U.S. patent application number 14/980874 was filed with the patent office on 2016-06-23 for vaccine and drug delivery by intranasal application of vector and vector extracts.
The applicant listed for this patent is UAB RESEARCH FOUNDATION. Invention is credited to Zhongkai Shi, De-Chu C. Tang, Kent Rigby van Kampen.
Application Number | 20160175428 14/980874 |
Document ID | / |
Family ID | 32995712 |
Filed Date | 2016-06-23 |
United States Patent
Application |
20160175428 |
Kind Code |
A1 |
Tang; De-Chu C. ; et
al. |
June 23, 2016 |
VACCINE AND DRUG DELIVERY BY INTRANASAL APPLICATION OF VECTOR AND
VECTOR EXTRACTS
Abstract
Disclosed and claimed is a method of non-invasive immunization
in an animal and/or a method of inducing a systemic immune response
or systemic therapeutic response to a gene product. The skin of the
animal is contacted with a non-replicative vector chosen from the
group of bacterium, virus, and fungus, wherein the vector comprises
and expresses a nucleic acid molecule encoding the gene product, in
an amount effective to induce the response.
Inventors: |
Tang; De-Chu C.;
(Birmingham, AL) ; Shi; Zhongkai; (Birmingham,
AL) ; van Kampen; Kent Rigby; (Hoover, AL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UAB RESEARCH FOUNDATION |
Birmingham |
AL |
US |
|
|
Family ID: |
32995712 |
Appl. No.: |
14/980874 |
Filed: |
December 28, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13961439 |
Aug 7, 2013 |
9248177 |
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14980874 |
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10346021 |
Jan 16, 2003 |
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13961439 |
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10116963 |
Apr 5, 2002 |
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10346021 |
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10052323 |
Jan 18, 2002 |
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10116963 |
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09563826 |
May 3, 2000 |
6348450 |
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10052323 |
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09533149 |
Mar 23, 2000 |
6716823 |
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09563826 |
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09402527 |
Jan 3, 2000 |
6706693 |
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09533149 |
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60132216 |
May 3, 1999 |
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Current U.S.
Class: |
128/200.22 ;
128/200.14; 128/200.21; 424/199.1 |
Current CPC
Class: |
A61K 2039/54 20130101;
A61K 39/001182 20180801; A61M 15/08 20130101; A61K 2039/53
20130101; A61K 2039/5254 20130101; A61K 2039/522 20130101; A61K
38/1774 20130101; Y02A 50/30 20180101; A61K 2039/521 20130101; A61K
2039/543 20130101; A61K 39/12 20130101; C12N 2760/16034 20130101;
A61K 2039/55555 20130101; C12N 2710/10343 20130101; C12N 2799/022
20130101; A61K 39/39 20130101; A61K 39/08 20130101; C12N 2760/16134
20130101; A61K 38/27 20130101; A61K 2039/541 20130101; A61K
2039/523 20130101; C12N 7/00 20130101; C12N 2710/10043 20130101;
A61M 11/02 20130101; A61M 15/009 20130101; A61M 2210/0618 20130101;
C12N 2760/16071 20130101; A61K 39/0011 20130101; A61K 2039/55516
20130101; A61K 2039/542 20130101; A61K 2039/55522 20130101; A61M
11/008 20140204; A61K 39/00 20130101; A61K 39/145 20130101; A61K
2039/5256 20130101; A61K 38/193 20130101; Y02A 50/412 20180101;
A61K 38/27 20130101; A61K 2300/00 20130101; A61K 38/193 20130101;
A61K 2300/00 20130101; A61K 38/1774 20130101; A61K 2300/00
20130101 |
International
Class: |
A61K 39/145 20060101
A61K039/145; A61M 15/08 20060101 A61M015/08; A61M 11/02 20060101
A61M011/02; A61M 15/00 20060101 A61M015/00; C12N 7/00 20060101
C12N007/00; A61M 11/00 20060101 A61M011/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] Research carried out in connection with this invention may
have been supported in part by grants from the National Institutes
of Health, grant numbers 2-R42-AI44520-02, 1-R41-AI44520-01 and
1-R43-AI-43802-01; Office of Naval Research grant N00014-01-1-0945;
and U.S. Army grant DAMD-17-98-1-8173. The United States government
may have certain rights in the invention.
Claims
1-10. (canceled)
11. A pharmaceutical dosage for intranasal administration,
comprising: a pharmaceutical acceptable carrier in a liquid form
admixed with a non-replicating adenoviral vector that expresses one
or more influenza antigens, one or more influenza epitopes, or a
combination thereof, wherein the vector is configured to
non-invasively induce a protective immune response against
influenza.
12. The pharmaceutical dosage of claim 11, further comprising a
squeeze spray dispenser, a pump dispenser, or an aerosol
dispenser.
13. The pharmaceutical dosage of claim 11, wherein the adenoviral
vector is defective in its E1 and/or E3 and/or E4 regions.
14. The pharmaceutical dosage of claim 11, wherein the adenoviral
vector is defective in its E1/E3 region.
15. The pharmaceutical dosage of claim 11, wherein the adenoviral
vector is defective in all adenoviral genes.
16. The pharmaceutical dosage of claim 11, further comprising an
adjuvant.
17. The pharmaceutical dosage of claim 11, wherein the influenza
antigen is influenza hemagglutinin or influenza nuclear
protein.
18. A pharmaceutical dosage for intranasal administration,
comprising: a pharmaceutical acceptable carrier in a liquid form
admixed with a non-replicating adenoviral vector that expresses one
or more heterologous antigens of interest, wherein the vector is
configured to non-invasively induce a protective immune response
against a pathogen.
19. The pharmaceutical dosage of claim 18, wherein the one or more
heterologous antigens of interest is selected from the group
consisting of influenza hemagglutinin, influenza nuclear protein,
influenza M2, tetanus toxin C-fragment, rabies glycoprotein, HBV
surface antigen, HIV gp120, HIV gp160, human carcinoembryonic
antigen, malaria CSP, malaria SSP, malaria MSP, malaria pfg,
botulinum toxin A and mycobacterium tuberculosis HSP.
20. The pharmaceutical dosage of claim 18, further comprising a
squeeze spray dispenser, a pump dispenser, or an aerosol
dispenser.
21. The pharmaceutical dosage of claim 18, wherein the adenoviral
vector is defective in its E1 and/or E3 and/or E4 regions.
22. The pharmaceutical dosage of claim 18, wherein the adenoviral
vector is defective in its E1/E3 region.
23. The pharmaceutical dosage of claim 18, wherein the adenoviral
vector is defective in all adenoviral genes.
24. The pharmaceutical dosage of claim 18, further comprising an
adjuvant.
25. A pharmaceutical dosage for intranasal administration,
comprising: a pharmaceutical acceptable carrier in a liquid form
admixed with a non-replicating adenoviral vector that expresses one
or more heterologous antigens of interest, wherein the vector is
configured to non-invasively induce a protective immune response
against a pathogen; and, a dispenser.
26. The pharmaceutical dosage of claim 25, wherein the dispenser is
a squeeze spray dispenser, a pump dispenser, or an aerosol
dispenser.
27. The pharmaceutical dosage of claim 25, wherein the one or more
heterologous antigens of interest is selected from the group
consisting of influenza hemagglutinin, influenza nuclear protein,
influenza M2, tetanus toxin C-fragment, rabies glycoprotein, HBV
surface antigen, HIV gp120, HIV gp160, human carcinoembryonic
antigen, malaria CSP, malaria SSP, malaria MSP, malaria pfg,
botulinum toxin A and mycobacterium tuberculosis HSP.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 10/116,963, filed Apr. 5, 2002, which is a
continuation-in-part of U.S. patent application Ser. No.
10/052,323, filed Jan. 18, 2002, which is a continuation-in-part of
U.S. patent application Ser. No. 09/563,826, filed May 3, 2000
(issued Feb. 19, 2002 as U.S. Pat. No. 6,348,450), which claims
priority from U.S. Provisional Application No. 60/132,216, filed
May 3, 1999, and is also a continuation-in-part of U.S. patent
application Ser. No. 09/533,149, filed Mar. 23, 2000, which in turn
is a continuation of U.S. patent application Ser. No. 09/402,527,
filed on Aug. 13, 2000. Each of these applications and each of the
documents cited in each of these applications ("application cited
documents"), and each document referenced or cited in the
application cited documents, either in the text or during the
prosecution of those applications, as well as all arguments in
support of patentability advanced during such prosecution, are
hereby incorporated herein by reference. Various documents are also
cited in this text ("application cited documents"). Each of the
application cited documents, and each document cited or referenced
in the application cited documents, is hereby incorporated herein
by reference.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the fields of
immunology and vaccine technology. The present invention also
relates to techniques of skin-targeted non-invasive delivery of to
elicit immune responses and uses thereof. The invention further
relates to methods of non-invasive immunization in an animal and/or
methods of inducing an immunological, e.g., systemic immune
response or a therapeutic, e.g., a systemic therapeutic response,
in an animal, products therefrom and uses for the methods and
products therefrom. The invention yet further relates to such
methods comprising contacting skin of the animal with a vector in
an amount effective to induce the response, e.g., systemic immune
response, in the animal. Even further, the invention relates to
such methods wherein the vector comprises and expresses an
exogenous nucleic acid molecule encoding an epitope or gene product
of interest, e.g., an antigen or therapeutic. Still further, the
invention relates to such methods wherein the response, e.g.,
systemic immune or therapeutic response, can be to or from the
epitope or gene product. Even further still, the invention relates
to such methods wherein the vector is non-replicative.
[0004] The invention yet further relates to such methods wherein
the response is induced by contacting the skin of an animal with
cell-free extracts in an amount effective to induce the response,
wherein the extracts are prepared by filtration of disrupted cells
chosen from the group consisting of bacterium, fungus, cultured
animal cells, and cultured plant cells, wherein the cell comprises
and expresses a nucleic acid molecule encoding the gene
product.
[0005] The invention still further relates to such methods wherein
the response is enhanced by methods comprising contacting skin of
the animal with vaccines, wherein the vaccines are admixed with
heat-shock protein 27, in an amount effective to induce the
response.
[0006] The invention yet further still relates to such methods
wherein the nucleic acid molecule can encode an epitope of interest
and/or an antigen of interest and/or a nucleic acid molecule that
stimulates and/or modulates an immunological response and/or
stimulates and/or modulates expression, e.g., transcription and/or
translation, such as transcription and/or translation of an
endogenous and/or exogenous nucleic acid molecule. The invention
additionally relates to such methods wherein the nucleic acid
molecule can be exogenous to the vector. The invention also relates
to such methods wherein the exogenous nucleic acid molecule encodes
one or more of an antigen or portion thereof, e.g., one or more of
an epitope of interest from a pathogen, e.g., an epitope, antigen
or gene product which modifies allergic response, an epitope
antigen or gene product which modifies physiological function,
influenza hemagglutinin, influenza nuclear protein, influenza M2,
tetanus toxin C-fragment, anthrax protective antigen, anthrax
lethal factor, anthrax germination factors, rabies glycoprotein,
HBV surface antigen, HIV gp120, HIV gp160, human carcinoembryonic
antigen, malaria CSP, malaria SSP, malaria MSP, malaria pfg,
botulinum toxin A, and mycobacterium tuberculosis HSP; and/or a
therapeutic or an immunomodulatory gene, a co-stimulatory gene
and/or a cytokine gene.
[0007] Even further, the invention relates to such methods wherein
the immune response can be induced by the vector expressing the
nucleic acid molecule in the vector or in the animal's cells, e.g.,
epidermal cells including but not limited to keratinocytes,
melanocytes, langerhans cells, merkel cells and hair matrix cells.
The invention still further relates to such methods wherein the
immune response can be against a pathogen or a neoplasm.
[0008] Also, the invention relates to compositions used in the
methods. For instance, the invention relates to a prophylactic
vaccine or a therapeutic vaccine or an immunological composition
comprising the vector, wherein the vector can be replicative or
non-replicative. Additionally, the invention relates to
compositions comprising the cell-free extract obtained from the
vector. The invention also comprises compositions comprising the
replicative vector, the non-replicative vector, the cell-free
extract, or the cell-free extract in combination with an adjuvant
to enhance the effectiveness of the composition. The invention also
comprises the above compositions wherein the adjuvant is heat shock
protein 27.
[0009] The invention additionally relates to such methods and
compositions therefor wherein the animal can be a vertebrate, e.g.,
a fish, bird, reptile, amphibian or mammal, advantageously a mammal
such as a human or a companion or domesticated or food- or
feed-producing or livestock or game or racing or sport animal, for
instance, a cow, a horse, a dog, a cat, a goat, a sheep or a pig,
or fowl such as chickens, duck, turkey.
[0010] The invention further relates to such methods and
compositions therefor wherein the vector can be one or more of a
viral, including viral coat, e.g., with some or all viral genes
deleted therefrom, bacterial, protozoan, transposon, and
retrotransposon, and DNA vector, e.g., a recombinant vector; an
adenovirus, such as an adenovirus defective in its E1 and/or E3
and/or E4 region(s) and/or all adenoviral genes.
[0011] The invention further relates to such methods and
compositions therefore wherein the vector can be
non-replicative.
[0012] The invention further relates to mucosal, intranasal,
perlingual, buccal, oral, oral cavity, administration of adenovirus
defective in its E1 and/or E3 and/or E4 region(s) and/or all
adenoviral genes, advantageously defective in its E1 and E3
regions, e.g., such an adenovirus comprising an exogenous or
heterologous nucleic acid molecule, such as an exogenous or
heterologous nucleic acid molecule encoding an epitope of interest
of an influenza, e.g., one or more influenza epitopes of interest
and/or one or more influenza antigens. Such an administration can
be a method to induce an immunological response, such as a
protective immunological response. The adenovirus in this instance
can be a human adenovirus. The adenovirus can be another type of
adenovirus, such as a canine adenovirus. Thus, if the host or
animal is other than a human, the adenovirus can be matched to the
host; for example, in veterinary applications wherein the host or
animal is a canine such as a dog, the adenovirus can be a canine
adenovirus.
[0013] The invention accordingly further relates to methods of the
invention wherein the vector or cell can be matched to the host or
can be a vector that is interesting to employ with respect to the
host or animal because the vector can express both heterologous or
exogenous and homologous gene products of interest in the animal;
for instance, in veterinary applications, it can be useful to use a
vector pertinent to the animal, for example, in canines one can use
canine adenovirus; or more generally, the vector can be an
attenuated or inactivated pathogen of the host or animal upon which
the method is being performed.
[0014] The invention further relates to methods of the invention
wherein the vector is chosen from yeast vectors, insect cells
transduced with baculovirus vectors, bacterial vectors, and tissue
culture cells expressing antigens of interest. Preferably, the
vector is a bacterial vector, wherein the bacteria are Escherichia.
Preferably, the invention relates to such methods wherein the
bacteria are Escherichia coli.
[0015] The invention still further relates to methods of the
invention wherein the vector is a bacterial vector, wherein the
bacteria are of the genus Clostridium. Preferably, the invention
relates to such methods wherein the bacteria are Clostridium tetuni
or Clostridium botulinum.
[0016] The invention still further relates to such methods
encompassing applying a delivery device including the vector to the
skin of the animal, as well as such a method further including
disposing the vector in and/or on the delivery device; and, to such
delivery devices.
[0017] The invention yet further relates to such methods wherein
the vector can have all viral genes deleted therefrom, as well as
to such vectors.
[0018] The invention yet further relates to such methods wherein
the vector can be non-replicative, for example, wherein the vector
has been irradiated.
[0019] The invention even further still relates to such methods
wherein the vector can induce an anti-tumor effect in the animal,
e.g., by expressing an oncogene, a tumor-suppressor gene, or a
tumor-associated gene.
[0020] In addition, the invention relates to immunological products
generated by the expression and the expression products, as well as
in in vitro and ex vivo uses thereof.
[0021] Still further, the invention relates to non-invasive methods
of administering vectors for purposes other than immunization, as
in the administration of botulinum toxin A for cosmetic
applications.
[0022] Even further still, the invention relates to non-invasive
methods of administering botulinum toxin A as a therapeutic in the
treatment of muscular or nervous system conditions including, but
not limited to, migraine headaches, spasms, and excessive
sweating.
BACKGROUND OF THE INVENTION
[0023] Activation of the immune system of vertebrates is an
important mechanism for protecting animals against pathogens and
malignant tumors. The immune system consists of many interacting
components including the humoral and cellular branches. Humoral
immunity involves antibodies that directly bind to antigens.
Antibody molecules as the effectors of humoral immunity are
secreted by B lymphocytes. Cellular immunity involves specialized
cytotoxic T lymphocytes (CTLs) which recognize and kill other cells
which produce non-self antigens. CTLs respond to degraded peptide
fragments that appear on the surface of the target cell bound to
MHC (major histocompatibility complex) class I molecules. It is
understood that proteins produced within the cell are continually
degraded to peptides as part of cellular metabolism. These
fragments are bound to the MHC molecules and are transported to the
cell surface. Thus the cellular immune system is constantly
monitoring the spectra of proteins produced in all cells in the
body and is poised to eliminate any cells producing non-self
antigens.
[0024] Vaccination is the process of priming an animal for
responding to an antigen. The antigen can be administered as
purified protein, protein contained in killed/attenuated pathogens,
or as a gene which then expresses the antigen in host cells
(genetic immunization). The process involves T and B lymphocytes,
other types of lymphoid cells, as well as specialized antigen
presenting cells (APCs) which can process the antigen and display
it in a form which can activate the immune system. Current modes
for the administration of vaccines has focused on invasive
procedures including needle injections, scarification, and gene
gun-mediated penetration. Inoculation of vaccines in an invasive
mode requires equipment and personnel with special medical
training, and is usually associated with discomfort and potential
hazards (bleeding, infection).
[0025] The efficacy of a vaccine is measured by the extent of
protection against a later challenge by a tumor or a pathogen.
Effective vaccines are immunogens that can induce high titer and
long-lasting protective immunity for targeted intervention against
diseases after a minimum number of inoculations. For example,
genetic immunization is an approach to elicit immune responses
against specific proteins by expressing genes encoding the proteins
in an animal's own cells. The substantial antigen amplification and
immune stimulation resulting from prolonged antigen presentation in
vivo can induce a solid immunity against the antigen. Genetic
immunization simplifies the vaccination protocol to produce immune
responses against particular proteins because the often difficult
steps of protein purification and combination with adjuvant, both
routinely required for vaccine development, are eliminated. Since
genetic immunization does not require the isolation of proteins, it
is especially valuable for proteins that may lose conformational
epitopes when purified biochemically. Genetic vaccines may also be
delivered in combination without eliciting interference or
affecting efficacy (Tang et al., 1992; Barry et al., 1995), which
may simplify the vaccination scheme against multiple antigens.
