WO2004020592A2 - Vaccination genetique retrocanalaire par l'intermediaire des glandes salivaires - Google Patents

Vaccination genetique retrocanalaire par l'intermediaire des glandes salivaires Download PDF

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Publication number
WO2004020592A2
WO2004020592A2 PCT/US2003/026872 US0326872W WO2004020592A2 WO 2004020592 A2 WO2004020592 A2 WO 2004020592A2 US 0326872 W US0326872 W US 0326872W WO 2004020592 A2 WO2004020592 A2 WO 2004020592A2
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salivary gland
antigen
dna
nucleic acid
lipid
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PCT/US2003/026872
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English (en)
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WO2004020592A3 (fr
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Sean Tucker
Michael Bennett
Yen-Ju Chen
David Olson
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Genteric, Inc.
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Priority to AU2003262922A priority Critical patent/AU2003262922A1/en
Publication of WO2004020592A2 publication Critical patent/WO2004020592A2/fr
Publication of WO2004020592A3 publication Critical patent/WO2004020592A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/55505—Inorganic 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Cellular immunity is enhanced because the protein being encoded by the DNA vector is processed and presented in a way that is analogous to the processing of viral antigens (see, e.g., Corr et al, J. Exp. Med. 184:1555 (1996)).
  • plasmid-based vectors are much simpler to manufacture than protein- based or whole, live organism vaccines, and are considered safer to use.
  • Protective immunity following DNA vaccination has been demonstrated in a variety of mouse models ⁇ see, e.g., Manickan et al, J. Immunol. 155:259 (1995) and Fynan et al, Proc.
  • Mucosal immune responses tend to be intense at the site where the antigen is delivered, and are sometimes not well distributed throughout all mucosal sites (see, e.g., Johansson et al, Infect. Immun. 69:7481 (2001) and Forrester et al, Vaccines for Enteric Diseases T. Vesikari, ed., Tampere, Finland (2001)).
  • inrranasal immunization tends to produce active mucosal responses in the lungs, some responses in the vagina, but more limited responses in the colon ⁇ see, e.g., Forrester et al, 2001, supra and Kuklin et al, J. Virol. 71:3138 (1997)).
  • the present invention provides compositions and methods for genetic immunization, whereby a broad immune response is generated.
  • the present invention provides compositions and methods for transfecting antigen presenting cells.
  • antigen presenting cells associated with the mucosal immune system are transfected.
  • the present invention provides a method for eliciting an immune response.
  • An immunogenically effective amount of a composition comprising a nucleic acid encoding an immunogenic polypeptide is retroductally introduced, whereby an immune response is generated.
  • the step of introducing is by cannulation.
  • the composition further comprises an adjuvant, such as, for example, cholera toxin or Al(OH) 3 .
  • the composition is administered multiple times, h some embodiments, the nucleic acid is operably linked to an expression control sequence, h some embodiments, the nucleic acid is in a viral vector.
  • the immunogenic polypeptide is a cancer antigen.
  • the immunogenic polypeptide is a viral antigen such as, for example, HIV envelope protein or a portions thereof ⁇ e.g., gp 160 or a portion thereof, gp 120 or a portion thereof, or gp41 or a portion thereof), h even other embodiments, the immunogenic polypeptide is a bacterial antigen, such as, for example anthrax protective antigen.
  • the composition further comprises a lipid ⁇ e.g., N,N,N',N'-tetramethyl-N,N'- bis(2-hydroxyethyl)-2-3-di(oleoyloxy)-l,4-butanediammonium iodide.), whereby the lipid facilitates uptake of the nucleic acid by antigen presenting cells.
  • the salivary gland is a submandibular salivary gland, a parotid salivary gland, or a sublingual salivary gland.
  • the subject is a mammal, such as, for example, a primate, such as, for example, a human.
  • the immune response comprises a mucosal immune response.
  • the present invention provides a method for transfecting antigen presenting cells.
  • An immunogenically effective amount of a composition comprising a nucleic acid encoding an immunogenic polypeptide retroductally introduced into the lumen of a salivary gland duct of a subject.
  • the step of introducing is by cannulation.
  • the composition is administered multiple times, i some embodiments, the nucleic acid is operably linked to an expression control sequence.
  • the nucleic acid is in a viral vector, i some embodiments, the immunogenic polypeptide is a cancer antigen.
  • the immunogenic polypeptide is a viral antigen such as, for example, HIV envelope protein or a portions thereof (e.g., gp 160 or a portion thereof, gpl20 or a portion thereof, or gp41 or a portion thereof).
  • the immunogenic polypeptide is a bacterial antigen, such as, for example anthrax protective antigen
  • the composition further comprises a lipid, whereby the lipid facilitates uptake of the nucleic acid by the antigen presenting cells.
  • the salivary gland is a submandibular salivary gland, a parotid salivary gland, or a sublingual salivary gland.
  • the subject is a mammal, such as, for example, a primate, such as, for example, a human.
  • antigen presenting cells ⁇ e.g., dendritic cells
  • the proximal lymph node is a draining lymph node.
  • the draining lymph node is a cervical lymph node or a submandibular lymph node.
  • the present invention provides a pharmaceutical composition comprising a nucleic acid encoding an immunogenic polypeptide, a lipid, and a transition metal enhancer. The pharmaceutical composition elicits an immune response.
  • the lipid is N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2-3- di(oleoyloxy)-l,4-butanediammonium iodide and the transition metal enhancer is ZnCl 2 .
  • An "immunogenic composition” is one that elicits or modulates an immune response, preferably the composition induces or enhances an immune response in response to a particular antigen. Immune responses include humoral immune responses and cell-mediated immune responses. An immunogenic composition can be used therapeutically or prophylactically to treat or prevent disease at any stage.
  • a "salivary gland” is a gland of the oral cavity which secretes saliva, including the glandulae salivariae majores of the oral cavity (the parotid, sublingual, and submandibular glands) and the glandulae salivariae minores of the tongue, lips, cheeks, and palate (labial, buccal, molar, palatine, lingual, and anterior lingual glands).
  • “Retroductally introducing” refers to introduction of a composition through a duct in a salivary gland, wherein the composition flows through the salivary gland duct in a retrograde manner. Suitable ducts include all major and minor salivary gland ducts.
  • an “adjuvant” is a non-specific immune response enhancer. Suitable adjuvants include, for example, cholera toxin, Al(OH) , and polyionic organic acids.
  • a "polyionic organic acid' (POD) as used herein, is typically a polyprotic polyaromatic organic compound wherein the compound contains at least two aromatic components.
  • Polyionic compounds refer to compounds comprising one or more ionizable units, either as in the protonated form or as the conjugate salt. In certain embodiments, the PODS has associated therewith, such as complexed with, a transition metal enhancer as described below.
  • a polyionic organic acid is a dye.
  • a dye is a compound that absorbs radiation in the ultraviolet, visible and/or infrared regions of the electromagnetic spectrum. These regions of the electromagnetic spectrum correspond to radiation having wavelengths of 10 " to 4x10 " , 4 - 7xl0 "7 and 7xl0 “7 to 10 "4 meters, respectively.
  • Dyes which are useful in the present invention include, but are not limited to, an acid dye, a disperse dye, a direct dye and a reactive dye. a preferred embodiment, an acid dye is used.
  • Suitable acid dyes include, but are not limited to, direct red dye, direct blue dye, acid black dye, an acid blue dye, an acid orange dye, an acid red dye, an acid violet dye, and an acid yellow dye.
  • suitable acid dyes include, but are not limited to, Evans Blue, Congo Red, Ponceau S, Congo Corinth, Sirius red F3B, Ponceau 6R, amido black 10B, biebrich scarlet and aurintricarboxylic acid.
  • a direct dye is used.
  • Preferred direct dyes include direct red, direct blue, direct yellow and direct green. More preferably, direct blue 15 (Light Blue), direct red 28 (Congo Red) and direct blue 53 (Evans Blue) are used.
  • the dye absorbs in the visible light spectrum, between about 400 nm to 700 nm.
  • a "cationic lipid” refers to any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH.
  • a "charge neutral lipid” or a “neutral lipid” refers to any of a number of lipid species which carry a net neutral charge at a selective pH, such as physiological pH.
  • An “anionic lipid” refers to any of a number of lipid species which carry a net negative charge at a selective pH, such as physiological pH.
  • Mucosal immune responses refers to immune responses generated in the mucosas of the gastrointestinal system ⁇ e.g., intestine, jejunum, ileum, duodenum, ), the respiratory system; ⁇ e.g., lungs, trachea), and the urogenital tract ⁇ e.g., vagina, urethra) ⁇ see, e.g., Bannister et al. ed. (1995) Gray 's Anatomy).
  • Components of the mucosal immune system include, for example, tonsils, adenoids, Peyer's patches, appendix, and single lypmphoid follicles.
  • a mucosal immune response includes both humoral aspects and cell mediated aspects.
  • Human immune responses or Th2-type responses are mediated by cell free components of the blood, i.e., plasma or serum; transfer of the serum or plasma from one individual to another transfers immunity. Humoral immune responses include, for example, production of antigen-specific antibodies ⁇ e.g., neutralizing antibodies).
  • Cell mediated immune responses or “Thl-type responses” are mediated by antigen specific lymphocytes; transfer of the antigen specific lymphocytes from one individual to another transfers immunity.
  • Cell mediated immune responses include, for example, development of antigen specific cytotoxicity, i.e., stimulation or activation of antigen-specific cytotoxic T cells.
