Intranasal influenza vaccine based on virosomes

[0001] The present invention relates, for example, to compositions for inactivated influenza vaccines and routes of administration, in which a single intranasal or inhalational administration gives a systemic immune response that is positively correlated with clinical protection.

Background of the invention [0002] Various concepts of immunization against influenza by the nasal or oropharynx and using inactivated influenza antigen have been sought as a needle-free alternative to subcutaneous or intramuscular immunization. Experimental data supporting no-needle approaches were obtained on animal models. Concepts using inactivated influenza antigen (such as chemically inactivated whole virus particle or further processed viral components, such as split virus or purified surface antigens, hemagglutinin (HA) and / or neuraminidase (NA)) for nasal immunization, which is supported by data animal, include either the use of an adjuvant or immune stimulator in combination with an inactivated viral antigen, or require multiple vaccinations. An adjuvant is any substance that increases the immunogenicity of antigens mixed with it. In humans, only effective vaccination against influenza by nasal route has been reported for (a) live (cold-adapted) flu vaccines (FluMist ™, MedImmune Vaccinies Inc) (Ref. 1,2,3), (b) a virosomal influenza vaccine assisted with thermolabile toxin from E. coli (NasalFlu, Berna Biotech Ltd.) (Ref. 4) or (c) using large amounts of antigen and re-vaccination (Ref. 5, 10, 11). Although live vaccines are capable of eliciting a satisfactory immune response, their peculiar nature - that they are live viruses raises additional safety concerns and is likely to cause side effects due to the required round of viral replication in the upper respiratory tract. The commercialization of these products also requires storage conditions. Strong association between the use of E. Nasal Flu vaccine. HLT coli as an adjuvant for facial nerve palsy (Bell's Palsy) led to the withdrawal of HLT-assisted virosomal vaccine from the market (Ref. 6). [0003] The efficacy of influenza vaccines in a given population can be determined by assessing immunogenicity parameters against the amount of influenza antibodies that are produced after vaccination. These immunogenicity parameters, generally defined as CHMP criteria, are used for the annual re-licensing of inactivated influenza vaccines (Ref. 7). To date, no effective immunization of humans against influenza meeting these immunological requirements or CHMP criteria has been described (Ref.7) with one single intranasal administration of an inactivated vaccine and without the addition of an adjuvant as an additional vaccine component that does not come from an infectious agent against which the vaccine is intended to prevent, and which is added to the vaccine formulation to increase the immune response to the antigen. Therefore, it was recognized that there was still a need in the art for inact1 composition

EP 1996 229 B1 of an influenza vaccine that is capable of eliciting a satisfactory systemic immune response after a single intranasal administration, does not contain an adjuvant and meets the CHMP criteria (Ref. 7) for a single administration.

[0004] "CHMP criteria" are defined as follows. In CHMP (Committee for Medicinal Products for Human Use ”) Note for Guidance on Harmonization of Requirements for Influenza Vaccinnes, the following serological parameters are determined to assess the immunogenicity of inactivated influenza vaccines:

- seroprotection rate with seroprotection defined as haemagglutination inhibition (HI) titer> 40,

- seroconversion rate with seroconversion defined as HI titer before vaccination <10, and titer after vaccination>, 40 or HI titer before vaccination> 10 and at least a 4-fold increase in HI titer,

- average fold growth, which is the geometric mean of intra-individual growths (i.e. HI post-vaccination / HI pre-vaccination).

[0005] The CHMP's requirement for the immunogenicity of the influenza vaccine is that at least one criterion for each of the three viral strains in the vaccine is met:

Senior adult criterion seroprotection rate:> 70%> 60% seroconversion rate:> 40%> 30% average growth rate:> 2.5> 2.0 [0006] In WO2004 / 110486 a vaccine containing influenza virosomes containing reconstituted virus envelopes is disclosed. The virus shells are derived from influenza virus particles. The lipid components in virosomes can be derived from natural (viral) lipids or from external sources, e.g. synthetic lipids. The virosomes contain HA and / or neuraminidase and the composition is suitable for nasal oral or parenteral delivery. In an intranasal immunization experiment, 5 μg of influenza proteins were administered to Balb / c mice. The compositions of WO 2004/110486 contain lipoproteins as a separately added adjuvant.

[0007] The invention also applies to children who have been shown to respond in a comparable manner to adults (Ref. 8). The invention also applies to older individuals. "Elders" means they are over sixty years old.

Description of the invention [0008] The present invention provides the uses of influenza viruses according to claim 1 and compositions according to claim 2. Preferred embodiments for such a use or composition are set out in the dependent claims. The present invention further provides a vaccine formulation comprising this composition and devices for intranasal or inhalational administration2

EP 1996 229 B1 containing such a vaccine formulation and a mechanism for producing a vaccine aerosol.

[0009] Surprisingly and contrary to preclinical data in rats and literature on human clinical experience, we found that the immune response in humans following a single intranasal vaccination with inactivated influenza vaccine containing reconstituted influenza envelopes was consistent with all three CHMP criteria for efficacy of influenza vaccine for the age group 18-60 years old. One single intranasal administration is vaccination with the vaccine formulation to one or both nostrils without the need to repeat administration of the vaccine formulation to meet the above-mentioned CHMP criteria for the immunogenicity of the inactivated influenza vaccine. One single administration of the vaccine (through the nose, by inhalation, orally, subcutaneously or by the intramuscular route) is generally a vaccination schedule that does not include multiple administrations of the vaccine that are separated by days or weeks known in the art as initial and booster. A formulation designed as a formulation for nasal or inhalational administration includes a mixture of one or more active ingredients and excipients prepared in such a way as to permit intranasal or inhalational administration. The present invention provides pathways for inducing a systemic immune response (circulating immunoglobulins or antibody-producing B cells) which is in line with CHMP criteria, preferably with a single intranasal or inhalational administration of a virosomal influenza vaccine. The present invention also provides a pathway for inducing a local or mucosal immune response including an increase in the secretion of immunoglobulins known as IgA on the mucosal surface, preferably with a single intranasal or inhalational administration of a virosomal influenza vaccine. Induction of specific IgG and IgA responses after intranasal administration includes lymphoid tissue activity in the nasal cavity (Ref. 12). Such tissue is known as lymphoid tissue associated with the nasal cavity and nasopharynx (NALT) and has also been shown to be a mucosal site of a cellular immune response (Ref. 13). Since virosomes are known to have the potential to elicit a cellular immune response (Ref. 14, 15), the present invention also provides a method of inducing specific cytotoxic lymphocytes (CTL).

