WO1994019013A1 - Compositions de vaccin contre la grippe, contenant un lipide a de monophosphoryle desacyle en position 3-o - Google Patents

Compositions de vaccin contre la grippe, contenant un lipide a de monophosphoryle desacyle en position 3-o Download PDF

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Publication number
WO1994019013A1
WO1994019013A1 PCT/EP1994/000448 EP9400448W WO9419013A1 WO 1994019013 A1 WO1994019013 A1 WO 1994019013A1 EP 9400448 W EP9400448 W EP 9400448W WO 9419013 A1 WO9419013 A1 WO 9419013A1
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Prior art keywords
mpl
vaccine composition
composition according
protein
vaccine
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PCT/EP1994/000448
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English (en)
Inventor
Susan Dillon
Hirotoshi Nishikawa
Paul Dal Monte
Robert J. Gyurick
Nathalie Marie-Josephe Claude Garcon-Johnson
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Smithkline Beecham Corporation
Smithkline Beecham Biologicals S.A.
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Priority to JP6518456A priority Critical patent/JPH08506592A/ja
Priority to EP94908327A priority patent/EP0684838A1/fr
Priority to CN94191720.7A priority patent/CN1124013A/zh
Priority to AU61410/94A priority patent/AU6141094A/en
Publication of WO1994019013A1 publication Critical patent/WO1994019013A1/fr

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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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • 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
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • 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
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention relates to vaccines useful in 0 preventing infection with influenza in humans.
  • Influenza virus infection causes acute respiratory disease in man, horses and fowl, sometimes of pandemic 5 proportions.
  • Influenza viruses belong to the orthomyxovirus family of RNA viruses and, as such, have enveloped virions of 80 to 120 nanometers in diameter, with two external glycoprotein spikes, hemagglutinin (HA) and neuraminidase (NA) , and five internal proteins, 0 nucleoprotein, matrix protein and three polymerases.
  • Influenza viral RNA also codes for two non-structural proteins (NS1 and NS2) which are produced in infected cells, but are not incorporated into infectious virions. Three types of influenza virus, Type A, Type B and 5 Type C, infect humans.
  • Type A viruses have been responsible for the majority of human epidemics in modern history, although there are also sporadic outbreaks of Type B infections.
  • Known swine, equine and fowl viruses have mostly been Type A, although Type C viruses have 0 also been isolated from swine.
  • H1N1, H2N2 and H3N2 are important subtypes, designated H1N1, H2N2 and H3N2.
  • subtypes HI wine flu
  • H2 asian flu
  • H3 Haong 5 Kong flu
  • Influenza viruses continually undergo genetic change in their surface glycoproteins which affects antigenic variation. This is most pronounced within the A virus type, where major genetic changes in the HA or NA proteins have already occurred ("antigenic shifts") . The emergence of these new virus subtypes have caused a pandemic spread of infection resulting in significant mortality and morbidity. For example, the H1N1 viruses, prevalent before 1957, were replaced by the H2N2 virus subtype which remained predominant until 1968, when they were, in turn, replaced by the H3N2 subtype. Currently, H3N2 strains are still circulating, but since 1977, H1N1 viruses have re-emerged.
  • antigenic drift The HAs within a given subtype also undergo smaller genetic changes (point mutations) every year or two (“antigenic drift") . These are largely restricted to antigenic determinants clustered around the sialic acid binding site in the HA1 and result in the emergence of new virus strains. Although this antigenic drift does not cause serious mortality and morbidity to the extent caused by antigenic shift, it is responsible for yearly influenza epidemics.
  • Influenza vaccines are classified into three types, whole-virion, split, and subunit.
  • Whole-virion vaccines based on intact viral particles, although generally more immunogenic, tend to be more reactogenic and are therefore being replaced by split and subunit vaccines which are prepared from purified viral components obtained after disruption of the virus by treatment with various chemical agents.
  • split and subunit vaccines resides in the fact that subunit vaccine contain almost exclusively haemagglutinin and neuraminidase, the surface antigens of the virus, whereas split vaccines contain in addition variable amounts of internal components of the virus such as the ribonucleoprotein and the matrix protein.
  • influenza vaccines are based on the principle that antibody to HA or NA confers protection. They consist of non-adjuvanted, inactivated, whole, or split virus products utilizing virus grown in embryonated hen's eggs. All influenza vaccines currently contain preparations from H1N1, H3N2, and type B virus strains. Due to the annual antigenic variation, specific virus strains are updated on a yearly basis according to WHO recommendations, which are based on epidemiological surveillance of prelevent circulating virus strains.
  • Influenza vaccines are under utilized for a variety of reasons including doubts about efficacy, fear of side effects, need for annual revaccination, and lack of interest among providers.
  • Present vaccines have demonstrated efficacy ranging from approximately 60-80% against infection with influenza viruses that are antigenically closely related to the virus strains used in the vaccine. This rate of protective efficacy tends to decrease when the HA antigen of the epidemic strain HA "drifted" away from the vaccine strain and would fall to zero if a "shift" in subtype occurs.
  • the present invention provides a vaccine composition capable of stimulating an enhanced immune and protective response in a vaccinated animal against influenza, the composition comprising a selected influenza antigen or antigenic polypeptide and an effective amount of 3-o-deacylated monophosphoryl lipid A (3D-MPL) .
  • a vaccine composition capable of stimulating an enhanced immune and protective response in a vaccinated animal against influenza, the composition comprising a selected influenza antigen or antigenic polypeptide and an effective amount of 3-o-deacylated monophosphoryl lipid A (3D-MPL) .
  • the invention provides a vaccine composition comprising a selected influenza antigen or antigenic polypeptide, an effective amount of 3D-MPL and a liposome preparation.
  • the liposome preparation is defined herein and, in addition to acting as a carrier, acts as an adjuvant and offers significant manufacturing and formulation advantages.
  • the invention provides a method for enhancing a vaccinee's immune response to a selected influenza antigen. This method involves administering to a mammal, preferably a human, a vaccine composition described above.
  • Fig. 1 is a bar graph illustrating cross-protection for H1N1 and H2N2 subtype influenza viruses in mice immunized with Flu D protein (SK&F 106160) in aluminum plus 3D-MPL, as described in Example 18.
  • Fig. 2A is a bar graph illustrating splenic proliferative responses pre-challenge in mice vaccinated with flu D formulations and controls. See Example 20.
  • Fig. 2B is a bar graph illustrating splenic proliferative responses post-challenge in mice vaccinated with flu D formulations and controls. See Example 20.
  • Fig. 3A is a bar graph illustrating lymph node proliferative responses obtained on day 4 in mice vaccinated with 20 ⁇ g flu D in aluminum (open bars) or aluminum and 3D-MPL (cross-hatched bars) . See Example 21.
  • Fig. 3B is a bar graph illustrating lymph node proliferative responses obtained on day 4 in mice vaccinated with 5 ⁇ g flu D in aluminum (open bars) or aluminum and 3D-MPL (cross-hatched bars) . See Example 21 .
  • Fig. 3C is a bar graph illustrating lymph node proliferative responses obtained on day 4 in mice vaccinated with 1 ⁇ g flu D in aluminum (open bars) or aluminum and 3D-MPL (cross-hatched bars) . See Example 21.
  • Fig. 4A is a bar graph illustrating proliferation on day 2 by immune lymph nodes in mice immunized with 1 ⁇ g D protein vaccine formulation. See Example 21.
  • Fig. 4B is a bar graph illustrating proliferation on day 3 by immune lymph nodes in mice immunized with 1 ⁇ g D protein vaccine formulation. See Example 21.
  • Fig. 4C is a bar graph illustrating proliferation on day 4 by immune lymph nodes in mice immunized with 1 ⁇ g D protein vaccine formulation. See Example 21.
  • Fig. 4D is a bar graph illustrating IL-2 production on day 2 by immune lymph nodes in mice immunized with 1 ⁇ g D protein vaccine formulation. See Example 21.
  • Fig. 4E is a bar graph illustrating IL-2 production on day 3 by immune lymph nodes in mice immunized with 1 ⁇ g D protein vaccine formulation. See Example 21.
  • Fig. 4F is a bar graph illustrating IL-2 production on day 4 by immune lymph nodes in mice immunized with 1 ⁇ g D protein vaccine formulation. See Example 21.
  • Fig. 5A is a graph demonstrating interferon levels in antigen-stimulated cultures from mice immunized as described in Example 24 below.
  • Fig. 5B is a graph demonstrating IL-2 levels in antigen-stimulated cultures obtained from mice immunized as described in Example 24 below.
  • Fig. 6A is graph showing virus titers determined in the nose by MDCK microassay on days 1, 3, 5, 7 and 9 post-challenge (5 mice per group) for a control containing alum and 3D-MPL (—square—) , influenza monovalent split vaccine containing A/PR/8 strain with no adjuvant (—triangle—) , and a strain A/PR/8 adjuvanted with 3D-MPL (solid line and circle) . See Example 28.
  • Fig. 6B is graph showing virus titers determined in the nose by MDCK microassay on days 1, 3, 5, 7 and 9 post-challenge (5 mice per group) for a control containing alum and 3D-MPL (—square—) , influenza monovalent split vaccine containing Singapore strain with no adjuvant (—triangle—) , and the Singapore strain adjuvanted with 3D-MPL (solid line and circle) . See Example 28.
  • Fig. 6C is graph showing virus titers determined in the trachea by MDCK microassay on days 1, 3, 5, 7 and 9 post-challenge (5 mice per group) for the same three vaccine formulations as in Fig. 6A.
  • Fig. 6D is graph showing virus titers determined in the trachea by MDCK microassay on days 1, 3, 5, 7 and 9 post-challenge (5 mice per group) for the same three vaccine formulations as in Fig. 6B.
  • Fig. 6E is graph showing virus titers determined in the lung by MDCK microassay on days 1, 3, 5, 7 and 9 post-challenge (5 mice per group) for the same three vaccine formulations as in Fig. 6A.
  • Fig. 6F is graph showing virus titers determined in the lung by MDCK microassay on days 1, 3, 5, 7 and 9 post-challenge (5 mice per group) for the same three vaccine formulations as in Fig. 6B.
  • a vaccine composition of this invention is characterized by containing an effective amount of a selected influenza antigen or antigenic polypeptide and 3-o-deacylated monophosphoryl lipid A (3D-MPL) .
  • a liposome preparation may also be a component of the vaccine compositions of this invention.
  • the inventors have discovered that the combination of 3D-MPL and certain influenza antigens are effective in achieving protective responses against influenza, which are not achieved by the influenza antigen alone.
  • the vaccinated host produces a stronger cellular immune response (protective T lymphocyte production) to the vaccine composition of the invention than is or would be produced by the host in response to the selected antigen when not adjuvanted, or when adjuvanted with other conventional adjuvants suitable for internal administration.
  • An increased antibody (B cell) response is also anticipated by this enhanced response.
  • immunologically effective amount or
  • an effective amount as used herein is meant that amount of antigen which induces a protective immune response.
  • selected antigen By the terms “selected antigen”, “antigenic polypeptide or protein” or “immunogen” as used herein is meant a whole or inactivated pathogen, an immunogenic protein, peptide or fragment from the pathogen, which is optionally fused to another peptide or protein which is of homologous or heterologous origin. These terms also include a split virus, defined below. These terms may also include non-proteinaceous biological materials from the pathogen. The pathogens are preferably disease- causing organisms which infect humans, although animal pathogens may also be employed in these vaccines, where desired for veterinary purposes. These terms refer to the ability of the whole pathogen, split virus, peptide or fusion protein to elicit a protective immune response in a vaccinated host.
  • a vaccine containing antigens from a single type or subtype of influenza virus, e.g., H1N1, H2N2, H3N2 of Type A, Type B and Type C.
