WO2004009032A2

WO2004009032A2 - Method and composition for treating and preventing hepatitis b infection and symptoms thereof

Info

Publication number: WO2004009032A2
Application number: PCT/US2003/022961
Authority: WO
Inventors: Scott Morham; Kenton Zavitz; Adrian Hobden
Original assignee: Myriad Genetics, Inc
Priority date: 2002-07-19
Filing date: 2003-07-21
Publication date: 2004-01-29
Also published as: AU2003254129A1; WO2004009032A3; AU2003254129A8

Abstract

The present invention provides methods for preventing and treating HBV infection and symptoms thereof by introducing cells displaying a HBV altered budding phenotype into a patient, or by administering to a patient nucleic acids, polypeptides and small organic compounds to cause the formation of cells displaying a HBV altered budding phenotype in the body of the patient.

Description

METHOD AND COMPOSITION FOR TREATING AND PREVENTING HEPATITIS B INFECTION AND SYMPTOMS THEREOF

Field of the Invention The present invention relates generally to treating and preventing viral infections and associated diseases, and particularly to methods and compositions for treating and preventing hepatitis B infection.

Background of the Invention Viral infection of humans is a major health problem. Combating viral infection has proven to be highly effective in some cases like smallpox where the disease was essentially eradicated with the advent of the smallpox vaccination. Other viral infections have been much more difficult to fight. Hepatitis B and C, human immunodeficiency virus (HIY), and herpes viruses are just a few prominent members of a list of viruses that pose significant health threats worldwide. Treatments available for these viruses are associated with adverse side-effects. In some cases the viruses mutate and become resistant to a particular treatment. Accordingly, there is a clear need for new antiviral treatments.

Hepatitis virus B (HBV) is estimated to be responsible for approximately 4000- 5000 deaths each year in the United States alone. The problem is substantially greater in the rest of the world and accounts for about 1 million deaths worldwide each year. According to the Center for Disease Control, about one in twenty people in the United States will get infected with HBV. About two billion people worldwide have been infected with HBV, and 350 million of those have chronic infection. It is estimated that upwards of 10% of the population of the developing world (large parts of Asia, the Pacific, and sub-Saharan Africa) are chronically infected with HBN. Chronically infected individuals are at a much greater risk for hepatocellular carcinoma (liver cancer) and cirrhosis of the liver. Liver cancer caused by HBN is one of the leading causes of death in men by cancer in the developing world. By any analysis, HBN is one of the more serious health problems facing the world.

HBN is transmitted by contact with blood or other body fluids of an infected person and is similar to the human immunodeficiency virus in that sense, but it is about two orders of magnitude more infectious than HIN. The main paths of transmission are from mother to infant at birth, child-to-child, unsafe injections and transfusions, and unsafe sexual contact.

HBN was probably first reported in the late 19* century in shipyard workers in Bremen who had been vaccinated against smallpox. Over the next one hundred years the association of hepatitis with the use of needles and syringes used for treating diseases like syphilis, diabetes, and the administration of vaccines for yellow fever, led scientists to discover HBN. Eventually a vaccine was developed from a viral envelope protein that was purified from the plasma of individuals with a chronic HBN infection. Later, a recombinant system was used to produce the protein used in the vaccines because the original source of the vaccine was those individuals infected with HBN and this same population was at high-risk for HIN infection.

The HBN vaccine was shown to be about 95% effective in preventing chronic infection in individuals if they were not previously infected. Unfortunately, the HBN vaccine does not cure the disease and there is a need from new treatments.

The mechanisms that viruses use to depart an infected cell are only beginning to be understood on a general level. Recently several proteins involved in the egress of certain viruses from infected cells have been discovered. Studies with the HIN-1 gag protein have demonstrated an interaction between a human protein, tsglOl, and gag which is critical for viral budding from the host cell (Garrus et al. Cell 107:55-65 (2001)). Related studies have established a common budding theme among retro virus. It appears that protein-protein interactions between specific host cell proteins and viral proteins are critical for viral budding. Rous sarcoma virus (RSN), equine infectious anemia virus (EIAN), as well as other retroviruses, were shown to have late domains with characteristic amino acid sequence motifs; the late-domain motifs are critical binding motifs that facilitate the interaction of the viral proteins with host cell proteins. See, e.g., Puffer et al. J. Virol. 72:10218-10221 (1998) and Campbell et al. J. Virol. 71:4425-35 (1997). Unexpectedly, it was discovered that specific hepatitis B proteins have late- domain motifs which can be manipulated or modulated to affect viral budding and egress. This discovery now provides an entirely new class of HBN antivirals.

Summary of the Invention

The present invention relates generally to treating and preventing HBV infection. The invention provides methods for treating and preventing HBV infection in an individual in need of such treatment by enhancing or inducing an immune response against HBV. The invention also provides compositions for use in treating and preventing HBV in individuals in need of such treatment.

The invention provides a method of enhancing or inducing an immune response in an individual against HBV. The method of the invention comprises enhancing or inducing an immune response in an individual by affecting viral budding. According to the method, viral budding is affected by modulating a viral-protein host-protein protein- protein interaction that is involved in viral budding. Modulation of a viral-protein host- protein protein-protein interaction that is involved in viral budding can stimulate or lead to an enhanced immune response against the virus.

Modulation of a viral-protein host protein protein-protein interaction involved in viral budding can lead to a HBV altered budding phenotype in host cells. A HBV altered budding phenotype can enhance or induce an immune response against HBV by, e.g., making the cells immunogenic and particularly effective in stimulating cytotoxic T lymphocytes (CTL) response, which is an important immune response for inhibiting viral replication. The HBV altered budding phenotype can also cause the cell to display molecular markers that tag the cells for an immune response or enhancement thereof. The altered budding phenotype can also lead to apoptosis and cellular death which can also lead to an enhanced immune response against HBN and cells infected with HBN. Therefore, the invention provides a method of enhancing or inducing any immune response against HBN or HBN infected cells.

In one embodiment, the method of the present invention comprises inducing an enhanced immune response in a patient against HBN. Enhancement of an immune response can be accomplished by administering to a patient, in need of therapeutic or prophylactic treatment, an effective amount of a mutant HBV protein that is capable of affecting a protein-protein interaction involved in viral budding. Alternatively, the enhancement can be achieved by administering to the patient a nucleic acid encoding such a mutant HBN polypeptide. In one aspect of this embodiment, a mutant HBN core protein which is sufficient for viral particle assembly and is capable of affecting a protein-protein interaction involved in viral budding in cells can be provided by modifying the HBN protein sequence that interacts with a host protein to affect viral budding. Such a HBN protein can have a modified late-domain motif. In a preferred aspect of this embodiment, a nucleic acid encoding a mutant HBN core protein sufficient for viral particle assembly but devoid of late-domain motifs is incorporated into a plasmid vector which is injected directly into a patient. The nucleic acid can also be delivered into a patient by infecting the patient with a recombinant live vector (e.g., recombinant viral or bacterial vector) carrying the nucleic acid. Preferably, the nucleic acid is administered to a patient by gene gun or powder jet or an equivalent device thereof.

In another embodiment, a mutant HBN core protein which is sufficient for viral particle assembly and is capable of affecting a protein-protein interaction involved in viral budding in cells can be provided by modifying the HBV protein sequence that interacts with a host protein to affect viral budding. Such a HBV protein can have a modified late-domain motif. In a prefeπed aspect of this embodiment, a nucleic acid encoding a mutant HBV core protein sufficient for viral particle assembly but devoid of late-domain motifs is incorporated into a plasmid vector which is injected directly into a patient. The nucleic acid can also be delivered into a patient by infecting the patient with a recombinant live vector (e.g., recombinant viral or bacterial vector) carrying the nucleic acid. Preferably, the nucleic acid is administered to a patient by gene gun or powder jet or an equivalent device thereof.

In yet another embodiment, a mutant HBV envelope protein which is sufficient for viral particle assembly and is capable of affecting a protein-protein interaction involved in viral budding in cells can be provided by modifying the HBV envelope protein sequence that interacts with a host protein to affect viral budding. Such a HBV envelope protein can have a modified late-domain motif. In a prefeπed aspect of this embodiment, a nucleic acid encoding a mutant HBV envelope protein sufficient for viral particle assembly but devoid of late-domain motifs is incorporated into a plasmid vector which is injected directly into a patient. The nucleic acid can also be delivered into a patient by infecting the patient with a recombinant live vector (e.g., recombinant viral or bacterial vector) carrying the nucleic acid. Preferably, the nucleic acid is administered to a patient by gene gun or powder jet or an equivalent device thereof.

In another embodiment, the invention provides a method of ameliorating the symptoms of HBV infection. In this embodiment, a HBV carrier having HBV symptoms is treated with an effective amount of a composition that affects viral budding. A composition that affects viral budding can have (or encode), e.g., a variant HBV core protein. The variant HBV core protein is modified to nullify the late-domain motif. Preferably, the variant HBV core protein is sufficient for virus particle formation. The administration of a composition having (or encoding) the modified core protein enhances an immune response against HBV and the enhanced immune response modulates the amelioration of the symptoms of HBV.

In still another embodiment, the invention provides a method of ameliorating the symptoms of HBV infection. In this embodiment, a HBV carrier having HBV symptoms is treated with an effective amount of a composition that affects viral budding. A composition that affects viral budding can have (or encode), e.g., a variant HBV envelope protein. The variant HBV envelope protein is modified to nullify the late-domain motif. Preferably, the variant HBV envelope protein is sufficient for virus particle formation. The administration, to an individual, a composition having (or encoding) the modified envelope protein enhances an immune response against HBV and the enhanced immune response modulates the amelioration of the symptoms of HBV.

In yet another embodiment, the invention relates to a method of reducing the side- effects related to interferon and/or lamivudine treatment. According to this aspect of the invention, a composition that leads to a HBV altered budding phenotype is administered to an individual infected with HBV and who is cuπently receiving interferon and/or lamivudine treatment. The composition enhances or induces an immune response against HBV or HBV containing cells. The enhancement or induction of the immune response allows for the administration of lower amounts of interferon and/or lamivudine, thereby reducing the side-effects associated with their administration.

The invention further provides a method of identifying compounds that modulate the activity of a viral protein host cell protein protein-protein interaction that is involved in a viral egress and/or budding pathway. More particularly, the method involves identifying compounds that modulate an interaction between a viral protein having a late- domain motif and a host cell protein that interacts with the late-domain motif containing protein. According to this embodiment an assay system is employed to detect compounds and/or compositions that affect the ability of the virus to propagate itself. The assay system targets host cell proteins that are utilized by the virus for budding that interact with the viral late-domain motif containing protein(s). In one aspect of this embodiment, the assay is configured to identify compounds that modulate the activity of a specific protein involved in a viral budding and/or egress pathway. In another aspect of this embodiment, the assay is configured to identify compounds that modulate a protein-protein interaction that is involved in the viral budding and/or egress pathway. In one aspect of this embodiment, the host protein that is modulated is selected from the group consisting of TSG 101 , NEDD4, a NEDD4-like protein, AIP 1 , a host cell protein containing a WW domain, or fragments, derivatives, or homologs thereof. In another aspect of this embodiment the viral late-domain motif containing protein has a YXXL, PXXP, or PPXY late-domain motif, or fragments, derivatives, or homologs thereof. As the skilled artisan is aware, the screening assays of the invention can be accomplished in any manner as long as they examine the affect of a particular treatment on a host cell viral protein protein-protein interaction involved in viral budding (or egress) or a host cell biochemical pathway involved in viral egress.

Detailed Description of the Invention The present invention relates to inducing an HBN altered budding phenotype in cells. The HBN altered budding phenotype is capable of stimulating an immune response against HBN and HBN infected cells. In particular, HBN contains at least one late- domain motif, so-called because it is required for viral budding. Without wishing to be bound by theory, it is believed that a protein-protein interaction between a HBN late- domain motif containing protein and a host protein is required for normal budding. Thus, according to the method of the invention, the viral budding process is impaired, and the host cell displays an altered budding phenotype. The HBV altered budding phenotype is used to induce and/or enhance an immune response.

The present invention relates to the discovery that HBV contains a motif that is thought to be involved in the budding process. In particular, it was discovered that the HBV core protein has a proline-proline-alanine-tyrosine (PPAY) motif which is thought to be critical to the budding process. The PPAY motif in the HBV core protein fits within the consensus sequence of PPXY, where X can be any amino acid. PPXY motifs are found in numerous eukaryotic proteins and are found in membrane-associated proteins from certain enveloped viruses. See Craven et al, J. Virol, 73:3359-3365

(1999); Harty et al, Proc. Natl. Acad. Sci. USA, 97:13871-13876 (2000); Harty et al, J. Virol, 73:2921-2929 (1999); and Jayakar et al, J. Virol, 74:9818-9827 (2000). The cellular target for one PY motif is Νedd4 which also contains a Hect ubiquitin E3 ligase domain. See Harvey and Kumar, Trends Cell Biol, 9: 166-169 (1999); Hicke, Trends Cell Biol, 9: 107-112 (1999). The PY motif binds Nedd4 via one or more of the type I WW-domains inNedd4. See Kanelis et al, Nat. Struct. Biol, 8:407-412 (2001); Lu et al, Science, 283:1325-1328 (1999).

It is contemplated that proteins other than the HBV core protein have a late- domain motif and can be involved in viral budding. Therefore, the methods and compositions of the invention include using these late-domain containing proteins and genes to enhance or induce an immune response in manner similar to that as described herein for the HBV core protein. For example, the hepatitis B presl/pres2/S envelope protein has a late-domain motif (see, e.g., GenBank Accession No. BAA85340). This protein has a PTAP late-domain motif. The host cell binding partner for the PTAP late domain motif is TSGIOI. The PTAP motif in the HBV presl/pres2/S protein is followed immediately by a PPAS motif that is similar to the PPAY late-domain motif. The PPAY late-domain motif is known to bind to WW domain containing proteins, particularly Nedd4. Thus, the method and compositions of the invention relate to these late-domain motifs in the HBV pres l/pres2/S protein. For example, variant HBV pres l/pres2/S protein can be used to induce an altered budding phenotype. The altered budding phenotype can be taken advantage of to stimulate or enhance an immune response in an individual in need of such treatment.