[0026] Although topical application of protein-based vaccines in
conjunction with cholera toxin may also immunize animals in a
non-invasive mode (Glenn et al., 1998), skin-targeted non-invasive
genetic vaccines activate the immune system via a different
mechanism than protein-based vaccines. These two vaccination
modalities may complement each other as they may induce different
immune profiles. Although U.S. Pat. No. 3,837,340 relates to a
method for vaccinating animals by contacting skin with dried
viruses, the viruses that are employed therein are not genetic
vectors capable of expressing transgenes or heterologous or
exogenous nucleic acid molecules. In addition, the immunogen may be
protein in the viral coat, instead of protein produced from
recombinant DNA or expression of exogenous genes in the animals'
own cells, and ergo U.S. Pat. No. 3,837,340 is non-analogous to the
present invention.
[0027] Vaccination using live bacteria has been studied, and often
utilizes a live bacteria strain in which a mutation has been
induced to knock out the lethal gene. However, this method requires
extreme safety precautions to ensure that a further mutation does
not occur that would allow the bacterium to return to virulence. A
more reliable method is to utilize a weakened bacterium to express
a protein to which the host can then produce antibodies against.
Often, a bacterial vector is studied for oral administration of a
vaccine; for example, Salmonella-based vaccines are being
researched for oral administration to protect against HIV, Lyme
disease, and Epstein-Barr virus.
[0028] In addition, baculovirus, yeast and tissue culture cells
have also been studied for use in vaccines, Examples are shown in
U.S. Pat. No. 6,287,759 where baculovirus is employed to produce a
protein used in a vaccine against Hepatitis E; U.S. Pat. No.
6,290,962 wherein yeast is used as a vector to produce a
Helicobacter polypeptide for use in a vaccine; and U.S. Pat. No.
6,254,873 wherein vertebrate tissue culture cells are used to
propagate purified inactivated dengue virus for use in a vaccine.
In all of these examples, the vectors were used to produce a
protein of interest, after which the protein would then be used in
the vaccine.
[0029] Additionally, it has now been demonstrated (as evidenced by
the following examples) that it can be advantageous to utilize
irradiated bacterial vectors that are non-replicative.
Non-replicative vectors are by nature safer than live vectors
because there is no danger of mutations causing the vector to
return to virulence.
[0030] Furthermore, it has now also been demonstrated (as evidenced
by the following examples) that it can be advantageous to utilize
cell-free extracts, wherein the extracts are prepared by filtration
of disrupted cells chosen from the group consisting of bacterium,
fungus, cultured animal cells, and cultured plant cells, and
wherein the cell comprises and expresses a nucleic acid molecule
encoding the gene product. These cell-free extracts can be applied
directly to the skin, and are by nature safer than the use of live
vectors.
[0031] Vaccines are often augmented through the use of adjuvants.
Vaccine adjuvants are useful for improving an immune response
obtained with any particular antigen in a vaccine composition.
Adjuvants are used to increase the amount of antibody and effector
T cells produced and to reduce the quantity of antigen and the
frequency of injection. Although some antigens are administered in
vaccines without an adjuvant, there are many antigens that lack
sufficient immunogenicity to stimulate a useful immune response in
the absence of an effective adjuvant. Adjuvants also improve the
immune response from "self-sufficient" antigens, in that the immune
response obtained can be increased or the amount of antigen
administered can be reduced.
[0032] Heat shock proteins are a class of molecular chaperones
which function by associating with cellular proteins and regulating
their conformation. Heat shock proteins are located in all major
cellular compartments and function as monomers, multimers, or are
complexed with other cellular proteins. Heat shock proteins bind to
steroid hormone receptors, repress transcription in the absence of
the ligand, and provide the proper folding of the ligand-binding
domain in the presence of the hormone. Specific heat shock proteins
bind immunosuppressive drugs and can play a role in modulation of
immune responses. In the present invention, it is demonstrated that
the use of heat shock protein 27 can be used as a vaccine adjuvant
to modulate immune responses.
[0033] The prior art of vaccination usually requires equipment,
e.g., syringe needles or a gene gun, and special skill for the
administration of vaccines. There is a great need and desire in the
art for the inoculation of vaccines by personnel without medical
training and equipment. A large number of diseases could
potentially be immunized against through the development of
non-invasive vaccination onto the skin (NIVS) because the procedure
is simple, effective, economical, painless, and potentially safe.
As a consequence, NIVS can boost vaccine coverages in developing
countries where medical resources are in short supply, as well as
in developed countries due to patient comfort. Infectious diseases
caused by viruses, including AIDS and flu, by bacteria, including
tetanus and TB, and by parasites, including malaria, and malignant
tumors including a wide variety of cancer types may all be
prevented or treated with skin-targeted non-invasive vaccines
without requiring special equipment and medical personnel. The
present invention addresses this longstanding need and desire in
the art.
[0034] Additionally, the present invention also addresses the
problems associated with new plastic surgery techniques involving
the bacteria Clostridium (C.) botulinum. In 2002, the Food and Drug
Administration (FDA) approved the use of botulinum toxin A (Botox)
for cosmetic treatment of glabellar lines. However, the current
procedure requires multiple injections associated with a number of
undesirable side effects.
[0035] The anaerobic, gram-positive bacterium Clostridium botulinum
produces a potent polypeptide neurotoxin, botulinum toxin, which
causes a neuroparalytic illness in humans and animals referred to
as botulism.
[0036] Seven immunologically distinct botulinum neurotoxins have
been characterized, these being respectively botulinum neurotoxin
serotypes A, B, C.sub.1, D, B, F and G each of which is
distinguished by neutralization with type-specific antibodies. The
different serotypes of botulinum toxin vary in the animal species
that they affect and in the severity and duration of the paralysis
they evoke.
[0037] The neuroparalytic syndromes of tetanus and botulism are
both caused by these neurotoxins produced by the bacteria. After
binding to the presynaptic membrane of motoneurons, tetanus
neurotoxin is internalized and transported retroaxonally to the
spinal cord, where it blocks neurotransmitter release from spinal
inhibitory interneurons. In contrast, the seven botulinum
neurotoxins act at the periphery and inhibit acetylcholine release
from peripheral cholinergic nerve terminals, inducing a flaccid
paralysis due to intoxication of the neuromuscular junction. The
clostridial neurotoxins responsible for tetanus and botulism are
both metallo-proteases that enter nerve cells and block
neurotransmitter release via zinc-dependent cleavage of protein
components of the neuroexocytosis apparatus.
[0038] Besides the use of botulinum toxin A for cosmetic
applications, botulinum toxins have been used in clinical settings
for the treatment of neuromuscular disorders characterized by
hyperactive skeletal muscles. Botulinum toxin type A has been
previously approved by the U.S. Food and Drug Administration for
the treatment of blepharospasm, strabismus and hemifacial spasm.
Botulinum toxin type A is also being studied as a treatment for
other neuro/muscular disorders including spasmodic dysphonia,
dystonias in general, hyperhidrosis, and cerebal palsy.
[0039] Non-type A botulinum toxin serotypes apparently have a lower
potency and/or a shorter duration of activity as compared to
botulinum toxin type A. Clinical effects of peripheral
intramuscular botulinum toxin type A are usually seen within one
week of injection. The typical duration of symptomatic relief from
a single intramuscular injection of botulinum toxin type A averages
about three months.
[0040] The demonstration that topical application of a patch
containing irradiated C. tetani cells could induce tetanus provides
evidence and rationale in support of a novel protocol for the
delivery of proteins capable of triggering beneficial
pharmacological effects by topical application of irradiated
bacterial cells containing the proteins using a patch. Topical
application of a Botox patch will improve the degree of patient
comfort and can eliminate some of the side effects associated with
the contemporary needle-dependent method.
OBJECTS AND SUMMARY OF THE INVENTION
[0041] Non-invasive vaccination onto the skin (NIVS) can improve
vaccination schemes because skin is an immunocompetent tissue and
this non-invasive procedure requires no specially trained
personnel. Skin-targeted non-invasive gene delivery can achieve
localized transgene expression in the skin and the elicitation of
immune responses (Tang et al., 1997) These results indicate that
vector-based NIVS is a novel and efficient method for the delivery
of vaccines. The simple, effective, economical and painless
immunization protocol of the present invention should make
vaccination less dependent upon medical resources and, therefore,
increase the annual utilization rate of vaccinations.
[0042] Accordingly, an object of the invention can be any one or
more of providing a method for inducing an immunological response,
e.g., protective immunological response, and/or a therapeutic
response in a host or animal, e.g., vertebrate such as mammal,
comprising topically administering a vector that comprises and
expresses a nucleic acid molecule encoding a gene product that
induces or stimulates the response; such a method wherein the
nucleic acid molecule is heterologous and/or exogenous with respect
to the host; mucosal, e.g., intranasal, perlingual, buccal, oral,
oral cavity administration of adenovirus defective in its E1 and/or
E3 and/or E4 region(s) and/or all adenoviral genes, advantageously
defective in its E1 and E3 and E4 regions, e.g., such an adenovirus
comprising an exogenous or heterologous nucleic acid molecule, such
as an exogenous or heterologous nucleic acid molecule encoding an
epitope of interest of an influenza, e.g., one or more influenza
epitopes of interest and/or one or more influenza antigens; such an
administration wherein an immunological response, such as a
protective immunological response is induced; products for
performing such methods; uses for such methods and products, inter
alia.
[0043] The present invention provides a method of non-invasive
immunization in an animal, comprising the step of: contacting skin
of the animal with a vector in an amount effective to induce an
immune response in the animal. The invention also provides a method
for immunizing animals comprising the step of skin-targeted
non-invasive delivery of a preparation comprising vectors, whereby
the vector is taken up by epidermal cells and has an immunogenic
effect on vertebrates. The invention further provides a method for
immunizing animals by a delivery device, comprising the steps of
including vectors in the delivery device and contacting the naked
skin of a vertebrate with a uniform dose of genetic material
confined within the device, whereby the vector is taken up by
epidermal cells for expressing and/or presenting a specific antigen
in the immunocompetent skin tissue. The vector may be adenovirus
recombinants, DNA/adenovirus complexes, DNA/liposome complexes,
bacterial vectors containing recombinant plasmids, or any other
vectors capable of expressing antigens in the skin of a
vertebrate.
[0044] In a preferred embodiment of the present invention, the
genetic vector is on-replicative. For example, the vector can be
irradiated.
[0045] In another preferred embodiment of the present invention,
the invention comprises a method of non-invasive immunization in an
animal and/or a method of inducing a systemic immune response or
systemic therapeutic response to a gene product, in an animal,
comprising contacting skin of the animal with cell-free extracts in
an amount effective to induce the response. The extracts are
prepared by filtration of disrupted cells wherein the cell
comprises and expresses a nucleic acid molecule encoding the gene
product.
[0046] In an embodiment of the present invention, there is provided
a method of inducing an immune response, comprising the step of:
contacting skin of an individual or animal in need of such
treatment by topically applying to said skin an immunologically
effective concentration of a recombinant vector encoding a gene of
interest.
[0047] In a further embodiment, the immune response can be enhanced
by admixing the vaccine or vector with heat shock protein 27.
[0048] In another embodiment of the present invention, there is
provided a method of inducing a protective immune response in an
individual or animal in need of such treatment, comprising the step
of contacting the skin of said animal by topically applying to said
skin an immunologically effective concentration of a vector
encoding a gene which encodes an antigen which induces a protective
immune effect in said individual or animal following
administration.
[0049] In another embodiment, the invention presents a method for
co-expressing transgenes in the same cell by contacting naked skin
with DNA/adenovirus complexes. This protocol allows the
manipulation of the immune system by co-producing cytokines,
costimulatory molecules, or other immune modulators with antigens
within the same cellular environment.
[0050] The invention thus provides methods of non-invasive
immunization in an animal and/or methods of inducing an immune,
e.g., systemic immune, or therapeutic response in an animal,
products therefrom and uses for the methods and products therefrom.
The invention further provides such methods comprising contacting
skin of the animal with a vector in an amount effective to induce
the response, e.g., immune response such as systemic immune
response or therapeutic response, in the animal. Even further, the
invention provides such methods wherein the vector comprises and
expresses an exogenous nucleic acid molecule encoding an epitope or
gene product of interest. Still further, the invention provides
such methods wherein the systemic immune response can be to or from
the epitope or gene product.
[0051] The invention yet further still provides such methods
wherein the nucleic acid molecule can encode an epitope of interest
and/or an antigen of interest and/or a nucleic acid molecule that
stimulates and/or modulates an immunological response and/or
stimulates and/or modulates expression, e.g., transcription and/or
translation, such as transcription and/or translation of an
endogenous and/or exogenous nucleic acid molecule; and/or elicits a
therapeutic response.
[0052] The invention additionally provides such methods wherein the
nucleic acid molecule can be exogenous to the vector. The invention
also provides such methods wherein the exogenous nucleic acid
molecule encodes one or more of an antigen of interest or portion
thereof, e.g., an epitope of interest, from a pathogen; for
instance, one or more of an epitope of interest from or the antigen
comprising influenza hemagglutinin, influenza nuclear protein,
influenza. M2, tetanus toxin C-fragment, anthrax protective
antigen, anthrax lethal factor, anthrax germination factors, rabies
glycoprotein, HBV surface antigen, HIV gp120, HIV gp160, human
carcinoembryonic antigen, malaria CSP, malaria SSP, malaria MSP,
malaria pfg, botulinum toxin A, and mycobacterium tuberculosis HSP;
and/or a therapeutic and/or an immunomodulatory gene, such as a
co-stimulatory gene and/or a cytokine gene. See also U.S. Pat. No.
5,990,091, WO 99/60164 and WO 98/00166 and documents cited
therein.
[0053] Even further, the invention provides such methods wherein
the immune response can be induced by the vector expressing the
nucleic acid molecule in the vector and/or in the animal's cells,
e.g., epidermal cells. The invention still further provides such
methods wherein the immune response can be against a pathogen or a
neoplasm.
[0054] Also, the invention provides compositions used in the
methods. For instance, the invention provides a prophylactic
vaccine or a therapeutic vaccine or an immunological or a
therapeutic composition comprising the vector, e.g., for use in
inducing or stimulating a response via topical application and/or
via mucosal and/or nasal and/or perlingual and/or buccal and/or
oral and/or oral cavity administration. The invention also provides
compositions comprising a non-replicative vector or a cell-free
extract. Additionally, the invention also provides compositions
comprising a vector, a non-replicative vector, or a cell-free
extract in combination with an adjuvant. It is provided that the
adjuvant can be heat shock protein 27.
[0055] The invention additionally provides to such methods and
compositions therefor wherein the animal can be a vertebrate, e.g.,
a fish, amphibian, reptile, bird, or mammal, such as human, or a
domesticated or companion or feed-producing or food-producing or
livestock or game or racing or sport animal such as a cow, a dog, a
cat, a goat, a sheep, a horse, or a pig; or, fowl such as turkeys,
ducks and chicken.
[0056] The invention further provides such methods and compositions
therefor wherein the vector can be one or more of a viral,
including viral coat, e.g., with some or all viral genes deleted
therefrom, bacterial, protozoan, transposon, retrotransposon, and
DNA vector, e.g., a recombinant vector; an adenovirus, such as an
adenovirus defective in its E1 and/or E3 and/or E4 region(s) and/or
all adenoviral genes. The invention further provides such methods
and compositions therefore wherein the vector can be chosen from
yeast vectors, insect cells transduced with baculovirus vectors, or
tissue culture cells, and wherein the vector is non-replicative.
For example, the vector can be irradiated.
[0057] The invention further provides such methods and compositions
therefor wherein the vector can be an Escherichia bacterial vector.
Further still, the invention provides such methods and compositions
therefor wherein the vector is preferably an Escherichia coli
bacterial vector.
[0058] The invention further provides methods of the invention
wherein the bacterial vector is altered such that the vaccination
process can be controlled. For example, a Salmonella vector could
be modified such that the bacterium is deficient in making
enterochelin, p-aminobenzoic acid and aromatic acids such that
bacteria are unable to thrive in mammalian tissues.
[0059] The invention further provides intranasal and/or mucosal
and/or perlingual and/or buccal and/or oral and/or oral cavity
administration of adenovirus defective in its E1 and/or E3 and/or
E4 region(s) and/or all adenoviral genes, advantageously defective
in its E1 and E3 and E4 regions, e.g., such an adenovirus
comprising an exogenous or heterologous nucleic acid molecule, such
as an exogenous or heterologous nucleic acid molecule encoding an
epitope of interest of an influenza, e.g., one or more influenza
epitopes of interest and/or one or more influenza antigens. Such an
administration can be a method to induce an immunological response,
such as a protective immunological response. The adenovirus in this
instance can be a human adenovirus. The adenovirus can be another
type of adenovirus, such as a canine adenovirus. Thus, if the host
or animal is other than a human, the adenovirus can be matched to
the host; for example, in veterinary applications wherein the host
or animal is a canine such as a dog, the adenovirus can be a canine
adenovirus.
[0060] The invention accordingly further relates to methods of the
invention wherein the vector can be matched to the host or can be a
vector that is interesting to employ with respect to the host or
animal because the vector can express both heterologous or
exogenous and homologous gene products of interest in the animal;
for instance, in veterinary applications, it can be useful to use a
vector pertinent to the animal, for example, in canines one can use
canine adenovirus; or more generally, the vector can be an
attenuated or inactivated natural pathogen of the host or animal
upon which the method is being performed. One skilled in the art,
with the information in this disclosure and the knowledge in the
art, can match a vector to a host or animal without undue
experimentation.
[0061] The invention still further provides such methods
encompassing applying a delivery device including the vector to the
skin of the animal, as well as such a method further including
disposing the vector in and/or on the delivery device; and, to such
delivery devices.
[0062] The invention yet further provides such methods wherein the
vector can have all viral genes deleted therefrom, as well as to
such vectors.
[0063] The invention still further provides such methods wherein
the vector can be non-replicative. For example, the vector can be
irradiated.
[0064] The invention even further still provides such methods
wherein the vector can induce a therapeutic effect, e.g., an
anti-tumor effect in the animal, for instance, by expressing an
oncogene, a tumor-suppressor gene, or a tumor-associated gene.