[0010] Virosomes are lipid bilayers containing viral glycoproteins. Virosomes are generally produced by detergent extraction of membrane proteins and lipids from virus envelopes with subsequent reconstitution of characteristic bilayers by removing this detergent. The present invention also provides influenza virosome compositions containing reconstituted influenza envelopes (in particular reconstituted without further addition of lipids and without the addition of an immunomodulator or immunostimulant (generally referred to as an adjuvant) for use in vaccination as an aerosol which is applied to the mucosa or nasopharynx or the oropharynx through one or both nostrils to achieve systemic and local od3

EP 1996 229 B1 against influenza. A single administration by inhalation is also possible. Single submucosal oral administration is also possible.

[0011] Reconstituted influenza virosomes can be produced from an inactivated virus that can be solubilized by a non-dialyzed detergent that is removed by adsorption to hydrophobic beads. The preparation may contain a purified suspension of one or more influenza antigens selected from haemagglutinin (HA), neraminidase (NA), hemagglutinin derivative and neuraminidase derivative. Viral membrane proteins, hemagglutinin and neuraminidase may be reconstituted in a membrane composed of viral lipids containing low levels of endotoxin and ovalbumin (see Ref. 9). Hemagglutinin and / or neuraminidase derivatives are hemagglutinin and / or neuraminidase molecules with modified amino acid sequences and / or structures. Amino acids can, for example, be deleted, changed or added to a sequence. The glycosylation pattern may also be changed. Derivatives, when introduced into a host, retain the ability to elicit an immune response.

[0012] The influenza virus used to produce reconstituted virosomes can grow, for example, on fertilized chicken eggs or in cell culture, or on adherent cells or on suspended cells. The virus may be, for example, a wild-type or rearranged virus or a genetically modified strain. The virus type may be, for example, any subtype of influenza A or B, including pandemic strains.

[00013] The present invention also provides vaccines. The term "vaccine" is understood to mean an immunoactive pharmaceutical preparation. In some embodiments, the vaccines may contain harmless variants or derivatives of pathogenic microorganisms that, for example, stimulate the immune system to increase defense against a particular pathogen. In some embodiments, the vaccine, for example, induces adaptive immunity when administered to a host. The vaccine may contain a dead or attenuated form of the pathogen or a pathogen component, such as an antigenic component of the pathogen. The vaccine preparation may further comprise a pharmaceutical carrier, which may be designed for the particular method by which the vaccine is to be administered, such as a pharmaceutical carrier designed for intranasal or inhalational administration. The influenza vaccine may contain one or more non-denatured influenza antigens, one or more of which are capable of eliciting an influenza-specific immune response.

[0014] The present invention provides a composition comprising influenza virosomes comprising reconstituted envelopes of said virus, wherein the composition is designed as a formulation for intranasal or inhalational administration. The invention also provides a composition in which virosomes include influenza antigens: hemagglutinin and / or neuraminidase or derivatives thereof. The invention also provides a composition in which the viral envelopes are entirely derived from viral particles. The invention also provides a composition in which no reconstituted virosomes

EP 1996 229 B1 is added lipid from an external source. The invention also provides a composition in which no separate adjuvant and / or immunostimulant is added. The invention also provides a composition in which a single intranasal or inhalational administration of the formulation to a subject is capable of eliciting a systemic immune response. The invention also provides a composition in which a single intranasal or inhalational administration of the formulation to a subject is also capable of eliciting a local immune response. The invention also provides a composition in which a single intranasal or inhalational administration of the formulation to a subject is also capable of eliciting cytotoxic lymphocyte responses. The invention also provides a composition whose ability to elicit a systemic immune response and / or local immune response and / or cytotoxic lymphocyte response is demonstrated in humans. The invention also provides a composition in which the immune response comprises an immune response directed against influenza antigens: hemagglutinin and / or neuraminidase or a derivative thereof. In a preferred embodiment, the invention also provides a composition whose immune response complies with the CHMP criteria for influenza vaccine. The invention also provides a composition whose immune response provides one or more seroprotection rates> 70% for adults and / or> 60% for older people and seroconversion rates> 40% for adults and / or> 30% for older people and the average fold increase is > 2.5 for adults and> 2.0 for older people. In a particularly preferred embodiment, the invention also provides a composition wherein the dose of haemagglutinin per viral strain per intranasal or inhalational administration is equal to or less than 30 μg. Finally, the invention also provides a composition that is a vaccine formulation comprising a pharmaceutical carrier for intranasal or inhalational administration.

[0015] The present invention also provides the use of influenza virosomes including reconstituted viral envelopes for the preparation of compositions for intranasal or inhalational administration. The invention also provides a use in which influenza virosomes include viral antigens, hemagglutinin and neuraminidase or derivatives thereof. The invention also provides a use in which the coatings are entirely derived from viral particles. The invention also provides a use in which no lipid from an external source is added to the virosomes in the composition. The invention also provides a use in which a separate adjuvant and / or immunostimulant is not added to the composition. The invention also provides a use in which a single intranasal or inhalational administration of the composition to a subject is sufficient to elicit a systemic immune response. The invention also provides a use in which a single additive or inhalational administration of the composition also elicits a local immune response. The invention also provides a use in which a single intranasal or inhalational administration of the composition elicits a cytotoxic lymphocyte response. The invention also provides a use in which the subject receiving this administration is a human. The invention also provides a use in which the composition elicits a response

An immune response comprising an immune response directed against influenza antigens, hemglutinin and / or neuraminidase or a derivative thereof. In a preferred embodiment, the invention also provides a use wherein the composition elicits an immune response that complies with the CHMP criteria for influenza vaccine. The invention also provides a use in which the immune response provides one or more seroprotection rates> 70% for adults and / or> 60% for older people, seroconversion rates> 40% for adults and / or> 30% for older people and average fold growth > 2.5 for adults and> 2.0 for older people. In a particularly preferred embodiment, the invention also provides a use wherein the dose of haemagglutinin per viral strain per intranasal or inhalational administration is equal to or lower than 30 μg. Finally, the invention also provides a use in which the composition being prepared is a vaccine formulation.

