Note: Descriptions are shown in the official language in which they were submitted.
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Novel Compounds
FIELI3 OF T>EIE INVENTION
This invention relates to polynucleotides, (herein referred to as "BASB207
polynucleotide(s)"), polypeptides encoded by them (referred to herein as
°°BASB207" or
"BASB207 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.
BACKGROI~ND OF THE INVENTION
Haemophilus influenzae is a non-motile Gram negative bacterium. Man is its
only
natural host.
H. inflasenzae isolates are usually classified according to their
polysaccharide capsule.
Six different capsular types designated a through f have been identif ed.
Isolates that
fail to agglutinate with antisera raised against one of these six serotypes
are classified
as non typeable, and do not express a capsule.
The H. influenzae type b is clearly different from the other types in that it
is a major
cause of bacterial meningitis and systemic diseases. Non typeable X in,
fh~enzae
(NTHi) are only occasionally isolated from the blood of patients with systemic
disease.
NTHi is a common cause of pneumonia, exacerbation of chronic bronchitis,
sinusitis
and otitis media.
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 children have experienced at
least one
episode of otitis before reaching the age of 3 (1). Left untreated, or
becoming
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chronic, this disease may lead to hearing loss that can 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 delayed
speech
learning.
Three bacterial species are primarily isolated from the middle ear of children
with
otitis media: Streptococcus pnea~moniae, NTHi and M. catarrhalis. These are
present
in 60 to 90 % of eases. A review of recent studies shows that S. pnea~moniae
and
NTHi each represent about 30 °~°, and M. catarrhalis about 15 %
of otitis media
cases (2). Other bacteria can be isolated from the middle ear (H. inJluenzae
type B, S.
pyogenes, ...) but at a much lower frequency (2 % of the cases or less).
Epidemiological data indicate that, for the pathogens found in the middle ear,
the
colonization of the upper respiratory tract is an absolute prerequisite for
the
1 S development of an otitis; other factors are however also required to lead
to the
disease (3-9). 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 other 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
(5). An
alternative explanation is that the exposure to environmental factors allows 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 (2).
Various proteins of H. infla~enzae have been shown to be involved in
pathogenesis or
have been shown to confer protection upon vaccination in animal models.
Adherence of NTHi to human nasopharygeal epithelial cells has been reported
(10).
Apart from fimbriae and pili (11-15), many adhesins have been identified in
NTHi.
Among them, two surface exposed high-molecular-weight~proteins designated
2
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HMW 1 and HMW2 have been shown to mediate adhesion of NTHi to epithelial cells
(16). Another family of high molecular weight proteins has been identified in
NTHi
strains that lack proteins belonging to HMW IlHMW2 family. The NTHi 115 kDa
Hia protein (17) is highly similar to the Hsf adhesin expressed by H.
influenzae type
b strains ( 18). Another protein, the Hap protein shows similarity to IgA 1
serine
proteases and has been shown to be involved in both adhesion and cell entry (
19).
Five major outer membrane proteins (OMP) have been identified and numerically
numbered.
Original studies using H. influenzae type b strains showed that antibodies
specific for
P1 and P2 protected infant rats from subsequent challenge (20-21). P2 was
found to
be able to induce bactericidal and opsonic antibodies, which are directed
against the
variable regions present within surface exposed loop structures of this
integral OMP
(22-23). The lipoprotein P4 also could induce bactericidal antibodies (24).
P6 is a conserved peptidoglycan-associated lipoprotein making up 1-5 % of the
outer
membrane (25). Later a lipoprotein of about the same mol. wt. was recognized,
called
PCP (P6 crossreactive protein) (26). A mixture of the conserved lipoproteins
P4, P6
and PCP did not reveal protection as measured in a chinchilla otitis-media
model
(27). P6 alone appears to induce protection in the chinchilla model (28).
P5 has sequence homology to the integral Escherichia coli OmpA (29-30). PS
appears to undergo antigenic drift during persistent infections with NTHi
(31).
However, conserved regions of this protein induced protection in the
chinchilla
model of otitis media.
In line with the observations made with gonococci and meningococci, NTHi
expresses a dual human transferrin receptor composed of TbpA and TbpB when
grown under iron limitation. Anti-TbpB protected infant rats. (32). Hemoglobin
l
haptoglobin receptors have also been described for NTHi (33). A receptor for
Haem:
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Hemopexin has also been identified (34). A lactofernn receptor is also present
in
NTHi, but is not yet characterized (35).
A 80kDa OMP, the D 15 surface antigen, provides protection against NTHi in a
mouse challenge model. (36). A 42kDa outer membrane lipoprotein,LPD is
conserved amongst Haemophilus influenzae and induces bactericidal antibodies
(37).
A minor 98kDa OMP (38), was found to be a protective antigen, this OMP may
very
well be one of the Fe-limitation inducible OMPs or high molecular weight
adhesins
that have been characterized. H. influenzae produces IgAI-protease activity
(39).
IgA 1-proteases of NTHi reveals a high degree of antigenic variability (40).
Another OMP of NTHi, OMP26, a 26-kDa protein has been shown to enhance
pulmonary clearance in a rat model (41). The NTHi HfrA protein has also been
shown to be a protective antigen. Indeed, this protein protected Chinchilla
against
otitis media and protected infant rats against H. influenzae type b bacteremia
(42)
Background References
1. Klein, JO ( 1994) Clin.Inf.Dis 19:823
2. Murphy, TF (1996) Microbiol.Rev. 60:267
3. Dickinson, DP et al. (1988) J. Infect.Dis. 158:205
4. Faden, HL et al. (1991) Ann.Otorhinol.Laryngol. 100:612
5. Faden, HL et al (1994) J. Infect.Dis. 169:1312
6. Leach, AJ et al. (1994) Pediatr.Infect.Dis.J. 13:983
7. Prellner, KP et al. (1984) Acta Otolaryngol. 98:343
8. Stenfors, L-E and Raisanen, S. ( 1992) J.Infeet.Dis. 165:1148
9. Stenfors, L-E and Raisanen, S. ( 1994) Acta Otolaryngol. 113:191
10. Read, RC. et al. (1991) J. Infect. Dis. 163:549
11. Brinton, CC. et al. (1989) Pediatr. Infect. Dis. J. 8:S54
12. Kar, S. et al. (1990) Infect. Immun. 58:903
13. Gildorf, JR. et al. (1992) Infect. Immun. 60:374
14. St. Genre, JW et al. (1991) Infect. Immun. 59:3366
15. St. Genre, JW et al. ( 1993) Infect. Immun. 61: 2233
4
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
16. St. Genre, JW. et al. (1993) Proc. Natl. Acad. Sci. USA 90:2875
17. Barenkamp, SJ. et JW St Genre ( 1996) Mol. Microbiol. (In press)
18. St. Genre, JW. et al. ( 1996) J. Bact. 178:6281
19. St. Genre, JW. et al. (1994) Mol. Microbiol. 14:217
20. Loeb, MR. et al. ( 1987) Infect. Immun. 55:2612
21. Musson, RS. Jr. et al. (1983) J. Clin. Invest. 72:677
22. Haase, EM. et al. (1994) Infect. Immun. 62:3712
23. Troelstra, A. et al. ( 1994) Infect. Immun. 62:779
24. Green, BA. et al. (1991) Infect.Immun.59:3191
25. Nelson, MB. et al. (1991) Infect. Immun. 59:2658
26. Deich, RM. et al. (1990) Infect. Immun. 58:3388
27. Green, BA. et al. (1993) Infect.immun. 61:1950
28. Demaria, TF. et al. ( 1996) Infect. Immun. 64:5187
29. Miyamoto, N., Bakaletz, LO (1996) Microb. Pathog. 21:343
30. Munson, RS.j.r. et al. (1993) Infect. Immun. 61:1017
31. Duim, B. et a1. (1997) Infect. Immun. 65:1351
32. Loosmore, SM. et a1(1996) Mol.Microbiol. 19:575
33. Maciver, I. et al. (1996) Infect. Immun. 64:3703
34. Cope, LD. et al. (1994) Mol.Microbiol. 13:868
35. Schryvers, AB. et al. (1989) J. Med. Microbiol. 29:121
36. Flack, FS. et al. (1995) Gene 156:97
37. Akkoyunlu, M. et al. (1996) Infect. Immun. 64:4586
38. Kimura, A. et al. (1985) Infect. Immun. 47:253
39. Mulks, MH. et Shoberg, RJ (1994) Meth. Enzymol. 235:543
40. Lomholt, H. Alphen, Lv, Kilian, M. (1993) Infect. Immun. 61:4575
41. Kyd, J.M. and Cripps, A. W. ( 1998) Infect. Immun. 66:2272
42. Loosmore, S.M. et al. (199$) Infect. Immun. 66:899
The frequency of NTHi infections has risen dramatically in the past few
decades. This
phenomenon has created an unmet medical need for new anti-microbial agents,
vaccines, drug screening methods and diagnostic tests for this organism. The
present
invention aims to meet that need.
5
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SUMMARY OF THE INVENTION
The present invention relates to BASB207, in particular BASB207 polypeptides
and
BASB207 polynueleotides, 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 BASB207
polynucleotides or polypeptides.
Various changes and modifications within the spirit and scope of the disclosed
invention
will become readily apparent to those skilled in the art from reading the
following
descriptions and from reading the other parts of the present disclosure.
DESCRIPTION OF THE INVENTION
The invention relates to BASB207 polypeptides and polynucleotides as described
in
greater detail below. In particular, the invention relates to polypeptides and
polynucleotides of BASB207 of non typeable H. influenzae. The BASB207
polypeptide has some characteristics of an autotransporter protein. BASB207
has a
signal sequence localised from residue 1 to residue 45. This signal sequence
is
cleaved in the mature protein and it could thus be exposed at the surface of
the
bacterium. The mature protein has three distinct domains. The hF-terminal
domain is
located from residue 46 to residue 117 of the BASB207 polypeptide. This domain
contains a (3-wrap that has adhesin function in some autotransporter proteins.
This
domain is likely to be surface-exposed. The second domain of the mature
protein is
likely to contain an hydrophobic a-helix located from residue 1175 to the
residue 1211
of the BASB207 polypeptide. The third domain is likely to contain four anti-
parallel
(3-strands located from residue 1237 to the end of the BASB207 polypeptide.
These (3-
6
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WO 02/32946 PCT/EPO1/11983
strands are predicted to span the outer-membrane, forming a pore through which
the
N-terminal domain is secreted.
The invention relates especially to BASB207 polynucleotides and encoded
polypeptides listed in table 1. Those polynucleotides and encoded polypeptides
have
the nucleotide and amino acid sequences set out in SEQ ID NO:1 to SEQ ID N0:8
as
described in table A.
Table A
Strain isolated from Nucleotidic peptidic
in sequence sequence
3224A USA Otitis mediaSEQ ID NO:1 SEQ ID N0:2
ATCC PTA-1816
322~A USA Otitis mediaSEQ ID N0:3 SEQ ID NO:~
32190 USA Otitis mediaSEQ ID NO:S SEQ ID N0:6
A84016~ NL Carrier strainSEQ ID N0:7 SEQ ID N0:8
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 ribopolynueleotides.
The sequences of the BASB207 polynucleotides are set out in SEQ ID NO: l, 3,
5, 7.
SEQ Group 1 refers herein to any one of the polynucleotides set out in SEQ ID
NO:1,
3, 5, 7.
The sequences of the BASB207 encoded polypeptides are set out in SEQ ID N0:2,
4, 6,
8. SEQ Group 2 refers herein to any one of the encoded polypeptides set out in
SEQ ID
N0:2, 4, 6, 8.
Polypeptides
In one aspect of the invention there are provided polypeptides of non typeable
H.
influenzae referred to herein as "BASB207" and "BASB207 polypeptides" as well
as
biologically, diagnostically, prophylaetically, clinically or therapeutically
useful variants
thereof, and compositions comprising the same.
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The present invention further provides for:
(a) an isolated polypeptide which comprises an amino acid sequence which has
at least
85% identity, preferably at least 90% identity, more preferably at least 95%
identity,
most preferably at least 97-99% or exact identity, to that of any sequence of
SEQ
Group 2;
(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 any sequence of SEQ Group 1 over the entire length of the selected sequence
of
SEQ Group 1; or
(c) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence encoding a polypeptide which has at least 85% identity, preferably at
least
90% identity, more preferably at least 95% identity, even more preferably at
least 97-
99% or exact identity, to the amino acid sequence of any sequence of SEQ Group
2.
The BASB207 polypeptides provided in SEQ Group 2 are the BASB207
polypeptides from non typeable H. influenzae strains as described in tableA.
The invention also provides an immunogenic fragment of a BASB207 polypeptide,
that is, a contiguous portion of the BASB207 polypeptide which has the same or
substantially the same immunogenic activity as the polypeptide comprising the
corresponding amino acid sequence selected from SEQ Group 2 ; That is to say,
the
fragment (if necessary when coupled to a carrier) is capable of raising an
immune
response which recognises the BASB207 polypeptide. Such an immunogenic
fragment may include, for example, the BASB207 polypeptide lacking an N-
terminal
leader sequence, andlor a transmembrane domain and/or a C-terminal anchor
domain.
In a preferred aspect the immunogenic fragment of BASB207 according to the
invention comprises substantially all of the extracellular domain of a
polypeptide
which has at least $5% identity, preferably at least 90% identity, more
preferably at
8
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WO 02/32946 PCT/EPO1/11983
least 95% identity, most preferably at least 97-99% identity, to that a
sequence
selected from SEQ Group 2 over the entire length of said sequence.
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
BASB207 polypeptides, fragments may be "free-standing," or comprised within a
larger
polypeptide of which they form a part or region, most preferably as a single
continuous
region in a single larger polypeptide.
Preferred fragments include, for example, truncation polypeptides having a
portion of an
amino acid sequence selected from SEQ Group 2 or of variants thereof, such as
a
continuous series of residues that includes an amino- and/or carboxyl-terminal
amino
acid sequence. Degradation forms of the polypeptides of the invention produced
by or in
a host cell, are also preferred. Further preferred are fragments characterized
by structural
or functional attributes such as fragments that comprise alpha-helix and alpha-
helix
forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-
forming
regions, coil and coil-forming regions, hydrophilic regions, hydrophobic
regions, alpha
amphipathic regions, beta amphipathic regions, flexible regions, surface-
forming
regions, substrate binding region, and high antigenic index regions.
Further preferred fragments include an isolated polypeptide comprising an
amino acid
sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from
an
amino acid sequence selected from SEQ Group 2 or an isolated polypeptide
comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100
contiguous amino acids truncated or deleted from an amino acid sequence
selected
from SEQ Group 2 .
BASB207 has a signal sequence localised from residue 1 to residue 45. The
mature
protein has three distinct domains. The N-terminal domain is located from
residue X16 to
residue 1174 of the BASB207 polypeptide. This domain contains a j3-wrap that
has
adhesin function in some autotransporter proteins. This domain is likely to be
surface-
9
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
exposed. The second domain of the mature protein is likely to contain an
hydrophobic a-
helix located from residue 1175 to the residue 1211 of the BASB207
polypeptide.°'fhe
third domain is likely to contain four anti-parallel (3-strands located from
residue 1237 to
the end of the BASB207 polypeptide. These (3-strands are predicted to span the
outer-
membrane, forming a pore through which the N-terminal domain is secreted. Each
of
these peptides/domains (comprised within any of the SEQ Group 2 polypeptides)
are
preferred fragments of the invention, as are truncates, variants, conjugates
and
recombinant fusions as described herein. The N-terminal domain is particularly
preferred
as a vaccine candidate. Furthermore full length polypeptides missing one or
more of the
above peptides/domains are further peptides/polypeptides of the invention (for
instance a
polypeptide lacking the signal peptide).
Still further preferred fragments are those which comprise a B-cell or T-
helper epitope,
for example those fragments/peptides described in Example 13.
Fragments of the polypeptides of the invention may be employed for producing
the
corresponding full-length polypeptide by peptide synthesis; therefore, these
fragments
may be employed as intermediates for producing the full-length polypeptides of
the
invention.
Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1
amino
acids are substituted, deleted, or added in any combination.
The polypeptides, or immunogenic fragments, of the invention may be in the
form of
the "mature" protein or may be a part of a larger protein such as a precursor
or a
fusion protein. It is often advantageous to include an additional amina 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.
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In one aspect, the invention relates to genetically engineered soluble fusion
proteins
comprising a polypeptide of the present invention, or a fragment thereof, and
various
portions of the constant regions of heavy or light chains of immunoglobulins
of
various subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the
constant part of the heavy chain of human IgG, particularly IgGI, where fusion
takes
place at the hinge region. In a particular embodiment, the Fc part can be
removed
simply by incorporation of a cleavage sequence which can be cleaved with blood
clotting factor Xa.
Furthermore, this invention relates to processes for the preparation of these
fusion
proteins by genetic engineering, and to the use thereof for drug screening,
diagnosis
and therapy. A further aspect of the invention also relates to polynucleotides
encoding such fusion proteins. Examples of fusion protein technology can be
found
in International Patent Application Nos. W094129458 and W094/22914.
The proteins may be chemically conjugated, or expressed as recombinant fusion
proteins allowing increased levels to be produced in an expression system as
compared to non-fused protein. The fusion partner may assist in providing T
helper
epitopes (immunological fusion partner), preferably T helper epitopes
recognised by
humans, or assist in expressing the protein (expression enhancer) at higher
yields
than the native recombinant protein. Preferably the fusion partner will be
both an
immunological fusion partner and expression enhancing partner.
Fusion partners include protein D from Haemophilats influenzae and the non-
structural protein from influenza virus, NS 1 (hemagglutinin). Another fusion
partner
is the protein known as Omp26 (WO 97101638). Another fusion partner is the
protein known as LytA. Preferably the G terminal portion of the molecule is
used.
LytA is derived from Streptococcus pneurnoniae which synthesize an N-acetyl-L-
alanine amidase, amidase LytA, (coded by the lvt~t gene tGene, 43 (19$6) page
265-
272 f ) an autolysin that specifically degrades certain bonds in the
peptidoglycan
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backbone. The C-terminal domain of the LytA protein is responsible for the
affinity
to the choline or to some choline analogues such as DEAF. This property has
bee$
exploited for the development ofE.coli C-LytA expressing plasmids useful for
expression of fusion proteins. Purification of hybrid proteins containing the
C-LytA
fragment at its amino terminus has been described {Biotechnology: 10, (1992)
page
795-798 } . It is possible to use the repeat portion of the LytA molecule
found in the
C terminal end starting at residue 178, for example residues 188 - 305.
The present invention also includes variants of the aforementioned
polypeptides, that
is polypeptides that vary from the referents by conservative amino acid
substitutions,
whereby a residue is substituted by another with like characteristics. Typical
such
substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the
acidic
residues Asp and Glu; among Asn and Gln; and among the basic residues Lys and
Arg; or aromatic residues Phe and Tyr.
Polypeptides of the present invention can be prepared in any suitable manner.
Such
polypeptides include isolated naturally occurring polypeptides, recombinantly
produced polypeptides, synthetically produced polypeptides, or polypeptides
produced
by a combination of these methods. Means for preparing such polypeptides are
well
understood in the art.
It is most preferred that a polypeptide of the invention is derived from non
typeable H.
influenzae, however, it may preferably be obtained from other organisms of the
same
taxonomic genus. A polypeptide of the invention may also be obtained, for
example,
from organisms of the same taxonomic family or order.
Polynucleotides
It is an object of the invention to provide polynucleotides that encode
BASB207
polypeptides, particularly polynucleotides that encode the polypeptides herein
designated BASB207.
12
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In a particularly preferred embodiment of the invention the polynucleotides
comprise a
region encoding BASB207 polypeptides comprising sequences set out in SEQ
Group° 1
which include full length gene, or a variant thereof.
The BASB207 polynucleotides provided in SEQ Group 1 are the BASB207
polynucleotides from non typeable H. inJlasenzae strains as described in table
A.
As a further aspect of the invention there are provided isolated nucleic acid
molecules encoding andlor expressing BASB207 polypeptides and polynucleotides,
particularly non typeable H. influenzae BASB207 polypeptides and
polynucleotides,
including, for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs,
genomic DNAs, B- and Z-DNAs. Further embodiments of the invention include
biologically, diagnostically, prophylactically, clinically or therapeutically
useful
polynucleotides and polypeptides, and variants thereof, and compositions
comprising the same.