  • multivalent vaccine a vaccine containing antigens from more than a single type or subtype of influenza virus, i.e., a trivalent vaccine may contain antigens from any three influenza types or subtypes, e.g., H1N1, H2N2, H3N2 of Type A, Type B and Type C.
  • split virus an influenza virus suspension, obtained from embryonated hens' eggs inoculated with seed lot material, which is partially purified and concentrated.
  • the concentrated virus suspension is treated with a detergent, such as sodium- desoxylcholate, to disrupt the virus particles. Removal of viral phospholipids during this splitting process produces an inactivated influenza antigen for which the reactogenicity potential is greatly reduced.
  • the split virus suspension is completely inactivated by the combined effect of detergent and formaldehyde.
  • the following disclosure of the compositions and methods of this invention specifically describes vaccine compositions for prophylactic use against influenza virus.
  • a vaccine composition capable of eliciting an enhanced immune response protective against infection with influenza virus contains at least a selected influenza antigenic polypeptide, such as NS1 1 _ 81 HA2 65 _ 222 (referred to herein as Flu D, the D protein or the Flu D protein) , adjuvanted with 3D-MPL.
  • a selected influenza antigenic polypeptide such as NS1 1 _ 81 HA2 65 _ 222 (referred to herein as Flu D, the D protein or the Flu D protein) , adjuvanted with 3D-MPL.
  • flu D protein is one preferred influenza antigenic polypeptide for use in the vaccine compositions of this invention because it is the most easily purified of the influenza fusion proteins which contain the entire carboxy-terminal region of HA2 portion of the hemagglutinin region.
  • D protein comprises the first 81 amino acids of NS1 fused to amino acid 65 of the truncated HA2 subunit (amino acids 65-222) .
  • a linker sequence may be inserted between the two fused sequences.
  • the DNA coding sequence for flu D protein is prepared by as described in EP 0366238 by restricting the HA2 coding sequence with
  • influenza antigenic polypeptides in addition to flu D may be used in the vaccine compositions of this invention including those described in European Patent Applications 366,238 and EP 366,239, both published May 2, 1990 and in co-pending U. S. Patent Applications SN 07/751,898, 07/751,896 and 07/837,773.
  • Such proteins include ⁇ M, ⁇ M+, A, C, C13, C13 short, and ⁇ D.
  • Other suitable include Cys-less D, HA2 66 _ 222 , and NS1H3HA2 constructs, such as those described in co- pending U.S. Patent Applications Ser. No.
  • H3HA2 constructs referenced above.
  • NS1-H1HA2 and NS1-H3HA2 fusion proteins were protected from lethal challenge with both HI and H3-virus subtypes.
  • Coding sequences for the HA2, NS1 and other viral proteins of influenza virus can be prepared synthetically or can be derived from viral RNA by known techniques, or from available cDNA-containing plasmids as described in the above-incorporated published European applications.
  • a DNA coding sequence for HA from the A/Japan/305/57 strain was cloned, sequenced and reported by Gething et al. Nature. 287:301-306 (1980); an HA coding sequence for strain A/NT/60/68 was cloned as reported by Sleigh et al, and by Both et al, both in Developments in Cell Biology, Elsevier Science Publishing Co., pages 69-79 and 81-89, (1980) ; an HA coding sequence for strain A/WSN/33 was cloned as reported by Davis et al, Gene, 1Q_:205-218 (1980); and by Hiti et al.
  • Virology, 111:113-124 (1981) An HA coding sequence for fowl plague virus was cloned as reported by Porter et al and by Emtage et al, both in Developments in Cell Biology, cited above, at pages 39-49 and 157-168.
  • Systems for cloning and expressing the vaccinal polypeptide in various microorganisms and cells, including, for example, ___.. coli, Bacillus, Streptomyce ⁇ , Saccharomyces, mammalian and insect cells, are known and available from private and public laboratories and depositories and from commercial vendors.
  • the D protein can be purified by the process described below in Example 13.
  • Various conventional procedures can be employed in connection with the purification of the proteins of the compositions of the present invention, although such other procedures are not necessary to achieve a highly purified, pharmaceutical grade product. Such procedures can be employed between, before or after the above described process steps.
  • One such optional step is diafiltration, a form of continuous dialysis which is extremely effective in achieving many buffer exchanges. Diafiltration is preferably carried out across a cellulosic membrane or ultrafilter. Suitable membranes/filters are those having from about a 1000 molecular weight (MW) cut-off to those having pore size up to 2.4 ⁇ m diameter.
  • diafiltration using a 20 mM Tris buffer at about pH 8 can be effectively employed in the purification and subsequent concentration of the polypeptide to be employed in the vaccine composition of this invention.
  • the antigen used in a vaccine composition of the invention is a whole inactivated pathogen, such as a split virus, described in detail in Example 25.
  • a monovalent split influenza vaccine containing a split virus or a multivalent (e.g. trivalent) split influenza vaccine containing more than one split virus may also be adjuvanted with 3D-MPL.
  • the vaccine contains the split virus prepared from an H1N1 strain, such as Singapore/6/86 [Sachsisches Serumtechnikl GmbH (SSW) , Dresden, German and the National Institute for Biological Standards and Control (NIBSC, London, England) ] which is currently used in conventional flu vaccines.
  • H1N1 split viruses may be prepared from A/PR/8/34 (also called A/PR/8) described in T. Francis, Proc. Soc. Exp. Bio . Med. , 22.' * - -7 (1935) and available from the American Type Culture Collection, Rockville, Maryland, USA under ATCC
  • the monovalent vaccine contains antigens from one strain of influenza type virus.
  • the vaccine may be multivalent, containing more than one influenza antigen, e.g., two or three split viruses, to increase the reactivity against more than one influenza type virus.
  • An example of a trivalent vaccine includes, for example, the H1N1 Singapore/6/86 strain, with an H3N2 strain Beijing/32/92 [SSW, Dresden, Germany], and a Type B strain, Panama/45/90 [SSW, Dresden, Germany] .
  • Influenza viruses which can be prepared as split viruses by known means as described in Example 25 include any strains, subtypes and types, particularly those recommended by WHO, many of which are available from clinical specimens and from public depositories, such as the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852, USA (ATCC) and NIBSC.
  • ATCC American Type Culture Collection
  • NIBSC NIBSC
  • other suitable H3N2 virus strains include, without limitation, A/Victoria/H3/75 described in Fiers et al, SnSnl , 12: 683-696 (1980); A/Udorn described in C. J. Lai et al, Proc. Nat! . A ⁇ art. Sci.. USA.
  • Type B strains include, without limitation, those known as B/Lee/40 described by Krystal et al, Proc. Natl. Acad. Sci.. USA. 22.: 804-4900 (1982) and B/Taiwan, available from the ATCC as No. VR-295, Panama/45/90, and B/Yamaghta strains. H2N2 viruses may also be useful in these vaccines.
  • 3D-MPL is the adjuvant, 3D-MPL.
  • 3D-MPL is described in detail in U. S. Patent No. 4,912,094, incorporated by reference herein, and is commercially available from RIBI Immunochem Research Inc., Hamilton, Montana. Briefly,
  • 3D-MPL is a derivative of the endotoxin, mono-phosphoryl Lipid A (MPL) , a Lipid A analog isolated from a heptoseless, RE mutant of a Gram-negative bacteria, such as Salmonella innesota .
  • MPL lacks a phosphate group at the C-l position of glucosamine.
  • 3D-MPL is non-toxic, in contrast to other enterobacterial lipopolysaccharides, but retains the antigenic activity of the parent endotoxin. This molecule is useful in preventing Gram- negative sepsis and endotoxemia.
  • 3D-MPL can be dissolved in water to yield solutions of vesicular aggregates, which are presumably composed of lipid bilayer membranes. Thus, it is not likely that 3D- MPL is seen as individual molecules by the immune system, but rather interacts by membrane-membrane contacts, involving cell surfaces and vesicle surfaces.
  • the particle size of the MPL is 'small' and in general does not exceed 120nm.
  • the size of the particles is in the range 60-120nm.
  • the particle size is below lOOnm.
  • an influenza antigenic polypeptide e.g., flu D protein
  • the protein when adjuvanted with 3D-MPL, as described herein and illustrated in Examples 14-24 below, the protein is capable of inducing protection against influenza infection.
  • the selected antigen is a split virus in a monovalent or trivalent composition
  • a composition of the present invention demonstrates an increase in immunogenicity and cross-reactivity.
  • an influenza antigenic polypeptide for example, flu D protein
  • 3D-MPL 3D-MPL
  • flu D protein an influenza antigenic polypeptide
  • a lower dose of flu D protein is required to achieve the same level of immune response obtained when the protein is adjuvanted with an aluminum adjuvant only.
  • the selected antigen is a split influenza virus in a monovalent or trivalent composition, it is anticipated that reactogenicity of the composition will be decreased by the use of less antigen in this embodiment of the invention.
  • Additional adjuvants may also be included in the vaccine compositions of the invention.
  • One desirable additional adjuvant is alum, or aluminum hydroxide or aluminum phosphate.
  • CFA Complete Freunds Adjuvant
  • a desired amount of the flu D protein in admixed with a suitable amount of the 3D-MPL, as described in more detail below.
  • lyophilized lipid A is admixed with the pre-liposome gel described in detail below prior to the antigen.
  • a molar ratio of phosphatides to lipid A is 66:1.
  • the density of the agent may be varied to the desired level.
  • Suitable agents for addition to the vaccine composition include, for example, IL-2, QS21 [C. R.
  • muramyl dipeptides may also be used at similar ratios as described above or as desired.
  • Other drugs which may form part of this vaccine may include any substance that when taken into the vaccinee modifies one or more of its functions, for example as recited in an official pharmacopeia, or a substance used in the treatment or prevention of an infection.
  • the vaccine compositions of this invention may further contain suitable carriers which are well known to those of skill in the vaccine art and can be readily selected.
  • suitable carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil, squalene and water.
  • the carrier or diluent may include a time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax.
  • suitable chemical stabilizers may be used to improve the stability of the pharmaceutical preparation.
  • Suitable chemical stabilizers are well known to those of skill in the art and include, for example, citric acid and other agents to adjust pH, chelating or sequestering agents, and antioxidants. Alternatively, when a liposomal delivery system is part of the vaccine composition, no carriers are necessary.
  • Another preferred vaccine composition of this invention comprises a selected antigen, e.g., the flu D protein as described above, and 3D-MPL, which components are entrapped or intercalated in a liposome preparation.
  • the liposome, flu D protein, and 3D-MPL- containing vaccine composition may also contain one or more additional influenza antigens or other antigens or desirable adjuvants and agents as described above.
  • the vaccine contains the antigenic polypeptide Flu D protein and 3D-MPL placed into a carrier liposome.
  • the vaccine contains a mono- or multi-valent influenza antigen similarly involved with a carrier liposome.
  • the liposome preparations described herein are capable of functioning not only as carriers, but also as adjuvants, and are particularly advantageous because making them does not require organic solvents or high shear fields which is a significant advantage for protein drugs, and all ingredients for the liposomes are considered safe for internal administration. Because of the simplicity and flexibility of the preparative method, these liposome preparations are suitable for large scale manufacturing.
  • 3D-MPL can be readily incorporated into the liposomal structures described herein in combination with influenza antigens to obtain results superior to that found when influenza antigens are combined with known conventional adjuvants.
  • a vaccine composition containing D protein in a 3D-MPL and liposome formulation has even greater potency than D protein in CFA. See, e.g., Examples 20 and 22 below.
  • liposome preparations useful in the vaccine compositions and methods of this invention are described in co-pending United States Patent Application Ser. No. 07/714,984, (US 5230899) incorporated herein by reference.
  • liposome has been proposed and accepted as the term to be used in the scientific literature to describe synthetic, oligolamellar lipid vesicles.
  • Such vesicles are usually comprised of one or more concentric natural or synthetic lipid bilayers surrounding an internal aqueous phase.
