Note: Descriptions are shown in the official language in which they were submitted.
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Novel Compounds
FIELD OF THE INVENTION
This invention relates to polynucleotides, (herein referred to as "BASB121
polynucleotide(s)"), polypeptides encoded by them (referred to herein as
"BASB121" or
"BASB 121 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
Moraxella catarrhalis (also named Branhamella catarrhalis) is a Gram negative
bacteria
frequently isolated from the human upper respiratory tract. It is responsible
for several
pathologies the main ones being otitis media in infants and children, and
pneumonia in
elderlies. It is also responsible of sinusitis, nosocomial infections and less
frequently of
invasive diseases.
Otitis media is an important childhood disease both by the number of cases and
its potential
sequelae. More than 3.5 millions cases are recorded every year in the United
States, and it is
estimated that 80 % of the children have experienced at least one episode of
otitis before
reaching the age of 3 (Klein, JO (1994) Clin.Inf.Dis 19:823). Left untreated,
or becoming
chronic, this disease may lead to hearing losses that could be temporary (in
the case of fluid
accumulation in the middle ear) or permanent (if the auditive nerve is
damaged). In infants,
such hearing losses may be responsible for a delayed speech learning.
Three bacterial species are primarily isolated from the middle ear of children
with otitis
media: Streptococcus pneumoniae, non typeable Haemophilus influenza (NTHi) and
M.
catarrhalis. They are present in 60 to 90 % of the cases. A review of recent
studies shows
that S. pneumoniae and NTHi represent both about 30 %, and M. catarrhalis
about 15 % of
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the otitis media cases (Murphy, TF (1996) Microbiol.Rev. 60:267). Other
bacteria could be
isolated from the middle ear (H. influenza type B, S. pyogenes etc) but at a
much lower
frequency (2 % of the cases or less).
Epidemiological data indicate that, for the pathogens found in the middle ear,
the
colonization of the upper respiratory tract is an absolute prerequisite for
the development of
an otitis; other are however also required to lead to the disease (Dickinson,
DP et al. (1988)
J. Infect.Dis. 158:205, Faden, HL et al. (1991) Ann.Otorhinol.Laryngol.
100:612). These are
important to trigger the migration of the bacteria into the middle ear via the
Eustachian
tubes, followed by the initiation of an inflammatory process. These factors
are unknown
todate. It has been postulated that a transient anomaly of the immune system
following a
viral infection, for example, could cause an inability to control the
colonization of the
respiratory tract (Faden, HL et al (1994) J. Infect.Dis. 169:1312). An
alternative explanation
is that the exposure to environmental factors allow a more important
colonization of some
children, who subsequently become susceptible to the development of otitis
media because
of the sustained presence of middle ear pathogens (Murphy, TF (1996)
Microbiol.Rev.
60:267).
The immune response to M. catarrhalis is poorly characterized. The analysis of
strains
isolated sequentially from the nasopharynx of babies followed from 0 to 2
years of age,
indicates that they get and eliminate frequently new strains. This indicates
that an
efficacious immune response against this bacteria is mounted by the colonized
children
(Faden, HL et al (1994) J. Infect.Dis. 169:1312).
In most adults tested, bactericidal antibodies have been identified (Chapman,
AJ et al.
(1985) J. Infect.Dis. 151:878). Strains ofM. catarrhalis present variations in
their capacity
to resist serum bactericidal activity: in general, isolates from diseased
individuals are more
resistant than those who are simply colonized (Hol, C et al. (1993) Lancet
341:1281, Jordan,
KL et al. (1990) Am.J.Med. 88 (suppl. 5A):285). Serum resistance could
therfore be
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considered as a virulence factor of the bacteria. An opsonizing activity has
been observed in
the sera of children recovering from otitis media.
The antigens targetted by these different immune responses in humans have not
been
identified, with the exception of OMP B 1, a 84 kDa protein which expression
is regulated
by iron, and that is recognized by the sera of patients with pneumonia (Sethi,
S, et al. (1995)
Infect.Immun. 63:1516) , and of UspAl and UspA2 (Chen D. et a1.(1999),
Infect.Immun.
67:1310).
A few other membrane proteins present on the surface of M. catarrhalis have
been
characterized using biochemical method, or for their potential implication in
the induction of
a protective immunity (for review, see Murphy, TF ( 1996) Microbiol.Rev.
60:267). In a
mouse pneumonia model, the presence of antibodies raised against some of them
(UspA,
CopB) favors a faster clearance of the pulmonary infection. Another
polypeptide (OMP CD)
is highly conserved among M. catarrhalis strains, and presents homologies with
a porin of
Pseudomonas aeruginosa, which has been demonstrated efficacious against this
bacterium
in animal models.
The frequency of Moraxella catarrhalis 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 Moraxella catarrhalis 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
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The present invention relates to BASB121, in particular BASB121 polypeptides
and
BASB 121 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 BASB 121 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
The invention relates to BASB 121 polypeptides and polynucleotides as
described in
greater detail below. The BASB121 polypeptide has the characteristics of a
lipoprotein
and is therefore predicted to be surface expressed. The invention relates
especially to
BASB 121 having the nucleotide and amino acid sequences set out in SEQ ID NO:1
or 3
and SEQ ~ N0:2 or 4 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.
Polypeptides
In one aspect of the invention there are provided polypeptides of Moraxella
catarrhalis
referred to herein as "BASB121" and "BASB121 polypeptides" as well as
biologically,
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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, preferably at least 90% identity, more preferably at least 95%
identity, most
preferably at least 97-99% or exact identity, to that of SEQ ID N0:2 or 4;
(b) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence which has at least 85% identity, preferably at least 90% identity,
more
preferably at least 95% identity, even more preferably at least 97-99% or
exact identity to
SEQ ID NO:1 or 3 over the entire length of SEQ ID NO:l or 3 respectively; or
(c) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence encoding a polypeptide which has at least 85% identity, preferably at
least 90%
identity, 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 or 4.
The BASB 121 polypeptides provided in SEQ ID N0:2 or 4 are the BASB 121
polypeptides from Moraxella catarrhalis strain Mc2931 (ATCC 43617).
