Note: Descriptions are shown in the official language in which they were submitted.
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BASB055 POLYNUCLEOTIDE AND POLYPEPTIDE FROM NEISSERIA MENINGITIDS. USES
THEREOF
FIELD OF THE INVENTION
This invention relates to polynucleotides, (herein referred to as "BASBO55
polynucleotide(s)"), polypeptides encoded by them (referred to herein as
"BASBO55" or
"BASBO55 polypeptide(s)"), recombinant materials and methods for their
production. In
another aspect, the invention relates to methods for using such polypeptides
and
polynucleotides, including vaccines against bacterial infections. In a further
aspect, the
invention relates to diagnostic assays for detecting infection of certain
pathogens.
BACKGROUND OF THE INVENTION
Neisseria meningitides (meningococcus) is a Gram-negative bacterium frequently
isolated
from the human upper respiratory tract. It occasionally causes invasive
bacterial diseases
such as bacteremia and meningitis. The incidence of meningococcal disease
shows
geographical seasonal and annual differences (Schwartz, B., Moore, P.S.,
Broome, C.V.;
Clin. Microbiol. Rev. 2 (Supplement), S 18-S24, 1989). Most disease in
temperate countries
is due to strains of serogroup B and varies in incidence from 1-
10/100,000/year total
population sometimes reaching higher values (Kaczmarski, E.B. (1997), Commun.
Dis.
Rep. Rev. 7: R55-9, 1995; Scholten, R.J.P.M., Bijlmer, H.A., Poolman, J.T. et
al. Clin.
Infect. Dis. 16: 237-246, 1993; Cruz, C., Pavez, G., Aguilar, E., et al.
Epidemiol. Infect.
105: 119-126, 1990).
Epidemics dominated by serogroup A meningococci, mostly in central Africa, are
encountered, sometimes reaching levels up to 1000/100.000/year (Schwartz, B.,
Moore,
P.S., Broome, C.V. Clin. Microbiol. Rev. 2 (Supplement), S18-524, 1989).
Nearly all cases
as a whole of meningococcal disease are caused by serogroup A, B, C, W-135 and
Y
meningococci and a tetravalent A, C, W-135, Y polysaccharide vaccine is
available
(Armand, J., Arminjon, F., Mynard, M.C., Lafaix, C., J. Biol. Stand. 10: 335-
339, 1982).
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The polysaccharide vaccines are currently being improved by way of chemical
conjugating
them to Garner proteins (Lieberman, J.M., Chiu, S.S., Wong, V.K., et al. JAMA
275 : 1499-
1503, 1996).
A serogroup B vaccine is not available, since the B capsular polysaccharide
was found to be
nonimmunogenic, most likely because it shares structural similarity to host
components
(Wyle, F.A., Artenstein, M.S., Brandt, M.L. et al. J. Infect. Dis. 126: 514-
522, 1972; Finne,
J.M., Leinonen, M., Makela, P.M. Lancet ii.: 35~-357, 1983).
For many years efforts have been initiated and carried out to develop
meningococcal outer
membrane based vaccines (de Moraes, J.C., Perkins, B., Cam«rgo, M.C. et al.
Lancet 340:
1074-1078, 1992; Bjune, G., Hoiby, E.A. Gronnesby, J.K. et al. 338: 1093-1096,
1991).
Such vaccines have demonstrated efficacies from 57% - 85% in older children
(>4 years)
and adolescents.
Many bacterial outer membrane components are present in these vaccines, such
as PorA,
PorB, Rmp, Opc, Opa, FrpB and the contribution of these components to the
observed
protection still needs father definition. Other bacterial outer membrane
components have
been defined by using animal or human antibodies to be potentially relevant to
the induction
of protective immunity, such as TbpB and NspA (Martin, D., Cadieux, N., Hamel,
J.,
Brodeux, B.R., J. Exp. Med. 185: 1173-1183, 1997; Lissolo, L., Maitre-
Wilmotte, C.,
Dumas, p. et al., Inf. Immun. 63: 884-890, 1995). The mechanisms of protective
immunity
will involve antibody mediated bactericidal activity and opsonophagocytosis.
A bacteremia animal model has been used to combine all antibody mediated
mechanisms
(Saukkonen, K., Leinonen, M., Abdillahi, H. Poolman, J. T. Vaccine 7: 325-328,
1989). It is
generally accepted that the late complement component mediated bactericidal
mechanism is
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crucial for immunity against meningococcal disease (Ross, S.C., Rosenthal
P.J., Berberic,
H.M., Densen, P. J. Infect. Dis. 155: 1266-1275, 1987).
The frequency of Neisseria meningitides infections has risen dramatically in
the past few
~ decades. This has been attributed to the emergence of multiply antibiotic
resistant strains
and an increasing population of people with weakened immune systems. It is no
longer
uncommon to isolate Neisseria meningitides strains that are resistant to some
or all of the
standard antibiotics. This phenomenon has created an unmet medical need and
demand for
new anti-microbial agents, vaccines, drug screening methods, and diagnostic
tests for this
organism.
SUMMARY OF THE INVENTION
The present invention relates to BASBO55, in particular BASBOS~ polypeptides
and
BASBO55 polynucleotides, recombinant materials and methods for their
production. In
another aspect, the invention relates to methods for using such polypeptides
and
polynucleotides, including prevention and treatment of microbial diseases,
amongst others.
In a further aspect, the invention relates to diagnostic assays for detecting
diseases
associated with microbial infections and conditions associated with such
infections, such
as assays for detecting expression or activity of BASBOS~ polynucleotides or
polypeptides.
Various changes and modifications within the spirit and scope of the disclosed
invention
will become readily apparent to those skilled in the art from reading the
following
descriptions and from reading the other parts of the present disclosure.
DESCRIPTION OF THE INVENTION
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The invention relates to BASBO55 polypeptides and polynucleotides as described
in greater
detail below. In particular, the invention relates to polypeptides and
polynucleotides of
BASBO55 of Neisseria meningitides, which is related by amino acid sequence
homology to
Neisseria gonorrhoeae MtrC protein. The invention relates especially to
BASBO55
having the nucleotide and amino acid sequences set out in SEQ ID NO: l and SEQ
ID N0:2
respectively. It is understood that sequences recited in the Sequence Listing
below as
"DNA" represent an exemplification of one embodiment of the invention, since
those of
ordinary skill will recognize that such sequences can be usefully employed in
polynucleotides in general, including ribopolynucleotides.
Potypeptides
In one aspect of the invention there are provided polypeptides of Neisseria
meningitides
referred to herein as "BASBO55" and "BASBOS~ polypeptides" as well as
biologically,
diagnostically, prophylactically, clinically or therapeutically useful
variants thereof, and
compositions comprising the same.
The present invention further provides for:
(a) an isolated polypeptide which comprises an amino acid sequence which has
at least
85% identity, more preferably at least 90% identity, yet more preferably at
least 95%
identity, most preferably at least 97-99% or exact identity, to that of SEQ ID
N0:2;
(b) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence which has at least 85% identity, more preferably at least 90%
identity, yet more
preferably at least 95% identity, even more preferably at least 97-99% or
exact identity to
SEQ ID NO:1 over the entire length of SEQ ID NO:1; or
(c) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence encoding a polypeptide which has at least 85% identity, more
preferably at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity, to the amino acid sequence of SEQ ID N0:2.
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The BASBO55 polypeptide provided in SEQ ID N0:2 is the BASBO55 polypeptide
from
Neisseria meningitides strains ATCC 13090.
The invention also provides an immunogenic fragment of a BASBO55 polypeptide,
that
S is, a contiguous portion of the BASBO55 polypeptide which has the same or
substantially
the same immunogenic activity as the polypeptide comprising the amino acid
sequence of
SEQ ID N0:2. That is to say, the fragment (if necessary when coupled to a
carrier) is
capable of raising an immune response which recognises the BASBO55
polypeptide.
Such an immunogenic fragment may include, for example, the BASBO55 polypeptide
lacking an N-terminal leader sequence, and/or a transmembrane domain and/or a
C-
terminal anchor domain. In a preferred aspect the immunogenic fragment of
BASBOS~
according to the invention comprises substantially all of the extracellular
domain of a
polypeptide which has at least 85% identity, more preferably at least 90%
identity, yet
more preferably at least 95% identity, most preferably at least 97-99%
identity, to that
of SEQ ID N0:2 over the entire length of SEQ ID N0:2.
A fragment is a polypeptide having an amino acid sequence that is entirely the
same as part
but not all of any amino acid sequence of any polypeptide of the invention. As
with
BASBO55 polypeptides, fragments may be "free-standing," or comprised within a
larger
polypeptide of which they form a part or region, most preferably as a single
continuous
region in a single larger polypeptide.
Preferred fragments include, for example, truncation polypeptides having a
portion of an
amino acid sequence of SEQ ID N0:2 or of variants thereof, such as a
continuous series of
2~ residues that includes an amino- and/or carboxyl-terminal amino acid
sequence.
Degradation forms of the polypeptides of the invention produced by or in a
host cell, are
also preferred. Further preferred are fragments characterized by structural or
functional
attributes such as fragments that comprise alpha-helix and alpha-helix forming
regions,
beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil
and coil-
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forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic
regions, beta
amphipathic regions, flexible regions, surface-forming regions, substrate
binding region,
and high antigenic index regions.
Further preferred fragments include an isolated polypeptide comprising an
amino acid
sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from
the amino
acid sequence of SEQ ID N0:2, or an isolated polypeptide comprising an amino
acid
sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids
truncated or
deleted from the amino acid sequence of SEQ ID N0:2.
Fragments of the polypeptides of the invention may be employed for producing
the
corresponding full-length polypeptide by peptide synthesis; therefore, these
fragments
may be employed as intermediates for producing the full-length polypeptides of
the
invention.
Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1
amino acids
are substituted, deleted, or added in any combination.
The polypeptides, or immunogenic fragments, of the invention may be in the
form of
the "mature" protein or may be a part of a larger protein such as a precursor
or a fusion
protein. It is often advantageous to include an additional amino acid sequence
which
contains secretory or leader sequences, pro-sequences, sequences which aid in
purification such as multiple histidine residues, or an additional sequence
for stability
during recombinant production. Furthermore, addition of exogenous polypeptide
or
lipid tail or polynucleotide sequences to increase the immunogenic potential
of the final
molecule is also considered.
In one aspect, the invention relates to genetically engineered soluble fusion
proteins
comprising a polypeptide of the present invention, or a fragment thereof, and
various
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portions of the constant regions of heavy or light chains of immunoglobulins
of various
subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the
constant part of
the heavy chain of human IgG, particularly IgGI, where fusion takes place at
the hinge
region. In a particular embodiment, the Fc part can be removed simply by
incorporation
of a cleavage sequence which can be cleaved with blood clotting factor Xa.
Furthermore, this invention relates to processes for the preparation of these
fusion
proteins by genetic engineering, and to the use thereof for drug screening,
diagnosis and
therapy. A further aspect of the invention also relates to polynucleotides
encoding such
fusion proteins. Examples of fusion protein technology can be found in
International
Patent Application Nos. W094/29458 and W094/22914.
The proteins may be chemically conjugated, or expressed as recombinant fusion
proteins allowing increased levels to be produced in an expression system as
compared
to non-fused protein. The fusion partner may assist in providing T helper
epitopes
(immunological fusion partner), preferably T helper epitopes recognised by
humans, or
assist in expressing the protein (expression enhancer) at higher yields than
the native
recombinant protein. Preferably the fusion partner will be both an
immunological
fusion partner and expression enhancing partner.
Fusion partners include protein D from Haemophilus influenzae and the non-
structural
protein from influenzae virus, NS 1 (hemagglutinin). Another fusion partner is
the
protein known as LytA. Preferably the C terminal portion of the molecule is
used.
LytA is derived from Streptococcus pneumoniae which synthesize an N-acetyl-L-
alanine amidase, amidase LytA, (coded by the lytA gene {Gene, 43 (1986) page
265-
272}) an autolysin that specifically degrades certain bonds in the
peptidoglycan
backbone. The C-terminal domain of the LytA protein is responsible for the
affinity to
the choline or to some choline analogues such as DEAE. This property has been
exploited for the development of E.coli C-LytA expressing plasmids useful for
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expression of fusion proteins. Purification of hybrid proteins containing the
C-LytA
fragment at its amino terminus has been described {Biotechnology: 10, (1992)
page
795-798 } . It is possible to use the repeat portion of the LytA molecule
found in the C
terminal end starting at residue 178, for example residues 188 - 305.
The present invention also includes variants of the aforementioned
polypeptides, that is
polypeptides that vary from the referents by conservative amino acid
substitutions,
whereby a residue is substituted by another with like characteristics. Typical
such
substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the
acidic
residues Asp and Glu; among Asn and Gln; and among the basic residues Lys and
Arg; or
aromatic residues Phe and Tyr.
Polypeptides of the present invention can be prepared in any suitable manner.
Such
polypeptides include isolated naturally occurnng polypeptides, recombinantly
produced
polypeptides, synthetically produced polypeptides, or polypeptides produced by
a
combination of these methods. Means for preparing such polypeptides are well
understood in the art.
It is most preferred that a polypeptide of the invention is derived from
Neisseria
meningitides, however, it may preferably be obtained from other organisms of
the same
taxonomic genus. A polypeptide of the invention may also be obtained, for
example, from
organisms of the same taxonomic family or order.
Polynucleotides
It is an object of the invention to provide polynucleotide that encode BASBO55
polypeptide,
particularly polynucleotides that encode the polypeptide herein designated
BASBO55.
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In a particularly preferred embodiment of the invention the polynucleotide
comprises a
region encoding BASBO55 polypeptide comprising a sequence set out in SEQ ID
NO:1
which includes a full length gene, or a variant thereof.
The BASBO55 polynucleotide provided in SEQ ID NO:1 is the BASBOS~
polynucleotide from Neisseria meningitides strains ATCC 13090.
As a further aspect of the invention there are provided isolated nucleic acid
molecules
encoding and/or expressing BASBO55 polypeptides and polynucleotides,
particularly
Neisseria meningitides BASBO55 polypeptides and polynucleotides, including,
for
example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B-
and Z-DNAs. Further embodiments of the invention include biologically,
diagnostically, prophylactically, clinically or therapeutically useful
polynucleotides and
polypeptides, and variants thereof, and compositions comprising the same.
Another aspect of the invention relates to isolated polynucleotides, including
at least one full
length gene, that encodes a BASBO55 polypeptide having a deduced amino acid
sequence of
SEQ ID N0:2 and polynucleotides closely related thereto and variants thereof.
In another particularly preferred embodiment of the invention there is a
BASBOS~
polypeptide from Neisseria meningitides comprising or consisting of an amino
acid
sequence of SEQ ID N0:2 or a variant thereof.
