TW200808345A - Intranasal or inhalational administration of virosomes - Google Patents

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 on 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 on 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 pg. The invention also provides the use of reconstituted influenza virosomes for the manufacture of said composition, and accordingly manufactured vaccine formulations.

Description

200808345 IX. OBJECTS OF THE INVENTION: FIELD OF THE INVENTION The present invention relates to, for example, a composition for inactivating an influenza vaccine and a 5-administration method in which a single intranasal or inhalation administration is obtained. Clinical protection is a positively related systemic immune response. [Prior Art] Background of the Invention • Various concepts for immunizing influenza by intranasal or oropharyngeal route, as well as the use of inactivated influenza antigen needle 10, have been used as a non-injection (needle-less) replacement for subcutaneous or intramuscular immunization. The pathway was studied. Experimental data supporting non-injection methods have been generated in animal models. Obtaining animal data-supported use of inactivated influenza antigens (eg, chemically inactivated vims particles, or further processed viral components, such as viruses that are cleaved, or purified surface antigen blood cells) The idea of lectin (HA) and/or divine-alaninase (NA) to immunize by intranasal route includes: using a rumo adjuvant or an immunostimulant (immime stimulator) in combination with inactivated influenza antigen, or Multiple immunizations. An adjuvant is any substance that enhances the immunogenicity of an antigen to be mixed therewith. In humans, successful vaccination against influenza 2 by intranasal routes has only been reported: (a) live (cold-modified strain) influenza vaccine (FluMistTM, Medlmmime Vaccines Inc) (Refs. 1, 2, 2) 3), (b) Viral granule influenza vaccine with heat labile toxin of E. coli (NasalFlu, Berna Biotech)

Ltd) (Reference 4) or (c) High dose antigen and repeated vaccination (Ref. 5 200808345, references 5, 10, 11). Although live vaccines can induce a satisfactory immune response, the specific nature of their ability to become live viruses leads to additional safety concerns that may trigger side effects due to the need for viral replication in the vicinity of the upper respiratory tract. In addition, the required storage conditions also limit the commercialization of these products. 5 The strong correlation between the intranasal flu vaccine with the large fat cup HLT as an adjuvant and the Bell's Palsy resulted in the withdrawal of the HLT-containing viral particle vaccine from the market (Reference 6). The efficacy of influenza vaccines in a given population can be assessed by assessing the immunogenicity of the number of anti-influenza antibodies produced after vaccination. These immunogenic parameters are commonly referred to as the CHMP standard, which is used for annual re-licensing of inactivated influenza vaccines (Reference 7). So far, by using a single intranasal administration of an inactivated vaccine, no adjuvant (which is an additional component in the vaccine, which is not derived from an infectious agent to be prevented by a vaccine) and is intended to enhance the antigen The purpose of the immune response was added to the 15 vaccine formulation), and the successful immunization against human influenza against these immunological requirements or the CHMP standard (Reference 7) has not been documented. Thus, it is recognized that there remains a need in the art for inactivated influenza vaccine compositions that are capable of inducing a satisfactory systemic immune response after a single intranasal administration, and without adjuvants. And the CHMP standard can be achieved by the single administration of 20 drugs (Reference 7). The "CHMP standard" is defined as described below. In the Note for Guidance on Harmonisation of Requirements for Influenza Vaccines of CHMP (Committee for Medicinal Products for Human Use), the following serological parameters are defined to assess the immunogenicity of the inactivated 200808345 influenza vaccine. Among them, seroprotection is defined as Haemagglutination Inhibition (HI) titer > 40, - seroconversion rate, wherein seroconversion is defined as m titer 5 < 10 before inoculation, HI titer after inoculation > 40 , or, before vaccination ΉΙ potency > 1 〇 and m valence at least 4 times increase, - average multiple increase value, which is the geometric mean of the increase within the individual (ie 'HI after vaccination / before vaccination hi potency). The CHMP requirement for the immunogenicity of influenza vaccines is that for each of the 10 vaccines, at least one of the following criteria is met:

