U.S. patent application number 13/966631 was filed with the patent office on 2013-12-05 for adipogenic adenoviruses as a biomarker for disease.
This patent application is currently assigned to OBETECH, LLC. The applicant listed for this patent is Richard L. Atkinson. Invention is credited to Richard L. Atkinson.
Application Number | 20130323273 13/966631 |
Document ID | / |
Family ID | 38610014 |
Filed Date | 2013-12-05 |
United States Patent
Application |
20130323273 |
Kind Code |
A1 |
Atkinson; Richard L. |
December 5, 2013 |
Adipogenic Adenoviruses as a Biomarker for Disease
Abstract
A vaccine composition may be administered to a subject to
increase immunity to an adipogenic adenovirus-related cancer. The
vaccine composition stimulates the production of adipogenic
adenovirus neutralizing antibodies in the subject, which may
increase immunity to the adipogenic adenovirus-related cancer in
the subject.
Inventors: |
Atkinson; Richard L.;
(Mechanicsville, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Atkinson; Richard L. |
Mechanicsville |
VA |
US |
|
|
Assignee: |
OBETECH, LLC
Richmond
VA
|
Family ID: |
38610014 |
Appl. No.: |
13/966631 |
Filed: |
August 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13022219 |
Feb 7, 2011 |
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13966631 |
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12257967 |
Oct 24, 2008 |
7910310 |
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13022219 |
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11616799 |
Dec 27, 2006 |
7442511 |
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12257967 |
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60753402 |
Dec 27, 2005 |
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Current U.S.
Class: |
424/186.1 ;
424/233.1 |
Current CPC
Class: |
G01N 33/574 20130101;
C12N 2710/10022 20130101; A61P 11/00 20180101; A61P 3/10 20180101;
A61K 39/235 20130101; A61P 37/04 20180101; A61P 31/20 20180101;
A61K 39/00 20130101; C12Q 1/701 20130101; G01N 2333/075 20130101;
C12N 2710/10322 20130101; A61P 5/38 20180101; A61P 5/48 20180101;
A61P 25/28 20180101; C12N 7/00 20130101; A61P 1/18 20180101; C07K
14/005 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/186.1 ;
424/233.1 |
International
Class: |
A61K 39/235 20060101
A61K039/235; C07K 14/005 20060101 C07K014/005 |
Claims
1. A method of increasing immunity to an adipogenic
adenovirus-related cancer in a subject, said method comprising the
steps of: administering a vaccine composition including a
pharmaceutically effective dose of an active ingredient selected
from the group consisting of a killed adenovirus type 36, an
inactivated adenovirus type 36, a protein or peptide sequence
encoding an adenovirus type 36 coat protein or fragment thereof,
and an adenovirus type 36 E1A protein, to the subject; and
stimulating the production of adipogenic adenovirus neutralizing
antibodies in the subject such that the adipogenic adenovirus
neutralizing antibodies increase immunity to the adipogenic
adenovirus-related cancer in the subject.
2. The method of claim 1, wherein the vaccine composition in said
administering step is administered one or more of intranasally,
orally, intravenously, intramuscularly, subcutaneously, and
peritoneally.
3. The method of claim 1, wherein the subject is a human.
4. The method of claim 1, wherein the subject is a non-human
animal.
5. The method of claim 1, wherein the administration of the vaccine
composition is a primary vaccination.
6. The method of claim 5, further comprising the step of
administering at least one booster vaccination after the primary
vaccination to maintain protective immunity against adipogenic
adenovirus in the vaccinated subject.
7. The method of claim 1, wherein the active ingredient has a
concentration in the range of about 1 ng/ml to about 1 mg/ml.
8. The method of claim 1, wherein the vaccine composition is a
solution having a volume in the range of about 0.1 ml to about 10
ml and wherein the active ingredient is inactivated adipogenic
adenovirus in an amount in the range of 1 ng/ml to about 1
mg/ml.
9. The method of claim 1, wherein the vaccine composition is a
solution having a volume in the range of about 0.1 ml to about 10
ml and wherein the active ingredient is an adenovirus type 36 coat
protein or fragment thereof in an amount in the range of about 1
ng/ml to about 1 mg/ml.
10. The method of claim 9, wherein the adenovirus type 36 coat
protein fragment is a peptide having a length of about 6 amino
acids to about 30 amino acids.
11. The method of claim 9, wherein the adenovirus type 36 coat
protein is an adenovirus type 36 fiber protein or an adenovirus
type 36 fiber protein fragment thereof encoded by a nucleic acid
selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and SEQ ID
No. 7.
12. The method of claim 1, wherein the adipogenic adenovirus is
adenovirus type 36.
13. The method of claim 1, wherein the cancer is selected from the
group consisting of breast cancer, prostate cancer, uterine cancer,
ovarian cancer, colon cancer, kinder cancer, pancreatic cancer, and
lung cancer.
14. The method of claim 1, wherein the cancer is breast cancer.
15. The method of claim 1, wherein the cancer is prostate
cancer.
16. The method of claim 1, wherein cancer cells in the subject
overexpress fatty acid synthetase (FAS) compared to non-cancer
cells from the patient.
17. The method of claim 1, further comprising screening the subject
for the presence of an adipogenic adenovirus prior to the
administering.
18. The method of claim 1, further comprising screening the subject
for the presence of an adipogenic adenovirus after the
administering.
19. The method of claim 1, wherein the subject has cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
13/022,219, filed Feb. 7, 2011, which is divisional application of
application Ser. No. 12/257,967, filed Oct. 24, 2008, which is a
divisional of application Ser. No. 11/616,799, filed Dec. 27, 2006,
which in turn is related to and claims benefit under 35 U.S.C.
.sctn.119(e) to Provisional Application Ser. No. 60/753,402, filed
Dec. 27, 2005, the disclosures of which are herein incorporated in
their entirety herein.
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED AS AN ASCII TEST
[0002] The Sequence Listing written in file 91375-884433 Sequence
Listing, created on Aug. 13, 2013, 2,675 bytes, machine format
IBM-PC, MS-Windows operating system, is hereby incorporated by
reference in its entirety for all purposes.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention relates to the relationship between infection
with adipogenic adenoviruses, such as, for example, adenovirus-36
(Ad-36), and the etiologies of obesity and obesity-related cancers
and other diseases. More specifically, the invention relates to
preventing obesity-related disease in a subject by administering a
vaccine composition including a pharmaceutically effective dose of
an active ingredient selected from the group consisting of a killed
adenovirus type 36, an inactivated adenovirus type 36, a protein or
peptide sequence encoding an adenovirus type 36 coat protein or
fragment thereof, and an adenovirus type 36 E1A protein,
stimulating the production of adipogenic adenovirus neutralizing
antibodies in the subject.
[0005] 2. Related Art
[0006] There has been a dramatic simultaneous increase in the
prevalence of obesity and of certain types of cancer. A worldwide
epidemic of obesity accelerated dramatically starting about 1980.
In the USA the prevalence of obesity in adults more than doubled in
the 20 years from 1980 to 2000 (from 15% to 31%), whereas the
prevalence increased only slightly in the prior 20 years from 1960
to 1980 (from 13.5% to 15%). The prevalence of obesity in children
tripled from about 1970 to 2000. Likewise, cancers of the breast,
prostate, colon, and liver have also rapidly increased in
prevalence in recent years.
[0007] Changes in reproductive hormones in obesity have been
suggested to play a role in the association of breast and prostate
cancer (among others) and in the aggressiveness of these cancers.
However, changes in reproductive hormones cannot explain cancers
such as colon, renal, or pancreatic cancer that are not under
hormonal control.
[0008] Another possibility for the link between obesity and cancer
is the decreased immune function seen in obese individuals. Obese
people have a lower antibody response to vaccination with hepatitis
B than vaccine people who are not obese. The immune system is
critical in inhibiting the growth of neoplasms, so it would not be
surprising if this were the mechanism of increased cancers of many
types in obesity. However, adenoviruses are well known to decrease
immune function as a way to enhance their replication within the
host, including human hosts. More relevant, the SMAM-1 avian
adenovirus, which has been reported to cause obesity, had a major
impact by decreasing immune function of chickens. Thus, there is a
direct link between an adenovirus that causes obesity and that also
impairs immune function. The inventor has reported that SMAM-1 is
associated with obesity in humans, adding a link from adenovirus to
human obesity.
[0009] Some human adenovirus serotypes are known to be oncogenic,
and induce tumors in rats or hamsters. Adenovirus serotypes are
divided by Groups. Group A adenoviruses (e.g., Ad-12, Ad-18) are
highly oncogenic, producing tumors in most animals within 4 months;
group B adenoviruses (e.g., Ad-3, Ad-7) are weakly oncogenic,
inducing tumors in most animals within 4 to 18 months; group D
viruses are thought to be less oncogenic, but serotype-9
efficiently induces mammary tumors within 3 to 5 months. There has
been extensive work on adenovirus-induced cancer, but to date,
there has been no evidence that adenoviruses cause human
cancer.
