WO2000027423A2

WO2000027423A2 - Methods and compositions for treating common cold symptoms

Info

Publication number: WO2000027423A2
Application number: PCT/US1999/027003
Authority: WO
Inventors: Ronald B. Turner
Original assignee: Musc Foundation For Research Development
Priority date: 1998-11-12
Filing date: 1999-11-12
Publication date: 2000-05-18
Also published as: AU2149600A; WO2000027423A3

Abstract

The present invention provides methods of inhibiting replication of cold-causing viruses in respiratory epithelium cells and elaboration of IL-8 in the cells of subjects exposed to the viruses, by interfering with the virus binding to the cell membrane receptor that stimulates elaboration of IL-8 and viral replication. A polypeptide that binds to a virus and interferes with the binding of the virus to the cell is provided. An antibody that binds to a cell membrane-bound cytochrome and interferes with the virus binding to the cytochrome is also provided. Further, a chemical inhibitor of a membrane-bound cytochrome, that inhibitis viral replication in and elaboration of IL-8 by the cell, is provided. Moreover, the present invention provides methods of treating and reducing cold symptoms in a subject comprising administering to a subject any of the compositions of this invention. Further, the invention provides methods of screening for a compound which inhibits the interaction of a cold-causing virus and a respiratory epithelium cell.

Description

METHODS AND COMPOSITIONS FOR TREATING COMMON COLD

SYMPTOMS

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates to the treatment of viral infections of the upper respiratory tract. In particular, the present invention relates to a method of treating cold symptoms in a subject by inhibiting the interaction between the virus and respiratory epithelium membrane-bound cytochrome. More specifically, the invention relates to methods of inhibiting viral replication and elaboration of IL-8 in a subject by administering a polypeptide, antibody or a chemical that inhibits the interaction between the virus and respiratory epithelium membrane-bound cytochrome.

BACKGROUND ART

Infection with a variety of different upper respiratory pathogens and rhinoviruses in particular, may result in common cold symptoms, such as rhinorrhea, nasal obstruction, sneezing, cough, sore throat, malaise, headache and chills (47). The pathogenesis of the symptoms of these infections is not known, but the host response to the virus may cause at least some of the manifestations. Kinins appear in nasal washes from symptomatic human volunteers with rhinovirus colds (48,49) and challenge of human subjects with bradykinin causes rhinorrhea and nasal obstruction (50). Similarly, an increased level of interleukin-lβ (IL-lβ) has been reported in the nasal secretions of subjects with symptomatic rhinovirus colds (51). In experimental colds in human volunteers, polymorphonuclear leukocytes (PMNs) appear in the nasal mucosa early in the course of infection (52). Examination of nasal secretions during experimental colds revealed an influx of PMNs in rhinovirus-infected volunteers who became ill that was not seen in infected non-ill or uninfected volunteers (48). Although this observed association between PMNs and symptomatic infections does not establish a role for PMNs in the pathogenesis of rhinovirus colds, identification of the mechanism by which PMNs are attracted to the nasal mucosa may provide insight into the pathogenesis of rhinovirus induced symptoms.

Interleukin-8 (IL-8) is a proinflammatory, leukocyte-derived cytokine with chemoattractant activity for PMNs (53,54). Infection of human respiratory epithelium with respiratory syncytial virus (RSV) or influenza A virus results in increased production of IL-8 (55, 56, 57). There is increasing evidence that virus-induced elaboration of proinflammatory cytokines plays a role in the pathogenesis of viral upper respiratory illness. The concentrations of the interleukins IL-6 and IL-8 increase in the nasal secretions of subjects with symptomatic rhinovirus infection (20, 22, 63, 19) and there is a direct correlation between the severity of the common cold symptoms and the concentration of IL-8 and IL-6 in the secretions (22, 63) . It has also been reported that intranasal challenge of normal subjects with IL-8 produces a symptom complex that mimics in some respects the common cold (23) .

Previous efforts to treat the common cold have used two general approaches- antiviral therapy, which has been uniformly unsuccessful except as prophylaxis, and symptomatic therapy, which requires combination therapy and is generally of limited efficacy. Thus, there exists a need for an effective therapy to relieve the symptoms of the common cold. Inhibition of IL-8 elaboration has a potential advantage since inhibition of the effects of viral infection early in the cascade of events that results in symptoms has the potential to affect different symptoms with a single agent.

The present invention provides an improved method for alleviating common cold symptoms and a method of inhibiting viral replication and IL-8 elaboration by administering a polypeptide, antibody or chemical that interferes with the interaction of the cold-causing virus and a membrane-bound cytochrome.

SUMMARY OF THE INVENTION

The present invention provides a method of inhibiting the elaboration of IL-8 from a cell in a subject, by blocking the interaction between an IL-8-inducing virus and membrane-bound cytochrome of the cell. A polypeptide that binds to the virus, an antibody that specifically binds to a cell membrane-bound cytochrome, or a chemical inhibitor of the membrane-bound cytochrome activity interferes with the virus interacting with the cell. This interference of the virus binding to the cell inhibits IL-8 elaboration.

Further, the present invention provides a composition comprising a polypeptide comprising an amino acid sequence weighing 65-67 kDa and a pharmaceutically acceptable carrier.

The present invention provides a method of inhibiting viral replication in a cell of a subject, by blocking the interaction of the cell with an IL-8-inducing virus. A polypeptide that binds to the virus, an antibody that specifically binds to a cell membrane-bound cytochrome, or a chemical inhibitor of the membrane-bound cytochrome activity interferes with the virus interaction with the cell. This interference of the virus binding to the cell inhibits viral replication in the cell.

The present invention provides a method of screening for a chemical compound that inhibits the interaction of a cold-causing virus with a cell, comprising the steps of a) contacting a cell that expresses a cell membrane-bound cytochrome with the compound and b) determining whether the compound binds the membrane-bound cytochrome on the cell. The binding to the cytochrome indicates that the compound inhibits the interaction of virus and the cell.

Further, the present invention provides a method of screening for a chemical compound that inhibits the interaction of a cold-causing virus and a cell membrane- bound cytochrome, comprising the steps of a) contacting a virus or cell with the compound, b) contacting a cell that expresses a cell membrane-bound cytochrome with the virus of step a), and c) determining whether the virus of step a) is inhibited from interacting in the usual manner with the membrane-bound cytochrome of the cell. Moreover, the present invention provides a method of screening for a chemical compound that inhibits the interaction of a cold-causing virus with a cell, comprising the steps of a) contacting a cell that expresses a cell membrane-bound cytochrome with the compound and b) determining that the compound inhibits the virus from stimulating the cytochrome. The inhibition of stimulation of the cytochrome is shown by decreased production of superoxide or decreased elaboration of IL-8 by the cell.

The present invention provides compositions comprising a polypeptide that binds to a virus, an antibody that specifically binds to a cell membrane-bound cytochrome and a chemical inhibitor of a membrane-bound cytochrome, for example NADPH-oxidase activity, in a pharmaceutically acceptable carrier.

Further, the invention provides a method of treating cold symptoms by administering to a subject a polypeptide, antibody or chemical inhibitor of a membrane- bound cytochrome, for example NADPH-oxidase, that interferes with the viral interaction with the cell and the NADPH-oxidase or membrane-bound cytochrome activity of the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Replication of rhinovirus and IL-8 elaboration in cells pre-incubated with antibody to ICAM-1

Figure 2. Virus replication and IL-8 elaboration associated with the variant strain of rhinovirus 39 (RV39vs)

Figure 3. Effect of diphenylene iodonium (DPI, 40 μM), phenylarsine oxide (PAO, 500 nM), allopurinol (500 μM), and rotenone (10 μM) on virus induced IL-8 elaboration and rhinovirus replication in fibroblast cells.

Figure 4. IL-8 elaboration and virus replication in skin fibroblast cells from a patient with chronic granulomatous disease (CGD) and from normal skin. Figure 5. Effect of anti-ICAM-1 antibody on rhinovirus replication and rhinovirus-induced IL-8 elaboration.

Figure 6. IL-8 elaboration induced by challenge of MRC-5 cells and BEAS-2B cells with "wild-type" and variant strain rhinovirus.

Figure 7. Effect of diphenylene iodonium (DPI, 40 μM) on NBT dye reduction and IL-8 elaboration in response to challenge of MRC-5 cells with rhinovirus type 39.

