WO2004098636A2 - Vaccinating against infectious diseases using proteosomes - Google Patents
Vaccinating against infectious diseases using proteosomes Download PDFInfo
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- WO2004098636A2 WO2004098636A2 PCT/US2004/014236 US2004014236W WO2004098636A2 WO 2004098636 A2 WO2004098636 A2 WO 2004098636A2 US 2004014236 W US2004014236 W US 2004014236W WO 2004098636 A2 WO2004098636 A2 WO 2004098636A2
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- antigen
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- immunogenic composition
- liposaccharide
- immune response
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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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
- A61K39/02—Bacterial antigens
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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
- A61K39/02—Bacterial antigens
- A61K39/07—Bacillus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
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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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6068—Other bacterial proteins, e.g. OMP
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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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]
Definitions
- This disclosure relates generally to nasally-delivered vaccines for protecting against infectious disease or toxic agents and, in particular, to Proteosome- based compositions and methods of use thereof for treating or preventing various diseases associated with microbial pathogens or toxic agents, such as Yersiniapestis, Variola virus, ricin toxin and botulinum toxin.
- T lymphocytes are important for coordinating the adaptive immune response by controlling the release of effector molecules.
- T helper (Th) 1 cells produce interleukin-2 (IL-2), tumor necrosis factor alpha (TNF- ⁇ ), and interferon gamma (IFN- ⁇ ), which are important for the development of cell-mediated immunity (Mosmann et al., J. Immunol. 136: 2348, 1986; Street and Mosmann, FASEB J. 5: 171, 1991).
- Th2 cells produce IL-4, IL-13, IL-5, IL-9, IL-6 and IL-10, which are important for the stimulation of IgE production, mucosal mastocytosis, and eosinophilia (Mosmann et al.; Street and Mosmann). While a shift toward a Thl (type 1 response) or Th2 (type 2 response) phenotype may be important for the defense against pathogens, a shift in one direction or another can also be associated with the induction of autoimmune disease (Thl) or inflammatory disease (Th2).
- Thl autoimmune disease
- Th2 inflammatory disease
- vaccines have been developed and used to induce an adaptive immune response.
- Vaccines typically include an attenuated microbe or a microbial antigen (i.e., a microbe component such as a protein or nucleic acid) to activate a specific immune response.
- a microbial antigen i.e., a microbe component such as a protein or nucleic acid
- the ability of an antigen to induce a protective immune response in a host sometimes must be enhanced by formulating the antigen with an immunostimulant or an adjuvant.
- aluminum-based mineral salts (generically known as alum) have been the only adjuvants approved for human use. Historically, alum has been an effective adjuvant by significantly enhancing responses to adsorbed proteins when injected intramuscularly.
- alum adjuvants has come under scrutiny due to reports (1) that alum is a weak adjuvant for the new generation of vaccine antigens, which are based on highly purified recombinant proteins; (2) that alum can reportedly induce IgE antibodies and has been associated with allergic reactions in some vaccine recipients; and (3) that alum can induce persistent inflammatory responses which are manifested as an adverse reaction at injection sites. These adverse reactions have been found to be associated with the tendency of alum-adsorbed vaccines to polarize antigen-specific immune responses towards a type 2 phenotype (i.e., inflammatory response). Furthermore, alum-based vaccines have only been approved for use in humans by the injectable route.
- adjuvants include Freund's adjuvant (complete or incomplete), lipopolysaccharide (LPS, also referred to as endotoxin), and pertussis adjuvant (a saline suspension of killed Bordatella pertussis organisms).
- Freund's adjuvant consists of a mixture of mycobacteria in an oil/water emulsion.
- LPS stimulates the immune system by triggering an innate immune response, but LPS is too toxic to be a viable adjuvant.
- Molecules that are structurally related to endotoxin such as monophosphoryl lipid A (“MPL” or detoxified LPS), are being tested as adjuvants in clinical trials.
- MPL monophosphoryl lipid A
- Other potential adjuvants being investigated include saponins (see U.S. Patent Nos. 6,080,725; 6,262,029; and 5,977,081); amphipathic aldehydes (see U.S. Patent No. 6,649,172); proteosomes (see U.S. Patent Nos. 5,726,292 and 5,985,284; U.S. Patent Application Nos. 2001/0053368 and 2003/0044425); and cyclic aminoalkyl glucosaminide phosphates (see U.S. Patent No. 6,525,028).
- the present disclosure provides adjuvant formulations for antigens, in particular Proteosome-based adjuvants for formulation with various antigens for use in a variety of therapeutic settings, such as in treating or preventing, for example, infectious disease or the action of toxic agents.
- the present invention provides a method for eliciting an immune response to an antigen, comprising administering to a subject in need thereof a therapeutically effective amount of an immunogenic composition, wherein said immunogenic composition comprises Proteosomes, liposaccharide and an antigen.
- the present invention provides a method for treating or preventing a microbial infection, comprising administering to a subject in need thereof a therapeutically effective amount of an immunogenic composition, wherein said immunogenic composition comprises Proteosomes, liposaccharide and an antigen.
- the antigen of the immunogenic compositions comprises one or more from Bacillus anthracis, Chlamydia trachomatis, Staphylococcus aureus, Clostridium perfringens, or Yersiniapestis. In certain other embodiments, the antigen of the immunogenic compositions comprises one or more from Poxvirus, Alphavirus, Flavivirus, Arenavirus, Bunyavirus, Filovirus, or Picornavirus. In yet other embodiments, the antigen of the immunogenic compositions comprises one or more toxins. In still other embodiments, the antigen is recombinant. In yet other embodiments, the immunogenic composition comprises a plurality of different antigens. In a related embodiment, the plurality of antigens can comprise viral, bacterial, parasitic, toxin, or a combination thereof.
- the immunogenic composition is administered by a route selected from mucosal, enteral, parenteral, transdermal, transmucosal, nasal, or inhalation, or a combination thereof.
- the immunogenic composition elicits a protective immune response.
- the antigen comprises a Protective Antigen from Bacillus anthracis, filamentous hemagglutinin (FHA) of Boretella pertussis, an FI antigen or a V antigen from Yersiniapestis, an Fl- V antigen fusion protein antigen, or a combination thereof.
- the antigen comprises one or more antigens from a Variola virus or from a Vaccinia virus. .
- the antigen comprises one or more of botulinum toxin, ricin toxin, Staphylococcus enterotoxin B, or fragment or variant thereof.
- any of the aforementioned immunogenic compositions have a liposaccharide final content by weight as a percentage of Proteosome protein ranges from about 1% to about 500% in the immunogenic composition.
- the Proteosomes and liposaccharide are obtained from the same bacteria or from different bacteria, such as Gram-negative bacteria.
- the liposaccharide is from Gram-negative bacteria selected from Shigella species, Chlamydia species, Yersinia species, Pseudomonas species, Plesiomonas species, Escherichia species, Salmonella species, or a combination thereof.
- the Proteosomes are from Neisseria species.
- the immunogenic compositions of this disclosure have Proteosomes are from Neisseria meningitides and the liposaccharide is from Shigella flexneri.
- any of the aforementioned immunogenic compositions have a ratio of Proteosomes to antigen that ranges from about 5: 1 to about 1 :500, or the ratio is at least 1:2, 1:5, or 1:10.
- any of the aforementioned immunogenic compositions further comprise a pharmaceutically acceptable carrier, excipient or diluent.
- the instant disclosure provides a method for eliciting an immune response to an antigen, comprising administering to a subject in need thereof a therapeutically effective amount of an immunogenic composition, wherein said immunogenic composition comprises an adjuvant and an antigen, wherein said adjuvant comprises Proteosomes and liposaccharide and said adjuvant to antigen ratio ranges from about 1 :5 to about 1 :500 or is at least 1 :5, such that an immune response to said antigen is elicited.
