COMPOSITIONS AND METHODS FOR TREATING SARS FIELD OF THE INVENTION The present invention is in the field of severe acute respiratory syndrome (SARS).

BACKGROUND OF THE INVENTION Recently, the World Health Organization and U. S. Centers for Disease Control and Prevention warned of a new respiratory illness, termed severe acute respiratory syndrome ("SARS"), a type of atypical viral pneumonia. The illness generally begins with a fever greater than 38°C, which is sometimes accompanied by chills or other symptoms, including headache, malaise, or body aches. Eventually, the infected individual may develop a dry, nonproductive cough, and the infected person may experience difficulty breathing. In 10- 20% of cases thus far, patients will require mechanical ventilation. Approximately 3-5% of the individuals that contract this disease die. There is currently no treatment for this disease.

The SARS virus is a member of the coronavirus family of viruses. Coronaviruses are characterized by: 1) irregularly shaped particles 60-220 nm in diameter, with an outer envelope bearing distinctive, club-shaped peplomers, the outer envelope giving the virus a crown-like appearance from which the family name is derived. Coronaviridae includes the genuses Coronavirus and Torovirus. The genus Coronavirus includes avian infectious bronchitis virus, bovine coronavirus, canine coronavirus, human coronavirus 299E, human coronavirus OC43, murine hepatitis virus, rat coronavirus, porcine hemagglutinating encephalomyelitis virus, etc.; while the genus Torovirus includes Berne virus and Breda virus. Members of the Coronaviridae family infect a wide variety of mammals and birds, and cause diseases such as respiratory infections and enteric infections.

There is a need in the art for methods of treating SARS. The present invention addresses these needs.

References of interest include USPN 6,506, 554, PCT publication WO 99/59615, Lu et al, J. Biomol. Str. And Dyn. 1997 15: 465-471, Lu et al Nature Str. Bio. 1995 2: 1075- 1082, Chan et al., Cell 1997 89: 263-273, Lamb Mol. Memb. Biol. 1999 16: 11-19.

SUMMARY OF THE INVENTION The present invention provides compositions and methods of treating severe acute respiratory syndrome (SARS), and methods of reducing SARS viral load, reducing the time to SARS viral clearance, and reducing morbidity or mortality in the clinical outcomes, in patients suffering from a SARS. The present invention further provides methods of reducing the risk that an individual will develop SARS. The methods generally involve administering a therapeutically effective amount of an agent, e. g. a peptide or a monoclonal antibody, that interacts with the E2 core structure and prevents viral entry into a susceptible cell. The cell may be in vitro or in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the sequence of an exemplary SARS E2 protein (SEQ ID NO : 1).

Fig. 2 shows a prediction of the secondary structure of an exemplary SARS E2 protein (SEQ ID NO : 1), showing alpha-helical structure at various positions in the protein.

Fig. 3 is a schematic showing an arrangement of E2 alpha-helices in an E2 complex that forms during viral entry.

Fig. 4 shows the sequence of two alpha-helices useful in the subject methods. SEQ ID NO : 2 is a N-terminal-alpha helix and SEQ ID NO : 3 is a C-terminal-alpha helix.

Fig. 5 shows exemplary amino acid sequences that therapeutic peptides may comprise. The sequence are present in the sequence listing, from top to bottom in each column with the left hand column first as SEQ ID NOS: 4-70.

Fig. 6 shows exemplary multimer structures that may be produced recombinantly to make antigen suitable for use in the subject methods.

DEFINITIONS The terms"individual, ""host,""subject,"and"patient"are used interchangeably herein, and refer to a mammal, including, but not limited to, primates, including simians and humans.

As used herein, the term"SARS virus"includes any member of the family Coronaviridae, that is a causative agent of SARS. The term"SARS virus"further includes <BR> <BR> naturally-occurring (e. g. , wild-type) SARS virus ; naturally-occurring SARS virus variants ; and SARS virus variants generated in the laboratory, including variants generated by selection, variants generated by chemical modification, and genetically modified variants <BR> <BR> (e. g. , coronavirus modified in a laboratory by recombinant DNA methods).<BR> <P>As used herein, the terms"treatment, ""treating,"and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. "Treatment, "as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet <BR> <BR> been diagnosed as having it; (b) inhibiting the disease, i. e. , arresting its development ; and (c)<BR> relieving the disease, i. e. , causing regression of the disease.<BR> <P>In many embodiments, "treating"SARS refers to reduction of a symptom of SARS.

