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TWI397419B - Intranasal or inhalational administration of virosomes

Publication number

TWI397419B

TWI397419B TW096109319A TW96109319A TWI397419B TW I397419 B TWI397419 B TW I397419B TW 096109319 A TW096109319 A TW 096109319A TW 96109319 A TW96109319 A TW 96109319A TW I397419 B TWI397419 B TW I397419B

Authority

TW

Taiwan

Prior art keywords

composition

hemagglutinin

inhalation

influenza

intranasal

Prior art date

2006-03-22

• Espacenet
• Global Dossier
• Discuss

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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/145—Orthomyxoviridae, e.g. influenza virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
  • A61K2039/543—Mucosal route intranasal
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Virology (AREA)
• Mycology (AREA)
• Epidemiology (AREA)
• Immunology (AREA)
• Medicinal Chemistry (AREA)
• Microbiology (AREA)
• Veterinary Medicine (AREA)
• Pharmacology & Pharmacy (AREA)
• Chemical & Material Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Pulmonology (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicinal Preparation (AREA)

本發明涉及，例如，用於滅活流感疫苗的組合物以及給藥方式，其中，單次鼻內或吸入給藥獲得了與臨床保護正相關的全身性免疫應答。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.

用於通過鼻內或口咽途徑，以及使用滅活流感抗原針對流感進行免疫的多種觀念已作為皮下或肌肉內免疫的非注射(無針頭needle－less)替代途徑被研究。已在動物模型中產生了支持非注射手段的實驗數據。得到動物數據支持的、使用滅活流感抗原(例如化學方式滅活的完整病毒粒子(virus particles)，或者被進一步加工的病毒組分，例如被切開的病毒，或者經純化的表面抗原血球凝集素(HA)和/或神經氨酸酶(NA))以通過鼻內途徑進行免疫的思路包括：使用佐劑或免疫刺激劑(immune stimulator)與滅活流感抗原組合，或者需要多次免疫。佐劑是能增強與其混合的抗原的免疫原性的任何物質。在人類中，通過鼻內途徑抵抗流感的成功疫苗接種僅報導過：(a)活的(經冷改造的毒株)流感疫苗(FluMist^TM ，MedImmune Vaccines Inc)(參考文獻1、2、3)，(b)病毒顆粒型流感疫苗，其佐有大腸桿菌(E.coli)的熱不穩定毒素(heat labile toxin)(NasalFlu，Berna Biotech Ltd)(參考文獻4)或(c)使用高劑量抗原和重複疫苗接種(參考文獻5、10、11)。雖然活疫苗能誘導滿意的免疫應答，但其能變成活病毒的特定性質導致了額外的安全顧慮，由於需要在上呼吸道附近的病毒複製，這可能會誘導出副作用。此外，所需的貯藏條件也限制了這些產品的商業化。使用用大腸桿菌HLT作為佐劑的鼻內流感疫苗與面癱(Bell’s Palsy)之間的強烈相關性導致佐有HLT的病毒顆粒疫苗從市場撤出(參考文獻6)。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).

流感疫苗在給定人群中的療效可通過下述方法來評估：評定接種後產生的抗流感抗體的數量相關的免疫原性參數。這些免疫原性參數通常被稱為CHMP標率，其用於對滅活流感疫苗進行每年重新許可認證(參考文獻7)。至今為止，通過使用滅活疫苗的一次單次鼻內施予，不加佐劑(其是疫苗中的附加成分，其不來自將用疫苗來預防的感染性試劑，並且是為了增強對抗原的免疫應答的目的被加入到疫苗配方製劑中)，就達到這些免疫學要求或者CHMP標準(參考文獻7)的、對人類抵抗流感的成功免疫尚未有記載。因此，人們認識到，在本領域中仍有對下述滅活流感疫苗組合物的需求，所述組合物能在單次鼻內給藥之後誘導滿意的全身性免疫反應，並且不含佐劑，且通過所述單次給藥能達到CHMP標準(參考文獻7)。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).

所述“CHMP標準”按照下文所述被定義。在CHMP(Committee for Medicinal Products for Human Use)的Note for Guidance on Harmonisation of Requirements for Influenza Vaccines中，定義了下述血清學參數，以評定滅活流感疫苗的免疫原性：－血清保護率，其中血清保護被定義為紅血球凝聚抑制(Haemagglutination Inhibition,HI)效價40，－血清轉化率，其中血清轉化被定義為接種前HI效價<10，接種後HI效價40，或者，接種前HI效價10以及HI效價至少4倍的增加，－平均倍數增加值，這是對個體內增加的幾何平均值(即，接種後HI效價/接種前HI效價)。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).

針對流感疫苗免疫原性的CHMP要求是：對於疫苗中三種病毒毒株中的每種而言，達到下述標準中的至少一項： 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:

本發明還應用於兒童，從他們顯示出，他們以與成年人相當的方式作出免疫應答(參考文獻8)。本發明還應用於老年個體。老年人為超過60歲的。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.

