U.S. patent application number 11/079834 was filed with the patent office on 2005-07-21 for interrupting the interaction of intercellular adhesion molecule-1 and respiratory syncytial virus for prevention and treatment of infection.
Invention is credited to Behera, Aruna, Kumar, Mukesh, Matsuse, Hiroto, Mohapatra, Shyam S..
Application Number | 20050158327 11/079834 |
Document ID | / |
Family ID | 34594190 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158327 |
Kind Code |
A1 |
Mohapatra, Shyam S. ; et
al. |
July 21, 2005 |
Interrupting the interaction of intercellular adhesion molecule-1
and respiratory syncytial virus for prevention and treatment of
infection
Abstract
There is provided a method of preventing a respiratory infection
by administering an effective amount of an agent for regulating
ICAM-1 expression. Also provided is a composition for the
prevention of respiratory infection including an agent which
regulates ICAM expression. Also provided is a method of preventing
RSV infection by administering an effective amount of an agent that
interferes with the binding of RSV to ICAM-1. A method of
preventing RSV infection by administering an effective amount of an
agent that down regulates the expression of ICAM-1, thereby
decreasing RSV binding to ICAM-1 is also provided. There is
provided a method of treating RSV infection by administering an
effective amount of an agent for down regulating ICAM-1 expression.
A method of blocking RSV-ICAM-1 interaction by administering an
effective amount of agents for blocking ICAM sites of binding is
provided. Also provided is a compound for blocking RSV-ICAM-1
interaction including an agent for blocking ICAM sites of
binding.
Inventors: |
Mohapatra, Shyam S.; (Tampa,
FL) ; Behera, Aruna; (Tampa, FL) ; Matsuse,
Hiroto; (Nagasaki, JP) ; Kumar, Mukesh;
(Tampa, FL) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK
A PROFESSIONAL ASSOCIATION
PO BOX 142950
GAINESVILLE
FL
32614-2950
US
|
Family ID: |
34594190 |
Appl. No.: |
11/079834 |
Filed: |
March 14, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11079834 |
Mar 14, 2005 |
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10440000 |
May 16, 2003 |
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6900299 |
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10440000 |
May 16, 2003 |
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09523054 |
Mar 10, 2000 |
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60123999 |
Mar 11, 1999 |
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Current U.S.
Class: |
424/159.1 ;
514/44A |
Current CPC
Class: |
A61P 11/00 20180101;
A61P 31/12 20180101; C12N 15/111 20130101; C12N 2310/11 20130101;
C12N 15/1138 20130101; C12N 2320/32 20130101 |
Class at
Publication: |
424/159.1 ;
514/044 |
International
Class: |
A61K 048/00; A61K
039/42 |
Claims
We claim:
1. A method of treating or preventing a respiratory infection
comprising administering an effective amount of an agent to a
patient, wherein the agent down-regulates ICAM-1 expression or
interferes with ICAM-1 binding sites.
2. The method according to claim 1, wherein the infection is a
viral infection.
3. The method according to claim 1, wherein the infection is a
respiratory syncytial virus (RSV) infection.
4. The method according to claim 1, wherein the agent comprises an
antibody to ICAM-1 or an antisense oligonucleotide for ICAM-1.
5. The method according to claim 1, wherein the infection is a
respiratory syncytial virus (RSV) infection, and wherein the agent
interferes with the binding of RSV to ICAM-1.
6. The method according to claim 5, wherein ICAM-1 has an RSV-F
binding site, and wherein the agent blocks the RSV-F binding site
on ICAM-1.
7. The method according to claim 1, wherein the agent comprises an
antibody to ICAM-1.
8. The method according to claim 1, wherein the infection is a
respiratory syncytial virus (RSV), and wherein the agent comprises
an antibody to an RSV epitope.
9. The method according to claim 1, wherein the patient is
human.
10. The method according to claim 1, wherein said administering
comprises administering the agent to airway epithelial cells.
11. The method according to claim 10, wherein said administering
comprises administering the agent orally.
12. The method according to claim 1, wherein said administering
comprises administering the agent intranasally.
13. The method according to claim 1, wherein said administering
comprises administering the agent by inhalation.
14. The method according to claim 1, wherein said administering
comprises injecting the agent.
15. A composition comprising an agent that down-regulates ICAM-1
expression or interferes with ICAM-1 binding sites.
16. The composition of claim 15, wherein said agent is selected
from the group consisting of an antibody to ICAM-1, an antibody to
an respiratory syncytial virus (RSV) epitope, and an antisense
oligonucleotide for ICAM-1.
17. The composition of claim 15, wherein said agent interferes with
the binding of respiratory syncytial virus (RSV) to ICAM-1.
18. The composition of claim 15, wherein said agent comprises an
antibody to ICAM-1.
19. The composition of claim 15, wherein said agent comprises an
antibody to an RSV epitope.
20. The composition of claim 15, wherein said agent blocks the
RSV-F binding site on ICAM-1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending patent
application Ser. No. 09/523,054, filed Mar. 10, 2000; which claims
the benefit of provisional patent application Ser. No. 60/123,999,
filed Mar. 11, 1999; both of which are hereby incorporated by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a method and compound for
prevention and/or treatment of respiratory viral infection. More
specifically, the present invention relates to blocking Respiratory
Syncytial Virus binding to ICAM-1 via agents that interfere with
binding or block the expression of ICAM-1
[0004] 2. Description of Related Art
[0005] Respiratory viruses such as respiratory syncytial virus
(RSV), the parainfluenza viruses (PIV), and the influenza viruses
cause severe lower respiratory tract diseases in infants and
children throughout the world. It is also an important cause of
disease in adults and is responsible for a significant amount of
excess morbidity and mortality in the elderly. It also can be
devastating in immunosuppressed populations (Murray et al., 1997;
Pullen et al. 1982; Hall et al. 1984).
[0006] Experimental live attenuated vaccines for each of these
viruses are being developed for intranasal administration in the
first weeks or months of life, but none are currently FDA approved.
A variety of RSV, PIV-3, and influenza virus vaccine strains have
been developed by classical biological methods, evaluated
extensively in preclinical and clinical studies, and shown to be
attenuated and genetically stable. However, a major remaining
obstacle to successful immunization of infants against respiratory
virus associated disease may be the relatively poor immune response
of very young infants to primary virus infection. (Crowe J E Jr
Vaccine 1998 Aug-Sep;16(14-15):1423-32 Immune responses of infants
to infection with respiratory viruses and live attenuated
respiratory virus candidate vaccines.)
[0007] Moreover, even if one or more vaccines are approved, they
may not be suitable for some populations vulnerable to RSV (e.g.
very young infants and the immunosuppressed). Ribavirin and
immunoglobulin preparations with high titers of RSV-specific
neutralizing antibodies are currently approved for use to treat and
prevent RSV infection. However, neither of these methods are
cost-effective or simple to administer. New agents are needed to
reduce the impact of RSV. (Wyde P R Antiviral Res 1998
Aug;39(2):63-79 Respiratory syncytial virus (RSV) disease and
prospects for its control.
[0008] Data obtained from the National Respiratory and Enteric
Virus Surveillance System demonstrates the seasonal pattern of RSV
infection, with peak rates of 30-40% occurring at the beginning of
each year (Murray et al., 1997; Pullen et al. 1982; Hall et al.,
1984). RSV infection is commonly associated with interstitial lung
diseases, such as bronchiolitis and asthma. It is a major risk
factor for a number of other disease conditions, such as
immunodeficiency, cardiac arrhythmia, congenital heart disease, and
unusual atrial tachycardia (Sly, et al., 1989; Robinson et al.
1997; Armstrong et al. 1993; Fixler, 1996; Lemen, 1995; Persson,
1997; Shelhamer et al. 1995).
[0009] Although the severity of the disease decreases with repeated
infection, previous RSV infection renders no or limited immunity to
subsequent RSV infection (Hal, 1991).
[0010] Despite the above serious implications of RSV infection, the
progress in the knowledge of the viral genes and gene products
(Collins, 1991; Collins et al., 1996; Barik, 1992), an effective
vaccine, or treatment against RSV, is yet to be developed.
[0011] Additionally, previous attempts to develop a vaccine using
formalin inactivated RSV not only failed but exacerbated the
disease when subsequent RSV infection occurred (Chanock, et al.
1992; Hall, 1994). An effective vaccine or treatment for RSV would
be highly desirable.
[0012] Additionally, human nasal, airway, and lung epithelial cells
constitute a major target for respiratory infections. Viral
infection alters the expression of genes encoding a number of
cytokines, chemokines and inflammatory mediators (Sabauste, et al.