[0010] If adenoviruses cause human cancers, it is likely that they
do so by altering expression of genes in the host that allow
unregulated cell growth to occur. Many such tumor markers have been
identified. Some are due to genetic variants. Hereditary breast
cancer has been linked to germline mutations in one allele of high
penetrance susceptibility genes such as BRCA1, BRCA2, CHEK 2, TP53
or PTEN. It is possible that adenovirus infections facilitate
cancer in these genetically susceptible individuals. However,
adenoviruses may contribute to spontaneous oncogenesis by inducing
expression of various oncogenes or suppressing expression of tumor
suppressor genes of the host. Among the alterations due to
adenoviruses that are thought to contribute to cancer are changes
in DNA-dependent protein kinase, fatty acid binding protein, mTOR,
p16, p53, PDZ protein, phosphatidylinositol 3-kinase, PML,
thymidine kinase, and Zip kinase. Of particular interest are those
tumor related factors that are altered by the adenovirus E4 region,
and specifically the E4orf1 gene. The E4 oncogenes include
DNA-dependent protein kinase, p53, PDZ protein,
phosphatidylinositol 3-kinase, PML, thymidine kinase, and Zip
kinase. As will be described below, the Ad-36 E4orf1 gene has been
shown to be involved in producing obesity by a direct effect on
adipocyte metabolism. The E4orf1 region of human adenovirus-5 has
been shown to be an oncogene, and a recent paper reported that Ad-5
produces obesity in mice. These findings show a direct link between
obesity and cancer, with both being due to a human adenovirus, and
provide the likely mechanism via a viral gene.
[0011] The general consensus of many investigators in the field of
cancer research has been that adenoviruses do not contribute to the
etiology of human cancer. Therefore, it would be a major
technological advance to demonstrate that adipogenic adenoviruses,
such as Ad-36, are associated with obesity-related cancers, such as
breast and prostate cancers.
BRIEF SUMMARY OF THE INVENTION
[0012] The invention provides a method of increasing immunity to an
adipogenic adenovirus-related cancer in a subject by administering
to the subject a vaccine composition including a pharmaceutically
effective dose of an active ingredient selected from the group
consisting of a killed adenovirus type 36, an inactivated
adenovirus type 36, a protein or peptide sequence encoding an
adenovirus type 36 coat protein or fragment thereof, and an
adenovirus type 36 E1A protein. The vaccine composition stimulates
the production of adipogenic adenovirus neutralizing antibodies in
the subject such that the adipogenic adenovirus neutralizing
antibodies increase immunity to the adipogenic adenovirus-related
cancer in the subject. The invention may be implemented in a number
of ways.
[0013] In one aspect, the vaccine composition is administered one
or more of intranasally, orally, intravenously, intramuscularly,
subcutaneously, and peritoneally.
[0014] In one aspect, the subject is a human. In another aspect,
the subject is a non-human animal.
[0015] In some embodiments, the administration of the vaccine
composition is a primary vaccination. In another aspect, the method
further comprises the step of administering at least one booster
vaccination after the primary vaccination to maintain protective
immunity against adipogenic adenovirus in the vaccinated
subject.
[0016] In one aspect, the active ingredient has a concentration in
the range of about 1 ng/ml to about 1 mg/ml.
[0017] In another aspect, the vaccine composition is a solution
having a volume in the range of about 0.1 ml to about 10 ml and the
active ingredient is inactivated adipogenic adenovirus in an amount
in the range of 1 ng/ml to about 1 mg/ml.
[0018] In yet another aspect, the vaccine composition is a solution
having a volume in the range of about 0.1 ml to about 10 ml and the
active ingredient is an adenovirus type 36 coat protein or fragment
thereof in an amount in the range of about 1 ng/ml to about 1
mg/ml.
[0019] In some embodiments, the adenovirus type 36 coat protein
fragment is a peptide having a length of about 6 amino acids to
about 30 amino acids. In other embodiments, the adenovirus type 36
coat protein is an adenovirus type 36 fiber protein or an
adenovirus type 36 fiber protein fragment encoded by a nucleic acid
selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2,
SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and SEQ ID
No. 7.
[0020] In one aspect, the adipogenic adenovirus is adenovirus type
36.
[0021] In another aspect, the cancer is selected from the group
consisting of breast cancer, prostate cancer, uterine cancer,
ovarian cancer, colon cancer, kinder cancer, pancreatic cancer, and
lung cancer. In some embodiments, the cancer is breast cancer. In
other embodiments, the cancer is prostate cancer.
[0022] In one aspect, the cancer cells in the subject overexpress
fatty acid synthetase (FAS) compared to non-cancer cells from the
patient.
[0023] In some embodiments aspect, the method further comprises
screening the subject for the presence of an adipogenic adenovirus
prior to the administering. In other embodiments, the method
further comprising screening the subject for the presence of an
adipogenic adenovirus after the administering.
[0024] Additional features, advantages, and embodiments of the
invention may be set forth or apparent from consideration of the
following detailed description, drawings, and claims. Moreover, it
is to be understood that both the foregoing summary of the
invention and the following detailed description are exemplary and
intended to provide further explanation without limiting the scope
of the invention as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The accompanying drawings, which are included to provide a
further understanding of the invention, are incorporated in and
constitute a part of this specification, illustrate embodiments of
the invention and together with the detailed description serve to
explain the principles of the invention. No attempt is made to show
structural details of the invention in more detail than may be
necessary for a fundamental understanding of the invention and
various ways in which it may be practiced.
[0026] FIG. 1 is a BODIPY staining of 3T3-L1 cells 5 days post MDI
treatment. This figure shows triglycerides in 3T3-L1 cells infected
with Ad-36 versus uninfected in vitro. Control cells show a
moderate BODIPY fat stain whereas Ad-36 infected cells have about
twice as much triglyceride, showing adipocyte biochemistry has
changed.
[0027] FIG. 2 is a graph showing the effects of spontaneous
infection with Ad-36 in ad libitum fed rhesus monkeys. As compared
to the period before a monkey became infected, once infection was
noted (designated .uparw.) there was a steady rise in body weight
that was still rising at about 18 months.
[0028] FIG. 3 is a graph showing an increase in body weight in
monkeys infected with Ad-36. As compared to uninfected monkeys,
infected monkeys gained about four times as much weight in about
seven months.
[0029] FIG. 4 is a graph showing a decrease in serum cholesterol in
Ad-36 infected marmoset monkeys. Serum cholesterol began dropping
immediately after infection and by about 10 weeks. This is
significantly different than baseline in infected monkeys. There is
no change in uninfected monkeys.
[0030] FIG. 5 is a gel showing the presence of Ad-36 DNA in adipose
tissue of infected marmosets using a nested PCR assay. Lane 1 is
Ad-36 DNA, lanes 2-4 show no Ad-36 DNA in fat of uninfected
monkeys, and lanes 5-7 shows the presence of Ad-36 DNA in all
infected monkeys.
[0031] FIG. 6 is gel showing Ad-36 DNA in liver (upper lanes) and
muscle tissue (lower lanes) of infected marmosets using nested PCR
assay. Ad-36 DNA from infected monkeys is seen in lanes 7-9 of
liver tissue and muscle tissue and is not present in lanes 4-6 from
uninfected monkeys. Lane 2 is Ad-36 from culture, lane 3 is
marker.
[0032] FIG. 7 is a gel showing Ad-36 DNA in brain and muscle tissue
of infected animals by nested PCR assay. Upper lanes are brain
tissue from marmosets, lower lanes are muscle tissue. Ad-36 DNA
from infected marmosets is seen clearly in lanes 8-9 and more
faintly in lane 7. Ad-36 DNA is not present in lanes 4-6 from
non-infected marmosets. Lane 2 is positive control from Ad-36
culture.
[0033] FIG. 8 is a gel showing Ad-36 DNA in adipose tissue of
humans using nested PCR assay (bottom lanes). This gel shows Ad-36
DNA in adipose tissue of marmosets in the upper lanes (repeat of
data in FIG. 5: lanes 4-6 negative from control marmosets, lanes
7-9 Ad-36 DNA from infected marmosets). In bottom lanes Ad-36 DNA
is seen in 2 of 6 samples of human visceral adipose tissue obtained
from cadavers at autopsy (lane 2 is positive control, lanes 6-7 are
Ad-36 positive, lanes 4,5,8,9 are negative).
[0034] FIG. 9 is a chart showing the effects of multiple human
adenoviruses on total body fat in chickens. Ad-2 and Ad-31 did not
increase body fat, but Ad-37 had a marked effect.
[0035] FIG. 10 is a chart showing the effects of multiple human
adenoviruses on visceral fat in chickens. Ad-2 and Ad-31 did not
increase visceral fat, but Ad-37 had a marked effect.
[0036] FIG. 11 is a chart showing food intake in animals exposed to
multiple human adenoviruses. There were no differences in
cumulative food intake among groups, yet individuals infected with
Ad-37 became obese while individuals infected with Ad-2 or Ad-31
were not.
[0037] FIG. 12 is a table showing human Ad-5 produced obesity in
mice. Also, body fat increased by almost 3 fold.
[0038] FIG. 13 is a table showing the presence of serum antibodies
to human Ad-36 in people from three US cities. An average of about
30% of obese people were infected with As-36 versus about 11% of
non-obese who were not infected with Ad-36.
[0039] FIG. 14 is a chart showing body mass index in 502
individuals from three US cities according to status of infection
with Ad-36. Overall, BMI was about 9 units higher in infected
versus uninfected people (p<0.0001). Infected individuals of
both obese and non-obese groups were significantly heavier than the
uninfected in each group.
[0040] FIG. 15 is a table showing a comparison of twin pairs
discordant for infection with Ad-36. Out of 89 twin pairs, 26 were
discordant. The twins infected with Ad-36 were heavier and fatter
than their uninfected co-twins.
[0041] FIG. 16 is a gel showing the presence of Ad-36 DNA in
prostate cancer tissue.
[0042] FIG. 17 is a gel showing the presence of Ad-36 DNA in
monkeys.
