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WO1990001948A1 - Vaccin contre la grippe et nouveaux adjuvants - Google Patents

Vaccin contre la grippe et nouveaux adjuvants Download PDF

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Publication number
WO1990001948A1
WO1990001948A1 PCT/US1989/003658 US8903658W WO9001948A1 WO 1990001948 A1 WO1990001948 A1 WO 1990001948A1 US 8903658 W US8903658 W US 8903658W WO 9001948 A1 WO9001948 A1 WO 9001948A1
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WO
WIPO (PCT)
Prior art keywords
optionally
liposome
dosage form
immunogen
sterol
Prior art date
Application number
PCT/US1989/003658
Other languages
English (en)
Inventor
Mircea C. Popescu
Marie S. Recine
Leonard F. Estis
Lynn D. Keyes
Carl R. Alving
Andrew S. Janoff
Original Assignee
The Liposome Company, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Liposome Company, Inc. filed Critical The Liposome Company, Inc.
Priority to AU41861/89A priority Critical patent/AU627226B2/en
Publication of WO1990001948A1 publication Critical patent/WO1990001948A1/fr
Priority to KR1019900700839A priority patent/KR0137360B1/ko

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    • 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
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with 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
    • 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
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—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
    • 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
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with 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
    • A61K2039/55505—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
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention in the vaccine arts is concerned with an influenza immunizing dosage form comprising a liposome and an antigen of Influenza, particularly the hemagglutinin or bromelain fragment, wherein said liposome and antigen are present in an immunization dose.
  • a dosage form including such form particularlyadapted to producing an immune response, comprising a salt form of an organic acid derivative of a sterol and an antigen wherein said organic acid derivative of a sterol and antigen are present in an immunization dose, and method of use.
  • a dosage form, including such form particularly adapted to producing an immune response comprising dimyristolyphosphatidylcholine
  • DMPC DMPC/cholesterol liposomes
  • an antigen wherein said DMPC/cholesterol and antigen are present in an immunization dose, and method of use.
  • antigens are introduced into an organism in a manner so as to stimulate an immune response in the host organism.
  • the induction of an immune response depends on many factors among which are believed to include the chemical composition and
  • An immune response has many facets some of which are exhibited by the cells of the immune system, (e.g.,B-lymphocytes,
  • Immune system cells may participate in the immune response through interaction with antigen, interaction with other cells of the immune system, the release of cytokines and reactivity to those cytokines. Immune response is conveniently (but arbitrarily) divided into two main categories — humoral and cell-mediated.
  • the humoral component of the immune response includes production of immunoglobulins specific for the antigen.
  • the cell-mediated component includes the
  • immune response is the result of an initial or priming dose of an antigen that is followed by one or more booster exposures to the antigen. Priming with relatively strong immunogens and liposomes is discussed in "Liposomal Enhancement of the
  • an antigen will exhibit two properties, the capacity to stimulate the formation of the corresponding antibodies and the propensity to react specifically with these antibodies.
  • Immunogens bear one or more epitopes which are the smallest part of an antigen recognizable by the combining site of an antibody or
  • immunogloublin In particular instances antigens or fractions of antigens or with particular presenting conditions the immune response precipitated by the desired antigen is inadequate or nonexistent and insufficient immunity is produced. This is particularly the case with peptide or other small molecules used as Immunogens.
  • adjuvants to potentiate an immune response when used in conjunction with an antigen.
  • adjuvants are further used to elicit immune response sooner, or a greater response, or with less antigen or to increase production of certain antibody subclasses that afford immunological protection, or to enhance components of the immune response (e.g., humoral, cellular).
  • Well known adjuvants are Freund's Adjuvants (and other oil
  • adjuvant will be understood to mean a substance or material administered together or in conjunction with an antigen which increases the immune response to that antigen.
  • adjuvants may be in a number of forms including emulsion (e.g., Freund's adjuvant) gels (aluminum hydroxide gel) and particles (liposomes) or as a solid material. Liposomal vaccines and adjuvancy are further discussed in U.S. Patent Application Ser. No. [Docket TLC-172] toffy filed on date even herewith the teachings of which are incorporated herein by reference.
  • adjuvant activity can be effected by a number of factors. Among such factors are (a) carrier effect, (b) depot formation, (c) altered lymphocyte recirculation, (d) stimulation of T-lymphocytes, (e) direct stimulation of B-lymphocytes and (f) stimulation of macrophages. With many adjuvants adverse reactions are seen. In some instances adverse reactions include granuloma formation at the site of injection, severe inflammation at the site of injection,
  • adjuvants are comprised of liposomes.
  • the present invention adopts conditions of DMPC/cholesterol liposomes that yield a therapeutically effective immunological response.
  • SRID Single Radial Immunodifussion Assay
  • EIA Enzyme Immunoassay
  • HAI Hemagglutination Inhibition Assay
  • EIA also known as ELISA (Enzyme Linked Immunoassay) is used to determine total antibodies in a sample.
  • the antigen is adsorbed to the surface of a microtiter plate.
  • the test serum is exposed to the plate followed by an enzyme linked immunogloublin, such as IgG.
  • the enzyme activity adherent to the plate is quantified by any combination of enzyme linked immunogloublin, such as IgG.
  • HAI utilizes the capability of an antigen such as viral proteins to agglutinate chicken red blood cells (or the like).
  • the assay detects neutralizing antibodies, i.e. those antibodies able to inhibit hemagglutination. Dilutions of the test serum are incubated with a standard concentration of antigen, followed by the addition of the red blood cells. The presence of neutralizing antibodies will inhibit the agglutination of the red blood cells by the antigen.
  • Tests to measure itic and cellular immune response include determination of delayed-type hypersensitivity or measuring the proliferative response of lymphocytes to target antigen. Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume.
  • Liposomes may be unilamellar vesicles (possessing a single bilayer membrane ) or multilameller vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer).
