Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE OF THE INVENTION
ELECTRICALLY-MEDIATED ENHANCEMENT
OF DNA VACCINE
IMMUNITY AND EFFICACY IN VIVO
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. Provisional Patent
Applications Serial Numbers 60/118996 (filed February 7, 1999) and 60/129189
(filed April 14, 1999), both of which applications are herein incorporated by
reference in their entirety.
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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.
( 1997) Ann. Rev. Immunol. 15:617-648).
Although 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. 18:449-484). DNA vaccines given orally have been reported to be
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incapable of eliciting an immune response (see Manikan et al. ( 1997) Crit.
Rev.
Immunol. 17:139-154). 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. 18:449-484). Introduction of DNA into muscle cells has been effective
to confer immunity in some cases, however, it has been reported that muscle
cells
do not seem capable of expressing molecules required for efficient antigen
presentation (Goebels et al. (1992) J. Immunol. 149:661-667; Hohfield and
Engel
( 1994) Immunol. Today 15:269-274; Michaelis et al. ( 1993) Amer. J. Pathol.
143:1142-1149). Accordingly, a need exists to enhance DNA vaccine efficacy.
The use of electric current has facilitated gene delivery in vitro and in
vivo.
Transient discontinuities in the plasma membranes of cells can be induced by
short
pulses of high-voltage electric current. These discontinuities allow
substances,
such as DNA to passively enter cells directly into the cytoplasm, thereby
avoiding
the indirect and inefficient route of endocytosis. As a consequence, more DNA
is
delivered inside cells and a greater degree of transfection occurs. This
process,
termed electroporation is widely used for facilitation of transfection of
cells in
vitro.
Recently, the use of electric current to mediate transfer of genes in vivo has
been reported. Successful transfer of genes has been accomplished for cells of
the
skin (Titomirov et al. (1991) Biochirn. Biophys. Acta 1088: 131-134; Nomura et
al.
( 1996) J. Immunol. Meth. 193: 41-49), liver (Heller et al. ( 1996) FEBS Lett.
389:225-228; Suzuki et al. ( 1998) FEBS Lett. 425: 436-440), tumors (Nishi et
al.
( 1996) Cancer Res. 56: 1050-1055; Nishi et al. (1997) Hum. Cell 10: 81-86;
Rols
et al. ( 1998) Nature Biotechnol. 16: 168-171 ), oviduct (Ochiai et al. (
1998) Poult
Sci. 77:299-302), and muscle (Aihara and Miyazaki ( 1998) Nature Biotechnol:
16:
867-870). In most cases, protein expression was demonstrated, and in some
cases
biological effects were noted, such as regression of tumors or increased
hematocrit
after inoculation of erythropoietin DNA (Rizutto et al. ( 1999) Proc. Natl.
Acad.
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Sci. (USA) 96:6417-6422). In one case, 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).
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.
The efficacy of DNA vaccines in preclinical models has been well
documented (for review see Donnelly et al. ( 1997) Ann. Rev. Immunol. 15:617-
648). The magnitude of immune responses, however, induced in primates is
generally lower than that in small animals, and the amount of DNA required for
effective immunization of primates is much higher (mg versus pg) (for example,
see Kent et al. ( 1998) J. Virol. 72:10180-10188; Gramzinski et al. ( 1998)
Molec.
Med. 4:109:118; Richmond et al. ( 1998) J. Virol. 72: 9092-9100). In addition,
several phase I human clinical studies have been conducted with little or no
immune responses reported (Calarota et al. (1998) Lancet 351: 1320-1325;
MacGregor et al. (1998) J. Infect. Dis. 178:92-100; McClements-Mann et al.
(1997) Amer. Soc. Virol. Ann. Meet. Abstr. (Vancouver, Canada), p. 115). ). In
one case, however, cytotoxic T lymphocytes were induced in human volunteers by
a malaria DNA vaccine, but no antibodies were detected (Wang et al. ( 1998)
Science 282:476-480. Therefore, the potency of DNA vaccines must be increased
to enable this technology for successful human application. The present
invention
demonstrates the enhancement of DNA vaccine potency in animals using
electrically-mediated delivery of DNA.
