TWI397419B - Intranasal or inhalational administration of virosomes - Google Patents

Description

Intranasal or inhaled administration of viral particles Field of invention

The present invention relates to, for example, compositions for inactivating influenza vaccines and modes of administration wherein a single intranasal or inhaled administration achieves a systemic immune response that is positively associated with clinical protection.

Background of the invention

A variety of concepts for immunization against influenza by the intranasal or oropharyngeal route, as well as the use of inactivated influenza antigens, have been investigated as a non-injection (needle-less) alternative pathway for subcutaneous or intramuscular immunization. Experimental data supporting non-injection means has been generated in animal models. Obtaining animal data-supported use of inactivated influenza antigens (eg, chemically inactivated whole virus particles, or further processed viral components, such as cleaved viruses, or purified surface antigen hemagglutinin) The idea of (HA) and/or neuraminidase (NA) to immunize by intranasal route includes the use of an adjuvant or an immunostimulant in combination with inactivated influenza antigen, or multiple immunizations. An adjuvant is any substance that enhances the immunogenicity of an antigen mixed therewith. In humans successful vaccination against influenza via the intranasal route reported only through: (a) live (cold transformation by strains) influenza vaccines ^{(FluMist TM, MedImmune Vaccines Inc)} ( Ref. 2,3) (b) Viral granule influenza vaccine with E. coli heat labile toxin (NasalFlu, Berna Biotech Ltd) (Reference 4) or (c) High dose antigen And repeated vaccination (Refs. 5, 10, 11). Although live vaccines can induce a satisfactory immune response, the specific nature of their ability to become a live virus leads to additional safety concerns that may trigger side effects due to viral replication in the vicinity of the upper respiratory tract. In addition, the required storage conditions also limit the commercialization of these products. The strong correlation between the intranasal influenza vaccine with E. coli HLT as an adjuvant and 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 immunogenic parameters associated with the amount of anti-influenza antibodies produced after vaccination. These immunogenic parameters are commonly referred to as CHMP rates, which are 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 immunity against antigen) The purpose of the response was added to the 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 (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 influenza vaccine: - seroprotection rate, wherein serum Protection is defined as Haemagglutination Inhibition (HI) titer 40, - seroconversion rate, where seroconversion is defined as HI titer <10 before inoculation, HI titer after inoculation 40, or, before the inoculation HI titer 10 and an increase in HI titer of at least 4 fold, an average fold increase value, which is an increased geometric mean for the individual (ie, HI titer after inoculation/HI titer before inoculation).

The CHMP requirement for immunogenicity of influenza vaccines is that for each of the three viral strains in the vaccine, at least one of the following criteria is reached:

The invention is also applicable to children from which they have shown that they respond immunely in a manner comparable to that of adults (Reference 8). The invention is also applicable to elderly individuals. The elderly are over 60 years old.

Description of the invention

Surprisingly, as opposed to preclinical rodent data and literature on human clinical experience, we found that for the 18-60 age group, a single inactivated influenza vaccine containing a reconstituted influenza virus coat was used. After intranasal inoculation, the human immune response reached all three CHMP criteria for the efficacy of influenza vaccines. A single intranasal administration is to vaccinate the vaccine formulation through one or two nostrils to achieve the above-described CHMP criteria for inactivated influenza vaccine immunogenicity without repeated administration of the vaccine formulation. A single vaccination (by nasal, inhalation, oral, subcutaneous or intramuscular route) is usually the vaccination schedule described below, which does not include: known in the art as priming and boosting, in number Multiple doses of the vaccine are administered at intervals of days or weeks. Formulations designed for intranasal or inhaled formulation include a mixture of one or more active ingredients and excipients prepared in such a manner as to allow 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 using a single intranasal or intracytoplasmic influenza vaccine Inhalation administration is carried out. The invention also provides a method for inducing a local or mucosal immune response comprising an increase in secretory immunoglobulin known as IgA on the surface of a mucosal membrane, advantageously, said method using a vaccine against a viral particle influenza It is administered by single intranasal or inhalation administration. The induction of specific IgG and IgA responses following intranasal administration involves the activity of lymphoid tissue in the nasal cavity (Ref. 12). Such tissues are known as nasal associated lymphoid tissue (NALT), which has also been 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 (Refs. 14, 15), the present invention also provides a method of inducing specific cytotoxic lymphocytes (CTL).

