U.S. patent application number 10/878939 was filed with the patent office on 2004-11-25 for immunogenic compounds.
This patent application is currently assigned to Smithkline Beecham. Invention is credited to Ruelle, Jean-Louis.
Application Number | 20040235114 10/878939 |
Document ID | / |
Family ID | 10848648 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040235114 |
Kind Code |
A1 |
Ruelle, Jean-Louis |
November 25, 2004 |
Immunogenic compounds
Abstract
The invention provides BASB081 polypeptides and polynucleotides
encoding BASB081 polypeptides and methods for producing such
polypeptides by recombinant techniques. Also provided are
diagnostic, prophylactic and therapeutic uses.
Inventors: |
Ruelle, Jean-Louis; (Limal,
BE) |
Correspondence
Address: |
DECHERT
ATTN: ALLEN BLOOM, ESQ
4000 BELL ATLANTIC TOWER
1717 ARCH STREET
PHILADELPHIA
PA
19103
US
|
Assignee: |
Smithkline Beecham
Rixensart
BE
|
Family ID: |
10848648 |
Appl. No.: |
10/878939 |
Filed: |
June 28, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10878939 |
Jun 28, 2004 |
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09914168 |
Jan 28, 2002 |
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6770459 |
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09914168 |
Jan 28, 2002 |
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PCT/EP00/01468 |
Feb 23, 2000 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/324; 530/325; 530/326; 536/23.5 |
Current CPC
Class: |
C12R 2001/38 20210501;
A61K 2039/53 20130101; C12N 1/205 20210501 |
Class at
Publication: |
435/069.1 ;
435/320.1; 435/325; 530/324; 530/325; 530/326; 536/023.5 |
International
Class: |
C07K 014/47; C07H
021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 6, 1999 |
GB |
9904559.3 |
Claims
What is claimed is:
1. An isolated polypeptide comprising a member selected from the
group consisting of (a) an amino acid sequence matching one of SEQ
ID NOs:2 or 4; (b) an immunogenic polypeptide comprising a fragment
sequence of at least 15 amino acids that matches an aligned
contiguous segment of one of SEQ ID NOs:2 or 4; wherein the
isolated polypeptide, when administered to a subject in a suitable
composition which can include an adjuvant, or a suitable carrier
coupled to the polypeptide, induces an antibody or T-cell immune
response to a polypeptide having the sequence of one of SEQ ID
NOs:2 or 4.
2. An isolated polynucleotide encoding a polypeptide of claim 1 or
the full complement to the isolated polynucleotide.
3. The isolated polypeptide of claim 1, wherein the polypeptide is
according to (a).
4. An isolated polynucleotide encoding a polypeptide of claim 3 or
the full complement to the isolated polynucleotide.
5. The isolated polypeptide of claim 1, wherein the polypeptide is
according to (b).
6. An isolated polynucleotide encoding a polypeptide of claim 5 or
the full complement to the isolated polynucleotide.
7. The isolated polypeptide of claim 1, wherein the immunogenic
fragment of (b) comprises at least 20 amino acids.
8. The isolated polypeptide of claim 1 wherein the isolated
polypeptide of (a) consists of one of SEQ ID NOs: 2 or 4.
9. An isolated polynucleotide encoding a polypeptide of claim 8 or
the full complement to the isolated polynucleotide.
10. A process for expressing the polynucleotide of claim 9
comprising transforming a host cell with an expression vector
comprising the polynucleotide and culturing the host cell under
conditions sufficient for expression of the polynucleotide.
11. A fusion protein comprising the isolated polypeptide of claim
1.
12. An isolated polynucleotide comprising the polynucleotide of one
of SEQ ID NOs:1 or 3.
13. An isolated polynucleotide segment comprising a polynucleotide
sequence or the full complement of the entire length of the
polynucleotide sequence, wherein the polynucleotide sequence
hybridizes to the full complement of one of SEQ ID NOs:1 or 3 minus
the complement of any stop codon, wherein the hybridization
conditions include incubation at 42.degree. C. in a solution
comprising: 50% formamide, 5.times.SSC (150 mM NaCl, 15 mM
trisodium citrate), 50 mM sodium phosphate (pH7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml
denatured, sheared salmon sperm DNA, followed by washing in
0.1.times.SSC at 65.degree. C.; and, wherein the polynucleotide
sequence is identical to one of SEQ ID NOs:1 or 3 minus any
terminal stop codon, except that, over the entire length
corresponding to one of SEQ ID NOs:1 or 3 minus any terminal stop
codon, n.sub.n nucleotides are substituted, inserted or deleted,
wherein n.sub.n satisfies the following
expressionn.sub.n.ltoreq.x.sub.n-(x.sub.n.multidot.y)wherein
x.sub.n is the total number of nucleotides in one of SEQ ID NOs:1
or 3 minus any terminal stop codon, y is at least 0.95, and wherein
any non-integer product of x.sub.n and y is rounded down to the
nearest integer before subtracting the product from x.sub.n; and
wherein the polynucleotide sequence detects Moraxella
catarrhalis.
14. An expression vector comprising the isolated polynucleotide of
claim 2.
15. A host cell transformed with the expression vector of claim
14.
16. A vaccine comprising the polypeptide of claim 1 and a
pharmaceutically acceptable carrier.
17. The vaccine of claim 16, wherein the vaccine comprises at least
one other Moraxella catarrhalis antigen.
18. An antibody immunospecific for the polypeptide or immunogenic
fragment of claim 1.
19. A method for inducing an immune response in a mammal comprising
administration of the polypeptide of claim 1.
20. A method of diagnosing a Moraxella catarrhalis infection,
comprising identifying a polypeptide of claim 1, or an antibody
that is immunospecific for the polypeptide, present within a
biological sample from an animal suspected of having such an
infection.
21. A method for inducing an immune response in a mammal comprising
administration of the isolated polynucleotide of claim 2.
22. A therapeutic composition useful in treating humans with
Moraxella catarrhalis comprising at least one antibody directed
against the polypeptide of claim 1 and a suitable pharmaceutical
carrier.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/914,168, filed Jan. 28, 2002, which is the National
Stage Application of International Application No. PCT/EP00/01468,
filed Feb. 23, 2000, which claims the benefit of Great Britain
Patent Application No. 9904559.3, filed Feb. 26, 1999. The
disclosure of these applications are herein incorporated by
reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to polynucleotides, (herein referred
to as "BASB081 polynucleotide(s)"), polypeptides encoded by them
(referred to herein as "BASB081" or "BASB081 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
[0003] 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.
[0004] 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, J O (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.
[0005] 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 the otitis media cases (Murphy, T F (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).
[0006] 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, D P et
al. (1988) J. Infect.Dis. 158:205, Faden, H L 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, H L 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, T F (1996) Microbiol.Rev.
60:267).
[0007] 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, H L et al (1994) J.
Infect.Dis. 169:1312).
[0008] In most adults tested, bactericidal antibodies have been
identified (Chapman, A J et al. (1985) J. Infect.Dis. 151:878).
Strains of M. 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, K L et al.
(1990) Am.J.Med. 88 (suppl. 5A):28S). Serum resistance could
therfore be considered as a virulence factor of the bacteria. An
opsonizing activity has been observed in the sera of children
recovering from otitis media.
[0009] The antigens targetted by these different immune responses
in humans have not been identified, with the exception of OMP B1, 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 UspA1 and UspA2 (Chen D. et
al.(1999), Infect.Immun. 67:1310).
[0010] 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, T F (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.
[0011] 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
[0012] The present invention relates to BASB081, in particular
BASB081 polypeptides and BASB081 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
BASB081 polynucleotides or polypeptides.
[0013] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A-1N show consecutive, aligned sequences for two
BASB081 polynucleotides.
[0015] FIGS. 2A-2E show consecutive, aligned sequences for two
BASB081 polypeptides.
[0016] FIG. 3 shows an SDS-PAGE analysis of BASB081 expression in
non induced (N.I.) or induced (I) Escherichia coli Top 10
cells.
DESCRIPTION OF THE INVENTION
[0017] The invention relates to BASB081 polypeptides and
polynucleotides as described in greater detail below. In
particular, the invention relates to polypeptides and
polynucleotides of BASB081 of Moraxella catarrhalis, which is
related by amino acid sequence homology to Neisseria meningitidis
omp85 outer membrane protein. The invention relates especially to
BASB081 having the nucleotide and amino acid sequences set out in
SEQ ID NO:1 or 3 and SEQ ID NO: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.
[0018] Polypeptides
[0019] In one aspect of the invention there are provided
polypeptides of Moraxella catarrhalis referred to herein as
"BASB081" and "BASB081 polypeptides" as well as biologically,
diagnostically, prophylactically, clinically or therapeutically
useful variants thereof, and compositions comprising the same.
[0020] The present invention further provides for:
[0021] (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 NO:2 or 4;
[0022] (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:1
or 3 respectively; or
[0023] (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 NO:2
or 4.
