WO2006113429A2 - Viral inhibitors - Google Patents

Abstract

The present invention claims and discloses methods of identifying proteins and/or peptide that inhibit viral infection for the treating, preventing and/or ameliorating of an infection caused by a virus. The application also claims and discloses formulations and specific peptides identified using the disclosed methods.

Description

Viral Inhibitors

This Application claims priority to provisional applications 60/672, 117, filed April 13, 2005, 60/675,157, filed April 27, 2005, 60/675,226, filed April 27, 2005, 60/675,134, filed April 27, 2005, 60/748, 332, filed December 7, 2005 and 60/776,239 filed February 24, 2006, all of which are incorporated herein by reference in their entireties for all proposes.

FIELD OF INVENTION

This application relates to methods of identifying proteins and/or peptides that inhibit viral infection for treating, preventing and/or ameliorating an infection caused by a virus. The application also discloses formulations and specific peptides identified using the disclosed methods.

BACKGROUND OF THE INVENTION Viruses are the smallest of parasites, and are completely dependent on the cells they infect for their reproduction. Viruses are generally composed of at least one outer coat protein, which is sometimes surrounded by a lipid envelope, and an inner nucleic acid core consisting of either RNA or DNA. Generally, after docking with the plasma membrane of a susceptible cell, the viral core penetrates the cell membrane to initiate the viral infection. After infecting cells, viruses take over the cell's molecular machinery to direct their own replication and packaging. The "replicative phase" of the viral lifecycle may begin immediately upon entry into the cell, or may occur after a period of dormancy or latency. After the infected cell synthesizes sufficient amounts of viral components, the "packaging phase" of the viral life cycle begins and new viral particles are assembled. Some viruses reproduce without killing their host cells, and many of these bud from host cell membranes. Other viruses cause their host cells to lyse or burst, releasing the newly assembled viral particles into the surrounding environment, where they can begin the next round of their infectious cycle.

The first step toward viral fusion with the host cell involves binding of viral receptors on the viral membrane with receptors on the host cell membrane. These host cell receptors are normal surface molecules involved in routine cellular function. However, viruses take advantage of these receptors to attach to the host cell's surface. For example, HIV adsorbs to CD4 molecules and chemokine receptors found on the surface of human T4-lymphocytes and macrophages. CD4 molecules are normally involved in immune recognition while chemokine receptors play a role in initiating inflammation and recruiting. During penetration, the viral envelope fuses with host cell membrane and the nucleocapsid enters the host cell. This is followed by uncoating during which the viral capsid is enzymatically degraded and the viral genome is released. Fusion of the virus to the cell is essential for viral replication.

Several hundred different types of viruses are known to infect humans, however, since many of these have only recently been recognized, their clinical significance is not fully understood. Of these viruses that infect humans, many infect their hosts without producing overt symptoms, while others (e.g., influenza) produce a well-characterized set of symptoms. Importantly, although symptoms can vary with the virulence of the infecting strain, identical viral strains can have drastically different effects depending upon the health and immune response of the host. Despite remarkable achievements in the development of vaccines for certain viral infections (i.e., polio and measles), and the eradication of specific viruses from the human population (e.g., smallpox), viral diseases remain an important medical and public health problems. Indeed, viruses are responsible for several "emerging" (or reemerging) diseases (e.g., West Nile encephalitis & Dengue fever), and also for the largest pandemic in the history of mankind (HIV and AIDS).

Viruses that primarily infect humans are spread mainly via respiratory and enteric excretions. These viruses are found worldwide, but their spread is limited by inborn resistance, prior immunizing infections or vaccines, sanitary and other public health control measures, and prophylactic antiviral drugs. Zoonotic viruses pursue their biologic cycles chiefly in animals, and humans are secondary or accidental hosts. These viruses are limited to areas and environments able to support their nonhuman natural cycles of infection (animals or arthropods or both). However, with increased global travel by humans, and the likely accidental co-transport of arthropod vectors bearing viral payloads, many zoonotic viruses are appearing in new areas and environments as emerging diseases. For example, West Nile virus, which is spread by the bite of an infected mosquito, and can infect people, horses, many types of birds, and other animals, was first isolated from a febrile adult woman in the West Nile District of Uganda in 1937. The virus made its first appearance in the Western Hemisphere, in the New York City area in the autumn of 1999, and during its first year in North America, caused the deaths of 7 people and the hospitalization of 62.

Treatment of viral diseases presents unique challenges to modern medicine. Since viruses depend on host cells to provide many functions necessary for their multiplication, it is difficult to inhibit viral replication without at the same time affecting the host cell itself. Consequently, antiviral treatments are often directed at the functions of specific enzymes of particular viruses. However, such antiviral treatments that specifically target viral enzymes (e.g., HIV protease, or HIV reverse transcriptase) often have limited usefulness, because resistant strains of viruses readily arise through genetic drift and mutation. For example, a number of reverse-transcriptase-targeted drugs, including 2',3'-dideoxynucleoside analogs such as AZT, ddl, ddC, and d4T have been developed which have been shown to been active against HIV (Mitsuya, H. et al, 1991). While beneficial, these nucleoside analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander, B. et al, 1989). In addition, the drugs often exhibit toxic side effects such as bone marrow suppression, vomiting, and liver function abnormalities.

Attempts are also being made to develop drugs which can inhibit viral entry into the cell. In HIV, for example, the focus has thus far been on CD4, the cell surface receptor for HIV. Recombinant soluble CD4, for example, has been shown to inhibit infection of CD-4⁺ T-cells by some HIV-I strains (Smith, D. H. et al, 1987). Certain primary HIV-I isolates, however, are relatively less sensitive to inhibition by recombinant CD-4 (Daar, E. et al, 1990). In addition, recombinant soluble CD-4 clinical trials have produced inconclusive results (Schooley, R. et al, 1990; Yarchoan, R. et al, 1989).

Recently, the Food and Drug administration approved a peptide, enfuvirtide, which inhibits the fusion of HIV-I with the CD4 cells. Enfuviride is a linear 36 amino acid synthetic peptide. Evfuviride interferes with the entry of HIV-I into cells by inhibiting fusion of viral and cellular membranes. Enfuvirtide binds to the first heptad-repeat in the gp- 41 subunit of the viral envelope glycoprotein and prevents the conformational changes required for the fusion of viral and cellular membranes. The peptide is currently sold under the trade name Fuzeon®.

SUMMARY OF THE INVENTION

This invention comprises a method for identifying a viral protein or peptide that inhibits viral infection comprising, searching a database for a viral protein with a W X_nlW motif and analyzing the region around said motif to further identify an I X₁₁₂ I motif, thereby identifying a viral protein or peptide that inhibits viral infection, wherein X is any amino acid and nl and n2 is a number from 0 to 20. The invention also encompasses aligning said identified protein or peptide with a known protein sequence that inhibits viral infection, identifying homology between said known sequence and identified protein or peptide and isolating the homologous amino acid sequence comprising the aforementioned motifs.

This invention also comprises a viral protein or peptide identified according to the method disclosed. In one embodiment, a viral protein or peptide is attached to another molecule or fused to another protein, In another embodiment, said viral protein or peptide inhibits viral infection.

The invention also comprises compositions comprising viral proteins or peptides identified according to the method disclosed, wherein said composition treats, prevents and/or ameliorates a viral infection in an animal when administered to said animal. The invention also comprises methods for treating, preventing and/or ameliorating an infection caused by a virus, comprising administering to an animal the viral protein or peptide of identified according to the methods disclosed.

The invention also comprises antigenic foπnulations comprising the viral protein or peptide identified according to the methods disclosed. In one embodiment, the antigenic formulation stimulates an immune response against said viral protein or peptide in an animal. In another embodiment, the antigenic formulation may comprise an adjuvant or immune stimulator. In another embodiment, the antigenic formulation maybe administered to an animal orally, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or subcutaneously. The invention also comprises methods of formulating a vaccine capable of delivering an effective dose that induces substantial immunity to a viral infection or at least one symptom thereof to an animal, comprising adding to said formulation the viral protein or peptide identified according to the method disclosed. In one embodiment, said substantial immunity to viral infection or at least one symptom thereof is delivered in one dose. In another embodiment, said substantial immunity to viral infection or at least one symptom thereof is delivered in multiple doses.

The invention also comprises a peptide consisting essentially of any one of the peptides selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 34. In one embodiment, said peptide is attached to another molecule or fused to another protein or polypeptide. In another embodiment, said peptide of inhibits viral transmission of a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus. In another embodiment, said peptide inhibits infection of a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus.

The invention also comprises compositions comprising a peptide consisting essentially of the peptide selected from the group consisting of group consisting of SEQ ID NO. 1 to SEQ ID NO. 34. hi one embodiment, the composition comprising said peptide treats, prevents and/or ameliorates a viral infection caused by a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus in an animal, when administered to said animal. In another embodiment, the composition comprising said peptide inhibits viral transmission of a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus in an animal, when administered to said animal, hi another embodiment, the composition comprising said peptide inhibits infection of a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus in an animal, when administered to said animal. The invention also comprises methods for treating, preventing and/or ameliorating a viral infection caused by a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus, comprising administering to an animal a peptide consisting essentially of a peptide selected from the group consisting of group consisting of SEQ ID NO. 1 to SEQ ID NO. 34. In one embodiment, said methods comprise a peptide which inhibits viral transmission of a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus. In another embodiment, said methods comprise a peptide which inhibits infection of a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus.

The invention also comprises antigenic formulations comprising the peptide consisting essentially of a peptide selected from the group consisting of group consisting of SEQ ID NO. 1 to SEQ ID NO. 34. In one embodiment, said antigenic formulation stimulates an immune response against said peptide in an animal. In another embodiment, said formulation is administered to an animal orally, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or subcutaneously.

The invention also comprises methods of formulating a vaccine capable of delivering an effective dose that induces substantial immunity to a viral infection caused by a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus or at least one symptom thereof to an animal, comprising adding to said formulation the peptide consisting essentially of the peptide selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 34. In another embodiment, said substantial immunity to a viral infection caused by a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus or at least one symptom thereof is delivered in one dose. In another embodiment, said substantial immunity to a viral infection caused by a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus is delivered in multiple doses.

The invention also comprises a polypeptide or peptide comprising the motifs W X_nl W and I X₁₁₂ I, wherein X is any amino acid and nl and n2 is a number from 0 to 20, and wherein the polypeptide or peptide is less than about 100 amino acids in length.

DETAILED DESCRIPTION

Definitions

Peptides are defined herein as organic compounds comprising two or more amino acids co valently joined by peptide bonds. Peptides may be referred to with respect to the number of constituent amino acids, i.e., a dipeptide contains two amino acid residues, a tripeptide contains three, etc. Peptides containing ten or fewer amino acids may be referred to as oligopeptides, while those with more than ten amino acid residues are polypeptides or peptides.