1995; Choi, et al. 1992; Becker et al. 1993
[0013] The secretion of cytokines by airway epithelial cells can
either initiate local inflammatory responses or amplify an
inflammatory event that was previously initiated by activated
macrophages, eosinophils, mast cells or lymphocytes (Shelhamer et
al., 1995; Holtzman, et al. 1991; Churchill, et al. 1989; Marini,
et al. 1992; Churchill, et al. 1992; Kwon, et al. 1994; Sousa, et
al. 1994; Cromwell, et al. 1992; Jin, et al. 1997). The epithelial
cell-mediated inflammation by involve a number of cytokines and
chemokines including IL-1.beta., IL-6, IL-8, IL-11, IFN-.GAMMA.,
TNF-.alpha., GM-CSF, GRO-.alpha., PLA-2, C3, inducible nitric oxide
synthase (iNOS), MCP-1, endothelin-1 (ET-1), mucin,
elastase-specific inhibitors, and secretory leukocyte proteinase
inhibitor.
[0014] The rhinovirus infection of a transformed HBE cell line,
BEAS-2B, caused the release of the granulocyte macrophage colony
stimulating factor (GM-CSF), IL-6, and IL-8 (Sabauste et al. 1995).
The influenza virus infection of primary cultures of human
bronchial epithelial (HBE) cells induced the expression of IL-8
(Choi, et al. 1992). Also, in response to RSV infection, nasal
epithelial cells and BEAS-2B cells generated IL-8 (Becker et al.,
1993; Merolla et al., 1995; Noah, et al., 1993; Garofalo et al.
1996).
[0015] It is known that, when infected bronchial epithelial cells
secrete several pro-inflammatory cytokines, as set forth above.
Some of these cytokines (IL-1 .beta., TNF-2) up-regulate ICAM-1
expression on these cells (Persson et al., 1997; Becker et al.,
1993; Noah et al., 1993; Sabauste et al., 1995). ICAM-1, a member
of the immunoglobulin gene super family, is a cell surface receptor
for the lymphocyte function-associated antigen (LFA-1) adhesion
molecule (Makgoba et al., 1998). ICAM-1 mediates the integration of
leukocytes into inflammatory sites and facilitates interaction
between lymphocytes and target cells. ICAM-1 is also the major cell
surface receptor for many of the rhinoviruses (Greve et al., 1989;
Staunton et al., 1989; Tomassini et al., 1989).
[0016] RSV, though phylogenetically different from rhinovirus,
induces a similar profile of cytokines in epithelial cells and also
NF-.kappa.B, which regulates expression of ICAM-1 that plays a role
in neutrophil and eosinophil adhesion to epithelial cells (Arnold
et al., 1995; Chini et al., 1998; Stark et al., 1996). Also an
elevated expression of ICAM-1 in nasal epithelial cells of
asthmatics has been reported (Vignola et al., 1993).
SUMMARY OF THE INVENTION
[0017] According to the present invention, there is provided a
method of preventing a respiratory infection by administering an
effective amount of an agent for regulating ICAM-1 expression. Also
provided is a composition for the prevention of respiratory
infection including an agent which regulates ICAM expression method
of preventing RSV infection by administering an effective amount of
an agent that interferes with the binding of RSV to ICAM-1. A
method of preventing RSV infection by administering an effective
amount of an agent that down regulates the expression of ICAM-1,
thereby decreasing RSV binding to ICAM-1 is also provided. There is
provided a method of treating RSV infection by administering an
effective amount of an agent for down regulating ICAM-1 expression.
A method of blocking RSV-ICAM-1 interaction by administering an
effective amount of agents for blocking ICAM sites of binding is
provided. Also provided is a compound for blocking RSV-ICAM-1
interaction including an agent for blocking ICAM sites of
binding.
DESCRIPTION OF THE DRAWINGS
[0018] Other advantages of the present invention will be readily
appreciated as the same becomes better understood by reference to
the following detailed description when considered in connection
with the accompanying drawings wherein:
[0019] FIG. 1 is a photograph showing the topography of ICAM-1
expression in RSV infected HEp2 cells by confocal microscopy;
[0020] FIGS. 2A-B are graphs showing the interaction of ICAM-1 with
RSV; (A) shows RSV bound to and immobilized ICAM-1 in a dose
dependent manner; (B) shows the inhibition of RSV binding to ICAM
which is examined by pre-incubation of RSV with mAbs;
[0021] FIGS. 3A-C are photographs showing the anti-ICAM-1 mAb
treatment and how it inhibits RSV infection in airway epithelial
cells; and
[0022] FIG. 4 A-B show the role of ICAM-1 in a murine model of RSV
infection; the C57B1/6 wildtype and ICAM-1 KO mice were inoculated
i.n. with RSV then after 4 days the mice were sacrificed and the
lungs were taken out; RT-PCR was done to check the replication of
RSV in the lung tissue of ICAM-1 KO and the wild type mice using
the method set forth in the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Generally, the present invention provides a compound and
method for protection and prevention against respiratory infection.
More specifically, the method provides protection against RSV
infection which can be accomplished by administering a
pharmaceutically effective amount of an agent.
[0024] The agent can be in the form of antibodies to either ICAM-1
or RSV epitopes that interact with the other, antisense
oligonucleotides for ICAM-1 or other agents that down regulate
ICAM-1 expression, or agents that block RSV interaction with
ICAM-1. Specifically, the agent should interfere with the binding
of RSV to ICAM-1. By blocking the ICAM sites of binding there is
provided protection from RSV infection. Alternatively, the compound
of the present invention can be used for the treatment of an RSV
infection. In the preferred embodiment, the agent of the present
invention blocks the RSV-F binding site on ICAM-1. This has been
proven to be the most effective method of prevention and
treatment.
[0025] The method of the present invention involves administering
an effective amount of an agent to a patient sufficient to either
prevent the RSV infection or treat the RSV infection. Applicable
methods are known to those of skill in the art. Preferably, the
agent will be administered to the airway epithelial cells either
intranasally or orally. Other methods of administration, as are
known to those of skill in the art can be used without departing
from the present invention, such as injection or other means of
inhalation. Specifically, the present invention provides a method
of blocking RSV-ICAM-1 interaction thereby providing protection
against the RSV infection by administering an agent with blocking
capabilities to a patient in need of such treatment.
[0026] Intercellular adhesion molecule-1 (ICAM-1), which is
expressed on nasal epithelial cells, is a receptor for rhinovirus
and is shown by the inventors to play a role in RSV infection. The
role of ICAM-1 in RSV infection was examined using in vitro and in
vivo models of RSV infection. Conofocal microscopy indicates that
RSV is co-localized with membrane ICAM-1 on cultured, RSV-infected
HEp-2 human bronchial epithelial cells.
[0027] RSV is also shown by inventors to bind to immobilized
ICAM-1. The antibody to RSV-F protein inhibits this binding by 80%,
whereas an antibody to RSV-G protein only inhibits by 36%. In a
mammalian two-hybrid assay, the RSV-F but not RSV-G gene product
interacts with ICAM-1 in transfected NIH3T3 cells. Furthermore,
preincubation of HEp-2 cells with a neutralizing monoclonal
antibody to ICAM-1 inhibits RSV infection of these cells in a dose
dependent manner. This antibody also inhibits RSV infection of
primary normal human bronchial epithelial cells and transformed
epithelial (A549) and macrophage (U937) cell lines. Moreover, mice
deficient in ICAM-1 or treated with antibodies and anti-sense
oligonucleotides for ICAM-1 are significantly (.rho.<0.01)
protected against RSV infection. Thus antibodies to RSV, ICAM-1 and
ICAM-1 anti-sense oligonucleotides inhibit RSV-ICAM-1 binding and
prevents infection.
[0028] The compounds of the present invention are administered and
dosed in accordance with good medical practice, taking into account
the clinical condition s of the individual patient, the site and
method of administration, scheduling of administration, patient
age, sex, body weight and other factors known to medical
practitioners. The pharmaceutically "effective amount" for purposes
herein is thus determined by such considerations as are known in
the art. The amount must be effective to achieve improvement
including but not limited to improved survival rate or more rapid
recovery, or improvement or elimination of symptoms and other
indicators as are selected as appropriate measures by those skilled
in the art. Additionally, when the compound is being administered
orally, there must be included a compound for preventing the
degradation of the agent.
[0029] The agent is administered and dosed in accordance with good
medical practice, taking into account the clinical condition of the
individual patient, the site and method of administration,
scheduling of administration, and other factors known to medical
practitioners. The "effective amount" for purposes herein is thus
determined by such considerations as are known in the art.
[0030] A gene therapy according to the present invention is
administered to the airways, e.g. nose, sinus, throat and lung, for
example, as nose drops, by nebulization, vaporization or other
methods known in the art.
[0031] In another embodiment of the present invention, the
treatment includes administering to the patient an effective amount
of a composition containing a recombinant construct comprising a
nucleic acid sequence encoding the agent, the nucleic acid sequence
being operatively linked to one or more transcription control
sequences. Further, the nucleic acid sequence is expressed at or
adjacent to respiratory epithelial cells and the agent results in
reduced proliferation of the respiratory infection.
[0032] Another embodiment of the present invention relates to a
method of protecting a host against respiratory infection by
administering to the host an effective amount of a vector
containing a construct having a nucleotide sequence encoding the
agent with a promoter sequence operatively attached thereto.