DETAILED DESCRIPTION OF THE INVENTION
[0043] It is understood that the present invention is not limited
to the particular methodology, protocols, devices, apparatus,
materials, and reagents, etc., described herein, as these may vary.
It is also to be understood that the terminology used herein is
used for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention. It
must be noted that as used herein and in the appended claims, the
singular forms "a," "an," and "the" include plural reference unless
the context clearly dictates otherwise. Thus, for example, a
reference to "a virus particle" is a reference to one or more virus
particles and equivalents thereof known to those skilled in the art
and so forth.
[0044] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Preferred methods, devices, and materials are described, although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention. All references cited herein are incorporated by
reference herein in their entirety.
[0045] Any numerical values recited herein include all values from
the lower value to the upper value in increments of one unit
provided that there is a separation of at least two units between
any lower value and any higher value. As an example, if it is
stated that the concentration of a component or value of a process
variable such as, for example, dosage, dilution, and the like, is,
for example, from 1 to 90, specifically from 20 to 80, more
specifically from 30 to 70, it is intended that values such as 15
to 85, 22 to 68, 43 to 51, 30 to 32 etc., are expressly enumerated
in this specification. For values which are less than one, one unit
is considered to be 0.0001, 0.001, 0.01 or 0.1 as appropriate.
These are only examples of what is specifically intended and all
possible combinations of numerical values between the lowest value
and the highest value enumerated are to be considered to be
expressly stated in this application in a similar manner.
[0046] Moreover, provided immediately below is a "Definition"
section, where certain terms related to the invention are defined
specifically for clarity, but all of the definitions are consistent
with how a skilled artisan would understand these terms. Particular
methods, devices, and materials are described, although any methods
and materials similar or equivalent to those described herein can
be used in the practice or testing of the invention.
DEFINITIONS
[0047] Ad-2 is adenovirus type 2.
[0048] Ad-5 is adenovirus type 5.
[0049] Ad-31 is adenovirus type 31.
[0050] Ad-36 is adenovirus type 36.
[0051] Ad-37 is adenovirus type 37.
[0052] BMI is body mass index.
[0053] FAS is fatty acid synthetase.
[0054] PPAR is peroxisome proliferator activated receptors.
[0055] CEBP is CCAAT-enhancer binding protein.
[0056] The term "adipogenic adenovirus" as used herein generally
refers to adenoviruses that are capable of stimulating increase
lipid production in cells, tissues, and/or organs by turning on the
cellular machinery in infected hosts to turn on the host's
production of lipogenic enzymes which then produce excess fatty
acids and promote fat storage with the infected cells. The
adipogenic adenoviruses include without limitation Ad-5, Ad-36, and
Ad-37.
[0057] The term "BMI" as used herein generally refers to a
statistical measure of the weight of a person scaled according to
height. BMI may be defined as the individual's body weight divided
by the square of the height and may be expressed in the unit
kg/m.sup.2. BMI may be used as a screening tool to identify
possible weight problems for adults and children. However, in order
to determine if excess weight is a health risk, a healthcare
provider may need to perform further assessments, such as skinfold
thickness measurements, evaluations of dies, physical activity,
family history, hip to waist ratio, infection with a adipogenic
adenovirus, and other appropriate health screenings. For adults 20
years old and older, BMI may be interpreted using standard weight
status categories that are the same for all ages and for both men
and women. Alternatively, for children and teens, the
interpretation of BMI is both age- and sex-specific. For example,
an adult having a (i) a BMI less than about 18.5 percent mat be
considered underweight, (ii) a BMI is the range of about 18.5 to
about 24.9 may be considered normal weight, (iii) a BMI in the
range of about 25 to about 29.9 may be considered overweight, and
(iv) a BMI greater than about 30.0 may be considered obese.
[0058] The term "hip to waist ratio" refers to a measurement that
may be used to help determine obesity. The distribution of fat is
evaluated by dividing the waist size by the hip size. For example,
an individual with about a 30 inch waist and about a 40 inch hip
size would have a ratio of about 0.75 and an individual with about
a 41 inch waist size and about a 39 inch hip size would have a
ratio of about 1.05. The higher the ratio, the higher the risk of
heart disease and other obesity-related disorders.
[0059] A "biological sample" refers to a sample of tissue or fluid
from a human or animal including, but not limited to plasma, serum,
spinal fluid, lymph fluid, the external sections of the skin,
respiratory, intestinal and genitourinary tracts, tears, saliva,
blood cells, tumors, organs, tissue and sample of in vitro cell
culture constituents.
[0060] An "isolated" or "substantially pure," nucleic acid (e.g.,
DNA, RNA, or a mixed polymer) for example, is one which is
substantially separated from other cellular components which
naturally accompany a native human or animal sequence or protein,
e.g., ribosomes, polymerases, many other human or animal genome
sequences and proteins. The term embraces a nucleic acid sequence
or protein which has been removed from its naturally occurring
environment, and includes recombinant or cloned DNA isolates and
chemically synthesized analogs or analogs biologically synthesized
by heterologous systems.
[0061] The term "immunogenic," generally refers to an anti-obesity
vaccine that has the capability to provoke in an immunized animal,
an immune response that yields neutralizing antibodies against an
obesity-causing, live virus that might infect the person after
administration of the vaccine.
[0062] The term "antibody" refers to antibodies, digestion
fragments, specified portions and variants thereof, including
antibody mimetics or comprising portions of antibodies that mimic
the structure and/or function of an antibody or specified fragment
or portion thereof, including single chain antibodies and fragments
thereof. The invention encompasses antibodies and antibody
fragments capable of binding to a biological molecule (such as an
antigen or receptor), such as the fiber coat protein of
adenoviruses, and specifically, Ad-36, or portions thereof.
[0063] The term "nucleic acid sequence," includes an
oligonucleotide, nucleotide, or polynucleotide, and fragments
thereof, and to DNA or RNA of genomic or synthetic origin which may
be single- or double-stranded, and represent the sense or antisense
strand, to peptide nucleic acid (PNA), or to any DNA-like or
RNA-like material, natural or synthetic in origin.
[0064] Fragments: include any portion of a heterologous peptide or
nucleic acid sequence. Heterologous peptide fragments retain at
least one structural or functional characteristic of the subject
heterologous polypeptides. Nucleic acid sequence fragments are
greater than about 60 nucleotides in length, and most preferably
includes fragments that are at least about 100 nucleotides, at
least about 1000 nucleotides, and at least about 10,000 nucleotides
in length.
[0065] Complementary or complementarity: as used herein, include
the natural binding of polynucleotides under permissive salt and
temperature conditions by base-pairing. For example, the sequence
"A-G-T" binds to the complementary sequence "T-C-A."
[0066] Complementarity between two single-stranded molecules may be
"partial," in which only some of the nucleic acids bind, or it may
be complete when total complementarity exists between the single
stranded molecules. The degree of complementarity between nucleic
acid strands has significant effects on the efficiency and strength
of hybridization between nucleic acid strands. This is of
particular importance in amplification reactions, which depend upon
binding between nucleic acids strands and in the design and use of
molecules.
[0067] Functional equivalent: a protein or nucleic acid molecule
that possesses functional or structural characteristics that are
substantially similar to a heterologous protein, polypeptide,
enzyme, or nucleic acid. A functional equivalent of a protein may
contain modifications depending on the necessity of such
modifications for the performance of a specific function. The term
"functional equivalent" is intended to include the "fragments,"
"mutants," "hybrids," "variants," "analogs," or "chemical
derivatives" of a molecule.
[0068] Protein purification: broadly defined, any process by which
proteins are separated from other elements or compounds on the
basis of charge, molecular size, or binding affinity.
[0069] Substantially purified: as used herein, includes nucleic or
amino acid sequences that are removed from their natural
environment, isolated or separated, and are at least 60% free,
preferably at least 75% free, and most preferably at least 90% free
from other components with which they are naturally associated.
[0070] Inhibition: as used herein, refers to a reduction in the
parameter being measured, whether it be adenovirus type 36 growth
or viability. The amount of such reduction is measured relative to
a standard (control). "Reduction" is defined herein as a decrease
of at least around 25% relative to control, preferably at least
around 50%, and most preferably of at least around 75%.
[0071] The anti-adipogenic adenovirus vaccines of the invention,
wherein the immunogenic component is live, inactivated virus,
killed virus, coat protein per se, epitope-comprising coat protein
segment, or coat protein (or epitope-comprising segment thereof)
provided with use of a non-pathogenic, genetically modified carrier
virus such as a vaccinia virus or a fowl pox virus, are prepared
using methods well known in the art. Thus, the vaccines will
include carriers, excipients, adjuvants, antimicrobials,
preservatives and the like as well understood in the art. Thus, in
addition to the active ingredient, the vaccines will have suitable
compositions, usually aqueous buffers, such as phosphate-buffered
saline or the like, in which the active ingredient will be
suspended along with, optionally, any of various immune-system
stimulating adjuvants used in human vaccine preparations,
antimicrobial compositions, and other compositions to stabilize the
preparations. All compositions included with the vaccine
preparation will be suitable for administration to humans. The
vaccine preparation may be stored in lyophilized form and then
combined with solution soon before administration. For oral
administration, the vaccine preparation may be in solution, tablet
or pill form optionally with an enteric coating as understood in
the art. The concentration of active (immunogenic or
immunogen-providing) component in solution with which it is
administered typically will be between about 1 ng and about 1
mg/ml.
[0072] The anti-adipogenic adenovirus vaccines of the invention
will be administered intranasally, orally, or by injection
intravenously, intramuscularly, subcutaneously or peritoneally.
Administration of the vaccines of the invention is to be under the
guidance of a physician.
