Note: Descriptions are shown in the official language in which they were submitted.
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.
21 96892
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.
2 1 9 6 8 9 2
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
11
(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
13
RM recognizes the EBV polypeptides EBNA1 and p21
in the BCBL-1 cells. Figure 27B shows reactivity
of untreated and chemically treated cells with
serum 01-03 from a patient with KS. Cells were
treated with TPA and n-butyrate for 48 hrs. For
description of the cell lines see Materials and
Methods. The immunoblots were prepared from 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
.. . .. . . . . . . . . . . . . .. . . ...
21 96892
~ 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
21 96892
W096~6159 PCTNS95/101
16
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
21 96892
~ WO96/06159 = PCT~S9~10194
17
"reference sequence" is a defined sequence used as a
basis for a sequence comparison; a reference sequence
may be a subset of a larger sequence, for example, as
a segment of a full-length cDNA or gene sequence given
in a sequence listing or may comprise a complete cDNA
or gene sequence.
Optimal alignment of sequences for aligning a
comparison window may be conducted by the local
homology algorithm of Smith and Waterman (1981) Adv.
Appl. Math. 2:482, by the 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
18
acid substitutions. For example, the substitution of
amino acids having similar=chemical properties such as
charge or polarity are not likely to effect the
properties=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
19
amount to other antigens present in the sample.
Specific binding to an antibody under such conditions
may require an antibody that is selected for its
specificity for a particular protein, For example,
antibodies raised to the human 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
21
This invention provides an isolated polypeptide
encoded by ORF20 (SEQ ID NOs: 22 and 23), ORF21 (SEQ
ID NOs:14 and 15), ORF22 (SEQ ID NOs:16 and 17), ORF23
(SEQ ID NOs:18 and 19), ORF24 (SEQ ID NOs: 20 and 21),
ORF25 (SEQ ID 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
2 1 9 6 8 9 2
Wo96106159 PCT~S95/lO
22
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
23
This invention provides an isolated DNA virus wherein
the viral DNA is about 270 kb in size, wherein the
viral DNA encodes a thymidine kinase, and wherein the
viral DNA is capable of selectively hybridizing to a
nucleic acid probe selected from the group consisting
of SEQ ID NOs: 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
24
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.
1 96892
WO96~61~9 ' ~' ~ PCT~S95/l0l9
26
Infected cells are observed by demonstrating
morphological changes, as well as:being positive for
antigens from the human herpesvirus by using
monoclonal antibodies immunoreactive with the _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
27
filtered to form a single cell suspension by standard
techniques [49, 66]. The cells are separated by
standard Ficoll-Plaque centrifugation and lymphocyte
layer is removed. The lymphocytes are then placed at
~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
28
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
29
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
33
(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 ~
34
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
36
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
W096/06159
37
the desired antibody. Alternatively, monoclonal
antibodies may be produced by in vitro techniques
known to a person of ordinary skill in the art. These
antibodies are useful to detect the expression of
~ 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
38
the use of antibodies coupled to a solid phase. The
EMIT system depends on deactivation of the enzyme in
the tracer-antibody complex; the activity can thus be
measured without the need for a separation step.
Additionally, chemiluminescent compounds may be used
as labels. Typical chemiluminescent compounds include
luminol, isoluminol, aromatic acridinium esters,
imidazoles, acridinium salts, and oxalate esters.
Similarly, 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
39
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
21 96892
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,
41
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
,
21 96892
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
43
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
44
Alternatively, one may take serum from a patient to
see which variant the patient responds to the best.
This invention provides an antagonist capable of
blocking the expression of the peptide or polypeptide
encoded by the isolated DNA molecule. In one
embodiment the antagonist is capable of hybridizing
with a double stranded DNA molecule. In another
embodiment the antagonist is a triplex 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
21 96892
- 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
46
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 ~
48
mRNA is obtained from the cell to detect expression of
the DNA virus.
~.
The suitable bodily fluid sample is any bodily fluid
sample which would contain 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.
21 96892
~ 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~
52
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
21 96892
~ 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
56
intervals and thawed onto 3-aminopropyltriethoxysilane
treated slides and allowed to air dry. The slides are
then be fixed in 4~ freshly prepared paraformaldehyde,
rinsed in water. Formalin-fixed, paraffin ~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
58
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
59
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
61
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
62
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
63
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-
2 1 96892
WO96/06159 ~ S
64
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 ~
66
Merck)) as well as other enzymes. It will be apparent
to one of ordinary skill in the art that there are
additional viral proteins, both characterized and as
yet to be discovered, that can serve as target for
antiviral agents.
iv) Other agents and modes of antiviral
action.
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
68
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
69
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
21 96892
W096/06159 r - ~ r~l/~ ~/1~i91
72
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
73
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 ~
74
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 ~
. . . -. . .
76
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
77
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 ~ .
78
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
79
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.
21 96892
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
81
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
83
Other vaccines may be prepared according to methods
well-known to those skilled in the art.
.-
Those of skill will readily recognize that it is only
necessary to expose a mammal to appropriate epitopes
in order to elicit effective immunoprotection. The
epitopes are typically segments of amino acids which
are a =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
84
being evaluated for the~treatment of KS, which are
targeted to modulating the immune response (i.e.
treatment with copolymer-l, antiidiotypic monoclonal
antibodies, T cell ~vaccination~). Antibodies to
human herpesvirus can be administered to the patient
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~
86
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
87
after several days by IFA for antigens or Southern
blotting for viral genome or Northern blotting for:
MRNA and compared to control cells. This assay can
quickly screen large numbers of chemical compounds
that may be useful against KS.
Further, this invention provides an assay system that
is employed to identify drugs or other molecules
capable of binding to the DNA molecule or proteins,
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
88
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
89
~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
91
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 ~
92
~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
93
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
21 96892
WO96106159 r~ S~
94
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
WO96106159 PCTNS95110194
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
96
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
97
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
21 96892
WO96/06159 ' PCT~S95/10194
98
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.
1 0
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
99
occur in the larger genome. Individual fragments from
the digests failed to simultaneously hybridize with
both KS330Bam and KS627Bam, demonstrating that these
two Bam HI restriction 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.
6 8 9 2
- WO96~6159 PCT~S95110194
100
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
101
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
103
sequencing of ~ double- stranded DNA using
oligonucleotide primers synthesized commercially to
~walk" along the fragments by the dideoxy-nucleotide
chain termination method. Junctions between clones
are ~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
104
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
105
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
106
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
107
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.
.
21 96892
WO96~61~9
108
~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
109
~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~
110
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
112
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
113
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 ~
114
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
115
The herpesvirus may be isolated from the cell DNA in
the following manner. An infected cell line, which
can be lysed using standard methods such as hyposmotic
shocking and Dounce homogenization, is first pelleted
at 2000xg ~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
116
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~ W O 96106159 2 1 ~ 6 8 9 2 P~rruss5llol94
121
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W096/06159 - i 2 1 9 6 ~ 9 2 r~ J ~cls~
122
~ 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
123
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
1 96892
Wo96tO6159 ' ' ' ' PCTtUS9s/lOI9
124
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
125
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
126
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
127
Sera from AIDS-KS patients were drawn between 1 and
31 months after initial KS diagnosis, sera from
intravenous drug user and homosexual/bisexual
; controls were drawn after non-KS AIDS diagnosis, and
sera from HIV-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
2 1 96892
WO96/06159 . ~ : PCT~595/l0l9
128
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,
129
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
21 96892
WO96106159 ~ ~ PCT~S9~/1019
130
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~
131
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~
.......
132
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
W096/06159
133
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
134
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
.
135
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 ~
136
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,
137
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 ~
138
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
WO96/06159 PCTNS95/10194
139
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 ~
140
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~ 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~ ~
2196892 148
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
149
~ . .
~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
150
~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
151
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 ~
152
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
154
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
155
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 ~
156
~ 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
157
~, 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 ~
158
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~
159
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~
.,
160
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
161
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~ ~
162
an EBV genome detectable with several different EBV DNA
probes. B95-8 is an EBV producer marmoset cell line
that can be efficiently induced into EBV lytic cycle
gene expression by phorbol esters (TPA) [33,34]. ~ 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
163
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
165
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
166
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
167
.