[0016] Thus, an embodiment of the present invention provides compositions of influenza virosomes comprising reconstituted viral envelopes, wherein the viral envelopes are entirely derived from influenza virus particles, no external source lipids are added to the reconstituted virosomes, and the virosomes include influenza antigens, hemagglutinin and / or neuraminidase or their derivatives, no separate adjuvant and / or immunostimulant are added to the composition, the composition is designed as a formulation for intranasal or inhalational administration, which composition is characterized in that a single intranasal or inhalational administration of this formulation to a human is capable of eliciting a systemic and / or local immune response against these influenza antigens, which systemic response matches the criteria CHMP for influenza vaccine, admixture of haemagglutinin per strain for intranasal or inhalational administration is less than or equal to 30 pg.

[0017] According to another embodiment, the present invention provides the use of influenza virosomes comprising reconstituted viral envelopes for producing compositions for intranasal or inhalational administration, wherein the viral envelopes are entirely derived from influenza viral particles, no external source lipids are added to the reconstituted virosomes, and the virosomes include influenza antigens, hemagglutinin and / or neuraminidase or derivatives thereof, no separate adjuvant and / or immunostimulant is added to the composition, which use of influenza virosomes for the preparation of compositions for intranasal or inhalational administration is characterized in that a single intranasal or inhalational administration of the composition to a human is sufficient to elicit a systemic or local immune response against these antigenomic influenza, which answer is in line with the CHMP criteria for influenza vaccine, an admixture of hemagglutinin per viral strain per intranasal or inhalational administration is less than or equal to 30 μg.

[0018] According to another embodiment, the present invention provides a vaccine formulation comprising a virosomal influenza composition comprising reconstituted envelopes of said virus,

Wherein the virus envelopes are entirely derived from influenza virus particles, no reconstituted lipids from an external source are added to the reconstituted virosomes, and the virosomes include influenza antigens, hemagglutinin and / or neuraminidase or derivatives thereof, no separate adjuvant and / or are added to the composition or an immunostimulant, which vaccine is characterized in that it is designed for single intranasal or inhalational administration to a human being, and the dose of haemagglutinin per viral strain per intranasal or inhalational administration is less than or equal to 30 μg. Preferably, this single intranasal or inhalational administration of the formulation is capable of eliciting a systemic or local immune response in a human being. A device containing an amount of this vaccine formulation for single intranasal or inhalational administration is also provided in accordance with the present invention.

[0019] The applied dose of haem agglutinin per virus strain per intranasal or inhalational administration in accordance with the present invention may also be less than or equal to 25 μg, 20 μg, 15 μg, 10 μg or 5 pg,

Literature cited [0020] (1) Maassab HF. Adaptation and growth characteristics of influenza virus at 25 ° C. Nature 213, 612-14 (1967) (2) Maassab HF, Bryant ML. The development of live attenuated cold-adapted influenza virus vaccine for Ihumans. Rev.Med.Virol. 1999 Oct-Dec; 9 (4): 237-44 (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 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 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. Uman Vaccines 1: 2, 85-90; March / April 2005 (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 (7) Note for Guidance on Harmonization of Requirements for Influenza Vaccines. EMEA / CpMP / BWP / 214/96 (8) Daubeney, P., Taylor, CJ, McGaw, J., 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 J, Nolan C, OBrien D. Burt D, Lowell G, Linden J, Fries L. Intranasal administration of a proteosomeinfluenza vaccine is well-tolerated and induces serum and nasal secretion influenza antibodies in healthy human subjects. Vaccine 2006; 24 (3): 254-62.

(11) Read RC, NaylorS.C., PotterC.W., Bond J. Jabbal-Gill l., FisherA., Lllum L. Jennings R. Effective nasal influenza vaccine delivery using chitosan. Vaccine 2005: 23 (35): 4367-74 (12) Kuper CF, Koomstra PJ, Hameleers DM, Biewenga J, Spit BJ. Duijvestein AM, van Breda Vriesman PJ. Sminia T. The role of nasopharyngeal lymphoid tissu e. Im mu nol.Today 1992 13: 219-24 (13) Zuercher AW, Coffin SE, Thurnheer MC, Fundova P, Cebra JJ. Nasal-associated lymphoid tissue isa mucosal inductive site for virus-specific humoral and cellular immune responses. J. Immunol. 2002 168: 1796-803 (14) Huckriede A. Bungener L. Stegmann T. Daemen T. Medema J, Palache AM. Wilschut J. The virosome concept for influenza vaccines. Vaccine 2005 23 (Suppl 1): S26-38 (15) Gluck R, Burri KG, Metcalfe I. Adjuvant and antigen delivery properties of virosomes. Curr. Drug Deliv. 2005 2: 395-400

EP 1996 229 B1

EXAMPLES

Example 1

Virosomal LPP vaccine in 8-week-old Balb / c mice; intranasal comparison of different HA / LPP ratios at suboptimal HA dose levels [0021] Groups of 10 seronegative female Balb / c mice received the virosomal LPP (lipopeptide) vaccine by intranasal administration, with a HA / LPP ratio of 1: 1.5; 1: 0.7; 1: 0.4; 1: 0 (i.e. without LPP) and with 2 μg HA per dose. In addition, a control group of 10 female mice received 0 μg HA / dose (intranasal administration of the vehicle).