SARS symptoms include: a fever of greater than 38°C, headache, chills, body aches, a dry cough, shortness of breath, difficulty breathing, hypoxia, or radiographic or other findings of either pneumonia or acute respiratory distress syndrome. Death is also a symptom of SARS.

The term, "E2"is a peptide, sometimes named"spike glycoprotein"or"E2 glycoprotein"that is encoded by the SARS virus genome. E2 functions during viral entry into the cell. Exemplary E2 proteins are provided by the sequences of Genbank Accession number AAP13441, AAP13567, andNP828851. Based on these sequences, one of skill in the art could recognize SARS virus E2 proteins as they become available.

These terms are well understood by those in the field, and refer to a protein consisting of one or more polypeptides that specifically binds an antigen. One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of antibody chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.

The recognized immunoglobulin polypeptides include the kappa and lambda light chains and the alpha, gamma (IgG,, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains or equivalents in other species. Full-length immunoglobulin"light chains" (of about 25 kDa or about 214 amino acids) comprise a variable region of about 110 amino acids at the NH2- terminus and a kappa or lambda constant region at the COOH-terminus. Full-length immunoglobulin"heavy chains" (of about 50 leDa or about 446 amino acids), similarly comprise a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e. g., gamma (of about 330 amino acids).

The terms"antibodies"and"immunoglobulin"include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen- binding portion of an antibody and a non-antibody protein. The antibodies may be detectably labeled, e. g. , with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like. The antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e. g., biotin (member of biotin- avidin specific binding pair), and the like. The antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. Also encompassed by the terms are Fab', Fv, F (ab') 2, and or other antibody fragments that retain specific binding to antigen.

Antibodies may exist in a variety of other forms including, for example, Fv, Fab, and <BR> <BR> (Fab') 2, as well as bi-functional (i. e. bi-specific) hybrid antibodies (e. g. , Lanzavecchia et al.,<BR> Eur. J. Immunol. 17,105 (1987) ) and in single chains (e. g. , Huston et al. , Proc. Natl. Acad.<BR> <P>Sci. U. S. A. , 85,5879-5883 (1988); Bird et al., Science, 242,423-426 (1988); see Hood et<BR> al. , "Immunology", Benjamin, N. Y. , 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986)).

An immunoglobulin light or heavy chain variable region consists of a"framework" region interrupted by three hypervariable regions, also called"complementarity determining regions"or CDRs. The extent of the framework region and CDRs have been precisely <BR> <BR> defined (see, "Sequences of Proteins of Immunological Interest, "E. Kabat et al., U. S.<BR> <P>Department of Health and Human Services, (1983) ). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen.

Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, the variable segments of the genes from a rabbit monoclonal antibody may be joined to human constant segments, such as gamma 1 and gamma 3. An example of a therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a rabbit antibody and the constant or effector <BR> <BR> domain from a human antibody (e. g. , the anti-Tac chimeric antibody made by the cells of A. T. C. C. deposit Accession No. CRL 9688), although other mammalian species may be used.

As used herein, unless otherwise indicated or clear from the context, antibody domains, regions and fragments are accorded standard definitions as are well known in the art. See, e. g. , Abbas, A. K. , et al. , (1991) Cellular and Molecular Immunology, W. B.

Saunders Company, Philadelphia, Pa.

As used herein, the term"humanized antibody"or"humanized immunoglobulin" refers to an antibody comprising one or more CDRs from an animal antibody, the antibody having being modified in such a way so as to be less immunogenic in a human than the parental animal antibody. An animal antibody can be humanized using a number of methodologies, including chimeric antibody production, CDR grafting (also called reshaping), and antibody resurfacing.

As used herein, the term"murinized antibody"or"murinized immunoglobulin"refers to an antibody comprising one or more CDRs from an animal antibody, the antibody having being modified in such a way so as to be less immunogenic in a mouse than the parental animal antibody. An animal antibody can be murinized using a number of methodologies, including chimeric antibody production, CDR grafting (also called reshaping), and antibody resurfacing.

The terms"polypeptide"and"protein", used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e. g. , fusion proteins including as a fusion partner a fluorescent protein, (3-galactosidase, luciferase, etc.; and the like.

