for elderly, and an average fold increase of >2.5 for adults and/or >2.0 for elderly. In a particularly preferred embodiment, the invention also provides such a use, wherein the administered dose of haemagglutinin per viral strain per intranasal or inhalational administration is equal to or lower than 30 μ g. Finally, the invention also provides said use wherein the composition prepared is a vaccine formulation.

Thus, in one embodiment, the invention provides a composition of influenza virosomes comprising a reconstituted envelope of said virus, wherein the viral envelope is obtained entirely from influenza virions, wherein no lipid is added from an external source to the reconstituted virions, wherein the viral particles comprise the influenza antigens haemagglutinin and/or neuraminidase or derivatives thereof, wherein no separate adjuvant and/or immunostimulant is added to the composition, and wherein the composition is designed as an intranasal or inhalational administration formulation, said composition being characterized in that a single intranasal or inhalational administration of said formulation to a human being is capable of inducing a systemic immune response and/or a local immune response against said influenza antigen, said systemic response being capable of complying with the CHMP criteria for influenza vaccines, and wherein the dose of haemagglutinin per viral strain per intranasal or inhalational administration is equal to or lower than 30 μ g.

According to another embodiment, the present invention provides the use of an influenza virosome comprising a reconstituted envelope of said virus, wherein said viral envelope is entirely obtained from influenza virosomes, wherein no lipid is added from an external source to the reconstituted virosome, wherein the virosome comprises the influenza antigens haemagglutinin and/or neuraminidase or derivatives thereof, wherein no separate adjuvant and/or immunostimulant is added to said composition, for the preparation of a composition for intranasal or inhalational administration, characterized in that a single intranasal or inhalational administration of said composition to a human is sufficient to induce a systemic and/or local immune response against said influenza antigens, said response being capable of complying with the CHMP standard for influenza vaccines, and wherein the dose of haemagglutinin per viral strain per intranasal or inhalational administration is equal to or lower than 30 μ g.

According to another embodiment, the invention provides a vaccine formulation comprising a composition of influenza virosomes comprising reconstituted envelopes of said viruses, wherein the viral envelopes are completely obtained from influenza virosomes, 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 immunostimulant is added to the composition, wherein the vaccine is characterized in that the vaccine is designed for a single intranasal or inhalational administration to a human, and wherein the dose of haemagglutinin per viral strain per intranasal or inhalational administration is equal to or lower than 30 μ g. Advantageously, said single intranasal or inhalational administration of said formulation is capable of inducing a systemic and/or local immune response in said human. There is also provided according to the invention a device comprising an amount of said vaccine formulation for a single intranasal or inhalational administration.

The dose of haemagglutinin according to the invention per viral strain per intranasal or inhalational administration may also be lower than or equal to 25 μ g, 20 μ g, 15 μ g, 10 μ g or 5 μ g.

Cited documents

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(2)Maassab HF.Bryant ML.The development of live attenuatedcold-adapted influenza virus vaccine for humans.Rev.Med.Virol.1999Oct-Dec；9(4)：237-44

(3)Keitel W，Piedra PA.Live cold-adapted，reassortant ininfluenza vaccines(USA).In：Textbook of Influenza.NicholsonKG，Webster RG，Hay AJ(Ed)，Blackwel Science Oxford，UK，373-390(1998)

(4)Gluck U，Gebbers JO，Gluck R，Phase 1 evaluation ofintranasal virosomal influenza vaccine with and withoutEscherichia coli heat-labile toxin in adult volunteers.J Virol.1999Sep；73(9)：7780-6

(5)Samdal HH，Bakke H，Of tung F，Holst J，Haugen IL，Korsvold GE，Kristoffersen AC，Krogh G，Nord K，Rappuoli R，Berstad AKH，Haneberg B，Anon-Living Nasal Influenza VaccineCan Induce Major Humoral and Cellular Immune Responses in Humanswithout the Need for Adjuvants.Human Vaccines 1:2，85-90；March/April2005

