WO1999000142A1 - Methode pour provoquer une reponse immunitaire systemique a un antigene de l'hepatite ou du vih - Google Patents

Methode pour provoquer une reponse immunitaire systemique a un antigene de l'hepatite ou du vih Download PDF

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Publication number
WO1999000142A1
WO1999000142A1 PCT/US1998/013194 US9813194W WO9900142A1 WO 1999000142 A1 WO1999000142 A1 WO 1999000142A1 US 9813194 W US9813194 W US 9813194W WO 9900142 A1 WO9900142 A1 WO 9900142A1
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Prior art keywords
liposomes
antigen
hepatitis
hiv
preparation
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PCT/US1998/013194
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English (en)
Inventor
Jackie R. See
Darryl M. See
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Bio-Sphere Technology, Inc.
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Publication date
Priority claimed from US08/948,568 external-priority patent/US6207185B1/en
Priority claimed from US09/007,297 external-priority patent/US6117449A/en
Application filed by Bio-Sphere Technology, Inc. filed Critical Bio-Sphere Technology, Inc.
Priority to AU81674/98A priority Critical patent/AU8167498A/en
Publication of WO1999000142A1 publication Critical patent/WO1999000142A1/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
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4891—Coated capsules; Multilayered drug free capsule shells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16234—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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24234—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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32311—Enterovirus
    • C12N2770/32334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention relates to a method for inducing a systemic immune response to an HIV, hepatitis B or hepatitis C antigen and to vaccines suitable for oral administration.
  • the immune system is a complicated system.
  • the epithelial surfaces of the body serve as a barrier to antigenic material. However, those surfaces are by no means impenetrable.
  • the mucosal immune system provides the next maj or line of defense against a maj ority of human pathogens .
  • the mucosal immune system includes gut-associated lymphoid tissue (GALT), bronchus-associated lymphoid tissue, the salivary glands, the conjunctiva, the mammary gland, parts of the urogenital tract, and the middle ear.
  • GALT gut-associated lymphoid tissue
  • bronchus-associated lymphoid tissue the salivary glands
  • the conjunctiva the mammary gland
  • parts of the urogenital tract and the middle ear.
  • GALT consists of two types of lymphoid aggregates. The first is referred to as Peyer's patches and the second consists of isolated lymphoid follicles.
  • Peyer's patches have a defined micro- structure including a central B cell dependent follicle and T cell dependent regions adjacent to the follicle.
  • the lymphocytes in Peyer's patches are heterogeneous, including B cells which express IgM, IgG, IgA, and IgE and various regulatory and cytotoxic T cells.
  • Peyer's patches also contain specialized macrophages.
  • the Peyer's patches are covered by M cells, which are specialized lympho- epithelium cells.
  • ingested antigens produce a local immune response.
  • the antigens are taken up by the M cells, which deliver the antigen to the underlying lymphocytes in the tissue. This results in the production of IgA at various secretory effector sites following the migration of activated lymphocytes through the efferent, lymphatic and circulatory system.
  • the absorption of antigens by the Peyer's patches can induce a systemic immune response if the antigen is taken up by macrophages in the Peyer's patches.
  • Macrophages induce a systemic response by processing antigens and presenting them to lymphocytes. The lymphocytes then become activated and cause the production of systemic antibodies specific to the antigens.
  • Childers et al. (Oral Microbiol. Immunol. 1994:9:146-153) reported that lyophilized liposomes containing S. mutatis antigen can be administered orally to human patients and will be absorbed by GALT to elicit a local immune response. No systemic response was observed however.
  • orally administered vaccines when administered alone, are destroyed in the GI tract with digestive juices and enzymes.
  • the antigen can be delivered with a non-destructible carrier, such as an adjuvant, destruction in the GI tract is greatly minimized.
  • the antigen be associated with an adjuvant.
  • the presence of the adjuvant permits the antigen/adjuvant combination to be recognized by the CD4 cells, which send signals to B cells to produce antibodies, and by the cytotoxic lymphocytes, which kill the infecting organism in affected host cells. Without the presence of the adjuvant, the CD4 cells and cytotoxic lymphocytes ignore the free antigen.
  • Typical adjuvants include alum, Freund's adjuvant, incomplete Freund's adjuvant and indotoxin. These adjuvants typically induce an inflammatory response.
  • Other typical adjuvants are immuno stimulating complexes (iscoms) that contain Quil A. These adjuvants typically cause clumping of antigens.
  • the present invention provides a method for inducing a systemic immune response to an HIV, hepatitis B or hepatitis C antigen in a mammal and does not require the presence of an adjuvant.
  • the lyophilized antigen-containing liposomes do not directly target the CD4 cells and cytotoxic lymphocytes, but instead are taken up by the macrophages in the Peyer's patches.
