CA2196892A1 - Unique associated kaposi's sarcoma virus sequences and uses thereof - Google Patents

Unique associated kaposi's sarcoma virus sequences and uses thereof

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Publication number
CA2196892A1
CA2196892A1 CA002196892A CA2196892A CA2196892A1 CA 2196892 A1 CA2196892 A1 CA 2196892A1 CA 002196892 A CA002196892 A CA 002196892A CA 2196892 A CA2196892 A CA 2196892A CA 2196892 A1 CA2196892 A1 CA 2196892A1
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sarcoma
kaposi
dna
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nucleic acid
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Yuan Chang
Patrick S. Moore
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Columbia University in the City of New York
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Priority claimed from US08/343,101 external-priority patent/US5830759A/en
Priority claimed from US08/420,235 external-priority patent/US5801042A/en
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Publication of CA2196892A1 publication Critical patent/CA2196892A1/en
Abandoned legal-status Critical Current

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    • C12N2710/16011Herpesviridae
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    • C12N2710/16434Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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Abstract

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 an antibody specific to the peptide. Antisense and triplex oligonucleotide molecules are also provided. 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.

Description

J ~ WO96/06159 ~21 96892 P~ 0l3~

TT~TTO~E ASSOCIATED RAPOSI'S SARCONA VIRUS ~u- _~:S AND
USES TUEREOF

The invention disclosed herein was made with Government support under a co-operative agreement CCU210852 from the Centers for Disease Control and Prevention, of the Department of Health and Human Services. Accordingly, the U.S. Government has certain rights in this invention.

This application is a continuation-in-part application of U.S. Serial No. 08/420,235, filed on April 11, 1995 which is a r~ntinn~tion-in-part application of U.S.
Serial No. 08/343,101, filed on November 21, 1994, a continuation-in-part application of U.S. Serial No.
08/292,365, filed on August 18, 1994, which is hereby incorporated by reference.

Throughout this application, various publications may be referenced by Arabic numerals in brackets. Full citations for these pnhl;cati~n~ may be found at the end of each Exp~r;-- t~ Details Section. The disclosures of the publicatlons 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.

BAC~GROUND OF ~TTe lN V ~
Kaposi's sarcoma (KS) is the most common neoplasm occurring in persons with ac~uired immunodeficiency syndrome (AIDS). Approximately 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) . W096/06159 ~' 3; ~ 1 q68~2 PCT~S951101 ~

has an infectious etiology. Gay and bisexual AIDS
patients are approximately twenty times more likely than h ~h; 1 iac AIDS patientg to develop KS, and KS
may be associated with speciflc sexuaI practices among gay men with AIDS [6, 15, 55, 83~. KS is nn~
among adult AIDS~ ~patieh~s. infected through heterosexual or parenteral ~IV transmis~ion, or among pediatric AIDS patients infected through vertical HIV
transmission [77~. Agents previously suspected of causing KS include cytomegalovirus, hepatitis B virus, human papillomavirus, ~pstein-Barr =virus, human herpesvirus 6, human immunodeficiency virug (~IV), and Mycoplasma penetrans Ll8, 23, 85, 9l, 92]. Non-infectious enviL~ ~1 agents, such as ni~rite i nh~ 1 ~nt ~, also have been propoged 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, 90].

~ W096/06159 2 1 ~ 6 8 9 2 F~ "~iSI

S~M~ARY OF TEE 1~V~1~L_ 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 provi~es a method of vaccinating a 0 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.

WO96/06159 ~ PCTNS95/10l RRT~ n~r~TpTIoN OF T~E FI~n Firure 1: =~
Agarose gel electrophoregig 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 subse~uent 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 regUlted in a single band at 540 bp (lane 5). 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 nonsper;f;r~lly to both KS and non-KS human DNA.
Lane M is a molecular weight marker.

F$oure~ 2A-2B:
Hybridization of3~p-l~h~ 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 KS~27Bam 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 ~ WO96/06159 2 1 9 6 ~ 9 2 PCT~S95/10194 specimen. Seven of 8 additional RS DNA samples also hybridized to both sequences.

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

Fioures 4A-4B:
PCR amplification of a representative set of KS-derived DNA samples using KS33023~ 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 '~P end-labelled 25 bp internal oligonucleotide (Figure 3B) after transfer of the gel to a nitrocellulose filter. Negative samples in lanes 3 and 15 respectively lacked microscopically detectable K~3 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 KS33023;. Lane 20 is a negative control and Lane M is a molecular weight marker.
Fi~ure 5: _ _ Southern blot hybridization of KS330Bam and KS627Bam to AIDS-KS genomic DNA extracted from three subjects (lanes 1, 2, and 3) and digested 3D- 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: 1), at bp 134 in KS330Bam (Figure 3B, SEQ ID NO: 2) and bp 414 in KS627Bam (Figure 3C, SEQ ID NO: 3). RS330Bam and KS627Bam failed to hybridize to the same fragments in the digests indicating that the two sequences are WO96/061~9 ~ 2 1 q 6 8 92 P~ l9~

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.

Fiqure 6:
Comparison of amino ~acid homologies between EBV
ORF BDLF1, ~SVSA ORP 26 and-a 918 bp reading frame o~ the Kapo~si's sarcama agent which includes KS330Bam. A~ino acid identity is denoted by reverse lettering. ln HSVSA, ORF 26 enccdes a minor capsid VP23 which is a late gene ~ product.

F~qure 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.

Fiqure 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 ;~nt;~Al orientation, except ~or the region corresponding to the ORF 28 o~ HSVSA (middle schematic section). The shadiny for each open reading frame corresponds to the approximate ~
amino acid identity for the KSHV ORF compared to =this homolog i~ HSVSA. Noteworthy homologs that are present in this section o~ DNA include homologs to thymidine kinase (ORP21), gH

~ WO96106159 ' ~ 2 1 9 6 ~ 9 2 PCT~59~10194 ylycoprotein (ORF22), major capsid protein (ORF25) and the VP23 protein ~ORF26) which contains the original KS330Bam sequence derived by representational difference analysis.
Fi~re 9:
The ~200 kD antigen band appearing on a Western blot of KS patient sera against BCBL1 lysate (Bl) and Raji lysate (RA). ~ is molecular weight marker. The antigen is a doublet between ca. 210 kD and 240 kD.

Fi~ure 10:
5 control patient sera without KS (AlN, A2~, A3N, A4N and A5N). B1=BCBLl lysate, RA=Raji lysate.
The 220 kD band is absent from the Western blots using patient sera without KS.

Fiaure 11:
In this figure, 0.5 ml aliquots of the gradient have been fract;n~to~ ~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 using standard techniques. The figure shows that the major solubilized fraction of the KS~V genome bands ~i.e. is isolated) in - f~actions 42 through 48 of the gradient with a high concentration of the genome being present in rr~rt; nn 44. ~ gecond band of solubilized KS~V
DNA occurs in fractions 26 through ~2.

Fiaure 12:
Location, feature, and relative homologies of KS5 open reading frames compared to translation WO96/06159 ~ ' 2 1 9 68 92 PCT~595/l0l9 ~

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 BX~-6 and P3~3 prior to~and after adsorption with P3H3.
:' Fioure 14:
Genetic map of KS5, a 20.7 kb lambda phage clone insert derived from a human genomic library prepared from an ~IDS-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 in~i~ate~ 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 (B, 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. =~

Fl~ures 15A-15B:
Phylogenetic trees of~KSHV based on comparison of aligned amino acid sequences between herpesviruses for the MCP gene and for a ~WO96/06159 ~ ! 2 1 9 6 8 9 2 I~1l~,5/l~sl concatenated ~ine-gene set. The comparison of MCP Eequences (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 , h~rpesvirus 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 ~hadinovirus. 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 basi~ 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) Al ph~h~rpesvirinae: HSV1 and HSV2, herpes simplex virus types 1 and 2; EHV1, equine herpesvirus 1; PRV, pseudorabies virus; and VZV, varicella-zoster virus, 2) Betaherpesvirinae:
~CMV, 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.
W096~6l59 P~~ S

Fi~es 16A-16B: , , OE F gel electrophoresis of BCBL-l 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 dif~use 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 termina~l 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 autoradiogra,phs.
e 17~
Induction of KSHV and EBV replication in BCBL-1 with increasing ~nc~nt~ations of TPA. Each ~t~rm;n~ti~n was made in triplicate after 48 h o~ 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 increa3e in hybridizing genome for EBV and KSHV induced by TPA compared to uninduced BCBL-1. TPA at 20 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.

Fi~e~ 18A-l~C:
I~ situ hybridization with an ORF26 oligomer to BCBL-1, Raji and RCC-l cells. Hybridization occurred to nuclei~of KSHV infected BCB~-1 ~ WO96/06159 , - 2 ~1 9 6 8 9 2 PCT~595110194 (Figure 18A~, but not to uninfected Raji cells (Figure 18B). RCC-l, a Raji cell li~e derived by cultivation of Raji with BCBL-l in c ;r~ting chambers separated 4y a 0.45 ~ filter, shows rare cells with positive hybridlzation to the KSHV
ORF26 probe (Figure 18C).

Fir~ure~ l9A-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 l9A) and control (Figure l9B) sera show homogeneous ~taining of BHL-6 at l: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 l9C). P3H3 adsorption of control sera eliminates immunofluorescent staining of BHL-6 2Q (Figure l9D).

Ficures 2OA-2OB:
Longitudinal PCR examination for KSHV DNA of paired PBMC samples from AIDS-KS patients (A) and homosexual/bisexual AIDS patients without KS (B).
Time 0 is the date of KS onset for cases or other AIDS-~f;n;ng illness for controls. All samples were randomized and PY~m; n~ blindly. Overall, 7 of the KS patients were KS~V positive at both examinatiQn 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 r~m~'n;nr, KS patients were negative at both timepoints (open bars). Two homosexual/bisexual control PBMC samples without WO96/06159 ~ 2 ~tl ~9 6 8 9 2 PCT~595/lo KS converted from negative to positive and one control patient reverted from PCR positive to negative for KSHV DNA

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

Fiqure 22: :
PCR analysis of KS330233 in DN~ samples from patients with Kaposi's sarcoma and tumor controls.

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

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

Fi ~re 25:
Comparison of KS patients with and without a~tibody to KSHV p40.

Fiqure 26: ___ _ Prevalence of antibo~y ~t~tA~l~ by indirect ' In~fluorescence to KSHV antigens in chemically induced BCBL-l cells in HIY-1 positive patients with and without KS.

Fiqures 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.

i 1~1 96~92 WO96/061~9 PCT~S9~10194 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 lO~
SDS polyacrylamide gels.

Fiqures 28A-28D:
D~t~rt;r~ 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 (r~nt~;n;ng only EBV) that were uninduced 15 ~ or treated for 48 hrs with chemical ;n~llr;ng agents, n-butyrate, TPA, or a combination of the two chemicals. T -1-lots 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.
_ _ _ Fi~ures 29A-29F:
Detection of KSHV lytic cycle antigens by ;n~;rert 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.

~; t ! ~ 1 9 6 8 9 2 WO 96/06159 PCrlUSgS/IOI!~

1~
n~T~ Tr.r~n L)~;S-:Kl~ ~ lU-. OF T~ lN V~l~ L lUN

Def; n; tions The following standard abbreviations are used throughout the specification to indicate specific nucleotides: :

C=cytosine A=adenosine T=thymidine C=guanosine The term "nucleic acids~, as used herein, refers to either DNA or RNA. "Nucleic acid ser~uence~ or "polynucleotide sequence" refers to a single- or aouble-stranded polymer of deoxyribonucleotide or r;hnnnrlrntide 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 seriuence ~homologous to" or "complementary to", it is meant a nucleic acid that selectively hybridizes, duplexes or binds to viral DNA
sequences ~nro~nr proteins or portions thereof when the DNA sequences rnro~lnr~ the viral protein are present in a human genomic or cDNA library. A DNA
sequence which is homologous to a target sequence can include seriuences which are shorter or longer than the target sequence so long as they meet the functional test set forth. ~ybridization 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, 65OC 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

.. . .. . . . . . . . . . . . . .. . . ...

~ WO96/06159 PCT~595/10194 refers to a solution having a aodium rhl ~r; ~ and sodium citrate n~n~pntration of 6 times this amount or 0.9 M sodium chloride and l20 mM sodium citrate.
0 2XSSC referc to a solution 0.2 timeC the SSC
concentration or 0.03 M sodium chloride and 4 mM
sodium citrate.

The phrase "nucleic acid molecule ~n~ing" refers to a nucleic acid molecule which directs the expression of a~epecific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the R~A sequence that is translated into protein The nncl~;~ acid molecule include both the full length nucIeic 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 casgette~, 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 ~crr1 h~ hereir 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 NA sequence he term ~vector~, refers to viral expression systems, autonomous self-replicating circular DNA (plasmids), and ;n~ln~pc both expression and nonexpression plasmids Where a re~ 1;ncnt microorganism or cell W096~6159 PCTNS95/101 culture i8 described as hosting an "expres6ion vector,~ this includes both extrachromosomal circular D~A and D~A that has been incorporated into the host chromosome(s). Where a vector-is being m~;nt,7;n~r7. by r7 a host cell, the vector may either be stably replicated by the cells during mitosis as an Antnn~. ~s gtructure, or is incorporated within the host's genome.

The term ~'plasmid'~ refers:to an autQnomous circular DNA molecule capable of =~eplicatlon in a cell, and includes both the expression and nQnexpression types.
Where a recombinant microorganism or cell culture is described as hosting an~expression plasmid", this includes latent viral DNA integrated into the host chl, - (9). Where a plasmid is being m lntA;n~,'by a host cell, the plasmid is either being stably rep1icated 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~7 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 d~L ~pL iate nucleic acid sequence. The recombinant protein will not be found in association with proteins and other subcellular cnmpnn~ntc 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

~ WO96/06159 = PCT~S9~10194 "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 homolo~y 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 ~yL~ms GAP or BESTFIT using default gap which share at least go 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 appro~imately 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 95~
amino acid identity. Preferably, residue positions which are not i~ntn~l differ ky conservative amino WO96106159 ' ~ 9 ~ 8 9 2 PCT~S95/1019 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=o~ a protein. Examples include glutamine for asparagine or glutamiç acid for aspartic acid.

The phrase "substantially purifiedn or nisolated" when referring to~a herpesvirus peptide or protein, means a chemical compocition which ic essentially free of other cellular cnmpnn~nt~. It is preferably in a homogeneous state althougk 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 pr~ ;n~nt 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 i8 purified to greater than 95~, and most preferably the protein is purified to essential h~ eity, wherein other macromolecular species are not detected by conventional techniques.

The phrase "specifically binds to an antibodyr 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 herpesviru6. Thus, under designated 1 n~a~y conditions, the specified ~nt;ho~ bind to the herpesvirus antigenc and do not bind in a 6ignificant _ _ _ _ _ _ _ _ _ _ _ _ . _ ~ W096106159 '2 1 9 6 8 92 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 herpegvirus immnnng~n 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 ; ~nnA~say formats may be used to select antibodies gpe~lf;~l1y immunoreactive with a particular protein. For example, solid-phase ELISA ;r~nnnA~S~yS are rout;n~ly used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Tvane [32] for a description of ; ~no~cq~y formats and conditions that can be used to determine specific immunoreactivity.

~Biological sample" as used herein reiers 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. Ka~osis's Sarcoma (KS) - As80ciated Heroesvirus.

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 N0 1). In another embodiment the isolated DNA molecule is a 330 base pair ~ (bp) sequence. In another ~mho~ the isolated DNA molecule is a 12-50 bp sequence. In W096~6l59 ~i ~ zl 9 6 8 9 2 ~ 9~

another Pmh~; ! the isolated DNA molecule i9 a 30-37 bp se~uence.

In another embodiment the isolated DNA molecule is genomic DNA. In another embodiment the isolated DNA
molecule is cDNA. In another ~mho~irent a RNA i5 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 non-lQ coding regions of the isolated nucleic acid molecule.

Further, the DNA molecule above may be asbociated with lymphoproliferative diseases including, but not limited to: ~odgkin's disease, non-~odgkin's lymphoma, lymphatic leukemia, lymphosarcoma, splenomegaly, reticular cell sarcoma, Sezary's ~y~ldL , mycosis fungoides, central nervous system lymphoma, AIDS
related central nervous system lymphoma, post-transplant 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:
20 and 21), ORF25 (SEQ ID NOs: 2 and 3), ORF26 (SEQ ID
NOs a4 and 25), ORF27 (SEQ ID NOs_26 and 27), ORFa3 (SEQ ID NOs:a8 and 29), ORF29A (SEQ ID NOs:30 and 31), oRFagB (SEQ m NOs:4 and 5), ORF30 (SEQ ID''~0s:6~and 7~, ORF31 (SEQ ID NOs:8 and 9~, ORF32 (SEQ ID NOs:32 and 33), ORF33 (SBQ ID NOs: 10 and 11~, ORF34 (SEQ ID
NOs: 34 and 35~, or ORF35 lSEQ ID NOs:12 AND 13).

2 1 9~892 WO96~06159 PCT~S95/10194 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 NO3: 2 and 3), ORF26 (SEQ ID NOs:24 and 25), ORF27 (SEQ ID NOs:26 and 27), ORF28 (SFQ ID
NOs:28 and 29), ORF29A ~SEQ I~ NOs:30 and 31), ORF29B
~SEQ ID NOs:4 and 5), ORF30 (SEQ ID NOs:6 and 7), ORF31 (SEQ ID NOs:8 and 9), OR~32 (SEQ ID NOs:32 and 33), ORF33 (SEQ ID NOs: 10 and 11), ORF34 (SEQ ID NOs:
3g and 35), or ORF35 (SEQ ID NOs:12 AND 13).

For Example, TK i8 encoded by ORF 21; glycoprotein H
(gH) by ORF 22; major capsid protein (MCP) by ORF 25;
= 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, ~ phage or yeast arti~iri~' chromosome (YAC) which ~nnt~in.c 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 dige~ted 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 Wo96106159 PCT~S95/lO

and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector ~nrln~ a promoter such as the lac promoter a~d for transcription initiation the Shine-Dalgarno sequence and the start codon A~G. Similarly, a eukaryotic expression vector includes a heterologous or homologous promoter for =2NA polymerase II, a downstrea~ polyadenylation- signal, the~start codon AUG, and a termination codon ior detachment oi 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 ior constructing vectors in general.

This invention provides a host cell rnntA;n1ng 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 ~P~ yeast cells, fungal cells, insect cells and animal cells. Suitable animal cells include, but are not limited to Vero cells, ~eLa cells, Cos cells, CVl cells and varioufi primary ~ liAn cells.

2~ 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 Fig~ures 3A (SEQ ID N0:
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 ~ ~rrFesvirinae~ The classification of the herpesvirus may vary based on the=phenotypic or molecular characteristics which are known to those ~ skilled in the art.

~ WO96~6159 21 q6~'~2 PCT~59~10194 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: 33-40 The KS-associated human herpesvirus of the invention is associated with KS and is involved in the etiology of the disease. The t~y~n~r;c 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 g~r~PrpeSVirinae subfamily, on the basis of nucleic acid homology.

A Se~lPn~p ;~pnt;tv of ~he vir~l n~A ~n~ its protP; nf~, 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 l (SEQ ID NO:38) AGCCGAAAGG ATTCCACCAT TGTGCTCGAA TCCAACGGAT TTGACCCCGT
GTTCCCCATG GTCGTGCCGC AGCAACTGGG GCACGCTATT CTGCAGCAGC
CCACATCTAC TrrAAAATAT CGGCCGGGGC CCCGGATGAT
GTAAATATGG CGGAACTTGA TCTATATArc ACCAATGTGT CATTTATGGG
GCGCACATAT CGTCTGGACG TAr.~rAAr~ GGA

,', ~2l 96892 . WO96106159 'i ' ~ ' PCT~S9~1101 Probe 2 ~SEQ ID NO:39):
GA~ATTACCC ACGAGATCGC TTCCCTGCAC ~rrGr~rTTG GCTACTCATC
AGTCATCGCC CCGGCCCACG TGr.rrr.rr~T AACTACAGAC ATGGGAGTAC
ATTGTCAGGA CCTCTTTATG AL11"LC~AG GGGACGCGTA TCAGGACCGC
CAGCTGCATG ACTATATCAA AATGA~AGCG GGCGTGCAAA CCGGCTCACC
r.r.r.~r~r.~ ATGGATCACG TGGGATACAC TGCTGGGGTT CCTCGCTGCG
AGAACCTGCC CGGTTTGAGT CATGGTCAGC TGGCAACCTG rr.~r.~T~TT
Crr~rr~rrGG TCACATCTGA CGTTGCCT ~ =

Probe 3 ~SEQ ID NO: 40~: =
AACACGTCAT GTGCAGGAGT GA~CATTGTGC CGCGGAGA~A CTCAGACCGC
ATrrrr.T~r CACACTGAGT GGGA~AATCT GCTGGCTATG 1111 TTATCTATGC CTTAGATCAC ~ACTGTCACC CG
~ybridization o~ a viral DNA to the nucleic acid probes listed above i8 determined by using standard nucleic acid hybr;~;7At;~n techni~ues as described herein. In particular, PCR amplification of a viral genome can be carried out using the following three sets of PCR primers:

1) AGrrr~ r~r.~TTCCACQT;
1C~1~11~1~1ACGTCCAG (SEQ ID NO 41) 2) GA~ATT~rrr~rr.~r~TCGC;
AGGCAACGTCAGATGTGA ~SEQ ID NO 42) 3) AACACGTCATr.TGr~r-r.~r~TGAC;
CGGGTGACAGTTGTGATCTAAGG (SEQ ID NO 43) In PCR techniques, oligonucleotide primers, a~ listed above, complementary to the two 3' boraers of the DNA
region to be amplified are synthesized. The -- ' " 21 96~92 ~ Wo961061~9 ' ' PCT~S9~10194 polymerase chain reaction is then carried out using the two primers. See PCP Protocols: A Guide to ~ethod6 and Application6 L74]. 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 primer6 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 ~1; g~nll~l eotides is by either native acrylamide gel electrophoresi6 or by anion-exchange HPLC as described in Pearson, J.D. and Regnier, F.E.
[75A]. The sequence of the synthetic oligonucleotide can be verified using the chemical degradation method of Maxam, A.M. and Gilbert, W. [63].

93. Isolation and ~ro~aqation of RS-inducinq str~; nC of the Human Her~esvirus Using conventional methods, the human herpesvirus can be propagated L~ vitro. For example, standard techniques for growing herpes viruse6 are described in Ablashi, D.V. [1]. Briefly, PHA stimulated cord blood mononuclear cells, macrophage, neuronal, or glial cell line6 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 ~g/ml polybrene for 2 hours at 37~ C prior to infection.

WO96~61~9 ' ~' ~ PCT~S95/l0l9 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 _uman 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 (KSXV) employing the following protocol. ~ong-term es~hl;R' -n~ of a B lymphoid cell line infected with the KSHV from body-cavity based ly ~h~--R (RCC-1 or BH~=6) is prepared extracting DNA from the ~ymphoma tissue:using standard techniriues [27, 49, 66].

The KS associated herpesvirus may be isolated from the cell DNA in the iollowing manner. An infected cell line (BHB-6 RCC-1), which can be lysed using standard methods such as lly~Gs~ tic Rhnrki nrj and Dounce homogrn;7~1rn, is first pelleted at 2000xg for 10 minutes, the supernatant:is removed and centrifuged again at 10,000xg for 15 minutes to removetnuclei and organelles. The supernatant is filtered through a 0.4~ filter and centrifuged again at 100,000xg for l hour to pellet the virus. The virus can then be washed and centrifuged again at 100,000xg for l 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 ~ WO96/06159 2 1 9 6 8 9 2 PCT~S95/10194 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 ~lxlo6cells/ml into standard lymphocyte tissue culture medium, such as RMP 164Q supplemented with 10~ fetal calf serum. Immortalized lymphocytes ~nt~lning the KSHV virus are ;n~f;nitely grown in the culture media while nonimmortilized cells die during course of prolo~ged~cultivation.

Further, the virus may be propagated in a new cell line by removing media supernatant containing the virus from a c~nt;n-~usly infected cell line at a c~n~n~ration of >lx106 cells/ml. The media is centrifuged at 2000xg for 10 minutes and filtered through a 0.45~ filter to remove cells. The media is applied in a 1:1 volume with cells growing at >1X106 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=12~s were deposited on October 19, 1994 under ATC 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.

W096/06l59 ~ 9 6 8 9 2 PCT~S9~/l0Ig C. lmmunolo~ic~l Identitv of the Virus The KS-associated human herpesvirus can also be described immunologically. KS-associated human herpesviruses are selectively immunoreactive to r 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 ; n~cSay 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 i n~hsorbtion prior to use in the i -,~c~y.

In order to produce ~antisera for use in an ; n~Rsay, 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 ~L~duced 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 ; l7Ation protocol (see [32], 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 ; Inn~ y, for example, a solid phase i n~csay with the immunogen immobilized on a solid support. PolyclonaI antisera with a titer of 10~ or greater are selected and tested for their cross reactivity against other viruses o~
the ~ hPrpesvirinae subfamily, particularly human herpes virus types 1-7, by using a standard ~ W096~06ls9 21 96892 PCT~S95/~0194 i n~gay as degcribed in [32], supra. These other g~ rpesvirinae 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 i n~hsnrption with the above-listed viruses.
The i lnn~hsorbed and pooled antisera are then used in a competitive binding i -~ y u~uuedu~ as described above to compare an unknown virus preparation to the specific KS herpesvirus preparation described :herein and cnnt~ining the nucleic acid sequence described in SEÇ ID NOs: 2-37. In order to make this comparison, the ; ,~l protein which is encoded by the amino acid sequence or nucleic acid of 5EQ 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 illllllUllU~I consisting of the protein of SEQ ID NOs: 2-37 are those virus where the amount of - vi~us needed to inhibit 50~ of the binding to the protein does not exceed an e~tablished amount. This amount is no more than 10 times the amount of the virus that is needed for 50~ inhibition for the KS-associated herpesvirus cnnt~;n;ng the DNA sequence of SEQ ID NO: 1. Thus, the XS-associated herpesviruses 21 96~92 WO96/06159 ~ r PCT~S95/lOI9 of the invention can be defined by ; ~logica 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 anather embodiment, the molecule is RNA. In another embodiment the nucleic acid molecule may be 14-20 nucleotides in length. In another ~mhn~; t 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: 1, 10-17, and 38-40)-High stringent hybridization conditions are selected at about 5~ C lower than the thermal melting point ~Tm) for the specific sequence at a defined ionic strength and pX 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 pX 7 and the temperature is at least about 60~C. As other factors may significantly affect the ~ WO96/06159 2 1 9 6 8 9 2 . ~I~u~ ~ l0l3 ~

stringency of hybridization, including, among others, base compositio~n 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~C in a 6x SSC
solution, washing at room temperature with 6x SSC
solution, followed by washing at about 68~C in a 6x S5C 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, O.lM Tris buffer at Ph 7.5, 5x Denhardt's solution; 2.) pre-hybridization at 37~C for 4 hours; 3) hybridization at 37~C with amount of labelled probe egual to 2~ 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~C for 30 minutes each;
and 6) dry and expose to film.

The phrase ~selectively hybridizing to" refers to a nucleic acid probe that hybridize~, duplexes or binds only to a particular target D~A or RNA se~uence when the target sequences are present in a preparation of total ~ lar 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 se~uence- 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 ~nn~l;ng conditions depend, for example, upon a probe's length, base W096/06159 S~ 9 6 8 9 2 PCT~S9511019 composition, and the number of mismatches and their position on the probe, and must often be determined empirically For discussions of ~ucleic acid probe design and ~nnP~l;ng conditions, see, for example, Sambrook et al , [81] or Ausubel, F., et al., [8]

It will hP rP~;ly 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 ~nhpt~nt;~lly correspond to its complementary sequence and those described including allowances for minor sequencing errors, single ba~e 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 radi~isotope or fluorescent dye, to facilitate detection of the probe.
DNA probe molecules may be produced by insertion of a 2~ 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
synt~P~;-~r8.

DNA virus nucleic acid reaL,~ny ts/mutations may be detected by Southern blotting, single stranded conformational polymorphism gel electrophoresis ~ W096/~6159 =~ - 2 1 9 6 8 9 2 PCT~S95/10194 (SSCP), PCR or other DNA based techniques, or for RNA
specie9 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 f~ 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 synth~; 7ed by either the phosphoramidite method described by Beaucage and Carruthers, [19], 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 ~nn~l;ng the rhPmi~ly synthesized single strands together under appropriate conditions or by 2~ 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 i~nt;f1~ 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 3~ also encompassea for use~ as a probe.

WO96~6159 ~ 2 1 9 6 8 9 2 PCT~S9~1019 ~

The DNA molecules of the subject invention also include DNA molecules coding for polypeptide analogs, fragments or derivatives of antigenic polypeptides which differ ~rom naturally-occurring forms in terms o~ 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 naturally-occurring forms. These molecules include: the incorporation of codons "pre~erred" for expression by selected non-mammalian hosts; the provision of-sites for cleavage by restriction ~n~nnnrlease enzymes; and the provision of additional initial, terminal or intermediate DNA se~uences that f~cillt~te 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 o~ producing a polypeptide encoded by isolated DNA molecule, which comprises growing the above host vector system under suitable ~nn~;t;nn~ permitting production of the polypeptide and recovering the polypeptide so pro.duced.

This invention provides an isolated peptide encoded by ~he isolated DNA moleoule associated with Kaposi's sarcoma. In one embodiment the peptide may be a polypeptide. ~urther, this invention provides a host ., .

~ Wo96iO6l59 2 1 9 6 8 9 2 r~ 3, cell which expresses the polypeptide of isolated DNA
molecule.

In one ~;m~nt 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 se~lence 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 forr, a fusion protein by expression in a suitable host cell In one embodiment the second nucleic acid molecule encodes beta-galactosidase. 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 bind~3 to the peptide or polypeptide encoded by the isolated DNA molecule. In one ~ir t 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 nnlnri -tric, a luminescent, or a fluorescent marker, or gold. Radioactive labels include, but are not limited to: 3H, 14C, 32p, 33p; 35S, Cl, Cr, s'Co, 59Co, s9Fe, 90y, l~sI l3lI and l36R
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 monoclo~al antibody are known to those of ordinary skill in the art.

WO96106159 i~ 6 8 9 2 PCT~S95/l019 Further, the antibody or nucleic acid molecule complex may be detected by a second antiboay which may be linked to an enzyme, such as ~lk~l;n~ phosphatase or horseradish peroxidase. Other enzymes which may be employed are welI 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 genera~e 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 mem,brane 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 zo 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 ~nt;hn~ies 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 ;mmnn;7;ng animals using the selected peptides. Monoclonal antibodies are prepared using 3~ hybridoma technology by fusing antibody producing s cells f" 1 i~ed anlmals with myeloma cells and selecting the resulting hybridoma cell li~e producing 'J ~'' 2196892 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 ~ 5 polypeptide encoded by the isolated DNA molecule of the DNA virus in livi~g animals, in humans, or in h;nlng; r~l ~issues or fluids isolated from animals or humans.

II_ Tmm7lnn~7c7savs The antibodies raised against the viral strain or peptides may be detectably labelled, utilizing conventional l,7h~11 ;ng techni~ues well-known to the art. Thus, the antibodies may be radiolabelled using, for example, radioactive isotopes such as 3~, ~23I, 131I, and 33S

The antibodies may also be labelled using fluorescent labels, enzyme labels, free radical labels, or bacteriophage labels, using techni~ues known in the art. Typical fluorescent labels include ~luorescein isothiocyanate, rhnn',7m;nP, 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 pror'--~t;~n of tracer materials. Suitable enzymes include -r71k;71;n~ phosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase, maleate dehydrDgenase, and peroxidase. Two principal types of:
e~zyme i n~C7say are the enzy~7e-linked immunosorbent assay (E~ISA), and the homogeneoug enzy~7e ; 7nn,7q~7y, also known as enzyme-multiplied ; ~;~c~5y (EMIT, Syva Corporation, Palo Alto, CA). In the ELISA
system, separation may be achieved, for example, by c~ 1 96~92 ~ ~u ; ~
WO96/06159 PCT~S95/lOI9 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, bioln~;n~cc~nt compounds may be utilized for labelling, the bioluminescent ~UILI~UU1ldS including luciferin, luciferase, and aequorin_ Once labeled, the antibody may be employed to identify and quantify immunologic counterparts (antibody or antigenic polypeptide~ l?t; 1; z; ng tech~iques well-known to the art.

A description of a radio~ ~csay (RIA) may be found in Laooratory Techniques in Biochemistry and Molecular Biology [52l, with particular reference to the chapter entitled "An Introduction to R~;oi ~ Assay and Related Techniques" by Chard, T., incorporated by reference herein.
A description of general ; triC 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 antiqens In addition to the detection of the causal agent using nucleic acid hybridization technology, one can use immunoassays to detect for theS virus, specific peptides, or for cnt;ho~;~C to the virus or peptides.
A general overview of the applicable technology is in ~ WO96~6159 2 1 9 6 ~ 9 2 PCT~S95110194 Harlow and Lane [32], 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 recomhin~nt antibodies) specific for the gene product can be used in various immunoassays Such assays include competitive ; 1nn~cqAys/ radi~; 1nn~qqayS, Western hlots/ ELISA, indirect immunofluorescent assays and the like For competitive ;mmnnn~A~q~yS, see ~arlow and Lane [32] at pages 567-573 and 584-589.

Monoclonal antibodies or recombinant antibodies may be obtained by various techniques f~m; 1; ~r to those skilled in the art. Bri~_ly, 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 [50], 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 pro~i-rt;nn of ant;hoA;~q of the desired specificity and affinaty 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 recomhinant phage antibody expression systems can also be used to generate monoclonal ~nt;hn~;Pq See for example:~McCafferty, J et al WO96/06159 ~j\ PCT~S95/1019 [64]; Hoogenboom, H.R. et al. [39]; and Marks, J.D. et al. [60].

Such peptides may be produced by expressing the specific se~uence in a recombinantly engineered cell such as bacteria,-yeast, f;l tnus 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 se~uence or portion thereof to a promoter (which is either constitutive or inducible), and i1~uur~ol~ted 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, in~ ;hle or regulatable promoter regions, and translation terminators that are useful for the expression of viral genes.

Methods for the expression of clo~ed 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 D~A vectors transformed in E coli is also useful.
Examples of such markers include genes specifying resistance to antibiotics. See [81] supra, for details cnn~Prn;ng selection markers and promoters~for use in E. coli. Suitable eukaryote hosts may include plant cells, insect cells, mammalian cells, yeast, and ~;1 tous fungi.

~ WO96/06159 21 96892 .~ 9, Methods for characterizing naturally processed peptides bound to MHC (major histocompatibility complex) I molecules have been developed. See, Falk et al [24¦, and PCT publication No. WO 92/21033 p~lhl;~h~d November 26, 1992, both of which are incorporated by reference herein. Typically, these methods involve lsolation 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 ~ractions by known automatic sequencing o~ peptides eluted from Class I molecules of the B cell type (Jardetzkey, et al. [45], incorporated by reference herein, and of the human MHC
_class I molecule, HLA-A2.1 type by mass spectrometry (Hunt, et al. [40], incorporated by refere~ce herein).
See also, Rotzschke and Falk [79], 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 bindirg moti~s can be applied to the identification o~ potential viral immunogenic peptides ' vitrc The peptides described herein produced by r~ ;nAnt 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 (e.g., ~n;~t;on) 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 , WO96!06l59 r ~ ~ PCTNS95/lOI9 ' 42 ~elective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, Scopes, R. [84], incorporated herein by reference. -B. Serolo~ioal te8ts for the ~tre~ence~ ofAntihQ~;es to the human her~tesvirus.

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 instr~~tinrt~l 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 Ant;ho~;eS 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 rnntAin~r ~nntA;n;ng a nucleic acid se~uence specific for the human herpesvirus and instructional material for detecting the KS-associated herpesvirus are also included. ~ ~nntA;n~r ~nntztining nucleic acid primers to any one of such se~uences is optionally included aE
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 i nAztcAy methods that are similar to the procedures described above for meabuL~ t of antigens. Por a review of immunological and i nAccAy procedure~
applicable to the meabuL, ~ of ~antibodies by ~ W096~6159 ~ 2 1 9 6 8 9 2 PCTNS95/10194 immunoassay techniques, see Basic and Clinical Immunology 7th Fdition [12], and [32], supra.

In brief, ; t~ccay8 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 com~petes with a labeled analyte for specific binding sites on a capture agent bound to a solid suriace. Preferably the capture agent is a purified rett ~in~nt 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 ~t~nc ~ctitive 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 ;~nnn~say iormats, separation tethn;t~lt~q and labels can be also be used similar to those described above for the measurement of the human herpesvirus antigens.

~emagglutin~tit~n Inhibition (HI) and r ~lt~t~nt 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.

erological 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 r~pt~ntlt~t~ to the new variant.

ci ~2~ 96892 W096/06159 . : ~ PCT~S95/1019 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 ol;g~nll~leotide capable of hybridizing to the DNA molecule. In another ~ t the triplex oli~onucleotide is capable of binding to at least a portion of the isolated DNA molecule with a nucleotide se~uence as shown in Figure 3A-3F (SEQ ~ 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 ~mho~i L the antisense moIecule is RNA.
--The antisense molecule may be DNA or RNA or variants thereof (i.e. 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 RWA 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 - WOg6~6159 ' PCT~S95/10194 hybridize to the AU~ initiation codon are particularly e_iicient, 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 nnnl~l1r-n mammal which comprises at least a portion oi the isolated DNA
molecule introduced into the mammal at an embryonic stage. Methods of producing a transgenic nnnhn~~n mammal are known to those skilled in the art.

This invention provides a cell line cnnt~7;n;ng the isolated KS associated herpesvirus oi 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- OEM (ATCC CC~ ll9); B-cells, such as BJAB and Raji (ATCC CC~ 86); and myeloid cells such as K562 (ATCC CC~ 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-l cell line.

III. In v~tro diaonostic assaYs for the detection of 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 ~b) contacting the nucleic acid m~l~rl7l~ with a labelled nucleic acid molecule of at least 15 nucleotides capable of spec;f;n~l1y hybridizing with the isolated DNA, under hyhr;~'.; 7; ng conditions; and (c) determining the presence of the 5~?~ 21 ,~6892 W096/06159 PCT~S9511019 nucleic acid molecule hybridized, the presence of which is indicative of Kaposi's sarcoma in the subject, thereby diagnosing Kaposi' 8 sarcoma in the subject.
In one ~mhn~;m~nt the DNA molecule from the tumor lesion is amplified before step ~b~. 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 pe~rson of ordinary skill in the art will be able to obtain appropriate DNA sample for diagnosing Kaposi's barcoma 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 ~mhn~ the size fractionation is effected by an agarose gel. Further, transferring the DNA Ll~y~ s Into a solid matrix may be employed before a hybridization step. One examp~le of such solid matrix is nitrocellulose paper This invention provides a method of diagnosing 3~ Kaposi's sarcoma in a subject which comprises: (a) obtaining a nucleic acid molecule from a suitable bodily fluid of the subject; (b) contacting the nucleic acid molecule wlth a l~h~ nucleic acid molecules of at least 15 nucleotides capable of specifically hybridizing with the isolated DNA, under hybridizing ~nn~;~;nnC; and (c) determining the presence of the nucleic acid molecule hybridizedr the ~ W096/06159 , 2 1 9 6 8 9 2 ~ i5 ~

presen~e of which is indicative of KapoEi'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 la) obtaining a suitable bodily fluid sample from the subject, (b) crntAct;nr the suitable bodily fluid of the subject to a support having already bound thereto a Kaposi's sarcDma 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 (d) ~ptpr~;n;nr 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 cf diagnosing Kaposi's sarcoma in a subject, which comprises (a) obtaining a suitable bodily fluid sample from the subject, (b) cr~nt-Art;nJ 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, (c) removing unbound bodily fluid from the support, and (d) 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 ~PtPrt;ng expression of a DNA virus associated with Kaposi's sarcoma in a cell which comprises obtaining total cDNA
obtained from the cell, rrntArt;nr~ the CDNA so obtained with a lAhPllP~ DNA molecule under hybridizing conditions, ~ptprm;n;ng the presence of cDNA hybridized to the molecule, and thereby detecting the expression of the DNA virus. In one embodiment W096/06159 ~ 2 1 9 6 8 9 2 PCT~S9~1019 ~

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 Raposi' 8 sarcoma antibody, antigen or f,~, ts there~Df A suitable bodily fluid ;nmln~, but is not limited to: serum, plasma, cerebrospinal fluid, lymp~ocytes, 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 ~t~rml nl ng the level of antibody or antigen include, but are not limited to: E~ISA, 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 RS 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, i.e., 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 PCT~7595/10194 Samples can be taken from patients with KS or from patients at risk for KS, such ag AIDS pat;~nt~.
Typically the sample6 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 indi-cated 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 ~ucleic acid sequence of the human herpesvirus described herein.

Accepted means for conducting hybridization assay~ are known and general overviews of the technology can be had from a review of: Nucleic Aci~7 ~ybridization: A
Practical Approach [72]; Hyhridization of Nucleic Acid6 T: hi 7 ; 7~7 on Solid 5upports [41]; Analytical iochemi6try [4] and Innis et al., PCR Protocols [74], supra, all of which are incorporated by reference herein If ~CR 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 25 homologue - ORF 32 homologue. From the information provided herein, those of~skill in the art will be able to select appropriate specific primers.

WO96/06159 2 1 9 6 ~ 9 2 PCT~Sg~lolg ~

Target specific probes may be used in the nucleic acid hybridization diagnostic assays for ~S. 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 lO00 nucleotides, most preferably about 200 to about 400 nucleotides.

A sequence i8 "specific" for a target organism of interest if it includes a nucleic acid sequence which when detected is determinative of the preaence of~the organism in the presence of a heterogeneous population of proteins and other biologics. A specific nucleic acid probe is ~argeted to that portion of the sequence which is det~rm,~tive 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 (e.g., nick translation, primer extension, reverse transcription, the polymerase chain reaction, and others) or chemically (e.g., by methods such as the phosphoramidite method described by Beaucage and Carruthers [19], or by the triester method according to Matteucci, et al. [62], 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, i.e., at least about 14 nucleotides, and ~ W096/06159 s ~ ; 2 1 9 6 ~ 9 2 PC~S9~10194 may be longer (e g., at least about 50 or 100 bases in length). Often t~e 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 hybridizationr occurs when a probe hybridizes to a target nucleic acid, as evi~SI~nrr~ hy a detectable signal, under conditions in -which the probe does not hybridize to other nucleic acids ( e . g., 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 mugt often be determined empirically. For discussions of nucleic acid probe design and ~nn~l ing conditions, see, for example, [81], supra, Ausubel, F., et al. [8] [hereinafter re~erred to as Sambrook], Methods in Enzymology [67]
or ~ybridization with Nucleic Aci~ Probes [42] all o~
which are incorporated herein by reference.

Usually, at least a part o~ the probe will have r~nci~Prable se~uence identity with the target nucleic acid. ~lthrugh the extent of the sequence identity re~uired or speci~ic 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 80%
identity, still more usually at least 90% identity and most usually at least 9~ or 100% identity.

t Q 1~i r ~ 2 1 9 6 8 9 2 W096/06159 ~ : ~' ' " rcT~ss~lols~

A probe can be i~nt;f;~ as capable of hybridizing Gpecifically to it~ target nucleic acid by hybridizing the probe to a sample treated according the protocol of this invention where=the sample cnntA;nA both target virus and animal cells (e.g., nerve cells~. A
probe is specific if the probe's characteristic signal is associated with the herpe6virus DNA in~the sample and ~ot generally with the DNA of the host cells and non-biological materials (e.g., substrate) in a sample.

The following stringent ~hybridization and washing conditions will be ade~uate to distinguish'a specific probe (e.g., a fluorescently labeled DNA probe) from a probe that is not specific: incubation of the probe with the sample for 12 hQurs at 37~C in a solution cnntA; n;ng denatured probe, 50~ formamide, 2X SSC, and 0.1~ (w/v) dextran sulfate, followed by washing in lX
SSC at 70~C for 5 minutes; 2X SSC at 37~C for 5 minutes; 0.2X SSC at room temperature for 5 minutes, and ~2~ at room temperature for 5 minutes. Those of skill will be aware that it will often be advantageous in nucleic acid hybridizations (i.e., in situ, Southern, or other) to include detergents (e.g., sodium dodecyl sulfate), chelating agents (e.g., EDTA) or other reagents (e.g., buffers, Denhardt's solution, dextran sulfate) in the hybri~i7Ptinn or wash solutions. To test the specificity of the virus specific probes, the probes can be tested on host cells n'n=nt-Ai=n;ns the ~S_AAAn~;AtP~ 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 conve~ient method for ~et~rm;n;ng whether a probe is specific for a XS-associated viral nucleic acid utilizes a Southern blot (or Dpt blot) using DNA

~ W096/061~9 2 1 9 6 8 9 2 PCT~S95/10194 ~L~a~d from one or more KS-associated human herpesviruses of the invention. Briefly, to identify a target specific probe DNA ls isolated from the virus. Test DNA either viral or cellular is transferred to a solid ~e.g., charged nylon) matrix.
The probes are labelled following co~ventional 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 T= (melting temperature~ of the hybridized probe (see, e.g., Sambrook for a description of cAlrnlAt;nn of the T=). For radioactively-labeled DNA
or RNA probes an example of stringent hybridization conditions is hybridization in a solution containing denatured probe and 5x SSC at 65~C for 8-2~ hours followed by washes in O.lx SSC, 0.1~ SDS ~sodium dodecyI sulfate) at 50-65~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 ~articular salt rr~nr~ntraticn the temperature may be selected that is 5~C below the TM or conversely, for a particular temperature, the salt crJncpntration is chosen to provide a TM for the hybrid that is 5~C
warmer than the wash temperature. Following stringent hybr;~;7at;rn and washing, a probe that hybridizes to the XS-associated viral DNA but not to the non-KS
associa~ed viral 3~A, 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 sp~r;~;r; ty and in nt; 1; 7; ng the method of this invention to detect Ks-associated herpesvirus, a certain amount of background signal is typical and can 2l 96892 W09~0~159 .. ~ ; PCT~S95/lo 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 absencerof 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.
Visualizatio~ of the hybridized portions allows the qualitative detrrmin~t;~ 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 det~rm;ning the presence of the human herpesvirus is in situ hybridization, or 2~ more recently, in situ polymerase chain reaction In ~i~~ PCR is described in Neuvo et al. [71], Intracellular l nr~l 1 7ation of poIymerase chain reaction (PCR)-amplified ~epatitis C cDNA; Bagasra et al. [10], Detection of Human Immunodeficiency virus type 1 provirus in I n~lmrlear cells by n situ polymerase chain reaction; and Heniford et al. [35], Variation in cellular EGF receptor mRNA expression demonstrated by n situ reverse transcriptase polymerase chain reaction. In situ hybridization assays are well known and are generally described in Methods Enzymol. ~67J incorporated by reference herein. In an in situ hybridization, cells are fixed ~ W096/061~9 ~ PCT~59~10194 ~5 to a solid support, typically a glass slide. The cells are then contacted with a hybridization solution at a moderate temperature to permit ~nn~l;ng 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 expre6s 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 1nr~1;7ation method which is not dependent on expression of antigens or native vs denatured conditions.

Oligonucleotide =(oligo) probes, synthetic olig~nllrl~rtide probes or riboprobes made from RSHV
phagemids/plasmids, are relatively homogeneous reagents and successful:hybridization conditions in tissue ~ections is readily transferable from one probe to another Commercially synthesized olig~nnrl~rtide probes are prepared against the i~nti~;~d genes These prcbes are chosen for length ~45-65 mers), high G-C cont~nt (50-70~) and are screened for uniqueness against other viral sequences in GenBank.