(6)Mutsch M，Zhou W，Rhodes P，Bopp M，Chen RT，Linder T，Spyr C，Steffen R.Use of the inactivated intranasal influenzavaccine and the risk of Bell’s palsy in Switzerl and.N EnglJ Med，2004Feb26；350(9)：896-903

(7)Note for Guidance on Harmonisation of Requirements forInfluenza Vaccines.EMEA/CpMP/BWP/214/96

(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 influenzasubunit vaccine(Influvac^R)in high-risk children aged 6 monthsto 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 reconstitutionof influenza viris envelopes.EMBO Journal1987，6(9)：2651-2659

(10)Treanor J，Nolan C，O’Brien D，Burt D，Lowell G，LindenJ，Fries L.Intranasal administration of a proteosome-influenzavaccine is well-tolerated and induces serum and nasal secretioninfluenza antibodies in healthy human subjects.Vaccine2006；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 nasalinfluenza vaccine delivery using chitosan.Vaccine2005；23(35)：4367-74

(12)Kuper CF，Koornstra PJ，Hameleers DM，Biewenga J，SpitBJ，Duijvestein AM，van Breda Vriesman PJ，Sminia T.The roleof nasopharyngeal lymphoid tissue.Immunol.Today199213：219-24

(13)Zuercher AW，Coffin SE，Thurnheer MC，Fundova P，CebraJJ.Nasal-associated lymphoid tissue is mucosal inductive sitefor virus-specific humoral and cellular immune responses.J.Immunol.2002168：1796-803

(14)Huckriede A，Bungener L，Stegmann T，Daemen T，MedemaJ，Palache AM，Wilschut J.The virosome concept for influenzavaccines.Vaccine200523(Suppl 1)：S26-38

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Examples

Example 1 LPP virion vaccine in 8 week old BALB/C mice; intranasal comparison of various HA/LPP ratios at suboptimal HA dose levels

Groups of influenza seronegative female Balb/c mice (10 per group) were given an LPP (lipopeptide) -virosome vaccine by intranasal administration with HA/LPP ratios of 1:1.5, 1:0.7, 1:0.4, 1:0 (i.e. no LPP) and 2 μ g HA per dose. In addition, a control group of 10 female mice received 0 μ g HA/dose (intranasal administration of vehicle).

Four preparations of LPP-containing viral particles were prepared. Briefly, inactivated influenza virus in a 30-40% sucrose solution was precipitated by centrifugation. The virus was resuspended and dissolved in a buffer containing the detergent octapolyethylene glycol monododecyl ether (OEG). Subsequently, the viral nucleocapsid was removed by ultracentrifugation. The supernatant containing OEG was divided into 4 aliquots and different amounts of lipopeptide P3CSK4(P3CSK 4: N-palmitoyl-S- [2, 3-bis (palmitoyloxy) - (2RS) -propyl ] - [ R ] -cysteinyl- [ S ] -seryl- [ S ] -lysyl- [ S ] -lysine) in OEG-containing buffer were added. The volume was adjusted with OEG containing buffer. The OEG was removed by adsorption onto a hydrophobic resin. This results in the formation of LPP-containing viral particles, a reconstituted viral vesicle containing HA and NA in their membrane and (optionally) LPP in their membrane. After OEG removal, the virus particles were filtered through a PVDF membrane with a pore size of 0.22 m.

The starting material was 20mg of HA from influenza A/Wyoming/3/2003X-147 (A/Fujian/411/200 (H3N2) strain) containing 252I.U. endotoxin/100. mu.g HA. After dissolution, 4 batches were prepared as outlined in table 1.

TABLE 1 preparation of viral particles

Batches of	Amount of HA (mg) as starting Material	Amount of P3CSK4 added (mg)	HA/LPP ratio
				VIR-2004-11	5	7.5	1：1.5
VIR-2004-12	5	3.5	1：0.7
				VIR-2004-13	5	2.0	1：0.4
VIR-2004-14	5	0	1：0

The vector consisted of 5mM Hepes, 145mM NaCl, 1mM EDTA (pH 7.4). For group E (see Table 2), the support was filtered through a PVDF membrane having a pore size of 0.22. mu.m. Batches of 4 prepared virus particles were diluted to a concentration of 200. mu.g/ml for intranasal immunization and 67. mu.g/ml for intramuscular immunization and dispensed into 1ml vials (2 vials per group) as described in Table 2. These vaccine groups were used as outlined in table 4.

