Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • 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
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• • 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
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/513—Organic macromolecular compounds; Dendrimers
  • A61K9/5169—Proteins, e.g. albumin, gelatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • 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
  • A61K2039/55522—Cytokines; Lymphokines; Interferons
• • 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

Definitions

• This invention is related to improved methods of immunization of mammals to achieve cell-mediated and humoral immune responses.
• plasmid DNA encoding genes for viral and bacterial antigens can direct the synthesis of the antigens in a native form and effectively present it to the immune system. For instance, potent humoral and cell-mediated immune responses have been induced by HIV-1 DNA vaccination in rodents and non-human primates [1, 2].
• the plasmids are typically administered as bolus injections into the muscle or through a gene gun.
• DNA immunization can elicit immune responses to viral proteins and confer protective immunity against challenge of the virus in rodent models have stimulated strong interest in optimizing this strategy for developing HIV vaccines [3-5].
• DNA immunization offers the advantage of expressing the antigen in its native form that may lead to optimal processing and presentation to antigen presenting cells for induction of both humoral and cellular immune responses.
• vaccinia vectors are efficient means of delivering the foreign genes in vivo, lingering concerns over the safety of these vectors remain.
• cytokines to enhance an immune response to vaccines has attracted intense attention, particularly in the field of cancer immunotherapy.
• One approach is to introduce cytokine genes directly into the tumor cells [6-23]. The cytokines either enhance the presentation of antigens to T cells or provide additional co-stimulatory signals for T cell activation.
• the locally secreted cytokines elicit an inflammatory reaction that leads to the rejection of the injected tumor cells.
• these genetically-altered tumor cells can generate systemic immunity against subsequent challenge of parental tumor cells, and occasionally even against established micrometastases.
• GM-CSF and TNF- ⁇ have been found to synergize with IL-12 to enhance induction of cytotoxic T lymphocytes against HIV-1 MN vaccine constructs [1].
• a solid nanosphere of less than 5 ⁇ m for genetic immunization of a mammal comprising a coacervate of a polymeric cation and a polyanion, wherein the polyanion consists of nucleic acids encoding an antigen, wherein the polymeric cation is selected from the group consisting of gelatin and chitosan, and wherein a cytokine is encapsulated in the coacervate.
• a method for immunizing a mammal to raise an immune response to an antigen comprises: administering to a mammal a solid nanosphere of less than 5 ⁇ m comprising a coacervate of a polymeric cation and a polyanion, wherein the polyanion consists of nucleic acids encoding an antigen, wherein the polymeric cation is selected from the group consisting of gelatin and chitosan, and wherein a cytokine is encapsulated in the coacervate.
• a method of forming solid nanospheres for immunization of a mammal comprises the step of: forming solid nanospheres by coacervation of a polyanion consisting of nucleic acids encoding an antigen and a polymeric cation, wherein the polymeric cation is selected from the group consisting of chitosan and gelatin, wherein said coacervation is done in the presence of a cytokine, whereby the cytokine is encapsulated in said solid nanospheres.
• FIG. 1 Proliferation of CT-4S cells in response to nanosphere-delivered IL- 4.
• DNA-gelatin nanospheres were synthesized with different loading levels of murine IL-4 (denoted as 0, 100, and 1000U added to the coacervating reaction), were subsequently purified, and incubated with CT-4S cells. 30 hrs. after the addition of nanospheres, the cells were labeled with 3 H-thymidine, then radioactivity associated with the cells was measured 18 hrs later. Nanosphere doses are indicated as total DNA. Data points are average of triplicates +/- s.d.
• DNA-gelatin nanospheres synthesized with either 0, 1.5, 15 U IL-4 per ug p43-clacZ DNA were injected intramuscularly into groups of 4 mice every two weeks for a total of three immunizations.
• ELISA assays for anti- ⁇ -gal IgG antibody responses were carried out on pooled mice serum at week 4 (after two immunizations).