[0065] In addition, the invention provides gene products, e.g.,
expression products, as well as immunological products (e.g.,
antibodies), generated by the expression, cells from the methods,
as well as in in vitro and ex vivo uses thereof. The expression
products and immunological products therefrom can be used in
assays, diagnostics, and the like; and, cells that express the
immunological products and/or the expression products can be
isolated from the host, expanded in vitro and re-introduced into
the host.
[0066] Even further still, while non-invasive delivery is desirable
in all instances of administration, the invention can be used in
conjunction with invasive deliveries; and, the invention can
generally be used as part of a prime-boost regimen. For instance,
the methods of the present invention can be used as part of a
prime-boost regimen wherein vaccines are administered prior to or
after or concurrently with another administration such as a
non-invasive or an invasive administration of the same or a
different immunological or therapeutic ingredient, e.g., before,
during or after prime vaccination, there is administration by
injection or by non-invasive methods described in this invention of
a different vaccine or immunological composition for the same or
similar pathogen such as a whole or subunit vaccine or
immunological composition for the same or similar pathogen whose
antigen or epitope of nterest is expressed by the vector in the
non-invasive administration.
[0067] The present invention further comprises the use of the
topical application of recombinant vectors as previously described
for use in the administration of genes encoding antigens of
interest, expression products, or immunological products, all of
which can be used to induce a therapeutic or cosmetic effect. The
genetic vectors can be used to induce a cosmetic effect including
the reduction of facial wrinkles, including glabellar lines. The
present invention further comprises the use of recombinant and
natural vectors, including genetic vectors, to provide a
therapeutic effect, wherein the vector provides a therapy or
treatment for use in the management of neurological or muscular
conditions, including the treatment of migraine headaches, tremors
or spasms including blepharospasm, strabismus spasm, hemifacial
spasm, spasmodic dysphonia, dystonias in general, cerebral palsy or
excessive sweating (hyperhidrosis).
[0068] The present invention also encompasses delivery devices
(bandages, adhesive dressings, spot-on formulation and its
application devices, pour-on formulation and its application
devices, roll-on formulation and its application devices, shampoo
formulation and its application devices or the like) for the
delivery of skin-targeted and other non-invasive vaccines or
immunological compositions and uses thereof, as well as
compositions for the non-invasive delivery of vectors; and, kits
for the preparation of compositions for the non-invasive delivery
of vectors. Such a kit comprises the vector and a pharmaceutically
acceptable or suitable carrier or diluent and an optional delivery
device, each in its own packaging; the packaging may be included in
a unitary container or the packaging may each be in separate
containers or each may be in its own separate container; the kit
can optionally include instructions for admixture of the
ingredients and/or administration of the composition.
[0069] Pour-on and spot-on formulations are described in U.S. Pat.
Nos. 6,010,710 and 5,475,005. A roll-on device is also described in
U.S. Pat. No. 5,897,267. The contents of U.S. Pat. Nos. 6,010,710,
5,475,005 and 5,897,267 are hereby incorporated herein by
reference, together with documents cited or referenced therein and
all documents cited or referenced in such documents. Moreover, a
skilled artisan also knows how to make shampoo formulation as well
as devices to apply the formulation to an animal.
[0070] Thus, the present invention also includes all recombinant
vectors for all of the uses contemplated in the methods described
herein.
[0071] It is noted that in this disclosure, terms such as
"comprises", "comprised", "comprising" and the like can have the
meaning attributed to it in U.S. patent law; e.g., they can mean
"includes", "included", "including" and the like.
[0072] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF FIGURES
[0073] The following Detailed Description, given by way of example,
but not intended to limit the invention to specific embodiments
described, may be understood in conjunction with the accompanying
Figures, incorporated herein by reference, in which:
[0074] FIG. 1 shows the transgene expression from adenovirus
recombinants in the skin by topical application of the vectors;
[0075] FIGS. 2a and 2b show the characterization of potential
target cells that can be transduced by topically-applied adenovirus
recombinants;
[0076] FIGS. 3a and 3b show the detection of specific antibodies in
the sera of mice immunized by adenovirus-mediated NIVS;
[0077] FIG. 4 shows the percent survival of control versus
immunized mice that were challenged by a lethal dose of tumor
cells;
[0078] FIG. 5 shows the characterization of tumor-infiltrating T
lymphocytes;
[0079] FIG. 6 shows the characterization of tumor-infiltrating
CTLs;
[0080] FIG. 7 shows the western blot analysis of antibodies to the
human CEA protein in mice immunized by topical application of
vaccine bandages;
[0081] FIG. 8a shows the detection of specific antibodies in the
serum of a mouse immunized by DNA/adenovirus-mediated NIVS;
[0082] FIG. 8b shows the detection of specific antibodies in the
serum of a mouse immunized by DNA/liposorne-mediated NIVS;
[0083] FIG. 9 shows the co-expression of DNA-encoded and
adenovirus-encoded transgenes in target cells;
[0084] FIG. 10 shows relative transgene expression from
topically-applied adenovirus recombinants, DNA/adenovirus
complexes, and DNA/liposome complexes;
[0085] FIG. 11 shows a device for the administration of
skin-targeted non-invasive vaccines.
[0086] FIG. 12 shows anti-influenza antibodies generated by
skin-targeted noninvasive vaccines in mice;
[0087] FIG. 13 shows protection of mice from death following virus
challenge.
[0088] FIG. 14 shows ELISA antibodies generated in a pigtail
macaque by a skin patch containing an adenovirus vector encoding
influenza HA;
[0089] FIG. 15 shows relocation of antigen spots in skin after
topical application of an adenovirus vector;
[0090] FIG. 16 shows amplification of foreign. DNA in various
tissues after localized gene delivery in a noninvasive mode;
[0091] FIG. 17 shows that a depilatory agent such as NAIR is not
essential for NIVS;
[0092] FIG. 18 shows protection from death following Clostridium
tetani challenge by topical application or intranasal inoculation
of an adenovirus-based tetanus vaccine.
[0093] FIG. 19 shows anti-tetC antibodies in mice following oral
inoculation, intranasal instillation, and topical application of a
Salmonella-based vector expressing the tetanus toxin C-fragment
(tetC).
[0094] FIG. 20 shows anti-tetC antibodies in mice following topical
administration of Escherichia-based vectors containing a
recombinant plasmid expressing the tetanus toxin C-fragment, driven
by the nirB promoter and another plasmid expressing the tetanus
toxin C-fragment, driven by the cytomegalovirus early promoter.
[0095] FIG. 21 shows antibodies raised against at least two C.
tetani proteins in mice three weeks after topical application of
irradiated C. tetani cells
[0096] FIG. 22 shows the survival rate for animals after topical
application of irradiated C. tetani cells.
[0097] FIG. 23 shows the anti-tetC antibodies in mice following
topical application of live and irradiated E. coli-vectored
vaccines at 3 weeks and 3 months after application.
[0098] FIG. 24 shows the survival rate for animals challenged by a
lethal dose of C. tetani cells three months after topical
application of live or irradiated E. coli vectored vaccines.
[0099] FIG. 25 shows anti-tetC antibodies is mice three weeks after
topical application of cell free extracts prepared by filtration of
sonicated E. coli vectors expressing tetC. A, live E. coli cells;
B, E. coli cells sonicated for 5 min; C, cell-free extract from E.
coli cells sonicated for 5 min; D, E. coli cells sonicated for 60
min; E, cell-free extract from E. coli cells sonicated for 60
min.
[0100] FIG. 26 shows anti-tetC antibodies in mice six months after
topical application of AdCMV-tetC (an adenovirus vector encoding
tetC) or E. coli DH10B cells harboring the plasmid pTET-nir
encoding tetC, with or without HSP27. Open bar, vectors alone
without HSP27; stippled bar, vectors admixed with 1 .mu.g of HSP27
prior to topical application; solid bar, vectors admixed with 3
.mu.g of HSP27 prior to topical application.
DETAILED DESCRIPTION
[0101] Inoculation of vaccines in an invasive mode is unnecessary
(Tang et al., 1997; Glenn et al., 1998). Because the skin
interfaces directly with the external environment and is in
constant contact with potential pathogens, the immune system must
constantly keep a mobilized biological army along the skin border
for warding off potential infections. As a consequence, the outer
layer of skin is an immunocompetent tissue. Immunologic components
present in the skin for the elicitation of both humoral and
cytotoxic cellular immune responses include epidermal Langerhans
cells (which are MHC class II positive antigen-presenting cells),
keratinocytes, and both CD4.sup.+ and CD8.sup.+ T lymphocytes.
These components make the skin an ideal site for administration of
vaccine. The large accessible area of skin and its durability are
other advantages for applying vaccines to this tissue. Expression
of a small number of antigens in the outer layer of skin without
physical penetration can thus elicit a potent immune response by
alarming the immune surveillance mechanism.
[0102] It is herein demonstrated that vectored vaccines can be
inoculated in a novel way as skin-targeted non-invasive vaccines,
or immunological or therapeutic compositions. The combination of
vectored vaccines with a non-invasive delivery mode results in a
new class of "democratic" vaccine, or immunological or therapeutic
compositions that require little or no special skill and equipment
for administration. Thus, one can administer such compositions to
the skin of himself or herself (and, this administration can
advantageously be under the direction of a medical practitioner,
e.g., to ensure that dosage is proper) or to the skin of an animal
(e.g., advantageously a shaved area of skin if the animal is a
mammal, although as demonstrated herein, hair removal is not
necessary, and more advantageously at a region where the animal
will not remove the administration by rubbing, grooming or other
activity); and, the present invention thus provides advantages in
the administration of vaccine, or immunological, or therapeutic
compositions comprising a vector that expresses a gene product,
especially with respect to administering such compositions to
newborns, young animals, animals generally, children and the like,
to whom invasive, e.g., needle, administration can be difficult or
inconvenient or painful or harmful.
[0103] The present invention is directed to a method of
non-invasive immunization or treatment in an animal, comprising the
step of: contacting skin of the animal with a recombinant vector in
an amount effective to induce immune response in the animal.
[0104] As used herein, a vector is a tool that allows or
facilitates the transfer of an entity from one environment to
another. By way of example, some vectors used in recombinant DNA
techniques allow entities, such as a segment of DNA (such as a
heterologous DNA segment, such as a heterologous cDNA segment)
and/or heterologous protein, to be transferred into a target cell.
In an advantageous embodiment, the vector includes a viral vector,
a bacterial vector, a protozoan vector, a DNA vector, or a
recombinant thereof.
[0105] As used herein, "AdCMV-tetC" represents an adenovirus vector
encoding the Clostridium tetani toxin C-fragment; "pCMV-tetC"
represents a plasmid expression vector encoding the Clostridium
tetani toxin C-fragment.
[0106] Reference is made to U.S. Pat. No. 5,990,091 issued Nov. 23,
1999, Einat et al. or Quark Biotech, Inc., WO 99/60164, published
Nov. 25, 1999 from PCT/US99/11066, filed May 14, 1999, Fischer or
Rhone Merieux, Inc., WO98/00166, published Jan. 8, 1998 from
PCT/US97/11486, filed Jun. 30, 1997 (claiming priority from U.S.
application Ser. Nos. 08/675,556 and 08/675,566), van Ginkel et al,
J. Immunol 159(2):685-93 (1997) ("Adenoviral gene delivery elicits
distinct pulmonary-associated T helper cell responses to the vector
and to its transgene"), Osterhaus et al., Immunobiology
184(2-3):180-92 (1992) ("Vaccination against acute respiratory
virus infections and measles in man"), Briles et al. or UAB, WO
99/53940, published Oct. 28, 1999 from PCT/US99/08895, filed Apr.
23, 1999, and Briles et al. or UAB, U.S. Pat. No. 6,042,838, issued
Mar. 28, 2000, and Briles et al. or UAB U.S. Pat. No. 6,004,802,
for information concerning expressed gene products, antibodies and
uses thereof, vectors for in vivo and in vitro expression of
exogenous nucleic acid molecules, promoters for driving expression
or for operatively linking to nucleic acid molecules to be
expressed, method and documents for producing such vectors,
compositions comprising such vectors or nucleic acid molecules or
antibodies, dosages, and modes and/or routes of administration
(including compositions for mucosal, nasal, oral, oral cavity,
buccal, perlingual administration), inter alia, which can be
employed in the practice of this invention; and thus, U.S. Pat. No.
5,990,091 issued Nov. 23, 1999, Einat et al. or Quark Biotech,
Inc., WO 99/60164, published Nov. 25, 1999 from PCT/US99/11066,
filed May 14, 1999, Fischer or Rhone Merieux, Inc., WO98/00166,
published Jan. 8, 1998 from PCT/US97/11486, filed Jun. 30, 1997
(claiming priority from U.S. application Ser. Nos. 08/675,556 and
08/675,566), van Ginkel et al., J. Immunol 159(2):685-93 (1997)
("Adenoviral gene delivery elicits distinct pulmonary-associated T
helper cell responses to the vector and to its transgene"),
Osterhaus et al., Immunobiology 184(2-3):180-92 (1992)
("Vaccination against acute respiratory virus infections and
measles in an"), Briles et al. or UAB, WO 99/53940, published Oct.
28, 1999 from PCT/US99/08895, filed Apr. 23, 1999, and Briles et
al. or UAB, U.S. Pat. No. 6,042,838, issued Mar. 28, 2000 and
Briles et al. or UAB, U.S. Pat. No. 6,004,802, and all documents
cited or referenced therein and all documents cited or referenced
in documents referenced or cited in each of U.S. Pat. No. 5,990,091
issued Nov. 23, 1999, Einat et al. or Quark Biotech, Inc., WO
99/60164, published Nov. 25, 1999 from PCT/US99/11066, filed May
14, 1999, Fischer or Rhone Merieux, Inc., WO98/00166, published
Jan. 8, 1998 from PCT/US97/11486, filed Jun. 30, 1997 (claiming
priority from U.S. application Ser. Nos. 08/675,556 and
08/675,566), van Ginkel et al., J. Immunol 159(2):685-93 (1997)
("Adenoviral gene delivery elicits distinct pulmonary-associated T
helper cell responses to the vector and to its transgene"),
Osterhaus et al., Immunobiology 184(2-3):180-92 (1992)
("Vaccination against acute respiratory virus infections and
measles in man"), Briles et al. or UAB, WO 99/53940, published Oct.
28, 1999 from PCT/US99/08895, filed Apr. 23, 1999, and Briles et
al. or UAB, U.S. Pat. No. 6,042,838, issued Mar. 28, 2000, and
Briles et al. or UAB U.S. Pat. No. 6,004,802, are hereby
incorporated herein by reference.
[0107] Reference is also made to U.S. Pat. Nos. 5,643,771,
5,695,983, 5,792,452, 5,843,426, 5,851,519, 6,136,325, and
6,251,406, the contents of which are hereby incorporated herein by
reference. These U.S. patents can be relied upon to provide
background information on the use of bacteria as a vector for
inducing a systemic immune response or systemic therapeutic
response.
[0108] Specifically, the bacterial vectors, according to the
present invention, can be absorbed by mammalian hosts. Examples of
these include members of the genera Salmonella, Bordetella, Vibrio,
Haemophilus, Escherichia. Information in U.S. Pat. No. 5,990,091
issued. Nov. 23, 1999, WO 99/60164, WO98/00166, van Ginkel et al.,
J. Immunol 159(2):685-93 (1997), Osterhaus et al., Immunobiology
184(2-3):180-92 (1992), WO 99/53940 and U.S. Pat. Nos. 6,042,838
and 6,004,802, can be relied upon for the practice of this
invention (e.g., expressed products, antibodies and uses thereof,
vectors for in vivo and in vitro expression of exogenous nucleic
acid molecules, exogenous nucleic acid molecules encoding epitopes
of interest or antigens or therapeutics and the like, promoters,
compositions comprising such vectors or nucleic acid molecules or
expressed products or antibodies, dosages, inter alia). It is noted
that immunological products and/or antibodies and/or expressed
products obtained in accordance with this invention can be
expressed in vitro and used in a manner in which such immunological
and/or expressed products and/or antibodies are typically used, and
that cells that express such immunological and/or expressed
products and/or antibodies can be employed in in vitro and ex vivo
applications, e.g., such uses and applications can include
diagnostics, assays, ex vivo therapy (e.g., wherein cells that
express the gene product and/or immunological response are expanded
in vitro and reintroduced into the host or animal), etc., see U.S.
Pat. No. 5,990,091, WO 99/60164, WO 98/00166, WO 99/53940, and U.S.
Pat. Nos. 6,042,838, and 6,004,802, and documents cited therein and
documents cited or referenced in such documents. Further, expressed
antibodies or gene products that are isolated from herein methods,
or that are isolated from cells expanded in vitro following herein
administration methods, can be administered in compositions, akin
to the administration of subunit epitopes or antigens or
therapeutics or antibodies to induce immunity, stimulate a
therapeutic response and/or stimulate passive immunity. The
quantity to be administered will vary for the patient (host) and
condition being treated and will vary from one or a few to a few
hundred or thousand micrograms, e.g., 1 .mu.g to 1 mg, from about
100 ng/kg of body weight to 100 mg/kg of body weight per day and
preferably will be from 10 pg/kg to 10 mg/kg per day. A vector can
be non-invasively administered to a patient or host in an amount to
achieve the amounts stated for gene product (e.g., epitope,
antigen, therapeutic, and/or antibody) compositions. Of course, the
invention envisages dosages below and above those exemplified
herein, and for any composition to be administered to an animal or
human, including the components thereof, and for any particular
method of administration, it is preferred to determine therefor;
toxicity, such as by determining the 50% lethal dose (LD.sub.50) in
a suitable animal model e.g., rodent such as mouse; and, the dosage
of the composition(s), concentration of components therein and
timing of administering the composition(s), which elicit a suitable
response, such as by titrations of sera and analysis thereof, e.g.,
by ELISA and/or seroneutralization analysis. Such determinations do
not require undue experimentation from the knowledge of the skilled
artisan, this disclosure and the documents cited herein. And, the
invention also comprehends sequential administration of inventive
compositions or sequential performance of herein methods, e.g.,
periodic administration of inventive compositions such as in the
course of therapy or treatment for a condition and/or booster
administration of immunological compositions and/or in prime-boost
regimens; and, the time and manner for sequential administrations
can be ascertained without undue experimentation. Further, the
invention comprehends compositions and methods for making and using
vectors, including non-replicative vectors, including methods for
producing gene products and/or immunological products and/or
antibodies in vivo and/or in vitro and/or ex vivo (e.g., the latter
two being, for instance, after isolation of cells from a host that
has had a non-invasive administration according to the invention,
e.g., after optional expansion of such cells), and uses for such
genes and/or immunological products and/or antibodies, including in
diagnostics, assays, therapies, treatments, and the like. Vector
compositions are formulated by admixing the vector with a suitable
carrier or diluent; and, gene product and/or immunological product
and/or antibody compositions are likewise formulated by admixing
the gene and/or immunological product and/or antibody with a
suitable carrier or diluent; see, e.g., U.S. Pat. No. 5,990,091, WO
99/60164, WO 98/00166, WO 99/53940, and U.S. Pat. Nos. 6,042,838
and 6,004,802, documents cited therein, and other documents cited
herein, and other teachings herein (for instance, with respect to
carriers, diluents and the like).
