WO1999008689A1 - Immunisation des muqueuses au moyen de techniques d'apport utilisant des particules - Google Patents

Immunisation des muqueuses au moyen de techniques d'apport utilisant des particules Download PDF

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WO1999008689A1
WO1999008689A1 PCT/US1998/017637 US9817637W WO9908689A1 WO 1999008689 A1 WO1999008689 A1 WO 1999008689A1 US 9817637 W US9817637 W US 9817637W WO 9908689 A1 WO9908689 A1 WO 9908689A1
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mammal
mucosal
nucleic acid
dna
cells
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PCT/US1998/017637
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Dennis E. Mccabe
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Powderject Vaccines, Inc.
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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
    • A61K39/145—Orthomyxoviridae, e.g. influenza 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
    • 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
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates generally to methods of immunization. More particularly, the invention pertains to the delivery of nucleic acid molecules or peptide antigens into mucosal tissue using particle-mediated delivery techniques .
  • live vaccines include attenuated microbes and recombinant molecules based on a living vector.
  • the dead vaccines include those based on killed whole pathogens, and subunit vaccines, e.g., soluble pathogen subunits or protein subunits.
  • Live vaccines are generally successful in providing an effective immune response in immunized subjects; however, such vaccines can be dangerous in immunocompromised or pregnant subjects, can revert to pathogenic organisms, or can be contaminated with other pathogens. Hassett et al .
  • Dead vaccines avoid the safety problems associated with live vaccines; however such vaccines often fail to provide an appropriate and/or effective immune response in immunized subjects. More recently, direct injection of DNA and mRNA into mammalian tissue for the purpose of eliciting an immune response has been described. See, e.g., U.S. Patent No. 5,589,466. The method, termed "naked DNA immunization,” has been reported to elicit both humoral and cell-mediated immune responses following DNA delivery to muscle.
  • mice immunized with a human immunodeficiency virus type 1 (HIV-1) DNA construct encoding the envelope glycoprotein, gpl60 were reported to react with recombinant gpl60 in immunoassays and lymphocytes from the injected mice were shown to proliferate in response to recombinant gpl20 (Wang et al . (1993) Proc. Natl . Acad. Sci . USA 0 :4156-4160) , and mice immunized with a plasmid containing a genomic copy of the human growth hormone (hGH) gene demonstrated a humoral immune response (Tang et al . (1992) Nature 356 ; 152-154) .
  • HMV-1 human immunodeficiency virus type 1
  • a number of delivery techniques can be used to deliver nucleic acids for immunizations, including particle- mediated (gene gun) techniques which accelerate nucleic acid-coated microparticles directly into the interior of cells in the target tissue.
  • Gene gun-based nucleic acid immunization has been shown to elicit both humoral and cytotoxic T lymphocyte immune responses following epidermal delivery of nanogram quantities of DNA.
  • Particle-mediated delivery techniques have been compared to other types of nucleic acid inoculation, and found markedly superior. Fynan et al. (1995) Int . J. Immunopharmacology 12:79-83, Fynan et al.
  • Mucosal immunity provides an important first line of defense in protection against pathogens which enter through mucosal tissues.
  • the mucosal surfaces of the gastrointestinal, respiratory and genitourinary tracts are continuously exposed to foreign antigen, including potentially infectious bacterial, viral and sometimes parasitic organisms .
  • Mucosal immune responses may protect against such challenges, and have distinct and specialized characteristics. Holmgren et al . (1994) Am. J " . Trop . Med . Hyg. 5.0:42-54.
  • Mucosal immunity includes both a humoral (antibody) response and a cytotoxic T lymphocyte (CTL) response, similar to non-mucosal immunity except localized to mucosal tissue.
  • CTL cytotoxic T lymphocyte
  • the current dogma holds as follows. 1. The principal immunoglobin produced by the mucosal immune system is secretory IgA, which is the most abundant immunoglobin class in humans. 2. Specialized antigen uptake cells in the Peyer's Patches of intestinal tract or nasopharyngeal lymphoid tissues, termed microfold or M cells, transport antigen to the underlying mucosal associated lymphoid tissues (MALT) . 3. In other areas of the mucosal epithelium, such as the pseudo-stratified airway epithelium, dendritic cells serve as antigen-presenting cells and migrate to local lymph nodes or MALT. Antigen processing and presentation occurs in the MALT, resulting in activation of antigen-specific IgA B cells.
  • MALT mucosal associated lymphoid tissues
  • the mucosal immune system is uniquely suited to respond to the types of antigenic challenge encountered by mucosal surfaces, and may provide the most effective type of immune response against pathogens that initially infect or enter the body through mucosal surfaces. It is difficult to achieve effective mucosal immune response using most prior art techniques .