TABLE 2 preparation of vaccines

Group number	Preparation of
		A	VIR-2004-11
B	VIR-2004-12
		C	VIR-2004-13
D	VIR-2004-14
		E	Carrier^*

^*Carrier: filtration through 5mM Hepes, 145mM NaCl, 1mM EDTA (pH7.4) of a PVDF membrane having a pore size of 0.22. mu.m.

Analysis of the formulations

The formulations used in this study were analyzed for several variables shown in table 3.

TABLE 3 analytical data for the virus particles used for the preparation of vaccines

Analyte	VIR-2004-11	VIR-2004-12	VIR-2004-13	VIR-2004-14
					Protein (mg/ml)^a	1.7	1.6	1.5	1.4
HA(μg/ml)^b	776	759	697	757
					Phospholipids (mmol/l)^c	0.658	0.692	0.658	0.682
Endotoxin (I.U per 100. mu.g HA)^d	3.1	1.5	1.9	1.0
					Ovalbumin (μ g of HA per 100 μ g)^e	0.047	0.050	0.055	0.050
Purity of^f	Mainly HA	Mainly HA	Mainly HA	Mainly HA

^aLowry test, principle: after treatment with basic copper sulfate and Folin-ciocalteu phenol reagent, the protein developed a blue color. Protein content was determined from absorbance at 750nm using albumin BSA standard as reference.

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 inEnzymology182：50-69(1990).

Hartree，EF.Anal Biochem48：422-427(1972).

^bPhEur: section 2053 and 2.7.1 of the topical articles

^cThe principle is as follows: each phospholipid contains a single phosphorus atom, which can be used to quantify the phospholipids. Phospholipid destruction by perchloric acid, phosphate generation by molybdate complexationThe molybdate is reduced by ascorbic acid to produce a blue colored product. The color was determined with a spectrophotometer at 812 nm. The amount of phospholipid in the sample was quantified by including a phosphate calibrator.

Ames BN.Assay of inorganic phosphate，total phosphate andphosphatases.Meth.Enzymol.1966；8：115-118

Bttcher CJF，van Gent CM & Pries C.A rapid and sensitivesub-micro phosphorus determination.Anal.Chim.Acta1961；24：203-204

^dPh.Eur.2.6.14

^eOvalbumin ELISA is a direct sandwich enzyme immunoassay method using immobilized polyclonal anti-ovalbumin antibody for capture and an anti-ovalbumin-HRP conjugate as the detection system. The combination and the sample are incubated simultaneously. Unbound components are removed by a washing step. The substrates (TMB and H)₂O₂) Added to the wells. The presence of specifically bound binder in the wells is indicated by the development of blue color. Sulfuric acid is added to the substrate to stop the reaction, which results in a color change in the product to yellow. Absorbance (OD) was read at 450 nm. For optimal results, a reference filter was used at 620 nm. A standard curve was established from the responses of ovalbumin standards (0.3-20.0ng/ml) included in the experiment. The concentration of the unknown sample is read by interpolation from the standard curve.

^fAccording to the topical articles 0869 and 2053: the purity of the monovalent pooled (monovalent pooled) pools was checked by polyacrylamide gel electrophoresis. Electrophoresis: performed as ph. eur 2.2.31.

Test system

Test animals

Seven groups of animals were used, ten female Balb/c mice per group (BALB/cAnNCrl).

At the beginning of the treatment, the mice were 8-9 weeks old and weighed 17-19 g.

Animals were vaccinated intranasally with monovalent LPP virion influenza vaccine (a/Wyoming) on days 0 and 14, and necropsies were performed 21 days after the second vaccination.

Intranasal: mild isoflurane/O on the back₂/N₂O-anesthetized animals were inoculated intranasally with the test substance (10 μ l, performed in two nostrils).