Using the information provided herein, such as a polynucleotide sequence set
out in SEQ ID
NO:1 a polynucleotide of the invention encoding BASB0~5 polypeptide may be
obtained
using standard cloning and screening methods, such as those for cloning and
sequencing
chromosomal DNA fragments from bacteria using Neisseria meningitides cells as
starting
material, followed by obtaining a full length clone. For example, to obtain a
polynucleotide
sequence of the invention, such as a polynucleotide sequence given in SEQ ID
NO:1
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typically a library of clones of chromosomal DNA of Neisseria meningitides in
E. coli or
some other suitable host is probed with a radiolabeled oligonucleotide,
preferably a 17-
mer or longer, derived from a partial sequence. Clones carrying DNA identical
to that of
the probe can then be distinguished using stringent hybridization conditions.
By
sequencing the individual clones thus identified by hybridization with
sequencing primers
designed from the original polypeptide or polynucleotide sequence it is then
possible to
extend the polynucleotide sequence in both directions to determine a full
length gene
sequence. Conveniently, such sequencing is performed, for example, using
denatured
double stranded DNA prepared from a plasmid clone. Suitable techniques are
described
by Maniatis, T., Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A
LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York (1989). (see in particular Screening By Hybridization 1.90
and
Sequencing Denatured Double-Stranded DNA Templates 13.70). Direct genomic DNA
sequencing may also be performed to obtain a full length gene sequence.
Illustrative of
the invention, each polynucleotide set out in SEQ ID NO:1 was discovered in a
DNA
library derived from Neisseria meningitides.
Moreover, each DNA sequence set out in SEQ ID NO:1 contains an open reading
frame
encoding a protein having about the number of amino acid residues set forth in
SEQ ID
N0:2 with a deduced molecular weight that can be calculated using amino acid
residue
molecular weight values well known to those skilled in the art.
The polynucleotide of SEQ ID NO:1, between the start codon at nucleotide
number 1 and
the stop codon which begins at nucleotide number 1237 of SEQ ID NO:1, encodes
the
polypeptide of SEQ ID N0:2.
In a further aspect, the present invention provides for an isolated
polynucleotide
comprising or consisting of
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(a) a polynucleotide sequence which has at least 85% identity, more preferably
at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least
97-99% or exact identity to SEQ ID NO:1 over the entire length of SEQ ID NO:I;
or
S (b) a polynucleotide sequence encoding a polypeptide which has at least 85%
identity,
more preferably at least 90% identity, yet more preferably at least 95%
identity, even
more preferably at least 97-99% or 100% exact, to the amino acid sequence of
SEQ ID
N0:2 over the entire length of SEQ ID N0:2.
A polynucleotide encoding a polypeptide of the present invention, including
homologs and
orthologs from species other than Neisseria meningitides, may be obtained by a
process
which comprises the steps of screening an appropriate library under stringent
hybridization
conditions (for example, using a temperature in the range of 45 - 65°C
and an SDS
concentration from 0.1-1%) with a labeled or detectable probe consisting of or
comprising
the sequence of SEQ ID NO: l or a fragment thereof; and isolating a full-
length gene and/or
genomic clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire
length to a coding
sequence (open reading frame) in SEQ ID NO:1. Also provided by the invention
is a coding
sequence for a mature polypeptide or a fragment thereof, by itself as well as
a coding
sequence for a mature polypeptide or a fragment in reading frame with another
coding
sequence, such as a sequence encoding a leader or secretory sequence, a pre-,
or pro- or
prepro-protein sequence. The polynucleotide of the invention may also contain
at least one
non-coding sequence, including for example, but not limited to at least one
non-coding 5'
and 3' sequence, such as the transcribed but non-translated sequences,
termination signals
(such as rho-dependent and rho-independent termination signals), ribosome
binding sites,
Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation
signals.
The polynucleotide sequence may also comprise additional coding sequence
encoding
additional amino acids. For example, a marker sequence that facilitates
purification of the
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fused polypeptide can be encoded. In certain embodiments of the invention, the
marker
sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen,
Inc.) and
described in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or
an HA peptide
tag (Wilson et al., Cell 37: 767 (1984), both of which may be useful in
purifying
polypeptide sequence fused to them. Polynucleotides of the invention also
include, but are
not limited to, polynucleotides comprising a structural gene and its naturally
associated
sequences that control gene expression.
The nucleotide sequence encoding BASBO55 polypeptide of SEQ ID N0:2 may be
identical to the polypeptide encoding sequence contained in nucleotides 1 to
1236 of SEQ
ID NO:1. Alternatively it may be a sequence, which as a result of the
redundancy
(degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID N0:2.
The term "polynucleotide encoding a polypeptide" as used herein encompasses
polynucleotides that include a sequence encoding a polypeptide of the
invention,
particularly a bacterial polypeptide and more particularly a polypeptide of
the Neisseria
meningitidis BASBO55 having an amino acid sequence set out in SEQ ID N0:2. The
term
also encompasses polynucleotides that include a single continuous region or
discontinuous
regions encoding the polypeptide (for example, polynucleotides interrupted by
integrated
phage, an integrated insertion sequence, an integrated vector sequence, an
integrated
transposon sequence, or due to RNA editing or genomic DNA reorganization)
together with
additional regions, that also may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described
herein that encode
variants of a polypeptide having a deduced amino acid sequence of SEQ ID N0:2.
Fragments of polynucleotides of the invention may be used, for example, to
synthesize full-
length polynucleotides of the invention.
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Further particularly preferred embodiments are polynucleotides encoding
BASBO55
variants, that have the amino acid sequence of BASBO55 polypeptide of SEQ ID
N0:2 in
which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues
are substituted,
modified, deleted and/or added, in any combination. Especially preferred among
these are
silent substitutions, additions and deletions, that do not alter the
properties and activities of
BASBO55 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at
least 85%
identical over their entire length to a polynucleotide encoding BASBO55
polypeptide having
an amino acid sequence set out in SEQ ID N0:2, and polynucleotides that are
complementary to such polynucleotides. In this regard, polynucleotides at
least 90%
identical over their entire length to the same are particularly preferred, and
among these
particularly preferred polynucleotides, those with at least 95% are especially
preferred.
Furthermore, those with at least 97% are highly preferred among those with at
least 95%,
and among these those with at least 98% and at least 99% are particularly
highly preferred,
with at least 99% being the more preferred.
Preferred embodiments are polynucleotides encoding polypeptides that retain
substantially
the same biological function or activity as the mature polypeptide encoded by
a DNA of
SEQ ID NO:1.
In accordance with certain preferred embodiments of this invention there are
provided
polynucleotides that hybridize, particularly under stringent conditions, to
BASBO55
polynucleotide sequences, such as those polynucleotides in SEQ ID NO:1.
The invention further relates to polynucleotides that hybridize to the
polynucleotide
sequences provided herein. In this regard, the invention especially relates to
polynucleotides that hybridize under stringent conditions to the
polynucleotides described
herein. As herein used, the terms "stringent conditions" and "stringent
hybridization
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conditions" mean hybridization occurring only if there is at least 95% and
preferably at least
97% identity between the sequences. A specific example of stringent
hybridization
conditions is overnight incubation at 42°C in a solution comprising:
50% formamide, Sx
SSC (150mM NaCI, lSmM trisodium citrate), 50 mM sodium phosphate (pH7.6), Sx
Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured,
sheared
salmon sperm DNA, followed by washing the hybridization support in O.lx SSC at
about
65°C. Hybridization and wash conditions are well known and exemplified
in Sambrook,
et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor,
N.Y., (1989), particularly Chapter 11 therein. Solution hybridization may also
be used
with the polynucleotide sequences provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a
polynucleotide
sequence obtained by screening an appropriate library containing the complete
gene for a
polynucleotide sequence set forth in SEQ ID NO: l under stringent
hybridization
1 S conditions with a probe having the sequence of said polynucleotide
sequence set forth in
SEQ ID NO: l or a fragment thereof; and isolating said polynucleotide
sequence.
Fragments useful for obtaining such a polynucleotide include, for example,
probes and
primers fully described elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the
invention, for
instance, the polynucleotides of the invention, may be used as a hybridization
probe for
RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones
encoding
BASBO55 and to isolate cDNA and genomic clones of other genes that have a high
identity,
particularly high sequence identity, to the BASBO55 gene. Such probes
generally will
comprise at least 15 nucleotide residues or base pairs. Preferably, such
probes will have at
least 30 nucleotide residues or base pairs and may have at least 50 nucleotide
residues or
base pairs. Particularly preferred probes will have at least 20 nucleotide
residues or base
pairs and will have less than 30 nucleotide residues or base pairs.
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A coding region of a BASBO55 gene may be isolated by screening using a DNA
sequence
provided in SEQ ID NO:1 to synthesize an oligonucleotide probe. A labeled
oligonucleotide having a sequence complementary to that of a gene of the
invention is then
used to screen a library of cDNA, genomic DNA or mRNA to determine which
members of
the library the probe hybridizes to.
There are several methods available and well known to those skilled in the art
to obtain
full-length DNAs, or extend short DNAs, for example those based on the method
of
Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al.,
PNAS
USA 85: 8998-9002, 1988). Recent modifications of the technique, exemplified
by the
MarathonTM technology (Clontech Laboratories Inc.) for example, have
significantly
simplified the search for longer cDNAs. In the MarathonTM technology, cDNAs
have
been prepared from mRNA extracted from a chosen tissue and an 'adaptor'
sequence
ligated onto each end. Nucleic acid amplification (PCR) is then carried out to
amplify the
"missing" 5' end of the DNA using a combination of gene specific and adaptor
specific
oligonucleotide primers. The PCR reaction is then repeated using "nested"
primers, that
is, primers designed to anneal within the amplified product (typically an
adaptor specific
primer that anneals further 3' in the adaptor sequence and a gene specific
primer that
anneals further 5' in the selected gene sequence). The products of this
reaction can then
be analyzed by DNA sequencing and a full-length DNA constructed either by
joining the
product directly to the existing DNA to give a complete sequence, or carrying
out a
separate full-length PCR using the new sequence information for the design of
the 5'
primer.
The polynucleotides and polypeptides of the invention may be employed, for
example, as
research reagents and materials for discovery of treatments of and diagnostics
for diseases,
particularly human diseases, as further discussed herein relating to
polynucleotide assays.
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The polynucleotides of the invention that are oligonucleotides derived from a
sequence of
SEQ ID NOS:1 - 2 may be used in the processes herein as described, but
preferably for
PCR, to determine whether or not the polynucleotides identified herein in
whole or in part
are transcribed in bacteria in infected tissue. It is recognized that such
sequences will also
have utility in diagnosis of the stage of infection and type of infection the
pathogen has
attained.
The invention also provides polynucleotides that encode a polypeptide that is
the mature
protein plus additional amino or carboxyl-terminal amino acids, or amino acids
interior to
the mature polypeptide (when the mature form has more than one polypeptide
chain, for
instance). Such sequences may play a role in processing of a protein from
precursor to a
mature form, may allow protein transport, may lengthen or shorten protein half
life or may
facilitate manipulation of a protein for assay or production, among other
things. As
generally is the case in vivo, the additional amino acids may be processed
away from the
mature protein by cellular enzymes.
For each and every polynucleotide of the invention there is provided a
polynucleotide
complementary to it. It is preferred that these complementary polynucleotides
are fully
complementary to each polynucleotide with which they are complementary.
A precursor protein, having a mature form of the polypeptide fused to one or
more
prosequences may be an inactive form of the polypeptide. When prosequences are
removed
such inactive precursors generally are activated. Some or all of the
prosequences may be
removed before activation. Generally, such precursors are called proproteins.
In addition to the standard A, G, C, TlCT representations for nucleotides, the
term "N" may
also be used in describing certain polynucleotides of the invention. "N" means
that any of
the four DNA or RNA nucleotides may appear at such a designated position in
the DNA
or RNA sequence, except it is preferred that N is not a nucleic acid that when
taken in
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WO 00/43517 PCT/EP00/00425
combination with adjacent nucleotide positions, when read in the correct
reading frame,
would have the effect of generating a premature termination codon in such
reading frame.
In sum, a polynucleotide of the invention may encode a mature protein, a
mature protein
plus a leader sequence (which may be referred to as a preprotein), a precursor
of a mature
protein having one or more prosequences that are not the leader sequences of a
preprotein,
or a preproprotein, which is a precursor to a proprotein, having a leader
sequence and one or
more prosequences, which generally are removed during processing steps that
produce
active and mature forms of the polypeptide.
In accordance with an aspect of the invention, there is provided the use of a
polynucleotide of the invention for therapeutic or prophylactic purposes, in
particular
genetic immunization.
The use of a polynucleotide of the invention in genetic immunization will
preferably
employ a suitable delivery method such as direct injection of plasmid DNA into
muscles
(Wolff et al., Hum Mol Genet (1992) 1: 363, Manthorpe et al., Hum. Gene Ther.
(1983) 4:
419), delivery of DNA complexed with specific protein carriers (Wu et al.,
JBiol Chem.
(1989) 264: 16985), coprecipitation of DNA with calcium phosphate (Benvenisty
&
Reshef, PNAS USA, (1986) 83: 9551), encapsulation of DNA in various forms of
liposomes (Kaneda et al., Science (1989) 243: 375), particle bombardment (Tang
et al.,
Nature (1992) 356:152, Eisenbraun et al., DNA Cell Biol (1993) 12: 791) and in
vivo
infection using cloned retroviral vectors (Seeger et al., PNAS USA (1984) 81:
5849).
Vectors, Host Cells, Expression Systems
The invention also relates to vectors that comprise a polynucleotide or
polynucleotides of
the invention, host cells that are genetically engineered with vectors of the
invention and the
production of polypeptides of the invention by recombinant techniques. Cell-
free
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translation systems can also be employed to produce such proteins using RNAs
derived
from the DNA constructs of the invention.
Recombinant polypeptides of the present invention may be prepared by processes
well
known in those skilled in the art from genetically engineered host cells
comprising
expression systems. Accordingly, in a further aspect, the present invention
relates to
expression systems that comprise a polynucleotide or polynucleotides of the
present
invention, to host cells which are genetically engineered with such expression
systems, and
to the production of polypeptides of the invention by recombinant techniques.
For recombinant production of the polypeptides of the invention, host cells
can be
genetically engineered to incorporate expression systems or portions thereof
or
polynucleotides of the invention. Introduction of a polynucleotide into the
host cell can be
effected by methods described in many standard laboratory manuals, such as
Davis, et al.,
BASIC METHODS IN MOLECULAR BIOLOGY, ( 1986) and Sambrook, et al.,
MOLECULAR CLONING: A LABORATORYMANUAL, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate
transfection, DEAF-dextran mediated transfection, transvection,
microinjection, cationic
lipid-mediated transfection, electroporation, transduction, scrape loading,
ballistic
introduction and infection.
Representative examples of appropriate hosts include bacterial cells, such as
cells of
streptococci, staphylococci, enterococci, E. coli, streptomyces,
cyanobacteria, Bacillus
subtilis, Moraxella catarrhalis, Haemophilus influenzae and Neisseria
meningitides; fungal
cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete,
Candida
albicans and Aspergillus; insect cells such as cells of Drosophila S2 and
Spodoptera Sue;
animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-l and Bowes
melanoma
cells; and plant cells, such as cells of a gymnosperm or angiosperm.