Peptide sequences defined herein are represented by one-letter symbols for amino acid residues as follows: A (alanine), R (arginine) , N (asparagine), D (aspartic acid), C (cysteine), Q (glutamine), E (glutamic acid), G (glycine), H (histidine), I (isoleucine), L (leucine), K (lysine), M (methionine), F (phenylalanine), P (proline), S (serine), T (threonine), W (tryptophan), Y (tyrosine), V (valine). It is contemplated that both L and D forms of the amino acids can comprise the viral protein or peptide of the invention. As used herein the term "substantial immunity" refers to a therapeutically effective amount of a peptide or viral protein of the invention that prevents viral infection, as measured by standard procedures for a said virus, that results in amelioration of at least one symptom related to a viral infection in an animal. As used herein the term "adjuvant" or "immune stimulator" refers to a compound that, when used in combination with a specific immunogen (e.g. a peptide of the invention) in a formulation, augments or otherwise alters or modifies the resultant immune response. Modification of the immune response includes intensification or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain antigen-specific immune responses.

As used herein an "effective dose" refers to that amount of the viral protein or peptide of the invention sufficient to treat or manage viral infections or to enhance the efficacy of another dose of a viral protein or peptide. An effective dose may refer to the amount of the viral protein or peptide sufficient to delay or minimize the onset of a viral infection. An effective dose amount may also refer to the amount of the viral protein or peptide that provides a therapeutic benefit in the treatment or management of viral infection. Further, an effective dose is the amount with respect to the viral protein or peptide of the invention alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a viral infection.

As used herein the term "substantially protective antibody response" refers to an immune response mediated by antibodies against a virus, which is exhibited by an animal (e.g., a human), that may protect or at least reduce a symptom of a virus in said animal. The antibody response stimulates the production of antibodies (e.g., neutralizing antibodies that block viruses from entering cells and/or replicating by binding to the virus, typically protecting cells from infection and destruction).

As used herein the term "substantially protective cellular response" refers to an immune response that is mediated by T-lymphocytes and/or other white blood cells against viruses exhibited by an animal (e.g., a human), that may protect or at least reduce a symptom of a virus in said animal. One important aspect of cellular immunity involves an antigen- specific response by cytolytic T-cells ("CTL"s). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular viruses, or the lysis of cells infected with such viruses. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A "cellular immune response" also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.

As used herein the term "vaccine" refers to a suspension or solution of an immunogen (e.g. viral protein or peptide) that is administered to an animal to produce active immunity. A vaccine is administered to provide immunity to a disease.

As used herein, the term "immunospecifically binds" and analogous terms refer to antibodies or fragments thereof that specifically bind to a viral protein or peptide of the invention or fragment thereof and do not specifically bind to other viral protein or peptides. As used herein, the terms "antibody" and "antibodies" refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies of the invention), bispecific, and epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.

As used herein the terms "viral proteins or peptides" refer to a protein or peptide that exhibits a W X_nl W motif and an I X_n2 I motif and which measurably inhibits viral infection (e.g. by inhibiting viral fusion to a cell, inhibiting viral penetration into a cell and/or which inhibiting viral transmission), wherein X is any amino acid and nl and n2 is a number from 0 to 20. nl and n2 may or may not be the same number, these variables are not related. Thus, throughout the specification, each nl and n2 is a number from 0 to 20, but nl and n2 can be the same number or can be a different number.

As used herein "inhibition of viral infection" or analogous terms refers to inhibiting a virus from entering a cell and/or limiting viral replication and/or limiting propagation of a virus in an infected a cell. Inhibition of viral infection can occur by, but not limited to, inhibiting viral fusion, replication, penetration and/or viral propagation. As used herein, the terms "prevent," "preventing" and "prevention" refer to the prevention of the recurrence, onset, or development of a disorder or one or more symptoms of a disorder in a subject resulting from the administration of a therapy (e.g., a prophylactic or therapeutic agent), or the administration of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents).

As used herein, "consisting essentially of in relation to a protein or peptide refers to the sequence of the peptide, said sequences including additional amino acids on either the C- or N- terminus of the protein or peptide as well as allowing for substitutions within the peptide that do not substantially alter said protein or peptide activity. Such peptides typically do not include a natural full length protein from a virus.

As used herein the term "analog" in the context of a protein or peptide refers to a protein or peptide that possesses a similar or identical function as a second protein or peptide but does not necessarily comprise a similar or identical amino acid sequence as the second protein or peptide. The term "analog" may also refer to a protein or peptide that does possess a similar or identical amino acid sequence of a second protein or peptide. A protein or peptide has a similar amino acid sequence if said protein or peptide has at least 70 percent, preferably at least 80 percent or 85 percent, more preferably at least about 86%, 87%, 88%, 89%, 905, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous amino acid sequence of the second protein or peptide. The term "analog" also refers to a protein or peptide with a similar secondary, tertiary or quaternary structure to a second protein or peptide. The structure of a protein or peptide can be determined by methods known to those skilled in the art, including but not limited to, sequencing, X-ray crystallography, nuclear magnetic resonance, circular dichroism, and crystallographic electron microscopy.

As used herein, the term "derivative" in the context of protein or peptide refers to a protein or peptide that comprises an amino acid sequence that has been altered by the introduction of amino acid residue substitutions, deletions, and/or additions. The term "derivative" as used herein also refers to a protein or peptide which has been modified, i.e., by the covalent attachment of any type of molecule to the protein or peptide.

Methods of Identifying Viral Proteins or Peptides Which Inhibit Viral Infections

The first step toward viral fusion with the host cell involves binding of viral receptors on the viral membrane with receptors on the host cell membrane. These host cell receptors are normal surface molecules involved in routine cellular function. However, viruses take advantage of these receptors to attach to the host cell's surface. The present invention claims and discloses methods of identifying proteins and/or peptides that inhibit viral infection at its earliest stages of infections for the treating, preventing and/or ameliorating of an infection caused by a virus.

The present invention provides a method identifying a viral protein or peptide that substantially inhibits viral infection, which comprises inhibiting viral fusion, penetration and/or viral transmission. The method comprises scanning a viral protein sequence for a W X _ni W motif. This can be accomplished by using a computer script or by visually analyzing the sequence and locating each W in the protein sequence and analyzing the region for said motif. If said W X_n] W motif is present, the next step is to locate an I X₁₁₂ I motif in the region around the W X_n[ W motif. Again, this can accomplished using a computer script (can be in the same script as above) or can be accomplished by visually analyzing the sequences for said motif. If the W X_nl W motif is near (i.e. within about 50 amino acids) the I X_n2 1 motif then a peptide that may substantially inhibits viral infection has been identified (putative inhibitor). However, the I X „₂ I motif need not be present in the region for this peptide to be a putative inhibitor. Other hydrophobic amino acids can be present as well. For example, I X₁₁₂ L, L X₁₁₂ L, V X_n2V, II, IL, LI, and LL are non-limiting examples of motifs that may be present. These identified peptides can be tested for inhibition of viral infection by standard procedures for a particular virus.

The putative inhibitor can be further confirmed by aligning said identified peptide with a known peptide that inhibits viral infection. For example, the peptide encoded by SEQ ID NO. 13 (enfuvirtide) can be used. By analyzing the alignment and confirming homology and/or structural similarity between a known viral inhibitor and the putative inhibitor, a peptide of the invention has been identified. Another method to confirm if these identified peptides can inhibit viral infection is by analyzing the tertiary structure of the putative inhibitor. This can be accomplished by several methods known in the art (X-ray crystallography, nuclear magnetic resonance, circular dichroism, crystallographic electron microscopy or any computer program available for such purposes). Often peptides that inhibit viral infections are alpha helical structures. Thus, if said motifs are present in the putative inhibitor, if the putative inhibitor has homology with a known viral inhibitor and/or the predicted structure of the putative inhibitor is an alpha helical motif, then a peptide of the invention has been identified. These identified peptides can be tested for inhibition of viral infection, which comprises inhibition of viral penetration into a cell, and/or inhibition of viral viral transmission, by standard procedures for a particular virus. Thus, this invention comprises a method for identifying a viral protein or peptide that inhibits infection, comprising searching a database for a viral protein with a W X_nlW motif and analyzing the region around said motif to further identify an I X₁₁₂ I motif thereby identifying a viral protein or peptide that inhibits viral infection, wherein X is any amino acid and nl and n2 is a number from 0 to 20.

In another embodiment, the method further comprises aligning said identified protein or peptide comprising a W X₁₁₁W and I X₁₁₂ I motif with a known protein sequence that inhibits viral infection, identifying homology between said known sequence and identified protein or peptide and optionally isolating the homologous amino acid sequence comprising the motifs. In another embodiment, said identified peptide does not include Fuzeon^® as exemplified in SEQ ID NO. 13. In another embodiment, said identified peptide has a length of about 10 to about 200, about 20 to about 150, about 45 to about 100, amino acids. In another embodiment, said method identifies a peptide of a length of about 20 to 100 amino acids. For example, the peptide maybe aboutlO, 15, 20, 30, 40, 50 or 60 amino acids, preferably about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more amino acids. Preferred examples of peptides of the invention include, but are not limited to, SEQ ID NO. 1 to SEQ ID NO. 34. In another embodiment, the identified peptide of the invention has an amino acid sequence that is at least 70 percent, preferably at least 80 percent or 85 percent, more preferably at least about 86%, 87%, 88%, 89%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous amino acid sequence of any of the above identified peptides or a peptide with a motif of W X_nlW and I X₁₁₂1, wherein X is any amino acid and nl and n2 is a number from 0 to 20 and said peptide inhibits viral infection.

Said identified viral protein of the invention comprises amino acids sequences of 150 amino acids or greater. In one embodiment, the viral protein of the invention has an amino acid sequence that is at least 70 percent, preferably at least 80 percent or 85 percent, more preferably at least about 86%, 87%, 88%, 89%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous amino acid sequence which comprise the motifs of W X_nlW and I X₁₁₂1, wherein X is any amino acid and nl and n2 is a number from 0 to 20 and said protein inhibits viral infection.

In calculating percent sequence identity, the BLAST algorithm performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01 , and most preferably less than about 0.001.