Ol;grn1]~leotideg are 3~end-labeled with [~-3~S]dATP to specific activities in the range of l x l~3~ dpm/ug using terminal deoxynucleotidyl transferase.
TTn;nrn~rorated labeled nucleotide~ are removed from - the oligo probe by centrifugation through a Sephadex G-25 column or by elution from a Waters Sep Pak C-18 column.
RS tissue embedded in OCT compound and snap frozen in freezing isopentane cooled with dry ice is cut at 6 ~m WO96~6159 _t "~ 9 6 ~ 9 2 PCT~S95/lOI9 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 ~mhe~ KS
tissues cut at 6 ~m 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~C is folIowed by hybridization overnight in a solution of 50~ formamide (v/v), 10~ dextran sulfate (w/v), 20mM
sodium phosphate (Ph 7.4), 3X SSC, lX Denhardt's solution, 100 ug/ml salmon sperm DNA, 125 ug/ml yeast tRNA and the oligo probe (106cpm/ml) at 42~C overnight.
The slides are washed twice with 2X SSC and twice with lX SSC for 15 minutes each at room temperature and visualized by autoradiography. Briefly, sections are dehydrated through graded alcohols c~nt~ining 0.3M
ammonium acetate and air dried. The slides are dipped in Kodak NTB2 emulsion, exposed for days to weeks, developed, and counterstained with hematoxyli~ and eoxin Alternative ; ~hi~tochemical protocols may be employed which are known to those skilled in the art.
I~. Treatment of hnm~n 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.

~ W0961061~9 2 t 9 6 3 9 2 PCT~59~/10194 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 ef~ective 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.
1 0 ,, Further, this invention provides a method of prophylaxis or treatment for Kaposi's sarcoma tKS) by administering to a patient at risk for KS, an antibody that binds to the human herpesvirus in a pharmaceutically acceptable :carrier. In one ~ the antiviral drug is used to treat a subject with the DNA herpesvirus of the subject invention.

The use of combinations of antiviral drugs and sequential treatments are useful ~or treatment of herpesvirus infections and will also be use~ul for the treatment of herpesvirus-induced KS. For example, Snoeck et al. [88], found additive or synergistic 2~ effects against CMV when cl 'ining antiherpes drugs (e.g., combinations of zidow dine [3~-azido-3~-deoxythymidine, AZT] with HPMPC, ganciclovir, foscarnet or acyclovir or of UPMPC 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 minimi7inr~ the adverge side effects of either treatment alone. An anti-herpetic composition that rrnt~in~ acyclovir and, e.g., 2-acetylpyridine-5-((2-pyridylamino)thiocarbonyl)-thiocarbonohydrazone is described_ in U.S. Pat. 5,175,165 (assigned to 2~ 968~2 WO96~6159 ~ PCT~S95/101 Burroughs Wellcome Co.). Comb;n~tinnc of TS-inhibitors and viral TK-inhibitors in antiherpetic medicines are disclosed in U.S. Pat. 5,137,724, assigned to St; rht; ng Rega VZW. ~ A synergistic inhibitory effect on EBV replication using certain ratios of combinations of HPMPC with AZT was reported by Li~ et al. 156].

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. [11]) describes the use of thymilydate synthase inhibitors (e.g., 5-fluoro-uracil and 5-fluro-2~-deoxyuridine) in combination with compounds having viral thymidine kinase inhibiting activity.

With the discovery of a disease causal agent for KS
ncw 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, S.L., 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 i~clude immunoregulatory agents that do not directly affect ~ WO96/06159 2 ! 9 6 ~ 9 2 PCT~S95/10194 viral titer or bind to viral products Antiviral agents are effective if they inactivate the virus, oth~rwiq~ 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 grou~ed into broad classes based on their presumed modes of action.
These classes include agents that act (i) by inhibition of viral DNA polymerase, (ii) by targeting other viral enzymes and proteins, (iii) by m;rcell~n~ous or incompletely undergtood merh~n;s~q, or (iv) by binding a target nucleic acid (i.e., inhibitory nucleic acid therapeutics). Antiviral agents may also be used in combination (i e , together or ser~1~nt;~11y) to achieve synergistic or additive effects or~other benefits.
~
Although it is convenient to group antiviral agents by their supposed r--~h~niqm of action, tAe applicants do not intend to be bound by any particular m~rh~n;rm of antiviral action :So~uv~" it will be understood by those of skill that an agent may act on more than one target in a virus or virus-;nfPrt~d 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 inAibition of viral DNA replication, ~ especially through inhibition of viral DNA polymerase~
3~ ~hese nucleoside analogs act as alternative substrates for the viral DNA polymerase or as competitive inhibitors of DNA polymerase substrates. Usually WO96/06159 ;~. , 21 96892 PCT~S95/1019 ~

these agents are preferentially phosphorylated by viral thymidine kinase (TK), if one i8 present, and/or have higher affinity for viral DNA polymerase than for the rrll~ r 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 ter~inator, cause increased lability (e.g., susceptibility to brea~age) of analogue-cnnt~;ning DNA, and/or impair the ability of the substituted DNA
to act as template for tra~scription or replication (see, e.g., Balzarini et=al. [ll]).

It will be known to one of skill that, like many drugs, many of the agents useful for treatment of herpes virus infections are modified (i.e., ~activated~) by the host, host cell, or virus-infected host cell r-t~hol;c 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 c , JU~ld ~OE 602 to ganciclovir in a three-step metabolic pathway (Winkler et al. [95]) and the phosphorylation of g~nr;rlnvir to its active form by, e.g., 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 ~ n;~m 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 K5 include, but are not limited to, - WO96/06159 ~i 96892 PCT~S95/10194 nucleoside ~ analogs including acyclic nucleoside p h Q s p h o n a t e a n a 1 o g 8 ( e . g . , phosphonylmethoxyalkylpurines and -pyrimidines), and cyclic nucleoside analogs. These include drugs such 5 ~ as: vidarabine (9-~-D-arabinofuranosyl~nin~;adenine arabinoside, ara-A, Vira-A, Parke-Davis); 1-~-D-arabinofuranosyluracil (ara-U); 1-~-D-arabinofuranosyl-cytosine (ara-C); HPMPC [(S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine(e.g.,GS
504 Gilead Science)] and its cyclic form (c~PMPC);
H P M P A [ ( S ) - 9 - ( 3 - h y d r o x y - 2 -phosphonylmethoxypropyl)adenine] and its cyclic form (cHPMPA); (S)-HPMPDAP [(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diaminopurine]; PMEDAP
[9-(2-phncr~nnyl-methoxyethyl)-2,6-~i ~m; nnpllrine]; HOE
602 [2-amino-9-(1,3-bis(isopropoxy)-2-propoxymethyl)purine] ; PMEA [9- (2-phosphonylmethoxyethyl)adenine]; bromovinyl-deoxyuridine (Burns and S~n~fnr~. [21]); l-~-D-arabinofuranosyl-E-5-(2-bromovinyl)-uridine or -2'-deQxyuridine; BVaraU ~ -D-arabinofuranosyl-E-5-(2-bromovinyl)-uracil, brovavir, Bristol-Myers Squibb, Yamsa Shoyu); BVDU [~E)-5-(2-bromovinyl)-2'-deoxyuridine, brivudin, e.g., ~elpin] and its carbocyclic analogue (in which the sugar moiety is replaced by a cyclopentane ring);_ IVDU [(E)-5-(2-iodovinyl)-2'-deoxyuridine] and its carbocyclic analogue, C-IVDU (Balzarini et al. [11])]; and 5-mercutithio analogs of 2'-deoxyuridine (Holliday, J., a~d w;l1;~mc, M.V. [38]); acyclovir [9-([2-hydroxyethoxy]methyl)guanine; e.g., Zovirax (Burroughs - Wellcome)]; penciclovir (9-[4-hydroxy-2-(hydroxymethyl)butyl]-guanine); ganciclovir [(9-[1,3-dihydroxy-2 propoxymethyl]-guanine) e.g., Cymevene, Cytovene ~Syntex), DHPG (Stals et al. [89]];
isopropylether derivatives of ganciclovir (see, e.g., W;nk~lr~nn et al. [94] ); cygalovir; famciclovir [2-W096/06159 '~ 9 6 8 9 2 PCT~S95/lOI9 amino-9-(4-acetoxy-3-(acetoxymethyl)but-1-yl)purine (Smithkline Beecham)]; valacyclovir (Burroughs Wellcome); desciclovir :[(2-amino-9-(2-ethoxymethyl)purine)] and ~2-amino-9-(2-hydroxyethoxymethyl)-9H-purine, prodrugs of acyclovir]; CDG (carbocyclic 2'-deoxyguanosine); and purine ~ucleosides with the pentafuranosyl ring replaced by a cyclo butane ring (e.~., cyclobut-A [(+-)-9-[1~,2~,3~)-2,3-his(hydroxymethyl)-1-cyclobutyl]adenine], cyclobut-G ~(+-)-9-~1~,2~,3~)-2,3-bis(hydroxymethyl)-1-cyclobutyl]guanine], BHCG
[ ( R ) - ( 1 ~ , 2 ~ , 1 ~ ) - 9 - ( 2 , 3 -bis(hydroxymethyl)cyclobutyl]guanine], and an active isomer of racemic BHCG, SQ 34,514 [lR-1~,2~,3~)-2-amino-9-~2,3-bis(llydLu~y -~yl)cyclobutyl]-6H-purin-6-one (see, Braitman et al.(1991) [20]]. Certain of these antiherpesviral agents are discussed in Gorach et al. [28]; Saunders et al. [82]; Yamanaka et al., [96]; Greenspan: et al. [29], all: of 'which are incorporated by reference herein.

Tri r i ri hi n ~ and tri r; ri hi n~ monophosphate~are potent inhibitors against herpes viruses. (Ickes et al. [43], incorporated by reference herein), HIV-l and HIV-2 (Kucera et al. [51], incorporated by reference herein) and are additional nucleoside analogs that may be used to treat KS. An exemplary protocol for these agents is an intrave~ous injection of about 0.3S mg/meter~
(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 rhrsr~rrylated by a herpesvirus thymidine kinase and undergoes further phosphorylation to be i~corporated as a chain ~ WO96/06159 2 1 9 6 8 9 2 terminator by the viral DNA polymerase during viral replication. It has therapeutic activity against a broad range of herpesviruses, Herpes simplex Types l and 2, Varicella- Zoster, ~Cytomegalovirus, and J 5 Epstein-Barr Virus, and is used to treat disease such as herpes PnrPph~1itis, neonatal herpesvirus infections, chickenpox in immunoc~ L, ;~ed hosts, herpes zoster recurrences, CMV retinitis, EBV
infections, chronic fatigue s,vndrome, and hairy : leukoplakia in AIDS patients. Exemplary intravenous dosages or oral dosages are 250 mg/kg/m' 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, e.g., Oren and Soble [73]. Treatment protocols for ganciclovir are 5 mglkg twice a day IV or 2.5 mg/kg three times a day for l0-14 days. ~;ntPn~nnP doses are 5-6 mg/kg ~or 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-l, HSV-2, T~- ~SV, VZV or CMV infections in animal models ~[22], supra).

Nucleoside analogs such as BVaraU are potent inhibitors of ~SV-l, EBV, and VZV that have greater activity than acyclovir in animal models oi PncPrh~l;tis. FIAC (flurni~n~h;nnsyl cytosine) and its related fluroethyl and iodo compounds (e.g., FEAU, - FIAU) have potent selective :activity against herpesviruses, and HPMPA ((S)-l-([3-hydroxy-2-phosphorylmethoxy]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-WO96/06159 ~ S

chlorodeoxyadenosine) is another nucleoside analogueknown as a highly specific antilym~hocyte agent (i.e., a immunosuppressive drugl.

Other useful antiviral agents include: 5-thien-2-yl-2'-deoxyuridine derivatives, e.g., BTDU [5-5(5-bromothien-2-yl)-2'-deoxyuridine] and CTDU [b-(5-chlorothien-2-yl)-2'-deoxyuridine]; and OXT-A [9-(2-deoxy-2-hydroxymethyl-~-D-erythro-n~ nncyl)adenine]
and OXT-G [9-(2-deoxy-2-hydroxymethyl-~-D-erythro-oxetanosyl)guanine]. Although OXT-G is=b~lieved to act by inhibiting viral DNA synthesis its mechanism of -action has not yet been elucidated. These and other ~nmpolln~ are described in Andrei et al. r5] 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 P.L.C.). 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 [e.g., idoxuridine (5-ido-2'-deoxyuridine)] and triflurothymidine) have antiherpes viral activity, but due to their systemic toxicity, are largely used for toploal ~erpesviral infections, incIuding ~SV stromal keratitis and uveitis, ard 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 phosp~nnn~netic acid (PAA). Foscarnet is an inorganic pyrophosphate analogue that acts by competitively blocking the pyrophosphate-binding site of DNA polymerase. ~hese agents which block DNA

' 21 96892 WO96/06159 .~ ,J,'i~l94 polymerase directly without processing by viral thymidine kinase. F4scarnet i6 reported to be less toxic than PAA.

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

Although applicants do not intend to be bound by a particular : r -~n;~m of antiviral action, the antiherpe6-virus agents described above are believed to act through inhibition of viral DNA poly~erase.
P,owever, viral replication re~uires not only the replication of the viral nucleic acid but also the productio~ oi viral proteins and other essential ~, ~nt~. Accordingly, the present invention contemplates treatment of KS by the inhibition of viral proliferation by targeting viral proteins other than DNA polymerase (e.g., by inhibition of their synthesis or activity, or destruction of viral proteins after their synthegis). For example, administration of agents that inhibit a viral serine protease, e.g., 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, e.g., parazofurin), CTP
synthetase inhibitors (targets o~, e.g., cyclopentenylcytosine), IMP dehydrogenase, ribonucleotide reductase (a target of, e.g., carboxyl-- ~nt~;n;ng N-alkyldipeptides as described in U.S.
Patent No. 5,110,7~9 (Tolman et al., Merck)), thymidine kinase (a target o~, e.g., 1-[2-(hydroxymethyl)cycloalkylmethyl~-5-substituted -uracils and -guanines as described in, e.g., U.S.
Patent Nos. 4,863,927 and 4,782,062 (Tolman et al.;

.

r ~ ~ r 2 1 9 6 8 9 2 WO96~6159 ~

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.

lD 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~C12U), 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/m2 are administered two to three times weekly to adults averaging 150 pounds. It is best to administer at least 200 mg/m2 per~week.
_ = _ _ __ other antiviral agents ~reported to show activity against herpes viruses (e.g., varicella zoster and herpes simplex) and will be useful for the treatment of herpesvirus-induced KS include mappicine ketone 30 ~ ~ (SmithKline Beecham); Compounds A,79296 and A,73209 (Abbott) for varicella zoster, and Compound 832C07 (3urroughs Wellcome) [see, The p~r Sheet 55(20) May 17, 1993].

Interferon is known inhibit replication of herpes viruses. See [73], supra. Interferon has known toxicity problems and it is expected that second ~ WO96/061~9 ;' ' 2i q6~92 PCT~S9~10194 yeneration derivatives will soon be available that will retain interferon's antiviral properties but have reduced side affects.

It is also contemplated that herpes virus-i~duced 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 (e.g., as discussed in PCT Application W0 93/04683).
Reactivating agents include agents such as estrogen, phorbol esters, forskolin and ~-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 d e r i v a t i v e s , i n c 1 u d i n g 9 - ( 2 -hydroxyethylmethyl)adenine, of the type described in U.S. Pat. Nos. 4,287,188, 4,294,831 a~d 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.

. W O 96/06159 ', '',: ~ 1 9 6892 PC~r/U595110194 Brovavir is an example of an antiviral deoxyuridine derivative of the type aescribed in US Patent Nos.
4,5g2,210 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 5,126,3g5.

HPMPC is an example of an antiviral phosphonyl methoxyalkyl derivative with of the type described in US Patent No. 5,1g2,051.

CDG ~Carbocyclic 2~-deoxyguanosine) is an example of an antiviral carbocyclic nucleoside analogue of the type described in US Patent Nos :g,5g3,255, g,855,g66, and g,89g,458.

Foscarnet is described in US Patent No. g,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 ~r~tin~ 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 describ d in U.S. Patent Nos. 5,286,6g9 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-1-~
propoxymethyl)guanine.

~ W O 96/06159 2 1 9 6 8 9 2 PC~rAUS95/10194 U.S. Patent No. 4,708,935 (Suhadolnik et al.; Research Corporation) describes a 3'-deoxyadenosine compound effective i~ 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 8 u s e o f (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 5~-zmino-5'-deoxythymidine and 5-iodo-5'-amino-2',5'-dideoxycytidine as potent inhibitors of herpes simplex virus. U.S. Patent No. 4,069,382 (Baker et al.; Parke, Davis & Company) describes 9-(5-O-Acyl-beta-D-arabinofuranosyl)adenine compounds useful as antiviral agents. U.S. Patent No. 3,927,216 (Witkowski et al.) describes the use of 1 , 2 , 4 - t r i a z o l e - 3 - c a r b o x a m i d e a n d 2~ 1,2,4-triazole-3-thior~rhn~n;de for inhibitingherpes virus infections. Patent No. 5,179,093 (Afonso et al., Schering) describes ~linn7in~-2,4-dione derivatives active against herpes~simplex virus 1 and 2, cytomegalovirus and Epstein Barr virus.
v) I~hibitory nucleic acid therapeutics Also cnn~pl~ted he ~ 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 W096/06159 _\ ~ , 2 1 9 6 8 9 2 . ~ 3llol3l ~

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.
s 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 me~h~nicTnc of the cells, such as promoting RNA degradation. Inhibitory nucleic acid methods therefore ~1 ~ cs 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. [34], which is hereby incorporated by reference and is referred to hereinafter as "Helene and Toulme. T
In brief, inhibitory nucleic acid therapy approaches can be classified into those that target DNA
se~uences, 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 categorie9.
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 T~PC; gn~ to bind to regions of single stranded D~A resulting from the opening of the duplex DNA during replication or transcription. See c Helene and Toulme.

W096/06159 7l PCT~S95/10194 More commonly, inhibitory nucleic acids are designed to bind to m.~NA or mRNA precursors. Inhibitory nucleic acids are used to prevent maturation of pre-m~NA. 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 ~nro~;ng critical proteins. Eo~
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 ~v~ esses the c-myc proto-oncogene. 5ee Wickstrom E.L., et al. [93] and 2Q Harel-Bellan, A., et al. [31A]. As de6cribed in Xelene and Toulme, inhibitory nucleic acids targeting m.~NA have been shown to work by several different me~h~nirm~ to inhibit translation of the encoded protein(s).
The inhibitory nucleic acids introduced into the cell can also ~n~ R the "sense" strand of the gene~or m~NA to trap or compete for the enzymes or binding proteins involved in mRNA translation. See Xelene and Toulme.

- Lastly, the inhibitory nucleic aci~s can be used to induce chemical inactivation or cleavage of the target genes or mRNA. Chemical inactivation can occur by the induction of crosslinXs between the inhibitory nucleic àcid and the target nucleic acid within the cell.
Other chemical modifications of the target nucleic W096/06159 r - ~ r~l/~ ~/1~i91 acids induced by appropriately derivatizea 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 in~nr~ 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 ~n~o~r~ses 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 se~uences 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, Bio~rhnrlrrJy News 14(14) p. 5].
:: :
A problem associated with inhibitory nucleic acid therapy is the effective delivery of the inhibitory n~ acid to the target cell n vivo and the subser~uent int~rn~l;7~ion 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 ~ W096~61~9 , 2 l 9 6 8 9 2 PCT~ss~/l0l94 receptor on the surface of the target infected cell, and which is ;nt~r~l;7~d 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, pigL 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 re~uired 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 subse~uent 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 rrntinnrusly or intermittently such that the therapeutic agent in the patient is - effectlve 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 herpesvirus-induced KS are preferably administered to human WO96/06159 , ~ 2 ~ 9 5 8 9 2 PCT~S9~/10194 ~ J ~

patients via oral, intravenous or parenteral administrations and other systemic forms. Those of skill in the art will understand ~ u~,iate 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 suc~ as, e.g., 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, non-toxic carriers or diluents, which are defined asvehicles commonly used to formulate p~rr-ceutical 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, physiologioal 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, nnni ~genic stabilizers and the like. Effective amounts of such diluent or carrier are those amounts which are effective to obtain a pharmaceutically acceptable formulation i~ terms of solubility of components, or biological activity, etc.
~ == = .= = ===~ .= . =~ .
V. T Inoloqical APProaches to TheraPv.

Having i~n~ i fied a primary causal agent of ~S 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 , . . . . _ _ . _ _ _ . _ _ . . .. _ _ _ W096/06159 ' PCT~S9~10194 -- ' 21 96892 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 ~nt~ining two or more antibodies, other therapeutic age~ts, compositions, or the like, including, but not limited to, immuno-suppressive agents, potentiators and side-effect re-lieving agents. Of particular interest are immuno-suppressive agents useful in suppressing allergic re-actio~s of a host. Immunosuppressive agents of inter-est include prednisone, prednisolone, DECADRON (Merck, Sharp & Dohme, West Point, PA), cyclorb~srh~m;del cyclosporine, 6-mercaptopurine, methotrexate, azathioprine and i.v. gamma globulin or their combination. Potentiators of interest include monensin, ammonium chloride and chloro~uine. All of these agents are administered in generally accepted efficacious dose ranges such as those disclosed in the Physician Des~ ~eference, 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 z 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 .

W096~6159 i 2 ~1 ,9~6 8 9 2 ~ 94 ~
. . . -. . .

donors are pooled. Alternatively, plasmas from donors are pooled and then tested for antibodies to the human herpesvirus of the inve~tion; high-titered pools are then selected for use in KS patients.
s 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 antibodie3 or immunotoxins are either sterile liquid solutions, liquid suspensions or lyophilized versions and optionally contain stabilizers or excipients.
~yophilized compositions are reconstituted ~with suitable diluents, e.g., 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 nnnt~;n;ng the antibodies or immunotoxins will be administered in a therapeutically effective dose in a range of from about .01 mg/kg to about 5 mg/kg of the treated mammal. A preferred therapeutically effective dose of the pharmaceutical composition ~nnt~;n;ng 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 se~uential patient dose-escalation regimen.
Antibody may be administered systemically by injection i.m., subcutaneously or intraperitoneally or directly into ~3 lesions. The dose will be ~p~n~nt upon the properties of the antibody or immunotoxin emplQyed, e.g., its activity and biological half-life, the concentration of antibody in the formulation, the site and rate of dosage, the clinical tolerance of the ~ WO96~6159 2 i 9 6 8 9 2 PCT~S95/10194 patient involved, the disease afflicting the patient and the like as is well within the skill of the ~ physician.

t 5 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
6.5 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 l to lO0 mM. The solution of antibody may also contain a salt, such as sodium chloride or potassium chloride in a c~nrpntr~tion of 50 to 150 mM.
An effective amount of a stabili~ing agent such as an albumin, a globulin, a gelatin, a protamine or a salt of protamine may also be i nrl 11~rd and may be added to a solution c~ntA;n;ng antibody or immunotoxin or to the composition ~rom 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 c~mrQlln~ and the condition being A treated. The age, weight, and rl;nir~l condition of the recipient patient; and the experience and judgment of the rl; n i r; ~n or practitioner administering the therapy are among the factors affecting the selected W096~6159 ~ 21 9 6 8 9 2 PCTN395/10194 _ ~ v ;j ~ .

dosage. For example, the dosage of an immunoglobulin can range from about 0.l milligram per kilogram of body weight per day to about lO 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 infusiQn. Preferably, the dosage is repeated daily until ea~her 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 ef~ective 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 ~ualified observer. The dosing range varies with the compound used, the route of administration and the potency of the particular compound.

VI. ~accines ~n~ Pro~hv~ for KS

This invention provides a method of vaccinating a subject against Kaposl~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 ~ WO961061~9 2 1 9 6 8 9 2 PCT~S95110194 comprises administering to the subject an effective ; i7;ng dosé of the isolated herpesvirus vaccine.

A. Vaccines The invention also provides substances suitable for use as vaccines for the preventi~n of KS and methods for administering them. The v~ccines are directed against the human herpesvirus of the invention, and O most preferably comprise antigen obtained from the KS-associated human herpesvirus.

Vaccines can be made recombinantly. Typically, a vaccine will include from about l to about 50 mi~luyrdll.D of antigen or antigenic protein or peptide.
More preferably, the amount of protein is from about 15 to about 45 mi.Lu~Ld1..D. Typically, the vaccine is formulated so that a dose includes about 0.5 milliliters. The vaccine may be administered by any route known in the art. Preferably, the route is parenteral. More preferably, ït is subcutaneous or intramuscular.

There are a number of strategies for amplifying an antigen's effective~ess, 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 conv~nt;~n~lly, 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.

WO96~6159 ~ ~JI A J~

For parenteral administration, such as subcutaneous injection, examples of .suitable carriers are the tetanus toxoid, the ~;phth~ria toxoid, serum albumin and lamprey, or keyhole limpet, hemocyanin because they provide the resultant conjugate with minimum s genetic restriction ~ ~onjugates including these universal carriers can function as T cell clone activators in individuals having very different gene sets.
1 0 _ The conjugation between a peptide and a carrier can be accompliched using one of the methods known in the art. Specifically, the conjugation can use bifunctional cross-linkers as binding agent_ as detailed, for example, by Means and Feeney, "A recent review of protein modification techni~ues,"
Bioco~jugate Chem. 1:2-12 (1990).

Vaccines against a number of the Herpesviruses have been successfully developed. Vaccines against Varicella-~oster Virus using a live attenuated Oka strain is effective in preventing herpes zoster in the elderly, and in preventing ~h;~k~nrn~ in both immunovv",~, ;ced and normal children (Hardy, I., et al [30]; Hardy, I. et al. [31]; Levin, M.J. et al.
[54]; Gershon, A.A. [26]. Vaccines against Herpes simplex Types 1 and 2 are also commercially available with some succes6 in protection against primary disease, but have been less successful ' n preventing the estAhl;cl t of latent infection in sensory ganglia (Roizman, B. [78]; Skinner, G.R. et al. [B7]).

Vaccines against the human herpesvirus can be made by isolating ~trA~llular viral particles from infected cell rcultures, inactivating the viruc ~ with formaldehyde followed -by ultracentrifugation to concentrate the viral particles and remove the ~ WO96/06159 2 1 9 6 8 9 2 PCT~S95/10194 formaldehyde, and ; ;7;ng individuals with 2 or 3 dose6 cnnt~;n;ng 1 x 109 virus particles (Skinner, G.R.
et al. [86]). Alter atively, envelope glycoproteins can be expressed in E. coli or transfected into stable ~ 5 mammalian cell lines, the proteins can be purified and used for vaccination ~Lasky, L.~. [53]). MHC -binding peptides from cells infected with the human herpesvirus can be 1~Pnt; f; ed _or_vaccine candidates per the methodology of [61], supra.

The antigen may be combined or mixed with various solutions and other compounds as iB known in the art.
For example, it may be ~;n;Rtered 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-in-oi~ P~nlR;nnq, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (2ropinn;h~terium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, D~AE-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, N.J.) 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 oi 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 W096106159 ~ '2! 96892 PCT~S9~/10194 can be present in an amount of about Q.5~ of the vaccine mixture (Al203 basis). On a per-dose basis, the amount of the antigen can range from about O.l ~g to about lOO ~g protein per patient. A preferable range is from about l ~g to about 50 ~g per dose. A
more preferred range is about l~ ~g to about 45 ~g.
A suitable dose size is about 0.5 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 0.5 ml rnnt~;nlng 45 ~g of antigen in admixture with 0.5~ Alnm;n1-m hydroxide. After formulation, the vaccine may be inouL~uLdted into a sterile rnnt~;n~ which is then sealed and stored at a low temperature, for ~example ~~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 : (e.g., 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 ~, h;n~ with appropriate doses of c~...~ou11ds including influenza antigens, such as influenza type A antigens.
Also, the antigen could be a rl , ~nt of a recombinant vaccine which could be adaptable for oral administration.

Vaccines of the invention may be combined with other vaccines for other disease6 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.

~ WO96/06159 21 96892 PCT~S95110194 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 =smail 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 additiors 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 oi passive immunotherapy of herpesviral diseases including perir~tal varicella and CMV. Immune globulin from , persons previously infected with the human herpesvirus and bearing a suitably high titer oi antibodies against the virus can be given in combination with antiviral agents (e.g. ganciclovir), or in combination with other modes of immunotherapy that are currently 21 9~892 W096/06159 ~ j PCT~S95/10194 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 a6 described herein. Antibodies specific for an epitope expres~ed on ceIls 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 ~alt 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, mandeIic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potasslum, ;l1m, 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 aetermining in a first sample from a subject with Kaposi's sarcoma the presence of the ;~ol~ted 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 .. _: ... _~_..... :.. _, ._ .... _ __ . __. _ .

~ Wo96~6159 2 1 9 6 8 9 2 PCT~Sg5/10194 the treated subject, and comparing the amount of isolated D~ molecule determined in the first sample with the amount determined in the second sample, a difference ~n~ t;ng 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.
1~
VII Screeninq Assavs For Pharmaceutical Aqents of Intere8t in Alleviatinq the Svm~toms of KS.

Since an agent involved in the causation or progression of KS has been ;~nt;f;ed 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 ~t~r~;n~ whether or not a drug has activity against the virus described herein are contemplated ir this invention_ Such assays comprise incubating a compound to be evaluated for use in KS
treatment with cells which express the XS associated human herpesvirus proteins or peptides and determining t~r~from 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 n vivo animal models would also be effective 38 Compounds with activity against the agent of interest or peptides from such agent can be screened in n vit~o as well as Ln vivo assay systems. In vitro assays include infecting peripheral blood leukocytes or susceptible T ~ell l;nes 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 WO96/06159 ~ 2 l 9 6 8 9 2 ~ ,.JI94~

terminators, antisense olig~nnrlpr~tides and random polypeptides (Asada, H. et al. [7]; Kikuta et al. [48]
both incorporated by reference herein). Infected cultures and their supernatants can be assayed for the total amount o~ virus including the presence of the viral genome by riuantitative PCR, by dot blot assays, or by using immunologic methods. For example, a culture of susceptible cells could be i~fected with the human herpesvirus in the presence of various rrnrPntrations of drug, fixed on slides after a period of days, ana Pr~minP~ for viral antigen by indirect immunofluorescence with monoclonal antibodies to viral peptides (t48], 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. [36], incorporated by reference herein).

As an alternative to whole cell i vitro assays, purified enzymes isolated from the human herpesvirus can be used as targets for rational drug design to de~ermine the ef~ect of the potential drug on enzyme activity, such as thymidine phosphotransferase or D~A
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 W0 94/04920 (UL13 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 (e.g. acyclo-guanosine).
The level o~ Yirus in the cells is then determined WO96/06159 ~ '~~' 2 1 q 6 8 9 2 PCT~595110194 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, 10 ~;th~r 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 lS 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 D3TAILS SECTION I:
Experiment l: Rnyl~es L~tional difference analy~is (RDA) to identify and characterize unique DNA sequ~n~e~ in KS tissue To search for foreign DNA sequences belonging to an infectious~ agent in AIDS-RS, representational di~erence 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-~lR~ced tissue obtained from the same patient [58~. This method can detect adenovirus genome added in single copy to human DNA but has not been used to WO96106159 ~t 2~ 6 8 q 2 PCT~S95/10194 identify previously uncultured infectious agents. RDA
i5 performed by making simplified "representations" of genomes from dis=eased and normal tissues from the same individual through PCR amplification of short restriction fragments. The DNA r~prp~nt~tion from the diseased tissue is then ligated to a priming sequence and hybridized to an excess of unligated, normal tis6ue DNA representation. Only unique sequences found in the diseased tissue have priming sequences o1l 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 (l0 ~g) extracted from both the KS lesion and unaffected tissue were separately digested to completion with Bam HI (20 units/~g) at 37~ C for 2 hours and 2 ~g of digestion fL~I nt~ were ligated to NBaml2 and NBam24 priming seguences [primer sequences described in 58]. Thirty cycles of PCR amplification were performed to amplify "representations" of both genomes. After construction of the genomic repre-sentations, RS tester amplicons between 150 and 1500 bp were isolated from an agarose gel and NBam priming sequences were removed by digestion with Bam ~I. To search for unigue 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 l, lane l). 0.2 ~g of ligated KS lesion amplicons were~hybridized to 20 ~g of unligated, normal tissue representational amplicons. An aliquot of the hybridization product t was then subjected to l0 cycles of PCR amplification using JBam24, followed by mung bean nuclease digestion~ A~ aliquot of the mung bean-treated ~ WO96/06159 2 1 ~ 6 8 9 2 PCT~S9~/10194 ~iff~r~n~P product wa~ then subjected to 15 more cycles of PCR with the JBam24 primer (Figure 1, lane 2). Amplification products were redigested with Bam HI and 2DD ng of the digested product was ligated to RBaml2 and RBam24 primer sets for a second round of hybridi~ation and PC~ ampli~ication (Figure 1, lane 3). This enrichment procedure was repeated a third time using the JBam primer set (Figure 1, lane 4).
Both the original driver and the tester D~A samples ~Table 2, Patient A) were subsequently found to contain the AIDS-KS specific 6equences 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-hybr;~;7Ati~n from KS and normal tissue resulted in a diffuse banding pattern (Figure 1, lane 2), but four bands at approximately 380, 450, 540 and 680 bp were i~nt;fiAhle after the second amplification-hybri~;_Ati~n (Figure 1, lane 3). These bands became discrete after a third round of amplification-hybridization (Figure 1, lane 4). Control RDA, per~ormed by hybridizing DNA extracted from AIDS-KS
tissue against itself, produced a single band at approximately 540 bp (Figure 1, lane 5). The four KS-associated bands (designated ~5330Bam, KS390Bam, KS480Bam, KS627Bam after digestion of the two flanking 2B.bp ligated priming sequences with 3am 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).

W096/061~9 ~ 2 1 q 6 8 9 2 1 ~ JIj~I94 ~

Ex~eri~ent 2: Det~rm;n-t;nn of the ~pee;f;r;ty of AIDS-RS uni~ue E ~

To determine the specificity of these sequences for AIDS-KS, random-primed 32P-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 ~Y~m; n~
microscopica-lly for morphologic confirmation of KS and ;mmnnn~;~tochemically for Factor VIII, U~ex 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, 5 vascular tumors from nonAIDS patients and 13 tissues infected with ~OL~Iistic 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 1, were collected from diagnostic biopsies and autopsies between 1983 and 1993 and stored at -70~C. Each tissue sample was ~rom a different patient, except as noted in Table 1. Most o~ the ~7 KS specimens were ~rom Iymph nodes dissected under surgical conditions which ~;m;n;c~ possible cnnt~m;n~tion with normal skin ~10ra. All specimens were digested with B~m ~I prior to hybridization ~ WO96~6159 ~ / " 2 1 9 6 8 92 PCT~59511~194 KS390Bam and KS480Bam hybridized nonspecifically to both KS and non-KS tissues and were not further characterized. 2Q of 27 (74~) 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 l).
In contrast to AIDS-KS lesions, only 6 of 39 (15~) non-KS tissues from patients with AIDS hybridized to the KS330Bam and KS627Bam inserts (Table l).
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, ;nrlu~;ng a hemangiopericytoma, two angio~r, q and a lymphangioma, were also negative by Southern blot hybridization DNA extracted from tissues with u~LLullistic infections common to AIDS patients, including 7:acid-fast bacillus (undetermined species), l cytomegalovirus, l cat-scratch bacillus, 2 cryptococcus and l toxoplasmosis infected tissues, were negative by Southern blot hybridization to KS330Bam and KS627Bam (Table l).
~ -W096106159 ~ ~ ' 2 ~ 9 6 8 9 2 PCT~S95110194 ~

~able 1. Southern blot hybridization for XS330Bam and KS627Bam and PCR amplification for KS330,3;
in human tissues from individual patients.

~i~~ n KS330Bam Southern RS627Bam Southern RS33D,3~
hYbridization n(t) hybr;sl73tio~ l PCR QQsitive AIDS-RS 27~ 20 (74) 21 ~78) 25 ~93) AIDS 27t 3 (11) 1 ~11) 3 (11) lymphomas AIDS 12 3 (25) 3 (25) 3 (25) lYmph nodes Non-AIDS 29 0 (0) 0 (0) 0 ~0) LYmphomas Non-AIDS 7 0 ~0) 0 ~0l 0 ~0) lYmph nodes Vascular 4i 0 ~0) 0 (0) 0 ~0) ~umors Opportunistic 13~ 0 ~0) 0 (0) 0 ~0) iniections Consecutive 49~ 0 ~0) o ~0) o ~0) sur5~ical biopsies ~ WO96/06159 2 1 9 6 8 9 2 PCTN59~l0194 Leqend 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 hybri~ t;nn to KS330Bam and KS627Bam and by PCR
amplification for the KS330234 amplicon.

tIncludes 7 small non-cleaved cell lymphomas, 20 diffuse large cell and ; lnr,hl~qtic lymphomas. Three of the lymphomas with ; nhl~ctic morphology were positive for KS330Bam and KS627Bam.

~ t Inclu~es 13 anaplastic large cell lymphomas, 4 diffuse large cell lymphomas, 4 small lymphocytic lymphomas/chronic lymphocytic l~nk~m;~ql 3 hairy cell leukemias, 2 monocytoid B-cell lymphomas, 1 frll;clll~r small cleaved cell lymphoma, 1 Burkitt's lymphoma, l plasmacytoma.

rnrln~pq 2 angiosarcomas, l hemangiopericytoma and l lymphangioma.

~ Includes 2 cryptococcus, l toxoplasmosis, l cat-scratch bacillus, l cytomegalovirus, l 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 Myrr,plnrm- penetrans were negative by PC~

Tissues included skin, appendix, kidney, prostate, hernia sac, lung, fibrous tissue, g~l lhl ~ r, colon, foreskin, thyroid, small bowel, adenoid, vein, axillary tissue, lipoma, heart, mouth, hemorrhoid, psen~r~n~llrysm and fistula track. Tissues were WO96106159 r~ S~

collected ~rom a consecutive series of biopsies on patients without AIDS but with unknown XIV serostatus.

**Apparent nonspeci~ic hybridization at approximately 20 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 ior these sve~uences and were PCR
negative ior KS330234.

21 96~92 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 (74~) AIDS-f 5 KS specimens hybridized to KS330Bam and 21 of 27 (78~) AIDS-KS specimens hybridized to KS627Bam, compared to only 6 of 142 (4~) non-KS human DNA control specimens (X7=85.02, p< 10-7 and'X2=92.4, p< 1o'7 respectively).

The sequence copy number in the AIDS-KS tissues was estimated by simultaneous hybridization:with KS330Bam and a ~40 bp probe for the constant region of the T
cell receptor ~ gene [76~. 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 L~ ;n;ng tissues had weaker hybridization signals for the KS330Bam probe.

Ex~eriment 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 manufactureris instructions. Nucleotides sequences were ~nf; ~~ with an Applied Biosystems 373A
Sequencer ln the DNA Sequencing ~;l;t;~c 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 BD~Fl open reading frame (O~F) of Epstein-Barr virus (EBV). Further analysis revealed that both RS330Bam and KS627Bam code for _ _ _ . ~. / 21 96892 WO96/06159 ~ ' PCT~S95/10194 amino acid sequences with homology to polypeptides of viral origin. SwissProt and PIR protein databases were searched for homologous ORF using BL~3TX [3].

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 [2], a ~ h~rpe5virus which causes fulminant lymphoma in New world monkeys.
This iragment 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) [9]. The amino acid sequence encoded by KS627Bam is homologous with weaker identity (31~) to the tegument protein, gpl40 (ORF 29, NCBI g.i. 60396, bplO8782-112~81~ of herpesvirus saimiri.

Sequence data from KS330Bam was used to construct PCR
primers to amplify a 234bp fragment designated KS330~34 (Figure 3B). The c~n~it;~n~ for PCR analyses were as follows: 94~C for 2 min (l cycle); 94~C for 1 min, 58~C for 1 min, 72~C for l min (35 cycles); 72~C
~t~nci~n for 5 min (1 cycle). ~ach PCR reaction used 0.1 ~g of genomic DNA, 50 pmoles of each primer, 1 unit of Taq polymerase, i00 ~M of each deoxynucleotide tr;ph~crh~te~ 50 mM KCl, 10mM Tris-HCl (pH 9.0), and 0_1~ Triton-X-100 in a final volume of 25 ~l. 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 KS330234 (Figures 4A-4B) demonstrating that KS330Bam is present in some KS lesions at levels below the WO96106159 ~ 2 1 9 6 8 9 2 PCrlUS95/10194 threshold for detection by Southern blot hybridization. All KS330,3, PCR products hybridized to a 32p end-labelled 25 bp internal oligomer, confirming the specificity of the PCR (Figure 4B). Of the two AIDS-KS specimens negative for KS33023~j, both ~pecimens appeared to be negative~ for technical reasons: one had no microscopically detectable KS tissue in the frozen sample ~Figures 4A-4B, lane 3), and the other (Figu=res 4A-4B,-lane 15) was negative in the control : PCR amplification for the p53 gene indicating either DNA degradation or the presence of PrR inhibitors in the sample. PCR amplification of the p53 tumor suppressor gene was used as a control for D~iA quality.
Sequences of p53 primers from P6-5, 5'-AcAGGGcTGGTTGrrrAr~r~r~T-3~(sEQ ID No: 44~; and P6-3. 5'-AGTTr~ 7~rr~rr-TcAG-3~(sEQ ID NO: 45) [25].

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. KS330,34. A11 of these specimens were amplifiable for the p53 gene, indicating that inadequate PCR amplification was not the reason for lack of detection of KS330,3, in the control tissues. Samples rnnt~;n;ng DNA from two r~n~l;rl~te XS 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 KS330234. In addition, several KS specimens were tested using commercial PCR primers (Stratagene, Ba Jolla, CA) specific for mycoplasmata and primers specific for the EBNA-2, EBNA-3C and EBER regions of EBV and were negative [57].
Overall, D~A from 25 (939~) of 27 AIDS-KS tissues were positive by PCR compared with DNA from 6 (496) of 1~2 WO96/06159 ' PCT~S95/10194 control tissues, including 6 (15~) of 39 non-KS lymph nodes and lymphomas from AIDS patients (X2=38.2, P ~
10-6), 0 of 36 lymph nodes and lymphomas from nonAIDS
patients (x'=55.2, p < 10-7) and 0 of~ 49 consecutive biopsy specimens (X2=67.7, p < 10-7) . Thus, KS31023~ 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 pa~ients 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 L. ining 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 i ~hlARtiC
lymphomas from AIDS patients. It is possible that the putative KS agent is also a cofactor for a subset of AIDS-associated lyl ~ R [16, 17, 80].
__ _ _ _ To determine whether KS330Bam and KS627Bam are portions of~a larger genome and to ~tPrmin~ 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 5). These sequences hybridized to various sized fL _ t~ of the digested KS DNA indicating that both sequences are fragments of larger genomes. Differences in the KS330Bam hybridization pattern to~Pw II digests of the three AIDS-KS specimens indicate that polymorphisms may . .__ _ _ ._ . ._: : : . . _ . ._ . . . ._ . ..

2'1 96892 WO96106159 p~ 194 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 f,d~ t~ are not adjacent to 5 one another.

If KS330Bam and KS627Bam are heritable polymorphic DNA
markers for KS, these 6equences should be uniformly detected at non-KS tissue sites in patients with AIDS-KS. Alternatively, if KS330Bam and KS627Bam aresequences specific for an exogen~ous 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 32P-labelled KS330Bam and KS627Bam probes as weIl as analyzed by PCR using the KS330~34 primers (Table 2). While KS
lesion D~A -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 2~ negative for these sequences. Uninvolved stomach lining adiacent 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.

- WO96~6159 PCT~S95110194 Table 2: Differenti l detection of RS330Bam, KS627Bam and KS3301,~ sequences in KS-involved and non-involved tissues from three patient~
with AIDS-KS.

KS330Bam KS627Bam KS330234 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: S~~~cl~nin3 and ~o~l~nr~n~ of KS~V

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 r~ntR;n;n~ the KS330Bam se~uence was ;~nt;~;ed~ The 20.kb clone digested with PwII
(which cuts in the middle of ~he KS330Bam sequence) produced l l kb and 3 kb fragments that hybridized to KS330Bam. The l.l kb 6ubcloned insert and -goo bp from the 3 kb 6ubcloned insert resulting in 9404 bp of 2~ 96892 WO96~61S9 = r~ i5 contiguous sequence was entirely sequenced. This sequence rrnt~;n~ partiaI and complete open reading ~ frames homologous to regions in gamma herpesviruses.

f 5 The KS33DBam 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 6). In HSVSA, the VP23 protein is a late structural protein involved in capsid construction. Reverse transcriptase (RT)-PCR o~ mRNA
from a KS Iesion is positive for transcribed KS330Bam mRNA and that indicates that this ORF is transcribed in KS lesions. Additional cvidence 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 o~ the KS agent lies immediately 5' to the BDLFl/ORF26 homolog which is a conserved orientation ~Lmong 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 ~our loci in two separate regions with homologies to gamma herpesviral genomes have been ir~rnt; f j ~
i In addition to fLay~ Ls obtained from Pvu II digest o_ the 21 Kb phage insert described above, fragments obtained ~rom a BamHI/NotI digest were also subcloned into pBluescript (Stratagene, La Jolla, CA). The WO96/06159 ~ 8 ~ ~ PCT~S95/1019 io2 termini of these subcloned fragments were sequenced and were also found to be homologous to nucleic acid sequence EBV and ~SVSA genes. These homologs have been used to develop a pr~l ;min~ry map of subcloned fragments (Figure 9). Thus, sequencing has revealed that the KS agent m~in~in.~ co=linear homology to gamma herpesvlruses over the length of the 21 Kb phage insert.

Ex~eriment 5: Determination of the phylogeny of KS~V

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 pslue~cript (Stratagene, ~a Jolia, 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 uni~ue 3' and 5' end-fragments from positive phage clones as probes, and following the strategy above a KS genomic library are ~r~ d by ~tandard methods for additional contiguous seguences.

For~sequencing purposes, restriction fragments are subcloned into phagemid pBluescript KS+, pBluescript KS-, pBS+, or pBS- (Stratagene) or into plasmid pUCla or pUC19 Recombinant DNA was purificd through CsCl density gradients or by anion-exchange chromatography (Qiagen).
: _ _ _ _ Nucleotide seguenced by standard screening methods of cloned fragments of KSHV were done by direct .. .. , . . , , . . . . _ . . _ ., .. , _ .

~ WO96/06159 2 1 9 6 8 9 2 PCT~59~10194 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 ~nnf; ~ by sequencing overlappin~ clones Targeted homologous genes in regions flanking KS330Bam include, but are not limited to: Il-10 homolog, thymidine kinase ~TR), 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 se~uence oi this gene will aid in the pr~;ct;~ of chemotherapeutic agents useful against KSHV. TK is encoded by the EBV BXBF1 ORF located -9700 bp rightward of BDLFl and by the HSVSA ORF 21 -9200 bp rightward of the ORF 26. A
subcloned fragment of KS5 was ;~nt;f;P~ with strong homology to the EBV and HSVSA TK open reading frames.

g85 is a late glycoprotein involved in membrane fusion homologous to gH in HSVl. In EBV, this protein is encoded by BLXF2 ORF located -7600 bp rightward of BD~Fl, and in HSVSA it is encoded by ORF 22 located -710~ bp rightward of ORF26.

g35 is a late EBV glycoprotein _ounsi in virion and plasma membrane. It is encoded by BDLF3 ORF which is 1300 bp leftward of BDLF1 in EBV. There is no BD~F3 ~ homolog in HSVSA. A subcloned fragment has already been i~nt;f;~d with strong homology to the EBV gp~5 open reading frame.
Major capsid protein (MCP) is a conserved 150 KDa protein which is the major component of herpesvirus ~ ~ 2 ~ 96892 WO96/06159 ~ r~ .l0i9 capsid. A~tibodies are generated against the MCP
during natural infection with most herpesviruses. The terminal 1026 br 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 repeat~, 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 torm;n~l repeat region is ;r~~~;At~ly adjacent to BNRF1 i~n EBV and ORF
75 in ~SVSA. 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 cirrnlAr;7od genome. There is no LMP1 homolog in ~SVSA.