- a method for treating or preventing a microbial infection comprising administering to a subject in need thereof a therapeutically effective amount of an immunogenic composition, wherein said immunogenic composition comprises an adjuvant and an antigen, wherein said adjuvant comprises Proteosomes and liposaccharide and said adjuvant to antigen ratio ranges from about 1 :5 to about 1 : 500 or is at least 1:5, such that a microbial infection is treated or prevented.
- the antigen is at least one of a bacterial, viral, or toxin antigen.
- Figure 1 shows serum IgG titers from mice immunized twice intranasally with 50, 20 or 5 ⁇ g of Fl-V with or without Protollin (2.5, 1 or 0.25 ⁇ g), or injected intramuscularly with 20 ⁇ g Fl-V adsorbed onto alum (Alhydrogel ). Half the mice were euthanized on day 35 post-primary immunization, and the other half on day 55. Titers are expressed as the geometric mean of specific antibody concentrations ( ⁇ g/ml for serum IgG; ng/ml for lung IgA and IgG) and 95% confidence limits are shown.
- Figure 2 shows lung IgG titers from mice immunized as described for
- Figure 3 shows lung IgA titers from mice immunized as described for Figure 1.
- Figures 4A and 4B show the survival of mice against challenge with lethal doses of aersolized Yersiniapestis.
- Mice immunized twice with 20 ⁇ g Fl-V intranasally with (0.25, 1, or 2.5 ⁇ g) or without Protollin, or intramuscularly adsorbed onto Alhydrogel were challenged by whole body exposure to 170 LD 50 of Y. pestis (A) 35 days or (B) 55 days post-primary immunization.
- all control mice that received Protollin only) died when challenged with Y. pestis, confirming the inoculum used was lethal.
- Figures 5 A and 5B show the survival of mice against challenge with lethal doses of aersolized Yersinia pestis. Mice immunized twice with 50 ⁇ g Fl-V intranasally with or without 1 ⁇ g of Protollin, or intramuscularly adsorbed onto
- Alhydrogel were challenged by whole body exposure to (A) 255 LD 50 of Y. pestis
- mice 35 and day 55 (different groups of mice) post-primary immunization with 170 lethal doses of aersolized Y. pestis.
- Mice were immunized twice with 5 ⁇ g Fl-V intranasally with (0.25, 1, or 2.5 ⁇ g) or without Protollin. All the control mice (those that received
- Protollin only died when challenged with 7 pestis, confirming the inoculum used was lethal.
- Figure 6 shows the amounts of IFN- ⁇ , TNF- ⁇ and IL-5 released from splenocytes stimulated in vitro with antigen Fl-V.
- Splenocytes were harvested from mice immunized intranasally with 50 ⁇ g of Fl-V with or without 1 ⁇ g Protollin, or injected intramuscularly with 20 ⁇ g of Fl-V adsorbed onto Alhydrogel , on day 35 after immunization.
- Figures 7A and 7B show serum IgG and lung IgA levels, respectively, in mice immunized nasally (on days 0 and 14) with 5 ⁇ g or 25 ⁇ g of recombinant Protective Antigen (rPA) from Bacillus anthracis admixed with (1 ⁇ g) or without
- rPA recombinant Protective Antigen
- FIGs 8 A and 8B show results of anthrax neutralization assays using serum and lung lavage fluid from mice immunized with rPA admixed with Protollin.
- Figure 9 shows serum IgG in mice immunized (on days 1, 21 and 31) with 15 ⁇ g of recombinant filamentous hemagglutinin (rFHA) from Bordetella pertusis admixed with liposome (intranasally, i.n.), Protollin (i.n.), or Alhydrogel
- rFHA recombinant filamentous hemagglutinin
- mice were immunized i.p. with QuadracelTM
- Figure 10 shows salivary IgA in mice immunized (on days 1, 21 and 31) with 15 ⁇ g of recombinant filamentous hemagglutinin (rFHA) from Bordetella pertusis formulated in the same compositions as described for Figure 9.
- rFHA recombinant filamentous hemagglutinin
- FIG 11 shows anti-rFHA serum IgG subclasses (IgG2a and IgGl) found in pooled sera from mice immunized as described for Figure 9.
- Figure 12 shows the level of Bordetella pertusis found in the lungs of immunized mice as described for Figure 9 when challenged with aerosolized B. pertusis eleven days after the final immunization (day 42). Lung counts were measured on days 1, 3, 7, and 14 post-aerosol challenge.
- Proiuvant refers to preparations of outer membrane proteins (OMPs, also known as porins) from Gram-negative bacteria, such as Neisseria species (see, e.g., Lowell et al., J. Exp. Med. 167:658, 1988; Lowell et al., Science 240:800, 1988; Lynch et al., Biophys. J. 45:104, 1984; Lowell, in "New Generation Vaccines” 2nd ed., Marcel Dekker, Inc., New York, Basil, Hong Kong, page 193, 1997; U.S.
- OMPs outer membrane proteins
- Patent No. 5,726,292; U.S. Patent No. 4,707,543) which are useful as a carrier or an adjuvant for immunogens, such as bacterial or viral antigens.
- Proteosomes are hydrophobic and safe for human use, and comparable in size to certain viruses. Proteosomes have the interesting ability to auto-assemble into vesicle or vesicle-like OMP clusters of 20-800 nm, and to noncovalently incorporate, coordinate, associate (e.g. , electrostatically or hydrophobically), or otherwise cooperate with protein antigens (Ags), particularly antigens that have a hydrophobic moiety.
- Ags protein antigens
- Proteosomes may be prepared, for example, as described in the art (see, e.g., U.S. Patent Nos. 5,726,292 or 5,985,284).
- LPS lipopolysaccharide or lipooligosaccharide
- Gram- negative bacteria such as Shigella flexneri or Plesiomonas shigelloides, or other Gram- negative bacteria (including Alcaligenes, Bacteroides, Bordetella, Borrellia, Brucella, Campylobacter, Chlamydia, Citrobacter, Edwardsiella, Ehrlicha, Enterobacter, Escherichia, Francisella, Fusobacterium, Gardnerella, Hemophillus, Helicobacter, Klebsiella, Legionella, Leptospira (including Leptospira interrogans), Moraxella, Morganella, Neiserria, Pasteurella, Proteus, Providencia, other Plesiomonas, Porphyromonas (including Porphyromonas (including Porphy
- the liposaccharide may be in a detoxified form (i.e., having the Lipid A core removed) or may be in a form that has not been detoxified. In the instant disclosure, liposaccharide need not be and preferably are not detoxified.
- the liposaccharide may be prepared, for example, as described in U.S. Patent Application Publication No. 2003/0044425.
- Proteosome:LPS or Protollin or IVX or IVX-908 refers to preparations of projuvant admixed as described herein with at least one kind of liposaccharide to provide an OMP -LPS composition (which can function as an immunostimulatory composition).
- the OMP-LPS adjuvant can be comprised of two of the basic components of Protollin, which include (1) an outer membrane protein preparation of Proteosomes (i.e., Projuvant) prepared from Gram-negative bacteria, such as Neisseria meningitides, and (2) a preparation of one or more liposaccharides.
- Protollin should also be understood to optionally include lipids, glycolipids, glycoproteins, small molecules, or the like, and combinations thereof.
- the Protollin may be prepared, for example, as described in U.S. Patent Application Publication No. 2003/0044425.
- Projuvant is generally used in conjunction with antigens that possess a , (naturally-occurring or modified) hydrophobic moiety (also referred to as a "foot” or “anchor”).
- Protollin (with exogenously added LPS), which although can be used with an antigen that do not contain a hydrophobic foot domain and which are largely hydrophilic in nature, or can be associated with a antigen containing a hydrophobic foot, or a combination thereof.