A viral particle is a lipid bilayer containing a viral glycoprotein. Viral particles are typically 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 present invention also provides a composition of influenza virus particles comprising a reconstituted influenza virus outer membrane (specifically, no lipids, and no immunostimulant (commonly referred to as an adjuvant) immunomodulator Reconstitution) 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 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 (HA), neuraminidase (NA), a derivative of hemagglutinin and a derivative of neuraminidase Things. 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). Derivatives of serum lectin and/or neuraminidase are hemagglutinin and/or neuraminidase molecules 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 influenza virus used to prepare the reconstituted viral particles can be cultured in, for example, eggs containing embryos or in cell culture of cells in attached cells or suspensions. The virus may, for example, be a wild-type or reassortant or genetically modified strain. The type of virus can be, for example, any influenza A or B subtype, including epidemic strains.

The invention also provides a vaccine. The term vaccine is to be understood as an immunologically active pharmaceutical preparation. In certain embodiments, the vaccine may comprise a harmless variant or derivative of the pathogenic microorganism, for example, stimulating the immune system to produce resistance to a real pathogen. In certain embodiments, the vaccine is, for example, capable of inducing adaptive immunity when administered to a host. The vaccine may contain a dead or attenuated form of the pathogen or 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 mode in which the vaccine will be administered, such as 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 viral particle comprises an influenza antigen hemagglutinin and/or a neuraminidase or a derivative thereof. The invention also provides such compositions wherein the viral envelope is obtained entirely from virions. The present invention also provides such compositions in which no lipids are added from an external source to the reconstituted viral particles. The invention also provides such compositions in which no separate adjuvant and/or immunostimulatory agent 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 invention also provides such compositions wherein a single intranasal or inhalation administration to an individual can also induce a local immune response. The invention also provides for the administration of a composition that induces a cytotoxic lymphocyte response in a single intranasal or inhalation administration to an individual. The invention also provides such compositions which are shown in humans in an ability to induce a systemic immune response and/or a local immune response and/or a cytotoxic lymphocyte response. The invention also provides such compositions wherein the immune response comprises an immune response against the influenza antigen hemagglutinin and/or neuraminidase or a derivative thereof. In a preferred embodiment, the invention also provides such a composition wherein the immune response reaches the CHMP criteria for influenza vaccine. The invention also provides the composition wherein the immune response provides one or more of the following: >70% for adults and/or >60% for elderly people, for adults > 40% and/or seroconversion rate >30% for the elderly, and an average fold increase of >2.5 for adults and/or >2.0 for the elderly. In a particularly preferred embodiment, the present invention also provides the composition wherein the amount of hemagglutinin administered by each intranasal or inhalation of each viral strain is equal to or lower than 30 μg. Finally, the invention also provides such compositions 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 the manufacture of a composition for intranasal or inhaled administration. The invention also provides the use wherein the influenza virus particle comprises an influenza antigen hemagglutinin and/or a neuraminidase or a derivative thereof. The invention also provides the use wherein the viral envelope is obtained entirely from influenza virions. The invention also provides such use wherein no lipid is added from an external source to the reconstituted viral particle. The invention also provides such use wherein no separate adjuvant and/or immunostimulant is added to the composition. The invention also provides such use wherein a single intranasal or inhaled administration to an individual is sufficient to induce a systemic immune response. The invention also provides such use wherein a single intranasal or inhaled administration to an individual also induces a local immune response. The invention also provides such use wherein a single intranasal or inhaled administration to an individual also induces a cytotoxic lymphocyte response. The invention also provides the use wherein the subject to be administered is a human. The invention also provides such use: the induced immune response comprises an immune response against the influenza antigen hemagglutinin and/or neuraminidase or a derivative thereof. In a preferred embodiment, the invention also provides such use wherein the composition induces an immune response that meets the CHMP criteria for influenza vaccines. The invention also provides such use wherein the immune response provides the composition of one or more of the following: >70% for adults and/or >60% for elderly people Protection ratio, >40% for adults and/or >30% for older adults, and >2.5 for adults and/or an average fold increase for older adults >2.0. In a particularly preferred embodiment, the invention also provides the use wherein the hemagglutinin administered intranasally or by inhalation is administered at a dose equal to or lower than 30 μg per intraviral strain. . Finally, the invention also provides said use wherein the composition produced is a vaccine formulation.