[0109] Methods and compositions of the invention also comprise the
administration of a cell-free extract to provide non-invasive
immunization in an animal and/or a method of inducing a systemic
immune response or systemic therapeutic response to a gene product.
The response can comprise an immune response against a pathogen or
a neoplasm. The cell-free extract is prepared by filtration of
disrupted cells or vectors. The cells or vectors can comprise and
express an exogenous or heterologous nucleic acid molecule encoding
the gene product. The gene product can be botulinum neurotoxins,
insulin, erythropoietin, tetanus toxin C-fragment or growth
hormone. The cells or vectors can comprise and express an antigen
or a therapeutic product. The nucleic acid molecule may encodes an
epitope of interest and/or an antigen or interest and/or a nucleic
acid molecule that stimulates and/or modulates an immunological
response and/or stimulates and/or modulates expression comprising
transcription and/or translation of an endogenous and/or exogenous
nucleic acid molecule. The exogenous nucleic acid molecule may
encode one or more of an antigen or portion thereof, or one or more
of an epitope of interest, from a pathogen. The exogenous nucleic
acid molecule may encode one or more of: influenza hemagglutinin,
influenza nuclear protein, influenza M2, tetanus toxin C-fragment,
anthrax protective antigen, anthrax lethal factor, anthrax
germination factors, rabies glycoprotein, HBV surface antigen, HIV
gp120, HIV gp160, human carcinoembryonic antigen, malaria CSP,
malaria SSP, malaria MSP, malaria pfg, botulinum toxin A, and
mycobacterium tuberculosis HSP. The exogenous nucleic acid molecule
can encode an immunomodulator.
[0110] The vector or cell may comprise any of the vectors described
hereinabove, or may be selected from the group consisting of
bacterium, fungus, cultured animal cells and cultured plant cells.
The vector or cell may be a bacterium, wherein bacteria are
selected from Clostridium, Escherichia, Salmonella, and Bacillus.
In a preferred embodiment, the bacterium is an Escherichia. In a
most preferred embodiment, the bacterium is Escherichia coli.
[0111] The vector or cells are temporarily disrupted by chemical or
mechanical means, such that the vector or cell remains intact and
viable and does not lyse. The disruption can be facilitated by
methods known in the art, including, but not limited to,
sonication. Ultrasonic cell disruption occurs when sound waves
having a frequency in the order of about 20,000 cps (20 kHz) are
converted to very rapid vibration in a liquid, thereby producing a
phenomenon called "cavitation.". Cavitation occurs when the rapid
vibration produces low pressure areas within the liquid. Gas
bubbles can form in areas where the pressure drops below the vapor
pressure of the liquid. However, these bubbles collapse when local
pressure rises again, sending a shock wave and creating shear
forces through the liquid which will disrupt cells. Sonication can
be performed at repeated short time intervals, i.e., 10 to 15
seconds with an appropriate resting period between each cycle.
Preferably, the total time of sonication is below 60 minutes.
Preferably, the sonication time intervals does not approach those
levels were complete disruption of the cell would be expected by
one of skill in the art, i.e. 2 minutes at a 50% power output.
[0112] The extract collected after the sonication of the vectors or
cells can then be filtered by means known to those of skill in the
art. The cell-free extract then contains the gene product. The gene
product can be applied directly to the skin of an animal as herein
described, or can be applied through the application of a delivery
device including the extract to the skin of the animal. The animal
may be a vertebrate, including birds and mammals. The bird or
mammal may be a human or a companion or domesticated or, food- or
feed-producing or livestock or game or racing or sport animal.
[0113] If nasal or respiratory (mucosal) administration is desired,
compositions may be in a form and dispensed by a squeeze spray
dispenser, pump dispenser or aerosol dispenser. Such dispensers may
also be employed to deliver the composition to oral or oral cavity
(e.g., buccal or perlingual) mucosa. Aerosols are usually under
pressure by means of a hydrocarbon. Pump dispensers can preferably
dispense a metered dose or, a dose having a particular particle
size.
[0114] Compositions of the invention can contain pharmaceutically
acceptable flavors and/or colors for rendering them more appealing,
especially if they are administered orally (or buccally or
perlingually); and, such compositions can be in the form of tablets
or capsules that dissolve in the mouth or which are bitten to
release a liquid for absorption buccally or perlingually (akin to
oral, perlingual or buccal medicaments for angina such as
nitroglycerin or nifedimen), The viscous compositions may be in the
form of gels, lotions, ointments, creams and the like (e.g., for
topical and/or mucosal and/or nasal and/or oral and/or oral cavity
and/or perlingual and/or buccal administration), and will typically
contain a sufficient amount of a thickening agent so that the
viscosity is from about 2500 to 6500 cps, although more viscous
compositions, even up to 10,000 cps may be employed. Viscous
compositions have a viscosity preferably of 2500 to 5000 cps, since
above that range they become more difficult to administer. However,
above that range, the compositions can approach solid or gelatin
forms which are then easily administered as a swallowed pill for
oral ingestion and/or a pill or capsule or tablet for holding in
the mouth, e.g., for buccal or perlingual administration.
[0115] Liquid preparations are normally easier to prepare than
gels, other viscous compositions, and solid compositions.
Additionally, liquid compositions are somewhat more convenient to
administer, especially by injection or orally or buccally or
perlinually, to animals, children, particularly small children, and
others who may have difficulty swallowing a pill, tablet, capsule
or the like, or in multi-dose situations. Viscous compositions, on
the other hand, can be formulated within the appropriate viscosity
range to provide longer contact periods with mucosa, such as the
lining of the stomach or nasal mucosa or for perlingual or buccal
or oral cavity absorption.
[0116] Obviously, the choice of suitable carriers and other
additives will depend on the exact route of administration and the
nature of the particular dosage form, e.g., liquid dosage form
(e.g., whether the composition is to be formulated into a solution,
a suspension, gel or another liquid form), or solid dosage form
(e.g., whether the composition is to be formulated into a pill,
tablet, capsule, caplet, time release form or liquid-filled
form).
[0117] Solutions, suspensions and gels, normally contain a major
amount of water (preferably purified water) in addition to the
antigen, lipoprotein and optional adjuvant. Minor amounts of other
ingredients such as pH adjusters (e.g., a base such as NaOH),
emulsifiers or dispersing agents, buffering agents, preservatives,
wetting agents, jelling agents, (e.g., methylcellulose), colors
and/or flavors may also be present. The compositions can be
isotonic, i.e., it can have the same osmotic pressure as blood and
lacrimal fluid.
[0118] The desired isotonicity of the compositions of this
invention may be accomplished using sodium chloride, or other
pharmaceutically acceptable agents such as dextrose, boric acid,
sodium tartrate, propylene glycol or other inorganic or organic
solutes. Sodium chloride is preferred particularly for buffers
containing sodium ions.
[0119] Viscosity of the compositions may be maintained at the
selected level using a pharmaceutically acceptable thickening
agent. Methylcellulose is preferred because it is readily and
economically available and is easy to work with. Other suitable
thickening agents include, for example, xanthan gum, carboxymethyl
cellulose, hydroxypropyl cellulose, carbomer, and the like. The
preferred concentration of the thickener will depend upon the agent
selected. The important point is to use an amount which will
achieve the selected viscosity. Viscous compositions are normally
prepared from solutions by the addition of such thickening
agents.
[0120] A pharmaceutically acceptable preservative can be employed
to increase the shelf-life of the compositions. Benzyl alcohol may
be suitable, although a variety of preservatives including, for
example, parabens, thimerosal, chlorobutanol, or benzalkonium
chloride may also be employed. A suitable concentration of the
preservative will be from 0.02% to 2% based on the total weight
although there may be appreciable variation depending upon the
agent selected.
[0121] Those skilled in the art will recognize that the components
of the compositions must be selected to be chemically inert with
respect to the vector or antigen or epitope of interest and
optional adjuvant or other active or immunity-enhancing
ingredients. This will present no problem to those skilled in
chemical and pharmaceutical principles, or problems can be readily
avoided by reference to standard texts or by simple experiments
(not involving undue experimentation), from this disclosure and the
documents cited herein.
[0122] The methods and compositions herein can include the admixing
the vector, vaccine, or cell-free extract with an immunomodulator
to increase the immune or immune system response. One such
immunomodulator is heat shock protein 27, which can be admixed as
described in the following examples. One of skill in the art will
recognize that the components of the compositions, including
immunomodulators such as heat shock protein 27 may require
adjustments based on the vector, antigen, epitope of interest or
cell-free extract being used in such a composition. This will
present no problem to those skilled in chemical and pharmaceutical
principles, and one may be guided in this by referencing standard
texts or by simple experiments as described above.
[0123] The immunologically effective compositions of this invention
are prepared by mixing the ingredients following generally accepted
procedures. For example the selected components can be simply mixed
in a blender, or other standard device to produce a concentrated
mixture which can then be adjusted to the final concentration and
viscosity by the addition of water or thickening agent and possibly
a buffer to control pH or an additional solute to control tonicity.
Generally the pH may be from about 3 to 7.5. Compositions can be
administered in dosages and by techniques well known to those
skilled in the medical and veterinary arts taking into
consideration such factors as the age, sex, weight, and condition
of the particular patient or animal, and the composition form used
for administration (e.g., solid vs. liquid). Dosages for humans or
other mammals can be determined without undue experimentation by
the skilled artisan, from this disclosure, the documents cited
herein, the Examples below and from the applications, patents and
other documents cited herein and documents cited or referenced in
documents cited herein, all of which are incorporated herein by
reference.
[0124] Suitable regimes for initial administration and booster
doses or for sequential administrations also are variable, and may
include an initial administration followed by subsequent
administrations; but nonetheless, may be ascertained by the skilled
artisan, from this disclosure, the documents cited and incorporated
by reference herein, including applications and patents cited
herein and documents referenced or cited herein, all of which are
hereby incorporated herein by reference, as well as the Examples
below. The compositions can be administered alone, or can be
co-administered or sequentially administered with other
compositions of the invention or with other prophylactic or
therapeutic compositions.
[0125] In another advantageous embodiment, the vector or cell
expresses a gene which encodes influenza hemagglutinin, influenza
nuclear protein, influenza M2, tetanus toxin C-fragment, anthrax
protective antigen, anthrax lethal factor, anthrax germination
factors, rabies glycoprotein, HBV surface antigen, HIV gp120, HIV
gp160, human carcinoembryonic antigen, malaria CSP, malaria SSP,
malaria MSP, malaria pfg, botulinum toxin A, mycobacterium
tuberculosis HSP or a mutant thereof.
[0126] In an embodiment of the invention, the immune response in
the animal is induced by recombinant vectors expressing genes
encoding antigens of interest in the vector or in the animal's
cells. In another embodiment of the invention, the antigen of
interest is selected from the group comprising influenza
hemagglutinin, influenza nuclear protein, influenza M2, tetanus
toxin. C-fragment, anthrax protective antigen, anthrax lethal
factor, anthrax germination factors, rabies glycoprotein, HBV
surface antigen, HIV gp120, HIV gp160, human carcinoembryonic
antigen, malaria CSP, malaria SSP, malaria MSP, malaria pfg,
botulinum toxin A, and mycobacterium tuberculosis HSP. In another
embodiment of the method, the animal's cells are epidermal cells.
Epidermal cells may include, but are not limited to, keratinocytes,
Langerhans cells, merkel cells, hair matrix cells and melanocytes.
In another embodiment of the method, the immune response is against
a pathogen or a neoplasm. In another embodiment of the method, the
recombinant vector is used as a prophylactic vaccine or a
therapeutic vaccine. In another embodiment of the invention, the
recombinant vector comprises vectors capable of expressing an
antigen of interest in the vector. In another embodiment of the
invention, the recombinant vector vectors capable of expressing an
antigen of interest in the animal's cells. In a further embodiment
of the method, the animal is a vertebrate.
[0127] With respect to exogenous DNA for expression in a vector
(e.g., encoding an epitope of interest and/or an antigen and/or a
therapeutic) and documents providing such exogenous DNA, as well as
with respect to the expression of transcription and/or translation
factors for enhancing expression of nucleic acid molecules, and as
to terms such as "epitope of interest", "therapeutic", "immune
response", "immunological response", "protective immune response",
"immunological composition", "immunogenic composition", and
"vaccine composition", inter glia, reference is made to U.S. Pat.
No. 5,990,091 issued Nov. 23, 1999, and WO 98/00166 and WO
99/60164, and the documents cited therein and the documents of
record in the prosecution of that patent and those PCT
applications; all of which are incorporated herein by reference.
Thus, U.S. Pat. No. 5,990,091 and WO 98/00166 and WO 99/60164 and
documents cited therein and documents or record in the prosecution
of that patent and those PCT applications, and other documents
cited herein or otherwise incorporated herein by reference, can be
consulted in the practice of this invention; and, all exogenous
nucleic acid molecules, promoters, and vectors cited therein can be
used in the practice of this invention. In this regard, mention is
also made of U.S. Pat. Nos. 6,004,777, 5,997,878, 5,989,561,
5,976,552, 5,972,597, 5,858,368, 5,863,542, 5,833,975, 5,863,542,
5,843,456, 5,766,598, 5,766,597, 5,762,939, 5,756,102, 5,756,101,
5,494,807, 6,042,838, 6,004,802 and WO 99/53940.
[0128] In another embodiment of the invention, the animal is
advantageously a vertebrate such as a mammal, bird, reptile,
amphibian or fish; more advantageously a human, or a companion or
domesticated or food-producing or feed-producing or livestock or
game or racing or sport animal such as a cow, a dog, a cat, a goat,
a sheep or a pig or a horse, or even fowl such as turkey, ducks or
chicken. In an especially advantageous another embodiment of the
invention, the vertebrate is a human. In another embodiment of the
invention, the recombinant vector is a viral vector, a bacterial
vector, a protozoan vector, a retrotransposon, a transposon, a
virus shell, or a DNA vector. In another embodiment of the
invention, the immune response is against influenza A. In another
embodiment of the invention, the immune response against influenza
A is induced by the recombinant vector expressing a gene encoding
an influenza hemagglutinin, an influenza nuclear protein, an
influenza M2 or a fragment thereof in the animal's cells. In
another embodiment of the invention, the recombinant vector is
selected from the group consisting of viral vector and plasmid DNA.
In another embodiment of the invention, the recombinant vector is
an adenovirus. In another embodiment of the invention, the
adenovirus vector is defective in its E1 region. In another
embodiment of the invention, the adenovirus vector is defective in
its E3 region. In another embodiment of the invention, the
adenovirus vector is defective in its E1 and E3 regions. In another
embodiment of the invention, the adenovirus vector is defective in
all adenoviral genes. In another embodiment of the invention, the
DNA is in plasmid form. In another embodiment of the invention, the
contacting step further comprises disposing the recombinant vector
containing the gene of interest on a delivery device and applying
the device having the recombinant vector containing the gene of
interest therein to the skin of the animal. In another embodiment
of the invention, the recombinant vector encodes an
immunomodulatory gene, a co-stimulatory gene or a cytokine gene. In
another embodiment of the invention, the recombinant viral vector
has all viral genes deleted. In another embodiment of the
invention, the recombinant vector induces an anti-tumor effect in
the animal. In a further embodiment of the invention, the
recombinant vector expresses an oncogene, a tumor-suppressor gene,
or a tumor-associated gene.
[0129] The present invention also provides a method of non-invasive
immunization in an animal, comprising the step of: contacting skin
of the animal with a recombinant vector in an amount effective to
induce immune response in the animal.
[0130] Representative examples of antigens which can be used to
produce an immune response using the methods of the present
invention include influenza hemagglutinin, influenza nuclear
protein, influenza M2, tetanus toxin C-fragment, anthrax protective
antigen, anthrax lethal factor, anthrax germination factors, rabies
glycoprotein, HBV surface antigen, HIV gp1.20, HIV gp160, human
carcinoembryonic antigen, malaria CSP, malaria SSP, malaria MSP,
malaria pfg, botulinum toxin A, and mycobacterium tuberculosis HSP,
etc. Most preferably, the immune response produces a protective
effect against neoplasms or infectious pathogens.
[0131] The present invention also includes a method of inducing a
systemic therapeutic response to a gene product, in an animal,
comprising contacting skin of the animal with a non-replicative
vector chosen from the group of bacterium, virus, and fungus,
wherein the vector comprises and expresses a nucleic acid molecule
encoding the gene product, in an amount effective to induce the
response.
[0132] In one embodiment of the present invention, a system
therapeutic response is induced to a gene product, wherein the
nucleic acid molecule encodes botulinum toxin A. The induced
systemic therapeutic response can be a therapeutic or cosmetic
effect. Such a cosmetic effect includes the reduction of facial
wrinkles, including glabellar lines. A further embodiment includes
the induction of a therapeutic effect, wherein the therapeutic
effect is used in the therapy or treatment or the management of
neurological or muscular conditions, including the treatment of
migraine headaches, tremors or spasms including blepharospasm,
strabismus spasm, hemifacial spasm, spasmodic dysphonia, dystonias
in general, cerebral palsy or excessive sweating
(hyperhidrosis).
[0133] The practice of the present invention includes delivering
recombinant vectors operatively coding for a polypeptide into the
outer layer of skin of a vertebrate by a non-invasive procedure for
immunizing the animal or for administering a therapeutic. These
recombinant vectors can be administered to the vertebrate by direct
transfer of the vector material to the skin without utilizing any
devices, or by contacting naked skin utilizing a bandage or a
bandage-like device. In preferred applications, the recombinant
vector is in aqueous solution. Vectors reconstituted from
lyophilized powder are also acceptable. The vector may encode a
complete gene, a fragment of a gene or several genes, gene
fragments fused with immune modulatory sequences such as ubiquitin
or CpG-rich synthetic DNA, together with transcription/translation
signals necessary for expression.
