WO2006078567A2 - Vaccins et ligands de ciblage des muqueuses permettant de faciliter l'administration de vaccins - Google Patents

Vaccins et ligands de ciblage des muqueuses permettant de faciliter l'administration de vaccins Download PDF

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WO2006078567A2
WO2006078567A2 PCT/US2006/001346 US2006001346W WO2006078567A2 WO 2006078567 A2 WO2006078567 A2 WO 2006078567A2 US 2006001346 W US2006001346 W US 2006001346W WO 2006078567 A2 WO2006078567 A2 WO 2006078567A2
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trefoil
mtl
vaccine
protein
fusion protein
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WO2006078567A3 (fr
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David W. Pascual
Massimo Maddaloni
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Montana State University
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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
    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • 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
    • A61K2039/55544—Bacterial toxins
    • 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
    • A61K2039/6075—Viral proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present disclosure relates to immune stimulatory compositions, including for instance vaccines, as well as the production of and methods of using and delivering them.
  • the mucous membranes are one of the largest organs of the body. Collectively, they cover a surface area of more than 400 square meters and comprise the linings of the gastrointestinal, urogenital and respiratory tracts (Ogra et ah, Clin Microbiol Rev. 14(2):430-445, 2001). Most mammalian pathogens invade the host through the mucosal surface. Thus, one of the important functions of the mucous is to keep invading pathogens out.
  • Mucosal immune system is divided into two sites.
  • foreign antigens are encountered and selectively taken up for the initiation of immune responses.
  • Mucosal inductive sites include the Peyer's patches of the gut-associated lymphoreticular tissue (GALT) and the Waldeyer's ring of tonsils and adenoids of the nasopharyngeal-associated lymphoreticular tissue (NALT), which collectively comprise the mucosal-associated lymphoreticular tissues (MALT) for continuous supply of memory B- and T-cells to mucosal effector sites.
  • GALT gut-associated lymphoreticular tissue
  • NALT nasopharyngeal-associated lymphoreticular tissue
  • antigens foreign proteins or materials referred to as antigens
  • antigens are sampled and used to trigger a host immune response. Once antigens are sampled and processed, they will induce memory lymphocyte responses in mucosal effector tissues. These mucosal effector sites ultimately determine the outcome of the immune response. How mucosal tissues encounter antigens determines the rate and the quality of the immune response, which ranges from antibody production and T-cell mediated immunity to mucosally induced tolerance.
  • mucosa forms the first layer that must be breached by most viral and bacterial pathogens in order to enter the body.
  • a vaccine should be administered directly to the mucosa where it can eliminate pathogens before they ever gain a foothold in the human host.
  • mucosal surfaces throughout the body are connected, there is an added advantage in that immunity induced at one mucosal site would also be effective at other unconnected locations.
  • Mucosal vaccines have several advantages over traditional systemic vaccines. For instance, they can be administered orally or nasally rather than via injection, making the vaccine simpler to administer and distribute. Mucosal immunity can be accomplished by facilitating vaccine uptake at mucosal inductive tissues.
  • Vaccines have traditionally been used as a means to protect against disease caused by infectious agents. However, with the advancement of vaccine technology, vaccines have been used in additional applications which include, but are not limited to, control of mammalian fertility, modulation of hormone action, and prevention or treatment of tumors.
  • the primary purpose of vaccines used to protect against a disease is to induce immunological memory to a particular pathogen. More generally, vaccines are needed to induce an immune response to specific antigens, whether they belong to a pathogen or are expressed by tumor cells. Both specificity and memory are generated by division and differentiation of B- and T-lymphocytes which have surface receptors specific for the antigen.
  • a vaccine In order for a vaccine to induce a protective immune response, it should fulfill the following requirements: 1) include the specific antigen(s) or fragment(s) thereof that will be the target of protective immunity following vaccination; 2) present such antigens in a form that can be recognized by the immune system, i.e., a form resistant to degradation prior to immune recognition; and 3) activate antigen presenting cells to present the antigen to CD8 + T cells or CD4 + T-cells, which in turn induce B -cell differentiation and other immune effector functions .
  • vaccines contain suspensions of attenuated or killed microorganisms, such as viruses or bacteria, incapable of inducing severe infection by themselves, but capable of counteracting the unmodified (or virulent) species when inoculated into a host.
  • vaccine has been extended to include essentially any preparation intended for active immune prophylaxis (e.g., preparations of killed microbes of virulent strains or living microbes of attenuated (variant or mutant) strains; microbial, fungal, plant, protozoal, or metazoan derivatives or products; synthetic vaccines).
  • vaccines include, but are not limited to, cowpox virus for inoculating against smallpox, tetanus toxoid to prevent tetanus, whole inactivated bacteria to prevent whooping cough (pertussis), polysaccharide subunits to prevent streptococcal pneumonia, and recombinant proteins to prevent hepatitis B.
  • Attenuated vaccines are usually immunogenic, their use has been limited because their efficacy generally requires specific, detailed knowledge of the molecular determinants of virulence. Moreover, the use of attenuated pathogens in vaccines is associated with a variety of risk factors that in most cases prevent their safe use in humans.
  • the problem with synthetic vaccines is that they are often non- immunogenic or non-protective.
  • the use of available adjuvants to increase the immunogenicity of synthetic vaccines is often not an option because of unacceptable side effects induced by the adjuvants themselves.
  • Adjuvants are defined as any substance that increases the immunogenicity of admixed antigens. The best adjuvants are those that mimic the ability of pathogens to activate the innate immune system.
  • the present disclosure is related to molecules that bind to the mucosal epithelium, referred to as mucosal targeting ligands (MTLs), which include viral attachment proteins. These molecules can mimic viral vector delivery in the absence of viral-like particles, potentiating host immunity to protein, DNA, carbohydrate, or lipid vaccines, or therapeutics.
  • MTLs mucosal targeting ligands
  • One provided example shows that protein expressed from a genetic fusion between a relevant vaccine antigen derived from ⁇ -trefoil domain of the heavy chain (Hc) of Clostridium botulinum strain A toxin (BoNT/A) and the binding domain of Adenovirus type 2 fiber protein elicits an immune response similar to that achieved using the ⁇ -trefoil domain of Hc in combination with the adjuvant, cholera toxin.
  • Hc heavy chain
  • BoNT/A Clostridium botulinum strain A toxin
  • adenovirus fiber protein or derivatives thereof and other structurally related proteins can act as ligands for the mucosal epithelium, as can other molecules.
  • embodiments disclosed herein exclude the need for exogenous co-delivery of mucosal adjuvants by effective targeting of vaccine antigens either genetically or chemically attached to MTLs.
  • Such delivery platform using a MTL has practical applications in, for instance, vaccine delivery, delivery of desired mucosal adjuvants, delivery of immune toleragens, and delivery of therapeutics for autoimmune diseases.
  • MTLs such as viral attachment proteins or proteins or peptides derived therefrom
  • MTLs can be used as a mucosal delivery platform and can act to supplant or reproduce vaccine adjuvancy when administered as a genetic or chemical fusion to other unrelated antigens.