Average multiple increase value > 2.5 The present invention is also applied to children, from which they have shown that they make an immune response in a manner comparable to that of an adult (Reference 8). This issue (4) is applied to elderly individuals. The elderly are over 60 years old. 15 SUMMARY OF THE INVENTION Description of the Invention 乂 Surprisingly, and contrary to preclinical rodent data and literature on human clinical experience, we have found that for age-old, inactivated influenza containing a reconstituted influenza virus envelope After the vaccine was vaccinated within 20 weeks, the human immune response reached the three CHMP criteria for influenza vaccine = two. - The single intranasal administration is: in order to achieve the above-mentioned CHMP standard for the immunogenicity of the inactivated influenza vaccine, the inoculation of the vaccine formulation is carried out in the nostrils of the Tong, Shang-, Qian, Qian, k or one of the 7 200808345 There is no need to repeatedly administer the vaccine formulation. - Single single dose administration (by nasal, inhalation, oral, subcutaneous or muscle 7 routes) is generally the following vaccination schedule, which does not include: known in the art as primary (pnmmg) and boosting, Multiple doses of the vaccine were administered at intervals of 5 days or weeks. Formulations designed for intranasal or inhalation formulation include a mixture of one or more active ingredients and excipients which are prepared for intranasal or inhalation administration. The present invention provides a method for inducing a systemic immune response (circulating immunoglobulin or antibody-producing B cells) that meets the CHMP standard, advantageously, the method 10 uses a single intranasal injection of a viral particle influenza vaccine Or by inhalation administration. The present invention also provides a method for inducing local or mucosal immunity, wherein the response comprises an increase in secretory immunoglobulin known as IgA on the surface of the membrane, advantageously using the virus against influenza virus A single intranasal or inhaled administration of the field is carried out. Induction of specific 15 kG and IgA responses following intranasal administration involves lymphoid activity in the nasal cavity (Ref. 12). Such tissues are known as nasal associated lymphoid tissue (NALT), which is also not shown to be a mucosal induction site for cellular immune responses (Ref. 13). Since virus particles are known to have the possibility of inducing an intracellular immune response (References 14, 15), the present invention also provides a method of inducing specific cell 20 toxic lymphocytes (CTL). A viral particle is a lipid bilayer containing a viral glycoprotein. Viral particles are produced by extracting membrane proteins and lipids from the enveloped virus with a detergent, followed by removal of the characteristic bilayer by removal of the detergent. The invention also provides a composition of influenza virus particles comprising a reconstituted influenza 8 200808345 viral outer membrane (specifically, no lipids are added, and immunomodulation without an immunostimulant (commonly referred to as an adjuvant) Reconstituted with the agent) for inoculation by aerosol, which is administered to the mucosa of the nasopharynx or oropharynx through one or both nostrils to obtain systemic and local immunity against influenza. A single administration by inhalation into 5 rows is also possible. A single oral mucosal administration is also feasible. The reconstituted influenza virus particles can be prepared from the inactivated virus, and the inactivated virus can be dissolved with a non-dialyzable detergent to remove the detergent by adsorption onto the hydrophobic beads. The preparation may comprise a purified suspension of one or more influenza antigens selected from the group consisting of hemagglutinin 10 (HA), neuraminidase (NA), a derivative of hemagglutinin and a neuraminidase derivative. The viral membrane proteins hemagglutinin and neuraminidase can be reconstituted in a membrane composed of viral lipids (containing low levels of endotoxin and ovalbumin) (see Reference 9). The derivative of serum lectin and/or neuraminidase is a hemagglutinin 15 and/or neuraminidase molecule having a modified amino acid sequence and/or structure. Amino acids can, for example, be deleted, replaced or added to the sequence. In addition, the glycosylation pattern can be altered. Derivatives retain the ability to induce an immune response when introduced into a host. The 20 influenza virus used to prepare the reconstituted virus particles can be cultured in, for example, an egg containing an embryo, or a cell culture of cells in an attached cell or suspension. The virus may, for example, be a wild-type or reass〇rtant or a genetically modified strain. The type of virus can be, for example, any influenza A*B subtype, including epidemic strains. The invention also provides a vaccine. The term vaccine is to be understood as a pharmaceutical preparation having immunological activity. In certain embodiments, the vaccine may comprise a non-hazardous variant or derivative of the pathogenic 9 200808345 microorganism, for example, stimulating the immune system to produce resistance to a real pathogen. In certain embodiments, the vaccine is administered, e.g., to induce adaptive immunity. The vaccine may contain a dead or weakened form of the pathogen or pathogen component, such as an antigenic component of the pathogen. The vaccine 5 preparation may further comprise a pharmaceutical carrier which may be designed for the particular mode in which the vaccine will be administered, e.g., a pharmaceutical carrier designed for intranasal or inhalation administration. The influenza vaccine may comprise one or more undenatured influenza antigens, one or more of which induce an immune response specific for influenza. The present invention provides a composition comprising influenza virus particles comprising a reconstituted outer shell of the virus, wherein the composition is designed to be administered intranasally or by inhalation formulation. The present invention also provides the composition wherein the virus particles comprise influenza antigen hemagglutinin and/or neuraminidase or a derivative thereof. The invention also provides such compositions wherein the viral envelope is completely obtained from viral particles. The present invention also provides such compositions in which no lipids are added from 15 external sources to the reconstituted viral particles. The present invention also provides such compositions in which no separate adjuvant and/or immunostimulant is added to the composition. The invention also provides such compositions wherein a single intranasal or inhaled administration to an individual induces a systemic immune response. The present invention also provides such compositions wherein a single intranasal or inhalation administration to an individual can also induce a local ankle immune response. The present invention also provides that the group of sigma cumin, which also induces a cytotoxic lymphocyte response in a single vr or inhalation to an individual, also provides a systemic immune response and/or a local immune response. / or the ability of a cytotoxic lymphocyte response to show the composition in humans. The present invention also provides such a composition wherein the immune response comprises an immune response against 10 200808345 Sonic antigen hemagglutinin and/or neuraminidase or a derivative thereof. In a preferred embodiment, the invention also provides said, wherein the immune response meets the cHMp criteria for influenza vaccines. The present invention also provides a composition in which the immune response provides one or five of the following compositions, the seroprotection rate of the adult and the oral 60/〇 for the elderly, far Ten pairs of adults. And/or 30% blood conversion rate for elderly people, and an average multiple increase value for adults > 2.5 and/or for elderly people > 2.0. In a particularly preferred embodiment I! X Mingtong provides the composition in which the blood lignin agent 1 of each of the virus strains is intranasally or inhaled to give 10 drugs equal to or less than % Μ. Most preferably, the composition is a composition wherein the composition is a vaccine formulation comprising a pharmaceutical carrier for intranasal or inhaled administration. The invention also provides the use of influenza virus particles comprising a reconstituted outer shell of a virus for use in compositions for intranasal or inhaled administration. The invention also provides the use of wherein the influenza virus particle comprises influenza antigen hemagglutinin and/or neuraminidase or a derivative thereof. The present invention also provides a k-like material: its towel, the viral outer shell is completely obtained from influenza virus particles. The invention also provides such use wherein no lipid is added to the reconstituted viral particle from an external source. The present invention also provides such use as described above, wherein no separate adjuvant and/or immunostimulant is added to the composition. The present invention also provides such a use wherein, intranasal or inhalation administration to an individual is sufficient to induce a systemic immune response. The use of the present invention is also provided by the present invention in which a single intranasal or inhalation administration to an individual induces a local immune response. The invention also provides the use of such 11 200808345: complex

The single intranasal or inhaled administration of cytotoxic cytotoxic pf' to an individual also induces fine, in response to a cell response. The invention also provides such use: The individual of the drug of use is a human. The present invention also provides such a fine/guided domain response containing a flu (4) hemaginary agglutinin and/or a fascinating (four) free money answer. In a preferred embodiment, the induction is also provided by the use wherein the composition provides an immune response to the CHMP standard for influenza vaccines. The invention further employs the use, wherein the immune response provides a composition of the following: a composition of the species: > 70% for adults and/or for the elderly. Serum protection rate of 6 〇 / ° for adults > 40% and / or for the elderly. The blood π conversion rate, as well as the average fold increase for adults > 2.5 and / or for senile sputum. In a particularly preferred embodiment, the present invention provides such a use in which the hemagglutinin of each viral virulence or inhaled is administered at a dose equal to or lower.