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-goo0 bp leftward of the BNRFl ORF in EBV; there are no corresponding regions in ~SVSA. ~

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 rnntAm;nAtion or to incidental co-infection with a known herpesvirus since ~ WO96/06159 ~ ~ 21 96892 PCT~S95/10194 the sequences are distinct from all sequenced herpesviral genomes (including EBV, CMV, HHV6 and HSVSA) and are associated spçcifically with KS in three separate comparative studies. Furthermore, PCF~
testing of KS DNA with primers specific for EBV-l and EBV-2 fail~d to demonstrate these viral genomes in these:tissues. Although KSHV is homologous to EBV
regions, the sequence does not match any other=known se~uence and thus provides evidence for a new viral genome, related to but distinct from known members of the herpesvirus family.

ExPerime t 6: Serological stud$es Tn~1re~t immunofluorescence as8av (IFA) Virus-c~nt~;n;ng 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 ~nt; ho~;es are washed off. An ~nt;h11r-n immunoglobulin linked to a fluolu~1.L- ~, such as fluorescein, is then incubated with the slides, and then excess fluorescent immunoglobulin is washed off The slides are then P~m;n~ under a microscope and if the cells fluoresce, then this indicates that the sera cnnt~inc antibodies directed against the antigens present in the cells, such as the virus.
An indirect immunofl~ c~n~ assay (IFA) was - periormed on the Body Cavity-Based Lymphoma cell line (BCB~-l), which is a naturally transformed EBV
;n~t~ (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 antibDdy binding. To remove ~, ;~. ? 2 1 9 6 8 9 2 W096/06159 ' ~ PCT~S95/1019 non6pecific antibodies directed against EBV and lymphocyte antigens, sera at 1:25 ~ tinn were pre-adsorbed using 3X106 196 paraformaldehyde-fixed Raji cells per ml Qf sera. BCBLl cells were ~ixed with ethanol/acetone, incubated with dilutionc of patient sera, washed and incubated with fluorescein-conjuyated goat anti-human IgG. Indir~c~ immuno~luorescent staining was determinçd.

Table 3 shows that unabsorbed case and control sera have similar end-point dilution indirect immunofluorescence as6ay (IFA1 titers against the BCBLl cell line. After Raji ad60rption,~:ca6e ~sera have four-fold higher IFA titers against BCBLl cells than control sera. Results indicated that pre-adsorption against paraformaldehyde-fixed Raji cells reduces fluorescent antibQody binding in control sera but do not Pl;m;n~tP antibody binding to KS case sera.
These results indicate that subject6 with KS have specific antibodies directed against the KS agent that can be detected in serological assays such as IFA, Western blot and Fnzyme i nn~ yS ~Table 3).

' ' 2 1 9 68 92 WO96106159 PCT~S9~10194 Table 3: Indirect; ~flll~escence end-point titers or KS case and non-~S control ~er~ again~t the BCPL-1 cell line . ~ ~ = = ~ ~
Sera No. Status* Pre-adsorPtion Po6t-adsorPtion**
1 XS > 1:400 ~ 1:400 2 KS 1:100 1:100 3 KS 1:200 1:100 g ~S > 1:400 1:200 Control > 1:400 1:50 6 Control 1:50 1:50 7 Control 1:100 1:50 0 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 Tmmnn~hlottin~ ("Western blot") Virus-c~nt~;n;ng cells or purified virus (or a portion of the .vi~us, 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 nitr~ lose 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 ~nt;h~;es in patient sera ~ 40 shows up as a band on the~ membrane at the corresponding molecular weight region.

.

WO96~61~9 ~n7vme imm~1n~cSaV (~TA or ~T,~q~) 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 ~lk~l;n~ 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 ~nt;hllm~n immunoglobulin attached to a reporter enzyme, such as a peroxidase. The plate is washed again to remove nonspecific antibody and then developed. Wells c~n~;n;ns antigen that is specifically recognized by antibodies in the patients sera change color and can be detected by an E~ISA plate reader (a spectrophotomer).

AllSthree 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 antibodie~
can potentially give a falsely positive test result (i.e. the patient is actually not infected with the virus but has a positive test result because of cross-reacting 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-adsorp~ion that bind to antigens in the preparation should be directed against the virus.

~ WO96/06159 2 1 9 6 8 9 2 PCT~S9~10194 ~Y~eriment 7: =

BCBL~l, from lymphomatous tissues belonging to a rare iniiltrating, anaplastic body cavity lymphoma occurring in AIDS patients has been placed in rnnt;nnnus cell culture and shown to be continuously infected with the ~S agent This cell line is also naturally infected with Epstein-Barr Virus (EBV). The BCBL cell li~e was used as an antigen substrate to detect specific KS ~nt;ho~;es 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 lOmin. and su6pended in sample buffer rnnt~in;ng 50 mM Tris-~Cl p~ 6.8, 2~ SDS (w/v), 15~ glycerol ~v/v), 5~ ~-mercaptoethanol (v/v) and 0.001~ bromophenol (w/v) with protease inhibitor, 100 ~M phenylmethylsulfonyl fluoride (PMSF). The sample was boiled at 100~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 5 (3). ~ Pr~ct~;n~ protein standards were included:
myosin, 200 kDa; ~-rJ~l~rtoc;dase~ 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).
T~nnnhlotting experiments were performed according to the method of Towbin et al. (4). Briefly, the proteins were electrophorectically transferred to WO96/061~9 ' ~ ' 2 l 9 6 8 9 2 PCT~S9~1019~

Hybon-C extra membranes (Pharmacia~ at 24 V for 70 min. The membranes were then dried at 37~C for 30 min, saturated with 5~ skim milk in Tris-buffered saline, pH 7.4 (TBS) containing 50 mM Tris-HCl and 200 mM NaCl, at room temperature for 1 h. The membranes were subse~uently incubated with human sera at dilution 1:200 in 1% skim milk overnight at room temperature, washed 3 times with a solution C~nt~ining 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 1:5Q00 in 1% skim milk. After repeating the.washing,the membranes were stained with nitroblue tetranolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (Gibco BRL).

Two bands of approximately 226 kDa and 234 kDa were i~nt;f;e~ to be specifically present on the Wester-blot 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.
5 sera from AIDS gay man patients without KS and 2 sera from AIDS woman patients without KS as well as l sera from nasopharyncel carcinoma patient were not able to detect these 2 bands in BCBL l, P3~3, Raji and Bjab cell lysates. In a blinded experiment, using the 226 kDa and 234 kDa markers, 15 out of 16 sera from KS
patier,ts 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 ~uclei fraction of BCBLl. 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

~ WO96106159 2 1 9 6892 PCT~S95/10194 ,~ 111 at 5XlOs cells/ml can be pelleted at low speed. The pellet is placed in 10 mM NaCl, 10 mM Tris pH 7.8, 1.5 mM MgCl2 (equi volume) + 1.0~ NP-40 on ice for 20 min to lyse cells~ The lysate is then spun at 1500 rpm for lD min. to pellet nn~lP;r The pellet is used as the starting fraction for the antigen preparation for the ~estern blot. This will reduce cross- reactive cytoplasmic antigens.

~Yr~; t 8: TrAnr~; Rsi~n studies Co-;nfection ex~eriments BCBLl cells were co-cultivated with Raji cell lines separated by a 0.45 ~ tissue filter insert.
Approximately, 1-2 x 106 BCBL1 and 2x106 Raji cells were co-cultivated for 2-20 days in supplemented RPMI
alone, in 10 ~g/ml 5'-b~ yuridine (BUdR) and 0.6 ~g/ml 5'-flourodeoxyuridine or 20 ng/~l 12-O-tetradeca~oylphorbol-13-acetate ITPA). 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 BCBLl in 20 ng/ml TPA
for 2 days survived and has been kept in continuous suspension culture for ~lO weeks. This cell line, designated RCC1 (Raji Co-Culture, No. 1) remains PCR
positive for the KS33023~ sequence after multiple passages. This cell line is identical to its parental Raji cell line by flow cytometry using EMA, B1, B4 and BerH2 lymphocyte-flow cytometry (approximately 2~).
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 WO96/06159 ~ ~ ?-1 ~ 6 ~ ~ 2 PCT~S9511019 AIDS-KS (>90~), and the low prevalence of the agent in non-KS tissues from immunocompromised AIDS patients, indicates that this agent has a causal role in ATDS-KS
[47, 68].
ExPer~ment l0: Isolation of ~SEV

Crude virus preparations are made from either the supernatant or low speed pelleted cell fraction of BCBLl cultures. Approximately 650ml or more of~log phase cells should be used (~5Xl06 cells/ml).

Eor bonding whole virion from supernatant, the cell free supernatant is spun at l0,000 rpm in a GSA rotor for l0 min to remove debris. PEG-8000 is added to 7~, dissolved and placed on ice for ,2.5 hours. The PEG-supernatant is then spun at l0,000 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.l M NaCl, 0.0l M Tris, pX 7.5).
Thi6 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 (e.g. using a fractionator) and each fraction is ~hen tested by dot blotting using specific hybridizing primer sequences to determine the gradient fraction rrnt~in;ng 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 i6 washed and pelleted in PBS, then lysed using hypotonic shock and/or repeated cycles of freezing and thawing in a small v~lume (~3 ml).

~ WO96/06159 ~ 2 1 9 6 8 9 2 PCT~S95/10194 Nuclei and other cytoplasmic debris are removed by centrifugation at lO,OOOg for 10 min, filtration through a 0.45 m filter and then repeat centrifugation at lO,OOOg for 10 min. This crude preparation ~nnt~in~ viral genome and soluble cell components.
The genome preparation can then be gently chloroform-phenol extracted to remove associated proteins or can be placed in neutral DNA buffer-~1 M NaCl, 50 mM Tris, 10 mM EDTA, pH 7.2-7.6) with 2~ sodium dodecylsulfate (SDS) and 1~ sarco~yl. 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 RSHV genome from RSHV infected cell cultures (97 and 98).
Approximately 800 ml of BCBL1 cells are pelletea, washed with saline, and pelleted by low speed centrifugation. :~The cell pellet is lysed with an equal volume of RSB (10 mM NaCl, 10 mM Tris-HCl, 1.5 mM MgCl2, pH 7.8) with 1~ NP-40 on ice for lO 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 dodecy~sulfate and EDTA to a final concentration of 10 mM. The supernatant is loaded on a 10-30~ sucrose gradient in 1.0 M NaCl, lmM 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 gradie~t 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 KS~V DNA
frd~ ~, KS631Bam has been hybridized to the membrane WO96106159 ~ 21 96892 PCT~59~/l0l9 ~

using standard techniques. Figure 11 shows that the major solubilized fraction of the KSHV genome bands (i.e. 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 Qccurs in fractions 26 through 32.

FxPeriment 11: Purifiaation o~ KS~V

DNA is extracted using standard techniques from the RCC-1 or RCC_12F5 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 z Fresh lymphoma tissue ~nnt~;n;ng 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 2Q ~lxlQScells/ml into standard lymphocyte tissue culture medium, such as RMP 1640 supplemented with 10% fetal calf serum. Immortalized lymphocytes cnnt~in;ng the KSHV virus are in~f;n;tely 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 cnnt~;n;ng the virus from a cn~t;nnnllcly infected cell line at a concentration o~ >lxlQG cells/ml. The media is centrifuged at 2000xg for 10 minutes and filtered~
through a 0.45~ filter tOE remove cells The media is applied in a l:l volume~with cells growing at ~lxlQ6 cells/ml for 48 hours ~ The cells are washed and pelleted and placed~in fresh culture medium, and tested after 14 days of growth.

~ WO96/06159 2 1 9 6 8 9 2 PCT~S9S/10194 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 ~or 10 minutes, the supernatant is removed and centrifuged again at lO,OOOxg for 15 minutes to remove nuclei and organelles. The supernatant is filtered through a 0.45~ filter and centrifuged again at lOO,OOOxg for 1 hour to pellet the virus. The virus~ can then be washed and centrifuged again at lOO,OOOxg for 1 hour. :1 ~, 21q,6,892 WO96/061~9 '' ' '' PCT~S95/lOI9 1. Ablashi, D.V., et al. Viroloqv 184:545-552.
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WO96/06159 ~ 2 ~ 9 6 8 9 2 PCT~S9~1019 ~

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W096/06159 - i 2 1 9 6 ~ 9 2 r~ J ~cls~

~ KIM~l~LAL DETAILS SBC~ION II:

Se~uencinq Studies: A lambda phage (KS5) from a KS
lesion genomic library identified by positive hybridization with KS330Bam was digested with 3amHI
and Not I (Boehringer-Mannheim, Tn~;~n~polis IN~; five fragments were ~gel isolated and subcloned into Bluescript II KS (Stratagene, La ~olla CA). The entire sequence was determined by bidir~rt;nn~l sequencing at a seven iold average r~nn~n~y 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 3-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 identifie~ ; ng Motif (Genetics Computer Group, Madison WI).

Sources of Her~esvirus Gene Seauence Com~arisons:
Complete genomic sequences of three ~ pesviruses were ava~lable: Epstein-Barr virus (EBV), a herpesvirus of humans [4]; herpesvirus saimiri (BVS), a herpesvirus of the New World monkey Saimiri sciureus [l]; and equine herpesvirus 2 (EHV2 [49]). Additional thymidine kinase gene se~uences were obtained for alcP~ ~r~; n~ herpesvirus 1 (AHVl [22]) and bovine herpesvirus 4 ~BHV4 [31]). Sequences for the ma~or capsid protein genes of human herpesvirus 6B and human herpesvirus 7 (HHV7) were from Mukai et al. [34]. The sources of all other sequences used are listed previously in McGeoch and Cook [31] and McGeoch et al.
[32].

' 21 96892 WO96/06159 PCT~595110194 Phvlo~enetic Inferen~e: Predicted amino acid sequence~
used for tree construction were based on previous experience-with herpesviral phylogenetic analyses [31]. Alignments of homologous sets of amino acid sequences were made with the AMPS [5] 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 cn~t~;nr~ inserted gaps in one or more se~uences were removed before use for tree construction. Phylogenetic ;nf~r~nc~ ~L~L~ were from the Phylip set, version 3 5c [14] and from the GC5 set [16]. Trees were built with t_e maximum parsimony (MP), neig-h-bcr 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 [46]. 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 l;k~l;hnod (ML) method. Protml was obtained form J. Adachi, Department of Statistica Science,-The Graduate University for Advanced Study, Tokyo 106, Japan. Because of computational constraints, Protml was used only with the 4-species CS1 alignment.
C1~mned Xomogeneou6 Electric Field (~ Cel - Electro~horesis: Agarose plugs were prepared by resuspending BCBL-1 cells in 1~ LMP agarose (Biorad, Hercules CA) and 0.9~ NaC1 at 42~C to a final ~nnc~ntration of 2 5 x 107 cells/ml. Solidified agaros~ plugs were transferred into lysis buffer (0.5M
EDTA p~ 3.0, 1~ sarcosyl, proteinase K at l mg/ml Wo96tO6159 ' ' ' ' PCTtUS9s/lOI9 final concentration) and incubated for 24 hours.
Approximately 10' 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 pul6ed field gel electrophoresis~apparatus (Biorad, Hercules CA), Southern blotted and hybridized to KS627Bam, KS330Bam and an EBV t~rm;n~l repeat se~uence [40].

TPA Induction of Genome Re~lication: Late log phase BCB~-1 cells (5xlOs cPlls per ml) were incubated with varying amounts of ~ 12-0-tetradecanoylphorbol-13-acetate (TPA, Sigma Chemical Co , St. Louis MO) for 48 h, cells were then harvested and wa6hed with phosphate-buffered saline (PBS) and D~A was isolated by chloroform-phenol extraction. D~A r~r~ntrations were determined by W absorbance; 5 ~g of whole cell DNA was quantitatively dot blot hybridized in triplicate (Manifold I, Sr~l ~; r~r and Schuell, Keene N~) KS631Bam, EBV terminal repeat and beta-actin sequences were random-primer labeled with 3~P
[13]. Specific hybridization was quantitated on a Molecular Dynamics PhosphorImager 425E

6ell ~1l1 tures ~n~ Tr~n~ sion Studies: Cells_were r~;nt~;n~ at 5~105 cells per ml in RPMI 1640 with 20~ fetal calf serum (FCS, Gibco-BRL, Gaithersburg MD~ and periodically ~r~m;n~ for c~nt;nn~d KSHV
infection by PCR and dot hybr;~i7~t;~n The T cell line Molt-3 (a gift from Dr Jodi Black, Centers for Disease Control and Prevention), Raji cells (American Type Culture ~ rt; on, Rockville MD) and RCC-1 cells were cultured in RPMI 1640 with 107i 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 96ro6lsg 2 1 9 6 8 9 2 Pcrrus9sll0l94 To produce the RCC-1 cell line, 2xl06Raji cells were cultivated with 1.4x106BCBL-1 cells in the presence of 20 ng/ml TPA for 2 days in rh: ' ~ separated by Falcon 0.45 ~g filter tissue culture inserts to prevent contamination of Raji with BCBL-1.
Demonstration that RCC-1 was not cnn~Amin~ted with BCBL-l was obtained by PCR typing of HLA-DR alleles [27] (Raji and RCC-1: DR~1*0310, DR~3*02; BCBL-1:
DR~104,*07, Dr~4*01) and confirmed by flow cytometry to ~tPrm;n~ the presence (Raji, RCC1) or absence (BCBL-1) of EMA membrane antigen. Clonal sublines of RCC-l 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 ~Am;n~d to ensure that each was derived from a single cluster of growing cells.

In situ hybridization was performed with a previ~ously described 25 bp oligomer located in ORF26 which was 5' labeled with fluorescein (Operon, Alameda CA) and hybridized to cytospin preparations of BCBL-I , RCC-l and Raji cells using the methods of Lungu et al. [29]. Slides were both directly visualized by W microscopy and by incubating slides with anti-fluorescein-alkaline phosphatase (AP)-conjugated antibody (Boehringer-Mannheim, Tn~ AnA~olis IN), allowing ; nh; stochemical detection of bound probe. Positive control hybridization was performed using a 26 bp TET-labeled EBV DNA polymerase gene oligomer (Applied Biosystems, Alameda CA) which was visualized by W
miuLuscu~y only and negative control hybridization was performed using a 25 bp 5' fluorescein-labeled ~SVl ~47 gene oligomer (Operon, Alameda CA) which was visualized in a similar manner as the ~SHV ORF26 21 96~92 WO96~061~9 ~ PCT~59S/1019 probe. All ~uclei o~ BCBL-1, RCC-l and Raji d~u~iately stained with the EBV hybr;~iz~tinn probe whereas .no specific staining of the cells occurred after hybri~i7~tinr with the HSV1 probe.
5=
The rr--ining suspension celL lines used in transmission experiments were pelleted, and resuspended in 5 ml of 0.22 or 0.45 ~ filtered BCBL-l tissue culture snrPrnct~nt for 16 h BCBL-1 sUpernCt~ntS were either from unstimulated cultures or from cultures stimlllAtPd with 20 ng/ml TPA. No difference in tr~ncmicsion to rPcipient 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 RP~I 1640 with 10~ fetal calf serum.
~h~rPnt 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 sllrPrn~t~nt. After incubation with BCBL-1 media supPrn~t~nt, cells were washed three times with sterile B SS, and suspended in fresh media.
Cells were subsequently L~.' ChP~ 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 in~ection was performed using nested and unnested primers from ORF 26 and ORF 25 as previously described [10, 35].

Tn~rect I~-nnnfluoresce~ce 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).

~ WO96/06159 2 1 9 6 8 9 2 PCT~595110194 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-in~ected hemophiliac controls were drawn at various times after ~IV 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 ~l serum diluted 1:10 in phosphate-buffer saline (PBS), pH
7.4, were adsorbed with 1-3x107 paraformaldehyde-fixed P3H3 cells for 4-10 h at 25~ C and removed by low speed centrifugation. P3~3 were induced prior to fixation with 20 ng/ml TPA for 48 h , fixed with 1~ paraformaldehyde in PBS for 2 h at 4~ C, and washed three times in PBS prior to adsorption.

RESV~TS

Se~uence ~n~lvsis of a 20.7 kb KSHV DNA Seauence:
To demonstrate that KS330sam and KS631Bam are genomic fragments from a new and previously uncharacterized herpesvirus, a lambda phage clone ; (KS5) derived from an AIDS-KS genomic DNA library was identified by hybri~izatinn to the KS330Bam sequence. The KS5 insert was 5nhr] nnr~ after NotI/Bdm~I digestion into five subfrdyl s and both strands of each fragment were sequenced by primer walking or nested deletion with a 7-fold average WO96/06159 . ~ : PCT~595/l0l9 r~lm~An~y. 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 15 complete ORFs with codin~ 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 CodorPreference which identified 17 regions having ~ nt 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 (~VS) Codon preference values for all o~ 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), glycoprotei~ H (g~), thymidine kinase (TK), and the putative DNA p~k~ging protein (ORF29a/ORF29b) genes The translated sequence of each ORF was used to search GenBank/E~3L databases with BLAS~X and FastA
algorithms [2, 38]. All of the putative KS5 ORFs, except one, have sequence and col1in~n positional homology to ORFs from gamma-2 herpesviruses, especially ~VS and equine herpesvirus 2 (E~V2).
Because of-the high degree of r~l lin~nity and amino acid sequence similarity between KSHV and HVS, KS~V
ORFs have been ramed according to their HVS

~ WO96/06159 2 1 9 6 8 9 2 . ~ ol, positional homologs (i.e. KSHV ORF25 i5 ~amed after HVS ORF 25).

; 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 whic~ encode herpesvirus virion structural proteins and enz-ymes involved in DNA
metabolism and replication. Amino acid identities between KS5 ORFs and HVS ORFs range from 30~ to 60~, with the conserved MCP ORF25 and ORF29b genes having the highest percentage amino acid identity to homologs in other g hprpesviruses~ KSHV ORF28, which has no detectabIe sequence homology to HVS or EBV genes, has positional homology to HVS ORF28 and EBV BDBF3. ORF28 lies at the junction of two gene blocks (Figure 14); these jllnrti~n~ tend to exhibit greater sequence divergence than intrablock regions among herpesviral genomes [17]. Two ORFs were ldentified with sequence homology to the putative spliced protein packaging genes of HVS
(ORF29a/ORF29b) and herpes simplex virus type 1 (UL15). The KS330Bam sequence is located within KSHV ORF26, whose HSV-1 counterpart, VP23, is a minor:virion structural r ~ ~nt, 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 rr,nt~in~ a proline-rich domain at it~ amino ; terminus (nt 20343-19636; aa 1-236) that is not conserved in other herpesvirus ~K 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 WO96106159 ~ ~ PCT~S9~/1019 fou~d 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 Baircch~ 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 (CL~CHI;
aa 257-261), (ii) an immunoglo~ulin and ma~or histocompatibility complex protein signature in ORF25 (FICQAKH; aa 1024-1030), (iii) a mitochondrial energy transfer protein motif in ORF26 (PDDITRMRV; aa 260-268), and (iv) the purine nucleotide bir,ding site i~Pn~;fiP~ in ORF21. The purine binding motif i8 the only motif with obvious functional sign;f;~n~p-- 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 [1].

Phvloqenetic Analvsis 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 ~ ts for ORFs 21, 22, 23, 24, 25, 26, 29a, 29b, 31 and 34 were suitable for phylogenetic analyses.

To demonstrate the phylogenetic rPl~ n~h;p of KSHV to other herpesviruses, a single-gene comparison was made fcr 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 ~ W096/06l59 21 9 6 8 92 r~

level of gapping, however, the overall similarity between viruses i8 relatively low t33]. The MCP set gave stable trees with high bootstrap scores and assigned the KSHV homolog to the gamma-2 sublineage (genus Rhadinovirus ), nnnt~;ning XVS, EHV2 and BVH4 [20, 33, 43]. KSHV was most closely associated with HVS. Similar results were obtained for single-gene alignments oi 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 gammaherpe6virus gene set (CS1) cnnt~;ning EBV, EHV2, HVS and KSHV amino acid sequences. The total length of CSl was 4247 residues after removal of positions cnnt~in;ng gaps introduced by the alignment process in one or ~ore of the sequences. The CS1 alignment was analyzed by the ML method, giving the tree shown in Figure 15B and by the MP and N~ methods used with the aligned herpesvirus MCP se~uences. 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 previou81y estimated that divergence of the HVS and EHV2 lineages may have been contemporary with divergence of the primate and ungulate host lineages [331. The results for the CS1 set suggest that HVS and ~SHV represent a iineage of primate herpesviruses and, based on the distance between KSXV and HVS relative to the position of EHV2, divergence between HVS and KSHV lines is ancient.

Genomic Stn~;es sf KSHV:
CXEF electrophore8is performed on BCBL-1 cells embedded in agaro8e plugs demonstrated the presence of a nonintegrated KSXV genome as well as a high molecular weight species (Figures 16A-16B). KS631Bam (Figure 16A) 2l 96892 WO96/06159 ' ' PCT~S95/l0l9~
.......

and ~S330Bam 6peclfically hybridized to a single CXEF
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 [24]. 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 [49]. To determine if TPA induces repl;rat;nn of KS~V and EBV in BCBD-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 inc ~ ase in KSHV genome abundance occurred a~ter 20-80 ng/ml TPA incubation for 48 h.

Tr~n~ 8ion Studies:
Prior to determining that the agent was likely to be a member of ~erpesviridae by sequence analysis, BCBD-1 cells were cultured with Raji cells, a nonlytic EBV
transformed B cell line, in cham~ers separated by a 0.45 ~ tissue culture filter. Recipient Raji cells generally demonstrated rapid cytolysis suggesting transmission of a cytotoxic r , n~rnt from the BCBD-1 cell line. One Ra~i line cultured in 10 ng/ml TPA for 2 days, underwent an initial period of cytolysis before reco~ery and resumption of logarithmic growth. This cell line (RCC-1) is a monoculture derived from Raji nnrnntAm;n~ted byBCB~-1 as determined by PCR amplification of ~LA-DR
sequences.

- ' 2196892 RCC-1 has ~ ;n~d positive for the KS330233 PCR product for ~6 months in rn~t;nnmlc culture (approximately 70 passages), but KSXV 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 demongtrate persistent lorAl;7At;nn of KSXV D~A to RCC-1 nuclei. As indicated in Figures 18A-18C, nuclei o~ BCBL-1 and RCC-1 (from passage ~65) cells had detectable hybridization with the ORF26 oligomer, whereas no speci~ic hybridization occurred with parental Raji cells ~Figure 18B). KSXV serluences were detectable in 65S of BCBB-1 and 2.6~ of RCC-1 cells under these rnn~;t;nnc In addition, forty-five monoclonal cultures were subcultured by serial dilution from RCC-1 at passage 50, of which eight (18~) clones were PCR positive by KS330,33. While PCR detection using unnested KS330233 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 KSXV genome is lost over time in RCC-1 and its clones.

Low but persistent levels of KS330i33 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 ~f eight human fetal cord blood lymphocyte (FCBL) cultures after inoculation with 0.2-0.45 filtered BCBL-1 SllpPrnAtAntC~ Among the PCR positive cultures, PCR detectable genome was lost after 2-6 weeks and muItiple washings. Five FCB~ cultures developed cell clusters characteristic of EBV immortalized ; lymphocytes and were positive for EBV by PCR using EBER
primers [23); three of these cultures were also ~ 35 initially KS330i33 positive. None of the recipient cell lines had detectable KSHV genome by dot blot hybridization.

;' i(~'. ~2196892 W096~6159 PCT~S9511019 Se~oloqlc Studies:
Indirect ; ~flnnregcence antibody assays (IFA~ were used to assess the presence of specific antibodies against the KSHV- and EBV-infected cell line B~L-6 in the sera from AIDS-RS patients and control patients with ~IV infection or AIDS. BBL-6 was substituted for BCBL-l for reasons of convenience; preliminary studies showed no significant differences in IFA re8ults between B~L-6 and BCBL-1. BHL-6 have diffuse immunofluorescent cell staining with most RS patient and control unabsorbed sera sugyesting nonspecific antibody binding (Figures l9A-19D). After ad80rption with paraformaldehyde-fixed, TPA=induced P3~3 ~an EBV producer subline of P3J-~X1, a gift of Dr. George Miller) to remove cross-reacting antibodies a~ainst 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 l9B and l9D~. Staining was localized primarily to the nucleus but weak cytoplasmic staining was also pre~ent at low sera dil~tions.

With unadsorbed sera, the initial endpoint geometric mean titers (GMT) against BHL-6 cell antigens for the = sera from AIDS-RS patients (GMT=1:1153, range: 1:150 to 1:12,150) were higher than for sera from control, non-RS
patients (GMT=1:342; range 1:50 to 1:12,150; p=0.04) (Figure 13). While AIDS-RS patients and EIV-infected gay/bisexual and intravenous drug user control patients had similar endpoint titers to B~L-6 antigens (GMT=1:1265 and GMT=1:157B, 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 P3X3.
The difference in endpo~nt GMT between case and control titers against B~L-6 antigens increased after adgorption with P3~3. After adsorption, case GMT ~l;n~ to 1:780 and control GMT ~1;n~ to 1:81 ~p=O.OOOO9). Similar _ . _ _ _ _ ; ~ " 2 1 9 68 92 ~ WO96/061S9 P~ ,J/~ i~t .

results were obtained by using BCBL-1 instead of BHL-6 cells, by prç-adsorbing with EBY-ini-ected nonproducer Raji cells instead of P3H3 and by using sera from a homosexual male KS patient without HIV infection, in complete remission for RS for S months (BHL-6 titer 1:450, P3H3 titer 1:150). Paired sera taken 8-14 months prior to RS onset and after KS onset were available for three KS patients: KS patie~ts.8 and 13 had eight-fold ri6es and patient 8 had a three-fold fall in P3H3-adsorbed BCBL-1 titers from pre-onset sera to post-KS
sera.

DISC~SSION
These studies demonstrate that specific~DNA sequences found in KS lesions by repr~c~nt~ti~nAl difference analysis belong to a newly i~rntirlPd human herpesvirus.
The current studies define this agent as a human gamma-2 herpesvirus that can be cnnt;mlnnyly 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 la~ger herpesvirus genome. KS5 has a 54.0~
G+C =content which is considerably higher than the corresponding HVS region (34.3~ G+C). While there is no CpG dinucleotide suppression in the KS5 sequence, the c~lL~ ;nJ HVS region has a 0.33 expected:observed CpG ainucleotide ratio [1]. 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 L21]. CpG
J 35 suppression among herpesviruses [21, 30, 44] has been hypot~rri7ed to reflect co-replication of latent genome in actively dividing host cells, but it is unknown whether or not KSHV is primarily ~1ntA;nr~ by a lytic r~pl;rAt;~n cycle in vivo.

.... _ _ . ... .... _ _ . , . . . _ _ _ _ _ _ _ _ _ W096~61sg ~ ' - ' ' ?~96892 PCT~S9511019 ~

The 20,705 bp RS5 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 cnll;n~r positional homology to 15 previously identified herpesviral genes ;n~ ing the highly conserved spliced gene. The conserved positional and sequence homology for RSHV 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 [41]. 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 RSHV belongs to the gamma-2 sublineage of the Gammaherpesvirinae subfamily, and is thus the first human gamma-2 herpesvirus id~nt;~ Its closest known relative based on available sequence comparisons is HVS, a squirrel monkey gamma-2 herpesvirus that causes fnlmin~nt polyclonal T cell lymphoprnl;~r~t;ve 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 piaced on the phylogenetic tree with precision (the sublineage also contains murine herpesvirus 68 and BHV4 [33~). Given the limitation in r~snl-~tinn imposed by this thin background, RSHV and ~VS appear to represent a lineage of primate gamma-2 viruses. Previonsly, 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

~ WO96/06159 2 ~ 9 6 8 9 2 T~J/~ ~Jioig, and HVS suggests that these viruses diverged at an ancient time, possibly n~nt~ ~nr~n~mlqly 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 [33]. The biologic behavior of KSHV is consistent with its phylogenetic designation in that RSHV can be found ir. in vitro lymphocyte cultures and in in vivo samples of lymphocytes [3].

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 BCBB-l 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 [Z4]. 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 p~qsagPd through prolonged ti3sue culture [23].
The EBV strain infecting Raji, for example, is an BABF-2 flP~iniPnt :mutant [19]; virus replication is not ;n~n~;h;lP with TPA and its genome is --;nt~;nPfl only as a latent circular form [23, 33]. The EBV strain coinfecting BCB~-l does not appear to be replication deficient bec~use TPA induces eight-fold increa6es in DNA content and has an apparent linear form on CHEF
electrophoresis. RSHV replication, however, is only v 35 marginally induced by comparable TPA treatment ; n~; r~t; ns either ingensitivity to TPA ; n~n~t; nn 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 , _ .. . . _ . . _ . . .. ... . . . _ _ _ .

WO961061~9 ~ ; ~ 2 1 9 6 8 9 2 PCT~59511019 ~

speed centrifugation of filtered organelle-free, DNase I-protected BCBL-l cell extracts, which is consistent with KSHV encapsidation.

Transmission of KSHV DNA from BCBL-l to a variety of recipient cell lines i8 po~ssible and RSHV DNA can be ~-int~;nPd at low levels in~recipient cells for up to 70 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 lo~li7At;rn of the ORF26 sequence in RCC-l. rf transmission of infectious virus from BCBL-l occurs, it is apparent that the viral genome 15 ~rl ;n~c in Ahun~Anr~ 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 ~te-rtAhle KSHV genome when first explanted, but rapidly lose viral genome 20 after initial passages and established spindle cell cultures generally do not have detectable KSHV sequehces [3] .

Infections with the human herpesviruses are generally 25 ubiquitous in that nearly all humans are infected by early adulthood with six of the seven previously identified human herpesviruses [42] . Universal infection with EBV, for example, is the primary reason for the ~iffir111ty ln clearly establishing a causal role 30 for this virus in EBV-associated human tumors. The serologic studies identified nuclear antigen in BCBL-l and BHL-6 which ls recognized by sera from AIDS-KS
patients but generally not by sera from control AIDS
patients without KS after removal of EBV-reactive 35 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 2~ 96892 epidemiologically similar to ~SV2 rather than the other known human herpesviruses. An alternative possibility i8 that elevated IFA titers against BCB~-l reflect disease status rather than in~ection with the virus.

-- WO96/06159 2 1 9 6 8 9 2 F~~ S ~

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44. Schwartz, R. M., and M. O. Dayhoff. 1978.
Matrices for detecting distant relationships, p. 353-8. Tn M. O. Dayhoff (ed.), Atlas of protein sequence and structure, vol. 5, supple 3. National Biomedical Research Foundation, ~ashington.
45. Su, I.-J., Y.-S. Hsu, Y.-C. Chang, and I.-N.
~ang. 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. R., 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, H., F. J. O'Neill, and U. K.
Freese. 1978. Persisting oncogenlc herpesvirus induced by the tumor promoter TPA. Nature. 272:373-375.

~ WO96~6159 2 t 9 6 ~ 9 2 PCT~595110194 147 ~ ' ' ,! -a DE~AILS 6ECTION III:

RS Patient Enrollment: Çases 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 t4 months)~and at least four~ months prior to KS
diagnosl-s (median -13 months), while the ll ~in;ng 7 had paired PBMC taken at the study visit ; ~;Ately prior to RS diagnosis (median -3 months) and at entry into their cohort study (median -51 months prior to KS
diagnosis).

~emo~hilic and r,~70mo8exual/Bi8exual Male AIDS Patient Control En~gllment: Two control groups of AIDS patients were ~Am~n~: 23 homosexual/bisexual men with A7.'DS
followed until death who did not develop KS (~high risk~
control group) from the Multicenter AIDS Cohort Study [16]), and 19 h Ih;l;c men ("low risk" control group) enrolled from joint projectg of the National 7T ~h;l;A
Foundation and the Centers for Disease Control and Pr~v~nt;~n Of the 16 hemophilic controls with available follow-up information, none are known to have developed RS and c2~ of hemophilic AID5 patients historically develop KS [2]. 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 ~;Agn~
(median Br~-6 months prior.to AIDS onset).

DNA 7l~trA~t;~n and Anal~ses: DNA from 106-107 PBMC in each specimen was extracted and quantitated by spectrophotometry. Samples were prepared i7~ physically isolated laboratories from the laboratory where polymerase chain reaction (PCR) analyses were performed.

WO96/06159 PCT~S95/1019~ ~

All samples were tested for amplifiability using primers specific for either the H~A-DQ locus tGH26/GH27) or ~-globin [18]. PCR detection of KSHV DNA was performed as previously described [7] with the following nested primer sets: No. 1 outer 5'-AGCACTCGCAGGGCAGTACG-3', 5'-GA~cllcG~l~ATGAACTGG-3'; No. 1 inner 5'-IC~~ ~lo~ACGTCCAG-3',5'-AGCCGA~AGGATTCCACCAT-3' 7 No.
2 outer ~'-AGGCAACGTCAGATGTGAC-3', 5'-GAAATTACCCACGAGATCGC-~'; No. 2 inner 5~-CATG~ T~c~TTGTCAGGACCTC-3~, 5'-GGAATTATCTCGCAGGTTGCC-3'; No. 3 outer 5'-GGCGACATTCATCAACCTCAGGG-3', 5'-ATATCATCCTGTGCGTTCACGAC-3'; No. 3 iDner 5'-CATGGGAGTACATTGTCAGGACCTC-3', 5'-GGAATTATCTCGCAGGTTGCC-3'. The outer primer set was amplified for 35 cycles at 94~ C for 30 seconds, 60~ C for 1 minute and 72~ C for 1 minute with a 5 minute flnal extension cycle at 72~ C.
One to three ml of the PCR product was added to the inner PCR reaction mixture and amplified for 25 additional cycles with a 5 minute final extension cycle.
Primary determination of sample positivity was made with primer 3et No. 1 and rnnf; 1 with either primer sets 2 or-3 which amplify nonoverlapping regions of the KSHV
hypothetical major capsid gene. Sampling two portions of the KS~V genome decreased the l; kPl; hnnd of intraexperimental PCR cnnt~minAtion. These nested primer sets are 2-3 logs more sensitive for detecting KSXV se~uences than the previously published KS330233 primers [6] and are estimated to be able to detect ~10 copies of KSHV genome under optimal rnn~;t;nnc Sample preparations were preali~uoted and amplified with alternating neyative control samples without DNA to monitor and control possible rnnt~m;n~tion. 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-s~uared estimates and exact confidence intervals using Epi-Info ver. 6 (USD Inc., Stone Mt. GA).

21 ~872 ~ W O 96/06159 PC~rrUS95/10194 ~ . .
~ES~TS

Ksav Positivitv of Case and Control PBMC Sam~les:
Paired PBMC samples were available from each KS patient and h( 5A~l/h;q~n~l control patient; a Eingle sample waE available from each hl ~hil;r control patient.
.

To ~t~rm;n~ the RSaV positivity rate for each group of AIDS patients, a Eingle specimen~from each participant taken closest to KS or other AIDS-defining illnesE
(~second sample~) was analyzed. Overall, 12 of 21 ~57~) of PBMC specimens from KS patients taken from 6 months prior~to ~S diagnosis to 20 months after RS diagnosis were KSEV positive. There was no apparent difference in positivity rate between immediate pre-diagnosis and post-diagnoEis visit Ep~ri- q (4 of 7 (57~) vs. 8 of 14 (57~) respectively).

The number o~ KSHV positive control PBMC specimens from both homoEexual/biEexual (second visit) and h~ L h;l;c patient controlE waE Eignificantly lower. Only 2 of 19 (11%) h ~h;l;c PBMC gamples were positive (odds ratio 11.3, g5 ~ confidence interval 1.8 to 118) and only 2 of 23 (9~) PBMC samples from homosexual/h;q~n~l men who did not develop KS were positive (odds ratio 14.0, 95~
confidence interval 2.3 to 144) . Ii' all KS patient PBMC
samples taken immediately prior to or after diagnosis were truly infected, the PCR assay was at leaEt 57~
senEitive in ~t ~ct i ng KS~V infection among PBMC
samples. No Eignifir~nt differenceE in CD4+ counts were iound 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 Eingle sample from h- ,h;l;c AIDS patients were higher than CD4+ counts from KS patients (Kruskall-Wallis p=0.004), although both groups showed evidence o~ EIV-related immunosuppression.

2 l j~,6,,~ ,?~
W096l06l59 PCT~S95110194 ~onqitudinal 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 grPup, initial PBMC
samples were taken four to 87 months (median 13 months) prior to the onset Pf RS. Initial PBMC samples from the control group were drawn .13 to 106 months (median 55 months) prior to onset of ~irst npnKS AIDS-defining illness (1987 CDC surveill=ance definition). 11 of 21 (52~) of KS patients had detectable KSHV DNA in PBMC
samples taken prior to KS onset compared to 2 of 19 (11~, p=0.005) hemophilic control samples, and 1 (4~, p=0.0004) and 2 (9~, p=0.002) of 23 homosexual/bisexual control samples taken at the first an-d 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 posltive and two KS patients and one control patient reverted from positive to negative between visits. The " ;n;n3 7 KS patients and 20 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 ~S diagnosis, the median length of time between the first sample and the KS ~; agn~; a 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 cowQrkers have found evidence that KSHV
preferentially infects CD19+ B cells by PBMC subset examination of ~ three Ipatients [19]. Other gammaherpesviruses, such as ~pstein-Barr virus (~BV) and herpesvirus saimiri are also lymphotrophic herpesviruses ~ W096/06159 ' 2196a~2 PCT~S95110194 and can cause lymphoprrlifrr~tive disorders in primates [11, 20].
~ .
It is possible that RSHV, like most human herpesviruses, ~ 5 is a ubiquitous infection of adults [21]. EBV, ~or example, is detectable by PCR in CD19+ B lymphocytes from virtually all seropositive persons [22] and approximatè~y 98~ MACS study participants had EBV VCA
~ntihr~ at entry into the cohort study [23]. The ~ f;n~;ngs, however, are most consistent with control patients having lower RSHV infection rates than cases and that RSHV is specifically associated with the subsequent development of XS. While it is possible that control patients are infected but have an undetectably low RSUV virai 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 10% of these patients were RSHV infected and did not develop KS. It is unknown whether or not this is similar to the RS~V infection rate for the general human population.

This study demonstrates that RSHV infection is both strongly associated with RS and precedes onset of disease in the majority of patients. 57% of RS patients had detectable RSHV infection at their second follow-up visit (52% prior to the onset of RS] 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 RS cases and controls, RSHV
DNA positivity rates were s;rn;f;r~ntly higher for cases at both the first (p=0.005) and second sample visits indicating that ; _u~lession alone was not responsible for_these elevated detection rates. It is also unlikely that KSHV simply colonizes existing RS
lesions in AIDS patients since neither patient group had RS at the time the initial sample was obtained. Five RS
patients and two homosexual/bisexual control patients WO96/06159 2 1 q 6 8 9 2 PCTNS95/10194 ~

converted from a negative to a positive, possibly due to new infection ac~uired during the study period.

The findings are in contrast to PCR detection of RSXV
DNA in all lO PBMC samples from KS patients by Ambroziak et al. [l9]. It is possible that the aasay was not sensitive enough to detect virus in all samples since it was rer~uired 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 RS patients. The PCR conditions test a DNA amount e~uivalent to approximately 2x103 lymphocytes; an average viral load less than l copy per 2x103 cells may be negative in the assay. Two KS
patients and a homosexual/biaexual control patient initially positive for RSXV PCR amplification reverted to negative in samples drawn after ~;~n~nnciC. These results probably reflect inability to detect KSHV DNA in peripheral blood rather than true loss of infection although more detailed studies of the natura~l history of infection are needed The study was designed to answer the fnn~ ~1 r~uestion o~ whether or not infection with KSHV precedes de~r~l~ of the KS phenotype. The findings indicate that there is a strong antecedent AC~nr;~inn between RSHV infection and RS. This temporal relationahip is an absolute re~uirement for est~hl;C~;nr, that RSXV is central to the causal pathway for developing RS. This study contributes additional evidence for a possible causal role for this virus in the development of KS.

-21 96P~92 t WO961061S9 ~ PCT~S95/1~194 153 ~ ~
~ ~.: ,. .
1. Katz MH, Hessol NA, B~ h;n~r 5P, Hirozawa A, O'Malley P, Holmberg SD. Temporal trends of opportunistic infections and malignancies in - S 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. Arc_ibald CP, Schechter MT, Le TN, Craib RJP, Mnnt~n~r JSG, O'Shaughnessy MV. Evidence for a _ sexually transmitted cofactor for AIDS-related Kaposi's sarcoma in a cohort of homosexual men.
Epidemiol . 1992j3:203-209 4. Beral V, Bull D, Jaffe H, Evans B, Gill N, Tillett X et al. Is risk of Kaposi's sarcoma in AIDS
patients in Britain increased if sexual partners came from ~nited States or Africa? BMJ.
1991;302:624-5.

25 5 Beral Y_ Epidemiology of Kaposi's sarcoma.
Cancer, ~IV and AIDS London: Imperial Cancer Research Eund; 1991:5-22 6 Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, ~nowles DM, et al. Identification of herpesvirus-like 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 XIV infection. New England J ~ed. 1995;332:1181-1185.

WO96106159 ~ ~ 9 ~ g 9 2 PCTNS95/l019 8. Boshoff C, Whitby D, Hatziionnou T, Fisher C, van der Walt J, Hatzakis A et al. Kaposi's sarcoma-associated herpesvirus in HIV-negative Kaposi's sarcoma. ~ancet. 1995j345:1043-44.
9. Su I-~T, Hsu Y-S, Chang Y-C, Wang I-n. ~erpesvirus-like DNA sequence in Eaposi's sarcoma from AIDS and non-AIDS patients in Taiwan. ~ance~. 1995j345:722-23.
10. Dupin N, Grandadam M, Calvez V, Gorin I, Aubin JT, Harvard S, et al. Herpe~svirus-like DNA in patients with Mediterranean Kaposi ' 5 sarcoma. Bancet.
1995j345:751-2.
15_ _ _ _ _ _ 11. Miller G. Oncogenicity of Epstein-Barr virus. T
Infect Dis. 1974jl30:187-205.

12. Hill AB. Environment and disease: association or 20causation? Proc Roy Soc Med. 1965jS8:295-300.

13. Susser M. Judgment and causal inference: criteria in epidemiologic studies. Am J Epid. 1977jlO5:1-15.

2514. Fishbein DB, Kaplan JE, Spira TJ, Miller B, Schonberger ~B, Pinsky PF, et al. TTn~yrl~;nod lymphadenopathy in homosexual men: a longitudinal study. JAMA. 1985j254:930-5.

3015. Holmberg SD. Possible cofactors for the development of AIDS-related n~pl~l . Cancer Detection and Prevention. 1990j14:331-336.

16. Raslow RA, Ostrow DG, ~etels R, Phair JP, Polk BF, 35Rinaldo CR. The Multicenter AIDS Cohort Study:
rationale, organization and selected characteristics of the participants. Am J
Epidemiol. 1987j126:310-318.

21 968~2 ~ W096/06l59 ~ PCT~S95ll0l9 17. Wolinsky S, Rinaldo C, Kwok S, Sinsky J, Gupta P, Imagawa D, et al. Human immunodeficiency virus type 1 (HIV-l) infection a median of 18 month6 before a diagnostic Western blot. Ann Internal Med.
lg89;111:961.

18. Bauer ~M, Ting Y, Greer ~E, Chambers JC, Tashiro CJ, Chimera J, et al. Genital papillomavirus infection in iemale universlty students a6 ~etermined by a PCR-based method. JAMA.
1991j265:2809-10.

19. Ambroziak JA, Blackbourn DJ, Herndier BG, Glogau RG, Gullett JH, McDonald AR, et al. Herpes-like se~uences in HIV-infected and uninfected Kaposl's 6arcoma patients. Science. l99S;268:582-583.

20. Roizman B. The family Herpesviridae. In: Roizman B, Whitley RJ, Lopez C, eds. The Human Herpeviruses.
New York: Raven Pre6s, Ltd.; 1993:1-9.

21. Roizman B. New ~viral footprints in Kaposi'6 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 Ep6tein-Barr virus latency in normal B cells in vivo. Cell.
1995;80:S93-601.

