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Title:  Spliced gene of KSHV / HHV8, its promoter and monoclonal antibodies specific for LANA2

United States Patent:  6,653,465

Issued:  November 25, 2003

Inventors:  Chang; Yuan (Irvington, NY); Moore; Patrick S. (Irvington, NY)

Assignee:  The Trustees of Columbia University in the City of New York (New York, NY)

Appl. No.:  733728

Filed:  December 8, 2000

Abstract

This invention provides an isolated nucleic acid which encodes a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide (LANA2) or a fragment thereof and also provides the LANA2 polypeptide. This invention provides an isolated nucleic acid comprising consecutive nucleotides having the sequence of a promoter of Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 transcription. This invention also provides a method of inhibiting p53 mediated apoptosis of a cell and a method of producing an antibody which comprises introducing into a cell a replicable vector of the subject invention.

SUMMARY OF THE INVENTION

This invention provides an isolated nucleic acid which encodes a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide (LANA2) or a fragment thereof.

This invention provides a replicable vector which comprises the isolated nucleic acid which encodes a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide.

This invention provides a host vector system which comprises the above vector and a suitable host cell. In one embodiment of the above host vector system, the host cell includes but is not limited to a eukaryotic cell, a hematopoietic cell, a B cell, a bacterial cell and E. Coli.

This invention provides a method of producing a polypeptide which comprises growing the above host vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.

This invention further provides an isolated nucleic acid comprising consecutive nucleotides having the sequence of a promoter of Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 transcription.

This invention provides a replicable vector which comprises the isolated nucleic acid comprising consecutive nucleotides having the sequence of a promoter of latency-associated nuclear antigen 2 transcription operably linked to a second nucleic acid.

This invention provides a host vector system which comprises a replicable vector which comprises the nucleic acid comprising consecutive nucleotides having the sequence of a promoter of latency-associated nuclear antigen 2 transcription operably linked to a second nucleic acid and a suitable host cell.

This invention provides a method of producing a polypeptide which comprises growing the above host vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.

This invention provides an isolated nucleic acid capable of specifically hybridizing to the isolated nucleic acid which encodes a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof.

This invention provides a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof.

This invention also provides an antibody capable of specifically binding to the above polypeptide.

This invention provides a composition comprising the above antibody and an agent conjugated to the antibody.

This invention provides a method of determining whether a subject is afflicted with a disease associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection of a B cell which comprises: (a) obtaining a suitable sample from the subject; (b) contacting the suitable sample with a detectable antibody capable of binding to Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof so as to form a complex between the antibody and any Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof present in the sample;(c) removing any unbound antibody; and (d) detecting any antibody which is bound to any Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof in the sample, wherein the presence of antibody indicates that the subject is afflicted with the disease associated with Kaposi's sarcoma-associated herpesvirus infection of a B cell.

This invention provides a method of determining whether a subject is afflicted with a disease associated with Kaposi's sarcoma-associated herpesvirus infection of a B cell which comprises:(a) obtaining a suitable sample from the subject; (b) immobilizing a capturing antibody wherein the capturing antibody is capable of binding to Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 to a support; (c) removing any unbound capturing antibody; (d) contacting the capturing antibody with the suitable sample so as to form a complex between the antibody and any Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 present in the sample; (e) removing any unbound sample; (f) contacting the complex obtained in step (d) with a detectable antibody of Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof so as to form a complex between the detectable antibody and the complex; (g) removing any unbound detectable antibody; and (h) detecting any detectable antibody which is bound to the complex wherein the presence of detectable antibody indicates that the subject is afflicted with the disease associated with Kaposi's sarcoma-associated herpesvirus infection of a B cell.

This invention provides a method of determining whether a subject is infected with Kaposi's sarcoma-associated herpesvirus which comprises:(a) obtaining a suitable sample from the subject; (b) contacting the suitable sample with the detectable antibody of Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof so as to form a complex between the antibody and any polypeptide or fragment thereof present in the sample;(c) removing any unbound antibody; and (d) detecting any antibody which is bound to any Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof in the sample, wherein the presence of antibody indicates that the subject is infected with Kaposi's sarcoma-associated herpesvirus.

This invention provides a method of determining whether a subject is infected with Kaposi's sarcoma-associated herpesvirus which comprises:(a) obtaining a suitable sample from the subject; (b) immobilizing a capturing antibody wherein the capturing antibody is capable of binding to polypeptide or fragment thereof to a support; (c) removing any unbound capturing antibody;(d) contacting the capturing antibody with the suitable sample so as to form a complex between the antibody and Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof present in the sample; (e) removing any unbound sample; (f) contacting the complex obtained in step (d) with the detectable antibody which is bound to Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof so as to form a complex between the detectable antibody and the complex; (g) removing any unbound detectable antibody; and (h) detecting any detectable antibody which is bound to the complex wherein the presence of detectable antibody indicates that the subject is infected with Kaposi's sarcoma-associated herpesvirus.