  • the present invention provides an effective method for eliciting an immune response in a mammalian subject using mucosal immunization and particle-mediated delivery techniques .
  • the invention is drawn to a method for eliciting a mucosal immune response or a systemic immune response against a virus in a mammalian subject.
  • the method includes the steps of (a) providing a particle coated with a nucleotide sequence encoding an antigen derived from the virus, wherein the nucleotide sequence is operably linked to control sequences that direct the expression thereof in a suitable recipient cell; and (b) administering the particle to mucosal tissue of the mammal using particle-mediated delivery techniques, whereby the particle is delivered into a recipient cell in said tissue, and the nucleotide sequence expressed at sufficient levels to elicit a mucosal immune response against said antigen.
  • a method in another embodiment, includes the steps of (a) providing a particle coated with an antigen derived from a virus; and (b) administering the particle to mucosal tissue of the mammal using particle-mediated delivery techniques, whereby the particle is delivered into a recipient cell in said tissue.
  • FIG. 1 is a schematic representation of the hemagglutinin (HA) expression vector pWRG1638.
  • This plasmid vector was constructed from pWRG7054, a mammalian expression vector based on a pUC19 backbone, and thus contains the cytomegalovirus (CMV) immediate early transcriptional enhancer, promoter and intron A regulatory elements, and the polyA signal of bovine growth hormone, operably linked to the full length cDNA encoding the HA gene from swine influenza virus A/Swine/Indiana/1726/88 (H1N1) .
  • CMV cytomegalovirus
  • Figure 2 depicts the geometric mean titers of nasal viral shedding profiles in porcine subjects after challenge with the swine influenza virus A/Swine/Indiana/1726/88 (Sw/IN) as described in Example 1.
  • the animals were vaccinated using nucleic acid immunization by a prime and booster administration with: a control plasmid DNA (open squares) ; the pWRG1638 construct to the epidermis (open diamond) ; the pWRG1638 construct to mucosal tissue (open triangles) ; or the pFluNP construct to epidermis (open circles) .
  • Control animals were vaccinated using parenteral injection by a prime and booster administration with a commercial inactivated whole virus vaccine (crossed squares) . All animals were challenged two weeks after the booster immunization.
  • an "antigen” refers to any agent, generally a macromolecule, which can elicit an immunological response in an individual .
  • the immunological response may be mediated by B- and/or T-lymphocytic cells.
  • the term may be used to refer to an individual macromolecule or to a homogeneous or heterogeneous population of antigenic macromolecules .
  • "antigen” is generally used to refer to a protein molecule or portion thereof which contains one or more epitopes .
  • B cell epitope generally refers to the site on an antigen to which a specific antibody molecule binds.
  • the identification of epitopes which are able to elicit an antibody response is readily accomplished using techniques well known in the art. See, e.g., Geysen et al . Proc .
  • T cell epitopes are generally those features of a peptide structure capable of inducing a T cell response.
  • T cell epitopes comprise linear peptide determinants that assume extended conformations within the peptide-binding cleft of MHC molecules, (Unanue et al . (1987) Science 236:551-557) .
  • a T cell epitope is generally a peptide having about 3-5, preferably 5-10 or more amino acid residues .
  • Gene delivery refers to methods or systems for reliably delivering foreign DNA into host cells. Such methods can result in the expression of the foreign DNA in the host cells.
  • nucleotide sequence or a “nucleic acid molecule” refers to single or double stranded DNA and RNA sequences.
  • the term captures molecules that include any of the known base analogues of DNA and RNA.
  • a "coding sequence” or a sequence which "encodes” a particular polypeptide antigen is a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vi tro or in vivo when placed under the control of appropriate regulatory sequences .
  • DNA “control sequences” refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a recipient cell.
  • control sequences for eukaryotes and prokaryotes can differ significantly, and for the present invention eukaryotic, and preferably, mammalian or mammalian virus control sequences are most suitable.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • a method for achieving mucosal immunity and/or systemic immunity against an antigen for a pathogen which normally enters the subject body through mucosal tissue.
  • the mucosal immunity which is contrasted to the humoral immunity obtained through prior DNA vaccination protocols, is achieved by the intracellular delivery of the DNA directly to target mucosal tissues. It has been found that delivery of DNA encoding a pathogenic antigen into the mucosal tissue of a mammal will result in a mucosal immunity expressed by mucosal tissues of the patient including those quite distant from the tissues into which the DNA vaccine is delivered.