The invention also provides an immunogenic fragment of a BASB 121 polypeptide,
that
is, a contiguous portion of the BASB 121 polypeptide which has the same or
substantially
the same immunogenic activity as the polypeptide comprising the amino acid
sequence of
SEQ ID N0:2 or 4; That is to say, the fragment (if necessary when coupled to a
Garner) is
capable of raising an immune response which recognises the BASB 121
polypeptide.
Such an immunogenic fragment may include, for example, the BASB 121
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 BASB
121
according to the invention comprises substantially all of the extracellular
domain of a
polypeptide which has at least 85% identity, preferably at least 90% identity,
more
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preferably at least 95% identity, most preferably at least 97-99% identity, to
that of SEQ
ID N0:2 or 4 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
BASB 121 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 4 or of variants thereof, such as a
continuous series
of 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-
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 4, 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 or 4.
Fragments of the polypeptides of the invention may be employed for producing
the
corresponding full-length polypeptide by peptide synthesis; therefore, these
fragments
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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
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.
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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 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.
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Polypeptides of the present invention can be prepared in any suitable manner.
Such
polypeptides include isolated naturally occurring 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
Moraxella
catarrhalis, 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 polynucleotides that encode
BASB121
polypeptides, particularly polynucleotides that encode the polypeptide herein
designated
BASB 121.
In a particularly preferred embodiment of the invention the polynucleotide
comprises a
region encoding BASB121 polypeptides comprising a sequence set out in SEQ ID
NO:1 or
3 which includes a full length gene, or a variant thereof.
The BASB 121 polynucleotides provided in SEQ ID NO:1 or 3 are the BASB 121
polynucleotides from Moraxella catarrhalis strain Mc2931 (ATCC 43617).
As a further aspect of the invention there are provided isolated nucleic acid
molecules
encoding and/or expressing BASB 121 polypeptides and polynucleotides,
particularly
Moraxella catarrhalis BASB 121 polypeptides and polynucleotides, including,
for
example, unprocessed IRNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B-
and Z-DNAs. Further embodiments of the invention include biologically,
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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 BASB 121 polypeptide having a deduced amino acid
sequence of
SEQ ID N0:2 or 4 and polynucleotides closely related thereto and variants
thereof.
In another particularly preferred embodiment of the invention there is a BASB
121
polypeptide from Moraxella catarrhalis comprising or consisting of an amino
acid
sequence of SEQ ID N0:2 or 4 or a variant thereof.
Using the information provided herein, such as a polynucleotide sequence set
out in SEQ ID
NO: l or 3, a polynucleotide of the invention encoding BASB 121 polypeptide
may be
obtained using standard cloning and screening methods, such as those for
cloning and
sequencing chromosomal DNA fragments from bacteria using Moraxella catarrhalis
Catlin
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 or 3, typically a library of clones of chromosomal DNA of
Moraxella
catarrhalis Catlin 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
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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: l or 3
was discovered in a DNA library derived from Moraxella catarrhalis.
Moreover, each DNA sequence set out in SEQ ID NO:1 or 3 contains an open
reading frame
encoding a protein having about the number of amino acid residues set forth in
SEQ ID
N0:2 or 4 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 613 of SEQ ID NO:1, encodes
the
polypeptide of SEQ ID N0:2.
The polynucleotide of SEQ ID N0:3, between the start codon at nucleotide
number 1 and
the last codon which begins at nucleotide number 610 of SEQ ID N0:3, encodes
the
polypeptide of SEQ ID N0:4.
In a further aspect, the present invention provides for an isolated
polynucleotide
comprising or consisting o~
(a) a polynucleotide sequence which has at least 85% identity, preferably at
least 90%
identity, more preferably at least 95% identity, even more preferably at least
97-99% or
exact identity to SEQ ID NO:1 or 3 over the entire length of SEQ ID NO:1 or 3
respectively; or
(b) a polynucleotide sequence encoding a polypeptide which has at least 85%
identity,
preferably at least 90% identity, 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
or 4, over the entire length of SEQ ID N0:2 or 4 respectively.
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A polynucleotide encoding a polypeptide of the present invention, including
homologs and
orthologs from species other than Moraxella catarrhalis, 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:1 or 3 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 or 3. 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
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 3,': 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.
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The nucleotide sequence encoding BASB 121 polypeptide of SEQ ID N0:2 or 4 may
be
identical to the polypeptide encoding sequence contained in nucleotides 1 to
612 of SEQ
ID NO:1 or the polypeptide encoding sequence contained in nucleotides 1 to 612
of SEQ
ID N0:3 respectively. 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 or 4.
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 Moraxella
catarrhalis BASB 121 having an amino acid sequence set out in SEQ ID N0:2 or
4. 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
or 4.
Fragments of polynucleotides of the invention may be used, for example, to
synthesize full-
length polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding BASB
121
variants, that have the amino acid sequence of BASB121 polypeptide of SEQ ID
N0:2 or 4
in which several, a few, S to 10, 1 to S, 1 to 3, 2, 1 or no amino acid
residues are substituted,
modified, deleted andJor added, in any combination. Especially preferred among
these are
silent substitutions, additions and deletions, that do not alter the
properties and activities of
BASB 121 polypeptide.
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Further preferred embodiments of the invention are polynucleotides that are at
least 85%
identical over their entire length to a polynucleotide encoding BASB 121
polypeptide having
an amino acid sequence set out in SEQ ID N0:2 or 4, and polynucleotides that
are
complementary to such polynucleotides. Alternatively, most highly preferred
are
polynucleotides that comprise a region that is at least 90% identical over its
entire length to
a polynucleotide encoding BASB 121 polypeptide and polynucleotides
complementary
thereto. In this regard, polynucleotides at least 95% identical over their
entire length to the
same are particularly 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 or 3.
In accordance with certain preferred embodiments of this invention there are
provided
polynucleotides that hybridize, particularly under stringent conditions, to
BASB 121
polynucleotide sequences, such as those polynucleotides in SEQ ID NO:1 or 3.
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
conditions" mean
hybridization occurnng 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,
5x SSC (150mM
NaCI, lSmM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x 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.
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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:1 or 3 under stringent
hybridization
conditions with a probe having the sequence of said polynucleotide sequence
set forth in
SEQ ID NO:l or 3 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
BASB 121 and to isolate cDNA and genomic clones of other genes that have a
high identity,
particularly high sequence identity, to the BASB 121 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.
A coding region of a BASB 121 gene may be isolated by screening using a DNA
sequence
provided in SEQ ID NO:1 or 3 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.