  • the bilayer is composed of two lipid monolayers having a hydrophobic "tail” region and a hydrophilic "head” region.
  • the structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer while the hydrophilic "head” orient towards the aqueous phase.
  • Small unilamellar vesicles have a diameter of about 100nm or less.
  • Unilamellar vesicles may be produced using an extrusion apparatus by a method described in Cullis et al., PCT Application No. WO
  • LUVETS Vesicles made by this technique, called LUVETS, are extruded under pressure once or a number of times through a membrane filter. LUVETs will be understood to be included in the term
  • multilamellar liposomes are those characterized as having substantially equal lamellar solute distribution.
  • This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Patent No. 4,522,803 to Lenk, et al., monophasic vesicles as described in U.S. Patent No. 4,588,578 to Fountain, et al. and frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are exposed to at least one freeze and thaw cycle; this procedure is described in Bally et al., PCT Publication No.
  • this invention includes an influenza immunizing dosage form comprising a liposome and an antigen of Influenza wherein said liposome and antigen are present in an immunization dose.
  • the antigen comprises the hemagglutinin fragment or the bromelain fragment.
  • the liposome comprises a salt form of an organic acid derivative of a sterol.
  • the antigen is entrapped in the liposome, preferably a multilamellar vesicle, and further preferably at least about 1 micron in diameter.
  • a particularly useful liposome comprises a tris (hydroxymethyl) aminomethane salt form of an organic acid derivative of a sterol.
  • Another particularly useful liposome comprises DMPC/cholesterol with particular reference to a mole ratio of from about 80 to about 20 DMPC to from about 20 to about 80 cholesterol, and more particularly wherein said ratio is from about 40:60 to about 60:40 and further wherein said liposome is a multilamellar vesicle such as one of substantially equal lamellar solute distribution (SPLV).
  • This invention includes a dosage form comprising a salt form of, an organic acid derivative of a sterol and an antigen wherein said organic acid derivative of a sterol and an antigen are present in an immunization dose.
  • the dosage form is a liposome such as a multilamellar vesicle, particularly those multilamellar vesicles at least about 1 micron in diameter.
  • the antigen is entrapped in the liposome.
  • the dosage form includes the salt form of an organic acid derivative of a sterol is a tris (hydroxymethyl) aminomethane.
  • the salt form is a carboxylic acid derivative of a sterol (such as an aliphatic carboxylic acid, particularly those up to five carbon atoms), a salt form of a dicarboxylic acid derivative of a sterol (such as an aliphatic dicarboxylic acid, particularly those up to seven carbon atoms), an hydroxy acid derivative of a sterol (such as citric acid), an amino acid derivative of a sterol or a salt form of a polyamino acid derivative of a sterol, or a salt form of a polycarboxylic acid derivative of a sterol.
  • the aliphatic dicarboxylic acid is succinate.
  • the immunogen is selected from the group comprising proteins, peptides, polysaccharides, nucleic acids, lipids, glycolipids, lipoproteins, lipopolysaccharides, synthetic peptides or bacterial fractions, viral fractions, protozal fractions, tissue fractions, or cellular fractions.
  • Specific antigens are influenza fractions such as hemagglutinin, parainfluenza 3 (fusion and
  • hemagglutinin-neuraminidase hemagglutinin-neuraminidase
  • malaria sporozoite fractions hepatitis (A, B, and non-A/non-B) fractions
  • hepatitis (A, B, and non-A/non-B) fractions meningococcus
  • the dosage form of the invention may further include an
  • immunomodulator such as a cytokine (e.g., interferons, thrombocytic derived factors, monokines and lymphokines such as IL2).
  • cytokine e.g., interferons, thrombocytic derived factors, monokines and lymphokines such as IL2.
  • Another aspect of this invention is a method of potentiating an immune response in an animal, including a human, comprising the, step of administering to such animal an immunization dose of a
  • composition comprising an organic acid derivative of a sterol and an antigen.
  • the method for potentiating the immune response includes using a dose which is a liposome such as a multilamellar vesicle, particularly those multilamellar vesicles at least about 1 micron in diameter.
  • the antigen is entrapped in the liposome.
  • the salt form is a carboxylic acid derivative of a sterol (such as an aliphatic carboxylic acid, particularly those up to five carbon atoms), a salt form of a dicarboxylic acid derivative of a sterol (such as an aliphatic dicarboxylic acid, particularly those up to seven carbon atoms), an hydroxy acid derivative of a sterol (such as citric acid), an amino acid
  • the aliphatic dicarboxylic acid is succinate.
  • the antigen is selected from the group comprising proteins, peptides, polysaccharides, nucleic acids, lipids, glycolipids, lipoproteins, lipopolysaccharides, synthetic peptides or bacterial fractions, viral fractions, protozal
  • the method of potentiating an immune response of the invention may further include using an immunomodulator such as a cytokine.
  • potentiating an immune response in an animal comprising the use of an adjuvant wherein the adjuvant comprises a salt form of an organic acid derivative of a sterol.
  • the method of potentiating immune response by use of an adjuvant uses a dose that includes the salt form of an organic acid derivative of a sterol being a tris (hydroxymethyl) aminomethane or a sodium salt.
  • the salt form is a carboxylic acid derivative of a sterol (such as an aliphatic carboxylic acid, particularly those up to five carbon atoms), a salt form of a dicarboxylic acid derivative of a sterol (such as an aliphatic dicarboxylic acid, particularly those up to seven carbon atoms), an hydroxy acid derivative of a sterol (such as citric acid), an amino acid derivative of a sterol or a salt form of a polyamino acid derivative of a sterol, or a salt form of a
  • polycarboxylic acid derivative of a sterol polycarboxylic acid derivative of a sterol.
  • the aliphatic dicarboxylic acid is succinate.