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A great variety of expression systems can be used to produce the polypeptides
of the
invention. Such vectors include, among others, chromosomal-, episomal- and
virus-derived
vectors, for example, vectors derived from bacterial plasmids, from
bacteriophage, from
transposons, from yeast episomes, from insertion elements, from yeast
chromosomal
elements, from viruses such as baculoviruses, papova viruses, such as SV40,
vaccinia
viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses,
retroviruses,
and alphaviruses and vectors derived from combinations thereof, such as those
derived from
plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The
expression system constructs may contain control regions that regulate as well
as engender
expression. Generally, any system or vector suitable to maintain, propagate or
express
polynucleotides and/or to express a polypeptide in a host may be used for
expression in this
regard. The appropriate DNA sequence may be inserted into the expression
system by any
of a variety of well-known and routine techniques, such as, for example, those
set forth in
Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, (supra).
In recombinant expression systems in eukaryotes, for secretion of a translated
protein into
the lumen of the endoplasmic reticulum, into the periplasmic space or into the
extracellular
environment, appropriate secretion signals may be incorporated into the
expressed
polypeptide. These signals may be endogenous to the polypeptide or they may be
heterologous signals.
Polypeptides of the present invention can be recovered and purified from
recombinant
cell cultures by well-known methods including ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange chromatography,
phosphocellulose
chromatography, hydrophobic interaction chromatography, affinity
chromatography,
hydroxylapatite chromatography and lectin chromatography. Most preferably, ion
metal
amity chromatography (IMAC) is employed for purification. Well known
techniques
for refolding proteins may be employed to regenerate active conformation when
the
polypeptide is denatured during intracellular synthesis, isolation and or
purification.
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The expression system may also be a recombinant live microorganism, such as a
virus
or bacterium. The gene of interest can be inserted into the genome of a live
recombinant
virus or bacterium. Inoculation and in vivo infection with this live vector
will lead to in
vivo expression of the antigen and induction of immune responses. Viruses and
bacteria
used for this purpose are for instance: poxviruses (e.g; vaccinia, fowlpox,
canarypox),
alphaviruses (Sindbis virus, Semliki Forest Virus, Venezuelian Equine
Encephalitis
Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus,
rhinovirus),
herpesviruses (varicella zoster virus, etc), Listeria, Salmonella , Shigella,
Neisseria,
BCG. These viruses and bacteria can be virulent, or attenuated in various ways
in order
to obtain live vaccines. Such live vaccines also form part of the invention.
Diagnostic, Prognostic, Serotyping and Mutation Assays
This invention is also related to the use of BASBO55 polynucleotides and
polypeptides of
the invention for use as diagnostic reagents. Detection of BASBO55
polynucleotides and/or
polypeptides in a eukaryote, particularly a mammal, and especially a human,
will provide a
diagnostic method for diagnosis of disease, staging of disease or response of
an infectious
organism to drugs. Eukaryotes, particularly mammals, and especially humans,
particularly
those infected or suspected to be infected with an organism comprising the
BASBO55 gene
or protein, may be detected at the nucleic acid or amino acid level by a
variety of well
known techniques as well as by methods provided herein.
Polypeptides and polynucleotides for prognosis, diagnosis or other analysis
may be obtained
from a putatively infected and/or infected individual's bodily materials.
Polynucleotides
from any of these sources, particularly DNA or RNA, may be used directly for
detection or
may be amplified enzymatically by using PCR or any other amplification
technique prior to
analysis. RNA, particularly mRNA, cDNA and genomic DNA may also be used in the
same ways. Using amplification, characterization of the species and strain of
infectious or
resident organism present in an individual, may be made by an analysis of the
genotype of a
CA 02360609 2001-07-20
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selected polynucleotide of the organism. Deletions and insertions can be
detected by a
change in size of the amplified product in comparison to a genotype of a
reference sequence
selected from a related organism, preferably a different species of the same
genus or a
different strain of the same species. Point mutations can be identified by
hybridizing
amplified DNA to labeled BASBO55 polynucleotide sequences. Perfectly or
significantly
matched sequences can be distinguished from imperfectly or more significantly
mismatched
duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by
detecting
differences in melting temperatures or renaturation kinetics. Polynucleotide
sequence
differences may also be detected by alterations in the electrophoretic
mobility of
polynucleotide fragments in gels as compared to a reference sequence. This may
be carned
out with or without denaturing agents. Polynucleotide differences may also be
detected by
direct DNA or RNA sequencing. See, for example, Myers et al., Science, 230:
1242 (1985).
Sequence changes at specific locations also may be revealed by nuclease
protection assays,
such as RNase, V 1 and S 1 protection assay or a chemical cleavage method.
See, for
example, Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
In another embodiment, an array of oligonucleotides probes comprising BASBO>j
nucleotide sequence or fragments thereof can be constructed to conduct
efficient screening
of, for example, genetic mutations, serotype, taxonomic classification or
identification.
Array technology methods are well known and have general applicability and can
be used to
address a variety of questions in molecular genetics including gene
expression, genetic
linkage, and genetic variability (see, for example, Chee et al., Science,
27.1: 610 (1996)).
Thus in another aspect, the present invention relates to a diagnostic kit
which comprises:
(a) a polynucleotide of the present invention, preferably the nucleotide
sequence of SEQ
ID NO:1, or a fragment thereof ;
(b) a nucleotide sequence complementary to that of (a);
(c) a polypeptide of the present invention, preferably the polypeptide of SEQ
ID N0:2 or
a fragment thereof; or
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(d) an antibody to a polypeptide of the present invention, preferably to the
polypeptide of
SEQ ID N0:2.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise
a substantial
component. Such a kit will be of use in diagnosing a disease or susceptibility
to a disease,
among others.
This invention also relates to the use of polynucleotides of the present
invention as
diagnostic reagents. Detection of a mutated form of a polynucleotide of the
invention,
preferable, SEQ ID NO:1, which is associated with a disease or pathogenicity
will provide a
diagnostic tool that can add to, or define, a diagnosis of a disease, a
prognosis of a course of
disease, a determination of a stage of disease, or a susceptibility to a
disease, which results
from under-expression, over-expression or altered expression of the
polynucleotide.
Organisms, particularly infectious organisms, carrying mutations in such
polynucleotide
may be detected at the polynucleotide level by a variety of techniques, such
as those
described elsewhere herein.
Cells from an organism carrying mutations or polymorphisms (allelic
variations) in a
polynucleotide and/or polypeptide of the invention may also be detected at the
polynucleotide or polypeptide level by a variety of techniques, to allow for
serotyping, for
example. For example, RT-PCR can be used to detect mutations in the RNA. It is
particularly preferred to use RT-PCR in conjunction with automated detection
systems, such
as, for example, GeneScan. RNA, cDNA or genomic DNA may also be used for the
same
purpose, PCR. As an example, PCR primers complementary to a polynucleotide
encoding
BASBO55 polypeptide can be used to identify and analyze mutations.
The invention further provides primers with l, 2, 3 or 4 nucleotides removed
from the 5'
and/or the 3' end. These primers may be used for, among other things,
amplifying
BASBO55 DNA and/or RNA isolated from a sample derived from an individual, such
as a
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CA 02360609 2001-07-20
WO 00/43517 PCT/EP00/00425
bodily material. The primers may be used to amplify a polynucleotide isolated
from an
infected individual, such that the polynucleotide may then be subject to
various techniques
for elucidation of the polynucleotide sequence. In this way, mutations in the
polynucleotide
sequence may be detected and used to diagnose and/or prognose the infection or
its stage or
course, or to serotype and/or classify the infectious agent.
The invention further provides a process for diagnosing disease, preferably
bacterial
infections, more preferably infections caused by Neisseria meningitidis,
comprising
determining from a sample derived from an individual, such as a bodily
material, an
increased level of expression of polynucleotide having a sequence of SEQ ID
NO: l .
Increased or decreased expression of a BASBO55 polynucleotide can be measured
using
any on of the methods well known in the art for the quantitation of
polynucleotides, such
as, for example, amplification, PCR, RT-PCR, RNase protection, Northern
blotting,
spectrometry and other hybridization methods.
1~
In addition, a diagnostic assay in accordance with the invention for detecting
over-
expression of BASBOS~ polypeptide compared to normal control tissue samples
may be
used to detect the presence of an infection, for example. Assay techniques
that can be used
to determine levels of a BASBO55 polypeptide, in a sample derived from a host,
such as a
bodily material, are well-known to those of skill in the art. Such assay
methods include
radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody
sandwich
assays, antibody detection and ELISA assays.
The polynucleotides of the invention may be used as components of
polynucleotide
arrays, preferably high density arrays or grids. These high density arrays are
particularly useful for diagnostic and prognostic purposes. For example, a set
of spots
each comprising a different gene, and further comprising a polynucleotide or
polynucleotides of the invention, may be used for probing, such as using
hybridization
or nucleic acid amplification, using a probe obtained or derived from a bodily
sample,
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WO 00/43517 PCT/EP00/00425
to determine the presence of a particular polynucleotide sequence or related
sequence in
an individual. Such a presence may indicate the presence of a pathogen,
particularly
Neisseria meningitides, and may be useful in diagnosing and/or prognosing
disease or a
course of disease. A grid comprising a number of variants of the
polynucleotide
sequence of SEQ ID NO:1 are preferred. Also preferred is a grid comprising a
number
of variants of a polynucleotide sequence encoding the polypeptide sequence of
SEQ ID
N0:2.
Antibodies
The polypeptides and polynucleotides of the invention or variants thereof, or
cells
expressing the same can be used as immunogens to produce antibodies
immunospecific for
such polypeptides or polynucleotides respectively.
In certain preferred embodiments of the invention there are provided
antibodies against
BASBO55 polypeptides or polynucleotides.
Antibodies generated against the polypeptides or polynucleotides of the
invention can be
obtained by administering the polypeptides and/or polynucleotides of the
invention, or
epitope-bearing fragments of either or both, analogues of either or both, or
cells expressing
either or both, to an animal, preferably a nonhuman, using routine protocols.
For
preparation of monoclonal antibodies, any technique known in the art that
provides
antibodies produced by continuous cell line cultures can be used. Examples
include various
techniques, such as those in Kohler, G. and Milstein, C., Nature 256: 495-497
(1975);
Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in
MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. Less, Inc. (1985).
Techniques for the production of single chain antibodies (U.S. Patent No.
4,946,778) can be
adapted to produce single chain antibodies to polypeptides or polynucleotides
of this
invention. Also, transgenic mice, or other organisms or animals, such as other
mammals,
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WO 00/43517 PCT/EP00/00425
may be used to express humanized antibodies immunospecific to the polypeptides
or
polynucleotides of the invention.
Alternatively, phage display technology may be utilized to select antibody
genes with
binding activities towards a polypeptide of the invention either from
repertoires of PCR
amplified v-genes of lymphocytes from humans screened for possessing anti-
BASBO55 or
from naive libraries (McCafferty, et al., (1990), Nature 348, 552-554; Marks,
et al.,
(1992) Biotechnology 10, 779-783). The affinity of these antibodies can also
be improved
by, for example, chain shuffling (Clackson et al., (1991) Nature 352: 628).
The above-described antibodies may be employed to isolate or to identify
clones expressing
the polypeptides or polynucleotides of the invention to purify the
polypeptides or
polynucleotides by, for example, affinity chromatography.
Thus, among others, antibodies against BASBO55-polypeptide or BASBO55-
polynucleotide
may be employed to treat infections, particularly bacterial infections.
Polypeptide variants include antigenically, epitopically or immunologically
equivalent
variants form a particular aspect of this invention.
Preferably, the antibody or variant thereof is modified to make it less
immunogenic in the
individual. For example, if the individual is human the antibody may most
preferably be
"humanized," where the complimentarity determining region or regions of the
hybridoma-
derived antibody has been transplanted into a human monoclonal antibody, for
example
as described in Jones et al. (1986), Nature 321, 522-525 or Tempest et al.,
(1991)
Biotechnology 9, 266-273.
Antagonists and Agonists - Assays and Molecules
CA 02360609 2001-07-20
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Polypeptides and polynucleotides of the invention may also be used to assess
the binding of
small molecule substrates and ligands in, for example, cells, cell-free
preparations, chemical
libraries, and natural product mixtures. These substrates and ligands may be
natural
substrates and ligands or may be structural or functional mimetics. See, e.g.,
Coligan et al.,
Current Protocols in Immunology 1 (2): Chapter 5 ( 1991 ).
The screening methods may simply measure the binding of a candidate compound
to the
polypeptide or polynucleotide, or to cells or membranes bearing the
polypeptide or
polynucleotide, or a fusion protein of the polypeptide by means of a label
directly or
indirectly associated with the candidate compound. Alternatively, the
screening method
may involve competition with a labeled competitor. Further, these screening
methods
may test whether the candidate compound results in a signal generated by
activation or
inhibition of the polypeptide or polynucleotide, using detection systems
appropriate to the
cells comprising the polypeptide or polynucleotide. Inhibitors of activation
are generally
assayed in the presence of a known agonist and the effect on activation by the
agonist by
the presence of the candidate compound is observed. Constitutively active
polypeptide
and/or constitutively expressed polypeptides and polynucleotides may be
employed in
screening methods for inverse agonists or inhibitors, in the absence of an
agonist or
inhibitor, by testing whether the candidate compound results in inhibition of
activation of
the polypeptide or polynucleotide, as the case may be. Further, the screening
methods
may simply comprise the steps of mixing a candidate compound with a solution
containing a polypeptide or polynucleotide of the present invention, to form a
mixture,
measuring BASB055 polypeptide and/or polynucleotide activity in the mixture,
and
comparing the BASB055 polypeptide and/or polynucleotide activity of the
mixture to a
standard. Fusion proteins, such as those made from Fc portion and BASB055
polypeptide, as hereinbefore described, can also be used for high-throughput
screening
assays to identify antagonists of the polypeptide of the present invention, as
well as of
phylogenetically and and/or functionally related polypeptides (see D. Bennett
et al., J Mol
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WO 00/43517 PCT/EP00/00425
Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16):9459-
9471
( 1995)).
The polynucleotides, polypeptides and antibodies that bind to and/or interact
with a
polypeptide of the present invention may also be used to configure screening
methods for
detecting the effect of added compounds on the production of mRNA and/or
polypeptide
in cells. For example, an ELISA assay may be constructed for measuring
secreted or cell
associated levels of polypeptide using monoclonal and polyclonal antibodies by
standard
methods known in the art. This can be used to discover agents which may
inhibit or
enhance the production of polypeptide (also called antagonist or agonist,
respectively)
from suitably manipulated cells or tissues.