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SUMMARY OF THE I1V'VENTION
It is a primary object of the invention to provide electrically-mediated
enhancement of DNA vaccine efficacy in vivo. This object is achieved through
the
use of electrical current to facilitate gene delivery to cells and tissue. In
accordance
with an embodiment of the invention, DNA encoding the immunogen of interest is
administered parenterally followed by the application of electrical current in
either
the iontophoresis or electroporation range.
It is a further object of the invention to provide electrically-mediated
enhancement of HIV DNA vaccine efficacy in vivo. Preferably, the HIV DNA is
HIV gag DNA. In embodiments of the invention, such DNA is incorporated into a
plasmid and is injected either via an intramuscular (i.m.) or intradermal
(i.d.) route.
In detail, the invention provides, a method of enhancing an immune
response generated in an animal comprising the steps of:
(A) administering to the animal DNA encoding one or more immunogen
of interest; and
(B) applying an electric field to at least the site of such DNA
administration.
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 invention is particularly
concerned
with the embodiment of the above method wherein the pathogen is the retrovirus
HIV, and wherein 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.
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The invention additionally provides an apparatus for enhancing an immune
response in an animal comprising:
(A) DNA encoding one or more immunogen of interest;
(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 invention is
particularly
concerned with the embodiment of the above apparatus wherein the pathogen is
the
retrovirus HIV, and wherein 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.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure lA and Figure 1B show the expression of (3-galactosidase in mouse
muscles that had received (3-galactosidase-encoding DNA either without
additional
treatment (Figure lA) or after electroporation (Figure 1B).
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.
DETAILED DESCRIPTION OF THE INVENTION
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. As used herein, 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. As used herein, a treatment or procedure is said to enhance an immune
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response if the treatment or procedure increases the extent, duration or
degree of
the response beyond that observed in the absence of such treatment or
procedure.
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.
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. 15:617-648; Manikan et al. (1997) Crit. Rev. Immunol. 17:139-154;
Alarcon et al. ( 1999) Adv. Parasitol. 42: 343-410; Lai and Bennett ( 1998)
Crit.
Rev. Immunol. 18:449-484; Tuteja (1999) Crit. Rev. Biochem. Molec. Biol. 34:1-
24). In a preferred embodiment, the DNA vaccine of the present invention will
encode more than one epitope. Thus, for example, the administered DNA may
encode all of the epitopes of a protein associated with HIV (such as the gag
or env
protein). Alternatively, the administered DNA may encode only a peptide of
such
protein that contains one (or fewer than all) of the protein's epitopes.
The present invention contemplates that the immunogens encoded by the
DNA vaccine of the present invention may comprise a protein or peptide of a
pathogen. Such 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. gonorrhoeae), Trepanoma, etc.), 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.
(especially Dermatophytes, Pneumocystis, Trypanosoma, Plasmodium, Candida,
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Cryptococcus, Histoplasma, Coccidioides, amoeba, schistosomes, etc.).
Alternatively, 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. As used herein, immunogens are related to one another if the immune
responses that they induce elicit antibodies that bind to the same cell,
microbe,
virus, etc. For example, 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. For example, individuals suffering from Acquired Immunodeficiency
Syndrome (AIDS) may develop infections caused by the bacterium Listeria
monocytogenes, and by the yeast Candida. DNA that encodes epitopes of a
Listeria monocytogenes protein and a Candida protein would encode clinically
related immunogens. Alternatively, 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.
Most preferably, 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. Alternatively, the administered DNA will not
contain such regulatory elements, and will require cellular processes (such as
recombination or integration into nuclear or mitochondria) 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
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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. For example, 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).
In a particularly preferred embodiment, DNA, preferably in the form of
plasmid DNA, is administered (especially by injection) into tissue and voltage
pulses are applied between electrodes disposed in the tissue, thus applying
electric
fields to cells of the tissue. 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.
