AU700913B2 - Unique associated kaposi's sarcoma virus sequences and uses thereof - Google Patents

Description

-1- UNIQUE ASSOCIATED KAPOSI'S SARCOMA VIRUS SEQUENCES AND USES

THEREOF

ooooo S"Throughout this application, various publications may be referenced by Arabic numerals in brackets. Full citations for these publications may be found at the end of each 20 Experimental Details Section. The disclosures of the publications cited herein are in their entirety hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers." BACKGROUND OF THE INVENTION Kaposi's sarcoma (KS) is the most common neoplasm occurring in persons with acquired immunodeficiency syndrome (AIDS).

SApproximately 15-20% of AIDS patients develop this neoplasm which rarely occurs in immunocompetent individuals [13, 14]. Epidemiologic evidence suggests that AIDS-associated KS (AIDS-KS) has an infectious etiology. Gay and bisexual AIDS patients are approximately twenty times more likely than hemophiliac AIDS patients to develop KS, and KS may be associated with specific sexual practices among gay men with AIDS 15, 55, 83]. KS is uncommon among adult AIDS patients infected through heterosexual or parenteral HIV transmission, or among pediatric AIDS patients infected through vertical HIV transmission Agents previously suspected of causing KS include cytomegalovirus, hepatitis B virus, human papillomavirus, Epstein-Barr virus, human herpesvirus 6, human immunodeficiency virus (HIV) and Mycoplasma penetrans [18, 23, 85, 91, 92]. Non-infectious 15 environmental agents, such as nitrite inhalants, also have been proposed to play a role in KS tumorigenesis [33].

Extensive investigations, however, have not demonstrated an etiologic association between any of these agents and AIDS- KS [37, 44, 46, WO 96/06159 PCT/US95/10194 3 SUMMARY OF THE INVENTION This invention provides an isolated DNA molecule which is at least 30 nucleotides in length and which uniquely defines a herpesvirus associated with Kaposi's sarcoma. This invention provides an isolated herpesvirus associated with Kaposi's sarcoma.

This invention provides a method of vaccinating a subject for KS, prophylaxis diagnosing or treating a subject with KS and detecting expression of a DNA virus associated with Kaposi's sarcoma in a cell.

WO 96/06159 PCT/US95/10194 4 BRIEF DESCRIPTION OF THE FIGURES Fiqure 1: Agarose gel electrophoresis of RDA products from AIDS-KS tissue and uninvolved tissue. RDA was performed on DNA extracted from KS skin tissue and uninvolved normal skin tissue obtained at autopsy from a homosexual man with AIDS-KS. Lane 1 shows the initial PCR amplified genomic representation of the AIDS-KS DNA after Bam HI digestion. Lanes 2-4 show that subsequent cycles of ligation, amplification, hybridization and digestion of the RDA products resulted in amplification of discrete bands at 380, 450, 540 and 680 bp. RDA of the extracted AIDS-KS DNA performed against itself resulted in a single band at 540 bp (lane Bands at 380 bp and 680 bp correspond to KS330Bam and KS627Bam respectively after removal of 28 bp priming sequences. Bands at 450 and 540 bp hybridized nonspecifically to both KS and non-KS human DNA.

Lane M is a molecular weight marker.

Fiqures 2A-2B: Hybridization of 32 P-labelled KS330Bam (Figure 2A) and KS627Bam (Figure 2B) sequences to a representative panel of 19 DNA samples extracted from KS lesions and digested with Bam HI.

KS330Bam hybridized to 11 of the 19 and KS627Bam hybridized to 12 of the 19 DNA samples from AIDS- KS lesions. Two additional cases (lanes 12 and 13) were shown to have faint bands with both KS330Bam and KS627Bam probes after longer exposure. One negative specimen (lane 3) did not have microscopically detectable KS in the tissue WO 96/06159 PCT/US95/10194 specimen. Seven of 8 additional KS DNA samples also hybridized to both sequences.

Fiqures 3A-3F: Nucleotide sequences of the DNA herpesvirus associated with KS (KSHV).

Figures 4A-4B: PCR amplification of a representative set of KSderived DNA samples using KS330 23 4 primers.

Figure 4A shows the agarose gel of the amplification products from 19 KS DNA samples (lanes 1-19) and Figure 4B shows specific hybridization of the PCR products to a 32 P endlabelled 25 bp internal oligonucleotide (Figure 3B) after transfer of the gel to a nitrocellulose filter. Negative samples in lanes 3 and respectively lacked microscopically detectable KS in the sample or did not amplify the constitutive p53 exon 6, suggesting that these samples were negative for technical reasons. An additional 8 AIDS-KS samples were amplified and all were positive for KS330 234 Lane 20 is a negative control and Lane M is a molecular weight marker.

Figure Southern blot hybridization of KS330Bam and KS627Bam to AIDS-KS genomic DNA extracted from three subjects (lanes 1, 2, and 3) and digested with PvuII. Based on sequence information (Figure 3A), restricted sites for Pvu II occur between bp 12361-12362 of the KSHV sequence (Figure 3A, SEQ ID NO: at bp 134 in KS330Bam (Figure 3B, SEQ ID NO: 2) and bp 414 in KS627Bam (Figure 3C, SEQ ID NO: KS330Bam and KS627Bam failed to hybridize to the same fragments in the digests indicating that the two sequences are WO 96/06159 PCTfUS95/10194 6 separated from each other by one or more intervening Bam HI restriction fragments.

Digestion with Pvu II and hybridization to KS330Bam resulted in two distinct banding patterns (lanes 1 and 2 vs. lane 3) suggesting variation between KS samples.

Figure 6: Comparison of amino acid homologies between EBV ORF BDLF1, HSVSA ORF 26 and a 918 bp reading frame of the Kaposi's sarcoma agent which includes KS330Bam. Amino acid identity is denoted by reverse lettering. In HSVSA, ORF 26 encodes a minor capsid VP23 which is a late gene product.

Fiqure 7: Subculture of Raji cells co-cultivated with BCBL- 1 cells treated with TPA for 2 days. PCR shows that Raji cells are positive for KSHV sequences and indicate that the agent is a transmissible virus.

Figure 8: A schematic diagram of the orientation of KSHV open reading frames identified on the KS5 20,710 bp DNA fragment. Homologs to each open reading frame from a corresponding region of the herpesvirus saimiri (HSVSA) genome are present in an identical orientation, except for the region corresponding to the ORF 28 of HSVSA (middle schematic section). The shading for each open reading frame corresponds to the approximate amino acid identity for the KSHV ORF compared to this homolog in HSVSA. Noteworthy homologs that are present in this section of DNA include homologs to thymidine kinase (ORF21), gH WO 96/06159 PCT/US95/10194 7 glycoprotein (ORF22), major capsid protein and the VP23 protein (ORF26) which contains the original KS330Bam sequence derived by representational difference analysis.

Figure 9: The -200 kD antigen band appearing on a Western blot of KS patient sera against BCBL1 lysate (Bl) and Raji lysate M is molecular weight marker. The antigen is a doublet between ca. 210 kD and 240 kD.

Fiqure control patient sera without KS (A1N, A2N, A3N, A4N and A5N). Bl=BCBL1 lysate, RA=Raji lysate.

The 220 kD band is absent from the Western blots using patient sera without KS.

Ficure 11: In this figure, 0.5 ml aliquots of the gradient have been fractionated (fractions 1-62) with the gradient fraction being at fraction No. 1 and the 10% gradient fraction being at fraction No.

62. Each fraction has been dot hybridized to a nitrocellulose membrane and then a "P-labeled KSHV DNA fragment, KS631Bam has been hybridized to the membrane using standard techniques. The figure shows that the major solubilized fraction of the KSHV genome bands is isolated) in fractions 42 through 48 of the gradient with a high concentration of the genome being present in fraction 44. A second band of solubilized KSHV DNA occurs in fractions 26 through 32.

Fiqure 12: Location, feature, and relative homologies of open reading frames compared to translation WO 96/06159 PCTUS95/10194 8 products of herpesvirus saimiri (HSV), equine herpesvirus 2 (EHV2) and Epstein-Barr virus

(EBV).

Fiqure 13: Indirect immunofluorescence end-point and geometric mean titers (GMT) in AIDS-KS and AIDS control sera against BHL-6 and P3H3 prior to and after adsorption with P3H3.

Figure 14: Genetic map of KS5, a 20.7 kb lambda phage clone insert derived from a human genomic library prepared from an AIDS-KS lesion. Seventeen partial and complete open reading frames (ORFs) are identified with arrows denoting reading frame orientations. Comparable regions of the Epstein- Barr virus (EBV) and herpesvirus saimiri (HVS) genomes are shown for comparison. Levels of amino acid similarity between KSHV ORFs are indicated by shading of EBV and HVS ORFs (black, over 70% similarity; dark gray, 55-70% similarity; light gray, 40-54% similarity; white, no detectable homology). Domains of conserved herpesvirus sequence blocks and locations of restriction endonuclease sites used in subcloning are shown beneath the KSHV map Bam HI site; N, Not I site). The small Bam HI fragment (black) in the VP23 gene homolog corresponds to the KS330Bam fragment generated by representational difference analysis which was used to identify the KS5 lambda phage clone.

Fiqures 15A-15B: Phylogenetic trees of KSHV based on comparison of aligned amino acid sequences between herpesviruses for the MCP gene and for a WO 96/06159 PCTI/US95/10194 9 concatenated nine-gene set. The comparison of MCP sequences (Figure 15A) was obtained by the neighbor-joining method and is shown in unrooted form with branch lengths proportional to divergence (mean number of substitution events per site) between the nodes bounding each branch.

Comparable results were obtained by maximum parsimony analysis. The number of times out of 100 bootstrap samplings the division indicated by each internal branch was obtained are shown next to each branch; bootstrap values below 75 are not shown. Figure 15B is a phylogenetic tree of gammaherpesvirus sequences based on a nine-gene set CS1 (see text) and demonstrates that KSHV is most closely related to the gamma-2 herpesvirus sublineage, genus Rhadinovirus. The CS1 amino acid sequence was used to infer a tree by the Protml maximum likelihood method; comparable results, not shown were obtained with the neighbor-joining and maximum parsimony methods.

The bootstrap value for the central branch is marked. On the basis of the MCP analysis, the root must lie between EBV and the other three species. Abbreviations for virus species used in the sequence comparisons are 1) Alphaherpesvirinae: HSV1 and HSV2, herpes simplex virus types 1 and 2; EHV1, equine herpesvirus 1; PRV, pseudorabies virus; and VZV, varicella-zoster virus, 2) Betaherpesvirinae: HCMV, human cytomegalovirus; HHV6 and HHV7, human herpesviruses 6 and 7, and 3) Gammaherpesvirinae: HVS, herpesvirus saimiri; EHV2, equine herpesvirus 2; EBV, Epstein-Barr virus; and Kaposi's sarcoma-associated herpesvirus.

WO 96/06159 PCT[UTS95/10194 Fiqures 16A-16B: CHEF gel electrophoresis of BCBL-1 DNA hybridized to KS631Bam (Figure 16A) and EBV terminal repeat (Figure 16B). KS631Bam hybridizes to a band at 270 kb as well as to a diffuse band at the origin. The EBV termini sequence hybridizes to a 150-160 kb band consistent with the linear form of the genome. Both KS631Bam (dark arrow) and an EBV terminal sequence hybridize to high molecular weight bands immediately below the origin indicating possible concatemeric or circular DNA.

The high molecular weight KS631Bam hybridizing band reproduces poorly but is visible on the original autoradiographs.

Fiqure 17: Induction of KSHV and EBV replication in BCBL-1 with increasing concentrations of TPA. Each determination was made in triplicate after 48 h of TPA incubation and hybridization was standardized to the amount of cellular DNA by hybridization to beta-actin. The figure shows the mean and range of relative increase in hybridizing genome for EBV and KSHV induced by TPA compared to uninduced BCBL-1. TPA at ng/ml induced an eight-fold increase in EBV genome (upper line) at 48 h compared to only a 1.4 fold increase in KSHV genome (lower line).

Despite the lower level of KSHV induction, increased replication of KSHV genome after induction with TPA concentrations over 10 ng/ml was reproducibly detected.

Fiqures 18A-18C: In situ hybridization with an ORF26 oligomer to BCBL-1, Raji and RCC-1 cells. Hybridization occurred to nuclei of KSHV infected BCBL-1 WO 96/06159 PCT/US95/10194 11 (Figure 18A), but not to uninfected Raji cells (Figure 18B). RCC-1, a Raji cell line derived by cultivation of Raji with BCBL-1 in communicating chambers separated by a 0.45 A filter, shows rare cells with positive hybridization to the KSHV ORF26 probe (Figure 18C).

Figures 19A-19D: Representative example of IFA staining of BHL-6 with AIDS-KS patient sera and control sera from HIV-infected patients without KS. Both AIDS-KS (Figure 19A) and control (Figure 19B) sera show homogeneous staining of BHL-6 at 1:50 dilution.

After adsorption with paraformaldehyde-fixed P3H3 to remove cross-reacting antibodies directed against lymphocyte and EBV antigens, antibodies from AIDS-KS sera localize to BHL-6 nuclei (Figure 19C). P3H3 adsorption of control sera eliminates immunofluorescent staining of BHL-6 (Figure 19D).

Figures 20A-20B: Longitudinal PCR examination for KSHV DNA of paired PBMC samples from AIDS-KS patients and homosexual/bisexual AIDS patients without KS Time 0 is the date of KS onset for cases or other AIDS-defining illness for controls. All samples were randomized and examined blindly. Overall, 7 of the KS patients were KSHV positive at both examination dates (solid bars) and 5 converted from a negative to positive PBMC sample (forward striped bars) immediately prior to or after KS onset. Two previously positive KS patients were negative after KS diagnosis (reverse striped bars) and the remaining KS patients were negative at both timepoints (open bars). Two homosexual/bisexual control PBMC samples without WO 96/06159 PCTUS95/10194 12 KS converted from negative to positive and one control patient reverted from PCR positive to negative for KSHV DNA.

Figure 21: Sample collection characteristics for AIDS-KS patients, gay/bisexual AIDS patients and hemophilic AIDS patients.

Figure 22: PCR analysis of KS330 233 in DNA samples from patients with Kaposi's sarcoma and tumor controls.

Figure 23: Characteristics of the study population of patients with KS and without KS.

Figure 24: Prevalence of antibody to KSHV p40 in HIV-1 positive patients with and without KS.

Figure Comparison of KS patients with and without antibody to KSHV Figure 26: Prevalence of antibody detectable by indirect immunofluorescence to KSHV antigens in chemically induced BCBL-1 cells in HIV-1 positive patients with and without KS.

Figures 27A-27B: Specific recognition of KSHV polypeptides in chemically treated BCBL-1 cells. Figure 27A shows reactivity of untreated BCBL-1 and B95-8 cells with RM, a reference human antibody to EBV.

WO 96/06159 PCT[S95/10194 13 RM recognizes the EBV polypeptides EBNA1 and p21 in the BCBL-1 cells. Figure 27B shows reactivity of untreated and chemically treated cells with serum 01-03 from a patient with KS. Cells were treated with TPA and n-butyrate for 48 hrs. For description of the cell lines see Materials and Methods. The immunoblots were prepared from SDS polyacrylamide gels.

Figures 28A-28D: Detection of KSHV p40 by sera from patients with KS. Extracts were prepared from BCBL-1 cells (containing KSHV and EBV) and Clone HH514-16 cells (containing only EBV) that were uninduced or treated for 48 hrs with chemical inducing agents, n-butyrate, TPA, or a combination of the two chemicals. Immunoblots prepared from 12% SDS polyacrylamide gels were reacted with a 1:200 dilution of serum from HIV-1 positive patients.

Figure 28A shows serum 01-06 from a patient with KS. Figure 28B shows serum 01-07 from a patient without KS. Figure 28C shows serum 04-01 from a patient with KS. Figure 28D shows serum 01-03 from a patient with KS.

Fiqures 29A-29F: Detection of KSHV lytic cycle antigens by indirect immunofluorescence. BCBL-1 cells were untreated (Figures 29A, 29C, and 29E) or treated with n-butyrate (Figures 29B, 29D, and 29F) for 48 hrs. Indirect immunofluorescence with a 1:10 dilution of serum from two patients with KS, 04- 18 (Figures 29A, and 29B) and 04-38 (Figures 29E, and 29F) and a serum, 04-37 (Figures 29C, and 29D), from a patient without KS.

WO 96/06159 PCT/US95/10194 14 DETAILED DESCRIPTION OF THE INVENTION Definitions The following standard abbreviations are used throughout the specification to indicate specific nucleotides: C=cytosine A=adenosine T=thymidine G=guanosine The term "nucleic acids", as used herein, refers to either DNA or RNA. "Nucleic acid sequence" or "polynucleotide 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 self-replicating plasmids, infectious polymers of DNA or RNA and nonfunctional DNA or RNA.

By a nucleic acid sequence "homologous to" or "complementary to", it is meant a nucleic acid that selectively hybridizes, duplexes or binds to viral DNA sequences encoding proteins or portions thereof when the DNA sequences encoding the viral protein are present in a human genomic or cDNA library. A DNA sequence which is homologous to a target sequence can include sequences which are shorter or longer than the target sequence so long as they meet the functional test set forth. Hybridization conditions are specified along with the source of the CDNA library.

Typically, the hybridization is done in a Southern blot protocol using a 0.2XSSC, 0.1% SDS, 650C wash.

The term "SSC" refers to a citrate-saline solution of 0.15 M sodium chloride and 20 Mm sodium citrate.

Solutions are often expressed as multiples or fractions of this concentration. For example, 6XSSC WO 96/06159 PCT/US95/10194 refers to a solution having a sodium chloride and sodium citrate concentration of 6 times this amount or 0.9 M sodium chloride and 120 mM sodium citrate.

0.2XSSC refers to a solution 0.2 times the SSC concentration or 0.03 M sodium chloride and 4 mM sodium citrate.

The phrase "nucleic acid molecule encoding" refers to a nucleic acid molecule which directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein. The nucleic acid molecule include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length protein. 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.

The phrase "expression cassette", refers to nucleotide sequences which are capable of affecting expression of a structural gene in hosts compatible with such sequences. Such cassettes include at least promoters and optionally, transcription termination signals.

Additional factors necessary or helpful in effecting expression may also be used as described herein.

The term "operably linked" as used herein refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence.

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 WO 96/06159 PCTIUS95/10194 16 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.

The term "plasmid" refers to an autonomous circular DNA molecule capable of replication in a cell, and includes both the expression and nonexpression types.

Where a recombinant microorganism or cell culture is described as hosting an "expression plasmid", this includes latent viral DNA integrated into the host chromosome(s) Where a plasmid is being maintained by a host cell, the plasmid is either being stably replicated by the cells during mitosis as an autonomous structure or is incorporated within the host's genome.

The phrase "recombinant protein" or "recombinantly produced protein" refers to a peptide or protein produced using non-native cells that do not have an endogenous copy of DNA able to express the protein.

The cells produce the protein because they have been genetically altered by the introduction of the appropriate nucleic acid sequence. The recombinant protein will not be found in association with proteins and other subcellular components normally associated with the cells producing the protein.

The following terms are used to describe the sequence relationships between two or more nucleic acid molecules or polynucleotides: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A WO 96/06159 PCT/US95/10194 17 "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing or may comprise a complete cDNA or gene sequence.

Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv.

Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA) 85:2444, or by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI).

As applied to polypeptides, the terms "substantial identity" or "substantial sequence identity" mean that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap which share at least 90 percent sequence identity, preferably at least 95 percent sequence identity, more preferably at least 99 percent sequence identity or more.

"Percentage amino acid identity" or "percentage amino acid sequence identity" refers to a comparison of the amino acids of two polypeptides which, when optimally aligned, have approximately the designated percentage of the same amino acids. For example, "95% amino acid identity" refers to a comparison of the amino acids of two polypeptides which when optimally aligned have amino acid identity. Preferably, residue positions which are not identical differ by conservative amino WO 96/06159 PCTIUS95/10194 18 acid substitutions. For example, the substitution of amino acids having similar chemical properties such as charge or polarity are not likely to effect the properties of a protein. Examples include glutamine for asparagine or glutamic acid for aspartic acid.

The phrase "substantially purified" or "isolated" when referring to a herpesvirus peptide or protein, means a chemical 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.

The phrase "specifically binds to an antibody" or "specifically immunoreactive with", when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the herpesvirus of the invention in the presence of a heterogeneous population of proteins and other biologics including viruses other than the herpesvirus. Thus, under designated immunoassay conditions, the specified antibodies bind to the herpesvirus antigens and do not bind in a significant WO 96/06159 PCT/US95/10194 19 amount to other antigens present in the sample.

Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised to the human herpesvirus immunogen described herein can be selected to obtain antibodies specifically immunoreactive with the herpesvirus proteins and not with other proteins. These antibodies recognize proteins homologous to the human herpesvirus protein. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane [32] for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

"Biological sample" as used herein refers to any sample obtained from a living organism or from an organism that has died. Examples of biological samples include body fluids and tissue specimens.

I. Kaposis's Sarcoma (KS) Associated Herpesvirus.

This invention provides an isolated DNA molecule which is at least 30 nucleotides in length and which uniquely defines a herpesvirus associated with Kaposi's sarcoma.

In one embodiment the isolated DNA molecule comprises at least a portion of the nucleic acid sequence as shown in Figure 3A (SEQ ID NO: In another embodiment the isolated DNA molecule is a 330 base pair (bp) sequence. In another embodiment the isolated DNA molecule is a 12-50 bp sequence. In WO 96/06159 PCT/US95/10194 another embodiment the isolated DNA molecule is a 37 bp sequence.

In another embodiment the isolated DNA molecule is genomic DNA. In another embodiment the isolated DNA molecule is cDNA. In another embodiment a RNA is derived form the isolated nucleic acid molecule or is capable of hybridizing with the isolated DNA molecule.

As used herein "genomic" means both coding and noncoding regions of the isolated nucleic acid molecule.

Further, the DNA molecule above may be associated with lymphoproliferative diseases including, but not limited to: Hodgkin's disease, non-Hodgkin's lymphoma, lymphatic leukemia, lymphosarcoma, splenomegaly, reticular cell sarcoma, Sezary's syndrome, mycosis fungoides, central nervous system lymphoma, AIDS related central nervous system lymphoma, posttransplant lymphoproliferative disorders, and Burkitt's lymphoma. A lymphoproliferative disorder is characterized as being the uncontrolled clonal or polyclonal expansion of lymphocytes involving lymph nodes, lymphoid tissue and other organs.

This invention provides an isolated nucleic acid molecule encoding an ORF20 (SEQ ID NOs: 22 and 23), ORF21 (SEQ ID NOs:14 and 15), ORF22 (SEQ ID NOs:16 and 17), ORF23 (SEQ ID NOs:18 and 19), ORF24 (SEQ ID NOs: and 21), ORF25 (SEQ ID NOs: 2 and ORF26 (SEQ ID NOs:24 and 25), ORF27 (SEQ ID NOs:26 and 27), ORF28 (SEQ ID NOs:28 and 29), ORF29A (SEQ ID NOs:30 and 31), ORF29B (SEQ ID NOs:4 and ORF30 (SEQ ID NOs:6 and ORF31 (SEQ ID NOs:8 and ORF32 (SEQ ID NOs:32 and 33), ORF33 (SEQ ID NOs: 10 and 11), ORF34 (SEQ ID NOs: 34 and 35), or ORF35 (SEQ ID NOs:12 AND 13).

WO 96/06159 PCT/US95/10194 21 This invention provides an isolated polypeptide encoded by ORF20 (SEQ ID NOs: 22 and 23), ORF21 (SEQ ID NOs:14 and 15), ORF22 (SEQ ID NOs:16 and 17), ORF23 (SEQ ID NOs:18 and 19), ORF24 (SEQ ID NOs: 20 and 21), ORF25 (SEQ ID NOs: 2 and ORF26 (SEQ ID NOs:24 and ORF27 (SEQ ID NOs:26 and 27), ORF28 (SEQ ID NOs:28 and 29), ORF29A (SEQ ID NOs:30 and 31), ORF29B (SEQ ID NOs:4 and ORF30 (SEQ ID NOs:6 and 7), ORF31 (SEQ ID NOs:8 and ORF32 (SEQ ID NOs:32 and 33), ORF33 (SEQ ID NOs: 10 and 11), ORF34 (SEQ ID NOs: 34 and 35), or ORF35 (SEQ ID NOs:12 AND 13).

For Example, TK is encoded by ORF 21; glycoprotein H (gH) by ORF 22; major capsid protein (MCP) by ORF virion polypeptide (VP23) by ORF 26; and minor capsid protein by ORF 27.

This invention provides for a replicable vector comprising the isolated DNA molecule of the DNA virus.

The vector includes, but is not limited to: a plasmid, cosmid, X phage or yeast artificial chromosome (YAC) which contains at least a portion of the isolated nucleic acid molecule.

As an example to obtain these vectors, insert and vector DNA can both be exposed to a restriction enzyme to create complementary ends on both molecules which base pair with each other and are then ligated together with DNA ligase. Alternatively, linkers can be ligated to the insert DNA which correspond to a restriction site in the vector DNA, which is then digested with the restriction enzyme which cuts at that site. Other means are also available and known to an ordinary skilled practitioner.

Regulatory elements required for expression include promoter or enhancer sequences to bind RNA polymerase WO 96/06159 PCT/US95/10194 22 and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors may be obtained commercially or assembled from the sequences described by methods well-known in the art, for example the methods described above for constructing vectors in general.

This invention provides a host cell containing the above vector. The host cell may contain the isolated DNA molecule artificially introduced into the host cell. The host cell may be a eukaryotic or bacterial cell (such as E.coli), yeast cells, fungal cells, insect cells and animal cells. Suitable animal cells include, but are not limited to Vero cells, HeLa cells, Cos cells, CV1 cells and various primary mammalian cells.

This invention provides an isolated herpesvirus associated with Kaposi's sarcoma. In one embodiment the herpesvirus comprises at least a portion of a nucleotide sequence as shown in Figures 3A (SEQ ID NO: 1).

In one embodiment the herpesvirus may be a DNA virus.

In another embodiment the herpesvirus may be a Herpesviridae. In another embodiment the herpesvirus may be a gammaherpesvirinae. The classification of the herpesvirus may vary based on the phenotypic or molecular characteristics which are known to those skilled in the art.

WO 96/06159 PCT/US95/10194 23 This invention provides an isolated DNA virus wherein the viral DNA is about 270 kb in size, wherein the viral DNA encodes a thymidine kinase, and wherein the viral DNA is capable of selectively hybridizing to a nucleic acid probe selected from the group consisting of SEQ ID NOs: 38-40.

The KS-associated human herpesvirus of the invention is associated with KS and is involved in the etiology of the disease. The taxonomic classification of the virus has not yet been made and will be based on phenotypic or molecular characteristics known to those of skill in the art. However, the novel KS-associated virus is a DNA virus that appears to be related to the Herpesviridae family and the gammaherpesvirinae subfamily, on the basis of nucleic acid homology.

A. Seauence identity of the viral DNA and its proteins.

The human herpesvirus of the invention is not limited to the virus having the specific DNA sequences described herein. The KS-associated human herpesvirus DNA shows substantial sequence identity, as defined above, to the viral DNA sequences described herein. DNA from the human herpesvirus typically selectively hybridizes to one or more of the following three nucleic acid probes: Probe 1 (SEQ ID NO:38) AGCCGAAAGG ATTCCACCAT TGTGCTCGAA TCCAACGGAT TTGACCCCGT GTTCCCCATG GTCGTGCCGC AGCAACTGGG GCACGCTATT CTGCAGCAGC TGTTGGTGTA CCACATCTAC TCCAAAATAT CGGCCGGGGC CCCGGATGAT GTAAATATGG CGGAACTTGA TCTATATACC ACCAATGTGT CATTTATGGG GCGCACATAT CGTCTGGACG TAGACAACAC GGA WO 96/06 159 PTU9/09 PCTfUS95/10194 Probe 2 (SEQ ID NO:39): GAAATTACCC ACGAGATCGC TTCCCTGCAC ACCGCACTTG AGTCATCGCC CCGGCCCACG TGGCCGCCAT AACTACAGAC ATTGTCAGGA CCTCTTTATG ATTTTCCCAG GGGACGCGTA CAGCTGCATG ACTATATCAA AATGAA.AGCG GGCGTGCAAA GGGAAACAGA ATGGATCACG TGGGATACAC TGCTGGGGTT AGAACCTGCC CGGTTTGAGT CATGGTCAGC TGGCAACCTG CCCACGCCGG TCACATCTGA CGTTGCCT

GCTACTCATC

ATGGGAGTAC

TCAGGACCGC

CCGGCTCACC

CCTCGCTGCG

CGAGATAATT

Probe 3 (SEQ ID NO: AACACGTCAT GTGCAGGAGT GACATTGTGC CGCGGAGAAA CTCAGACCGC ATCCCGTAAC CACACTGAGT GGGAAAATCT GCTGGCTATG TTTTCTGTGA TTATCTATGC CTTAGATCAC AACTGTCACC CG Hybridization of a viral DNA to the nucleic acid probes listed above is determined by using standard nucleic acid hybridization techniques as described herein. In particular, PCR amplification of a viral genome can be carried out using the following three sets of PCR primers: 1) AGCCGAAAGGATTCCACCAT; TCCGTGTTGTCTACGTCCAG (SEQ ID NO: 41) 2) GAAATTACCCACGAGATCGC; AGGCAACGTCAGATGTGA (SEQ ID NO: 42) 3) AACACGTCATGTGCAGGAGTGAC; CGGGTGACAGTTGTGATCTAAGG (SEQ ID NO:43) In PCR techniques, oligonucleotide primers, as listed above, complementary to the two 3' borders of the DNA region to be amplified are synthesized. The WO 96/06159 PCTIUTS95/10194 polymerase chain reaction is then carried out using the two primers. See PCR Protocols: A Guide to Methods and Applications Following PCR amplification, the PCR-amplified regions of a viral DNA can be tested for their ability to hybridize to the three specific nucleic acid probes listed above.

Alternatively, hybridization of a viral DNA to the above nucleic acid probes can be performed by a Southern blot procedure without viral DNA amplification and under stringent hybridization conditions as described herein.

Oligonucleotides for use as probes or PCR primers are chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Carruthers [19] using an automated synthesizer, as described in Needham-VanDevanter [69].

Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson, J.D. and Regnier, F.E.

The sequence of the synthetic oligonucleotide can be verified using the chemical degradation method of Maxam, A.M. and Gilbert, W. [63].

B. Isolation and propagation of KS-inducinq strains of the Human Herpesvirus Using conventional methods, the human herpesvirus can be propagated in vitro. For example, standard techniques for growing herpes viruses are described in Ablashi, D.V. Briefly, PHA stimulated cord blood mononuclear cells, macrophage, neuronal, or glial cell lines are cocultivated with cerebrospinal fluid, plasma, peripheral blood leukocytes, or tissue extracts containing viral infected cells or purified virus. The recipient cells are treated with 5 Ag/ml polybrene for 2 hours at 370 C prior to infection.

WO 96/06159 PCT/US95/10194 26 Infected cells are observed by demonstrating morphological changes, as well as being positive for antigens from the human herpesvirus by using monoclonal antibodies immunoreactive with the human herpes virus in an immunofluorescence assay.

For virus isolation, the virus is either harvested directly from the culture fluid by direct centrifugation, or the infected cells are harvested, homogenized or lysed and the virus is separated from cellular debris and purified by standard methods of isopycnic sucrose density gradient centrifugation.

One skilled in the art may isolate and propagate the DNA herpesvirus associated with Kaposi's sarcoma (KSHV) employing the following protocol. Long-term establishment of a B lymphoid cell line infected with the KSHV from body-cavity based lymphomas (RCC-1 or BHL-6) is prepared extracting DNA from the Lymphoma tissue using standard techniques [27, 49, 66].

The KS associated herpesvirus may be isolated from the cell DNA in the following manner. An infected cell line (BHL-6 RCC-1), which can be lysed using standard methods such as hyposomatic shocking and Dounce homogenization, is first pelleted at 2000xg for minutes, the supernatant is removed and centrifuged again at 10,000xg for 15 minutes to remove nuclei and organelles. The supernatant is filtered through a 0.45A filter and centrifuged again at 100,000xg for 1 hour to pellet the virus. The virus can then be washed and centrifuged again at 100,000xg for 1 hour.

The DNA is tested for the presence of the KSHV by Southern blotting and PCR using the specific probes as described hereinafter. Fresh lymphoma tissue containing viable infected cells is simultaneously WO 96/06159 PCT/US95/10194 27 filtered to form a single cell suspension by standard techniques [49, 66]. The cells are separated by standard Ficoll-Plaque centrifugation and lymphocyte layer is removed. The lymphocytes are then placed at >1x10 6 cells/ml into standard lymphocyte tissue culture medium, such as RMP 1640 supplemented with 10% fetal calf serum. Immortalized lymphocytes containing the KSHV virus are indefinitely grown in the culture media while nonimmortilized cells die during course of prolonged cultivation.

Further, the virus may be propagated in a new cell line by removing media supernatant containing the virus from a continuously infected cell line at a concentration of >1x10 6 cells/ml. The media is centrifuged at 2000xg for 10 minutes and filtered through a 0.45A filter to remove cells. The media is applied in a 1:1 volume with cells growing at >1x10 6 cells/ml for 48 hours. The cells are washed and pelleted and placed in fresh culture medium, and tested after 14 days of growth.

RCC-1 and RCC-1 2

F

5 were deposited on October 19, 1994 under ATCC Accession No. CRL 11734 and CRL 11735, respectively, pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A.

BHL-6 was deposited on November 18, 1994 under ATCC Accession No. CRL 11762 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A.

WO 96/06159 PCTUS95/10194 28 C. Immunological Identity of the Virus The KS-associated human herpesvirus can also be described immunologically. KS-associated human herpesviruses are selectively immunoreactive to antisera generated against a defined immunogen such as the viral major capsid protein depicted in Seq. ID No.

12, herein. Immunoreactivity is determined in an immunoassay using a polyclonal antiserum which was raised to the protein which is encoded by the amino acid sequence or nucleic acid sequence of SEQ ID NOs: 18-20. This antiserum is selected to have low crossreactivity against other herpes viruses and any such crossreactivity is removed by immunoabsorbtion prior to use in the immunoassay.

In order to produce antisera for use in an immunoassay, the protein which is encoded by the amino acid sequence or nucleic acid of SEQ ID NOs: 18-20 is isolated as described herein. For example, recombinant protein can be produced in a mammalian cell line. An inbred strain of mice such as balb/c is immunized with the protein which is encoded by the amino acid sequence or nucleic acid of SEQ ID NOs: 2- 37 using a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see supra). Alternatively, a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen. Polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 104 or greater are selected and tested for their cross reactivity against other viruses of the gammaherpesvirinae subfamily, particularly human herpes virus types 1-7, by using a standard WO 96/06159 PCTfS95/10194 29 immunoassay as described in supra. These other gammaherpesvirinae virus can be isolated by standard techniques for isolation herpes viruses as described herein.

The ability of the above viruses to compete with the binding of the antisera to the immunogen protein is determined. The percent crossreactivity for other viruses is calculated, using standard calculations.

Those antisera with less than 10% crossreactivity with each of the other viruses listed above is selected and pooled. The cross-reacting antibodies are then removed from the pooled antisera by immunoabsorption with the above-listed viruses.

The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay procedure as described above to compare an unknown virus preparation to the specific KS herpesvirus preparation described herein and containing the nucleic acid sequence described in SEQ ID NOs: 2-37. In order to make this comparison, the immunogen protein which is encoded by the amino acid sequence or nucleic acid of SEQ ID NOs: 2-37 is the labeled antigen and the virus preparations are each assayed at a wide range of concentrations. The amount of each virus preparation required to inhibit 50% of the binding of the antisera to the labeled immunogen protein is determined. Those viruses that specifically bind to an antibody generated to an immunogen consisting of the protein of SEQ ID NOs: 2-37 are those virus where the amount of virus needed to inhibit 50% of the binding to the protein does not exceed an established amount. This amount is no more than 10 times the amount of the virus that is needed for 50% inhibition for the KSassociated herpesvirus containing the DNA sequence of SEQ ID NO: 1. Thus, the KS-associated herpesviruses WO 96/06159 PCT/US95/10194 of the invention can be defined by immunological comparison to the specific strain of the KS-associated herpesvirus for which nucleic acid sequences are provided herein.

This invention provides, a nucleic acid molecule of at least 14 nucleotides capable of specifically hybridizing with the isolated DNA molecule. In one embodiment, the molecule is DNA. In another embodiment, the molecule is RNA. In another embodiment the nucleic acid molecule may be 14-20 nucleotides in length. In another embodiment the nucleic acid molecule may be 16 nucleotides in length.

This invention provides, a nucleic acid molecule of at least 14 nucleotides capable of specifically hybridizing with a nucleic acid molecule which is complementary to the isolated DNA molecule. In one embodiment, the molecule is DNA. In another embodiment, the molecule is RNA.

The nucleic acid molecule of at least 14 nucleotides may hybridize with moderate stringency to at least a portion of a nucleic acid molecule with a sequence shown in Figures 3A-3F (SEQ ID NOs: i, 10-17, and 38- High stringent hybridization conditions are selected at about 50 C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about As other factors may significantly affect the WO 96/06159 PCT/US95/10194 31 stringency of hybridization, including, among others, base composition and size of the complementary strands, the presence of organic solvents, ie. salt or formamide concentration, and the extent of base mismatching, the combination of parameters is more important than the absolute measure of any one. For Example high stringency may be attained for example by overnight hybridization at about 68 0 C in a 6x SSC solution, washing at room temperature with 6x SSC solution, followed by washing at about 68 0 C in a 6x SSC in a 0.6x SSX solution.

Hybridization with moderate stringency may be attained for example by: 1) filter pre-hybridizing and hybridizing with a solution of 3x sodium chloride, sodium citrate (SSC), 50% formamide, 0.1M Tris buffer at Ph 7.5, 5x Denhardt's solution; prehybridization at 37 0 C for 4 hours; 3) hybridization at 37 0 C with amount of labelled probe equal to 3,000,000 cpm total for 16 hours; 4) wash in 2x SSC and 0.1% SDS solution; 5) wash 4x for 1 minute each at room temperature at 4x at 60 0 C for 30 minutes each; and 6) dry and expose to film.

The phrase "selectively hybridizing to" refers to a nucleic acid probe 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 probe 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, in a different "Complementary" or "target" nucleic acid sequences refer to those nucleic acid sequences which selectively hybridize to a nucleic acid probe. Proper annealing conditions depend, for example, upon a probe's length, base WO 96/06159 PCT/UJS95/10194 32 composition, and the number of mismatches and their position on the probe, and must often be determined empirically. For discussions of nucleic acid probe design and annealing conditions, see, for example, Sambrook et al., [81 or Ausubel, et al., It will be readily understood by those skilled in the art and it is intended here, that when reference is made to particular sequence listings, such reference includes sequences which substantially correspond to its complementary sequence and those described including allowances for minor sequencing errors, single base changes, deletions, substitutions and the like, such that any such sequence variation corresponds to the nucleic acid sequence of the pathogenic organism or disease marker to which the relevant sequence listing relates.

Nucleic acid probe technology is well known to those skilled in the art who readily appreciate that such probes may vary greatly in length and may be labeled with a detectable label, such as a radioisotope or fluorescent dye, to facilitate detection of the probe.

DNA probe molecules may be produced by insertion of a DNA molecule having the full-length or a fragment of the isolated nucleic acid molecule of the DNA virus into suitable vectors, such as plasmids or bacteriophages, followed by transforming into suitable bacterial host cells, replication in the transformed bacterial host cells and harvesting of the DNA probes, using methods well known in the art. Alternatively, probes may be generated chemically from DNA synthesizers.

DNA virus nucleic acid rearrangements/mutations may be detected by Southern blotting, single stranded conformational polymorphism gel electrophoresis WO 96/06159 PCTIUS95/10194 33 (SSCP), PCR or other DNA based techniques, or for RNA species by Northern blotting, PCR or other RNA-based techniques.

RNA probes may be generated by inserting the full length or a fragment of the isolated nucleic acid molecule of the DNA virus downstream of a bacteriophage promoter such as T3, T7 or SP6. Large amounts of RNA probe may be produced by incubating the labeled nucleotides with a linearized isolated nucleic acid molecule of the DNA virus or its fragment where it contains an upstream promoter in the presence of the appropriate RNA polymerase.

As defined herein nucleic acid probes may be DNA or RNA fragments. DNA fragments can be prepared, for example, by digesting plasmid DNA, or by use of PCR, or synthesized by either the phosphoramidite method described by Beaucage and Carruthers, or by the triester method according to Matteucci, et al., [62], both incorporated herein by reference. A double stranded fragment may then be obtained, if desired, by annealing the chemically synthesized single strands together under appropriate conditions or by synthesizing the complementary strand using DNA polymerase with an appropriate primer sequence. Where a specific sequence for a nucleic acid probe is given, it is understood that the complementary strand is also identified and included. The complementary strand will work equally well in situations where the target is a double-stranded nucleic acid. It is also understood that when a specific sequence is identified for use a nucleic probe, a subsequence of the listed sequence which is 25 basepairs or more in length is also encompassed for use as a probe.

WO 96/06159 PCT/US95/10194 34 The DNA molecules of the subject invention also include DNA molecules coding for polypeptide analogs, fragments or derivatives of antigenic polypeptides which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (deletion analogs containing less than all of the residues specified for the protein, substitution analogs wherein one or more residues specified are replaced by other residues and addition analogs where in one or more amino acid residues is added to a terminal or medial portion of the polypeptides) and which share some or all properties of naturallyoccurring forms. These molecules include: the incorporation of codons "preferred" for expression by selected non-mammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences that facilitate construction of readily expressed vectors.

This invention provides for an isolated DNA molecule which encodes at least a portion of a Kaposi's sarcoma associated herpesvirus: virion polypeptide 23, major capsid protein, capsid proteins, thymidine kinase, or tegument protein.

This invention also provides a method of producing a polypeptide encoded by isolated DNA molecule, 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 peptide encoded by the isolated DNA molecule associated with Kaposi's sarcoma. In one embodiment the peptide may be a polypeptide. Further, this invention provides a host WO 96/06159 PCT/US95/10194 cell which expresses the polypeptide of isolated DNA molecule.

In one embodiment the isolated peptide or polypeptide is encoded by at least a portion of an isolated DNA molecule. In another embodiment the isolated peptide or polypeptide is encoded by at least a portion of a nucleic acid molecule with a sequence as set forth in (SEQ ID NOs: 2-37) Further, the isolated peptide or polypeptide encoded by the isolated DNA molecule may be linked to a second nucleic acid molecule to form a fusion protein by expression in a suitable host cell. In one embodiment the second nucleic acid molecule encodes betagalactosidase. Other nucleic acid molecules which are used to form a fusion protein are known to those skilled in the art.

This invention provides an antibody which specifically binds to the peptide or polypeptide encoded by the isolated DNA molecule. In one embodiment the antibody is a monoclonal antibody. In another embodiment the antibody is a polyclonal antibody.

The antibody or DNA molecule may be labelled with a detectable marker including, but not limited to: a radioactive label, or a colorimetric, a luminescent, or a fluorescent marker, or gold. Radioactive labels include, but are not limited to: 3 H, 14C, 32 P, 33 P; 35

S,

36 C1, s 5 Cr, "5Co, 59 Co, 59 Fe, 90 125 131, and 1 86 Re.

Fluorescent markers include but are not limited to: fluorescein, rhodamine and auramine. Colorimetric markers include, but are not limited to: biotin, and digoxigenin. Methods of producing the polyclonal or monoclonal antibody are known to those of ordinary skill in the art.

WO 96/06159 PCTIUS95/10194 36 Further, the antibody or nucleic acid molecule complex may be detected by a second antibody which may be linked to an enzyme, such as alkaline phosphatase or horseradish peroxidase. Other enzymes which may be employed are well known to one of ordinary skill in the art.

This invention provides a method to select specific regions on the polypeptide encoded by the isolated DNA molecule of the DNA virus to generate antibodies.

The protein sequence may be determined from the cDNA sequence. Amino acid sequences may be analyzed by methods well known to those skilled in the art to determine whether they produce hydrophobic or hydrophilic regions in the proteins which they build.

In the case of cell membrane proteins, hydrophobic regions are well known to form the part of the protein that is inserted into the lipid bilayer of the cell membrane, while hydrophilic regions are located on the cell surface, in an aqueous environment. Usually, the hydrophilic regions will be more immunogenic than the hydrophobic regions. Therefore the hydrophilic amino acid sequences may be selected and used to generate antibodies specific to polypeptide encoded by the isolated nucleic acid molecule encoding the DNA virus.

The selected peptides may be prepared using commercially available machines. As an alternative, DNA, such as a cDNA or a fragment thereof, may be cloned and expressed and the resulting polypeptide recovered and used as an immunogen.

Polyclonal antibodies against these peptides may be produced by immunizing animals using the selected peptides. Monoclonal antibodies are prepared using hybridoma technology by fusing antibody producing B cells from immunized animals with myeloma cells and selecting the resulting hybridoma cell line producing WO 96/06159 PCT/US95/10194 37 the desired antibody. Alternatively, monoclonal antibodies may be produced by in vitro techniques known to a person of ordinary skill in the art. These antibodies are useful to detect the expression of polypeptide encoded by the isolated DNA molecule of the DNA virus in living animals, in humans, or in biological tissues or fluids isolated from animals or humans.

II. Immunoassays The antibodies raised against the viral strain or peptides may be detectably labelled, utilizing conventional labelling techniques well-known to the art. Thus, the antibodies may be radiolabelled using, for example, radioactive isotopes such as 3 H, 1251, 131I, and 3

"S.

The antibodies may also be labelled using fluorescent labels, enzyme labels, free radical labels, or bacteriophage labels, using techniques known in the art. Typical fluorescent labels include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, alophycocyanin, and Texas Red.

Since specific enzymes may be coupled to other molecules by covalent links, the possibility also exists that they might be used as labels for the production of tracer materials. Suitable enzymes include alkaline phosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase, maleate dehydrogenase, and peroxidase. Two principal types of enzyme immunoassay are the enzyme-linked immunosorbent assay (ELISA), and the homogeneous enzyme immunoassay, also known as enzyme-multiplied immunoassay (EMIT, Syva Corporation, Palo Alto, CA). In the ELISA system, separation may be achieved, for example, by WO 96/06159 PCT/US95/10194 38 the use of antibodies coupled to a solid phase. The EMIT system depends on deactivation of the enzyme in the tracer-antibody complex; the activity can thus be measured without the need for a separation step.

Additionally, chemiluminescent compounds may be used as labels. Typical chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

Similarly, bioluminescent compounds may be utilized for labelling, the bioluminescent compounds including luciferin, luciferase, and aequorin.

Once labeled, the antibody may be employed to identify and quantify immunologic counterparts (antibody or antigenic polypeptide) utilizing techniques well-known to the art.

A description of a radioimmunoassay (RIA) may be found in Laboratory Techniques in Biochemistry and Molecular Biology with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, incorporated by reference herein.

A description of general immunometric assays of various types can be found in the following U.S. Pat.

Nos. 4,376,110 (David et al.) or 4,098,876 (Piasio).

A. Assays for viral antigens In addition to the detection of the causal agent using nucleic acid hybridization technology, one can use immunoassays to detect for the virus, specific peptides, or for antibodies to the virus or peptides.

A general overview of the applicable technology is in WO 96/06159 PCT/US95/10194 39 Harlow and Lane incorporated by reference herein.

In one embodiment, antibodies to the human herpesvirus can be used to detect the agent in the sample. In brief, to produce antibodies to the agent or peptides, the sequence being targeted is expressed in transfected cells, preferably bacterial cells, and purified. The product is injected into a mammal capable of producing antibodies. Either monoclonal or polyclonal antibodies (as well as any recombinant antibodies) specific for the gene product can be used in various immunoassays. Such assays include competitive immunoassays, radioimmunoassays, Western blots, ELISA, indirect immunofluorescent assays and the like. For competitive immunoassays, see Harlow and Lane [32] at pages 567-573 and 584-589.

Monoclonal antibodies or recombinant antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells or other lymphocytes from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein [501, incorporated herein by reference). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. New techniques using recombinant phage antibody expression systems can also be used to generate monoclonal antibodies. See for example: McCafferty, J et al.

WO 96/06159 PCT/US95/10194 Hoogenboom, H.R. et al. [391; and Marks, J.D. et al. Such peptides may be produced by expressing the specific sequence in a recombinantly engineered cell such as bacteria, yeast, filamentous fungal, insect (especially employing baculoviral vectors), and mammalian cells. Those of skill in the art are knowledgeable in the numerous expression systems available for expression of herpes virus protein.

Briefly, the expression of natural or synthetic nucleic acids encoding viral protein will typically be achieved by operably linking the desired sequence or portion thereof to a promoter (which is either constitutive or inducible), and incorporated into an expression vector. The vectors are suitable for replication or integration in either prokaryotes or eukaryotes. Typical cloning vectors contain antibiotic resistance markers, genes for selection of transformants, inducible or regulatable promoter regions, and translation terminators that are useful for the expression of viral genes.

Methods for the expression of cloned genes in bacteria are also well known. In general, to obtain high level expression of a cloned gene in a prokaryotic system, it is advisable to construct expression vectors containing a strong promoter to direct mRNA transcription. The inclusion of selection markers in DNA vectors transformed in E. coli is also useful.

Examples of such markers include genes specifying resistance to antibiotics. See [81] supra, for details concerning selection markers and promoters for use in E. coli. Suitable eukaryote hosts may include plant cells, insect cells, mammalian cells, yeast, and filamentous fungi.

WO 96/06159 PCT/US95/10194 41 Methods for characterizing naturally processed peptides bound to MHC (major histocompatibility complex) I molecules have been developed. See, Falk et al. and PCT publication No. WO 92/21033 published November 26, 1992, both of which are incorporated by reference herein. Typically, these methods involve isolation of MHC class I molecules by immunoprecipitation or affinity chromatography from an appropriate cell or cell line. Other methods involve direct amino acid sequencing of the more abundant peptides in various HPLC fractions by known automatic sequencing of peptides eluted from Class I molecules of the B cell type (Jardetzkey, et al. incorporated by reference herein, and of the human MHC class I molecule, HLA-A2.1 type by mass spectrometry (Hunt, et al. [401, incorporated by reference herein) See also, R6tzschke and Falk incorporated by reference herein for a general review of the characterization of naturally processed peptides in MHC class I. Further, Marloes, et al. [61], incorporated by reference herein, describe how class I binding motifs can be applied to the identification of potential viral immunogenic peptides in vitro.

The peptides described herein produced by recombinant technology may be purified by standard techniques well known to those of skill in the art. Recombinantly produced viral sequences can be directly expressed or expressed as a fusion protein. The protein is then purified by a combination of cell lysis sonication) and affinity chromatography. For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired peptide.

The proteins may be purified to substantial purity by standard techniques well known in the art, including WO 96/06159 PCT/US95/10194 42 selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, Scopes, R. [84] incorporated herein by reference.

B. Serological tests for the presence of antibodies to the human herpesvirus.

This invention further embraces diagnostic kits for detecting the presence of a KS agent in biological samples, such as serum or solid tissue samples, comprising a container containing antibodies to the human herpesvirus, and instructional material for performing the test. Alternatively, inactivated viral particles or peptides or viral proteins derived from the human herpesvirus may be used in a diagnostic kit to detect for antibodies specific to the KS associated human herpesvirus.

Diagnostic kits for detecting the presence of a KS agent in tissue samples, such as skin samples or samples of other affected tissue, comprising a container containing a nucleic acid sequence specific for the human herpesvirus and instructional material for detecting the KS-associated herpesvirus are also included. A container containing nucleic acid primers to any one of such sequences is optionally included as are antibodies to the human herpesvirus as described herein.

Antibodies reactive with antigens of the human herpesvirus can also be measured by a variety of immunoassay methods that are similar to the procedures described above for measurement of antigens. For a review of immunological and immunoassay procedures applicable to the measurement of antibodies by WO 96/06159 PCT/US95/10194 43 immunoassay techniques, see Basic and Clinical Immunology 7th Edition and supra.

In brief, immunoassays to measure antibodies reactive with antigens of the KS-associated human herpesvirus can be either competitive or noncompetitive binding assays. In competitive binding assays, the sample analyte competes with a labeled analyte for specific binding sites on a capture agent bound to a solid surface. Preferably the capture agent is a purified recombinant human herpesvirus protein produced as described above. Other sources of human herpesvirus proteins, including isolated or partially purified naturally occurring protein, may also be used.

Noncompetitive assays are typically sandwich assays, in which the sample analyte is bound between two analyte-specific binding reagents. One of the binding agents is used as a capture agent and is bound to a solid surface. The second binding agent is labelled and is used to measure or detect the resultant complex by visual or instrument means. A number of combinations of capture agent and labelled binding agent can be used. A variety of different immunoassay formats, separation techniques and labels can be also be used similar to those described above for the measurement of the human herpesvirus antigens.

Hemagglutination Inhibition (HI) and Complement Fixation (CF) which are two laboratory tests that can be used to detect infection with human herpesvirus by testing for the presence of antibodies against the virus or antigens of the virus.

Serological methods can be also be useful when one wishes to detect antibody to a specific variant. For example, one may wish to see how well a vaccine recipient has responded to the new variant.

WO 96/06159 PCT/US95/10194 44 Alternatively, one may take serum from a patient to see which variant the patient responds to the best.

This invention provides an antagonist capable of blocking the expression of the peptide or polypeptide encoded by the isolated DNA molecule. In one embodiment the antagonist is capable of hybridizing with a double stranded DNA molecule. In another embodiment the antagonist is a triplex oligonucleotide capable of hybridizing to the DNA molecule. In another embodiment the triplex oligonucleotide is capable of binding to at least a portion of the isolated DNA molecule with a nucleotide sequence as shown in Figure 3A-3F (SEQ ID NOs: 1-37).

This invention provides an antisense molecule capable of hybridizing to the isolated DNA molecule. In one embodiment the antisense molecule is DNA. In another embodiment the antisense molecule is RNA.

The antisense molecule may be DNA or RNA or variants thereof DNA or RNA with a protein backbone).

The present invention extends to the preparation of antisense nucleotides and ribozymes that may be used to interfere with the expression of the receptor recognition proteins at the translation of a specific mRNA, either by masking that MRNA with an antisense nucleic acid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific MRNA molecule. In the cell, they hybridize to that MRNA, forming a double stranded molecule. The cell does not translate an MRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of MRNA into protein. Oligomers of about fifteen nucleotides and molecules that WO 96/06159 PCTIUS95/10194 hybridize to the AUG initiation codon are particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules upon introduction to cells.

This invention provides a transgenic nonhuman mammal which comprises at least a portion of the isolated DNA molecule introduced into the mammal at an embryonic stage. Methods of producing a transgenic nonhuman mammal are known to those skilled in the art.

This invention provides a cell line containing the isolated KS associated herpesvirus of the subject invention. In one embodiment the isolated DNA molecule is artificially introduced into the cell.

Cell lines include, but are not limited to: fibroblasts, such as HFF, NIH/3T3; Epithelial cells, such as 5637; lymphocytes, such as FCB; T-cells, such as CCRF-CEM (ATCC CCL 119); B-cells, such as BJAB and Raji (ATCC CCL 86); and myeloid cells such as K562 (ATCC CCL 243); Vero cells and carcinoma cells.

Methods of producing such cell lines are known to those skilled in the art. In one embodiment the isolated KS associated herpesvirus is introduced into a RCC-1 cell line.

III. In vitro diagnostic assays for the detection of

KS

This invention provides a method of diagnosing Kaposi's sarcoma in a subject which comprises: (a) obtaining a nucleic acid molecule from a tumor lesion of the subject; contacting the nucleic acid molecule with a labelled nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with the isolated DNA, under hybridizing conditions; and determining the presence of the WO 96/06159 PCT/US95/10194 46 nucleic acid molecule hybridized, the presence of which is indicative of Kaposi's sarcoma in the subject, thereby diagnosing Kaposi's sarcoma in the subject.

In one embodiment the DNA molecule from the tumor lesion is amplified before step In another embodiment PCR is employed to amplify the nucleic acid molecule. Methods of amplifying nucleic acid molecules are known to those skilled in the art.

A person of ordinary skill in the art will be able to obtain appropriate DNA sample for diagnosing Kaposi's sarcoma in the subject. The DNA sample obtained by the above described method may be cleaved by restriction enzyme. The uses of restriction enzymes to cleave DNA and the conditions to perform such cleavage are well-known in the art.

In the above described methods, a size fractionation may be employed which is effected by a polyacrylamide gel. In one embodiment, the size fractionation is effected by an agarose gel. Further, transferring the DNA fragments into a solid matrix may be employed before a hybridization step. One example of such solid matrix is nitrocellulose paper.

This invention provides a method of diagnosing Kaposi's sarcoma in a subject which comprises: (a) obtaining a nucleic acid molecule from a suitable bodily fluid of the subject; contacting the nucleic acid molecule with a labelled nucleic acid molecules of at least 15 nucleotides capable of specifically hybridizing with the isolated DNA, under hybridizing conditions; and determining the presence of the nucleic acid molecule hybridized, the WO 96/06159 PCT/US95/10194 47 presence of which is indicative of Kaposi's sarcoma in the subject, thereby diagnosing Kaposi's sarcoma in the subject.

This invention provides a method of diagnosing a DNA virus in a subject, which comprises obtaining a suitable bodily fluid sample from the subject, (b) contacting the suitable bodily fluid of the subject to a support having already bound thereto a Kaposi's sarcoma antibody, so as to bind the Kaposi's sarcoma antibody to a specific Kaposi's sarcoma antigen, (c) removing unbound bodily fluid from the support, and determining the level of Kaposi's sarcoma antibody bound by the Kaposi's sarcoma antigen, thereby diagnosing the subject for Kaposi's sarcoma.

This invention provides a method of diagnosing Kaposi's sarcoma in a subject, which comprises (a) obtaining a suitable bodily fluid sample from the subject, contacting the suitable bodily fluid of the subject to a support having already bound thereto a Kaposi's sarcoma antigen, so as to bind Kaposi's sarcoma antigen to a specific Kaposi's sarcoma antibody, removing unbound bodily fluid from the support, and determining the level of the Kaposi's sarcoma antigen bound by the Kaposi's sarcoma antibody, thereby diagnosing Kaposi's sarcoma.

This invention provides a method of detecting expression of a DNA virus associated with Kaposi's sarcoma in a cell which comprises obtaining total cDNA obtained from the cell, contacting the cDNA so obtained with a labelled DNA molecule under hybridizing conditions, determining the presence of cDNA hybridized to the molecule, and thereby detecting the expression of the DNA virus. In one embodiment WO 96/06159 PCTUS95/10194 48 mRNA is obtained from the cell to detect expression of the DNA virus.

The suitable bodily fluid sample is any bodily fluid sample which would contain Kaposi's sarcoma antibody, antigen or fragments thereof. A suitable bodily fluid includes, but is not limited to: serum, plasma, cerebrospinal fluid, lymphocytes, urine, transudates, or exudates. In the preferred embodiment, the suitable bodily fluid sample is serum or plasma. In addition, the bodily fluid sample may be cells from bone marrow, or a supernatant from a cell culture.

Methods of obtaining a suitable bodily fluid sample from a subject are known to those skilled in the art.

Methods of determining the level of antibody or antigen include, but are not limited to: ELISA, IFA, and Western blotting. Other methods are known to those skilled in the art. Further, a subject infected with a DNA virus associated with Kaposi's sarcoma may be diagnosed with the above described methods.

The detection of the human herpesvirus and the detection of virus-associated KS are essentially identical processes. The basic principle is to detect the virus using specific ligands that bind to the virus but not to other proteins or nucleic acids in a normal human cell or its environs. The ligands can either be nucleic acid or antibodies. The ligands can be naturally occurring or genetically or physically modified such as nucleic acids with non-natural or antibody derivatives, Fab or chimeric antibodies. Serological tests for detection of antibodies to the virus may also be performed by using protein antigens obtained from the human herpesvirus, and described herein.

WO 96/06159 PCTfS95/10194 49 Samples can be taken from patients with KS or from patients at risk for KS, such as AIDS patients.

Typically the samples are taken from blood (cells, serum and/or plasma) or from solid tissue samples such as skin lesions. The most accurate diagnosis for KS will occur if elevated titers of the virus are detected in the blood or in involved lesions. KS may also be indicated if antibodies to the virus are detected and if other diagnostic factors for KS is present.

A. Nucleic acid assays.

The diagnostic assays of the invention can be nucleic acid assays such as nucleic acid hybridization assays and assays which detect amplification of specific nucleic acid to detect for a nucleic acid sequence of the human herpesvirus described herein.

Accepted means for conducting hybridization assays are known and general overviews of the technology can be had from a review of: Nucleic Acid Hybridization: A Practical Approach [72] Hybridization of Nucleic Acids Immobilized on Solid Supports Analytical Biochemistry and Innis et al., PCR Protocols [74], supra, all of which are incorporated by reference herein.

If PCR is used in conjunction with nucleic acid hybridization, primers are designed to target a specific portion of the nucleic acid of the herpesvirus. For example, the primers set forth in SEQ ID NOs: 38-40 may be used to target detection of regions of the herpesvirus genome encoding ORF homologue ORF 32 homologue. From the information provided herein, those of skill in the art will be able to select appropriate specific primers.