- Toxin or Toxic Agent refers to is a noxious or poisonous substance that is antigenic and is formed or elaborated as an integral part of a cell or tissue (e.g., endotoxin), as an extracellular product (e.g., exotoxin), or as a combination of the two, during the metabolism or growth of certain microorganisms or some higher plant and animal species.
- endotoxin a cell or tissue
- exotoxin extracellular product
- a toxin or toxic agent will be in the form of a "toxoid" so that the immunogenic compositions are not toxic or poisonous.
- a "toxoid” form is a toxin that has been treated so as to destroy the toxic property of the agent while still retaining antigenicity (i. e. , capable of eliciting anti-toxin or a neutralizing immune response).
- the "treatment" of a toxin to generate a toxoid includes chemical treatment (e.g., formaldehyde), mutations, fragments, and combinations thereof.
- Exemplary toxins include ricin toxin, botulinum toxin, alfatoxin, episolntoxin, Staphylococcus enterotoxin B, tetrodotoxin, snae venom toxin, diptheria toxin, cholera toxin, saxitoxin, trichothecene mycotoxins, etc.
- Immunogenic composition refers to any one or more compounds or agents capable of priming, potentiating, activating, stimulating, augmenting, boosting, amplifying, or enhancing an adaptive (specific) immune response, which may be cellular (T cell) or humoral (B cell), or a combination thereof.
- an adaptive immune response will be protective, neutralizing, or both.
- a representative example of an immunogen is a microbial antigen (such as one or more bacterial, viral, or parasite proteins of interest) or a non-microbial antigen (e.g., antigens obtained from other sources, such as from plants (ricin), from dinoflagellates (saxitoxin), and the like).
- Specific or Acquired or Adaptive immune response refers to a resistance mediated by a mammalian immune system resulting from previous exposure to an infectious agent or antigen.
- specific immunity can be a result of a naturally acquired (patent or latent) infection or from an intentional vaccination.
- specific immunity may be passively and transitorily acquired from the transfer of antibodies from another naturally (e.g., maternally inherited), or from transfer of antibodies or immune cells by intentional inoculation (e.g., antibody mediated immunotherapy).
- any concentration range, percentage range, ratio range or other integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
- the use of the alternative (e.g., "or”) should be understood to mean one, both, or any combination thereof of the alternatives.
- an indefinite article, such as “a” or “an” should be understood to refer to the singular and the plural of a noun or noun phrase.
- compositions, formulations, or compounds, or groups of compositions, formulations, or compounds, derived from the various components or combinations of the composition or sequences, structures, and substituents described herein, are disclosed by the present application to the same extent as if each composition or compound or group of compositions or compounds was set forth " individually. Thus, selection of particular sequences, structures, or substituents is within the scope of the present invention.
- the present invention provides compositions and methods for using and making immunogenic compositions to treat or prevent a variety of infectious diseases or to treat or prevent pathology associated with toxic agents.
- immunogenic compositions comprising an adjuvant (such as Proteosomes or Proteosomes with liposaccharide) and an antigen (such as microbial antigens or toxins) formulated with, for example, limited amounts of adjuvant and excess amounts of antigen.
- an adjuvant such as Proteosomes or Proteosomes with liposaccharide
- an antigen such as microbial antigens or toxins
- compositions comprising Proteosome:LPS or Proteosomes formulated with one or more microbial antigens.
- the compositions of the instant description are suitable for therapeutic uses such as treating or preventing a microbial infection by inducing a specific immune response, or eliciting an immune response capable of providing protective immunity (against infection) or neutralizing immunity (against toxic agents).
- PROTEOSOME-BASED VACCINE ADJUVANTS - ("PROJUVANT” AND “PROTOLLIN")
- the invention relates to immunogenic compositions that contain one or more antigen capable of eliciting an immune response.
- many antigens are poorly immunogenic unless formulated with an adjuvant.
- the only adjuvant licensed for use in humans is alum, but this adjuvant has limitations as described above.
- this adjuvant has limitations as described above.
- Despite the multiplicity of efforts to identify alternative adjuvants there remains a need for effective compositions to immunize individuals in need thereof, particularly against infectious diseases and toxic agents.
- the Proteosome-based adjuvant of the instant disclosure can be used in vaccine formulations, which may include a variety of antigen sources. For example, live attenuated microbes, killed microbes, split antigens, subunit antigens, toxic agent antigens, and combinations thereof. To maximize the effectiveness of a subunit vaccines, the antigens should be combined with a potent immunostimulant or adjuvant.
- exemplary adjuvants include alum (aluminum hydroxide, REHYDRAGEL ® ), aluminum phosphate, Proteosome adjuvant (see, e.g., U.S. Patent Nos. 5,726,292 and 5,985,284, and U.S. Patent Application Publication Nos. 2001/0053368 and
- virosomes liposomes with and without Lipid A, Detox (Ribi/Corixa), MF59, or other oil and water emulsions type adjuvants, such as nanoemulsions (see, e.g., U.S. Patent No. 5,716,637) or submicron emulsions (see, e.g., U.S. Patent No. 5,961,970), and Freund's complete and incomplete adjuvant.
- a particularly preferred adjuvant is a Proteosome or Protollin.
- Proteosomes are comprised of outer membrane proteins (OMP) from Neisseria species typically, but can be derived from other Gram-negative bacteria (see, e.g., Lowell et ah, J. Exp. Med. 167:658, 1988; Lowell et al., Science 240:800, 1988; Lynch et al, Biophys. J. 45:104, 1984; U.S. Patent No. 5,726,292; U.S. Patent No. 4,707,543).
- OMP outer membrane proteins
- Proteosomes have the interesting ability to auto-assemble into vesicle or vesicle-like OMP clusters of 20-800 nm, and to noncovalently incorporate, coordinate, associate, or otherwise cooperate with protein antigens (Ag), particularly antigens that have a hydrophobic moiety.
- Proteosomes are hydrophobic and safe for human use, and comparable in size to certain viruses.
- a protein e.g., antigen
- mixing of Proteosomes with a protein e.g., antigen
- a protein e.g., antigen
- Proteosomes may be prepared as described in the art or as described in U..S. Patent Application Nos. 2001/0053368 and 2003/0044425.
- Proteosomes are from Neisseria species, and more preferably from Neisseria meningitides. In certain embodiments, Proteosomes are not a carrier but are an adjuvant. In certain other embodiments, Proteosomes may be an adjuvant and an antigen delivery composition.
- an immunogenic composition comprises one or more antigens (e.g., bacterial, viral, parasitic, fungal, toxin antigens, and fragments or variants thereof) as described herein and an adjuvant, wherein the adjuvant comprises Projuvant or Protollin.
- the antigens can be from a recombinant source or comprise, for example, a (detergent) split antigen.
- the invention provides an immunogenic composition that further comprises an immunostimulant, such as a liposaccharide. That is, the adjuvant may be prepared to include an additional immunostimulant.
- the projuvant may be mixed with a liposaccharide to provide an OMP-LPS adjuvant.
- the OMP-LPS (Protollin) adjuvant can be comprised of two basic components.
- the first component is an outer membrane protein preparation of Proteosomes (i.e., Projuvant) prepared from Gram-negative bacteria, such as Neisseria meningitidis.
- the second component is a preparation of liposaccharide.
- the liposaccharide may be prepared as described in U.S. Patent Application Nos. 2001/0053368 and 2003/0044425. It is also contemplated that the second component may include lipids, glycolipids, glycoproteins, small molecules or the like, and combinations thereof.
- the two components of an OMP-LPS adjuvant may be formulated at specific initial ratios to optimize interaction between the components resulting in stable association and formulation of the components for use in the preparation of an immunogenic composition of the invention.