As used herein the term "hepatitis B" or "HBV" refers to any of the strains and isolates of hepatitis B that have been identified or are identifiable according to known classifications. The term "hepatitis B" or "HBV" is intended to encompass all cuπently known strains, types and subtypes of HBV as wells those discovered and classified as HBV in the future. The identification of HBV is well within the purvey of an ordinary skilled artisan. Numerous HBV strains, types, and subtypes are known with their sequences being deposited in public databases. As used herein, the term "diagnosed with HBV" refers to an individual in which a

HBV marker has been detected. A variety of HBV markers are known in the art and can be readily measured by a skilled artisan. For example, HBV can be detected by testing for antibodies against HBV proteins (anti-HBV) in a patient or suspected carrier's blood serum. Another approach to detecting HBV is to test for HBV DNA in the serum of a patient or suspected carrier using a PCR based assay. Recombinant immunoblots can also be used to detect HBV. In this test serum is incubated with four recombinant viral proteins that are blotted on nitrocellulose strips. A simple color change indicates antibodies are present in the serum that bind to the viral proteins. Furthermore, new methods for detecting HBV are continually being developed and the methods can be employed for diagnosing HBV infection. As used herein, the term "alleviate or ameliorate a symptom associated with HBV infection" refers to a lessening of: jaundice, fatigue, abdominal pain, loss of appetite, nausea, vomiting, malaise, fever, and joint pain. Also included within the scope of this term is a lessening or improvement in the level of an HBV marker, e.g., HBV protein detected in an immunoassay.

As used herein, the term "HBN altered budding phenotype" refers to the phenotype that a host cell or tissue displays upon modulation of a viral-protein host- protein protein-protein interaction involved in viral budding. The term "HBV altered budding phenotype" is in comparison to the normal HBV budding phenotype which is one characterized by normal viral budding and extracellular virus release. Thus, in a cell infected with a wild-type HBV strain, the virus will bud normally. In an "HBV altered budding phenotype" cell, HBV particles may never reach maturity, i.e., form wild-type viral particles, and/or have an accumulation of virus in a particular cellular compartment. Cells having a HBV altered budding phenotype can also display different markers on the cell surface or within the cell as compared to normal uninfected cells or cells infected with wild-type viruses. The difference in markers serves as a tag for the immune system to recognize these cells or tissues with the altered phenotype. The HBV altered budding phenotype is readily determined by one of skill in the art. Markers in control host cells, wild-type HBV infected cells, and cells which have had a viral protein-host protein protein-protein interaction modulated, can be quantitated using standard techniques to determine the altered budding phenotype. A non-limiting example is a comparison of the type and number of HBV particles found in, e.g., the cytoplasm, the uninfected host is expected to have no HBV particles in the cytoplasm, the wild-type HBV infected host cell is expected to a given amount of virus in the cytoplasm, and an altered budding phenotype cell is expected to a substantially different amount of virus in the cytoplasm. Electron microscopy is another useful tool for determining a HBV altered budding phenotype by examination of the cellular localization of viral particles and proteins. Thus, an altered budding phenotype is recognizable by one of ordinary skill in the art by making such comparisons using art-known techniques. As used herein, the term "altered egress phenotype" refers to the phenotype that the host cell displays upon modulation of a host cell protein-protein interaction involved in viral egress. The term "altered egress phenotype" is in comparison to the normal egress phenotype which is one characterized by normal viral egress and extracellular virus release. Thus, in a cell infected with a wild-type strain, the virus will egress normally. In a cell having an "altered egress phenotype", the virus may never reach maturity, i.e., form wild-type virus, or alternatively, have an accumulation of virus in a particular cellular compartment. Altered egress phenotype displaying cells may also display different molecular markers on the cell surface or within the cell as compared to normal uninfected cells or cells infected with wild-type viruses. The difference in markers can serve as a signal for the immune system to recognize the cells or tissues with the altered phenotype. The altered egress phenotype is readily determined by one of skill in the art. For example, markers in uninfected control host cells, virus infected cells, and infected cells which have had a protein-protein interaction modulated, or a protein modulated, can be quantitated using standard techniques to determine the altered egress phenotype. A non-limiting example is a comparison of the type and number of viral particles found in, e.g., the cytoplasm, the uninfected host is expected to have no viral particles in the cytoplasm, the wild-type virus infected host cell is expected to have a given amount of virus in the cytoplasm, and an altered egress phenotype cell is expected to have a substantially different amount of virus in the cytoplasm. Koshel et al. J. Virol. 74 : 1 -7 (2000) describe methods of detecting altered egress HBV phenotypes . Electron microscopy is another useful tool for determining an altered egress phenotype by examination of the cellular localization of viral particles and proteins. Thus, an altered egress phenotype is recognizable by one of ordinary skill in the art by making such comparisons.

As used herein, the term "alpha interferon" refers to the family of interferon proteins that inhibit viral replication, inhibit cellular proliferation, and modulate immune response. The term "alpha interferon" encompasses a variety of commercially available alpha interferons, including, but not limited to, Roferon A interferon (Hoffman-La Roche, Nutley, NJ), Berofor alpha 2 (Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, CT), Sumiferon (Sumitomo, Japan), Wellferon interferon alpha-nl (Glaxo- Wellcome Ltd., London, Great Britain). U.S. patent no. 4,530,901 (which is hereby incorporated by reference in its entirety) provides a description of the manufacture of alpha interferon 2b. The term alpha interferon is also intended to encompass PEG interferon.

As used herein, the term "lamivudine" refers to the drug known as 3TC and has a chemical name of (2R, cis)-4-amino-l -(2-hydroxymethyl-l,3-oxathiolan-5-yl) - (1H)- pyrimidin-2-one. It is also known as (-)2',3'-dideoxy, 3'-thiacytidine.

As used herein, the term "side effects of alpha interferon treatment" include fatigue, muscle aches, headaches, nausea, vomiting, low-grade fever, weight loss, irritability, depression, mild bone marrow suppression, and hair loss associated with treatment with alpha interferon. As used herein, the term "side effects of lamivudine treatment" refers to nausea, vomiting, headaches, hair loss, peripheral neuropathy, and pancreatitis associated with treatment with lamivudine.

As used herein, the term "viral-like particle" refers to nucleocapsids or any HBV protein containing particles which in their wild-type form are capable of being secreted or are capable of budding.

As used herein, the term "individual in need of treatment" encompasses individuals who have symptoms of HBV infection, those who have been diagnosed with HBV, and those in need of protection against HBV infection, i.e., prophylaxis. An "individual in need of treatment" can have antibodies to HBV antigens, HBV DNA, elevated serum aminotransferase levels, and/or evidence of chronic hepatitis.

The terms "polypeptide," "protein," and "peptide" are used herein interchangeably to refer to amino acid chains in which the amino acid residues are linked by peptide bonds or modified peptide bonds. The amino acid chains can be of any length of greater than two amino acids. Unless otherwise specified, the terms "polypeptide," "protein," and "peptide" also encompass various modified forms thereof. Such modified forms may be naturally occurring modified forms or chemically modified forms. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, prenylated forms, etc. Modifications also include infra-molecular crosslinking and covalent attachment to various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, etc. In addition, modifications may also include cyclization, branching and cross-linking. Further, amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide.

As used herein, the term "homologue," when used in connection with a first native protein or fragment thereof that is discovered, according to the present invention, to interact with a second native protein or fragment thereof, means a polypeptide that exhibits an amino acid sequence homology and/or structural resemblance to the first native interacting protein, or to one of the interacting domains of the first native protein such that it is capable of interacting with the second native protein. Typically, a protein homologue of a native protein may have an amino acid sequence that is at least 50%, preferably at least 75%, more preferably at least 80%, 85%, 86%, 87%, 88% or 89%, even more preferably at least 90%, 91%, 92%, 93% or 94%, and most preferably 95%, 96%, 97%, 98% or 99% identical to the native protein. Examples of homologues may be the ortholog proteins of other species including animals, plants, yeast, bacteria, and the like. Homologues may also be selected by, e.g., mutagenesis in a native protein. For example, homologues may be identified by site-specific mutagenesis in combination with assays for detecting protein-protein interactions, e.g., the yeast two-hybrid system described below, as will be apparent to skilled artisans apprised of the present invention. Other techniques for detecting protein-protein interactions include, e.g., protein affinity chromatography, affinity blotting, in vitro binding assays, and the like.

As used herein, the term "derivative," when used in connection with a first native protein (or fragment thereof) that is discovered, according to the present invention, to interact with a second native protein (or fragment thereof), means a modified form of the first native protein prepared by modifying the side chain groups of the first native protein without changing the amino acid sequence of the first native protein. The modified form, i.e., the derivative should be capable of interacting with the second native protein. Examples of modified forms include glycosylated forms, phosphorylated forms, myristylated forms, ribosylated forms, ubiquitinated forms, prenylated forms, and the like. Derivatives also include hybrid or fusion proteins containing a native protein or a fragment thereof. Methods for preparing such derivative forms should be apparent to skilled artisans. The prepared derivatives can be easily tested for their ability to interact with the native interacting partner using techniques known in the art, e.g., protein affinity chromatography, affinity blotting, in vitro binding assays, yeast two-hybrid assays, and the like.

As used herein, the term "interacting" or "interaction" means that two protein domains, fragments or complete proteins exhibit sufficient physical affinity to each other so as to bring the two "interacting" protein domains, fragments or proteins physically close to each other. An extreme case of interaction is the formation of a chemical bond that results in continual and stable proximity of the two entities. Interactions that are based solely on physical affinities, although usually more dynamic than chemically bonded interactions, can be equally effective in co-localizing two proteins. Examples of physical affinities and chemical bonds include but are not limited to, forces caused by electrical charge differences, hydrophobicity, hydrogen bonds, van der Waals force, ionic force, covalent linkages, and combinations thereof. The state of proximity between the interaction domains, fragments, proteins or entities may be transient or permanent, reversible or irreversible. In any event, it is in contrast to and distinguishable from contact caused by natural random movement of two entities. Typically, although not necessarily, an "interaction" is exhibited by the binding between the interaction domains, fragments, proteins, or entities. Examples of interactions include specific interactions between antigen and antibody, ligand and receptor, enzyme and substrate, and the like.

An "interaction" between two protein domains, fragments or complete proteins can be determined by a number of methods. For example, an interaction can be determined by functional assays such as the two-hybrid systems. Protein-protein interactions can also be determined by various biophysical and biochemical approaches based on the affinity binding between the two interacting partners. Such biochemical methods generally known in the art include, but are not limited to, protein affinity chromatography, affinity blotting, immunoprecipitation, and the like. The binding constant for two interacting proteins, which reflects the strength or quality of the interaction, can also be determined using methods known in the art. See Phizicky and Fields, Microbiol. Rev., 59:94-123 (1995). The term "protein fragment" as used herein means a polypeptide that represents a portion of a protein. When a protein fragment exhibits interactions with another protein or protein fragment, the two entities are said to interact through interaction domains that are contained within the entities.

As used herein, the term "domain" means a functional portion, segment or region of a protein, or polypeptide. "Interaction domain" refers specifically to a portion, segment or region of a protein, polypeptide or protein fragment that is responsible for the physical affinity of that protein, protein fragment or isolated domain for another protein, protein fragment or isolated domain.

1. General

The invention provides for the treatment or prevention of HBV infection by stimulating an immune response against the virus, virus infected cells, or antigens preparations obtained from virus infected cells displaying an HBV altered budding (or egress) phenotype. The method can be performed by treating an individual with a composition that affects a viral-protein host-protein protein-protein interaction, or by providing to the individual cells (or purified fractions thereof) that were prepared in vitro to have an altered budding (or egress) phenotype. The treatments of the invention can be in combination with other agents, as in combination therapy, or with the use of adjuvants. The invention also provides a method for screening for modulators of a viral-protein host-protein protein-protein interaction involved in viral budding. The screen can be used to identify molecules that are capable of producing a HBN altered budding phenotype. These representative embodiments of the invention are discussed in further detail in sections 2-7.

2. Formation of Immunogenic Cells In Vivo

According to the present invention, a method for treating and/or preventing HBN infection or symptoms thereof, is provided that includes a step of inducing, in a patient, an enhanced immune response against HBN. According to one aspect of the invention, a viral protein involved in budding is modified such that it has an altered binding affinity for its host protein interacting partner. Modulation of a host-protein viral-protein interaction involved in viral budding can lead to a stimulation of the patient's immune system and elicit cellular immune responses against HBV. Cells which have had the viral-protein host-protein interaction involved in budding disrupted, can enhance or induce a humoral immune response in the patient against the HBV viral proteins carried by the cells, thus eliciting antibodies against the HBV proteins.

Any method for inducing or enhancing an immune response against HBV in a patient can be used for purposes of this aspect of the present invention as long as it affects a viral-protein host-protein interaction involved in viral budding. In some embodiments, the induction or enhancement of an immune response in a patient can be achieved by administering to a patient in need of treatment a mutant HBV core protein which affects a protein-protein interaction involved in viral budding, or by administering to a patient a nucleic acid encoding a mutant HBV core protein. In a preferred aspect, the mutant HBV core protein that is administered to the patient is sufficient for viral particle formation. In other embodiments, the induction or enhancement of an immune response in a patient can be achieved by administering to a patient in need of treatment a mutant HBV envelope protein which affects a protein-protein interaction involved in viral budding, or by administering to a patient a nucleic acid encoding a mutant HBV envelope protein. For example, the hepatitis B presl/pres2/S envelope protein has a late-domain motif. In a prefeπed aspect, the mutant HBV envelope protein that is administered to the patient is sufficient for viral particle formation. Therefore the methods of the invention include the use of the late domain motif in other HBV proteins like that in the presl/pres2/S region. In a preferred embodiment, a mutant HBV core protein can be provided based on any of the wild-type or modified HBN core proteins known in the art to be sufficient for the assembly of virus-like particles. Specifically, a mutant core protein that affects viral budding and is sufficient for viral particle assembly can be generated by modifying a wild-type or modified core protein such that it can mediate virus-like particle assembly and disrupts viral budding. Thus, in one embodiment, a mutant HBV core protein that is sufficient for viruslike particle assembly and affects viral budding can be provided by modifying a wild- type HBV core protein to nullify the functional late domain therein. Also, if a modified HBV core protein sufficient for virus-like particle assembly contains a functional late domain, that late domain can be nullified to provide a mutant core protein that is sufficient for viral particle assembly and affects viral budding.