[0073] Appropriate dosing of the anti-adipogenic adenovirus vaccine
is well within the skill of medical practitioners and will depend
on a number of factors including the age of the person being
treated, the urgency of the person's developing protective
immunity, the status of the person's immune system, and other
factors known to the skilled. The vaccine typically will be
administered in several steps in order to cause and maintain
protective immunity against obesity-causing virus in the person
being vaccinated. Thus, after the primary vaccination, there
typically will be between one and about ten booster vaccinations
separated by periods between about 1 week and 10 years.
[0074] A single dose of an anti-obesity vaccine of the invention
(in solution form) will have a volume of between about 0.1 ml and
10 ml and, in any form, will have between about 1 ng and 10 mg of
killed or inactivated adipogenic virus, between about 1 ng and 10
mg of genetically modified, non-pathogenic virus, or between about
1 ng and 10 mg of coat protein (e.g., fiber protein) or 6-30 amino
acid peptide (in its form as modified to be immunogenic).
[0075] An anti-adipogenic adenovirus vaccine of the invention,
wherein the active ingredient is nucleic acid, will also be a
standard preparation for vaccines of that type. With vaccines of
this type, the nucleic acid is not the immunogen but is expressed
in vivo after administration of the vaccine as a peptide or protein
which in turn is immunogenic. Vaccines of this type will be
administered by techniques known in the art for such vaccines
(e.g., intramuscular injection). Dosing will also be according to
procedures known in the art to cause and maintain protective
immunity against viral obesity in the vaccinated individual.
[0076] Note that an anti-adipogenic adenovirus vaccine according to
the invention may include active ingredients based on more than one
adipogenic virus (or the coat protein (e.g. fiber protein) or
epitopic segments of the coat protein thereof).
[0077] In yet another aspect, the invention is a method of
preventing adipogenic adenovirus related disease caused by a virus
in a human susceptible thereto which comprises administering to the
human an amount of an anti-adipogenic adenovirus vaccine of the
invention that is effective to raise and maintain a protective
immune response against an adipogenic adenovirus.
[0078] This invention generally relates to the relationship between
infection with human adipogenic adenoviruses, such as Ad-5, Ad-36,
and Ad-37, the etiologies of obesity, and the dysfunction of cells,
tissues and/or organs, such as cancers, dysfunction of the
pancreas, dysfunction of skeletal and cardiovascular muscle,
pulmonary dysfunction, dysfunction of the brain and nervous system,
and adrenal disease and dysfunction. Moreover, the invention
relates to utilizing adipogenic adenovirus status as a predictor
for disease status, prognosis, treatment outcome, and prevention of
infection and disease.
[0079] The main mechanism of adipogenic adenovirus-related disease
is the production of lipids within cells. The adipogenic
adenoviruses are capable of changing the cellular machinery in
infected subjects to turn on the host's production of lipogenic
enzymes. As a result, the lipogenic enzymes make excess fatty acids
and promote fat storage within the cells of multiple organs.
[0080] Multiple cells of the body are known to have the capacity to
make fatty acids within their cells. Fatty acid synthetase (FAS) is
one of the most important lipogenic enzymes and is expressed in a
number of adult and fetal cells, which suggests that these tissues
are capable of producing fatty acids. For example, FAS is expressed
in adult cells such as epithelial cells of the duodenum and
stomach, hemopoietic cells, appendix, ganglion cells of alimentary
tract, hepatocytes, mast cells, seminal vesicle, umbrella cells of
urinary bladder, adrenal zona fasciculate cells, adipocytes,
anterior pituitary cells, basket cells of cerebellum, cerebral
cortical neurons, deciduas, decidualized stromal cells of
endometrium, epithelial cells of apocrine gland, duct and acinus of
breast, prostate, and sebaceous gland, letein cells, and Type II
alveolar cells of lung.
[0081] Additionally, FAS is also expressed in fetal cells such as
anterior pituitary cells, chondrocytes of tracheobronchial wall,
endothelium of blood vessels and heart, epithelial cells of
bronchus, esphagogastrointestinal tract, lung, pancreas, prostate,
thyroid, tongue, trachea, proximal tubules of kidney, fibroblasts,
nodal lymphocytes, neuroblasts in adrenal medulla, thymocytes,
striated myocytes of tongue, epithelial cells of salivary glans and
tracheobronchial glands, hemopoietic cells, heptocytes, Lanhans
cells of chorionic villi, osteoblasts, perivertebral fibroblastic
cells, Schwann cells of sympathetic ganglion and Auerbach plexus,
subcapsular cells of adrenal, adipocytes, Leidig cells of testis,
mast cells, uroepithelium of urinary tract, and adrenocortical
cells of upper layer.
[0082] While fatty acids are critical to the intracellular milieu
for many biochemical processes, excess fatty acids within the cells
profoundly alter cellular biochemistry. Excess fatty acids within
cells may lead to abnormal functioning of a number of intracellular
processes. For example, excess fatty acids in pancreatic tissue
reduce insulin secretion, and fatty acids in cancer cells provide
the major source of energy for cancer growth. Moreover, fatty acids
within the lung cells stimulate attraction of macrophages to the
area and are associated with asthma and emphysema. Therefore,
adverse results occur when cells are exposed to excess to fatty
acids.
[0083] According to one embodiment. adipogenic adenoviruses may be
a mechanism by which excess fatty acids and triglycerides are
produced in cells and tissues. Studies have demonstrated that
adipogenic adenoviruses can alter lipogenic enzymes in adipose
tissue and in liver. For example, other studies have shown that
infection with the adipogenic adenovirus, Ad-36, stimulates the
rapid appearance of differentiation factors including
glycerol-3-phospodehydrogenase, PPAR-gamma, CEBP-alpha and beta,
and lipoprotein lipase in 3T3-L1 cells. Since multiple tissues
accumulate adipogenic adenoviral DNA, excess lipogenic enzymes may
be present in all infected tissues that are capable of producing
these enzymes.
[0084] According to another embodiment of the invention, adipogenic
adenovirus infection may be used to determine the presence of an
obesity-related cancer. In a further embodiment, adipogenic
adenovirus infection may be used to determine whether a subject is
predisposed to developing an obesity-related cancer.
[0085] The prevalence of multiple types of cancers is increased in
obese people. These cancers include without limitation breast,
prostate, uterus, ovary, colon, kidney, pancreas and lung.
Hepatocellular cancer may also be linked to obesity and the
metabolic syndrome. Additionally, obesity may be linked to the
aggressiveness of some types of cancer and to a poorer prognosis.
For example, obesity may be associated with a higher grade of
prostate cancer and higher recurrence rates after radical
prostatectomy. Also, in non-Hispanic White women, breast tumor size
correlates with obesity. Specifically, this is most notable for the
highest quartile of waist circumference where the odds ration is
2.76.
[0086] Studies have shown that fatty acids are the substrate for
energy expenditure in cancer cells and that blocking FAS can block
cancer growth. Moreover, it has been shown that FAS is increased in
many obesity-related cancers and that FAS favors tumor growth.
Adipogenic adenovirus infection in cancer cells may stimulate fatty
acids in cell thereby promoting cancer growth by providing an
energy source for the cancer cells.
[0087] FAS expression has been detected in various tumors and
associated with histological subtype, histopathological grade and
tumor aggressiveness. Studies have demonstrated that FAS is highly
expressed in human neoplasm such as breast, prostate, ovarian,
colorectal, and endometrial cancers. It has been determined that
FAS (OA-519) was a predictor of prostate cancer. It was observed
that OA-519 immunoreactivity was seen in 56 (57%) of the 99 primary
prostate cancers examined. OA-519 cancers were more likely to
progress than the non-OA-519 cancers.
[0088] Indeed, the presence of Ad-36 was confirmed in 61% of human
prostate cancer tissues and it was observed that 50% and 51% of
breast and prostate cancer patients, respectively, have antibodies
to Ad-36 in comparison to 13.8% of the normal population.
[0089] Therefore, the presence of FAS in prostate cancer tissue in
approximately the same number of cancer patients as were observed
to be infected with Ad-36 in combination with the findings that
adipogenic adenoviruses increase FAS in cells and that inhibition
of FAS may inhibit cancer, suggest that infection of an adipogenic
adenovirus cause both obesity and obesity-related cancer in a
subject.
[0090] According to an embodiment of the invention, the prevalence
of Ad-36 infection may be statistically higher in patients with
breast and prostate cancer than in individuals without cancer. In a
further embodiment, Ad-36 status may serve as a marker for breast
and prostate cancer. Yet a further embodiment relates to patients
with breast or prostate cancer who have Ad-36 DNA in their tissues
will have more aggressive cancer and/or a poorer prognosis than
those without Ad-36 DNA.
[0091] According to another embodiment of the invention, adipogenic
adenovirus infection may be used as a predictor for disease
prognosis, treatment prognosis, or as a predictor of tumor
aggressiveness. In breast, prostate and ovarian carcinomas, high
levels of FAS are often associated with poor prognosis. The
association between FAS expression and tumor size or a marker of
proliferative activity of tumor cells may suggest that FAS is
related to growth and proliferation of these malignant tumors.
[0092] For example, studies have shown that overexpression of FAS
increased heptacarcinogenesis in rat. Other studies have shown that
about 30% of patients with early breast cancers expressed FAS. The
patients with breast cancer expressing FAS had significantly higher
tumor grade, larger tumor volume, advancing clinical stage, and
Gleason's score, one of the most powerful predictors. FAS
expression has also been studied in ovarian neoplasia, where it was
seen to be associated with histological tumor grade and shorter
survival. Moreover, studies have shown that overweight and obese
individuals had significantly increased mortality from multiple
types of cancer in humans.