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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 carned 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.
The polynucleotides of the invention that are oligonucleotides derived from a
sequence of
SEQ ID NOS:l or 3 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.
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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, T/U 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
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
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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) l: 363, Manthorpe et al., Hum. Gene Thef-.
(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
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
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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 METHODSINMOLECULAR 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, DEAE-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, Neisseria meningitidis and Moraxella catarrhalis; fungal cells, such
as cells of a
yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and
Aspergillus;
insect cells such as cells ofDrosophila S2 and Spodoptera Sfi3; animal cells
such as CHO,
COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plant
cells, such
as cells of a gymnosperm or angiosperm.
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
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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 canon exchange chromatography,
phosphocellulose
chromatography, hydrophobic interaction chromatography, affinity
chromatography,
hydroxylapatite chromatography and lectin chromatography. Most preferably, ion
metal
affinity 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.
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
CA 02380498 2002-O1-23
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Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus,
rhinovirus),
herpesviruses (varicella zoster virus, etc), Listeria, Salmonella , Shigella,
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 BASB 121 polynucleotides and
polypeptides of
the invention for use as diagnostic reagents. Detection of BASB121
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. Euka_ryotes, particularly mammals, and especially humans,
particularly
those infected or suspected to be infected with an organism comprising the
BASB 121 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 andlor 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
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 BASB121 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
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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 BASB 121
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, 274:
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 3, 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
4 or a fragment thereof; or
(d) an antibody to a polypeptide of the present invention, preferably to the
polypeptide of
SEQ ID N0:2 or 4.
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.
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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,
preferably SEQ ID NO:1 or 3, 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
BASB 121 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
BASB 121 DNA and/or PSlA isolated from a sample derived from an individual,
such as a
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.
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The invention further provides a process for diagnosing, disease, preferably
bacterial
infections, more preferably infections caused by Moraxella catarrhalis,
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:1 or 3.
Increased or decreased expression of a BASB 121 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.
In addition, a diagnostic assay in accordance with the invention for detecting
over-
expression of BASB 121 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 BASB 121 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 probes obtained or derived from a
bodily sample,
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
Moraxella catarrhalis, 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 or 3 are preferred. Also preferred is a comprising a
number
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of variants of a polynucleotide sequence encoding the polypeptide sequence of
SEQ ID
N0:2 or 4.
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. The term "immunospecific"
means that
the antibodies have substantially greater affinity for the polypeptides of the
invention than
their affinity for other related polypeptides in the prior art.
In certain preferred embodiments of the invention there are provided
antibodies against
BASB 121 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. Liss, 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,
may be used to express humanized antibodies immunospecific to the polypeptides
or
polynucleotides of the invention.
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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-BASB
121 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 BASB121-polypeptide or BASB121-
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
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
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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 BASB 121 polypeptide and/or polynucleotide activity in the mixture,
and
comparing the BASB 121 polypeptide and/or polynucleotide activity of the
mixture to a
standard. Fusion proteins, such as those made from Fc portion and BASB 121
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
Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16):9459-
9471
(1995)).
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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 BASB 121 polypeptides or
polynucleotides, particularly those compounds that are bacteriostatic 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 BASB 121
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 BASB 121 agonist or
antagonist. The
ability of the candidate molecule to agonize or antagonize the BASB 121
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
BASB 121 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
converted into product, a reporter gene that is responsive to changes in BASB
121
polynucleotide or polypeptide activity, and binding assays known in the art.
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Another example of an assay for BASB 121 agonists is a competitive assay that
combines
BASB121 and a potential agonist with BASB121-binding molecules, recombinant
BASB 121 binding molecules, natural substrates or ligands, or substrate or
ligand mimetics,
under appropriate conditions for a competitive inhibition assay. BASB 121 can
be labeled,
such as by radioactivity or a colorimetric compound, such that the number of
BASB 121
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
BASB 121-induced activities, thereby preventing the action or expression of
BASB 121
polypeptides and/or polynucleotides by excluding BASB 121 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 BASB 121.
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
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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
BASB 121 proteins that mediate tissue damage and/or; to block the normal
progression of
pathogenesis in infections initiated other than by the implantation of in-
dwelling devices
or by other surgical techniques.
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In accordance with yet another aspect of the invention, there are provided
BASB 121
agonists and antagonists, preferably bacteristatic or bactericidal agonists
and antagonists.
The antagonists and agor~ists 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 Garner 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 tie performed to create a beneficial derivative, for
example to
enhance stability of the peptide.
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 B1). This technique, generates a
large number
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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 BASB 121 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 Moraxella catarrhalis 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 BASB121 polynucleotide and/or polypeptide, or a fragment or a
variant
thereof, for expressing BASB 121 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.
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
BASB 121 polynucleotide and/or polypeptide encoded therefrom, wherein the
composition
comprises a recombinant BASB 121 polynucleotide and/or polypeptide encoded
therefrom
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and/or comprises DNA and/or RNA which encodes and expresses an antigen of said
BASB 121 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 BASB 121 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
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 influenzae, 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 BASB 121 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.
Also suitable are non-live vectors for the BASB 121 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,
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Brucella melitensis, Brucella ovis, Esherichia coli, Haemophilus influenza,
Legionella
pneumophila, Moraxella catarrhalis, 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 BASB 121 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 BASB 121 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
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
BASB 121 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
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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 earned 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
expression includes the promoters porA, porB, lbpB, tbpB, p 110, 1st, hpuAB
from N.
meningitidis or N. gonorroheae; ompCD, copB, lbpB, ompE, UspAl; UspA2; TbpB
from
M. Catarrhalis; p1, 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
CA 02380498 2002-O1-23
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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 BASB
121 gene,
which modified upstream region contains a heterologous regulatory element
which alters
the expression level of the BASB 121 protein located at the outer membrane.
The
upstream region according to this aspect of the invention includes the
sequence upstream
of the BASB 121 gene. The upstream region starts immediately upstream of the
BASB 121
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.
Thus, the invention provides a BASB 121 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
BASB 121 gene having a modified upstream region containing a heterologous
regulatory
element.
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Further provided by the invention are processes to prepare the host cells and
bacterial blebs
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 Moraxella
catarrhalis.
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 Moraxella catarrhalis 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
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, bacteriostatic 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
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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 THl 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 THl-type responses (cell-mediated response), and TH2-type
immune
responses (humoral response).