Further, this construct contains a noninfectious, nonintegrating
DNA sequence which controls the expression of the agent.
Additionally, administration of the agent is in an amount
sufficient to increase levels of the agent in the respiratory tract
thus providing a protective response.
[0033] The above discussion provides a factual basis for the use of
the compound and method for prevention of respiratory viral
infection. The methods used with a utility of the present invention
can be shown by the following non-limiting examples and
accompanying figures.
EXAMPLES
[0034] General Methods:
[0035] Gene Therapy:
[0036] By gene therapy as used herein refers to the transfer of
genetic material (e.g DNA or RNA) of interest into a host with
subsequent expression to treat or prevent a disease. The genetic
material of interest encodes a product, more specifically it
encodes an anti-sense molecule that binds to mucleic acids encoding
ICAM-1, thereby preventing its translation. For a review see, in
general, the text "Gene Therapy" (Advances in Pharmacology 40,
Academic Press, 1997).
[0037] According to the method of the present invention, a
vehicle/method (transfection, transduction, homologous
recombination, etc.) and an expression system as needed and then
the modified cells are expanded in culture and returned to the
host/patient. These genetically reimplanted cells have been shown
to express the transfected genetic material
[0038] In in vivo gene therapy, the genetic material to be
transferred is introduced into the cells of the recipient organism
in situ, that is within the recipient. These genetically altered
cells have been shown to express the transfected genetic material
in situ.
[0039] The gene expression vehicle is capable of delivery/transfer
of heterologous nucleic acid into a host cell. The expression
vehicle may include elements to control targeting, expression and
transcription of the nucleic acid in a cell selective manner as is
known in the art. It should be noted that often the 5'UTR and/or
3'UTR of the gene may be replaced by the 5'UTR and/or 3'UTR of the
expression vehicle.
[0040] The expression vehicle can include a promotor for
controlling transcription of the heterologous material and can be
either a constitutive or inducible promotor to allow selective
transcription. Enhancers that may be required to obtain necessary
transcription levels can optionally be included. Enhancers are
generally any non-translated DNA sequence which works contiguously
with the coding sequence (in cis) to change the basal transcription
level dictated by the promoter. The expression vehicle can also
include a selection gene as described herein below.
[0041] Vectors can be introduced into cells or tissues by any one
of a variety of known methods within the art. Such methods can be
found generally described in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989,
1992), in Ausubel et al., Current Protocols in Molecular Biology,
John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic
Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene
Targeting, CRC Press, Ann Arbor, Mich. (1995), Vectors: A Survey of
Molecular Cloning Vectors and Their Uses, Butterworths, Boston
Mass. (1988) and Gilboa et al (1986) and include, for example,
stable or transient transfection, lipofection, electroporation and
infection with recombinant viral vectors. In addition, see U.S.
Pat. No. 4,866,042 for vectors involving the central nervous system
and also U.S. Pat. Nos. 5,464,764 and 5,487,992 for
positive-negative selection methods.
[0042] Features that limit expression to particular cell types can
also be included. Such features include, for example, promoter and
regulatory elements that are specific for the desired cell
type.
[0043] In addition, recombinant viral vectors are useful for in
vivo expression of a desired nucleic acid because they offer
advantages such as lateral infection and targeting specificity.
Lateral infection is inherent in the life cycle of, for example,
retrovirus and is the process by which a single infected cell
produces many progeny virions that bud off and infect neighboring
cells. The result is that a large area becomes rapidly infected,
most of which was not initially infected by the original viral
particles. This is in contrast to vertical-type of infection in
which the infectious agent spreads only through daughter progeny.
Viral vectors can also be produced that are unable to spread
laterally. This characteristic can be useful if the desired purpose
is to introduce a specified gene into only a localized number of
targeted cells.
[0044] As described above, viruses are very specialized infectious
agents that have evolved, in many cases, to elude host defense
mechanisms. Typically, viruses infect and propagate in specific
cell types. The targeting specificity of viral vectors utilizes its
natural specificity to specifically target predetermined cell types
and thereby introduce a recombinant gene into the infected cell.
The vector to be used in the methods of the invention will depend
on desired cell type to be targeted and will be known to those
skilled in the art.
[0045] Retroviral vectors can be constructed to function either as
infectious particles or to undergo only a single initial round of
infection. In the former case, the genome of the virus is modified
so that it maintains all the necessary genes, regulatory sequences
and packaging signals to synthesize new viral proteins and RNA.
Once these molecules are synthesized, the host cell packages the
RNA into new viral particles which are capable of undergoing
further rounds of infection. The vector's genome is also engineered
to encode and express the desired recombinant gene. In the case of
non-infectious viral vectors, the vector genome is usually mutated
to destroy the viral packaging signal that is required to
encapsulate the RNA into viral particles. Without such a signal,
any particles that are formed will not contain a genome and
therefore cannot proceed through subsequent rounds of infection.
The specific type of vector will depend upon the intended
application. The actual vectors are also known and readily
available within the art or can be constructed by one skilled in
the art using well-known methodology.
[0046] The compound of the present invention is administered and
dosed in accordance with good medical practice, taking into account
the clinical condition of the individual patient, the site and
method of administration, scheduling of administration, patient
age, sex, body weight and other factors known to medical
practitioners. The pharmaceutically "effective amount" for purposes
herein is thus determined by such considerations as are known in
the art. The amount must be effective to achieve improvement
including but not limited to improved survival rate or more rapid
recovery, or improvement or elimination of symptoms and other
indicators as are selected as appropriate measures by those skilled
in the art.
[0047] The recombinant vector can be administered in several ways.
It should be noted that it can be administered as the compound or
as pharmaceutically acceptable salt and can be administered alone
or as an active ingredient in combination with pharmaceutically
acceptable carriers, diluents, adjuvants and vehicles. Local
administration can provide a quicker and more effective treatment.
Administration can also be performed by, for intranasally orally,
subcutaneously or parenterally including intravenous,
intraarterial, intramuscular, intraperitoneally, and intranasal
administration as well as intrathecal and infusion techniques.
Implants of the compounds are also useful. The patient being
treated is a warm-blooded animal and, in particular, mammals
including man. The pharmaceutically acceptable carriers, diluents,
adjuvants and vehicles as well as implant carriers generally refer
to inert, non-toxic solid or liquid fillers, diluents or
encapsulating material not reacting with the active ingredients of
the invention.
[0048] It is noted that humans are treated generally longer than
the mice or other experimental animals exemplified herein which
treatment has a length proportional to the length of the disease
process and drug effectiveness. The doses may be single doses or
multiple doses over a period of several days, but single doses are
preferred.
[0049] The doses may be single doses or multiple doses over a
period of several days. The treatment generally has a length
proportional to the length of the disease process and drug
effectiveness and the patient species being treated.
[0050] When administering the compound of the present invention
parenterally, it will generally be formulated in a unit dosage
injectable form (solution, suspension, emulsion). The
pharmaceutical formulations suitable for injection include sterile
aqueous solutions or dispersions and sterile powders for
reconstitution into sterile injectable solutions or dispersions.
The carrier can be a solvent or dispersing medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, liquid polyethylene glycol, and the like), suitable
mixtures thereof, and vegetable oils.
[0051] Proper fluidity can be maintained, for example, by the use
of a coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil,
olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and
esters, such as isopropyl myristate, may also be used as solvent
systems for compound compositions. Additionally, various additives
which enhance the stability, sterility, and isotonicity of the
compositions, including antimicrobial preservatives, antioxidants,
chelating agents, and buffers, can be added. Prevention of the
action of microorganisms can be ensured by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol,
phenol, sorbic acid, and the like. In many cases, it will be
desirable to include isotonic agents, for example, sugars, sodium
chloride, and the like. Prolonged absorption of the injectable
pharmaceutical form can be brought about by the use of agents
delaying absorption, for example, aluminum monostearate and
gelatin. According to the present invention, however, any vehicle,
diluent, or additive used would have to be compatible with the
compounds.
[0052] Sterile injectable solutions can be prepared by
incorporating the compounds utilized in practicing the present
invention in the required amount of the appropriate solvent with
various of the other ingredients, as desired.
[0053] A pharmacological formulation of the present invention can
be administered to the patient in an injectable formulation
containing any compatible carrier, such as various vehicle,
adjuvants, additives, and diluents; or the compounds utilized in
the present invention can be administered parenterally to the
patient in the form of slow-release subcutaneous implants or
targeted delivery systems such as monoclonal antibodies, vectored
delivery, iontophoretic, polymer matrices, liposomes, and
microspheres. Examples of delivery systems useful in the present
invention include: U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616;
4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224;
4,439,196; and 4,475,196. Many other such implants, delivery
systems, and modules are well known to those skilled in the
art.
[0054] A pharmacological formulation of the compound utilized in
the present invention can be administered orally to the patient.
Conventional methods such as administering the compounds in
tablets, suspensions, solutions, emulsions, capsules, powders,
syrups and the like are usable. Known techniques which deliver it
orally or intravenously and retain the biological activity are
preferred.
