Note: Descriptions are shown in the official language in which they were submitted.
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Novel Compounds
FIELD OF THE INVENTION
This invention relates to polynucleotides, (herein referred to as "BASB208
polynucleotide(s)"), polypeptides encoded by them (referred to herein as
"BASB208" or
"BASB208 polypeptide(s)"), recombinant materials and methods for their
production. In
another aspect, the invention relates to methods for using such polypeptides
and
polynucleotides, including vaccines against bacterial infections. In a further
aspect, the
invention relates to diagnostic assays for detecting infection of certain
pathogens.
BACKGROUND OF THE INVENTION
Haemophilus iufluenzae is a non-motile Gram negative bacterium. Man is its
only
natural host.
H. influenzae isolates are usually classified according to their
polysaccharide capsule.
Six different capsular types designated a through f have been identified.
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. infl'uenzae type b is clearly different from the other types in that it
is a major
cause of bacterial meningitis and systemic diseases. Non typeable H.
influenzae (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
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otitis before reaching the age of 3 (1). Left untreated, or becoming 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 pneumohiae, NTHi and M. catarrhalis. These are present in
60 to
90 % of cases. A review of recent studies shows that S. pneumoniae and NTHi
each
represent about 30 %, and M. catar~halis about 15 % of otitis media cases (2).
Other
bacteria can be isolated from the middle ear (H, influenzae 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 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. influenzae 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.
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Among them, two surface exposed high-molecular-weight proteins designated HMWl
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 HMWI/I~VIW2 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 IgAl 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.i~fluenzae 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).
PS 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.
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In line with the observations made with gonococci and meningococci, NTHi
expresses a
dual human transfernn receptor composed of TbpA and TbpB when grown under iron
limitation. Anti-TbpB protected infant rats. (32). Hemoglobin / haptogloliin
receptors
have also been described for NTHi (33). A receptor for Haem: Hemopexin has
also been
identified (34). A lactoferrin 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 IgAl-protease activity (39). IgAl-
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 HtrA 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.Infect.Dis. 165:1148
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WO 02/32944 PCT/EPO1/11557
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. 8:554
12. Kar, S. et al. (1990) Infect. Immun. 58:903
5 13. Gildorf, JR. et al. (1992) Infect. Immun. 60:374
14. St. Genre, JW et al. (1991) Infect. I~nmun. 59:3366
15. St. Genre, JW et al. (1993) Infect. Immun. 61: 2233
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. Genie, 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 al. (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
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WO 02/32944 PCT/EPO1/11557
38. Kimura, A. et al. (1985) Infect. Immun. 47:253
39. Mulles, MH. et Shoberg, RJ (1994) Meth. Enzymol. 235:543
40. Lomholt, H. Alphen, Lv, Kilian, M. (1993) Infect. Immun. 61:4575
41. I~yd, J.M. and Cripps, A.W. (1998) Infect. Immun. 66:2272
42. Loosmore, S.M. et al. (1998) 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.
SL1MNMY OF THE INVENTION
The present invention relates to BASB208, in particular BASB208 polypeptides
and
BASB208 polynucleotides, recombinant materials and methods for their
production. In
another aspect, the invention relates to methods for using such polypeptides
and
polynucleotides, including prevention and treatment of microbial diseases,
amongst others.
In a further aspect, the invention relates to diagnostic assays for detecting
diseases
associated with microbial infections and conditions associated with such
infections, such
as assays for detecting expression or activity of BASB208 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 BASB208 polypeptides and polynucleotides as described
in
greater detail below. In particular, the invention relates to polypeptides and
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7
polynucleotides of BASB208 of non typeable H. influe~zae. The BASB208
polypeptide
has some characteristics of an integral outer membrane protein, and could thus
be exposed
at the surface of the bacterium. The BASB208 polypeptide has a signal sequence
located
from residue 1 to the residue 19.
The invention relates especially to BASB208 polynucleotides and encoded
polypeptides
from differents strains as listed in table, A. Those polynucleotides and
encoded
polypeptides have the nucleotide and amino acid sequences set out in SEQ ID
NO:1 to
SEQ ID NO:10 as described in table A.