A polypeptide is typically substantially identical to a second polypeptide, for example, where the two polypeptides differ only by conservative substitutions. Amino acid substitutions may be of a conserved or non-conserved nature. Conserved amino acid substitutions consist of replacing one or more amino acids of the proteins or peptides of the invention, including, but not limited to SEQ ID NO. 1 to SEQ ID NO. 34, or a viral protein or peptide sequence with amino acids of similar charge, size, and/or hydrophobicity characteristics, such as, for example, a glutamic acid (E) to aspartic acid (D) amino acid substitution. When only conserved substitutions are made, the resulting peptide is functionally equivalent to viral proteins or peptides of the invention from which it is derived. Non-conserved substitutions consist of replacing one or more amino acids of the viral protein or peptide of the invention sequence with amino acids possessing dissimilar charge, size, and/or hydrophobicity characteristics, such as, for example, a glutamic acid (E) to valine (V) substitution.

Amino acid insertions may consist of single amino acid residues or stretches of residues ranging from 2 to 15 amino acids in length. One or more insertions may be introduced into the viral proteins or peptide of the invention.

The viral proteins or peptides of the invention can be modified to increase solubility in an aqueous solution. One or more hydrophobic amino acids can be substituted to hydrophilic amino acids to increase solubility of the peptides. If hydrophobic amino acids are substituted for a hydrophilic amino acid, said peptide should be tested to ensure that the antigencity of the peptide or ability to prevent viral infection is not affected. In addition, one or more hydrophilic amino acids can be added to a terminal of the peptides of the invention to increase solubility. In one embodiment, one or more lysines are added to the carboxy terminal of the peptides to increase solubility. In a preferred embodiment, two or three lysines are added to the carboxy terminal of the peptides to increase solubility. If one or more hydrophilic amino acids are added to a terminal of the peptides, said peptide should be tested to ensure that the antigencity of the peptide or ability to prevent viral infection is not affected. In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also "conservatively modified variations."

Some specific examples of viruses and viral infections, which can be protected against or treated by using the viral proteins, peptides, methods or compositions of the invention are: hepatitis viruses A, B, C, D & E3, herpes viruses 1, 2, 6 & 7, cytomegalovirus, coronavirus, varicella zoster, papilloma virus, Epstein Barr virus, adenoviruses, bunya viruses {e.g. lianta virus), coxsakie viruses, picoma viruses, rotaviruses, respiratory syncytial viruses, rhinoviruses, rubella virus, papovavirus, mumps virus, marlburg, ebola virus, measles virus, polio virus (multiple types), adenovirus (multiple types), parainfluenza virus (multiple types), avian influenza (various types), sendai virus, simian virus, fer-de-lance virus, flaviviruses, including yellow fever, Japanese encephalitis, dengue, and Saint Louis encephalitis, shipping fever virus, Western and Eastern equine encephalomyelitis, Japanese B. encephalomyelitis, Russian Spring Summer encephalomyelitis, yokose virus, tick-borne virus. Additional viruses include hog cholera virus, Newcastle disease virus, fowl pox, rabies, feline and canine distemper and the like viruses, slow brain viruses, rous sarcoma virus (RSV), Arboviruses, Papovaviridae, Parvoviridae, Picomaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, Junin, Chikungunya, Rift Valley fever, meningitis, opportunistic infections, pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, the common cold, Polio, leukemia, Rubella, Severe Acute Respiratory Syndrome (SARS), sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia.

Viral Proteins and Peptides of the Invention

The invention also encompasses a viral protein or peptide identified according to the method described above. Said viral proteins or peptides may have a length of about 10 to about 200, about 20 to about 150, about 45 to about 100 amino acids. In another embodiment, said peptide has a length of about 15 to about 60 amino acids. For example, the peptide may be about 20, 30, 40, 50 or 60 amino acids, preferably 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more amino acids. Preferably, the peptide has less than 100, more preferably no more than 60 amino acids. Examples of peptide of the invention include, but are not limited to, SEQ ID NO. 1 to SEQ ID NO. 34. In another embodiment, the peptide of the invention has an amino acid sequence that is at least 70 percent, preferably at least 80 percent or 85 percent, more preferably at least about 86%, 87%, 88%, 89%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% percent identical to a contiguous amino acid sequence which comprise the motifs W X_nlW and I X₁₁₂1, wherein X is any amino acid and nl and n2 is a number from 0 to 20 and said protein inhibits viral infection, which includes inhibiting viral fusion or viral transmission. In another embodiment, said identified peptide does not include Fuzeon^® as exemplified in SEQ ID NO. 13.

Said identified viral protein of the invention comprises amino acids sequences of 150 amino acids or greater. In one embodiment, the viral protein of the invention has an amino acid sequence that is at least 70 percent, preferably at least 80 percent or 85 percent, more preferably at least about 86%, 87%, 88%, 89%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous amino acid sequence which comprises the motif of W X_nlW and I X_n21, wherein X is any amino acid and nl and n2 is a number from 0 to 20 and said protein inhibits viral infection, which includes inhibiting viral fusion or viral transmission.

In another embodiment of the invention, a viral protein or peptide identified by the method described above is attached to another molecule. In one embodiment the viral protein or peptide of the invention attached to said molecule increases the in vivo half-life of said protein or peptide.

The invention also comprises attaching viral proteins or peptides of the invention with molecules that may serve a stabilizing function (e.g., to increase half-life in solution and/or in vzVo, to make the polypeptides more water soluble, to increase the polypeptides hydrophilic or hydrophobic character, etc.). Polymers useful as stabilizing materials may be of natural, semi-synthetic (modified natural) or synthetic origin. Exemplary natural polymers include naturally occurring polysaccharides, such as, for example, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans (such as, for example, inulin), levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, pulmlan, glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose, polyglucose, polydextrose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum, starch and various other natural homopolymer or heteropolymers, such as those containing one or more of the following aldoses, ketoses, acids or amines: erythose, tlireose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, and naturally occurring derivatives thereof. Accordingly, suitable polymers include, for example, proteins, such as albumin, polyalginates, and polylactide-coglycolide polymers. Exemplary semi-synthetic polymers include carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose. Exemplary synthetic polymers include polyphosphazenes, hydroxyapatites, fluoroapatite polymers, polyethylenes (such as, for example, polyethylene glycol, polyoxyethylene, and polyethylene terephthlate), polypropylenes (such as, for example, polypropylene glycol), polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinyl chloride and polyvinylpyrrolidone), polyamides including nylon, polystyrene, polylactic acids, fmorinated hydrocarbon polymers, fluorinated carbon polymers (such as, for example, polytetrafluoroethylene), acrylate, methacrylate, and polymethylmethacrylate, and derivatives thereof. Methods for the preparation of viral proteins or peptides of the invention which employ polymers as stabilizing compounds will be readily apparent to one skilled in the art, in view of the present disclosure, when coupled with information known in the art, such as that described and referred to in Unger, U.S. Pat. No. 5,205,290, the disclosure of which is hereby incorporated by reference herein in its entirety.

The peptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers. In another embodiment, viral proteins or peptides of the invention are fused to another protein or polypeptide. Such fusions may increase said protein's or peptide's in vivo half-life, make said protein and/or peptide more soluble and/or facilitated purification. As one of skill in the art will appreciate, the viral proteins or peptides of the invention can be fused to other amino acid sequences. For example, viral proteins or peptides of the present invention may be fused with albumin, human serum albumin or with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHl, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides. Well known examples of fusion partners include hexahistidine (6x-HIS)-tag, N-Flag, glutathione-5- transferase (GST) and maltose binding protein (MBP), which are particularly useful for isolation of the fusion polypeptide by affinity chromatography. For the purposes of fusion polypeptide purification by affinity chromatography, relevant matrices for affinity chromatography may include nickel-conjugated or cobalt-conjugated resins, fusion polypeptide specific antibodies, qlutathione-conjugated resins, and amylose-conjugated resins respectively. Viral proteins or peptides of the invention may have its serum half-life increase by fusing said protein or peptide to proteins that can increase its serum half-life (e.g. albumin fusion proteins).

The peptides of the invention may be synthesized or prepared by techniques well known in the art. See, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman and Co., N.Y., which is incorporated herein by reference in its entirety for all purposes. Short peptides, for example, can be synthesized on a solid support or in solution. Longer peptides or protein may be made using recombinant DNA techniques. Here, the nucleotide sequences encoding the viral proteins or peptides of the invention may be synthesized, and/or cloned, and expressed according to techniques well known to those of ordinary skill in the art. See, for example, Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, VoIs. 1-3, Cold Spring Harbor Press, N.Y.

The viral proteins or peptides of the invention may alternatively be synthesized such that one or more of the bonds which link the amino acid residues of the peptides are non- peptide bonds. These alternative non-peptide bonds may be formed by utilizing reactions well known to those in the art, and may include, but are not limited to imino, ester, hydrazide, semicarbazide, and azo bonds, to name but a few. hi yet another embodiment, peptides comprising the sequences described above may be synthesized with additional chemical groups present at their amino and/or carboxy termini, such that, for example, the stability, bioavailability, and/or inhibitory activity of the peptides is enhanced. For example, hydrophobic groups such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups, may be added to the peptides' amino termini. Likewise, an acetyl group or a 9-fiuorenyhnethoxy- carbonyl group may be placed at the peptides' amino termini. Additionally, the hydrophobic group, t-butyloxycarbonyl, or an amido group may be added to the peptides' carboxy termini. Further, the peptides of the invention may be synthesized such that their steric configuration is altered. For example, the D-isomer of one or more of the amino acid residues of the peptide may be used, rather than the usual L-isomer. Still further, at least one of the amino acid residues of the peptides of the invention may be substituted by one of the well known non-naturally occurring amino acid residues. Alterations such as these may serve to increase the stability, bioavailability and/or inhibitory action of the peptides of the invention.

Any of the peptides described above may have a non-peptide macromolecular carrier group covalently attached to their amino and/or carboxy termini. Such macromolecular carrier groups may include, for example, lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.

The invention also comprises a polypeptide or peptide comprising the motifs W X_nl W and I X₁₁₂ I, wherein X is any amino acid and nl and n2 is a number from 0 to 20, and wherein the polypeptide or peptide is less than about 100 amino acids in length. In one embodiment, said polypeptide or peptide of is less than about 50 amino acids in length. In another embodiment, said polypeptide or peptide of is less than about 20 amino acids in length.

The protein or peptide of the invention can also be used to find additional agents that can inhibit viral infections. By utilizing the viral protein or peptide of the invention, a person with skill in the art can screen a library {e.g. a chemical library (including a small molecule library), peptide library, cDNA or other library) to find an agent that, e.g., competes with, binds to, or enhances the activity of the protein or peptide of the invention.