Typically, the late-domain motif in the HBV core protein has a PPAY sequence motif in the core protein. Without wishing to be bound by theory, the PPAY sequence motif is thought to bind to the proteins in the host cell containing a WW domain, particularly Nedd4 (or a Nedd4 like protein). The binding of the PPAY sequence motif to proteins containing a WW domain (particularly Nedd4) is thought to be required for HBV viral budding from a host cell membrane system. Mutations in the PPAY sequence motif that abolish the binding to a late-domain motif binding partner can lead to the disruption of viral budding. For purposes of the present invention, the HBV core protein late-domain motif in a wild-type or modified HBV core protein can be nullified by any means so long as the resultant protein is incapable of functionally binding proteins containing a late-domain motif binding domain, i.e., WW domain (particularly Nedd4, or a Nedd4 like protein). Being incapable of functionally binding refers to the inability to complete wild-type viral budding. In one embodiment, the functional late domain motif can be nullified by deleting the entire PPAY sequence motif in a wild-type or modified HBV core protein. Alternatively, amino acid substitution mutations in or near the PPAY sequence motif can be created so that the resultant HBV core protein is incapable of binding proteins containing a WW domain (particularly Nedd4). For example, substitution of the amino acid alanine (A) for the first amino acid P in the PPAY sequence motif, substitution of the amino acid leucine (L) for the second amino acid P in the PPAY sequence motif, and/or substitution of the amino acid aspartate (D) for the last amino acid Y in the PPAY sequence motif can be introduced to affect the HBV core protein binding affinity to proteins containing a WW domain (particularly Nedd4). Additionally, it is contemplated that deletion of one, two, or three of the four amino acids in the PPAY sequence motif affects the binding affinity of an HBV core protein to proteins containing a WW domain (particularly Nedd4) and therefore its interaction with its host protein binding partner. Insertion of amino acids into the PPAY sequence motif may also be useful. Further, a combination of deletion, insertion and/or substitution mutations can also nullify the HBV late domain. Deletions, insertions and substitutions can be achieved by standard molecular biology techniques or created during standard chemical synthesis of the proteins, as will be apparent to skilled artisans.

The HBV late domain in a wild-type or modified HBV envelope polypeptide (i.e., presl/pres2/S envelope protein) can be nullified by any means so long as the resultant polypeptide is incapable of binding TsglOl, particularly the UEV domain of TsglOl . In one embodiment, the functional late domain can be nullified simply by deleting the entire P(T/S)AP sequence motif in a wild-type or modified HBV envelope polypeptide. Alternatively, amino acid substitution mutations in or near the P(T/S)AP sequence motif can be created so that the resultant HBV envelope polypeptide polypeptide is incapable of binding TsglOl. For example, as disclosed in commonly assigned U.S. Provisional Application Serial No. 60/276,259, substitution of the amino acid leucine (L) for the first amino acid P in the P(T/S)AP sequence motif, substitution of the amino acid arginine (R) for the third amino acid A in the P(T/S)AP sequence motif, and substitution of the amino acid leucine (L) for the last amino acid P in the P(T/S)AP sequence motif each individually abolishes the GAGp6 binding affinity to TsglOl. The same mutations in the PTAP motif of the HBV envelope polypeptide prevent HBV particles from properly budding. See Huang et al, J. Virol, 69:6810-6818 (1995). Additionally, deletion of one, two or three of the four amino acids in the P(T/S)AP sequence motif can also lead to the abolishment of the binding affinity of an HBV envelope polypeptide to TsglOl and cause altered budding (or egress) phenotypes in the host cells. Insertion of amino acids into the P(T/S)AP sequence motif may also be useful. Further, a combination of deletion, insertion and/or substitution mutations can also nullify the HBV late domain. Deletions, insertions and substitutions can be achieved by standard molecular biology techniques or created during standard chemical synthesis of the proteins, as will be apparent to skilled artisans. As is known in the art, distinct late domain motifs have been identified in the structural proteins of several other enveloped viruses. See Vogt, Proc. Natl Acad. Sci. USA, 97:12945-12947 (2000). The "YL" motif (YPDL or YxxL) was found in the GAG protein of equine infectious anemia virus (ELAN). Puffer et al, J. Virol, 71:6541-6546 (1997); Puffer et al, J. Virol, 72:10218-10221 (1998). The cellular receptor for the "YL" motif appears to be the AP-50 subunit of AP-2. Puffer et al, J. Virol, 72: 10218- 10221 (1998). Alternatively the YXXL may bind A1P1 (Vincent et al. Mol Cell. Bio. 23: 1647-1655 (2003)). Another late-domain motif was found in the HIV gag protein. The HIV gag protein late domain motif is PTAP or PSAP. Interestingly, the late domains such as the P(T/S)AP motif, PY motif and the YL motif can still function when moved to different positions within retro viral GAG proteins. Parent et al, J. Virol, 69:5455-5460 (1995); Yuan et al, EMBO J, 18:4700-4710 (2000). Moreover, the late domains such as the P(T/S)AP motif, PY motif and the YL motif can function interchangeably. That is, one late domain motif can be used in place of another late domain motif without affecting viral budding. Parent et al, J. Virol, 69:5455-5460 (1995); Yuan et al, EMBO J., 18:4700-4710 (2000); Srrack et al, Proc. Natl. Acad. Sci. USA, 97:13063-13068 (2000). Accordingly, in providing a mutant HBV protein that induces an altered budding phenotype in cells, it is important that no such PY motif, YL motif or new P(T/S)AP motif is created anywhere in the HBV protein. That is, the mutant HBV protein should be devoid of late-domain motifs. As used herein, the term "devoid of late-domain motifs" is intended to mean that the mutant HBV protein does not contain any late domain motifs, i.e., any amino acid sequence that, when placed in a mutant HBV protein that otherwise cannot drive the budding of viral particles in the host cell.

In another embodiment, a nucleic acid encoding an effective amount of a mutant HBV protein that affects an interaction involved in viral budding is administered to a patient for purposes of preventing and/or treating HBV infection and symptoms thereof. The mutant HBV protein encoded by the administered nucleic acid can be in any form as described above. In a preferred aspect of this embodiment, the mutant HBV protein is HBV core protein with a modified late-domain motif.

In addition to a nucleic acid encoding mutant HBV protein according to the present invention, another nucleic acid encoding one or more other HBV proteins, e.g., envelope proteins, non-structural proteins, and the like may also be administered to a patient. For example, a DΝA or RΝA molecule including an HBV genome devoid of the sequence encoding the PPAY motif in the core protein can be administered. A nucleic acid containing a portion of an HBN genome that includes the core protein-encoding sequence devoid of the sequence encoding the PPAY motif in the core protein will also be useful. For example, the nucleic acid can be a modified HBN genome devoid of the PPAY motif-encoding sequence. In another example, the nucleic acid is a modified HBN genome devoid of the HBV core protein PPAY domain-encoding sequence. The nucleic acid encoding the mutant HBV protein, particularly the core protein, according to the present invention and the nucleic acid(s) encoding other HBV protein(s) can be linked together and incorporated into the same expression vector. They can also be provided for by separate expression vectors. Thus, the present invention also provides a composition that includes one expression vector containing a nucleic acid encoding the mutant HBV protein according to the present invention and one or more other expression vectors caπying a nucleic acid encoding another protein, preferably another HBV protein or homologue or fragment thereof. Examples of HBV proteins include, but are not limited to, envelope proteins, non-structural proteins, polymerase, and the like.

In one embodiment, the nucleic acid of the present invention encoding a mutant HBV core protein that is sufficient for viral particle assembly and induces an altered budding phenotype in cells can be delivered into a patient by any suitable methods known in the art. For example, the nucleic acid can be delivered by various gene therapy methods known in the art. Successes in gene therapy have been reported recently. See e.g., Kay et al, Nature Genet., 24:257-61 (2000); Cavazzana-Calvo et al, Science, 288:669 (2000); and Blaese et al, Science, 270: 475 (1995); Kantoff, et al, J. Exp. Med., 166:219 (1987).

Any suitable gene therapy method may be used for purposes of the present invention. Generally, the exogenous nucleic acid of the present invention is incorporated into a suitable expression vector and is operably linked to a promoter in the vector such that the promoter can drive the transcription from the exogenous nucleic acid. Suitable promoters include but are not limited to viral transcription promoters derived from adenovirus, simian virus 40 (SV40) (e.g., the early and late promoters of SV40), Rous sarcoma virus (RSV), and cytomegalovirus (CMV) (e.g., CMN immediate-early promoter), human immunodeficiency virus (HIN) (e.g., long terminal repeat (LTR)), vaccinia virus (e.g., 7.5K promoter), and herpes simplex virus (HSN) (e.g., thymidine kinase promoter). Where tissue-specific expression of the exogenous gene is desirable, tissue-specific promoters may be operably linked to the exogenous gene. In this regard, a CD ⁺ T cell-specific promoter will be most desirable. In one embodiment, the exogenous nucleic acid is incorporated into a plasmid

DΝA vector. Many commercially available expression vectors may be useful for the present invention, including, e.g., pCEP4, pcDΝAI, pIΝD, pSecTag2, pNAXl, pcDΝA3.1, pBI-EGFP, pBlueScript, and pDisplay.

Various viral vectors may also be used. Typically, in a viral vector, the viral genome is engineered to eliminate the disease-causing capability, e.g., the ability to replicate in the host cells. The exogenous nucleic acid to be introduced into a patient may be incorporated into the engineered viral genome, e.g., by inserting it into a viral gene that is non-essential to the viral infectivity. Viral vectors are convenient to use as they can be easily introduced into tissue cells by way of infection. Once in the host cell, the recombinant virus typically is integrated into the genome of the host cell. In rare instances, the recombinant virus may also replicate and remain as extrachromosomal elements.

A large number of retroviral vectors have been developed for gene therapy. These include vectors derived from oncoretroviruses (e.g., MLV), lentiviruses (e.g., HIV and STV) and other refroviruses. For example, gene therapy vectors have been developed based on murine leukemia virus (See, Cepko, et al, Cell, 37:1053-1062 (1984), Cone and Mulligan, Proc. Natl. Acad. Sci. U.S.A., 81:6349-6353 (1984)), mouse mammary tumor virus (See, Salmons et al, Biochem. Biophys. Res. Commun.,l59:l l9l-l 198 (1984)), gibbon ape leukemia virus (See, Miller et al, J. Virology, 65:2220-2224 (1991)), HIN, (See Shimada et al, J. Clin. Invest., 88:1043-1047 (1991)), and avian refroviruses (See Cosset et al, J. Virology, 64: 1070-1078 (1990)). In addition, various retroviral vectors are also described in U.S. Patent Νos. 6,168,916; 6,140,111; 6,096,534; 5,985,655; 5,911,983; 4,980,286; and 4,868,116, all of which are incorporated herein by reference.

Adeno-associated virus (AAN) vectors have been successfully tested in clinical trials. See e.g., Kay et al, Nature Genet. 24:257-61 (2000). NAN is a naturally occurring defective virus that requires other viruses such as adenoviruses or herpes viruses as helper viruses. See Muzyczka, Curr. Top. Microbiol Immun., 158:97 (1992). A recombinant AAN virus useful as a gene therapy vector is disclosed in U.S. Patent No. 6,153,436, which is incorporated herein by reference. Adenoviral vectors can also be useful for purposes of delivering the exogenous nucleic acid in accordance with the present invention. For example, U.S. Patent No. 6,001,816 discloses an adenoviral vector, which is used to deliver a leptin gene intravenously to a mammal to treat obesity. Other recombinant adenoviral vectors may also be used, which include those disclosed in U.S. Patent Nos. 6,171,855; 6,140,087; 6,063,622; 6,033,908; and 5,932,210, and Rosenfeld et al, Science, 252:431-434 (1991); and Rosenfeld et al, Cell, 68:143-155 (1992).

Other useful viral vectors include recombinant hepatitis viral vectors (See, e.g., U.S. Patent No. 5,981,274), and recombinant entomopox vectors (See, e.g., U.S. Patent Nos. 5,721,352 and 5,753,258). In addition, alphavirus vectors may also be desirable. Alphaviruses are positive stranded RNA viruses. The sequence encoding replica structural proteins can be replaced by the exogenous nucleic acid. The alphavirus vectors carrying the exogenous nucleic acid can be targeted to dendritic cells to facilitate the stimulation of cytotoxic T lymphocytes. See Polo et al, Proc. Natl. Acad. Sci. USA, 96:4598-4603 (1996). In addition to viral vectors, bacterial vectors can also be used. That is, the exogenous nucleic acid to be delivered can be incorporated into attenuated bacteria such as attenuated salmonella and shigella. See Levine et al, J. Biotechnol, 44:193-196 (1996); Tacket et al, Infect. Immun., 65:3381-3385 (1997); and Sizemore et al, Vaccine, 15:804-807 (1997). The exogenous nucleic acid itself or a plasmid or viral or other expression vector caπying the exogenous nucleic acid can be introduced into a patient by various methods known in the art. For example, as will be apparent to skilled artisans, the exogenous nucleic acid incorporated into a viral or bacterial vector can be administered to patients by direct infection. The nucleic acid carried by plasmid vectors can be used as DNA or RNA vaccines, i.e., in the form of naked DNA or RNA, that are administered directly into an appropriate tissue or organ of a patient. The naked DNA or RNA can be delivered by injection into skin, muscle or other tissues. Alternatively, a gene gun or an equivalent device thereof can be used for delivery into skin or mucous membrane or other tissues. Some success has been achieved using such genetic vaccines in the art. The use of this approach for purposes of the present invention will be apparent to a skilled artisan apprised of the present disclosure. See Ulmer et al, Science, 259:1745 (1993); Weiner & Kennedy, Scientific American, July, 1999; Barouch et al, Science, 290:486-492 (2000), all of which are incorporated herein by reference.

Alternatively, catheters or like devices may be used for delivery into a target organ or tissue. Suitable catheters are disclosed in, e.g., U.S. Patent Nos. 4,186,745;

5,397,307; 5,547,472; 5,674,192; and 6,129,705, all of which are incorporated herein by reference.

Other non-traditional vectors may also be used for purposes of this invention. For example, International Publication No. WO 94/18834 discloses a method of delivering DNA into mammalian cells by conjugating the DNA to be delivered with a polyelectrolyte to form a complex. The complex may be microinjected into or taken up by cells.

The exogenous nucleic acid or plasmid DNA vector containing the exogenous gene may also be introduced into cells by way of receptor-mediated endocytosis. See e.g, U.S. Patent No. 6,090,619; Wu and Wu, J. Biol. Chem., 263:14621 (1988); Curiel et al, Proc. Natl. Acad. Sci. USA, 88:8850 (1991). For example, U.S. Patent No. 6,083,741 discloses introducing an exogenous nucleic acid into mammalian cells by associating the nucleic acid to a polycation moiety (e.g., poly-L-lysine, having 3-100 lysine residues), which is itself coupled to an integrin receptor binding moiety (e.g., a cyclic peptide having the amino acid sequence RGD).