At 3 〇. Finally, the invention also provides said use wherein the composition produced is a vaccine formulation. Accordingly, in one embodiment, the invention provides a composition comprising an influenza virus surface of a reconstituted outer shell of a virus, wherein the viral outer shell is obtained entirely from influenza virions, wherein no reconstitution from an external source is made 20 virus particles are added to the lipid, wherein the viral particles comprise influenza antigen hemagglutinin and/or neuraminidase or a derivative thereof, wherein no separate adjuvant and/or immunostimulant is added to the composition, and Wherein the composition is designed as an intranasal or inhaled formulation, the composition being characterized in that a single intranasal or inhaled administration of the formulation to humans induces 12 200808345 resistance to the influenza antigen. A systemic immune response and/or a local immune response that is consistent with the CHMP criteria for influenza vaccines, and wherein the hemagglutinin dose per intranasal or inhaled dose of each viral strain is equal to or low At #g. 5 According to another Beth mode, the present invention provides reconstruction including viruses

The use of a shell of influenza virus particles for the manufacture of a composition for intranasal or inhalation administration, wherein the viral envelope is obtained entirely from influenza virions, wherein no lipids are added to the reconstituted viral particles from an external source, Wherein the viral particle comprises influenza antigen hemagglutinin and/or neuraminidase or a 10 derivative thereof, wherein no separate adjuvant and/or immunostimulant is added to the composition, the influenza virus particle is used for The use of a composition for the manufacture of intranasal or inhaled administration is characterized in that a single intranasal or inhalation of the composition to a human is sufficient to induce a systemic immune response and/or local immunity against the influenza antigen. In response, the response can achieve the CHMP criteria for influenza vaccines, and wherein the hemagglutinin dose of each viral strain administered intranasally or by inhalation is equal to or lower than 30/zg. 20 According to another embodiment, a combination of influenza virus particles (4) a vaccine formulation, which is difficult to contain a reconstituted outer shell of the virus, wherein the virus is obtained by obtaining a virus, and wherein There is no reconstitution of patient lipids from external sources, where 'viral age contains domain antigen hepatocytes (tetra) and/or neuraminidase or derivatives thereof, wherein no separate adjuvant and/or immunization is added to the composition A stimulant, wherein the vaccine is characterized in that a single intranasal or inhaled administration of a human is designed to design a vaccine 'and its towel, each viral strain 13 200808345, each intranasal or inhaled hemagglutinin The dose is equal to or lower than 3〇# g. Advantageously, said single intranasal or inhaled administration of said formulation formulation induces a systemic and/or local immune response in said human. Also provided in accordance with the invention is an apparatus comprising a quantity of the vaccine formulation formulation for single intranasal or inhalation administration. The hemagglutinin administered intranasally or by inhalation of each virus strain may also be used at a dose lower than or equal to 25^8, 20//g, 15, 〇#g or 5 vg according to the present invention. Citation 10 (1) Maassab HF. Adaptation and growth characteristics of influenza virus at 25〇C, Nature 213, 612-14 (1967) (2) Maassab HF. Bryant ML. The development of live attenuated cold-adapted influenza virus Vaccine for humans.

Rev.Med.Virol. 1999 Oct-Dec;9(4):237-44 15

(3) Keitel W? Piedra PA. Live cold-adapted, reassortant in influenza vaccines (USA). In: Textbook of Influenza. Nicholson KG, Webster RG, Hay AJ (Ed), Blackwel Science Oxford, UK, 373-390 ( 1998) (4) Gluck U, Gebbers JO, Gluck R, Phase 1 evaluation 20 of intranasal virosomal influenza vaccine with and without Escherichia coli heat-labile toxin in adult volunteers. J Virol. 1999 Sep; 73(9): 7780-6 (5) Samdal HH, Bakke H, Oftung F, Holst J, Haugen IL, Korsvold GE, Kristoffersen AC, Krogh G, Nord K, Rappuoli 14 200808345 R, Berstad AKH, Haneberg B, Anon-Living Nasal Influenza Vaccine Can Induce Major Humoral and Cellular Immune

Responses in Humans without the Need for Adjuvants. • Human Vaccines 1:2? 85-90; March/April 2005 - 5 (6) Mutsch M, Zhou W, Rhodes P, Bopp M, Chen RT,

Linder T, Spyr C, Steffen R. Use of the inactivated intranasal influenza vaccine and the risk of Bell 's palsy in Switzerland. N Engl J Med? 2004 Feb 26;350(9):896-903 Lu (7) Note for Guidance on Harmonisation of 10 Requirements for Influenza Vaccines. EMEA/CpMP/B WP/214/96 15

20 (8) Daubeney, P., Taylor, CJ·, McGaw, J., Brown, Brown, EM, Ghosal, S., Keeton, BR., 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, HWM·, Booy, FP·, Van Breemen, JFL·, Scherphof, G_, Wilschut, J. Functional reconstitution of influenza viris envelopes. EMBO Journal 1987, 6(9): 2651-2659 (10) Treanor J5 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 15 200808345 human subjects. Vaccine 2006; 24(3): 2 54-62 (11) Read RC·, Naylor SC· Potter CW·, Bond J” Jabbal-Gill I·, Fisher A., Ilium L·, Jennings R· Effective nasal influenza vaccine delivery using chitosan. Vaccine 5 2005; 23(35): 4 367- 74