23. Rinaldo ~R, Kingsley LA, Lyter DW, Rabin BS, Atchison RW, Bodner AJ, et al. Association of HTLV-III with Epstein-Barr ~Lnus infection and abnormalitie6 of T lymphocytes in homosexual men.
~ J Infect Di6. 1986;154:556-61 WO96~6159 2 1 9 ~ 8 q ~ PCT~S95/10194 ~

~ ar ~ETAILS SECTION IV:

To determine if the KHV-KS virus iB also present in both endemic and HIV-associated B lesions from African patients, formalin-fixed, paraffin-embedded tissues from both HIV seropositive and XIV seropositive Uganda~n KS
patients were compared to cancer ti6sues from patients without KS in a blinded case-control study.

Pati~nt Enrollment: Archival KS biopsy specimens were selected from approximately equal numbers of HIV-associated and endemic HIV-negative B patients enrolled in an ongoing case-control study of cancer and HIV
infection at Makerere University, Kampala Uganaa.
Control tissues were consecutive archival biopsies from patients with various malignanci~s enrolled in the same study, chosen without prior knowledge of HIV serostatus.
All patients were tested for HIV antibody (measured by Cambridge i3ioscience Recombigen Elisa assay).
Tissue PreParation: Each sample examined was from an individual patient. Approximately ten tissue sections were cut (l0 micron) from each paraffin block using a cleaned knife blade for each specimen. Tissue sections were depar~ff;n;7o~ by extracting the sections twice with l ml xylene for 15 min. followed by two extractions with 100% ethanol for 15 mir. The , ining pellet was then resuspended and incubated overnight at 50~ C in 0.5 ml of lysis buffer (25 mM KCl, l0 mM Tris-HCl, pH 8.3, 1.4 mM MgCl2, 0.0l~ gelatin, l mg/ml proteinase K). DNA
was extracted with phenol/chloroform, ethanol precipitated and resuspended in l0 mM Tris-HC:l, 0.l mM
EDTA, pH 8.3.

~CR AmPlification: 0.2-0.4 ug of DNA was used in PCR
reactions with KS330l3, primers as previously described [7]. The samples which were negative were retested by nested PCR amplification, which is approximately l03-l03 fold more sensitive in detecting KS330l33 sequence than t WO96/06159 2 1 9 6 8 9 2 PCT~S9~10l94 ~, 1.
the previously published KS3302" primer set [7]. These samples were tested twice and samples showing discordant results were retested a third time. 51 of 74 samples initially PY~m;n~d were available for independent extraction and testing at Chester Beatty Laboratories, - ~ondon using identical nested P~R 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.

RES~LTS:
~f 66 ~issues ~Y~m;n~d, 24 were from AIDS-KS cases, 20 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 15 were HrV s~rrn~rJat;ve (Figure 22). Tumors examined in the control group included carcinomas of the breast, ovaries, rectum, stomach, and color" fibrosarcoma, lymphocytic lymphomas, Hodgkin's lymphomas, choriocarcinoma and anaplastic r~rr;n~ 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 (B9%) were positive for KS330233 PCR product, including KS tissues from 22 of 24 (92~) HIV seropositive and 17 of 20 (85%) HIV
seronegative patients. In comparison, 3 of 22 (14~) nonES cancer control tissues were positive, including 1 of 7 (14~) HIV seropositive and 2 of 15 (13%) HIV
seronegative control patients (Figure 19). These control patients included a 73 year old HIV seronegative male and a 29 year old HIU seronegative female with breast carcinomas, and a 36 year old HIV seropositive female with ovarian carcinoma. The odds ratios for WO961061~9 2 1 9 ~ 8 q 2: PCT~S95/10194 ~

detecting the se~uençes in tissues from HIV seroposi~ive and XIV seronegative cases and controls was 66 ~95%
confidence interval (95% C.I.) 3.8-3161) and 36.8 (95%
C.I. 4.3-428) respectively. The overall weighted Mantel-Xaenzel odds ratio stratified by XIV serostatus was 49.2 (95% C.I. 9.l-335). KS tissues from f our XIV
seropositive children (ages 3, 5, 6,~and 7 years) and four XIV seronegative children (ages 3, 4, 4, and 12 years) were all positive for KS330l33.
All discordant results (i.e. KSXV negative KS or KSXV
positive nonKS cancers) were reviewed microscopically.
All KS330~33 ~CR negative KS samples were c~nfi ~d to be KS. ~ikewise, all KS330233 PCR positive nonKS cancers were found not to have occult KS histopathologically.

DISC~SSION
These results indicate that KSXV DNA sequences are found not only in AIDS-KS [5], classical KS [6] and transplant KS [7] but also in African KS from both XIV seropositive and seronegative patients. Despite differences in clinical and epidemiological features, KSXV DNA
s3e~uences are preser,t in all major clinical subtypes of KS from widely dispersed geographic settings.
~his study was performed on banked, formalin-fixed tissues which prevented the use of specific detectio~
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 5 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 ;n~p~n~ntly 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 se~uenced and found to be more than 98$ identical to the published KS330233 ~ W096/06159 21 9 68 q2 r~ Jllal~

se~uence confirming their specific~nature and, because of minor sequence variation, making the possibility of ~nt~m;n~tion unlikely.

~ 5 In contrast to previous studies in North American and European populations, it was found 3 of 22 control tissues to have evidence of KS~V infection Since these cancers represent a variety of tissue types, it is unlikely that KSHV has an etiologic role in these tumors. One pnc.cihlP explanation for the findings is that these results reflect the rate of XS~V infection in the nonKS population in Uganda. Four In~PpPn~Pnt controlled studies from North America [5 and9 ] Europe [7] and Asia [8] have failed to detect evidence of KS~V
. infectlon in over 200 cancer control tissues, with the exception of an unusual AIDS-associated, body-cavity-based lymphoma [9]. Taken together, these studies indicate that DNA-based ~PtP~ n of KSHV infection is rare in ~most nonKS cancer tissues from developed countries. KS~V infection has been reported in post-transplant skin tumors, although well-controlled studies are needed to confirm that these findings are not due to PCR ~nt~m;n~tion [10]. Since the rate of ~TV-negative KS is much more frequent in Uganda than the United States, detection of KSXV in control tissues from cancer patients in the study may reflect a relatively high prevalence infection in the general Ugandan population.

While RS is extremely rare among children in developed countries [2], the rate of KS in Ugandan children has risen dramatically over the past 3 decades: age-standardized rates (per 100,000) for boys age 0-14 years were 0.25 in 196g-68 and 10.1 ln 1992-93. Detection of KS~V genome in KS lesions from prepubertal children suggests that the virus has a n~nqP~n~l mode of tr~nrm;cfiion 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 tr~n~;C~n W096/06159 21 96892 : PCT~sgs/lo!9~
., accounts for the more fulminant form of KS occurring in African children remains to be investigated.

i~L..'_~:S
1. Cettle A.C. Geographic and raclal differences in the frequency of Kaposi's sarcoma as evidence of enviL~ ~1 or genetic causes. Acta Un Int Cancer 1962j18:33~-363. ~ ~~

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

3. Wabinga H.R., Parkin D.M., Wabwire-Mangen F., Mugerwa J. Cancer in Kampala, Uganda, in 1989-91.
changes in ~ n~ n~ in the era oi 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.

5. Chang Y. et al. Identification of herpe6virus-like DNA sequences in AIDS-associated Kaposi's sarcoma.
Science 1994; 266:1865-9.

6. Moore P~S. and Chang Y. Detection of herpesvirus-like DNA se~uences in Kaposi's sarcoma lesions from persons with and without XIV infection. New England J Med 1995; 332:il81-85.

7. Boshof~ C et al. Kaposi's sarcoma-associated herpesvirus in HIV negative Kaposi's sarcoma (letter). Lancet 1995; 345:1043-44.
= = ~
8. Su, I.-J., ~su, Y.-S. r Chang, Y.-C., Wang, I.-W.
Herpevirus-like DNA sequence in Kaposi's sarcoma from AIDS and non-AIDS patients in Taiwan. Lancet 1995;345 722-3.

t WO96/06159 2 1 ~ 6 8 9 2 PCTNS95/10194 9. Cesarman E., Chang Y., Moore P.S., Said J.W., Knowles D.M. Kaposi's sarcoma-associated herpesvirus-like DNA sequences are present in AIDS-related body cavity based lymphomas. New England J
~ 5 Med 199~; 332:1186-ll91.

10. ~ady P.L., et al. Xerpesvirus-like DNA sequences in nonRaposi's sarcoma skin lesions of transplant patients. Lancet 1995;348:1339-~Q.
L DETAILS SECTION V:

Serglo~ic rirkPr for KSHV infection.

~E~XODS

Patients Serum was collected from a convenience sample of 89 XIV-infected patients seen at several clinical sites in Connecticut, New York, and California.
Demographic and clinical information was recorded on st~n~Ars; 7eS 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 ~;~gn~q;q of RS
2~ was unequivocal on clinical grounds. Eighty six (97~) were male; 90 of the 86 men (93~) were homosexual or bisexual. Forty seven patients, all male, had KS. The characteristics of the study population are found in Figure 23].
Cell ~; nPc - The BCBL-1 line was estAhl;qh~ from an AIDS-associated body cavity B cell non-Hodgkin's lymphoma [30]. 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 rea.~Gn~ ~q that are rhArActPristic of B cells [31]. RSXV DNA sequences can be detected in BCBL-1 cells by DNA representational difference analysis [23,32~. BCBL-1 cells also contain WO96/06159 2 1 9 6 8 9 2 P~ ,5l,Di9~ ~

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]. ~ 514-16 is an EBV rnntA;ning cell line, originally from aBurkitt 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 lF40 medium containing 8% fetal calf serum.

T ~h~o~tinq Assavs Extracts of ~l~nin~llrP~ BCB~-1 cells or BCBL-1 cells that had been treated with 20ngfml 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 105 cells in SDS sample bufferi electrophoresis, transfer to nitrocellulose and blocking with skim milk followed standard protocols [38]. Sera were screened at 1~100 dilution. The reaction was developed by 1.0 ~Ci of lZ5I Staphylococcal protein A.
Radloautographs were exposed to film for 24-48 hrs.
Immunoblotting assays were performed and interpreted on coded sera.

T - n~luoresCent assaY The antigens were BC=BL-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 ; nhl otting assays.

~ W096106159 2 !;9 6 ~ 92 ~ J~IVI9 RESULTS

~h~m;cal In~nction of lvtic cvcle KSHV ~roteins in BCBL
cell8: Initial experiments using the immunoblotting technique were designed to determine whether BCBL-1 cells expre6sed unique antigenic polypeptides that might be specific ~or KSEV 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 es6ential to distinguish EBV polypeptides from those encoded or induced by KSHV. Figures 27A-27B, an ; nhlot prepared from BCBL-1 cells reacted with a reference EBV antiserum, show6 that BCBL-1 cells expressed two polypeptides, representing the latent nuclear antigen EBNA1 and p21, a late antigen complex [39], that were present in other EBV producer cell lines, such as B95-8 (Figure 27A) and HX514-16 (Figure 2~B 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 pQlypeptides that were not also seen in the EBV producer cell lines. However, if extracts were ~Lep~Led 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 ~L~ 'n~nt o~ these many polypeptides were estimated at about 27 ~Da, 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 5 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 WO96/06159 2 1 9 6 8 9 2 PCT~S95/lOI9 ~
;

164 ~
and appeared after treatment with chemicals they likely represented lytic cycle rather than latent cycle polypeptides of ~SHV.

p40 ~n~ ~60 are ~5HV s~ecific: Figures 27A-27B shows that antigenic polypeptides corresponding in molecular weight to p40 were not observed in two EBV producer lines, B95-8 and HX514-16, 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-l cells~but had little or no effect or 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 RSHV lytic cycle mRNA. TPA, by contrast, induced the EBV lytic cycle efficiently' treatment with TPA caused an increa6e in the ~hlln~n~ of the EBV p21 protein and minimal ;n~netinn of KSHV p40. These findings suggested that latency to lytic cycle switch of the two gamma herpes viru6es carried by BCBL-1 cells was under separate control and that the p40 complex was specific to the KSHV genome.

~40 as a sexoloqic marker for KSHV: Whlle a few highly reactive sera, such as XS 01-03, (Figure 27B) recognized multiple antigenic proteins unique to the chemically induced BCBL-l cells, ;n~ln~;ns p27, p60 and p40, sera from other patients with KS did not react with p27 or p60 but still recognized p40 (Figure 28A and 28B).
Therefore re ogrition of p40 was investigated as a serologic marker for infection with KSHV. Sera from 89 ~IV-l infected patients from Connecticut, New York and California were examined for presence of ~n~;hn~;~c to p40; only 3 of 42 patients (7~) 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 s~rnlogi~ marker for the presence of KS were 84~ and ~ WO96/06159 2 ~ q 6 8 q 2 PCT~S95~10l94 78~ respectively. Three HIV-1 infected men from New York with non-Hodgkin's lymphoma but without KS were s non-reactive to the KSHV p40 antigen. Figure 25 compares the patients with KS whose serum did or did not ~ 5 contain antibodies to KS~V p40. ~either CD4 cell number nor the extent of KS disease preaicted the presence or absence of a serologic response to p40.

,T ~luorescence assavs: Immunoblots showed that n-butyrate induced ex~ression of KS~ 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 Z7B, that contains antibodies to EBV but not KSHV there were about 2% antigen positive untreated BCBL-l c~lls and a similar number of antigen positive BCBL cell that had been treated with n-butyrate. Serum 01-03 that is EBV-positive and XS-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 n-butyrate treated population 6erved as the basis of an ; ~1uorescence screening assay for antibodies to KSHV lytic cycle antigens ~Figures 29A-29F). The results of the immunofluorescence :assay were nearly identical to the i -~lotting assay (Figure 26).
Among 89 sera there were only 4 (3~) 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-l infected patients without KS were reactive by indirect ~ ~fluorescence assay (IFA). Thus using two differe~t assays, antibodies to KSHV lytic cycle 2 1 96~92 WO96/06159 P~ Cl'74 antigens were found 6 to 9 times more frequently among patient6 with KS than among EIV-l infected patients without RS. Stated another way, among individuals who were seropositive to RSEV p40 32/35 (9l~) had RS. Among those seropositive by the immunofluorescence assay 32/37 (86~) had KS. Thus infection with RSXV, as defined by these serologic markers, sarries a high risk of development of KS.

DISC~SSION

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 RS.
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 XIV-l, other forms of immunosuppression, geographic factors, sex differences, the role of cytokines and growth factors, and the O~ULL~C~ Of distinct clinical variants must all be eventually understood. By identifying the infection rate in different populations a serologic marker for infection with RSHV would be great aid in unravelling the significance of the new virus in this complicated puzzle.

One possibility is that KSEV, 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 l;k~7;h~od of devel4ping disease, whereas immunocompetent individuals would remain healthy. This pathogenetic model is similar to that postulated for the role that EBV plays in non-Xodgkin's lymphoma or cytomegalovirus in retinitis in patients with AIDS. If this model is correct a very high proportion of- the adult human 2~ 96892 Wo96106159 ~ PCT~S95/~0194 .
population might be found to be seropositive for KSXV
The model of a ubiquitous virus selectively causing disease in ; ndpficient individuals does not account for classical KS affecting patients who are not e r; ; ~~ ~ Aed nor does it account for the observations that endemic KS in Africa preceded the XIV-l epidemic. Since many African patients with KS are HIV-l negative other co-factors must be implicated.

The other pncc;hility is that KSXV 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 s~Lu~r~v~lence of KSHV would be expected to be higher in 15 HIV-l se~opositive and XIV-l seronegatIve homosexual men than in other populations. If the virus alone were capable of inducing disease, acquisition of KSXV
infection, as monitored by the presence of antibody, would be associated with a high rate of nl;n;~lly 20 evident KS. Xowever, if KSXV infection needed to a~ ~ ied by other co-factors to cause disease, the prevalence of antibody of KSXV might be similar among patients with and without KS The other co-factors would not be id~nt;f;ed in a serologic test for 25 ~ntiho~;es to KSXV antigens.

The findings, using tests for antibodies to KSXV lytic cycle antigens, are consistent with the general model in which infection with KSXV is infrequent but associated with a hig~ rate of apparent disease. Only a few XIV-l infected patients without KS had ~ntihn~ies to the KSXV
lytic cycle antigens; by contrast a very high proportion of XIV-l infected men who had clinically evident KS were seropositive. This finding suggests that a high proportion of individuals who are dually infected with HIV-l and KSHV develop KS. Xowever, 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 W096~61~9 2 1 9 6 8 9 2 PCT~Sg~lo?gS~

KSEV might be ubiriuitous~ Ant;ho~;oq 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 de~cribed would indicate reactivated ;n~rt;~n 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 KSX~ ~eronegative Regardless of which of these two lnterpretations is correct, the serologic studies provide a strong correlation between the presence of Ant;hr~;rc to KS~V
lytic cycle gene products and clinical KS. ~onetheless there are two groups of patients whose serologic results reriuire further explanation. One group consi~ts of the few patients with positive serology for KS~V p40 without rl;n;rAl 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 rr,n~; 1 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 p40 may reflect the extent of Iytic viral replication which ~may vary during different phases of the disease. To ~t~rm;n~ whether this is true prospective studies including serial bleedings are reriuired.

p40 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 BCB~-l cell~ cannot be induced to express these antigens, or because the antigens are , 21968q2 W096/06159 PCT~S95/10!94 of low ~hlln~noP or denatured on the immunoblots. In some individuals serum antibodies to p40 may be consumed J in immune complexes with p40 antigen in the circulation.
Thus detection of p40 on immun~blots may not be of optimal~sensitivity. In this conrection three sera recognized antigens in immunofluorescence tests but did not react with p40 on western blots. The serologic test employing whole BCBL-l 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 ~41]. An association between the degree of immunosuppression, as monitored by the numoer of CD4 cells, and the presence or absence of antibody p40 among patients with KS was not found (Figure 25). Furthermore all the patient6 with or without ~ntiho~;es 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 ma~ority, 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 r 35 the pathogenesis of KS is a multifactorial process. It has been observed that the product of the HIY-tat gene stimulates growth of KS tissue culture cells [42] and can induce KS-like lesions in mice [43]. These findings suggest a direct role for HIV-1 in the pathogenesis of ~ _ _ _ _ _ _ , _ . .... . . ..

W0961061~9 ~ 1 9 6 3 9 2 PCT~S9511019~ ~

KS, at least in HIV-in.fected 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 [44]. Interleukin-6 is also produced in AIDS-KS
derived r~ 1lture [44]. Thus, KS pathogenesls may involve autocrine and paracrine growth factors together with infection with KSHV in some-patients or with certain. strains of HIV-l in other patients. If ;nr~t;~n with KSHV is the sine gua 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 l~ screening assay for detection of antibodies to lytic cycle antigens of KSHV i6 disclosed. These assays should permit detaile~ serQepidemiologic 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 _Qf. development of clinical KS.

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~ WO96/06159 2 1 ~ 6 8 q 2 PCT~S9~1~19~

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==~ = ~ . =
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21 96~92 WO96/06159 '' PCT~S95/10l9 27. Dupin N, Grandadam M, Calvez V, Gorin I, Aubin JT, Harvard S, et al. Herpesviurs-like DNA sequence sin pateints with Medit~rr~n~n Kaposi's sarcoma.
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2 1 96~ 92~
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~ W 096/06159 2 i 9 6 ~ 9 ~ L5I

SEQUENCE LISTING

~1) GENERAL INFORMATION:
. ~i) APPLICANT: The Trustees of Columbia University in the City of - New York City (ii) TITLE OF INVENTION: UNIQUE D.q.q~rT~TRT1 YAPOSI'S SARCOMA VIRUS
SEQUENCES AND TTCRC T~TRTiR~R
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

TCGAGTCGGA GAGTTr-r~r~r Dr~rrTTGAG ~L.4~L~L~ CGTTCTCACG ~L~LL44LL4 60 GGATCAGCTG GTGACTCAGA CAAGTCrIGA GCTCTASA~LC ~TD~r~TPrr GGCTGATGCC 120 rDrrrr.DTDr cAGAATTL~cG r~r-TCr-rr~ IL~L4L4~e TAGAGTCACC TrD~ T~ 180 AL~L4L44L4 Trrr~r-~ V44LL~L444 r.rrrr.rTDrT T~ ~rrr CATAGATCGG 240 , -- . : ' s . ~ . .

W096/06159 ' 2 ~ 96892 PCTAUS95/10194 ~

GCAGGGTGGA GTACTTGAGG prrrrrrrGT AGGTGGCCAG L-L~44CL~44 TTACCTGCTC 300 lLllQLL,~L TGCTGGAAGC CTGCTCAGGG ATTTCTTAAC L1~LLLL1~L 411~L-~L41A ~ 360 CCATGGCAGA AGGCGGTTTT C~'rrrr~rT ~~L'1~L-4~L rr~r~rr~r-pr~A APGGCCTCTG 420 rrDrrprrr:p TATGGACGAC CTCCCTGAGG rr~rr~rrrc PrT~rrr~r~r.P AAGTCTGTAA 540 AaAccTcGTA rPTpTprr~r GTGCCCACCG TCCCGACCAG CA~GCCGTGG CATTTAATGC ~600 ACGACAACTC CCTCTACGCA ACGCCTAGGT TTCCGCCCAG ACCTCTCATA CGGCACCCTT ..~660 rrr~rprDrG CAGCATTTTT GCCAGTCGGT TGTCAGCGAC TGACGACGAC TCGGGAGACT 720 A rr.rrrrP r~T GGATCGCTTC GCCTTCCAGA GCCCCAGGGT ~l~lvLl~4L ~ Lll- 780 CGCCTCCA~A TCACCCACCT rrrGrp~rTp GGrrGGrpnr- CGCGTCAaTG rrrr~rrTrr 840 L~1~L-WL'~ TCTGCAGGGA CTCAAGAGGA rrrrPp~rr~ ATTTTTA M A ACATCTACCA .- 900 ArGr~GGr-rPr TCTCA M GCC CGTGGACGCG ATGTAGGTGA CCGTCTCAGG r~rr.r.rrrrT 960 TTGCCTTTAG TCCTAGGGGC GTGM ATCTG CCATAGGGCA AAliCATTALr~ 1~l V~ 1~4 1020 GGATCGGAGA ATCATCGGCG ACTGCTGTCC CCGTCACCAC GCAGCTTATG GTPrrr~r~TGr 1080 ACCTCATTAG p~rrrrTr~Tr ACCGTGGACT ACAGGAATGT TTATTTGCTT TACTTAGAGG 1140 GGGTAATGGG TGTG w CAAA TCAACGCTGG TCAACGCCGT GTGCGGGATC TTGCCCCAGG I200 AGAGAGTGAC AAGTTTTCCC r~rrrpTr~r~ TGTACTGGAC GAGGGCATTT ACAGATTGTT 1260 Prr~r~LAAT TTCCCACCTG ATGAAGTCTG rTA~~crrr AGACCCGCTG ACGTCTGCCA 1320 AaATATACTC ATGCCALAAC AAGTTTTCGC 1~LLL1~LL- rPrrAPrrrr ACCGCTATCC 1380 TGCGAATGAT GCAGCCCTGG AACGTTGGGG 41~W1~1~L r~rrr~r~rprT CACTGGTGCG 1440 TCTTTGATAG GCATCTCCTC Trrrrprrpr~ 1L-L1~11LL'L TCTCATGCAC CTGAAGCACG 1500 GCCGCCTATC TTTTGATCAC TTCTTTCAAT TACTTTCCAT CTTTAGAGCC prAr~rr 1560 ACGTGGTCGC CATTCTCACC CTCTCCAGCG CCGAGTCGTT 4L~L-~WL-1~ pr~r-GrrTrrG 1620 rPrr~pAr~r rr~rnrC~rr GTGGAGcAaA ACTACATCAG AGAATTGGCG TG w CTTATC 1680 ACGCCGTGTA LI~ll~A'l~Q ATCATGTTGC AGTACATCAC TGTGGAGCAG ATGGTACPAC 1740 TATGCGTACA Prrrprrr~T ATTCCGGAaA 1~1VL'11~LL CAGCGTGCGC rTr~rrArprp 1800 Pr~r~rr TTTGApAaAc CTTCACGAGC AGAGCATGCT ACCTATGATC ACCGGTGTAC 1860 TGGATCCCGT GAGACATCAT LLL41~41~ TCGAGCTTTG L~LlL'-'lLL TTCACAGAGC 1520 TGAGA M ATT ACAATTTATC rTpr.rrr~~r. CGGATAaGTT rrPrr~rrrr GTATGCGGCC 1580 TGTGGACCGA AATCTACAGG CAGATCCTGT CCAATCCGGC TATTAAACCC Pr~r~r~rrpTrp 2040 ACTGGCCAGC ATTAGAGAGC.CAGTCTAaAG CAGTTAATCA rrTr~r~ ACATGCAGGG 2100 TCTAGCCTTC ~1~4L4~LLL TTGCATGCTG GCGATGCATA TCGTTGACAT r~Trr~prrrpr 2160 1~4LQL4l~ rrr~rr~rr,r. rr~rr~-r~T AACCCGCTCC GrrArr-rprr TCATCAATGG 2220 r~r~PrrP~r CTCTCCATAG AACTGGPATT rArrr~rrprT AGTTTTTTTC TAaATTGGcA 22bO

W O96/06159 _ PCTrUS95rlO194 ADATCTGTTG ~TTr~Tr~pTr~ rrrnr-rrrr CCTGACAGAG TTGTGGACCT CCGCCGAAGT 2340 rrrrrnr-nr CTCAGGGTAA CTCTGAA~AA r'--~rDDnrT ~lllllll - L rrPDrnn - n- 2400 AGTTGTGATC TCTGGAGACG GCCATCGCTA TACGTGCGAG rTGrrrn-GT CGTCGCAAAC 2460 TTATAACATC Prr~n--CCT TTAACTATAG ~ GGGCACCTTG GCGGATTTGG 2520 rrn-PrrrrD GAGTATCGGG TGTTTTprrr DnTGAATGTC Al~4~l-~ AGTTTTCCAT 2640 ATCCATTGGC p~r~nrrDrT CCGGCGTAGC GCTCTATGGA ~l~ lG~ AAGATTTCGT 2700 GGTCGTCACG CTCCACAACD GGTCCP~AGA rrrT~nrrnr ACGGCGTCCC Al.ll.l~ll 2760 C~1~L~ GATTCACTGC CATCTCTGAA GGGCCATGCC ACCTATGATG AACTCACGTT 2820 rr7rrrrnnnr GCAAAATATG CGCTAGTGGC GATCCTGCCT lLUAGATTCTT Prrzrn-DrT 2880 CCTTACAGAG AATTPrPrTr ~ L.l GAACATGACG GAGTCGACGC CCCTCGAGTT 2940 rDrrrrr~rr ATCCAGACCA GGATCGTATC AATCGAGGCC PrrrrrrrrT GCGCDGCTCA 3000 prn-r,rrr,rr rr~r~n~PTPT ~L-LL~L~LL GTTTCAGATG TTGGTGGCAC ACTTTCTTGT 3060 l~W~ ATTGCCGAGC ACCGATTTGT GGAGGTGGAC l~l~l~l~ GGCAGTATGC 3120 GGAACTGTAT 'lLl~ GCATCTCGCG TrTrTGrpTr CCCACGTTCA CCACTGTCGG 3180 rT~Tnnrr~r ~CCPLCCCTTG ~ L~ rr,rr~rPrnn ATAGCTCGCG TrTrrrrrDr 3240 GAAGTTGGCC AGTTTGCCCC ~l.LL.~A c-nnnrPr-Tr~ rTrrrrPTrr TCCAGCTTGG 3300 CGCCCGTGAT ~ W~L~ ~LL~LL~T TCTGGAGGGC ATTGCTATGG TCGT w AP~A 3360 TATGTATACC GCC~ACACTT PTrTrTDr~r ~rTrr-r~cr~pT ACTGAAAGAA AATTA~TGTT 3420 r~rprpTprpr ACGGTCCTCA crrprprrTr~ rrrr.rrr~nn GACTC w GAG TATCAGAAAA 3480 GCTACTGAGA ACATATTTGA TGTTCACATC AATGTGTACC pnrpT~-n-- Trrnrrn~nT 3s40 GATCGCCCGC TTTTCCaAAC rr~n~Pr-rrT TAACATCTAT AGGGCATTCT C~ L~LL 3600 TCTAGGACTA ~rr~T~rr~TT TGrDTrrprr r~-TTGrrr rrrrn--rrr CGCAGTCGTC 3660 CGCTCTGACG CGGACTGCCG TTr,rrDrn-r ppr~Trr-Grp TTrrrPrnnT TGCTCCA w C 3720 GCTGCACCTC r~PTprrTT~n ~TTTnTTTrr rrrrDTTpnr TGTTC_AAGA TT~rprrrr~p 3780 r~nr~TPpTp GCTACGGTAC ~ CGTCACGTAT ATCATCAGTT rrr~Drr~rT 3840 CTCGAACGCT GTTGTCTACG AGGTGTCGGA GATCTTCCTC Dnrn-TGrrp TGTTTATATC 3900 TGCTATCPi~ rrrrPTTr,rT rrrrrTTT~n rTTTTcTrpr PTTrPTPrrr ACATTCCCAT 3960 AGTCTACAAC ATCAGCACAC rD~-nnrn-- Il~ll TGTGACTCTG TPpTrpTrrr 4020 CTACGATGAG AG w ATGGCC TGCAGTCTCT C~TGTATGTC prT~nT-n~n GGGTGCPGAC 4080 C~ACCTCTTT TTPrPTP~r~T CACCTTTCTT TrPTPpT~nr ~nrrTPrpr~ TTCATTATTT 4140 GTGGCTGAGG rnrP~rrrr~ rrrTPrTrrp rpTr~rrrcc PTr7TPT~rnn rnrrrrrPrr 4200 CAGTGCTTTG TTTCTAATTC ~L~L~LLLLILT L~LL.L.~ ~LL~I~L iU TTTCTTTA 4260 CAGACTGTTT TCCATCCTTT PTTPrnrr,r,T rPnTpnnrrr TAGATTTTTA AAAGGTTTCC 4320 W096106159 ' 2 1 q ~ 9 ~ PCTr~595/1019 TGTGCATTCT TTTTGTATGG GCATATACTT Qrrr~r~rrT rrr~rDrrT rDr~rTGr 4380 ATTGCCGTCA CATATCAGTT rrDrrDrrrr TGCACCTAGC CATGCGGCGC TTTGACGGTC 4440 lllV~VV~l~ CACATCATAA AGTACTTTTC CAlVV~ TDrrrDrrT TGGAACAATC 4500 'lVVVV~ll~V CGAATGGGTT rrrTrr~rQr GADATCCTCT ATGGTATTCA QQrDQDDr-~ 4560 ~V~Vl~l~C ACCCGACGTT TGAGTCTTTC TDQrDnDQCQ rrrDDrDrrT ~ W ~I~l~ 4620 lVlll..v~A GGGGCAAGTT ~l~v~lA rpr~rQDTr~Q DDrrDrr~rD CGATGTTTTC 4680 CAGCCCCATG rTQrr,rDQrD ACACGTGCTT rDQQDDrDQQ TGTTGTAGCC GGTTCAGTTT 4740 TAGCTTGGGT AGAP~4AGTTA TCGAGTTGTT AGCACGCTCC ATGATGGTAA CGGTGTTGAA 4800 GTCACAGACC VV~ lV CGAGTCTCGG CCGCCTGAGT CCA~TCATGT rr~DrDTDGp 4860 W ~V~-'~ llVl~ TA~GTGACAC GATATCCCGT TCGCAPJ~CCT GTGCGATGTT 4920 GTGTTTCAGT ATAGATCTGG TCTGACCGGC ACGGGGTGTT ATGGGGTGAC QrGr~TD'~Q 4980 CGACTCTGGG TCAaACACCT TTATGCGGTT vb-Gv~l~ TCGATGACGA CACGCTTGTT 5040 ~~ - VV~VlVl ATGGGGACGC GACGGCATCC CGCTGGCAGA TCTATAATCT TAD~AGTTGGT 5100 ATAAGACTGG l v~l~vllA TGGCCAGCCG GCACTCCGGT AGTATCTGCG TGTCCTCGAA 5160 Il~vlv~v CGTACGACTG GCTTGGAGTG rPQr-TDDrrr CCAAGAGATa ~vvl-~ll~ 5220 QrrTDrrrDr ~AGTGGCTTC TTA~DCGCGTA VVVVlV~VVl r'~rraTr~ TCCGTAGCAA 5280 CGATAGTTCC vv~lv~l~G rrQrrTrr~r TGGCAGGGTA GACGAGTCCG GAGTCCCAPA 5340 QQrrQr-r~- GTCAP~CGT r~rnrDrr,rr ~l~V~lvl~ c~rpr~r,~rr~rQ C~v~vL~ 5460 TPrTPQDrTD GCCTTCACGT CCGGaACTCG T~DrpTDrrT Tr~-~rDnrr. ~-rr~-r,rp 5520 prr.TPrrrQr GGATCGGCTG V~vlvl.l~ CTCGTTGGAC G~VV~V11~ ~lVV~ A 5580 GTGCAGGCCT AGTTTGCGAA TGGCGTGACG GACAATTTGT GGCTTTAGAG rrr'rrDprrQ 5640 ATGACCCGTG QTGGrr~rD ACGADATGAA GTTTGCATTG rrQCCrP~rT CGTCTAGCCT 5700 GV1~11~11V TTTCGGGCAT AGATTTTCGG GATTAGGTTA CACTTTTTAT ATCCCAGTAC 5760 TGCGCACTCG IVlllV~lll TAGTGTGACT GATTATCTTC TTTGAGAAGT rrrrrDrrrr 5820 ~G~Vuv GCTCGCCTA~ TQrr~rrrDr GTCAAGCCTG Pr~rr~rr AGCATTCCAC -5880 CAGACACTCC AGGAACCTTT TGTGTAGCGT ~lvl~lllvv GAACGGTTTC TGTGCTCAAG 5940 TDrGrDr~DT ATTCTATTTT l~lll~Vl~ GATGCGCGCG l~lvvl~ TGAGAATGGG 6000 CGCCAGCTCG TGGCGAATCT GTTCCACAAG ~VV~lV~ V TACACTTTAG ADATCGTGGC 6060 lvl~vWv~ TTP~rrPrr ACACGTTTAG ~~I~LIV rTrr~rDrrp rPQpTr~rDD~ 6120 V~llVlVVl~ rrr~rTPrGT ~l~l~V~ CPTTCTCACC ATGTACTGGT TTTCCAGTCC ~6180 rDr~D~rrpT TTCAGCGTAC CCATTGCGAA GAGAaAGTGC AGCATGTCCC CACTGATGTT 6300 GATGTTTATT V~VVlV~ll GACACATGTT GTCGGAaAPA AACACGCTTA TGGTAP~AGA 6360 ~ W 096~61~9 2 1 9 ~8'9 2 - PCTNS9~10194 ' 5 AGGTTCCTTT Prrr~TPrT TTrRTpT~r r~TTrTT5 GTCAATCTGG GGATGTTTAA 6420 GGTGTTGAAT ATGGTGATCT Tr-Ppr-TTTTr rp~prTr-prr- l~LLlL~L~ GTTCCAGCAT 6540 GTCTGACACT GTAGAGCTGC rrPrPrTrrr ~j~L~4L~jIj(lij(r_,~l~ r~TTrr~rrp 6600 CGCCTGCAA~ LLL~LiL~'~ L~~ ~L~i~ j4L~LLL~ qrrrrrTDrr GGATTCTTGA 6660 AAGCGTCGCC rrrPrr~r~r ~ AAAAAGTTTG CGC~GGGGTG 6720 cAGTccGcTG-rprr-pr-Trnr rrATrrPrTr Trrr~rTrrr ATprprpTr~ r~ -TrTnTP 6780 GATGGCCGGT GTGCCCGGAT prprTDr~lTp GTAGGTACAA TCTGGGGTAC Tnprr~rrpr 6840 CCTGTATGGC LLl~j4L~44 ~4L~LL~j~4 Llj~iA~LLLLL DrrTrr~rPr r~rr rprr~r 6900 CTGGTTTAGA GCCAGCTGAA PrcrrPrrpr Al~4L.CG TTAACCTTGA ~'4L~L~j41~j 6960 CTTACTC GT TTCGACAGGT TrTTrDr~rpr GGTGGGCAGT CGCTCTACGT TrTr~Lr~rr-AT 7020 rrrPrr,rrrr ~rrr~r~rrP 4~L~1~4L~j rrDrrrrrPr GTGGCCATGA prrTr-rTr-pT 7080 GTTAPACTTT ~ PPTrTD 4 L~jL~4L~ TrrlnrATrrr GGTGGCATTA TTr~P~nrrD 7140 GAGATGCTTC AGGCTCTCCA GGAGTGCAPA ATAATTTTGA TAGATTGTGG rTTrT~r~rT 7200 ATGGGGCAAC Prrrrrpr~D ACGCATGAAA ACACTGTTCG p~rTrrr~r-p prTrrDr-r-TA 7260 CCTGCACACT PTrrT~rP ~ - A~ATATGGTG CACGTTAGTA rrr-rrrr~~ 7320 ATDrprrrpr CGTAGCTCCC TGAATTCGCA W ~LLLAl~A CAATCATCGG TAAGTTCCCA 7380 TGATCCCACC GCAGGTAGGT AGTTGTCGGT GTCTATCTGT rrnrrrrT~ ACACTCCACC 7440 ACCGTCAATT ATTAA_CCTT ~ ~GTCGACCC ACTTTTCCCA APAGAGTCCC 7~00 TTCTTGATGT pTp~rrrT GGAGGCGTTC rrrrprr~T AGTCTGCGTA l'~ v~ 7860 rrrrPp~r ~L~jW LL~4~j GCTGCATCAT CTTATCAAGA CCTTCTAAGG TCAGCTCTGC 7620 rTrrPrrTrr GAGTTGGTGG rrDrPrPrrD r'~TPTTTCC DrrTrTrPTT rrr~rTrrr _ 76B0 TTGATAP~CAC /-~ i[(i GACTCGTCGT rPrrr~-rr ~ i(;( 'L r7TAr-TprGrn 7740 GrrrTrr~rr rrTrr~rPTrr Prrrr~rTT n~DrrD~rrD ~LLL~ i DrrTrnrrDr 7800 rrDrrrrD~r rTrrTP~rrr AGATTAAGGA ~L~iLL~iCC GA~ÇXLPLCTCT TCA~GAGCTT 7860 TCAGCTATTG rTrGrrDpnri DrrrrDr~r~ Dnr~rDr-TriTr rrTTTrr~r rrrrDrTrrr 7920 rrTDTPTPrr D~TrjTrrTrr AGTTTGTTAA r-TTTrTr~Dr- DrrrrrrTrr ~4~b~LL~j 7980 CGTCAATACC GAGTTrD~ DrrTrrrr~r APTGATAGAT rr ~ TPr AGTTTADAAT 8040 TTCAATGCCC ACTA~TGCCC Drrr~r~rrr rDrrLrrrrr p~rD~rrPr~p GACAGTATAT 8100 CGTCATGAAG rrTTr~r~7~Tp prrDrrDrLT rrrTrrrr~r ATTnprrTTr~ rrrrrrrDrD . 8160 rDTrr~rrTT l, ~ r~r~r~r GCCCTTGGAC TTCACAGAGT Drr~rrrrTrjr 8220 rPTr~r~rr I~TACGTC W CTTTGCAGTT TrrTDTnr~r G~rrrTrr~r rrnrrrTDrT 8280 GGACACGGTT rTrr~rPrTTD AACTTCGGCA CGCTCCACCC ~ Tp~r~rr~rT 8340 GGGCGATCCC rTrTDrTrTr ~r~rrcrcrT rDD~rrrr GTCAAGTCTG ACATGGTATC 8400 WO 96106159 2 1 9 6 ~ 9 2 PCTI~JS95110194~

CATGTTCAAG GCACACCTCA TAGAACATTC A'l 1111~ nDTDDGrrrn ~rrTrDTrAr 8460 AAGGGGGA~G CAGTATGTCC TAACCATGCT CTCCGACATG ~ 'J( :'' TGTGCGAGGA 8520 TBCCGTCTTT AAGGGTGTCA GrDrGT~rDr CACGGCCTCT rr~rrDrrDr~r~ l~v - ~V~Vl T8580 CCTGGAGACG ArrrTrDrrr~ TCATGAGACG GCTGATGAAC ~lV W v~ AAGTGGAAAG 8640 TGCCATGTCC i~ ~, rrTDrrcrDr CTACGTTGTC AGGGGTGCCA ACCTCGTCAC 8700 CGCCGTTAGC TDrrr~ T rrc CGATGAGAAA CTTTGAACAG TTTATGGCAC GCATAGTGGA 8760 CCATCCCAAC G~L~1V~1 CTGTGGAAGG TrTrD~rrr ~ V~V~ DrrrT~DrrD 8820 CGAGATTCAG DrT~rrrr~rD 1~4~L~ TCTCGTCAAG DT~Gr'rr'DT~ AGTTTGTGGC 8880 CATCCAGTAC ACCTATTTCT l-L-~llvG CCTTCACCTT ~lvCC---GCTACTCGAC 9OOD

ATCCGTCTCA GTCAGGGGCG TAGAATCCCC GGCCATCCAG TCGACCGAGA ~l VV~lV~l 9060 T~DTP~Dr AACGTGCCTC lllV~ll.V~ TTACCAAAAC GrrrTrDD~ GCATATGCCA 9120 CCCTCGAATG rDr~rrrrD CCCAGTCAGC rrDrrrDrTD AACCAAGCTT TTCCCGATCC 9180 rr~rGGrr,r~D CAT wGTACG GTCTCAGGTA Trr~-~rTrG CCAAACATGA ACCTATTCAG 9240 AGCGCTCGTA DrrDrrrT-r~ ATCTACTGCA CCCAACCTCT CACCGTCTCC=TCAGATTGGA 9360 GGTCCACCCC 'll~llL~hll IL~1L~1V~A C~L1~LV~1 rrTGrr~rTr GATCGTACCG 9420 rr~rrDrrrDr AGAACAATGG TTGGAAATAT DrrDrD~rrr CTCGCTCCAA GGGAGTTTCA 9480 rr~r~T~r~D G9Gr~rr~rDr~T TCGACGCTGT GACGAATATG ACACACGTCA TArArrDrrT -9540 AACTATTGAC GTCATACAGG AGACGGCATT TGACCCCGCG T~1.~1V1 TCTGCTATGT 9600 AATCGAAGCA ATGATTCACG r~ r~rrTT~T AAAATTCGTG ATGAACATGC CCCTCA;TTGC 9660 CCTGGTCATT r~D~rrTDrT GGGTCAACTC GGGAAAACTG V~lll~lV~ ACAGTTATCA 9720 CATGGTTAGA TTCATCTGTA CGCATATTGG GAATGGAAGC ATCCCTAAGG DrGcr~r~rr-r~ 9780 rrDrT~rrr~n AAAATCTTAG GCGAGCTCAT CGCCCTTGAG CAGGCGCTTC TCAAGCTCGC 9840 r~rrTrDrrDr ACGGTGGGTC GrTrrrrnDT CACACATCTG ~lll~W - l- TCCTCGACCC 9900 GCATCTGCTG G~L-C~111V rrTDrrDrr'D TGTCTTTACG GATCTTATGC AGAAGTCATC 9960 r~r.~rPDrrr ATAATCA~GA TCW GGATCA DD~rTArrTr AACCCTCAAA DTDr-r~GrrT- 10020 GACAAGGGTC AATGAGGACC ATGACGAGAG ACACGTCCTG GACGTGGCGC rrrTrrT~rD 10140 GAATGACTAC AACCCGGTCC T w AGAAGCT ATTCTACTAT GTTTTAATGC CGGTGTGCAG 10200 T~rr.r.rrDr ATGTGCGGTA lVV W~l-~ CTATCAAAAC Vl iV~--lV~ CGCTGACTTA 10260 r~nrr.rrrrr ~L-LllV W ~ ~CGTCGT~AA rr~rDrDr~r'DT GATATTCTAC TGCACCTGGA 10320 rTDrrr~rc TTGAAGGACA TTrTGrDr-r~r Dr-GcrDrDTD rrcrcrT-rr~ TW ACATGAT 10380 CAGGGTGCTG TGCACCTCGT TTrTrT~rTr~ ~111~1~ ~rrrDr~rrrr~ ~l-Oi~lUAT 10440 ~ W 096106159 2 1 9 68 ~2 PCr~US95~10194 ,i 'I i ~;

rDr~--er nDrrrrrrrr AGAGTTTTGC rDrrrDrr~ TDrnrrrDr- D'l.~ 10500 GACCGTGCTT nTT~Tr'GrT L ' Ubl~i bl 1 ~b~bbL~ibLb GACCGCTCTC Gr~-~rr-rr 10560 GGAGACTATG TTTTAT ~GG TPrrrTTTDD rr~ - - TrTDr r~rTr~DrrrrT ~lbbl~ib ~iC 10620 CACACTGCAT l_lb( L~L~il' r~ rTDTrT rDrrDr~GrTr rrrD7~rrDr~D r~ rGrr~r~T 10680 GGTCTTTAAC r.TGrrDTrrD ~TrTrDTr.rr AGAATATGAG r~DDTr~r~rDrD Dr~Trr~rrrrT 10740rnrrrrrTDT jl~ rTrDrnrrDr r.~li-i-,--ill DTTDnrrrrD TrrTr~rrDT 108QQ
rrDrrDD~D rTDTrTrrrr ~rcAGTTTcAT TTGCCAGGCA DD~rDrrr,rD TGCACCCTGG 10860 TTTTGCCATG ACAGTCGTCA rrD~r--~-r~D GGTTCTAGCA c~-~rarDTrr TDTDrTGrTr 10920 rAr~rr~rrTrr DrDTrrDTr~T 'ILUL~ibb~LL bU_LL~bb'~i rTDrr~Grr~rr Dr~nTDrrTTr 10980 GGACGCGGTG ACTTTTGAAA TTACCCACGA GATCGCTTCC rTr~rDrDrrr rDrTTrGrTA 11040 CTCATCAGTC A'~b~bb rrrDrrTr-rc rr~rrDTD~rT Dr~-~ - DTGr GAGTACATTG 11100 TCAGGACCTC TTTATGATTT TrrrDrr~rrD ~b~bL~I~G r~LrrrrrDr~r TGrDTr~TD 11160 TATCM AATG ~Dr'rr'rrrr TGCM ACCGG rTrDrrGrr~D AACAGAATGG ATCACGTGGG 11220 DTDrDrTGrT ~3i1ii1 1~'~'11 GrTGrr'-~ ~-'Uj~bbL TTGAGTCATG GTCAGCTGGC 11280 AACCTGCGAG ATAATTCCCA CGCCGGTCAC ATCTGACGTT b~L~L ' L~ ~r~ - rrrrDr~ 11340 rPDrrrrrrr~ 1 j '(ili I bL~ibl~bl GTCGTGTGAT r~rTTDrDr~TD Dr-~rrnr 11400 AGAGCGTTTG TTCTACGACC DTTr~TDrr ~-~rrrGrr. TDrr~Tr'rr GGTCCACCAA 11460 CM CCCGTGG GCTTCGCAGC ~LUili~LU~l rnr~rnDrnTr~ rT~TDrr~Ta TCACCTTTCG 11520 CCAGACTGCG (l~ Tr~TDrDrTcc TTGTCGGCAG TTCTTCCACA ~ r~ - pT 11580 TATGCGGTAC DDTDrrr,r~nT TGTDr~rTTT GGTTAATGAG ._l,~lU~ ~ ''i' "''- 11640 b~ DrrDr~rDrTD. r~-~-rTrrD GTACGTCGTG GTCAACGGTA CAGACGTGTT 11700 TTTGGACCAG CCTTGCCATA TGrTGrDrrD 13~ '1' DrrrTrnrrn crDnrrDrDr 11760 AGTTATGCTT rCrr~DrTDrD Tr.Tr~r~ GrD-~-DrDr r~rrrrDr~TDr DrDTr~r~GcrD . 11820 GTATCTCATT -~ --TGG CGccnDTr~ GAGACTATTA D~--TCC-~ prD~ - - TrinT 11880 GTATTAGCTA ACCCTTCTAG rnTTnnrTDr' TrDTGGrDrT rnDrD~r~-T ATAGTGGTTA 11940 ACTTCACCTC CAGACTCTTC r~rTrDTr~Dr l'i'i"'i" '~ TrDr~TrD~ DTDnnr~~~r~ 12000 TACTGCCGCT rr-~-TTTnr CACCGTTTAC ~ TDTDrD GGCATTGGGC ~ 12060 TATGCTCACG T-~-~rDTrT rrr'nDrTDrD Trr~TTDT GCAGTATCTA TCCM GTGCA 12120 CACTCGCTGT rrTGr~Dr~ i PrPr~rrTnrr~ rrTPDrnrr~r~ ATGGATCCCT 12180 CTGACAACCT TrPnDTP~D AACGTATATG ~bLLLLL TCAGTGGGAC Pr~rr~rPrrr 12240 PnrTPrrDr.T rrTprrcrrD~ TTTTTTPr.rr GADAGGATTC CACCATTGTG CTCGMATCCA . 12300 ACGGATTTGA ~bLU~L~ CCCAJGGTCG Tr~rrr~rDnrp DrTrr.nr.rPr ~L~-lL~Lli~ 12360 pr.rprrTr~TT GGTGTACCAC.ATCTACTCCA D~TDTrr~Gc ~ 'b~ ' GATGATGTAA 12420 ATATGGCGGA ACTTrPTrTD TDTPrrDrrD PTnTr~TCATT TpTr~nrr~rrr ACATATCGTC 12480 W O96/06159 2 ~ 9 6 8 9 2 PCTNSgS/l0l94~