Pharrnacol. 13:353-359; Singh and Maibach ( 1993) Derrnatology. 187:235-238;
Singh and Maibach ( 1994) Crit. Rev. Ther. Drug Carrier Syst. 11:161-213; Su
et
al. ( 1994) J. Pharm. Sci. 83:12-17; Costello et al. ( 1995) Phys. Ther.
75:554-563;
Howard et al. ( 1995) Arch. Phys. Med. Rehabil. 76:463-466; Kassan et al. (
1996)
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J. Amer. Acad. Dermatol. 34:657-666; Riviere et al. (1997) Pharm. Res. 14:687-
697; Zempsky et al. ( 1998) Amer. J. Anesthesiol. 25:158-162; Muramatsu et al.
( 1998) Int. J. Mol. Med. 1:55-62; Garrison J. ( 1998) Med. Device Technol.
9:32-36;
Banga et al. ( 1998) Trends Biotechnol. 16:408-412; Banga et al. ( 1999) Int.
J.
Pharm. 179:1-19; Singh et al. ( 1999) Anticancer Drugs. 10:139-146; Neumann et
al. (1999) Bioelectrochem. Bioenerg. 48:3-16; and Heiser (2000) Methods Mol.
Biol. 130:117-134. Whereas any suitable route of inoculation may be employed,
of
intra-muscular (i.m.), intra-dermal (i.d.), or sub-cutaneous (s.c.), i.m.
injection is
the most efficacious. Enhanced immune responses are, however, also seen after
i.d. injections.
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. Examples of 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
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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 lmm. 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.Smm. The needles are insulated from direct contact with the
stainless steel block.
Preferred electrical field conditions for i.m. administration are as follows:
SOmA for lOmsec for 5 pulses then rotated 90° (i.e., orthogonally) for
5 additional
pulses; 120V for 10 msec for 5 pulses then rotate orthogonally for 5
additional
pulses when using the bipolar probe; and 80V for lOmsec for 5 pulsed then
rotate
orthogonally for 5 additional pulses when using the combination plate and
needle
probe. Preferred electrical field conditions for i.d. administration are as
follows:
SOmA for SOmsec 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.
Examples of 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
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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.
Alternatively, or in addition, infiltration of inflammatory cells (in response
to the
electric current) could have an "adjuvant" effect on the produced antigen. The
present invention demonstrates that DNA vaccine potency can be increased by
application of electric current. The results indicate that a significant
limitation to
efficient transfection of cells in vivo by naked DNA vaccines in the past
(possibly
accounting for the lack of efficacy of DNA vaccines in larger animals, such as
primates, in the past) has been the distribution of the introduced DNA within
tissue
and/or uptake of DNA by cells. Iontophoresis and electroporation (as well as
equivalent means for facilitating the delivery of DNA into cells and tissue
can be
used to surmount this problem and enable the development of DNA vaccines.
Having now fully described the invention, the same will be further
illustrated by way of the following examples, which are meant solely to
illustrate
the invention and are not to be construed to limit the invention in any way.
Those
skilled in the art will recognize modifications that are within the spirit and
scope of
the invention.
Example 1
Materials And Methods For In Vivo
Electrical-Enhanced Delivery Of DNA.
Bacterial Strain and plasmid preparation
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). In brief, 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
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manufacturer instructions and resuspended in 0.9% sodium chloride (Abbott
Laboratories, North Chicago, 1L).
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 l, 1999). The plasmid
was grown in E. coli strain HB 101, purified using a Qiagen Endofree 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.
Expression of the encoded antigens was verified by transient expression
studies in B 16 cells. One pg of each plasmid DNA was used for Lipofectin
(Gibco/BRL) transfection following the manufacturers protocol; 5x 105 cells
were
used per 3 cm tissue culture dish; incubation time for DNA/Lipofectin on cells
was
for 4 hours. Supernatants were harvested 36 hours after removal of the
DNA/Lipofectin solution and cells were lysed in 500 ~l phosphate buffered
saline
(PBS)/0.5% TritonX100 (Mallinckrodt). Luciferase activity in cell lysates was
detected by commercial Luciferase Reporter Gene Assay (Roche, Indianapolis,
IN).