令人吃驚地，以及與臨床前齧齒類數據和關於人類臨床經歷的文獻相反地，我們發現，對於18－60歲年齡組而言，用包含重構流感病毒外殼的滅活流感疫苗進行單次鼻內接種後，人類的免疫應答達到了針對流感疫苗療效的全部三個CHMP標準。一次單次鼻內施予是：為達到針對滅活流感疫苗免疫原性的上述CHMP標準，通過一個或兩個鼻孔進行疫苗配方製劑的接種，而無需重複施予疫苗配方製劑。一次單次疫苗施予(通過鼻、吸入、口服、皮下或肌內途徑)通常是下述接種安排，其不包括：在本領域作為初次(priming)和強化(boosting)而已知的、以數天或數周的時間間隔對疫苗的多次施予。被設計為鼻內或吸入給藥配方製劑的配方製劑包含一種或多種活性組分和賦形劑的混合物，是按照允許鼻內或吸入給藥的方式製備的。本發明提供了一種方法，用於誘導達到CHMP標準的全身性免疫應答(循環的免疫球蛋白或產生抗體的B細胞)，有利地，所述方法用對病毒顆粒流感疫苗的單次鼻內或吸入給藥來進行。本發明還提供了一種方法，用於誘導局部或粘膜免疫應答，所述應答包括粘膜膜表面上作為IgA已知的分泌免疫球蛋白的增加，有利地，所述方法用對病毒顆粒流感疫苗的單次鼻內或吸入給藥來進行。鼻內施予之後對特異性IgG和IgA應答的誘導涉及鼻腔中淋巴組織的活性(參考文獻12)。此類組織作為鼻相關淋巴組織(NALT)已知，其還被顯示為用於細胞免疫應答的粘膜誘導位點(參考文獻13)。因為已知病毒顆粒具有誘導細胞內免疫應答的可能性(參考文獻14、15)，因此本發明還提供了誘導特異性細胞毒性淋巴細胞(CTL)的方法。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).

病毒顆粒(virosome)是含有病毒糖蛋白的脂質雙層。通常通過用去垢劑從帶包膜病毒提取膜蛋白和脂類，接著通過除去所述去垢劑來重構特徵性雙層來生產病毒顆粒。本發明還提供了流感病毒顆粒的組合物，其包含重構的流感病毒外膜(具體而言，不再加脂類，並且不加免疫刺激劑(通常稱為佐劑)的免疫調節劑而重構)，用於通過氣溶膠進行接種，所述氣溶膠通過一個或兩個鼻孔施予鼻咽或口咽的粘膜，以獲得抵抗流感的全身性和局部免疫性。通過吸入進行的單次施予也是可行的。單次口服粘膜施予也是可行的。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.

可從滅活病毒來製備重構流感病毒顆粒，可以用不可透析的去垢劑對滅活病毒加以溶解，通過吸附到疏水珠粒上來除去所述去垢劑。製備物可包含一種或多種流感抗原的經過純化的懸浮液，所述流感抗原選自血球凝集素(HA)、神經氨酸酶(NA)、血球凝集素的衍生物和神經氨酸酶的衍生物。可以在病毒脂類(含有低水平的內毒素和卵清蛋白)構成的膜中，對病毒膜蛋白血球凝集素和神經氨酸酶進行重構(見參考文獻9)。血清凝集素和/或神經氨酸酶的衍生物是具有經修飾的氨基酸序列和/或結構的血球凝集素和/或神經氨酸酶分子。氨基酸可例如被刪除、替換或添加到序列中。此外，糖基化方式可被改變。衍生物保留有導入宿主時誘導免疫應答的能力。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.

可在例如含有胚胎的雞蛋中，或在附著的細胞或懸浮液中的細胞的細胞培養物中培養用於製備重構病毒顆粒的流感病毒。病毒例如可以是野生型的或重配的(reassortant)或經過遺傳修飾的毒株。病毒類型可例如是任何流感A或B亞型，包括流行性的毒株。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.

本發明還提供疫苗。術語疫苗應被理解為具有免疫活性的藥物製備物。在某些實施方式中，疫苗可包含病原性微生物的無害變體或衍生物，例如，刺激免疫系統產生針對真實病原體的抵抗。在某些實施方式中，疫苗施予宿主時例如能誘導適應性免疫。疫苗可含有病原體或病原體組分的已死亡或削弱形式，例如病原體的抗原性組分。疫苗製備物可還含有藥物載體，可針對疫苗將由其被施予的特定模式對載體加以設計，例如針對鼻內或吸入給藥設計的藥物載體。流感疫苗可包含一種或多種未變性流感抗原，其一種或多種能誘導對流感特異的免疫應答。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.

本發明提供了含流感病毒顆粒的組合物，所述流感病毒顆粒包含所述病毒的重構外殼，其中所述組合物被設計為鼻內或吸入給藥配方製劑。本發明還提供了其中病毒顆粒包含流感抗原血球凝集素和/或神經氨酸酶或其衍生物的所述組合物。本發明還提供了其中病毒外殼完全從病毒粒子獲得的所述組合物。本發明還提供了其中沒有脂類從外界來源向重構病毒顆粒加入的所述組合物。本發明還提供了其中沒有向所述組合物加入單獨的佐劑和/或免疫刺激劑的所述組合物。本發明還提供了其中向個體的單次鼻內或吸入給藥能誘導全身性免疫應答的所述組合物。本發明還提供了其中向個體的單次鼻內或吸入給藥還能誘導局部免疫應答的所述組合物。本發明還提供了向個體的單次鼻內或吸入給藥還能誘導細胞毒性淋巴細胞應答的所述組合物。本發明還提供了其中誘導全身性免疫應答和/或局部免疫應答和/或細胞毒性淋巴細胞應答的能力在人類中顯示的所述組合物。本發明還提供了其中免疫應答包含針對流感抗原血球凝集素和/或神經氨酸酶或其衍生物的免疫應答的所述組合物。在一種優選的實施方式中，本發明還提供了其中免疫應答達到用於流感疫苗的CHMP標準的所述組合物。本發明還提供了其中免疫應答提供下述中的一種或多種的所述組合物：對成年人而言>70%和/或對老年人而言>60%的血清保護率，針對成年人>40%和/或針對老年人>30%的血清轉化率，以及針對成年人>2.5和/或針對老年人>2.0的平均倍數增加值。在一種特別優選的實施方式中，本發明還提供了其中每種病毒毒株每次鼻內或吸入給藥的血球凝集素劑量等於或低於30 μ g的所述組合物。最後，本發明還提供了其中組合物是包含用於鼻內或吸入給藥的藥物載體的疫苗配方製劑的所述組合物。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.

