Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/18—Applying electric currents by contact electrodes
  • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
  • A61N1/325—Applying electric currents by contact electrodes alternating or intermittent currents for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body
• • 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
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/18—Applying electric currents by contact electrodes
  • A61N1/20—Applying electric currents by contact electrodes continuous direct currents
  • A61N1/30—Apparatus for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body, or cataphoresis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • 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
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/02—Details
  • A61N1/04—Electrodes
  • A61N1/0404—Electrodes for external use
  • A61N1/0408—Use-related aspects
  • A61N1/0412—Specially adapted for transcutaneous electroporation, e.g. including drug reservoirs
  • A61N1/0416—Anode and cathode
  • A61N1/0424—Shape of the electrode
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/02—Details
  • A61N1/04—Electrodes
  • A61N1/0404—Electrodes for external use
  • A61N1/0408—Use-related aspects
  • A61N1/0428—Specially adapted for iontophoresis, e.g. AC, DC or including drug reservoirs
  • A61N1/0432—Anode and cathode
  • A61N1/044—Shape of the electrode
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/18—Applying electric currents by contact electrodes
  • A61N1/20—Applying electric currents by contact electrodes continuous direct currents
  • A61N1/30—Apparatus for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body, or cataphoresis
  • A61N1/303—Constructional details
  • A61N1/306—Arrangements where at least part of the apparatus is introduced into the body
• • 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
• • 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

Definitions

• the present invention relates generally to the use of electrical pulses to enhance DNA vaccine efficacy in vivo. More particularly, the present invention relates to the use of electrical pulses to enhance HIV DNA vaccine efficacy in vivo and even more particularly to the use of electrical pulses to enhance HIV gag DNA vaccine efficacy in vivo.
• Vaccines composed of live, attenuated pathogens have long been used to provide and/or enhance immunity. Recently, however, the ability to introduce DNA into cells and tissues has led to the proposal that DNA vaccines could be used in lieu of pathogens to provide immunity (for review, see Donnelly et al. (1991) Ann. Rev. Immunol. 75:617-648).
• DNA vaccines offer the potential for greater safety, efficacy and protection than that provided by conventional vaccines, the delivery of such DNA to cells has presented several problems (Lai and Bennett (1998) Crit. Rev. Immunol. 78:449-484). DNA vaccines given orally have been reported to be incapable of eliciting an immune response (see Mani an et al. (1997) Crit. Rev. Immunol. 77: 139-154). Likewise, introduction of DNA into the dermis has been found to be complicated both by the susceptibility of the basal cells of the epidermis to transformation, and by rapid turnover of epidermal cells that leads to the expulsion of much of the administered DNA (Lai and Bennett (1998) Crit. Rev. Immunol. 78:449-484).
• induction of an immune response was detected in mice after electroporation in vivo with DNA encoding a fusion protein containing a CTL epitope from influenza nucleoprotein (Nomura et al. (1996) J. Immunol. Meth. 193: 41-49).
• Iontophoresis A technology related to electroporation, termed iontophoresis, involves the application of an electric field to facilitate movement of charged molecules, such as "naked DNA,” in tissue and across biological membranes. Iontophoresis, which involves lower electric current than what is required for electroporation, has been widely used for transdermal delivery of drugs and oligonucleotides.
• DNA encoding the immunogen of interest is administered parenterally followed by the application of electrical current in either the iontophoresis or electroporation range.
• the HIV DNA is HIV gag DNA.
• such DNA is incorporated into a plasmid and is injected either via an intramuscular (i.m.) or intradermal (i.d.) route.
• the invention provides, a method of enhancing an immune response generated in an animal comprising the steps of:
• the invention particularly concerns the embodiment of the above method in which the immunogen is a protein or peptide of a pathogen (especially a bacterium, a fungus, a yeast, a protozoan, or a virus).
• the pathogen is the retrovirus HIV
• the DNA administered in step (A) encodes one or more HIV protein or peptide (especially the HIV gag and/or env proteins or a peptide fragment of either, and most preferably codon-optimized DNA molecules encoding these immunogens).
• the invention particularly concerns the embodiment of the above method in which the electrical field is applied under electroporation conditions or under iontophoresis conditions.