[0093] FAS is associated with colon cancer and with its severity.
Studies have shown that only 2 (5%) of 43 adenomas with low-grade
dysplasia showed reactivity for FAS. However, positive FAS
immunostaining was seen in 7 (17%) of 40 cases of adenomas with
moderate-grade dysplasia, in 9 (53%) of 17 cases of adenomas with
high-grade dysplasia, and in 81% of adenocarcinomas
(p<0.0001).
[0094] Therefore, one embodiment of the invention is directed to a
diagnostic screening test for the presence of adipogenic adenovirus
infection in a subject. If the subject tests positive for
adipogenic adenovirus, FAS expression may be increased in cancer
cells resulting in a more aggressive cancer and/or a poorer
prognosis. Other information linking Ad-36 and cancer is the
finding of unique sequences of Ad-36 DNA in obesity-related
cancers, such as prostate cancer tissue by polymerase chain
reaction test (PCR). The unique sequences of DNA from Ad-36 fiber
protein, which are described in Assignee's previous U.S. Pat. Nos.
6,664,050 and 6,127,113 were detected in 11 of 18 samples of
prostate cancer tissue. Exemplary screening techniques are
described below.
[0095] The adipogenic adenovirus test will be useful in two ways.
If a subject with a breast lump or an enlarged prostate has a
positive adipogenic adenovirus test (tested by, for example, either
a positive serum test or a positive PCR tissue test), there is a
higher risk that the subject has cancer and tests should be done to
evaluate for cancer (e.g., biopsy) rather than "watchful waiting."
Conversely, if a person has a positive adipogenic adenovirus test,
then the subject should be screened for cancer at more regular
intervals than a person with a negative Ad-36 test.
[0096] A vaccine against Ad-36 has been developed in rabbits using
killed virus. Serum from the rabbits contains antibodies that
prevent Ad-36 growth in tissue culture in up to 17 serial dilutions
(a dilution of 1:131,072 of the original concentration). It is
known that the only effective antibodies against adenovirus growth
are directed against segments of fiber protein of the various
adenoviruses (neutralizing antibodies). Ad-36 antibodies do not
cross react with any other adenoviruses, so unique DNA sequences in
Ad-36 fiber protein are responsible for this specificity. Three
sequences in Ad-36 fiber protein that are unique to Ad-36 have been
identified. Using standard technology in the field, peptides from
these sequences will be used to make a highly purified vaccine that
should have minimal allergic reactions. The peptides will be bound
to adjuvants commonly used in vaccines to enhance effectiveness.
These techniques are known by those of skill in the art.
[0097] In a particular embodiment, a vaccine against adipogenic
adenoviruses, such as Ad-36 to prevent cancer may be made. Since
adipogenic adenoviruses are associated with cancer, a vaccine
against adipogenic adenoviruses may be used to prevent cancer. For
example, a vaccine against Ad-36 using unique DNA sequences
contained in the fiber protein DNA to make peptides may be
developed. This technology is known by those of skill in the
art.
[0098] In a further embodiment, a novel concept of sequencing the
fiber protein DNA from all of the adipogenic adenoviruses to
determine their structures may be performed. Following sequencing,
the DNA sequences that are present in many or all of the fiber
proteins will be identified using DNA comparison databases commonly
used in the field. This may be accomplished by, for example,
employing GenBank to identify the unique sequences in Ad-36 fiber
protein DNA. DNA sequences of about 30-75 base pairs that code for
peptides of about 10-25 amino acids are then selected. It is known
that peptides in the knob portion of the fiber protein are
effective, so these sequences will be examined. Following
identification, the peptides may be coupled with adjuvants to make
a vaccine that will prevent infection from any of the human
adenoviruses. The usefulness of a common vaccine for the adipogenic
adenoviruses will be to prevent obesity, cancer, and other human
diseases that are due to adipogenic adenoviruses. For example, a
significant percentage of military recruits contract adenovirus
infections during basic training Also, many of the mild illnesses
associated with fever, cough, diarrhea, or conjunctivitis in the
first two years of life are due to adenoviruses.
[0099] According to one embodiment of the invention, adipogenic
adenovirus infection may be used as a predictor of the development
of obesity and diseases due to the complications of obesity and the
outcome of regimens that affect body weight. Complications of
obesity may include, inter alia, diabetes mellitus, hypertension,
hyperlipoproteinemia, cardiac disease such as atherosclerotic
disease and congestive heart failure, pulmonary diseases such as
sleep apnea and asthma, cerebrovascular accidents, cancers such as
breast, uterus colon and prostate cancer, gall bladder disease such
as stones and infection, toxemia during pregnancy, risks during
surgery, gout, decreased fertility, degenerative arthritis, and
early mortality.
[0100] It has been demonstrated by the Assignee that human Ad-36
stimulates the storage of fat and formation of new fat cells in
mouse preadipocytes (3T3-L1 cells) (FIG. 1) and in human
preadipocytes. Additionally, Assignee has demonstrated in U.S. Pat.
Nos. 6,127,113 and 6,664,050, herein incorporated in their entirety
by reference, that Ad-36 infection is associated with obesity. For
example, four experiments were conducted in chickens, one
experiment in mice, and two experiments in monkeys, all showing
that infection with Ad-36 increased body fat and lowered serum
cholesterol and triglycerides. Most notable, food intake measured
in chickens, mice, and rats was not difference between infected and
control animals indicating that energy expenditure was different.
The mechanism of Ad-36 has a direct effect on adipocytes to
increase lipogenic enzymes and differentiation factors. Cells
infected with Ad-36 exhibited about 2 times as much stored
triglyceride at 5 days.
[0101] A large number of studies were also performed with the
adipogenic adenovirus, Ad-36. Rhesus monkeys that were
spontaneously infected with Ad-36 showed a significant weight gain
in 18 months after infection (FIG. 2). Marmosets were also infected
with Ad-36 and a four-fold fold weight gain was observed in
infected marmosets in comparison to uninfected marmosets (FIG. 3).
Moreover, body fat increased by about 70% and serum cholesterol
dropped by about 35 mg/dl after infection (FIG. 4). Notably, Ad-36
DNA was observed in multiple tissues of the monkey, such as brain,
liver, lung, muscle and adipocyte tissue seven months following
initial infection and was no longer able to grow the virus from the
blood or feces of the infected monkeys after two months (FIGS.
5-7). Ad-36 DNA has also been observed in human adipocyte tissue as
seen in FIG. 8.
[0102] Other studies have demonstrated that Ad-37 caused obesity in
chickens, but Ad-2 and Ad-31 did not (FIGS. 9-11). Cumulative food
intake was not different among the groups studied. Moreover,
studies have also demonstrated that Ad-5 caused obesity in mice
(FIG. 12). These studies show that stimulation of lipogenic enzymes
and obesity are not non-specific effects of all adenovirus
infections, but multiple adenoviruses may do so.
[0103] Another embodiment of the invention includes using
adipogenic adenoviruses to predict diabetes and other pancreatic
dysfunction. Additionally, another embodiment is directed to a
vaccine against adenoviruses that may prevent pancreatic disease,
including some cases of diabetes.
[0104] Pancreatic beta cells make lipids and lipid metabolism in
the beta-cell is critical for the regulation of insulin secretion.
Enzymes that are associated with lipid synthesis or oxidation cause
a decrease in the amount of insulin produced and secreted. Studies
have demonstrated that overexpression of SREBP-1 in beta cells
decreases insulin synthesis. SREBP-1 is a direct precursor of FAS
and has been found to be increased by adenovirus infection in vitro
and in vivo in hepatocytes. Other studies have demonstrated that
carbohydrate responsive element-binding protein (ChREBP) binding to
fatty acid synthase and L-type pyruvate kinase genes is stimulated
by glucose in pancreatic beta-cells. Higher carnitine
palmitoyltransferase I (CPT I) protein levels in beta cells causes
a decrease in secretion of insulin in response to glucose.
Adipogenic adenoviruses may cause diabetes because they increase
obesity and produce insulin resistance, but the changes in
lipogenic enzymes due to virus infection of the pancreas also may
play a role.
[0105] An additional embodiment of the invention is for using
adipogenic adenoviruses status to predict liver disease, cirrhosis,
and other liver dysfunction. Also, a vaccine against adenoviruses
may be used to prevent liver diseases due to adipogenic
adenoviruses.
[0106] Obesity is associated with liver disease. The spectrum of
liver disease with obesity starts with fat infiltration of the
liver, progresses to steatohepatitis (non-alcoholic
steatohepatitis=NASH), and a sizeable percentage of patients with
NASH go on to develop cryptogenic cirrhosis. The frequency and
severity of fatty infiltration of the liver goes up with increasing
body weight and is present in the vast majority of patients with
morbid obesity. It has been reported that obesity is present in
about 30% to about 100% of the cases of non-alcoholic fatty liver
disease (NAFLD). Other studies have demonstrated that greater than
about 95% of subjects with severe obesity had various degrees of
fatty infiltration of the liver and about 65% had NASH.
[0107] Adipogenic adenoviruses produce increased levels of
lipogenic enzymes and an accumulation of fat in the liver. As noted
above, fatty infiltration of the liver may lead to cirrhosis. There
are a number of causes of fatty infiltration of the liver, and
additional insults to the liver may increase the chance that
cirrhosis will occur. Individuals with risk factors for liver
disease or with abnormal liver function tests should be tested for
adipogenic adenoviruses as they may need special attention to
prevent progression of fatty liver to cirrhosis. A vaccine against
adipogenic adenoviruses may reduce or prevent many cases of fatty
liver and cirrhosis.