Extreme THl-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 IgGl, IgA, and IgM.
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
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THl or predominantly TH2. However, it is often convenient to consider the
families of
cytokines in terms of that described in murine CD4 +ve T cell clones by
Mosmann and
Coffinan (Mosmann, T R. and Coffman, R.L. (1989) THl and TH2 cells: different
patterns of lymphokine secretion lead to different functional properties.
Annual Review
oflmmunology, 7, p145-173). Traditionally, TH1-type responses are associated
with
the production of the INF-y and IL-2 cytokines by T-lymphocytes. Other
cytokines
often directly associated with the induction of THl-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
either TH1 or TH2 - type cytokine responses. Traditionally the best indicators
of the
THl:TH2 balance of the immune response after a vaccination or infection
includes
direct measurement of the production of THl or TH2 cytokines by T lymphocytes
in
vitro after restimulation with antigen, and/or the measurement of the IgGl
:IgG2a ratio
of antigen specific antibody responses.
Thus, a THl-type adjuvant is one which preferentially stimulates isolated T-
cell
populations to produce high levels of THl-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 TH1-type isotype.
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, 5 or 6 acylated chains and is manufactured by
Ribi
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Immunochem, Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid
A is disclosed in European Patent 0 689 454 B 1 (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 l Opg - 100pg preferably 25-SOpg 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 an
Garner.
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.
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 TH1 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 : 1;
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.
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Preferably a Garner 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 lpg - 200pg, such as 10-100~g, preferably lOp,g - SO~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.
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 Garner. 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
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polyvalent vaccine composition may include a TH-1 inducing adjuvant as
hereinbefore
described.
While the invention has been described with reference to certain BASB 121
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.
Compositions, kits and administration
In a further aspect of the invention there are provided compositions
comprising a BASB 121
polynucleotide and/or a BASB 121 polypeptide for administration to a cell or
to a
multicellular organism.
The invention also relates to compositions comprising a polynucleotide and/or
a
polypeptides discussed herein or their agonists or antagonists. The
polypeptides and
polynucleotides of the invention may be employed in combination with a non-
sterile or
sterile carrier or carriers 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 carrier 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.
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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 andlor polynucleotide of the present
invention, agonist
or antagonist peptide or small molecule compound, in combination with a
pharmaceutically
acceptable carrier 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
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 inj ection,
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.
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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-S
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
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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.
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
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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;
Biocomputing:
Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York,
1993;
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., J. Molec. Biol. 215: 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;
46
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Altschul, S., et al., J. Mol. Biol. 215: 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
Gap Penalty: 50
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:1, wherein said
polynucleotide
sequence may be identical to the reference sequence of SEQ ID NO: l or may
include up
to a certain integer number of nucleotide alterations as compared to the
reference
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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 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 xn and y
is rounded
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
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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:1, 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
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)
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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
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 NO: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.
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"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.
"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
S1
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acid residue may or may not be one encoded by the genetic code. A variant of a
polynucleotide or polypeptide may be a naturally occurnng such as an allelic
variant, or
it may be a variant that is not known to occur naturally. Non-naturally
occurnng
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, otitis media in infants and children, pneumonia in elderlies,
sinusitis,
nosocomial infections and invasive diseases, chronic otitis media with hearing
loss, fluid
accumulation in the middle ear, auditive nerve damage, delayed speech
learning, infection
of the upper respiratory tract and inflammation of the middle ear.
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EXAMPLES:
The examples below are carned 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: DNA sequencing of the BASB121 gene from Moraxella catarrhalis
strain ATCC 43617.
A: BASB 121 in Moraxella catarrhalis strain.
The BASB 121 gene of SEQ ID NO:1 is from Moraxella catarrhalis strain ATCC
43617. The translation of the BASB121 polynucleotide sequence is showed in SEQ
ID
N0:2.
B: BASB121 in Moraxella Catarrhalis strain 43617.
The sequence of the BASB 121 gene was confirmed in Moraxella Catarrhalis
strain
ATCC 43617. For this purpose, plasmid DNA (see example 2A) containing the gene
region encoding the mature BASB 121 from Moraxella Catarrhalis. strain ATCC
43617
used as a PCR template. This material was then submitted to Polymerase Chain
Reaction DNA amplification using primers pTLZ F 5' TGA CAA TTA ATC ATC GGC
TCG-3' [SEQ ID NO:S] and reverse pTLZ RV.2 5' GGC TGA AAA TCT TCT CTC
ATC C-3' [SEQ ID N0:6]. The PCR amplicon was then submitted to DNA sequencing
using the Big Dyes kit (Applied biosystems) and analyzed on a ABI 373/A DNA
sequencer in the conditions described by the supplier. As a result, the
polynucleotide
and deduced polypeptide sequences, referred to as SEQ ID N0:3 and SEQ ID N0:4
respectively, were obtained. These sequences do not comprise the signal
sequence as the
signal sequence was from the plasmid.
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Using the MegAlign program from the DNASTAR software package, an alignment of
the polynucleotide sequences of SEQ ID NO:1 and 3 was performed, and is
displayed in
Figure l; a pairwise comparison of identities shows that the two BASB121
polynucleotide gene sequences are 100% identical in the region coding for the
mature
protein. Using the same MegAlign program, an alignment of the polypeptide
sequences
of SEQ ID N0:2 and 4 was performed, and is displayed in Figure 2; a pairwise
comparison of identities shows that the two BASB 121 protein sequences are
100%
identical in the region of the mature protein.
Example 2: Construction of Plasmid to Express Recombinant BASB121
A: Cloning of BASB 121.
The EcoRI and SaII restriction sites engineered into the MC-Lipl3-Fn/t-RI (5'-
AGG
CAG AGG GAA TTC ATG AAA ATC CGA GTA AAG TGG CC -3') [SEQ ID N0:7]
forward and MC-Lipl3RCh/t-Sal (5'-AGG CAG AGG GTC GAC TTA ATG GTG
ATG GTG ATG GTG TTT ATA ATC ACT TAA TAA TTT ATA-3') [SEQ ID N0:8]
reverse amplification primers, respectively, permitted directional cloning a
PCR product
into the E. coli expression plasmid pTLZ2 such that a mature BASB 121 protein
could be
expressed as a fusion protein containing a (His)6 affinity chromatography tag
at the C-
terminus. The BASB 121 PCR product was purified from the amplification
reaction
using silica gel-based spin columns (QiaGen) according to the manufacturers
instructions. To produce the required EcoRI and SaII termini necessary for
cloning,
purified PCR product was sequentially digested to completion with EcoRI and
SaII
restriction enzymes as recommended by the manufacturer (Life Technologies).