(12) Kuper CF? Koornstra PJ? Hameleers DM? Biewenga J? Spit BJ9 Duijvestein AM? van Breda Vriesman PJ? Sminia T. The role of nasopharyngeal lymphoid tissue. Immunol. Today 1992 13:219-24 10 (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? Bimgener L? Stegmann T9 Daemen T? 15 Mederna J? Palache AM, Wilschut J. The virosome concept for influenza vaccines. Vaccine 2005 23(Suppl 1): S26-38 (15) Gliick R? Burri KG, Metcalfe I, Adjuvant and antigen delivery properties of virosomes. Curr. Drug Deliv. 2005 2:395-400 20 [Embodiment] Example 1 LPP virus particle vaccine in 8 week old BALB/C mice; intranasal comparison of various HA/LPP ratios to influenza sera negative at suboptimal HA dose level Group of female Balb/c mice (1 group per group) passed 16 200808345 Intranasal administration of LPP (lipopeptide)-viral vaccines with a HA/LPP ratio of 1:1.5, 1:0.7, 1 : 0.4, 1: 〇 (ie no LPP) 'and each dose has 2 / / g of HA. In addition, a control group of 10 female mice obtained a dose of 〇#gHA/' (intranasal administration of the vehicle). • 5 Preparation of four preparations of LPP-containing viral particles. Briefly, the 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 octaethylene glycol monododecyl ether (OEG). Subsequently, the viral nucleocapsid was removed by ultracentrifugation. The #0 containing OEG supernatant was divided into 4 equal volumes, and different amounts of lipopeptide P3CSK4 containing OEG buffer 10 were added (P3CSK4: N-palmium-S-[2,3-double (palm oxime H2RS) )-propyl]-[R]-cysteine-[S]-serine-[S]-lysine_[S]_lysine-[S]-lysine-[S] -Lysine). Adjust the volume with OEG-containing buffer. The OEG is removed by adsorption onto a hydrophobic resin. This results in the formation of LPP-containing viral particles containing HA and NA in their membranes and (optionally) 15 reconstituted viral vesicles containing LPP in their membranes. After the OEG was removed, the virus particles were filtered through a PVDF membrane having a pore size of 0.22 / / m. _ Starting material is 20 mg of HA from influenza A/Wyoming/3/2003 X-147 (class A/Fujian/411/200 (H3N2) strain) containing 252 LU·endotoxin/100 HA. After dissolution, prepare 4 batches according to 20 as outlined in Table 1. Table 1 Preparation of virus particles Batch of HA as starting material (mg) 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 17 200808345 The vector consisted of 5 mM Hepes, 145 mM NaCh 1 mM EDTA (pH 7 4). For group E (see Table 2), the vector was filtered through a PVDF membrane with a pore size of 〇22"m. The 4 batches of virus particles prepared were diluted to 200//g for intranasal immunization as described in Table 2. The concentration of ml, for intramuscular immunization, was diluted to "g/ml concentration and dispensed into 1 ml vials (2 bottles per group). These vaccine sets were used as outlined in Table 4. Table 2 Preparation of vaccines No. Preparation A VIR-2004-11 B VIR-2004-12 C VIR-2004-13 D VIR-2004-14 E Vector* 1 mM EDTA (pH 7.4)

10 Analysis of Formulations The formulations used in this study were analyzed for several variables shown in Table 3. Table 3 Analytical data for virus particles used to prepare 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 ΗΑ (β g/ml)b 776 759 697 757 Stone resin (mmol/l)c 0.658 0.692 0.658 0.682 Endotoxin (IU per 100 /zg HA) d 3.1 1.5 1.9 1.0 Ovalbumin (per 100 μg HA // g)e 0.047 0.050 0.055 0.050 Purity f Mainly HA Mainly HA Mainly HA Mainly HA Lowry test, principle: use domain type sulfuric acid j _*Folin-cioc; alteu regules after L treatment, egg to 4 eggs 2 content

Lowry, OH, NJ Roseb

Rosebrough, AL Farr, and RJ Randall. J. Biol. Chem. 193:265. 1951. 15

Biochem. 89: 31. 1978.

Moscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990). Hartree, EF. Anal Biochem 48: 422-427 (1972) 18 20 200808345 bPhEur: Feature Articles 2053 and 2·7·1 Section e Principle: Each phospholipid contains a single phosphorus atom that can be used to quantify phospholipids. The phospholipid is destroyed by perchloric acid, and the 5 phosphate/ester produced by the combination of molybdate, the molybdate is reduced by ascorbic acid to produce a blue product. The color was measured at 812 nm using a spectrophotometer. The amount of phospholipid in the sample is quantified by including a dish acid calibration.

Ames BN. Assay of inorganic phosphate, total phosphate and phosphatases. Meth. EnzymoL 1966; 8:115-118 10 Bottcher CJF5 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 6 Ovalbumin ELISA is a direct sandwich enzyme immunization method in which 15 immobilized polyclonal anti-ovalbumin antibodies and anti-printing proteins are used for capture - The HRP conjugate is used as a detection system. The conjugate and the sample are simultaneously cultured. Unbound components are removed by a washing step. The substrate (TMB and 〇2) was added to the wells. The presence of a conjugate that specifically binds in the well is indicated by the color development of the blue. Sulfuric acid was added to the substrate to terminate the reaction, which resulted in a yellow color change in the product. The absorbance (OD) was read at 450 nm. For best results, use a reference filter at 62〇. A standard curve was prepared from the response of the ovalbumin standard (〇.3-2〇 ng/ml) included in the test. The concentration of the unknown sample is interpolated from the standard curve. 19 200808345 f According to feature articles 0869 and 2053: the purity of the monovalent pooled collection was examined by polypropylene gel electrophoresis. Electric swimming: Follow Ph.Eur 2.2.31. Test System - 5 Test Animals Seven groups of animals, each group of ten female Balb/c mice (BALB/cAnNCrl) were used. At the beginning of the treatment, the mice were 8-9 weeks old and weighed 17-19 g. # On-day vaccination of animals with monovalent LPP virion vaccine 10 (A/Wyoming) on day 0 and day 14 and necropsy 21 days after the second vaccination. The intranasal test substance was inoculated intranasally (10//1, divided into two nostrils), which was performed on animals anesthetized with mild isoflurane/〇2/N2〇 on the back. Table 4 treatment arrangement group number administration route A intranasal B intranasal C intranasal D intranasal ~ E intranasal 2 / / 11 18, 11 eight, 1 ^? ratio is 〇: 〇 vaccine formulation 2 # hydroxy 11 Eight '11 eight, 〇>? ratio is 1:0.7 01-10 11-20

41·50 15 2 Private Palace only eight, 11 eight, 〇 ^ ratio is 1:1" Before the first vaccination and 14 days after the first vaccination, collect blood samples under isoflurane / ο2 / ν2 o anesthesia . On the 35th day, the animals were sacrificed and blood samples were collected (Blood by abdominal aorta or cardiac puncture under 〇2/C〇2 anesthesia)