[0108] Yet another embodiment of the invention includes using
adipogenic adenoviruses to predict muscle dysfunction. Also, a
vaccine against adenoviruses may be used to prevent muscle
dysfunction.
[0109] Muscles are known to have lipogenic enzymes present and to
store intracellular lipids. Obesity is associated with abnormal
lipid metabolism and accumulation of intramuscular lipid. Studies
have shown that a lipogenic gene, stearoyl-CoA desaturase 1 (SCD1),
is up-regulated in skeletal muscle from extremely obese humans.
High intramuscular fat is related to poor muscle function in obese
people and is related to insulin resistance and diabetes. Other
studies have showed that insulin resistance and type 2 diabetes
result from the accumulation of lipids in tissues not suited for
fat storage, such as skeletal muscle and the liver. FFAs stimulated
the de novo synthesis of ceramide and sphingosine, two
sphingolipids shown previously to inhibit insulin action. The
inability to transition from fat to glucose as the primary source
of fuel may be associated with insulin resistance, metabolic
dysregulation, and cardiovascular risk. Since leptin has been shown
to go down in adipocytes infected with Ad-36, this might produce
insulin resistance.
[0110] Obese people complain of pain with exercise. The reasons for
this are not clear, but obese individuals have more intramuscular
lipids and a lower exercise capacity. The intramuscular lipids
alter the substrate utilization of skeletal muscles. Increased
intramuscular lipids due to adipogenic adenoviruses may limit
exercise capacity, increase pain, and alter substrate utilization.
This would be particularly important in animals used for
performance or endurance, such as race horses or racing dogs. An
animal (or person) infected with an adipogenic adenovirus may not
be able to achieve as good a performance as an uninfected, so
testing for adipogenic adenoviruses may be used as a predictor of
performance quality.
[0111] Finally, increased intramuscular lipid in heart muscle may
alter cardiac function, produce cardiomyopathy and congestive heart
failure. Studies have showed that accumulation of excess fatty
acids and triglycerides within heart muscle is associated with
cardiac insulin resistance and cardiac dysfunction.
[0112] A further embodiment of the invention is related to using
adipogenic adenoviruses status to predict pulmonary dysfunction.
Also, a vaccine against adenoviruses will prevent pulmonary
dysfunction.
[0113] It has been known for many years that obesity is associated
with pulmonary disease such as asthma and emphysema. The mechanisms
of this association are not clear. It is known that the lungs make
FAS and synthesize lipids. Therefore, the finding that adipogenic
adenoviruses can cause lung disease may be associated with the
production of FAS in the lungs by adenoviruses. Studies have
indicated that Ad-5 infection doubles the number of macrophages in
the lung of guinea pigs. The combination of smoking and Ad-5
quadruples the number of macrophages. Latent viral infections were
present in patients with asthma and emphysema and Ad-5 E1A protein
in the lungs was correlated with inflammation in asthma and
emphysema patients.
[0114] An embodiment of the invention is using adipogenic
adenovirus status to predict brain and nervous system dysfunction.
Also, a vaccine against adenoviruses may be used to prevent brain
and nervous system dysfunction.
[0115] Adipogenic viruses infect brain tissue. It is well known
that the nerves and brain make FAS. Obesity is linked to adverse
neurocognitive outcome, including reduced cognitive functioning and
Alzheimer's disease. Studies have demonstrated that BMI was
inversely related to performance on all cognitive tests and there
was no evidence of a BMI.times.age interaction. There is an
increased prevalence of Alzheimer's disease in obesity. Abeta42, a
type of amyloid that is deposited in Alzheimer's patients,
correlated with BMI. Obese people may have about a 74% higher
chance of dementia. Other studies have demonstrated that an Ad-5
vector that has been used in gene therapy caused inflammation of
the brain. Wild type Ad-5 has been reported to cause obesity and we
have noted that an Ad-5 vector that is used for gene transfer
retains its ability to stimulate SREBP-1 and FAS synthesis in
hepatocytes. Accordingly, these data support the concept that
adipogenic adenoviruses may increase lipogenic enzymes in the brain
and nerves, produce excess fatty acid and triglycerides, and result
in dysfunction.
[0116] An aspect of the invention is that testing for adipogenic
adenoviruses may be used to predict adrenal dysfunction. Also, a
vaccine against adenoviruses may be used to prevent adrenal
dysfunction.
[0117] Obesity is known to significantly affect adrenal gland
function. There is an increased secretion of aldosterone, but
without renin, which may be associated with hypertension. Studies
have demonstrated that adrenal function was not normal in obese
women, but differed with abdominal fat distribution. Adrenal
hormones use cholesterol as a substrate for production, and with
the changes of adipogenic adenoviruses in both intracellular fatty
acids and cholesterol metabolism, the use of a test to detect
adipogenic adenoviruses may be a predictor of adrenal
dysfunction.
[0118] Exemplary screening immunoanalytical techniques include
without limitation, standard virus neutralization assay techniques
or enzyme immunoassay techniques well known in the art. Techniques
for raising and purifying antibodies against these viruses or
fragments thereof (e.g., fiber protein or fragments thereof), or
proteins (or fragments thereof) from these viruses for use in these
immunoassay techniques may be prepared by conventional techniques
are well known in the art. In a specific embodiment of the
invention, antibodies will immunoprecipitate adenovirus virus or
adenovirus proteins from solution as well as react with these
proteins on Western or immunoblots or polyacrylamide gels. In
another specific embodiment, antibodies will detect the presence of
adenovirus or adenovirus proteins in frozen tissue sections, using
immunocytochemical techniques. Specific embodiments relating to
methods for detecting adenovirus or adenovirus proteins include
enzyme linked immunosorbent assays (ELISA), radioimmunoassay (RIA),
immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA),
including sandwich assays using monoclonal and/or polyclonal
antibodies.
[0119] Similarly, the nucleic acid probe hybridization assay
techniques used in these methods of the invention will be standard
techniques (optionally after amplification of DNA or RNA extracted
from a sample of blood, other body fluid, feces, tissue or organ)
using nucleic acid probes (and primers if amplification is
employed) made available by the obesity-causing viruses identified
and made available by the present invention. The sequences of
nucleic acids characteristic of these viruses can be determined by
standard techniques once the viruses are conventionally isolated,
and probes and primers that are specific for the viruses and that
provide the basis for nucleic acid probes and primers that can be
used in nucleic acid based assays for the viruses are prepared
using conventional techniques on the basis of the sequences.
[0120] For example, in order to detect the presence of an
adenovirus predisposing an individual to obesity, a biological
sample such as blood is prepared and analyzed for the presence or
absence of adenovirus proteins, such as the Ad-36 fiber coat
protein sequences. Results of these tests and interpretive
information are returned to the health care provider for
communication to the tested individual. Such diagnoses may be
performed by diagnostic laboratories, or alternatively, diagnostic
kits are manufactured and sold to health care providers or to
private individuals for self-diagnosis.
[0121] Initially, screening involves amplification of the relevant
adenovirus sequences. In a specific embodiment of the invention,
the screening method involves a non-PCR based strategy. Such
screening methods include two-step label amplification
methodologies that are well known in the art. Both PCR and non-PCR
based screening strategies can detect target sequences with a high
level of sensitivity.
[0122] One embodiment of the invention relates to target
amplification. Here, the target nucleic acid sequence is amplified
with polymerase. One specific method using polymerase-driven
amplification is the polymerase chain reaction (PCR). The
polymerase chain reaction and other polymerase-driven amplification
assays can achieve over a million-fold increase in copy number
through the use of polymerase-driven amplification cycles. Once
amplified, the resulting nucleic acid can be sequenced or used as a
substrate for DNA probes.
[0123] When the probes are used to detect the presence of the
target sequences the biological sample to be analyzed, such as
blood or serum, may be treated, if desired to extract the nucleic
acids. The sample nucleic acid may be prepared in various ways to
facilitate detection of the target sequence, e.g., denaturation,
restriction digestion, electrophoresis or dot blotting. The
targeted region of the analyte nucleic acid usually must be at
least partially single-stranded to form hybrids with the targeting
sequence of the probe. If the sequence is naturally
single-stranded, denaturation will not be required. However, if the
sequence is double-stranded, the sequence will probably need to be
denatured. Denaturation can be carried out by various techniques
well known in the art.
[0124] Analyte nucleic acid and probe are incubated under
conditions which promote stable hybrid formation of the target
sequence in the analyte. The region of the probes which is used to
bind to the analyte can be made completely complementary to the
targeted region of the adenovirus of interest, and in particular
the fiber coat protein. Therefore, high stringency conditions are
desirable in order to prevent false positives. However, conditions
of high stringency are used only if the probes are complementary to
regions of the adenovirus. The stringency of hybridization is
determined by a number of factors during hybridization and during
the washing procedure, including temperature, ionic strength, base
composition, probe length, and concentration of formamide.
[0125] Detection, if any, of the resulting hybrid is usually
accomplished by the use of labeled probes. Alternatively, however,
the probe may be unlabeled, but may be detectable by specific
binding with a ligand which is labeled, either directly or
indirectly. Suitable labels, and method for labeling probes and
ligands are well known in the art, and include, for example,
radioactive labels which may be incorporated by known methods
(e.g., nick translation, random priming or kinasing), biotin,
fluorescent groups, chemiluminescent groups (e.g., dioxetanes)
enzymes, antibodies, gold nanoparticles and the like. Variations of
this basic scheme are known in the art, and include those
variations that facilitate separation of the hybrids to be detected
from extraneous materials and/or that amplify the signal from the
labeled moiety.