[0022] Four preparations of LPP containing virosomes were prepared. Briefly, inactivated influenza virus in a 30-40% sucrose solution was sedimented by centrifugation. The virus was resuspended and solubilized in a buffer containing detergent, octaethyl monododecyl ether (OEG). Then, the virus nucleocapsid was removed by ultracentrifugation. The OEG-containing supernatant was divided into 4 equal volumes and various amounts of P3CSK4 lipopeptide were added in OEG-containing buffer (P3CSK4: N-palmitoyl-S - [2.3 bis (palmitoyloxy) - (2RS) -propyl] - [R] cysteinyl- [ S] -seryl- [S] -lysyl- [S] -lysyl- [S] -lysyl- [S] -lysine). The volume is adjusted with a buffer containing OEG. OEG was removed by adsorption on a hydrophobic resin. As a result, LPP-containing virosomes, reconstituted viral vesicles containing HA and NA and (optionally) LPP in their membranes were formed. After OEG removal, the virosomes were filtered through a PVDF membrane with a pore size of 0.22 μm.

[0023] The starting material was 20 mg HA influenza A / Wyoming / 3 / 20003X-147 (from the strain similar to A / Fujian / 411/200 (H3N2) containing 252 IU endotoxin / 100 μg HA. After the solubilization, 4 batches were produced as are shown in Table 1.

Table 1 Production of virosomes

<td>Party</td><td>Amount of HA as starting material (mg)</td><td>Amount of P3CSK4 added (mg)</td><td>HA / LPP ratio</td>

<td>VIR-2004-11</td><td> 5</td><td> 7,5</td><td> 1:1,5</td>

<td>VIR-2004-12</td><td> 5</td><td> 3,5</td><td> 1:0,7</td>

<td>VIR-2004-13</td><td> 5</td><td> 2,0</td><td> 1:0,4</td>

<td>VIR-2004-14</td><td> 5</td><td> 0</td><td> 1:0</td>

[0024] The support consisted of 5 mM Hepes, 145 mM NaCl, 1 mM EDTA (pH 7.4). For group E (see Table 2) the support was filtered through a PVDF membrane with a pore size of 0.22 μm. The 4 batches of generated virosomes were diluted to a concentration of 200 μg / ml for intranasal immunization and 67 μg for inhalation, and separated samples in 1 ml vials (2 per group) as indicated in Table 2. These vaccine groups were used as shown in Table 4.

EP 1996 229 B1

Table 2 Preparation of vaccines

<td>Group No.</td><td>Preparation</td>

<td>AND</td><td>VIR-2004-11</td>

<td>B</td><td>VIR-2004-12</td>

<td>C</td><td>VIR-2004-13</td>

<td>D</td><td>VIR-2004-14</td>

<td>E</td><td>Carrier*</td>

<td>* Carrier: 5 mM Hepes, 145 mM NaCl, 1 mM E PVDE with 0.22 μm pore size.</td><td>DTA (pH 7.4) filtered through the membrane</td>

Formulation Analysis [0025] The formulations used in this study were analyzed for a number of variables as indicated in Table 3.

Table 3. Analytical data on virosomes used to make vaccines

<td>analyte</td><td>VIR-2004-11</td><td>VIR-2004-12</td><td>VIR-2004-13</td><td>VIR-2004-14</td>

<td>Protein (mg / ml)^and</td><td> 1,7</td><td> 1,6</td><td> 1,5</td><td> 1,4</td>

<td>HA ^ g / ml) ^b</td><td> 776</td><td> 759</td><td> 697</td><td> 757</td>

<td>Phospholipid (mmol / L)^c</td><td> 0,658</td><td> 0,692</td><td> 0,658</td><td> 0,682</td>

<td>Endotoxin ^d (IU per 100 μg HA)^d</td><td> 3,1</td><td> 1,5</td><td> 1,9</td><td> 1,0</td>

<td>Ovalbumin ^ g on 100 μg HA)^e ^ g on 100 vgHA)^e</td><td> 0,047</td><td> 0,050</td><td> 0,055</td><td> 0,050</td>

<td>Cleanliness ^f</td><td>Mainly HA</td><td>Mainly HA</td><td>Mainly HA</td><td>Mainly HA</td>

<td colspan="5">^and Lowry analysis, principle: Proteins form a blue color after treatment with basic copper sulfate and Folin-Ciocalteu phenolic reagent. The protein content is determined by absorbance at 750 nm using BSA albumin as standard 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, Quatitation of Protein. Methods in Enzymology 182: 50-69 (1990), Hartree EF. Anal. Biochem 48: 422-427 (1972).</td>

<td colspan="2">^b Ph. Eur. monograph 2053 and section 2.7.1</td><td></td><td></td><td></td>

<td colspan="5">^c Principle: Each phospholipid contains a single phosphorus atom that can be used to quantify phospholipids. Phospholipids are destroyed by perchloric acid and the phosphate produced is complexed by molybdate, which is reduced by ascorbic acid to obtain a blue colored product. The color is determined using</td>

EP 1996 229 B1 spectrophotometer at 812 nm. The amount of phospholipids in a sample is quantified by a phosphate calibrator. Ames BN. Asssay of inorganic phosphate, Total phosphate and phosphatases. Meth. Enzymol. 1966 8: 115-118 Bottcher CJF, van Gent CM &amp; Pries C. A rapid and sensitive sub-micro phosphorus determination. Anal. Chim. Acta 1961; 24: 203-204^dPh Eur. 2.6.14^e Ovalbumin ELISA is a direct enzymatic sandwich immunoassay using immobilized polyclonal anti-ovalbumin antibodies for uptake and HRP-anti-albumin conjugate as capture system. Conjugates and samples are incubated simultaneously. Unbound components are removed in the washing step. Substrate (TMB and H2O2) is added to the wells. The presence of specifically bound conjugates in the wells is indicated by the formation of a blue color. Sulfuric acid is added to the substrate to stop the reaction, which results in the product turning yellow. Absorbances (OD) are read at 450 nm. For optimal results, a reference filter at 620 nm is used. The standard curve is formed from the ovalbumin standard responses (0.3-20.0 ng / ml) indicated in the assay. The concentrations of unknown samples can be interpolated from a standard curve.

^f. According to monographs 0869 and 2053: the purity of the monovalent pool is examined by polyacrylamide gel electrophoresis. Electrophoresis according to Ph.Eur. 2.2.31.