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~
168
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
169
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~ ~
170
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.
21 96892
~ W096/06159 ~ ! ' PCT~S95/10194
171
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177
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
~iii) NUMBER OF SEQUEN OES: 45
~iV) ~K~K~NJ~ ADDRESS:
A) DnT)RRc.qRR: Cooper & Dunham LLP
:B) STREET: l18s Avenue o~ the Americas
C) CITY: New York
D) STATE: New York
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(A) APPLICATION NUMBER:
(B) FILING DATE:
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~viii) ATTORNEY/AGENT INFORMATION:
~A) NAME: White, ~ohn P.
~B) REGISTRATION NCMBER: 28,678
~C) ~s~K~N~/DOCRET N~MBER: 45185-C-PCT/~PW/MSC
~ix) TRT.K~I... IN I ~ATION INFORMATION
~A) TELEP~ONE: (212) 278-0400
(B) TELEFAX: (212) 391-0525
~2) INFORMATION FOR SEQ ID NO:I:
(i) SEQUENCE r~rTTT1TcTIcs
(A) LENGTU: 20710 base pairs
(B) TYPE: nucleic acid
(C) STR~R~qq: single
(D) TOPOLOGY: linear ~~
(ii) MOLECULE TYPE: DNA (genomic)
(iii) ~Y~L~ll~AL: N
(iv) ANTI-SENSE: N
(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 ~
178
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
TGACTAGGGG AGGCAGGTGG GACTTGGGGA GCTCGGACGA CGAATCAAGC ACCTCCACAA 480
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
21 96892
W O96/06159 _ PCTrUS95rlO194
179
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
GATCAACGCG CGTCTGGTAC TGGGTGATAT CTTCGCATCA ADATGGTCGC TATTCGCGAG 2580
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
180
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
CTTTTCGAAC AACAGTGGCA TCGGGACTTC AGGATTAGAG ACTCCCACCA TGGCCGCCAC 5400
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
GTGCAGGTCC AACGTGGAGT TCCAATTTGC TATCGATACA GGAIDATATGT GCCTGATTGG 6240
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
181
AGGTTCCTTT Prrr~TPrT TTrRTpT~r r~TTrTT5 GTCAATCTGG GGATGTTTAA 6420
AATAGTCTTT TGCAGGGTGT TAGGAPCGTG GCAGCTTATC TTAGTGTTAA TCACCATGTT 6480
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~
182
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
CATTGAAAGT TTGCAGCGCA TGTACAACGA GACTCAGTTT CCCTGCCCAC TGAACCGGCG 8940
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
AACGTTCCAC CAGTATTACA TGGGGAAAAA CGTGGCATTT GTTCCCGATG TGGCCCAAAA 9300
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
ATTCATCAAC CTCAGGGGTC GCATGGAGGA CCTAGTCAAT AACCTTGTTA ACATTTACCA 10080
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 ~;
183
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~
184
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
185
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~ ~
186
~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
GTCGCCATTC CTCGCGACGC GGAAATCGCA GACGCAGTTA AATCGCACTT TTTAGAGGCG 17400
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~
I88
rTP~ r~ GTTTATTAAG T~UUUlulvG Ar~rrr~r~T r~r~r.r.~r.r. GCAGCTGTAT 20700
CGCTATTTGA 20710
(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:
ATG GAG GCG ACC TTG GAG CAA CGA CCT TTC CCG TAC CTC GCC ACG GAG 48
Met Glu Ala Thr Leu Glu Gln Arg Pro Phe Pro Tyr Leu Ala Thr Glu
5 10 15
GCC AAC CTC CTA ACG CAG ATT AAG GAG TCG GCT GCC GAC GGA CTC TTC 96
Ala Asn Leu Leu Thr Gln Ile LYG Glu Ser Ala Ala Asp Gly Leu Phe
20 25 30
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
CGT TTC GAA GCG CTA CTG GGC GTA TAT ACC AAT GTG GTG GAG TTT GTT 192
Arg Phe Glu Ala Leu Leu Gly Val Tyr Thr Asn Val Val Glu Phe Val
50 55 60
AAG TTT CTG GAG ACC GCC CTC GCC GCC GCT TGC GTC AAT ACC GAG TTC 240
Lys Phe Leu Glu Thr Ala Leu Ala Ala Ala Cys Val Asn Thr Glu Phe
65 70 75 60
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
85 90 95
ATG CCC ACT ATT GCC CAC GGA GAC GGG AGG AGG CCC AAC AAG CAG AGA 336
Met Pro Thr Ile Ala ~is Gly Asp Gly Arg Arg Pro Asn Lys Gln Arg
100 105 110
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
115 120 125
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
130 135 140
ACG CCC TTG GAC TTC ACA GAG TAC GCG GGT GCC ATC AAG ACG ATT ACG 480
Thr Pro Leu Asp Phe Thr Glu Tyr Ala Gly Ala Ile Lys Thr Ile Thr
145 l50 155 160
21 96892
~ W O96106159 ; ; PCTrUS95~10194
.
189
TCG GCT TTG CAG TTT GGT ATG GAC GCC CTA GAA CGG GGG CTA GTG GAC 528
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
180 185 190
.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
195 200 205
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
TCA TTT TTT CTA GAT AAG GCC GAG CTC ATG ACA AGG GGG AAG CAG TAT 720
Ser Phe Phe Leu Asp Lys Ala Glu Leu Met Thr Arg Gly Lys Gln Tyr
225 : -- 230 235 240
GTC CTA ACC ATG CTC TCC GAC ATG CTG GCC GCG GTG TGC GAG GAT ACC 768
Val Leu Thr Met Leu Ser Asp Met Leu Ala Ala Val Cys Glu Asp Thr
245 250 255
GTC TTT AAG GGT GTC AGC ACG TAC ACC ACG GCC TCT GGG CAG CAG GTG 816
Val Phe Lys Gly Val Ser Thr Tyr Thr Thr Ala Ser Gly Gln Gln Val
260 265 270
GCC GGC GTC CTG GAG ACG ACG GAC AGC GTC ATG AGA CGG CTG ATG AAC 864
Ala Gly Val Leu Glu Thr Thr Asp Ser Val Met Arg Arg Leu Met Asn
275 280 285
CTG CTG GGG 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
290 295 300
AGC TAC GTT:GTC AGG GGT GCC AAC CTC GTC ACC GCC GTT AGC TAC GGA 960
Ser Tyr Val Val Arg Gly Ala Asn Leu Val Thr Ala Val Ser Tyr Gly
305 ::. ~310 315 320
AGG GCG ATG AGA AAC TTT GAA CAG TTT ATG GCA CGC ATA GTG GAC CAT 1008
Arg Ala Met Arg :Asn Phe Glu Gl~ Phe Met Ala Arg Ile Val Asp His
325 330 335
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
340 345 350
GGA CAC GAC GAG ATT CAG AGA ACC CGC ATC GCC GCC TCT CTC GTC AAG 1104
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
370 375 380
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
TTC TTC CCT GTT GGC CTT CAC CTT CCC GTG CCC CGC TAC TCG ACA TCC 1248
Phe Phe Pro Val Gly Leu His Leu Pro Val Pro Arg Tyr Ser Thr Ser
405 410 415
GTC TCA GTC AGG GGC 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
420 425 430
WO 96/06159 2 -1 9 6 8 ~ 2 r~ s,i~is~--
190
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
435 440 445
GCC CTC AAA AGC ATA TGC CAC CCT CGA ATG CAC AAC CCC ACC CAG TCA 139Z