WO 96/06159 PCT/US95/10194 Target specific probes may be used in the nucleic acid hybridization diagnostic assays for KS. The probes are specific for or complementary to the target of interest. For precise allelic differentiations, the probes should be about 14 nucleotides long and preferably about 20-30 nucleotides. For more general detection of the human herpesvirus of the invention, nucleic acid probes are about 50 to about 1000 nucleotides, most preferably about 200 to about 400 nucleotides.

A sequence is "specific" for a target organism of interest if it includes a nucleic acid sequence which when detected is determinative of the presence of the organism in the presence of a heterogeneous population of proteins and other biologics. A specific nucleic acid probe is targeted to that portion of the sequence which is determinative of the organism and will not hybridize to other sequences especially those of the host where a pathogen is being detected.

The specific nucleic acid probe can be RNA or DNA polynucleotide or oligonucleotide, or their analogs.

The probes may be single or double stranded nucleotides. The probes of the invention may be synthesized enzymatically, using methods well known in the art nick translation, primer extension, reverse transcription, the polymerase chain reaction, and others) or chemically by methods such as the phosphoramidite method described by Beaucage and Carruthers or by the triester method according to Matteucci, et al. both incorporated herein by reference) The probe must be of sufficient length to be able to form a stable duplex with its target nucleic acid in the sample, at least about 14 nucleotides, and WO 96/06159 PCT/US95/10194 51 may be longer at least about 50 or 100 bases in length). Often the probe will be more than about 100 bases in length. For example, when probe is prepared by nick-translation of DNA in the presence of labeled nucleotides the average probe length may be about 100- 600 bases.

As noted above, the probe will be capable of specific hybridization to a specific KS-associated herpes virus nucleic acid. Such "specific hybridization" occurs when a probe hybridizes to a target nucleic acid, as evidenced by a detectable signal, under conditions in which the probe does not hybridize to other nucleic acids animal cell or other bacterial nucleic acids) present in the sample. A variety of factors including the length and base composition of the probe, the extent of base mismatching between the probe and the target nucleic acid, the presence of salt and organic solvents, probe concentration, and the temperature affect hybridization, and optimal hybridization conditions must often be determined empirically. For discussions of nucleic acid probe design and annealing conditions, see, for example, supra, Ausubel, et al. [81 [hereinafter referred to as Sambrook], Methods in Enzymology [67] or Hybridization with Nucleic Acid Probes [42] all of which are incorporated herein by reference.

Usually, at least a part of the probe will have considerable sequence identity with the target nucleic acid. Although the extent of the sequence identity required for specific hybridization will depend on the length of the probe and the hybridization conditions, the probe will usually have at least 70% identity to the target nucleic acid, more usually at least identity, still more usually at least 90% identity and most usually at least 95% or 100% identity.

MMIMMMM

WO 96/06159 PCT/US95/10194 52 A probe can be identified as capable of hybridizing specifically to its target nucleic acid by hybridizing the probe to a sample treated according the protocol of this invention where the sample contains both target virus and animal cells nerve cells). A probe is specific if the probe's characteristic signal is associated with the herpesvirus DNA in the sample and not generally with the DNA of the host cells and non-biological materials substrate) in a sample.

The following stringent hybridization and washing conditions will be adequate to distinguish a specific probe a fluorescently labeled DNA probe) from a probe that is not specific: incubation of the probe with the sample for 12 hours at 370C in a solution containing denatured probe, 50% formamide, 2X SSC, and 0.1% dextran sulfate, followed by washing in 1X SSC at 700C for 5 minutes; 2X SSC at 37 0 C for minutes; 0.2X SSC at room temperature for 5 minutes, and H 2 0 at room temperature for 5 minutes. Those of skill will be aware that it will often be advantageous in nucleic acid hybridizations in situ, Southern, or other) to include detergents sodium dodecyl sulfate), chelating agents EDTA) or other reagents buffers, Denhardt's solution, dextran sulfate) in the hybridization or wash solutions. To test the specificity of the virus specific probes, the probes can be tested on host cells containing the KS-associated herpesvirus and compared with the results from cells containing non- KS-associated virus.

It will be apparent to those of ordinary skill in the art that a convenient method for determining whether a probe is specific for a KS-associated viral nucleic acid utilizes a Southern blot (or Dot blot) using DNA WO 96/06159 PCT/US95/10194 53 prepared from one or more KS-associated human herpesviruses of the invention. Briefly, to identify a target specific probe DNA is isolated from the virus. Test DNA either viral or cellular is transferred to a solid charged nylon) matrix.

The probes are labelled following conventional methods. Following denaturation and/or prehybridization steps known in the art, the probe is hybridized to the immobilized DNAs under stringent conditions. Stringent hybridization conditions will depend on the probe used and can be estimated from the calculated Tm (melting temperature) of the hybridized probe (see, Sambrook for a description of calculation of the Tm). For radioactively-labeled DNA or RNA probes an example of stringent hybridization coni ti otI is h v ybridiziation in a olution containing denatured probe and 5x SSC at 65 0 C for 8-24 hours followed by washes in 0.1x SSC, 0.1% SDS (sodium dodecyl sulfate) at 50-65 0 C. In general, the temperature and salt concentration are chosen so that the post hybridization wash occurs at a temperature that is about 5°C below the TM of the hybrid. Thus for a particular salt concentration the temperature may be selected that is 5 0 C below the TM or conversely, for a particular temperature, the salt concentration is chosen to provide a TM for the hybrid that is 5 0

C

warmer than the wash temperature. Following stringent hybridization and washing, a probe that hybridizes to the KS-associated viral DNA but not to the non-KS associated viral DNA, as evidenced by the presence of a signal associated with the appropriate target and the absence of a signal from the non-target nucleic acids, is identified as specific for the KS associated virus. It is further appreciated that in determining probe specificity and in utilizing the method of this invention to detect KS-associated herpesvirus, a certain amount of background signal is typical and can WO 96/06159 PCT/US95/10194 54 easily be distinguished by one of skill from a specific signal. Two fold signal over background is acceptable.

A preferred method for detecting the KS-associated herpesvirus is the use of PCR and/or dot blot hybridization. The presence or absence of an KS agent for detection or prognosis, or risk assessment for KS includes Southern transfers, solution hybridization or non-radioactive detection systems, all of which are well known to those of skill in the art.

Hybridization is carried out using probes.

Visualization of the hybridized portions allows the qualitative determination of the presence or absence of the causal agent.

Similarly, a Northern transfer may be used for the detection of message in samples of RNA or reverse transcriptase PCR and cDNA can be detected by methods described above. This procedure is also well known in the art. See [81] incorporated by reference herein.

An alternative means for determining the presence of the human herpesvirus is in situ hybridization, or more recently, in situ polymerase chain reaction. In situ PCR is described in Neuvo et al. [71], Intracellular localization of polymerase chain reaction (PCR)-amplified Hepatitis C cDNA; Bagasra et al. Detection of Human Immunodeficiency virus type 1 provirus in mononuclear cells by in situ polymerase chain reaction; and Heniford et al. Variation in cellular EGF receptor mRNA expression demonstrated by in situ reverse transcriptase polymerase chain reaction. In situ hybridization assays are well known and are generally described in Methods Enzymol. [67] incorporated by reference herein. In an in situ hybridization, cells are fixed WO 96/06159 PCT/US95/10194 to a solid support, typically a glass slide. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of target-specific probes that are labelled. The probes are preferably labelled with radioisotopes or fluorescent reporters.

The above described probes are also useful for in-situ hybridization or in order to locate tissues which express this gene, or for other hybridization assays for the presence of this gene or its MRNA in various biological tissues. In-situ hybridization is a sensitive localization method which is not dependent on expression of antigens or native vs. denatured conditions.

Oligonucleotide (oligo) probes, synthetic oligonucleotide probes or riboprobes made from KSHV phagemids/plasmids, are relatively homogeneous reagents and successful hybridization conditions in tissue sections is readily transferable from one probe to another. Commercially synthesized oligonucleotide probes are prepared against the identified genes.

These probes are chosen for length (45-65 mers), high G-C content (50-70%) and are screened for uniqueness against other viral sequences in GenBank.

Oligonucleotides are 3'end-labeled with [a-3S]dATP to specific activities in the range of 1 x 10 10 dpm/ug using terminal deoxynucleotidyl transferase.

Unincorporated labeled nucleotides are removed from the oligo probe by centrifugation through a Sephadex column or by elution from a Waters Sep Pak C-18 column.

KS tissue embedded in OCT compound and snap frozen in freezing isopentane cooled with dry ice is cut at 6 im WO 96/06159 PCT/US95/10194 56 intervals and thawed onto 3 -aminopropyltriethoxysilane treated slides and allowed to air dry. The slides are then be fixed in 4% freshly prepared paraformaldehyde, rinsed in water. Formalin-fixed, paraffin embedded KS tissues cut at 6 Am and baked onto glass slides can also be used. The sections are then deparaffinized in xylenes and rehydrated through graded alcohols.

Prehybridization in 20mM Tris Ph 7.5, 0.02% Denhardt's solution, 10% dextran sulfate for 30 min at 37 0 C is followed by hybridization overnight in a solution of formamide 10% dextran sulfate sodium phosphate (Ph 3X SSC, 1X Denhardt's solution, 100 ug/ml salmon sperm DNA, 125 ug/ml yeast tRNA and the oligo probe (10 6 cpm/ml) at 42 0 C overnight.

The slides are washed twice with 2X SSC and twice with 1X SSC fr 15 mnuts eac-L at oom tempe-atue and Q O .L I jL)LLL L.CLLL Je.cLi.L..L e dJUL visualized by autoradiography. Briefly, sections are dehydrated through graded alcohols containing 0.3M ammonium acetate and air dried. The slides are dipped in Kodak NTB2 emulsion, exposed for days to weeks, developed, and counterstained with hematoxylin and eoxin. Alternative immunohistochemical protocols may be employed which are known to those skilled in the art.

IV. Treatment of human herpesvirus-induced KS This invention provides a method of treating a subject with Kaposi's sarcoma, comprising administering to the subject an effective amount of the antisense molecule capable of hybridizing to the isolated DNA molecule under conditions such that the antisense molecule selectively enters a tumor cell of the subject, so as to treat the subject.

WO 96/06159 PCTIUS95/10194 57 This invention provides a method for treating a subject with Kaposi's sarcoma (KS) comprising administering to the subject having a human herpesvirus-associated KS a pharmaceutically effective amount of an antiviral agent in a pharmaceutically acceptable carrier, wherein the agent is effective to treat the subject with KS-associated human herpes virus.

Further, this invention provides a method of prophylaxis or treatment for Kaposi's sarcoma (KS) by administering to a patient at risk for KS, an antibody that binds to the human herpesvirus in a pharmaceutically acceptable carrier. In one m 4e ,4ral dr ug it tieat a subject with the DNA herpesvirus of the subject invention.

The use of combinations of antiviral drugs and sequential treatments are useful for treatment of herpesvirus infections and will also be useful for the treatment of herpesvirus-induced KS. For example, Snoeck et al. found additive or synergistic effects against CMV when combining antiherpes drugs combinations of zidovudine [3'-azido-3'deoxythymidine, AZT] with HPMPC, ganciclovir, foscarnet or acyclovir or of HPMPC with other antivirals). Similarly, in treatment of cytomegalovirus retinitis, induction with ganciclovir followed by maintenance with foscarnet has been suggested as a way to maximize efficacy while minimizing the adverse side effects of either treatment alone. An anti-herpetic composition that contains acyclovir and, 2-acetylpyridine-5-((2pyridylamino)thiocarbonyl)-thiocarbonohydrazone is described in U.S. Pat. 5,175,165 (assigned to WO 96/06159 PCT/US95/10194 58 Burroughs Wellcome Combinations of TSinhibitors and viral TK-inhibitors in antiherpetic medicines are disclosed in U.S. Pat. 5,137,724, assigned to Stichting Rega VZW. A synergistic inhibitory effect on EBV replication using certain ratios of combinations of HPMPC with AZT was reported by Lin et al. [56].

U.S. Patent Nos. 5,164,395 and 5,021,437 (Blumenkopf; Burroughs Wellcome) describe the use of a ribonucleotide reductase inhibitor (an acetylpyridine derivative) for treatment of herpes infections, including the use of the acetylpyridine derivative in combination with acyclovir. U.S. Patent No. 5,137,724 (Balzari et al. describes the use of thymilydate J. a* .f j AM J UJJ\t fluro-2'-deoxyuridine) in combination with compounds having viral thymidine kinase inhibiting activity.

With the discovery of a disease causal agent for KS now identified, effective therapeutic or prophalactic protocols to alleviate or prevent the symptoms of herpes virus-associated KS can be formulated. Due to the viral nature of the disease, antiviral agents have application here for treatment, such as interferons, nucleoside analogues, ribavirin, amantadine, and pyrophosphate analogues of phosphonoacetic acid (foscarnet) (reviewed in Gorbach, et al. [28]) and the like. Immunological therapy will also be effective in many cases to manage and alleviate symptoms caused by the disease agents described here.

Antiviral agents include agents or compositions that directly bind to viral products and interfere with disease progress; and, excludes agents that do not impact directly on viral multiplication or viral titer. Antiviral agents do not include immunoregulatory agents that do not directly affect WO 96/06159 PCT/US95/10194 59 viral titer or bind to viral products. Antiviral agents are effective if they inactivate the virus, otherwise inhibit its infectivity or multiplication, or alleviate the symptoms of KS.

A. Antiviral Agents.

The antiherpesvirus agents that will be useful for treating virus-induced KS can be grouped into broad classes based on their presumed modes of action.

These classes include agents that act by inhibition of viral DNA polymerase, (ii) by targeting other viral enzymes and proteins, (iii) by miscellaneous or incompletely understood mechanisms, or (iv) by binding a target nucleic acid L1 L V L L CL _LQ agents may also be used in combination together or sequentially) to achieve synergistic or additive effects or other benefits.

Although it is convenient to group antiviral agents by their supposed mechanism of action, the applicants do not intend to be bound by any particular mechanism of antiviral action. Moreover, it will be understood by those of skill that an agent may act on more than one target in a virus or virus-infected cell or through more than one mechanism.

i) Inhibitors of viral DNA polymerase Many antiherpesvirus agents in clinical use or in development today are nucleoside analogs believed to act through inhibition of viral DNA replication, especially through inhibition of viral DNA polymerase.

These nucleoside analogs act as alternative substrates for the viral DNA polymerase or as competitive inhibitors of DNA polymerase substrates. Usually WO 96/06159 PCT/US95/10194 these agents are preferentially phosphorylated by viral thymidine kinase if one is present, and/or have higher affinity for viral DNA polymerase than for the cellular DNA polymerases, resulting in selective antiviral activity. Where a nucleoside analogue is incorporated into the viral DNA, viral activity or reproduction may be affected in a variety of ways.

For example, the analogue may act as a chain terminator, cause increased lability susceptibility to breakage) of analogue-containing DNA, and/or impair the ability of the substituted DNA to act as template for transcription or replication (see, Balzarini et al. It will be known to one of skill that, like many drugs, many of the agnts useful for treatment of herpes virus infections are modified "activated") by the host, host cell, or virus-infected host cell metabolic enzymes. For example, acyclovir is triphosphorylated to its active form, with the first phosphorylation being carried out by the herpes virus thymidine kinase, when present. Other examples are the reported conversion of the compound HOE 602 to ganciclovir in a three-step metabolic pathway (Winkler et al. and the phosphorylation of ganciclovir to its active form by, a CMV nucleotide kinase. It will be apparent to one of skill that the specific metabolic capabilities of a virus can affect the sensitivity of that virus to specific drugs, and is one factor in the choice of an antiviral drug. The mechanism of action of certain anti-herpesvirus agents is discussed in De Clercq [22] and in other references cited supra and infra, all of which are incorporated by reference herein.

Anti-herpesvirus medications suitable for treating viral induced KS include, but are not limited to, WO 96/06159 PTU9/09 PCTIUS95/10194 G61 nucleoside analogs including acyclic nucleoside p h os ph on ate a n alo gs g. phosphonylmethoxyalkylpurines and -pyrimidines) and cyclic nucleoside analogs. These include drugs such as: vidarabine (9-0-D-arabinofuranosyladenine; adenine arabinoside, ara-A, Vira-A, Parke-Davis); 1-f3-Darabinofuranosyluracil (ara-U); arabinofuranosyl-cytosine (ara-C) HPMPC [3hydroxy- 2- (phosphonylmethoxy) propyl I cytos ine g. GS 504 Gilead Science)]I and its cyclic form (cHPMPC) HP M PA S) 9- h y dr o xy -2 phosphonylmethoxypropyl) adenine] and its cyclic form (cHPMPA); (S)-HPMPDAP [(S)-9-(3-hydroxy-2phosphonylmethoxypropyl) 6-diaminopurine]; PMEDAP (2-phosphonyl-methoxyethyl) 6-diaminopurine]; HOE r, 9 r inn-r -f h i iso roo oxv)-2 propoxymethyl)purinel PMEA 9- (2phosphonylmethoxyethyl) adenine] bromovinyldeoxyuridine (Burns and Sandford. [211); 1-03-Darabinofuranosyl-E-5- (2-bromovinyl) -uridine or deoxyuridine; BVaraU (l-/-D-arabinofuranosyl-E-5- (2bromovinyl) -uracil, brovavir, Bristol-Myers Squibb, Yamsa Shoyu); BVDU [(E)-5-(2-bromovinyl)-2'deoxyuridine, brivudin, Helpin] and its carbocyclic analogue (in which the sugar moiety is replaced by a cyclopentane ring); IVDTJ iodovinyl) -deoxyuridinel and its carbocyclic analogue, C-IVDU (Balzarini et al. [11] and mercutithio analogs of 2'-deoxyuridine (Holliday, J., and Williams, M.V. [381); acyclovir hydroxyethoxy] methyl) guanine; e. g. Zovirax (Burroughs Wellcome)]; penciclovir (9-[4-hydroxy-2- (hydroxymethyl)butyll -guanine); ganciclovir [1,3dihydroxy-2 propoxymethyll -guanine) Cymevene, Cytovene (Syntex), DHPG (Stals et al. [8911 isopropylether derivatives of ganciclovir (see, e.g., Winkelmann et al. [941 cygalovir; famciclovir [2- WO 96/06159 PCT/US95/10194 62 amino-9-(4-acetoxy-3-(acetoxymethyl)but-l-yl)purine (Smithkline Beecham)]; valacyclovir (Burroughs Wellcome); desciclovir [(2-amino-9-(2ethoxymethyl)purine)] and 2-amino-9-(2hydroxyethoxymethyl)-9H-purine, prodrugs of acyclovir]; CDG (carbocyclic 2'-deoxyguanosine); and purine nucleosides with the pentafuranosyl ring replaced by a cyclo butane ring cyclobut-A 2a,30)-2, 3-bis (hydroxymethyl)-1cyclobutyl]adenine], cyclobut-G 2,3-bis(hydroxymethyl)-1-cyclobutyl]guanine],

BHCG

R 1 a 2 g 1 a 9 2 3 bis(hydroxymethyl)cyclobutyl]guanine], and an active isomer of racemic BHCG, SQ 34,514 [1R-l a, 26,3a)-2amino-9-[2,3-bis(hydroxymethyl)cyclobutyl]-6H-purin-6one (see, Braitman et al.(1991) Certain of these antiherpesviral agents are discussed in Gorach et al. Saunders et al. Yamanaka et al., Greenspan et al. all of which are incorporated by reference herein.

Triciribine and triciribine monophosphate are potent inhibitors against herpes viruses. (Ickes et al. [43], incorporated by reference herein), HIV-1 and HIV-2 (Kucera et al. incorporated by reference herein) and are additional nucleoside analogs that may be used to treat KS. An exemplary protocol for these agents is an intravenous injection of about 0.35 mg/meter 2 (0.7 mg/kg) once weekly or every other week for at least two doses, preferably up to about four to eight weeks.

Acyclovir and ganciclovir are of interest because of their accepted use in clinical settings. Acyclovir, an acyclic analogue of guanine, is phosphorylated by a herpesvirus thymidine kinase and undergoes further phosphorylation to be incorporated as a chain WO 96/06159 PCT/US95/10194 63 terminator by the viral DNA polymerase during viral replication. It has therapeutic activity against a broad range of herpesviruses, Herpes simplex Types 1 and 2, Varicella- Zoster, Cytomegalovirus, and Epstein-Barr Virus, and is used to treat disease such as herpes encephalitis, neonatal herpesvirus infections, chickenpox in immunocompromised hosts, herpes zoster recurrences, CMV retinitis, EBV infections, chronic fatigue syndrome, and hairy leukoplakia in AIDS patients. Exemplary intravenous dosages or oral dosages are 250 mg/kg/m 2 body surface area, every 8 hours for 7 days, or maintenance doses of 200-400 mg IV or orally twice a day to suppress recurrence. Ganciclovir has been shown to be more active than acyclovir against some herpesviruses. See, Oren and Soble Treatment protocols for ganciclovir are 5 mg/kg twice a day IV or 2.5 mg/kg three times a day for 10-14 days. Maintenance doses are 5-6 mg/kg for 5-7 days.

Also of interest is HPMPC. HPMPC is reported to be more active than either acyclovir or ganciclovir in the chemotherapy and prophylaxis of various HSV-1, HSV-2, TK- HSV, VZV or CMV infections in animal models supra).

Nucleoside analogs such as BVaraU are potent inhibitors of HSV-1, EBV, and VZV that have greater activity than acyclovir in animal models of encephalitis. FIAC (fluroidoarbinosyl cytosine) and its related fluroethyl and iodo compounds FEAU, FIAU) have potent selective activity against herpesviruses, and HPMPA ((S)-1-([3-hydroxy-2phosphorylmethoxy]propyl)adenine) has been demonstrated to be more potent against HSV and CMV than acyclovir or ganciclovir and are of choice in advanced cases of KS. Cladribine (2- WO 96/06159 PCT/US95/10194 64 chlorodeoxyadenosine) is another nucleoside analogue known as a highly specific antilymphocyte agent a immunosuppressive drug).

Other useful antiviral agents include: 5-thien-2-yl- 2'-deoxyuridine derivatives, BTDU [5-5(5bromothien-2-yl)-2'-deoxyuridine] and CTDU chlorothien-2-yl)-2'-deoxyuridine]; and OXT-A deoxy-2-hydroxymethyl-3-D-erythro-oxetanosyl)adenine] and OXT-G [9-(2-deoxy-2-hydroxymethyl-5-D-erythrooxetanosyl)guanine] Although OXT-G is believed to act by inhibiting viral DNA synthesis its mechanism of action has not yet been elucidated. These and other compounds are described in Andrei et al. which is incorporated by reference herein. Additional antiviral purine derivatives useful in treating herpesvirus infections are disclosed in US Pat.

5,108,994 (assigned to Beecham Group 6- Methoxypurine arabinoside (ara-M; Burroughs Wellcome) is a potent inhibitor of varicella-zoster virus, and will be useful for treatment of KS.

Certain thymidine analogs idoxuridine 2'-deoxyuridine)] and triflurothymidine) have antiherpes viral activity, but due to their systemic toxicity, are largely used for topical herpesviral infections, including HSV stromal keratitis and uveitis, and are not preferred here unless other options are ruled out.

Other useful antiviral agents that have demonstrated antiherpes viral activity include foscarnet sodium (trisodium phosphonoformate, PFA, Foscavir (Astra)) and phosphonoacetic acid (PAA). Foscarnet is an inorganic pyrophosphate analogue that acts by competitively blocking the pyrophosphate-binding site of DNA polymerase. These agents which block DNA WO 96/06159 PCT/US95/10194 polymerase directly without processing by viral thymidine kinase. Foscarnet is reported to be less toxic than PAA.

ii) Agents that target viral proteins other than DNA polymerase or other viral functions.

Although applicants do not intend to be bound by a particular mechanism of antiviral action, the antiherpes-virus agents described above are believed to act through inhibition of viral DNA polymerase.

However, viral replication requires not only the replication of the viral nucleic acid but also the production of viral proteins and other essential components. Accordingly, the present invention contemplates treatment of KS by the inhibition of viral proliferation by targeting viral proteins other than DNA polymerase by inhibition of their synthesis or activity, or destruction of viral proteins after their synthesis). For example, administration of agents that inhibit a viral serine protease, such as one important in development of the viral capsid will be useful in treatment of viral induced KS.

Other viral enzyme targets include: OMP decarboxylase inhibitors (a target of, parazofurin), CTP synthetase inhibitors (targets of, e.g., cyclopentenylcytosine), IMP dehydrogenase, ribonucleotide reductase (a target of, carboxylcontaining N-alkyldipeptides as described in U.S.

Patent No. 5,110,799 (Tolman et al., Merck)), thymidine kinase (a target of, 1-[2- -uracils and -guanines as described in, U.S.

Patent Nos. 4,863,927 and 4,782,062 (Tolman et al.; WO 96/06159 PCT/US95/10194 66 Merck)) as well as other enzymes. It will be apparent to one of ordinary skill in the art that there are additional viral proteins, both characterized and as yet to be discovered, that can serve as target for antiviral agents.

iv) Other agents and modes of antiviral action.

Kutapressin is a liver derivative available from Schwarz Parma of Milwaukee, Wisconsin in an injectable form of 25 mg/ml. The recommended dosage for herpesviruses is from 200 to 25 mg/ml per day for an average adult of 150 pounds.

Poly(I)-Poly(C 12 an accepted antiviral drug known as Ampligen from HEM Pharmaceuticals of Rockville, MD has been shown to inhibit herpesviruses and is another antiviral agent suitable for treating KS. Intravenous injection is the preferred route of administration.

Dosages from about 100 to 600 mg/m 2 are administered two to three times weekly to adults averaging 150 pounds. It is best to administer at least 200 mg/m 2 per week.

Other antiviral agents reported to show activity against herpes viruses varicella zoster and herpes simplex) and will be useful for the treatment of herpesvirus-induced KS include mappicine ketone (SmithKline Beecham); Compounds A,79296 and A,73209 (Abbott) for varicella zoster, and Compound 882C87 (Burroughs Wellcome) [see, The Pink Sheet 55(20) May 17, 1993].

Interferon is known inhibit replication of herpes viruses. See supra. Interferon has known toxicity problems and it is expected that second WO 96/06159 PCT/US95/10194 67 generation derivatives will soon be available that will retain interferon's antiviral properties but have reduced side affects.

It is also contemplated that herpes virus-induced KS may be treated by administering a herpesvirus reactivating agent to induce reactivation of the latent virus. Preferably the reactivation is combined with simultaneous or sequential administration of an anti-herpesvirus agent. Controlled reactivation over a short period of time or reactivation in the presence of an antiviral agent is believed to minimize the adverse effects of certain herpesvirus infections as discussed in PCT Application WO 93/04683).

Reactivating agents include agents such as estrogen, phorbol esters, forskolin and g-adrenergic blocking agents.

Agents useful for treatment of herpesvirus infections and for treatment of herpesvirus-induced KS are described in numerous U.S. Patents. For example, ganciclovir is an example of a antiviral guanine acyclic nucleotide of the type described in US Patent Nos. 4,355,032 and 4,603,219.

Acyclovir is an example of a class of antiviral purine derivatives, including 9- (2hydroxyethylmethyl)adenine, of the type described in U.S. Pat. Nos. 4,287,188, 4,294,831 and 4,199,574.

Brivudin is an example of an antiviral deoxyuridine derivative of the type described in US Patent No.

4,424,211.

Vidarabine is an example of an antiviral purine nucleoside of the type described in British Pat.

1,159,290.

WO 96/06159 PCT/US95/10194 68 Brovavir is an example of an antiviral deoxyuridine derivative of the type described in US Patent Nos.

4,542,210 and 4,386,076.

BHCG is an example of an antiviral carbocyclic nucleoside analogue of the type described in US Patent Nos. 5,153,352, 5,034,394 and 5,126,345.

HPMPC is an example of an antiviral phosphonyl methoxyalkyl derivative with of the type described in US Patent No. 5,142,051.

CDG (Carbocyclic 2'-deoxyguanosine) is an example of an antiviral carbocyclic nucleoside analogue of the type described in US Patent Nos. 4,543,255, 4,855,466, and 4,894,458.

Foscarnet is described in US Patent No. 4,339,445.

Trifluridine and its corresponding ribonucleoside is described in US Patent No. 3,201,387.

U.S. Patent No. 5,321,030 (Kaddurah-Daouk et al.; Amira) describes the use of creatine analogs as antiherpes viral agents. U.S. Patent No. 5,306,722 (Kim et al.; Bristol-Meyers Squibb) describes thymidine kinase inhibitors useful for treating HSV infections and for inhibiting herpes thymidine kinase.

Other antiherpesvirus compositions are described in U.S. Patent Nos. 5,286,649 and 5,098,708 (Konishi et al., Bristol-Meyers Squibb) and 5,175,165 (Blumenkopf et al.; Burroughs Wellcome). U.S. Patent No.

4,880,820 (Ashton et al.; Merck) describes the antiherpes virus agent (S)-9-(2,3-dihydroxy-1propoxymethyl)guanine.

WO 96/06159 PCT/US95/10194 69 U.S. Patent No. 4,708,935 (Suhadolnik et al.; Research Corporation) describes a 3'-deoxyadenosine compound effective in inhibiting HSV and EBV. U.S. Patent No.

4,386,076 (Machida et al.; Yamasa Shoyu Kabushiki K a i s h a d e s c r i b e s u s e of (E)-5-(2-halogenovinyl)-arabinofuranosyluracil as an antiherpesvirus agent. U.S. Patent No. 4,340,599 (Lieb et al.; Bayer Aktiengesellschaft) describes phosphonohydroxyacetic acid derivatives useful as antiherpes agents. U.S. Patent Nos. 4,093,715 and 4,093,716 (Lin et al. Research Corporation) describe and as potent inhibitors of herpes simplex virus. U.S. Patent No. 4,069,382 (Baker et al.; Parke, Davis Company) describes compounds useful as antiviral agents. U.S. Patent No. 3,927,216 (Witkowski et al.) describes the use of 1 2 4-triazole-3-carboxamide and 1,2,4-triazole-3-thiocarboxamide for inhibiting herpes virus infections. Patent No. 5,179,093 (Afonso et al., Schering) describes quinoline-2,4-dione derivatives active against herpes simplex virus 1 and 2, cytomegalovirus and Epstein Barr virus.

v) Inhibitory nucleic acid therapeutics Also contemplated here are inhibitory nucleic acid therapeutics which can inhibit the activity of herpesviruses in patients with KS. Inhibitory nucleic acids may be single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex or triplex is formed. These nucleic acids are often termed "antisense" because they are usually complementary to the sense or coding strand of the WO 96/06159 PCT/US95/10194 gene, although recently approaches for use of "sense" nucleic acids have also been developed. The term "inhibitory nucleic acids" as used herein, refers to both "sense" and "antisense" nucleic acids.

By binding to the target nucleic acid, the inhibitory nucleic acid can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking DNA transcription, processing or poly(A) addition to mRNA, DNA replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradation. Inhibitory nucleic acid methods therefore encompass a number of different approaches to altering expression of herpesvirus genes. These different types of inhibitory nucleic acid technology are described in Helene, C. and Toulme, J. which is hereby incorporated by reference and is referred to hereinafter as "Helene and Toulme.' In brief, inhibitory nucleic acid therapy approaches can be classified into those that target DNA sequences, those that target RNA sequences (including pre-mRNA and mRNA), those that target proteins (sense strand approaches), and those that cause cleavage or chemical modification of the target nucleic acids.

Approaches targeting DNA fall into several categories.

Nucleic acids can be designed to bind to the major groove of the duplex DNA to form a triple helical or "triplex" structure. Alternatively, inhibitory nucleic acids are designed to bind to regions of single stranded DNA resulting from the opening of the duplex DNA during replication or transcription. See Helene and Toulme.

WO 96/06159 PCTIUS95/10194 71 More commonly, inhibitory nucleic acids are designed to bind to mRNA or mRNA precursors. Inhibitory nucleic acids are used to prevent maturation of premRNA. Inhibitory nucleic acids may be designed to interfere with RNA processing, splicing or translation.

The inhibitory nucleic acids can be targeted to mRNA.

In this approach, the inhibitory nucleic acids are designed to specifically block translation of the encoded protein. Using this approach, the inhibitory nucleic acid can be used to selectively suppress certain cellular functions by inhibition of translation of mRNA encoding critical proteins. For example, an inhibitory nucleic acid complementary to regions of c-myc mRNA inhibits c-myc protein expression in a human promyelocytic leukemia cell line, HL60, which overexpresses the c-myc protooncogene. See Wickstrom et al. [93] and Harel-Bellan, et al. [31A]. As described in Helene and Toulme, inhibitory nucleic acids targeting mRNA have been shown to work by several different mechanisms to inhibit translation of the encoded protein(s).

The inhibitory nucleic acids introduced into the cell can also encompass the "sense" strand of the gene or mRNA to trap or compete for the enzymes or binding proteins involved in mRNA translation. See Helene and Toulme.

Lastly, the inhibitory nucleic acids can be used to induce chemical inactivation or cleavage of the target genes or mRNA. Chemical inactivation can occur by the induction of crosslinks between the inhibitory nucleic acid and the target nucleic acid within the cell.

Other chemical modifications of the target nucleic WO 96/06159 PCT/US95/10194 72 acids induced by appropriately derivatized inhibitory nucleic acids may also be used.

Cleavage, and therefore inactivation, of the target nucleic acids may be effected by attaching a substituent to the inhibitory nucleic acid which can be activated to induce cleavage reactions. The substituent can be one that affects either chemical, or enzymatic cleavage. Alternatively, cleavage can be induced by the use of ribozymes or catalytic RNA.

In this approach, the inhibitory nucleic acids would comprise either naturally occurring RNA (ribozymes) or synthetic nucleic acids with catalytic activity.

The targeting of inhibitory nucleic acids to specific cells of the immune system by conjugation with targeting moieties binding receptors on the surface of these cells can be used for all of the above forms of inhibitory nucleic acid therapy. This invention encompasses all of the forms of inhibitory nucleic acid therapy as described above and as described in Helene and Toulme.

This invention relates to the targeting of inhibitory nucleic acids to sequences the human herpesvirus of the invention for use in treating KS. An example of an antiherpes virus inhibitory nucleic acid is ISIS 2922 (ISIS Pharmaceuticals) which has activity against CMV [see, Biotechnology News 14(14) p. A problem associated with inhibitory nucleic acid therapy is the effective delivery of the inhibitory nucleic acid to the target cell in vivo and the subsequent internalization of the inhibitory nucleic acid by that cell. This can be accomplished by linking the inhibitory nucleic acid to a targeting moiety to form a conjugate that binds to a specific WO 96/06159 PCT/US95/10194 73 receptor on the surface of the target infected cell, and which is internalized after binding.

iii) Administration The subjects to be treated or whose tissue may be used herein may be a mammal, or more specifically a human, horse, pig, rabbit, dog, monkey, or rodent. In the preferred embodiment the subject is a human.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each subject.

Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration.

As used herein administration means a method of administering to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, administration topically, parenterally, orally, intravenously, intramuscularly, subcutaneously or by aerosol. Administration of the agent may be effected continuously or intermittently such that the therapeutic agent in the patient is effective to treat a subject with Kaposi's sarcoma or a subject infected with a DNA virus associated with Kaposi's sarcoma.

The antiviral compositions for treating herpesvirusinduced KS are preferably administered to human WO 96/06159 PCT/US95/10194 74 patients via oral, intravenous or parenteral administrations and other systemic forms. Those of skill in the art will understand appropriate administration protocol for the individual compositions to be employed by the physician.

The pharmaceutical formulations or compositions of this invention may be in the dosage form of solid, semi-solid, or liquid such as, suspensions, aerosols or the like. Preferably the compositions are administered in unit dosage forms suitable for single administration of precise dosage amounts. The compositions may also include, depending on the formulation desired, pharmaceutically-acceptable, nontoxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants; or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. Effective amounts of such diluent or carrier are those amounts which are effective to obtain a pharmaceutically acceptable formulation in terms of solubility of components, or biological activity, etc.

V. Immunological Approaches to Therapy.

Having identified a primary causal agent of KS in humans as a novel human herpesvirus, there are immunosuppressive therapies that can modulate the immunologic dysfunction that arises from the presence of viral infected tissue. In particular, agents that WO 96/06159 PCT/US95/10194 block the immunological attack of the viral infected cells will ameliorate the symptoms of KS and/or reduce the disease progress. Such therapies include antibodies that specifically block the targeting of viral infected cells. Such agents include antibodies which bind to cytokines that upregulate the immune system to target viral infected cells.

The antibody may be administered to a patient either singly or in a cocktail containing two or more antibodies, other therapeutic agents, compositions, or the like, including, but not limited to, immunosuppressive agents, potentiators and side-effect relieving agents. Of particular interest are immunosuppressive agents useful in suppressing allergic reactions of a host. Immunosuppressive agents of interest include prednisone, prednisolone, DECADRON (Merck, Sharp Dohme, West Point, PA), cyclophosphamide, cyclosporine, 6-mercaptopurine, methotrexate, azathioprine and i.v. gamma globulin or their combination. Potentiators of interest include monensin, ammonium chloride and chloroquine. All of these agents are administered in generally accepted efficacious dose ranges such as those disclosed in the Physician Desk Reference, 41st Ed. (1987), Publisher Edward R. Barnhart, New Jersey.

Immune globulin from persons previously infected with human herpesviruses or related viruses can be obtained using standard techniques. Appropriate titers of antibodies are known for this therapy and are readily applied to the treatment of KS. Immune globulin can be administered via parenteral injection or by intrathecal shunt. In brief, immune globulin preparations may be obtained from individual donors who are screened for antibodies to the KS-associated human herpesvirus, and plasmas from high-titered WO 96/06159 PCT/US95/10194 76 donors are pooled. Alternatively, plasmas from donors are pooled and then tested for antibodies to the human herpesvirus of the invention; high-titered pools are then selected for use in KS patients.

Antibodies may be formulated into an injectable preparation. Parenteral formulations are known and are suitable for use in the invention, preferably for i.m. or i.v. administration. The formulations containing therapeutically effective amounts of antibodies or immunotoxins are either sterile liquid solutions, liquid suspensions or lyophilized versions and optionally contain stabilizers or excipients.

Lyophilized compositions are reconstituted with suitable diluents, water for injection, saline, 0.3% glycine and the like, at a level of about from .01 mg/kg of host body weight to 10 mg/kg where appropriate. Typically, the pharmaceutical compositions containing the antibodies or immunotoxins will be administered in a therapeutically effective dose in a range of from about .01 mg/kg to about mg/kg of the treated mammal. A preferred therapeutically effective dose of the pharmaceutical composition containing antibody or immunotoxin will be in a range of from about 0.01 mg/kg to about 0.5 mg/kg body weight of the treated mammal administered over several days to two weeks by daily intravenous infusion, each given over a one hour period, in a sequential patient dose-escalation regimen.

Antibody may be administered systemically by injection subcutaneously or intraperitoneally or directly into KS lesions. The dose will be dependent upon the properties of the antibody or immunotoxin employed, its activity and biological half-life, the concentration of antibody in the formulation, the site and rate of dosage, the clinical tolerance of the WO 96/06159 PCT/US95/10194 77 patient involved, the disease afflicting the patient and the like as is well within the skill of the physician.

The antibody of the present invention may be administered in solution. The pH of the solution should be in the range of pH 5 to 9.5, preferably pH to 7.5. The antibody or derivatives thereof should be in a solution having a suitable pharmaceutically acceptable buffer such as phosphate, tris (hydroxymethyl) aminomethane-HCl or citrate and the like. Buffer concentrations should be in the range of 1 to 100 mM. The solution of antibody may also contain a salt, such as sodium chloride or potassium chloride in a concentration of 50 to 150 mM.

An effective amount of a stabilizing agent such as an albumin, a globulin, a gelatin, a protamine or a salt of protamine may also be included and may be added to a solution containing antibody or immunotoxin or to the composition from which the solution is prepared.

Systemic administration of antibody is made daily, generally by intramuscular injection, although intravascular infusion is acceptable. Administration may also be intranasal or by other nonparenteral routes. Antibody or immunotoxin may also be administered via microspheres, liposomes or other microparticulate delivery systems placed in certain tissues including blood.

In therapeutic applications, the dosages of compounds used in accordance with the invention vary depending on the class of compound and the condition being treated. The age, weight, and clinical condition of the recipient patient; and the experience and judgment of the clinician or practitioner administering the therapy are among the factors affecting the selected WO 96/06159 PCT/US95/10194 78 dosage. For example, the dosage of an immunoglobulin can range from about 0.1 milligram per kilogram of body weight per day to about 10 mg/kg per day for polyclonal antibodies and about 5% to about 20% of that amount for monoclonal antibodies. In such a case, the immunoglobulin can be administered once daily as an intravenous infusion. Preferably, the dosage is repeated daily until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. Generally, the dose should be sufficient to treat or ameliorate symptoms or signs of KS without producing unacceptable toxicity to the patient.

An effective amount of the compound is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. The dosing range varies with the compound used, the route of administration and the potency of the particular compound.

VI. Vaccines and Prophylaxis for KS This invention provides a method of vaccinating a subject against Kaposi's sarcoma, comprising administering to the subject an effective amount of the peptide or polypeptide encoded by the isolated DNA molecule, and a suitable acceptable carrier, thereby vaccinating the subject. In one embodiment naked DNA is administering to the subject in an effective amount to vaccinate a subject against Kaposi's sarcoma.

This invention provides a method of immunizing a subject against a disease caused by the DNA herpesvirus associated with Kaposi's sarcoma which WO 96/06159 PCT/US95/10194 79 comprises administering to the subject an effective immunizing dose of the isolated herpesvirus vaccine.

A. Vaccines The invention also provides substances suitable for use as vaccines for the prevention of KS and methods for administering them. The vaccines are directed against the human herpesvirus of the invention, and most preferably comprise antigen obtained from the KSassociated human herpesvirus.

Vaccines can be made recombinantly. Typically, a vaccine will include from about 1 to about micrograms of antigen or antigenic protein or peptide.

More preferably, the amount of protein is from about to about 45 micrograms. Typically, the vaccine is formulated so that a dose includes about milliliters. The vaccine may be administered by any route known in the art. Preferably, the route is parenteral. More preferably, it is subcutaneous or intramuscular.

There are a number of strategies for amplifying an antigen's effectiveness, particularly as related to the art of vaccines. For example, cyclization or circularization of a peptide can increase the peptide's antigenic and immunogenic potency. See U.S.

Pat. No. 5,001,049 which is incorporated by reference herein. More conventionally, an antigen can be conjugated to a suitable carrier, usually a protein molecule. This procedure has several facets. It can allow multiple copies of an antigen, such as a peptide, to be conjugated to a single larger carrier molecule. Additionally, the carrier may possess properties which facilitate transport, binding, absorption or transfer of the antigen.

WO 96/06159 PCT/US95/10194 For parenteral administration, such as subcutaneous injection, examples of suitable carriers are the tetanus toxoid, the diphtheria toxoid, serum albumin and lamprey, or keyhole limpet, hemocyanin because they provide the resultant conjugate with minimum genetic restriction. Conjugates including these universal carriers can function as T cell clone activators in individuals having very different gene sets.

The conjugation between a peptide and a carrier can be accomplished using one of the methods known in the art. Specifically, the conjugation can use bifunctional cross-linkers as binding agents as detailed, for example, by Means and Feeney, "A recent review of protein modification techniques," Bioconjugate Chem. 1:2-12 (1990).

Vaccines against a number of the Herpesviruses have been successfully developed. Vaccines against Varicella-Zoster Virus using a live attenuated Oka strain is effective in preventing herpes zoster in the elderly, and in preventing chickenpox in both immunocompromised and normal children (Hardy, et al. Hardy, I. et al. Levin, M.J. et al.

Gershon, A.A. Vaccines against Herpes simplex Types 1 and 2 are also commercially available with some success in protection against primary disease, but have been less successful in preventing the establishment of latent infection in sensory ganglia (Roizman, B. Skinner, G.R. et al. Vaccines against the human herpesvirus can be made by isolating extracellular viral particles from infected cell cultures, inactivating the virus with formaldehyde followed by ultracentrifugation to concentrate the viral particles and remove the WO 96/06159 PCT/US95/10194 81 formaldehyde, and immunizing individuals with 2 or 3 doses containing 1 x 109 virus particles (Skinner, G.R.

et al. Alternatively, envelope glycoproteins can be expressed in E. coli or transfected into stable mammalian cell lines, the proteins can be purified and used for vaccination (Lasky, L.A. MHC binding peptides from cells infected with the human herpesvirus can be identified for vaccine candidates per the methodology of supra.

The antigen may be combined or mixed with various solutions and other compounds as is known in the art.

For example, it may be administered in water, saline or buffered vehicles with or without various adjuvants or immunodiluting agents. Examples of such adjuvants or agents include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-inoil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionibacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan). Other suitable adjuvants are Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel.

Only aluminum is approved for human use.

The proportion of antigen and adjuvant can be varied over a broad range so long as both are present in effective amounts. For example, aluminum hydroxide WO 96/06159 PCT/US95/10194 82 can be present in an amount of about 0.5% of the vaccine mixture (A1 2 0 3 basis). On a per-dose basis, the amount of the antigen can range from about 0.1 gg to about 100 gg protein per patient. A preferable range is from about 1 gg to about 50 Ag per dose. A more preferred range is about 15 gg to about 45 Ag.

A suitable dose size is about 0.5 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 0.5 ml containing 45 Ag of antigen in admixture with 0.5% aluminum hydroxide. After formulation, the vaccine may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4 0 C, or it may be freeze-dried. Lyophilization permits long-term storage in a stabilized form.

The vaccines may be administered by any conventional method for the administration of vaccines including oral and parenteral subcutaneous or intramuscular) injection. Intramuscular administration is preferred. The treatment may consist of a single dose of vaccine or a plurality of doses over a period of time. It is preferred that the dose be given to a human patient within the first 8 months of life. The antigen of the invention can be combined with appropriate doses of compounds including influenza antigens, such as influenza type A antigens.

Also, the antigen could be a component of a recombinant vaccine which could be adaptable for oral administration.

Vaccines of the invention may be combined with other vaccines for other diseases to produce multivalent vaccines. A pharmaceutically effective amount of the antigen can be employed with a pharmaceutically acceptable carrier such as a protein or diluent useful for the vaccination of mammals, particularly humans.

WO 96/06159 PCT/US95/10194 83 Other vaccines may be prepared according to methods well-known to those skilled in the art.

Those of skill will readily recognize that it is only necessary to expose a mammal to appropriate epitopes in order to elicit effective immunoprotection. The epitopes are typically segments of amino acids which are a small portion of the whole protein. Using recombinant genetics, it is routine to alter a natural protein's primary structure to create derivatives embracing epitopes that are identical to or substantially the same as (immunologically equivalent to) the naturally occurring epitopes. Such derivatives may include peptide fragments, amino acid substitutions, amino acid deletions and amino acid additions of the amino acid sequence for the viral proteins from the human herpesvirus. For example, it is known in the protein art that certain amino acid residues can be substituted with amino acids of similar size and polarity without an undue effect upon the biological activity of the protein. The human herpesvirus proteins have significant tertiary structure and the epitopes are usually conformational.

Thus, modifications should generally preserve conformation to produce a protective immune response.

B. Antibody Prophylaxis Therapeutic, intravenous, polyclonal or monoclonal antibodies can been used as a mode of passive immunotherapy of herpesviral diseases including perinatal varicella and CMV. Immune globulin from persons previously infected with the human herpesvirus and bearing a suitably high titer of antibodies against the virus can be given in combination with antiviral agents ganciclovir), or in combination with other modes of immunotherapy that are currently WO 96/06159 PCT/US95/10194 84 being evaluated for the treatment of KS, which are targeted to modulating the immune response (i.e.

treatment with copolymer-l, antiidiotypic monoclonal antibodies, T cell "vaccination"). Antibodies to human herpesvirus can be administered to the patient as described herein. Antibodies specific for an epitope expressed on cells infected with the human herpesvirus are preferred and can be obtained as described above.

A polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.

Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

C. Monitoring therapeutic efficacy This invention provides a method for monitoring the therapeutic efficacy of treatment for Kaposi's sarcoma, which comprises determining in a first sample from a subject with Kaposi's sarcoma the presence of the isolated DNA molecule, administering to the subject a therapeutic amount of an agent such that the agent is contacted to the cell in a sample, determining after a suitable period of time the amount of the isolated DNA molecule in the second sample from WO 96/06159 PCTIUS95/10194 the treated subject, and comparing the amount of isolated DNA molecule determined in the first sample with the amount determined in the second sample, a difference indicating the effectiveness of the agent, thereby monitoring the therapeutic efficacy of treatment for Kaposi's sarcoma. As defined herein "amount" is viral load or copy number. Methods of determining viral load or copy number are known to those skilled in the art.

VII. Screening Assays For Pharmaceutical Agents of Interest in Alleviating the Symptoms of KS.

Since an agent involved in the causation or progression of KS has been identified and described here, assays directed to identifying potential pharmaceutical agents that inhibit the biological activity of the agent are possible. KS drug screening assays which determine whether or not a drug has activity against the virus described herein are contemplated in this invention. Such assays comprise incubating a compound to be evaluated for use in KS treatment with cells which express the KS associated human herpesvirus proteins or peptides and determining therefrom the effect of the compound on the activity of such agent. In vitro assays in which the virus is maintained in suitable cell culture are preferred, though in vivo animal models would also be effective.

Compounds with activity against the agent of interest or peptides from such agent can be screened in in vitro as well as in vivo assay systems. In vitro assays include infecting peripheral blood leukocytes or susceptible T cell lines such as MT-4 with the agent of interest in the presence of varying concentrations of compounds targeted against viral replication, including nucleoside analogs, chain WO 96/06159 PCT/US95/10194 86 terminators, antisense oligonucleotides and random polypeptides (Asada, H. et al. Kikuta et al. [48] both incorporated by reference herein). Infected cultures and their supernatants can be assayed for the total amount of virus including the presence of the viral genome by quantitative PCR, by dot blot assays, or by using immunologic methods. For example, a culture of susceptible cells could be infected with the human herpesvirus in the presence of various concentrations of drug, fixed on slides after a period of days, and examined for viral antigen by indirect immunofluorescence with monoclonal antibodies to viral peptides supra. Alternatively, chemically adhered MT-4 cell monolayers can be used for an infectious agent assay using indirect immunofluorescent antibody staining to search for focus reduction (Higashi, K. et al. incorporated by reference herein).

As an alternative to whole cell in vitro assays, purified enzymes isolated from the human herpesvirus can be used as targets for rational drug design to determine the effect of the potential drug on enzyme activity, such as thymidine phosphotransferase or DNA polymerase. The genes for these two enzymes are provided herein. A measure of enzyme activity indicates effect on the agent itself.

Drug screens using herpes viral products are known and have been previously described in EP 0514830 (herpes proteases) and WO 94/04920 (UL1 3 gene product).

This invention provides an assay for screening anti-KS chemotherapeutics. Infected cells can be incubated in the presence of a chemical agent that is a potential chemotherapeutic against KS acyclo-guanosine).

The level of virus in the cells is then determined WO 96/06159 PCT/US95/10194 87 after several days by IFA for antigens or Southern blotting for viral genome or Northern blotting for MRNA and compared to control cells. This assay can quickly screen large numbers of chemical compounds that may be useful against KS.

Further, this invention provides an assay system that is employed to identify drugs or other molecules capable of binding to the DNA molecule or proteins, either in the cytoplasm or in the nucleus, thereby inhibiting or potentiating transcriptional activity.

Such assay would be useful in the development of drugs that would be specific against particular cellular activity, or that would potentiate such activity, in time or in level of activity.

This invention is further illustrated in the Experimental Details section which follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter.

EXPERIMENTAL DETAILS SECTION I: Experiment i: Representational difference analysis (RDA) to identify and characterize unique DNA sequences in KS tissue To search for foreign DNA sequences belonging to an infectious agent in AIDS-KS, representational difference analysis (RDA) was employed to identify and characterize unique DNA sequences in KS tissue that are either absent or present in low copy number in non-diseased tissue obtained from the same patient This method can detect adenovirus genome added in single copy to human DNA but has not been used to WO 96/06159 PCT/US95/10194 88 identify previously uncultured infectious agents. RDA is performed by making simplified "representations" of genomes from diseased and normal tissues from the same individual through PCR amplification of short restriction fragments. The DNA representation from the diseased tissue is then ligated to a priming sequence and hybridized to an excess of unligated, normal tissue DNA representation. Only unique sequences found in the diseased tissue have priming sequences oi both DNA strands and are preferentially amplified during subsequent rounds of PCR amplification. This process can be repeated using different ligated priming sequences to enrich the sample for unique DNA sequences that are only found in the tissue of interest.

DNA (10 jig) extracted from both the KS lesion and unaffected tissue were separately digested to completion with Bam HI (20 units/g) at 370 C for 2 hours and 2 Ag of digestion fragments were ligated to NBaml2 and NBam24 priming sequences [primer sequences described in 58]. Thirty cycles of PCR amplification were performed to amplify "representations" of both genomes. After construction of the genomic representations, KS tester amplicons between 150 and 1500 bp were isolated from an agarose gel and NBam priming sequences were removed by digestion with Bam HI. To search for unique DNA sequences not found in non-KS driver DNA, a second set of priming sequences (JBaml2 and JBam24) was ligated onto only the KS tester DNA amplicons (Figure 1, lane 0.2 Ag of ligated KS lesion amplicons were hybridized to 20 Ag of unligated, normal tissue representational amplicons. An aliquot of the hybridization product was then subjected to 10 cycles of PCR amplification using JBam24, followed by mung bean nuclease digestion. An aliquot of the mung bean-treated WO 96/06159 PCT/US95/10194 89 difference product was then subjected to 15 more cycles of PCR with the JBam24 primer (Figure 1, lane Amplification products were redigested with Bam HI and 200 ng of the digested product was ligated to RBaml2 and RBam24 primer sets for a second round of hybridization and PCR amplification (Figure 1, lane This enrichment procedure was repeated a third time using the JBam primer set (Figure 1, lane 4).

Both the original driver and the tester DNA samples (Table 2, Patient A) were subsequently found to contain the AIDS-KS specific sequences KS330Bam and KS631Bam (previously identified as KS627Bam) indicating that RDA can be successfully employed when the target sequences are present in unequal copy number in both tissues.

The initial round of DNA amplification-hybridization from KS and normal tissue resulted in a diffuse banding pattern (Figure 1, lane but four bands at approximately 380, 450, 540 and 680 bp were identifiable after the second amplificationhybridization (Figure 1, lane These bands became discrete after a third round of amplificationhybridization (Figure 1, lane Control RDA, performed by hybridizing DNA extracted from AIDS-KS tissue against itself, produced a single band at approximately 540 bp (Figure 1, lane The four KSassociated bands (designated KS330Bam, KS390Bam, KS480Bam, KS627Bam after digestion of the two flanking 28 bp ligated priming sequences with Bar HI) were gel purified and cloned by insertion into the pCRII vector. PCR products were cloned in the pCRII vector using the TA cloning system (Invitrogen Corporation, San Diego, CA).

WO 96/06159 PCTIUS95/10194 Experiment 2: Determination of the specificity of AIDS-KS unique sequences.

To determine the specificity of these sequences for AIDS-KS, random-primed 32 P-labeled inserts were hybridized to Southern blots of DNA extracted from cryopreserved tissues obtained from patients with and without AIDS. All AIDS-KS specimens were examined microscopically for morphologic confirmation of KS and immunohistochemically for Factor VIII, Ulex europaeus and CD34 antigen expression. One of the AIDS-KS specimens was apparently mislabeled since KS tissue was not detected on microscopic examination but was included in the KS specimen group for purposes of statistical analysis. Control tissues used for comparison to the KS lesions included 56 lymphomas from patients with and without AIDS, 19 hyperplastic lymph nodes from patients with and without AIDS, vascular tumors from nonAIDS patients and 13 tissues infected with opportunistic infections that commonly occur in AIDS patients. Control DNA was also extracted from a consecutive series of 49 surgical biopsy specimens from patients without AIDS.

Additional clinical and demographic information on the specimens was not collected to preserve patient confidentiality.

The tissues, listed in Table i, were collected from diagnostic biopsies and autopsies between 1983 and 1993 and stored at -700C. Each tissue sample was from a different patient, except as noted in Table i. Most of the 27 KS specimens were from lymph nodes dissected under surgical conditions which diminishes possible contamination with normal skin flora. All specimens were digested with Bam HI prior to hybridization.

WO 96/06159 PCT/US95/10194 91 KS390Bam and KS480Bam hybridized nonspecifically to both KS and non-KS tissues and were not further characterized. 20 of 27 AIDS-KS DNAs hybridized with variable intensity to both KS330Bam and KS627Bam, and one additional KS specimen hybridized only to KS627Bam by Southern blotting (Figure 2 and Table 1).

In contrast to AIDS-KS lesions, only 6 of 39 non-KS tissues from patients with AIDS hybridized to the KS330Bam and KS627Bam inserts (Table 1).

Specific hybridization did not occur with lymphoma or lymph node DNA from 36 persons without AIDS or with control DNA from 49 tissue biopsy specimens obtained from a consecutive series of patients. DNA extracted from several vascular tumors, including a hemangiopericytoma, two angiosarcomas and a lymphangioma, were also negative by Southern blot hybridization. DNA extracted from tissues with opportunistic infections common to AIDS patients, including 7 acid-fast bacillus (undetermined species), 1 cytomegalovirus, 1 cat-scratch bacillus, 2 cryptococcus and 1 toxoplasmosis infected tissues, were negative by Southern blot hybridization to KS330Bam and KS627Bam (Table 1).

WO 96/06159 PCT/US95/10194 Table 1. Southern blot hybridization for KS330Bam and KS627Bam and PCR amplification for KS330 234 in human tissues from individual patients.

Tissue

AIDS-KS

n KS330Bam Southern hybridization 27* 20 (74) KS627Bam Southern KS330 234 hybridization PCR positive

AIDS

lymphomas

AIDS

lymph nodes Non-AIDS Lymphomas Non-AIDS lymph nodes Vascular tumors 27t 3 (11) 12 3 (25) 29 0 (0) 7 0 (0) 4§ 0 (0) 21 (78) 3 (11) 3 (25) 0 (0) 0 (0) 0 (0) 0 (0) 25 (93) 3 (11) 3 0 (0) 0 (0) 0 (0) 0 (0) Opportunistic 131 infections Consecutive 49 surgical biopsies 0 (0) 0 0 (0) 0 (0) WO 96/06159 PCT/US95/10194 93 Legend to Table 1: *Includes one AIDS-KS specimen unamplifiable for p53 exon 6 and one tissue which on microscopic examination did not have any detectable KS tissue present. Both of these samples were negative by Southern blot hybridization to KS330Bam and KS627Bam and by PCR amplification for the KS330 234 amplicon.

tIncludes 7 small non-cleaved cell lymphomas, diffuse large cell and immunoblastic lymphomas. Three of the lymphomas with immunoblastic morphology were positive for KS330Bam and KS627Bam.

Includes 13 anaplastic large cell lymphomas, 4 diffuse large cell lymphomas, 4 small lymphocytic lymphomas/chronic lymphocytic leukemias, 3 hairy cell leukemias, 2 monocytoid B-cell lymphomas, 1 follicular small cleaved cell lymphoma, 1 Burkitt's lymphoma, 1 plasmacytoma.

Includes 2 angiosarcomas, 1 hemangiopericytoma and 1 lymphangioma.

H Includes 2 cryptococcus, 1 toxoplasmosis, 1 catscratch bacillus, 1 cytomegalovirus, 1 Epstein-Barr virus, and 7 acid-fast bacillus infected tissues. In addition, pure cultures of Mycobacterium avium-complex were negative by Southern hybridization and PCR, and pure cultures of Mycoplasma penetrans were negative by

PCR.

Tissues included skin, appendix, kidney, prostate, hernia sac, lung, fibrous tissue, gallbladder, colon, foreskin, thyroid, small bowel, adenoid, vein, axillary tissue, lipoma, heart, mouth, hemorrhoid, pseudoaneurysm and fistula track. Tissues were WO 96/06159 PCT/US95/10194 94 collected from a consecutive series of biopsies on patients without AIDS but with unknown HIV serostatus.

**Apparent nonspecific hybridization at approximately Kb occurred in 4 consecutive surgical biopsy DNA samples: one colon and one hernia sac DNA sample hybridized to KS330Bam alone, another hernia sac DNA sample hybridized to KS627Bam alone and one appendix DNA sample hybridized to both KS330Bam and KS627Bam.

These samples did not hybridize in the 330-630 bp range expected for these sequences and were PCR negative for KS330 234 WO 96/06159 PCT/US95/10194 In addition, DNA from Epstein-Barr virus-infected peripheral blood lymphocytes and pure cultures of Mycobacterium avium-complex were also negative by Southern hybridization. Overall, 20 of 27 AIDS- KS specimens hybridized to KS330Bam and 21 of 27 (78%) AIDS-KS specimens hybridized to KS627Bam, compared to only 6 of 142 non-KS human DNA control specimens

(X

2 =85.02, p< 10- 7 and X 2 =92.4, p< 10- 7 respectively).