[0024] The BASB081 polypeptides provided in SEQ ID NO:2 or 4 are
the BASB081 polypeptides from Moraxella catarrhalis strain Mc2931
(ATCC 43617).
[0025] The invention also provides an immunogenic fragment of a
BASB081 polypeptide, that is, a contiguous portion of the BASB081
polypeptide which has the same or substantially the same
immunogenic activity as the polypeptide comprising the amino acid
sequence of SEQ ID NO:2 or 4; That is to say, the fragment (if
necessary when coupled to a carrier) is capable of raising an
immune response which recognises the BASB081 polypeptide. Such an
immunogenic fragment may include, for example, the BASB081
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 BASB081 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 preferably at least 95% identity, most
preferably at least 97-99% identity, to that of SEQ ID NO:2 or 4
over the entire length of SEQ IDNO:2
[0026] 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 BASB081
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.
[0027] Preferred fragments include, for example, truncation
polypeptides having a portion of an amino acid sequence of SEQ ID
NO: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.
[0028] 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 NO: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 NO:2 or 4.
[0029] Fragments of the polypeptides of the invention may be
employed for producing the corresponding full-length polypeptide by
peptide synthesis; therefore, these fragments may be employed as
intermediates for producing the full-length polypeptides of the
invention.
[0030] 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.
[0031] 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.
[0032] 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 IgG1, 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.
[0033] 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.
WO94/29458 and WO94/22914.
[0034] 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.
[0035] Fusion partners include protein D from Haemophilus influenza
and the non-structural protein from influenzae virus, NS1
(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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] Polynucleotides
[0040] It is an object of the invention to provide polynucleotides
that encode BASB081 polypeptides, particularly polynucleotides that
encode the polypeptide herein designated BASB081.
[0041] In a particularly preferred embodiment of the invention the
polynucleotide comprises a region encoding BASB081 polypeptides
comprising a sequence set out in SEQ ID NO:1 or 3 which includes a
full length gene, or a variant thereof.
[0042] The BASB081 polynucleotides provided in SEQ ID NO:1 or 3 are
the BASB081 polynucleotides from Moraxella catarrhalis strain
Mc2931 (ATCC 43617).
[0043] As a further aspect of the invention there are provided
isolated nucleic acid molecules encoding and/or expressing BASB081
polypeptides and polynucleotides, particularly Moraxella
catarrhalis BASB081 polypeptides and polynucleotides, including,
for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic
DNAs, B- and Z-DNAs. Further embodiments of the invention include
biologically, diagnostically, prophylactically, clinically or
therapeutically useful polynucleotides and polypeptides, and
variants thereof, and compositions comprising the same.
[0044] Another aspect of the invention relates to isolated
polynucleotides, including at least one full length gene, that
encodes a BASB081 polypeptide having a deduced amino acid sequence
of SEQ ID NO:2 or 4 and polynucleotides closely related thereto and
variants thereof.
[0045] In another particularly preferred embodiment of the
invention there is a BASB081 polypeptide from Moraxella catarrhalis
comprising or consisting of an amino acid sequence of SEQ ID NO:2
or 4 or a variant thereof.
[0046] Using the information provided herein, such as a
polynucleotide sequence set out in SEQ ID NO:1 or 3, a
polynucleotide of the invention encoding BASB081 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 LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989). (see in
particular Screening By Hybridization 1.90 and Sequencing Denatured
Double-Stranded DNA Templates 13.70). Direct genomic DNA sequencing
may also be performed to obtain a full length gene sequence.
Illustrative of the invention, each polynucleotide set out in SEQ
ID NO:1 or 3 was discovered in a DNA library derived from Moraxella
catarrhalis.
[0047] 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 NO: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.
[0048] The polynucleotide of SEQ ID NO:1, between the start codon
at nucleotide number 1 and the stop codon which begins at
nucleotide number 2758 of SEQ ID NO:1, encodes the polypeptide of
SEQ ID NO:2.
[0049] The polynucleotide of SEQ ID NO:3, between the start codon
at nucleotide number 1 and the stop codon which begins at
nucleotide number 2668 of SEQ ID NO:3, encodes the polypeptide of
SEQ ID NO:4.
[0050] In a further aspect, the present invention provides for an
isolated polynucleotide comprising or consisting of:
[0051] (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
[0052] (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 NO:2 or
4, over the entire length of SEQ ID NO:2 or 4 respectively.
[0053] 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.degree. 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.
[0054] 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 377: 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.
[0055] The nucleotide sequence encoding BASB081 polypeptide of SEQ
ID NO:2 or 4 may be identical to the polypeptide encoding sequence
contained in nucleotides 1 to 2757 of SEQ ID NO:1 or the
polypeptide encoding sequence contained in nucleotides 1 to 2667 of
SEQ ID NO: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 NO:2 or 4.
[0056] 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 BASB081 having an amino acid sequence set out in SEQ ID
NO: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.
[0057] 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 NO:2 or
4. Fragments of polynucleotides of the invention may be used, for
example, to synthesize full-length polynucleotides of the
invention.
[0058] Further particularly preferred embodiments are
polynucleotides encoding BASB081 variants, that have the amino acid
sequence of BASB081 polypeptide of SEQ ID NO:2 or 4 in which
several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid
residues are substituted, modified, deleted and/or added, in any
combination. Especially preferred among these are silent
substitutions, additions and deletions, that do not alter the
properties and activities of BASB081 polypeptide.
[0059] Further preferred embodiments of the invention are
polynucleotides that are at least 85% identical over their entire
length to a polynucleotide encoding BASB081 polypeptide having an
amino acid sequence set out in SEQ ID NO: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 BASB081 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.
[0060] 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.
[0061] In accordance with certain preferred embodiments of this
invention there are provided polynucleotides that hybridize,
particularly under stringent conditions, to BASB081 polynucleotide
sequences, such as those polynucleotides in SEQ ID NO:1 or 3.
[0062] The invention furtherer 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
occurring only if there is at least 95% and preferably at least 97%
identity between the sequences. A specific example of stringent
hybridization conditions is overnight incubation at 42.degree. C.
in a solution comprising: 50% formamide, 5.times.SSC (150 mM NaCl,
15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of
denatured, sheared salmon sperm DNA, followed by washing the
hybridization support in 0.1.times.SSC at about 65.degree. C.
Hybridization and wash conditions are well known and exemplified in
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11
therein. Solution hybridization may also be used with the
polynucleotide sequences provided by the invention.
[0063] 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:1 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.
[0064] 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 BASB081 and to isolate cDNA and genomic clones of other
genes that have a high identity, particularly high sequence
identity, to the BASB081 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.
[0065] A coding region of a BASB081 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.
[0066] 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 Marathon.TM. technology (Clontech Laboratories
Inc.) for example, have significantly simplified the search for
longer cDNAs. In the Marathon.TM. technology, cDNAs have been
prepared from mRNA extracted from a chosen tissue and an `adaptor`
sequence ligated onto each end. Nucleic acid amplification (PCR) is
then carried out to amplify the "missing" 5' end of the DNA using a
combination of gene specific and adaptor specific oligonucleotide
primers. The PCR reaction is then repeated using "nested" primers,
that is, primers designed to anneal within the amplified product
(typically an adaptor specific primer that anneals further 3' in
the adaptor sequence and a gene specific primer that anneals
further 5' in the selected gene sequence). The products of this
reaction can then be analyzed by DNA sequencing and a full-length
DNA constructed either by joining the product directly to the
existing DNA to give a complete sequence, or carrying out a
separate full-length PCR using the new sequence information for the
design of the 5' primer.
[0067] 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.
[0068] The polynucleotides of the invention that are
oligonucleotides derived from a sequence of SEQ ID NOS:1 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] In sum, a polynucleotide of the invention may encode a
mature protein, a mature protein plus a leader sequence (which may
be referred to as a preprotein), a precursor of a mature protein
having one or more prosequences that are not the leader sequences
of a preprotein, or a preproprotein, which is a precursor to a
proprotein, having a leader sequence and one or more prosequences,
which generally are removed during processing steps that produce
active and mature forms of the polypeptide.
[0074] 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.
[0075] The use of a polynucleotide of the invention in genetic
immunization will preferably employ a suitable delivery method such
as direct injection of plasmid DNA into muscles (Wolff et al., Hum
Mol Genet (1992) 1: 363, Manthorpe et al., Hum. Gene Ther. (1983)
4: 419), delivery of DNA complexed with specific protein carriers
(Wu et al., J Biol 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).
[0076] Vectors, Host Cells, Expression Systems
[0077] 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.
[0078] Recombinant polypeptides of the present invention may be
prepared by processes well known in those skilled in the art from
genetically engineered host cells comprising expression systems.
Accordingly, in a further aspect, the present invention relates to
expression systems that comprise a polynucleotide or
polynucleotides of the present invention, to host cells which are
genetically engineered with such expression systems, and to the
production of polypeptides of the invention by recombinant
techniques.