  • Antigen presenting cells refers to cells that are able to present immunogenic peptides or fragments thereof to T cell to activate or enhance an immune response.
  • APCs include, for example, dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs.
  • Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver ⁇ i.e., matched HLA haplotype).
  • APCs may be from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.
  • Lymph nodes refers to any of the masses of lymphoid tissue which filter the flow of lymph ⁇ i.e., a body fluid that comprises lymphocytes). Lymph nodes are typically surrounded by a capsule of connective tissue, are distributed along the lymphatic vessels, and contain numerous lymphocytes, including antigen presenting cells.
  • Lymph nodes include, for example, submandibular nodes, parotid nodes ⁇ i.e., superficial), buccal nodes, occipital nodes, cervical nodes ⁇ i.e., upper deep, lower deep, anterior, and superficial) submental nodes, infrahyoid nodes, retro-aurical nodes, jugulo-omohyoid nodes, jugulodigastric nodes, prelaryngeal nodes, pretracheal nodes, inguinal nodes, and intestinal mesentery nodes.
  • a "draining lymph node” is a lymph node to which antigens or antigenic fragments are filtered by the lymph.
  • the immunogenic compositions ⁇ i.e., pharmaceutical compositions) of the present invention are administered to a subject in an amount sufficient to elicit an immune response in the subject. An amount adequate to accomplish this is defined as “immunogenically effective dose or amount.”
  • the term “protein” is used herein interchangeably with “polypeptide” and “peptide.”
  • the terms “promoter” and “expression control sequence” are used herein to refer to an array of nucleic acid control sequences that direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • a "constitutive” promoter is a promoter that is active under most environmental and developmental conditions.
  • An “inducible” promoter is a promoter that is active under environmental or developmental regulation.
  • the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence. DNA regions are “operably linked” when they are functionally related to each other.
  • DNA for a signal peptide is operably linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is "operably linked" to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is “operably linked” to a coding sequence if it is positioned so as to permit translation.
  • "operably linked” means contiguous and, in the case of secretory leaders, in reading frame.
  • DNA sequences encoding immunogenic polypeptides which are to be expressed in a microorganism will preferably contain no introns that could prematurely terminate transcription of DNA into mRNA.
  • heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
  • a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature ⁇ e.g., a fusion protein).
  • An "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
  • the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
  • the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
  • Antibody refers to a polypeptide encoded by an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond.
  • the F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially Fab with part of the hinge region ⁇ see, e.g. Fundamental Immunology (Paul ed., 4th ed. 1999).
  • the term antibody as used herein, also includes antibody fragments either produced by the modification of whole antibodies.
  • Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates. methyl phosphonates, chiral-methyl phosphonates, 2- O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • PNAs peptide-nucleic acids
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; Rossolini et al. (1994) Mol. Cell. Probes 8:91-98).
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions ⁇ i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identity of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 25% sequence identity. Alternatively, percent identity can be any integer from 25% to 100%.
  • More preferred embodiments include at least: 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or higher, compared to a reference sequence using the programs described herein, preferably BLAST using standard parameters, as described below.
  • BLAST BLAST using standard parameters
  • More preferred embodiments include at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
  • Polypeptides which are "substantially similar" share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.
  • Optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math. 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always > 0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • nucleotide sequences are substantially identical is if two molecules hybridize to each other, or to a third nucleic acid, under moderately, and preferably highly, stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes ⁇ e.g., 10 to 50 nucleotides) and at least about 60°C for long probes ⁇ e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5X SSC, and 1% SDS, incubating at 42°C, or, 5X SSC, 1% SDS, incubating at 65°C, with wash in 0.2X SSC, and 0.1% SDS at 65°C.
  • suitable "moderately stringent conditions” include, for example, prewashing in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridizing at 50°C-65°C, 5X SSC overnight, followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC (containing 0.1% SDS).
  • Transition metal enhancer refers to compounds having one or more transition metal atoms selected from the elements in Groups IIIB, IVB context VB, VIIB, VIIIB, IB, and IIB of the periodic table (i.e., the J-block) ⁇ see, e.g., Huheey, INORGANIC CHEMISTRY, Harper & Row, New York, 1983).
  • the transition metals of the present invention also include those lanthanides (i.e., the first row of the /-block of the periodic table) and main group metals ⁇ i.e., groups IIIA, IV A, VA, and VILA, of the periodic table), having chemical properties similar to transition metal complexes.
  • Figure 1 illustrates expression of hGH within the submandimular gland with retreatment.
  • the right and left submandibular glands of rats were each treated with 200 ⁇ L of water containing 175 ⁇ g of hGH plasmid DNA on day 0.
  • Retreated animals received a second DNA dose on Day 21.
  • the animals were sacrificed and their submandibular glands were harvested on either Day 7 or Day 28.
  • Figure 2 illustrates antibody responses following salivary gland DNA administration. Fig.
  • FIG. 2 A illustrates measurement of IgG and IgA antibody isotypes 3 weeks after 350 ⁇ g of plasmid DNA encoding hGH was infused retroductally into the salivary glands (submandibular) or injected intramuscular.
  • 15 ⁇ g of hGH protein was injected subcutaneously (s.c.) in a 50/50 mixture of sterile saline/ Freund's complete adjuvant.
  • N 6 for DNA groups
  • N 4 animals for the s.c. group
  • N 3 for the untreated group.
  • Fig. 2B illustrates IgG plasma titers over time following DNA administration.
  • submucosal refers to needle injection into the tissue below the tongue.
  • Salivary gland SG
  • intramuscular i.m.
  • the submucol groups were given one dose of 175 ⁇ g DNA at the time shown by the arrow.
  • FIG. 3 illustrates antibody responses to gpl20 following salivary gland and intramuscular DNA administration. Antibody responses were measured after plasmid DNA was retroductally infused into the salivary gland (SG) or injected intramuscular (i.m.).
  • Fig. 3 A illustrates measurement of anti-HIV gpl20 in the plasma of rats treated by two doses of 175 ⁇ g DNA given four weeks apart. The plasma titers were measured 2 weeks after the last dose. These experiments were performed with either DNA and water (SG) or DNA and sterile saline (i.m.).
  • hGH DNA administered to the SMG represents an irrelevant DNA control in this experiment, and controls for any non-specific immune responses resulting due to DNA delivery to the SG.
  • FIG. 4 illustrates anti-g l20 T cell responses to antigen.
  • Fig. 4A illustrates T cell activity specific to gpl20 measured by ⁇ -IFN ELISA of the cell supernate.
  • the stimulation index represents the ratio between the ⁇ -IFN secreted by g l20 stimulated cells divided by the ⁇ -IFN secreted by cultured but not antigen stimulated cells.
  • Two concentrations of antigen were provided to the cultured splenocytes, 0.2 ⁇ g gpl20 (solid) and 1.0 ⁇ g gpl20 (open).
  • DNA vaccinated groups were treated 3 times with 175 ⁇ g gpl20 DNA per treatment, with the last DNA dose given 1 week before spleen harvest.
  • Fig. 4B illustrates CD4 and CD8 T cell responses measured by intracellular ⁇ -IFN. The percentage of ⁇ -IFN+ cells from each T cell subset was determined by flow cytometry.
  • DNA vaccinated animals were treated with 175 ⁇ g gpl20 (or 175 ⁇ g hGH) on weeks 0, 4, and 8 weeks and harvested on week 9.
  • FIG. 5 illustrates mucosal immune responses within saliva following salivary gland DNA vaccination.
  • Fig. 5 A illustrates anti-hGH IgA was measured by ELISA.
  • Four vaccinated animals (1-4) were compared to two untreated animals (5-6) at 3 weeks after a single dose given to the SG.
  • Each saliva sample was diluted to a concentration of 12.5 ng/ml total IgA prior to the anti-hGH IgA ELISA.
  • One saliva sample, shown with an * was diluted to 6.25 ng/ml total IgA due to insufficient material.
  • the O.D. values were then plotted for individual animals.
  • Fig. 5B illustrates anti-hGH secretory component measured by ELISA.
  • FIG. 6A-C illustrate immune responses following salivary gland DNA vaccination.
  • FIG. 7 illustrates plasma antibody titers in dogs following parotid gland retroductal DNA delivery.
  • plasmid DNA encoding hGH or 2.5 mg of plasmid DNA encoding secreted alkaline phosphatase was retroductally delivered to the parotid salivary glands of 10 kg dogs in a total volume of 700 ⁇ l with 2 mg/ml Evans Blue.
  • 5 mg of plasmid DNA encoding hGH or 5.25 mg of plasmid DNA encoding secreted alkaline phosphatase (SEAP) was retroductally delivered to the parotid salivary glands of 10 kg dogs in a total volume of 3000 ⁇ l with 2 mg/ml Evans Blue.
  • Figure 8 illustrates enhancement of genetic immunization by co-formulation of the nucleic acid with lipid.
  • DNA encoding hGH human growth hormone
  • 88 ⁇ g DNA encoding hGH was administered per submandibular salivary gland on weeks 0 and 6.
  • Anti-hGH IgA was measured on week 9 after normalizing the amount of total IgA in each sample. The response at two different concentrations of total IgA is shown. Results show that 3 out of 3 rats had high responds in the Lipid/Zn group and 2 out of 6 responded in the no lipid/Zn group.
  • Figure 10 illustrates distal mucosal immune response following genetic immunization.