Alternatively, the exogenous nucleic acid or vectors containing it can also be delivered into cells via amphiphiles. See e.g., U.S. Patent No. 6,071,890. Typically, the exogenous nucleic acid or a vector containing the nucleic acid forms a complex with the cationic amphiphile. Cells in a patient's body contacted with the complex can readily absorb the complex. To deliver the mutant HBN protein or other peptide compounds into cells in a patient's body, the mutant HBN protein or peptide compounds are preferably associated with a "transporter" capable of increasing the uptake of the molecules by human cells. As used herein, the term "transporter" refers to an entity (e.g., a compound or a composition or a physical structure formed from multiple copies of a compound or multiple different compounds) that is capable of facilitating the uptake of the mutant HBN protein of the present invention by human cells. Typically, the cell uptake of the mutant HBV protein of the present invention in the presence of a "transporter" is at least 20% higher, preferably at least 40%, 50%, 75%, and more preferably at least 100% higher than the cell uptake of the polypeptide in the absence of the "transporter."

Many molecules and structures known in the art can be used as "transporter." In one embodiment, a penefratin is used as a transporter. For example, the homeodomain of Antennapedia, a Drosophila transcription factor, can be used as a transporter to deliver a polypeptide of the present invention. Indeed, any suitable member of the penefratin class of peptides can be used to cany a polypeptide of the present invention into cells.

Penetratins are disclosed in, e.g., Derossi et al, Trends Cell Biol, 8:84-87 (1998), which is incorporated herein by reference. Penetratins transport molecules attached thereto across cytoplasm membranes or nucleus membranes efficiently in a receptor- independent, energy-independent, and cell type-independent manner. Methods for using a penefratin as a carrier to deliver oligonucleotides and polypeptides are also disclosed in U.S. Patent No. 6,080,724; Pooga et al, Nat. Biotech., 16:857 (1998); and Schutze et al, J. Immunol, 157:650 (1996), all of which are incorporated herein by reference. U.S. Patent No. 6,080,724 defines the minimal requirements for a penefratin peptide as a peptide of 16 amino acids with 6 to 10 of which being hydrophobic. The amino acid at position 6 counting from either the N- or C-terminal is tryptophan, while the amino acids at positions 3 and 5 counting from either the N- or C-terminal are not both valine. Preferably, the helix 3 of the homeodomain of Drosophila Antennapedia is used as a transporter. More preferably, a peptide having a sequence of the amino acids 43-58 of the homeodomain Antp is employed as a transporter. In addition, other naturally occurring homologs of the helix 3 of the homeodomain of Drosophila Antennapedia can also be used. For example, homeodomains of Fushi-tarazu and Engrailed have been shown to be capable of transporting peptides into cells. See Han et al, Mol Cells, 10:728-32 (2000). As used herein, the term "penefratin" also encompasses peptoid analogs of the penefratin peptides. Typically, the penefratin peptides and peptoid analogs thereof are covalently linked to a compound to be delivered into cells thus increasing the cellular uptake of the compound.

3. Formation of Immunogenic Cells In Vitro

In accordance with another aspect of the present invention, a method for treating and/or preventing HBV infection and symptoms thereof is provided comprising administering to a patient in need of treatment an effective amount of cells having a viral- host protein-protein interaction involved in viral budding disrupted (i.e., cells displaying an altered budding phenotype). That is, cells with a HBV protein-host protein interaction disrupted are prepared in vitro and delivered to a patient in need of treatment. Furthermore, this aspect of the invention includes the use of antigen preparations derived from infected cells with an altered budding phenotype.

The administration of such cells can lead to an induction or enhancement of an immune response against HBV. Although cells from a variety of sources may be employed to prepare cells displaying HBV altered budding phenotype, mammalian cells including human cells and others may be preferable. In a prefeπed embodiment, the cells are of the same tissue type as those which the virus normally infects. As will be apparent to skilled artisans, the methods described above for modulating, in the body, a protein- protein interaction involved in viral budding can be used in in vitro procedures to create cells with a disruption in the protein-protein interaction. For example, a mutant HBV core protein sufficient for viral particle assembly but devoid of the PPAY motif in the HBV core protein domain containing the motif or devoid of the entire domain may be introduced into cells in vitro to initiate the assembly of virus-like particle and form cells displaying an HBV disrupted budding phenotype. Alternatively, a nucleic acid encoding a mutant HBV core protein sufficient for virus-like particle assembly but devoid of the PPAY motif in the HBV core protein domain containing the motif or devoid of the entire domain is introduced into cells in vitro to express the mutant HBV core protein in the cells.

In another embodiment, a wild-type or mutant HBV core protein sufficient for viral particle assembly or a nucleic acid encoding such a polypeptide can be administered to cells defective in respect to one or more proteins required for HBV budding. For example, human cells with the gene of a protein containing a WW domain (i.e., Nedd4) being knocked out may be used for this purpose. Methods for creating WW domain- deficient (i.e., Nedd4 WW domain) cells are apparent to skilled artisans.

A nucleic acid encoding a wild-type or mutant HBV core protein or vectors containing the nucleic acid can be introduced into cells in vitro using any known techniques such as calcium phosphate precipitation, microinjection, lipofection, electroporation, gene gun, receptor-mediated endocytosis, and the like. The wild-type or mutant HBV core protein can be introduced into cells in vitro by attaching the polypeptide to a transporter as described above capable of increasing the cell uptake of the polypeptide. The cells displaying or capable of displaying HBV late-domain phenotype can be administered to a patient by, e.g., injection or cell transplantation. The appropriate amount of cells delivered to a patient will vary with patient conditions, and desired effect, which can be determined by a skilled artisan. See e.g., U.S. Patent Nos. 6,054,288; 6,048,524; and 6,048,729.

4. Compounds and Methods for Identifying Compounds that Disrupt Viral Budding

Any compounds capable of interfering with the protein-protein interaction between host cell proteins containing a WW domain (particularly Nedd4 or a Nedd4 like protein) and HBV core protein may be used. This means useful compounds include those that interfere with, block, disrupt or destabilize the protein-protein interaction; block or interfere with the formation of a protein complex by proteins containing a WW domain (particularly Nedd4) and HBV core protein or fragment thereof; or destabilize, disrupt or dissociate an existing protein complex comprising proteins containing a WW domain (particularly Nedd4) and HBV core protein or fragment thereof. Such compounds can be selected by various screening assays known in the art. For example, test compounds may be screened in an in vitro assay to select interaction antagonists. A complex containing a Nedd4-HBV core protein complex can be contacted with a test compound and disruption or destabilization of the protein complex can be detected. For example, the presence or absence of the protein complex can be detected by an antibody selectively immunoreactive with the protein complex. Thus, after incubation of the protein complex with a test compound, an immunoprecipitation assay can be conducted with the antibody. If the test compound disrupts the protein complex, then the amount of immunoprecipitated protein complex in this assay will be significantly less than that in a confrol assay in which the same protein complex is not contacted with the test compound. Various other detection methods may be suitable in the dissociation assay, as will be apparent to skilled artisan apprised of the present disclosure. In one embodiment, one of the interacting partners with a detectable marker fused thereto is fixed to a solid support. For example, a GST-HBV core protein fusion protein is attached to a solid support. Then the other interacting partner with a detectable marker fused thereto (e.g., a myc-tagged protein containing a WW domain fragment, e.g., Nedd4) is contacted with the immobilized first interacting partner in the presence of one or more test compounds. If binding between the two interacting partners occurs, the myc-tagged protein containing a WW domain (particularly Nedd4) fragment is also immobilized, which can be detected using an anti-myc antibody after the binding reaction mixture is washed to remove unbound myc-tagged proteins containing a WW domain (particularly Nedd4) fragment.

Alternatively, test compounds can also be screened in any in vivo assays to select compounds capable of interfering with the interaction between proteins containing a WW domain (particularly Nedd4) and HBV core protein. Any in vivo assays known in the art useful in selecting compounds capable of interfering with the stability of the protein complexes of the present invention may be used.

In a preferred embodiment, one of the yeast two-hybrid systems or their analogous or derivative forms is used. Examples of suitable two-hybrid systems known in the art include, but are not limited to, those disclosed in U.S. Patent Nos. 5,283,173; 5,525,490; 5,585,245; 5,637,463; 5,695,941; 5,733,726; 5,776,689; 5,885,779; 5,905,025; 6,037,136; 6,057,101; 6,114,111; and Bartel and Fields, eds., Tlie Yeαst Two-Hybrid System, Oxford University Press, New York, NY, 1997, all of which are incorporated herein by reference.

Typically, in a classic transcription-based two-hybrid assay, two chimeric genes are prepared encoding two fusion proteins: one contains a transcription activation domain fused to an interacting protein member of a protein complex of the present invention or an interacting domain of the interacting protein member, while the other fusion protein includes a DNA binding domain fused to another interacting protein member of the protein complex or an interacting domain thereof.

In a screening assay for dissociators, a protein containing a WW domain (particularly Nedd4 or a Nedd4 like protein)), a mutant form or a binding domain thereof, and HBV core protein, or a mutant form or a binding domain thereof, are used as test proteins expressed in the form of fusion proteins as described above for purposes of a two-hybrid assay. The fusion proteins are expressed in a host cell and allowed to interact with each other in the presence of one or more test compounds. In a preferred embodiment, a counterselectable marker is used as a reporter such that a detectable signal (e.g., appearance of color or fluorescence, or cell survival) is present only when the test compound is capable of interfering with the interaction between the two test proteins. In this respect, the reporters used in various "reverse two- hybrid systems" known in the art may be employed. Reverse two-hybrid systems are disclosed in, e.g., U.S. Patent Nos. 5,525,490; 5,733,726; 5,885,779; Vidal et al, Proc. Natl. Acad. Sci. USA, 93:10315-10320 (1996); and Vidal et al, Proc. Natl. Acad. Sci. USA, 93:10321-10326 (1996), all of which are incorporated herein by reference.

Examples of suitable counterselectable reporters useful in a yeast system include the URA3 gene (encoding orotidine-5'-decarboxylase, which converts 5-fluroorotic acid (5-FOA) to the toxic metabolite 5-fluorouracil), the CAN1 gene (encoding arginine permease, which transports toxic arginine analog canavanine into yeast cells), the GAL1 gene (encoding galactokinase, which catalyzes the conversion of 2-deoxygalactose to toxic 2-deoxygalactose- 1 -phosphate), the LYS2 gene (encoding α-aminoadipate reductase, which renders yeast cells unable to grow on a medium containing - aminoadipate as the sole nitrogen source), the MET15 gene (encoding O- acetylhomoserine sulfhydrylase, which confers on yeast cells sensitivity to methyl mercury), and the CYH2 gene (encoding L29 ribosomal protein, which confers sensitivity to cycloheximide). In addition, any known cytotoxic agents including cytotoxic proteins such as the diphtheria toxin (DTA) catalytic domain can also be used as counterselectable reporters. See U.S. Patent No. 5,733,726. DTA causes the ADP-ribosylation of elongation factor-2 and thus inhibits protein synthesis and causes cell death. Other examples of cytotoxic agents include ricin, Shiga toxin, and exotoxin A of Pseudomonas aeruginosa.

For example, when the URA3 gene is used as a counterselectable reporter gene, yeast cells containing a mutant URA3 gene can be used as host cells (Ura^" Foa^R phenotype) for the in vivo assay. Such cells lack f/i 43-encoded functional orotidine-5 '- phosphate decarboxylase, an enzyme required for the biosynthesis of uracil. As a result, the cells are unable to grow on media lacking uracil. However, because of the absence of a wild-type orotidine-5' -phosphate decarboxylase, the yeast cells cannot convert non- toxic 5-fluoroorotic acid (5-FOA) to a toxic product, 5-fluorouracil. Thus, such yeast cells are resistant to 5-FOA and can grow on a medium containing 5-FOA. Therefore, for example, to screen for a compound capable of disrupting interaction between a protein containing a WW domain (particularly Nedd4) and HBV core protein, a protein containing a WW domain (particularly Nedd4) can be expressed as a fusion protein with a DNA-binding domain of a suitable transcription activator while HBV core protein is expressed as a fusion protein with a transcription activation domain of a suitable transcription activator. In the host strain, the reporter URA3 gene may be operably linked to a promoter specifically responsive to the association of the transcription activation domain and the DNA-binding domain. After the fusion proteins are expressed in the Ura^" Foa^R yeast cells, an in vivo screening assay can be conducted in the presence of a test compound with the yeast cells being cultured on a medium containing uracil and 5-FOA. If the test compound does not disrupt the interaction between a protein containing a WW domain (particularly Nedd4) and HBV core protein, active URA3 gene product, i.e., orotidine-5 '-decarboxylase, which converts 5-FOA to toxic 5-fluorouracil, is expressed. As a result, the yeast cells cannot grow. On the other hand, when the test compound disrupts the interaction between a protein containing a WW domain (particularly Nedd4) and HBV core protein, no active orotidine-5 '-decarboxylase is produced in the host yeast cells. Consequently, the yeast cells will survive and grow on the 5-FOA-containing medium. Therefore, compounds capable of interfering with or dissociating the interaction between a protein containing a WW domain (particularly Nedd4) and HBV core protein can thus be identified based on colony formation.

As will be apparent, the screening assay of the present invention can be applied in a format appropriate for large-scale screening. For example, combinatorial technologies can be employed to construct combinatorial libraries of small organic molecules or small peptides. See generally, e.g., Kenan et al, Trends Biochem. Sc, 19:57-64 (1994); Gallop et al, J. Med. Chem., 37:1233-1251 (1994); Gordon et al, J. Med. Chem., 37:1385-1401 (1994); Ecker et al, Biotechnology, 13:351-360 (1995). Such combinatorial libraries of compounds can be applied to the screening assay of the present invention to isolate specific modulators of particular protein-protein interactions. In the case of random peptide libraries, the random peptides can be co-expressed with the fusion proteins of the present invention in host cells and assayed in vivo. See e.g., Yang et al, Nucl. Acids Res., 23 : 1152-1156 (1995). Alternatively, they can be added to the culture medium for uptake by the host cells.

Conveniently, yeast mating is used in an in vivo screening assay. For example, haploid cells of a-mating type expressing one fusion protein as described above is mated with haploid cells of alpha-mating type expressing the other fusion protein. Upon mating, the diploid cells are spread on a suitable medium to form a lawn. Drops of test compounds can be deposited onto different areas of the lawn. After culturing the lawn for an appropriate period of time, drops containing a compound capable of modulating the interaction between the particular test proteins in the fusion proteins can be identified by stimulation or inhibition of growth in the vicinity of the drops.