  • the presence of such a targeting molecule (MTL) redirects the vaccine (or other passenger molecule) to mucosal tissues, where for instance the vaccine will be retained for processing and presentation to B and T lymphocytes.
  • MTL vaccine delivery is that in some instances, adjuvants may not be needed, or the amount of adjuvant required will be lessened.
  • the MTL can also be applied to adjuvant delivery to nonspecif ⁇ cally activate the innate immune system.
  • MTL redirection can also be adapted for vaccination for autoimmune diseases in which an objective is to tolerize the host against immune reactivity towards "self, or to tolerize the host/subject against an allergen.
  • the present disclosure provides compositions and methods for use in subjects, including for instance humans, livestock, and wildlife.
  • the present disclosure provides the ability to produce previously unknown proteins using the cloned nucleic acid molecules derived from any given infectious agent (e.g., bacterial, fungal, viral, or parasitic agents).
  • infectious agent e.g., bacterial, fungal, viral, or parasitic agents.
  • the MTL also can be adapted by genetic, chemical, or en2ymatic modifications for the delivery of proteins, nucleic acids, carbohydrates, or lipids as a vaccine or as a toleragen.
  • MTLs targeting molecules
  • vaccines or other passenger molecules
  • a vaccine for botulinum toxin A can supplant the use of a mucosal adjuvant because the same secretory (S)-IgA antibody titers are obtained when the Hc- ⁇ tre- MTL is administered without or with the potent mucosal adjuvant, cholera toxin (CT).
  • S secretory
  • CT cholera toxin
  • the delivery platform (MTL) (e.g., vaccine delivery platform) of the present disclosure acts as a potent adjuvant or replaces the need for adjuvants, for instance when the MTL is administered mucosally.
  • the described vaccine vehicle can be a specific protein, chemical, or lectin molecule that serves as an epithelial cell ligand to facilitate entry into mucosal inductive tissues.
  • recombinant proteins that contain a ⁇ - trefoil structure for a specific antigenic type; these can be used as an immune stimulant (vaccine) against that antigenic type. Since this ⁇ -trefoil structure is conserved among the seven BoNTs, immunization with one is expected to stimulate cross-protection against other BoNTs.
  • the recombinant protein contains more than one ⁇ -trefoil structure from two or more antigenic types. Such proteins (and nucleic acids encoding them) are useful for specifically stimulating immune response in a subject to multiple antigenic types.
  • nucleic acids encoding ⁇ -trefoil domains and in particular the optimized encoding sequences provided herein (see, e.g., SEQ ID NOs: 7, 9, 11, 13, 15, and 17, as well as nucleic acids 670 to 1353 of SEQ ID NO: 5).
  • FIG. 1 Schematic representation of a MTL used to deliver the ⁇ -trefoil domain to botulinum A (BoNT/A) heavy chain (Hc).
  • FIG. 2 Intranasal immunization with a mucosal targeting ligand (MTL; Ad2 fiber protein) genetically fused to Hc/A trefoil stimulates elevated (A) copro-IgA and (B) serum IgG antibodies to Hc ⁇ -trefoil when co-administered with or without the mucosal adjuvant, cholera toxin (CT).
  • MTL mucosal targeting ligand
  • CT cholera toxin
  • BALB/c mice were dosed three times with 50 ⁇ g of Hc( ⁇ -trefoil)-MTL or Hc ⁇ -trefoil and antibody responses were measured by Hc ⁇ -trefoil-specific ELISA. In the absence or presence of CT, elevated mucosal IgA were obtained.
  • Hc ⁇ -trefoil by itself stimulated poor IgG and S-IgA responses, and these S-IgA responses were delayed even when CT was used.
  • FIG. 3 Intranasal immunization with Hc ⁇ Tre-Ad2 fiber protein + CT enhances IgA (Figure 3A) and IgG ( Figure 3B) antibody-forming cells (AFC) 7 wks after primary immunization when compared to mice similarly immunized with Hc ⁇ Tre + CT.
  • Intestinal lamina limbal (iLP), Peyer's patches (PP), spleen, submaxillary gland (SMG), nasal passages (NP), and nasal-associated lymphoid tissue (NALT) were harvested and analyzed for Hc- ⁇ tre-specific and total IgA AFC (top) and Hc- ⁇ tre-specific and total IgG AFC (bottom).
  • ⁇ -trefoil/A protein is immunogenic and able to elicit neutralizing antibodies despite the inability to recognize BoNToxoid/A.
  • Figure 5B The supportive Th cell responses were measured by T cell ELISPOT method and showed that IL-4 and EFN- ⁇ were induced in the head and neck lymph nodes.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids, as defined in 37 C.F.R. ⁇ 1.822. Only one strand of each nucleic acid sequence is shown, but as appropriate in context the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ED NO: 1 shows the nucleic acid sequence encoding adenovirus 2 fiber protein (HAD278923).
  • SEQ ID NO: 2 shows the protein sequence of adenovirus 2 fiber protein.
  • SEQ ID NO: 3 shows the nucleic acid sequence encoding reovirus type 3 sigma 1 (haemagglutinin) (RET3S1).
  • SEQ ID NO: 4 show's the amino acid sequence of reovirus type 3 sigma 1 (haemagglutinin)
  • SEQ ID NO: 5 shows the nucleic acid sequence encoding the fusion protein comprising adenovirus type 2 fiber protein (amino acids 378-582) genetically fused to the ⁇ trefoil of BoNT/A.
  • Nucleic acids 715 to 1353 encode BoNT/A ⁇ -trefoil; this sequence has been optimized for expression as discussed herein.
  • SEQ ID NO: 6 shows the amino acid sequence of the fusion protein comprising adenovirus type 2 attachment protein (amino acids 378-582) fused to ⁇ trefoil of BoNT/A.
  • SEQ ID NO: 7 shows the nucleic acid sequence encoding synthetic (optimized) BoNT/B ⁇ -trefoil (CLOBOTB).
  • SEQ ID NO: 8 shows the amino acid sequence of synthetic BoNT/B ⁇ -trefoil (CLOBOTB).
  • SEQ ID NO: 9 shows the nucleic acid sequence encoding synthetic (optimized)
  • SEQ ID NO: 10 shows the amino acid sequence of synthetic BoNT/C ⁇ -trefoil (CBCPHGCl).
  • SEQ ID NO: 11 shows the nucleic acid sequence encoding synthetic (optimized) BoNT/D ⁇ -trefoil (S49407).
  • SEQ ID NO: 12 shows the amino acid sequence of synthetic BoNT/D ⁇ -trefoil (S49407).
  • SEQ DD NO: 13 shows the nucleic acid sequence encoding synthetic (optimized) BoNT/E ⁇ -trefoil (CBNTTE).
  • SEQ ID NO: 14 shows the amino acid sequence of synthetic BoNT/E ⁇ -trefoil (CBNTTE).
  • SEQ ID NO: 15 shows the nucleic acid sequence encoding synthetic (optimized) BoNT/F ⁇ -trefoil (CLONEUTOXF).