Test system

Test animals [0026] Seven groups of ten female Balb / c mice (BALB / cAnNCrl) each were used.

[0027] At the start of treatment, the mice were 8-9 weeks old and weighed 17-19 g.

[0028] Animals were intranasally vaccinated on day 0 and on day 14 with a monovalent virosomal LPP vaccine (A / Wyoming) and sacrificed 21 days after the second vaccination.

[0029] Intranasally: animals in dorsal position inoculated nasally with test substances (10 mg divided into two nostrils) under light anesthesia with Isoflurane / O2 / N2O.

EP 1996 229 B1

Table 4. Treatment scheme

<td>Group No.</td><td>Route of administration</td><td>Vaccine formulation</td><td>Number of females</td><td>Animal no</td>

<td>AND</td><td>intranasal</td><td>2 mg HA HA / LPP 1: 1.5 ratio</td><td> 10</td><td> 01-10</td>

<td>B</td><td>intranasal</td><td>2 mg HA HA / LPP ratio 1: 0.7</td><td> 10</td><td> 11-20</td>

<td>C</td><td>intranasal</td><td>2 mg HA HA / LPP 1: 0.4 ratio</td><td> 10</td><td> 21-30</td>

<td>D</td><td>intranasal</td><td>2 mg HA HA / LPP 1: 0 ratio</td><td> 10</td><td> 31-40</td>

<td>E</td><td>intranasal</td><td>2 mg HA HA / LPP 0: 0 ratio</td><td> 10</td><td> 41-50</td>

[0030] Prior to the first vaccination and 14 days after the first vaccination, peripheral blood samples were collected under anesthesia with Isoflurane / O2 / N2O. On day 35, the animals were sacrificed and blood samples were collected (bleeding under O2 / CO2 anesthesia via the abdominal aorta or cardiac puncture). Serum from all samples was collected, deep frozen, and stored in polypropylene tubes at temperatures below -10^aboutC until processing.

[0031] The influenza virus agglutinates red blood cells (RBC), which is blocked in the presence of a virus sufficiently specific for the virus. This phenomenon provides the basis for a haemagglutination inhibition (HI) test that is used to detect and quantify the serum of specific antiviral antibodies. Sera were added to influenza virus and RBC turkey. A number of dilutions were tested (titer). The HI titer is defined as the reciprocal of the highest dilution that still inhibits hemagglutination. The Geometric Mean Mian (GMT) is calculated as follows:

1) calculated unit - log (titer) as an arithmetic mean of two repetitions: [log (titer1) + [log (titer 2) / 2

2) calculation of the arithmetic mean of the log unit (titer) a

3) GMT (groups) = 10 EXP (average log (titer) in the group

Statistics [0032] HI titers were summarized in the vaccinated group and on the day of vaccination using the geometric mean titers. Log-transformed HI titers for day 35 groups were analyzed by linear regression means to examine the dose-response relationships between the amount of LPP in the vaccine and GMT.

Results

HI titer analysis [0033] GMTs are shown in Table 5. Table 5. Geometric mean titers

EP 1996 229 B1

<td>Group</td><td>Route of administration</td><td>Ratio HA / LPP</td><td>Day 0</td><td>Day 14</td><td>Day 35</td>

<td>AND</td><td>intranasal</td><td> 1:1,5</td><td> 5</td><td> 8</td><td> 415</td>

<td>B</td><td>intranasal</td><td> 1:0,7</td><td> 5</td><td> 6</td><td> 161</td>

<td>C</td><td>intranasal</td><td> 1:0,4</td><td> 5</td><td> 7</td><td> 97</td>

<td>D</td><td>intranasal</td><td> 1:0</td><td> 5</td><td> 7</td><td> 12</td>

<td>E</td><td>intranasal</td><td> 0:0</td><td> 5</td><td> 5</td><td> 5</td>

[0034] On day 0, HA-specific antibodies (ie all HI &lt;10 titers) could not be detected in the mice. On day 14, no HA-specific antibodies could be detected in most intranasal (in) mice. All HI titers were £ 10, except for 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).

[0035] On day 35, a dose-response was observed in the production of HA-specific antibodies, i.e. the addition of more LPP to the vaccine led to higher antibody titers.

[0036] Log-transformed HI titers on day 35 were compared between groups by linear regression. The adjusted regression decrease was highly significant (P &lt;0.0001). Thus, the observed dose-response relationship between the amount of LPP in the vaccine and GMT was statistically significant.

Proposal:

[0037] Repeated intranasal vaccination in mice with non-adjuvanted reconstituted influenza virosomes not inducing an measurable systemic immune response. Repeated intranasal vaccination using non-adjuvanted LPP reconstituted virosomes at the same HA dose level (2 mg HA / dose) and with raised LPP showed a systemic immune response in a dose dependent manner. In clear contrast to the present invention (see Example 2 below), these data were previously considered by consensus opinion in the art to support the claim that the use of an immunostimulant (in this case LPP) is essential for intranasal vaccination with an inactivated influenza vaccine, even when the influenza antigen (HA ) is presented in a reconstituted virosome.

Example 2 [0038] RANDOMIZED PARALLEL GROUP TEST WITH DOUBLE BLIND SAMPLE FOR THE DETERMINATION AND SAFETY OF POPEPTIDENT ADJUVAN AND ITS EFFECTIVENESS OF VIRSOMAL VEHICLEE SUBSTANKS

and £ 40 YEARS.

[0039] Healthy human volunteers were vaccinated intranasally with non-adjuvanted reconstituted influenza viruses LPP (lipopeptide) in a 0.2 ml (0.1 ml per nostril) dose volume containing 150mcg HA / ml per strain and 315 mcg LPP / ml. A similar group was intranasally inoculated with reconstituted influenza virosomes without LPP in a 0.2 ml (0.1 ml per nostril) dose volume containing 150 mcg HA / ml per strain. The aim of the study was to confirm in humans a sample of the concept that has been demonstrated in mice that an adjuvant (e.g. LPP) is required to obtain a satisfactory systemic immune response after intranasal vaccination with inactivated influenza vaccine.