  • This invention yet further comprises a method of priming an immune response in an animal, including a human, comprising the step of administering to the animal a priming immunization dose of a composition comprising an a liposome adjuvant — any type of liposome — and particularly a liposome which is an organic acid derivative of a sterol and an adjuvant-obligatory immunogen such that administration of a booster dose of adjuvant-obligatory immunogen absent adjuvant further potentiates immune response.
  • the method of priming an immune response in an animal uses a salt form of an organic acid derivative of a sterol wherein the salt form is a tris (hydroxymethyl) aminomethane or sodium salt form of an organic acid derivative of a sterol.
  • the liposome is salt form is a carboxylic acid derivative of a sterol (such as an aliphatic carboxylic acid, particularly those up to five carbon atoms), a salt form of a dicarboxylic acid derivative of a sterol (such as an aliphatic dicarboxylic acid, particularly those up to seven carbon atoms), an hydroxy acid derivative of a sterol (such as citric acid), an amino acid
  • SPLV liposomes or multilamellar liposomes, especially those of about 1 micron or more are used as well as liposomes that comprises phosphatidylcholine, cholesterol,
  • a preferred embodiment of the method of priming further includes immunizing a primed animal by the step of administering to said animal at least one booster dose of adjuvant-obligatory immunogen absent adjuvant.
  • An additional aspect of this invention is a method of conferring immunity on an animal, including a human, comprising the step of administering to such animal a therapeutically effective
  • immunization course at least one element of which is administering an immunization dose of a composition comprising an antigen and an organic acid derivative of a sterol.
  • a composition comprising an antigen and an organic acid derivative of a sterol.
  • composition further comprises a liposome (including multilamellar vesicles) and preferable wherein the antigen is entrapped in the liposome as well as liposomes at least about 1 micron in diameter.
  • the composition further can comprise a tris (hydroxymethyl) aminomethane salt form of an organic acid derivative of a sterol, or a salt form of a carboxylic acid derivative of a sterol such as an aliphatic carboxylic acid (optionally up to 5 carbon atoms), a salt form of a dicarboxylic acid derivative of a sterol such as an aliphatic dicarboxylic acid optionally up to seven carbon atoms (e,g, succinate), or a salt form of a polycarboxylic acid derivative of a sterol.
  • the composition further comprises a salt form of an hydroxy acid derivative of a sterol such as citric acid.
  • this invention includes a dosage form comprising an immunogen and a multilamellar liposome comprising DMPC/cholesterol in an immunization dose, in one embodiment further including aluminum adjuvants such as aluminum hydroxide gel.
  • a dosage form comprising an immunogen and a multilamellar liposome comprising DMPC/cholesterol in an immunization dose, in one embodiment further including aluminum adjuvants such as aluminum hydroxide gel.
  • the liposome of the dosage form comprises a mole ratio of from about 80 to about 20 DMPC to from about 20 to about 80 cholesterol, and particularly wherein the ratio is from about 30:70 to about 70:30 and preferably 70:30.
  • the dosage form multilamellar liposome is a of equal solute distribution (SPLV) and/or at least 1 micron in diameter and particularly a 70:30 mole ratio DMPC/cholesterol SPLV.
  • the antigen is selected from the group comprising proteins, peptides, polysaccharides, bacterial fractions, viral fractions, protozal fractions, synthetic peptides or lipopolysaccharides.
  • the dosage form may comprise an immunomodulator including a cytokine. Additionally the dosage form may comprise a suitable pharmaceutical carrier.
  • Another aspect of this invention includes a method of potentiating an immune response in an animal, including a human, comprising the step of administering to such animal an immunization dose of a composition comprising an antigen and a multilamellar liposome comprising DMPC/cholesterol, and optionally further including aluminum adjuvants such as aluminum hydroxide gel.
  • a composition comprising an antigen and a multilamellar liposome comprising DMPC/cholesterol, and optionally further including aluminum adjuvants such as aluminum hydroxide gel.
  • the liposomes comprise a mole ratio of from about 80 to about 20 DMPC to from about 20 to about 80 cholesterol, and particularly wherein the ratio is from about 30:70 to about 70:30.
  • the multilamellar liposome is of equal solute distribution (e.g., SPLV) and/or at least about 1 micron in diameter and particularly a 70:30 mole ratio
  • the antigen is selected from the group comprising proteins, peptides, polysaccharides, bacterial fractions, viral fractions, protozal fractions, synthetic peptides or lipopolysaccharides.
  • the dose may comprise an immunomodulator including a cytokine.
  • the dosage form may comprise a suitable pharmaceutical carrier.
  • a method of potentiating an immune response in an animal including a human comprising the use of an adjuvant wherein the adjuvant comprises a liposome comprising DMPC/cholesterol, in one embodiment further including aluminum adjuvants such as aluminum hydroxide gel.
  • the liposomes comprise a mole ratio of from about 80 to about 20 DMPC to from about 20 to about 80 cholesterol, and particularly wherein the ratio is from about 30:70 to about 70:30.
  • the multilamellar liposome is an SPLV and/or at least about 1 micron in diameter and particularly a 70:30DMPC/cholesterol SPLV.
  • the antigen is selected from the group comprising proteins, peptides, polysaccharides, bacterial fractions, viral fractions, protozal fractions, synthetic peptides or lipopolysaccharides.
  • the dose may comprise an immunomodulator including a cytokine.
  • a further embodiment of the invention is a method of priming an immune response in an animal, including a human, comprising the step of administering to the animal a priming immunization dose of a composition comprising an adjuvant which is a multilamellar liposome comprising DMPC/cholesterol and an adjuvant-obligatory immunogen (and optionally aluminum adjuvants such as aluminum hydroxide gel) such that administration of a booster dose of adjuvant-obligatory immunogen absent adjuvant further potentiates immune response.
  • a priming immunization dose of a composition comprising an adjuvant which is a multilamellar liposome comprising DMPC/cholesterol and an adjuvant-obligatory immunogen (and optionally aluminum adjuvants such as aluminum hydroxide gel) such that administration of a booster dose of adjuvant-obligatory immunogen absent adjuvant further potentiates immune response.