- the process generally involves the mixing of components in a selected detergent solution (e.g., Empigen BB,
- an immunogenic composition comprises one or more antigens as described herein and an adjuvant, wherein the adjuvant comprises a Projuvant (i.e., Proteosome) and liposaccharide.
- a Projuvant i.e., Proteosome
- the final liposaccharide content by weight as a percentage of the total Proteosome protein can be in a range from about 1% to about 500%, more preferably in range from about 10% to about 200%, or in a range from about 30% to about 150%.
- Another embodiment of the instant invention includes an adjuvant wherein the Proteosomes are prepared from Neisseria meningitides and the liposaccharide is prepared from Shigella flexneri or Plesiomonas shigelloides, and the final liposaccharide content is between 50% to 150% of the total Proteosome protein by weight.
- Proteosomes are prepared with endogenous lipooligosaccharide (LOS) content ranging from about 0.5% up to about 5% of total OMP.
- LOS endogenous lipooligosaccharide
- Another embodiment of the instant invention provides Proteosomes with endogenous liposaccharide in a range from about 12% to about 25%, and in a preferred embodiment between about 15% and about 20% of total OMP.
- the instant invention also provides a composition containing liposaccharide derived from any Gram-negative bacterial species, which may be from the same Gram-negative bacterial species that is the source of Proteosomes or is a different bacterial species.
- the Proteosome or Protollin to antigen ratio in the mixture is greater than 1:1, greater than 2:1, greater than 3 : 1 or greater than 4:1.
- the ratio can be as high as 8: 1 or higher.
- the ratio of Proteosomes to antigen of the immunogenic composition ranges from about 1:1 to about 1 :500, preferably the ratio is at least 1 :5, at least 1 : 10, at least 1 :20, at least 1 :50, or at least 1:100.
- the invention relates to immunogenic compositions that contain one or more antigens, which can be used to elicit an immune response, such as a protective immune response.
- the invention further relates to methods for treating and preventing microbial infections by administering to a subject one or more antigens or fragments or variant thereof, fusion protein, multivalent immunogen, or a mixture of such immunogens at a dose sufficient to elicit an immune response (cellular or humoral) specific for the antigen (which may be a protective immune response), as described herein.
- the antigens are preferably from clinically relevant microorganisms, such as bacteria, viruses (e.g., Influenza, Measles, Coronavirus), parasites (e.g., Trypansome, Plasmodium, Leishmania, pathogenic bacteria) fungi (e.g., Aspergillus, Candida, Coccidioides, Cryptococcus), and the like.
- viruses e.g., Influenza, Measles, Coronavirus
- parasites e.g., Trypansome, Plasmodium, Leishmania, pathogenic bacteria
- fungi e.g., Aspergillus, Candida, Coccidioides, Cryptococcus
- the antigen may be from bacteria, such as the causative agent of anthrax (Bacillus anthracis), plague (Yersinia pestis), stomach cancer (Helicobacter pylori), Q fever (Coxiella burneti ⁇ ), whooping cough (Bordetella pertussis, e.g., FHA), botulism (Clostridium botulinum, types A, B,C, D, and E), gas gangrene (Clostridium perfringens), tularemia (Francisella tularensis), meliodiosis (Pseudomonas pseudomalle ⁇ ), brucellosis (Brucella suis, B.
- bacteria such as the causative agent of anthrax (Bacillus anthracis), plague (Yersinia pestis), stomach cancer (Helicobacter pylori), Q fever (Coxiella burneti ⁇ ), whooping cough (Bordetella pert
- abortus B. canis, B. melitensis
- sexually transmitted diseases Cholamydia trachomatis or Neisseria gonorrhea
- toxin producing organisms such as Clostridium perfringens (epsilon toxin, one of twelve protein toxins produced by these gram positive, anaerobic spore-forming bacteria).
- Epsilon toxin is known as a performing polypeptide that causes potassium and fluid leakage from cells.
- the A through E types may produce one or more of Alpha, beta, epsilon or iota toxin.
- the epsilon toxin can be disseminated by ingestion of contaminated food or water, or by inhalation of aerosolized toxin.
- Staphylococcal enterotoxin B (SEB) Staphylococcal enterotoxin B (SEB) produced by Staphylococcus aureus.
- SEB is known to cause illness if inhaled and maybe the cause of food poisoning.
- the immune system of SEB intoxicated individuals is compromised and although not frequently associated with death, there is significant and wide spread incapacitating illness, such as high fever, chills, headache, muscle aches, inflammation of the lining of the eyelids, nausea, vomiting and diarrhea.
- isolated LPS may be an antigen, for example, LPS isolated from P. gingivalis, which may be formulated with Proteosomes for use in stimulating an immune response to P. gingivalis for treating or preventing gum disease, periodontal disease, tooth decay, and the like.
- Filoviruses include Marburg virus and Ebola virus
- Flaviviruses include Dengue virus, Yellow Fever virus, Japanese encephalitis virus, Russian spring-summer encephalitis virus, St.
- Arenaviruses include Junin (Argentine hemorrhagic fever) virus; Lassa fever virus, lymphocytic choriomeningitis virus, Machupo (Bolivian hemorrhagic fever) virus; examples of Bunyaviruses include Crimean-Congo hemorrhagic fever virus, Hantaan (Korean hemorrhagic fever) virus, Rift Valley fever virus; examples of Picornavirus includes Hepatitis A virus, polio virus; examples of orthomyxo virus includes influenza virus A, B, C (e.g., HA antigen), Thogoto virus; examples of alphaviruses include Chikungunya virus, Eastern encephalitis virus, Venezuelan encephalitis virus, Western encephalitis virus.
- Exemplary fungi include Candida albicans, Aspergillus spp., Coccidioides immites, Histoplasma capsulatum, Norcardia farcinica; and exemplary parasites include the causative agents of trypanosomiasis, leishmania, pneumonic plague, and lyme disease (Borrellia burgdorferi).
- antigens of interest may be toxic agents (e.g., ricin, botulinum).
- the antigens can be prepared recombinantly, synthetically, isolated from a biological source, recombinantly or chemically modified, and any combination thereof.
- Microbial antigens or fragments thereof can be prepared from a variety of biological sources, such as tissues of an infected subject or cultured cell lines. Primary isolation may be from, for example, peripheral blood cells or from respiratory secretions. Preferably, the isolated microbes are amplified on primary cell cultures or on established cell lines known in the art. In certain embodiments, the antigens or fragments thereof are isolated from intact microbial particles. As used herein, the term “isolated” or "derived from” means that the material is removed from its original or natural environment.
- nucleic acid molecules or polypeptide present in a living animal or cell, or virus is not isolated, but the same nucleic acid molecule or polypeptide is isolated when separated from some or all of the co-existing materials in the natural system.
- nucleic acid molecules could be contained in a vector and/or such nucleic acids could be part of a composition and still be isolated in that such a vector or composition is not part of the original nucleic acid molecule's natural environment.
- peptides or polypeptides, such as antigens or variants and fragments thereof may be either partially purified or purified to homogeneity.
- the present invention further provides methods for producing synthetic antigens, including fusion proteins.
- the immunogenic polypeptide components may be synthesized by standard chemical methods, including synthesis by automated procedure. In general, immunogenic polypeptides or peptides are synthesized based on the standard solid-phase Fmoc protection strategy with HATU as the coupling agent.
- the immunogenic peptide can be cleaved from the solid-phase resin with trifluoroacetic acid containing appropriate scavengers, which also deprotects side chain functional groups. Crude immunogenic peptide may be further purified using preparative reverse phase chromatography. Other purification methods, such as partition chromatography, gel filtration, gel electrophoresis, or ion-exchange chromatography may be used.
- the microbial antigens or fragments thereof of the invention may be recombinant, wherein a desired antigen is expressed from a polynucleotide that is operably linked to an expression control sequence (e.g., promoter) in a nucleic acid expression construct.