  • the macrophages express the antigen in conjunction with self major histocompatibility antigen I and II (SMH I and SMH II).
  • SMH I and SMH II self major histocompatibility antigen I and II
  • the CD4 cells recognize the antigen expressed with SMH I
  • the cytotoxic lymphocytes recognize the antigen expressed with SMH II.
  • the present methods involve an intermediate step, being taken up by the macrophages, which is different from the process that occurs when an antigen/adjuvant combination is orally administered.
  • the present invention involves a method whereby the antigen containing liposomes can be orally administered without an adjuvant to induce a systemic immune response.
  • the inventive methods do not generate an adjuvant effect, e.g., an inflammatory response or clumping of antigens.
  • the inventive method comprises first incorporating at least one antigen selected from HIV I, HIV II, hepatitis B and hepatitis C antigens into liposomes, preferably multilamellar liposomes having a size from about 20 nm to about 20 microns or greater, preferably from about 200 nm to about 10 microns and more preferably from about 1 micron to about 5 microns.
  • the antigen- containing liposomes are then lyophilized and packaged in a suitable form, such as a pill or capsule, for oral ingestion.
  • Means, such as an enteric coating are provided for preventing breakdown of the preparation in the stomach but allowing digestion in the gut, i.e., small intestine.
  • the preparation passes through the stomach into the gut wherein antigen-containing liposomes are absorbed in the Peyer's patches of the gut.
  • antigen-containing liposomes are taken up by macrophages to induce a systemic immune response and preferably a long-term systemic immune response to the antigen(s).
  • the invention further provides a preparation suitable for oral ingestion for inducing a systemic response, and preferably a long-term systemic immune response, to one or more antigens selected from HIV, hepatitis B and hepatitis C antigens.
  • the composition comprises lyophilized, preferably multilamellar, liposomes that contain the antigen(s).
  • the liposomes have a size, before lyophilization, of from about 20 nm to about 20 microns or greater, preferably from about 200 nm to about 10 microns, and more preferably from about one to about five microns.
  • a particularly preferred composition comprises liposomes of varying sizes including small liposomes, i.e., about 20 nm to about 1 micron, medium liposomes, i.e., about 1 to about 3 microns, and large liposomes, i.e., about 3 to about 20 microns or greater and preferably about 3 to about 5 microns. It is presently preferred that such a composition comprise at least 5% by volume small liposomes, at least 10% by volume medium liposomes, and at least 20% by volume large liposomes.
  • the composition preferably comprises means for preventing breakdown of the preparation in the stomach but for allowing digestion of the liposomes in the gut. In the gut, the liposomes are absorbed by Peyer's patches and sufficient liposomes are taken up by macrophages to stimulate a long term systemic immune response.
  • Fig. 1 is an electron photomicrograph (magnification: 100,000x) of liposomes in lymphoid tissue of a Peyer's patch.
  • Fig. 2 is an electron photomicrograph (magnification: 10,000x) of lymphoid tissue within the
  • Fig. 3 is an electron photomicrograph (magnification: 20,000x) of splenic lymphoid cells.
  • Fig. 4 is an electron photomicrograph (magnification: 60,000x) of splenic lymphoid cells.
  • Fig. 5 is an electron photomicrograph (magnification: 15,000x) of a macrophage in the Peyer's patch.
  • Fig. 6 is an electron photomicrograph (magnification: 10,000x) of an extracellular space in the Peyer's patches.
  • Fig. 7 is an electron photomicrograph (magnification: 15,000x) of an extracellular space in the Peyer's patches.
  • Fig. 8 is an electron photomicrograph (magnification: 10,000x) of liposomes surrounding a white blood cell in a venule of the Peyer's patch.
  • Fig. 9 is an electron photomicrograph (magnification: 50,000x) of the cytoplasm and cellular membrane of a macrophage in the Peyer's patch.
  • Fig. 10 is an electron photomicrograph (magnification: 40,000x) showing liposomes at the cellular membrane and inside a macrophage in the Peyer's patch.
  • Fig. 11 is an electron photomicrograph (magnification: 70,000x) showing liposomes inside a macrophage in the Peyer's patch.
  • Fig. 12 is an electron photomicrograph (magnification: 75,000x) showing liposomes adhering to a venule wall in the lymphoid cells of the Peyer's patch.
  • Fig. 13 is an electron photomicrograph (magnification 25,000x) showing 980 nm liposomes in a macrophage vacuole 7 days after oral inoculation.
  • Fig. 14 is an electron photomicrograph (magnification 12,500x)showing 10 micron liposomes in a macrophage 21 days after oral inoculation.
  • Fig. 15 is an electron photomicrograph (magnification 40,000x) showing 2 micron liposomes in a macrophage vacuole 60 days after oral inoculation.