TABLE 4 processing arrangement

Group number	Administration route	Vaccine formulations	Female numbering	Animal numbering
					A	In nose	2 ug HA/LPP ratio of 1:1.5	10	01-10
B	In nose	2 ug HA/LPP ratio of 1:0.7	10	11-20
					C	In nose	2 ug HA/LPP ratio of 1:0.4	10	21-30
D	In nose	2 ug HA/LPP ratio of 1:0	10	31-40
					E	In nose	2 ug HA/LPP ratio of 0:0	10	41-50

Before and 14 days after the first inoculation, at isoflurane/O₂/N₂Orbital blood samples were collected under O-anesthesia. On day 35, animals were sacrificed and blood samples were collected (at O)₂/CO₂Bleeding under anesthesia through the abdominal aorta or cardiac puncture). Serum from all samples was collected, deep frozen, and stored in polypropylene tubes at below-10 ℃ until processing.

Influenza viruses agglutinate Red Blood Cells (RBCs), which are prevented in the presence of sufficient virus-specific antibodies. This phenomenon provides the basis for a Hemagglutination Inhibition (HI) assay, which is used to detect and quantify specific anti-viral antibodies in serum. Serum was added to influenza virus and turkey RBCs. Several dilutions were tested (titer analysis). HI titer was defined as the reciprocal of the highest dilution that still inhibited hemagglutination. Geometric Mean Titers (GMTs) were calculated as follows:

1) the log (titer) was calculated for each replicate to give the arithmetic mean of the two replicates:

[ log (potency)₁) + log (potency)₂)]/2

2) Calculate the arithmetic mean of the respective logs (titers)

3)GMT_(group)10EXP (group mean log (titer))

Statistical analysis

HI titers were summarized by inoculation and day number using geometric mean titers. The log transformed day 35 groups were analyzed for HI titers by linear regression to investigate the dose response relationship between the amount of LPP and GMT in the vaccine.

Results

HI potency assay

GMT is shown in table 5.

TABLE 5 geometric mean Titers

Group of	Administration route	HA/LPP ratio	Day 0	Day 14	Day 35
						A	In nose	1:1.5	5	8	415
B	In nose	1:0.7	5	6	161
						C	In nose	1:0.4	5	7	97
D	In nose	1:0	5	7	12
						E	In nose	0:0	5	5	5

On day 0, no HA-specific antibodies could be detected in mice (i.e., all HI titers < 10).

On day 14, HA-specific antibodies could not be detected in the majority of mice vaccinated by the intranasal route (i.n.). All titers were <10, except one mouse in group A (HI titer: 80), one mouse in group C (HI titer: 35) and one mouse in group D (HI titer: 160).

On day 35, a dose response in HA-specific antibody production was observed, i.e. adding more LPP to the vaccine resulted in higher antibody titers.

HI titers at day 35 (log transformed) were compared between groups by linear regression. The fitted regression slopes were highly significantly different (P < 0.0001). Thus, the dose-response relationship between LPP content and GMT observed in the vaccine was statistically significant.

And (4) conclusion:

repeated intranasal vaccinations of mice with reconstituted influenza virus particles without adjuvant did not induce measurable systemic immune responses. Repeated intranasal vaccinations with reconstituted influenza virus particles adjuvanted with LPP at the same HA dose level (2 μ g HA/dose) with ascending LPP dose showed LPP dose-dependent immune responses. In stark contrast to the present invention (see example 2 below), these data were previously believed to support the art-recognized conclusion that the use of an immunostimulant (LPP in this case) is necessary for intranasal vaccination with inactivated influenza vaccine, even if influenza antigen (HA) is present in the reconstituted viral particles.