Serum from all samples was collected, deep-cold, and placed in a poly-20 tube until it was processed. The K flu virus agglutinates red blood cells (10)Cs), which is prevented in the presence of sufficient disease 20 200808345 toxic specific antibodies. This phenomenon provides the basis for a hemagglutination inhibition (HI) assay that is used to detect and quantify specific antiviral antibodies in serum. Serum was added to influenza virus and turkey RBCs. Several dilutions were tested (potency analysis). Η丨 valence is defined as the reciprocal of the highest dilution that still inhibits 5 hemagglutination. Calculate the geometric mean titer (GMT) as described below: 1) Calculate each log (potency) to get the arithmetic mean of two replicates: [log (potency ο+log (potency 2)] /2 10 2) Calculate the arithmetic mean of each log (potency) 3) GMT (group) = 10 EXP (group mean l〇g (potency)) Statistical analysis by vaccination group and number of days, using geometric mean Potency to summarize the price of Hi sister. The HI titer of the log transformed day 35 group was analyzed by linear regression to study the dose response relationship between the amount of LPP in the vaccine and GMT. Results HI titer analysis GMTtf is shown in Table 5. Table 5 Geometric mean titer group Administration route HA/LPP ratio Day 0 Day 14 Day A Intranas 1:1.5 5 8 B Intranas 1:0.7 5 6 161^ C Intranas 1:0.4 5 7 D Nasal Within 1:0 5 7 E Intranasal 0:0 5 5 On day 0, HA-specific antibodies were not detected in mice (ie, all 21 200808345 HI titers < 10). On day 14, no HA-specific antibodies were detected in most of the mice vaccinated by the intranasal route (i.n.). The effective price of 10, except for one mouse in group A (HI titer: 80), one mouse in group C (HI titer: 35) * 5 and one mouse in group D (HI titer: 160). On day 35, a dose response in the production of HA-specific antibodies was observed, i.e., the addition of more LPP to the vaccine resulted in higher antibody titers. The HI effect price on day 35 of the log-transformed comparison between groups by linear regression. A suitable regression slope is highly significant (P < 0.0001). Therefore, the dose-response relationship between the LPP content and the GMT in the vaccine of 10 was observed to be statistically significant. Conclusion: Repeated intranasal inoculation of mice with unadjuvanted reconstituted influenza virus particles does not induce a systemic immune response that can be measured. Lpp dose-dependent immune response was demonstrated by repeated intranasal vaccination with Lpp-reconstituted influenza virus particles at the same HA dose of water 15 (2//g HA/dose) with increasing lpp dose . Contrary to the present invention (see Example 2 below), these data were previously considered to support the accepted conclusions in the art that the use of immunostimulants (in this case LPP) for intranasal inactivated influenza vaccines 20 vaccination is necessary even if the influenza antigen (HA) is present in the reconstituted virus particles. Example 2 A double-blind, randomized, parallel group study to study the safety of lipopeptide adjuvants and their efficacy against the efficacy of viral particle subunit influenza vaccines (intranasal in healthy young adults of age - 丨8 and Φ40) After shipment) 22 200808345 Reconstituted influenza virus particles containing LPP (lipopeptide) containing 150 meg HA/mL and 315 meg LPP/mL per strain in a dose volume of 0.2 ml (1 ml per nostril) Intranasal inoculation of healthy human volunteers. A similar group was intranasally inoculated with a reconstituted influenza virus particle containing 150 meg HA/mL per strain at a dose volume of 5 0.2 ml (each nostril ι·ι mi). The aim of the study was to validate the view expressed in mice in men that an adjuvant (e.g., LPP) is required to obtain a satisfactory systemic immune response after intranasal vaccination with an inactivated influenza vaccine. #研究设计: 10 This is a double-blind, randomized, parallel-group study of healthy young individuals of age >18 and S40. The study was conducted at a research center: Swiss Phamia Contract Ltd. Basel, Switzerland. The researcher of the principle is Dr. Seiberlmg. The study has two parts. In the section, the virus particles with LPP are evaluated in 12 individuals. Safety of subunit influenza vaccine. Inoculate nine individuals with 15 LPP_RVM (LPP-reconstituted viral membrane; influenza vaccine_surface antigen, inactivated 'viral particles-with LPP), using RVM (flu vaccine_ ® surface antigen Inactivated, viral particles·) to inoculate three individuals. In the part of π of the study, the efficacy and safety of Lpp_RVM was assessed in one hundred individuals (5 per group). 20 Study in healthy individuals In addition, in the three years prior to the start of the study, some of the individuals involved in the study did not vaccinate against the flu. This increased the number of individuals with pre-existing antibodies against influenza by increasing the number of individuals in the II study population. The homogeneity. Part I = 23, 8〇8345, 14 days before the vaccination (1st visit), in the individual has given the approval of the needle inclusion and exclusion criteria to screen him or her And he or she has a physical examination. In this interview, a sample of nasal epithelial cells was collected for 5 cytological analysis, and a saccharin test was used to measure baseline cilia activity. Days), taking 4-6 mL of blood samples for standard hematology analysis. Take 6-1〇mL blood samples for standard biochemical analysis and assess vital signs. After randomization, use one of the two vaccine formulations. To inoculate the individual's individual stay in place for the first 24 hours after vaccination to monitor immediate local and systemic reactions as well as adverse events. Vital signs were assessed at 4 and 14 hours after inoculation. After 24 hours, two blood samples were taken for standard hematology (4-6 mL) and biochemical (6-10 mL) analysis; after inoculation, samples of nasal epithelial cells were collected for cytological analysis and inoculated with saccharin test. Post-Ciliary Activity Analysis. Issue questionnaires (investigations I) to individuals and bring them home to assess local and systemic 15 responses on the next day (Day 3). Individuals must return two weeks after they are released home. Research point Visit 3 (Day 4). In this visit, assess local and systemic reactions, record any spontaneous adverse events that occurred between the previous and current visits. Also take two blood samples for standard hematology ( 4-6 mL) and biochemistry (6·ι〇mL) 20 analysis and assessment of vital signs. Two weeks after the first vaccination, on the 15th day, the individual returned to the study site (4th visit). In this visit, samples of nasal epithelial cells were collected for cytological analysis, ciliary activity was measured by saccharin test, and adverse events occurred between the 3rd visit and the 4th visit were recorded. 24 200808345 Part II: Blood collection in the first time And before the nasal wash (sampling) ^天丨 "under-visit" - after the individual has given a consensus, he or she is screened for inclusion and exclusion criteria, and his or her health is checked by physical examination. . 5 On the 2nd visit (-1 day; the visit can be combined with the first visit) 'take 6_1〇眺 blood sample for baseline hemagglutination inhibition (m) titer and take blood samples for standard hematology ( 4-6 mL) and standard biochemical mL) knife analysis. Nasal wash samples were collected for determination of baseline nasal gA antibody titers. 10 ίγ days On the third visit (Day 1), after the assessment of vital signs, the individuals were randomized and vaccinated with a single dose of one of the two formulations of the nasal flu vaccine. Any immediate local reactions, systemic reactions, and adverse events were monitored in situ during the first hour after inoculation. Afterwards, the vital signs were reassessed and the individual was taken home with the questionnaire and local and systemic responses were recorded daily for the first seven days after vaccination. Two weeks later (4th visit; day 15), 6-10 mL of blood samples were taken for HI titer determination, and two additional blood samples were taken for standard hematology (4 _ 6 m L) and biochemistry (6 _ 〇 mL) analysis, nasal wash samples were taken for nasal IgA antibody titer analysis. 0 20 Effect evaluation for pingding effect ' Blood samples and nasal wash samples were collected on day 1 (baseline) and day 15. Blood samples 6-10 mL of blood were collected to determine the hemagglutination inhibition (HI) antibody effect 25 200808345 price. 1 After blood collection and coagulation (at least 30 minutes at room temperature), serum was separated and kept frozen (-20 ° C) until titer analysis. Antibody titer analysis was performed in duplicate. The titer of the sample is the geometric mean of the two measurements. Pre- and post-inoculation sera were simultaneously analyzed for potency. 5 nasal wash samples