Study design:

[0040] This is a randomized, double-blind, parallel group study in healthy juveniles&gt; 18 and &lt;40 years old. This study was carried out at one research center: Swiss Pharma Contract Ltd., Basel, Switzerland. The head of research was Dr. M. Seiberling. The study had two parts. Part I defines the safety of the LPP-assisted viral subunit viral vaccine in 12 individuals. Nine individuals were vaccinated with LPP-RVM (reconstituted LPP viral membranes; influenza vaccine surface antigen, inactivated LPP adjuvanted virosome), and three individuals were vaccinated with RVM (influenza vaccine - surface antigen, inactivated virosome). Part II of the study determined the efficacy and safety of LPP-RVM in hundreds of individuals (50 per group). The study was carried out in healthy individuals. In addition, subjects participating in Part II subjects were vaccinated against influenza during the three years preceding the study. This increased the homogeneity of the studied population in Part II by reducing the number of individuals with pre-existing anti-influenza antibodies.

Part I [0041] During 14 days before vaccination (Visit 1), after the subject received the informed consent form, he or she was screened for inclusion and exclusion criteria and underwent a medical examination. At this visit, a nasal epithelial cell sample was taken to perform cytology and measure related cilia activity with a saccharin test. At Visit 2 (Day 1), a 4-6 ml blood sample was taken for standard hematological analysis, a 6-10 ml blood sample was taken for standard biochemical analysis, and signs of vitality were assessed. After randomization, the subject was vaccinated with one of two vaccine formulations and left on site for the first twenty-four hours after vaccination to monitor immediate local and systemic reactions and adverse events. Signs of vitality were assessed four and twenty-four hours after vaccination. In addition, after twenty-four hours two blood samples were taken for standard hematological (4-6 ml) and biochemical (6-10 ml) analysis; after vaccination, a sample of nasal epithelial cells was taken to perform cytology and measure cilia activity with a saccharin test.

EP 1996 229 B1 [0042] Subjects received a questionnaire (Questionnaire I) to take him home to assess local and systemic reactions the next day (Day 3).

[0043] The subject had to return to the study site two days after they left: Visit 3 (Day 4). At this visit, local and systemic reactions were assessed and any spontaneous adverse events that occurred between the previous and current visits were recorded. In addition, two blood samples were taken for standard hematological (4-6 ml) and biochemical (6-10 ml) analysis and signs of vitality were determined.

[0044] Subjects returned to the study site two weeks after the first vaccination on Day 15 (Visit 4). At this visit, a sample of nasal epithelial cells was taken for cytology, cilia activity was measured with a saccharin test, and adverse events that occurred between Visit 3 and Visit 4 were recorded.

Part II [0045] During 14 days before the first blood sampling and nasal washing (Visit 1) after receiving the informed consent form, he or she was screened for inclusion and exclusion criteria and he or she was checked by a medical examination.

[0046] At Visit 2 (day 1; this visit can be combined with Visit 1), a 6 to 10 ml blood sample was taken to determine the baseline hemagglutination inhibition titer and blood samples were taken for analysis: standard hematology (4-6 ml) and standard biochemistry (6-10 ml). Nasal washes were collected to determine the nasal IgA antibody titers.

[0047] The next day at Visit 3 (Day 1) after determining the signs of vitality, the subject was randomized to be vaccinated with a single dose of one of the two nasal influenza vaccine formulations. Any immediate local and systemic reactions and adverse events were monitored on site during the first hour after vaccination. Shortly thereafter, signs of viability (vitality) were reassessed and the subject received a questionnaire to take home to record daily local and systemic reactions for the first seven days after vaccination.

[0048] Two weeks later (Visit 4; Day 15) a blood sample (6-10 ml) was taken to determine HI titer, two additional blood samples were taken for standard hematological (4-6 ml) and biochemical (6-10) analysis ml), nasal washes sample, nasal IgA antibody titre.

EP 1996 229 B1

Assessing efficacy [0049] To assess efficacy on Day 1 (baseline) and Day 15, blood and nasal lavage samples were collected.

Blood samples [0050] Six to ten ml of blood was collected to determine the titre of anti-hemagglutination antibodies. After collecting blood and coagulation (at least 30 minutes at room temperature), the sera were separated and kept frozen (-20^aboutC) up to titration. Antibody titrations were performed in duplicate. The titre assigned to the sample was the geometric mean of two determinations. Pre- and post-vaccine sera were titrated simultaneously.

Nasal washes samples [0051] 6 ml of preheated saline (37 ml) was used to collect nasal washes (37^aboutC) with a sight glass control of one of the nostrils. The subject was asked to tilt his head at an angle of 60^aboutC so that the flushing fluid can flow. The collected washes were applied to the second nostril, which was washed under the same conditions. A preservative solution (1/100 sample volume) was added to the samples. The preservative solution contained 10 mg / ml bovine serum albumin dissolved in 100 mM Tris-HCl buffer, pH 8. Samples were directly purified by low speed centrifugation (10 min at 800x g) and separated into equal volumes (to avoid re-melting the sample later) and placed on dry ice before transferring to -80^aboutC.

¹Palmer DF, Dowdle WR, Coleman MT, Schild GC. Hemagglutination inhibition Test. Advanced laboratory techniques for influenza diagnosis. US Dep. Hlth Ed. Welfare, PHS Atlanta, 1975: 25-62.

[0052] IgA levels in nasal washes were determined by ELISA and statistically analyzed by Wilcoxon test. The influenza vaccine was used as a coating antigen in 96 well plates. Non-specific binding sites were blocked by incubation with blocking buffer. Twice dilution (twelve dilutions per sample) nasal washes in blocking buffer were used to absorb influenza-specific antibodies on antigens in 96-well plates. 96-well plates were washed prior to incubation with enzyme conjugated anti-human antibodies (horseradish peroxidase or alkaline phosphatase conjugated). Unbound anti-human antibodies were removed by washing, and the amount of influenza-specific antibodies was determined by measuring the optical density after addition of the substrate for the enzymatic reaction.