As used herein the term"isolated, "when used in the context of an isolated antibody, refers to an antibody of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which the antibody is associated with prior to purification.

Techniques for determining nucleic acid and amino acid"sequence identity"also are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. In general, "identity"refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their"percent identity. "The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3: 353-358, National Biomedical <BR> Research Foundation, Washington, D. C. , USA, and normalized by Gribskov, Nucl. Acids Res. 14 (6): 6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, WI) in the"BestFit"utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, WI). A preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S.

Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the"Match"value reflects"sequence <BR> <BR> identity. "Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both ; cutoff = 60; expect = 10 ; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs can be found at the following internet address: http ://www. ncbi. nhn. gov/cgi-bin/BLAST.

Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease (s), and size determination of the digested fragments. Two DNA, or two polypeptide sequences are"substantially homologous"to each other when the sequences exhibit at least about 80%-85%, preferably at least about 85%- 90%, more preferably at least about 90%-95%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules, as determined using the methods above. As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e. g. , Sambrook et al., supra ; DNA Cloning, supra ; Nucleic Acid Hybridization, supra.

Two nucleic acid fragments are considered to"selectively hybridize"as described herein. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit a completely identical sequence from hybridizing to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e. g., Southern blot, Northern blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning : A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, <BR> <BR> N. Y. ). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate <BR> <BR> conditions the probe and the target sequence"selectively hybridize, "or bind, to each other to form a hybrid molecule. A nucleic acid molecule that is capable of hybridizing selectively to a target sequence under"moderately stringent"typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe. Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe.

Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J.

Higgins, (1985) Oxford; Washington, DC ; IRL Press).

With respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e. g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions.

The selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning : A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N. Y. ). An example of stringent hybridization conditions is hybridization at 50°C or higher and 0. 1XSSC (15 mM sodium chloride/1. 5 mM sodium citrate). Another example of stringent hybridization conditions is overnight incubation at 42°C in a solution: 50 % formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65°C. Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above representative conditions, where conditions are considered to be at least as stringent if they are at least about 80% as stringent, typically at least about 90% as stringent as the above specific stringent conditions.

Other stringent hybridization conditions are known in the art and may also be employed to identify nucleic acids of this particular embodiment of the invention.

A first polynucleotide is"derived from"a second polynucleotide if it has the same or substantially the same nucleotide sequence as a region of the second polynucleotide, its cDNA, complements thereof, or if it displays sequence identity as described above.

A first polypeptide is"derived from"a second polypeptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide, or (ii) displays sequence identity to the second polypeptides as described above.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, the singular forms "a","and", and"the"include plural referents unless the context clearly dictates otherwise.

Thus, for example, reference to"a dose"includes a plurality of such doses and reference to "the method"includes reference to one or more methods and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides compositions and methods of treating severe acute respiratory syndrome (SARS), and methods of reducing SARS viral load, reducing the time to SARS viral clearance, and reducing morbidity or mortality in the clinical outcomes, in patients suffering from a SARS. The present invention further provides methods of reducing the risk that an individual will develop SARS. The methods generally involve administering a therapeutically effective amount of an agent, e. g. a peptide or a monoclonal antibody, that interacts with the E2 core structure and prevents viral entry into a susceptible cell.

In many embodiments, the agent binds to the SARS virus E2 protein to prevent the formation of a trimer of E2 proteins that is essential for viral entry to a cell.

In describing the invention, the subject compositions are described first, and the methods in which the subject compositions may be used are then described.

Compositions The SARS virus E2 protein is encoded by the SARS genome and is described in general by Genbank Accession numbers AY274119, AY278741, AY278554, NC004718, AY278491. SARS E2 proteins may be identified by sequence comparison, e. g. , by using the BLAST algorithm to the E2 proteins identified in these Genbank accessions. The sequence of an exemplary SARS E2 protein, SEQ ID NO : 1, is provided in Fig. 1.