Ala Leu LYA Ser Ile Cys Hi6 prD Arg Met His Asn Pro Thr Gln Ser
450 455 460
GCC CAG GCA CTA AAC CAA GCT TTT CCC GAT CCC GAC GGG GGA CAT GGG 1440
Ala Gln Ala Leu Asn Gln Ala Phe Pro Asp Pro Asp Gly Gly His Gly
465 470 475 480
TAC GGT CTC AGG TAT GAG CAG ACG CCA AAC-ATG AAC CTA TTC AGA ACG 1488
Tyr Gly Leu Arg Tyr Glu Gln Thr Pro Asn Met Asn Leu Phe Arg Thr
485 490 495
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
500 505 510
GCC CAA AAA GCG CTC GTA ACC ACG GAG GAT CTA CTG CAC CCA ACC TCT 1584
Ala G:ln Lys Ala Leu Val Thr Thr Glu Asp Leu Leu His Pro Thr Ser
515 ~ 520 525
CAC CGT CTC CTC AGA TTG GAG GTC CAC CCC TTC TTT GAT TTT TTT GTG 1632
His Arg Leu Leu Arg Leu Glu Val His Pro Phe Phe Asp Phe Phe Val
530 535 540
CAC CCC TGT CCT GGA GCG AGA GGA TCG TAC CGC GCC ACC CAC AGA ACA 1680
His Pro Cys Pro Gly Ala Arg Gly Ser Tyr Arg Ala Thr His Arg Thr
545 550 555 560
ATG GTT GGA AAT ATA CCA CAA CCG CTC GCT CCA AGG GAG TTT CAG GAA 172B
Met Val Gly Asn Ile Pro Gln Pro Leu Ala Pro Arg Glu Phe Gln Glu
565 . 570 575
AGT AGA GGG GCG CAG TTC GAC GCT GTG ACG AAT ATG ACA CAC GTC ATA -1776
Ser Arg Gly Ala Gln Phe Asp Ala Val Thr Asn Met Thr His Val Ile
580 585 590
GAC CAG CTA ACT ATT GAC GTC ATA CAG GAG ACG GCA TTT GAC CCC GCG ~ 1824Asp Gln Leu Thr Ile Asp Val Ile Gln Glu Thr Ala Phe Asp Pro Ala
sg5 ~ 600 605
TAT CCC CTG TTC TGC TAT GTA ATC GAA GCA ATG ATT CAC GGA CAG GAA 1872
Tyr Pro Leu Phe Cys Tyr Val Ile Glu Ala Met Ile His Gly 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
625 630 635 640
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
645 650 655
GTT AGA TTC ATC TGT ACG CAT ATT GGG AAT GGA AGC ATC CCT AAG GAG Z016
Val Arg Phe Ile Cys Thr His Ile Gly Asn Gly Ser Ile Pro Lys Glu
660 665 670
GCG CAC GGC CAC TAC CGG 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
675 680 685
CAG GCG CTT CTC AAG CTC GCG GGA CAC GAG ACG GTG GGT CGG ACG CCG 2112
Gln Ala Leu Leu Lys Leu Ala Gly His Glu Thr val Gly Arg Thr Pro
690 695 700
~ W 096/06159 21 96892 ~ 91
191
ATC ACA CAT CTG QTT TCG GCT CTC CTC GAC CCG CAT CTG CTG CCT CCC 216P
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
725 730 735
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
740 745 750
AGG GCG ACA TTC ATC AAC CTC AGG GQT CGC ATG QAG GAC CTA GTC AAT 2304
Arg Ala Thr Phe Ile Asn Leu Arg Gly Arg Met Glu Asp Leu Val Asn
AAC CTT GTT AAC ATT TAC CAG ACA AGG GTC AAT GAQ GAC CAT GAC GAQ 2352
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
785 790 795 800
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
805 810 815
GGC CAC ATG TGC GGT ATG GGG GTC GAC TAT CAA AAC GTG GCC CTG ACG 2496
Gly ~is Met Cys Gly Met Gly Val Asp Tyr Gln Asn Val AIa Leu Thr
820 825 830
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
GAT ATT CTA CTG CAC CTG GAG AAC GGA ACC TTG AAG GAC ATT CTG CAG 2592
Asp Ile Leu Leu ~is Leu Glu Asn Gly Thr Leu Lys Asp Ile Leu Gln
850 855 860
GCA GGC GAC ATA CGC CCG ACG GTG GAC ATG ATC AGG GTG CTG TGC ACC 2640
Ala Gly Asp Ile Arg Pro Thr Val Asp Met Ile Arg Val Leu Cys Thr
865 ~ :87P 875 880
TCG TTT CTG ACG TGC CCT TTC GTC ACC CAQ GCC GCT CGC GTG ATC ACA 2688
Ser Phe Leu Thr Cys Pro Phe Val Thr Gln Ala Ala Arg Val Ile Thr
885 890 895
AAG CGG GAC CCG GCC CAQ AQT TTT GCC ACG CAC GAA TAC GGG AAQ GAT 2736
Lys Arg Asp Pro Ala Gln Ser Phe Ala Thr ~is Glu Tyr Gly Lys Asp
900 905 910
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
915 920 925
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
CTG CCA AAC TAT GTC ACC AGG CTC CCC AAC CAQ AGA AAC GCG GTG GTC 2928
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 ~
192
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
980 985 990
TCG CCC GTC GCG GCG TAT GCC GCG TCT TGT CAG GCC ACC CCG GGC GCC 3024
3er Pro Val Ala Ala Tyr Ala Ala Ser Cys Gln Ala Thr Pro Gly Ala
995 1000 1005
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
1025 1030 1035 - 1040
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
1060 1065 1070
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
1075 1080 1085
CTG CAC ACC GCA CTT GGC TAC TCA TCA GTC ATC GCC CCG GCC CAC GTG 3312
Leu His Thr Ala Leu Gly Tyr Ser Ser Val Ile Ala Pro Ala His Val
1090 1095 - 1100
GCC GCC ATA ACT ACA GAC ATG GGA GTA CAT TGT CAG GAC CTC TTT ATG ~360
Ala Ala Ile Thr Thr AGP Met Gly Val HiG Cys Gln Asp Leu Phe Met
llOS 1110 lllS 1120
ATT TTC CCA GGG GAC GCG TAT CAG GAC CGC CAG CTG CAT GAC TAT ATC 3408
Ile Phe Pro Gly Asp Ala Tyr Gln AGP Arg Gln Leu HiG AGp Tyr Ile
1125 1130 1135
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
CAC GTG GGA TAC ACT GCT-GGG GTT CCT CGC TGC GAG AAC CTG CCC GGT 3504
His val Gly Tyr Thr Ala Gly Val Pro Arg Cy9 Glu AGn Leu Pro Gly
llSS 1160 1165
TTG AGT CAT GGT CAG CTG GCA ACC TGC GAG ATA ATT CCC ACG CCG GTC 3552
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
GCG GCG TCG GTC GTG TCG TGT GAT GCT TAC AGT APC GAA AGC GCA GAG 3648
Ala Ala Ser Val Val Ser CYG Asp Ala Tyr Ser Asn Glu Ser Ala Glu
1205 1210 1215
CGT TTG TTC TAC GAC CAT TCA ATA CCA GAC CCC GCG TAC GAA TGC CGG 3696
Arg Leu Phe Tyr AGP HiG Ser Ile Pro Asp Pro Ala Tyr Glu CYG Arg
1220 1225 1230
TCC ACC AAC AAC CCG TGG GCT TCG CAG CGT GGC TCC CTC GGC GAC GTG 3744
Ser Thr AGn Asn Pro Trp Ala Ser Gln Arg Gly Ser Leu Gly Asp Val
1235 1240 1245
~ W O 96/06159 2 1 9 6 8 9 ~ PC~r/US95J10194
193
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.
GGG TTG TAC ACT TTG GTT AAT GAG TAT TCT GCC AGG CTT GCT GGG GCC 3888
Gly Leu Tyr Thr Leu Val Asn Glu Tyr Ser Ala Arg Leu Ala Gly Ala
1285 1290 1295
CCC GCC ACC AGC ACT 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
1300 1305 1310
GAC GTG TTT TTG GAC CAG CCT TGC CAT ATG CTG CAG GAG GCC TAT CCC 3984
Asp Val Phe Leu Asp 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
1345 1350 1355 1360
GTG GCG CCG ATG AAG AGA CTA TTA AAG CTC GGA AAC AAG GTG GTG TAT 4128
Val Ala Pro Met Lys Arg Leu Leu Lys Leu Gly Asn Lys Val Val Tyr
1365 1370 1375
TAG 4131
(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
1 5 10 15
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
0 55 60
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
100 105 110
. .