Figure 8. Effect of diphenylene iodonium (DPI, 40 μM), ibuprofen (100 μM), allopurinol (500 μM), and rotenone (10 μM) on rhinovirus replication and rhinovirus-induced IL-8 elaboration in MRC-5 cells.

Figure 9. Effect of diphenylene iodonium (DPI, 40 μM) on virus replication and virus-induced IL-8 elaboration following challenge of MRC-5 cells with respiratory syncytial virus (A) or coronavirus 229E (B). The quantitative coronavirus titer in the presence of DPI is the mean of two experiments.

Figure 10. Superoxide anion production as measured by NBT dye reduction (A) and H2O2 production (B) in response to rhinovirus challenge in normal skin fibroblasts, skin fibroblasts from a patient deficient in gp9l-phox, and skin fibroblasts from a patient deficient in p41-phox both with (RV/DPI) and without (RV) preincubation of the cells with DPI (40 μM).

Figure 11. IL-8 elaboration induced by rhinovirus, coronavirus and respiratory syncytial virus (RSV) in normal skin fibroblasts, skin fibroblasts from a patient deficient in gp9\-phox, and skin fibroblasts from a patient deficient in p41-phox. DETAILED DESCRIPTION OF THE INVENTION

As used herein, "a" or "an" may mean one or more. For example, "a" cell may mean one cell or more than one cell.

The present invention comprises several ways of treating or reducing common cold symptoms that arise after respiratory epithelium cells are affected by cold- producing viruses. One embodiment of the invention is the inhibition of elaboration of IL-8 by respiratory epithelium cells, by blocking the interaction of the cell with an IL- 8-inducing virus. For example, a polypeptide that binds to the IL-8-inducing virus at a site of contact between the IL-8-inducing virus and a cell membrane-bound cytochrome interferes with the virus binding to the membrane-bound cytochrome and thereby inhibits elaboration of IL-8.

As used herein, a "respiratory epithelium cell" is a cell which lines the upper respiratory tract of mammals and in particular human beings. The cell is in direct contact with the upper airway passages of the nose. Moreover, it is contemplated that respiratory fibroblast cells are acted upon by this invention.

As used herein, a "cold-producing virus" or "cold-causing virus" is any one of many viruses that can interact with the upper respiratory tract of mammals, in particular human beings, to produce symptoms of rhinorrhea (runny nose), congestion with difficulty breathing through the nose, malaise, headache and fever. There are many viruses known to cause infection of the upper respiratory epithelium of human beings. Rhinoviruses, respiratory syncytial virus (RSV) and corona viruses are known common cold pathogens. These are IL-8-inducing viruses. Rhinoviruses are the principal cause of colds in human beings.

As used herein, an "IL-8-inducing virus" is one which stimulates an affected cell to produce interleukin-8 cytokine. "Inhibit," as used herein, means to restrain, block, or suppress. In the embodiments of the invention, "inhibition" means that a process may be totally blocked or partially blocked. Moreover, the process may be lessened in severity or frequency.

As used herein, "blocking the interaction" means to interfere with or impede the interaction of two entities. For example, something that prevents a virus from making contact with a target cell is said to block the interaction between the virus and the cell. The present method provides a means of blocking a specific interaction between the virus and the cell. The blocking or interfering can be via specific binding, steric interaction or chemical action.

In this invention, "site of contact" means the location on the surface of a virus that normally is exposed and touches or binds with a target cell.

As used herein, a "cell membrane-bound cytochrome" is a heme-containing protein that transfers electrons during cellular respiration and is located, at least partially, within the external membrane of the cell. Gp91-phox is one specific example of a membrane-bound cytochrome and part of NADPH-oxidase. NADPH-oxidase is a membrane-bound protein and is composed of five subunits, one of which is gp91-phox, a membrane-bound cytochrome. The gp91-phox subunit comprises two extracellular domains.

A 65-67 kDa membrane-bound protein is another example of a membrane- bound cytochrome. A person skilled in the art recognizes that this membrane-bound protein is approximately 67 kDa. Because the electrophoresis technique used to measure the protein may vary from one laboratory to another, the protein may actually be as low as 63 kDa and as high as 69 kDa. This 65-67 kDa protein may be p67-phox, a protein currently recognized as a cytoplasmic component of NADPH-oxidase. The 65-67 kDa protein may be a distinct membrane-bound cytochrome involved in virus replication and IL-8 elaboration. The 65-67 kDa protein is shown herein to bind to DPI and to be present in fibroblast membranes. "Binding to" in the embodiments of this invention means "linked to" in any of several ways including, but not limited to, covalently, ionically, sterically and mechanically.

"Isolated" as used herein means the polypeptide of this invention is sufficiently free of contaminants or cell components with which polypeptides normally occur and is present in such concentration as to be the only significant polypeptide present in the sample. "Isolated" does not require that the preparation be technically pure (homogeneous), but it is sufficiently pure to provide the peptide or polypeptide in a form in which it can be used therapeutically.

Cold-causing viruses produce symptoms in subjects when the viruses contact cells that line the upper respiratory tract in mammals. These cells are respiratory epithelium cells and respiratory fibroblast cells. The present invention is based on the unexpected and surprising discovery that cold-causing viruses, contacting a respiratory epithelium cell membrane-bound cytochrome, for example the 65-67 kDa protein, stimulate viral replication and elaboration of IL-8 by the cell, causing typical cold symptoms of rhinorrhea, congestion, malaise and fever.

Moreover, the influenza viruses and adenoviruses, although not commonly referred to as cold-producing viruses, generate oxidative stress and induce IL-8 elaboration. There is evidence that inhibiting influenza viral replication decreases the incidence and lessens the severity of secondary otitis media and sinusitis. Further, asthmatic subjects with higher levels of IL-8 in their respiratory secretions have more severe symptoms of bronchial obstruction (58). Thus, the present invention can have a beneficial effect in treating these viruses as well as with rhinovirus, RSV and corona virus.

The present data teach that viral interaction with membrane-bound cytochrome is responsible for the induction of oxidative stress and IL-8 elaboration. Because this interaction between rhinovirus and the cell is responsible for triggering the cytokine response of the cell and may also be necessary for viral replication, there is a need to interfere with that interaction. As described in detail below, treatment of cells with inhibitors of NADPH-oxidase, diphenylene iodonium or phenylarsine oxide, inhibits viral replication as well as IL-8 elaboration. The simultaneous interruption of both viral replication and virus-induced cytokine elaboration by inhibiting the interaction between rhinovirus and NADPH-oxidase or a membrane-bound cytochrome is the target for the present anti-rhinovirus therapy. The invention is a significant improvement over the prior art because it provides a method of treating cold symptoms caused by a large number of viruses, particularly rhinoviruses.

The present invention provides a method of reducing cold symptoms in a subject by blocking the interaction of the subject's respiratory epithelium cells with an IL-8-inducing cold virus that infects those cells. An antibody of this invention can be used to contact the cells under conditions that allow the antibody to bind a cell membrane-bound cytochrome. By binding to the cytochrome, the antibody prevents the virus from interacting with the cytochrome as it would in the untreated subject. By blocking virus interaction with the cell, the antibody inhibits the virus from replicating and inducing IL-8 elaboration in the cell, and the subject would suffer fewer and milder cold symptoms, such as rhinorrhea, congestion, malaise and fever.

The antibody to the membrane-bound ctyochrome may be polyclonal or monoclonal and can be prepared by immunology protocols that are standard in the art, such as, for example, as set forth in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988. Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells can then be fused with an immortal cell line and screened for monoclonal antibody secretion. Humanized and chimeric antibodies are also contemplated in this invention. Heterologous antibodies can be made by well known methods (See, for example, US Patents 5545806, 5569825,5625126, 5633425, 5661016, 5770429, 5789650, and 5814318). An antibody of the present invention is described in Example 3. In another embodiment of the method of reducing cold symptoms in a subject by blocking the interaction of the subject's respiratory epithelium cells and an IL-8- inducing virus that infects those cells, a polypeptide can be administered that binds to the virus. The polypeptide of this invention binds to the virus at a site of contact between the IL-8-inducing virus and a cell membrane-bound cytochrome. This binding makes the viral binding sites unavailable for contact with the cell membrane-bound cytochrome of the respiratory epithelium cell. The virus is then inhibited from replicating and inducing IL-8 elaboration in the cell, and the subject suffers fewer and milder cold symptoms, such as rhinorrhea, congestion, malaise and fever.