This invention provides a kit for diagnosing Kaposi's sarcoma-associated herpesvirus infection comprising the labeled antibody capable of specifically binding to the Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof.

This invention provides a method of inhibiting p53 mediated apoptosis of a cell which comprises introducing into the cell an effective amount of the replicable vector which comprises the isolated nucleic acid which encodes Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof, so as to thereby inhibit p53 mediated apostosis of the cell.

This invention provides a method of immortalizing a cell which comprises introducing into the cell an amount of the replicable vector which comprises the isolated nucleic acid which encodes Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof effective to inhibit p53 mediated apoptosis of the cell, so as to thereby immortalize the cell.

This invention provides a method of producing an antibody which comprises introducing into a cell an amount of the replicable vector which comprises the isolated nucleic acid which encodes Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide effective to inhibit p53 mediated apoptosis of the cell producing the antibody and thereby immortalizing the cell, so as to thereby produce the antibody.

This invention provides a method of determining whether a subject is infected with Kaposi's sarcoma-associated herpesvirus which comprises: (a) obtaining a suitable sample from the subject; (b) contacting the suitable sample with a detectable nucleic acid capable of hybriding to a nucleic acid which encodes Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof under hybridizing conditions so as to form a complex between the detectable nucleic acid and any nucleic acid which encodes a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof which is present in the sample; (c) removing any unbound detectable nucleic acid; and (d) detecting any detectable nucleic acid which is bound to the complex, wherein the presence of detectable nucleic acid indicates that the subject is infected with Kaposi's sarcoma-associated herpesvirus.

This invention provides a kit for diagnosing Kaposi's sarcoma-associated herpesvirus infection comprising a labeled nucleic acid which encodes a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof.

This invention provides a transgenic non-human animal which has stably integrated into the genome of its germ cells or somatic cells an exogenous nucleic acid construct wherein the nucleic acid construct comprises a B-cell specific promoter of Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 operably linked to a second nucleic acid which encodes a gene of interest and is introduced into the transgenic non-human animal, or an ancestor, at an embryonic stage. In one embodiment of the above transgenic animal, the animal is a mammal.

This invention provides a method for evaluating in a non-human transgenic animal the potential therapeutic effect of an agent for treating Kaposi's sarcoma-associated herpesvirus infection in a human, which comprises: (a) providing an agent to a transgenic non-human animal whose cells comprise the nucleic acid which encodes a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide; and (b) determining the therapeutic effect of the agent on the transgenic non-human animal by monitoring Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 expression, wherein a decrease in Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 indicates that the agent would have a potential therapeutic effect on Kaposi's sarcoma-associated herpesvirus infection in a human. In one embodiment of the above method, the animal is a mammal.

This invention provides a method of treating Kaposi's sarcoma-associated herpesvirus infection in a subject, which comprises introducing into the subject's cells an effective amount of the nucleic acid capable of specifically hybridizing to the isolated nucleic acid which encodes Kaposi's sarcoma-associated latency-associated nuclear antigen 2 polypeptide or fragment thereof to hybridize to any of the above nucleic acid which is present in the subject's cells, so as to thereby treat Kaposi's sarcoma-associated herpesvirus infection.

This invention provides a method of treating Kaposi's sarcoma-associated herpesvirus infection in a subject, which comprises introducing into the subject's cells an effective amount of a nucleic acid capable of specifically hybridizing to an isolated nucleic acid comprising nucleotides having the sequence of a promoter of latency-associated nuclear antigen 2 transcription to hybridize to any of this nucleic acid which is present in the subject's cells, so as to thereby treat the subject.

This invention provides a composition comprising the antibody capable of specifically binding to the polypeptide encoded by the isolated nucleic acid which encodes Kaposi's sarcoma-associated latency-associated nuclear antigen 2 polypeptide and a carrier.

This invention provides a method of treating a subject infected with Kaposi's sarcoma-associated herpesvirus, which comprises administering to the subject an amount of the above composition under conditions such that the antibody binds to any LANA2 present in the subject, so as to thereby treat the subject.

This invention provides a composition comprising the polypeptide encoded by the isolated nucleic acid which encodes Kaposi's sarcoma-associated latency-associated nuclear antigen 2 polypeptide or fragment thereof and a carrier.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following standard abbreviations are used throughout the specification to indicate specific amino acids:

        A = ala = alanine                R = arg = arginine
        N = asn = asparagine             D = asp = aspartic acid
        C = cys = cysteine               Q = gln = glutamine
        E = glu = glutamic acid          G = gly = glycine
        H = his = histidine              I = ile = isoleucine
        L = leu = leucine                K = lys = lysine
        M = met = methionine             F = phe = phenylalanine
        P = pro = proline                S = ser = serine
        T = thr = threonine              W = trp = tryptophan
        Y = tyr = tyrosine               V = val = valine
        B = asx = asparagine or aspartic acid
        Z = glx = glutamine or glutamic acid

As used herein, the following standard abbreviations are used throughout the specification to indicate specific nucleotides: C=cytosine; A=adenosine; T=thymidine; G=guanosine; and U=uracil.