The invention also provides a method of screening compounds to identify those
which
enhance (agonist) or block (antagonist) the action of BASBO55 polypeptides or
polynucleotides, particularly those compounds that are bacteristatic and/or
bactericidal. The
method of screening may involve high-throughput techniques. For example, to
screen for
agonists or antagonists, a synthetic reaction mix, a cellular compartment,
such as a
membrane, cell envelope or cell wall, or a preparation of any thereof,
comprising BASBO55
polypeptide and a labeled substrate or ligand of such polypeptide is incubated
in the absence
or the presence of a candidate molecule that may be a BASBO55 agonist or
antagonist. The
ability of the candidate molecule to agonize or antagonize the BASBOS~
polypeptide is
reflected in decreased binding of the labeled ligand or decreased production
of product from
such substrate. Molecules that bind gratuitously, i.e., without inducing the
effects of
BASBO55 polypeptide are most likely to be good antagonists. Molecules that
bind well
and, as the case may be, increase the rate of product production from
substrate, increase
signal transduction, or increase chemical channel activity are agonists.
Detection of the rate
or level of, as the case may be, production of product from substrate, signal
transduction, or
chemical channel activity may be enhanced by using a reporter system. Reporter
systems
that may be useful in this regard include but are not limited to colorimetric,
labeled substrate
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WO 00/43517 PCT/EP00/00425
converted into product, a reporter gene that is responsive to changes in
BASB055
polynucleotide or polypeptide activity, and binding assays known in the art.
Another example of an assay for BASB055 agonists is a competitive assay that
combines
BASB055 and a potential agonist with BASB055-binding molecules, recombinant
BASB055 binding molecules, natural substrates or ligands, or substrate or
ligand mimetics,
under appropriate conditions for a competitive inhibition assay. BASB055 can
be labeled,
such as by radioactivity or a colorimetric compound, such that the number of
BASB055
molecules bound to a binding molecule or converted to product can be
determined
accurately to assess the effectiveness of the potential antagonist.
Potential antagonists include, among others, small organic molecules,
peptides,
polypeptides and antibodies that bind to a polynucleotide and/or polypeptide
of the
invention and thereby inhibit or extinguish its activity or expression.
Potential antagonists
also may be small organic molecules, a peptide, a polypeptide such as a
closely related
protein or antibody that binds the same sites on a binding molecule, such as a
binding
molecule, without inducing BASB055-induced activities, thereby preventing the
action or
expression of BASB055 polypeptides and/or polynucleotides by excluding BASB055
polypeptides and/or polynucleotides from binding.
Potential antagonists include a small molecule that binds to and occupies the
binding site of
the polypeptide thereby preventing binding to cellular binding molecules, such
that normal
biological activity is prevented. Examples of small molecules include but are
not limited to
small organic molecules, peptides or peptide-like molecules. Other potential
antagonists
include antisense molecules (see Okano, J. Neurochem. 56: 560 ( 1991 );
OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION,
CRC Press, Boca Raton, FL (1988), for a description of these molecules).
Preferred
potential antagonists include compounds related to and variants of BASB055.
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In a further aspect, the present invention relates to genetically engineered
soluble fusion
proteins comprising a polypeptide of the present invention, or a fragment
thereof, and
various portions of the constant regions of heavy or light chains of
immunoglobulins of
various subclasses (IgG, IgM, IgA, IgE). Preferred.as an immunoglobulin is the
constant
part of the heavy chain of human IgG, particularly IgGl, where fusion takes
place at the
hinge region. In a particular embodiment, the Fc part can be removed simply by
incorporation of a cleavage sequence which can be cleaved with blood clotting
factor Xa.
Furthermore, this invention relates to processes for the preparation of these
fusion
proteins by genetic engineering, and to the use thereof for drug screening,
diagnosis and
therapy. A further aspect of the invention also relates to polynucleotides
encoding such
fusion proteins. Examples of fusion protein technology can be found in
International
Patent Application Nos. W094/29458 and W094/22914.
Each of the polynucleotide sequences provided herein may be used in the
discovery and
development of antibacterial compounds. The encoded protein, upon expression,
can be
used as a target for the screening of antibacterial drugs. Additionally, the
polynucleotide
sequences encoding the amino terminal regions of the encoded protein or Shine-
Delgarno
or other translation facilitating sequences of the respective mRNA can be used
to
construct antisense sequences to control the expression of the coding sequence
of interest.
The invention also provides the use of the polypeptide, polynucleotide,
agonist or
antagonist of the invention to interfere with the initial physical interaction
between a
pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible
for
sequelae of infection. In particular, the molecules of the invention may be
used: in the
prevention of adhesion of bacteria, in particular gram positive and/or gram
negative
bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins
on in-
dwelling devices or to extracellular matrix proteins in wounds; to block
bacterial adhesion
between eukaryotic, preferably mammalian, extracellular matrix proteins and
bacterial
BASBO55 proteins that mediate tissue damage and/or; to block the normal
progression of
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pathogenesis in infections initiated other than by the implantation of in-
dwelling devices
or by other surgical techniques.
In accordance with yet another aspect of the invention, there are provided
BASBO55
agonists and antagonists, preferably bacteristatic or bactericidal agonists
and antagonists.
The antagonists and agonists of the invention may be employed, for instance,
to prevent,
inhibit and/or treat diseases.
In a further aspect, the present invention relates to mimotopes of the
polypeptide of the
invention. A mimotope is a peptide sequence, sufficiently similar to the
native peptide
(sequentially or structurally), which is capable of being recognised by
antibodies which
recognise the native peptide; or is capable of raising antibodies which
recognise the
native peptide when coupled to a suitable carrier.
Peptide mimotopes may be designed for a particular purpose by addition,
deletion or
substitution of elected amino acids. Thus, the peptides may be modified for
the purposes
of ease of conjugation to a protein Garner. For example, it may be desirable
for some
chemical conjugation methods to include a terminal cysteine. In addition it
may be
desirable for peptides conjugated to a protein Garner to include a hydrophobic
terminus
distal from the conjugated terminus of the peptide, such that the free
unconjugated end
of the peptide remains associated with the surface of the carrier protein.
Thereby
presenting the peptide in a conformation which most closely resembles that of
the
peptide as found in the context of the whole native molecule. For example, the
peptides
may be altered to have an N-terminal cysteine and a C-terminal hydrophobic
amidated
tail. Alternatively, the addition or substitution of a D-stereoisomer form of
one or more
of the amino acids may be performed to create a beneficial derivative, for
example to
enhance stability of the peptide.
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WO 00/43517 PCT/EP00/00425
Alternatively, peptide mimotopes may be identified using antibodies which are
capable
themselves of binding to the polypeptides of the present invention using
techniques such
as phage display technology (EP 0 552 267 Bl). This technique, generates a
large number
of peptide sequences which mimic the structure of the native peptides and are,
therefore,
capable of binding to anti-native peptide antibodies, but may not necessarily
themselves
share significant sequence homology to the native polypeptide.
Vaccines
Another aspect of the invention relates to a method for inducing an
immunological
response in an individual, particularly a mammal, preferably humans, which
comprises
inoculating the individual with BASBO55 polynucleotide and/or polypeptide, or
a
fragment or variant thereof, adequate to produce antibody and/ or T cell
immune response
to protect said individual from infection, particularly bacterial infection
and most
particularly Neisseria meningitides infection. Also provided are methods
whereby such
immunological response slows bacterial replication. Yet another aspect of the
invention
relates to a method of inducing immunological response in an individual which
comprises
delivering to such individual a nucleic acid vector, sequence or ribozyme to
direct
expression of BASBO55 polynucleotide and/or polypeptide, or a fragment or a
variant
thereof, for expressing BASBO55 polynucleotide and/or polypeptide, or a
fragment or a
variant thereof in vivo in order to induce an immunological response, such as,
to produce
antibody and/ or T cell immune response, including, for example, cytokine-
producing T
cells or cytotoxic T cells, to protect said individual, preferably a human,
from disease,
whether that disease is already established within the individual or not. One
example of
administering the gene is by accelerating it into the desired cells as a
coating on particles
or otherwise. Such nucleic acid vector may comprise DNA, RNA, a ribozyme, a
modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA-
protein
complex.
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WO 00/43517 PCT/EP00/00425
A further aspect of the invention relates to an immunological composition that
when
introduced into an individual, preferably a human, capable of having induced
within it an
immunological response, induces an immunological response in such individual
to a
BASBOS~ polynucleotide and/or polypeptide encoded therefrom, wherein the
composition comprises a recombinant BASBO55 polynucleotide and/or polypeptide
encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses
an
antigen of said BASBO55 polynucleotide, polypeptide encoded therefrom, or
other
polypeptide of the invention. The immunological response may be used
therapeutically
or prophylactically and may take the form of antibody immunity and/or cellular
immunity, such as cellular immunity arising from CTL or CD4+ T cells.
A BASBO55 polypeptide or a fragment thereof may be fused with co-protein or
chemical
moiety which may or may not by itself produce antibodies, but which is capable
of
stabilizing the first protein and producing a fused or modified protein which
will have
1 ~ antigenic and/or immunogenic properties, and preferably protective
properties. Thus
fused recombinant protein, preferably further comprises an antigenic co-
protein, such as
lipoprotein D from Haemophilus in_fluenzae, Glutathione-S-transferase (GST) or
beta-
galactosidase, or any other relatively large co-protein which solubilizes the
protein and
facilitates production and purification thereof. Moreover, the co-protein may
act as an
adjuvant in the sense of providing a generalized stimulation of the immune
system of the
organism receiving the protein. The co-protein may be attached to either the
amino- or
carboxy-terminus of the first protein.
In a vaccine composition according to the invention, a BASBO55 polypeptide
and/or
polynucleotide, or a fragment, or a mimotope, or a variant thereof may be
present in a
vector, such as the live recombinant vectors described above for example live
bacterial
vectors.
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Also suitable are non-live vectors for the BASBO55 polypeptide, for example
bacterial
outer-membrane vesicles or "blebs". OM blebs are derived from the outer
membrane of
the two-layer membrane of Gram-negative bacteria and have been documented in
many
Gram-negative bacteria (Zhou, L et al. 1998. FEMS Microbiol. Lett. 163:223-
228)
including C. trachomatis and C. psittaci. A non-exhaustive list of bacterial
pathogens
reported to produce blebs also includes: Bordetella pertussis, Borrelia
burgdorferi,
Brucella melitensis, Brucella ovis, Esherichia coli, Haemophilus influenza,
Legionella
pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas
aeruginosa
and Yersinia enterocolitica.
Blebs have the advantage of providing outer-membrane proteins in their native
conformation and are thus particularly useful for vaccines. Blebs can also be
improved
for vaccine use by engineering the bacterium so as to modify the expression of
one or
more molecules at the outer membrane. Thus for example the expression of a
desired
immunogenic protein at the outer membrane, such as the BASBO55 polypeptide,
can be
introduced or upregulated (e.g. by altering the promoter). Instead or in
addition, the
expression of outer-membrane molecules which are either not relevant (e.g.
unprotective
antigens or immunodominant but variable proteins) or detrimental (e.g. toxic
molecules
such as LPS, or potential inducers of an autoimmune response) can be
downregulated.
These approaches are discussed in more detail below.
The non-coding flanking regions of the BASBO55 gene contain regulatory
elements
important in the expression of the gene. This regulation takes place both at
the
transcriptional and translational level. The sequence of these regions, either
upstream or
downstream of the open reading frame of the gene, can be obtained by DNA
sequencing.
This sequence information allows the determination of potential regulatory
motifs such as
the different promoter elements, terminator sequences, inducible sequence
elements,
repressors, elements responsible for phase variation, the shine-dalgarno
sequence, regions
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WO 00/43517 PCT/EP00/00425
with potential secondary structure involved in regulation; as well as other
types of
regulatory motifs or sequences. This sequence is a further aspect of the
invention.
This sequence information allows the modulation of the natural expression of
the
BASBO55 gene. The upregulation of the gene expression may be accomplished by
altering the promoter, the shine-dalgarno sequence, potential repressor or
operator
elements, or any other elements involved. Likewise, downregulation of
expression can be
achieved by similar types of modification. Alternatively, by changing phase
variation
sequences, the expression of the gene can be put under phase variation
control, or it may
be uncoupled from this regulation. In another approach, the expression of the
gene can be
put under the control of one or more inducible elements allowing regulated
expression.
Examples of such regulation include, but are not limited to, induction by
temperature
shift, addition of inductor substrates like selected carbohydrates or their
derivatives, trace
elements, vitamins, co-factors, metal ions, etc.
Such modifications as described above can be introduced by several different
means. The
modification of sequences involved in gene expression can be carried out in
vivo by
random mutagenesis followed by selection for the desired phenotype. Another
approach
consists in isolating the region of interest and modifying it by random
mutagenesis, or
site-directed replacement, insertion or deletion mutagenesis. The modified
region can then
be reintroduced into the bacterial genome by homologous recombination, and the
effect
on gene expression can be assessed. In another approach, the sequence
knowledge of the
region of interest can be used to replace or delete all or part of the natural
regulatory
sequences. In this case, the regulatory region targeted is isolated and
modified so as to
contain the regulatory elements from another gene, a combination of regulatory
elements
from different genes, a synthetic regulatory region, or any other regulatory
region, or to
delete selected parts of the wild-type regulatory sequences. These modified
sequences can
then be reintroduced into the bacterium via homologous recombination into the
genome.
A non-exhaustive list of preferred promoters that could be used for up-
regulation of gene
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WO 00/43517 PCT/EP00/00425
expression includes the promoters porA, porB, lbpB, tbpB, p 110, 1st, hpuAB
from N.
meningitides or N. gonorroheae; ompCD, copB, lbpB, ompE, UspAl; UspA2; TbpB
from
M. Catarrhalis; pl, p2, p4, p5, p6, lpD, tbpB, D15, Hia, Hmwl, Hmw2 from H.
influenzae.
In one example, the expression of the gene can be modulated by exchanging its
promoter
with a stronger promoter (through isolating the upstream sequence of the gene,
in vitro
modification of this sequence, and reintroduction into the genome by
homologous
recombination). Upregulated expression can be obtained in both the bacterium
as well as
in the outer membrane vesicles shed (or made) from the bacterium.
In other examples, the described approaches can be used to generate
recombinant
bacterial strains with improved characteristics for vaccine applications.
These can be, but
are not limited to, attenuated strains, strains with increased expression of
selected
antigens, strains with knock-outs (or decreased expression) of genes
interfering with the
immune response, strains with modulated expression of immunodominant proteins,
strains with modulated shedding of outer-membrane vesicles.
Thus, also provided by the invention is a modified upstream region of the
BASB055
gene, which modified upstream region contains a heterologous regulatory
element which
alters the expression level of the BASB055 protein located at the outer
membrane. The
upstream region according to this aspect of the invention includes the
sequence upstream
of the BASB055 gene. The upstream region starts immediately upstream of the
BASB055
gene and continues usually to a position no more than about 1000 by upstream
of the gene
from the ATG start codon. In the case of a gene located in a polycistronic
sequence
(operon) the upstream region can start immediately preceding the gene of
interest, or
preceding the first gene in the operon. Preferably, a modified upstream region
according to
this aspect of the invention contains a heterologous promotor at a position
between 500 and
700 by upstream of the ATG.