  • the liposome preparations useful in the vaccine composition of the invention are prepared by dispersing in an aqueous medium in a manner adequate to form liposomes, a composition comprising a liposome- forming material containing a long chain aliphatic or aromatic-based acid or amine; a hydrating agent of charge opposite to that of the acid or amine, which agent is present in a molar ratio of between 1:20 and 1:0.05 relative to the acid or amine; and water in an amount up to 300 moles relative to the solids.
  • the preparation of the liposomal adjuvant carriers useful in the invention follows.
  • liposome- forming materials include saponifiable and non- saponifiable lipids, e.g., the acyl glycerols, the phosphoglycerides, the sphingolipids, the glycolipids, etc.
  • the fatty acids include saturated or unsaturated alkyl (C 8 ⁇ C 24 ) carboxylic acids, mono-alkyl (C 8 ⁇ C 27 ) esters of C 4 ⁇ C 10 dicarboxylic acids (e.g., cholesterol hemi- succinic acid and fatty acid derivatives of amino acids in which any N-acyl carboxylic acids also are included (e.g., N-oleoyl threonine, N-linoleoyl serine, etc.) .
  • Mono- or di-alkyl (C 8 ⁇ C 24 ) sulfonate esters and mono- or di-alkyl (C 8 ⁇ C 24 ) phosphate esters can be substituted for the fatty acids.
  • mono- or di-acyl (C 8 ⁇ C 24 ) glycerol derivatives of phosphoric acids and mono- or di- acyl (C 8 ⁇ C 24 ) glycerol derivatives of sulfuric acids can be used in place of the fatty acids.
  • the fatty acids also can be replaced by amines (e.g., C 8 ⁇ C 24 NH 2 ) , C 8 ⁇ C 24 fatty acid derivatives of amines (e.g., C 8 ⁇ C 24 CONH ⁇ NH 2 ) , C 8 ⁇ C 24 fatty alcohol derivatives of amino acids (e.g., C 8 ⁇ C 24 OOC ⁇ NH 2 ) , and C 8 ⁇ C 24 fatty acid esters of amines (e.g., C 8 ⁇ C 24 COO ⁇ NH 2 ) .
  • amines e.g., C 8 ⁇ C 24 NH 2
  • C 8 ⁇ C 24 fatty acid derivatives of amines e.g., C 8 ⁇ C 24 CONH ⁇ NH 2
  • C 8 ⁇ C 24 fatty alcohol derivatives of amino acids e.g., C 8 ⁇ C 24 OOC ⁇ NH 2
  • C 8 ⁇ C 24 fatty acid esters of amines e.g., C 8 ⁇ C
  • Photopolymerizable lipids and/or fatty acids (or amines) e.g., diacetylenic fatty acids
  • Photopolymerizable lipids and/or fatty acids (or amines) e.g., diacetylenic fatty acids
  • photopolymerizable lipids and/or fatty acids (or amines) also can be included, which can provide a sealed liposome with cross- linked membrane bilayers upon photo-initiation of polymerization.
  • a long chain aliphatic and/or aromatic-based acid or amine includes an acid or amine having an open chain structure and consisting of paraffin, olefin and acetylene hydrocarbons and their derivatives, i.e., saturated and unsaturated hydrocarbons or the backbone of such chain contains an where aromatic substituent. Such acids and amines may have more than one such function.
  • the term "long chain” means that the backbone of the aliphatic chain of the acid or amine has ten or more carbon atoms. If the chain contains an aromatic group, such as phenyl, the chain will comprise at least a five carbon backbone in conjunction with that aromatic group.
  • the chain of carbon atoms comprising the backbone may be variously substituted with saturated or unsaturated aliphatic or aromatic functions.
  • an acid function may be a carboxylate acid, or a phosphorous or sulfur derived acid function such as phosphate, phosphite or pyrophosphate or sulfate, sulfite, thiosulfate, or similarly constituted phosphorous or sulfur-based acid.
  • Amines must be sufficiently basic so as to have an ionizable hydrogen or be capable of forming quaternary salts which have an ionization constant such that they are capable of forming the requisite hydrate complex.
  • solids refers to the liposome-forming materials and the acid or amine component, hydrating agents, the 3D-MPL, alum, and the selected antigenic material to be encapsulated.
  • a hydrating agent means a compound having at least two ionizable groups, preferably of opposite charge, one of which is capable of forming an easily dissociative ionic salt, which salt can complex with the ionic functionality of the acid or amine in the liposome-forming material.
  • the hydrating agent inherently does not form liposomes in and of itself. Such an agent will also be physiologically acceptable, i.e., it will not have any untoward or deleterious physiological effect on the host to which it is administered in the context of its use in this invention.
  • the preferred hydrating agents are alpha amino acids having an ionizable omega substitution such as a carboxylate, amino, and guanidino function and those compounds represented by the formula: X-(CH 2 )n-Y I wherein
  • X is H 2 N-C(NH)-NH-, H 2 N-, Z0 3 S-, Z 2 0 3 P-, or Z0 2 C- wherein Z is H or an inorganic or organic cation;
  • Y is -CH(NH 2 )-C0 2 H, -NH 2 , -NH-C (NH) -NH 2 -COOH, CH(NH 2 )S0 3 Z or ZH(NH 2 )P0 3 Z 2 wherein Z is defined above; and n is the integer 1-10; or a pharmaceutically acceptable salt thereof.
  • n is the integer 1-10; or a pharmaceutically acceptable salt thereof.
  • More preferred hydrating agents are the omega- substituted, alpha amino acids such as arginine, its N- acyl derivatives, homoarginine, gamma-aminobutyric acid, asparagine, lysine, ornithine, glutamic acid, aspartic acid or a compound represented by the following formulas:
  • the most preferred compounds are arginine, homoarginine, gamma-aminobutyric acid, lysine, ornithine, glutamic acid or aspartic acid.
  • the hydrating agents of this invention may be used alone or as a mixture. No limitation is implied or intended in the use of mixtures of these hydrating materials.
  • Arginine, homoarginine, lysine, glutamic acid, aspartic acid, and other naturally occurring amino acids may be obtained by the hydrolysis of protein and separation of the individual amino acids or from bacterial sources.
  • the compounds of formula II can be made by the method of Eisele, K. et al, Justusliebigs. Ann. Chern., p. 2033 (1975) . Further information on several representative examples of these compounds is available through their respective Chemical Abstracts Service numbers as follows: nonarginine, CAS #14191-90-3; arginine, CAS #74-79-3; and homoarginine, CAS #151-86-5.
  • nonarginine CAS #14191-90-3
  • arginine CAS #74-79-3
  • homoarginine CAS #151-86-5.
  • formula III see for 2,4- diaminobutonic acid CAS #305-62-4 and for lysine CAS #56- 87-1.
  • glutamic acid is well known in the art and is available from many commercial sources. Descriptions of how to make other representative compounds is contained in the literature, for example: 2-aminohexandioc acid - CAS # 62787-49-9 and 2-aminoheptandioc acid - CAS # 32224-51-0.
  • Glutamic acid, the compound of formula VII where n is 2 is well known in the art and can be made by the method of Maryel and Tuley, Org. Syn. f ⁇ :69 (1925) .
  • Hydrate complex means the complex formed between the hydrating agent and the acid or amine in the liposome-forming material.
  • Complexing in this context denotes the formation of dissociative ionic salts where one functionality associates with the ionic functionality of the liposome- forming material and the other functionality has hydrophilic properties which impart water-solubility to the resulting complex. While the liquid crystal structure of the hydrate complex varies with pH and amount of hydrating agent, the liquid crystal structure remains.
  • NMR spectroscopy confirms that this crystal structure consists of multilamellar lipid bilayers and hydrophilic layers stacked together in alternating fashion.
  • the 31 P-NMR spectrum exhibits an anisotropic peak, confirming the existence of multilamellar bilayers.
  • the primary components of these liposomes will be lipids, phospholipids, other fatty acids, there may also be added various other components to modify the liposomes 1 permeability.
  • non-ionic lipid components such as polyoxy alcohol compounds, polyglyceral compounds or esters of polyoles, polyoxyalcolinolated alcohols; the esters of polyoles and synthetic lipolipids, such as cerebrosides.
  • composition of the liposome can be made of more than one component of the various kinds of lipids, the fatty acids, alkyl amines, or the like, and the hydrating agents.
  • the lipid composition may not require the inclusion of the fatty acids (or the amines) or the hydrating agents to form the "pre-liposome gel” or liposomes.
  • the mixture of dipalmitoylphosphatidylcholine (DPPC) and distearoyl phosphatidylethanolamine forms the "pre-liposome gel” or liposomes with aqueous glutamic acid solution and the mixture of DPPC and oleic acid with aqueous epinephrine solution forms the "pre-liposome gel” and liposomes.
  • DPPC dipalmitoylphosphatidylcholine
  • distearoyl phosphatidylethanolamine forms the "pre-liposome gel” or liposomes with aqueous glutamic acid solution
  • the mixture of DPPC and oleic acid with aqueous epinephrine solution forms the "pre-liposome gel” and liposomes.
  • the liposome preparation preferably includes phospholipids, oleic acid (or phosphatidyl-ethanolamine) and arginine or lysine (or glutamic acid and/or aspartic acid) .
  • About 1:20 molar ratio of hydrating agent relative to the liposome-forming material will provide the salutory effects of this invention with an upper limit of about 1:0.05.
  • the preferred concentration range for the hydrating agent is between a 1:2 to 1:0.5 molar ratio of the hydrating relative to the liposome-forming material.
  • liposomes are prepared with a long chain aliphatic or aromatic-based acid
  • hydrating agents which contain at least one ionizable nitrogen, such as arginine, homoarginine, lysine, ornithine, and the like.
  • the amphipatic materials used to form the liposomes contain a long chain aliphatic or aromatic-based amine
  • a di-acid such as glutamic acid, aspartic acid
  • any of the alkyl di-acids such as the simple di-acids such as valeric acid, caprylic, caproic, capric or the like; or those di- acids having two phosphate, or sulfate functionalities; or those di-acids having mixed -COOH/-S0 3 H or -COOH/-P0 3 H 2 functions.
  • Certain aliphatic and aromatic-based acids and amines are preferred in the practice of this invention.
  • Such compounds can have multiple functions such as having two or more acid or amine groups or combinations thereof.
  • the preferred compounds are those with one or two acid or amine functions. More preferred are the fatty mono-acids of 10-20 carbons, saturated and unsaturated. Most preferred are the alkyl and alkenyl acids of 10 to 20 carbon atoms, particularly oleic acid.
  • Liposome-forming materials Mixtures of liposome-forming materials, a long chain aliphatic or aromatic-based acid or amine, one or more hydrating agents with up to 300 moles of water relative to the total solids, with or without a selected amount of antigenic material, produces a gel which forms liposomes directly therefrom upon addition of an aqueous solution.
  • This gel can be labelled a pre-liposome gel because its structural characteristics are essentially those of liposomes and, the gel has the facility for being converted into liposomes upon dilution with an aqueous solution. Aqueous solution in excess of about 300 molar equivalents cause the beginning of liposome formation.
  • the structure of this gel is a highly ordered liquid crystal which forms an optically clear solution.
  • the X, Y, and Z dimensions of the liquid crystal vary with the concentrations of hydrating agent at constant pH as well as with the pH of the solution.
  • concentrations of hydrating agent at constant pH By varying the hydrating agent concentration at constant pH or changing the pH while maintaining the percentage of hydrating agent, the size and number of lamellae structures of the lipid bilayers of the subsequent liposome vesicles can be controlled.
  • the gel structure itself can accommodate up to approximately 300 moles of water per mole of solid without disturbing the stability of the gel structure.
  • the structure of the gel as determined by proton nuclear magnetic resonance (NMR) spectroscopy is comprised of multilamellae lipid bilayers and hydrophilic layers stacked together in an alternating fashion.