This invention provides an isolated nucleic acid which encodes a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide (LANA2) or a fragment thereof.

In one embodiment of the above nucleic acid, the polypeptide comprises consecutive amino acids having the amino acid sequence set forth in SEQ ID NO: 2. In a further embodiment of the above nucleic acid, the isolated nucleic acid is designated ORFK10.5 and comprises consecutive nucleotides having the sequence set forth in SEQ ID NO: 1.

As used herein, the term "nucleic acid" refers to either DNA or RNA, including complementary DNA (cDNA), genomic DNA and messenger RNA (mRNA). As used herein, "genomic" means both coding and non-coding regions of the isolated nucleic acid molecule. "Nucleic acid sequence" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes both replicating vectors, infectious polymers of DNA or RNA and nonfunctional DNA or RNA.

The nucleic acids of the subject invention also include nucleic acids coding for polypeptide analogs, fragments or derivatives which differ from the naturally-occurring forms in terms of the identity of one or more amino acid residues (deletion analogs containing less than all of the specified residues; substitution analogs wherein one or more residues are replaced by one or more residues; and addition analogs, wherein one or more resides are added to a terminal or medial portion of the polypeptide) which share some or all of the properties of the naturally-occurring forms.

As used herein, the phrase "nucleic acid encoding" refers to a nucleic acid which directs the expression of a specific polypeptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA, the complementary DNA strand, and the RNA sequence that is translated into protein. The nucleic acid includes both the full length nucleic acid sequence as well as non-full length sequences. It being further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.

As used herein, "peptide" and "polypeptide" are used to denote two or more amino acids linked by a peptidic bond between the .alpha.-carboxyl group of one amino acid and the .alpha.-amino group of the next amino acid. Peptide includes not only the full-length protein, but also partial-length fragments. Peptides may be produced by solid-phase synthetic methods that are well-known to those skilled in the art. In addition to the above set of twenty-two amino acids that are used for protein synthesis in vivo, peptides may contain additional amino acids, including but not limited to hydroxyproline, sarcosine, and .beta.-carboxyglutamate. The peptides may contain modifying groups including but not limited to sulfate and phosphate moieties. Peptides can be comprised of L- or D-amino acids, which are mirror-image forms with differing optical properties. Peptides containing D-amino acids have the advantage of being less susceptible to proteolysis in vivo.

Peptides may by synthesized in monomeric linear form, cyclized form or as oligomers such as branched multiple antigen peptide (MAP) dendrimers (Tam et al. Biopolymers 51:311, 1999). Nonlinear peptides may have increased binding affinity by virtue of their restricted conformations and/or oligomeric nature. Peptides may also be produced using recombinant methods as either isolated peptides or as a portion of a larger fusion protein that contains additional amino acid sequences.

Peptides may be chemically conjugated to proteins by a variety of well-known methods. Such peptide-protein conjugates can be formulated with a suitable adjuvant and administered parenterally for the purposes of generating polyclonal and monoclonal antibodies to the peptides of interest. Alternatively, unconjugated peptides can be formulated with adjuvant and administered to laboratory animals for the purposes of generating antibodies. Methods for generating and isolating such antibodies are well-known to those skilled in the art.

The nucleic acids of the subject invention include but are not limited to DNA, RNA, mRNA, synthetic DNA, genomic DNA, and cDNA.

In one embodiment, the above nucleic acid is detectable. In one embodiment of the above nucleic acid, the nucleic acid is labeled with a detectable marker. As used herein, "detectable marker" includes but is not limited to a radioactive label, or a calorimetric, a luminescent, or a fluorescent marker. As used herein, "labels" include radioactive isotopes, fluorescent groups and affinity moieties such as biotin that facilitate detection of the labeled peptide. Other labels and methods for attaching labels to compounds are well-known to those skilled in the art.

This invention provides a replicable vector which comprises the isolated nucleic acid which encodes a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide. In one embodiment, the above vector includes but is not limited to plasmid, cosmid, .lambda. phage and YAC. As used herein, the term "vector" refers to viral expression systems, autonomous self-replicating circular DNA (plasmids), and includes both expression and nonexpression plasmids. Where a recombinant microorganism or cell culture is described as hosting an "expression vector," this includes both extrachromosomal circular DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.

This invention provides a host vector system which comprises the above vector and a suitable host cell. In one embodiment of the above host vector system, the host cell includes but is not limited to a eukaryotic cell, a hematopoietic cell, a B cell, a bacterial cell and E. Coli.

This invention provides a method of producing a polypeptide which comprises growing the above host vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.