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.
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B: Production of Expression Vector.
To prepare the expression plasmid pTLZ2 for ligation, it was similarly
digested to
completion with both EcoRI and SaII 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 ~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 (~5 ~l) was used to transform electro-competent JM109 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
ampicillin (100 ~g/ml). Antibiotic was included in the selection. 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
BASB 121
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 Beckmann
microcentrifuge (~3 min., room temperature, 12,000 X g). The cell pellet was
suspended in ~200~1 of sterile water and a ~10?l aliquot used to program a
~50~1 final
volume PCR reaction containing both BASB 121 forward and reverse amplification
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 BASB121
fragment from the
lysed transformant samples. Following thermal amplification, a ~20~1 aliquot
of the
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reaction was analyzed by agarose gel electrophoresis (0.8 % agarose in a Tris-
acetate-
EDTA (TAE) buffer). DNA fragments were visualized by LJV 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 size PCR product were identified as strains containing a BASB 121
expression
construct. Expression plasmid containing strains were then analyzed for the
inducible
expression of recombinant BASB 121.
C: Expression Analysis of PCR-Positive Transformants.
For each PCR-positive transformant identified above, ~5.0 ml of LB broth
containing
ampicillin (100 ug/ml) was inoculated with cells from the patch plate and
grown
overnight at 37 °C with shaking 0250 rpm). An aliquot of the overnight
seed culture
(~l .0 ml) was inoculated into a 125 ml erlenmeyer flask containing ~25 of LB
Ap broth
and grown at 37 °C with shaking 0250 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 012.5 ml) was transferred to a second 125 ml flask and
expression of
recombinant BASB121 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 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
Laemelli
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
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(SeeBlue, Novex) under conventional conditions using a standard
SDS/Tris/glycine
running buffer (BioRad). Following electrophoresis, one gel was stained with
commassie brilliant blue 8250 (BioRad) and then destained to visualize novel
BASB 121 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-RGS (His)3 antibody, followed by a second
rabbit
anti-mouse antibody conjugated to HRP (QiaGen), was used to confirm the
expression
and identity of the BASB 121 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: Production of Recombinant BASB121
Bacterial strain
A recombinant expression strain of E. coli JM109 containing a plasmid (pTLZ2)
encoding BASB 121 from M. catarrhalis. was used to produce cell mass for
purification
of recombinant protein. The expression strain was cultivated on LB agar plates
containing 100pg/ml ampicillin ("Ap") to ensure that the pTLZ2 was maintained.
For
cryopreservation at -80 °C, the strain was propagated in LB broth
containing the same
concentration of antibiotics 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 BASB
121
recombinant protein, IPTG (Isopropyl 13-D-Thiogalactopyranoside) was added to
the
fermentor (1 mM, final).
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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
150rpm (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
antibiotics. 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.
Chemicals and Materials.
Imidazole and Triton X-100 were purchased from Merck. Guanidine hydrochloride
was
from Fluka. Aprotinin was obtained from Sigma Chemical Company. Urea and
AEBSF were from ICN-Biochemicals. All other chemicals were reagent grade or
better.
Ni-NTA Superflow resin and Penta-His Antibody, BSA free were obtained from
QiaGen. MicroBCA assay was obtained from Pierce; Amicon 3 filters from
Millipore.
Dialysis membrane (MWC012-14000) were from MFPI, USA. Molecular mass marker
(BenchMark ladder) was from Life-technologies.
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Example 4: Purification of recombinant BASB121 from E. coli.
Extraction-Purification
Cell paste from 750 ml 1PTG induced culture (~4 hours, OD620= 0.5) was
resuspended
in 60 ml of phosphate buffer pH 7.5 containing 1mM AEBSF and 1mM Aprotinin as
protease inhibitors. Cells were lysed in a cell disruptor. Lysate was
centrifuged at
27,OOOg for 20 minutes. Pellet was washed once with phosphate buffer pH 7.5
and
centrifuged again at 27,OOOg for 20 minutes. Pellet was suspended in 100 mM
NaH2P04, 10 mM Tris-HCl buffer pH 8 containing 6M Guanidium Chloride (buffer
A)
and left for 1 hour at room temperature. Total extract was centrifuged at
27,OOOg for 20
minutes. Supernatant was incubated for 1 hour at room temperature with Ni-NTA
superflow resin equilibrated in buffer A. Resin was washed twice with 100 mM
NaH2P04, 10 mM Tris-HCl buffer pH 6.3, containing 8M Urea (buffer B). Elution
was
performed with buffer B containing successively 50 mM, 100 mM, and 200 mM
Imidazole. Fractions containing BASB 121 protein were pooled, 3 fold diluted
with
buffer B and applied to a Q-Sepharose resin in a batch mode. Flowthrough was
dialyzed against 100 mM NaH2P04, 10 mM Tris-HCl buffer pH 6.3, containing 4M
Urea, then 2M Urea, and finally against PBS buffer pH 7.4 containing 0.1 %
Triton X-
100. Attempts to concentrate the protein failed as precipitation occurred when
the
concentration reached 50 ~g/ml.
Purified BASB121 protein was quantified using Micro BCA assay reagent.
0.5 mg of purified protein were obtained, at a final concentration of 50
ug/ml.
As shown in figure 3-A, purified BASB 121 protein appeared in SDS-PAGE
analysis as
a major band migrating at around 23 kDa (estimated relative molecular mass).
Purity
was estimated to more than 50 %. BASB121protein was reactive against a mouse
monoclonal antibody raised against the 6-Histidine motif (figure 3-B).
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Example 5: Production of Antisera to Recombinant BASB121
Polyvalent antisera directed against the BASB 121 protein are generated by
vaccinating
rabbits with the purified recombinant BASB 121 protein. Animals are bled prior
to the
first immunization ("pre-bleed") and after the last immunization.
Anti-BASB 121 protein titres are measured by an ELISA using purified
recombinant
BASB 121 protein. The titre is defined as mid-point titers calculated by 4-
parameter
logistic model using the XL Fit software.