[0126] As noted above, non-PCR based screening assays are also
contemplated by this invention. This procedure hybridizes a nucleic
acid probe (or analog such as a methyl phosphonate backbone
replacing the normal phosphodiester) to the low level DNA target.
This probe may have an enzyme covalently linked to the probe, such
that the covalent linkage does not interfere with the specificity
of the hybridization. The enzyme-probe-conjugate-target nucleic
acid complex can then be isolated away from the free probe
conjugate and a substrate is added for enzyme detection. Enzymatic
activity is observed as a change in color development or
luminescent output resulting in about a 10.sup.3 to about a
10.sup.6 increase in sensitivity.
[0127] Two-step label amplification methodologies are known in the
art. These assays work on the principle that a small ligand (such
as digioxigenin, biotin, or the like) is attached to a nucleic acid
probe capable of specific binding the adenovirus sequence region of
interest. In one example, the small ligand attached to the nucleic
acid probe is specifically recognized by an antibody-enzyme
conjugate. In one embodiment of this example, digioexigenin is
attached to the nucleic acid probe. Hybridization is detected by an
antibody-alkaline phosphatase conjugate which turns over a
chemiluminescent substrate. In a second example, the small ligand
is recognized by a second ligand-enzyme conjugate that is capable
of specifically complexing to the first ligand. A well known
embodiment of this example is the biotin-avidin type
interactions.
[0128] It is also contemplated within the scope of this invention
that the nucleic acid probe assays of this invention will employ a
cocktail of nucleic acid probes capable of detecting various
species of adenoviruses. Thus, in one example to detect the
presence of ad-36, ad-37 and/or ad-5, for example, in a biological
sample, more than one probe complementary of the targeted regions
of interest in the various types of adenovirus may be employed.
[0129] As the skilled will understand, more than one strain of
obesity-causing virus may be tested for simultaneously in an
immunological or nucleic acid-based assay method for testing for
virus in accordance with the invention and kits may be assembled to
facilitate carrying out the methods for a particular virus or a
plurality of them.
[0130] The invention has been disclosed broadly and illustrated in
reference to representative embodiments described above. Those
skilled in the art will recognize that various modifications can be
made to the present invention without departing from the spirit and
scope thereof. Without further elaboration, it is believed that one
skilled in the art, using the preceding description, can utilize
the present invention to the fullest extent. The following examples
are illustrative only, and not limiting of the remainder of the
disclosure in any way whatsoever.
EXAMPLES
Specific Example 1
[0131] Blood samples from 502 humans in separate populations from
three cities: Madison, Wis., Naples, Fla., and New York City, N.Y.,
were measured for Ad-36 infection. The definition of obesity was a
BMI greater than about 30 kg/m.sup.2 and the population was divided
into obese and non-obese subjects. The prevalence of Ad-36
antibodies in serum obese subjects was about 30% and in non-obese
was about 11% (i.e., a 3:1 ratio) (FIG. 13).
[0132] When the subjects were divided into antibody positive versus
antibody negative, the antibody positive were observed to be about
9 BMI units heavier (i.e., greater than about 50
pounds)(p<0.001) (FIG. 14).
[0133] Moreover, serum cholesterol and triglycerides were lower in
antibody positive humans (less than about -35 mg/dl, p<0.001),
exactly the same reductions observed in prospectively infected
monkeys. There were no correlations of antibodies to Ad-2 or Ad-31
with either body composition or serum cholesterol and triglycerides
in humans, implying that the effects of Ad-36 are specific and are
not common to all human adenoviruses.
[0134] A second study was performed in 28 twin pairs discordant for
Ad-36 antibodies. Normally twins track body weight and BMI closely,
but the Ad-36 antibody positive twins were fatter than their Ad-36
antibody negative co-twin (p<0.04) (FIG. 15).
[0135] In conclusion, these findings show that Ad-36 causes obesity
in animals and humans.
Specific Example 2
[0136] Very strong evidence of an association of Ad-36 with cancer
comes from studies at the University of Wisconsin. Banked serum
from 128 subjects with breast cancer and 37 subjects with prostate
cancer was obtained, and assayed for Ad-36 antibodies using a serum
neutralization assay. It was observed that 51% of the patients with
prostate cancer and 50% of the breast cancer patients were positive
for Ad-36 antibodies. However, since the samples were anonymous, no
data on obesity, type or stage of cancer, or presence of
metastases. From data on Ad-36 antibodies in obese and non-obese
individuals it was estimated that about 14% of the general
population had Ad-36 antibodies. Thus, there is an almost four fold
increase in the prevalence of Ad-36 antibodies in both breast and
prostate cancer patients.
Specific Example 3
[0137] Prostate cancer tissue from 18 patients was obtained and
assayed for Ad-36 DNA by nested polymerase chain reaction assay
(PCR) using primers made to unique DNA sequences in the Ad-36 fiber
protein genome. Eleven of the 18, or 61%, had Ad-36 DNA in the
cancer tissue. FIG. 16 shows a PCR gel demonstrating the presence
of Ad-36 DNA in prostate cancer tissues. As noted above, Ad-36 DNA
in multiple tissues of experimental animals was found, particularly
in adipose tissue using nested PCR with primers made from unique
DNA sequences in the Ad-36 fiber protein genome. FIG. 17 shows a
PCR gel from DNA extracted from adipose tissue of monkeys infected
with Ad-36. All three of the infected monkeys, but none of the
controls, have viral DNA present.
Specific Example 4
[0138] This example shows the use of Ad-36 lab test as a marker to
identify individuals at risk of developing cancer: People with
cancer have a much higher prevalence of a positive Ad-36 lab test
than do non-cancer patients. We tested 128 women with breast cancer
and 37 men with prostate cancer and compared the results to 502
non-cancer patients. Of the total of 165 cancer patients, 83 (50%)
had a positive Ad-36 test (50% of the breast cancer patients and
51% of the prostate cancer patients). Based on the prevalence of
obesity in the US population, the data of the 502 non-cancer
subjects showed that 17% of the non-cancer subjects had a positive
Ad-36 test. If only those non-cancer subjects from Wisconsin are
considered (since all the cancer patients were from Wisconsin, this
is a better comparison), 14% of the non-cancer subjects had a
positive Ad-36 test. Thus, almost 4 times as many cancer patients
were positive compared to the non-cancer patients.
Specific Example 5
[0139] Adipose tissue from 9 humans at autopsy at the University of
Wisconsin Hospital was obtained and assayed for Ad-36 DNA. Seven of
the 9 had Ad-36 DNA in the adipose tissue. These data demonstrate
that Ad-36 DNA may isolated from human and animal adipose tissue
and more importantly, that the presence of Ad-36 DNA in tissues is
a marker for post Ad-36 infection. Since the Ad-36 DNA appeared in
multiple tissues of infected animals, it seems clear that the
initial viremia results in infection of most of the tissues of the
body. Therefore, as described in the methods Section as described
in Specific Example 6, below, cancer tissues or adipose tissue from
cancer may be obtained from patients to test for the presence of
Ad-36 DNA.
Specific Example 6
[0140] Samples are assayed for Ad-36 DNA in samples of breast and
prostate tissue from cancer patients and non-cancer patients to
determine if the prevalence of infection with Ad-36 is greater in
cancer patients. Ad-36 status may be correlated with the presence
of cancer and may be correlated with stage of cancer, presence of
metastases, and prognosis of cancer victims. A vaccine against
Ad-36 may prevent Ad-36 induced cancers.
Experimental Design:
[0141] Anonymous samples of tissue and complete medical information
are obtained from the NCI resource banks, the NCI Cooperative Human
Tissue Network (CHTN) and the NCI Cooperative Breast Cancer Tissue
Resource (CBCTR). The samples are assayed for Ad-36 DNA by nested
PCR assay as described below. Alternatively, if DNA samples not are
available, they are may ordered. If DNA has not been extracted from
the samples, it may be as described below.
[0142] To evaluate breast cancer, samples of breast cancer tissue
or adipose tissue near the breast from 100 women with cancer are
compared with breast tissue or adipose tissue from 50 women
undergoing breast surgery who do not have cancer. As noted above,
if breast cancer tissue is not available, adipose tissue from near
the breast is acceptable. Actual breast tissue is not critical as
Ad-36 DNA appears in all tissues after infection, so adipose tissue
is suitable.
[0143] To evaluate prostate cancer, samples of prostate cancer from
100 men is compared to prostate tissue from men undergoing surgery
for benign prostatic hypertrophy (BPH).
[0144] Both NCI tissue banks have information on age, menopausal
status of female patients, height, weight, family history of breast
or prostate cancer, type and stage of cancer, presence of
metastases, and in some cases, survival time. Because breast cancer
associated with obesity is more prevalent in postmenopausal women,
non-cancer subjects are matched as well as possible for age. It may
be expected that prostate cancer patients from whom we obtain
samples will be in the older age group (>50 years) because this
is the population in which prostate cancer and BPH occurs. It may
be very unlikely that we will have any tissues from pediatric
patients as breast and prostate cancers are so rare in them.
[0145] Laboratory techniques: The procedures that will be needed
for this protocol are published in the medical literature are
understood by one of relevant skill in the art. A brief summary of
each is given below:
[0146] Culture of Ad-36: Viral preparations are grown in tissue
culture as previously described using A549 human bronchial
carcinoma cells. A549 cells are obtained from American Type Culture
Collection (ATCC, Rockville, Md.). Minimum Essential Media Eagle
(MEM) (Cat # M-0643, Sigma Chemicals) with non-essential amino
acids, Earle's salts and L-glutamine are used for growing A549
cells. Work stocks are grown in A549 cells using MEM with 10% fetal
bovine serum (FBS) and 2.9% NaHCO.sub.3 (v/v) at pH 7.4.