The sequence copy number in the AIDS-KS tissues was estimated by simultaneous hybridization with KS330Bam and a 440 bp probe for the constant region of the T cell receptor 3 gene Samples in lanes 5 and 6 of Figures 2A-2B showed similar intensities for the two probes indicating an average copy number of approximately two KS330Bam sequences per cell, while remaining tissues had weaker hybridization signals for the KS330Bam probe.

Experiment 3: Characterization of KS330Bam and KS627Bam To further characterize KS330Bam and KS627Bam, six clones for each insert were sequenced. The Sequenase version 2.0 (United States Biochemical, Cleveland, OH) system was used and sequencing was performed according to manufacturer's instructions. Nucleotides sequences were confirmed with an Applied Biosystems 373A Sequencer in the DNA Sequencing Facilities at Columbia University.

KS330Bam is a 330 bp sequence with 51% G:C content (Figure 3B) and KS627Bam is a 627 bp sequence with a 63% G:C content (Figure 3C). KS330Bam has 54% nucleotide identity to the BDLF1 open reading frame (ORF) of Epstein-Barr virus (EBV). Further analysis revealed that both KS330Bam and KS627Bam code for WO 96/06159 PCT/US95/10194 96 amino acid sequences with homology to polypeptides of viral origin. SwissProt and PIR protein databases were searched for homologous ORF using BLASTX KS330Bam is 51% identical by amino acid homology to a portion of the ORF26 open reading frame encoding the capsid protein VP23 (NCBI g.i. 60348, bp 46024 46935) of herpesvirus saimiri a gammaherpesvirus which causes fulminant lymphoma in New world monkeys.

This fragment also has a 39% identical amino acid sequence to the theoretical protein encoded by the homologous open reading frame BDLF1 in EBV (NCBI g.i.

59140, bp 132403 -133307) The amino acid sequence encoded by KS627Bam is homologous with weaker identity to the tegument protein, gpl40 (ORF 29, NCBI g.i. 60396, bpl08782-112681) of herpesvirus saimiri.

Sequence data from KS330Bam was used to construct PCR primers to amplify a 234bp fragment designated KS330 234 (Figure 3B). The conditions for PCR analyses were as follows: 94 0 C for 2 min (1 cycle); 94°C for 1 min, 58 0 C for 1 min, 720C for 1 min (35 cycles) 720C extension for 5 min (1 cycle). Each PCR reaction used 0.1 pg of genomic DNA, 50 pmoles of each primer, 1 unit of Taq polymerase, 100 pM of each deoxynucleotide triphosphate, 50 mM KC1, 10mM Tris-HCl (pH and 0.1% Triton-X-100 in a final volume of A1. Amplifications were carried out in a Perkin- Elmer 480 Thermocycler with 1-s ramp times between steps.

Although Southern blot hybridization detected the KS330Bam sequence in only 20 of 27 KS tissues, 25 of the 27 tissues were positive by PCR amplification for KS330 234 (Figures 4A-4B) demonstrating that KS330Bam is present in some KS lesions at levels below the WO 96/06159 PCT/US95/10194 97 threshold for detection by Southern blot hybridization. All KS330 2 34 PCR products hybridized to a 2 "P end-labelled 25 bp internal oligomer, confirming the specificity of the PCR (Figure 4B). Of the two AIDS-KS specimens negative for KS330 234 both specimens appeared to be negative for technical reasons: one had no microscopically detectable KS tissue in the frozen sample (Figures 4A-4B, lane and the other (Figures 4A-4B, lane 15) was negative in the control PCR amplification for the p53 gene indicating either DNA degradation or the presence of PCR inhibitors in the sample. PCR amplification of the p53 tumor suppressor gene was used as a control for DNA quality.

Sequences of p53 primers from P6-5, ACAGGGCTGGTTGCCCAGGGT-3' (SEQ ID No: 44); and P6-3. AGTTGCAAACCAGACCTCAG-3'(SEQ ID NO: 45) Except for the 6 control samples from AIDS patients that were also positive by Southern blot hybridization, none of the other 136 control specimens were positive by PCR for KS330 234 All of these specimens were amplifiable for the p53 gene, indicating that inadequate PCR amplification was not the reason for lack of detection of KS330 234 in the control tissues. Samples containing DNA from two candidate KS agents, EBV and Mycoplasma penetrans (ATCC Accession No. 55252), a pathogen commonly found in the genital tract of patients with AIDS-KS [59] were also negative for amplification of KS330 234 In addition, several KS specimens were tested using commercial PCR primers (Stratagene, La Jolla, CA) specific for mycoplasmata and primers specific for the EBNA-2, EBNA-3C and EBER regions of EBV and were negative [57].

Overall, DNA from 25 of 27 AIDS-KS tissues were positive by PCR compared with DNA from 6 of 142 WO 96/06159 PCT/US95/10194 98 control tissues, including 6 of 39 non-KS lymph nodes and lymphomas from AIDS patients (X 2 =38.2, p 10-6), 0 of 36 lymph nodes and lymphomas from nonAIDS patients (X 2 =55.2, p 10- 7 and 0 of 49 consecutive biopsy specimens (X 2 =67.7, p 10-7). Thus, KS330 234 was found in all 25 amplifiable tissues with microscopically detectable AIDS-KS, but rarely occurred in non-KS tissues, including tissues from AIDS patients.

Of the six control tissues from AIDS patients that were positive by both PCR and Southern hybridization, two patients had KS elsewhere, two did not develop KS and complete clinical histories for the remaining two patients were unobtainable. Three of the six positive non-KS tissues were lymph nodes with follicular hyperplasia taken from patients with AIDS. Given the high prevalence of KS among patients with AIDS, it is possible that undetected microscopic foci of KS were present in these lymph nodes. The other three positive tissue specimens were B cell immunoblastic lymphomas from AIDS patients. It is possible that the putative KS agent is also a cofactor for a subset of AIDS-associated lymphomas [16, 17, To determine whether KS330Bam and KS627Bam are portions of a larger genome and to determine the proximity of the two sequences to each other, samples of KS DNA were digested with Pvu II restriction enzymes. Digested genomic DNA from three AIDS-KS samples were hybridized to KS330Bam and KS627Bam by Southern blotting (Figure These sequences hybridized to various sized fragments of the digested KS DNA indicating that both sequences are fragments of larger genomes. Differences in the KS330Bam hybridization pattern to Pvu II digests of the three AIDS-KS specimens indicate that polymorphisms may WO 96/06159 PCT/US95/10194 99 occur in the larger genome. Individual fragments from the digests failed to simultaneously hybridize with both KS330Bam and KS627Bam, demonstrating that these two Bam HI restriction fragments are not adjacent to one another.

If KS330Bam and KS627Bam are heritable polymorphic DNA markers for KS, these sequences should be uniformly detected at non-KS tissue sites in patients with AIDS- KS. Alternatively, if KS330Bam and KS627Bam are sequences specific for an exogenous infectious agent, it is likely that some tissues are uninfected and lack detectable KS330Bam and KS627Bam sequences. DNA extracted from multiple uninvolved tissues from three patients with AIDS-KS were hybridized to 32 P-labelled KS330Bam and KS627Bam probes as well as analyzed by PCR using the KS330 234 primers (Table While KS lesion DNA samples were positive for both bands, unaffected tissues were frequently negative for these sequences. KS lesions from patients A, B and C, and uninvolved skin and muscle from patient A were positive for KS330Bam and KS627Bam, but muscle and brain tissue from patient B and muscle, brain, colon, heart and hilar lymph node tissues from patient C were negative for these sequences. Uninvolved stomach lining adjacent to the KS lesion in patient C was positive by PCR, but negative by Southern blotting which suggests the presence of the sequences in this tissue at levels below the detection threshold for Southern blotting.

WO 96/06159 PCTIUS95/10194 100 Table 2: Differential detection of KS330Bam, KS627Bam and KS330 234 sequences in KS-involved and non-involved tissues from three patients with AIDS-KS.

KS330Bam KS627Bam KS330 2 3 4 Patient A KS, skin nl skin nl muscle Patient B KS, skin nl muscle nl brain Patient C KS, stomach nl stomach adjacent to KS nl muscle nl brain nl colon nl heart nl hilar lymph nodes Experiment 4: Subcloning and sequencing of KSHV KS330Bam and KS627Bam are genomic fragments of a novel infectious agent associated with AIDS-KS. A genomic library from a KS lesion was made and a phage clone with a 20 kb insert containing the KS330Bam sequence was identified. The 20 kb clone digested with PvuII (which cuts in the middle of the KS330Bam sequence) produced 1.1 kb and 3 kb fragments that hybridized to KS330Bam. The 1.1 kb subcloned insert and -900 bp from the 3 kb subcloned insert resulting in 9404 bp of WO 96/06159 PCT/US95/10194 101 contiguous sequence was entirely sequenced. This sequence contains partial and complete open reading frames homologous to regions in gamma herpesviruses.

The KS330Bam sequence is an internal portion of an 918 bp ORF with 55-56% nucleotide identity to the ORF26 and BDLF1 genes of HSVSA and EBV respectively. The EBV and HSVSA translated amino acid sequences for these ORFs demonstrate extensive homology with the amino acid sequence encoded by the KS-associated 918 bp ORF (Figure In HSVSA, the VP23 protein is a late structural protein involved in capsid construction. Reverse transcriptase (RT)-PCR of mRNA from a KS lesion is positive for transcribed KS330Bam mRNA and that indicates that this ORF is transcribed in KS lesions. Additional evidence for homology between the KS agent and herpesviruses comes from a comparison of the genomic organization of other potential ORFs on the 9404 bp sequence (Figure 3A) The 5' terminus of the sequence is composed nucleotides having 66-67% nucleotide identity and 68- 71% amino acid identity to corresponding regions of the major capsid protein (MCP) ORFs for both EBV and HSVSA. This putative MCP ORF of the KS agent lies immediately 5' to the BDLF1/ORF26 homolog which is a conserved orientation among herpesvirus subfamilies for these two genes. At the 3' end of this sequence, the reading frame has strong amino acid and nucleotide homology to HSVSA ORF 27. Thus, KS-associated DNA sequences at four loci in two separate regions with homologies to gamma herpesviral genomes have been identified.

In addition to fragments obtained from Pvu II digest of the 21 Kb phage insert described above, fragments obtained from a BamHI/NotI digest were also subcloned into pBluescript (Stratagene, La Jolla, CA) The I- WO 96/06159 PCT/US95/10194 102 termini of these subcloned fragments were sequenced and were also found to be homologous to nucleic acid sequence EBV and HSVSA genes. These homologs have been used to develop a preliminary map of subcloned fragments (Figure Thus, sequencing has revealed that the KS agent maintains co-linear homology to gamma herpesviruses over the length of the 21 Kb phage insert.

Experiment 5: Determination of the phylogeny of KSHV Regions flanking KS330Bam were sequenced and characterized by directional walking. This was performed by the following strategy: 1) KS genomic libraries were made and screened using the KS330Bam fragment as a hybridization probe, 2) DNA inserts from phage clones positive for the KS330Bam probe were isolated and digested with suitable restriction enzyme(s), 3) the digested fragments were subcloned into pBluescript (Stratagene, La Jolla, CA), and 4) the subclones were sequenced. Using this strategy, the major capsid protein (MCP) ORF homolog was the first important gene locus identified. Using sequenced unique 3' and 5' end-fragments from positive phage clones as probes, and following the strategy above a KS genomic library are screened by standard methods for additional contiguous sequences.

For sequencing purposes, restriction fragments are subcloned into phagemid pBluescript KS+, pBluescript KS-, pBS+, or pBS- (Stratagene) or into plasmid pUC18 or pUC19. Recombinant DNA was purified through CsC1 density gradients or by anion-exchange chromatography (Qiagen).

Nucleotide sequenced by standard screening methods of cloned fragments of KSHV were done by direct WO 96/06159 PCT/US95/10194 103 sequencing of double- stranded DNA using oligonucleotide primers synthesized commercially to "walk" along the fragments by the dideoxy-nucleotide chain termination method. Junctions between clones are confirmed by sequencing overlapping clones.

Targeted homologous genes in regions flanking KS330Bam include, but are not limited to: 11-10 homolog, thymidine kinase g85, g35, gH, capsid proteins and MCP. TK is an early protein of the herpesviruses functionally linked to DNA replication and a target enzyme for anti-herpesviral nucleosides. TK phosphorylates acyclic nucleosides such as acyclovir which in turn inhibit viral DNA polymerase chain extension. Determining the sequence of this gene will aid in the prediction of chemotherapeutic agents useful against KSHV. TK is encoded by the EBV BXLF1 ORF located -9700 bp rightward of BDLF1 and by the HSVSA ORF 21 -9200 bp rightward of the ORF 26. A subcloned fragment of KS5 was identified with strong homology to the EBV and HSVSA TK open reading frames.

is a late glycoprotein involved in membrane fusion homologous to gH in HSV1. In EBV, this protein is encoded by BLXF2 ORF located -7600 bp rightward of BDLF1, and in HSVSA it is encoded by ORF 22 located -7100 bp rightward of ORF26.

is a late EBV glycoprotein found in virion and plasma membrane. It is encoded by BDLF3 ORF which is 1300 bp leftward of BDLF1 in EBV. There is no BDLF3 homolog in HSVSA. A subcloned fragment has already been identified with strong homology to the EBV open reading frame.

Major capsid protein (MCP) is a conserved 150 KDa protein which is the major component of herpesvirus WO 96/06159 PCT/US95/10194 104 capsid. Antibodies are generated against the MCP during natural infection with most herpesviruses. The terminal 1026 bp of this major capsid gene homolog in KSHV have been sequenced.

Targeted homologous genes/loci in regions flanking KS627Bam include, but are not limited to: terminal reiterated repeats, LMPI, EBERs and Ori P. Terminal reiterated sequences are present in all herpesviruses.

In EBV, tandomly reiterated 0.5 Kb long terminal repeats flank the ends of the linear genome and become joined in the circular form. The terminal repeat region is immediately adjacent to BNRF1 in EBV and ORF in HSVSA. Since the number of terminal repeats varies between viral strains, identification of terminal repeat regions may allow typing and clonality studies of KSHV in KS legions. Sequencing through the terminal repeat region may determine whether this virus is integrated into human genome in KS.

LMPI is an latent protein important in the transforming effects of EBV in Burkitt's lymphoma.

This gene is encoded by the EBV BNRF1 ORF located -2000 bp rightward of tegument protein ORF BNRF1 in the circularized genome. There is no LMP1 homolog in

HSVSA.

EBERs are the most abundant RNA in latently EBV infected cells and Ori-P is the origin of replication for latent EBV genome. This region is located between -4000-9000 bp leftward of the BNRF1 ORF in EBV; there are no corresponding regions in HSVSA.

The data indicates that the KS agent is a new human herpesvirus related to gamma herpesviruses EBV and HSVSA. The results are not due to contamination or to incidental co-infection with a known herpesvirus since WO 96/06159 PCT/US95/10194 105 the sequences are distinct from all sequenced herpesviral genomes (including EBV, CMV, HHV6 and HSVSA) and are associated specifically with KS in three separate comparative studies. Furthermore,

PCR

testing of KS DNA with primers specific for EBV-1 and EBV-2 failed to demonstrate these viral genomes in these tissues. Although KSHV is homologous to EBV regions, the sequence does not match any other known sequence and thus provides evidence for a new viral genome, related to but distinct from known members of the herpesvirus family.

Experiment 6: Serological studies Indirect immunofluorescence assay

(IFA)

Virus-containing cells are coated to a microscope slide. The slides are treated with organic fixatives, dried and then incubated with patient sera.

Antibodies in the sera bind to the cells, and then excess nonspecific antibodies are washed off. An antihuman immunoglobulin linked to a fluorochrome, such as fluorescein, is then incubated with the slides, and then excess fluorescent immunoglobulin is washed off. The slides are then examined under a microscope and if the cells fluoresce, then this indicates that the sera contains antibodies directed against the antigens present in the cells, such as the virus.

An indirect immunofluorescence assay (IFA) was performed on the Body Cavity-Based Lymphoma cell line (BCBL-1), which is a naturally transformed

EBV

infected (nonproducing) B cell line, using 4 KS patient sera and 4 control sera (from AIDS patients without KS). Initially, both sets of sera showed similar levels of antibody binding. To remove WO 96/06159 PCT/US95/10194 106 nonspecific antibodies directed against EBV and lymphocyte antigens, sera at 1:25 dilution were preadsorbed using 3x10 6 1% paraformaldehyde-fixed Raji cells per ml of sera. BCBL1 cells were fixed with ethanol/acetone, incubated with dilutions of patient sera, washed and incubated with fluorescein-conjugated goat anti-human IgG. Indirect immunofluorescent staining was determined.

Table 3 shows that unabsorbed case and control sera have similar end-point dilution indirect immunofluorescence assay (IFA) titers against the BCBL1 cell line. After Raji adsorption, case sera have four-fold higher IFA titers against BCBL1 cells than control sera. Results indicated that preadsorption against paraformaldehyde-fixed Raji cells reduces fluorescent antibody binding in control sera but do not eliminate antibody binding to KS case sera.

These results indicate that subjects with KS have specific antibodies directed against the KS agent that can be detected in serological assays such as IFA, Western blot and Enzyme immunoassays (Table 3).

WO 96/06159 PCTIUS95/10194 107 Table 3: Indirect immunofluorescence end-point titers for KS case and non-KS control sera against the BCBL-1 cell line Sera No. Status* Pre-adsorption Post-adsorption** 1 KS 1:400 1:400 2 KS 1:100 1:100 3 KS 1:200 1:100 4 KS 1:400 1:200 Control 1:400 1:50 6 Control 1:50 1:50 7 Control 1:100 1:50 8 Control 1:200 1:50 Legend Table 3: KS=autopsy-confirmed male, AIDS patient Control=autopsy-confirmed female, AIDS patient, no KS Adsorbed against RAJI cells treated with 1% paraformaldehyde Immunoblotting ("Western blot") Virus-containing cells or purified virus (or a portion of the virus, such as a fusion protein) is electrophoresed on a polyacrylamide gel to separate the protein antigens by molecular weight. The proteins are blotted onto a nitrocellulose or nylon membrane, then the membrane is incubated in patient sera. Antibodies directed against specific antigens are developed by incubating with a anti-human immunoglobulin attached to a reporter enzyme, such as a peroxidase. After developing the membrane, each antigen reacting against antibodies in patient sera shows up as a band on the membrane at the corresponding molecular weight region.

WO 96/06159 PCT/US95/10194 108 Enzyme immunoassay ("EIA or ELISA") Virus-containing cells or purified virus (or a portion of the virus, such as a fusion protein) is coated to the bottom of a 96-well plate by various means (generally incubating in alkaline carbonate buffer).

The plates are washed, then the wells are incubated with patient sera. Antibodies in the sera directed against specific antigens stick on the plate. The wells are washed again to remove nonspecific antibody, then they are incubated with a antihuman immunoglobulin attached to a reporter enzyme, such as a peroxidase. The plate is washed again to remove nonspecific antibody and then developed. Wells containing antigen that is specifically recognized by antibodies in the patients sera change color and can be detected by an ELISA plate reader (a spectrophotomer) All three of these methods can be made more specific by pre-incubating patient sera with uninfected cells to adsorb out cross-reacting antibodies against the cells or against other viruses that may be present in the cell line, such as EBV. Cross-reacting antibodies can potentially give a falsely positive test result the patient is actually not infected with the virus but has a positive test result because of crossreacting antibodies directed against cell antigens in the preparation). The importance of the infection experiments with Raji is that if Raji cells, or another well-defined cell line, can be infected, then the patient's sera can be pre-adsorbed against the uninfected parental cell line and then tested in one of the assays. The only antibodies left in the sera after pre-adsorption that bind to antigens in the preparation should be directed against the virus.

WO 96/06159 PCT/US95/10194 109 Experiment 7: BCBL 1, from lymphomatous tissues belonging to a rare infiltrating, anaplastic body cavity lymphoma occurring in AIDS patients has been placed in continuous cell culture and shown to be continuously infected with the KS agent. This cell line is also naturally infected with Epstein-Barr Virus (EBV). The BCBL cell line was used as an antigen substrate to detect specific KS antibodies in persons infected with the putative virus by Western-blotting. Three lymphoid B cell lines were used as controls. These included the EBV genome positive cell line P3H3, the EBV genome defective cell line Raji and the EBV genome negative cell line Bjab.

Cells from late-log phase culture were washed 3 time with PBS by centrifugation at 500 g for 10min. and suspended in sample buffer containing 50 mM Tris-HCl pH 6.8, 2% SDS 15% glycerol 5% 0mercaptoethanol and 0.001% bromophenol (w/v) with protease inhibitor, 100 AM phenylmethylsulfonyl fluoride (PMSF). The sample was boiled at 100 0 C for 5 min and centrifuged at 14,000 g for 10 min. The proteins in the supernatant was then fractionated by sodium, dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions with a separation gel of 15% and a stacking gel of Prestained protein standards were included: myosin, 200 kDa; /-galactosidase, 118 kDA; BSA, 78 kDa; ovalbumin, 47.1 kDa; carbonic anhydrase, 31.4 kDa; soybean trypsin inhibitor, 25.5 kDa, lysozyme, 18.8 kDa and aprotinin, 8.3 kDa (Bio-Rad).

Immunoblotting experiments were performed according to the method of Towbin et al. Briefly, the proteins were electrophorectically transferred to WO 96/06159 PCT/US95/10194 110 Hybon-C extra membranes (Pharmacia) at 24 V for min. The membranes were then dried at 37 0 C for min, saturated with 5% skim milk in Tris-buffered saline, pH 7.4 (TBS) containing 50 mM Tris-HC1 and 200 mM NaC1, at room temperature for 1 h. The membranes were subsequently incubated with human sera at dilution 1:200 in 1% skim milk overnight at room temperature, washed 3 times with a solution containing TBS, 0.2% Triton X-100 and 0.05% skim milk and then 2 times with TBS. The membranes were then incubated for 2 h at room temperature with alkaline phosphatase conjugated goat anti-mouse IgG IgM IgA (Sigma) diluted at 1:5000 in 1% skim milk. After repeating the washing,the membranes were stained with nitroblue tetranolium chloride and 5-bromo-4-chloro-3indolylphosphate p-toluidine salt (Gibco BRL).

Two bands of approximately 226 kDa and 234 kDa were identified to be specifically present on the Westerblot of BCBL cell lysate in 5 sera from AIDS gay man patients infected with KS. These 2 bands were absent from the lysates of P3H3, Raji and Bjab cell lysates.

sera from AIDS gay man patients without KS and 2 sera from AIDS woman patients without KS as well as 1 sera from nasopharyncel carcinoma patient were not able to detect these 2 bands in BCBL 1, P3H3, Raji and Bjab cell lysates. In a blinded experiment, using the 226 kDa and 234 kDa markers, 15 out of 16 sera from KS patients were correctly identified. In total, the 226 kDa and 234 kDa markers were detected in 20 out of 21 sera from KS patients.

The antigen is enriched in the nuclei fraction of BCBL1. Enriched antigen with low background can be obtained by preparing nucleic from BCBC as the starting antigen preparation using standard, widely available protocols. For example, 500-750ml of BCBL WO 96/06159 PCT/US95/10194 111 at 5X10 5 cells/ml can be pelleted at low speed. The pellet is placed in 10 mM NaC1, 10 mM Tris pH 7.8, mM MgC12 (equi volume) 1.0% NP-40 on ice for 20 min to lyse cells. The lysate is then spun at 1500 rpm for 10 min. to pellet nucleic. The pellet is used as the starting fraction for the antigen preparation for the Western blot. This will reduce cross- reactive cytoplasmic antigens.

Experiment 8: Transmission studies Co-infection experiments BCBL1 cells were co-cultivated with Raji cell lines separated by a 0.45 A tissue filter insert.

Approximately, 1-2 x 106 BCBL1 and 2x10 6 Raji cells were co-cultivated for 2-20 days in supplemented

RPMI

alone, in 10 pg/ml 5'-bromodeoxyuridine (BUdR) and 0.6 Ag/ml 5'-flourodeoxyuridine or 20 ng/ml 12-0tetradecanoylphorbol-13-acetate (TPA). After 2,8,12 or 20 days co-cultivation, Raji cells were removed, washed and placed in supplemented RPMI 1640 media. A Raji culture co-cultivated with BCBL1 in 20 ng/ml TPA for 2 days survived and has been kept in continuous suspension culture for >10 weeks. This cell line, designated RCC1 (Raji Co-Culture, No. 1) remains

PCR

positive for the KS330 234 sequence after multiple passages. This cell line is identical to its parental Raji cell line by flow cytometry using EMA, Bl, B4 and BerH2 lymphocyte-flow cytometry (approximately RCC1 periodically undergo rapid cytolysis suggestive of lytic reproduction of the agent. Thus, RCC1 is a Raji cell line newly infected with KSHV.

The results indicate the presence of a new human virus, specifically a herpesvirus in KS lesions. The high degree of association between this agent and WO 96/06159 PCT/US95/10194 112 AIDS-KS and the low prevalence of the agent in non-KS tissues from immunocompromised AIDS patients, indicates that this agent has a causal role in AIDS-KS [47, 68].

Experiment 10: Isolation of KSHV Crude virus preparations are made from either the supernatant or low speed pelleted cell fraction of BCBL1 cultures. Approximately 650ml or more of log phase cells should be used (>5X106 cells/ml) For bonding whole virion from supernatant, the cell free supernatant is spun at 10,000 rpm in a GSA rotor for 10 min to remove debris. PEG-8000 is added to 7%, dissolved and placed on ice for >2.5 hours. The PEGsupernatant is then spun at 10,000 xg for 30 min.

supernatant is poured off and the pellet is dried and scraped together from the centrifuge bottles. The pellet is then resuspended in a small volume (1-2 ml) of virus buffer (VB, 0.1 M NaC1, 0.01 M Tris, pH This procedure will precipitate both naked genome and whole virion. The virion are then isolated by centrifugation at 25,000 rpm in a 10-50% sucrose gradient made with VB. One ml fractions of the gradient are then obtained by standard techniques using a fractionator) and each fraction is then tested by dot blotting using specific hybridizing primer sequences to determine the gradient fraction containing the purified virus (preparation of the fraction maybe needed in order to detect the presence of the virus, such as standard DNA extraction).

To obtain the episomal DNA from the virus,the pellet of cells is washed and pelleted in PBS, then lysed using hypotonic shock and/or repeated cycles of freezing and thawing in a small volume ml).

WO 96/06159 PCT/US95/10194 113 Nuclei and other cytoplasmic debris are removed by centrifugation at 10,000g for 10 min, filtration through a 0.45 m filter and then repeat centrifugation at 10,000g for 10 min. This crude preparation contains viral genome and soluble cell components.

The genome preparation can then be gently chloroformphenol extracted to remove associated proteins or can be placed in neutral DNA buffer (1 M NaC1, 50 mM Tris, mM EDTA, pH 7.2-7.6) with 2% sodium dodecylsulfate (SDS) and 1% sarcosyl. The genome is then banded by centrifugation through 10-30% sucrose gradient in neutral DNA buffer containing 0.15% sarcosyl at 20,000 rpm in a SW 27.1 rotor for 12 hours (for 40,000 rpm for 2-3 hours in an SW41 rotor). The band is detected as described above.

An example of the method for isolating KSHV genome from KSHV infected cell cultures (97 and 98).

Approximately 800 ml of BCBL1 cells are pelleted, washed with saline, and pelleted by low speed centrifugation. The cell pellet is lysed with an equal volume of RSB (10 mM NaC1, 10 mM Tris-HCl, mM MgCl2, pH 7.8) with 1% NP-40 on ice for 10 minutes.

The lysate is centrifuged at 900xg for 10 minutes to pellet nuclei. This step is repeated. To the supernatant is added 0.4% sodium dodecylsulfate and EDTA to a final concentration of 10 mM. The supernatant is loaded on a 10-30% sucrose gradient in M NaC1, 1mM EDTA, 50mM Tris-HCl, pH 7.5. The gradients are centrifuged at 20,000 rpm on a SW 27.1 rotor for 12 hours. In figure 11, 0.5 ml aliquots of the gradient have been fractionated (fractions 1-62) with the 30% gradient fraction being at fraction No.

1 and the 10% gradient fraction being at fraction No.

62. Each fraction has been dot hybridized to a nitrocellulose membrane and then a "P-labeled KSHV DNA fragment, KS631Bam has been hybridized to the membrane WO 96/06159 PCT/US95/10194 114 using standard techniques. Figure 11 shows that the major solubilized fraction of the KSHV genome bands is isolated) in fractions 42 through 48 of the gradient with a high concentration of the genome being present in fraction 44. A second band of solubilized KSHV DNA occurs in fractions 26 through 32.

Experiment 11: Purification of KSHV DNA is extracted using standard techniques from the RCC-1 or RCC-1 2 Fl cell line [27, 49, 66]. The DNA is tested for the presence of the KSHV by Southern blotting and PCR using the specific probes as described hereinafter. Fresh lymphoma tissue containing viable infected cells is simultaneously filtered to form a single cell suspension by standard techniques [49, 66]. The cells are separated by standard Ficoll-Plaque centrifugation and lymphocyte layer is removed. The lymphocytes are then placed at >1x10 6 cells/ml into standard lymphocyte tissue culture medium, such as RMP 1640 supplemented with 10% fetal calf serum. Immortalized lymphocytes containing the KSHV virus are indefinitely grown in the culture media while nonimmortilized cells die during course of prolonged cultivation.

Further, the virus may be propagated in a new cell line by removing media supernatant containing the virus from a continuously infected cell line at a concentration of >1x10 6 cells/ml. The media is centrifuged at 2000xg for 10 minutes and filtered through a 0.45A filter to remove cells. The media is applied in a 1:1 volume with cells growing at >1x10 6 cells/ml for 48 hours. The cells are washed and pelleted and placed in fresh culture medium, and tested after 14 days of growth.

WO 96/06159 PCT/US95/10194 115 The herpesvirus may be isolated from the cell DNA in the following manner. An infected cell line, which can be lysed using standard methods such as hyposmotic shocking and Dounce homogenization, is first pelleted at 2000xg for 10 minutes, the supernatant is removed and centrifuged again at 10,000xg for 15 minutes to remove nuclei and organelles. The supernatant is filtered through a 0.45L filter and centrifuged again at 100,000xg for 1 hour to pellet the virus. The virus can then be washed and centrifuged again at 100,000xg for 1 hour.

WO 96/06159 PCT/US95/10194 116

REFERENCES:

1. Ablashi, et al. Viroloqv 184:545-552.

2. Albrecht, et al. (1992) J. Virol. 66:5047.

3. Altshul, et al. (1990) J. Molec. Biol.

215:403.

4. Analytical Biochemistry (1984) 238:267-284.

Andrei, et al. (1992) Eur. J. Clin. Microbiol.

Infect. Dis. 11(2):143-51.

6. Archibald, et al. (1992) Epidemiol. 3:203.

7. Asada, et al (1989) J. Clin. Microbiol.

27(10):2204.

8. Ausubel, et al. (1987) Current Protocols in Molecular Biology, New York.

9. Baer, et al. (1984) Nature 310:207.

Bagasra, et al. (1992) J. New England Journal of Medicine 326(21):1385-1391.

11. Balzarini, et al. (1990) Mol. Pharm. 37,402-7.

12. Basic and Clinical Immunoloqy 7th Edition

D.

Stites and A. Terr ed.

13. Beral, et al. (1990) Lancet 335:123.

14. Beral, et al. (1991) Brit. Med. J. 302:624.

Beral, et al. (1992) Lancet 339:632.

16. Bends6e, et al. (1990) Eur. J. Cancer 26:699.

17. Biggar, et al. (1994) Am. J. Epidemiol.

139:362.

18.

19.

Bovenzi, et al. (1993) Lancet 341:1288.

Beaucage and Carruthers (1981) Tetrahedron Lett.

22:1859-1862.

20. Braitman, et al. (1991) Antimicrob. Agents and Chemotherapy 35(7):1464-8.

21. Burns and Sanford, (1990) J. Infect. Dis.

162(3):634-7.

22. De Clercq, (1993) Antimicrobial Chemotherapy 32, Suppl. A, 121-132.

23. Drew, et al. (1982) Lancet ii:125.

24. Falk, et al. (1991) Nature 351:290.

WO 96/06159 PCT/US95/10194 117 Gaidano, et al. (1991) Proc. Natl. Acad. Sci.

USA 88:5413.

26. Gershon, (1992) J. Inf. Des.

166(Suppl):563.

27. Glick, (1980) Fundamentals of Human Lymphoid Culture, Marcel Dokker, New York.

28. Gorbach, et al. (1992) Infectious Disease Ch.35:289, W.B. Saunders, Philadelphia, Pennsylvania.

29. Greenspan, et al. (1990) J. Acquir. Immune Defic.

Syndr. 3 (6):571.

Hardy, et al. (1990) Inf. Dis. Clin. N. Amer.

4(1):159.

31. Hardy, et al. (1991) New Engl. J. Med. 325 (22):1545.

31A. Harel-Bellan, et al. (1988) Exp. Med.

168:2309-2318 32. Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publication, New York.

33. Haverkos, et al. (1985) Sexually Transm.

Dis. 12:203.

34. Helene, C. and Toulme, J. (1990) Biochim.

Biophys. Acta. 1049:99-125.

Heniford, et al. (1993) Nucleic Acids Research 21(14):3159-3166.

36. Higashi, et al. (1989) J. Clin. Micro.

27(10):2204.

37. Holmberg, et al. (1990) Cancer Detection and Prevention 14:331.

38. Holliday, and Williams, (1992) Antimicrob. Aqents Chemother. 36(9):1935.

39. Hoogenboom, et al. (1991) Nuc. Acids Res.

19:4133.

Hunt, et al. (1991) Eur. J. Immunol. 21:2963- 2970.

41. Hybridization of Nucleic Acids Immobilized on Solid SuDports Meinkoth, J. and Wahl, G.

WO 96/06159 PCTIUS95/10194 118 42. Hybridization with Nucleic Acid Probes pp. 495- 524, (1993) Elsevier, Amsterdam.

43. Ickes, et al. (1994) Antiviral Research 23, Seventh International Conf. on Antiviral Research, Abstract No. 122, Supp. 1.

44. Jahan, et al. (1989) AIDS Research and Human Retroviruses 5:225.

Jardetzkey, et al. (1991) Nature 353:326.

46. Johnston, et al. (1990) Cancer Detection and Prevention 14:337.

47. Jung, et al. (1991) Proc. Natl. Acad. Sci.

USA 88:7051.

48. Kikuta, et al. (1989) Lancet Oct. 7:861.

49. Knowles, et al. (1989) Blood 73:792-798.

50. Kohler and Milstein, (1976) Eur. J. Immunol.

6:511-519.

51. Kucera, et al. (1993) AIDS Res. Human Retroviruses 9:307-314.

52. Laboratory Techniques in Biochemistry and Molecular Biology (1978) North Holland Publishing Company, New York.

53. Lasky, (1990) J. Med. Virol. 31(1):59.

54. Levin, et al. (1992) J. Inf. Dis.

166(2):253.

55. Lifson, et al. (1990) Am. J. Epidemiol.

131:221.

56. Lin, et al. (1991) Antimicrob Agents Chemother 35(11):2440-3.

57. Lin, et al. (1993) Blood 81:3372.

58. Lisitsyn, et al. (1993) Science 259:946.

59. Lo, S et al. (1992) Internat. J. Systematic Bacteriol. 42:357.

Marks, et al. (1991) J. Mol Biol. 222:581- 597.

61. Marloes, et al. (1991) Eur. J. Immunol. 21:2963- 2970.

62. Matteucci, et al. (1981) Am. Chem. Soc. 103:3185.

WO 96/06159 PCT/US95/10194 119 63. Maxam, A.M. and Gilbert, W. Methods in Enzymoloqy (1980) Grossman, L. and Moldave, eds., Academic Press, New York, 65:499-560.

64. McCafferty, et al. (1990) Nature 348:552.

65. Means and Feeney, (1990) Bioconjugate Chem. A recent reveiw of protein modification techniques, 1:2-12.

66. Metcalf, D. (1984) Clonal Culture of Hematopoeitic Cells:Techniques and Applications, Elvier, New York.

67. Methods in Enzvmology Vol. 152, (1987) Berger,

S.

and Kimmel, A. ed., Academic Press, New York 68. Miller, Virology (1990) B. N. Fields,

D.M.

Knipe eds., Raven Press, New York, 2:1921.

69. Needham-VanDevanter, et al., (1984) Nucelic Acids Res. 12:6159-6168.

Needleman and Wunsch, (1970) J. Mol. Biol.

48:443.

71. Neuvo, et al. (1993) American Journal of Surgical Pathology 17(7), 683-690.

72. Nucleic Acid Hybridization: A Practical Approach (1985) Ed. Hames, B.D. and Higgins,

IRL

Press.

73. Oren and Soble, (1991) Clinical Infectious Diseases 14:741-6.

74. PCR Protocols: A Guide to Methods and Applications. (1990) Innis, Gelfand D., Sninsky, J. and White, eds., Academic Press, San Diego.

75. Pearson and Lipman, (1988) Proc. Natl. Acad. Sci.

(USA) 85:2444.

Pearson, and Regnier, (1983) J.

Chrom. 255:137-14976.

76. Pellici, et al. (1985) J. Exp. Med.

162:1015.

77. Peterman, et al. (1991) Cancer Surveys Imperial Cancer Research Fund, London, 10:23-37.

WO 96/06159 PCT/US95/10194 120 78. Roizman, B. (1991) Rev. Inf. Disease 13 Suppl.

11:S892.

79. R6tzschke and Falk, (1991) Immunol. Today 12:447.

Safai, et al. (1980) Cancer 45:1472.

81. Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2nd Cold Spring Harbor Laboratory, Vols. 1-3.

82. Saunders, et al. (1990) J. Acquir. Immune Defic.

Svndr. 3 (6):571.

83. Schecter, et al. (1991) Am. J. Epidemiol.

134:485.

84. Scopes, (1982) Protein Purification: Principles and Practice Springer-Verlag, New York.

85. Siddiqui, et al. (1983) Proc. Natl. Acad.

Sci. USA 80:4861.

86. Skinner, et al. (1991) Comp. Immuno.

Microbiol. Inf. Dis. 14(2):13.

87. Skinner, et al. (1992) Med. Microbiol.

Immunol. 180(6):305.

Smith and Waterman (1981) Adv. Appl. Math. 2:482.

88. Snoeck, et al. (1992) Eur. J. Clin. Micro.

Infect. Dis. 11(12):1144-55.

89. Stals, et al. (1993) Antimicrobial Agents Chemother. 37(2):218-23.

van den Berg, F. et al. (1989) J. Clin. Pathol.

42:128.

91. Vogel, et al. (1988) Nature 335:606.

92. Wang, R.H. et al. (1993) Clin. Infect. Dis.

17:724.

93. Wickstrom, et al. (1988) PNAS (USA) 85:1028-1032.

94. Winkelmann, et al. (1988) Drug Res. 38, 1545-48.

Winkler, et al. (1990) Antiviral Research 14:61- 74.

96. Yamandaka, et al. (1991) Mol. Pharmacol.

40(3):446.

WO 96/06159 PCT[US95/10 194 121 97. Pellicer, A. et al. (1978) Cell 14:133-141.

98. Gibson, W. and Roizmann B. (1972) J. Virol.

10:1044-52.

WO 96/06159 PCT/US95/10194 122 EXPERIMENTAL DETAILS SECTION

II:

Sequencing Studies: A lambda phage (KS5) from a KS lesion genomic library identified by positive hybridization with KS330Bam was digested with BamHI and Not I (Boehringer-Mannheim, Indianapolis IN); five fragments were gel isolated and subcloned into Bluescript II KS (Stratagene, La Jolla CA). The entire sequence was determined by bidirectional sequencing at a seven fold average redundancy by primer walking and nested deletions.

DNA sequence data were compiled and aligned using ALIGN (IBI-Kodak, Rochester NY) and analyzed using the Wisconsin Sequence Analysis Package Version 8-UNIX (Genetics Computer Group, Madison WI) and the GRAIL Sequence Analysis, Gene Assembly and Sequence Comparison System v. 1.2 (Informatics Group, Oak Ridge TN). Protein site motifs were identified using Motif (Genetics Computer Group, Madison WI).

Sources of Herpesvirus Gene Sequence Comparisons: Complete genomic sequences of three gammaherpesviruses were available: Epstein-Barr virus (EBV), a herpesvirus of humans herpesvirus saimiri (HVS), a herpesvirus of the New World monkey Saimiri sciureus and equine herpesvirus 2 (EHV2 Additional thymidine kinase gene sequences were obtained for alcelaphine herpesvirus 1 (AHV1 and bovine herpesvirus 4 (BHV4 Sequences for the major capsid protein genes of human herpesvirus 6B and human herpesvirus 7 (HHV7) were from Mukai et al. The sources of all other sequences used are listed previously in McGeoch and Cook [31] and McGeoch et al.

[32].

WO 96/06159 PCT/US95/10194 123 Phyloqenetic Inference: Predicted amino acid sequences used for tree construction were based on previous experience with herpesviral phylogenetic analyses Alignments of homologous sets of amino acid sequences were made with the AMPS and Pileup [16] programs. Regions of alignments that showed extreme divergence with marked length heterogeneity, typically terminal sections, were excised. Generally, positions in alignments that contained inserted gaps in one or more sequences were removed before use for tree construction. Phylogenetic inference programs were from the Phylip set, version 3.5c [14] and from the GCG set Trees were built with the maximum parsimony neighbor joining (NJ) methods. For the NJ method, which utilizes estimates of pairwise distances between sequences, distances were estimated as mean numbers of substitution events per site with Protdist using the PAM 250 substitution probability matrix of Schwartz Dayhoff Bootstrap analysis [15] was carried out for MP and NJ trees, with 100 sub-replicates of each alignment, and consensus trees obtained with the program Consense.

In addition the program Protml was used to infer trees by the maximum likelihood (ML) method. Protml was obtained form J. Adachi, Department of Statistical Science, The Graduate University for Advanced Study, Tokyo 106, Japan. Because of computational constraints, Protml was used only with the 4-species CS1 alignment.

Clamped Homoeneous Electric Field (CHEF) Gel Electrophoresis: Agarose plugs were prepared by resuspending BCBL-1 cells in 1% LMP agarose (Biorad, Hercules CA) and 0.9% NaCl at 42 0 C to a final concentration of 2.5 x 10 7 cells/ml. Solidified agarose plugs were transferred into lysis buffer EDTA pH 8.0, 1% sarcosyl, proteinase K at 1 mg/ml WO 96/06159 PCT/US95/10194 124 final concentration) and incubated for 24 hours.

Approximately 107 BCBL-1 cells were loaded in each lane. Gels were run at a gradient of 6.0 V/cm with a run time of 28 h 28 min. on a CHEF Mapper XA pulsed field gel electrophoresis apparatus (Biorad, Hercules CA), Southern blotted and hybridized to KS627Bam, KS330Bam and an EBV terminal repeat sequence TPA Induction of Genome Replication: Late log phase BCBL-1 cells (5x10 5 cells per ml) were incubated with varying amounts of 1 2 -O-tetradecanoylphorbol-13acetate (TPA, Sigma Chemical Co., St. Louis MO) for 48 h, cells were then harvested and washed with phosphate-buffered saline (PBS) and DNA was isolated by chloroform-phenol extraction. DNA concentrations were determined by UV absorbance; 5 Ag of whole cell DNA was quantitatively dot blot hybridized in triplicate (Manifold I, Schleicher and Schuell, Keene NH). KS631Bam, EBV terminal repeat and betaactin sequences were random-primer labeled with 32

P

Specific hybridization was quantitated on a Molecular Dynamics PhosphorImager 425E.

Cell Cultures and Transmission Studies: Cells were maintained at 5x10 5 cells per ml in RPMI 1640 with fetal calf serum (FCS, Gibco-BRL, Gaithersburg MD) and periodically examined for continued

KSHV

infection by PCR and dot hybridization. The T cell line Molt-3 (a gift from Dr. Jodi Black, Centers for Disease Control and Prevention), Raji cells (American Type Culture Collection, Rockville MD) and RCC-1 cells were cultured in RPMI 1640 with 10% FCS.

Owl monkey kidney cells (American Type Culture Collection, Rockville MD) were cultured in MEM with 10% FCS and 1% nonessential amino acids (Gibco-BRL, Gaithersburg

MD).

WO 96/06159 PCT/US95/10194 125 To produce the RCC-1 cell line, 2x10 6 Raji cells were cultivated with 1.4x10 6 BCBL-1 cells in the presence of 20 ng/ml TPA for 2 days in chambers separated by Falcon 0.45 gg filter tissue culture inserts to prevent contamination of Raji with BCBL-1.

Demonstration that RCC-1 was not contaminated with BCBL-1 was obtained by PCR typing of HLA-DR alleles [27] (Raji and RCC-1: DRgl*0310, DR03*02; BCBL-1: DRg104,*07, DrP4*01) and confirmed by flow cytometry to determine the presence (Raji, RCC1) or absence (BCBL-1) of EMA membrane antigen. Clonal sublines of RCC-1 were obtained by dilution in 96 well plates to 0.1 cells/well in RPMI 1640, 20% FCS and 30% T- STIM culture supplement (Collaborative Biomedical Products, Bedford MA). Subcultures were examined to ensure that each was derived from a single cluster of growing cells.

In situ hybridization was performed with a previously described 25 bp oligomer located in ORF26 which was 5' labeled with fluorescein (Operon, Alameda CA) and hybridized to cytospin preparations of BCBL-1 RCC-1 and Raji cells using the methods of Lungu et al. Slides were both directly visualized by UV microscopy and by incubating slides with anti-fluorescein-alkaline phosphatase (AP)conjugated antibody (Boehringer-Mannheim, Indianapolis IN), allowing immunohistochemical detection of bound probe. Positive control hybridization was performed using a 26 bp TETlabeled EBV DNA polymerase gene oligomer (Applied Biosystems, Alameda CA) which was visualized by UV microscopy only and negative control hybridization was performed using a 25 bp 5' fluorescein-labeled HSV1 a47 gene oligomer (Operon, Alameda CA) which was visualized in a similar manner as the KSHV ORF26 WO 96/06159 PCT/US95/10194 126 probe. All nuclei of BCBL-1, RCC-1 and Raji appropriately stained with the EBV hybridization probe whereas no specific staining of the cells occurred after hybridization with the HSV1 probe.

The remaining suspension cell lines used in transmission experiments were pelleted, and resuspended in 5 ml of 0.22 or 0.45 p filtered BCBL- 1 tissue culture supernatant for 16 h. BCBL-1 supernatants were either from unstimulated cultures or from cultures stimulated with 20 ng/ml TPA. No difference in transmission to recipient cell lines was noted using various filtration or stimulation conditions. Fetal cord blood lymphocytes (FCBL) were obtained from heparinized fresh post-partum umbilical cord blood after separation on Ficoll- Paque (Pharmacia LKB, Uppsala Sweden) gradients and cultured in RPMI 1640 with 10% fetal calf serum.

Adherent recipient cells were washed with sterile Hank's Buffered Salt Solution (HBSS, Gibco-BRL, Gaithersburg MD) and overlaid with 5 ml of BCBL-1 media supernatant. After incubation with BCBL-1 media supernatant, cells were washed three times with sterile HBSS, and suspended in fresh media.

Cells were subsequently rewashed three times every other day for six days and grown for at least two weeks prior to DNA extraction and testing. PCR to detect KSHV infection was performed using nested and unnested primers from ORF 26 and ORF 25 as previously described [10, Indirect Immunofluorescence Assay: AIDS-KS sera were obtained from ongoing cohort studies (provided by Drs. Scott Holmberg, Thomas Spira and Harold Jaffe, Centers for Disease Control, and Prevention, and Isaac Weisfuse, New York City Department of Health).

WO 96/06159 PCT/US95/10194 127 Sera from AIDS-KS patients were drawn between 1 and 31 months after initial KS diagnosis, sera from intravenous drug user and homosexual/bisexual controls were drawn after non-KS AIDS diagnosis, and sera from HIV-infected hemophiliac controls were drawn at various times after HIV infection.

Immunofluorescence assays were performed using an equal volume mixture of goat anti-human IgG-FITC conjugate (Molecular Probes, Eugene OR) and goat anti-human IgM-FITC conjugate (Sigma Chemical Co., St. Louis MO) diluted 1:100 and serial dilutions of patient sera. End-point titers were read blindly and specific immunoglobulin binding was assessed by the presence or absence of a specular fluorescence pattern in the nuclei of the plated cells. To adsorb cross-reacting antibodies, 20 Al serum diluted 1:10 in phosphate-buffer saline (PBS), pH 7.4, were adsorbed with 1-3x10 7 paraformaldehydefixed P3H3 cells for 4-10 h at 250 C and removed by low speed centrifugation. P3H3 were induced prior to fixation with 20 ng/ml TPA for 48 h fixed with 1% paraformaldehyde in PBS for 2 h at 40 C, and washed three times in PBS prior to adsorption.

RESULTS

Sequence Analysis of a 20.7 kb KSHV DNA Sequence: To demonstrate that KS330Bam and KS631Bam are genomic fragments from a new and previously uncharacterized herpesvirus, a lambda phage clone derived from an AIDS-KS genomic DNA library was identified by hybridization to the KS330Bam sequence. The KS5 insert was subcloned after NotI/BamHI digestion into five subfragments and both strands of each fragment were sequenced by primer walking or nested deletion with a 7-fold average WO 96/06159 PCT/US95/10194 128 redundancy. The KS5 sequence is 20,705 bp in length and has a G+C content of 54.0%. The observed/expected CpG dinucleotide ratio is 0.92 indicating no overall CpG suppression in this region.

Open reading frame (ORF) analysis identified complete ORFs with coding regions ranging from 231 bp to 4128 bp in length, and two incomplete ORFs at the termini of the KS5 clone which were 135 and 552 bp in length (Figure 12). The coding probability of each ORF was analyzed using GRAIL 2 and CodonPreference which identified 17 regions having excellent to good protein coding probabilities.

Each region is within an ORF encoding a homolog to a known herpesvirus gene with the exception of one ORF located at the genome position corresponding to ORF28 in herpesvirus saimiri (HVS). Codon preference values for all of the ORFs were higher across predicted ORFs than in non-coding regions when using a codon table composed of KS5 homologs to the conserved herpesvirus major capsid (MCP), glycoprotein H thymidine kinase and the putative DNA packaging protein (ORF29a/ORF29b) genes.

The translated sequence of each ORF was used to search GenBank/EMBL databases with BLASTX and FastA algorithms 38]. All of the putative KS5 ORFs, except one, have sequence and collinear positional homology to ORFs from gamma-2 herpesviruses, especially HVS and equine herpesvirus 2 (EHV2).

Because of the high degree of collinearity and amino acid sequence similarity between KSHV and HVS, KSHV ORFs have been named according to their HVS WO 96/06159 PCT/US95/10194 129 positional homologs KSHV ORF25 is named after HVS ORF The KS5 sequence spans a region which includes three of the seven conserved herpesvirus gene blocks (Figure 14) [10] ORFs present in these blocks include genes which encode herpesvirus virion structural proteins and enzymes involved in DNA metabolism and replication. Amino acid identities between KS5 ORFs and HVS ORFs range from 30% to with the conserved MCP ORF25 and ORF29b genes having the highest percentage amino acid identity to homologs in other gammaherpesviruses. KSHV ORF28, which has no detectable sequence homology to HVS or EBV genes, has positional homology to HVS ORF28 and EBV BDLF3. ORF28 lies at the junction of two gene blocks (Figure 14); these junctions tend to exhibit greater sequence divergence than intrablock regions among herpesviral genomes Two ORFs were identified with sequence homology to the putative spliced protein packaging genes of HVS (ORF29a/ORF29b) and herpes simplex virus type 1 The KS330Bam sequence is located within KSHV ORF26, whose HSV-1 counterpart, VP23, is a minor virion structural component.

For every KSHV homolog, the HVS amino acid similarity spans the entire gene product, with the exception of ORF21, the TK gene. The KSHV TK homolog contains a proline-rich domain at its amino terminus (nt 20343-19636; aa 1-236) that is not conserved in other herpesvirus TK sequences, while the carboxyl terminus (nt 19637-18601; aa 237-565) is highly similar to the corresponding regions of HVS, EHV2, and bovine herpesvirus 4 (BHV4) TK. A purine binding motif with a glycine-rich region WO 96/06159 PCT/US95/10194 130 found in herpesviral TK genes, as well as other TK genes, is present in the KSHV TK homolog (GVMGVGKS; aa 260-267).

The KS5 translated amino acid sequences were searched against the PROSITE Dictionary of Protein Sites and Patterns (Dr. Amos Bairoch, University of Geneva, Switzerland) using the computer program Motifs. Four sequence motif matches were identified among KSHV hypothetical protein sequences. These matches included: a cytochrome c family heme-binding motif in ORF33 (CVHCHG; aa 209-214) and ORF34 (CLLCHI; aa 257-261), (ii) an immunoglobulin and major histocompatibility complex protein signature in (FICQAKH; aa 1024-1030), (iii) a mitochondrial energy transfer protein motif in ORF26 (PDDITRMRV; aa 260- 268), and (iv) the purine nucleotide binding site identified in ORF21. The purine binding motif is the only motif with obvious functional significance. A cytosine-specific methylase motif present in HVS ORF27 is not present in KSHV ORF27. This motif may play a role in the methylation of episomal DNA in cells persistently infected with HVS Phylogenetic Analysis of KSHV: Amino acid sequences translated from the KS5 sequence were aligned with corresponding sequences from other herpesviruses. On the basis of the level of conserved aligned residues and the low incidence of introduced gaps, the amino acid alignments for ORFs 21, 22, 23, 24, 25, 26, 29a, 29b, 31 and 34 were suitable for phylogenetic analyses.

To demonstrate the phylogenetic relationship of KSHV to other herpesviruses, a single-gene comparison was made for ORF25 (MCP) homologs from KS5 and twelve members of Herpesviridae (Figures 15A-15B). The thirteen available MCP amino acid sequences are large (1376 a.a. residues for the KSHV homolog) and alignment required only a low WO 96/06159 PCT/US95/10194 131 level of gapping; however, the overall similarity between viruses is relatively low The MCP set gave stable trees with high bootstrap scores and assigned the KSHV homolog to the gamma-2 sublineage (genus Rhadinovirus containing HVS, EHV2 and BVH4 33, 43]. KSHV was most closely associated with HVS. Similar results were obtained for single-gene alignments of TK and UL15/ORF29 sets but with lower bootstrap scores so that among gamma-2 herpesvirus members branching orders for EHV2, HVS and KSHV were not resolved.

To determine the relative divergence between KSHV and other gammaherpesviruses, alignments for the nine genes listed above were concatenated to produce a combined gammaherpesvirus gene set (CS1) containing EBV, EHV2, HVS and KSHV amino acid sequences. The total length of CS1 was 4247 residues after removal of positions containing gaps introduced by the alignment process in one or more of the sequences. The CS1 alignment was analyzed by the ML method, giving the tree shown in Figure 15B and by the MP and NJ methods used with the aligned herpesvirus MCP sequences. All three methods identified KSHV and HVS as sister groups, confirming that KSHV belongs in the gamma-2 sublineage with HVS as its closest known relative. It was previously estimated that divergence of the HVS and EHV2 lineages may have been contemporary with divergence of the primate and ungulate host lineages The results for the CS1 set suggest that HVS and KSHV represent a lineage of primate herpesviruses and, based on the distance between KSHV and HVS relative to the position of EHV2, divergence between HVS and KSHV lines is ancient.

Genomic Studies of KSHV: CHEF electrophoresis performed on BCBL-1 cells embedded in agarose plugs demonstrated the presence of a nonintegrated KSHV genome as well as a high molecular weight species (Figures 16A-16B). KS631Bam (Figure 16A) WO 96/06159 PCT/US95/10194 132 and KS330Bam specifically hybridized to a single CHEF gel band comigrating with 270 kilobase (kb) linear DNA standards. The majority of hybridizing DNA was present in a diffuse band at the well origin; a low intensity high molecular weight (HMW) band was also present immediately below the origin (Figure 16A. arrow). The same filter was stripped and probed with an EBV terminal repeat sequence [40] yielding a 150-160 kb band (Figure 16B) corresponding to linear EBV DNA The HMW EBV band may correspond to either circular or concatemeric EBV DNA [24].

The phorbol ester TPA induces replication-competent EBV to enter a lytic replication cycle To determine if TPA induces replication of KSHV and EBV in BCBL-1 cells, these cells were incubated with varying concentrations of TPA for 48 h (Figure 17). Maximum stimulation of EBV occurred at 20 ng/ml TPA which resulted in an eight-fold increase in hybridizing EBV genome. Only a 1.3-1.4 fold increase in KSHV genome abundance occurred after 20-80 ng/ml TPA incubation for 48 h.

Transmission Studies: Prior to determining that the agent was likely to be a member of Herpesviridae by sequence analysis, BCBL-1 cells were cultured with Raji cells, a nonlytic EBV transformed B cell line, in chambers separated by a 0.45 y tissue culture filter. Recipient Raji cells generally demonstrated rapid cytolysis suggesting transmission of a cytotoxic component from the BCBL-1 cell line. One Raji line cultured in 10 ng/ml TPA for 2 days, underwent an initial period of cytolysis before recovery and resumption of logarithmic growth. This cell line (RCC- 1) is a monoculture derived from Raji uncontaminated by BCBL-1 as determined by PCR amplification of HLA-DR sequences.

WO 96/06159 PCT/US95/10194 133 RCC-1 has remained positive for the KS330 233 PCR product for >6 months in continuous culture (approximately passages), but KSHV was not detectable by dot or Southern hybridization at any time. In situ hybridization, however, with a 25 bp KSHV ORF26-derived oligomer was used to demonstrate persistent localization of KSHV DNA to RCC-1 nuclei. As indicated in Figures 18A-18C, nuclei of BCBL-1 and RCC-1 (from passage cells had detectable hybridization with the ORF26 oligomer, whereas no specific hybridization occurred with parental Raji cells (Figure 18B). KSHV sequences were detectable in 65% of BCBL-1 and 2.6% of RCC-1 cells under these conditions. In addition, forty-five monoclonal cultures were subcultured by serial dilution from RCC-1 at passage 50, of which eight clones were PCR positive by KS330 233 While PCR detection using unnested KS330 233 primer pairs was lost by passage 15 in each of the clonal cultures, persistent KSHV genome was detected in 5 clones using two more sensitive nonoverlapping nested PCR primer sets [33] suggesting that KSHV genome is lost over time in RCC-1 and its clones.

Low but persistent levels of KS330 233 PCR positivity were found for one of four Raji, one of four Bjab, two of three Molt-3, one of one owl monkey kidney cell lines and three of eight human fetal cord blood lymphocyte (FCBL) cultures after inoculation with 0.2-0.45 g filtered BCBL-1 supernatants. Among the PCR positive cultures, PCR detectable genome was lost after 2-6 weeks and multiple washings. Five FCBL cultures developed cell clusters characteristic of EBV immortalized lymphocytes and were positive for EBV by PCR using EBER primers three of these cultures were also initially KS330 233 positive. None of the recipient cell lines had detectable KSHV genome by dot blot hybridization.

WO 96/06159 PCT/US95/10194 134 Serologic Studies: Indirect immunofluorescence antibody assays (IFA) were used to assess the presence of specific antibodies against the KSHV- and EBV-infected cell line BHL-6 in the sera from AIDS-KS patients and control patients with HIV infection or AIDS. BHL-6 was substituted for BCBL-1 for reasons of convenience; preliminary studies showed no significant differences in IFA results between BHL-6 and BCBL-1. BHL-6 have diffuse immunofluorescent cell staining with most KS patient and control unabsorbed sera suggesting nonspecific antibody binding (Figures 19A-19D) After adsorption with paraformaldehyde-fixed, TPA-induced P3H3 (an EBV producer subline of P3J-HR1, a gift of Dr. George Miller) to remove cross-reacting antibodies against EBV and lymphocyte antigens, patient sera generally showed specular nuclear staining at high titers while this staining pattern was absent from control patient sera (Figures 19B and 19D). Staining was localized primarily to the nucleus but weak cytoplasmic staining was also present at low sera dilutions.

With unadsorbed sera, the initial endpoint geometric mean titers (GMT) against BHL-6 cell antigens for the sera from AIDS-KS patients (GMT=1:1153, range: 1:150 to 1:12,150) were higher than for sera from control, non-KS patients (GMT=1:342; range 1:50 to 1:12,150; p=0.04) (Figure 13). While AIDS-KS patients and HIV-infected gay/bisexual and intravenous drug user control patients had similar endpoint titers to BHL-6 antigens (GMT=1:1265 and GMT=1:1578, respectively), hemophilic AIDS patient titers were lower (GMT=1:104). Both case and control patient groups had elevated IFA titers against the EBV infected cell line P3H3.

The difference in endpoint GMT between case and control titers against BHL-6 antigens increased after adsorption with P3H3. After adsorption, case GMT declined to 1:780 and control GMT declined to 1:81 (p=0.00009). Similar WO 96/06159 PCT/US95/10194 135 results were obtained by using BCBL-1 instead of BHL-6 cells, by pre-adsorbing with EBV-infected nonproducer Raji cells instead of P3H3 and by using sera from a homosexual male KS patient without HIV infection, in complete remission for KS for 9 months (BHL-6 titer 1:450, P3H3 titer 1:150). Paired sera taken 8-14 months prior to KS onset and after KS onset were available for three KS patients: KS patients 8 and 13 had eight-fold rises and patient 8 had a three-fold fall in P3H3adsorbed BCBL-1 titers from pre-onset sera to post-KS sera.