本發明還提供了包含病毒的重構外殼的流感病毒顆粒用於製造用於鼻內或吸入給藥的組合物的用途。本發明還提供了這樣的所述用途：其中，流感病毒顆粒包含流感抗原血球凝集素和/或神經氨酸酶或其衍生物。本發明還提供了這樣的所述用途：其中，病毒外殼完全從流感病毒粒子獲得。本發明還提供了這樣的所述用途：其中，沒有脂類從外界來源向重構病毒顆粒加入。本發明還提供了這樣的所述用途：其中，沒有向所述組合物加入單獨的佐劑和/或免疫刺激劑。本發明還提供了這樣的所述用途：其中，向個體的單次鼻內或吸入給藥足以誘導全身性免疫應答。本發明還提供了這樣的所述用途：其中，向個體的單次鼻內或吸入給藥還誘導局部免疫應答。本發明還提供了這樣的所述用途：其中，向個體的單次鼻內或吸入給藥還誘導細胞毒性淋巴細胞應答。本發明還提供了這樣的所述用途：其中，接受給藥的個體是人類。本發明還提供了這樣的所述用途：誘導的免疫應答包含針對流感抗原血球凝集素和/或神經氨酸酶或其衍生物的免疫應答。在一種優選的實施方式中，本發明還提供了這樣的所述用途，其中，組合物誘導達到針對流感疫苗的CHMP標準的免疫應答。本發明還提供了這樣的所述用途，其中，免疫應答提供下述中的一種或多種的所述組合物：對成年人而言>70%和/或對老年人而言>60%的血清保護率，針對成年人>40%和/或針對老年人>30%的血清轉化率，以及針對成年人>2.5和/或針對老年人>2.0的平均倍數增加值。在一種特別優選的實施方式中，本發明還提供了這樣的所述用途，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素被施予劑量等於或低於30 μ g。最後，本發明還提供了其中製造的組合物是疫苗配方製劑的所述用途。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.

因此，在一種實施方式中，本發明提供了包含病毒的重構外殼的流感病毒顆粒的組合物，其中，所述病毒外殼完全從流感病毒粒子獲得，其中，沒有從外界來源向重構病毒顆粒加入脂類，其中，病毒顆粒包含流感抗原血球凝集素和/或神經氨酸酶或其衍生物，其中，沒有向所述組合物加入單獨的佐劑和/或免疫刺激劑，以及其中，所述組合物作為鼻內或吸入給藥配方製劑來設計，所述組合物特徵在於，所述配方製劑向人類的單次鼻內或吸入給藥能誘導抵抗所述流感抗原的全身性免疫應答和/或局部免疫應答，所述全身性應答能符合用於流感疫苗的CHMP標準，以及其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素劑量等於或低於30 μ g。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.

根據另一種實施方式，本發明提供了包含病毒的重構外殼的流感病毒顆粒用於製造用於鼻內或吸入給藥的組合物的用途，其中，所述病毒外殼完全從流感病毒粒子獲得，其中，沒有從外界來源向重構病毒顆粒加入脂類，其中，病毒顆粒包含流感抗原血球凝集素和/或神經氨酸酶或其衍生物，其中，沒有向所述組合物加入單獨的佐劑和/或免疫刺激劑，所述流感病毒顆粒用於製造用於鼻內或吸入給藥的組合物的用途的特徵在於，所述組合物向人類的單次鼻內或吸入給藥足以誘導抵抗所述流感抗原的全身性免疫應答和/或局部免疫應答，所述應答能達到針對流感疫苗的CHMP標準，以及其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素劑量等於或低於30 μ g。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.

根據另一種實施方式，本發明提供了一種包含流感病毒顆粒的組合物的疫苗配方製劑，所述病毒顆粒包含所述病毒的重構外殼，其中，所述病毒外殼完全從流感病毒粒子獲得，其中，沒有從外界來源向重構病毒顆粒加入脂類，其中，病毒顆粒包含流感抗原血球凝集素和/或神經氨酸酶或其衍生物，其中，沒有向所述組合物加入單獨的佐劑和/或免疫刺激劑，其中，疫苗特徵在於，針對向人類的單次鼻內或吸入給藥來設計疫苗，並且，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素劑量等於或低於30 μ g。有利地，所述配方製劑的所述單次鼻內或吸入給藥能在所述人類中誘導全身性和/或局部免疫應答。根據本發明還提供了包含一定量的用於單次鼻內或吸入給藥的所述疫苗配方製劑的設備。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.

每種病毒毒株每次鼻內或吸入給藥的血球凝集素的根據本發明的應用劑量還可以低於或等於25 μ g、20 μ g、15 μ g、10 μ g或5 μ g。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.

(1)Maassab HF.Adaptation and growth characteristics of influenza virus at 25℃,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,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 Harmonisation of Requirements for Influenza Vaccines.EMEA/CpMP/BWP/214/96(8)Daubeney,P.,Taylor,C.J.,McGaw,J.,Brown,Brown,E.M.,Ghosal,S.,Keeton,B.R.,Palache,B.,Kerstens,R.Immunogenicity and tolerability of a trivalent influenza subunit vaccine(InfluvacR)in high－risk children aged 6 months to 4 years.BJCP 1997 March,51(2)：87－90(9)Stegmann,T.,Morselt,H.W.M.,Booy,F.P.,Van Breemen,J.F.L.,Scherphof,G.,Wilschut,J.Functional reconstitution of influenza viris envelopes.EMBO Journal 1987,6(9)：2651－2659(10)Treanor J,Nolan C,O’ Brien D,Burt D,Lowell G,Linden J,Fries L.Intranasal administration of a proteosome－influenza vaccine is well－tolerated and induces serum and nasal secretion influenza antibodies in healthy human subjects.Vaccine 2006；24(3)：2 54－62(11)Read R.C.,Naylor S.C.Potter C.W.,Bond J.,Jabbal－Gill I.,Fisher A.,Illum L.,Jennings R.Effective nasal influenza vaccine delivery using chitosan.Vaccine 2005；23(35)：4367－74(12)Kuper CF,Koornstra PJ,Hameleers DM,Biewenga J,Spit BJ,Duijvestein AM,van Breda Vriesman PJ,Sminia T.The role of nasopharyngeal lymphoid tissue.Immunol.Today 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)Glck R,Burri KG,Metcalfe I,Adjuvant and antigen delivery properties of virosomes.Curr.Drug Deliv.2005 2：395－400(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

流感血清陰性雌性Balb/c小鼠組成的組(每組10只)通過鼻內施予獲得LPP(脂肽)－病毒顆粒疫苗，其HA/LPP比例為1：1.5、1：0.7、1：0.4、1：0(即沒有LPP)，並且每份劑量具有2 μ g的HA。此外，10只雌性小鼠的對照組獲得0 μ g HA/劑量(載體的鼻內施予)。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).