EP 1996 229 B1

Vaccine formulation [0053] Two different influenza vaccine formulations were used in the study. Both formulations contained viral antigens as recommended by the WHO for the southern hemisphere year 2005² at a dose level of 30 mcg per strain, a dose of 0.2 ml.

-Strain similar to A / New Caledonia / 20/99 (H1N-1)

- Strain similar to strain A / Wellington / 1/2004 (H3N2)

- strain similar to strain B / Snanghai / 361/2002 ²WHO. Recommended influenza virus vaccine composition for use in the 2005 influenza season. Weekly Epidem. Rec. 2004; 79: 369-376.

[0054] Briefly, inactivated influenza virus in a 30-40% sucrose solution was sedimented by centrifugation. The virus was re-suspended and solubilized in a buffer containing detergent monododecyl ether octaethyl glycol (OEG). Then the viral neocapsid was removed by ultracentrifugation. The OEG-containing supernatant was adjusted with P3CSK4 lipopeptide in OEG-containing buffer or OEG-containing buffer only for reconstituted LPP-free viral membranes (P3CSK4: N-palmitoyl-S- [2,3 bis (palmitoyloxy) (2RS) -propyl] - [ R] -cysteinyl- [S] -seryl- [S]-lysyl- [S]-lysyl - [S]-lysyl- [S]-lysine). OEG was removed by adsorption to a hydrophobic resin. The result is reconstituted viral membranes containing LPP or LPP-free (reconstituted viral vesicles with HA and NA in membranes and possibly LPP in membranes). After OEG removal, the virosomes were filtered through a 0.22 mm PPVDF membrane.

[0055] For each virus strain, a separate formulation was prepared with or without LPP (Table 6). The amounts of LPP added correspond to the ratio HA / LPP 1: 0.7 (by weight).

<td>Series</td><td>LPP</td><td>Virus strain</td>

<td>VIR-2005-09</td><td>Present</td><td>Influenza B / Jiangsu / 10/2003</td>

<td>VIR-2005-11</td><td>Present</td><td>Influenza A / New Caledonia / 20/1999 IVR116 reassortant</td>

<td>VIR-2005-13</td><td>Present</td><td>Influenza A / Wellington / 1/2004 IVR-139 reassortant</td>

<td>VIR-2005-10</td><td>Absent</td><td>Influenza B / Jiangsu / 10/2003</td>

<td>VIR-2005-12</td><td>Absent</td><td>Influenza A / New Caledonia / 20/1999 IVIR116 reassortant</td>

<td>VIR-2005-14</td><td>Absent</td><td>Influenza A / Wellington / 1/2004 IVR-139 reassortant</td>

EP 1996 229 B1

Table 7 Formulation analysis

<td>analyte</td><td>VIR-200509</td><td>VIR-20051o</td><td>VIR-200511</td><td>VIR-200512</td><td>VIR-200 513</td><td>VIR-200 514</td>

<td>Protein (Mg / ml)^and</td><td> 1,51</td><td> 1,54</td><td> 1,86</td><td> 1,83</td><td> 1,37</td><td> 1,18</td>

<td>HA ^ g / ml) ^b</td><td> 805</td><td> 854</td><td> 711</td><td> 784</td><td> 704</td><td> 644</td>

<td>phospholipid (Mmol / l)^c</td><td> 0,494</td><td> 0,563</td><td> 0,820</td><td> 1,03</td><td> 0,717</td><td> 0,695</td>

<td>Endotoxin ^d (IU per 100 μg HA)^d</td><td> &lt;0,4</td><td> &lt;0,4</td><td> &lt;0,4</td><td> &lt;0,4</td><td> &lt;0,4</td><td> &lt;0,5</td>

<td>ovalbumin ^ g per 100 μg HA)^e</td><td> 0,058</td><td> 0,088</td><td> 0,037</td><td> 0,036</td><td> 0,132</td><td> 0,126</td>

<td>Cleanliness ^f</td><td>Mainly HA</td><td>Mainly HA</td><td>Mainly HA</td><td>Mainly HA</td><td>Mainly HA</td><td>Mainly HA</td>

<td colspan="5">^and Lowry analysis, principle: Proteins form a blue color with copper sulfate and phenolic Folin-Ciocalteu reagent. Zave based on absorbance at 750 nm using reference Lowry, OH, NJRosebrough, AL. Farr, and RJ Randall. J. Biol. Chem. 19 GM, NS Mathewson and GN Catravas, Anal. Biochem., 89; 31. 1978. Sion of Protein. Methods in Enzymology 182; 50-69 (1990), Hartree E 422-427 (1972).</td><td colspan="2">alkaline targeting Protein mercury is determined by BSA ard albumin. 3: 265, 1951. Oostra, toscheck, CM, QuantitaF. Anal. Biochem 48:</td>

<td colspan="3">^b Ph. Eur: monograph 2053 and section 2.7.1</td><td></td><td></td><td></td><td></td>

<td colspan="7">^c Principle: Each phospholipid contains a single phosphorus atom that can be used to quantify phospholipids. The phospholipidin is destroyed with perchloric acid and the phosphate produced is complexed by molybdate, which is reduced by ascorbic acid to obtain a blue colored product. The color is determined by means of a spectrophotometer at 812 nm. The amount of phospholipids in the sample is determined by a phosphate calibrator. Ames BN. Asssay of inorganic phosphate, total phosphate and phosphatases. Meth. Enzymol. 1966 8: 115-118 Bottcher CJF, van Gent CM &amp; Pries C. A rapid and sensitive submicro phosphorus determination. Anal. Chim. Acta 1961; 24: 203-204</td>

<td colspan="2">^dPh Eur. 2.6.14</td><td></td><td></td><td></td><td></td><td></td>

<td colspan="7">^e Ovalbumin ELISA is a direct enzyme sandwich immunoassay using immobilized polyclonal antibodies to ovalbumin to</td>

EP 1996 229 B1 uptake and conjugate HRP-antialbumin as a detection system. Conjugates and samples are incubated simultaneously. Unbound components are removed in the washing step. Substrate (TMB and H2O2) is added to the wells. The presence of specifically bound conjugates in the wells is indicated by the formation of a blue color. Sulfuric acid is added to the substrate to stop the reaction, which results in the product turning yellow. Adsorbances (OD) are read at 450 nm. A reference filter at 620 nm is used for optimal results. The standard curve is formed from the ovalbumin standard responses (0.3-20.0 ng / ml) indicated in the assay. The concentrations of unknown samples can be interpolated from a standard curve.