In one example of the present method, the polypeptide used in the invention comprises an amino acid sequence corresponding to at least part of the membrane- bound cytochrome. Thus, the polypeptide can comprise an amino acid sequence comprising part or all of a 65-67 kDa protein. In the embodiment in which the membrane-bound cytochrome comprises p67-phox, the peptide can be SEQ ID NO:l or a fragment thereof. It is contemplated that the polypeptide used in the method can interact sterically with other regions of the virus adjacent to the normal site of contact with the cell, thus specifically interfering with the interaction of the virus with the cell.

As used herein, a "polypeptide" is a chain of amino acids which corresponds to those encoded by a nucleic acid. A polypeptide usually describes a chain of amino acids having more than about 5 amino acids. The term "polypeptide" can refer to a linear chain of amino acids or it can refer to a chain of amino acids which has been processed and folded into a functional protein. The polypeptide of the present invention can be obtained by isolation and purification of the polypeptide from cells where they are produced naturally or by expression of exogenous nucleic acid encoding the polypeptide. The polypeptide of this invention can be obtained by well-known methods of chemical synthesis, by proteolytic cleavage of a polypeptide and/or by synthesis from nucleic acid encoding the polypeptide.

It is contemplated that other membrane-bound cytochromes with an amino acid sequence similar to the 65-67 kDa protein or having a similar steric conformation may also be the site of interaction between the virus and the cell. Moreover, the other membrane-bound cytochromes would also bind to DPI. Thus, additional examples of a polypeptide that binds to the cold-causing virus and inhibits the virus from contacting the cell can be obtained from the membrane-bound cytochrome.

Another embodiment of this invention is a method of reducing cold symptoms in a subject by inhibiting activity of a membrane-bound cytochrome of a respiratory epithelium cell. Contacting the cell with a chemical inhibitor of cytochrome activity blocks the effect of an IL-8-inducing cold virus on the cell. Because the chemical inhibitor of the cytochrome will reduce the ability of the virus to stimulate elaboration of IL-8 in the cell, the subject will have fewer and milder cold symptoms, such as rhinorrhea, congestion, malaise and fever. One example of the membrane-bound cytochrome is the 65-67 kDa protein.

The present invention also provides a method of inhibiting elaboration of IL-8 from a cell by blocking the interaction of the cell and the cold-causing virus by contacting the cell with an antibody that binds to a cell membrane-bound cytochrome under conditions that permit the antibody to contact the cell. Once the antibody has bound to the cytochrome, the cold-causing virus is blocked from binding to the membrane-bound cytochrome, and the virus can not stimulate the cell to elaborate IL-8. The membrane-bound cytochrome can be a 65-67 kDa protein.

The invention provides a method of inhibiting replication of an IL-8-inducing virus in a cell by contacting the virus with a polypeptide of this invention. The polypeptide binds to the IL-8-inducing virus at a site of contact between the virus and a cell membrane-bound cytochrome, thus interfering with virus interaction with the membrane-bound cytochrome. This interference inhibits replication of the virus in the cell.

The present invention provides a method of inhibiting replication of IL-8- inducing virus, by blocking the interaction of the cell with an IL-8-inducing virus, by contacting the cell with an antibody of this invention. The antibody binds a cell membrane-bound cytochrome and blocks the virus from binding to the membrane- bound cytochrome, thus inhibiting viral replication. The antibody of this invention can bind the region of a membrane-bound cytochrome which comprises an amino acid sequence of a 65-67 kDa protein.

The present invention provides a method of inhibiting elaboration of IL-8 from a cell of a subject by inhibiting a membrane-bound cytochrome, by contacting the cell with a chemical inhibitor of the membrane-bound cytochrome. The chemical inhibitor of the cytochrome activity results in decreased elaboration of IL-8. The chemical inhibitor of the cytochrome can be diphenylene iodonium or phenylarsine oxide. Further, any chemical which inhibits the cytochrome can be used in this method. Moreover, the present invention provides a composition comprising a chemical inhibitor of the membrane-bound cytochrome and a pharmaceutically acceptable carrier. One example of a membrane-bound cytochrome which is inhibited by a chemical inhibitor is a 65-67 kDa protein.

The present invention provides a method of inhibiting replication of an IL-8- inducing virus in a cell of a subject already infected by the virus, by inhibiting the activity of a membrane-bound cytochrome with a chemical inhibitor of the cytochrome. The chemical inhibition of the cytochrome results in inhibition of viral replication in the cell. The chemical inhibitor of the membrane-bound cytochrome can be diphenylene iodonium or phenylarsine oxide. Further, any chemical which inhibits the activity of the cytochrome can be used in this method. Moreover, the present invention provides a composition comprising a chemical inhibitor of membrane-bound cytochrome activity and a pharmaceutically acceptable carrier. One example of a membrane-bound cytochrome is a 65-67 kDa protein.

The present invention provides a method of treating cold symptoms comprising administering to a cell of a subject a composition comprising an isolated nucleic acid in a vector and a pharmacologically acceptable carrier. The composition may be introduced into the upper nasal passages by intranasal spray or by any other inhalation method so that the nucleic acid can contact and enter the cells of the respiratory tract and be transcribed by the cell to make an inhibitory polypeptide of the invention. It is contemplated that the polypeptide produced will migrate to the cell surface and interfere with a cold-causing virus and its interaction with the cell.

The invention also provides a polypeptide, comprising all or part of a 65-67 kDa protein, that blocks the interaction of a cold-causing virus and a cell, by binding to the virus at a site where the virus would normally make contact with the membrane-bound cytochrome. A fragment of the 65-67 kDa protein also binds to the virus at a site where the virus would normally make contact with the membrane-bound cytochrome. The polypeptide or fragment can be designed based on the crystalline structure or conformation studies of the cytochrome. The peptide may bind to a site adjacent to the cytochrome and sterically interfere with the virus binding to the membrane-bound cytochrome.

The present invention provides a composition comprising a polypeptide comprising an amino acid sequence comprising all or part of a 65-67 kDa protein and a pharmaceutically acceptable carrier. An example of a sequence of this protein is deposited in GenBank at AAA36379. Science, 1990, Vol. 248, p. 727-730.

It is also understood that the polypeptide of this invention may also contain conservative substitutions where a naturally occurring amino acid is replaced by one having similar properties and which does not alter the function of the polypeptide. Such conservative substitutions are well known in the art. Thus, it is understood that, where desired, modifications and changes may be made in the nucleic acid and/or amino acid sequence of the polypeptide of the present invention and still obtain a polypeptide having like or otherwise desirable characteristics. Such changes may occur in natural isolates or may be synthetically introduced using site-specific mutagenesis, the procedures for which, such as mis-match polymerase chain reaction (PCR), are well known in the art.

"Nucleic acid" as used herein refers to single- or double-stranded molecules which may be DNA, comprised of the nucleotide bases A, T, C and G, or RNA, comprised of the bases A, U (substitutes for T) , C, and G. The nucleic acid may represent a coding strand or its complement. Nucleic acids may be identical in sequence to the sequence which is naturally occurring or may include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence. Furthermore, nucleic acids may include codons which represent conservative substitutions of amino acids as are well known in the art.

As used herein, the term "isolated nucleic acid" means a nucleic acid separated or substantially free from at least some of the other components of the naturally occurring organism, for example, the cell structural components commonly found associated with nucleic acids in a cellular environment and/or other nucleic acids. The nucleic acids of this invention can be isolated from cells according to methods well known in the art for isolating nucleic acids. Alternatively, the nucleic acids of the present invention can be synthesized according to standard protocols well described in the literature for synthesizing nucleic acids. Modifications to the nucleic acids of the invention are also contemplated, provided that the essential structure and function of the polypeptide encoded by the nucleic acid are maintained.

The nucleic acid encoding the polypeptide of this invention can be part of a recombinant nucleic acid construct comprising any combination of restriction sites and/or functional elements as are well known in the art which facilitate molecular cloning and other recombinant DNA manipulations. Thus, the present invention further provides a recombinant nucleic acid construct comprising a nucleic acid encoding a polypeptide of this invention.