Another aspect of the invention relates to isolated polynucleotides, including
at least one
full length gene, that encodes a BASB207 polypeptide having a deduced amino
acid
sequence of SEQ Group 2 and polynucleotides closely related thereto and
variants
thereof.
In another particularly preferred embodiment of the invention relates to
BASB207
polypeptide from non typeable H. inflzienzae comprising or consisting of an
amino acid
sequence selected from SEQ Group 2 or a variant thereof.
Using the information provided herein, such as a polynucleotide sequences set
out in
SEQ Group 1 , a polynucleotide of the invention encoding BASB207 polypeptides
may
be obtained using standard cloning and screening methods, such as those for
cloning and
sequencing chromosomal DNA fragments from bacteria using non typeable H
influenzae strain322~A cells as starting material, followed by obtaining a
full length
clone. For example, to obtain a polynucleotide sequence of the invention, such
as a
13
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WO 02/32946 PCT/EPO1/11983
polynucleotide sequence given in SEQ Group l, typically a library of clones of
chromosomal DNA of non typeable H. influenzae strain 3224A 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 LtIBOR~4TORYMANU~1L, 2nd Ed.; Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York (1989). (see in particular
Screening
By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates
13.70). Direct genomic DNA sequencing may also be performed to obtain a full
length gene sequence. Illustrative of the invention, each polynucleotide set
out in SEQ
Group 1 was discovered in a DNA library derived from non typeable H.
influenzae.
Moreover, each DNA sequence set out in SEQ Group 1 contains an open reading
frame
encoding a protein having about the number of amino acid residues set forth in
SEQ
Group 2 with a deduced molecular weight that can be calculated using amino
acid
residue molecular weight values well known to those skilled in the art.
The polynucleotides of SEQ Group l, between the start codon and the stop
codon,
encode respectively the polypeptides of SEQ Group 2. The nucleotide number of
start
codon and first nucleotide of stop codon are listed in table B for each
polynucleotide of
SEQ Group 1.
Table B
nucleotidic encoded peptidicStart codon 1st nucleotide
of
sequence sequence stop codon
1 ~1
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WO 02/32946 PCT/EPO1/11983
SEQ ID NO:1 SEQ ID N0:2 1 3895
SEQ ID N0:3 SEQ ID N0:4 1 3892*
SEQ ID N0:5 SEQ iD N0:6 1 3892*
SEQ ID N0:7 SEQ ID N0:8 1 3892**
* first nucleotide of the last codon of encoding sequence
** first nucleotide of the last codon of partial encoding sequence
In a further aspect, the present invention provides for an isolated
polynucleotide
comprising or consisting of:
(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 any polynucleotide sequence from
SEQ Group 1 over
the entire length of the polynucleotide sequence from SEQ Group 1; or
(b) a polynucleotide sequence encoding a polypeptide which has at least 85%
identity,
preferably at least 90% identity, more preferably at least 95% identity, even
more
preferably at least 97-99% or 100% exact identity, to any amino acid sequence
selected from SEQ Group 2 , over the entire length of the amino acid sequence
from
SEQ Group 2.
A polynucleotide encoding a polypeptide of the present invention, including
homologs
and orthologs from species other than non typeable H. infla~enzae, 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°G and
an SDS concentration from 0.1 - 1%) with a labeled or detectable probe
consisting of or
comprising any sequence selected from SEQ Group 1 or a fragment thereof; and
isolating a full-length gene andlor genomie clones containing said
polynucleotide
sequence.
The invention provides a polynucleotide sequence identical over its entire
length to a
coding sequence (open reading frame) set out in SEQ Group 1. Also provided by
the
invention is a coding sequence for a mature polypeptide or a fragment thereof,
by itself
as well as a coding sequence for a mature polypeptide or a~fragment in reading
frame
CA 02426413 2003-04-08
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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 polynueleotide
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., ZISA 86: 821-824 (1989), or an HA peptide tag
(Wilson et al.,
Cell 37: 767 (1984), both of which may be useful in purifying polypeptide
sequence
fused to them. Polynucleotides of the invention also include, but are not
limited to,
polynucleotides comprising a structural gene and its naturally associated
sequences that
control gene expression.
The nucleotide sequence encoding the BASB207 polypeptide of SEQ Group 2 may be
identical to the corresponding polynucleotide encoding sequence of SEQ Group
1. The
position of the first and last nucleotides of the encoding sequences of SEQ
Goup 1 are
listed in table C. Alternatively it may be any sequence, which as a result of
the
redundancy (degeneracy) of the genetic code, also encodes a polypeptide of SEQ
Group 2.
Table C
nueleotidic encoded peptidicStart codon Last nucleotide
sequence sequence of encoding
sequence
SEQ ID NO:1 SEQ ID N0:2 1 3894
SEQ ID N0:3 SEQ ID N0:4 1 3894
SEQ ID NO:S SEQ ID N0:6 1 3894
SEQ ID N0:7 SEQ ID N0:8 1 3893*
last nucleotide of the last codon of partial encoding sequence
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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 non
typeable H. influenzae BASB207 having an amino acid sequence set out in any of
the
sequences of SEQ Group 2 . The term also encompasses polynucleotides that
include a
single continuous region or discontinuous regions encoding the polypeptide
(for
example, polynueleotides interrupted by integrated phage, an integrated
insertion
sequence, an integrated vector sequence, an integrated transposon sequence, or
due to
RNA editing or genomic DNA reorganization) together with additional regions,
that also
may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described
herein that
encode variants of a polypeptide having a deduced amino acid sequence of any
of the
sequences of SEQ Group 2 . Fragments of polynucleotides of the invention may
be
used, for example, to synthesize full-length polynucleotides of the invention.
Preferred fragments are those polynucleotides which encode a B-cell or T-
helper
epitope, for example the fragments/peptides described in Example 13, and
recombinant,
chimeric genes comprising said polynucleotide fragments.
Further particularly preferred embodiments are polynucleotides encoding
BASB207
variants, that have the amino acid sequence of BASB207 polypeptide of any
sequence
from SEQ Group 2 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no
amino acid
residues are substituted, modified, deleted and/or added, in any combination.
Especially
preferred among these are silent substitutions, additions and deletions, that
do not alter
the properties and activities of BASB207 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at
least 85°l0
identical over their entire length to a polynucleotide encoding BASB207
polypeptide
having an amino acid sequence set out in any of the sequences of SEQ Group 2 ,
and
polynucleotides that are complementary to such polynucleotides. Alternatively,
most
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highly preferred are polynucleotides that comprise a region that is at least
90% identical
over its entire length to a polynucleotide encoding BASB207 polypeptide and
polynucleotides complementary thereto. In this regard, polynucleotides at
least 95%
identical over their entire length to the same are particularly preferred.
Furthermore,
those with at least 97% are highly preferred among those with at least 95%,
and among
these those with at least 98% and at least 99% are particularly highly
preferred, with at
least 99% being the more preferred.
Preferred embodiments are polynucleotides encoding polypeptides that retain
substantially the same biological function or activity as the mature
polypeptide encoded
by a DNA sequence selected from SEQ Group 1.
In accordance with certain preferred embodiments of this invention there are
provided
polynucleotides that hybridize, particularly under stringent conditions, to
BASB207
polynucleotide sequences, such as those polynucleotides of SEQ Group 1.
The invention further relates to polynucleotides that hybridize to the
polynucleotide
sequences provided herein. In this regard, the invention especially relates to
polynucleotides that hybridize under stringent conditions to the
polynucleotides
described herein. As herein used, the terms "stringent conditions" and
"stringent
hybridization conditions" mean hybridization occurring only if there is at
least 95% and
preferably at least 97% identity between the sequences. A specific example of
stringent
hybridization conditions is overnight incubation at 42°C in a solution
comprising:
50°l° formamide, 5x SSC (150mM NaGI, lSmM trisodium citrate), 50
mM sodium
phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20
micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the
hybridization support in 0. lx SSG at about 65°C. Hybridization and
wash conditions
are well known and exemplified in Sambrook, et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),
particularly
Chapter 11 therein. Solution hybridization may also be used with the
polynucleotide
sequences provided by the invention.
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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 any of the sequences
of SEQ
Group 1 under stringent hybridization conditions with a probe having the
sequence of
said polynucleotide sequence set forth in the corresponding sequence of SEQ
Group 1
or a fragment thereof; and isolating said polynucleotide sequence. Fragments
useful
for obtaining such a polynucleotide include, for example, probes and primers
fully
described elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the
invention, for
instance, the polynucleotides of the invention, may be used as a hybridization
probe for
RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones
encoding BASB207 and to isolate cDNA and genomic clones of other genes that
have a
high identity, particularly high sequence identity, to the BASB207 gene. Such
probes
generally will comprise at least 15 nucleotide residues or base pairs.
Preferably, such
probes will have at least 30 nucleotide residues or base pairs and may have at
least 50
nucleotide residues or base pairs. Particularly preferred probes will have at
least 20
nucleotide residues or base pairs and will have less than 30 nucleotide
residues or base
pairs.
A coding region of a BASB207 gene may be isolated by screening using a DNA
sequence provided in SEQ Group 1 to synthesize an oligonucleotide probe. A
labeled
oligonucleotide having a sequence complementary to that of a gene of the
invention is
then used to screen a library of cDNA, genomic DNA or mRNA to determine which
members of the library the probe hybridizes to.
There are several methods available and well known to those skilled in the art
to
obtain full-length DNAs, or extend short DNAs, for example those based on the
method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman,
et
al., PNAS ClS~1 85: 8998-9002, 1988). Recent modifications of the technique,
19
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WO 02/32946 PCT/EPO1/11983
exemplified by the MarathonTM technology (Clontech Laboratories Inc.) for
example,
have significantly simplified the search for longer cDNAs. In the MarathonTM
technology, cDNAs have been prepared from mRNA extracted from a chosen tissue
and an'adaptor' sequence ligated onto each end. Nucleic acid amplification
(PCR) is
then carried out to amplify the "missing" 5' end of the DNA using a
combination of
gene specific and adaptor specific oligonucleotide primers. The PCR reaction
is then
repeated using "nested" primers, that is, primers designed to anneal within
the
amplified product (typically an adaptor specific primer that anneals further
3' in the
adaptor sequence and a gene specific primer that anneals further 5' in the
selected gene
sequence). The products of this reaction can then be analyzed by DNA
sequencing
and a full-length DNA constructed either by joining the product directly to
the existing
DNA to give a complete sequence, or carrying out a separate full-length PCR
using
the new sequence information for the design of the 5' primer.
The polynucleotides and polypeptides of the invention may be employed, for
example,
as research reagents and materials for discovery of treatments of and
diagnostics for
diseases, particularly human diseases, as further discussed herein relating to
polynucleotide assays.
The polynucleotides of the invention that are oligonucleotides derived from a
sequence of SEQ Group 1 may be used in the processes herein as described, but
preferably for PCR, to determine whether or not the polynucleotides identified
herein
in whole or in part are transcribed in bacteria in infected tissue. It is
recognized that
such sequences will also have utility in diagnosis of the stage of infection
and type of
infection the pathogen has attained.
The invention also provides polynucleotides that encode a polypeptide that is
the mature
protein plus additional amino or carboxyl-terminal amino acids, or amino acids
interior
to the mature polypeptide (when the mature form has more than one polypeptide
chain,
for instance). Such sequences may play a role in processing of a protein from
precursor
to a mature form, may allow protein transport, may lengthen or shorten protein
half life
CA 02426413 2003-04-08
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or may facilitate manipulation of a protein for assay or production, among
other things.
As generally is the case in vivo, the additional amino acids may be processed
away from
the mature protein by cellular enzymes.
For each and every polynueleotide of the invention there is provided a
polynucleotide
complementary to it. It is preferred that these complementary polynucleotides
are fully
complementary to each polynucleotide with which they are complementary.
A precursor protein, having a mature form of the polypeptide fused to one or
more
prosequences may be an inactive form of the polypeptide. When prosequences are
removed such inactive precursors generally are activated. Some or all of the
prosequences may be removed before activation. Generally, such precursors are
called
proproteins.
In addition to the standard A, G, C, TILT representations for nucleotides, the
term "N"
may also be used in describing certain polynucleotides of the invention. "N"
means
that any of the four DNA or RNA nucleotides may appear at such a designated
position in the DNA or RNA sequence, except it is preferred that N is not a
nucleic
acid that when taken in combination with adjacent nucleotide positions, when
read in
the correct reading frame, would have the effect of generating a premature
termination
codon in such reading frame.
In sum, a polynucleotide of the invention may encode a mature protein, a
mature protein
plus a leader sequence (which may be referred to as a preprotein), a precursor
of a
mature protein having one or more prosequences that are not the leader
sequences of a
preprotein, or a preproprotein, which is a precursor to a proprotein, having a
leader
sequence and one or more prosequences, which generally are removed during
processing
steps that produce active and mature forms of the polypeptide.
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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
particu'Iar
genetic immunization.
The use of a polynucleotide of the invention in genetic immunization will
preferably
employ a suitable delivery method such as direct injection of plasmid DNA into
muscles (Wolff et al., Hum Mol Genet (1992) 1: 363, Manthorpe et al., Hum.
Gene
Ther. (1983) 4: 419), delivery of DNA complexed with specific protein carriers
(Wu et
al., JBiol Chern. (1989) 264: 16985), coprecipitation of DNA with calcium
phosphate
(Benvenisty & Reshef, PNAS US~1, (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., DN~1 Cell
Biol
(1993) 12: 791) and in vivo infection using cloned retroviral vectors (Seeger
et al.,
PN~iS ZIS~1 (1984) 81: 5849).
Vectors, Lost Cells, Expression Systems
The invention also relates to vectors that comprise a polynucleotide or
polynucleotides
of the invention, host cells that are genetically engineered with vectors of
the invention
and the production of polypeptides of the invention by recombinant techniques.
Gell-
free translation systems can also be employed to produce such proteins using
RNAs
derived from the DNA constructs of the invention.
Recombinant polypeptides of the present invention may be prepared by processes
well
known in those skilled in the art from genetically engineered host cells
comprising
expression systems. Accordingly, in a further aspect, the present invention
relates to
expression systems that comprise a polynucleotide or polynucleotides of the
present
invention, to host cells which are genetically engineered with such expression
systems,
and to the production of polypeptides of the invention by recombinant
techniques.
For recombinant production of the polypeptides of the invention, host cells
can be
genetically engineered to incorporate expression systems or portions thereof
or
polynucleotides of the invention. Introduction of a polynucleotide into the
host cell can
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WO 02/32946 PCT/EPO1/11983
be effected by methods described in many standard laboratory manuals, such as
Davis, et
al., B~1SICMETHODSINMOLECULAR BIOLOGY, (1986) and Sambrook, et al.,
MOLECULAR CLONING: ~ LABORATORYMANUAL, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate
transfection, DEAE-dextran mediated transfection, transvection,
microinjection, cationic
lipid-mediated transfection, electroporation, conjugation, transduction,
scrape loading,
ballistic introduction and infection.
Representative examples of appropriate hosts include bacterial cells, such as
cells of
streptococci, staphylococci, enterococci, E. coli, streptomyces,
cyanobacteria, Bacillus
subtilis, Neisseria meningilidis, Haemophilus influenzae and Moraxella
catarrhalis;
fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, Pichia, a
basidiomycete, Candida albicans and ~Ispergillzas; insect cells such as cells
of
Drosophila S2 and Spodoptera Sf~3; animal cells such as CHO, COS, HeLa, C 127,
3T3,
BHK, 293, CV-1 and Bowes melanoma cells; and plant cells, such as cells of a
gymnosperm or angiosperm.
A great variety of expression systems can be used to produce the polypeptides
of the
invention. Such vectors include, among others, chromosomal-, episomal- and
virus
derived vectors, for example, vectors derived from bacterial plasmids, from
bacteriophage, from transposons, from yeast episomes, from insertion elements,
from
yeast chromosomal elements, from viruses such as baculoviruses, papova
viruses, such
as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies
viruses,
picornaviruses, retroviruses, and alphaviruses and vectors derived from
combinations
thereof, such as those derived from 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
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WO 02/32946 PCT/EPO1/11983
techniques, such as, for example, those set forth in Sambrook et al.,
MOLECULAR
CLONING, A LABORATORYMANUtIL, (supra).
In recombinant expression systems in eukaryotes, for secretion of a translated
protein
into the lumen of the endoplasmic reticulum, into the periplasmic space or
into the
extracellular environment, appropriate secretion signals may be incorporated
into the
expressed polypeptide. These signals may be endogenous to the polypeptide or
they
may be heterologous signals.
Polypeptides of the present invention can be recovered and purified from
recombinant
cell cultures by well-known methods including ammonium sulfate or ethanol
precipitation, acid extraction, anion or canon exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity
chromatography, hydroxylapatite chromatography and lectin chromatography. Most
preferably, ion metal affinity chromatography (IMAC) is employed for
purification.
Well known techniques for refolding proteins may be employed to regenerate
active
conformation when the polypeptide is denatured during intracellular synthesis,
isolation and or purification.
The expression system may also be a recombinant live microorganism, such as a
virus or bacterium. The gene of interest can be inserted into the genome of a
live
recombinant virus or bacterium. Inoculation and in vivo infection with this
live
vector will lead to in vivo expression of the antigen and induction of immune
responses. Viruses and bacteria used for this purpose are for instance:
poxviruses
(e.g; vaccinia, fowlpox, canarypox), alphaviruses (Sindbis virus, Semliki
Forest
Virus, Venezuelian Equine Encephalitis Virus), adenoviruses, adeno-associated
virus, picornaviruses (poliovirus, rhinovirus), herpesviruses (varicella
zoster virus,
ete), Listeria, Salmonella , Shigella, BGG, streptococci. 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.
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Diagnostic, Prognostic, Serotyping and Mutation Assays
This invention is also related to the use of BASB207 polynucleotides and
polypepticf~s
of the invention for use as diagnostic reagents. Detection of BASB207
polynucleotides
andlor 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 BASB207 gene or protein, may be detected at the nucleic acid or
amino
acid level by a variety of well known techniques as well as by methods
provided herein.
Polypeptides and polynucleotides for prognosis, diagnosis or other analysis
may be
obtained from a putatively infected andlor infected individual's bodily
materials.
Polynucleotides from any of these sources, particularly DNA or RNA, may be
used
directly for detection or may be amplified enzyrnatically 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 BASB207 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.' 122 (1985). Sequence changes at specific locations also may be revealed
by
CA 02426413 2003-04-08
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nuclease protection assays, such as RNase, V 1 and S 1 protection assay or a
chemical
cleavage method. See, for example, Cotton et al., Proc. Natl. ~lcad. Sci.,
USA, 85: 4~97-
4401 (1985).
In another embodiment, an array of oligonucleotides probes comprising BASB207
nucleotide sequence or fragments thereof can be constructed to conduct
efficient
screening of, for example, genetic mutations, serotype, taxonomic
classification or
identification. Array technology methods are well known and have general
applicability
and can be used to address a variety of questions in molecular genetics
including gene
expression, genetic linkage, and genetic variability (see, for example, Chee
et al.,
Science, 274: 610 (1996)).
Thus in another aspect, the present invention relates to a diagnostic kit
which
compnses:
(a) a polynucleotide of the present invention, preferably any of the
nucleotide
sequences of SEQ Group 1, or a fragment thereof ;
(b) a nucleotide sequence complementary to that of (a);
(c) a polypeptide of the present invention, preferably any of the polypeptides
of SEQ
Group 2 or a fragment thereof; or
(d) an antibody to a polypeptide of the present invention, preferably to any
of the
polypeptides of SEQ Group 2 .
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise
a
substantial component. Such a kit will be of use in diagnosing a disease or
susceptibility to a Disease, among others.
This invention also relates to the use of polynucleotides of the present
invention as
diagnostic reagents. Detection of a mutated form of a polynucleotide of the
invention,
preferably any sequence of SEQ Group 1 , which is associated with a disease or
pathogenicity will provide a diagnostic tool that can add to, or define, a
diagnosis of a
disease, a prognosis of a course of disease, a determination of a stage of
disease, or a
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CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
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 polynueleotide may be detected at the
polynucleotide level by
a variety of techniques, such as those described elsewhere herein.
Cells from an organism carrying mutations or polymorphisms (allelic
variations) in a
polynucleotide andlor 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 BASB207 polypeptide can be used to identify and
analyze
mutations.
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
BASB207 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.