製備含有LPP的病毒顆粒的四種製備物。簡言之，通過離心對30－40%蔗糖溶液中的滅活流感病毒進行沈澱。重新懸浮病毒，溶解於含有去垢劑八聚乙二醇單十二烷基醚(OEG)的緩衝液中。隨後，通過超離心除去病毒核殼體。將含有OEG的上清液分為4等分體積，加入不同量的含OEG緩衝液中的脂肽P3CSK4(P3CSK4：N－棕櫚醯－S－[2,3－雙(棕櫚醯氧)－(2RS)－丙基]－[R]－半胱氨醯－[S]－絲氨醯－[S]－賴氨醯－[S]－賴氨醯－[S]－賴氨醯－[S]－賴氨酸)。用含OEG的緩衝液調節體積。通過吸附到疏水樹脂上除去OEG。這導致了含LPP的病毒顆粒的形成，在它們膜中含有HA和NA以及(可選地)重構的病毒泡囊在它們膜中含有LPP。OEG去除之後，將病毒顆粒濾經孔徑為0.22 μm的PVDF膜。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.

起始原料為20 mg的HA，其來自甲型流感/Wyoming/3/2003 X－147(類A/Fujian/411/200(H3N2)毒株)，含有252 I.U.內毒素/100 μ g HA。溶解後，按照表1所概述的製備4批。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.

載體由5 mM Hepes、145 mM NaCl、1 mM EDTA(pH 7.4)構成。對組E(見表2)來說，將載體濾經孔徑為0.22 μm的PVDF膜。按照表2所述，針對鼻內免疫，將製備的4批病毒顆粒稀釋至200 μ g/ml的濃度，針對肌內免疫，稀釋至67 μ g/ml的濃度，分裝進1 ml的小瓶(每組2瓶)。按照表4所概述的來使用這些疫苗組。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.

針對表3所示的若干種變數來分析用於本研究的配方製劑。Formulation formulations for this study were analyzed for several variables shown in Table 3.

^a Lowry試驗，原理：用堿式硫酸銅和Folin－ciocalteu酚試劑處理之後，蛋白質形成藍色。使用白蛋白BSA標準作為參考，從750 nm處的吸光度來測定蛋白質含量。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：專題文章2053和2.7.1節^c 原理：每個磷脂含有單個磷原子，其可用於對磷脂進行定量。通過高氯酸來破壞磷脂，通過鉬酸鹽配合產生的磷酸鹽/酯，鉬酸鹽被抗壞血酸還原產生藍色的產物。用分光光度計在812 nm處測定顏色。通過包括磷酸校準物來對樣品中磷脂的量加以定量。Ames BN.Assay of inorganic phosphate,total phosphate and phosphatases.Meth.Enzymol.1966；8：115－118 Bttcher CJF,van Gent CM & Pries C.A rapid and sensitive sub－micro phosphorus determination.Anal.Chim.Acta 1961；24：203－204^d Ph.Eur.2.6.14^e 卵清蛋白ELISA是直接的三明治式酶免疫方法，其中使用用於捕捉的被固定的多克隆抗卵清蛋白抗體以及抗卵清蛋白－HRP結合物作為檢測系統。對結合物和樣品進行同時培養。通過洗滌步驟去除未結合的組分。將底物(TMB和H₂ O₂ )加入到孔中。通過藍色的顯色來指示孔中特異性結合的結合物的存在。向底物中加入硫酸來終止反應，其導致產物中顏色變化為黃色。在450 nm讀取吸光度(OD)。為獲得最優結果，使用620 nm處的參照濾光器。從試驗中包括的卵清蛋白標準(0.3－20.0 ng/ml)的應答來製造標準曲線。未知樣品的濃度從標準曲線內插來讀取。^f 根據專題文章0869和2053：通過聚丙烯醯胺凝膠電泳來檢查單價合併的(monovalent pooled)收集物的純度。電泳：按照Ph.Eur 2.2.31來進行。 ^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.

使用七組動物，每組十隻雌性Balb/c小鼠(BALB/cAnNCrl)。Seven groups of animals, ten female Balb/c mice (BALB/cAnNCrl), were used.

在處理的一開始，小鼠為8－9周齡，重量為17－19 g。At the beginning of the treatment, the mice were 8-9 weeks old and weighed 17-19 g.

在第0天和第14天用單價LPP病毒顆粒流感疫苗(A/Wyoming)對動物進行鼻內接種，在第二次接種後21天進行屍體檢查。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.

鼻內：測試物質被鼻內接種(10 μ l，分成兩個鼻孔上進行)，這用在背部輕度異氟烷/O₂ /N₂ O麻醉的動物進行。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.

在第一次接種之前以及第一次接種14天之後，在異氟烷/O₂ /N₂ O麻醉下收集眼窩血樣。第35天，處死動物，收集血樣(在O₂ /CO₂ 麻醉下通過腹部大動脈或心臟穿刺放血)。收集來自全部樣品的血清，深度冷凍，於低於－10℃貯存在聚丙烯管中直到進行處理。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.