- an expression control sequence e.g., promoter
- host cells such as baculovirus and mammalian cell lines
- the antigens may be further fused or conjugated to an amino acid sequence, which sequence may be a hydrophobic anchor or foot to facilitate or otherwise enhance non-covalent association with Projuvant or Protollin.
- the antigen polypeptides may comprise any portion of such polypeptides that have at least one epitope capable of eliciting a protective immune response (cellular or humoral) against microbial infection.
- Immunogenic polypeptides of the instant invention may also be arranged or combined in a linear form, and each immunogen may or may not be reiterated, wherein the reiteration may occur once or multiple times.
- a plurality of different immunogenic polypeptides e.g., protein variants, or fragments thereof
- Plague also known as the Black Death, is a disease with a long history.
- the plague pandemic of the Middle Ages killed up to 30 million people in Europe.
- the present invention provides a method for eliciting an immune response against plague, comprising administering to a subject in need thereof a therapeutically effective amount of an immunogenic composition, wherein said immunogenic composition comprises Proteosomes, liposaccharide and one or more plague antigens, or variants or fragments thereof.
- the present invention provides a method for treating or preventing a plague infection, comprising administering to a subject in need thereof a therapeutically effective amount of an immunogenic composition, wherein said immunogenic composition comprises Proteosomes, liposaccharide and one or more plague antigens or variants or fragments thereof, wherein the plague infection is treated or prevented.
- the plague antigen used in the immunogenic composition can comprise an FI antigen or a V antigen from Yersinia pestis, or an Fl-V antigen fusion protein antigen, or a combination thereof.
- Protollin formulations comprising viral antigens that evoke a protective immune response against infection by, for example, variola virus causing smallpox disease in man is contemplated herein.
- Variola virus is the most notorious member of the virus family termed the Poxviridae and has historically had a significant and dramatic effect on human civilization.
- the Poxviridae is a large family of DNA viruses, which replicate in the cytoplasm of infected host cells. Infection with Variola virus kills 20-30% of those infected, and is highly contagious.
- Virus particles purified from infected host cells are composed of as many as 30 or more discreet polypeptide bands when resolved by polyacrylamide gel electrophoresis.
- the genomic sequence of the Copenhagen strain of vaccinia virus contains about 185 unique, nonoverlapping open reading frames (ORFs) of more than 65 amino acids.
- ORFs open reading frames
- the naming of the orthopoxvirus ORFs consists of using the Hindll restriction endonuclease fragment letter followed by the ORF number (from left to right) and L or R depending on the direction of the ORF.
- the complete and nearly identical sequences of the India and Bangladesh strains of variola virus also contain about 187 ORFs of which 150 have more than 90% sequence identity to those of vaccinia virus.
- One or more of he polypeptides encoded by vaccinia virus ORFs with 90% sequence identity to variola virus encoded polypeptides is assumed to mediate the immune response responsible for protection following vaccinia virus immunization.
- ORFs are known to encode extracellular virus-specific polypeptides that may be involved in eliciting a protective immune response.
- the immune response to one or more of these proteins is expected to elicit a humoral or cell mediated immune response which may protect from variola virus infection or reduce the pathological effects of infection.
- An infected host immune response preferentially involving a balanced or fine tuned TH1/Th2 cytokine profile is expected to be effective in preventing or treating variola virus infection and occurrence of smallpox disease.
- Cowpox and vaccinia virus have been widely used to vaccinate humans against smallpox infection because they produced less serious complications compared to infection by variola virus.
- smallpox has been eradicated from being a serious health threat, and continued wide spread vaccination of the general population has been halted.
- reemergence of a natural virus or the rapid and wide deployment of a smallpox virus as a biological threat agent, and the possible unwanted side effects resulting from vaccination with live virus creates the need for the development of alternative vaccines against small pox.
- the present invention provides a method for eliciting an immune response against small pox, comprising administering to a subject in need thereof a therapeutically effective amount of an immunogenic composition, wherein said immunogenic composition comprises Proteosomes, liposaccharide and one or more small pox antigens or a small pox viral particle extract.
- the present invention provides a method for treating or preventing a small pox infection, comprising administering to a subject in need thereof a therapeutically effective amount of an immunogenic composition, wherein said immunogenic composition comprises Proteosomes, liposaccharide and one or more small pox antigens or a small pox viral particle extract, wherein the small pox infection is treated or prevented.
- Proteosome-based vaccine compositions capable evoking an immune response to non-infectious, toxic or poisonous agents are contemplated herein.
- non-infectious agents include, for example, ricin toxin produced by the bean plant Ricinus communis, which is poisonous to humans, animals and insects.
- Ricin is a cytotoxin produced by castor beans, poisoning by inhalation or ingestion is caused by ricin.
- Ricin poisoning can, depending on dose, begin within a few hours of ingestion, causing abdominal pain, vomiting, diarrhea.
- Ricin poisoning if unchecked, develops within several days into severe dehydration and a decrease in blood pressure resulting in death within 3-5 days after ingestion (or inhalation).
- Ricin is a potent toxin that is fairly easily produced and can cause poisoning (toxicity) by inhalation as a small particle or following ingestion.
- Ricin is also quite stable.
- Symptoms of ricin poisoning include weakness, fever, cough and pulmonary edema followed by severe respiratory distress and death from hypoxemia.
- the histopathology of aerosol exposure is characterized by necrotizing airway lesions causing tracheitis, bronchitis and interstitial pneumonia with perivascular and aleveolar edema.
- ricin In rodent, ricin is more toxic by inhalation than by other routes of exposure. Symptoms of ricin poisoning in a significant number of geographically localized individuals may suggest exposure to aerosolized ricin. Cytotoxicity is mediated by the inhibition of protein synthesis through a mechanism that specifically and irreversibly inactivates the eukaryotic ribosome. Accordingly, ricin is also known a ribosome-inactivating protein (RIP).
- the RIPs such as ricin are generally made up of two disulfide bonded polypeptide chains. One polypeptide chain mediates binding to cells and uptake into the cytoplasm while the other chain functions to inhibit protein synthesis.
- the present invention provides a method for eliciting an immune response against ricin toxin, comprising administering to a subject in need thereof a therapeutically effective amount of an immunogenic composition, wherein said immunogenic composition comprises Proteosomes, liposaccharide and one or more ricin toxin antigens or fragments or variant thereof, wherein the ricin toxin antigen elicits a neutralizing antibody response.
- an immunogenic composition comprises Proteosomes, liposaccharide and one or more ricin toxin antigens or fragments or variant thereof, wherein the ricin toxin antigen elicits a neutralizing antibody response.
- proteosome vaccine formulations comprising ricin immunogenic polypeptides that modulate the host Thl/Th2 immune response, and inactivates or attenuates toxin activity, are contemplated herein.
- Botulism is caused by intoxication with any of the seven distinct neurotoxins produced by the bacillus, Clostridium botulinum.
- the botulinum toxins are proteins with molecular mass of approximately 150,000 KDa, which bind to the presynaptic membrane of neurons at peripheral cholinergic synapses to prevent release of acetylcholine and block neurotransmission.
- the blockade is most evident clinically in the cholinergic autonomic nervous system and at the neuromuscular junction. Symptoms of inhalation botulism may begin as early as 24-36 hours following exposure or as late as several days. Initial signs and symptoms include ptosis, generalized weakness, lassitude, and dizziness. Development of respiratory failure may be abrupt.
- the present invention provides a method for eliciting an immune response against botulinum toxin, comprising administering to a subject in need thereof a therapeutically effective amount of an immunogenic composition, wherein said immunogenic composition comprises Proteosomes, liposaccharide and one or more botulinum toxin antigens or fragments or variant thereof, wherein the botulinum toxin antigen elicits a neutralizing antibody response.