Table A
nucleotidic peptidic Strain Isolatedfrom
sequence sequence in
SEQ ID NO:1 SEQ ID N0:2 3224A / USA Otitis media
ATCC
PTA-1816
SEQ ID N0:3 SEQ ID N0:4 3224A USA Otitis media
SEQ ID NO:S SEQ ID N0:6 810956 NL Meningitidis
SEQ ID NO:7 SEQ ID N0:8 27W11679 DK Cystic Fibrosis
SEQ ID N0:9 SEQ ID NO:10A840164 NL Carrier strain
It is understood that sequences recited in the Sequence Listing below as "DNA"
represent
an exemplification of one embodiment of the invention, since those of ordinary
skill will
recognize that such sequences can be usefully employed in polynucleotides in
general,
including ribopolynucleotides.
The sequences of the BASB208 polynucleotides are set out in SEQ ID NO:1, 3, 5,
7, 9.
SEQ Group 1 refers herein to any one of the polynucleotides set out in SEQ ID
NO: l, 3,
5,7or9.
The sequences of the BASB208 encoded polypeptides are set out in SEQ ID N0:2,
4, 6, 8,
10. SEQ Group 2 refers herein to any one of the encoded polypeptides set out
in SEQ ID
N0:2a, Zb, 4, 6, 8 or 10.
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Polypeptides
In one aspect of the invention there are provided polypeptides of non typeable
H. influehzae
referred to herein as "BASB208" and "BASB208 polypeptides" as well as
biologically,
diagnostically, prophylactically, clinically or therapeutically useful
variants thereof, and
compositions comprising the same.
The present invention further provides for:
(a) an isolated polypeptide which comprises an amino acid sequence which has
at least
85% identity, 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.
(d) any of the (a), (b) or (c) polypeptides, with a N terminal end of the
general formula
MKLE(ASKQ)x, where ( )x means x tandem repeats of what is inside the
parenthesis,
which will arise from the translation of any polynucleotide of SEQ Group:l,
where a
variable, but multiple of 3, number of the tetranucleotide repeat AAGC would
be present,
starting after position 10 in any sequence of SEQ Group 1.
The BASB208 polypeptides provided in SEQ Group 2-are the BASB208 polypeptides
from non typeable H. influenzae strains as listed in table A.
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9
The invention also provides an immunogenic fragment of a BASB208 polypeptide,
that
is, a contiguous portion of the BASB208 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
BASB208 polypeptide. Such an immu~ogenic fragment may include, for example,
the
BASB208 polypeptide lacking an N-terminal leader sequence, and/or a
transmembrane
domain and/or a C-terminal anchor domain. In a preferred aspect the
immunogenic
fragment of BASB208 according to the invention comprises substantially all of
the
extracellular domain of a polypeptide which has at least 85% identity,
preferably at least
90% identity, more preferably at least 95% identity, most preferably at least
97-99%
identity, to that 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
BASB208 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
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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
5 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
(for instance the mature polypeptide lacking the signal sequence of residues 1-
19).
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 amino acid sequence
which
contains secretory or leader sequences, pro-sequences, sequences which aid in
purification such as multiple histidine residues, or an additional sequence
for stability
during recombinant production. Furthermore, addition of exogenous polypeptide
or
lipid tail or polynucleotide sequences to increase the immunogenic potential
of the final
molecule is also considered.
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11
Tn 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 IgGl, where fusion takes place at
the hinge
region. In a particular embodiment, the Fc part can be removed simply by
incorporation
of a cleavage sequence which can be cleaved with blood clotting factor Xa.
Furthermore, this invention relates to processes for the preparation of these
fusion
proteins by genetic engineering, and to the use thereof for drug screening,
diagnosis and
therapy. A further aspect of the invention also relates to polynucleotides
encoding such
fusion proteins. Examples of fusion protein technology can be found in
International
Patent Application Nos. W094/29458 and W094/22914.
The proteins may be chemically conjugated, or expressed as recombinant fusion
proteins allowing increased levels to be produced in an expression system as
compared
to non-fused protein. The fusion partner may assist in providing T helper
epitopes
(immunological fusion partner), preferably T helper epitopes recognised by
humans, or
assist in expressing the protein (expression enhancer) at higher yields than
the native
recombinant protein. Preferably the fusion partner will be both an
immunological
fusion partner and expression enhancing partner.