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
115 120 125
Ile Glu Leu Ala Ala Ala Asp Ile Glu Leu Leu Phe Ala Glu Lys Glu
130 135 140
Thr Pro Leu Asp Phe Thr Glu Tyr Ala Gly Ala Ile Lys Thr Ile Thr
145 150 155 160
~er Ala Leu Gln Phe Gly Met Asp Ala Leu Glu Arg Gly Leu Val Asp
165 170 175
~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
195 200 205
Val Lys Ser Asp Met Val Ser Met Phe Lys Ala Eis 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
~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
260 265 270
Ala Gly Val Leu Glu Thr Thr Asp Ser Val Met Arg Arg Leu Met Asn
275 280 285
Leu Leu Gly Gln Val Glu Ser Ala Met Ser Gly Pro Ala Ala Tyr Ala
290 295 300
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
325 330 335
~ro Asn ~la Leu Pro ser Val Glu Gly Asp Lys Ala Ala Leu Ala Asp
340 345 350
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
370 375 380
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
405 410 415
~al Ser Val Arg Gly Val Glu Ser Pro Ala Ile Gl-~ Ser Thr Glu Thr
420 425 430
Trp Val Val Asn Lys Asn Asn Val Pro Leu Cys Phe Gly Tyr Gln Asn
435 440 445
Ala Leu Lys Ser Ile Cys His Pro Arg Met Eis Asn Pro Thr Gln Ser
450 455 460
Ala Gln Ala Leu Asn Gln Ala Phe Pro Asp Pro Asp Gly Gly His Gly
2l q6892'
WO 96/06159 ' ' r~ l34
195
465 470 475 480
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
515 520 525
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
580 585 590
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
660 665 670
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
725 730 735
Gln Pro Ile Ile Lys Ile Gly Asp Gln Asn Tyr Asp Asn Pro Gln Asn
740 745 750
Arg Ala Thr Phe Ile Asn Leu Arg Gly Arg ~et Glu Asp Leu Val Asn
755 760 765
Asn Leu Val Asn Ile Tyr Gln Thr Arg Val Asn Glu Asp Xis Asp Glu
770 775 780
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
805 - 810 815
Gly Pis Met Cys Gly Met Gly Val Asp Tyr Gln Asn Val Ala Leu Thr
820 825 330
,,~
21 96892
WO 96/06159 , PCI/US95/10194
196
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
850 855 860
Ala Gly Asp Ile Arg Pro Thr Val Asp Met Ile Arg Val Leu Cys Thr
865 870 875 880
~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
915 920 925
Asp Arg Ser Arg Glu Ala Ala Glu Thr Met Phe Tyr Pro Val Pro Phe
930 935 940
Asn Lys Leu Tyr Ala Asp Pro Leu Val Ala Ala Thr Leu Xis Pro Leu
945 950 955 960
~eu Pro Asn Tyr Val Thr Arg Leu Pro Asn Gln Arg Asn Ala Val Val
965 970 975
~he Asn Val Pro Ser Asn Leu Met Ala Glu Tyr Glu Glu Trp His Lys
9B0 9B5 990
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
1025 1030 1035 1040
~al Arg Thr Asp Glu Val Leu Ala Glu Xis Ile Leu Tyr Cys Ser Arg
1045 1050 1055
~la Ser Thr Ser Met Phe Val Gly Leu Pro Ser Val Val Arg Arg Glu
1060 1065 1070
Val Arg Ser Asp Ala Val Thr Phe Glu Ile Thr His Glu Ile Ala Ser
1075 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
1125 1130 1135
~ys Met Lys Ala Gly Val Gln Thr Gly Ser Pro Gly Asn Arg Met Asp
1140 1145 1150
Xis Val Gly Tyr Thr Ala Gly Val Pro Arg Cys Glu Asn Leu Pro Gly
1155 1160 1165
Leu Ser Xis Gly Gln Leu Ala Thr Cys Glu Ile Ile Pro Thr Pro Val
1170 1175 1180
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
1285 1290 1295
Pro Ala Thr Ser Thr Thr Asp Leu Gln Tyr Val Val Val Asn Gly Thr
1300 13Q5 I310
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
1365 1370 1375
(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
5 10 15
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
TCC ACG AGT TTC CTG CTT AAC CTC AGG AAC GCC CAG GAA AAG ATG CTG 192
Ser Thr Ser Phe Leu Leu Asn Leu Arg Asn Ala Gln Glu Lys Met Leu
50 55 60
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
65 70 75 80
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
115 120 125
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 ~
GAC ATG TGT CGG GTA GAC ACC ACC GCC CAG GAG GTT CAG AAG TGC CTT 480
Asp Met Cys Arg Val Asp Thr Thr Ala Gln Glu Val Gln Lys Cys Leu
145 150 155 160
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 =
GAG GCG TCC GGT ACT GGC GTG GGC GCC GTT GTC ACG AGT ACT CAG ACT 576
Glu Ala Ser~Gly Thr Gly Val Gly Ala Val Val Thr Ser Thr Gln Thr
180 185 190
CCC ACC AGA AGC CTC ATA TTG GGC ATG GAG CAT TTC TTC CTG CGC GAC 624
Pro Thr A_g Ser Leu Ile Leu Gly Met Glu His Phe Phe Leu Arg Asp
195 200 205
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
210 215 220
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
GCG GTC GAA GGC AAC AGC AGC CAA GAT TCT GGG GTG GCC ATT GCA ACC --768
Ala Val Glu Gly Asn Ser Ser Gln Asp Ser Gly Val Ala Ile Ala Thr
24s 250 : 255
GTC CTT AAC GAA ATA TGC CCG CTC CCC ATA CAT TTT CTA CAC TAT ACT 816
Val Leu Asn Glu Ile Cys Pro Leu Pro Ile His Phe Leu His Tyr Thr
260 265 : 270
GAC AAG AGC AGC GCC CTG CAG TGG CCA ATT TAC ATG TTG GGA GGC GAG 864
Asp Lys Ser Ser Ala Leu Gln Trp Pro Ile Tyr Met Leu Gly Gly Glu
275 280 285
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
290 295 300
~ W O96106159 2 1 9 6 ~ 9 2 PCT/US95/l0194
199
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
305 310 315 320
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
325 330 335
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
340 345 350
TCG GAC GAC TTA CTT GTG GCA GTT GTC ATG GCC CAT TTT ATG GCT ACC 1104
Ser Asp Asp Leu Leu Val Ala Val Val Met Ala ~is Phe Met ALa Thr
355 360 365
GAT GAT AGA CAC ATG TAC AAG CCC ATA TCC CCA CAA TAA 1143
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
1 5 10 15
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
- 55 60
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
130 135 140
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
165 170 175
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--
200
Pro Thr Arg Ser Leu Ile Leu Gly Met Glu Xis Phe Phe Leu Arg Asp
195 200 205
Leu Thr Gly Ala Ala Ala Tyr Glu Ile Ala Ser Cys Ala Cys Thr Met
210 215 -- 220
Ile Lys Ala Ile Ala Val Leu His Thr Thr Ile Glu Arg Val Asn Ala
225 230 235 240
~la Val Glu Gly Asn Ser Ser Glr. Asp Ser Gly Val Ala Ile Ala Thr
245 250 255
~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
275 280 2B5
Lys Ser Ser Ala Phe Glu Thr Phe Ile Tyr Ala Leu Asn Ser Gly Thr
290 295 300
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
325 330 335
~ro Leu Arg Asp Gly Gly Gln Ser Tyr Ser Ala Lys Gln Lys His Met
340 345 350
~er Asp Asp Leu Leu Val Ala Val Val Met Ala Xis Phe Met Ala Thr
355 360 365
~sp Asp Arg Xis Met Tyr Lys Pro Ile Ser Pro Gln
370 375 380
~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
GAG GAG TGT GAA CAA TTT TTC AGT CAA CCC CTT AGG GAG CAA GTG GTC 96
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,
201
GCG GGG GTC AGG GCA CTC GAC GGC CTC GGT CTC GCT GAC TCT CTA TGT 144
Ala Gly Val Arg Ala Leu Asp Gly Leu Gly Leu Ala Asp Ser Leu Cys
35 40 45
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
0 55 60
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
1 5 10 15