  • antigen processing cells such as macrophages or Kupffer cells
  • T lymphocytes T lymphocytes
  • This processed antigen is then displayed on the macrophage surface in association with HLA molecules and presented to the T cell to confer systemic immunity.
  • the macrophage also produces certain soluble cytokines that have an important role in T-cell activation, which confers systemic immunity as well.
  • the presenting antigen such as liposomal lyophilized antigen
  • Size and composition of the liposomes are important in determining the duration of the systemic immune response to the incorporated antigen.
  • Administration of liposomes of varying size and/or composition ensure a long lasting immune response, and thus avoid the need for repeated vaccine administrations. Since the half life of the macrophage is approximately 90 days, the presentation of an antigen taken up by GALT macrophages can last up to 180 days for conferring systemic immunity.
  • the presence of liposomal antigen in the Peyer's patches (outside of the macrophages) initiates a local immune response to the antigen as the liposomes breakdown and release the antigen.
  • the uptake of sufficient liposomal antigen in the macrophages stimulates a systemic immune response, and preferably a long-term systemic immune response, to the antigen(s) as the liposomes breakdown within the macrophages to release antigen.
  • local immune response refers to mucosal IgA, which confers protection from organisms in the bowel lumen and is characterized by secretion of local slgA.
  • systemic immune response refers to whole body production and circulation of organism specific humoral and cellular immune cells and is characterized by organism specific immune globulin (antibodies) and cytotoxic mononuclear cells.
  • long term systemic immune response means a detectible systemic immune response to an antigen that lasts at least 150 days after administration of the antigen.
  • sufficient liposomal antigen to stimulate a systemic immune (or long-term systemic immune) response means that amount of antigen-containing liposomes that affect a detectible systemic immune response (or long-term systemic immune response).
  • a systemic immune response may be confirmed by neutralizing antibody testing or other means of specific antibody testing, cytotoxic mononuclear cell assays and in vivo microbe challenge experiments, as is well known in the art.
  • antigens may be any substance that, when introduced into a mammal, will induce a detectable immune response, both humoral and cellular.
  • the term “antigen” also includes any portion of an antigen, e.g., the epitope, which can induce an immune response.
  • the antigen is an attenuated or killed microorganism, such as a virus or bacteria, rendering the preparation an oral vaccine against that microorganism.
  • HIV I and HIV II antigens include any substance that, when introduced into a mammal, will induce a detectable immune response, both humoral and cellular.
  • Typical HIV I and HIV II antigens include, but are not limited to, p24 antigen, gpl20, gp41, and envelope proteins.
  • hepatitis B and hepatitis C antigens include any substance that, when introduced into a mammal, will induce a detectable immune response, both humoral and cellular, to hepatitis B and hepatitis C.
  • Typical hepatitis B and hepatitis C antigens include, but are not limited to, hepatitis B surface antigen and hepatitis C NS-3 and NS-4 encoded antigens.
  • the liposomes of the present invention may be made of any suitable phospholipid, glycolipid, derived lipid, and the like.
  • suitable phospholipids include phosphatide choline, phosphatidyl serine, phosphatidic acid, phosphatidyl glycerin, phosphatidyl ethanolamine, phosphatidyl inositol, sphingomyelin, dicetyl phosphate, lysophosphatidyl choline and mixtures thereof, such as soybean phospholipids, and egg yolk phospholipids.
  • Suitable glycolipids include cerebroside, sulphur-containing lipids, ganglioside and the like.
  • Suitable derived lipids include cholic acid, deoxycholic acid, and the like.
  • the presently preferred lipid for forming the liposomes is egg phosphatidylcholine.
  • the liposomes may be formed by any of the known methods for forming liposomes and may be loaded with antigen according to known procedures.
  • Known methods for forming liposomal antigen are described, for example, in U.S. Patent No. 4,235,871 to Papahadjopoulos, et al., and Oral
  • Viral, bacterial and parasitic antigens may all be incorporated into liposomes and generate long-term immunity. In all cases, varying the size of the liposome for each antigen is crucial.
  • the antigens may first be individually incorporated into liposomes and then given individually or mixed with liposomes containing other antigens. Viral, bacterial and/or parasitic antigens may be combined.
  • the liposomes are loaded with p24 antigens.
  • the liposomes may also be loaded with other HIV antigens or whole virus.
  • the liposomes are loaded with hepatitis B surface antigen and/or hepatitis C NS-3 and NS-4 encoded antigens.
  • the liposomes may also be loaded with other hepatitis B and/or hepatitis C antigens or whole virus.
  • preparations may be prepared comprising a mixture of liposomes wherein each liposome contains only a single antigen. If desired, the liposomes may be loaded with a therapeutic drug in addition to the antigen.
  • the liposomes used in the present invention have an average mean diameter from about 20 nm to about 20 microns, preferably from about 200 nm to about 10 microns, and more preferably of from about 1 micron to about 5 microns.