The SARS E2 protein complex mediates endocytosis and membrane fusion during SARS viral entry to a susceptible cell. During viral entry, the SARS E2 trimerizes to form a complex. Each E2 protein has two alpha helices that participate in E2 complex formation: a N-terminal alpha helix and a C-terminal alpha helix that are predicted (see Fig. 2 for the prediction for SEQ ID NO : 1). The alpha helices of each polypeptide interact with each other to form an anti-parallel structure, and the anti-parallel structures of three E2 proteins interact to form an E2 complex that is a cylinder (schematically shown in Fig. 3). The sequence of the predicted N-terminal alpha helix and C-terminal alpha helix, SEQ ID NOS: 2 and 3 are described in Fig. 4.

The two alpha-helices that participate in E2 complex formation are known collectively as"E2-multimerization regions".

Therapeutic agents The invention provides agents for treating SARS. The compositions are either derived from an E2-multimerization region or are compositions that specifically bind to an E2-multimerization region and/or E2 core structure. In many embodiments, the agents bind to E2 and prevent E2 multimerization.

Peptides Subject therapeutic peptides are based on the sequences of the E2-multimerization regions described above. The peptides usually contain at least 10 contiguous amino acids (e. g. about 11, about 15, about 20, about 25, about 30, or the entire length) of an E2- multimerization region, and, in certain embodiments, are at least 15 (e. g. about 16, about 20, about 25, about 30, about 35, about 40, about 50 or more, usually up to about 100) amino acids in length. As is known in the art, the subject peptides may contain naturally occurring or non-naturally occurring amino acids, and the peptide may be modified, e. g. , PEGylated,<BR> fused to other moieties, or oligomerized (e. g. , is a tetramer or dimer).

Subject therapeutic peptides may also be based on variants of the E2-multimerization regions described, which variants include mutants, fragments, and fusions of a E2- multimerization region. Mutants can include amino acid substitutions, additions or deletions. The amino acid substitutions can be conservative amino acid substitutions (gly/ala; val/ile/leu ; asp/glu; asn/gln ; ser/thr ; lys/arg ; and phe/tyr) or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, a phosphorylation site or an acetylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function. Conservative amino acid substitutions are those that preserve the general charge, hydrophobicity/hydrophilicity, and/or steric bulk of the amino acid substituted.

Further modifications, e. g. , the addition of"enhancer"moieties to C-and N-termini, that can be made in preparation of a subject therapeutic peptide are described in PCT publications WO/00-55173, WO/01-07611, WO/02-16429 and W099/59615, the entire contents of which are hereby specifically incorporated herein by reference.

Subject therapeutic peptides may comprise any of the amino acid sequences shown in Fig. 5 (SEQ ID NOS: 4-70).

Antibodies Subject therapeutic antibodies usually specifically bind to an E2-multimerization region and/or to the core structure of an E2 complex. The subject therapeutic antibodies may bind to a particular structure that is formed by non-contiguous amino acids. As such, the subject therapeutic antibodies may not specifically bind to a particular sequence of contiguous amino acids. In other words, the subject antibodies may bind one of the peptides set forth in Fig. 5, for example. In certain other embodiments, the antibodies may bind to a structure formed by a non-contiguous sequence of amino acids (e. g. , a structure that only occurs when a subject polypeptide is folded or when two amino acid sequences interact), <BR> <BR> and, accordingly, may not significantly bind to a peptide (e. g. , any of the peptides listed in the sequence listing) while it is in denatured form.

Suitable antigens for antibody production may be made using a number of methods.

In most embodiments, a polypeptide is produced recombinantly in a host cell, and purified.

Methods for producing recombinant proteins are well known in the art (Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed. , Wiley &amp; Sons, 1995 and Sambrook, et al, Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold Spring Harbor, N. Y.). and, as such, are not described in any detail herein.

In one embodiment, a suitable antigen is a peptide containing at least 10 contiguous amino acids (e. g. about 11, about 15, about 20, about 25, about 30, or the entire length) of an E2-multimerization region. Examples of 10 contiguous amino acids may be found in Fig. 5 (SEQ ID NOS: 4-70).

In another embodiment, an antigen contains a mixture of peptides containing of at least 10 contiguous amino acids of each of the C-and N-E2-multimerization regions (e. g.

SEQ ID NOS: 2 and 3). In these embodiments, the peptides are produced using methods that allow the peptides to form secondary structure, e. g. alpha-helices that interact to form a three dimensional structure similar to the E2 core structure shown in Fig. 3. Typically, protein production in a cell e. g. , a bacterial, yeast or mammalian cell, is sufficient to provide a protein with sufficient structure for use in these methods.