TGGACGTAGA rA~rprr-nDT CCACGTACTG CCCTGCGAGT GCTTGACGAT CTGTCCATGT 12540 ACCTTTGTAT rrTPTr~rrr IlvhlLLL A LhLhh~jl~L L~Ll,L,jLl~ DrrrrrrTrr, 12600 TGCGGCACGA CAGGCBTCCT CTGACAGAGG TGTTTGAGGG hhl~l~jLLA GATGAGGTGA 12660 rrpr~r~pTpr~ TrTrr~rrDn TTGAGCGTCC CBGATGACAT CACCAGGATG CGCGTCATGT 12720 TCTCCTATCT TCAGAGTCTC AGTTCTATAT TTAATCTTGG CCCCAGACTG rprr~Tr~TDTr~ 12780 CCTACTCGGC AGAGACTTTG ~ l GTTGGTATTC rcrprr~rTDD CGATTTGAAG 12840 ~ ATGGCGTCAT CTGATATTCT ~l~'~hll~A AGGACGGATG ACGGCTCCGT 12900 CTGTGAAGTC lLL~.lj~Ll~j r~rrT~rrP~ pD~DrTprr GTCTACCTGC CGGACACTGA 12960 ACCCTGGGTG rTPr~r~rrr ACGCCATCAA AGACGCCTTC CTCAGCGACG GGATCGTGGA 13020 TATGGCTCGA AAGCTTCATC Ll~jl~LLLl rrrrTr~T TCTCACAACG GCTTGAGGAT 13080 hLl~jLlllll TGTTATTGTT ACTTGCAD~LA TTGTGTGTAC CTAGCCCTGT ll~l~jL~LLL 13140 CCTT~ATCCT TACTTGGTPA CTCCCTCA~G CATTGAGTTT I~ i TTrTGrrDrr 13200 TGAGjGTGCTC TTCCCACACC rrGrTr~r~T hl~lChLhLL TGCGATGACG LhAllll.l~ 13260 TAAACTGCCC TPTDrrr.TGr rTPTD~Tr~ CACCACGTTT GGACGCATTT Drrrr~DrTc 13320 Tprprrrr~n rr~nr7rD rr.rrTDrr~D TTACTCCATG GCCCTTAGAA (jL~j~LLLL~L 13380 AGTTATGGTT A~CACGTCAT r~Tr-r~r~rT GACATTGTGC rr,rr~rDr~ CTCAGACCGC 13440 ATCCCGTAAC CACACTGAGT GGGAAAATCT GCTGGCTATG 'lll~ l~A TTATCTATGC 13500 CTTAGATCAC AACTGTCACC CCr~PrT GTCTATCGCG AGCGGCATCT TTGACGAGCG 13560 TGACTATGGA TTATTCATCT CTCAGCCCCG GAGCGTGCCC TCGCCTACCC rTTrrr~rT 13620 GTCGTGGGAA GATATCTACA ACGGGACTTA rrTPnrTrrr CCTGGAAACT GTGACCCCTG 13680 GCCCAATCTA TCCACCCCTC CCTTGATTCT AAATTTTA~A TAAAGGTGTG TCACTGGTTA 13740 CACCACGATT ~ rrprT CACTGAGATG TCTTTTTADC rrrT~rrr~ TTATACCGGG 13800 ATTTAD~ACC GCCCACTGAT TTTTTTACGC TAAGAGTTGG hl~'~ h~G GGTTTTGCAT 13860 l~l~l~jll~ TP~rT~TAT ATAAGTTAAA CCAaAATTCG rDrrr~r~-~ AGGTGACGGT 13920 GGTGAGPACT CAGTTGAGAG TCAGAGAATA r~r.TGrTP~T rPrrr.T~rPT GAGCATGACT 13980 lL~LLL~l~l CCAGTCACCG r~rr~TrGT GGACGGCTCC ~l.Ll~hl~jL GAATGGCCAC 14040 CAAGCCTCCC GTGATTGGTC TTATAACAGT LLl~ll~LlL CTAGTCATAG LLhLL~jLLl 14100 CTACTGCTGC ATTCGCGTGT ICLl4hLjLC TCGACTGTGG rrrrrrPrrr rDrTDr~rr~r 14160 GGCCACCGTG GCGTATCAGG TCCTTCGCAC CCTGGGACCG rPr-rrrrGrT CACATGCACC 14220 Grrr~rr7nTG rrrpTDnrTP rrr~rr~rrr rTprrrTDr~ ATATACATGC CAGATTAGAA 14280 ~hh~Ll~l~l GrTDT~DTr-n ATGGCTATGG hh~ihh W ~l~ TAGATAATTG AGCGCTGTGC 14340 TTTTATTGTG GGGATATGGG CTTGTACATG TGTCTATCAT rnrT~nrrDT AAAATGGGCC 14400 ATGACAACTG rrPrpDr~Tp~ GTCGTCCGAC A'l~l~j~llll h~ll~LjLh~ GTATGACTGC 144E0 CCTCCATCCC TPPrrrr-n~r GCACTTGATC nrrCCC~rrT GTTCTACCAG GTAGGTCACC 14520 ~ W 096/061S9 2 1 q6892 PCTrUS95110194 r i GGGTCAAATG ATATT~TGAT GGTGTTGGAC ACCACw TCT ~L~I~G~L-~l CAGGGTGCCG 14580 GAGTTCAGAG CGTAGATGAA TGTcTr-AAAc GCGGAGGATT LLlLbLLlLL CAACATGTAA 14640 ATTGGCCACT rrDrrrrrrT GL~,11U1U~ GTATAGTGTA GAaaATGTAT r,;rr-----rrr 14700 CATATTTCGT TDT~~~~rrT TGCAATGGCC MrCrr~-~nT Lll~L-Ll~Ll 41l~LLllU'L- 14760 Drrnrrr.rnT TCACGCGCTC ~ATTGTGGTG Tr.rMrrDrDr CGATCGCCTT AATCATCGTG 14820 rDTGrr,rDr~- DrrrTMTrTr rTD~rirDr~rT GrrrrDr-TrD GGTCGCGCAG GAAGAAATGC 14880 LCL~'I~CLA ATATGAGGCT lUL~L-l~LL-A GTCTGAGTAC TCGTGACAAC r,r,rnrrrMrr. 14940 rrDrTDrrrir. ACGCCTCCGT 411~11L41A TDrr~rr~r~r~rT CGATGTAAAC AaACAGCTGT lS000 TTTCCAAGGC ACTTCTGAAC L-uLl~4bW 4~4~U~lA rrrGMrDrMT GTCAAACTGT 15060 GTCAGCGCTG CGTCACCCAC r-MrrrGQT~ Mrrr.T-Mr~rDT TTGACGACGC T~ [~ i 15120 CCCATTAGTT CGGTGTCGAA '4LLLLLlUL -MTD~ -T L41L~bl~4l TTTGATGGAT 15180 TCGTCGATGG TGATGTACGT CGGAATGTGC AGTCTGTAAC ~r~-MrrM CACTAGTGCG 15240 TCTTGCAGGT GGAaATCTTC 1~441~41UL r~r~rMr-Mrr~T DrrT - ~ - r-Mr ATTCAGCATC 15300 ~ ~Ll~LL CGTTCCTGAG GTTAAGCAGG AaACTCGTGG AGCGGTCTGA CGAGTTCACG 15360 GATGATATAA ATATAAGCTT 4LL41Ul~l~ TGAAGCATGA DDrrrDrrDT Dr.rrr.r.rDrT 15420 r7rDTrrTTTT T~TD~TT LL-L~L~4~1 DrGT~ - ~ - - D r~nTTDD~~~T Ll~luLLLbA 15480 A1~LL~L~ r.DrDrr-~D r~-Dr~ - DnrnrrTrMT ~-nr.rT~ rMr.T~ 15540 r~ -C-r-D ACAGTGCGTG LLlULl~411 CTTGGGaATA ~--crrrr l~Lul~CLLA 15600 TCGATCGTAT çr.r.Tr~--r-M GTGGATCCTG GACATGTGGT GAATGAGAAA GATTTTGAGG 15660 AGTGTGAACA ATTTTTCAGT CAACCCCTTA Gr~r~Mr~rD~ - T ~41U4L~44L- r~Tr-Mr~nr~rMr 15720 TCGACGGCCT ~ GACTCTCTAT GTCACAAAAC AGAAAGACTC L~~ .M 15780 Tnr~-rTnnT Gr-nrMrr-r-Mr~ Lll~L4A 4441~LL4 rrTL-~rDrr r7nTnrr~T 15840 GAAGAGTGTG GCGAGTCCCT TATGTCAGTT rrMrr-r-rr~Tr~ LL~LLlul ACCAGTGTCG 15900 rrDnTr.rrTr. r.r-MT~MrrMrr. TGTGTGATGG r.nGrr.rrr~ w ll~l~L TGrMTprr~rr 15960 GGAGAGCGTC DTrTGrr-DMr TD~rr~r~r~T~ ~L~L~Iu~Lu GGCAACATTC D~r~---rrD 16020 GTTTTTAGGG rrr-rTDrrr~T-~TcGGAcTTT rr.MT~rrMr. r~TTr~MrMrrr~ ACGCATATCA 16080 L444~L~LL~ ~LL1~LU1~ D~rrr-~- MT I ~ . I MT TTr~rDr~MrDT r~r~rrr-~-Dr .16140 CACCGTAATC rTGrDrr~D~ TAGCCCTGGG GGACGGCGTC MDrr--~DrrL TCTCGGCCAT 16200 TATAGATGAA ~rDTTrr~nTr DnTnTrTTrr cGTDrTr~r~r-n r~---rr~r nrnr-nTDrnr 16260 LLL~4LUL~L AGCATGTATC TGCACGTTAT CGTCTCCATC TDTTr-~-D~ AaACGGTGTA 16320 CAAcAGTATG rTMDTTTD~T r.rDrD~ ~ TD~ D n~_TDr GACTGCATTG. rrD~--rrrT 16380 rrr-~-D~ Tr~r~DTr~rr~rD TGCTATCaAC r~D~r~TDrG TAGGTCCT WG CTGCCACCGT 16440 TTGGCCCACG 'LV4LULL~LL TDrrMDrrTTT rTrrTGrDTr Drn~rrDTDrr rrTr~~~rr 16500 GAGATCATCT TTTCCACCT~ rDrrrnr~TTc AGCCGGTCGC CAGGG~rATC ~ ; 16560 W O96/06159 2 1 9 6 8 9 2 PCTrUS95/1Ol9~ ~

~i~l~l~l~i GGAAACGTGT CCTGCCAGGG r~ DDDrr AACTTGCGTC TTCACCTTCT 16620 Wlll~C TTAGCCTGCC i~L~LLLLLL CACGATGGGA ACTTTCATCC ATTTGACATC 16680 TCGGTACTGC GCATTTCCTG LLL 1 ~i 11 L 1 AATCTTAGTC TTACTGTCAG Al 11 ~L ~ l~T 16740 CTATCTCTGG TGGTGGCTAT '''~-l3i":':':~ CGGAATAATG L~iL~iL-A~l-- GACCGTTGAC 16800 GGGGTATCGC rr.rr~~~~~~ rGrrrTpr-rr CACCCTTTGG AGGAACTGCA GAGGCTGGCG 16860 CGTGCTACGC rC~~-rrr-Gr ACTCACCCGT GGACCGTTGC AGGTCCTGAC ~b~iLLll-lL 16920 rr~rr-rDr~r~T rD~~~c~~~~ rrr-rnrrDrT CACCACATGG CGCTCGAGGC TCCGGGAACC 16980 GTGCGTGGAG ~r~~rTarD LLL~iCLl~ll TCACAGAAGG rrrrArrrrr rDrDrrirrDr 17040 ~rnrrDrrrr rrrTrrrDrT GAGCTTCAAC CCCGTCAATG CCGATGTACC ~i~LA~l~ 17100 rr-~~~rcrD CTAACGTGTA Ll~i~l~LL rrrTDrTDTr~ l~l~l~Lll~ rr~rGrr~rT 17160 GGCCGTCAGG AAGACGACTG G - ~j~L~'1A CCACTGAGCT TrrrD~~~rD i~C~vl~i-- - 17220 rrrrr~rrrr GCTTAGTGTT rDTGr~rr~r TTGTTCATTA DrDrr~ GTGCGACTTT 17280 GTGGACACGC T~~~~~~rGr rTr-TrGrDrr CAAGGCTACA CGTTGAGACA ~LLL~ iLLl 77340 TGCCTAGTGT TDrrrrrrrT GGCTTCGGAG GCTAGTGCCT R,rDTD~-~~r L~LLALL-l~L D17460 CLbL'LLLll7 rrrrrrDrrr rTr-rTr-r-DTr- GACGTGTTAG GATTATGGGA ~~~rrrrrr 17520 CACACTCTAG GTTTGGAGTT DrrrGrrrTA AACTGTGGCG rrDrr~~~r~r TGACTGGTTA 17580 GAGATTTTAA AArDnrrrrD TGTGCAPAAG ACAGTCAGCG GGAGTCTTGT GGCATGCGTG 17640 ATCGTCACAC CCGCATTGGA AGCCTGGCTT GTGTTACCTG L~i~llll~L TATTA~AGCC 17700 rGrTDTDrrr CGTCGAAGGA GGATCTGGTG TTCATTCGAG L-LLLLlAI~iL rTDrrrr-r~~ 17760 rrrrpD~rTT CGGAATTTCC T~rDD~~~ ATGCATATGG ACTGTTAACC CAATGTCAGG I7820 GGACCATATC AAGGTCTTTA ACGCCTGCAC ~l.l~I.~.L- CCGGTGTATG DrrrTr~~~T 17880 GGTAACCAGC TDrnrDrTr-A ~i~Ll~l~ TTACAATGTG I~1~ jL~1~ I.11~L1~jLA 17940 TA~AGT QTG GGACCGTGTG lL-L-L1~71~W AATTA~CGGA GA~ATGATCA TGTACGTCGT 18000 AAGCCAGTGT ~lll-~l~C L-LLLL~l~LL GrrrrrrrDT ~ TCAT~TACTT 18060 TGGACAGTTT CTGGAGGAAG CATCCGGACT GAGATTTCCC TACATTGCTC LLLL~LL~l~ 18120 rrr,rr~rDr rTDrrTr~~r T~~crDr~~D AGAATTAGTT CATACCTCCC AGGTGGTGCG 18180 rrrrrrrr~~ rTr~~r~TT GCACTATGGG TCTCGAATTC AGGAATGTGA ACCCTTTTGT 18240 'll~i~iLlL~iLL GGCGGATCGG I~1~L1~L1 ~ll~ll~WL GTGGACTACA TL~L~11~1~ 18300 TCLbi~l~lL GACGGAATGC L~l~lL~iC AD~GAGTGGCC LLLLl~iLll~ CCAGGTGCGA 18360 rrDrrrD~~~ TGTGTCCACT GCCATGGACT CCGTGGACAC GTTAATGTAT ll~L-l~lA 18420 Ll~ll~l~LL rDnTrnrrrr GTCTATCTAA CATCTGTCCC TGTATCAAAT CATGTGGGAC 18480 CGGGAATGGA GTGACTAGGG TCACTGGAAA CAGAAATTTT Ll~iL-l.llL TGTTCGATCC 18540 CATTGTCCAG Dr~rDrrrTDA CAGCTCTGAA rATD~rTDnr rDrrrDDrcr CCACGCACGT 18600 ~ W 096/06159 2 1 9~82 PCTnU595110194 1~7 CGAGAATGTG rT2~r~-r~r TGCTCGACGA CGGCACCTTG bi~'L4L~L4 TCC2AGGC2C 18660 LLL~ w L~LL rTT2rr~Tr TrTr-DrT2rT TC2GCCGCTT rrTr2T2T2T r~rTr.T2~ 18720 A2CTTAAGGC ~-L~LL~L~ G~GC2TGTT r,rr,r2rDTra r~rr~rrTrn2 18780 LLLl~LL~L4 L4L~4L~L~L 2r.2TT2T,r,r.T ,.~ , TTrTTr2TrT TT~2TTTTT 18840 ar~-r.rr.r.2~r r2rrr~ra~2 il~ T r,2TTTrrr.rr 2~r2rrr~rT TGGCTACGTG 18900 LLLLL~4L~ GrT2rrT2rr ,C2~TGTTAAT GTTCTCTACG r.2Tr.rr2rT2 r.r2Tr.rTr.~T 18960 GATCGCCACC 2rT2Tcr2Tr~ L~LLL~L4L~ ~--~L~L~4~ 2TT~ nT2 LLLLL~LLl 19020 TTGCTTAAAC GTCTGTAP~A r2rTr~TTTrr 2rTTTrr~T 2~rrr.22rT ACTGCTTA2A 19080 CP~TCC2~ C P2rTGrTGrr 'L~LLLL~L~iL GGCCTTGATT r-~rr2~2~ 2r~ rT 19140 GTGC,2TTACT 2r.rTrrTr.TT rr~rrccTr r2rrr2nTn~c Lrrrrrrr.~ rr.T2~r2rrr 19200 GTTC,2~1AAAG ~'rr'2~rrT T~rr2r-~p AGCCTGAAGT Trr~rrr~T2r~2 r2r~rr2rr.r 19260 GTGC2GGGAG 'I~L~L~ J' ~ ' CTGGTACTCG 2rr2nTTr.2T "i,." ,.,.. i~ 19320 GACGTGCGCG ~L ~ r2r2rrGrDT ,rTr,r~2r,T2T riTTr~2T2r~r~r~ ACTCC2ATAG 19380 GL~L~L1L rrnrRr'rrT l~l~l~i~ ~l~l~i~i~i GTTCCCLCGT CGGGATTTGC 19440 TGACGTGGGC ~l~iW LL-~T ~l~L~L~L GrPr-TPTrTT Trr~rr7 - rn AACTGTATGA l9500 GTTTATTCTG TGCACCACGC r2~TP~r~r- rTGrnrrpTc ~LLLLLL Trrr~rprrr. 19560 TCGCGTGAAT r~TrGr~GGrpr Trpr~TTrrrp LLL~L~L~LL G~ ~- L~4~L~LL~ 19620 CAGGTTGGCG Gr~7'r.rirGrT rrrTGTrDrn r:rTr~rrPnr D~ .. ., TnPr.rTrr.rT 19680 rnTnTrrr-r GGTGACCCGG pnnTn2~rrpr TDnnTPrnTr 2~rr~-rnTpr a~rTTr-rrrT 19740 GGACCTTAGC r~r~2rpr~r CTGGAC2ATT T~rTTrPT2 r~2rTrrrr TGAACPGCTT 19800 ~L~L~V~LL TrrD~rr-Tnp Tr~rrrrpr-nT CC2GCC2~ATC TGC_GTGGCC WiLLLG~LLL 19860 Grrr.rr~r~r TTTAGTAATC TCCACTTGCC T2r~rTrr~r p2r~cTcrpr2 GAGTCCTCGG 19920 GCPGGGTTTC l;lilil~ r~Tr~ T rr~rprTcr~r CCGTCTCACG T2r.~r2rp 19980 rn2a~2rr.r.r r2r~rTnTTrT Prr~rrprTp TnrT2rrr.Pn r.PnTrr~rnT GGGCTTTGAC 20040 TCTGAATAAG ~I~L~jLL~L TTCG w AGGC T~TAGATGGC rTrTrTr~rr CCGGAACTTG 20100 GAAGGGTCTT rTTrrTr.2rn PrrrrrTTrr 4, ~L~rLL~iL LL~LL~L~ Drr.rPrrrrc 20160 ~L~LLLll~l Crrr,rrr'rT ~ l r2'rr2rrDr TnrrnTT2rr l'lli~ _l 20220 GCTACTTCCA r~rTrDrpTriT ACGCTCCCPU~ ACGGG~TCTT lL~il~ll-~i TTA2TC2TGC 20280 rrTrr~rT2r ACC2~GTTTC T2TPrrr'r' L~ ~L~4 2rPTnrnrr.r ~i~Lll~LL 20340 CCCGCCATTC GrTprTTcTr r.r2T2r22~r GnT2r~Tr~rT C2GATGA21~A TCALTAGATGC 20400 TTrrr~r~rT TA~A~TTCCC 2r2rrTnrrT rTTnTnTrPr PT2TPTrPnr ~2~TPnr2T 20460 ~ATTGCGGGT rPrGGr~Prrr 2rnTrnnTrr ~2TrrT2rTr TTGAGTGGAP. aDr~rrPrrr2 20520 rTrT2TaarP r.rr~Tr.TTr arprrr~r r7Tr.Trra~rT arrr,r,rr~rT aTrT2~TraT 20580 CCCATCGTAT r~rPTarrrn 5GATCrLTC,2~C C2TGATCAAG rDn2pTrr~r TC2ACCAACT 20640 WO 96/06159 2 ~ 9 6 8 9 2 PCT/US95/101941~

rTP~ r~ GTTTATTAAG T~UUUlulvG Ar~rrr~r~T r~r~r.r.~r.r. GCAGCTGTAT 20700 (2~ INFORMATION FOR SEQ ID NO:2: ~
(i) SEQ-JENCE r~rT~cTIcs:
~A LENGT~: 4131 ~ase pairs ~B TYPE: nucleic acid C sT~np~-qq single D TOPOLOGY: linear (ii) MOLEC~LE TYPE: DNA (genomic) (iii~ ~Y~U~ -~L: N
(i~) ANTI-SENSE: N
(ix) FEATURE:
(A) NAME/XEY: CDS
(B) LOCATION: 1..4131 (D) OTLER INFORMATION:

(xi) SEQ~ENCE ~Su~I~Ilu~: SEQ ID NO:2:

Met Glu Ala Thr Leu Glu Gln Arg Pro Phe Pro Tyr Leu Ala Thr Glu Ala Asn Leu Leu Thr Gln Ile LYG Glu Ser Ala Ala Asp Gly Leu Phe A~G AGC TTT CAG CTA TTG CTC GGC AAG GAC GCC AGA GAA GGC AGT GTC 144 Lys Ser Phe Gln Leu Leu Leu Gly Lys Asp Ala Arg Glu Gly Ser Val Arg Phe Glu Ala Leu Leu Gly Val Tyr Thr Asn Val Val Glu Phe Val Lys Phe Leu Glu Thr Ala Leu Ala Ala Ala Cys Val Asn Thr Glu Phe AAG GAC CTG CGG AGA ATG ATA GAT GGA A~A ATA CAG TTT AAA ATT TCA 288 Lys Asp Leu Arg Arg Met Ile Asp Gly Lys Ile Gln Phe Lys Ile Ser Met Pro Thr Ile Ala ~is Gly Asp Gly Arg Arg Pro Asn Lys Gln Arg CAG TAT ATC GTC ATG A~G GCT TGC AAT AAG CAC CAC ~TC GGT GCG GAG 384 Gln Tyr Ile Val Met Lys Ala Cys Asn Lys ~is ~is Ile Gly Ala Glu ATT GAG CTT GCG GCC GCA GAC ATC GAG CTT CTC TTC GCC GAG A~A GAG 432 Ile Glu Leu Ala Ala Ala Asp~n e Glu Leu Leu Phe Ala Glu Lys Glu Thr Pro Leu Asp Phe Thr Glu Tyr Ala Gly Ala Ile Lys Thr Ile Thr 145 l50 155 160 ~ W O96106159 ; ; PCTrUS95~10194 .

ser ~la Leu Gln Phe Gly Met Asp Ala Leu Glu Arg Gly Leu Val Asp 165 ~ 170 175 ACG GTT CTC GCA GTT A~A 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 .AAG ACG CTG GGC GAT CCC GTC T~C TCT GAG AGG GGC CTC A~A AAG GCC 624 Lys Thr Leu Gly Asp Pro Val Tyr Ser Glu Arg Gly ,Leu Lys Lys Ala GTC AAG TCT GAC ATG GTA TCC ATG TTC AaG GCA CAC CTC ATA GAA CAT 672 Val Lys 8er Asp Met Val Ser Met Phe Lys Ala His Leu Ile Glu His 210 ~ ~ 215 220 Ser Phe Phe Leu Asp Lys Ala Glu Leu Met Thr Arg Gly Lys Gln Tyr 225 : -- 230 235 240 Val Leu Thr Met Leu Ser Asp Met Leu Ala Ala Val Cys Glu Asp Thr Val Phe Lys Gly Val Ser Thr Tyr Thr Thr Ala Ser Gly Gln Gln Val Ala Gly Val Leu Glu Thr Thr Asp Ser Val Met Arg Arg Leu Met Asn CTG CTG GGG C~A GTG GA~ AGT GCC ATG TCC GGG CCC GCG GCC TAC GCC 912 Leu Leu Gly Gln Val Glu Ser Ala Met Ser Gly Pro Ala Ala Tyr Ala 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 Arg Ala Met Arg :Asn Phe Glu Gl~ Phe Met Ala Arg Ile Val Asp His CCC AAC GCT CTG CCG TCT GTG GAA GGT GAC A~G GCC GCT CTG GCG GAC 1056 Pro Asn Ala Leu Pro Ser Val Glu Gly Asp Lys Ala Ala Leu Ala Asp Gly Eis Asp Glu Ile GLn Arg Thr Arg Ilç Ala Ala Ser Leu Val Lys 355 360 . 365 ATA GGG GAT A~G TTT GTG GCC ATT GAA A3T TTG CAG CGC ATG TAC A~C 1152 Ile Gly Asp Lys Phe Val Ala Ile Glu Ser Leu Gln Arg Met Tyr Asn GAG ACT CAG T~T CCC TGC CCA CTG AAC r~ rrr ~r r~. TAC ACC TAT 1200 Glu Thr Gln Phe Pro Cys Pro Leu Asn Arg Arg Ile Gln Tyr Thr Tyr 385 39~ 395 400 Phe Phe Pro Val Gly Leu His Leu Pro Val Pro Arg Tyr Ser Thr Ser GTC TCA GTC AGG GGC GT~ GAA TCC CCG GCC ATC CAG TCG ACC GAG ACG 1296 Val Ser Val Arg Gly Val Glu Ser Pro ~la Ile Gln Ser Thr Glu Thr WO 96/06159 2 -1 9 6 8 ~ 2 r~ s,i~is~--TGG GTG GTT AAT A~A 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 Gln Asn Ala Leu LYA Ser Ile Cys Hi6 prD Arg Met His Asn Pro Thr Gln Ser Ala Gln Ala Leu Asn Gln Ala Phe Pro Asp Pro Asp Gly Gly His Gly Tyr Gly Leu Arg Tyr Glu Gln Thr Pro Asn Met Asn Leu Phe Arg Thr TTC CAC CAG TAT TAC ATG GGG A~A AAC GTG GCA TTT GTT CCC GAT GTG 1536 Phe His Gln Tyr Tyr Met Gly Lys Asn Val Ala Phe Val Pro Asp Val Ala G:ln 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 His Pro Cys Pro Gly Ala Arg Gly Ser Tyr Arg Ala Thr His Arg Thr Met Val Gly Asn Ile Pro Gln Pro Leu Ala Pro Arg Glu Phe Gln Glu 565 . 570 575 Ser Arg Gly Ala Gln Phe Asp Ala Val Thr Asn Met Thr His Val Ile GAC CAG CTA ACT ATT GAC GTC ATA CAG GAG ACG GCA TTT GAC CCC GCG ~ 1824Asp Gln Leu Thr Ile Asp Val Ile Gln Glu Thr Ala Phe Asp Pro Ala sg5 ~ 600 605 Tyr Pro Leu Phe Cys Tyr Val Ile Glu Ala Met Ile His Gly Gln Glu 610 615 ~ 620 GAA A~A 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 Gln Thr TAC TGG GTC AAC TCG GGA A~A CTG GCG TTT GTG AAC AGT TAT CAC ATG 1965 Tyr Trp Val Asn Ser Gly Lys Leu Ala Phe Val Asn Ser Tyr His Met Val Arg Phe Ile Cys Thr His Ile Gly Asn Gly Ser Ile Pro Lys Glu GCG CAC GGC CAC TAC CGG A~A 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 Gln Ala Leu Leu Lys Leu Ala Gly His Glu Thr val Gly Arg Thr Pro ~ W 096/06159 21 96892 ~ 91 Ile Thr ~is Leu Val Ser Ala Leu Leu Asp Pro ~i8 Leu Leu Pro Pro 705 , 71P 715 720 TTT GCC TAC CAC QAT GTC TTT ACG GAT CTT ATG cAr~ AAG TCA TCC AGA 2208 Phe Ala Tyr aiS ABp Val~Phe Thr Asp Leu Met Gln Lys Ser Ser Arg CAA CCC AT~ ATCI~G ATC GGG r~T r~ ~ TAC GAC AAC CCT CaA AAT 2256 Gln Pro Ile Ile Lys Ile Gly Asp Gln Asn Tyr Asp Asn Pro Gln Asn Arg Ala Thr Phe Ile Asn Leu Arg Gly Arg Met Glu Asp Leu Val Asn Asn Leu Val Asn Ile Tyr Gln Thr Arg Val Asn Glu Asp ~is Asp Glu AGA CAC GTC CTG GAC GTG GCG CCC CTG GAC G~G AAT GAC TAC AAC CCG 2400 Arg ~is Val Leu Asp Val Ala Pro Leu Asp Glu Asn Asp Tyr Asn Pro GTC CTC GAG AAQ CTA TTC TAC TAT GTT TT}sATG CCG GTG TGC AGT AAC 2448 Val Leu Glu Lys Leu Phe Tyr Tyr Val Leu Met Pro Val Cys Ser Asn Gly ~is Met Cys Gly Met Gly Val Asp Tyr Gln Asn Val AIa Leu Thr CTG ACT TAC AAC GGC CCC GTC TTT GCG GAC GTC GTG AAC.GCA CAG GAT 2544 Leu Thr Tyr Asn Gly Pro Val Phe Ala Asp Val Val Asn Ala Gln Asp 835 ~ 84P 845 Asp Ile Leu Leu ~is Leu Glu Asn Gly Thr Leu Lys Asp Ile Leu Gln Ala Gly Asp Ile Arg Pro Thr Val Asp Met Ile Arg Val Leu Cys Thr 865 ~ :87P 875 880 Ser Phe Leu Thr Cys Pro Phe Val Thr Gln Ala Ala Arg Val Ile Thr Lys Arg Asp Pro Ala Gln Ser Phe Ala Thr ~is Glu Tyr Gly Lys Asp GTG GCG CAG ACC GTG CTT GTT AAT GGC TT~ QGT GCG TTC GrQ GTG GCG 2784 Val Ala Gln Thr Val Leu Val Asn Gly Phe Gly Ala Phe Ala Val Ala GAC CGC TCT CGC GAQ QCQ GCG GAQ.ACT ATG TTT TAT CCG r-TA CCC TTT 2832 Asp Arg Ser Arg Glu Ala Ala Glu Thr Met Phe Tyr Pro Val Pro Phe 930 : 935 940 AAC AAG CTC TAC GCT QAC CCG TTG GTG GCT GCC ACA.CTG CAT CCG CTC 2880 Asn Lys Leu Tyr Ala Asp Pro Leu Val Ala Ala Thr Leu ~is Pro Leu 945 . 950= gss = 960 Leu Pro Asn Tyr Val Thr Arg Leu Pro Asn Gln Arg Asn Ala Val Val 965 97p 975 WO 96/06159 2 1 ~ 6 8 ~ 2 PCT/US9~/10194 ~

TTT AAC GTG CCA TCC A~T CTC ATG GCA GAA TAT GAG GAA TGG CAC AAG -2976 Phe AGn Val Pro Ser Asn Leu Met Ala Glu Tyr Glu Glu Trp His LyG

3er Pro Val Ala Ala Tyr Ala Ala Ser Cys Gln Ala Thr Pro Gly Ala ATT AGC GCC ATG GTG AGC ATG CAC CAA ~AA CTA TCT GCC CCC AGT TTC 3072 Ile Ser Ala Met Val ser Met HiG Gln Lys Leu Ser Ala Pro Ser Phe 1010 lOlS : io20 ATT TGC CAG GCA AAA CAC CGC ATG CAC CCT GGT TTT GCC ATa ACA GTC 3120 Ile cy5 Gln Ala Lys HiG Arg Met HiG Pro Gly Phe Ala Met Thr Val GTC AGG ACG GAC GAG GTT CTA GCA GAG CAC ATC CTA TAC TGC TCC AGG -:3168Val Arg Thr Asp Glu Val Leu Ala Glu HiG Ile Leu Tyr Cys Ser Arg 1045 lOS0 lOSS
GCG TCG ACA TCC ATG TTT=GTG GGC TTG CCT TCG GTG GTA CGG CGC ~AG 3216 Ala ser Thr Ser Met Phe Val Gly Leu Pro Ser Val Val Arg Arg Glu GTA CGT TCG GAC GCG GTG ~CT TTT GAA ATT ACC CAC GAG ATC GCT ~CC 3264 Val Arg Ser Asp Ala Val Thr Phe Glu Ile Thr His Glu Ile Ala Ser Leu His Thr Ala Leu Gly Tyr Ser Ser Val Ile Ala Pro Ala His Val GCC GCC ATA ACT ACA GAC ATG GGA GTA CAT TGT CAG GAC CTC TTT ATG ~360 Ala Ala Ile Thr Thr AGP Met Gly Val HiG Cys Gln Asp Leu Phe Met llOS 1110 lllS 1120 Ile Phe Pro Gly Asp Ala Tyr Gln AGP Arg Gln Leu HiG AGp Tyr Ile AP~A ATG A~A GCG GGC GTG CAA ACC GGC TCA CCG GGA A~C AGA ATG GAT 3456 Lys Met LYG Ala Gly Val Gln Thr Gly Ser Pro Gly Asn Arg Met Asp 1140 1145 -llS0 His val Gly Tyr Thr Ala Gly Val Pro Arg Cy9 Glu AGn Leu Pro Gly llSS 1160 1165 Leu Ser His Gly Gln Leu Ala Thr CYG 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 Gln Thr Pro Ser Asn Pro Arg Gly Arg 1185 ll9D ll9S 1200 Ala Ala Ser Val Val Ser CYG Asp Ala Tyr Ser Asn Glu Ser Ala Glu Arg Leu Phe Tyr AGP HiG Ser Ile Pro Asp Pro Ala Tyr Glu CYG Arg Ser Thr AGn Asn Pro Trp Ala Ser Gln Arg Gly Ser Leu Gly Asp Val ~ W O 96/06159 2 1 9 6 8 9 ~ PC~r/US95J10194 CTA TAC AAT ATC ACC TTT.CGC CAG ACT GCG CTG CCG GGC ~TG TAC AGT 3792 Leu Tyr Asn Ile Thr Phe Arg Gln 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 Gln Phe Phe His Lys Glu Asp Ile Met Arg Tyr Asn Arg 1265 1270 ~ 1275 . 12ao.

Gly Leu Tyr Thr Leu Val Asn Glu Tyr Ser Ala Arg Leu Ala Gly Ala CCC GCC ACC AGC ACT IU~A GAC CTC CAG TAC GTC GTG GTC AAC.GGT ACA 3936 Pro Ala Thr Ser Thr Thr Asp Leu Gln Tyr Val Val Val Asn Gly Thr Asp Val Phe Leu Asp Gln Pro Cys His Met Leu Gln Glu Ala Tyr Pro ~ 1315 . 1320 1325 ACG CTC GCC GCC AGC CAC A~I~ 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.eAA GAG 4080 Lys Gln Thr His Ala Pro Val Eis Met Gly Gln Tyr Leu Ile Glu Glu Val Ala Pro Met Lys Arg Leu Leu Lys Leu Gly Asn Lys Val Val Tyr (2) INFORMATION FOR SEQ ID NO:3:
ti) SEQJENOE CHARACTERISTICS:
(A) LENGTH: 1376 amino acids tB) TYPE: amino acid ~D) TOPOLOGY: linear (ii) MOLECUL~ TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Glu Ala Thr Leu Glu Gl~ Arg Pro Phe Pro Tyr Leu Ala Thr Glu Ala Asn Leu Leu Thr Gl~ Ile Lys Glu Ser Ala Ala Asp Gly Leu Phe Lys Ser Phe Gln Leu Leu Leu Gly Lys Asp Ala Arg Glu Gly Ser Val ~ 40 45 Arg Phe Glu Ala Leu Leu Gly Val Tyr Thr Asn Val Val Glu Phe Val Lys Phe Leu Glu Thr Ala Leu Ala Ala Ala Cys Val Asn Thr Glu Phe Lys Asp Leu Arg Arg Met Ile Asp Gly Lys Ile Gln Phe Lys Ile Ser Met Pro Thr Ile Ala His Gly Asp Gly Arg Arg Pro Asn Lys Gln Arg . .

2~ 96892 WO96/061~9 . PCT/U59~110194 194~ -Gln Tyr Ile Val Met Lys Ala Cys Asn Lys Eis His Ile Gly Ala Glu Ile Glu Leu Ala Ala Ala Asp Ile Glu Leu Leu Phe Ala Glu Lys Glu Thr Pro Leu Asp Phe Thr Glu Tyr Ala Gly Ala Ile Lys Thr Ile Thr ~er Ala Leu Gln Phe Gly Met Asp Ala Leu Glu Arg Gly Leu Val Asp ~hr Val Leu Ala Val Lys Leu Arg Eis Ala Pro Pro Val Phe Ile Leu 180 185 = . .= 190 Lys Thr Leu Gly Asp Pro Val Tyr Ser Glu Arg Gly Leu Lys Lys Ala Val Lys Ser Asp Met Val Ser Met Phe Lys Ala Eis Leu Ile Glu His Ser Phe Phe Leu Asp Lys Ala Glu Leu Met Thr Arg Gly Lys Gln Tyr ~al Leu Thr Met Leu Ser Asp Met Leu Ala Ala Val Cys Glu Asp Thr a4s 250 255 ~al Phe Lys Gly Val Ser Thr Tyr Thr Thr Ala Ser Gly Gln Gln Val Ala Gly Val Leu Glu Thr Thr Asp Ser Val Met Arg Arg Leu Met Asn Leu Leu Gly Gln Val Glu Ser Ala Met Ser Gly Pro Ala Ala Tyr Ala ser Tyr Val Val Arg Gly Ala Asn Leu Val Thr Ala Val Ser Tyr Gly 305 310 .315 320 ~rg Ala Met Arg Asn Phe Glu Gln Phe Met Ala Arg Ile Val Asp His ~ro Asn ~la Leu Pro ser Val Glu Gly Asp Lys Ala Ala Leu Ala Asp Gly Xis Asp Glu Ile Gln Arg Thr Arg Ile Ala Ala Ser Leu Val Lys 3sS 360 365 Ile Gly Asp Lys Phe Val Ala Ile Glu Ser Leu Gln Arg 3~et Tyr Asn Glu Thr Gln Phe Pro Cys Pro Leu Asn Arg Arg Ile Gln Tyr Thr Tyr 385 390 395 . _ 400 ~he Phe Pro Val Gly Leu His Leu Pro Val Pro Arg Tyr Ser Thr Ser ~al Ser Val Arg Gly Val Glu Ser Pro Ala Ile Gl-~ Ser Thr Glu Thr Trp Val Val Asn Lys Asn Asn Val Pro Leu Cys Phe Gly Tyr Gln Asn Ala Leu Lys Ser Ile Cys His Pro Arg Met Eis Asn Pro Thr Gln Ser Ala Gln Ala Leu Asn Gln Ala Phe Pro Asp Pro Asp Gly Gly His Gly 2l q6892' WO 96/06159 ' ' r~ l34 Tyr Gly Leu Arg Tyr Gl~u Gln Thr Pro Asn Met Asn Leu Phe Arg Thr " 485 490 495 Phe His Gln Tyr Tyr Met Gly Lys Asn Val Ala Phe Val Pro Asp Val . 500 505 510 Ala Gln Lys Ala Leu Val Thr Thr Glu Asp Leu Leu }lis Pro Thr Ser Pis Arg Leu Leu Arg Leu Glu Val Pis Pro Phe Phe Asp Phe Phe Val 530 535 ~ . =. 540 Pis Pro Cys Pro Gly Ala Arg Gly Ser Tyr Arg Ala Thr Ptis Arg Thr 545 550 _ 555 _ _ _ 5~0 Met Val Gly Asn Ile Pro Gln Pro Leu Ala Pro ~rg Glu Phe Gln Glu 565 . 570 575 Ser ~:g Gly Ala Gln Phe Asp Ala Val Thr Asn Met Thr His Val Ile Asp Gln Leu Thr Ile Asp Val Ile Gln Glu Thr Ala Phe ASp Pro Ala s9s 600 605 Tyr Pro Leu Phe Cys Tyr Val Ile Glu Ala Met Ile Pis Gly Gln Glu 610 ~ ~ :~ 615 620 Glu Lys Phe Val Met Asn Met Pro Leu Ile Ala Leu Val Ile Gln Thr 625 . 63Q ~ . 635 640 Tyr Trp Val Asn Ser Gly Lys Leu Ala Phe Val Asn Ser Tyr }lis Met 645 : 650 655 Val Arg Phe Ile Cys Thr Pis Ile Gly Asn Gly Ser Ile Pro Lys Glu Ala Pis Gly E~is Tyr Arg Lys Ile Leu Gly Glu Leu Ile Ala Leu Glu 675 ~ ~ 680 685 Gln Ala LeU Leu Lys Leu'Ala Gly ~is Glu Thr Val Gly Arg Thr Pro Ile Thr ~is Leu Val Ser Ala Leu Leu Asp Pro His ~eu Leu Pro Pro 70s 7I0 =. := 715 720 Phe Ala Tyr Pis Asp Val Phe Thr Asp Leu Met Gln Lys Ser Ser Arg Gln Pro Ile Ile Lys Ile Gly Asp Gln Asn Tyr Asp Asn Pro Gln Asn Arg Ala Thr Phe Ile Asn Leu Arg Gly Arg ~et Glu Asp Leu Val Asn Asn Leu Val Asn Ile Tyr Gln Thr Arg Val Asn Glu Asp Xis Asp Glu Arg E!is 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 Gly Pis Met Cys Gly Met Gly Val Asp Tyr Gln Asn Val Ala Leu Thr ,,~

WO 96/06159 , PCI/US95/10194 Leu Thr Tyr Asn Gly Pro Val Phe Ala Asp Val Val Asn Ala Gln Asp 835 ~ 840 845 Asp Ile Leu Leu E~i3 Leu Glu Asn Gly Thr Leu Lys Asp Ile Leu Gln Ala Gly Asp Ile Arg Pro Thr Val Asp Met Ile Arg Val Leu Cys Thr ~er Phe Leu Thr Cys Pro Phe Val Thr Gln Ala Ala Arg Val Ile Thr 8B5 B90 89s ~ys Arg Asp Pro Ala Gln Ser Phe Ala Thr His Glu Tyr Gly Lys Asp 900 905 . . 910 Val Ala Gln Thr Val Leu Val Asn Gly Phe Gly Ala Phe Ala Val Ala Asp Arg Ser Arg Glu Ala Ala Glu Thr Met Phe Tyr Pro Val Pro Phe Asn Lys Leu Tyr Ala Asp Pro Leu Val Ala Ala Thr Leu Xis Pro Leu ~eu Pro Asn Tyr Val Thr Arg Leu Pro Asn Gln Arg Asn Ala Val Val ~he Asn Val Pro Ser Asn Leu Met Ala Glu Tyr Glu Glu Trp His Lys Ser Pro Val Ala Ala Tyr Ala Ala Ser Cys Gln Ala Thr Pro Gly Ala 995 lO00 1005 Ile Ser Ala Met Val Ser Met Xis Glr. Lys Leu ser Ala Pro Ser Phe lOlO 1015 1020 Ile Cys Gln Ala I.ys Xis Arg Met Xis Pro Gly Phe Ala Met Thr Val ~al Arg Thr Asp Glu Val Leu Ala Glu Xis Ile Leu Tyr Cys Ser Arg ~la Ser Thr Ser Met Phe Val Gly Leu Pro Ser Val Val Arg Arg Glu Val Arg Ser Asp Ala Val Thr Phe Glu Ile Thr His Glu Ile Ala Ser 1075 lOB0 lOB5 Leu }~is Thr Ala Leu Gly Tyr Ser Ser Val Ile Ala Pro Ala Xis Val lOgO 1095 : 1100 Ala Ala Ile Thr Thr Asp Met Gly Val Xis Cys Gln Asp l.eu Phe Met 1105 1110 1115 .. - -1120 ~le Phe Pro Gly Asp Ala Tyr Gln Asp Arg Gln Leu Xis Asp Tyr Ile ~ys Met Lys Ala Gly Val Gln Thr Gly Ser Pro Gly Asn Arg Met Asp Xis Val Gly Tyr Thr Ala Gly Val Pro Arg Cys Glu Asn Leu Pro Gly Leu Ser Xis Gly Gln Leu Ala Thr Cys Glu Ile Ile Pro Thr Pro Val Thr Ser Asp Val Ala Tyr Phe Gln Thr Pro Ser Asn Pro Arg Gly Arg ~ W 096106159 2 t q 6 ~ ~ 2 PCT~US95/10194 ~ 197 1185 ~ n ~195~ 1200 Ala Ala Ser Val Val Ser Cys Asp Ala Tyr Ser Asn Glu Ser Ala Glu f 1205 1210 1215 Arg Leu Phe Tyr Asp ~is Ser Ile Pro Asp Pro Ala Tyr Glu Cys Arg 122Q ~ lZZ5 :_ 1230 Ser Thr Asn Asn Pro Trp Ala Ser Gln Arg Gly Ser Leu Gly Asp Val 1235 :' :1240 ~1245 Leu Tyr Asn Ile Thr Phe Arg Gln Thr Ala Leu Pro Gly Met Tyr Ser 1250 ~ 1255 _ _ i2~n _ _ Pro Cys Arg Gln Phe Phe ~i6 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 Pro Ala Thr Ser Thr Thr Asp Leu Gln Tyr Val Val Val Asn Gly Thr Asp Val Phe Leu Asp Gln Pro Cys ~is Met Leu Gln Glu Ala Tyr Pro 1315 ~ =1320 1325 Thr Leu Ala Ala Ser His Arg Val Met Leu Ala GIu Tyr Met Ser Asn 1330 1335 _ 1340 Lys Gln Thr His Ala Pro Val ~is Met Gly Gln Tyr Leu Ile Glu Glu 1345 _ 1350 . 1355 1350 Val Ala Pro Met Lys Arg Leu~Leu Lys Leu Gly Asn Lys Val Val Tyr (2) INFORMATION FOK SEQ ID NO:4:
(i) SEQ~EN OE r~D~ I r.~1 ~11~
(A) LENGTH: 1143 hase pairs (B) TYPE: nucleic acid (C) sT~Nn~n~R~ single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iil) ~Yr~l~ll~L: N
(i~) ANTI=SENSE: N
(ix) FEA~RE:
(A) NAME/~EY: CDS
(;3) LOCATION: 1..1143 (D) OTHER INFORMATION:

(xi) SEQ~ENCE ~Kl~ll~: SEQ ID NO:4:
AGC ATT CGG GGA CA ~CC TTT AAC CTG CTC TAC OE A GAC G~G GCG AAT 48 Ser Ile Arg Gly Gln Thr Phe Asn Leu Leu Tyr Val Asp Glu Ala Asn TTT ATT AAA AAG GAT GCA CTG CCG GCT AT~ CTG GGT TTC ~lG CTT CAG 96 Phe Ile Lys Lys Asp Ala Leu Pro Ala Ile Leu Gly Phe Met Leu Gln 21 968~9'2 W 096~6159 _ .. . PCTnUS95/lOl9 19~
AAA GAC GCC AAG CTT ATA TTT ATA TCA TCC GTG A~C TCG._TCA GAC CGC 144 Lys Asp Ala Lys Leu Ile Phe Ile Ser Ser Val Asn Ser Ser Asp Arg 35 ~ 40 45 Ser Thr Ser Phe Leu Leu Asn Leu Arg Asn Ala Gln Glu Lys Met Leu AAT GTG GTC AGT TAC GTG TGT GCG GAC CAC CGA GAA GAT TTC CAC CTG ~ 240 Asn Val Val ser Tyr val Cys Ala Asp Hi6 Arg Glu Asp Phe His Leu CAA GAC GCA CTA aTG TCC TGT CCT TGT TAC AGA CTG CAC ATT CCG ACG -. 288Gln Asp Ala Leu Val Ser Cys Pro Cys Tyr Arg Leu His Ile Pro Thr 85 90 . 95 TAC ATC ACC ATC GAC GAA TCC ATC A~A ACC ACC ACC AAC CTC TTT ATG 336 Tyr Ile Thr Ile Asp Glu Ser Ile Lys Thr Thr Thr Asn Leu Phe Met 100 105 llQ
GAG GGG GCA TTC GAC ACC gAA CTA ATG GGC GAG GGA GCA GCG TCG TCA 384 Glu Gly Ala Phe Asp Thr Glu Leu Met Gly Glu Gly Ala Ala Ser Ser AAT GCT ACG CTT TAC CGC GTG GTG GGT GAC GCA GCG CTG ACA CAG TTT ~=432 Asn Ala Thr Leu Tyr Arg Val Val Gly Asp Ala Ala Leu Thr Gln Phe 130 135 140 ~

Asp Met Cys Arg Val Asp Thr Thr Ala Gln Glu Val Gln Lys Cys Leu GGA AAA CAG CTG TTT GTT TAC ATC GAC CCC GCG TAT ACG AAC A~C ACG 528 Gly Lys Gln Leu Phe Val Tyr Ile Asp Pro Ala Tyr Thr Asn Asn Thr 165 170 175 =

Glu Ala Ser~Gly Thr Gly Val Gly Ala Val Val Thr Ser Thr Gln Thr Pro Thr A_g Ser Leu Ile Leu Gly Met Glu His Phe Phe Leu Arg Asp CTC ACT GGC GCA GCT GCT-TAC GAG ATA GCG TCC TGC GCA TGC ACG ATG - 672Leu Thr Gly Ala Ala Ala Tyr Glu Ile Ala Ser Cys Ala Cys Thr Met ATT AAG GCG ATC GCT GTG CTC CAC ACC ACA ATT GAG CGC GTG AAC GCG ~ 720 Ile Lys Ala Ile Ala val Leu His Thr Thr Ile Glu Arg Val Asn Ala 22s 230 235 . .240 Ala Val Glu Gly Asn Ser Ser Gln Asp Ser Gly Val Ala Ile Ala Thr 24s 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 Gln Trp Pro Ile Tyr Met Leu Gly Gly Glu A~A TCC TCC GCG TTT GAG ACA TTC ATC TAC GCT CTG AAC TCC GGC ACC 912 Lys Ser Ser Ala Phe Glu Thr Phe Ile Tyr Ala Leu Asn Ser Gly Thr ~ W O96106159 2 1 9 6 ~ 9 2 PCT/US95/l0194 CTG AGC GCC AGC CAG ACG GTG GTG TCC AAC ACC ATC A~A ATA TCA TTT 960 Leu Ser Ala 8er Gln Thr Val Val Ser Asn Thr Ile Lys Ile Ser Phe GAC CCG GTG ACC TAC CTG GTA GAA C~LG GTC CGC GCG ATC AAG TGC GTC 1008 Asp Pro Val Thr Tyr Leu Val Glu Gln Val Arg Ala Ile Lys Cys Val CCG CTT AGG GPLT GGA GGG CAG TCA TAC AGC GCC A~G CAA A~G CAC ATG 1056 Pro Leu Arg Asp Gly Gly Gln Ser Tyr Ser Ala Lys Gln Lys ~is Met Ser Asp Asp Leu Leu Val Ala Val Val Met Ala ~is Phe Met ALa Thr ALP Asp Arg ~is Met Tyr Lys Pro Ile Ser Pro Gln 370 375 380 .