The invention further provides a process for diagnosing, disease, preferably
bacterial
infections, more preferably infections caused by non typeable H. influen~ae,
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 any of
the
sequences of SEQ Group 1. Increased or decreased expression of BASB207
polynueleotide 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-
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WO 02/32946 PCT/EPO1/11983
PCR, RNase protection, Northern blotting, spectrometry and other hybridization
methods.
In addition, a diagnostic assay in accordance with the invention for detecting
over-
expression of BASB207 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 BASB207 polypeptide, in a sample derived from a
host, such
as a bodily material, are well-known to those of skill in the art. Such assay
methods
include radioimmunoassays, competitive-binding assays, Western Blot analysis,
antibody sandwich assays, antibody detection and ELISA assays.
The polynucleotides of the invention may be used as components of
polynucleotide
arrays, preferably high density arrays or grids. These high density arrays are
particularly useful for diagnostic and prognostic purposes. For example, a set
of
spots each comprising a different gene, and further comprising a
polynucleotide or
polynucleotides of the invention, may be used for probing, such as using
hybridization or nucleic acid amplification, using a probes obtained or
derived from
a bodily sample, to determine the presence of a particular polynucleotide
sequence
or related sequence in an individual. Such a presence may indicate the
presence of a
pathogen, particularly non-typeable H. influenzae, and may be useful in
diagnosing
and/or prognosing disease or a course of disease. A grid comprising a number
of
variants of any polynucleotide sequence of SEQ Group 1 is preferred. Also
preferred is a number of variants of a polynucleotide sequence encoding any
polypeptide sequence of SEQ Group 2 .
Antibodies
The polypeptides and polynucleotides of the invention or variants thereof, or
cells
expressing the same can be used as immunogens to produce antibodies
immunospecific
for such polypeptides or polynucleotides respectively. Alternatively,
mimotopes,
3p particularly peptide mimotopes, of epitopes within the polypeptide sequence
may also be
used as immunogens to produce antibodies immunospecifrc for the polypeptide of
the
2$
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
invention. The term "immunospecific" means that the antibodies have
substantially
greater affinity for the polypeptides of the invention than their amity for
other relate
polypeptides in the prior art.
In certain preferred embodiments of the invention there are provided
antibodies against
BASB207 polypeptides or polynucleotides.
Antibodies generated against the polypeptides or polynueleotides 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., Natarre 256:
495-497
(1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96
in
MONOCLONAL ANTIBODIES AND G~INCER THERAPY, Alan R. Liss, Inc. (1985).
Techniques for the production of single chain antibodies {LT.S. Patent No.
4,946,778) can
be adapted to produce single chain antibodies to polypeptides or
polynucleotides of this
invention. Also, transgenic mice, or other organisms or animals, such as other
mammals, may be used to express humanized antibodies immunospecific to the
polypeptides or polynucleotides of the invention.
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-
BASB207 or from naive libraries (McGafferty, et al., (1990), Nature 34$, 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)
Natzrre
352:628).
29
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WO 02/32946 PCT/EPO1/11983
The above-described antibodies may be employed to isolate or to identify
clones
expressing the polypeptides or polynucleotides of the invention to purify the
polypeptides or polynucleotides by, for example, affinity chromatography.
Thus, among others, antibodies against BASB207 polypeptide or BASB207
polynucleotide may be employed to treat infections, particularly bacterial
infections.
Polypeptide variants include antigenically, epitopically or immunologically
equivalent
variants form a particular aspect of this invention.
Preferably, the antibody or variant thereof is modified to make it less
immunogenic in
the individual. For example, if the individual is human the antibody may most
preferably be "humanized," where the complimentarity determining region or
regions
of the hybridoma-derived antibody has been transplanted into a human
monoclonal
antibody, for example as described in Jones et al. (1986), Natasre 321, 522-
525 or
Tempest et al., (1991) Biotechnology 9, 266-273.
Antagonists and Agonists - Assays and Molecules
Polypeptides and polynucleotides of the invention may also be used to assess
the binding
of small molecule substrates and ligands in, for example, cells, cell-free
preparations,
chemical libraries, and natural product mixtures. These substrates and ligands
may be
natural substrates and ligands or may be structural or functional mimetics.
See, e.g.,
Coligan et al., Current Protocols in Imrnitnology 1 (2): Chapter 5 (1991).
The screening methods may simply measure the binding of a candidate compound
to
the polypeptide or polynucleotide, or to cells or membranes bearing the
polypeptide or
polynucleotide, or a fusion protein of the polypeptide by means of a label
directly or
indirectly associated with the candidate compound. Alternatively, the
screening
method may involve competition with a labeled competitor. Further, these
screening
methods may test whether the candidate compound results in a signal generated
by
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
activation or inhibition of the polypeptide or polynucleotide, using detection
systems
appropriate to the cells comprising the polypeptide or polynucleotide.
Inhibitors o~'
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 BASB207
polypeptide and/or polynucleotide activity in the mixture, and comparing the
BASB207 polypeptide andlor polynucleotide activity of the mixture to a
standard.
Fusion proteins, such as those made from Fc portion and BASB207 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):959-9471 ( 1995)).
The polynucleotides, polypeptides and antibodies that bind to and/or interact
with a
polypeptide of the present invention may also be used to configure screening
methods
for detecting the effect of added compounds on the production of mRI~A andlor
polypeptide in cells. For example, an ELISA assay may be constructed for
measuring
secreted or cell associated levels of polypeptide using monoclonal and
polyclonal
antibodies by standard methods known in the art. This can be used to discover
agents
which may inhibit or enhance the production of polypeptide (also called
antagonist or
agonist, respectively) from suitably manipulated cells or tissues.
The invention also provides a method of screening compounds to identify those
which
enhance (agonist) or block (antagonist) the action of BASB207 polypeptides or
31
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
polynucleotides, particularly those compounds that are bacteriostatic andlor
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
BASB207 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 BASB207
agonist
or antagonist. The ability of the candidate molecule to agonize or antagonize
the
BASB207 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 BASB207 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 transduetion, or increase
chemical
channel activity are agonists. Detection of the rate or level of, as the ease
may be,
production of product from substrate, signal transduction, or chemical channel
activity
1 S 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 BASB207
polynucleotide or
polypeptide activity, and binding assays known in the art.
Another example of an assay for BASB207 agonists is a competitive assay that
combines BASB207 and a potential agonist with BASB207 binding molecules,
recombinant BASB207 binding molecules, natural substrates or ligands, or
substrate or
ligand mimetics, under appropriate conditions for a competitive inhibition
assay.
BASB207 can be labeled, such as by radioactivity or a colorimetric compound,
such that
the number of BASB207 molecules bound to a binding molecule or converted to
product
can be determined accurately to assess the effectiveness of the potential
antagonist.
Potential antagonists include, among others, small organic molecules,
peptides,
polypeptides and antibodies that bind to a polynucleotide and/or polypeptide
of the
invention and thereby inhibit or extinguish its activity or expression.
Potential
antagonists also may be small organic molecules, a peptide, a polypeptide such
as a
32
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WO 02/32946 PCT/EPO1/11983
closely related protein or antibody that binds the same sites on a binding
molecule, such
as a binding molecule, without inducing BASB207 induced activities, thereby
preventing the action or expression of BASB207 polypeptides and/or
polynucleotides by
excluding BASB207 polypeptides andlor polynueleotides from binding.
Potential antagonists include a small molecule that binds to and occupies the
binding site
of the polypeptide thereby preventing binding to cellular binding molecules,
such that
normal biological activity is prevented. Examples of small molecules include
but are not
limited to small organic molecules, peptides or peptide-like molecules. Other
potential
antagonists include antisense molecules (see Okano, J. Nez~rochern. 56: 560
(1991);
OLIGODEOXYNUCLEOTIDES ~S ~NTISENSE INHIBITORS OF GENE
EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these
molecules). Preferred potential antagonists include compounds related to and
variants of
BASB207.
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
IgGI, where fusion takes place at the hinge region. In a particular
embodiment, the Fc
part can be removed simply by incorporation of a cleavage sequence which can
be
cleaved with blood clotting factor Xa. Furthermore, this invention relates to
processes
for the preparation of these fusion proteins by genetic engineering, and to
the use
thereof for drug screening, diagnosis and therapy. A further aspect of the
invention
also relates to polynucleotides encoding such fusion proteins. Examples of
fusion
protein technology can be found in International Patent Application Nos.
W09~129~58 and W094/22914.
Each of the polynucleotide sequences provided herein may be used in the
discovery
and development of antibacterial compounds. The encoded protein, upon
expression,
33
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
can be used as a target for the screening of antibacterial drugs.
Additionally, the
polynucleotide sequences encoding the amino terminal regions of the encoded
protein
or Shine-Delgarno or other translation facilitating sequences of the
respective mRNA
can be used to construct antisense sequences to control the expression of the
coding
sequence of interest.
The invention also provides the use of the polypeptide, polynucleotide,
agonist or
antagonist of the invention to interfere with the initial physical interaction
between a
pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible
for
sequelae of infection. In particular, the molecules of the invention may be
used: in the
prevention of adhesion of bacteria, in particular gram positive andlor 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 BASB207 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.
In accordance with yet another aspect of the invention, there are provided
BASB207
agonists and antagonists, preferably bacteristatic or bactericidal agonists
and antagonists.
The antagonists and agonists of the invention may be employed, for instance,
to prevent,
inhibit and/or treat diseases.
In a further aspect, the present invention relates to mimotopes of the
polypeptide of
the invention. A mimotope is a peptide sequence, sufficiently similar to the
native
peptide (sequentially or structurally), which is capable of being recognised
by
antibodies which recognise the native peptide; or is capable of raising
antibodies
which recognise the native peptide when coupled to a suitable carrier.
3~
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
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
G-
terminal hydrophobic amidated tail. Alternatively, the addition or
substitution of a
D-stereoisomer form of one or more of the amino acids (inverso sequences) may
be
performed to create a beneficial derivative, for example to enhance stability
of the
peptide. Mimotopes may also be retro sequences of the natural peptide
sequences, in
that the sequence orientation is reversed. Mimotopes may also be retro-inverso
in
character. Retro, inverso and retro-inverso peptides are described in WO
95124916
and WO 94/05311.
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.
Vaccines
Another aspect of the invention relates to a method for inducing an
immunological
response in an individual, particularly a mammal, preferably humans, which
comprises
inoculating the individual with BASB207 polynucleotide andlor polypeptide, or
a
fragment or variant thereof, adequate to produce antibody andl or T cell
immune
response to protect said individual from infection, particularly bacterial
infection and
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
most particularly non typeable H. influenzae 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 BASB207 polynucleotide andlor
polypeptide, or a fragment or a variant thereof, for expressing BASB207
polynucleotide andlor polypeptide, or a fragment or a variant thereof in vivo
in order
to induce an immunological response, such as, to produce antibody andl 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 DNAIRNA hybrid, a DNA-protein complex or an RNA-protein complex.
A further aspect of the invention relates to an immunological composition that
when
introduced into an individual, preferably a human, capable of having induced
within it
an immunological response, induces an immunological response in such
individual to
a BASB207 polynucleotide and/or polypeptide encoded therefrom, wherein the
composition comprises a recombinant BASB207 polynucleotide andlor polypeptide
encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses
an antigen of said BASB207 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.
BASB207 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/ar immunogenic properties, and preferably~protective properties.
Thus
36
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
fused recombinant protein, preferably further comprises an antigenic co-
protein, such
as lipoprotein D from Haemophilus inJluenzae, Glutathione-S-transferase (GST)
off'
beta-galactosidase, or any other relatively large co-protein which solubilizes
the
protein and facilitates production and purification thereof. Moreover, the co-
protein
may act as an adjuvant in the sense of providing a generalized stimulation of
the
immune system of the organism receiving the protein. The co-protein may be
attached
to either the amino- or carboxy-terminus of the first protein.
In a vaccine composition according to the invention, a BASB207 polypeptide
andlor
polynucleotide, or a fragment, or a mimotope, or a variant thereof may be
present in a
vector, such as the live recombinant vectors described above for example live
bacterial
vectors.
Also suitable are non-live vectors for the BASB207 polypeptide, for example
bacterial
outer-membrane vesicles or "blebs". OM blebs are derived from the outer
membrane
of the two-layer membrane of Gram-negative bacteria and have been documented
in
many Gram-negative bacteria (Zhou, L et al. 1998. FEMS tL~Iicrobiol. Lett.
163:223-
228) including C. trachomatis and C. psittaci. A non-exhaustive list of
bacterial
pathogens reported to produce blebs also includes: Bordetella pertussis,
Borrelia
burgdorferi, Brucella ntelitensis, Brucella ovas, Esherichia coli, Haemophilus
influenzae, Legionella pneumophila, ~Yloraxella catarrhalis, Neisseria
gonorrhoeae,
Neisseria meningitidis, Psettdomonas aeruginosa and Yersinia enterocolitica.
Blebs have the advantage of providing outer-membrane proteins in their native
conformation and are thus particularly useful for vaccines. Blebs can also be
improved for vaccine use by engineering the bacterium so as to modify the
expression
of one or more molecules at the outer membrane. Thus for example the
expression of
a desired immunogenic protein at the outer membrane, such as the BASB207
polypeptide, can be introduced or upregulated (e.g. by altering the promoter).
Instead
or in addition, the expression of outer-membrane molecules which are either
not
relevant (e.g. unprotective antigens or immunodominant but variable proteins)
or
37
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
detrimental (e.g. toxic molecules such as LPS, or potential inducers of an
autoimmune
response) can be downregulated. These approaches are discussed in more detail
below.
The non-coding flanking regions of the BASB207 gene contain regulatory
elements
important in the expression of the gene. This regulation takes place both at
the
transcriptional and translational level. The sequence of these regions, either
upstream or downstream of the open reading frame of the gene, can be obtained
by
DNA sequencing. This sequence information allows the determination of
potential
regulatory motifs such as the different promoter elements, terminator
sequences,
inducible sequence elements, repressors, elements responsible for phase
variation,
the shine-dalgarno sequence, regions with potential secondary structure
involved in
regulation, as well as other types of regulatory motifs or sequences. This
sequence is
a further aspect of the invention. Furthermore, SEQ ID NO: 9 is the non
typeable
Haemophilus influenzae upstream sequence (upstream of the predicted initiation
codon of the preferred genes) comprising approximately 1000bp.
This sequence information allows the modulation of the natural expression of
the
BASB207 gene. The upregulation of the gene expression may be accomplished by
altering the promoter, the shine-dalgarno sequence, potential repressor or
operator
elements, or any other elements involved. Likewise, downregulation of
expression
can be achieved by similar types of modification. Alternatively, by changing
phase
variation sequences, the expression of the gene can be put under phase
variation
control, or it may be uncoupled from this regulation. In another approach, the
expression of the gene can be put under the control of one or more inducible
elements
allowing regulated expression. Examples of such regulation include, but are
not
limited to, induction by temperature shift, addition of inductor substrates
like selected
carbohydrates or their derivatives, trace elements, vitamins, co-factors,
metal ions, etc.
Such modifications as described above can be introduced by several different
means.
The modification of sequences involved in gene expression can be earned out in
vivo
38
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
by random mutagenesis followed by selection for the desired phenotype. Another
approach consists in isolating the region of interest and modifying it by
random
mutagenesis, or site-directed replacement, insertion or deletion mutagenesis.
The
modified region can then be reintroduced into the bacterial genome by
homologous
recombination, and the effect on gene expression can be assessed. In another
approach, the sequence knowledge of the region of interest can be used to
replace or
delete all or part of the natural regulatory sequences. In this ease, the
regulatory region
targeted is isolated and modified so as to contain the regulatory elements
from another
gene, a combination of regulatory elements from different genes, a synthetic
regulatory region, or any other regulatory region, or to delete selected parts
of the
wild-type regulatory sequences. These modified sequences can then be
reintroduced
into the bacterium via homologous recombination into the genome. A non-
exhaustive
list of preferred promoters that could be used for up-regulation of gene
expression
includes the promoters porA, porB, lbpB, tbpB, p 110, 1st, hpuAB from N.
meningitides
or N. gonorroheae; ompCD, copB, lbpB, ompE, UspAl; UspA2; TbpB from M.
Catarrhalis; p l, p2, p~, p5, p6, lpD, tbpB, D 15, Hia, Hmw 1, Hmw2 from H.
influenzae.
In one example, the expression of the gene can be modulated by exchanging its
promoter with a stronger promoter (through isolating the upstream sequence of
the
gene, in vitro modification of this sequence, and reintroduction into the
genome by
homologous recombination). Upregulated expression can be obtained in both the
bacterium as well as in the outer membrane vesicles shed (or made) from the
bacterium.
In other examples, the described approaches can be used to generate
recombinant
bacterial strains with improved characteristics for vaccine applications.
These can be,
but are not limited to, attenuated strains, strains with increased expression
of selected
antigens, strains with knock-outs (or decreased expression) of genes
interfering with
the immune response, strains with modulated expression of immunodominant
proteins, strains with modulated shedding of outer-membrane vesicles.
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WO 02/32946 PCT/EPO1/11983
Thus, also provided by the invention is a modified upstream region of the
BASB2a7
gene, which modified upstream region contains a heterologous regulatory
element
which alters the expression level of the BASB207 protein located at the outer
membrane. The upstream region according to this aspect of the invention
includes the
sequence upstream of the BASB207 gene. The upstream region starts immediately
upstream of the BASB207 gene and continues usually to a position no more than
about
1000 by upstream of the gene from the ATG start codon. In the case of a gene
located in
a polycistronic sequence (operon) the upstream region can start immediately
preceding
the gene of interest, or preceding the first gene in the operon. Preferably, a
modified
upstream region according to this aspect of the invention contains a
heterologous
promotor at a position between 500 and 700 by upstream of the ATG.
The use of the disclosed upstream regions to upregulate the expression of the
BASB207 gene, a process for achieving this through homologous recombination
(for
instance as described in WO 01109350 incorporated by reference herein), a
vector
comprising upstream sequence suitable for this purpose, and a host cell so
altered are
all further aspects of this invention.
Thus, the invention provides a BASB207 polypeptide, in a modified bacterial
bleb.
The invention further provides modified host cells capable of producing the
non-live
membrane-based bleb vectors. The invention further provides nucleic acid
vectors
comprising the BASB207 gene having a modified upstream region containing a
heterologous regulatory element.
Further provided by the invention are processes to prepare the host cells and
bacterial
blebs according to the invention.
Also provided by this invention are compositions, particularly vaccine
compositions,
and methods comprising the polypeptides and/or polynucleotides of the
invention and
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
immunostimulatory DNA sequences, such as those described in Sato, Y. et al.
Science
273: 352 (1996).
Also, provided by this invention are methods using the described
polynucleotide or
particular fragments thereof, which have been shown to encode non-variable
regions
of bacterial cell surface proteins, in polynucleotide constructs used in such
genetic
immunization experiments in animal models of infection with non typeable H.
influenzae. 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 non typeable H.
inflZrenzae
infection, in mammals, particularly humans.
The invention also includes a vaccine formulation which comprises an
immunogenic
recombinant polypeptide andlor polynucleotide of the invention together with a
suitable earner, 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 mufti-
dose
containers, for example, sealed ampoules and vials and may be stored in a
freeze-dried
condition requiring only the addition of the sterile liquid earner immediately
prior to
use.
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WO 02/32946 PCT/EPO1/11983
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 TH 1 type of response.
An immune response may be broadly distinguished into two extreme catagories,
being a humoral or cell mediated immune responses (traditionally characterised
by
antibody and cellular effector mechanisms of protection respectively). These
categories of response have been termed TH1-type responses (cell-mediated
response), and TH2-type immune responses (humoral response).
Extreme TH1-type immune responses may be characterised by the generation of
antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural
killer cell
responses. In mice TH1-type responses are often characterised by the
generation of
antibodies of the IgG2a subtype, whilst in the human these correspond to IgGI
type
antibodies. TH2-type immune responses are characterised by the generation of a
broad range of immunoglobulin isotypes including in mice IgGI, IgA, and IgM.
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.
The distinction of TH 1 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 CDR
+ve T
cell clones by Mosmann and Coffman (Mosmann, T.R. and Coffman, R.L. (1989
THl and TH2 cells: different patterns of lyrnphokine secretion lead to
different
functional properties. ~lnnaral Review oflrnrnasnologv, 7, p145-173).