流感病毒使紅血球細胞(RBCs)凝集，在存在足夠的病毒特異性抗體時這會被阻止。該現象提供了血細胞凝集抑制(HI)試驗的基礎，該試驗被用於探測和定量血清中的特異性抗病毒抗體。將血清加入到流感病毒和火雞RBCs中。對若干稀釋液加以測試(效價分析)。HI效價被定義為仍能抑制血細胞凝集的最高稀釋度的倒數。按下文所述來計算幾何平均值效價(GMT)：1)對各個log(效價)進行計算，得到兩次重複的算術平均值：[log(效價₁ )＋log(效價₂ )]/2 2)計算各個log(效價)的算術平均值3)GMT_(組) ＝10 EXP(組平均值log(效價))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))

通過接種組和天數，使用幾何平均值效價來概括HI效價。通過線性回歸來分析經log轉化的第35天組的HI效價，以研究疫苗中LPP的量與GMT之間的劑量應答關係。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.

GMT示於表5中。The GMT is shown in Table 5.

第0天，在小鼠中不能探測到HA特異性抗體(即，所有HI效價<10)。On day 0, HA-specific antibodies were not detectable in mice (i.e., all HI titers < 10).

第14天，在通過鼻內途徑(i.n.)接種的小鼠中的大多數中不能探測到HA特異性抗體。所有效價10，除了組A中的一隻小鼠(HI效價：80)、組C中的一隻小鼠(HI效價：35)和組D中的一隻小鼠(HI效價：160)之外。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.

第35天，觀察到了HA特異性抗體產生中的劑量應答，即，向疫苗中加入更多的LPP導致更高的抗體效價。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.

通過線性回歸在組間比較(經log轉換的)第35天的HI效價。適合的回歸斜率高度顯著(P<0.0001)。因此，觀察到的疫苗中的LPP含量與GMT之間的劑量應答關係在統計上是顯著的。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.

用無佐劑的重構流感病毒顆粒對小鼠進行重複鼻內接種不誘導可被測量到的全身性免疫應答。以同樣HA劑量水平(2μg HA/劑量)，採用上升的LPP劑量，用佐以LPP的重構流感病毒顆粒進行的重複鼻內接種顯示出了LPP劑量依賴型免疫應答。與本發明完全相反(見下面的實施例2)，這些數據之前被認為支持本領域的公認結論，即，使用免疫刺激劑(在這種情況下LPP)對於用滅活流感疫苗進行鼻內接種是必要的，即使流感抗原(HA)存在於重構病毒顆粒中。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.

用含有每毒株150 mcg HA/mL和315 mcg LPP/mL的、佐有LPP(脂肽)的重構流感病毒顆粒以0.2 ml的劑量體積(每個鼻孔0.1 ml)對健康人類志願者進行鼻內接種。用含有每毒株150 mcg HA/mL的、沒有LPP的重構流感病毒顆粒以0.2 ml的劑量體積(每個鼻孔0.1 ml)對類似的組進行鼻內接種。研究目的是在男人中驗證小鼠中展示的觀點，即，為在用滅活流感疫苗進行鼻內接種後獲得滿意的全身性免疫應答，需要使用佐劑(例如LPP)。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.

這是在年齡18和40的健康年輕個體中進行的雙盲、隨機平行組研究。該研究在一個研究中心進行：Swiss Pharma Contract Ltd.,瑞士巴塞爾。原理的研究者是M.Seiberling博士。研究具有兩個部分。在部分I中，在12個個體中評定佐有LPP的病毒顆粒亞基流感疫苗的安全性。用LPP－RVM(LPP重構的病毒膜；流感疫苗－表面抗原，滅活的，病毒顆粒－佐有LPP)接種九個個體，用RVM(流感疫苗－表面抗原，滅活的，病毒顆粒－)來接種三個個體。在研究的部分II中，在一百個個體中(每組50個)評定LPP－RVM的療效和安全性。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).

研究在健康個體中進行。此外，在研究開始前三年，參與研究的部分II的個體都沒有針對流感進行接種。這通過儘量減小具有預先存在的針對流感的抗體的個體的數目，增加了部分II中研究人群的同質性。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.

在接種前14天(第1次訪問)，在個體已經給出贊成意見後，針對包括和排除標準對他或她加以篩選，並對他或她進行身體檢查。在該次訪問中，採集鼻上皮細胞的樣品用於細胞學分析，用糖精試驗來測量基線纖毛(cilia)活性。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.

在第2次訪問(第1天)，取4－6 mL血樣，用於標準血液學分析，取6－10 mL血樣用於標準生物化學分析，並評定生命體征。經過隨機化後，用兩種疫苗配方製劑之一來接種個體，在接種之後個體就地停留第一個24小時，以監測立即產生的局部和全身性反應以及不利的事件。在接種後第四和二十四小時評定生命體征。此外，24小時後，取兩份血樣用於標準血液學(4－6 mL)和生物化學(6－10 mL)分析；接種後，採集鼻上皮細胞的樣品用於細胞學分析，用糖精試驗進行接種後纖毛活性分析。向個體發放調查問卷(調查問卷I)，讓他們帶回家，評定下一天(第3天)的局部和全身性反應。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).

個體必須在他們被釋放回家後兩周回到研究點：第3次訪問(第4天)。在該次就診中，評定局部和全身性反應，記錄前次和本次就診之間發生的任何自發不利事件。此外取兩份血樣用於標準血液學(4－6 mL)和生物化學(6－10 mL)分析，並評定生命體征。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.

在第一次接種後兩周，在第15天，個體回到研究點(第4次訪問)。在該次就診中，收集鼻上皮細胞的樣品用於細胞學分析，用糖精試驗測量纖毛活性，記錄第3次訪問和第4次訪問間發生的不利事件。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.

在第一次採血和鼻洗出物(wash)採樣前14天(第1次訪問)，在個體已經給出贊成意見後，針對包括和排除標準對他或她加以篩選，通過身體檢查來檢測他或她的健康。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.