[0079] For recombinant production of the polypeptides of the
invention, host cells can be genetically engineered to incorporate
expression systems or portions thereof or polynucleotides of the
invention. Introduction of a polynucleotide into the host cell can
be effected by methods described in many standard laboratory
manuals, such as Davis, et al., BASIC METHODS IN MOLECULAR BIOLOGY,
(1986) and Sambrook, et al., MOLECULAR CLONING: A LABORATORY
MANUAL, 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.
[0080] 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 of Drosophila S2 and Spodoptera Sf9; 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.
[0081] 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,
picomaviruses, retroviruses, and alphaviruses and vectors derived
from combinations thereof, such as those derived from plasmid and
bacteriophage genetic elements, such as cosmids and phagemids. The
expression system constructs may contain control regions that
regulate as well as engender expression. Generally, any system or
vector suitable to maintain, propagate or express polynucleotides
and/or to express a polypeptide in a host may be used for
expression in this regard. The appropriate DNA sequence may be
inserted into the expression system by any of a variety of
well-known and routine techniques, such as, for example, those set
forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,
(supra).
[0082] 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.
[0083] Polypeptides of the present invention can be recovered and
purified from recombinant cell cultures by well-known methods
including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Most preferably, ion
metal 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.
[0084] The expression system may also be a recombinant live
microorganism, such as a virus or bacterium. The gene of interest
can be inserted into the genome of a live recombinant virus or
bacterium. Inoculation and in vivo infection with this live vector
will lead to in vivo expression of the antigen and induction of
immune responses. Viruses and bacteria used for this purpose are
for instance: poxviruses (e.g; vaccinia, fowlpox, canarypox),
alphaviruses (Sindbis virus, Semliki Forest Virus, Venezuelian
Equine Encephalitis Virus), adenoviruses, adeno-associated virus,
picomaviruses (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.
[0085] Diagnostic, Prognostic, Serotyping and Mutation Assays
[0086] This invention is also related to the use of BASB081
polynucleotides and polypeptides of the invention for use as
diagnostic reagents. Detection of BASB081 polynucleotides and/or
polypeptides in a eukaryote, particularly a mammal, and especially
a human, will provide a diagnostic method for diagnosis of disease,
staging of disease or response of an infectious organism to drugs.
Eukaryotes, particularly mammals, and especially humans,
particularly those infected or suspected to be infected with an
organism comprising the BASB081 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.
[0087] Polypeptides and polynucleotides for prognosis, diagnosis or
other analysis may be obtained from a putatively infected and/or
infected individual's bodily materials. Polynucleotides from any of
these sources, particularly DNA or RNA, may be used directly for
detection or may be amplified enzymatically by using PCR or any
other amplification technique prior to analysis. RNA, particularly
mRNA, cDNA and genomic DNA may also be used in the same ways. Using
amplification, characterization of the species and strain of
infectious or resident organism present in an individual, may be
made by an analysis of the genotype of a 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 BASB081 polynucleotide sequences.
Perfectly or significantly matched sequences can be distinguished
from imperfectly or more significantly mismatched duplexes by DNase
or RNase digestion, for DNA or RNA respectively, or by detecting
differences in melting temperatures or renaturation kinetics.
Polynucleotide sequence differences may also be detected by
alterations in the electrophoretic mobility of polynucleotide
fragments in gels as compared to a reference sequence. This may be
carried 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, V1 and S1 protection assay or a
chemical cleavage method. See, for example, Cotton et al, Proc.
Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
[0088] In another embodiment, an array of oligonucleotides probes
comprising BASB081 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)).
[0089] Thus in another aspect, the present invention relates to a
diagnostic kit which comprises:
[0090] (a) a polynucleotide of the present invention, preferably
the nucleotide sequence of SEQ ID NO:1 or 3, or a fragment
thereof;
[0091] (b) a nucleotide sequence complementary to that of (a);
[0092] (c) a polypeptide of the present invention, preferably the
polypeptide of SEQ ID NO:2 or 4 or a fragment thereof; or
[0093] (d) an antibody to a polypeptide of the present invention,
preferably to the polypeptide of SEQ ID NO:2 or 4.
[0094] 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.
[0095] 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.
[0096] 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
BASB081 polypeptide can be used to identify and analyze
mutations.
[0097] The invention further provides primers with 1, 2, 3 or 4
nucleotides removed from the 5' and/or the 3' end. These primers
may be used for, among other things, amplifying BASB081 DNA and/or
RNA 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.
[0098] 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 BASB081 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.
[0099] In addition, a diagnostic assay in accordance with the
invention for detecting over-expression of BASB081 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 BASB081 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.
[0100] 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 of variants of a polynucleotide
sequence encoding the polypeptide sequence of SEQ ID NO:2 or 4.
[0101] Antibodies
[0102] 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.
[0103] In certain preferred embodiments of the invention there are
provided antibodies against BASB081 polypeptides or
polynucleotides.
[0104] 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).
[0105] Techniques for the production of single chain antibodies
(U.S. Pat. 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.
[0106] 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-BASB081 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).
[0107] 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.
[0108] Thus, among others, antibodies against BASB081-polypeptide
or BASB081-polynucleotide may be employed to treat infections,
particularly bacterial infections.
[0109] Polypeptide variants include antigenically, epitopically or
immunologically equivalent variants form a particular aspect of
this invention.
[0110] 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.
[0111] Antagonists and Agonists--Assays and Molecules
[0112] Polypeptides and polynucleotides of the invention may also
be used to assess the binding of small molecule substrates and
ligands in, for example, cells, cell-free preparations, chemical
libraries, and natural product mixtures. These substrates and
ligands may be natural substrates and ligands or may be structural
or functional mimetics. See, e.g., Coligan et al., Current
Protocols in Immunology 1(2): Chapter 5 (1991).
[0113] 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 BASB081 polypeptide and/or
polynucleotide activity in the mixture, and comparing the BASB081
polypeptide and/or polynucleotide activity of the mixture to a
standard. Fusion proteins, such as those made from Fc portion and
BASB081 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)).
[0114] 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.
[0115] The invention also provides a method of screening compounds
to identify those which enhance (agonist) or block (antagonist) the
action of BASB081 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
BASB081 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 BASB081 agonist or antagonist. The
ability of the candidate molecule to agonize or antagonize the
BASB081 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 BASB081 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
BASB081 polynucleotide or polypeptide activity, and binding assays
known in the art.
[0116] Another example of an assay for BASB081 agonists is a
competitive assay that combines BASB081 and a potential agonist
with BASB081-binding molecules, recombinant BASB081 binding
molecules, natural substrates or ligands, or substrate or ligand
mimetics, under appropriate conditions for a competitive inhibition
assay. BASB081 can be labeled, such as by radioactivity or a
colorimetric compound, such that the number of BASB081 molecules
bound to a binding molecule or converted to product can be
determined accurately to assess the effectiveness of the potential
antagonist.
[0117] 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 BASB081-induced activities, thereby
preventing the action or expression of BASB081 polypeptides and/or
polynucleotides by excluding BASB081 polypeptides and/or
polynucleotides from binding.
[0118] 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,
Fla. (1988), for a description of these molecules). Preferred
potential antagonists include compounds related to and variants of
BASB081.
[0119] In a further aspect, the present invention relates to
genetically engineered soluble fusion proteins comprising a
polypeptide of the present invention, or a fragment thereof, and
various portions of the constant regions of heavy or light chains
of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
Preferred as an immunoglobulin is the constant part of the heavy
chain of human IgG, particularly IgG1, 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. WO94/29458 and WO94/22914.
[0120] 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.
[0121] 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 BASB081
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.
[0122] In accordance with yet another aspect of the invention,
there are provided BASB081 agonists and antagonists, preferably
bacteristatic or bactericidal agonists and antagonists.
[0123] The antagonists and agonists of the invention may be
employed, for instance, to prevent, inhibit and/or treat
diseases.
[0124] 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.
[0125] 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 carrier. 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 carrier to include a hydrophobic terminus distal from
the conjugated terminus of the peptide, such that the free
unconjugated end of the peptide remains associated with the surface
of the carrier protein. Thereby presenting the peptide in a
conformation which most closely resembles that of the peptide as
found in the context of the whole native molecule. For example, the
peptides may be altered to have an N-terminal cysteine and a
C-terminal hydrophobic amidated tail. Alternatively, the addition
or substitution of a D-stereoisomer form of one or more of the
amino acids may be performed to create a beneficial derivative, for
example to enhance stability of the peptide.
[0126] 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 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.
[0127] Vaccines
[0128] 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 BASB081 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 BASB081 polynucleotide and/or polypeptide, or
a fragment or a variant thereof, for expressing BASB081
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.
[0129] 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 BASB081 polynucleotide and/or polypeptide encoded
therefrom, wherein the composition comprises a recombinant BASB081
polynucleotide and/or polypeptide encoded therefrom and/or
comprises DNA and/or RNA which encodes and expresses an antigen of
said BASB081 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.