Glu Glu Cys Glu Gln Phe Phe Ser Gln Pro Leu Arg Glu Gln Val Val
2D 25 30
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
65 70 75
(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 -
202
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
35 40 45
ACG GGT AAC TGC ATG CTC GGC AAC ATT CAA GAG GGC CAG TTT TTA GGG 192
Thr Gly Asn Cys Met Leu Gly Asn Ile Gln Glu Gly Gln Phe Leu Gly
so SS 60
CCG GTA CCG TAT CGG ACT TTG GAT AAC CAG GTT GAC AGG GAC GCA TAT 240
Pro Val Pro Tyr Arg Thr Leu Asp Asn Gln Yal Asp Arg Asp Ala Tyr
65 70 75 80
CAC GGG ATG CTA GCG TGT CTG AAA CGG GAC ATT GTG CGG TAT TTG CAG 288
Hi6 Gly Met Leu Ala Cys Leu Lys Arg Asp Ile Val Arg Tyr LeY Gln
85 90 . 9S
ACA TGG CCG GAC ACC ACC GTA ATC GTG CAG GAA ATA GCC CTG GGG GAC 336
Thr Trp Pro ArP Thr Thr Val Ile Val Gln Glu Ile Ala Leu Gly Asp
100 105 110
GGC GTC ACC GAC ACC ATC TCG GCC ATT ATA GAT GAA ACA TTC GGT GAG - 384
Gly Val Thr Asp Thr Ile Ser Ala Ile Ile Asp Glu Thr Phe Gly Glu
llS 120 125
TGT CTT CCC GTA CTG GGG GAG GCC CAA GGC GGG TAC GCC CTG GTC TGT 432
Cys Leu Pro Val Leu Gly Glu Ala Gln Gly Gly Tyr Ala Leu Val Cys
130 135 140
AGC ATG TAT CTG CAC GTT ATC GTC TCC ATC TAT TCG ACA AAA ACG GTG 480
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
165 170 175
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
180 185 190
GAT ACG TAG 585
Asp Thr
195 - -
(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
40 45
~ W 096/061~9 2 1 96~q2 PCTrUS9~/10194
203
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
100 105 110
Gly Val Thr Asp Thr Ile Ser Ala Ile Ile Asp Glu Thr Phe Gly Glu
115 120 125
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
145 150 155 160
Tyr Asn Ser Met Leu Phe Lys Cys Thr Lys Asn Lys Lys Tyr Asp Cys
165 170 175
Ile Ala Lys Arg Val Arg Thr Lys Trp Met Arg Met Leu Ser Thr Lys
180 185 190
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
5 10 15
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
20 25 30
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
35 40 45
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
50 55 60
. _~ . _ . . _.... . ... ... . _ . . __ ___ __ ___ ___ _ _
WO96/06159 2 1 9 ~ 8 9 2. . PCT~US95/1019 ~
204
CAT AAA GTC ATG GGA CCG TGT GTG GCT GTG GGA ATT AAC GGA GAA ATG 240
~is Lys Val Met Gly Pro Cys Val Ala Val Gly Ile Asn Gly Glu Met
65 70 75 80
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
85 90 95
CGC GAT GGT ATG GCG CTC ATC TAC TTT GGA CAG TTT CTG GAG GAA GCA 336
Arg Asp Gly Met Ala Leu Ile Tyr Phe Gly Gln Phe Leu GlU Glu Ala
100 105 - 110
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
130 135 - - - 140
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
165 170 175
TTG GGC GTG GAC TAC ATG GCG TTC TGT CCG GGT GTC GAC GGA ATG CCG 576
Leu Gly Val Asp Tyr Met Ala Phe Cys Pro Gly Val Asp Gly Met Pro
180 185 l9O
TCG TTG GCA AGA GTG GCC GCC CTG CTT ACC AGG TGC GAC CAC CCA GAC 624
Ser Leu Ala Arg Val Ala Ala Leu Leu Thr Arg Cys ASp ~is Pro ASp
195 200 205
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
210 215 - 220
TAC TGT TCT GCG CAG TCG CCG GGT CTA TCT AAC ATC TGT CCC TGT ATC 720
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
245 250 255
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
275 280 285
CTA ACA GGA GTG CTC GAC GAC GGC ACC TTG GTG CCG TCC GTC CAA GGC 915
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
305 310
~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
100 105 110
Ser Gly Leu Arg Phe Pro Tyr Ile Ala Pro Pro Pro Ser Arg Glu His
115 ~ 120 125
Val Pro Asp Leu Thr Arg Gln Glu Leu Val His Thr Ser Gln Val Val
130 135 140
Arg Arg Gly Asp Leu Thr Asn Cys Thr Met Gly Leu Glu Phe Arg Asn
145 150 155 160
Val Asn Pro Phe Val Trp Leu Gly Gly GIy Ser VaI Trp Leu Leu Phe
165 170 175
Leu Gly Val Asp Tyr Met Ala Phe Cys Pro Gly Val Asp Gly Met Pro
180 185 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
210 215 Z20
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
260 265 270
Ala Leu Lys Ile Thr Ser His Pro Thr Pro Thr His Val Glu Asn Val
275 280 285
Leu Thr Gly Val Leu Asp Asp Gly Thr Leu Val Pro Ser Val Gln Gly
290 295 300
Thr Leu Gly Pro Leu Thr Asn Val
''21 96892
W096/06l59 PCTrUS95/l0l9
206
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
5 10 15
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~
2Q7
(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:
ATG GCA GAA GGC GGT TTT GGA GCG GAC TCG GTG GG3 CGC GGC GGA GAA 48
Met Ala Glu &ly Gly Phe Gly Ala Asp Ser Val Gly Arg Gly Gly Glu
5 10 15
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
20 25 30
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
35 40 45
GAG GAG AGG AAA CCA CTA ACG GGA AAG TCT GTA AAA ACC TCG TAC ATA 192
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
165 170 175
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
180 185 190
GAC CGT CT- ACG GAC GGC GG- TTT GCC TTT AGT CCT AGG GGC GTG AAA 624
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
210 215 220
TCG GCG ACT GCT ~TC CCC GTC ACC ACG CaG CTT ATG GTA CCG GTG CAC 720
21 96892
WO 96/06159 PZ~rlUS9511019
200
Ser Ala Thr Ala Val Pro val Thr Thr Gln Leu Met Val Pro Val iqis
225 230 235 -240
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
260 265 270
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
ATG GTG TAC TGG ACG AGG GCA TTT ACA GAT TGT TAC AAG GAA ATT TCC 912
Met Val Tyr Trp Thr Arg Ala Phe Thr Asp Cys Tyr Lys Glu Ile Ser
290 295 30D
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
325 330 335
ACC GCT ATC CTG CGA ATG ATG CAG CCC TGG AAC GTT GGG GGT GGG TCT 1056
Thr Ala Ile Leu Arg Met Met G1n Pro Trp Asn Val Gly Gly Gly Ser
340 345 350
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
370 375 380
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
420 425 430
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
435 440 445
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
450 455 46C
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
485 490 495
~ W 096/06159 2 1 9 6 8 ~ 2 PCTAUS9~1019~
209
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
50D SD5 510
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
565 570 575
ACA TGC AGG GTC TAG 1743
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
- 40 45
Glu Glu Ars Lys Pro Leu Thr Gly Lys Ser Val Lys Thr Ser Tyr Ile
6D
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
94 95
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
115 120 125
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
145 150 155 160
Trp Ala Asp Leu Gln Gly Leu Lys Arg Thr Pro Lys Gly Phe Leu Lys
WO96106159 ' 21 96892 PCTIUS95/10191--
210
165 170 175
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
245 250 255
~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
275 2B0 285
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
325 - 330 335
~hr Ala Ile Leu Arg Met Met Gln Pro Trp Asn val Gly Gly Gly Ser. ,.