在第2次訪問(-1天；該就診可以與第1次就診合併進行)，取6-10 mL血樣用於基線血細胞凝集抑制(HI)效價測定，並且取血樣進行標準血液學(4-6 mL)和標準生物化學(6-10 mL)分析。收集鼻洗出物樣品用於測定基線鼻IgA抗體效價。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.

下一天，在第3次訪問(第1天)，在對生命體征的評定之後，對個體進行隨機化，用鼻流感疫苗的兩種配方製劑之一的單一劑量進行接種。在接種後的第一個小時中，就地監測任何立即的局部反應、全身性反應和不利事件。之後，重新評定生命體征，個體獲得調查問卷帶回家，在接種後的第一個七天，記錄每天的局部和全身性反應。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.

兩周後(第4次訪問；第15天)，取6-10 mL血樣用於HI效價測定，取兩份額外血樣用於標準血液學(4-6 mL)和生物化學(6-10 mL)分析，取鼻洗出物樣品用於鼻IgA抗體效價分析。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.

為評定效果，在第1天(基線)和第15天收集血樣和鼻洗出物樣品。To assess the effect, blood samples and nasal wash samples were collected on day 1 (baseline) and day 15.

收集6-10 mL血，以測定血細胞凝集抑制(HI)抗體效價。¹ 血液收集和凝固之後(室溫下至少30分鐘)，分離血清，並保持冷凍(－20℃)，直到進行效價分析。以雙份重複進行抗體效價分析。樣品的效價是兩次測定的幾何平均值。接種前和接種後的血清被同時進行效價分析。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.

為收集鼻洗出物樣品，於一個鼻孔，在鼻鏡檢查控制下，應用6 mL預熱的鹽水(37℃)。個體被要求將頭傾60°角，使得洗液可以流動。收集的洗液應用於第二個鼻孔，其在相同的條件下被洗滌。向樣品中加入防腐溶液(樣品體積的1/100)。防腐溶液含有10 mg/ml溶解於100 mM Tris HCl緩衝液中的牛血清白蛋白，pH 8。通過低速離心(800 x g，10分鐘)來直接澄清樣品，將其分為小份(以避免今後對樣品的重複解凍)，將其放置在乾冰上，直到轉移進－80℃。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.

通過ELISA來測定鼻洗滌樣品中的IgA水平，用Wilcoxon試驗來進行統計分析。流感疫苗在96孔板中被用作塗敷抗原。通過與封閉緩衝液一起溫育來封閉非特異性結合位點。將鼻洗出物以封閉緩衝液兩倍稀釋(每份樣品12份稀釋液)，用於將流感特異性抗體吸附到96孔板的抗原上。在與酶結合的抗人抗體(結合有馬辣根過氧化物酶或鹼性磷酸酶)一起溫育之前對96孔板加以洗滌。通過洗滌除去未結合的抗人抗體，通過在加入用於酶反應的底物之後測量光密度來測定流感毒株特異性抗體的量。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.

兩種不同的流感疫苗配方製劑用於本項研究。兩種配方製劑都含有WHO為2005年南半球推薦的病毒抗原² ，其劑量水平為每0.2 ml的劑量每毒株30 mcg。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)樣毒株－A/Wellington/1/2004(H3N2)樣毒株－B/Shanghai/361/2002樣毒株-A/New Caledonia/20/99/(H1N1)-like strain-A/Wellington/1/2004 (H3N2)-like strain-B/Shanghai/361/2002-like strain

簡言之，通過離心對30－40%蔗糖溶液中的滅活流感病毒進行沈澱。重新懸浮病毒，溶解於含有去垢劑，八聚乙二醇單十二烷基醚(OEG)的緩衝液中。隨後，通過超離心除去病毒核殼體。用含OEG緩衝液中的脂肽P3CSK4來調節含OEG的上清液，或者在不含LPP的重構病毒膜的情況下，僅用含OEG的緩衝液來調節(P3CSK4：N－棕櫚醯－S－[2,3－雙(棕櫚醯氧)－(2RS)－丙基]－[R]－半胱氨醯－[S]－絲氨醯－[S]－賴氨醯－[S]－賴氨醯－[S]－賴氨醯－[S]－賴氨酸)。通過吸附到疏水樹脂上除去OEG。這導致了含LPP或不含LPP的病毒膜(在膜中含有HA和NA以及可選地在膜中含有LPP的重構病毒泡囊)的形成。OEG去除之後，將病毒顆粒濾經孔徑為0.22 μm的PVDF膜。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.

對於病毒的每種毒株而言，製備具有或不具有LPP的單獨的製備物(表6)。加入的LPP的量相應於1：0.7(w/w)的HA/LPP比例。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試驗，原理：用堿式硫酸銅和Folin－ciocalteu酚試劑處理之後，蛋白質形成藍色。使用白蛋白BSA標準作為參考，從750 nm處的吸光度來測定蛋白質含量。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：專題文章2053和2.7.1節^c 原理：每個磷脂含有單個磷原子，其可用於對磷脂進行定量。通過高氯酸來破壞磷脂，通過鉬酸鹽配合產生的磷酸鹽/酯，鉬酸鹽被抗壞血酸還原產生藍色的產物。用分光光度計在812 nm處測定顏色。通過包括磷酸校準物來對樣品中磷脂的量加以定量。Ames BN.Assay of inorganic phosphate,total phosphate and phosphatases.Meth.Enzymol.1966；8：115－118 Bttcher CJF,van Gent CM & Pries C.A rapid and sensitive sub－micro phosphorus determination.Anal.Chim.Acta 1961；24：203－204 dPh.Eur.2.6.14,^e 卵清蛋白ELISA是直接的三明治式酶免疫方法，其中使用用於捕捉的被固定的多克隆抗卵清蛋白抗體以及抗卵清蛋白－HRP結合物作為檢測系統。對結合物和樣品進行同時培養。通過洗滌步驟去除未結合的組分。將底物(TMB和H₂ O₂ )加入到孔中。通過藍色的顯色來指示孔中特異性結合的結合物的存在。向底物中加入硫酸來終止反應，其導致產物中顏色變化為黃色。在450 nm讀取吸光度(OD)。為獲得最優結果，使用620 nm處的參照濾光器。從試驗中包括的卵清蛋白標準(0.3－20.0 ng/ml)的應答來製造標準曲線。未知樣品的濃度從標準曲線內插來讀取。^f 根據專題文章0869和2053：通過聚丙烯醯胺凝膠電泳來檢查單價合併的收集物的純度。電泳：按照Ph.Eur 2.2.31來進行。 ^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.