  • the 31 P-NMR spectrum of the same gel exhibits an anisotropic peak further confirming that the gel consists of a multilamellar bilayer.
  • This gel can be autoclaved, a convenient means of sterilization. Furthermore, the gel shows no discoloration and remains clear at room temperature for at least one year after being autoclaved. If desirable and feasible, the gel can optionally further be sterilized by filtration through an appropriate sterilization filter. Upon dispersion of the gel into an aqueous solution, liposomes are efficiently and spontaneously produced.
  • the pre-liposome gel with or without the 3D-MPL and antigenic material to be encapsulated, also can be dehydrated (e.g. lyophilized) and the powder rehydrated to form liposomes spontaneously, even after a long period of storage.
  • This capability provides the vaccine compositions particularly useful for administering water- sensitive antigenic materials where long term pre-use storage is needed.
  • the pre-liposomal gel is prepared as follows.
  • a semisolid liquid crystalline gel referred to as the pre- liposomal gel is prepared by combining three basic ingredients: a phospholipid, a fatty acid, and a hydrating agent in water.
  • a variety of phospholipids or mixtures thereof ranging in gel-liquid transition temperatures (Tm) may be employed.
  • the fatty acid can be chosen based on degree of saturation or chain length and is usually mixed with the phospholipid into a thick paste.
  • Cholesterol may be added to the lipid mix to control the bilayer character. (Cholesterol in some instances increases the Tm of the membrane thereby influencing its permeability to entrapped agents) .
  • the hydrating agent preferably an alpha amino acid, such as arginine
  • arginine is added to the lipid mix as a solution in water at a slow rate until a homogeneous paste is achieved.
  • One desirable formulation employs egg phosphatidyl choline:oleic acid at a 1:1.2 molar ratio.
  • the arginine is added in water at approximately 1:1.2 phospholipid to arginine molar ratio.
  • the concentration of L-arginine in the aqueous phase component of the gel governs the diameter of the stable liposome particle which eventually forms.
  • the size of the particle depends on the route of administration and whether or not it is desired to target macrophage cells. For example, for oral administration, a liposome particle size of between about 1 to about 5 micrometers is desired. To target to lymphocytes, a particle size of between about 200 to 500 nm is preferred. For a depot effect, a liposome particle size between about 5 to about 10 micrometers is desired. Various size particles may be easily tested and selected by one of skill in the art.
  • the final pre-liposomal gel can contain up to about 65 to about 70% water by weight, has the consistency of an ointment, and has the appearance of a typical liquid crystal when observed under a polarizing light microscope.
  • the pre-liposomal gel can be stored under an inert atmosphere, or lyophilized to a dry powder for long term storage.
  • the same steps of liposome preparation are followed with the addition of an immunologically effective amount of the selected antigen, e.g., an antigenic polypeptide, particularly the flu D protein, and 3D-MPL, and optionally, one or more additional immunogenic proteins, peptides or fragments from a selected pathogen mixed with the liposome preparation.
  • the selected antigen is encapsulated or intercalated within the liposome preparation by mixing therewith.
  • the 3D-MPL and the selected antigen can be incorporated into the liposome composition to prepare a vaccine composition according to this invention.
  • One method involves the incorporation of the antigen with liposomes by hydration of the pre- liposomal gel, or the hydrated lyophilized powder, with a solution of the antigen in an appropriate buffer.
  • the other method involves the physical mixing of the pre- liposomal gel with a lyophilized preparation of the antigen. This mixture is subsequently hydrated which causes the spontaneous formation of liposomes.
  • the method selected is the one which results in the largest level of antigen-liposome association with the smallest loss of antigen as unassociated fraction and is dependent upon the physico-chemical properties of the antigen in the presence of the lipid.
  • the ratios of the components in such a vaccine mixture are 20 mg antigenic protein: 2 mg 3D-MPL: 300 g liposomal gel.
  • the antigen is flu D
  • the second method is employed because the antigen has significant hydrophobic character and a low aqueous solubility and, as below described, to form a flu-D composition according to this invention, 300 mg of pre- liposomal gel was mixed with 20 to 30 mg flu-D protein and 0.5 to 2 mg 3D-MPL.
  • agents may be added co-entrapped with the selected antigen, e.g., flu D protein, and 3D- MPL in a liposome composition. Suitable other agents are described above.
  • any unassociated antigen may be removed by various means.
  • the liposomes are centrifuged serially at approximately 100,000 x g and the supernatants are discarded.
  • the final liposome pellet is reconstituted in an acceptable liquid, for example, 5% dextrose, normal saline, or buffered solution, for injection at a desired lipid or antigen concentration.
  • Other methods known to those of skill in the art, such as gel filtration or dialysis, may be employed for removal of unassociated antigen.
  • the description of the selected antigen, 3D-MPL and liposome formulation also encompasses the use of the split viruses described herein in place of, or in addition to. Flu D.
  • the present invention also provides a method of enhancing an immune response, particularly a T-cell mediated response, in a human or other mammal, to the selected antigen in the vaccine composition.
  • This method involves administering to the human or other mammal a vaccine composition of the invention containing 3D-MPL and the selected antigen.
  • a liposome preparation may be part of the composition as described above.
  • the method is not limited to any particular antigen exemplified herein.
  • the method is useful in eliciting an enhanced protection effective against influenza infection.
  • the vaccine composition comprises an effective amount of flu D and 3D-MPL, as described above.
  • the vaccine composition comprises an effective amount of one or more split influenza viruses and 3D-MPL, as described above.
  • Other vaccines may be prepared and administered in effective amounts in a similar manner.
  • the vaccine composition employing split viruses adjuvanted with 3D-MPL results in superior virus clearance particularly in the lung, the stimulation of higher neutralizing antibody titers; the occurrence of heterosubtypic (H ⁇ ) cross-reactivity in the absence of neutralizing activity; and altered progression of disease from upper to lower respiratory track (via trachea) .
  • the dosage and administration protocols can be optimized in accordance with standard vaccination practices for these vaccine compositions.
  • the vaccines will be administered intramuscularly, although other routes of administration may be used, such as the subcutaneous, colonic, oral, pulmonary, intradermal, intraperitoneal, or intravenous routes. The route chosen may be dictated by the type of immune response desired.
  • a subcutaneous route may provide the classical "depot effect" or more prolonged stimulation than others. It is also possible that a given route of administration may generate a stronger antibody response than a cellular response, or vice versa. For example, the oral route of administration may permit generation of an enhanced IgA response that is useful for local immunity.
  • a useful single dosage for average adult humans of an influenza antigenic polypeptide vaccine such as the flu D protein containing vaccine of this invention is in the range of between about 1 to about 1000 micrograms of D protein, preferably about 50 to about 500 micrograms protein, and most preferably about 100 ⁇ g, in admixture with suitable amounts of 3D-MPL adjuvant.
  • the amounts of 3D-MPL in the vaccine composition are between about 1 to about 500 micrograms 3D-MPL/ ⁇ g viral protein, more preferably about 10 to about 50 ⁇ g 3D-MPL/ ⁇ g viral protein, and most preferably about 50 ⁇ g 3D-MPL.
  • the preferred amount of D protein in the vaccine composition is between about 50 to about 500 ⁇ g and the amount of 3D-MPL is between about 10 to about 50 ⁇ g.
  • Such a 3D-MPL adjuvanted flu D vaccine composition will contain about 1 to about 10 mg, and preferably about 3 mg, liposome material per about 0.2 mg antigenic protein.
  • the flu D and 3D-MPL vaccine formulation optionally contains another adjuvant, such as aluminum or aluminum hydroxide
  • the preferred dosage of D protein is between about 10 to about 500 ⁇ g protein; the preferred amount of aluminum or aluminum hydroxide is between about 10 to about 500 ⁇ g.
  • a useful single dosage for average adult humans of an influenza monovalent or multivalent split virus vaccine is about 15 micrograms of hemagglutinin (HA) per strain, with total protein ranging from about 80-300 ⁇ g/ml, in admixture with suitable amounts of 3D-MPL adjuvant.
  • the amount of 3D-MPL in the vaccine composition is preferably about 50 micrograms 3D-MPL per dose.
  • these amounts of virus protein and 3D- MPL may be altered by one of skill in the art.
  • each split virus in the vaccine composition is likely to be less than about 15 ⁇ g HA/strain and the amount of 3D-MPL is between about 10 to about 50 ⁇ g.
  • Such a 3D-MPL adjuvanted influenza split virus vaccine composition will contain about 1 to about 10 mg, and preferably about 3 mg, liposome material per about 0.2 mg viral protein.
  • the preferred dosage of each split virus in the vaccine composition may be adjusted downward.
  • a preferred amount of aluminum or aluminum hydroxide is similar to that described above for the antigenic polypeptide.
  • Any of the vaccines described by this invention can be administered (preferably in 0.5 mis dosage units) initially in late summer or early fall and can be readministered two to six weeks later, if desirable, or periodically as immunity wanes, for example, every two to five years .
  • Dipalmitoylphosphatidylcholine 3.0 grams, was weighed into a 50 ml beaker. Oleic acid 1.2 grams was added and mixed together to form a uniform paste.
  • Arginine 0.72 grams in 30 ml of distilled deionized water was added to the dipalmitoylphosphatidyl- cholineoleic acid paste and heated to 45°C. With mixing by hand, the mixture formed a clear stable gel. The gel was stored and liposomes later formed by diluting the get with phosphate buffered saline.
  • the arginine-saline solution was added to the paste and heated to 40°C for M hour, or until a slightly turbid solution was observed.
  • Example 3 Lar ⁇ e Scale Gel and Liposome Preparation i) Gel Manufacture: To 50 grams of egg phosphatide powder type 20 (Asahi Chemicals) was added 20 grams of oleic acid N.F. Mixing gave a white paste which was cooled to 4°C and ground into a fine powder. This powder was added to an aqueous solution containing 20 grams of arginine and 500 grams of distilled deionized water. The mixture was mixed with a spatula as the solution was heated to about 35°C to help hydrate phospholipids. A homogeneous, slightly yellow get was formed. This gel can be autoclaved and stored at 4°C or can be frozen and later reconstituted.
  • a homogenous paste of 1.0 gram of dipalmitoylphosphatidycholine (DPPC) and 400 mg of oleic acid was formed. Then 300 mg arginine was mixed in 10 mL of phosphate buffered saline, heated to 45°C and added to the DPPC/oleic acid paste to form liposomes.
  • DPPC dipalmitoylphosphatidycholine
  • Example 5 Pre-T.iposome Gel
  • DPPL dipalmitoylphosphatidylcholine
  • oleic acid oleic acid
  • 300 mg of arginine was mixed with 2 ml of water at 45°C until dissolved.
  • the arginine solution was mixed with the DPPC/oleic acid paste at about 45°C to give a thick gel. Liposomes formed when this gel was diluted with phosphate buffered saline.
  • Cholesterol Containing Liposomes Cholesterol, 15 mg, was mixed with 100 mg dipalmitoylphosphatidylcholine (DPPC) to form a homogeneous powder. Then 23 mg of oleic acid was added to the powder and thoroughly mixed to form a homogeneous paste. To make liposomes, 30 mg of arginine was added to 10 ml of phosphate buffered saline, heated to 40°C and added to the DPPC/cholesterol/oleic acid paste. The combination was mixed at 40°C to obtain liposomes.
  • DPPC dipalmitoylphosphatidylcholine
  • DPPC Palmitic Acid-Containing Liposomes
  • Dipalmitoylphosphatidylcholine (DPPC) 250 mg was mixed with 25 mg of palmitic acid to form a uniform powder.
  • 80 mg of oleic acid was mixed with this powder and heated to 45°C with constant stirring until a uniform paste was formed.