(2) INFORMATION FO~ SEQ ID NO:5:
(i) SEQUEN OE ~F~5.. r.~ Ll~a:
(A) LENGT : 380 amino acids ~S) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENOE ~L5~lLllU~: SEQ ID NO:5:
Ser Ile Arg Gly Gln Thr Phe Asn Leu Leu Tyr Val Asp Glu Ala Asn Phe Ile Lys Lys Asp Ala Leu Pro Ala Ile Leu Gly Phe Met Leu Gln Lys ASp Ala Lys Leu Ile~Phe Ile Ser Ser Val Asn Ser 5er Asp Arg Ser Thr Ser Phe Leu Leu Asn Leu Arg Asn Ala Gln Glu Lys Met Leu Asn Val Val Ser Tyr Val Cy9 Ala P~p ~is Arg Glu Asp Phe ~is Leu : ~70 75 80 Gln Asp Ala Leu Val Ser Cys Pro Cys Tyr Arg Leu ~is Ile Pro Thr Tyr Ile Thr Ile Asp Glu Ser Ile Lys Thr Thr Thr Asn Leu Phe Met 100 1~5 _ 110 Glu Gly Ala Phe Asp Thr Glu Leu Met Gly Glu Gly Ala Ala Ser Ser 115 1? 0 125 Asn Ala Thr Leu Tyr Arg Val Val Gly Asp Ala Ala Leu Thr Gln Phe Asp Met Cys Arg Val Asp Thr Thr Ala Gln Glu Val Gln Lys Cy6 Leu 145 150_ . . 155_ ..~_ . 160 Gly Lys Gln Leu Phe Val Tyr Ile Asp Pro Ala Tyr Thr Asn Asn Thr Glu Ala Ser Gly Thr Gly Val Gly Ala Val Val Thr 5er Thr Gln Thr lB0 185 190 2 1 96,892 Wo 96/06159 i ' PcrruS95~10194--Pro Thr Arg Ser Leu Ile Leu Gly Met Glu Xis Phe Phe Leu Arg Asp Leu Thr Gly Ala Ala Ala Tyr Glu Ile Ala Ser Cys Ala Cys Thr Met Ile Lys Ala Ile Ala Val Leu His Thr Thr Ile Glu Arg Val Asn Ala ~la Val Glu Gly Asn Ser Ser Glr. Asp Ser Gly Val Ala Ile Ala Thr ~al Leu Asn Glu Ile Cys Pro Leu Pro Ile ~is Phe Leu Xis Tyr Thr 260 265 ~ 270 Asp Lys Ser Ser Ala Leu Gln Trp Pro Ile Tyr Met Leu Gly Gly Glu Lys Ser Ser Ala Phe Glu Thr Phe Ile Tyr Ala Leu Asn Ser Gly Thr Leu ser Ala Ser Gln Thr Val Val Ser Asn Thr Ile Lys Ile ser Phe 305 310 315 3ao Asp Pro Val Thr Tyr Leu Val Glu Gln Val Arg Ala Ile Lys cys yal ~ro Leu Arg Asp Gly Gly Gln Ser Tyr Ser Ala Lys Gln Lys His Met ~er Asp Asp Leu Leu Val Ala Val Val Met Ala Xis Phe Met Ala Thr ~sp Asp Arg Xis Met Tyr Lys Pro Ile Ser Pro Gln ~2~ INFORMATION FO~ SEQ ID NO:6:
(i) SEQUENCE ~ ~T~T~TICS:
A) LENGTH: Z34 base pairs B) TYPE: nucleic acid C) ST~ : single D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) XYPOTXETICAL: N
(iv) ANTI-SENSE: N
(ix) FEATURE:
(A) NAME/~EY: CDS
(B) LOCATION:~ 234 (D) OTXER INFORNATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

ATG GGT GAG CCA GTG GAT CCT GGA CAT GTG GTG AAT GAG A~A GAT TTT 48 Met Gly Glu Pro Val Asp Pro Gly Pis Val Val Asn Glu Lys Asp Phe 1 5 10 ~ 15 Glu Glu Cys Glu Gln Phe Phe Ser Gln Pro Leu Arg Glu Gln Val Val ~ W 096/061~9 21 968 ~2 r~l~L~ s, Ala Gly Val Arg Ala Leu Asp Gly Leu Gly Leu Ala Asp Ser Leu Cys CAC A~A ACA GAA AGA CTC TGC CTG CTG ATG GBC ~TG.GTG.GGC ACG GAG 192 His Lys Thr Glu Arg Leu Cys Leu Leu Met Asp Leu Val Gly Thr Glu TGC TTT GCG AGG GTG TG.C.CGC CTA.GDC iiCC GGT GcG~aAA TGA . 234 Cys Phe Ala Arg Val Cys Arg Leu Asp Thr Gly Ala Lys 65 70 . ~ 75 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE r~D~DrTF~TqTTrc (A) LENGTH: 77 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ~ii) MOLEC~LE TYPE: protein (xi) SEQJEN OE J~18LlUN: SEQ ID NO:7:
Met Gly Glu Pro Val Asp Pro Gly His Val Val Asn Glu Lys Asp Phe Glu Glu Cys Glu Gln Phe Phe Ser Gln Pro Leu Arg Glu Gln Val Val Ala Gly Val Arg Ala Leu Asp Gly Leu Gly Leu Ala Asp Ser Leu Cys His Lys Thr Glu Arg Leu Cys Leu Leu Met Asp Leu Val Gly Thr Glu Cys Phe Ala Arg Val Cys Arg Leu Asp Thr Gly Ala Lys (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQ'UENCE r~D~DrTF7TqTIcs:
A LENGTX: 585 base pairs B TYPE: nucleic acid C sT7D~nRnN~q~q: single .D TOPOLOGY:~linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(ix) FEATURE:
(A) NAME/KBY: CDS
(B) LOCATION: 1..585 (D) OTHER INFORMATION:

(xi) SEQ~ENCE ~1811~N: SEQ ID NO:8:
ATG A~ DrT aTr. r.rr AGT CCC TTA TGT CAG TTC C~E5GGC OE G.TTT.TGC 48 Met Lys Ser Val Ala Se~ Pro ~eu Cys Gln Phe His Gly Val Phe Cys 5 10 ~- 15 CTG TAC CAG TGT CGC ~G TGC CTG GCA TAC CAC GTG TGT.GAT GGG GGC 96 -2 1 96~92 W O96/06159 PCTNS95/10!94 -Leu Tyr Gln Cys Arg Gln Cy8 Leu Ala Tyr His Val Cy9 A6p Gly Gly GCC GAA TGC G~T CTC CTG CAT ACG CCG GAG AGC GTC ATC TGC GAA CTA 144 Ala Glu Cys val Leu Leu His Thr Pro Glu Ser Val Ile Cys Glu Leu Thr Gly Asn Cys Met Leu Gly Asn Ile Gln Glu Gly Gln Phe Leu Gly so SS 60 Pro Val Pro Tyr Arg Thr Leu Asp Asn Gln Yal Asp Arg Asp Ala Tyr Hi6 Gly Met Leu Ala Cys Leu Lys Arg Asp Ile Val Arg Tyr LeY Gln 85 90 . 9S

Thr Trp Pro ArP Thr Thr Val Ile Val Gln Glu Ile Ala Leu Gly Asp Gly Val Thr Asp Thr Ile Ser Ala Ile Ile Asp Glu Thr Phe Gly Glu llS 120 125 Cys Leu Pro Val Leu Gly Glu Ala Gln Gly Gly Tyr Ala Leu Val Cys Ser Met Tyr Leu His Val Ile Val Ser Ile Tyr Ser Thr Lys Thr Val 145 150 lSS 160 TAC AAC AGT ATG CTA TTT AaA TGC ACA AAG AAT AAA AaG TAC GAC TGC 528 Tyr Asn Ser Met Leu Phe Lys Cys Thr Lys A6n Lys Lys Tyr A6p Cys ATT GCC AAG CGG GTG CGG ACA AaA TGG ATG CGC ATG CTA TCA ACG AaA 576 Ile Ala Lys Arg Val Arg Thr Lys Trp Met Arg Met Leu Ser Thr Lys Asp Thr (2) INFORMATION FOE SEQ ID NO:g:
(i) SEQ~ENCE ~R~T.CTICS:
(A) LHNGTH: 194 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECCLE TYPE: protein (xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:g:
~et Lys Ser Val Ala Ser Pro Leu Cys Gln Phe His Gly Val Phe Cys . 15 ~eu Tyr Gln Cys Arg Gln Cys Leu Ala Tyr His Val Cys Asp Gly Gly Ala Glu Cys Val Leu Leu His Thr Pro Glu Ser Val Ile Cys Glu Leu ~ W 096/061~9 2 1 96~q2 PCTrUS9~/10194 Thr Gly Asn Cys Met Leu Gly Asn Ile Gln Glu Gly Gln Phe Leu Gly . 60 Pro Val Pro Tyr Arg Thr Leu Asp Asn Gln Val Asp Arg Asp Ala Tyr Uis Gly Met Leu ~la Cys Leu Lys Arg Asp Ile Val Arg Tyr Leu Gln Thr Trp Pro Asp Thr Thr Val Ile Val Gln Glu Ile Ala Leu Gly Asp Gly Val Thr Asp Thr Ile Ser Ala Ile Ile Asp Glu Thr Phe Gly Glu Cys Leu Pro Val Leu Gl:y Glu Ala Gln Gly Gly Tyr Ala Leu Val Cys 130 ~_L35 140 Ser Met Tyr Leu Xis Val Ile Val Ser Ile Tyr Ser Thr Lys Thr Val Tyr Asn Ser Met Leu Phe Lys Cys Thr Lys Asn Lys Lys Tyr Asp Cys Ile Ala Lys Arg Val Arg Thr Lys Trp Met Arg Met Leu Ser Thr Lys Asp Thr (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQ-JENCE r~D~T~T~TICS:
(A LENGT~: 939 base pairs (B TYPE: nucleic acid (C ST~ : sir,gle (D TOPOLOGY: linear (ii) MOLECaLE TYPE. DNA (genomic) (iii) ~Y~Ul~rLl AL: N
(iv) ANTI-SENSE: N
(ix~ PEATURE:
~A~ NAME/~EY. CDS
(B) LOCATION: 1..939 (D) OT~ER INPORMATION:

(xi) SEQ~ENOE U~C~l~llUN: SEQ ID NO:10:
ATG GCT AGC CGG AGG CGC A~A CTT CGG A~T TTC CTA A~C AAG GAA TGC 48 Met Ala Ser Arg Arg Arg Lys Leu Arg A5n Phe Leu Asn Lys Glu Cys ATA TGG ACT GTT A~C CC~ ~TG TCA GGG GAC CAT ATC A~G GTC TTT ~AC 96 Ile Trp Thr Val Asn Pro Met Ser Gly Asp ~is Ile Lys Val Phe Asn GCC TGC ACC TCT ATC TCG CCG GTG TAT GAC CCT. GAG CTG GTA ACC AGC 144 Ala Cys Thr Ser Ile Ser Pro Val Tyr Asp Pro Glu Leu Val Thr Ser TAC GCA CTG ~C GTG CCT GCT TAC A~T.GTG TCT GTG GCT ~TC TTG CTG 192 Tyr Ala Leu Ser Val Pro Ala Tyr Asn Val Ser Val Ala Ile Leu Leu . _~ . _ . . _.... . ... ... . _ . . __ ___ __ ___ ___ _ _ WO96/06159 2 1 9 ~ 8 9 2. . PCT~US95/1019 ~

~is Lys Val Met Gly Pro Cys Val Ala Val Gly Ile Asn Gly Glu Met ATC ATG TAC GTC GTA AGC CAG TGT GTT TCT GTG CGG CCÇ GTC CCG GGG 28B
Ile Met Tyr Val Val Ser Gln Cys Val Ser Val Arg Pro Val Pro Gly Arg Asp Gly Met Ala Leu Ile Tyr Phe Gly Gln Phe Leu GlU Glu Ala TCC GGA CTG AGA TTT CCC TAC ATT GCT CCG CCG CCG T~G CGC GAA CAC 384 Ser Gly Leu Arg Phe Pro Tyr Ile Ala Pro Pro Pro Ser Arg Glu ~is~
llS ~ 120 125 GTA CCT GAC CTG ACC AGA CAA GAA TTA GTT CAT A-C TC_ CAG GTG GTG 432 Val Pro Asp Leu Thr Arg Gln Glu Leu Val Xis Thr Se- Gln Val Val CGC CGC GGC GAC CTG ACC ~AT TGC ACT ATG GGT CTC GAA TTC AGG AAT 480 Arg Arg Gly Asp Leu Thr Asn Cys Thr Met Gly Leu Glu Phe Arg Asn 145 150 lSS = 160 GTG AAC CCT TTT GTT TGG CTC GGG GGC GGA TCG GTG TGG CTG CTG TTC = 523 Val Asn Pro Phe Val Trp Leu Gly Gly Gly Ser Val Trp Leu Leu Phe Leu Gly Val Asp Tyr Met Ala Phe Cys Pro Gly Val Asp Gly Met Pro 180 185 l9O

Ser Leu Ala Arg Val Ala Ala Leu Leu Thr Arg Cys ASp ~is Pro ASp TGT GTC CAC TGC CAT GGA CTC CGT GGA CAC GTT AAT GTA TTT CGT:GGG 672 Cys Val ~is Cys ~is Gly Leu Arg Gly ~is Val Asn Val Phe Arg,Gly Tyr CYS Ser Ala Gln Ser Pro Gly Leu Ser Asn Ile Cys PrQ Cys Ile 22s ~ 230 2is 2~0 AAA TCA TGT GGG ~CC GGG AAT GGA GTG ACT AGG GTC ACT-GGA AAC AGA 768 Lvs Ser Cys Gly Thr Gly Asn Gly Val Thr Arg Val Thr Gly Asn Arg AAT T~T CTG GGT CTT CTG TTC GAT CCC ~TT GTC CAG AGC AGG GTA ACA 816 Asn Phe Leu Gly Leu Leu Phe Asp Pro Ile Val Gln Se- Arg Val Thr 260 265 ~270 GCT CTG AAG ATA ACT AGC CAC CCA ACC CCC AcG CAC GTC GAG AAT GTG 864 Ala Leu Lvs Ile Thr Ser ~is Pro Thr Pro Thr ~is V~l Glu Asn Val Leu Thr Gly Val Leu Asp Asp Gly Thr Leu Val Pro Se~ Val Gln Gly 290 29s 300 ACC CTG GGT CCT CTT ACG ~AT GT. TGA 939 Th- Leu Gly Pro Leu Thr Asn Val ~2) INFORMATION FOR SSQ ID NO 11 2'1 q6892 ~ WO 96106159 PCI'IUS95/l0~9 ~0:~
(i) SE01~ENCE r~ rTF~T.~TICS:
(A) LENGTH: 312 amino acids (9) TYPE: amino a~id r (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: protein ~xi) SEQ-OENCE L)~::lw~LL~L~ SEQ ID NO:11:
Met Ala Ser ~rg }Irg Arg Lys Leu Arg Asn Phe Leu Asn Lys Glu Cys Ile Trp Th~ Va~ Asn Pro Met Ser Gly Asp His Ile Lys Val Phe Asn ~la Cys Thr Ser Ile Ser Pro Val Tyr Asp Pro Glu Leu Val Thr Ser 9.5 Tyr ALa Leu Ser Val Pro Ala Tyr Asn Val Ser Val Ala Ile Leu Leu : 55 60 His Lys Val Met Gly Pro Cys Val Ala Val Gly Ile Asn Gly Glu Met 6s 70 75 : 80 Ile Met Tyr Val Val Ser Gln Cys Val Ser Val Ars Pro Val Pro Gly Arg Asp Gly Met Ala Leu Ile Tyr Phe Gly Gln Phe Leu Glu Glu Ala 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 Gln Glu Leu Val His Thr Ser Gln Val Val Arg Arg Gly Asp Leu Thr Asn Cys Thr Met Gly Leu Glu Phe Arg Asn Val Asn Pro Phe Val Trp Leu Gly Gly GIy Ser VaI Trp Leu Leu Phe Leu Gly Val Asp Tyr Met Ala Phe Cys Pro Gly Val Asp Gly Met Pro 180 185 l9O
Ser Leu Ala Arg Val Ala Ala Leu Leu Thr Arg Cys Asp His Pro Asp 195 :: Z00 : 205 Cys Val Xis Cys Xis Gly Leu Arg Gly Xis Val Asn Val Phe Arg Gly Tyr Cys Ser Ala Gln Ser Pro Gly Leu Ser Asr. Ile Cys Pro Cys Ile 225 230~ 235 240 Lys Ser Cys Gly Thr Gly Asn Gly Val Thr Arg Val Th- Gly Asn Arg 245 250 : 255 Asn Phe Leu Gly Leu Leu Phe Asp Pro Ile Val Gln Ser Arg Val Thr Ala Leu Lys Ile Thr Ser His Pro Thr Pro Thr His Val Glu Asn Val Leu Thr Gly Val Leu Asp Asp Gly Thr Leu Val Pro Ser Val Gln Gly Thr Leu Gly Pro Leu Thr Asn Val ''21 96892 W096/06l59 PCTrUS95/l0l9 30s 310 (2) INFORMATION FOR SEQ ID NO:12: G
(i) SEQUENCE rM~v~TF~TcTIcs~
(A) LENGT~: 86 base palrs (B) TYPE: nuclei- acid (C) STv7~ Fn~c: single (D) TOPOLOGY: lirear (ii) MOLECULE TYPE: DNA (genomir) (iii) MYPOTEETICAL: N
(iv) ANTI-SENSE: N
(ix) FEATUP~E:
(A) NAME/REY: CDS
(B) LOCATION: 1..86 (D) OTFEP. INFORMATION:

(xi) SEQUENCE ~C~l~llUN: SEQ ID NO:12: ~=
ATG GAC TCA ACC AAC TCT AAA AGA GAG TTT ATT AAG TCG GC~ CTG GAG 48 Met Asp Ser Thr Asn Ser Lys Arg Glu Phe Ile Lys Ser Ala Leu Glu GCC Aac ATC A~C AGG AGG GCA GCT GTA TCG CTA TTT GA , 86 Ala Asr. Ile Asn Arg Arg Ala Ala Val Ser Leu Phe (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CMU~ACTERISTICS:
(A) LENGT,M: 28 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (il) MOLECULE TYPE: protein (Y.i) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Asp Ser Th- Asn Ser Lys Arg Glu Phe Ile Lvs Ser Ala Leu Glu 5 . 10 1 Ala Asn Ile Asr. Ary Arg Ala Ala Val Ser Leu Phe ~~) INFORMATION FOR SEQ ID NO:14:
(_~ SEQUENCE rM~v~rT~TcTIcs:
~A) LENGT~: 1743 base pairs tB) TYPE: nucleic acid ~C) ST~N~T'nNFC~: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~u~~ll~AL: N

~ W O 96/06159 2 1 9 6 8 9 2 PCTrUS9~ll019~

(iv~ ANTI-SENSE: N
(ix) EEATURE:
J (A) NAME/REV. CDS
(B) LOCATION. 11743 (D) OTBER INFORMATION:

(xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:14:

Met Ala Glu &ly Gly Phe Gly Ala Asp Ser Val Gly Arg Gly Gly Glu AAG GCC TC~m GTG ACT.AGG.GG~.GGC AGG TGG G~C TTG GGG AGC TCG GAC 9Ç
Lys Ala Ser Val Thr Ar~ Gly Gly Arg Trp Asp Leu Gly Ser Ser Asp GAC GaA TCA AGC.~CC TCC ACA ACC AGC ACG G~T Am,G GAC GAC CTC CCT 144 Asp Glu Ser 5er~Thr Ser T~r Thr Ser Thr Asp Met Asp Asp Leu Pro Glu Glu A_g Lys Pro Leu Thr Gly Lys Ser Val Lys Thr Ser Tyr Ile 50 SS . , 6Q _.
TAC GAC GTG rrr ~rr rmr CCG ~rr ~rr ~ CCG TGG r~m TTA ATG C~C 240 Tyr Asp Val Pro Thr Val Pro Thr Ser Lys Pro Trp Uis Leu Met ~is 65 . ~70 = 75 =~ ~.= 80 GAC AAC TCC CTC T r G~A 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 CGG CAC CCT TCC G~A AAA GGC AGC ~TT lTT GCC ~GT CGG TTG T~A GCG 336 Arg Uis Pro Ser Glu Lys Gly Ser Ile Phe Ala Ser Arg Leu Ser Ala lQQ 105 .. ~ ~ 110 ACT GAC GAC GAC TCG Gca GAC ~AC GCG CCA ATG GA~ CGC TTC GCC.TTC 384 Thr Asp Asp Asp Ser Gly Asp Tyr Ala Pro Met Asp Arg Phe Ala Phe llS 12Q 125 CAG AGC CCC AGG GTG TGT GGT CGC CCT CCC CTT CCG CCT CCA AAT CAC 43' Gln Ser Pro Arg Val Cys Gly Arg Pro Pro Leu Pro Pro Pro Asn Bis 13~ = - 135 ,~ l~Q~
CCA CCT CCG GCa ACT AGG CCG GCA GaC GCG TC~ ~TG GGG GAC GTG GGC 480 Pro Pro Pr~ ~la Thr Arg Pro Ala Asp Ala Ser Me~ Gly Asp Val Gly 145 lSQ lSS =~ ~160 TGG GCG GAT r~r. rAr~ rr.~ CTC AAG AGG ACC CCA AAG GGA TTT TTA AAA 528 Tro Ala Asp Leu Gln Gly Leu Lys Arg Thr Pro Lys Gly Phe Leu Lys ACA TCT ACC AAG GGG GGC AGT CTC.AA~ GCC CGT GGA CGC G~T GTA GGT 576 Thr Ser Thr Lys Gly Gly Ser Leu Lys Ala Arg G~y Arg Asp Val Gly Asp Arg Leu Arg Asp Gly Gly Phe Ala Phe Ser Pro Arg Glv Val Lys l9S : 200 2QS
TCT GCC ATA GGG CA~ AAC ATT AAA TCA TGG TTG GGG ATC.GGA GAA TCA 672 Ser ALa Ile ~Gly Gln Asn Ile Lys Ser Trp Leu Gly Ile Gly Glu Ser TCG GCG ACT GCT ~TC CCC GTC ACC ACG CaG CTT ATG GTA CCG GTG CAC 720 WO 96/06159 PZ~rlUS9511019 Ser Ala Thr Ala Val Pro val Thr Thr Gln Leu Met Val Pro Val iqis CTC ~TT AGA ACG CCT GTG ACC GTG GAC TAC AGG AAT GTT TAT TTG CTT 768 s Leu Ile Arg Thr Pro Val Thr Val Asp Tyr Arg Asn Val Tyr Leu Leu TAC TTA GAG GGG GTA ATG GGT GTG GGC A~A TCA ACG CTG GTC AAC ~GCC ~ 816 Tyr Leu Glu Gly Val Met Gly Val Gly Lys Ser Thr Leu Val Asn Ala GTG TaC GGG ATC TTG CCC CAG~ GAG AGA GTG ~ACA AGT TTT= CCC GAG CCC ; 864 Val Cys Gly Ile Leu Pro Gln Gl~u Arg Val ,~Thr Ser Phe Pro Glu Pro 275 ~ 280 ' 285 Met Val Tyr Trp Thr Arg Ala Phe Thr Asp Cys Tyr Lys Glu Ile Ser CAC CTG ATG A;~G TCT GGT AAG GCG GGA GAC -CCG CTG ACG TCT GCC ~A 960 iqis Leu Met Lys Ser Gly ~ys Ala Gly Asp Pro Leu Thr Ser Ala Lys 30s ~ 31C ~ 315 ' ' '' ' '320 ATA TAC TCA TGC CAA AAC AAG TTT TCG CTC CCC TTC CGG ACG A~C GCC 1008 Ile Ty- Ser Cys Gln Asn Lys Phe Ser ,Leu Pro Phe Arg Thr Asn Ala Thr Ala Ile Leu Arg Met Met G1n Pro Trp Asn Val Gly Gly Gly Ser GGG AGG GGC A''T CAC TGG TGC GTC TTT GAT AGG CAT CTC CTC TCC CCA 1104 Gly Arg Gly Thr i is Trp Cys Val Phe Asp Arg iqis Leu Leu Ser Pro 35s 360 365 GCA GTG GTG TTC CCT CTC l~TG CAC CTG A~G CAC GGC CGC CTA TCT TTT 1152 Ala Val Val Phne Pro Leu Met qis Leu Lys llis Gly Arg Leu Ser Phe GAT CAC TTC TTT Ci.A T~ CTT TCC ATC TTT AGA GCC ACA GA~ GGC GAC 1200 Asp q Zs Phe Phe Gln Leu Leu Ser Ile Phe Arg Ala Thr Glu Gly Asp 38~ 390 395 400 GTG G C GCC ATT CTC ACC: CTC TCC ~GC GCC ~ZG TCG TTG CGG CGG GTC 1248 Val Val Ala Ile Leu Thr Leu Ser Ser Ala Glu Ser Leu Arg Arg Val 40s 410 415 AGG G''G AGG GGA AGA A;Z.ZG AAC Gl C GGG ACG GTG GAG C~ AAC TAC ATC 1296 Arg Ala A-g Gly Arg Lys Asn Asp Gly Thr Val Glu Gln Asn Tyr Ile AGA GAA TTG GCG TGG GCT TAT ~AC GCC GTG TAC TGT TCA TGG ATC ATG 1344 Arg Glu Leu Ala Trp Ala Tyr Xis Ala Val ~ r Cys Ser Trp Ile Met TTG CAG TAC A--C ACT GTG GAG C~G ATG GTA CAA CTA TGC GTA CAA ACC ~ 1392Leu Gln Tyr Ile Thr Val Glu Gln Met Val Glr. Leu Cys Val Gin Thr ACA AAT ATT CCG GAA ATC TGCTTC CGC AGC GTG CG" CTG GC~ CAC AAG -1440 Thr Asn Ile Pro Glu Ile Cys Phe Arg Ser yal Arg Leu Ala His Lys 465 4?0 475 480 GAG GAA ACT TTG AAA AAC CTT CAC GAG CAG AGC ATG CTA CCT ATG ATC : 1488Glu Glu Thr Leu Lys Asn Leu Pis Glu Gln Ser Met Leu Pro Met Ile ~ W 096/06159 2 1 9 6 8 ~ 2 PCTAUS9~1019~

ACC GGT GTA CTG GAT CCC GTG AGA CAT CaT CCC GTC GTG ATC GAG CTT 1536 Thr Gly Val Leu Asp Pro Val Arg UiS uis Pro Val Val Ile Glu Leu TGC TTT TGT TTC TTr ~r~ CTG ~GA lUU~ TTA CAA TTT ATC GTA GCC 1584 Cys Phe Cys Phe Phe Thr Glu Leu Arg Lys Leu Gln Phe Ile Val Ala 515 : 520 525 GAC GCG GAT AAG TTC CAC GAC G~C GTA TGC GGC CTG TGG ACC GAA ATC 1632 Asp Ala Asp Lys Phe ,uis Asp Asp Val Cys Gly Leu Trp Thr Glu Ile 530 535 : 54n TAC AGG C~ ATC.-CTG ~CC AAT CCG GCT ATT A~A CCC AGG GCC ATC AAC 16a0 Tyr Arg Gln Ile Leu Ser Asn Pro Ala Ile Lys Fro Arg~Ala Ile Asn 545 _55n _ 555 ,: . .560 TGG CCA GCA TTA GAG AGC CAG TCT A~A GCA GTT AAT CAC CTA GAG GAG 172a Trp Pro Ala Leu Glu Ser Gln Ser Lys Ala Val Asn uis Leu Glu Glu Thr Cys Arg Val sao ~2) INFORMATION FOR SEQ ID NO:15:
~i~ SEQ~ENCE ru~rT~TqTIcs ~A) LENGT5: 580 amino 7cids ~3) TYPE: amino acid iD) TOPOLOGY: linear ~ii) MOLEC~LE TYPE: protein ~Xi) SEQ~EN-OE J~OKl~l~N: SEQ ID NO:15:
Met Ala Glu Gly Gly Phe Gly Ala Asp Ser Val Gly Arg Gly Gly Glu 5 ~ 10 15 Lys Ala Ser Val Thr Arg Gly Gly Arg Trp Asp Leu Gly Ser Ser Asp ~ 25 30 Asp Glu Ser 5e~ Thr Ser Thr Thr Ser Thr Asp Met Asp Asp Leu Pro Glu Glu Ars Lys Pro Leu Thr Gly Lys Ser Val Lys Thr Ser Tyr Ile Tyr Asp Val Pro Thr Val Pro Thr Ser Lys Pro Trp uis Leu Met uiS
~ D=~ 75 ~ ,~ , 80 Asp Asn Ser Leu Tyr Ala Thr Pro Arg Phe Pro Pro Arg Pro Leu Ile Arg Uis Pro Ser Glu ~s Gly Ser Ile Phe Ala Ser Arg ~eu Se_ Ala 100 iDs 110 Thr Asp Asp Asp Ser Gly Asp Tyr Ala Pro Met Asp Arg Phe Ala Phe Gln Ser Pro Arg Val Cys Gly Arg Pro Pro Leu Pro Pro Pro Asn uiS
130 : 135 140 Pro Pro Pro Ala Thr Arg Pro Ala Asp Ala Ser Met Gly Asp Val Gly Trp Ala Asp Leu Gln Gly Leu Lys Arg Thr Pro Lys Gly Phe Leu Lys WO96106159 ' 21 96892 PCTIUS95/10191--Thr Ser Thr Lys Gly Gly Ser Leu Lys Ala Arg Gly Arg Asp Val Gly 180 185 190 s Asp Arg Leu Arg Asp Gly Gly Phe Ala Phe Ser Pro Al g Gly Val Lys lg5 Z00 205 Ser Ala Ile Gly Gln Asn Ile Lys Ser Trp Leu Gly Ile Gly Glu Ser 21~ 215 220 Ser Ala Thr Ala Val Pro Val ThI Thr Gln Leu Met Val Pro Val His 225 ~ 230 = 235 240 ~eu Ile Arg Thr Pro Val Thr Val Asp Tyr Arg A9n Val Tyr Leu Leu ~yr Leu Glu Gly Val Met Gly Val Gly Lys Ser Thr Leu Val Asn Ala 2~0 265 2io ~al Cy~ Gly Ile Leu Pro Gln Glu Arg Val Thr Ser Phe Pro Glu Pro Met Val Tyr Trp Thr Arg Ala Phe Thr Asp Cy5 Tvr Lys Glu Ile Ser 290 295 . 300 His Leu Met Lys Ser Gly Lys Ala Gly Asp Pro Leu Thr Ser Ala Lys 305 ~ 31~) 315 ~ 320 ~le Tyr ser Cys Gln Asn Lys Phe Ser Leu Pro Phe Arg Thr Asn Ala ~hr Ala Ile Leu Arg Met Met Gln Pro Trp Asn val Gly Gly Gly Ser. ,.

~ly Arg Gly Thr His T==rp Cys Val Phe Asp Arg His Leu Leu Ser Pro 355 . 360 365 Ala Val val Phe Pro Leu Met His Leu LYL His Gly Arg Leu Ser Phe 370 375 ~ 380 Asp His Phe Phe Gln Leu Leu Ser Ile Phe Arg Ala Thr Glu Gly Asp 385 390 395 ~ 400 ~al Val Ala Ile LeU Thr Leu Ser Ser Ala Glu 5er Leu Arg P.rg Val ~rg Ala Arg Gly Arg Lys Asn Asp Gly Thr Val Glu Gln Asn Tyr Ile ~rg Glu Leu Ala Trp Ala Tyr His Ala Val Tyr Cys Ser Trp Ile Met Leu Gln Tyr Ile Thr Val Glu Gln Met Val Gln Leu Cys Val Gln Thr Th- As-. Ile Pro Glu Ile Cys Phe Arg Ser Val Arg Leu Ala His Lys ~lu Glu Thr Leu Lys Asn Leu His Glu Gln Ser Met Leu Pro Met Ile ~hr Gly Val Leu Asp Pro Val Arg His His Pro Val Val Ile Glu Leu ~ys Phe Cys Phe Phe Thr Glu Leu Arg Lys Leu Gln Phe Ile Val Ala 515 5Z0 52;

2-1 9~89~
~ W O96/06159 _ , ' PCT~US9S/I0l9~
. .

Asp Ala Asp Lys Phe His Asp Asp Val Cys Gly Leu Trp Thr Glu Ile 530 ~35 540 t Tyr Arg Gln Ile Leu Se~r.Asn Pro Ala Ile Lys Pro Arg Ala Ile Asn 545 ~ 55,=4 ~ , _ 555 = 560 Trp Pro Ala Leu Glu Ser Gln Ser Lys Ala Val Asn His Leu Glu Glu 565 .' . 570 S~S
Thr Cys Arg Val SSO
t2~ INFORMATION FOR SEQ ID NO:16:
(i~ SECf~ENCE rTTpRprTFR~.cTIcs ~A' LENGT~.: 2193 base pairs B TYPE: nucleic acid C STR~nrAnN~.~c single Dl TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genoPPic) (iii) HYPOTHETICAL: N . _ (iv) ANTI-SENSE: N
(ix~ FEATURE:
(A) NAME/~EY: CDS
(B1 LOCATION: 1.,2193 (D) OTHER INFORMATION:

(xi) SEri~ENOE ~SU~lrll~N: SEO ID NO:16:

Me~ Gln Gly Leu Ala Phe Leu Ala Ala Leu Ala Cys Trp Arg Cys Ile 1 5 = ~ 10 , lS
TCG TTG ACA TGT QrA-Gcc ACT QGC 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 20 25 , 30 ATA ACC CGC TCC QC_ ACQ CAG CTC ATC A~T GQG AQA ACC AAC CTC TCC 144 Ile Thr Arg Ser ~la ThI Ql~ Leu Ile Asn Gly Arg Thr Asn Leu Ser ATA GAA CTG GAA TTC A~C GGC ACT AGT TTT TTT,CTA AA~ TGG ca~ AAT 192 Ile Glu Leu Glu Phe Asn Gly Thr Ser Phe Phe Leu Asn Trp Gln Asn 50 55 60 ~
CTG TTG AAT GTG ATr ACG GAG CCQ GCC CTG ACA QAG TTG TGG ACC.TCC 24C
Leu Leu Asn Val Ile Thr Glu Pro Ala Leu Thr Glu Leu Trp Thr Ser 65 : 70 ~ 75 , 80 GCC GA~ GT. r,rr r~r Rpr CTC AGG GTA ACT CTG AAA AA- AGG CAA AGT 28B
- Ala Glu Val Ala Glu Asp Leu Arg Val Thr Leu Lys Lys Arg Gln Ser c 85 90 95 CTT TTT ~TC rrr ~r ~Pr ACA GTT GTG ATC TCT GGA ~A_ GG_ CAT CGC 336 Leu Phe Phe Pro Asr, Lys Th- Val Val Ile Ser Gly Asp Gly Eis Arg 100 105 ~ 110 .:
TAT ACG TGC QAG GTG CCG ACG TCG TCG C~A ACT.TAT A~C AT_ ACC A~G 384 Tyr Thr rys Glu Val Pro Thr Ser Ser Gln Thr Tyr Asn Ile Thr Lys llS 129 125 GGC TTT Aa TAT AGC GCT CTG CCC GGG CAC CTT GGC GGA TTT GGG ATC 43~

. W 096/06l59 2 i 9 6 8 9 2 PCTNS951101~ ~

Gly Phe Asn Tyr Ser Ala Leu Pro Gly His Leu Gly Gly Phe Gly Iie AAC GCG CGT CTG GTA CTG GGT GAT ATC TTr GCA TCA.3~Ll TGG TCG CTA 480 Asn Ala Arg Leu Val Leu Gly Asp Ile Phe Ala Ser Lys Trp Ser Leu 145 150 :155 160 TTC GCG AGG GAC ACC CCA GAG TAT CGG GTG TTT TAC CCA ATG AAT GTC = 528 165 170 ~ - 175 ATG GCC GTC AAG TTT TCC ATA TCC ATT GGC:AAC AAC GAG TCC GGC:GTA _ ~76 Mee Ala Val Lys Phe Ser Ile Ser Ile Gly Asn Asn Glu~Ser Gly~Val GCG CTC TAT GGA GTG GTG TCG GAA GAT TTC,GTG GTC GTC ACG CTC-CAC 624 Ala Leu Tyr Gly Val Val Ser Glu Asp Phe Val Val Val Thr Leu His 195 20D 20s AAC AGG TCC A~A GAG GCT AAC GAG ACG GCG TCC CAT CTT CTG TTC GGT 67 Asn Arg Ser,Lys Glu Ala Asn Glu Thr Ala Ser His Leu Leu Phe Gly CTC CCG GAT TCA CTG CCA.TCT CTG AAG GGC CAT GCC ACC T~T GAT GAA 72D
Leu Pro Asp Ser Leu Pro Ser Leu Lys Gly His Ala Thr Tyr Asp Glu 225 23~ ._--23~ 240 CTC ACG TTC GCC CGA AAC GCA A~A TAT GCG CTA GTG GCG ATC CTG CCT 768 Leu Thr Phe Ala Arg Asn Ala Lys Tyr Ala Leu Val Ala Ile Leu Pro Lys Asp Ser Tyr Gln Thr Leu Leu Thr Glu Asn Tyr Thr Arg Ile Phe CTG AAC ATG ACG GAG TCG ACG CCC CTC GAG TTC ACG CGG ACG ~TC_CAG 864 Leu Asn Met Thr Glu ser Thr Pro Leu Glu Phe Thr Arg Thr Ile~Gln ACC AGG ATC GTA TCA ATC GAG GCC AGG CGC GCC TGC GCA GCT CAA GAG 91' Thr Arg Ile Val Ser Ile Glu Ala Arg Arg Ala Cys Ala Ala Gln Glu 29Q ~ 295 .~ 300 GCG GCG CCG GAC ATA TTC TTG GTG TTG,TTT CAG ATG TTG GTG GCA CAC 960 Ala Ala Pro Asp Ile Phe Leu Val Leu Phe Gln Met Leu Val Ala His 305 310 315 :320 TTT CTT GTT GCG CGG GGC:.ATT GCC,GAG CAC CGA TTT GTG GAG GTG GAC ~1008 Phe Leu Val Ala Arg Gly Ile Ala Glu His Arg Phe Val Glu Val Asp 325 330 = 335 ~

Cys Val Cys Arg Gln Tyr Ala Glu Leu Tyr Phe Leu Arg Arg Ile Ser 340 345 35~

A-g Leu Cys Met Pro Thr Phe ~hr Thr Val Gly Tyr Asn Hi5 Thr:Thr CTT GGC GCT GTG GCC GCC ACA ~AA ATA GCT CGC GTG TCC GCC ACG ~AG 1152 Leu Gly Ala Val Ala Ala Thr Gln Ile Ala Arg Val Ser Ala Thr Lys TTG GCC AGT TTG CCC CGC TCT TCC CAG GAA ACA GTG CTG GCC ATO GTC . 1200 Leu Ala Ser Leu Pro Arg Ser Ser Gln Glu Thr Val Leu Ala Met Val ~ W 096/06159 ~ PCT~U59511019~
~ .;

CAG CTT GGC GCC CGT GAT GGC GCC GTC CCT TCC TCC A~T CTG GAG GGC 1248 Gln Leu Gly Ala Arg Asp Gly Ala Val Pro Ser Ser Ile Leu Glu Gly 40s 410 415 ATT GCT ATG GTC GTC GAA CAT ATG TAT ACC GCC TAC ACT TAT GTG TAC 12g6 Ile Ala Met Val Val Glu His Met Tyr Thr Ala Tyr Thr Tyr Val Tyr Thr Leu Gly Asp Thr Glu Arg Lys Leu Met Leu Asp Ile His Thr Val 435 440 44;
CTC ACC GAC AGC TGC CCG CCC AAa GAC TCC GGA GTA TCA GAA A~G CTA 13g2 Leu Thr Asp Ser Cys Pro Pro Lys Asp Ser Gly Val Ser Glu Lys Leu CTG AGA ACA TA~ TTG ~TG TTC ACA TCA ATG TGT ACC A~. ATA GAG CTG 1440 Leu Arg Thr Tyr Leu Met Phe Thr Ser Met Cys Thr Asr. Ile Glu Leu 465 . 470 g75 480 GGC GAA ATG ATC GCC CGC TTT TCC AaA CCG GAC AGC CTT AAC ATC TAT 1488 Gly Glu Met Ile Ala Arg Phe Ser Lys Pro Asp Ser Leu Asn Ile Tyr 485 4gO 4gS
AGG GCA TTC TCC CCC TGC TTT CTA GGA CTA AGG TAC GA~ TTG CAT CCA 1536 Arg Ala Phe Ser Pro Cys Phe Leu Gly Leu Arg Tyr Asp Leu His Pro Ala Lys Leu Arg Ala Glu Ala Pro Gln ser Ser Ala Leu Tkr Arg Thr Ala Val Ala Arg Gly Thr Ser Gly Phe Ala Glu Leu Leu His Ala Leu CAC CTC GAT AGC TTA AAT TTA ATT CCG GCG ATT AAC TGT TCA A~G ATT 1680 His Leu Asp Ser Leu Asn Leu Ile Pro Ala Ile Asn Cys Ser Lys Ile 545 = ~ = . = 550 555 - 560 AC~ G~C GAC AAG ATA ATA GCT ACG GT~ CCC TTG CCT CAC GTC ACG TAT 1728 Thr Ala Asp Lys Ile lle Ala Thr Val Pro Leu Pro His Val Thr Tyr ATC ATC AGT TCC ~AA GCA CTC TCG ~AC GCT GTT GTC TAC GAG GTG TCG 1776 Ile Ile S-r ~er ~lu Ala Leu Ser Asn Ala Val Val Tyr G~u Val Se~
580 585 s90 G;u Ile Phe Leu Lys Ser ~la Met Phe Ile Ser Ala Ile Lys Pro Asp 5g5 600 _ 605 Cys Ser Gly Phe Asn Phe Ser Gln Ile Asp Arg His Ile Pro Ile Val TA- AA~ ~TC AGC ACA CCA AGA AGA GGT TGC CCC CTT TGT GA~ TC" GTA lg20 Tyr Asn Ile Ser Thr Pro Arg Arg Gly Cys Pro~ Leu CYS Asp Ser Val 6~5 . : 6~ _ _ : 63 _ : _ 640 .