The present invention further provides a vector comprising a nucleic acid encoding a polypeptide of this invention. The vector can be an expression vector which contains all of the genetic components required for expression of the nucleic acid in cells into which the vector has been introduced, as are well known in the art. The expression vector can be a commercial expression vector or it can be constructed in the laboratory according to standard molecular biology protocols. The expression vector can comprise viral nucleic acid including, but not limited to, vaccinia virus, adenovirus, retrovirus and/or adeno-associated virus nucleic acid. The nucleic acid or vector of this invention can also be in a liposome or a delivery vehicle which can be taken up by a cell via receptor-mediated or other type of endocytosis.

The nucleic acid of this invention can be in a cell, which can be a cell expressing the nucleic acid whereby a polypeptide of this invention is produced in the cell. In addition, the vector of this invention can be in a cell, which can be a cell expressing the nucleic acid of the vector whereby a polypeptide of this invention is produced in the cell. It is also contemplated that the nucleic acids and/or vectors of this invention can be present in a host animal (e.g., a transgenic animal) which expresses the nucleic acids of this invention and produces the polypeptide of this invention.

The nucleic acid encoding the polypeptide of this invention can be any nucleic acid that functionally encodes the polypeptide of this invention. The nucleic acid may be identical in sequence to the sequence which is naturally occurring or may include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence. Furthermore, nucleic acids may include codons which represent conservative substitutions of amino acids. Thus, modification to the nucleic acids of the invention are also contemplated as long as the essential structure and function of the polypeptide encoded by the nucleic acid is maintained. To functionally encode the polypeptide (i.e., allow the nucleic acids to be expressed), the nucleic acid of this invention can include, for example, expression control sequences, such as an origin of replication, a promoter, an enhancer and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcriptional terminator sequences.

The present invention also provides a vector comprising the nucleic acid of the invention and a cell comprising the vector. Moreover, the invention provides a composition comprising the nucleic acid, vector, and a pharmaceutically acceptable carrier. The recognized methods of administering nucleic acids to a subject can be used. The present invention provides an antibody which binds an amino acid sequence of a membrane-bound cytochrome. For example, the membrane-bound cytochrome can be a 65-67 kDa protein. For example, the antibody can bind to a region of SEQ ID NO:l, particularly an extracellular region of SEQ ID NO:l. In another example, the antibody binds an epitope of a similar membrane-bound protein and identifies another membrane-bound cytochrome. The antibody can be monoclonal or polyclonal and is produced according to methods known to a person skilled in the art (62).

An antibody of the present methods and compositions may bind to the cell membrane adjacent to the cytochrome, and not specifically to the cytochrome. This binding of the antibody to the cell membrane close to the cytochrome produces a steric interference and prevents the virus from binding to the cytochrome, thereby inhibiting elaboration of IL-8.

The antibody of the invention is used not only to inhibit IL-8 elaboration by the cell but also to inhibit replication of the IL-8-inducing virus in the cell. The antibody is used in any of the methods and compositions of the invention.

The present invention provides a composition comprising an antibody, as described above, and a pharmaceutically acceptable carrier.

The present invention provides a method of screening for a compound that inhibits the interaction of a cold-causing virus with a respiratory epithelium cell, by contacting a cell that expresses a membrane-bound cytochrome with the putative inhibitory compound and determining whether the compound inhibits the function or activity of the membrane-bound cytochrome. If the compound blocks the interaction between a cold-causing virus and the cytochrome, there is reduced elaboration of IL-8 by the cell. The specificity of this blockade can be verified by showing that the addition of hydrogen peroxide to the treated cell increases oxidative stress in the cell and elaboration of IL-8. Moreover, the present invention provides a method of screening for a compound that inhibits the interaction of a cold-causing virus with a respiratory epithelium cell membrane-bound cytochrome, by contacting an IL-8-inducing virus with the test compound and then contacting a cell that expresses a membrane-bound cytochrome with the virus that has been contacted with the compound. The compound is shown to inhibit the interaction of the virus and cell when the compound blocks the binding of the virus to the cytochrome. One measure of this inhibition is a reduction of IL-8 elaboration.

To measure the effect of blocking the membrane-bound cytochrome, hydrogen peroxide levels in the cell can be measured. Blocking cytochrome activity results in lower levels of hydrogen peroxide formation in the cell. Further, Nitroblue Tetrazolium (NTB) dye reduction measures superoxide anion production in the cell. Blocking cytochrome activity results in lower levels of dye reduction (59).

Moreover, looking for carbonyl groups in cells by staining the cells with a fluorescent antibody is an indirect method for measuring the activity of the membrane- bound cytochrome activity. Blocking cytochrome activity results in fewer carbonyl groups produced in the cell (60).

Further, the effectiveness of blocking the membrane-bound cytochrome can be measured by looking at viral replication. Blocking the cytochrome causes less viral replication. Quantification of virus was performed by methods known to a person skilled in the art (61). See Examples.

The present invention also provides a method for identifying other membrane- bound cytochromes with which cold-causing viruses may interact and cause viral replication and elaboration of IL-8 in the cell. After separating proteins from the cell membrane, labeled DPI and PAO can be mixed with the proteins. The proteins bound to labeled DPI and PAO are then identified as other membrane-bound cytochromes. The present invention provides methods of administration of the compositions of this invention that include intranasally and by inhalation. Approximately 4.4 mg of the polypeptide or antibody in three divided doses over 18 hours is administered. Alternatively, the 4.4 mg can be administered in six doses over 18 hours. Although lower doses can be effective, the preferred dose is greater than 1 mg/day. The chemical inhibitors of membrane-bound cytochrome activity must be solubilized (for example, in DMSO) and diluted to a 1 mM concentration. A single metered dose of 0.2 ml in each nostril per day can be administered.

The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLE 1

Oxidative stress and IL-8 elaboration are independent of virus replication: RV39 is a major group rhinovirus that requires attachment to ICAM-1 to produce infection. Incubation of cells with anti-ICAM prior to virus challenge completely inhibited virus replication; however, this treatment had no effect on either NBT dye reduction or on the concentration of IL-8 elaborated into the supernatant media six hours after virus challenge (Figure 1). Viral replication was measured by quantitative viral cultures in serial dilutions, looking for cytopathic effect. Virus titrations were done in 96-well microtiter plates (Falcon Labware, Oxnard, CA). Serial 10-fold dilutions of each specimen were made and then 2 x 10⁴ MRC-5 cells were added to each well. The plates were incubated at 33° C for 7 days and then examined for viral cytopathic effects (CPE). The virus titers were calculated by standard methods.

Cell culture. All experiments were done in human respiratory cell lines maintained at 37 °C in 5% CO₂. Human embryonic lung fibroblast cells (MRC-5, Biowhittaker, Walkersville, MD) were grown in Eagle's Minimal Essential Medium (EMEM) supplemented with 10% fetal calf serum , 5 units/ml penicillin G sodium, and 5 μg/ml streptomycin. Cells were used for experiments at passage 21-26 within two days of the time the monolayers became confluent. Human bronchial epithelial cells (Beas-2b, ATCC, Rockville, MD) were grown in bronchial epithelial growth media (BEGM, Clonetics, Minneapolis, MN) supplemented with human recombinant epithelial growth factor (0.5 ng/ml), insulin (5 μg/ml), hydrocortisone (0.5 μg/ml), epinephrine (0.5 μg/ml), transferrin (10 μg/ml), gentamicin (50 μg/ml), and amphotericin B (50 ng/ml). All experiments with Beas-2b cells were done with cells at passage 35-55 when the monolayers were 85-95% confluent.

Measurement of IL-8 protein. Beas-2b and MRC-5 cells were grown in 24-well tissue culture plates. The virus challenge was 100 TCID₅₀/cell for experiments in Beas-2b cells and 10 TCID₅₀/cell for experiments in MRC-5 cells. The cells were washed three times with media and then incubated for 120 min. at 37 °C with either media alone or with media containing 5, 10, 20, and 30 mM of NAC. The cells were then challenged with virus in a final volume of 1 ml/well and incubated at 33 °C for 1 hr. to allow for the absoφtion of the virus. The cells were washed 3x with media and then incubated for six hours at 33 °C. Supernatants were then collected and stored at -80°C until analyzed for IL-8 protein. For experiments with hydrogen peroxide, supernatants were collected after the cells were challenged with H₂O₂ at concentrations between 0.0625 and 0.5 mM for six hours at 33 °C. The IL-8 concentrations in the cell culture supernatant specimens were determined by ELISA using commercially available assays (Quantikine, Minneapolis, MN). All assays were done in duplicate on an automated spectrophotometric plate reader (Anthos HTII, Anthos Labtec Instruments Co., Salzburg, Germany). Sample concentrations were determined from OD values using a standard curve based on a linear regression. Specimens with IL-8 concentrations below the detection limit of the assay were assigned a value of 3 pg/mL for calculations.