Traditionally,
TH 1-type responses are associated with the production of the INF-y and II,-2
42
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
cytokines by T-lymphocytes. Other cytokines often directly associated with the
induction of THl-type immune responses are not produced by T-cells, such as IL-
12. In contrast, TH2- type responses are associated with the secretion of IL-
4, IL-5,
IL-6 and IL-13 .
It is known that certain vaccine adjuvants are particularly suited to the
stimulation of
either TH 1 or TH2 - type cytokine responses. Traditionally the best
indicators of the
TH l :TH2 balance of the immune response after a vaccination or infection
includes
direct measurement of the production of THl or TH2 cytokines by T lymphocytes
in
vitro after restimulation with antigen, andlor the measurement of the IgGI
:IgG2a
ratio of antigen specific antibody responses.
Thus, a THl-type adjuvant is one which preferentially stimulates isolated T-
cell
populations to produce high levels of TH 1-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 THl-type
isotype.
Adjuvants which are capable of preferential stimulation of the THl cell
response are
described in International Patent Application No. WO 94100153 and WO 95!17209.
3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant. This is
known from GB 2220211 (Ribi). Chemically it is a mixture of 3 De-O-acylated
monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by
Ribi
Immunochem, Montana. A preferred form of 3 De-O-acylated monophosphoryl
lipid A is disclosed in European Patent 0 689 454 B 1 (SmithKline Beecham
Biologicals SA).
Preferably, the particles of 3D-MPL are small enough to be sterile filtered
through a
0.22micron membrane (European Patent number 0 689 454).
3D-MPL will be present in the range of 10~g - 100~tg preferably 25-SOpg per
dose
wherein the antigen will typically be present in a range 2-SOpg per dose.
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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.
The method of production of QS21 is disclosed in US patent No. 5,057,540.
Non-reactogenic adjuvant formulations containing QS21 have been described
previously (WO 96133739). Such formulations comprising QS21 and cholesterol
have been shown to be successful TH1 stimulating adjuvants when formulated
together with an antigen.
Further adjuvants which are preferential stimulators of TH1 cell response
include
immunomodulatory oligonucleotides, for example unmethylated CpG sequences as
disclosed in WO 96/02555.
Combinations of different TH1 stimulating adjuvants, such as those mentioned
hereinabove, are also contemplated as providing an adjuvant which is a
preferential
stimulator of TH1 cell response. For example, QS21 can be formulated together
with 3D-MPL. The ratio of QS21 : 3D-MPL will typically be in the order of 1 :
10
to 10 : l; preferably 1:5 to 5 : 1 and often substantially 1 : 1. The
preferred range
for optimal synergy is 2.5 : 1 to 1 : 1 3D-MPL: QS21.
Preferably a carrier is also present in the vaccine composition according to
the
invention. The earner may be an oil in water emulsion, or an aluminium salt,
such
as aluminium phosphate or aluminium hydroxide.
A preferred oil-in-water emulsion comprises a metabolisible oil, such as
squalene,
alpha tocopherol and Tween 80. In a particularly preferred aspect the antigens
in the
vaccine compositian accarding to the invention are camhined with QS21 and 3D-
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MPL in such an emulsion. Additionally the oil in water emulsion may contain
span
85 andlor lecithin and/or tricaprylin.
Typically for human administration QS21 and 3D-MPL will be present in a
vaccine
in the range of 1 pg - 200pg, such as 10-100p.g, preferably l Op.g - SOp,g per
dose.
Typically the oil in water will comprise from 2 to 10% squalene, from 2 to 10%
alpha tocopherol and from 0.3 to 3% tween 80. Preferably the ratio of
squalene:
alpha tocopherol is equal to or less than 1 as this provides a more stable
emulsion.
Span 85 may also be present at a level of 1%. In some cases it may be
advantageous that the vaccines of the present invention will further contain a
stabiliser.
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, fox example, phosphate buffered saline.
A particularly potent adjuvant formulation involving QS21, 3D-MPL and
tocopherol in an oil in water emulsion is described in WO 95117210.
While the invention has been described with reference to certain BASB207
polypeptides and polynucleotides, it is to be understood that this covers
fragments of
the naturally occurnng 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. Preferred fragments/peptides are described in Example 13.
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 otiti,r media. Such a polyvalent vaccine
composition may
include a TH-1 inducing adjuvant as hereinbefore described.
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WO 02/32946 PCT/EPO1/11983
In a preferred embodiment, the polypeptides, fragments and immunogens of the
invention are formulated with one or more of the following groups of antigens:
a) bne
or more pneumococcal capsular polysaccharides (either plain or conjugated to a
carrier
protein); b) one or more antigens that can protect a host against M.
catarrhalis
infection; c) one or more protein antigens that can protect a host against
Streptococcus
pneurnoniae infection; d) one or more further non typeable Haemophilus
inJluenzae
protein antigens; e) one or more antigens that can protect a host against RSV;
and f)
one or more antigens that can protect a host against influenza virus.
Combinations
with: groups a) and b); b) and c); b), d), and a) and/or c); b), d), e), f),
and a) andlor c)
are preferred. Such vaccines may be advantageously used as global otitis media
vaccines.
The pneumococcal capsular polysaccharide antigens axe preferably selected from
serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 1 1A, 12F, 14, 15B, 17F, 18C,
19A,
19F, 20, 22F, 23F and 33F (most preferably from serotypes 1, 3, 4, 5, 6B, 7F,
9V,
14, 18C, 19F and 23F).
Preferred pneumococcal protein antigens are those pneumococcal proteins which
are
exposed on the outer surface of the pneumococcus (capable of being recognised
by a
host's immune system during at least part of the life cycle of the
pneumococcus), or
are proteins which are secreted or released by the pneumococcus. Most
preferably,
the protein is a toxin, adhesin, 2-component signal tranducer, or lipoprotein
of
Streptococcus pneZCrnoniae, or fragments thereof. Particularly preferred
proteins
include, but are not limited to: pneumolysin (preferably detoxified by
chemical
treatment or mutation) [Mitchell et al. Nucleic Acids Res. 1990 Jul 11;
18(13): 4010
"Comparison of pneumolysin genes and proteins from Streptococcus pneumoniae
types 1 and 2.", Mitchell et al. Biochim Biophys Acta 19$9 Jan 23; 1007(1): 67-
72
"Expression of the pneumolysin gene in Escherichia coli: rapid purification
and
biological properties.", WO 96/05859 {A. Cyanamid), WO 90/06951 {Paton et al),
WO 99103884 {NAVA)]; PspA and transmembrane deletion variants thereof {WO
92/14488; WO 99153940; US 5804193 - Briles et al.); PspC and transmembrane
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deletion variants thereof (WO 99153940; WO 97/09994 - Briles et al); PsaA and
transmembrane deletion variants thereof (Berry & Paton, Infect Immun 196
Dec;64(12):5255-62 "Sequence heterogeneity of PsaA, a 37-kilodalton putative
adhesin essential for virulence of Streptococcus pneumoniae"); pneumococcal
choline binding proteins and transmembrane deletion variants thereof; CbpA and
transmembrane deletion variants thereof (WO 97/41151; WO 99/51266);
Glyceraldehyde-3-phosphate - dehydrogenase (Infect. Immun. 1996 64:3544);
HSP70 (WO 96/40928); PcpA (Sanchez-Beato et al. FEMS Rlicrobiol Lett 1998,
164:207-14); M like protein, SB patent application No. EP 0837130; and adhesin
18627, SB Patent application No. EP 0834568. Further preferred pneumococcal
protein antigens are those disclosed in WO 98/18931, particularly those
selected in
WO 98118930 and PGTlUS99/30390.
Preferred Moraxella catarrhalis protein antigens which can be included in a
combination vaccine (especially for the prevention of otitis media) are: OMP
106
[WO 97141731 (Antex) & WO 96/34960 (PMG)]; OMP21; LbpA &/or LbpB [WO
98/55606 (PMC)]; TbpA &/or TbpB [WO 97/13785 & WO 97/32980 (PMC)];
CopB [Helminen ME, et al. (1993) Infect. Immun. 61:2003-2010]; UspAl and/or
UspA2 [WO 93/03761 (University of Texas)]; OmpCD; HasR (PCT/EP99/03824);
PiIQ (PCT/EP99/03823); OMP85 (PGT/EP00/01468); lipo06 (GB 9917977.2);
lipol0 (GB 9918208.1); lipoll (GB 9918302.2); lipol8 (GB 9918038.2); P6
(PGT/EP99/03038); D15 (PCT/EP99/03822); OmplAl (PCT/EP99/06781); Hly3
(PCT/EP99/03257); and OmpE.
Preferred further non-typeable Haemophilus inflatenzae protein antigens which
can
be included in a combination vaccine (especially for the prevention of otitis
media)
include: Fimbrin protein [(US 5766608 - Ohio State Research Foundation)] and
fusions comprising peptides therefrom [cg LB1(f) peptide fusions; US 5843464
(OSU) or WO 99/64067]; OMP26 [WO 97/01638 (Gortecs)]; P6 [EP 281673 (State
University of New York)]; protein D (EP 594610); TbpA andlor TbpB; Hia; Hsf;
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WO 02/32946 PCT/EPO1/11983
Hin47; Hif; Hmw l; Hmw2; Hmw3; Hmw4; Hap; D 15 (WO 94/ 12641 ); P2; and PS
(WO 94/26304).
Preferred influenza virus antigens include whole, live or inactivated virus,
split
influenza virus, grown in eggs or MDCK cells, or Vero cells or whole flu
virosomes
(as described by R. Gluck, Vaccine, 1992, 10, 915-920) or purified or
recombinant
proteins thereof, such as HA, NP, NA, or M proteins, or combinations thereof.
Preferred RSV (Respiratory Syncytial Virus) antigens include the F
glycoprotein,
the G glycoprotein, the HN protein, or derivatives thereof.
Compositions, kits and administration
In a further aspect of the invention there are provided compositions
comprising a
BASB207 polynucleotide andlor a BASB207 polypeptide for administration to a
cell or
to a multicellular organism.
The invention also relates to compositions comprising a polynucleotide andlor
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 earner suitable for administration to an individual. Such
compositions
comprise, for instance, a media additive or a therapeutically effective amount
of a
polypeptide andlor 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.
Polypeptides, polynucleotides and other compounds of the invention may be
employed
alone or in conjunction with other compounds, such as therapeutic compounds.
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The pharmaceutical compositions may be administered in any effective,
convenient
manner including, for instance, administration by topical, oral, anal,
vaginal,
intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or
intradermal
routes among others.
In therapy or as a prophylactic, the active agent may be administered to an
individual
as an injectable composition, for example as a sterile aqueous dispersion,
preferably
isotonic.
In a further aspect, the present invention provides for pharmaceutical
compositions
comprising a therapeutically effective amount of a polypeptide andlor
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.
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
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WO 02/32946 PCT/EPO1/11983
these compounds may also be topical andlor localized, in the form of salves,
pastes, gels,
solutions, powders and the like.
For administration to mammals, and particularly humans, it is expected that
the daily
dosage level of the active agent will be from 0.01 mg/kg to 10 mglkg,
typically around
1 mglkg. The physician in any event will determine the actual dosage which
will be
most suitable for an individual and will vary with the age, weight and
response of the
particular individual. The above dosages are exemplary of the average case.
There
can, of course, be individual instances where higher or lower dosage ranges
are
merited, and such are within the scope of this invention.
The dosage range required depends on the choice of peptide, the route of
administration,
the nature of the formulation, the nature of the subject's condition, and the
judgment of
the attending practitioner. Suitable dosages, however, are in the range of 0.1-
100 pg/kg
of subject.
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 microgramlkg of antigen, and such dose is preferably
administered 1-3 times and with an interval of 1-3 weeks. With the indicated
dose
range, no adverse toxicological effects will be observed with the compounds of
the
invention which would preclude their administration to suitable individuals.
Wide variations in the needed dosage, however, are to be expected in view of
the variety
of compounds available and the differing efficiencies of various routes of
administration.
For example, oral administration would be expected to require higher dosages
than
administration by intravenous injection. Variations in these dosage levels can
be
adjusted using standard empirical routines for optimization, as is well
understood in the
art.
3p
SO
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Sequence Databases, Sequences in a Tangible Medium, and Algorithms
Polynucleotide and polypeptide sequences form a valuable information resource
wit~l
which to determine their 2- and 3-dimensional structures as well as to
identify further
sequences of similar homology. These approaches are most easily facilitated by
storing
the sequence in a computer readable medium and then using the stored data in a
known
macromolecular structure program or to search a sequence database using well
known
searching tools, such as the GCG program package.
Also provided by the invention are methods for the analysis of character
sequences or
strings, particularly genetic sequences or encoded protein sequences.
Preferred
methods of sequence analysis include, for example, methods of sequence
homology
analysis, such as identity and similarity analysis, DNA, RNA and protein
structure
analysis, sequence assembly, cladistic analysis, sequence motif analysis, open
reading
frame determination, nucleic acid base calling, codon usage analysis, nucleic
acid base
trimming, and sequencing chromatogram peak analysis.
A computer based method is provided for performing homology identification.
This
method comprises the steps of: providing a first polynucleotide sequence
comprising
the sequence of a polynucleotide of the invention in a computer readable
medium; and
comparing said first polynucleotide sequence to at least one second
polynucleotide or
polypeptide sequence to identify homology.
A computer based method is also provided for performing homology
identification,
said method comprising the steps of: providing a first polypeptide sequence
comprising the sequence of a polypeptide of the invention in a computer
readable
medium; and comparing said first polypeptide sequence to at least one second
polynucleotide or polypeptide sequence to identify homology.
All publications and references, including but not limited to patents and
patent
applications, cited in this specification are herein incorporated by reference
in their
entirety as if each individual publication or reference wer-a specifically and
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WO 02/32946 PCT/EPO1/11983
individually indicated to be incorporated by reference herein as being fully
set forth.
Any patent application to which this application claims priority is also
incorporate2l by
reference herein in its entirety in the manner described above for
publications and
references.
DEFINITIONS
"Identity," as known in the art, is a relationship between two or more
polypeptide
sequences or two or more polynucleotide sequences, as the case may be, as
determined
by comparing the sequences. In the art, "identity" also means the degree of
sequence
relatedness between polypeptide or polynucleotide sequences, as the case may
be, as
determined by the match between strings of such sequences. "Identity" can be
readily
calculated by known methods, including but not limited to those described in
(Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press,
New
IS 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., Na~cleic Acids Research 12(1): 387
(1984)),
BLASTP, BLASTN (Altschul, S.F. et al., .l. 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 NLH Bethesda, MD 20894;
Altschul,
3p S., et al., J. Mol. Biol. 215: 403-410 (1990). The well known Smith
Waterman
algorithm may also be used to determine identity.
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Parameters for polypeptide sequence comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,
Proc. Natl. Acad. Sci. USA. 89:1091 S-10919 ( 1992)
Gap Penalty: 8
Gap Length Penalty: 2
A program useful with these parameters is publicly available as the "gap"
program
from Genetics Computer Group, Madison WI. The aforementioned parameters are
the
default parameters for peptide comparisons (along with no penalty for end
gaps).
Parameters for polynucleotide comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix: matches = +10, mismatch = 0
Gap Penalty: 50
Gap Length Penalty: 3
Available as: The "gap" program from Genetics Computer Group, Madison WI.
These are the default parameters for nucleic acid comparisons.
A preferred meaning for "identity" for polynucleotides and polypeptides, as
the ease
may be, are provided in (1) and (2) below.
(1) Polynucleotide embodiments further include an isolated polynucleotide
comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90,
95, 97
or 100% identity to the reference sequence of SEQ ID NO:1, wherein said
polynucleotide sequence may be identical to the reference sequence of SEQ ID
NO: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
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WO 02/32946 PCT/EPO1/11983
the reference nucleotide sequence or anywhere between those terminal
positions,
interspersed either individually among the nucleotides in the reference
sequence o~ 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: l, or:
nn ~ xn ' ~xn ' Y)
wherein nn is the number of nucleotide alterations, xn is the total number of
nucleotides in SEQ ID NO:1, y is 0.50 for SO%, 0.60 for 60%, 0.70 for 70%,
0.80 for
80%, 0.85 for 85°~°, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or
1.00 for 100%, and
~ is the symbol for the multiplication operator, and wherein any non-integer
product of
xn and y is rounded down to the nearest integer prior to subtracting it from
xn.
Alterations of polynucleotide sequences encoding the polypeptides of SEQ ID
N0:2
may create nonsense, missense or frameshift mutations in this coding sequence
and
thereby alter the polypeptide encoded by the polynucleotide following such
alterations.
By way of example, a polynucleotide sequence of the present invention may be
identical to the reference sequences of SEQ ID NO: l, 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
polynueleotide 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
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WO 02/32946 PCT/EPO1/11983
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 irk'
SEQ ID NO: l, or:
nn ~ xn ' ~xn ' Y)
wherein nn is the number of nucleic acid alterations, xn is the total number
of nucleic
acids in SEQ ID NO:1, y is, for instance 0.70 for 70°~0, 0.80 for 80%,
0.85 for 85%
etc., ~ is the symbol for the multiplication operator, and wherein any non-
integer
product of xn and y is rounded down to the nearest integer prior to
subtracting it from
xn.
(2) Polypeptide embodiments further include an isolated polypeptide comprising
a
polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity
to the
polypeptide reference sequence of SEQ ID N0:2, wherein said polypeptide
sequence
may be identical to the reference sequence of SEQ ID N0:2 or may include up to
a
certain integer number of amino acid alterations as compared to the reference
sequence, wherein said alterations are selected from the group consisting of
at least
one amino acid deletion, substitution, including conservative and non-
conservative
substitution, or insertion, and wherein said alterations may occur at the
amino- or
carboxy-terminal positions of the reference polypeptide sequence or anywhere
between those terminal positions, interspersed either individually among the
amino
acids in the reference sequence or in one or more contiguous groups within the
reference sequence, and wherein said number of amino acid alterations is
determined
by multiplying the total number of amino acids in SEQ ID N0:2 by the integer
defining the percent identity divided by 100 and then subtracting that product
from
said total number of amino acids in SEQ ID N0:2, or:
na ~ xa' ~xa' Y)
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WO 02/32946 PCT/EPO1/11983
wherein na is the number of amino acid alterations, xa is the total number of
amino
acids in SEQ ID N0:2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for
80/0,
0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and ~
is the
symbol for the multiplication operator, and wherein any non-integer product of
xa and
y is rounded down to the nearest integer prior to subtracting it from xa.
By way of example, a polypeptide sequence of the present invention may be
identical
to the reference sequence of SEQ ID N0:2, that is it may be 100% identical, or
it may
include up to a certain integer number of amino acid alterations as compared
to the
reference sequence such that the percent identity is less than 100% identity.
Such
alterations are selected from the group consisting of at least one amino acid
deletion,
substitution, including conservative and non-conservative substitution, or
insertion,
and wherein said alterations may occur at the amino- or carboxy-terminal
positions of
the reference polypeptide sequence or anywhere between those terminal
positions,
interspersed either individually among the amino acids in the reference
sequence or in
one or more contiguous groups within the reference sequence. The number of
amino
acid alterations for a given % identity is determined by multiplying the total
number of
amino acids in SEQ ID N0:2 by the integer defining the percent identity
divided by
100 and then subtracting that product from said total number of amino acids in
SEQ
ID N0:2, or:
na ~ xa - ~xa ~ Y)
wherein na is the number of amino acid alterations, xa is the total number of
amino
acids in SEQ ID N0:2, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for
85%
etc., and ~ is the symbol for the multiplication operator, and wherein any non-
integer
product of xa and y is rounded down to the nearest integer prior to
subtracting it from
xa.
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"Individual(s)," when used herein with reference to an organism, means a
multicellular
eukaryote, including, but not limited to a metazoan, a mammal, an ovid, a
bovid, a°
simian, a primate, and a human.
"Isolated" means altered "by the hand of man" from its natural state, i.e., if
it occurs in
nature, it has been changed or removed from its original environment, or both.
For
example, a polynucleotide or a polypeptide naturally present in a living
organism is not
"isolated," but the same polynucleotide or polypeptide separated from the
coexisting
materials of its natural state is "isolated", as the term is employed herein.
Moreover, a
polynucleotide or polypeptide that is introduced into an organism by
transformation,
genetic manipulation or by any other recombinant method is "isolated" even if
it is still
present in said organism, which organism may be living or non-living.