This invention further provides an isolated nucleic acid comprising consecutive nucleotides having the sequence of a promoter of latency-associated nuclear antigen 2 transcription. In one embodiment of this nucleic acid, the nucleic acid comprises consecutive nucleotides having the sequence set forth in SEQ ID NO: 3. The promoter of the subject invention is capable of driving the expression of any gene in a B cell. Accordingly, this permits one skilled in the art to study gene expression in certain cells, such as B cells, since there will be expression of the protein in the B cell. In a transgenic animal, the gene would be expressed in the B cells of the animal.

This invention provides a replicable vector which comprises the isolated nucleic acid comprising consecutive nucleotides having the sequence of a promoter of latency-associated nuclear antigen 2 transcription operably linked to a second nucleic acid. This second nucleic acid is one which encodes a protein or gene of interest. As used herein, the term "operably linked" refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence.

The vector of the subject invention includes but is not limited to a plasmid, cosmid, .lambda. phage and YAC.

This invention provides a host vector system which comprises a replicable vector which comprises the nucleic acid comprising consecutive nucleotides having the sequence of a promoter of latency-associated nuclear antigen 2 transcription operably linked to a second nucleic acid and a suitable host cell. The host cell includes but is not limited to a eukaryotic cell, a hematopoietic cell, a B cell, a bacterial cell and E. Coli.

This invention provides a method of producing a polypeptide which comprises growing the above host vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.

This invention provides an isolated nucleic acid capable of specifically hybridizing to the isolated nucleic acid which encodes a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof. This invention also provides a nucleic acid capable of specifically hybridizing to the isolated nucleic acid comprising nucleotides having the sequence of a promoter of latency-associated nuclear antigen 2 transcription. The above nucleic acids include but are not limited to DNA, RNA, mRNA, synthetic DNA, genomic DNA, and cDNA. The phrase "specifically hybridizing" and the phrase "selectively hybridizing" describe a nucleic acid that hybridizes, duplexes or binds only to a particular target DNA or RNA sequence when the target sequences are present in a preparation of total cellular DNA or RNA. By selectively hybridizing it is meant that a nucleic acid binds to a given target in a manner that is detectable in a different manner from non-target sequence under high stringency conditions of hybridization. "Complementary", "antisense" or "target" nucleic acid sequences refer to those nucleic acid sequences which selectively and specifically hybridize to a nucleic acid. Proper annealing conditions depend, for example, upon a nucleic acid's length, base composition, and the number of mismatches and their position on the nucleic acid, and must often be determined empirically. For discussions of nucleic acid design and annealing conditions for hybridization, see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, Vols. 1-3 or Ausubel, F., et al. (1987) Current Protocols in Molecular Biology, New York. The above hybridizing nucleic acids may vary in length. The hybridizing nucleic acid length includes but is not limited to a nucleic acid of at least 15 nucleotides in length, of at least 25 nucleotides in length, or at least 50 nucleotides in length.

This invention provides a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof. This invention also provides a purified and/or an isolated polypeptide. In one embodiment of the above polypeptide, the nucleic acid comprises consecutive nucleotides having the sequence set forth in SEQ ID NO: 1. This invention provides an isolated polypeptide comprising consecutive amino acids having the amino acid sequence set forth in SEQ ID NO:2.

The phrase "isolated" or "purified" when referring to a polypeptide, means a composition which is essentially free of other cellular components. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified. Generally, a substantially purified or isolated protein will comprise more than 80% of all macromolecular species present in the preparation. Preferably, the protein is purified to represent greater than 90% of all macromolecular species present. More preferably the protein is purified to greater than 95%, and most preferably the protein is purified to essential homogeneity, wherein other macromolecular species are not detected by conventional techniques.

This invention also provides an antibody capable of specifically binding to the above polypeptide. The antibody includes but is not limited to a monoclonal antibody or a polyclonal antibody. The polyclonal and monoclonal antibodies of the invention are immunoreactive with the peptides or immunogenic fragments of the peptides or functionally capable of binding an epitopic determinant of the peptides.

As used herein, "antibody" means an immunoglobulin molecule comprising two heavy chains and two light chains and which recognizes an antigen. The immunoglobulin molecule may derive from any of the commonly known classes, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgG1, IgG2, IgG3 and IgG4. It includes, by way of example, both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.

The phrase "specifically binding" refers to a binding reaction which is determinative of the presence of the LANA2 polypeptide of the invention in the presence of a heterogeneous population of polypeptides and other biologics including viruses other than KSHV. Thus, under designated immunoassay conditions, the specified antibodies bind to the LANA2 antigen and do not bind in a significant amount to other antigens present in the sample.

In one embodiment of the above antibody, the antibody is humanized. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgG1, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody would retain a similar antigenic specificity as the original antibody, i.e., in the present invention, the ability to bind CCR5.

One skilled in the art would know how to make the humanized antibodies of the subject invention. Various publications, several of which are hereby incorporated by reference into this application, also describe how to make humanized antibodies. For example, the methods described in U.S. Pat. No. 4,816,567 (45) comprise the production of chimeric antibodies having a variable region of one antibody and a constant region of another antibody.