  • Arginine 100 mg was dissolved in 25 ml of distilled deionized water and heated to 45°C. This arginine solution was added to the paste at 45°C and mixed until a uniform homogeneous gel was formed. The gel was diluted ten fold with phosphate buffered saline to form liposomes.
  • Dipalmitoylphosphatidylcholine 100 mg was mixed with 50 mg of isostearic acid to form a uniform homogeneous paste.
  • An arginine solution of 50 mg of arginine in 2.0 ml of distilled deionized water was made and added to the isostearic acid paste and heated to 45°C. This mixture was mixed until a clear gel was formed. Liposomes are formed upon dilution with phosphate-buffered saline.
  • Dipalmitoylphosphatidylcholine 192 mg was added to 72 mg of myristyl amine and heated with constant mixing until a uniform paste was formed.
  • Glutamic acid 65 mg in 5 ml of distilled deionized water was added to the paste and heated until a gel was formed.
  • Phosphate buffered saline was added to the get to form liposomes.
  • Dilaurylphosphatidylcholine (DLPC) 50 mg was mixed with 20 mg oleic acid to form a homogeneous paste.
  • Arginine 20 mg was added to 10 ml of phosphate buffered saline, added to the paste and hand mixed until a turbid liposome solution formed.
  • Phosphatidyl thanolamine-Glutamic Acid Liposomes L-glutamic acid 32 mg was dissolved in 2.0 ml of distilled deionized water and the pH adjusted to 5.2 with 1.0 N sodium hydroxide. This solution was heated to 60°C, and 100 mg of phosphatidylethanolamine added. The solution was kept to 60°C with constant mixing until a uniform viscous gel was observed. The phosphatidylethanolamine-glutamic acid gel was diluted 1/10 by phosphate buffered saline. Vesicular- like structures are observed under phase contrast light microscopy.
  • dipalmitoylphosphatidylcholine DPPC
  • (2-palmitoyl-l-C 14 ) 0.1 mCi/ml
  • dipalmitoylphosphatidylcholine Chloroform was added to effect complete mixing of the radioactivity and then evaporated.
  • Oleic acid (OA) 195 mg, was then mixed into the lipid to form a paste.
  • Five ml of distilled water containing 119 mg of arginine was added and mixed at 45°C to form a clear gel.
  • PCS photon correlation spectroscopy
  • the vesicle size can be varied by varying the pH of the aqueous buffer solution.
  • DPPC dipalmitoylphosphatidylcholine
  • 2-palmitoyl-l-C 14 2-palmitoyl-l-C 14
  • Chloroform was added to effect complete mixing of the radioactivity and then evaporated.
  • Oleic acid (OA) 40.1 mg, was then mixed into the lipid to form a paste.
  • One ml of a solution containing 24 mg/ml arginine in water was added to the lipid mixture and mixed at 45°C to form a clear gel.
  • a desired size of the liposomal vesicles can be prepared by varying the arginine concentration or the pH of the aqueous buffer solution.
  • Example 9 Liposome Stability Sterile liposomes may be prepared from the heat sterilized pre-liposome gel. Alternatively, the liposome gel or the liposomes may be sterile filtered through an appropriate sterilizing filter.
  • Liposomes prepared from DPPC:OA:Arg (1:1:2) at pH 8.0 were heat sterilized and stored at room temperature for approximately one year without adding antimicrobial agents and anti-oxidants. No bacterial growth, discoloration and precipitation were observed. Negative stain electron microscopic examination of the one year old liposomes revealed that the liposomal vesicles are stable.
  • Encapsulated sucrose latency was measured using C 14 - sucrose encapsulated with the DPPC:OA:Arg (1:1:1) liposome system in aqueous phosphate buffer solution at pH 7.8. The result was presented in the following Table 4. Table 4
  • the present liposome system has an excellent latency for drug delivery.
  • Example 11 Lyophilized Liposomes Oleic acid, 30.0 gm, and 7.5 gm of cholesterol
  • Plasmid pAPR701 is a pBR322-derived cloning vector which carries coding regions for the Ml and M2 influenza virus proteins (A/PR/8/34) . It is described by Young et al, in The Origin of Pandemic Influenza Viruses, 1983, edited by W. G. Laver, Elsevier Science Publishing Co.
  • Plasmid pAPR801 is a pBR322-derived cloning vector which carries the NS1 coding region (A/PR/8/34) . It is described by Young et al, cited above.
  • Plasmid pASl is a pBR322-derived expression vector which contains the P L promoter, an N utilization site (to relieve transcriptional polarity effects in the presence of N protein) and the cll ribosome binding site including the cll translation initiation codon followed immediately by a BamHI site. It is described by Rosenberg et al, Methods Enzvmol.. 11:123-138 (1983) .
  • Plasmid pASldeltaEH was prepared by deleting a non- essential EcoRI-Hindlll region of pBR322 origin from pASl.
  • the resulting plasmid, pASldeltaEH/801 expresses authentic NSl (230 amino acids) .
  • This plasmid has an Ncol site between the codons for amino acids 81 and 82 and an Nrul site 3' to the NS sequences. The BamHI site between amino acids 1 and 2 is retained.
  • Plasmid pASl ⁇ EH/801 was cut with Bglll, end-filled with DNA polymerase I (DNApolI; Klenow) and ligated closed, thus eliminating the Bglll site.
  • the resulting plasmid pBgl " was digested with Ncol, end-filled with DNApolI (Klenow) and ligated to a Bglll linker.
  • the resulting plasmid, pB4 contains a Bglll site within the NSl coding region. Plasmid pB4 was digested with Bglll and ligated to a synthetic DNA linker as described in Example 4 of EP 0366238.
  • Plasmid pB4+ permits insertion of DNA fragments within the linker following the coding region for the first 81 amino acids of NSl followed by termination codons in all three reading frames.
  • Plasmid pB4+ was digested with Xmal (cuts within linker) , end- filled (Klenow) and ligated to a 520 base pair PvuII/Hindlll, end-filled fragment derived from the HA2 coding region.
  • the resulting plasmid, pD codes for a protein comprised of the first 81 amino acids of NSl, three amino acids derived from the synthetic DNA linker (Gln-Ile-Pro) , followed by amino acids 65-222 of the HA2, as shown in Fig. 2 of published European Patent Application No. 366,238.
  • a BamHI fragment derived from the pD expression plasmid encoding the recombinant flu D protein was ligated into the BamHI site of a pASl plasmid derivative containing a kanamycin resistance gene from Tn903 for selection [Berg et al, Microbiology, ed. D.
  • pOTS207 is a pAS derived cloning vector which carries the kanamycin resistance gene from Tn903 [Berg, cited above; Nomura, cited above; Castellazzi, cited above] . It was constructed by digesting plasmid pUC8 [Yanisch-Perron et al. Gene, 21:103-119 (1985)], with BamHI and ligated to a Bell fragment containing the kanamycin gene from Tn903.
  • the resulting plasmid is pOTS207.
  • a 520 bp fragment encoding the HA2 coding sequence was isolated from pJZ102 [a pBR322-derived cloning vector which carries a coding region for the entire HA protein (A/PR/8/34) (described by Young et al, The Origin of Pandemic Influenza V ruses, ed. W.G. Laver, Elsevier Science Publishing Co. (1983) ] and inserted into pB4+ which had been cut with Xmal and end filled.
  • the resulting plasmid, pC 13 H 65 _ 222 was digested with BamHI and the fragment encoding the flu D protein isolated from this fragment was then ligated into the BamHI site of pOTS207 to produce the plasmid pC 13 (H 65 _ 222 )Kn [SmithKline Beecham] .
  • nucleotide positions 1-31, 3136-3964, 4021-4343, 4533-7166 were derived from pBR322 [Young et al, cited above]; nucleotide positions 32-1879, 4344-4532 were derived from ⁇ phage, nucleotide positions 1880-2122, 2682-3135 were derived from the NSl gene, nucleotide positions 2123- 2132, 2660-2681 were derived from a synthetic linker, nucleotide positions 2133-2659 were derived from the HA2 gene, nucleotide positions 3965-4020 were derived from the pCVi polylinker, and nucleotide positions 7167-8601 were derived from the pUCKanl2 (Tn903:Kn r ) .
  • the above applications are incorporated for reference for their description of other appropriate vectors.
  • the pC 13 (H 65 _ 222 )Kn plasmid was transformed into E. coli expression strain AR58 [SmithKline Beecham]; and production of the flu D protein was confirmed by immunoblot analysis [Towbin et al, Proc. Natl. Acad. Sci. HS ⁇ , 2£:4350 (1979)] which revealed a major immunoreactive species at the predicted molecular weight of 27.7 kD.
  • the NSli. 8j -HA2 65 . 222 or D protein (MW 27.7 kD) , was purified as described in detail in published European
  • Patent Application NO. 366,239 corresponding to U.S.
  • This suspension was then lysed on a Manton Gaulin homogenizer [APV Gaulin, Inc., Everett, Massachusetts] at 8,000 psi in two passes.
  • the resulting suspension was treated with Triton X-100 (1% final concentration) and deoxycholate (DOC; 0.1% final concentration) .
  • the pellet containing D protein was suspended in 50 mM Gly-NaOH buffer containing 2 mM EDTA and 5% glycerol at pH 10.5 using a Turrax homogenizer.
  • the resulting suspension after addition of Triton X-100 (1% final concentration) was stirred for about 1 hour in a 4°C cold room, then centrifuged.
  • the pellet containing D protein was dissolved in 8 M urea, 50 mM Tris at pH 8.0, overnight at 4°C. The supernatant contained the D protein.
  • the resulting solution was loaded onto a Superose-12 column [Pharmacia] , equilibrated with 25 mM Tris-Glycine buffer at pH 8,6, containing 1% SDS.
  • the D protein was eluted at its predicted monomer MW in an isocratic gradient of equilibration buffer. Fractions containing D protein of adequate purity were pooled, concentrated, treated with 10 mg SDS per mg of protein and DTT (to 50 mM final concentration) and then chromatographed on a Superose-12 column under the same conditions described immediately above.
  • Vaccine compositions according to this invention containing superose purified D protein (Example 13) in aluminum hydroxide adjuvant, with or without 3D-MPL were prepared as follows: 3D-MPL (RIBI Immunochemical, Hamilton, MT) was reconstituted in sterile water for injection to a working concentration of 1 mg/ml. This stock solution was sonicated for 30 minutes, and subsequent dilutions, made in 5% dextrose, were sonicated for an additional 10 minutes prior to addition to mixtures of the D protein in aluminum hydroxide, prepared as described previously [Dillon et al, Vaccine, .10 . (5) :309-318 (1992)] .
  • 3D-MPL RIBI Immunochemical, Hamilton, MT
  • the antigen doses were titrated from 0.01, 0.1, 1.0, 10, 20, to 100 ⁇ g/dose of injection; and the 3D-MPL doses were titrated from 0.025, 0.25, 2.5, 25, to 50 ⁇ g/dose of injection.
  • the ratio of 3D-MPL:antigen was maintained at 2.5:1 (w/w) for all antigen doses except the 100 ⁇ g dose, as shown in Table 5 below.
  • Aluminum hydroxide was 100 ⁇ g per dose in all cases.
  • a control vaccine composition contained 20 ⁇ g antigen mixed with CFA, as described in Dillon et al. Vaccine, 1H(5) :309-318 (1992). The final injection volumes were 0.2 ml per mouse.
  • mice (CB6F J ; H-2 dxb ) were injected subcutaneously with a selected vaccine dose, at weeks 0 and 3, and challenged intranasally (under metofane anesthesia) at week 7 with 2 to 5 LD 50 doses of A/Puerto Rico/8/34 [A/PR/8/34 (influenza type A, H1N1) ] virus.
  • Example 15 Evaluation of Flu D Vaccine Compositions This experiment was performed essentially as 5 described in Example 14 above except that the dose of 3D- MPL was titrated and administered either alone, with the minimal effective dose of flu D antigen (1 ⁇ g) and the aluminum adjuvant (100 ⁇ g) , or with flu D antigen (lO ⁇ g) and the aluminum adjuvant (100 ⁇ g) .