  • Liposomes larger than about 20 microns are generally not preferred because they tend not to be taken up by the macrophages and only affect a local secretory antibody response. That is, the presence of large antigen-containing liposomes in the lymphoid tissue of the Peyer's patches will induce gut-associated lymphoid tissue (GALT) to produce IgA antibodies to destroy the antigen. However, no systemic immune response is induced.
  • GALT gut-associated lymphoid tissue
  • Liposomes smaller than about 20 nm are generally not preferred because they also tend not to be processed adequately by macrophages. These smaller liposomes tend to reside in the lymphoid tissue until they eventually are absorbed into the bloodstream and are destroyed by the reticulo- endothelial (RE) system. The smaller liposomes may induce a low grade production of secretory IgA, but do not stimulate systemic immunity.
  • RE reticulo- endothelial
  • antigen-containing liposomes of from about 20 nm to about 20 microns, preferably from about 200 nm to about 10 microns and more preferably from about 1 micron to 5 microns tend to be absorbed by macrophages in the Peyer's patches.
  • the macrophages digest the liposomes to release the antigen, which is then presented or displayed at the surface of the macrophage.
  • the macrophages act as antigen-presenting cells which process and present the antigen to systemic lymphocytes thereby inducing a systemic immune response to the antigen.
  • the macrophages display the antigen in conjunction with the major histocompatibility complex II (MHC II) glycoproteins to T-helper cells.
  • MHC II major histocompatibility complex II glycoproteins to T-helper cells.
  • T-helper cells activate B cells, which proliferate and differentiate into mature plasma cells that secrete copious amounts of immunoglobulins. In the systemic response, the immunoglobulins secrete
  • the liposomes be a mixture of sizes. Such heterogeneous sizes of liposomes are preferred as they are broken down over a period of time, e.g., up to 180 days or more by the macrophages.
  • the mixture of sizes will include liposomes having a size of about 20 nm to about 1 micron (small liposomes), liposomes having a size of about 1 micron to about 3 microns (medium liposomes) and liposomes having a size of about 3 to about 20 microns (large liposomes).
  • Preferred large liposomes are those having a size of from about 3 to about 5 microns.
  • a particularly preferred composition comprises about 10% by volume small liposomes, about 25% by volume medium liposomes and about 65% by volume large liposomes.
  • composition containing a heterogeneous population of liposomes there may be a uniform distribution of sizes or two or more discrete, homogeneous populations.
  • a combination of small, medium and large sizes is preferred because a smoother amnestic antibody curve is generated producing the most effective and dependable long-term immunity.
  • compositions comprising liposomes of various sizes allow antigens to be released in the macrophages over a long period of time, thereby continuing to stimulate a systemic immune response over a period of time.
  • the small size liposomes are taken up by the macrophages quickly and provide an immediate systemic immune response.
  • Medium size liposomes are taken up by the macrophages, but at a slower pace.
  • These liposomes act as a booster, i.e., provide an amnestic response.
  • the larger size liposomes take even longer to be taken up by the macrophages and act as a second booster, i.e., provide a second amnestic response.
  • liposomes of varying sizes enables a single dose of the antigen-containing liposomes to be sufficient to result in long term, and even permanent, immunity to the antigen.
  • the liposomes may be unilamellar or multilamellar. Production of unilamellar and multilamellar liposomes is also well known in the art and is described, for example, in U.S. Patent No. 5,008,050 to Cullis et al. and U.S. Patent Nos. 5,030,453 and 9,522,803 both to Lenk, et al., the disclosures of which are incorporated herein by reference.
  • Preparation of a homogeneous population may be accomplished by conventional techniques such as extrusion through a filter, preferably of 200 nm to 20 micron pore size, the filter being either the straight path or tortuous path type.
  • Other methods of treating liposomes to form a homogenous size distribution are ultrasonic exposure, the French press technique, hydrodynamic shearing, homogenization using, for example, a colloid mill or Gaulin homogenizer, and microfluidization techniques. Microfluidization is one presently preferred method. Other techniques involving sonication are also preferred. Microfluidization is described, for example, in U.S. Patent No. 4,533,254 to Cook, et al., which is incorporated herein by reference. In a preferred microfluidization procedure, the liposomal emulsion is forced at high pressure through a small diameter opening and splattered onto a wall and then collected.
  • the liposomes are passed one to ten, and preferably four, times through an M-110 Series Laboratory Microfluidizer manufactured by Microfluidics Corporation at a pressure of, e.g., 14,000 pounds per square inch, to achieve a generally homogenous population of liposomes having an average mean diameter of about 1 micron.
  • Liposomes of other sizes can be prepared using the same method by adjusting the number of runs through the microfluidizer, the pressure, and flow rate.