"Polynucleotide(s)" generally refers to any polyribonucleotide or
polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or
DNA including single and double-stranded regions.
"Variant" refers to a polynucleotide or polypeptide that differs from a
reference
polynucleotide or polypeptide, but retains essential properties. A typical
variant of a
polynueleotide 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
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WO 02/32946 PCT/EPO1/11983
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
an$
polypeptides may be made by mutagenesis techniques or by direct synthesis.
"Disease(s)" means any disease caused by or related to infection by a
bacteria,
including, for example, otitis media in infants and children, pneumonia in
elderlies,
sinusitis, nosocomial infections and invasive diseases, chronic otitis media
with
hearing loss, fluid accumulation in the middle ear, auditive nerve damage,
delayed
speech learning, infection of the upper respiratory tract and inflammation of
the
middle ear.
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EXAMPLES:
The examples below are 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 BASB207 gene from Non typable
Haemoplaila~s ira fluenzae strain 3224A.
A: BASB207 in Non typableHaemophilus influenzae strain 322~A
The DNA sequence of the BASB207 polynucleotide from the Non typable
Haemophilus Influenzae strain 3224A ( also referred to as strain ATCC PT-1816)
is
shown in SEQ ID N0:1. The translation of the BASB207 polynucleotidic sequence
is showed in SEQ ID N0:2.
B: BASB207 in Non typable Haemophilus in fluenzae strain 3224A
The sequence of the BASB207 polynucleotide was confirmed in Non Typable
Haemophilus influenzae strain 322~A. For this purpose, plasmid DNA (see
example
3A) containing the gene region encoding BASB207 from Non Typable Haemophilus
influenzae strain 3224A was 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 M13 Universal Sequence Primer(S'-GTA AAA
CGA CGG CCA GT-3') [SEQ ID NO:10] and M13 Reverse Sequence Primer (5'-
CAG GAA ACA GCT ATG AC-3') [SEQ ID NO:11] specific for the vector and
additional internal primers specific for the BASB207 polynucleotide. As a
result, the
polynucleotide and deduced polypeptide sequences, respectively, were obtained.
Using the Clustalx 1.8 program, the polynucleotide sequence SEQ ID NO: 3 was
aligned with SEQ ID NO:1; a pairwise comparison of identities showed that the
polynucleotide sequence was 99 % identical to SEQ ID NO:1 (fig 1) . Using the
same Clustalx 1.8 program, the polypeptide sequence SEQ ID NO: 4 was aligned
with SEQ ID N0:2; a pairwise comparison of identities showed that the
polypeptide
sequence was 99 % identical to SEQ ID N0:2 (FIG 2)
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Example 2:
Variability analysis of the BASB207 gene among Non Typable Haemophilus
influenzae strains.
Genomic DNA was extracted from 2 further NT Haemophilus inflatenzae strains
(presented in Table 1) as follows. A SOOmI erlenmeyer flask containing 100 ml
of
BHI broth was inoculated with the seed culture and grown for ~12-16 hours at
37 °C
in a shaking incubator, 175 rpm, to generate cell mass for DNA isolation.
Cells
' were collected by centrifugation in a Sorvall GSA rotor at 2000 X g for 15
minutes
at 4°C. The supernatant was removed. Genomie DNA was extracted from the
pellet
of the NT Haemophilzts influehzae cells using the QIAGEN genomic DNA
extraction kit (Qiagen Gmbh). leg of this material was submitted to Polymerase
Chain Reaction DNA amplification using primers MCM023 (5'- ATG ACA AAA
CAA CTA CAA CCA TC-3') [SEQ ID N0:12] and MCM024 (5'- ATT CAA ATT
TAT AAA GTA AAT CAA ACA-3') [SEQ ID N0:13]. This PCR product was
purified using the High Pure PCR Product Purification Kit (Roche) , subjected
to
DNA sequencing using the Big Dyes kit (Applied biosystems) and analyzed on a
ABI PRISM 310 Genetic Analyser by means of the primers MCM023 [SEQ ID
N0:12] and MCM024 [SEQ ID N0:13] and additional internal primers in the
conditions described by the supplier. Using the Clustalx 1.8 program, an
alignment
of the polynucleotide sequences was performed, and is displayed in Figure 3. A
pairwise comparaison of identities showed that the polynucleotidic sequences
SEQ
ID NO: 5 and 7 turned out to be between 93 and 95 % identical to SEQ ID N0:3
(Table 2). Using the Clustalx 1.8 program, an alignment of the polypeptidic
sequences was performed, and is displayed in Figure 4. A pairwise comparaison
of
identities showed that the polypeptidic sequences SEQ ID NO: 6 and 8 turned
out to
be between 94 and 95 °ro identical to SEQ ID N0:4 (Table 3).
'Table 1: Features of the NT Haemophilus influenzae strains used in this study
Strain isolated in from nucleotidic peptidic
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sequence sequence
3224A USA Otitis mediaSEQ ID N0:3 SEQ ID NO:~
3219C USA Otitis mediaSEQ ID NO:S SEQ ID N0:6
A840164 NL Garner strainSEQ ID N0:7 SEQ ID N0:8
Table 2: Pairwaise comparison of polynucleiotidic sequences
SEQ ID SEQ ID SEQ ID N0:7
N0:3 NO:S
SEQ ID 95 95
N0;3
SEQ ID 93
NO:S
SEQ ID
N0;7 i
Table 3: Pairwaise comparison of polypeptidic sequences
SEQ ID N0:4SEQ ID SEQ ID
N0:6 NO:$
SEQ ID 95 95
NO;~
SEQ ID
N0:6
SEQ ID
N0:8
Example 3 : Construction of Plasmid to Express Recombinant BASB207
A: Cloning of BASB207.
The BSpHI and BgIII restriction sites engineered into oli 1 nt YtfN (5'-TC ATG
ACA
AAA GAA CTA CAA CC-3') [SEQ ID N0:14] forward and oli 6 nt YtfN (5'-AGA
TGT AAA TTC AAA TTT ATA AAGTAAATG -3') [SEQ ID NO:15] reverse
amplification primers, respectively, permitted directional cloning of the PCR
product into the E.coli expression plasmid pQE60 such that BASB207 protein
could
be expressed as a fusion protein containing a (His)6 affinity chromatography
tag at
the C-terminus. Due to the large size of the gene, three parallel PCR
experiments
were carried out to amplify the entire coding sequence using oli 1 nt YtfN (5'-
TC
ATG ACA AAA CAA CTA CAA CC-3') [SEQ ID N0:14] and oli2 nt YtfN (5'-G
GGG TTT GAA GAG AAT GTT CG-3') [SEQ ID N0:16] for one amplification,
oli3 nt YtfN (5'iCAG GCG GTT TTG CAG GCTG-3') [SEQ ID N0:17] and oli4
nt YtfN (S'- CAG TTG TTC TTT GCT GTT GTG-3') [SEQ ID N0:18] for the
second amplification, and oli5nt YtfN (5'-GCG AGTCAG CGA AGA TGA AGT
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G-3') [SEQ ID N0:19]and oli 6 nt YtfN (5'-AGA TCT AAA TTC AAA TTT ATA
AAGTAAATC -3') [SEQ ID NO: I S] for the third amplification . The fragments
amplified using oli 1 nt YtfN -oli2 nt YtfN and oli3 nt Ytfn -oli4 nt YtfN
share a
common region with a FokI restriction site, while the fragments amplified
using
oli3 nt YtfN - olio nt YtfN and oli5nt YtfN - oli6 nt YtfN share a common
region
with a HaeIII restriction site. The three PCR products were first introduced
into the
pCRIITOPO cloning vector (In vitrogen) using Top 10 bacterial cells, according
to
the manufacturer's instructions. These intermediate constructs were realized
to
facilitate further cloning into the expression vector. Transformants
containing the
fragments were selected by restriction enzyme analysis. Following digestion, a
~20q1 aliquot of each reaction was analyzed by agarose gel electrophoresis
(0.8
agarose in a Tris-acetate-EDTA (TAE) buffer). DNA fragments were visualized by
LTV illumination after gel electrophoresis and ethidium bromide staining. A
DNA
molecular size standard (1 Kb ladder, Life Technologies) was electrophoresed
in
parallel with the test samples and was used to estimate the size of the DNA
fragments. Plasmid purified from selected transformants far each cloning were
then
sequentially digested to completion with either BSpHI and Fokl or Fokl and
Haelll
or HaeIIII and BgIII restriction enzymes as recommended by the manufacturer
(Life
Technologies). The digested DNA fragments were then purified using silica gel-
based spin columns prior to ligation with the pQE60 plasmid.
B: Production of expression vector.
To prepare the expression plasmid pQE60 for ligation, it was similarly
digested to
completion with both NcoI and BgIII. An approximately 5-fold molar excess of
the
digested fragments to the prepared vector was used to program the ligation
reaction.
A standard ~20 Itl ligation reaction (~16°C, ~16 hours), using methods
well known
in the art, was performed using T~ DNA ligase (~2.0 units / reaction, Life
Technologies). An aliquot of the ligation (~5 q1) was used to transform
M15(pREP4) electro-competent cells according to methods well known in the art.
Following a ~2-3 hour outgrowth period at 37°C in ~l .0 ml of LB
broth,
transformed cells were plated on LB agar plates containing ampicillin (100
~g/ml)
and kanamycin (30~.g/m1). Antibiotic was included in the selection. Plates
were
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incubated overnight at 37°G for ~16 hours. Individual ApRIKanR colonies
were
picked with sterile toothpicks and used to "patch" inoculate fresh LB ApR/KanR
plates as well as a ~1.0 ml LB Apl Kan broth culture. Both the patch plates
and the
broth culture were incubated overnight at 37°C in either a standard
incubator (plates)
or a shaking water bath. A whole cell-based PCR analysis was employed to
verify
that transformants contained the BASB207 DNA insert. Here, the ~1.0 ml
overnight
LB Ap/Kan broth culture was transferred to a 1.5 ml polypropylene tube and the
cells collected by centrifugation in a Beckmann microcentrifuge (~3 min., room
temperature, ~ 12,000 X g). The cell pellet was suspended in ~200p 1 of
sterile water
and a ~lOpl aliquot used to program a ~50p1 final volume PCR reaction
containing
both BASB207 forward and reverse amplification primers. The initial
95°C
denaturation step was increased to 3 minutes to ensure thermal disruption of
the
bacterial cells and liberation of plasmid DNA. An ABI Model 9700 thermal
cycler
and a 32 cycle, three-step thermal amplification profile, i.e. 95°C,
45sec; 55-58°C,
45sec, 72°C, lmin., were used to amplify the BASB207 fragment from the
lysed
transformant samples. Following thermal amplification, a ~20p1 aliquot of the
reaction was analyzed by agarose gel electrophoresis (0.8 % agarose in a Tris-
acetate-EDTA (TAE) buffer). DNA fragments were visualized by UV illumination
after gel electrophoresis and ethidium bromide staining. A DNA molecular size
standard (1 Kb ladder, Life Technologies) was electrophoresed in parallel with
the
test samples and was used to estimate the size of the PCR products.
Transformants
that produced the expected size PCR product were identified as strains
containing a
BASB207 expression construct. Expression plasmid containing strains were then
analyzed for the inducible expression of recombinant BASB207.
C: Expression Analysis of PCR-Positive Transformants
An aliquot of the overnight seed culture (~1.0 ml) was inoculated into a 125
ml
erlenmeyer flask containing ~25 ml of LB AplKan broth and grown at 37
°C with
shaking 0250 rpm) until the culture turbidity reached O.D.600 of ~0.5, i.e.
mid-log
phase (usually about 1.5 - 2.0 hours). At this time approximately half of the
culture
012.5 ml) was transferred to a second 125 ml flask and expression of
recombinant
BASB207 protein induced by the addition of IPTG (1.0 M stock prepared in
sterile
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water, Sigma) to a final concentration of 1.0 mM. Incubation of both the IPTG-
induced and non-induced cultures continued for an additional ~4 hours at 37
°C w~'th
shaking. Samples (~ 1.0 ml) of both induced and non-induced cultures were
removed after the induction period and the cells collected by centrifugation
in a
microcentrifuge at room temperature for ~3 minutes. Individual cell pellets
were
suspended in ~SOp I of sterile water, then mixed with an equal volume of 2X
Laemelli SDS-PAGE sample buffer containing 2-mercaptoethanol, and placed in
boiling water bath for -~-3 min to denature protein. Equal volumes (~ 15 p1)
of both
the crude IPTG-induced and the non-induced cell lysates were loaded onto
duplicate
12°,~o Tris/glycine polyacrylamide gel (1 mm thick Mini-gels, Novex).
The induced
and non-induced lysate samples were electrophoresed together with prestained
molecular weight markers (SeeBlue, Novex) under conventional conditions using
a
standard SDS/Tris/glycine running buffer (BioRad). Following electrophoresis,
one
gel was stained with commassie brilliant blue 8250 (BioRad) and then destained
to
visualize novel BASB207 TPTG-inducible protein(s). The second gel was
electroblotted onto a PVDF membrane (0.45 micron pore size, Novex) for ~2 hrs
at
4 °C using a BioRad Mini-Protean II blotting apparatus and Towbin's
methanol (20
%) transfer buffer. Blocking of the membrane and antibody incubations were
performed according to methods well known in the art. A monoclonal anti-RGS
(His)3 antibody, followed by a second rabbit anti-mouse antibody conjugated to
HRP (QiaGen), was used to confirm the expression and identity of the BASB207
recombinant protein . Visualization of the anti-His antibody reactive pattern
was
achieved using either an ABT insoluble substrate or using Hyperfilm with the
Amersham ECL chemiluminescence system.
Example 4: Production of Recombinant BASB207
Bacterial strain
A recombinant expression strain ofE. coli M15(pREP4) containing a plasmid
(pQE60) encoding BASB207 from NT haemophilus inJluenzae was used to produce
cell mass for purification of recombinant protein. The expression strain was
cultivated on LB agar plates containing 100pg/ml ampicillin ("Ap") and 30pg/ml
kanamycin ("Km") to ensure that pQE60 and pREP4 were maintained. For
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eryopreservation at -80 °C, the strain was propagated in LB broth
containing the
same concentration of antibiotics then mixed with an equal volume of LB broth
containing 30% (w/v) glycerol.
Media
The growth medium used for the production of recombinant protein consisted of
LB
broth (Difco) containing 100pglml Ap and 30 p.glml Km. To induce expression of
the BASB207 recombinant protein, IPTG (Isopropyl 13-D-Thiogalactopyranoside)
was added to the culture {1 mM, final).
Fermentation
A 100-ml erlenmeyer seed flask, containing l Oml working volume, was
inoculated
with 0.3 ml of rapidly thawed frozen culture, or several colonies from a
selective
agar plate culture, and incubated for approximately 12 hours at 37 ~
1°C on a
shaking platform at 150rpm (Innova 2100, New Brunswick Scientific). This seed
culture was then used to inoculate a 500m1 working volume erlen containing LB
broth and both Ap and Km antibiotics. IPTG ( 1.0 M stock, prepared in sterile
water)
was added to the erlen when the culture reached mid-log of growth (~0.5
O.D.600
units). Cells were induced for 4 hours then harvested by centrifugation using
either a
28RS Heraeus {Sepatech) or RCSC superspeed centrifuge (Sorvall Instruments).
Cell paste was stored at -20 C until processed.
Example 5: Expression and purification of recombinant BASB207 protein in
Escherichia coli.
The construction of the pET-BASB207 cloninglexpression vector is described in
Example 1. This vector harbours the BASB207 gene isolated from the non
typeable
Haemophilus influenzae strain 3224A in fusion with a stretch of 6 Histidine
residues,
placed under the control of the strong bacteriophage T7 gene 10 promoter. For
expression study, this vector is introduced into the Escherichia coli strain
hFovablue
(DE3) (Novagen), in which, the gene for the T7 polymerase is placed under the
control
of the isopropyl-beta-D thiogalactoside (IPTG)-regulatable lac promoter.
Liquid
cultures (100 ml) of the Novablue (DE3) [pET-BASB207] E. coli recombinant
strain
are grown at 37°C under agitation until the optical density at 600nm
(0D600) reached
0.6. At that time-point, IPTG is added at a final concentration of 1 mM and
the culture
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is grown for 4 additional hours. The culture is then centrifuged at 10,000 rpm
and the
pellet is frozen at -20°C for at least 10 hours. After thawing, the
pellet is resuspe$ded
during 30 min at 25°C in buffer A (6M guanidine hydrochloride, O.1M
NaH2P04,
0.01 M Tris, pH 8.0), passed three-times through a needle and clarified by
centrifugation (20000rpm, 15 min). The sample is then loaded at a flow-rate of
lml/min on a Ni2+ -loaded Hitrap column (Pharmacia Biotech). After passsage of
the
flowthrough, the column is washed succesively with 40m1 of buffer B (8M Urea,
O.IMNaH2P0~1, O.OIM Tris, pH 8.0), ~Oml of buffer C (8M Urea, O.IMNaH2P0~1,
0.01 M Tris, pH 6.3). The recombinant protein BASB207/His6 is then eluted from
the
column with 30m1 of buffer D (8M Urea, O.IMNaH2P04, O.O1M Tris, pH 6.3)
containing 500mM of imidazole and 3ml-size fractions are collected. Highly
enriched
BASB207lHis6 protein can be eluted from the column. This polypeptide is
detected
by a mouse monoclonal antibody raised against the 5-histidine motif. Moreover,
the
denatured, recombinant BASB207-His6 protein is solubilized in a solution
devoid of
urea. For this purpose, denatured BASB207-His6 contained in 8M urea is
extensively
dialyzed (2 hours) against buffer R (NaCI 150mM, IOmM NaH2P04, Arginine O.SM
pH6.8) containing successively 6M, ~1M, 2M and no urea. Alternatively, this
polypeptide is purified under non-denaturing conditions using protocoles
described in
the Quiexpresssionist booklet (Qiagen Gmbh).
Example 6: Production of Antisera to Recombinant BASB207
Polyvalent antisera directed against the BASB207 protein are generated by
vaccinating rabbits with the puribed recombinant BASB207 protein. Polyvalent
antisera directed against the BASB207 protein are also generated by
vaccinating
mice with the purified recombinant BASB207 protein. Animals are bled prior to
the
first immunization ("pre-bleed") and after the last immunization.
Anti-BASB207 protein titers are measured by an ELISA using purified
recombinant
BASB207 protein as the coating antigen. The titer is defined as mid-point
titers
calculated by ~I-parameter logistic model using the JCL Fit so~rivare.The
antisera are
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also used as the first antibody to identify the protein in a western blot as
described in
example 8 below.
Example 7: Immunological characterization: Surface exposure of BASB207
S Anti-BASB207 protein titres are determined by an ELISA using formalin-killed
whole cells of non typable Haemophilus influenzae (NTHi). The titer is defined
as
mid-point titers calculated by 4-parameter logistic model using the XL Fit
software.
Example 8. Immunological Characterisation: Western Blot Analysis
Several strains of NTHi, as well as clinical isolates, are grown on Chocolate
agar
I0 plates for 24 hours at 36°C and S% CO,. Several colonies are used to
inoculate
Brain Heart Infusion (BHI) broth supplemented by NAD and hemin, each at 10
pg/ml. Cultures are grown until the absorbance at 620nm is approximately 0.4
and
cells are collected by centrifugation. Cells are then concentrated and
solubilized in
PAGE sample buffer. The solubilized cells are then resolved on 4-20%
1 S polyacrylamide gels and the separated proteins are electrophoretically
transferred to
PVDF membranes. The PVDF membranes are then pretreated with saturation buffer.
All subsequent incubations are carried out using this pretreatment buffer.
PVDF membranes are incubated with preimmune serum or rabbit or mouse immune
20 serum. PVDF membranes are then washed.
PVDF membranes are incubated with biotin-labeled sheep anti-rabbit or mouse
Ig.
PVDF membranes are then washed 3 times with wash buffer, and incubated with
streptavidin-peroxydase. PVDF membranes are then washed 3 times with wash
buffer and developed with 4-chloro-1-naphtol.
2S
Example 9: Immunological characterization' Bactericidal Activity
Complement-mediated cytotoxie activity of anti-BASB207 antibodies is examined
to determine the vaccine potential of BASB207 protein antiserum that is
prepared as
described above. The activities of the pre-immune serum and the anti-BASB207
30 antiserum in mediating complement killing of NTI-Ii are examined.