Accordingly, in one embodiment, the invention provides a composition of influenza virus particles comprising a reconstituted outer shell of a virus, wherein the viral outer shell is obtained entirely from influenza virions, wherein no reconstituted viral particles are obtained from an external source Adding a lipid, wherein the viral particle comprises an influenza antigen hemagglutinin and/or a 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 which is characterized in that a single intranasal or inhaled administration of the formulation to a human induces a systemic immune response against the influenza antigen and / or a local immune response, which is consistent with the CHMP criteria for influenza vaccines, and wherein the hemagglutinin dose per intranasal or inhaled administration of each viral strain is equal to or lower than 30 μg.

According to another embodiment, the invention provides the use of an influenza virion comprising a reconstituted outer shell of a virus for the manufacture of a composition for intranasal or inhaled administration, wherein the viral envelope is obtained entirely from influenza virions, Wherein, no lipid is added 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 adjuvant is added to the composition. And/or an immunostimulating agent, the use of said influenza virus particle for the manufacture of a composition for intranasal or inhalation administration, characterized in that said composition is administered to a human in a single intranasal or inhalation sufficient to induce resistance a systemic immune response and/or a local immune response of the influenza antigen that achieves the CHMP criteria for influenza vaccines, and wherein the hemagglutinin dose per intranasal or inhaled administration of each viral strain is equal to Or less than 30 μg.

According to another embodiment, the present invention provides 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 The lipid is not added to the reconstituted virus particles from an external source, wherein the viral particles comprise the influenza antigen hemagglutinin and/or neuraminidase or a derivative thereof, wherein no separate adjuvant is added to the composition and / or an immunostimulating agent, 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 is administered intravesically or by inhaled hemagglutinin dose Equal to or below 30 μg. Advantageously, said single intranasal or inhaled administration of said formulated formulation induces a systemic and/or local immune response in said human. Also provided according to the invention is an apparatus comprising a quantity of said vaccine formulation for single intranasal or inhalation administration.

The dose of the hemagglutinin administered intranasally or by inhalation of each virus strain according to the present invention may also be lower than or equal to 25 μg, 20 μg, 15 μg, 10 μg or 5 μg.

Cited literature

(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 humans.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. Human Vaccines 1:2,85-90; March/April 2005(6)Mutsch M,zhou W,Rhodes P, Bopp M, Chen RT, Linde r 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 Harmonisation Of Requirements for Influenza Vaccines.EMEA/CpMP/BWP/214/96(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 J, Nolan C, O' Brien D, Burt D, Lowell G, Linden J, Fries L. Intranasal administration of a proteosome-influenza vaccine is well-tolerated and induces serum and nasal secretion influenza antibodies in healthy human subjects. Vaccine 2006;24(3): 2 54-62(11)Read RC,Naylor SCPotter CW,Bond J.,Jabbal-Gill I., Fisher A., Illum L., Jennings R. Effective nasal influenza vaccine delivery using chitosan. Vaccine 2005; 23(35): 4367-74(12) Kuper CF, Koornstra PJ, Hameleers DM, Biewenga J, Spit BJ, Duijvestein AM ,van Breda Vriesman PJ,Sminia T.The role of nasopharyngeal lymphoid tissue.Immunol.Today 1992 13:219-24(13)Zuercher AW,Coffin SE,Thurnheer MC,Fundova P,Cebra JJ.Nasal-associated lymphoid tissue is mucosal Inductive site for virus-specific humoral and cellular immune responses. J.Immunol.2002 168:1796-803(14)Huckriede A,Bungener L,Stegmann T,Daemen T,Medema J,Palache AM,Wilschut J.The virosome concept for Influenza vaccines.Vaccine 2005 23(Suppl 1):S26-38(15)Gl Ck R, Burri KG, Metcalfe I, Adjuvant and antigen delivery properties of virosomes. Curr.Drug Deliv.2005 2:395-400