  • FIG. 11 illustrates HIV neutralization following genetic immunization. 88 ⁇ g DNA encoding HIV envelope protein gpl20 in 200 ⁇ l distilled, deionized H 0 was retroductally delivered to the submandibular salivary glands of Sprague Dawley rats on weeks 0 and 3.
  • the DNA was delivered alone or in a formulation comprising: Congo Red (6 mg/ml), Congo Red (6 mg/ml )/DOHBD:DOPE (3:1)/Zn (0.125 mM), or aurintricarboxylic acid/Zn (0.125 mM).
  • Congo Red (6 mg/ml)
  • Congo Red (6 mg/ml )/DOHBD:DOPE (3:1)/Zn (0.125 mM)
  • aurintricarboxylic acid/Zn (0.125 mM
  • Figure 12 illustrates a comparison of anti-anthrax protective antigen (PA) plasma IgG titers from retroductal introduction of formulations with DNA encoding PA with or without a polyionic organic acid into the salivary gland of rats.
  • Figure 13 illustrates a time course comparing anti-anthrax protective antigen (PA) plasma IgG titers using different introduction methods and positive (PA protein) and negative (hGH DNA ) controls.
  • Antibody titers were measured following retroductal delivery of PA DNA to the salivary gland (SG/PA DNA), injection of PA DNA into the muscle (i.m./PA DNA), or retroductal delivery of hGH DNA to the salivary gland (SG/hGH DNA).
  • Subcutaneous PA protein plus CFA vaccination (s.c./Prtn+CFA), and naive animals served as positive and negative controls respectively. Arrows indicate when DNA or protein was administered.
  • Figures 14A-B illustrate hGH expression in tissue and anti-hGH responses in the plasma of rats following salivary gland retroductal delivery.
  • DNA was formulated with either a ZnLipid combination (0.125mM Zn, 3:1 DOHBD.-DOPE Lipid) or a 3.6mM Zn formulation and administered at 0 and 6 weeks to rat submandibular glands (SMG). The antibody responses were measured at 8 wks post initial DNA administration for either IgG or IgA.
  • SMG rat submandibular glands
  • the antibody responses were measured at 8 wks post initial DNA administration for either IgG or IgA.
  • FIGs 15A-B illustrate mucosal immune responses in saliva and lungs following salivary gland vaccination.
  • Salivary gland vaccination was compared using different DNA formulations. Either Znlipid, or Zn was co-formulated with plasmid DNA before vaccinating at weeks 0 and 3.
  • Figures 16A-B illustrate mucosal immune responses in plasma, saliva, and fecal samples following salivary gland vaccination using different adjuvants. Either CR, ZnLipid, CTb, or water (ddH2O) were co-formulated with DNA encoding gpl20 before vaccinating on weeks 0, 3.
  • Saliva samples were normalized to use equivalent amounts of total IgA before measuring the specific saliva response at weeks 6 and 9 by ELISA.
  • Fecal samples were measured on week 9 by ELISA.
  • Figure 17 illustrates antibody (IgA) responses in saliva samples following salivary gland vaccination. Rat salivary glands were vaccinated by retroductal DNA administration on weeks 0 and 3.
  • Plasmid DNA was co-formulated with either LipidZn, CR, or EB. Stimulated saliva samples were normalized to use equivalent amounts of total IgA before measuring the specific IgA response to HIV gpl20. Data are plotted for the 6 week time point.
  • the present invention provides methods and compositions for eliciting immune responses and for transfecting antigen presenting cells by retroductal delivery of compositions comprising nucleic acids encoding an immunogenic polypeptide to the lumen of a salivary gland duct.
  • the invention is based on the surprising discovery that retroductal delivery of nucleic acids is particularly effective for eliciting immune responses specific for the immunogenic peptide encoded by the nucleic acid and for transfecting antigen presenting cells.
  • Salivary glands have been used as depot organs for gene transfer and therapeutic protein expression (see, e.g., Goldfme et al, Nat. Biotechnol. 15:1378 (1997)). Unlike the skin or muscle, the anatomy and physiology of the salivary glands makes them ideal candidates as platforms for gene delivery and enhanced protein expression ⁇ see, e.g., Goldfme et al, 1997, supra and Baum and O'Connell, Crit. Rev. Oral Biol. Med. 10:276 (1999)). These organs produce and secrete large amounts of protein. The secreted protein is detected both in the blood and the saliva ⁇ see, e.g., Hoque et al, Hum. Gene Ther.
  • the major glands can be accessed by non-surgical means through the duct that opens into the oral cavity.
  • the ductal nature of these glands allows for simple and direct application of aqueous material, because retroductal infusion provides for a near complete exposure of the target cells to the gene vector without dilution.
  • retroductal delivery perfuses the entire organ regardless of the size of the animal, so delivery is scalable from mice to men.
  • retroductal administration of compositions comprising nucleic acids encoding immunogenic polypeptides as disclosed herein results in both direct and indirect priming of T cells.
  • the retroductally introduced nucleic acids encoding immunogenic polypeptides directly transfect APC, e.g., dendritic cells, which then present the immunogenic peptides to T lymphocytes, thereby generating an immune response specific for the immunogenic peptide.
  • the retroductally introduced nucleic acids encoding immunogenic polypeptides transfect non-professional antigen presenting cells, which then express the immunogenic polypeptides.
  • the expressed immunogenic polypeptides are picked up by APC, e.g., dendritic cells, which then present the immunogenic peptides to T lymphocytes, thereby generating an immune response specific for the immunogenic peptide.
  • APC e.g., dendritic cells
  • the transfection may occur within the salivary gland or within a proximal lymph node.
  • a professional APC expresses an antigen and is able to stimulate or activate a T cell
  • the stimulation or activation is referred to as direct priming of the T cell.
  • a non- professional antigen presenting cell ⁇ i.e., any cell expressing Class I MHC
  • the stimulation or activation is referred to as indirect priming of the T cell since the cell that is stimulating the T cell is not the same cell that is expressing the antigen.
  • a composition comprising a nucleic acid encoding an immunogenic polypeptide is retroductally delivered to the lumen of a salivary gland duct such that the immunogenic polypeptide or a fragment thereof is presented on the surface of the antigen presenting cell.
  • Any APC may be transfected, including, professional APC such as for example, dendritic cells, B cells or macrophages, and nonprofessional APC. Typically dendritic cells or progenitors thereof are transfected by the methods of the present invention. Dendritic cells are highly potent APCs (Banchereau et al. (1998) Nature 392:245-251).
  • dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency, and their ability to activate na ⁇ ve T cell responses.
  • Dendritic cells and their progenitors are found at low levels in peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood, lamina intestinal and Peyer's patches. Dendritic cells are conveniently categorized as "immature” and “mature” cells, which allows a simple way to discriminate between two well characterized phenotypes.
  • Immature dendritic cells are characterized as APCs with a high capacity for antigen uptake and processing, which correlates with the high expression of Fc ⁇ receptor and mannose receptor.
  • the mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules ⁇ e.g., CD54 and CD11) and costimulatory molecules ⁇ e.g., CD40, CD80, CD86 and 4-1BB).
  • the present invention overcomes the problems associated with isolation and transfection of dendritic cells by providing a method to transfect antigen presenting cells ⁇ e.g., dendritic cells) in vivo by retroductally delivering compositions comprising nucleic acids encoding immunogenic polypeptides to the lumen of the salivary gland duct.
  • the methods and compositions of the present invention are particularly useful for transfecting dendritic cells associated with the mucosal immune system.
  • compositions of the Present Invention provides compositions (z. e. , pharmaceutical compositions) comprising a nucleic acid encoding an immunogenic polypeptide.
  • the compositions may further comprise a lipid and/or a non-lipid compound, and/or a transition metal enhancer.
  • Nucleic acids encoding suitable immunogenic polypeptides may be derived from antigens, such as, for example, cancer antigens, bacterial antigens, viral antigens, fungal antigens, or parasite antigens.
  • Cancer antigens include, for example, antigens expressed, for example, in colon cancer, stomach cancer, liver cancer, pancreatic cancer, lung cancer, ovarian cancer, prostate cancer, breast cancer, skin cancer ⁇ e.g., melanoma), leukemia, lymphoma, or myeloma.
  • Exemplary cancer antigens include, for example, HPV LI, HPV L2, HPV El, HPV E2, PSA, placental alkaline phosphatase, AFP, BRCA1, Her2/neu, CA 15-3, CA 19-9, CA-125, CEA, hCG, urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor and MAGE-1.
  • Bacterial antigens may be derived from, for example, Staphylococcus aureus, Staphylococcus epidermis, Helicobacter pylori, Streptococcus bovis, Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium leprae, Corynebacterium diphtheriae, Borrelia burgdorferi, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Salmonella typhi, Vibrio chloerae, Haemophilus influenzae, Bordetella pertussis, Yersiniapestis, Neisseria gonorrhoeae, Treponema pallidum, Mycoplasm sp., Neisseria meningitidis, Legionella pneumophila, Rickettsia
  • Viral antigens may be derived from, for example, human immunodeficiency virus, human papilloma virus, Epstein Barr virus, herpes simplex virus, human herpes virus, rhinoviruses, cocksackieviruses, enteroviruses, hepatitis A, hepatitis B, hepatitis C, and hepatitis E, rotaviruses, mumps virus, rubella virus, measles virus, poliovirus, smallpox virus, influenza virus, rabies virus, and Variella-zoster virus.