  • the results reported 10 in Table 6 identify 2 ⁇ g 3D-MPL as the minimal effective dose.
  • 3D-MPL was compared to liposomes (Lipo) without 3D-MPL [Ribi Immunochem], and to Al (OH) , or CFA, by determining the level of protection seen after challenge with 2, 10 or 50 LD 50 doses of virus.
  • the vaccine used was the same as in Table 9 below. Mice were injected at weeks 0 and 3 and challenged with A/PR/8/34 at week 7. Survival is shown in Table 7 below through day 21.
  • Example 17 Evaluation of Flu D Vaccine Compositions
  • a vaccine composition of the invention containing flu D protein, 3D-MPL, and liposomes was evaluated in comparison to a vaccine composition containing flu D, 3D-MPL and aluminum adjuvant.
  • the actual amount of 3D-MPL incorporated into liposomes was measured.
  • the flu D protein (superose purified) in 25 mM Tris/1 mM EDTA, pH 8, at a total protein concentration of 2.49 mg/mL was dialyzed against 20 mM ammonium bicarbonate pH 8 and lyophilized to a powder in 20 mg aliquots.
  • the lyophilized protein was combined with the pre- liposomal gel (0.06-0.08 protein/gel (w/w) ratio) and mixed at room temperature until a homogeneous paste is formed.
  • Monophosphoryl lipid A (Ribi/Immunochem) was added at a ratio of 1 ⁇ mole of lipid A to 66 ⁇ mole of phosphatides. This was a transparent colorless system composed of nanoparticulate (submicron in diameter) structures of aggregated 3D-MPL monomers.
  • the resulting liposome suspension was further diluted with 25 mM Tris/1 mM EDTA, pH 8.0 buffer and homogenized.
  • the liposome preparation was then serially centrifuged (3x) to remove unincorporated protein and 3D-MPL. Each time the pellet was resuspended with the Tris/EDTA buffer. The final liposome suspension was analyzed and kept stored under N 2 at 5°C while not in use.
  • the liposomes were subsequently assayed to determine protein content by the modified Lowery colorimetric assay, the phospholipid concentration by the Barlett assay [Bartlett, G. R., J. Bio . Che .. 211:466-468 (1959) ] and liposome size by photon correlation spectroscopy [Malvern Model 4700] . All assays were performed by standard techniques. Liposomes were not analyzed for lipid A content, however, it is likely that all of the lipid A remains in the final liposome fraction.
  • D protein LA and control LA-I was prepared by incorporating solid lipid A into the lipid phase.
  • Control LA-II was prepared by hydrating with a solution of lipid A for incorporation.
  • Liposome Phospholipid Protein 3D-MPL Diameter Z- Sample ( ⁇ mole/mL) (mg/mL) ( ⁇ g/mL) Avg Mean (nm)
  • mice were immunized s.c. with 0.2 ml of the antigen/carrier/3D-MPL formulation at weeks 0 and 3, and challenged intranasally on week 7 with 5 LD 50 of A/PR/8/34 virus.
  • the "carrier” was either aluminum hydroxide (Al) or liposomes (Lipo) . Survival was monitored for 21 days.
  • mice (12 per group) were injected sc at weeks 0 and 3 with a vaccine composition comprising 100 ⁇ g flu D in aluminum (100 ⁇ g) plus 3D-MPL (10 ⁇ g) .
  • a vaccine composition comprising 100 ⁇ g flu D in aluminum (100 ⁇ g) plus 3D-MPL (10 ⁇ g) .
  • the mice were challenged with 3-5 LD 50 doses of the viruses shown in Fig. 1. Survival was monitored for 21 days post-challenge.
  • Fig. 1 demonstrate that the antigenic specificity of the vaccine was equivalent to that demonstrated earlier with the Al +3 adjuvant [S. B. Dillon et al, cited above] (i.e., cross-protective for both HI and H2 subtypes, but lack of efficacy for H3 or Type B) . Survival was also greater in the vaccinated group challenged with the heterologous H1N1 virus, A/Tai/86, although the results were not significant, p ⁇ 0.09. (See Fig. 1) .
  • Example 1 - Evaluation of Flu D Vaccine Composition CB6F 2 mice were injected subcutaneously (sc) with a vaccine composition comprising 1 ⁇ g D protein in aluminum (100 ⁇ g) plus 3D-MPL (2.5 ⁇ g) or with aluminum only at 0 and 3 weeks. Eighteen mice were challenged at week 7 with 5 LD 50 doses of A/PR/8/34 virus. Five mice were sacrificed at day 7 post-challenge for lung titers.
  • mice immunized with 1 ⁇ g of D protein in Al vs A1/3D-MPL (2.5 ng 3D-MPL) adjuvants were monitored in the spleens of mice immunized with 1 ⁇ g of D protein in Al vs A1/3D-MPL (2.5 ng 3D-MPL) adjuvants (from the study shown in Table 10) pre- and post-challenge.
  • two mice were sacrificed 6 days after virus challenge. Spleens were co-cultured with D protein and pulsed with 3 H-thymidine on day 3. Culture supernatants were harvested 8 hours later. Overall, splenic proliferative responses were similar pre- vs.
  • Lymph nodes from mice given a single injection of 1, 5 or 20 ⁇ g D protein in A1/3D-MPL (ratio of 3D- MPL:antigen at 2.5:1 w/w) or Al + adjuvant (100 ⁇ g) 7 days earlier were cultured with 0-30 ⁇ g/ml purified D protein.
  • Fig. 3 maximal cpm in unstimulated cultures equalled 1368 cpm.
  • maximal cpm in unstimulated cultures equalled 1600 cpm; and maximal cpm of CTLL (an IL-2 dependent CTL line) cells cultured with supernatants from unstimulated cultures equalled 195 cpm) .
  • Peak (48 hours) IL-2 activity was greater in the groups vaccinated with the A1/3D-MPL formulations, although the levels were generally low in all cultures (Fig. 4) .
  • Table 12 provides the results of the analysis of interferon-gamma in supernatants from the same antigen- stimulated cultures. Interferon levels in supernatants from adjuvant control cultures were all ⁇ 1 ng/ml. Interferon-gamma was measured with Gibco-BRL ELISA kit. This cytokine, which was highest at day 4 of culture, was up to 5-fold greater in the A1/3D-MPL group. Table 12
  • Example 23 The results in Table 13 below (shown in Example 23) show that the incorporation of 3D-MPL (125 ⁇ g) into a vaccine formulation with 50 ⁇ g of antigen significantly increased survival in mice given either one or two injections when compared to the same dose of the vaccine protein adsorbed to aluminum. Again, survival in the Al/MPL group was comparable to that seen in the CFA.
  • Culture medium was RPMI 1640 supplemented with 10% fetal calf serum [Hyclone Laboratories, Logan, UT] , 2 mM glutamine, 5 x 10 "5 M 2-ME, 10 mM HEPES, penicillin and streptomycin.
  • mice The spleens from mice given one or two sc injections of 50 ⁇ g D protein (0 and 3 weeks) , were removed at week seven and restimulated in vitro as described above.
  • One lytic unit (LU 35 ) is defined as the number of effector cells in the chromium release assay required for 35% lysis (determined by regression analysis) .
  • Table 13 reports the results of HlNl-specific CTL activity in these spleens of mice immunized with SK&F 106160 (D Protein) in aluminum hydroxide and 3D-MPL.
  • 3D-MPL did not potentiate class I restricted memory CD8+ CTL relative to the response generated with aluminum hydroxide (indicated as Al t3 ) as reported in Table 13.
  • Al t3 aluminum hydroxide
  • the results in Table 13 show that splenic CTL were comparable in mice given 1 vs. 2 sc injections, whereas protection was clearly improved in mice given a second injection. Therefore it appears that a mechanism other than CD8+ CTL must be responsible for the improved immunity seen when 3D-MPL is included in the vaccine composition containing the antigenic peptide and aluminum adjuvant, or when mice are given a booster injection.
  • Example 24 Depletion Studies To further determine which T cell subset is best correlated with the activity of the 3D-MPL adjuvant, depletion studies were done with anti-CD-4 or anti-CD-8 monoclonal antibodies (Mabs) .
  • mice were treated with Mabs to deplete T cell subsets prior to the first injection of vaccine (pre-vaccination protocol) on days -3, -2, -1. After the vaccination on day 0, mice were further treated with Mabs on days, 7, 14, 18, 19 and 20, and then boosted with vaccine on day 21. Mice were further treated with Mabs on days 28, 35, 39, 40 and 41 prior to virus challenge on day 42. Mice were further treated with Mabs post- challenge on days 43, 49 and 54. The results of these T cell subset depletion experiments are shown in Table 14 below.
  • mice were depleted of CD4 + or CD8 + T cell svibsets by injecting anti-CD4 or anti-CD8 Mabs ip daily for 3 days (300 ⁇ g/injection) .
  • mice were immunized with 20 ⁇ g flu D protein in the aluminum hydroxide (100 ⁇ g) and 3D-MPL (20 ⁇ g) formulation, and lymph nodes were removed 7 days later.
  • the lymph node cells were restimulated in vitro with 0, 1 or 10 ⁇ g/ml D protein, and supernatants collected on days 1-4 for interferon and IL-2 assays.
  • the results in Fig. 5 show that IL-2 production was completely eliminated by anti-CD4 treatment, but was also partially reduced in anti-CD-8 treated mice (Fig. 5B) .
  • Peak IFN ⁇ production (day 4 supernatants) was reduced by approximately 50% in anti-CD4 or anti-CD8 treated mice (Fig. 5C) . Therefore, both CD4 + and CD8 + T cell subsets produced IL-2 and IFN ⁇ .
  • Serumwerkl GmbH (SSW) (Dresden, Germany) may be prepared by well known methods, such as those documented in, for example, European Pharmacopoeia PA/PH/Exp 3 (1992) ; DAB 10 "Vaccines Influenzae ex virorum fragmentis praeparatum” and the World Health Organization draft revised requirements for Influenza vaccines (Inactivated) (1990) .
  • split viruses are prepared using one or more influenza virus strains recommended by WHO and the EEC, such as A/Singapore/6/86, A/Beijing/32/92 and B/Panama/45/90. These strains can change depending on the strains popular in a particular year.
  • influenza viruses are obtained from embryonated hens' eggs inoculated with seed lot material. These virus suspensions are partially purified and concentrated. The concentrated virus suspension is treated with a detergent, sodium desoxylcholate, to disrupt (or "split") the virus particles. Following the removal of viral phospholipids during the splitting process, the reactogenicity potential is greatly reduced. The split virus suspension is completely inactivated by the combined effect of the detergent and formaldehyde.
  • the process for producing a split virus of this invention is as follows.
  • a fresh inoculum is prepared by mixing the influenza working seed lot with a phosphate buffer containing gentamycin sulphate at 0.5 mg/ml and hydrocortisone at 25 ⁇ g/ml.
  • the virus inoculum is kept at 2-8°C.
  • Nine to eleven day old embryonated eggs are used for virus replication.
  • the eggs are incubated at the farms before arrival at the manufacturing plant and enter into the production rooms after decontamination of the shells.
  • the eggs are inoculated with 0.2 ml of the virus inoculum on an automatic egg inoculation apparatus.
  • the inoculum is injected at a pressure of ⁇ 0.03 MPa.
  • the inoculated eggs are incubated at the appropriate temperature (virus strain-dependent) for 50 to 96 hours.
  • the embryos are killed by cooling the eggs and storage for 12-60 hours at 2-8°C.
  • the allantoic fluid from the chilled embryonated eggs is harvested by appropriate egg harvesting machines. Usually, 8 to 10 ml of crude allantoic fluid can be collected per egg. To the crude monovalent virus bulk is add 0.100 mg/ml thiomersal.