DISCUSSION

These studies demonstrate that specific DNA sequences found in KS lesions by representational difference analysis belong to a newly identified human herpesvirus.

The current studies define this agent as a human gamma-2 herpesvirus that can be continuously cultured in naturally-transformed, EBV-coinfected lymphocytes from AIDS-related body-cavity based lymphomas.

Sequence analysis of the KS5 lambda phage insert provides clear evidence that the KS330Bam sequence is part of a larger herpesvirus genome. KS5 has a 54.0% G+C content which is considerably higher than the corresponding HVS region (34.3% While there is no CpG dinucleotide suppression in the KS5 sequence, the corresponding HVS region has a 0.33 expected:observed CpG dinucleotide ratio The CpG dinucleotide frequency in herpesviruses varies from global CpG suppression among gammaherpesviruses to local CpG suppression in the betaherpesviruses, which may result from deamination of 5'-methylcytosine residues at CpG sites resulting in TpG substitutions CpG suppression among herpesviruses [21, 30, 44] has been hypothesized to reflect co-replication of latent genome in actively dividing host cells, but it is unknown whether or not KSHV is primarily maintained by a lytic replication cycle in vivo.

WO 96/06159 PCT/US95/10194 136 The 20,705 bp KS5 fragment has 17 protein-coding regions, 15 of which are complete ORFs with appropriately located TATA and polyadenylation signals, and two incomplete ORFs located at the phage insert termini. Sixteen of these ORFs correspond by sequence and collinear positional homology to 15 previously identified herpesviral genes including the highly conserved spliced gene. The conserved positional and sequence homology for KSHV genes in this region are consistent with the possibility that the biological behavior of the virus is similar to that of other gammaherpesviruses. For example, identification of a thymidine kinase-like gene on KS5 implies that the agent is potentially susceptible to TK-activated DNA polymerase inhibitors and like other herpesviruses possesses viral genes involved in nucleotide metabolism and DNA replication The presence of major capsid protein and glycoprotein H gene homologs suggest that replication competent virus would produce a capsid structure similar to other herpesviruses.

Phylogenetic analyses of molecular sequences show that KSHV belongs to the gamma-2 sublineage of the Gammaherpesvirinae subfamily, and is thus the first human gamma-2 herpesvirus identified. Its closest known relative based on available sequence comparisons is HVS, a squirrel monkey gamma-2 herpesvirus that causes fulminant polyclonal T cell lymphoproliferative disorders in some New World monkey species. Data for the gamma-2 sublineage are sparse: only three viruses (KSHV, HVS and EHV2) can at present be placed on the phylogenetic tree with precision (the sublineage also contains murine herpesvirus 68 and BHV4 Given the limitation in resolution imposed by this thin background, KSHV and HVS appear to represent a lineage of primate gamma-2 viruses. Previously, McGeoch et al.

[33] proposed that lines of gamma-2 herpesviruses may have originated by cospeciation with the ancestors of their host species. Extrapolation of this view to KSHV WO 96/06159 PCT/US95/10194 137 and HVS suggests that these viruses diverged at an ancient time, possibly contemporaneously with the divergence of the Old World and New World primate host lineages. Gammaherpesviruses are distinguished as a subfamily by their lymphotrophism [41] and this grouping is supported by phylogenetic analysis based on sequence data The biologic behavior of KSHV is consistent with its phylogenetic designation in that KSHV can be found in in vitro lymphocyte cultures and in in vivo samples of lymphocytes This band appears to be a linear form of the genome because other "high molecular weight" bands are present for both EBV and KSHV in BCBL-1 which may represent circular forms of their genomes. The linear form of the EBV genome, associated with replicating and packaged DNA [41] migrates substantially faster than the closed circular form associated with latent viral replication While the 270 kb band appears to be a linear form, it is also consistent with a replicating dimer plasmid since the genome size of HVS is approximately 135 kb. The true size of the genome may only be resolved by ongoing mapping and sequencing studies.

Replication deficient EBV mutants are common among EBV strains passaged through prolonged tissue culture [23].

The EBV strain infecting Raji, for example, is an BALF-2 deficient mutant virus replication is not inducibile with TPA and its genome is maintained only as a latent circular form [23, 33] The EBV strain coinfecting BCBL-1 does not appear to be replication deficient because TPA induces eight-fold increases in DNA content and has an apparent linear form on CHEF electrophoresis. KSHV replication, however, is only marginally induced by comparable TPA treatment indicating either insensitivity to TPA induction or that the genome has undergone loss of genetic elements required for TPA induction. Additional experiments, however, indicate that KSHV DNA can be pelleted by high WO 96/06159 PCT/US95/10194 138 speed centrifugation of filtered organelle-free, DNase I-protected BCBL-1 cell extracts, which is consistent with KSHV encapsidation.

Transmission of KSHV DNA from BCBL-1 to a variety of recipient cell lines is possible and KSHV DNA can be maintained at low levels in recipient cells for up to passages. However, detection of virus genome in recipient cell lines by PCR may be due to physical association of KSHV DNA fragments rather than true infection. This appears to be unlikely given evidence for specific nuclear localization of the ORF26 sequence in RCC-1. If transmission of infectious virus from BCBL-1 occurs, it is apparent that the viral genome declines in abundance with subsequent passages of recipient cells. This is consistent with studies of spindle cell lines derived from KS lesions. Spindle cell cultures generally have PCR detectable KSHV genome when first explanted, but rapidly lose viral genome after initial passages and established spindle cell cultures generally do not have detectable KSHV sequences Infections with the human herpesviruses are generally ubiquitous in that nearly all humans are infected by early adulthood with six of the seven previously identified human herpesviruses Universal infection with EBV, for example, is the primary reason for the difficulty in clearly establishing a causal role for this virus in EBV-associated human tumors. The serologic studies identified nuclear antigen in BCBL-1 and BHL-6 which is recognized by sera from AIDS-KS patients but generally not by sera from control AIDS patients without KS after removal of EBV-reactive antibodies. These data are consistent with PCR studies of KS and control patient lymphocytes suggesting that KSHV is not ubiquitous among adult humans, but is specifically associated with persons who develop Kaposi's sarcoma. In this respect, it appears to be WO 96/06159 PCT/US95/10194 139 epidemiologically similar to HSV2 rather than the other known human herpesviruses. An alternative possibility is that elevated IFA titers against BCBL-1 reflect disease status rather than infection with the virus.

WO 96/06159 PCT/US95/10194 140

REFERENCES

1. Albrecht, J. Nicholas, D. Biller, K.

R. Cameron, B. Biesinger, C. Newamn, S.

Wittmann, M. A. Craxton, H. Coleman, B.

Fleckenstein, and R. W. Honess. 1992. Primary structure of the Herpesvirus saimiri genome.

J Virol. 66:5047-5058.

2. Altschul, S. W. Gish, W. Miller, E. W.

Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J Mol Biol. 215:403- 410.

3. Ambroziak, J. D. J. Blackbourn, B. G.

Herndier, R. G. Glogau, J. H. Gullett, A. R.

McDonald, E. T. Lennette, and J. A. Levy.

1995. Herpes-like sequences in HIV-infected and uninfected Kaposi's sarcoma patients.

Science. 268:582-583.

4. Baer, A. T. Bankier, P. L. Biggin, P. L.

Deininger, P. J. Farrell, T. J. Gibson, G.

Hatfull, G. S. Hudson, S. C. Satchwell, C.

S6guin, P. S. Tuffnell, and B. G. Barrell.

1984. DNA sequence and expression of the 8 Epstein-Barr virus genome. Nature. 310:207- 211.

5. Barton, G. and M. J. E. Sternberg. 1987.

A strategy for the rapid multiple alignment of protein sequences. Confidence levels from tertiary structure comparisons. J Mol Biol.

198:327-37.

6. Beral, T. A. Peterman, R. L. Berkelman, and H. W. Jaffe. 1990. Kaposi's sarcoma among persons with AIDS: a sexually transmitted infection? Lancet. 335:123-128.

WO 96/06159 PCT/US95/10194 141 7. Boshoff, D. Whitby, T. Hatziionnou, C.

Fisher, J. van der Walt, A. Hatzakis, R.

Weiss, and T. Schulz. 1995. Kaposi's sarcomaassociated herpesvirus in HIV-negative Kaposi's sarcoma. Lancet. 345:1043-44.

8. Cesarman, Y. Chang, P. S. Moore, J. W.

Said, and D. M. Knowles. 1995. Kaposi's sarcoma-associated herpesvirus-like DNA sequences are present in AIDS-related body cavity based lymphomas. New England J Med.

332:1186-1191.

9. Chang, E. Cesarman, M. S. Pessin, F. Lee, J. Culpepper, D. M. Knowles, and P. S. Moore.

1994. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science. 265:1865-69.

10. Chee, M. S. B. Bankier, C. M. Bohni, R.

C. Brown, T. Horsnell, C. A. Hutchison, T.

Kouzarides, J. A. Martignetti, E. Preddie, S.

C. Satchwell, P. Tomlinson, K. M. Weston, and B. G. Barrell. 1990. Analysis of the protein coding content of the sequence of cytomegalovirus strain AD169. Curr Top Microbiol Immunol. 154:125-69.

11. Collandre, S. Ferris, O. Grau, L.

Montagnier, and A. Blanchard. 1995. Kaposi's sarcoma and new herpesvirus. Lancet.

345:1043.

12. Dupin, M. Grandadam, V. Calvez, I. Gorin, J. T. Aubin, S. Harvard, F. Lamy, M.

Leibowitch, J. M. Huraux, J. P. Escande, and H. Agut. 1995. Herpesvirus-like DNA in patients with Mediterranean Kaposi's sarcoma.

Lancet. 345:761-2.

WO 96/06159 PCT/US95/10194 142 13. Feinberg, A. and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 132:6.

14. Felsenstein, J. 1989. PHYLIP-phylogeny inference package (ver Cladistics.

5:164-6.

15. Felsenstein, J. 1988. Phylogenies from molecular sequences: inferences and reliability. Annual Rev Microbiol. 22:521-65.

16. Genetics Computer Group. 1994. Program manual for the GCG package, version 8, Madison, Wisconsin.

17. Gompels, U. J. Nicholas, G. Lawrence, M.

Jones, B. J. Thomson, M. E. D. Martin, S.

Efstathiou, M. Craxton, and H. A. Macaulay.

1995. The DNA sequence of human herpesvirus- 6: Structure, coding content and genome evolution. Virology. 209:29-51.

18. Hatfull, A. T. Bankier, B. G. Barrell, and P. J. Farrell. 1988. Sequence analysis of Raji Epstein-Barr virus DNA. Virol. 164:334- 19. Holmberg, S. D. 1990. Possible cofactors for the development of AIDS-related neoplasms.

Cancer Detection and Prevention. 14:331-336.

20. Honess, R. W. 1984. Herpes simplex and 'the herpes complex': diverse observations and a unifying hypothesis. J Gen Virol. 65:2077- 2107.

WO 96/06159 PCT/US95/10194 143 21. Honess, R. U. A. Gompels, B. G. Barrell, M. Craxton, K. R. Cameron, R. Staden, Y.-N.

Chang, and G. S. Hayward. 1989. Deviations from expected frequencies of CpG dinucleotides in herpesvirus DNAs may be diagnostic of differences in the states of their latent genomes. J Gen Virol. 70:837-55.

22. Hsu, L. M. Shih, and Y. C. Zee. 1990.

Nucleotide sequence of a 3.5 nucleotide fragment of malignant catarrhal fever virus strain WC11. Arch Virol. 113:53-60.

23. Kieff, and D. Liebowitz. 1990. Epstein- Barr virus and its replication, p. 1889-1920.

In B. N. Fields and D. M. Knipe Virology, vol. 2. Raven Press, New York.

24. Kolman, J. C. J. Kolman, and G. Miller.

1992. Marked variation in the size of genomic plasmids among members of the family of related Epstein-Barr viruses. Proc Natl Acad Sci, USA. 89:7772-7776.

25. Lebb6, P. de Cr6moux, M. Rybojad, C.

Costa da Cunha, P. Morel, and F. Calvo. 1995.

Kaposi's sarcoma and new herpesvirus. Lancet.

345:1180.

26. Lin, J. S. C. Lin, B. K. De, W. P. Chan, and B. L. Evatt. 1993. Precision of genotyping of Epstein-Barr virus by polymerase chain reaction using three gene loci (EBNA-2, EBNA-3C and EBER): predominance of type A virus associated with Hodgkin's disease. Blood. 81:3372-81.

27. Liu, S. Yu-Kai, Xi, P. Harris, and N. Suciu-Foca. 1992. T cell recognition of WO 96/06159 PCT/US95/10194 144 self-human histocompatibility leukocyte antigens (HLA)-DR peptides in the context of syngeneic HLA-DR molecules. J Exp Med.

175:1663-8.

28. Lomonte, M. Bublot, P-P. Pastoret, and E.

Thiry. 1992. Location and characterization of the bovine herpesvirus type 4 thymidine kinase gene; comparison with thymidine kinase of other herpesviruses. Arch. Virol. 127:327- 337.

29. Martin, M. E. J. Nicholas, B. J. Thomson, C. Newman, and R. W. Honess. 1991.

Identification of a transactivating function mapping to the putative immediate-early locus of human herpesvirus 6. J Virol. 65:5381- 5390.

30. McGeoch, D. and S. Cook. 1994. Molecular phylogeny of the Alphaherpesvirinae subfamily and a proposed evolutionary timescale. J Mol Biol. 238:9-22.

31. McGeoch, D. S. Cook, A. Dolan, F. E.

Jamieson, and E. A. R. Telford. 1995.

Molecular phylogeny and evolutionary timescale for the family of mammalian herpesviruses. J Molec Biol. 247:443-58.

32. Miller, G. 1990. Epstein-Barr virus: Biology, pathogenesis and medical aspects, p.

1921-1957. In B. N. Fields and D. M. Knipe Virology, 2nd ed, vol. 2. Raven Press, New York.

33. Moore, P. and Y. Chang. 1995. Detection of herpesvirus-like DNA sequences in Kaposi's sarcoma lesions from persons with and without WO 96/06159 PCT/US95/10194 145 HIV infection. New England J Med. 332:1181- 1185.

34. Mukai, Y. Isegawa, and K. Yamanishi.

1995. Identification of the major capsid protein gene of human herpesvirus 7. Virus Res. 37:55-62.

35. Oettle, A. G. 1962. Geographic and racial differences in the frequency of Kaposi's sarcoma as evidence of environmental or genetic causes, vol. 18. Symposium on Kaposi's sarcoma: Unio Internationalis Contra Cancrum, Karger, Basel.

36. Pearson, W. and D. J. Lipman. 1988.

Improved tools for biological sequence analysis. Proc Natl Acad Sci, USA. 85:2444-8.

37. Peterman, T. H. W. Jaffe, A. E. Friedman- Kien, and R. A. Weiss. 1991. The aetiology of Kaposi's sarcoma, p. 23-37, Cancer, HIV, and AIDS, vol. 10. Imperial Cancer Research Fund.

London.

38. Raab-Traub, and K. Flynn. 1986. The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation. Cell. 47:883-889.

39. Roizman, B. 1993. The family Herpesviridae, p. 1-9. In B. Roizman and R. J. Whitley and C. Lopez The Human Herpeviruses. Raven Press, Ltd., New York.

Roizman, B. 1995. New viral footprints in Kaposi's sarcoma. N Engl J Med. 332:1227- 1228.

WO 96/06159 PCT/US95/10194 146 41. Roizman, R. C. Desrosiers,

B.

Fleckenstein, C. Lopez, A. C. Minson, and M.

J. Studdert. 1992. The family Herpesviridae: an update. Arch Virol. 123:425-449.

42. Sandford, G. K. Ho, and W. H. Burns.

1993. Characterization of the major locus of immediate-early genes of rat cytomegalovirus.

J Virol. 67:4093-4103.

43. Schalling, M. Ekman, E. E. Kaaya, A.

Linde, and P. Bieberfeld. 1995. A role for a new herpesvirus (KSHV) in different forms of Kaposi's sarcoma. Nature Med. 1:707-8.

44. Schwartz, R. and M. O. Dayhoff. 1978.

Matrices for detecting distant relationships, p. 353-8. In M. O. Dayhoff Atlas of protein sequence and structure, vol. supple 3. National Biomedical Research Foundation, Washington.

Su, Hsu, Chang, and I.-W.

Wang. 1995. Herpesvirus-like DNA sequence in Kaposi's sarcoma from AIDS and non-AIDS patients in Taiwan. Lancet. 345:722-23.

46. Telford, E. A. M. S. Watson, H. C. Aird, J. Perry, and A. J. Davison. 1995. The DNA sequence of equine herpesvirus 2. J Molec Biol. 249:520-8.

47. zur Hausen, F. J. O'Neill, and U. K.

Freese. 1978. Persisting oncogenic herpesvirus induced by the tumor promoter TPA. Nature. 272:373-375.

WO 96/06159 PCT/US95/10194 147 EXPERIMENTAL DETAILS SECTION III: KS Patient Enrollment: Cases and controls were selected from ongoing cohort studies based on the availability of clinical information and appropriate PBMC samples. 21 homosexual or bisexual men with AIDS who developed

KS

during their participation in prospective cohort studies were identified [14-16] Fourteen of these patients had paired PBMC samples collected after KS diagnosis (median +4 months) and at least four months prior to KS diagnosis (median -13 months), while the remaining 7 had paired PBMC taken at the study visit immediately prior to KS diagnosis (median -3 months) and at entry into their cohort study (median -51 months prior to KS diagnosis) Hemophilic and Homosexual/Bisexual Male AIDS Patient Control Enrollment: Two control groups of AIDS patients were examined: 23 homosexual/bisexual men with AIDS followed until death who did not develop KS ("high risk" control group) from the Multicenter AIDS Cohort Study and 19 hemophilic men ("low risk" control group) enrolled from joint projects of the National Hemophilia Foundation and the Centers for Disease Control and Prevention. Of the 16 hemophilic controls with available follow-up information, none are known to have developed KS and of hemophilic AIDS patients historically develop KS [21 For homosexual/bisexual AIDS control patients who did not develop KS, paired PBMC specimens were available at entry into their cohort study (median -35 months prior to AIDS onset) and at the study visit immediately prior to nonKS AIDS diagnosis (median BHL-6 months prior to AIDS onset) DNA Extraction and Analyses: DNA from 106-107 PBMC in each specimen was extracted and quantitated by spectrophotometry. Samples were prepared in physically isolated laboratories from the laboratory where polymerase chain reaction (PCR) analyses were performed.

WO 96/06159 PCT/US95/10194 148 All samples were tested for amplifiability using primers specific for either the HLA-DQ locus (GH26/GH27) or bglobin PCR detection of KSHV DNA was performed as previously described with the following nested primer sets: No. 1 outer 5'-AGCACTCGCAGGGCAGTACG-3', 5'-GACTCTTCGCTGATGAACTGG-3'; No. 1 inner TCCGTGTTGTCTACGTCCAG-3',5'-AGCCGAAAGGATTCCACCAT-3';No.

2 outer 5'-AGGCAACGTCAGATGTGAC-3', GAAATTACCCACGAGATCGC-3'; No. 2 inner CATGGGAGTACATTGTCAGGACCTC-3', No. 3 outer 5'-GGCGACATTCATCAACCTCAGGG-3', ATATCATCCTGTGCGTTCACGAC-3'; No. 3 inner CATGGGAGTACATTGTCAGGACCTC-3', The outer primer set was amplified for 35 cycles at 940 C for 30 seconds, 600 C for 1 minute and 720 C for 1 minute with a 5 minute final extension cycle at 720 C.

One to three ml of the PCR product was added to the inner PCR reaction mixture and amplified for additional cycles with a 5 minute final extension cycle.

Primary determination of sample positivity was made with primer set No. 1 and confirmed with either primer sets 2 or 3 which amplify nonoverlapping regions of the KSHV hypothetical major capsid gene. Sampling two portions of the KSHV genome decreased the likelihood of intraexperimental PCR contamination. These nested primer sets are 2-3 logs more sensitive for detecting KSHV sequences than the previously published KS330 233 primers and are estimated to be able to detect copies of KSHV genome under optimal conditions. Sample preparations were prealiquoted and amplified with alternating negative control samples without DNA to monitor and control possible contamination. All samples were tested in a blinded fashion and a determination of the positivity/negativity made before code breaking.

Significance testing was performed with Mantel-Haenszel chi-squared estimates and exact confidence intervals using Epi-Info ver. 6 (USD Inc., Stone Mt. GA).

WO 96/06159 PCT/US95/10194 149

RESULTS

KSHV Positivity of Case and Control PBMC Samples: Paired PBMC samples were available from each KS patient and homosexual/bisexual control patient; a single sample was available from each hemophilic control patient.

To determine the KSHV positivity rate for each group of AIDS patients, a single specimen from each participant taken closest to KS or other AIDS-defining illness ("second sample") was analyzed. Overall, 12 of 21 (57%) of PBMC specimens from KS patients taken from 6 months prior to KS diagnosis to 20 months after KS diagnosis were KSHV positive. There was no apparent difference in positivity rate between immediate pre-diagnosis and post-diagnosis visit specimens (4 of 7 vs. 8 of 14 respectively).

The number of KSHV positive control PBMC specimens from both homosexual/bisexual (second visit) and hemophilic patient controls was significantly lower. Only 2 of 19 hemophilic PBMC samples were positive (odds ratio 11.3, 95 confidence interval 1.8 to 118) and only 2 of 23 PBMC samples from homosexual/bisexual men who did not develop KS were positive (odds ratio 14.0, confidence interval 2.3 to 144). If all KS patient PBMC samples taken immediately prior to or after diagnosis were truly infected, the PCR assay was at least 57% sensitive in detecting KSHV infection among PBMC samples. No significant differences in CD4+ counts were found for KS patients and homosexual/bisexual patients without KS at the second sample evaluation (Kruskall- Wallis p=0.15) (Figure 21). CD4+ counts from the single sample from hemophilic AIDS patients were higher than CD4+ counts from KS patients (Kruskall-Wallis p=0.004), although both groups showed evidence of HIV-related immunosuppression.

WO 96/06159 PCT/US95/10194 150 Longitudinal Studies: Paired specimens were available from all 21 KS patients and 23 homosexual/bisexual male AIDS control patients who did not develop KS. For the KS group, initial PBMC samples were taken four to 87 months (median 13 months) prior to the onset of KS. Initial PBMC samples from the control group were drawn 13 to 106 months (median months) prior to onset of first nonKS AIDS-defining illness (1987 CDC surveillance definition). 11 of 21 of KS patients had detectable KSHV DNA in PBMC samples taken prior to KS onset compared to 2 of 19 p=0.005) hemophilic control samples, and 1 p=0.0004) and 2 p=0.002) of 23 homosexual/bisexual control samples taken at the first and second visits respectively (Figures 20A-20B). The figure shows that 7 of the paired KS patient samples were positive at both visits, 5 KS patients and 2 control patients converted from negative to positive and two KS patients and one control patient reverted from positive to negative between visits. The remaining 7 KS patients and control patients were negative at both visits.

For the 5 KS patients that converted from an initial negative PBMC result to a positive result at or near to KS diagnosis, the median length of time between the first sample and the KS diagnosis was 19 months. Three of the 6 KS patients that were negative at both visits had their last PBMC sample drawn 2-3 months prior to onset of illness. It is unknown whether these patients became infected between their last study visit and the KS diagnosis date.

DISCUSSION

Ambroziak and coworkers have found evidence that KSHV preferentially infects CD19+ B cells by PBMC subset examination of three patients Other gammaherpesviruses, such as Epstein-Barr virus (EBV) and herpesvirus saimiri are also lymphotrophic herpesviruses WO 96/06159 PCT/US95/10194 151 and can cause lymphoproliferative disorders in primates [11, It is possible that KSHV, like most human herpesviruses, is a ubiquitous infection of adults EBV, for example, is detectable by PCR in CD19+ B lymphocytes from virtually all seropositive persons [22] and approximately 98% MACS study participants had EBV VCA antibodies at entry into the cohort study The findings, however, are most consistent with control patients having lower KSHV infection rates than cases and that KSHV is specifically associated with the subsequent development of KS. While it is possible that control patients are infected but have an undetectably low KSHV viral PBMC load, the inability to find evidence of infection in control patients under a variety of PCR conditions suggests that the majority of control patients are not infected. Nonetheless, approximately of these patients were KSHV infected and did not develop KS. It is unknown whether or not this is similar to the KSHV infection rate for the general human population.

This study demonstrates that KSHV infection is both strongly associated with KS and precedes onset of disease in the majority of patients. 57% of KS patients had detectable KSHV infection at their second follow-up visit (52% prior to the onset of KS] compared to only 9% of homosexual/bisexual (p=0.002) and 11% of hemophilic control patients (p=0.005). Despite similar CD4+ levels between homosexual/bisexual KS cases and controls, KSHV DNA positivity rates were significantly higher for cases at both the first (p=0.005) and second sample visits indicating that immunosuppression alone was not responsible for these elevated detection rates. It is also unlikely that KSHV simply colonizes existing KS lesions in AIDS patients since neither patient group had KS at the time the initial sample was obtained. Five KS patients and two homosexual/bisexual control patients WO 96/06159 PCT/US95/10194 152 converted from a negative to a positive, possibly due to new infection acquired during the study period.

The findings are in contrast to PCR detection of KSHV DNA in all 10 PBMC samples from KS patients by Ambroziak et al. It is possible that the assay was not sensitive enough to detect virus in all samples since it was required that each positive sample to be repeatedly positive by two independent primers in blinded PCR assays. This appears unlikely, however, given the sensitivity of the PCR nested primer sets. The 7 KS patients who were persistently negative on both paired samples may represent an aviremic or low viral load subpopulation of KS patients. The PCR conditions test a DNA amount equivalent to approximately 2x10 3 lymphocytes; an average viral load less than 1 copy per 2x10 3 cells may be negative in the assay. Two KS patients and a homosexual/bisexual control patient initially positive for KSHV PCR amplification reverted to negative in samples drawn after diagnosis. These results probably reflect inability to detect KSHV DNA in peripheral blood rather than true loss of infection although more detailed studies of the natural history of infection are needed.

The study was designed to answer the fundamental question of whether or not infection with KSHV precedes development of the KS phenotype. The findings indicate that there is a strong antecedent association between KSHV infection and KS. This temporal relationship is an absolute requirement for establishing that KSHV is central to the causal pathway for developing KS. This study contributes additional evidence for a possible causal role for this virus in the development of KS.

WO 96/06159 PCT/US95/10194 153

REFERENCES

1. Katz MH, Hessol NA, Buchbinder SP, Hirozawa A, O'Malley P, Holmberg SD. Temporal trends of opportunistic infections and malignancies in homosexual men with AIDS. J Infect Dis.

1994;170:198-202.

2. Beral V, Peterman TA, Berkelman RL, Jaffe HW.

Kaposi's sarcoma among persons with AIDS: a sexually transmitted infection? Lancet.

1990;335:123-128.

3. Archibald CP, Schechter MT, Le TN, Craib KJP, Montaner JSG, O'Shaughnessy MV. Evidence for a sexually transmitted cofactor for AIDS-related Kaposi's sarcoma in a cohort of homosexual men.

Epidemiol. 1992;3:203-209.

4. Beral V, Bull D, Jaffe H, Evans B, Gill N, Tillett H et al. Is risk of Kaposi's sarcoma in AIDS patients in Britain increased if sexual partners came from United States or Africa? BMJ.

1991;302:624-5.

5. Beral V. Epidemiology of Kaposi's sarcoma.

Cancer, HIV and AIDS. London: Imperial Cancer Research Fund; 1991:5-22.

6. Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM, et al. Identification of herpesviruslike DNA sequences in AIDS-associated Kaposi's sarcoma. Science. 1994;265:1865-69.

7. Moore PS, Chang Y. Detection of herpesvirus-like DNA sequences in Kaposi's sarcoma lesions from persons with and without HIV infection. New England J Med. 1995;332:1181-1185.

WO 96/06159 PCT/US95/10194 154 8. Boshoff C, Whitby D, Hatziionnou T, Fisher C, van der Walt J, Hatzakis A et al. Kaposi's sarcomaassociated herpesvirus in HIV-negative Kaposi's sarcoma. Lancet. 1995;345:1043-44.

9. Su I-J, Hsu Y-S, Chang Y-C, Wang I-W. Herpesviruslike DNA sequence in Kaposi's sarcoma from AIDS and non-AIDS patients in Taiwan. Lancet. 1995;345:722- 23.

Dupin N, Grandadam M, Calvez V, Gorin I, Aubin JT, Harvard S, et al. Herpesvirus-like DNA in patients with Mediterranean Kaposi's sarcoma. Lancet.

1995;345:761-2.

11. Miller G. Oncogenicity of Epstein-Barr virus. J Infect Dis. 1974;130:187-205.

12. Hill AB. Environment and disease: association or causation? Proc Roy Soc Med. 1965;58:295-300.

13. Susser M. Judgment and causal inference: criteria in epidemiologic studies. Am J Epid. 1977;105:1-15.

14. Fishbein DB, Kaplan JE, Spira TJ, Miller B, Schonberger LB, Pinsky PF, et al. Unexplained lymphadenopathy in homosexual men: a longitudinal study. JAMA. 1985;254:930-5.

15. Holmberg SD. Possible cofactors for the development of AIDS-related neoplasms. Cancer Detection and Prevention. 1990;14:331-336.

16. Kaslow RA, Ostrow DG, Detels R, Phair JP, Polk BF, Rinaldo CR. The Multicenter AIDS Cohort Study: rationale, organization and selected characteristics of the participants. Am J Epidemiol. 1987;126:310-318.

WO 96/06159 PCT/US95/10194 155 17. Wolinsky S, Rinaldo C, Kwok S, Sinsky J, Gupta P, Imagawa D, et al. Human immunodeficiency virus type 1 (HIV-1) infection a median of 18 months before a diagnostic Western blot. Ann Internal Med.

1989;111:961.

18. Bauer HM, Ting Y, Greer CE, Chambers JC, Tashiro CJ, Chimera J, et al. Genital papillomavirus infection in female university students as determined by a PCR-based method. JAMA.

1991;265:2809-10.

19. Ambroziak JA, Blackbourn DJ, Herndier BG, Glogau RG, Gullett JH, McDonald AR, et al. Herpes-like sequences in HIV-infected and uninfected Kaposi's sarcoma patients. Science. 1995;268:582-583.

Roizman B. The family Herpesviridae. In: Roizman B, Whitley RJ, Lopez C, eds. The Human Herpeviruses.

New York: Raven Press, Ltd.; 1993:1-9.

21. Roizman B. New viral footprints in Kaposi's sarcoma. N Engl J Med. 1995;332:1227-1228.

22. Miyashita EM, Yang B, Lam KMC, Crawford DH, Thorley-Lawson DA. A novel form of Epstein-Barr virus latency in normal B cells in vivo. Cell.

1995;80:593-601.

23. Rinaldo CR, Kingsley LA, Lyter DW, Rabin BS, Atchison RW, Bodner AJ, et al. Association of HTLV- III with Epstein-Barr virus infection and abnormalities of T lymphocytes in homosexual men.

J Infect Dis. 1986;154:556-61.

WO 96/06159 PCT/US95/10194 156 EXPERIMENTAL DETAILS SECTION IV: To determine if the KHV-KS virus is also present in both endemic and HIV-associated KS lesions from African patients, formalin-fixed, paraffin-embedded tissues from both HIV seropositive and HIV seropositive Ugandan KS patients were compared to cancer tissues from patients without KS in a blinded case-control study.

Patient Enrollment: Archival KS biopsy specimens were selected from approximately equal numbers of HIVassociated and endemic HIV-negative KS patients enrolled in an ongoing case-control study of cancer and HIV infection at Makerere University, Kampala Uganda.

Control tissues were consecutive archival biopsies from patients with various malignancies enrolled in the same study, chosen without prior knowledge of HIV serostatus.

All patients were tested for HIV antibody (measured by Cambridge Bioscience Recombigen Elisa assay) Tissue preparation: Each sample examined was from an individual patient. Approximately ten tissue sections were cut (10 micron) from each paraffin block using a cleaned knife blade for each specimen. Tissue sections were deparaffinized by extracting the sections twice with 1 ml xylene for 15 min. followed by two extractions with 100% ethanol for 15 min. The remaining pellet was then resuspended and incubated overnight at 500 C in ml of lysis buffer (25 mM KC1, 10 mM Tris-HC1, pH 8.3, 1.4 mM MgC12, 0.01% gelatin, 1 mg/ml proteinase DNA was extracted with phenol/chloroform, ethanol precipitated and resuspended in 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.3.

PCR Amplification: 0.2-0.4 ug of DNA was used in PCR reactions with KS330 233 primers as previously described The samples which were negative were retested by nested PCR amplification, which is approximately 102-103 fold more sensitive in detecting KS330 233 sequence than WO 96/06159 PCT/US95/10194 157 the previously published KS330 233 primer set These samples were tested twice and samples showing discordant results were retested a third time. 51 of 74 samples initially examined were available for independent extraction and testing at Chester Beatty Laboratories, London using identical nested PCR primers and conditions to ensure fidelity of the PCR results. Results from eight samples were discordant between laboratories and were removed from the analysis as uninterpretable (four positive samples from each laboratory). Statistical comparisons were made using EPI-INFO ver. 5 (USD, Stone Mt. GA, USA) with exact confidence intervals.

RESULTS:

Of 66 tissues examined, 24 were from AIDS-KS cases, were from endemic HIV seronegative KS cases, and 22 were from cancer control patients without KS. Seven of the cancer control patients were HIV seropositive and were HIV seronegative (Figure 22). Tumors examined in the control group included carcinomas of the breast, ovaries, rectum, stomach, and colon, fibrosarcoma, lymphocytic lymphomas, Hodgkin's lymphomas, choriocarcinoma and anaplastic carcinoma of unknown primary site. The median age of AIDS-KS patients was 29 years (range 3-50) compared to 36 years (range 3-79) for endemic KS patients and 38 years (range 21-73) for cancer controls.

Among KS lesions, 39 of 44 were positive for KS330 233 PCR product, including KS tissues from 22 of 24 HIV seropositive and 17 of 20 HIV seronegative patients. In comparison, 3 of 22 (14%) nonKS cancer control tissues were positive, including 1 of 7 HIV seropositive and 2 of 15 HIV seronegative control patients (Figure 19). These control patients included a 73 year old HIV seronegative male and a 29 year old HIV seronegative female with breast carcinomas, and a 36 year old HIV seropositive female with ovarian carcinoma. The odds ratios for WO 96/06159 PCT/US95/10194 158 detecting the sequences in tissues from HIV seropositive and HIV seronegative cases and controls was 66 confidence interval (95% 3.8-3161) and 36.8 C.I. 4.3-428) respectively. The overall weighted Mantel-Haenzel odds ratio stratified by HIV serostatus was 49.2 (95% C.I. 9.1-335). KS tissues from four HIV seropositive children (ages 3, 5, 6, and 7 years) and four HIV seronegative children (ages 3, 4, 4, and 12 years) were all positive for KS330 233 All discordant results KSHV negative KS or KSHV positive nonKS cancers) were reviewed microscopically.

All KS330 233 PCR negative KS samples were confirmed to be KS. Likewise, all KS330 233 PCR positive nonKS cancers were found not to have occult KS histopathologically.

DISCUSSION

These results indicate that KSHV DNA sequences are found not only in AIDS-KS classical KS and transplant KS but also in African KS from both HIV seropositive and seronegative patients. Despite differences in clinical and epidemiological features, KSHV DNA sequences are present in all major clinical subtypes of KS from widely dispersed geographic settings.

This study was performed on banked, formalin-fixed tissues which prevented the use of specific detection assays such as Southern hybridization. DNA extracted after such treatment is often fragmented which reduces the detection sensitivity of PCR and may account for the PCR negative KS samples found in the study. The results, however, are unlikely to be due to PCR contamination or nonspecific amplification. Specimens were tested blindly and a subset of samples were independently extracted and tested at a physically separate laboratory. Specimen blinding is essential to ensure the integrity of results based solely on PCR analyses. A subset of amplicons was sequenced and found to be more than 98% identical to the published KS330 233 WO 96/06159 PCTIUS95/10194 159 sequence confirming their specific nature and, because of minor sequence variation, making the possibility of contamination unlikely.

In contrast to previous studies in North American and European populations, it was found 3 of 22 control tissues to have evidence of KSHV infection. Since these cancers represent a variety of tissue types, it is unlikely that KSHV has an etiologic role in these tumors. One possible explanation for the findings is that these results reflect the rate of KSHV infection in the nonKS population in Uganda. Four independent controlled studies from North America [5 and9 I Europe and Asia have failed to detect evidence of KSHV infection in over 200 cancer control tissues, with the exception of an unusual AIDS-associated, body-cavitybased lymphoma Taken together, these studies indicate that DNA-based detection of KSHV infection is rare in most nonKS cancer tissues from developed countries. KSHV infection has been reported in posttransplant skin tumors, although well-controlled studies are needed to confirm that these findings are not due to PCR contamination Since the rate of HIV-negative KS is much more frequent in Uganda than the United States, detection of KSHV in control tissues from cancer patients in the study may reflect a relatively high prevalence infection in the general Ugandan population.

While KS is extremely rare among children in developed countries the rate of KS in Ugandan children has risen dramatically over the past 3 decades: agestandardized rates (per 100,000) for boys age 0-14 years were 0.25 in 1964-68 and 10.1 in 1992-93. Detection of KSHV genome in KS lesions from prepubertal children suggests that the virus has a nonsexual mode of transmission among Ugandan children. That five of these children were 5 years old or less raises the possibility that the agent can be transmitted perinatally. Whether or not immune tolerance due to perinatal transmission WO 96/06159 PCT/US95/10194 160 accounts for the more fulminant form of KS occurring in African children remains to be investigated.

REFERENCES

1. Oettle A.G. Geographic and racial differences in the frequency of Kaposi's sarcoma as evidence of environmental or genetic causes. Acta Un Int Cancer 1962;18:330-363.

2. Beral V. Epidemiology of Kaposi's sarcoma. In: Cancer, HIV and AIDS. London: Imperial Cancer Research Fund, 1991: 5-22.

3. Wabinga Parkin Wabwire-Mangen F., Mugerwa J. Cancer in Kampala, Uganda, in 1989-91: changes in incidence in the era of AIDS. Int J Cancer 1993;54:26-36.

4. Kestens L. et al. Endemic Kaposi's sarcoma is not associated with immunodeficiency. Int. J. Cancer 1985;36:49-54.

Chang Y. et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.

Science 1994; 266:1865-9.

6. Moore P.S. and Chang Y. Detection of herpesviruslike DNA sequences in Kaposi's sarcoma lesions from persons with and without HIV infection. New England J Med 1995; 332:1181-85.

7. Boshoff C. et al. Kaposi's sarcoma-associated herpesvirus in HIV negative Kaposi's sarcoma (letter). Lancet 1995; 345:1043-44.

8. Su, Hsu, Chang, Wang, I.-W.

Herpevirus-like DNA sequence in Kaposi's sarcoma from AIDS and non-AIDS patients in Taiwan. Lancet 1995;345: 722-3.

WO 96/06159 PCT/US95/10194 161 9. Cesarman Chang Moore Said J.W., Knowles D.M. Kaposi's sarcoma-associated herpesvirus-like DNA sequences are present in AIDSrelated body cavity based lymphomas. New England J Med 1995; 332:1186-1191.

Rady et al. Herpesvirus-like DNA sequences in nonKaposi's sarcoma skin lesions of transplant patients. Lancet 1995;345:1339-40.

EXPERIMENTAL DETAILS SECTION V: Serologic marker for KSHV infection.

METHODS

Patients Serum was collected from a convenience sample of 89 HIV-infected patients seen at several clinical sites in Connecticut, New York, and California.

Demographic and clinical information was recorded on standardized forms which were linked to samples by a numerical code. Patients were classified as having KS if the diagnosis was histologically confirmed or, in the opinion of the primary clinician, the diagnosis of KS was unequivocal on clinical grounds. Eighty six (97%) were male; 90 of the 86 men were homosexual or bisexual. Forty seven patients, all male, had KS. The characteristics of the study population are found in Figure 23].

Cell lines The BCBL-1 line was established from an AIDS-associated body cavity B cell non-Hodgkin's lymphoma Neither BCBL-1 cells, nor the tumor from which they were derived, express surface immunoglobulin or B cell specific surface markers; however BCBL-1 cells contain immunoglobulin gene rearrangements that are characteristic of B cells KSHV DNA sequences can be detected in BCBL-1 cells by DNA representational difference analysis [23,32]. BCBL-1 cells also contain WO 96/06159 PCT/US95/10194 162 an EBV genome detectable with several different EBV DNA probes. B95-8 is an EBV producer marmoset cell line that can be efficiently induced into EBV lytic cycle gene expression by phorbol esters (TPA) [33,34]. HH 514- 16 is an EBV containing cell line, originally from a Burkitt lymphoma, that is optimally inducible into EBV lytic cycle gene expression by n-butyrate [35,36]. B141 is an EBV-negative Burkitt lymphoma cell line [37].

B95-8, HH514-16 and BL41 do not hybridize with the KSHV probes. All cell lines were cultured in RPMI 1640 medium containing 8% fetal calf serum.

Immunoblottinq Assays Extracts of uninduced BCBL-1 cells or BCBL-1 cells that had been treated with TPA and 3 mM n-butyrate for 48 hrs were prepared by sonication. HH514-16 cells, treated similarly, served to control for antibody reactivity to EBV polypeptides.

Each lane of a 10% or 12% polyacrylamide gel was loaded with extract of 5 X 10 5 cells in SDS sample buffer; electrophoresis, transfer to nitrocellulose and blocking with skim milk followed standard protocols Sera were screened at 1"100 dilution. The reaction was developed by 1.0 Ci of 125I Staphylococcal protein A.

Radioautographs were exposed to film for 24-48 hrs.

Immunoblotting assays were performed and interpreted on coded sera.

Immunofluorescent assay The antigens were BCBL-1 cells that were untreated or treated with 3mM n-butyrate for 48 hrs. Cells were dropped onto slides that were fixed in acetone and methanol. Sera were tested at 1:10 dilution, followed by 1:30 dilution of fluoresceinated goat anti-human Ig. The reactivity of a serum was compared on untreated and n-butyrate treated BCBL-1 cells. Reactivity with 30-50% of the chemically treated BCBL-1 cells was considered a positive reaction. All immunofluorescence tests were performed on coded sera.

The two readers were blinded to disease status or results of immunoblotting assays.

WO 96/06159 PCT/US95/10194 163

RESULTS

Chemical Induction of Ivtic cycle KSHV proteins in BCBL cells: Initial experiments using the immunoblotting technique were designed to determine whether BCBL-1 cells expressed unique antigenic polypeptides that might be specific for KSHV infection. Since sera from HIV-1 infected patients with or without KS would be expected to contain antibodies to EBV polypeptides and since BCBL-1 cells are dually infected with KSHV and EBV it was essential to distinguish EBV polypeptides from those encoded or induced by KSHV. Figures 27A-27B, an immunoblot prepared from BCBL-1 cells reacted with a reference EBV antiserum, shows that BCBL-1 cells expressed two polypeptides, representing the latent nuclear antigen EBNA1 and p21, a late antigen complex that were present in other EBV producer cell lines, such as B95-8 (Figure 27A) and HH514-16 (Figure 27B and Figures 28A-28D). When sera from patients with KS were used as a source of antibody they failed to identify in extracts from untreated BCBL-1 cells additional antigenic polypeptides that were not also seen in the EBV producer cell lines. However, if extracts were prepared from BCBL-1 cells that had first been treated witha combination of phorbol ester, TPA, and n-butyrate, KS patient sera now recognized a number of novel polypeptides that were present int eh BCBL-1 cell line but not in standard EBV producer cell lines(Figure 27B). The molecular weights of the most prominent of these many polypeptides were estimated at about 27 KDa, 40 KDa and 60 KDa on 10% polyacrylamide gels. These polypeptides were detected within 24 hrs after addition of the chemical inducing agents, but were not evident in BCBL-1 held in culture for as long as days without chemical treatment. Further experiments showed that n-butyrate was the chemical agent primarily responsible for induction of p40, whereas p60 could be induced by TPA or n-butyrate (Figures 28A-28D). Since p27, p40 and p60 were not detected in untreated cells WO 96/06159 PCT/US95/10194 164 and appeared after treatment with chemicals they likely represented lytic cycle rather than latent cycle polypeptides of KSHV.

p40 and p60 are KSHV specific: Figures 27A-27B shows that antigenic polypeptides corresponding in molecular weight to p40 were not observed in two EBV producer lines, B95-8 and HH514-16, that were induced into the EBV lytic cycle by the same chemicals or in comparably treated EBV-negative BL41 cells. Furthermore n-butyrate strongly induced expression of p40 in BCBL-1 cells but had little or no effect on the level of expression of the EBV p21 complex in the same cells. In related experiments it was found that n-butyrate also induced an increase in the abundance of KSHV DNA and KSHV lytic cycle mRNA. TPA, by contrast, induced the EBV lytic cycle efficiently' treatment with TPA caused an increase in the abundance of the EBV p21 protein and minimal induction of KSHV p40. These findings suggested that latency to lytic cycle switch of the two gamma herpes viruses carried by BCBL-1 cells was under separate control and that the p40 complex was specific to the KSHV genome.

p40 as a serologic marker for KSHV: While a few highly reactive sera, such as KS 01-03, (Figure 27B) recognized multiple antigenic proteins unique to the chemically induced BCBL-1 cells, including p27, p60 and p 4 0, sera from other patients with KS did not react with p27 or p 60 but still recognized p40 (Figure 28A and 28B).

Therefore recognition of p40 was investigated as a serologic marker for infection with KSHV. Sera from 89 HIV-1 infected patients from Connecticut, New York and California were examined for presence of antibodies to p40; only 3 of 42 patients without KS had antibodies to p40 (p<0.0001 by Chi square). These three patients were homosexual or bisexual men from New York city. The positive and negative predictive values of the serologic marker for the presence of KS were 84% and WO 96/06159 PCT/US95/10194 165 78% respectively. Three HIV-1 infected men from New York with non-Hodgkin's lymphoma but without KS were non-reactive to the KSHV p40 antigen. Figure compares the patients with KS whose serum did or did not contain antibodies to KSHV p40. Neither CD4 cell number nor the extent of KS disease predicted the presence or absence of a serologic response to Immunofluorescence assays: Immunoblots showed that nbutyrate induced expression of KSHV lytic cycle polypeptides in BCBL-1 cells without significantly affecting expression of EBV polypeptides (Figure 28A).

Therefore it was reasoned that n-butyrate might also induce many more BCBL-1 cells into the KSHV lytic cycle than into the EBV lytic cycle. Using indirect immunofluorescence with a reference human antiserum, RM in Figure 27B, that contains antibodies to EBV but not KSHV there were about 2% antigen positive untreated BCBL-1 cells and a similar number of antigen positive BCBL cell that had been treated with n-butyrate. Serum 01-03 that is EBV-positive and KS-positive (Figure 27B) detected 2% antigen positive cells in the untreated BCBL population, presumably the EBV expressing cells, while it detected 50% antigen positive BCBL-1 cells that had been treated with n-butyrate. This increase in the number of antigen positive BCBL-1 cells among the nbutyrate treated population served as the basis of an immunofluorescence screening assay for antibodies to KSHV lytic cycle antigens (Figures 29A-29F). The results of the immunofluorescence assay were nearly identical to the immunoblotting assay (Figure 26).

Among 89 sera there were only 4 that were discordant in the two assays. Three sera scored positive by IFA and negative by immunoblotting: one was considered positive by immunoblotting and negative by IFA. 68% of patients with KS and 12% of HIV-1 infected patients without KS were reactive by indirect immunofluorescence assay (IFA). Thus using two different assays, antibodies to KSHV lytic cycle WO 96/06159 PCT/US95/10194 166 antigens were found 6 to 9 times more frequently among patients with KS than among HIV-1 infected patients without KS. Stated another way, among individuals who were seropositive to KSHV p40 32/35 had KS. Among those seropositive by the immunofluorescence assay 32/37 had KS. Thus infection with KSHV, as defined by these serologic markers, carries a high risk of development of KS.

DISCUSSION

The recent discovery of genetic sequences representative of a new human herpes virus in KS tumor tissue, taken together with past epidemiologic observations, strongly implicate this novel agent in the pathogenesis of KS.

However, these observations, by themselves, do not permit the construction of a unified theory of pathogenesis that accounts for the many mysterious features of KS. For example, the relative contribution of HIV-1, other forms of immunosuppression, geographic factors, sex differences, the role of cytokines and growth factors, and the occurrence of distinct clinical variants must all be eventually understood. By identifying the infection rate in different populations a serologic marker for infection with KSHV would be great aid in unravelling the significance of the new virus in this complicated puzzle.

One possibility is that KSHV, the putative etiologic agent is, like all the other human herpes viruses, a ubiquitous, or at least widespread virus which infects large segments of the human population. Individuals who are immunosuppressed would have a greater likelihood of developing disease, whereas immunocompetent individuals would remain healthy. This pathogenetic model is similar to that postulated for the role that EBV plays in non-Hodgkin's lymphoma or cytomegalovirus in retinitis in patients with AIDS. If this model is correct a very high proportion of the adult human WO 96/06159 PCT/US95/10194 167 population might be found to be seropositive for KSHV.

The model of a ubiquitous virus selectively causing disease in immunodeficient individuals does not account for classical KS affecting patients who are not immunocompromised nor does it account for the observations that endemic KS in Africa preceded the HIV- 1 epidemic. Since many African patients with KS are HIV-1 negative other co-factors must be implicated.

The other possibility is that KSHV infection occurs selectively in the human population. Transmission may be promoted by sexual behavior that also carries a high risk of acquiring HIV-1. In this scenario seroprevalence of KSHV would be expected to be higher in HIV-1 seropositive and HIV-1 seronegative homosexual men than in other populations. If the virus alone were capable of inducing disease, acquisition of KSHV infection, as monitored by the presence of antibody, would be associated with a high rate of clinically evident KS. However, if KSHV infection needed to accompanied by other co-factors to cause disease, the prevalence of antibody of KSHV might be similar among patients with and without KS. The other co-factors would not be identified in a serologic test for antibodies to KSHV antigens.

The findings, using tests for antibodies to KSHV lytic cycle antigens, are consistent with the general model in which infection with KSHV is infrequent but associated with a high rate of apparent disease. Only a few HIV-1 infected patients without KS had antibodies to the KSHV lytic cycle antigens; by contrast a very high proportion of HIV-1 infected men who had clinically evident KS were seropositive. This finding suggests that a high proportion of individuals who are dually infected with HIV-1 and KSHV develop KS. However, another interpretation of the data is possible, though this interpretation is novel and no other examples are known among the human herpes virus family. Infection with WO 96/06159 PCT/US95/10194 168 KSHV might be ubiquitous, antibodies to the virus would not normally be detected in healthy infected individuals. Antibodies would only appear after the virus has been reactivated from the latent into the lytic cycle as might occur during the course of immunosuppression. Thus the two serologic tests that are described would indicate reactivated infection but would not be an index of past exposure to the virus. If this interpretation is correct, it should be possible to demonstrate KSHV DNA sequences or tot isolate the virus from healthy individuals who are KSHV seronegative.

Regardless of which of these two interpretations is correct, the serologic studies provide a strong correlation between the presence of antibodies to KSHV lytic cycle gene products and clinical KS. Nonetheless there are two groups of patients whose serologic results require further explanation. One group consists of the few patients with positive serology for KSHV p40 without clinical KS. They may have subclinical or visceral disease, or they may develop KS in the future. The other group is the approximately 30% of patients with KS whose sera lacked antibody to p40. The patients with KS who were p40 seronegative were not misclassified since the diagnosis was confirmed in all of them by biopsy (Figure 25). It is possible that the antibodies being measured are variable and wax and wane with time following infection. The appearance of antibody to may reflect the extent of lytic viral replication which may vary during different phases of the disease. To determine whether this is true prospective studies including serial bleedings are required.

is likely to be only one among a number of KSHV antigens recognized by the infected patients. Antibody recognition of other KSHV antigens may not be possible on immunoblots because they comigrate with EBV polypeptides, because the BCBL-I cells cannot be induced to express these antigens, or because the antigens are WO 96/06159 PCT/US95/10194 169 of low abundance or denatured on the immunoblots. In some individuals serum antibodies to p40 may be consumed in immune complexes with p40 antigen in the circulation.

Thus detection of p40 on immunoblots may not be of optimal sensitivity. In this connection three sera recognized antigens in immunofluorescence tests but did not react with p40 on western blots. The serologic test employing whole BCBL-1 cells as antigen are clearly first generation assays to be improved by better characterization of the KSHV gene products and preparation of recombinant antigens.

Lack of a serologic response to p40 could also reflect severely impaired humoral immunity. Although humoral immunity is usually relatively intact in HIV infection, examples of impaired antibody response have been described. For instance, some individuals are known to have impaired antibody responses to parvovirus B19(40 and others have been observed to lose antibodies to hepatitis B surface antigen An association between the degree of immunosuppression, as monitored by the number of CD4 cells, and the presence or absence of antibody p40 among patients with KS was not found (Figure 25) Furthermore all the patients with or without antibodies to KSHV p40 had antibodies to EBV p21 suggesting an intact humoral immune response.

In these serologic studies, as in the genetic probe studies previously reported, KSHV infection was found in the majority, but not all, patients with KS. Assuming that methodologic explanations do not account exclusively for the seronegative patients, other pathways, in addition to infection with KSHV, may lead to development of KS. In fact, most data suggest that the pathogenesis of KS is a multifactorial process. It has been observed that the product of the HIV-tat gene stimulates growth of KS tissue culture cells [42] and can induce KS-like lesions in mice These findings suggest a direct role for HIV-1 in the pathogenesis of WO 96/06159 PCT/US95/10194 170 KS, at least in HIV-infected hosts. In other settings, other growth factors may play a similar or complementary function. Interleukin-6 and basic fibroblast growth factor are both known stimulate growth of KS cells invitro Interleukin-6 is also produced in AIDS-KS derived cell culture Thus, KS pathogenesis may involve autocrine and paracrine growth factors together with infection with KSHV in some patients or with certain strains of HIV-1 in other patients. If infection with KSHV is the sine qua non of this process on would expect to see evidence of KSHV infection in all patients with KS.

In summary, an immunoblotting and a immunofluorescence screening assay for detection of antibodies to lytic cycle antigens of KSHV is disclosed. These assays should permit detailed seroepidemiologic investigations of KSHV. The findings support the notion of a strong association between infection with KSHV and the development of KS in HIV-infected patients. Infection with KSHV, as defined by these serologic assays, appears to carry an extremely high risk of development of clinical KS.

WO 96/06159 PCT/US95/10194 171

REFERENCES:

1. Kaposi M. Idiopathic multiple pigmented sarcoma of the skin. Cancer 1892; 31:3 2. Safai B, Good RA. Kaposi's sarcoma: A review and recent developments. Clin Bull 1980; 10:62-8.

3. Loethe R. Kaposi's sarcoma in Ugandan Africans.

Acta Pathol Microbiol Scand 1963 (suppl); 161:1-70.

4. Gordon JA. Clinical features of Kaposi's sarcoma amongst Rhodesian Africans. Central African Journal of Medicine 1972; 19:1-6.

Penn I. Kaposi's sarcoma in organ transplant recipients. Transplantation 1979; 27:8-1.

6. Friedman-Kien AE. Kaposi's sarcoma and Pneumocystis carinii pneumonia among homosexual men New York City and California. MMWR. 1981; 30:305-8.

7. Friedman-Kien AE, Laubensin LJ, Rubenstein P, et al. Disseminated Kaposi's sarcoma in homosexual men. Ann Intern Med 1982; 96:693-700.

8. Berel V, Peterman TA, Berkel RL, Jaffe HW. Kaposi's sarcoma among persons with AIDS: a sexually transmitted disease? Lancet 1990; 335:123-128.

9. Drew WL, Mills J, Hauer LB, et al. Declining prevalence of Kaposi's sarcoma in homosexual AIDS patients paralleled by a fall in cytomegalovirus transmission. Lancet 1988; 66.

Beral V, Peterman TA, Berkelman RL, et al. Kaposi's sarcoma among persons with AIDS: A sexually transmitted infection? Lancet 1990; 2:123.

WO 96/06159 PCT/US95/10194 172 11. Barley AC, Downng RG, Cheingson-Popov R et al.

HTLV-III serology distinguishes atypical and endemic Kaposi's sarcoma in Africa. Lancet 1985; 1:359.

12. Ziegler, J. J.A. Beckstead, P.A. Volberding, et al. 1984. Non-Hodgkin's lymphoma in 90 homosexual men: Relation to generalized lymphoadenopathy and the acquired immunodeficiency syndrome. N. Engl. J.

Med. 311:565-570.

13. Levine AM. Non-Hodgkins lymphomas and other malifnancies in the acquired immunodeficiency syndrome. Semin Oncol 1987; 14:34-9.

14. Knowles DM, Chamulak M, Subar M, et al. Lymphoid neoplasia associated with the acquired immunodeficiency syndrome. Ann Intern Med 1988; 108:744-53.

MacMahon EME, Glass JD, Hayward SD, Mann RB, Becher PS, Charache P, McArthur JC, Ambender RF. Epstein- Barr virus in AIDS-related primary central nervous system lymphoma. Lancet 1991; 338:969-73.

16. Sillman FH, Sedlis A. Anogenital papillomavirus infection adn neoplasia in immunodeficiency women.

Obstet Gynecol Clin North Am 1987; 260:348-53.

17. Drew WL, Mills J. Hauer LB, Miner RC, et al.

Cytomegalovirus and Kaposi's sarcoma in young homosexual men. Lancet. 1982; 2:125-127.

18. Callant JE, Moore RD, Richman DD, Keruly J, Chaisson RE. Risk factors for Kaposi's sarcoma in patients with advanced human immunodeficiency virus disease treated wiht zidovudine. Arch Inter Med 1994; 154:566-72.

WO 96/06159 PCT/US95/10194 173 19. Giraldo, E. Beth, and E. Huang. 1980. Kaposi's sarcoma and its relationship to cytomegalovirus

III

CMV DNA and CMV early antigens in Kaposi's sarcoma.

Int. J. Cancer 26:23-29.

Ambinder, Newman, Hawyard, G.S. et al.

1987 Lack of association of cytomegalovirus with endemic African Kaposi's sarcoma J. Inf. Dis.

156:193-7.

21. Jahan N, Razzaque A, Greenspan J et al. Analysis of human KS biopsies and cloned cell lines for cytomegalovirus, HIV-1 and other selected DNA virus sequences. ADIS Res Human Retrovirus 1989; 5:225.

22. Wang RY-H, Shih, JW-k, Weiss SH, et al. Mycoplasma penetrans infections in male homosexuals with AIDS: high seroprevalence and association with Kaposi's sarcoma. Clin Infect Dis 1993; 17:724-29.

23. Chang YE, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM, Moore PS. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science 1994: 266; 1865-9.

24. Moore PS, Cahng Y. Dectection of Herpes virus-like DNA sequences in Kaposi's sarcoma inpateints with and those without HIV infection. N Engl J Med 1995; 332"1181-5.

Su IJ, Hsu YS, Chang YC, Wang IW. Herpesvirus-like DNA sequences in Kaposi's sarcoma from AIDS and non-AIDS pateints in Taiwan. Lancet 1995; 345:722- 3.

26. Huang YQ, Li JJ, Kaplan MH, Polesz B, Katabira WC, Zhang D, et al. Human herpesvirus-like nucleic acid in various forms of Kaposi's sarcoma. Lancet 1995; 345:759-61.

WO 96/06159 PCT/US95/10194 174 27. Dupin N, Grandadam M, Calvez V, Gorin I, Aubin JT, Harvard S, et al. Herpesviurs-like DNA sequence sin pateints with Mediterranean Kaposi's sarcoma.

Lancet 1995; 345:761-2.

28. Collandre H, Ferris S, Grau O, MontagnierL, Blanchard A, Kaposi's sarcoma and new herpes virus.

Lancet 1995;345:1043.

29. Boshoff C, Whitby D, Hatziioannu T, Fisher C, van der Walt J, Hatzakis A, Weiss R Schultz T. Kaposi's sarcoma associated herpes virus in HIV-negative Kaposi's Sarcoma. Lancet 1995; 345:1043-4.

30. Cesarman E, Chang Y, Moore PS, Said JW< Knowles DM.

Kaposi's sarcoma associated herpes virus-like DNA sequences in aIDS-related body-cavity-based lymphomas. N Engl J Med 1995; 332:1186-91.

31. Knowles, D. G. Inghirami, A. Ubriaco, and R.

Dalla-Favera. 1989. Molecular genetic analysis of three AIDS-associated neoplamss of uncertain lineage demonstrate their B-cell derivation and the possible pathogenetic role of the Epstein-Barr virus. Blod 74:792-799.

32. Lisitsyn, N. Lisitsyn, and M. Wigler. 1993.

Cloning the differences between two complex genomes. Science 259:946-951.