Example 1 Intranasal comparison of multiple HA/LPP ratios in LPP virion vaccines in 8-week-old BALB/C mice; suboptimal HA dose levels

A group consisting of influenza-negative female Balb/c mice (10 mice per group) received an intranasal administration of LPP (lipopeptide)-viral vaccine with a HA/LPP ratio of 1:1.5, 1:0.7, 1: 0.4, 1:0 (ie no LPP) and 2 μg HA per dose. In addition, a control group of 10 female mice obtained 0 μg HA/dose (intranasal administration of the vehicle).

Four preparations of LPP-containing viral particles were prepared. 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 supernatant containing OEG was divided into 4 aliquots, and different amounts of lipopeptide P3CSK4 (P3CSK4: N-palm 醯-S-[2,3-bis(palmitine oxime)-) (containing OEG buffer) were added. 2RS)-propyl]-[R]-cysteine-[S]-serine-[S]-lysine-[S]-lysine-[S]-lysine-[S ]-lysine). The volume was adjusted with a buffer containing OEG. 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) 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 μm.

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

The vector consisted of 5 mM Hepes, 145 mM NaCl, 1 mM EDTA (pH 7.4). For Group E (see Table 2), the carrier was filtered through a PVDF membrane having a pore size of 0.22 μm. According to Table 2, for the intranasal immunization, the prepared 4 batches of virus particles were diluted to a concentration of 200 μg/ml, diluted to a concentration of 67 μg/ml for intramuscular immunization, and dispensed into a 1 ml vial. (2 bottles per group). These vaccine sets were used as outlined in Table 4.

Analysis of formulated formulations

Formulation formulations for this study were analyzed for several variables shown in Table 3.

^a Lowry test, principle: After treatment with bismuth sulfate and Folin-ciocalteu phenol reagent, the protein forms blue. 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. 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). ^b PhEur: Feature Article 2053 and 2.7.1 ^c Principle: Each phospholipid contains a single phosphorus atom, It can be used to quantify phospholipids. The phospholipid is destroyed by perchloric acid, and the phosphate 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 phosphate calibrator. Ames BN.Assay of inorganic phosphate,total phosphate and phosphatases.Meth.Enzymol.1966;8:115-118 B Ttcher CJF, van Gent CM & Pries CA rapid and sensitive sub-micro phosphorus determination.Anal.Chim.Acta 1961;24:203-204 ^d Ph.Eur.2.6.14 ^e ovalbumin ELISA is a direct sandwich enzyme immunization A method in which an immobilized polyclonal anti-ovalbumin antibody for capture and an anti-ovalbumin-HRP conjugate are used as a detection system. Simultaneous culture of the conjugate and sample. Unbound components are removed by a washing step. Substrates (TMB and H ₂ O ₂ ) were 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 is added to the substrate to terminate the reaction, which results in a color change in the product which is yellow. The absorbance (OD) was read at 450 nm. For best results, use a reference filter at 620 nm. A standard curve was prepared from the response of the ovalbumin standard (0.3-20.0 ng/ml) included in the assay. 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 checked by polypropylene gel electrophoresis. Electrophoresis: Follow Ph.Eur 2.2.31.

Test system Test animal

Seven groups of animals, 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.

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

Intranasal: The test substance was inoculated intranasally (10 μl, divided into two nostrils), which was performed on animals anesthetized with mild isoflurane/O ₂ /N ₂ O on the back.

The orbital blood samples were collected under isoflurane/O ₂ /N ₂ O anesthesia before the first vaccination and 14 days after the first vaccination. On day 35, animals were sacrificed and blood samples were collected (blood bleeding by abdominal aorta or cardiac puncture under O ₂ /CO ₂ anesthesia). Serum from all samples was collected, deep frozen, stored in polypropylene tubes below -10 °C until processing.