• the invention additionally provides an apparatus for enhancing an immune response in an animal comprising:
• (B) means for administering the DNA to the animal; and (C) means for applying an electric field to at least the site of such DNA administration.
• the invention particularly concerns the embodiment of the above apparatus in which the immunogen is a protein or peptide of a pathogen (especially a bacterium, a fungus, a yeast, a protozoan, or a virus).
• the pathogen is the retrovirus HIV
• the administered DNA encodes one or more HIV protein or peptide (especially the HIV gag and/or env proteins or a peptide fragment of either and most preferably codon-optimized DNA molecules encoding these immunogens).
• the invention additionally concerns the embodiment of the above apparatus in which the means for administering the DNA to the animal accomplishes the intramuscular or intradermal administration of the DNA.
• the invention additionally concerns the embodiment of the above apparatus in which the electrical field is produced under electroporation or iontophoresis conditions.
• the invention additionally concerns the embodiment of the above apparatus in which the means for administering the DNA is a device selected from the group consisting of a single needle probe, a bipolar probe and a combination needle and plate probe.
• Figure 1A and Figure IB show the expression of ⁇ -galactosidase in mouse muscles that had received ⁇ -galactosidase-encoding DNA either without additional treatment ( Figure 1A) or after electroporation ( Figure IB).
• Figure 2 shows the ability of electroporation and iontophoresis to enhance the antibody responses of mammals after a single inoculation with DNA encoding the HIV gag protein.
• Figure 3 shows the effect of vaccine boosting on antibody responses in mammals inoculated with DNA encoding the HIV gag protein. Note the enhanced immune responses induced by electroporation and iontophoresis even after the booster immunization.
• Figure 4 shows the efficacy of electroporation on the anti-HIV gag antibody response of mammals inoculated with a DNA vaccine encoding HIV gag, followed by immunization with recombinant gag protein. Note the enhanced levels of booster response in rabbits that had been primed with DNA and electroporation compared to animals primed with DNA alone.
• the present invention provides a method for the enhancement of DNA vaccine efficacy by electrically-mediated administration of the DNA in vivo.
• the recipient of the DNA vaccine may be any mammal (especially a cat, a dog, a horse, a human, a rabbit or a rodent).
• the invention particularly contemplates that the recipient of the DNA vaccine may be a human.
• the DNA vaccine that is administered in accordance with the present invention encodes one or more immunogens.
• an immunogen is a protein or a peptide (i.e., a fragment of a protein) that contains at least one epitope such that the immunogen induces an enhanced immune response in a recipient mammal.
• a treatment or procedure is said to enhance an immune - 1 -
• the enhanced immune responses of the present invention include the enhanced production of antibody that is specifically reactive with the immunogen, and the enhanced production of lymphocytes that produce such antibody.
• An antibody is said to be specifically reactive with an immunogen if it binds to the immunogen in an immunologically relevant manner.
• DNA vaccines Any of a variety of DNA vaccines may be used in accordance with the present invention include those (for review, see Donnelly et al. (1997) Ann. Rev. Immunol. 75:617-648; Manikan et al. (1997) Crit. Rev. Immunol. 77: 139-154; Alarcon et al. (1999) Adv. Parasitol. 42: 343-410; Lai and Bennett (1998) Crit. Rev. Immunol. 78:449-484; Tuteja (1999) Crit. Rev. Biochem. Molec. Biol. 34: 1- 24).
• the DNA vaccine of the present invention will encode more than one epitope.
• the administered DNA may encode all of the epitopes of a protein associated with HIV (such as the gag or env protein).
• the administered DNA may encode only a peptide of such protein that contains one (or fewer than all) of the protein's epitopes.
• the immunogens encoded by the DNA vaccine of the present invention may comprise a protein or peptide of a pathogen.
• pathogen may be any of a wide group of bacteria (e.g., E. coli strains and strains of other enterics (e.g., Salmonella), Clostridria, Vibrio, Corynebacteria, Listeria, Nocardia, Legionella, Bacilli (especially B. anthracis), Staphylococcus, Streptococci (especially beta-hemolytic Streptococci and S. pneumoniae), Borrelia, Mycobacterium (especially M. tuberculosi), Neisseria (especially N.