  • SEQ ID NO: 16 shows the amino acid sequence of synthetic BoNT/F ⁇ -trefoil
  • SEQ ID NO: 17 shows the nucleic acid sequence encoding synthetic (optimized) BoNT/G ⁇ -trefoil (CBBONTG).
  • SEQ DD NO: 18 shows the amino acid sequence of synthetic BoNT/G ⁇ -trefoil (CBBONTG).
  • SEQ ID NO: 19 shows the nucleic acid sequence encoding adenovirus 16 fiber protein (AX034843).
  • SEQ ID NO: 20 shows the amino acid sequence of adenovirus 16 fiber protein.
  • SEQ ID NO: 21 shows the nucleic acid sequence encoding adenovirus 35 fiber (fiber) protein (30827 to 31798 of BK005236).
  • SEQ ID NO: 22 shows the amino acid sequence of adenovirus 35 fiber protein (30827 to 31798 of BK005236).
  • SEQ ID NO: 23 shows the nucleic acid sequence encoding adenovirus 37 fiber protein (x94484).
  • SEQ DD NO: 24 shows the amino acid sequence of adenovirus 37 fiber protein
  • SEQ DD NO: 25 shows the nucleic acid sequence (V00383) encoding ovalbumin.
  • SEQ DD NO: 26 shows the amino acid sequence of ovalbumin.
  • the present disclosure is based, in part, on the ability of the described formulations to target mucosal epithelium.
  • Previously known vaccines introduced onto mucosal surfaces following nasal, oral, rectal, or vaginal application are lost due to their inability to be retained on the epithelia for sufficient time to be taken up by antigen-presenting cells. Consequently, the host does not become vaccinated.
  • targeting molecules are provided that will bind the mucosal epithelia which we term as mucosal targeting ligands (MTLs).
  • MTLs mucosal targeting ligands
  • the genetic, chemical, or enzymatic coupling or association of MTLs to vaccines will enable vaccination of the host to produce elevated mucosal S-IgA responses and mucosal and systemic IgG responses.
  • a binding event is required to occur, in which the MTL binds to the host epithelia that eventually will result in vaccination. These events include the uptake of the vaccine and ultimately presented to host B and T lymphocytes
  • the present disclosure in certain embodiments specifically provides vaccine compounds useful in treating or preventing infections by pathogens that invade the host through the mucosal surface, e.g., viral influenza, human immunodeficiency virus, bacterial agents and toxins (as described here using a component of the heavy chain from botulinum toxin A), and parasitic and fungal infectious agents.
  • MTLs can be adapted for the delivery of adjuvants to stimulate the host innate immune system or the delivery of auto- antigens to tolerize the host against autoimmune diseases.
  • compositions ⁇ e.g., vaccines
  • proteins that contain at least one ⁇ -trefoil domain from a botulinum heavy chain of serotype A, B, C, D, E, F, or G.
  • fusions proteins that comprise a mucosal targeting ligand (MTL) and at least one ⁇ -trefoil component of botulinum neurotoxin A, B, C, D, E, F, and/or G.
  • MTL mucosal targeting ligand
  • any of such proteins may contain two or more ⁇ -trefoil domains, for instance from different serotypes.
  • Compositions comprising one or more ⁇ -trefoil containing proteins (with or without a MTL) are also provided, including for instance pharmaceutical compositions.
  • isolated fusion proteins comprising a mucosal targeting ligand (MTL), which MTL is a viral protein which targets the mucosa, or a fragment thereof that retains the ability or function of such targeting, and an antigen.
  • MTL mucosal targeting ligand
  • representative antigens comprise at least one ⁇ -trefoil component of a botulinum neurotoxin (BoNT), for instance a ⁇ -trefoil component is from BoNT A, B, C, D, E, F, and/or G.
  • the antigen of the fusion protein is a composite or complex antigen (designed to contain a selection of epitopes), which is capable of stimulating immunity to a number of different targets.
  • complex antigens can contain ⁇ -trefoil domains from two or different serotypes of botulinum.
  • One such resultant fusion protein comprises an MTL and the following as antigen: BoNT/A ⁇ -trefoil, BoNT/B ⁇ -trefoil, BoNT/C ⁇ -trefoil, BoNT/D ⁇ -trefoil, BoNT/E ⁇ - trefoil, BoNT/F ⁇ -trefoil, and BoNT/G ⁇ -trefoil.
  • the MTL comprises an adenoviral fiber protein, a reoviral protein ⁇ l, a GP2 of Ebola virus, retroviral envelope glycoprotein, a F protein of Respiratory Syncytial Virus, a rabies G protein, a HAl of influenza virus, a HA2 of influenza virus, or a fragment of any one of these that retains mucosal targeting function.
  • the MTL is recombinantly coupled or associated to the antigen in the fusion protein.
  • the MTL is chemically coupled or associated to the antigen.
  • the MTL is enzymatically coupled or associated to the antigen.
  • compositions for stimulating mucosal immunity which compositions comprise at least one fusion protein as described.
  • compositions for providing in a subject ⁇ e.g., a human or non-human animal) immunity against a disease are also described, which compositions include a fusion protein in which the antigen is specific for the disease.
  • the MTL-containing fusion proteins also are useful as vaccine delivery systems for eliciting a protective immune response in subject against a disease.
  • this vaccine delivery system in some embodiments is used for prevention of or therapeutic control of a specific infectious agent. In other embodiments, it is used for prevention of or therapeutic control of an autoimmune disease or allergy.
  • a method of preparing a vaccine delivery system comprising a vaccine antigen attached to a MTL.
  • the vaccine delivery system is in some instances used to deliver adjuvant to stimulate host innate immune system.
  • Also described are methods of increasing vaccine retention in a subject which method comprises administering to the subject an immune stimulatory composition comprising a fusion protein comprising a MTL and an antigen, wherein the MTL of the fusion protein increases vaccine retention in the subject.
  • the composition is administered in the form of nasal drops, or a nasal spray or dry powder for inhalation, formulated for oral ingestion, or absorption via vaginal, rectal, or genitor-urinary tract mucosa.
  • Vaccines comprising at least on fusion protein described herein, or comprising a nucleic acid encoding such fusion protein, wherein the vaccine does not comprise an adenoviral vector are also contemplated.
  • Immune stimulatory compositions comprising at least one ⁇ -trefoil domain from the heavy chain of botulinum serotype A, B, C, D, E, F, or G are described. In some embodiments, these compositions are vaccines. Also provided are nucleic acid sequences optimized for expression of such a ⁇ -trefoil domain. For instance, examples of such nucleic acid sequences are shown in SEQ ID NOs: 1, 9, 11, 13, 15, and 17, as well as residues about 670 to 1353 of SEQ E) NO: 5, and residues about 715 to 1353 of SEQ ID NO: 5. All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference (including those so indicated).
  • GALT gut-associated lymphoreticular tissues iLP, small intestinal lamina propria
  • OVA-p ⁇ l OVA genetically fused to protein sigma one of rebvirus
  • the term "adjuvant” refers to a substance sometimes included in a vaccine formulation to enhance or modify the immune-stimulating properties of a vaccine.