A number of different screening protocols can be utilized to identify agents that compete with, bind to, or enhance the activity of viral proteins or peptides of the invention. In general terms, the screening methods involve screening a plurality of agents to identify an agent that competes with, binds to, or enhances the activity of a protein or peptide of the invention (test agent) by, e.g., competition binding assays. The test agent can be any type of molecule, including, for example, a peptide, a peptidomimetic, a polynucleotide, or a small organic molecule, that one wishes to examine for the ability to compete with, bind to, or enhance the activity of the protein or peptide of the invention. It will be recognized that said screening can be accomplish by any method known in the art that is convenient for screening a plurality of test agents either serially or in parallel. A test agent can be one of a plurality of test agents, for example, a library of test agents produced by a combinatorial method. Methods for preparing a combinatorial library of molecules that can be tested for are well known in the art and include, for example, methods of making a phage display library of peptides, which can be constrained peptides, a peptide library, a peptidomimetic library, an oligosaccharide library, a lipoprotein library, a glycoprotein or glycolipid library, or a chemical library.

Pharmaceutical Compositions

The viral proteins or peptides of the invention are useful for preparing pharmaceutical compositions that treat, prevent and/or ameliorate a viral infection in an animal when administered to said animal. Said compositions can prevent and/or ameliorate a viral infection in an animal when administered to said animal because said viral protein or peptide inhibits a virus from infecting a cell, prevents a virus from propagating and/or prevents viral transmission. Said viral proteins or peptides of the invention can also treat, prevent and/or ameliorate a viral infection in an animal by stimulating an immune response that confers immunity to viruses. In one embodiment, said animal is a human. In another embodiment, said viral proteins or peptides of the invention is artificially synthesized. Examples of peptides of the invention, include but are not limited to, SEQ ID NO. 1 to SEQ ID NO. 34. In another embodiment, said identified peptide does not include Fuzeon^® as exemplified in SEQ ID NO. 13. The pharmaceutical compositions useful herein contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce a harmful reaction to the animal receiving the composition, and which may be administered without undue toxicity. These compositions can be useful as a composition for delivering viral proteins or peptides of the invention. In preferred embodiments, the pharmaceutical compositions comprise viral proteins or peptides of the invention and a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers include but are not limited to saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in Remington's Pharmaceutical Sciences (Mack Pub. Co. NJ. current edition). The pharmaceutical composition should suit the mode of administration. In a preferred embodiment, the pharmaceutical composition is suitable for administration to humans, preferably is sterile, non-particulate and/or non-pyrogenic. The invention also comprises a method of formulating a pharmaceutical composition comprising a viral protein or peptide of the invention capable of delivering an effective dose that inhibits viral infection, which includes inhibiting viral fusion, viral penetration into a cell and/or viral transmission to an animal. Selection for an effective dose in a human can be determined (e.g. via clinical trials) by a skilled artisan based upon the consideration of several factors which will be known to one of skill in the art. Some factors include age, physical condition, body weight, sex, diet, time of administration, and other clinical factors. The precise dose to be employed will also depend on the route of administration. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal test systems. In one embodiment, inhibition of viral infection is delivered in a single dose. A single dose may be best for prophylactic treatment after accidental exposure to a virus, hi another embodiment, inhibition of viral infection is delivered in multiple doses or a dose extended over a period of time (e.g. slow release). This type of treatment may be best for a chronic viral infection of an animal (e.g. hepatitis C infection). The dosage of the pharmaceutical formulation can be determined readily by the skilled artisan, for example, by first identifying doses effective to inhibit viral fusion, viral penetration and/or viral transmission of a virus. Methods to measure viral inhibition are known in the art. For example, after a viral challenge and administration of the protein or peptide of the invention, viral load can be measured by collecting serum or sputum (depending on the virus) and processing to isolate and count viruses.

The compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The compositions can be a solid form, such as a lyophilized powder suitable for reconstitution, a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

The invention also provides for a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the viral proteins or peptides of the invention and/or formulations of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The invention also provides that the viral proteins or peptides of the invention may be packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of composition. In one embodiment, the viral proteins or peptides of the invention are supplied as a liquid, in another embodiment, as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container that can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject. Preferably, the viral proteins or peptides of the invention are supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of an amount which can deliver an effective dose to an animal. The viral proteins or peptides of the invention composition should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted from the lyophilized powder.

Methods of administering a pharmaceutical composition comprising viral proteins or peptides of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral or pulmonary routes or by suppositories). In a specific embodiment, compositions of the present invention are administered intramuscularly, intravenously, subcutaneously, transdermally or intradermally. The compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucous, colon, conjunctiva, nasopharynx, oropharynx, vagina, urethra, urinary bladder and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

Formulations comprising viral protein and/or peptides of the invention may also be administered on a dosage schedule, for example, an initial administration of the composition comprising viral protein or peptides of the invention with subsequent administrations. In particular embodiments, additional doses of the pharmaceutical composition are administered anywhere from two weeks to one year, preferably from one to six months, after the initial administration. In a one embodiment, additional doses are administered approximately one month apart. In another embodiment, additional doses are administered approximately two, three and/or four months apart. The invention also comprises a method of formulating a vaccine capable of delivering an effective dose that induces substantial immunity to a viral infection or at least one symptom thereof to an animal, comprising adding to said formulation the viral protein or peptide of the invention. Selection for an effective dose in a human can be determined (e.g. via clinical trials) by a skilled artisan based upon the consideration of several factors which will be known to one of skill in the art. Some factors include age, physical condition, body weight, sex, diet, time of administration, and other clinical factors. The precise dose to be employed will also depend on the route of administration. Effective doses may be extrapolated from does-response curves derived from in vitro or animal test systems. In one embodiment, said substantial immunity to viral infection or at least one symptom thereof is delivered in one dose. In another embodiment, said substantial immunity to viral infection or at least one symptom thereof is delivered in multiple doses. In another embodiment, said viral protein or peptide is formulated with an adjuvant or immune stimulator. Substantial immunity to viral infections can be induced by administering vaccines or antigenic formulations comprising said viral protein or peptides that inhibit viral infection via the development in a subject of a substantially protective antibody response and/or a substantially protective cellular response to an epitope present on the protein or peptide of the invention. For purposes of the present invention, an antibody response refers to an immune response mediated by antibody molecules, while a cellular response is one mediated by T- lymphocytes and/or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells ("CTL"s). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A cellular response also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.

The ability of a viral proteins or peptides of the invention to stimulate a substantially protective cellular response may be determined by a number of assays, including challenge assays, lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for T-lymphocytes specific for the antigen in a sensitized subject. Such assays are well known in the art. See, e.g., Erickson et at, 1993; Doe et at, 1994. Recent methods of measuring cell-mediated immune response include measurement of intracellular cytokines or cytokine secretion by T-cell populations, or by measurement of epitope specific T-cells {e.g., by the tetramer technique)(reviewed by McMichael, A. J., and O'Callaglian, C. A 1998; Mcheyzer- Williams et al, 1996; Lalvani et al, 1997).

A substantially protective antibody response as used herein is an immune response that stimulates the production of antibodies that protects an animal or at least reduces a symptom of a viral infection in an animal {e.g. , neutralizing antibodies that block influenza viruses from entering cells and/or replicating by binding to the virus, typically protecting cells from infection and destruction). Viral proteins or peptides of the invention may also elicit production of CTLs. Hence, said immunological response may include one or more of the following effects: the production of antibodies by B-cells; and/or the activation of suppressor T-cells directed specifically to vaccines or antigenic formulations comprising said viral proteins of the invention. These responses may serve to neutralize infectivity, and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays, well known in the art. See, e.g., Montefiori et al., 1988; Dreyer et al. , 1999.

In another embodiment, the pharmaceutical formulation can comprise an adjuvant (or immune stimulator if an immune response to viral protein or peptides of the invention is desired. It is well known in the art that the immunogenicity of a particular composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants (immune stimulators). Adjuvants have been used experimentally to promote a generalized increase in immunity against unknown antigens (e.g., U.S. Pat. No. 4,877,611). Immunization protocols have used adjuvants to stimulate responses for many years, and as such, adjuvants are well known to one of ordinary skill in the art. Some adjuvants affect the way in which antigens are presented. For example, the immune response is increased when protein antigens are precipitated by alum. Emulsification of antigens also prolongs the duration of antigen presentation. Any adjuvant described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2^nd Edition)," herein incorporated by reference in its entirety for all purposes, is envisioned within the scope of this invention. Exemplary, adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant. Other adjuvants that may also be used include IL-I, IL-2, IL- 4, IL-7, IL-12, interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion also is contemplated. MF-59, Novasomes^®, MHC antigens may even be used. Amphipathic and surface active agents, e.g., saponin and derivatives such as QS21 (Cambridge Biotech), form yet another group of adjuvants for use with the immunogens of the present invention. Nonionic block copolymer surfactants (Rabinovich et al, 1994) may also be employed. Oligonucleotides are another useful group of adjuvants (Yamamoto et al, 1988). Quil A and lentinen are other adjuvants that may be used in certain embodiments of the present invention. Another group of adjuvants are the detoxified endotoxins, such as the refined detoxified endotoxin of U.S. Pat. No. 4,866,034. These refined detoxified endotoxins are effective in producing adjuvant responses in animals. Of course, the detoxified endotoxins may be combined with other adjuvants to prepare multi-adjuvant-incorporated cells. For example, combination of detoxified endotoxins with trehalose dimycolate is particularly contemplated, as described in U.S. Pat. No. 4,435,386. Combinations of detoxified endotoxins with trehalose dimycolate and endo toxic glycolipids is also contemplated (U.S. Pat. No. 4,505,899), as is combination of detoxified endotoxins with cell wall skeleton (CWS) or CWS and trehalose dimycolate, as described in U.S. Pat. Nos. 4,436,727, 4,436,728 and 4,505,900. Combinations of just CWS and trehalose dimycolate, without detoxified endotoxins, is also envisioned to be useful, as described in U.S. Pat. No. 4,520,019.

Those of skill in the art will know the different kinds of adjuvants that can be conjugated to viral proteins and/or peptides of the invention and these include alkyl lysophosphilipids (ALP); BCG; and biotin (including biotinylated derivatives) among others. Certain adjuvants particularly contemplated for use are the teichoic acids from Gram-cells. These include the lipoteichoic acids (LTA), ribitol teichoic acids (RTA) and glycerol teichoic acid (GTA). Active forms of their synthetic counterparts may also be employed in connection with the invention (Takada et al, 1995).

Various adjuvants, even those that are not commonly used in humans, may still be employed in other animals, where, for example, one desires to raise antibodies or to subsequently obtain activated T cells. The toxicity or other adverse effects that may result from either the adjuvant or the cells, e.g., as may occur using non-irradiated tumor cells, is irrelevant in such circumstances. The dosage of the pharmaceutical formulation can be determined readily by the skilled artisan, eliciting a prophylactic or therapeutic immune response, e.g., by measuring the serum titer of vaccine specific immunoglobulins or by measuring the inhibitory ratio of serum samples, or urine samples, or mucosal secretions.