[0134] In another embodiment of the present invention, the vector
further contains a gene selected from the group consisting of
co-stimulatory genes and cytokine genes. In this method the gene is
selected from the group consisting of a GM-CSF gene, a B7-1 gene, a
B7-2 gene, an interleukin-2 gene, an interleukin-12 gene and
interferon genes.
[0135] In a further embodiment of the present invention, the
response is against Clostridium tetani infection and the exogenous
nucleic acid molecule encodes tetanus toxin C-fragment as described
(Shi et al, 2001).
[0136] The present invention also provides for a method of
non-invasively inducing an immune response to influenza virus
comprising the step of: contacting skin of a subject in need of
such treatment topically by applying to the skin an immunologically
effective amount of a recombinant vector encoding for
influenza-specific antigens or fragments thereof which induce an
anti-influenza effect in the animal following administration. In
one embodiment of the method, the recombinant vector is selected
from the group consisting of viral vector and plasmid. DNA, In
another embodiment of the method, the vector is an adenovirus. In
another embodiment of the method, the adenovirus vector is
defective in its E1 and E3 regions. In a further embodiment of the
method, the DNA is in plasmid form. In still another embodiment of
the method, the contacting step further comprises disposing the
recombinant vector containing the gene of interest on a delivery
device and applying the device having the recombinant vector
containing the gene of interest therein to the skin of the
animal.
[0137] Embodiments of the invention that employ adenovirus
recombinants, may include E1-defective, E3-defective, and/or
E4-defective adenovirus vectors, or the "gutless" adenovirus vector
in which all viral genes are deleted. The E1 mutation raises the
safety margin of the vector because E1-defective adenovirus mutants
are replication incompetent in non-permissive cells. The E3
mutation enhances the immunogenicity of the antigen by disrupting
the mechanism whereby adenovirus down-regulates MHC class I
molecules. The E4 mutation reduces the immunogenicity of the
adenovirus vector by suppressing the late gene expression, thus
allowing repeated re-vaccination utilizing the same vector. The
"gutless" adenovirus vector is the latest model in the adenovirus
vector family. Its replication requires a helper virus and a
special packaging cell line, a condition that does not exist in
natural environment; the vector is deprived of all viral genes,
thus the vector as a vaccine carrier is non-immunogenic and may be
inoculated for multiple times for re-vaccination. The "gutless"
adenovirus vector also contains 36 kb space for accommodating
transgenes, thus allowing co-delivery of a large number of antigen
genes into cells. Specific sequence motifs such as skin-binding
ligands may be inserted into the H-I loop of an adenovirus vector
to enhance its efficiency in transducing specific components in the
skin. An adenovirus recombinant is constructed by cloning specific
transgenes or fragments of transgenes into any of the adenovirus
vectors such as those described above. The adenovirus recombinant
is used to transduce epidermal cells of a vertebrate in a
non-invasive mode for use as an immunizing agent.
[0138] Embodiments of the invention that use DNA/adenovirus
complexes can have the plasmid. DNA complexed with adenovirus
vectors utilizing a suitable agent therefor, such as either PEI
(polyethylenimine) or polylysine. The adenovirus vector within the
complex can be either "live" or "killed" by UV or gamma
irradiation. The irradiation-inactivated adenovirus vector as a
receptor-binding ligand and an endosomolysis agent for facilitating
DNA-mediated transfection (Cotten et al., 1992) can raise the
safety margin of the vaccine carrier. The DNA/adenovirus complex is
used to transfect epidermal cells of a vertebrate in a non-invasive
mode for use as an immunizing agent.
[0139] Embodiments of the invention that use DNA/liposome complexes
can have materials for forming liposomes, and DNA/liposome
complexes be made from these materials. The DNA/liposome complex is
used to transfect epidermal cells of a vertebrate in a non-invasive
mode for use as an immunizing agent.
[0140] Recombinant vectors provided by the invention can also code
for immunomodulatory molecules to provoke a humoral and/or cellular
immune response. Such molecules include cytokines, co-stimulatory
molecules, or any molecules that may change the course of an immune
response. One can conceive of ways in which this technology can be
modified to enhance still further the immunogenicity of
antigens.
[0141] The recombinant vector used for NIVS can take any number of
forms, and the present invention is not limited to any particular
genetic material coding for any particular polypeptide. All forms
of recombinant vectors including viral vectors, bacterial vectors,
protozoan vectors, transposons, retrotransposons,
virus-like-particles, and DNA vectors, when used as skin-targeted
non-invasive vaccine carriers, are within the methods contemplated
by the invention.
[0142] The genes can be delivered by various methods including
device-free topical application or coating the genes on the surface
of the skin of an animal by a device such as a pad or bandage;
e.g., an adhesive bandage. Referring to FIG. 11, a device for
non-invasive vaccination is shown. This vaccine delivery device
includes a non-allergenic, skin adhesive patch having a bleb
disposed therein. In one embodiment, the patch is further comprised
of plastic, approximately 1 cm in diameter. The vaccine can be
disposed within the bleb. In another embodiment, the bleb contains
approximately 1 mL of vaccine (as liquid, lyophilized powder with
reconstituting fluid, and variants thereof). In a preferred
embodiment, the surface of the bleb in contact with the skin is
intentionally weaker than the opposite surface, such that when
pressure is applied to the opposite surface, the lower surface
breaks and releases the vaccine contents of the bleb onto the skin.
The plastic patch traps the vaccine against the skin surface.
[0143] Dosage forms for the topical administration of the
recombinant vector and gene of interest of this invention can
include liquids, ointments, powders, and sprays. The active
component can be admixed under sterile conditions with a
physiologically acceptable carrier and any preservatives, buffers,
propellants, or absorption enhancers as may be required or desired.
Reference is made to documents cited herein, e.g., U.S. Pat. Nos.
5,990,091, 6,042,838, and 6,004,802, and WO 98/00166 and WO
99/60164, and WO 99/53940, and documents cited therein for methods
to construct vectors, as well as for compositions for topical
application, e.g., viscous compositions that can be creams or
ointments, as well as compositions for nasal and/or mucosal and/or
oral cavity and/or buccal and/or perlingual administration.
[0144] In terms of the terminology used herein, an immunologically
effective amount is an amount or concentration of the recombinant
vector encoding the gene of interest, that, when administered to an
animal, produces an immune response to the gene product of
interest.
[0145] Various epitopes, antigens or therapeutics can be delivered
topically by expression thereof at different concentrations.
Generally, useful amounts for adenovirus vectors are at least
approximately 100 pfu and for plasmid DNA at least approximately 1
ng of DNA. Other amounts can be ascertained from this disclosure
and the knowledge in the art, including documents cited and
incorporated herein by reference, without undue
experimentation.
[0146] Furthermore, in the present description of the invention,
the term vector can be a replicative vector or a non-replicative
vector. Furthermore, all methods and compositions described herein
as using a vector can also use the cell-free extract herein
described.
[0147] The methods of the invention can be appropriately applied to
prevent diseases as prophylactic vaccination or treat diseases as
therapeutic vaccination.
[0148] The vaccines of the present invention can be administered to
an animal either alone or as part of an immunological
composition.
[0149] Beyond the human vaccines described, the method of the
invention can be used to immunize animal stocks. The term animal
means all animals including humans. Examples of animals include
humans, cows, dogs, cats, goats, sheep, horses, pigs, turkey, ducks
and chicken, etc. Since the immune systems of all vertebrates
operate similarly, the applications described can be implemented in
all vertebrate systems.
[0150] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion.
EXAMPLES
Protocols
Mice and Cell Cultures
[0151] Mice were maintained at the University of Alabama at
Birmingham. Cells were cultured in RPMI 1640 or DMEM media
containing 2% fetal bovine serum and 6% calf serum.
Topical Application of Recombinant Vectors
[0152] Mice were anesthetized and hair and cornified epithelium
covering a restricted area of abdominal or neck skin were removed
by a brush (Shi et al, 2001) or a depilatory (e.g., NAIR) (Tang et
al, 1997). Recombinant vectors were pipetted onto the preshaved
skin and kept in contact with naked skin for varying amounts of
time (e.g., 10 minutes to 18 hours). Vectors can be pipetted
directly onto naked skin.
Preparation of Adenovirus Vectors
[0153] High titer adenovirus stocks were prepared from human 293
cells infected with specific adenovirus recombinants. Lysates were
subjected to ultracentrifugation through a cesium chloride
gradient. Viral bands were extracted and dialyzed against 10 mM
Tris (pH 7.5)/135 mM NaCl/5 mM KCl/1 mM MgCl.sub.2. Purified
viruses were filter sterilized with glycerol added to 10%, and
stored in aliquots at -80.degree. C. Titer for adenovirus stocks
was determined by plaque assay.
Luciferase Assay
[0154] The amount of luciferase in the skin was determined as
previously described (Tang, 1994). Briefly, a piece of excised skin
was homogenized with a Kontes glass tissue grinder in lysis buffer.
After removing tissue debris by centrifugation, luciferase activity
in the skin extract was determined with a luminometer by
measurement of integrated light emission in the presence of excess
ATP and luciferin.
.beta.-Galactosidase Assay
[0155] A piece of excised skin was quickly frozen in Tissue-Tek
O.C.T. compound (Miles Laboratories Inc.) in liquid nitrogen and
stored at -80.degree. C. until use. The frozen tissue was cross
sectioned at 4 .mu.m, fixed in 4% paraformaldehyde, and stained for
.beta.-galactosidase activity by incubation in X-gal staining
solution as previously described (Tang et al., 1994). Sections were
counterstained with haematoxylin and eosin.
Preparation of DNA/Adenovirus Complexes
[0156] DNA/adenovirus complexes were prepared by mixing 100 .mu.g
plasmid DNA with 1.times.10.sup.11 particles of adenovirus in the
presence of a condensing agent such as PEI or polylysine for each
application. The titer of adenovirus was determined by
absorbance.
Preparation of DNA/Liposome Complexes
[0157] DNA/liposome complexes were prepared by mixing 100 .mu.g
plasmid DNA with 100 .mu.g DOTAP/DOPE (1:1; Avanti) for each
application. Plasmids were prepared using Qiagen Plasmid Maxi
Kits.
Western Blot Analysis
[0158] Sera from tail bleeds were diluted 1:250 to 1:500 and
reacted with purified proteins that had been separated in a
SDS-polyacrylamide gel and transferred to an Immobilon-P membrane
(Millipore). Reaction was visualized using the ECL kit
(Amersham).
ELISA Analysis
[0159] Following coating 96-well plates with the capture antigen,
serum samples and peroxidase conjugated goat anti-mouse IgG
(Promega Corp., Madison, Wis.) were incubated sequentially on the
plates with extensive washing between each incubation.
Example 1
[0160] The present invention demonstrates that antigen genes can be
delivered into the skin of mice in a simplified manner by
skin-targeted non-invasive delivery of a genetic vector without
using sophisticated equipment. FIG. 1 shows that substantial
amounts of luciferase enzyme was produced after delivery of limited
amounts of AdCMV-luc (an adenovirus vector encoding the firefly
luciferase) (Tang et al., 1994) onto the skin. Ad, adenovirus; pfu,
plaque-forming units; LU, light units. Results are the mean log [LU
per cm.sup.2 skin].+-.SE (n is shown on top of each column). Mice
mock-applied or coated with an adenovirus vector that did not
encode luciferase produced no detectable luciferase activity in the
skin. The level of transgene expression from the adenovirus vector
in the skin did not appear to correlate with the titer of the
virus. It is possible that only a small number of cells can be
transduced by the virus in a restricted subset of skin, and
10.sup.8 plaque-forming units (pfu) of adenovirus recombinants may
have saturated the target cells. This variability could also be
due, in part, to variations of individual mice. In addition, some
of the variability probably arose from the procedure for removing
cornified epithelium which had not been standardized (Johnston and
Tang, 1994). The amount of antigen produced may potentially be
amplified by applying more vectors onto a larger area.
Example 2
[0161] Without wishing to be necessarily bound by any one
particular theory, target cells for non-invasive vaccination onto
the skin appear to be epidermal cells, including but not limited
hair matrix cells within hair follicles (FIG. 2a) and keratinocytes
within the outermost layer of epidermis (FIG. 2b), as shown by
staining frozen sections with X-gal substrates after skin-targeted
non-invasive delivery of an adenovirus vector encoding the E. coli
.beta.-galactosidase gene (AdCMV-.beta.gal) (Tang et al., 1994). No
physical abrasions were found in the skin tissue subjected to the
treatment, and there was no inflammation induced. The skin tissue
subjected to non-invasive gene delivery was excised from animals 1
day after pipetting 10.sup.8 pfu of AdCMV-.beta.gal onto the skin,
cross sectioned, fixed, and stained with X-gal substrates as
described (Tang et al., 1994). FIG. 2a shows the
adenovirus-transduced epidermal cells, e.g. hair matrix cells
within a hair follicle, .times.150. FIG. 2b shows the
adenovirus-transduced keratinocytes within the outermost layer of
epidermis, .times.150. No blue cells were found in control animals
that were either mock-applied or coated with AdCMV-luc.
Example 3
Elicitation of Humoral Immune Responses by Adenovirus-Mediated
NIVS
[0162] NIVS is a novel method for vaccinating animals. To
demonstrate that the procedure can elicit a specific immune
response against the antigen encoded by the vector, AdCMV-hcea [an
adenovirus vector encoding the human carcinoembryonic antigen
(CEA)] was pipetted onto the skin of the C57BL/6 strain mice. Serum
from a vaccinated mouse a month after skin-targeted non-invasive
delivery of 10.sup.8 pfu AdCMV-hcea was diluted 1:500 and reacted
with purified human CEA protein and adenoviral proteins that had
been separated in a 5% SDS-polyacrylamide gel, and transferred to
Immobilon-P membranes (Millipore). Referring to FIG. 3a, lane 1,
0.5 .mu.g of human CEA; lane 2, 0.5 .mu.g of BSA; lane 3, 10.sup.7
pfu of adenovirus. FIG. 3a shows that the test sera from a
vaccinated animal reacted in western blots with purified human CEA
protein, but not with bovine serum albumin (BSA), which supports
the conclusion that specific antibodies have been produced against
exogenous proteins encoded by adenovirus vectors as a result of
skin-targeted non-invasive gene delivery.
[0163] To test whether this technique might be generally
applicable, AdCMV-hgmcsf (an adenovirus vector encoding the human
granulocyte macrophage colony stimulating factor (hGM-CSF)) was
applied onto the skin. To detect antibodies against the human
GM-CSF protein, the animal was vaccinated by skin-targeted
non-invasive delivery of 10.sup.8 pfu of AdCMV-hgmcsf. Purified
human GM-CSF protein (CalBiochem) separated in a 15%
SDS-polyacrylamide gel was transferred to membranes and allowed to
react with diluted serum. Other treatments were carried out as
described in FIG. 3a. Referring to FIG. 3b, lane 1, 0.25 .mu.g of
human GM-CSF; lane 2, 0.25 .mu.g of BSA; lane 3, 10.sup.7 pfu of
adenovirus. The replication-defective human adenovirus serotype 5
derived AdCMV-hcea and AdCMV-hgmcsf were produced in human 293
cells. A cassette containing the human CEA gene or the human GM-CSF
gene, driven by the cytomegalovirus (CMV) early enhancer-promoter
element was inserted in place of the E1a deletion. Since the
sequences in the E1a region were deleted, the ability of these
viruses to replicate autonomously in nonpermissive cells was
impaired.
[0164] Results (Tang et al., 1997) show that 96% (23/24) of the
C57BL16 strain mice produced antibodies against the human CEA
protein a month after skin-targeted non-invasive delivery of
AdCMV-hcea, and 43% (6/14) of the same strain mice produced
antibodies against the human GM-CSF protein after skin-targeted
non-invasive delivery of AdCMV-hgmcsf. Both pre-immune sera
collected before NIVS and sera from naive animals failed to react
with the human CEA and GM-CSF proteins. The possibility of oral
vaccination by ingesting vectors through grooming was eliminated by
(1) rinsing vectors away from the skin before animals recovered
from anesthesia, (2) pipetting vectors onto unshaved skin, and (3)
mixing naive and vaccinated animals in the same cage. No
cross-vaccination between naive and vaccinated mice was ever
observed. Thus, adenovirus-mediated NIVS is capable of eliciting a
humoral immune response against an antigen encoded by the
vector.
Example 4
[0165] To demonstrate that the techniques of the present invention
can elicit a protective antitumor immune response, syngeneic tumor
cells that express the human carcinoembryonic antigen (CEA) gene
(MC38-CEA-2) (Conry et al., 1995) were inoculated into naive
C57BL/6 strain mice and the same strain mice that had been
vaccinated by topical application of an adenovirus vector encoding
the human CEA gene (AdCMV-hcea). Animals subjected to tumor
challenges were observed for survival (FIG. 4). In the control
group, 90% (9/10) of the animals developed palpable tumor nodules
and died within 30 days after tumor cell implantation. In the
vaccinated group, only 10% (1/10) of the animals died, and 70%
(7/10) of them remained totally tumor-free. Mice were euthanized
when the tumor exceeded 1 cm in diameter. The interval between
tumor cell injection and euthanization is used as the individual
survival time. Referring to FIG. 4, control mice (no vaccines were
administered) and animals immunized by NIVS (10.sup.8 pfu of
AdCMV-hcea were topically applied a month before) were subjected to
tumor challenges. Numbers in parentheses represent the number of
animals for each treatment. Results show that non-invasive delivery
of genetic vaccines onto the skin is able to elicit protective
immune responses against tumor cells expressing a specific
antigen.
Example 5
Construction of Recombinant Adenovirus Vectors Encoding Cytokine
and Co-Stimulatory Genes
[0166] Adenovirus vectors encoding co-stimulatory and cytokine
genes were constructed for the co-delivery of these
immune-modulatory genes with antigen genes into skin cells in an
attempt to direct the immune profile in vaccinated animals. The
adenovirus vector AdCMV-mB7.1 encoding the murine B7-1 gene and the
adenovirus vector AdCMV-mgmcsf encoding the murine GM-CSF gene were
constructed by homologous recombination between two transfected
plasmids in human 293 cells following a standard procedure for
generating new adenovirus vectors (Gomez-Foix et al., 1992). All
transgenes in these vectors were transcriptionally driven by the
CMV early enhancer-promoter element. AdCMV-mB7.1 was characterized
by staining transduced human lung carcinoma SCC-5 cells with the
anti-CD80 antibody (PharMingen), followed by flow cytometric
analysis. AdCMV-mgmcsf was characterized by measuring murine GM-CSF
secreted from transduced SCC-5 cells with an ELISA kit
(Ameisham).