Example 2 double-blind, randomized, parallel group study to investigate the safety of lipopeptide adjuvants and their efficacy for virion subunit influenza vaccine therapy (after intranasal delivery in healthy young adults aged 18 and 40 or more)

Healthy human volunteers were inoculated intranasally with reconstituted influenza virus particles containing 150mcg HA/mL and 315mcg LPP/mL per strain, supplemented with LPP (lipopeptide), in a dose volume of 0.2mL (0.1 mL per nostril). Similar groups were inoculated intranasally with reconstituted influenza virus particles containing 150mcg HA/mL per strain without LPP in a dose volume of 0.2mL (0.1 mL per nostril). The aim of the study was to verify in men the view shown in mice, i.e. to obtain a satisfactory systemic immune response after intranasal vaccination with an inactivated influenza vaccine, the use of an adjuvant (e.g. LPP) was required.

Research and design:

this was a double-blind, randomized, parallel group study in healthy young subjects aged 18 and 40. The study was conducted in a research center: swiss Pharma contiract ltd, basell, switzerland. The main investigator was doctor m. The study has two parts. In section I, the safety of LPP-adjuvanted virion subunit influenza vaccines was assessed in 12 subjects. Nine subjects were vaccinated with LPP-RVM (LPP reconstituted viral membrane; influenza vaccine-surface antigen, inactivated, virosome-adjuvanted with LPP) and three subjects were vaccinated with RVM (influenza vaccine-surface antigen, inactivated, virosome-). In part II of the study, efficacy and safety of LPP-RVM was assessed in one hundred subjects (50 per group).

The study was performed in healthy subjects. Furthermore, none of the subjects participating in part II of the study were vaccinated against influenza during the three years before the study began. This increased the homogeneity of the study population in part II by minimizing the number of subjects with pre-existing antibodies to influenza.

Part I:

at 14 days prior to vaccination (visit 1), after the subject has given informed consent, he or she is screened for inclusion and exclusion criteria and subjected to physical examination. In this visit, a sample of nasal epithelial cells was collected for cytological analysis, and the saccharin assay was used to measure baseline ciliary (cilia) activity.

At visit 2 (day 1), 4-6mL blood samples were taken for standard hematological analysis, 6-10mL blood samples were taken for standard biochemical analysis, and vital signs were assessed. After randomization, subjects were vaccinated with one of the two vaccine formulations and left in situ for the first 24 hours after vaccination to monitor the immediate local and systemic responses and adverse events. Vital signs were assessed at the fourth and twenty-four hours post inoculation. In addition, after 24 hours, two blood samples were taken for standard hematology (4-6mL) and biochemical (6-10mL) analysis; after inoculation, samples of nasal epithelial cells were collected for cytological analysis and post-inoculation ciliary activity analysis was performed using the saccharin assay. Subjects were issued a questionnaire (questionnaire I) and allowed to go home to assess local and systemic responses the following day (day 3).

Subjects must return to the study site two weeks after they are released home: visit 3 (day 4). During this visit, local and systemic responses were assessed and any spontaneous adverse events that occurred between the previous and current visits were recorded. Additionally, two blood samples were taken for standard hematology (4-6mL) and biochemistry (6-10mL) analysis and vital signs were assessed.

Two weeks after the first vaccination, on day 15, the subjects returned to the study point (visit 4). In this visit, samples of nasal epithelial cells were collected for cytological analysis, ciliary activity was measured using the saccharin assay, and adverse events occurring between visit 3 and visit 4 were recorded.

Part II:

14 days before the first blood draw and nasal eluate (wash) sampling (visit 1), the subject was screened for inclusion and exclusion criteria after he or she had given informed consent, and his or her health was checked by physical examination.

At visit 2 (-1 day; this visit may be combined with visit 1), 6-10mL blood samples were taken for baseline Hemagglutination Inhibition (HI) titer determination, and blood samples were taken for standard hematological (4-6mL) and standard biochemical (6-10mL) analyses. Nasal eluate samples were collected for determination of baseline nasal IgA antibody titers.

On the following day, at visit 3 (day 1), after assessment of vital signs, the subjects were randomized and vaccinated with a single dose of one of the two formulations of the nasal influenza vaccine. During the first hour after inoculation, any immediate local reactions, systemic reactions and adverse events were monitored in situ. Thereafter, vital signs were re-assessed and subjects were taken home on a questionnaire and daily local and systemic responses were recorded the first seven days after vaccination.