  • the term “antibody” refers to a large Y shaped protein molecule made by B-cells of the immune system which very selectively binds to other specific protein molecules called antigens.
  • the term "antigen" refers to a foreign substance that that when introduced into the body triggers an immune system response, resulting in production of an antibody as part of the body's defense against disease.
  • DNA vaccine refers to a eukaryotic expression system encoding the molecular machinery for the expression of the subunit vaccine encoded in plasmid nucleic acids.
  • expression refers to the vaccine vector which is responsible for producing the vaccine.
  • the term "immunization” refers to a process by which a person or animal becomes protected against a disease; the process of inducing immunity by administering an antigen (vaccine) to allow the immune system to prevent infection or illness when it subsequently encounters the infectious agent.
  • an antigen vaccine
  • mucosal means any membrane surface covered by mucous.
  • MTL refers in many embodiments to a viral protein or adhesin that specifically binds to the epithelia to enable or enhance uptake, for instance uptake of a passenger protein domain attached to the MTL, such as an antigen, which optionally can be used as a vaccine or toleragen.
  • MTLs are not restricted only to proteins, but can a protein derivatized with carbohydrates and/or lipids. Likewise, it is contemplated that carbohydrate, lipid, or nucleic acids found to bind to the epithelia can also be included as mucosal targeting ligands.
  • the term "vaccine” means anything intended for immune stimulation and/or active immune prophylaxis of any cell, tissue, organ or whole organism. This term includes, but is not limited to, a protein or the protein encoded by nucleic acids for the expressed vaccine, optionally conjugated with carbohydrates, or lipids.
  • a vaccine works by inducing the vaccinated host to produce antibodies or cell- mediated immune responses specific for the vaccine. The production of these antibodies or cell-mediated immune responses will protect the host upon subsequent exposure to the infectious agent.
  • any given antigen or any given gene encoded by nucleic acids that can be used for eliciting a host response can be enhanced through effective targeting mediated by mucosal targeting ligands.
  • One embodiment of the present disclosure is based in part on the unexpected discovery that vaccines comprising chimeric constructs of a MTL and an antigen (e.g., a ⁇ - trefoil domain to botulinum A heavy chain) exhibit the essential immunological characteristics or properties expected of a conventional vaccine supplemented with an adjuvant.
  • the present disclosure is based on the finding that the described vaccine for botulinum toxin A (Hc- ⁇ -trefoil) can supplant the use of a mucosal adjuvant because the same secretory (S)-IgA antibody titers are obtained when the Hc- ⁇ Tre-MTL is administered without or with the potent mucosal adjuvant, cholera toxin (CT).
  • Hc- ⁇ -trefoil the described vaccine for botulinum toxin A
  • CT cholera toxin
  • Yet another aspect of the present disclosure is based on the finding that the intranasal immunization with a mucosal targeting ligand (MTL) genetically fused to Hc/A ⁇ -trefoil stimulates elevated (A) copro-IgA and (B) serum IgG antibodies to Hc ⁇ -trefoil when co-administered with or without the mucosal adjuvant, CT.
  • MTL mucosal targeting ligand
  • ⁇ -trefoil vaccine is protective against intoxication with native botulinum neurotoxin A. It is believed that the provided vaccines also will induce cross-reactive antibodies against other serotypes for botulinum neurotoxins.
  • an "antigen” is any substance that induces a state of sensitivity and/or immune responsiveness after any latent period (normally, days to weeks in humans) and that reacts in a demonstrable way with antibodies and/or immune cells of the sensitized subject in vivo or in vitro.
  • antigens include, but are not limited to, infectious agents including viruses, fungi and bacteria; cellular antigens including cells containing normal transplantation antigens and/or malignant transplantation antigens; RR Rh antigens; and antigens specific to particular cells or tissues.
  • antigens include, but are not limited to, antigens selected from the group consisting of Herpes Simplex Virus type 1, Herpes Simplex Virus type 2, Human cytomegalovirus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E, Respiratory Syncytial Virus, Human papilloma Virus, Influenza Virus, Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, Plasmodium and Toxoplasma, Prostate-specific antigen (PSA), gp 120, p24, gp41 , gp 160, Envl 0 and Envl 3 (HIV-I and HIV-2).
  • PSA Prostate-specific antigen
  • Antigens induce immune enhancement, which refers to any increase in an organism's capacity to respond to foreign antigens, which includes an increased activity and ability of the immune system to detect and destroy foreign antigens, in those cells primed to attack foreign antigens.
  • the strength of an immune response can be measured by standard tests including, but not limited to, the following: direct measurement of peripheral blood lymphocytes by means known to the art; natural killer cell cytotoxicity assays (Provinciali et ah, J. Immunol. Meth. 155:19-24, 1992), cell proliferation assays (Vollenweider et al, J. Immunol. Meth.
  • MTLs are molecules that bind to the mucosal epithelium. They can mimic viral vector delivery in the absence of viral-like particles, potentiating host immunity to protein, DNA, carbohydrate, or lipid vaccines or therapeutics.
  • MTLs include, but are not limited to nucleic acid, carbohydrate, lipid, viral attachment proteins such as the adenovirus fiber protein, reoviral protein ⁇ l, GP2 of Ebola virus, the retroviral envelope glycoproteins, the F protein of Respiratory Syncytial Virus, rabies G protein, and the HAl and 2 of influenza virus.
  • the present disclosure contemplates fragments, portions, parts, or peptides of MTLs that bind to the mucosal epithelium.
  • fragments of MTLs all refer to an immunostimulatory part of an entire MTL molecule.
  • protein ⁇ l proteins that attach to M cells via a protein called "protein ⁇ l”.
  • These attachment proteins of adenovirus ssp. andreovirus ssp. share a strikingly structural similarity despite lack of homology at the primary structure level. Both proteins are composed by a N-terminal shaft followed by a C-terminal globular domain, sometimes referred to as "head” or "knob".
  • the shaft inserts into the viral capsids, while the globular domains contain the cell-specific targeting regions. For both of these viruses, the shaft contains a domain that causes the protein to form homotrimers, the active form of the protein.
  • Binding to sialic acid is a feature shared in common by distantly related viruses. Influenza virus hemagglutinin, polyoma virus VPl protein, rotavirus VP4, adenovirus sppD, and reovirus protein ⁇ l all bind sialic acid. However, no common binding domain can be identified. This observation is paralleled by the finding that, for example, Ad37 fiber knob and VPl share the absence of interaction with the glycerol group of sialic acid itself, while forming a salt bridge with the carboxyl group of sialic acid. Conversely, influenza virus hemagglutinin and rotavirus VP4 form hydrogen bonds with the carboxyl group (discussed in Burmeister et al, J.
  • reovirus protein ⁇ l binds sialic acid through residues in its shaft region
  • Ad37 fiber protein binds sialic acid through residues located at the very top of the knob.
  • certain MTL contemplated herein (and the resultant fusion proteins) contain a sialic acid binding domain.