[0147] Plaque forming units assay: Titers of Ad-36 virus are
determined using A549 cells by this assay. Starting with 100 .mu.l
of virus suspension and 900 .mu.l of media, serial 10 fold
dilutions are made. A549 cells are grown to confluence in 6 well
plates and 3 wells are used for each dilution. Three wells are used
as the blank control and are not infected with the virus
suspension. Media is removed from each cell and 100 .mu.l of the
serially diluted virus suspension are pipetted out in the wells.
The plates are incubated at 37.degree. C., shaking gently every 15
min. After 1 h of incubation, the viral suspension from the wells
is removed and discarded. The wells are overlaid with about 3 ml of
1% agar in media per well, with 1.times.antibiotic-antimycotic
solution. The plates are inverted and incubated at 37.degree. C.
for 8 days until plaques appear. After 8 days, wells are stained
overnight with about 1 ml of crystal violet per well. The next day,
the number of plaques formed is counted after removing the agar.
The number of plaques formed.times.dilution of viral suspension
used gives PFU/100 .mu.l of inoculum used. This is multiplied by 10
to express PFU/mL.
[0148] Tissue culture infectivity dose (TCID50)(66-72): The titer
of the work stocks that cause a cytopathic effect (CPE) in 50% of
the wells containing A549 cells are calculated and expressed as
tissue culture infectivity dose (TCID-50) units per ml. TCID-50 of
the work stocks are determined using serial ten fold dilutions of
the virus work stock. TCID50 is calculated by serially diluting the
virus stock solution and inoculating cells with the dilutions to
find out the reciprocal of the highest dilution of virus which
causes cytopathic effect (CPE) in 50% of the cells inoculated.
Titers are calculated after the cells inoculated with the virus
dilutions are incubated at 37.degree. C. for 8 days.
[0149] Nested PCR assay: The nested PCR detects Ad-36 DNA in
biological samples. As previously described, four primers were
designed to unique regions of the Ad-36 fiber protein gene for use
in a nested PCR assay for detection of viral DNA. Sequences of
primers:
TABLE-US-00001 outer forward primer (SEQ ID No: 1
(5'-GTCTGGAAAACTGAGTGTGGATA), outer reverse primer (SEQ ID No: 2)
(5 = -ATCCAAAATCAAATGTAATAGAGT), inner forward primer (SEQ ID No.
3) (5 = -TTAACTGGAAAAGGAATAGGTA), inner reverse primer (SEQ ID No.
4) (5 = -GGTGTTGTTGGTTGGCTTAGGATA).
[0150] DNA is isolated from human breast and prostate tissues using
a QIAamp Tissue Kit (Qiagen, Valencia, Calif., USA; Cat # 29304).
Negative PCR controls are water and DNA from A549 cells. Positive
PCR control is DNA from Ad-36 infected A-549 cells. DNA is
denatured for 2 min at 95.degree. C. and subjected to 35 cycles of
PCR (94.degree. C. for 1 min, 55.degree. C. for 1 min, 72.degree.
C. for 2 min) followed by incubation at 72.degree. C. for 5 min.
PCR products are visualized on a 1% agarose gel with a size
marker.
[0151] This assay was developed at the University of Wisconsin and
at that time the conditions were optimized for temperature,
magnesium concentration, and number of cycles in samples consisting
of animal and human tissues, and of 3T3-L1 cells. Samples of the
PCR products are extracted from the gels and are sent to the
University of Wisconsin Biotech Center for sequencing to insure
that the amplified DNA sequences were from the targeted regions.
The Biotech Center confirmed their accuracy. These quality control
procedures are repeated to conform to CLIA specifications and good
laboratory practice. DNA sequencing of two positive samples each of
breast cancer and of prostate cancer and the A549 positive control
DNA samples are performed to insure accuracy of the
amplifications.
[0152] Statistical analyses and Power calculations: Statistical
assistance is available from the Department of Statistical Sciences
and Operations Research of the Virginia Commonwealth
University.
[0153] Power calculations: Power analysis was performed using
preliminary data on the prevalence of Ad-36 antibodies in the
general population versus in cancer patients in Madison, Wis. It
was shown that 50% and 51% of breast cancer and prostate cancer
patients, respectively, at the University of Wisconsin Hospital had
antibodies to Ad-36. In a sample of volunteers from the general
population and the Obesity Treatment Program at the University of
Wisconsin, 247 subjects were evaluated, of whom 183 were obese and
64 were non-obese. The prevalence of Ad-36 antibodies was 20% and
11%, respectively. CDC figures show that approximately 31% of adult
Americans are obese and 69% are non-obese. Using these numbers, it
was estimated that the prevalence of Ad-36 antibodies in the
general population of Madison, Wis., was 13.8%. Thus, the
prevalence of Ad-36 in cancer patients is almost fourfold higher.
Using these figures, the power calculations revealed that 32
subjects per group is necessary to achieve a power of 80% at the
0.05 level to determine a difference in prevalence of Ad-36 in
cancer vs non-cancer patients. About 50% of both breast and
prostate cancer patients may be expected to be Ad-36 antibody
positive based on the preliminary data. However, it may be possible
that the Ad-36 prevalence may be lower in the banked samples from
the NCI tissue banks (or Asterand tissue bank), and this would
affect the power significantly. Therefore, 100 cancer subjects and
50 non-cancer subjects may be certain to have sufficient power.
[0154] Statistical analyses: Chi-square analysis will be used to
determine if the prevalence of Ad-36 DNA in cancer subjects is
greater than in non-cancer subjects. The number of subjects is
relatively small for multiple regression analyses and we do not
expect to see significant correlations unless the effect is
powerful.
Specific Example 7
[0155] The cDNA sequence of the Ad-36 genome was screened against
all known cDNA sequences and two 25-base sequences and one 28-base
sequence were found, all lying in the fiber-encoding sequence that
were unique to Ad-36. These three sequences are as follows:
TABLE-US-00002 SEQ ID NO: 5: 5'-AGTTGAAACAGCAAGAGACTCAAAG SEQ ID
NO: 6 5'-GGTACTGGATCAAGTGCACATGGAG SEQ ID NO: 7
5'-TTGAAACAGCAAGAGACTCAAAGCTAAC
[0156] Sequence 3 above was employed a probe for Ad-36 in a
conventional nuclei acid probe hybridization assay of DNA isolated
from four chickens, two of which had been infected with the virus
and became obese and two of which had not been infected and were
not obese. DNA hybridizing to the probe was observed with only the
DNA from the two infected chickens. The assay involved direct
detection and was by capillary electrophoresis using laser-induced
fluorescence for detection. More particularly, a replaceable
polyacrylamide matrix was employed in the electrophoretic
separation and detection employed a dual system with 5'-labeling of
the oligo and thiazole orange intercalator in the buffer
system.
[0157] The skilled will understand that probes, and primers when
amplification is also used, of between about 15 and 30 bases in
length are advantageously employed to provide suitable specificity
and sensitivity. Amplification methods using PCR and variations
thereof maybe employed, as well known in the art.
[0158] The examples given above are merely illustrative and are not
meant to be an exhaustive list of all possible embodiments,
applications or modifications of the invention. Thus, various
modifications and variations of the described methods and systems
of the invention will be apparent to those skilled in the art
without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific embodiments, it should be understood that the invention as
claimed should not be unduly limited to such specific embodiments.
Indeed, various modifications of the described modes for carrying
out the invention which are obvious to those skilled in molecular
biology or related fields are intended to be within the scope of
the appended claims.
[0159] The disclosures of all references and publications cited
above are expressly incorporated by reference in their entireties
to the same extent as if each were incorporated by reference
individually.
Sequence CWU 1
1
7123DNAArtificial SequenceDescription of Artificial
Sequencesynthetic nested PCR outer forward primer to detect unique
region of adenovirus type 36 (Ad-36) fiber protein gene 1gtctggaaaa
ctgagtgtgg ata 23224DNAArtificial SequenceDescription of Artificial
Sequencesynthetic nested PCR outer reverse primer to detect unique
region of adenovirus type 36 (Ad-36) fiber protein gene 2atccaaaatc
aaatgtaata gagt 24322DNAArtificial SequenceDescription of
Artificial Sequencesynthetic nested PCR inner forward primer to
detect unique region of adenovirus type 36 (Ad-36) fiber protein
gene 3ttaactggaa aaggaatagg ta 22424DNAArtificial
SequenceDescription of Artificial Sequencesynthetic nested PCR
inner reverse primer to detect unique region of adenovirus type 36
(Ad-36) fiber protein gene 4ggtgttgttg gttggcttag gata
24525DNAArtificial SequenceDescription of Artificial
Sequencesynthetic fiber-encoding sequence unique to adenovirus type
36 (Ad-36) cDNA 5agttgaaaca gcaagagact caaag 25625DNAArtificial
SequenceDescription of Artificial Sequencesynthetic fiber-encoding
sequence unique to adenovirus type 36 (Ad-36) cDNA 6ggtactggat
caagtgcaca tggag 25728DNAArtificial SequenceDescription of
Artificial Sequencesynthetic fiber-encoding sequence unique to
adenovirus type 36 (Ad-36) cDNA 7ttgaaacagc aagagactca aagctaac
28
* * * * *