  • the method of priming in specific instances includes the liposome being an SPLV multilamellar vesicle and/or the liposome being at least about 1 micron in diameter, and preferably about 70:30
  • influenza fractions such as hemagglutinin, parainfluenza 3 (fusion and hemagglutinin-neuraminidase), malaria sporozoite fractions, hepatitis (A, B, and non-A/non-B) fraction, meningococcus fractions, HIV fractions (all strains), and melanoma fractions.
  • influenza fractions such as hemagglutinin, parainfluenza 3 (fusion and hemagglutinin-neuraminidase), malaria sporozoite fractions, hepatitis (A, B, and non-A/non-B) fraction, meningococcus fractions, HIV fractions (all strains), and melanoma fractions.
  • the invention in a further embodiment comprises a method of conferring immunity on an animal, including a human, comprising the step of administering to such animal a therapeutically effective immunization course at least one element of which is administering an immunization dose of a composition comprising an antigen and a multilamellar liposome comprising DMPC/cholesterol.
  • the composition further comprises aluminum adjuvant such as aluminum hydroxide gel.
  • a particular liposome comprises a mole ratio of from about 80 to about 20 DMPC to from about 20 to about 80 cholesterol, preferably from about 70:30 to about 30:70 and most preferably about 70:30 particularly wherein the liposome is of equal solute distribution (e.g.,SPLV) and in cluding the lipo some b eing at l east about 1 micron in d iameter.
  • the antigen can be selected from the group comprising proteins, peptides, polysaccharides, bacterial fractions, viral fractions, protozal fractions, synthetic peptides or lipopolysaccharides.
  • the method can further comprising an immunomodulator such as a
  • cytokine The method can further comprise a suitable pharmaceutical carrier.
  • this invention includes a dosage form comprising an antigen and a liposome comprising DMPC/cholesterol 70:30 +/-5 (mole) in an immunization dose.
  • the dosage form can further include aluminum adjuvant such as aluminum hydroxide gel.
  • the immunogen is selected from the group comprising proteins, peptides, polysaccharides, bacterial fractions, viral fractions, protozal fractions, synthetic peptides or lipopolysaccharides and further an immunomodulator such as cytokine and a suitable pharmaceutical carrier.
  • the invention includes a method of
  • composition of the method can further comprise aluminum adjuvant such as aluminum hydroxide gel.
  • the liposomes of the method include multilamellar liposomes such as SPLVs and unilamellar liposomes, and preferably wherein the liposomes are at least about 1 micron in diameter.
  • the antigen is selected from the group comprising proteins, peptides, polysaccharides, bacterial fractions, viral fractions, protozal fractions, synthetic peptides or lipopolysaccharides and further an immunomodulator such as cytokine and a suitable pharmaceutical carrier.
  • the invention includes a method of potentiating an immune response in an animal including as human comprising the use of an adjuvant wherein the adjuvant comprises a liposome comprising DMPC/cholesterol 70:30 +/-5 (mole).
  • the method can further comprise aluminum adjuvant such as aluminum hydroxide gel.
  • the liposomes of the method include mutilamellar liposomes such as SPLVs and unilamellar liposomes, and preferably wherein the
  • liposomes are at least about 1 micron in diameter.
  • a method of priming an immune response in an animal comprising the step of administering to the animal a priming immunization dose of a composition comprising an adjuvant which is a liposome comprising DMPC/cholesterol 70:30 +/-5 (mole) and an adjuvant-obligatory immunogen such that administration of a booster dose of
  • the method can further comprise aluminum adjuvant such as aluminum hydroxide gel.
  • the liposomes of the method include multilamellar liposomes such as SPLVs and unilamellar liposomes, and preferably wherein the liposomes are at least about 1 micron in diameter.
  • this invention includes a method of conferring immunity on an animal, including a human, comprising the step of
  • the composition of the method can further comprise aluminum adjuvant such as aluminum hydroxide gel.
  • the liposomes of the method include multilamellar liposomes such as SPLVs and unilamellar liposomes, and preferably wherein the liposomes are at least about 1 micron in diameter.
  • the antigen is selected from the group comprising proteins, peptides, polysaccharides, bacterial fractions, viral fractions, protozal fractions, synthetic peptides or
  • lipopolysaccharides and further an immunomodulator such as cytokine and a suitable pharmaceutical carrier.
  • an immunomodulator such as cytokine and a suitable pharmaceutical carrier.
  • adjuvants of this invention comprise an influenza immunizing dosage form comprising a liposome and an antigen of Influenza wherein said liposome and antigen are present in an immunization dose.
  • antigens of Influenza are the hemagglutinin fragment or the bromelain fragment.
  • salt forms of organic acid derivatives of sterols are particularly useful pharmaceutical adjuvants (and particularly in the form of liposomes and (ii) that DMPC/cholesterol liposomes (particularly multilamellar liposomes) are particularly useful pharmaceutical adjuvants (such adjuvants are advantageously used in aluminum hydroxide gels).
  • DMPC/cholesterol liposomes both multilamellar and unilamellar wherein the DMPC/cholesterol ratio is 70:30 +/-5 (mole).
  • Antigen shall mean a substance or material that is recognized specifically by antibody and/or combines with an antibody.
  • Immunogen shall mean a substance or material to potentiate an immune response when used in conjunction with an antigen. Adjuvants are further used to elicit immune response sooner, or a greater response, or with less antigen. Immunogen-obligatory adjuvant refers to an antigen which alone is not immunogenic but becomes, immunogenic with adjuvant. "Immunogen” shall mean a substance or material (including antigens) that is able to induce an immune response alone or in conjunction with an adjuvant. Both natural and synthetic substances may be immunogens.
  • An immunogen will generally be a protein, peptide, polysaccharide, nucleoprotein, lipoprotein, synthetic polypeptide, or hapten linked to a protein, peptide, polysaccharide, nucleoprotein, lipoprotein or synthetic polypeptide or other bacterial, viral or protozal fractions.