33. Miller G, Lipman M, Release of infectious Epstein- Barr virus by transformed marmoset leukocytes.

Proc Nat Acad Sci USA 1973; 70:190-4.

34. zur Hausen, F. J. O'Neill, U. K. Freese, and E.

Hecker. 1978. Persisting oncogenic herpesvirus induced bytumor pomoter TPA. Naturer 272:373-375.

WO 96/06159 PCT/US95/10194 175 Rabson, L. Heston, and G, Miller. 1983.

Identification of a rare Epstein-Barr virus variant which enhances early antigen expression in Raji cells. Proc. Natl. aCad. Sci. USA 80:2762-2766.

36. Luka, B. Kallin, and G. Klien. 1979. Induction of the Epstein-Barr virus (EBV) cycle in latently infected cells by n-butyrate. Virology 94:228-231.

37. Calendar A, Billaud M, Aubry J-P, Bauchereau J, Vuillaume M, Lenoir GM. Epstein-Barr virus (EBV) induces expression of B-cell activation markers on in vitro infection of EBV-neative B-lymphoma cells.

Proc nat Acad Sci USA 1987; 84;8060-4.

38. Twobin, Staehelin, Gordon, J.

Electrophoeretic transfer of proteins from polyacrylamide gels to nitrocullulose sheets: procedure and some applications Proc Natl Acad Sci USA 1979; 76: 4350-4354.

39. van Grunsven, E. C. van Heerde, H.J.W. de Haard, W.J.M. Spaan, and J Middledorp. 1993. Gene mapping and expression of two immunodominant Epstein-Barr virus capsid proteins. J. Virol.

67:3908-3916.

Frickhofen N, Abkowitz JL, Safford M, Berry M, Antunez-de-Mayolo J, et al. Persistent B19 Parvovirus infection in pateints infected with human immunodeficiency virus type 1 (HIV-1): a treatable cause of anemia in ADIS. Ann Intern Med 1990; 113:926-33.

41. Biggar RJ, Goedert JJ, Hoofnagle J: Accelerated loss of antibody to hepatitis B surface antigen among immunodeficient homosexual men infected with HIV. N Engl J Med 1987; 316:630-31.

I

WO 96/06159 PCT/US95/10194 176 42. Ensoli B, Barillari G, Salahuddin SZ et al. Tat protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients.

Nature 1990; 345:84.

43. Vogel J, Hinrichs SH, Reynolds RK, Luciw PA, Jay G.

The HIV tat gene induces dermal lesions resembling Kaposi's sarcoma in transgenic mice. Nature 1994; 335:601-11.

44. Miles SA, Rezai AR, Salazar-Gonzalez JF, Stevens RH, Logan DM, Mistuyasu RT, Taga T, et al. AIDS Kaposi's sarcoma-derived cells produce and resond to interleukin-6. Proc Nath Acad Sci USA 1990; 87: 4068-72.

WO 96/06159 PCT/US95/10194 177 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: The Trustees of Columbia University in the City of New York City (ii) TITLE OF INVENTION: UNIQUE ASSOCIATED KAPOSI'S SARCOMA VIRUS SEQUENCES AND USES THEREOF iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Cooper Dunham LLP STREET: 1185 Avenue of the Americas CITY: New York STATE: New York COUNTRY: U.S.A.

ZIP: 10036 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: White, John P.

REGISTRATION NUMBER: 28,678 REFERENCE/DOCKET NUMBER: 45185-C-PCT/JPW/MSC (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (212) 278-0400 TELEFAX: (212) 391-0525 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 20710 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TCGAGTCGGA GAGTTGGCAC AGGCCTTGAG CTCGCTGTGA CGTTCTCACG GTGTTGGTTG GGATCAGCTG GTGACTCAGA CAAGTCTTGA GCTCTACAAC GTAACATACG GGCTGATGCC CACCCGATAC CAGAATTACG CAGTCGGCAA TTCTGTGCCC TAGAGTCACC TCAAAGAATA ATCTGTGGTG TCCAAGGGGA GGGTTCTGGG GCCGGCTACT TAGAAACCGC CATAGATCGG WO 96/06159 WO 9606159PCTIUS95/10194 178

GCAGGGTGGA

TTTTGCGTGC

CCATGGCAGA

TGACTAGGGG

CCAGCACGGA

AA-ACCTCGTA

ACGACAACTC

CCGAAAAAGG

ACGCGCCAAT

CGCCTCCAAA

GCTGGGCGGA

AGGGGGGCAG

TTGCCTTTAG

GGATCGGAGA

ACCTCATTAG

GGGTAATGGG

AGAGAGTGAC

ACAAGGAAAT

AAATATACTC

TGCGAATGAT

TCTTTGATAG

GCCGCCTATC

ACGTGGTCGC

GAAGAAAGAA

ACGCCGTGTA

TATGCGTACA

AGGAGGAAAC

TGGATCCCGT

TGAGAAAATT

TGTGGACCGA

ACTGGCCAGC

TCTAGCCTTC

TGGCGCGTTG

GAGAACCAAC

GTACTTGAGG

TGCTGGAAGC

AGGCGGTTTT

AGGCAGGTGG

TATGGACGAC

CATATACGAC

CCTCTACGCA

CAGCATTTTT

GGATCGCTTC

TCACCCACCT

TCTGCAGGGA

TCTCAAAGCC

TCCTAGGGGC

ATCATCGGCG

AACGCCTGTG

TGTGGGCAAA

AAGTTTTCCC

TTCCCACCTG

ATGCCAAAAC

GCAGCCCTGG

GCATCTCCTC

TTTTGATCAC

CATTCTCACC

CGACGGGACG

CTGTTCATGG

AACCACAAAT

TTTGAAAAAC

GAGACATCAT

ACAATTTATC

AATCTACAGG

ATTAGAGAGC

TTGGCGGCCC

CCGACAACGG

CTCTCCATAG

AGCCGGCGGT

CTGCTCAGGG

GGAGCGGACT

GACTTGGGGA

CTCCCTGAGG

GTGCCCACCG

ACGCCTAGGT

GCCAGTCGGT

GCCTTCCAGA

CCGGCAACTA

CTCAAGAGGA

CGTGGACGCG

GTGAAATCTG

ACTGCTGTCC

ACCGTGGACT

TCAACGCTGG

GAGCCCATGG

ATGAAGTCTG

AAGTTTTCGC

AACGTTGGGG

TCCCCAGCAG

TTCTTTCAAT

CTCTCCAGCG

GTGGAGCAAA

ATCATGTTGC

ATTCCGGAAA

CTTCACGAGC

CCCGTCGTGA

GTAGCCGACG

CAGATCCTGT

CAGTCTAAAG

TTGCATGCTG

CGACGACAAT

AACTGGAATT

AGGTGGCCAG

ATTTCTTAAC

CGGTGGGGCG

GCTCGGACGA

AGAGGAAACC

TCCCGACCAG

TTCCGCCCAG

TGTCAGCGAC

GCCCCAGGGT

GGCCGGCAGA

CCCCAAAGGG

ATGTAGGTGA

CCATAGGGCA

CCGTCACCAC

ACAGGAATGT

TCAACGCCGT

TGTACTGGAC

GTAAGGCGGG

TCCCCTTCCG

GTGGGTCTGG

TGGTGTTCCC

TACTTTCCAT

CCGAGTCGTT

ACTACATCAG

AGTACATCAC

TCTGCTTCCG

AGAGCATGCT

TCGAGCTTTG

CGGATAAGTT

CCAATCCGGC

CAGTTAATCA

GCGATGCATA

AACCCGCTCC

CAACGGCACT

GTGGGCCCGG

CTCGGCCTCG

CGGCGGAGAA

CGAATCAAGC

ACTAACGGGA

CAAGCCGTGG

ACCTCTCATA

TGACGACGAC

GTGTGGTCGC

CGCGTCAATG

ATTTTTAAAA

CCGTCTCAGG

AAACATTAAA

GCAGCTTATG

TTATTTGCTT

GTGCGGGATC

GAGGGCATTT

AGACCCGCTG

GACGAACGCC

GAGGGGCACT

TCTCATGCAC

CTTTAGAGCC

GCGGCGGGTC

AGAATTGGCG

TGTGGAGCAG

CAGCGTGCGC

ACCTATGATC

CTTTTGTTTC

CCACGACGAC

TATTAAACCC

CCTAGAGGAG

TCGTTGACAT

GCCACGCAGC

AGTTTTTTTC

TTACCTGCTC

GTTGGACGTA

AAGGCCTCTG

ACCTCCACAA

AAGTCTGTAA

CATTTAATGC

CGGCACCCTT

TCGGGAGACT

CCTCCCCTTC

GGGGACGTGG

ACATCTACCA

GACGGCGGCT

TCATGGTTGG

GTACCGGTGC

TACTTAGAGG

TTGCCCCAGG

ACAGATTGTT

ACGTCTGCCA

ACCGCTATCC

CACTGGTGCG

CTGAAGCACG

ACAGAAGGCG

AGGGCGAGGG

TGGGCTTATC

ATGGTACAAC

CTGGCACACA

ACCGGTGTAC

TTCACAGAGC

GTATGCGGCC

AGGGCCATCA

ACATGCAGGG

GTGGAGCCAC

TCATCAATGG

TAAATTGGCA

300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 WO 96/06159 WO 9606159PCTJUS95/10194 179

AAATCTGTTG

CGCCGAGGAC

AGTTGTGATC

TTATAACATC

GATCAACGCG

GGACACCCCA

ATCCATTGGC

GGTCGTCACG

CGGTCTCCCG

CGCCCGAAAC

CCTTACAGAG

CACGCGGACG

AGAGGCGGCG

TGCGCGGGGC

GGAACTGTAT

GTATAACCAC

GAAGTTGGCC

CGCCCGTGAT

TATGTATACC

GGACATACAC

GCTACTGAGA

GATCGCCCGC

TCTAGGACTA

CGCTCTGACG

GCTGCACCTC

CAAGATAATA

CTCGAACGCT

TGCTATCAAA

AGTCTACAAC

CTACGATGAG

CAACCTCTTT

GTGGCTGAGG

CAGTGCTTTG

CAGACTGTTT

AATGTGATCA

CTCAGGGTAA

TCTGGAGACG

ACCAAGGGCT

CGTCTGGTAC

GAGTATCGGG

AACAACGAGT

CTCCACAACA

GATTCACTGC

GCAAAATATG

AATTACACTC

ATCCAGACCA

CCGGACATAT

ATTGCCGAGC

TTTCTCCGCC

ACCACCCTTG

AGTTTGCCCC

GGCGCCGTCC

GCCTACACTT

ACGGTCCTCA

ACATATTTGA

TTTTCCAAAC

AGGTACGATT

CGGACTGCCG

GATAGCTTAA

GCTACGGTAC

GTTGTCTACG

CCCGATTGCT

ATCAGCACAC

AGCGATGGCC

TTAGATAAGT

GACAACGGGA

TTTCTAATTC

TCCATCCTTT

CGGAGCCGGC

CTCTGAAAAA

GCCATCGCTA

TTAACTATAG

TGGGTGATAT

TGTTTTACCC

CCGGCGTAGC

GGTCCAAA.GA

CATCTCTGAA

CGCTAGTGGC

GCATATTTCT

GGATCGTATC

TCTTGGTGTT

ACCGATTTGT

GCATCTCGCG

GCGCTGTGGC

GCTCTTCCCA

CTTCCTCCAT

ATGTGTACAC

CCGACAGCTG

TGTTCACATC

CGGACAGCCT

TGCATCCAGC

TTGCCAGAGG

ATTTAATTCC

CCTTGCCTCA

AGGTGTCGGA

CCGGCTTTAA

CAAGAAGAGG

TGCAGTCTCT

CACCTTTCTT

CCGTAGTGGA

TCTCTTTTAT

ATTAGACGGT

CCTGACAGAG

GAGGCAAAGT

TACGTGCGAG

CGCTCTGCCC

CTTCGCATCA

AATGAATGTC

GCTCTATGGA

GGCTAACGAG

GGGCCATGCC

GATCCTGCCT

GAACATGACG

AATCGAGGCC

GTTTCAGATG

GGAGGTGGAC

TCTGTGCATG

CGCCACACAA

GGAAACAGTG

TCTGGAGGGC

ACTCGGCGAT

CCCGCCCAAA

AATGTGTACC

TAACATCTAT

CAAGTTGCGC

AACATCGGGA

GGCGATTAAC

CGTCACGTAT

GATCTTCCTC

CTTTTCTCAG

TTGCCCCCTT

CATGTATGTC

TGATAATAAC

GATAAGGGGC

TGGGTTCTCG

CAATAAAGCG

TTGTGGACCT

CTTTTTTTCC

GTGCCGACGT

GGGCACCTTG

AAATGGTCGC

ATGGCCGTCA

GTGGTGTCGG

ACGGCGTCCC

ACCTATGATG

AAAGATTCTT

GAGTCGACGC

AGGCGCGCCT

TTGGTGGCAC

TGCGTGTGTC

CCCACGTTCA

ATAGCTCGCG

CTGGCCATGG

ATTGCTATGG

ACTGAAAGAA

GACTCCGGAG

AACATAGAGC

AGGGCATTCT

GCCGAGGCGC

TTCGCAGAAT

TGTTCAAAGA

ATCATCAGTT

AAGAGTGCCA

ATTGATAGGC

TGTGACTCTG

ACTAATGAAA

AACCTACACA

ATGTATAGAA

GGGGTTATCT

TAGATTTTTA

CCGCCGAAGT

CCAACAAGAC

CGTCGCAAAC

GCGGATTTGG

TATTCGCGAG

AGTTTTCCAT

AAGATTTCGT

ATCTTCTGTT

AACTCACGTT

ACCAGACACT

CCCTCGAGTT

GCGCAGCTCA

ACTTTCTTGT

GGCAGTATGC

CCACTGTCGG

TGTCCGCCAC

TCCAGCTTGG

TCGTCGAACA

AATTAATGTT

TATCAGAAAA

TGGGCGAAAT

CCCCCTGCTT

CGCAGTCGTC

TGCTCCACGC

TTACAGCCGA

CCGAAGCACT

TGTTTATATC

ACATTCCCAT

TAATCATGAG

GGGTGCAGAC

TTCATTATTT

GACGCGCAGC

ACTTTCTTTA

AAAGGTTTCC

2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 WO 96/06159 WO 96/6 159PCT/US95110194 180 TGTGCATTCT TTTTGTATGG GCATATACTT GGCAAGAAAT CCGAGCACCT CAGAAAGTGG

ATTGCCGTCA

TTTGGGGCTA

TGGGGGTTGG

CGCGTCCTCC

TGTTTTCGCA

CAGCCCCATG

TAGCTTGGGT

GTCACAGACC

CGCGGCCTCG

GTGTTTCAGT

CGACTCTGGG

CGCGGCGTGT

ATAAGACTGG

TTCGTGGCCG

GCCTACGCAC

CGATAGTTCC

CTTTTCGAAC

CGCCGGAGAG

TACTAGACTA

ACGTACGCGG

GTGCAGGCCT

ATGACCCGTG

GGTCTTCTTG

TGCGCACTCG

CCGGGCGGCG

CAGACACTCC

TAGGGAGAAT

CGCCAGCTCG

TGTCGCGGCC

GTTTGTGGTC

GTGCAGGTCC

CAGAAAGCAT

GATGTTTATT

CATATCAGTT

CACATCATAA

CGAATGGGTT

ACCCGACGTT

GGGGCAAGTT

CTGCGCAGCA

AGAAAAGTTA

GGGCTTTCTC

TTGTCTGTGT

ATAGATCTGG

TCAAACACCT

ATGGGGACGC

TCGCTCGTTA

CGTACGACTG

AAGTGGCTTC

GGGTGCCTAG

AACAGTGGCA

GTCAAGACGT

GCCTTCACGT

GGATCGGCTG

AGTTTGCGAA

GTGGCGACGA

TTTCGGGCAT

TGTTTGCTTT

GCTCGCCTAA

AGGAACCTTT

ATTCTATTTT

TGGCGAATCT

TTAAACCAGG

CAAAATACGT

AACGTGGAGT

TTCAGCGTAC

GCGGTGCCTT

CGACCACCCC

AGTACTTTTC

CCCTAAACGG

TGAGTCTTTC

CTGCGCCGTA

ACACGTGCTT

TCGAGTTGTT

CGAGTCTCGG

TAAGTGACAC

TCTGACCGGC

TTATGCGGTT

GACGGCATCC

TGGCCAGCCG

GCTTGGAGTG

TTAACGCGTA

CCGCGTAGAG

TCGGGACTTC

GAAACACGCG

CCGGAACTCG

GCGGTGTCTG

TGGCGTGACG

ACGAAATGAA

AGATTTTCGG

TAGTGTGACT

TGCAAGCCAC

TGTGTAGCGT

TGTTTCCGTC

GTTCCACAAG

ACACGTTTAG

TTTTTCGCCC

TCCAATTTGC

CCATTGCGAA

GACACATGTT

TGCACCTAGC

CATGGCTTCT

GAAATCCTCT

TAGCAGAGCG

CAGCGATGAG

CAGGAACAGG

AGCACGCTCC

CCGCCTGAGT

GATATCCCGT

ACGGGGTGTT

GGCGGCCTCG

CGCTGGCAGA

GCACTCCGGT

CAGGTAAACG

GGGGTGCGGT

TGGCAGGGTA

AGGATTAGAG

CTCGCCTGTC

TAACATAGCT

CTCGTTGGAC

GACAATTTGT

GTTTGCATTG

GATTAGGTTA

GATTATCTTC

GTCAAGCCTG

CTGTATTTGG

GATGCGCGCG

AGGCTGCCCG

CCCATCCTTG

CATTCTCACC

TATCGATACA

GAGAAAGTGC

GTCGGAAAAA

CATGCGGCGC

ATAAGCACCT

ATGGTATTCA

CCGAAGAACT

AAACACGACA

TGTTGTAGCC

ATGATGGTAA

CCAATCATGT

TCGCAAACCT

ATGGGGTGAC

TCGATGACGA

TCTATAATCT

AGTATCTGCG

CCAAGAGATG

GAGAGCATGA

GACGAGTCCG

ACTCCCACCA

GACAGGCGCG

TAGACCAGCG

GCGGCCGTTC

GGCTTTAGAG

CGGCCCAACT

CACTTTTTAT

TTTGAGAAGT

AGAAACGAAC

GAACGGTTTC

TGCTGGTCCG

TACACTTTAG

CTGGAGACCA

ATGTACTGGT

GGAAATATGT

AGCATGTCCC

AACACGCTTA

TTTGACGGTC

TGGAACAATC

GGCAGAAGAC

CCCGCTCGTG

CGATGTTTTC

GGTTCAGTTT

CGGTGTTGAA

AGAACATAGA

GTGCGATGTT

GCGGTAAAGG

CACGCTTGTT

TAAAGTTGGT

TGTCCTCGAA

CGGTCTCTTC

TCCGTAGCAA

GAGTCCCAAA

TGGCCGCCAC

CCGCGCCCTC

GACGGACGCA

GGTGGCGCCA

CGGCGAACCG

CGTCTAGCCT

ATCCCAGTAC

CAAACAGGCC

AGCATTCCAC

TGTGCTCAAG

TGAGAATGGG

AAATCGTGGC

CAGATGGAAA

TTTCCAGTCC

GCCTGATTGG

CACTGATGTT

TGGTAAA.AGA

4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 WO 96/06159 WO 9606159PCTIUS95/10194 181

AGGTTCCTTT

AATAGTCTTT

GGTGTTGAAT

GTCTGACACT

CGCCTGCAAA

AAGCGTCGCC

CAGTCCGCTG

GATGGCCGGT

CCTGTATGGC

CTGGTTTAGA

CTTACTCTGT

GGCACGGCGC

GTTAAACTTT

GAGATGCTTC

ATGGGGCAAC

CCTGCACACT

ATACAGCGAG

TGATCCCACC

ACCGTCAATT

TTCTTGATGT

GGCGAAAAAG

CTGCAGGTGC

TTGATAACAC

GCCCTCGAGC

GGAGGCCAAC

TCAGCTATTG

CGTATATACC

CGTCAATACC

TTCAATGCCC

CGTCATGAAG

CATCGAGCTT

CATCAAGACG

GGACACGGTT

GGGCGATCCC

ACGGAGTACT

TGCAGGGTGT

ATGGTGATCT

GTAGAGCTGC

TTTCCTTTCA

GCCAGGAGAC

CACGAGTGGC

GTGCCCGGAT

TTTGGTCCGG

GCCAGCTGAA

TTCGACAGGT

AGCGAGACCA

AAAAATGTA

AGGCTCTCCA

ACCGCCAGAA

ATCCTGAACA

CGTAGCTCCC

GCAGGTAGGT

ATTAAACCTT

ATAAAAGGGT

GTGGGCTCGG

GAGTTGGTGG

GTGGTCTGCG

GCTGCCATGG

CTCCTAACGC

CTCGGCAAGG

AATGTGGTGG

GAGTTCAAGG

AC TATTGCC C

GCTTGCAATA

CTCTTCGCCG

ATTACGTCGG

CTCGCAGTTA

GTCTACTCTG

TTCGTATAAC

TAGGAACGTG

TGAAGTTTTC

CCAGAGTCCG

TGGCTGCTCG

GCGGTGTCTC

CGATGCAGTC

ACACTAGATA

GGTCCTTGCG

AGCCCACCAG

TCTTCAGCAC

GCTCTCCGTG

GCTGTGCGTC

GGAGTGCAAA

ACGCATGAA&

TGGCTTTGTA

TGAATTCGCA

AGTTGTCGGT

CGCCGCTGTA

GGAGGCGTTC

GCTGCATCAT

CCAGACAGCA

GACTCGTCGT

AGGCGACCTT

AGATTAAGGA

ACGCCAGAGA

AGTTTGTTAA

ACCTGCGGAG

ACGGAGACGG

AGCACCACAT

AGAAAGAGAC

CTTTGCAGTT

AACTTCGGCA

AGAGGGGCCT

AAAATTGTTG

GCAGCTTATC

CAAACTGACG

CGCGTCCGTG

CCGGTCTTTC

GTGGGTGCCT

TGCCACTGCC

GTAGGTACAA

TTGGATTTTT

ATCCCGTCCG

GGTGGGCAGT

CCACCCCCAC

TGGGGATGCG

ATAATTTTGA

ACACTGTTCG

ACATATGGTG

GGGTTTATCA

GTCTATCTGT

CCGTCGACCC

CCCCAGGAGT

CTTATCAAGA

GAATATTTCC

CAGGGAGGCG

GGAGCAACGA

GTCGGCTGCC

AGGCAGTGTC

GTTTCTGGAG

AATGATAGAT

GAGGAGGCCC

CGGTGCGGAG

GCCCTTGGAC

TGGTATGGAC

CGCTCCACCC

CAAAAAGGCC

GTCAATCTGG

TTAGTGTTAA

TGTTTTGTGG

GCCGCGTATC

GGCGCGTACC

AAAAAGTTTG

ATACACATGA

TCTGGGGTAC

ACGTGCAGAC

TTAACCTTGA

CGCTCTACGT

GTGGCCATGA

GGTGGCATTA

TAGATTGTGG

AACTCCCAGA

CACGTTAGTA

CAA.TCATCGG

CCGCGCGTAA

ACTTTTCCCA

AGTCTGCGTA

CCTTCTAAGG

AGCTGTGATT

CTCGGTGGCA

CCTTTCCCGT

GACGGACTCT

CGTTTCGAAG

ACCGCCCTCG

GGAAAAATAC

AACAA.GCAGA

ATTGAGCTTG

TTCACAGAGT

GCCCTAGAAC

GTCTTTATTT

GTCAAGTCTG

GGATGTTTAA

TCACCATGTT

GTTCCAGCAT

GTTGGAAGCA

GGATTCTTGA

CGCAGGGGTG

CGAGTCTGTA

TGACGACCAC

GGGACACGAG

CGTCCTGGTG

TGTGAGCGAT

AGCTGCTGAT

TTGAAAACGA

GTTGTAGACT

ACTCCAGGTA

GCGCGGGAAG

TAAGTTCCCA

ACACTCCACC

AAAGAGTCCC

TCGCTCTGCA

TCAGCTCTGC

CCCAAGTCGC

GTAGTAGGGG

ACCTCGCCAC

TCAAGAGCTT

CGCTACTGGG

CCGCCGCTTG

AGTTTAAAAT

GACAGTATAT

CGGCCGCAGA

ACGCGGGTGC

GGGGGCTAGT

TAAAGACGCT

ACATGGTATC

6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 WO 96/06159 PTU9/09 PCTfUS95110194 182

CATGTTCAAG

AAGGGGGAAG

TACCGTCTTT

CCTGGAGACG

TGCCATGTCC

CGCCGTTAGC

CCATCCCAAC

CGAGATTCAG

CATTGAAAGT

CATCCAGTAC

ATCCGTCTCA

TAATAAAAAC

CCCTCGAATG

CGACGGGGGA

AA.CGTTCCAC

AGCGCTCGTA

GGTCCACCCC

CGCCACCCAC

GGAA.AGTAGA

AACTATTGAC

AATCGAAGCA

CCTGGTCATT

CATGGTTAGA

CCACTACCGG

GGGACACGAG

GCATCTGCTG

CAGACAACCC

ATTCATCAAC

GACAAGGGTC

GAATGACTAC

TAACGGCCAC

CAACGGCCCC

GAACGGAACC

CAGGGTGCTG

GCACACCTCA

CAGTATGTCC

AAGGGTGTCA

ACGGACAGCG

GGGCCCGCGG

TACGGAAGGG

GCTCTGCCGT

AGAACCCGCA

TTGCAGCGCA

ACCTATTTCT

GTCAGGGGCG

AACGTGCCTC

CACAACCCCA

CATGGGTACG

CAGTATTACA

ACCACGGAGG

TTCTTTGATT

AGAACAATGG

GGGGCGCAGT

GTCATACAGG

ATGATTCACG

CAAACCTACT

TTCATCTGTA

AAAATCTTAG

ACGGTGGGTC

CCTCCCTTTG

ATAATCAAGA

CTCAGGGGTC

AATGAGGACC

AACCCGGTCC

ATGTGCGGTA

GTCTTTGCGG

TTGAAGGACA

TGCACCTCGT

TAGAACATTC

TAACCATGCT

GCACGTACAC

TCATGAGACG

CCTACGCCAG

CGATGAGAAA

CTGTGGAAGG

TCGCCGCCTC

TGTACAACGA

TCCCTGTTGG

TAGAATCCCC

TTTGCTTCGG

CCCAGTCAGC

GTCTCAGGTA

TGGGGAAAAA

ATCTACTGCA

TTTTTGTGCA

TTGGAAATAT

TCGACGCTGT

AGACGGCATT

GACAGGAT4GA

GGGTCAACTC

CGCATATTGG

GCGAGCTCAT

GGACGCCGAT

CCTACCACGA

TCGGGGATCA

GCATGGAGGA

ATGACGAGAG

TCGAGAAGCT

TGGGGGTCGA

ACGTCGTGAA

TTCTGCAGGC

TTCTGACGTG

ATTTTTTCTA

CTCCGACATG

CACGGCCTCT

GCTGATGAAC

CTACGTTGTC

CTTTGAA.CAG

TGACAAGGCC

TCTCGTCAAG

GACTCAGTTT

CCTTCACCTT

GGCCATCCAG

TTACCAA.AAC

CCAGGCACTA

TGAGCAGACG

CGTGGCATTT

CCCAACCTCT

CCCCTGTCCT

ACCACAACCG

GACGAATATG

TGACCCCGCG

AAAATTCGTG

GGGAAAACTG

GAATGGAAGC

CGCCCTTGAG

CACACATCTG

TGTCTTTACG

AAACTACGAC

CCTAGTCAAT

ACACGTCCTG

ATTCTACTAT

CTATCAAAAC

CGCACAGGAT

AGGCGACATA

CCCTTTCGTC

GATAAGGCCG

CTGGCCGCGG

GGGCAGCAGG

CTGCTGGGGC

AGGGGTGCCA

TTTATGGCAC

GCTCTGGCGG

ATAGGGGATA

CCCTGCCCAC

CCCGTGCCCC

TCGACCGAGA

GCCCTCAAAA

AACCAAGCTT

CCAAACATGA

GTTCCCGATG

CACCGTCTCC

GGAGCGAGAG

CTCGCTCCAA

ACACACGTCA

TATCCCCTGT

ATGAACATGC

GCGTTTGTGA

ATCCCTAAGG

CAGGCGCTTC

GTTTCGGCTC

GATCTTATGC

AACCCTCAAA

AACCTTGTTA

GACGTGGCGC

GTTTTAATGC

GTGGCCCTGA

GATATTCTAC

CGCCCGACGG

ACCCAGGCCG

AGCTCATGAC

TGTGCGAGGA

TGGCCGGCGT

AA.GTGGAAAG

ACCTCGTCAC

GCATAGTGGA

ACGGACACGA

AGTTTGTGGC

TGAACCGGCG

GCTACTCGAC

CGTGGGTGGT

GCATATGCCA

TTCCCGATCC

ACCTATTCAG

TGGCCCAAAA

TCAGATTGGA

GATCGTACCG

GGGAGTTTCA

TAGACCAGCT

TCTGCTATGT

CCCTCATTGC

ACAGTTATCA

AGGCGCACGG

TCAAGCTCGC

TCCTCGACCC

AGAAGTCATC

ATAGGGCGAC

ACATTTACCA

CCCTGGACGA

CGGTGTGCAG

CGCTGACTTA

TGCACCTGGA

TGGACATGAT

CTCGCGTGAT

8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 WO 96/06159 WO 9606159PCTJUS95IIO194

CACAAAGCGG

GACCGTGCTT

GGAGACTATG

CACACTGCAT

GGTCTTTAAC

CGCGGCGTAT

GCACCAAAAA

TTTTGCCATG

CAGGGCGTCG

GGACGCGGTG

CTCATCAGTC

TCAGGACCTC

TATCAAAATG

ATACACTGCT

AACCTGCGAG

CAACCCCCGG

AGAGCGTTTG

CAACCCGTGG

CCAGACTGCG

TATGCGGTAC

GGCCCCCGCC

TTTGGACCAG

AGTTATGCTT

GTATCTCATT

GTATTAGCTA

ACTTCACCTC

TACTGCCGCT

TATGCTCACG

CACTCGCTGT

CTGACAACCT

AGCTAGCAGT

ACGGATTTGA

AGCAGCTGTT

ATATGGCGGA

GACCCGGCCC

GTTAATGGCT

TTTTATCCGG

CCGCTCCTGC

GTGCCATCCA

GCCGCGTCTT

CTATCTGCCC

ACAGTCGTCA

ACATCCATGT

ACTTTTGAAA

ATCGCCCCGG

TTTATGATTT

AAAGCGGGCG

GGGGTTCCTC

ATAATTCCCA

GGGCGTGCGG

TTCTACGACC

GCTTCGCAGC

CTGCCGGGCA

AATAGGGGGT

ACCAGCACTA

CCTTGCCATA

GCCGAGTACA

GAAGAGGTGG

ACCCTTCTAG

CAGACTCTTC

CGGAGATTGC

TGAGACATCT

CCTGGAGGAG

TCAGATAAAA

GCTACCCCCA

CCCCGTGTTC

GGTGTACCAC

ACTTGATCTA

AGAGTTTTGC

TTGGTGCGTT

TACCCTTTAA

CAAACTATGT

ATCTCATGGC

GTCAGGCCAC

CCAGTTTCAT

GGACGGACGA

TTGTGGGCTT

TTACCCACGA

CCCACGTGGC

TCCCAGGGGA

TGCAAACCGG

GCTGCGAGAA

CGCCGGTCAC

CGTCGGTCGT

ATTCAATACC

GTGGCTCCCT

TGTACAGTCC

TGTACACTTT

CAGACCTCCA

TGCTGCAGGA

TGTCA74.CAA

CGCCGATGAA

CGTTGGCTAG

GCTGATGAAC

CACCGTTTAC

CCGGACTACA

GTTCGCCCGG

AACGTATATG

TTTTTTAGCC

CCCATGGTCG

ATCTACTCCA

TATACCACCA

CACGCACGAA

CGCGGTGGCG

CAAGCTCTAC

CACCAGGCTC

AGAATATGAG

CCCGGGCGCC

TTGCCAGGCA

GGTTCTAGCA

GCCTTCGGTG

GATCGCTTCC

CGCCATAACT

CGCGTATCAG

CTCACCGGGA

CCTGCCCGGT

ATCTGACGTT

GTCGTGTGAT

AGACCCCGCG

CGGCGACGTG

TTGTCGGCAG

GGTTAATGAG

GTACGTCGTG

GGCCTATCCC

GCAGACACAC

GAGACTATTA

TCATGGCACT

TGGCCGCCCT

AAA.ATATACA

TCCAAATTAT

ACAGCCTGCG

CCCCCTTTTT

GAAAGGATTC

TGCCGCAGCA

AAATATCGGC

ATGTGTCATT

TACGGGAAGG

GACCGCTCTC

GCTGACCCGT

CCCAACCAGA

GAATGGCACA

ATTAGCGCCA

AAACACCGCA

GAGCACATCC

GTACGGCGCG

CTGCACACCG

ACAGACATGG

GACCGCCAGC

AACAGAATGG

TTGAGTCATG

GCCTATTTCC

GCTTACAGTA

TACGAATGCC

CTATACAATA

TTCTTCCACA

TATTCTGCCA

GTCAACGGTA

ACGCTCGCCG

GCCCCAGTAC

AAGCTCGGAA

CGACAAGAGT

TCAGTCAAAA

GGCATTGGGC

GCAGTATCTA

CCTAACGCGG

TCAGTGGGAC

CACCATTGTG

ACTGGGGCAC

CGGGGCCCCG

TATGGGGCGC

ATGTGGCGCA

GCGAGGCGGC

TGGTGGCTGC

GAAACGCGGT

AGTCGCCCGT

TGGTGAGCAT

TGCACCCTGG

TATACTGCTC

AGGTACGTTC

CACTTGGCTA

GAGTACATTG

TGCATGACTA

ATCACGTGGG

GTCAGCTGGC

AGACCCCCAG

ACGAAAGCGC

GGTCCACCAA

TCACCTTTCG

AGGAAGACAT

GGCTTGCTGG

CAGACGTGTT

CCAGCCACAG

ACATGGGCCA

ACAAGGTGGT

ATAGTGGTTA

ATAGGGAGCG

CTGGGGTGCG

TCCAAGTGCA

ATGGATCCCT

AGCAACACCC

CTCGAATCCA

GCTATTCTGC

GATGATGTAA

ACATATCGTC

10500 10560 10620 10680 10740 10800 10860 10920 10980 11040 11100 11160 11220 11280 11340 11400 11460 11520 11580 11640 11700 11760 11820 11880 11940 12000 12060 12120 12180 12240 12300 12360 12420 12480 WO 96/06159 WO 9606159PCT/US95/10194 184 TGGACGTAGA CAACACGGAT CCACGTACTG CCCTGCGAGT GCTTGACGAT CTGTCCATGT

ACCTTTGTAT

TGCGGCACGA

CCAGGATAGA

TCTCCTATCT

CCTACTCGGC

CGGGGGGGGT

CTGTGAAGTC

ACCCTGGGTG

TATGGCTCGA

GGTGCTTTTT

CCTTAATCCT

TGAGGTGCTC

TAAACTGCCC

TACACGCGAG

AGTTATGGTT

ATCCCGTAAC

CTTAGATCAC

TGACTATGGA

GTCGTGGGAA

GCCCAATCTA

CACCACGATT

ATTTAAAACC

TGCTCTGTTG

GGTGAGAACT

TTCCCCGTCT

CAAGCCTCCC

CTACTGCTGC

GGCCACCGTG

GCCGACGGTG

CGGGGTGTGT

TTTTATTGTG

ATGACAACTG

CCTCCATCCC

CCTATCAGCC

CAGGCATCCT

TCTCGACCAG

TCAGAGTCTC

AGAGACTTTG

ATGGCGTCAT

TCCCTGCGTG

GTAGAGACCG

AAGCTTCATC

TGTTATTGTT

TACTTGGTAA

TTCCCACACC

TATACCGTGC

CCGGACGGCA

AACACGTCAT

CACACTGAGT

AACTGTCACC

TTATTCATCT

GATATCTACA

TCCACCCCTC

AAAA.ACCACT

GCCCACTGAT

TAAACTATAT

CAGTTGAGAG

CCAGTCACCG

GTGATTGGTC

ATTCGCGTGT

GCGTATCAGG

GGCATAGCTA

GCTATAATGG

GGGATATGGG

CCACAAGTAA

TAAGCGGGAC

TTGGTTCCCA

CTGACAGAGG

TTGAGCGTCC

AGTTCTATAT

GCGGCCTCCT

CTGATATTCT

GAGGTAGGAA

ACGCCATCAA

GTGGTGCCCT

ACTTGCAAAA

CTCCCTCAAG

CGGCTGAGAT

CTATAATCAA

GGCCTACGGA

GTGCAGGAGT

GGGAAAATCT

CGGAAGCACT

CTCAGCCCCG

ACGGGACTTA

CCTTGATTCT

CACTGAGATG

TTTTTTACGC

ATAAGTTAAA

TCAGAGAATA

GAGGAATGGT

TTATAACAGT

TCCTGGCGGC

TCCTTCGCAC

CCCAGGAGCC

ATGGCTATGG

CTTGTACATG

GTCGTCCGAC

GCACTTGATC

GGGGGTGTCT

TGTTTGAGGG

CAGATGACAT

TTAATCTTGG

GTTGGTATTC

GTCGGTTGCA

AAAAACTACC

AGACGCCTTC

GCCCTCAAAT

TTGTGTGTAC

CATTGAGTTT

GTCTCGCGGT

CACCACGTTT

TTACTCCATG

GACATTGTGC

GCTGGCTATG

GTCTATCGCG

GAGCGTGCCC

CCTAGCTCGG

AAATTTTAAA

TCTTTTTAAC

TAAGAGTTGG

CCAAAATTCG

CAGTGCTAAT

GGACGGCTCC

GCTCTTCCTC

TCGACTGTGG

CCTGGGACCG

CTACCGTACA

GGGGGGGCTG

TGTCTATCAT

ATGTGCTTTT

GCGCGGACCT

CCGTCTGCTC

GGTGGTGCCA

CACCAGGATG

CCCCAGACTG

CCCACGCTAA

AGGACGGATG

GTCTACCTGC

CTCAGCGACG

TCTCACAACG

CTAGCCCTGT

GCCGAGCCCG

TGCGATGACG

GGACGCATTT

GCCCTTAGAA

CGCGGAGAAA

TTTTCTGTGA

AGCGGCATCT

TCGCCTACCC

CCTGGAAACT

TAAAGGTGTG

CGCTAAGGGA

GTGCTTGGGG

CAGGGAGACA

CAGGGTAGAT

GTCCTGGTGC

CTAGTCATAG

CGCGCCACCC

CAGGCCGGGT

ATATACATGC

TAGATAATTG

CGGTAGCCAT

GCTTGGCGCT

GTTCTACCAG

ACGGCGCTCG

GATGAGGTGA

CGCGTCATGT

CACGTGTATG

CGATTTGAAG

ACGGCTCCGT

CGGACACTGA

GGATCGTGGA

GCTTGAGGAT

TTCTGTGCCC

TTGTGGCACC

CGATTTTCTG

ACCCGAACTC

GGGCTTTTGC

CTCAGACCGC

TTATCTATGC

TTGACGAGCG

CTTGCGACGT

GTGACCCCTG

TCACTGGTTA

TTATACCGGG

GGTTTTGCAT

AGGTGACGGT

GAGCATGACT

GAATGGCCAC

GCGCCTGCGT

CACTAGGCAG

CACATGCACC

CAGATTAGAA

AGCGCTGTGC

AAAATGGGCC

GTATGACTGC

GTAGGTCACC

12540 12600 12660 12720 12780 12840 12900 12960 13020 13080 13140 13200 13260 13320 13380 13440 13500 13560 13620 13680 13740 13800 13860 13920 13980 14040 14100 14160 14220 14280 14340 14400 14460 14520 WO 96/06 159 PTU9/09 PCTIUS95/10194 2185

GGGTCAAATG

GAGTTCAGAG

ATTGGCCACT

CATATTTCGT

ACCGCCGCGT

CATGCGCAGG

TCCATGCCCA

CCAGTACCGG

TTTCCAAGGC

GTCAGCGCTG

CCCATTAGTT

TCGTCGATGG

TCTTGCAGGT

TTTTCCTGGG

GATGATATAA

GCATCCTTTT

ATGCTCTGCA

GAGGAGGCGA

TCGATCGTAT

AGTGTGAACA

TCGACGGCCT

TGGACCTGGT

GAAGAGTGTG

CCAGTGCCTG

GGAGAGCGTC

GTTTTTAGGG

CGGGATGCTA

CACCGTAATC

TATAGATGAA

CCTGGTCTGT

CAACAGTATG

GCGGACAAAA

TTGGCCCACG

GAGATCATCT

ATATTTTGAT

CGTAGATGAA

GCAGGGCGCT

TAAGGACGGT

TCACGCGCTC

ACGCTATCTC

ATATGAGGCT

ACGCCTCCGT

ACTTCTGAAC

CGTCACCCAC

CGGTGTCGAA

TGATGTACGT

GGAAATCTTC

CGTTCCTGAG

ATATAAGCTT

TAATAAAATT

GACACGGAAA

ACAGTGCGTG

GGGTGAGCCA

ATTTTTCAGT

CGGTCTCGCT

GGGCACGGAG

GCGAGTCCCT

GCATACCACG

ATCTGCGAAC

CCGGTACCGT

GCGTGTCTGA

GTGCAGGAAA

ACATTCGGTG

AGCATGTATC

CTATTTAAAT

TGGATGCGCA

TGGTGCTGCC

TTTCCACCTA

GGTGTTGGAC

TGTCTCAAAC

GCTCTTGTCA

TGCAATGGCC

AATTGTGGTG

GTAAGCAGCT

TCTGGTGGGA

GTTGTTCGTA

CTCCTGGGCG

CACGCGGTAA

TGCCCCCTCC

CGGAATGTGC

TCGGTGGTCC

GTTAAGCAGG

GGCGTCTTTC

CGCCTCGTCT

GACACAAAAG

GCTCTTGGTT

GTGGATCCTG

CAACCCCTTA

GACTCTCTAT

TGCTTTGCGA

TATGTCAGTT

TGTGTGATGG

TAACGGGTAA

ATCGGACTTT

AACGGGACAT

TAGCCCTGGG

AGTGTCTTCC

TGCACGTTAT

GCACAAAGAAi

TGCTATCAAC

TAGGACCTTT

CACCCGGTTC

ACCACCGTCT

GCGGAGGATT

GTATAGTGTA

ACCCCAGAAT

TGGAGCACAG

GCGCCAGTGA

GTCTGAGTAC

TACGCGGGGT

GTGGTGTCTA

AGCGTAGCAT

ATAAAGAGGT

AGTCTGTAAC

GCACACACGT

AAACTCGTGG

TGAAGCATGA

ACGTAGAGCA

AGGGGCTCAT

CTTGGGAATA

GACATGTGGT

GGGAGCAAGT

GTCACAAAAC

GGGTGTGCCG

CCACGGCGTG

GGGCGCCGAA

CTGCATGCTC

GGATAACCAG

TGTGCGGTAT

GGACGGCGTC

CGTACTGGGG

CGTCTCCATC

TAAAAAGTAC

GAAAGATACG

CTGCTGCATC

AGCCGGTCGC

GGCTGGCGCT

TCTCGCCTCC

GAAAATGTAT

CTTGGCTGCT

CGATCGCCTT

GGTCGCGCAG

TCGTGACAAC

CGATGTAAAC

CCCGACACAT

TTGACGACGC

TGGTGGTGGT

AAGGACAGGA

AACTGACCAC

AGCGGTCTGA

AACCCAGAAT

GGTTAAAGGT

AAGCGGCTAA

AAAGGGGGCG

GAATGAGAAA

GGTCGCGGGG

AGAAAGACTC

CCTAGACACC

TTTTGCCTGT

TGCGTTCTCC

GGCAACATTC

GTTGACAGGG

TTGCAGACAT

ACCGACACCA

GAGGCCCAAG

TATTCGACAA

GACTGCATTG

TAGGTCCTCG

ACGCCATACC

CAGGGTCATC

CAGGGTGCCG

CAACATGTAA

GGGGAGCGGG

GTTGCCTTCG

AATCATCGTG

GAAGAAATGC

GGCGCCCACG

AAACAGCTGT

GTCAAACTGT

TGCTCCCTCG

TTTGATGGAT

CACTAGTGCG

ATTCAGCATC

CGAGTTCACG

AGCCGGCAGT

CTGTCCCCGA

CAGTAAAGGA

TGTGTGCCGA

GATTTTGAGG

GTCAGGGCAC

TGCCTGCTGA

GGTGCGAAAiT

ACCAGTGTCG

TGCATACGCC

AAGAGGGCCA

ACGCATATCA

GGCCGGACAC

TCTCGGCCAT

GCGGGTACGC

AAACGGTGTA

CCAAGCGGGT

CTGCCACCGT

CCTGGAGCCC

CCGCCGGTTG

14580 14640 14700 14760 14820 14880 14940 15000 15060 15120 15180 15240 15300 15360 15420 15480 15540 15600 15660 15720 15780 15840 15900 15960 16020 16080 16140 16200 16260 16320 16380 16440 16500 16560 WO 96/06159 PTU9/09 PCTIUS95/10194 186

GTGGTGTGTG

GGTTTGGCGC

TCGGTACTGC

CTATCTCTGG

GGGGTATCGC

CGTGCTACGC

CGCGCAGGGT

GTGCGTGGAG

AGGCCACCCC

CGAGACGCCA

GGCCGTCAGG

CCGCCACCGG

GTGGACACGC

GTCGCCATTC

TGCCTAGTGT

CCGCCCCTTG

CACACTCTAG

GAGATTTTAA

ATCGTCACAC

CGCTATAGGG

GCGCAAACTT

GGACCATATC

GGTAACCAGC

TAAAGTCATG

AAGCCAGTGT

TGGACAGTTT

GCGCGAACAC

CCGCGGCGAC

TTGGCTCGGG

TCCGGGTGTC

CCACCCAGAC

CTGTTCTGCG

CGGGAATGGA

CATTGTCCAG

GGAA.ACGTGT

TTAGCCTGCC

GCATTTCCTG

TGGTGGCTAT

CGCCAGAGGG

CGGACCCGGC

CAGACGGAGA

AAAiGCCTAGA

CCGTGCGACT

CTAACGTGTA

AAGACGACTG

GCTTAGTGTT

TAGAGGCCGC

CTCGCGACGC

TACGGGGGCT

GCCGCCACGC

GTTTGGAGTT

AACAGCCCGA

CCGCATTGGA

CGTCGAALGGA

CGGAATTTCC

AAGGTCTTTA

TACGCACTGA

GGACCGTGTG

GTTTCTGTGC

CTGGAGGAAG

GTACCTGACC

CTGACCAATT

GGCGGATCGG

GACGGAATGC

TGTGTCCACT

CAGTCGCCGG

GTGACTAGGG

AGCAGGGTAA

CCTGCCAGGG

TCTGTTTTCC

CCCTGGTTCT

GGGGGCGGGA

CGCCGTAGCC

ACTCACCCGT

CCGCGCCACT

CCCGCCTGTT

GAGCTTCAAC

CTCGGGTGCT

GCTGCCGATA

CATGGACGAC

CTGTCGCACG

GGAAATCGCA

GGCTTCGGAG

CTGCTGGATG

ACGCGGCGTA

TGTGCAAAAG

AGCCTGGCTT

GGATCTGGTG

TAAACAAGGA

ACGCCTGCAC

GCGTGCCTGC

TGGCTGTGGG

GGCCCGTCCC

CATCCGGACT

TGACCAGACA

GCACTATGGG

TGTGGCTGCT

CGTCGTTGGC

GCCATGGACT

GTCTATCTAA

TCACTGGAAA

CAGCTCTGAA

GAGGAAAAcc

CACGATGGGA

AATCTTAGTC

CGGAATAATG

CACCCTTTGG

GGACCGTTGC

CACCACATGG

TCACAGAAGG

CCCGTCAATG

CCCTACTATG

CCACTGAGCT

TTGTTCATTA

CAAGGCTACA

GACGCAGTTA

GCTAGTGCCT

GACGTGTTAG

AACTGTGGCG

ACAGTCAGCG

GTGTTACCTG

TTCATTCGAG

ATGCATATGG

CTCTATCTCG

TTACAATGTG

AATTAACGGA

GGGGCGCGAT

GAGATTTCCC

AGAATTAGTT

TCTCGAATTC

GTTCTTGGGC

AAGAGTGGCC

CCGTGGACAC

CATCTGTCCC

CAGAAATTTT

GATAACTAGC

AACTTGCGTC

ACTTTCATCC

TTACTGTCAG

CGCGGAGTCC

AGGAACTGCA

AGGTCCTGAC

CGCTCGAGGC

GGCCAGCGCG

CCGATGTACC

TGTGTGTTTA

TCCCAGAAGA

ACACGAAGCA

CGTTGAGACA

AATCGCACTT

GGATAAGAGC

GATTATGGGA

GCACGGACGG

GGAGTCTTGT

GGGGTTTTGC

GCCGCTATGG

ACTGTTAACC

CCGGTGTATG

TCTGTGGCTA

GAAATGATCA

GGTATGGCGC

TACATTGCTC

CATACCTCCC

AGGAATGTGA

GTGGACTACA

GCCCTGCTTA

GTTAATGTAT

TGTATCAAAT

CTGGGTCTTC

CACCCAACCC

TTCACCTTCT

ATTTGACATC

ATTTCTCTAT

GACCGTTGAC

GAGGCTGGCG

CGGCCTTCTC

TCCGGGAACC

CACACGCCAC

CGCTACCTGG

CGAACGCGGT

GCCCGTGCCC

GTGCGACTTT

GCGCGTGCCT

TTTAGAGGCG

TGCCACGTCC

AAGCCGCCCC

TGACTGGTTA

GGCATGCGTG

TATTAAAGCC

CTAGCCGGAG

CAATGTCAGG

ACCCTGAGCT

TCTTGCTGCA

TGTACGTCGT

TCATCTACTT

CGCCGCCGTC

AGGTGGTGCG

ACCCTTTTGT

TGGCGTTCTG

CCAGGTGCGA

TTCGTGGGTA

CATGTGGGAC

TGTTCGATCC

CCACGCACGT

16620 16680 16740 16800 16860 16920 16980 17040 17100 17160 17220 17280 17340 17400 17460 17520 17580 17640 17700 17760 17820 17880 17940 18000 18060 18120 18180 18240 18300 18360 18420 18480 18540 18600 WO 96/06159 WO 9606159PCTfUS95/10194 187

CGAGAATGTG

CCTGGGTCCT

AACTTAAGGC

CCGTCCTCCG

GGGGGGGAAC

CTTTTGGTGG

GATCGCCACC

TTGCTTAAAC

CAATCCAAAC

GTGCATTACT

GTTCAGAAAG

GTGCAGGGAG

GACGTGCGCG

GCGCGGCTTT

TGACGTGGGC

GTTTATTCTG

TCGCGTGAAT

CAGGTTGGCG

CGTGTCCGAG

GGACCTTAGC

CCTCTTGGTT

GCGGCCAGAC

GCAGGGTTTC

CGAAAAGGGC

TCTGAATAAG

GAAGGGTCTT

CTCTTTTTGT

GCTACTTCCA

CCTGAAGTAC

CCCGCCATTC

TTCCGACACT

AATTGCGGGT

GTATATAACA

CCCATCGTAT

CTAACAGGAG

CTTACGAATG

CCTGGGCTTA

GGTCGCGTGT

CACCGACAAA

GCTACGTACC

ACTATCCATG

GTCTGTAAAA

AACTGGTGCG

AGCTGCTGTT

GACGAAAGGT

TCGTGTGTTT

TCCTCGCGCA

GCGGGGACGT

GTGGCGGGAT

TGCACCACGC

GTCGGGGCAC

GCAAGGCGCT

GGTGACCCGG

GAGAACACAC

TCCAACGTGA

TTTAGTAATC

GGGGCGGCGG

CAGGTGTTCT

GATGCGCTCC

CTTCCTGACG

CGGGCCGACT

GGTCACATGT

ACCAAGTTTC

GCTACTTCTC

TACATTTCCC

CAGGGGACCC

GGCAATGTTC

GACATACCGG

TGCTCGACGA

TCTGACTACT

CGTTCTTATT

AGATTATGGT

GCGTCTTTAT

CAATGTTAAT

TCTTTCCGTG

CACTGTTTGG

TCTTTTGTGG

GGAAGGGCTC

TAACCAGAAA

TTCTGCCCGC

CACACCGCAT

TGTCCTCGGA

GGTGCCGTGT

CAATAAAAGG

TCAGTTCCCA

CCCTGTGACG

AGGTGACCAG

CTGGACAATT

TGCCCGAGGT

TCCACTTGCC

GTGAGGAAAT

ACAACCACTA

TTCGGGAGGC

ACCCCCTTCC

GTTGCCTGTA

ACGCTCCCAA

TATACGGAGA

GGATACAAAG

ACACCTGCCT

ACGTGGGTGG

AGACCCAAAG

CGATCATCAC

CGGCACCTTG

TCAGCCGCTT

GAAGCATGTT

TCCGTTCTCC

GATTTCCGCG

GTTCTCTACG

TCTCCTTGGT

AGTTTCAAAT

GGCCTTGATT

CAGCCAGTGC

AGCCTGAAGT

CTGGTACTCG

CTGCAAGTAT

CGGTCTGGGG

GCAGTATGTT

GTGCGCCATC

CCTCTCTCCG

GCTGAGCAGC

TAGGTACGTC

TAAGTTGATA

CCAGCCAATC

TAGACTGGAG

CGCACTGGAC

TGCTACCGAG

TGTAGATGGC

GTTGCTATGG

CAAGCAGCAC

ACGGGATCTT

TTTTTCCGGG

GGTAGTGAGT

CTTGTGTCAC

AP.TCCTACTG

GTGTCCAACT

CATGATCAAG

GTGCCGTCCG

GCTGATATAT

GCGCACATCA

TTCTTGATGT

AACACGGAGT

GATGCCAGTA

ATTAGGAATA

AAACCGAAGT

GAAACCAAA

ACCCCGGGAA

TCGCGGTAGA

ACCAGTTGAT

GTTGATAGGG

GTTCCCACGT

TCCAGGACCG

CGTGCCGTTT

GCGTCTTTGG

ATGTTTGCTT

AAGGGCGTAC

GAAACTCCCC

TGCAGTGGCC

AAGCTCCAGA

CCGTCTCACG

GAGTGGACGT

CTGTGTGACC

CTGCTGTTCA

TGCGGTTACC

TTGTCGTTCG

ACATGGGCGG

CAGATGAAAA

ATATATCAGC

TTGAGTGGAA

ACGGGCGACT

GAGAATGGAC

TCCAAGGCAC

GAGTGTAAAA

GCGAGCTGGA

TTAAATTTTT

TGGCTACGTG

GCATGCTGAT

CGCTTGCCTT

ACTGCTTAAA

AGAAAAAAGT

CGTAACAGCC

CAGAGCAGGC

CGGCCGTGGA

ACTCCAATAG

CGGGATTTGC

AACTGTATGA

TGGGACAGTG

CGGTCTCCTC

TGAGCTCGCT

AACTTGCCCT

TGAACAGCTT

GGCCGGCCTT

GAGTCCTCGG

TAGAAACACA

GGGCTTTGAC

CCGGAACTTG

ACGGACCCGC

CGGGCCCGGT

TTAATCATGC

CGGCTTGCCG

TCATAGATGC

AAA.ATAGCAT

AAGGGACCCA

ATCTAATCAT

TCAACCAACT

18660 18720 18780 18840 18900 18960 19020 19080 19140 19200 19260 19320 19380 19440 19500 19560 19620 19680 19740 19800 19860 19920 19980 20040 20100 20160 20220 20280 20340 20400 20460 20520 20580 20640 WO 96/06159 PCTIUS95/10194 CTAAAAGAGA GTTTATTAAG TCGGCTCTGG AGGCCAACAT CAACAGGAGG GCAGCTGTAT 20700 CGCTATTTGA 20710 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 4131 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..4131 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

ATG

Met 1 GAG GCG ACC Glu Ala Thr

TTG

Leu 5 GAG CAA CGA CCT Glu Gln Arg Pro

TTC

Phe 10 CCG TAC CTC GCC Pro Tyr Leu Ala ACG GAG Thr Glu GCC AAC CTC Ala Asn Leu AAG AGC TTT Lys Ser Phe ACG CAG ATT AAG Thr Gln Ile Lys TCG GCT GCC GAC Ser Ala Ala Asp GGA CTC TTC Gly Leu Phe GGC AGT GTC Gly Ser Val CAG CTA TTG CTC Gln Leu Leu Leu AAG GAC GCC AGA Lys Asp Ala Arg

GAA

Glu 96 144 192 240 CGT TTC Arg Phe GAA GCG CTA CTG Glu Ala Leu Leu

GGC

Gly GTA TAT ACC AAT Val Tyr Thr Asn GTG GAG TTT GTT Val Glu Phe Val

AAG

Lys TTT CTG GAG ACC Phe Leu Glu Thr

GCC

Ala 70 CTC GCC GCC GCT Leu Ala Ala Ala GTC AAT ACC GAG Val Asn Thr Glu

TTC

Phe AAG GAC CTG CGG Lys Asp Leu Arg ATG ATA GAT GGA Met Ile Asp Gly

AAA

Lys ATA CAG TTT AAA Ile Gin Phe Lys ATT TCA Ile Ser ATG CCC ACT Met Pro Thr CAG TAT ATC Gin Tyr Ile 115

ATT

Ile 100 GCC CAC GGA GAC Ala His Gly Asp GGG AGG AGG CCC AAC AAG CAG AGA Gly Arg Arg Pro Asn Lys Gin Arg 105 110 GTC ATG AAG GCT Val Met Lys Ala AAT AAG CAC CAC Asn Lys His His GGT GCG GAG Gly Ala Glu ATT GAG Ile Glu 130 CTT GCG GCC GCA Leu Ala Ala Ala