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Thus, the invention provides a BASB055 polypeptide, in a modified bacterial
bleb. The
invention further provides modified host cells capable of producing the non-
live membrane-
based bleb vectors. The invention further provides nucleic acid vectors
comprising the
BASB055 gene having a modified upstream region containing a heterologous
regulatory
element.
Further provided by the invention are processes to prepare the host cells and
bacterial blebs
15
according to the invention.
Also provided by this invention are compositions, particularly vaccine
compositions, and
methods comprising the polypeptides and/or polynucleotides of the invention
and
immunostimulatory DNA sequences, such as those described in Sato, Y. et al.
Science
273: 352 (1996).
Also, provided by this invention are methods using the described
polynucleotide or
particular fragments thereof, which have been shown to encode non-variable
regions of
bacterial cell surface proteins, in polynucleotide constructs used in such
genetic
immunization experiments in animal models of infection with Neisseria
meningitides.
Such experiments will be particularly useful for identifying protein epitopes
able to
provoke a prophylactic or therapeutic immune response. It is believed that
this approach
will allow for the subsequent preparation of monoclonal antibodies of
particular value,
derived from the requisite organ of the animal successfully resisting or
clearing infection,
for the development of prophylactic agents or therapeutic treatments of
bacterial
infection, particularly Neisseria meningitides infection, in mammals,
particularly humans.
The invention also includes a vaccine formulation which comprises an
immunogenic
recombinant polypeptide and/or polynucleotide of the invention together with a
suitable
carrier, such as a pharmaceutically acceptable carrier. Since the polypeptides
and
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WO 00/43517 PCT/EP00/00425
polynucleotides may be broken down in the stomach, each is preferably
administered
parenterally, including, for example, administration that is subcutaneous,
intramuscular,
intravenous, or intradermal. Formulations suitable for parenteral
administration include
aqueous and non-aqueous sterile injection solutions which may contain anti-
oxidants,
buffers, bacteristatic compounds and solutes which render the formulation
isotonic with
the bodily fluid, preferably the blood, of the individual; and aqueous and non-
aqueous
sterile suspensions which may include suspending agents or thickening agents.
The
formulations may be presented in unit-dose or mufti-dose containers, for
example, sealed
ampoules and vials and may be stored in a freeze-dried condition requiring
only the
addition of the sterile liquid carrier immediately prior to use.
The vaccine formulation of the invention may also include adjuvant systems for
enhancing the immunogenicity of the formulation. Preferably the adjuvant
system
raises preferentially a TH1 type of response.
An immune response may be broadly distinguished into two extreme catagories,
being a
humoral or cell mediated immune responses (traditionally characterised by
antibody and
cellular effector mechanisms of protection respectively). These categories of
response
have been termed TH1-type responses (cell-mediated response), and TH2-type
immune
responses (humoral response).
Extreme TH1-type immune responses may be characterised by the generation of
antigen
specific, haplotype restricted cytotoxic T lymphocytes, and natural killer
cell responses.
In mice TH1-type responses are often characterised by the generation of
antibodies of
the IgG2a subtype, whilst in the human these correspond to IgGl type
antibodies. TH2-
type immune responses are characterised by the generation of a broad range of
immunoglobulin isotypes including in mice IgGI, IgA, and IgM.
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It can be considered that the driving force behind the development of these
two types of
immune responses are cytokines. High levels of THl-type cytokines tend to
favour the
induction of cell mediated immune responses to the given antigen, whilst high
levels of
TH2-type cytokines tend to favour the induction of humoral immune responses to
the
antigen.
The distinction of TH1 and TH2-type immune responses is not absolute. In
reality an
individual will support an immune response which is described as being
predominantly
THl or predominantly TH2. However, it is often convenient to consider the
families of
I 0 cytokines in terms of that described in murine CD4 +ve T cell clones by
Mosmann and
Coffman (Mosmann, T. R. and Coffrnan, R.L. (1989) THI and TH2 cells: different
patterns of lymphokine secretion lead to different functional properties.
Annual Review
oflmmunology, 7, p145-173). Traditionally, THl-type responses are associated
with
the production of the INF-y and IL-2 cytokines by T-lymphocytes. Other
cytokines
15 often directly associated with the induction of TH1-type immune responses
are not
produced by T-cells, such as IL-12. In contrast, TH2- type responses are
associated with
the secretion of IL-4, IL-5, IL-6 and IL-13.
It is known that certain vaccine adjuvants are particularly suited to the
stimulation of
20 either THl or TH2 - type cytokine responses. Traditionally the best
indicators of the
TH1:TH2 balance of the immune response after a vaccination or infection
includes
direct measurement of the production of TH1 or TH2 cytokines by T lymphocytes
in
vitro after restimulation with antigen, and/or the measurement of the IgGI
:IgG2a ratio
of antigen specific antibody responses.
Thus, a THI-type adjuvant is one which preferentially stimulates isolated T-
cell
populations to produce high levels of THI-type cytokines when re-stimulated
with
antigen in vitro, and promotes development of both CD8+ cytotoxic T
lymphocytes and
antigen specific immunoglobulin responses associated with THI-type isotype.
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Adjuvants which are capable of preferential stimulation of the TH1 cell
response are
described in International Patent Application No. WO 94/00153 and WO 95/17209.
3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant. This is
known from GB 2220211 (Ribi). Chemically it is a mixture of 3 De-O-acylated
monophosphoryl lipid A with 4, S or 6 acylated chains and is manufactured by
Ribi
Immunochem, Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid
A is disclosed in European Patent 0 689 454 B1 (SmithKline Beecham Biologicals
SA).
Preferably, the particles of 3D-MPL are small enough to be sterile filtered
through a
0.22micron membrane (European Patent number 0 689 454).
3D-MPL will be present in the range of 10~g - 100~.g preferably 25-SO~g per
dose
wherein the antigen will typically be present in a range 2-SOpg per dose.
Another preferred adjuvant comprises QS21, an Hplc purified non-toxic fraction
derived from the bark of Quillaja Saponaria Molina. Optionally this may be
admixed
with 3 De-O-acylated monophosphoryl lipid A (3D-MPL), optionally together with
a
carrier.
The method of production of QS21 is disclosed in US patent No. 5,057,540.
Non-reactogenic adjuvant formulations containing QS21 have been described
previously (WO 96/33739). Such formulations comprising QS21 and cholesterol
have
been shown to be successful TH1 stimulating adjuvants when formulated together
with
an antigen.
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Further adjuvants which are preferential stimulators of TH1 cell response
include
immunomodulatory oligonucleotides, for example unmethylated CpG sequences as
disclosed in WO 96/02555.
Combinations of different TH1 stimulating adjuvants, such as those mentioned
hereinabove, are also contemplated as providing an adjuvant which is a
preferential
stimulator of TH 1 cell response. For example, QS21 can be formulated together
with
3D-MPL. The ratio of QS21 : 3D-MPL will typically be in the order of 1 : 10 to
10 : l;
preferably 1:5 to 5 : 1 and often substantially 1 : 1. The preferred range for
optimal
synergy is 2.5 : 1 to 1 : 1 3D-MPL: QS21.
Preferably a carrier is also present in the vaccine composition according to
the
invention. The carrier may be an oil in water emulsion, or an aluminium salt,
such as
aluminium phosphate or aluminium hydroxide.
A preferred oil-in-water emulsion comprises a metabolisible oil, such as
squalene, alpha
tocopherol and Tween 80. In a particularly preferred aspect the antigens in
the vaccine
composition according to the invention are combined with QS21 and 3D-MPL in
such
an emulsion. Additionally the oil in water emulsion may contain span 85 and/or
lecithin and/or tricaprylin.
Typically for human administration QS21 and 3D-MPL will be present in a
vaccine in
the range of lp,g - 200~g, such as 10-100p,g, preferably lOp,g - SOp.g per
dose.
Typically the oil in water will comprise from 2 to 10% squalene, from 2 to 10%
alpha
tocopherol and from 0.3 to 3% tween 80. Preferably the ratio of squalene:
alpha
tocopherol is equal to or less than 1 as this provides a more stable emulsion.
Span 85
may also be present at a level of 1 %. In some cases it may be advantageous
that the
vaccines of the present invention will further contain a stabiliser.
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Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g.
squalane or
squalene, an emulsifier, e.g. Tween 80, in an aqueous carrier. The aqueous
carrier may
be, for example, phosphate buffered saline.
A particularly potent adjuvant formulation involving QS21, 3D-MPL and
tocopherol
in an oil in water emulsion is described in WO 95/17210.
The present invention also provides a polyvalent vaccine composition
comprising a
vaccine formulation of the invention in combination with other antigens, in
particular
antigens useful for treating cancers, autoimmune diseases and related
conditions. Such a
polyvalent vaccine composition may include a TH-1 inducing adjuvant as
hereinbefore
described.
While the invention has been described with reference to certain BASBO55
polypeptides
and polynucleotides, it is to be understood that this covers fragments of the
naturally
occurring polypeptides and polynucleotides, and similar polypeptides and
polynucleotides
with additions, deletions or substitutions which do not substantially affect
the
immunogenic properties of the recombinant polypeptides or polynucleotides.
The antigen can also be delivered in the form of whole bacteria (dead or
alive) or as
subcellular fractions, these possibilities do include N. meningitidis itself.
Compositions, kits and administration
In a further aspect of the invention there are provided compositions
comprising a BASBO55
polynucleotide and/or a BASBO55 polypeptide for administration to a cell or to
a
multicellular organism.
The invention also relates to compositions comprising a polynucleotide and/or
a polypeptide
discussed herein or their agonists or antagonists. The polypeptides and
polynucleotides of
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WO 00/43517 PCT/EP00/00425
the invention may be employed in combination with a non-sterile or sterile
carrier or Garners
for use with cells, tissues or organisms, such as a pharmaceutical carrier
suitable for
administration to an individual. Such compositions comprise, for instance, a
media additive
or a therapeutically effective amount of a polypeptide and/or polynucleotide
of the invention
and a pharmaceutically acceptable Garner or excipient. Such Garners may
include, but are
not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol
and combinations
thereof. The formulation should suit the mode of administration. The invention
further
relates to diagnostic and pharmaceutical packs and kits comprising one or more
containers
filled with one or more of the ingredients of the aforementioned compositions
of the
invention.
Polypeptides, polynucleotides and other compounds of the invention may be
employed
alone or in conjunction with other compounds, such as therapeutic compounds.
1 ~ The pharmaceutical compositions may be administered in any effective,
convenient manner
including, for instance, administration by topical, oral, anal, vaginal,
intravenous,
intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes
among others.
In therapy or as a prophylactic, the active agent may be administered to an
individual as
an injectable composition, for example as a sterile aqueous dispersion,
preferably
isotonic.
In a further aspect, the present invention provides for pharmaceutical
compositions
comprising a therapeutically effective amount of a polypeptide and/or
polynucleotide, such
as the soluble form of a polypeptide and/or polynucleotide of the present
invention, agonist
or antagonist peptide or small molecule compound, in combination with a
pharmaceutically
acceptable Garner or excipient. Such carriers include, but are not limited to,
saline, buffered
saline, dextrose, water, glycerol, ethanol, and combinations thereof. The
invention further
relates to pharmaceutical packs and kits comprising one or more containers
filled with one
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WO 00/43517 PCT/EP00/00425
or more of the ingredients of the aforementioned compositions of the
invention.
Polypeptides, polynucleotides and other compounds of the present invention may
be
employed alone or in conjunction with other compounds, such as therapeutic
compounds.
The composition will be adapted to the route of administration, for instance
by a systemic or
an oral route. Preferred forms of systemic administration include injection,
typically by
intravenous injection. Other injection routes, such as subcutaneous,
intramuscular, or
intraperitoneal, can be used. Alternative means for systemic administration
include
transmucosal and transdermal administration using penetrants such as bile
salts or fusidic
acids or other detergents. In addition, if a polypeptide or other compounds of
the present
invention can be formulated in an enteric or an encapsulated formulation, oral
administration may also be possible. Administration of these compounds may
also be
topical and/or localized, in the form of salves, pastes, gels, solutions,
powders and the like.
For administration to mammals, and particularly humans, it is expected that
the daily
dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg,
typically around 1
mg/kg. The physician in any event will determine the actual dosage which will
be most
suitable for an individual and will vary with the age, weight and response of
the particular
individual. The above dosages are exemplary of the average case. There can, of
course,
be individual instances where higher or lower dosage ranges are merited, and
such are
within the scope of this invention.
The dosage range required depends on the choice of peptide, the route of
administration, the
nature of the formulation, the nature of the subject's condition, and the
judgment of the
attending practitioner. Suitable dosages, however, are in the range of 0.1-100
~g/kg of
subj ect.
A vaccine composition is conveniently in injectable form. Conventional
adjuvants may be
employed to enhance the immune response. A suitable unit dose for vaccination
is 0.5-5
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WO 00/43517 PCT/EP00/00425
microgram/kg of antigen, and such dose is preferably administered 1-3 times
and with an
interval of 1-3 weeks. With the indicated dose range, no adverse toxicological
effects will
be observed with the compounds of the invention which would preclude their
administration to suitable individuals.
Wide variations in the needed dosage, however, are to be expected in view of
the variety of
compounds available and the differing efficiencies of various routes of
administration. For
example, oral administration would be expected to require higher dosages than
administration by intravenous injection. Variations in these dosage levels can
be adjusted
using standard empirical routines for optimization, as is well understood in
the art.
Sequence Databases, Sequences in a Tangible Medium, and Algorithms
Polynucleotide and polypeptide sequences form a valuable information resource
with which
to determine their 2- and 3-dimensional structures as well as to identify
further sequences of
similar homology. These approaches are most easily facilitated by storing the
sequence in a
computer readable medium and then using the stored data in a known
macromolecular
structure program or to search a sequence database using well known searching
tools, such
as the GCG program package.
Also provided by the invention are methods for the analysis of character
sequences or
strings, particularly genetic sequences or encoded protein sequences.
Preferred methods
of sequence analysis include, for example, methods of sequence homology
analysis, such
as identity and similarity analysis, DNA, RNA and protein structure analysis,
sequence
assembly, cladistic analysis, sequence motif analysis, open reading frame
determination,
nucleic acid base calling, codon usage analysis, nucleic acid base trimming,
and
sequencing chromatogram peak analysis.
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A computer based method is provided for performing homology identification.
This
method comprises the steps of: providing a first polynucleotide sequence
comprising the
sequence of a polynucleotide of the invention in a computer readable medium;
and
comparing said first polynucleotide sequence to at least one second
polynucleotide or
polypeptide sequence to identify homology.
A computer based method is also provided for performing homology
identification, said
method comprising the steps of: providing a first polypeptide sequence
comprising the
sequence of a polypeptide of the invention in a computer readable medium; and
comparing said first polypeptide sequence to at least one second
polynucleotide or
polypeptide sequence to identify homology.