U.S. Pat. No. 5,225,539 (46) describes another approach for the production of a humanized antibody. This patent describes the use of recombinant DNA technology to produce a humanized antibody wherein the CDRs of a variable region of one immunoglobulin are replaced with the CDRs from an immunoglobulin with a different specificity such that the humanized antibody would recognize the desired target but would not be recognized in a significant way by the human subject's immune system. Specifically, site directed mutagenesis is used to graft the CDRs onto the framework.

Other approaches for humanizing an antibody are described in U.S. Pat. No. 5,585,089 (47) and U.S. Pat. No. 5,693,761 (48) and WO 90/07861 which describe methods for producing humanized immunoglobulins. These have one or more CDRs and possible additional amino acids from a donor immunoglobulin and a framework region from an accepting human immunoglobulin. These patents describe a method to increase the affinity of an antibody for the desired antigen. Some amino acids in the framework are chosen to be the same as the amino acids at those positions in the donor rather than in the acceptor. Specifically, these patents describe the preparation of a humanized antibody that binds to a receptor by combining the CDRs of a mouse monoclonal antibody with human immunoglobulin framework and constant regions. Human framework regions can be chosen to maximize homology with the mouse sequence. A computer model can be used to identify amino acids in the framework region which are likely to interact with the CDRs or the specific antigen and then mouse amino acids can be used at these positions to create the humanized antibody.

The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 (49) also propose four possible criteria which may used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 .ANG. of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies.

In one embodiment, the antibody of the subject invention is detectable. In one embodiment of the above antibody, the detectable antibody is labeled with a detectable marker as described above.

This invention provides a composition comprising the above antibody and an agent conjugated to the antibody. In one embodiment, the agent is a radioactive isotope or toxin.

This invention provides a method of determining whether a subject is afflicted with a disease associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection of a B cell which comprises: (a) obtaining a suitable sample from the subject; (b) contacting the suitable sample with a detectable antibody capable of binding to Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof so as to form a complex between the antibody and any Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof present in the sample; (c) removing any unbound antibody; and (d) detecting any antibody which is bound to any Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof in the sample, wherein the presence of antibody indicates that the subject is afflicted with the disease associated with Kaposi's sarcoma-associated herpesvirus infection of a B cell.

This invention provides a method of determining whether a subject is afflicted with a disease associated with Kaposi's sarcoma-associated herpesvirus infection of a B cell which comprises:(a) obtaining a suitable sample from the subject; (b) immobilizing a capturing antibody wherein the capturing antibody is capable of binding to Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 to a support; (c) removing any unbound capturing antibody; (d) contacting the capturing antibody with the suitable sample so as to form a complex between the antibody and any Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 present in the sample; (e) removing any unbound sample; (f) contacting the complex obtained in step (d) with a detectable antibody of Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof so as to form a complex between the detectable antibody and the complex; (g) removing any unbound detectable antibody; and (h) detecting any detectable antibody which is bound to the complex wherein the presence of detectable antibody indicates that the subject is afflicted with the disease associated with Kaposi's sarcoma-associated herpesvirus infection of a B cell.

The disease in the above methods includes but is not limited to Castleman's disease and Primary Effusion Lymphoma. The disease may also be one not associated with a B cell.

This invention provides a method of determining whether a subject is infected with Kaposi's sarcoma-associated herpesvirus which comprises:(a) obtaining a suitable sample from the subject; (b) contacting the suitable sample with the detectable antibody of Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof so as to form a complex between the antibody and any polypeptide or fragment thereof present in the sample; (c) removing any unbound antibody; and (d) detecting any antibody which is bound to any Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof in the sample, wherein the presence of antibody indicates that the subject is infected with Kaposi's sarcoma-associated herpesvirus.

This invention provides a method of determining whether a subject is infected with Kaposi's sarcoma-associated herpesvirus which comprises:(a) obtaining a suitable sample from the subject; (b) immobilizing a capturing antibody wherein the capturing antibody is capable of binding to polypeptide or fragment thereof to a support; (c) removing any unbound capturing antibody;(d) contacting the capturing antibody with the suitable sample so as to form a complex between the antibody and Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof present in the sample; (e) removing any unbound sample; (f) contacting the complex obtained in step (d) with the detectable antibody which is bound to Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof so as to form a complex between the detectable antibody and the complex; (g) removing any unbound detectable antibody; and (h) detecting any detectable antibody which is bound to the complex wherein the presence of detectable antibody indicates that the subject is infected with Kaposi's sarcoma-associated herpesvirus.

The suitable sample includes but is not limited to tonsil tissue, lymph nodes, spleen, skin lesions, blood, serum, plasma, cerebrospinal fluid, lymphocytes, urine, transudates, exudates, bone marrow cells, or supernatant from a cell culture.

In one embodiment of the above method, the antigen bound by the antibody is detected by an immunoassay. The immunoassay of the above method includes but is not limited to ELISA, IFA, and Western blotting.