  • immunogen includes substances which do not generate an immune response (or generate a only therapeutically ineffective immune response) unless associated with an adjuvant (e.g., small peptides) which will be referred to as "adjuvant-obligatory" immunogens.
  • Immune response shall mean a specific response of the immune system of an animal to antigen or immunogen. Immune response may include the production of antibodies.
  • Immunization conditions shall mean factors which affect an immune response including amount and kind of immunogen or adjuvant delivered to a subject animal including a htiman, method of delivery, number of inoculations, interval of inoculations, the type of subject animal and its condition.
  • Vaccine shall mean a pharmaceutical formulation able to induce immunity.
  • Immunity shall mean a state of resistance of an subject animal including a human to a infecting organism or substance. It will be understood that infecting organism or substance is defined broadly and includes parasites, toxic substances, cancers and cells as well as bacteria and viruses. A Therapeutically Effective Immunization Course will produce the immune response such as that exhibited by production of specific antibodies and/or reactivity of immune cells to antigen.
  • Immunization dose shall mean the amount of antigen or immunogen needed to precipitate an immune response. This amount will vary with the presence and effectiveness of various adjuvants. This amount will vary with the animal and immunogen or antigen or adjuvant but will generally be between about 0. lug/ml or less to about 500ug per inoculation.
  • the immunization dose is easily determined by methods well known to those skilled in the art, such as by conducting statistically valid host animal immunization and challenge studies. See, for example, Manual of Clinical Immunology, H.R. Rose . Friedman, American Society for Microbiology,
  • Primary shall mean the stimulation of a primary (as opposed to a secondary or later) response by an animal to an antigen.
  • the primary response is characterized by the manufacture by the animal of antibody to the antigen, and ideally by the generation of a population of B-lymphocytes and T-lymphocytes that respond to secondary or later immunogenic challenge — even absent adjuvant — with a rapid and substantive production of antibodies. Based upon such response 1, 2, 3 or more booster doses of immunogen, absent adjuvant, will generate a therapeutically effective immune response to the antigen.
  • the liposomes will have a net charge or be neutral. Charged and particularly negatively charged liposomes may display superior adjuvancy to neutral
  • a preferred class of lipids for forming liposomes are those of cholesterol hemisuccinate ("CHS”), such as those with sodium
  • CHS sodium tris(hydroxymethyl) aminomethane
  • CHS tris tris(hydroxymethyl) aminomethane
  • Salt forms of an organic acid derivative of a sterol may be used in the practice of the invention.
  • any sterol which can be modified by the attachment of an organic acid may be used in the practice of the present invention.
  • sterols include but are not limited to cholesterol, vitamin D, phytosterols (including but not limited to sitosterol, campesterol, stigmasterol, and the like), steroid hormones, and the like.
  • Organic acids which can be used to derivatize the sterols include but are not limited to the carboxylic acids, dicarboxylic acids, polycarboxylic acids, hydroxy acids, amino acids and polyamino acids.
  • any organic acid may be used to derivatize the sterols; however an advantage may be obtained if the organic acid moiety itself is water soluble.
  • water soluble organic acid moieties include but are not limited to water-soluble aliphatic carboxylic acids such acetic, propionic, butyric, valeric acids and the like (N.B., up to four-carbon acids are miscible with water; the five-carbon free acid is partly soluble and the longer chain free acids are virtually insoluble); water-soluble aliphatic dicarboxylic acids such as malonic, succinic glutaric, adipic, pimelic, maleic and the like (N.B., the shorter chains are appreciable more soluble in water; borderline solubility in water occurs at G 6 to C 7 );
  • hemimellitic, trimesic, succinimide, and the like polycarboxylic acids
  • water-soluble hydroxy acids such as glycolic, lactic, mandelic, glyceric, malic, tartaric, citric, and the like (N.B., alpha-hydroxy acids containing a branched chain attached to the alpha-carbon of the carbonyl group would be less susceptible to hydrolysis and, therefore, advantageous in the practice of the present invention)
  • any of the amino acids and polyamino acids any of the amino acids and polyamino acids.
  • the organic acid can be linked to an hydroxyl group of the sterol via an ester or an ether bond using conventional methods (see, for example, U.S. Pat. Nos. 3,859,047; 4,040,784; 4,042,330;
  • the salt forms of the derivatized sterols can be prepared by dissolving both the organic acid derivative of the sterol and the counterion of the salt (e.g., the free base of the salt) in an appropriate volatile solvent, and removing the solvent by evaporation or a similar technique leaving a residue which consists of the salt form of the organic acid derivative of the sterol.
  • Counterions that may be used include, but are not limited to, tris, 2-amino-2-methyl-1,3-propanediol,
  • CHS forms liposomes when added to an aqueous material. This can conveniently be performed at 20°-25°C (room temperature) and atmospheric pressure. Agitation accelerates the process of liposome formation and is preformed by such methods as vortexing, sonication or other methods well known in the art . If desired the resulting liposomes may be filtered or sized such as by passing through a filter stack such as a 0.4 or 0.2um filter (Nucleopore, Pleasanton, CA).
  • Immunogens which partition into the liposome lamellae such as melanoma antigen in CHS liposomes may yield an insufficient immunogenic responses without repeated inoculations and additional immuno stimulator. Without being bound by any particular theory it is believed that this partitioning results in the limitation of exposure of epitopes externally to the adjuvant liposomes. Immunogens may be modified by a number of methods well known in the art such as by amino acid addition or subtraction or conjugation with other moieties.
  • a preferred class of lipids for forming liposomes are those of dimyristoylphosphatidylcholine and cholesterol ("DMPC/cholesterol").