Example 6
Detection of Antitumor Immunity by In Viva Cytotoxicity Assay
[0167] An in vivo cytotoxicity assay was developed in which target
cells were implanted as monolayers onto the muscle tissue of mice
(Tang et al., 1996). Implantation of target cells as monolayers
allowed for an efficient retrieval of target cells for assessing
their fates after a few days of in viva growth. This assay was
particularly useful for detecting weak immune responses that are
not potent enough for eradicating target cells. Immune responses
can be characterized by histological analysis of the implantation
bed. Without an immune response, target cells would grow. With a
potent immune response, target cells would be eradicated in the
presence of a large number of immune effector cells at the
implantation bed, probably by virtue of migration to and in situ
sensitization around growing target cells. With a weak immune
response, growing target cells would intermingle with infiltrating
immune effector cells at the implantation bed. Implanting
5.times.10.sup.5 RM1-luc cells (RM1 prostate tumor cells expressing
the luciferase gene) as a monolayer into naive C57BL/6 mice
resulted in a tumor layer due to proliferation of RM1-luc cells in
vivo, with no evidence of immune intervention. In contrast to
control animals, RM1-luc cells were intermingled with a large
number of immune effector cells at the implantation bed in animals
vaccinated by skin-targeted non-invasive delivery of AdCMV-luc.
Example 7
Characterization of Immune Effector Cells Recruited by Tumor
Cells
[0168] The in vivo cytotoxicity assay was able to concentrate a
large number of immune effector cells at the implantation bed by
implanting a small number of target cells as a monolayer onto
muscle. Characterization of specific immune effector cells at the
implantation bed may provide evidence as to whether a cell-mediated
immune response has been elicited for killing target cells. For
characterizing T cells that were recruited by luciferase-expressing
tumor cells in animals vaccinated by skin-targeted non-invasive
delivery of AdCMV-luc, tissue sections of the implantation bed were
stained with an anti-CD3 monoclonal antibody (mAb). RM1-luc cells
were produced by lipofecting pHBA-luc DNA (a plasmid encoding
luciferase driven by the human .beta.-actin promoter) into RIM
prostate tumor cells (provided by T. Thompson at the Baylor College
of Medicine), followed by selection in medium containing G418.
Clones expressing luciferase were characterized by luciferase
assay. Five.times.10.sup.5 RM1-luc cells were implanted as a
monolayer into a mouse that had been vaccinated by skin-targeted
non-invasive delivery of 10.sup.8 pfu AdCMV-luc. Five days after
implantation, the implantation bed was frozen in O.C.T. and
sections were cut at 4 .mu.m, dried in 100% acetone, and stained
with an anti-CD3 mAb (clone F500A2, provided by P. Bucy at UAB),
via the ABC immunoperoxidase procedure with diaminobcnzidine as the
chromogen.
[0169] As shown in FIG. 5, a large number of T cells infiltrated
into the implantation bed after 5 days of in vivo growth of RM1-luc
cells in a mouse vaccinated by skin-targeted non-invasive delivery
of AdCMV-luc (.times.150) while only a few T cells were found in
naive animals. It appeared that the same number of RM1-luc target
cells could recruit more T lymphocytes to the implantation bed in
vaccinated animals than in naive animals.
[0170] For characterizing CTLs that were recruited by target cells,
frozen sections of the implantation bed were subjected to in situ
hybridization using an antisense granzyme A RNA molecule as the
probe. Five.times.10.sup.5 RM1-luc cells were implanted as a
monolayer into either a naive C57BL/6 mouse or a mouse that had
been vaccinated by skin-targeted non-invasive delivery of 10.sup.8
pfu AdCMV-luc. Five days after implantation, the implantation bed
was frozen in O.C.T. and sections were cut at 4 pill. Frozen
sections were fixed in 3% paraformaldehyde, incubated in 0.2 M HCl
for inhibiting endogenous alkaline phosphatase activity, and
hybridized with a heat-denatured antisense granzyme A RNA probe.
Probes for in situ hybridization were single-stranded RNA molecules
produced by transcription from a plasmid containing bacteriophage
promoters. During the transcription, digoxigenin-UTP was directly
incorporated into the sequence. Sense sequence probes were used as
negative controls. After hybridizing with probes, sections were
washed and incubated with alkaline phosphatase-conjugated
anti-digoxigenin antibody, followed by incubation in the NBT/BCIP
enzyme substrate solution.
[0171] CTLs that express granzyme A are activated CTLs and have
been used as predictive markers for tissue rejection during
transplantation. Granzyme-positive CTLs were found within the
RM1-luc implantation bed only in animals that had been vaccinated
by skin-targeted non-invasive delivery of AdCMV-luc (FIG. 6). Their
presence at the bed suggests that a cell-mediated immune response
against tumor cells expressing a specific antigen may have been
induced by NIVS.
Example 8
Topical Application of Recombinant Vaccines by Adhesive
Bandages
[0172] It was demonstrated, for the first time, that bandages could
be used for the administration of vaccines. This development may
allow personnel without medical training to deliver a uniform dose
of non-invasive vaccines onto the skin. To transduce skin by
bandage, 50 .mu.l of the AdCMV-luc vector described in Example 7
was pipetted into the pad of an adhesive bandage (Johnson &
Johnson). The vector-containing bandage was subsequently adhered to
pre-shaved skin of a mouse. The vector was kept in contact with
naked skin for 18 hours. To detect transgene expression from
genetic vectors delivered by a bandage, the skin was assayed for
luciferase (Table 1). While the results show substantial variation,
transgene expression in the skin was achievable using adhesive
bandages.
[0173] To demonstrate that animals could be vaccinated with
non-invasive adhesive bandages, sera from tail bleeds were assayed
for anti-CEA antibodies two months after adhering bandages
containing AdCMV-hcea onto the skin of mice. As shown in FIG. 7,
anti-CEA antibodies were detected in 100% (10/10) of mice that
received non-invasive vaccines through adhesive bandages.
Example 9
DNA/Adenovirus-Mediated NIVS
[0174] Adenovirus-based vectors can be made more versatile by
binding plasmid DNA to the exterior of an adenovirus. The resulting
vector system mediates high-efficiency gene delivery to a wide
variety of target cells. This approach allows greatly enhanced
flexibility in terms of the size and design of foreign genes.
DNA/adenovirus complexes may thus be able to deliver antigen genes
into the skin via the same adenovirus receptor-mediated endocytosis
pathway with more flexibility.
[0175] To demonstrate the feasibility of DNA/adenovirus-mediated
NIVS, plasmid DNA encoding the human growth hormone (pCMV-GH) (Tang
et al., 1992) was allowed to complex with an E4-defective
adenovirus. Mice (strain C57BL16) were vaccinated by contacting
DNA/adenovirus complexes with naked skin for one day. Immunized
animals were subsequently monitored for the production of
antibodies against the human growth hormone protein (hGH) by
assaying sera from tail-bleeds. As shown in FIG. 8a, lane 1, hGH
(0.5 .mu.g); lane 2, BSA (0.5 .mu.g), the test sera reacted in
western blots with purified hGH, but not with irrelevant proteins.
Of ten mice vaccinated by DNA/adenovirus complexes, eight (80%)
produced antibodies against hGH within three months, indicating
that specific antibodies could be produced against exogenous
proteins encoded by plasmid DNA that is complexed with adenovirus
and administered in a non-invasive mode. Pre-immune sera collected
before treatment, sera from untreated animals, and sera from
animals vaccinated with irrelevant vectors all failed to react with
hGH. Thus, DNA/adenovirus complexes, like adenovirus recombinants,
appear as a legitimate vector system for NIVS.
Example 10
DNA/Liposome-Mediated NIVS
[0176] In addition to developing genetic vectors involving
adenovirus as carriers for non-invasive vaccines, it has also been
demonstrated that mice could be vaccinated by topical application
of DNA/liposome complexes without viral elements. It is apparent
that many different vectors can be applied in a creative way for
the administration of skin-targeted non-invasive vaccines. As shown
in FIG. 8b, lane 1, hGH (0.5 .mu.g); lane 2, BSA (0.5 .mu.g), the
test serum from a mouse immunized by topical application of
DNA/liposome complexes encoding hGH reacted with hGH but not with
BSA. Of 10 mice vaccinated by DNA/liposome complexes, the test sera
reacted with purified hGH in 9 (90%) treated animals within 5
months. Thus, the DNA/liposome complex, like the adenovirus and the
DNA/adenovirus complex, appears as another legitimate vector system
for NIVS.
Example 11
Co-Expression of DNA-Encoded and Adenovirus-Encoded Transgenes
[0177] Strategies of augmenting the immune system's response can
potentially improve the clinical outcomes of vaccines. Local
production of immune-modulatory molecules involved in the
activation and expansion of lymphocyte populations may
significantly improve the vaccination effects. Adenovirus vectors
encoding the murine B7-1 and GM-CSF genes have been made. Topical
application of DNA/adenovirus complexes may thus be able to
co-express DNA-encoded antigens or immune modulatory molecules with
adenovirus-encoded antigens or immune modulatory molecules in
individual skin cells for enhancing the immune response against the
antigen.
[0178] FIG. 9 shows that the expression of transgenes from plasmid
DNA in target cells is dependent upon the presence of adenovirus,
thus allowing plasmid-encoded and adenovirus-encoded transgenes to
be co-expressed in the same cell. pVR-1216 plasmid DNA (provided by
Vical), AdCMV-.beta.gal particles and polylysine were mixed at
specific ratios as shown in the figure. The complex was applied to
2.times.10.sup.5 SCC-5 cells in a well and incubated for 2 hours.
The complex was then removed and cells were harvested for
luciferase and .beta.-galactosidase assays the next day. Open
column: luciferase activity; stippled column: .beta.-galactosidase
activity. Results show that DNA-encoded transgenes are not
expressed in target cells in the absence of adenovirus, and
adenovirus-encoded transgenes can be expressed in the presence of
DNA. It is also possible that DNA may be condensed onto the surface
of other viruses for targeting different cell types. Accordingly,
this protocol provides a simple but versatile gene delivery system
which allows the expression of transgenes from both a virus
recombinant and an externally-bound plasmid, simultaneously.
Example 12
Relative Transgene Expression in the Skin from Different Genetic
Vectors by Topical Application
[0179] It has been shown that adenovirus recombinants,
DNA/adenovirus complexes, DNA/Liposome complexes, and perhaps many
other genetic vectors can all be applied as carriers for
non-invasive vaccines. It is conceivable that the higher the
efficiency for transgene expression, the more powerful the carrier
will be. To define the relative efficiencies for the vectors
utilized, adenovirus recombinants, DNA/adenovirus complexes, or
DNA/liposome complexes were allowed to contact mouse skin by
topical application for 18 hr. The treated skin was subsequently
removed from the animal and assayed for luciferase activity with a
luminometer by measurement of integrated light emission for 2 min
using the Promega's luciferase assay system, and background was
subtracted from the readings. As shown in FIG. 10, adenovirus
recombinants were found to be the most efficient vector system for
skin-targeted non-invasive gene delivery. Mice mock-treated
produced no detectable luciferase activity in the skin. LU, light
units; Ad, AdCMV-luc; DNA/Ad, pVR-1216 DNA complexed with Ad
d11014; DNA/liposome, pVR-1216 DNA complexed with DOTAP/DOPE.
Results are the mean log(LU per cm.sup.2 skin).+-.SE (n is shown on
top of each column). Although the efficiency of DNA/adenovirus
complex is lower than that of adenovirus recombinant, it is
significantly higher than that of DNA/liposome complex. In
addition, adenovirus may be inactivated by UV or gamma irradiation
before complexing with DNA to prevent viable viral particles from
disseminating. Thus, DNA/adenovirus complexes may appear as a
promising carrier system for the delivery of non-invasive vaccines
when efficiency and safety factors are both considered in
formulating a new generation of vaccines.
Example 13
Construction of an Expression Vectors Encoding Influenza
Antigens
[0180] An E1/E3-defective adenovirus recombinant encoding the
A/PR/8/34 HA gene (AdCMV-PR8.ha) was constructed as described
(Gomez-Foix et al., 1992). Briefly, an 1.8 kb BamHI fragment
containing the entire coding sequence for HA was excised from the
plasmid pDP122B [American Type Culture Collection (ATCC)] and
subsequently inserted into the BamHI site of pACCMV.PLPA in the
correct orientation under transcriptional control of the human
cytomegalovirus (CMV) early promoter. The resulting plasmid
encoding HA was co-transfected with the plasmid pJM17 into human
293 cells for generating E1/E3-defective adenovirus recombinants.
An E1/E3-defective adenovirus recombinant encoding the A/PR/8/34
nuclear protein (NP) gene (AdCMV-PR8.np) was constructed by cloning
the NP gene (provided by Merck) into pACCMV.PLPA, followed by
homologous recombination in 293 cells as described above.
[0181] A plasmid expression vector encoding HA (pCMV-PR8.ha) and
another encoding NP (pCMV-PR8.np) were constructed by cloning the
HA and NP genes into pVR1012 (provided by Vical), respectively.
Example 14
Anti-Influenza Antibodies Generated by Topical Application and
Intranasal Inoculation of Adenovirus-vectored vaccines in mice
[0182] As shown in FIG. 12, BALB/c mice (3 months old) were
immunized by a variety of vaccination modalities including
intramuscular injection of DNA, intranasal inoculation of
adenovirus vectors, and topical application of an adenovirus-based
vaccine patch. Skin-targeted noninvasive vaccination was carried
out by pipetting adenovirus vectors onto pre-shaved abdominal skin
followed by covering the vector as a thin film over naked skin with
a piece of the Tegaderm patch (3M) Unabsorbed vectors were washed
away in an hour. All animals were immunized 3 times at intervals of
3 weeks. Serum samples were assayed for anti-influenza antibodies 1
week after the last boost. Titers of anti-influenza IgG were
determined by ELISA using purified A/PR/8/34 virus as the capture
antigen. Serum samples and peroxidase-conjugated goat anti-mouse
IgG (Promega) were incubated sequentially on the plates for 1 hour
at room temperature with extensive washing between each incubation.
The end-point was calculated as the dilution of serum producing the
same OD.sub.490 as a 1/100 dilution of preimmune serum. Sera
negative at the lowest dilution tested were assigned endpoint
titers of 1. Hemagglutination inhibition (HI) assay was carried out
for measuring the ability of anti-HA antibodies to inhibit the
agglutination of red blood cells (RBC) by virus, possibly by
blocking cell surface binding. Serum samples preabsorbed with
chicken RBCs were diluted and mixed with 4 HA units of influenza
A/PR/8/34. Chicken RBCs were then added to a final concentration of
0.5%. Agglutination was determined by visual examination. The titer
was defined as the dilution being the limit of inhibition. All
preimmune sera had titers of .ltoreq.20. Group 1, intranasal
inoculation of 2.5.times.10.sup.7 pfu wild-type adenovirus serotype
5 followed by topical application of 10.sup.8 pfu AdCMV-PR8.ha and
10.sup.8 pfu AdCMV-PR8.np 2 weeks later (n=9); Group 2, intranasal
inoculation of 2.5.times.10.sup.7 pfu wild-type adenovirus serotype
5 followed by intramuscular injection of 100 .mu.g pCMV-PR8.ha DNA
and 100 .mu.g pCMV-PRS.np DNA 2 weeks later (n=10); Group 3,
intranasal inoculation of 2.5.times.10.sup.7 pfu wild-type
adenovirus serotype 5 followed by intranasal inoculation of
2.5.times.10.sup.7 pfu AdCMV-PR8.ha and 2.5.times.10.sup.7 pfu
AdCMV-PR8.np 2 weeks later (n=8); Group 4, topical application of
10.sup.8 pfu AdCMV-PR8.ha and 10.sup.8 pfu AdCMV-PR8.np (n=10);
Group 5, topical application of 10.sup.8 pfu AdCMV-PR8.np (n=10);
Group 6, topical application of 10.sup.8 pfu AdCMV-PR8.ha (n=10);
Group 7, intramuscular injection of 100 .mu.g pCMV-PR8.ha DNA and
100 .mu.g pCMV-PR8.np DNA (n=10); Group 8, intranasal inoculation
of 2.5.times.10.sup.7 pfu AdCMV-PR8.ha and 2.5.times.10.sup.7 pfu
AdCMV-PR8.np (n=9). The data was plotted as geometric mean endpoint
titers. In the naive control group (n=7), no anti-influenza
antibodies were detectable. The analysis of variance (ANOVA)
approach was utilized to compare the differences in ELISA and HI
titers. Multiple pairwise comparisons were made with Tukey's
procedure with the overall alpha level set at 0.05. The analyses
were performed in log scale of the measurements to meet the
constant variance assumption required by the ANOVA approach. The
differences in ELISA and HI titers, among the 8 groups were
significant (P<0.0001). The ELISA titer in group 8 was
significantly higher than that in other groups (P<0.02). The
average ELISA titer in group 1 was the lowest but was not
significantly different from that in group 5 or 6. The HI titer in
group 8 was the highest and that in group 3 was the second highest.
The HI titer values in groups 1, 2, 4, 5, and 6 were not
significantly different.