• enterics e.g., Salmonella
• Clostridria e.g., Vibrio, Corynebacteria, Listeria, Nocardia, Legionella, Bacilli (especially B. anthracis), Staphylococcus, Streptococci (especially beta-hemolytic Strept
• viruses e.g., parvoviruses, orthomyxoviruses (especially those causing influenza), paramyxoviruses, picornaviruses (especially rhinoviruses or polioviruses), papoviruses, herpesviruses, togaviruses, retroviruses (especially HIV), rhabdoviruses, etc.), and lower eukaryotes (e.g., fungi, protozoa, yeast, helminths, nematodes, etc.
• viruses e.g., parvoviruses, orthomyxoviruses (especially those causing influenza), paramyxoviruses, picornaviruses (especially rhinoviruses or polioviruses), papoviruses, herpesviruses, togaviruses, retroviruses (especially HIV), rhabdoviruses, etc.
• lower eukaryotes e.g., fungi, protozo
• the immunogens of the present invention may encode antigens that are produced by aberrant or diseased cells of the recipient (e.g., cancer cells, etc.), such that the recipient animal will form antibodies that will attack such cells.
• the immunogens encoded by the DNA vaccine of the present invention may be related to one another, may be clinically related, or may be unrelated to one another.
• immunogens are related to one another if the immune responses that they induce elicit antibodies that bind to the same cell, microbe, virus, etc.
• DNA that encodes epitopes of the gag or env protein would encode related immunogens.
• Immunogens are said to be clinically related to one another if the immune responses that they induce elicit antibodies that bind to different cells, microbes, viruses, etc. that are associated with the same clinical condition.
• DNA that encodes epitopes of a Listeria monocytogenes protein and a Candida protein would encode clinically related immunogens.
• the DNA vaccine of the present invention may encode an epitope of a poliovirus and an epitope of a measles virus, and thus provide unrelated immunogens.
• the DNA of the DNA vaccine of the present invention will contain regulatory elements (promoters, translation initiation sites, etc.) operably linked to the immunogen-encoding sequences and sufficient to permit the protein expression of the immunogen.
• the administered DNA will not contain such regulatory elements, and will require cellular processes (such as recombination or integration into nuclear or mitochondrial DNA, etc.) in order to produce the encoded immunogen.
• the DNA vaccine of the present invention may comprise more than one molecular species of DNA. Such multiples species may contain the same DNA sequence (e.g., a mixture of circular and linearized plasmids), or may contain different DNA sequences encoding the same immunogen (e.g., a mixture of DNA molecules of different length all of which contain a particular immunogen- encoding sequence), or may contain DNA sequences encoding different immunogens.
• the administered DNA can be either "naked" DNA (i.e., free of associated protein or lipids), or may be complexed with protein or lipids or other molecules.
• the DNA can be administered with a local anesthetic such as bupivicaine or a myotoxin such as cardiotoxin, or with proteins that assist in the efficient presentation of antigen (e.g., CD80, CD86, etc.) (Tuteja (1999) Crit. Rev. Biochem. Molec. Biol. 34: 1-24).
• the DNA may encode only the desired immunogen or immunogens, or may encode other additional proteins or peptides that may be linked or unlinked to the immunogen and that enhance immunogen stability or immunogenicity.
• the DNA may also encode protein extraneous to the immunogenicity of the immunogen that is encoded by the DNA; such extraneous protein may likewise be linked or unlinked to the immunogen.
• the DNA of the DNA vaccine of the present invention may contain untranslated or untranscribed DNA.
• the DNA can be incorporated into a recombinant expression vector such as a chimeric virus, a plasmid DNA, etc.
• the DNA is preferably dissolved or suspended in a buffer or other solution (e.g., 5% dextrose).
• DNA preferably in the form of plasmid DNA
• the electrically-mediated enhancement covers administration using either iontophoresis or electroporation in vivo. Suitable techniques of electroporation and iontophoresis are provided by Singh et al. (1989) Drug Des. Deliv. 4: 1-12; Theiss U et al. (1991) Methods Find. Exp. Clin. Pharmacol. 73:353-359; Singh and Maibach (1993) Dermatology. 787:235-238; Singh and Maibach (1994) Crit. Rev. Ther. Drug Carrier Syst.