  • the raw materials for the liposomes e.g., phospholipids
  • a sonicator e.g., phospholipids
  • antigens placed in a sonicator, and sonicated for a time, at a temperature and at a speed sufficient to obtain liposomes of the desired size.
  • raw materials are placed in a Brinkman Inc. or Beckman Inc. Sonicator and sonicated at 1,000 to 10,000 meters per second at 50°C for 20, 5 and 2 minutes to obtain small, medium and large liposomes, respectively.
  • larger sonication times result in smaller liposomes.
  • the emulsion is lyophilized. Lyophilized liposomal antigen can be stored at room temperature for one half to three years without degradation of the liposomes or antigen.
  • Lyophilization may be accomplished by any method known in the art. Such procedures are disclosed, for example, in U.S. Patent No. 4,880,836 to Janoff, et al., the disclosure of which is incorporated herein by reference. Lyophilization procedures preferably include the addition of a drying protectant to the liposome suspension. The drying protectant stabilizes the liposome suspension. The drying protectant stabilizes the liposomes so that the size and content are maintained during the drying procedure and through rehydration.
  • Preferred drying agents are saccharide sugars including dextrose, sucrose, maltose, manose, galactose, raffinose, trehalose, lactose, and triose sugars which are preferably added in amounts of about 5% to about 20%, and preferably about 10%, by weight of the aqueous phase of the liposomal suspension.
  • Dextrose, sucrose and maltose are presently preferred.
  • Manitol may be used in conjunction with any of the saccharides. Additional preservatives such as BHT, EDTA, urea, albumin, dextran or polyvinyl alcohol may also be used.
  • the lyophilized liposomal antigen may be packaged for oral administration in either a pill form or a capsule.
  • An enteric coating is preferably applied to the liposomal antigen to prevent breakdown in the stomach.
  • the enteric coating may be made of any suitable composition. Suitable enteric coatings are described, for example, in U.S. Patent Nos. 4,311,833 to Namikoshi, et al.; 4,377,568 to Chopra;
  • enteric coating compositions include alkyl and hydroxyalkyl celluloses and their aliphatic esters, e.g., methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxybutylcellulose, hydroxyethylethylcellulose, hydroxyprophymethylcellulose, hydroxybutylmethylcellulose, hydroxypropylcellulose phthalate, hydroxypropylmethylcellulose phthalate and hydroxypropylmethylcellulose acetate succinate; carboxyalkylcelluloses and their salts, e.g., carboxymethylethylcellulose; cellulose acetate phthalate; polycarboxymethylene and its salts and derivatives; polyvinylalcohol and its esters, polycarboxymethylene copolymer with sodium formaldehyde carboxylate; acrylic polymers and copolymers, e.g., methacrylic acid-methyl methacrylic acid copolymer and methacrylic acid-methyl acrylate copo
  • enteric coatings include polyvinylacetate esters, e.g., polyvinyl acetate phthalate; alkyleneglycolether esters of copolymers such as partial ethylene glycol monomethylether ester of ethylacrylate-maleic anhydride copolymer or diethyleneglycol monomethylether ester of methylacrylate- maleic anhydride copolymer, N-butylacrylate-maleic anhydride copolymer, isobutylacrylate-maleic anhydride copolymer or ethylacrylate-maleic anhydride copolymer; and polypeptides resistant to degradation in the gastric environment, e.g., polyarginine and polylysine.
  • Mixtures of two or more of the above compounds may be used as desired.
  • the enteric coating material may be mixed with various excipients including plasticizers such as triethyl citrate, acetyl triethyl citrate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, dibutyl tartrate, dibutyl maleate, dibutyl succinate and diethyl succinate and inert fillers such as chalk or pigments.
  • plasticizers such as triethyl citrate, acetyl triethyl citrate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, dibutyl tartrate, dibutyl maleate, dibutyl succinate and diethyl succinate and inert fillers such as chalk or pigments.
  • composition and thickness of the enteric coating may be selected to dissolve immediately upon contact with the digestive juice of the intestine.
  • the composition and thickness of the enteric coating may be selected to be a time-release coating which dissolves over a selected period of time, as is well known in the art.
  • Example 1 To establish the effective absorption of lyophilized liposomes by Peyer's patches and uptake by macrophages, the following protocol was followed: Preparation of antigen-containing liposomes: Antigen-containing liposomes having a diameter of approximately 142 nanometers used in the experimental study described below were prepared according to the following procedure.
  • Microfluidizer Four (4) passes through the microfluidizer at 110°F: Weight of Materials to be Used
  • mice 100 micrograms of lyophilized liposomes 142 nanometers in diameter were suspended in 0.3 ml of 0.5% xanthum gum aqueous solution. The mixture was given via a gavage tube to four week old male CD-I mice. Five mice were given the liposomal preparation and five mice were given 0.5% xanthum only as controls. For one week the ten mice were kept on ad lib diet and water ad lib. On day seven the mice were anesthetized with methyloxyfluorane, and through a mid-line abdominal incision the peritoneum was entered. The small bowel was resected and examined for the Peyer's patches. The Peyer's patches from the small bowel were removed and placed in one molar phosphate buffer minced with a straight razor into less than 1 mm sections on wax paper.