Methods of Administering and Treating Viral Infections

The invention also encompasses a method for treating, preventing and/or ameliorating an infection caused by a virus, comprising administering to an animal the viral protein or peptide of the invention. In one embodiment, said animal is a human. In one embodiment, said viral protein or peptide is artificially synthesized. In another embodiment, said viral protein or peptide is attached to another molecule. In another embodiment, said molecule increases the in vivo half-life of said viral protein or peptide. In another embodiment, said viral protein or peptide is fused to another protein or polypeptide.

The invention also comprises a method of formulating a composition capable of delivering an effective dose that treats, prevents and/or ameliorated a viral infection or at least one symptom thereof to an animal, comprising adding to said composition the viral protein or peptide of the invention. Selection for an effective dose in a human can be determined (e.g. via clinical trials) by a skilled artisan based upon the consideration of several factors which will be known to one of skill in the art. Some factors include age, physical condition, body weight, sex, diet, time of administration, and other clinical factors. The precise dose to be employed will also depend on the route of administration. Effective doses may be extrapolated from does-response curves derived from in vitro or animal test systems. In one embodiment, said substantial immunity to viral infection or at least one symptom thereof is delivered in one dose. In another embodiment, said substantial immunity to viral infection or at least one symptom thereof is delivered in multiple doses. In another embodiment, said viral protein or peptide is formulated with an adjuvant or immune stimulator.

The invention also comprises a method of formulating a vaccine capable of delivering an effective dose that induces substantial immunity to a viral infection or at least one symptom thereof to an animal, comprising adding to said formulation the viral protein or peptide of the invention. Selection for an effective dose in a human can be determined (e.g. via clinical trials) by a skilled artisan based upon the consideration of several factors which will be known to one of skill in the art. Some factors include age, physical condition, body weight, sex, diet, time of administration, and other clinical factors. The precise dose to be employed will also depend on the route of administration. Effective doses may be extrapolated from does-response curves derived from in vitro or animal test systems. In one embodiment, said substantial immunity to viral infection or at least one symptom thereof is delivered in one dose. In another embodiment, said substantial immunity to viral infection or at least one symptom thereof is delivered in multiple doses. In another embodiment, said viral protein or peptide is formulated with an adjuvant or immune stimulator.

Once the viral protein or peptide of the invention is formulated, it can be administered to the animal. Methods of administering viral proteins or peptides of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In a specific embodiment, the viral protein and/or peptide of the invention are administered intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985, 20; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, each of which is incorporated herein by reference in its entirety.

In one embodiment, the dosage of the viral protein or peptide of the invention is administered to a patient are 0.01 mg to 1000 mg, when used as single agent therapy. In another embodiment, the viral proteins or peptides of the invention are used in combination with other therapeutic compositions and the dosage administered to a patient are lower than when said therapeutics agents are used as a single agent therapy.

In another embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.

In another embodiment, the compositions can be delivered in a vesicle, in particular a liposome (See Langer, 1990); Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez- Berestein, ibid., pp. 3 17-327; see generally ibid.).

In yet another embodiment, the compositions can be delivered in a controlled release or sustained release system. Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or viral protein or peptide of the invention. See, e.g., U.S. Pat. No. 4,526,938; PCT publication WO 91/05548; PCT publication WO 96/20698; Ning et al, 1996, Song et al, 1995, Cleek et al, 1997, and Lam et al, 1997. In one embodiment, a pump may be used in a controlled release system (See Langer, supra; Sefton, 1987, Buchwald et al, 1980, and Saudek et al, 1989,). In another embodiment, polymeric materials can be used to achieve controlled release of viral proteins or peptides of the invention (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, Levy et al, 1985, Howard et al, 1989, U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, ρoly(2- hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene- co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly( vinyl alcohol), polyacrylamide, poly( ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). In another embodiment, polymeric compositions useful as controlled release implants are used according to Dunn et al (See U.S. Pat. No. 5,945,155). Controlled release systems are discussed in the review by Langer (1990). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more therapeutic agents of the invention. See, e.g., U.S. Pat. No. 4,526,938; International Publication Nos. WO 91/05548 and WO 96/20698;

Ning et al., 1996, Song et al., 1995, Cleek et al, 1997, and Lam et al, 1997, each of which is incorporated herein by reference in its entirety for all purposes.

Dosage treatment may be a single dose schedule or a multiple dose schedule. A multiple dose schedule is one in which a primary course of administration and/or vaccinations may be with 1-10 separate doses, followed by other doses given at subsequent time intervals, chosen to maintain viral protein or peptides of the invention present in the serum and/or reinforce the immune response, for example at 1 month to 4 months for a second dose, and if needed, a subsequent dose(s) after several months. The boost may be with the compositions of the invention given for the primary immune response, or may be for preventing infection of a virus. The dosage regimen will also, at least in part, be determined by the need of the subject and be dependent on the judgment of the practitioner. Furthermore, if prevention of disease is desired, the antigenic composition and/or vaccines are generally administered prior to primary infection with the pathogen of interest. If treatment is desired, e.g., the reduction of symptoms or recurrences, viral proteins or peptides of the invention are generally administered subsequent to primary infection. Viral proteins or peptides of the invention can also be administered to prevent viral infection, comprising inhibiting viral fusion, inhibiting viral penetration and/or inhibiting viral viral transmission, of a virus to an animal at risk for said virus.

The dosages and methods for treating, preventing and/or ameliorating an infection caused by a virus, can be determined by standard research techniques. For example, the dosage of the composition, which will be effective in eliciting a protective immune response or prevention of viral infection, can be determined by administering the agents to an animal model or in vitro model such as, e.g. the animal models known those skilled in the art or cell culture models. In addition, in vitro assays may optionally be employed to help identify optimal dose ranges. Selection for the preferred effective dose in a human can be determined (e.g. via clinical trials) by a skilled artisan based upon the consideration of several factors which will be known to one of skill in the art. Some factors include age, physical condition, body weight, sex, diet, time of administration, and other clinical factors. The precise dose to be employed will also depend on the route of administration. Effective doses may be extrapolated from does-response curves derived from in vitro or animal test systems.

Antibodies and Antibody Generation The antibodies of the invention or fragments thereof can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. The antibodies of the invention can be used for diagnosis, research and/or prevention, amelioration or treatment of a viral infection in an animal. In addition, the antibody of the invention can also bind to, inhibits, compete with or enhance the activity or the viral protein or peptides of the invention

As used herein, an "antibody" is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 IcD) and one "heavy" chain (about 50-7O kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab') ₂, a dimer of Fab which itself is a light chain joined to VH-CHl by a disulfide bond. The F(ab') ₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (FaV)₂ dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region {see, Fundamental Immunology, W. E. Paul, ed., Raven Press, TSf .Y. (1999), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, includes antibodies or fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Antibodies include, e.g., polyclonal antibodies, monoclonal antibodies, multiple or single chain antibodies, including single chain Fv (sFv or scFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide, and humanized or chimeric antibodies.

Polyclonal antibodies to viral proteins or peptide of the invention can be produced by various procedures well known in the art. For example, viral proteins or peptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, ferrets etc. to induce the production of sera containing polyclonal antibodies specific for a virus.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al, in: Monoclonal Antibodies and T-CeIl Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties for all purposes).

The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. Briefly, mice can be immunized with a viral proteins or peptide of the invention and once an immune response is detected, e.g., antibodies specific for a specific virus or an antibody that binds to, inhibits, competes with or enhances the activity or the viral protein or peptides of the invention are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries {e.g. , human or murine cDNA libraries of lymphoid tissues). The DNA encoding the VH and VL domains are recombined together with a scFv linker by PCR and cloned into a phagemid vector {e.g., pCANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and Ml 3 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to viral proteins or peptide of the invention or an antigen binding domain that competes with, binds to, or enhances the activity of viral proteins or peptides of the invention can be selected or identified with a viral proteins or peptide of the invention, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead or specific assays. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al, 1995; Ames et al, 1995; Kettleborough et al; Persic et al, 1997; Burton et al, 1994; PCT application No. PCT/GB91/O1134; PCT publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and W097/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety for all purposes. As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. Techniques to recombinantly produce Fab, Fab' and F(ab')₂ fragments can also be employed using methods known in the art such as those disclosed in PCT publication No. WO

92/22324; Mullinax et al, 1992; Sawai et al, 1995; and Better et al, 1988 (said references incorporated by reference in their entireties for all purposes).

To generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, e.g., the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions. Preferably, the vectors for expressing the VH or VL domains comprise an EF- lα promoter, a secretion signal, a cloning site for the variable domain, constant domains, and a selection marker such as neomycin. The VH and VL domains may also cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.

For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use human or chimeric antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, W098/16654, WO 96/34096, WO 96/33735, and WO 91/10741 ; each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the J_H region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., viral proteins or peptide of the invention. Monoclonal antibodies directed against the viral proteins or peptide of the invention or antibodies that compete with, binds to, or enhances the activity of viral proteins or peptides of the invention can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, 1995. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporated by reference herein in their entirety for all purposes.

A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a human antibody and a non-human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, 1985; Oi et al, 1986; Gillies et al, 1989; and U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference in their entirety for all purposes. Chimeric antibodies comprising one or more CDRs from human species and framework regions from a non- human immunoglobulin molecule can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991; Studnicka et al, 1994; and Roguska et al, 1994), and chain shuffling (U.S. Pat. No. 5,565,332). Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al, U.S. Pat. No. 5,585,089; and Riechmann et al, 1988, Nature 332:323, which are incorporated herein by reference in their entireties.)

The present invention provides antibodies or fragments thereof which immunospecifically bind to one or more one or more viral proteins or peptides of the invention and have an apparent dissociation constant of less than 100 ng/mL as determined by a sandwich ELISA. The present invention provides antibodies or fragments thereof which immuospecifically bind to one or more viral proteins or peptides of the invention and have an apparent dissociation constant of about InM to aboutlO nM as measured by surface plasmon resonance (SPR) using a BIAcore sensor. The present invention provides antibodies or fragments thereof which immuospecifically bind to one or more viral proteins or peptides of the invention and have an on rate of about IxIO⁴, about 5x10⁴, about IxIO⁵, about 5x10⁵, about 1x10⁶, or about 5x10⁶ and an off rate of about IxIO^"3, about 5xlO^"4, about IxIO^"4, about 5xlO^~5, about IxIO^"5, about 5xlO^"6, as measured by surface plasmon resonance (SPR) using a BIAcore sensor.

Antibodies of the invention encompass antibodies conjugated to a label capable of producing a detectable signal. These conjugated antibodies are useful, for example, in detection systems such as a diagnostic, quantization or imaging of virus. Such labels are known in the art and include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds, and other antibodies. The labels may be covalently or conjugated to said antibodies through a secondary reagent, such as a second antibody, protein A, or a biotin-avidin complex. Methods of labeling antibodies are known in the art and need not be described in detail herein.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are incorporated herein by reference for all purposes.