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Strains of NTHi are grown on plates. Several colonies are added to liquid
medium.
Cultures are grown and collected until the A620 is approximately 0.4. After
one
wash step, the pellet is suspended and diluted.
Preimmune sera and the anti-BASB207 sera are deposited into the first well of
a 96-
wells plate and serial dilutions are deposited in the other wells of the same
line. Live
diluted NTHi is subsequently added and the mixture is incubated. Complement is
added into each well at a working dilution defined beforehand in a toxicity
assay.
Each test includes a complement control (wells without serum containing active
or
inactivated complement source), a positive control (wells containing serum
with a
know titer of bactericidal antibodies), a culture control (wells without serum
and
complement) and a serum control (wells without complement).
Bactericidal activity of rabbit or mice antiserum (50% killing of homologous
strain)
is measured.
Example 10: Presence of Antibod to BASB207 in Human Convalescent Sera
Western blot analysis of purified recombinant BASB207 is performed as
described
in Example 5 above, except that a pool of human sera from children infected by
NTHi is used as the first antibody preparation.
Example 11: Efficacy of BASB207 vaccine enhancement of lung clearance of
NTHi in mice.
This mouse model is based on the analysis of the lung invasion by NTHi
following a
standard intranasal challenge to vaccinated mice.
Groups of mice are immunized with BASB207 vaccine. After the booster, the mice
are challenged by instillation of bacterial suspension into the nostril under
anaesthesia. Mice are killed between 30 minutes and 24 hours after challenge
and
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the lungs are removed aseptically and homogenized individually. The 1og10
weighted mean number of CFU/lung is determined by counting the colonies grown
on agar plates after plating of dilutions of the homogenate. The arithmetic
mean of
the 1og10 weighted mean number of CFU/lung and the standard deviations are
calculated for each group.
Results are analysed statistically.
In this experiment groups of mice are immunized either with BASB207 or with a
killed whole cells (kwc) preparation of NTHi or sham immunized.
Example 12: Inhibition of NTHi adhesion onto cells by anti-BASB207
antiserum.
This assay measures the capacity of anti BASB207 sera to inhibit the adhesion
of
NTHi bacteria to epithelial cells. This activity could prevent colonization of
the
nasopharynx by NTHi.
One volume of bacteria is incubated on ice with one volume of pre-immune or
anti-
BASB207 immune serum dilution. This mixture is subsequently added in the wells
of a 24 well plate containing a confluent cells culture that is washed once
with
culture medium to remove traces of antibiotic. The plate is centrifuged and
incubated.
Each well is then gently washed. After the last wash, sodium glycocholate is
added
to the wells. After incubation, the cell layer is scraped and homogenised.
Dilutions
of the homogenate are plated on agar plates and incubated. The number of
colonies
on each plate is counted and the number of bacteria present in each well
calculated.
Example 13: Useful Epitopes
The B-cell epitopes of a protein are mainly localized at its surface. To
predict B-cell
epitopes of BASB207 polypeptide two methods were combined: 2D-structure
prediction and antigenic index prediction. The 2D-structure prediction was
made
using the PSIPRED program (from David Jones, Brunei Bioinformatics Group,
Dept.
Biological Sciences, Brunei University, Uxbridge UB8 3PH, UK) (Fig.S). The
antigenic index was calculated on the basis of the method described by Jameson
and
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Wolf (CABIOS x:181-186 X1988]). The parameters used in this program are the
antigenic index and the minimal length for an antigenic peptide. An antigenic
inde~c
of 0.9 for a minimum of 5 consecutive amino acids was used as threshold for
the
program. Peptides comprising good, potential B-cell epitopes are listed in
table 4
These can be useful (preferably conjugated or recombinantly joined to a larger
protein) in a vaccine composition for the prevention of ntHi infections, as
could
similar peptides comprising conservative mutations (preferably 70, 80, 95, 99
or
100% identical to the sequences of table 4) or truncates comprising 5 or more
(e.g. 6,
7, 8, 9, 10, 1 l, 12, 15 or 20) amino acids therefrom or extensions comprising
e. g. l,
2, 3, 5, 10 further amino acids at either or both ends from the native context
of
BASB207 polypeptide which preserve an effective epitope which can elicit an
immune response in a host against the BASB207 polypeptide.
Table 4: Potential B-cell epitopes from SEQ TI3 N0:2
PositionSequence
1 MTKQLQPSDTSPKAPKQANKKH
134 STPDNSKSSQMK
183 LNNEKG
207 SEVKSELKAEQPNKPVD
252 EISGKNWQ
264 NEKGET
304 SSQGK
324 HLEPLKSDGK
374 AQDKM
439 ADGKL
466 ANWKNGAN
500 LHSRGF
551 RYGENYLK
584 DLKGRVK
'647 HYGENI
662 SGDEQNH
687 GHFDRTLEQWK
722 YDNKQTQ
735 CWQNTDV
750 NAGKQGN
776 NSL,KG
792 TDKPF
,812 KLDYRTF
830 DIQNNN
845 HNQGR i
915 RNIRTKLK
937 FNGNRSTL
969 WTTEL
10 0 ~ ANpKETrNL
0
1024 ~ PDTAEPVSE
'1039 GPHKSKEELI
1056 TKSGMDI
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1092 KQDKGN
1106 LTKGRY
1142 RNPETMEDSK
1173 PSKPQDQA
1188 RSLESSG
1284 SSTNQ
The T-helper cell epitopes are peptides bound to HLA class II molecules and
recognized by T-helper cells. The prediction of useful T-helper cell epitopes
of
BASB207 polypeptide was based on the TEPITOPE method describe by Sturniolo at
al. (Nature Biotech. 17: 555-561 [1999]). Peptides comprising good, potential
T-cell
epitopes are listed in table 5. These can be useful (preferably conjugated to
peptides,
polypeptides or polysaccharides) for vaccine purposes, as could similar
peptides
comprising conservative mutations (preferably 70, 80, 95, 99 or 100°~o
identical to
the sequences below) or truncates comprising 5 or more (e.g. 6, 7, 8, 9, 10,
11, 12,
14, 16, 18, 20, 22, 2~1 or 30) amino acids therefrom or extensions comprising
e. g. l,
2, 3, 5, 10 further amino acids at either or both ends from the native context
of
BASB207 polypeptide which preserve an effective T-helper epitope from BASB207
polypeptide.
Table 5: Potential T-helper cell epitopes from SEQ ID N0:2
PositionSequence
5 LQPSDTSPK
23 WVRKVICIGSAVTFIPVLGVAGA
56 IQLADKMLD
79 LVLKNVRYQTAGIE
100 LQLDFGCLLrSREVCLR
117 FTLNKPTIA
144 MKRISLPISINAES
159 WQNLSVNIDQTNIT
177 FKSAVNLNNEKGLTIAPTEINDISVIAKKLS
235 FLGNVSEIILPFDLHT
258 WQYQAVNEK
273 VEMSSLIAQANTVD
289 LQLQKLAVESSLGNL,SSQGK
315 MPLDLTLKS
328 LKSDGKEIL
349 ~LKKSTALSLKTKGVLDA
I 3 6 LrNGNVQLAQ I
7
378 MPLNLTLNVAK
391 YTFVNTMTPLKINDVTLrKLrTGDf~
427 MNYIPASQVELNADGKLYE
462 FVGNANWKNGANWDI
488 FVPVMPATLS
500 LHSRGFAGS
531 ~f~KGSATLNQ
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550 TRYGENYLK
569 FALDINAPNLRGLWSDLKGRVKGRV
615 LQGFQLAKASIKGHINNAS
639 LNIKAEQLHYGENIKLHLLDLD
670 LILKSQGAPVA
683 LQINGHFDR
696 WKGTISQVK
712 VKSNQAIAVSYDNKQTQ
736 WQNTDVELC
757 IPFQFKRVNLDLVNKLIEQN
790 WFTDKPFQFTANIDG
836 LVLKTDINL
850 IVGDIRLNDFVKTRQLGGTAIERLNLSIANQL
889 VNGKWSKLSFGG
912 FNTRNIRTKLKSMPVNIT
935 LRFNGNRSTLQ
957 LTGRANWAN
969 WTTELNAQA
980 FNVDTPSMAKLRFSPDITIKANPKELNLSGTVDI
1018 IKIDSLPDT
1035 VILNGPHKS
1052 FAAKTKSGM
1062 IRSDLRINIGKDVSLDAYGLKTNLDG
1098 LGLFGQINLTK
1118 LLIRKGLTSFSGQAA
1141 IRNPETMED
1156 VRVIGIADSPEV
1169 IFSEPSKPQ
1181 LSYLLTGRSLES
1206 LIGLGISKSGKLVGSIGEVFGIQDLNLG
1241 FKVTVSGNI
1253 LQIKYGVGLFDGLAEVTLRYRLMPQLYFQSVSSTNQ
1290 FDLLYKFEF
All identified regions containing epitopes as defined above are in respect of
SEQ ID
N0:4. The corresponding regions in SEQ ID NO: 6, 8 as defined by position in
table
4&5 with respect to SEQ ID N0:4 and by its corresponding peptide in the
alignment
of figure 4 for SEQ ID N0:6, 8 are also preferred peptides of the invention as
described in this example.
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Deposited materials
A deposit of strain 3 (strain 3224A) has been deposited with the American Type
Culture
Collection (ATCC) on May 5 2000 and assigned deposit number PTA-1816.
The non typeable Haemophilus influenzae-strain deposit is referred to herein
as "the
deposited strain" or as "the DNA of the deposited strain."
The deposited strain contains a full length BASB207 gene.
The sequence of the polynueleotides contained in the deposited strain, as well
as the
amino acid sequence of any polypeptide encoded thereby, are controlling in the
event of
any conflict with any description of sequences herein.
The deposit of the deposited strain has been made under the terms of the
Budapest
Treaty on the International Recognition of the Deposit of Micro-organisms for
Purposes
of Patent Procedure. The deposited strain will be irrevocably and without
restriction or
condition released to the public upon the issuance of a patent. The deposited
strain is
provided merely as convenience to those of skill in the art and is not an
admission that a
deposit is required for enablement, such as that required under 35 U.S.C. ~
112. A
license may be required to make, use or sell the deposited strain, and
compounds derived
therefrom, and no such license is hereby granted.
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Applicant's or agent's file NIJLlBM454?4 International application No.
reference number
INDICATIONS RELATING TO A DEP05ITED MICROORGANISM
(PCT Rule l3bis)
A. The indications made below
relate to the microorganism
referred to in the description
on page 73 ,lines 1-2?
B. IDENTIFICATION OF DEPOSIT
Further deposits are identified
on an additional sheet
Name of depository institution
AMERICAN TYPE CULTURE COLhECTION
Address of depository institution
(including postal code and
couniry)
10801 UNIVERSITY BLVD, MANASSAS,
VIRGINIA 20110-2209, UNITED
STATES OF
AMERICA
Date of deposit 05/0512000 Accession Number
PTA 1816
C. ADDITIONAL INDICATIONS (leave
blank fnot applicable) This
information is continued on
an additional sheet
In respect of those designations
where a European Patent is
sought, a sample of the deposited
microorganisms
will be made available until
the publication of the mention
of the grant of the European
Patent or until the
date on which the application
has been refused or withdrawn,
only by issue of such a sample
to an expert
nominated by the person requesting
the sample
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE tifthe
indications are not for all
designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blankifrtot applicable)
The indications listad below
will be submitted to the International
Bureau later (specify the general
nahtre of tire indications
e.g.,
"Accession Number of Deposit')
For receiving Office use only For International Bureau use only
This sheet was received with the ittlernational ~ This sheet was received by
the International Bureau
application on~
Authorized officer
Authorized officer
t~
. :~'
~l~ ~tt 3 i"~ t''~ ~ : .... ...~~ ~ : - :,_, a i
Form PCTlR01134 (July 199Z)
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SEQUENCE INFORMATION
BASB207 Polynucleotide and Polypeptide Sequences
SEQ ID NO:I polynucleotide sequence of BASB207
J ATGACAAAACAACTACAACCATCAGATACTTCACCAAAAGCACCAAAACAAGCAAATAAAAAACACTG
GGTACGCAAAGTTATTTGTATTGGAAGTGCGGTCATTTTTATACCTGTTTTAGGTGTTGCTGGGGCAA
TTTCTTTCGATGCGGGACAAAAAAGTTTAATTCAACTAGCCGACAAAATGCTCGATAGTTTTTCCGTT
GAGCAAATTGAAGGCGGATTACAAAATGGCTTGGTGTTAAAGAATGTTCGTTATCAAACCGCTGGGAT
TGAGACACATATTGCACAAGCACGTTTACAGCTCGATTTCGGTTGTTTGCTTTCACGTGAAGTTTGTT