[0055] In one embodiment, the compound of the present invention can
be administered initially by intravenous injection to bring blood
levels to a suitable level. The patient's levels are then
maintained by an oral dosage form, although other forms of
administration, dependent upon the patient's condition and as
indicated above, can be used. The quantity to be administered will
vary for the patient being treated and will vary from about 100
ng/kg of body weight to 100 mg/kg of body weight per day and
preferably will be from 10 mg/kg to 10 mg/kg per day.
[0056] Local administration is advantageous because there is no
dilution effect and, therefore, a smaller dose is required to
achieve expression in a majority of the targeted cells.
Additionally, local inoculation can alleviate the targeting
requirement required with other forms of administration since a
vector can be used that infects all cells in the inoculated area.
If expression is desired in only a specific subset of cells within
the inoculated area, then promoter and regulatory elements that are
specific for the desired subset can be used to accomplish this
goal. Such non-targeting vectors can be, for example, viral
vectors, viral genome, plasmids, phagemids and the like.
Transfection vehicles such as liposomes can also be used to
introduce the non-viral vectors described above into recipient
cells within the inoculated area. Such transfection vehicles are
known by one skilled within the art.
Example 1
[0057] Materials and Methods
[0058] Cell Culture, Virus and Infection.
[0059] Bronchial epithelial cells, HEp-2 (ATCC CCL-23), and RSV A2
strain (VR-1302) were obtained from the ATCC, Rockville, Md. HEp-2
cells were grown at 37.degree. C. with 5% CO.sub.2 in a minimum
essential medium with Hank's salt, supplemented with 5% fetal
bovine serum (FBS). HEp-2 cells were infected with RSV at
5.times.10.sup.5 pfu/ml and left for absorbtion for 2 h at
37.degree. C. with 5% CO.sub.2, after which the virus inoculum was
replaced by complete medium. Cells were trypsinized and harvested
at various hours post infection. RSV infection was detectable as
early as 4 hours post infection by RT-PCR using primers for RSV-N
gene (Hegele et al., 1994). Also, the RSV-infected cells could be
enumerated by fluorescence microscopy using FITC-conjugated
antibodies to RSV.
[0060] Confocal Microscopy.
[0061] RSV infected (5.times.10.sup.5 pfu/ml) HEp-2 cells grown on
coverslips for 24 hours were fixed in ethanol, blocked for 1 hour
with 1% BSA in PBS, pH 7.4 and double stained with goat anti-RSV
polyclonal Abs and mouse anti-human [cam-1 MaB (bba-4) Each at 4
.mu.g/ml (R & D Systems, Minneapolis, Minn.) for 1 hour at
37.degree. C. The cells were subsequently incubated for 1 hour with
secondary antibodies: rabbit anti-goat IgG-PE conjugate and sheep
anti-mouse Igg-FITC conjugate, and were finally mounted in DAPI
antifade (Oncor, Gaithersburg, Md.). The cells were scanned using
an Oncor digital confocal microscope at 525 nm for FITC, at 580 nm
for PE, at 350 nm for DAPI for counter staining of DNA; all three
images were merged to confirm the co-localization of ICAM-1 and RSV
in the same cell.
[0062] Assay for RSV-ICAM-1 Interaction.
[0063] Soluble CAM-1 protein (R & D Systems, Minneapolis,
Minn.) was coated onto high affinity ELISA plates (Costar,
Cambridge, Mass.) at 4.degree. C. over night at 100 ng/well in
carbonate bicarbonate buffer, pH 9.5. All other incubations were
done for 2 hours at 37.degree. C. Wells were blocked in 1% BSA in
PBS, pH 7.4 and then incubated with different dilutions of RSV
(from 2.times.10.sup.5 to 4.times.19.sup.6 pfu/ml). For
competition, RSV was either pre-incubated or simultaneously
incubated with either anti-RSV-G mAb or anti-RSV-F mAb (20 to 100
.mu.g/ml). Wells were washed in BSA-Tween-20 (0.1%), pH 7.4 and
sequentially incubated with goat anti-RSV polyclonal Ab at 90
.mu.g/ml (Chemicon, Temecula, Calif.) and anti-goat IgG HEP
conjugate (Sigma, St. Louis, Mo.). Wells were washed and developed
with the TMB (substrate). Color development was stopped after 30
minutes by addition of 0.2 M sulfuric acid. The optical density was
measured at 450 nm.
[0064] Flow Cytometry.
[0065] HEp-2 cells were infected with RSV, which had been
preincubated with mAb to either F or G, or mouse IgG at 4.degree.
C. overnight with gentle shaking. Cells were harvested 24 hours
post infection, washed in old PBS, pH 7.4 and incubated with goat
anti-RSV polyclonal Ab (Chemicon, Temecula, Calif.) at 1:100
dilution for 1 hour on ice. Cells were then washed with 1 ml of
FACS buffer (PBS, pH 7.4: 0.5% FBS; 1 mM EDTA) and incubated with
rabbit anti-goat IgG PE conjugate for 1 hour on ice. Cells were
again washed in FACS buffer and analyzed for RSV infected cells on
a FACScan instrument (Becton Dickinson, Mountain View, Calif.).
[0066] Mammalian Two-Hybrid Assay.
[0067] Full-length cDNAs encoding human [CAM-1, RSV-F and RSV-G
were amplified from mRNA of RSV infected HEp-2 cells using gene
specific primers, each of which included an appropriate restriction
site for subsequent cloning [ICAM-1: Forward-5'CCT GGC GAA TTC CAG
ACA TCT GTG TCC CCC TCA, Reverse-5'GTG TGG ATC CAC TGC CAC CAA TAT;
F gene: Forward-5' CAA GAA TTC ATG GAG TTG CTA ATC CTC AAA CA,
Reverse-5' CTA TGT CGA CTT AGT TAC TAA ATG CAA TAT TAT TTA; and G
gene: Forward-5' AAT GAA TTC ATG TCC AAA AAC AAG GAC CAA CGC,
Reverse-5'GTT GTC GAC TAA CTA CTG GCG TGG TGT GTT]. The ICAM-1 cDNA
was cloned in-frame with the activation domain (AD) derived from
VP16 protein of herpes simplex virus in the vector pVP16 (Clontech,
Palo Alto, Calif.). The cDNA encoding F or G protein was amplified,
and cloned in frame to the GAL4 DNA-binding domain (DBD) of the pM
vector. The orientation and the reading frame of all these fusion
constructs were verified to be correct by restriction endonuclease
analysis. The pG5CAT vector was used to detect protein-protein
interaction by expression of CAT enzyme. CAT activity was assayed
using ELISA (Boehringer Mannheim, Indianapolis, Ind.) following the
manufacturer's protocol. The vectors, pM, pVP16 and pG5CAT, were
transfected into NIH 3T3 fibroblast cells to determine the basal
level of CAT activity.
[0068] ICAM-1 Blocking in vitro.
[0069] HEp-2 cells (10.sup.5 cells/ml) were treated with different
concentrations (100 to 400 .mu.g/ml) of anti-ICAM-1 mAb (BBA 4) (R
& D Systems, Minneapolis, Minn.) or purified mouse IgG.sub.1
antibodies (isotype control) (Sigma, St. Louis, Mo.) for 3 hours at
37.degree. C. and were subsequently infected with RSV at
5.times.10.sup.5 n pfu/ml. Cells were harvested 24 hours
post-infection.
[0070] Reverse Transcriptase PCR Analysis.
[0071] The total RNA was isolated from the harvested cells and
tissue samples using Trizol (Life Tech., Gaithersburg, Md.). Random
primed cDNA was prepared using superscript II RNAse H-reverse
transcriptase (Life Tech., Gaithersburg, Md. The first strand cDNA
product (1 .mu.l) was amplified using Taq polymerase (Life Tech.,
Gaithersburg, Md.). Forward and reverse primers used are as
follows: RSV-N forward: 5'-GCG ATG TCT AGG TTA GGA AGA A-3';
reverse: 5'-GCT ATG TCC TTG GGT AGT AAG CCT-3' (Vignola et al.,
1993); ICAM-1 forward: 5'-ATG GCT CCC AGC AGC CCC-3'; reverse:
5'-CAC CTG GCA GCG TAG GGT-3' and .beta.-actin forward: 5'-CGC GAG
AAG ATG ACC CAG-3'; reverse: 5'-ATC ACG ATG CCA GTC GTA C-3'. ALL
PCR reactions were denatured at 95.degree. C. for 1', annealed at
56.degree. C. for 1' and extended at 72.degree. C. for 1 minute for
40 cycles. All amplifications were RNA specific, as no bands were
seen in the control (no template) PCR samples. The reaction
products were separated on 1.5% agarose gels.
[0072] Immunofluroscence.