Such peptides may be produced separately and then mixed, denatured, and renatured, or, alternatively, may be produced in a suitable cell, allowing cellular machinery to correctly fold the polypeptides into a suitable E2 core protein structure. Strategies for expressing two or more polypeptides in a single cell are known in the art. peptides are produced as multimers fused to a flexible "linker"peptide that permits the peptides to form a"hairpin"structure. Suitable exemplary arrangements of multimers are shown in Fig. 6. In these embodiments, trimers of"C-C-C" and"N-N-N"may be mixed, e. g., equimolarly, to produce an E2 core structure. Similarly, <BR> <BR> monomers containing C-N fusion may be mixed, e. g. , equimolarly, to produce an E2 core<BR> structure. "C"and"N"in the previous embodiments refer to peptides having at least 10 contiguous amino acids of the C and N E2-multimerization regions, and the arrows represent the polarity, e. g. , C to N, or N to C of the polypeptide.

As noted above, the subject polypeptides typically include at least one sequence that is flexible, allowing an amino acid chain to bend or rotate freely. Suitable linkers include glycine polymers (G) n, glycine-serine polymers (including, for example, (GS) n, (GSGGS) n and (GGGS) n, where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers such as the tether for the shaker potassium channel, and a large variety of other flexible linkers, as will be appreciated by those in the art. Glycine and glycine-serine polymers are preferred since both of these amino acids are relatively unstructured, and therefore may be able to serve as a neutral tether between components.

Glycine polymers are the most preferred as glycine accesses significantly more phi-psi space than even alanine, and is much less restricted tan residues with longer side chains (see <BR> <BR> Scheraga, Rev. Computational Chem. 11173-142 (1992) ). Secondly, serine is hydrophilic and therefore able to solubilize what could be a globular glycine chain. Similar chains have been shown to be effective in joining subunits of recombinant proteins such as single chain antibodies. In many embodiments, a flexible linker comprises about 6, about 8, about 10, about 12, about 15, about 20, about 25 or about 30 amino acids or more.

Suitable antibodies are obtained by immunizing a host animal with these compositions. Suitable host animals include mouse, rat, sheep, goat, hamster, rabbit, etc. In certain embodiments, the compositions may be used as a vaccine, and, as such, may be used to immunize a human directly.

For preparation of polyclonal antibodies, the first step is immunization of the host animal with an antigen, where the antigen will preferably be in substantially pure form, comprising less than about 1% contaminant. The antigen may comprise complete E2 multimerization region or E2 core structure, fragments or derivatives thereof. To increase the immune response of the host animal, the antigen may be combined with an adjuvant, where suitable adjuvants include alum, dextran, sulfate, large polymeric anions, oil & water emulsions, e. g. Freund's adjuvant, Freund's complete adjuvant, and the like. The antigen may also be conjugated to synthetic carrier proteins or synthetic antigens. A variety of hosts may be immunized to produce the polyclonal antibodies. Such hosts include rabbits, guinea pigs, rodents, e. g. mice, rats, sheep, goats, and the like. The antigen is administered to the host, usually intradermally, with an initial dosage followed by one or more, usually at least two, additional booster dosages. Following immunization, the blood from the host will be collected, followed by separation of the serum from the blood cells. The Ig present in the resultant antiserum may be further fractionated using known methods, such as ammonium salt fractionation, DEAE chromatography, and the like.

Monoclonal antibodies are produced by conventional techniques. Generally, the spleen and/or lymph nodes of an immunized host animal provide a source of plasma cells.

The plasma cells are immortalized by fusion with myeloma cells to produce hybridoma cells.

Culture supernatant from individual hybridomas is screened using standard techniques to identify those producing antibodies with the desired specificity. Suitable animals for production of monoclonal antibodies include mouse, rat, hamster, etc. The antibody may be purified from the hybridoma cell supernatants or ascites fluid by conventional techniques, e. g. affinity chromatography using the antigen bound to an insoluble support, protein A sepharose, etc.

The antibody may be produced as a single chain, instead of the normal multimeric structure. Single chain antibodies are described in Jost et al. (1994) J. B. C. 269: 26267-73, and others. DNA sequences encoding the variable region of the heavy chain and the variable region of the light chain are ligated to a spacer encoding at least about 4 amino acids of small neutral amino acids, including glycine and/or serine. The protein encoded by this fusion allows assembly of a functional variable region that retains the specificity and affinity of the original antibody.