340 345 350
~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
405 410 415
~rg Ala Arg Gly Arg Lys Asn Asp Gly Thr Val Glu Gln Asn Tyr Ile
420 425 430
~rg Glu Leu Ala Trp Ala Tyr His Ala Val Tyr Cys Ser Trp Ile Met
435 440 445
Leu Gln Tyr Ile Thr Val Glu Gln Met Val Gln Leu Cys Val Gln Thr
450 455 - 460
Th- As-. Ile Pro Glu Ile Cys Phe Arg Ser Val Arg Leu Ala His Lys
465 470 475 480
~lu Glu Thr Leu Lys Asn Leu His Glu Gln Ser Met Leu Pro Met Ile
485 490 495
~hr Gly Val Leu Asp Pro Val Arg His His Pro Val Val Ile Glu Leu
500 505 510
~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~
. .
211
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:
ATG CAG GGT CTA GCC TTC TTG GCG GCC CTT GCA TGC TGG CGA TGC ATA 48
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
35 40 45
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~ ~
212
Gly Phe Asn Tyr Ser Ala Leu Pro Gly His Leu Gly Gly Phe Gly Iie
130 135 140
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
180 185 190
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
21D 215 22D
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
245 250 255
AAA GAT TCT TAC CAG ACA CTC CTT ACA GAG AAT TAC ACT CGC ATA TTT 816
Lys Asp Ser Tyr Gln Thr Leu Leu Thr Glu Asn Tyr Thr Arg Ile Phe
260 265 27D
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
275 28D 285
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 ~
TG- GTG TGT CGG CAG TAT GCG GAA CTG TAT TTT CTC CGC CGC ATC TCG 1056
Cys Val Cys Arg Gln Tyr Ala Glu Leu Tyr Phe Leu Arg Arg Ile Ser
340 345 35~
CGT CTG TGC ATG CCC ACG TTC ACC ACT GTC GGG TAT AAC CAC ACC ACC 1104
A-g Leu Cys Met Pro Thr Phe ~hr Thr Val Gly Tyr Asn Hi5 Thr:Thr
355 360 - 365
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
370 375 38D
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
385 390 395 400
21 96892
~ W 096/06159 ~ PCT~U59511019~
~ .;
213
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
420 425 430
ACA CTC GGC GAT ACT GAA AGA AAA TTA ATG TTG GAC ATA CAC ACG GTC 1344
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
450 455 460
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
500 505 510
GCC AAG TTG CGC GCC GAG GCG CCG CAG TCG TCC GCT CTG ACG CGG ACT 1584
Ala Lys Leu Arg Ala Glu Ala Pro Gln ser Ser Ala Leu Tkr Arg Thr
515 520 525
GCC GTT GCC AGA GGA ACA TCG GGA TTC GCA GAA TTG CTC CAC GCG CTG 1632
Ala Val Ala Arg Gly Thr Ser Gly Phe Ala Glu Leu Leu His Ala Leu
530 535 540
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
565 570 575
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
GAG ATC TTC CTC AAG AGT GCC ATG TTT ATA TCT GCT ATC AAA CCC GAT 1824
G;u Ile Phe Leu Lys Ser ~la Met Phe Ile Ser Ala Ile Lys Pro Asp
5g5 600 _ 605
TGC TCC GGC TTT AAC TTT TCT CAG ATT GAT AGG CAC ATT CCC ATA GTC 187
Cys Ser Gly Phe Asn Phe Ser Gln Ile Asp Arg His Ile Pro Ile Val
610 615 620
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
645 650 655
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
660 665 670
~ ~ 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
675 680 685
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
7Q5 71Q 715 72Q
TTT CTT IAC AGA CTG TTT TCC ATC CTT TAT TAG ~ 2193
Phe Leu Tyr Arg Leu Phe Ser Ile Leu TYr
725 73Q
~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 ,~
8Q
~la Glu Val Ala Glu Asp Leu Arg Val Thr Leu Lys Lys Arg Gln Ser
ES 9Q 95
~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
13Q 135 14Q
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
165 170 17
~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
2I5
A6n Arg Ser Lys Glu Ala Asn Glu Thr Ala Ser ~;is Leu Leu Phe Gly
210 215 220
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
260 265 270
Leu Asn Met Thr Glu Ser D~r Pro Leu Glu Phe Thr Arg Thr Ile Gln
275 - - - 230 2B5
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
325 330 335
~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
355 360 365
Leu Gly Ala Val Ala Ala Thr Gln Ile Ala Arg Val Ser Ala Thr Lys
370 375 380
Leu Ala Ser Leu Pro Brg Ser Ser Gln Glu Thr Val Leu Ala Met Val
3B5 390 395 - 400
~ln Leu Gly Ala Arg Asp Gly Ala Val Pro Ser Ser Ile Leu Glu Gly
405 410 415
~le Ala Met Val Val G1U }{is Met Tyr Thr Ala Tyr Thr Tyr Val Tyr
420 425 430
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
455 470 475 480
~ly Glu Met Ile Aia Arg Phe Ser Lys Pro Asp Se- Leu Asn Ile Tyr
4B5 490 495
~rg Ala Phe Ser Pro Cys Phe Leu Gly Leu Arg Tyr Asp Leu E~is Pro
500 505 510
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
530 535 540
~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~ .