  • the preparation was then fixed at room temperature with 4% glutaraldehyde in two molar phosphate buffer, washed three times with one molar phosphate buffer and taken to the electronmicroscopy facility.
  • the preparation was then dehydrated and mounted in epoxy resin, cut with a microtome, stained with osmium tetroxide, then examined under a Zeis CR10 electron microscope. The Peyer's patches were then photographed and labeled as noted.
  • the spleen and Peyer's patches of the gut were sectioned and slides were prepared. Photomicrographs were taken and are presented here as Figures 1-12.
  • the photomicrographs show liposomes (Fig. 1) residing in venules and extracellular tissue of the Peyer's patch (Figs. 2, 6, 7, 8, 12). They also show that the liposomes were not present in the splenic lymphoid tissue which indicate that the liposomes were staying in the Peyer's patches and not circulating through the blood stream in the mouse. (Figs. 3, 4). Finally, the photomicrographs show liposomes being absorbed and digested by macrophages (Figs. 5, 9, 10, 11).
  • Virus stocks of CVB5 strain C59 were prepared in monolayers of monkey kidney (MK) cells using an inoculum giving an MOI of 1 pfu/cell in supplemental Leibovitz's L 15 medium as described in See D.M., Tilles J.G., "Efficacy of a Polyvalent Inactivated-virus Vaccine in Protecting Mice from Infection with Clinical Strains of Group B Coxsackieviruses.” Scand. J. Infect. Dis. 26: 739-747, 1994, the disclosure of which is incorporated herein by reference. Flasks were observed daily for cytopathic effect (cpe) until cpe reached 4+. At that time, the virus was harvested, aliquoted and frozen at -80 ° C until further use. Animals
  • mice 16-18 g were obtained from Charles Rivers Farms, Wilmington, Massachusetts.
  • Preparation of Viral Antigen One strain of coxsackieviruses groups Bl-6 were absorbed to monolayers of MK cells at a multiplicity of 1 pfu/cell and incubated as described. When maximal cytopatic effect was observed, the virus-containing media for a single strain was harvested and pooled. Aliquots were stored and tested for viral titer as previously described in See, D.M., Tilles, J.G., "Efficacy of a Polyvalent Inactivated-virus Vaccine in Protecting Mice from Infection with Clinical Strains of Group B Coxsackieviruses.” Scand. J. Infect. Dis. 26: 739-747, 1994.
  • Viral proteins were encapsulated with 3 different particle size liposomes as follows: Before beginning, 3 round bottom flasks were labeled A (2 minutes), B (5 minutes) and C (20 minutes). 783 mg of diphosphatidylchoxene (DPPC) (Avanti), 180 mg cholesterol (Sigma), and 36mg dicetyl-phosphate (Sigma) were added to each of the flasks.
  • DPPC diphosphatidylchoxene
  • 180 mg cholesterol Sigma
  • 36mg dicetyl-phosphate Sigma
  • the resultant 3 liposomes (2 ⁇ m, 10 ⁇ m, 908nm) were orally administered to male CD-I mice weighing 16-18g either alone or mixed for either 1, 2 or 3 doses over the same number of weeks.
  • 30 mg liposomes were given orally in 0.3 cc containing sodium acetate buffer, pH 9.0.
  • 120 mg of mixed liposomes were given.
  • the final set of mice were given either mixed liposomes or a placebo and then infected with CVB5/C59.
  • mice were then sacrificed. Blood samples, Peyer's patches and spleens were taken for microtiter neutralization, antibody titration assays and electron microscopy work respectively. Pancreas samples were taken only from infected mice to run viral titer assays.
  • n 5 for each group. Titers - ⁇ 3 were assigned a value of 2 for purposes of determining the mean. Means are for all 6 coxsackie B serogroups. * 30mg + 120mg
  • titers of virus were determined in the pancreas of mice killed 3 days after infection. Two groups of 5 mice were used; one group was given 3 doses of mixed liposomes and the other group was given buffer 4 placebo. The placebo group ended up with a mean titer of 5.3 x 10 (pfu/mg) while the vaccine
  • Fig. 13 seven days after final oral inoculation, 980 nm liposomes are visible in vacuoles within macrophages of the Peyer's Patches. As shown in Fig. 14, 21 days after oral inoculation, a 10 micron liposome was observed in a macrophage of the Peyer's patches. As shown in Fig. 15, 60 days after oral inoculation, 2 micron liposomes were observed in a vacuole within a macrophage of the Peyer's Patches.