The screening assays for selecting compounds capable of interfering with protein- protein interactions can also be fine-tuned by various techniques to adjust the thresholds or sensitivity of the positive and negative selections. Mutations can be introduced into the reporter proteins to adjust their activities. The uptake of test compounds by the host cells can also be adjusted. For example, yeast high uptake mutants such as the ergo mutant strains can facilitate yeast uptake of the test compounds. See Gaber et al, Mol Cell. Biol, 9:3447-3456 (1989). Likewise, the uptake of the selection compounds such as 5-FOA, 2-deoxygalactose, cycloheximide, α-aminoadipate, and the like can also be fine-tuned. Any test compounds may be screened in the screening assays of the present invention to select compounds capable of interfering with the interaction between a protein containing a WW domain (particularly Nedd4) and HBV core protein. The test compounds may include, by way of example, proteins (e.g., antibodies, small peptides, artificial or natural proteins), nucleic acids, and derivatives, mimetics and analogs thereof, and small organic molecules having a molecular weight of no greater than 10,000 daltons, more preferably less than 5,000 daltons. Preferably, the test compounds are provided in library formats known in the art, e.g., in chemically synthesized libraries, recombinant expression libraries (e.g., phage display libraries), and in vitro translation- based libraries (e.g., ribosome display libraries). In prefeπed embodiments, the compound capable of interfering with the interaction between a protein containing a WW domain (particularly Nedd4) and HBV core protein used in the methods of the present invention comprises an amino acid that is a variant of the sequence motif of PPXY and capable of binding a protein containing a WW domain, wherein X is any amino acid. Preferably, a variant of the sequence motif of PPXY is where both prolines and the tyrosine are mutated to different amino acids. The compound which comprises the variant of the amino acid sequence motif PPXY and is capable of binding a protein containing a WW domain (particularly Nedd4) can be of any type of chemical compounds so long as the compound is capable of binding a protein containing a WW domain. For example, the compound can be a peptide, a modified peptide, an oligonucleotide-peptide hybrid (e.g., PNA), etc.

In one embodiment, in the compound comprising a variant of the amino acid sequence motif PPAY capable of binding a protein containing a WW domain (particularly Nedd4), is selected from the group consisting of APAY, PPAA, and PAAY. In another embodiment, the compound comprises an amino acid sequence selected from the group PDAY, DP AY, PPAD. In yet another embodiment, the compound administered comprises the amino acid sequence selected form the group XPAY, PXAY, and PPAZ, wherein X is not proline and Z is not tyrosine, tryptophan, phenylalanine or histidine.

The compound can also include a longer peptide comprising a variant of the amino acid sequence motif PPXY and is capable of binding a protein containing a WW domain (particularly Nedd4). For example, the compound may include a peptide of 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15 or more amino acids. In a prefeπed embodiment, the compound includes a peptide that contains a contiguous span of at least 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15 or more amino acids of a naturally occurring HBV core protein sequence. However, generally speaking, the contiguous span has less than 50, preferably less than 40, more preferably less than 30 amino acids. The contiguous span should span the HBV late domain motif which can be the PPXY motif or a variation thereof. Preferably, the late domain motif in the contiguous span is the PPAY motif. In another embodiment, the PPXY motif in the compound according to the present invention is within an amino acid sequence that is at least 70 percent, preferably at least 80 percent or 85 percent, more preferably at least 90 percent or 95 percent identical to a contiguous span of at least 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15 or more amino acids, but preferably less than 50 or 40, more preferably less than 30 amino acids, of a naturally occurring HBV core protein sequence, which contiguous span of amino acids spans the HBV late domain motif. In this respect, the percentage identity is determined by the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-77 (1993), which is incorporated into the various BLAST programs. Specifically, the percentage identity is determined by the "BLAST 2 Sequences" tool, which is available at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. See Tatusova and Madden, FEMS Microbiol Lett., 174(2):247-50 (1999). For pairwise protein-protein sequence comparison, the BLASTP 2.1.2 program is employed using default parameters (Matrix: BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 15; expect: 10.0; and wordsize: 3, with filter). It should be understood that such homologue peptides should retain the ability to bind a protein containing a WW domain (particularly Nedd4). The homologues can be made by site-directed mutagenesis based on a late- domain motif-containing HBV core protein sequence of HBV. The site-directed mutagenesis can be designed to generate amino acid substitutions, insertions, or deletions. Methods for conducting such mutagenesis should be apparent to skilled artisans in the field of molecular biology. The resultant homologues can be tested for their binding affinity to a protein containing a WW domain (particularly Nedd4).

The peptide portion in the compounds according to the present invention can also be in a modified form. Various modifications may be made to improve the stability and solubility of the compound, and/or optimize its binding affinity to a protein containing a WW domain. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, prenylated forms, etc. Modifications also include intramolecular crosslinking and covalent attachment to various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, etc. hi addition, modifications may also include cyclization, and branching. Amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide sequence in the compound of the present invention. For example, the compounds may include D-amino acids in place of L-amino acids.

To increase the stability of the peptidic compounds according to the present invention, various protection groups can also be incorporated into the amino acid residues of the compounds. In particular, terminal residues are preferably protected. Carboxyl groups may be protected by esters (e.g., methyl, ethyl, benzyl, p-nifrobenzyl, t-butyl or t- amyl esters, etc.), lower alkoxyl groups (e.g., methoxy, ethoxy, propoxy, butoxy, etc.), aralkyloxy groups (e.g., benzyloxy, etc.), amino groups, lower alkylamino or di(lower alkyl)amino groups. The term "lower alkoxy" is intended to mean an alkoxy group having a straight, branched or cyclic hydrocarbon moiety of up to six carbon atoms. Protection groups for amino groups may include lower alkyl, benzyloxycarbonyl, t- butoxycarbonyl, and sobornyloxycarbonyl. "Lower alkoxy" is intended to mean an alkyl group having a straight, branched or cyclic hydrocarbon moiety of up to six carbon atoms. In one example, a 5-oxo-L-prolyl residue may be used in place of a prolyl residue. A 5-oxo-L-prolyl residue is especially desirable at the N-terminus of a peptide compound. In another example, when a proline residue is at the C-terminus of a peptide compound, a N-ethyl-L-prolinamide residue may be desirable in place of the proline residue. Various other protecting groups known in the art useful in increasing the stability of peptide compounds can also be employed. Additionally, as will be apparent to skilled artisans apprised of the present disclosure, peptide mimetics can be designed based on the above-described variant PPXY motif-containing compounds according to the present invention. However, it is noted that the mimetics must be capable of binding a protein containing a WW domain (particularly Nedd4). For example, peptoid analogs of the PPAY motif can be prepared using known methods. Peptoids are oligomeric N-substituted glycines. Typically, various side chain groups can be included when forming an N-substituted glycine (peptoid monomer) that mimics a particular amino acid. Peptoid monomers can be linked together to form an oligomeric N-substituted glycines - peptoid. Peptoids are easy to synthesize in large amounts. In contrast to peptides, the backbone linkage of peptoids are resistant to hydrolytic enzymes. In addition, since a variety of functional groups can be presented as side chains off of the oligomeric backbone, peptoid analogs corresponding to any peptides can be produced with improved characterics. See Simon et al, Proc. Natl. Acad. Sci. USA, 89:9367-9371 (1992); Figliozzi et al, Methods Enzymol., 267:437-447 (1996); Horwell, Trends Biotechnol., 13:132-134 (1995); and Horwell, Drug Des. Discov., 12:63-75 (1994), all of which are incorporated herein by reference.

Thus, peptoid analogs of the above-described variant PPXY motif-containing compounds of the present invention can be made using methods known in the art. The prepared peptoid analogs can be tested for their binding affinity to a protein containing a WW domain (particularly Nedd4). They can also be tested in anti-viral assays for their ability to inhibit virus budding from infected host cells and ability to inhibit virus propagation. In particular, they can be tested for ability to suppress HBV budding from infected cells and inhibit HBV propagation.

Mimetics of the compounds of the present invention can also be selected by rational drug design and/or virtual screening. Methods known in the art for rational drug design can be used in the present invention. See, e.g., Hodgson et al, Bio/Technology, 9:19-21 (1991); U.S. Patent Nos. 5,800,998 and 5,891,628, all of which are incorporated herein by reference. Structural information on a protein containing a WW domain (particularly Nedd4) and/or the binding complex formed by a protein containing a WW domain and the HBV core protein PPAY motif are obtained. The interacting complex can be studied using various biophysics techniques including, e.g. , X-ray crystallography, NMR, computer modeling, mass spectrometry, and the like. Likewise, structural information can also be obtained from protein complexes formed by a protein containing a WW domain (particularly Nedd4) and a variation of the PPAY motif.

Computer programs are employed to select compounds based on structural models of the binding complex formed by a protein containing a WW domain

(particularly Nedd4) and the HBV core protein containing the PPAY motif. In addition, once an effective compound is identified, structural analogs or mimetics thereof can be produced based on rational drug design with the aim of improving drug efficacy and stability, and reducing side effects. In addition, understanding of the interaction between a protein containing a WW domain and compounds of the present invention can also be derived from mutagenesis analysis using yeast two-hybrid system or other methods for detection protein-protein interaction. In this respect, various mutations can be introduced into the interacting proteins and the effect of the mutations on protein-protein interaction is examined by a suitable method such as in vitro binding assay or the yeast two-hybrid system.

Various mutations including amino acid substitutions, deletions and insertions can be infroduced into the protein sequence of a protein containing a WW domain (particularly Nedd4) and/or a compound of the present invention using conventional recombinant DNA technologies. Generally, it is particularly desirable to decipher the protein binding sites. Thus, it is important that the mutations introduced only affect protein-protein interaction and cause minimal structural disturbances. Mutations are preferably designed based on knowledge of the three-dimensional structure of the interacting proteins. Preferably, mutations are infroduced to alter charged amino acids or hydrophobic amino acids exposed on the surface of the proteins, since ionic interactions and hydrophobic interactions are often involved in protein-protein interactions. Alternatively, the "alanine scanning mutagenesis" technique is used. See Wells, et al, Methods Enzymol, 202:301-306 (1991); Bass et al, Proc. Natl. Acad. Sci. USA, 88:4498- 4502 (1991); Bennet et al, J. Biol. Chem., 266:5191-5201 (1991); Diamond et al, J. Virol, 68:863-876 (1994). Using this technique, charged or hydrophobic amino acid residues of the interacting proteins are replaced by alanine, and the effect on the interaction between the proteins is analyzed using e.g., an in vitro binding assay. In this manner, the domains or residues of the proteins important to compound-target interaction can be identified.

Based on the structural information obtained, structural relationships between a protein containing a WW domain (particularly Nedd4) and a compound of the present invention are elucidated. The moieties and the three-dimensional structures critical to the interaction are revealed. Medicinal chemists can then design analog compounds having similar moieties and structures.

The residues or domains critical to the modulating effect of the identified compound constitute the active region of the compound known as its "pharmacophore."

Once the pharmacophore has been elucidated, a structural model can be established by a modeling process that may incorporate data from NMR analysis, X-ray diffraction data, alanine scanning, spectroscopic techniques and the like. Various techniques including computational analysis, similarity mapping and the like can all be used in this modeling process. See e.g., Peπy et al, in OSAR: Quantitative Structure-Activity Relationships in Drug Design, pp.189-193, Alan R. Liss, Inc., 1989; Rotivinen et al, Acta Pharmaceutical Fennica, 97:159-166 (1988); Lewis et al, Proc. R. Soc. Lond., 236:125- 140 (1989); McKinaly et al, Annu. Rev. Pharmacol. ToxicioL, 29:111-122 (1989). Commercial molecular modeling systems available from Polygen Corporation, Waltham, MA, include the CHARMm program, which performs the energy minimization and molecular dynamics functions, and QUANTA program which performs the construction, graphic modeling and analysis of molecular structure. Such programs allow interactive construction, visualization and modification of molecules. Other computer modeling programs are also available from BioDesign, Inc. (Pasadena, CA.), Hypercube, Inc. (Cambridge, Ontario), and Allelix, Inc. (Mississauga, Ontario, Canada). A template can be formed based on the established model. Various compounds can then be designed by linking various chemical groups or moieties to the template. Various moieties of the template can also be replaced. These rationally designed compounds are further tested. In this manner, pharmacologically acceptable and stable compounds with improved efficacy and reduced side effect can be developed. The compounds identified in accordance with the present invention can be incorporated into a pharmaceutical formulation suitable for administration to an individual.

The mimetics including peptoid analogs can exhibit optimal binding affinity to a protein containing a WW domain (particularly Nedd4) or animal orthologs thereof. Various known methods can be utilized to test WW domain-binding characteristics of a mimetics. For example, an entire a protein containing a WW domain (e.g., Nedd4) or a fragment thereof containing the WW domain may be recombinantly expressed, purified, and contacted with the mimetics to be tested. Binding can be determined using a surface plasmon resonance biosensor. See e.g., Panayotou et al, Mol Cell. Biol, 13:3567-3576 (1993). Other methods known in the art for estimating and determining binding constants in protein-protein interactions can also be employed. See Phizicky and Fields, et al, Microbiol Rev., 59:94-123 (1995). For example, protein affinity chromatography may be used. First, columns are prepared with different concentrations of an interacting member, which is covalently bound to the columns. Then a preparation of its interacting partner is run through the column and washed with buffer. The interacting partner bound to the interacting member linked to the column is then eluted. The binding constant is then estimated based on the concentrations of the bound protein and the eluted protein. Alternatively, the method of sedimentation through gradients monitors the rate of sedimentation of a mixture of proteins through gradients of glycerol or sucrose. At concentrations above the binding constant, the two interacting members sediment as a complex. Thus, a binding constant can be calculated based on the concentrations. Other suitable methods known in the art for estimating binding constant include, but are not limited to, gel filtration columns such as nonequilibrium "small-zone" gel filtration columns (See e.g., Gill et al, J. Mol. Biol, 220:307-324 (1991)), the Hummel-Dreyer method of equilibrium gel filtration (See e.g., Hummel and Dreyer, Biochim. Biophys. Acta, 63:530-532 (1962)) and large-zone equilibrium gel filtration (See e.g., Gilbert and Kellett, J. Biol. Chem., 246:6079-6086 (1971)), sedimentation equilibrium (See e.g., Rivas and Minton, Trends Biochem., 18:284-287 (1993)), fluorescence methods such as fluorescence spectrum (See e.g., Otto-Bruc et al, Biochemistry, 32:8632-8645 (1993)) and fluorescence polarization or anisofropy with tagged molecules (See e.g., Weiel and Hershey, Biochemistry, 20:5859-5865 (1981)), and solution equilibrium measured with immobilized binding protein (See e.g., Nelson and Long, Biochemistry, 30:2384-2390 (1991)).

The compounds capable of interfering with the interaction between a protein containing a WW domain (e.g., Nedd4) and HBV core protein can be delivered into cells by direct cell internalization, receptor mediated endocytosis, or via a "transporter." It is noted that a compound preferably is delivered into patient's cells in order to achieve optimal results. Preferably, peptidic transporters as described above such as penetratins and HIV tat protein are employed. Fusion proteins can be conveniently made by recombinant expression to contain a transporter peptide covalently linked by a peptide bond to a peptide having the variant of the PPXY motif. Alternatively, conventional methods can be used to chemically synthesize a transporter peptide or a peptide of the present, invention or both.