  • a systemic immune response as used herein may include a cell-mediated immune response characterized by peripheral blood CTL or a humoral immunity characterized by increased levels of circulating antibodies, such as IgG, in post immunization blood sera.
  • the tongue and the buccal tissue of the interior of the mouth, or cheek are the most convenient tissues into which to direct a particle-mediated DNA vaccine delivery protocol.
  • the tissues are readily accessible through relatively non- invasive procedures.
  • both tongue and buccal tissue are capable of engendering a sufficient immune response to introduce mucosal immunity by antigen encoding DNA delivered to these tissues.
  • the delivery of antigen encoding DNA to the cheek or buccal tissues results in a systemic mucosal immune response shared by ?immune? mucosal tissues throughout the body.
  • the present invention provides a method for eliciting, in a mammalian subject, an immune response against mucosally transmitted pathogens using nucleic acid immunization and particle-mediated delivery techniques.
  • the method can thus be used in a variety of mammalian subjects to provide a suitable immune response against infection by a pathogen which would normally enter the subject through a mucosal tissue.
  • Mucosal tissues are the preferred entry site into the body for a wide variety of pathogens.
  • Pathogens which enter the body through mucosal tissues include Human Pappiloma Viruses (HPV) , HIV, HSV2/HSV1, influenza virus (types A, B, and C) , Polio virus, RSV virus, Rhinoviruses, Rotaviruses, Hepatitis A virus, Norwalk Virus Group, Enteroviruses, Astroviruses, Measles virus, Para Influenza virus, Mumps virus, Varicella-Zoster virus, Cytomegalovirus , Epstein-Barr virus, Adenoviruses, Rubella virus, Human T-cell Lymphoma type I virus (HTLV-I) , Hepatitis B virus (HBV) , Hepatitis C virus (HCV) , Hepatitis D virus, Pox virus, Marbug and Ebola; bacteria including M.
  • HPV Human Pappiloma Viruses
  • HIV HSV2/HSV1
  • influenza virus types A, B, and C
  • Polio virus
  • tuberculosis Chlamydia, N. Gonorrhea, Shigella, Salmonella, Vibrio Cholera, Treponema pallidua, Pseudomonas , Bordetella pertussis, Brucella, Franciscella tulorensis, Helicobacter pylori , Leptospria interrogaus , Legionella pneomophila, Yersinina pestis, Streptococcus (types A and B) , Pneumococcus , Meningococcus , Hemophilus influenza (type b) , Toxoplasma gondic, Complylobacteriosis , Moraxella catarrhalis , Legionella pneumophlia, Pseudomonas aeruginosa, Donovanosis, and
  • Actinomycosis ; fungal pathogens including Candidiasis and Aspergillosis; parasitic pathogens including Taenia,
  • the present invention can be used to provide a suitable immune response against numerous veterinary diseases, such as Foot and Mouth diseases, Coronavirus,
  • Pasteurella mul tocida Helicobacter, Strongylus vulgaris, Actinobacillus pleuropneumonia , Bovine viral virus diarrhea (BVDV) , Klebsiella pneumoniae , E. coli , Bordetella pertussis, Bordetella parapertussis and brochiseptica .
  • the invention is broadly applicable for providing an immune response against any pathogen which would normally enter through mucosal tissue.
  • influenza virus and immunodeficiency virus DNA Both of these are intended only as examples of viruses which enter the body through mucosal tissues. It is here thought that a suitable mucosal immune response can be created following delivery of DNA encoding antigens from these viruses to mucosal tissues.
  • suitable immune response it is meant that the methods of the invention can bring about in an immunized subject an immune response characterized by the stimulation and clonal expansion of B and/or T lymphocytes specific for a virus antigen, wherein the immune response can protect the subject against subsequent infection with homologous or heterologous viral strains, reduce viral burden and/or shedding during an infection, bring about resolution of infection in a shorter amount of time relative to a non-immunized subject, or prevent or reduce clinical manifestation of disease symptoms .
  • nucleic acid molecules used in the subject methods contain coding regions with suitable control sequences and, optionally, ancillary therapeutic nucleotide sequences.
  • the nucleic acid molecules are prepared in the form of vectors which include the necessary elements to direct transcription and translation in a recipient cell.
  • the nucleic acids may be the entire genome of the virus less only sequences necessary for viral pathogenicity .
  • the antigen-encoding nucleic acid molecules can be administered in conjunction with ancillary substances, such as pharmacological agents, adjuvants, cytokines, or in conjunction with delivery of vectors encoding cytokines.
  • ancillary substances such as pharmacological agents, adjuvants, cytokines, or in conjunction with delivery of vectors encoding cytokines.