  • Example 3 Antigen Stock hepatitis B surface antigen (400 ⁇ g/ml) was purchased from Advanced
  • mice Male CD-I mice (16-18 g) were obtained from Charles Rivers Farms, Wilmington, Massachusetts.
  • Hepatitis B and/or hepatitis C antigen was encapsulated in 3 different particle size liposomes as follows: A solution of PyS DHPE was prepared in a test tube by dissolving 25 mg of PyS DHPE in 0.1 ml of chloroform. Lipid solution was then prepared in a separate test tube by combining 313 mg of DPPC, 72 mg of cholesterol, 14 mg of dicetylphosphate, 144 ⁇ L PyS DHPE solution, and 1.056 ml of chloroform, for a total volume of approximately 1.20 ml.
  • lipid solution 360 ⁇ l was aliquoted into three glass tubes labeled "B", “C” and "B + C". The solvent in each tube was evaporated to dryness with nitrogen gas.
  • a maltose solution was prepared by dissolving 200 mg of maltose in 2.0 ml of water. A final concentration of 166.6 ⁇ g of hepatitis antigen (B, C or B + C) per tube was prepared.
  • Each antigen was aliquotted evenly (3 ml/tube) into the labeled tubes.
  • B-1, C-l and B+C-l solutions were warmed at 50°C for 2 minutes then sonicated at level 1 at 50°C for 10 minutes.
  • B-2, C-2 and B+C-2 solutions were warmed at 50°C for 2 minutes then sonicated at level 1 at 50°C for 2 minutes.
  • B-10, C-10 and B+C- 10 solutions were warmed at 50 °C for 2 minutes with no sonication.
  • Like antigens e.g., B-1, B-2 and B-10) were mixed for a total of 9.0 ml. 0.9 ml of each combined antigen solution was aliquotted into ten vials, which were placed in a freezer at 10°C overnight and subsequently lyophilized.
  • the resultant lyophilized liposome mixtures of three different-sized liposomes (1 ⁇ m, 2 ⁇ m, and 10 ⁇ m) were each orally administered to male CD- 1 mice weighing 16- 18g.
  • 30 mg liposome mixtures were orally administered in 0.3cc containing sodium acetate buffer, pH 9.0, so that each mouse received 50 ⁇ g of hepatitis antigen.
  • Two mice from each of B, C, and B+C groups were sacrificed at biweekly intervals. Blood samples, Peyer's patches and spleens were taken for antibody to hepatitis B or hepatitis C antigen by ELISA, Electron Microscopy work, and lymphocyte proliferation assay, respectively.
  • Peyer's patches and spleens were diced into pieces ⁇ 1 mm with a single edged blade on a wax sheet and kept moist in O.IM phosphate buffer.
  • the pieces were then added to a vial of gluteraldehyde solution prepared by mixing 0.2M phosphate buffer, pH 7 (28ml 0.2M NaH PO 4 + 72ml 0.2M Na 2 HP0 4 ) 1 : 1 with 8% gluteraldehyde (Ted Pella Inc.). Tissue was fixed 2-4 hours at room temperature and then washed 3 times with O.IM phosphate buffer.
  • ELISA assays to hepatitis B and hepatitis C antigen developed in the laboratory using antigen- coated plates from East Coast biologies were conducted at weekly intervals from 5ml aliquots of serum for 5 weeks. By the second week and thereafter, all mice assayed had developed antibodies to the antigens. Lymphocyte Proliferation Assay
  • a lymphocyte proliferation assay was performed after weeks two to four.
  • a plate was laid by setting up two rows, each consisting of twelve wells for each of B cells, C cells and B+C cells. 100 ⁇ L of cells were added in each well.
  • 20 ⁇ L of RPMI (no antigen) was added to the first six wells in each group of cells.
  • 20 ⁇ L of 10 ⁇ g of B antigen was added to the next six wells in each group of cells.
  • 20 ⁇ L of 10 ⁇ g of B antigen was added to the next six wells in each group of cells.
  • the proliferation index indicates the extent of cellular proliferation resulting from prior exposure to hepatitis antigens compared to a control not previously exposed to the antigen, and takes into account the amount of hepatitis antigen added to the assay. A higher proliferation index indicates more cellular proliferation and therefore a better cellular immune response. A proliferation index of at least 1 indicates a very active immune response.
  • Example 4 Antigen p24 antigen was purchased from Biodesign International, Kennebunk, Maine. Animals
  • Microencapsulation p24 antigen was encapsulated in 3 different particle size liposomes as follows: A solution of
  • PyS DHPE was prepared in a test tube by dissolving 25 mg of PyS DHPE in 1.0 mL of chloroform. Lipid solution was then prepared in a separate test tube by combining 313 mg of DPPC, 72 mg of cholesterol, 14 mg of dicetylphosphate, 144 ⁇ L PyS DHPE solution, and 1.056 ml of chloroform, for a total volume of approximately 1.20 mL.