Challenge of fibroblast cells with a mutant strain of rhinovirus (RV39vs) resulted in normal viral replication as assessed by quantitative viral cultures of supernatant media collected 48 hours after challenge (Figure 2). In spite of this normal viral replication, however, challenge with RV39vs does not result in NBT dye reduction or elaboration of IL-8 (Figure 2). Pretreatment of cells with diphenylene iodonium or phenylarsine oxide but not allopurinol or rotenone inhibits IL-8 elaboration and virus replication. Diphenylene iodonium is an inhibitor of cellular oxidases with potent activity against NADPH oxidase. Pretreatment of fibroblast cells with DPI (40 μM) reduced IL-8 elaboration to concentrations comparable to those from unstimulated cells (Figure 3). Inhibition of IL-8 elaboration was also seen following pretreatment of cells with phenylarsine oxide another inhibitor of NADPH oxidase (Figure 3). The reduction in IL-8 elaboration after treatment with these inhibitors was associated with a significant reduction in virus replication as measured by quantitative viral cultures done on supernatant media collected 48 hours after virus challenge (Figure 3).

In contrast to the results seen with pretreatment of cells with DPI or PAO, treatment of cells with allopurinol, an inhibitor of xanthine oxidase, or with rotenone an inhibitor of mitochondrial superoxide generation, had no effect on either IL-8 elaboration or virus replication (Figure 3).

Fibroblast cells from a patient with chronic granulomatous disease have reduced elaboration of IL-8 compared to normal skin fibroblasts. Chronic granulomatous disease (CGD) is a congenital disease caused by dysfunction of leukocyte NADPH-oxidase. Skin fibroblasts from a patient with CGD were cultured and analyzed for the presence of gp91-phox and for the ability to reduce NBT dye. gp91-phox was present by Western blotting but NBT dye reduction was reduced compared to normal fibroblasts. IL-8 concentrations in supernatant media from CGD fibroblasts were approximately 33% of the concentrations in supernatant from normal fibroblasts six hours after virus challenge and were comparable to the levels seen in supernatants from unchallenged normal fibroblasts (Figure 4). CGD fibroblasts supported viral replication but the quantity of virus present in supernatant media after 48 hours was decreased compared to normal skin fibroblast controls (Figure 4).

Investigation of the effect of chemical inhibitors of NADPH-oxidase on RSV and

CV -induced IL-8 elaboration and virus replication. Diphenylene iodonium and phenylarsine oxide are commonly used and well-known inhibitors of NADPH-oxidase. Purified pools of RSV and CV were used to challenge cell culture monolayers of MRC- 5 and BEAS-2b cells after a preincubation with the inhibitor. Viral replication was assessed by quantitative culture and IL-8 concentrations were determined by ELISA (Quantikine, R&D Systems, Minneapolis, MN).

One potential problem is that these inhibitors are not specific for NADPH- oxidase and any observed effects on IL-8 elaboration or virus replication could be due to a variety of other known effects of these agents. The likelihood that any observed effects are due to inhibition of NADPH-oxidase is enhanced in two ways. The mechanism of action of diphenylene iodonium involves binding to flavoproteins while phenylarsine oxide binds to gp91-phox (42, 43). The use of two inhibitors with different mechanisms of action makes it more likely that the observed effects are due to the desired action of the agents. The second way in which the specificity of any inhibitory effects is assessed is by comparison with inhibitors of other sources of superoxide anion that appear to have greater specificity. Effects of diphenylene iodonium and phenylarsine oxide were compared with the effects of allopurinol, an inhibitor of xanthine oxidase, rotenone, an inhibitor of mitochondrial metabolism, and indomethacin, a cyclooxygenase inhibitor. The absence of effects of these compounds enhances the probability that observed effects are due to the desired inhibition of NADPH-oxidase.

Evaluation of RSV and CV -induced IL-8 elaboration and virus replication in fibroblast cells with functionally deficient NADPH-oxidase. Chronic granulomatous disease is an inherited syndrome associated with various defects in neutrophil NADPH- oxidase. A similar NADPH-oxidase is expressed in non-myeloid cells (36). There are conflicting data regarding the function of fibroblast NADPH-oxidase in patients with CGD (44,45). Primary skin fibroblasts from a patient with CGD were cultured. Preliminary experiments with this cell line suggest that rhinovirus replication and IL-8 elaboration are reduced compared to normal skin fibroblasts. A clearly defined defect in the intracytoplasmic p47-phox subunit of NADPH-oxidase has been found. Because other membrane-bound cytochromes may use p47-phox, it is possible to use p47-phox to label other cytochromes. Fibroblast cells with demonstrated defects in NADPH- oxidase function as assessed by evaluation of diphenylene iodonium-inhibitable superoxide anion generation were used to assess the role of NADPH-oxidase in RSV and CV- induced IL-8 elaboration and viral replication. Preliminary data suggest that the cell line that has already been produced has deficient superoxide anion generation and was a suitable cell line for definitive experiments.

EXAMPLE 2

Reagents. Monoclonal antibody to ICAM-1 (RRl/1.1.1) was provided by

Robert Rothlein (Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT). This antibody was used at a final concentration of 10 ng/mL of medium. Diphenylene iodonium (DPI) and MnTBAP were purchased from Calbiochem (La Jolla, CA), allopurinol and rotenone were purchased from Sigma (St. Louis, MO) and ibuprofen was purchased from BioMol (Plymouth Meeting, PA).

Cell culture. Human embryonic lung fibroblast cells (MRC-5, Biowhittaker, Walkersville, MD) were grown in Eagle's Minimal Essential Medium (EMEM) supplemented with 10% fetal calf serum, 5 units/ml penicillin G sodium, and 5 μg/ml streptomycin. Cells were used for experiments at passage 21-25 within two days of the time the monolayers became confluent. Human bronchial epithelial cells (BEAS-2B, ATCC, Rockville, MD) were grown in bronchial epithelial growth media (BEGM, Clonetics, Minneapolis, MN) supplemented with human recombinant epithelial growth factor (0.5 ng/ml), insulin (5 μg/ml), hydrocortisone (0.5 μg/ml), epinephrine (0.5 μg/ml), transferrin (10 μg/ml), gentamicin (50 μg/ml), and amphotericin B (50 ng/ml). All experiments with BEAS-2B cells were done with cells at passage 40-55 when the monolayers were 85-95% confluent.

Primary skin fibroblast cell lines were prepared from a normal volunteer and from volunteers with chronic granulomatous disease (CGD). Skin fibroblast cultures were prepared from biopsy specimens obtained from one volunteer with a deficiency of p41-phox demonstrated by Western blotting of neutrophils with antibodies to p47-phox (provided by Thomas L. Leto, Ph.D., NIAID, Bethesda, MD) and from a second volunteer with a deficiency of gp9l-phox. The skin fibroblast cells from these volunteers and from the normal volunteer were used for experiments at passage 5-15. For all experiments control cells and CGD cells were used at the same passage.

Viral preparation and purification. Rhinovirus, type 39 (RV39) was grown in HeLa-I cells, a HeLa cell clone with increased surface expression of ICAM-1 (provided by F.G. Hayden, University of Virginia HSC, Charlottesville, VA). HeLa-I cells infected with RV39 were mechanically collected, lysed by a freeze-thaw and the supernatants clarified by centrifugation at 2000 g (Beckman GPR centrifuge, Beckman Instruments, Inc., Palo Alto, CA). The supernatants were then centrifuged at 125,000 g at 4°C for 45 min. (Ti45 rotor, Beckman L8-70M centrifuge, Beckman Instruments, Inc., Palo Alto, CA) . Partially purified virus was produced by a modification of a published method [63] . Briefly, after ultracentrifugation, the resulting viral pellet was resuspended in 200 μl PBS buffer and overlaid onto a two layer sucrose cushion containing 60% sucrose in PBS on the bottom layer and 30% sucrose in PBS on the top layer. Following centrifugation at 110,000 g (SW28 rotor) for 135 min. at 4°C, the interface containing the virus was collected and resuspended in 50 ml of EMEM. The virus suspension was again centrifuged at 125,000 g for 45 min. at 4°C and the resulting pellet was resuspended in EMEM with 1% BSA and aliquots were snap frozen in liquid nitrogen and stored at -70°C. Respiratory syncytial virus and coronavirus 229E grown in MRC-5 cells were also partially purified as described.