The antisera are also used as the first antibody to identify the protein in a
western blot as
described in example 7 below. The western-blot is used to demonstrate the
presence of
anti-BASB 121 antibody in the sera of immunized animals.
Example 6: Immunological characterization: Surface exposure of BASB121
Anti-BASB 121 protein titres are determined by an ELISA using formalin-killed
whole
cells of Moraxella catarrhalis. The titre is defined as mid-point titers
calculated by 4-
parameter logistic model using the XL Fit software.
The titre observed with the rabbit or mouse immune sera demonstrates that the
BASB 121 protein is present at the surface of M. catarrhalis cells.
Example 7. Immunological Characterisation: Western Blot Anal sis
Several strains of M. catarrhalis including ATCC 43617, as well as clinical
isolates
from various geographic regions, are grown on Muller Hinton agar plates for 24
hours at
36°C. Several colonies are used to inoculate broth. Cultures are grown
until the A620
is approximately 0.6 and cells are collected by centrifugation. Cells are then
concentrated and solubilized in PAGE sample buffer. The solubilized cells are
then
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resolved on 4-20% polyacrylamide gels and the separated proteins are
electrophoretically transferred to PVDF membranes. The PVDF membranes are then
pretreated with saturation buffer. All subsequent incubations are carned out
using this
pretreatment buffer.
PVDF membranes are incubated with preimmune serum or rabbit or mouse immune
seru. PVDF membranes are then washed.
PVDF membranes are incubated with biotin-labeled sheep anti-rabbit or mouse
Ig.
PVDF membranes are then washed 3 times with wash buffer, and incubated with
streptavidin-peroxydase. PVDF membranes are then washed 3 times with wash
buffer
and developed with 4-chloro-1-naphtol.
Detection of a protein corresponding to BASB 121 expected molecular weight
that is
reactive with the antisera is used to demonstrate that this protein is
produced by and
conserved in all Moraxella strains tested.
Example 8: Immunological characterization: Bactericidal Activity
Complement-mediated cytotoxic activity of anti-BASB 121 antibodies is examined
to
determine the vaccine potential of BASB 121 protein antiserum that is prepared
as
described above. The activities of the pre-immune serum and the anti-BASB 121
antiserum in mediating complement killing of M. catarrhalis are examined.
Strains ofM.catarrhalis are grown on plates. Several colonies are added to
liquid
medium. Cultures are grown and collected until the A620 is approximately 0.4.
After
one wash step, the pellet is suspended and diluted.
Preimmune sera and the anti-BASB 121 sera is deposited into the first well of
a 96-wells
plate and serial dilutions are deposited in the other wells of the same line.
Live diluted
M catarrahlis is subsequently added and the mixture is incubated. Complement
is added
into each well at a working dilution defined beforehand in a toxicity assay.
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Each test includes a complement control (wells without serum containing active
or
inactivated complement source), a positive control (wells containing serum
with a know
titer of bactericidal antibodies), a culture control (wells without serum and
complement)
and a serum control (wells without complement).
Bactericidal activity of rabbit or mice antiserum (50% killing of homologous
strain) is
measured.
Example 9: Presence of Antibody to BASB121 in Human Convalescent Sera
Western blot analysis of purified recombinant BASB 121 is performed as
described in
Example 7 above, except that a pool of human sera from children infected by M.
catarrhalis is used as the first antibody preparation. This is used to show
that antisera
from naturally infected individuals react to the purified recombinant protein.
Example 10: Efficacy of BASB121 vaccine: enhancement of lung clearance of M.
catarrhalis in mice.
This mouse model is based on the analysis of the lung invasion by M.
catarrhalis
following a standard intranasal challenge to vaccinated mice.
Groups of mice are immunized with BASB 121 vaccine. After the booster, the
mice are
challenged by instillation of bacterial suspension into the nostril under
anaesthesia.
Mice are killed between 30 minutes and 24 hours after challenge and the lungs
are
removed aseptically and homogenized individually. The 1og10 weighted mean
number
of CFU/lung is determined by counting the colonies grown on agar plates after
plating
of dilutions of the homogenate. The arithmetic mean of the 1og10 weighted mean
number of CFU/lung and the standard deviations are calculated for each group.
Results are analysed statistically.
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In this experiment groups of mice are immunized either with BASB 121 or with a
killed
whole cells (kwc) preparation of M. catarrhalis or sham immunized.
This is used to show that the kwc preparation and the BASB 121 vaccine induce
significant lung clearance as compared to the control group.
Example 11: Inhibition of M. catarrhalis adhesion onto cells b anti-BASB121
antiserum.
This assay measures the capacity of anti BASB121 sera to inhibit the adhesion
of
Moraxella bacteria to epithelial cells. This activity could prevent
colonization of f.i. the
nasopharynx by Moraxella.
One volume of bacteria is incubated on ice with one volume of pre-immune or
anti-
BASB 121 immune serum dilution. This mixture is subsequently added in the
wells of a
24 well plate containing a confluent cells culture that is washed once with
culture
medium to remove traces of antibiotic. The plate is centrifuged and incubated.
Each well is then gently washed. After the last wash, sodium glycocholate is
added to
the wells. After incubation, the cell layer is scraped and homogenised.
Dilutions of the
homogenate are plated on agar plates and incubated. The number of colonies on
each
plate is counted and the number of bacteria present in each well calculated.
This is used to show that bacteria incubated with anti-BASB 121 antiserum are
inhibited
in their adherence capacity to the Hep-2 cells.
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Deposited materials
A deposit containing a Moraxella catarrhalis Catlin strain has been deposited
with the American
Type Culture Collection (herein "ATCC") on June 21, 1997 and assigned deposit
number 43617.
The deposit was described as Branhamella catarrhalis (Frosch and Kolle) and is
a freeze-dried, 1.5-
2.9 kb insert library constructed from M. catarrhalis isolate obtained from a
transtracheal aspirate of
a coal miner with chronic bronchitits. The deposit is described in Antimicrob.
Agents Chemother.
21: 506-508 ( 1982).
The Moraxella catarrhalis strain deposit is referred to herein as "the
deposited strain" or as "the
DNA of the deposited strain."
The deposited strain contains a full IengthBASB121 gene.