  • ancillary therapeutic nucleic acid sequences coding for peptides known to stimulate, modify, or modulate a host's immune response can be coadministered with the above-described antigens.
  • genes encoding one or more of the various cytokines (or functional fragments thereof) such as the interleukins, interferons, and colony stimulating factors, will find use with the instant invention.
  • the gene sequences for a number of these substances are known.
  • mucosal nucleic acid immunization is coupled with codelivery of one or more of the following immunological response modifiers: IL-2; IL-4; IL-6; IL-10; IL-12; and IFN- ⁇ .
  • Nucleotide sequences selected for use in the present invention can be derived from known sources, for example, by isolating the same from infected cells or viral particles containing a desired gene or nucleotide sequence using standard techniques. The nucleotide sequences for many, if not most, pathogen antigens have been identified to assist in vaccine and therapy design. It is now possible to construct DNA molecules of significant length once DNA sequence information is available.
  • sequences for desired antigens can be cloned into any suitable vector or replicon.
  • Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Ligations to other sequences are performed using standard procedures, known in the art.
  • Selected nucleotide sequences can be placed under the control of regulatory sequences such as a promoter or ribosome binding site (collectively referred to herein as "control" elements) , so that the sequence encoding the desired antigen is transcribed into RNA in the host tissue transformed by a vector containing this expression construct .
  • regulatory sequences such as a promoter or ribosome binding site (collectively referred to herein as "control" elements)
  • control elements will depend on the host being treated and the type of preparation used. Thus, if the host's endogenous transcription and translation machinery will be used to express the proteins, control elements compatible with the particular host will be utilized.
  • promoters for use in mammalian systems include, but are not limited to, promoters derived from SV40, CMV, HSV, RSV, MMTV, among others .
  • regulatory sequences which allow for regulation of the expression of antigens encoded by the delivered nucleotide sequences.
  • Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a coding sequence to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate control and, optionally, regulatory sequences such that the positioning and orientation of the coding sequence with respect to the control sequences allows the coding sequence to be transcribed under the "control" of the control sequences (i.e., RNA polymerase, which binds to ,the DNA molecule at the control sequences, transcribes the coding sequence) .
  • control i.e., RNA polymerase, which binds to ,the DNA molecule at the control sequences, transcribes the coding sequence
  • Modification of the sequences encoding the particular antigen of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it is attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.
  • control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • Conventional mammalian expression vectors and elements can be enhanced for use as DNA vaccines. For example, it has been found that the addition of signal peptide sequences directing secretion of expressed proteins can enhance CTL immune response . The use of a Kozak ATG sequence can enhance the translational efficiency of a DNA vaccine.
  • the inclusion of a mono/poly ubiquitination sequence in the expression vector can enhance the MHC Class I presentation signal while alternatively the use of an invariant chain sequence can enhance MHC Class II presentation signal. The use of such elements is within the abilities of those of skill in the art.
  • Particle-mediated methods for delivering nucleic acid preparations are known in the art.
  • the above-described nucleic acid molecules can be coated onto carrier particles using a variety of techniques known in the art.
  • Carrier particles are selected from materials which have a suitable density in the range of particle sizes typically used for intracellular delivery from a gene gun device. The optimum carrier particle size will, of course, depend on the diameter of the target cells.
  • tungsten, gold, platinum and iridium carrier particles can be used.
  • Tungsten and gold particles are preferred.
  • Tungsten particles are readily available in average sizes of 0.5 to 2.0 ⁇ m in diameter. Although such particles have optimal density for use in particle acceleration delivery methods, and allow highly efficient coating with DNA, tungsten may potentially be toxic to certain cell types and may degrade DNA over time.
  • Gold particles provide uniformity in size (available from Alpha Chemicals in particle sizes of 1-3 ⁇ m, or available from Degussa, South Plainfield, NJ in a range of particle sizes including 0.95 ⁇ m) and reduced toxicity.
  • Microcrystalline gold provides a diverse particle size distribution, typically in the range of 0.5-5 ⁇ m.
  • a number of methods are known and have been described for coating or precipitating DNA or RNA onto gold or tungsten particles. Most such methods generally combine a predetermined amount of gold or tungsten with plasmid DNA, CaCl 2 and spermidine .
  • the coated particles can be transferred to suitable membranes and allowed to dry prior to use, coated onto surfaces of a sample module or cassette, or loaded into a delivery cassette for use in particular gene gun instruments.
  • carrier particles coated with either nucleic acid preparations, or peptide or protein antigen preparations are delivered to mucosal tissue using particle-mediated delivery techniques.