Immunization Procedure:
Female 6-8 week old CB6F1 or BalbC mice (Charles River) were
anesthetized using 4 parts ketamine HCI, 100mg/ml stock solution, (Fort Dodge
Animal Health, Fort Dodge, Iowa) 1 part xylazine, 20mg/ml, (LLoyd Labs,
Shenandoah, Iowa). The mice received 1 ~1 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 ~1. To control
needle depth, the syringe was covered with polyethylene tubing (i.d. 0.38) to
expose only the bevel. 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
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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.
Chemi-luminescent ELISA
MicroLite 2, 96 well flat bottom plates (Dynes Technologies, Chantilly,
VA) were coated with HN p24 protein at 5~g/ml in IOmM tris pH=7.5, 50 ~l per
well and incubated at 4°C overnight. The plates were washed 3X with
wash buffer
[1X 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
pl/well blocking buffer [1X 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
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Sciences, Inc. Bogart, GA), diluted 1/500 in wash buffer, 50 pl/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 1X 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.
Colormetric ELISA
Wells of Immulon 2 HB "U" bottom microtiter plates (Dynex
Technologies, Chantilly, VA) were coated with HN p55 protein at 5 ~1/ml in
PBS,
50 p,l per well, and incubated at 4°C overnight. The plates were washed
6X with
wash buffer [PBS, 0.1 % tween (Sigma, St. Louis, MO)] and blocked at
37°C for 1
hour with 150g1/well blocking buffer [PBS, 0.1% tween 20 (Sigma, St. Louis
MO),
1 % goat serum]. Test sera were diluted 1/25 followed by serious 3-fold
dilutions
in blocking buffer. The block solution was aspirated the plates were incubated
at
37° for 2 hours with 150p1/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 (O.1M citric acid, O.1M dibasic sodium
phosphate), Spl 30% H~O~. The reaction was stopped with 501 per well 4H
HZS04 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 1Ø
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Example 2
Enhancement Of Luciferase Gene Expression
In Muscle Cells In Mammals
Previous reports have demonstrated that application of electric current after
injection of plasmid DNA has resulted in increased expression of the encoded
proteins in the injected tissues (for example see Mir et al. ( 1999) CR Acad.
Sci. III
321:893-899; Mathieson ( 1999) Gene Ther. 6: 508-514). In order to demonstrate
the ability of electoporation and iontophoresis to facilitate the distribution
and/or
uptake of DNA into mammalian cells and tissue, mice were injected with DNA
encoding the readily discernable marker enzyme luciferase (Luc).
Immunization Procedure
Female 6-8 week old CB6F1 mice (Charles River) were anesthetized using
4 parts ketamine HCI, 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 p,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 pl. To control needle depth, a 0.3 cc insulin
syringe was covered with polyethylene tubing (i.d. 0.38) to expose only the
bevel.
In some instances, electric current was applied to the injected muscles as
follows. For constant current deliveries (iontophoresis), 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. Following plasmid
injection, 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. For constant voltage deliveries
(electroporation), plasmid DNA in PBS was injected into the right tibialis
anterior
muscle as previously described. Electrical energy delivery was performed
through
a bipolar needle probe that was placed over the site of plasmid injection. The
probe needles had a separation distance of 0.4 cm and a needle length of 0.3
cm.
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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-l, were applied in
one
orientation, the probe was rotated 90 degrees and 5 additional pulses were
applied.
Measurement of 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 1X 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 pl of luciferase assay
reagent
(Promega, Madison, WI) into wells containing 20 p,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 QuantiLumO Recombinant Luciferase (Promega, Madison, WI).
Results are expressed as ng luciferase per mg muscle protein, with protein
determination by BCA Protein Assay Reagent (Pierce).
The results of this experiment are shown in Table l, and indicate that
electoporation and iontophoresis facilitated the distribution andlor uptake of
DNA
into mammalian cells and tissue. In Table 1, results are expressed as ng
luciferase
activity per mg muscle protein. Numbers in parentheses indicate standard
deviation of the mean (sd).