565 570 575
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
675 680 685
Gly Thr Val Val Glu Ile Arg Gly Met Tyr Arg Arg Arg Ala Ala Ser
690 695 ~ -- 700
Ala Leu Phe Leu Ile Leu Ser Phe Ile Gl~ Phe Ser Gly Val Ile Tyr
705 710 715 720
~he Leu Tyr Arg Leu Phe Ser Ile Leu Tyr
725 730
~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
5 10 15
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
35 40 -45
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~
GAC CAG TCT TAT ACC AAC TTT AAG ATT ATA GAT CTG CCA GCG GGA TGC 432
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
165 170 175
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
195 200 205
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
260 265 270
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
290 Z95 300
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
218
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
340 345 350
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
TTA TTG ACC GTC TAA 1215
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
9S
~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
130 135- 140
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~
219
165 170 175
His Pro Ile ThL~ Pro Arg Ala Gly Gln Thr Arg Ser Iie Leu Lys His
- 180 185 190
Asn Ile Ala GIn Val Cys Glu Arg Asp Ile Val Ser Leu Asn Thr Asp
195 200 205
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
245 250 255
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
325 330 335
Val Leu lle Glu Ala Met Glu Lys Tyr Phe Met Met Cys Ser Pro Lys
340 345 350
Asp Arg Gln Ser Ala Ala Trp Leu Gly Ala Gly Val Val Glu Leu Ile
355 360 365
Cys Asp Gly Asn Pro Leu Ser Glu Val Leu Gly Phe Leu Ala Lys Tyr
370 375 3E0
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 ~
22
(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
35 40 45
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 . =_
TTC GCC TGC AGA GCG ATA CGC AGA CTA CTC CTG GGG GAA CGC CTC CAC 240
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
165 17D 175
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
180 185 I90
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
21Q 215 220
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
AAC GGA CGG GAT CTG GTG GGC TTT CAG CTG GCT CTA AAC CAG CTC GTG 864
Asn Gly Arg Asp Leu Val Gly Phe Gln Leu Ala Leu Asn Gln Leu Val
.~ 275 280 285
TCC CGT CTG CAC GTA AAA ATC CAA CGC AAG GAC CCC GGA CCA AAG CCA 912
ser Arg Leu His Val Lys Ile Gln Arg Lys Asp Pro, Gllr Pr,o Lys Pro
290 255 300
TAC AGG GTG GTC GTC AGT ACC CCA GAT TGT ACC TAC TAT CTA GTG TAT 960
Tyr Ar~ Val Val Val Ser Thr Pro Asp Cys Thr Ty- Tyr Leu Val Tyr
305 . .= = 31~ ~ ~ ,= 315 . . 320
CCG GGC ACA CCG GCC ATC TAC AGA CTC GTC ATG TGT ATG GCA GTG GCA 1008
Pro Gly Thr Pro Ala Ile Tyr Arg Leu Val Met Cys Me~ Ala Val Ala
325 330 335
GAC TGC ATC GGC CAC TCG TGC AGC GGA CTG CAC CCC TGC GCA AAC TTT 1056
Asp Cys Ile Gly His Ser Cys Ser Gly Leu His Pro Cys Ala Asn Phe
340 345 350
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
370 375 380
CAG GCG TGC TTC CAA CGA TAC GCG GCC ACG GAC GCG CGG ACT CTG GGC 1200
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
405 410 415
GAA AAC TTC AAG ATC ACC ATA TTC AAC ACC AAC ATG GTG ATT AAC ACT 1296
Glu Asn Phe Lys Ile Thr Ile Phe Asn Thr Asn Met Val Ile Asn Thr
420 425 - 430
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
450 455 460
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
465 470 475 480
- 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
485 490 495
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
500 505 - 510
CCT GTA TCG ATA GCA AAT TGG AAC TCC ACG TTG GAC CTG CAC GGA CTG 1584
Pro Val Ser Ile Ala Asn Trp Asn Ser Thr Leu Asp Leu His Gly Leu
515 520 525
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
53D 535 540
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
580 585 590
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
740 745 750
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
223
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
100 105 110
Gly Gln Ile Asp Thr ASp Asn Tyr Leu Pro Ala Val Gly Ser Trp Glu
115 120 125
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
165 170 175
Phe ~lis Ala Phe Leu Ala Val Leu Pro His Ser Leu Gln Pro Thr Ile
180 185 190
Tvr Gln Asn Tyr Phe Ala Leu Leu Glu Ser Leu Lys ~lis Leu Ser Phe
195 200 205
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
245 250 255
Val Leu Lys Asn Leu Ser Lys Gln Ser Lys ~is Gln Asp Val Lys Val
260 265 270
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
290 295 300
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
325 330 335
WO 96/06159 ~ ;! 2 i qi 6 8 9 2 PCTIUS95/1019~
224
Asp Cys Ile Gly Pis Ser Cys Ser Gly Leu Xis Pro Cys Ala Asn Phe
340 345 350
Leu Gly Thr His Glu Thr Pro Arg Leu Leu Ala Ala Thr Leu Ser Arg
355 360 365
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
405 410 415
~lu Asn Phe Lys Ile Thr Ile Phe Asn Thr Asn Met Val Ile Asn Thr
420 425 430
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
530 535 540
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
565 570 575
~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
645 650 655
~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
675 680 685
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~
690 695 700
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
740 745 750
~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
5 10 15
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
20 25 30
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
50 SS 60
GAG GTG ACT CTA GGG CAC AGA ATT GCC GAC TGC GTA ATT CTG GTA TCG 240
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
AGT CAC CAG CTG ATC CCA ACC AAC ACC GTG AGA ACG TCA CAG CGA GCT 336
Ser Xis Gln Leu Ile Pro Thr Asn Thr Val Arg Thr Ser Gln Arg Ala
100 105 110
CAA GGC CTG TGC CAA CTC TCC GAC TCG A 364
Gln Gly Leu Cys Gln Leu Ser Asp Ser
llS 120
.21 9689~
W O96/06159 ' ' ' I ~ PCTnUS9S/1019
226
(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
100 105 110
Gln Gly Leu Cys Gln Leu Ser Asp Ser
115 120
(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
227
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
35 40 45
TGC GTA TGC TCA CGT GAG ACA TCT CCG GAC TAC ATC CAA ATT ATG CAG 192
Cy6 Val Cys Ser Arg Glu Thr Ser Pro Asp Tyr IIe Gln Ile Met Gln
50 55 60
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
85 90 95
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
100 105 - 110
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
145 150 155 160
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
165 170 175
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
180 185 190
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
210 215 220
CTG CTC ACG GCG CTC OEG CGG CAC GA- AGG CAT CCT CTG ACA GAG GTG 720
Leu Leu Thr Ala Leu Val Arg ~is Asp Arg Pis Pro Leu ~hr Glu Val
225 . .~ ~ 23~ 235 ~ 240
TTT GAG GGG GTG OE G CCA GAT GAG GTG ACC AGG ATA GAT CTC GAC CAG 768
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
260 265 270
CTT CAG AGT CTC AGT TCT ATA TTT AAT CTT GGC CCC AGA CTG CAC GTG 864
Leu Gln Ser Leu Ser Ser Ile Phe Asn Leu Gly Pro Arg Leu ~ls Val
275 280 285
TAT GCC TAC TCG GCA GAG ACT TTG GCG GCC TCC T OE TGG TAT TCC CCA 912
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 ~
228
.
CGC TAA - 918
Arg
305
(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
5 10 15
Ala Asp Glu Leu Ala Ala Leu Gln Ser Lys Ile Gly Se- Val Leu Pro
20 25 30
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
50 55 60
Tyr Leu Ser Lys Cys Thr Leu Ala Val Leu Glu Glu Val Arg Pro Asp
65 70 75 80
Ser Leu Arg Leu Thr Arg Met Asp Pro Ser Asp Asn Leu Gln Ile Lys
85 90 9S
Asn Val Tyr Ala Pro Phe Phe Gln Trp Asp Ser Asn Thr Gln Leu Ala
100 105 110
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
165 170 175
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
260 265 270
2l q68q2
~ W 096l06159 PCT~U595~10194
22~
Leu Gln Ser Leu Ser Ser Ile Phe A5n Leu Gly Pro Arg Leu ~is Val
275 280 285
Tyr Ala Tyr Ser Ala Glu Thr Leu Ala Ala Ser Cys Trp Tyr Ser Pro
290 295 300
Arg
305
(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
5 10 15
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
20 25 30
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
35 40 45
GCC TTC CTC AGC GAC GGG ATC GTG GAT ATG GCT CGA AAG CTT CAT CGT 192
Ala Phe Leu Ser Asp Gly Ile Val Asp Met Ala Arg Lys Leu ~is Arg
50 55 60
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
65 70 75 80
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
85 90 95
~ 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
100 105 110
CCC GTT GTG GCA CCT GAG GTG CTC TTC CCA CAC CCG GCT GAG ATG TCT 384
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 ~
230