Additionally, the screening methods of the invention include the use of other late- domain motif containing proteins of HBV such as that in the presl/pres2/Sl region which is likely to have a host cell binding partner which is TSGIOI or a homologue thereof.

5. Adjuvants

Although not required, it is prefeπed that an adjuvant capable of stimulating immune response is also administered to a patient who is treated with cells displaying HBV altered budding phenotype or with a compound (nucleic acids, polypeptides or small organic compounds) capable of causing the formation, in a patient's body, of cells displaying HBV altered budding phenotype. As used herein, the term "adjuvant" means any substance that is not a component of HBV but is capable of stimulating or enhancing immune responses to an immunogen administered to a patient. The disruption or interference with the protein-protein interaction between the host cellular protein containing a WW domain (particularly Nedd4) and HBV core protein is contemplated in itself be effective in treating and/or preventing HBV infection and symptoms thereof by way of inhibiting HBV viral budding and propagation. However, the disruption or interference with the protein-protein interaction between the host cellular protein containing a WW domain (particularly Nedd4) and HBV core protein may also induce the formation of cells displaying HBV altered budding phenotype which can act as immunogens in eliciting immune responses in the patient. Therefore, the compounds administered to a patient capable of disrupting or interfering with the protein-protein interaction between a protein containing a WW domain

(particularly Nedd4) and HBV core protein can also stimulate immune responses in the patient against HBV viruses, particularly cytotoxic T lymphocytes (CTL) response. In this respect, the administration of an adjuvant capable of enhancing the patient's immune response, particularly cytotoxic T lymphocytes (CTL) response, along with the administration to the patient of a compound capable of disrupting or interfering with the protein-protein interaction between a protein containing a WW domain (particularly Nedd4) and HBV core protein will significantly bolster the anti-HBV immune response in the patient and result in a treatment efficacy significantly greater than the administration of the compound alone. It is believed that the methods of the invention are capable of stimulating and/or enhancing humoral immune responses. The invention includes the use of adjuvant for enhancing and stimulating humoral immune responses.

Similarly, an adjuvant capable of enhancing patient immune response, particularly CTL response, would also significantly boost the prophylactic and/or therapeutic effect when coupled with a mutant HBV core protein that is sufficient for viral particle assembly and causes an altered budding phenotype in cells, or a nucleic acid encoding the mutant HBV core protein, or with cells displaying HBV altered budding phenotype.

Any adjuvant may be used so long as the adjuvant is capable of stimulating immune response against cells displaying HBV altered budding phenotype or against the HBV viral proteins in such cells, and thus can enhance immune response against HBV viruses in a patient. For example, alum has long been used as an adjuvant for human use. Another example of suitable adjuvant is MF59. See Minutello et al, Vaccine, 17:99-104 (1999). Another useful adjuvant in development that may also be used is LTR72, a mutant of E. coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity. See Giuliani et al, J. Exp. Med., 187:1123-1132 (1998). Polyphosphazine adjuvant may also be used. In addition, cytokines and lymphokines may also be used as adjuvants along with the cells, polypeptides, nucleic acids and/or a compound capable of disrupting or interfering with the protein-protein interaction between a protein containing a WW domain (particularly Nedd4) and HBV core protein. Examples of cytokines and lymphokines include, but are not limited to, interleukins such as IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12, interferons such as alpha-interferon and gamma-interferon, tumor necrosis factor (TNF), platelet derived growth factor (PDGF), GCSF, granulocyte- macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), and the like.

Preferably, adjuvants capable of stimulating cellular immune responses, particularly cytotoxic T lymphocytes (CTL) responses, are used. A synergistic effect on the induction of HBV-specific CTL response can be achieved when adjuvants capable of stimulating CTL response are used in the presence of cells displaying HBV altered budding phenotype. This is especially beneficial to the prevention and treatment of HBV infection and symptoms thereof because it is known that a potent killer T cell response is critical to effective inhibition of viral infection in humans. Many adjuvants capable of stimulating cellular immune responses are known in the art. For example, cytokines secreted by helper T cells called Thl cells have been shown to promote cellular responses. Such Thl type cytokines include interleukm-2 (IL- 2), interleukin-4, and interleukin-12 (IL-12), interleukin-18, among others. In addition, fusion proteins having one of such Thl type cytokines (e.g., IL-2) fused to the Fc portion of immunoglobulin G (IgG) may also be used. See e.g., Barouch et al, Science, 290:486- 492 (2000), which is incorporated herein by reference. Other preferred adjuvants capable of stimulating cell-mediated immune responses include interferons such as alpha- interferon, beta-interferon and gamma-interferon. Certain chemokines may also be used to enhance cellular immune responses. Such chemokines are small molecules that attract T cells to infected tissues. Additionally, certain short bacterial immunostimulatory DNA sequences (ISSs) have also been discovered as CTL response-promoting adjuvants that potently stimulate immune responses to co-administered antigens. See Roman et al, Nat. Med., 3:849-854 (1997), which is incorporated herein by reference. Thus, noncoding, ISS-enriched plasmid DNAs or ISS oligonucleotides (ISS-ODNs) can also be used in the present invention as adjuvants to enhance cellular immunity against cells displaying HBV altered budding phenotype.

One or more adjuvants can be used for purposes of the present invention. The adjuvant(s) can be administered in the same composition as the cells or compounds as described above. Alternatively, the adjuvant(s) can also be given to a patient separately. Accordingly, the present invention also provides compositions comprising cells displaying HBV altered budding phenotype and one or more adjuvants capable of enhancing immune responses in a patient. In a prefeπed embodiment, the compositions include cells displaying HBV altered budding phenotype and one or more adjuvants capable of enhancing cellular immune response, cell-mediated immune response or CTL response. As is apparent from the above description, many of the suitable adjuvants are proteins or peptides. Such adjuvants can be administered to patients in the form of nucleic acids (e.g., DNA, RNA, and the like) so long as the administered nucleic acids are capable of expressing the adjuvants they encode. Thus, the term "administering an adjuvant to a patient" or a paraphrase thereof also encompasses administering a nucleic acid encoding a protein adjuvant and capable of expressing the adjuvant in a patient's body. Methods for administering nucleic acids are described above.

Accordingly, the present invention provides a composition comprising at least (1) a first nucleic acid encoding a mutant HBV core protein sufficient for viral particle assembly but devoid of late domain motifs, and (2) a second nucleic acid encoding an adjuvant capable of stimulating cytotoxic T lymphocyte (CTL) response. The two nucleic acids can be incorporated into separate expression vectors. Preferably, the two nucleic acids are carried within the same expression vector. In one embodiment, each of the two nucleic acids is operably linked to a transcriptional promoter, which drives the transcription from the nucleic acid linked to the promoter. In preferred embodiments, the nucleic acid encoding a mutant HBV core protein sufficient for viral particle assembly but devoid of late domain motifs is capable of expressing the mutant HBV core protein. For example, the nucleic acid may contain one or more mutations that decrease the effect of an inhibitory/instability sequence that is present in the coπesponding nucleotide sequence of the native HBN core protein encoding nucleic acid. Methods for making the expression vectors and compositions and for administering them to a patient are as described elsewhere in the present disclosure.

6. Combination Therapy

The methods and compositions according to the present invention are distinct from other known or commercially available approaches for treating or preventing HBN infection and symptoms thereof. Currently, interferon and lamivudine are used to treat HBN infection. Therefore, it may be desirable to employ combination therapies to combine a method of the present invention with other prophylactic or treatment methods so as to increase the prophylactic or therapeutic effect. Thus, any other methods useful in treating or preventing HBV infection and symptoms combined with the methods of the present invention. The therapeutic and/or prophylactic materials in the methods of the present invention and in other methods may be combined in the same pharmaceutical composition or administered separately. In addition, one or more adjuvants described above may be included in the same composition or administered separately in the combination therapy approaches. However, it is to be understood that such other anti-HBV agents should be pharmaceutically compatible with the cells, nucleic acids, polypeptides or other active agents, and/or adjuvants of the present invention. By "pharmaceutically compatible" it is intended that the other anti-viral agent(s) will not interact or react with the cells, nucleic acids, polypeptides or other active agents, and/or adjuvants of the present invention, directly or indirectly, in such a way as to substantially adversely affect the effect of the treatment, or to cause any significant adverse side reaction in the patient.

7. Dosage, Formulation and Administration Typically, the active agents (cells, polypeptides, small organic molecules, nucleic acids in plasmid vectors or recombinant vectors) of the present invention are administered to a patient in a pharmaceutical composition, which typically includes one or more pharmaceutically acceptable carriers that are inherently nontoxic and non- therapeutic.

The pharmaceutical composition according to the present invention may be administered to a subject needing treatment or prevention through any appropriate routes such as parenteral, oral, mucosal or topical administration. The active agents of this invention are administered at a therapeutically or prophylactically effective amount to achieve the desired therapeutic and/or prophylactic effect without causing any serious adverse effects in the patient treated. Generally, the toxicity profile and therapeutic or prophylactic efficacy of the active agents can be determined by standard pharmaceutical procedures in suitable cell models or animal models or human clinical trials. As is known in the art, the LD₅₀ represents the dose lethal to about 50% of a tested population. The ED₅₀ is a parameter indicating the dose therapeutically or prophylactically effective in about 50% of a tested population. Both LD₅₀ and ED₅₀ can be determined in cell models and animal models. In addition, the IC₅₀ may also be obtained in cell models and animal models, which stands for the circulating plasma concentration that is effective in achieving about 50% of the maximal inhibition of the symptoms of a disease or disorder. Such data may be used in designing a dosage range for clinical trials in humans.

Typically, as will be apparent to skilled artisans, the dosage range for human use should be designed such that the range centers around the ED₅₀ and/or IC₅₀, but significantly below the LD₅₀ obtained from cell or animal models.

Typically, the cells displaying HBV altered budding phenotype may be effective at an amount of from about 10 cells to about 20x10⁶ per dosage with an administration frequency of once per year, once per month up to once per day. The cells can be injected in a composition having a suitable pharmaceutically acceptable carrier. For example, such a composition may include cells suspended in a standard sterile cell culture medium, preferably devoid of serum. Alternatively, the cells may be simply suspended in a saline solution, e.g., PBS, or other standard cell transplantation carrier before administration. Typically, the composition should contain one or more agents for maintaining optimal isotonicity, isomocity and pH. Suitable adjuvant(s) may also be included in the composition. The cell-containing composition can be administered to a patient through any suitable routes, e.g., by parenteral injection or transplantation. Preferably, the cells are injected intravenously. When a nucleic acid (either alone or in a plasmid vector or a recombinant live vector) is used as an active agent in the present invention, the nucleic acid can be administered in an amount of from about 0.1 microgram to about 5000 milligram, preferably from about 1 microgram to about 500 milligram per dosage. The appropriate amount can be administered daily, weekly, monthly, bimonthly, semi-annually or annually. The nucleic acid can be administered to a patient in a manner as described above in Section 1 or by a standard procedure known in the art, as will be apparent to skilled artisans.

Typically, the mutant HBV core protein and the other active peptidic or small organic compounds of the present invention capable of causing the formation of cells displaying HBV late-domain phenotype can be effective at an amount of from about 0.01 microgram to about 5000 mg per day, preferably from about 1 microgram to about 2500 mg per day. However, the amount can vary with the body weight of the patient treated and the state of disease conditions. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at predetermined intervals of time. The suitable dosage unit for each administration of the compounds of the present invention can be, e.g., from about 0.01 microgram to about 2000 mg, preferably from about 1 microgram to about 1000 mg.

In the case of combination therapy, a therapeutically or prophylactically effective amount of another anti-HBV compound can be administered in a separate pharmaceutical composition, or alternatively included in the pharmaceutical composition that contains a compound according to the present invention. The pharmacology and toxicology of many of such other anti-HBV compounds are known in the art. See e.g., Physicians Desk Reference, Medical Economics, Montvale, NJ; and The Merck Index, Merck & Co., Rahway, NJ. The therapeutically or prophylactically effective amounts and suitable unit dosage ranges and route of administration of such compounds used in art can be equally applicable in the present invention.

It should be understood that the dosage ranges set forth above are exemplary only and are not intended to limit the scope of this invention. The therapeutically or prophylactically effective amount for each active agent can vary with factors including but not limited to the activity of the agent used, stability of the active agent in the patient's body, the severity of the conditions to be alleviated, the purpose of the treatment (prophylactic vs. therapeutic), the total weight of the patient treated, the route of adminisfration, the ease of absorption, distribution, and excretion of the active agent by the body, the age and sensitivity of the patient to be treated, and the like, as will be apparent to a skilled artisan. The amount of administration can also be adjusted as the various factors change over time.

The active agents according to this invention (cells, nucleic acids, polypeptides and small organic compounds) can be administered to patients to be treated through any suitable routes of administration. Advantageously, the active agents are delivered to the patient parenterally, i.e., by intravenous, intramuscular, intraperiotoneal, intracisternal, subcutaneous, or intraarticular injection or infusion.

For parenteral adminisfration, the non-cell active agents can be formulated into solutions or suspensions, or in lyophilized forms for conversion into solutions or suspensions before use. Lyophilized compositions may include pharmaceutically acceptable carriers such as gelatin, DL-lactic and glycolic acids copolymer, D-mannitol, etc. To convert the lyophilized forms into solutions or suspensions, diluent containing, e.g., carboxymethylcellulose sodium, D-mannitol, polysorbate 80, glycerine, and water may be employed. Lyophilized forms may be stored in, e.g., a dual chamber syringe with one chamber containing the lyophilized composition and the other chamber containing the diluent. In addition, the active ingredient(s) can also be incorporated into sterile lyophilized microspheres for sustained release. Methods for making such microspheres are generally known in the art. See U.S. Patent Nos. 4,652,441; 4,728,721; 4,849,228; 4,917,893; 4,954,298; 5,330,767; 5,476,663; 5,480,656; 5,575,987; 5,631,020; 5,631,021; 5,643,607; and 5,716,640. In a solution or suspension form suitable for parenteral administration, the pharmaceutical composition can include, in addition to a therapeutically or prophylactically effective amount of an active agent of the present invention, a buffering agent, an isotonicity adjusting agent, a preservative, and/or an anti-absorbent. Examples of suitable buffering agent include, but are not limited to, citrate, phosphate, tartrate, succinate, adipate, maleate, lactate and acetate buffers, sodium bicarbonate, and sodium carbonate, or a mixture thereof. Preferably, the buffering agent adjusts the pH of the solution to within the range of 5-8. Examples of suitable isotonicity adjusting agents include sodium chloride, glycerol, mannitol, and sorbitol, or a mixture thereof. A preservative (e.g., anti-microbial agent) may be desirable as it can inhibit microbial contamination or growth in the liquid forms of the pharmaceutical composition. Useful preservatives may include benzyl alcohol, a paraben and phenol or a mixture thereof. Materials such as human serum albumin, gelatin or a mixture thereof may be used as anti- absorbents. In addition, conventional solvents, surfactants, stabilizers, pH balancing buffers, and antioxidants can all be used in the parenteral formulations, including but not limited to dextrose, fixed oils, glycerine, polyethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, and the like. The parenteral formulation can be stored in any conventional containers such as vials, ampoules, and syringes.