  • the harvested allantoic fluid is clarified by flow through centrifugation at a volume of 100-200 L/hour and a centrifugal force of 10-17,000 g.
  • This preclarified liquid can be further clarified on a 6-8 ⁇ m membrane filter.
  • a CaHP0 4 gel in the clarified virus pool 0.5 M Na 2 HP0 4 and 0.5 M CaCl 2 solutions are added to reach a final concentration of CaHP0 4 of 1.5 g to 3.5 g CaHP0 4 /liter depending on the virus strain. After sedimentation for at least 10 hours, the supernatant is removed and the sediment containing the Influenza virus is resolubilized by addition of a 0.26 M EDTA solution. The concentration of EDTA varies between 4.5 and 10 g/liter of the original harvest volume.
  • the resuspended sediment is filtered on a 6 to 8 ⁇ m filter membrane.
  • the Influenza virus is concentrated by isopycnic centrifugation in a linear sucrose gradient (0-55%) at 90,000 g.
  • the flow through volume is from 8-12 liters/hour.
  • the content of the rotor is recovered by four different fractions (the sucrose is measured in a refractometer) :
  • Fraction 3 is diluted in order to reduce the sucrose content to approximately 6%.
  • the Influenza virus present in this diluted fraction is pelleted at 53,000 g to remove soluble contaminants.
  • the pellet is resuspended and thoroughly mixed to obtain a homogenous suspension.
  • Fraction 2 and the resuspended pellet of fraction 3 are pooled and phosphate buffer is added to obtain a volume of 30 liters. At this stage, the product is called "monovalent whole virus concentrate".
  • the selected Influenza virus preferably the monovalent whole virus concentrate of Part A above, is disrupted by centrifugation at 70,000 g through a Nadoc linear sucrose gradient of 0-55% containing a linear distribution of sodiumdesoxycholate from 1,2-1,5%. Tween 80 is present at 0.1% in the gradient.
  • the virus concentrate is pumped at the rate of 5 liters/hour. At the end of the centrifugation, the content of the rotor is collected in 3 different fractions:
  • the diluted fraction 2 is filtered on filter membranes ending with a 0.2 ⁇ m membrane.
  • the filters are washed with phosphate buffer containing 0.01% thiomersal and 0.01% Tween 80.
  • the final volume of the filtered fraction 2 is 5 times the original fraction volume.
  • a brief sonication of the split virus material can be introduced to facilitate the sterile filtration.
  • the filtered monovalent split material is incubated at 22 ⁇ 2°C for at least 84 hours to allow inactivation of viruses and mycoplasma by the effect of sodium desoxycholate.
  • phosphate buffer containing 0.01% thiomersal and 0.01% Tween 80 is added in order to bring the total protein content down to maximum of 250 ⁇ g/ml.
  • Formaldehyde is added at the rate of 50 ⁇ g/ml and the inactivation takes place at 4°C ⁇ 2°C for at least 72 hours.
  • the inactivated split virus material is ultrafiltered on membranes having a mean pore size of 20,000 daltons. During ultrafiltration, the content of formaldehyde, NaDOC and saccharose is greatly reduced. The volume remains constant during ultrafiltration (diafiltration) by adding phosphate buffer containing 0.01% thiomersal and 0.01% Tween 80.
  • the ultrafiltered split material is filtered on membranes ending with a 0.2 ⁇ m, depending on the virus strain the last filtration membrane can be 0.8 ⁇ m.
  • the product is called: "monovalent final bulk".
  • the monovalent final bulk is stored at 2-8°C for a maximum of 18 months.
  • Example 26 Split Virus Vaccine Composition (a) Preparation of MPL with a particle size of 60 - 120 nm
  • Suspensions can in some cases be stored at 4 degrees C without significant aggregation up to 5 months.
  • Isotonic NaCl (0.15M) or isotonic NaCl plus lOmM phosphate induces a rapid aggregation (size >3-5 ⁇ m) .
  • the split virus vaccine composition of this invention is prepared as follows.
  • a final bulk-buffer is prepared by adding to water for injection the concentrated salt solutions: NaCl 4 mg; Na 2 HPOforce 0.52 mg; KH 2 P0 4 0.19 mg; KCl 0.1 mg; and MgCl 2 0.05 mg, and Thiomersal 50 ⁇ g. The resulting solution is stirred 15 minutes before further use.
  • Monovalent split virus bulk (15 ⁇ g HA) is then mixed with 3D-MPL 50 ⁇ g and the resulting mixture is stirred for about 1 hour at room (or ambient) temperature.
  • the buffer mixture and the virus bulk mixture are then mixed together. After 30 minutes stirring at room temperature, the pH is brought to 7.15 ⁇ 0.1.
  • the resulting final vaccine is stored at +2 - +8°C.
  • monovalent split virus pools of the selected strains prepared as described above are mixed to constitute the final multivalent vaccine.
  • the resulting pooled material is stirred for 30 minutes at room temperature (pH 7.15 ⁇ 0.1) .
  • the resulting final vaccine may be stored at temperatures between about 2°C to about 8°C.
  • the vaccine is a colourless, light opalescent aqueous suspension of purified split influenza virus.
  • a vaccine preparation was prepared without the adjuvant.
  • a final monovalent bulk-buffer is prepared by adding to water for injection the concentrated salt solutions: NaCl 4 mg; Na 2 HP0 4 0.52 mg; KH 2 P0 4 0.19 mg; KCl 0.1 mg; MgCl 2 0.05 mg; and Thiomersal 50 ⁇ g.
  • the resulting solution is stirred 15 minutes before further use.
  • the mixture is stirred for 15 minutes at room temperature.
  • Monovalent bulk (15 ⁇ g HA) is added, followed by 30 minutes stirring at room temperature (pH 7.15 ⁇ 0.1) .
  • the resulting final vaccine is stored at temperatures between +2°C - +8°C.
  • the immunogenicity of the monovalent vaccine formulation of this invention has been evaluated and compared to the immunogenicity of the monovalent non- adjuvanted formula and to the adjuvant 3D-MPL without antigen in a lethal influenza challenge in mice.
  • mice For each vaccine, CB6F 1 mice (30 per group) were immunized subcutaneously with 2 injections given 3 weeks apart with a preparation that contains 5 ⁇ g of indicated strain ⁇ 5 ⁇ g 3D-MPL. Seven weeks after the first injection, mice were intranasally challenged under metofane anaesthesia with 5 LD 50 of A/PR/8. Survival and clinical signs of illness were monitored for 15 mice up to 3 weeks after challenge. Lung virus titers were determined by MDCK microassay [A. L. Frank et al, . Clin. Microbiol., 12:426-432 (1980)] on the remaining animals (5 mice per group) .
  • the two strains induce a certain level of protection.
  • the survival rate for both groups was markedly higher than that of the control preparation devoid of antigen (Alum + 3D-MPL) , and lung virus clearance was faster.
  • A/PR/8- vaccinated group performed better than the Singapore-vaccinated group (heterologous with respect to challenge) .
  • both strains supplemented with 3D-MPL induced 100% survival, practically no lung viral titer, and no clinical symptoms.
  • the 3D-MPL adjuvanted vaccine of the invention induced superior protection against lower respiratory track infection and therefore serious clinical manifestations such as viral pneumonia. From this experiment it therefore appears that 3D-MPL adjuvantation improves homosubtypic and heterosubtypic activity of the split vaccine and is therefore anticipated to provide broader protection against antigenic shifts and drifts than unadjuvanted vaccine.
  • mice were not anesthesized before intranasal challenge.
  • Virus titration in the respiratory tract, virus neutralization assays and scanning electron microscopy of trachea were performed.
  • Viral neutralization titration against A/PR/8 virus was carried out on days 1, 5 and 9 post-challenge (Table 16) .
  • Both A/PR/8 groups produced neutralizing antibodies, with a titer consistently higher in the presence of 3D- MPL.
  • the vaccination with Singapore did not induce neutralization antibodies, even with the addition of 3D-MPL. This indicated that the heterosubtypic activity observed previously with Singapore and 3D-MPL was not due to antibody and provides strong evidence that cell-mediated immunity was responsible.
  • lung titers were differentiated: A/PR/8 + 3D- MPL and Singapore + 3D-MPL titers were always lower or equal to the values of the antigen alone, or of 3D-MPL alone (Figs. 6A through 6F) . This indicates that 3D-MPL adjuvantation confers a better lung protection in mice.
  • CB6F X mice (15 per group) were immunized subcutaneously with 2 injections given 3 weeks apart of a preparation that contains one tenth of a human dose of conventional unadjuvanted Singapore monovalent split vaccine or a vaccine composition of this invention containing the Singapore strain with 3D-MPL.
  • a human dose is defined as a 0.5 ml injection containing 15 ⁇ g HA of each viral strain.
  • the formulation used herein contained a hemaglutinin (HA) to 3D-MPL ratio of 1.5 ⁇ g HA per 5 ⁇ g 3D-MPL to 5 ⁇ g HA per 5 ⁇ g 3D-MPL.
  • mice received only 3D-MPL (5 ⁇ g) .
  • mice were bled and sera antibodies were individually assayed by inhibition of haemagglutination. Titers were calculated against a calibrated reference. Mice were considered responders if they have antibody titers greater than the cut-off value.
  • cut-off value is lower than 25
  • This experiment indicates that antibody response in mice is improved by the use of the 3D-MPL adjuvanted vaccine of this invention.
  • Trivalent Influsplit was prepared as described in Example 25 and contained an HlNl strain Singapore/6/86, an H3N2 strain Beijing/353/84 and a Type B strain, B/Yamaghta/16/88.
  • the sensitizing agent [allantoic fluid 0.5 or 2.5 mg ⁇ 3D-MPL 50 ⁇ g; one human dose (0.5 ml injection containing 15 ⁇ g HA for each of these strains of influenza virus) Trivalent Influsplit ⁇ 3D-MPL 50 ⁇ g] was given intraperitoneally by six injections, at days 0, 3, 5, 7, 10, and 12. Guinea pigs were allowed to rest for 4 weeks and then challenged intravenously, under anesthesia, with the challenging agent (allantoic fluid 0.45 mg; one human dose Trivalent Influsplit ⁇ 3D-MPL 50 ⁇ g) . The animals were observed 30 minutes after challenge, and again after 2 or 3 hours. The observed symptoms (scratching, breathing problems, convulsions, death) were recorded. Where no symptoms arose from the first challenge, the animals were re-challenged with allantoic fluid (1.3 mg) 24 hours later.

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Abstract

La présente invention se rapporte à des compositions de vaccins susceptibles de renforcer une réaction protectrice par rapport à un antigène de la grippe choisi, la composition contenant au moins cet antigène et le lipide A de monophosphoryle désacylé en position 3-O (3D-MPL). Des procédés de renforcement de la réponse immunitaire à la grippe à l'aide de ces compositions sont également décrits.