^f. According to monographs 0869 and 2053: the purity of the monovalent pool is examined by polyacrylamide gel electrophoresis. Electrophoresis according to Ph.Eur. 2.2.31

Efficacy [0056] Log-transformed HI antibody titers per viral strain on Day 15 were compared with nasal IgA titers on Day 15 between two vaccinated groups by the rankWilcoxon sum test at a two-sided significance level of 0.05.

[0057] HI antibody titer on Day 15 was also analyzed by calculating the following three parameters per viral strain and graft group:

- seroprotection rate with seroprotection defined as HI&gt; 40 titer

- seroconversion rate with seroconversion defined as HI titer before vaccination &lt;10 and HI titer after vaccination&gt; 40 or HI titer before vaccination&gt; 10, and after vaccination HI titer increases at least 4 times

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

[0058] Efficacy data were analyzed both according to the previous protocol and the intended treatment principle. However, given that this was a so-called proof-of-principle study (prognostic study), analyzes of the previous protocol were considered first. An intent-to-treat sample was created by vaccinated individuals with some post-vaccination efficacy data. The pre-protocol sample was created by vaccinated individuals who completed the protocol and did not have major primary violations. Major violations include (among others) violation of inclusion and exclusion criteria and use of a prohibited drug. In addition, individuals with laboratory-confirmed concurrent influenza infection and individuals for whom no initial effective data are available were also excluded from the per-protocol sample. The subject was excluded from the per-protocol sample or was not decided before the test database was declassified.

EP 1996 229 B1

Results [0059]

Table 8. CHMP assessment of humoral immune response following intranasal vaccination with virosomal influenza vaccine (RVM)

Seroprotection rates

<td rowspan="2">Statistics</td><td colspan="2" rowspan="2">A (H3N2) a similar LPP-RVM (N = 48)</td><td rowspan="2">RVM (N = 43)</td><td rowspan="2">A (H1N1) a similar LPP-RVM (N = 48)</td><td colspan="3">B-like</td>

<td>RVM (N = 43)</td><td>LPP-RVM (N = 48)</td><td>RVM N = 43)</td>

<td colspan="2">After vaccination (Day 15)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>seroprotection</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Yes</td><td>n (%)</td><td> 48(100%)</td><td> 42(97,7%)</td><td> 41(85,4%)</td><td> 38(88,4%)</td><td> 35(72,9%)</td><td> 35(81,4%)</td>

<td>No</td><td>n (%)</td><td> 0</td><td> 1(2,3%)</td><td> 7(14,6%)</td><td> 5(11,6%)</td><td> 13(27,1%)</td><td> 8(18,6%)</td>

<td>Whole</td><td>n</td><td> 48</td><td> 43</td><td> 48</td><td> 43</td><td> 48</td><td> 43</td>

<td>seroconversion</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Yes</td><td>n (%)</td><td> 37(77,1%)</td><td> 25(58,1%)</td><td> 25(52,1%)</td><td> 33(76,7%)</td><td> 24(50,0%)</td><td> 22(51,2%)</td>

<td>No</td><td>n (%)</td><td> 11(22,9%)</td><td> 18(41,9%)</td><td> 23(47,9)</td><td> 10(23,3%)</td><td> 24(50,0%)</td><td> 21(48,8%)</td>

<td>Whole</td><td>n</td><td> 48</td><td> 43</td><td> 48</td><td> 43</td><td> 48</td><td> 43</td>

<td colspan="4">mean fold increase in HI antibody titers</td><td></td><td></td><td></td><td></td>

<td>Statistics</td><td></td><td>A (H3N2) -</td><td></td><td>A (H1N1) -</td><td></td><td>B-like</td><td></td>

<td></td><td></td><td>like LPP-</td><td></td><td>like LPP-</td><td></td><td></td><td></td>

<td></td><td></td><td>RVM</td><td>RVM</td><td>RVM</td><td>RVM</td><td>LPP-RVM</td><td>RVM</td>

<td></td><td></td><td>(N = 48)</td><td>(N = 43)</td><td>(N = 48)</td><td>(N = 43)</td><td>(N = 48)</td><td>(N = 43)</td>

After vaccination (Day 15), average fold increase n 48 43 48 43 48 43

Average * 12.14 8.52 4.78 10.07 3.64 4.51

Annotation * geometric mean

RVM: Virosomal Flu Vaccine

LPP-RVM: Lipopeptide Assisted Virosomal Vaccine

EP 1996 229 B1

Table 9. IgA titers in nasal washes (GMT)

<td></td><td colspan="2">H3N2</td><td colspan="2">H1N1</td><td colspan="2">B</td>

<td></td><td>LPP-RVM N = 48</td><td>RVM N = 43</td><td>LPP-RVM N = 48</td><td>RVM N = 43</td><td>LPP-RVM N = 48</td><td>RVM N = 43</td>

<td>Day 1</td><td> 93,88</td><td> 96,45</td><td> 80,93</td><td> 85,97</td><td> 65,84</td><td> 55,21</td>

<td>Day 15</td><td> 104,51</td><td> 126,96</td><td> 89,16</td><td> 96,94</td><td> 87,93</td><td> 102,10</td>

Conclusion [0060] Unexpectedly and in contradiction to preclinical data obtained for the same vaccine series (example 1), and as described in WO 04/110486 and clinical data (GluckU, Gebbers JQ, Gluck R, Phase 1 evaluation of internanasal virosomal influenza vaccine with and without Escherichia coli heat-labile toxin in adult volunteers. J. Virol. 1999 Vulture; 73 (9) 7780-6), a satisfactory immune response in accordance with the CHMP criteria for influenza vaccines was observed in the group of people vaccinated only once with non-assisted reconstituted virosomal influenza vaccine.