Classifications

• • 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
  • 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—Liposomes
• • 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
• • 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 relates to a method for inducing a systemic immune response to an antigen and more particularly to vaccines suitable for oral administration.
• 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 major line of defense against a majority 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 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.
• GALT 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 abso ⁇ tion 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.
• the present invention provides a method for inducing a systemic immune response to one or more antigens in a mammal.
• the method comprises first inco ⁇ orating the antigen(s) into liposomes, preferably multilaminar 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(s)-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(s)-containing liposomes are absorbed in the Peyer's patches of the gut.
• antigen(s)-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.
• the composition comprises lyophilized, multilaminar liposomes which 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, 10% by volume medium liposomes and 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 Peyer's patch.
• 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. 1 1 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.
• Some antigens require intracellular processing by antigen processing cells such as macrophages or Kupffer cells before being presented to T lymphocytes as a processed antigen. 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 enter the macrophage of the GALT for processing to confer systemic immunity and this is dependent upon the size of the liposome presented to the GALT.
• Size and composition of the liposomes are important in determining the duration of the systemic immune response to the inco ⁇ orated antigen. Administration of liposomes of varying size and 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 nobule 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 which 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 effect 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 which, 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.
• 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 emanolamine, 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 Microbiology and Immunology, 1994, 9:146-153 which are inco ⁇ orated herein by reference. What is formed is an emulsion comprising liposomal antigen. Viral, bacterial and parasitic antigens may all be inco ⁇ orated into liposomes and generate long-term immunity. Ln all cases, varying the size of the liposome for each antigen is crucial.
• the antigens may first be individually inco ⁇ orated into liposomes and then given individually or mixed with liposomes containing other antigens. Viral, bacterial and/or parasitic antigens may be combined.
• Current candidate antigens include: polio 1, 2, 3; hepatitis A through N; coxsackie B1-B6; mumps; measles, rubella; respiratory syncytial (RS) virus, parainfluenza 1-4; influenza A, B and C; adenovirus types 1-41; mycoplasma pneumonia; streptococcus pneumonia; chlamydia trachomatis; pneumoniae and psittacocci; hemophilus, influenza, meningococcus, the four types of malaria, leishmanie species, brucella species, trypanosoma brucei strains, mycobacterium tuberculosis, pseudomonas species, escherichia coli species, salmonella species, trypanasom
• the liposomes are loaded with each of the three polio viral antigens.
• Other preferred embodiments include liposomes loaded with each of the known hepatitis antigens, i.e., A through H; and liposomes loaded with several unrelated antigens such as mumps, measles and rubella. It is understood that various other combinations, including combinations involving more than one type of viral antigen, e.g. coxsackie B antigens and hepatitis antigens may be loaded into a single liposome, as desired.
• preparations may be prepared comprising a mixture of liposomes wherein each liposome contains only a single antigen. If desired, the liposome 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 20 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 effect 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. It has been found that 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 Payer'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 secreted are initially IgM followed by IgG.
• 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.
• each size of liposomes i.e., small, medium and large
• a particularly preferred composition comprises about 10% by volume small liposomes, about 25% by volume medium liposomes and about 65% by volume large liposomes.
• compositions 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.
• 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.
• use of 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 unilaminar or multilaminar. Production of unilaminar and multilaminar liposomes is also well known in the art and is described, for example, in U.S. Patent Nos. 5,008,050 to Cullis et al. and 5,030,453 and 9,522,803 both to Lenk, et al., which are all inco ⁇ orated 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 or 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 inco ⁇ orated herein by reference.
• 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 4 times through an M-l 10 Series Laboratory Microfiuidizer manufactured by Microfluidics Co ⁇ oration 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 microfiuidizer, the pressure, and flow rate.
• the raw materials for the liposomes e.g., phospholipids
• a sonicator and sonicated for a time and 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. Typically, 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. 4,880,835 to Janoff, et al., which is inco ⁇ orated 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
• dextrose 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 or 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. 4,31 1,833 to Namikoshi, et al.; 4,377,568 to Chopra; 4,385,078 to Onda, et al.; 4,457,907 to Porter; 4,462,839 to McGinley, et al.; 4,518,433 to McGinley, et al.; 4,556,552 to Porter, et al.; 4,606,909 to Bechgaard, et al.; 4,615,885 to
• enteric coating compositions include alkyl and hydroxyalkyi 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.
• alkyleneglycolether esters of copolymers such as partial ethylene glycol monomethylether ester of ethylacrylate-maleic anhydride copolymer or diethyleneglycol mono
• 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.
• the 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
• Antigen-containing liposomes having a diameter of approximately 142 nanometers used in the experimental study described below were prepared according to the following procedure. 1. 2250 ml of water (double distilled) to beaker (keep cool) and set with a nitrogen sparge for at least 30 minutes.
• Microfiuidizer Four (4) passes through the microfiuidizer at 1 10°F:
• 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, 1 1).
• Example 2 Example 2
• 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 L15 medium as described in DM, Tilles JG., "Efficacy of a Polyvalent Inactivated-virus Vaccine in Protecting Mice from Infection with Clinical Strains of Group B Coxsackie viruses.” Acand J Infect Dis 26: 739-747, 1994, which is inco ⁇ orated 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 deg C until further use. Animals Male CD-I Mice 16-18 g were obtained from Charles Rivers Farms, Wilmington, Massachusetts. Preparation of Viral Antigen
• the resultant 3 liposomes (2um, lOu , 908nm) were given to male CD-I mice weighing 16-18g obtained from Charles Rivers Farms, Wilmington, Mass. orally either alone or mixed for either 1 , 2 or 3 doses over the same number of weeks.
• 30 mg liposomes were given orally in 0.3cc containing sodium acetate buffer, ph 9.0.
• 120mg of mixed liposomes were given.
• the final set of mice were given either mixed liposomes or a placebo and then infected with CVB5/C59. The mice were then sacrificed.
• n 5 for each group. Titers ⁇ 3 were assigned a value of 2 for pu ⁇ oses 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 placebo. The placebo group ended up with a mean titer of 5.3 x IO 4 (pfu/mg) while the vaccine group's mean titer was only 2.2 x IO 2 (pfu/mg). (Titers of ⁇ 2 (the lower limit of sensitivity of the assay) were assigned a value of 1 for the pu ⁇ ose of calculating the mean.) Electron Microscopy

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