• the nature of the electric field generated in accordance with the present invention is determined by the nature of the tissue, the size of the selected tissue and its location. It is desirable that the field be as homogeneous as possible and of the correct amplitude. The use of insufficient or excessive field strength is to be avoided. As used herein, a field strength is excessive if it results in the lysing of cells. A field strength is insufficient if it results in a reduction of efficacy of 90% relative to the maximum efficacy obtainable.
• the electrodes may be mounted and manipulated in many ways known in the art.
• the waveform of the electrical signal provided by the pulse generator can be an exponentially decaying pulse, a square pulse, a unipolar oscillating pulse train or a bipolar oscillating pulse train.
• the waveform, electric field strength and pulse duration are dependent upon the type of cells and the DNA that are to enter the cells via electrical-mediated delivery and thus are determined by those skilled in the art in consideration of these criteria.
• any number of known devices may be used for delivering the DNA vaccine and generating the desired electric field.
• suitable devices include, but are not limited to, a single needle probe, a bipolar probe and a combination needle and plate probe.
• the single needle probe exemplified herein is a single stainless steel needle, with an insulation stop that provides preferably about 3mm of active zone.
• the single needle serves as the negative electrode and the plasmid delivery device.
• the positive electrode is a hypodermic needle located in the opposite leg or arm of the recipient patient or test animal.
• the bipolar probe exemplified herein contains two stainless steel needles preferably about 3mm in length and separated by a distance of preferably about 0.4cm. One needle carries a positive charge and one needle carries a negative charge.
• the combination needle and plate probe exemplified herein contains two stainless steel needles preferably about 3 mm in length and separated by a distance of preferably about 0.4cm.
• the needles are insulated except for the distal 1mm. Both needles serve as the negative electrodes.
• the needles protrude from a stainless steel block. The block sits on the surface of the skin and serves as the positive electrode.
• the separation distance between the nearest active area on the block to the nearest active area on the needles is preferably about 2.5mm.
• the needles are insulated from direct contact with the stainless steel block.
• Preferred electrical field conditions for i.m. administration are as follows:
• Preferred electrical field conditions for i.d. administration are as follows: 50mA for 50msec for 5 pulses then rotate orthogonally for 5 additional pulses; and 120 V for 50 msec for 5 pulses then rotate orthogonally for 5 additional pulses when using the bipolar probe.
• Preparations of DNA for parenteral administration include but are not limited to sterile or aqueous or non-aqueous solutions, suspensions, and emulsions.
• non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
• the increased DNA vaccine potency observed after iontophoresis or electroporation may reflect a facilitation, by the electric current, of the distribution of DNA within the injected tissue and/or uptake of DNA by cells, leading to increased transfection.
• the ensuing increase in the amount of antigen expressed by cells is likely to have played a role in the elevated immune responses.
• infiltration of inflammatory cells in response to the electric current
• the present invention demonstrates that DNA vaccine potency can be increased by application of electric current.
• the bacteria Escherichia coli strain HB 101 were transformed with the plasmids pCMV HIV gag prepared as described in U.S. Provisional Patent Application 60/114495, filed 31 December 1998, or pCMV KM LUC encoding firefly luciferase reporter gene (LUC).
• a luciferase expression plasmid was obtained from Promega Corporation (Madison, WI).
• E. coli strain XL-1 Blue (Stratagene, La Jolla), carrying the expression plasmid, was grown in LB; antibiotic selection employed 50 ⁇ g/ml of ampicillin.
• Plasmids were purified using Qiagen Endo Free Plasmid Maxi Kits (Qiagen, Inc., Chatsworth, CA) according to the manufacturer instructions and resuspended in 0.9% sodium chloride (Abbott Laboratories, North Chicago, IL).
• the plasmid pCMV HIV gag was used as a source of gag-encoding DNA.
• the plasmid expresses high levels of HIV- 1 gag, due to a potent CMV promoter with intron A and a codon-optimized gag encoding region (see U.S. Provisional Patent Application Serial No. 60/168,471, filed December 1, 1999).