Fusion partners include protein D from Haemophilus influenzae and the non-
structural
protein from influenza virus, NS 1 (hemagglutinin). Another fusion partner is
the protein
known as Omp26 (WO 97/01638). Another fusion partner is the protein known as
LytA. Preferably the C terminal portion of the molecule is used. LytA is
derived from
Streptococcus pneumoniae which synthesize an N=acetyl-L-alanine amidase,
amidase
LytA, (coded by the lytA gene {Gene, 43 (1986) page 265-272}) an autolysin
that
specifically degrades certain bonds in the peptidoglycan backbone. The C-
terminal
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12
domain of the LytA protein is responsible for the affinity to the choline or
to some
choline analogues such as DEAF. This property has been exploited for the
development
of E. colt 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
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13
It is an object of the invention to provide polynucleotides that encode
BASB208
polypeptides; particularly polynucleotides that encode the polypeptides herein
designated
BASB208.
In a particularly preferred embodiment of the invention the polynucleotides
comprise a
region encoding BASB208 polypeptides comprising sequences set out in SEQ Group
1
which include full length gene, or a variant thereof.
The BASB208 polynucleotides provided in SEQ Group 1 are the BASB208
I O polypeptides from non typeable H. influe~zzae strains as listed in table
A.
As a further aspect of the invention there are provided isolated nucleic acid
molecules
encoding and/or expressing BASB208 polypeptides and polynucleotides,
particularly
non typeable H. influehzae BASB208 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 BASB208 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
BASB208
polypeptide from non typeable H. influe~zae 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 BASB208 polypeptides may
be
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14
obtained using standard cloning and screening methods, such as those for
cloning and
sequencing chromosomal DNA fragments from bacteria using non typeable H.
iuflueuzae
strain3224A cells as starting material,~followed by obtaining a full length
clone. For
example, to obtain a polynucleotide sequence of the invention, such as a
polynucleotide
sequence given in SEQ Group 1, typically a library of clones of chromosomal
DNA of
non typeable H. in, fluenzae 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 LABORATORYMANUAL, 2nd Ed.; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). (see in
particular
Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA
Templates 13.70). Direct genomic DNA sequencing may also be performed to
obtain a
full length gene sequence. Illustrative of the invention, each polynucleotide
set out in SEQ
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.
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Table B
nucleotidic encoded peptidicStart codori1 st nucleotide
sequence sequence of stop codon
SEQ ID NO:1 SEQ ID N0:2 1 529
SEQ ID N0:3 SEQ ID N0:4 1 526*
SEQ ID NO:S SEQ ID N0:6 1 529
SEQ ID N0:7 SEQ ID N0:8 1 529
SEQ ID N0:9 SEQ ID NO:10 1 529
* first nucleotide of last coding codon of partial gene.
5 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
10 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
15 2.
A polynucleotide encoding a polypeptide of the present invention, including
homologs and
orthologs from species other than non typeable H. i~flue~zae, may be obtained
by a process
which comprises the steps of screening an appropriate library under stringent
hybridization
conditions (for example, using a temperature in the range of 45 - 65°C
and an SDS
concentration from 0.1-1%) with a labeled or detectable probe consisting of or
comprising
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16
any sequence selected from SEQ Group 1 or a fragment thereof; and isolating a
full-length
gene and/or genomic clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire
length to a coding
sequence (open reading frame) 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 with another
coding
sequence, such as a sequence encoding a leader or secretory sequence, a pre-,
or pro- or
prepro-protein sequence. The polynucleotide of the invention may also contain
at least one
non-coding sequence, including for example, but not limited to at least one
non-coding 5'
and 3' sequence, such as the transcribed but non-translated sequences,
termination signals
(such as rho-dependent and rho-independent termination signals), ribosome
binding sites,
Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation
signals.
The polynucleotide sequence may also comprise additional coding sequence
encoding
additional amino acids. For example, a marker sequence that facilitates
purification of the
fused polypeptide can be encoded. In certain embodiments of the invention, the
marker
sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen,
Inc.) and
described in Gentz et al., Proc. Natl. Acad. Sci., LISA 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 BASB208 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.
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Table C
nucleotidic encoded peptidicStart codon last nucleotide
sequence sequence of encoding
sequence
SEQ ID NO:1 SEQ ID N0:2 1 528
SEQ ID N0:3 SEQ ID N0:4 1 528*
SEQ ID NO:S SEQ ID N0:6 1 528
SEQ ID N0:7 SEQ ID N0:8 1 528
SEQ ID N0:9 SEQ ID NO:10 1 528
* last coding nucleotide of partial gene
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. in, flue~czae BASB208 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,
polynucleotides
interrupted by integrated phage, an integrated insertion sequence, an
integrated vector
sequence, an integrated transposon sequence, or due to RNA editing or genomic
DNA
reorganization) together with additional regions, that also may contain coding
andlor 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.