For in vivo use, particularly for injection into humans, it is desirable to decrease the antigenicity of the antibody. An immune response of a recipient against the blocking agent will potentially decrease the period of time that the therapy is effective. Methods of humanizing antibodies are known in the art. The humanized antibody may be the product of an animal having transgenic human immunoglobulin constant region genes (see for example International Patent Applications WO 90/10077 and WO 90/04036). Alternatively, the antibody of interest may be engineered by recombinant DNA techniques to substitute the CH1, CH2, CH3, hinge domains, and/or the framework domain with the corresponding human sequence (see WO 92/02190).

The use of Ig cDNA for construction of chimeric immunoglobulin genes is known in the art (Liu et al. (1987) P. N. A. S. 84: 3439 and (1987) J. Immunol. 139: 3521). mRNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA.

The cDNA of interest may be amplified by the polymerase chain reaction using specific primers (U. S. Patent nos. 4,683, 195 and 4,683, 202). Alternatively, a library is made and screened to isolate the sequence of interest. The DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences. The sequences of human constant regions genes may be found in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, N. I. H. publication no. 91-3242. Human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity. Preferred isotypes are IgGl, IgG3 and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. The chimeric, humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F (ab') 2 and Fab may be prepared by cleavage of the intact protein, e. g. by protease or chemical cleavage. Alternatively, a truncated gene is designed. For example, a chimeric gene encoding a portion of the F (ab') 2 fragment would include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments. C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence. Monoclonal antibodies may be humanized, if desired.

Suitable antibodies may be screened for using a variety of assays, including binding assays and in vitro assays for SARS. Antibodies that have an activity to reduce levels of the SARS virus in in vitro cultured cells infected with the SARS virus, or an in vitro SARS virus protective activity have desirable properties. In many assays, antibodies are tested for their ability to bind specifically to a substrate. The term"specifically"in the context of antibody binding, refers to high avidity and/or high affinity binding of an antibody to a specific antigen i. e. , a polypeptide, or epitope. In many embodiments, the specific antigen is an antigen (or a fragment or subtraction of an antigen) used to immunize the animal host from which the antibody-producing cells were isolated. Antibody specifically binding an antigen or fragment thereof is stronger than binding of the same antibody to other antigens.

Antibodies which bind specifically to a polypeptide may be capable of binding other <BR> <BR> polypeptides at a weak, yet detectable, level (e. g. , 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to a subject polypeptide, e. g. by use of appropriate controls. In general, specific antibodies bind to an antigen with a binding affinity of 10-7 M <BR> <BR> or more, e. g., 10-8 M or more (e. g., 10-9 M, 10-1°, 10-11, etc. ). In general, an antibody with a binding affinity of 10-6 M or less is not useful in that it will not bind an antigen at a detectable level using conventional methodology currently used.

The therapeutic agents described above find use in a variety of methods, represented protocols of which are described below.

Methods The therapeutic agents described above find use in various in vitro and in vivo methods. In some in vitro embodiments, the methods involve contacting cells that are infected with SARS virus or cells that are to be infected with SARS virus, and determining a <BR> <BR> SARS virus related phenotype, e. g. , virus titer, number of cells infected, protection against<BR> SARS virus, etc. , of the cells.

The therapeutic compositions described above also find use as a treatment for SARS.

In some embodiments, the subject treatment is administered to an individual prophylactically, e. g. , is initiated before the appearance of symptoms. Such prophylactic treatment is administered in the case of individuals who are asymptomatic and who may or may not yet be infected, but who have come into close contact with an individual who has been diagnosed with SARS ; individuals who are asymptomatic and who are not yet be infected, but who expect to come into contact with an individual who has been diagnosed with SARS (e. g. , health care workers working in a facility in which individuals who have been diagnosed with SARS are being cared for); individuals who are asymptomatic and who are not yet be infected, and who are traveling to a location known to have a relatively high incidence of SARS cases; and the like.

In other embodiments, the subject treatment is initiated after the appearance of clinical signs of SARS, e. g., the appearance of a fever often exceeding 38°C. An advantage <BR> <BR> of the subject methods is that the severity of SARS symptoms is reduced, e. g. , the viral load is reduced, and/or the time to viral clearance is reduced, and/or the morbidity or mortality is reduced.