EXAMPLES

Using the methods described above, several peptides were identified that may inhibit viral infection.

Example 1: Hepatitis C Viral Infections

Hepatitis C is an infectious disease of the liver caused by the hepatitis C virus (HCV). Currently, an estimated 170 million people worldwide are chronically infected with HCV. In the United States, HCV infection is more common than hepatitis B virus (HBV) infection. There are 175,000 new cases of hepatitis C in the U.S. per year. About 50-85% of these new cases develop persistent HCV infection in which the virus continues to replicate and remains in their blood lifelong, in contrast to 10% for adults infected with HBV. Some studies suggest that liver cirrhosis develops in about 20% of patients with chronic hepatitis C within 20 years and once cirrhosis is established, one-fourth of them will ultimately suffer from liver cancer or liver failure. HCV is a small (40-60 nm in diameter), enveloped, single-stranded RNA virus of the family flaviviridae with a genetic sequence of 9,700 nucleotides. The genome encodes one large polyprotein of approximately 3000 amino acids that is then cleaved into several functional proteins. These functional proteins include structural proteins and enzymes, such as protease, helicase, and RNA-dependent RNA polymerase. HCV replicates at a very high rate and has a half-life of 2.7 hours. The polymerase has no proofreading mechanisms, and therefore, mutations are common. Frequent new mutations help the virus escape the immune system and chronic infection occurs in most cases. The virus is spread primarily by contact with infected blood and blood products. It is also transmitted by sexual contact and from mother to infant. With the implementation of anti-HCV screening in blood donors, the risk of post-transfusion hepatitis C has been dramatically reduced, but the infection continues to occur via other modes of transmission. Several recent studies have established a strong association between HCV and hepatocellular carcinoma (HCC). Anti-HCV antibodies, in the absence of HBV markers, were detected in 44.5% of HCC patients in Spain, 43% in Japan, 16% in Italy, 7% in South Africa. Since HBV carriers often have chronic HCV infection, it is conceivable that the two viruses may act together to cause HCC. However, the epidemiological data linking HBV to HCC may require reassessment in terms of the magnitude of the risk involved with the availability of testing for chronic HCV infection. HCV may prove to be as important as HBV in the causation of hepatocellular carcinoma worldwide. It has recently been suggested that the risk of developing HCC is approximately 5% year in cirrhotic patients with chronic hepatitis C, greater than the risk from hepatitis B. The standard therapy for chronic hepatitis C is combination therapy with alpha interferon (IFN) and ribavirin. However, this intervention has only been demonstrated to have an effect on virological, biochemical, and histological responses for 20-30% of patients. Furthermore, IFN and ribavirin are connected with a plethora of adverse events such as anorexia, nausea, dry skin, and leukopenia and are costly. Therefore, new medications and approaches to treatment are needed. Using the methods described above, SEQ ID NO. 1 and SEQ ID NO. 2 where identified from Hepatitis C virus. In vitro antiviral cellular assays may be used to demonstrate potent anti-HCV activities of the polypeptides (SEQ ID: 1 and SEQ ID: 2) and their analogs or derivatives. These compounds are useful as anti-HCV treatment agents for administration to patients in different disease stages or vaccine to prevent human from HCV infections.

SEQ ID: 1 AAARVTQILSSLTITQLLKRLHQWLNEDCSTPCSGSWLRDVWDWICTVLT SEQ ID: 2

ASQRVTQLLGSLTITSLLRRLHNWITEDCPIPCSGSWLRD VWDWVCTILT

Example 2: Hepatitis B Viral Infections

Hepatitis B is caused by the hepatitis B virus (HBV), which can lead to lifelong infection, cirrhosis (scarring) for the liver, liver cancer, liver failure and death. It is estimated that there are 280 million HBV carriers worldwide representing more that 5% of the global population, HBV infection is very common in Asia and the Middle East. In Europe and North America, the incidence of known carriers is about 1 person in about 1000 people.

Hepatitis B is transmitted by the exchange of body fluids e.g. blood, semen and in some circumstances saliva. People most at risk include: anybody who has unprotected sexual intercourse; IV drug users who share needles and syringes; health care workers in contact with potentially contaminated blood or body fluids; babies born to mothers with the hepatitis B virus. Many cases of acute hepatitis B occur sporadically with no known source. It is possible that a person infected with the hepatitis B virus does not develop illness at all. Common symptoms include: yellow skin or yellowing of the whites of the eyes (jaundice), tiredness, loss of appetite, nausea, abdominal discomfort, dark urine, clay-colored bowel movements and joint pain. Approximately, 90% of the people infected with hepatitis B recover completely and become immune to the virus. However, about 10% of the people infected with hepatitis B develop chronic infection, may have ongoing symptoms and they continue to be infectious for a variable length of time. Chronic infection is defined as having hepatitis B present for six months or more. People with a chronic hepatitis infection are at risk of liver damage and 20% to 30% of those will progress to cirrhosis.

A safe and effective genetically engineered vaccine for hepatitis B is available. It is given in three subcutaneous injections generally over a period of six months and conveyed immunity in 90 to 95% of the people injected. At the end of the course of injections, a blood test is taken to ensure that those vaccinated have developed the required antibodies. For the 5% to 10% of the people who did not respond to the vaccination, recent studies have shown that a repeat in course injections given intramuscularly can create an immune response in about 62% to 98% (depending on several factors) of those who did not respond or whose response did not last when the vaccine was administered subcutaneously. Hepatitis B virus is a DNA virus of the hepadnaviridae family of viruses. It replicates within infected liver cells (hepatocytes). HBV is a spherical particle with a diameter of 42 nm and consist of an inner core plus an outer surface coat. The surface coat or envelope composes of several proteins known collectively as HBs or surface proteins. The outer surface coat surrounds an inner protein shell, composed of HBc protein. This inner shell is referred to as the core particle or capsid. Finally, the core particles surround the viral DNA and the enzyme DNA polymerase.

The life cycle of HBV begins when the virus attaches to liver cell membranes via its envelope proteins. Then the viral membrane fuses with the cell membrane and the viral particle is released in the liver cell. The core particle then releases its contents of DNA and DNA polymerase into the liver cell nucleus. Within the cell nucleus, HBV DNA polymerase converts the partially double stranded DNA genome into covalently closed circle DNA (cccDNA), which serves as the template for synthesis of viral DNA an messenger RNA. Then, the liver cells produces surface (HBs) proteins, the core (HBc) protein, DNA polymerase, the HBe protein, HBx protein and possibly other as yet undetected proteins, via messenger RNA. HBV core particles are assembled in the cytosol and then transported to the Golgi for further modifications of glucans in the surface proteins before secreted out of the host cell to finish the life cycle.

Using the methods described above, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, where identified from Hepatitis B virus. In vitro antiviral cellular assays may be used to demonstrate potent anti-HBV activities of these peptides and their analogs or derivatives. These compounds are useful as anti-HBV treatment agents for administration to patients in different disease stages or vaccine to prevent human from HBV infections.

SEQ ID NO. 3

ASVRFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYS

SEQ ID NO. 4

NSTFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASARF

SEQ ID NO. 5 NSTFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASARFSWLSLLVPFV Example 2: Influenza Viral Infections

Influenza, also known as flu, is a contagious disease that is caused by the influenza virus. It attacks the respiratory tract in humans (nose, throat and lungs). Influenza usually comes on suddenly and may include the following symptoms: fever, headache, tiredness (can be extreme), dry cough, sore throat, nasal congestion, body aches. These symptoms are usually referred to as "flu like symptoms."

Influenza virus infections rank as one of the most common infectious diseases in humankind. Approximately 21 million people worldwide in the 1918-1919 influenza pandemic, with 549,000 deaths in the United States. The Centers for Disease Control (CDC) estimates that about 20,000 deaths occur annually as a result of influenza virus infection. Influenza results from infection with one of three basic types of virus: A, B, or C. Influenza viruses are classified with the family Orthomyxoviridae, thus, influenza viruses have a segmented single stranded RNA genome. Influenza viruses are classified by their antigenic characteristics. These subtypes differ because of changes of the hemagglutinin

(HA) and neuraminidase (NA) on their surface. Many different combinations of HA and NA proteins are possible.

Influenza A and B are the most common influenza infections in humans. Influenza A is a zoonotic infection that also infects pigs, birds, horses, and seals. Indeed, the 1918 pandemic that resulted in millions of deaths worldwide is believed to have originated from pigs. The virion is generally rounded, but may be long and filamentous. A single stranded RNA genome is closely associated with a helical nucleoprotein (NP), and is present in eight separate segments of ribonucleoprotein (RNP), each of which has to be present for successful replication. The segmented genome is enclosed within an outer lipoprotein envelope. An antigenic protein called the matrix protein (MP-I) lines the inside of the envelope and is chemically bound to the RNP.

The envelope carries two types of protruding spikes. One is the box-shaped protein, called neuraminidase (NA), of which there are nine major antigenic types, and which has enzymatic properties as the name implies. The other type of envelope spike is a trimeric protein called hemagglutinin (HA) of which there are 13 major antigenic types. The HA functions during the attachment of the virus particle to the cell membrane and can combine with specific receptors on a variety of cells, including red blood cells. From a clinical viewpoint, the most significant surface protein are HA and NA. The viruses are typed based on these proteins. For example, influenza A (H3N2) expresses HA 3 and NA2. The most common prevailing human influenza A subtypes are HlNl and H3N2. Each year, the distributed vaccine contains A strain from HlNl and H3N2, along with an influenza B strain (trivalent formulation). Recently, avian influenza virus jumped from a bird to a human in Hong Kong during an outbreak of bird flu in poultry. This virus was identified as influenza virus H5N1. The virus caused severe respiratory illness in 18 people, six of whom died. Since that time, many more cases of known H5N1 infections have occurred among humans worldwide; approximately half of those people have died. This influenza virus is a type A strain. Avian influenza viruses do not normally infect other species other than birds or pigs.

Of the several avian influenza subtypes, H5N1 is of particular concern for several reasons. H5N1 mutates rapidly and has documented propensity to acquire genes from viruses infecting other animal species. Its ability to cause severe disease in humans has now been documented on several occasions. In addition, laboratory studies have demonstrated that isolates from this virus have high pathogenicity and can cause severe disease in humans. Birds that survive infection excrete virus for at least 10 days, orally and in feces, thus facilitating further spread at live poultry markets and by migratory birds.