Influenza viruses agglutinate red blood cells (RBCs), which are prevented in the presence of sufficient virus-specific antibodies. This phenomenon provides the basis for the Hemagglutination Inhibition (HI) assay, which 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). HI titer is defined as the reciprocal of the highest dilution that still inhibits hemagglutination. The geometric mean titer (GMT) is calculated as follows: 1) Calculate each log (potency) to obtain the arithmetic mean of two replicates: [log (potency ₁ ) + log (potency ₂ )] /2 2) Calculate the arithmetic mean of each log (potency) 3) GMT _(group) = 10 EXP (group mean log (potency))

Statistical analysis

The geometric mean titer was used to summarize the HI titer by vaccination group and number of days. The HI titer of the log transformed day 35 group was analyzed by linear regression to investigate the dose response relationship between the amount of LPP in the vaccine and GMT.

result HI titer analysis

The GMT is shown in Table 5.

On day 0, HA-specific antibodies were not detectable in mice (i.e., all HI titers < 10).

On day 14, no HA-specific antibodies were detected in most of the mice vaccinated by the intranasal route (in). Effective price 10, except one mouse in group A (HI titer: 80), one mouse in group C (HI titer: 35), and one mouse in group D (HI titer: 160) Outside.

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

The HI titer on day 35 of the log-transformed (log-transformed) was compared by linear regression. A suitable regression slope is highly significant (P < 0.0001). Therefore, the dose response relationship between the observed LPP content in the vaccine and GMT is statistically significant.

in conclusion:

Repeated intranasal vaccination of mice with unadjuvanted reconstituted influenza virions does not induce a systemic immune response that can be measured. Repeated intranasal vaccination with reconstituted influenza virions adjuvanted with LPP at the same HA dose level (2 [mu]g HA/dose) with increasing LPP dose showed an LPP dose-dependent immune response. 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 inoculation with inactivated influenza vaccines It 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 (after intranasal delivery in healthy young adults aged 18 and 40)

Healthy human volunteers were dosed with reconstituted influenza virus particles containing LPP (lipopeptide) containing 150 mcg HA/mL and 315 mcg LPP/mL per strain in a dose volume of 0.2 ml (0.1 ml per nostril) Intranasal inoculation. A similar group was intranasally inoculated with a reconstituted influenza virus particle containing 150 mcg HA/mL per strain at a dose volume of 0.2 ml (0.1 ml per nostril). 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.

Research design:

This is in age 18 and A double-blind, randomized, parallel-group study of 40 healthy young individuals. The study was conducted at a research center: Swiss Pharma Contract Ltd., Basel, Switzerland. The researcher of the principle is Dr. M. Seiberling. The study has two parts. In Part I, the safety of the viral particle subunit influenza vaccine entrained with LPP was assessed in 12 individuals. Inoculate nine individuals with 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 Part II of the study, the efficacy and safety of LPP-RVM was assessed in one hundred individuals (50 in each group).

The study was conducted in healthy individuals. In addition, individuals who participated in Part II of the study did not vaccinate the flu three years prior to the start of the study. This increases the homogeneity of the study population in Part II by minimizing the number of individuals with pre-existing antibodies to influenza.

Part I:

14 days prior to vaccination (1st visit), after the individual has given an approval, he or she is screened for inclusion and exclusion criteria and his or her physical examination is performed. During this visit, samples of nasal epithelial cells were collected for cytological analysis and baseline cilia activity was measured using the saccharin test.

On the second visit (Day 1), 4-6 mL of blood samples were taken for standard hematology analysis, and 6-10 mL of blood samples were taken for standard biochemical analysis and vital signs were assessed. After randomization, the individual was vaccinated with one of two vaccine formulations, and the individual stayed on site 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 24 hours after inoculation. In addition, 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 using saccharin test Ciliary activity analysis after inoculation was performed. Questionnaires (questionnaire I) were distributed to individuals and taken home to assess local and systemic reactions on the next day (Day 3).