The active agents (particularly nucleic acids, polypeptides and small organic compounds) can also be delivered orally in enclosed gelatm capsules or compressed tablets. Capsules and tablets can be prepared in any conventional techniques. For example, the active agents can be incorporated into a formulation which includes pharmaceutically acceptable carriers such as excipients (e.g., starch, lactose), binders (e.g., gelatin, cellulose, gum tragacanth), disintegrating agents (e.g., alginate, Primogel, and corn starch), lubricants (e.g., magnesium stearate, silicon dioxide), and sweetening or flavoring agents (e.g., glucose, sucrose, saccharin, methyl salicylate, and peppermint). Various coatings can also be prepared for the capsules and tablets to modify the flavors, tastes, colors, and shapes of the capsules and tablets. In addition, liquid carriers such as fatty oil can also be included in capsules. Other forms of oral formulations such as chewing gum, suspension, syrup, wafer, elixir, and the like can also be prepared containing the active compounds used in this invention. Various modifying agents for flavors, tastes, colors, and shapes of the special forms can also be included. In addition, for convenient administration by enteral feeding tube in patients unable to swallow, the active agents can be dissolved in an acceptable lipophilic vegetable oil vehicle such as olive oil, corn oil and safflower oil. The active agents (e.g. , nucleic acids in recombinant live vectors, polypeptides and small organic compounds) can also be administered topically through rectal, vaginal, nasal, bucal, or mucosal applications. Topical formulations are generally known in the art including creams, gels, ointments, lotions, powders, pastes, suspensions, sprays, drops and aerosols. Typically, topical formulations include one or more thickening agents, humectants, and/or emollients including but not limited to xanthan gum, petrolatum, beeswax, or polyethylene glycol, sorbitol, mineral oil, lanolin, squalene, and the like.

A special form of topical administration is delivery by a transdermal patch. Methods for preparing transdermal patches are disclosed, e.g., in Brown, et al, Annual Review of Medicine, 39:221-229 (1988), which is incorporated herein by reference. The active agents (e.g., cells, nucleic acids, polypeptides and small organic molecules) can also be delivered by subcutaneous implantation for sustained release. This may be accomplished by using aseptic techniques to surgically implant the active agents in any suitable formulation into the subcutaneous space of the anterior abdominal wall. See, e.g., Wilson et al, J. Clin. Psych. 45:242-247 (1984). Sustained release can be achieved by incorporating the active ingredients into a special carrier such as a hydrogel. Typically, a hydrogel is a network of high molecular weight biocompatible polymers, which can swell in water to form a gel like material. Hydrogels are generally known in the art. For example, hydrogels made of polyethylene glycols, or collagen, or poly(glycolic-co-L-lactic acid) are suitable for this invention. See, e.g., Phillips et al., J Pharmaceut. Sci., 73:1718-1720 (1984).

Alternatively, other forms controlled release or protection including microcapsules and nanocapsules generally known in the art, and hydrogels described above can all be utilized in oral, parenteral, topical, and subcutaneous administration of the active agents. Another preferable delivery form is using liposomes as carrier. Liposomes are micelles formed from various lipids such as cholesterol, phospholipids, fatty acids, and derivatives thereof. Active compounds can be enclosed within such micelles. Methods for preparing liposomal suspensions containing active ingredients therein are generally known in the art and are disclosed in, e.g., U.S. Pat. No. 4,522,811, and Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq., both of which are incorporated herein by reference. Several anticancer drugs delivered in the form of liposomes are known in the art and are commercially available from Liposome Inc. of Princeton, New Jersey, U.S.A. It has been shown that liposomes can reduce the toxicity of the active agents, and increase their stability.

8. Examples

Example 1: Analysis of the PPAY Motif.

A yeast two-hybrid assay can be utilized to determine the effect of amino acid substitution mutations in the PPAY motif of HBV core protein on the interaction between Nedd4 and HBV core protein. A yeast two-hybrid activation domain-Nedd4 construct can be prepared from a DNA fragment encompassing the full-length coding sequence for a protein containing Nedd4 (or fragments thereof) (GenBank accession D42055) by PCR from a human cDNA library and can be subsequently cloned into the appropriate sites of the activation domain parent plasmid GADpN2 (LEU2, CEN4, ARS 1 , ADH lp-

SV40NLS-GAL4 (768-881)-MCS (multiple cloning site)-PGKlt, AmpR, ColEl_ori).

The yeast two-hybrid DNA binding domain-HBV core protein construct can be prepared from a fragment coπesponding to the HBV core protein derived from any HBV strain. This HBV core protein gene or fragment can be obtained by PCR and can be cloned into the appropriate sites of the binding domain parent plasmid pGBT.Q.

Any amino acid substitution mutations can be introduced by PCR into the HBV core protein sequence in the yeast two-hybrid DNA binding domain-HBV core protein construct described above. The mutations are verified by DNA sequence analysis. Non- limiting examples of mutations that can be introduced into the DNA binding domain- HBV core protein construct are summarized in Tables I-III below.

Table I: Mutations in HBV Core Protein

The effect of the mutations can be tested in yeast cells of the strain Y189 purchased from Clontech (ura3-52 his3*200 ade2-101 trpl-901 leu2-3,l 12 met gal4 gal80 URA3::GALlp-lacZ) by co-transforming with the activation domain-Nedd4 construct and one of the binding domain-mutant HBN core protein constructs or the binding domain- wild type core protein construct. Filter lift assays for β-Gal activity can be conducted by lifting the transformed yeast colonies with filters, lysing the yeast cells by freezing and thawing, and contacting the lysed cells with X-Gal. Positive β-Gal activity indicates that the core protein wild-type or mutant protein interacts with the Νedd4 WW domain. All binding domain constructs can be also tested for self-activation of β-Gal activity.

Interactions between a Nedd4 WW domain (or fragments thereof) and wild-type HBN core protein (WT)(or fragments thereof) or the core protein mutants can be further quantitated by performing liquid culture β-galactosidase assays. Cultures are grown overnight in synthetic media (-Leu, -Trp, + glucose) in 96 well plates, normalized for optical density, and lysed by addition of 6X lysis/substrate solution in 6X Z-buffer (60mM KC1, 6mM MgSO₄, 360mM Νa₂HPO₄, 240 mM NaH₂PO₄, 6mg/ml CPRG, 0.12U/ml lyticase, 0.075% NP-40). Cultures are incubated for 2 hr at 37°C, clarified by centrifugation, and the optical absorbance of each supernatant is measured (575 nm). Full length protein containing a Nedd4 bound to wild-type HBN core protein in the two- hybrid liquid culture assay, will result in higher levels of β-galactosidase activity (e.g., greater than 10-fold, more preferably greater than 100-fold, more preferably greater than 300-fold over background). Different core protein point mutants can be used to test whether the Nedd4 WW domain binding interaction requires the core protein late domain motif within HBN core protein, and will result in reduced β-galactosidase activity.

Example 2: In Vitro Binding Assay.

A fusion protein with a GST tag fused to the HBN core protein or fragment thereof can be recombinantly expressed and purified by chromatography. In addition, a HBV core protein fragment can be synthesized chemically by standard peptide synthesis methods. The peptide can be purified by conventional protein purification techniques, e.g. , by chromatography.

Νunc/Νalgene Maxisorp plates are incubated overnight at 4°C or for 1-2 hrs at room temperature in 100 μl of a protein coupling solution containing purified GST-HBV core protein and 50mM Carbonate, pH=9.6. This allows the attachment of the GST-HBV core protein fusion protein to the plates. Liquids in the plates are then emptied and the wells are filled with 400 μl/well of a blocking buffer (SuperBlock; Pierce-Endogen, Rockford, IL). After incubation for 1 hr at room temperature, 100 μl of a mixture containing Drosophila S2 cell lysate myc-tagged Νedd4 (or a fragment or homolg thereof) and a specific amount of the HBV core protein fragment peptide is applied to the wells of the plate. This mixture is allowed to react for 2 hr at room temperature to form HBV core protein:Nedd4 WW domain protein-protein complexes.

Plates are then washed 4 x lOOμl with 1 x PBST solution (Invitrogen; Carlsbad, CA). After washing, lOOμl of lμg/ l solution of anti-myc monoclonal antibody (Clone 9E10; Roche Molecular Biochemicals; Indianapolis, IN) in 1 x PBST is added to the wells of the plate to detect the myc-epitope tag on the a Nedd4 WW domain containing polypeptide. Plates are then washed again with 4 x lOOμl with 1 x PBST solution and lOOμl of lμg/ml solution of horseradish peroxidase (HRP) conjugated Goat anti-mouse IgG (Jackson Immunoresearch Labs; West Grove, Pennsylvania) in 1 x PBST was added to the wells of the plate to detect bound mouse anti-myc antibodies. The plates are then washed again with 4 x lOOμl with 1 x PBST solution and 100 μl of fluorescent substrate (QuantaBlu; Pierce-Endogen, Rockford, IL) is added to all wells. After 30 min, 100 μl of stop solution is added to each well to inhibit the function of HRP. Plates are then analyzed on a Packard Fusion instrument at an excitation wavelength of 325nm and an emission wavelength of 420nm. The presence of fluorescent signals indicates binding of Nedd4 WW domain to the fixed GST-HBV core protein. In contrast, the absence of fluorescent signals indicates that the PPAY-containing analog short peptide is capable of disrupting the interaction between a Nedd4 WW domain and HBV core protein.

Different concentrations of the HBV core protein peptide are tested, and the relative intensities of the fluorescence signals obtained at different concentrations are plotted against the peptide concentrations.

Example 3: Yeast Screen To Identify Small Molecule Inhibitors Of The Interaction Between HBV Core Protein And Nedd4.

Beta-galactosidase is used as a reporter enzyme to signal the interaction between yeast two-hybrid protein pairs expressed from plasmids in Saccharomyces cerevisiae. Yeast strain MY209 (ade2 his3 leu2 trpl cyh2 ura3::GALl-p-lacZ gal4 gal80 lys2::GALlτp-HIS3) bearing a plasmid containing a gene (or fragment thereof) for a Nedd4 (LEU2 CEN4ARS1 ADHlp-$V40^~NLS-GAL4 (76S-SSl)-Nedd4-PGKltAmpR ColEljm) and a plasmid containing a gene for the HBV core protein (or fragment thereof) (TRP1 CEN4 ARS ADHlp-GAL4(l-l47)-HBV-coreprotein-ADHltAmpR

ColEl m) is cultured in synthetic complete media lacking leucine and tryptophan (SC - Leu -Trp) overnight at 30°C. This culture is diluted to 0.01 OD₆₃₀ units/ml using SC - Leu -Trp media. The diluted MY209 culture is dispensed into 96-well microplates. Compounds from a library of small molecules are added to the microplates; the final concenfration of test compounds is approximately 60μM. The assay plates are incubated at 30°C overnight.

The following day an aliquot of concentrated substrate/lysis buffer is added to each well and the plates incubated at 37°C for 1-2 hours. At an appropriate time an aliquot of stop solution is added to each well to halt the beta-galactosidase reaction. For all microplates an absorbance reading is obtained to assay the generation of product from the enzyme substrate. The presence of putative inhibitors of the interaction between HBN core protein and Νedd4 results in inhibition of the beta-galactosidase signal generated by MY209. Additional testing eliminates compounds that decreased expression of beta-galactosidase by affecting yeast cell growth and non-specific inhibitors that affected the beta-galactosidase signal generated by the interaction of an unrelated protein pair.

Once a hit, i.e., a compound which inhibits the interaction between the viral and cellular proteins, is obtained, the compound is identified and subjected to further testing wherein the compounds are assayed at several concentrations to determine an IC₅₀ value, this being the concentration of the compound at which the signal seen in the two-hybrid assay described in this Example is 50% of the signal seen in the absence of the inhibitor.

Example 4: Detection of Modulators of Viral Budding Using FRET A homogeneous biochemical assay can be utilized for the detection of compounds that disrupt the interaction between a host cell protein and a viral protein containing a late-domain sequence. Briefly, tagged components of the interaction (GST-host protein or fragment thereof) and Biotin-viral-protein (or peptide fragment thereof) are incubated in the presence of cognate labels (GST-Europium [donor fluor] and streptavidin-APC [acceptor chromophore]). Upon interaction of all assay components, the donor, europium, is brought in close proximity to the acceptor, APC; excitation of the donor at 360nm results in emission fluorescence at 620nm, which in turn is absorbed by APC and is subsequently emitted at 665nm. The ratio of the APC emission at 665nm and the europium emission at 620nm is known as FRET (Fluorescence Resonance Energy Transfer) and can be utilized as a direct measure of protein-protein interaction. In the absence of an interaction between GST-host-protein and Biotin-viral-protein, the europium and APC are not brought into close proximity and thus excitation of europium at 360nm results only in a fluorescence emission at 620nm; detection of fluorescence emission at 620nm would indicate that APC did not receive any excitation at this wavelength. Detection reagents: LANCE Eu-W1024-labelled Anti-GST Antibody (PerkinElmer Life Sciences) is provided as a 3.13 μM stock and is stored at 4 °C and used at a final concenfration of 2nM in the assay. Streptavidin conjugated to SureLight™- Allophycocyanin (APC) (PerkinElmer Life Sciences) is provided as a lyophilizate and should be reconstituted with diH2O to a final concentration of lmg/ml and stored at 4 °C in the dark. SureLight™-APC is used at a final concentration of 1 μg/ml (~10nM) in the assay.

Summary of analytical method: First pass read establishes europium signal by excitation at 360nm and emission detection at 620nm (400nm diochroic mirror, Z-height 3mm (set), Attenuator out, 200 μsec delay after flash, 1000 μsec integration time, 100 flashes/well using 1000V flash lamp with 10msec between flashes, Raw data units=counts, PMT=digital, plate setting time=10 msec). Second pass read establishes APC signal by excitation at 360nm and emission detection at 665nm (same settings as europium with exceptions: 100 μsec delay after flash, 150 μsec integration time). FRET is determined by 665nm reading/620nm reading multiplied by 10000.