Example 15
Protection of Mice from Death Following Virus Challenge
[0183] As shown in FIG. 13, BALB/c mice (3 months old) were
immunized by a variety of vaccination modalities including
intramuscular injection of DNA, intranasal inoculation of
adenovirus vectors, and topical application of an adenovirus-based
vaccine patch. Skin-targeted noninvasive vaccination was carried
out by pipetting adenovirus vectors onto pre-shaved abdominal skin
followed by covering the vector as a thin film over naked skin with
a piece of the Tegaderm patch (3M). Unabsorbed vectors were washed
away in an hour. All animals were immunized 3 times at intervals of
3 weeks. One week after the last boost, mice were challenged
intranasally with a lethal dose of influenza virus A/PR/8/34 (1,000
HA units) and monitored daily for survival. The data was plotted as
% survival versus days after challenge. Naive Control, naive mice
without exposure to adenovirus; Group 1, intranasal inoculation of
2.5.times.10.sup.7 pfu wild-type adenovirus serotype 5 followed by
topical application of 10.sup.8 pfu. AdCMV-PR8.ha and 10.sup.8 pfu
AdCMV-PR8.np 2 weeks later; Group 2, intranasal inoculation of
2.5.times.10.sup.7 pfu wild-type adenovirus serotype 5 followed by
intramuscular injection of 100 .mu.g pCMV-PR8.ha DNA and 100 .mu.g
pCMV-PR8.np DNA 2 weeks later; Group 3, intranasal inoculation of
2.5.times.10.sup.7 pfu wild-type adenovirus serotype 5 followed by
intranasal inoculation of 2.5.times.10.sup.7 pfu AdCMV-PR8.ha and
2.5.times.10.sup.7 pfu AdCMV-PR8.np 2 weeks later; Group 4, topical
application of 10.sup.8 pfu AdCMV-PR8.ha and 10.sup.8 pfu
AdCMV-PR8.np; Group 5, topical application of 10.sup.8 pfu
AdCMV-PR8.np; Group 6, topical application of 10.sup.8 pfu
AdCMV-PR8.ha; Group 7, intramuscular injection of 100 .mu.g
pCMV-PR8.ha DNA and 100 .mu.g pCMV-PR8.np DNA; Group 8, intranasal
inoculation of 2.5.times.10.sup.7 pfu AdCMV-PR8.ha and
2.5.times.10.sup.7 pfu AdCMV-PR8.np. AdCMV-PR8.ha, an adenovirus
vector encoding the A/PR/8/34 hemagglutinin; AdCMV-PR8.np, an
adenovirus vector encoding the A/PR/8/34 nuclear protein;
pCMV-PR8.ha, a plasmid expression vector encoding the A/PR/8/34
hemagglutinin; pCMV-PR8.np, a plasmid expression vector encoding
the A/PR/8/34 nuclear protein. Numbers in parentheses represent the
number of animals for each treatment.
[0184] Results suggested that protection may be mediated
principally by a Immoral immune response when animals were
immunized by intranasal inoculation of adenovirus recombinants. In
contrast to the intranasal route, animals immunized by topical
application of AdCMV-PR8.ha and AdCMV-PR8.np were afforded 71%
protection from the challenge. However, animals with pre-exposure
to adenovirus failed to be protected by NIVS (noninvasive
vaccination onto the skin).
Example 16
Elicitation of Anti-HA Antibodies in a Pigtail Macaque by NIVS
[0185] Although NIVS could reproducibly elicit systemic immune
responses in mice (FIGS. 12 and 13), it may not be possible for
NIVS to immunize humans if transdermal diffusion of vectors should
be required for vaccination to occur, because human skin is thicker
than its murine counterpart. However, non-invasive vaccine patches
may be able to immunize humans or other animals with thick skin if
all that is required is a transient but productive wave of antigen
expression in cells within the outer layer of skin. To address
these issues, we have immunized a pigtail macaque by AdCMV-PR8.ha
in a non-invasive mode. As shown in FIG. 14, the immunized animal
produced antibodies against HA in 4 weeks. The result provides
evidence that non-invasive vaccine patches may be able to immunize
many different species in addition to mice.
[0186] In FIG. 14, a pigtail macaque was immunized in a
non-invasive mode by pipetting pfu of AdCMV-PR8.ha onto pre-shaved
abdominal skin followed by covering the vector as a thin film over
naked skin with the Tegaderm patch (3M). Unabsorbed vectors were
washed away in 5 hours. Serum samples were assayed for anti-HA
antibodies 4 weeks post-immunization. Titers of anti-HA IgG were
determined by ELISA using purified A/PR/8/34 virus as the capture
antigen. Serum samples and peroxidase-conjugated goat anti-monkey
IgG (Bethyl Laboratories, Inc.) were incubated sequentially on the
plates for 1 hour at room temperature with extensive washing
between each incubation. The end-point was calculated as the
dilution of serum producing the same OD.sub.490 as a 1/100 dilution
of preimmune serum. Sera negative at the lowest dilution tested
were assigned endpoint titers of 1.
Example 17
Relocation of Luciferase Spots in the Skin after Localized Gene
Delivery in a Non-Invasive Mode
[0187] In an attempt to determine whether antigen genes delivered
onto the surface of the skin could diffuse into deep tissues and
express antigens in cells beyond epidermis, we incubated neck skin
with AdCMV-luc (an adenovirus vector encoding luciferase) (Tang et
al., 1997). As shown in FIG. 15, luciferase activity could be
detected in ears (or as discrete luciferase spots in other areas
within the skin) in some of the treated animals one day after
non-invasive delivery of AdCMV-luc onto neck skin. Luciferase was
undetectable in any of the internal organs including lymph nodes,
liver, spleen, heart, lung and kidney.
[0188] In FIG. 15, 1.times.10.sup.8 pfu of AdCMV-luc was incubated
with neck skin for an hour. Neck skin as, well as ears were
harvested for luciferase assay as described (Tang et al., 1994) one
day after inoculation. Numbers represented light units with
background subtracted from the readings.
[0189] In a further attempt to identify and characterize the target
cells that are able to express the transgene from a
topically-applied adenovirus vector, and the putative mobile cells
containing the protein expressed from the transgene, we stained
skin sections with X-gal after topical application of AdCMV-.beta.
gal (an adenovirus vector encoding .beta.-galactosidase) (Tang et
al., 1994). By examining histological sections in search of dark
blue cells, we identified labeled cells within hair follicles
epidermal cells, e.g. hair matrix cells within hair follicles, and
labeled keratinocytes in the outermost layer of epidermis as the
principal target cells for adenovirus-mediated transduction when
the vector was inoculated in a noninvasive mode. None of the dermal
fibroblasts were transduced by this procedure, although these cells
were highly transducible when AdCMV-.beta.gal was injected
intradermally using a needle, Results suggested that few, if any,
of the adenovirus particles that were topically applied could
penetrate into dermis beyond the outer layer of epidermis (further
research has indicated that this may have been a spurious
conclusion, as shown by the following examples). Microscopic
examination of histologic sections did not reveal any physical
abrasions of the transduced skin. Macroscopically, there was no
inflammation associated with the treated skin. However, transduced
cells could only be visualized within the inoculation area (e.g.,
neck skin), We were unable to identify dark blue cells in ears or
other areas within the skin when luciferase activities could be
detected in those areas, probably because luciferase assay is more
sensitive than X-gal-mediated .beta.-galactosidase assay. We
hypothesize that some antigen-presenting cells (APCs) may respond
to antigens expressed on the surface of the skin by acquiring the
antigen. The protein may be degraded rapidly, hence undetectable
from internal organs including lymph nodes.
Example 18
Amplification of Foreign DNA in Various Tissues after Localized
Gene Delivery in a Noninvasive Mode
[0190] In an attempt to determine whether topical application of an
adenovirus vector can also deliver exogenous DNA beyond the
inoculation area, we extracted DNA from various tissues, followed
by amplification of the transgene as well as the adenovirus type 5
fiber gene by polymerase-chain reaction (PCR) after noninvasive
delivery of AdCMV-PR8.ha onto skin. As shown in FIG. 16, the
full-length HA and fiber genes could be amplified from skin 3 hours
post-inoculation. The full-length gene was usually undetectable in
skin DNA after 1 day or in DNA extracted from other tissues.
However, subfragments of both HA and fiber genes could be amplified
from liver, whole blood, ear, abdominal skin, or pooled lymph nodes
using different sets of primers. No foreign DNA was detectable in
any of the tissues 4 weeks post-inoculation. Results suggested that
topical application of an adenovirus vector could deliver exogenous
DNA into a localized area in skin, although foreign DNA may be
rapidly acquired by some putative antigen-presenting cells,
degraded, and relocated into deep tissues. The elimination of
foreign DNA in 4 weeks highlights the safety of NIVS. In FIG. 16,
AdCMV-PR8.ha and AdCMV-luc were inoculated onto preshaved skin in a
noninvasive mode. DNA was extracted by DNAZOL (GIBCOBRL), and
amplified by the following sets of primers:--
TABLE-US-00001 Ha5.1: (SEQ ID NO: 1) 5'-A T G A A G G C A A A C C T
A C T G G T-3' Ha3.1: (SEQ ID NO: 2) 5'-G A T G C A T A T T C T G C
A C T G C A-3' Ha5.2: (SEQ ID NO: 3) 5'-G T G G G G T A T T C A T C
A C C C G T-3' Ha3.2: (SEQ ID NO: 4) 5'-T G C A T A G C C T G A T C
C C T G T T-3' Luc5.1: (SEQ ID NO: 5) 5'-G C G C C A T T C T A T C
C T C T A G A-3' Luc3.1: (SEQ ID NO: 6) 5'A C A A T T T G G A C T T
T C C G C C C-3' Luc5.2: (SEQ ID NO: 7) 5'-G T A C C A G A G T C C
T T T G A T C G-3' Luc3.2: (SEQ ID NO: 8) 5'-C C C T C G G G T G T
A A T C A G A A T-3' Fb5.1: (SEQ ID NO: 9) 5'-C C G T C T G A A G A
T A C C T T C A A-3' Fb3.1: (SEQ ID NO: 10) 5'-A C C A G T C C C A
T G A A A A T G A C-3' Fb5.2: (SEQ ID NO: 11) 5'-G G C T C C T T T
G C A T G T A A C A G-3' Fb3.2: (SEQ ID NO: 12) 5'-C C T A C T G T
A A T G G C A C C T G T-3'
Ha5.1 and Ha3.1 amplified the nearly full-length 1.7 kb HA gene;
Ha5.2 and Ha3.2 amplified an 0.6 kb subfragment encompassing 33% of
the HA gene; Luc5.1 and Luc3.1 amplified the nearly full-length 1.7
kb luciferase gene; Luc5.2 and Luc3.2 amplified an 0.52 kb
subfragment encompassing 30% of the luciferase gene; Fb5.1 and
Fb3.1 amplified the nearly full-length 1.7 kb adenovirus type 5
fiber gene; Fb5.2 and Fb3.2 amplified an 0.55 kb subfragment
encompassing 32% of the fiber gene. Lane M, Molecular weight marker
(Lambda DNA cleaved with HindIII); lane 1, the nearly full-length
luciferase gene amplified by Luc5.1 and Luc3.1 from skin DNA 3
hours after NIVS; lane 2, the nearly full-length luciferase gene
amplified by Luc5.1 and Luc3.1 from skin DNA 1 day after NIVS; lane
3, a subfragment of luciferase DNA amplified by Luc5.2 and Luc3.2
from mouse ear DNA 1 day after NIVS; lane 4, a subfragment of
luciferase DNA amplified by Luc5.2 and Luc3.2 from lymph node DNA,
1 day after NIVS; lane 5, a subfragment of luciferase DNA amplified
by Luc5.2 and Luc3.2 from liver DNA 1 day after NIVS; lane 6, a
subfragment of luciferase DNA amplified by Luc5.2 and Luc3.2 from
DNA extracted from whole blood 1 day after NIVS; lane 7, the nearly
full-length HA gene amplified by Ha5.1 and Ha3.1 from skin DNA 3
hours after NIVS; lane 8, a subfragment of HA gene amplified by
Ha5.2 and Ha3.2 from skin DNA 1 day after NIVS; lane 9, a
subfragment of HA gene amplified by Ha5.2 and Ha3.2 from lymph node
DNA 1 day after NIVS; lane 10, a subfragment of HA gene amplified
by Ha5.2 and Ha3.2 from liver DNA 1 day after NIVS; lane 11, a
subfragment of HA gene amplified by Ha5.2 and Ha3.2 from kidney DNA
1 day after NIVS; lane 12, a subfragment of HA gene amplified by
Ha5.2 and Ha3.2 from DNA extracted from whole blood 1 day after WS;
lane 13, the nearly full-length fiber gene amplified by Fb5.1 and
Fb3.1 from skin DNA 3 hours after NIVS; lane 14, the nearly
full-length fiber gene amplified by Fb5.1 and Fb3.1 from skin DNA 1
day after NIVS; lane 15, a subfragment of fiber gene amplified by
Fb5.2 and Fb3.2 from skin DNA 1 day after NIVS; lane 16, a
subfragment of fiber gene amplified by Fb5.2 and Fb3.2 from ear DNA
1 day after NIVS; lane 17, a subfragment of fiber gene amplified by
Fb5.2 and Fb3.2 from lymph node DNA 1 day after NIVS; lane 18, a
subfragment of fiber gene amplified by Fb5.2 and Fb3.2 from liver
DNA 1 day after NIVS; lane 19, a subfragment of fiber gene
amplified by Fb5.2 and Fb3.2 from DNA extracted from whole blood 1
day after NIVS. DNA from lymph nodes was extracted by pooling
inguinal, cervical, and brachial lymph nodes in DNAZOL solution.
DNA was amplified for 35 cycles at optimized annealing temperatures
in a Stratagene Robocycler gradient 40 thermal cycler. Amplified
DNA fragments were fractionated in 1% agarose gel and stained with
ethidium bromide.
Example 19
A Depilatory Agent is not Required for NIVS
[0191] To determine whether a depilatory agent such as NAIR (Tang
et al., 1997) is essential for NIVS, we have compared antibody
titers elicited by vaccine patches with or without pre-treatment
using NAIR. FIG. 17 shows that antibody titers in mice without NAIR
pre-treatment are as high as their counterparts with NAIR
pre-treatment. The elimination of NAIR simplifies the NIVS
procedure.
[0192] In FIG. 17, mice were either injected intradermally (ID)
with a dose of 10.sup.8 pfu, or immunized in a non-invasive mode
(NIVS) by pipetting 10.sup.8 pfu of AdCMV-hcea (Tang et al., 1997)
onto abdominal skin followed by covering the vector as a thin film
over naked skin with a piece of the Tegaderm patch (3M). Unabsorbed
vectors were washed away, Serum samples were assayed for anti-CEA
antibodies at 4 weeks after inoculation. Titers of anti-CEA IgG
were determined by ELISA using purified human CEA (CalBiochem) as
the capture antigen. Serum samples and peroxidase-conjugated goat
anti-mouse IgG (Promega) were incubated sequentially on the plates
for 1 hour at room temperature with extensive washing between each
incubation. The end-point was calculated as the dilution of scrum
producing the same OD.sub.490 as a 1/100 dilution of preimmune
serum. Sera negative at the lowest dilution tested were assigned
endpoint titers of 1. The data was plotted as geometric mean
endpoint ELISA titers, where n=4 for ID, n=14 for 1 hr, n=10 for
NAIR(-), and n=15 for NAIR/clip(-). ID, intradermal injection; 1
hr, vectors were in contact with the outer layer of skin for an
hour with shaving and NAIR pre-treatment; NAIR(-), vectors were in
contact with the outer layer of skin overnight with shaving but
without NAIR pre-treatment; NAIR/clip(-), vectors were in contact
with the outer layer of skin overnight with neither shaving nor
NAIR pre-treatment. Shaving was performed with an electric
trimmer.
Example 20
Protection Against Tetanus by Topical Application of an
Adenovirus-Vectored Vaccine
[0193] As shown in FIG. 18, BALB/c mice (3 months old) were
immunized by a variety of vaccination modalities including
intramuscular injection of DNA, topical application or intranasal
inoculation of an adenovirus-based tetanus vaccine. Skin-targeted
noninvasive vaccination was carried out by pipetting approximately
10.sup.8 pfu AdCMV-tetC onto pre-shaved abdominal skin followed by
covering the vector as a thin film over naked skin with a piece of
the Tegaderm patch (3M). Unabsorbed vectors were washed away in an
hour. Nasal vaccines were administered by pipetting approximately
10.sup.7 pfu AdCMV-tetC into the nasal cavity. All animals were
immunized 3 times at intervals of 3 weeks. One week after the last
boost, mice were challenged by injecting a lethal dose of
Clostridium tetani into the footpad and monitored daily for
survival. The data was plotted as % survival versus days after
challenge. Naive Control, naive mice without vaccination prior to
challenge. Ad-tetC:NIVS, mice immunized by topical application of
AdCMV-tetC; Ad-tetC:IN, mice immunized by intranasal inoculation of
AdCMV-tetC; pCMV-tetC:IM, mice immunized by intramuscular injection
of 100 .mu.g pCMV-tetC DNA. AdCMV-tetC, an adenovirus vector
encoding the Clostridium tetani toxin C-fragment; pCMV-tetC, a
plasmid expression vector encoding the Clostridium tetani toxin
C-fragment. Numbers in parentheses represent the number of animals
for each treatment.
Example 21
Immunization by Topical Application of a Salmonella-Based
Vector
[0194] As shown in FIG. 19, three-month old ICR mice (Harlan,
Indianapolis, Ind.) were vaccinated with the Salmonella typhimurium
strain BRD847 (Chatfield et al., 1992) expressing the tetanus toxin
C-fragment. Vaccination was accomplished by oral inoculation,
intranasal instillation, or topical application as described in Shi
et al. (2001). Briefly, mouse skin was prepared by depilation with
an electric trimmer paired with gentle brushing using a
soft-bristle brush (erythema was not induced). Topical application
was carried out by pipetting the recombinant vector as a thin film
onto the prepared skin followed by coverage with a Tegaderm patch
(3M). After 1 hour, unabsorbed vectors were washed away. The
possibility of oral or nasal immunization through grooming was
eliminated as described above (see for example, Example 19) and as
known in the art. Oral and intranasal instillation consisted of
pipetting the recombinant vector into the mouth or one of the
nostrils of an anesthetized mouse, respectively. Oral inoculation
consisted of approximately 10.sup.9 BRD847 cells (n=6), intranasal
instillation consisted of approximately 10.sup.8 BRD847 cells
(n=9), and topical application consisted of approximately 10.sup.10
BRD847 cells (n=10).
[0195] One month after vaccination, serum samples were obtained and
titers of anti-tetC IgG were determined by ELISA using purified
TetC protein (CalBiochem, San Diego, Calif.) as the capture
antigen, as described above and in Shi et al. (1999). Briefly,
serum samples and peroxidase-conjugated goat anti-mouse IgG
(Promega Corp., Madison, Wis.) were incubated sequentially on the
plates with extensive washing between each incubation. The
end-point was calculated as the dilution of serum producing the
same OD.sub.490 as a 1/100 dilution of pre-immune serum. Sera
negative at the lowest dilution tested were assigned endpoint
titers of 1.
[0196] Animals immunized by all three methods [ORAL, IN
(intranasal), and NIVS (noninvasive vaccination on the skin)]
produced anti-tetC antibodies one month after vaccination.
Quantitative results are shown in FIG. 19.
[0197] As shown by the figure, topical application of the vector
caused similar production of anti-tetC antibodies as did intranasal
instillation under specified experimental conditions.