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

Two weeks after the first vaccination, on day 15, the individual returned to the study site (4th visit). At this visit, samples of nasal epithelial cells were collected for cytological analysis, ciliary activity was measured by the saccharin test, and adverse events occurred between the 3rd visit and the 4th visit were recorded.

Part II:

14 days before the first blood collection and nasal wash sampling (1st visit), after the individual has given a consensus, he or she is screened for inclusion and exclusion criteria and examined by physical examination. His or her health.

On the second visit (-1 day; this visit can be combined with the first visit), take 6-10 mL of blood samples for baseline hemagglutination inhibition (HI) titer determination, and take blood samples for standard hematology (4 -6 mL) and standard biochemical (6-10 mL) analysis. Nasal wash samples were collected for determination of baseline nasal IgA antibody titers.

On the next day, on the third visit (Day 1), after the assessment of vital signs, the individuals were randomized and inoculated with a single dose of one of the two formulations of the nasal influenza 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 mL) and biochemistry (6-10) mL) analysis, nasal wash samples were taken for nasal IgA antibody titer analysis.

Effect evaluation

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

Blood sample

6-10 mL of blood was collected to determine the hemagglutination inhibition (HI) antibody titer. ^{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 repeated in duplicate. The titer of the sample is the geometric mean of the two measurements. Serum before and after vaccination was simultaneously analyzed for potency.

Nasal wash sample

To collect nasal wash samples, 6 mL of pre-warmed saline (37 ° C) was applied to one nostril under nose control. The individual is required to tilt the head at an angle of 60° so that the lotion can flow. The collected lotion was applied to the second nostril, which was washed under the same conditions. A preservative solution (1/100 of the sample volume) was added to the sample. The preservative solution contained 10 mg/ml bovine serum albumin dissolved in 100 mM Tris HCl buffer, pH 8. The sample was directly clarified by low speed centrifugation (800 x g, 10 minutes) and divided into small portions (to avoid repeated thawing of the sample in the future), which was placed on dry ice until transferred to -80 °C.

IgA levels in nasal wash samples were determined by ELISA and statistical analysis was performed using the Wilcoxon test. The influenza vaccine was used as a coating antigen in a 96-well plate. Non-specific binding sites are blocked by incubation with blocking buffer. The nasal washes were diluted two-fold in blocking buffer (12 dilutions per sample) for adsorption of influenza-specific antibodies to antigens in 96-well plates. The 96-well plates were washed prior to incubation with an enzyme-conjugated anti-human antibody (conjugated with horseradish peroxidase or alkaline phosphatase). The amount of influenza strain-specific antibodies was determined by washing to remove unbound anti-human antibodies by measuring the optical density after addition of the substrate for the enzymatic reaction.

Vaccine formulation

Two different influenza vaccine formulations were used for this study. Both formulations contained WHO's recommended viral antigen ^{2 for the} southern hemisphere in 2005 at a dose level of 30 mcg per strain per 0.2 ml dose.

-A/New Caledonia/20/99/(H1N1)-like strain-A/Wellington/1/2004 (H3N2)-like strain-B/Shanghai/361/2002-like strain

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 OEG-containing supernatant was adjusted with the lipopeptide P3CSK4 in OEG buffer, or in the absence of LPP-reconstituted viral membrane, only with OEG-containing buffer (P3CSK4: N-palm- S-[2,3-bis(palmitooxime)-(2RS)-propyl]-[R]-cysteine-[S]-serine-[S]-lysine-[S] - Lysine-[S]-lysine-[S]-lysine). The OEG is removed by adsorption onto a hydrophobic resin. This results in the formation of a viral membrane containing LPP or no LPP (reconstituted viral vesicles containing HA and NA in the membrane and optionally LPP in the membrane). After the OEG was removed, the virus particles were filtered through a PVDF membrane having a pore size of 0.22 μm.