[0130] A BASB081 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.
[0131] 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).
[0132] 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.
[0133] 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 formulations may be
presented in unit-dose or multi-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.
[0134] The vaccine formulation of the invention may also include
adjuvant systems for enhancing the immunogenicity of the
formulation. Preferably the adjuvant system raises preferentially a
TH1 type of response.
[0135] An immune response may be broadly distinguished into two
extreme catagories, being a humoral or cell mediated immune
responses (traditionally characterised by antibody and cellular
effector mechanisms of protection respectively). These categories
of response have been termed TH1-type responses (cell-mediated
response), and TH2-type immune responses (humoral response).
[0136] Extreme TH1-type immune responses may be characterised by
the generation of antigen specific, haplotype restricted cytotoxic
T lymphocytes, and natural killer cell responses. In mice TH1-type
responses are often characterised by the generation of antibodies
of the IgG2a subtype, whilst in the human these correspond to IgG1
type antibodies. TH2-type immune responses are characterised by the
generation of a broad range of immunoglobulin isotypes including in
mice IgG1, IgA, and IgM.
[0137] It can be considered that the driving force behind the
development of these two types of immune responses are cytokines.
High levels of TH1-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.
[0138] 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 TH1 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 Coffman (Mosmann, T. R. and Coffman, R. L. (1989)
TH1 and TH2 cells: different patterns of lymphokine secretion lead
to different functional properties. Annual Review of Immunology, 7,
p145-173). Traditionally, TH1-type responses are associated with
the production of the INF-.gamma. and IL-2 cytokines by
T-lymphocytes. Other cytokines often directly associated with the
induction of TH1-type immune responses are not produced by T-cells,
such as IL-12. In contrast, TH2-type responses are associated with
the secretion of IL-4, IL-5, IL-6 and IL-13.
[0139] 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 TH1:TH2 balance
of the immune response after a vaccination or infection includes
direct measurement of the production of TH1 or TH2 cytokines by T
lymphocytes in vitro after restimulation with antigen, and/or the
measurement of the IgG1:IgG2a ratio of antigen specific antibody
responses.
[0140] Thus, a TH1-type adjuvant is one which preferentially
stimulates isolated T-cell populations to produce high levels of
TH1-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.
[0141] 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.
[0142] 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 Immunochem, Montana. A
preferred form of 3 De-O-acylated monophosphoryl lipid A is
disclosed in European Patent 0 689 454 B1 (SmithKline Beecham
Biologicals SA).
[0143] Preferably, the particles of 3D-MPL are small enough to be
sterile filtered through a 0.22 micron membrane (European Patent
number 0 689 454).
[0144] 3D-MPL will be present in the range of 10 .mu.g -100 .mu.g
preferably 25-50 .mu.g per dose wherein the antigen will typically
be present in a range 2-50 .mu.g per dose.
[0145] 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
carrier.
[0146] The method of production of QS21 is disclosed in U.S. Pat.
No. 5,057,540.
[0147] 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.
[0148] Further adjuvants which are preferential stimulators of TH1
cell response include immunomodulatory oligonucleotides, for
example unmethylated CpG sequences as disclosed in WO 96/02555.
[0149] 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 1:13D-MPL: QS21.
[0150] Preferably a carrier is also present in the vaccine
composition according to the invention. The carrier may be an oil
in water emulsion, or an aluminium salt, such as aluminium
phosphate or aluminium hydroxide.
[0151] 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.
[0152] Typically for human administration QS21 and 3D-MPL will be
present in a vaccine in the range of 1 .mu.g -200 .mu.g, such as
10-100 .mu.g, preferably 10 .mu.g-50 .mu.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.
[0153] Non-toxic oil in water emulsions preferably contain a
non-toxic oil, e.g. squalane or squalene, an emulsifier, e.g. Tween
80, in an aqueous carrier. The aqueous carrier may be, for example,
phosphate buffered saline.
[0154] A particularly potent adjuvant formulation involving QS21,
3D-MPL and tocopherol in an oil in water emulsion is described in
WO 95/17210.
[0155] The present invention also provides a polyvalent vaccine
composition comprising a vaccine formulation of the invention in
combination with other antigens, in particular antigens useful for
treating cancers, autoimmune diseases and related conditions. Such
a polyvalent vaccine composition may include a TH-1 inducing
adjuvant as hereinbefore described.
[0156] While the invention has been described with reference to
certain BASB081 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.
[0157] Compositions, Kits and Administration
[0158] In a further aspect of the invention there are provided
compositions comprising a BASB081 polynucleotide and/or a BASB081
polypeptide for administration to a cell or to a multicellular
organism.
[0159] 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 carriers 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.
[0160] Polypeptides, polynucleotides and other compounds of the
invention may be employed alone or in conjunction with other
compounds, such as therapeutic compounds.
[0161] 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.
[0162] 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.
[0163] In a further aspect, the present invention provides for
pharmaceutical compositions comprising a therapeutically effective
amount of a polypeptide and/or polynucleotide, such as the soluble
form of a polypeptide and/or polynucleotide of the present
invention, agonist or antagonist peptide or small molecule
compound, in combination with a pharmaceutically acceptable 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.
[0164] The composition will be adapted to the route of
administration, for instance by a systemic or an oral route.
Preferred forms of systemic administration include injection,
typically by intravenous injection. Other injection routes, such as
subcutaneous, intramuscular, or intraperitoneal, can be used.
Alternative means for systemic administration include transmucosal
and transdermal administration using penetrants such as bile salts
or fusidic acids or other detergents. In addition, if a polypeptide
or other compounds of the present invention can be formulated in an
enteric or an encapsulated formulation, oral administration may
also be possible. Administration of these compounds may also be
topical and/or localized, in the form of salves, pastes, gels,
solutions, powders and the like.
[0165] 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.
[0166] 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
of0.1-100 .mu.g/kg of subject.
[0167] A vaccine composition is conveniently in injectable form.
Conventional adjuvants may be employed to enhance the immune
response. A suitable unit dose for vaccination is 0.5-5
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.
[0168] 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.
[0169] Sequence Databases, Sequences in a Tangible Medium, and
Algorithms
[0170] 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.
[0171] 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.
[0172] 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.
[0173] 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.
[0174] All publications and references, including but not limited
to patents and patent applications, cited in this specification are
herein incorporated by reference in their entirety as if each
individual publication or reference were specifically and
individually indicated to be incorporated by reference herein as
being fully set forth. Any patent application to which this
application claims priority is also incorporated by reference
herein in its entirety in the manner described above for
publications and references.
Definitions
[0175] "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; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990).
The well known Smith Waterman algorithm may also be used to
determine identity.
[0176] Parameters for polypeptide sequence comparison include the
following:
[0177] Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453
(1970)
[0178] Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,
[0179] Proc. Natl Acad. Sci. USA. 89:10915-10919 (1992)
[0180] Gap Penalty: 8
[0181] Gap Length Penalty: 2
[0182] A program useful with these parameters is publicly available
as the "gap" program from Genetics Computer Group, Madison Wis. The
aforementioned parameters are the default parameters for peptide
comparisons (along with no penalty for end gaps).
[0183] Parameters for polynucleotide comparison include the
following:
[0184] Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453
(1970)
[0185] Comparison matrix: matches=+10, mismatch=0
[0186] Gap Penalty: 50
[0187] Gap Length Penalty: 3
[0188] Available as: The "gap" program from Genetics Computer
Group, Madison Wis. These are the default parameters for nucleic
acid comparisons.
[0189] A preferred meaning for "identity" for polynucleotides and
polypeptides, as the case may be, are provided in (1) and (2)
below.
[0190] (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:1 or may
include up to a certain integer number of nucleotide alterations as
compared to the reference sequence, wherein said alterations are
selected from the group consisting of at least one nucleotide
deletion, substitution, including transition and transversion, or
insertion, and wherein said alterations may occur at the 5' or 3'
terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually
among the nucleotides in the reference sequence or in one or more
contiguous groups within the reference sequence, and wherein said
number of nucleotide alterations is determined by multiplying the
total number of nucleotides in SEQ ID NO:1 by the integer defining
the percent identity divided by 100 and then subtracting that
product from said total number of nucleotides in SEQ ID NO:1,
or:
n.sub.n.ltoreq.x.sub.n-(x.sub.n.multidot.y),
[0191] wherein n.sub.n is the number of nucleotide alterations,
x.sub.n 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
.multidot. is the symbol the multiplication operator, and wherein
any non-integer product of x.sub.n and y is rounded down to the
nearest integer prior to subtracting it from x.sub.n. Alterations
of a polynucleotide sequence encoding the polypeptide of SEQ ID
NO:2 may create nonsense, missense or frameshift mutations in this
coding sequence and thereby alter the polypeptide encoded by the
polynucleotide following such alterations.