Two weeks later (visit 4; day 15), 6-10mL blood samples were taken for HI titer determination, two aliquots of external blood samples were taken for standard hematology (4-6mL) and biochemical (6-10mL) analysis, and nasal eluate samples were taken for nasal IgA antibody titer analysis.

Evaluation of effects

To assess the effect, blood samples and nasal eluate samples were collected on day-1 (baseline) and day 15.

Blood sample

6-10mL of blood was collected to determine Hemagglutination Inhibition (HI) antibody titers.¹After blood collection and coagulation (at least 30 minutes at room temperature), the serum was separated and kept frozen (-20 ℃) until titer analysis was performed. Antibody titer analysis was performed in duplicate. The titer of the sample is the geometric mean of the two determinations. Sera before and after inoculation were simultaneously subjected to titer analysis.

Nasal wash sample

To collect a nasal eluate sample, 6mL of pre-heated saline (37 ℃) was applied to one nostril under nasoscopy control. The subject was asked to tilt the head at an angle of 60 ° so that the wash solution could flow. The collected wash solution was applied to the second nostril, which was washed under the same conditions. To the sample was added a preservative solution (1/100 sample volume). The preservative solution contained 10mg/ml bovine serum albumin dissolved in 100mM Tris HCl buffer, pH 8. The sample was clarified directly by low speed centrifugation (800Xg, 10 minutes), divided into small portions (to avoid repeated thawing of the sample in the future), placed on dry ice until transferred to-80 ℃.

IgA levels in nasal wash samples were determined by ELISA and statistical analysis was performed using the Wilcoxon test. Influenza vaccines were used as coating antigens in 96-well plates. Through the reaction with a blocking buffer solution IIncubation is initiated to block non-specific binding sites. The nasal eluate was buffered with blocking bufferFold dilutions (12 dilutions per sample) were used to adsorb influenza specific antibodies to antigens on 96-well plates. The 96-well plates were washed prior to incubation with enzyme-bound anti-human antibodies (conjugated with horse radish peroxidase or alkaline phosphatase). Unbound anti-human antibody was removed by washing, and the amount of influenza strain-specific antibody was determined by measuring optical density after adding a substrate for enzyme reaction.

Vaccine formulations

Two different influenza vaccine formulations were used in this study. Both formulations contained viral antigens recommended by WHO for the 2005 southern hemisphere²The dose level was 30mcg per strain per 0.2ml dose.

-A/New Caledonia/20/99/(H1N1) sample strain

A/Wellington/1/2004(H3N2) -like strain

-B/Shanghai/361/2002-like Strain

Briefly, inactivated influenza virus in a 30-40% sucrose solution was precipitated by centrifugation. The virus was resuspended and dissolved in a buffer containing detergent, octapolyethylene glycol monododecyl ether (OEG). Subsequently, the viral nucleocapsid was removed by ultracentrifugation. The supernatant containing OEG was conditioned with lipopeptide P3CSK4 in OEG-containing buffer or, in the case of reconstituted viral membranes without LPP, only OEG-containing buffer (P3CSK 4: N-palmitoyl-S- [2, 3-bis (palmitoyloxy) - (2RS) -propyl ] - [ R ] -cysteinyl- [ S ] -seryl- [ S ] -lysyl- [ S ] -lysine). The OEG was removed by adsorption onto a hydrophobic resin. This results in the formation of LPP-containing or LPP-free viral membranes (reconstituted viral vesicles containing HA and NA in the membrane and optionally LPP in the membrane). After OEG removal, the virus particles were filtered through a PVDF membrane with a pore size of 0.22 μm.

For each strain of virus, separate preparations with or without LPP were prepared (table 6). The amount of LPP added corresponds to a HA/LPP ratio of 1:0.7 (w/w).