- the invention also relates to immunogenic compositions that contain one or more antigens, which can be used to elicit an immune response, such as a protective immune response.
- the invention further relates to methods for treating and preventing microbial infections or effects of toxic agents by administering to a subject one or more antigens or fragments thereof, fusion protein, multivalent immunogen, or a mixture of such antigens at a dose sufficient to elicit an immune response (cellular or humoral) specific for the antigen(s) (which may be a protective immune response), as described herein.
- Antigens and variants thereof, or a cocktail of such immunogens are preferably part of a composition comprising an adjuvant, such as Projuvant or Protollin, when used in the methods of the present invention.
- the immunogenic compositions of the instant invention may further comprise one or more additional microbial antigens, such as viral antigens, bacterial antigens, parasitic antigens, or a combination thereof.
- additional microbial antigens such as viral antigens, bacterial antigens, parasitic antigens, or a combination thereof.
- an immunogenic composition for plague or anthrax may also include antigens for rubella and mumps antigens.
- the immunogenic compositions may further include a pharmaceutically acceptable vehicle, carrier, diluent, or excipient, in addition to one or more antigen or fragment thereof and, optionally, other components.
- pharmaceutically acceptable carriers or other components suitable for use with an immunogenic composition of this invention include a thickening agent, a buffering agent, a solvent, a humectant, a preservative, a chelating agent, an additional adjuvant, and the like, and combinations thereof.
- the pharmaceutical composition of the instant invention may further include a diluent such as water or phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- diluent is PBS with a final phosphate concentration range from about 0.1 mM to about 1 M, more preferably from about 0.5 mM to about 500 mM, even more preferably from about 1 mM to about 50 mM, and most preferably from about 2.5 mM to about 10 mM; and the final salt concentration ranges from about 100 mM to about 200 mM and most preferably from about 125 mM to about 175 mM.
- the final PBS concentration is about 5 mM phosphate and about 150 mM salt (such as NaCl).
- any of the aforementioned immunogenic compositions comprising a cocktail of antigens and an adjuvant (such as Projuvant or Protollin) of the instant invention are preferably sterile.
- compositions can be sterilized either by preparing them under an aseptic environment or they can be terminally sterilized using methods available in the art.
- Many pharmaceuticals are manufactured to be sterile and this criterion is defined by the USP XXII ⁇ 1211>.
- Sterilization in this embodiment may be accomplished by a number of means accepted in the industry and listed in the USP XXII ⁇ 1211>, including gas sterilization, ionizing radiation or filtration. Sterilization may be maintained by what is termed aseptic processing, defined also in USP XXII ⁇ 1211>. Acceptable gases used for gas sterilization include ethylene oxide.
- Acceptable radiation types used for ionizing radiation methods include gamma, for instance from a cobalt 60 source and electron beam.
- a typical dose of gamma radiation is 2.5 MRad.
- filtration may be accomplished using a filter with suitable pore size, for example 0.22 ⁇ m and of a suitable material, for instance Teflon ® .
- the term "USP" refers to U.S. Pharmacopeia (see www.usp.org; Rockville, MD). Due to the fact that Proteosomes or OMP-LPS result in particles small enough that the immunogenic compositions of the invention can be filtered through a 0.8 ⁇ filter, a 0.45 ⁇ filter, or a 0.2 ⁇ filter.
- the immunogenic compositions of this invention are sterilized by filtration. This is highly advantageous as it is desirable to eliminate any complications by virtue of the presence of such contaminants.
- the present invention also pertains to methods for treating or preventing a microbial infection, comprising administering to a subject in need thereof an immunogenic composition comprising an adjuvant and one or more antigens, wherein the adjuvant comprises either Proteosomes or OMP-LPS.
- the immunogenic compositions of this invention may be used to elicit an immune response (cellular or humoral or both, which may favor a Type 1 or Type 2 cellular response).
- a subject suitable for treatment or for eliciting an immune response with an antigen formulation may be identified by well-established indicators of risk for developing a disease or well-established hallmarks of an existing disease.
- the terms “treat” and “ameliorate” refer to the therapeutic administration of a desired composition or compound, in an amount or for a time sufficient to treat, inhibit, attenuate, ameliorate, reduce, prevent or alter at least one aspect or marker of a disease, in a statistically significant manner.
- Infections that may be treated, ameliorated or prevented with an antigen of the subject invention include those caused by or due to any of the microbes described herein, whether the infection is primary, secondary, opportunistic, or the like.
- Other exemplary antigens are the toxins or variants thereof described herein.
- the antigens can be prepared recombinantly, synthetically, isolated from a biological source, recombinantly or chemically modified, and any combination thereof.
- Microbial antigens or fragments thereof can be prepared from a variety of biological sources, such as tissues of an infected subject or cultured cell lines. Primary isolation' may be from, for example, peripheral blood cells or from respiratory secretions. Preferably, the isolated microbes are amplified on primary cell cultures or on established cell lines known in the art. In certain embodiments, the antigens or fragments thereof are isolated from intact microbial particles.
- isolated or "derived from” means that the material is removed from its original or natural environment.
- nucleic acid molecules or polypeptide present in a living animal or cell, or virus is not isolated, but the same nucleic acid molecule or polypeptide is isolated when separated from some or all of the co-existing materials in the natural system.
- nucleic acid molecules could be contained in a vector or such nucleic acids could be part of a composition and still be isolated in that such a vector or composition is not part of the original nucleic acid molecule's natural environment.
- peptides or polypeptides such as antigens or variants and fragments thereof, may be either partially purified or purified to homogeneity.
- the present invention further provides methods for producing synthetic antigens, including fusion proteins.
- the immunogenic polypeptide components may be synthesized by standard chemical methods, including synthesis by automated procedure. In general, immunogenic polypeptides or peptides are synthesized based on the standard solid-phase Fmoc protection strategy with HATU as the coupling agent.
- the immunogenic peptide can be cleaved from the solid-phase resin with trifluoroacetic acid containing appropriate scavengers, which also deprotects side chain functional groups. Crude immunogenic peptide may be further purified using preparative reverse phase chromatography. Other purification methods, such as partition chromatography, gel filtration, gel electrophoresis, or ion-exchange chromatography may be used.
- the microbial antigens or fragments thereof of the invention may be recombinant, wherein a desired antigen is expressed from a polynucleotide that is operably linked to an expression control sequence (e.g., promoter) in a nucleic acid expression construct.
- an expression control sequence e.g., promoter
- host cells such as baculovirus and mammalian cell lines
- the antigens may be further fused or conjugated to an additional amino acid sequence, which may be a hydrophobic anchor or foot to facilitate or otherwise enhance non-covalent association of the antigen fusion with Projuvant or Protollin.
- the antigen polypeptides may comprise any portion of such polypeptides that have at least one epitope capable of eliciting a protective immune response (cellular or humoral) against a microbial infection.
- Immunogenic polypeptides of the instant invention may also be arranged or combined in a linear form, and each immunogen may or may not be reiterated, wherein the reiteration may occur once or multiple times.
- a plurality of different immunogenic polypeptides e.g., protein variants, or fragments thereof
- a mixture of at least one antigen with a Proteosome (projuvant) or Protollin is prepared in the presence of detergent, and reducing or removal of the detergent from the mixture by diafiltration/ultrafiltration leads to association (or coordination) of the antigens with the adjuvant.
- the Protollin (or Proteosome) to antigen ratio in the mixture ranges from about 1 : 1 to about 5:1. The ratio can be as high as 8 : 1 or higher.
- the Protollin (or Proteosome) to antigen ratio in the mixture ranges from about 1:1 to about 1:500, or in a range of about 1:2 to about 1:200, or in a range of about 1:2 to about 1:100, or in a range of about 1:5 to about 1:50, or in a range of about 1:2 to about 1 :20, or at least 1:5, 1:10, 1:20 or 1:100.