[0073] RSV infected HEp-2 cells 24 hours post-infection were fixed
in chilled acetone for 10 minutes and air-dried. Cells were stained
for 30 minutes at 37.degree. C. with FITC-labeled anti-RSV mAbs
(Chemicon, Temecula, Calif.) in a humid chamber. The unbound
antibodies were removed by washing three times in PBS-Tween-20
(0.2%) buffer, pH 7.4. The slides were air dried again and mounted
on Fluromount G (Fisher, Pittsburgh, Pa.) and observed under
fluorescent microscope. RSV positive cells (green fluorescence)
were counted randomly from 15 different spots and from two
different slides for the same treatment group and the percent of
infected cells were plotted against the concentration of ICAM-1
mAb.
[0074] Animals and Virus Infection.
[0075] All animal experiments were performed in accordance with
protocols approved by the University of South Florida and James A.
Haley Veteran's Hospital Animal Care Committee. Mice were obtained
from Jackson Laboratory (Bar Harbor, Me.) and maintained in
pathogen free condition. Mice were infected with 50 .mu.l RSV
suspension (5.times.10.sup.5 pfu/ml) under light anesthesia of
Nembutal (Abbot Laboratories, North Chicago, Ill.) by intranasal
administration. The pattern of RSV induced lung infection in mice
was similar to the published reports (Anderson et al., 1990;
Oppenshaw et al., 1995; Hsu et al., 1998).
[0076] ICAM-1 Blocking in vivo.
[0077] Mice were first intra-tracheally administered with the
mixture of ICAM-1 pholphothioate anti-sense ODNs (300 .mu.g/mouse)
complexed with the cationic lipid, lipofectamine (2.5 .mu.g
ODN/.mu.g lipofectamine), and goat anti-mouse ICAM-1 mAb (40 .mu.g
mouse). After 2 hours the same mice were incolulated intranasally
with goat anti-mouse ICAM-1 mAb (50 .mu.g mouse) and infected with
RSV 2 hours post-treatment. Mice were sacrificed two days
post-infection and lungs were collected to examine RSV replication
in lung cells by RT-PCR.
[0078] Histopathology.
[0079] Viral infection, immunohistopathology and scoring
inflammatory lesions were performed as described (Tayler et al.,
1984; Graham et al., 1988) with modifications. Mice were sacrificed
on the fourth day post infection (pi) by overdose (0.6 g/kg) of
pentobarbital. Lungs were inflated via trachea, fixed in Zamboni's
PAF fixative at 4.degree. C. for 24 hours and subsequently
transferred to 80% ethanol followed by paraffin embedding. The
sections were stained with hematoxylin and eosin (HE). The slides
were coded and scored in blind fashion twice each by three
different individuals. The severity of lung pathology was scored on
a scale of 0-3 indicating the degree of inflammation. The entire
lung section was reviewed and pathological changes were evaluated
for epithelial damage, peri-bronchovascular cell infiltrate and
interstitial-alveolar cell infiltrate from both the ICAM-1.sup.+/+
and ICAN-1.sup.-/- mice. Epithelial damage was scored as: 0=no
damage, 1=increased cytoplasm of epithelial cells without
desquamation, 2=epithelial desquamation without bronchial exudate
composed of inflammatory cells, 3=bronchial exudate composed with
desquamated epithelial cells and inflammatory cells.
Peribronchovascular cell infiltrate was scored as: 0=no infiltrate,
1=infiltrate up to four cells, 2=infiltrate five to ten cells,
3=infiltrate more than ten cells. Interstitial-alveolar cell
infiltrate was scored as: 0=no infiltrate, 1=mild, generalized
increase in cell mass of the alveolar septa without thickening of
the septa or significant airspace consolidation, 2=dense septal
infiltrate with thickening of septa, 3=significant alveolar
consolidation in addition to interstitial inflammation.
Pathological scores were expressed as mean.+-.standard error of
mean (SEM). lntraobserver variation was<5%.
[0080] Statistical Analysis.
[0081] The difference between the treated and control cells was
analyzed by Student's t test. The percent infection between groups
of mice was compared with chi-square test.
[0082] Pathological scores were expressed as mean.+-.standard error
of mean (SEM) and statistical comparisons between two groups were
made with Mann-Whittney Test. Differences between groups were
considered significant as p values less than 0.05. All analyses
were performed on a Macintosh Computer (Apple Computer Inc.,
Cupertino, Calif.) with Startview II software (Abacus Concepts,
Berkely, Calif.).
[0083] Results and Discussion
[0084] Surface ICAM-1 expression on HEp-2 cells infected with RSV
was assayed following RSV infection by flow cytometry. The
uninfected HEp-2 cells showed constitutive expression (3.6%) of
ICAM-1. Surface ICAM-1 expression increased by four and eight fold,
respectively, 24 hours and 48 hours post RSV infection. RSV antigen
expression was detected both on the plasma membrane and in the
cytoplasm as determined by confocal microscopy, whereas ICAM-1
expression was localized mostly to the plasma membrane (FIG. 1).
The co-localization of RSV And ICAM-1 ON HEp-2 cell surfaces (FIG.
1D) suggested that RSV directly binds to ICAM-1 on epithelial
cells
[0085] More specifically, FIG. 1 shows the topography of ICAM-1
expression in RSV-infected HEp-2 cells by confocal microscopy as
described (Fixler, 1996). FIG. 1A shows that RSV was distributed on
the cell surface and in the cell cytoplasm (red), FIG. 1B shows
that ICAM-1 was localized mostly on the cell surface by FITC
staining (green), and FIG. 1C shows that the nuclei were visualized
by staining with DAPI (blue). FIG. 1D shows that superimposition of
all the three images indicated the co-localization of RSV and
ICAM-1 on the plasma membrane of RSV infected cells.
[0086] The direct binding of RSV to ICAM-1 was examined by ELISA.
RSV bound to immobilized ICAM-1 in a dose-dependent manner (FIG.
2A). The lack of ICAM-1 binding to conditioned medium in absence of
RSV (control) indicated that the binding between ICAM-1 and RSV in
specific (FIG. 2A). Pneumoviruses encode two major surface
glycoproteins, the G protein that presumably attaches to the cell
surface receptor (Levine et al., 1987) and the F protein that
mediates fusion of viral envelope to cell membrane (Hall et al.,
1991). Antibodies to either G or F protein neutralize virus
infectivity and seem to play a major role in protective immunity
against RSV both in human and mice (Olmsted et al., 1986; Stott et
al., 1987). To determine which of these RSV proteins interacts with
ICAM-1, the RSV suspension was incubated with mAb to either F or G
and the degree of inhibition of RSV binding to ICAM-1 was assayed.
Incubation with mAb to either F or G decreased the RSV-ICAM-1
binding by 80% and 36%, respectively (FIG. 2B). These results show
that RSV can bind to ICAM-1 in the absence of other cellular
factors and that the RSV binds to ICAM-1 primarily via its F
protein.
[0087] The role of F vis-a-vis G protein on the binding of RSV to
ICAM-1 on HEp-2 cells was investigated. RSV was incubated with mAb
to F or G protein or mouse IgG.sub.1 (control) and then used to
infect HEp-2 cells and the percent of infected cells estimated by
flow cytometry. Compared to control Abs, the mAb to F significantly
inhibited (73.8%) the RSV infection of HEp-2 cells G protein,
whereas the mAb to G did not. Furthermore, direct interaction of F
or G protein with ICAM-1 protein was assayed by estimating
chloramphenicol acetyl transferase (CAT) expression in a mammalian
two-hybrid system. The transfection of NIH3T3 fibroblast cells with
cDNA constructs expressing G, wherein ICAM-1 and CAT (pG5CAT)
resulted in CAT activity similar to the basal level. In contrast,
co-transfection of these cells with cDNA constructs expressing F,
wherein ICAM-1 and CAT vectors resulted in a two-fold increase
compared to basal CAT activity showing that interaction of RSV with
ICAM-1 is mediated by F protein. Together, these results show that
F protein binds to ICAM-1. The finding that the F and not G protein
binds to ICAM-1 is consistent with the result noted and with other
reports that antibodies to F protein have greater neutralizing
ability than antibodies to G protein (Graham et al., 1988; Levine
et al., 1987; Hall et al., 1991; Olmsted et al., 1986; Stott et
al., 1987).
[0088] To explore the possibility that blocking of ICAM-1 would
inhibit the initiation of RSV infection. HEp-2 cells were infected
with RSV after having been incubated with various concentrations of
a mAb to human ICAM-1. Since RSV infection begins when live RSV
replicates in the host cell; the intracellular viral replication
was determined 24 hours post infection using RT-PCR analysis for
the expression of mRNA of the RSV nucleocapsid (N) gene.
Preincubation of HEp-2 cells with anti-ICAM-1 inhibited amplifiable
mRNA levels in a dose-dependent manner, with the greatest
inhibition (70% compared to control) observed using a concentration
of 400 .mu.g of anti-ICAM-1 mAb/ml medium (FIG. 3A). The reduction
in virus replication was correlated with the RSV titers in the
supernatant as determined by ELISA.