GAC

Asp 135 ATC GAG CTT CTC Ile Glu Leu Leu

TTC

Phe 140 GCC GAG AAA GAG Ala Glu Lys Glu 432 480

ACG

Thr 145 CCC TTG GAC TTC Pro Leu Asp Phe

ACA

Thr 150 GAG TAC GCG GGT Glu Tyr Ala Gly ATC AAG ACG ATT Ile Lys Thr Ile WO 96/06159 PCTIUS95/10194 189 TCG GCT TTG CAG TTT GGT ATG GAC GCC CTA GAA CGG GGG CTA GTG GAC 528 Ser Ala Leu Gin Phe Gly Met Asp Ala Leu Glu Arg Gly Leu Val Asp 165 170 175 ACG GTT CTC GCA GTT AAA CTT CGG CAC GCT CCA CCC GTC TTT ATT TTA 576 Thr Val Leu Ala Val Lys Leu Arg His Ala Pro Pro Val Phe Ile Leu 180 185 190 AAG ACG CTG GGC GAT CCC GTC TAC TCT GAG AGG GGC CTC AAA AAG GCC 624 Lys Thr Leu Gly Asp Pro Val Tyr Ser Glu Arg Gly Leu Lys Lys Ala 195 200 205 GTC AAG TCT GAC ATG GTA TCC ATG TTC AAG GCA CAC CTC ATA GAA CAT 672 Val Lys Ser Asp Met Val Ser Met Phe Lys Ala His Leu Ile Glu His 210 215 220 TCA TTT TTT CTA GAT AAG GCC GAG CTC ATG ACA AGG GGG AAG CAG TAT 720 Ser Phe Phe Leu Asp Lys Ala Glu Leu Met Thr Arg Gly Lys Gin Tyr 225 230 235 240 GTC CTA ACC ATG CTC TCC GAC ATG CTG GCC GCG GTG TGC GAG GAT ACC 768 Val Leu Thr Met Leu Ser Asp Met Leu Ala Ala Val Cys Glu Asp Thr 245 250 255 GTC TTT AAG GGT GTC AGC ACG TAC ACC ACG GCC TCT GGG CAG CAG GTG 816 Val Phe Lys Gly Val Ser Thr Tyr Thr Thr Ala Ser Gly Gin Gin Val 260 265 270 GCC GGC GTC CTG GAG ACG ACG GAC AGC GTC ATG AGA CGG CTG ATG AAC 864 Ala Gly Val Leu Glu Thr Thr Asp Ser Val Met Arg Arg Leu Met Asn 275 280 285 CTG CTG GGG CAA GTG GAA AGT GCC ATG TCC GGG CCC GCG GCC TAC GCC 912 Leu Leu Gly Gin Val Glu Ser Ala Met Ser Gly Pro Ala Ala Tyr Ala 290 295 300 AGC TAC GTT GTC AGG GGT GCC AAC CTC GTC ACC GCC GTT AGC TAC GGA 960 Ser Tyr Val Val Arg Gly Ala Asn Leu Val Thr Ala Val Ser Tyr Gly 305 310 315 320 AGG GCG ATG AGA AAC TTT GAA CAG TTT ATG GCA CGC ATA GTG GAC CAT 1008 Arg Ala Met Arg Asn Phe Glu Gin Phe Met Ala Arg Ile Val Asp His 325 330 335 CCC AAC GCT CTG CCG TCT GTG GAA GGT GAC AAG GCC GCT CTG GCG GAC 1056 Pro Asn Ala Leu Pro Ser Val Glu Gly Asp Lys Ala Ala Leu Ala Asp 340 345 350 GGA CAC GAC GAG ATT CAG AGA ACC CGC ATC GCC GCC TCT CTC GTC AAG 1104 Gly His Asp Glu Ile Gin Arg Thr Arg Ile Ala Ala Ser Leu Val Lys 355 360 365 ATA GGG GAT AAG TTT GTG GCC ATT GAA AGT TTG CAG CGC ATG TAC AAC 1152 Ile Gly Asp Lys Phe Val Ala Ile Glu Ser Leu Gin Arg Met Tyr Asn 370 375 380 GAG ACT CAG TTT CCC TGC CCA CTG AAC CGG CGC ATC CAG TAC ACC TAT 1200 Glu Thr Gin Phe Pro Cys Pro Leu Asn Arg Arg Ile Gin Tyr Thr Tyr 385 390 395 400 TTC TTC CCT GTT GGC CTT CAC CTT CCC GTG CCC CGC TAC TCG ACA TCC 1248 Phe Phe Pro Val Gly Leu His Leu Pro Val Pro Arg Tyr Ser Thr Ser 405 410 415 GTC TCA GTC AGG GGC GTA GAA TCC CCG GCC ATC CAG TCG ACC GAG ACG 1296 Val Ser Val Arg Gly Val Glu Ser Pro Ala Ile Gin Ser Thr Glu Thr 420 425 430 WO 96/06159 PCTIUS95/10194 190 TGG GTG GTT AAT AAA AAC AAC GTG CCT CTT TGC TTC GGT TAC CAA AAC 1344 Trp Val Val Asn Lys Asn Asn Val Pro Leu Cys Phe Gly Tyr Gin Asn 435 440 445 GCC CTC AAA AGC ATA TGC CAC CCT CGA ATG CAC AAC CCC ACC CAG TCA 1392 Ala Leu Lys Ser Ile Cys His Pro Arg Met His Asn Pro Thr Gin Ser 450 455 460 GCC CAG GCA CTA AAC CAA GCT TTT CCC GAT CCC GAC GGG GGA CAT GGG 1440 Ala Gin Ala Leu Asn Gin Ala Phe Pro Asp Pro Asp Gly Gly His Gly 465 470 475 480 TAC GGT CTC AGG TAT GAG CAG ACG CCA AAC ATG AAC CTA TTC AGA ACG 1488 Tyr Gly Leu Arg Tyr Glu Gin Thr Pro Asn Met Asn Leu Phe Arg Thr 485 490 495 TTC CAC CAG TAT TAC ATG GGG AAA AAC GTG GCA TTT GTT CCC GAT GTG 1536 Phe His Gin Tyr Tyr Met Gly Lys Asn Val Ala Phe Val Pro Asp Val 500 505 510 GCC CAA AAA GCG CTC GTA ACC ACG GAG GAT CTA CTG CAC CCA ACC TCT 1584 Ala Gin Lys Ala Leu Val Thr Thr Glu Asp Leu Leu His Pro Thr Ser 515 520 525 CAC CGT CTC CTC AGA TTG GAG GTC CAC CCC TTC TTT GAT TTT TTT GTG 1632 His Arg Leu Leu Arg Leu Glu Val His Pro Phe Phe Asp Phe Phe Val 530 535 540 CAC CCC TGT CCT GGA GCG AGA GGA TCG TAC CGC GCC ACC CAC AGA ACA 1680 His Pro Cys Pro Gly Ala Arg Gly Ser Tyr Arg Ala Thr His Arg Thr 545 550 555 560 ATG GTT GGA AAT ATA CCA CAA CCG CTC GCT CCA AGG GAG TTT CAG GAA 1728 Met Val Gly Asn Ile Pro Gin Pro Leu Ala Pro Arg Glu Phe Gin Glu 565 570 575 AGT AGA GGG GCG CAG TTC GAC GCT GTG ACG AAT ATG ACA CAC GTC ATA 1776 Ser Arg Gly Ala Gin Phe Asp Ala Val Thr Asn Met Thr His Val Ile 580 585 590 GAC CAG CTA ACT ATT GAC GTC ATA CAG GAG ACG GCA TTT GAC CCC GCG 1824 Asp Gin Leu Thr Ile Asp Val Ile Gin Glu Thr Ala Phe Asp Pro Ala 595 600 605 TAT CCC CTG TTC TGC TAT GTA ATC GAA GCA ATG ATT CAC GGA CAG GAA 1872 Tyr Pro Leu Phe Cys Tyr Val Ile Glu Ala Met Ile His Gly Gin Glu 610 615 620 GAA AAA TTC GTG ATG AAC ATG CCC CTC ATT GCC CTG GTC ATT CAA ACC 1920 Glu Lys Phe Val Met Asn Met Pro Leu Ile Ala Leu Val Ile Gin Thr 625 630 635 640 TAC TGG GTC AAC TCG GGA AAA CTG GCG TTT GTG AAC AGT TAT CAC ATG 1968 Tyr Trp Val Asn Ser Gly Lys Leu Ala Phe Val Asn Ser Tyr His Met 645 650 655 GTT AGA TTC ATC TGT ACG CAT ATT GGG AAT GGA AGC ATC CCT AAG GAG 2016 Val Arg Phe Ile Cys Thr His Ile Gly Asn Gly Ser Ile Pro Lys Glu 660 665 670 GCG CAC GGC CAC TAC CGG AAA ATC TTA GGC GAG CTC ATC GCC CTT GAG 2064 Ala His Gly His Tyr Arg Lys Ile Leu Gly Glu Leu Ile Ala Leu Glu 675 680 685 CAG GCG CTT CTC AAG CTC GCG GGA CAC GAG ACG GTG GGT CGG ACG CCG 2112 Gin Ala Leu Leu Lys Leu Ala Gly His Glu Thr Val Gly Arg Thr Pro 690 695 700 WO 96/06159 PCT/US95/10194 191

ATC

Ile 705

TTT

Phe

CAA

Gin

AGG

Arg

AAC

Asn

AGA

Arg 785

GTC

Val

GGC

Gly

CTG

Leu

GAT

Asp

GCA

Ala 865

TCG

Ser

AAG

Lys

GTG

Val

GAC

Asp

AAC

Asn 945

CTG

Leu

ACA

Thr

GCC

Ala

CCC

Pro

GCG

Ala

CTT

Leu 770

CAC

His

CTC

Leu

CAC

His

ACT

Thr

ATT

Ile 850

GGC

Gly

TTT

Phe

CGG

Arg

GCG

Ala

CGC

Arg 930

AAG

Lys

CCA

Pro CAT CTG GTT His Leu Val TAC CAC GAT Tyr His Asp 725 ATA ATC AAG Ile Ile Lys 740 ACA TTC ATC Thr Phe Ile 755 GTT AAC ATT Val Asn Ile GTC CTG GAC Val Leu Asp GAG AAG CTA Glu Lys Leu 805 ATG TGC GGT Met Cys Gly 820 TAC AAC GGC Tyr Asn Gly 835 CTA CTG CAC Leu Leu His GAC ATA CGC Asp Ile Arg CTG ACG TGC Leu Thr Cys 885 GAC CCG GCC Asp Pro Ala 900 CAG ACC GTG Gin Thr Val 915 TCT CGC GAG Ser Arg Glu CTC TAC GCT Leu Tyr Ala AAC TAT GTC Asn Tyr Val 965 TCG GCT CTC CTC GAC CCG Ser Ala Leu Leu Asp .Pro i 710

GTC

Val

ATC

Ile

AAC

Asn

TAC

Tyr

GTG

Val 790

TTC

Phe

ATG

Met

CCC

Pro

CTG

Leu

CCG

Pro 870

CCT

Pro

CAG

Gin

CTT

Leu

GCG

Ala

GAC

Asp 950

ACC

Thr

TTT

Phe

GGG

Gly

CTC

Leu

CAG

Gin 775

GCG

Ala

TAC

Tyr

GGG

Gly

GTC

Val

GAG

Glu 855

ACG

Thr

TTC

Phe

AGT

Ser

GTT

Val

GCG

Ala 935

CCG

Pro

ACG

Thr

GAT

Asp

AGG

Arg 760

ACA

Thr

CCC

Pro

TAT

Tyr

GTC

Val

TTT

Phe 840

AAC

Asn

GTG

Val

GTC

Val

TTT

Phe

AAT

Asn 920

GAG

Glu

TTG

Leu

GAT

Asp

CAA

Gin 745

GGT

Gly

AGG

Arg

CTG

Leu

GTT

Val

GAC

Asp 825

GCG

Ala

GGA

Gly

GAC

Asp

ACC

Thr

GCC

Ala 905

GGC

Gly

ACT

Thr

GTG

Val 715 CTT ATG Leu Met 730 AAC TAC Asn Tyr CGC ATG Arg Met GTC AAT Val Asn GAC GAG Asp Glu 795 TTA ATG Leu Met 810 TAT CAA Tyr Gin GAC GTC Asp Val ACC TTG Thr Leu ATG ATC Met Ile 875 CAG GCC Gin Ala 890 ACG CAC Thr His TTT GGT Phe Gly ATG TTT Met Phe GCT GCC Ala Ala 955

CAT

His

CAG

Gin

GAC

Asp

GAG

Glu

GAG

Glu 780

AAT

Asn

CCG

Pro

AAC

Asn

GTG

Val

AAG

Lys 860

AGG

Arg

GCT

Ala

GAA

Glu

GCG

Ala

TAT

Tyr 940

ACA

Thr CTG CTG CCT CCC Leu Leu Pro Pro

AAG

Lys

AAC

Asn

GAC

Asp 765

GAC

Asp

GAC

Asp

GTG

Val

GTG

Val

AAC

Asn 845

GAC

Asp

GTG

Val

CGC

Arg

TAC

Tyr

TTC

Phe 925

CCG

Pro

CTG

Leu

TCA

Ser

CCT

Pro 750

CTA

Leu

CAT

His

TAC

Tyr

TGC

Cys

GCC

Ala 830

GCA

Ala

ATT

Ile

CTG

Leu

GTG

Val

GGG

Gly 910

GCG

Ala

GTA

Val

CAT

His

TCC

Ser 735

CAA

Gin

GTC

Val

GAC

Asp

AAC

Asn

AGT

Ser 815

CTG

Leu

CAG

Gin

CTG

Leu

TGC

Cys

ATC

Ile 895

AAG

Lys

GTG

Val

CCC

Pro

CCG

Pro

AGA

Arg

AAT

Asn

AAT

Asn

GAG

Glu

CCG

Pro 800

AAC

Asn

ACG

Thr

GAT

Asp

CAG

Gin

ACC

Thr 880

ACA

Thr

GAT

Asp

GCG

Ala

TTT

Phe

CTC

Leu 960 2160 2208 2256 2304 2352 2400 2448 2496 2544 2592 2640 2688 2736 2784 2832 2880 2928 AGG CTC CCC AAC CAG AGA AAC GCG GTG GTC Arg Leu Pro Asn 970 Gin Arg Asn Ala Val Val WO 96/06159 PCTUS95/10194 192 TTT AAC GTG CCA TCC AAT CTC ATG GCA GAA TAT GAG GAA TGG CAC AAG 2976 Phe Asn Val Pro Ser Asn Leu Met Ala Glu Tyr Glu Glu Trp His Lys 980 985 990 TCG CCC GTC GCG GCG TAT GCC GCG TCT TGT CAG GCC ACC CCG GGC GCC 3024 Ser Pro Val Ala Ala Tyr Ala Ala Ser Cys Gin Ala Thr Pro Gly Ala 995 1000 1005 ATT AGC GCC ATG GTG AGC ATG CAC CAA AAA CTA TCT GCC CCC AGT TTC 3072 Ile Ser Ala Met Val Ser Met His Gin Lys Leu Ser Ala Pro Ser Phe 1010 1015 1020 ATT TGC CAG GCA AAA CAC CGC ATG CAC CCT GGT TTT GCC ATG ACA GTC 3120 Ile Cys Gin Ala Lys His Arg Met His Pro Gly Phe Ala Met Thr Val 1025 1030 1035 1040 GTC AGG ACG GAC GAG GTT CTA GCA GAG CAC ATC CTA TAC TGC TCC AGG 3168 Val Arg Thr Asp Glu Val Leu Ala Glu His Ile Leu Tyr Cys Ser Arg 1045 1050 1055 GCG TCG ACA TCC ATG TTT GTG GGC TTG CCT TCG GTG GTA CGG CGC GAG 3216 Ala Ser Thr Ser Met Phe Val Gly Leu Pro Ser Val Val Arg Arg Glu 1060 1065 1070 GTA CGT TCG GAC GCG GTG ACT TTT GAA ATT ACC CAC GAG ATC GCT TCC 3264 Val Arg Ser Asp Ala Val Thr Phe Glu Ile Thr His Glu Ile Ala Ser 1075 1080 1085 CTG CAC ACC GCA CTT GGC TAC TCA TCA GTC ATC GCC CCG GCC CAC GTG 3312 Leu His Thr Ala Leu Gly Tyr Ser Ser Val Ile Ala Pro Ala His Val 1090 1095 1100 GCC GCC ATA ACT ACA GAC ATG GGA GTA CAT TGT CAG GAC CTC TTT ATG 3360 Ala Ala Ile Thr Thr Asp Met Gly Val His Cys Gin Asp Leu Phe Met 1105 1110 1115 1120 ATT TTC CCA GGG GAC GCG TAT CAG GAC CGC CAG CTG CAT GAC TAT ATC 3408 Ile Phe Pro Gly Asp Ala Tyr Gin Asp Arg Gin Leu His Asp Tyr Ile 1125 1130 1135 AAA ATG AAA GCG GGC GTG CAA ACC GGC TCA CCG GGA AAC AGA ATG GAT 3456 Lys Met Lys Ala Gly Val Gin Thr Gly Ser Pro Gly Asn Arg Met Asp 1140 1145 1150 CAC GTG GGA TAC ACT GCT GGG GTT CCT CGC TGC GAG AAC CTG CCC GGT 3504 His Val Gly Tyr Thr Ala Gly Val Pro Arg Cys Glu Asn Leu Pro Gly 1155 1160 1165 TTG AGT CAT GGT CAG CTG GCA ACC TGC GAG ATA ATT CCC ACG CCG GTC 3552 Leu Ser His Gly Gin Leu Ala Thr Cys Glu Ile Ile Pro Thr Pro Val 1170 1175 1180 ACA TCT GAC GTT GCC TAT TTC CAG ACC CCC AGC AAC CCC CGG GGG CGT 3600 Thr Ser Asp Val Ala Tyr Phe Gin Thr Pro Ser Asn Pro Arg Gly Arg 1185 1190 1195 1200 GCG GCG TCG GTC GTG TCG TGT GAT GCT TAC AGT AAC GAA AGC GCA GAG 3648 Ala Ala Ser Val Val Ser Cys Asp Ala Tyr Ser Asn Glu Ser Ala Glu 1205 1210 1215 CGT TTG TTC TAC GAC CAT TCA ATA CCA GAC CCC GCG TAC GAA TGC CGG 3696 Arg Leu Phe Tyr Asp His Ser Ile Pro Asp Pro Ala Tyr Glu Cys Arg 1220 1225 1230 TCC ACC AAC AAC CCG TGG GCT TCG CAG CGT GGC TCC CTC GGC GAC GTG 3744 Ser Thr Asn Asn Pro Trp Ala Ser Gin Arg Gly Ser Leu Gly Asp Val 1235 1240 1245 WO 96/06159 PCTIUS95/10194 193 CTA TAC AAT ATC ACC TTT CGC CAG ACT GCG CTG CCG GGC ATG TAC AGT 3792 Leu Tyr Asn Ile Thr Phe Arg Gin Thr Ala Leu Pro Gly Met Tyr Ser 1250 1255 1260 CCT TGT CGG CAG TTC TTC CAC AAG GAA GAC ATT ATG CGG TAC AAT AGG 3840 Pro Cys Arg Gin Phe Phe His Lys Glu Asp Ile Met Arg Tyr Asn Arg 1265 1270 1275 1280 GGG TTG TAC ACT TTG GTT AAT GAG TAT TCT GCC AGG CTT GCT GGG GCC 3888 Gly Leu Tyr Thr Leu Val Asn Glu Tyr Ser Ala Arg Leu Ala Gly Ala 1285 1290 1295 CCC GCC ACC AGC ACT ACA GAC CTC CAG TAC GTC GTG GTC AAC GGT ACA 3936 Pro Ala Thr Ser Thr Thr Asp Leu Gin Tyr Val Val Val Asn Gly Thr 1300 1305 1310 GAC GTG TTT TTG GAC CAG CCT TGC CAT ATG CTG CAG GAG GCC TAT CCC 3984 Asp Val Phe Leu Asp Gin Pro Cys His Met Leu Gin Glu Ala Tyr Pro 1315 1320 1325 ACG CTC GCC GCC AGC CAC AGA GTT ATG CTT GCC GAG TAC ATG TCA AAC 4032 Thr Leu Ala Ala Ser His Arg Val Met Leu Ala Glu Tyr Met Ser Asn 1330 1335 1340 AAG CAG ACA CAC GCC CCA GTA CAC ATG GGC CAG TAT CTC ATT GAA GAG 4080 Lys Gin Thr His Ala Pro Val His Met Gly Gin Tyr Leu Ile Glu Glu 1345 1350 1355 1360 GTG GCG CCG ATG AAG AGA CTA TTA AAG CTC GGA AAC AAG GTG GTG TAT 4128 Val Ala Pro Met Lys Arg Leu Leu Lys Leu Gly Asn Lys Val Val Tyr 1365 1370 1375 TAG 4131 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1376 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Glu Ala Thr Leu Glu Gin Arg Pro Phe Pro Tyr Leu Ala Thr Glu 1 5 10 Ala Asn Leu Leu Thr Gin Ile Lys Glu Ser Ala Ala Asp Gly Leu Phe 25 Lys Ser Phe Gin Leu Leu Leu Gly Lys Asp Ala Arg Glu Gly Ser Val 40 Arg Phe Glu Ala Leu Leu Gly Val Tyr Thr Asn Val Val Glu Phe Val 55 Lys Phe Leu Glu Thr Ala Leu Ala Ala Ala Cys Val Asn Thr Glu Phe 70 75 Lys Asp Leu Arg Arg Met Ile Asp Gly Lys Ile Gin Phe Lys Ile Ser 90 Met Pro Thr Ile Ala His Gly Asp Gly Arg Arg Pro Asn Lys Gin Arg 100 105 110 WO 96/06159 PCT/US95/10194 194 Gin Tyr Ile Glu 130 Thr Pro 145 Ser Ala Thr Val Lys Thr Val Lys 210 Ser Phe 225 Val Leu Val Phe Ala Gly Leu Leu 290 Ser Tyr 305 Arg Ala Pro Asn Gly His Ile Gly 370 Glu Thr 385 Phe Phe Val Ser Trp Val Ala Leu 450 Ala Gin Ile Val 115 Leu Ala Leu Asp Leu Gin Leu Ala 180 Leu Gly 195 Ser Asp Phe Leu Thr Met Lys Gly 260 Val Leu 275 Gly Gin Val Val Met Arg Ala Leu 340 Asp Glu 355 Asp Lys Gin Phe Pro Val Val Arg 420 Val Asn 435 Lys Ser Ala Leu Met Lys Ala Ala Phe Thr 150 Phe Gly 165 Val Lys Asp Pro Met Val Asp Lys 230 Leu Ser 245 Val Ser Glu Thr Val Glu Arg Gly 310 Asn Phe 325 Pro Ser Ile Gin Phe Val Pro Cys 390 Gly Leu 405 Gly Val Lys Asn Ile Cys Asn Gin Ala Asp 135 Glu Met Leu Val Ser 215 Ala Asp Thr Thr Ser 295 Ala Glu Val Arg Ala 375 Pro His Glu Asn His 455 Ala Cys 120 Ile Tyr Asp Arg Tyr 200 Met Glu Met Tyr Asp 280 Ala Asn Gin Glu Thr 360 Ile Leu Leu Ser Val 440 Pro Phe Asn Lys Glu Leu Ala Gly Ala Leu 170 His Ala 185 Ser Glu Phe Lys Leu Met Leu Ala 250 Thr Thr 265 Ser Val Met Ser Leu Val Phe Met 330 Gly Asp 345 Arg Ile Glu Ser Asn Arg Pro Val 410 Pro Ala 425 Pro Leu Arg Met Pro Asp His Leu Ala 155 Glu Pro Arg Ala Thr 235 Ala Ala Met Gly Thr 315 Ala Lys Ala Leu Arg 395 Pro Ile Cys His Pro His Phe 140 Ile Arg Pro Gly His 220 Arg Val Ser Arg Pro 300 Ala Arg Ala Ala Gin 380 Ile Arg Gin Phe Asn 460 Asp Ile 125 Ala Lys Gly Val Leu 205 Leu Gly Cys Gly Arg 285 Ala Val Ile Ala Ser 365 Arg Gin Tyr Ser Gly 445 Pro Gly Gly Ala Glu Lys Thr Ile Leu Val 175 Phe Ile 190 Lys Lys Ile Glu Lys Gin Glu Asp 255 Gin Gin 270 Leu Met Ala Tyr Ser Tyr Val Asp 335 Leu Ala 350 Leu Val Met Tyr Tyr Thr Ser Thr 415 Thr Glu 430 Tyr Gin Thr Gin Gly His Glu Glu Thr 160 Asp Leu Ala His Tyr 240 Thr Val Asn Ala Gly 320 His Asp Lys Asn Tyr 400 Ser Thr Asn Ser Gly WO 96/06159 PCT/US95/10194 195 465 470 475 480 Tyr Gly Leu Arg Tyr Glu Gin Thr Pro Asn Met Asn Leu Phe Arg Thr 485 490 495 Phe His Gin Tyr Tyr Met Gly Lys Asn Val Ala Phe Val Pro Asp Val 500 505 510 Ala Gin Lys Ala Leu Val Thr Thr Glu Asp Leu Leu His Pro Thr Ser 515 520 525 His Arg Leu Leu Arg Leu Glu Val His Pro Phe Phe Asp Phe Phe Val 530 535 540 His Pro Cys Pro Gly Ala Arg Gly Ser Tyr Arg Ala Thr His Arg Thr 545 550 555 560 Met Val Gly Asn Ile Pro Gin Pro Leu Ala Pro Arg Glu Phe Gin Glu 565 570 575 Ser Arg Gly Ala Gin Phe Asp Ala Val Thr Asn Met Thr His Val Ile 580 585 590 Asp Gin Leu Thr Ile Asp Val Ile Gin Glu Thr Ala Phe Asp Pro Ala 595 600 605 Tyr Pro Leu Phe Cys Tyr Val Ile Glu Ala Met Ile His Gly Gin Glu 610 615 620 Glu Lys Phe Val Met Asn Met Pro Leu Ile Ala Leu Val Ile Gin Thr 625 630 635 640 Tyr Trp Val Asn Ser Gly Lys Leu Ala Phe Val Asn Ser Tyr His Met 645 650 655 Val Arg Phe Ile Cys Thr His Ile Gly Asn Gly Ser Ile Pro Lys Glu 660 665 670 Ala His Gly His Tyr Arg Lys Ile Leu Gly Glu Leu Ile Ala Leu Glu 675 680 685 Gin Ala Leu Leu Lys Leu Ala Gly His Glu Thr Val Gly Arg Thr Pro 690 695 700 Ile Thr His Leu Val Ser Ala Leu Leu Asp Pro His Leu Leu Pro Pro 705 710 715 720 Phe Ala Tyr His Asp Val Phe Thr Asp Leu Met Gin Lys Ser Ser Arg 725 730 735 Gin Pro Ile Ile Lys Ile Gly Asp Gin Asn Tyr Asp Asn Pro Gin Asn 740 745 750 Arg Ala Thr Phe Ile Asn Leu Arg Gly Arg Met Glu Asp Leu Val Asn 755 760 765 Asn Leu Val Asn Ile Tyr Gin Thr Arg Val Asn Glu Asp His Asp Glu 770 775 780 Arg His Val Leu Asp Val Ala Pro Leu Asp Glu Asn Asp Tyr Asn Pro 785 790 795 800 Val Leu Glu Lys Leu Phe Tyr Tyr Val Leu Met Pro Val Cys Ser Asn 805 810 815 Gly His Met Cys Gly Met Gly Val Asp Tyr Gin Asn Val Ala Leu Thr 820 825 830 WO 96/06159 PCT/US95/10194 196 Leu Thr Tyr Asn Gly Pro Val Phe Ala Asp Val Val Asn Ala Gin Asp 835 840 845 Asp Ile Leu Leu His Leu Glu Asn Gly Thr Leu Lys Asp Ile Leu Gin 850 855 860 Ala Gly Asp Ile Arg Pro Thr Val Asp Met Ile Arg Val Leu Cys Thr 865 870 875 880 Ser Phe Leu Thr Cys Pro Phe Val Thr Gin Ala Ala Arg Val Ile Thr 885 890 895 Lys Arg Asp Pro Ala Gin Ser Phe Ala Thr His Glu Tyr Gly Lys Asp 900 905 910 Val Ala Gin Thr Val Leu Val Asn Gly Phe Gly Ala Phe Ala Val Ala 915 920 925 Asp Arg Ser Arg Glu Ala Ala Glu Thr Met Phe Tyr Pro Val Pro Phe 930 935 940 Asn Lys Leu Tyr Ala Asp Pro Leu Val Ala Ala Thr Leu His Pro Leu 945 950 955 960 Leu Pro Asn Tyr Val Thr Arg Leu Pro Asn Gin Arg Asn Ala Val Val 965 970 975 Phe Asn Val Pro Ser Asn Leu Met Ala Glu Tyr Glu Glu Trp His Lys 980 985 990 Ser Pro Val Ala Ala Tyr Ala Ala Ser Cys Gin Ala Thr Pro Gly Ala 995 1000 1005 Ile Ser Ala Met Val Ser Met His Gin Lys Leu Ser Ala Pro Ser Phe 1010 1015 1020 Ile Cys Gin Ala Lys His Arg Met His Pro Gly Phe Ala Met Thr Val 1025 1030 1035 1040 Val Arg Thr Asp Glu Val Leu Ala Glu His Ile Leu Tyr Cys Ser Arg 1045 1050 1055 Ala Ser Thr Ser Met Phe Val Gly Leu Pro Ser Val Val Arg Arg Glu 1060 1065 1070 Val Arg Ser Asp Ala Val Thr Phe Glu Ile Thr His Glu Ile Ala Ser 1075 1080 1085 Leu His Thr Ala Leu Gly Tyr Ser Ser Val Ile Ala Pro Ala His Val 1090 1095 1100 Ala Ala Ile Thr Thr Asp Met Gly Val His Cys Gin Asp Leu Phe Met 1105 1110 1115 1120 Ile Phe Pro Gly Asp Ala Tyr Gin Asp Arg Gin Leu His Asp Tyr Ile 1125 1130 1135 Lys Met Lys Ala Gly Val Gin Thr Gly Ser Pro Gly Asn Arg Met Asp 1140 1145 1150 His Val Gly Tyr Thr Ala Gly Val Pro Arg Cys Glu Asn Leu Pro Gly 1155 1160 1165 Leu Ser His Gly Gin Leu Ala Thr Cys Glu Ile Ile Pro Thr Pro Val 1170 1175 1180 Thr Ser Asp Val Ala Tyr Phe Gin Thr Pro Ser Asn Pro Arg Gly Arg WO 96/06159 PCT/US95/10194 197 1185 1190 1195 1200 Ala Ala Ser Val Val Ser Cys Asp Ala Tyr Ser Asn Glu Ser Ala Glu 1205 1210 1215 Arg Leu Phe Tyr Asp His Ser Ile Pro Asp Pro Ala Tyr Glu Cys Arg 1220 1225 1230 Ser Thr Asn Asn Pro Trp Ala Ser Gin Arg Gly Ser Leu Gly Asp Val 1235 1240 1245 Leu Tyr Asn Ile Thr Phe Arg Gin Thr Ala Leu Pro Gly Met Tyr Ser 1250 1255 1260 Pro Cys Arg Gin Phe Phe His Lys Glu Asp Ile Met Arg Tyr Asn Arg 1265 1270 1275 1280 Gly Leu Tyr Thr Leu Val Asn Glu Tyr Ser Ala Arg Leu Ala Gly Ala 1285 1290 1295 Pro Ala Thr Ser Thr Thr Asp Leu Gin Tyr Val Val Val Asn Gly Thr 1300 1305 1310 Asp Val Phe Leu Asp Gin Pro Cys His Met Leu Gin Glu Ala Tyr Pro 1315 1320 1325 Thr Leu Ala Ala Ser His Arg Val Met Leu Ala Glu Tyr Met Ser Asn 1330 1335 1340 Lys Gin Thr His Ala Pro Val His Met Gly Gin Tyr Leu Ile Glu Glu 1345 1350 1355 1360 Val Ala Pro Met Lys Arg Leu Leu Lys Leu Gly Asn Lys Val Val Tyr 1365 1370 1375 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 1143 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1143 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AGC ATT CGG GGA CAG ACC TTT AAC CTG CTC TAC GTA GAC GAG GCG AAT 48 Ser Ile Arg Gly Gin Thr Phe Asn Leu Leu Tyr Val Asp Glu Ala Asn 1 5 10 TTT ATT AAA AAG GAT GCA CTG CCG GCT ATT CTG GGT TTC ATG CTT CAG 96 Phe Ile Lys Lys Asp Ala Leu Pro Ala Ile Leu Gly Phe Met Leu Gin 25 WO 96/06159 WO 96/6 159PCI'1US95/10194 198

AAA

Lys

TCC

Ser

AAT

Asn

CAA

Gin

TAC

Tyr

GAG

Giu

AAT

Asn

GAC

Asp 145

GGA

Gly

GAG

Glu

CCC

Pro

CTC

Leu

ATT

Ile 225

GCG

Al a

GTC

Val

GAC

Asp

GAC

Asp

ACG

Thr

GTG

Val

GAC

Asp

ATC

Ile

GGG

Gly

GCT

Al a 130

ATG

Met

AAA

Lys

GCG

Al a

ACC

Thr

ACT

Thr 210

AAG

Lys

GTC

Val

CTT

Leu

AAG

Lys

GCC

Al a

AGT

Ser

GTC

Val

GCA

Ala

ACC

Thr

GCA

Ala 115

ACG

Thr

TGT

Cys

CAG

Gin

TCC

Ser

AGA

Arg 195

GGC

Gly

GCG

Ala

GAA

Glu

AAC

Asn

AGC

Ser 275 AAG CTT ATA TTT ATA TCA TCC GTG AAC TCG TCA Lys

TTC

Phe

AGT

Ser

CTA

Leu

ATC

Ile 100

TTC

Phe

CTT

Leu

CGG

Arg

CTG

Leu

GGT

Gly 180

AGC

Ser

GCA

Ala

ATC

Ile

GGC

Gly

GAA

Giu 260

AGC

Ser Leu

CTG

Leu

TAC

Tyr

GTG

Val

GAC

Asp

GAC

Asp

TAC

Tyr

GTA

Val

TTT

Phe 165

ACT

Thr

CTC

Leu

GCT

Al a

GCT

Ala

AAC

Asn 245

ATA

Ile

GCC

Ala Ile

CTT

Leu

GTG

Val 70

TCC

Ser

GAA

Glu

ACC

Thr

CGC

Arg

GAC

Asp 150

GTT

Vai

GGC

Gly

ATA

Ile

GCT

Ala

GTG

Val 230

AGC

Ser

TGC

Cys

CTG

Leu Phe

AAC

Asn 55

TGT

Cys

TGT

Cys

TCC

Ser

GAA

Glu

GTG

Val 135

ACC

Thr

TAC

Tyr

GTG

Val

TTG

Leu

TAC

Tyr 215

CTC

Leu

AGC

Ser

CCG

Pro

CAG

Gln Ile 40

CTC

Leu

GCG

Ala

CCT

Pro

ATC

Ile

CTA

Leu 120

GTG

Val

ACC

Thr

ATC

Ile

GGC

Gly

GGC

Gly 200

GAG

Giu

CAC

His

CAA

Gin

CTC

Leu

TGG

Trp 280 Ser

AGG

Arg

GAC

Asp

TGT

Cys

AAA

Lys 105

ATG

Met

GGT

Gly

GCC

Al a

GAC

Asp

GCC

Ala 185

ATG

Met

ATA

Ile

ACC

Thr

GAT

Asp

CCC

Pro 265

CCA

Pro Val

GCC

Al a

CGA

Arg 75

AGA

Arg

ACC

Thr

GAG

Glu

GCA

Al a

GAG

Giu 155

GCG

Ala

GTC

Val

CAT

His

TCC

Ser

ATT

Ile 235

GGG

Gly

CAT

His

TAC

Tyr Asn

CAG

Gin

GAA

Glu

CTG

Leu

ACC

Thr

GGA

Gly

GCG

Ala 140

GTT

Val

TAT

Tyr

ACG

Thr

TTC

Phe

TGC

Cys 220

GAG

Giu

GTG

Val

TTT

Phe

ATG

Met Ser

GAA

Giu

GAT

Asp

CAC

His

AAC

Asn

GCA

Al a 125

CTG

Leu

CAG

Gin

ACG

Thr

AGT

Ser

TTC

Phe 205

GCA

Al a

CGC

Arg

GCC

Ala

CTA

Leu

TTG

Leu 285 Ser

AAG,

Lys

TTC

Phe

ATT

Ile

CTC

Leu 110

GCG

Ala

ACA

Thr

AAG

Lys

AAC

Asn

ACT

Thr 190

CTG

Leu

TGC

Cys

GTG

Val

ATT

Ile

CAC

His 270

GGA

Gly

GAC

Asp

ATG

Met

CAC

His

CCG

Pro

TTT

Phe

TCG

Ser

CAG

Gin

TGC

Cys

AAC

Asn 175

CAG

Gin

CGC

Arg

ACG

Thr

AAC

Asn

GCA

Ala 255

TAT

Tyr

GGC

Gly

CGC

Arg

CTG

Leu

CTG

Leu

ACG

Thr

ATG

Met

TCA

Ser

TTT

Phe

CTT

Leu 160

ACG

Thr

ACT

Thr

GAC

Asp

ATG

Met

GCG

Ala 240

ACC

Thr

ACT

Thr

GAG

Glu

ACC

Thr 144 192 240 288 336 384 432 480 AAA TCC TCC GCG TTT GAG ACA TTC ATC TAC GCT CTG AAC TCC GGC Lys Ser Ser Ala Phe Giu Thr Phe Ile Tyr Ala 290 295 Leu 300 Asn Ser Gly WO 96/06159 PCT/US95/10194 199 CTG AGC GCC AGC CAG ACG GTG GTG TCC AAC ACC Leu Ser Ala Ser Gin Thr Val Val Ser Asn Thr 305 310 315 GAC CCG GTG ACC TAC CTG GTA GAA CAG GTC CGC Asp Pro Val Thr Tyr Leu Val Glu Gin Val Arg 325 330 CCG CTT AGG GAT GGA GGG CAG TCA TAC AGC GCC Pro Leu Arg Asp Gly Gly Gin Ser Tyr Ser Ala 340 345 TCG GAC GAC TTA CTT GTG GCA GTT GTC ATG GCC Ser Asp Asp Leu Leu Val Ala Val Val Met Ala 355 360 GAT GAT AGA CAC ATG TAC AAG CCC ATA TCC CCA Asp Asp Arg His Met Tyr Lys Pro Ile Ser Pro 370 375 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 380 amino acids TYPE: amino acid TOPOLOGY: linear

ATC

Ile

GCG

Ala

AAG

Lys

CAT

His

CAA

Gin 380 AAA ATA TCA TTT Lys Ile Ser Phe 320 ATC AAG TGC GTC Ile Lys Cys Val 335 CAA AAG CAC ATG Gin Lys His Met 350 TTT ATG GCT ACC Phe Met Ala Thr 365

TAA

960 1008 1056 1104 1143 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Ser 1 Phe Lys Ser Asn Gin Tyr Glu Asn Asp 145 Gly Ile Arg Gly Gin Thr Phe Asn Leu I Ile Asp Thr Val Asp Ile Gly Ala 130 Met Lys Lys Ala Ser Val Ala Thr Ala 115 Thr Cys Gin Lys Asp Lys Leu Phe Leu Ser Tyr Leu Val Ile Asp 100 Phe Asp Leu Tyr Arg Val Leu Phe 165 Ala Ile Leu Val 70 Ser Glu Thr Arg Asp 150 Val Leu Phe Asn 55 Cys Cys Ser Glu Val 135 Thr Tyr Pro Ala 25 Ile Ser 40 Leu Arg Ala Asp Pro Cys Ile Lys 105 Leu Met 120 Val Gly Thr Ala Ile Asp

I

S

A

H

T

T

G

A

G

P

1 jeu Tyr 10 :le Leu ;er Val hsn Ala [is Arg 75 'yr Arg 90 hr Thr ly Glu .sp Ala In Glu 155 ro Ala 70 Gly Asn Gin Glu Leu Thr Gly Ala 140 Val Tyr Phe Ser Glu Asp His Asn Ala 125 Leu Gin Thr Met Ser Lys Phe Ile Leu 110 Ala Thr Lys Asn Leu Asp Met His Pro Phe Ser Gin Cys Asn 175 Gin Arg Leu Leu Thr Met Ser Phe Leu 160 Thr Val Asp Glu Ala Asn Glu Ala Ser Gly 180 Thr Gly Val Gly Ala Val Val Thr Ser Thr Gin Thr WO 96/06159 PCT/US95/10194 200 Pro Thr Arg Ser Leu Ile Leu Gly Met Glu His Phe Phe Leu Arg Asp 195 200 205 Leu Thr Gly Ala Ala Ala Tyr Glu Ile Ala Ser Cys Ala Cys Thr Met 210 215 220 Ile Lys Ala Ile Ala Val Leu His Thr Thr Ile Glu Arg Val Asn Ala 225 230 235 240 Ala Val Glu Gly Asn Ser Ser Gin Asp Ser Gly Val Ala Ile Ala Thr 245 250 255 Val Leu Asn Glu Ile Cys Pro Leu Pro Ile His Phe Leu His Tyr Thr 260 265 270 Asp Lys Ser Ser Ala Leu Gin Trp Pro Ile Tyr Met Leu Gly Gly Glu 275 280 285 Lys Ser Ser Ala Phe Glu Thr Phe Ile Tyr Ala Leu Asn Ser Gly Thr 290 295 300 Leu Ser Ala Ser Gin Thr Val Val Ser Asn Thr Ile Lys Ile Ser Phe 305 310 315 320 Asp Pro Val Thr Tyr Leu Val Glu Gin Val Arg Ala Ile Lys Cys Val 325 330 335 Pro Leu Arg Asp Gly Gly Gin Ser Tyr Ser Ala Lys Gin Lys His Met 340 345 350 Ser Asp Asp Leu Leu Val Ala Val Val Met Ala His Phe Met Ala Thr 355 360 365 Asp Asp Arg His Met Tyr Lys Pro Ile Ser Pro Gin 370 375 380 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 234 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..234 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATG GGT GAG CCA GTG GAT CCT GGA CAT GTG GTG AAT GAG AAA GAT TTT 48 Met Gly Glu Pro Val Asp Pro Gly His Val Val Asn Glu Lys Asp Phe 1 5 10 GAG GAG TGT GAA CAA TTT TTC AGT CAA CCC CTT AGG GAG CAA GTG GTC 96 Glu Glu Cys Glu Gin Phe Phe Ser Gin Pro Leu Arg Glu Gin Val Val 25 WO 96/06159 PCT/US95/10194 201 GCG GGG GTC AGG GCA CTC GAC GGC CTC GGT CTC GCT GAC TCT CTA TGT 144 Ala Gly Val Arg Ala Leu Asp Gly Leu Gly Leu Ala Asp Ser Leu Cys 40 CAC AAA ACA GAA AGA CTC TGC CTG CTG ATG GAC CTG GTG GGC ACG GAG 192 His Lys Thr Glu Arg Leu Cys Leu Leu Met Asp Leu Val Gly Thr Glu 55 TGC TTT GCG AGG GTG TGC CGC CTA GAC ACC GGT GCG AAA TGA 234 Cys Phe Ala Arg Val Cys Arg Leu Asp Thr Gly Ala Lys 70 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 77 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Met Gly Glu Pro Val Asp Pro Gly His Val Val Asn Glu Lys Asp Phe 1 5 10 Glu Glu Cys Glu Gln Phe Phe Ser Gln Pro Leu Arg Glu Gin Val Val 25 Ala Gly Val Arg Ala Leu Asp Gly Leu Gly Leu Ala Asp Ser Leu Cys 40 His Lys Thr Glu Arg Leu Cys Leu Leu Met Asp Leu Val Gly Thr Glu 55 Cys Phe Ala Arg Val Cys Arg Leu Asp Thr Gly Ala Lys 70 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 585 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..585 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ATG AAG AGT GTG GCG AGT CCC TTA TGT CAG TTC CAC GGC GTG TTT TGC 48 Met Lys Ser Val Ala Ser Pro Leu Cys Gin Phe His Gly Val Phe Cys 1 5 10 CTG TAC CAG TGT CGC CAG TGC CTG GCA TAC CAC GTG TGT GAT GGG GGC 96 WO 96/06159 PCT/US95/10194 202 Leu

GCC

Ala

ACG

Thr

CCG

Pro

CAC

His

ACA

Thr

GGC

Gly

TGT

Cys

AGC

Ser 145

TAC

Tyr

ATT

Ile

GAT

Asp Tyr

GAA

Glu

GGT

Gly

GTA

Val

GGG

Gly

TGG

Trp

GTC

Val

CTT

Leu 130

ATG

Met

AAC

Asn

GCC

Ala

ACG

Thr Gln Cys Arg TGC GTT CTC Cys Val Leu AAC TGC ATG Asn Cys Met CCG TAT CGG Pro Tyr Arg ATG CTA GCG Met Leu Ala CCG GAC ACC Pro Asp Thr 100 ACC GAC ACC Thr Asp Thr 115 CCC GTA CTG Pro Val Leu TAT CTG CAC Tyr Leu His AGT ATG CTA Ser Met Leu 165 AAG CGG GTG Lys Arg Val 180

TAG

195 Cys

CAT

His

GGC

Gly 55

TTG

Leu

CTG

Leu

GTA

Val

TCG

Ser

GAG

Glu 135

ATC

Ile

AAA

Lys Leu

ACG

Thr 40

AAC

Asn

GAT

Asp

AAA

Lys

ATC

Ile

GCC

Ala 120

GCC

Ala

GTC

Val

TGC

Cys Ala 25

CCG

Pro

ATT

Ile

AAC

Asn

CGG

Arg

GTG

Val 105

ATT

Ile

CAA

Gin

TCC

Ser

ACA

Thr Tyr

GAG

Glu

CAA

Gln

CAG

Gin

GAC

Asp 90

CAG

Gln

ATA

Ile

GGC

Gly

ATC

Ile

AAG

Lys 170 His

AGC

Ser

GAG

Glu

GTT

Val 75

ATT

Ile

GAA

Glu

GAT

Asp

GGG

Gly

TAT

Tyr 155

AAT

Asn Val

GTC

Val

GGC

Gly

GAC

Asp

GTG

Val

ATA

Ile

GAA

Glu

TAC

Tyr 140

TCG

Ser

AAA

Lys Cys

ATC

Ile

CAG

Gin

AGG

Arg

CGG

Arg

GCC

Ala

ACA

Thr 125

GCC

Ala

ACA

Thr

AAG

Lys Asp

TGC

Cys

TTT

Phe

GAC

Asp

TAT

Tyr

CTG

Leu 110

TTC

Phe

CTG

Leu

AAA

Lys

TAC

Tyr Gly

GAA

Glu

TTA

Leu

GCA

Ala

TTG

Leu

GGG

Gly

GGT

Gly

GTC

Val

ACG

Thr

GAC

Asp 175 Gly

CTA

Leu

GGG

Gly

TAT

Tyr

CAG

Gln

GAC

Asp

GAG

Glu

TGT

Cys

GTG

Val 160

TGC

Cys CGG ACA AAA TGG ATG CGC ATG CTA TCA ACG AAA Arg Thr Lys Trp 185 Met Arg Met Leu Ser Thr Lys 190 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 194 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Met Lys Ser Val Ala Ser Pro Leu Cys Gin Phe His Gly Val Phe Cys 1 5 10 Leu Tyr Gin Cys Arg Gin Cys Leu Ala Tyr His Val Cys Asp Gly Gly 25 Ala Glu Cys Val Leu Leu His Thr Pro Glu Ser Val Ile Cys Glu Leu 40 WO 96/06159 PCT/US95/10194 Thr Pro His Thr Gly Cys Ser 145 Tyr Ile Asp (2)

ATG

Met 1

ATA

Ile

GCC

Ala

TAC

Tyr 203 Gly Asn Cys Met Leu Gly Asn Ile Gin Glu 55 Val Pro Tyr Arg Thr Leu Asp Asn Gin Val 70 75 Gly Met Leu Ala Cys Leu Lys Arg Asp Ile 90 Trp Pro Asp Thr Thr Val Ile Val Gin Glu 100 105 SVal Thr Asp Thr Ile Ser Ala Ile Ile Asp 115 120 Leu Pro Val Leu Gly Glu Ala Gin Gly Gly 130 135 Met Tyr Leu His Val Ile Val Ser Ile Tyr 150 155 Asn Ser Met Leu Phe Lys Cys Thr Lys Asn 165 170 Ala Lys Arg Val Arg Thr Lys Trp Met Arg 180 185 Thr INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 939 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..939 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID GCT AGC CGG AGG CGC AAA CTT CGG AAT TTC Ala Ser Arg Arg Arg Lys Leu Arg Asn Phe 5 10 TGG ACT GTT AAC CCA ATG TCA GGG GAC CAT Trp Thr Val Asn Pro Met Ser Gly Asp His 25 TGC ACC TCT ATC TCG CCG GTG TAT GAC CCT Cys Thr Ser Ile Ser Pro Val Tyr Asp Pro 40 GCA CTG AGC GTG CCT GCT TAC AAT GTG TCT Ala Leu Ser Val Pro Ala Tyr Asn Val Ser 55 Gly Asp Val Ile Glu Tyr 140 Ser Lys Met Gin Arg Arg Ala Thr 125 Ala Thr Lys Leu Phe Asp Tyr Leu 110 Phe Leu Lys Tyr Ser 190 Leu Ala Leu Gly Gly Val Thr Asp 175 Thr Gly Tyr Gin Asp Glu Cys Val 160 Cys Lys CTA AAC AAG GAA TGC Leu Asn Lys Glu Cys ATC AAG GTC TTT AAC Ile Lys Val Phe Asn GAG CTG GTA ACC AGC Glu Leu Val Thr Ser GTG GCT ATC TTG CTG Val Ala Ile Leu Leu WO 96/06159 WO 9606159PCTIUS95/10194 204 CAT AAA GTC ATG GGA CCG TGT His Lys Val Met Gly Pro Cys 70

ATC

Ile

CGC

Arg

TCC

Ser

GTA

Val CG C Arg 145

GTG

Val

TTG

Leu

TCG

Ser

TGT

Cys

TAC

Tyr 225

AAA

Lvs

AAT

Asn

GOT

Al a

CTA

Leu

ACC

Thr 305

ATG

Met

GAT

Asp

GGA

Gly

OCT

Pro 130

CGC

Arg

AAO

Asn

GGC

Gly

TTG

Leu

GTC

Val 210

TGT

Cys

TCA

S er

TT

Phe

CTG

Leu

ACA

Thr 290

CTG

Leu

TAO

Tyr

GGT

Gly

CTG

Leu 115

GAO

Asp

GGC

Gly

CCT

Pro

GTG

Val

GCA

Al a 195

CAC

His

TCT

Ser

TGT

Cys

CTG

Leu

AAG

Lvs 275

GGA

Gly

GGT

Gly

GTC

Val

ATG

Met 100

AGA

Arg

CTG

Leu

GAC

Asp

TTT

Phe

GAC

Asp 180

AGA

Arg

TGC

Cys

GCG

Al a

GGG

Gly

GGT

Gly 260

ATA

Ile

GTG

Val

CCT

Pro

GTA

Val

GCG

Al a

TTT

Phe

ACC

Thr

CTG

Leu

GTT

Val 165

TAC

Tyr

GTG

Val

CAT

His

CAG

Gin

ACC

Thr 245 C TT Leu

ACT

Thr

CTC

Leu

CTT

Leu

AGO

Ser

CTC

Leu

CCC

Pro

AGA

Arg

ACC

Thr 150

TGG

Trp

ATG

Met

GCC

Al a

GGA

Gly

TCG

Ser 230

GGG

Gly

CTG

Leu

AGO

Ser

GAC

Asp

ACG.

Thr 310

CAG

Gin

ATO

Ile

TAO

Tyr

CAA

Gin 135

AAT

Asn

CTC

Leu

GCG

Ala

GCC

Al a

CTC

Leu 215

CCG

Pro

AAT

Asn

TTC

Phe

CAC

His

GAO

Asp 295

AAT

Asn GTG GCT GTG GGA ATT Val Ala Val Gly Ile 75 TGT GTT TCT GTG CGG Cys Val Ser Val Arg 90 TAO TTT GGA CAG TTT Tyr Phe Gly Gin Phe 105 ATT GCT CCG CCG CCG Ile Ala Pro Pro Pro 120 GAA TTA GTT CAT ACC Giu Leu Val His Thr 140 TGO ACT ATG GGT CTC Cys Thr Met Gly Leu 155 GGG GGC GGA TCG GTG Gly Gly Gly Ser Val 170 TTC TGT CCG GGT GTC Phe Cys Pro Gly Val 185 CTG CTT ACC AGG TGC Leu Leu Thr Arg Cys 200 CGT GGA CAC GTT AAT Arg Gly His Val Asn 220 GGT CTA TOT AAO ATC Gly Leu Ser Asn Ile 235 GGA GTG ACT AGG GTC Gly Val Thr Arg Val 250 GAT CCC ATT GTC CAG Asp Pro Ile Val Gin 265 CCA ACC CCC ACOG CAC Pro Thr Pro Thr His 280 GGC ACC TTG GTG COG Gly Thr Leu Val Pro 300 GTC TGA Val AAC GGA GAA ATG Asn Gly Giu Met

CCC

Pro

CTG

Leu

TCG

Ser 125 Ser

GAA

Giu

TGG

Trp

GAO

Asp

GAO

Asp 205

GTA

Val

TGT

Cys

ACT

Thr

AGO

Ser

GTC

Val 285

TCC

Ser 240

GTC

Val

GAG

Glu 110

CGC

Arg

CAG

Gin TT C Phe

CTG

Leu

GGA

Gly 190

CAC

His

TTT

Phe

CCC

Pro

GGA

Gly

AGG

Arg 270

GAG

Glu GT1O Val

CCG

Pro 95

GAA

Glu

GAA

Giu

GTG

Val

AGG

Arg

CTG

Leu 175

ATG

Met

CCA

Pro

CGT

Arg

TGT

Cys

AAC

Asn 255

GTA

Val1

AAT

Asn

CAA

Gin

GGG

Gly

GCA

Ala

CAC

His

GTG

Val1

AAT

Asn 160

TTC

Phe

CCG

Pro

GAC

Asp

GGG

Gly

ATO

Ile 240

AGA

Arg

ACA

Thr

GTG

Val

GGC

Gly 288 3 3 C 384 432 480 528 576 624 672 720 768 816 864 912Z INFORM4ATION FOR SEQ ID NO:11: WO 96/06159 PCT/US95/10194 205 SEQUENCE CHARACTERISTICS: LENGTH: 312 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Met Ala Ser Arg Arg Arg Lys Leu Arg Asn Phe Leu Asn Lys Glu Cys 1 5 10 Ile Trp Thr Val Asn Pro Met Ser Gly Asp His Ile Lys Val Phe Asn 25 Ala Cys Thr Ser Ile Ser Pro Val Tyr Asp Pro Glu Leu Val Thr Ser 40 Tyr Ala Leu Ser Val Pro Ala Tyr Asn Val Ser Val Ala Ile Leu Leu 55 His Lys Val Met Gly Pro Cys Val Ala Val Gly Ile Asn Gly Glu Met 70 75 Ile Met Tyr Val Val Ser Gin Cys Val Ser Val Arg Pro Val Pro Gly 90 Arg Asp Gly Met Ala Leu Ile Tyr Phe Gly Gin Phe Leu Glu Glu Ala 100 105 110 Ser Gly Leu Arg Phe Pro Tyr Ile Ala Pro Pro Pro Ser Arg Glu His 115 120 125 Val Pro Asp Leu Thr Arg Gin Glu Leu Val His Thr Ser Gin Val Val 130 135 140 Arg Arg Gly Asp Leu Thr Asn Cys Thr Met Gly Leu Glu Phe Arg Asn 145 150 155 160 Val Asn Pro Phe Val Trp Leu Gly Gly Gly Ser Val Trp Leu Leu Phe 165 170 175 Leu Gly Val Asp Tyr Met Ala Phe Cys Pro Gly Val Asp Gly Met Pro 180 185 190 Ser Leu Ala Arg Val Ala Ala Leu Leu Thr Arg Cys Asp His Pro Asp 195 200 205 Cys Val His Cys His Gly Leu Arg Gly His Val Asn Val Phe Arg Gly 210 215 220 Tyr Cys Ser Ala Gin Ser Pro Gly Leu Ser Asn Ile Cys Pro Cys Ile 225 230 235 240 Lys Ser Cys Gly Thr Gly Asn Gly Val Thr Arg Val Thr Gly Asn Arg 245 250 255 Asn Phe Leu Gly Leu Leu Phe Asp Pro Ile Val Gin Ser Arg Val Thr 260 265 270 Ala Leu Lys Ile Thr Ser His Pro Thr Pro Thr His Val Glu Asn Val 275 280 285 Leu Thr Gly Val Leu Asp Asp Gly Thr Leu Val Pro Ser Val Gin Gly 290 295 300 Thr Leu Gly Pro Leu Thr Asn Val WO 96/06159 PCT/US95/10194 206 305 310 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 86 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..86 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: ATG GAC TCA ACC AAC TCT AAA AGA GAG TTT ATT AAG TCG GCT CTG GAG 48 Met Asp Ser Thr Asn Ser Lys Arg Glu Phe Ile Lys Ser Ala Leu Glu 1 5 10 GCC AAC ATC AAC AGG AGG GCA GCT GTA TCG CTA TTT GA 86 Ala Asn Ile Asn Arg Arg Ala Ala Val Ser Leu Phe INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 28 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Asp Ser Thr Asn Ser Lys Arg Glu Phe Ile Lys Ser Ala Leu Glu 1 5 10 Ala Asn Ile Asn Arg Arg Ala Ala Val Ser Leu Phe INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 1743 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N WO 96/06159 PCT/US95/10194 207 (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1743 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: ATG GCA GAA GGC GGT TTT GGA GCG GAC TCG GTG GGG CGC GGC GGA GAA 48 Met Ala Glu Gly Gly Phe Gly Ala Asp Ser Val Gly Arg Gly Gly Glu 1 5 10 AAG GCC TCT GTG ACT AGG GGA GGC AGG TGG GAC TTG GGG AGC TCG GAC 96 Lys Ala Ser Val Thr Arg Gly Gly Arg Trp Asp Leu Gly Ser Ser Asp 25 GAC GAA TCA AGC ACC TCC ACA ACC AGC ACG GAT ATG GAC GAC CTC CCT 144 Asp Glu Ser Ser Thr Ser Thr Thr Ser Thr Asp Met Asp Asp Leu Pro 40 GAG GAG AGG AAA CCA CTA ACG GGA AAG TCT GTA AAA ACC TCG TAC ATA 192 Glu Glu Arg Lys Pro Leu Thr Gly Lys Ser Val Lys Thr Ser Tyr Ile 55 TAC GAC GTG CCC ACC GTC CCG ACC AGC AAG CCG TGG CAT TTA ATG CAC 240 Tyr Asp Val Pro Thr Val Pro Thr Ser Lys Pro Trp His Leu Met His 70 75 GAC AAC TCC CTC TAC GCA ACG CCT AGG TTT CCG CCC AGA CCT CTC ATA 288 Asp Asn Ser Leu Tyr Ala Thr Pro Arg Phe Pro Pro Arg Pro Leu Ile 90 CGG CAC CCT TCC GAA AAA GGC AGC ATT TTT GCC AGT CGG TTG TCA GCG 336 Arg His Pro Ser Glu Lys Gly Ser Ile Phe Ala Ser Arg Leu Ser Ala 100 105 110 ACT GAC GAC GAC TCG GGA GAC TAC GCG CCA ATG GAT CGC TTC GCC TTC 384 Thr Asp Asp Asp Ser Gly Asp Tyr Ala Pro Met Asp Arg Phe Ala Phe 115 120 125 CAG AGC CCC AGG GTG TGT GGT CGC CCT CCC CTT CCG CCT CCA AAT CAC 432 Gln Ser Pro Arg Val Cys Gly Arg Pro Pro Leu Pro Pro Pro Asn His 130 135 140 CCA CCT CCG GCA ACT AGG CCG GCA GAC GCG TCA ATG GGG GAC GTG GGC 480 Pro Pro Pro Ala Thr Arg Pro Ala Asp Ala Ser Met Gly Asp Val Gly 145 150 155 160 TGG GCG GAT CTG CAG GGA CTC AAG AGG ACC CCA AAG GGA TTT TTA AAA 528 Trp Ala Asp Leu Gln Gly Leu Lys Arg Thr Pro Lys Gly Phe Leu Lys 165 170 175 ACA TCT ACC AAG GGG GGC AGT CTC AAA GCC CGT GGA CGC GAT GTA GGT 576 Thr Ser Thr Lys Gly Gly Ser Leu Lys Ala Arg Gly Arg Asp Val Gly 180 185 190 GAC CGT CTO AGG GAC GGC GGC TTT GCC TTT AGT CCT AGG GGC GTG AAA 624 Asp Arg Leu Arg Asp Gly Gly Phe Ala Phe Ser Pro Arg Gly Val Lys 195 200 205 TCT GCC ATA GGG CAA AAC ATT AAA TCA TGG TTG GGG ATC GGA GAA TCA 672 Ser Ala Ile Gly Gin Asn Ile Lys Ser Trp Leu Gly Ile Gly Glu Ser 210 215 220 TCG GCG ACT GCT GTC CCC GTC A AC ACG CAG CTT ATG GTA CCG GTG CAC 720 WO 96/06159 WO 9606159PCT[US95/10194 Ser 225

CTC

Leu

TAC

Tyr

GTG

Val

ATG

Met

CAC

His 305

ATA

Ile

ACC

Thr

GGG

Gly

GCA

Ala

CAT

Asp 385

GTG

Val

AGG

Arg

AGA

Ar;

TTG

Leu

ACA

Thr 465

GAG

Glu Ala

ATT

Ile

TTA

Leu

TGC

Cys

GTG

Val 290

CTG

Leu

TAC

Tyr

GCT

Al a

AGG

Arg

GTG

Val1 370

CAC

His

GTC

Val

GCG

Ala

GAA

Giu

CAC

Gin 450

AAT

Asn

GAA

Giu Thr

AGA

Arg

GAG

Giu

GGG

Gly 275

TAC

Tyr

ATG

Met

TCA

Ser

ATC

Ile

GC

Gly 355

GTC

Val

TTC

Phe

CC

Al a

AGG

Ar;

TTG

Leu 435

TAC

Tyr

ATT

Ile

ACT

Thr Ala

ACC

Thr

CCC

Cly 260

ATC

Ile

TCG

Trp

AAG

Lys

TGC

Cys

CTG

Leu 340

ACT

Thr

TTC

Phe

TTT

Phe

ATT

Ile

CGA

Gly 420

CC

Al a

ATC

Ile

CCC

Pro

TTG

Leu Val

CCT

Pro 245

GTA

Val

TTC

Leu

ACC

Thr

TCT

Ser

CAA

Gin 325

CCA

Arg

CAC

His

CCT

Pro

CAA

Gin

CTC

Leu 405

AGA

Arg

TCC

Trp

ACT

Thr

CAA

Clu

AAA

Lys 485 Pro 230

CTC

Val

ATC

Met

CCC

Pro

AG

Arg

GGT

Cly 310

AAC

Asn

ATC

Met

TCC

Trp,

CTC

Leu

TTA

Leu 390

ACC

Thr

AAG

Lys

CCT

Al a

CTG

Val

ATC

Ile 470

AAC

Asn Val

ACC

Thr

CCT

Gly

CAC

Gin

CCA

Ala 295

AAG

Lys

AAG

Lys

ATC

Met

TC

Cys

ATG

Met 375

CTT

Leu CT C Leu

AAC

Asn

TAT

Tyr

GAG

Clu 455

TGC

Cys

CTT

Leu Thr

CTG

Val

CTC

Val

GAG

Glu 280

TTT

Phe

CC

Al a

TTT

Phe

CAG

Gin

GTC

Val 360

CAC

His

TCC

Ser

TCC.