- the detergent- based solutions of the two components may contain the same detergent or different detergents, and more than one detergent may be present in the mixture subjected to ultrafiltration/diafiltration. Suitable detergents include Triton , Empigen BB, and Mega-10.
- detergents can also be used (e.g., octoglucoside).
- the detergents serve to solubilize the components used to prepare the composition.
- the use of a mixture of detergents may be particularly advantageous. This mixture is, of course, removed or the concentration is reduced by diafiltration/ultrafiltration prior to final formulation.
- the immunogenic compositions that contain one or more antigens and a Proteosome-based adjuvant of the invention may be in any form that allows for the composition to be administered to a subject, such as a human or animal.
- immunogenic compositions of the present invention may be prepared and administered as a liquid solution or prepared as a solid form (e.g., lyophilized), which may be administered in solid form, or resuspended in a solution in conjunction with administration.
- the immunogenic polypeptide compositions are formulated to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a subject or patient or bioavailable via slow release.
- compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a drop may be a single dosage unit, and a container of one or more compounds of the invention in aerosol form may hold a plurality of dosage units.
- any of the aforementioned pharmaceutical compositions comprising an immunogen or cocktail of immunogens (i. e. , anitgens) of the invention are in a container, preferably in a sterile container.
- the design of a particular protocol for administration, including dosage levels and timing of dosing are determined by optimizing such procedures using routine methods well known to those having ordinary skill in the art.
- the immunogenic composition is administered nasally.
- enteral is a route of administration in which the immunogenic composition is absorbed through the gastrointestinal tract or oral mucosa, including oral, rectal, and sublingual.
- parenteral describes administration routes that bypass the gastrointestinal tract, including intraarterial, intradermal, intramuscular, intranasal, intraocular, intraperitoneal, intravenous, subcutaneous, submucosal, and intravaginal injection or infusion techniques.
- transdermal/transmucosal is a route of administration in which the immunogenic composition is administered through or by way of the skin, including topical.
- nasal and “inhalation” encompass techniques of administration in which an immunogenic composition is introduced into the pulmonary tree, including intrapulmonary or transpulmonary.
- the compositions of the present invention are administered nasally.
- intranasally administered Fl-V combined with Protollin elicited comparable serum anti-Fl-V IgG titers to those induced by intramuscular injection of Fl-V formulated with Alhydrogel®.
- Protollin/Fl-V also elicited specific IgA and IgG responses as detected in lung lavage fluids.
- Immunized mice were subsequently challenged with aerosolized 7 pestis.
- Protollin/Fl-V immunized mice were 80% protected against high dose (255 LD 50 ) challenge as compared to 60% in the Alhydrogel/Fl-V group, while both vaccines were 90-100% protective against challenge with a lower dose (170 LD 50 ) of the organism.
- ELISAs were used to determine total and antigen-specific IgG, IgA and IgM titers in biosamples obtained from animals immunized with test immunostimulatory or immunogenic formulations according to the instant application, and controls. Samples include serum, nasal and lung mucosal washes in mice and rabbits. A standard ELISA protocol was used to determine linearity, specificity, sensitivity and reproducibility. Briefly, serial dilutions of the test samples (serum and lavage fluids) were added to the wells of ELISA plates coated with purified antigen, or derivatives thereof. Antigen-specific antibodies that adhere to the immobilized antigen were detected with animal and antibody subtype specific horse radish peroxidase (HRP) conjugated antibodies.
- HRP horse radish peroxidase
- HRP antibody was detected following incubation with a HRP substrate (TMB) and measuring absorbance at 490 nm.
- Antibody concentrations in the test samples were calculated from standard curves, run in parallel, using purified standard antibodies for IgA, IgM, IgG, IgGl and IgG2a. Where appropriate, specific antibody levels in mucosal wash fluid samples are standardized and normalized by expressing the specific antibodies detected in comparison to the total amount of IgA or IgG in the sample as measured in a separate assay. This technique has resulted in superior reproducibility and consistency in assays performed on nasal wash fluids collected in human clinical trials. ELISA data is expressed as geometric means at 95% confidence levels with statistical analysis using log-transformed data.
- mice and rabbits were collected to analyze the immune response to immunostimulatory or immunogenic formulations according to the instant application.
- nasal washes and lung lavage were performed by cannulating the trachea and 1 ml PBS supplemented with 0.1 % bovine serum albumin and protease inhibitors (General Use Protease Inhibitor Cocktail; Sigma Chemicals containing 0.2 mM AEBSR, l ⁇ g/mL aprotinin, 3.25 ⁇ M bestatin, 10 ⁇ M Leupeptin) were pumped upwards through the trachea. Fluid emerging from the nostrils was collected, vortexed and then centrifuged to remove tissue and cell debris.
- the supernatants were stored at -70°C until assayed.
- Lungs were lavaged twice with l.O mL protease supplemented PBS, the fluid was collected and vortexed, and the cell debris removed by centrifugation.
- Lung and nasal washes were stored at -70°C until being assayed. Rabbit mucosal fluids were similarly collected, adjusting the volumes as appropriate.
- cervical and mediastinal lymph nodes were surgically removed and mononuclear cells isolated and cultured for ELISPOT antibody, cytokine and CMI assays as described herein.
- Proteosome:LPS Protollin
- Fl-V plague antigen
- the Fl-V immune response was assessed by immunizing groups of twenty 6-8 week old female Swiss- Webster mice (Charles River, St-Constant, Quebec) on days 0 and 21. For immunization, freshly thawed aliquots of Protollin and Fl-V solutions were mixed ⁇ 16 hrs prior to immunization.
- mice were first lightly anesthetized by isoflurane inhalation, then 25 ⁇ l of vaccine or appropriate control samples (Protollin or Fl-V alone) was applied to the nares (12.5 ⁇ l per nostril) of each mouse.
- mice were immunized intramuscular (i.m.) with 25 ⁇ l ) of Fl-V adsorbed to 500mg of Alhydrogel injected into hind limbs. Control i.m. injections were also performed. Thirty-five and 55 days thereafter, 10 mice from each group were euthanized by asphyxiation with CO 2 and exsanguination. Serum was obtained and stored at -80°C until assay.
- Nasal wash and lung lavage samples were obtained and stored at -80°C until assay. Spleens were processed for in viti-o restimulation and assessment of released cytokines. The remaining 10 mice from each group were challenged on day 35 or 55 by inhalation of 170-250 LD50 of aerosolized 7 pestis (Colorado 92 strain) to assess protection. Mice were monitored for 28 days after challenge for determination of morbidity and mortality.
- Antibodies present in serum and lung lavage fluid samples obtained from mice immunized intranasally with two doses of Fl-V antigen formulated with Protollin were compared with those from mice immunized i.n. with Fl-V alone or with mice
- Non-adjuvanted Fl-V administered i.n., elicited IgA levels that were barely detectable; no secretory IgA was detected in samples from mice injected i.m. with Alhydrogel -adsorbed Fl-V.
- lung lavage samples also contained significant titers of Fl-V specific IgG, even though the titers represented only a small percentage of the serum titers (range 0.11% - 0.56%; median 0.175%).
- mice immunized intranasally with 5, 20 or 50 ⁇ g Fl-V plus 1 or 2.5 ⁇ g Protollin were 100% protected against death, as were the mice injected with Fl- V adsorbed onto Alhydrogel.
- Mice immunized i.n. with 5, 20 or 50 ⁇ g Fl-V and 0.25 ⁇ g Protollin were 90%, 100%o and 90% protected, respectively, while mice immunized i.n. with the same doses of Fl-V without Protollin were only 30%, 40% and 40%) protected, respectively. None of the control mice, which received Protollin alone, survived longer than 4 days post challenge.
