U.S. patent application number 12/390246 was filed with the patent office on 2011-03-03 for compositions comprising anti-icam-1 antibodies.
This patent application is currently assigned to University of South Florida. Invention is credited to Aruna Behera, Mukesh Kumar, Hiroto Matsuse, Shyam S. Mohapatra.
Application Number | 20110052601 12/390246 |
Document ID | / |
Family ID | 34594190 |
Filed Date | 2011-03-03 |
United States Patent
Application |
20110052601 |
Kind Code |
A1 |
Mohapatra; Shyam S. ; et
al. |
March 3, 2011 |
COMPOSITIONS COMPRISING ANTI-ICAM-1 ANTIBODIES
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. 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.; (Lutz,
FL) ; Behera; Aruna; (Watertown, MA) ;
Matsuse; Hiroto; (Nagasaki, JP) ; Kumar; Mukesh;
(Watertown, MA) |
Assignee: |
University of South Florida
Tampa
FL
|
Family ID: |
34594190 |
Appl. No.: |
12/390246 |
Filed: |
February 20, 2009 |
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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12390246 |
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10440000 |
May 16, 2003 |
6900299 |
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11079834 |
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09523054 |
Mar 10, 2000 |
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10440000 |
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60123999 |
Mar 11, 1999 |
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Current U.S.
Class: |
424/159.1 |
Current CPC
Class: |
A61P 11/00 20180101;
A61P 31/12 20180101; C12N 2310/11 20130101; C12N 2320/32 20130101;
C12N 15/1138 20130101; C12N 15/111 20130101 |
Class at
Publication: |
424/159.1 |
International
Class: |
A61K 39/42 20060101
A61K039/42; A61P 11/00 20060101 A61P011/00; A61P 31/12 20060101
A61P031/12 |
Claims
1. A method of preventing a respiratory syncytial virus (RSV)
infection, comprising administering an effective amount of an
antibody to ICAM-1 to a patient sufficient to prevent the RSV
infection.
2. The method according to claim 1, wherein the antibody blocks the
RSV-F binding site on ICAM-1.
3. The method according to claim 1, wherein the patient is
human.
4. The method according to claim 1, wherein said administering
comprises administering the antibody to airway epithelial
cells.
5. The method according to claim 1, wherein said administering
comprises administering the antibody orally.
6. The method according to claim 1, wherein said administering
comprises administering the antibody intranasally.
7. The method according to claim 1, wherein said administering
comprises administering the antibody by inhalation.
8. The method according to claim 1, wherein said administering
comprises injecting the antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/079,834, filed Mar. 14, 2005, which is a continuation of
U.S. application Ser. No. 10/440,000, filed May 16, 2003, now U.S.
Pat. No. 6,900,299, which is a continuation of U.S. application
Ser. No. 09/523,054, filed Mar. 10, 2000, now abandoned; which
claims the benefit of U.S. Provisional Application Ser. No.
60/123,999, filed Mar. 11, 1999; each 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 August-September; 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 August;
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; Bank, 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 (Sheihamer 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 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
General Methods
Gene Therapy:
[0034] 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).
[0035] 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
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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
Materials and Methods
[0055] Cell culture, virus and infection. 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. is 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.
[0056] Confocal microscopy. 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.
[0057] Assay for RSV-ICAM-1 Interaction. 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.
[0058] Flow cytometry. 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.).
[0059] Mammalian two-hybrid assay. 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.
[0060] ICAM-1 blocking in vitro. 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.
[0061] Reverse transcriptase PCR analysis. 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.
[0062] Immunofluoroscence. 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 Fluoromount 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.
[0063] Animals and Virus Infection. 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 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).
[0064] ICAM-1 blocking in vivo. 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.
[0065] Histopathology. 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). Intraobserver variation was <5%.
[0066] Statistical analysis. 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.
[0067] 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 .rho. 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.).
Results and Discussion
[0068] 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-10N HEp-2 cell surfaces (FIG.
1D) suggested that RSV directly binds to ICAM-1 on epithelial
cells
[0069] 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. 10 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.
[0070] 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 it F
protein.
[0071] 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).
[0072] 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.
[0073] 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-1mAb 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.
[0074] 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.sub.1 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.
[0075] 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% (13/25) of
C57BL6-ICAM-1.sup.-/-, 88% (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.
[0076] 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.
[0077] 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.-/-; open triangles,
RSV-infected ICAM-1.sup.+/+; closed triangles, RSV-infected
ICAM-1.
[0078] 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.
[0079] 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; Hail 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.
[0080] 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.
[0081] 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.
[0082] 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.
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Sequence CWU 1
1
12133DNAArtificial sequenceArtificial sequence(1)..(33)Primer
1cctggcgaat tccagacatc tgtgtccccc tca 33224DNAArtificial
sequenceArtificial sequence(1)..(24)reverse primer for ICAM-1
2gtgtggatcc actgccacca atat 24332DNAArtificial sequenceArtificial
sequence(1)..(32)Forward primer for F gene 3caagaattca tggagttgct
aatcctcaaa ca 32436DNAArtificial sequenceArtificial
sequence(1)..(36)Reverse primer for F gene 4ctatgtcgac ttagttacta
aatgcaatat tattta 36533DNAArtificial sequenceArtificial
sequence(1)..(33)Forward primer for G gene 5aatgaattca tgtccaaaaa
caaggaccaa cgc 33630DNAArtificial sequenceArtificial
sequence(1)..(30)Reverse primer for G gene 6gttgtcgact aactactggc
gtggtgtgtt 30722DNAArtificial sequenceArtificial
sequence(1)..(22)Forward primer for RSV-N 7gcgatgtcta ggttaggaag aa
22824DNAArtificial sequenceArtificial sequence(1)..(24)Reverse
primer for RSV-N 8gctatgtcct tgggtagtaa gcct 24918DNAArtificial
sequenceArtificial sequence(1)..(18)Forward primer for ICAM-1
9atggctccca gcagcccc 181018DNAArtificial sequenceArtificial
sequence(1)..(18)Reverse primer for ICAM-1 10cacctggcag cgtagggt
181118DNAArtificial sequenceArtificial sequence(1)..(18)Forward
primer for Beta-actin 11cgcgagaaga tgacccag 181219DNAArtificial
sequenceArtificial sequence(1)..(19)primer 12atcacgatgc cagtcgtac
19
* * * * *