Where a subject treatment method is prophylactic, the methods reduce the risk that an individual will develop pathological infection with a SARS virus. Effective amounts of a subject agent are amounts that, alone or in combination therapy, reduce the risk or reducing the probability that an individual will develop a pathological infection with a SARS virus.

For example, an effective amount reduces the risk that an individual will develop a pathological infection by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the risk of developing a pathological infection with the virus in the absence of the treatment.

In some embodiments, effective amounts of a subject agent are amounts that, alone or in combination therapy, reduce SARS viral load by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the viral load in the absence of treatment.

In some embodiments, effective amounts of a subject agent are amounts that, alone or in combination therapy, reduce the time to viral clearance, by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the time to viral clearance in the absence of treatment.

In some embodiments, effective amounts of a subject agent are amounts that, alone or in combination therapy, reduce morbidity or mortality due to a SARS infection by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the morbidity or mortality in the absence of treatment.

Whether a subject treatment method is effective in reducing the risk of a pathological coronavirus infection, reducing viral load, reducing time to viral clearance, or reducing morbidity or mortality due to a coronavirus infection is readily determined by those skilled in the art. Viral load is readily measured by measuring the titer or level of virus in serum. <BR> <BR> <P>The number of virus (e. g. , the number of viral particles or the number of viral genomes) in<BR> the serum can be determined using any known assay, including, e. g. , a quantitative polymerase chain reaction assay using oligonucleotide primers specific for the SARS virus being assayed. Whether morbidity is reduced can be determined by measuring any symptom <BR> <BR> associated with a SARS infection, including, e. g. , fever, respiratory symptoms (e. g. , cough, ease or difficulty of breathing, and the like).

As such, the invention provides formulations, including pharmaceutical formulations, that include an agent which inhibits multimerization of the SARS virus E2 protein. In general, a formulation comprises an effective amount of an agent that inhibits SARS virus E2 multimerization in a host. An"effective amount"refers to an amount that is sufficient to produce a desired result, e. g. , reduction SARS virus E2 multimerization, reduction of a SARS symptom, reduction of viral load, reduced time for viral clearance, etc. In many embodiments, the desired result is at least a reduction or increase in a phenotype as compared to a control such that the phenotype is more similar to normal.

Formulations In the subject methods, the active agent (s) may be administered to the host using any convenient means capable of resulting in the desired reduction in of a SARS phenotype.

Thus, the agent can be incorporated into a variety of formulations for therapeutic administration. More particularly, the agents of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

In pharmaceutical dosage forms, the agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch ; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose ; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

The agents can be utilized in aerosol formulation to be administered via inhalation.

The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor (s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

The term"unit dosage form, "as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

Other modes of administration will also find use with the subject invention. For instance, an agent of the invention can be formulated in suppositories and, in some cases, aerosol and intranasal compositions. For suppositories, the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%.

Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention. The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.

An agent of the invention can be administered as injectables. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles.

Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e. g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985; Remington: The Science and Practice of Pharmacy, A. R. Gennaro, (2000) Lippincott, Williams &amp; Wilkins. The composition or formulation to be administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

Dosages Although the dosage used will vary depending on the clinical goals to be achieved, a suitable dosage range is one which provides up to about 1 ug to about 1,000 llg or about 10, 000 ug of an agent that reduces a symptom of SARS a subject animal.

Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

Routes of administration Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, intratumoral, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral and other parenteral routes of administration.

Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect. The composition can be administered in a single dose or in multiple doses.

The agent can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated by the invention include, but are not necessarily limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i. e. , any route of administration other than through the alimentary canal. Parenteral administration can be carried to effect systemic or local delivery of the agent. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.

The agent can also be delivered to the subject by enteral administration. Enteral <BR> <BR> routes of administration include, but are not necessarily limited to, oral and rectal (e. g. , using a suppository) delivery.

Methods of administration of the agent through the skin or mucosa include, but are not necessarily limited to, topical application of a suitable pharmaceutical preparation, transdermal transmission, injection and epidermal administration. For transdermal transmission, absorption promoters or iontophoresis are suitable methods. Iontophoretic transmission may be accomplished using commercially available"patches"which deliver their product continuously via electric pulses through unbroken skin for periods of several days or more.