• the plasmid was grown in E. coli strain HB101, purified using a Qiagen ⁇ ndofree Plasmid Giga kit, (Qiagen, Inc.) and resuspended in 0.9% sodium chloride (Abbott Laboratories, North Chicago, IL). Plasmid concentrations were analyzed by measuring absorbance at 260 nm.
• mice Female 6-8 week old CB6F1 or BalbC mice (Charles River) were anesthetized using 4 parts ketamine HC1, lOOmg/ml stock solution, (Fort Dodge Animal Health, Fort Dodge, Iowa) 1 part xylazine, 20mg/ml, (LLoyd Labs, Shenandoah, Iowa). The mice received l ⁇ l per gm of body weight intramuscularly in the posterior thigh. The anterior tibialis (TA) muscle was shaved and the animals were injected with 10 ⁇ g of plasmid in a volume of 50 ⁇ l. To control needle depth, the syringe was covered with polyethylene tubing (i.d.
• the animals were injected intramuscularly, intradermally or subcutaneously. For each of the types of injections, an electrical field was then applied to the animals except to the control group of animals.
• One group of animals received an electrical field in the iontophoresis range. That is. using a single needle probe set-up 50 mA at a 10 msec pulse width, 1 Hz frequency for a total of 60 pulses were delivered.
• Another group of animals received an electrical field in the low electroporation range. That is, 40 V at 10 msec pulse width, 1 Hz frequency for 5 pulses were delivered plus 5 additional pulses were delivered after the probe was turned in an orthogonal direction to the first set of 5 pulses.
• Another group of animals received an electrical field in the high electroporation range. That is 80 V, at a 10 msec pulse width, 1 Hz frequency for 5 pulses were delivered plus 5 additional pulses were delivered after the probe was turned in an orthogonal directed to the first set of 5 pulses. Serum samples were collected at 2, 4, 8 and 12 week intervals and analyzed by the below-outlined procedures. The results of this experiment showed enhanced antibody titers in the animals inoculated by the i.m. route with enhancement ranging from 8- to 20-fold.
• Immunoassays The mouse anti-p55 IgG antibodies were measured by one of two methods, chemi-luminescent or colormetric ELISA assays.
• the plates were washed 3X with wash buffer [IX AquaLite® Wash Buffer (SeaLite Sciences, Inc. Bogart, GA) containing 0.3% Tween 20 (Sigma, St. Louis, MO)], and blocked at 37°C for 1 hour with 150 ⁇ l/well blocking buffer [IX Streptavidin AquaLite® Assay buffer (SeaLite Sciences, Inc. Bogart, GA) containing 5% goal serum].
• the plates were washed 3X and the test sera were diluted 1/300 or 1/9000 followed by serial 3-fold dilutions in the blocking buffer. A volume of 50 ⁇ l of each dilution was added per well and the plates were incubated at 37°C for 1 hour. The plates were washed 6X and incubated for 1 hour at 37°C with 50 ⁇ l/well of Goat anti-mouse IgG -Biotin (Sigma St. Louis, MO), diluted 1/1000 in block buffer. After washing 6X, the plates were incubated at 37°C for 1 hour with Streptavidin-Aqualite® (SeaLite Sciences, Inc. Bogart, GA), diluted 1/500 in wash buffer, 50 ⁇ l/well.
• the plates were washed 6X and stored in wash buffer until reactivity was measured on the luminometer (MLX, Dynex Technologies, Chantilly, VA). Setting for the luminometer - mode: Integrate Flash, Gain: High, Data: Table, Delay window: 0.00 sec, Integrate window: 3.00 sec, Before peak: 0.10 sec, After peak: 2.00 sec, calibrate on each well.
• the plates were tapped dry and put into the luminometer. Fifty microliters of IX AquaLite® Trigger Buffer (SeaLite Sciences, Inc. Bogart, GA) were automatically dispensed per well and the relative light units (RLU) measured. Endpoint titers were calculated as the inverse of the dilution that yields an RLU equal to the background plus 5 times the standard deviation.
• the block solution was aspirated the plates were incubated at 37° for 2 hours with 150 ⁇ l/well of Goat anti-mouse IgG-HRP (Caltag, Burlingame, CA) diluted 1/40,000 in block buffer. Following a final 6 washes, the plates were developed with OPD for 30 min.