[0089] Inhibition of RSV infection was also determined by
immunofluorescence by scoring the RSV-infected cells stained with
FITC-labeled mAb to RSV. Cells were counted randomly from fifteen
different spots from two different slides of each group and the
percentage of infected cells plotted against the concentration of
anti-ICAM-1 mAb (FIG. 3B). A dose-dependent reduction in the number
of infected cells occurred with increasing concentrations of
anti-ICAM-1 mAb and the greatest inhibition of infection (96%) was
observed at 400 .mu.g/ml of anti-ICAM-1 mAb. In contrast,
pre-incubation with the same concentration of isotype matched
control antibodies showed no significant reduction in RSV
replication. Two other cell lines, A549 (ATCC) and NHBE (Clonetics,
San Diego, Calif.), were examined for inhibition of RSV infection
by anti-ICAM-1 mAb, to exclude the inhibition an HEp-2 cell
specific phenomenon NHBE is a normal human primary cell line
derived from bronchial explants, whereas A549 is a transformed lung
type II alveolar epithelial cell line. NHBE and A549 exhibited
significant reductions (54% and 67%, respectively) in RSV infection
when they were preincubated with 400 .mu.g of anti-ICAM-1 mAb/ml
(FIG. 3B). These two cell lines differed from HEp-2 cells in the
magnitude of RSV infection and inhibition of RSV infection. HEp-2
cells showed the greatest infection and inhibition of infection
among the cell lines examined. The fact that preincubation of
epithelial cells with anti-ICAM-1 mAb significantly inhibited both
RSV replication and RSV infection indicates that ICAM-1 may be the
principal molecule required for the initiation of RSV infection in
these cells.
[0090] More specifically, FIG. 3 shows anti-ICAM-1 mAb treatment
inhibits RSV infection in airway epithelial cells. In FIG. 3(A),
preincubation of HEp-2 cells with anti-ICAM-1 mAb inhibited the
expression of the RSV N gene. HEp-2 cells were treated either with
anti-ICAM-1 mAb 100 .mu.g/ml (lane 4), 200 .mu.g/ml (lane 5) or 400
.mu.g/ml (lane 6), or mouse IgG, antibody (control) 400 .mu.g/ml
(lane 3), and subsequently infected with RSV (lanes 2-6 (Staunton
et al., 1989). Total RNA was subjected to RT-PCR analysis using
primers for the RSV N gene and .beta.-actin (as internal control).
RT-PCR products were quantified by densitometry, and the band
intensity relative to .beta.-actin was plotted (lower panel). In
FIG. 3(B), anti-ICAM-1 mAb treatment inhibited RSV infection of
epithelial cells. The percent of RSV infected HEp-2, NHBE and A549
cells after pre-incubation with anti-ICAM-1 mAb or an isotype
control Ab was estimated as described (Tomassini et al., 1989).
Each value represents mean.+-.SEM. The experiment was repeated for
each cell line with similar results. A representative experiment
for each of the cell lines is shown. FIG. 3C shows inhibition of
mRNA expression for RANTES and ET-1 by pretreatment of HEp-2 cells
with anti-ICAM-1 mAb prior to infection. Total RNA was subjected to
RT-PCR analysis using primers for RANTES, ET-1 and .beta.-actin for
each treatment (lanes 1-6 as described in A) was plotted.
[0091] To examine the in vivo role of ICAM-1 in the initiation of
RSV infection, a murine model of RSV infection was adapted using
C57B1/6 and BALB/c mice. Intranasal administration of RSV to C57BL6
and BALB/c mice induced lung infection, which peaked between day
three and seven post infection. RSV replication was detectable in
lung cells two to four days post infection, utilizing RT-PCR, as
described for HEp-2 cells. ICAM-1 deficient (ICAM-1.sup.-1-) mice
were used to evaluate the role of ICAM-1 in the initiation of RSV
infection (Sligh et al., 1993). The RT-PCR analysis of RSV N gene
mRNA demonstrated that in contrast to 52% ({fraction (13/25)}) of
C57BL6-ICAM-1.sup.-/-, 88% ({fraction (22/25)}) of the wild type
(ICAM-1.sup.+/+) mice permitted RSV replication in their lung
tissue (FIG. 4A). These results indicate a significant role of
ICAM-1 in the initiation of RSV infection.
[0092] ICAM-1 antibodies and anti-sense oligodeoxynucleotides
(ODNs) were previously found to protect mice from septic shock by
decreasing pulmonary inflammation (Kumasaka et al., 1996) and the
magnitude of protection ranged from 58 to 75% of control. However,
the role of anti-ICAM-1 mAbs and ODNs against the initiation of RSV
infection has not been previously examined. To confirm the in vivo
role of ICAM-1 in RSV infection, BALB/c mice were intra-tracheally
administered a combination of anti-ICAM-1 mAb and ICAM-1 anti-sense
ODNs and subsequently infected with RSV. The RSV infection was
monitored by RT-PCR of RSV-N gene mRNA from lung homogenate. The
results demonstrate that pretreatment of BALB/c mice with a
combination of ICAM-1 anti-sense ODNs and anti-ICAM-1 mAb decreased
RSV N mRNA expression by 84% anti-ICAM-1 mAb decreased RSV N mRNA
expression by 84% in RSV infected mice (FIG. 4B). These results
confirm that the presence of ICAM-1 enhances the successful
initiation of RSV infection in vivo.
[0093] More specifically, FIG. 4 shows the analysis of RSV
infection in mice. FIG. 4(A) shows the RT-PCR analysis of the lung
mRNA of RSV infected ICAM-1.sup.+/+ and ICAM-1.sup.-/- C57BL6 mice
(n=25 each) using RSV-N gene specific primers. FIG. 4(B) shows the
RT-PCR analysis of RSV infection in the lung RNA of BALB/c mice
pre-treated with ICAM-1 anti-sense ODNs and anti-ICAM-1 mAb (lanes
1-4, PBS treated; and lanes 5-8, treated with mAb and ODNs) and
subsequently infected with RSV (Oppenshaw 1995). Lung tissues were
examined for replication by RT-PCR for RSV-N and .beta.-actin
(upper panel). RT-PCR products were quantified by densitometry, and
the band intensity relative to .beta.-actin for each mouse was
plotted (Lower panel). FIG. 4(C) shows RSV-induced lung pathology
was evaluated for epithelial cell damage, interstitial-alveolar and
peribronchovascular infiltrations (Vignola et al., 1993). The bars
represent mean.+-.SEM; the asterics indicate the level significance
at .rho.<0.05; open circles, sham-infected ICAM-1.sup.+/+;
closed circles, sham-infected ICAM-1.sup.-1; open triangles,
RSV-infected ICAM-1.sup.+/+; closed triangles, RSV-infected
ICAM-1.
[0094] Histopathology of the lung sections of the ICAM-1.sup.-/-
and ICAM-1.sup.+/+ mice [for each infected (n=13) and sham infected
(n=8)] were analyzed to determine the pattern of inflammation
induced by RSV. Pathological changes were determined by scoring for
epithelial damage, interstitial-alveolar infiltrate and
peribronchovascular infiltrates (FIG. 4C). RSV infected
ICAM-1.sup.-/- mice exhibited significantly less (.rho.<0.05)
epithelial damage and interstitial-alveolar cellular infiltration
compared to the ICAM-1.sup.+/+ mice. No significant difference was
seen in peri-bronchovascular infiltration between ICAM-1.sup.-/-
and ICAM-1.sup.+/+ mice. These results show that ICAM-1 deficiency
provides a significant but only partial protection against
inflammation due to RSV infection, similar to attenuated responses
reported for neutrophil emigration in chemical peritonitis, in
ischemic injury and septic shock in ICAM-1.sup.-/- mice (Sligh et
al. 1993; Kelly et al., 1996; Xu et al., 1994). The protection in
all of these disease models is believed to be primarily due to the
role of ICAM-1 in mediating infiltration of inflammatory cells into
the lung.
[0095] A significant finding from these studies is that ICAM-1
plays a pivotal role in the initial steps of RSV infection. RSV
binds to ICAM-1 on the cell surface via its F protein. Blocking
ICAM-1 with a neutralizing anti-ICAM-1 mAb or RSV with a mAb to F
protein significantly inhibits RSV infection both in in vitro and
in vivo models. Although these studies were confined to the RSV
strain A.sub.2, RSV are considered serologically monotypic, i.e.,
antiserum to A.sub.2 neutralizes heterologous strains in in vitro
assays and in experimental animals (Graham et al., 1988; Levine et
al., 1987; Hall et al., 1991). The inventor's demonstration that a
combination of anti-ICAM-1 mAb and ICAM-1 antisense ODNs prevented
RSV infection of the lung cells in mice suggests that ICAM-1
mediation of RSV infection is not an epithelial cell specific
phenomenon. Preincubation of a human macrophage cell line, U937,
also inhibited RSV infection of these cells. Since RSV F-ICAM-1
binding is pivotal to RSV entry into the host cells, RSV can be
neutralized using a soluble ICAM-1 may be potentially useful.