  • Fungal antigens maybe derived from, for example, Tinea pedis, Tinea corporus, Tinea cruris, Tinea unguium, Cladosporium carionii, Coccidioides immitis, Candida sp.,
  • nucleic acids encoding immunogenic polypeptides are typically produced by recombinant DNA methods (see, e.g., Ausubel, et al. ed. (2001) Current Protocols in Molecular Biology).
  • the DNA sequences encoding the immunogenic polypeptide can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, or amplified from cDNA using appropriate primers to provide a synthetic gene which is capable of being inserted in a recombinant expression vector ⁇ i. e. , a plasmid vector or a viral vector) and expressed in a recombinant transcriptional unit.
  • a recombinant expression vector ⁇ i. e. , a plasmid vector or a viral vector
  • Recombinant expression vectors contain a DNA sequence encoding an immunogenic polypeptide operably linked to suitable transcriptional or translational regulatory elements derived from mammalian or viral genes.
  • suitable transcriptional or translational regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation.
  • An origin of replication and a selectable marker to facilitate recognition of transformants may additionally be incorporated.
  • the transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells in vivo may be provided by viral sources.
  • promoters and enhancers are derived, e.g., from adenovirus, simian virus (SV40), and human cytomegalo virus.
  • vectors allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in mammalian cells are suitable.
  • Further viral genomic promoter, control and/or signal sequences may be used, provided such control sequences are compatible with the host cell chosen.
  • Suitable vectors include, for example, herpes simplex virus vectors as described in Lilley et al, Curr. Gene Ther. l(4):339-58 (2001), alphavirus DNA and particle replicons as decribed in e.g., Polo et al., Dev. Biol. (Basel) 104:181-5 (2000), Epstein-Barr virus (EBV)- based plasmid vectors as described in, e.g., Mazda, Curr. Gene Ther. 2(3):379-92 (2002), EBV replicon vector systems as described in e.g., Otomo et al, J. Gene Med.
  • EBV Epstein-Barr virus
  • adeno-virus associated viruses from rhesus monkeys as described in e.g., Gao et al, PNAS USA. 99(18): 11854 (2002), adenoviral and adeno-associated viral vectors as described in , e.g., Nicklin and Baker, Curr. Gene Ther. 2(3):273-93 (2002).
  • AAV adeno- associated virus
  • Additional suitable vectors include E1B gene-attenuated replicating adenoviruses described in, e.g., Kim et al, Cancer Gene Ther.9 ⁇ 9):725-36 (2002) and nonreplicating adenovirus vectors described in e.g., Pascual et al, J. Immunol. 160(9):4465-72 (1998) Exemplary vectors can be constructed as disclosed by Okayama et al. (1983) Mol. Cell. Biol. 3:280. [0071] Molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al. (1993) J. Biol. Chem. 268:6866-6869 and Wagner et al.
  • retroviruses provide a convenient and effective platform for gene delivery systems.
  • a selected nucleotide sequence encoding an immunogenic polypeptide can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to a subject.
  • Suitable vectors include lentiviral vectors as described in e.g., Scherr and Eder, Curr. Gene Ther. 2(l):45-55 (2002).
  • the composition further comprises a transition metal enhancer.
  • Suitable transition metal enhancers include, for example, zinc chloride, zinc acetate, zinc bromide, zinc carbonate, zinc citrate, zinc fluoride, zinc halide, zinc hydroxide, zinc iodide, zinc nitrate, zinc oxide, zinc selenide, zinc sulfate, zinc telluride, or mixtures thereof.
  • Other suitable transition metal enhancers include, for example, CuCl 2 , CoCl 2 , NiCl 2 , and MgSO (Shiokawa et al, Biochem J. 326:675 (1997) and Torriglia et al, Biochimie 79:435 (1997)).
  • Transition metals enhancers that are useful include copper containing compounds such as, for example..
  • the transition metal enhancer is a nickel, cobalt, copper, aluminum or gallium halide.
  • the transition metal enhancer is NiCl 2 , CoCl 2 , CuCl 2 , AIC1 2 , or GaCI 2 .
  • the transition metal enhancers is a zinc ammonium complex together with its counter ion, zinc antimonide, zinc arsenate, zinc arsenide, zinc arsenite, zinc benzoate, zinc borate (Zn 2 Z 6 O ⁇ ), zinc perborate, zinc bromide, zinc butyrate, zinc carbonate, zinc chromate, zinc chrome, zinc chromite, zinc citrate, zinc decanoate, zinc dichromate, zinc dimer, zinc ethylenebis(dithiocarbarnate), zinc fluoride, zinc formate, zinc gluconate, zinc glycerate, zinc glycolate, zinc hydroxide, zinc iodide, zinc lactate, zinc methoxyethoxide, zinc naphthenate, zinc nitrate, zinc nitrate hexahydrate, zinc nitrate trihydrate, zinc octanoate, zinc oleate, zinc oxide, zinc pentanoate, zinc perchlorate hexahydrate
  • Additional transition metal enhancers that may be used according to the methods of the present invention include, for example, cobaltous nitrate, cobaltous oxide, cobaltic oxide, cobalt nitrite, cobaltic phosphate, cobaltous chloride, cobaltic chloride, cobaltous carbonate, chromous acetate, chromic acetate, chromic bromide, chromous chloride, chromic fluoride, chromous oxide, chromium dioxide, chromic oxide, chromic sulf ⁇ te, chromous sulfate heptahydrate, chromic sulfate, chromic formate, chromic hexanoate, chromium oxychloride, chromic phosphite, cuprous oxide, cupric oxide, cupric chloride, cuprous acetate, cuprous oxide, cuprous chloride, cupric acetate, cupric bromide, cupric chloride, cupric i
  • the transition metal enhancers of the present invention are free metals, complexes, adducts, clusters, and/or salts of zinc, copper, nickel, cobalt, aluminum or gallium.
  • the compositions further comprise a lipid or a non-lipid compound that binds to the nucleic acid.
  • a lipid or a non-lipid compound that binds to the nucleic acid it is proposed that the lipids or other compounds enhance transfection efficiency by serving as an adjuvant or by enhancing target cell absorption of the nucleic acid.
  • the lipid is a cationic lipid.
  • Suitable cationic lipids include, for example, N,N,N',N'-teframethyl-N,N'-bis(2-hydroxyethyl)-2-3-di(oleoyloxy)-l,4-butanediammonium iodide (DOHBD), N,N[bis (2-hydroxyethyl)-N-methyl-N-[2,3- di(tetradedecanoyloxy)propyl] ammonium chloride, N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”), N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTMA”), N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”), N-(2,3- dioleoyloxy) ⁇ ropyl)-N,
  • cationic lipids suitable for use in the present invention are disclosed in for example, U.S. Patent Nos. 5,527,928, 5,744,625, 5,892,071, 5,869,715, 5,824,812, 5,925,623 and 6,043,390. Additional suitable lipids are described in U.S. Patent Application No. 09/766,320, filed January 18, 2001, and WO 01/52903, filed January 19, 2001.
  • Poly(lactide-co-glycolide) (PLG)-based cationic microparticles as described in Singh et al, 2000, supra, can also be used for retroductal delivery of compositions comprising nucleic acids according to the methods of the present invention..
  • the cationic lipid may be used alone, or complexed with a charge neutral lipid before co-formulation with the nucleic acid.
  • the cationic lipid is DOHBD.
  • the cationic may be used alone or complexed with a charge neutral lipid such as, for example, dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), cholesterol.
  • DOPE dioleoylphosphatidylethanolamine
  • POPC palmitoyloleoylphosphatidylcholine
  • EPC egg phosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • cholesterol distearoylphosphatidylcholine
  • the charge neutral lipid is DOPE.
  • the cationic lipidxharge neutral lipid ratio in the complex may be about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:19, or 1:10.
  • the cationic lipid.xharge neutral lipid is 3:1.
  • the amount of lipid present in the cationic lipid/DNA/transition metal is expressed in terms of the cationic lipidDNA phosphate charge ratio. Suitable charge ratios include, for example, about 0.1, 0.25, 0.35, 0.4, 0.5, 0.75, 1.0, 1.5, and 2.0.
  • Suitable non-lipid compounds include molecules having opposite properties on each end of the molecule, for example, a protein, a polypeptide, a polypeptide fragment, a carbohydrate, a dendrimer, a receptor, a hormone, a toxin, and an amphipathic lipid.
  • Suitable non-lipid compounds include, cationic polymers such as, for example, polyethyleneimine, polylysine, polyarginine, and polyo ⁇ iithine and natural DNA-binding proteins of a polycationic nature, such as histones and protamines or analogues or fragments thereof. Additional suitable non-lipid compounds include polyamines such as, for example, spermidine and spermine, and polycations having two or more different positively charged amino acids or basic proteins. Suitable polypeptides include, for example, ID2 and peptides based on it such as, for example ID2-2, ID2-3, ID2-4 (Sperinde et al, J. Gene Med. 3:101 (2001)).
  • compositions further comprise an adjuvant.
  • adjuvants include, for example, the lipids and non-lipid compounds described above, cholera toxin (CT), CT subunit B, CT derivative CTK63, E. coli heat labile enterotoxin (LT), LT derivative LTK63, Al(OH) 3 , and polyionic organic acids as defined above and described in e.g., United States patent application 60/402,811, filed August 12, 2002 (Bennett et al, "Polyionic Organic Acid Formulations," Atty. Docket No. 020714- 000600), Anderson and Crowle, Infect. Immun.