  • a sialic acid binding domain might be present, but the position of this binding domain may vary.
  • adenovirus fiber protein and reovirus protein ⁇ l share overall structural homologies with other viral attachment proteins that protrude as trimeric spikes from the virion surface. Examples of these include GP2 of Ebola virus, the retroviral envelope glycoproteins, the F protein of Respiratory Syncytial Virus, rabies G protein, and the HAl and 2 of influenza virus. Many, if not all, the mentioned proteins bind sialic acid as the rotavirus VP4 does, albeit the latter protein spikes from the virion forms a dimer.
  • the present disclosure contemplates peptide mimetics of non-protein MTLs, such as lipopolysaccharides and peptidoglycans, for vaccine preparations.
  • the present disclosure contemplates in some embodiments using phage selection methods to identify peptide mimics of these non-protein MTLs.
  • an antibody raised against a non-protein MTL can be used to screen a phage library containing randomized short-peptide sequences. Selected sequences are isolated and assayed to determine their usefulness as vaccines.
  • Such peptide mimetics or mimics are useful to produce the recombinant vaccines disclosed herein.
  • This disclosure also includes peptide mimetics which mimic the three-dimensional structure of MTLs.
  • Such peptide mimetics might have significant advantages over naturally-occurring peptides, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
  • mimetics are peptide-containing molecules that mimic elements of protein secondary structure (see, for example, Johnson et al., Peptide Turn Mimetics, in Biotechnology and Pharmacy, Pezzuto et al., (editors) Chapman and Hall, 1993).
  • a peptide mimetic is expected to permit molecular interactions similar to the natural molecule.
  • analogs of peptides are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of a subject peptide.
  • These types of non-peptide compounds are also referred to as "peptide mimetics” or “peptidomimetics” (Fauchere, A dv. DrugRes ⁇ 5, 29-69, 1986; Veber et al, Trends Neurosci. 8:392-396, 1985; Evans et al, J. Med. Chem. 30:1229-1239, 1987) and are usually developed with the aid of computerized molecular modeling.
  • Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.
  • Labeling of peptide mimetics usually involves covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering position(s) on the peptide mimetic that are predicted by quantitative structure-activity data and molecular modeling.
  • Such non-interfering positions generally are positions that do not form direct contacts with the macromolecule(s) (e.g., in the present example they are not contact points in MTL-PRR complexes) to which the peptide mimetic binds to produce the therapeutic effect.
  • Derivatization (e.g., labeling) of peptide mimetics should not substantially interfere with the desired biological or pharmacological activity of the peptide mimetic.
  • MTL peptide mimetics can be constructed by structure-based drug design through replacement of amino acids by organic moieties (Hughes, Philos. Trans. R. Soc. Lond. 290: 387-394, 1980; Hodgson, Biotechnol. 9:19-21, 1991; Suckling, ScL Prog. 75:323-359, 1991).
  • the design of peptide mimetics can be aided by identifying amino acid mutations that increase or decrease binding of MTL to its PRR. Approaches that can be used include the yeast two-hybrid method (Chien et al., Proc. Natl. Acad. ScL USA 88: 9578-9582, 1991) and using the phage display method.
  • the two-hybrid method detects protein-protein interactions in yeast (Fields et al, Nature 340:245-246, 1989).
  • the phage display method detects the interaction between an immobilized protein and a protein that is expressed on the surface of phages such as lambda and Ml 3 (Amberg et al, Strategies 6: 2-4, 1993; Hogrefe et al, Gene 128: 119-126, 1993). These methods allow positive and negative selection for protein-protein interactions and the identification of the sequences that determine these interactions.
  • Conventional methods of peptide synthesis peptide are known in the art. See, e.g.,
  • the present disclosure also contemplates conservative variants of naturally- occurring protein MTLs, peptides of MTLs, and peptide mimetics of MTLs that target mucosal epithelium.
  • conservative mutations include mutations that substitute one amino acid for another within one of the following groups:
  • the types of substitutions selected may be based on the analysis of the frequencies of amino acid substitutions among the MTLs of different species (Schulz et al, Principles of Protein Structure, Springer- Verlag, 1978, pp. 14-16) on the analyses of structure-forming potentials developed by Chou and Fasman (Chou et al. , Biochemistry 13:211, 1974; Schulz et al, Principles in Protein Structure, Springer-Verlag, pp. 108-130, 1978) and on the analysis of hydrophobicity patterns in proteins developed by Kyte and Doolittle (Kyte et al., J. MoI. Biol. 157: 105-132, 1982).
  • the present disclosure also contemplates conservative variants that do not affect the ability of the MTL to bind to the mucosal epithelium.
  • the present disclosure includes MTLs with altered overall charge, structure, hydrophobicity/hydrophilicity properties produced by amino acid substitution, insertion, or deletion that retain and/or improve the ability to bind to their receptor.
  • the mutated MTL has at least about 70% sequence identity, more preferably at least about 80% sequence identity, even more preferably at least about 85% sequence identity, yet more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity to its corresponding wild-type MTL.
  • A/T content had to be decreased from the original 75%.
  • A/T rich genes are not efficiently expressed in eukaryotes, and they might not express at all.
  • A/T rich stretches were removed to where no more than four A/Ts were adjacent, except when there was no other option available that left five adjacent A/Ts.
  • Codons were also selected such that the pool of tRNAs would not be depleted for any particular molecule.
  • the present disclosure provides fusion proteins comprising at least one antigen molecule and at least one MTL, for use as vaccines, immunogens or toleragens.
  • the fusion protein has maximal immunogenicity and induces minimal inflammatory response. This may be achieved by increasing the number of antigens, by using multiple antigen proteins and/or multiple domains of the same antigenic protein and/or a combination of both or increasing the number of MTLs. It is within the skill of the artisan to determine the optimal ratio of MTL to antigen molecules to maximize immunogenicity and minimize inflammatory response.
  • a fusion system for the production of recombinant polypeptides.
  • heterologous proteins and peptides are often degraded by host proteases; this may be avoided, especially for small peptides, by using a gene fusion expression system.
  • general and efficient purification schemes are established for several fusion partners. The use of a fusion partner as an "affinity handle" allows rapid isolation of the recombinant peptide.
  • the recombinant product may be localized to different cellular compartments or it might be secreted; such strategy could lead to facilitation of purification of the fusion partner and/or directed compartmentalization of the fusion protein.
  • the present disclosure also contemplates modified fusion proteins having affinity for metal (metal ion) affinity matrices, whereby one or more specific metal-binding or metal-chelating amino acid residues are introduced, by addition, deletion, or substitution, into the fusion protein sequence as a tag.
  • the fusion partner i.e., the antigen or MTL sequence
  • the antigen or MTL could also be altered to provide a metal-binding site if such modifications could be achieved without adversely effecting a ligand-binding site, an active site, or other functional sites, and/or destroying important tertiary structural relationships in the protein.
  • metal-binding or metal-chelating residues may be identical or different, and can be selected from the group consisting of cysteine, histidine, aspartate, tyrosine, tryptophan, lysine and glutamate, and are located so to permit binding or chelation of the expressed fusion protein to a metal. Histidine is the preferred metal-binding residue.