• lymphocytes from the spleens and lymph nodes from each group were harvested, pooled, and subjected to CTL assays for anti- ⁇ -gal responses.
• a) Anti- ⁇ -gal antibody response b) Anti- ⁇ -gal CTL response. The results are average of triplicates +/- s.d.
• FIG. 3 The anti- ⁇ -gal antibody response in mice injected with IL-4 given as nanosphere-encapsulated vs. free form.
• Groups of 4 mice were injected i.m. with nanospheres mixed with 20 U of free IL-4,. nanospheres encapsulated with 20 U IL-4 per 1 mg p43-clacZ DNA, DNA, or DNA mixed with 20 U of free IL-4.
• Each group was vaccinated twice at four weeks intervals with 1 mg total DNA.
• the sera from individual groups were pooled then assayed for total IgG anti- ⁇ -gal antibody. Data are average of triplicates +/- s.d.
• FIG. 4 Potentiation of CTL responses using nanosphere-encapsulated IL-2 and ⁇ -INF.
• Groups of 4 mice were injected once i.m. with 2 mg of total p43- clacZ DNA either as nanospheres, nanospheres encapsulated with IL-2 (20 U per 1 mg p43-clacZ DNA), nanospheres encapsulated with IL-2 and ⁇ -INF (100 U per 1 mg pg 43-clacZ DNA), or 'naked' DNA.
• Standard CTL assays were carried out at week 4. Data are average +/- s.d.
• mice 8-wk old BALB/c mice (10 mice per group) were immunized i.m. in the tibialis anterior with three monthly injections of nanospheres containing 0.5 ⁇ g (- ⁇ -) or 3 ⁇ g (- ⁇ -) Ebola NP pDNA; 0.5 ⁇ g (-o-) or 3 ⁇ g (-•-) Ebola GP pDNA; or 3 ⁇ g control WRG7077 pDNA (- ⁇ -) (vector without the Ebo NP or GP gene insert).
• the mouse-adapted Ebola virus challenge system and Ebola pDNA vector contructs are described in detail in Vanderzanden, 1998, Virology
• nanospheres synthesized by the salt-induced complex coacervation of nucleic acids with either gelatin or chitosan.
• Cytokines are co-encapsulated in the nanospheres to enhance the efficacy of genetic vaccination and to produce a desired immune response.
• the ultimate immune response elicited by the vaccine is influenced by the interaction between the specific and non-specific immune mechanisms.
• the type of immune cells attracted to the site of vaccination for example, can determine the eventual antitumor immunity induced by a tumor-associated antigen.
• the repertoire of these cells is in turn governed by the temporal and spatial distribution of cytokines.
• the temporal and spatial distribution of the cytokines can be further altered. This can direct the immune response toward a specific immune arm, such as the Thl or Th2 pathway. This strategy can be used, for example, to modulate the immune response against HIV infection by emphasizing the humoral or cellular arm.
• ligands can be conjugated to the nanosphere to stimulate receptor-mediated endocytosis and potentially to target cell/tissue; 2) lysosomolytic agents can be incorporated to promote escape of intact DNA into cytoplasm; 3) other bioactive agents such as RNA, oligonucleotides, proteins, or multiple plasmids can be co-encapsulated.
• the protein antigen may also be co-encapsulated in the nanosphere for potential augmentation of immune response through class II presentation, and as suggested by recent findings, as well as class I presentation when the nanosphere is internalized by antigen presenting cells; 4) bioavailability of the nucleic acids can be improved because of protection from serum nuclease degradation by the matrix, and there is little release of the nucleic acids until the nanosphere is sequestered into the endolysosomal pathway. There is also the potential of intracellular sustained release of nucleic acids that may provide more prolonged expression of the gene product; 5) the nanosphere is stable in plasma electrolytes, and can be lyophilized without loss of bioactivity. Hence the nanospheres can be handled more like conventional pharmaceutical formulations in terms of production, reproducibility, and storage.
• the electrostatic interaction between the two species of macromolecules results in the separation of a coacervate (polymer-rich phase) from the supernatant (polymer-poor phase).
• This phenomenon can be used to form nanospheres and encapsulate a variety of compounds.
• the encapsulation process can be performed entirely in aqueous solution and at low temperatures, and has a good chance, therefore, of preserving the bioactivity of the encapsulant.
• gelatin or other polymeric cation having a similar charge density to gelatin is used to complex with nucleic acids to form nanospheres.
• the source of gelatin is not thought to be critical; it can be from bovine, porcine, human, or other animal source.
• the polymeric cation has a molecular weight of between 19,000-30,000.
• Poly-L-lysine or chitosan may be particularly useful as the polymeric cation of the present invention.