All publications and references, including but not limited to patents and
patent
applications, cited in this specification are herein incorporated by reference
in their
entirety as if each individual publication or reference were specifically and
individually
indicated to be incorporated by reference herein as being fully set forth. Any
patent
application to which this application claims priority is also incorporated by
reference
herein in its entirety in the manner described above for publications and
references.
DEFINITIONS
"Identity," as known in the art, is a relationship between two or more
polypeptide sequences
or two or more polynucleotide sequences, as the case may be, as determined by
comparing
the sequences. In the art, "identity" also means the degree of sequence
relatedness between
polypeptide or polynucleotide sequences, as the case may be, as determined by
the match
between strings of such sequences. "Identity" can be readily calculated by
known
methods, including but not limited to those described in (Computational
Molecular
Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988;
Biocomputircg:
Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York,
1993;
CA 02360609 2001-07-20
WO 00143517 PCT/EP00/00425
Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G.,
eds.,
Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von
Heine,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and
Devereux, J.,
eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM
J.
Applied Math., 48: 1073 (1988). Methods to determine identity are designed to
give the
largest match between the sequences tested. Moreover, methods to determine
identity are
codified in publicly available computer programs. Computer program methods to
determine identity between two sequences include, but are not limited to, the
GAP
program in the GCG program package (Devereux, J., et al., Nucleic Acids
Research 12(1):
387 (1984)), BLASTP, BLASTN (Altschul, S.F. et al., .I. Mol. Biol. 21~: 403-
410 (1990),
and FASTA( Pearson and Lipman Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988).
The BLAST family of programs is publicly available from NCBI and other sources
(BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894;
Altschul, S.,
et al., J. Mol. Biol. 21~: 403-410 (1990). The well known Smith Waterman
algorithm
may also be used to determine identity.
Parameters for polypeptide sequence comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,
Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)
Gap Penalty: 8
Gap Length Penalty: 2
A program useful with these parameters is publicly available as the "gap"
program from
Genetics Computer Group, Madison WI. The aforementioned parameters are the
default
parameters for peptide comparisons (along with no penalty for end gaps).
Parameters for polynucleotide comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix: matches = +10, mismatch = 0
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Gap Penalty: SO
Gap Length Penalty: 3
Available as: The "gap" program from Genetics Computer Group, Madison WI.
These
are the default parameters for nucleic acid comparisons.
A preferred meaning for "identity" for polynucleotides and polypeptides, as
the case may
be, are provided in (1) and (2) below.
( 1 ) Polynucleotide embodiments further include an isolated polynucleotide
comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90,
95, 97 or
100% identity to the reference sequence of SEQ ID NO:l, wherein said
polynucleotide
sequence may be identical to the reference sequence of SEQ ID NO:1 or may
include up
to a certain integer number of nucleotide alterations as compared to the
reference
sequence, wherein said alterations are selected from the group consisting of
at least one
nucleotide deletion, substitution, including transition and transversion, or
insertion, and
wherein said alterations may occur at the 5' or 3' terminal positions of the
reference
nucleotide sequence or anywhere between those terminal positions, interspersed
either
individually among the nucleotides in the reference sequence or in one or more
contiguous groups within the reference sequence, and wherein said number of
nucleotide
alterations is determined by multiplying the total number of nucleotides in
SEQ ID NO:1
by the integer defining the percent identity divided by 100 and then
subtracting that
product from said total number of nucleotides in SEQ ID NO:1, or:
nn ~ Xn ' ~Xn' Y)
wherein nn is the number of nucleotide alterations, xn is the total number of
nucleotides
in SEQ ID NO:1, y is 0.50 for SO%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%,
0.85 for
85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and ~ is the
symbol for
the multiplication operator, and wherein any non-integer product of xn and y
is rounded
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WO 00/43517 PCT/EP00/00425
down to the nearest integer prior to subtracting it from xn. Alterations of a
polynucleotide
sequence encoding the polypeptide of SEQ ID N0:2 may create nonsense, missense
or
frameshift mutations in this coding sequence and thereby alter the polypeptide
encoded by
the polynucleotide following such alterations.
By way of example, a polynucleotide sequence of the present invention may be
identical
to the reference sequence of SEQ ID NO:1, that is it may be 100% identical, or
it may
include up to a certain integer number of nucleic acid alterations as compared
to the
reference sequence such that the percent identity is less than 100% identity.
Such
alterations are selected from the group consisting of at least one nucleic
acid deletion,
substitution, including transition and transversion, or insertion, and wherein
said
alterations may occur at the 5' or 3' terminal positions of the reference
polynucleotide
sequence or anywhere between those terminal positions, interspersed either
individually
among the nucleic acids in the reference sequence or in one or more contiguous
groups
within the reference sequence. The number of nucleic acid alterations for a
given percent
identity is determined by multiplying the total number of nucleic acids in SEQ
ID NO:1
by the integer defining the percent identity divided by 100 and then
subtracting that
product from said total number of nucleic acids in SEQ ID NO:l, or:
nn <_ xn - (xn ~ Y),
wherein nn is the number of nucleic acid alterations, xn is the total number
of nucleic
acids in SEQ ID NO:1, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for
85% etc.,
is the symbol for the multiplication operator, and wherein any non-integer
product of xn
and y is rounded down to the nearest integer prior to subtracting it from xn.
(2) Polypeptide embodiments further include an isolated polypeptide comprising
a
polypeptide having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity
to a
polypeptide reference sequence of SEQ ID N0:2, wherein said polypeptide
sequence may
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be identical to the reference sequence of SEQ ID N0:2 or may include up to a
certain
integer number of amino acid alterations as compared to the reference
sequence, wherein
said alterations are selected from the group consisting of at least one amino
acid deletion,
substitution, including conservative and non-conservative substitution, or
insertion, and
wherein said alterations may occur at the amino- or carboxy-terminal positions
of the
reference polypeptide sequence or anywhere between those terminal positions,
interspersed either individually among the amino acids in the reference
sequence or in one
or more contiguous groups within the reference sequence, and wherein said
number of
amino acid alterations is determined by multiplying the total number of amino
acids in
SEQ ID N0:2 by the integer defining the percent identity divided by 100 and
then
subtracting that product from said total number of amino acids in SEQ ID N0:2,
or:
na ~ xa ' ~xa' Y)
wherein na is the number of amino acid alterations, xa is the total number of
amino acids
in SEQ ID N0:2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%,
0.85 for
85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and ~ is the
symbol for
the multiplication operator, and wherein any non-integer product of xa and y
is rounded
down to the nearest integer prior to subtracting it from xa.
By way of example, a polypeptide sequence of the present invention may be
identical to
the reference sequence of SEQ ID N0:2, that is it may be 100% identical, or it
may
include up to a certain integer number of amino acid alterations as compared
to the
reference sequence such that the percent identity is less than 100% identity.
Such
alterations are selected from the group consisting of at least one amino acid
deletion,
substitution, including conservative and non-conservative substitution, or
insertion, and
wherein said alterations may occur at the amino- or carboxy-terminal positions
of the
reference polypeptide sequence or anywhere between those terminal positions,
interspersed either individually among the amino acids in the reference
sequence or in one
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WO 00/43517 PCT/EP00/00425
or more contiguous groups within the reference sequence. The number of amino
acid
alterations for a given % identity is determined by multiplying the total
number of amino
acids in SEQ ID N0:2 by the integer defining the percent identity divided by
100 and
then subtracting that product from said total number of amino acids in SEQ ID
N0:2, or:
na ~ xa - (xa' Y)
wherein na is the number of amino acid alterations, xa is the total number of
amino acids
in SEQ ID N0:2, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85%
etc., and ~ is
the symbol for the multiplication operator, and wherein any non-integer
product of xa and
y is rounded down to the nearest integer prior to subtracting it from xa.
"Individual(s)," when used herein with reference to an organism, means a
multicellular
eukaryote, including, but not limited to a metazoan, a mammal, an ovid, a
bovid, a
simian, a primate, and a human.
"Isolated" means altered "by the hand of man" from its natural state, i.e., if
it occurs in
nature, it has been changed or removed from its original environment, or both.
For example,
a polynucleotide or a polypeptide naturally present in a living organism is
not ''isolated," but
the same polynucleotide or polypeptide separated from the coexisting materials
of its natural
state is "isolated", as the term is employed herein. Moreover, a
polynucleotide or
polypeptide that is introduced into an organism by transformation, genetic
manipulation or
by any other recombinant method is "isolated" even if it is still present in
said organism,
which organism may be living or non-living.
"Polynucleotide(s)" generally refers to any polyribonucleotide or
polydeoxyribonucleotide,
which may be unmodified RNA or DNA or modified RNA or DNA including single and
double-stranded regions.
CA 02360609 2001-07-20
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"Variant" refers to a polynucleotide or polypeptide that differs from a
reference
polynucleotide or polypeptide, but retains essential properties. A typical
variant of a
polynucleotide differs in nucleotide sequence from another, reference
polynucleotide.
Changes in the nucleotide sequence of the variant may or may not alter the
amino acid
sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide
changes may result in amino acid substitutions, additions, deletions, fusions
and
truncations in the polypeptide encoded by the reference sequence, as discussed
below.
A typical variant of a polypeptide differs in amino acid sequence from
another,
reference polypeptide. Generally, differences are limited so that the
sequences of the
reference polypeptide and the variant are closely similar overall and, in many
regions,
identical. A variant and reference polypeptide may differ in amino acid
sequence by
one or more substitutions, additions, deletions in any combination. A
substituted or
inserted amino acid residue may or may not be one encoded by the genetic code.
A
variant of a polynucleotide or polypeptide may be a naturally occurring such
as an
allelic variant, or it may be a variant that is not known to occur naturally.
Non-naturally
occurring variants of polynucleotides and polypeptides may be made by
mutagenesis
techniques or by direct synthesis.
"Disease(s)" means any disease caused by or related to infection by a
bacteria, including ,
for example, upper respiratory tract infection, invasive bacterial diseases,
such as bacteremia
and meningitis.
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EXAMPLES:
The examples below are earned out using standard techniques, which are well
known and
routine to those of skill in the art, except where otherwise described in
detail. The examples
are illustrative, but do not limit the invention.
Example 1: Sequence of BASBO55 in N. meningitidis serogroup B strain
ATCC13090.
The sequence of the BASBO55 gene of N. meningitidis strain ATCC 13090 is shown
in
SEQ ID NO:l. The translation of the BASBO55 polynucleotide sequence. showed in
SEQ ID N0:2, is related by amino acid sequence homology to Neisseria
gonorrhoeae
MtrC protein. The BASBOS~ polypeptide contains a signal sequence
characteristic of a
lipoprotein.
Example 2: Construction of Plasmid to Express Recombinant BASBOS~
A: Cloning of BASBO55.
The NdeI and XhoI restriction sites engineered into the forward Lipl 1-Fm/p
(~'- AGG
CAG AGG CAT ATG GCT TTT TAT GCT TTT AAG GCG ATG CG -3') ([SEQ ID
N0:3]) and reverse Lipl 1-RCf/p ( ~'- AGG CAG AGG CTC GAG TTC CGC TTC
AGA AGC AGT TTT GGC TTC -3')([SEQ ID N0:4]) amplification primers,
respectively, permitted directional cloning of a BASBO55 PCR product into the
low
copy number E. coli expression plasmid pTLZ2 such that a BASBO55 protein could
be
expressed as a fusion protein containing a (His)6 affinity chromatography tag
at the C-
terminus. The BASBOS~ PCR product was purified from the amplification reaction
using silica gel-based spin columns (QiaGen) according to the manufacturers
instructions. To produce the required NdeI and XhoI termini necessary for
cloning,
purified PCR product was sequentially digested to completion with NdeI and
XhoI
restriction enzymes as recommended by the manufacturer (Life Technologies).
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Following the first restriction digestion, the PCR product was purified via
spin column
as above to remove salts and eluted in sterile water prior to the second
enzyme
digestion. The digested DNA fragment was again purified using silica gel-based
spin
columns prior to ligation with the pTLZ2 plasmid.
B: Production of Expression Vector.
To prepare the expression plasmid pTLZ2 for ligation, it was similarly
digested to
completion with both NdeI and XhoI and then treated with calf intestinal
phosphatase
(CIP, 0.02 units / pmole of 5' end, Life Technologies) as directed by the
manufacturer
to prevent self ligation. An approximately 5-fold molar excess of the digested
fragment
to the prepared vector was used to program the ligation reaction. A standard
~20 p,l
ligation reaction (~16°C, ~16 hours), using methods well known in the
art, was
performed using T4 DNA ligase (~2.0 units / reaction, Life Technologies). An
aliquot
of the ligation (~~ pl) was used to transform electro-competent BL21 DE3 cells
according to methods well known in the art. Following a ~2-3 hour outgrowth
period at
37°C in ~1.0 ml of LB broth, transformed cells were plated on LB agar
plates
containing ampicilline (100 pg/ml). The antibiotic was included in the
selection media
to ensure that all transformed cells carried the pTLZ2 plasmid (ApR). Plates
were
incubated overnight at 37°C for ~16 hours. Individual ApR colonies were
picked with
sterile toothpicks and used to "patch" inoculate fresh LB ApR plates as well
as a ~1.0
ml LB ApR broth culture. Both the patch plates and the broth culture were
incubated
overnight at 37°C in either a standard incubator (plates) or a shaking
water bath.
A whole cell-based PCR analysis was employed to verify that transformants
contained
the BASBO55 DNA insert. Here, the ~1.0 ml overnight LB Ap broth culture was
transferred to a 1.5 ml polypropylene tube and the cells collected by
centrifugation in a
Beckman microcentrifuge (~3 min., room temperature, 12,000 X g). The cell
pellet
was suspended in ~200~.1 of sterile water and a ~l Opl aliquot used to program
a ~SOpI
final volume PCR reaction containing both BASBO55 forward and reverse
amplification
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primers. Final concentrations of the PCR reaction components were essentially
the
same as those specified in example 2 except ~5.0 units of Taq polymerase was
used.
The initial 95°C denaturation step was increased to 3 minutes to ensure
thermal
disruption of the bacterial cells and liberation of plasmid DNA. An ABI Model
9700
thermal cycler and a 32 cycle, three-step thermal amplification profile, i.e.
95°C, 45sec;
55-58°C, 45sec, 72°C, lmin., were used to amplify the BASB055
PCR fragment from
the lysed transformant samples. Following thermal amplification, a ~201
aliquot of the
reaction was analyzed by agarose gel electrophoresis (0.8 % agarose in a Tris-
acetate-
EDTA (TAE) buffer). DNA fragments were visualized by UV illumination after gel
electrophoresis and ethidium bromide staining. A DNA molecular size standard (
1 Kb
ladder, Life Technologies) was electrophoresed in parallel with the test
samples and was
used to estimate the size of the PCR products. Transformants that produced the
expected PCR product were identified as strains containing a BASB055
expression
construct. Expression plasmid containing strains were then analyzed for the
inducible
expression of recombinant BASB055.