PCT/EP1994/000448 1993-02-19 1994-02-15 Compositions de vaccin contre la grippe, contenant un lipide a de monophosphoryle desacyle en position 3-o WO1994019013A1 (fr)

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JP6518456A JPH08506592A (ja) 1993-02-19 1994-02-15 ３−ｏ−脱アシル化モノホスホリル脂質Ａ含有のインフルエンザワクチン組成物
EP94908327A EP0684838A1 (fr) 1993-02-19 1994-02-15 Compositions de vaccin contre la grippe, contenant un lipide a de monophosphoryle desacyle en position 3-o
CN94191720.7A CN1124013A (zh) 1993-02-19 1994-02-15 含3-邻位-脱酰基的单磷酰基脂质a的流感疫苗组合物
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WO1996033739A1 (fr) * 1995-04-25 1996-10-31 Smithkline Beecham Biologicals S.A. Vaccins contenant une saponine ainsi qu'un sterol
EP0689454B1 (fr) * 1993-03-23 1997-09-10 SMITHKLINE BEECHAM BIOLOGICALS s.a. Compositions vaccinales renfermant le lipide a monophosphorylique 3-o desacetyle
US6027730A (en) * 1991-03-21 2000-02-22 Smithkline Beecham Biologicals Herpes simplex vaccine comprising HSV glycoprotein GD and 3 deacylated monophosphoryl lipid A
WO2000047222A2 (fr) * 1999-02-11 2000-08-17 Baxter Vaccine Aktiengesellschaft Vaccin a virus grippal inactive pour application nasale ou orale
WO2001022992A3 (fr) * 1999-09-30 2001-05-17 Smithkline Beecham Biolog Nouvelle composition
EP1124578A1 (fr) * 1998-10-30 2001-08-22 Avant Immunotherapeutics, Inc. Nouveaux immunoadjuvants
US6306404B1 (en) 1998-07-14 2001-10-23 American Cyanamid Company Adjuvant and vaccine compositions containing monophosphoryl lipid A
WO2002067983A1 (fr) * 2001-02-23 2002-09-06 Glaxosmithkline Biologicals S.A. Nouveau vaccin
WO2002074336A3 (fr) * 2001-02-23 2003-08-14 Glaxosmithkline Biolog Sa Vaccin
US6635261B2 (en) 1999-07-13 2003-10-21 Wyeth Holdings Corporation Adjuvant and vaccine compositions containing monophosphoryl lipid A
GB2341801B (en) * 1998-09-21 2003-10-29 Allergy Therapeutics Ltd Formulation
EP1381344A2 (fr) * 2001-04-27 2004-01-21 GlaxoSmithKline Biologicals S.A. Nouveau vaccin
WO2006062637A2 (fr) 2004-11-03 2006-06-15 Novartis Vaccines And Diagnostics Inc. Vaccination antigrippale
WO2008134830A1 (fr) * 2007-03-22 2008-11-13 Fundação Butantan Procédé pour obtenir le lipide monophosphorylé a à partir de bordetella pertussis comme sous-produit de la production du vaccin cellulaire anti-coquelucheux
WO2009115917A2 (fr) * 2008-03-18 2009-09-24 Novartis Ag Améliorations dans la préparation des antigènes pour le vaccin contre le virus de la grippe
EP2236155A1 (fr) 2004-09-09 2010-10-06 Novartis Vaccines and Diagnostics GmbH Diminution des risques iatrogéniques potentiels associés aux vaccins contre la grippe
WO2012032482A1 (fr) 2010-09-07 2012-03-15 Novartis Ag Tests génériques pour la détection de réovirus de mammifères
EP2653156A1 (fr) * 2012-10-01 2013-10-23 College of Pharmacy of Taif University Composition d'émulsion pharmaceutique et son utilisation
WO2015147899A1 (fr) * 2014-03-25 2015-10-01 The Government Of The United States Of America As Represented By The Secretary Of The Army Procedes pour l'amelioration de l'activite d'immunostimulation de vaccins adsorbes sur sel d'aluminium
US9278127B2 (en) 2006-07-17 2016-03-08 Glaxosmithkline Biologicals, Sa Influenza vaccine
US9452209B2 (en) 2007-04-20 2016-09-27 Glaxosmithkline Biologicals Sa Influenza vaccine
US9730999B2 (en) 2005-03-23 2017-08-15 Glaxosmithkline Biologicals Sa Adjuvanted influenza virus compositions
WO2020010394A1 (fr) * 2018-07-10 2020-01-16 Seqirus Pty Ltd Élimination d'agglomérats
CN112361796A (zh) * 2020-11-13 2021-02-12 安徽省天长市周氏羊业有限公司 一种秸秆回收储存前破碎干燥装置
CN114989269A (zh) * 2022-06-30 2022-09-02 天康制药(苏州)有限公司 一种牛赤羽免疫原性抗原及疫苗

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EP1175912A1 (fr) * 1993-03-23 2002-01-30 SmithKline Beecham Biologics SA Compositions vaccinales renfermant le lipide à monophosphorylique 3-O désacétylé
EP0689454B1 (fr) * 1993-03-23 1997-09-10 SMITHKLINE BEECHAM BIOLOGICALS s.a. Compositions vaccinales renfermant le lipide a monophosphorylique 3-o desacetyle
EP0812593A1 (fr) * 1993-03-23 1997-12-17 SMITHKLINE BEECHAM BIOLOGICALS s.a. Compositions vaccinales renfermant de lipide A monophosphorylique 3-0-desacétylé
EA000839B1 (ru) * 1995-04-25 2000-04-24 Смитклайн Бичем Байолоджикалз С.А. Липосомальная вакцинная композиция, применение липосомальной вакцинной композиции и способ лечения млекопитающего
EP0884056A1 (fr) * 1995-04-25 1998-12-16 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccins contenant une saponine ainsi qu'un sterol
EP0955059A3 (fr) * 1995-04-25 2000-07-12 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccins contenant une saponine ainsi qu'un sterol
WO1996033739A1 (fr) * 1995-04-25 1996-10-31 Smithkline Beecham Biologicals S.A. Vaccins contenant une saponine ainsi qu'un sterol
US6306404B1 (en) 1998-07-14 2001-10-23 American Cyanamid Company Adjuvant and vaccine compositions containing monophosphoryl lipid A
GB2341801B (en) * 1998-09-21 2003-10-29 Allergy Therapeutics Ltd Formulation
EP1124578A1 (fr) * 1998-10-30 2001-08-22 Avant Immunotherapeutics, Inc. Nouveaux immunoadjuvants
EP1124578A4 (fr) * 1998-10-30 2005-01-19 Avant Immunotherapeutics Inc Nouveaux immunoadjuvants
WO2000047222A3 (fr) * 1999-02-11 2001-10-11 Baxter Vaccine Ag Vaccin a virus grippal inactive pour application nasale ou orale
WO2000047222A2 (fr) * 1999-02-11 2000-08-17 Baxter Vaccine Aktiengesellschaft Vaccin a virus grippal inactive pour application nasale ou orale
US7052701B2 (en) 1999-02-11 2006-05-30 Baxter Healthcare S.A. Inactivated influenza virus vaccine for nasal or oral application
US6861244B2 (en) 1999-02-11 2005-03-01 Baxter Healthcare S.A. Inactivated influenza virus vaccine for nasal or oral application
US6635246B1 (en) * 1999-02-11 2003-10-21 Baxter Healthcare S.A. Inactivated influenza virus vaccine for nasal or oral application
US6635261B2 (en) 1999-07-13 2003-10-21 Wyeth Holdings Corporation Adjuvant and vaccine compositions containing monophosphoryl lipid A
EP1618889A2 (fr) * 1999-09-30 2006-01-25 SmithKline Beecham Biologicals s.a. Vaccin contre l'influenza
EP2269637A1 (fr) * 1999-09-30 2011-01-05 SmithKline Beecham Biologicals S.A. Vaccin contre l'influenza
EP1618889A3 (fr) * 1999-09-30 2006-06-14 SmithKline Beecham Biologicals s.a. Vaccin contre l'influenza
EP3103473A1 (fr) * 1999-09-30 2016-12-14 GlaxoSmithKline Biologicals S.A. Vaccin contre l'influenza
US7238349B1 (en) 1999-09-30 2007-07-03 Smithkline Beecham Biologicals, S.A. Composition
WO2001022992A3 (fr) * 1999-09-30 2001-05-17 Smithkline Beecham Biolog Nouvelle composition
WO2002074336A3 (fr) * 2001-02-23 2003-08-14 Glaxosmithkline Biolog Sa Vaccin
WO2002067983A1 (fr) * 2001-02-23 2002-09-06 Glaxosmithkline Biologicals S.A. Nouveau vaccin
US8557251B2 (en) 2001-02-23 2013-10-15 Glaxosmithkline Biologicals, Sa Non-live trivalent influenza vaccine for one-dose intradermal delivery
EP2281573A3 (fr) * 2001-02-23 2011-12-07 GlaxoSmithKline Biologicals s.a. Formulations de vaccins contre la grippe dispositif pour administration intradermique
EP2269639A3 (fr) * 2001-02-23 2011-12-07 GlaxoSmithKline Biologicals s.a. Formulations de vaccins contre la grippe dispositif pour administration intradermique
EP1381344A2 (fr) * 2001-04-27 2004-01-21 GlaxoSmithKline Biologicals S.A. Nouveau vaccin
EP1381344A4 (fr) * 2001-04-27 2008-06-11 Glaxosmithkline Biolog Sa Nouveau vaccin
EP2236155A1 (fr) 2004-09-09 2010-10-06 Novartis Vaccines and Diagnostics GmbH Diminution des risques iatrogéniques potentiels associés aux vaccins contre la grippe
EP2471551A2 (fr) 2004-09-09 2012-07-04 Novartis Vaccines and Diagnostics GmbH Diminution des risques iatrogéniques potentiels associés aux vaccins contre la grippe
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EP2923711A1 (fr) 2004-11-03 2015-09-30 Novartis Vaccines and Diagnostics, Inc. Vaccin contre la grippe
WO2006062637A2 (fr) 2004-11-03 2006-06-15 Novartis Vaccines And Diagnostics Inc. Vaccination antigrippale
US9730999B2 (en) 2005-03-23 2017-08-15 Glaxosmithkline Biologicals Sa Adjuvanted influenza virus compositions
US11564984B2 (en) 2006-07-17 2023-01-31 Glaxosmithkline Biologicals Sa Influenza vaccine
US9943588B2 (en) 2006-07-17 2018-04-17 Glaxosmithkline Biologicals S.A. Influenza vaccine
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WO2008134830A1 (fr) * 2007-03-22 2008-11-13 Fundação Butantan Procédé pour obtenir le lipide monophosphorylé a à partir de bordetella pertussis comme sous-produit de la production du vaccin cellulaire anti-coquelucheux
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WO2009115917A3 (fr) * 2008-03-18 2010-02-04 Novartis Ag Améliorations dans la préparation des antigènes pour le vaccin contre le virus de la grippe
WO2012032482A1 (fr) 2010-09-07 2012-03-15 Novartis Ag Tests génériques pour la détection de réovirus de mammifères
EP3012330A1 (fr) 2010-09-07 2016-04-27 Novartis AG Dosages génériques de détection de réovirus de mammifère
EP2653156A1 (fr) * 2012-10-01 2013-10-23 College of Pharmacy of Taif University Composition d'émulsion pharmaceutique et son utilisation
WO2015147899A1 (fr) * 2014-03-25 2015-10-01 The Government Of The United States Of America As Represented By The Secretary Of The Army Procedes pour l'amelioration de l'activite d'immunostimulation de vaccins adsorbes sur sel d'aluminium
CN106535929A (zh) * 2014-03-25 2017-03-22 美国政府陆军部 增强铝盐吸附疫苗的免疫刺激效力的方法
WO2020010394A1 (fr) * 2018-07-10 2020-01-16 Seqirus Pty Ltd Élimination d'agglomérats
US11596684B2 (en) 2018-07-10 2023-03-07 Seqirus Pty Ltd Removal of agglomerates
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CN114989269A (zh) * 2022-06-30 2022-09-02 天康制药(苏州)有限公司 一种牛赤羽免疫原性抗原及疫苗
CN114989269B (zh) * 2022-06-30 2023-09-19 天康制药股份有限公司 一种牛赤羽免疫原性抗原及疫苗

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AU6141094A (en) 1994-09-14
MA23118A1 (fr) 1994-10-01
AP9400621A0 (en) 1994-04-30
AP431A (en) 1995-11-15
IL108681A0 (en) 1994-05-30
SI9400085A (en) 1994-09-30
EP0684838A1 (fr) 1995-12-06
MX9401225A (es) 1994-08-31

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