  • Various particle acceleration devices suitable for particle-mediated delivery are known in the art, and are all suited for use in the practice of the invention.
  • Current particle acceleration device designs employ an explosive, electric or gaseous discharge to propel coated carrier particles toward target cells.
  • the coated carrier particles can themselves be releasably attached to a movable carrier sheet, or removably attached to a surface along which a gas stream passes, lifting the particles from the surface and accelerating them toward the target.
  • An example of a gaseous discharge device is described in U.S. Patent No. 5,204,253.
  • An explosive-type device is described in U.S. Patent No. 4,945,050.
  • the coated particles are administered to the subject to be treated in a manner compatible with the dosage formulation, and in an amount that will be effective to bring about a desired immune response.
  • the amount of the composition to be delivered which, in the case of nucleic acid molecules is generally in the range of from 0.001 to 10.0 ⁇ g, more preferably 0.25 to 5.0 ⁇ g of nucleic acid molecule per dose, depends on the subject to be treated.
  • dose it is meant to refer to a single event of DNA delivery by gene gun.
  • a prime immunization or a boost it is common for a single immunization procedure, whether a prime immunization or a boost, to include more than one gene gun dose.
  • a prime might consist of two to six gene gun doses to the tongue .
  • the additional doses are appropriate to, in essence, treat more tissue.
  • a gene gun design which is capable of treating more tissue in a single operation would lower the number of doses in a single vaccination.
  • the total amount of DNA delivered in the entire immunization will be in the range of about l-30 ⁇ g total for all doses.
  • a prime immunization and either one or two boost immunizations will be appropriate to achieve the desired level of immune response.
  • the exact amount necessary will vary depending on the age and general condition of the individual being immunized and the particular nucleotide sequence or peptide antigens selected, as well as other factors. An appropriate effective amount can be readily determined by one of skill in the art upon reading the instant specification.
  • over-dosages of DNA have been used. This was done because optimization of dosages for the particular antigens and the particular animals have not yet been done. It has been previously found that mild over-dosing of delivered DNA is not harmful to the immune response and thus, to err in dosing to achieve the desired immune response, it was decided to err on the high side. For a practical nucleotide vaccine for a given antigen, optimization studies would be performed to determine the minimum dosing required and such studies are well within the skill of those in the art.
  • an effective amount of the antigens herein described, or rather nucleic acids coding therefor will be sufficient to bring about a suitable immune response in an immunized subject, and will fall in a single to double digit microgram range of DNA that can be optimized through routine trials for a particular DNA and mammal.
  • coated particles are delivered to suitable recipient cells in mucosal tissue in order to bring about mucosal, humoral and/or cellular immune responses in the treated subject.
  • Weanling pigs (10-15 kg) sero-negative for swine influenza by hemagglutination inhibition (HI) , (Palmer et al. (1975) U.S. Department of Health, Education and Welfare Immunology Series) and ELISA (Sheerar (1989) J. Gen . Virol . 20.:3297-3303) were housed in a Biosafety level 2-N facility for immunizations, and then housed in a Biosafety level 3-N facility for viral challenge. The animal subjects were cared for in accordance with the guidelines prescribed by the University of Wisconsin Research Animal Resource Center.
  • A/swine influenza isolate A/Swine/lndiana/1726/88 (H1N1) (Sw/IN) was obtained from the influenza repository at the University of Wisconsin School of Veterinary Medicine.
  • the virus was cultured in 10-day-old embryonated hens' eggs and stored at -70 °C as previously described (Sheerar et al . , supra) except that the allantoic fluid was concentrated by the addition of PEG-8000 to 8%.
  • Precipitated virus was centrifuged at 8000 X g prior to purification on 30-60% sucrose gradients at 24,000 rpm in an SW28 rotor (Beckman) . Plasmid constructs and DNA preparations
  • the hemagglutinin expression plasmid pWRG1638 depicted in Figure 1 was constructed by ligating the cloned cDNA encoding the HA of swine influenza virus (SW/IN/1726/88) into the mammalian expression cassette pWRG7054.
  • the cDNA synthesis of the HA gene was done in a one-step PCR method according to Wentworth et al . (1994) J. Virol . £jl_2051- 2058.
  • PWRG1638 is a pUC19-based vector and includes the human cytomegalovirus immediate early transcriptional enhancer/promoter (CMVie) to drive transcription of the HA coding region.
  • CMVie human cytomegalovirus immediate early transcriptional enhancer/promoter
  • the plasmid also contains the polyadenylation region from the bovine growth hormone bGH gene (Chapman et al . (1991) Nucleic Acids Res . 19:3979- 3986) .