Example 22
Immunization by Topical Application of an Escherichia-Based
Vector
[0198] Three-month old ICR mice (Harlan, Indianapolis, Ind.) (3
animals per group) were vaccinated with either the Escherichia (E.)
coli strain DH10B (Stratagene, La Jolla, Calif.) expressing the
tetanus toxin C-fragment (tetC) driven by the nirB promoter
(pTET-nir) (Chatfield et al., 1002), or with DH10B expressing a
plasmid encoding tetC driven by the cytomegalovirus (CMV) early
promoter (pCMV-tetC) (Shi et al., 2001). Vaccination was
accomplished by topical application of 5*10.sup.9 cfu (colony
forming-units). As described in Shi et al. (2001) topical
application involved preparing mouse skin by depilation with an
electric trimmer paired with gentle brushing using a soft-bristle
brush (erythema was not induced). Topical application was carried
out by pipetting the recombinant vector as a thin film onto the
prepared skin followed by coverage with a Tegaderm patch (3M).
After 1 hour, unabsorbed vectors were washed away. As above,
precautions were taken to avoid accidental oral or nasal
immunization.
[0199] Three weeks after immunization, serum samples were obtained
and titers of anti-tetC IgG were determined by ELISA as described
above and in Shi et al. (2001), using purified tetC protein
(CalBiochem, San Diego, Calif.) as the capture antigen. Briefly,
serum samples and peroxidase-conjugated goat anti-mouse IgG
(Promega Corp., Madison, Wis.) were incubated sequentially on the
plates with extensive washing between each incubation. The
end-point was calculated as the dilution of serum producing the
same OD490 as a 1/100 dilution of preimmune serum. Sera negative at
the lowest dilution tested were assigned endpoint titers of 1.
[0200] Quantitative results are shown in FIG. 20. As depicted in
FIG. 20, vaccination with E. coli cells harboring pTET-nir was
significantly more potent in eliciting an anti-tetC humoral immune
response than was vaccination with E. coli cells harboring
pCMV-tetC.
Example 23
Detection of Anti-Clostridium (C.) Tetani Antibodies in Mice
Following Topical Application of Irradiated C. tetani Cells
[0201] A toxigenic strain of C. tetani (ATCC number 9441; American
Type Culture Collection, Manassas, Va.) was cultivated in the ATCC
38 beef liver medium for anaerobes at 37.degree. C. under an
anaerobic gas mixture (80% N.sub.2-10% CO.sub.2-10% H.sub.2).
Gram-stained cells were counted under a light microscope using a
hemacytometer, C. tetani cells were .gamma.-irradiated at a lethal
dose (20,000 Gy) prior to administration.
[0202] The abdominal skin of young (2 to 3 months old) ICR mice
(Harlan, Indianapolis, Ind.) was prepared by depilation with an
electric trimmer in conjunction with gentle brushing using a
soft-bristle brush as described (1). Topical application of
irradiated C. tetani cells was carried out by pipetting 10.sup.6
irradiated bacterial cells onto the preshaved skin of the ICR mice
and covering the cells with a piece of Tegaderm.TM. patch (3M). The
irradiated bacterial cells were in contact with naked skin as a
thin film under the Tegaderm patch (3M), which prevented the
animals from ingesting or intranasally absorbing the C. tetani
during grooming. After 1 hour, the patch was removed and the skin
was washed to remove bacterial cells that did not adhere to the
skin or which were not taken up by the skin.
[0203] Three weeks after immunization, sera were collected and
pooled from 4 immunized animals. The sera were diluted 1:100 and
reacted with C. tetani proteins separated on a 13%
SDS-polyacrylamide gel. The products were transferred to membranes.
As a negative control, pooled pre-immune sera from the same strain
of mice did not react with any proteins on the membrane.
[0204] The membranes showed that antibodies against at least two C.
tetani proteins were elicited in mice at three weeks after topical
application of irradiated C. tetani cells (See FIG. 21). This
demonstrated that animals can be immunized against
pathogen-specific antigens by topical application of irradiated
bacterial pathogens that are made into non-replicative vectors.
Example 24
Induction of Tetanus in Mice Following Topical Application of
Irradiated Clostridium tetani
[0205] C. tetani cells were .gamma.-irradiated at a lethal dose of
20,000 Gy and administered onto mouse skin as described in Example
23. Topical application of irradiated C. tetani cells was carried
out by pipetting an escalating dose (10.sup.5-10.sup.8) of
irradiated bacterial cells onto the preshaved abdominal skin of
mice. Animals were monitored daily for symptoms of tetanus and
euthanized at the onset of muscular fasciculation.
[0206] One hundred % of the animals that received topical
application of irradiated C. tetani cells in a dose
.gtoreq.10.sup.7 developed tetanus symptoms within three days of
the application. One hundred % of the animals who received a dose
of 10.sup.5 irradiated C. tetani cells survived without tetanus
symptoms. Some animals surviving the challenge developed antibodies
against C. tetani-specific antigens (Example 23).
[0207] Quantitative data is shown in FIG. 22. The data were plotted
as percent survival versus number of days after inoculation. The
number of animals in each treatment group are shown in parentheses
in the figure legend.
[0208] This data demonstrates that the tetanus neurotoxin in
irradiated C. tetani cells can be taken up by the outer layer of
skin and trigger a biological response.
Example 25
Elicitation of Anti-tetC Antibodies in Mice by Topical Application
of Irradiated E. coli Vectors Expressing the Tetanus Toxin
C-Fragment (tetC)
[0209] E. coli cells harboring the plasmid pTET-nir encoding the
tetanus toxin C-fragment were .gamma.-irradiated at a lethal dose
of 20,000 Gy and administered onto prepared mouse skin by topical
application as described above at doses of 10.sup.7 and 10.sup.9
cfu. Non-irradiated cells were administered to the control animals
at a dose of 10.sup.9 cfu. At three weeks post immunization and
three months post immunization, sera were harvested for ELISA-based
anti-tetC analysis.
[0210] Quantitative data is shown in the graph in FIG. 23, where
the open bar is the titer three weeks post-immunization and the
solid bar is titer three months post-immunization. The data shown
are Geometric mean ELISA titers for anti-tetC antibodies, and show
that the potency of irradiated. E. coli vector is comparable to
that of its non-irradiated counterpart. This demonstrates that
animals can be immunized against a specific antigen by topical
application of irradiated, non-replicative E. coli vectors
expressing the protein.
Example 26
Protection of Mice Against Tetanus by Topical Application of
Irradiated E. coli Vectors Expressing the Tetanus Toxin
C-Fragment
[0211] E. coli cells harboring the plasmid pTET-nir encoding the
tetanus toxin C-fragment were .gamma.-irradiated at a lethal dose
of 20,000 Gy and administered onto prepared mouse skin by topical
application as described above at doses of 10.sup.5, 10.sup.7 and
10.sup.9 cfu. Control animals received non-irradiated E. coli cells
at a dose of 10.sup.9 au. Animals were challenged by footpad
injection of a lethal dose of C. tetani cells three months after
immunization. Challenged mice were monitored daily for symptoms of
tetanus and were euthanized at the onset of muscular
fasciculation.
[0212] Quantitative data are depicted in FIG. 24. The data were
plotted as percent survival versus number of days after challenge.
Results show that mice immunized with irradiated vectors at a dose
of 10.sup.9 cfu had a higher percent survival than all other
groups, including those immunized with non-irradiated vectors at a
dose of 10.sup.9 cfu. This again demonstrates that animals can be
protected against a lethal dose of pathogen by topical application
of irradiated bacterial vectors expressing the pathogen's
antigen.
Example 27
Vaccination by Topical Application of Cell-Free Extracts Prepared
by Filtration of Disrupted Cells Expressing a Specific Antigen
[0213] E. coli vectors expressing tetC (E. coli-DH10BpnirB-tetC)
were generated by transforming E. coli DH10B cells with the plasmid
pTET-nir encoding tetC driven by the nirB promoter. Immunization
products were prepared in one of three methods: (1) resuspending
10.sup.10 cfu of live E. coli-DH10BpnirB-tetC cells in phosphate
buffered saline (PBS); (2) resuspending 10.sup.10 cfu of live E.
coli-DH10BpnirB-tetC cells in PBS and subjecting the cells to
sonication in PBS; and (3) resuspending 10.sup.10 cfu of live E.
coli-DH10BpnirB-tetC cells in PBS, subjecting the cells to
sonication in PBS, and preparing cell-free extracts by filtration
of the sonicated E. coli cells. Sonication was conducted with a
Misonix Sonicator 3000 (Labcaire, North Somerset, UK) at output
level 10 using a microtip which was inserted into a tube containing
the resuspended cells on ice in a cold room for 5 min (20 cycles of
15-sec sonication at intervals of 1 min) or 60 min (240 cycles of
15-sec sonication at intervals of 1 min). Cell-free extracts were
prepared by filtering sonicated. E. coli cells through 0.2 .mu.m
pores of a Gelman Acrodisc PF syringe filter.
[0214] One of the above preparations of cells or extracts in PBS
was inoculated onto prepared mice by topical application (NIVS) as
described above; each treatment group contained 10 animals. Animals
were immunized only once. Sera were harvested for ELISA-based
anti-tetC analysis three weeks postimmunization.
[0215] FIG. 25 shows the GMTs for anti-tetC antibodies in each of
the five groups (live cells, cells sonicated for 5 minutes, cell
free extracts prepared from cells sonicated for 5 minutes, cells
sonicated for 60 minutes, and cell free extracts prepared from
cells sonicated for 60 minutes). As shown in the graph, mild
sonication (i.e., 5 min) enhances the immunogenicity of tetC
produced in E. coli cells following topical application. Filtrate
from mildly-sonicated E. coli cells is even more effective in
eliciting an immune response against the antigen than its
non-filtered counterpart. However, extensive sonication (i.e., 60
min) abolishes the immunogenicity of the antigen.
Example 28
Heat-Shock Protein 27 as an Adjuvant for Epicutaneous Vaccines
[0216] 5.times.10.sup.8 pfu AdCMV-tetC (an adenovirus vector
encoding tetC) or 1.times.10.sup.10 cfu E. coli DH10B cells
harboring the plasmid pTET-nir encoding tetC were administered onto
prepared (as described above) mouse skin by topical application as
described with or without mixing with mouse heat-shock protein
(HSP) 27.
[0217] Sera were harvested for ELISA-based anti-tetC analysis six
months postimmunization. FIG. 26 shows the average GMTs (4-5
animals per group) for anti-tetC antibodies for vectors alone
without HSP27, vectors mixed with 1 .mu.g of HSP27 prior to topical
application, and vectors mixed with 3 .mu.g of HSP27 prior to
topical application.
[0218] As shown in FIG. 26, HSP 27 enhances E. coli-vectored
epicutaneous vaccines, whereas suppresses adenovirus-vectored
counterparts.
[0219] The herein examples involving topical administration further
illustrate that one can achieve a suitable response via non-mucosal
administration.
[0220] Thus, the invention includes the application of bacterial
vectors containing one or more genetic inserts that encode an
antigen or epitope of interest or an immune stimulus, or a
gene-product to the skin of an animal, whereby the product(s)
encoded by the inserted gene(s) produce an immunological response
that may be protective or therapeutic against an infectious
disease. The invention further comprehends such bacterial vectors
or gene-product of a bacterial vector incorporated onto, into or
adhered to a matrix, forming a carrier mechanism from which the
products for immunization may be released onto the skin. The
invention yet further includes such embodiments wherein the matrix
into which the product for immunization is incorporated may be
bioactive or inactive and composed of materials which maintain the
integrity of the products for immunization; for instance, the
matrix material may be composed of polymeric substances such as
glucose or other sugars which are biodegradable, or other
biodegradable substances, or materials that are disposable, but may
not be biodegradable.
TABLE-US-00002 TABLE 1 Detection of transgene expression from
genetic vectors delivered by a bandage, the skin was assayed for
luciferase Incubation time (hours) LU per cm.sup.2 skin 1 0 1 2,100
2 0 2 0 2 6,200 2 7,300 2 13,000 2 48,000 2 1,800 2 13,000 18 830
18 2,400 18 260 18 630 18 1,300,000 18 24,000 18 2,700 18 280
AdCMV-luc (an adenovirus vector encoding luciferase) was
administered onto the surface of mouse abdominal skin using a
bandage. The vectored bandage was allowed to cover a restricted
subset of skin for 1, 2, or 18 hours. At the end of each incubation
period, the skin underneath the bandage was resected for luciferase
assay.
TABLE-US-00003 TABLE 2 Summary of AdCMV-PR8.ha DNA relocation
following topical application Time point Ear pinna Abdominal
skin.sup.a Lymph nodes.sup.b Spleen Liver Kidney Blood Muscle.sup.c
I. Nearly full-length HA gene 3 hr 0/2 2/2 0/2 0/2 0/2 012 0/2 0/2
1 day 0/3 2/3 0/3 0/3 0/3 0/3 0/3 0/3 1 month 0/2 0/2 0/2 0/2 0/2
0/2 0/2 0/2 II. Subfragment of HA gene 3 hr 0/2 2/2 0/2 0/2 0/2 0/2
0/2 0/2 1 day 1/3 3/3 3/3 1/3 2/3 2/3 2/3 2/3 1 month 0/2 0/2 0/2
0/2 0/2 0/2 0/2 0/2 III. Nearly full-length fiber gene 3 hr 0/2 2/2
0/2 0/2 0/2 0/2 0/2 0/2 1 day 1/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3 1
month 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 IV. Subfragment of fiber gene
3 hr 0/2 2/2 0/2 0/2 0/2 0/2 012 0/2 1 day 1/3 3/3 0/3 0/3 0/3 0/3
0/3 0/3 1 month 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 Mice were immunized
by topical application of AdCMV-PRS.ha as described in the
foregoing Examples and Figures, e.g., description pertaining to FIG
1. At indicated time points, total DNA was extracted from the
tissues and amplified by PCR using specific primer sets as
described in the foregoing Examples and Figures. The data were
presented as the number of animals containing detectable signals
for a specific tissue per total number of animals analyzed.
.sup.aAdministration site; .sup.bpooled lymph nodes; .sup.chind leg
quadriceps.
TABLE-US-00004 TABLE 3 Summary of pCMV-PR8.ha DNA relocation
following intramuscular injection Time point Ear pinna Abdominal
skin Lymph nodes.sup.a Spleen Liver Kidney Blood Muscle.sup.b I.
Nearly full-length HA gene 3 hr 2/3 0/3 3/3 1/3 0/3 0/3 1/3 3/3 1
day 0/3 0/3 0/3 013 0/3 1/3 0/3 0/3 1 month 0/2 0/2 0/2 0/2 0/2 0/2
0/2 0/2 II. Subfragment of HA gene 3 hr 3/3 1/3 3/3 2/3 3/3 2/3 3/3
3/3 1 day 2/3 1/3 2/3 1/3 3/3 2/3 2/3 3/3 1 month 1/2 1/2 2/2 1/2
1/2 0/2 0/2 1/2 Mice were immunized by intramuscular injection of
pCMV-PR8.ha DNA as described in the foregoing Examples and Figures,
e.g., description pertaining to FIG. 1. At indicated time points,
total DNA was extracted from the tissues and amplified by PCR using
specific primer sets as described the foregoing Examples and
Figures. The data were presented as the number of animals
containing detectable signals for a specific tissue per total
number of animals analyzed. .sup.aPooled lymph nodes; .sup.bhind
leg quadriceps (administration site).
TABLE-US-00005 TABLE 4 Summary of AdCMV-PR8.ha DNA relocation
following, administration of heat- inactivated adenovirus vectors
Time point Ear pinna Abdominal Skin.sup.a Lymph Nodes.sup.b Spleen
Liver Kidney Blood Muscle.sup.c I. Nearly full-length HA gene 1 day
0/3 1/3 0/3 0/3 0/3 0/3 0/3 0/3 (3/7) (7/7) (1/7) (0/7) (0/7) (0/7)
(0/7) (0/7) II. Subfragment of HA gene 1 day 0/3 3/3 0/3 0/3 0/3
0/3 0/3 0/3 (4/7) (7/7) (2/7) (1/7) (1/7) (0/7) (0/7) (0/7) III.
Nearly full-length fiber gene 1 day 0/3 2/3 0/3 013 0/3 0/3 0/3 0/3
(2/7) (6/7) (1/7) (0/7) (1/7) (0/7) (0/7) (0/7) IV. Subfragment of
fiber gene 1 day 0/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3 (2/7) (7/7) (2/7)
(0/7) (2/7) (1/7) (1/7) (0/7) AdCMV-PR8.ha particles were
inactivated by heating at 95.degree. C. for 10 min. Vectors were
administered to mice either by topical application as described in
the foregoing Examples and Figures, e.g., description pertaining to
FIG. 1, or by intradermal injection of an equivalent amount of
vectors using a needle. One day following localized gene delivery,
total DNA was extracted from various tissues. Nearly full-length HA
and fiber genes and their subfragment counterparts were amplified
by PCR using specific primer sets. The data were presented as the
number of animals containing detectable signals for a specific
tissue per total number of animals analyzed. Numbers without
parentheses represent topical application; numbers in parentheses
represent intradermal injection. .sup.aAdministration site;
.sup.bpooled lymph nodes; .sup.chind leg quadriceps.
[0221] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the appended claims is not to be limited by particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope thereof.
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Sequence CWU 1
1
12120DNAArtificial SequencePrimer used to amplify DNA 1atgaaggcaa
acctactggt 20220DNAArtificial SequencePrimer used to amplify DNA
2gatgcatatt ctgcactgca 20320DNAArtificial SequencePrimer used to
amplify DNA 3gtggggtatt catcacccgt 20420DNAArtificial
SequencePrimer used to amplify DNA 4tgcatagcct gatccctgtt
20520DNAArtificial SequencePrimer used to amplify DNA 5gcgccattct
atcctctaga 20620DNAArtificial SequencePrimer used to amplify DNA
6acaatttgga ctttccgccc 20720DNAArtificial SequencePrimer used to
amplify DNA 7gtaccagagt cctttgatcg 20820DNAArtificial
SequencePrimer used to amplify DNA 8ccctcgggtg taatcagaat
20920DNAArtificial SequencePrimer used to amplify DNA 9ccgtctgaag
ataccttcaa 201020DNAArtificial SequencePrimer used to amplify DNA
10accagtccca tgaaaatgac 201120DNAArtificial SequencePrimer used to
amplify DNA 11ggctcctttg catgtaacag 201220DNAArtificial
SequencePrimer used to amplify DNA 12cctactgtaa tggcacctgt 20
* * * * *