ATC ATG AGC TAC GAT GAG AGC GAT GGC CTG CAG TCT C~ ~TG T ~ GTC lg68 Ile Me~ Ser Tyr Asp Glu Ser Asp Gly Leu Gln Ser Leu Met Tyr Val ACT AAT GAA AGG GTG CAG ACC A~C CTC TTT TTA GAT AAG TCA CCT TTC 2016 Thr Asn Glu Arg Val GIn Thr Asn Leu Phe Leu Asp Lys Ser Pro Phe ~ ~ G ~
WO 96/06159 2 ~ 9 6 8 9 2 PCTrUS95/1019 ~

21~
TTT GAT AAT DDr pD~ rTD CPc ~TT CAT T~T TTG TGG CTG AGG GAC AAC 2064 Phe As~ Asn Asn Asn Leu His Ile His Tyr Leu Trp Leu Arg Asp Asn GGG ACC GTA GTG ~AG ATA AGG GGC ATG TAT AGA AGA CGC GC~ GCC AGT 2112 Gly Thr Val Val Glu Ile Arg Gly Met Tyr Arg Arg Arg Ala Ala Ser 690 69~ ~ 70Q
GCT TTG TTT CTA ATT CTC TCT:TTT ATT GGG TTC TCG GGG GTT ATC TAC - 216QAla Leu Phe Leu Ile Leu Ser Phe Ile Gly Phe Ser Gly Val Ile Tyr TTT CTT IAC AGA CTG TTT TCC ATC CTT TAT TAG ~ 2193 Phe Leu Tyr Arg Leu Phe Ser Ile Leu TYr ~2) INFORMATION FOR SEQ ID NO:17:
(i~ SEQQENCE CHARACTERISTICS:
~A) LENGT~: 73Q amino acids ~B~ TYPE: amino acid tD) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi~ SEQUENOE DESCRIPTION: SEQ ID NO:17:
Met Gln Gly Leu Ala Phe Leu Ala Ala Leu Ala Cys Trp Prg Cys Ile 1 S lQ lS
~er Leu Thr Cys Gly Ala Thr Gly Ala Leu Pro Thr Thr Ala Thr Thr Ile Thr Arg Ser Ala Thr Gln Leu Ile Asn Gly Arg Thr Asn Leu Ser Ile Glu Leu Glu Phe Asn Gly Thr Ser Phe Phe Leu Asn Trp Gln Asn SS - -. 60 Leu Leu Asn Val Ile Thr Glu Pro Ala Leu Thr Glu Leu Trp Thr Ser ,~

~la Glu Val Ala Glu Asp Leu Arg Val Thr Leu Lys Lys Arg Gln Ser ~eu Phe Phe Pro Asn Lys Thr Val Val Ile Ser Gly Asp Gly His Arg lQQ lQS llQ
Tyr Thr Cys Glu Val Pro Thr Ser Ser Gln Thr Tyr Asn Ile Thr Lys llS =~= 720 125 Gly Phe Asn Tyr Ser Ala Leu Pro Gly Pis Leu Gly Gly Phe Gly Ile Asn Ala Arg Leu Val Leu Gly Asp Ile Phe Ala Ser Lys Trp Ser Leu 145 lSQ . . lSS ~ ~, ..................................... 160 _ ~he Ala Arg Asp Thr Pro Glu Tyr Arg Val Phe Tyr Pro Met Asn Val ~et Ala Val Lys Phe Ser Ile Ser Ile Gly Asn Asn Glu Ser Gly Val 18Q lES l9Q
~la Leu Tyr Gly Val Val Ser Glu Asp Phe Val Val Val Thr Leu His l9S 200 205 ~ WO96106159 ~ 2~ 968 92 PCI'IUS95/lOl9-1 A6n Arg Ser Lys Glu Ala Asn Glu Thr Ala Ser ~;is Leu Leu Phe Gly Leu Pro Asp Ser Leu Pro Ser Leu Lys Gly Pis Ala Thr T~rr Asp Glu 2z5 ~230 235 ~ a40 ~eu Thr Phe Ala Arg Asn Ala Lys Tyr Ala Leu Val Ala Ile Leu Pro 245 - 250 2s5 ~ys Asp Ser Tyr Gln Thr Leu Leu Thr Glu Asn Tyr Thr Arg Ile Phe Leu Asn Met Thr Glu Ser D~r Pro Leu Glu Phe Thr Arg Thr Ile Gln Thr Arg Ile Val Ser Ile Glu Ala Arg Arg Ala Cys Ala Ala Gln Glu 290 ~ 295~ 300 Ala Ala Pro Asp Ile Phe Leu Val Leu Phe Gln Met Leu Val Ala Hls 305 3~0 315 320 ~he Leu Val Ala Arg Gly Ile Ala Glu Pis Arg Phe Val Glu Val Asp ~ys Val Cys Arg Gln Tyr Ala Glu Leu Tyr Phe Leu Arg Arg Ilc Ser 340 ~ 345 35D
Arg Leu Cys Met Pro Thr Phe Thr Thr Val Oly Tyr Asn ~is Thr Thr Leu Gly Ala Val Ala Ala Thr Gln Ile Ala Arg Val Ser Ala Thr Lys Leu Ala Ser Leu Pro Brg Ser Ser Gln Glu Thr Val Leu Ala Met Val ~ln Leu Gly Ala Arg Asp Gly Ala Val Pro Ser Ser Ile Leu Glu Gly ~le Ala Met Val Val G1U }{is Met Tyr Thr Ala Tyr Thr Tyr Val Tyr Th- Leu Gly Asp Thr Glu Arg Lys Leu Met Leu Asp Ile E~is Thr Val 435 ~ 440 445 Leu Thr Asp Ser Cys Pro Pro Lys Asp Ser Gly Val Ser Glu Lys Leu 450 ~55 460 Leu Arg Thr Tyr Leu Met Phe Thr Ser Met Cys Thr Asn Ile Glu Leu ~ly Glu Met Ile Aia Arg Phe Ser Lys Pro Asp Se- Leu Asn Ile Tyr ~rg Ala Phe Ser Pro Cys Phe Leu Gly Leu Arg Tyr Asp Leu E~is Pro Ala Lys Leu Arg Ala Glu~Ala Pro Gln Ser Ser Ala Leu Thr Arg Thr 515 ~ -- 520 525 Ala Val Ala Arg Gly Thr=Ser Gly Phe Ala Glu Leu Leu }iis Ala Leu ~is Leu Asp Ser Leu Asn Leu Ile Pro Ala Ile Bsn Cys Ser Lys Ile 545 _550 555 560 Thr Ala Asp Lys Ile Ile Ala Thr Val Pro Leu Pro Mis Val Thr Tyr _ _ _ _ . _ _ _ _ W O 96/06159 ' ~2 f q 6 8 9 2 PCT~DS9SIl0l9~ ~
21~ .

Ile Ile Ser Ser Glu Ala Leu Ser Asn Ala Val Val Tyr Glu Val Ser 580 585 sgo Glu Ile Phe Leu Lys Ser Ala Met Phe Ile Ser Ala Ile Lys Pro:Asp sg, . 6~0 ~ ~ =605 Cys Ser Gl}r Phe Asn Phe Ser Gln Ile Asp Arg His Ile Pro Ile Val 610 615 ~ 620 Tvr Asn Ile Ser Thr Pro Arg Arg Gly CYR Pro Leu Cys As~ Ser:.Val 625 630 635~ 64 ~le Met Ser Tyr Asp Glu Ser Asp Gly ~eu Gln Ser Leu Met Tyr Val 6~5 650 - 655 ~hr Asn Glu Arg Val Gln Thr Asn Leu Phe Leu Asp Lvs Ser Pro Phe 660 '~ 665 670 Phe Asp Asn Asn Asn Leu His Ile His Tyr Leu Trp Leu Arg Asp Asn 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 Gl~ Phe Ser Gly Val Ile Tyr ~he Leu Tyr Arg Leu Phe Ser Ile Leu Tyr ~2) INFORMATION FOR SEQ ID NO:18:
~i) SEQ~ENCE r~T~LrTTTlT.cTIcs A) LENGTH: 1215 base pairs B) TYPE: nucleie a~id C~ 5TT~NnFnR~cC: single D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic~
liii) HYPOTHETIChL N
(i~) ANTI-SENSE: N
(ix) FEATURE:
(A) NAME/~EY: CDS
(B) LOCATION: 1 .1215 ID) OTHER INFORMATION:

(xi! SEQUENCE DEs~ ~N: SEQ ID NO:18-ATG TTA CGA G~T CCG GAC GTG AAG GCT AGT CTA GTA GAG GGC GCG GCG 48 Met Le~l Arg Val Pro Asp Val Lys Ala Se~ Leu Val G.u Glv Ala Ala CGC CTG TCG ACA GGC GAG CG_ GTG TTT CAC GTC.TTG AC- TCT CCG GCG 96 Arg Leu Ser Th- Gly Glu Arg Val Phe His Val Leu Th_ Ser Pro Ala 20 25 ~ 30 GTG GCG GCC ATG G7'G GGA GTC TCT AAT CCT EaA GTC CCG ATG CCA CTG 14i Val Ala Ala Met Val Giy Val Ser Asn Pro Glu Val Pro Met Pro Leu TTG TT- EAA AAG TTT GGG A~T CCG GA_ TCG TC~ ACC..CTE.CCh CT~ TAC . 192 WO 96/06159 ' ' '' ~ I q 6 8 9 2 PCT/US95/lOI9.

~ 217 Leu Phe Glu Lys Phe Gly Thr Pro Asp Ser Ser Thr Leu Pro Leu Tyr . 55 . 60 ~ ..
GCG GCT AGG C AC CCG GA CT~ TCG TTG CTA CGG ATC. ATG CTC T~A . CCG . 240 Ala Ala Arg Xis Pro Glu Leu Ser Leu Leu Arg Ile Met Leu Ser Pro 65 : 70 ~75 80 CAC CCC Tac GCG .TTA.AGA AGC CAC TTG TGC GTA GGC GhA GAG ACC GCA 288 Xis Pro Tyr Ala Leu Arg Ser Xis Leu Cys Val Gly Glu Glu Thr Ala 85 . 90 ~ 95 TCT CTT Gr.r rTT Tar CTG. CAC TCC AAG CCA.. GTC GTA CGC r~GC.. CAC GaA 336 Ser Leu Gly Val Tyr Leu Xis Ser Lys Pro Val Val Arg=Gly E~is Glu 100 105 .: 110 TTC GAG GaC ACG r~r. ~T- rT~ rrr r~-, Trr CGG rTr r.rr :~TZ~ ~rr. I-rr 384 Phe Glu Asp Thr Gln Ile Leu Pro Glu Cys Arg Leu Ala Ile Thr Ser 115 : ~ . 120 ~ 12~

Asr Gln Ser Tyr Thr Asn Phe Lys Ile Ile Asp Leu Pro Ala Gly Cys 130 ~ ~ 135 . . 140 CGT CGC GTC CCC:ATA CACGCC GCGbAC AAG CGT GTC GTC ATC GaC GAG . 480 Arg Arg Val Pro Ile Xls= Ala Ala Asn Lys Arg Val Val Ile Asp Glu 145 150 155 . ~ _ 16D
GCC GCC AAC CGC ATA AaG GTG TTT GAC CCA GAG TCG CCT TTA CCG CGT 528 Ala Ala Asn Arg Ile Lys Val Phe Asp Pro Glu Ser Pro Leu Pro Arg CAC CCC ATA ACA C''C CGT GCC GGT ~aG ACC AGA TCT ATA CTG ~ CAC 576 Xis Pro Ile Thr Pro Arg Ala Gly Gln Thr Arg Ser Ile Leu Lys Xis 180 185 l9D
Aac ATC GCA CAG GTT ~CGC GAA CGG GAT ATC GTG TCA CTT AAC ACA GAC 624 Asn Ile Ala Gln Val Cys Glu Arg Asp Ile Val Ser Leu Asn Thr Asp AAC GAG GCC GCG TrT ~Tr. TTr T~r ~Tr aTT r.r.a rTr AGG ~GG CCG ~GA 672 Asn Glu Ala Ala Ser Met Phe Tyr Met Ile Gly Leu Arg Arg Pro Arg 21D :' ~ 2215 , ~ T : :: ~: 22D~
CTC GGA C~ ~rr crr r.Tr Tr~T r~ TTC AAC ACC GTT.ACC ATC ATG GAG 720 Leu Glv Glu Ser :Pro V ~:~ Cys Asp Phe Asn Thr Val Thr Ile Met Glu 225 ~230, _ _~ 235 ~= ~= =240 CGT GCT AAC AaC TCG TA ACT TTT rT~ rrr ~r. rT~ CTG AAC CGG 768 Arg Ala Asn Asn Ser Ile Thr Phe Leu Pro Lys Leu Lys Leu Asn Arg 245 250 _ 255 CTA CaA CAC CTG TTC CTG AAG CAC GTQ TTG CTG CG~ I~GC ATG GG CTG 816 Leu Gln Xis Leu Phe Leu Lys Xis Val Leu Leu Arg Ser Met Gly Leu GAA ~~ ATC GTG :TCG. TGT TTC TCA ~CG CTg TAC GGC GCa GAA CTT GCC 864 Glu As- ~le Val Ser Cys Phe Ser Ser Leu Tyr Gly Ala Glu Leu Ala 275 ~ :: _ 280 . 285 CC I GCG AaA AcA-cac GAr- CGG GAG TTC TTC GGC GCT CTG CTA GaA AGA 912 Pro Ala Lys ~hr Xis Glu Arg Glu Phe Phe Gly AIa Leu Leu Glu Arg CTC AAA CGT CGG .GTG. ~AG Ga~ GCG GTC TTC . TGC . CTG }~T ACC ATA GAG 960 Leu Lys Arg Arg Val Glu Asp Ala Val Phe Cys Leu Asn Thr Ile Glu 30s 310 315 320 WO 96/06159 2 1 9 6 8 9 2 PCTNS95/l019-1 GAT TTC CCG TTT P~GG GAA CCC ATT CGC CAA CCC CCA GAT TGT TCC AAG ~ 100B
Asp Phe Pro Phe Arg Glu Pro Ile Arg Gln Pro Pro Asp Cys Ser Lys 325 330 335 s GTG CTT ATA GAA GCC ATG GAA AAG TAC TTT ATG ATG TGT AGC CCC AAA ~ 105Ç
Val Leu IIe Glu Ala Met Glu Lys Tyr Phe Met Met Cvs 5er PrQ Lys GAC CGT CPA AGC GCC GC~ TGG CTA GGT G12A GGG GTQ QTC GAA CTG ATA 1104 Asp Arg Gln Ser ~la Ala Trp Leu Gly Ala Gly Val Val GIu Leu Ile 355 _ 360 ~: 31~5 TGT GAC GGC ~AT CCD-CTT TCT GAG GTG C~C~ GGA TTT CTT QCC AAG TAT llS_ Cys Asp Gly Asn Pro Leu Ser Glu Val Leu Gly Phe Leu AIa Lys Tyr 370 ' 375 ~ 380 ATG CCC AT~ CaA AaP, GAA TGC ACA GGA AAC CTT TTA AAA AT- TAC GrT ~ 1200 Met Pro Ile Gln Lys Glu Cys Thr Gly Asn Leu Leu Lys Ile Tyr Ala 385 ~ 390 ~ 395 - _ 400 Leu Leu Thr Val (2) INFOPMATION FOR SEQ ID NO:lg:
(i) SEQHENCE r~TDl~DrT~cTIcs:
(A) LENGTX 404 amino acids (E) TYPE: amino acid (D) TOPOLOGY lir,ear (ii) MOLECULE TYPE: protein (xi~ SEQ~ENCE IJ~:~LICl~lUN: SEQ ID NO l9 ~et Leu Arg Val Pro Asp Val Lys Ala Ser Leu Val Glu Gly Ala Ala ~rg Leu Ser Thr Gly Glu Arg Val Phe His Val Leu Thr Ser Pro Ala ~a' Ala Ala Met VaI GTy Val Ser Asn Pro_Glu Val Pro Met Pro Leu Leu Phe Glu Lys Phe Gly Thr Pro Asp Ser Ser Thr Leu Pro Leu Tyr so SS e 60 Ala Ala Arg Xis Pro Glu Leu Ser Leu Leu Arg Ile Met Leu Ser~Pro ~ 70 75 _ 80 ~is Pro Tyr Ala Leu Arg Ser Xis Leu Cys Val Gly Glu Glu Tkr Ala ~e- Leu Gl} Val Ty- Leu His Ser Lys Pro Val V..l Ar~ G1y His Glu 100 lQ5 liQ =
Phe Glu Asp .'hr Gln Ile Leu Pro Glu Cys Arg Leu Ala Ile Th-~ 5er llS lZQ = 125 Asp Gln Ser Tyr Thr Asn Phe Lys Ile Ile_Asp Leu Pro Ala Gly Cys Arg Arg Val Pro Ile Xis Ala Ala Asn Lys Arg Val Val Ile Asp Glu 145 150 - ~ - -lSS-- _ -,-160 Ala Ala Asn Arg Ile Lys Val Phe Asp Pro Glu Ser Pro Leu Pro Arg ~ W 096106159 2 1 9 6 8 q 2 PCTrDS95/1019~

His Pro Ile ThL~ Pro Arg Ala Gly Gln Thr Arg Ser Iie Leu Lys His Asn Ile Ala GIn Val Cys Glu Arg Asp Ile Val Ser Leu Asn Thr Asp Asn Glu Ala Ala Ser Met Phe Tyr Met ILe Gly Leu Arg AFg Pro Arg 21D :~ ~ 215 - 220 Leu Gly GIu Ser Pro Val Cys Asp Fhe Asn Thr Val Thr Ile Met Glu 225 230 235 2io Arg Ala Asn Asn Ser Ile Thr Phe Leu Pro Lys Leu Lys Leu Asn Arg Leu Gln His Leu Phe Leu Lys His Val Leu Leu Arg Ser Met Gly Leu 260 265 ~ 270 Glu Asn Ile Val Ser Cys Phe Ser Ser Leu Tyr Gly Ala Glu Leu Ala Pro Ala Lys Thr ~is Glu Arg Glu Phe Phe Gly Ala Leu Leu Glu Arg 290 295 30~
Leu Lys Arg Arg Val Glu Asp Ala Val Phe Cys Leu Asn Thr Ile Glu 305 . ~ 310 315 320 Asp Phe Pro Phe Arg Glu Pro Ile Arg Gln Pro Pro ASp Cys Ser Lys Val Leu lle Glu Ala Met Glu Lys Tyr Phe Met Met Cys Ser Pro Lys Asp Arg Gln Ser Ala Ala Trp Leu Gly Ala Gly Val Val Glu Leu Ile Cys Asp Gly Asn Pro Leu Ser Glu Val Leu Gly Phe Leu Ala Lys Tyr Met Pro Ile Gln Lys Glu Cys Thr Gly Asn Leu Leu hys Ile Tyr Ala 385 ~ 390 395 400 Leu Leu Thr Val ~2) INFORMATION FOR SEQ ID NO:20:
~i~ SEQ~EN OE ~H~TT~TcTICS:
AI LENGTH: 2259 ~ase pairs ~B TYPE: nucleic acid C sT~Nn~nNFss single D~ TOPOLOGY: linear ~ (ii) MOLECULE TYPE: DNA (genomie~
(iii) HYPOTHETICAL: N

) A~TI-SENSE: N
(i~) FEAT~RE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..2259 (D) OT~ER INFORMATION:

W 096/06159 ~' ~ ~ 9 ~ 8 9 2 PCTNS9S/1019 ~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
ATG GCA.GCG CTC GAG GGC CCC CTA CTA CTG CCA CCG AGC GCC TCC CTG 48 Met Ala Ala Leu Glu Gly Pro Leu Leu LeU Pro Pro Ser Ala Ser Leu 1 5 10, lS
ACG ACG AGT CCG CAq ACC ACG TGT TAT C~A qCG ACT TGG GDA TCA CAG ~ 96 Thr T~r Ser Pro Gln Thr Thr Cys Tyr Gln Ala Thr Trp Glu Ser Gln 20 25 ~o CTG GAD~ ATA TTC TGC TGT CTG GCC ACC ADC TCG CAC CTG ClG GCA GAG 144 Leu Glu Ile Phe Cys Cys Leu Ala Thr Asn Ser Qis Leu Gln Ala Glu CTG ACC TTA GAA GGT CTT G~T AAG ATG ATG CAG CCC GAG CCC ACC TTT . 192 Leu Thr Leu Glu Gly Leu Asp Lys Met Met Gln Pro Glu Pro Thr Phe so ~ ~ ~ ~ SS ~ 60 . =_ Phe Ala Cys Arg Ala Ile Arg Arg Leu Leu~Leu Gly~Glu Arg Leu His CCT TTT ATA CAT ~A GP~A GGG ACT CTT ~TQ GGA APl~GT~ GGT CGA CGG 288 Pro Phe Ile Qis Gln Giu Gly Thr Leu Leu Gly Lvs Val Gly Arg Arg 85 . 90 : ~ 9S
TAC AGC GGC GAA GGT TTA AT~ ATT GAC GGT~GGT GGD GT~ T~T ACG CGC :. 336 Tyr Ser Gly Glu Gly Leu Ile Ile Asp Gly Gly Gly Val Phe Thr Arg 100 105 : llQ
GGA CAG ATA GAC ACC GAC ~AC TAC CTA CCT GCG GTG GqA T~A TGG GAA 384 Gly Gln Ile Asp Thr Asp Asn Tyr Leu Pro Ala Val Gly Ser Trp Glu 115 - 120 _ 125 .
CTT ACC GAT qAT TGT GAT A~4A CCC TGC GAP. TTC AGG GAG CTA CGC TCG , 432 Leu Th- Asp Asp Cys Asp Lys Pro Cys Glu Phe Arg Glu Leu Arg Ser 13~ 13~ 140 CTG TAT CTT CCC GCG CTA CTA ACG TGC ACC ATA TGT TAC DD~ r~rr ATG 480 Leu Tyr Leu Pro Ala Leu Leu Thr Cys Thr Ile Cys Tyr Lys Ala Met 145 150 ~ ~ 155 16Q
TTC AGG ATA GTG TGC AGG TAC CTG GAG TTC.TGG GAG TTr GlA CAG TGT 528 Phe Arg Ile Val Cys Arg Tyr Leu Glu Phe Trp Glu Phe Glu Gln Cys TTT CAT GCG TTT CTG GCG GTG TTG CCC C~T AGT CTA CDJ~ CCC ACA ATC = 576 Phe Q~ 5 Aia Phe Leu Ala Val Leu Pro Qis Ser Leu Gln Pro Thr Ile TAT CDA A~T TAT TTT GCA CTC CTG GAG AGC CTG.AAG C~I CTC_TCG TTT 624 T,vr Gln Asn Ty_ Phe Ala Leu Leu Glu Ser Leu Lys ~is Leu Ser Phe l9S ~ 200 , 2~5 TCA ATA ATG CCA.CCC GCA TCC CCA GAC GCA CAG CTA C~T TTT TTA AAG ~ 672 Ser Ile Met Pro Pro Aia Ser Pro Asp Ala Gln Leu ~is Phe Leu Lys TTT AAC ATC AG- AGC TT~ ATG. OE C ACG TGR GGG TGG.CAC qGA GAG CTG _ 720 Phe Asn Ile Ser Ser Phe Met Aia Thr Trp Gly Trp ~is Gly Glu Leu ~25 .30 _ ~ 235 = 240 GTC TCG CTG cqc CGT GCC ATC GCT CAC AAC GTA~lG CGA CTG CCC ACC - 768 Val Ser Leu Arg Arg Ala Ile Ala ~is Asn Val Glu Ars Leu Pro Thr 245 25D~ , 255 GTG CTG AAG AAC CTG TCG AAA CAG AGT AAQ C_C CAG GAC.G~C ADG GTT ... 816 ~ WO96106159 ~ . 2;1 96892 PCTIUS95/10191 f ~
2~1 val Leu Lys Asn Leu Ser Lys Gln Ser Lys His Gln Asp Val Lys Val 26D 265 ~ 70 Asn Gly Arg Asp Leu Val Gly Phe Gln Leu Ala Leu Asn Gln Leu Val .~ 275 280 285 ser Arg Leu His Val Lys Ile Gln Arg Lys Asp Pro, Gllr Pr,o Lys Pro Tyr Ar~ Val Val Val Ser Thr Pro Asp Cys Thr Ty- Tyr Leu Val Tyr 305 . .= = 31~ ~ ~ ,= 315 . . 320 Pro Gly Thr Pro Ala Ile Tyr Arg Leu Val Met Cys Me~ Ala Val Ala Asp Cys Ile Gly His Ser Cys Ser Gly Leu His Pro Cys Ala Asn Phe TTA GGC }~CC CAC GAG ACI~ CCG CGT CTC CTG GCG GCG ACG CTT TCA AGA 1104 Leu Gly Thr His Glu Thr Pro Arg Leu Leu Ala Ala Thr Leu Ser Arg 355 ~ 360 365 ATC CGG TAC GCG CCG .alllL GAC CGG CGA GCA GCC ATG AAA GGA AAT TTG 1152 Ile Arg Tyr Ala Pro Lys Asp Arg Arg Ala Ala Met Lys Gly Asn Leu Gln Ala Cys Phe Gln Arg Tyr Ala Ala Thr Asp Ala Arg Thr Leu Gly 385 = . = ~390 395 ~ - 400 AGC TCT ACA GTG TCA GAC ATG CTG GAA CCC ACA A~A CAC GTC AGT TTG 124B
Ser Ser Thr Val Ser Asp Met Leu Glu Pro Thr Lys His Val Ser Leu Glu Asn Phe Lys Ile Thr Ile Phe Asn Thr Asn Met Val Ile Asn Thr AAG ATA AGr TGC CAC GTT CCT AAC ACC CTG CAA AAG ACT ATT TTA AAC 1344 Lys Ile Ser Cys Hls Val Pro Asn Thr Leu Gln Lys Thr Ile Leu Asn 435 . - - 440 445 ATC CCC AGA TTG AC0 ~C AAT TTT GTT ATA CGA AAG TAr TCC GTA AAG 1392 Ile Prc~ Arg Leu Thr Asn Asn Phe Val Ile Arg Lys Tyr Ser Val Lys GAA CCT TCT TTT ACC ATA AGC GTG TTT TTT TCC GA- A~ ATG TGT CAA 1440 Glu Pro Ser Phe Thr Ile Ser Val Phe Phe Ser Asp Asn Met Cys Gln - GGC A''--:GCA AT, AAC ATC AAC ATC AGT GGG GAC ATG CTG CA-- TTT CTC 1488 Gly Tr.- Ala Ile Asn Ile Asn Ile Ser Gly Asp Met Leu His Phe Leu TTC G~A ATG GGT ACG CTG AAA TG-- TTT CTG CCA A-'C AGG CAC ATA TTT 1536 Phe Ala Met Gl}~ Thr Leu Lys Cys Phe Leu Pro Ile Arg Hls Ile Phe Pro Val Ser Ile Ala Asn Trp Asn Ser Thr Leu Asp Leu His Gly Leu W 096/06l59 2 î 9 6 8 9 2 PCTrDS95/10l9 ~

22~ ~
GAA AAC CAG TAC ATG GTG AGA ATG GGG CGA A~A AAC GTA TTT TGG ACC 1632 Glu Asn Gln Tyr Met Val Arg Met Gly Arg Ly5 Asn Val Phe Trp Thr ACA AAC TTT CCA TCT GTG GTC TCC AGC AAG GAT GGG CT~ AAC GTG TCC ....... 1680 Thr Asn Phe Pro Ser Val Val Ser Ser Lys Asp Gly Leu Asn Val Ser :.
545 : 550 SSS 560 ~ -TGG TTT AAG GCC GCG ACA qCC ACG ATT TCT A~A GTG TAC GG.G CAG~CCT ~ 1728 Trp Phe Lvs Ala Ala Thr Ala Thr Ile Ser:Lys Val Tyr Gly Gln Pro 565 57D 57s CTT GTG GA~ C~G ATT CGC CAC GAG CTG GCG CCC ATT CTC ACG.GAC CAG . 1776 Leu Val GIu Glr. Ile Arg~Lis Glu Ieu Ala Pro Ilc Leu Thr Asp Gln CAC GCG CGC ATC GAC G~A AAC ~AA AAT AG~ ATA TTC TCC CTA CTT GAG 1324 His Ala Arg Ile Asp Gly Asn Lys Asn Arg Ile Phe Ser Leu Leu Glu S9S _ 600 . . _ 6Q~ _ CAC AGA AAC CGT.TCC C ATA CAG ACG CTA CAC AAA AGG TT~ CTG GAG ......... 1872 His Arg Asn Arg Ser Gln Ile Gl~. Thr Leu His Lys Arg Phe Leu Glu 61Q : : ~615 620 " ~ ~:
TGT CTG GTG GAA TGC TGT TCG TTT CTC AGG CTT GAC GTG GCT ~GC ATT _ 1920.
Cys Leu Val Glu Cys Cys Ser Phe Leu Arg Leu Asp Val Ala Cys Ile 625 _ 630 635 ~ ~ ~ 640 AGG CGA GCC GCC.GCC CGG GGC CTG TTT GAC TTC TCA AAG AAG ATA ATC _ 1968 Arg Arg Ala Ala Ala Arg Gly Leu Phe Asp Phe Ser Lys Lys Ile Ile 645 650 655 ~~
AGT CAC ACT AAA AGC A~ CAC GAG TGC GCA GTA CTG GGA TAT AaA AAG 2Q16 Ser His Thr Lys Ser Lys His Glu Cys Ala Val Leu Gly Tyr Lys Lys 660' : 665 ~ 670 ~
TGT AAC CTA ATC CCG AaA ATC TAT GCC CGA AaC ALG.AAG ACC AGG CTA . 2064 Cys Asn Leu Ile.Pro Lys Ile Tyr Ala Arg Asn Lys Lys Thr~Arg Leu 675 : . ~680 _ _685 GAC GAG TTG GGC CGC.A~T.GCA AAC TTC ATT TCG TTC GTC.GCC.ACC ACG _ 2112 Asp Glu Leu Gly Arg Asn Ala Asn Phe Ile Ser Phe Val Ala Thr Thr 690 695 700 ~
GGT CAT CGG TTC GCC GCT CTA A~G CCA CAA ATT GTC CGT CAC GCC ATT ._ 2160 Gly His Arg Phe Ala Ala Leu Lys Pro Gln Ile Val Arg His Ala Ile 705 ~ 710 ,= _ _ 715 ~ 720 CG- A~ CTA GGC CTG CAC TGG CGC CAC CGA ACG GCC GCG TCC AAC GAG 22Q8 Arg L~s Leu Gly Leu Lis Trp Arg His Arg Thr AIa Ala Ser Asn Glu . 725 . 73Q 735 CAG ACA CCG CCA=GCC GAT CCC CGC.GTA CGT TG. GTC CGT CCG CTG GTC 2256 Gln Thr Pro Pro Ala Asp Pro Arg Val Arg Cys Val Arg Pro Leu Val TAA ' 2259 ~2~ INFORMATION FOR SEQ ID NO:21:
~i) SEQUENCE ~5rTT~TcTIcs (A) LENGTH: 752 amino asias (B) TYPE: amino aoid (D~ TOPOLOGY: lir~ear .. . .
_ _ _ ~ WO96/06159 2 1 96892 pCI/US95/10191 iii) MOLECULE TYPE: protein ~xi) SEQIE~IOE DESCRIPTION: SEQ ID NO:21:
Met Ala Ala Leu Glu Gly Pro Leu Leu Leu Pro Pro Ser Ala Ser Leu Thr Thr Ser Pro Gln Thr Thr Cys Tyr Gln Ala Thr Trp Glu Ser Gln Leu Glu Ile Phe Cys Cys Leu Ala Thr Asn Ser Uis Leu Gln Ala Glu : 40 45 Leu Thr Leu Glu Gly Leu Asp Lys Met Met Gln Pro Glu Pro Thr Phe Phe Ala Cys Arg Ala Ile Arg Arg LeY Leu Leu Gly Glu Arg Leu ~lis 6, 70 75 80 Pro Phe Ile His Gln Glu Gly Thr LeU Leu Gly Lys Val Gly Arg Arg Tyr Ser Gly Glu Gly Leu Ile Ile Asp Gly Gly Gly Vai Phe Thr Arg Gly Gln Ile Asp Thr ASp Asn Tyr Leu Pro Ala Val Gly Ser Trp Glu Leu Thr ASp Asp Cys Asp Lys Pro Cys Glu Phe Arg Glu Leu Arg Ser 130 ~ 135 140 Leu Tyr Leu Pro Ala Leu Leu Thr Cy8 Thr Ile Cys Tyr Lys Ala Met 145 15a 155 160 Phe Arg Ile Val Cys Arg Tyr Leu Glu Phe Trp Glu Phe Glu Gln Cys Phe ~lis Ala Phe Leu Ala Val Leu Pro His Ser Leu Gln Pro Thr Ile Tvr Gln Asn Tyr Phe Ala Leu Leu Glu Ser Leu Lys ~lis Leu Ser Phe Ser Ile Met Pro Pro Ala Ser Pro Asp Ala Gln Leu Uis Phe Leu Lys 210 _ 215 220 Phe Asn Ile Ser Ser Phe Met Ala Thr Trp Gly Trp ~Iis Gly Glu Leu ~ 5 230 235 240 Val Ser Leu Arg ~r=g Z~l a Ile Ala ~Iis Asn Val Glu Arg l:,eu Pro Thr Val Leu Lys Asn Leu Ser Lys Gln Ser Lys ~is Gln Asp Val Lys Val A5n Gly Arg Asp Leu val GlY Phe Gln Leu Ala Leu Asn Gln Leu Val 275 280 2es Ser Arg Leu 31i~a Val Lys Ile Gln Arg Lys Asp Pro Gly Pro Lys Pro Tyr Arg Val Val Val Ser Thr Pro Asp Cys Thr Tyr Tyr Leu Val Tyr 30s .=~=._31D 315 320 Pro Gly Thr Pro Ala Ile Tyr Arg Leu Val Met Cys Met Ala Val Ala WO 96/06159 ~ ;! 2 i qi 6 8 9 2 PCTIUS95/1019~

Asp Cys Ile Gly Pis Ser Cys Ser Gly Leu Xis Pro Cys Ala Asn Phe Leu Gly Thr His Glu Thr Pro Arg Leu Leu Ala Ala Thr Leu Ser Arg Ile A_g Tyr Ala Pro Lys Asp Arg Arg Ala Ala Met Lys Gly Asn Leu 370 375 380 ~ ~
Gln Ala Cys Phe Gl~ Arg Tyr Ala Ala Thr. Asp Ala Arg Thr Leu Gly 355 39D : 395 ~ 400 ~er Ser~ Thr Val Ser Asp Met Leu Glu Pro: Thr Lys ilis Val Ser Leu ~lu Asn Phe Lys Ile Thr Ile Phe Asn Thr Asn Met Val Ile Asn Thr Lys Ile Ser Cys Pis Val Pro Asn Thr Leu Gln Lys Thr Ile Leu Asn 435 440 ~ 445 Ile Pro Arg Leu Thr Asn Asn Phe Val Il~ Arg Lys Tyr Ser Val Lys 450 ~ : 455 ~ 460 ~ -Glu Pro Ser Phe Thr Ile Ser Yal Phe Phe Ser Asp Asn Met Cys Gln 465 ~ 470 475 480 ~ly Thr Ala Ile Asn Ile Asn Ile Ser Gly Asp Met Leu lIis Phe Leu ~85 490 495 ~he Ala Met Gly Thr Leu Lys Cys Phe Leu Pro Ile Arg Xis Ile Phe 500 505 , 510 Pro VA1 Ser Ile ala Ash Trp Asn Ser Thr Leu Asp Leu Pis Gly Leu 5~5 ~ 520 ~ 525 ~
Glu Asn Gln Tyr Met Val Arg Met Gly Ar~ Lys Asn Val Phe Trp Thr Thr Asn Phe Pro Ser Val Val Ser Ser Lys Asp Gly Leu Asn Val Ser 545 550 _ 555 560 ~rp Phe Lys Ala Ala Thr Ala Thr Ile Ser Lys Val Tyr Gly Gln Pro ~eu Val Glu Gln Ile Arg Xis Glu Leu Ala Pro Ile Leu Thr Asp Gln 580 585 ~ 590 Xis Ala Arg Ile Asp Gly Asn Lys Asn Arg Ile Phe Ser Leu Leu Glu 595 ~ .. 600 605 _ _ Xis Arg Asn Arg Ser Gln Ile Gln Thr Leu Xis Lys Arg Phe Leu Glu 610 : _~ 615 ~ 620 Cys Leu Val Glu Cys Cys Ser Phe Leu Arg Leu Asp Val Ala Cys Ile 625 630 635 -~ 640 ~rg Arg Ala Ala zla Arg Gly Leu Phe Asp Phe Ser Lys Lys Ile Ile ~er Pis Thr Lys Ser Lys Xis Glu Cys Ala Val Leu Gly Tyr Lys Lys 660 665 ~ 670 Cys Asn Leu Ile Pro Lys Ile Tyr Ala Arg Asn Lys Lys Thr Arg Leu Asp Glu Leu Gly Arg Asn Ala Asn Phe Ile Ser Phe Val Ala Thr Thr ~ W 096l06l59 2 1 9 6 8 9 2 PCTA~S95~1019~
22~

Gly Xis Arg Phe Ala Ala Leu Lys Pro Gln Ile Val Arg Xis Ala Ile 705 710 71~ 720 ~rg Lys Leu Gly Leu Xis Trp Arg Xis Arg Thr Ala Ala Ser Asn Glu 725 730 7~5 ~ln Thr Pro Pro Ala Asp Pro Arg Val Arg Cys Val Arg Pro Leu Val ~2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE rE~T~rTE~TcTIcs-~A) LENGTX: 364 base pairs (B) TYPE: nucleic acid (C) STT~N~NECC: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (geno~ir) (iii1 ~l~hll~AL: N
(iv) ANTI-SENSE: N
( iY. ) FEATUEE:
(A) NAME/REY: CDS
(B) LOCATION: 1..364 (D) OTXER INFORMATION:

~xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
ATG GTA CGT CCA ACC GAG GCC GAG GTT A~G ALA TCC CTG AGC AGG CTT 48 Met Val Arg Pro Thr Glu Ala Glu Val Lys Lys Ser Leu Ser Arg Leu CCA GCA QCA CGC AAA AGA GCA GGT AAC CGG GCC CAC CTG GCC ACC TAr 96 Prr Ala Ala Arg Lys Arg Ala Gly Asn Arg Ala Xis Leu Ala Thr Tyr CGC CGG CTC CTC ~ALLT~C TCC ACC CTG CCC GAT CTA~TGG CGG TTT CTA 144 A-g Arg Leu Leu Lys Tyr Ser Thr Leu Pr~ Asp Leu Trp Arg Phe Leu AGT AGC CGG CCC CA~ AAC CCT CCC CTT GGA CAC CAC AGA TTA TTC TTT 192 Ser Ser Arg Pro Gln Asn Pro Pro Leu Gly Xis Xis Arg Leu Phe Phe Glu Val Thr Leu Gly Xis Arg Ile Ala Asp Cys Val Ile Leu Val 5er = ..~=70 75 50 GGT GGG CAT CAG CCC GTA TGT TAC GTT GTA GAG CTr AaG ACT TGT CTG 2a8 Gly Gly Xis Gln Pro Val Cys Tyr Val Val Glu Leu Lys Thr Cys Leu 85 9o 9S

Ser Xis Gln Leu Ile Pro Thr Asn Thr Val Arg Thr Ser Gln Arg Ala Gln Gly Leu Cys Gln Leu Ser Asp Ser llS 120 .21 9689~
W O96/06159 ' ' ' I ~ PCTnUS9S/1019 (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE ~ llW' Y
(A) LENGTH: lZl amino acids (B) TYPE: amino acid , lD) TOPOLOGY: linear (ii) MOLEC~LE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Met Val Arg Pro Thr Glu Ala Glu Val Lys Lys Ser Leu Ser Arg~Leu 1 5 10 ~ lS
~ro Ala Ala Arg Lys Arg Ala Gly Asn Arg Ala His Leu Ala Thr Tyr Arg Arg Leu Leu Lys Tyr Ser Thr Leu Pro Asp Leu Trp Arg Phe Leu ~ ~ =i5 ~er Ser Arg Pro Gln Asn Pro Pro Leu Gly His His Arg Leu Phe Phe 5Q ~5 . ~ ~ 60 } =~= ~ ~~~
:
Glu Val Thr Leu Gly His Arg Ile Ala Asp Cys Val Ile Leu Val Ser . :
~ly Gly His Gln Pro Val Cy9 Tyr Val Val Glu Leu Lys Thr Cys Leu '~ 95 ~er His Gln Leu Ile Pro Thr Asn Thr Val Arg Thr Ser Gln Arg Ala Gln Gly Leu Cys Gln Leu Ser Asp Ser (2~ INFORMATION FOR SEQ ID NO:24:
(i) SEQ~ENCE r~a~rTR~TcTTrc (A LENGTH: 91B ~ase pairs (B TYPE: nucleic acid (C ST~n~n~cc: single (D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iiS) HYPOTHETIC~L: N
(i~) ANTI-SENSE: N
( iY ) EE~TURE:
(A) NAME/~EY: CDS
(B) LocATIoN 1..918 (D) 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 48 Met Ala Leu Asp Lys Ser Ile Val Val Asn Phe Thr Ser. Arg Leu Phe 1 5 10 ~ ~5 GCT GAT GAA CTG GCC GCC CTT CaG TCa A~A ATA GGG AGC GTA CTG CCG 96 Ala Asp Glu Leu Ala Ala Leu Gln Ser Lys Ile Gly Ser Val Leu Pro 20 25 = 30 _ _ : . _ . . _ . . : . . . _ _ W 096/06159 . . '2 1 9 6 8 9 2 PCTAUS95/lO

CTC GGA GAT TGC r~r rr~T TTA C~A iL~T ATA CaG rr~ TTr r~rr CTG GGG 144 Leu Gly Asp Cys His Arg Leu Gln Asn Ile Gln Ala Leu Gly Leu Gly Cy6 Val Cys Ser Arg Glu Thr Ser Pro Asp Tyr IIe Gln Ile Met Gln TAT CTA TCC AAG T~ACA rTC GCT GTC CTG GAG GAG GTT CGC CCG GAC 240 Tyr Leu Ser Lys Cys Thr Leu Ala Val Leu Glu Glu Val Arg Pro Asp = ~0 75 ,_ , ~ 80 hGC CTG CGC CTA ACG CGG ATG GAT CCC ~CT GAC~L~C CTT CAG ATA AAA 288 Ser Leu Arg Leu Thr Arg Met Asp Pro Ser Asp Asn Leu Gln Ile Lys AAC GTA TAT G~C CCC TTT TTT CAG TGG G~C AGC AAC ACC CaG CTA GCA 336 Asn Val Tyr Ala Pro Phe Phe Gln Trp Asp Ser Asn Thr Gln Leu Ala GTG CTA rrr rr~ TTT TTT aGC_CGA Aa~LGAT TCC ACC ATT GTG_CTC GAA 384 Val Leu Pro Pro Phe Phe Ser Arg Lys Asp Ser Thr Ile Val Leu Glu lI5 := ~ 12D . ._ _- .123. . _ TCC AAC GGA TTT GAC CCC GTG TTC CCC ATG GTC GTG CCG caG caA CTG 432 Ser Asn Gly Phe Asp Pro Val Phe Pro Met Val Val Pro Gln Gln Leu 130 ~ 5 . . 140 GGG cac GCT ATT CTG CAG C~G CTG TTG GTG TAC cac ATC TAC TCC AAA 480 Gly ~is Ala Ile Leu Gln Gln Leu Leu Val Tyr Pls Ile Tyr Ser Lys ATA TCG GCC ~GG GCC CCG GAT GAT GTA AAT ATG GCG GAA CTT GAT CTA 528 Ile Ser Ala Gly Ala Pro Asp Asp Val Asn Met Ala Glu Leu Asp Leu TAT ACC ACC AAT GTG TCA TTT ATG GGG CGC ACA TAT CGT CTG GAr GTA 576 Tyr Thr Thr Asn Val Ser Phe Met Gly Arg Thr Tyr Arg Leu Asp Val GAC Aa-- ACG GAT CCA CG~ ACT GCC CTG CGA rTr. rTT r~r GAT CTG TCC 624 Asp Asn Thr Asp Pro Arg Thr Ala Leu Arg Val Leu Asp Asp Leu Ser 195 : 200 205 ATG TA- CTT TOE ATC CTA Tca GCC TTG GTT CCC_hGG GGG TGT CTC CGT 672 Met Ty~ Leu Cys Ile Leu Ser Ala Leu Val Pr~ Arg Gly Cys Leu Arg Leu Leu Thr Ala Leu Val Arg ~is Asp Arg Pis Pro Leu ~hr Glu Val 225 . .~ ~ 23~ 235 ~ 240 Phe Glu Gly Val Val Pro Asp Glu Val Thr A_g Ile Asp Leu Asp Gln 245 2~0 25~
TTG AG- GTC CCA GAT GAC ATC ACC AGG ATG CGC GTC ATG TTC TCC.TAT 816 Leu Se- Val Pro Asp Asp Ile Thr Arg Met Arg Val Met Phe Se- Tyr Leu Gln Ser Leu Ser Ser Ile Phe Asn Leu Gly Pro Arg Leu ~ls Val Tyr Ala Tyr Ser Ala Glu Thr Leu Ala Ala Ser Cys Trp Tyr Ser Pro 290 2gS 300 W 096/06159 ' 3~ 2 ~ 9 6 8 9 2 PCTGUS95/1019 ~

.