[0192] By way of example, a polynucleotide sequence of the present
invention may be identical to the reference sequence of SEQ ID
NO:1, that is it may be 100% identical, or it may include up to a
certain integer number of nucleic acid alterations as compared to
the reference sequence such that the percent identity is less than
100% identity. Such alterations are selected from the group
consisting of at least one nucleic acid deletion, substitution,
including transition and transversion, or insertion, and wherein
said alterations may occur at the 5' or 3' terminal positions of
the reference polynucleotide sequence or anywhere between those
terminal positions, interspersed either individually among the
nucleic acids in the reference sequence or in one or more
contiguous groups within the reference sequence. The number of
nucleic acid alterations for a given percent identity is determined
by multiplying the total number of nucleic acids in SEQ ID NO:1 by
the integer defining the percent identity divided by 100 and then
subtracting that product from said total number of nucleic acids in
SEQ ID NO:1, or:
n.sub.n.ltoreq.x.sub.n-(x.sub.n.multidot.y),
[0193] wherein n.sub.n is the number of nucleic acid alterations,
x.sub.n 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.,
.multidot. is the symbol for the multiplication operator, and
wherein any non-integer product of x.sub.n and y is rounded down to
the nearest integer prior to subtracting it from x.sub.n.
[0194] (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 NO:2, wherein said polypeptide sequence may be
identical to the reference sequence of SEQ ID NO: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 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 NO:2, or:
n.sub.a.ltoreq.x.sub.a-(x.sub.a.multidot.y),
[0195] wherein n.sub.a is the number of amino acid alterations,
x.sub.a is the total number of amino acids in SEQ ID NO: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
.multidot. is the symbol for the multiplication operator, and
wherein any non-integer product of x.sub.a and y is rounded down to
the nearest integer prior to subtracting it from x.sub.a.
[0196] By way of example, a polypeptide sequence of the present
invention may be identical to the reference sequence of SEQ ID
NO: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 NO:2, or:
n.sub.a.ltoreq.x.sub.a-(x.sub.a.multidot.y),
[0197] wherein n.sub.a the number of amino acid alterations,
x.sub.a is the total number of amino acids in SEQ ID NO:2, y is,
for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and
.multidot. is the symbol for the multiplication operator, and
wherein any non-integer product of x.sub.a and y is rounded down to
the nearest integer prior to subtracting it from x.sub.a.
[0198] "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.
[0199] "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.
[0200] "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.
[0201] "Variant" refers to a polynucleotide or polypeptide that
differs from a reference polynucleotide or polypeptide, but retains
essential properties. A typical variant of a polynucleotide differs
in nucleotide sequence from another, reference polynucleotide.
Changes in the nucleotide sequence of the variant may or may not
alter the amino acid sequence of a polypeptide encoded by the
reference polynucleotide. Nucleotide changes may result in amino
acid substitutions, additions, deletions, fusions and truncations
in the polypeptide encoded by the reference sequence, as discussed
below. A typical variant of a polypeptide differs in amino acid
sequence from another, reference polypeptide. Generally,
differences are limited so that the sequences of the reference
polypeptide and the variant are closely similar overall and, in
many regions, identical. A variant and reference polypeptide may
differ in amino acid sequence by one or more substitutions,
additions, deletions in any combination. A substituted or inserted
amino acid residue may or may not be one encoded by the genetic
code. A variant of a polynucleotide or polypeptide may be a
naturally occurring such as an allelic variant, or it may be a
variant that is not known to occur naturally. Non-naturally
occurring variants of polynucleotides and polypeptides may be made
by mutagenesis techniques or by direct synthesis.
[0202] "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.
EXAMPLES
[0203] The examples below are carried 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 BASB081 Gene from Moraxella catarrhalis
Strain ATCC 43617
[0204] A: BASB081 in Moraxella catarrhalis Strain.
[0205] The BASB081 gene of SEQ ID NO:1 is from Moraxella
catarrhalis strain ATCC 43617. The translation of the BASB081
polynucleotide sequence is showed in SEQ ID NO:2.
[0206] B: BASB081 in Moraxella Catarrhalis Strain 43617.
[0207] The sequence of the BASB081 gene was confirmed in Moraxella
Catarrhalis strain ATCC 43617. For this purpose, plasmid DNA (see
example 2A) containing the gene region encoding the mature BASB081
from Moraxella Catarrhalis. strain ATCC 43617 used as a PCR
template. This material was then submitted to Polymerase Chain
Reaction DNA amplification using primers Moraxella catarrhalis
MCD15b-01 (5'-CAT GCC ATG GGT CAA CAA AAT AAC CCT GCA AAC -3') [SEQ
ID NO:5] and reverse MCD15b-02 (5'CTA GTC TAG ATT AAA ATG GTG TGC
CAA TAA AAA AAT G -3') [SEQ ID NO:6] specific for the BASB081 gene.
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 NO:3 and SEQ ID NO:4 respectively, were obtained. These
sequences do not comprise the signal sequence as the signal
sequence was from the plasmid.
[0208] 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 FIG. 1; a pairwise
comparison of identities shows that the two BASB081 polynucleotide
gene sequences are 99.9% identical in the region coding for the
mature protein. Using the same MegAlign program, an alignment of
the polypeptide sequences of SEQ ID NO:2 and 4 was performed, and
is displayed in FIG. 2; a pairwise comparison of identities shows
that the two BASB081 protein sequences are 99.9% identical in the
region of the mature protein.
Example 2
Construction of Plasmid to Express Recombinant BASB081
[0209] A: Cloning of BASB081.
[0210] The NcoI and XbaI restriction sites (underlined) engineered
into the primers Moraxella catarrhalis MCD15b-01 (5'-CAT GCC ATG
GGT CAA CAA AAT AAC CCT GCA AAC -3') and reverse MCD15b-02 (5'CTA
GTC TAG ATT AAA ATG GTG TGC CAA TAA AAA AAT G 3') amplification
primers, respectively, permitted directional cloning of a BASB081
PCR product into the commercially available E. coli expression
plasmid pBADgIII Calmodulin (Invitrogen, USA, ampicillin
resistant). This plasmid provides the signal peptide from the
bacteriophage fd pIII protein such that a mature BASB081 protein
could be targeted to the periplasm of E. coli. The BASB081 PCR
product was purified from the amplification reaction using Wizard
PCR prep.TM. (Promega) according to the manufacturers instructions.
To produce the required NcoI and XbaI termini necessary for
cloning, purified PCR product was sequentially digested to
completion with NcoI and XbaI restriction enzymes as recommended by
the manufacturer (Boehringer Mannheim). Digested BASB081 PCR
products and pBAD were gel-purified and ligated together using an
approximately 5-fold molar excess of the digested fragment to the
vector. A standard .about.20 .mu.l ligation reaction
(.about.16.degree. C., .about.16 hours), using methods well known
in the art, was performed using T4 DNA ligase (.about.2.0
units/reaction, Boehringer Mannheim). An aliquot of the ligation
was used to transform electro-competent E. coli Top10 cells
according to methods well known in the art. Following a .about.2-3
hour outgrowth period at 37.degree. C. in .about.1.0 ml of LB
broth, transformed cells were plated on LB agar plates containing
Ampicillin (50 .mu.g/ml). Individual ampicillin-resistant colonies
were selecteded and analyzed by whole cell-based PCR to verify that
transformants contained the BASB081 DNA insert. Transformants that
produced the expected PCR product were identified as strains
containing a BASB081 expression construct. Expression plasmid
containing strains were then analyzed for the inducible expression
of recombinant BASB081.
[0211] B: Expression Analysis of PCR-Positive Transformants.
[0212] For each PCR-positive transformant identified above,
.about.5.0 ml of LB broth containing ampicillin (50 .mu.g/ml) was
inoculated with cells from the patch plate and grown overnight at
37.degree. C. with shaking (.about.250 rpm). An aliquot of the
overnight seed culture (.about.1.0 ml) was inoculated into a 125 ml
erlenmeyer flask containing .about.25 ml of LB ampicilline broth
and grown at 37.degree. C. with shaking (.about.250 rpm) until the
culture turbidity reached O.D.600 of .about.0.5, i.e. mid-log phase
(usually about 1.5-2.0 hours). At this time approximately half of
the culture (.about.12.5 ml) was transferred to a second 125 ml
flask and expression of recombinant BASB081 protein induced by the
addition of L-Arabinose to a final concentration of 0.2% (w/v).
Incubation of both the arabinose-induced and non-induced cultures
continued for an additional .about.4 hours at 37.degree. C. with
shaking. Samples (.about.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 .about.3 minutes. Individual cell pellets were
suspended in .about.50 .mu.l of sterile water, then mixed with an
equal volume of 2.times. Laemelli SDS-PAGE sample buffer containing
2-mercaptoethanol, and placed in boiling water bath for .about.3
min to denature protein. Equal volumes (.about.15 .mu.l) of both
the crude arabinose-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 under conventional conditions using a standard
SDS/Tris/glycine running buffer. Following electrophoresis, one gel
was stained with commassie brilliant blue R250 (BioRad) and then
destained to visualize novel BASB081 arabinose-inducible protein(s)
(FIG. 3).