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

^a Lowry test, principle: After treatment with bismuth sulfate and Folin-ciocalteu phenol reagent, the protein forms blue. 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. 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). ^b PhEur: Feature Article 2053 and Section 2.7.1 ^c Principle: Each phospholipid contains a single phosphorus atom, It can be used to quantify phospholipids. The phospholipid is destroyed by perchloric acid, and the phosphate 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 phosphate calibrator. Ames BN.Assay of inorganic phosphate,total phosphate and phosphatases.Meth.Enzymol.1966;8:115-118 B Ttcher CJF, van Gent CM & Pries CA 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 sandwich enzyme immunization A method in which an immobilized polyclonal anti-ovalbumin antibody for capture and an anti-ovalbumin-HRP conjugate are used as a detection system. Simultaneous culture of the conjugate and sample. Unbound components are removed by a washing step. Substrates (TMB and H ₂ O ₂ ) were 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 is added to the substrate to terminate the reaction, which results in a color change in the product which is yellow. The absorbance (OD) was read at 450 nm. For best results, use a reference filter at 620 nm. A standard curve was prepared from the response of the ovalbumin standard (0.3-20.0 ng/ml) included in the assay. 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 checked by polypropylene gel electrophoresis. Electrophoresis: Follow Ph.Eur 2.2.31.

effect

The log-transformed HI antibody titer of each virus strain on day 15 was compared to the nasal IgA antibody titer on day 15 between the two vaccinated groups by the Wilcoxon's ranking sum test at a significant level of 0.05 on both sides. .

The HI antibody titer on day 15 was also analyzed by calculating the following three parameters for each virus strain and each vaccination group.

- seroprotection rate, in which serum protection is defined as red blood cell aggregation inhibition (HI) titer 40, - seroconversion rate, where seroconversion is defined as HI titer <10 before inoculation, HI titer after inoculation 40, or, before the inoculation HI titer 10 and an increase in the HI titer of at least 4 fold, the average fold increase, ie the geometric mean of the increase in the HI titer multiple.

The effect data was analyzed according to the principles of pre-protocol and intent-to-treat. However, since this is a so-called proof-of-principle 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-program sample consisted of vaccinated individuals who completed the protocol and did not experience major program bias. The main deviations include (not limited to): inclusion or exclusion of standard deviations, banned use, etc. In addition, laboratory-verified 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.

result

in conclusion

Unexpectedly, 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, in a human population that was vaccinated only once with an unadjuvanted reconstituted viral particle influenza vaccine, observed A satisfactory systemic immune response to the CHMP standard for influenza vaccines is reached.

Claims (40)