• the OPD developer consists of 1 tablet (10 mg) o-phenylenediamine, 12 ml buffer (0.1M citric acid, 0.1M dibasic sodium phosphate), 5 ⁇ l 30% H 2 O .
• the reaction was stopped with 50 ⁇ l per well 4H H 2 SO and optical density was measured at dual wavelengths 492-690.
• the reported titers correspond to the reciprocal of the serum dilution producing an absorbance value of 1.0.
• mice were injected with DNA encoding the readily discernable marker enzyme luciferase (Luc).
• mice Female 6-8 week old CB6F1 mice (Charles River) were anesthetized using 4 parts ketamine HC1, 100 mg/ml stock solution (Fort Dodge Animal Health, Fort Dodge, Iowa), 1 part xylazine, 20 mg/ml (Lloyd Labs, Shenandoah, Iowa). The mice received 1 ⁇ l per mg of body weight intramuscularly in the posterior thigh.
• the tibialis anterior (TA) muscle was shaved and the animals were injected with 10 ⁇ g of plasmid in a volume of 50 ⁇ l.
• TA tibialis anterior
• a 0.3 cc insulin syringe was covered with polyethylene tubing (i.d. 0.38) to expose only the bevel.
• plasmid DNA in 5% dextrose was injected into the right tibialis anterior muscle using a single needle delivery probe, which has a functional length of 3 mm.
• the plasmid delivery needle was attached to the negative lead from the controller and a needle electrode placed in the contralateral leg was attached to the positive lead.
• Constant current pulses of 5 mA in amplitude, 10 msec in width, were given at a frequency of 1 Hz for 1 min.
• plasmid DNA in PBS was injected into the right tibialis anterior muscle as previously described.
• the probe needles had a separation distance of 0.4 cm and a needle length of 0.3 cm.
• the probe was connected to a constant voltage power supply and 5 constant voltage pulses, 50 msec in width, either 100 or 200 V cm-1, were applied in one orientation, the probe was rotated 90 degrees and 5 additional pulses were applied.
• Luciferase Activity Mice were sacrificed up to 14 days post vaccination, and TA muscles were collected and flash frozen in liquid nitrogen. The frozen tissue was homogenized with a mortar and pestle (on dry ice), lysed with 0.5 ml IX reagent lysis buffer (Promega, Madison, WI), and vortexed for 15 minutes at room temperature. The samples were subjected to 3 freeze thaws and centrifuged for 10 minutes at 10,000 X g. Supernatants were collected and stored at -80°C until assayed.
• the ML3000 microplate luminometer (Dynex Technologies, Chantilly, VA) measured the luciferase activity by automatically dispensing 100 ⁇ l of luciferase assay reagent (Promega, Madison, WI) into wells containing 20 ⁇ l of supernatant, and measuring the relative light units (RLU).
• the setting for the luminometer were the following, Mode: enhanced flash, Gain: medium, Delay time: 1 sec, Integrate time: 5 sec, calibrate each run. Sample values were extrapolated from a standard curve prepared from QuantiLum® Recombinant Luciferase (Promega, Madison, WI). Results are expressed as ng luciferase per mg muscle protein, with protein determination by BCA Protein Assay Reagent (Pierce).
• results of this experiment are shown in Table 1, and indicate that electoporation and iontophoresis facilitated the distribution and/or uptake of DNA into mammalian cells and tissue.
• results are expressed as ng luciferase activity per mg muscle protein. Numbers in parentheses indicate standard deviation of the mean (sd).
• mice were inoculated with 10 ⁇ g of luciferase (Luc) DNA in the TA muscle of one leg.
• One group of mice was not further treated and one group was treated with electroporation (Electro).
• Electro electroporation
• the muscles were collected and luciferase activity was measured and expressed as ng luciferase activity per mg muscle protein.
• Table 2 shows a significant enhancement of luciferase gene expression in mammalian tissue that had been subjected to electroporation, relative to non-electroporated, control animals.
• numbers in brackets indicate standard deviation of the mean (sd).
• mice were injected with DNA encoding a different readily discernable marker enzyme ( ⁇ -galactosidase).