IO TACGTGACTTTACCTTAAATAAACCGACAATTGCGATTAATACTGCATTATTGCCTCCATCAACGCCT
GATAATTCCAAGTCTAGCCAGATGAAGCGTATTTCCTTGCCAATTAGTATCAATGCAGAAAGTTTGGT
AGTGCAAAATTTATCTGTAAATATTGATCAGACTAATATTACACTTGGAAATTTTAAAAGTGCGGTAA
ATTTAAATAATGAAAAAGGTTTAACTATTGCACCGACTGAAATTAACGATATTTCAGTGATTGCAAAA
AAATTGTCTGAAGTTAAATCAGAGCTGAAAGCCGAACAGCCGAATAAACCTGTAGATTGGGCTACGAT
IS TGAGCAATCTTTAACGCCTGCTTTTTTAGGTAATGTATCAGAAATCATTCTTCCTTTTGATTTACATA
TTCCTGAAATTTCAGGCAAAAACTGGCAGTATCAAGCAGTGAATGAAAAAGGCGAAACGCTTCAATCT
GTTGAAATGTCTAGTTTAATTGCACAGGCGAATACCGTCGATAACCAACTGCAATTACAAAAATTAGC
GGTAGAAAGCTCGTTGGGTAATCTTTCTTCACAAGGTAAATTACAACTCGATGGCGATATGCCTCTCG
ATTTAACTTTAAAATCCCATTTGGAACCGTTGAAATCCGATGGAAAAGAAATTCTACCCGCGAGTGAT
2O GTGGATCTTACGCTTTCAGGATCATTGAAAAAATCTACCGCTCTTTCTTTGAAAACaaaaggCGTATT
AGATGCTGAACTGAATGGCAACGTGCAATTAGCGCAAGACAAAATGCCCTTAAACCTCACATTAAATG
TGGCAAAAGGACAATACACTtttgtaAACACAATGACGCCACTCAAAATTAATGATGTGACACTTAAA
CTCACGGGGGAT'E'TACTAAATTATCACGCCGAACTTAAAGGCGATGTAGCAGGAATGAATTATATTCC
CGCATCTCAAGTGGAACTTAATGCCGACGGTAAACTTTATGAAGTTACCGTCAATAAATTGGGGATAG
ZS CTTCATTAGACGGTAAAAGTGAATTTGTGGGTAATGCGAATTGGAAAAATGGTGCAAACTGGGATATT
CAAGCCGATTTAGAAAAAATGAATATTGGTTTTTTTGTTCCTGTCATGCCTGCAACACTATCTGGTAA
ATTGCATTCACGTGGTTTTGCAGGCTCTCAAGGATGGCAAGTGGAAGTGCCAGTAGCCGATTTAAATG
GAACATTGTCTTCAAACCCCATCAGTTTAAAAGGCTCCGCAACACTTAATCAAAATGTACTTCTAACA
GTACCTGATTTACAGATTAGATATGGCGAAAATTATCTTAAAGCCAGTGGCGTATTAGATGATCATTC
3O TGATTTTGCGCTAGATATAA.ATGCGCCAAATTTACGTGGATTATGGTCAGATTTAAAAGGGCGAGTAA
AAGGTCGAGTAGCGATTTCTGGTCAAATTACGACACCTAATCTTGATCTTGATTTAACTTCATCTAAT
TTACATTTACAGGGTTTTCAGTTAGCCAAAGCAAGTATAAAAGGTCATATAAACAATGCCTCACTCTC
GAGTGGAAAATTGAATATTAAAGCAGAACAACTTCATTATGGCGAAAATATTAAACTCCATTTACTTG
ATCTTGATTTATCAGGCGATGAACAAAATCACAAACTAATCCTAAAATCTCAAGGCGCGCCTGTTGCT
3S GCAAATTTACAAATTAACGGACATTTTGACCGCACTTTGGAACAGTGGAAAGGCACAATTTCACAGGT
AAAATTTGAAACTCCGATTGGCGATGTAAAATCCAACCAAGCGATTGCAGTGTCCTACGATAACAAAC
AAACACAAGCCAATA'F'TGCCTCACATTGCTGGCAAAACACCGATGTAGAATTATGTTTCCCACAAGCA
TTCAATGCAGGCAAGCAAGGCAATATTCCTTTCCAATTTAAACGTGTTAATTTGGATTTAGTGAATAA
ACTTATCGAACAAAACAGCCTCAAAGGCAATTTACAAGTGCAAGGAAATGTTGCTTGGTTTACGGATA
AACCATTCCAATTTACCGCCAACATAGATGGCAATCATTTAGCCTTCTCTCAAAAATTGGATTACCGC
ACGTTTAAATTAGATATTCCAAAATTAACCCTTAATGCCGACATTCAAAATAATp,ATTTGGTTTTAAA
AACAGACATCAATTTGCATAATCAAGGCAGAATTGTAGGCGATATTCGTTTAAATGATTTTGTTAAAA
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CTCGCCAACTTGGCGGAACCCTTGCGATAGAACGATTAAACTTGTCGATCGCAAATCAGCTACTTACT
AGCGGTGAATCAGTGAATGGTAAGGTAGTCTCGAAGTTGAGTTTCGGCGGGAATTTAGAGAAACCATT
a
ATTAAATGGTGACTTCAATATTCGTAATATTCGTACCAAATTGAAATCTATGCCAGTCAATATTACAG
ATGGCGATATTGCCCTACGCTTTAATGGTAACCGTTCAACATTGCAAGGGAAAATCGAAACAGTAGAT
S AGTCACTTAAACTTAACTGGACGTGCAAACTGGGCGAATATTGAACATTGGACGACAGAATTGAATGC
ACAAGCAAATAATTTCAACGTGGATATTCCATCTATGGCGAAGCTACGTTTCAGTCCAGATATCACGA
TAAAAGCCAATCCTAAGGAATTGAATTTAAGCGGAACAGTGGATATTCCTTGGGCGCGTATTAAAATT
GACAGTCTGCCAGATACTGCTGAGCCAGTCAGCGAAGATGAAGTGATTTTAAACGGCCCGCACAAGAG
CAAAGAAGAACTGATTAAACGTGAATTTGCCGCAAAAACGAAATCAGGAATGGATATTCGCTCTGATT
IO TACGCATCAATATTGGTAAAGACGTCAGCCTTGATGCCTATGGCTTAAAAACCAATCTTGATGGTTTA
CTTTCTGTGAAACAAGACAAAGGCAATTTAGGTTTATTCGGGCAAATCAATTTAACCAAAGGTCGCTA
TGCTTCTTTCGGACAAGATTTACTCATTCGTAAAGGGCTTATTAGTTTTTCTGGGCAAGCGGCACAAC
CTACGTTGAATATTGAGGCTATTCGTAATCCTGAAACAATGGAAGACAGCAAAATTACTGCTGGAGTG
AGAGTGATTGGCATAGCCGATAGCCCAGAAGTCACTATTTTCAGTGAACCAAGCAAACCACAAGATCA
IS AGCACTTTCTTATTTATTAACAGGTCGTTCGTTAGAAAGTAGTGGAGAAGTGGGTTCAACGGGATCAG
TTGGTGCAGCATTAATTGGTTTAGGCATTTCAAAGAGCGGTAAATTAGTGGGCAGTATTGGCGAAGTT
TTTGGTATTCAAGACTTAAACCTTGGTACATCTGGCGTGGGCGATAAATTTAAGGTTACAGTAAGTGG
CAATATTACCAATCGCTTACAAATAAAATATGGCGTAGGTTTATTTGATGGGTTAGCAGAAGTTACAT
TACGTTATCGCCTAATGCCACAGCTTTATTTTCAATCCGTTTCTAGCACAAACCAAGTGTTTGATTTA
ZO CTTTATAAATTTGAATTTTAG
SEQ ID N0:2 polypeptide sequence of BASB207
MTKQLQPSDTSPKAPKQANKKHWVRKVICIGSAVIFIPVLGVAGAISFDAGQKSLIQLADKMLDSFSV
EQIEGGLQNGLVLKNVRYQTAGIETHIAQARLQLDFGCLLSREVCLRDFTLNKPTIAINTALLPPSTP
~S DNSKSSQMKRTSLPISINAESLWQNLSVNIDQTNITLGNFKSAVNI~NNEKGT~TIAPTEINDTSVIAK
KLSEVKSELKAEQPNKPVDWATIEQSLTPAFLGNVSEIILPFDLHIPEISGKNWQYQAVNEKGETLQS
VEMSSLTAQANTVDNQLQLQKLAVESSLGNLSSQGKLQLDGDMPLDLTLKSHLEPLKSDGKEILPASD
VDLTLSGSLKKSTDLSLKTKGVLDAELNGNVQLAQDKMPLNLTLNVAKGQYTFWTMTPLKINDVTLK
LTGDLLNYHAELKGDVAGMNYIPASQVELNADGKLYEVTWKLGIASIsDGKSEFVGNANWKNGANWDI
3O QADLEKMNIGFFVPVMPATLSGKLHSRGFAGSQGWQVEVPVADLNGTLSSNPISLKGSATLNQNVIrLT
VPDLQIRYGENYLKASGVLDDHSDFALDINAPNLRGLWSDLKGRVKGRVAISGQITTPNLDLDLTSSN
LHLQGFQLrAKASTKGHINNASLSSGKLNIKAEQLHYGENIKLHLLDLDLSGDEQNHKLILKSQGAPVA
ANLQINGHFDRTLEQWKGTISQVKFETPIGDVKSNQAIAVSYDNKQTQANIASHCWQNTDVELCFPQA
FNAGKQGNIPFQFKRVNLDLVNKLIEQNSLrKGNLQVQGNVAWFTDKPFQFTANIDGNHLAFSQKLDYR
3S TFKLDIPKLTLNADIQNNNF~VT~KTDINLHNQGRIVGDIRLNDFVKTRQLGGTLAIERLrNLSIANQLLT
SGESVNGKWSKLSFGGNLEKPLLNGDFNIRNIRTKLKSMPVNTTDGDIALRFNGNRSTLQGKIETVD
SHLNLTGRANWANIEHWTTELNAQANNFNVDIpSMAKLRE'SPDITIKANPKELNLSGTVDIpWARIKI
DSLPDTAEPVSEDEVILNGPHKSKEELIKREFAAKTKSGMDIRSDLRINIGKDVSLDAYGLKTNLDGL
LSVKQDKGNLGLrFGQINLTKGRYASFGQDLLIRKGL,ISFSGQAAQPTLNIEAIRNPETMEDSKITAGV
RVIGIADSPEVTTFSEPSKPQDQALSYLLTGRSLESSGEVGSTGSVGAALIGLGISKSGKLVGSIGEV
FGTQDLNLGTSGVGDKSKVTVSGNITNRLQIKYGVGLFDGLAEVTLRYRLMPQLYFQSVSSTNQVFDL
LYKFEF
SEQ ID N0:3 polynucleotide sequence of BASB207
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ATGACAAAACAACTACAACCATCAGATACTTCACCAAAAGCACCAAAACAAGCAAATAAAAAACACTG
GGTACGCAAAGTTATTTGTATTGGAAGTGCGGTCATTTTTATACCTGTTTTAGGTGTTGCTGGGGC~AA
TTTCTTTCGATGCGGGACAAAAAAGTTTAATTCAACTAGCCGACAAAATGCTCGATAGTTTTTCCGTT
GAGCAAATTGAAGGCGGATTACAAAATGGCTTGGTGTTAAAGAATGTTCGTTATCAAACCGCTGGGAT
S TGAGACACATATTGCACAAGCACGTTTACAGCTCGATTTCGGTTGTTTGCTTTCACGTGAAGTTTGTT
TACGTGACTTTACCTTAAA'." ACCGACAATTGCGATTAATACTGCATTATTGCCTCCATCAACGCCT
GATAATTCCAAGTCTAGCCAGATGAAGCGTATTTCCTTGCCAATTAGTATCAATGCAGAAAGTTTGGT
AGTGCAAAATTTATCTGTAAATATTGATCAGACTAATATTACACTTGGAAATTTTAAAAGTGCGGTAA
ATTTAAATAATGAAAAAGGTTTAACTATTGCACCGACTGAAATTAACGATATTTCAGTGATTGCAAAA
1O AAATTGTCTGAAGTTAAATCAGAGCTGAAAGCCGAACAGCCGAATAAACCTGTAGATTGGGCTACGAT
TGAGCAATCTTTAACGCCTGCTTTTTTAGGTAATGTATCAGAAATCATTCTTCCTTTTGATTTACATA
TTCCTGAAATTTCAGGCAAAAACTGGCAGTATCAAGCAGTGAATGAAAAAGGCGAAACGCTTCAATCT
GTTGAAATGTCTAGTTTAATTGCACAGGCGAATACCGTCGATAACCAACTGCAATTACAAAAATTAGC
GGTAGAAAGCTCGTTGGGTAATC'.~'TTCTTCACAAGGTAAATTACAACTCGATGGCGATATGCCTCTCG
LS ATTTAACTTTAAAATCCCATTTGGAACCGTTGAAATCCGATGGAAAAGAAATTCTACCCGCGAGTGAT
GTGGATCTTACGCTTTCAGGATCATTGAAAAAATCTACCGCaCTTTCTTTGAAAACaaaaggCGTATT
AGATGCTGAACTGAATGGCAACGTGCAATTAGCGCAAGACAAAATGCCCTTAAACCTCACATTAAATG
TGGCAAAAGGACAATACACTtttgtaAACACAATGACGCCACTCAAAATTAATGATGTGACACTTAAA
CTCACGGGGGATTTACTAAATTATCACGCCGAACTTAAAGGCGATGTAGCAGGAATGAATTATATTCC
ZO CGCATCTCAAGTGGAACTTAATGCCGACGGTAAACTTTATGAAGTTACCGTCAATAAATTGGGGATAG
CTTCATTAGACGGTAAAAGTGAATTTGTGGGTAATGCGAATTGGAAAAATGGTGCAAACTGGGATATT
CAAGCCGATTTAGAAAAAATGAATATTGGTTTTTTTGTTCCTGTCATGCCTGCAACACTATCTGGTAA
ATTGCATTCACGTGGTTTTGCAGGCTCTCAAGGATGGCAAGTGGAAGTGCCAGTAGCCGATTTAAATG
GAACATTGTCTTCAAACCCCATCAGTTTAAAAGGCTCCGCAACACTTAATCAAAATGTACTTCTAACA
2S GTACCTGATTTACAGATTAGATATGGCGAAAATTATCTTAAAGCCAGTGGCGTATTAGATGATCATTC
TGATTTTGCGCTAGATATAAATGCGCCAAATTTACGTGGATTATGGTCAGATTTAAAAGGGCGAGTAA
AAGGTCGAGTAGCGATTTCTGGTCAAATTACGACACCTAATCTTGATCTTGATTTAACTTCATCTAAT
TTACATTTACAGGGTTTTCAGTTAGCCAAAGCAAGTATAAAAGGTCATATAAACAATGCCTCACTCTC
GAGTGGAAAATTGAATATTAAAGCAGAACAACTTCATTATGGCGAAAATATTAAACTCCATTTACTTG
3O ATCTTGATTTATCAGGCGATGAACAAAATCACAAACTAATCCTAAAATCTCAAGGCGCGCCTGTTGCT
GCAAATTTACAAATTAACGGACATTTTGACCGCACTTTGGAACAGTGGAAAGGCACAATTTCACAGGT
AAAATTTGAAACTCCGATTGGCGATGTAAAATCCAACCAAGCGATTGCAGTGTCCTACGATAACAAAC
AAACACAAGCCAATATTGCCTCACATTGCTGGCAAAACACCGATGTAGAATTATGTTTCCCACAAGCA
TTCAATGCAGGCAAGCAAGGCAATATTCCTTTCCAAT'h'TAAACGTGTTAATTTGGATTTAGTGAATAA
35 ACTTATCGAACAAAACAGCCTCAAAGGCAATTTACAAGTGCAAGGAAATGTTGCTTGGTTTACGGATA
AACCATTCCAATTTACCGCCAACATAGATGGCAATCATTTAGCCTTCTCTCAA.AAATTGGATTAGCGC
ACGTTTAAATTAGATATTCCAAAATTAACCCTTAATGCCGACATTCAAAATAATAATTTGGTTTTAAA
AACAGACATCAATTTGCATAATCAAGGCAGAATTGTAGGCGATATTCGTTTAAATGATTTTGTTAAAA
CTCGCCAACTTGGCGGAACCCTTGCGATAGAACGATTAAACTTGTCGATCGCAAATCAGCTACTTACT
AGCGGTGAATCAGTGAATGGTAAGGTAGTCTCGAAGTTGAGTTTCGGCGGGAATTTAGAGAAACCATT
ATTAAATGGTGACTTCAATATTCGTAATATTCGTACCAAATTGAAATCTATGCCAGTCAATATTACAG
ATGGCGATATTGCCCTACGCTTTAATGGTAACCGTTCAACATTGCAAGGGAAAATCGAAACAGTAGAT
AGTCACTTAAACTTAACTGGACGTGCAAACTGGGCGAATATTGAACATTGGACGACAGAATTGAATGC
ACAAGCAAATAATTTCAACGTGGATA"~'TCCATCTATGGCGAAGCTACGTTTCAGTCCAGATATCAGGA
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TAAAAGCCAATCCTAAGGAATTGAATTTAAGCGGAACAGTGGATATTCCTTGGGCGCGTATTAAAATT
GACAGTCTGCCAGATACTGCTGAGCCAGTCAGCGAAGATGAAGTGATTTTAAACGGCCCGCACAA~G
CAAAGAAGAACTGATTAAACGTGAATTTGCCGCAAAAACGAAATCAGGAATGGATATTCGCTCTGATT
TACGCATCAATATTGGTAAAGACGTCAGCCTTGATGCCTATGGCTTAAAAACCAATCTTGATGGTTTA
S CTTTCTGTGAAACAAGACAAAGGCAATTTAGGTTTATTCGGGCAAATCAATTTAACCAAAGGTCGCTA
TGCTTCTTTCGGACAAGATTTACTCATTCGTAAAGGGCTTATTAGTTTTTCTGGGCAAGCGGCACAAC
CTACGTTGAATATTGAGGCTATTCGTAATCCTGAAACAATGGAAGACAGCAAAATTACTGCTGGAGTG
AGAGTGATTGGCATAGCCGATAGCCCAGAAGTCACTATTTTCAGTGAACCAAGCAAACCACAAGATCA
AGCACTTTCTTATTTATTAACAGGTCGTTCGTTAGAAAGTAGTGGAGAAGTGGGTTCAACGGGATCAG
IO TTGGTGGAGCATTAATTGGTTTAGGCATTTCAAAGAGCGGTAAATTAGTGGGCAGTATTGGCGAAGTT
TTTGGTATTCAAGACTTAAACCTTGGTACATCTGGCGTGGGCGATAAATcTAAGGTTACAGTAAGTGG
CAATATTACCAATCGCTTACAAATAAAATATGGCGTAGGTTTATTTGATGGGTTAGCAGAAGTTACAT
TAGGTTATCGCCTAATGCCACAGCTTTATTTTCAATCCGTTTCTAGCACAAACCAAGTGTTTGATTTA
CTTTATAAATTTGAATTT
15 SEQ ID NO:~ polypeptide sequence of BASB207
MTKQLQPSDTSPKAPKQANKKHWVRKVICTGSAVIFIPVLGVAGAISFDAGQKSLIQLADKMLDSFSV
EQIEGGLQNGLVLKNVRYQTAGIETHIAQARLQLDFGCLLSREVCLRDFTLNKPTIAINTALLPPSTP
DNSKSSQMKRISLPISINAESLVVQNLSVNIDQTNITLGNFKSAVNLNNEKGLTIAPTETNDISVIAK
KLSEVKSELKAEQPNKPVDWATIEQSLTPAFLGNVSEIILPFDLHIPEISGKNWQYQAVNEKGETLQS
ZO VEMSSLIAQANTVDNQLQLQKLAVESSLGNLSSQGKLQLDGDMPLDLTLKSHLEPLKSDGKEILPASD
VDLTLSGSLKKSTALSLKTKGVLDAELNGNVQLAQDKMPLNLTLNVAKGQYTFVNTMTPLKINDVTLK
LTGDLLNYHAELKGDVAGMNYIPASQVELNADGKLYEVTVNKLGIASLDGKSEFVGNANWKNGANWDI
QADLEKMNTGFFVPVMPATLSGKLHSRGFAGSQGWQVEVPVADLNGTLSSNPISLKGSATLNQNVLLT
VPDLQIRYGENYLKASGVLDDHSDFALDINAPNLRGLWSDLKGRVKGRVAISGQITTPNLDLDLTSSN
ZS LHLQGFQLAKASIKGHINNASLSSGKLNIKAEQLHYGENIKLHLLDLDLSGDEQNHKLILKSQGAPVA
ANLQINGHFDRTLEQWKGTISQVKFETPIGDVKSNQAIAVSYDNKQTQANIASHCWQNTDVELCFPQA
FNAGKQGNIPFQFKRVNLDLVNKLIEQNSLKGNLQVQGNVAWFTDKPFQFTANIDGNHLAFSQKLDYR
TFKLDIPKLTLNADIQNNNLVLKTDINLHNQGRIVGDTRLNDFVKTRQLGGTLAIERLNLSIANQLLT
SGESVNGKWSKLSFGGNLEKPLLNGDFNIRNIRTKLKSMPVNITDGDIALRFNGNRSTLQGKIETVD
3O SHLNLTGRANWANIEHWTTELNAQANNFNVDIPSMAKLRFSPDITIKANPKELNLSGTVDIPWARTKI
DSLPDTAEPVSEDEVILNGPHKSKEELIKREFAAKTKSGMDIRSDLRINIGKDVSLDAYGLKTNLDGL
LSVKQDKGNLGLFGQINLTKGRYASFGQDLLIRKGLISFSGQAAQPTLNIEAIRNPETMEDSKITAGV
RVIGIADSPEVTIFSEPSKPQDQALSYLLTGRSLESSGEVGSTGSVGAALIGLGISKSGKLVGSIGEV
FGIQDLNLGTSGVGDKSKVTVSGNITNRLQIKYGVGLFDGLAEVTLRYRLMPQLYFQSVSSTNQVFDL
3S LYKFEF
SEQ ID NO:S polynucleotide sequence of BASB207
ATGACAAAACAACTACAACCATCAGAAACTTCGCCAAAAGCACCAGAGCAACCAAGTAAAAAACATTG
GATGCGTAAAGCTGTTTGTATCGGAAGTGCGGTCATTTTTATACCTGTTTTAGGTGTTGCTGGGGCGC
TTTCTTTCGATGCAGGACAAAAAAGTTTAATTCAACTTGCCGATAAAATGCTTGATAGTTTTTCCGTT
~O GAGCAAATtGAAGGCGGATTaCAAAAtGGCttGGTGTTAAAGAATGTTCAATACCAAACTGCTGGGAT
TGAGACACATATTGCACAAGCACGGT'FACAGATTGATTTCGCTTGTTTGTTTTCACGTGAAGTTTGTT
TACGTGACTTTACCTTAAATAAACCGACGATTGCGATTAATACTGCATTATTGCCTCCATCGACAACT
GATAATTCCAAGTCTAGCCCGATGAAGCGtATTTCCTTGCCAATTGGTATCAATGCAGAAAATTTGGC
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GGTGCAAGATTTATCTGTAAACATTGATCAGACTAGTATTACGCTTGGAAATTTTAAAAGTGCGGTAA
GTTTAAATAATGAAAAAGGCTTAACTATTGCACCGACTGAAATTAACGATATTTCAGTGATTGCAAAA
a
AAATTGCCCGAAGGTAAACCTGAGCTGAAAGCCGAACAGCCGAATAAGCCTGTAGATTGGGCTGCGAT
TGAGCAATCTTTAACGCCTGCTTTTTTAGGTAATGTATCAGAAATAATTCTTCCTTTTGATTTACATA
S TTCCTGAAATTTCAGGCAAAAACTGGCAGTATCAAGCCGTGAATGAAAAAGGCGAAACGCTTCAATCT
GTTGAAATGTCTAGTTTAATTGCACAGGCGGATACCGTCGATAACCAACTGCAATTACAAAAATTAGC