• Polyamino acids synthetic or naturally occurring, can also be used, such as polylysine, poly-lysine-poly-arginine, polyarginine, protamine, spermine, spermidine, etc.
• Polysaccharides may also be used.
• Desirably sodium sulfate is used to induce the coacervation of polymeric cation and nucleic acids.
• Ethanol can also be used at a concentration of about 40 to 60% to induce coacervation.
• Chondroitin sulfate can also be incorporated into the nanosphere, which is especially beneficial if one desires other substances such as drugs and lysosomolytic agents to be incorporated in the nanosphere.
• the concentration of chondroitin sulfate is between about 0.005% and 0.1%. It is preferred that the nanospheres be less than 5 microns. More preferred are nanospheres of less than 3 microns, and even more preffered are nanospheres which are less than 2, 1, 0.5, and 0.1 microns. While size can be effected by the conditions of coacervation and the size of the component polyanion and polycation, nanospheres of the desired size can also be size selected using a technique which separates the nanospheres on the basis of size.
• Targeting ligands can be directly bound to the surface of the nanosphere or can be indirectly attached using a "bridge” or "spacer". Because of the amino groups provided by the lysine groups of the gelatin, the surface of the nanospheres can be easily derivatized for the direct coupling of targeting moieties. For example, carbo-diimides can be used as a derivatizing agent. Alternatively, spacers (linking molecules and derivatizing moieties on targeting ligands) such as avidin-biotin can be used to indirectly couple targeting ligands to the nanospheres.
• Biotinylated antibodies and/or other biotinylated ligands can be coupled to the avidin-coated nanosphere surface efficiently because of the high affinity of biotin (k a ⁇ 10 15 M "1 ) for avidin (Hazuda, et al., 1990, Processing of precursor interleukin 1 beta and inflammatory disease, J. Biol. Chem., 265:6318-22; Wilchek, et al., 1990, Introduction to avidin-biotin technology, Methods In Enzymology, 184:5-13).
• Orientation-selective attachment of IgGs can be achieved by biotinylating the antibody at the oligosaccharide groups found on the F c portion (O'Shannessy, et al., 1984, A novel procedure for labeling immunoglobulins by conjugation to oligosaccharides moieties, Immunol. Lett., 8:273-277). This design helps to preserve the total number of available binding sites and renders the attached antibodies less immunogenic to F c receptor-bearing cells such as macrophages.
• Spacers other than the avidin-biotin bridge can also be used, as are known in the art.
• Staphylococcal protein A can be coated on the nanospheres for binding the F c portions of immunoglobulin molecules to the nanospheres.
• Cross-linking of linking molecules or targeting ligands to the nanosphere is used to promote the stability of the nanosphere as well as to covalently affix the linking molecule or targeting ligand to the nanosphere.
• the degree of cross-linking directly affects the rate of nucleic acids released from the nanospheres.
• Cross-linking can be accomplished using glutaraldehyde, carbodiimides such asEDC (l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, DCC (NjN'-dicyclohexylcarbodiimide), carboxyls (peptide bond) linkage, bis
• Targeting ligands are any molecules which bind to specific types of cells in the body. These may be any type of molecule for which a cellular receptor exists. Preferably the cellular receptors are expressed on specific cell types only. Examples of targeting ligands which may be used are hormones, antibodies, cell-adhesion molecules, saccharides, drugs, and neurotransmitters.
• the nanospheres of the present invention have good loading properties. Typically, following the method of the present invention, nanospheres having at least 5% (w/w) nucleic acids can be achieved. Preferably the loading is greater than 10 or 15% nucleic acids. Often nanospheres of greater than 20 or 30%, but less than 40 or 50% nucleic acids can be achieved. Typically loading efficiencies of nucleic acids into nanospheres of greater than 95% can be achieved.
• the method of the present invention involves the coacervation of polymeric cations and nucleic acids. Because this process depends on the interaction of the positively charged polymeric cations and the negatively charged nucleic acids it can be considered as a complex coacervation process.
• nucleic acids are present in the coacervation mixture at a concentration of between 1 ng/ml to 500 ⁇ g/ml. Desirably the nucleic acids are at least about 2-3 kb in length.