通過Wilcoxon’s排名總和試驗以0.05的兩側顯著水平，在兩個接種組之間對第15天每種病毒毒株的經log轉化的HI抗體效價與第15天的鼻IgA抗體效價加以比較。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. .

還通過對每種病毒毒株和每個接種組計算下述三個參數來分析第15天的HI抗體效價。The HI antibody titer on day 15 was also analyzed by calculating the following three parameters for each virus strain and each vaccination group.

－血清保護率，其中血清保護被定義為紅血球凝聚抑制(HI)效價40，－血清轉化率，其中血清轉化被定義為接種前HI效價<10，接種後HI效價40，或者，接種前HI效價10以及HI效價至少4倍的增加，－平均倍數增加值，即，HI效價倍數增加的幾何平均值。- 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.

根據預方案(pre－protocol)和治療意向(intent－to－treat)的原理對效果數據加以分析。但是，鑒於這是所謂的原理驗證型研究，預方案分析被認為是基本的一種。治療意向的樣品由被接種個體的一些接種後效果數據組成。預方案樣品由完成了方案並且沒有發生主要的方案偏差的被接種個體組成。主要的偏差包括(不限於)：包含或排除標準的偏差，禁藥使用等。此外，實驗室驗證的並發流感感染的個體以及失去了基本效果數據的個體也被從預方案樣品中排除。無論從方案樣品中排除的個體是否是之前決定的，研究數據庫都不是盲的。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.

出人意料地，以及與用同樣的疫苗批次獲得的臨床前數據(實施例1)和WO 04/110486以及臨床數據(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)相反地，在用無佐劑的重構病毒顆粒流感疫苗僅進行一次接種的人類群體中，觀察到了令人滿意的、達到針對流感疫苗的CHMP標準的全身性免疫應答。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.