  • Suitable polyionic organic acids include for example, 6,6'-[3,3'-demithyl[l,l'-biphenyl]-4,4'-diyl)bis(azo)bis[4- amino-5-hydroxy-l,3-naphthalene-disulfonic acid] (Evans Blue) and 3,3'-[l,l'bi ⁇ henyl]- 4,4'-diylbis(azo)bis[4-amino-l-naphthalenesulfonic acid] (Congo Red). It will be appreciated by those of skill in the art that the polyionic organic acids may be used for any genetic vaccination method in conjunction with any type of administration. Additional suitable polyionic organic acids are described in, e.g., U.S. Patent Application No. , filed August 12, 2003 (Bennett et al, "Polyionic Organic Acid
  • Suitable adjuvants include topical immunomodulators such as, members of the imidazoquinoline family such as, for example, imiquimod and resiquimod ⁇ see, e.g., Hengge et al, Lancet Infect. Dis. 1(3): 189-98 (2001).
  • Additional suitable adjuvants are commercially available as, for example, additional alum-based adjuvants ⁇ e.g., Alhydrogel, Rehydragel, aluminum phosphate, Algammulin); oil based adjuvants (Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI), Specol, RIBI, TiterMax, Montanide ISA50 or Seppic MONTANIDE ISA 720); nonionic block copolymer-based adjuvants, cytokines ⁇ e.g., GM-CSF or Flat3-ligand); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres
  • Cytokines such as GM- CSF or interleukin-2, -7, or -12, are also suitable adjuvants.
  • Hemocyanins ⁇ e.g., keyhole limpet hemocyanin
  • Polysaccharide adjuvants such as, for example, chitin, chitosan,, and deacetylated chitin are also suitable as adjuvants.
  • Other suitable adjuvants include muramyl dipeptide (MDP, N acetylmuramyl L alanyl D isoglutamine) bacterial peptidoglycans and their derivatives ⁇ e.g., threonyl-MDP, and MTPPE).
  • BCG and BCG cell wall skeleton may also be used as adjuvants in the invention, with or without trehalose dimycolate.
  • Trehalose dimycolate may be used itself ⁇ see, e.g., U. S. Patent No. 4,579,945).
  • Detoxified endotoxins are also useful as adjuvants alone or in combination with other adjuvants (see, e.g., U. S. Patent Nos. 4,866,034; 4,435,386; 4,505,899; 4,436,727; 4,436,728; 4,505,900; and 4,520,019.
  • the saponins QS21, QSI7, QS7 are also useful as adjuvants (.see, e.g., US Patent No. 5,057,540; EP 0362 279; WO 96/33739; and WO 96/11711).
  • Other suitable adjuvants include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants ⁇ e.g., SB AS-2, SBAS-4 or SBAS-6 or variants thereof, available from SmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton, MT), and RC-529 (Corixa, Hamilton, MT).
  • Superantigens are also contemplated for use as adjuvants in the present invention.
  • Superantigens include Staphylococcus exoproteins, such as the ⁇ , ⁇ , ⁇ and ⁇ enterotoxins from S. aureus and S. epidermidis, and the ⁇ , ⁇ , ⁇ and ⁇ E. coli exotoxins.
  • Staphylococcus enterotoxins are known as staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB), with enterotoxins through E (SEE) being described (Rott et al, 1992).
  • the adjuvant composition can be designed to induce, e.g., an immune response predominantly of the Thl or Th2 type .
  • Thl -type cytokines e.g., IFN- ⁇ , TNF ⁇ , IL-2 and IL-12
  • Th2-type cytokines e.g., IL-4, IL-5, IL-6 and IL-10
  • an immune response that includes Thl- and Th2-type responses will typically be elicited.
  • compositions within the scope of the present invention may also contain other compounds, which may be biologically active or inactive.
  • Polypeptides may, but need not, be conjugated to other macromolecules as described, for example, within U.S. Patent Nos. 4,372,945 and 4,474,757.
  • Pharmaceutical compositions may generally be used for prophylactic and therapeutic purposes.
  • a pharmaceutical composition or vaccine may contain a polynucleotide encoding an immunogenic polypeptide.
  • a polynucleotide may comprise DNA, RNA, a modified nucleic acid or a DNA/RNA hybrid.
  • a polynucleotide may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland (1998) Crit. Rev. Therap. Drug Carrier Systems 15:143-198, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal).
  • a vaccine may contain pharmaceutically acceptable salts of the polynucleotides encoding immunogenic polypeptides.
  • Such salts may be prepared from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).
  • Suitable carriers include, for example, water, saline, alcohol, a fat, a wax, a buffer, a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, or biodegradable microspheres ⁇ e.g., polylactate polyglycolate).
  • Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883.
  • the immunogenic polypeptide maybe encapsulated within the biodegradable microsphere or associated with the surface of the microsphere.
  • compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates ⁇ e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives.
  • buffers e.g., neutral buffered saline or phosphate buffered saline
  • carbohydrates ⁇ e.g., glucose, mannose, sucrose or dextrans
  • mannitol proteins
  • polypeptides or amino acids such as glycine
  • antioxidants e.g., antioxidants, bacteriostats, chelating
  • Any vaccine provided herein may be prepared using well known methods that result in a combination of antigen, immune response enhancer and a suitable carrier or excipient.
  • the compositions described herein may be administered as part of a sustained release formulation ⁇ i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration).
  • a sustained release formulation ⁇ i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration.
  • Such formulations may generally be prepared using well known technology (-fee, e.g., Coombes et al. (1996) Vaccine 14:1429-1438).
  • Sustained- release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane.
  • Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release.
  • Such carriers include microparticles of poly(lactide-co-glycolide), as well as polyacrylate, latex, starch, cellulose and dextran.
  • Other delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core ⁇ e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid ⁇ see, e.g., U.S. Patent No.
  • compositions may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use.
  • formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles.
  • a pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.
  • a composition comprising a nucleic acid encoding an immunogenic polypeptide is retroductally introduced into the lumen of a salivary gland duct.
  • the nucleic acid may be a in a vector as described above or may be "naked” as described in, e.g., Ulmer et al. (1993) Science 259:1745-1749 and reviewed by Cohen (1993) Science 259:1691-1692.
  • the composition may be introduced alone or with an adjuvant as described above.
  • the adjuvant is administered at the same time as the composition.
  • the adjuvant is administered after the composition, e.g., 6, 12, 18, 24, 36, 48, 60, or 72 hours after administration of the composition.
  • Suitable methods of retroductal introduction of the composition to the salivary gland duct include, for example, cannulation or injection of the composition into the salivary gland duct using a syringe, cannula, catheter, or shunt.
  • the type of syringe, cannula, catheter, or shunt used is not a critical part of the invention.
  • One of skill in the art will appreciate that multiple types of syringes, cannulas, catheters, or shunts maybe used to administer compositions according to the methods of the present invention.
  • Retroductal delivery of the composition using the methods of the present invention may be via gravity or an assisted delivery system.
  • Suitable assisted delivery systems include controlled release pumps, time release pumps, osmotic pumps, and infusion pumps.
  • the particular delivery system or device is not a critical aspect of the invention.
  • One of skill in the art will appreciate that multiple types of assisted delivery systems may be used to deliver compositions according to the methods of the present invention. Suitable delivery systems and devices are described in U.S. Patent Nos. 5,492,534, 5,562,654, 5,637,095, 5,672,167, and 5,755,691.
  • the infusion rate for delivery of the composition may be varied.
  • Suitable infusion rates may be from about 0.005 ml/min to about 1 ml/minute, preferably from about 0.01 ml/min to about 0.8 ml/min., more preferably from about 0.025 ml/min. to about 0.6 ml/min. It is particularly preferred that the infusion rate is about 0.05 ml min.
  • polynucleotides that encode immunogenic polypeptides are used to generate an immune response ⁇ i.e., a mucosal, humoral, or cell-mediated immune response) to antigens, such as, for example, cancer antigens, bacterial antigens, viral antigens, fungal antigens, or parasite antigens.
  • antigens such as, for example, cancer antigens, bacterial antigens, viral antigens, fungal antigens, or parasite antigens.
  • cancer antigens include antigens expressed, for example, in colon cancer, stomach cancer, liver cancer, pancreatic cancer, lung cancer, ovarian cancer, prostate cancer, breast cancer, skin cancer (e.g., melanoma), leukemia, lymphoma, or myeloma.
  • Exemplary cancer antigens include, for example, HPV LI, HPV L2, HPV El, HPV E2, PSA, placental alkaline phosphatase, AFP, BRCA1, Her2/neu, CA 15-3, CA 19-9, CA-125, CEA, hCG, urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor and MAGE-1.
  • Bacterial antigens may be derived from, for example, Staphylococcus aureus,
  • Staphylococcus epidermis Helicobacter pylori, Streptococcus bovis, Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium leprae, Corynebacterium diphtheriae, Borrelia burgdorferi, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Salmonella typhi, Vibrio chloerae, Haemophilus influenzae, Bordetella pertussis, Yersinia pestis, Neisseria gonorrhoeae, Treponema pallidum, Mycoplasm sp., Neisseria meningitidis, Legionella pneumophila, Rickettsia typhi, Chlamydia trachomatis, and Shigella dysenteriae.
  • Viral antigens may be derived from, for example, human immunodeficiency virus, human papilloma virus, Epstein Barr virus, herpes simplex virus, human herpes virus, rhinoviruses, cocksackieviruses, enteroviruses, hepatitis A, hepatitis B, hepatitis C, and hepatitis E, rotaviruses, mumps virus, rubella virus, measles virus, poliovirus, smallpox virus, influenza virus, rabies virus, and Variella-zoster virus.