A composition comprising influenza virus particles, the viral particles comprising reconstituted envelopes of the virus, wherein the viral envelope is obtained entirely from influenza virions, wherein no external source is present The reconstituted viral particle is added to a lipid, wherein the viral particle comprises an influenza antigen hemagglutinin and/or neuraminidase or a derivative thereof, wherein no separate (separate) is added to the composition. An adjuvant 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 can be induced. The composition of claim 1, wherein the amount of hemagglutinin administered intranasally or by inhalation per viral strain is equal to or lower than 30 μg. The composition of claim 1, wherein the single intranasal or inhaled administration of the formulation further induces a cytotoxic lymphocyte response. The composition of claim 2, wherein a single intranasal or inhaled administration of the formulation further induces a cytotoxic lymphocyte response. The composition of any one of claims 1 to 4, wherein the immune response conforms to the CHMP standard for influenza vaccines. The composition of claim 5, wherein the immune response provides one or more of the following: >70% for adults and/or >60% for elderly people. Rates, >40% for adults and/or >30% for older adults, and a multiple for adults >2.5 and/or for adults >2.0. The composition of claim 2, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 25 μg. The composition of claim 2, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 20 μg. The composition of claim 2, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 15 μg. The composition of claim 2, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 10 μg. The composition of claim 2, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 5 μg. The composition of any one of claims 1 to 4, wherein the composition is a vaccine formulation comprising a pharmaceutical carrier for intranasal or inhalation administration. The composition of claim 5, wherein the combination A vaccine formulation comprising a pharmaceutical carrier for intranasal or inhalation administration. The composition of claim 6, wherein the composition is a vaccine formulation comprising a pharmaceutical carrier for intranasal or inhalation administration. Use of an influenza virus particle comprising a reconstituted outer shell of the virus for the manufacture of a composition for intranasal or inhalation administration, wherein the viral envelope is obtained entirely from influenza virions, wherein no external source is used The reconstituted viral particle is added to a lipid, wherein the viral particle comprises an influenza antigen hemagglutinin and/or a neuraminidase or a derivative thereof, wherein no separate adjuvant and/or a separate adjuvant is added to the composition An immunostimulatory agent, characterized in that said composition is administered to a human in a single intranasal or inhalation sufficient to induce systemic resistance against said influenza antigen hemagglutinin and/or neuraminidase or a derivative thereof Immune response and / or local immune response. The use according to claim 15, wherein the hemagglutinin dose per intranasal or inhalation of each virus strain is equal to or lower than 30 μg. The use of claim 15, wherein a single intranasal or inhaled administration of the composition also induces a cytotoxic lymphocyte response. The use of claim 16, wherein a single intranasal or inhaled administration of the composition also induces a cytotoxic lymphocyte response. The use of any one of clauses 15 to 18, wherein the immune response conforms to the CHMP standard for influenza vaccines. The use of claim 19, wherein the immune response provides one or more of the following: >70% for adults and/or >60% for older persons. , a seroconversion rate of >40% for adults and/or >30% for the elderly, and a fold increase for adults >2.5 and/or for the elderly >2.0. The use according to claim 16, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 25 μg. The use according to claim 16, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 20 μg. The use according to claim 16, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 15 μg. The use according to claim 16, wherein the dose of hemagglutinin administered intranasally or by inhalation is less than or equal to 10 μg per virus strain. The use according to claim 16, wherein the dose of hemagglutinin administered intranasally or by inhalation is less than or equal to 5 μg per virus strain. The use according to any one of claims 15 to 18, wherein the composition produced is a vaccine formulation. The use of claim 19, wherein the composition produced is a vaccine formulation. The use of claim 20, wherein the composition produced is a vaccine formulation. A vaccine formulation comprising a composition of 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 reconstituted viral particles are obtained from an external source Adding a lipid, wherein the viral particle comprises an influenza antigen hemagglutinin and/or a neuraminidase or a derivative thereof, wherein no separate adjuvant and/or immunostimulant is added to the composition, wherein the vaccine characteristic The vaccine is designed for a single intranasal or inhaled administration to humans, and wherein each viral strain has a hemagglutinin dose of 30 μg or less per intranasal or inhaled administration, and Wherein the single intranasal or inhalation administration of the composition to a human induces a systemic immune response and/or a local immune response against the influenza antigen hemagglutinin and/or neuraminidase or a derivative thereof. The vaccine formulation according to claim 29, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 25 μg. The vaccine formulation according to claim 29, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 20 μg. The vaccine formulation according to claim 29, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 15 μg. A vaccine formulation as described in claim 29, wherein each The dose of hemagglutinin administered intranasally or by inhalation of the virus strain is less than or equal to 10 μg. The vaccine formulation according to claim 29, wherein the dose of hemagglutinin administered intranasally or by inhalation per viral strain is less than or equal to 5 μg. The vaccine formulation according to any one of claims 29 to 34, wherein the single intranasal or inhalation administration of the composition to a human can also induce a cytotoxic lymphocyte-mediated immune response. The vaccine formulation according to claim 35, wherein the immune response conforms to the CHMP standard for influenza vaccine. The vaccine formulation according to claim 36, wherein the immune response provides one or more of the following: >70% for adults and/or >60% for elderly people. Protection ratio, >40% for adults and/or >30% for seniles for adults, and a multiple for adults >2.5 and/or for adults >2.0. A device for intranasal or inhalation administration, the device comprising a vaccine formulation as described in any one of claims 29 to 37, and a mechanism for aerosolizing the vaccine. The device of claim 38, wherein the device comprises a quantity of a vaccine formulation for a single intranasal or inhaled administration. The device of claim 38, wherein the device is disposable.