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
145 150 155 160
CCG GAC GGC AGG CCT ACG GAT TAC TCC ATG GCC CTT AGA AGG GCT TTT 528
Pro Asp Gly Arg Pro Thr Asp Tyr Ser Met Ala Leu Arg Arg Ala Phe
165 170 175
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
180 185 190
GAA ACT CAG ACC GCA TCC CGT AAC CAC ACT GAG TGG GAA AAT CTG CTG 624
Glu Thr Gln Thr Ala Ser Arg Asn Hls Thr Glu Trp Glu Asn Leu Leu
19~ ~ 200 205
GCT ATG TTT TCT GTG ATT ATC TAT GCC TTA GAT CAC AAC TGT CAC CCG 672
Ala Met Phe Ser Val Ile Ile Tyr Ala Leu Asp Uis Asn Cys ~is Pro
210 215 22D ~ ,
GAA GCA CTG TCT ATC GCG AGC GGC ATC TTT GAC GAG CGT GAC TAT GGA 720
Glu Ala Leu Ser Ile Ala Ser Gly Ile Phe Asp Glu Arg Asp Tyr Gly
225 230 235 240
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
245 250 255 -- -
GTG TCG TGG GAA GAT ATC TAC AAC GGG ACT TAC CTA GCT CGG CCT GGA 816
Val Ser Trp Glu Asp Ile Tyr Asn Gly Thr Tyr Leu Ala Arg Pro Gly
260 265 270
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
275 280 285
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
180 185 190
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
245 250 255
~al Ser Trp Glu Asp Ile Tyr Asn Gly Thr Tyr Leu Ala Arg Pro Gly
260 265 270
275 28D 285
~he Lys
290
~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~ ~
232
CTC CGT CCT GGT GCG AAT GGC CAC CAA GCC TCC CGT GAT TGG TCT TAT = 96
Leu Arg Pro Gly Ala Asn Gly His 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
50 SS 60
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
85 90 9S
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
115 120
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
SS - - - - 60
Gl}~ His Arg Gly Val Ser Gly Pro Ser~~i5 Pro Gly Thr~Ala Gl} Arg
7D 75 80
~al Thr Cys Thr Ala Asp Gly Gly His Ser Tyr Pro Gly Ala Leu Pro
9S
~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:
ATG CTG CTC AGC CGT CAC AGG GAG CGC CTT GCC GCC AAC CTG GAG GAG 48
Met Leu Leu Ser Ary ~is Arg Glu Arg Leu Ala AIa Asn Leu Glu Glu
1 S 10 15
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
35 40 45
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
50 55 60
ACA CGG CAC CAT CCC GCC ACG CCC ACG TCA GCA AAT CCC GAC GTG GGA 240
Thr Arg Bis His Pro Ala Thr Pro Thr Ser Ala Asn Pro Asp Val Gly
65 70 75 80
ACC CCC AGA CCG TCC GAG GAC AAC GTC CCC GCA AAG CCG CGC CTA TTG 288
Th- Pro Arg Pro Ser Glu Asp Asn Val Pro Ala Lys Pro Arg Leu Leu
85 90 95
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
100 105 110
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
130 135 140
-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
145 150 155 160
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
165 170 175
TT- TTT TTG GTT TCA ATC AAG GCC CCA CAA AAG ACG CAC CAG TTG TTT 576
Phe Phe Leu Val Ser Ile Lys Ala Pro Gln Lys Thr Bis Gln Leu Phe
180 185 190
WO96/06159 i 2 1 9 6 8 9 2 PcT~usgS/I0!9J--
234
GGA TTG TTT AAG CAG TAC TTC GGT TTA TTT GAA ACT CCA AAC AGT GTT 624
Gly Leu Phe Lys Gln Tyr Phe Gly Leu Phe Glu Thr Pro Asn Ser Val
l9S 200 205
TTA CAG ACG TTT AAG CAA AAG GCA AGC GTA TTC CTA ATA CCA AGG AGA 672
Leu Gln Thr Phe Lys Gln Lys Ala Ser Val Phe Leu Ile Pro Arg Arg
210 -215 2ao
CAC GGA AAG ACA TGG ATA GTG GTG GCG ATC ATC AGC ATG CTA CTG GCA 720
His Gly Lys Thr Trp Ile Val Val Ala Ile Ile 5er Met Leu Leu Ala
225 230 235 240
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
260 265 270
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
275 280 285
ACG CGA CCC GGA GGA CGG TCC AGC TCG CTG ATG TGC GCA ACA TGC TTC 912
Thr Arg Pro Gly Gly Arg Ser Ser Ser Leu Met Cys Ala Thr Cys Phe
290 295 300
AAT A~G AAC 921
Asn Lys Asn
305
(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
SS 60
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
9S
~lu Ser Leu Ser Thr Tyr Leu Gln Met Arg Cys Val Arg Glu Asp Ala
100 105 110
~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
165 170 175
~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
245 250 255
~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
290 295 300
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
65 70 75 - 80
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
100 105 110
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
130 135 140
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
165 170 175
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
180 185 190
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
CAG AAG GGG CCA GCG CGC ACA CGC CAC AGG CCA CCC CCC GTG CGA CTG 672
Gln Lys Gly Proi~la Ar~ Thr Arg ~is Arg Pro Pro Pro Val Arg~Leu
210 215 220
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
245 250 255
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
275 280 285
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
325 330 335
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
340 345 350
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
385 390 395 - 400
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
405 410 415
GTG ATC GTC ACA CCC GCA TTG GAA GCC TGG CTT GTG TTA CCT GGG GGT 1296
Val Ile Val Thr Pro Ala Leu Glu Ala Trp Leu Val Leu Pro Gly Gly
420 425 430
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
435 440 445
ATT CGA GGC CGC TAT GGC T~G 1365
Ile Arg Gly Arg Tyr Gly
450
~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
7D 75 - 80
WO96/06159 ' 2 1 96892 PCT/US95/10194~
238
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
115 120 125
Asn Acn Ala Arg Ser Pro~Thr Val Asp Gly Val Ser Pro prQ Glu Gly
130 135 140
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
165 170 175
~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
195 200 205
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
245 250 255
~ly Gly Arg Gln Glu Asp Asp Trp Leu Pro Ile Pro Leu Ser Phe Pro
260 265 270
Glu Glu Pro Val Pro Pro Pro Pro Gly Leu Val Phe Met Asp Asp Leu
275 280 285
Phe Ile Asn Thr Lys 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
340 345 350
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
370 375 380
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
420 425 430
~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
239
435 440 445
.Ile Arg Gly Arg Tyr Gly
450
(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
20 25 30
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
TTG GTT TCC AAC GTG ATG CCC GAG GTC CAG CCA ATC TGC AGT GGC CGG 192
Leu Val Ser Asn Val Met Pro Glu Val 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
85 90 9S
ATC GCA CTG GAC CCG TCT CAC GTA GAA ACA CAC GAA AAG GGC CAG GTG 336
Ile Ala Leu Asp Pro Ser His Val Glu Thr His Glu Lys Gly Gln Val
100 105 110
TTC TAC AAC CAC TAT GCT ACC GAG GAG TGG ACG TGG GCT TTG ACT CTG 384
Phe Tyr Asn His Tyr Ala Thr Glu Glu Trp Thr lrp Ala Leu Thr Leu
llS : 12~ ~: 125
AAT AAG GAT GCG CTC CTT CGG GAG GCT GTA GAT GGC CTG TGT GAC CCC 432
Asn Lys Asp Ala Leu Leu Arg Glu Ala Val Asp Gly Leu Cys Asp Pro
130 135 140
GGA ACT TGG AAG GGT CTT CTT CCT GAC GAC CCC CTT CCG TTG CTA TGG 480
Gly Thr Trp Lys Gly Leu Leu Pro Asp ASp Pro Leu Pro Leu Leu Trp
145 lSO lSS 160
WO 96/06159 ~, 2 1 9 6 8 9 2 PCT/US95/1019--
240
CTG CTG TTC AAC GGA CCC GCC TCT TTT TGT CGG GCC GAC TGT TGC CTG 528
Leu Leu Phe Asn Gly Pro Ala Ser Phe Cys Arg Ala Asp Cys Cys Leu
165 170 175
TAC AAG CAG CAC TGC GGT TAC CCG GGC CCG GTG CTA CTT CCA GGT CAC 576
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
195 200 205
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
210 215 220
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
245 250 255
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
260 265 270
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
275 280 285
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
325
~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
Z0 25 30
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
130 -135 140
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
165 170 175
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
195 200 205
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
245 250 255
Leu Leu Cys His Ile Tyr Gln Gln Asn Ser Ile Ile Ala Gly Gln Gly
260 265 270
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
290 295 300
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
325
~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
242
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
GCATTCTTTG GAAGTAGTGG TAGAGATGGA GCAGACTGCC AGGGCGTTGC AGGAGTGGTG 360
GCGATGGTGC GCACCGTTTT T~ rrr rrrDr-Q~rTr~r~ rr~rTrrrr~r TCCCTGCAGC 420
ATCTCGGCCT GCTGTACGTC CTTGGCGAAT ATGCGACGAA ATCGGCTGTG CGCACGGGGT 480
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~
243
~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 ~
244
(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:
AACACGTCAT GTGCAGGAGT GACCGGGTGA CAGTTGTGAT CTAAGG 46
(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