[0096] Throughout this application, various publications, including
United States patents, are referenced by author and year and
patents by number. Full citations for the publications are listed
below. The disclosures of these publications and patents in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
[0097] The invention has been described in an illustrative manner,
and it is to be understood that the terminology which has been used
is intended to be in the nature of words of description rather than
of limitation.
[0098] Obviously, many modifications and variations of the present
invention are possible in light of the above teachings. It is,
therefore, to be understood that within the scope of the described
invention, the invention may be practiced otherwise than as
specifically described.
REFERENCES
[0099] Burke and Olson, "Preparation of Clone Libraries in Yeast
Artificial-Chromosome Vectors" in Methods in Enzymology, Vol. 194,
"Guide to Yeast Genetics and Molecular Biology", eds. C. Guthrie
and G. Fink, Academic Press, Inc., Chap. 17, pp. 251-270
(1991).
[0100] Capecchi, "Altering the genome by homologous recombination"
Science 244:1288-1292 (1989).
[0101] Davies et al., "Targeted alterations in yeast artificial
chromosomes for inter-species gene transfer", Nucleic Acids
Research, Vol. 20, No. 11, pp. 2693-2698 (1992).
[0102] Dickinson et al., "High frequency gene targeting using
insertional vectors", Human Molecular Genetics, Vol. 2, No. 8, pp.
1299-1302 (1993).
[0103] Duff and Lincoln, "Insertion of a pathogenic mutation into a
yeast artificial chromosome containing the human APP gene and
expression in ES cells", Research Advances in Alzheimer's Disease
and Related Disorders, 1995.
[0104] Huxley et al., "The human HPRT gene on a yeast artificial
chromosome is functional when transferred to mouse cells by cell
fusion", Genomics, 9:742-750 (1991).
[0105] Jakobovits et al., "Germ-line transmission and expression of
a human-derived yeast artificial chromosome", Nature, Vol. 362, pp.
255-261 (1993).
[0106] Lamb et al., "Introduction and expression of the 400
kilobase precursor amyloid protein gene in transgenic mice", Nature
Genetics, Vol. 5, pp. 22-29 (1993).
[0107] Pearson and Choi, Expression of the human b-amyloid
precursor protein gene from a yeast artificial chromosome in
transgenic mice. Proc. Natl. Acad. Sci. USA, 1993. 90:10578-82.
[0108] Rothstein, "Targeting, disruption, replacement, and allele
rescue: integrative DNA transformation in yeast" in Methods in
Enzymology, Vol. 194, "Guide to Yeast Genetics and Molecular
Biology", eds. C. Guthrie and G. Fink, Academic Press, Inc., Chap.
19, pp. 281-301 (1991).
[0109] Schedl et al., "A yeast artificial chromosome covering the
tyrosinase gene confers copy number-dependent expression in
transgenic mice", Nature, Vol. 362, pp. 258-261 (1993).
[0110] Strauss et al., "Germ line transmission of a yeast
artificial chromosome spanning the murine a.sub.1 (I) collagen
locus", Science, Vol. 259, pp. 1904-1907 (1993).
[0111] Gilboa, E, Eglitis, M A, Kantoff, P W, Anderson, W F:
Transfer and expression of cloned genes using retroviral vectors.
BioTechniques 4(6):504-512, 1986.
[0112] Cregg J M, Vedvick T S, Raschke W C: Recent Advances in the
Expression of Foreign Genes in Pichia pastoris, Bio/Technology
11:905-910, 1993
[0113] Culver, 1998. Site-Directed recombination for repair of
mutations in the human ADA gene. (Abstract) Antisense DNA & RNA
based therapeutics, February, 1998, Coronado, Calif.
[0114] Huston et al, 1991 "Protein engineering of single-chain Fv
analogs and fusion proteins" in Methods in Enzymology (J J Langone,
ed.; Academic Press, New York, N.Y.) 203:46-88.
[0115] Johnson and Bird, 1991 "Construction of single-chain Fvb
derivatives of monoclonal antibodies and their production in
Escherichia coli in Methods in Enzymology (J J Langone, ed.;
Academic Press, New York, N.Y.) 203:88-99.
[0116] Mernaugh and Mernaugh, 1995 "An overview of phage-displayed
recombinant antibodies" in Molecular Methods In Plant Pathology (RP
Singh and US Singh, eds.; CRC Press Inc., Boca Raton, Fla.) pp.
359-365.
[0117] Collins, P. L. 1991. The molecular biology of human
respiratory syncytial virus (RSV) of the genus Pneumovirus. In The
Paramyxoviruses. D. W. Kingsbury, Editor, Plenum, N.Y., 1991.
103-162.
[0118] Murray, A. R., and S. F. Dowell. 1997. Respiratory syncytial
virus: not just for kids. Hospital Practice, July 15:87-104.
[0119] Center for Disease Control and Prevention: Respiratory
syncytial virus activity: United States. 1996-1997 season, MMW
45:1053, 1996.
[0120] Sly, P. D., and M. E. Hibbrt. 1989. Childhood asthma
following hospitalization with acute viral bronchiolitis in
infancy. Pediatr. Pulmonol. 7:153-158.
[0121] Jeng, M. J., and R. J. Lemen 1997. Respiratory syncytial
virus and bronchiolitis. Am. Fam. Physician. 55:1139-1146.
[0122] Armstrong, D. S., and S. Menahem. 1993. Cardiac arrythmias
as manifestation of acquired heart disease in association with
pediatric respiratory syncytial virus infection. J. Ped. Child
Health 29:309-311.
[0123] Fixler, D. E. 1996. Respiratory syncytial virus infection in
children with congenital heart disease. Ped. Cardiol.
17:163-168.
[0124] Persson, C. G., J. S. Erjefalt, M. Anderson, I. Erjefalt, L.
Greiff, M. Korsgren, M. Linden, F. Sundler, and C. Svensson. 1997.
Epithelium, microcirculation, and eosinophils-new aspects of the
allergic airway in vivo. Allergy 52:241-255.
[0125] Becker, S., H. S. Koren, and D. C. Henke. 1993.
Interleukin-8 expression in normal nasal epithelium and its
modulation by infection with respiratory syncytial virus and
cytokines tumor necrosis factor, interleukin-1 and interleukin-6.
Am. J. Respir. Cell Mol. Biol. 8:20-27.
[0126] Noah, T. L., and S. Becker. 1993. Respiratory syncytial
virus-induced cytokine production by a human bronchial epithelial
cell line. Am. J. Physiol. L472-L478.
[0127] Sabauste, M. C., D. B. Jacoby, S. M. Richards, and D. Proud.
1995. Infection of a human respiratory epithelial cell line with
rhinovirus. Induction of cytokine release and modulation of
susceptibility to infection by cytokine exposure. J. Clin. Invest.
96:549-557.
[0128] Makgoba, M. W., M. E. Sanders, G. E. Ginther Luce, E. A.
Gugel, M. L. Dustin, T. A. Springer, and S. Shaw. 1998. Functional
evidence that intercellular adhesion molecule-1 (ICAM-1) is a
ligand for LFA-1 dependent adhesion in T cell-mediated
cytotoxicity. Eur. J. Immunol. 18:637-640.
[0129] Greve, J. M., G. Davis, A. M. Meyer, C. P. Forte, S. C.
Yost, C. W. Marlor, M. E. Kamarck, and A. McClelland. 1989. The
major human rhinovirus receptor is ICAM-1. Cell 56:839-847.
[0130] Staunton, D. E., J. Merluzzi, R. Rothlein, R. Barton, S. D.
Marlin, and T. A. Springer. 1989. A cell adhesion molecule, ICAM-1,
is the major surface receptor for rhinoviruses. Cell
56:849-853.
[0131] Tomassini, J. E., D. Graham, C. M. Dewitt, D. W. Lineberger,
J. A. Rodkey, and R. J. Colonno. 1989. CDNA cloning reveals that
the major group rhinovirus receptor on HeLa cells in intercellular
adhesion molecule 1. Proc. Natl. Acad. Sci. USA. 86:4907-4911.
[0132] Chini, B. A., M. A. Fiedler, L. Milligan, T. Hopkins, and J.
M. Stark. 1998. Essential roles of NF-kappa B and c/EBP in the
regulation of intercellular adhesion molecule-1 after respiratory
syncytial virus infection of human respiratory epithelial cell
cultures. J. Virol. 72:1623-1626.
[0133] Stark, J. M., V. Godding, J. B. Sedgwick, and W. W. Busse.
1996. Respiratory syncytial virus infection enhances neutrophil and
eosinophil adhesion to intercellular adhesion molecule-1. J.
Immunol. 156:4774-4782.
[0134] Arnold, R., H. Werchau, and W. Konig. 1995. Expression of
adhesion molecules (ICAM-1, LFA-3) on human epithelial cells (A549)
after respiratory syncytial virus infection. Int. Arch. Allergy
Immunol. 107:392-393.