• Sodium sulfate is present at between 7 and 43 mM.
• Gelatin or other polymeric cation is present at between about 2 and 7% in the coacervation mixture. 99/36089
• An attractive nanosphere delivery system requires a delicate balance among factors such as the simplicity of preparation, cost effectiveness, nucleic acids loading level, controlled release ability, storage stability, and immunogenicity of the components.
• the gene delivery system described here may offer advantages compared to other particulate delivery systems, including the liposomal system.
• the problems of instability, low loading level, and controlled release ability are better resolved with the polymeric nanosphere systems.
• Gelatin has received increasing biologic use ranging from surgical tissue adhesive (Weinschelbaum, et al., 1992, Surgical treatment of acute type A dissecting aneurysm with preservation of the native aortic valve and use of biologic glue.
• nucleic acid molecules of greater than about 2 kb can be used, and nucleic acid molecules even greater than 10 kb may be used. Typically the nucleic acid will be greater than 300 bases, and typically geater than 0.5, 1, 2, 5, or 10 kb. Typically the nucleic acid molecule will be less than 200, 100, or
• the range of possible targets is dependent on the route of injection, e.g., intravenous or intraarterial, subcutaneous, intra-peritoneal, intrathecal, etc.
• the specificity of this delivery system is affected by the accessibility of the target to blood borne nanospheres, which in turn, is affected by the size range of the particles. Size of the particles is affected by temperature, component concentration, and pH in the coacervation mixture.
• the particles can also be size-fractionated, e.g., by sucrose gradient ultracentrifijgation. Particles with size less than 150 nanometers can access the interstitial space by traversing through the fenestrations that line most blood vessels walls.
• the range of cells that can be targeted is extensive.
• An abbreviated list of cells that can be targeted includes the parenchymal cells of the liver sinusoids, the fibroblasts of the connective tissues, the cells in the Islets of Langerhans in the pancreas, the cardiac myocytes, the Chief and parietal cells of the intestine, osteocytes and chondrocytes in the bone, keratinocytes, nerve cells of the peripheral nervous system, epithelial cells of the kidney and lung, Sertoli cells of the testis, etc.
• the targets for particles with sizes greater than 0.2 microns will be confined largely to the vascular compartment.
• the targetable cell types include erythrocytes, leukocytes (i.e. monocytes, macrophages, B and T lymphocytes, neutrophils, natural killer cells, progenitor cells, mast cells, eosinophils), platelets, and endothelial cells.
• the p43-clacZ vector still out-performed the pCI-clacZ vector, although the difference in responses were significantly smaller as compared to that observed at the low dose (1 mg).
• the neomycin resistance gene ('neo') on pCI-clacZ the only difference between pCI-clacZ and p43-clacZ is the AAV-ITR flanking the CMV/lacZ cassette. Since the neo gene does not contribute to lacZ gene expression, and it has no known immunological consequences in studies where it has been employed, these results suggest that the observed differences in anti- ⁇ -gal immunological responses may be attributable to the presence of the AAV-ITR. In vitro transfection studies using Lipofectamine reagents carried out on 293 cells showed no differences in the gene expression levels between these two vectors (data not shown), suggesting that the differences in immune response was not related to the level of gene expression.
• EXAMPLE 2 Immunological responses of pCI-clacZ plasmid DNA vectors. Anti ⁇ -gal immune responses elicited by two lacZ expression vectors differing primarily by the presence of the adeno-associated virus inverted terminal repeats (AAV-
• neomycin resistance gene ('neo') on pCI-clacZ
• the only difference between pCI-clacZ and p43-clacZ is the AAV-ITR flanking the CMV/lacZ cassette. Since the neo gene does not contribute to lacZ gene expression, and it has no known immunological consequences in studies where it has been employed, these results suggest that the observed differences in anti- ⁇ -gal immunological responses may be attributable to the presence of the AAV-ITR. In vitro transfection studies using Lipofectamine reagents carried out on 293 cells showed no differences in the gene expression levels between these two vectors (data not shown), suggesting that the differences in immune response was not related to the level of gene expression.