  • DMPC/cholesterol forms the required multilamellar liposomes over a wide range of proportions from about 100:1 (mole) to about 20:80. More preferred is about 70:30 to about 30:70, and yet further preferred is about 70:30. Additionally other lipids may be admixed with DMPC/cholesterol, such as dimyristoyl phosphatidylglycerol, dicetyl phosphate, phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine and cholesterol hemisuccinate ("CHS”), such as those with sodium (“CHS sodium ”) or tris(hydroxymethyl)
  • CHS cholesterol hemisuccinate
  • CHS tris aminomethane
  • Aluminum compounds are adjuvants well known in the art, and include aluminum hydroxide, aluminum phosphate, aluminum oxide or aluminum sulfate and will be termed collectively aluminum adjuvants.
  • aluminum hydroxide is widely used in diptheria and tetanus toxoid vaccines as well as in veterenary applications.
  • Aluminum hydroxide powder spontaneously forms a gel upon hydration.
  • a vaccine containing aluminum hydroxide commonly immunogen in aqueous buffer is added to the preformed gel.
  • Such vaccines are referred to as being aluminum-adsorbed.
  • DMPC/cholesterol multilamellar liposomes of the SPLV process are preferred but any other type of liposome may be used.
  • the SPLV process generally involves . rotoevaporation of lipids in solvent in a round bottom flask to form a thin film. The lipid film is then dispersed in a non-water miscible solvent such as ether or methylene chloride to which the aqueous solute (containing immunogen) is then added. The mixture is then sonicated while being dried by a stream of nitrogen gas which drives off the organic solvent. The resultant liposome paste is resuspended in aqueous buffer.
  • U.S. Patent No. 4,522,803 to Lenk, et al. further describes this process and is incorporated herein by reference. If desired the resulting
  • liposomes may be filtered or sized such as by passing through a filter stack such as a 0.4 or 0.2um filter (Nucleopore, Pleasanton, CA).
  • a filter stack such as a 0.4 or 0.2um filter (Nucleopore, Pleasanton, CA).
  • the resulting liposomes are conveniently administered in aqueous material.
  • the volume of aqueous material will vary with the particular liposome to be administered and is not critical.
  • 0.5ml is a convenient liposome dosage volume.
  • Suitable aqueous material for either sterol or DMPC/cholesterol liposomes is saline solution, phosphate buffer or other well known aqueous pharmaceutical diluents. These liposomes are conveniently associated with an immunogenic amount of antigen or immunogen. This association is engendered by mixing, adsorption, encapsulation, co-formation or other methods well known in the art.
  • the adjuvant effect of the instant invention is seen from the results in Tables 1, 2 and 3 as to sterol liposomes and Tables 4, 5, and 6 as to DMPC/cholesterol liposomes and Table 7 as to both sterol and DMPC/cholesterol liposomes.
  • Table 1A compares the antibody response in guinea pigs immunized with Influenza B/Ann Arbor hemagglutinin (HA) alone or formulations with CHS tris liposomes.
  • HA elicits a particular level of antibody response without adjuvant, but this response is enhanced by adjuvant.
  • CHS tris liposomes are seen from the results to potentiate this antibody production.
  • neutralizing antibody responses as detected by HAI to 5 or 0.5 ug of HA are increased up to about 50-fold and 70-fold respectively when HA is administered with the steroidal adjuvant.
  • Table 1B shows the antibody response in guinea pigs immunized with the bromelain fragment of HA (HAB) in various formulations.
  • HAB is generally non-immunogenic when administered alone and is exemplary of an adjuvant-obligatory immunogen.
  • Use of steroidal adjuvant in the form of CHS increases the immune response on a par or better than complete Freund's adjuvant, especially the production of protective neutralizing antibodies as detected by HAI. This increase may be about 1400-fold.
  • increasing the amount of adjuvant increases the immune response.
  • Table 2 demonstrates the importance of priming in generation of the immune response.
  • HAB is seen to generate a weak immune response that is not greatly potentiated on secondary challenge, even with adjuvant of this invention.
  • HAB is administered in association with the adjuvant of this invention, here in the form of a CHS tris liposome a substantial immune response is generated upon application of a second inoculation of HAB, here in solution.
  • priming of the subject animal with an immunogenic dose of adjuvant-obligatory immunogen and adjuvant permitted later booster doses to be highly effective wherein such booster doses did not contain adjuvant but were merely adjuvant-obligatory immunogen in solution.
  • the priming of immune response with the adjuvant of this invention permits booster administrations of adjuvant-obligatory immunogen without adjuvant, even when this immunogen would not have initially generated an immune response without coadministration with an adjuvant.
  • any liposome can be used as a priming adjuvant, not just steroidal liposomes, for use with adjuvant-obligatory immunogens. Table 3 discloses that superior results are obtained when the antigen is entrapped in an adjuvant liposome of the present
  • HAB hemagglutinin
  • HAB administered with aluminum hydroxide gel.
  • the immunogenicity is greatly increased when HAB is entrapped within DMPC/cholesterol SPLVs at various mole ratios and lipid concentrations.
  • Total anti-HA IgG responses detected by EIA are increased up to 5000 fold and protective neutralizing antibodies detected by HAI are increased up to 35 fold at 6 weeks when HAB is administered in
  • HA administered in free form is a relatively good immunogen at 5ug dose but elicits low antibody titers when administered at 0.5ug.
  • Liposomal HA at both of these dosages generates responses which are up to 80 fold higher than free HA.
  • Table 6 demonstrates the adjuvant effect of DMPC/cholesterol SPLVs and aluminum hydroxide gel.
  • Table 6 demonstrates the adjuvant effect of DMPC/cholesterol SPLVs and aluminum hydroxide gel.
  • Table 6 (Experiment 1), administration of HAB, with DMPC/cholesterol liposomes (200 mg starting lipid concentration) generates a strong anti-HA IgG response up to 100 fold greater and an HAI (neutralizing) antibody titer 10 fold greater than HAB alone.
  • HAI neutralizing antibody titer 10 fold greater than HAB alone.
  • Increasing the starting lipid concentration to 500 mg increases the adjuvant effect even more (500 fold and 30 fold respectively).