  • An influenza nucleoprotein (NP) expression plasmid, pFluNP, that encodes the nucleoprotein of influenza A strain PR/8/34 was obtained from Dr. K. Irvine at the
  • Plasmid DNA was coated onto gold particles normally in the range of 1-3 ⁇ m in size (Degussa Corp., South Plainfield, NJ) using techniques described by Eisenbraun et al. (1993) DNA Cell Biol . 12:791-797.
  • the D ⁇ A-coated gold particles were then loaded into Tefzel ® tubing as described in U.S. Patent No. 5,584,807 to McCabe, and the tubing was cut into 1.27 cm lengths to serve as cartridges in the ACCELL ® gene gun delivery device.
  • the helium-pulse ACCELL ® gene gun device was obtained from Geniva, Madison, WI .
  • each 1.27 cm cartridge contained 0.5 mg gold particles coated with 1.25 ⁇ g of plasmid DNA.
  • Chinese hamster ovary (CHO) cells were transfected with the pWRG1638 construct, or with control plasmid pWRG1630 which codes for the mature form of epidermal growth factor (Andree et al . (1994) Proc . Natl . Acad. Sci . USA 11:12188-12192), using the electric discharge ACCELL ® gene gun delivery device (Geniva, Madison, WI) .
  • the CHO cells were cultured as monolayers on 22x22 mm glass cover slips. For transfection, growth medium was aspirated and the cells treated as previously described (Christou et al . (1990) Trends Biotech. 8 . : 145-151.
  • CHO cells that were transfected with the pWRG1638 construct showed intense staining, indicating that the cells were expressing influenza HA.
  • CHO cells transfected with the pWRG1630 control plasmid were not immunoreactive in the assay.
  • Animal subjects receiving nucleic acid immunizations in the present study included: (1) a first experimental group of three pigs that were vaccinated by particle-mediated delivery to the epidermis with the NP expression vector pFluNP; (2) a second experimental group of four pigs that were vaccinated by particle-mediated delivery to the epidermis with the HA expression vector pWRG1638; (3) a third experimental group of five pigs that were vaccinated by particle-mediated delivery to the inferior surface of the tongue (mucosal immunization) with the HA expression vector pWRG1638; and (4) a fourth experimental group of four pigs that were vaccinated by particle mediated delivery to the epidermis with a negative control plasmid pWRG3510 (a plant expression vector encoding ⁇ -glucuronidase
  • Animals in the first and second experimental groups were immunized using ACCELL ® gene gun transfer of either the pFluNP, the pWRG1638 construct, or the control plasmid pWRG3510, into the epidermis in different anatomical regions including the dorsal surface of the ear, the inguinal region, and the lateral thoracic region. Treatment typically included six target sites at each location. Hair was removed with clippers prior to treatment of the lateral thoracic region, but other regions were treated without prior preparation. Delivery was conducted at 500 or 600 psi helium pressure.
  • a fifth experimental group of four pigs received a 2 ml parenteral (intramuscular) injection of a commercial swine influenza vaccine (MaxiVacTM-FLU, SyntroVet, Kenexa, KS) as directed by the manufacturer.
  • the MaxiVacTM-FLU vaccine is an oil- in-water vaccine containing inactivated whole Influenza A (H1N1) virus. Vaccination consisted of a priming administration followed by a booster injection four weeks later.
  • a sixth experimental group of four pigs was infected with swine influenza and allowed to recover from infection to provide a comparison between protection afforded by conventional vaccine and by natural infection.
  • serum samples were collected prior to vaccination, prior to booster administration, and one week after booster administration. All blood samples were collected from the superior vena cava. After these serum collections were completed, the animals were challenged with virus, the course of infection monitored, and sera was again collected two weeks after the challenge .
  • Viral challenge consisted of intranasal instillation of 2 x 10 4 or 2 x 10 6 EID 50 (50% egg infectious dose) of
  • SW/IN virus SW/IN virus. Challenged animals were monitored daily for clinical signs of influenza infection (e.g., lethargy, coryza and elevated body temperature) . Nasal swabs were collected from each pig on days 1, 3, 5, and 7 post infection, and viral titers were determined by limiting- dilution egg inoculation assays (Wentworth et al . (1994) J. Virol . £8:2051-2058). Ten days after challenge, convalescent sera were taken.
  • influenza infection e.g., lethargy, coryza and elevated body temperature
  • Nasal swabs were collected from each pig on days 1, 3, 5, and 7 post infection, and viral titers were determined by limiting- dilution egg inoculation assays (Wentworth et al . (1994) J. Virol . £8:2051-2058).