  • Fungal antigens may be derived from, for example, Tinea pedis, Tinea corporus, Tinea cruris, Tinea unguium, Cladosporium carionii, Coccidioides immitis, Candida sp., Aspergillus fumigatus, and Pneumocystis carinii.
  • Parasite antigens may be derived from, for example, Giardia lamblia, Leishmania sp., Trypanosoma sp., Trichomonas sp., Plasmodium sp., and Schistosoma sp.
  • An immune response to the immunogenic polypeptides can be long-lived and can be detected long after immunization, regardless of whether the protein is present or absent in the body at the time of testing.
  • An immune response to the immunogenic polypeptide can be detected by examining for the presence or absence, or enhancement, of specific activation of CD4 + or CD8 + T cells or by antibodies. For instance, T cells isolated from an immunized individual by routine techniques (e.g., by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes) are incubated with the immunogenic polypeptide.
  • T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37°C with an immunogenic polypeptide (typically, about 0.2 to about 5 ⁇ g/ml). It may be desirable to incubate another aliquot of a T cell sample in the absence of the immunogenic polypeptide to serve as a control.
  • an immunogenic polypeptide typically, about 0.2 to about 5 ⁇ g/ml
  • Specific activation of CD4 + or CD8 + T cells associated with a mucosal, humoral, or cell-mediated immune response may be detected in a variety of ways.
  • Methods for detecting specific T cell activation include, but are not limited to, detecting the proliferation of T cells, the production of cytokines (e.g. , lymphokines), or the generation of cytolytic activity ⁇ i. e. , generation of cytotoxic T cells specific for the immunogenic polypeptide).
  • cytokines e.g. , lymphokines
  • cytolytic activity ⁇ i. e. , generation of cytotoxic T cells specific for the immunogenic polypeptide.
  • a preferred method for detecting specific T cell activation is the detection of the proliferation of T cells.
  • a preferred method for detecting specific T cell activation is the detection of the generation of cytolytic activity using 51 Cr release assays ⁇ see, e.g., Brossart and Be ⁇ an, Blood 90(4): 1594-1599 (1997) and Lenz et /., J. Exp. Med. 192(8):1135-1142 (2000)).
  • T cell proliferation can be detected by measuring the rate of DNA synthesis.
  • T cells which have been stimulated to proliferate exhibit an increased rate of DNA synthesis.
  • a typical way to measure the rate of DNA synthesis is, for example, by pulse- labeling cultures of T cells with tritiated thymidine, a nucleoside precursor which is incorporated into newly synthesized DNA. The amount of tritiated thymidine incorporated can be determined using a liquid scintillation spectrophotometer.
  • T cell proliferation examples include measuring increases in interleukin-2 (IL-2) production, Ca 2+ flux, or dye uptake, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.
  • IL-2 interleukin-2
  • dye uptake such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.
  • synthesis of lymphokines ⁇ e.g., interferon- gamma) can be measured or the relative number of T cells that can respond to the immunogenic polypeptide may be quantified.
  • Humoral immune responses, including mucosal humoral responses can be detected using immunoassays known in the art. Suitable immunoassays include the double monoclonal antibody sandwich immunoassay technique of David et al. (U.S. Patent No.
  • Monoclonal antibodies to the immunogenic peptides can be generated using methods known in the art ⁇ see, e.g., Kohler and Milstein, Nature 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)). Generation of monoclonal antibodies has been previously described and can be accomplished by any means known in the art. (Buhring et al. in Hybridoma 1991, Vol. 10, No. 1, pp. 77-78).
  • an animal such as a guinea pig or rat, preferably a mouse is immunized with an immunogenic polypeptide
  • the antibody- producing cells preferably splenic lymphocytes
  • a stable, immortalized cell line preferably a myeloma cell line
  • Binding of a monoclonal antibody to the immunogenic polypeptide presented on the surface the transfected cell may be detected by direct (in the case of labeled antibodies) or indirect (in the case of unlabeled antibodies) methods known in the art and described in e.g., Ausubel et al, supra and Harlow and Lane, 1999, supra.
  • flow cytomerry or enzyme linked immunosorbent assays can be used to detect MHC Class II/ ⁇ eptide complexes or MHC Class I/peptide complexes on the surface of antigen presenting cells.
  • the particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody to the immunogenic polypeptide.
  • the detectable group can be any material having a detectable physical or chemical property.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, electrical, optical or chemical means.
  • a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • Useful labels in the present invention include magnetic beads (e.g., DYNABEADSTM), fluorescent dyes ⁇ e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).
  • the molecules can be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
  • the label may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence.
  • the fluorescence maybe detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge coupled devices
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected simply by observing the color associated with the label.
  • conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • Incubation steps can vary from about 5 seconds to several hours, optionally from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, antigen, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C.
  • immunoassays One of skill in the art will appreciate that it is often desirable to minimize nonspecific binding in immunoassays. Particularly, where the assay involves an antigen or antibody immobilized on a solid substrate it is desirable to minimize the amount of nonspecific binding to the substrate. Means of reducing such non-specific binding are well known to those of skill in the art. Typically, this technique involves coating the substrate with a proteinaceous composition. In particular, protein compositions such as bovine serum albumin (BSA), nonfat powdered milk, and gelatin are widely used with powdered milk being most preferred.
  • BSA bovine serum albumin
  • immunogenic polypeptides ⁇ i.e., antigens
  • immunogenic polypeptides may either be labeled or unlabeled.
  • immunogenic polypeptides find use in agglutination assays.
  • unlabeled immunogenic polypeptides can be used in combination with labeled molecules that are reactive with immunocomplexes, or in combination with labeled antibodies (second antibodies) that are reactive with the antibody directed against the immunogenic polypeptide.
  • the immunogenic polypeptide can be directly labeled.
  • the reporter group can include, e.g., radioisotopes, fluorophores, enzymes, luminescers, dye particles and the like.
  • the immunogenic polypeptide is adsorbed to the surface of a microtiter well. Residual protein-binding sites on the surface are then blocked with an appropriate agent, such as bovine serum albumin (BSA), heat-inactivated normal goat serum (NGS), or BLOTTO (buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent).
  • BSA bovine serum albumin
  • NGS heat-inactivated normal goat serum
  • BLOTTO bidirectional antifoaming agent
  • the well is then incubated with a sample (e.g., a biological sample from the subject to whom the composition comprising a nucleic acid encoding the immunogenic polypeptide was administered) suspected of containing specific antibody.
  • a sample e.g., a biological sample from the subject to whom the composition comprising a nucleic acid encoding the immunogenic polypeptide was administered
  • the sample can be applied neat, or, more often, it can be diluted, usually in a buffered solution which contains a small amount (0.1%-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO.
  • a buffered solution which contains a small amount (0.1%-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO.
  • the reporter group can be chosen from a variety of enzymes, including, e.g., horseradish peroxidase, beta-galactosidase, alkaline phosphatase, and glucose oxidase. Sufficient time is allowed for specific binding to occur, then the well is again washed to remove unbound conjugate, and the substrate for the enzyme is added. Color is allowed to develop and the optical density of the contents of the well is determined visually or instrumentally.
  • enzymes including, e.g., horseradish peroxidase, beta-galactosidase, alkaline phosphatase, and glucose oxidase.
  • a reporter group is bound to the immunogenic polypeptide of interest.
  • the step of detecting immunocomplexes involves removing substantially any unbound immunogenic polypeptide and then detecting the presence or absence of the reporter group.
  • a reporter group is bound to a second antibody capable of binding to the antibodies specific for immunogenic polypeptide.
  • the step of detecting immunocomplexes involves (a) removing substantially any unbound antibody, (b) adding the second antibody, (c) removing substantially any unbound second antibody and then (d) detecting the presence or absence of the reporter group.
  • the second antibody is an anti-human antibody.
  • a reporter group is bound to a molecule capable of binding to the immunocomplexes.
  • the step of detecting involves (a) adding the molecule, (b) removing substantially any unbound molecule, and then (c) detecting the presence or absence of the reporter group.
  • An example of a molecule capable of binding to the immunocomplexes is protein A.
  • Reporter groups suitable for use in any of the methods include, e.g., radioisotopes, fluorophores, enzymes, luminescers, and dye particles.
  • One aspect of the present invention involves using the immunogenic compositions described herein to elicit an antigen specific immune response from a subject or patient with a disease such as, for example, a viral infection, bacterial infection, a parasitic infection, a fungal infection, or cancer.
  • a "subject” or a “patient” refers to any warmblooded animal, such as, for example, a rodent, a feline, a canine, or a primate, preferably a human.
  • the immunogenic compositions may be used to treat at any stage of the disease, i.e., at the pre-cancer, cancer, or metastatic stages, or to prevent disease.
  • compositions described herein may be used for immunotherapy ⁇ i.e., prevention or treatment) of cancer, such as breast, ovarian, colon, lung and prostate cancer.
  • pharmaceutical compositions are typically administered to a patient.
  • a patient may or may not be afflicted with cancer.
  • the above pharmaceutical compositions may be used to prevent the development of a cancer or to treat a patient afflicted with a cancer.
  • a cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor.
  • Pharmaceutical compositions may be administered either prior to or following surgical removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemotherapeutic drugs.
  • compositions described herein may be used for immunotherapy ⁇ i.e., prevention or treatment) of bacterial, viral, fungal, or parasitic diseases and disorders.
  • pharmaceutical compositions are typically administered to a patient.