  • the same formulations are administered with aluminum hydroxide gel, even these high titers are increased up to 3 times greater.
  • the combined adjuvant effect for liposomes and aluminum hydroxide is clearly evident in (Table 6, Experiment II).
  • liposomal HAB increases antibody responses up to 200 fold over free
  • Table 7 shows the results obtained with HA used for vaccination after splitting the influenza virus with detergent.
  • the results of Table 7 show that 5 mg HA in CHS tris liposomes induced a strong adjuvant effect regardless whether this amount of antigen was entrapped in 13.6 or 1.9 mg of lipid.
  • the same observation can be made in the case of DMPC/chol formulations.
  • non-entrapped antigen control at 0.5 or 5 ug HA.
  • Aluminum adjuvants are used in forms and proportions well known to those skilled in the art. Commercial preparations of aluminum hydroxide gel containing vaccines such as tetanus toxoids range from about 0.2 to about 1mg of aluminum/ml. The safe upper range is far higher for humans vaccines with as much as 15 mg or more of aluminum hydroxide per dose are known with no limit for veterinary
  • Vaccines are conveniently administered in a dosage form.
  • a "dosage form” will be understood to mean any pharmaceutically form of administering a vaccine including subcutaneous, oral, intramuscular, and ocular administration and utilizing vaccines in live, attenuated or synthetic or partial forms along with adjuvants and optionally immunomodulators such as cytokines.
  • the combinations of the foregoing elements are prepared so that the dosage form is adapted to produce an immune response in the subject animal including a human as easily and effectively as possible.
  • the dosage forms including liposomal dosage forms resulting from the method of the present invention can be used therapeutically in animals such as mammals, including man, in the treatment of
  • infections or conditions which require the delivery of immunogen in its bioactive form include but are not limited to disease states such as those that can be treated with vaccines.
  • Extracorporeal treatment of immunoresponsive tissues is also contemplated.
  • Dosage forms also include micelle forms of the adjuvant as well as adjuvant incorporated into gel such as aluminum gels, liquid crystals, powders, precipitates and solutions.
  • the dosage form can be a unit dosage form configured and adapted to a single administration.
  • the mode of administration of the dosage form may determine the sites and cells in the organism to which the dosage form will be delivered.
  • the dosage forms including liposomal dosage forms of the present invention can be administered alone but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the dosage forms may be injected
  • parenterally for example, intra-muscularly or subcutaneously.
  • the dosage forms may also be administered via the oral route.
  • parenteral administration they can be used, for example, in the form of a sterile aqueous solution which may contain other solutes, for example, enough salts or glucose to make the solution isotonic.
  • Other uses depending upon the particular properties of the preparation, may be envisioned by those skilled in the art.
  • the prescribing physician will ultimately determine the appropriate therapeutically effective dosage for a given human subject, and this can be expected to vary according to the age, weight, and response of the individual as well as the nature and severity of the patient's disease.
  • the dosage of the antigen in liposomal dosage form will generally be about that or less than that employed for the free antigen. In some cases, however, it may be necessary to administer dosages outside these limits.
  • Liposomes containing HAB were prepared as in Example 2.
  • Entrapment values were determined by SRID and the liposomes were diluted in saline to a concentration of 10 ug protein/ml and were sealed in a glass vial with a rubber stopper and crimp seal.
  • EIA Enzyme Immunoassay
  • HAI Hemagglutination Inhibition Assay
  • Adjuvant Liposomes with Antigen 92mg of cholesterol and 108mg of DMPC were placed into a 100ml round bottom flask and suspended in 3ml chloroform and dried to a film by rotoevaporation. 10ml of anhydrous ether was added to the flask followed by 2.0ml of HAB in aqueous buffer was added (1,100ug HAB, 0.01M phosphate buffered saline in 0.9%NaCl). The mixture was covered loosely with foil and sonicated in a 40oC water bath while concurrently evaporating the ether with a gentle stream of nitrogen gas. The resultant lipid paste was thoroughly dried under nitrogen until no trace of ether was noted by smell.
  • the mixture was sealed in a glass vial with rubber stopper and crimp seal.
  • Liposomes containing HAB were prepared as in Example 9.
  • Entrapment values were determined by SRID and the liposomes were diluted in saline to a concentration of 10 ug protein/ml and were sealed in a glass vial with a rubber stopper and crimp seal.
  • the blood was allowed to clot at room temperature overnight. The blood was centrifuged, and the serum was drawn off and stored at 4°C until tested. The day after bleeding, the guinea pigs were again injected i.m. with 0.5 ml of free or liposomal HAB (5ug), this time in the left hind leg. Blood was collected in the same manner up to one year after the initial injection.
  • EIA Enzyme Immunoassay
  • HAI Hemagglutination Inhibition Assay
  • GD 3 antigen suspended in phosphate buffered saline (PBS but without Ca ++ or Mg ++ ) at pH 7.2 was entrapped in either
  • CHS tris lipid liposomes or DMPC/cholesterol (70:30 mole percent) lipid liposomes were made by Method A (below) and DMPC/cholesterol/GD 3 SPLV liposomes by Method B
  • guinea-pigs were inoculated with liposome formulations at 5 or 0.5 ug HA each entrapped in various amount of lipid (Table, 7).
  • Example 2 Two CHS tris formulations were prepared as described in Example 2 to give a dose of 5.0 by HA and either 13.6 mg lipid (MLV, CHS tris , H) or 1.9 mg lipid as multilamellae vesicles oc CHS (MLV, CHS tris , L).
  • the "H” and “L” designations refer to high and low amount of lipid in particular dosages.
  • DMPC/CHOL formulations were prepared as described in Example 8 to give a dose of 5.0 mg HA and either 9.7 mg lipid of stable plurilamellar vesicles, dimyristoylphosphatidylcholine/cholesterol (SPLV, DMPC/C, H) or 2.9 mg lipid (SPLV, DMPC/C, L).