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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
BASB208
variants, that have the amino acid sequence of BASB208 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 BASB208 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at
least 85%
identical over their entire length to a polynucleotide encoding BASB208
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 highly
preferred are polynucleotides that comprise a region that is at least 90%
identical over its
entire length to a polynucleotide encoding BASB208 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.
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In accordance with certain preferred embodiments of this invention there are
provided
polynucleotides that hybridize, particularly under stringent conditions, to
BASB20~
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% formamide, Sx
SSC (150mM NaCI, lSmM trisodium citrate), 50 mM sodium phosphate (pH7.6), Sx
Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured,
sheared
salmon sperm DNA, followed by washing the hybridization support in O.lx SSC at
about
65°C. Hybridization and wash conditions axe well known and exemplified
in Sambrook,
et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor,
N.Y., (199), particularly Chapter 11 therein. Solution hybridization may also
be used
with the polynucleotide sequences provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a
polynucleotide
sequence obtained by screening an appropriate library containing the complete
gene for a
polynucleotide sequence set forth in 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
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RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones
encoding
BASB208 and to isolate cDNA and genomic clones of other genes that have a high
identity,
particularly high sequence identity, to the BASB208 gene. Such probes
generally will
comprise at least 1 S nucleotide residues or base pairs. Preferably, such
probes will have at
S least 30 nucleotide residues or base pairs and may have at least SO
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 BASB208 gene may be isolated by screening using a DNA
sequence
10 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.
1 S There are several methods available and well known to those skilled in the
art to obtain
full-length DNAs, or extend short DNAs, for example those based on the method
of
Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al.,
PNAS
USA ~5: 8998-9002, 1988). Recent modifications of the technique, exemplified
by the
MarathonTM technology (Clontech Laboratories Inc.) for example, have
significantly
20 simplified the search for longer cDNAs. In the MarathonTM technology, cDNAs
have
been prepared from mRNA extracted from a chosen tissue and an'adaptor'
sequence
ligated onto each end. Nucleic acid amplification (PCR) is then carned out to
amplify the
"missing" S' 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
2S 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 S' 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
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21
separate full-length PCR using the new sequence information for the design of
the 5'
pruner.
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 or may
facilitate manipulation of a protein for assay or production, among other
things. As
generally is the case in vivo, the additional amino acids may be processed
away from the
mature protein by cellular enzymes.
For each and every polynucleotide ofthe 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
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such inactive precursors generally are activated. Some or all of the
prosequences may be
removed before activation. Generally, such precursors are called proproteins.
In addition to the standard A, G, C, T/LJ 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.
In accordance with an aspect of the invention, there is provided the use of a
polynucleotide of the invention for therapeutic or prophylactic purposes, in
particular
genetic immunization.
The use of a polynucleotide of the invention in genetic immunization will
preferably
employ a suitable delivery method such as direct injection of plasmid DNA into
muscles
(Wolff et al., Hum Mol Genet (1992) 1: 363, Manthorpe et al., Hum. Gene Ther.
(1983) 4:
419), delivery of DNA complexed with specific protein carriers (Wu et al.,
JBiol Chem.
(1989) 264: 16985), coprecipitation of DNA with calcium phosphate (Benvenisty
&
Reshef, PNAS USA, (1986) 83: 9551), encapsulation of DNA in various forms of
liposomes (Kaneda et al., Science (1989) 243: 375), particle bombardment (Tang
et al.,
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Nature (1992) 356:152, Eisenbraun et al., DNA Cell Biol (1993) 12: 791) and zn
vivo
infection using cloned retroviral vectors (Seeger et al., PNAS USA (1984) 81:
5849).
Yectors, Host Cells, Expression Systems
The invention also relates to vectors that comprise a polynucleotide or
polynucleotides of
the invention, host cells that are genetically engineered with vectors of the
invention and the
production of polypeptides of the invention by recombinant techniques. Cell-
free
translation systems can also be employed to produce such proteins using RNAs
derived
from the DNA constructs of the invention.
Recombinant polypeptides of the present invention may be prepared by processes
well
known in those skilled in the art from genetically engineered host cells
comprising
expression systems. Accordingly, in a further aspect, the present invention
relates to
expression systems that comprise a polynucleotide or polynucleotides of the
present
invention, to host cells which are genetically engineered with such expression
systems, and
to the production of polypeptides of the invention by recombinant techniques.