Viral infection. Cells were grown in 24-well tissue culture plates (= 10^ cells/well). Unless specified, the virus challenge was 100 TCID5_Q/cell for experiments in BEAS-2B cells and 10 TCID5()/cell for experiments in fibroblast cells. The cells were challenged with virus in a final volume of 1 ml/well and incubated at 33°C for 1 hour to allow for the absoφtion of virus. The cells were then washed 3 times with media and further incubated with the fresh media at 33°C. Supernatants were then collected at the specified time (6, 24 or 48 hours) and stored at -70°C until analyzed for IL-8 protein or viral titers. For experiments involving inhibition with antibody or chemical reagents the inhibitor was incubated on the cells at an appropriate concentration in medium for 30 minutes at 37°C. Virus was then added and incubated at 33°C for 1 hour to allow for the absoφtion of virus. The cells were then washed 3 times with media and further incubated with the fresh media containing the inhibitor until specimens were collected at the specified times. All results are presented as the mean ± standard deviation of at least three separate experiments.

Cell cytotoxicity. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma Chemical Co., St Louis, MO) was dissolved in water at a concentration of 5 mg/ml then syringe filtered through a 0.2 μm filter and stored at 5°C. After experimental treatment of cells the media was poured off and 50 μl of MTT stock solution in 450 μl of media was added to each well. The cells were then incubated at 37°C for 3-4 hrs. The media were then discarded and the MTT salt was extracted with 500 μl/well of acidic isopropanol ( 0.1 N HC1 in absolute isopropanol) and the absorbance of the converted dye was measured at a wavelength of 570 nm with a reference wavelength of 620 nm using an automated spectrophotometric plate reader. For all experiments, the absence of cell toxicity at the concentrations of inhibitor that are reported was demonstrated by the absence of cytopathic effect and MTT dye reduction comparable to control monolayers.

Measurement of IL-8 protein. The IL-8 concentrations in cell culture supernatant specimens were determined by ELISA using commercially available assays (R&D Systems, Minneapolis, MN). All assays were done in duplicate on an automatic spectrophotometric plate reader (Anthos HTII, Anthos Labtec Instrument Co., Salzburg, Austria). Sample concentrations were determined from OD values using a standard curve based on a linear regression. All data presented represent the mean ± SD of at least three separate experiments.

Quantitative nitroblue tetrazolium (NBT) assay. The NBT assay was done in fibroblast cells grown to confluency in 96-well flat bottom plates. The cells were washed with 2% EMEM and then preincubated with various inhibitors or control media for 30 min. at 37°C. The cells were then challenged with RV (MOI=10 TCID₅o/cell) and NBT solution was added to each well at a final concentration of 1 mg/ml. After incubation for 6 hr., the cells were washed 3X with methanol and the reduced NBT was extracted by adding a mixture of 92 μl of 10 M KOH and 108 μl DMSO. Optical density was measured at 620 nm on an automated optical reader. All data presented represent the mean ± SD of at least three separate experiments.

Quantitative hydrogen peroxide assay. The concentration of hydrogen peroxide was determined in cell culture supernatants with a commercially available colorimetric assay (Bioxytech H2O2.56O, OXIS International, Inc., Portland, OR). For this assay, cell culture supernatants were collected one hour after challenge of cells with virus. All assays were done in triplicate on an automatic spectrophotometric plate reader (Anthos HTII, Anthos Labtec Instrument Co., Salzburg, Austria). Sample concentrations were determined from OD values using a standard curve based on a linear regression. The data presented represent the mean ± SD of three separate experiments.

Quantitation of virus. Virus titrations were done in 96-well microtiter plates

(Falcon Labware, Oxnard, CA). Serial 10-fold dilutions of each specimen were made and then 2 x 10^ MRC-5 cells were added to each well. The plates were incubated at 33°C for 7 days and then examined for viral cytopathic effects (CPE). The virus titers were calculated by standard methods [61] . All data presented represent the mean ± SD of at least three separate experiments except as noted.

Oxidative stress and IL-8 elaboration are independent of rhinovirus replication. RV39 is a major group rhinovirus that requires attachment to ICAM-1 to produce infection. As expected, incubation of MRC-5 cells with anti-ICAM antibody inhibited virus replication as assessed by development of cytopathic effect and quantitative viral cultures of supernatant media (Figure 11). In contrast, incubation with anti-ICAM antibody prior to virus challenge had no effect on either NBT dye reduction (data not shown) or on the concentration of IL-8 elaborated into the supernatant media six hours after virus challenge (Figure 11).

A variant strain of RV39 (RV39vs), identified during the course of these studies, did not induce IL-8 elaboration. This variant strain replicated normally as assessed by cytopathic effect and quantitative viral cultures compared to the "wild type" virus. After 48 hours of incubation, virus titers in the supernatant media from MRC-5 cells infected with "wild type" virus were 2.8 ± 0.4 TCID5o/ml compared to 2.4 ± 0.5 TCID5o/ml in media from cells infected with the variant strain. The variant strain was neutralized by type specific antiserum to RV39 and replication was inhibited by pretreatment of cells with anti-ICAM antibody (data not shown). The variant strain of rhinovirus did not induce oxidative stress in MRC-5 cells as determined by hydrogen peroxide production. Hydrogen peroxide concentrations in supernatant media from control and rhinovirus challenged MRC-5 cells were 1.18 ± 0.43 μM and 5.3 ± 1.4 μM, respectively, while the supernatant from MRC-5 cells challenged with RV39vs contained 0.89 ± 0.18 μM H2O2. Similarly, the RV39vs did not produce oxidative stress in BEAS-2B cells as determined by NBT dye reduction or by fluorescence staining of carbonyl groups (data not shown). In contrast to "wild type" RV39, challenge of cells with this virus in vitro was not associated with activation of NF-κB (data not shown) or with IL-8 elaboration in either MRC-5 cells or BEAS-2B cells (Figure 9). The observations that inhibition of virus replication with anti-ICAM antibody does not affect IL-8 elaboration and that the variant strain replicates normally but does not produce an IL-8 response suggest that virus-induced oxidative stress and IL-8 elaboration are independent of attachment to ICAM-1 or viral replication.

Pretreatment of cells with diphenylene iodonium but not ibuprofen, allopurinol or rotenone inhibits rhinovirus-induced IL-8 elaboration and virus replication. Diphenylene iodonium is a flavoprotein inhibitor with potent activity against NADPH oxidase. Pretreatment of fibroblast cells with DPI (40 μM) reduced superoxide anion production and virus induced IL-8 elaboration (Figure 10). The reduction in oxidative stress was also demonstrated by decreased H2O2 production. Hydrogen peroxide concentrations in supernatant media from control and rhinovirus challenged MRC-5 cells were 1.18 ± 0.43 μM and 5.3 ± 1.4 μM, respectively. The supernatant from MRC-5 cells challenged with rhinovirus in the presence of DPI (40 μM) contained 0.48 ± 0.18 μM H2O2. A similar inhibitory effect of DPI was seen in BEAS-2B cells. The IL-8 concentration in media from BEAS-2B cells challenged with rhinovirus was reduced from 246 ± 25 pg/ml to 121 ± 20 pg/ml by pretreatment with 40 μM DPI. The IL-8 concentrations in media from BEAS-2B cells without virus challenge were 144 ± 15 pg/ml and 97 ± 11 pg/ml, respectively. The reduction in IL-8 elaboration after treatment with DPI was associated with a reduction in virus replication as measured by quantitative viral cultures done on supernatant media collected 48 hours after virus challenge (Figure 11). Treatment of the cells with MnTBAP, which mimics the activity of superoxide dismutase, inhibited oxidative stress and IL-8 elaboration in response to virus challenge but had no effect on virus replication. These observations suggest that the inhibition of virus replication by DPI was not a result of the inhibition of oxidative stress. Similar effects of DPI on virus induced IL-8 elaboration and virus replication were seen following challenge of fibroblast cells with either RSV or coronavirus 229E (Figure 9).

In contrast to the effect of pretreatment of cells with DPI, inhibitors of other cellular sources of ROS had no effect. Treatment of cells with ibuprofen, a cyclooxygenase inhibitor, allopurinol, an inhibitor of xanthine oxidase, or rotenone, an inhibitor of mitochondrial superoxide generation, had no effect on either IL-8 elaboration or virus replication (Figure 11).