C: Expression Analysis of PCR-Positive Transformants.
For each PCR-positive transformant identified above, ~5.0 ml of LB broth
containing
ampicilline ( 100 ~.g/ml) was inoculated with cells from the patch plate and
grown
overnight at 37 °C with shaking (~250 rpm). An aliquot of the overnight
seed culture
(~1.0 ml) was inoculated into a 125 ml erlenmeyer flask containing ~25 ml of
LB Ap
broth and grown at 37 °C with shaking (~250 rpm) until the culture
turbidity reached
O.D.600 of ~0.5, i.e. mid-log phase (usually about 1.5 - 2.0 hours). At this
time
approximately half of the culture (~12.5 ml) was transferred to a second 125
ml flask
and expression of recombinant BASB055 protein induced by the addition of IPTG
(1.0
M stock prepared in sterile water, Sigma) to a final concentration of 1.0 mM.
Incubation of both the IPTG-induced and non-induced cultures continued for an
additional ~4 hours at 37 °C with shaking. Samples (~1.0 ml) of both
induced and non-
induced cultures were removed after the induction period and the cells
collected by
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centrifugation in a microcentrifuge at room temperature for ~3 minutes.
Individual cell
pellets were suspended in ~50~1 of sterile water, then mixed with an equal
volume of
2X Laemmli SDS-PAGE sample buffer containing 2-mercaptoethanol, and placed in
boiling water bath for ~3 min to denature protein. Equal volumes (~15~1) of
both the
crude IPTG-induced and the non-induced cell lysates were loaded onto duplicate
12%
Tris/glycine polyacrylamide gel ( 1 mm thick Mini-gels, Novex). The induced
and non-
induced lysate samples were electrophoresed together with prestained molecular
weight
markers (SeeBlue, Novex) under conventional conditions using a standard
SDS/Tris/glycine running buffer (BioRad). Following electrophoresis, one gel
was
stained with commassie brilliant blue R2~0 (BioRad) and then destained to
visualize
novel BASBO55 IPTG-inducible protein(s). The second gel was electroblotted
onto a
PVDF membrane (0.45 micron pore size, Novex) for ~2 hrs at 4 °C using a
BioRad
Mini-Protean II blotting apparatus and Towbin's methanol (20 %) transfer
buffer.
Blocking of the membrane and antibody incubations were performed according to
methods well known in the art. A monoclonal anti- (His)5 antibody, followed by
a
second rabbit anti-mouse antibody conjugated to HRP (QiaGen), was used to
confirm
the expression and identity of the BASBO55 recombinant protein. Visualization
of the
anti-His antibody reactive pattern was achieved using either an ABT insoluble
substrate
or using Hyperfilm with the Amersham ECL chemiluminescence system.
Example 3: Analysis of Recombinant BASBO55 lipidation status
To determine if the BASBO55 recombinant protein is lipidated, radiolabelling
experiments using 3H-glyceral and 3H-palmitic acid as lipid-specific
incorporation tag
have been initiated. Cultures of the BL21 DE3 E. coli strain containing the
expression
plasmid was grown on M9 minimal medium and pulse-labelled with either SOqCi of
3H-
glyceral or 3H-palmitic acid during the 3 hours of IPTG induction. Following a
wash
step to remove unincorporated isotope, whole cell lysates of radiolabelled
cultures were
run on gradient polyacrylamide gels (4-20%), fixed and then dried under
vacuum. An
autoradiograph was produced by exposing the dried gel to Hyperfilm (Amersham)
at -
CA 02360609 2001-07-20
WO 00/43517 PCT/EP00/00425
70°C for ~ about 7 days. A visible band of the same size as the IPTG-
inducible
coomassie stainable protein was detected by both labelling. That 3H-label
incorporated
into the IPTG inducible protein support the conclusion that the BASBO55
protein is
lipidated to some extent in E. coli.
Example 4: Production of Recombinant BASB055
Bacterial strain
A recombinant expression strain of E. coli BL21 DE3 containing a pTLZ2 plasmid
encoding BASBO55 from N. meningitides. was used to produce cell mass for
purification of recombinant protein. The expression strain was cultivated on
LB agar
plates containing 100~,g/ml ampicilline ("Ap") to ensure plasmid maintenance.
For
cryopreservation at -80 °C, the strain was propagated in LB broth
containing the same
concentration of antibiotic then mixed with an equal volume of LB broth
containing
30% (w/v) glycerol.
Media
The fermentation medium used for the production of recombinant protein
consisted of
2X YT broth (Difco) containing 100~.g/ml Ap. Antifoam was added to medium for
the
fermentor at 0.25 ml/L (Antifoam 204, Sigma). To induce expression of the
BASBO55
recombinant protein, IPTG (Isopropyl I3-D-Thiogalactopyranoside) was added to
the
fermentor (1 mM, final).
Fermentation
A 500-ml erlenmeyer seed flask, containing SOmI working volume, was inoculated
with
0.3 ml of rapidly thawed frozen culture, or several colonies from a selective
agar plate
culture, and incubated for approximately 12 hours at 37 ~ 1°C on a
shaking platform at
1 SOrpm (Innova 2100, New Brunswick Scientific). This seed culture was then
used to
inoculate a 5-L working volume fermentor containing 2X YT broth and both Ap
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WO 00/43517 PCT/EP00/00425
antibiotic. The fermentor (Bioflo 3000, New Brunswick Scientific) was operated
at 37
~ 1°C, 0.2 - 0.4 VVM air spurge, 250 rpm in Rushton impellers. The pH
was not
controlled in either the flask seed culture or the fermentor. During
fermentation, the pH
ranged 6.5 to 7.3 in the fermentor. IPTG ( 1.0 M stock, prepared in sterile
water) was
added to the fermentor when the culture reached mid-log of growth (~0.7
O.D.600
units). Cells were induced for 2 - 4 hours then harvested by centrifugation
using either a
28RS Heraeus (Sepatech) or RCSC superspeed centrifuge (Sorvall Instruments).
Cell
paste was stored at -20 C until processed.
Purification
Imidazole and biotechnology grade or better reagents were all obtained from
Ameresco
Chemical, Solon, Ohio. Triton X-100 (t-Octylphenoxypolyethoxy-ethanol), Triton
X-
114, sodium phosphate, monobasic, and urea were reagent grade or better and
obtained
from Sigma Chemical Company, St. Louis, Missouri. Dulbecco's Phosphate
Buffered
Saline(lx PBS) was obtained from Quality Biological, Inc., Gaithersburg,
Maryland.
Dulbecco's Phosphate Buffered Saline (lOx PBS) was obtained from BioWhittaker,
Walkersville, Maryland. Penta-His Antibody, BSA free was obtained from QiaGen,
Valencia, California. Peroxidase-conjugated AffmiPure Goat Anti-mouse IgG was
obtained from Jackson Immuno Research, West Grove, Penn. All other chemicals
were
reagent grade or better.
Ni-chelating Sepharose Fast Flow resin was obtained from Pharmacia, Sweden.
Precast
Tris-Glycine 4-20% and 10-20% polyacrylamide gels, all running buffers and
solutions,
SeeBlue Pre-Stained Standards, MultiMark Multi-Colored Standards and PVDF
transfer
2~ membranes were obtained from Novex, San Diego, California. SDS-PAGE Silver
Stain
kits were obtained from Daiichi Pure Chemicals Company Limited, Tokyo, Japan.
Coomassie Stain Solution was obtained from Bio-Rad Laboratories, Hercules,
California. Acrodisc~ PF 0.2 m syringe filters were obtained from Pall Gelman
Sciences, Ann Arbor, Michigan. GD/X 25mm disposable syringe filters were
obtained
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WO 00/43517 PCT/EP00/00425
from Whatman Inc., Clifton, New Jersey. Dialysis tubing 8,000 MWCO was
obtained
from BioDesign Inc. Od New York, Carmal New York. BCA Protein Assay Reagents
and Snake Skin dialysis tubing 3,500 MWCO were obtained from Pierce Chemical
Co.,
Rockford, Illinois.
Extraction Protocol
Cell paste was thawed at room temperature for 30 to 60 minutes. Five to six
grams of
material was weighed out into a 50-ml disposable centrifuge tube. Recombinant
BASBO55 antigen was purified by extraction of cell membranes with 1.0 % Triton
Xl 14, and allowing phase partitioning based on Triton X114 cloud point at
37°C. The
Triton X114 phase was diluted with 50 mM Tris-HCl containing 10% glycerol, 5%
ethylene glycol and 0.5% Triton X100. This was applied to nickel-chelating
Sepharose
Fast Flow. The protein is afterwards eluted with 200 mM imidazole to affinity
purify
the histidine-tagged protein and yielded greater than 90% pure protein.
Final Formulation
BASBO55 was formulated by dialysis overnight against, three changes of 0.1 %
Triton
X-100 and lx PBS, pH 7.4. The purified protein was characterized and used to
produce
antibodies as described below.
Biochemical Characterizations : SDS-PAGE and Western Blot Analysis
The recombinant purified protein was resolved on 4-20 % polyacrylamide gels
and
electrophoretically transferred to PVDF membranes at 100 V for 1 hour as
previously
described (Thebaine et al. 1979, Proc. Natl. Acad. Sci. USA 76:4350-4354). The
2~ PVDF membranes were then pretreated with 25 ml of Dulbecco's phosphate
buffered
saline containing 5 % non-fat dry milk. All subsequent incubations were
carried out
using this pretreatment buffer.
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PVDF membranes were incubated with a dilution of anti-His tail antibodies for
1 hour
at room temperature. PVDF membranes were then washed twice with wash buffer
(20
mM Tris buffer, pH 7.5, containing 1 SO mM sodium chloride and 0.05 % Tween-
20).
PVDF membranes were incubated with 25 ml of a 1:5000 dilution of peroxidase-
labeled
species specific conjugate for 30 minutes at room temperature. PVDF membranes
were
then washed 4 times with wash buffer, and were developed with 3-amino-9-
ethylcarbazole and urea peroxide as supplied by Zymed (San Francisco, CA) for
10
minutes each.
The results of an SDS-PAGE (Figure lA) show a protein about ~0 kDa purified to
greater than 90 % and that is reactive to an anti-tetra (His) antibody by
western blots
(Figure 1 B) of the SDS-PAGE.
Example 5 : Immunization of mice with recombinant BASBOS~
Partially purified recombinant BASBO55 protein expressed in E. coli has been
injected
three times in Balb/C mice on days 0, 14 and 28 (10 animals/group). Animals
were
injected by the subcutaneous route with around 5 ~.g of antigen in two
different
formulations: either adsorbed on 100 ~g A1P04 or formulated in SBAS2 emulsion
(SB62 emulsion containing 5 pg MPL and S~g QS21 per dose). A negative control
group consisting of mice immunized with the SBAS2 emulsion only has also been
added in the experiment. Mice were bled on days 28 (14 days Post II) and 35 (7
days
Post III) in order to detect specific anti-BASBO55 antibodies. Specific anti-
BASBO55
antibodies were measured by Elisa on partially purified BASBO55 protein as
well as on
E. coli proteins. Antibody responses were also evaluated by western-blotting
when
2~ tested against different Neisseria meningitides B strains. Pooled sera
(from 10
mice/group) from both formulations (on day 7 Post III only) were tested in
these assays.
Results illustrated in Figure 2 clearly indicate that the antibody response is
quite good,
while the anti-E. coli antibody response (Figure 3), which is clearly
positive, is much
lower than the specific BASBO55 response (Figure 2). The A1P04 formulation
induces
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the highest antibody levels. Western-blots illustrated in Figures 4 and ~
confirm that
the BASBO55 protein is well recognized at the expected MW of 50 kDa by
immunized
mice sera.
CA 02360609 2001-07-20
WO 00/43517 PCT/EP00/00425
Recognition of the BASBO55 epitopes on different Neisseria meningitides
serogroup B strains by western-blotting
In this test, immunized mice sera (pooled) have been tested by western-
blotting for
recognition of the BASB055 epitopes on seven different Neisseria meningitides
B
strains: H44/76 (B:15:P1.7, 16, lineage ET-5), M97 250687 (B:4:P1.15), BZ10
(B:2b:P1.2, lineage A4), BZ198 (B:NT*: -, lineage 3), EG328 (B:NT*, lineage ST-
18),
NGP165 (B:2a:P1.2, ET 37 cluster) and the ATCC 13090 (B:15:P1.15) Neisseria
meningitides B strains , as well as on partially purified recombinant BASB055
protein.
(* : NT : Not Typed).
Briefly, 10 ~l (> lOx cells/lane) of each sample treated with sample buffer
(10 min at
95°C) are put into a SDS-PAGE gradient gel (Tris-glycine 4-20%, Novex,
code
n°EC6028). Electrophoretic migration occurs at 125 volts for 90 min.
(35mA/gel).
Afterwards, proteins are transferred to nitrocellulose sheet (0.45 Vim, Bio-
rad code n°
162-0114) at 100 volts for 1:30 hour using a Bio-rad Trans-blot system (code
n° 170-
3930). Filter was blocked with PBS - 0.05 % Tween 20 overnight at room
temperature,
before incubation with the mice sera containing the anti-BASB055 antibodies
from both
A1P04 and SBAS2 formulations. These sera are diluted 100 times in PBS - 0.05
Tween 20, and incubated on the nitrocellulose sheet for two hours at room
temperature
with gentle shaking. After three repeated washing steps in PBS - 0.05 % Tween
20 for 5
min., the nitrocellulose sheet is incubated at room temperature for 1 hour
under gentle
shaking with the appropriate conjugate (biotinylated anti-mouse Ig antibodies
from
sheep, Amersham code n°RPN 1001 ) diluted at 1 /500 in the same washing
buffer. The
membrane is washed three times as previously, and incubated for 30 min with
agitation
using the streptavidin-peroxidase complex (Amersham code n°1051)
diluted at 1/1000
in the washing buffer. After the last three repeated washing steps, the
revelation occurs
during the 20 min incubation time in a 50 ml solution containing 30 mg 4-
chloro-1-
naphtol (Sigma), 10 ml methanol, 40 ml PBS, and 30 ~.1 of H,Oz. The staining
is
stopped while washing the membrane several times in distillated water.
61
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Results illustrated in Figures 4 and 5 show that all strains tested present a
clear
reactivity against the major BASBO55 band around 50 kDa, plus few other bands
at
lower MW, which are most probably related to the antigen, as they are not
present on
the E. coli preparation (Figure 5). These results suggest that the BASBO55
protein is
expressed in probably all Neisseria meningitidis B strains. In both Figures 4
and 5, the
recombinant BASBO55 protein is recognized by mice sera at the same MW.
However,
the lowest band recognized by mice sera (< 9 kDa) appears to be related to an
E. coli
contaminant, as demonstrated in the last lane (Figure 5) with E. coli
exctract.