  • the metal- binding/chelating residues are situated with reference to the overall tertiary structure of the fusion protein to maximize binding/chelation to the metal and to minimize interference with the expression of the fusion protein or with the protein's biological activity.
  • a fusion sequence of an antigen, a MTL and (optionally) a tag may also optionally contain a linker peptide that separates the tag from the antigen sequence or the MTL sequence.
  • the linker peptide so used encodes a sequence that is selectively cleavable or digestible by chemical or enzymatic methods, then the tag can be separated from the rest of the fusion protein, e.g., after purification.
  • a selected cleavage site within the linker or tag may be an enzymatic cleavage site.
  • Suitable enzymatic cleavage sites include sites for cleavage by a proteolytic enzyme, such as enterokinase, Factor Xa, trypsin, collagenase, or thrombin.
  • the cleavage site in the linker may be a site capable of cleavage upon exposure to a selected chemical, e.g., cyanogen bromide, hydroxylamine, or low pH. Cleavage at the selected cleavage site enables separation of the tag from the antigen/MTL fusion protein. The antigen/MTL fusion protein may then be obtained in purified form, substantially free from any peptide fragment to which it was previously linked for ease of expression or purification.
  • the cleavage site if inserted into a linker useful in the fusion sequences of this disclosure, does not limit this disclosure or any of its embodiments. Any desired cleavage site, of which many are known in the art, may be used for this purpose.
  • the optional linker peptide in a fusion protein of the present disclosure might serve a purpose other than the provision of a cleavage site.
  • the linker peptide might be inserted between the MTL and the antigen to prevent or alleviate steric hindrance between the two domains.
  • the linker sequence might provide for post-translational modification including, but not limited to, e.g., phosphorylation sites, biotinylation sites, acetylation sites, carboxylation sites, and the like.
  • the fusion protein of this disclosure contains an antigen sequence fused directly at its amino or carboxyl terminal end to the sequence of a MTL.
  • the fusion protein comprising an antigen and a MTL sequence, is fused directly at its amino or carboxyl terminal end to the sequence of a tag.
  • the resulting fusion protein is a soluble cytoplasmic fusion protein.
  • the fusion sequence further comprises a linker sequence interposed between the antigen sequence and a MTL sequence or sequence of a tag. This fusion protein is also produced as a soluble cytoplasmic protein.
  • BoNTs Botulinum neurotoxins
  • the recombinant protein (with or without a MTL) contains more than one ⁇ -trefoil structure from two or more antigenic types.
  • Such proteins and nucleic acids encoding them) are useful for specifically stimulating immune response in a subject to multiple antigenic types.
  • the ⁇ -trefoil structure there are several linear (contiguous) protective epitopes against BoNTs. In addition, there are conformational (discontiguous) epitopes that also elicit neutralizing immunity. These collectively impart the capacity of the ⁇ -trefoil structure to serve as a protective vaccine. While additional protective epitopes are present within the H chain and which the MTL can co-deliver, the ⁇ -trefoil structure contains the binding domain of BoNT. Host immune responses, primarily antibody responses, directed to this structure, will prevent binding of the BoNT to host tissues, and thus, neutralizing protection will occur. Thus, the development of a multivalent vaccine containing each of two or more (or all) of the antigenic types, A-G, will protect against multiple (or all) BoNTs.
  • the ⁇ -trefoil structure can be used to prevent immune reactivity.
  • the fusion proteins of the present disclosure contain one or more MTL (a domain that binds to mucosal epithelia) and one or more antigen, i.e., a domain recognized by the innate or adaptive immune system.
  • MTL a domain that binds to mucosal epithelia
  • antigen i.e., a domain recognized by the innate or adaptive immune system.
  • the present disclosure contemplates vaccines comprising chimeric constructs including at least one antigen domain and at least one MTL domain.
  • the vaccines of the present disclosure comprise a Hc- ⁇ Tre-MLT fusion protein.
  • compositions comprising the chimeric constructs of the present disclosure can be formulated according to known methods for preparing pharmaceutically useful compositions, whereby the chimeric constructs are combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition is said to be a "pharmaceutically acceptable carrier” if its administration can be tolerated by the recipient and if that composition renders the active ingredient(s) accessible at the site where its action is required.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
  • Suitable carriers are well known to those in the art (Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 5 th Edition (Lea & Febiger 1990); Gennaro (ed.), Remington 's Pharmaceutical Sciences 18th Edition (Mack Publishing Company 1990)). Examples of several other excipients that can be contemplated may include, water, dextrose, glycerol, ethanol, and combinations thereof. Vaccines of the present disclosure may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or stabilizers.
  • the chimeric constructs provided herein can be used as vaccines by conjugating to soluble immunogenic carrier molecules or other vaccines.
  • the chimeric construct can be conjugated to the carrier molecule using standard methods
  • compositions comprising a pharmaceutically acceptable injectable vehicle with or without adjuvant(s).
  • adjuvants include, but are limited to, liposomes, aluminum phosphate, aluminum hydroxide, polynucleotides, polyelectrolytes, muramyl dipeptides.
  • Preparation of injectable vaccines in certain embodiments includes mixing the chimeric/fusion/engineered construct with muramyl dipeptides or other adjuvants.
  • the resultant mixture may be emulsified in a mannide monooleate/squalene or squalane vehicle.
  • Four parts by volume of squalene and/or squalane are used per part by volume of mannide monooleate.
  • Methods of formulating vaccine compositions are well known to those of ordinary skill in the art (RoIa, "Immunizing Agents and Diagnostic Skin Antigens," In: Remington 's Pharmaceutical Sciences, 18th Edition, Gennaro (ed.) (Mack Publishing Company, 1990) pages 1389-1404).
  • Control release preparations can be prepared through the use of polymers to complex or adsorb chimeric construct.
  • biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid (Sherwood et ah, Bio/Technology 10: 1446, 1992).
  • the rate of release of the chimeric construct from such a matrix depends upon the molecular weight of the protein, the amount of the protein within the matrix, and the size of dispersed particles (Saltzman et al., Biophys. J.
  • the chimeric construct can also be conjugated to polyethylene glycol (PEG) to improve stability and extend bioavailability times ⁇ e.g., U.S. Patent 4,766,106).
  • PEG polyethylene glycol
  • compositions of this disclosure may be administered parenterally.
  • the usual modes of administration of the vaccine are intramuscular, subcutaneous, and intra-peritoneal injections.
  • the administration may be by continuous infusion or by single or multiple boluses.
  • the vaccine of the present disclosure may be formulated and delivered in a manner designed to evoke an immune response at a mucosal surface.
  • the vaccine compositions may be administered to mucosal surfaces by, for example, nasal or oral
  • Intranasal, oral, or alternative mucosal immunization using a vaccine construct of the present disclosure can be readily administered to stimulate protective immunity by the production of mucosal and systemic antibodies and cell-mediated immune responses against the encoded vaccine.
  • Other modes of administration include suppositories and oral formulations.