• CB6 FI mice were inoculated with 100 ⁇ g of pCMV ⁇ -gal, a ⁇ - galactosidase-encoding DNA, in the TA muscle of one leg.
• the plasmid uses the same promoter as that used for HIV gag and env to express ⁇ -galactosidase.
• One group of mice was not further treated, one group was treated with electroporation, and another with iontophoresis.
• the data ( Figure 1A (untreated); Figure IB (electroporation)) indicated that electroporation had substantially facilitated the distribution and/or uptake of DNA into mammalian cells and tissue.
• the number of muscle fibers detectably transfected after inoculation of ⁇ - galactosidase DNA was found to have been substantially increased by iontophoresis and electroporation, as compared to untreated muscles, as judged by ⁇ -galactosidase staining of muscle tissue sections.
• application of electric current appears to decrease the variability of reporter gene expression in muscle cells. Therefore, application of electric current facilitates delivery of DNA to muscle cells in situ promotes efficient transfection.
• mice were inoculated a single time with 10 ⁇ g of DNA encoding the HIV gag protein.
• the plasmid pCMV HIV p55 gag grown in E. coli strain HB 101 , as described above, was employed as the source of the gag-encoding DNA.
• the DNA was inoculated into the TA muscle of one leg.
• One group of mice was not further treated, one group was treated with iontophoresis and another with electroporation.
• Sera from mice were analyzed for anti-gag antibody titer at 2, 4, 8 and 12 weeks after inoculation. The data are shown in Figure 2. In Figure 2, data are plotted as geometric mean ELISA titer and error bars indicate SEM.
• mice were inoculated with 10 ⁇ g of DNA encoding the HIV gag protein. Inoculation was into the TA muscle of one leg of the animals at 3 and 6 weeks. One group of mice was not further treated (Figure 3, open bars), one group was treated with iontophoresis (Figure 3, solid bars) and another with electroporation ( Figure 3, shaded bars). Sera were collected at 3 weeks after each immunization and analyzed for antibody responses. Data are plotted as geometric mean ELISA titer and error bars indicate SEM. Antibody titers were elevated in all groups after the booster injection, but the approximately 10-fold enhancement in titers observed in mice receiving electric current was maintained even after the boost ( Figure 3).
• mice were inoculated with 10 ⁇ g of HIV gag DNA (obtained as described above) in the TA muscle of one leg at 3 weeks. Groups of mice were treated as indicated in Table 3. Sera were collected at 3 weeks and analyzed for antibody responses. In Table 3, data are tabulated as geometric mean ELISA titer and as fold increase over titers achieved in vaccinated but untreated mice. The results show that enhancement of DNA vaccine potency is achieved across a wide range of conditions.
• mice were inoculated with 10 ⁇ g of HIV gag DNA intradermally on the backs.
• DNA control One group was treated with iontophoresis and another with electroporation at the conditions indicated in Table 4.
• Sera were collected at 3 weeks after immunization and analyzed for antibody responses.
• data are tabulated as geometric mean ELISA titer and fold increase over titers achieved in vaccinated but untreated mice.
• electroporation and iontophoresis are also effective for the intradermal route of administration of DNA vaccines.
• mice were inoculated with 10 ⁇ g of HIV gag DNA in the TA muscle of one leg. Groups of mice were treated as indicated in Table 5.
• the combination needle and plate electrode system consists of 3 electrically conducting components, plus electrical leads for connections, and a holder apparatus. Two of the electrically conductive components represent needle electrodes, of the same polarity (typically negative). These needle electrodes are fabricated of stainless steel (cylindrical, grade 316). Needle lengths were 3mm. The needles were encapsulated within insulation, and were retained in the electrode assembly, surrounded by the plate electrode.
• the plate electrode consisted of a stainless steel block, with dimensions of 1 x 1 x 1 cm.
• the needle electrodes extended through the plate electrode, with approximately 3 mm length extending beyond the surface of the electrode. Insulation around the needle prevented passage of electric current from the needle directly to the plate electrode.
• the electric current path was from the power source through the connector cable to the needle electrodes. Electric current was then transmitted from the end of the needle electrodes through biological tissue, to the plate electrode, and thus through a connecting cable to the power source, completing the circuit.