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SEQUENCE INFORMATION
BASBO55 Polynucleotide and Polypeptide Sequences
SEQ ID NO:1
S Neisseria meningitides BASBO55 polynucleotide sequence from strain ATCC
13090
ATGGCTTTTTATGCTTTTAAGGCGATGCGTGCGGCCGCGTTGGCTGCCGCCGTTGCATTG
GTACTGTCGTCTTGCGGTAAAGGCGGAGACGCGGCGCAGGGCGGGCAGCCTGCTGGTCGG
GAAGCCCCTGCGCCCGTCGTCGGTGTCGTAACCGTCCATCCGCAAACCGTCGCATTGACC
GTCGAGTTGCCGGGGCGTTTGGAATCGCTGCGTACCGCCGATGTCCGCGCCCAAGTCGGC
IO GGCATCATCCAAAAACGCCTGTTCCAAGAAGGCAGTTATGTCCGTGCCGGACAGCCGCTG
TATCAGATCGACAGTTCCACTTATGAAGCAAATCTGGAAAGCGCGCGCGCGCAACTGGCA
ACGGCTCAGGCAACGCTTGCCAAAGCGGATGCGGATTTGGCGCGATACAAGCCTTTGGTT
GCCGCCGAAGCCGTCAGCCGGCAGGAATACGATGCTGCGGTAACGGCGAAACGTTCTGCC
GAGGCAGGTGTCAAAGCAGCACAGGCGGCAATCAAATCTGCCGGCATTAATCTGAACCGT
IS TCGCGCATTACCGCGCCGATTTCCGGCTTTATCGGTCAGTCCAAAGTTTCCGAAGGTACG
CTGTTGAATGCGGGCGATACGACCGTGCTGGCAACCATCCGCCAAACCAATCCGATGTAT
GTGAACGTTACCCAGTCTGCATCCGAAGTGATGAAATTGCGCCGTCAGATAGCCGAAGGC
AAACTGCTGGCGGCGGATGGTGTGATTGCGGTCGGCATCAAATTTGACGACGGCACAGTT
TACCCTGAAAAAGGCCGCCTGCTGTTTGCCGATCCGGTCGTCAACGAATCGACCGGTCAG
ZO ATTACCCTGCGCGCCGCCGTACCGAACGATCAGAATATCCTGATGCCCGGTCTGTATGTG
CGCGTGCTGATGGACCAAGTGGCGGTGGATAACGCATTTGTTGTGCCGCAGCAGGCGGTA
ACGCGCGGTGCGAAAGATACCGTGATGATTGTGAATGCCCAAGGCGGTATGGAACCCCGC
GAGGTAACGGTTGCGCAACAGCAGGGTACGAATTGGATTGTTACGTCGGGTCTGAAGGAC
GGGGACAAGGTGGTTGTGGAAGGCATCAGTATCGCCGGTATAACGGGTGCGAAAAAGGTA
ZS ACGCCCAAAGAATGGGCGTCGTCTGAAAACCAAGCCGCCGCGCCTCAATCCGGCGTTCAG
ACGGCATCTGAAGCCAAAACTGCTTCTGAAGCGGAATAA
SEQ ID N0:2
Neisseria meningitides BASBO55 polypeptide sequence deduced from the
polynucleotide of
30 SeQ ID NO:1
MAFYAFKAMRAAALAAAVALVLSSCGKGGDAAQGGQPAGREAPAPWGVVTVHPQTVALT
VELPGRLESLRTADVRAQVGGIIQKRLFQEGSYVRAGQPLYQIDSSTYEANLESARAQLA
TAQATLAKADADLARYKPLVAAEAVSRQEYDAAVTAKRSAEAGVKAAQAAIKSAGINLNR
SRITAPISGFIGQSKVSEGTLLNAGDTTVLATIRQTNPMYVNVTQSASEVMKLRRQIAEG
3S KLLAADGVIAVGIKFDDGTVYPEKGRLLFADPVVNESTGQITLRAAVPNDQNILMPGLW
RVLMDQVAVDNAFWPQQAVTRGAKDTVMIVNAQGGMEPREVTVAQQQGTNWIVTSGLKD
GDKVVVEGISIAGITGAKKVTPKEWASSENQAAAPQSGVQTASEAKTASEAE
40 SEQ ID N0:3
AGG CAG AGG CAT ATG GCT TTT TAT GCT TTT AAG GCG ATG CG
SEQ ID N0:4
AGG CAG AGG CTC GAG TTC CGC TTC AGA AGC AGT TTT GGC TTC
4S
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Deposited materials
A deposit containing a Neisseria meningitides Serogroup B strain has been
deposited with
the American Type Culture Collection (herein "ATCC") on June 22, 1997 and
assigned
deposit number 13090. The deposit was described as Neisseria meningitides
(Albrecht and
Ghon) and is a freeze-dried, 1.5-2.9 kb insert library constructed from N.
meningitides
isolate. The deposit is described in Int. Bull. Bacteriol. Nomencl. Taxon. 8:
1-1 ~ (1958).
The Neisseria meningitides strain deposit is referred to herein as "the
deposited strain" or as
"the DNA of the deposited strain."
The deposited strain contains the full length BASBO55 gene. The sequence of
the
polynucleotides contained in the deposited strain, as well as the amino acid
sequence of any
polypeptide encoded thereby, are controlling in the event of any conflict with
any
description of sequences herein.
l~
The deposit of the deposited strain has been made under the terms of the
Budapest Treaty on
the International Recognition of the Deposit of Micro-organisms for Purposes
of Patent
Procedure. The strain will be irrevocably and without restriction or condition
released to the
public upon the issuance of a patent. The deposited strain is provided merely
as
convenience to those of skill in the art and is not an admission that a
deposit is required for
enablement, such as that required under 35 U.S.C. ~ 112.
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INDICATIONS RELATING TO DEPOSITED MICROORGANISM
OR OTHER BIOLOGICAL MATERIAL
(PCT Rule 136is)
A. The indications made below relate
to the deposited microorganism
or other biological material referred
to in the description
on pave H~, , line 2-21
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depository institution
AMERICAN TYPE CULTURE COLLECTION
Address of depository institution
(including postal code and country)
10801 UNIVERSITY BLVD, MANASSAS,
VIRGINIA 20110-2209,
UNITED STATES OF AMERICA
Date of deposit 22 June 1997 ( Accession Number 13090
22 . 06. 97 )
C. ADDITIONAL INDICATIONS (leave
blank if not applicable) This
information is continued on an
additional sheet
In respect of those designations
where a European Patent is sought,
a sample
of the deposited microorganism
will be made available until the
publication
of the mention of the grant of
the European Patent or until the
date on which
the application has been refused
or withdrawn, only by issue of
such a sample
to an expert nominated by the person
requesting_thevsample.
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (if the indicatioru
are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank if not applicable)
The indications listed below will
be submitted to the International
Bureau later(spec~thegeneralnaauroftheindicationse.g.,
'Accession
Number of Deposit')
--- For receiving Office use only For International Bureau use only
This sheet was received with the international application D This sheet was
received by the International Bureau on:
Authorized officer ~ ~ Authorized officer
Form PCT/RO/l34 (lulu19981 65
CA 02360609 2001-07-20
FOR THE PURPOSES OF INFORMATION ONLY
Codes used to identify States party to the PCT on the front pages of pamphlets
publishing international applications under the PCT.
AL Albania ES Spain LS Lesotho SI Slovenia
AM Armenia FI Finland LT Lithuania SK Slovakia
AT Austria FR France LU Luxembourg SN Senegal
AU Australia GA Gabon LV Latvia SZ Swaziland
AZ Azerbaijan GB United KingdomMC Monaco TD Chad
BA Bosnia and GE Georgia MD Republic of TG Togo
Herzegovina Moldova
BB Barbados GH Ghana MG Madagascan TJ Tajikistan
BE Belgium GN Guinea MK The former TM Turkmenistan
Yugoslav
BF Burkina Faso GR Greece Republic of TR Turkey
Macedonia
BG Bulgaria HU Hungary ML Mali TT Trinidad
and Tobago
BJ Benin IE Ireland MN Mongolia UA Ukraine
BR Brazil IL Israel MR Mauritania UC Uganda
BY Belams IS Iceland MW Malawi US United States
of America
CA Canada IT Italy MX Mexico UZ Uzbekistan
CF Central AfricanJP Japan NE Niger VN Viet Nam
Republic
CG Congo KE Kenya NL Netherlands YU Yugoslavia
CH Switzerland KG Kyrgyzstan NO Norway ZW Zimbabwe
CI CBte d'lvoireKP Democratic NZ New Zealand
People's
CM Cameroon Republic PL Poland
of Korea
CN China KR Republic PT Portugal
of Korea
CU Cuba KZ Kazakstan RO Romania
CZ Czech RepublicLC Saint Lucia RU Russian Federation
DE Gem~any LI LiechtensteinSD Sudan
DK Denmark LK Sri Lanka SE Sweden
EE Estonia LR Liberia SG Singapore
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SEQUENCE LISTING
<110> SmithKline Beecham Biologicals S.A.
<120> Novel compounds
<130> BM45353
<160> 4
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 1239
<212> DNA
<213> Neisseria meningitidis
<400> 1
atggcttttt atgcttttaaggcgatgcgtgcggccgcgttggctgccgccgttgcattg60
gtactgtcgt cttgcggtaaaggcggagacgcggcgcagggcgggcagcctgctggtcgg120
gaagcccctg cgcccgtcgtcggtgtcgtaaccgtccatccgcaaaccgtcgcattgacc180
gtcgagttgc.cggggcgtttggaatcgctgcgtaccgccgatgtccgcgcccaagtcggc240
ggcatcatcc aaaaacgcctgttccaagaaggcagttatgtccgtgccggacagccgctg300
tatcagatcg acagttccacttatgaagcaaatctggaaagcgcgcgcgcgcaactggca360
acggctcagg caacgcttgccaaagcggatgcggatttggcgcgatacaagcctttggtt420
gccgccgaag ccgtcagccggcaggaatacgatgctgcggtaacggcgaaacgttctgcc480
gaggcaggtg tcaaagcagcacaggcggcaatcaaatctgccggcattaatctgaaccgt540
tcgcgcatta ccgcgccgatttccggctttatcggtcagtccaaagtttccgaaggtacg600
ctgttgaatg cgggcgatacgaccgtgctggcaaccatccgccaaaccaatccgatgtat660
gtgaacgtta cccagtctgcatccgaagtgatgaaattgcgccgtcagatagccgaaggc720
aaactgctgg cggcggatggtgtgattgcggtcggcatcaaatttgacgacggcacagtt780
taccctgaaa aaggccgcctgctgtttgccgatccggtcgtcaacgaatcgaccggtcag840
attaccctgc gcgccgccgtaccgaacgatcagaatatcctgatgcccggtctgtatgtg900
cgcgtgctga tggaccaagtggcggtggataacgcatttgttgtgccgcagcaggcggta960
acgcgcggtg cgaaagataccgtgatgattgtgaatgcccaaggcggtatggaaccccgc1020
gaggtaacgg ttgcgcaacagcagggtacgaattggattgttacgtcgggtctgaaggac1080
ggggacaagg tggttgtggaaggcatcagtatcgccggtataacgggtgcgaaaaaggta1140
acgcccaaag aatgggcgtcgtctgaaaaccaagccgccgcgcctcaatccggcgttcag1200
1
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acggcatctg aagccaaaac tgcttctgaa gcggaataa 1239
<210> 2
<211> 412
<212> PRT
<213> Neisseria meningitidis
<400> 2
Met Ala Phe Tyr Ala Phe Lys Ala Met Arg Ala Ala Ala Leu Ala Ala
1 5 10 15
Ala Val Ala Leu Val Leu Ser Ser Cys Gly Lys Gly Gly Asp Ala Ala
20 25 30
Gln Gly Gly Gln Pro Ala Gly Arg Glu Ala Pro Ala Pro Val Val Gly
35 40 45
Val Val Thr Val His Pro Gln Thr Val Ala Leu Thr Val Glu Leu Pro
50 55 60
Gly Arg Leu Glu Ser Leu Arg Thr Ala Asp Val Arg Ala Gln Val Gly
65 70 75 80
Gly Ile Ile Gln Lys Arg Leu Phe Gln Glu Gly Ser Tyr Val Arg Ala
85 90 95
Gly Gln Pro Leu Tyr Gln Ile Asp Ser Ser Thr Tyr Glu Ala Asn Leu
100 105 110
Glu Ser Ala Arg Ala Gln Leu Ala Thr Ala Gln Ala Thr Leu Ala Lys
115 120 125
Ala Asp Ala Asp Leu Ala Arg Tyr Lys Pro Leu Val Ala Ala Glu Ala
130 135 140
Val Ser Arg Gln Glu Tyr Asp Ala Ala Val Thr Ala Lys Arg Ser Ala
145 150 155 160
Glu Ala Gly Val Lys Ala Ala Gln Ala Ala Ile Lys Ser Ala Gly Ile
165 170 175
Asn Leu Asn Arg Ser Arg Ile Thr Ala Pro Ile Ser Gly Phe Ile Gly
180 185 190
Gln Ser Lys Val Ser Glu Gly Thr Leu Leu Asn Ala Gly Asp Thr Thr
195 200 205
Val Leu Ala Thr Ile Arg Gln Thr Asn Pro Met Tyr Val Asn Val Thr
210 215 220
G1n Ser Ala Ser Glu Val Met Lys Leu Arg Arg Gln Ile Ala Glu Gly
225 230 235 240
Lys Leu Leu Ala Ala Asp Gly Val Ile Ala Val Gly Ile Lys Phe Asp
245 250 255
2
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Asp Gly Thr Val Tyr Pro Glu Lys Gly Arg Leu Leu Phe Ala Asp Pro
260 265 270
Val Val Asn Glu Ser Thr Gly Gln Ile Thr Leu Arg Ala Ala Val Pro
275 280 285
Asn Asp Gln Asn Ile Leu Met Pro Gly Leu Tyr Val Arg Val Leu Met
290 295 300
Asp Gln Val Ala Val Asp Asn Ala Phe Val Val Pro Gln Gln Ala Val
305 310 315 320
Thr Arg Gly Ala Lys Asp Thr Val Met Ile Val Asn Ala Gln Gly Gly
325 330 335
Met Glu Pro Arg Glu Val Thr Val Ala Gln Gln Gln Gly Thr Asn Trp
340 345 350
Ile Val Thr Ser Gly Leu Lys Asp Gly Asp Lys Val Val Val Glu Gly
355 360 365
Ile Ser Ile Ala Gly Ile Thr Gly Ala Lys Lys Val Thr Pro Lys Glu
370 375 380
Trp Ala Ser Ser Glu Asn Gln Ala Ala Ala Pro Gln Ser Gly Val Gln
385 390 395 400
Thr Ala Ser Glu Ala Lys Thr Ala Ser Glu Ala Glu
405 410
<210> 3
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 3
aggcagaggc atatggcttt ttatgctttt aaggcgatgc g 41
<210> 4
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
3
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<400> 4
aggcagaggc tcgagttccg cttcagaagc agttttggct tc 42