A deposit of the vector pMC-D15 consisting ofMoraxella catarrhalis DNA
inserted in pQE30 has
been deposited with the American Type Culture Collection (ATCC) on February 12
1999 and
assigned deposit number 207105.
The sequence of the polynucleotides contained in the deposited strain / clone,
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.
The deposit of the deposited strains have 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
deposited strains will be irrevocably and without restriction or condition
released to the public upon
the issuance of a patent. The deposited strains are provided merely as
convenience to those of skill
in the art and are 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 A DEPOSITED MICROORGANISM
(PCT Rule l3bis)
A. The indications made below relate
to the microorganism referred
to in the description
on page 64 , lines 1-2, 10-12
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depositary institution
AMERICAN TYPE CULTURE COLLECTION
Address of depositary institution
(including postal code and country)
10801 UNIVERSITY BLVD, MANASSAS,
VIRGINIA 20110-2209, UNITED STATES
OF
AMERICA
Date of deposit 21/06/97 12/02/99 Accession Number
21 June 1997 12 February 1999 43617 & 207105
C. ADDITIONAL INDICATIONS (leave
blank ifnot 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 microorganisms
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
the sample
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (if the indications
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 (specify the general
nature of the indications e.g.,
"Accession Number of Deposit "~
For receiving Office use only For International Bureau use only -
This sheet was received with the international ~ This sheet was received by
the International Bureau
application on:
Authorized officer / / Authorized o~cer
~.t~c3~
Form PCT/RO/134 (July 1992)
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SEQUENCE INFORMATION
BASB121 Polynucleotide and Polypeptide Sequences
SEQ ID NO:1
Moraxella catarrhalis BASB121 polynucleotide sequence from strain ATCC43617
ATGAAAATCCGAGTAAAGTGGCCTATGGTAATGGCGATGGGGCTGGTTTTAAGTGCCTGTCAAAGTACACCAATACCGC
C
TAAAAATAATCCGCAATTGGCACAAATCCGAACCCAAATTGCAATCTCGCTACTGGACATGGGTAAGCTTGATCAAGCC
A
AACAGCAGTTGGATGCTGCTTTATCGGCTGATCGGCAGTTTGCACCTGCCTATCGCACCTTGGCAAAGGTTTATCAAGC
T
TCGGAGGATGCCACTCACCAAACCAAAGCTCAACGCTTGTTTGAAAAGGCGATTGAACTAAATCCTAAAGATATGCAAA
G
TTATATGGATTATGGATTTTATTTGGTGCAGATGGGGGACTTGTCAGGTGCGTTGATTTATTTTGATAAACCTAGCCGA
G
CCATCGGTTATGAAGGGCGTGTGGTAGCCATCGAAAATATGGCATATATTTATTATCATCAATATGAAGCTGCCAAATC
A
CCAACAAAAGATGACTATAATAACGCCAAATCAGCACTTGAGCGTGCGTTAATTTCAGGCACTCAACATGATGAAATCA
A
AAAGTCTTATGACAAATTATTAAGTGATTATAAATTATTAAGTGATTATAAATAG
SEQ ID N0:2
Moraxella catarrhalis BASB121 polypeptide sequence deduced from the
polynucleotide of
SeQ ID NO:1
MKIRVKWPMVMAMGLVLSACQSTPIPPKNNPQLAQIRTQIAISLLDMGKLDQAKQQLDAALSADRQFAPAYRTLAKVYQ
A
SEDATHQTKAQRLFEKAIELNPKDMQSYMDYGFYLVQMGDLSGALIYFDKPSRAIGYEGRVVAIENMAYIYYHQYEAAK
S
PTKDDYNNAKSALERALISGTQHDEIKKSYDKLLSDYKLLSDYK
SEQ ID N0:3
Moraxella catarrhalis BASB121 polynucleotide sequence from strain ATCC43617
ATGAAAATCCGAGTAAAGTGGCCTATGGTAATGGCGATGGGGCTGGTTTTAAGTGCCTGTCAAAGTACACCAATACCGC
C
TAAAAATAATCCGCAATTGGCACAAATCCGAACCCAAATTGCAATCTCGCTACTGGACATGGGTAAGCTTGATCAAGCC
A
AACAGCAGTTGGATGCTGCTTTATCGGCTGATCGGCAGTTTGCACCTGCCTATCGCACCTTGGCAAAGGTTTATCAAGC
T
TCGGAGGATGCCACTCACCAAACCAAAGCTCAACGCTTGTTTGAAAAGGCGATTGAACTAAATCCTAAAGATATGCAAA
G
TTATATGGATTATGGATTTTATTTGGTGCAGATGGGGGACTTGTCAGGTGCGTTGATTTATTTTGATAAACCTAGCCGA
G
CCATCGGTTATGAAGGGCGTGTGGTAGCCATCGAAAATATGGCATATATTTATTATCATCAATATGAAGCTGCCAAATC
A
CCAACAAAAGATGACTATAATAACGCCAAATCAGCACTTGAGCGTGCGTTAATTTCAGGCACTCAACATGATGAAATCA
A
AAAGTCTTATGACAAATTATTAAGTGATTATAAATTATTAAGTGATTATAAA
SEQ ID N0:4
Moraxella catarrhalis BASB121 polypeptide sequence deduced from the
polynucleotide of
SeQ ID N0:3
MKIRVKWPMVMAMGLVLSACQSTPIPPKNNPQLAQIRTQIAISLLDMGKLDQAKQQLDAALSADRQFAPAYRTLAKVYQ
A
SEDATHQTKAQRLFEKAIELNPKDMQSYMDYGFYLVQMGDLSGALIYFDKPSRAIGYEGRVVAIENMAYIYYHQYEAAK
S
PTKDDYNNAKSALERALISGTQHDEIKKSYDKLLSDYKLLSDYK
SEQ ID NO:S
TGA CAA TTA ATC ATC GGC TCG
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SEQ ID N0:6
GGC TGA AAA TCT TCT CTC ATC C
SEQ ID N0:7
AGG CAG AGG GAA TTC ATG AAA ATC CGA GTA AAG TGG CC
SEQ ID N0:8