By treatment is meant at least an amelioration of the symptoms associated with the pathological condition afflicting the host, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e. g. symptom, associated with the pathological condition being treated, such as an sebaceous gland disorder and psychological trauma associated therewith. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e. g. prevented from happening, or stopped, e. g. terminated, such that the host no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition.

A subject polynucleotide can be delivered as a naked polynucleotide, or associated with (complexed with) a delivery vehicle."Associated with", or"complexed with", encompasses both covalent and non-covalent interaction of a polynucleotide with a given delivery vehicle.

Additional therapeutic agents Any of the above-described treatments can be used in conjunction with administration of an additional antiviral agents, e. g. , a specific antiviral agent that is effective in treating a pathological SARS infection. Additional antiviral agents that are suitable for use in combination therapy include, but are not limited to, nucleotide and nucleoside analogs. Non-limiting examples include AZT (zidovudine), DDI (didanosine), DDC (dideoxycytidine), D4T (stavudine), combivir, abacavir, adefovir dipoxil, cidofovir, ribavirin, ribavirin analogs, and the like.

In some embodiments, the method further includes administration of ribavirin.

Ribavirin, l-, B-D-ribofuranosyl-lH-1, 2,4-triazole-3-carboxamide, available from ICN <BR> <BR> Pharmaceuticals, Inc. , Costa Mesa, Calif. , is described in the Merck Index, compound No.

8199, Eleventh Edition. Its manufacture and formulation is described in U. S. Pat. No.

4,211, 771. The invention also contemplates use of derivatives of ribavirin (see, e. g., U. S.

Pat. No. 6,277, 830). The ribavirin may be administered orally in capsule or tablet form, or in the same or different administration form and in the same or different route as the therapeutic compositions described above. Of course, other types of administration of both medicaments, as they become available are contemplated, such as by nasal spray, transdermally, intravenously, by suppository, by sustained release dosage form, etc. Any form of administration will work so long as the proper dosages are delivered without destroying the active ingredient.

Ribavirin is generally administered in an amount ranging from about 30 mg to about 60 mg, from about 60 mg to about 125 mg, from about 125 mg to about 200 mg, from about 200 mg to about 300 gm, from about 300 mg to about 400 mg, from about 400 mg to about 1200 mg, from about 600 mg to about 1000 mg, or from about 700 to about 900 mg per day, or about 10 mg/kg body weight per day.

In some embodiments, an additional antiviral agent is administered during the entire course of treatment. In other embodiments, an additional antiviral agent is administered for a period of time that is overlapping with that of the subject treatment, e. g. , the additional antiviral agent treatment can begin before the subject treatment begins and end before the subject treatment ends; the additional antiviral agent treatment can begin after the treatment begins and end after the subject treatment ends; the additional antiviral agent treatment can begin after the treatment begins and end before the subject treatment ends; or the additional antiviral agent treatment can begin before the subject treatment begins and end after the subject treatment ends.

The methods described above find use in treating an individual in need thereof.

Individuals who are to be treated according to the methods of the invention include individuals who have been clinically diagnosed with SARS, as well as individuals who exhibit one or more of the signs and the symptoms of SARS but have not yet been diagnosed with SARS. Individuals who are to be treated according to the methods of the invention also include individuals with anticipated exposure to individuals diagnosed with SARS (e. g., health care professionals; individuals traveling to areas with a relatively high incidence of SARS; and the like); individuals with suspected exposure to an individual diagnosed with SARS; and individuals with known exposure to an individual with SARS. "Exposure" includes contact that is sufficiently close as to allow the etiologic agent to be transmitted from an infected individual to the exposed individual.

Animal models for SARS, e. g. rats, mice, monkeys and the like, may also be treated using the subject compositions.

Kits Also provided by the subject invention are kits containing therapeutic compositions for practicing the subject methods, as described above. The subject kits at least include one or more of : a monoclonal antibody that specifically bind to an E2-multimerization region and/or E2 core structure or a peptide derived from an E2-multimerization region. The various components of the kit may be present in separate containers or certain compatible components may be precombined into a single container, as desired. In many embodiments, kits with unit doses of the active agent, e. g. in oral or injectable doses, are provided.

In addition to above-mentioned components, the subject kits typically further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i. e. , associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e. g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e. g. via the internet, are provided.

An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.