To collect nasal wash samples, apply 6 mL of pre-warmed saline (37 〇) in one nostril under nose control. Individuals are required to tilt their heads 6 times. The angle allows the lotion to flow. The collected lotion was applied to the second nostril, which was washed under the same conditions. A preservative solution (10 1/100 of the sample volume) was added to the sample. The preservative solution contained 10 mg/ml bovine serum albumin, pH 8 dissolved in 1 mM Tris HC1 buffer. The sample was directly clarified by low speed centrifugation (800 xg, 1 〇 minutes) and divided into small portions (to avoid repeated thawing of the sample in the future). Place it on dry ice until it is transferred. 15 20 The private level in the nasal wash samples was determined by ELISA and statistical analysis was performed using the Wilc_ test. The influenza vaccine was used as a coating antigen in a % well plate. By blocking the non-specific, "site wash" with the blocking buffer to (4) double-test (two copies per sample), used to absorb influenza-specific antibodies _% plate The antigen is washed in a 96-well plate before the "antigen," anti-human antibody (in combination with horseradish peroxidase or an assay phytase). The amount of antibodies specific for attenuated strains was determined by measuring the sensation of the substrate after the substrate was touched. The vaccine formulation was used in this study. Two formulations with two different influenza vaccine formulations 26 200808345 Contains WHO's recommended viral antigen 2 for the southern hemisphere in 2005, with a dose level of 30 meg per strain of 0.2 ml per dose. -A/New Caledonia/20/99/(HlNl)-like strain-A/Wellington/1 /2004(H3N2)-like strain 5 - B/Shanghai/361/2002-like strain Briefly, the inactivated influenza virus in 30-40% of the cane toxin solution was precipitated by centrifugation. The virus was resuspended and dissolved in the containing Detergent, in octaethylene glycol monododecyl ether (OEG) buffer. Subsequently, the virus is removed by ultracentrifugation. Nuclear capsid. The supernatant containing 10 〇 EG was adjusted with the lipopeptide P3CSK4 in OEG buffer or in the case of a reconstituted viral membrane without LPP, only with OEG-containing buffer (P3CSK4) : N-palm 醯-S-[2,3-double (brown oxime H2RS)·propyl HR]-cysteine-[S]-serine-[S]-lysine-[S ] lysine-[S]-lysine-[S]-lysine). OEG is removed by adsorption onto a hydrophobic resin. This results in a viral membrane containing LPP or no LPP (15 in the membrane) Formation of HA and NA and optionally reconstituted viral vesicles containing LPP in the membrane. After OEG removal, the virions were filtered through a PVDF membrane with a pore size of 0.22/m. For the time, a separate preparation with or without LPP was prepared (Table 6). The amount of LPP added corresponds to a ratio of 20 HA/LPP of 1:0.7 (w/w). 27 200808345 Table 6 Batch of preparation of virus particles Sub-LPP virus strain VIR-2005-09 Influenza B/Jiangsu/10/2003 VIR-2005-11 Influenza A/New Caledonia/20/1999 IVR-116 reassortant VIR-2005-13 Influenza type flu/ Wellington/1/2004 IVR-139 reassortant VIR- 2005-10 No influenza B/Jiangsu/10/2003 VIR-2005-12 No influenza A/New Caledonia/20/1999 IVR-116 reassortant VIR-2005-14 No influenza A/Wellington/1/ 2004 IVR-139 reassortant Table 7 Analysis of 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( β g /ml)b 805 854 711 784 704 644 phospholipid (mmol/l)c 0.494 0.563 0.820 1.03 0.717 0.695 endotoxin (IU per 100 //g HA) d <0.4 <0.4 <0.4 <0.4 <0.4 0.4 <0.5 Ovalbumin (W per 100 β g HA) e 0.068 0.088 0.037 0.036 0.132 0.126 Purity f Mainly HA Mainly HA Mainly HA Mainly HA Mainly HA Mainly HA aLowry test, principle: use domain After treatment with copper sulphate and Folin-ciocalteu reagent, the protein forms a blue color. The protein content was determined from the absorbance at 750 nm using the albumin BSA standard as a reference.

Lowry, OH? NJ Rosebrough, AL Farr, and RJ Randall. J. Biol. Chem. 193:265. 1951. 10 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). 28 200808345 bPhEur: Feature Article 2053 and Section 2.7.1 ° Principle: Each phospholipid contains a single constitutive atom' which can be used to quantify the lipid. The phospholipids are destroyed by perchloric acid. The acid salt produced by the combination of the acid salt and the molybdate is reduced by ascorbic acid to produce a blue product. The color was measured at 812 nm using a spectrophotometer -5. The amount of phospholipid in the sample is quantified by including a phosphate calibrator.