As used herein, "capturing antibody" refers to an antibody capable of binding a polypeptide, a second antibody or a complex comprising an antibody and a polypeptide as described above. In one embodiment, a capturing antibody binds to a different epitope on the target protein than the detecting antibody.

As used herein, "support" includes but is not limited to a solid surface, a bead, a column, a plastic dish, a plastic plate, a microscope slide, and a nylon membrane. The use of these and other supports are known by one skilled in the art.

This invention provides a kit for diagnosing Kaposi's sarcoma-associated herpesvirus infection comprising the labeled antibody capable of specifically binding to the Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof. In one embodiment of the above kit, the kit further comprises a means for determining the level of Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof bound by an antibody. In one embodiment of the above kit, the labeled antibody capable of specifically binding to the polypeptide encoded by the isolated nucleic acid of Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof is bound to a support.

Studies have shown that LANA2 polypeptide can inhibit p53 mediated apoptosis. Accordingly, this invention provides a method of inhibiting p53 mediated apoptosis of a cell which comprises introducing into the cell an effective amount of the replicable vector which comprises the isolated nucleic acid which encodes Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof, so as to thereby inhibit p53 mediated apostosis of the cell.

This invention provides a method of immortalizing a cell which comprises introducing into the cell an amount of the replicable vector which comprises the isolated nucleic acid which encodes Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof effective to inhibit p53 mediated apoptosis of the cell, so as to thereby immortalize the cell.

As used herein, "immortalizing" refers to the action of LANA2 polypeptide in a B cell wherein the LANA2 polypeptide interacts with the p53 mediated apoptosis pathway to inhibit the action of p53 in the cell. The above interaction does not allow the cell to die, thereby creating an "immortalized" cell.

This invention provides a method of producing an antibody which comprises introducing into a cell an amount of the replicable vector which comprises the isolated nucleic acid which encodes Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide effective to inhibit p53 mediated apoptosis of the cell producing the antibody and thereby immortalizing the cell, so as to thereby produce the antibody. An application of this method is to immortalize a cell which produces an antibody so to thereby increase production of the antibody.

The cell in the above methods includes but is not limited to a hematopoietic tissue cell, and a B cell.

As used herein, the term "introducing into a cell" includes but is not limited to transduction and transfection. Transfection can be achieved by calcium phosphate co-precipitates, conventional mechanical procedures such as micro-injection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors or any other method known to one skilled in the art. This invention provides an antibody produced by the above method.

This invention provides a method of determining whether a subject is infected with Kaposi's sarcoma-associated herpesvirus which comprises: (a) obtaining a suitable sample from the subject; (b) contacting the suitable sample with a detectable nucleic acid capable of hybridizing to a nucleic acid which encodes Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof under hybridizing conditions so as to form a complex between the detectable nucleic acid and any nucleic acid which encodes a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof which is present in the sample; (c) removing any unbound detectable nucleic acid; and (d) detecting any detectable nucleic acid which is bound to the complex, wherein the presence of detectable nucleic acid indicates that the subject is infected with Kaposi's sarcoma-associated herpesvirus.

In one embodiment of the above method, the suitable sample includes but is not limited to tonsil tissue, lymph nodes, spleen, skin lesions, blood, serum, plasma, cerebrospinal fluid, lymphocytes, urine, transudates, exudates, bone marrow cells, or supernatant from a cell culture.

In one embodiment of the above methods, the subject is a mouse, rat, dog, guinea pig, ferret, rabbit, primate, and human. As used herein, "subject" means any animal or artificially modified animal capable of becoming KSHV infected. Artificially modified animals include, but are not limited to, SCID mice with human immune systems. The subjects include but are not limited to mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates. In the preferred embodiment, the subject is a human being.

This invention provides a kit for diagnosing Kaposi's sarcoma-associated herpesvirus infection comprising a labeled nucleic acid which encodes a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof. In one embodiment of the above kit, the kit further comprises a means for determining the level of sample bound to the above labeled nucleic acid. In one embodiment of the above kit, the above labeled nucleic acid is bound to a support.

This invention provides a transgenic non-human animal which has stably integrated into the genome of its germ cells or somatic cells an exogenous nucleic acid construct wherein the nucleic acid construct comprises a B-cell specific promoter of Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 operably linked to a second nucleic acid which encodes a gene of interest and is introduced into the transgenic non-human animal, or an ancestor, at an embryonic stage. In one embodiment of the above transgenic animal, the animal is a mammal. In one embodiment of the above transgenic animal, the non-human animal is a mouse, a rat, a sheep, a dog, a primate, or a reptile.

This invention provides a method for evaluating in a non-human transgenic animal the potential therapeutic effect of an agent for treating Kaposi's sarcoma-associated herpesvirus infection in a human, which comprises: (a) providing an agent to a transgenic non-human animal whose cells comprise the nucleic acid which encodes a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide; and (b) determining the therapeutic effect of the agent on the transgenic non-human animal by monitoring Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 expression, wherein a decrease in Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 indicates that the agent would have a potential therapeutic effect on Kaposi's sarcoma-associated herpesvirus infection in a human. In one embodiment of the above method, the animal is a mammal. In one embodiment of the above method, the non-human animal is a mouse, a rat, a sheep, a dog, a primate, or a reptile.