[0213] Deposited Materials
[0214] A deposit containing a Moraxella catarrhalis Catlin strain
has been deposited with the American Type Culture Collection
(herein "ATCC") on Jun. 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).
[0215] The Moraxella catarrhalis strain deposit is referred to
herein as "the deposited strain" or as "the DNA of the deposited
strain."
[0216] The deposited strain contains a full length BASB081
gene.
[0217] A deposit of the vector pMC-D15 consisting of Moraxella
catarrhalis DNA inserted in pQE30 has been deposited with the
American Type Culture Collection (ATCC) on Feb. 12, 1999 and
assigned deposit number 207105.
[0218] 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.
[0219] 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.
.sctn.112.
Sequence CWU 1
1
6 1 2760 DNA Moraxella catarrhalis 1 atgtcaaagc ccgttttgtt
tgcaaatcgc agttttatgc ctgtcgcatt ggcggcttat 60 ttgcctttga
tgacatcgca agcattggca caacaaaata accctgcaaa catcatcaat 120
catgtacccg ctcatgacac cgccatcaat caagcaaagg caggcaatcc gcctgttttg
180 ctaacacctg agcagataca agcacgcctt aatgctgctg gactgaatgc
taagccccaa 240 tcacaagctt tggatgttgt caattttgat gatcaatcgc
cgatatctcg tatcggtgag 300 caatcacccc ctttgggttt ggatatgtcg
gtcatcgaag aaaccacacc gctaagcttg 360 gaggaattat ttgctcaaga
atctactgag atgggaatca atccaaatga ttatattcca 420 gaatatcaag
gcgagcaacc taatagtgag gtggttgtac caccgacatt agaacctgaa 480
aaaccaggtt tgatcaagcg tctttatgca cgcctattta atgatggtgt caataaggtg
540 cctaggctta aggcaaaatt ttatcaatca tcgcaatcag gcgaaaccag
tgcgattggg 600 tcatcgcatc aaaaaacaga gccttatgca aatatcaaag
cagcacttga agacatcacc 660 caagagtcag cgatggattt gaatggctct
atcccacgcc taaggcaaac tgctttggtg 720 gcagcgcgtg ctgtcggtta
ttatgatatt gatttatcaa tcataagaaa tagcatcgga 780 gaggtggatg
tcatcatcca tgatttaggt gaacctgttt atattgatta tcgagcggtg 840
gaggtacgag gtgaaggtgc tgatgataaa gcatttacta ccgtggcgga tgaggtgcca
900 ttgctgatcg gcgatgtctt tcatcatggc aagtacgaaa ccaaaaaaaa
tctcatcgaa 960 aatgccagtg ctgaacatgg atattttgat gggcgttggc
tggatcgttc agttgatgta 1020 attttgccag ataataccgc tgatgtcagc
ttaatttatg atacaggtac gcagtatcgc 1080 tttgatgagg tggtattttt
taccattgat cctaaaacca atcaattgac aaccgatcca 1140 gataagctgc
cagttaaacg agaattactt gagcagttac tcaccgttaa catgggagag 1200
gcttacaatt tacaggcggt gcgtgcactt tcaaatgatt tgattgccac acggtatttt
1260 aatatggtga ataccgagat tgtctttcca gagcgtgaac agatccaaaa
cgaccaagtg 1320 agctttgagc agtcttcaag tagccgtact gaaccagcac
aagttgatga aagcacactt 1380 gaacctgtca ttgaaaccgt tgagctaacg
gatgggatat taatggatat ttcgcccatc 1440 gaatttagtg catctaatct
gattcaagac aagctaaatt tggtggctgc caaggctcgc 1500 catttatatg
acatgcctga tgatagggtg cttgccatca atcatgatga tggcgtaaat 1560
cgctctattt tgggcagaat cagcgatgcc gtatctgccg ttgcacgtgc tattttacct
1620 gatgaatctg aaaatgaggt aatagatttg cccgagcgta ccgcattggc
taatcgcaag 1680 acccctgctg atgtctatca aagtaaaaaa gtgccgctat
atgtctttgt ggcgagtgat 1740 aaaccacgag atggtcaaat tggtttgggc
tggggatcgg acacaggtac ccgcctagtc 1800 acaaaatttg agcataattt
gattaatcgt gatggctatc aagcaggcgc tgagctaaga 1860 ctgtctgagg
ataaaaaagg ggtcaagtta tatgccacca aaccgcttag ccaccctcta 1920
aatgatcagc taagagcaac tttgggttat caacaagaag tttttggtca ctctaccaat
1980 ggttttgatt tatccacacg caccctagag catgagatta gccgcagtat
tatccaaaat 2040 ggtggctgga atcgtactta ttcattgcgt tatcgtcttg
ataagcttaa aacccaagca 2100 ccccctgaaa catggcagga tttaccagtg
gattttgtca atggtaagcc aagccaagag 2160 gcgttattgg caggtgttgc
tgtgcataaa acggttgcag ataatttggt taatccgatg 2220 cgtggctatc
gtcagcgata ttctttagag gttggctcaa gcggtttggt atcggatgct 2280
aatatggcta ttgctcgagc tggtattagt ggcgtgtata gttttgggga taatgcttat
2340 ggcagcaatc gtgcccatca gatgactggt ggcatacaag caggatacat
ttggtcggat 2400 aattttaatc atgtgccata tcgtttgcgt ttttttgctg
gtggcgacca aagtattcgt 2460 ggatatgcac atgacagttt atcacctata
tcagataagg gttatctgac aggcggtcaa 2520 gtattggcgg ttggtacagc
tgaatataat tatgaattta tgaaagattt gcgtttggcg 2580 gtttttggtg
atattggtaa tgcttatgat aaaggcttta ctaatgatac caaaattggt 2640
gcaggtgtcg gtgttcgctg ggcatcacct gtcggtcaag ttcgtgttga tgtggcaact
2700 ggtgtcaaag aagagggcaa tcccattaag ctgcattttt ttattggcac
accattttaa 2760 2 919 PRT Moraxella catarrhalis 2 Met Ser Lys Pro
Val Leu Phe Ala Asn Arg Ser Phe Met Pro Val Ala 1 5 10 15 Leu Ala
Ala Tyr Leu Pro Leu Met Thr Ser Gln Ala Leu Ala Gln Gln 20 25 30
Asn Asn Pro Ala Asn Ile Ile Asn His Val Pro Ala His Asp Thr Ala 35
40 45 Ile Asn Gln Ala Lys Ala Gly Asn Pro Pro Val Leu Leu Thr Pro
Glu 50 55 60 Gln Ile Gln Ala Arg Leu Asn Ala Ala Gly Leu Asn Ala
Lys Pro Gln 65 70 75 80 Ser Gln Ala Leu Asp Val Val Asn Phe Asp Asp
Gln Ser Pro Ile Ser 85 90 95 Arg Ile Gly Glu Gln Ser Pro Pro Leu
Gly Leu Asp Met Ser Val Ile 100 105 110 Glu Glu Thr Thr Pro Leu Ser
Leu Glu Glu Leu Phe Ala Gln Glu Ser 115 120 125 Thr Glu Met Gly Ile
Asn Pro Asn Asp Tyr Ile Pro Glu Tyr Gln Gly 130 135 140 Glu Gln Pro
Asn Ser Glu Val Val Val Pro Pro Thr Leu Glu Pro Glu 145 150 155 160
Lys Pro Gly Leu Ile Lys Arg Leu Tyr Ala Arg Leu Phe Asn Asp Gly 165
170 175 Val Asn Lys Val Pro Arg Leu Lys Ala Lys Phe Tyr Gln Ser Ser
Gln 180 185 190 Ser Gly Glu Thr Ser Ala Ile Gly Ser Ser His Gln Lys
Thr Glu Pro 195 200 205 Tyr Ala Asn Ile Lys Ala Ala Leu Glu Asp Ile
Thr Gln Glu Ser Ala 210 215 220 Met Asp Leu Asn Gly Ser Ile Pro Arg
Leu Arg Gln Thr Ala Leu Val 225 230 235 240 Ala Ala Arg Ala Val Gly
Tyr Tyr Asp Ile Asp Leu Ser Ile Ile Arg 245 250 255 Asn Ser Ile Gly
Glu Val Asp Val Ile Ile His Asp Leu Gly Glu Pro 260 265 270 Val Tyr