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Table 1
Luc DNA Luc Mean Fold
Treatment Activit (sd) Increase
6.76
0.74 3.794 1.00
(control)
0,44
(3.91)
(10 fig) 9.11
1.92
26.63
10.54 17.35 4.57
Ionto
23.46
(895)
(10 pg) 5.51
20.61
18.5
35.02 27.764 7.32
Electro
39.02
(11.30)
(10 pg) 33.22
13.06
Example 3
Enhancement Of Luciferase Gene Expression
In Muscle Cells In Mammals
In order to assess the duration of luciferase gene expression in mammalian
tissue, groups of 6 CB6 F1 mice were inoculated with 10 ltg 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). At 4 and 14 days after
inoculation, the muscles were collected and luciferase activity was measured
and
expressed as ng luciferase activity per mg muscle protein. The data (Table 2)
showed a significant enhancement of luciferase gene expression in mammalian
tissue that had been subjected to electroporation, relative to non-
electroporated,
control animals. In Table 2, numbers in brackets indicate standard deviation
of the
mean (sd).
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Table 2
4 Days 14 Days
Luc DNA
Treatment Luc Mean Luc Mean
activit (sd) activit (sd)
0 0
5.02 1.32 0 0
(control)
0 (2.03) o (0)
(25 pg) 0.
17
0.05 0
0 0
17.3 5.26
42.9 50.8 3 12.7
63
(Electro) .
9.38 (33.2) 18.9
(14.9)
(10 fig) 69.7 7.52
92.8 40.4
72.9 0.71
Example 4
Enhancement Of (3-Galactosidase Gene Expression
In Muscle Cells In Mammals
To further demonstrate the ability of electoporation and iontophoresis to
facilitate the distribution and/or uptake of DNA into mammalian cells and
tissue,
mice were injected with DNA encoding a different readily discernable marker
enzyme ((3-galactosidase).
CB6 F1 mice were inoculated with 100 pg of pCMV (3-gal, a (3-
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 (3-galactosidase.
One
group of mice was not further treated, one group was treated with
electroporation,
and another with iontophoresis. At 1 day after inoculation, the muscles were
collected and prepared for microscopy (magnification = X). The data (Figure lA
(untreated); Figure 1B (electroporation)) indicated that electroporation had
substantially facilitated the distribution and/or uptake of DNA into mammalian
cells and tissue. A similar result was observed in mouse tissue that had been
subjected to iontophoresis.
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Thus, DNA plasmids encoding the reporter genes luciferase and (3-
galactosidase were employed to measure transfection of muscles cells in vivo.
At 4
and 14 days after a single inoculation of DNA, luciferase expression was found
to
be higher in muscles treated with electric current (as compared to untreated
muscles (see Example 2, Table 1)). This was true for muscles that had been
subjected to both iontophoresis (4.6-fold) and electroporation (7.3-fold).
Similarly,
the number of muscle fibers detectably transfected after inoculation of (3-
galactosidase DNA was found to have been substantially increased by
iontophoresis and electroporation, as compared to untreated muscles, as judged
by
(3-galactosidase staining of muscle tissue sections. In addition, as noted
previously
(Mir et al. ( 1999) CR Acad. Sci. III 321:893-899), 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.
Example 5
Enhancement Of Antibody Responses In Mammals
In order to demonstrate the ability of electroporation and iontophoresis to
enhance the antibody responses of mammals, groups of 4-6 CB6 Fl mice were
inoculated a single time with 10 p.g of DNA encoding the HIV gag protein.
The plasmid pCMV HIV p55 gag, grown in E. coli strain HB101, 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. At all time points
tested,
antibody titers in mice that had been subjected to iontophoresis and
electroporation
were 8- to 20-fold higher than in animals receiving no further treatment
(Figure 2).
As with luciferase expression levels, in general, electroporation conditions
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appeared slightly superior to iontophoresis for enhancement of antibody
responses.
The data indicate a pronounced enhancement of antibody response in animals
subjected to electroporation and iontophoresis, relative to the response of
control
animals.