Ser

CAC

Asp

CAC

His 440

CAC

Gin

TTC

Phe

CAC

His Thr

CAC

Asp

CGC

Gly 265

AGA

Arg

ACA

Thr

CGA

Gly

TCC

Ser

CCC

Pro 345

TTT

Phe

CTG

Leu

ATC

Ile AG C Ser

CCC

Gly 425

GCC

Al a

ATG

Met

CC

Arg

GAG

Glu 208 Gin

TAC

Tyr 250

AAA

Lys

GTC

Val

CAT

Asp

GAC

Asp

CTC

Leu 330

TGG

Trp

CAT

Asp

AAC

Lys

TTT

Phe

CC

Al a 410

ACGC

Thr

GTG

Val

GTA

Val

AGC

Ser

CAG

Gin 490 Leu 235

AGG

Arg

TCA

Ser

ACA

Thr

TCT

Cys

CCC

Pro 315

CCC

Pro

AAC

Asn

AGG

Arg

CAC

His

AGA

Arg 395

GAG

Clu

CTG

Val

TAC

Tyr

CAA

Gin

GTG

Val 475

AC

Ser Met

AAT

Asn

ACG

Thr

ACT

Ser

TAC

Tyr 300

CTG

Leu

TTC

Phe

CTT

Val

CAT

His

GCC

Cly 380

CC

Al a

TCC

Ser

GAG

Clu

TCT

Cys

CTA

Leu 460

CC

Ar;

ATG

Met Val

GTT

Val1

CTC

Leu

TTT

Phe 285

AAC

Lys

ACC

Thr

CCC

Ar;

CCC

Cly

CTC

Leu 365

CC

Arg

ACA

Thr

TTG

Leu

CAA

Gin

TCA

Ser 445

TC

Cys

CTG

Leu

CTA

Leu Pro

TAT

Tyr

CTC

Val 270

CCC

Pro

CAA

Giu

TCT

Ser

ACG

Thr

CCT

Cly 350

CTC

Leu

CTA

Leu

CAA

Clu

CCC

Arg

AAC

Asn 430

TGC

Trp GT A Val1

CCA

Al a

CCT

Pro Val His 240 TTG CTT Leu Leu 255 AAC CC Asn Ala GAG CCC Clu Pro ATT TCC Ile Ser CCC AAA Ala Lys 320 AAC CC Asn Ala 335 CCC TCT Cly Ser TCC CCA Ser Pro TCT TTT Ser Phe CCC CAC Cly Asp 400 CCC CTC Arg Val 415 TAC ATC Tyr Ile ATC ATC Ile Met CAA ACC Gin Thr CAC AAG His Lys 480 ATG ATC Met Ile 495 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 WO 96/06159 WO 9606159PCTfUS95/10194

ACC

Thr

TGC

Cys

GAC

Asp

TAC

Tyr 545

TGG

Trp

ACA

Thr

GGT

Gly

TTT

Phe

GCG

Al a 530

AGG

Arg

CCA

Pro

TGC

Cys

GTA

Val

TGT

Cys 515

GAT

Asp

CAG

Gin

GCA

Ala

AGG

Arg

CTG

Leu 500

TTC

Phe

AAG

Lys

ATC

Ile

TTA

Leu

GTC

Val 580

CCC

Pro

ACA

Thr

CAC

His

TCC

Ser 550

AGC

Ser

GTG

Val

GAG

Glu

GAC

Asp 535

AAT

Asn

CAG

Gin

AGA

Arg

CTG

Leu 520

GAC

Asp

CCG

Pro

TCT

Ser

CAT

His 505

AGA

Arg

GTA

Val

GCT

Ala

AAA

Lys 209

CAT

His

AAA

Lys

TGC

Cys

ATT

Ile

GCA

Al a 570

CCC

Pro

TTA

Leu

GGC

Gly

AAA

Lys 555

OTT

Val

GTG

Val

TTT

Phe 525

TGG

Trp

AGG

Arg

CA--

His

GAG

Glu

GTA

Val

GAA

Glu

ATC

Ile

GAG

Glu 575

CTT

Leu

GCC

Ala

ATC

Ile

AAC

Asn 560

GAG

Glu 1536 1584 1632 1680 1728 1743 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 580 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ala Glu Gly Lys Asp Glu Tyr Asp Arg Thr Gln Pro 145 Trp Al a Glu Glu Asp Asn His Asp) Ser 130 Pro Al a Ser Ser Arg Val Ser Pro Asp 115 Pro Pro Asp Val1 Ser Lys Pro Leu Ser 100 Asp Arg Al a Leu Gly Th Thr Pro Thr Tyr Glu Ser Val1 Thr Gln Arg Ser Leu Val 70 Al a Lys Gly Cys Arg 150 Gly Phe Gly Ala Asp Ser Val Gly Arg Gly Gly Thr Thr 55 Pro Thr Gly Asp Gly 135 P ro Leu Gly Thr 40 Gly Thr Pro Ser Tyr 120 Arg Ala Lys Arg 25 Ser Lys Ser Arg Ile 105 Ala Pro Asp Arg Trp, Thr Ser Lys Phe 90 Phe Pro Pro Al a Thr Asp Asp Val Pro 75 Pro Al a Met Leu Ser 155 Pro Leu Met Lys Trp Pro Ser Asp Pro 140 Met Lys Oly Asp Thr His Arg Arg Arg 125 Pro Gly Gly Ser Asp Ser Leu Pro Leu 110 Phe Pro Asp Phe Gly Ser Leu Tyr Met Leu Ser Ala Asn Val Leu Glu Asp Pro Ile His Ile Al a Phe His Gly 160 Lys WO 96/06159 PCTIUS95/10194 210 Thr Asp Ser Ser 225 Leu Tyr Val Met His 305 Ile Thr Gly Ala Asp 385 Val Arg Arg Leu Thr 465 Glu Thr Cys Se2 ArS Ala 210 Ala Ile Leu Cys Va1 290 Leu Tyr Ala Arg Val 370 His Va1 Ala lu G m 450 Asr.

Glu Gly Phe Thr Leu 195 Ile Thr Arg Glu Gly 275 Tyr Met Ser Ile Gly 355 Val Phe Ala Arg Leu 435 Tyr ile Thr Val Cys 515 Lys 180 Arg Gly Ala Thr Gly 260 Ile Trp Lys Cys Leu 340 Thr Phe Phe Ile Gly 420 Ala Ile Pro Leu Ueu 2 500 Phe I 16E Glj Asp Gin Val Pro 245 Va1 Leu Thr Ser Gin 325 Arg His Pro Gin Leu 405 Arg rrp Thr lu Lys 485 ksp ?he Gly Gly Asn Pro 230 Val Met Pro Arg Gly 310 Asn Met Trp Leu Leu 390 Thr Lys Ala Val Ile 470 Asn Pro Thr C Ser Gly Ile 215 Val Thr Gly Gin Ala 295 Lys Lys Met Cys Met 375 Leu Leu Asn Tyr lu 455 :ys eu Jal lu Leu Phe 200 Lys Thr Val Val Glu 280 Phe Ala Phe Gin Val 360 His Ser Ser Asp His 440 Gin I Phe 2 His Arg I Leu 520 Lys 185 Ala Ser Thr Asp Gly 265 Arg Thr Gly Ser Pro 345 Phe Leu Ile Ser Giy 425 kla Olet Lrg lu lis I j05 ~rg I 170 Ala Phe Trp Gin Tyr 250 Lys Va1 Asp Asp Leu 330 Trp Asp Lys Phe Ala 410 Thr Val Val Ser 3mn 490 iis ys Arg Ser Leu Leu 235 Arg Ser Thr Cys Pro 315 Pro Asn Arg His Arg 395 Glu Val Tyr Gin Val 2 475 Ser I Pro Leu C GlI Prc Glj 22C Met Asr Thr Ser Tyr 300 Leu Phe Val His Gly 380 Ala Ser 31u :ys eu 160 krg 'et lal ;In i Arg Arg 205 Ile Val Val Leu Phe 285 Lys Thr Arg Gly Leu 365 Arg Thr Leu Gin Ser 445 Cys Leu Leu Val Phe 525 Asl 19C Glj Gl Prc Tyr Val 270 Pro Glu Ser Thr Gly 350 Leu Leu Glu Arg Asn 430 Trp Val Ala Pro lie 510 lie 175 Val r Val Glu Val Leu 255 Asn Glu Ile Ala Asn 335 Gly Ser Ser Gly Arg 415 Tyr Ile Gin His Met 495 Glu Val 4 Gly Lys Ser His 240 Leu Ala Pro Ser Lys 320 Ala Ser Pro Phe Asp 400 Val Ile Mlet Thr Lys 480 lie eu la WO 96/06159 PCT/US95/10194 211 Asp Ala Asp Lys Phe His Asp Asp Val Cys Gly Leu Trp Thr Glu Ile 530 535 540 Tyr Arg Gin Ile Leu Ser Asn Pro Ala Ile Lys Pro Arg Ala Ile Asn 545 550 555 560 Trp Pro Ala Leu Glu Ser Gin Ser Lys Ala Val Asn His Leu Glu Glu 565 570 575 Thr Cys Arg Val 580 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 2193 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..2193 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: ATG CAG GGT CTA GCC TTC TTG GCG GCC CTT GCA TGC TGG CGA TGC ATA 48 Met Gin Gly Leu Ala Phe Leu Ala Ala Leu Ala Cys Trp Arg Cys Ile 1 5 10 TCG TTG ACA TGT GGA GCC ACT GGC GCG TTG CCG ACA ACG GCG ACG ACA 96 Ser Leu Thr Cys Gly Ala Thr Gly Ala Leu Pro Thr Thr Ala Thr Thr 25 ATA ACC CGC TCC GCC ACG CAG CTC ATC AAT GGG AGA ACC AAC CTC TCC 144 Ile Thr Arg Ser Ala Thr Gin Leu Ile Asn Gly Arg Thr Asn Leu Ser 40 ATA GAA CTG GAA TTC AAC GGC ACT AGT TTT TTT CTA AAT TGG CAA AAT 192 Ile Glu Leu Glu Phe Asn Gly Thr Ser Phe Phe Leu Asn Trp Gin Asn 55 CTG TTG AAT GTG ATC ACG GAG CCG GCC CTG ACA GAG TTG TGG ACC TCC 240 Leu Leu Asn Val Ile Thr Glu Pro Ala Leu Thr Glu Leu Trp Thr Ser 70 75 GCC GAA GTC GCC GAG GAC CTC AGG GTA ACT CTG AAA AAG AGG CAA AGT 288 Ala Giu Val Ala Glu Asp Leu Arg Val Thr Leu Lys Lys Arg Gin Ser 90 CTT TTT TTC CCC AAC AAG ACA GTT GTG ATC TCT GGA GAC GGC CAT CGC 336 Leu Phe Phe Pro Asn Lys Thr Val Val Ile Ser Gly Asp Gly His Arg 100 105 110 TAT ACG TGC GAG GTG CCG ACG TCG TCG CAA ACT TAT AAC ATC ACC AAG 384 Tyr Thr Cys Glu Val Pro Thr Ser Ser Gin Thr Tyr Asn Ile Thr Lys 115 120 125 GGC TTT AAC TAT AGC GCT CTG CCC GGG CAC CTT GGC GGA TTT GGG ATC 432 WO 96/06159 WO 96/6 159PCTIUS95/10194 212 Gly

AAC

Asn 145

TTC

Phe

ATG

Met

OCG

Ala

AAC

Asn

CTC

Leu 225

CTC

Leu

AAA

Lys

CTO

Leu

ACC

Thr

GCO

Ala 305

TTT

Phe

TGC

Cys

COT

Arg

CTT

Leu

TTGC

Leu 385 Phe 130

GCG

Ala

GCG

Al a

GCC

Al a

CTC

Leu

AGG

Arg 210

CCG

Pro

ACG

Thr

GAT

Asp

AAC

Asn

AGG

Arg 290

GCG

Aa

CTT

Leu

"TG

,7ai ST G Aeu 3G C "1v ;cc l a Asn

CGT

Arg

AGG

Arg

GTC

Val

TAT

Tyr 195

TCC

Ser

OAT

Asp

TTC

Phe

TCT

Ser

ATG

Met 275

ATC

Ile

CCO

Pro

OTT

Val1

TOT

Cys

TOC

Cys 355

OCT

Ala

AGT

Ser Tyr

CTG

Leu

GAC

Asp

AAG

Lys 180

OGA

Gly

AAA

Lys

TCA

Ser 0CC Al a

TAC

Tyr 260

ACG

Thr

OTA

Val

GAC

Asp

GCG

Ala

COO

Arg 340

ATG

Met

OTO

Val1

TTO

Leu *Ser

GTA

*Val

ACC

Thr 165

TTT

Phe

GTG

Val

GAG

Glu

CTG

Leu

CGA

Arg 245

CAG

Oln

GAG

Glu

TCA

Ser

ATA

Ile

CG

Arg 325

CAG

Gin

CCC

Pro 0CC Ala

CCC

Pro Ala

CTG

Leu 150

CCA

*Pro

*TCC

Ser

OTO

Val

OCT

Ala

CCA

Pro 230

AAC

Asn

ACA

Thr

TCG

Ser

ATC

Ile

TTC

Phe 310

GOC

Gly

TAT

Tyr

ACO

Thr 0CC Al a

CGC

Arg 390 Leu 135

GT

Oly

GAG

Olu

ATA

Ile

TCO

Ser

AAC

Asn 215

TCT

Ser

OCA

Ala

CTC

Leu

ACO

Thr

GAO

Giu 295

TTO

Leu

ATT

Ile

OCO

Ala

TTC

Phe

ACA

Thr 375

TCT

Ser Pro

OAT

Asp

TAT

Tyr

TCC

Ser

GAA

Olu 200

GAG

Oiu

CTO

Leu

AAA

Lys

CTT

Leu

CCC

Pro 280 0CC Ala

OTO

Val 0CC Ala

GAA

Glu

ACC

Thr 360

CAA

Gln

TCC

Ser Gly His Leu Gly 140 ATC TTC OCA TCA Ile Phe Ala Ser 155 COO OTO TTT TAC Arg Val Phe Tyr 170 ATT GOC AAC AAC Ile Gly Asn Asn 185 OAT TTC OTO GTC Asp Phe Val Val ACG OCO TCC CAT Thr Ala Ser His 220 AAG GGC CAT 0CC Lys Gly His Ala 235 TAT OCO CTA GTO Tyr Ala Leu Val 250 ACA GAG AAT TAC Thr Giu Asn Tyr 265 CTC GAG TTC ACO Leu Glu Phe Thr AGO COC 0CC TOC Arg Arg Ala Cys 300 TTG TTT CAG ATO Leu Phe Gin Met 315 GAO CAC COA TTT Giu His Arg Phe 330 CTG TAT TTT CTC Leu Tyr Phe Leu 345 ACT GTC 000 TAT Thr Val Gly Tyr ATA OCT CGC OTG Ile Ala Arg Val 380 CAG GAA ACA OTG Gin Glu Thr Val 395 Oly Phe Gly Ile

AAA

Lys

CCA

Pro

GAO

Glu

GTC

Val 205

CTT

Leu

ACC

Thr

OCO

Ala

ACT

Thr

CG

Arg 285

OCA

Al a

TTG

Leu

GTG

Val

CGC

Arg

AAC

Asn 365

I'CC

Ser

CTG

Lieu

*TGG

Trp

ATG

Met

TCC

Ser 190

ACG

Thr

CTG

Leu

TAT

Tyr

ATC

Ile

CGC

Arg 270

ACG

Thr

OCT

Al a

GTG

Val

GAO

Giu

CGC

Arg 350

CAC

His

GCC

Ala 0CC2 Ala

TCC

Ser

AAI

Asn 175

GOC

Gly

CTC

Leu

TTC

Phe

OAT

Asp

CTG

Leu 255

ATA

Ile

ATC

Ile

CAA

Gin

GCA

Al a

GTG

Val 335

PSTC

Ile

A.CC

rhr

%CG

rhr

TG

4et

CTA

*Leu 160

GTC

Val

GTA

*Val

CAC

*His

GOT

Oly

GAA

Giu 240

CCT

Pro

TTT

Phe

CAG

Gin

GAG

Glu

CAC

His 320

GAC

Asp

TCO

Ser

ACC

Thr

AAG

Lys

GTC

Val1 400 480 528 576 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 WO 96/06159 WO 96/6 159PCTIUS95/10194 213

OCT

Pro CAG CTT GO GOC CGT GAT GGC GOC GTO Gin Leu Gly Ala Arg Asp Gly Ala Val 405 TCO TOO ATT OTG GAG GGC Ser Ser Ile Leu Giu Gly 410 415

ATT

Ile

ACA

Thr

CTO

Leu

CTG

Leu 465 000 Gly

AGO

Arg 0CC Ala

GCC

Al a

CAC

His 545

ACA

Thr

ATC

le

GAG

Giu

TOO

ON's

TAO

Tvr 625

ATC

le

GC'

CTC

Lei.

ACC

Thr 450

AGA

Arg

GAA

Glu

GCA

Al a

AAG

Lys

GTT

Val 530

CTO

Leu

GC

Al a

A.TO

Ile

A.TC

Ile ocC Ser 610 ksni vlt

ATG

Met

GGO

Glv 435

GAO

Asp

ACA

*Thr

*ATG

Met

TTO

Phe

TTG

Leu 515

GC

Ala

GAT.

Asp

GAO

Asp

ACT

Ser

TTC

Phe 595 000 Gly ATC I Ile

AGO

Ser 'I

GTC

Val 420

GATI

Asp

AGO

Ser

TAT

Tyr AT C Ile

TOO

Ser 500

OGO

Arg

AGA

Arg

AGO

S er

AAG

Lys Ser 580

CTC

,eu

PTT

?he er

'AC

7yr

GTC

Val

ACT

Thr

TGC

Oys

TTG

Leu 000 Al a 485 000 Pro 000 Al a

GGA

Oly

TTA

Leu

ATA

Ile 565

GAA

Giu

AAG

Lys

AAO

Asn

ACA

Thr

GAT

Asp 645

GAA

Oiu

GAA

Giu 000 Pro

ATG

Met 470

OGO

Arg

TGO

Oys

GAG

Giu

ACA

Thr

AAT

Asn 550

ATA

Ile

OOA

Ala

AGT

Ser

TTT

Phe OOA2 Pro2 630 GAG2 Glu

CAT

His

AGA

Arg 000 Pro 455

TTO

Phe

TTT

Phe

TTT

Phe

GOG

Al a

TOG

Ser 535

TTA

Leu

GCT

Al a CT0 Leu G00 ki a

POT

Ser 615

.GA

rg 1GC er

ATC

Met

AA

*Lys 440C Lys

ACA

Thr

TOO

Ser

OTA

Leu

OOG

Pro 520

GGA

Gly

ATT

Ile

AOG

Thr

TOG

Ser

ATG

Met 600

OAG

Gin

AGA

Arg

GAT

Asp

TAT

Tyr 425

TTA

Leu

GAO

Asp

TOA

Ser

AAA

Lys

GGA

Gly 505

OAG

Gin

TTO

Phe

OOG

Pro

GTA

Val

AAO

Asn 585

TTT

Phe

ATT

Ile

GT

Gly

GGO

Gly

ACC

Thr

ATG

Met

TOC

Ser

ATG

Met 000 Pro 490

OTA

Leu

TOG

Ser

GOA

Ala 000 Al a 000 Pro 570

GOT

Al a

ATA

Ile

GAT

ksp

TGO

7TG eu 550

GOO

Ala

TTG

Leu

OGA

Gly

TGT

Oys 475

GAO

Asp

AGO

Arg

TOO

Ser

GAA

Glu

ATT

Ile 555

TTG

Leu

OTT

Val

TOT

Ser

AGO

Arg 00C Pro 1 635 CAG 'I Gin S

TAC

Tyr

GAC

Asp

GTA

Val 460

ACC

Thr

AGO

Ser

TAO

Tyr

GOT

Aila

TTG

Leu 540

AAO

Asn

COT

Pro GT0 Val

GCT

a

:AO

lis 520

'TT

.eu

COT

~er

ACT

*Thr

ATA

Ile 445

*TOA

Ser AA2 Asr.

OTT

Leu

OAT.

Asp

OTO

Leu 525

OTO

Leu

TOT

Oys

CAC

His

TAO

Tyr ATO 2 Ile 605

ATTC

le I TGT C Ox's I

OTO

Leu ly

TA~

Ty 43(

CA(

HPE

GA

AT;

Ile

AAC

Asn

TTG

Leu 510

AOO

Thr

CAC

His

TOA

Ser GT0 Val

GAG

Glu 390 ?ro

;AC

~SD

TG

e t r OTO :Val

AOG

Thr

AAO

1Lys

GAG

Oiu

ATO

le 495

OAT

His 000 Arg 000 Al a

AAG

Lys

AOG

Thr 575

OTO

Val 000 Pro

ATA

Ile

TOT

Ser

TAT

Tyr 655

TAO

Tyr

GTO

Val

OTA

Leu

OTG

Leu 480

TAT

Tyr

OOA

Pro

ACT

Thr

OTO

Leu

ATT

Ile 560

TAT

Tyr

TOO

Ser

GAT

Asp

GTC

Val

GTA

Val 640 "T0 Val 1248 1296 1344 13 92 1440 1488 1536 1584 1632 1680 1728 1776 1824 1872 1920 1968 2016 ACT AAT OAA AGO OTO OAO ACC AAO OTO TTT TTA OAT AAG TOA OCT TTC Thr Asn Oiu Arg 660 Val Gin Thr Asn Leu Phe Leu Asp Lys Ser Pro Phe WO 96/06159 WO 9606159PCTIUS95/10194 214 TTT GAT AAT AAC AAC CTA CAC ATT CAT TAT TTG TGG CTG AGG GAC AAC Phe Asp Asn Asn Asfl Leu His Ile His Tyr Leu Trp Leu Arg Asp Asn 675 680 685 GGG ACC GTA GTG GAG ATA AGG GGC ATG TAT AGA AGA CGC GCA GCC AGT Gly Thr Val Val Glu Ile Arg Gly Met Tyr Arg Arg Arg Ala Ala Ser 690 695 700 GCT TTG TTT CTA ATT CTC TCT TTT ATT GGG TTC TCG GGG GTT ATC TAC Ala Leu Phe Leu Ile Leu Ser Phe Ile Gly Phe Ser Gly Val Ile Tyr 705 710 715 720 TTT CTT TAC AGA CTG TTT TCC ATC CTT TAT TAG Phe Leu Tyr Arg Leu Phe Ser Ile Leu Tyr 725 730 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 730 amino acids TYPE: amino acid TOPOLOGY: linear 2064 2112 2160 2193 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Met Gin Gly Leu Ala Phe Leu Ala Ala Leu Ala Cys Trp Arg Cys Ile Ser Ile Ile Leu Al a Leu Tyr Gly Asn 145 Phe Met Leu Thr Giu s0 Leu Glu Phe Thr Phe 130 Ala Al a Ala Thr Arg Leu Asn Val Phe Cys 115 Asn Arg Arg Val Cys Ser Glu Val Ala Pro 100 Glu Tyr Leu Asp Lys 180 Gly Al a Phe Ile Giu Asn Val Ser Val Thr 165 Phe Ala Thr Asn Thr 70 Asp Lys Pro Ala Leu 150 Pro Ser Thr Gin Gly 55 Giu Leu Thr Thr Leu 135 Gly Glu Ile Gly Leu 40 Thr Pro Arg Val Ser 120 Pro Asp Tyr Ser Ala 25 Ile Ser Ala Val Val 105 Ser Gly Ile Arg Ile 185 Leu Asn Phe Leu Thr Ile Gin His Phe Val 170 Gly Pro Gly Phe Thr 75 Leu Ser Thr Leu Al a 155 Phe Thr Arg Leu Glu Lys Gly Tyr Gly 140 Ser Tyr Asn Thr Thr Asn Leu Lys Asp Asn 125 Gly Lys Pro Glu Ala Asn Trp Trp Arg Gly 110 Ile Phe Trp Met Ser 190 Thr Leu Gin Thr Gin His Thr Gly Ser Asn 175 Gly Thr Ser Asn Ser Ser Arg Lys le Leu 160 Val1 Val Ala Leu Tyr 195 AlaLeuTyrGly Val Val Ser Glu Asp Phe Val Val Val Thr Leu His 195200 205 WO 96/06159 PCT/US95/10194 215 Asn Arg Ser Lys Glu Ala Asn Glu Thr 210 215 Leu 225 Leu Lys Leu Thr Ala 305 Phe Cys Arg Leu Leu 385 Gin Ile Thr Leu Leu 465 Gly Arg Ala Ala His 545 Pro Thr Asp Asn Arg 290 Ala Leu Val Leu Gly 370 Ala Leu Ala Leu Thr 450 Arg Glu Ala Lys Val 530 Leu Asp Phe Ser Met 275 Ile Pro Val Cys Cys 355 Ala Ser Gly Met Gly 435 Asp Thr Met Phe Leu 515 Ala Asp Ser Ala Tyr 260 Thr Val Asp Ala Arg 340 Met Val Leu Ala Val 420 Asp Ser Tyr Ile Ser 500 Arg Arg Ser Leu Arg 245 Gin Glu Ser Ile Arg 325 Gin Pro Ala Pro Arg 405 Val Thr Cys Leu Ala 485 Pro Ala Gly Leu Pro 230 Asn Thr Ser Ile Phe 310 Gly Tyr Thr Ala Arg 390 Asp Glu Glu Pro Met 470 Arg Cys Glu Thr Asn Ser Ala Leu Thr Glu 295 Leu Ile Ala Phe Thr 375 Ser Gly His Arg Pro 455 Phe Phe Phe Ala Ser 535 Leu Leu Lys Leu Pro 280 Ala Val Ala Glu Thr 360 Gin Ser Ala Met Lys 440 Lys Thr Ser Leu Pro 520 Gly Ile Lys Tyr Thr 265 Leu Arg Leu Glu Leu 345 Thr Ile Gin Val Tyr 425 Leu Asp Ser Lys Gly 505 Gin Phe Pro Ala Gly Ala 250 Glu Glu Arg Phe His 330 Tyr Val Ala Glu Pro 410 Thr Met Ser Met Pro 490 Leu Ser Ala Ala Ser His 235 Leu Asn Phe Ala Gin 315 Arg Phe Gly Arg Thr 395 Ser Ala Leu Gly Cys 475 Asp Arg Ser 31u Ile His 220 Ala SVal Tyr Thr Cys 300 Met Phe Leu Tyr Val 380 Val Ser Tyr Asp Val 460 Thr Ser Tyr Ala Leu 540 Asn Leu Thr Ala Thr Arg 285 Ala Leu Val Arg Asn 365 Ser Leu Ile Thr Ile 445 Ser Asn Leu Asp Leu 525 Leu Cys Leu Tyr Ile Arg 270 Thr Ala Val Glu Arg 350 His Ala Ala Leu Tyr 430 His Glu Ile Asn Leu 510 Thr His Ser SPhe Asp Leu 255 Ile Ile Gin Ala Val 335 Ile Thr Thr Met Glu 415 Val Thr Lys Glu Ile 495 His Arg Ala Lys Gly Glu 240 Pro Phe Gin Glu His 320 Asp Ser Thr Lys Val 400 Gly Tyr Val Leu Leu 480 Tyr Pro Thr Leu Ile 560 550 555 Thr Ala Asp Lys Ile Ile Ala Thr Val Pro Leu Pro His Val Thr Tyr 1 WO 96/06159 PCT/US95/10194 216 565 570 575 Ile Ile Ser Ser Glu Ala Leu Ser Asn Ala Val Val Tyr Glu Val Ser 580 585 590 Glu Ile Phe Leu Lys Ser Ala Met Phe Ile Ser Ala Ile Lys Pro Asp 595 600 605 Cys Ser Gly Phe Asn Phe Ser Gin Ile Asp Arg His Ile Pro Ile Val 610 615 620 Tyr Asn Ile Ser Thr Pro Arg Arg Gly Cys Pro Leu Cys Asp Ser Val 625 630 635 640 Ile Met Ser Tyr Asp Glu Ser Asp Gly Leu Gin Ser Leu Met Tyr Val 645 650 655 Thr Asn Glu Arg Val Gin Thr Asn Leu Phe Leu Asp Lys Ser Pro Phe 660 665 670 Phe Asp Asn Asn Asn Leu His Ile His Tyr Leu Trp Leu Arg Asp Asn 675 680 685 Gly Thr Val Val Glu Ile Arg Gly Met Tyr Arg Arg Arg Ala Ala Ser 690 695 700 Ala Leu Phe Leu Ile Leu Ser Phe Ile Gly Phe Ser Gly Val Ile Tyr 705 710 715 720 Phe Leu Tyr Arg Leu Phe Ser Ile Leu Tyr 725 730 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 1215 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1215 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ATG TTA CGA GTT CCG GAC GTG AAG GCT AGT CTA GTA GAG GGC GCG GCG 48 Met Leu Arg Val Pro Asp Val Lys Ala Ser Leu Val Glu Gly Ala Ala 1 5 10 CGC CTG TCG ACA GGC GAG CGC GTG TTT CAC GTC TTG ACC TCT CCG GCG 96 Arg Leu Ser Thr Gly Glu Arg Val Phe His Val Leu Thr Ser Pro Ala 25 GTG GCG GCC ATG GTG GGA GTC TCT AAT CCT GAA GTC CCG ATG CCA CTG 144 Val Ala Ala Met Val Gly Val Ser Asn Pro Glu Val Pro Met Pro Leu 40 TTG TTC GAA AAG TTT GGG ACT CCG GAC TCG TCT ACC CTG CCA CTC TAC 192 WO 96/06159 WO 9606159PCTIUS95/10194 217 Leu

GCG

Al a

CAC

His

TCT

Ser

TTC

Phe

GAC

AspD

CGT

Arg 145

GCC

Ala

CAC

His

AAC

Asn

AAC

Asn

CTC

Leu 225

CGT

Arg

CTA

Leu

GAA

Glu CCTr pro Phe

GCT

Ala

CCC

Pro

CTT

Leu

GAG

Glu

CAG

Gln 130

CGC

Arg

CC

Ala

CCC

Pro

ATC

Ile

GAG

Glu 210

GGA

Gly G C T Ala

CAA

Gln

AAC

Asr.

GCG

Ala 290 Glu Lys Phe Gly Thr Pro Asp Ser Ser Thr Leu Pro Leu Tyr

AGG

Arg

TAC

Tyr

GGC

Glv

GAC

Asp 115

TCT

Ser

GTC

Val1

AAC

Asn

ATA

Ile

GCA

Ala 195

GCC

Al a

GAA

Glu

AAC

Asn

CAC

His5

A.TC

11le 275 Lys

CAC

His

GCG

Ala

GTT

Val1 100

ACG

Thr

TAT

Tyr

CCC

Pro

CGC

Arg

ACA

Thr 180

CAG

Gln

GCG

Ala

AGC

Ser

AAC

Asn

CTG

Leu 260

GTG

Val

ACA

T hr

CCG

Pro

TTA

Leu

TAC

Tyr

CAG

Gln

ACC

Thr

ATA

Ile

ATA

Ile 165

CCC

Pro

GTT

Val

TCT

Ser

CCG

Pro

TCG

Ser 245

TTC

Phe

I'CG

Ser

CAC

H~is

GAA

Glu 70

AGA

Arg

CTG

Leu

ATA

Ile

AAC

Asn

CAC

His 150

AAG

Lys

CGT

Arg

TGC

Cys

ATG

Met

GTC

Val 230

ATA

Ile

CTG

Leu

TGT

Cys

GAG

Glu 55

CTA

Leu

AGC

Ser

CAC

His

CTA

Leu

TTT

Phe 135

CC

Al a

GTG

Val

GCC

Al a

GAA

Glu

TTC

Phe 215

TGT

Cys

ACT

Thr

AAG

Lys

TTC

Phe

CGG

Arg 295

TCG

Ser

CAC

His

TCC

Ser

CCG

Pro 120

AAG

Lys

GCG

Al a

TTT

Phe

GGT

Gly

CGG

Arg 200

TAC

Tyr

GAC

Asp

TTT

Phe

CAC

His

TCA

Ser 280

GAG

Glu

TTG

Leu

TTG

Leu

AAG

Lys 105

GAG

Glu

ATT

Ile

AAC

Asn

GAC

Asp

CAG

Gln 185

GAT

Asp

ATG

Met

TTC

Phe

CTA

Leu

GTG

Val 265

TCG

Ser

TTC

Phe C TP Leu

TGC

Cys 90

CCA

Prc

TGC

Cys

ATA

Ile

AAG

Lys

CCA

Pro 170

ACC

Thr

ATC

Ile

ATT

Ile

AAC

Asn

CCC

Pro 250

TTG

Leu

CTG

Leu

FTC

Phe

CGG

1Arg 75

GTA

*Val

-GTC

*Val

CGG

Arg

GAT

Asp

CGT

Arg 155

GAG

Glu

AGA

Arg

GTG

Val

GGA

Gly

ACC

Thr 235

AAG

Lys

CTG

Leu

TAC

Tyr

GGC

Gly

ATC

I le

GGC

Gly

GTA

Val

CTG

Leu

CTG

Leu 140

GTC

Val

TCG

Ser

TCT

Ser

TCA

Ser

CTC

Leu 220

GTT

Val

CTA

Leu

CC

Arg

GGC

Gly 3CT kl a 300

ATC

Met

GA-Z

*Glu

*CGC

Arg

GCC

Al a 125

CCA

Pro

GTC

Val

CCT

Pro

ATA

Ile

CTT

Leu 205

AG

Arg

ACC

Thr

AAA

Lys

AGC

Ser

GCA

Al a 285

CTG

Leu

CTC

Leu

GAG

Glu

GGC

Gly 110

ATA

Ile

GCG

Ala

ATC

Ile

TTA

Leu

CTG

Leu 190

AAC

Asn

CGG

Arg

ATC

Ile

CTG

Leu

ATG

Met 270

GAA

Glu

CTA

Leu T C.

Ser

ACC

Thr

CAC

His

ACG

Thr

GGA

Gly

GAC

Asp

CCG

Pro 175

AAA

Lys

ACA

Thr

CCG

Pro

ATG

Met 1AAC Asn 255

GGG

Gly

CTT

Leu 3AA "lu

CCG

Pro

GCA

*Ala

GAA

Glu

AC

Ser

TGC

Cys

GAG

Glu 160

CGT

Arg

CAC

His

GAC

Asp

AGA

Arg

GAG

Glu 240

CCC

Arg

CTG

Leu

GCC

Ala

AGA

Arg 240 288 336 384 432 480 528 576 624 672 720 768 816 864 912 960 CTC AAA CGT CGG GTG GAG GAC GCG GTC TTC TGC CTG AAT ACC ATA GAG Leu 305 Lys Arg Arg Val Glu Asp Ala Val Phe Cys 315 Leu Asn Thr Ile Glu 320 WO 96/06159 PCT/US95/10194 218 GAT TTC CCG TTT AGG GAA CCC ATT CGC CAA CCC CCA GAT TGT TCC AAG 1008 Asp Phe Pro Phe Arg Glu Pro Ile Arg Gin Pro Pro Asp Cys Ser Lys 325 330 335 GTG CTT ATA GAA GCC ATG GAA AAG TAC TTT ATG ATG TGT AGC CCC AAA 1056 Val Leu Ile Glu Ala Met Glu Lys Tyr Phe Met Met Cys Ser Pro Lys 340 345 350 GAC CGT CAA AGC GCC GCA TGG CTA GGT GCA GGG GTG GTC GAA CTG ATA 1104 Asp Arg Gin Ser Ala Ala Trp Leu Gly Ala Gly Val Val Glu Leu Ile 355 360 365 TGT GAC GGC AAT CCA CTT TCT GAG GTG CTC GGA TTT CTT GCC AAG TAT 1152 Cys Asp Gly Asn Pro Leu Ser Glu Val Leu Gly Phe Leu Ala Lys Tvr 370 375 380 ATG CCC ATA CAA AAA GAA TGC ACA GGA AAC CTT TTA AAA ATC TAC GCT 1200 Met Pro Iie Gin Lys Glu Cys Thr Gly Asn Leu Leu Lys Ile Tyr Ala 385 390 395 400 TTA TTG ACC GTC TAA 1215 Leu Leu Thr Val INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 404 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Met Leu Arg Val Pro Asp Val Lys Ala Ser Leu Val Glu Gly Ala Ala 1 5 10 Arg Leu Ser Thr Gly Glu Arg Val Phe His Val Leu Thr Ser Pro Ala 25 Val Ala Ala Met Val Gly Val Ser Asn Pro Glu Val Pro Met Pro Leu 40 Leu Phe Glu Lys Phe Gly Thr Pro Asp Ser Ser Thr Leu Pro Leu Tyr 55 Ala Ala Arg His Pro Glu Leu Ser Leu Leu Arg Ile Met Leu Ser Pro 70 75 His Pro Tyr Ala Leu Arg Ser His Leu Cys Val Gly Glu Glu Thr Ala 90 Ser Leu Gly Val Tyr Leu His Ser Lys Pro Val Val Arg Gly His Glu 100 105 110 Phe Glu Asp Thr Gin Ile Leu Pro Glu Cys Arg Leu Ala Ile Thr Ser 115 120 125 Asp Gin Ser Tyr Thr Asn Phe Lys Ile Ile Asp Leu Pro Ala Gly Cys 130 135 140 Arg Arg Val Pro Ile His Ala Ala Asn Lys Arg Val Val Ile Asp Glu 145 150 155 160 Ala Ala Asn Arg Ile Lys Val Phe Asp Pro Glu Ser Pro Leu Pro Arg WO 96/06159 PCT/US95/10194 219 His Asn Asn Leu 225 Arg Leu Glu Pro Leu 305 Asp Val Asp Cys Met 385 Leu Pro Ile Glu 210 Gly Ala Gin Asn Ala 290 Lys Phe Leu Arg Asp 370 Pro Leu Ile Thr 180 Ala Gin 195 Ala Ala Glu Ser Asn Asn His Leu 260 Ile Val 275 Lys Thr Arg Arg Pro Phe Ile Glu 340 Gin Ser 355 Gly Asn Ile Gin Thr Val Pro Arg Ala Gly Val Ser Pro Ser 245 Phe Ser His Val Arg 325 Ala Ala Pro Lys Cys Met Val 230 Ile Leu Cys Glu Glu 310 Glu Met Ala Leu Glu 390 Glu Arg 200 Phe Tyr 215 Cys Asp Thr Phe Lys His Phe Ser 280 Arg Glu 295 Asp Ala Pro Ile Glu Lys Trp Leu 360 Ser Glu 375 Gin Thr 185 Asp Ile Met Ile Phe Asn Leu Pro 250 Val Leu 265 Ser Leu Phe Phe Val Phe Arg Gin 330 Tyr Phe 345 Gly Ala Val Leu Arg Val Gly Thr 235 Lys Leu Tyr Gly Cys 315 Pro Met Gly Gly Ser Ser Leu 220 Val Leu Arg Gly Ala 300 Leu Pro Met Val Phe Ile Leu 205 Arg Thr Lys Ser Ala 285 Leu Asn Asp Cys Val 365 Leu Leu 190 Asn Arg Ile Leu Met 270 Glu Leu Thr Cys Ser 350 Glu Ala Lys Thr Pro Met Asn 255 Gly Leu Glu Ile Ser 335 Pro Leu Lys His Asp Arg Glu 240 Arg Leu Ala Arg Glu 320 Lys Lys Ile Tyr Ala 400 380 Cys Thr Gly Asn Leu 395 Leu Lys Ile Tyr INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 2259 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..2259 OTHER INFORMATION: WO 96/06159 WO 9606159PCTIUS95/10194 220 (xi) SEQUENCE DESCRIPTION: SEQ ID ATG GCA GCG CTC GAG GGC CCC CTA CTA CTG CCA CCG AGC Met Ala Ala Leu Glu Gly Pro Leu Leu Leu Pro Pro Ser GCC TCC CTG Ala Ser Leu 1

ACG

Thr

CTG

Leu

CTG

Leu

TTC

Phe

CCT

Pro

TAC

Tyr

GGA

Gly

CTT

Leu

CTG

Leu 145

TTC

Phe

TTT

Phe

TAT

Tyr

TCA

Ser

TTT

P'ne 225

GTC

Val

ACG

Thr

GAA

Glu

ACC

Thr 50

GCC

Al a

TTT

Phe

AGC

Ser

CAG

Gln

ACC

Thr 130

TAT

Tyr

AGG

Arg

CAT

His

CAA

Gln

ATA

Ile 210

A.AC

Asn

TCG

Ser

AGT

Ser

ATA

Ile 35

TTA

Leu

TGC

Cys

ATA

Ile

GGC

Gly

ATA

Ile 115

GAT

Asp

CTT

Leu

ATA

Ile

GCG

Al a

AAT

Asn 195

ATG

Met

ATC

Ile

CTG

Leu

CCG

Pro 20

TTC

Phe

GAA

Glu

AGA

Arg

CAT

His

GAA

Glu 100

GAC

Asp

GAT

Asp

CCC

Pro

GTG

Val

TTT

Phe 180

TAT

ryr

CCA

Pro k.GC Ser

CGC

krg

CAC

Gln

TGC

Cys

GGT

Gly

GCG

Ala

CAA

Gln

GGT

Gly

ACC

Thr

TGT

Cys

GCG

Ala

TGC

Cys 165

CTG

Leu

TTT

Phe

CCC

Pro

AGC

Ser

CGT

Arg 245

ACC

Thr

TGT

Cys

CTT

*Leu

*ATA

Ile 70

GAA

Glu

TTA

Leu

GAC

Asp

GAT

Asp

CTA

Leu 150

AGG

Arg

GCG

Ala

GCA

Ala

GCA

Ala TT2- Phe 230

GCC

Ala

ACG

Thr

CTG

Leu

GATI

Asp 55

CGC

Arg

GGG

Gly

ATA

Ile

AAC

Asri

AAA

Lys 135

CTA

Leu

TAC

Tyr

GTG

Val1

CTC

Leu

TCC

Ser 215

ATG

Me t kTC I le

TGT

-Cys

GCC

Ala 40

AAG

Lys

*AGA

Arg

ACT

Thr

ATT

Ile

TAC

Tyr 120

CCC

Pro

ACG

Thr

CTG

Leu

TTG

Leu

CTG

Leu 200

CCA

Pro

GCC

Ala

GCT

Ala

TATI

Tyr 25

ACC

Thr

ATG

Met

CTA

Leu

CTT

Leu

GAC

Asp 105

CTA

Leu

TGC

Cys

TGC

Cys

GAG

Glu

CCC

Pro 185

GAG

Glu

GAC

Asp kCG Thr

CAC

li 5 10

CAA

Gln

AAC

*Asn

ATG

Met

*CTC

Leu

TTG

Leu 90

GGT

Gly

CCT

Pro

GAA

Glu

ACC

Thr

TTC

Phe 170

CAT

His

AGC

Ser

GCA

Ala

TGG

Trp AAC C Asn 250

GCC

Al

TCC

Se2

CAC

Glr

CTC

Leu 75

GGA

Gly

GGT

Gly

GCG

Ala

TTC

Phe

ATA

Ile 155

TGG

Trp

PAGT

Ser

CTG

Leu

CAG

'ln 3GG ,ly

TA

Jal 3ACTI i Thr

;CAC

His

CCC

Pro 60

GGG

Gly

~AAA

*Lvs

GGA

*Gly

GTG

Val

AGG

Arg 140

TGT

Cys

GAG

Glu

CTA

Leu

AAG

Lys

CTA

Leu 220

TGG

Trp

GAG

Glu

TGG

-Trp

CTG

Leu 45

GAG

Glu

GAA

*Glu

GTG

Val

GTG

Val

GGA

Gly 125

GAG

Glu

TAC

Tyr

TTC

Phe

CAA

Gln

CAT

His 205

CAT

His

CAC

His

CGA

ArgI

GAP

Glu 30

CAG

Glr

CCC

Pro

CGC

Arg

GGT

Gly

TTT

Phe 110

TCA

Ser

CTA

Leu

AAA

Lys

GAA

Glu

CCC

Pro 190

CTC

Leu rTTT Phe 3GA Ily

:TG

,eu

TCA

Ser

GCA

Ala

ACC

Thr

CTC

Leu

CGA

Arg

ACG

Thr

TGG

Trp

CGC

Arg

GCC

Ala

CAG

Gln 175

ACA

Thr

TCG

Ser

TTA

Leu

GAGC

Glu I CCC Pro 'I

CAG

Gln

GAG

Glu

TTT

Phe

CAC

His

CGG

Arg

CGC

Arg

GAA

Glu

TCG

Ser

ATG

Met 160

TGT

2 kTC Ile

FTT

?he

:LAG

'TG

~eu 'hr 96 144 192 240 288 336 384 432 480 528 576 624 67"12 720 768 255 GTG CTG AAG AAC CTG TCG AAA CAG AGT AAG CAC CAG GAC GTC AAG GTT WO 96/06159 WO 9606159PCTfUS95/10194 221 Val Leu Lys Asn Leu Ser Lys Gin Ser 260 265 Lys His Gin Asp Vai Lys Val 270" CAG CTC GTG Gin Leu Val AAC GGA CGG GAT CTG GTG GGC TTT CAG CTG GCT OTA Asn Gly Arg Asp Leu Val Giy Phe Gin Leu Aia Leu 275 280

AAC

Asn 285 TCC CGT Ser Arg 290 CTG CAC GTA AAA Leu His Vai Lys

ATC

Ile 295 CAA CGC AAG GAC Gin Arg Lys Asp GGA CCA AAG CCA Giy Pro Lys Pro

TAO

Tyr 305 AGG GTG GTC GTC Arg Vai Vai Vai ACC OCA GAT TGT Thr Pro Asp Cys

ACC

Thr 315 TAC TAT CTA GTG Tyr Tyr Leu Vai

TAT

Tyr 320 CCG GGC ACA CCG Pro Giy Thr Pro

GCC

Al a 325 ATO TAC AGA CTC Ile Tyr Arg Leu

GTC

Vali 330 ATG TGT ATG GCA Met Cys Met Aia GTG GCA Vai Aia 335 GAC TGC ATC Asp Cys Ile TTA GGC ACC Leu Giy Thr 355

GGC

Giy 340 CAC TCG TGC AGC His Ser Cys Ser OTG CAC CC-C TGC Leu His Pro Cys GCA AAC TTT Aia Asn Phe 350 CTT TOA AGA Leu Ser Arg CAC GAG ACA CCG His Giu Thr Pro

CGT

Arg 360 OTC CTG CG GCG Leu Leu Ala Ala

ACG

Thr 365 ATC CGG Ile Arg 370 TAO GOG OCG AAA Tyr Aia Pro Lys

GAO

Asp 375 CGG CGA GCA GCC Arg Arg Aia Ala

ATG

Met 380 AAA GGA AAT TTG Lys Gly Asn Leu

CAG

Gin 385 GCG TGC TTC CAA Ala Cys Phe Gin TAC GCG GCC ACG Tyr Ala Ala Thr GCG CGG ACT CTG Ala Arg Thr Leu

GGC

Gly 400 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 AGC TCT ACA GTG Ser Ser Thr Val

TCA

Ser 405 GAC ATG CTG GAA Asp Met Leu Giu

CCC

Pro 410 ACA AAA CAC GTC Thr Lys His Val AGT TTG Ser Leu 415 GAA AAC TTC Giu Asn Phe AAG ATA AGO Lys Ile Ser 435

AAG

Lys 420 ATC ACC ATA TTC Ile Thr Ile Phe

AAC

Asn 425 ACC AAC ATG GTG Thr Asn Met Val ATT AAC ACT Ile Asn Thr 430 ATT TTA AAC Ile Leu Asn TGO CAC GTT COT Cys His Vai Pro

AAC

Asn 440 ACC- OTG CAA AAG Thr Leu Gin Lys

ACT

Thr 445 ATC CCC Ile Pro 450 AGA TTG ACC AAO Arg Leu Thr Asn

AAT

Asn 455 TTT GTT ATA CGA Phe Vai Ile Arg

AAG

Lys 460 TAO TCC GTA AAG Tyr Ser Val Lys

GAA

Giu 465 COT TOT TTT ACC Pro Ser Phe Thr

ATA

Ile 470 AGO GTG TTT TTT Ser Val Phe Phe TOO GAO AAO ATG TGT CAA Ser Asp Asn Met Cys Gin 475 480 GGO ACC OCA ATA Gly Thr Ala 'le ATO AAC ATO ACT Ile A- sn Ile Ser

GG

Gly 490 GAO ATG CTG CAC Asp Met Leu His TTT OTO Phe Leu 495 TTC GOA ATG Phe Ala Met OCT GTA TOG Pro Val Ser 52.5

CGT

Cl y 500 AOG CTC AAA TGC Thr Leu Lys Cys

TTT

Phe 505 CTGCOCA ATC AGG Leu Pro Ile Arg CAC ATA TTT Hi.s Ile Phe 510 1536 1584 ATA GCA AAT TGC AAC TOO ACG TTG GAO OTG CAC GGA OTG Ile Ala Asn Trp Asn 520 Ser Thr Leu Asp Leu His Cly Leu WO 96/06159 PCTJUS95/10194 222

CGA

GAA AAC CAG Glu Asn Gin 530 TAC ATG GTG AGA ATG GGG AAA AAC GTA Tyr Met Val Met Gly Arg Lys Asn Val TTT TGG ACC Phe Trp Thr

ACA

Thr 545

TGG

Trp

CTT

Leu

CAC

His

CAC

His

TGT

Cys 625

AGO

Arg

AGT

Ser

TGT

Cys

GAC

Asp

GGT

Gly I 705

CGC

Arg

AAC

Asr

TTT

Phe

GTG

Va1

GCG

Ala

AGA

Arg 610

CTG

Leu

CGA

Arg

CAC

His

AAC

Asn

GAG

Glu 590

CAT

His

U.AA

1,ys

TTT

1 Phe

AAG

Lvs

GAA

Glu

CGC

Arg 595

AAC

Asn

GTG

Val

GCC

Ala

ACT

Thr

CTA

Leu 675

TTG

Leu

CGG

Arg

CTA

Leu

CCA

Pro

GCC

Ala

CAG

Gin 580

ATC

Ile

CGT

Arg

GAA

Glu

GCC

Ala

AAA

Lys 660

ATC

Ile

GGC

Gly

TTC

Phe 3GC Gly

TCT

Ser

GCG

Ala 565

ATT

Ile

GAC

Asp

TCC

Ser

TGC

Cys

GCC

Ala 645

AGC

Ser

CCG

Pro

CGC

Arg

GCC

Ala

CTG

Leu I 725

GTC

Val 550

AC.P

Thr

CGC

Arg

GGA

Gly

CAA

Gin

TGT

Cys 630

CGG

Arg

AAA

Lys

AAA

Lys

AAT

Asn

GCT

kla 710

CAC

lis

GTC

Val

GCC

Ala

CAC

His

AAC

Asn

ATA

Ile 615

TCG

Ser

GGC

Gly

CAC

His

ATC

Ile

GCA

Ala 695

CTA

Leu

TGG

Trp 2

TCC

Ser

ACG

Thr

GAG

Glu

AAA

Lys 600

CAG

Gin

TTT

Phe

CTG

Leu

GAG

Glu

TAT

Tyr 680

AAC

Asn kAG Lys

CGC

%rg

AGC

Ser

ATT

Ile

CTG

Leu 585

AAT

Asn

ACG

Thr

CTC

Leu

TTT

Phe

TGC

Cys 665

GOC

Ala

TTC

Phe

CCA

Pro

CAC

His 2

AAG

Lys

TCI

Ser 570

GCG

Ala

AGA

Arg

CTA

Leu

AGG

Arg

GAC

Asp 650

GCA

Ala

CGA

Arg

ATT

Ile

CAA

GIn

GA

krg 730

GAT

Asp 555

AAA

Lys

CCC

Pro

ATA

Ile

CAC

His

CTT

Leu 635

TTC

Phe

GTA

Val

AAC

Asn

TCO

Ser

ATT

Ile 715 ACG C Thr I

GGG

Gly

GTG

Val

ATT

Ile

TTC

Phe

AAA

Lys 620

GAC

Asp

TCA

Ser

CTG

Leu

AAG

Lys rTC Phe 700

"TC

lal ;cc la

CT;

Leu

TAC

Tyr

CTC

Leu

TCC

Ser 605

AGO

Arg

GTG

Val

AAG

Lys

GGA

Gly

AAG

Lys 685

GTC

Val

CGT

Arg

GCG

Ala

AAC

Asn

GGG

Gly

ACG

Thr 590

CTA

Leu

TTC

Phe

GCT

Ala

AAG

Lys

TAT

Tyr 670

ACC

Thr

GCC

Ala

CAC

His 2 TCC 1 Ser I

GTC

Val

CAG

Gin 575

GAC

Asp

CTT

Leu

CTG

Leu

TGC

Cys

ATA

Ile 655

AAA

Lys

AGG

Arg kCC rhr 3CC kla

AC

~sn 735

TCC

Ser 560

CCT

Pro

CAG

Gin

GAG

Glu

GAG

Glu

ATT

Ile 640

ATC

Ile

AAG

Lys

CTA

Leu

ACO

Thr

ATT

Ile 720

GAG

Glu 1632 1680 1728 1776 1824 1872 1920 1968 2016 2064 2112 2160 2208 CAG ACA CCG CCA GCC GAT CCC CGC OTA Gin Thr Pro Pro Ala Asp Pro Arg Val 740 745

TAA

CGT TC GTC CGT CCG CTG OTC Arg Cys Val Arg Pro Leu Val 750 2256 2259 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 752 amino acids TYPE: amino acid TOPOLOGY: linear WO 96/06159 PCTIUS95/10194 223 (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:21: Met Ala Ala Leu Giu Giy Pro Leu Leu Leu Pro Pro Ser Ala Ser Leu Thr Leu Leu Phe Pro Tyr Gly Leu Leu 145 Phe Phe Tyr Ser Phe 225 Val Val Asn Ser 2 Tyr J 305 Thr Glu Thr Ala Phe Ser Gin Thr 130 Tyr Arg His Gin Ile 210 Asn Ser eu "ly krg krg Ser Ile Leu Cys Ile Gly Ile 115 Asp Leu Ile Ala Asn 195 Met Ile Leu Lys Arg 275 Leu Val Pro Phe Glu Arg His Glu 100 Asp Asp Pro Val Phe 180 Tyr Pro Ser Arg Asn 260 Asp His Val Glr Cys Gl Ala Gin Gly Thr Cys Ala Cys 165 Leu Phe Pro Ser Arg 245 Leu Leu Val Val 1 Thr Cvs Leu Ile 70 Glu Leu Asp Asp Leu 150 Arg Ala Ala Ala Phe 230 Ala Ser Val Lys Ser 310 Th2 Lei Asp 55 Arc Gly Ile Asn Lys 135 Leu Tyr Vai Leu Ser 215 Met Ile Lys Gly Ile 295 Thr Cys i Ala 40 Lys Arg Thr Ile Tyr 120 Pro Thr Leu Leu Leu 200 Pro Ala Ala Gin Phe 280 Gin Pro Tyr 25 Thr Met Leu Leu Asp 105 Leu Cys Cys Glu Pro 185 Glu Asp Thr His Ser 265 3Gm Arg ksp Gl Asr Met Leu Leu 90 Gly Pro Glu Thr Phe 170 His Ser Ala Trp Asn 250 Lys Leu Lys cvs i Ala 1 Ser Gin Leu 75 Gly Gly Ala Phe Ile 155 Trp Ser Leu Gin Gly 235 Val His Ala Asp Thr 315 Th2 His Pro Gly Lys Glv Val Arg 140 Cys Glu Leu Lys Leu 220 Trp Glu Gin Leu Pro 300 ryr Trp Leu Glu Glu Val Val Gly 125 Glu Tyr Phe Gin His 205 His His Arg Asp Asn 285 Gly Tyr I Gli 3C Glr Pro Arg Gly Phe 110 Ser Leu Lys Glu Pro 190 Leu Phe 1 y Leu Val 270 Gin ?ro 4eu i Ser Ala Thr Leu Arg Thr Trp Arg Ala Gin 175 Thr Ser Leu Glu Pro 255 Lys Leu Lys I Val Gin Glu Phe His Arg Arg Glu Ser Met 160 Cys Ile Phe Lys Leu 240 Thr Val ial ?ro yr 320 Pro Gly Thr Pro Ala Ile Tyr Arg Leu Val Met Cys Met Ala Val Ala 335 WO 96/06159 PCTIUS95/10194 Asp Cys IlE Leu Gly Thr 355 Ile Arg Tyr 370 Gin Ala Cvs 385 Ser Ser Thr Glu Asn Phe Lys Ile Ser 435 Ile Pro Arg 450 Glu Pro Ser 465 Gly Thr Ala Phe Ala Met Pro Val Ser 515 Glu Asn Gin 530 Thr Asn Phe 545 Trp Phe Lys Leu Val Glu His Ala Arg 595 His Arg Asn 610 Cys Leu Val 625 Arg Arg Ala Ser His Thr Cvs Asn Leu 675 Gly 340 His Ala Phe Val Lys 420 Cys Leu Phe Ile Gly 500 Ile Tyr i Pro Ala Gin 580 Ile 2 Arg c Glu C Ala 6 Lvs S 660 Ile P His Ser Cys Sei Gi.

Pro Gin Ser 405 Ile His Thr Thr Asn 485 Thr Ala Mlet Ser kla 565 Lie ksp er :ys ila ;45 ;er >ro Thr Lys Arg 390 Asp Thr Val Asn Ile 470 Ile Leu Asn Vai Val 550 Thr Arg Gly Gin Cvs 630 Arg Lys I Lys Pro Asp 375 Tyr Met Ile Pro Asn 455 Ser Asn Lys Trp Arg 535 Val Ala His Asn Ile 615 Ser ly lis le ArS 36C Arc Ala Leu Phe Asn 440 Phe Vai Ile Cys Asn 520 Met Ser Thr Glu Lys 600 Gin Phe Leu Glu Tyr 224 Gly Leu 345 Leu Leu Arg Ala Ala Thr Glu Pro 410 Asn Thr 425 Thr Leu Val Ile Phe Phe Ser Gly 490 Phe Leu 505 Ser Thr Gly Arg Ser Lys Ile Ser 570 Leu Ala 585 Asn Arg Thr Leu I Leu Arg I Phe Asp 1 650 Cys Ala 665 Ala Arg Hi Al Alz Asr 395 Th Asn Gir Arg Ser 475 Asp Pro Leu Lys Asp 555 Lys Pro lie iis ~eu ;35 !he Tal LSn s Pr( i Al Me 38( Al Lye Met Lys Lys 46C Asp Met Ile Asp Asn 540 Gly Val Ile Phe Lys 620 Asp Ser Leu Lys Cy Thl 365 t LyE a Arc His Val Thr 445 Tyr Asn Leu Arg Leu 525 Val Leu Tyr Leu Ser 605 Arg Val Lys Gly Lys 685 Ala Asn 350 Leu Ser 3 Gly Asn Thr Leu Val Ser 415 Ile Asn 430 Ile Leu Ser Val Met Cys His Phe 495 His Ile 510 His Gly Phe Trp Asn Val Gly Gin 575 Thr Asp 590 Leu Leu Phe Leu Ala Cys 1 Lys Ile I 655 Tyr Lys I 670 Thr Arg L Phe Arg Leu Gly 400 Leu Thr Asn Lys Gin 480 Leu Phe Leu Thr Ser 560 Pro 3Gl lu 'lu :le le ys leu 680 Asp Giu Leu Giy Arg Asn Ala Asn Phe Ile Ser Phe Val Ala Thr Thr WO 96/06159 PCT/US95/10194 225 690 695 700 Gly His Arg Phe Ala Ala Leu Lys Pro Gin Ile Val Arg His Ala Ile 705 710 715 720 Arg Lys Leu Gly Leu His Trp Arg His Arg Thr Ala Ala Ser Asn Glu 725 730 735 Gin Thr Pro Pro Ala Asp Pro Arg Val Arg Cys Val Arg Pro Leu Val 740 745 750 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 364 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..364 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: ATG GTA CGT CCA ACC GAG GCC GAG GTT AAG AAA TCC CTG AGC AGG CTT 48 Met Val Arg Pro Thr Glu Ala Glu Val Lys Lys Ser Leu Ser Arg Leu 1 5 10 CCA GCA GCA CGC AAA AGA GCA GGT AAC CGG GCC CAC CTG GCC ACC TAC 96 Pro Ala Ala Arg Lys Arg Ala Gly Asn Arg Ala His Leu Ala Thr Tyr 25 CGC CGG CTC CTC AAG TAC TCC ACC CTG CCC GAT CTA TGG CGG TTT CTA 144 Arg Arg Leu Leu Lys Tyr Ser Thr Leu Pro Asp Leu Trp Arg Phe Leu 40 AGT AGC CGG CCC CAG AAC CCT CCC CTT GGA CAC CAC AGA TTA TTC TTT 192 Ser Ser Arg Pro Gin Asn Pro Pro Leu Gly His His Arg Leu Phe Phe 55 GAG GTG ACT CTA GGG CAC AGA ATT GCC GAC TGC GTA ATT CTG GTA TCG 240 Glu Val Thr Leu Gly His Arg Ile Ala Asp Cys Val Ile Leu Val Ser 70 75 GGT GGG CAT CAG CCC GTA TGT TAC GTT GTA GAG CTC AAG ACT TGT CTG 288 Gly Gly His Gin Pro Val Cys Tyr Val Val Glu Leu Lys Thr Cys Leu 90 AGT CAC CAG CTG ATC CCA ACC AAC ACC GTG AGA ACG TCA CAG CGA GCT 336 Ser His Gin Leu Ile Pro Thr Asn Thr Val Arg Thr Ser Gin Arg Ala 100 105 110 CAA GGC CTG TGC CAA CTC TCC GAC TCG A 364 Gin Gly Leu Cys Gin Leu Ser Asp Ser 115 120 WO 96/06159 PCT/US95/10194 226 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 121 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met 1 Pro Arg Ser Glu Gly Ser Gin (xi) SEQUENCE Val Arg Pro Thr 5 Ala Ala Arg Lys Arg Leu Leu Lys Ser Arg Pro Gin Val Thr Leu Gly Gly His Gin Pro His Gin Leu Ile 100 Gly Leu Cys Gin 115 DESCRIPTION: SEQ ID Glu Ala Glu Val Lys 10 Arg Ala Gly Asn Arg 25 Tyr Ser Thr Leu Pro 40 Asn Pro Pro Leu Gly 55 His Arg Ile Ala Asp 70 Val Cys Tyr Val Val 90 Pro Thr Asn Thr Val 105 Leu Ser Asp Ser 120 NO:23: Lys Ser Ala His Asp Leu His His Cys Val 75 Glu Leu Arg Thr Leu Ser Leu Ala Trp Arg Arg Leu Ile Leu Lys Thr Ser Gin 110 Arg Leu Thr Tyr Phe Leu Phe Phe Val Ser Cys Leu Arg Ala INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 918 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..918 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: ATG GCA CTC GAC AAG AGT ATA GTG GTT AAC TTC ACC TCC AGA CTC TTC Met Ala Leu Asp Lys Ser Ile Val Val Asn Phe Thr Ser Arg Leu Phe 1 5 10 GCT GAT GAA CTG GCC GCC CTT CAG TCA AAA ATA GGG AGC GTA CTG CCG Ala Asp Glu Leu Ala Ala Leu Gin Ser Lys Ile Gly Ser Val Leu Pro 25 WO 96/06159 WO 96/6 159PCTIUS95/10194 227 CTO GGA GAT TGO CAC CGT TTA CAA AAT CAG GCA TTG GGC CTG GGG Leu Gly Asp Cys His Arg Leu Gin Asn Ile Gin Ala Leu Gly Leu Gly

TGC

Cys

TAT

Tyr

AGO

S er

AAO

Asn

GTG

Val

TC

Ser

GGG

Giy 145

ATA

Ile

TAT

Tyr

GAC

Asp

ATG

Met

OTG

Leu 225

TTT

Phe

TTG

Leu

CTT

Leu

GTI

Val

CT;

Leu

CTG

Leu

GTA

Va l

CTA

Leu

AAC

Asn 130

CAC

His

TOG

Ser

ACC

Thr

AAO

Asn

TAO

Tvr 210

CTC

Leu

GAG

Giu 1.GO Ser

"AG

"in

~TGO

-Cys

TOO

Ser

OGO

Arg

*TAT

Tyr 00 Pro 115

GGA

Giy

GOT

Ala

GC

Al a

ACC

Thr

ACG

Thr 195

OTT

Leu

ACG

Thr

GGG

Gly'

GTO

Val

AGT

Ser 275

TO)

Se2

AAC

Lys

CT;

Let

GOC

Al a 100

CCA

Pro

TTT

Phe

ATT

Ile

GGG

Gly

AAT

Asn 180

GAT

Asp

TGT

Cys

GOG

Al a

GTG

Val

CCA

Pro 260

CTC

Leu k. CGT Arg

TGO

Cys

AOG

Thr

*CCC

*Pro

*TTT

Phe

GAO

Asp

CTG

Leu

GC

Aia 165

GTG

Val

COA

Pro

ATO

Ile

OTO

Leu

GTG

Val 245

GAT

Asp

AGT

Ser

GAC

Gl.