TABLE 6 preparation of viral particles

Batches of	LPP	Viral strains
			VIR-2005-09	Exist of	Influenza B/Jiangsu/10/2003
VIR-2005-11	Exist of	Influenza A/New Caledonia/20/1999IVR-116 reassortant strain
			VIR-2005-13	Exist of	Influenza A/Wellington/1/2004 IVR-139 reassortant strain
VIR-2005-10	Is absent from	Influenza B/Jiangsu/10/2003
			VIR-2005-12	Is absent from	Influenza A/New Caledonia/20/1999IVR-116 reassortant strain
VIR-2005-14	Is absent from	Influenza A/Wellington/1/2004 IVR-139 reassortant strain

Table 7 analysis of the formulations

Analyte	VIR-2005-09	VIR-2005-10	VIR-2005-11	VIR-2005-12	VIR-2005-13	VIR-2005-14
							Protein (mg/ml)^a	1.51	1.54	1.86	1.83	1.37	1.18
HA(μg/ml)^b	805	854	711	784	704	644
							Phospholipids (mmol/l)^c	0.494	0.563	0.820	1.03	0.717	0.695
Endotoxin (I.U per 100. mu.g HA.)^d	<0.4	<0.4	<0.4	<0.4	<0.4	<0.5
							Ovalbumin (μ g of HA per 100 μ g)^e	0.068	0.088	0.037	0.036	0.132	0.126
Purity of^f	Mainly HA	Mainly HA	Mainly HA	Mainly HA	Mainly HA	Mainly HA

Oos tra，GM，NS Mathewson，and GN Catravas.Anal.Biochem.89：31.1978.

Stoscheck，CM.Quantitation of Protein.Methods inEnzymology182：50-69(1990).

Hartree，EF.AnalBiochem48：422-427(1972).

^bPhEur: section 2053 and 2.7.1 of the topical articles

^cThe principle is as follows: each phospholipid contains a single phosphorus atom, which can be used to quantify the phospholipids. The phospholipids are destroyed by perchloric acid, the molybdate is reduced by ascorbic acid to produce a blue colored product by the phosphate produced by molybdate complexation. The color was determined with a spectrophotometer at 812 nm. The amount of phospholipid in the sample was quantified by including a phosphate calibrator.

^dPh.Eur.2.6.14，

^eOvalbumin ELISA is a direct sandwich enzyme immunoassay method using immobilized polyclonal anti-ovalbumin antibody for capture and an anti-ovalbumin-HRP conjugate as the detection system. The combination and the sample are incubated simultaneously. Unbound components are removed by a washing step. The substrates (TMB and H)₂O₂) Added to the wells. The presence of specifically bound binder in the wells is indicated by the development of blue color. Sulfuric acid is added to the substrate to stop the reaction, which results in a color change in the product to yellow. Absorbance (OD) was read at 450 nm. For optimal results, a reference filter at 620nm was used. A standard curve was prepared from the response of ovalbumin standards (0.3-20.0ng/ml) included in the experiment. The concentration of the unknown sample is read by interpolation from the standard curve.

^fAccording to the topical articles 0869 and 2053: the purity of the monovalent pooled harvest was checked by polyacrylamide gel electrophoresis. Electrophoresis: performed as ph. eur 2.2.31.

Effect

Log transformed HI antibody titers for each strain at day 15 were compared to nasal IgA antibody titers at day 15 between the two vaccinated groups by Wilcoxon's sum of rank test at a two-sided significance level of 0.05.

Day 15 HI antibody titers were also analyzed by calculating the following three parameters for each strain and each vaccinated group.

-seroprotection rate, wherein seroprotection is defined as the erythrocyte coagulation inhibition (HI) titer 40,

seroconversion, wherein seroconversion is defined as pre-inoculation HI titre <10, post-inoculation HI titre >40, or, pre-inoculation HI titre 10 and at least a 4-fold increase of HI titre,

-mean fold increase, i.e. geometric mean of the fold increase in the HI titer.