Example 6
Effect Of Vaccine Boosting On Antibody Responses In Mammals
In order to demonstrate the effect of vaccine boosting on antibody
responses in mammals, groups of 6 CB6 F 1 mice were inoculated with 10 pg 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).
Example 7
Effect Of Conditions Of Iontophoresis And Electrophoresis
On Antibody Responses In Mammals
In order to demonstrate the effect of the conditions of iontophoresis and
electroporation on mammalian antibody responses, groups of 6 CB6 F1 mice were
inoculated with 10 p.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.
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Table 3
Treatment ConditionsNumber DurationGeometric Fold
of
Pulses (cosec) Mean Titer Increase
DNA control- - - 414 1
Ionto 50 mA 60 10 1071 2.59
Ionto 50 mA 10 10 2521 6.09
Ionto 50 mA 10 50 1738 4.20
Ionto 100 mA 10 10 1876 4.53
Ionto 100 mA 10 50 2293 5.54
Electro 25 V/cm 10 10 479 1.16
Electro 50 V/cm 10 10 1099 2.65
Electro 50 V/cm 10 50 2390 5.77
Electro 100 V/cm 10 SO 1800 4.35
Electro 200 V/cm 10 10 2208 5.33
Electro 200 V/cm 10 50 2079 5.02
Electro 300 V/cm 10 10 2834 6.85
Electro 400 V/cm 10 10 1359 3.28
Example 8
Efficacy Of Intradermal Administration
Of Iontophoresis And Electroporation In Mammals.
In order to assess the efficacy of intradermal administration of
iontophoresis and electroporation in mammals, groups of 6 CB6 F1 mice were
inoculated with 10 ~g of HIV gag DNA intradermally on the backs. One group of
mice was not further treated (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. In Table 4, data are tabulated as geometric mean ELISA titer and
fold
increase over titers achieved in vaccinated but untreated mice. As shown,
electroporation and iontophoresis are also effective for the intradermal route
of
administration of DNA vaccines.
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Table 4
Treatment ConditionsNumber Duration Geometric Fold
of
Pulses (cosec) Mean Increase
DNA control- - - 472 1
Ionto 50 mA 60 10 696 1.47
Ionto 50 mA 10 10 1291 2.74
~
Ionto 50 mA 10 50 626 1.33
Ionto 100 mA 10 10 2376 5.03
Ionto 100 mA 10 50 1134 2.40
Electro 150 V/cm 10 10 2768 5.86
Electro 300 V/cm 10 10 851 1.80
Electro 450 V/cm 10 10 132 0.28
Electro 600 V/cm 10 10 887 1.88
Electro 750 V/cm 10 10 480 1.02
Electro 75 V/cm 10 50 224 0.47
Electro 150 V/cm 10 50 728 1.54
Electro 225 V/cm 10 50 2202 4.67
Electro 300 V/cm 10 50 6125 12.98
Electro 375 V/cm 10 50 937 1.99
Example 9
Efficacy Of Plate Electrode For
Iontophoresis And Electroporation In Mammals
In order to demonstrate the efficacy of employing a plate electrode for
iontophoresis and electroporation in mammals, groups of 6 CB6 Fl mice were
inoculated with 10 pg 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
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directly to the plate electrode. For in vivo application, 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.
Table 5
Treatment ConditionsNumber Duration Geometric Fold
of (cosec) Mean Increase
Pulses
DNA control- - - 198 1
Ionto 50 mA 10 10 1596 8.06
Ionto 100 mA 10 10 1235 6.24
Electro 200 V/cm 10 10 1411 7.13
Electro 300 V/cm 10 10 1252 6.32
Example 10
Efficacy Of Electroporation On
Anti-HIV Gag Antibody Responses in Mammals
In order to demonstrate the efficacy of electroporation on the anti-HIV gag
antibody response of mammals, groups of 4-6 New Zealand white rabbits were
inoculated with a combination DNA vaccine consisting of S00 pg of DNA
encoding the HIV gag protein and 1 mg of DNA encoding the HIV env protein.
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The plasmid pCMV HIV gag was used as the source of the gag- encoding DNA.