  • ELISA serology was conducted using 200 hemagglutinin (HA) units/well of Sarksyl-disrupted purified SW/IN virus diluted in PBS as described (Sheerar et al . , supra) , with the swine antibodies being measured directly using a goat anti-swine IgG alkaline phosphatase conjugate (Kirkegaard and Perry Laboratories, Inc. Gaithersburg, MD) .
  • HI assays were performed using previously described techniques (Palmer et al. (1975) , supra) .
  • the animals that received mucosal vaccinations with the pWRG1638 HA DNA construct had higher ELISA titers (ranging from 1:800 to 1:6400), and lower HI titers (ranging from 1:20 to 1:80), relative to the animals of groups 1 and 2 that received epidermal vaccinations.
  • the animals of group 5 vaccinated with the inactivated whole virus exhibited the highest ELISA and HI titers relative to all other experimental groups, while the group receiving natural infection (group 6) had ELISA and HI titers similar to the groups vaccinated with the pWRG1638 HA DNA construct.
  • Control animals vaccinated with the plant expression vector (group 4) showed no evidence of an anti- influenza immune response.
  • Epidermal NP 1 ⁇ 100 ⁇ 10 1600 ⁇ 10 20
  • DNA Vaccine 2 ⁇ 100 ⁇ 10 800 ⁇ 10 80
  • animals vaccinated epidermally with the pFluNP DNA construct developed high antibody titers to NP, but showed no evidence of protection from viral infection in terms of nasal virus titer.
  • Animals receiving epidermal vaccination with the pWRG1638 HA DNA construct (group 2) became infected and shed lower levels of virus over the course of infection, and resolved infection approximately two days earlier than the control animals of group 6.
  • Animals receiving mucosal vaccination with the pWRG1638 HA DNA construct (group 3) developed weak HI titers, but were able to reduce viral shedding over the seven days of the study. Further, the mucosally vaccinated animals were able to reduce the initial infection, as evidenced by a decrease in the level of shedding by an order of magnitude on days 1 and 3, relative to the epidermally vaccinated animals of group 2 .
  • the animals of group 4 that received the commercial inactivated whole virus vaccine showed the highest titer antibody responses, as seen in the ELISA and HI results of Table 1.
  • this higher HI titer did not translate to a higher level of protection upon challenge ( Figure 2) .
  • the animal from group 4 having the highest HI titer in the entire study was the least protected when challenged with the influenza virus.
  • nucleic acid immunization to mucosal tissue via particle-mediated delivery techniques provides an immune response that is both quantitatively and qualitatively different than the responses generated by particle-mediated epidermal immunization with nucleic acids, or parenteral immunization with inactivated whole virus.
  • Particle- mediated mucosal immunization with the pWRG1638 construct induced higher ELISA but lower HI influenza-specific antibody titers relative to particle-mediated epidermal immunization with the same construct.
  • the ability of the mucosally vaccinated animals to reduce nasal shedding of virus on days 1 and 3 of infection is consistent with a systemic mucosal immune response.
  • Example 2 Particle-Mediated Nucleic Acid Immunization Directed to Equine Mucosal Tissue The work reported in this example was performed by a research group separate from that of the inventor here and is reported because it is supportive of the concept of the present invention.
  • the first experimental group received a 3 -dose course of particle-mediated nucleic acid immunization to epidermal tissue on days 0, 65 and 130 of the study.
  • the second experimental group received a 3- dose course of particle-mediated nucleic acid immunization to both epidermal and mucosal tissue, also on days 0, 65 and 130 of the study.
  • the mucosal tissue targeted was the lower side of the tongue as well as the conjunctiva and third eyelid of the animals.
  • Nucleic immunizations were carried out using an ACCELL ® (Geniva, Madison, WI) gene gun device.
  • Each immunization included multiple doses of DNA delivery by gene gun. Each gene gun application delivered .5 ⁇ g of DNA. For the epidermal delivery immunizations, each immunization included 14 doses to the inguinal epidermis and 10 doses to the perineum for each animal. For the immunizations to the skin and mucosal tissue, the animals received the same skin doses plus 10 doses to the tongue and 4 doses to the conjunctiva of the third eyelid.
  • a challenge infection with homologous virus was administered 28 days after the final administration (on day 160 of the study) to each experimental group, and to a third group of four seronegative control ponies.
  • monkeys were vaccinated by administering the nucleic acid to the skin using a gene gun. Each animal received multiple boosts to the skin. Immunized monkeys were tested to determine whether immunization induced a mucosal immune response or a systemic immune responses. The results are presented in Table 2. All four monkeys immunized mucosally showed an increase in CTL response in the mucosal gut tissue. These results indicates that the monkeys vaccinated either in the buccal or tongue tissues were able to elicit a system wide mucosal immune response as demonstrated by the existence of appropriate IgA-based CTL responses in a mucosal site, which was not a site of DNA injection.