  • a patient may or may not be afflicted with the disease or disorder.
  • the above pharmaceutical compositions may be used to prevent the development of a particular disease or disorder or to treat a patient afflicted with a disease or disorder.
  • a disease or disorder may be diagnosed using criteria generally accepted in the art.
  • Immunotherapy is typically active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against, e.g., tumors or bacterially or virally infected cells, with the administration of immune response-modifying agents (compositions comprising nucleic acids encoding immunogenic polypeptides as provided herein).
  • immune response-modifying agents compositions comprising nucleic acids encoding immunogenic polypeptides as provided herein.
  • Frequency of administration of the prophylactic or therapeutic compositions described herein, as well as dosage will vary from individual to individual, and may be readily established using standard techniques. Often between 1 and 10 doses may be administered over a 52 week period. Typically 6 doses are administered, at intervals of 1 month, more typically, 2-3 doses are administered every 2-3 months. Booster vaccinations may be given periodically thereafter.
  • a suitable dose is an amount of a compound that, when administered as described above, is capable of promoting, e.g., an anti-tumor, an anti- viral, or an antibacterial, immune response, and is at least 10-50% above the basal (t.e., untreated) level.
  • Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing, e.g., the patient's tumor cells, the patient's virally infected cells, or the patient's bacterially infected cells in vitro.
  • the amount of the nucleic acid encoding an immunogenic polypeptide present in a dose ranges from about 1 ⁇ g to 5 mg, preferably 100 ⁇ g to 5 mg, and most preferably 5 ⁇ g to 300 ⁇ g per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.01 ml to about 10 ml, more typically from about 0.025 to about 7.5 ml, most typically from about 0.05 to about 5 ml. Those of skill in the art will appreciate that the dose size may be adjusted based on the particular patient or the particular disease or disorder being treated.
  • an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit.
  • a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients.
  • Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays described above, which may be performed using samples obtained from a patient before and after treatment.
  • detection of immunocomplexes formed between immunogenic polypeptides and antibodies in body fluid which are specific for immunogenic polypeptides may be used to monitor the effectiveness of therapy, which involves a particular immunogenic polypeptide, for a disease or disorder in which the immunogenic polypeptide is associated.
  • Samples of body fluid taken from an individual prior to and subsequent to initiation of therapy may be analyzed for the immunocomplexes by the methodologies described above. Briefly, the number of immunocomplexes detected in both samples are compared. A substantial change in the number of immunocomplexes in the second sample (post-therapy initiation) relative to the first sample (pre-therapy) reflects successful therapy.
  • Example 1 Materials and Methods [0123] Animals and plasmids: Sprague-Dawley rats were obtained from Harlan
  • Plasmid encoding the human growth hormone (hGH) gene was originally obtained from M. German (see, e.g., Goldfme et al, Nat. Biotechnol. 15(13):1378-1382 (1997) and placed under the control of the CMV promoter to generate pFOXCMVhuGH-G3.
  • the DNA encoding HIV envelope (gpl20) was obtained from the NIH AIDS Research and Reference Reagent Program ⁇ see, e.g., Andre et al, J. Virol. 72(2): 1497-1503 (1998) and placed under control of the CMV promoter.
  • Plasmids were purified under endotoxin-reduced conditions using Qiagen's Gigaprep kits. Endotoxin less than 100 E.U./mg DNA as measured by clot LAL assay (Charles River's Endosafe).
  • DNA and protein vaccination Retroductal DNA delivery to the salivary glands has been described previously ⁇ see, e.g., Goldfme et al, 1997, supra and U.S. Patent No. 6,372,722). Briefly, after anesthesia polyethylene (PE) 10 tubes were inserted into the left and right duct openings of the submandibular glands. Aqueous solutions of DNA were instilled by retrograde infusion using a syringe pump.
  • PE polyethylene
  • Each infusion contained either 88 ⁇ g or 175 ⁇ g in 200 ⁇ l water per gland, depending on the experiment, hi order to enhance expression, DNA was co-formulated in one experiment with 3.6mM Zn in water and co- formulated in two experiments using a lipid and 0.125 mM Zn (Lipid/Zn) formulation ⁇ see, e.g., U.S. Patent No. 6,372,722 and Pichon et al, J. Gene Med. (2002), available at www3.interscience.wiley.com/cgi-bin/abstract/94518178/START).
  • the lipid was a 3:1 ratio of N,N,N' ,N' -tetramethyl-N,N' -bis(2-hydroxyethyl)-2,3-di(oleoyloxy)- 1 ,4-butanediaminium iodide: dioleoylphosphatidylethanolamine (DOHBD:DOPE) ⁇ see, e.g., U.S. Patent No. 5,527,928).
  • DOHBD dioleoylphosphatidylethanolamine
  • ELISA Assays To measure gene expression with the salivary glands, the glands were removed by dissection, added to phosphate buffered saline, and homogenized to make lysates. hGH protein was measured by commercial ELISA kit (Roche, Indianapolis, IN).
  • microtiter plates were coated in IX carbonate buffer with 1.0 ⁇ g/ml protein, either hGH (Fitzgerald, Concord, MA) or gpl20 (Fitzgerald). Plates were incubated overnight at 4 °C in a humidified chamber, then the plates were blocked in PBS + 0.05% Tween 20 (PBST) + 1% Bovine serum albumin (BSA) solution for 1 hour before washing. Plasma samples were serially diluted in PBST. After a two hour incubation, plates were washed with PBST at least 6 times.
  • PBST PBS + 0.05% Tween 20
  • BSA Bovine serum albumin
  • Antibodies were then added, either anti-rat IgG-Horse Radish Peroxidase (HRP) (Sigma) or anti-rat IgA-HRP (Bethyl labs. Montgomery, TX.), or anti-rat secretory component-HRP (Bethyl labs) each at 1 :2000 dilution. Plates were washed at least 6 times after a 1 hour incubation. Antigen specific rat antibodies were detected with 3,3',5,5'-tetramethyl-Benzidine (TMB) substrate (Dako) using a microplate reader. Antibody titers were reported as the reciprocal dilution giving an absorbance value greater than 2 standard deviations times the average background. O.D.
  • IgA salivary IgA
  • saliva was collected by cannulating the salivary duct followed by l.Orng/ml pilocarpine injection s.c.
  • Total IgA was measured by coating ELISA plates with mouse anti-rat IgA at 5 ⁇ g/ml (Biosource) and detecting with mouse anti-rat light chain-HRP at 1:1000 dilution (Serotec, Raleigh, NC) using a rat IgA standard (Biosource).
  • ELISPOTS To assess the numbers of antibody secreting cells (ASC), an enzyme- linked immunospot (ELISpot) was performed as described ⁇ see, e.g., Yamamoto et al, J. Immunol. 161(8):4l 15-4121 (1998)). Millipore HA plates were coated with 50 ⁇ l/well of lO ⁇ g/ml gpl20 protein under sterile conditions and incubated overnight at 4 °C in a humidified chamber. The next day, the plate was washed with sterile PBST and blocked with w/ RPMI+5% fetal bovine serum (FBS) for 1 hour at 37°C while shaking.
  • FBS w/ RPMI+5% fetal bovine serum
  • PBST+1%BSA at desired concentrations.
  • T cell assays Splenocytes were isolated by breaking the spleen capsula and then pushing the cells through a sterile strainer into NH C1 2 lysis solution. After a 5 minute incubation, the cells were washed in IMDM media (Invitrogen, San Diego, CA)+ 10% serum and counted.
  • IMDM media Invitrogen, San Diego, CA
  • ⁇ -IFN ⁇ -interferon
  • Example 2 Systemic IgG and IgA responses are elicited by salivary gland genetic vaccination.
  • Retroductal delivery of gene vectors to the salivary glands has been described as an efficient method of both local and systemic protein delivery.
  • plasmid DNA encoding human growth hormone (hGH) was delivered to the salivary glands (submandibular) of rats, significant levels of hGH protein was detected in the glands 7 days after DNA administration ( Figure 1).
  • the amount of protein present decreased substantially over 28 days, and that retreatment of the gland with plasmid did not restore expression ( Figure 1).
  • plasmid DNA encoding hGH protein was delivered to the salivary glands by retroductal delivery.
  • an equal amount of hGH DNA was injected i.m. and hGH protein, formulated with complete Freund's adjuvant (CFA), was injected s.c.
  • Plasma from the animals was collected 3 weeks after DNA or protein treatment. The plasma was analyzed by ELISA for anti-hGH IgG antibody titers.
  • RSGV HIV envelope protein gpl20
  • Rats were treated with equal amounts of gpl20 DNA by RSGV or by i.m. injection. Plasma samples were assayed 6 weeks after the first treatment, two weeks after the last DNA treatment for a total of two administrations. Rats were also treated with gpl20 protein plus CFA as a control. The plasma IgG and IgA responses to gpl20 were typically better with RSGV than with i.m., although the IgG titers were stastically insignificant in this experiment (Figure 3A).
  • the anti-gpl20 IgG and IgA titers resulting from protein plus CFA were similar to RSGV, with the average IgG slightly higher with protein.
  • the temporal pattern of IgG production was also evaluated ( Figure 3B).
  • the antibody responses to gpl20 protein at 9 weeks reached an average IgG titer above 8.0xl0 3 .
  • Example 3 Both CD8 and CD4 T cells responses are induced upon salivary gland vaccination.
  • RSGV resulted in significantly more ⁇ -IFN secreted by cultured splenocytes than either i.m. DNA or s.c. protein, and the ⁇ -IFN produced increased with increasing amounts of antigen (Figure 4A).