一種包含流感病毒顆粒(influenza virosomes)的組合物，所述病毒顆粒包含所述病毒的重構外殼(envelopes)，其中，所述病毒外殼完全從流感病毒粒子獲得，其中，沒有從外界來源向所述重構病毒顆粒加入脂類，其中，所述病毒顆粒包含流感抗原血球凝集素和/或神經氨酸酶(neuraminidase)或其衍生物，其中，沒有向所述組合物加入單獨的(separate)佐劑和/或免疫刺激劑，以及其中，所述組合物作為鼻內或吸入給藥配方製劑來設計，所述組合物特徵在於，所述配方製劑向人類的單次鼻內或吸入給藥能誘導抵抗所述流感抗原血球凝集素和/或神經氨酸酶或其衍生物的全身性免疫應答和/或局部免疫應答。 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. 如申請專利範圍第1項所述的組合物，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素劑量等於或低於30μg。 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. 如申請專利範圍第1項所述的組合物，其中，所述配方製劑的單次鼻內或吸入給藥還誘導細胞毒性淋巴細胞應答。 The composition of claim 1, wherein the single intranasal or inhaled administration of the formulation further induces a cytotoxic lymphocyte response. 如申請專利範圍第2項所述的組合物，其中，所述配方製劑的單次鼻內或吸入給藥還誘導細胞毒性淋巴細胞應答。 The composition of claim 2, wherein a single intranasal or inhaled administration of the formulation further induces a cytotoxic lymphocyte response. 如申請專利範圍第1至4項中任意一項所述的組合物，其中，所述免疫應答符合針對流感疫苗的CHMP標準。 The composition of any one of claims 1 to 4, wherein the immune response conforms to the CHMP standard for influenza vaccines. 如申請專利範圍第5項所述的組合物，其中，所述免疫應答提供下述中的一種或多種：對成年人而言>70%和/或對老年人而言>60%的血清保護率，針對成年人>40%和/或針對老年人>30%的血清轉化率，以及針對成年人>2.5和/或針對老年人>2.0的平均值增加倍數。 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. 如申請專利範圍第2項所述的組合物，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於25μg。 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. 如申請專利範圍第2項所述的組合物，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於20μg。 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. 如申請專利範圍第2項所述的組合物，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於15μg。 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. 如申請專利範圍第2項所述的組合物，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於10μg。 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. 如申請專利範圍第2項所述的組合物，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於5μg。 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. 如申請專利範圍第1至4項中任意一項所述的組合物，其中，所述組合物是包含用於鼻內或吸入給藥的藥物載體的疫苗配方製劑。 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. 如申請專利範圍第5項所述的組合物，其中，所述組合 物是包含用於鼻內或吸入給藥的藥物載體的疫苗配方製劑。 The composition of claim 5, wherein the combination A vaccine formulation comprising a pharmaceutical carrier for intranasal or inhalation administration. 如申請專利範圍第6項所述的組合物，其中，所述組合物是包含用於鼻內或吸入給藥的藥物載體的疫苗配方製劑。 The composition of claim 6, wherein the composition is a vaccine formulation comprising a pharmaceutical carrier for intranasal or inhalation administration. 一種包含所述病毒的重構外殼的流感病毒顆粒用於製造用於鼻內或吸入給藥的組合物的用途，其中，所述病毒外殼完全從流感病毒粒子獲得，其中，沒有從外界來源向所述重構病毒顆粒加入脂類，其中，所述病毒顆粒包含流感抗原血球凝集素和/或神經氨酸酶或其衍生物，其中，沒有向所述組合物加入單獨的佐劑和/或免疫刺激劑，所述組合物特徵在於，所述組合物向人類的單次鼻內或吸入給藥足以誘導抵抗所述流感抗原血球凝集素和/或神經氨酸酶或其衍生物的全身性免疫應答和/或局部免疫應答。 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. 如申請專利範圍第15項所述的用途，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素劑量等於或低於30μg。 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. 如申請專利範圍第15項所述的用途，其中，所述組合物的單次鼻內或吸入給藥還誘導細胞毒性淋巴細胞應答。 The use of claim 15, wherein a single intranasal or inhaled administration of the composition also induces a cytotoxic lymphocyte response. 如申請專利範圍第16項所述的用途，其中，所述組合物的單次鼻內或吸入給藥還誘導細胞毒性淋巴細胞應答。 The use of claim 16, wherein a single intranasal or inhaled administration of the composition also induces a cytotoxic lymphocyte response. 如申請專利範圍第15至18項中任意一項所述的用途，其中，所述免疫應答符合針對流感疫苗的CHMP標準。 The use of any one of clauses 15 to 18, wherein the immune response conforms to the CHMP standard for influenza vaccines. 如申請專利範圍第19項所述的用途，其中，所述免疫應答提供下述中的一種或多種：對成年人而言>70%和/或對老年人而言>60%的血清保護率，針對成年人>40%和/或針對老年人>30%的血清轉化率，以及針對成年人>2.5和/或針對老年人>2.0的平均值增加倍數。 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. 如申請專利範圍第16項所述的用途，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於25μg。 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. 如申請專利範圍第16項所述的用途，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於20μg。 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. 如申請專利範圍第16項所述的用途，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於15μg。 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. 如申請專利範圍第16項所述的用途，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於10μg。 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. 如申請專利範圍第16項所述的用途，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於5μg。 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. 如申請專利範圍第15至18項中任意一項所述的用途，其中，製造的組合物是疫苗配方製劑。 The use according to any one of claims 15 to 18, wherein the composition produced is a vaccine formulation. 如申請專利範圍第19項所述的用途，其中，製造的組合物是疫苗配方製劑。 The use of claim 19, wherein the composition produced is a vaccine formulation. 如申請專利範圍第20項所述的用途，其中，製造的組合物是疫苗配方製劑。 The use of claim 20, wherein the composition produced is a vaccine formulation. 一種包含流感病毒顆粒的組合物的疫苗配方製劑，所述病毒顆粒包含所述病毒的重構外殼，其中，所述病毒外殼完全從流感病毒粒子獲得，其中，沒有從外界來源向重構病毒顆粒加入脂類，其中，病毒顆粒包含流感抗原血球凝集素和/或神經氨酸酶或其衍生物，其中，沒有向所述組合物加入單獨的佐劑和/或免疫刺激劑，其中，疫苗特徵在於，針對向人類的單次鼻內或吸入給藥來設計所述疫苗，並且，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素劑量等於或低於30μg，並且，其中，所述組合物向人類的單次鼻內或吸入給藥能誘導抵抗所述流感抗原血球凝集素和/或神經氨酸酶或其衍生物的全身性免疫應答和/或局部免疫應答。 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. 如申請專利範圍第29項所述的疫苗配方製劑，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於25μg。 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. 如申請專利範圍第29項所述的疫苗配方製劑，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於20μg。 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. 如申請專利範圍第29項所述的疫苗配方製劑，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於15μg。 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. 如申請專利範圍第29項所述的疫苗配方製劑，其中，每 種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於10μg。 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. 如申請專利範圍第29項所述的疫苗配方製劑，其中，每種病毒毒株每次鼻內或吸入給藥的血球凝集素的劑量低於或等於5μg。 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. 如申請專利範圍第29至34項中任意一項所述的疫苗配方製劑，其中，所述組合物向人類的單次鼻內或吸入給藥還能誘導細胞毒性淋巴細胞介導的免疫應答。 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. 如申請專利範圍第35項所述的疫苗配方製劑，其中，所述免疫應答符合針對流感疫苗的CHMP標準。 The vaccine formulation according to claim 35, wherein the immune response conforms to the CHMP standard for influenza vaccine. 如申請專利範圍第36項所述的疫苗配方製劑，其中，所述免疫應答提供下述中的一種或多種：對成年人而言>70%和/或對老年人而言>60%的血清保護率，針對成年人>40%和/或針對老年人>30%的血清轉化率，以及針對成年人>2.5和/或針對老年人>2.0的平均值增加倍數。 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. 一種用於鼻內或吸入給藥的設備，所述設備包含如申請專利範圍第29至37項中任意一項所述的疫苗配方製劑以及用於將所述疫苗氣溶膠化的機構。 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. 如申請專利範圍第38項所述的設備，其中，所述設備包含一定量的用於單次鼻內或吸入給藥的疫苗配方製劑。 The device of claim 38, wherein the device comprises a quantity of a vaccine formulation for a single intranasal or inhaled administration. 如申請專利範圍第38或39項所述的設備，其中，所述設備是一次性的(disposable)。 The device of claim 38, wherein the device is disposable.

• 2007
  • 2007-03-19 TW TW096109319A patent/TWI397419B/en not_active IP Right Cessation
  • 2007-03-21 MX MX2008012046A patent/MX2008012046A/en active IP Right Grant
  • 2007-03-21 UA UAA200812366A patent/UA93236C2/en unknown
  • 2007-03-21 CN CN2007800102179A patent/CN101405027B/en not_active Expired - Fee Related
  • 2007-03-21 CN CN2012100495363A patent/CN102580077A/en active Pending
• 2008
  • 2008-09-12 ZA ZA200807880A patent/ZA200807880B/en unknown
• 2009
  • 2009-06-19 HK HK09105522.7A patent/HK1128077A1/en not_active IP Right Cessation

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