The effect data was analyzed according to per-protocol and intent-to-treat (intent-to-treat) principles. However, per-protocol analysis is considered to be an essential one in view of this being a so-called proof-of-principle type of study. The samples of the intended treatment consisted of some post-vaccination efficacy data for the vaccinated subjects. per-protocol samples consisted of vaccinated subjects who completed the protocol and did not develop major protocol deviations. Major deviations include (without limitation): inclusion or exclusion of standard deviation, drug use inhibition, and the like. In addition, laboratory validated subjects with concurrent influenza infection as well as subjects who lost essential efficacy data were also excluded from per-protocol samples. Before the study database was non-blind, it was decided whether to exclude the subject from per-protocol.

Results

Table 8 decides whether to exclude the subject from per-protocol. CHMP assessment of humoral immune response following intranasal vaccination with virosome influenza vaccine (RVM)

RVM: virosome influenza vaccines

LPP-RVM: lipopeptide adjuvanted virosome influenza vaccines

TABLE 8 CHMP assessment of humoral immune response following intranasal vaccination with virosome influenza vaccine (RVM)

RVM: virosome influenza vaccines

LPP-RVM: lipopeptide adjuvanted virosome influenza vaccines

RVM: virosome influenza vaccines

LPP-RVM: lipopeptide adjuvanted virosome influenza vaccines

TABLE 9 nasal washes IgA titres (GMT)

Conclusion

Surprisingly and in contrast to the preclinical data obtained with the same vaccine batches (example 1) and described in WO04/110486 and clinical data (Gluck U, Gebbers JO, GluckR, Phase 1 evaluation of Intra viromal inhibition with vaccenium vacutaine with and without Escherichia coli heat-lipid toxin in adultvolenteers. J Virol. 199Sep; 73 (9): 7780-6), a satisfactory systemic immune response was observed in the human population vaccinated only once with the unadjuvanted reconstituted virosome influenza vaccine.

Claims

1. A composition for single intranasal or inhalational administration to humans and capable of inducing a systemic and/or local immune response against influenza antigens haemagglutinin and/or neuraminidase, said composition comprising influenza virosomes formed from reconstituted envelopes of influenza viruses, wherein:

● the viral envelope is obtained entirely from influenza virions,

● no lipid is added from an external source to the reconstituted viral particles,

● the viral particle comprises influenza haemagglutinin and/or neuraminidase,

● the dose of haemagglutinin per virus strain per single intranasal or inhalational administration is 5 to 30 mug,

characterized in that no adjuvant and/or immunostimulant is added to the composition, respectively meaning that no such additional component is added to the composition: it does not originate from an infectious agent to be prevented with the composition and is added to the composition for the purpose of enhancing the immune response to an antigen;

and wherein the immune response provides one or more of: seroprotection > 70% for adults 18 to 40 years of age, seroconversion > 40% for adults 18 to 40 years of age, and a mean fold increase >2.5 for adults 18 to 40 years of age.

2. The composition according to claim 1, wherein the immune response meets the CHMP criteria for an influenza vaccine.

3. Use of influenza virosomes formed from reconstituted envelopes of influenza virus in the manufacture of a composition for single intranasal or inhalational administration to humans and capable of inducing a systemic and/or local immune response against the influenza antigens haemagglutinin and/or neuraminidase, said composition comprising influenza virosomes formed from reconstituted envelopes of said virus, wherein:

● the viral envelope is obtained entirely from influenza virions,

● the viral particle comprises influenza haemagglutinin and/or neuraminidase,

and wherein the immune response provides one or more of: for 18 to 40

Seroprotection rate > 70% for adults aged, and > 40% for blood from 18 to 40 years old

Clear conversion, and a mean fold increase of >2.5 for adults 18 to 40 years of age.

4. Use according to claim 3, wherein the immune response meets the CHMP criteria for influenza vaccines.

5. Use according to claim 3 or 4, wherein the composition prepared is a vaccine formulation.

6. A vaccine formulation comprising a composition according to claim 1 or 2.

7. A device for intranasal or inhalational administration comprising said vaccine formulation according to claim 6 and a mechanism for nebulization of said vaccine.

8. The device according to claim 7, wherein the device comprises an amount of the vaccine formulation for a single intranasal or inhalational administration.

9. The device according to claim 7 or 8, wherein the device is disposable.