  • T cell activity was examined using an intracellular ⁇ -IFN flow cytometry assay. This assay provided additional information as to which T cells could be contributing to the secreted ⁇ -IFN shown above. Animals were vaccinated 3 times, as described previously, and harvested one week after the last DNA inoculation.
  • Example 4 Mucosal immune responses are significantly heightened by salivary gland DNA vaccination.
  • Peyer's patches are part of the gut associated lymphoid tissue (GALT), and IgA secreted from the Peyer's patches contributes significantly to the total IgA found within the lumen of the small intestine ⁇ see, e.g., Brandzaeg et al, 1988, supra)).
  • Animals were treated with DNA encoding gpl20 by RSGV or by i.m. injection on weeks 0, 4, and 8 (same animals as described for the ⁇ -IFN intracellular T cell assay in Figure 4B). Cells were isolated from the Peyer's patches and the numbers of IgA producing cells from the Peyer's patches were measured one week after the last DNA delivery ⁇ see, e.g., Williams et al, J.
  • IgA and IgG responses to gpl20 were measured in lung lavages (Fig. 6C).
  • Antibodies that recognize gpl20 were present in the lung lavages of RSGV animals, with significantly higher O.D. values for IgA isotype responses over untreated animals.
  • Dimeric IgA at mucosal surfaces contains the secretory component, a remnant of active transport of the antibody through epithelial cells of the mucosa, whereas the majority of IgA in blood is monomeric in form and lacks the secretory component ⁇ see, e.g., Brandtzaeg, J Reprod. Immunol. 36(1-2): 23-50 (1997)).
  • vaginal washes in our experimental animals contained almost no IgA and significant amounts of total IgG.
  • the vaginal washes contained less than 4 ng/ml IgA whereas the amount of total IgG was above 350 ng/ml. For this reason, only specific IgG was measured in the vaginal washes.
  • Anti-g ⁇ l20 IgG values were found to be significantly above the values from the naive animals (Fig. 6A).
  • DNA encoding hGH was co-formulated with ZnCl 2 alone or with ZnCl and 200 ⁇ g DOHBD:DOPE (3:1), then retroductally delivered to submandibular salivary glands of Sprague-Dawley rats at weeks 0 and 6. 88 ⁇ g DNA encoding hGH was administered per submandibular salivary gland. Nine weeks after delivery, hGH specific IgA titers were analyzed. The results are shown in Figure 8.
  • Example 7 Dendritic Cells are Transfected by Retroductal Introduction of Compositions Comprising Nucleic Acids into the Lumen of the Salivary Gland Duct.
  • Anti-hGH IgA was detected using an ELISA as follows. Briefly, 96 well microtiter plates were coated with hGH at lug/ml. 25ng/mL of lung lavage supernatant was added to each well of the first row of hGH coated ELISA plate. Subsequent rows received serially diluted samples, diluted 1 :3 from 25ng/mL to O.Olng/mL with PBST. Sample dilution was based on the concentration of total IgA content of each lung lavage sample as determined by non-specific rat IgA ELISA. The secondary is goat anti-Rat IgG HRP at 1 :2000 dilution. The results are shown in Figure 10.
  • Adesanya et al. have reported inflammation of the salivary glands after retroductal delivery of adenoviral vectors to rats ⁇ see, e.g., Adesanya et al, Hum. Gene Ther. 7:1085 (1996)).
  • Kawabata et al. report that needle injection of DNA in mice, through the submucosal tissue and into the salivary gland, can produce humoral responses against the encoded protein ⁇ see, e.g., Kawabata et al, Infect. Immun. 67:5863 (1999)).
  • the needle injection technique described by Kawabata et al. is similar to i.m. or s.c. in that the DNA is likely poorly distributed within the target tissue.
  • the more robust immune responses e.g., T cell activity and distal mucosal responses
  • the more robust immune responses may be derived from the perfusion of the gland with DNA solution.
  • the results presented here reveal that needle injection into the submucosa yields immune activity that is inferior to retroductal perfusion of the gland ( Figure 2B).
  • Example 10 HIV Neutralization Following Genetic Immunization
  • DNA encoding anthrax protective antigen (PA) in 200 ⁇ l with 4 mg/ml Congo Red was (1) retroductally introduced (50 ⁇ l/min.) into the lumen of each submandibular salivary gland duct or (2) intramuscularly introduced into the leg of Sprague/Dawley rats on week 0, 3, 6, and 9.
  • DNA was formulated in compositions comprising water; Zn (0.125 mM)/DOHBD:DOPE lipid (3:1); Evans Blue (4 mg/ml); or Congo Red (4 mg/ml).
  • Anti-PA IgG titer was measured by ELISA over 14 weeks.
  • the Congo Red-treated animals were then challenged with 10X the minimum lethal dose of anthrax toxin. Table 1 shows that all animals with antibody titers above 5000 survived while the negative control animals all died within 2 hours.
  • mice were treated by (1) retroductally introducing DNA encoding anthrax protective antigen (PA) or human growth hormone (hGH) into the salivary gland (SG), (2) intramuscularly (i.m.) introducing DNA encoding PA, or (3) subcutaneously (s.c.) injecting PA protein with complete Freund's adjuvant (CFA).
  • PA anthrax protective antigen
  • hGH human growth hormone
  • CFA complete Freund's adjuvant
  • mice were challenged with 10X the minimum lethal dose of anthrax toxin. All intramuscularly vaccinated, negative control, and irrelevant DNA (hGH) vaccinated animals died within 3 hours (Table 2). In contrast, 4 out of 5 SG vaccinated animals with titers above 1000 survived for longer than 24 hours and had no clinical signs of exposure to the toxin. The results are shown in Table 2 below: Table 2.
  • Example 12 hGH Expression in Tissue and anti-hGH Responses in the Plasma of Rats Following RSGV
  • Example 13 Enhancement of Mucosal Immune Responses to HIV Envelope Protein following DNA vaccination to the SMG.
  • CTb Cholera toxin B subunit
  • ZnLipid Zn Lipid formulation
  • CR Congo Red
  • Fig. 16 A the fecal results appear to show potentially equivalent results between CTb and CR formulations. Many of these animals had elevated O.D. values (Fig. 16B).

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  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Mycology (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Communicable Diseases (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des compositions et des méthodes pour déclencher une réponse immunitaire, ainsi que des compositions et des méthodes pour transfecter des cellules présentatrices d'antigènes.
PCT/US2003/026872 2002-08-30 2003-08-26 Vaccination genetique retrocanalaire par l'intermediaire des glandes salivaires WO2004020592A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003262922A AU2003262922A1 (en) 2002-08-30 2003-08-26 Retroductal salivary gland genetic vaccination

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US40737502P 2002-08-30 2002-08-30
US60/407,375 2002-08-30
US45399903P 2003-03-11 2003-03-11
US60/453,999 2003-03-11
US63993503A 2003-08-12 2003-08-12
US10/639,935 2003-08-12

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WO2004020592A2 true WO2004020592A2 (fr) 2004-03-11
WO2004020592A3 WO2004020592A3 (fr) 2004-08-12

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011034950A1 (fr) 2009-09-16 2011-03-24 Vaxart, Inc. Stratégie d'immunisation pour empêcher une infection par le h1n1
EP3791859A1 (fr) 2015-06-12 2021-03-17 Vaxart, Inc. Formulations pour l'administration dans l'intestin grêle d'antigènes du rsv et des norovirus
EP4353255A2 (fr) 2014-02-20 2024-04-17 Vaxart, Inc. Formulations pour administration intestinale à faible volume

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5593972A (en) * 1993-01-26 1997-01-14 The Wistar Institute Genetic immunization
US5744460A (en) * 1996-03-07 1998-04-28 Novartis Corporation Combination for treatment of proliferative diseases
US5962429A (en) * 1996-11-22 1999-10-05 University Of Iowa Complexes of adenovirus with cationic molecules
US6004944A (en) * 1995-03-24 1999-12-21 The Regents Of The University Of California Protein delivery by secretory gland expression
US6372722B1 (en) * 2000-01-19 2002-04-16 Genteric, Inc. Method for nucleic acid transfection of cells

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5593972A (en) * 1993-01-26 1997-01-14 The Wistar Institute Genetic immunization
US6004944A (en) * 1995-03-24 1999-12-21 The Regents Of The University Of California Protein delivery by secretory gland expression
US5744460A (en) * 1996-03-07 1998-04-28 Novartis Corporation Combination for treatment of proliferative diseases
US5962429A (en) * 1996-11-22 1999-10-05 University Of Iowa Complexes of adenovirus with cationic molecules
US6372722B1 (en) * 2000-01-19 2002-04-16 Genteric, Inc. Method for nucleic acid transfection of cells

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011034950A1 (fr) 2009-09-16 2011-03-24 Vaxart, Inc. Stratégie d'immunisation pour empêcher une infection par le h1n1
EP4353255A2 (fr) 2014-02-20 2024-04-17 Vaxart, Inc. Formulations pour administration intestinale à faible volume
EP3791859A1 (fr) 2015-06-12 2021-03-17 Vaxart, Inc. Formulations pour l'administration dans l'intestin grêle d'antigènes du rsv et des norovirus

Also Published As

Publication number Publication date
AU2003262922A8 (en) 2004-03-19
AU2003262922A1 (en) 2004-03-19
WO2004020592A3 (fr) 2004-08-12

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