[0135] Vignola, A. M., A. M. Campbell, P. Chanez, J. Bousquet, P.
Paul-Lacoste, F. B. Michael, P. Godard. 1993. HLA-DR and ICAM-1
expression on bronchial epithelial cells in asthma and chronic
bronchitis. Am. Rev. Respir. Dis. 148: 689-94.
[0136] Hegele, R. G., S. Hayashi, A. M. Bramley, and J. C. Hogg.
1994. Persistence of respiratory syncytial virus genome and protein
after acute bronchiolitis in guinea pigs. Chest 105: 1848-1854.
[0137] Anderson J. J., J. Nordan, D. Saunders, G. L. Toms, and R.
Scott. 1990. Analysis of the local and systemic immune responses
induced in BALB/c mice by experimental respiratory syncytial virus
infection. J. Gen. Virol. 71, 1561-1570.
[0138] Oppenshaw, P. J. M. 1995. Immunity and immunopathology to
respiratory syncytial virus: the mouse model. Am. J. Res. Crit.
Care Med. 152, S59-62.
[0139] Hsu, S. C., D. Chargelegue, and M. W. Steward. 1998.
Reduction of respiratory syncytial virus titer in the lungs of mice
after intranasal immunization with a chimeric peptide consisting of
a single CTL epitope linked to a fusion peptide. Virology. 240,
376-381.
[0140] Tayler, G., E. J. Scott, M. Hugh, and A. P. Collins. 1984.
Respiratory syncytial virus infection in mice. Infection and
Immunity 43:649-655.
[0141] Graham, B. S., M. D. Perkins, P. F. Wright, and D. T.
Karzon. 1988. Primary respiratory syncytial virus infection in
mice. J. Med. Virol. 26:153-162.
[0142] Levine, S., R. Klaiber-Franco, and P. R. Paradiso. 1987.
Demonstration that glycoprotein G is the attachment protein of
respiratory syncytial virus. J. Gen. Virol. 68:2521-2524.
[0143] Hall, C. B., E. E. Walsh, C. E. Long, and K. C. Schnabel.
1991. Immunity to and frequency of reinfection with respiratory
syncytial virus. J. Infec. Dis. 163:693-698.
[0144] Olmsted, R. A., R. M. Chanock, and P. L. Collins. 1986.
Expression of the F glycoprotein of respiratory syncytial virus by
a recombinant vaccinia virus: comparison of the individual
contributions of the F and G glycoproteins to host immunity. Proc.
Natl. Acad. Sci. USA 83:7462-7466.
[0145] Stott, E. J., G. Taylor, L. A. Ball, K. Anderson, K. K.
Young, A. M. King, and G. W. Wertz. 1987. Immune and
histopathological responses in animals vaccinated with recombinant
vaccinia viruses that express individual genes of human respiratory
syncytial virus. J. Virol. 61:3855-3861.
[0146] Li, X., S. Sambhara, C. X. Li, M. Ewasyshyn, M. Parrington,
J. Caterini, O. James, G. Gates, R. P. Du, and M. Klein. 1998. J.
Exp. Med. 188:681-688.
[0147] Becker, S. W. Reed, F. W. Henderson, and T. L. Noah. 1997.
RSV infection of human airway epithelial cells causes production of
the beta-chemokine RANTES. Am. J. Physiol. 272 (Lung Cell. Mol.
Physiol. 16): L512-L520.
[0148] Saito, T., R. W. Deskin, A. Casola, H. Haeberle, B.
Olszewska, P. B. Ernst, R. Alam, P. L. Ogra, and R. Garofalo. 1997.
Respiratory syncytial virus induces selective production of the
chemokine RANTES by upper airway epithelial cells. J. Infect. Dis.
175:497-504.
[0149] Behera, A. K., M. Kumar, H. Matsuse, R. F. Lockey, and S. S.
Mohapatra. 1998. Respiratory syncytial virus induces the expression
of 5-lipoxygenase and endothelin-1 in bronchial epithelial cells.
Biochem. Biophys. Res. Commun. In pres.
[0150] Sligh, J. E., C. M. Ballantyne, and S. S. Rich, H. K.
Hawkins, C. W. Smith, A. Bradley, and A. L. Beaudet. 1993.
Inflammatory and immune responses are impaired in mice deficient in
intercellular adhesion molecule-1. Proc. Natl. Acad. Sci. USA.
90:8529-8533.
[0151] Kelly, K. J., W. W. Williams Jr., R. B. Colvin, S. M.
Meehan, T. A. Springer, J. C. Gutierrez-Ramos, and J. V. Bohventre.
1996. Intercellular adhesion molecule-1 deficient mice are
protected against ischemic renal injury. J. Clin. Invest.
97:1056-1063.
[0152] Xu, H., J. A. Gonzalo, Y. St. Pierre, I. R. Williams, T. S.
Kupper, R. S. Cotran, T. A. Springer, and J. C. Gutierrez-Ramos.
1994. Leukocytosis and resistance to septic shock in intercellular
adhesion molecule-1 deficient mice. J. Exp. Med. 180:95-109.
[0153] Kumasaka, T., W. M. Quinlan, N. A. Doyle, T. P. Condon, J.
Sligh, F. Takei, A. L. Beaudet, C. F. Bennett, and C. M. Doerschuk.
1996. Role of the intercellular adhesion molecule-1 (ICAM-1) in
endotoxin-induced pneumonia evaluated using ICAM-1 antisense
oligonucleotides, anti-ICAM-1 monoclonal antibodies, and ICAM-1
mutant mice. J. Clin. Invest. 97:2362-2369.
[0154] Almenar-queralt, A., A. Duperray, L. A. Miles, J. Felez, and
D. C. Altieri. 1995. Apical topography and modulation of ICAM-1
expression on activated endothelium. Am. J. Pathol.
147:1278-1288.
[0155] Staunton, D. E., A. Gaur, P. Y. Chan, and T. A. Springer.
1992. Internalization of a major group human rhinovirus does not
require cytoplasmic or transmembrane domains of ICAM-1. J. Immunol.
148:3271-3274.
[0156] Huguenel, E. D., D. Cohn, D. P. Dockum, J. M. Greve, M. A.
Fournel, L. Hammond, R. Irwin, J. Mahoney, A. McClelland, E.
Muchmore, A. C. Ohlin, and P. Scuderi. 1997. Prevention of
rhinovirus infection in chimpanzees by soluble intercellular
molecule-1. Am. J. Respir. Crit. Care Med. 155:1206-1210.
[0157] Bella, J., P. R. Kolatkar, C. W. Marlor, J. M. Greve, and M.
G. Rossmann. 1998. The structure of the two amino-terminal domains
of human ICAM-1 suggests how it functions as a rhinovirus receptor
and as an LFA-1 integrin ligand. Proc. Natl. Acad. Sci. USA
95:4140-4145.
[0158] Casanovas, J. M., T. Stehle, J. Liu, J. Wang, and T. A.
Springer, 1998. A dimeric crystal structure for the N-terminal two
domains of intercellular adhesion molecule-1 Proc. Natl. Acad. Sci.
USA 95:4134-4139.
Sequence CWU 1
1
12 1 33 DNA Artificial sequence Artificial sequence (1)..(33)
Primer 1 cctggcgaat tccagacatc tgtgtccccc tca 33 2 24 DNA
Artificial sequence Artificial sequence (1)..(24) reverse primer
for ICAM-1 2 gtgtggatcc actgccacca atat 24 3 32 DNA Artificial
sequence Artificial sequence (1)..(32) Forward primer for F gene 3
caagaattca tggagttgct aatcctcaaa ca 32 4 36 DNA Artificial sequence
Artificial sequence (1)..(36) Reverse primer for F gene 4
ctatgtcgac ttagttacta aatgcaatat tattta 36 5 33 DNA Artificial
sequence Artificial sequence (1)..(33) Forward primer for G gene 5
aatgaattca tgtccaaaaa caaggaccaa cgc 33 6 30 DNA Artificial
sequence Artificial sequence (1)..(30) Reverse primer for G gene 6
gttgtcgact aactactggc gtggtgtgtt 30 7 22 DNA Artificial sequence
Artificial sequence (1)..(22) Forward primer for RSV-N 7 gcgatgtcta
ggttaggaag aa 22 8 24 DNA Artificial sequence Artificial sequence
(1)..(24) Reverse primer for RSV-N 8 gctatgtcct tgggtagtaa gcct 24
9 18 DNA Artificial sequence Artificial sequence (1)..(18) Forward
primer for ICAM-1 9 atggctccca gcagcccc 18 10 18 DNA Artificial
sequence Artificial sequence (1)..(18) Reverse primer for ICAM-1 10
cacctggcag cgtagggt 18 11 18 DNA Artificial sequence Artificial
sequence (1)..(18) Forward primer for Beta-actin 11 cgcgagaaga
tgacccag 18 12 19 DNA Artificial sequence Artificial sequence
(1)..(19) primer 12 atcacgatgc cagtcgtac 19
* * * * *