Ames BN. Assay of inorganic phosphate, total phosphate and phosphatases. Meth. Enzymol. 1966; 8:115-118 • Bottcher CJF, van Gent CM & Pries C. A rapid and 10 sensitive sub-micro phosphorus determination. Anal. Chim Acta 1961; 24:203-204 dPh· Eur· 2.6.14, e ovalbumin ELISA is a direct sandwich enzyme immunoassay in which immobilized polyclonal anti-ovalbumin antibodies and anti-antigens are used for capture. 15 Albumin-HRP conjugate as a detection system. Simultaneous culture of the conjugate and sample. Unbound components are removed by a washing step. The substrate (TMB and fragrant H202) was added to the wells. The presence of a conjugate that specifically binds in the well is indicated by the color development of the blue. Sulfuric acid was added to the substrate to terminate the reaction, which resulted in a yellow color change in the product. The absorbance (OD) was read at 450 rnn. To get the best results for 20, use a reference filter at 620 nm. A standard curve was prepared from the response of the ovalbumin standard (〇·3-20.0 ng/ml) included in the test. The concentration of the unknown sample was interpolated from the standard curve to read. f According to feature articles 0869 and 2053: the purity of the monovalent pooled collection was examined by polypropylene gel electrophoresis. Electrophoresis: Follow Ph. Eur 2.2.31 29 200808345. The effect was determined by the Wilcoxon's ranking sum test with a significant level of 0.05 on both sides' between the two vaccinated groups on day 15 for the HI antibody titer of each of the viral strains on day 15 and the nasal IgA antibody on day 15 The titer is compared. The HI antibody titer at day 15 was also analyzed by calculating the following three parameters for each virus strain and each vaccination group. - seroprotection rate, wherein seroprotection is defined as erythrocyte aggregation inhibition (HI) titer ^40, 10 - seroconversion, wherein seroconversion is defined as pre-vaccination hi titer <10 'inoculation HI titer> 40, or, before the inoculation, the HI titer 210 and the HI titer increase by at least 4 times, the average fold increase value, that is, the geometric mean of the increase in the HI titer multiple. 15 The effect data was analyzed according to the principles of Pre-protocol and intem-t0_treat. However, since this is a so-called principle verification study, pre-plan analysis is considered to be a basic one. The treatment-intended sample consisted of some post-inoculation effect data from the inoculated individual. The pre-planned sample consisted of 20 inoculated bodies that had completed the protocol and did not have major program bias. The main deviations include (not limited to): inclusion or exclusion of standard deviations, banned drug use, etc. In addition, laboratory-confirmed individuals with concurrent influenza infections and individuals who lost data on basic effects were also excluded from the pre-program samples. The study database is not blind, regardless of whether the individuals excluded from the protocol sample were previously determined. 30 200808345 Results Table 8 CHMP assessment of humoral immune response after intranasal vaccination with viral particle flu vaccine (RVM) seroprotection rate / statistical Α (Η3Ν2) sample A (H1N1)-like B-like 〆 LPP-RVM ( N=48) RVM (N=43) LPP-RVM (N=48) RVM (N=43) LPP-RVM (N=48) RVM (N=43)〆 After inoculation (Day 15) Serum protection> Yes η(%) 48(100%) 42(97.7%) 41(85.4%) 38(88.4%) 35(72.9%) 35(81.4%)_ No η(%) 0 1(2.3%) 7(14.6 %) 5(11.6%) 13(27.1%) 8(18.6%)_ Total Ν 48 43 48 43 48 43 RVM: Viral flu vaccine 5 LPP-RVM: Viral flu vaccine with lipopeptide

Table 8 C Η Ρ Ρ evaluation of humoral immune response after intranasal vaccination with viral particle influenza vaccine (RV Μ) (continued) Seroconversion J ψ Statistical Α (Η3Ν2) Α (Ν1Ν1) Sample B-like LPP-RVM (Ν=48) RVM (Ν=43) LPP-RVM (N=48) RVM (N=43) LPP-RVM (N=48) RVM (N=43) After inoculation (Day 15) Blood = Conversion Yes η(%) 37(77.1%) 25(58.1%) 25(52.1%) 33(76.7%) 24(50.0%) 22(51.2%) No η(%) 11(22.9%) 18(41.9%) 23 (47.9%) 10 (23.3%) 24 (50.0%) 21 (48.8%) "Ιέ Number Ν 48 43 48 43 48 43 RVM: Viral flu vaccine 10 LPP-RVM · Contains fatty virus flu Vaccine 31 200808345 Table 8 CHMP assessment of body 5 immune response after intranasal vaccination with viral particle flu vaccine (RVM) (continued) Average fold increase in HI antibody titer " : -- Statistics A (H3N2) Sample A (H1N1)-like B-like LPP-RVM RVM LPP-RVM RVM LPP-RVM RVM (N=48) (N=43) (N=48) (N=43) (N=48) (N=43) After inoculation (Day 15) Average increase multiples N 48 43 48 43 48 43 Average * 12.14 8.52 4.78 10.07 3,64 4.51 Note: * Geometric mean RVM: Viral particles Sense vaccine LPP-RVM. Virus-borne influenza vaccine with lipoproteins Table 9 Nasal wash-off IgA titer (GMT) H3N2 H1N1 B LPP-RVM N=48 RVM N=43 LPP-RVM N=48 RVM N=43 LPP-RVM N-48 RVM N=43 Day 1 93.88 96.45 80.93 85.97 65.84 55.21 Day 15 104.51 126.96 89.16 96.94 87.93 102.10

The conclusions are unexpected, as well as preclinical data obtained with the same vaccine batch (Example 1) and WO 04/110486 and clinical data (Gluck U, 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) Conversely, the reconstituted viral particle influenza vaccine 15 with no adjuvant is only performed once In the vaccinated human population, a satisfactory systemic immune response to meet the CHMP criteria for influenza vaccines was observed.