- the observed protection was highly significant (P ⁇ 0.01 or better) compared to mice immunized with non-adjuvanted Fl-V (10-30%) protection) or the control group of mice in which there were no survivors.
- Mice immunized i.n. with 50 ⁇ g of Fl-V with or without l ⁇ g of Protollin, or injected with 20 ⁇ g of Fl-V adsorbed onto Alhydrogel were challenged on day 55 by whole body exposure to 255 LD50 aerosolized live 7 pestis. 80%, 20% and 60% respectively of the mice survived the high dose lethal challenge while control mice given Protollin alone all died.
- splenocytes from mice immunized by injection of Fl-V (20 ⁇ g) adsorbed onto Alhydrogel responded by secreting comparatively lower amounts of IFN- ⁇ and TNF- ⁇ , although a significant amount of IL-5 was detected.
- the cytokine profile elicited by i.n. administration of Protollin formulated (adjuvanted) with Fl-V antigen was consistent with eliciting a type 1 immune response, whereas the cytokine
- Protollin formulations with a recombinant Protective Antigen (rPA) of Bacillus anthracis were evaluated for the ability to induce an immune response capable eliciting a statistically significant reduction in PA-mediated macrophage killing, using a cell culture assay system.
- Mice immunized i.n. with 5 ⁇ g or 25 ⁇ g rPA (List Laboratories) admixed with 1 ⁇ g of Protollin (on days 0, 14) showed specific anti-rPA serum IgG and lung IgA levels significantly higher than those of mice that were i.n.
- An anthrax PA neutralization assay was also performed as described using serum and lung lavage fluid samples from these animals. For this assay, 2 x 105 RAW264.7 cells were plated out in sterile 96 well plates and incubated at 37°C for 24 h in 5% CO2. Serial dilutions of serum or lung lavage fluid samples (from PA immunized animals) were incubated with a PA solution for 1 h at 37°C, which was then added to the wells containing cells. Thereafter, a solution of LF was added to the wells and the plates incubated at 37°C in 5% CO 2 for 4 h.
- Nasal Protollin anthrax vaccines were made by admixing the anthrax PA antigens with soluble pre-formed Proteosome plus LPS (i.e., Protollin) prior to immunization. Both rPA and rPA-anchor antigens are evaluated with several versions of Protollin to determine the formulation(s) having the optimal Protollin to immunogenic antigen ratio. Control formulations include, for example, Protollin alone or mixed with one or more control antigens (as appropriate), such as recombinant streptococcal protein that contains or lacks a hydrophobic anchor sequence.
- Protollin containing LPS from different sources various Proteosome:LPS ratios, and various Protolli rPA antigen ratios are evaluated.
- rPA- anchor are formulated with Proteosomes with very low levels of LPS ( ⁇ 2% by weight).
- the Proteosome adjuvant preparations do not have exogenous LPS added.
- Proteosomes themselves have been used extensively in pre- clinical toxicity studies as well as in close to 500 persons in Phase 1 and Phase 2 safety, immunogenicity and experimental challenge trials of a Proteosome nasal influenza vaccine.
- LPS in terms of LPS bacterial source
- Protollin in terms of LPS source and Proteosome:LPS ratio
- Fermentation of the preferred bacterial source for LPS is followed by LPS purification and analysis.
- the selected purified LPS is then mixed with Proteosome OMP particles at the selected ratio to form a preferred Protollin.
- Protollin typically includes analyses of LPS content using KDO, NMR, and silver stain PAGE, for Proteosome OMP content using LC-MS, RP-HPLC, SDS-Page (C.
- T cell-derived cytokines are assessed on PA-re-stimulated purified or enriched T cells isolated from mouse spleen and/or mediastinal lymph nodes.
- Type 1 e.g., IFN- ⁇
- type 2 e.g., IL-4 and IL-5
- cytokines are determined by one or more methods including ELISA, ELISPOT and intracellular cytokines by fiow-cytometry.
- Proliferation of PA re-stimulated PBMC T cells from PBMC are used to evaluate CMI responses in rabbits due to lack of reagents specific for rabbit cytokines.
- T lymphocyte proliferation assays are used to measure the effect of immunization on clonal expansion and presence of memory lymphocytes in various animal models. Following animal sacrifice, mediastinal and cervical lymph nodes are removed surgically using standard techniques, the lymphocytes isolated and cultured and PA added to the isolated cells. Proliferation is measured by uptake of H -thymidine. Cells from animals immunized with sham vaccine are used as negative controls. Assay results are used to determine the effect of immunization on T cell differentiation in the lymph nodes involved in mucosal immunity, and correlated with efficacy of immunization as determined in anthrax challenge studies.
Abstract
Description
Claims
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CA002524485A CA2524485A1 (en) | 2003-05-05 | 2004-05-05 | Vaccinating against infectious diseases using proteosomes |
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EP (1) | EP1622641A2 (en) |
JP (1) | JP2006525373A (en) |
AU (1) | AU2004235815A1 (en) |
CA (1) | CA2524485A1 (en) |
WO (1) | WO2004098636A2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7255867B2 (en) | 2002-11-15 | 2007-08-14 | Id Biomedical Corporation Of Quebec | Vaccine |
EP1689433B1 (en) * | 2003-10-22 | 2010-06-30 | ID Biomedical Corporation of Quebec | Compositions and methods for activating innate and allergic immunity |
US20110159047A1 (en) * | 2006-09-08 | 2011-06-30 | Becton, Dickinson And Company | Stable powder formulations of alum-adsorbed vaccines |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001045639A2 (en) * | 1999-12-22 | 2001-06-28 | The Ohio State University Research Foundation | Methods for protecting against lethal infection with bacillus anthracis |
WO2002072012A2 (en) * | 2001-03-09 | 2002-09-19 | Id Biomedical Corporation Of Quebec | A novel proteosome-liposaccharide vaccine adjuvant |
-
2004
- 2004-05-05 CA CA002524485A patent/CA2524485A1/en not_active Abandoned
- 2004-05-05 JP JP2006514314A patent/JP2006525373A/en not_active Withdrawn
- 2004-05-05 EP EP04760725A patent/EP1622641A2/en not_active Withdrawn
- 2004-05-05 AU AU2004235815A patent/AU2004235815A1/en not_active Abandoned
- 2004-05-05 WO PCT/US2004/014236 patent/WO2004098636A2/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001045639A2 (en) * | 1999-12-22 | 2001-06-28 | The Ohio State University Research Foundation | Methods for protecting against lethal infection with bacillus anthracis |
WO2002072012A2 (en) * | 2001-03-09 | 2002-09-19 | Id Biomedical Corporation Of Quebec | A novel proteosome-liposaccharide vaccine adjuvant |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7255867B2 (en) | 2002-11-15 | 2007-08-14 | Id Biomedical Corporation Of Quebec | Vaccine |
EP1689433B1 (en) * | 2003-10-22 | 2010-06-30 | ID Biomedical Corporation of Quebec | Compositions and methods for activating innate and allergic immunity |
US8173140B2 (en) | 2003-10-22 | 2012-05-08 | Id Biomedical Corporation Of Quebec | Compositions and methods for activating innate and allergic immunity |
US8709447B2 (en) | 2003-10-22 | 2014-04-29 | Id Biomedical Corporation Of Quebec | Compositions and methods for activating innate and allergic immunity |
US20110159047A1 (en) * | 2006-09-08 | 2011-06-30 | Becton, Dickinson And Company | Stable powder formulations of alum-adsorbed vaccines |
Also Published As
Publication number | Publication date |
---|---|
AU2004235815A1 (en) | 2004-11-18 |
CA2524485A1 (en) | 2004-11-18 |
WO2004098636A8 (en) | 2005-08-18 |
WO2004098636A3 (en) | 2005-05-19 |
EP1622641A2 (en) | 2006-02-08 |
JP2006525373A (en) | 2006-11-09 |
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