Arg (2) INFORMATION FOR SEQ ID NO:25:
~i~ SEQ~ENCE rE~rT~TCTICS:
(A1 LENGT~: 305 amino acids ~B~ TYPE: a~ino acid ~D) TOPOLOGY: linear iii) MOLECULE TYPE: protein ~i) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Met Ala Leu ASp Lys Ser Ile Val Val Asn Phe ThF Se~ Arg Leu=Phe Ala Asp Glu Leu Ala Ala Leu Gln Ser Lys Ile Gly Se- Val Leu Pro Leu Gly Asp Cys P;is Arg Leu Gln Asn Ile Gln Ala Leu Gly Leu Gly 35 = 40 45 Cys Val Cys Ser Arg Glu Thr Ser Pro Asp Tyr Ile Gln Ile Met Gln Tyr Leu Ser Lys Cys Thr Leu Ala Val Leu Glu Glu Val Arg Pro Asp Ser Leu Arg Leu Thr Arg Met Asp Pro Ser Asp Asn Leu Gln Ile Lys Asn Val Tyr Ala Pro Phe Phe Gln Trp Asp Ser Asn Thr Gln Leu Ala Val Leu Pro Pro Phe Phe Ser Arg Lys Asp Ser ThF Ile Val Leu Glu llS 120 125 Ser Asn Gly Phe Asp Pro Val Phe Pro Met Val Val Pro Gln Gln Leu 130 = = ~ 135 140 Gly Eis Ala Ile Leu Gln Gln Leu Leu Val Tyr Eis Ile Tyr Ser Lys 145 150 lSS 160 Ile Ser Ala Gly Ala Pro Asp Asp Val Asn Met Ala Glu Leu Asp Leu Ty~ Thr Thr Asr Val Ser Phe Met Gly Arg Thr Tyr Arg Leu Asp Val lB0 185 190 Asp Asn Th- Asp Pro Arg Thr Ala Leu Arg val Leu Asp Asp Leu Ser l9S . 200 205 Met Tyr Leu Cys Ile Leu Ser Ala Leu Val Pro Arg Gly Cys Leu Arg 210 215 . i20 ' . . : :
Leu Leu Thr Ala Leu Val Arg ~is Asp Arg,U1s Pro Leu Thr Glu Val 7~5 . ~ 23~ 235 - - -240 ~he Glu Gly Val Val Pro Asp Glu Val Thr Arg Ile Asp Leu Asp Gln 245 25~ 255 ~eu Ser Val Pro Asp Asp Ile Thr Arg Met Arg Val Met Phe Ser Tyr 2l q68q2 ~ W 096l06159 PCT~U595~10194 22~
Leu Gln Ser Leu Ser Ser Ile Phe A5n Leu Gly Pro Arg Leu ~is Val Tyr Ala Tyr Ser Ala Glu Thr Leu Ala Ala Ser Cys Trp Tyr Ser Pro Arg (2~ INFORMATION FOR SEQ ID NO:26:
~i~ SEQ~ENCE r~aT~rTFT~T~TIcs A1 LPNGT~: S73 ~ase pairs B) TYPE: nucleic acid C~ STT~NnFn~PC~: single D~ TOPOLOGY_ linear (ii) MOLECULE TYPE: DNA (genomic) (iii~ HyrUl~ll~AL: N
(iv) ANTI-SENSE: N
(ix) FEATURE:
(A~ NAME/~EY: CDS
(B) LOCATION: l .S73 (D) O~ER IWFORMATION:

(xi) SEQ~EN OE U~b~ llU~: SEQ ID NO:26:
ATG GCG TCA TCT GAT ATT. CTG TCG GTT GCA AGG ACG G~T GAC GGC TCC 48 Met Ala Ser S r Asp Ile Leu Ser Vai Ala Arg Thr Asp Asp Gly Ser GTC TGT GAA GTC TCC CTG CGT GGA GGT AGG A~A AAA ACT ACC GTC TAC 96 Val Cys Glu Val Ser Leu Arg Gly Gly Arg Lys Lys Thr Thr Val Tyr CTG CCG Gac ACT GAA CCC TGG GTG GTA GAG ACC GAC GCC ATC A~A GAC 144 Leu Pro Asp Thr Glu Pro Trp Val Val Glu Thr Asp Ala Ile Lys Asp Ala Phe Leu Ser Asp Gly Ile Val Asp Met Ala Arg Lys Leu ~is Arg GGT GCC rTG-ccc TCA AAT T ~ CAC AAC GGC TTG AGG ATG GTG CTT TTT 240 Gly Ala Leu Pro Ser Asn Ser ~is Asn Gly Leu Arg Met Val Leu Phe TGT TAT TGT ~AC.TTG CAA AAT TGT GTG TAC CTA GCC CTG TTT CTG TGC 288 Cys Tyr Cys Tyr Leu Gln Asn Cys Val Tyr Leu Ala Leu Phe Leu Cys ~ CCC CTT AAT CCT TAC TTG GTA ACT CCC TCA AGC ATT GAG TTT GCC GAG 336 Pro Leu Asn Pro Tyr Leu Val Thr Pro Ser Ser Ile Glu Phe Ala Glu Pro Val Val Ala Pro Glu Val Leu Phe Pro Pis Pro Ala Glu Met Ser llS lZ0 ~ 125 CGC GGT TGC GAT GAC GCG ATT TTC TGT A~A CTG CCC TAT ACC GTG CCT 432 Arg Gly Cys Asp Asp Ala Ile Phe Cys Lys Leu Pro Tyr Thr Val Pro 130 135 ~ 140 WO96/06159 2 1 9 6 8 9 2 PCTrDS95/~019 ~

ATA ATC AAC ACC ACG TTT GGA CGC ATT TAC CCG AAC TCT ACA CGC GAG : 480 Ile Ile Asn Thr Thr Phe Gly Arg Ile Tyr Pro Asn Ser Thr Arg Glu Pro Asp Gly Arg Pro Thr Asp Tyr Ser Met Ala Leu Arg Arg Ala Phe GCA GTT ATG GTT ~AC ACG TCA TGT GCA GGA GTG ACA TTG TGC CGC G~A 576 Ala Val Met Val Asn Thr Ser Cys Ala Gly Val Thr Leu Cys Arg Gly Glu Thr Gln Thr Ala Ser Arg Asn Hls Thr Glu Trp Glu Asn Leu Leu 19~ ~ 200 205 Ala Met Phe Ser Val Ile Ile Tyr Ala Leu Asp Uis Asn Cys ~is Pro 210 215 22D ~ , Glu Ala Leu Ser Ile Ala Ser Gly Ile Phe Asp Glu Arg Asp Tyr Gly TTA TTC ATC TCT CAG CCC CGG AGC GTG CCC TCG CCT.ACC CCT TGC GAC ~ 768 Leu Phe Ile Ser Gln Pro Arg Ser Val Pro Ser PrD Thr Pro Cys Asp Val Ser Trp Glu Asp Ile Tyr Asn Gly Thr Tyr Leu Ala Arg Pro Gly AAC TGT GAC CCC TGG CCC~T CTA TCC ACC.CCT CCC TTG ATT CTA AAT 864 Asn Cys Asp Pro Trp Pro Asn Leu Ser Thr Pro Pro Leu Ile Leu Asn TTT A~A TAA 873 Phe Lys 2~0 t2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE r~TF~TcTIcs (A) LENGTH: 290 amino acids (B) TYPE: amino acid ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein ~ SE9UENCE DESCRIPTIQN: SEQ ID NO:27:
~et Ala Ser Ser Asp Ile Leu Ser Val Ala Arg Thr Asp Asp Gly Ser ~~l Cys Glu Val Ser Leu Arg Gly Gly Arg Lys Lys Thr Thr Val Tyr Leu Pro Asp Thr Glu Pro Trp Val Val Glu Thr Asp Ala Ile Lys Asp Ala Phe Leu Ser Asp Gly Ile Val Asp Met Ala Arg Lys Leu His Arg Gly Ala Leu Pro ser Asn Ser Uis Asn Gly Leu Arg Met Val Leu Phe ~ W 096/06159 ~ ~ 2 1 9 6 8 ~ 2 PCTrUS9S/1019~

~' 231 Cys Tyr Cy5 Tyr Leu G~ln Asn 5ys Val Tyr Leu Ala Leu Phe Leu Cys Pro Leu Asn Pro Tyr Leu Val Thr Pro Ser 5er Ile Glu Phe Ala Glu 10D lDS 110 Pro Val Val Ala Pro Glu Val Leu Phe Pro Xis Pro Ala Glu Met Ser llS ~ ~~ I2D 125 Arg Gly Cys Asp Asp Ala=Ile Phe Cys Lys Leu Pro Tyr Thr Val Pro 13~ ~:135 140 Ile Ile:Asn Thr Thr Phe Gly Arg n e Tyr Pro As~:Ser Thr Arg Glu 145 ~0~ 155 - 160 ~ro Asp Gly Arg Pro~Thr Asp Tyr Ser Met Ala Leu Arg Arg Ala Phe 165 = 170 : 175 ~la Val Met val Asn Thr ser Cys Ala Gly Val Thr Leu Cys Arg Gly Glu Thr Gln Thr Ala Ser Arg Asn His Thr Glu Trp Glu Asn Leu Leu l9S =~.= 200 205 Ala Met Phe Ser Val Ile Ile Tyr Ala Leu Asp Xis Asr. Cys Xis Pro 21Q -~ ~ 215 ~ 220 -Glu Ala Leu Ser Ile Ala Ser Gly Ile Phe Asp Glu Arg Asp Tyr Gly ~eu Phe Ile Ser Gln Pro Arg Ser Val Pro ser Pro Thr Pro Cys Asp ~al Ser Trp Glu Asp Ile Tyr Asn Gly Thr Tyr Leu Ala Arg Pro Gly ~he Lys ~2) I~FORM~TI4N FOR SEQ ID N4:28:
li) SEQ~EN OE r~rT~TqTICS:
(A LENGTX: 363 base pairs IB TYPE: nucleic acid (C ST~Nn~nNrCq: single (D T4P4L4GY: linear (i ) MOLEC~rE TYPE: DNA (genomic) (iii) ~Y~kll AL_ N
(iv) ANTI=SENSE: N
(ix) FEATURE:
(A) NAME/KEY: CDS
(E) LOCATI4N: 1..363 (D) 4TXER INF4RMATI4N:

(xi) SEQ~ENCE ~ ~l~llJN: SEQ ID NO:28:
ATG AGC ATG ACT TTC CCC GTC TCC ~GT CAC CGG AGG AAT GGT GGA CGG 48 Me~ Ser Met Thr Phe Pro Val Ser Ser Xis Arg Arg Asn Gly Gly Arg 1 S lQ =. lS

W 096/06159 ~ 2 ~ 9 6 8 q 2 PCT~US95/1019~ ~

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 Gln Ala Ser Arg Asp Trp Ser Tyr AAC AGT GCT CTT ~T 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 45 TCG CGT GTT CCT GGC GGC TCG ACT GTG GCG CGC CAC CCC ACT AGG CAG 192Ser Arg Val Pro Gly Gly Ser Thr Val Ala Arg His Pro Tnr Arg Gln GGC CAC CGT GGC GTA TCA GGT CCT TCG CAC CCT GGG ACC GCA GGC CGG 240Gly His Arg Gly Val Ser Gly Pro Ser His Pro Gly Thr Ala Gly Arg 65 70 75 , - 80 GTC ACA TGC ACC GCC GAC GGT GGG CAT AGC TAC CCA GGA GCC CTA CCG 288Val Thr Cy6 Thr Ala Asp Gly Gly His Ser Tyr Pro Gly Ala Leu Pro TAC AAT ATA CAT GCC AGA TTA GAA CGG GGT GTG TGC TAT AAT GGA TGG 336Tyr Asr. Ile His Ala Arg Leu Glu Arg Gly Val Cys Tyr~Asn Gly Trp lD0 l05 _ll0 CTA TGG GGG GGG GCT GTA GAT AAT TGA : 363 Leu Trp Gly Gly Ala Val Asp Asn i2) }NFORMATION FOR SEQ ID NO 29 ~:) SEQUENCE r~=~rT~TCTICS:
~A) LENGTH: 120 a~ino acids ~E) TYPE: amino acid ~D) TOPOLOGY: linear ~ii) MOLEC~LE TYPE: protein (xi~ SEQUENCE ~U~l~llUN: SEQ ID NO 29 ~et Ser Met Thr Phe Pro Val Ser Ser Xis Arg Arg Asn Gly Gly Arg ~eu Arg Pro Gly Ala Asn Gly His Gln Ala Ser Arg Asp Trp Ser Tyr Asn Ser Ala Leu Pro Pro Ser His Arg Arg Leu Arg Leu Leu Leu XiS
: 45~
Ser Arg Val Pro Gly Gly Sçr Thr Val Ala Arg xis Pro_Thr Arg Gln Gl}~ His Arg Gly Val Ser Gly Pro Ser~~i5 Pro Gly Thr~Ala Gl} Arg ~al Thr Cys Thr Ala Asp Gly Gly His Ser Tyr Pro Gly Ala Leu Pro ~yr Asr. Ile His Ala Arg Leu Glu Arg Gly V~al Cys Tyr Asn Gly Trp lD0 lD5 ~ =llQ ~ = _ ~eu Trp Gly Gly Ala Val Asp Asn ~2) INFORMATION FOR SEQ ID NO 30 (i) SEQUENOE r~r~T~TICS:

t W 096/06159 - 2=1 96892 PCTAUS95~1019~

~ 233 (A) LENGT~: 921 base pairs (B) TYPE: nucleic acid (C) ST~ ~PC~: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA ~genomic) N
~iv) ANTI-SENSE: N
(ix~ FEATU~E:
(A) NAME/~EY: CDS
(B) LOCATION: 1..921 (D) OT~ER INFORMATION:

(xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:30:

Met Leu Leu Ser Ary ~is Arg Glu Arg Leu Ala AIa Asn Leu Glu Glu ACC GCC A~A GAC GCC GGA GAG AGG TGG GAA CTG AGT GC- CCG ACA TTC 96 Thr Ala Lys Asp Ala Gly Glu Arg Trp Glu 1eu Ser Ala Pro Thr Phe 20 25 ~ 30 ACG CGA CAC TGT.CCC AAA ACG GCA CGG ATG GCG CAC CCT T~T ATT GGC 144 Thr Arg Bis Cys Pro Lys Thr Ala Arg Met Ala Bis Pro Phe Ile Gly GTG GTG CAC AGA ATA A~C TCA TAC AGT TCG GTC CTG GAA ACA TAC TGC 192 Val Val Bis Arg Ile Asn Ser Tyr Ser Ser val Leu Glu Thr Tyr Cys Thr Arg Bis His Pro Ala Thr Pro Thr Ser Ala Asn Pro Asp Val Gly Th- Pro Arg Pro Ser Glu Asp Asn Val Pro Ala Lys Pro Arg Leu Leu GAG TCC CTA TCA ACA TAC TTG C~G ATG CGG TGT GTG CGC GAG GAC GCG 336 Glu Ser Leu Ser Thr Tyr Leu Gln Met Arg Cys Val Arg Glu Asp Ala CAC GTC TCC ACG.~CC GAT CAA CTG GTC GAG TAC CAG GCG GGC AGA AAA 384 Lis Val Ser Thr Ala Asp Gln Leu Val Giu Tyr Gln Aia Gly Arg Lys lli : 120 125 ACA CAC GAC TCC CTG CAC GCC TGC TCT GTC TAC CGD GAA CTT CAG GCT 43~
T~- ~is Asp Ser Leu ~is Ala Cys Ser Val Tyr Arg Glu Leu Gln Ala -TT CTG GTT A~C CTT TCG TCC TTT CTG AAC GGC TG~ TAC G-T CCC GGG 480 Phe Leu Val Asn Leu Ser Ser Phe Leu Asn Gly Cys Tyr Val Pro Gly G-G CAC TGG CTG GAG CCC TTC CAA CAG C~G.CTA GTA ATG CAC ACT TTT 528 Val Bis Trp Leu Glu Pro Phe Gln Gln Gln Leu Val Met Bis Thr Phe Phe Phe Leu Val Ser Ile Lys Ala Pro Gln Lys Thr Bis Gln Leu Phe WO96/06159 i 2 1 9 6 8 9 2 PcT~usgS/I0!9J--Gly Leu Phe Lys Gln Tyr Phe Gly Leu Phe Glu Thr Pro Asn Ser Val l9S 200 205 Leu Gln Thr Phe Lys Gln Lys Ala Ser Val Phe Leu Ile Pro Arg Arg 210 -215 2ao His Gly Lys Thr Trp Ile Val Val Ala Ile Ile 5er Met Leu Leu Ala TCC GTA GAG A~C ATT AAC ATT GGG TAC GTA GCC CAC CAA A~G CAC GTA 76B
ser Val Glu Asn Ile Asn Ile Gly Tyr Val Ala Xls Gln Lys Xis Val 245 250 255::
GC_ AAC TCC GTG TTC GCG GA~ 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 CCC CCC AaA A~T TTA AAC ATC AAG AAG GAG ~AC GGA ACC ATA ATC TAC 864 Pro Pro Lys Asn Leu Asn Ile Lys Lys Glu Asn Gly Thr Ile Ile Tyr Thr Arg Pro Gly Gly Arg Ser Ser Ser Leu Met Cys Ala Thr Cys Phe AAT A~G AAC 921 Asn Lys Asn (2) INFORMATION FOR SEQ ID NO:31 (i) SEQUENCE rH~rTF~TCTICS:
(A~ LENGTH 307 amino acids (P) TYPE: ~mino aoid (D~ TOPOLOGY: linear (ii) MOLECULE TYPE protein (xi) SEQUENCE DESCRIPTION SEQ ID NO:31 ~et Leu Leu Ser Arg Xis Arg Glu Arg Leu Ala Ala Asn Leu Glu Glu ~hr Ala Lys Asp Ala Gly Glu Arg Trp Glu Leu Ser Ala Pro Thr Phe Thr Arg His Cys Pro Lys Thr Ala Arg Met Ala His Pro Phe Ile Gly Val Val His Arg Ile Asn Ser Tyr Ser Ser Val Leu Glu Thr Tyr Cys Thr Arg His His Pro Ala Thr Pro Thr Se- Ala Asn Pro Asp Val Gly 7s ao ~kr Pro Arg Pro Ser Glu Asp Asn Val Pro Ala Lys Pro Arg Leu Leu ~lu Ser Leu Ser Thr Tyr Leu Gln Met Arg Cys Val Arg Glu Asp Ala ~is Val Ser Thr Ala Asp Gln Leu Val Glu Tyr Gln Ala Gly Arg Lys llS 120 125 W 096/06159 2 ) 9 6 8 9 2 PCTAUS95/1019 23~
Thr His Asp Ser Leu His Ala Cys Ser.Val Tyr Arg Glu Leu Gln Ala 13Q . ~ ~ ~ ~ 135 - 14Q
Phe Leu Val Asn Leu Ser Ser Phe Leu Asn Gly Cys Tyr Val Pro Gly ~al His Trp Leu Glu Pro Phe Gln Gln Gln Leu Val Met His Thr Phe ~he Phe Leu Val Ser Ile.Lys Ala Pro Gln Lys Thr His Gln Leu Phe 180 , 135 . 190 Gly Leu Phe Lys Gln Tyr Phe Gly Leu Phe Glu Thr Pro Asn Ser Val 195 ~ 200 ~ 205 Leu Gln Thr Phe Lys Gln Lys Ala Ser Val Phe Leu Ile Pro Arg Arg 210 215 .220= .
His Gly Lys Thr Trp Ile Val Val Ala Ile Ile Se~Me~ Leu Leu Ala 225 = ~ ~ 23Q . 235 .~ - 240 ~er Val Glu Asn Ile Asn Ile Gly Tyr Val Ala His Glr. Lys His Val ~la Asn Ser Val Phe Ala Glu Ile Ile Lys Thr Leu Cys 2A7rg0 Trp Phe Pro Pro Lys Asn Leu Asn Ile Lys Lys Glu Asn Gly Thr Ile Ile Tyr 275 ~ 280 2bS
Thr Arg Pro Gly Gly Arg Ser~S~èr 3er Leu Met Cys Ala Thr Cys Phe Asn Lys Asn 305 : ~:
(2) INFORMATION FOR SEQ ID NO:32:
~i1 SEQUENCE C~ARACTERISTICS
A) LENGTH: 1365 base pairs 3) TYPE: nucleic acid C) STR~nFnNFC':: single D) TOPOLOGY: lirear (ii) MOLECULE TYPE: DNA (genomic) (iii) B~YPOTHETICAL: N
(iv) ANTI-SENSE: N
( iY. ~ FEATURE:
(A) NAME/REY: CDS
(B) LOCATION: 1 .1365 (D) QTHER INFORMATION:

(xil SEQUENCE DFscRIpTIoN: SEQ ID NO:32:
ATG G~T GCG CAT ~T ATc Aac GAA AGA TAC GTA GGT C_T;CGC TGC CAC 4b Me~ Asp Ala His Ala Ile Asn Glu Arg Tyr VaI Gly Pro Arg Cys His ~ 5 . 1 0 , 15 CGT TTG GCC CAC G~G.GI~LCTG CCT AGG ACC TTT CTG CTG CAT CAC GCC 96 Arg Leu Ala His Val Val Leu Pro Arg Thr Phe Leu Leu_His Hls Ala 20 25 .= 30 ATA CCC CTG GAG CCC ~AG ATC ATC TT TCC ACC TAC ACC CGG.TTC AGC 144 W 096/06159 ' 2 1 9 6 8 9 2 PCTAUS9SI1019 ~

2~6 Ile Pro Leu Glu Pro Glu Ile Ile Phe Ser Thr Tyr Thr Arg Phe Ser . 40 45 CGG TCG CC~ GGG TCA TCC CGC CGG TTG GTG GTG TGT GGa AAA CGT GTC 19' Arg Ser Pro Gly Ser Ser Arg Arg Leu Val Val Cys Gly Lys Arg Val So ~ SS 60 CTG CCA GGG GAG aAA AAC CAA CTT GCG TCT TCA C~T TCT GGT TTG GCG 240 Leu Pro Gly Glu Glu Asn Gln Leu Ala Ser Ser Pro Ser Gly Leu Ala CTT AGC CTG CCT CTG TTT TCC CAC GAT GaG~AAC TTT CAT- CCA TTT GAC 238 Leu Ser Leu Pro Leu Phe Ser ~is Asp Gly Asn Phe Pis Pro.Phe Asp 85 90' 9S
ATC TCG GTA CTG CGC ATT TCC TGC CCT GGT TCT AAT CTT ~GT CTT ACT 336 Ilc Ser Val Leu Arg Ile Ser Cys Pro Gly Ser Asn Leu Ser Leu Thr GTC AGA TTT CTC TAT CTA TCT CTG GTG GTG GCT'ATa GGa GCG GGA-CGG 384 Val Arg Phe Leu Tyr Leu Ser Leu Val Val~Ala~Met Gly Ala Gly Arg~ ~ :
llS ~ ~ ~ 120 125 AAT AAT GCG CGG AGT CCG ACC GTT GAC GGG GTA TCG CCG CCA GAG GGC : 432 , =
Asn Asn Ala Arg Ser Pro Thr Val Asp Gly Val Ser Pro Pro Glu Gly GCC GTA GCC CAC CCT TTG GAG aAA CTG CAG AGG CTG GCG CGT GCT~ ACG 480 Ala Val Ala ~is Pro Leu Glu Glu Leu Gln Arg Leu Ala Arg Ala Thr 145 lS0 lSS : 160 CCG GAC CCG GCA CTC ACC CGT GGA CCG TTG CAG GTC CTG ACC GGC CTT u: 528 Pro Asp Pro Ala Leu Thr Arg Gly Pro Leu Gln Val Leu Thr Gly Leu CTC CGC GCA GGG TCA GAC GGA GAC CGC GCC ACT CAC CAC ATa GCG CTC 576 Leu Arg Ala Gly Ser Asp Gly Asp Arg Ala Thr ~is ~is Met Ala Leu GAG aCT CCG GGA ACC GTG CGT GGA GAA AGC CTA -vAC CCG CCT GTT TCA : 624Glu Ala Pro Gly Thr Val: Arg Gly Glu Ser Leu Asp Pro Pro Val Ser 195 ~ 200 : 205 Gln Lys Gly Proi~la Ar~ Thr Arg ~is Arg Pro Pro Pro Val Arg~Leu AGC TTC AAC CCC GTC AAT GCC GAT GTA CCC~GCT ACC TGG CGA GAC acc 720 Ser Phe Asn Pro Val Asn Ala Asp Val Pro Ala Thr Trp Arg Asp Ala 225 230 235 - . ~240 ACT AA2 GTG TAC TCG GGT GCT CCC TAC TAT GTG TGT GTT TA~ GAA CGC -~ 768Thr Asn Val Tyr 5er Gly Ala Pro T~r Tyr Val Cys Val Tyr Glu Arg GGT GGC CGT CAG GAA GAC GAC TGG CTG CCG'ATA CCA CTG AG_ TTC CCA 816 Gly Gly Arg Gln Glu Asp Asp Trp Leu Pro Ile Pro~Leu 5er Phe~Pro 260 26s : ~ : 270 GAA GAG ~ C GTG C C CC~_CCA CCG GGC TTA GTG TTC ATG GAC GAC TTG 86i Glu Glu Pro Val Pro Pro Pro Pro GIy Leu Val Phe Met Asp Asp Leu TTC ATT AAC ACG AAG CAa TGC GAC TTT GTG GAC ACG CTA GAG GCC:GCC : 912 Phe Ile Asn Thr Lys Gln Cys Asp Phe Val Asp Thr Leu Glu Ala Ala 290 zg5 300 ~ WO 96/061S9 2 1 9 6 8 9 2 PCTrUS9~ll0l9~

._~ 2~7 TGT CGC ACG CA~ GGC TAC ACG TTG AGA CAG CGC GTG CCT GTC GCC ATT 960 Cys Arg Thr Gln Gly Tyr Thr Leu Arg Gln Arg Vai Pro Val Ala }le 30; 310 315 320 CCT CGC GAC GCG GAA AT~ GCA GAC GCA GTT A~A TCG CAC TTT TTA GAG lOOB
Pro Arg Asp Ala Glu Ile Ala Asp Ala Val Lys Ser His Phe Leu Glu GCG TGC CT~ GTG TTA CGG GGG CTG GCT TCG GAG GCT AGT GCC TGG ATA 1056 Ala CYB Leu~Val Leu Arg Gly Leu Ala Ser Glu Ala Ser Ala Trp Ile AGA GCT GCC ACG TCC CCG CCC CTT GGC CGC CAC GCC:TGC TGG ATG GAC 1104 Arg Ala Ala Thr Ser Pro Pro Leu Gl~ Arg Hls Ala Cys Trp Met Asp 355 :~ 360 ~ 365 GTG TTA GGA TTA TGG GAA AGC CGC CCC CAC ACT CTA GGT TTG G~G T~A 1152 Val Leu Gly Leu Trp G1u Ser Arg Pro His Thr Leu Giy Leu Glu Leu 370 . 3J5 38'0 CGC GGC GTA AAC TGT GGC GGC ACG GAC GGT GhC TGG TTA GAG ATT TTA 1200 Arg Gly Val Asn Cys Gly Gly Thr Asp Gly Asp Trp Leu Giu Ile Leu A~A E~G CCC ~AT GTG CAA AAG ACA GTC AGC GGG AGT CTT GTG GCA TGC 1248 Lys Gln Pro Asp Val GIn Lys Thr Val Ser Gly Ser Leu Val Ala Cys Val Ile Val Thr Pro Ala Leu Glu Ala Trp Leu Val Leu Pro Gly Gly TTT GCT ATT AaA GCC CGC TAT AGG GCG TCG AAG GAG GAT CTG GTG TTC 1344 Phe Ala Ile Lys Ala Arg Tyr Arg Ala Ser Lys Glu Asp Leu Val Phe ATT CGA GGC CGC TAT GGC T~G 1365 Ile Arg Gly Arg Tyr Gly ~21 INFORMATION FOR SEQ ID NO:33:
(i~ SEQUENCE r~
(A~ LENGTH: 454 amino acids (5) TYPE: amino aeid (D) TOPCi~OGY: linear ~iil MOLECCLE TYPÉ: proteiu ~xi) SEQUEN OE DESCRIPTION: SEQ ID NO:33:~':
Met Asp Ala His Ala Ile Asn Glu Arg Tyr Val Gl~ Pro Arg Cys His . , 10 ,. _ 15 Arg Leu Ala His Val Val Leu Pro Ar~ Thr Phe Leu Leu His His Ala 2s 30 Ile Pro Leu Glu Pro Glu Ile Ile Phe Ser Thr Tyr Thr Arg Phe Ser 4, Arg Ser Pro Gly Ser Ser Arg Arg Leu Val Val Cys Gly Lys Arg Val Leu Pro Gly Glu Glu Asn Gln Leu Ala Ser Ser Pro Ser Gly Leu Ala WO96/06159 ' 2 1 96892 PCT/US95/10194~

Leu Ser Leu Pro Leu Phe Ser }~is Asp Gly Asn Phe E~is Pro Phe Asp 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 Asn Acn Ala Arg Ser Pro~Thr Val Asp Gly Val Ser Pro prQ Glu Gly Ala Val Ala Elis Pro Leu Glu Glu Leu Gln Arg Leu Ala Arg Ala Thr 145 150 .155 - 160 ~ro Asp Pro Ala Leu Thr Arg Gly Pro Leu Gln Val Leu Thr Gly Leu ~eu Arg Ala Gly Ser Asp Gly Asp Arg AIa Thr Xis Elis Met Ala Leu 180 185 : ~[9o Glu Ala Pro Gly Thr Val Arg Gly Glu Ser ~Leu Asp Pro Pro Val ~ Ser ~1 Gln Lys Gly Pro ~la Arg Thr Arg }~is Arg Pro PrQ 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 ~: Z40 ~hr Asn Val Tyr Ser Gly Ala Pro Tyr Tyr Val Cys Val Tyr GIu Arg ~ly Gly Arg Gln Glu Asp Asp Trp Leu Pro Ile Pro Leu Ser Phe Pro Glu Glu Pro Val Pro Pro Pro Pro Gly Leu Val Phe Met Asp Asp Leu Phe Ile Asn Thr Lys Gln Cys Asp Phe Val Asp Thr Leu Glu Ala Ala 290 295 ~ 300 Cys Arg Thr Gln Gly Tyr Thr Leu Arg Gln Arg Val PrQ Val Ala Ile 305 310 ~315 - .320 ~ro Arg Asp Ala Glu Ile Ala Asp Ala Val Lys Ser ~las Phe Leu Glu 325 330 ~ 335 ~la Cys Leu Val Leu Arg Gly Leu Ala Ser Glu Ala Se~ Ala Trp Ile Arg Ala Ala Thr Ser Pro Pro Leu Gly Arg His Ala Cys Trp Met Asp 355 : , 360 365 ~
Val Leu Glv Leu Trp Glu Ser Arg Pro Pis Thr Leu Gly Leu Glu= Leu Arg Glv Val Asn Cys Gly Gly Thr Asp Gly Asp Trp Leu Glu Ile Leu 385 390 ~395 ~_ _ 400 ~ys Gln Pro Asp Val Gln Lvs Thr Val Ser Gly Ser Leu Val Ala Cys 405 4io : 415 ~al Ile Val Thr Pro Ala Leu Glu Ala Trp Leu Val Leu Pro Gly Gly ~he Ala Il~ Lys Ala Arg T~r Arg Ala Ser Lys Glu Asp Leu Val Phe ~ W O96/06159 2 1 9 6 8 9 2 PCTAU59~10194 .Ile Arg Gly Arg Tyr Gly (2) INFORMATION FOR SEQ ID NO:34:
(i) SE~ENCE r~rT~RTqTIcs:
A LENGTH: 984 base pairs B TYPE: nucleic acid C ST~Fn~ single ~D TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) ~iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(ix~ FEATCRE:
(A) NAME/~EY: CDS
(E) LOCATION 1 984 (D) OTHER INFORMATION:

(xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:34:
ATG TTT GCT TTG ~rr TCG CTC GTG Trc-~AG GGT GAC CCG G~G GTG ACC 48 Met Phe Ala Leu Ser Ser Leu Val Ser Glu Gly Asp Pro Glu Val Thr 5 10 . _ _ _ . . 15 AGT AGG TAC GTC AAG GGC GTA CAA CTT GCC ~G GAC CTT AGC GAG Aac 96 Ser Arg Tyr Val Lys Gly Val Gln Leu Ala Leu Asp Leu Ser Glu Asn ACA CCT GGA CAA T~T_~G TTG ATA GAA ACT CCC CTG AAC AGC TTC CTC 144 Thr Pro Gly Gln Phe Lys Leu Ile Glu Thr Pro Leu Asn Ser Phe Leu 35 . 40 45 Leu Val Ser Asn Val Met Pro Glu Val Gln Pro Ile Cys Ser Gly Arg 50 ~ SS 60 CCG GCC TTG CGG CCA GAC TTT 8U~T AAT CTC CAC TTG rrT ~r~ rTr. GAG 240 Pro Ala Leu Arg Pro Asp Phe Ser Asn Leu His Leu Pro Arg Leu Glu 65 ::~0 ~ : =~S = ~ 80 AAG CTC CAG AGA GTC CTC GGG CAG GGT TTC GGG GCG.GCG GGT GAG GAA 288 Lys Leu Gln Arg Val Leu Gly Gln Gly Phe Gly Ala Ala Gly Glu Glu Ile Ala Leu Asp Pro Ser His Val Glu Thr His Glu Lys Gly Gln Val Phe Tyr Asn His Tyr Ala Thr Glu Glu Trp Thr lrp Ala Leu Thr Leu llS : 12~ ~: 125 Asn Lys Asp Ala Leu Leu Arg Glu Ala Val Asp Gly Leu Cys Asp Pro Gly Thr Trp Lys Gly Leu Leu Pro Asp ASp Pro Leu Pro Leu Leu Trp 145 lSO lSS 160 WO 96/06159 ~, 2 1 9 6 8 9 2 PCT/US95/1019--Leu Leu Phe Asn Gly Pro Ala Ser Phe Cys Arg Ala Asp Cys Cys Leu Tyr Lys Gln ~is Cys Gly Tyr Pro Gly Pro Val Leu Leu Pro Gly His 180 185 ~ 190 ATG TAC GCT CCC A~A CGG GAT CTT TTG TCG TTC ~TT AAT CAT GCC CTG 624 Met Tyr Ala Pro Lys Arg Asp Leu Leu Ser Phe Val Asn ~is Ala Leu AAG TAC ACC A~G TTT CTl T~C GGA GAT TTT TCC GGG ACA TGG GCG GCG 672 Lys Tyr Thr Lys Phe Leu~=Tyr Gly Asp Phe Ser Gly Thr Trp Ala Ala GCT TGC CGC CCG..CCA TTC GCT ACT TCT CGG ATA CAA AGG GTA GTG AGT : 720 Ala Cys Arg Pro Pro Phe Ala Thr Ser Arg Ile Glr. Arg Val Val Ser 225 230 _ 235 ~240 CAG ATG A~A ATC ATA GAT GCT TCC GAC ACT TAC ATT TCC CAC ACC TGC 768 Gln Met Lys Ile Ile Asp Ala Ser Asp Thr Tyr Ile Ser His Thr Cys CTC TTG TGT CAC ATA TAT CAG CAA AAT AGC ATA ATT GCG GGT CAG GGG . 816 Leu Leu Cys ~is Ile Tyr Gln Gln Asn Ser Ile Ile Ala Gly Glr. Gly ACC CAC GTG GGT GGA ATC CTA CTG TTG AGT GGA A~A GGG ACC CAG TAT 864 Thr ~is Val Gly Gly Ile Leu Leu Leu Ser Gly Lys Gly Thr Gln Tyr ATA A~A GGC AAT GTT CAG ACC C~A AGG TGT CCA ACT ACG GGC GAC TAT 912 Ile Thr Gly Asn Val Gln Thr Gln Arg Cys Pro Thr Thr Gly Asp Tyr 290 29s 300 CTA ATC ATC CCA TCG T~T GAC ATA CCG GCG ATC ATC ACC ATG ATC AAG 960 Leu Ile Ile Pro Ser Tyr Asp Ile Pro Ala Ile Ile Thr Met Ile Lys 30s 310 315 320 GAG AAT GGA CTC AAC C~A CTC TAA 984 Glu Asn Gly Leu Asn Gln Leu ~2) INFORMATION FOR SEQ ID NO:35~
(i) SEQ~ENCE C~ARA-~ ~lS ' lOS:
(A) LENGTE: 327 amino acids (;3) TYPE: amino acid (D) TOPOLOG}': linear ~ii) MOL~C'~LF TYPE: protein ~i) SEQ~ENCE D~SCPIPTION: SEQ IC NO:35:
~et Pne Ala Leu Ser ~r Le~ Val 5er Glu Gly Asp Pro Glu Val Thr ~er Arg Tyr Val Lys Gly Val Gln Leu Ala Leu Asp Leu Ser Glu Asn Thr Pro Gly Gln Phe Lys Leu Ile Glu Thr Pro Leu Asn Ser Phe Leu Leu Val Ser Asn Val Met Pro Glu Val Gln Pro Ile Cys Ser Gly Arg ~ W 096106159 2 l 9 6 8 9 2 PCTrUS9511019~
}

~ 24~1 Pro Ala Leu Arg Pro Asp Phe Ser Asn Leu His Leu Pro Arg Leu Glu ~ 80 Lys Leu Gln Ar~ Val Leu,Gly Gln Gly Phe Gly Ala Ala Gly Glu Glu . 90 95 -Ile ~la Leu ~sp Pro~Ser~ His Val Glu Thr ~is Glu Lys Gly Gln Val100 105 110 Phe Tyr Asn Dis Tyr Ala Thr Glu Glu Trp Thr Trp Ala Leu Thr Leu 115 ~ 120 ~ ' i25 Asn Lys Asp Ala Leu Leu Arg Glu Ala Val Asp Gly Leu Cys Asp Pro Gly Thr Trp Lys Gly Leu Leu Pro Asp Asp Pro Leu Pro Leu Leu Trp 145 150 155 ~ 160 Leu Leu Phe Asn Gly Pro Ala Ser Phe Cys Arg Ala Asp Cys Cys Leu Tyr Lys Glr. Hls ,Cys_G.ly Tyr Pro Gly Pro Val Leu Leu Pro Gly His l~O 185 - 190 Met Tyr Ala Pro Lys Arg Asp Leu Leu Ser Phe Val Asn ~is Ala Leu LYB Tyr Thr Lys Phe Leu ~yr Gly Asp Phe Ser Gly Thr Trp Ala Ala 210 215 = 220-Ala Cys Arg Pro Pro Phe Ala Thr Ser Arg Ile Gln Arg Val Val Ser225 ~ ~ 230 235 240 Gln Met Lys Ile Ile Asp Ala Ser Asp Thr Tyr Ile Ser His Thr Cys Leu Leu Cys His Ile Tyr Gln Gln Asn Ser Ile Ile Ala Gly Gln Gly Thr His Val Gly Gly Ile~Leu Leu Leu Ser Gly Lys Gly Thr Gln Tyr Ile Thr Gly Asn Val Gl~,Thr Gln Arg Cys Pro Thr Thr Gly Asp Tyr Leu Ile Ila Pro Ser Tvr Asp Ile Pro Ala Ile Ile Thr Met Ile Lys 30s ~ 315 . _- . 320 Glu Asn Gl~ 1eu Asn Gln Leu ~2) INFORMATION FOR SEQ ID NO:36:

~i) SEQ~ENC3 r~T~T5TICS:
- A LE~GT~: 330 base pairs B TYP3: nucleic acid C , ~ C: single ~D TOPOLOGY linear MOL3C~LE TYPE: DNA (genomic) ~iii) ~Y~u~ ~L: N
(iv) ANTI-SENSE: N
~Y.i) SEQUENC3 ~.~U~l~llUN: SEQ ID NO:36:

. ~ . .
., ~. .

W O96/06159 '; 2 1 9 6 8 9 2 PCTrUS9~l019 GGATCCCTCT GACAACCTTC DrDT~D~ CGTATATGCC ~U~llLl~L~ DrTr~r7r~DrDr - 60 CAACACCCAG CTAGCAGTGC TACCCCCATT TTTTAGCCGA AAGGATTCCA ~uAI~vlvul 120 CGAATCCAAC GGATTTGACC 5~bl~ll~U C~TGGTCGTG rrrrDrrDDr TGGGGCACGC 180 TATTCTGCAG CAGCTGTTGG TGTACCACAT CTACTCCAaA~AT~TCGGCCG (~ [~ 240 TGATGTAAAT ATGrcGGAAc TTGATCTATA TDrrDrr~Dm GTGTCATTTA TGGGGCGCAC 300 ATATCGTCTG,G CGTAGACA ACACGGATCC 330 .) INFOKMATION FOR SEQ ID NO 37 li~ SEQUENCE r~DRDrT~RTcTIcs A) LENGT~: 627 base pairs B) TYPE: nucleic acid C) STRDNnFnNFcc single D) TOPOLOGY linear ~ii) MOLECU~E TYPE: DNA (genomic) ~iii) ~iY~Ul~l~llLI~: N
~iv) ANTI-SENSE: N
~xi) SEQUENCE ~S.K1~11~ SEQ ID NO 37:
GGATCCGCTG GCDGGTGGGC GCGCACCTCG TCGGGTAGCT Tr.r.Dr~ rDrrTrrDr~r~ 60 CCAGTCCGCG rrrTDrrrrr TGCAGGTGCC TCACCACCGG WU~Vl~A TGCGATCTGT 120 TTAGTCCGGA r~Dr~ATDrr~G CCCTTGGGAA rrrrrTr~r CAGCTCCAGG GTCTCCAAGA 180 TGCGCACCGG TTGTCGGAGC TGTCGCGATA GAGGTTAGGG TAGGTGTCCG ~l~U~l~U~l 240 GGGCTCAAAC CTGCCCAGAC ~rDrrDrTGT UlVUl~V~ ATCATCCTTC TCAGGGAGAT 300 GCGATGGTGC GCACCGTTTT T~ rrr rrrDr-Q~rTr~r~ rr~rTrrrr~r TCCCTGCAGC 420 rrrDrr.r.rrr ~ V~lVbU A~DrDrrrrr GTGAGGGCCC Culvv~l~lV 'lU~'V'~ VA 540 AACAGGGTGC TGTGAAACAA CAGGTTGCAA rGrrr~rr-DDT ACCCCTCTGC ACGCTGCTGT :600 GGACGTGGGT GTATGCTCCG TGGATCC ~ 627 ~2) INFOKMATION FOK SEQ ID NO 38 ~i) SEQUENCE C~DRDrT~RTqTICS
A) LENGT~: 233 base pairs B) TYPE: nucleic acid IC) STRDunFnNFcc single D) TOPOLOGY: linear (ii) MO~ECULE TYPE DNA (genomic) (iii) ~YPOT~ETICAL: N
(iv) ANTI-SENSE: N
(xi) SEQUENCE DESCRIPTION SEQ ID Nû:38 ~ W 096/06159 2 1 ~ 6 8 9 2 PCTnUS9i~1019~

~rrrrL~rir ATTCCACCAT TGTGCTCGAA TCC~CGGAT TTGACCCCGT L-~l~L~'lL 60 Ll~L~ ( , PrrLLrTr~ir GCACGCTATT rTrrLr~rLrr ,~ ~ CCACATCTAC 120 TCCAALATAT ~ CCCGGATGAT rTLD~T~Trr CGGAACTTGA TCTATATACC 180 ACCAATGTGT rLTTT~Tr~r~r~ rrrrLrLTLT CQTCTGGACG T~rrrLLrLr GGA 233 (2) INFORMATION FOR SEQ ID NO 39 (i) SEQ~ENCE r~L~LrT~TCTICS
(A) LENGT~ 323 base pairs (B) TYPE nucleiç acid (C) sTFD~nr~qq single (D) TOPOLOGY linear (iil MOLECULE TYPE DNA (genomic) (iii) ~Y~u,~llLAL N
(is-) ANTI-SlNSE N
(xi) SEQUENCE DESCRIPTION SEQ ID NO 39 GAAATTACCC ACGAGATCGC TTCCCTGCAC AccGcAcTTG GrTLrTrLTr LnTrLTrrrr 60 rrnr,rrrLrr TrrrrrrrLT L~rTLrrr r LTrrrLrTLr LTTrTrLrrL LLl.L~ 120 ATTTTCCC~G rnrLrrrrT~ TrrrrLrcrr CAGCTGCATG ACT~TATCAA AATGALAGCG 180 GGCGTGCP~A rrrnrTrLrr Cr~LLrLnL ATGGATCACG Trrr~TLrLr ~L~L-LI~ 240 LL~.GLl~LL AGALCCTGCC CGGTTTGAGT CATGGTCAGC Tr~r~rp~rrTr CGAGATAATT 300 rrrLrnrrr,n TC_C~TGA CGTTGCCT ~ _ 328 (2) INFORMATION FO~ SEQ ID NO 40 (i) SEQUENCE r~LrTFTTqTIcs A LENGT~ 132 ~ase pairs TYPE nucleic arid C ST~Fn'~CC: single D TOPOLOGY linear (ii) MOLECULE TYPE DNA (genomic) (iii) ~Y~oLd~llLAL N
(iv) ANTI-SENSE N
(xi) SEQUENCE DESCRIPTION SEQ ID NO 40 ~Lr~rnTrLT rTrrLrr~LnT GACATTGTGC rnrnrLr~ rTrLnLrrnr LTrrrr,TLLr 60 CACACTGAGT GGGAAAATCT LL~LL1~1~ Lll~l~L~A TT~TCTATGC CTTAGATCL- 120 AACTGTCACC CG ~ 132 (2) INFORMATION FOR SEQ ID NO 41 (i) SEQUENCE r~T~LrTFTTqTIcs:
(A) LENGTE 40 ~ase pairs ('O) TYPE nucleic acid (C) ~ .NI~ N~ : single W 096/06159 2 1 9 6 8 9 2 PCT~DS95/1019 ~

(D) TOPOLOGY: linear (ii) MOLEC~LE TYPE: DNA (genomic) (iii) ~YPOTHETICAL: N
(iv) ANTI-SENSE: N
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:il:
rr~ ATTCCACCAT 1~1~1L~l CTACGTCCAG =~~40 (2) INFORMATION FOR SEQ ID NO:42 (i) SEQUENCE CHARACTERISTIC5~
(A) LENGTH: 38 base pairs (E) TYPE: nucleic acid (C) ST~Fn~q.q: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOT ETICAL: N
(iv) ANTI-5ENSE: N
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
GAAATTACCC ACGAGATCGC AGGCAACGTC ~GATGTGA38 (2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE C~ARACTERISTICS:
~A LENGTH: 46 base pairs (B TYPE: nucleic acid (C ST~Nn~nN~qC: single (D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (:ii) ~Y~u~ L: N
~iv) ANTI-SENSE: N
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE ~rT~.qTICS:
A~ LENGTH: 21 base pairs E~ TYPE: nucleic acid C~ ST~Nn~nN~cq: single D TûPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(xi) SEQUENCE DESCRIPTIQN: SEQ ID NO:44:

~ W 096/06159 2 ~ 9 6 8 9 2 PCTrDS95/~019~

24~
~r~ ~Tr.~. TTGCCCAGGG T 21 (2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE r~a~TF~T.CTICS:
A LENGT~: 20 base pairs IB TYPE: nucleic acid ,C ST~Nn~NFcc single D. TOP0LOGY: lir,ear (ii) MOLECULE TYPE DNA (genomic) ~ :
( iil) liY ~U L~ .:All . N
(i~) ANTI-SENSE: N
(xi) SEQ~ENCE ~ ~lrllU~: SEQ ID NO:45:
AGTTGCA~AC CAGACCTCAG 20

Claims (42)

What is claimed is:
1. An isolated DNA molecule which is at least 30 nucleotides in length and uniguely 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.
5. The isolated DNA molecule of claim 1 which is labelled with a detectable marker.
6. 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, .lambda. phage or YAC containing at least a portion of the isolated DNA molecule of Claim 1.
9. A host cell containing the vector of claim 7.
10. 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.
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.
15. 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 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).
17. An isolated peptide encoded by at least a portion 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 17.
19. The isolated peptide of claim 17, wherein the peptide is linked to a second peptide to form a fusion protein.
20. 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.
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 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.
29. A triplex oligonucleotide capable of hybridizing with a double stranded isolated DNA molecule of claim 1.
30. A transgenic nonhuman mammal which comprises at least a portion 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: (a) obtaining a nucleic acid molecule 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 (c) 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 (b).
34. A method of diagnosing Kaposi's sarcoma which comprises: (a) 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 (c) 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 DNA virus associated with Kaposi's sarcoma which comprises (a) obtaining a suitable bodily fluid sample from a 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 Kaposi's sarcoma antibody to a specific Kaposi's sarcoma antigen, (c) 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.
36. A method of diagnosing a DNA virus associated with Kaposi's sarcoma which comprises (a) obtaining a suitable bodily fluid sample from a subject, (b) 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, (c) removing unbound bodily fluid from the support, and (d) 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 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 subject, 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 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.
40. A method of vaccinating a subject against Kaposi's sarcoma, comprising administering to the 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 12.
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 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 KS-associated human herpes virus of claim 12.
CA002196892A 1994-08-18 1995-08-11 Unique associated kaposi's sarcoma virus sequences and uses thereof Abandoned CA2196892A1 (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US29236594A 1994-08-18 1994-08-18
US08/343,101 US5830759A (en) 1994-08-18 1994-11-21 Unique associated Kaposi's sarcoma virus sequences and uses thereof
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US7932066B2 (en) 1994-08-18 2011-04-26 The Trustees Of Columbia University In The City Of New York Unique associated kaposi's sarcoma virus sequences and uses thereof
US5861240A (en) * 1996-02-28 1999-01-19 The Regents Of The University Of California Isolated human herpesvirus type 8 sequences and uses thereof
EA004402B1 (en) * 1996-05-24 2004-04-29 Байоджен, Инк. Modulators of tissue regeneration
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US6348586B1 (en) 1996-07-25 2002-02-19 The Trustees Of Columbia University In The City Of New York Unique associated Kaposi's sarcoma virus sequences and uses thereof
GB9618890D0 (en) * 1996-09-10 1996-10-23 Univ Liverpool An immunogenic determinant
ATE240398T1 (en) 1997-07-11 2003-05-15 Fleckenstein Bernard Prof Dr POLYPEPTIDE ENCODED BY KAPOSI SARCOMA ASSOCIATED HERPES VIRUS (KSHV,HHV-8) AND ITS USE IN DIAGNOSTICS AND THERAPY
US6653465B2 (en) 2000-12-08 2003-11-25 The Trustees Of Columbia University In The City Of New York Spliced gene of KSHV / HHV8, its promoter and monoclonal antibodies specific for LANA2

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