Plasmid pCMV HIV env was employed as the source of the env-encoding DNA.
The plasmid expresses high levels of HN-1 env, due to a potent CMV promoter
with intron A and a codon-optimized env-encoding region (see U.S. Provisional
Patent Application Serial No. 60/168,471, filed December l, 1999).
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. 15:124-132).
At 26 weeks, all rabbits were boosted with recombinant gag protein and
sera were collected at 2 weeks post-protein boost and analyzed for anti-gag
antibody responses. 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 S~g/ml in PBS, 50 pl per
well,
and incubated at 4°C overnight. The plates were washed 6X with wash
buffer
[PBS, 0.1 % Tween 20 (Sigma, St. Louis, MO)] and blocked at 37°C for 1
hour
with 150p1/well blocking buffer [PBS, 0.5% casein, and 5% goat serum]; the
dilution buffer was blocking buffer plus 0.3% Tween 20. The secondary antibody
was goat anti-rabbit IgG used at 1/20,000; and the OD cutoff used was 0.6.
Test
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sera were diluted 1/25 followed by serial 3-fold dilutions in blocking buffer.
The
blocking buffer was aspirated and the plates were incubated at 37°C for
2 hours
with 50~1/well of each dilution. After washing 6 times, the plates were
incubated
for 1 hour at 37°C with 50~1/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 (O.1M citric acid, O.1M
dibasic
sodium phosphate), 5p130% H20z. The reaction was stopped with 50p1 per well
4N H2S04 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 1Ø
For measurement of anti-env antibodies in rabbits and guinea pigs, Nunc
Immunoplate U96 Maxisorp plates (Nalge Nunc International, Rochester, NY)
were coated with 200ng per well of recombinant gp 120SF2 protein and incubated
for at least 14 hours at 4°C. Between steps, the plates were washed in
a buffer
containing 137mM NaCI and 0.05% Triton X100. Serum samples were initially
diluted 1:25 or 1:100 (in a buffer containing 100mM NaPO~, 0.1% Casein, 1mM
EDTA, 1% Triton X-100, 0.5M NaCI 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 NaCI, 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 HZS04 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.
The data is shown in Figure 4, and indicates that electroporation was
effective in enhancing the induced immune response. In Figure 4, data are
plotted
as geometric mean ELISA titer and error bars indicate SEM.
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Example 11
Efficacy Of Electroporation On
Anti-HIV Env Antibody Responses in Mammals
As a further demonstration of the efficacy of electroporation on the
antibody response of mammals, groups of 4 New Zealand white rabbits were
inoculated with a combination DNA vaccine consisting of 500 ~.g of HIV gag-
encoding DNA and 1 mg of HIV env-encoding DNA (obtained as described above)
in the hind leg muscles at 0 and 6 weeks. One group of rabbits received DNA
without further treatment and one group was treated with electroporation with
a 6-
needle electrode as described above. Sera were collected at 2 weeks post the
second DNA immunization and analyzed for anti-env antibody responses. The
data are shown in Table 6. In Table 6, data are tabulated as individual ELISA
titers, geometric mean ELISA titers (GMT) and fold increase over titers
achieved
in vaccinated but untreated rabbits. The data show a pronounced enhancement of
antibody titer in animals subjected to electroporation.
Table 6
Geometric Fold
ConditionsTiter Mean Titer Increase
36
DNA 8 44 1
control 141
86
2660
6-needle 762 833 18.93
Electro 821
289
It will be apparent to those skilled in the art that various modifications may
be made in the present invention without departing from the spirit and scope
of the
present invention. It will be additionally apparent to those skilled in the
art that the
basic construction of the present invention is intended to cover any
variations, uses
or adaptations of the invention following, in general, the principle of the
invention
and including such departures from the present disclosure as come within known
or
customary practice within the art to which the invention pertains. Therefore,
it will
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be appreciated that the scope of this invention is to be defined by the claims
appended hereto, rather than the specific embodiments which have been
presented
as examples. All references and documents cited herein are incorporated by
reference herein in their entirety.