H5N1 variants demonstrated a capacity to directly infect humans in 1997 and have done so again in Viet Nam in January 2004 and in China in 2005. The spread of infection in birds increases the opportunities for direct infection of humans. If more humans become infected over time, the likelihood also increases that humans, if concurrently infected with human and avian influenza strains, could serve as a mixing vessel for the emergence of a novel subtype with sufficient human genes to be easily transmitted from person to person. Such an event would mark the start of a flu pandemic. Symptoms of bird flu in humans ranged from typical flu-like symptoms to eye infections, pneumonia, severe respiratory diseases and other severe and life-threatening complications. The symptoms of bird flu may depend on which virus caused the infection.

Antiviral drugs, some of which can be used for both treatment and prevention, are clinically effective for influenza A virus strains in otherwise healthy adults, but have some limitations. Some of these drugs are also expensive and supplies are limited.

Influenza binds to the host receptor. Receptor bound viruses are then taken into the cell by endocytosis. In the low pH environment of the endosome, RNP is released from MP- 1 and the viral lipoprotein envelope fuses with the lipid-bilayer of the vesicle, releasing the viral RNP into the cell cytoplasm, from where it is transported to the nucleus. New viral proteins are translated from now transcribed messenger RNA. New viral RNA is encased in the capsid protein and together with new matrix protein is then transported to sites at the cell surface where envelope HA and NA components have been incorporated into the cell membrane. Progeny virions are formed and release by budding.

Using the methods described above, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, where identified from influenza virus. Also, SEQ ID NO. 9 was identified from an avian influenza virus. In vitro antiviral cellular assays may be used to demonstrate potent anti- influenza activities of these peptides and their analogs or derivatives. These compounds are useful as anti-influenza treatment agents for administration to patients in different disease stages or vaccine to prevent human from influenza infections.

SEQ ID NO. 6

YVELIRGRPKEDKVWWTSNSIVSMCSSTEFLGQWDWPDGAKIEYF

SEQ ID NO. 7 YVELIRGRPKESSVLWTSNSrVALCGSKERLGSWSWHDGAEITFY

SEQ ID NO. 8 YVELIRGRPKETRVWWTSNSrVVFCGTSGTYGTGSWPDGANINFM

SEQ ID NO. 9 WVELIRGRPKESTIWTSGSSISFCGVNSDTVGWSWP

Example 3: Parainfluenza Viral Infections

Parainfluenza viruses (PIVs) belong to the family of paramyxoviruses and rank second only to respiratory syncycial virus (RSV) as a common cause of lower respiratory tract disease in infants and children. These infections are usually manifested by an upper respiratory tract disease (such as a cold or sore throat). PIVs can also cause serious lower respiratory tract disease with repeat infection (including pneumonia, bronchitis and bronchiolitis), especially among the elderly and among patients with compromised immune systems. There are four serotypes of PIV (1 to 4). Each of the four PIVs has different clinical and epidemiologic features. The most distinctive clinical feature of PIV-I and PIV-2 is croup (laryngo tracheobronchitis); PIV-I is the leading cause of croup in children, whereas PIV-2 is less frequently detected. Both PIV-I and PIV-2 can cause other upper and lower respiratory tract illness. PIV-3 is more often associated with bonchiolitis and pneumonia. PIV-4 is infrequently detected, possibly because it is less likely to cause severe disease.

PIV are ubiquitous and infect most people during childhood. The highest rates of serous PIV illness occur among young children. Serologic surveys have shown that 90% to 100% of children aged 5 years and older have antibodies to PIV-3, and about 75% have antibodies to PIV-I and PIV-2. The different PIV serotypes differ in their clinical features and seasonality. PIV-I causes biennial outbreaks or croup in the fall. PIV-2 cause annual or biennial fall outbreaks. PIV-3 peak activity occurs during the spring and early summer months each year, but the virus can isolated throughout the year.

PrVs are negative sense, single stranded RNA viruses that possess fusion and hemagglutinin and neuraminidase glycoprotein "spikes" on their surface. The virion varies in size (average diameter between 150 nm and 300 nm), with a helical nucleoside 12 nm to 17 nm in diameter. A lipid bilayer covered with glycoprotein spikes surround the nucleocapsid. Each virus particle contains a single stranded, non-segmented, negative-sense RNA genome with nucleoprotein and P and L proteins. The diagnosis of PIV can be confirmed by to methods: (1) isolation and identification if the virus in cell culture or direct detection of the virus in the respiratory secretions using immunofluorescence, enzyme immunoassay, or polymerases chain reaction (PCR) assay, and (2) by demonstration of a significant rise in IgG antibodies between approximately collected paired serum specimens of specific IgM antibodies in a single serum specimen. No vaccine is currently available to protect against infection caused by any of the

PrVs, however, researchers are developing vaccines against PIV-I and PIV-3 infections. Passively acquired maternal antibodies may play a role in protection from PIV types 1 and 2 in the first few months of life, highlighting the probability of passive immunizations.

Using the methods described above, SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, where identified from PIV. In vitro antiviral cellular assays may be used to demonstrate potent anti-PIV activities of these peptides and their analogs or derivatives. These compounds are useful as anti-PIV treatment agents for administration to patients in different disease stages or vaccine to prevent human from PIV infections. SEQ ID NO. 10 AEGRLLKLGiαCIYIYTRSSGWHSNLQIGSLDINNPITINWAPHKVLS

SEQ ID NO. 11

SEGRLLLLGNKIYIYTRSTSWHSKLQLGIIDITDYSDIRIKWTWHNVLS

SEQ ID NO. 12

AEGRLYVIDNNLYYYQRSSSWWSASLFYRINTDFSKGIPPIIEAQWVPS

Example 4: Coronavirus Infections and SARS

The etiologic agent of SARS was identified in late March 2003, when laboratories in Hong Kong, the United States, and Germany found evidence of a novel coronavirus in patients with SARS. This evidence included isolation on cell culture, demonstration by electron microscopy, demonstration of specific genomic sequences by polymerase chain reaction (PCR) and by microarray technology, as well as indirect immunofluorescent antibody tests.

The coronaviruses (order Nidovirales, family Coronaviridae, genus Coronavirus) are members of a family of large, enveloped, positive-sense single-stranded RNA viruses that replicate in the cytoplasm of animal host cells.

The genomes of coronaviruses range in length from 27 to 32 kb, the largest of any of the RNA viruses. The virions measure between about 100 and 140 nanometers in diameter. Most, but not all, viral particles show the characteristic appearance of surface projections, giving rise to the virus' name. These spikes extend a further 20 nanometers from the surface. The Coronaviridae family has been divided up into three groups, originally based on serological cross-reactivity, but more recently based on genomic sequence homology. Groups one (canine, feline infectious peritonitis, porcine transmissible gastroenteritis and porcine respiratory viruses, human coronavirus 229E) and two (bovine, murine hepatitis, rat sialodacryoadenitis viruses, human coronavirus OC43) contain mammalian viruses, while group three contains only avian viruses (avian infectious bronchitis, turkey coronavirus). hi animals, coronaviruses can lead to highly virulent respiratory, enteric, and neurological diseases, as well as hepatitis, causing epizootics of respiratory diseases and/or gastroenteritis with short incubation periods (2-7 days), such as those found in SARS. Coronaviruses are generally highly species-specific. In immunocompetent hosts, infection elicits neutralizing antibodies and cell-mediated immune responses that kill infected cells.

Several coronaviruses can cause fatal systemic diseases in animals, including feline infectious peritonitis virus (FIPV), hemagglutinating encephalomyelitis virus (HEV) of swine, and some strains of avian infectious bronchitis virus (IBV) and mouse hepatitis virus (MHV). These coronaviruses can replicate in liver, lung, kidney, gut, spleen, brain, spinal cord, retina, and other tissues. Coronaviruses cause economically important diseases in domestic animals.

Human coronaviruses (HCoVs) were previously only associated with mild diseases. They are found in both group one (HCoV-229E) and group two (HCoV-OC43) and are a major cause of normally mild respiratory illnesses. They can occasionally cause serious infections of the lower respiratory tract in children and adults and necrotizing enterocolitis in newborns. The known human coronaviruses are able to survive on environmental surfaces for up to three hours. Coronaviruses may be transmitted from person-to-person by droplets, hand contamination, fomites, and small particle aerosols.

SARS-related CoV seems to be the first coronavirus that regularly causes severe disease in humans.

Using the methods described above, SEQ ID NO. 14 was identified from the SARS agent. In vitro antiviral cellular assays may be used to demonstrate potent anti-S ARS activities of these peptides and their analogs or derivatives. These compounds are useful as anti-SARS treatment agents for administration to patients in different disease stages or vaccine to prevent humans SARS infections.

SEQ ID NO. 14 NSVVNIQKEIDRLNEVAKNLNESLIDLGELGKYEGYIKWPWY

Example 5: Rotavirus Infections

Rotavirus most often infects infants and young children, ages 3 months to 2 years and is one of the most common causes of diarrhea in children. Rotavirus infections are responsible for approximately 3 million cases of diarrhea and 55,000 hospitalizations for diarrhea and dehydration in children under 5 years old each year in the United States. Although these infections cause relatively few deaths in the United States, diarrhea caused by rotavirus results in hundreds of thousands of deaths worldwide every year. This is especially true in developing countries, where nutrition and health care are not optimal.

Children with a rotavirus infection have fever, nausea, and vomiting, which are often followed by abdominal cramps and frequent, watery diarrhea. Children who are infected may also have a cough and runny nose. As with all viruses, some rotavirus infections cause few or no symptoms, especially in adults.

Using the methods described above SEQ ID NO. 15 was identified from rotavirus. In vitro antiviral cellular assays may be used to demonstrate potent anti-rotavirus activities of these peptides and their analogs or derivatives. These compounds are useful as an anti- rotavirus treatment agents for administration to patients in different disease stages or vaccine to prevent humans Rotavirus infections.

SEQ ID NO. 15 KLGPRENVAVIQVGGANILDITADPTTTPQTERMMRINWKKWWQVFYTVV

Example 6. Diseases caused by Flaviviruses

Flaviviridae are arboviruses (arthropod-borne virus) mainly transported by mosquitoes and blood-sucking ticks. They are small encapsulated viruses and their genomes consist of infectious single-stranded and linear RNA of positive polarity. In humans, flaviviruses cause deadly hemorrhagic fever or meningo-encephalitis. Yellow fever, dengue fever and Japanese encephalitis are the main tropical flaviviruses. Other important human flaviviruses are Saint Louis encephalitis, tick-born European encephalitis and West Nile fever. Using the methods described above, several peptides were identified that may prevent infection of these viruses. Although some of the peptides do not follow the motifs described above, these peptides are similar in structure and amino acid sequences to other peptides that do follow the motifs (see SEQ ID NO. 19).