• the shortest electrically conductive path through tissue is approximately 2.5 mm. This is accounted for by the 2 mm of insulated needle electrode extending above the plate electrode, and the diameter of the holes through the plate electrode, through which the needle electrodes extend.
• the electrode assembly was used to deliver a series of electrical energy pulses in either constant voltage (electroporation) or a constant current (iontophoresis) mode. Sera were collected at 6 weeks and analyzed for antibody responses. One group of mice was not further treated (DNA control). Other groups were treated with iontophoresis and electroporation at the indicated conditions. In Table 5, data are tabulated as geometric mean ELISA titer and fold increase over titers achieved in vaccinated but untreated mice. The results indicate that a significant increase in antibody titer could be obtained using the needle and plate electrode system to deliver current for electroporation or iontophoresis.
• Inoculations were into the hind leg gracilis muscles at 0, 6 and 12 weeks.
• One group of rabbits received DNA without further treatment (DNA control).
• Other groups were treated with electroporation with a 6-needle electrode or a 2- needle electrode.
• the two-needle array electrodes (BTX) were inserted into the muscle immediately after DNA delivery for electroporation. The distance between the electrodes was 5 mm and the array was inserted longitudinally relative to the muscle fibers.
• In vivo electroporation parameters were: 20V/mm distance between the electrodes, 50 msec pulse length, 6 pulses with reversal of polarity after three pulses, at 1 pulse per second, given by a BTX 820 square wave generator.
• the electroporation with a 6-needle electrode array formed a circle (Genetronics, Inc.).
• the diameter of the electrode array was 1 cm, with a needle length of 1 cm.
• Six electroporation pulses of 20V/mm, 50 msec pulse length, one pulse per second were given by a BTX 820 square wave generator, combined with an electronic switch (Genetronics, Inc.) to rotate the electric field in 60 degree increments after each discharge (Hofmann et al. (1996) IEEE Engineer. Med. Biol. 75: 124-132).
• Anti-HIV gag antibodies were measured by ELISA as follows. Wells of Immulon 2 HB "U" bottom microtiter plates (Dynex Technologies, Chantilly, VA) were coated with HIV p55 protein at 5 ⁇ g/ml in PBS, 50 ⁇ l per well, and incubated at 4°C overnight. The plates were washed 6X with wash buffer [PBS, 0.1% Tween 20 (Sigma, St.
• the plates were incubated for 1 hour at 37°C with 50 ⁇ l/ well of Goat anti-mouse IgG-HRP (Caltag, Burlingame, CA) diluted 1/40,000 in blocking buffer. Following a final 6 washes, the plates were developed with OPD for 30min.
• the OPD developer consists of 1 tablet (10 mg) o-phenylenediamine, 12 ml buffer (0.1M citric acid, 0.1M dibasic sodium phosphate), 5 ⁇ l 30% H 2 O 2 .
• the reaction was stopped with 50 ⁇ l per well 4N H SO 4 and optical density was measured at dual wavelengths 492-690.
• the reported titers correspond to the reciprocal of the serum dilution producing an absorbance value of 1.0.
• Nunc Immunoplate U96 Maxisorp plates (Nalge Nunc International, Rochester, NY) were coated with 200ng per well of recombinant gpl20SF2 protein and incubated for at least 14 hours at 4°C. Between steps, the plates were washed in a buffer containing 137mM NaCl and 0.05% Triton X100.
• Serum samples were initially diluted 1:25 or 1: 100 (in a buffer containing lOOmM NaPO 4 , 0.1% Casein, lmM EDTA, 1% Triton X-100, 0.5M NaCl and 0.01% Thimerosal, pH 7.5) and were serially diluted 3-fold. The plates were incubated for 50 minutes. After washing in a buffer containing 137mM NaCl, 0.05% Triton X-100, the samples were then reacted with an HRP-conjugated second antibody. The plates were then developed using a TMB substrate kit (Pierce, Rockford, IL). The plates were stopped with either 2N H 2 SO or 10% SDS, respectively and read at wavelengths of 450nm or 415nm, respectively. Anti-env antibody responses were measured as the dilution at which an OD of 0.6 was achieved.

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