  • binders and carriers may include polyalkalene glycols or triglycerides.
  • Oral formulations may include normally employed incipients such as pharmaceutical grades of saccharine, cellulose and magnesium carbonate. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 1 to 95% of the chimeric construct.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, protective and immunogenic dosages.
  • the quantity of vaccine employed will of course vary depending upon the patient's age, weight, height, sex, general medical condition, previous medical history, the condition being treated and its severity, and the capacity of the individual's immune system to synthesize antibodies, and produce a to produce a cell-mediated immune response.
  • a dosage of the chimeric construct which is in the range of from about 1 ⁇ g/kg to 10 mg/kg (amount of agent/body weight of patient), although a lower or higher dosage may also be administered.
  • Precise quantities of the active ingredient depend on the judgment of the practitioner.
  • Suitable dosage ranges are readily determinable by one skilled in the art and may be on the order of micrograms of the chimeric construct to milligrams of the chimeric construct, depending on the particular construct. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dosage may depend on the route of administration and will vary according to the size of the subject.
  • the present disclosure encompasses vaccines containing antigen and MTLs from a single organism, such as a specific pathogen.
  • the present disclosure also encompasses vaccines which contain antigenic material from several different sources and/or MTL material isolated from several different sources.
  • Such combined vaccines contain, for example, antigen and MTLs from various pathogens or from various strains of the same pathogen, or from combinations of various pathogens.
  • the antigen/MTL fusion proteins are administered to a mammal in a therapeutically effective amount.
  • a protein or nucleic acid preparation or composition e.g., an immune stimulatory composition or vaccine
  • a preparation or composition of the present disclosure is physiologically significant if it invokes a measurable humoral and/or cellular immune response in the recipient mammal.
  • the term "treatment” refers to both therapeutic treatment and prophylactic or preventative treatment.
  • the present disclosure contemplates using the disclosed vaccines to treat patients in need thereof.
  • the patients may be suffering from diseases such as, but not limited to, cancer, allergy, infectious disease, or a disease associated with an allergic reaction.
  • the present disclosure contemplates administering the disclosed vaccines to passively immunize patients against diseases such as but not limited to, cancer, allergy, infectious disease, or disease associated with an allergic reaction.
  • the present disclosure contemplates administering the disclosed vaccines to passively immunize patients against diseases in addition to those cited in the previous sentence in which the objective is to rid the body of specific molecules or specific cells.
  • a non-limiting example might be vaccination against autoimmune diseases or allergies.
  • compositions of the present disclosure can be used to enhance the immunity of subject animals, more specifically mammals, and even more specifically humans in need thereof. Enhancement of immunity is a desirable goal in the treatment of patients diagnosed with, for example, autoimmune diseases, cancer, immune deficiency syndrome, certain topical and systemic infections, leprosy, tuberculosis, shingles, warts, herpes, malaria, gingivitis, and atherosclerosis.
  • vaccines e.g., immune stimulant compositions
  • Advantages of vaccines include that they induce a strong immune response and exhibit minimal undesired inflammatory reaction.
  • the methods and compositions of the disclosure may be used as a vaccine in a subject in which immunity for the antigen(s) is desired.
  • antigens can be any antigen known in the art to be useful in a vaccine formulation.
  • the methods and compositions can be used to enhance the efficacy of any vaccine known in the art.
  • Vaccine described herein may be used to enhance an immune response to infectious agents and diseased or abnormal cells, such as, but not limited to, bacteria, parasites, fungi, viruses, tumors, and cancers.
  • the compositions may be used to either treat or prevent a disease or disorder amenable to treatment or prevention by generating an immune response to the antigen provided in the composition.
  • Diseases and disorders that can be treated using methods and compositions described herein include viral infections, such as an infection by HIV, CMB, hepatitis, herpes virus, measles virus; bacterial infections; fungal and parasitic infections; cancers; autoimmune diseases; and any other disease or disorder amenable to treatment or prevention by eliciting an immune response against a particular antigen or antigens.
  • the methods and compositions of the present disclosure may be used to elicit a humoral and/or a cell-mediated response against the antigen(s) of the vaccine in a subject.
  • the methods and compositions elicit a humoral response against the administered antigen in a subject.
  • the methods and compositions elicit a cell-mediated response against the administered antigen in subject.
  • both a humoral and a cell-mediated response are triggered.
  • the subjects to which dosage with a composition described herein is applicable may be any mammalian or vertebrate species, which include, but are not limited to, cows, horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice, rats, monkeys, rabbits, chimpanzees, and humans.
  • the subject is a human.
  • the ease of intranasal or oral administration using this vaccine delivery system can be applied to any number of enteric, respiratory, or sexually transmitted diseases.
  • the procedures of the present disclosure can be used to generate a chimeric construct comprising one or more antigens of interest and one or more MTLs.
  • a small, non- immunogenic epitope tag (such as His tag) is added to facilitate the purification of the fusion protein expressed in bacteria.
  • the combination of the antigen with a MTL, such as a viral fiber protein, provides both signals necessary for the activation of the antigen-specific adaptive and increased immune responses.
  • a large number of differing chimeric constructs comprising different combinations of antigens and MTLs can be readily generated using recombinant DNA technology.
  • recombinant protein product can be generated using a bacterial expression system.
  • the product can be purified from bacterial cultures using standard techniques. The approach is thus extremely economical and cost efficient.
  • recombinant vaccine product can be produced and purified from cultures of yeast, such as Pichiapastoris, or other eukaryotic cells including, without limitation, insect cells or mammalian cells.
  • yeast expression system will be the first choice as it lacks endotoxin and it can be easily and cheaply scaled up by fermentation.
  • DNA vaccines are produced using conventional eukaryotic plasmid expression systems for the encoded gene.
  • Other non-protein epitopes can be derived using conventional methods to glycosylate or lipidate or synthesize nucleic acids that bind the mucosal epithelia.
  • T-cell and B-cell antigens can be used to generate vaccines by this approach. Fusion of an antigen with a MTL such as a viral fiber protein optimizes the stoichiometry of the two signals thus minimizing the unwanted excessive inflammatory responses, which occur, for example, when antigens are mixed with adjuvants to increase , their immunogenicity.
  • a MTL such as a viral fiber protein
  • the present disclosure will allow the generation of vaccines against microbial and viral pathogens, as well as a specific infectious agent. Additionally, the approach can be used to construct vaccines that may suppress allergic reactions.
  • Example 1 Schematic representation of a vaccine antigen attached to a mucosal targeting Iigand (MTL)
  • Clostridium botulinum strain A toxin (BoNT/A) is genetically fused to the binding domain of Adenovirus type 2 fiber protein (MTL) ( Figure 1).
  • Botulinum neurotoxin A (BoNT/A) is one of seven serotypes of botulinum neurotoxins (BoNT) designated by letters A through G. It is produced by the bacterium Clostridium botulinum, a gram positive, obligate anaerobe, spore-former that causes the deadly botulism food poisoning.