[Simple description of the figure; J (none) [Description of main component symbols] (none) 32

Claims (1)

200808345 X. Patent application scope: 1. A composition comprising influenza virus particles, the virus particles comprising a reconstructed outer shell of the virus, wherein the diseased peripheral element is obtained from influenza virus particles, wherein, Adding lipids to the reconstituted viral particles from an external source, wherein the viral particles comprise influenza antigen hemagglutinin and/or neuraminidase or a derivative thereof, wherein no separate addition to the composition is added And/or an immunostimulating agent, and wherein the composition is designed as an intranasal or inhaled formulation, the composition being characterized by a single intranasal or inhalation administration of the formulation to a human A systemic immune response and/or a local immune response against the influenza antigen hemagglutinin and/or neuraminidase or a derivative thereof is induced. 2. The composition of claim 1, wherein the amount of hemagglutinin administered by intranasal or inhalation per virus strain is equal to or lower than 30/g. The composition of any one of claims 1 to 2, wherein a single intranasal or inhaled administration of the formulation of the formula induces a cytotoxic lymphocyte response. The composition of any one of claims 1 to 3, wherein the immune response conforms to the CHMP standard for influenza vaccines. 5. The composition of claim 4, wherein the immunization is such as to provide one or more of the following: for an adult > 70% and/or for an elderly> 60% seroprotection rate, for adults > 4% and/or for elderly > 3 % seroconversion rate, and for adults 33 200808345 彳 / or for the elderly > 280 average The value is increased by a multiple. The composition of any one of items 2 to 5, wherein the dose of hemagglutinin per intranasal or inhaled is less than or equal to 25/ for each virus strain. /g. A composition according to any one of claims 2 to 5, wherein the dose of hemagglutinin per intranasal or inhalation of each virus strain is lower than or equal to 20// g. The composition according to any one of claims 2 to 5, wherein each of the viral strains has a hemagglutination dose of less than or equal to 15/g per intranasal or inhaled administration. The composition of any one of claims 2 to 5, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 10 /zg. The composition of any one of claims 2 to 5, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 5/zg. The composition according to any one of claims 1 to 3, wherein the composition is a vaccine formulation comprising a drug carrier for intranasal or inhalation administration. 12. The use of influenza virus particles comprising a reconstituted outer shell of said virus for the manufacture of a composition for intranasal or inhalation administration, wherein said viral envelope is obtained entirely from influenza virions, wherein An external source adds a lipid to the reconstituted viral particle, wherein the viral particle comprises an influenza antigen hemagglutinin and/or neuraminidase or a derivative thereof; 2008 20084545 'where' is not added to the composition alone An adjuvant and/or an immunostimulating agent, said composition being characterized in that said composition is administered to a human in a single intranasal or inhalation sufficient to induce resistance to said influenza pathogen hemagglutinin and/or neuraminidase or A systemic immune response and/or a local immune response of the derivative. 13. The use according to claim 12, wherein the amount of hemagglutinin administered by intranasal or inhalation per viral strain is equal to or lower than 30//g 〇14. The use of any of 12 or 13, wherein a single intranasal or inhaled administration of the formulation also induces a cytotoxic lymphocyte response. The use according to any one of claims 12 to 14, wherein the immune response conforms to the CHMP standard for influenza vaccines. The use of claim 15, wherein the immune response provides one or more of the following: for an adult >7% and/or for an elderly> % seroprotection rate for adults > 4% and/or seroconversion rate for elderly > 3〇G/❾, as well as for adults > 2.5 and/or for seniors > 2 〇 The average value is increased by a multiple. The use according to any one of claims 13 to 16, wherein the dose of hemagglutinin administered intranasally or by inhalation is less than or equal to 25/zg per virus strain. The use according to any one of claims 13 to 16, wherein the dose of hemagglutinin per intranasal or inhalation is less than or equal to 20/g per virus strain. 35 200808345 19. The use according to any one of claims 13 to 16, wherein each of the fortified strains: the dose of the lectin is less than or equal to 15# g. The use according to any one of claims 13 to 16, wherein each of the virus strains has a dose of less than or equal to 10 #g per intranasal or inhaled _hemagglutinin. The use according to any one of claims 13 to 16, wherein the dose of hemagglutinin per intranasal or inhalation of each virus strain is lower than or equal to 5# g. The use according to any one of claims 12 to 21, wherein the composition produced is a vaccine formulation. 23. A vaccine formulation comprising a composition comprising influenza virus particles, the viral particle comprising a reconstituted outer shell of the virus, wherein the viral outer shell is obtained entirely from influenza virions, wherein no The viral particles are added to the lipid, wherein the viral particles comprise the influenza antigen hemagglutinin and/or neuraminidase or a derivative 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 inhaled administration to humans, and wherein each viral strain has a hemagglutinin dose equal to or lower than each intranasal or inhaled dose. 30 // g. The vaccine formulation according to claim 23, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 25/g. 25. The vaccine formulation according to claim 23, wherein the dose of hemagglutinin administered intranasally or by inhalation per 36 200808345 virus strains is less than or equal to 20/zg. The vaccine formulation according to claim 23, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 15 // g. The vaccine formulation according to claim 23, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 10 mg. 28. The vaccine formulation according to claim 23, wherein each virus strain has a hemagglutinin of less than or equal to 5 // g per intranasal or inhaled hemagglutinin. The vaccine formulation according to any one of claims 23 to 28, wherein a single intranasal or inhaled administration of the formulation induces an immune response in the human, the immunization Responses include: systemic immune responses against influenza antigen hemagglutinin and/or neuraminidase or derivatives thereof, local immune responses against influenza antigen hemagglutinin and/or neuraminidase or derivatives thereof, and cells One or more of a toxic lymphocyte-mediated immune response. The vaccine formulation according to claim 29, wherein the immune response conforms to the CHMP standard for influenza vaccine. The vaccine formulation according to claim 3, wherein the immune response provides one or more of the following: for adults > 70% and/or for the elderly > 60% seroprotection rate for adults > 40% and/or seroconversion rate for the elderly > 3〇%, and oblique pair 37 200808345 Adults > 2.5 and / or for the elderly > 2.0 The average value is increased by a multiple. 32. A device for intranasal or inhalation administration, the device comprising a vaccine formulation as described in any one of claims 23 to 31. 33. The device of claim 32, wherein the device comprises a quantity of a vaccine formulation for a single intranasal or inhaled administration. 34. The device of claim 32, wherein the device is disposable. 38 200808345 VII. Designated representative map: 〇 (none) (1) The representative representative of the case is: ( ) (2) A brief description of the symbol of the representative figure: 8. If there is a chemical formula in this case, please disclose the chemical formula that best shows the characteristics of the invention: