Note: Descriptions are shown in the official language in which they were submitted.
CA 02241023 1998-06-19
W097/23624 PCT/GB96/03221
Novel DolYnucleotides and ~olyDe~tides in Pathoaenic mYcobacteria
and their u~e as diaonostic~, ~accines and taraets for
chemothera~Y .
This invention relates to the novel polynucleotide sequence we
have designated "GS" which we have identified in pathogenic
mycobacteria. GS is a pathogenicity island within 8kb of DNA
comprising a core region of 5.75kb and an adjacent transmissable
element within 2.25kb. GS is contained within Mycobacterium
paratuberculosis, Mycobacterium avium subsp. silvaticum and some
pathogenic isolates of M.avium. Functional portions of the core
region of GS are also represented by regions with a high degree
of homology that we have identified in cosmids containing genomic
DNA from Mycobacterium tuberculosis.
Backqround to the invention
Mycobacterium tuberculosis (Mtb) is a major cause of global
diseases of humans as well as animals. Although conventional
methods of diagnosis including microscopy, culture and skin
testing exist for the recognition of these diseases, improved
methods particularly new immunodiagnostics and DNA-based
detection systems are needed. Drugs used to treat tuberculosis
are increasingly encountering the problem of resistant organisms.
New drugs targeted at specific pathogenicity determinants as well
as new vaccines for the prevention and treatment of tuberculosis
are required. The importance of Mtb as a global pathogen is
reflected in the commitment being made to sequencing the entire
genome of this organism. This has generated a large amount of
DNA sequence data of genomic DNA within cosmid and other
libraries. Although the DNA sequence is known in the art, the
functions of the vast majority of these sequences, the proteins
they encode, the biological significance of these proteins, and
the overall relevance and use of these genes and their products
as diagnostics, vaccines and targets for chemotherapy for
tuberculous disease, re~ns entirely unknown.
Mycobacterium avium subsp. silvaticum (Mavs) is a pathogenic
mycobacterium causing diseases of animals and birds, but it can
CA 02241023 1998-06-19
w097~3624 PcT/Gsg6lo322l
also affect humans. Mycobacterium paratu~erculosis (Mptb) causes
chronic inflammation of the intestine in many species of animals
including primates and can also cause Crohn's disease in humans.
Mpt~ is associated with other chronic inflammatory diseases of
h~ n.~ such as sarcoidosis. Subclinical Mptb infection is
widespread in domestic livestock and is present in milk from
infected animals. The organism is more resistant to
pasteurisation than Mtb and can be conveyed to humans in retail
milk supplies. Mptb i9 also present in water supplies,
particularly those contaminated with run-off from heavily grazed
pastures. Mptb and Mavs contain the insertion elements IS900 and
ISg02 respectively, and these are linked to pathogenicity in
these organisms. IS900 and IS902 provide convenient highly
specific multi-copy DNA targets for the sensitive detection of
these organisms using DNA-based methods and for the diagnosis of
infections in animals and humans. Much improvement is however
required in the immunodiagnosis of Mptb and Ma~s infections in
animals and humans. Mptb and Mavs are in general, resistant in
vivo to standard anti-tuberculous drugs. Although substantial
clinical improvements in infections caused by Mpt~, such as
Crohn's disease, may result from treatment of patients with
combinations of existing drugs such as Rifabutin, Clarithromycin
or Azithromycin, additional effective drug treatments are
required. Furthermore, there is an urgent need for effective
vaccines for the prevention and treatment of Mptb and MaYs
infections in animals and hl]m~n~ based upon the recognition of
specific pathogenicity determinants.
Pathogenicity islands are, in general, 7-9kb regions of DNA
comprising a core domain with multiple ORFs and an adjacent
transmissable element. The transmissable element also encodes
proteins which may be linked to pathogenicity, such as by
providing receptors for cellular recognition. Pathogenicity
islands are envisaged as mobile packages of DNA which, when they
enter an organism, assist in bringing about its convertion from
3~ a non-disease-causing to a disease-causing strain.
Descri~tion of the Drawinqs
CA 02241023 1998-06-19
WO 97/23624 PCT/(~B5~ 3221
Figure l(a) and (b) shows a linear map of the pathogenicity
island GS in Mavs (Fig la~ and in Mptb ( Fig lb). The main open
reading frames are illustrated as ORFs A to H. ORFs A to F are
found within the core region of GS. ORFs G and H are encoded by
the adjacent transmissable element portion of GS.
Disclosure of the invention
Using a DNA-based differential analysis technology we have
discovered and characterised a novel polynucleotide in Mpt~
(isolates 0022 from a Guernsey cow and 0021 from a red deer~.
This polynucleotide comprises the gene region we have designated
GS. GS is found in Mptb using the identifier DNA sequences
Seq.ID.No 1 and 2 where the Seq.ID No2 is the complementary
sequence of Seq.ID No 1. GS is also identified in Mavs. The
complete DNA sequence incorporating the positive strand of GS
from an isolate of Mavs comprising 7995 nucleotides, including
the core region of GS and adjacent transsmissable element, is
given in Seq.ID No. 3. DNA sequence comprising 4435 bp of the
positive strand of GS obtained from an isolate of Mptb including
the core region of GS (nucleotides 1614 to 6047 of GS in Mavs)
is given in Seq.ID No 4. The DNA sequence of GS from Mptb is
highly (99.4~) homologous to GS in Mavs. The remaining portion
of the DNA sequence of GS in Mptb, is readily obtainable ~y a
person skilled in the art using standard laboratory procedures.
The entire functional DNA sequence including core region and
transmisable element of GS in Mptb and Mavs as described above,
comprise the polynucleotide sequences of the invention.
There are 8 open reading frames (ORFs) in GS. Six of these
designated GSA, GSB, GSC, GSD, GSE and GSF are encoded by the
core DNA region of GS which, characteristically for a
pathogenicity island, has a different GC content than the rest
of the microbial genome. Two ORFs designated GSG and GSH are
encoded by the transmissable element of GS whose GC content
resembles that of the rest of the mycobacterial genome. The ORF
GSH comprises two sub-ORFs H1 H2 on the complementary DNA strand
linked by a programmed frameshifting site so that a single
polypeptide is translated from the ORF GSH. The nucleotide
CA 0224l023 l998-06-l9
W097/23624 PCTIGB96/03221
sequences of the 8 ORFs in GS and their translations are shown
in Seq. ID No 5 to Seq.ID No 29 as follows:
ORF A: Seq. ID No 5 Nucleotides 50 to 427 of GS from Mav~
Seq. ID No 6 Amino acid sequence encoded by Seq.ID No
5.
ORF B: Seq. ID No 7 Nucleotides 772 to 1605 of GS from Mavs
Seq. ID No 8 Amino acid sequence encoded by Seq.ID No
7.
ORF C: Seq. ID No 9 Nucleotides 1814 to 2845 of GS from Mavs
Seq. ID No 10 Amino acid sequence encoded by Seq.ID No
9.
Seq. ID No 11 Nucleotides 201 to 1232 of GS from Mptb
Seq. ID No 12 Amino acid sequence encoded by Seq.I~ No
11
ORF D: Seq. ID No 13 Nucleotides 2785 to 3804 of GS from Mavs
Seq. ID No 14 Amino acid sequence encoded by Seq.ID No
13.
Seq. ID No 15 Nucleotides 1172 to 2191 of GS from Mptb
Seq. ID No 16 Amino acid sequence encoded by Seq.ID No
15.
ORF E: Seq. ID No 17 Nucleotides 4080 to 4802 of GS from Mavs
Seq. ID No 18 Amino acid sequence encoded by Seq.ID No
17.
Seq. ID No 19 Nucleotides 2467 to 3189 of GS from Mptb
Seq. ID No 20 Amino acid sequence encoded by Seq.ID No
19 .
ORF F: Seq. ID No 21 Nucleotides 4947 to 5747 of GS from Mavs
Seq. ID No 22 Amino acid sequence encoded by Seq.ID No
21.
Seq. ID No 23 Nucleotides 3335 to 4135 of GS from Mptb
Seq. ID No 24 Amino acid sequence encoded by Seq.ID No
23.
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W097l23624 PCT/GB96/03221
ORF G: Seq. ID No 25 Nucleotides 6176 to 7042 of GS from Mavs
Seq. ID No 26 Amino acid sequence encoded by
Seq.ID No 25.
ORF H: Seq.ID No 27 Nucleotides 7953 to 6215 from Mavs.
5 ORF Hl: Seq.ID No 28 Amino acid sequence encoded by
nucleotides 7953 to 7006 of Seq.ID No 27
ORF H2: Seq.ID No 29 Amino acid sequence encoded by
nucleotides 7009 to 6215 of Seq.ID No 27
The polynucleotides in Mtb with homology to the ORFs B, C, E and
F of GS in Mptb and Mavs, and the polypeptides they are now known
to encode as a result of our invention, are as follows:
ORF B: Seq.ID No 30 Cosmid MTCY277 nucleotides 35493 to
34705
Seq.ID No 31 Amino acid sequence encoded by Seq.ID
No30.
ORF C: Seq.ID No 32 Cosmid MTCY277 nucleotides 31972 to 32994
Seq.ID No 33 Amino acid sequence encoded by Seq.ID
No32.
ORF E: Seq.ID No 34 Cosmid MTCY277 nucleotides 34687 to 33956
Seq.ID No 35 Amino acid sequence encoded by Seq.ID
No34.
ORF E: Seq.ID No 36 Cosmid MTO24 nucleotides lS934 to 15203
Seq.ID No 37 Amino acid sequence encoded by Seq.ID
No36.
ORF F: Seq.ID No38 Cosmid MTO24 nucleotides 15133 to ~4306
Seq.ID No 39 Amino acid sequence encoded by Seq.ID
No38.
The proteins and peptides encoded by the ORFs A to H in Mptb and
Mavs and the amino acid sequences from homologous genes we have
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WO 97n3624 PCTJGl~ 3221
discovered in Mtb given in Seq.ID Nos 3l, 33, 35, 37 and 39, as
described above and fragments thereof, comprise the polypeptides
of the invention. The polypeptides of the invention are believed
to be associated with specific immunoreactivity and with the
pathogenicity of the host micro-organisms from which they were
obtained.
The present invention thus provides a polynucleotide in
substantially isolated form which is capable of selectively
hybridising to sequence ID Nos 3 or 4 or a fragment thereof. The
polynucleotide fragment may alternatively comprise a sequence
selected from the group of Seq.ID.No: 5, 7, 9, ll, 13, 15, 17,
lg, 21, 23, 25 and 27. The invention further provides a
polynucleotide in substantially isolated form whose sequence
consists essentially of a sequence selected from the group Seq
ID Nos. 30, 32, 34, 36 and 38, or a corresponding sequence
selectively hybridizable thereto, or a fragment of said sequence
or corresponding sequence.
The invention further provides diagnostic probes such as a probe
which comprises a fragment of at least 15 nucleotides of a
polynucleotide of the invention, or a peptide nucleic acid or
similar synthetic sequence specific ligand, optionally carryiny
a revealing label. The invention also provides a vector carrying
a polynucleotide as defined above, particularly an expression
vector.
The invention further provides a polypeptide in substantially
isolated form which comprises any one of the sequences selected
from the group consisting Seq.ID.No: 6, 8, lO, 12, 14, 16, 18,
20, 22, 24, 26, 28, 29, 31, 33, 3~, 37 and 39, or a polypeptide
substantially homologous thereto. The invention additionally
provides a polypeptide fragment which comprises a fragment of a
polypeptide defined above, said fragment comprising at least lO
amino acids and an epitope. The invention also provides
polynucleotides in substantially isolated form which encode
polypeptides of the invention, and vectors which comprise such
polynucleotides, as well as antibodies capable of binding such
polypeptides. In an additional aspect, the invention provides
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WO 97/23624 PCT/(il~5GJ~3221
kits comprising polynucleotides, polypeptides, antibodies or
synthetic ligands of the invention and methods of using such kits
in diagnosing the presence or absence of mycobacteria in a
sample. The invention also provides pharmaceutical compositions
comprising polynucleotides of the invention, polypeptides of the
invention or antisense probes and the use of such compositions
in the treatment or prevention of diseases caused by
mycobacteria. The invention also provides polynucleotihe
prevention and treatment of infections due to GS-containing
pathogenic mycobacteria in animals and humans and as a means of
enhacing in vivo susceptibility of said mycobacteria to
antimicrobial drugs. The invention also provides bacteria or
viruses transformed with polynucleotides of the invention for use
as vaccines. The invention further provides Mptb or Mavs in
which all or part or the polynucleotides of the invention have
been deleted or disabled to provide mutated organisms of lower
pathogenicity for use as vaccines in animals and humans. The
invention further provides Mtb in which all or part of the
polynucleotides encoding polypeptides of the invention have been
deleted or disabled to provided mutated organisms or lower
pathogenicity for use as vaccines in ~nlm~l s and h-lm~s.
A further aspect of the invention is our discovery of homologies
between the ORFs B, C and E in GS on the one hand, and Mtb cosmid
MTCY277 on the other (data from Genbank database using the
~5 computer programmes BLAST and BLIXEM). The homologous ORFs in
MTCY277 are adjacent to one another consistent with the form of
another pathogenicity island in Mtb. A further aspect of the
invention is our discovery of homologies between ORFs E and F in
GS, and Mtb cosmid MTO24 (also Genbank, as above) with the
homologous ORFs close to one another. The use of polynucleotides
and polypeptides from Mtb tSe~. ID Nos 30,31, 32, 33, 34, 35, 36,
37, 38 and 39) in substantially isolated form as diagnostics,
vaccines and targets for chemotherapy, for the management and
prevention of Mtb infections in humans and animals, and the
processes involved in the preparation and use of these
diagnostics, vaccines and new chemotherapeutic agents, comprise
further aspects of the invention.
CA 02241023 1998-06-19
WO 97/23624 PCT/GB~61'03221
Detailed descri~tion of the invention.
A. Pol~nucleotides
Polynucleotides of the invention as defined herein may comprise
DNA or RNA. They may also be polynucleotides which include
within them synthetic or modified nucleotides or peptide nucleic
acids. A number of different types of modification to
oligonucleotides are known in the art. These include
methylphosphonate and phosphorothioate backbones, addition of
acridine or polylysine chains at the 3' and/or 5' ends of the
molecule. For the purposes of the present invention, it is to
be understood that the polynucleotides described herein may be
modified by any method available in the art. Such modifications
may be carried out in order to couple the said polynucleotide to
a solid phase or to enhance the recognition, the in vivo
activity, or the lifespan of polynucleotides of the invention.
A number of different types of polynucleotides of the invention
are envisaged. In the broadest aspect, polynucleotides and
fragments thereof capable of hybridizing to SEQ ID NO:3 or ~ form
a first aspect of the invention. This includes the
polynucleotide of SEQ ID NO: 3 or 4. Within this class of
polynucleotides various sub-classes of polynucleotides are of
particular interest.
One sub-class of polynucleotides which is of interest is the
class of polynucleotides encoding the open reading frames A, B,
C, D, E, F, G and H, including SEQ ID NOs:5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25 and 27. As discussed below, polynucleotides
encoding ORF H include the polynucleotide sequences 7953 to 7006
and 7009 to 6215 within SEQ ID NO: 27, as well as modified
sequences in which the frame-shift has been modified so that the
two sub-reading frames are placed in a single reading frame.
This may be desirable where the polypeptide is to be produced in
recombinant expression systems.
The invention thus provides a polynucleotide in substantially
isolated form which encodes any one of these ORFs or combinations
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W O 97/23624 PCT/G B96/03221
thereof. Combinations thereof includes combinations of 2, 3, 4,
5 or all of the ORFs. Polynucleotides may be provided which
comprise an individual ORF carried in a recombinant vector
including the vectors described herein. Thus in one preferred
aspect the invention provides a polynucleotide in substantially
isolated form capable of selectively hybridizing to the nucleic
acid comprising ORFs A to F of the core region of the Mptb and
Mavs pathogenicity islands of the invention. Fragments thereof
corresponding to ORFs A to E, B to F, A to D, B to E, A to C, B
to D or any two adjacent ORFs are also included in the invention.
Polynucleotides of the invention will be capable of selectively
hybridizing to the corresponding portion of the ~S region, or to
the corresponding ORFs of Mtb described herein. The term
"selectively hybridizing" indicates that the polynucleotides will
hybridize, under conditions of medium to high stringency (for
example 0.03 M sodium chloride and 0.03 M sodium citrate at from
about 50OC to about 60OC) to the corresponding portion of SEQ ID
NO:3 or 4 or the complementary strands thereof but not to genomic
DNA from mycobacteria which are usually non-pathogenic including
non-pathogenic species of M.avium. Such polynucleotides will
generally be generally at least 68~, e.g. at least 70%,
preferably at least 80 or 90% and more preferably at least 95~
homologous to the corresponding DNA of GS. The corresponding
portion will be of over a region of at least 20, preferably at
least 30, for instance at least 40, 60 or l00 or more contiguous
nucleotides.
By "corresponding portion" it is meant a sequence from the GS
region of the same or substantially similar size which has been
determined, for example by computer alignment, to have the
greatest degree of homology to the polynucleotide.
Any combination of the above mentioned degrees of homology and
minimum sizes may be used to define polynucleotides of the
invention, with the more stringent combinations ~i.e. higher
homology over longer lengths) being preferred. Thus for example
a polynucleotide which is at least 80~ homologous over 25,
preferably 30 nucleotides forms one aspect of the invention, as
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- 10 -
does a polynucleotide which is at least 90~ homologous over 40
nucleotides.
A further class of polynucleotides of the invention is the class
of polynucleotides encoding polypeptides of the invention, the
polypeptides of the invention being defined in section B below.
Due to the redundancy of the genetic code as such,
polynucleotides may be of a lower degree of homology than
required for selective hybridization to the GS region. However,
when such polynucleotides encode polypeptides of the invention
these polynucleotides form a further aspect. It may for example
be desirable where polypeptides of the invention are produced
recombinantly to increase the GC content of such polynucleotides.
This increase in GC content may result in higher levels of
expression via codon usage more appropriate to the host cell in
which recombinant expression is taking place.
An additional class of polynucleotides of the invention are those
obtainable from cosmids MTCY277 and MT024 ~containing Mtb genomic
sequences), which polynucleotides consist essentially of the
fragment of the cosmid containing an open reading frame encoding
any one of the homologous OR~s B, C, E or F respectively. Such
polynucleotides are referred to below as Mtb polynucleotides.
However, where reference is made to polynucleotides in general
such reference includes Mtb polynucleotides unless the context
is explicitly to the contrary. In addition, the invention
provides polynucleotides which encode the same polypeptide as the
abovementioned ORFs of Mtb but which, due to the re~-ln~ncy of
the genetic code, have different nucleotide sequences. These
form further Mtb polynucleotides of the invention. Fragments of
Mtb polynucleotides suitable for use as probes or primers also
form a further aspect of the invention.
The invention further provides polynucleotides in substantially
isolated form capable of selectively hybridizing (where
selectively hybridizing is as defined above) to the Mtb
polynucleotides of the invention.
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W097/23624 PcT/Gsg6/03221
The invention further provides the Mtb polynucleotides of the
invention linked, at either the 5~ and/or 3' end to
polynucleotide sequences to which they are not naturally
contiguous. Such sequences will typically be sequences found in
cloning or expression vectors, such as promoters, 5' untranslated
sequence, 3' untranslated sequence or termination sequences. The
sequences may also include further coding sequences such as
signal sequences used in recombinant production of proteins.
Further polynucleotides of the invention are illustrated in the
accompanying examples.
Polynucleotides of the invention may be used to produce a primer,
e.g. a PCR primer, a primer for an alternative amplification
reaction, a probe e.g. labelled with a revealing label by
conventional means using radioactive or non-radioactive labels
or a probe linked covalently to a solid phase, or the
polynucleotides may be cloned into vectors. Such primers,
probes and other fragments will be at least 15, preferably at
least 20, for example at least 25, 30 or 40 or more nucleotides
in length, and are also encompassed by the term polynucleotides
of the invention as used herein.
Primers of the invention which are preferred include primers
directed to any part of the ORFs defined herein. The ORFs from
other isolates of pathogenic mycobacteria which contain a GS
region may be determined and conserved regions within each
individual ORF may be identified. Primers directed to such
conserved regions form a further preferred aspect of the
invention. In addition, the primers and other polynucleotides
of the invention may be used to identify, obtain and isolate ORFs
capable of selectively hybridizing to the polynucleotides of the
invention which are present in pathogenic mycobacteria but which
are not part of a pathogenicity island in that particular species
of bacteria. Thus in addition to the ORFs B, C, E and F which
have been identified in Mtb, similar ORFS may be identified in
other pathogens and ORFs corresponding to the GS ORFs C, D, E,
F and H, may also be identified.
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W097/23624 PcT/Gss6/03221
Polynucleotide~ such as DNA polynucleotides and probes according
to the invention may be produced recombinantly, synthetically,
or by any means available to those of s~ill in the art. They may
also be cloned by standard techniques.
In general, primers will be produced by synthetic means,
involving a step-wise manufacture of the desired nucleic acid
sequence one nucleotide at a time. Techniques for accomplishing
this using automated techniques are readily available in the art.
Longer polynucleotides will generally be produced using
recombinant means, for example using a PCR (polymerase chain
reaction~ cloning techniques. This will involve making a pair
or primers (e.g. of about 15-30 nucleotides) to a region of GS,
which it is desired to clone, bringing the primers into contact
with genomic DNA from a mycobacterium or a vector carrying the
GS sequence, performing a polymerase chain reaction under
conditions which bring about amplification of the desired region,
isolating the amplified fragment (e.g. by purifying the reaction
mixture on an agarose gel) and recovering the amplified DNA. The
primers may be designed to contain suitable re~triction enzyme
recognition sites so that the amplified DNA can be cloned into
a suitable cloning vector.
Such techniques may be used to obtain all or part of the GS or
ORF sequences described herein, as well as further genomic clones
containing full open reading frames. Although in general such
techniques are well known in the art, reference may be made in
particular to Sambrook J., Fritsch EF., Maniatis T (1989~.
Molecular cloning: a Laboratory Manual, 2nd edn. Cold Spring
Harbor, New York, Cold Spring Harbor Laboratory.
Polynucleotides which are not l00~ homologous to the sequences
of the present invention but fall within the scope of the
invention can be obtained in a number of ways.
Other isolates or strains of pathogenic mycobacteria will be
expected to contain allelic variants of the GS sequences
described herein, and these may be obtained for example by
probing genomic DNA libraries made from such isolates or strains
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WO 97123624 PCTl(iL!~1'&3221
of bacteria using GS or ORF sequences as probes under conditions
of medium to high stringency (for example 0.03M sodium chloride
and 0.03M sodium citrate at from about 50~C to about 60~C).
A particularly preferred group of pathogenic mycobacteria are
isolates of M.paratuberculosis. Polynucleotides based on GS
regions from such bacteria are particularly preferred. Preferred
fragments of such regions include fragments encoding individual
open reading frames including the preferred groups and
combinations of open reading frames discussed above.
Alternatively, such polynucleotides may be obtained by site
directed mutagenesis of the GS or ORF sequences or allelic
variants thereof. This may be useful where for example silent
codon changes are required to sequences to optimise codon
preferences for a particular host cell in which the
1~ polynucleotide sequences are being expressed. Other sequence
changes may be desired in order to introduce restriction enzyme
recognition sites, or to alter the property or function of the
polypeptides encoded by the polynucleotides of the invention.
Such altered property or function will include the addition of
amino acid sequences of consensus signal peptides known in the
art to effect transport and secretion of the modified polypeptide
of the invention. Another altered property will include
metagenesis of a catalytic residue or generation of fusion
proteins with another polypeptide. Such fusion proteins may be
with an enzyme, with an antibody or with a cytokine or other
ligand for a receptor, to target a polypeptide of the invention
to a specific cell type in vitro or in vivo.
The invention further provides double stranded polynucleotides
comprising a polynucleotide of the invention and its complement.
Polynucleotides or primers of the invention may carry a revealing
label. Suitable labels include radioisotopes such as 32p or ~5S,
enzyme labels, other protein labels or smaller labels such as
biotin or fluorophores. Such labels may be added to
polynucleotides or primers of the invention and may be detected
using by techniques known per se.
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- 14 -
Polynucleotides or primers of the invention or fragments thereof
labelled or unlabelled may be used by a person skilled in the art
in nucleic acid-based tests for the presence or absence of Mptb,
Mavs, other GS-containing pathogenic mycobacteria, or Mtb applied
to samples of body fluids, tissues, or excreta from animals and
hllm~n~, as well as to food and environmental samples such as
river or ground water and domestic water supplies.
Human and animal body fluids include sputum, blood, serum,
plasma, saliva, milk, urine, csf, semen, faeces and infected
discharges. Tissues include intestine, mouth ulcers, skin, lymph
nodes, spleen, lung and liver obtained surgically or by a biopsy
technique. Animals particularly include commercial livestock
such as cattle, sheep, goats, deer, rabbits but wild animals and
animals in zoos may also be tested.
Such tests comprise bringing a human or animal body fluid or
tissue extract, or an extract of an environmental or food sample,
into contact with a probe comprising a polynucleotide or primer
of the invention under hybridising conditions and detecting any
duplex formed between the probe and nucleic acid in the sample.
Such detection may be achieved using techniques such as PCR or
by immobilising the probe on a solid support, removing nucleic
acid in the sample which is not hybridized to the probe, and then
detecting nucleic acid which has hybridized to the probe.
Alternatively, the sample nucleic acid may be immobilized on a
solid support, and the amount of probe bound to such a support
can be detected. Suitable assay methods of this any other
formats can be found in for example WO89/03891 and WO90/13667.
Polynucleotides of the invention or fragments thereof labelled
or unlabelled may also be used to identify and characterise
different strains of Mptb, Mavs, other GS-containing pathogenic
mycobacteria, or Mtb, and properties such as drug resistance or
susceptibility.
The probes of the invention may conveniently be packaged in the
form of a test kit in a suitable container. In such kits the
probe may be bound to a solid support where the assay format for
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WO 97n3624 PCT/GB96/03221
- 15 -
which the kit is desiyned requires such binding. The kit may
also contain suitable reagents for treating the sample to be
probed, hybridising the probe to nucleic acid in the sample,
control reagents, instructions, and the like.
The use of polynucleotides of the invention in the diagnosis of
inflammatory diseases such as Crohn's disease or sarcoidosis in
humans or Johne's disease in animals form a preferred aspect of
the invention. The polynucleotides may also be used in the
prognosis of these diseases. For example, the response of a
human or animal subject in response to antibiotic, vaccination
or other therapies may be monitored by utilizing the diagnostic
methods of the invention over the course of a period of treatment
and following such treatment.
The use of Mtb polynucleotides (particularly in the form of
probes and primers) of the invention in the above-described
methods form a further aspect of the invention, particularly for
the detection, diagnosis or prognosis of Mtb infections.
B. Poly~e~tides.
Polypeptides of the invention include polypeptides in
substantially isolated form encoded by GS. This includes the
full length polypeptides encoded by the positive and
complementary negative strands of GS. Each of the full length
polypeptides will contain one of the amino acid sequences set out
in Seq ID NOs:6, 8, lO, 12, 14, 16, 18, 20, 22, 24, 26, 28 and
29. Polypeptides of the invention further include variants of
such sequences, including naturally occurring allelic variants
and synthetic variants which are substantially homologous to said
polypeptides. In this context, substantial homology is regarded
as a sequence which has at least 70%, e.g. 80~, 90%, 95% or 98%
amino acid homology (identity) over 30 or more, e.g 40, 50 or lO0
amino acids. For example, one group of substantially homolgous
polypeptides are those which have at least 95% amino acid
identity to a polypeptide of any one of Seq ID NOs:6, 8, lO, 12,
14, 16, 18, 20, 22, 24, 26, 28 and 29 over their entire length.
35 Even more preferably, this homology is 98%.
CA 0224l023 l998-06-l9
wo97n3624 pcTlGs96lo322
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Polypeptides of the invention further include the polypeptide
se~uences of the homologous ORFs of Mtb, namely Seq TD Nos. 31,
33, 35, 37 and 39. Unless explicitly specified to the contrary,
reference to polypeptides of the invention and their fragments
include these Mtb polypeptides and fragments, and variants
thereof (substanially homologous to said sequences) as defined
herein.
Polypeptides of the invention may be obtained by the standard
techniques mentioned above. Polypeptides of the invention also
include fragments of the above mentioned full length polypeptides
and variants thereof, including fragments of the sequences set
out in SEQ I~ NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,
29, 31, 33, 35, 37 and 39. Such fragments for example of 8, 10,
12, 15 or up to 30 or 40 amino acids may also be obtained
synthetically using standard techniques known in the art.
Preferred fragments include those which include an epitope,
especially an epitope which is specific to the pathogenicity of
the mycobacterial cell from which the polypeptide is derived.
Suitable fragments will be at least about 5, e.g. 8, 10, 12, 15
or 20 amino acids in size, or larger. Epitopes may be determined
either by techniques such as peptide scanning techniques as
described by Geysen et al, Mol.Immunol., 23; 709-715 (1986), as
well as other techniques known in the art.
The term "an epitope which is specific to the pathogenicity of
the mycobacterial cell" means that the epitope is encoded by a
portion of the GS region, or by the corresponding ORF sequences
of Mtb which can be u~ed to distinguish mycobacteria which are
pathogenic by from related non-pathogenic mycobacteria including
non-pathogenic species of M.avium. This may be determined using
routine methodology. A candidate epitope from an ORF may be
prepared and used to immunise an animal such as a rat or rabbit
in order to generate antibodies. The antibodies may then be used
to detect the presence of the epitope in pathogenic mycobacteria
and to confirm that non-pathogenic mycobacteria do not contain
any proteins which react with the epitope. Epitopes may be
linear or conformational.
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- 17 -
Polypeptides of the invention may be in a substantially isolated
form. It will be understood that the polypeptide may be mixed
with carriers or diluents which will not interfere with the
intended purpose of the polypeptide and still be regarded as
substantially isolated. A polypeptide of the invention may also
be in a substantially purified form, in which case it will
generally comprise the polypeptide in a preparation in which more
than 90~, e.g. 95~, 98~ or 99% of the polypeptide in the
preparation is a polypeptide of the invention.
Polypeptides of the invention may be modified to confer a desired
property or function for example by the addition of Histidine
residues to assist their purification or by the addition of a
signal sequence to promote their secretion from a cell.
Thus, polypeptides of the invention include fusion proteins which
comprise a polypeptide encoding all or part of one or more of an
ORF of the invention fused at the N- or C-terminus to a second
sequence to provide the desired property or function. Sequences
which promote secretion from a cell include, for example the
yeast ~-factor signal sequence.
A polypeptide of the invention may be labelled with a revealing
label. The revealing label may be any suitable label which
allows the polypeptide to be detected. Suitable labels include
radioisotopes, e.g. lZsI, 35S enzymes, antibodies, polynucleotides
and ligands such as biotin. Labelled polypeptides of the
invention may be used in diagnostic procedures such as
immunoassays in order to determine the amount of a polypeptide
of the invention in a sample. Polypeptides or labelled
polypeptides of the invention may also be used in serological or
cell mediated immune assays for the detection of immune
reactivity to said polypeptides in animals and humans using
standard protocols.
A polypeptide or labelled polypeptide of the invention or
fragment thereof may also be fixed to a solid phase, for example
the surface of an immunoassay well, microparticle, dipstick or
biosensor. Such labelled and/or immobilized polypeptides may be
CA 02241023 1998-06-l9
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- 18 -
packaged into kits in a suitable container along with suitable
reagents, controls, instructions and the like.
Such polypeptides and kits may be used in methods of detection
of antibodies or cell mediated immunoreactivity, to the
mycobacterial proteins and peptides encoded by the ORFs of the
invention and their allelic ~ariants and fragments, using
immunoassay. Such host antibodies or cell mediated immune
reactivity will occur in humans or animals with an immune system
which detects and reacts against polypeptides of the in~ention.
The antibodies may be present in a biological sample from such
hl~m~ns or animals, where the biological sample may be a sample
as defined above particularly blood, milk or saliva.
Immunoassay methods are well known in the art and will generally
comprise:
(a) providing a polypeptide of the invention comprising an
epitope bindable by an antibody against said
mycobacterial polypeptide;
(b) incubating a biological sample with said polypeptide
under conditions which allow for the formation of an
antibody-antigen complex; and
(c) determining whether antibody-antigen complex
comprising said polypeptide is formed.
Immunoassay methods for cell mediated immune reacti~ity in
animals and humans are also well known in the art (e.g. as
described by Weir et al 1994, J.Immunol Methods 176; 93-l0l) and
will generally comprise
(a) providing a polypeptide of the invention comprising an
epitope bindable by a lymphocyte or macrophage or
other cell receptor;
(b) incubating a cell sample with said polypeptide under
conditions which allow for a cellular immune response
such as release of cytokines or other mediator to
occur; and
(c) detecting the presence of said cytokine or mediator in
the incubate.
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Polypeptides of the invention may be made by standard synthetic
means well known in the art or recombinantly, as described below.
Polypeptides of the invention or fragments thereof labelled or
unlabelled may also be used to identify and characterise
different strains of Mptb, Mavs, other 5S-containing pathogenic
mycobacteria, or Mtb, and properties such as drug resistance or
susceptibility.
The polypeptides of the invention may conveniently be packaged
in the form of a test kit in a suitable container. In such kits
the polypeptide may be bound to a solid support where the assay
format for which the kit is designed requires such binAlng. The
kit may also contain suitable reagents for treating the sample
to be examined, control reagents, instructions, and the like.
The use of polypeptides of the invention in the diagnosis of
~5 inflammatory diseases such as Crohn's disease or sarcoidosis in
m~nc or Johne~s disease in animals form a preferred aspect of
the invention. The polypeptides may also be used in the
prognosis of these diseases. For example, the response of a
human or animal subject in response to antibiotic or other
therapies may be monitored by utilizing the diagnostic methods
of the invention over the course of a period of treatment and
following such treatment.
The use of Mt~ polypeptides of the invention in the above-
described methods form a further aspect of the invention,
particularly for the detection, diagnosis or prognosis of Mtb
infections.
Polypeptides of the invention may also ~e used in assay methods
for identifying candidate chemical compounds which will be useful
in inhibiting, binding to or disrupting the function of said
polypeptides required for pathogenicity. In general, such assays
involve bringing the polypeptide into contact with a candidate
inhibitor compound and observing the ability of the compound to
disrupt, bind to or interfer with the polypeptide.
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- 20 -
There are a number of ways in which the assay may be formatted.
For example, those polypeptides which have an enzymatic function
may be assayed using labelled substrates for the enzyme, and the
amount of, or rate of, conversion of the substrate into a product
measured, e.g by chromatograpy such as HPLC or by a colourimetric
assay. Suitable labels include 35Sr 12~I, biotin or enzymes such
as horse radish peroxidase.
For example, the gene product of ORF C is believed to have GDP-
mannose dehydratase activty. Thus an assay for inhbitors of the
gene product may utilise for example labelled GDP-mannose, GDP
or mannose and the activity of the gene product followed. ORF
D encodes a gene related to the synthesis and regulation of
capuslar polysaccharides, which are often associated with
invasiveness and pathogenicity. Labelled polysaccharide
substrates may be used in assays of the ORF D gene product. The
gene product of ORF F encodes a protein with putative glucosyl
transferase activity and thus labelled amino sugars such as
3-N-acetylglucosamine may be used as substrates in assays.
Candidate chemical compounds which may be used may be natural or
synthetic chemical compounds used in drug screening programmes.
Extracts of plants which contain several characterised or
uncharacterised components may also be used.
Alternatively, the a polypeptide of the invention may be screened
against a panel of peptides, nucleic acids or other chemical
functionalities which are generated by combinatorial chemistry.
This will allow the definition of chemical entities which bind
to polypeptides of the invention. Typically, the polypeptide of
the invention will be brought into contact with a panel of
compounds from a combinantorial library, with either the panel
or the polypeptide being immobilized on a solid phase, under
conditions suitable for the polypeptide to bind to the panel.
The solid phase will then be washed under conditions in which
only specific interactions between the polypeptide and individual
members of the panel are retained, and those specific members may
be utilized in further assays or used to design further panels
of candidate compounds.
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- 21 -
For example, a number of assay methods to define peptide
interaction with peptides are known. For example, W086/00991
describes a method for determining mimotopes which comprises
making panels of catamer preparations, for example octamers of
amino acids, at which one or more of the positions is defined and
the remaining positions are randomly made up of other amino
acids, determining which catamer binds to a protein of interest
and re-screening the protein of interest against a further panel
based on the most reactive catamer in which one or more
additional designated positions are systematically varied. This
may be repeated throughout a number of cycles and used to build
up a sequence of a binding candidate compound of interest.
W089/03430 describes screening methods which permit the
preparation of specific mimotopes which mimic the immunological
activity of a desired analyte. These mimotopes are identified
by reacting a panel of individual peptides wherein said peptides
are of systematically varying hydrophobicity, amphipathic
characteristics and charge patterns, using an antibody against
an antigen of interest. Thus in the present case antibodies
against the a polypeptide of the inventoin may be employed and
mimotope peptides from such panels may be identified.
C. Vectors.
Polynucleotides of the invention can be incorporated into a
recombinant replicable vector. The vector may be used to
2~ replicate the nucleic acid in a compatible host cell. Thus in
a further embodiment, the invention provides a method of making
polynucleotides of the invention by introducing a polynucleotide
of the invention into a replicable vector, introducing the vector
into a compatible host cell, and growing the host cell under
conditions which bring about replication of the vector. The
vector may be recovered from the host cell. Suitable host cells
are described below in connection with expression vectors.
D. Ex~ression Vectors.
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WO 97/23624 PCT/GB96/03221
Preferably, a polynucleotide of the invention in a vector is
operably linked to a control sequence which is capable of
providing for the expression of the coding sequence by the host
cell, i.e. the vector is an expression vector. The term "operably
linked" refers to a juxtaposition wherein the components
described are in a relationship permitting them to function in
their intended manner. A control sequence "operably linked~ to
a coding sequence is ligated in such a way that expression of the
coding sequence is achieved under conditions compatible with the
control se~uences. Such vectors may be transformed into a
suitable host cell as described above to provide for expression
of a polypeptide of the invention. Thus, in a further aspect the
invention provides a process for preparing polypeptides according
to the invention which comprises cultivating a host cell
transformed or transfected with an expression vector as described
above, under conditions to provide for expression by the vector
of a coding sequence encoding the polypeptides, and recovering
the expressed polypeptides.
A further embodiment of the invention provides vectors for the
replication and expression of polynucleotides of the invention,
or fragments thereof. The vectors may be for example, plasmid,
virus or phage vectors provided with an origin of replication,
optionally a promoter for the expression of the said
polynucleotide and optionally a regulator of the promoter. The
vectors may contain one or more selectable marker genes, for
example an ampicillin resistance gene in the case of a bacterial
plasmid or a neomycin resistance gene for a mammalian vector
Vectors may be used in vitro, for example for the production of
RNA or used to transfect or transform a host cell. The vector
may also be adapted to be used in vivo, for example in a method
of naked DNA vaccination or gene therapy. A further embodiment
of the invention provides host cells transformed or transfected
with the vectors for the replication and expression of
polynucleotides of the invention, including the DNA of GS, the
open reading frames thereof and other corresponding ORFs
particularly ORFs B, C, E and F from Mtb. The cells will be
chosen to be compatible with the said vector and may for example
be bacterial, yeast, insect or mammalian.
CA 02241023 1998-06-19
W O 97123624 PCT/GB96103221
Expression vectors are widely available in the art and can be
obtained commercially. Mammalian expression vectors may comprise
a mammalian or viral promoter. ~mm~l ian promoters include the
metallothionien promoter. Viral promoters include promoters from
adenovirus, the SV40 large T promoter and retroviral LTR
promoters. Promoters compatible with insect cells include the
polyhedrin promoter. Yeast promoters include the alcohol
dehydrogenase promoter. Bacterial promoters include the
~-galactosidase promoter.
The expression vectors may also comprise enhancers, and in the
case of eukaryotic vectors polyadenylation signal sequence
downstream of the coding sequence being expressed.
Polypeptides of the invention may be expresse~ in suitable host
cells, for example bacterial, yeast, plant, insect and mammalian
cells, and recovered using standard purification techniques
including, for example affinity chromatography, HPLC or other
chromatographic separation techniques.
Polynucleotides according to the invention may also be inserted
into the vectors described above in an antisense orientation in
order to provide for the production of antisense ~NA. Antisense
RNA or other antisense polynucleotides or ligands may also ~e
produced by synthetic means. Such antisense polynucleotides may
be used in a method of controlling the levels of the proteins
encoded by the ORFs of the invention in a mycobacterial cell.
Polynucleotides of the invention may also be carried by vectors
suitable for gene therapy methods. Such gene therapy methods
include those designed to provide vaccination against diseases
caused by pathogenic mycobacteria or to boost the immune response
of a human or animal infected with a pathogenic mycobacteria.
For example, Ziegner et al, AIDS, l995, 9;43-50 describes the use
of a replication defective recombinant amphotropic retrovirus to
boost the immune response in patients with HIV infection. Such
a retrovirus may be modified to carry a polynucleotide encoding
a polypeptide or fragment thereof of the in~ention and the
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-24-
retrovirus delivered to the cells of a human or animal subject
in order to provide an immune response against said polypeptide.
The retrovirus may be delivered directly to the patient or may
be used to infecte cells eX-vivo, e.g. fibroblast cells, which
are then introduced into the patient, optionally after being
inactivated. The cells are desirably autologous or HLA-matched
cells from the human or animal subject.
Gene therapy methods including methods for boosting an immune
response to a particluar pathogen are disclosed generally in for
example W095/14091, the disclosure of which is incoporated herein
by reference. Recombinant viral vectors include retroviral
vectors, adenoviral vectors, adeno-associated ~iral vectors,
vaccinia virus vectors, herpes virus vectors and alphavirus
vectors. Alpha virus vectors are described in, for example,
W095/07994, the disclosure of which is incorporated herein by
reference.
Where direct administration of the recombinant viral vector is
contemplated, either in the form of naked nucleic acid or in the
form of packaged particles carrying the nucleic acid this may be
done by any suitable means, for example oral A~mi n; stration or
intravenous injection. From lOs to lO~ c.f.u of virus represents
a typical dose, which may be repeated for example weekly over a
period of a few months. Administration of autologous or HLA-
matched cells infected with the virus may be more convenient in
some cases. This will generally be achieved by administering
doses, for example from 105 to lO~ cells per dose which may be
repeated as described above.
The recombinant viral vector may further comprise nucleic acid
capable of expressing an accessory molecule of the immune system
designed to increase the immune response. Such a moleclue may
be for example and interferon, particularly interferon gamma, an
interleukin, for example IL-l~, IL-l~ or IL-2, or an HLA class
I or II moleclue. This may be particularly de~irable where the
vector is intended for use in the treatment of humans or animals
already infected with a mycobacteria and it is desired to boost
the immune response.
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W O 97~3624 PCTIG B96103221
E. Antibodies.
The invention also provides monoclonal or polyclonal antibodies
to polypeptides of the invention or fragments thereof. The
invention further provides a process for the production of
S monoclonal or polyclonal antibodies to polypeptides of the
invention. Monoclonal antibodies may be prepared by conventional
hybridoma technology using the polypeptides of the invention or
peptide fragments thereof, as immunogens. Polyclonal antibodies
may also be prepared by conventional means which comprise
inoculating a host ~ ?1, for example a rat or a rabbit, with
a polypeptide of the invention or peptide fragment thereof and
recovering immune serum.
In order that such antibodies may be made, the invention also
provides polypeptides of the invention or fragments thereof
haptenised to another polypeptide for use as immunogens in
~n~ ~1 S or humans.
For the purposes of this invention, the term "antibody", unless
specified to the contrary, includes fragments of whole antibodies
which retain their binding activity for a polypeptide of the
invention. Such fragments include Fv, F(ab') and F(ab' )2
fragments, as well as single chain antibodies. Furthermore, the
antibodies and fragments thereof may be humanised antibodien,
e.g. as described in EP-A-239400.
Antibodies may be used in methods of detecting polypeptides of
the invention present in biological samples (where such samples
include the human or animal body samples, and environmental
samples, mentioned above) by a method which comprises:
(a) providing an antibody of the invention;
(b) incubating a biological sample with said antibody
under conditions which allow for the formation of an
antibody-antigen complex; and
(c) determining whether antibody-antigen complex
comprising said antibody is formed.
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W097~3624 PCT/GB96/03221
-26-
Antibodies of the invention may be bound to a solid support for
example an immunoassay well, microparticle, dipstick or biosensor
and/or packaged into kits in a suitable container along with
suitable reagents, controls, instructions and the like.
Antibodies of the invention may be used in the detection,
diagnosis and prognosis of disea5es as descirbed above in
relation to polypeptides of the invention.
F. Com~ositions.
The present invention also provides compositions comprising a
polynucleotide or polypeptide of the invention together with a
carrier or diluent. Compositions of the invention also include
compositions comprising a nucleic acid, particularly and
expression vector, of the invention. Compositions further
include those carrying a recombinant virus of the invention.
Such compositions include pharmaceutical compositions in which
case the carrier or diluent will be pharmaceutically acceptable.
Pharmaceutically acceptable carriers or diluents include those
used in formulations suitable for inhalation as well as oral,
parenteral (e.g. intramuscular or intravenous or transcutaneous)
administration. The formulatlons may conveniently ~e presented
in unit dosage form and may be prepared by any of the methods
well known in the art of pharmacy. Such methods include the step
of bringing into association the active ingredient with the
carrier which constitutes one or more accessory ingredients. In
general the formulations are prepared by uniformly and intimately
bringing into association the active ingredient with liquid
carriers or finely divided solid carriers or both, and then, if
necessary, shaping the product.
For example, formulations suitable for parenteral administration
include aqueous and non-aqueous sterile injection solutions which
may contain anti-oxidants, buffers, bacteriostats and solutes
which render the formulation isotonic with the blood of the
intended recipient, and aqueous and non-aqueous sterile
suspensions which may include suspending agents and thickening
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W097/23624 pcTlGs96lo322l
agents, and liposomes or other microparticulate systems which are
designed to target the polynucleotide or the polypeptide of the
invention to blood components or one or more organs, or to target
cells such as M cells of the intestine after oral administration
G. Vaccines.
In another aspect, the invention provides novel vaccines for the
prevention and treatment of infections caused by Mptb, Mave,
other GS-containing pathogenic mycobacteria and Mtb in animals
and humans. The term "vaccine" as used herein means an agent
used to stimulate the immune system of a vertebrate, particularly
a warm blooded vertebrate including humans, so as to provide
protection against future harm by an organism to which the
vaccine is directed or to assist in the eradication of an
organism in the treatment of established infection. The immune
system will be stimulated by the production of cellular ~ ;ty
antibodies, desirably neutralizing antibodies, directed to
epitopes found on or in a pathogenic mycobacterium which
expresses any one of the ORFs of the invention. The antibody so
produced may be any of the immunological classes, such as the
immunoglobulins A, D, E, G or M. Vaccines which stimulate the
production of IgA are interest since this is the principle
immunoglobulin produced by the secretory system of warm-blooded
~nlm~lS~ and the production of such antibodies will help prevent
infection or colonization of the intestinal tract. However an
Ig~ and IgG response will also be desirable for systemic
infections such as Crohn's disease or tuberculosis.
Vaccines of the invention include polynucleotides of the
invention or fragments thereof in suitable vectors and
administered by injection of naked DNA using standard protocols.
Polynucleotides of the invention or fragments thereof in suitable
vectors for the expression of the polypeptides of the invention
may be given by injection, inhalation or by mouth. Suitable
vectors include M.bo~is BCG, M. s~egmatis or other mycobacteria,
Corynebacteria, Salmonella or other agents according to
established protocols.
CA 02241023 1998-06-19
WO 97123624 PCT/GB96103221
Polypeptides of the invention or fragments thereof in
substantially isolated form may be u~ed as vaccines by injection,
inhalation, oral administration or by transcutaneous application
according to standard protocols. Adjuvants (such as Iscoms or
polylactide-coglycolide encapsulation), cytokines such as IL-12
and other immunomodulators may be used for the selective
enhancement of the cell mediated or humoral immunological
responses. Vaccination with polynucleotides and/or polypeptides
of the invention may be undertaken to increase the susceptibility
of pathogenic mycobacteria to antimicrobial agents ln vi~o.
In instances wherein the polypeptide is correctly configured so
as to provide the correct epitope, but is too small to be
immunogenic, the polypeptide may be linked to a suitable carrier.
A number of techniques for obtaining such linkage are known in
the art, including the formation of disulfide linkages using N-
succinimidyl-3-(2-pyridylthio)propionate(SPDP) andsuccinimidyl
4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC) obtained
from Pierce Company, Rockford, Illinois, (if the peptide lacks
a sulfhydryl group, this can be provided by addition of a
cysteine residue). These reagents create a disulfide linkage
between themselves and peptide cysteine residues on one protein
and an amide linkage through the epsilon-amino on a lysine, or
other free amino group in the other. A variety of such
disulfide/amide-forming agents are known. See, for example,
Immun Rev (1982) 62:1B5. Other bifunctional coupling agents form
a thioether rather than a disulfide linkage. Many of these thio-
ether-forming agents are commercially available and include
reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid,
2-iodoacetic acid, 4-(N-maleimido-methyl)cycloh~x~ne-l-carboxylic
acid, and the like. The carboxyl group can be activated by
combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic
acid, sodium salt. Additional methods of coupling antigens
employs the rotavirus/"binding peptide" system described in EPO
Pub. No. 259,149, the disclosure of which is incorporated herein
by reference. The foregoing list is not meant to be exhaustive,
and modifications of the named compounds can clearly be used.
CA 02241023 1998-06-19
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- 29 -
Any carrier may be used which does not itself induce the
production of antibodies harmful to the host. Suitable carriers
are typically large, slowly metabolized macromolecules such as
proteins; polysaccharides, such as latex functionalized
Sepharose~, agarose, cellulose, cellulose beads and the like;
polymeric amino acids, such as polyglutamic acid, polylysine,
polylactide-coglycolide and the like; amino acid copolymers; and
inactive virus particles. Especially useful protein substrates
are serum albumins, keyhole limpet hemocyanin, immunoglobulin
molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other
proteins well known to those skilled in the art.
The immunogenicity of the epitopes may also be enhanced by
preparing them in m~mm~l ian or yeast systems fused with or
assembled with particle-forming proteins such as, for example,
that associated with hepatitis B surface antigen. See, e.g., US-
A-4,722,840. Constructs wherein the epitope is linked directly
to the particle-forming protein coding sequences produce hybrids
which are immunogenic with respect to the epitope. In addition,
all of the vectors prepared include epitopes specific to HBV,
having various degrees of immunogenicity, such as, for example,
the pre-S peptide.
In addition, portions of the particle-forming protein coding
sequence may be replaced with codons encoding an epitope of the
invention. In this replacement, regions which are not required
to mediate the aggregation of the units to form i~mllnogenic
particles in yeast or ~m~l S can be deleted, thus eliminating
additional HBV antigenic sites from competition with the epitope
of the invention.
Vaccines may be prepared from one or more immunogenic
polypeptides of the invention. These polypeptides may be
expressed in various host cells (e.g., bacteria, yeast, insect,
or m~mm~lian cells), or alternatively may be isolated from viral
preparations or made synthetically.
In addition to the above, it is also possible to prepare live
vaccines of attenuated microorganisms which express one or more
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-30-
recom~inant polypeptides of the invention. Suitable attenuated
microorganisms are known in the art and include, for example,
viruses (e.g., vaccinia virus), as well as bacteria.
The preparation of vaccines which contain an immunogenic
polypeptide(s) as active ingredients, is known to one skilled in
the art. Typically, such vaccines are prepared as injectables,
or as suitably encapsulated oral preparations and either liquid
solutions or suspensions; solid forms suitable for solution in,
or suspension in, liquid prior to injestion or injection may also
be prepared. The preparation may also be emulsified, or the
protein encapsulated in liposomes. The active immunogenic
ingredients are often mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients are, for example, water, saline,
dextrose, glycerol, ethanol, or the like and combinations
thereof. In addition, if desired, the vaccine may contain minor
amounts of auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, and/or adjuvants which enhance the
effectiveness of the vaccine. Examples of adjuvants which may
be effective include but are not limited to: aluminum hydroxide,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-
nor-muramyl-L-alanyl-D-isoglutamine (CGP ll637, referred to as
nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-
(l~-2~-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine
(CGP 19835A, referred to as MTP-PE), and RIBI, which contains
three components extracted from bacteria, monophosphoryl lipid
A, trehalose dimycolate and cell wall skeleton ~MPL+TDM+CWS) in
a 2~ squalene/Tween~ 80 emulsion. The effectiveness of an
adjuvant may be determined by measuring the amount of antibodies
directed against an immunogenic polypeptide containing an
antigenic sequence resulting from administration of this
polypeptide in vaccines which are also comprised of the various
adjuvants.
The vaccines are conventionally administered parenterally, by
injection, for example, either subcutaneously or intramuscularly.
Additional formulations which are suita~le for other modes of
administration include suppositories, oral formulations or as
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enemas. For suppositories, traditional binders and carriers may
include, for example, polyalkylene glycols or triglycerides; such
suppositories may be formed from mixtures containing the active
ingredient in the range of 0.5~ to 10~, preferably 1~ - 2%. Oral
formulations include such normally employed excipients as, for
example, pharmaceutical grades of mannitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, and the like. These compositions take the form of
solutions, suspensions, tablets, pills, capsules, su~tained
release formulations or powders and contain 10~ - 95~ of active
ingredient, preferably 25~ - 70~.
The proteins may be formulated into the vaccine as neutral or
salt forms. Pharmaceutically acceptable salts include the acid
addition salts (formed with free amino groups of the peptide) and
which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acid~, or such organic acids such as
acetic, oxalic, tartaric, maleic, and the like. Salts formed
with the free carboxyl groups may also be derived from inorganic
bases such as, for example, sodium, potassium, ammonium, calcium,
or ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine, and
the like.
The vaccines are administered in a manner compatible with the
dosage formulation, and in such amount as will be
prophylactically and/or therapeutically effective. The quantity
to be administered, which is generally in the range of 5~g to
250~g, of antigen per dose, depends on the subject to be treated,
capacity of the subject's immune system to synthesize antibodies,
mode of administration and the degree of protection desired.
Precise ~mounts of active ingredient required to be administered
may depend on the judgement of the practitioner and may be
peculiar to each subject.
The vaccine may be given in a single dose schedule, or preferably
in a multiple dose schedule. A multiple dose schedule is one in
which a primary course of vaccination may be with 1-10 separate
doses, followed by other doses given at subsequent time intervals
CA 02241023 1998-06-19
WO 97/23624 PCT/GB96i~3221
required to maintain and or reenforce the immune response, for
example, at 1-4 months for a second dose, and if needed, a
subsequent dose(s) after several months. The dosage regimen will
also, at least in part, be determined by the need of the
individual and be dependent upon the judgement of the
practitioner.
In a further aspect of the invention, there is provided an
attenuated vaccine comprising a normally pathogenic mycobacteria
which harbours an attenuating mutation in any one of the genes
encoding a polypeptide of the invention. The gene is selected
from the group of ORFs A, B, C, D, E, F, G and H, including the
homologous ORFs B, C, E and F in Mtb.
The mycobacteria may ~e used in the form of killed bacteria or
as a live attenuated vaccine. There are advantages to a live
attenuated vaccine. The whole li~e organism is used, rather than
dead cells or selected cell components which may exhibit modified
or denatured antigens. Protein antigens in the outer membrane
will maintain their tertiary and ~uaternary structures.
Therefore the potential to elicit a good protective long term
immunity should be higher.
The term "mutation" and the like refers to a genetic lesion in
a gene which renders the gene non-functional. This may be at
either the level of transcription or translation. The term thus
envisages deletion of the entire gene or substantial portions
thereof, and also point mutations in the coding sequence which
result in truncated gene products unable to carry out the normal
function of the gene.
A mutation introduced into a bacterium of the invention will
generally be a non-reverting attenuating mutation. Non-reverting
means that for practical purposes the probability of the mutated
gene being restored to its normal function is small, for example
less than 1 in 1o6 such as less than 1 in 109 or even less than
1 in 10l2.
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-33-
An attenuated mycobacteria of the invention may be in isolated
form. This is usually desirable when the bacterium is to be used
for the purposes of vaccination. The term "isolated~' means that
the bacterium is in a form in which it can be cultured, processed
or otherwise used in a form in which it can be readily identified
and in which it is substantially uncontaminated by other
bacterial strains, for example non-attenuated parent strains or
unrelated bacterial strains. The term "isolated bacterium~ thus
encompasses cultures of a bacterial mutant of the invention, for
example in the form of colonies on a solid medium or in the form
of a liquid culture, as well as frozen or dried preparation~ of
the strains.
In a preferred aspect, the attenuated mycobacterium further
comprises at least one additional mutation. This may be a
mutation in a gene responsible for the production of products
essential to bacterial growth which are absent in a human or
~n;m~l host. For example, mutations to the gene for aspartate
semi-aldehyde dehydrogenase (asd) have been proposed for the
production of attenuated strains of Salmonella. The asd gene is
described further in Gene ~1993) 129; 123-128. A lesion in the
asd gene, encoding the enzyme aspartate ~-semialdehyde
dehydrogenase would render the organism auxotrophic for the
essential nutrient diaminopelic acid (DAP), which can be provided
exogenously during bulk culture of the vaccine strain. Since
this compound is an essential constituent of the cell wall for
gram-negative and some gram-positive organisms and is absent from
mammalian or other vertebrate tissues, mutants would undergo
lysis after about three rounds of division in such tissues.
Analogous mutations may be made to the attenuated mycobacteria
of the invention.
In addition or in the alternative, the attenuated mycobacteria
may carry a recA mutation. The recA mutation knocks out
homologous recombination - the process which is exploited for the
construction of the mutations. Once the recA mutation has been
incorporated the strain will be unable to repair the constructed
deletion mutations. Such a mutation will provide attenuated
strains in which the possibility of homologous recombination to
CA 02241023 1998-06-19
W O 97123624 PCT/GB96103221
- 34 -
with DNA from wild-type strains has been minimized. RecA genes
have been widely studied in the art and their sequences are
available. Further modifications may be made for additional
sa~ety.
The invention further provides a process for preparing a vaccine
composition comprising an attenuated bacterium according to the
invention process comprises (a) inoculating a culture vessel
containing a nutrient medium suitable for growth of said
bacterium; (b) culturing said bacterium; (c) recovering said
bacteria and (d) mixing said bacteria with a pharmaceutically
acceptable diluent or carrier.
Attenuated bacterial strains according to the invention may be
constructed using recombinant DNA methodology which is known per
se. In general, bacterial genes may be mutated by a process of
targeted homologous recombination in which a DNA construct
containing a mutated form of the gene is introduced into a host
bacterium which it is desired to attenuate. The construct will
recombine with the wild-type gene carried by the host and thus
the mutated gene may be incorporated lnto the host genome to
provide a bacterium of the present invention which may then be
isolated.
The mutated gene may be obtained by introducing deletions into
the gene, e.g by digesting with a restriction enzyme which cuts
the coding sequence twice to excise a portion of the gene and
then religating under conditions in which the excised portion is
not reintroduced into the cut gene. Alternatively frame shift
mutations may be introduced by cutting with a restriction enzyme
which leaves overhanging 5' and 3' termini, filling in and/or
trimming back the overhangs, and religating. Similar mutations
may be made by site directed mutagenesis. These are only
examples of the types of techniques which will readily be at the
disposal of those of skill in the art.
Various assays are available to detect successful recombination.
In the case of attenuations which mutate a target gene necessary
3~ for the production of an essential metabolite or catabolite
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compound, selection may be carried out by screening for bacteria
unable to grow in the absence of such a compound. Bacteria may
also be screened with antibodies or nucleic acids of the
invention to determine the absence of production of a mutated
gene product of the invention or to confirm that the genetic
lesion introduced - e.g. a deletion - has been incorporated into
the genome of the attenuated strain.
The concentration of the attenuated strain in the vaccine will
be formulated to allow convenient unit dosage forms to be
prepared. Concentrations of from about 1 04 to lO9 bacteria per
ml will generally be suitable, e.g. from about lOs to lO~ such as
about 106 per ml. Live attenuated organisms may be administered
su~cutaneously or intramuscularly at up to lO~ organisms in one
or more doses, e.g from around lOs to lO8, e.g about 106 or 107
organisms in a single dose.
The vaccines of the invention may be administered to recipients
to treat established disea6e or in order to protect them against
diseases caused by the corresponding wild type mycobacteria, such
as inflammatory diseases such as Crohn's disease or sarcoidosis
in humans or Johne's disease in animals. The vaccine may be
administered by any suitable route. In general, subcutaneous or
intramuscular injection is most con~enient, but oral, intranasal
and colorectal administration may also be used.
The following Examples illustrates aspects of the invention.
2~ EXAMP~E 1
Tests for the presence of the GS identifier sequence were
performed on 5~1 bacterial DNA extracts (25 ~g/ml to 500 ~g/ml)
using polymerase chain reaction based on the oligonucleotide
primers 5'-GATGCCGTGAGGAGGTAAAGCTGC-3' (Seq ID No. 40) and 5'-
GATACGGCTCTTGAATCCTGCACG-3' ( Seq ID No. 4l) from within the
identifier DNA sequences ~Seq.ID Nos 1 and 2). PCR was performed
for 40 cycles in the presence of l.5 mM magnesium and an
annealing temperature of 58~C. The presence or absence of the
correct amplification product indicated the presence or absence
CA 02241023 1998-06-l9
W097123624 PCT/GB96/03221
of GS identifier sequence in the corresponding bacterium. GS
identifier sequence is shown to be present in all the laboratory
and field strains of Mptb and Mavs tested. This includes Mptb
isolates 002~ (bovine CVL Weybridge), 0021 (caprine, Moredun),
0022 (bovine, Moredun), 0139 (human, Chiodini 1984), 0209,
0208, 0211, 0210, 0212, 0207, 0204, 0206 (~ovine, Whipple 1990).
All Mptb strains were IS900 positive. The Mavs strains include
0010 and 0012 (woodpigeon, Thorel) 0018 (armadillo, Portae~s) and
0034, 0037, 0038, 0040 (AIDS, Hoffner). All Mavs strains were
IS902 positive. One pathogenic M.avium strain 0033 (AIDS,
Hoffner) also contained GS identifier sequence. GS identifier
sequence is absent from other mycobacteria including other
M.avium, M.malmoense, M.szulgai, M.gordonae, M.chelonei,
M.fo~tuitum, M.phlei, as well as E.coli, S.areus, Nocardia sp,
Streptococcus sp. S~ige71a sp. Pseudomonas sp.
Example 2:
To obtain the full sequence of GS in Mavs and Mptb we generated
a genomic li~rary of Mavs using the restriction endonuclease
EcoRI and cloning into the vector pUC18. This achieved a
representative library which was screened with 32P-labelled
identifier sequence yielding a positive clone containing a 17k~p
insert. We constructed a restriction map of this insert and
identified GS as fragments unique to Mavs and Mptb and not
occurring in laboratory strains of M. avium. These fragments
were sub-cloned into pUC18 and pGEM4Z. We identified GS
contained within an 8kb region. The full nucleotide sequence
was determined for GS on both DNA strands using primer walking
and automated DNA sequencing. DNA sequence for GS in Mptb was
obtained using overlapping PCR products generated using PwoDNA
polymerase, a proofreading thermostable enzyme. The final DNA
sequences were derived using the University of Wisconsin GCG gel
assembly software package.
Example 3:
The DNA sequence of GS in Mavs and Mptb was found to be more
than 99~ homologous. The ORFs encoded in GS were identified
using GeneRunner and DNAStar computer pro~rammes. Eight ORFs
were identified and designated GSA, GSB, GSC, GSD, GSE, GSF, GSG
- 37 -
and GSH. Database comparisons were carried out against the
GenEMBL Database release version 48.0 (9/96), using the BLAST and
BLIXEM programmes. GSA and GSB encoded proteins of 13.5kDa and
30.7kDa respectively, both of unknown functions. GSC encoded
a protein of 38.4kDa with a 65% homology to the amino acid
sequence of rfbd of V.cholerae, a 62% amino acid sequence
homology to gmd of E.coli and a 58% homology to gca of
Ps.aeruginosa which are all GDP-D-mannose dehydratases.
Equivalent gene products in H.influenzae, S. dysenteriae,
Salmonella enterica are all involved in 'O'-antigen processing
known to be linked to pathogenicity. GSD encoded a protein of
37.1kDa which showed 58% homology at the DNA level to wcaG from
E.coli, a gene involved in the synthesis and regulation of
capsular polysaccharides, also related to pathogenicity. GSE
was found to have a > 30% amino acid homology to rfbT of
V. cholerae, involved in the transport of specific LPS components
across the cell membrane. In V. cholerae the gene product causes
a servoconversion from the Inaba to the Ogawa 'epidemic' strain.
GSF encoded a protein of 30.2kDa which was homologous in the
range 25-40% at the amino acid level to several glucosyl
transferases such as rfpA of K. pneumoniae, rfbB of K. pneumoniae,
lgtD of H. influenzae, lsi of N. gonorrhoae. In E.coli and
equivalent gene galE adds .beta.-1-3 N-acetylglucosamine to galactose,
the latter only found in 'O' and 'M' antigens which are also
related to pathogenicity. GSH comprising the ORFs GSH1 and GSH2
encodes a protein totalling about 60kDa which is a putative
transposase with a 40 - 43% homology at the amino acid level to
the equivalent gene product of IS21 in E.coli. This family of
insertion sequences is broadly distributed amongst gram negative
bacteria and is responsible for mobility and transposition of
genetic elements. An IS21- like element in B.fragilis is split
either side of the .beta.-lactamase gene controlling its activation
and expression. We programmed an E.coli S30 cell-free extract
with plasmid DNA containing the ORF GSH under the control of a
lac promoter in the presence of a 35 S-methionine, and
demonstrated the translation of an abundant 60kDa protein.
The proteins homologous to GS encoded in other organisms are in
general highly antigenic. Thus the proteins encoded by the ORFs
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W097~3624 pcTlGs96/o322l
in GS may be used in immunoassays of antibody or cell mediated
immuno-reactivity for diagnosing infections caused by
mycobacteria, particularly Mptb, Mavs and Mtb. Enhancement of
host immune recognition of GS encoded proteins by vaccination
using naked specific ~NA or recombinant GS proteins, may be used
in the prevention and treatment of infections caused by Mptb,
Mavs and Mtb in humans and animals. Mutation or deletion of all
or some of the ORFs A to H in GS may be used to generate
attenuated strains of Mptb, Mavs or Mtb with lower pathogenicity
for use as living or killed vaccines in humans and animals. Such
vaccines are particularly relevant to Johne's disease in ~n;m~ls~
to diseases caused by Mpt~ in humans such as Crohn's disease, and
to the management of tuberculosis especially where the disease
is caused by multiple drug-resistant organisms.
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SEQU~NCE L I ST ING
Seq. ID No.l
5'- 1 GATCC M CTA M CCCGATGG M CCCCGCGC M ACTATTGG ACGTCTCCGC &CTACGCAGT
61 TGGGTTGGCG CCCGCG M TC GCACTGA M G AGGGCATCGA TGCM CGGTG TCGTGGTACC
121 GCAC M ATGC CGATGCCGTG AGGAGGT M A GCTGCGGGCC GGCCGATGTT ATCCCTCCGG
181 CCGGACGGGT AGGGCGACCT GCCATCGAGT GGTACGGCAG TCGCCTGGCC GGCGAGGCGC
241 ATGGCCTATG TGAGTATCCC ATAGCCTGGC TTGGCTCGCC CCTACGCATT ATCAGTTGAC
301 CGC m CGCG CCACGTCGCA GGCTTGCGGC AGCATCCCGT TCAGGTCTCC TCATGGTCCG
361 GTGTGGCACG ACCACGC M G CTCGAACCGA CTCG m CCC M m CGCAT GCTAATATCG
421 CTCGATGGAT TTTTTGCGCA ACGCCGGCTT GATGGCTCGT M CGTTAGCA CCGAGATGCT
481 GCGCCACTCC GAACG M AGC GCCTATTAGT AAACC M GTC G MGCATACG GAGTC MCGT
541 TGTTATTGAT GTCGGTGCTA ACTCCGGCCA GTTCGGTAGC GC m GCGTC GTGCAGGATT
601 C M GAGCCGT ATCG m CCT TTG M CCTCT TTCGGGGCCA m GCGC M C T M CGCGC M
661 GTCGGCATCG GATC -3
Seq. ID No.2
5'- 1 GATCCGATGC CGACTTGCGC GTTAGTTGCG CAAATGGCCC CGAAAGAGGT TrAMr~
61 Cr~T~rGGCT CTTGA~TCCT Gr~Cr-ArGCA AAGCGCTACC GAACTGGCCG GAGTTAGCAC
121 CGACATCAAT AACAACGTTG ACTCCGTATG CTTCGACTTG GTTTACTAAT AGGCGCTTTC
181 GTTCGGAGTG GCGCAGCATC ~'CGblvC~AA CGTTACGAGC CATCAAGCCG G~1~CGCA
241 AAAAATCCAT cr~.cr.~T~T TAGCATGCGA AATTGGGAAA CGAGTCGGTT CGAGCTTGCG
301 r~ .~cc ArPrCGG~rC ATr~Gr-Ar~r CTGAACGGGA ~ ~C~A AG~ C~AC
361 ~aGCGC~AA AGCGGTCAAC TGATAATGCG T~r~Gcr.~r Cr~r~Cr~r,G CTATGGGATA
421 CTr~r~TpGG CCATGCGCCT ~C~GC~AG GCGACTGCCG TACCACTCGA TGGCAGGTCG
481 CCCTACCCGT CCGGCCGGAG GGATAACATC GGCCGGCCCG CAGC m ACC TCCTCACGGC
541 ATCGGCATTT GTGCGGTACC ~rr~r~rCGT TGCATCGATG CC~ A GTGCGATTCG
601 CGGGC~C~AA CCCAACTGCG TAGCGCGGAG ACGTCCAATA GTTTGCGCGG GGTTCCATCG
661 GGTTTAGTTG GATC -3'
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- 40 -
Seq ID No 3
1 GAA~G TTGrAr.Arr.A CGTCGAACTC ~L~GL~G~LC TTGCTTCGAA
51 TGA.~GA~.G~l~G GC~.~C'~A rAr-r-AACCGT CGACTTGTCG
101 ACGATCACCT TGTACCGGTC GATGTATGAC CCAATGTCGT CCGCAACCGA
151 r~AAr~ArGTAC GTCAGGTCCG CC~CCCC~CT TTCACCCATG wGC~lC~GA
201 CGGCGATGAA AATGACGTCC GC~l~-~C~A TTCCGCGTTG CCG~lC~l~
251 GTGAAGTCAA TCAGCCCGTT CTCACGGTTC CTCGCAATCA ACTCCCAACC
301 Cw GCTCGAA AATCGGGACA C~ -L~CGA Gn~Gr~?C GA-~Ll~GCC
351 TGATCGATAT rGArArAr~AC GACATCGTTG CCGCTATCCG Cr.Ar-~GGC
401 GCCCGTGACG AGGCCTACAT AGCCTGATCC GACCACCGAA AT m CAAGA
451 TGACC~..C AA~CC~-~A rCG~.~ACG ArrATACTGC CGCAACTCTG
501 TACC~C~ L wGTAATTCG CAI~--~C~- TCGTAAGGAG CAGCCAGCGA
551 ~.CaG~ rG l~Cw~GAGA GAGTCGCAGG ACTACGAGGT TGCC~.G~
601 ATACATCACA G-~-GC~-C I~.~GG~AAC GATG QGCAA GAAccr~cGG
651 GGrA~rccTG AACTGCGCGC ATGACCGGTC ~ CACCTTTGAT
701 CGGCCACCGC TTCCATGCGA ACATGACCGG AATCCATAGC G~ ~LCAA
751 GCAGCGGGGA GGTAGACGTC GGTGTCATCT GCTCCAACCG r~ ~ c~ . GAT
B01 AACGATTTCG CTGAACGATC Tcr~Ar~Gr~ATT GA~AAGCACC GTGGAGAGCG
B51 ~-~CG~A GCGCTATGGG GGGCGAATCG AGCACATCGT CATCGACGGT
901 GGAlC~UGCG ACGCCGTCGT GGAGTATCTG TC~w ~ATC ~-~G~.-l~C
951 ATATTGGCAA TCTCAGCCCG ACAACGGGAG ATATGACGCG ATGAATCAGG
1001 GCATTGCCCA T.~ WGC GAC~.~l.~. GG m ATGCA CTCrACGrAT
1051 C~,.~ ATCCAGATGC AGTCGCTTCC GTGGTGGAGG CG~--LC~aG
1101 GCATGGACCA GTACGTGATT I~l~G~. ~A rr~ AAC~Ll~CG
1151 GACTCGACGG r~ r~rTT ~.CC~ GC CGT~CGGC?A TATGCCGTTT
1201 AAGATGCGGA AAl -G-- CGGCGCGACG ~ ~ATC AGGCGACATT
1251 ~--~7G~ LAG CCAAGTTGGG CGGTTACGAT CTTGATTTTG
1301 GACTCGAGGC wACCAGCTG TTCATCTACC ~LGC~ACT AATACGGCCT
1351 CCCGTCACGA TCGACCGCGT G~...~GGAC TTCGATGTCA CGGGACCTGG
1401 TTrAAccr7~r7 CCCATCCGTG AGCACTATCG GACC~G~G CGGC.. laGG
1451 ACCTGCATGG crArTArccG ~lGÇ~,-~GGC GCAGAGTGTC GTGGGCTTAC
1501 l-G~l~.~A AGGAGTACTT GATTCGGGCC GACCTGGCCG CATTCAACGC
1551 GGTAAAGTTC TTGCGAGCGA AGTTCGCCAG AG~ GG AAGCAAAATT
1601 rATAr7~7~A~c AACTTCTACT GCCTGACCTG AGCAGCGCCG AGG~ AG
1651 CGCGATCAGT GCGACCTGAA rGGcr~r~GTG GAAAGCGCCA CCGATCCCGG
1701 r~ccr~AGTGc CTGACGCTTC GGATCCCTTG r~rr~rAArr, AGAGTGAGAG
1751 CGCCATGATG AGr-~TATC GG~ bd AGTCAACGCC GGAGTGACAA
1801 AAGTGAGAAC CCGGTGAAGC GAGCGCTTAT Ar~M7Gr~Tc ACGGwCAGG
1851 A.G~. l~A CCTCGCCGAG CTACTACTGA GrAAr~7r7AT~ CGAGGTTCAC
1901 GGG~... C GTCGAGCTTC GACGTTTAAC A~ G~GA TCGATCACCT
1951 CTACGTTGAC ccAcArr~r CGGGCGCGCG ~ G CACTATGCAG
2001 ACCTCACTGA CGGCACCCGG TTGGTGACCC TGCTCAGCAG TATCGACCCG
2051 GATGAGGTCT ACAACCTCGC AGCGr~GTCC CAl~.GCGC'G TCAGCTTTGA
2101 CGAGCCAGTG CATACCGGAG ArArr7Crr,G CATGGGATCG ATCCGACTTC
2151 TGGAAGCAGT CCGCCTTTCT CGG~ACT GCCG~--~LA TCAGGCTTCC
2201 r~l~d,AGA l~ll~GG~GC ATCTCCGCCA CCGCAGAACG AATCGACGCC
2251 GTTCTATCCC C~llCGC~AT ACGGCGCGGC CAAGGTCTTC TCGTACTGGA
2301 CGACTCGCAA CTATCGAGAG GCGTACGGAT TATTCGCAGT GAATGGCATC
2351 TTGTTCAACC ATGAGTCCCC CCGGCGCGGC GAGACTTTCG TGACCCGAAA
2401 GATCACGCGT GCCGTGGCGC GCATCCGAGC TGGCGTCCAA TCGGAGGTCT
2451 ATATGwCAA CCTCGATGCG ATCCGCGACT GGGGCTACGC GCCCGAATAT
2501 GTCGAGGGGA TGTGGAGGAT GTTGCAAGCG CCTGAACCTG ATGACTACGT
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W O 97/23624 PCT/G B96/03221
- 41 -
2551 CCTGGCGACA GGGCGTGGTT ACACCGTACG TGAGTTCGCT CAAGCTGCTT
2601 TTGACCATGT CGGGCTCGAC TGGCAAAAGC GCGTCAAGTT TG~rr-~rcGc
2651 TAl.lGCG~C cr~rr-r~r-GT CGATTCGCTA GTArr~TG CCGACAAGGC
2701 GGCCCAGTCA ~l~G~l~GA AA~ll~G~l TCATACTGGT GAACTCGCGC
2751 GCATCATGGT GGACGCGGAC ATCGCCGCGT TGGAGTGCGA TGGCACACCA
2801 TGGATCGACA CGCCGATGTT GC~..GG GGCAGAGTAA GTTGACGACT
2851 ACAC~ GC CTCTGGACCG CGCAACGCCC GTGTATATCG CCGGTCATCG
2901 GGGGCTGGTC GGCTCAGCGC TCGTACGTAG ATTTGAGGCC GAGG~-~CA
2951 CCAATCTCAT TGTGCGATCA CGCGATGAGA TTGATCTGAC Gr~rcr~r~cc
3001 GCAACG m G A11~L~1C Tr.~r.ArAAr.~ CCACAGGTGA TCATCGATGC
3051 GGCCGCACGG ~,~GC~G~A TCATGGCGAA T~Ar~CCT~T C'CCGC~ACT
3101 L~ CC~A AAACCTCCGA ATCr~-~r~ A..lG~C~A CGCAGCTGTC
3151 GC~.G~.G l~C~GCT C~....C~lC G~ cAT GCATCTACCC
3201 GAAGTACGCT CCGCAACCTA TCr~rr.~r.~r. TGCTTTATTG A~.a~CC~.l
3251 TCrP,GCCrP.r CAACGACGCG TATGCGATCG CCAAGATCGC CGGTATCCTG
3301 CAAGTTCAGG CGGTTAGGCG CCAATATGGG ~ C~.G~A L~l~.G~AT
3351 Gccr~AcTA~r CTCTACGGAC CCGGCr-~r~A ~ CCCG -L~C~G--~C
3401 A..~.~.~CC ~GCGC- ~ATc CGTCGATATG Ar~r-~GCrAA AG~
3451 Gr~r~r-~GG TGACGAATTG r~Gar-~rCGGT ACTCCGCGGC GCGAACTTCT
3501 GCATGTCGAC GATCTGGCGA GCGCATGCCT ~ ....... 1~ GAACATTTCG
3551 A~ CC~AA CCACGTCAAC GTGGGCACCG GCGTCGATCA CAGCATTAGC
3601 GAGATCGCAG ACA~ C TrC~GCGGTG GGCT~r~TCG GCr-~ar~rG
3651 TTGGGATCCA ACTPP~rCCG ATGr.~CCCC GCGCAAACTA TTGGACGTCT
3701 C~ .ACG CGAGTTGGGT ~5~'GCCCGC GAATCGCACT G~r~GGC
3751 ATCGATGCAA C~ ~lG GT~rCGC~r~ AATGCCGATG CCGTGAGGAG
3801 GTAAAGCTGC GG~--~GGCCa ATGTTATCCC ~CCGf,~ A cG~aGaGc
3851 GAC'~.OCC~l CGAGTGGTAC GGCAGTCGCC .~C~GC~'A GGCGCGTGGC
3901 CTATGGGAGT ATCCAATAGC ~.b~L~.GGC TCGCCCCTAC GCATTATCAG
3951 TTGACCGCTT IC~C~AGC TCGCAGGCTT GCGGra~r~T CCCGl. AGG
4001 ~ ATG ~ lG Gr~r~ACrAr GCAAGCTCGA ~rCr~ArTCGT
4051 TTCCCAA m CGCATGCTAA TA.~G~-. ~A TGGATTTTTT GCGCAACGCC
4101 GGCTTGATGG C$CGTAACGT TAGTACCGAG AI~CGCC ACTTCGAACG
4151 AAAGCGCCTA TTAGTAAACC AATTCAAAGC ATArGr~AGTc AAC~ -lA
4201 TTGATGTCGG TGCTAACTCC GGCCAGTTCG GTAGCGCTTT GCGTCGTGCA
4251 GGATTCAAGA GCCGTATCGT ... C~-.. ~AA C~ ~G GGCCA m GC
4301 Gra~rTA~CG CGCAAGTCGG CATCGGATCC A~-A.~aGAG TGTCACCAGT
4351 ATGCCCTAGG CGACGCCGAT r~r~arr.ATTA CCATCAATGT GGCAGGCAAT
4401 GcGGGr~GraA GTAGTTCCGT G~-~OCCGATG CTTAAAAGTC ATCAAGATGC
4451 ~--.C~-.CCC GCGAATTATA TTOGrarrrA AGACGTTGCA ATArArcGcc
4501 TTGATTCGGT TGCATCAGAA m CTGAACC CT~Cr~aTGT TA~.-C-.G
4551 AAGATCGACG TACAGGGTTT CGAGAAGCAG GTTATCACGG GCAGTAAGTC
4601 AACGCTTAAC GAAAG ~ GCG TCGGCATGCA ACTCGAACTT TCTTTTATTC
4651 CGTTGTACGA AGGTGACATG CTGATTCATG AAGCGCTTGA A~ AT
4701 TCCCTAGaTT TCAGACTGAC Go~ lA CGGATCCGCG
4751 CAATGGTCGA ATGCTTCAAG CTGACGGCAT ll.--l.~C~l GGGr~rr~ATT
4801 r~ar~TA~TG ~C~l~GC AC~ GG TATCCAAACG GGCGATCTGG
4351 TGAGCCGGCC TCCCGGGCAC CTAATCGACT ATCTAAATTG AGGCGGCCGC
4901 GACGTGCGGC ArG~ar~rGT GGCCGGCTGC TAGCGTTACA CACGTCATGA
4951 ~ ~CCAGT ~l~lC~AATA ATTATCCCTA CCTTCAATGC AGCGGTGACG
5001 CTGCAAGCCT G~.. ~GAAG CA~-~lCG~G CAGACCTACC GGGAAGTGGA
5051 A~ C~l GTCGACGGCG GTTCGACCGA TCGGACCCTC GACATCGCGA
5101 AC~ C~G CCCGr~ArTC GG~-~C~AC r~..~.l~A CAGCGGGCCC
5151 GATGATGGCC CCTACGACGC CATGAACCGC GGC~-~GGCG TGGCCACAGG
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wo s7n3624 PCT/Gn96~'03221
-42-
5201 CGAATGGGTA ~ AG GCGCCGACGA CACCCTCTAC GAACCAACCA
5251 CGTTGGCCCA GGTAGCCGCT ll~.-lC~,GCG ACCATGCGGC AAGCCATCTT
5301 GTCTATGGCG A~ -,AT GCGTTCGACG AAAAGCCGGC ATGCCGGACC
5351 TTTCGACCTC GACCGCCTCC TA m GAGAC GAATTTGTGC CACCAATCGA
5401 T~5-1-rTACCG CCGTGAGCTT TTCGACGGCA TCGGCCCTTA CAACCTGCGC
5451 TACCGAGTCT GGGCGGACTG GGACTTCAAT Al.CGC~GCT TCTCCAACCC
5501 GGCGCTGATT ACCCGCTACA TGGACGTCGT GATTTCCGAA TArA~rr.~r~
5551 TGA~CGG~l CAGCATGAGG r'~r~GGr-~rTG ATA~Ar~r-TT CAGAAAACGG
5601 CTGCCAATGT A~ GG~,~ TGCAGGGTGG GAGACTTGCA GGCGCATGCT
5651 GGC~.l... ~ A~G'rAArG AGAATCGCCG TCTGGCCTTG CGTACGCGGT
5701 Tn~TAArGGT TAAGGC'C~-C TCr~ GAAGCGCAGA ACCGTAGTCG
5751 CGGATCCACA TTGGACTTCT TTAACGCGTT l.,C-,.C~l.,A TCCACCTTTC
5801 AAGCCC~-.C CGCGTAACGC GGr~r,~.r-~r~ GA~,..,~,..~,C ATATCGCATC
5851 A~ C~ TGCCAGTGCT TGGAAAGCGT CGAGCACTCT ~7~ 7~71~
5901 CTTGACGTTC GCGCCCGCTC CTAGAGGTAG CGTGTCACGT GACTGAAGCC
5951 AATGAGTGCA ACTCGGCGTC GCGAAAGGTT TCA~ ~CGG TTGAGCAAGA
6001 CACCGCAAGA CTACTGGAGT GCGTGCACAA GCGCCTCCAG CTCGCGGCTG
6051 AAAGCGGATG CAAAGGGATT CGAAGCTTGA GCAACATGCG AAGGGGAGAA
6101 CGGCCTATGA GGCTGGGACA G~".ll~,AT CCGCGCGCGA ATGCACTGTC
6151 AATGGCCAAG TAGAAGTCCC CG~l~l~,GC CAGCAGAAGT CCCCACTCCG
6201 ~.G~GG TTGGCTAATT ~l~GGCGG~ CC~ l.~l G~,.C~GC-,l~,
6251 GCGCATCCGG TAGGACTCGC CGGAGGTGAC GACGATGCTG GC~-~--7C~
6301 Gr~r.CCr-~TC GAGGATGCTG GCGGr~w ~GG l~ GG CAGGAATCGC
6351 CCCCATTGTT CGAAGGGCCA ATGCGAGGCG ATGGCrArGG AG~-~G~r,~l~
6401 GTAGCCGGCA GCCACGAGCC r,r~ pr~r, TTGAGTCCCG ~1~7~7 rc~A
6451 G~GGGGC(,AA GCCGATCTCG TCCAAGATGA CCAGATCCGC GCGr-AGr~rG
6501 GTGTCGATGA 1~ 7CCGAC G~..,ll.,lCG GCr~-~,CCGC GGTAGAGGAC
6551 CTCGATCAGG TC~,CGGCr~G TGAAGTAGCG GACTTTGAAT CCGGCGTGGA
6601 CGGCAGCGTG CCCGCAGCCG ATG~-rA-GT GA~.lll~,CC CGTACCAGGT
6651 GGGCCAATGA CCGCCAGGTT C;1.,.1.71~CC CGAATCCATT CCAGGCTCGA
6701 CAGGTAGTCG AAG~GG~ld CGGTGATCGA CGA.C~.., ACGTCGAACC
6751 CGTCGAGGGT ~.1~7~ACC GGGAAGGCTG CGGC~ ,AG A~GGl.C,GC~
6aO1 ~71~..GK,AGG CAL~,CC.GGC AGCGATCTCG GCCTCAACCA AC'~l~r_~CAG
6851 GAr~ lCC Gr~ CCAGC GTTGCGTCTT GGCGACTTGC AACACCTCGG
6901 ~GG~Ll.,~G GCGCACCGTG GCCAGCTTCA Ar~Cr.~ CGcc~,c~l~A
6951 AGGTCAGCAG CCAGrG-,l-,C CGCrr'~r~rAC GGTGCr~CrG GCTTGGCAGC
7001 G~71~,1iATG AGGC~-7L-'CC ~ LwlG TTGATCTTGT A~GC~l~AA
7051 CGAGCGG~,lC TCGACGGTGG GCAGATCGAG CACGAGTGCG l~cr~Gr~7
7101 GGC~d~ , lGG~h,-~,CC-7 G-~,CC~,G~G CCAGGATCGA GCGCACGTCG
7151 GrAr-cGrGr77 PCCGGCr-AA~ CGr'~CCGCC CGGCGCAGCG CGTCAATCAA
7201 AGC~ , CCGTGGGCGG rrCr~r.r,CC GArr7~AATG TCGAGTTCGG
7251 A m CAGTCG GGTGTTGCCG ATCGCAGCAG r~rCr-ArrAG GAACTGCTGC
7301 G~ C CCAATGCGCA GAA.~ lC T~ -llGGG llL..~7Ci,
7351 ~rrArrArrC GAGGGTGCGG GTCTGGGTCC GTCGTAGTGT TCATCGAGGA
7401 TGGACACCTC ACCTGGGCTG ACGAGCTCGT G~CGGC~AC GAT QCACCG
7451 GTCGCAGGTT CCAACAGGAT CAGGGCGCCA TGATCGACCA CrACCGCC~C
7501 GGTGGCACCG ~cr7i-7crrcT rArGr~!Crr' GTAACGAGCT GAGCCGTAAC
7551 GGATGCACGA GAGG~C~lCG ACCTTACGGC GC~rrnArCC CGAGCCGATC
760~ GGCCC,CA GCGAGGGCAG ~lCC~-lCAAG A~c~ GC~ CGTCAACCAA
7651 GCGATCGTTG GGCACGGCGC AGATCTCCGA GTGGACCGTG GCATTGACCT
7701 CGGCGCACCA TAGTTGCGCC lGGGC~,lLGA GGGCACGTAG GTCGACCTGC
7751 TCACCGGCTA ACGCAGCTTC GGTCAGCAGC GGCACCGCAA Gc,~lCC.G
7aol AGCGTAGCCA CAGAGGTTCT CCACGATGCC CTTCGATTGC GGATCCGCAC
CA 02241023 1998-06-19
WO 97/23624 PCT/GB96fO3221
- 43 -
7851 CGTGGCAGAA GTCCGGAACG AAGCCATAGT GGGACGCGAA TCGCACATAA
7901 1~W~7~7~i n~r~ArAAc ATTGGCGACG ACACCACCTT TGAGGCAGCC
7951 CA~ .~i GCCAGGATCT TGGCCGGAAC ccc~rrrATc GCCTC
Seq ID No 4
1 TTCTACTGCC TGACCTGAGC AGCGCCGAGG CGCGCAGCGC GATCACTGCG ACCTGAATGG
61 CCAGGTGGAA AGCGCCACCG A~CC~GCAC CGAGTGCCTG ACGATTCGGA lCC~ AC
121 rAr~-~crr~r~ GTr~r7~rcGc CATGATGACG AAATATCGGC TGGGCGGAGT r~rGrrr,r~
181 GTr~r-'U~G TGArAArCCG GTr~r~CG'r- CGCTTATAAC AGGGATCACG GGGCAGGATG
241 GTTCCTACCT CGCCGAGCTA CTACTGAGCA lr-Gr~TArG~ GGTTCACGGG ~ iL''l~il'C
301 GAGCTTCGAC GTTTAA Q CG TCGCGGATCG ATCACCTCTA CGTTGACCCA ~rr~ArrGCi
361 G w~ CGCTT ~ CAC TATGCAGACC TCACTGACGG CACC~ GTGACCCTGC
421 TCAGCAGTAT CGACCCGr-r~T GAGGTCTACA ACCTCGCAGC GCAGTCCCAT ~ CCi~i.CA
481 GCTTTGACGA GCCAGTGCAT ~CCGC~G~A CCACCGGCAT GGGATCGATC CGACTTCTGG
541 AAGCAGTCCG C~ CW GTGGACTGCC GGTTCTATCA GG-~C~ ~ TCGGAGATGT
601 TCGGCGCATC TCCGCCACCG r~Arr~AT CGACGCCGTT CTA ~CCCj TCGCCATACG
661 GCGC~GC~AA G~ ~ G TACTGGACGA CTCGCAACTA TCGAGAGGCG TACGGATTAT
721 TCGCAGTGAA TGGCATCTTG TTCAACCATG A~i CCCCCC~ GCGCGGCGAG A~ GA
781 CCCr~ T CA~G-~GCC ~"'~GC~ iCA TCCGAGCTGG CGTCCAATCG GAGGTCTATA
841 TGGGCAACCT CGATGCGATC CGCGACTGGG GCT~GCGCC CGAATATGTC r~GGr'TGT
901 GGAGGATGTT GCAAGCGC ~ GAACCTGATG ACTACGTCCT GGC~r~r-GG C'i G~i ACA
961 CCGTACGTGA ~l -~i~- ~AA G~ ll. .a ACCACGTCGG GCTCGACTGG r~ Gr~CG
1021 TCAAGTTTGA Cr~rCGCT~T ~G7CG~L~A CCGAGGTCGA TTCGCTAGTA GGAGATGCCG
10B1 Ar~iCGGC CCAGTCACTC GGCTGGAAAG ~ .C~llCA TACTGGTGAA CTCGCGCG Q
1141 TCATGGTGGA CGcGr~r~Tc GCC~,C~ AGTGCGATGG CACACCATGG ATCr-~r~rGc
1201 CGA.~ CC I~ l~C,GGC GGAGTAAGTT GACGACTACA ~- ~C,G~lC TGr~rC'GCGC
1261 AA~jC'~r~ 7 TATATCGCCG GTCATCGGGG G-l~~ C TCAGCGCTCG TACGTAGATT
1321 TGAGGCCGAG GGGTTCACCA ATCTCATTGT GCGATCACGC GATGAGATTG ATCTGACGGA
1381 ccr~GcrrjcA ACGTTTGATT 1~ A r~r~r~rrA CAGGTGATCA TCGATGCGGC
1441 cGrpcGGGTc GGCGGCATCA TGGCGAATAA CACCTATCCC GCGGACTTCT TGTCCGAAAA
1501 CCTCCGAATC r~GACrAATT TGCTCGACGC AGCTGTCGCC ~jlGC'il~lGC CiCiG~
1561 ~-- CG~jl TCGTCATGCA TCTArCCr~ GTACGCTCCG CAACCTATCC Arr~r~nTGC
1621 TTTATTGACT GGCC- l GG AGCCr~Cr~ CGACGCGTAT GCGATCGCCA AGATCGCCGG
16B1 TATCCTGCAA GTTCAGGCGG TTAGGCGCCA ATAq~Kj~G GCGTGGATCT CTGCGATGCC
1741 r~AcT~ArcTc TArCrA~ccCr7 GCr~r~TT ~lCCCC~CC CGKi ATC TCTTGCCGGC
1801 GCTCATCCGT CGATATGAGG AAGCCAAAGC ~ GG~j~A GAAGAGGTGA CGAATTGGGG
1861 GACCGGTACT C~ GCiCG AA~ A TGTCGACGAT CTGGCr'~GCG CA~GC~
1921 C~...GiGAA CATTTCGATG GTCCr~Cr' CGTCAACGTG GGCACCGGCG TCGATCACAG
1981 CATTAGCGAG ATCGCAGACA j~ C~j~ AC GGCG~ GGC TACATCGGCG ~A~rArGTTG
2041 GGATCCAACT P~C'CCrATG r~AccccGcG CAAACTATTG GACj.~lCCG CGcTArGcr~A
2101 ~ l~iG~llGG CGCCrGrnA~ TCGCACTGAA Pr'~r,Gr~TC GATGCAACGG l~ Gl~blA
2161 CCGCACAAAT GCCGATGCCG TGAGGAGGTA AAG~l~CGGG CCGGCCGATG TTATCCCTCC
2221 GGCCGr'rGG GTAGGGCGAC ~ GCCATCGA GTGGTACGGC A~7'C(jC~lGG CCGGrr-AGGC
2281 GCATGGCCTA TGGGAGTATC CCATAGCCTG GCTTGGCTCG CCCCTACGCA TTATCAGTTG
2341 ACCGCTTTCG CGCCAGCTCG CAGG~ -jC~j GCAGCATCCC GTTCAGGTCT CCTCATGGTC
2401 ~ CA Cr'ACr~CGCA AGCTCGAACC GA~ C CCAATTTCGC ATGCTAATAT
2461 CG ~ CGATGG Allll lGCG CAACGCCGGC TTGATGGCTC GTAACGTTAG CArrG~r-ATG
2521 ~ljCGC~ACT TCGAACGAAA GCGCCTATTA GT~r~rAAT TCAAAGCATA CGGAGTCAAC
2581 GTTGTTATTG AT~li~lGC TAACTCCGGC CA~J C~lA GCG~-..l~CG TCGTGCAGGA
2641 TTCAAGAGCC GTAl~l.lC CTTTGAACCT ~ CGGGC CATTTGCGCA ACTAACGCGC
2701 GAGTCGGCAT CGGATCCACT ATGGGAGTGT CACCAGTATG CCCTAGGCGA CGCCGATGAG
CA 0224l023 l998-06-l9
WO 97/23624 PCT/GL~'0~221
-44-
2761 ACGATTACCA TCAATGTGGC AGGCAATGCG GGGGCAAGTA bllC~bl~CT GCCGATGCTT
2821 AAAAGTCATC AAGATGCCTT ~c~-lCC~GCG AATTATATTG GrArrr.AAr.A CGTTGCAATA
28B1 CACCGC~llG ATTCGGTTGC ATCAGAATTT CTGAACCCTA CCGATGTTAC lllC~lbAAG
2941 ATCGACGTAC AGG~lllLbA GAAGCAGGTT ATCGCGGGCA GTAAGTCAAC GCTTAACGAA
3001 AG~lbCL-~c~ GCATGCAACT CGAACTTTCT TTTATTCCGT TGTACGAAGG TGACATGCTG
3061 ATTCATGAAG CGCTTGAACT TGTCTATTCC CTAGGTTTCA GACTGACGGG l~l~llb~'CC
3121 GGATTTACGG ATCCGCGCAA TGGTCGAATG CTTCAAGCTG ACGGCATTTT ~LlC~bl~GG
3181 GACGATTGAC ATAAATGCTT GCGTCGGCAC ~lGC~lA TCCAAACGGG CGAlL.GGlb
3241 AGCO~GC--lC CCGGGCACCT AATCGACTAT CTAAATTGAG G~'Gb~CG~ CGTGCGGCAC
3301 GAACAGGTGG CCGGCTGCTA GCGTTACACA CGTCATGACT GCGCCAGTGT TCTCGATAAT
3361 TATCCCTACC TTCAATGCAG CGGTGACGCT GCAAGCCTGC CTCGGAAGCA l-blCGG~A
3421 GACCTACCGG GAAGTGGAAG ~ -C~ l CGAC~CGbl TCr~CCnATC GGACCCTCGA
3481 CATCGCGAAC AblllL-~CL CGGAACTCGG CTCGCGACTG bl~ cACA GCGGGCCCGA
3541 TGA~laaCCCC TAcr-~-vccA TGAACCGCGG Cb~LGGCb~A GCr~rAGGrG AATGGGTACT
3601 TTTTTTAGGC GCCr~CrAr~ CCCTCTACGA ~CrParr~cG ~ 3CC(Ar,G TAGC~
3661 'l~lC~-~AC CATGCGGCAA GCCATCTTGT CTATGGCGAT GTTGTGATGC GTTCGACGAA
3721 a~rCCOr,rAT GCCGGACCTT TCGACCTCGA CCGC~lc~lA TTTr~ArrA Al~l~lGCcA
3781 CCAATCGATC TTTTACCGCC GTGAGCTTTT CGACGGCATC GbCC~l.ACA ACCTGCGCTA
3841 CCGAGTCTGG GCGGACTGGG ACTTCAATAT lCbC~-llC TCraarCCGG CGCTGATTAC
3901 CCGCTACATG GACb.cb~bA TTTCCGAATA r~arG~rATG ACCGGC~l A GCATGAGGCA
3961 GGGGACTGAT AAAGAGTTCA naaa~CGGCT GCCAATGTAC llc~G~b CAGGb~aWA
4021 GACTTGCAGG CGCATGCTGG Cb-l---~AA ~-r~aa~r~An AAlCGC~b~C ~C~Lb
4081 TAC~w ~G ATAAGGGTTA A~G~-~b~clL rAAAA~-~acr~ ~rCGCa-~AC CGTAGTCGCG
4141 GATCCACATT GGACTTCTTT AACGCGTTTG CGTCCTGATC CACCTTTCAA CCCCbl~CC'b
4201 CGTGACGCGG CGCGr~Ar~ b~Glcb~AT AI-~b~AC ~ CCAGTGCTTG
4261 GAAAGCGTCG AGCACTCTGG l~Cb~b~ TGACGTTCGC GCCCGCCCCT AGAGGTAGCG
4321 TGTCACGTGA CTGAAGCCAA TGAGTGCAAC ~ LGC GAAAGGTTTC AblObCbb~.
4381 r~~r~ ra CCGCAAGACT ACTGGAGTGC GTGCACAAGC GCCTCCAGCT CACGG
Seq ID No 5
1 atgatcgctg tgatctggtc ggcggtgccg acaggaaccg tcgacttgtc gacgatcacc
61 ttgtaccggt cgatgtatga cccaatgtcg tccgcaaccg agaagacgta cgtcaggtcc
121 gccyccccgc tttcacccat gggcgtcggg acggcgatga aaatgacgtc cgcgtgctcg
181 attccgcgtt gccggtcggt ggtgaagtca atcagcccgt tctcacggtt cctcgcaatc
241 aactcccaac ccgggctcga aaatcgggac actgcctgcg a~ -t cgatcttggc
301 ctgatcgata tcgscac~ cgacatcgtt gccgctatcc gc~__gsc~gg cgcccgtgac
361 gaggcctaca tagcctga
Seq ID No 6
1 M I A V I W S A V P T G T V D L S T I T L Y R S M Y D P M S
31 S A T E R T Y V R S A A P L S P M G V G T A M K M T S A C S
61 I P R C R S V V K S I S P F S R F L A I N S Q P G L E N R D
91 T A C E E Q I D L G L I D I D T D D I V A A I R E T G A R D
121 E A Y I A
CA 02241023 1998-06-19
WO 97/23624 PCT/GB96/03221
- ~5 -
Seq. ID No.7
1 gtgtcatctg ctccaaccgt gtcggtyata acgatttcgc tgaacgatct cgagggattg
61 aaaagcaccg tggagagcgt tcycycy~ag cgctatgggg ggcgaatcga gcacatcgtc
121 atcgacggtg gatcgggcga cgccgtcgtg gagtatctgt ccggcgatcc tggctttgca
181 tattggcaat ctcagcccga caacgggaga tatgacgcga tgaatcaggg cattgcccat
241 tcgtcgggcg acctgttgtg gtttatgcac tccacggatc gtttctccga tccagatgca
301 gtcgcttccg tggtggaggc gctctcgggg catggaccag tacgtgattt gtggggttac
361 ggg-A~aca accttgtcgg actcgacggc aaaccacttt tccctcggcc gtacggctat
421 atgccgttta agatgcggaa atttctgctc ggcgcgacgg ttgcgcatca ggcgacattc
481 ttcggcy~y- cgctggtagc caagttgggc ggttacgatc ttgattttgg actcgaggcg
541 gaccagctgt tcatctaccg tgccgcacta atacggcctc ccgtcacgat cgaccgcgtg
601 gtttgcgact tcgatgtcac gggacctggt tc~arccagc ccatccgtga gcactatcgg
661 accctgcggC ggctctggga cctgcatggc gactacccgc tgggtgggcg cagagtgtcg
721 tgggcttact tgcgtgtgaa ggagtacttg attcgggccg acctggccgc attcaacgcg
7al gtaaagttct tgcgagcgaa gttcgccaga gcttcgcgga agcaaaattc atag
Seq. ID No.8
1 V S S A P T V S V I T I S L N D L E G L K S T V E S V R A Q
31 R Y G G R I E H I V I D G G S G D A V V E Y L S G D P G F A
61 Y W Q S Q P D N G R Y D A M N Q G I A H S S G D L L W F M H
91 S T D R F S D P D A V A S V V E A L S G H G P V R D L W G Y
121 G K N N L V G L D G K P L F ~ R P Y G Y M P F R M R K F L L
151 G A T V A H Q A T ~ P G A S L V A K L G G Y D L D F G L E A
181 D Q L F I Y R A A L I R P P V T I D R V V C D F D V T G P G
211 S T Q P I R E H Y R T L R R L W D L H G D Y P L G G R R V S
241 W A Y L R V R E Y L I R A D L A A P N A V K F L R A K F A R
271 A S R R Q N S
Seq. ID No.9
1 gtgaagcg~g cgcttataac agggatcacg gggcaggatg gttcctacct cgccgagcta
61 ctactgagca agggatacga ggttcacggg ctcgttcgtc gagcttcgac gtttaacacg
121 tcgcggatcg atcacctcta cgttgaccca caccaaccgg y~yCyCy~Lt gttcttgcac
181 tatgcagacc tcactgacgg cacccggttg gtgaccctgc tcagcagtat cgacccggat
241 gaggtctaca acctcgcagc gcagtcccat gtgcgcgtca gctttgacga gccagtgcat
301 accggagaca c~accggcat gggatcgatc cgacttctgg aagcagtccg cctttctcgg
361 gtggactgcc ggttctatca ggcttcctcg tcggagatgt tcggcgcatc tccgccaccg
421 c~3 acgaat cgacgccgtt ctatccccgt tcgccatacg ycycyy~caa ggtcttctcg
481 tactggacga ctcgcaacta tcgagaggcg tacggattat tcgcagtgaa tggcatctty
541 ttcaaccatg agtccccccg gcgcyyl~y-g actttcgtga cccgaaagat cacgcgtgcc
601 gtgycy~yca tccgagctgg cgtccaatcg gaggtctata tgggc~cct cgatgcgatc
661 cgcgactggg gctacgcgcc cgaatatgtc gaggggatgt ggaggatgtt gcaagcgcct
721 gaacCtgatg actacgtcct ggcg?caggg cgtggttaca ccgtacgtga gttcgctcaa
781 gctgcttttg accatgtcgg gctcgactgg caaaagcgcg tcaagtttga cgaccgctat
841 ttgcgtccca ccgaggtcga ttcgctagta ggagatgccg acaaggcggc ccagtcactc
901 ggctggaaag cttcggttca tactggtgaa ctcgcgcgca tcatggtgga cgcggacatc
961 gccgcgttgg agtgcgatgg cacaccatgg atcgacacgc cgatgttgcc tggttggggc
1021 agagtaagtt ga
CA 02241023 1998-06-19
WO 97123624 PCT/G1~9S,'03221
- 46 -
Seq.IDNo.10
1 V K RALITGITGQDGSYLAELLLSKGYEVHG
31 LVRRASTFNTSRIDHLYVDPHQPGARLFLH
61 YADLTDGTRLVTLLSSIDPDEVYNLAAQSH
591 VRVSFDEPVHTGDTTGMGSIRLLEAVRLSR
121 VDCRFYQASSSEMFGASPPPQNESTPFYPR
151 SPYGAAKVFSYWTTRNYREAYGLFAVNGIL
181 FNHESPRRGETFVTRRITRAVARIRAGVQS
211 EVYMGNLDAIRDWGYAPEYVEGMWRMLQAP
10241 EPDDYVLATGRGYTVREFAQAAFDHVGLDW
271 QRRVKFDDRYLRPTEVDSLVGDADKAAQSL
301 G W K ASVHTGELARIMVDADIAALECDGTPW
331 IDTPMLPGWGRVS
Seq.IDNo.ll
151 gtgaagcgag cgcttataac agggatcacg gggcaggatg getcctacct cgccgagcta
61 ctactgagca agggatacga ggttcacggg ctcgttcgtc gagcttcgac gtttaacacg
121 tcgcggatcg atcacctcta cgttgaccca caccaaccgg y~ ycg.tt gttcttgcac
181 tatgcagacc tcactgacgg cacccggttg gtgaccctgc tcagcagtat cgacccggat
241 gaggtctaca acctcgcagc gcagtcccat gtgcgcgtca gctttgacga gccagtgcat
20301 accggagaca ccaccggcat gggatcgatc cgacttctgg aagcagtccg cctttctcgg
361 gtggactgcc ggttctatca ggcttcctcg tcggagatgt tcggcgcatc tccgc~ccg
421~ ,_acg~t cgacgccgtt ctatccccgt tcgccatacg gcgcggccl~ ggtcttctcg
481 tactggacga ctcgcaacta tcgagaggcg tacggattat tcgcagtgaa tggcatcttg
541 ttcaaccatg agtcccccl~y y~-ycyy¢ydg actttcgtga cccgaaagat cacgcgtgcc
25601 gtggcgcgca tccgagctgg cgtccaatcg gaggtctata tgggcaacct cgatgcgatc
661 cgcgactggg gctacgcgcc cgaatatgtc gaggggatgt ggaggatgtt gcaagcgcct
721 gaacctgatg actacgtcct ggcgacaggg cgtggttaca ccgtacgtga gttcgctcaa
781 gctgcttttg accacgtcgg gctcgactgg caaaagcacg tcaagtttga cgaccgctat
841 ttgcgcccca ccgaggtcga ttcgctagta ggagatgccg acagggcggc ccagtcactc
30gol ggctggaaag cttcggttca tactggtgaa ctcgcgcgca tcatggtgga cgcggacatc
961 gccgcgtcgg agtgcgatgg cacaccatgg atcgacacgc cgatgttgcc tggttggggc
1021 ggagtaagtt ga
Seq.ID No.12
1 V K RALITGITGQDGSYLAELLLSKGYEVHG
3531 LVRRASTFNTSRIDHLYVDPHQPGARLFLH
61 YADLTDGTRLvTLLssIDpDEvyNLAAQsH
91 VRVSFDEPVHTGDTTGMGSIRLLEAVRLSR
121 VDCRFYQASSSE M FGASPPPQNESTPFYPR
151 SPYGAA K V FSYWTTRNYREAYGLFAVNGIL
40181 FNHESPRRGETFV T R KITRAVARIRAGVQS
211 EVYMGNLDAIRDWGYAPEYVEGMWRMLQAP
241 EPDDYVLATGRGYTVREFAQAAFDHV G LDW
271 Q K HVKFDDRYLRPTEVDSLVGDADRAAQSL
301 G W RASVHTGELARIMVDADIAASECDGTPW
45 331 IDTP M LPGW G G V S
CA 02241023 1998-06-19
W O 97~3624 PCT/G B96/03221
-47 -
Seq. ID No.13
1 gtgegatggc acaccatgga tegacacgcc gatgttgcct ggttggggca gagtaagttg
61 aegaetacae ctgggeetet ggaecgcgca aegeccgtgt atategeegg teateggggg
121 etggtegget cagcgctegt acgtagattt gaggeegagg ggttcaeeaa teteattgtg
lBl egateacgeg atgagattga tetgacggac egagccgcaa cgtttgattt tgtgtetgag
241 aeA~g~eeae aggtgateat egatgcggce geaegggtcg gcggeateat ggegaataac
301 aeetateceg eggaettett gteeg?aa~c cteegaatec agaeeaattt getegaegea
361 getgtegeeg tgegtgtgee geggetcett tteeteggtt egteatgeat etaeeeg~Pg
421 taegeteege aaeetateea egagagtget ttattgaetg geeetttgga geeeaeeaae
4Bl gaegegtatg egatcgeeaa gategceggt ateetgeaag tteaggeggt tay~L~e~
541 tatgggetgg egtggatete tgegatgeeg aetaaeetet aegg~eegg cgaeaaettc
601 teeeegteeg ggtegeatet ettgeeggeg eteateegte gatat~a~ga ageeaaaget
661 ggtggtgeag aagaggtgae gaattggggg accggtactc ~ycyy~JLJa acttctgcat
721 gtegaCgate tggcgagege atgcctgtte cttttggaac atttegatgg teeg~~-rae
731 gteaaegtgg geaeeggegt egatcaeage attagegaga tcgeagaeat ggtegetaea
841 geggtggget aeateggega aaeaegttgg gateeaaeta aaecegatgg ~aeee-~Jc~J.
901 aaaetattgg aegteteege getaegegag ttgggttggc ycccy~yaat egeaetgaaa
961 g~egge~tcg atge~eggt gtegtggtae egeaeaaatg cegatgeegt gaggaggtaa
Seq. ID No.14
1 V R W H T M D R H A D V A W L G Q S K L T T T P G P L D R A
31 T P V Y I A G H R G L V G S A L V R R F E A E G F T N L I V
61 R S R D E I D L T D R A A T F D P V S E T R P Q V I I D A A
91 A R V G G I M A N N T Y P A D F L S E N L R I Q T N L L D A
121 A V A V R V P R L L F L G S S C I Y P R Y A P Q P I H E S A
151 L L T G P L E P T N D A Y A I A R I A G I L Q V Q A V R R Q
131 Y G L A W I S A M P T N L Y G P G D N F S P S G S H L L P A
211 L I R R Y E E A R A G G A E E V T N W G T G T P R R E L L H
241 V D D L A S A C L F L L E H F D G P N H V N V G T G V D H S
271 I S E I A D M V A T A V G Y I G E T R W D P T K P D G T P R
301 K L L D V S A L R E L G W R P R I A L K D G I D A T V S W Y
331 R T N A D A V R R
CA 02241023 1998-06-19
WO 97/23624 PCT/Gl~S~ 3221
- 48 -
Seq. ID No. 15
1 gtgcgatggc acaccatgga tcgacacgcc gatgttgcct ggttggggcg gagtaagttg
61 acgactacac ctgggcctct ggaccgcgca acgcccgtgt atatcgccgg tcatcggggg
121 ctggtcggct cagcgctcgt acgtagattt gaggccgagg ggttcaccaa tctcattgtg
5181 cgatcacgcg atgagattga tctgacggac cgagccgcaa cgtttgattt tgtgtctgag
241 acARg~ccAc aggtgatcat cgatgcggcc gcacgggtcg gcggcatcat ggcgaataac
301 acctatcccg cggacttctt gtccg~ac ctccgaatcc agaccaattt gctcgacgca
361 gctgtcgccg tgcgtgtgcc gcggctcctt ttcctcggtt cgtcatgcat ct~cccg~g
421 tacgctccgc aacctatcca cgagagtgct ttattgactg gccctttgga gcccacr~-r
10481 gacgcgtatg cgatcgccaa gatcgccggt atcctgcaag ttcaggcggt taggcgccaa
541 tatgggctgg cgtggatctc tgcgatgccg actaacctct ac~acccgg cgacaacttc
601 tccccgtccg ggtcgcatct cttgccggcg ctcatccgtc gatatgagga agccaaagct
661 ggtggtgcag aagaggtgac gaattggggg accggtactc cgcyy~gCyd acttctgcat
721 gtcgacgatc tggcgagcgc atgcctgttc cttttggaac atttcgatgg tccgaacca~
15781 gtcaacgtgg gcaccggcgt cgatcacagc attagcgaga tcgcagacat ggtcgctacg
841 gcggtgggct acatcggcga aacacgttgg gatccaacta aacccgatgg aaccccgcyc
901 aaactattgg acgtctccgc gctacgcgag ttgggttggc gcccy~y~at cgcactgaaa
961 gacggcatcg atgcaacggt gtcgtggtac cgc~ Atg ccgatgccgt gaggaggtaa
Seq. ID No. 16
201 V R W H T M D R H A D V A W L G R S K L T T T P G P L D R A
31 T P V Y I A G H R G L V G S A L V R R F E A E G F T N L I V
61 R S R D E I D L T D R A A T F D F V S E T R P Q V I I D A A
91 A R V G G IMANNTYPA D F L S E N L R I Q TNLL D A
121 A V A V R V P R LLPL G S S C I Y P R Y A P Q P I H E S A
151 L LTGPL E P T N D A Y A I A R I A G I L Q V Q A V R R Q
181 Y G L A W I S A M P T N LY G P G D N F S P S G S H LLP A
211 LIR R Y E E A K A G G A EEVTNWGTGTP R R ELLH
241 V D D L A S A C LF L L E H F D G P NHV N V G T G V D H S
271 I S EI A D M V A T A V GYI(:~ET R W DPTKPDG T P R
301 K L L D V S A L R E L G W R P R I A L K D GI D A T V S W Y
331 R T N A DAV R R
Seq. ID No.17
1 atggattttt tgcgcaacgc cggcttga~g gctcgtaacg ttagtaccga gatgctgcgc
61 cacttcgaac gaaagcgcct attagtaaac caattcaaag catacggagt caacgttgtt
121 attgatgtcg gtgctaacec cggccagttc ggtagcgctt tgcgtcgtgc aggattcaag
181 agccgtatcg tttcctttga acctctttcg gggccatttg cgcaactaac gcgcaagtcg
241 gcatcggatc cactatggga gtgtcaccag tatgccctag gcgacgccga tgagacgatt
301 accatcaatg tggcaggcaa tgcy~yyyca agtagttccg tgctgccgat gcttaaaagt
40361 catcaaga~g cctttcctcc cgcgaattat attggcaccg aagacgttgc aatacaccgc
421 ctegattcgg ttgcatcaga atttctgaac cctaccgatg ttactttcct gaagatcgac
481 gtacagggtt tcgagaagca ggttatcacg ggcagtaagt caacgcttaa cgaaagctgc
541 gtcggcatgc aactcgaact ttcttttatt ccgttgtacg aaggtgacat gctgattcat
601 gaagcgcttg aacttgtcta ttccctaggt ttcagactga cgggtttgtt gcccggcttt
45661 acggatccgc gcaatggtcg aatgcttcaa gctgacggca ttttcttccg tggggacgat
721 tga
CA02241023l998-06-l9
WO97/23624 PCT/G~g~3221
-49-
Seq.IDNo.18
l MDFLRNAGLMARNvsTEMLRHFERKRLLvN
31 QFKAyGvNvvIDvGANsGQFGsALRRAGFK
61 SRIvsFEpLsGpFAQLTRKsAsDpLwEcHQ
591 YA L GDADETITINVAGNAGASSSVLPMLKS
121 HQDAFPPANYIGTEDVAIHRLDSVASEF L N
151 PTDVTFLKIDVQGFEKQVITGSKST L NESC
1~1 VGMQ L ELSFIPLYEGDMLIHEALE L V Y S L G
211 FRLTGLLpGFTDpRNGRMLQADGIFFRGDD
Seq.IDNo.l9
1 atggattttt tgcgcaacgc cggcttgatg gctcgtaacg ttagcaccga gatgctgcgc
61 cacttcgaac gaaagcgcct attagtaaac caattcaaag cacacggagt caacgttgtt
121 attgatgtcg gtgctaactc cggccagttc ggtagcgctt tgcgtcgtgc aggattcaag
181 agccgtatcg tttcctttga acctctttcg gggccatttg cgcaactaac gcgcgagtcg
241 gcatcggatc cactatggga gtgtcaccag tatgccctag gcgacgccga tgagacgatt
301 accatcaatg tggcaggcaa tgcyyyyy~a agtagttccg tgctgccgat gcttaaaagt
361 catcaagatg cctttcctcc cgcgaattat attggcaccg aagacgttgc aatacaccgc
421 cttgatecgg ttgcatcaga atttctgaac cctaccgatg ttactttcct gaagatcgac
481 gtacagggtt tcqags~-gc~ ggttatcgcg ggcagtaagt caacgcttaa cgaaagctgc
541 gtcggcatgc aactcgaact ttcttttatt ccgttgtacg aaggtgacat gctgattcat
601 gaagcgcttg aacttgtcta ttccctaggt ttcagactga cgggtttgtt gcccggattt
661 acggatccgc gcaatggtcg aatgcttcaa gctgacggca ttttcttccg tgggg~cgat
721 tga
Seq.IDNo.20
1 MDFLRNAGLMARNVSTEMLRHFERKRLLVN
31 QFKAYGVNVVIDVGANSGQFGSALRRAGFK
61 SRIVSFEPLSGPFAQLTRESASDP L W ECHQ
91 YALGDADETITINVAGNAGA S S S V L P M L KS
121 HQDAFPPANYIGTEDVAIHRLDSVASEF L N
151 PTDVTFLKIDVQGFEKQVIAGSKSTLNESC
181 VGMQLELSFIPLYEGDMLIHEAL E L V Y S LG
211 FRLTGL L PGFTDPRNGRMLQADGIFFRGDD
CA 02241023 1998-06-19
WO 97/23624 PCT/G~ 3221
- ~0 -
Seq. ID No.21
1 atgaetgcgc cagtgttctc gataattatc cctaccttca atgeagcggt gacgctgeaa
61 geetgeeteg gaagcategt egggcagace taeegggaag tggaagtggt ccttgtegac
121 ggeggttcga ccgateggae eetegaeate gegaaeagtt tCCyCCCyyd aeteggeteg
181 egaetggteg tteaeagegg geeegatgat ggeeeetaeg aegeeatgaa cegeggegtc
241 ggegtggeea eaggegaatg ggtacttttt ttaggegccg acgaeaeeet ctaegaaeea
301 aeeaegttgg eeeaggtage egettttete ggeqaeeatg cggcaageca tettgtetat
361 ggegatgttg tgatgcgttc gaegaAAagc cggcatgceg gaeetttega eetegaeege
421 eteetatttg agaegaattt gtgeeaeeaa tegatetttt aeegeegtga gettttegae
4Bl ggeateggec cttacaaeet gegetaeega gtetgggcgg actgggactt caatattege
541 tgettetcca aeccggeget gattaccegc tacatggac~ tcgtgatttc cgaataeaae
601 gaeatgaccg getteagcat gaggeagggg aetgataaag agtteagaaa aeggetgeea
661 atgtaettet gggttgeagg gtgggagaet tgeaggegea tgetggegtt tttgaaagae
721 ~Aggag~Ate geegtetgge ettgegtaeg eggttgataa gggttaagge cgteteeaaa
7al gaaegaageg eagaaeegta g
Seq. ID No.22
1 M T A P V F S I I I P T F N A A V T L Q A C L G S I V G Q T
31 Y R E V E V V L V D G G S T D R T L D I A N S F R P E L G S
61 R L V V H S G P D D G P Y D A M N R G V G V A T G E W V L F
91 L G A D D T L Y E P T T L A Q V A A F L G D H A A S H L V Y
121 G D V V M R S T K S R H A G P F D L D R L L F E T N L C H Q
151 S I F Y R R E L F D G I G P Y N L R Y R V W A D W D F N I R
181 C F S N P A L I T R Y M D V V I S E Y N D M T G F S M R Q G
211 T D K E F R K R L P M Y F W V A G W E T C R R M L A F L K D
241 K E N R R L A L R T R L I R V K A V S K E R S A E P
Seq. ID No.23
1 atgaetgegc cagtgttete gataattate eetaeettca atgeageggt gacgctgeaa
61 geetgeeteg gaageategt egggeagaee taeegggaag tggaagtggt eettgtegae
121 ggeggttega eegateggae eetegaeate gcgaacagtt tccgcecyy~ aeteggeteg
181 egaetggteg tteaeagegg geeegatgat ggeeeetaeg aegeeatgaa ecy.yy~yLc
241 ggegtageea eaggegaatg ggtaettttt ttagyegccg aegaeaeeet etaeq~eea
301 aeeaegttgg ceeaggtage cgettttete ggegaeeatg eggeaageea tettgtetat
361 ggegatgttg tgatgegtte qaeq~A~ge eggeatgecg gaeetttega eetegaeege
421 eteetatttg agaegaattt gtgeeaecaa tegatetttt aeegeegtga gettttegae
481 ggeaeeggee cttaeaaeet gegctaecga gtctgggegg aetgggaett eaatattege
541 tgetteteea aecegycg~t gattaeeege taeatggaeg tegtgattte egaataeaae
601 gaeatgaeeg getteageat gaggeagggg aetgataaag agtteagaaa aeggetgeea
661 atgtaettct gggttgeagg gtgggagact tgcaggcgca tgctggegtt tttgaaagae
~21 aaggagaatc gcegtetgge cttgcgtacg cggttgataa gggttaagge egtctceaaa
781 ga~ega~Ageg eagaacegta g
CA 02241023 1998-06-19
W O 97~3624 PCTIG B96103221
- 51 -
Seq.IDNo.24
1 M T APVFSIIIPTFNAAVTLQACLGSIVGQT
31YREVEVVLVDGGSTDRTLDIANSFRPELGS
61RLVVHSGPDDGPYDAMNRGVGVATGEWVLF
91 LGAD3TLYEPTTLAQVAAFLGDHAASHLVY
121GDVV M RSTKSRHAGPFDLDRLLFETN L CHQ
151SIFYRRELFDGIGPYNLRYRVWADWDFNIR
181 CFSNPALITRYMDVVISEYNDMTGFSMRQG
211TDRBFRKRLPMYFWVAGWETCRRMLAFLRD
241KENRRLALRTRLIRVKAVSRERSAEP
Seq.IDNo.25
1 gtggccagca gaagtcccca ctccgctgcg ggtggttggc taattcttgg cggctccctt
61 cttgtggtcg gcgtggcgca tccggtagga ctcgccggag gtg~Cg?Cga tgctggcgtg
121 gtgc~ gc cgatcgagga tgctggcggc ggtggtgtgc tcgggc~gga atcgccccca
181 ttgttcgaag ggccaatgcg aggcgatggc r~gggagcgg cgctcgtagc cggcagccac
241gagcc~g~c aacagttgag tcccggtgtc gtcgagcggg gcgaagccga tctcgtccaa
301 gatg~cc~ga tccgc~.J-J~ gcagggtgtc gatgatcttg ccgacggtgt tgtcggccag
361 g~gcyy-ag aggacctcga tcaggtcggc ggcggtgaag tagcggactt tgaatccggc
421 gtgg-l~,, gcgtgcccgc agccgatgag caggtgactt ttgcccgtac caggtgggcc
4Bl aatgaccgcc aggttctgtt gtgcccgaat ccattccagg ctcgacaggt agtcgaacgt
541 ggctgcggtg atcgacg~tc cggtgacgtc gaacccytcg agggtcttgg tg~ccggg~
601 ggctgcggcc ttgagacggt tggcggtgtt ggaggcatcg cgggcagcga tctcggcctc
661 Aa~c~gtc cgcaggatct cctccggtgt ccagcgttgc gtcttggcga cttgr~c~c
721ctcgy.y~J~gttgcgyc9ca ccgtggccag cttcaaccgc cgc~gcgccg cgtcaaggtc
781agcagcc~gcggt9C-,y~Cy aggacggtgc caccggcttg gcagcggtgg tcatgaggcc
B41 gtcccgtcgg tggtgttgat cttgtag
Seq.IDNo.26
lVASRSPHSAAGGWLI L GGSLLVVGVAH e v G
31 LAGGDDDAGVVQQPIEDAGGGGVLGQESPP
61LFEGPMRGDGQGAALVAGSHEPEQQLSPGV
91VSRGEADLVQDDQIRAEQGVDDLADGVVGQ
121AAVEDLDQVGGGEVADFESGVDGSVPAADE
151QVTFARTRWANDRQVLLCPNPFQARQVVER
181GCGDRRSGDVEPVEGLGDREGCGLETVGGV
211GGIAGSDLGLNQRPQDLLRCPA L RLGDLQH
241LGGVAAHRGQLQPPQRRVRVSSQRCRRGRC
271HRLGSGGHEAVPSVVLIL
CA 02241023 1998-06-l9
WO 97/23624 PCT/GB96/03221
- 52 -
Seq. ID No.27
1 atgggctgcc tcaaaggtgg tgtcgtcgcc aatgttgttg ttccaacacc ggattatgtg
61 cgattcgcgt cccactatgg cttcgttccg gacttctgcc acggtgcgga tccgcaatcg
121 aagggcatcg tggagaacct ctgtggctac gctcaggacg accttgcggt gccgctgctg
181 accgansctg cgttagccgg tgagcaggtc gacctacgtg ccctcaacgc ccaggcgcaa
241 ctatggtgcg ccgaggtcaa tgccacggtc cactcggaga tctgcgccgt gcccaacgat
301 cgcttggttg acgagcgcac cgtcttgagg gagctgccct cgctgcggcc gacgatcggc
361 tcggggtcgg tgcgccgtaa ggtcgacggc ctctcgtgca tccgttacgg ctcagctcgt
421 tactcggtgc ctcagcggct cgtcggtgcc accgtggcgg tggtggtcga tcatggcgcc
481 ctgatcctgt tggaacctgc gaccggtgtg atcgtggccg agc~cgagct cgtcagccca
541 ggtgaggtgt ccatcctcga tgaacactac gacggaccca gacccgcacc ctcgcgtggt
601 cctcgcccga aaacccaagc agagaaacga ttctgcgcat tgggaaccga agcgcagcag
661 ttcctcgtcg gtgctgctgc gatcggcaac acccgactga aatccgaact cgacattctg
721 cteggccttg gcgccgccca cggcg~acag gctttgattg acgcgctgcg C~yyy~yLt
781 gcgtttcgcc ggttccgcgc tgccgacgtg cgctcgatcc tgy~cgccyy cgccygcacc
841 ccacaacccc yccccgccyy cgacgcactc gtgctcgatc tgcccaccgt cgagacccgc
901 tcgttggagg cctacaagat caacaccacc gacgggacgg cctcatgacc accgctgcca
961 agccggtggc accgtcctcg gcggcaccgc tggctgctga ccttgacgcg gcgctgcggc
1021 ggttgaagct ggccacggtg cgccgcaacg ccgccgaggt gttgcaagtc gcc~gPcgc
1081 aacgctggac accggaggag atcctgcgga cgttggttga ggccgagatc getgcc~g~g
1141 atgectecaa caccgceaac cgtctcaagg ccge~.gcctt cccggtcacc aagaccctcg
1201 acgggttcga cgtc~ccgq~ tcgtcgatca ccgcagccac gttcgactac ctgtcgagcc
1261 tggaatggat tcgggc~cafl cagaacctgg cggtcattgg cccacctggt acgggcaaaa
1321 gteacetgct catcggetgc gggcacgctg ecgtccacgc cggattcaaa gtccgctact
1381 traccgccgc cgacctgatc gaggtcctct aecgcggcct ggccg~e~c accgtcggca
1441 agatcatcga caccctgctc cgcy~yyatc tggtcatctt gg~cgag~tc ggcttcgccc
1501 cgctcgacga caccgggact caactgttgt tccggctcgt ggctgccggc t~rgagcgcc
1561 gctccctggc catcgcctcg cattggccct tcgaac~tg ggggcgatte ctgcccgagc
1621 acaccaccgc cgccagcatc ctcgategge tgctgcacca cycc~g~atc gtcgtcacct
1691 ccggcgagtc ctaccggatg cgccacgccg accaca~gaa ggg~ccgcc aagaattag
Seq. ID No.28
1 M G C L R G G V V A N V V V P T P D Y V R F A S H Y G F V P
31 D F C H G A D P Q S K G I V E N L C G Y A Q D D L A V P L L
61 T E A A L A G E Q V D L R A L N A Q A Q L W C A E V N A T V
91 H S E I C A V P N D R L V D E R T V L R E L P S L R P T I G
121 S G S V R R K V D G L S C I R Y G S A R Y S V P Q R L V G A
151 T V A V V V D H G A L I L L E P A T G V I V A E H E L V S P
181 G E V S I L D E H Y D G P R P A P S R G P R P K T Q A E K R
211 F C A L G T E A Q Q F L V G A A A I G N T R L K S E L D I L
241 L G L G A A H G E Q A L I D A L R R A V A F R R F R A A D V
271 R S I L A A G A G T P Q P R P A G D A L V L D L P T V E T R
301 S L E A Y R I N T T D G T A S
CA 0224l023 l998-06-19
W O 97~3624 PCT/G B96/03221
Seq.IDNo.29
1 MTTAAKPVAPSSAAPLAADLDAALRRLK L A
31 TVRRNAAEV LQV AKTQRWTPEEILRTLVEA
61 EIAARDASNTANRLKAAAFPVTKT L DGFDV
591 TGSSITAATPDYLSSLEWIRAQQN L AVIGP
121 PGTGKSH L LIGCGHAAVHAGFKVRYFTAAD
151 LIEVLYRGLADNTVGKIIDT L LRADLV IL D
lBl EIGFAP L DDTG TQ LLFRLVAAGYERRSLAI
211 ASHWPFEQWGRP LPE HTTAASI LD R LL HHA
10241 SIVVTSGESYRMRHADHKKGAAKN
Seq.IDNo.30
1 gtgacgectg ctccgaccgt ctcggtgata acgatctcgt tcaArg--ct cgacgggttg
61 cagcgcacgg tgaaaagtgt y~;yyy~:ycaa cgctaccggg gacgcatcga gcacatcgta
121 atcgacggtg gcagcggcga cgacgtggtg gcatacctgt ccgggtgtga accaggcttc
15131 gcgtattggc agtccgagcc cgacggcggg cggtacgacg cgatgaacca gggcatcgcg
241 cacgcatcgg gtgatctgtt gtggttcttg cactccgccg atcgtttttc ~gyy~ccy.~c
301 gtggtagccc aggccgtgga ggcgctatcc ggcaagggac cggtgtccga attgtggggc
361 ttcgggatgg atcgtctcgt cgggctcgat cgggtgcgcg gcccgatacc tttcagcctg
421 cgcaaattcc tyyccg~:aa gcaggttgtt ccgcatcaag catcgttctt cggatcatcg
20481 ctggtggcca agatcggtgg ctacgacctt gatttcggga tcy~cyccgd ccaggaattc
541 atattgcggg ccgcgctggt atgcgagccg gtcacgattc ggtgtgtgct gtgcgagttc
601 gacaccacgg gcgtcggctc gcaccgggaa cr~Agcgcgg tcttcggtga tctgcgccgc
661 atgggcgPrc ttcatcgccg ctacccgttc ggggga~ggc gaatatcaca tgcctaccta
721Cyc~Jy~,c~yy agttctacgc ctacaacagt cgattctggg aaaacgtctt cacgrgAAtg
25781 tcgaaatag
Seq.IDNo.31
1 MTSAPTVSVITISFND L DG LQ RTVKSVRAQ
31 RYRGRIEHIVIDGGSGDDVVAYLSGCEPGF
61 AYW Q SEPDGGRYDAMN Q GIAHASGD LLWF L
3091 HSADRFSGPDVVA Q AVEA L SGRGPVSELWG
121 FGMDR LV G L DRVRGPIP FSL RKF L AGK QVV
151 PHQASFFGSSLVAKIGGYD L DFGIAADQEF
181 ILRAA LVC EPVTIRCV L CEFDTTGVGSHRE
211 PSAVFGDLRRMGD L HRRYPFGGRRISHAYL
35 241 RGREFYAYNSRFWENVFTRMSK
CA 02241023 1998-06-19
W O 97~3624 PCT/~'~G~03221
-54 -
Seq. ID No.32
1 gtgaagcgag cgctcateac cggaatcace ggecaggacg gctcgtatet egccgaaetg
61 etgetggeca aggggtatga ggetcacggg etcatccggc gcgcttegac gttcaaeacc
121 tcgcggatcg atcacctcta cgtcgacccg caccaaccgg ycg~yeyy~t gtttctgcac
181 tatggtgacc tgatcgacgg aacccggttg gtgaccctgc tgagcaccat cgaacccgae
241 gaggtgtaea aeetggcgge gcagtcacac gtgcgggtga gcttcgacga acccgtgcac
301 accggtgaca ccaccggeat gggateeatg egaetgetgg aagcegttcg gctctctcgg
361 gtgcactgcc gcttctatca ggcgtcctcg tcggagatgt tCgy~y~LC gecgccaeeg
421 eagAAcgagc tgaegeegtt etaeecgegg teacegtatg ycg~egccaa ggtctattcg
481 tactgggcga cccgcaatta tegcgaagcg tacggattgt tcgccgttaa cggcatcttg
541 ttcaatcacg aatcaccgcg gcgcggtgag acgttcgtga ceegA,AAg~t caeeagggee
601 gtggcacgca teaaggeegg tatceagtcc gaggtetata tgggeaatet ggatgeggte
661 cgcgaetggg ggtacgegee cgaatacgtc gaAggcatgt ggcggatget geagaeegac
721 gagceegacg aettcgtttt ggcgaceggg egeggtttca eegtgegtga gttegegegg
781 geegegtteg ageatgeegg tttggaetgg eageagtaeg tgaaattega ceaaegetat
841 ctgcggeeea eegaggtgga ttegctgate ggcgaegcga ccaaggetgc egaattgctg
901 ggctggaggg cttcggtgca caetgaegag ttggetcgga teatggtcga egcggaeatg
961 g~yy-~y~Lgg agtgegaagg caagccgtgg atcgacaagc cgatgategc cyyCcyy ca
1021 tga
Seq. ID No.33
1 M K R A L I T G I T G Q D G S Y L A E L L L A ~ G Y E V H G
31 L I R R A S T F N T S R I D H L Y V D P H Q P G A R L F L H
61 Y G D L I D G T R L V T L L S T I E P D E V Y N L A A Q S H
91 V R V S F D E P V H T G D T T G M G S M R L L E A V R L S R
121 V H C R F Y Q A S S S E M F G A S P P P Q N E L T P F Y P R
151 S P Y G A A ~ V Y S Y W A T R N Y R E A Y G L F A V N G I L
181 F N H E S P R R G E T F V T R ~ I T R A V A R I K A G I Q S
211 E V Y M G N L D A V R D W G Y A P E Y V E G M W R M L Q T D
241 E P D D F V L A T G R G F T V R E F A R A A F E H A G L D W
271 Q Q Y V ~ F D Q R Y L R P T E V D S L I G D A T K A A E L L
301 G W R A S V H T D E L A R I M V D A D M A A L E C E G ~ P W
331 I D K P M I A G R T
Seq. ID No.34
1 atgaggetgg cccgtegcge tcggaacatc ttgcgtcgca acggeatega ggtgtcgegc
61 tactttgceg aactggactg ggaAcgcaat ttcttgcgcc aactgcaatc gcatcgggtc
121 agtgccgtgc tcgatgtcgg ggccaattcg gggeagtacg ccaggggtet g~ycyy~yc~
181 ggettcgegg gcegcatcgt ctcgttcgag ccgctgeecg ggccctttge egtettgeag
241 egcagegect ccacggaeee gttgtgggaa ty~cyycyct gtgcgctggg cgatgtcgat
301 gg~Aecatct cgatcaacgt cy.cyy.a~c gagggcgcca gcagttccgt cttgccgatg
361 ttg~acg~c atcaggacgc etttecaeca gceaaetacg tgggcgceea acgggtgeeg
421 ataeategac tcgattccgt ggctgcagac gttctgcggc ccaacgatat tgcgttcttg
401 aagatcgacg tteaaggatt egagaagcag gtgatcgcgg gtggegattc aaeggtgeae
541 gaeegatgcg teggcatgea getcgagctg tetttccagc cgttgtacga gggtggeatg
601 cteatccgeg aggegctcga tetcgtggat tcgttgggct ttaegctctc gggattgcaa
661 cccggtttca ccgacccccg eaaeggtcga atgetgeagg ccgatggcat cttetteegg
721 ggcagcgatt ga
CA 02241023 1998-06-19
W O 97n3624 PCT/G B96/03221
Seq.IDNo.35
1 M R L A R RARNILRRNGIEVSRYFAELDWERN
31 F L R Q LQSHRVS A V LDVGANSGQYARGLRGA
61 GFAGRIVSFEPLPGPFAVLQRSASTDP L W E
91 C R R CALGDVDGTISINVAGNEGASSSV L P M
121 L K R H Q DAFPPANYVGAQRVPIHR L DSVAAD
151 VLRPNDI A F L R I D V Q GFE K Q V I A GGDSTVH
181 DRCVG M Q L E L S FQPLYEGG M LIREA L D L V D
211 SLGFTLSGLQPGFTDPRNGR M L Q A DGIFFR
241 GS D
Seq.IDNo.36
1 gtgaaatcgt tgaaactcgc tcgtttcatc gcgcgtagcg ccgccttcga ggtttcgcgc
61 cgctattctg agcgagacct gaagcaccag tttgtgaagc aactcaaatc gcgtcgggta
121 gatgtcgttt tcgatgtcgg cgccaactca ggacaatacg ccgccggcct ccgccgagca
181 gcataeaagg gccgcattgt ctcgttcgaa ccgctatccg gaccgtttac gatcttggaa
241 agCA~g-J~ caacggatcc actttgggat egccggcagc atgcgttggg cgattctgat
301 ggAAcggtta cgatcaatat cg~agg~Ac gccggtcaga gcagttccgt cttgcccatg
361 ctgaaaagtc atcagaacgc ttttcccccg gcaaactatg tcggtaccca agaggcgtcc
421 atacatcgac ttgattccgt ggcgccagaa tttctaggca tgaacggtgt cgcttttctc
481 aaggtcgacg ttcaaggctt tgAA~AgcAg gtgctcgccg ggggrAAAtc aaccatagat
541 gaccattgcg tcggcatgca actcgaactg tccttcctgc cgttgtacga aggtggcatg
601 ctcattcctg aagccctcga tctcgtgtat tccttgggct tcacgttgac gggattgctg
661 ccttgtttca ttgatgcaaa taatggtcga atgttgcagg ccgacggcat ctttttccgc
721 gaggacgatt ga
Seq.IDNo.37
1 M K S L K LARFIARSAAFEVSRRYSERDL K HQ
31 FV K QLRSRRVDVVFDFTVGANSGQYAAG L R
61 RAAY K G RIVSFEPLS G PFTILES K ASTDP L
91 WDCRQHALGDSDGTVTINIA G NAG Q S S S V L
121 PML K S H Q NAFPPANYVGTQEASIHRLDSVA
151 PEFLG M N GV A F L K V DV Q GFEK Q V LAGG K S T
181 I D DHCVG M Q LE L S F LPLYEGG M L I PE A LD L
211 VYS L GFTLTG L L P C F I DANNGR M L Q A DGIF
241 FRED D
CA 0224l023 l998-06-l9
W O g7/23624 PCT/~B96/0322
-56 -
Seq. ID No.38
1 atggtgcaga cgaaacgata cy~cgy~ttg accgcagcta acacaaagaa agtcgccatg
61 gccgcaccaa tgttttcgat catcatcccc accttgaacg tggctgcggt attgcctgcc
121 tgcctcgaca gcatcgcccg tcagacctgc ggtgactccg agctggtact ggtcgacggc
181 ggctcgacgg acgaaaccct cgacatcgcc aacattttcg cccccaacct cggcgagcgg
241 ttgatcattc atcgcgacac cg?ccagggc gtctacgacg ccatgaaccg cggcgtggac
301 ctggccaccg gaacgtggtt gctctttctg gyLy~yy~cg acagcctgta cgaggctgac
361 accctggcgc gggtggccgc cttcattggc g~R~c~cgRgc ccagcgatct ggtatatggc
421 gacgtgatca tgcgctcaac caatttccgc tggggtggcg ccttcgacct cgaccgtctg
481 ttgttcaagc gcaacatctg ccatcaggcg atcttctacc gccgcggact cttcggcacc
541 atcggtccct acaacctccg ctaccgggtc ctggccgact gggacttcaa tattcgctgc
601 ttttccaacc cagcgctcgt cacccgctac atgcacgtgg tcgttgcaag c~Rc~Rcg~R
661 tt~yycyyy. tcagcaatac gatcgtcgac aaggagtttt tg~gcggct gccgatgtcc
721 acgagactcg gcataaggct ggtcatagtt ctggtgcgca ggtggccaaa ggtgatcagc
781 agggccatgg taatgcgcac cgtcatttct tyycyg~gcc gacgttag
Seq. ID No.39
1 M V Q T K R Y A G L T A A N T K K V A M A A P M F S I I I P
31 T L N V A A V L P A C L D S I A R Q T C G D F E L V L V D G
61 G S T D E T L D I A N I F A P N L G E R L I I N R D T D Q G
91 V Y D A M N R G V D L A T G T W L L F L G A D D S L Y E A D
121 T L A R V A A F I G E H E P S D L V Y G D V I M R S T N F R
lSl W G G A F D L D R L L F K R N I C H Q A I F Y R R G L F G T
lBl I G P Y N L R Y R V L A D W D F N I R C F S N P A L V T R Y
211 M H V V V A S Y N E F G G L S N T I V D K E F L K R L P M S
241 T R L G I R L V I V L V R R W P K V I S R A M V M R T V I S
271 W R R R R
Seq 40:
GAl~CC~l-~AGGAGGTAAAGCTGC
Seq 41:
GATACGGCTCTTGAATCCTGCACG