AC;

Thi.

70

CGG

Arg

TTI

Phe

TTT

Phe

COO

Pro

CAG

Gin 150

COG

Pro

TCA

Ser

CGT

Arg

OTA

Leu

GTG

Vai 230

CCA

Pro

GAC

%sp rCT 3er

ACA

SThr 55

CTO

Leu

ATG

Met

TTT

Phe

AGO

Ser

GTG

Val 135

CAG

Gin

GAT

Asp

TTT

Phe

ACT

Thr

TCA

Ser 215

CGG

Arg

GAT

Asp

ATO

Ile

ATA

Ile

TO']

Sex.

G C71 Aila

GATI

Asp

CAG

Gin

CGA

Arg 120

TTO

Phe

CTG

Leu

GAT

Asp

ATG

Met

GC

Ala 200

GC

Al a

CAC

His

GAG

Glu

%CC

rhr rT T ?he so0 ccC Prc

GTC

Val ccc Prc

TGG

*Trp 105

*AAG

Lys

COO

Pro

TTG

Leu

GTA

Val

GGG

Gly 185

OTG

Leu

TTG

Leu

GAO

Asp

GTG

Val

AGG

Arg 265

AAT

Asn .7 GAO Asp

CTG

-Leu

TOT

Ser 90

GAO

Asp

GAT

Asp

ATG

Met

GTG

Val

AAT

Asn 170

CGO

Arg

CGA

Arg

GTTC

Val

AGGC

Arg 1 ACC Thr 1 250 ATG C Met OTT G Leu G

TAC

Ty2

GAC

Gi.

75

GAC

Asp

AGC

Ser

TOO

Ser

GTC

Val

TAO

Tyr 155

ATG

Met

ACA

rhr

GTG

Jai ?ro

'AT

{is ~35

~GG

~rg

:GC

Lrg

~GC

liy

AT(

6(

GAC

1Gl

:AAC

Asr

:AAC

Asn

ACC

Thr

GTG

Val1 140

CAC

His

GCG

Al a

TAT

Tyr

CTT

Leu

AGG

Arg 220

OCT

Pro

ATA

Ile

GTO

Val

COO

Pro

CCAA

e Gin 3 GTT a Val

OTT

1Leu

ACC

Thr

ATT

Ile 125

CCG

Pro

ATO

Ile

GAA

Giu

CGT

Arg

GAC

Asp 205

GGG

GlyC CTG2 Leu IJ GAT C Asp I ATG TI Met P 2 AGA C Arg L 285

AT

11.

CGC

Arc

CAC

Gir

CAG

Gin 110

GTG

Val

CAG

Gin

TAC

Tyr

OTT

Leu

CTG

Leu 190

GAT

ksp

PGT

:ys

.CA

?hr

~TC

~eu

'TO

~he

TG

eu U ATG SMet

COG

Pro

ATA

Ile

OTA

Leu

OTO

Leu

CAA

Gin

TOO

Ser

GAT

Asp 175

GAO

Asp

OTG

Leu Leu2 GAG C GAO C Asp G 255 TOO TI Ser TI CAC G His v

CAG

Gin

GAO

Asp

AAA

Lys

GCA

Al a

GAA

Glu

CTG

Leu

AAA

Lys 160

CTA

Leu

GTA

Val roo Ser 7GT krg

TG

Tal

AG

in

'AT

yr

TG

'al 192 240 288 336 384 432 480 528 576 624 672 720 768 816 864 TAT GC Tyr Al a 290 TAO TOG GOA GAG Tyr Ser Ala Glu

ACT

Thr 295 TTG GOG GOO TOO Leu Ala Ala Ser TGG TAT TOO OCA Trp, Tyr Ser Pro WO 96/06159 228 CGC TAA Arg 305 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 305 amino acids TYPE: amino acid TOPOLOGY: linear PCT/US95/10194 91P (ii) MOLECULE TYPE: protein (xiJ) SEQUENCE DESCRIPTION: SEQ ID Met Ala Leu Asp Lys Ser Ile Val Val Asn Phe Thr Ser Arg Leu Phe Al a Leu Cys Tyr Ser Asn Val Ser Gly 145 Ile Tyr Asp Met Leu 225 Phe Asp Gly Val Leu Leu Val Leu Asn 130 His Ser Thr Asn Tyr 210 Leu Glu Glu Asp Cys Ser Arg Tyr Pro 115 Gly Al a Al a Thr Thr- 195 Leu Thr Gly Leu Cys Ser Lys Leu Al a 100 Pro Phe Ile Gly Asn 180 Asp Cvs Al a Val Al a His Arg Cys Thr Pro Phe Asp Leu Al a 165 Val Pro Ile Leu Val 245 Al a Arg Glu Thr 70 Arg Phe Phe Pro Gln 150 Pro Ser Arg Leu Val 230 Pro Leu Leu Thr Leu Met Phe Ser Val 135 Gln Asp Phe Thr Ser 215 Arg Asp Gln Gln 40 Ser Ala Asp Gln Arg 120 Phe Leu Asp Met Ala 200 Al a His Glu Ser 25 Asn Pro Val Pro Trp 105 Lys Pro Leu Val Gly 185 Leu Leu Asp Val Lys Ile Asp Leu Ser 90 Asp Asp Met Val Asn 170 Arg Arg Val Arg Thr 250 Ile Gln Tyr Glu 75 Asp Ser Ser Val Tyr 155 Met Thr Val Pro His 235 Arg Gly Al a Ile Glu Asn Asn Thr Val 140 His Ala Tyr Leu Arg 220 Pro Ile Ser Leu Gln Val Leu Thr Ile 125 Pro Ile Glu Arg Asp 205 Gl v Leu Asp Val Gly Ile Arg Gln Gln 110 Val Gln Tyr Leu Leu 190 Asp Cys Thr Leu Leu I Leu G Met G Pro A Ile L Leu A Leu G Gln L Ser L Asp L.

175 Asp V Leu S4 Leu A: Glu V~ Asp G 255 ~ro ;ly ~ln ,ys l1a lu eu ys eu al ln Leu Ser Val Asp Asp Ile Thr Arg Met Arg Val Met Phe Ser Tyr 265 270 WO 96/06159 PCT/US95/10194 229 Leu Gin Ser Leu Ser Ser Ile Phe Asn Leu Gly Pro Arg Leu His Val 275 280 285 Tyr Ala Tyr Ser Ala Glu Thr Leu Ala Ala Ser Cys Trp Tyr Ser Pro 290 295 300 Arg 305 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 873 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..873 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: ATG GCG TCA TCT GAT ATT CTG TCG GTT GCA AGG ACG GAT GAC GGC TCC Met Ala ser Ser 1 GTC TGT Val Cys CTG CCG Leu Pro GCC TTC Ala Phe GGT GCC Gly Ala TGT TAT Cys Tyr CCC CTT Pro Leu CCC GTT Pro Val GAA GTC Glu Val GAC ACT Asp Thr CTC AGC Leu Ser CTG CCC Leu Pro TGT TAC Cys Tyr AAT CCT Asn Pro 100 GTG GCA Val Ala 115 Asp 5

TCC

Ser

GAA

Glu

GAC

Asp

TCA

Ser

TTG

Leu

TAC

Tyr

CCT

Pro

CTG

Leu

CCC

Pro

GGG

Gly

AAT

Asn 70

CAA

Gin

TTG

Leu

GAG

Glu

CGT

Arg

TGG

Trp

ATC

Ile 55

TCT

Ser

AAT

Asn

GTA

Val

GTG

Val Ile Leu Ser Val Ala Arg Thr Asp Asp

GGA

Gly

GTG

Val 40

GTG

Val

CAC

His

TGT

Cys

ACT

Thr

CTC

Leu 120

GGT

Gly 25

GTA

Val

GAT

Asp

AAC

Asn

GTG

Val

CCC

Pro 105

TTC

Phe 10

AGG

Arg

GAG

Glu

ATG

Met

GGC

Gly

TAC

Tyr 90

TCA

Ser

CCA

Pro Gly Ser

AAA

Lys

ACC

Thr

GCT

Ala

TTG

Leu 75

CTA

Leu

AGC

Ser

CAC

His

AAA

Lys

GAC

Asp

CGA

Arg

AGG

Arg

GCC

Ala

ATT

Ilie

CCG

Pro

ACT

Thr

GCC

Ala

AAG

Lys

ATG

Met

CTG

Leu

GAG

Glu

GCT

Ala 125

ACC

Thr

ATC

Ile

CTT

Leu

GTG

Val

TTT

Phe

TTT

Phe 110

GAG

Glu GTC TAC Val Tyr AAA GAC Lys Asp CAT CGT His Arg CTT TTT Leu Phe CTG TGC Leu Cys GCC GAG Ala Glu ATG TCT Met Ser 96 144 192 240 288 336 384 CGC GGT TGC GAT GAC GCG ATT TTC TGT AAA CTG Arg Gly Cys Asp Asp Ala Ile Phe Cys Lys Leu 130 135

CCC

Pro 140 TAT ACC GTG CCT Tyr Thr Val Pro WO 96/06159 WO 9606159PCTfUS95/10194 230 CGC ATT TAC ATA ATC AAC ACC ACG TTT GGA Ile Ile Asn Thr Thr Phe Gly CCG AAC TCT ACA CGC GAG Arg Ile Tyr Pro Asn Ser Thr Arg Glu 145

CCG

Pro

GCA

Al a

GAA

Glu

GCT

Aila

GAA

Giu 225

TTA

Leu

GTG

Val

AAC

Asn

TTT

Phe

GAC

Asp

GTT

Val

ACT

Thr

ATG

Met 210

GCA

Al a

TTC

Phe

TCG

Ser

TGT

Cys

~AA

Lys 290

GGC

Gly

ATG

Met

CAG

Gin 195

TTT

Phe

CTG

Leu

ATC

Ile

TGG

Trp

GAC

Asp 275

TAA

AGG

Arg

GTT

Val 180

ACC

Thr

TCT

Ser

TCT

Ser

TCT

Ser

GAA

Giu 260 CCT ACG Pro Thr 165 AAC ACG Asn Thr GCA TCC Ala Ser GTG ATT Val Ile ATC GCG Ile Ala 230 CAG CCC Gin Pro 245 GAT ATC Asp Ile

GAT

Asp

TCA

Ser

CGT

Arg

ATC

Ile 215

AGC

Ser

CGG

Arg

TAC

Tyr

TAC

Tyr

TGT

Cys

AAC

Asn 200

TAT

Tyr

GGC

Gly

AGC

Ser

AAC

Asn

CTA

Leu 280

TCC

Ser

GCA

Al a 185

CAC

His

GCC

Al a

ATC

Ile

GTG

Val

GGG

Gly 265

ATG

Met 170

GGA

Gly

ACT

Thr

TTA

Leu

TTT

Phe

CCC

Pro 250

ACT

Thr

GCC

Ala

GTG

Val

GAG

Glu

GAT

Asp

GAC

Asp 235

TCG

Ser

TAC

Tyr

CTT

Leu

ACA

Thr

TGG

Trp

CAC

His 220

GAG

Giu

CCT

Pro

CTA

Leu

AGA

Arg

TTG

Leu

GAA

Glu 205

AAC

Asn

CGT

Arg

ACC

Thr

GCT

Al a

TTG

Leu 285

AGG

Arg

TGC

Cys 190

AAT

Asn

TGT

Cys

GAC

Asp

CCT

Pro

CGG

Arg 270 OCT TTT Ala Phe 175 CGC GGA Arg Gly CTG CTG Leu Leu CAC CCG His Pro TAT GGA Tyr Gly 240 TGC GAC Cys Asp 255 CCT GGA Pro Gly 528 576 624 672 720 768 816 864 CCC TOG CCC AAT Pro Trp, Pro Asn TCC ACC CCT CCC Ser Thr Pro Pro ATT CTA AAT Ile Leu Asn INFORMATION FOR SEQ ID NO:27: Wi SEQUENCE CHARACTERISTICS: LENGTH: 290 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Met Ala Ser Ser Asp Ile Leu Ser Val Ala Arg Thr Asp Asp Gly Ser 1 5 10 Val Cys Glu Val Ser Leu Arg Oly Oly Arg Lys Lys Thr Thr Val Tyr 25 Leu Pro Asp Thr Oiu Pro Trp Val Val Glu Thr Asp Ala Ile Lys Asp 40 Ala Phe Leu Ser Asp Gly Ile Val Asp Met Ala Arg Lys Leu His Arg 55 Gly Ala Leu Pro Ser Asn Ser His Asn Gly Leu Arg Met Val Leu Phe 70 75 WO 96/06159 WO 9606159PCTIUS95/10194 Cys Pro Pro Arg Ile 145 Pro Ala Glu Ala Giu 225 Leu Val Asn Phe Tyr Leu Val Gly 130 Ile Asp Val Thr Met 210 Al a Phe Ser Cys Lys 290 Cys Asn Val Cys Asn Gly Met Gin 195 Phe Leu Ile Trp Asp 27 c Tyr Pro 100 Al a Asp Thr Arg Val 180 Thr Ser Ser Ser Glu 260 Pro Leu Tyr Pro Asp Thr Pro 165 Asn Al a Val1 Ile Gin 245 Asp Trp Gin Asn Cys Val Leu Glu Ala Phe 150 Thr Thr Ser Ile Ala 230 Pro Ile Pro Val Val Ile 135 Gly Asp Ser Arg Ile 215 Ser Arg Tyr Asn Thr Leu 120 Phe Arg Tyr Cys Asn 200 Tyr Gly Ser Asn Leu 280 Pro 105 Phe Cys Ile Ser Al a 185 His Al a Ile Val Gly 265 Ser Tyr Ser Pro Lys Tyr Met 170 Gly Thr Leu Phe Pro 250 Thr Thr Leu Ala Leu Phe Leu Cys Ser His Leu Pro 155 Al a Val1 Glu Asp Asp 235 Ser Tyr Pro Ile Prc Pro 140 Asn Leu Thr Trp His 220 Glu Pro Leu Pro Glu Ala 125 Tyr Ser Arg Leu Glu 205 Asn Arg.

Thr Ala Leu 285 Phe 110 Giu Thr Thr Arg Cvs 190 Asn Cys Asp Pro Arg 270 Ile Al a Met Val Arg Ala 175 Arg Leu His Tyr Cys 255 Pro Leu Glu Ser Pro Glu 160 Phe Gly Leu Pro Gly 240 Asp Gly Asn INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 363 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .363 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: ATG AGC ATG ACT TTC CCC GTC TCC AGT CAC CGG AGG AAT GGT GGA CGG Met Ser Met Thr Phe Pro Val Ser Ser His Arg Arg Asn Gly Gly Arg 10 WO 96/06159 PCT/US95/10194 232 CTC CGT CCT GGT GCG AAT GGC CAC CAA GCC TCC CGT GAT TGG TCT TAT 96 Leu Arg Pro Gly Ala Asn Gly His Gin Ala Ser Arg Asp Trp Ser Tyr 25 AAC AGT GCT CTT CCT CCT AGT CAT AGG CGC CTG CGT CTA CTG CTG CAT 144 Asn Ser Ala Leu Pro Pro Ser His Arg Arg Leu Arg Leu Leu Leu His 40 TCG CGT GTT CCT GGC GGC TCG ACT GTG GCG CGC CAC CCC ACT AGG CAG 192 Ser Arg Val Pro Gly Gly Ser Thr Val Ala Arg His Pro Thr Arg Gin 55 GGC CAC CGT GGC GTA TCA GGT CCT TCG CAC CCT GGG ACC GCA GGC CGG 240 Gly His Arg Gly Val Ser Gly Pro Ser His Pro Gly Thr Ala Gly Arg 70 75 GTC ACA TGC ACC GCC GAC GGT GGG CAT AGC TAC CCA GGA GCC CTA CCG 288 Val Thr Cys Thr Ala Asp Gly Gly His Ser Tyr Pro Gly Ala Leu Pro 90 TAC AAT ATA CAT GCC AGA TTA GAA CGG GGT GTG TGC TAT AAT GGA TGG 336 Tyr Asn Ile His Ala Arg Leu Glu Arg Gly Val Cys Tyr Asn Gly Trp 100 105 110 CTA TGG GGG GGG GCT GTA GAT AAT TGA 363 Leu Trp Gly Gly Ala Val Asp Asn 115 120 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 120 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Met Ser Met Thr Phe Pro Val Ser Ser His Arg Arg Asn Gly Gly Arg 1 5 10 Leu Arg Pro Gly Ala Asn Gly His Gin Ala Ser Arg Asp Trp Ser Tyr 25 Asn Ser Ala Leu Pro Pro Ser His Arg Arg Leu Arg Leu Leu Leu His 40 Ser Arg Val Pro Gly Gly Ser Thr Val Ala Arg His Pro Thr Arg Gin 55 Gly His Arg Gly Val Ser Gly Pro Ser His Pro Gly Thr Ala Gly Arg 70 75 Val Thr Cys Thr Ala Asp Gly Gly His Ser Tyr Pro Gly Ala Leu Pro 90 Tyr Asn Iie His Ala Arg Leu Glu Arg Gly Val Cys Tyr Asn Gly Trp 100 105 110 Leu Trp Gly Gly Ala Val Asp Asn 115 120 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: WO 96/06159 WO 96/6 159PCTIUS95/10194 233 LENGTH: 922. base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .921.

OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID ATG CTG CTC AGC CGT CAC AGG GAG CGC CTT GCC GCC AAC CTG GAG Met Leu Leu Ser

ACC

Thr

ACG

Thr

GTG

Val

ACA

Thr

ACC

Thr

GAG

Glu

CAC

His

ACA

Thr

TTT

Phe 145 G T I Val

GCC

Al a

CGA

Arg

CTG

Val

CGG

Arg

CCC

Pro

TCC

Ser

GTC

Val

CAC

His 130

CTG

Leu

CAC

Hi s

AAA

Lys

CAC

His

CAC

His

CAC

His

AGA

Arg

CTA

Leu

TCC

Ser 115

GAC

Asp

GTT

Val

TGG

Trp

GAC

Asp

TGT

Cys

AGA

Arg

CAT

His

CCG

Pro

TCA

Ser 100

ACG

Thr

TCC

Ser

AAC

Asn

CTG

Leu *Arg

GC

*Ala

CCC

Pro

ATA

Ile

CCC

Pro

TCC

Ser

ACA

Thr

GCC

Ala

CTG

Leu

CTT

Leu

GAG

Clu 165 His Arg Glu Arg Leu Ala Ala Asn Leu Glu

GGA

Gly

AAA

Lys

AAC

Asn

GCC

Ala 70

GAG

Glu

TAC

Tyr

GAT

Asp

CAC

His

TCG

Ser 150

CCC

Pro

GAG

Glu

ACG

Thr

TCA

Ser 55

ACG

Thr

GAC

Asp

TTG

Leu

CAA

Gln

GCC

Al a 135

TCC

Ser

TTC

Phe

AGG

Arg

GCA

*Ala

*TAC

Tyr

CCC

Pro

AAC

Asn

CAG

Gln

CTG

Leu 120

TGC

Cys

TTT

Phe

CAA

Gin

TGG

Trp 25

CGG

Arg

ACT

Ser

ACG

Thr

CTC

Val

ATG

Met 105

CTC

Val

TCT

Ser

CTG

Leu

:AC

GAA CTG Glu Leu ATG GCC Met Ala TCG GTC Ser Val TCA GCA Ser Ala 75 CCC GCA Pro Ala 90 CGG TGT Arg Cys GAG TAC Glu Tyr GTC TAC Val Tyr AAC GGC Asn Gly 155 CAG CTA Gin Leu 170 Ser

CAC

Al a

CCT

CCG

Pro

TTT

Thr

ATT

GAG

Glu

TTC

Phe

GGC

His Pro Phe Ile Gly

CTG

Leu

AAT

Asn

AAC

Lys

GTG

Val

CAG

Gin

CC

Arg 140

TGT

Cys

GTA

Val

GAA

Clu

CCC

Pro

CCG

Pro

CGC

Arg

C

Al a 125

GAA

Giu

TAC

Tyr

ATG

Met

ACA

Thr

GAC

Asp

CC

Arg

GAG

Glu 110

GCC

Gly

CTT

Leu

CTT

Val

CAC

His

TAC

Tyr

CTG

Val1

CTA

Leu

CAC

Asp

AGA

Arg

CAG

Gin

CCC

Pro

ACT

Thr 175

TC

Cys

GGA

Gly

TTG

Leu

CC

Al a

AAA

Lys

GCT

Al a

GCC

Gly 160

TTT

Phe 48 96 144 192 240 288 336 384 43Z 480 528 TT C TTT TTG GTT TCA ATC AAC CCC CCA CAA AAG ACC CAC CAC Phne P'ne Leu Val 180 Ser Ile Lys Ala Pro 185 Gin Lys Thr His Cln 190 TTG TTT Leu Phe WO 96/06159 PCT/US95/10194 234 GGA TTG TTT AAG CAG TAC TTC GGT TTA TTT GAA ACT CCA AAC AGT GTT 624 Gly Leu Phe Lys Gin Tyr Phe Gly Leu Phe Glu Thr Pro Asn Ser Val 195 200 205 TTA CAG ACG TTT AAG CAA AAG GCA AGC GTA TTC CTA ATA CCA AGG AGA 672 Leu Gin Thr Phe Lys Gin Lys Ala Ser Val Phe Leu Ile Pro Arg Arg 210 215 220 CAC GGA AAG ACA TGG ATA GTG GTG GCG ATC ATC AGC ATG CTA CTG GCA 720 His Gly Lys Thr Trp Ile Val Val Ala Ile Ile Ser Met Leu Leu Ala 225 230 235 240 TCC GTA GAG AAC ATT AAC ATT GGG TAC GTA GCC CAC CAA AAG CAC GTA 768 Ser Val Glu Asn Ile Asn Ile Gly Tyr Val Ala His Gin Lys His Val 245 250 255 GCC AAC TCC GTG TTC GCG GAA ATC ATA AAG ACG CTT TGT CGG TGG TTC 816 Ala Asn Ser Val Phe Ala Glu Ile Ile Lys Thr Leu Cys Arg Trp Phe 260 265 270 CCC CCC AAA AAT TTA AAC ATC AAG AAG GAG AAC GGA ACC ATA ATC TAC 864 Pro Pro Lys Asn Leu Asn Ile Lys Lys Glu Asn Gly Thr Ile Ile Tyr 275 280 285 ACG CGA CCC GGA GGA CGG TCC AGC TCG CTG ATG TGC GCA ACA TGC TTC 912 Thr Arg Pro Gly Gly Arg Ser Ser Ser Leu Met Cys Ala Thr Cys Phe 290 295 300 AAT AAG AAC 921 Asn Lys Asn 305 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 307 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: Met Leu Leu Ser Arg His Arg Glu Arg Leu Ala Ala Asn Leu Glu Glu 1 5 10 Thr Ala Lys Asp Ala Gly Glu Arg Trp Glu Leu Ser Ala Pro Thr Phe 25 Thr Arg His Cys Pro Lys Thr Ala Arg Met Ala His Pro Phe Ile Gly 40 Val Val His Arg Ile Asn Ser Tyr Ser Ser Val Leu Glu Thr Tyr Cys 55 Thr Arg His His Pro Ala Thr Pro Thr Ser Ala Asn Pro Asp Val Gly 70 75 Thr Pro Arg Pro Ser Glu Asp Asn Val Pro Ala Lys Pro Arg Leu Leu 90 Glu Ser Leu Ser Thr Tyr Leu Gin Met Arg Cys Val Arg Glu Asp Ala 100 105 110 His Val Ser Thr Ala Asp Gin Leu Val Glu Tyr Gin Ala Gly Arg Lys 115 120 125 WO 96/06159 PCT/US95/10194 235 Thr His Asp Ser Leu His Ala Cys Ser Val Tyr Arg Glu Leu Gin Ala 130 135 140 Phe Leu Val Asn Leu Ser Ser Phe Leu Asn Gly Cys Tyr Val Pro Gly 145 150 155 160 Val His Trp Leu Glu Pro Phe Gin Gin Gin Leu Val Met His Thr Phe 165 170 175 Phe Phe Leu Val Ser Ile Lys Ala Pro Gin Lys Thr His Gin Leu Phe 180 185 190 Gly Leu Phe Lys Gin Tyr Phe Gly Leu Phe Glu Thr Pro Asn Ser Val 195 200 205 Leu Gin Thr Phe Lys Gin Lys Ala Ser Val Phe Leu Ile Pro Arg Arg 210 215 220 His Gly Lys Thr Trp Ile Val Val Ala Iie Ile Ser Met Leu Leu Ala 225 230 235 240 Ser Val Glu Asn Ile Asn Ile Gly Tyr Val Ala His Gin Lys His Val 245 250 255 Ala Asn Ser Val Phe Ala Glu Ile Ile Lys Thr Leu Cys Arg Trp Phe 260 265 270 Pro Pro Lys Asn Leu Asn Ile Lys Lys Glu Asn Gly Thr Ile Ile Tyr 275 280 285 Thr Arg Pro Gly Gly Arg Ser Ser Ser Leu Met Cys Ala Thr Cys Phe 290 295 300 Asn Lys Asn 305 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 1365 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1365 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: ATG GAT GCG CAT GCT ATC AAC GAA AGA TAC GTA GGT CCT CGC TGC CAC 48 Met Asp Ala His Ala Ile Asn Glu Arg Tyr Val Gly Pro Arg Cys His 1 5 10 CGT TTG GCC CAC GTG GTG CTG CCT AGG ACC TTT CTG CTG CAT CAC GCC 96 Arg Leu Ala His Val Val Leu Pro Arg Thr Phe Leu Leu His His Ala 25 ATA CCC CTG GAG CCC GAG ATC ATC TTT TCC ACC TAC ACC CGG TTC AGC 144 WO 96/06159 WO 9606159PCT/US95/10194 Ile

CGG

Arg

CTG

Leu

CTT

Leu

ATC

Ile

GTC

Val

AAT

Asn

GCC

Ala 1~45

CCG

Pro

CTC

Leu

GAG

Glu

CAG

Gin

AGO

Ser 225 ACT2 Thr2 GGT C Gly C GAA C Glu G Pr

TC

Se: Prc

AG(

Sei

TCC

Ser Arc

AAI

Asn 130

GTA

Val

GAO

Asp

CGC

Arg

GCT

Aa k.AG Lys 210

FTC

?he 1 AC ksn

;GC

fly

AG

~lu oLeu G CCA r Pro 0 )Gly

CTG

Leu

GTA

Val

TTT

Phe 115

GCG

*Ala Al a

CCG

Pro

GCA

Ala

CCG

Pro 195

GGG

Gly

AAO

Asn

GTG

Val

CT

Arg C Pro\ 275 Gli.

GGC

G1

GAG

Glu

CCT

Pro

CTG

Leu 100

CTC

Leu

CGG

Arg

CAC

His

GOA

Ala

GGG

Gly 180

GGA

Gly

CCA

Pro

CC

Pro rAC ryr

"AG

fIn

;TG

Tal 1Pro

TCA

SSer

GAA

Glu

CTG

Leu

CGC

Arg

TAT

Tyr

AGT

Ser

OCT

Pro

CTC

Leu 165

TCA

Ser

ACC

Thr

GCG

Ala

GTO

Val2 TOG C Ser C 245 GAA C Glu I CCC C Pro P Gli

TC(

Se

AAC

Asr 70 Phe

ATT

Ile

CTA

Leu

CG

Pro

TTG

Leu IS0

ACC

Thr

GAC

Asp

GTG

VJal CG0 %.rg

AT

.sn ~30

GT

fly

;AC

~sp

CG

'ro Arg 55 Gin

TOO

Ser

TOO

Ser

TOT

Ser

ACC

Thr 135

GAG

Glu

CT

Arg

GGA

Gly

CGT

Arg

ACA

Thr 215

GC

Ala2

GOT

Ala I GAO I2 Asp I2 OCA C Pro P 2 l

CGC

Arc Lei.

CAC

His

TGC

Cyc

OTG

Leu 120

GTT

Val.

GAA

Glu

GGA

Gly

GAC

Asp

GGA

G

1 y 200

CGC

krg 3AT ksp ~ro

'GG

'rp

CG

'ro :80 ePhf 0

TTC

SLeu

'GC

1Ala

GAT

Asp

CT

Pro 105

GTG

Val

GAO

Asp

CTG

Leu

COG

Pro

CGO

Arg 185

GAA

Glu

CAC

His

GTA

Val

TAO

Tyr

OTG

Leu 265

GGC

Gly 236 Ser

GTG

Val

TOT

*Ser

GGG

Gly 90

*GGT

Gly

GTG

Val

GGG

Gly

CAG

Gin

TTG

Leu 170 GC 2 Ala AGO C Ser I AGG C Arg P COO G Pro A 2 TAT G Tyr V 250 COG A Pro I TTA G Leu V Th:

GT(

Va T02 Se2 75

AAC

Asr TC2 Ser

GCT

Ala Val

A.GG

krg 155

CAG

.,In kCT rhr

:TA

~eu

CA

'ro

;CT

~la 35

TG

al1

TA

le

TG

al.

r Tyr Thr G TGT GGG O ys Giy SCOT TOT Pro Ser TTT CAT 1Phe His AAT OTT Asn Leu ATG GGG Met Gly 125 TOG COG Ser Pro 140 OTG GOG Leu Ala GTO OTG Val Leu CAC CAC His His GAO COG Asp Pro 205 000 000 Pro Pro 220 ACC TGG Thr Trp TGT GTT Cys Val OCA OTG 2 Pro Leu 1 TTC ATG C Phe Met 285 Ar

AA.

GG'

Gl Prc AG2 Se2

GOC

Al a oo; Pro 0GT Arg

ACC

Thr

ATG

Met 190

OCT

Pro

GTG

Val

CGA

krg rAC Fyr

.GC

er

AC

~sp gPhe C. GT s Arg F TTG {Leu k TTT D Phe E' TT Leu

GGA

Gly

GAG

Glu

GOT

Ala

GGC

Gly 175

GOG

Al a

GTT

Val

OGA

Arg

GAO

Asp2

GAAC

Giu 2 255 TTO C Phe F GAO T2 Asp L Ser

GTC

Val

GOG

Al a

GAO

Asp

ACT

Thr

CGG

Arg

GGO

Gly

AOG

Thr 160

OTT

Leu

OTO

Leu

TCA

Ser

CTG

Leu 3CO k1 a

'GC

~rg

:CA

~ro

~TG

~eu 192 240 288 336 384 432 480 528 57 6 624 672 720 768 sic 864 912 TTO ATT Phe Ile 290 AAC ACG AAG CAG Asn Thr Lys Gin TGC GAO TTT GTG GAO ACG OTA GAG GOC GC Cys Asp Phe Val Asp Thr 295 300 Leu Glu Ala Ala WO 96/06159 WO 96/6 159PCT/US95/10194 237 TGT CGC ACG CAA GGC TAO ACG TTG AGA CAG OGO GTG OCT GTC Cys 305

OCT

Pro

GCG

Al a

AGA

Arg

GTG

Val1

CGC

Arg 385

AAA

Lys

GTG

Val

TTT

Phe

ATT

Ile Arg Thr Gin Gly CG C Arg

TGC

Cys

GOT

Al a

TTA

Leu 370

GGC

Gly

CAG

Gin

ATC

Ile

GCT

Al a

CGA

Arg 450 GAO GCG Asp Ala OTA GTG Leu Val 340 GOC AOG Ala Thr 355 GGA TTA Gly Leu GTA AAO Val Asn 000 GAT Pro Asp GTO ACA Val Thr 420 ATT AAA Ile Lys 435 GGC CGO Gly Arg

GAA

Glu 325

TTA

Leu

TCO

Ser

TGG

Trp

TGT

Cys

GTG

Val 405

COO

Pro

GCC

Ala

TAT

Tyr Tyr 310

ATO

Ile

CGG

Arg

COG

Pro

GAA

Glu

GGC

Gly 390

CAA

Gin

GCA

Ala

CGC

Arg

GGC

Gly Thr Leu Arg Gin Arg 315 000 ATT Ala Ile Val Pro Val GCA GAC Ala Asp GGG CTG Gly Leu CCC CTT Pro Leu 360 AGO OGO Ser Arg 375 GGC ACG Giy Thr AAG ACA Lys Thr TTG GAA Leu Giu TAT AGG Tyr Arg 440

TAG

GCA OTT Ala Val 330 GCT TOO Ala Ser 345 GGO OGO Giy Arg CCC CAC Pro His GAO OGT Asp Gly GTC AGO Val Ser 410 GCC TG Ala Trp, 425

AAA

Lys

GAG

Giu

CAC

His

ACT

Thr

GAO

Asp 395

GG

Gly

CTT

Leu

TCO

Ser

GOT

Al a

GC

Ala

CTA

Leu 380

TGO

Trp

AGT

Ser

GTG

Val

CAC

His

AGT

Ser

TGC

Cys 365

GT

Gly

TTA

Leu

OTT

Leu

TTA

Leu TTT TTA Phe Leu 335 GOC TGG Ala Trp, 350 TGG ATG Trp Met TTG GAG Leu Giu GAG ATT Glu Ile GTG GOA Val Ala 415 OCT GGG Pro Gly 430 320

GAG

Glu

ATA

Ile

GAO

Asp

TTA

Leu

TTA

Leu 400

TGO

Cys

GGT

Gly 1008 1056 1104 1152 1200 1248 1296 1344 1365 GOG TOG AAG GAG GAT OTG OTG TTC Ala Ser Lys Glu Asp Leu Val Phe 445 INFORMATION FOR SEQ ID NO:33: Met Asp Arg Leu Ile Pro Arg Ser Leu Pro SEQUENCE CHARACTERISTICS: LENGTH: 454 amino acids TYPE: amino acid TOPOLOGY: linear ii) MOLECULE TYPE: protein xi) SEQUENCE DESCRIPTION: SEQ ID Ala His Ala Ile Asn Glu Arg Tyr 10 Ala His Val Val Leu Pro Arg Thr 1 25 Leu Glu Pro Glu Ile Ile Phe Ser 1 40 Pro Gly Ser Ser Arg Arg Leu Val 55 Gly Giu Glu Asn Gln Leu Ala Ser S 70 40: 33: Tal Gly Pro Arg Ovs His ~he Leu Leu His His Ala ~hr Tyr Thr Arg Phe Ser ~al Cys Gly Lys Arg Val er Pro Ser Gly Leu Ala 75 WO 96/06159 PCT/US95/10194 238 Leu Ser Leu Pro Leu Phe Ser His Asp Gly Asn Phe His Pro Phe Asp 90 Ile Ser Val Leu Arg Ile Ser Cys Pro Gly Ser Asn Leu Ser Leu Thr 100 105 110 Val Arg Phe Leu Tyr Leu Ser Leu Val Val Ala Met Gly Ala Gly Arg 115 120 125 Asn Asn Ala Arg Ser Pro Thr Val Asp Gly Val Ser Pro Pro Glu Gly 130 135 140 Ala Val Ala His Pro Leu Glu Glu Leu Gin Arg Leu Ala Arg Ala Thr 145 150 155 160 Pro Asp Pro Ala Leu Thr Arg Gly Pro Leu Gin Val Leu Thr Gly Leu 165 170 175 Leu Arg Ala Gly Ser Asp Gly Asp Arg Ala Thr His His Met Ala Leu 180 185 190 Glu Ala Pro Gly Thr Val Arg Gly Glu Ser Leu Asp Pro Pro Val Ser 195 200 205 Gin Lys Gly Pro Ala Arg Thr Arg His Arg Pro Pro Pro Val Arg Leu 210 215 220 Ser Phe Asn Pro Val Asn Ala Asp Val Pro Ala Thr Trp Arg Asp Ala 225 230 235 240 Thr Asn Val Tyr Ser Gly Ala Pro Tyr Tyr Val Cys Val Tyr Glu Arg 245 250 255 Gly Gly Arg Gin Glu Asp Asp Trp Leu Pro Ile Pro Leu Ser Phe Pro 260 265 270 Glu Glu Pro Val Pro Pro Pro Pro Gly Leu Val Phe Met Asp Asp Leu 275 280 285 Phe Ile Asn Thr Lys Gin Cys Asp Phe Val Asp Thr Leu Glu Ala Ala 290 295 300 Cys Arg Thr Gin Gly Tyr Thr Leu Arg Gin Arg Val Pro Val Ala Ile 305 310 315 320 Pro Arg Asp Ala Glu Ile Ala Asp Ala Val Lys Ser His Phe Leu Glu 325 330 335 Ala Cys Leu Val Leu Arg Gly Leu Ala Ser Glu Ala Ser Ala Trp Ile 340 345 350 Arg Ala Ala Thr Ser Pro Pro Leu Gly Arg His Ala Cys Trp Met Asp 355 360 365 Val Leu Gly Leu Trp Glu Ser Arg Pro His Thr Leu Gly Leu Glu Leu 370 375 380 Arg Gly Val Asn Cys Gly Gly Thr Asp Gly Asp Trp Leu Glu Ile Leu 385 390 395 400 Lys Gin Pro Asp Val Gin Lys Thr Val Ser Gly Ser Leu Val Ala Cys 405 410 415 Val Ile Val Thr Pro Ala Leu Glu Ala Trp Leu Val Leu Pro Gly Gly 420 425 430 Phe Ala Ile Lys Ala Arg Tyr Arg Ala Ser Lys Glu Asp Leu Val Phe WO 96/06159 PCT/US95/10194 239 435 440 445 Ile Arg Gly Arg Tyr Gly 450 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 984 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..984 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: ATG TTT GCT TTG AGC TCG CTC GTG TCC GAG GGT GAC CCG GAG GTG ACC 48 Met Phe Ala Leu Ser Ser Leu Val Ser Glu Gly Asp Pro Glu Val Thr 1 5 10 AGT AGG TAC GTC AAG GGC GTA CAA CTT GCC CTG GAC CTT AGC GAG AAC 96 Ser Arg Tyr Val Lys Gly Val Gin Leu Ala Leu Asp Leu Ser Glu Asn 25 ACA CCT GGA CAA TTT AAG TTG ATA GAA ACT CCC CTG AAC AGC TTC CTC 144 Thr Pro Gly Gin Phe Lys Leu Ile Glu Thr Pro Leu Asn Ser Phe Leu 40 TTG GTT TCC AAC GTG ATG CCC GAG GTC CAG CCA ATC TGC AGT GGC CGG 192 Leu Val Ser Asn Val Met Pro Glu Val Gin Pro Ile Cys Ser Gly Arg 55 CCG GCC TTG CGG CCA GAC TTT AGT AAT CTC CAC TTG CCT AGA CTG GAG 240 Pro Ala Leu Arg Pro Asp Phe Ser Asn Leu His Leu Pro Arg Leu Glu 70 75 AAG CTC CAG AGA GTC CTC GGG CAG GGT TTC GGG GCG GCG GGT GAG GAA 288 Lys Leu Gin Arg Val Leu Gly Gin Gly Phe Gly Ala Ala Gly Glu Glu 90 ATC GCA CTG GAC CCG TCT CAC GTA GAA ACA CAC GAA AAG GGC CAG GTG 336 Ile Ala Leu Asp Pro Ser His Val Glu Thr His Glu Lys Gly Gin Val 100 105 110 TTC TAC AAC CAC TAT GCT ACC GAG GAG TGG ACG TGG GCT TTG ACT CTG 384 Phe Tyr Asn His Tyr Ala Thr Glu Glu Trp Thr Trp Ala Leu Thr Leu 115 120 125 AAT AAG GAT GCG CTC CTT CGG GAG GCT GTA GAT GGC CTG TGT GAC CCC 432 Asn Lys Asp Ala Leu Leu Arg Giu Ala Val Asp Gly Leu Cys Asp Pro 130 135 140 GGA ACT TGG AAG GGT CTT CTT CCT GAC GAC CCC CTT CCG TTG CTA TGG 480 Gly Thr Trp Lys Gly Leu Leu Pro Asp Asp Pro Leu Pro Leu Leu Trp 145 150 155 160 WO 96/06159 WO 96/6 159PCT/US95/10194 240

TGT

CTG CTG TTC Leu Leu Phe AAC GGA CCC GCC TCT TTT CGG GCC GAC TGT TGC CTG Asn Gly Pro Ala Ser 165 Phe Cys Arg Ala Asp Cys Cys Leu 170 175

TAC

Tyr

ATG

Met Lys

GCT

Ala 225

CAG

Gin

CTC

Leu

ACC

Thr

ATA

Ile

CTA

Leu 305

GAG

Glu

AAG

Lys

TAC

Tyr

TAC

Tyr 210

TGC

Cys

ATG

Met

TTG

Leu

CAC

His

ACA

Thr 290

ATC

Ile

AAT

Asn

CAG

Gin

GCT

Ala 195

ACC

Thr

CGC

Arg

AAA

Lys

TGT

Cys

GTG

Val 275

GGC

Gly

ATC

Ile

GGA

Gly

CAC

His 180

CCC

Pro

AAG

Lys

CCG

Pro

ATC

le

CAC

His 260

GGT

Gly

AAT

Asn

CCA

Pro

CTC

Leu

TGC

Cys

AAA

Lys

TTT

Phe

CCA

Pro

ATA

Ile 245

ATA

Ile

GGA

Gly

GTT

Val

TCG

Ser

AAC

Asn 325

GGT

Gly

CGG

Arg

CTA

Leu

TTC

Phe 230

GAT

Asp

TAT

Tyr

ATC

Ile

CAG

Gin

TAT

Tyr 310

CAA

Gin

TAC

Tyr

GAT

Asp

TAC

Tyr 215

GCT

Al a

GCT

Al a

CAG

Gin

CTA

Leu

ACC

Thr 295

GAC

Asp

CTC

Leu

CCG

Pro

CTT

Leu 200

GGA

Giy

ACT

Thr

TCC

Ser

CA.A

Gin

CTG

Leu 280

CAA

Gin

GGC

Giy 185

TTG

Leu

GAT

Asp

TCT

Ser

GAC

Asp

AAT

Asn 265

TTG

Leu

AGG

Arg

CCG

Pro

TCG

Ser

TTT

Phe

CGG

Arg

ACT

Thr 250

AGC

Ser

AGT

Ser

TGT

Cys

GTG

Val

TTC

Phe

TCC

Ser

ATA

Ile 235

TAC

Tyr

ATA

Ile

GGA

Gly

CCA

Pro

CTA

Leu GT T Val

GGG

Gly 220

CAA

Gin

ATT

Ile

ATT

Ile

AAA

Lys

ACT

Thr 300

CTT

Leu

AAT

Asn 205

ACA

Thr

AGG

Arg

TCC

Ser

GCG

Al a

GGG

Gly 285

ACG

Thr

CCA

Pro 190

CAT

His

TGG

Trp

GTA

Val

CAC

His

GGT

Gly 270

ACC

Thr

GGC

Gly

GGT

Gly

GCC

Al a

GCG

Al a

GTG

Val1

ACC

Thr 255

CAG

Gin

CAG

Gin

GAC

Asp

CAC

His

CTG

Leu

GCG

Ala

AGT

Ser 240

TGC

Cys

GGG

Gly

TAT

Tyr

TAT

Tyr

AAG

Lys 320 576 624 672 720 768 816 864 912 960 ATA CCG GCG ATC Ile Pro Ala Ile 315

TAA

ATC ACC ATG ATC Ile Thr Met Ile INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 327 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:35: Met Phe Ala Leu Ser Ser Leu Val Ser Glu Gly Asp Pro Giu Val Thr 1 5 10 Ser Arg Tyr Val Lys Gly Val Gin Leu Ala Leu Asp Leu Ser Giu Asn 25 Thr Pro Gly Gin Phe Lys Leu Ile Glu Thr Pro Leu Asn Ser Phe Leu 40 Leu Val Ser Asn Val Met Pro Giu Val Gin Pro Ile Cys Ser Gly Arg 55 WO 96/06159 WO 96/6 159PCTIUS95/10194 Pro Ala Leu Arg Pro Lys Ile Phe Asn Giy 145 Leu Tyr Met Lys Ala 225 Gin Leu Thr Ile Leu 305 Glu Leu Ala Tyr Lys 130 Thr Leu Lvs Tyr Tyr 210 Cys Met Leu His Thr 290 Ile Asn Gin Leu Asn 115 Asp Trp Phe Gin Ala 195 Thr Arg Lys Cys Val 275 Gly Ile Gly Arg Asp 100 His Al a Lys Asn His 180 Pro Lys Pro Ile His 260 Gly Asn Pro Leu Val Pro Tyr Leu Gly Gly 165 Cys Lys Phe Pro Ile 245 Ile Gly Val Ser Asn 325 Asp) Leu Ser Ala Leu Leu 150 Pro Gly Arg Leu Phe 230 Asp Tyr Ile Gin Tvr 310 Gin Gly His Thr Arg 135 Leu Ala Tyr Asp Tyr 215 Al a Al a Gin Leu Thr 295 Asp Leu Gin Val Glu 120 Glu Pro Ser Pro Leu 200 Gly Thr Ser Gin Leu 280 Gin Gly Glu 105 Giu Ala Asp Phe Gly 185 Leu Asp Ser Asp Asn 265 Leu Arg Phe 90 Thr Trp, Val Asp Cys 170 Pro Ser Phe Arg Thr 250 Ser Ser Cys Phe Ser Asn Leu His Leu Pro Arg Leu Glu 75 Gly His Thr Asp Pro 155 Arg Val1 Phe Ser Ile 235 Tyr Ile Gly Pro Ile 315 Ala Glu Trp Gly 140 Leu Al a Leu Val Gly 220 Gin Ile Ile Lys Thr 300 Ala Lys Ala 125 Leu Pro Asp Leu Asn 205 Thr Arg Ser Al a Gly 285 Thr Gly Gly 110 Leu Cys Leu Cys Pro 190 His Trp Val His Gly 270 Thr Gly Giu Gin Thr Asp Leu Cys 175 Gly Ala Ala Val Thr 255 Gin Gin Asp Giu Val Leu Pro Trp 160 Leu His Leu Al a Ser 240 Cys Gly Tyr Tyr Lys 320 Ile Pro Ala Ile Thr Met Ile INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 330 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N SEQUENCE DESCRIPTION: SEQ ID NO:36: WO 96/06159 WO 9606159PCT/US95/10194 242

GGATCCCTCT

CAACACCCAG

CGAATCCAAC

TATTCTGCAG

TGATGTAAAT

ATATCGTCTG

GACAACCTTC

CTAGCAGTGC

GGATTTGACC

CAGCTGTTGG

ATGGCGGAAC

GACGTAGACA

AGATAAAAAA CGTATATGCC CCCTTTTTTC AGTGGGACAG TACCCCCATT TTTTAGCCGA AAGGATTCCA CCATTGTGCT CCGTGTTCCC CATGGTCGTG CCGCAGCAAC TGGGGCACGC TGTACCACAT CTACTCCAAA ATATCGGCCG GGGCCCCGGA TTGATCTATA TACCACCAAT GTGTCATTTA TGGGGCGCAC

ACACGGATCC

INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 627 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GGATCCGCTG GCAGGTGGGC GCGCACCTCG TCGGGTAGCT TGGAGACAAA CAGCTCCAGG

CCAGTCCGCG

TTAGTCCGGA

TGCGCACCGG

GGGCTCAAAC

GCATTCTTTG

GCGATGGTGC

ATCTCGGCCT

CCCAGGGCCG

AACAGGGTGC

GGACGTGGGT

CCGTAGCGCC

GAAGATAGGG

TTGTCGGAGC

CTGCCCAGAC

GAAGTAGTGG

GCACCGTTTT

GCTGTACGTC

GTCCGGTGGC

TGTGAAACAA

GTATGCTCCG

TGCAGGTGCC

CCCTTGGGAA

TGTCGCGATA

ACACCACTGT

TAGAGATGGA

TAAGAAACCC

CTTGGCGAAT

ATACAGGCCG

CAGGTTGCAA

TGGATCC

TCACCACCGG

GCCGCTGAAC

GAGGTTAGGG

CTGCTGGGGG

GCAGACTGCC

CCCAGGGTGG

ATGCGACGAA

GTGAGGGCCC

GGCCGGGTCA

CAGCTCCAGG

TAGGTGTCCG

ATCATCCTTC

AGGGCGTTGC

GGACTCCCGC

ATCGGCTGTG

CCTGGGTCTG

TGCGATCTGT

GTCTCCAAGA

GTCCGTCCGT

TCAGGGAGAT

AGGAGTGGTG

TCCCTGCAGC

CGCACGGGGT

TCCGCCTGGA

120 180 240 300 360 420 480 540 600 627 GGCCGCGAAT ACCCCTCTGC ACGCTGCTGT INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 233 base pairs TYPE: nucleic acid STRAN'DEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: WO 96/06159 PCT/US95/10194 243 AGCCGAAAGG ATTCCACCAT TGTGCTCGAA TCCAACGGAT TTGACCCCGT GTTCCCCATG GTCGTGCCGC AGCAACTGGG GCACGCTATT CTGCAGCAGC TGTTGGTGTA CCACATCTAC 120 TCCAAAATAT CGGCCGGGGC CCCGGATGAT GTAAATATGG CGGAACTTGA TCTATATACC 180 ACCAATGTGT CATTTATGGG GCGCACATAT CGTCTGGACG TAGACAACAC GGA 233 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 328 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: GAAATTACCC ACGAGATCGC TTCCCTGCAC ACCGCACTTG GCTACTCATC AGTCATCGCC CCGGCCCACG TGGCCGCCAT AACTACAGAC ATGGGAGTAC ATTGTCAGGA CCTCTTTATG 120 ATTTTCCCAG GGGACGCGTA TCAGGACCGC CAGCTGCATG ACTATATCAA AATGAAAGCG 180 GGCGTGCAAA CCGGCTCACC GGGAAACAGA ATGGATCACG TGGGATACAC TGCTGGGGTT 240 CCTCGCTGCG AGAACCTGCC CGGTTTGAGT CATGGTCAGC TGGCAACCTG CGAGATAATT 300 CCCACGCCGG TCACATCTGA CGTTGCCT 328 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 132 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID AACACGTCAT GTGCAGGAGT GACATTGTGC CGCGGAGAAA CTCAGACCGC ATCCCGTAAC CACACTGAGT GGGAAAATCT GCTGGCTATG TTTTCTGTGA TTATCTATGC CTTAGATCAC 120 AACTGTCACC CG 132 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 96/06159 PCT/US95/10194 244 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: AGCCGAAAGG ATTCCACCAT TCCGTGTTGT CTACGTCCAG INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: GAAATTACCC ACGAGATCGC AGGCAACGTC AGATGTGA 38 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 46 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: AACACGTCAT GTGCAGGAGT GACCGGGTGA CAGTTGTGAT CTAAGG 46 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: WO 96/06159 PCT/US95/10194 245 ACAGGGCTGG TTGCCCAGGG T 21 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID AGTTGCAAAC CAGACCTCAG 7n

Claims (33)

1. An isolated DNA molecule which is at least nucleotides in length and uniquely defines a herpesvirus associated with Kaposi's sarcoma.

2. The isolated DNA molecule of claim 1, wherein the isolated DNA molecule is cDNA.

3. The isolated DNA molecule of claim 1, wherein the isolated DNA molecule is genomic DNA.

4. An isolated RNA molecule which is derived from the isolated nucleic acid molecule of claim 1. 15 5. The isolated DNA molecule of claim 1 which is labelled with a detectable marker. The isolated DNA molecule of claim 5, wherein the marker is a radioactive label, or a calorimetric, a luminescent, or a fluorescent marker.

7. A replicable vector comprising the isolated DNA molecule of claim 1.

8. A plasmid, cosmid, X phage or YAC comprising at least 12 nucleotides of the isolated DNA molecule of claim 1.

9. A host cell containing the vector of claim 7. The cell of claim 9 which is a eukaryotic cell.

11. The cell of claim 9 which is a bacterial cell.

12. An isolated herpesvirus associated with Kaposi's sarcoma. -247-

13. A nucleic acid molecule of at least 14 nucleotides capable of specifically hybridizing with the isolated DNA molecule of claim 1.

14. A DNA molecule of claim 13. A nucleic acid molecule of at least 14 nucleotides capable of specifically hybridizing with a nucleic acid molecule which is complementary to the isolated DNA molecule of claim 1.

16. A nucleic acid molecule of claim 15 wherein the 15 nucleic acid molecule is capable of hybridizing :'with moderate stringency to at least a portion of a nucleotide sequence as shown in Figure 3A (SEQ ID NO: 1). 20 17. An isolated peptide encoded by at least 12 nucleotides of a nucleic acid molecule with a sequence as set forth in (SEQ ID NOs: 1-37)

18. A host cell which expresses the peptide of claim 25 17.

19. The isolated peptide of claim 17, wherein the peptide is linked to a second peptide to form a fusion protein. The fusion protein of claim 17, wherein the second peptide is beta-galactosidase.

21. An antibody which specifically binds to the peptide encoded by the isolated DNA molecule of claim 17. -248-

22. The antibody of claim 21, wherein the antibody is monoclonal antibody.

23. The antibody of claim 21, wherein the antibody is a polyclonal antibody.

24. The antibody of claim 21, wherein the antibody is labelled with a detectable marker.

25. The labelled antibody of claim 24, wherein the marker is a radioactive label, or a calorimetric, a luminescent, or a fluorescent marker.

26. An antisense molecule capable of hybridizing to S• 15 the isolated DNA molecule of claim 1.

27. The antisense molecule of claim 26, wherein the molecule is a DNA.

28. The antisense molecule of claim 26, wherein the molecule is a RNA. 9**e9*

29. A triplex oligonucleotide capable of hybridizing with a double stranded isolated DNA molecule of claim 1. A transgenic nonhuman mammal which comprises at least 12 nucleotides of the isolated DNA molecule of claim 1 introduced into the mammal at an embryonic stage.

31. A vaccine which comprises an effective immunizing amount of the isolated herpesvirus of claim 12 and a suitable pharmaceutical carrier.

32. A method of diagnosing Kaposi's sarcoma which comprises: obtaining a nucleic acid molecule -249- from a tumor lesion of the subject; (b) contacting the nucleic acid molecule with the labelled nucleic acid molecule of claim 13 under hybridizing conditions; and determining the presence of the nucleic acid molecule hybridized, the presence of which is indicative of Kaposi's sarcoma in the subject, thereby diagnosing Kaposi's sarcoma.

33. The method of claim 32 wherein the DNA molecule from the tumor lesion is amplified before step

34. A method of diagnosing Kaposi's sarcoma which comprises: obtaining a nucleic acid molecule from a suitable bodily fluid of a subject; (b) contacting the nucleic acid molecule with the labelled nucleic acid molecule of claim 13 under hybridizing conditions; and determining the presence of the nucleic acid molecule hybridized, the presence of which is indicative of Kaposi's sarcoma in the subject, thereby diagnosing Kaposi's sarcoma.

35. A method of diagnosing a herpesvirus associated with Kaposi's sarcoma which comprises (a) obtaining a suitable bodily fluid sample from a subject, contacting the suitable bodily fluid of the subject to a support having already bound thereto a Kaposi's sarcoma antibody, so as to bind Kaposi's sarcoma antibody to a specific Kaposi's sarcoma antigen, removing unbound bodily fluid from the support, and (d) determining the level of Kaposi's sarcoma antibody bound by the Kaposi's sarcoma antigen, thereby diagnosing Kaposi's sarcoma. S. S S 20 25 -250-

36. A method of diagnosing a herpesvirus associated with Kaposi's sarcoma which comprises (a) obtaining a suitable bodily fluid sample from a subject, contacting the suitable bodily fluid of the subject to a support having already bound thereto a Kaposi's sarcoma antigen, so as to bind Kaposi's sarcoma antigen to a specific Kaposi's sarcoma antibody, removing unbound bodily fluid from the support, and determining the level of the Kaposi's sarcoma antigen bound by the Kaposi's sarcoma antibody, thereby diagnosing Kaposi's sarcoma. .37. A method of treating a subject with Kaposi's 15 sarcoma, comprising administering to the subject 'an effective amount of an antisense molecule of claim 26 under conditions such that the antisense molecule selectively enters a tumor cell of the 0subject, so as to treat the subject.

38. A method for treating a subject with Kaposi's sarcoma (KS) comprising administering to the subject having a human herpesvirus-associated KS a pharmaceutically effective amount of an 25 antiviral agent in a pharmaceutically acceptable carrier, wherein the agent is effective to treat the subject with KS-associated human herpes virus of claim 12.

39. A method of prophylaxis or treatment for Kaposi's sarcoma (KS) by administering to a subject at risk for KS, an antibody that binds to the human herpesvirus of claim 12 in a pharmaceutically acceptable carrier. A method of vaccinating a subject against SKaposi's sarcoma, comprising administering to the -251- subject an effective amount of the peptide of claim 17, and a suitable acceptable carrier, thereby vaccinating the subject.

41. A method of immunizing a subject against a disease caused by the herpesvirus associated with Kaposi's sarcoma which comprises administering to the subject an effective immunizing dose of the vaccine of claim 31.

42. A method for preventing the development or transmission of herpesvirus associated Kaposi's sarcoma in a subject by treating a subject with Kaposi's sarcoma (KS) comprising administering to 15 the subject having a human herpesvirus-associated KS a pharmaceutically effective amount of an antiviral agent in a pharmaceutically acceptable carrier, wherein the agent is effective to preventing the development or transmission of the 20 KS-associated human herpes virus of claim 12. 4 DATED this 26TH day of NOVEMBER, 1998 4 The Trustees of Columbia University in the City of New York by DAVIES COLLISON CAVE Patent Attorneys for the Applicants