Ile Asp Tyr Arg Ala Val Glu Val Arg Gly Glu Gly Ala Asp 275 280 285
Asp Lys Ala Phe Thr Thr Val Ala Asp Glu Val Pro Leu Leu Ile Gly 290
295 300 Asp Val Phe His His Gly Lys Tyr Glu Thr Lys Lys Asn Leu Ile
Glu 305 310 315 320 Asn Ala Ser Ala Glu His Gly Tyr Phe Asp Gly Arg
Trp Leu Asp Arg 325 330 335 Ser Val Asp Val Ile Leu Pro Asp Asn Thr
Ala Asp Val Ser Leu Ile 340 345 350 Tyr Asp Thr Gly Thr Gln Tyr Arg
Phe Asp Glu Val Val Phe Phe Thr 355 360 365 Ile Asp Pro Lys Thr Asn
Gln Leu Thr Thr Asp Pro Asp Lys Leu Pro 370 375 380 Val Lys Arg Glu
Leu Leu Glu Gln Leu Leu Thr Val Asn Met Gly Glu 385 390 395 400 Ala
Tyr Asn Leu Gln Ala Val Arg Ala Leu Ser Asn Asp Leu Ile Ala 405 410
415 Thr Arg Tyr Phe Asn Met Val Asn Thr Glu Ile Val Phe Pro Glu Arg
420 425 430 Glu Gln Ile Gln Asn Asp Gln Val Ser Phe Glu Gln Ser Ser
Ser Ser 435 440 445 Arg Thr Glu Pro Ala Gln Val Asp Glu Ser Thr Leu
Glu Pro Val Ile 450 455 460 Glu Thr Val Glu Leu Thr Asp Gly Ile Leu
Met Asp Ile Ser Pro Ile 465 470 475 480 Glu Phe Ser Ala Ser Asn Leu
Ile Gln Asp Lys Leu Asn Leu Val Ala 485 490 495 Ala Lys Ala Arg His
Leu Tyr Asp Met Pro Asp Asp Arg Val Leu Ala 500 505 510 Ile Asn His
Asp Asp Gly Val Asn Arg Ser Ile Leu Gly Arg Ile Ser 515 520 525 Asp
Ala Val Ser Ala Val Ala Arg Ala Ile Leu Pro Asp Glu Ser Glu 530 535
540 Asn Glu Val Ile Asp Leu Pro Glu Arg Thr Ala Leu Ala Asn Arg Lys
545 550 555 560 Thr Pro Ala Asp Val Tyr Gln Ser Lys Lys Val Pro Leu
Tyr Val Phe 565 570 575 Val Ala Ser Asp Lys Pro Arg Asp Gly Gln Ile
Gly Leu Gly Trp Gly 580 585 590 Ser Asp Thr Gly Thr Arg Leu Val Thr
Lys Phe Glu His Asn Leu Ile 595 600 605 Asn Arg Asp Gly Tyr Gln Ala
Gly Ala Glu Leu Arg Leu Ser Glu Asp 610 615 620 Lys Lys Gly Val Lys
Leu Tyr Ala Thr Lys Pro Leu Ser His Pro Leu 625 630 635 640 Asn Asp
Gln Leu Arg Ala Thr Leu Gly Tyr Gln Gln Glu Val Phe Gly 645 650 655
His Ser Thr Asn Gly Phe Asp Leu Ser Thr Arg Thr Leu Glu His Glu 660
665 670 Ile Ser Arg Ser Ile Ile Gln Asn Gly Gly Trp Asn Arg Thr Tyr
Ser 675 680 685 Leu Arg Tyr Arg Leu Asp Lys Leu Lys Thr Gln Ala Pro
Pro Glu Thr 690 695 700 Trp Gln Asp Leu Pro Val Asp Phe Val Asn Gly
Lys Pro Ser Gln Glu 705 710 715 720 Ala Leu Leu Ala Gly Val Ala Val
His Lys Thr Val Ala Asp Asn Leu 725 730 735 Val Asn Pro Met Arg Gly
Tyr Arg Gln Arg Tyr Ser Leu Glu Val Gly 740 745 750 Ser Ser Gly Leu
Val Ser Asp Ala Asn Met Ala Ile Ala Arg Ala Gly 755 760 765 Ile Ser
Gly Val Tyr Ser Phe Gly Asp Asn Ala Tyr Gly Ser Asn Arg 770 775 780
Ala His Gln Met Thr Gly Gly Ile Gln Ala Gly Tyr Ile Trp Ser Asp 785
790 795 800 Asn Phe Asn His Val Pro Tyr Arg Leu Arg Phe Phe Ala Gly
Gly Asp 805 810 815 Gln Ser Ile Arg Gly Tyr Ala His Asp Ser Leu Ser
Pro Ile Ser Asp 820 825 830 Lys Gly Tyr Leu Thr Gly Gly Gln Val Leu
Ala Val Gly Thr Ala Glu 835 840 845 Tyr Asn Tyr Glu Phe Met Lys Asp
Leu Arg Leu Ala Val Phe Gly Asp 850 855 860 Ile Gly Asn Ala Tyr Asp
Lys Gly Phe Thr Asn Asp Thr Lys Ile Gly 865 870 875 880 Ala Gly Val
Gly Val Arg Trp Ala Ser Pro Val Gly Gln Val Arg Val 885 890 895 Asp
Val Ala Thr Gly Val Lys Glu Glu Gly Asn Pro Ile Lys Leu His 900 905
910 Phe Phe Ile Gly Thr Pro Phe 915 3 2670 DNA Moraxella
catarrhalis 3 caacaaaata accctgcaaa catcatcaat catgtacccg
ctcatgacac cgccatcaat 60 caagcaaagg caggcaatcc gcctgttttg
ctaacacctg agcagataca agcacgcctt 120 aatgctgctg gactgaatgc
taagccccaa tcacaagctt tggatgttgt caattttgat 180 gatcaatcgc
cgatatctcg tatcggtgag caatcacccc ctttgggttt ggatatgtcg 240
gtcatcgaag aaaccacacc gctaagcttg gaggaattat ttgctcaaga atctactgag
300 atgggaatca atccaaatga ttatattcca gaatatcaag gcgagcaacc
taatagtgag 360 gtggttgtac caccgacatt agaacctgaa aaaccaggtt
tgatcaagcg tctttatgca 420 cgcctattta atgatggtgt caataaggtg
cctaggctta aggcaaaatt ttatcaatca 480 tcgcaatcag gcgaaaccag
tgcgattggg tcatcgcatc aaaaaacaga gccttatgca 540 aatatcaaag
cagcacttga agacatcacc caagagtcag cgatggattt gaatggctct 600
atcccacgcc taaggcaaac tgctttggtg gcagcgcgtg ctgtcggtta ttatgatatt
660 gatttatcaa tcataagaaa tagcatcgga gaggtggatg tcatcatcca
tgatttaggt 720 gaacctgttt atattgatta tcgagcggtg gaggtacgag
gtgaaggtgc tgatgataaa 780 gcatttacta ccgtggcgga tgaggtgcca
ttgctgatcg gcgatgtctt tcatcacggc 840 aagtacgaaa ccaaaaaaaa
tctcatcgaa aatgccagtg ctgaacatgg atattttgat 900 gggcgttggc
tggatcgttc agttgatgta attttgccag ataataccgc tgatgtcagc 960
ttaatttatg atacaggtac gcagtatcgc tttgatgagg tgatattttt taccattgat
1020 cctaaaacca atcaattgac aaccgatcca gataagctgc cagttaaacg
agaattactt 1080 gagcagttac tcaccgttaa catgggagag gcttacaatt
tacaggcggt gcgtgcactt 1140 tcaaatgatt tgattgccac acggtatttt
aatatggtga ataccgagat tgtctttcca 1200 gagcgtgaac agatccaaaa
cgaccaagtg agctttgagc agtcttcaag tagccgtact 1260 gaaccagcac
aagttgatga aagcacactt gaacctgtca ttgaaaccgt tgagctaacg 1320
gatgggatat taatggatat ttcgcccatc gaatttagtg catctaatct gattcaagac
1380 aagctaaatt tggtggctgc caaggctcgc catttatatg acatgcctga
tgatagggtg 1440 cttgccatca atcatgatga tggcgtaaat cgctctattt
tgggcagaat cagcgatgcc 1500 gtatctgccg ttgcacgtgc tattttacct
gatgaatctg aaaatgaggt aatagatttg 1560 cccgagcgta ccgcattggc
taatcgcaag acccctgctg atgtctatca aagtaaaaaa 1620 gtgccgctat
atgtctttgt ggcgagtgat aaaccccgag atggtcaaat tggtttgggc 1680
tggggatcgg acacaggtac ccgcctagtc acaaaatttg agcataattt gattaatcgt
1740 gatggctatc aagcaggcgc tgagctaaga ctgtctgagg ataaaaaagg
ggtcaagtta 1800 tatgccacca aaccgcttag ccaccctcta aatgatcagc
taagagcaac tttgggttat 1860 caacaagaag tttttggtca ctctaccaat