The following U.S. Patents are hereby incorporated by reference: U.S. Pat. No. 6,025,539, IL-5 transgenic mouse; U.S. Pat. No. 6,023,010, Transgenic non-human animals depleted in a mature lymphocytic cell-type; U.S. Pat. No. 6,018,098, In vivo and in vitro model of cutaneous photoaging; U.S. Pat. No. 6,018,097, Transgenic mice expressing human insulin; U.S. Pat. No. 6,008,434, Growth differentiation factor-11 transgenic mice; U.S. Pat. No. 6,002,066; H2-M modified transgenic mice; U.S. Pat. No. 5,994,618, Growth differentiation factor-8 transgenic mice; U.S. Pat. No. 5,986,171, Method for examining neurovirulence of polio virus, U.S. Pat. No. 5,981,830, Knockout mice and their progeny with a disrupted hepsin gene; U.S. Pat. No. 5,981,829, .DELTA.Nur77 transgenic mouse; U.S. Pat. No. 5,936,138; Gene encoding mutant L3T4 protein which facilitates HIV infection and transgenic mouse expressing such protein; U.S. Pat. No. 5,912,411, Mice transgenic for a tetracycline-inducible transcriptional activator; U.S. Pat. No. 5,894,078, Transgenic mouse expressing C-100 app.

The methods used for generating transgenic animals are well known to one of skill in the art. For example, one may use the manual entitled "Manipulating the Mouse Embryo" by Brigid Hogan et al. (Ed. Cold Spring Harbor Laboratory) 1986.

See for example, Leder and Stewart, U.S. Pat. No. 4,736,866 for methods for the production of a transgenic mouse.

For sometime it has been known that it is possible to carry out the genetic transformation of a zygote (and the embryo and mature organism which result therefrom) by the placing or insertion of exogenous genetic material into the nucleus of the zygote or to any nucleic genetic material which ultimately forms a part of the nucleus of the zygote. The genotype of the zygote and the organism which results from a zygote will include the genotype of the exogenous genetic material. Additionally, the inclusion of exogenous genetic material in the zygote will result in a phenotype expression of the exogenous genetic material.

The genotype of the exogenous genetic material is expressed upon the cellular division of the zygote. However, the phenotype expression, e.g., the production of a protein product or products of the exogenous genetic material, or alterations of the zygote's or organism's natural phenotype, will occur at that point of the zygote's or organism's development during which the particular exogenous genetic material is active. Alterations of the expression of the phenotype include an enhancement or diminution in the expression of a phenotype or an alteration in the promotion and/or control of a phenotype, including the addition of a new promoter and/or controller or supplementation of an existing promoter and/or controller of the phenotype.

The genetic transformation of various types of organisms is disclosed and described in detail in U.S. Pat. No. 4,873,191, issued Oct. 10, 1989, which is incorporated herein by reference to disclose methods of producing transgenic organisms. The genetic transformation of organisms can be used as an in vivo analysis of gene expression during differentiation and in the elimination or diminution of genetic diseases by either gene therapy or by using a transgenic non-human mammal as a model system of a human disease. This model system can be used to test putative drugs for their potential therapeutic value in humans.

The exogenous genetic material can be placed in the nucleus of a mature egg. It is preferred that the egg be in a fertilized or activated (by parthenogenesis) state. After the addition of the exogenous genetic material, a complementary haploid set of chromosomes (e.g., a sperm cell or polar body) is added to enable the formation of a zygote. The zygote is allowed to develop into an organism such as by implanting it in a pseudopregnant female. The resulting organism is analyzed for the integration of the exogenous genetic material. If positive integration is determined, the organism can be used for the in vivo analysis of the gene expression, which expression is believed to be related to a particular genetic disease.

The "transgenic non-human animals" of the invention may be produced by introducing "transgenes" into the germline of the non-human animal. Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The zygote is the best target for micro-injection. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of 1-2 pl of DNA solution. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster, et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4438-4442). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene. Microinjection of zygotes is the preferred method for incorporating transgenes in practicing the invention.

Retroviral infection can also be used to introduce transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) Proc. Natl. Acad. Sci U.S.A. 73, 1260-1264). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan, et al. (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner, et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6927-6931; Van der Putten, et al. (1985) Proc. Natl. Acad. Sci U.S.A. 82, 6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart, et al. (1987) EMBO J. 6, 383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner, D., et al. (1982) Nature 298, 623-628). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by intrauterine retroviral infection of the midgestation embryo (Jahner, D. et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans, M. J., et al. (1981) Nature 292, 154-156; Bradley, M. O., et al. (1984) Nature 309, 255-258; Gossler, et al. (1986) Proc. Natl. Acad. Sci U.S.A. 83, 9065-9069; and Robertson, et al. (1986) Nature 322, 445-448). Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. For review see Jaenisch, R. (1988) Science 240, 1468-1474.