  • lipid solution 900 ⁇ L was aliquoted into a glass test tube. The solvent in each tube was evaporated to dryness with Nitrogen gas.
  • a maltose solution was prepared by dissolving 100 mg of maltose in 1.0 mL of water. 750 ⁇ L of maltose solution was measured into the three test tubes. 200 ⁇ L of p24 antigen was added to each tube, then 4550 ⁇ L of water was added.
  • the solution in the first tube was warmed at 50° C for 2 minutes then sonicated at 50° C for
  • liposomes have a diameter of approximately 5 ⁇ m.
  • the solution in the second tube was warmed at 50° C for 2 minutes then sonicated at 50° C for 5 minutes to obtain liposomes having a diameter of approximately 2 ⁇ m.
  • the solution in the third test tube was warmed at 50 °C for 2 minutes, giving liposomes having a size of approximately 5 ⁇ m.
  • Approximately 10 ⁇ l of liposomes was removed from each test tube for particle sizing.
  • the liposomes from all three test tubes were combined for a total volume of approximately 15 mL.
  • the solution was aliquoted into glass vials (1.5 mL/vial; 60 ⁇ g/vial). The vials were placed in a -10°C freezer overnight. The samples were then lyophilized.
  • the resultant lyophilized liposome mixtures of three different-sized liposomes (1 ⁇ m, 2 ⁇ m, and 5 ⁇ m) were orally administered to male CD-I mice weighing 16-18g obtained from Charles
  • a lymphocyte proliferation assay was performed after week two and week four. Proliferation of splenic mononuclear cells was significantly enhanced in liposomal p24 antigen-treated mice compared to untreated control mice, as shown in Table 2 below.
  • the proliferation index indicates the extent of cellular proliferation resulting from prior exposure to p24 antigen compared to a control not previously exposed to the antigen, and takes into account the amount of p24 antigen added to the assay. A higher proliferation index indicates more cellular proliferation and therefore a better cellular immune response. A proliferation index of at least 1 indicates a very active immune response.

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Abstract

L'invention concerne une méthode permettant de provoquer chez un mammifère une réponse immunitaire systémique à un antigène sélectionné parmi les antigènes du VIH I, du VIH II, de l'hépatite B et de l'hépatite C. La méthode consiste à administrer par voie orale des liposomes multilamellaires lyophilisés contenant l'antigène. La taille des liposomes est comprise entre 20 nm et 20 νm. Les liposomes contenant l'antigène sont absorbés dans les plaques de Peyer de l'intestin. Les liposomes contenant l'antigène sont repris par des macrophages dans les plaques de Peyer en quantités suffisantes pour provoquer une réponse immunitaire systémique à l'antigène.
PCT/US1998/013194 1997-06-26 1998-06-25 Methode pour provoquer une reponse immunitaire systemique a un antigene de l'hepatite ou du vih WO1999000142A1 (fr)

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US88296897A 1997-06-26 1997-06-26
US08/882,968 1997-06-26
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US08/948,568 US6207185B1 (en) 1996-03-22 1997-10-10 Method for inducing a systemic immune response to an HIV antigen
US09/007,297 US6117449A (en) 1996-03-22 1998-01-14 Method for inducing a systemic immune response to a hepatitis antigen
US09/007,297 1998-01-14

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2850872A1 (fr) * 2003-02-12 2004-08-13 Ethypharm Sa Composition vaccinale destinee a induire une reponse immunitaire contre le virus responsable du sida

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4997926A (en) * 1987-11-18 1991-03-05 Scripps Clinic And Research Foundation Deaminase-stable anti-retroviral 2-halo-2',3'-dideoxy
US5709879A (en) * 1990-06-29 1998-01-20 Chiron Corporation Vaccine compositions containing liposomes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4997926A (en) * 1987-11-18 1991-03-05 Scripps Clinic And Research Foundation Deaminase-stable anti-retroviral 2-halo-2',3'-dideoxy
US5709879A (en) * 1990-06-29 1998-01-20 Chiron Corporation Vaccine compositions containing liposomes

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2850872A1 (fr) * 2003-02-12 2004-08-13 Ethypharm Sa Composition vaccinale destinee a induire une reponse immunitaire contre le virus responsable du sida
WO2004073596A2 (fr) * 2003-02-12 2004-09-02 Ethypharm Composition vaccinale comprenant un antigene proteique du vih incorpore dans des vesicules lipidiques multilamellaires.
WO2004073596A3 (fr) * 2003-02-12 2004-10-28 Ethypharm Sa Composition vaccinale comprenant un antigene proteique du vih incorpore dans des vesicules lipidiques multilamellaires.

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