GGTAGAAAGCTCGTTGGGTAATCTTTATTCGCAAGGTAAATTACAACTTGATGGCGATATGCCGCTAG
ATTTAACTTTAAAATCCCATTTGGAGCCGTTGAAATCCGCTGGAAAAGAAATTCTACCCGCGAGTGAT
GTGGATCTTACGCTTTCGGGATCATTGAAAAAATCCACCGCACTTTCTTTAAAAACAAAAGGCGTATT
IO AGATGCTGAACTGAATGGCGACGTGCAATTAGCACAAGACAAAATGCCCTTAAACCTCACATTAAACG
tGGCAAAAGGACAATACGCTTTTGTAAACFCAATGATGCCACTCAAAATTAATGATGTGACACTTAAA
CTCACGGGGGATTTACTAAATTATCACGCTGAACTTAAGGGCAATGTAGTAGGAATGAATTATATTCC
TGCATCTCAAGTGGAACTTAATGCCGACGGTAAACTTTATGAAGTCACCATAAATAAATTGGGGATAG
CTTCATTAGATGGTAAAAGTGAATTTGTGGGTAATGCGAATTGGAAAAATGGTGCAAACTGGGATATT
IS CAAGCCGaTTTAGAAA.AAATGAATATTGGTTTTTTTGTTCCTGTAATGCCTGCAACACTATCTGgCAA
ATTACATTCGCGTGGTTTTGCAGGCTCTCAAGGATGGCAAGTGGAAGTGCCAGTAGCCGATTTAAACG
GAATACTTTCTGCAAAACC'FATCAGTTTAAAAGGTTCTGCAACGCTCAATCAAAAGGTACTTCTAACA
GTACCTGATTTACAGATTAAATATGGCGAAAATTATCTTAAAGCCAGTGGCGTATTAGATGATCATTC
TGATTTTGCGCTAGATATAAATGCGCCAAATTTACGTGGATTATGGTCAGATTTAAAAGGGCGAGTAA
ZO AAGGTCGAGTAGCGATTTCTGGTCAAATTACGACACCTAATCTTGATCTTGATTTAACTTCATCTGAT
TTACATTTACAGGGTTTTCAGTTAGCTAAAGCAAGTATAAAAGGTCATATAAACAATACCTCACTCTC
AAGTGGAAAATTGAATATTAAAGCAGAACAACTTCATTATGGCGAAAATATTAAACTCCATTTACTTG
ATCTTGATTTATCAGGCGATGAACAAAATCACAAACTAATCCTAAAATCTCAAGGCGAGCCTGTTGCT
GCAAATTTACAAATTAACGGCCATTTTGACCGCACTTTGGAACAGTGGAAAGGCACAATTTCACAGGT
ZS AAAATTTGAAACTCCGATTGGCGATGTAAAATCCAACCAAGCGATTGCGGTGTCCTACGATAACAAAC
AAACACAAGCCAATATTGCCTCACATTGCTGGCAA.AACACCGATGTAGAATTATGTTTCCCACAAGCA
TTCAATGCAGGCAAGCAAGGCAATATTCCTTTCCAATTTAAACGTGTTAATTTGGATTTAGTCAATAA
ACTTATCGAACAAGACAGCCTCAAAGGCAATTTACAAGTGCAAGGAAATGTGGCTTGGTTTACGGATA
AACCATTCCAATTTACCGCCAACGTAGATGGCAATCATTTAGCCTTCTCTCAAAAATTGGATTACCGT
3O aCATTTAAATTGGaTATTCCAAAATTAACCCTTAACGCCGACATTCAAAATAATAATTTGGTTTTAAA
AACAGACATCAATGTGCATAACCAAGGCAGAATTGTAGGCGATATTCGTTTAAACGATCTTGCGAAAA
ATCGCCAACTTAGCGGTAGCCTTGCGATAGAACGATTAAGTTTGAGTATTGCGAATCAACTACTCACT
CAAGGCGAATCCGTGAATGGAGAAGTGGTATCAAAATTGCGTTTCGGTGGGAATTTAGAGAAACCATT
ATTAAATGGTGACTTCAATATTCGTAATATTCGTACCAAATTAAAATCTATGCCAGTCAATATTACAG
3S ATGGCGATATTGCTCTACGCTTTAATGATAACCGTTCAACATTGCAAGGAAAAATTGAAACGGTAGAT
AGTCACTTAAACTTAACTGGACGTGCAAACTGGGCTAATATTGAACATTGGCCAACAGAATTAAATGC
GCAAGCAAATAATTTCAACGTGGATATTCCTTCTATGGCGAAGCTACGTTTCAGTCCAAATATCACGA
TAAAAGCCAATCCGAAGGAATTGAATTTAAGCGGAACAGTGGATATTCCTTGGGCACGTATTAAAATT
GACAGTCTGCCAGATACTGCTGAGCCAGTAAGCGAAGATGAAGTTATTTTAAATGGCCCTCATAAGAG
CAAAGAAGAACTGATTAAACGCGAATTTGCGGCAAAAACAAAATCAGGCATGGATATTCGCTCTGATT
TACGCATCAATATTGGTAAAGACGTTAGCCTTGATGCCTATGGCTTGAAAACCAATCTTGATGGTTTA
CTTTCTGTGAAACAAGACAAAGGCAATTTAGGTTTATTCGGTCAAATCAATTTAACCAAAGGTCGCTA
TGCTTCTTTCGGGCAAGATTTACTCATTCGTAAAGGGCTTATTAGCTTTTCTGGGCAAGCGACACAAC
CTACATTGAATATTGAGGCTATTCGTAATCCTGAAACAATGGAAGACAGCAAAATTACTGCTGGCGTG
79
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
AGAGTGATTGGCATAGCTGATAGCCCAGAAGTCACTATTTTCAGTGAACCAAGCAAACCACAAGATCA
AGCACTTTCTTATTTATTAACAGGTCGTTCGTTAGAAAGCAGCGGAGAAGTGGGTTTAACGGGATCAG
a
TTGGCGCAGCATTGATTGGTTTAGGCATTTCAAAGAGCGGTAAATTAGTGGGCAGTATTGGCGAAGTT
TTTGGTATTCAAGATTTAAACCTTGGTACATCTGGCGTGGGCGATAAATCCAAAGTGACGGTAAGTGG
AAATATTACCAATCGCTTACAAATAAAATATGGCGTAGGTTTATTTGATGGCTTAGCAGAAGTTACAT
TACGTTATCGCCTAATGCCACAGCTTTATTTTCAATCCGTTTCTAGCACAAACCAAGTGTTTGATTTA
CTTTATAAATTTGAATTA
SEQ ID N0:6 polypeptide sequence of BASB207
MTKQLQPSETSPKAPEQPSKKHWMRKAVCIGSAVIFIPVLGVAGALSFDAGQKSLIQLADKMLDSFSV
iO EQIEGGLQNGLVLKNVQYQTAGIETHTAQARLQIDFACT~FSREVCLRDFTLNKPTIAINTALLppSTT
DNSKSSPMKRISLPIGTNAENLAVQDLSVNIDQTSITLGNFKSAVSLNNEKGLTIAPTEINDISVIAK
KLPEGKPELKAEQPNKPVDWAATEQSLTPAFLGNVSEIILPFDLHIPEISGKNWQYQAVNEKGETLQS
VEMSSLIAQADTVDNQLQLQKLAVESSLGNLYSQGKLQLDGDMPLDLTLKSHLEPLKSAGKEILPASD
VDT~TLSGSLKKSTALSLKTKGVT~DAELrNGDVQLAQDKMpLrNLTLNVAKGQYAFVNTMMPDKINDVTLK
IS LTGDI~LNYHAELKGNWGMNYIPASQVELNADGKLYEVTINKLGIASLDGKSEFVGNANWKNGANWDI
QADLEKMNIGFFVPVMPATLSGKLHSRGFAGSQGWQVEVpVADLNGILSAKPISLKGSATLNQKVLrLT
VPDLQIKYGENYLKASGVLDDHSDFALDINAPNLRGLWSDLKGRVKGRVAISGQITTPNLDLDLTSSD
LHT~QGFQLAKASIKGHINNTSLSSGKLNIKAEQLHYGENIKLHLLDLDLSGDEQNHKLrILKSQGEPVA
ANLQINGHFDRTLEQWKGTISQVKFETPIGDVKSNQATAVSYDNKQTQANTASHCWQNTDVELCFPQA
FNAGKQGNIPFQFKRVNT~DLVNKLTEQDSLKGNLQVQGNVAWFTDKPFQFTANVDGNHLAFSQKLDYR
TFKLDIPKLTLNADIQNNNLVLKTDINVHNQGRIVGDIRLNDLAKNRQLSGSLAIERLSLSIANQI~LT
QGESVNGEVVSKLRFGGNLEKPLLNGDFNIRNIRTKLrKSMPVNITDGDIALRFNDNRSTLQGKIETVD
SHLNLTGRANWANIEHWpTELNAQANNFNVDIPSMAKT~RFSPNITIKANPKELNLSGTVDTPWARIKT
DSLrPDTAEPVSEDEVILNGPHKSKEEIiTKREFAAKTKSGMDIRSDLRTNTGKDVSLDAYGLhKTNLDGL
ZS LSVKQDKGNLGLFGQINLTKGRYASFGQDLLIRKGLISFSGQATQPTLNTEAIRNPETMEDSKITAGV
RVTGIADSPEVTIFSEPSKPQDQALSYLIrTGRSLESSGEVGLTGSVGAALIGLGISKSGKE~VGSIGEV
FGIQDLNLGTSGVGDKSKVTVSGNTTNRLQIKYGVGLrFDGLAEVTLRYRLMPQLYFQSVSSTNQVFDL
LYKFEL
SEQ ID N0:7 polynucleotide sequence of BASB207
3O ATGACAAAACAACTACAACCATCAGATACTTCACCAAAAGCACCAAAACAAGCAAATAAAAAACACTG
GGTACGCAAAGTTATTTGTATTGGAAGTGCGGTCATTTTTATACCTGTTTTAGGTGTTGCTGGGGCGA
TTTCTTTCGATGCGGGACAAAAAAGTTTAATTCAACTAGCCGACAAAATGCTCGATAGTTTTTCCGTT
GAGCAAATTGAAGGCGGATTACAAAATGGCTTGGTGTTAAAGAATGTTCGTTATCAAACCGCTGGGAT
TGAGACACATATTGCACAAGCACGGTTACAGCTTGATTTCGGTTGTTTGCTTTCACGTGAAGTTTGTT
3S TACGAGACTTTACTCTAAATAAACCGACGATTGCGATTAATACCGCATTGTTACCGCCATCAATGCCT
GATGATTCAAAGTCTAGCCCGATGAAGCGCATTTCCTTGCCAATTAGTATCAATGCAGAAAATTTAGT
GGTGCAAGATTTATCCGTAAATGTTGATCAGACTAATATTACGCTTGGAAATTTTAAAAGTGCGGTAA
GTTTAAATAATGAAAAAGGCTTAACTATTGCACCGACTGAAATTAACGATATTTCAGTGATTTCAAAA
AAATTGCCTGAAGTTAAATCAGAGGCGAAAGCCGAACAGCCGAATAAGCCTGTAGATTGGGCTGCGAT
O TGAGCAATCTTTAACTCCCGCTTTTTTAGGCAATGTATCAGAAATCATTCTTCCTTTTGATTTACATA
TTCCTGAAATTTCAGGCAAAAACTGGCAGTATCAAGCAGTCAATGAAAAAGGCGAAACGCTTCAATCT
ATTGAAATCCCAAGCCTGATTGCACAGGCGGATACCGTCGATAACCAACTGCAATTACAAAAATTATC
GGTTGAAAGTTCGCTCGGTAATCTTTATTCACAAGGTAAATTACAACTTGATGGCGATATGCCACTAG
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
ATTTAACCTTAAAATCCCATTTGGAGCCGTTGAAATCCGCTGGAAAAGAAATTCTGCCAGAGAGTGAT
GTGGATCTTACGCTTTCGGGAACATTGAAAAAATCCACCGCACTTTCTTTAAAAACAAAAGGCGT1~TT
AGATGCTGAACTGAATGGCGAAGTGCAATTAGCACAAGACAAAATGCCCTTAAACCTCACATTAAATG
TGGCAAAAGGACAATACGCTTTTGTAAACACAATGGCACCACTCAAAATTAATGATGTGACACTTAAA
S CTCACGGGGGATTTACTGAATTATCACGCCGAATTTAAGGGCAATGTGGCAGGAATGAATTATATTCC
CGCATCTCAAGTAGACTTTAATGCCGACGGCAAACTTTATGAAATAACCGTAAATAAATTGGGGATAG
CTTCATTAGACGGTAAAAGTGAATTTGTGGGTAATGCAAATTGGAAAAATGGTGCAAACTGGGATATT
CAAGCCGATTTAGAAAAAATGAATATTGGTTTTTTTGTTCCAGTTATGCCTGCAATATTATCGGGTAA
ATTACATTCTCGCGGTTTTGCGGACTCTCAAGGTTGGCAAGTGGAAGTGCCTACAGCCGATTTAAACG
IO GAACGCTTTCTGCAAAACCTATCAGTTTAAAAGGTTCTGCAACGCTCAATCAAAAGGTACTTCTAACC
GTGCCTGATTTACAGATTAGATATGGCGAAAATTATCTTAAAGCCAGTGGCGTATTAGATGATCATTC
TGATTTTGTGCTAGATATAGATGCGCCAAAT'FTACGTGGTTTATGGTCAGATTTAAAAGGGCGAGTAA
AAGGTCGTGTATCGATTTCTGGTCAAATTACGACACCTAATCTTGATCTTGATTTAACTTCATCCAAT
TTACATTTACAAGGTTTTCAGTTAGCTAAAGCAAGTATAAAAGGTCATATAAACAATACCCCACTCTC
IS GAGTGGAAAATTGAATATTAAAGCAGAACAACTTCATTATGGCGAAAATATTAAACTCCATTTACTTG
ATCTTGATTTATCAGGCGATGAACAAAATCACAAACTAATCCTAAAATCTCAAGGCGAGCCTGTTGCT
GCAAATTTACAAATTAACGGCCATTTTGACCGCACTTTGGAACAGTGGAAAGGCACACTTTCACAAGC
AAAATTTGAAACCCCGATTGGCGATGTAAAATCCAACCAAGCGATTGCGGTGTCTTACGATAACAAAC
AAACACAAGCCAATATTGCCGCACATTGCTGGCAAAATACAGATGTAGAATTATGTTTCCCACAAGCA
ZO TTCAATGCAGGCAAGCAAGGCAATATTCCTTTCCAATTCAAACGTGTTAATTTGGATTTAATGAATAA
ACTTATCGAACAAGATAGCCTCAAAGGAAACTTGCAAGCGCAAGGTAATGTGGCTTGGTTTACGGATA
AACCATTCCAATTTAGCACAAAAGTAGATGGCAATCATTTAGCTTTCTCTCAAAAATTGGATTACCGC
ACGTTTAAATTAGATA'~'TCCAAAATTAACCCTTAATGCCGACATTCAAAATAATAATTTGGTTTTAAA
AACAGACATCAATTTGCATAATCAAGGCAGAATTGTAGGCGATATTCGTGTAAATGATTTTGTTAAAA
2S CTCGCCAACTTGGTGGAACCCTTGCGATAGAACGATTAAACTTGTCGATCGCAAATCAGCTACTTACT
AGCGGTGAATCAGTGAATGGTAAGGTAGTCTCGAAGTTGAGTTTCGGCGGGAATTTAGAGAAACCATT
ATTAAATGGTGACTTCAATATTCGTAATATTCGTACCAAATTGAAATCTATGCCAGTCAATATTACTG
ATGGCGATATTGCCCTACGTTTCAATGGTACCCGTTCAACATTGCAAGGGAAAATCGAAACAGTAGAT
AGTCACTTAAACTTAACTGGACGTGCACACTGGGCTAATATTGAACATTGGACGACAGAATTGAATGC
3O GCAAGCAAATAATTTCAACGTGGATATTCCATCTATGGCAAAACTACGTTTCAGTCCAGATATCACGA
TAAAAGCCAATCCTAAGGAATTGAATTTAAGCGGAACAGTGGATATTCCTTGGGCGCGTATTAAAATT
GACAGTCTGCCAGATACTGCTGAGCCAGTCAGCGAAGATGAAGTGATTTTAAACGGCCCGCACAAGAG
CAAAGAAGAACTGATTAAACGTGAATTTGCCGCAAAAACGAAATCAGGAATGGATATTCGCTCTGATT
TACGCATCAATATTGGTAAAGACGTCAGCCTTGATGCCTATGGCTTAAAAACCAATCTTGATGGTTTA
3S CTTTCTGTGAAACAAGACAAAGGCAATTTAGGTTTATTCGGGCAAATCAATTTAACCAAAGGTCGCTA
TGCTTCTTTCGGACAAGATTTACTCATTCGTAAAGGGCTTATTAGTTTTTCTGGGCAAGCGGCACAAC
CTACGTTGAATATTGAGGCTATTCGTAATCCTGAAACAATGGAAGACAGCAAAATTACTGCTGGAGTG
AGAGTGATTGGCATAGCCGATAGCCCAGAAGTCACTATTTTCAGTGAACCAAGCAAACCACAAGATCA
AGCACTTTCTTATTTATTAACAGGTCGTTCGTTAGAAAGTAGCGGTGAAGCTGGCTCCAGTGGTTCGA
TTGGCGCAGCATTGATTGGTTTAGGCATTTCAAAGAGCGGTAAATTAGTGGGCAGTATTGGCGAAGTT
TTTGGTATTCAAGACTTAAACCTTGGTACATCTGGCGTGGGCGATAAATCTAAGGTTACAGTAAGTGG
AAATATTACCAATCGCTTACAAATAAAATATGGCGTAGGTTTATTTGATGGTTTAGCAGAAGTCACCT
TACGTTATCGCCTAATGCCACAGCTTTATTTTCAATCCGTTTCTAGCACAAACCAAGTGTTTGATTTA
CTTTATATTTTTGAATA
8l
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
SEQ ID N0:8 polypeptide sequence of BASB207
MTKQLQPSDTSPKAPKQANKKHWVRKVTCIGSAVIFIPVLGVAGAISFDAGQKSLIQLADKMLDSF'°5V
EQIEGGLQNGLVLKNVRYQTAGIETHIAQARLQLDFGCLLSREVCLRDFTLNKPTIAINTALLPPSMP
DDSKSSPMKRISLPISINAENLWQDLSVNVDQTNTTLGNFKSAVSLNNEKGLTIAPTETNDISVISK
S KLPEVKSEAKAEQPNKPVDWAAIEQSLTPAFLGNVSEITLPFDLHIPEISGKNWQYQAWEKGETLQS
IEIPSLIAQADTVDNQLQLQKLSVESSLGNLYSQGKLQLDGDMPLDLTLKSHItEPL~KSAGKEILPESD
VDLTLSGTLKKSTALSLKTKGVLDAELNGEVQLAQDKMPLNLTLNVAKGQYAFVNTMAPLKINDVTLK
LTGDLLNYHAEFKGNVAGMNYIPASQVDFNADGKLYEITVNKLGIASLDGKSEFVGNANWKNGANWDI
QADLEKMNIGFFVPWPAILSGKLHSRGFADSQGWQVEVPTADLNGTLSAKPISLKGSATLNQKVLLT
IO VPDl,QIRYGENYLKASGVLDDHSDFVLDIDAPNLRGLWSDLKGRVKGRVSISGQITTPNLDLDLTSSN
LHLQGFQLAKAS I KGHINNTPLSSGKT~NIKAEQLrHYGENIKT~HLLDIsDLSGDEQNHKLTLKSQGEPVA
ANLQTNGHFDRTLEQWKGTLSQAKFETPIGDVKSNQAIAVSYDNKQTQANIAAHCWQNTDVELCFPQA
FNAGKQGNIPFQFKRVNLDLMNKLTEQDSLKGNLQAQGNVAWFTDKPFQFSTKWGNHLAFSQKLDYR
TFKLDIPK1~TLNADTQNNNLVLrKTDINLHNQGRIVGDIRVNDFVKTRQLGGTLAIERLNLSIANQLLT
IS SGESVNGKWSKT~SFGGNLEKPLLNGDFNIRNIRTKLKSMPVNITDGDIALRFNGTRSTLQGKIETVD
SHLNLTGRAHWANIEHWTTELNAQANNFNVDIPSMAKLRFSPDITIKANPKEhNLSGTVDIPWARIKI
DSLPDTAEPVSEDEVILNGPHKSKEELIKREFAAKTKSGMDIRSDLRINIGKDVSLDAYGLKTNLDGIr
LSVKQDKGNLGLFGQINLTKGRYASFGQDLLTRKGLISFSGQAAQPTLNIEAIRNPETMEDSKITAGV
RVIGIADSPEVTIFSEPSKPQDQALSYItLTGRSLESSGEAGSSGSIGAALIGLGISKSGKLVGSIGEV
FGIQDLrNT~GTSGVGDKSKVTVSGNITNRLQIKYGVGLFDGLAEVTLRYRLMPQLYFQSVSSTNQVFDL
LYIFE
SEQ ID NO:9 polynucleotide sequence upstream of the predicted initiation
codon of polynucleotide BASB207
TGGAACTCGGTGTGGGCTTTGCTACTGATGGCGGCGTTCACGGACAAATAGGCTGGACAAAACCTTGG
ZS ATTAATAGCCGTGGACATAGTTTGCGTTCAAATCTTTATCTCTCTGCACCAAAACAAACTCTAGAGGC
AACTTATCGAATGCCACTGCTTAAAAATCCATTAAATTATTACTATGATTTTGCCGTCGGTTGGGAAG
GGGAAAAAGAGAACGATACCAATACGAGAGCGCTTACGTTGTCAGCGTTACGTTATTGGAATAATGCG
CGTGGTTGGCAATATTTTGGCGGACTTCGAGCAAGATACGATAGTTTTACACAAGCGGATATCACTGA
TAAAACCTTACTTCTTTATCCAACTGTTGGATTTACTCGTACTCGATTACGTGGTGGTTCCTTTGCCA
3O CTTGGGGCGATGTGCAAAAAATTACTTTTGATTTAAGCAAACGAATTTGGCTATCAGAATCTTCTTTT
ATAAAAGTGCAAGCATCAAGCGCGTGGATTCGTACTTATGCAGAAAATCATCGTATTGTTGCTCGTGC
TGAAATCGGGTATTTACATACAAAAGATATTGAAAAAATTCCGCCTACACTGCGTTTCTTTGCTGGTG
GCGATCGCAGTWGCGTGGTTACGGCTATAAAAAAATTGCGCCTAAAAATAAAAATGGGAAATTGGTG
GGTGGCTCACGTTTGCTTACAGGTTCTTTAGAATATCAATATCAAGTTTATCCGAAATGGTGGGTGGC
3S AACTTTTGCAGATAGTGGATTAGCCGCTAATATTACACAGCAAAAGAGCTGCGTTATGGCGCAGGCGT
TGGTGTGCGTTGGGCATCGCCAGTGGGCGCGATTAAATTTGATATTGCCACACCCATTCGCGATAAAG
ATAACAGCAAAAATATTCAATTTTACATTGGACTTGGTACAGAAATTTAAGGTGAATTTA
SEQ ID NO:10
GTA AAA CGA GGG CCA GT
40 SEQ ID NO:11
CAG GAA ACA GGT ATG AC
SEQ ID N0:12
8'_'
CA 02426413 2003-04-08
WO 02/32946 PCT/EPO1/11983
ATG ACA AAA CAA CTA CAA CCA TC
SEQ ID N0:13
ATT GAA ATT TAT AAA GTA AAT CAA ACA
SEQ ID N0:14
TC ATG AGA AAA CAA CTA CAA GC
SEQ ID NO:15
AGA TCT AAA TTC AAA TTT ATA AAGTAAATC
SEQ ID N0:16
G GGG TTT GAA GAC AAT GTT CC
SEQ ID N0:17
CAC GCG GTT TTG GAG GCTC
SEQ ID N0:18
CAG TTC TTG TTT GCT CTT GTG
SEQ ID N0:19
GCC AGTCAG CGA AGA TGA AGT G
$3