GST-host-protein, Biotin-viral-protein, LANCE-Eu and SureLightTM-APC are added concomitantly to 50mM HEPES, pH 7.0, 0.1% BSA, to final concentrations of 33nM, 330nM, 2nM and 1 μg/ml, respectively. Upon thorough mixing of reagents, 100 μl is aliquoted to each well of a 96-well plate. Compound addition can follow immediately and at least 30 minutes of incubation at room temperature is required prior to reading plate.

In the presence of compounds, average europium signal (620nm) is ~30-40000 counts, average APC signal (665nm) is ~2-3000 counts with the resultant FRET signal ~500-700. From competition experiments, the assay floor for FRET is ~50, allowing for a window of approximately 5-20 fold. An assessment of 144 compounds suggested that some non-specific fluorescent effects (from the compounds) can be mainly seen of europium fluorescence in the form of either quenching or hyper-excitation. A control protein (InM Biotin-GST) can be utilized to distinguish non-specific effects from actual positive hits. Example 5: Anti-HBV Assay

The assay used is similar to the assay described by Korba and Milman, Antiviral Res., 15:217-228 (1991) and Korba and Germ, Antiviral Res., 19:55-70 (1992), with the exception that viral DNA detection and quantification is simplified. Briefly, HepG2- 2.2.15 cells are plated in 96-well microtiter plates at an initial density of 2 x 10⁴ cells/100 μl in DMEM medium supplemented with 10% fetal bovine serum. To promote cell adherence, the 96-well plates have been pre-coated with collagen prior to cell plating. After incubation at 37°C in a humidified, 5% CO₂ environment for 16-24 hours, the confluent monolayer of HepG2-2.2.15 cells is washed and the medium is replaced with complete medium containing various concentrations of test compound. Every three days, the culture medium is replaced with fresh medium containing the appropriately diluted drug. Nine days following the initial administration of test compounds, the cell culture supernatant is collected and clarified by centrifugation (Sorvall RT-6000D centrifuge, 1000 rpm for 5 min). Three microliters of clarified supernatant is then subjected to real- time quantitative PCR using conditions described below.

Virion-associated HBV DNA present in the tissue culture supernatant is PCR amplified using primers derived from HBV strain ayw. Subsequently, the PCR-amplified HBV DNA is detected in real-time (i.e., at each PCR thermocycle step) by monitoring increases in fluorescence signals that result from exonucleolytic degradation of a quenched fluorescent probe molecule following hybridization of the probe to the amplified HBV DNA. The probe molecule, designed with the aid of Primer Express™ (PE- Applied Biosystems, Foster City, CA) software, is complementary to DNA sequences present in the HBV DNA region amplified.

Routinely, 3 μl of clarified supernate is analyzed directly (without DNA extraction) in a 50 μl PCR reaction. Reagents and conditions used are per the manufacturers suggestions (PE-Applied Biosystems). For each PCR amplification, a standard curve is simultaneously generated several log dilutions of a purified 1.2 kbp HBV ayw subgenomic fragment; routinely, the standard curve ranged from lxlO⁶ to lxlO¹ nominal copy equivalents per PCR reaction. In addition, HBV-infected cells treated with the compounds are also studied under electron microscope or similar devices to examine if the viruses are defective in viral egress and/or budding from the cells.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The mere mentioning of the publications and patent applications does not necessarily constitute an admission that they are prior art to the instant application.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. Use of cells displaying HBV altered budding phenotype for the manufacture of a medicament useful for treating HBV infection.

2. The use of Claim 1, wherein said medicament further comprises an adjuvant capable of stimulating cellular immune response.

3. The use of Claim 2, wherein said adjuvant is selected from the group consisting of IL-2, IL-4, IL-12, IL-18, and gamma-interferon.

4. A composition comprising: cells capable of displaying HBV altered budding phenotype; and an adjuvant capable of stimulating cytotoxic T lymphocytes (CTL) response.

5. The composition of Claim 4, wherein said cells are human cells and contain a nucleic acid encoding a mutant HBV core protein sufficient for virus particle assembly but devoid of late-domain motifs.

6. The composition of Claim 4 or 5, wherein said cells contain a mutant HBV core polypeptide sufficient for virus-like particle assembly but devoid of late-domain motifs.

7. The composition of any one of Claims 4-7, wherein said adjuvant is selected from the group consisting of IL-2, IL-4, IL-12, IL-18, and gamma-interferon.

8. Use of a composition for the manufacture of a medicament useful for the treatment of HBV infection, said composition comprising a compound capable of causing the formation of cells displaying HBV altered budding phenotype, and an adjuvant capable of stimulating cytotoxic T lymphocytes (CTL) response.

9. The use of Claim 8, wherein said adjuvant is a cytokine protein or a nucleic acid encoding the same.

10. The use of Claim 9, wherein said cytokine is selected from the group consisting of IL-2, IL-4, IL-12, IL-18, gamma-interferon, and a fusion protein thereof.

11. The use of any one of Claims 8-11, wherein said composition comprises a compound capable of interfering with the protein-protem interaction between a WW domain containing protein, Nedd4, or a Nedd4-like protein and HBV core protein.

12. The use of any one of Claims 8-11, wherein said composition comprises a mutant HBV core polypeptide devoid of late domain motifs and/or a nucleic acid encoding said mutant HBV core polypeptide.

13. The use of Claim 12, wherein said nucleic acid is within an HBV genome.

14. The use of Claim 13 wherein the genome is devoid of late domain motifs.

15. The use of any one of Claim 8-14, wherein said composition further comprises another HBV protein or an immunogenic fragment thereof, and/or a nucleic acid encoding said other HBV protein or said immunogenic fragment.

16. The use of Claim 15, wherein said other HBV protein is an envelope protein.

17. A method for identifying modulators of HBN budding comprising: providing a HBV protein comprising a late domain motif, or a fragment, derivative, or homologue thereof of said HBV protein; providing a host cell protein capable of binding a late domain motif binding, or a fragment, derivative or homologue thereof of said host cell protein; contacting said HBV protein and said host cell protein in the presence and absence of a test compound; wherein a modulator of HBV budding is identified if the amount of said HBV protein bound to said host cell protein more or less in the presence as compared to the amount of said HBV protein bound to said host cell protein in the absence of said test compound.

18. The method of claim 17 wherein said HBV protein is HBV core protein.

19. The method of claim 17 wherein said host cell protein is selected from the group consisting of ΝEDD4, a NEDD4-like protein, or a protein having a WW domain.

20. The method of claim 17 wherein said amount of said HBV protein bound to said host cell protein is less in the presence as compared to the amount of said HBV protein bound to said host cell protein in the absence of said test compound.

21. Use of a composition for the manufacture of a medicament useful for the treatment of HBV infection, said composition comprising a nucleic acid encoding a mutant HBV core polypeptide that is incapable of interacting with a human WW domain containing protein.

22. The use of Claim 21 wherein the human WW domain containing protein is Nedd4.

23. The use of Claim 21 , wherein said nucleic acid is incorporated into a plasmid expression vector.

24. The use of Claim 21 , wherein said nucleic acid is incorporated into a live vector.

25. The use of any one of Claims 21 -24, wherein the mutant HBV core polypeptide is sufficient for viral particle assembly but devoid of late-domain motifs.

26. The use of any one of Claims 21 -24, wherein the composition further comprising an adjuvant capable of stimulating cytotoxic T lymphocytes (CTL) response.

27. The use of Claim 26, wherein said adjuvant is: a protein selected from the group consisting of IL-2, IL-4, IL-12, IL-18, gamma- interferon, and a fusion protein thereof; and/or a nucleic acid encoding said protein.

28. The use of Claim 27, wherein said fusion protein comprises the Fc portion of immunoglobulin G (IgG).

29. The use of Claim 26, wherein said adjuvant is (a) IL-2, (b) a fusion protein containing IL-2 and the Fc portion of immunoglobulin G (IgG), or (c) a nucleic acid encoding (a) or (b).

30. The use of any one of Claims 21-29, wherein the composition further comprises another HBV protein or an immunogenic fragment thereof, and/or a nucleic acid encoding said other HBV protein or said immunogenic fragment.

31. The use of Claim 30, wherein said other HBV protein is an envelope protein.

32. The use of any one of Claims 21-31, wherein the medicament is suitable for administration by intravenous, intramuscular, intradermal, intranasal or intramucosal introduction.

33. Use of a composition comprising a mutant HBV core polypeptide sufficient for viral particle assembly but devoid of late-domain motifs, in the manufacture of a medicament useful for the treatment of HBV infection.

34. The use of Claim 33, wherein the mutant HBV core polypeptide is covalently linked to a transporter capable of increasing cellular uptake of said polypeptide.

35. The use of Claim 34, wherein said transporter is selected from the group consisting of penetratins, /-Tat _-5 ), d-Tat_{4 -5} , retro-inverso isomers of/- or c?-Tat_49-5 , L- arginine oligomers, D- arginine oligomers, L-lysine oligomers, D-lysine oligomers, L- histine oligomers, D-histine oligomers, L-omithine oligomers, D-omithine oligomers, short peptide sequences derived from fibroblast growth factor, Galparan, and HSV-1 structural protein VP22, and peptoid analogs thereof.

36. The use of any one of Claims 33-35, said composition further comprising an adjuvant capable of stimulating cytotoxic T lymphocytes (CTL) response.

37. The use of Claim 36, wherein said adjuvant is: a protein selected from the group consisting of IL-2, IL-4, IL-12, IL-18, gamma- interferon, and a fusion protein thereof; and/or a nucleic acid encoding said protein.

38. The use of Claim 37, wherein said fusion protein comprises the Fc portion of immunoglobulin G (IgG).

39. The use of any one of Claims 33-38, said composition further comprising another HBV protein or an immunogenic fragment thereof, and/or a nucleic acid encoding said other HBV protein or said immunogenic fragment.

40. A composition comprising: a compound capable of causing the formation of cells displaying HBV altered budding phenotype; and a pharmaceutically acceptable adjuvant.

41. The composition of Claim 40, wherein said adjuvant is capable of stimulating cytotoxic T lymphocytes (CTL) response.

42. The composition of Claim 40, wherein said adjuvant is a cytokine protein or a nucleic acid encoding the same.

43. The composition of Claim 42, wherein the cytokine is selected from the group consisting of IL-2, IL-4, IL- 12, IL- 18, gamma-interferon, and a fusion protein thereof.

44. The composition of any one of Claims 40-43, wherein said compound is capable of interfering with the protein-protein interaction between a human WW domain containing protein and HBN core protein.

45. The composition of any one of Claims 40-44, wherein said compound is a mutant HBN core polypeptide devoid of late domain motifs and/or a nucleic acid encoding said mutant HBV core polypeptide.

46. The composition of Claim 45, wherein said nucleic acid is within an HBV genome.

47. The composition of claim 46 wherein the HBV genome is devoid of functional late domain motifs.

48. The composition of Claim 46 or 47, wherein said composition further comprises another HBV protein or an immunogenic fragment thereof, and/or a nucleic acid encoding said other HBV protein or said immunogenic fragment.

49. The composition of Claim 48, wherein said other HBV protein is an envelope protein.

50. The composition of any one of Claims 40-44, wherein the compound is a nucleic acid encoding a mutant HBV core polypeptide that is incapable of interacting with a human WW domain containing protein.

51. The composition of Claim 50, wherein said mutant HBV core polypeptide is sufficient for viral particle assembly but devoid of late-domain motifs.

52. The composition of any one of Claims 45-51 , wherein the adjuvant is (a) IL-2, (b) a fusion protein containing IL-2 and the Fc portion of immunoglobulin G (IgG), or (c) a nucleic acid encoding (a) or (b).

53. A composition comprising: a first nucleic acid encoding a mutant HBV core polypeptide sufficient for viral particle assembly but devoid of late domain motifs; and a second nucleic acid encoding a cytokine capable of stimulating cytotoxic T lymphocyte (CTL) response.

54. The composition of Claim 53, wherein the cytokine is selected from the group consisting of IL-2, IL-4, IL-12, IL-18, gamma-interferon, and a fusion protein thereof.

55. The composition of Claim 54, wherein said adjuvant is IL-2 or a fusion protein containing IL-2 and the Fc portion of immunoglobulin G (IgG).

56. The composition of any one of Claims 53-55, further comprising a third nucleic acid encoding another HBV protein.

57. An expression vector comprising the first and second nucleic acids of Claim 56.

58. A composition comprising: a first nucleic acid encoding a mutant HBV core polypeptide sufficient for viral particle assembly but devoid of late-domain motifs; and another HBV protein or a second nucleic acid encoding said other HBV protein.

59. The composition of Claim 58, comprising said second nucleic acid.

60. The composition of Claim 58 or 59, wherein said other protein is selected from the group consisting of HBV polymerase, HBV envelope proteins, HBV surface proteins, and immunogenic fragments thereof.

61. An expression vector comprising said first and second nucleic acids of Claim 60.

62. A composition comprising: a first nucleic acid encoding a mutant HBV core polypeptide sufficient for viral particle assembly but devoid of late domain motifs; a second nucleic acid encoding an HBV envelope protein or an immunogenic fragment thereof; and a third nucleic acid encoding a cytokine capable of stimulating cytotoxic T lymphocyte (CTL) response.

63. The composition of Claim 62, wherein said cytokine is a fusion protein comprising IL-2 and the Fc portion of immunoglobulin G (IgG).

64. A composition comprising a mutant HBV core polypeptide sufficient for viral particle assembly but devoid of late-domain motifs covalently linked to a transporter capable of increasing cellular uptake of said polypeptide.

65. The composition of Claim 64, further comprising an adjuvant capable of stimulating CTL response.

66. The composition of Claim 65, wherein the adjuvant is a cytokine capable of stimulating cytotoxic T lymphocytes (CTL) response or a nucleic acid encoding the cytokine.

67. The composition of any one of Claims 64-66, further comprising another HBV protein or an immunogenic fragment thereof, and/or a nucleic acid encoding said other HBV protein or said immunogenic fragment.

68. The composition of any one of Claims 64-67, wherein the transporter is selected from the group consisting of penetratins, /-Tat_{4 -}s ), c?-Tat _9-57, refro-inverso isomers of /- or c?-Tat_{4 -57}, L-arginine oligomers, D- arginine oligomers, L-lysine oligomers, D-lysine oligomers, L-histine oligomers, D-histine oligomers, L-omithine oligomers, D-omithine oligomers, short peptide sequences derived from fibroblast growth factor, Galparan, and HSV-1 structural protein VP22, and peptoid analogs thereof.