• Cytokine production lymphocytes from nanosphere-vaccinated mice were harvested, stimulated with ⁇ -gal protein, then analyzed for IL-4 and ⁇ -INF production.
• Standard ELISA assays carried out on 3-day cultured serum revealed that mice immunized with nanospheres with increasing IL-4 content produced proportionately more IL-4 upon re-stimulation with antigens. In these serum, a dose-related decrease in ⁇ -INF production was observed.
• T h 2 helper cells Because the production of IL-4 is associated with actively proliferating T h 2 helper cells, while ⁇ -INF production is associated with T h l helper cells, it is concluded that in mice vaccinated with nanosphere IL-4, the T h 2 helper cells response was potentiated while T h l helper cell response was depressed. This result, in combination with a dose-related potentiation of anti- ⁇ -gal antibody and a concomitant inhibition of CTL responses, is consistent with an IL-4 mediated immunological switch form a T h l -weighed response to that of a T h 2.
• EXAMPLE 5 The effects ofIL-4 given as nanosphere-encapsulated vs. free form.
• the effectiveness of IL-4 in potentiating an antibody response was examined in mice vaccinated with nanospheres containing IL-4 either as microencapsulated or free form.
• ELISA assays carried out on mouse serum at week 8 showed that those injected with nanospheres mixed with free IL-4 failed to elicit an enhancement in anti- ⁇ -gal antibody response (Fig. 3).
• mice vaccinated with nanosphere-encapsulated IL-4 generated a marked enhancement of antibody response.
• EXAMPLE 6 Potentiation of the CTL response using nanosphere-encapsulated IL-2 and ⁇ - INF.
• the feasibility of immune response modulation using nanosphere co- delivered cytokines was further demonstrated by examining the effects of nanosphere-encapsulated LL-2 and ⁇ -INF on the CTL responses. Mice immunized with a single injection of nanospheres containing p43-clacZ DNA alone (2 mg total DNA), with IL-2, or with IL-2 and ⁇ -INF were examined for the generation of anti- ⁇ -gal CTL responses at week 4. Mice vaccinated with nanosphere alone or naked DNA generated poor CTL response (Fig. 4). This was consistent with our previous studies which showed at least two or three immunizations were necessary for strong CTL responses.
• INF in nanosphere improved the anti- ⁇ -gal CTL response from 25% lysis (at an E:T ratio of 64: 1) to at least 65% with just a single immunization.
• EXAMPLE 7 Protection against a lethal challenge dose of live Ebola virus in nanosphere- vaccinated mice.
• Ebola virus a negative sense RNA virus, causes severe hemorrhagic fever leading to high mortality rates in infected humans.
• Ebo NP Ebola virus ribonucleocapsid NP protein
• Ebo GP envelope GP glycoprotein
• mice vaccinated with 0.5 or 3 ⁇ g of nanosphere DNA encoding Ebola NP or Ebola GP were challenged with 30 x LD 50 of mouse-adapted live Ebola Zaire strain. The survival rate was better with each antigen than with the vector control and was significantly greater with the higher dose (p ⁇ 0.01) (Fig. 5). A higher degree of protection was achieved with Ebola NP vaccination than with Ebola GP (90% vs 40%). The geometric means anti-GP or anti-NP antibody titers of immunized mice were low, 1 +/- 0.1 x 10 2 . Vaccination with DNA nanospheres was at least as efficient as the gene gun vaccination method. A parallel challenge experiment using the NP antigen given as PowerJect-XrTM gene gun DNA (3 ⁇ g dose, three total vaccinations), showed a protection level of 80%.
• G-CSF Granulocyte colony-stimulating factor

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Epidemiology (AREA)
• Immunology (AREA)
• Molecular Biology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Nanotechnology (AREA)
• Genetics & Genomics (AREA)
• Physics & Mathematics (AREA)
• Biomedical Technology (AREA)
• Microbiology (AREA)
• Optics & Photonics (AREA)
• Biotechnology (AREA)
• Mycology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Organic Chemistry (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicinal Preparation (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=22103306

Family Applications (1)

Country Status (6)

Families Citing this family (15)

Family Cites Families (3)

• 1999
  • 1999-01-15 WO PCT/US1999/000860 patent/WO1999036089A1/en not_active Application Discontinuation
  • 1999-01-15 CA CA002318498A patent/CA2318498A1/en not_active Abandoned
  • 1999-01-15 EP EP99901486A patent/EP1045699A1/de not_active Withdrawn
  • 1999-01-15 AU AU21172/99A patent/AU743226B2/en not_active Ceased
  • 1999-01-15 JP JP2000539862A patent/JP2002509116A/ja not_active Withdrawn
  • 1999-01-15 NZ NZ505876A patent/NZ505876A/xx unknown

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events