AGG CAG AGG GTC GAC TTA ATG GTG ATG GTG ATG GTG TTT ATA ATC
ACT TAA TAA TTT ATA
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SEQUENCE LISTING
<110> SmithKline Beecham Biologicals SA
<120> Novel Compounds
<130> BM45398
<160> 8
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 615
<212> DNA
<213> Moraxella catarrhalis
<400> 1
atgaaaatccgagtaaagtggcctatggtaatggcgatggggctggttttaagtgcctgt60
caaagtacaccaataccgcctaaaaataatccgcaattggcacaaatccgaacccaaatt120
gcaatctcgctactggacatgggtaagcttgatcaagccaaacagcagttggatgctgct180
ttatcggctgatcggcagtttgcacctgcctatcgcaccttggcaaaggtttatcaagct240
tcggaggatgccactcaccaaaccaaagctcaacgcttgtttgaaaaggcgattgaacta300
aatcctaaagatatgcaaagttatatggattatggattttatttggtgcagatgggggac360
ttgtcaggtgcgttgatttattttgataaacctagccgagccatcggttatgaagggcgt420
gtggtagccatcgaaaatatggcatatatttattatcatcaatatgaagctgccaaatca480
ccaacaaaagatgactataataacgccaaatcagcacttgagcgtgcgttaatttcaggc540
actcaacatgatgaaatcaaaaagtcttatgacaaattattaagtgattataaattatta600
agtgattataaatag 615
<210> 2
<211> 204
<212> PRT
<213> Moraxella catarrhalis
<400> 2
Met Lys Ile Arg Val Lys Trp Pro Met Val Met Ala Met Gly Leu Val
1 5 10 15
Leu Ser Ala Cys Gln Ser Thr Pro Ile Pro Pro Lys Asn Asn Pro Gln
20 25 30
Leu Ala Gln Ile Arg Thr Gln Ile Ala Ile Ser Leu Leu Asp Met Gly
35 40 45
Lys Leu Asp Gln Ala Lys Gln Gln Leu Asp Ala Ala Leu Ser Ala Asp
50 55 60
Arg Gln Phe Ala Pro Ala Tyr Arg Thr Leu Ala Lys Val Tyr Gln Ala
65 70 75 80
Ser Glu Asp Ala Thr His Gln Thr Lys Ala Gln Arg Leu Phe Glu Lys
85 90 95
Ala Ile Glu Leu Asn Pro Lys Asp Met Gln Ser Tyr Met Asp Tyr Gly
100 105 110
Phe Tyr Leu Val Gln Met Gly Asp Leu Ser Gly Ala Leu Ile Tyr Phe
115 120 125
Asp Lys Pro Ser Arg Ala Ile Gly Tyr Glu Gly Arg Val Val Ala Ile
130 135 140
Glu Asn Met Ala Tyr Ile Tyr Tyr His Gln Tyr Glu Ala Ala Lys Ser
145 150 155 160
Pro Thr Lys Asp Asp Tyr Asn Asn Ala Lys Ser Ala Leu Glu Arg Ala
1
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165 170 175
Leu Ile Ser Gly Thr Gln His Asp Glu Ile Lys Lys Ser Tyr Asp Lys
180 185 190
Leu Leu Ser Asp Tyr Lys Leu Leu Ser Asp Tyr Lys
195 200
<210> 3
<211> 612
<212> DNA
<213> Moraxella catarrhalis
<400>
3
atgaaaatccgagtaaagtggcctatggtaatggcgatggggctggttttaagtgcctgt60
caaagtacaccaataccgcctaaaaataatccgcaattggcacaaatccgaacccaaatt120
gcaatctcgctactggacatgggtaagcttgatcaagccaaacagcagttggatgctgct180
ttatcggctgatcggcagtttgcacctgcctatcgcaccttggcaaaggtttatcaagct240
tcggaggatgccactcaccaaaccaaagctcaacgcttgtttgaaaaggcgattgaacta300
aatcctaaagatatgcaaagttatatggattatggattttatttggtgcagatgggggac360
ttgtcaggtgcgttgatttattttgataaacctagccgagccatcggttatgaagggcgt420
gtggtagccatcgaaaatatggcatatatttattatcatcaatatgaagctgccaaatca480
ccaacaaaagatgactataataacgccaaatcagcacttgagcgtgcgttaatttcaggc540
actcaacatgatgaaatcaaaaagtcttatgacaaattattaagtgattataaattatta600
agtgattataas 612
<210> 4
<211> 204
<212> PRT
<213> Moraxella catarrhalis
<400> 4
Met Lys Ile Arg Val Lys Trp Pro Met Val Met Ala Met Gly Leu Val
1 5 10 15
Leu Ser Ala Cys Gln Ser Thr Pro Ile Pro Pro Lys Asn Asn Pro Gln
20 25 30
Leu Ala Gln Ile Arg Thr Gln Ile Ala Ile Ser Leu Leu Asp Met Gly
35 40 45
Lys Leu Asp Gln Ala Lys Gln Gln Leu Asp Ala Ala Leu Ser Ala Asp
50 55 60
Arg Gln Phe Ala Pro Ala Tyr Arg Thr Leu Ala Lys Val Tyr Gln Ala
65 70 75 80
Ser Glu Asp Ala Thr His Gln Thr Lys Ala Gln Arg Leu Phe Glu Lys
85 90 95
Ala Ile Glu Leu Asn Pro Lys Asp Met Gln Ser Tyr Met Asp Tyr Gly
100 105 110
Phe Tyr Leu Val Gln Met Gly Asp Leu Ser Gly Ala Leu Ile Tyr Phe
115 120 125
Asp Lys Pro Ser Arg Ala Ile Gly Tyr Glu Gly Arg Val Val Ala Ile
130 135 140
Glu Asn Met Ala Tyr Ile Tyr Tyr His Gln Tyr Glu Ala Ala Lys Ser
145 150 155 160
Pro Thr Lys Asp Asp Tyr Asn Asn Ala Lys Ser Ala Leu Glu Arg Ala
165 170 175
Leu Ile Ser Gly Thr Gln His Asp Glu Ile Lys Lys Ser Tyr Asp Lys
180 185 190
Leu Leu Ser Asp Tyr Lys Leu Leu Ser Asp Tyr Lys
195 200
<210> 5
2
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<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 5
tgacaattaa tcatcggctc g 21
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 6
ggctgaaaat cttctctcat cc 22
<210> 7
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 7
aggcagaggg aattcatgaa aatccgagta aagtggcc 3g
<210> 8
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 8
aggcagaggg tcgacttaat ggtgatggtg atggtgttta taatcactta ataatttata 60
3