Using a similar method described above SEQ ID NO. 16 to SEQ ID NO. 22 were identified from different flaviviruses. In vitro antiviral cellular assays may be used to demonstrate potent anti- flaviviruses activities of these peptides and their analogs or derivatives. These compounds are useful to prevent, treat and/or ameliorate diseases caused by flaviviruses. Example 7. Adenoviral Infections

Adenoviruses are a group of viruses that infect the membranes (tissue linings) of the respiratory tract, the eyes, the intestines and the urinary tract. Adenoviruses account for about 10% of acute respiratory infections in children and are a frequent cause of diarrhea. Adenoviral infections affect infants and young children much more frequently than adults. Child-care centers and schools sometimes experience multiple cases of respiratory infections and diarrhea that are caused by adenovirus. Although these infections can occur at any time of the year, respiratory tract disease caused by adenovirus is more common in late winter, spring, and early summer. However, conjunctivitis and pharyngoconjunctival fever caused by adenovirus tend to affect older children mostly in the summer, spreading from swimming in pools and lakes.

Febrile respiratory disease, which is an infection of the respiratory tract that includes a fever, is the most common result of adenoviral infection in children. The illness often appears flu-like and can include symptoms of pharyngitis (inflammation of the pharynx, or sore throat), rhinitis (inflammation of nasal membranes, or a congested, runny nose), cough, and swollen lymph nodes (glands). Sometimes the respiratory infection leads to acute otitis media, an infection of the middle ear. Adenovirus often affects the lower respiratory tract as well, causing bronchiolitis, croup, or viral pneumonia, which is less common but can cause serious illness in infants. Adenovirus can also produce a dry, harsh cough that can resemble whooping cough (pertussis).

Other diseases caused by adenovirus include: Gastroenteritis; Urinary tract infections; Conjunctivitis (or pinkeye); Pharyngoconjunctival fever; and Keratoconjunctivitis.

Using the similar method described above SEQ ID NO. 23 to SEQ ID NO. 29 where identified from different adenoviruses. In vitro antiviral cellular assays may be used to demonstrate potent anti- Adenoviral activities of these peptides and their analogs or derivatives. These compounds are useful to prevent, treat and/or ameliorate diseases caused by adenovirus.

It should be understood that the foregoing discussion and examples merely present a detailed description of certain preferred embodiments. It therefore should be apparent to those of ordinary skill in the art that various modifications and equivalents can be made without departing from the spirit and scope of the invention. All articles, patents and texts that are identified above are incorporated by reference in their entirety.

Claims

Claims:

1. A method for identifying a viral protein or peptide that inhibits viral infection comprising:

(a) searching a database for a viral protein with a W X_niW motif; and

(b) analyzing the region around said motif in (a) to further identify an I X₁₁₂ I motif;

thereby identifying a viral protein or peptide that inhibits viral infection, wherein X is any amino acid and nl and n2 is a number from 0 to 20.

2. The method of claim 1, further comprising aligning said identified protein or peptide in claim 1 with a known protein sequence that inhibits viral infection, identifying homology between said known sequence and identified protein or peptide in claim 1 and isolating the homologous amino acid sequence comprising the motifs in (a) and (b).

3. The method of claim 2, wherein said peptide is artificially synthesized.

4. The method of claim 1, wherein said peptide has a length of 15 to 60 amino acids.

5. The method of claim 1, wherein said viral protein or peptide inhibits viral fusion, viral penetration and/or inhibits viral transmission of a virus.

6. The method of claim 1, wherein said viral protein or peptide inhibits viral infection of more than one virus.

7. A viral protein or peptide identified according to the method of claim 1.

8. The viral protein or peptide of claim 7, wherein said protein or peptide is attached to another molecule.

9. The viral protein or peptide of claim 8, wherein said molecule increases the in vivo half- life of said protein or peptide.

10. The viral protein or peptide of claim 7, wherein said protein or peptide is fused to another protein or polypeptide.

11 The viral protein or peptide of claim 10, wherein said protein or polypeptide increases the in vivo half-life of said viral protein or peptide.

12. The viral protein or peptide of claim 7, wherein said protein or peptide inhibits viral infection.

13. The viral protein or peptide of claim 12, wherein said viral protein or peptide inhibits viral fusion, viral penetration and/or viral transmission of a virus.

14. A composition comprising the viral protein or peptide of claim 7.

15. The composition of claim 14, wherein said composition treats, prevents and/or ameliorates a viral infection in an animal when administered to said animal.

16. The composition of claim 14, wherein said animal is a human.

17. The composition of claim 14, wherein said protein or peptide is artificially synthesized.

18. The composition of claim 14, wherein said viral protein or peptide inhibits viral infection.

19. The composition of claim 18, wherein said viral protein or peptide inhibits viral fusion, viral penetration and/or inhibits viral transmission of a virus.

20. A method for treating, preventing and/or ameliorating an infection caused by a virus, comprising administering to an animal the viral protein or peptide of claim 7.

21. The method of claim 20, wherein said animal is a human.

22. The method of claim 20, wherein said viral protein or peptide is artificially synthesized.

23. The method of claim 23, wherein said viral protein or peptide is attached to another molecule.

24. The method of claim 20, wherein said molecule increases the in vivo half-life of said viral protein or peptide.

25. The method of claim 20, wherein said viral protein or peptide is fused to another protein or polypeptide.

26. The method of claim 25, wherein said protein or polypeptide increases the in vivo half- life of said viral protein or peptide.

27. The method of claim 20, wherein said viral protein or peptide inhibits viral infection.

28. The method of claim 27, wherein said viral protein or peptide inhibits viral fusion, viral penetration and/or inhibits viral transmission of a virus.

29. An antigenic formulation comprising the viral protein or peptide of claim 7.

30. The antigenic formulation of claim 29, wherein said antigenic formulation stimulates an immune response against said viral protein or peptide in an animal.

31. The antigenic formulation of claim 29, wherein said formulation comprises an adjuvant or immune stimulator.

32. The antigenic formulation of claim 29, wherein said antigenic formulation is suitable for human administration.

33. The antigenic formulation claim 29, wherein the formulation is administered to an animal orally, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or subcutaneously.

34. A method of formulating a vaccine capable of delivering an effective dose that induces substantial immunity to a viral infection or at least one symptom thereof to an animal, comprising adding to said formulation the viral protein or peptide of claim 7.

35. The method of claim 34, wherein said substantial immunity to viral infection or at least one symptom thereof is delivered in one dose.

36. The method of claim 34, wherein said substantial immunity to viral infection or at least one symptom thereof is delivered in multiple doses.

37. The method of claim 34, wherein said viral protein or peptide is formulated with an adjuvant or immune stimulator.

38. An antibody that immunospecifically binds to the viral protein or peptide identified in claim 1.

39. A peptide consisting essentially of any one of the peptides selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 12 and SEQ ID NO. 14 to SEQ ID NO. 34

40. The peptide of claim 39, wherein said peptide is attached to another molecule.

41. The peptide of claim 40, wherein said molecule increases the in vivo half-life of said peptide.

42. The peptide of claim 39, wherein said peptide is fused to another protein or polypeptide.

43. The peptide of claim 42, wherein said protein or polypeptide increases the in vivo half- life of said peptide.

44. The peptide of claim 39, wherein said peptide inhibits infection of a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus.

45. The peptide of claim 39, wherein said viral protein or peptide inhibits viral fusion, viral penetration and/or inhibits viral transmission of a virus.

46. A composition comprising the peptide of claim 38.

47. The composition of claim 46, wherein said composition treats, prevents and/or ameliorates a viral infection cause by a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus in an animal, when administered to said animal.

48. The composition of claim 46, wherein said animal is a human.

49. The composition of claim 46, wherein said composition induces an immune response in an animal when administered to said animal.

50. The composition of claim 46, wherein said peptide inhibits infection of a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus in an animal, when administered to said animal.

51. The composition of claim 46, wherein said viral protein or peptide inhibits viral fusion, viral penetration and/or inhibits viral transmission of a virus.

52. A method for treating, preventing and/or ameliorating a viral infection caused by a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus, comprising administering to an animal the peptide of claim 39.

53. The method of claim 52, wherein said animal is a human.

54. The method of claim 52, wherein said method induces an immune response in an animal when administered to said animal.

55. The method of claim 52, wherein said peptide is attached to another molecule.

56. The method of claim 55, wherein said molecule increases the in vivo half-life of said peptide.

57. The method of claim 52, wherein said peptide is fused to another protein or polypeptide.

58. The method of claim 57, wherein said protein or polypeptide increases the in vivo half- life of said peptide.

59. The method of claim 52, wherein said peptide inhibits infection of a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus.

60. The method of claim 52, wherein said viral protein or peptide inhibits viral fusion, viral penetration and/or inhibits viral transmission of a virus.

61. An antigenic formulation comprising the peptide of claim 39.

62. The antigenic formulation of claim 61, wherein said antigenic formulation stimulates an immune response against said peptide in an animal.

63. The antigenic formulation of claim 61, wherein said formulation comprises an adjuvant or immune stimulator.

64. The antigenic formulation of claim 61, wherein said antigenic formulation is suitable for human administration.

65. The antigenic formulation of claim 61, wherein the formulation is administered to an animal orally, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or subcutaneously.

66. A method of formulating a vaccine capable of delivering an effective dose that induces substantial immunity to a viral infection cause by a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus or at least one symptom thereof to an animal, comprising adding to said formulation the peptide of claim 39.

67. The method of claim 66, wherein said substantial immunity to a viral infection caused by a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus or at least one symptom thereof is delivered in one dose.

68. The method of claim 66, wherein said substantial immunity to a viral infection caused by a virus selected from the group consisting of hepatitis C virus, hepatitis B virus, influenza virus, avian influenza virus and parainfluenza virus is delivered in multiple doses.

69. The method of claim 66, wherein said peptide is formulated with an adjuvant or immune stimulator.

70. An antibody that immunospecifically binds to the peptide identified in claim 39.

71. A polypeptide or peptide comprising the motifs W X_nl W and I X₁₁₂ I, wherein X is any amino acid and nl and n2 is a number from 0 to 20, and wherein the polypeptide or peptide is less than about 100 amino acids in length.

72. The polypeptide or peptide of claim 70, wherein said polypeptide or peptide is less than about 50 amino acids in length.

73. The polypeptide or peptide of claim 70, wherein said polypeptide or peptide is less than about 20 amino acids in length.

74. A method for identifying an agent that inhibits viral infection, comprising screening agents which will compete with, bind to, or enhance the activity of viral proteins or peptides of the invention.

75. The method of claim 74, wherein said method comprises the use of a library.

76. The method of claim 75, wherein said library is selected from the group consisting of a phage display library of peptides, a peptide library, a peptidomimetic library, an oligosaccharide library, a lipoprotein library, a glycoprotein or glycolipid library and chemical library.