Fibroblast cells from a volunteer without p41-phox have reduced production of superoxide anion and elaboration of IL-8 after rhinovirus challenge compared to normal skin fibroblasts. NBT dye reduction and H2O2 production were decreased in fibroblasts obtained from a p41-phox deficient individual compared to either the normal control or fibroblasts cells from a volunteer with gp9l-phox deficiency both at baseline and following challenge with rhinovirus (Figure 10). Similarly, IL-8 concentrations in supernatant media from these fibroblasts were approximately 33% of the concentrations in supernatant from normal fibroblasts both before and six hours after challenge with rhinovirus, respiratory syncytial virus or coronavirus 229E (Figure 11). In contrast, IL-8 elaboration by fibroblasts cells from a volunteer with gp9l-phox deficiency was comparable to that by fibroblasts from the normal control after challenge with each of the viral pathogens. Fibroblast cells from CGD patients supported viral replication although the quantity of virus present in supernatant media after 48 hours was slightly decreased compared to normal skin fibroblast controls. The quantitative viral titers were 3.6 ± 0.1 (mean ± SD) TCID5Q/ml in fibroblasts from normal skin compared to 2.5 ± 0.3 and 2.7 ± 0.3 TCID5o/ml in skin fibroblasts from p41-phox and gp9l-phox deficient patients, respectively.

Although the fibroblast cells from the p41-phox deficient patient had a reduced oxidative response and decreased production of IL-8 following RV challenge when compared to normal fibroblast cells, the p41-phox deficient cells did respond to RV challenge. Pretreatment of cells with DPI completely blocked the oxidative response to RV challenge as measured by NBT dye reduction and H2O2 production (Figure 10). Similarly, DPI-pretreatment inhibited RV-induced elaboration of IL-8. IL-8 concentrations in the pM-phox deficient cells without virus challenge were 40 ± 14 pg/ml compared to 158 ± 54 pg/ml after RV challenge. RV-challenge of these cells after pretreatment with DPI (40 μM), allopurinol (500 μM), or rotenone (10 μM) resulted in IL-8 concentrations of 13.4 ± 2.2, 323 ± 100, and 186 ± 104 pg/ml, respectively. DPI-pretreatment also inhibited virus replication in the fibroblast cells from the p41-phox deficient patient.

The data presented here show that production of reactive oxygen species and oxidative stress in cells in response to rhinovirus challenge is not a direct result of virus attachment to ICAM-1 or of virus replication. It is also clear thatvirus-induced production of reactive oxygen species is inhibited by pretreatment of cells with diphenylene iodonium, but not by inhibitors of xanthine oxidase, cyclooxygenase, or mitochondrial metabolism. The data also show that p41-phox, a cytoplasmic component of NADPH-oxidase, may be involved in virus induced superoxide production and IL-8 elaboration. EXAMPLE 3

Isolation and solubilization of fibroblast membranes. Control fibroblasts (MRC-5) or fibroblasts challenged with virus followed by one hour incubation at 33° C, were washed X 2 with TBS and then scraped into Buffer A (10 mM Tris Base, 1 mM EDTA, 340 mM sucrose, 1 mM AEBSF, pH 7.1). The cells were pelleted at 1200 φm for 5 minutes at 4° C, and the pellet was sonicated with 4 X 15 second bursts at 15 second interval. The sonicate was again centrifuged at 1200 φm for 5 minutes at 4° C and then the supernatant was layered onto a discontinuous gradient of sucrose consisting of 10 ml of 30% sucrose over 20 ml of 50%> sucrose. Following centrifugation at 140,000 g for 45 minutes at 4° C the interface between the two sucrose layers was collected, diluted in three volumes of distilled water and then centrifuged at 27,000 g for 30 minutes at 4° C. The membrane pellet was suspended in solubilizing buffer (20 mM glycine, 0.25% sodium deoxycholate, 0.25% Luberol PX, 1 mM NaN3, 1.7 mM CaCl2, and 25% glycerol), incubated on ice for 30 minutes with occasional mixing and then centrifuged at 100,000 g for 30 minutes at 4° C. The supernatant was then loaded on a column of 2'-5' ADP agarose and eluted with solubilizing buffer alone or with solubilizing buffer containing either DPI (100 nM) or DPI plus NADP (1 mM).

This procedure results in isolation of multiple membrane proteins in eluate with solubilizing buffer alone. Subsequent elution with either DPI or DPI plus NADP results in a single protein band of approximately 67 kDa apparent molecular weight that stains by Western blotting with polyclonal antibody to p61-phox.

EXAMPLE 4

Antibody Binding to Intact Cells. Cells are grown in 24-well tissue culture plates (= 10^ cells/well). The cells are contacted with polyclonal antibody serum raised against p67-phox and allowed to incubate for 30 minutes at 37°C. Binding of the antibody to the cells is then detected, either by detecting a label on the antibody or by staining with a labeled secondary antibody. These and other methods used to detect the antibody bound to the intact cell membrane are well known. Cells are grown in 24-well tissue culture plates (= 10^ cells/well). The cells are contacted with polyclonal antibody serum raised against p67-phox and allowed to incubate for 30 minutes at 37°C. After the cells are washed, the cell-antibody complexes are exposed to virus. Unless specified, the virus challenge is 100 TCID5()/cell for experiments in BEAS-2B cells and 10 TCID5o/cell for experiments in fibroblast cells. The cells are challenged with virus in a final volume of 1 ml/well and incubated at 33°C for 1 hour to allow for the absoφtion of virus. The cells are then washed 3 times with media and further incubated with the fresh media at 33°C. Supernatants are then collected at the specified time (6, 24 or 48 hours) and stored at -70°C until analyzed for IL-8 protein or viral titers. Decreased levels of IL-8 elaboration found after the cells are incubated with the polyclonal antibody indicate that the membrane-bound cytochrome is bound by the antibody, and virus-cell interaction has been blocked. Moreover, measurement of viral replication shows that the antibody bound to the membrane-bound cytochrome causes reduced replication of virus, indicating inhibition of virus-cell interaction. Similarly, decreased levels of viral replication found after the cells are incubated with the polyclonal antibody indicate that the membrane-bound cytochrome is bound by the antibody, and virus-cell interaction has been blocked.

Moreover, similar studies are carried out with cells incubated with a monoclonal antibody directed to an epitope of p67-phox. Binding of the monoclonal antibody to a receptor on the surface of the intact cell is detected by observing the staining of the antibody-membrane complex as described in the literature.

Further, measurement of decreased elaboration of IL-8 by the cells or reduced viral replication after incubation of the cells with the virus indicates that the monoclonal antibody has blocked the interaction of the virus and the cell at the membrane-bound cytochrome.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incoφorated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

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Claims

What is claimed is:

1. A method of inhibiting the elaboration of IL-8 from a cell in a subject, comprising blocking the interaction of the cell with an IL-8-inducing virus, by contacting the IL-8-inducing virus with an agent that binds to the IL-8-inducing virus at a site of contact between the IL-8-inducing virus and a cell membrane- bound cytochrome, thereby inhibiting virus interaction with the membrane- bound cytochrome and inhibiting elaboration of IL-8.

2. The method of claim 1, wherein the membrane-bound cytochrome is a 65- 67kDa protein that binds diphenylene iodonium.

3. The method of claim 1, wherein the subject is a mammal.

4. The method of claim 1, wherein the subject is human.

5. The method of claim 1, wherein the cell is a respiratory epithelial cell.

6. The method of claim 1, wherein the IL-8-inducing virus is selected from the group consisting of rhinoviruses, respiratory syncytial virus (RSV), coronavirus (CV) and other viruses infecting the respiratory epithelium.

7. The method of claim 1, wherein the agent is an isolated polypeptide comprising an amino acid sequence corresponding to at least a part of the membrane-bound cytochrome.

8. The method of claim 7, wherein the isolated polypeptide comprises an amino acid sequence of a 65-67 kDa protein that binds diphenylene iodonium.

9. The method of claim 8, wherein the 65-67 kDa protein has the amino acid sequence of SEQ ID NO: 1.

10. A composition comprising a polypeptide comprising an amino acid fragment of a 65-67 kDa protein that binds diphenylene iodonium, wherein the fragment binds diphenylene iodonium, and a pharmaceutically acceptable carrier.