ggttttgatt tatccacacg caccctagag 1920 catgagatta gccgcagtat
tatccaaaat ggtggctgga atcgtactta ttcattgcgt 1980 tatcgtcttg
ataagcttaa aacccaagca ccccctgaaa catggcagga tttaccagtg 2040
gattttgtca atggtaagcc aagccaagag gcgttattgg caggtgttgc tgtgcataaa
2100 acggttgcag ataatttggt taatccgatg cgtggctatc gtcagcgata
ttctttagag 2160 gttggctcaa gcggtttggt atcggatgct aatatggcta
ttgctcgagc tggtattagt 2220 ggcgtgtata gttttgggga taatgcttat
ggcagcaatc gtgcccatca gatgactggt 2280 ggcatacaag caggatacat
ttggtcggat aattttaatc atgtgccata tcgtttgcgt 2340 ttttttgctg
gtggcgacca aagtattcgt ggatatgcac atgacagttt atcacctata 2400
tcagataagg gttatctgac aggcggtcaa gtattggcgg ttggtacagc tgaatataat
2460 tatgaattta tgaaagattt gcgtttggcg gtttttggtg atattggtaa
tgcttatgat 2520 aaaggcttta ctaatgatac caaaattggt gcaggtgtcg
gtgttcgctg ggcatcacct 2580 gtcggtcaag ttcgtgttga tgtggcaact
ggtgtcaaag aagagggcaa tcccattaag 2640 ctgcattttt ttattggcac
accattttaa 2670 4 889 PRT Moraxella catarrhalis 4 Gln Gln Asn Asn
Pro Ala Asn Ile Ile Asn His Val Pro Ala His Asp 1 5 10 15 Thr Ala
Ile Asn Gln Ala Lys Ala Gly Asn Pro Pro Val Leu Leu Thr 20 25 30
Pro Glu Gln Ile Gln Ala Arg Leu Asn Ala Ala Gly Leu Asn Ala Lys 35
40 45 Pro Gln Ser Gln Ala Leu Asp Val Val Asn Phe Asp Asp Gln Ser
Pro 50 55 60 Ile Ser Arg Ile Gly Glu Gln Ser Pro Pro Leu Gly Leu
Asp Met Ser 65 70 75 80 Val Ile Glu Glu Thr Thr Pro Leu Ser Leu Glu
Glu Leu Phe Ala Gln 85 90 95 Glu Ser Thr Glu Met Gly Ile Asn Pro
Asn Asp Tyr Ile Pro Glu Tyr 100 105 110 Gln Gly Glu Gln Pro Asn Ser
Glu Val Val Val Pro Pro Thr Leu Glu 115 120 125 Pro Glu Lys Pro Gly
Leu Ile Lys Arg Leu Tyr Ala Arg Leu Phe Asn 130 135 140 Asp Gly Val
Asn Lys Val Pro Arg Leu Lys Ala Lys Phe Tyr Gln Ser 145 150 155 160
Ser Gln Ser Gly Glu Thr Ser Ala Ile Gly Ser Ser His Gln Lys Thr 165
170 175 Glu Pro Tyr Ala Asn Ile Lys Ala Ala Leu Glu Asp Ile Thr Gln
Glu 180 185 190 Ser Ala Met Asp Leu Asn Gly Ser Ile Pro Arg Leu Arg
Gln Thr Ala 195 200 205 Leu Val Ala Ala Arg Ala Val Gly Tyr Tyr Asp
Ile Asp Leu Ser Ile 210 215 220 Ile Arg Asn Ser Ile Gly Glu Val Asp
Val Ile Ile His Asp Leu Gly 225 230 235 240 Glu Pro Val Tyr Ile Asp
Tyr Arg Ala Val Glu Val Arg Gly Glu Gly 245 250 255 Ala Asp Asp Lys
Ala Phe Thr Thr Val Ala Asp Glu Val Pro Leu Leu 260 265 270 Ile Gly
Asp Val Phe His His Gly Lys Tyr Glu Thr Lys Lys Asn Leu 275 280 285
Ile Glu Asn Ala Ser Ala Glu His Gly Tyr Phe Asp Gly Arg Trp Leu 290
295 300 Asp Arg Ser Val Asp Val Ile Leu Pro Asp Asn Thr Ala Asp Val
Ser 305 310 315 320 Leu Ile Tyr Asp Thr Gly Thr Gln Tyr Arg Phe Asp
Glu Val Ile Phe 325 330 335 Phe Thr Ile Asp Pro Lys Thr Asn Gln Leu
Thr Thr Asp Pro Asp Lys 340 345 350 Leu Pro Val Lys Arg Glu Leu Leu
Glu Gln Leu Leu Thr Val Asn Met 355 360 365 Gly Glu Ala Tyr Asn Leu
Gln Ala Val Arg Ala Leu Ser Asn Asp Leu 370 375 380 Ile Ala Thr Arg
Tyr Phe Asn Met Val Asn Thr Glu Ile Val Phe Pro 385 390 395 400 Glu
Arg Glu Gln Ile Gln Asn Asp Gln Val Ser Phe Glu Gln Ser Ser 405 410
415 Ser Ser Arg Thr Glu Pro Ala Gln Val Asp Glu Ser Thr Leu Glu Pro
420 425 430 Val Ile Glu Thr Val Glu Leu Thr Asp Gly Ile Leu Met Asp
Ile Ser 435 440 445 Pro Ile Glu Phe Ser Ala Ser Asn Leu Ile Gln Asp
Lys Leu Asn Leu 450 455 460 Val Ala Ala Lys Ala Arg His Leu Tyr Asp
Met Pro Asp Asp Arg Val 465 470 475 480 Leu Ala Ile Asn His Asp Asp
Gly Val Asn Arg Ser Ile Leu Gly Arg 485 490 495 Ile Ser Asp Ala Val
Ser Ala Val Ala Arg Ala Ile Leu Pro Asp Glu 500 505 510 Ser Glu Asn
Glu Val Ile Asp Leu Pro Glu Arg Thr Ala Leu Ala Asn 515 520 525 Arg
Lys Thr Pro Ala Asp Val Tyr Gln Ser Lys Lys Val Pro Leu Tyr 530 535
540 Val Phe Val Ala Ser Asp Lys Pro Arg Asp Gly Gln Ile Gly Leu Gly
545 550 555 560 Trp Gly Ser Asp Thr Gly Thr Arg Leu Val Thr Lys Phe
Glu His Asn 565 570 575 Leu Ile Asn Arg Asp Gly Tyr Gln Ala Gly Ala
Glu Leu Arg Leu Ser 580 585 590 Glu Asp Lys Lys Gly Val Lys Leu Tyr
Ala Thr Lys Pro Leu Ser His 595 600 605 Pro Leu Asn Asp Gln Leu Arg
Ala Thr Leu Gly Tyr Gln Gln Glu Val 610 615 620 Phe Gly His Ser
Thr
Asn Gly Phe Asp Leu Ser Thr Arg Thr Leu Glu 625 630 635 640 His Glu
Ile Ser Arg Ser Ile Ile Gln Asn Gly Gly Trp Asn Arg Thr 645 650 655
Tyr Ser Leu Arg Tyr Arg Leu Asp Lys Leu Lys Thr Gln Ala Pro Pro 660
665 670 Glu Thr Trp Gln Asp Leu Pro Val Asp Phe Val Asn Gly Lys Pro
Ser 675 680 685 Gln Glu Ala Leu Leu Ala Gly Val Ala Val His Lys Thr
Val Ala Asp 690 695 700 Asn Leu Val Asn Pro Met Arg Gly Tyr Arg Gln
Arg Tyr Ser Leu Glu 705 710 715 720 Val Gly Ser Ser Gly Leu Val Ser
Asp Ala Asn Met Ala Ile Ala Arg 725 730 735 Ala Gly Ile Ser Gly Val
Tyr Ser Phe Gly Asp Asn Ala Tyr Gly Ser 740 745 750 Asn Arg Ala His
Gln Met Thr Gly Gly Ile Gln Ala Gly Tyr Ile Trp 755 760 765 Ser Asp
Asn Phe Asn His Val Pro Tyr Arg Leu Arg Phe Phe Ala Gly 770 775 780
Gly Asp Gln Ser Ile Arg Gly Tyr Ala His Asp Ser Leu Ser Pro Ile 785
790 795 800 Ser Asp Lys Gly Tyr Leu Thr Gly Gly Gln Val Leu Ala Val
Gly Thr 805 810 815 Ala Glu Tyr Asn Tyr Glu Phe Met Lys Asp Leu Arg
Leu Ala Val Phe 820 825 830 Gly Asp Ile Gly Asn Ala Tyr Asp Lys Gly
Phe Thr Asn Asp Thr Lys 835 840 845 Ile Gly Ala Gly Val Gly Val Arg
Trp Ala Ser Pro Val Gly Gln Val 850 855 860 Arg Val Asp Val Ala Thr
Gly Val Lys Glu Glu Gly Asn Pro Ile Lys 865 870 875 880 Leu His Phe
Phe Ile Gly Thr Pro Phe 885 5 33 DNA Artificial Sequence Primer 5
catgccatgg gtcaacaaaa taaccctgca aac 33 6 37 DNA Artificial
Sequence Primer 6 ctagtctaga ttaaaatggt gtgccaataa aaaaatg 37
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