As used herein, a "transgene" is a DNA sequence introduced into the germline of a non-human animal by way of human intervention such as by way of the above described methods.

This invention provides a method of treating Kaposi's sarcoma-associated herpesvirus infection in a subject, which comprises introducing into the subject's cells an effective amount of the nucleic acid capable of specifically hybridizing to the isolated nucleic acid which encodes Kaposi's sarcoma-associated latency-associated nuclear antigen 2 polypeptide or fragment thereof to hybridize to any of the above nucleic acid which is present in the subject's cells, so as to thereby treat Kaposi's sarcoma-associated herpesvirus infection. An application of this method is to inhibit transcription of Kaposi's sarcoma-associated latency-associated nuclear antigen 2 polypeptide or fragment thereof, thereby treating the subject.

As used herein, "effective amount" means an amount in sufficient quantities to either treat the subject or prevent the subject from becoming infected with Kaposi's sarcoma-associated herpesvirus. A person of ordinary skill in the art can perform simple titration experiments to determine what amount is required to treat the subject.

The subject invention has various applications which includes KSHV treatment such as treating a subject who has become afflicted with KSHV. As used herein, "afflicted with the disease" means that the subject has at least one cell which has been infected by KSHV. As used herein, "treating" means either slowing, stopping or reversing the progression of an HIV-1 disorder. In the preferred embodiment, "treating" means reversing the progression to the point of eliminating the disorder. As used herein, "treating" also means the reduction of the number of viral infections, reduction of the number of infectious viral particles, reduction of the number of virally infected cells, or the amelioration of symptoms associated with KSHV.

Another application of the subject invention is to prevent a subject from contracting KSHV. As used herein, "contracting KSHV" means becoming infected with KSHV, whose genetic information replicates in and/or incorporates into the host cells. Another application of the subject invention is to treat a subject who has become infected with KSHV.

As used herein, "KSHV infection" means the introduction of KSHV genetic information into a target cell, such as by fusion of the target cell membrane with KSHV or an KSHV envelope glycoprotein cell. The target cell may be a bodily cell of a subject. In the preferred embodiment, the target cell is a bodily cell from a human subject.

This invention provides a method of treating Kaposi's sarcoma-associated herpesvirus infection in a subject, which comprises introducing into the subject's cells an effective amount of a nucleic acid capable of specifically hybridizing to an isolated nucleic acid comprising nucleotides having the sequence of a promoter of latency-associated nuclear antigen 2 transcription to hybridize to any of this nucleic acid which is present in the subject's cells, so as to thereby treat the subject. An application of this method is to hybridize a nucleic acid to the above promoter, thereby inhibiting Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide expression, thereby treating Kaposi's sarcoma-associated herpesvirus infection in the subject.

This invention provides a composition comprising the antibody capable of specifically binding to the polypeptide encoded by the isolated nucleic acid which encodes Kaposi's sarcoma-associated latency-associated nuclear antigen 2 polypeptide and a carrier.

As used herein, "carriers" include but are not limited to aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. The carriers include but are not limited to an aerosol, intravenous, oral or topical carrier. Carriers are well known to those skilled in the art.

As used herein, "composition" means a mixture. The compositions include but are not limited to those suitable for oral, rectal, intravaginal, topical, nasal, opthalmic, or parenteral administration to a subject. As used herein, "parenteral" includes but is not limited to subcutaneous, intravenous, intramuscular, or intrasternal injections or infusion techniques.

This invention provides a method of treating a subject infected with Kaposi's sarcoma-associated herpesvirus, which comprises administering to the subject an amount of the above composition under conditions such that the antibody binds to any LANA2 present in the subject, so as to thereby treat the subject.

This invention provides a composition comprising the polypeptide encoded by the isolated nucleic acid which encodes Kaposi's sarcoma-associated latency-associated nuclear antigen 2 polypeptide or fragment thereof and a carrier.

In one embodiment of the methods of this invention, the cell is present in a subject and the contacting is effected by administering the compound to the subject.

The subject invention has therapeutic applications. For example, one skilled in the art can target a latency associated gene, such as the gene which encodes the LANA2 polypeptide, so as to inactivate the gene and thereby treat the KSHV infection or other diseases associated with KSHV infection, such as the B-cell associated diseases Castleman's disease or Primary Effusion Lymphoma. One can use antisense technology in order to inhibit the expression of a gene, such as LANA2. One can also use monoclonal antibody technology so as to degrade or sequester the protein, such as the LANA2 protein.

Claim 1 of 3 Claims

What is claimed is:

1. An isolated nucleic acid comprising the sequence of the promoter of latency-associated nuclear antigen 2 transcription as set forth in SEQ ID NO: 3.



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