11. A method of inhibiting the elaboration of IL-8 from a cell in a subject, comprising blocking the interaction of the cell with an IL-8-inducing virus, by contacting the cell with an antibody that binds a cell membrane-bound cytochrome, thereby inhibiting virus binding to the membrane-bound cytochrome and inhibiting elaboration of IL-8.

12. The method of claim 11, wherein the membrane-bound cytochrome is a 65-67 kDa protein that binds diphenylene iodonium.

13. The method of claim 11, wherein the subject is a mammal.

14. The method of claim 11, wherein the subject is human.

15. The method of claim 11, wherein the cell is a respiratory epithelial cell.

16. The method of claim 11, wherein the IL-8-inducing virus is selected from the group consisting of rhinoviruses, respiratory syncytial virus (RSV), coronavirus (CV) and other viruses infecting the respiratory epithelium.

17. The method of claim 11 , wherein the antibody is polyclonal.

18. The method of claim 11, wherein the antibody is monoclonal.

19. The method of claim 11, wherein the antibody binds an amino acid sequence of a 65-67 kDa protein that binds diphenylene iodonium.

20. An antibody that binds a cell membrane-bound cytochrome.

21. The antibody of claim 20, wherein the antibody specifically binds a 65-67 kDa membrane-bound protein that binds diphenylene iodonium.

22. The antibody of claim 20, wherein the antibody is polyclonal.

23. The antibody of claim 20, wherein the antibody is monoclonal.

24. A composition comprising the antibody of claim 20 and a pharmaceutically acceptable carrier.

25. A method of inhibiting viral replication in a cell in a subject, comprising blocking the interaction of the cell with an IL-8-inducing virus, by contacting the IL-8-inducing virus with an agent that binds to the IL-8-inducing virus at a site of contact between the IL-8-inducing virus and a cell membrane-bound cytochrome, thereby inhibiting virus binding to the membrane-bound cytochrome and inhibiting viral replication.

26. The method of claim 25, wherein the membrane-bound cytochrome is a 65-67 kDa protein that binds diphenylene iodonium.

27. The method of claim 25, wherein the subject is a mammal.

28. The method of claim 25, wherein the subject is human.

29. The method of claim 25, wherein the cell is a respiratory epithelial cell.

30. The method of claim 25, wherein the IL-8-inducing virus is selected from the group consisting of rhinoviruses, respiratory syncytial virus (RSV), coronavirus (CV) and other viruses infecting the respiratory epithelium.

31. The method of claim 25, wherein the agent is an isolated polypeptide comprising an amino acid sequence corresponding to at least a part of the membrane-bound cytochrome.

32. The method of claim 25, wherein the isolated polypeptide comprises an amino acid sequence comprising a 65-67 kDa protein that binds diphenylene iodonium.

33. The method of claim 26, wherein the 65-67 kDa protein has the amino acid sequence of SEQ ID NO: 1.

34. A method of inhibiting viral replication in a cell of a subject, comprising blocking the interaction of the cell with an IL-8-inducing virus, by contacting the cell with an antibody that binds a cell membrane-bound cytochrome, thereby inhibiting virus binding to the membrane-bound cytochrome and inhibiting viral replication.

35. The method of claim 34, wherein the membrane-bound cytochrome is a 65-67 kDa protein that binds diphenylene iodonium.

36. The method of claim 34, wherein the subject is a mammal.

37. The method of claim 34, wherein the subject is human.

38. The method of claim 34, wherein the cell is a respiratory epithelial cell.

39. The method of claim 34, wherein the IL-8-inducing virus is selected from the group consisting of rhinoviruses, respiratory syncytial virus (RSV), coronavirus (CV) and other viruses infecting the respiratory epithelium.

40. The method of claim 34, wherein the antibody is polyclonal.

41. The method of claim 34, wherein the antibody is monoclonal.

42. The method of claim 34, wherein the region of membrane-bound cytochrome comprises an amino acid sequence of a 65-67 kDa protein that binds diphenylene iodonium.

43. A method of inhibiting virus-induced elaboration of IL-8 from a cell of a subject, comprising inhibiting stimulation of a membrane-bound cytochrome by contacting the cell with a chemical inhibitor of the cytochrome, thereby inhibiting the membrane-bound cytochrome and inhibiting elaboration of IL-8.

44. The method of claim 43, wherein the chemical inhibitor of the membrane-bound cytochrome is diphenylene iodonium.

45. The method of claim 43, wherein the chemical inhibitor of the membrane-bound cytochrome is phenylarsine oxide.

46. A composition comprising a chemical inhibitor of a membrane cytochrome and a pharmaceutically acceptable carrier.

47. The method of claim 43, wherein the IL-8-inducing virus is selected from the group consisting of rhinoviruses, respiratory syncytial virus (RSV), coronavirus (CV) and other viruses infecting the respiratory epithelium.

48. A method of inhibiting viral replication in a cell of a subject, comprising inhibiting stimulation of a membrane-bound cytochrome by contacting the cell with a chemical inhibitor of the cytochrome, thereby inhibiting the membrane- bound cytochrome and inhibiting viral replication.

49. The method of claim 48, wherein the chemical inhibitor of the membrane-bound cytochrome is diphenylene iodonium.

50. The method of claim 48, wherein the chemical inhibitor of the membrane-bound cytochrome is phenylarsine oxide.

51. A composition comprising a chemical inhibitor of a membrane-bound cytochrome and a pharmaceutically acceptable carrier.

52. A method of reducing cold symptoms, comprising blocking the interaction of a cell of a subject with an IL-8-inducing virus, by contacting the cell with an antibody that binds a cell membrane-bound cytochrome of the cell, thereby inhibiting virus binding to the cytochrome and inhibiting elaboration of IL-8.

53. The method of claim 52, wherein the subject is human.

54. The method of claim 52, wherein the cell is a respiratory epithelial cell.

55. The method of claim 52, wherein the antibody is administered intranasally.

56. A method of reducing cold symptoms, comprising blocking the interaction of a cell of a subject with an IL-8-inducing virus, by contacting the IL-8-inducing virus with an isolated polypeptide that binds to the IL-8-inducing virus at a site of contact between the IL-8-inducing virus and a cell membrane-bound cytochrome, thereby inhibiting virus binding to the membrane-bound cytochrome and inhibiting elaboration of IL-8.

57. The method of claim 56, wherein the virus is selected from the group consisting of rhinoviruses, respiratory syncytial virus (RSV), coronavirus (CV) and other viruses infecting the respiratory epithelium.

58. The method of claim 56, wherein the subject is human.

59. The method of claim 56, wherein the polypeptide is administered intranasally.

60. A method of reducing cold symptoms, comprising inhibiting a membrane- bound cytochrome in a cell of a subject, by contacting the cell with a chemical inhibitor of the membrane-bound cytochrome, thereby inhibiting the membrane- bound cytochrome and inhibiting elaboration of IL-8.

61. The method of claim 60, wherein the subject is human.

62. The method of claim 60, wherein the cell is a respiratory epithelial cell.

63. The method of claim 60, wherein the cell is in vivo.

64. The method of claim 60, wherein the chemical inhibitor of the membrane-bound cytochrome is administered intranasally.

65. A method of screening for a compound that inhibits the interaction of a cold- causing virus with a cell, comprising the steps of a) contacting a cell that expresses a cell membrane-bound cytochrome with the compound; and b) determining whether the compound inhibits the virus from stimulating the cytochrome, the inhibition of stimulation of the cytochrome being indicated by decreased production of superoxide or decreased elaboration of IL-8 by the cell.

66. The method of claim 65, wherein the membrane-bound cytochrome comprises a 65-67 kDa protein.

67. The method of claim 65, wherein the binding of the compound to the membrane-bound cytochrome is indicated by a reduction in IL-8 elaboration.

68. A method of screening for a compound that inhibits the interaction of a cold- causing virus and a cell membrane-bound cytochrome, comprising the steps of a) contacting a virus with the compound; b) contacting a cell that expresses a cell membrane-bound cytochrome with the virus of step a); and c) determining whether the virus of step a) is inhibited from binding to the membrane-bound cytochrome of the cell.

69. The method of claim 68, wherein the membrane-bound cytochrome comprises a 65-67 kDa protein.

70. The method of claim 68, wherein the inhibition of the interaction between a cold-causing virus and a cell membrane-bound cytochrome is indicated by a reduction in IL-8 elaboration.