  • BoNT/A is produced as a single polypeptide chain that has a molecular weight of about 150 kDa. It is subsequently cleaved into two functional subunits held together by a disulfide bond.
  • the 50-kDa "light chain” is a zinc endoprotease that selectively cleaves synaptic proteins, therefore inhibiting neurotransmitter release.
  • the 100-kDa "heavy chain” is made up of an N-terminal half, believed to mediate cytoplasmic entry of the light chain, and a C-terminal half (BoNT/A- H c ), which exhibits a ⁇ -trefoil motif and mediates cellular adherence (Lalli et al, J. Cell ScL 112: 2715-2724, 1999).
  • MTL This targeting molecule
  • Example 2 Antibody responses induced by intranasal immunization with a MTL genetically fused to Hc/A trefoil
  • Hc/A trefoil in mice achieves similar results in inducing IgA and IgG as compared to the administration of a MTL with an adjuvant, cholera toxin.
  • the onset of the S-IgA response is more rapid in the mice treated with the fusion protein than treated with Hc/A trefoil with or without cholera toxin.
  • the BALB/c mice were dosed three times with 50 ⁇ g of a mucosal targeting ligand (MTL) genetically fused to Hc/A trefoil (Hc( ⁇ -trefoil)-MTL) or Hc ⁇ -trefoil.
  • MTL mucosal targeting ligand
  • Antibody responses including copra-IgA and serum IgG, were measured by Hc ⁇ -trefoil-specific ELISA.
  • Intranasal immunization with Hc( ⁇ -trefoil)-MTL stimulates elevated (A) copro-IgA and (B) serum IgG antibodies to Hc ⁇ -trefoil when co-administered with or without the mucosal adjuvant, cholera toxin (CT).
  • CT cholera toxin
  • Figure 2 elevated mucosal IgA were obtained ( Figure 2).
  • the Hc ⁇ -trefoil by itself stimulated poor IgG and S-IgA responses, and these S-IgA responses were delayed even when CT was used.
  • Example 3 Antibody-producing cell responses after intranasal immunization with a MTL genetically fused to
  • mice were intranasally immunized with Hc ⁇ Tre-MTL + CT or Hc ⁇ Tre +
  • BoNTs botulinum toxins
  • BoNTs are serologically divided into seven groups (A to G). Serotypes A, B, and, to a minor extent, E are typically associated with botulism in humans, whereas serotype C mostly affect domestic animals (Kobayashi et al, J. Immunol. 174: 2190-2195, 2005).
  • the current vaccine is a pentavalent vaccine with as little at 10% of the toxoid preparation representing neurotoxoid (Singh & DasGupta, Toxicon.
  • toxins are each naturally synthesized as a single 150 KD polypeptide, requiring post-translational proteolysis to yield a ⁇ 50 kDa fragment or light (L) chain, containing the catalytic activity, and a 100 KDa component or heavy (H) chain ( ⁇ 100 kDa).
  • This H chain contains the translocation domain in the N-terminus (H N ) and the cell binding domain in C-terminus (Hc).
  • the H N Upon binding the target cell, the H N promotes the translocation of the L chain to the cytosol and the L chain endopeptidase activity inactivates a group of proteins (termed SNARE proteins) required for release of acetylcholine at the neuromuscular junction resulting in flaccid paralysis (Arnon et al, Jama. 285: 1059-1070, 2001; Simpson, Annual Rev Pharma Tox 44: 167-193, 2004).
  • SNARE proteins proteins required for release of acetylcholine at the neuromuscular junction resulting in flaccid paralysis
  • Hc/A To assess the feasibility of generating a prototype vaccine for Hc/A, the Hc ⁇ -trefoil from serotype A was cloned and expressed. The ⁇ -trefoil structure was selected since each BoNT shares similar structure with its Hc. We hypothesize that Abs to this structure will be cross-protective against the various BoNT serotypes.
  • the Hc/A gene was synthesized to reduce its A/T content and codon optimized to allow for successful expression in yeast ( Figure 4).
  • primers were synthesized to PCR the synthetic Hc ⁇ -trefoil cDNA (encodes the last 210 amino acids of Hc/A), and when evaluated by SDS-PAGE, it migrated as a single band of the expected MW 22.5 kDa and was also recognized by a rabbit antiserum made to recombinant Hc/A.
  • mice were immunized nasally with 50 ⁇ g ⁇ -trefoil/A + cholera toxin (CT) on days 0, 7, and 14. Serum IgG and copro-IgA endpoint titers were measured by standard ELISA methods against ⁇ - trefoil ( Figure 5A). The ⁇ -trefoil vaccine was determined immunogenic as evidenced by the induction of S-IgA and IgG Abs. These Ab responses were supported by IL-4- and IFN- ⁇ - producing head and neck lymph node Th cells ( Figure 5B).

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Abstract

La présente invention relève du domaine du développement des vaccins et concerne plus spécifiquement des procédés permettant de concentrer des vaccins sur l'épithélium muqueux ainsi que des vaccins fondés sur le domaine β-trèfle de la toxine botulinique.
PCT/US2006/001346 2005-01-21 2006-01-13 Vaccins et ligands de ciblage des muqueuses permettant de faciliter l'administration de vaccins WO2006078567A2 (fr)

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WO2008148164A1 (fr) * 2007-06-08 2008-12-11 Ian Andrew Ferguson Vaccins administrés par voie nasale utilisant le ciblage nalt à détection multiple et des séquences de transport de polypeptide phagocytaire
WO2009015840A2 (fr) * 2007-07-27 2009-02-05 Merz Pharma Gmbh & Co. Kgaa Polypeptide utilisé pour cibler des cellules neuronales
US7910113B2 (en) 2006-03-27 2011-03-22 Montana State University Tolerizing agents
US8580274B2 (en) 2009-02-10 2013-11-12 University Of The Ryukyus Drug transporter, and adjuvant and vaccine each utilizing same
WO2017139558A1 (fr) * 2016-02-12 2017-08-17 Virtici, Llc Agent thérapeutique de tolérance pour le traitement d'une activité immunitaire induite par un polypeptide

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7910113B2 (en) 2006-03-27 2011-03-22 Montana State University Tolerizing agents
WO2008148164A1 (fr) * 2007-06-08 2008-12-11 Ian Andrew Ferguson Vaccins administrés par voie nasale utilisant le ciblage nalt à détection multiple et des séquences de transport de polypeptide phagocytaire
WO2009015840A2 (fr) * 2007-07-27 2009-02-05 Merz Pharma Gmbh & Co. Kgaa Polypeptide utilisé pour cibler des cellules neuronales
WO2009015840A3 (fr) * 2007-07-27 2009-07-09 Merz Pharma Gmbh & Co Kgaa Polypeptide utilisé pour cibler des cellules neuronales
US8580274B2 (en) 2009-02-10 2013-11-12 University Of The Ryukyus Drug transporter, and adjuvant and vaccine each utilizing same
WO2017139558A1 (fr) * 2016-02-12 2017-08-17 Virtici, Llc Agent thérapeutique de tolérance pour le traitement d'une activité immunitaire induite par un polypeptide

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