  • each H formulation exhibiting a relatively high amount of lipid per 0.5 ml inoculum was further diluted 1:10 in PBS and the resulting suspensions containing 0.5 ug HA and either 1.4 mg
  • Liposomes containing HA or HAB were prepared at the indicated lipid concentrations. (Example formulations are indicated) Entrapment values were determined by single radial immuno-diffusion (SRID) and liposomes were diluted in saline to a dosage of 0.5 or 5ug per 0.5 ml dose. Hartley guinea pigs were injected IM at 0 and 4 weeks. At the Indicated timepoints, blood was collected by cardiac puncture. Serum antibody titers (total antl-HA IgG and neutralizing antibodies) were determined by EIA and HAI assay, respectively. Values represent the geometric mean of 3-5 guinea pigs per group.
  • SRID single radial immuno-diffusion
  • Liposomes containing HAB were prepared from CHS as in example 3. Entrapment value was determined by SRID and liposomes were diluted in saline to a dosage of 5 or 15 ug protein per 0.5 ml dose. Hartley guinea pigs were injected IM at O time and boosted at 4 weeks . At the indicated timepoints , blood was collected by cardiac puncture. Serum antibody titers (total anti-HA IgG and neutralizing antibodies) were determined by EIA and HAI assay, respectively. Values represent the geometric mean of 4-5 guinea pigs per group.
  • HAB was entrapped In liposomes or alxed with empty liposomes. All liposomes were prepared using 500 mg of lipid. The inoculum contained 5 ug HAB or HA/0.5 ml. The experiment was conducted aa described In the legend of Table 1.
  • DMPC:Cholesterol SPLVs containing HAB were prepared at the indicated lipid concentrations and mole ratios. Entrapment values were determined by SRID and liposomes were diluted in saline to a dosage of 5ug per 0.5 ml dose. Hartley guinea pigs were injected i.m. at 0 and 4 weeks. At the indicated timepoints, blood was collected by cardiac puncture. Serum antibody titers (total antl-HA IgG and neutralizing antibodies) were determined by EIA and HAI assay, respectively. Values represent the geometric mean of 4-5 guinea pigs per group.
  • DMPC/choleaterol SPLV's (70:30 mole ratio) containing HA were prepared as In Example 1. Entrapment values were determined by SRID and Liposomes were diluted in saline to a dosage of 0.5 or 5.0 ug per 0.5 ml dose. Hartley guinea pigs were Injected i.m. at 0 and 4 weeks. At the Indicated tlae points, blood was collected by cardiac puncture. Serum antibody tltera (total anti-HA IgG and neutralizing antibodies were determined by EIA and HAI assay, respectively. Values represent the geometrlc mean of 4-5 guinea pigs per group.
  • DHPC:Cholesterol SPLV's (70:30 mole ratio) containing HAB were prepared at the indicated lipid concentrations.
  • the liposomes in Experiment II were sterile filtered through a 0.4/0.2 um filter stack.
  • Bntrapaent valuea were deteralned by SKID and llposoaes were diluted In saline or aluminum hydroxide gel and saline to a dosage of 0.5 or 5 ug per 0.5 al dose.
  • Hartley guinea pigs were injected i.a. at 0 and 4 weeks. At the indicated timepoints, blood was collected by cardiac puncture.
  • Serum antibody titers total anti-HA IgG and neutralizing antibodies
  • Vaues represent the geometric mean of 4-5 guinea pigs per group.
  • A/ Liposomes were prepared with either 500 mg (H) or 50 mg (L) of lipid (CHST or DMPC/C, 70:30 mole percent)
  • B/ Geometric mean titer (n-51) determined by enzyme-linked immuno assay (EIA) and hemagglutination imhibitlon (HAI)

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Abstract

Une forme de dosage d'immunisation contre la grippe comprend un liposome et un antigène d'Influenza, en particulier l'hémaglutinine ou un fragment de bromélaïne, le liposome et l'antigène étant présents dans une dose d'immunisation. De plus, une forme de dosage, comprenant cette forme particulièrement adaptée pour produire une réponse d'immunisation, comprend une forme de sel d'un dérivé d'acide organique d'un stérol et un antigène, qui sont présents dans une dose d'immunisation. L'invention concerne également un procédé d'utilisation. En outre, une forme de dosage, comprenant ladite forme particulièrement adaptée pour produire une réponse d'immunisation, comprend des liposomes de dimyristolyphosphatidylcholine (DMPC)/cholestérol, éventuellement dans un gel d'hydroxy d'aliminium, et un antigène, le DMPC/cholestérol et l'antigène étant présents dans une dose d'immunisation; et procédé d'utilisation.
PCT/US1989/003658 1988-08-25 1989-08-24 Vaccin contre la grippe et nouveaux adjuvants WO1990001948A1 (fr)

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US6096307A (en) * 1997-12-11 2000-08-01 A. Glenn Braswell Compositions for immunostimulation containing Echinacea angustofolia, bromelain, and lysozyme
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WO2000048630A1 (fr) * 1999-02-17 2000-08-24 Csl Limited Complexes immunogenes et methodes y relatives
HK1047892B (en) * 1999-11-19 2009-01-09 Csl Limited HCV vaccine ingredients

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DE4136553A1 (de) * 1991-11-06 1993-05-13 Biotechnolog Forschung Gmbh Impfstoff gegen schleimhauterreger und herstellungsverfahren
US6096307A (en) * 1997-12-11 2000-08-01 A. Glenn Braswell Compositions for immunostimulation containing Echinacea angustofolia, bromelain, and lysozyme
US8808686B2 (en) 2006-06-15 2014-08-19 Novartis Ag Adjuvant-sparing multi-dose influenza vaccination regimen
US9901630B2 (en) 2006-06-15 2018-02-27 Seqirus UK Limited Adjuvant-sparing multi-dose influenza vaccination regimen

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