  • Mucosally-immunized monkeys demonstrate systemic humoral and cell-mediated immune responses. Two of the three monkeys exhibited increased gag-specific peripheral or humoral blood (PBMC) CTL and three of the four monkeys showed increased IgG titers (200-400) . Systemic responses were also observed in monkeys immunized by skin.
  • PBMC peripheral or humoral blood

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Abstract

L'invention concerne un procédé pour induire chez un mammifère une réponse immunitaire contre un virus ou d'autres pathogènes. Le procédé consiste à prendre une particule, encapsidée dans un ADN qui code pour un antigène dérivé d'un virus, et à l'administrer au mammifère, dans son tissu muqueux, en utilisant des techniques d'apport au moyen de particules. La particule est livrée dans la cellule réceptrice du tissu en question. Chez les mammifères, cette technique peut induire une réponse immunitaire efficace au niveau des muqueuses.
PCT/US1998/017637 1997-08-21 1998-08-21 Immunisation des muqueuses au moyen de techniques d'apport utilisant des particules WO1999008689A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000014547A1 (fr) * 1998-09-04 2000-03-16 Powderject Research Limited Immunodiagnostics au moyen de procedes d'administration de particules
GB2397025A (en) * 2003-01-02 2004-07-14 Optinose As Devices to deliver substances to the mucosa of the nose or mouth
US6802826B1 (en) 1999-10-11 2004-10-12 Felton International, Inc. Universal anti-infectious protector for needleless injectors
WO2009058564A2 (fr) 2007-11-01 2009-05-07 Maxygen, Inc. Polypeptide immunosuppresseur et acides nucléiques
US7887506B1 (en) 1999-11-23 2011-02-15 Pulse Needlefree Systems, Inc. Safety mechanism to prevent accidental patient injection and methods of same
US8911742B2 (en) 1996-11-14 2014-12-16 The United States Of America As Represented By The Secretary Of The Army Transcutaneous immunization without heterologous adjuvant

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KELLER E T, ET AL.: "IN VIVO PARTICLE-MEDIATED CYTOKINE GENE TRANSFER INTO CANINE ORAL MUCOSA AND EPIDERMIS", CANCER GENE THERAPY, APPLETON & LANGE, GB, vol. 03, no. 03, 1 June 1996 (1996-06-01), GB, pages 186 - 191, XP002913796, ISSN: 0929-1903 *
STAATS H F, ET AL.: "MUCOSAL IMMUNITY TO INFECTION WITH IMPLICATIONS FOR VACCINE DEVELOPMENT", CURRENT OPINION IN IMMUNOLOGY., ELSEVIER, OXFORD., GB, vol. 06, no. 04, 1 August 1994 (1994-08-01), GB, pages 572 - 583, XP002913795, ISSN: 0952-7915, DOI: 10.1016/0952-7915(94)90144-9 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8911742B2 (en) 1996-11-14 2014-12-16 The United States Of America As Represented By The Secretary Of The Army Transcutaneous immunization without heterologous adjuvant
WO2000014547A1 (fr) * 1998-09-04 2000-03-16 Powderject Research Limited Immunodiagnostics au moyen de procedes d'administration de particules
US6802826B1 (en) 1999-10-11 2004-10-12 Felton International, Inc. Universal anti-infectious protector for needleless injectors
US7887506B1 (en) 1999-11-23 2011-02-15 Pulse Needlefree Systems, Inc. Safety mechanism to prevent accidental patient injection and methods of same
GB2397025A (en) * 2003-01-02 2004-07-14 Optinose As Devices to deliver substances to the mucosa of the nose or mouth
GB2397025B (en) * 2003-01-02 2007-09-05 Optinose As Delivery devices which provide for a reflex action to prevent inhalation
WO2009058564A2 (fr) 2007-11-01 2009-05-07 Maxygen, Inc. Polypeptide immunosuppresseur et acides nucléiques
EP2385065A1 (fr) 2007-11-01 2011-11-09 Perseid Therapeutics LLC Polypeptides immunosuppresseurs et acides nucléiques
EP2612867A1 (fr) 2007-11-01 2013-07-10 Perseid Therapeutics LLC Acides nucléiques et polypeptides immunosuppresseurs
EP2612868A1 (fr) 2007-11-01 2013-07-10 Perseid Therapeutics LLC Acides nucléiques et polypeptides immunosuppresseurs

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