For recombinant production of the polypeptides of the invention, host cells
can be
genetically engineered to incorporate expression systems or portions thereof
or
polynucleotides of the invention. Introduction of a polynucleotide into the
host cell can be
effected by methods described in many standard laboratory manuals, such as
Davis, et al.,
BASIC METHODSINMOLECULAR BIOLOGY, (1986) and Sambrook, et al.,
MOLECULAR CLONING: A LABORATORYMANUAL, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate
transfection, DEAF-dextran mediated transfection, transvection,
microinjection, cationic
lipid-mediated transfection, electroporation, conjugation, transduction,
scrape loading,
ballistic introduction and infection.
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Representative examples of appropriate hosts include bacterial cells, such as
cells of
streptococci,-staphylococci, enterococci, E. coli, streptomyces,
cyanobacteria, Bacillus
subtilis, Neisseria mer~ingitidis, Haemophilus influenzae and Moraxella
catarrhalis; fungal
cells, such as cells of a yeast, Kluveromyces, Saccharomyces, Pichia, a
basidiomycete,
Candida albicarcs and Aspergillus; insect cells such as cells of Drosophila S2
and
Spodoptera Sue; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-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 techniques, such as, for example, those
set forth in
Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, (supra).
In recombinant expression systems in eukaryotes, for secretion of a translated
protein into
the lumen of the endoplasmic reticulum, into the periplasmic space or into the
extracellular
environment, appropriate secretion signals may be incorporated into the
expressed
polypeptide. These signals may be endogenous to the polypeptide or they may be
--
heterologous signals.
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Polypeptides of the present invention can be recovered and purified from
recombinant
cell cultures by well-known methods including ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange chromatography,
phosphocellulose
chromatography, hydrophobic interaction chromatography, affinity
chromatography,
5 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.
10 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),
15 alphaviruses (Sindbis virus, Semliki Forest Virus, Venezuelian Equine
Encephalitis
Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus,
rhinovirus),
herpesviruses (varicella zoster virus, etc), Listeria, Salmonella , Shigella,
BCG,
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.
Diagnostic, Prognostic, Serotyping and Mutation Assays
This invention is also related to the use of BASB208 polynucleotides and
polypeptides of
the invention for use as diagnostic reagents. Detection of BASB208
polynucleotides and/or
polypeptides in a eukaryote, particularly a mammal, and especially a human,
will provide a
diagnostic method for diagnosis of disease, staging of disease or response of
an infectious
organism to drugs. Eukaryotes, particularly mammals, and especially humans,
particularly
those infected or suspected to be infected with an organism comprising the
BASB208 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.
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Polypeptides-and polynucleotides for prognosis, diagnosis or other analysis
may be obtained
from a putatively infected andlor infected individual's bodily materials.
Polynucleotides
from any of these sources, particularly DNA or RNA, may be used directly for
detection or
may be amplified enzymatically by using PCR or any other amplification
technique prior to
analysis. RNA, particularly mRNA, cDNA and genomic DNA may also be used in the
same ways. Using amplification, characterization of the species and strain of
infectious or
resident organism present in an individual, may be made by an analysis of the
genotype of a
selected polynucleotide of the organism. Deletions and insertions can be
detected by a
change in size of the amplified product in comparison to a genotype of a
reference sequence
selected from a related organism, preferably a different species of the same
genus or a
different strain of the same species. Point mutations can be identified by
hybridizing
amplified DNA to labeled BASB208 polynucleotide sequences. Perfectly or
significantly
matched sequences can be distinguished from imperfectly or more significantly
mismatched
duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by
detecting
differences in melting temperatures or renaturation kinetics. Polynucleotide
sequence
differences may also be detected by alterations in the electrophoretic
mobility of
polynucleotide fragments in gels as compared to a reference sequence. This may
be carried
out with or without denaturing agents. Polynucleotide differences may also be
detected by
direct DNA or RNA sequencing. See, for example, Myers et al., Science, 230:
1242 (1985).
Sequence changes at specific locations also may be revealed by nuclease
protection assays,
such as RNase, V l and S 1 protection assay or a chemical cleavage method.
See, for
example, Cotton et al., Proc. Natl. Acad. Sci., USA, ~5: 4397-4401 (1985).
In another embodiment, an array of oligonucleotides probes comprising BASB208
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
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27
address a variety of questions in molecular genetics including gene
expression, genetic
linkage, and genetic variability (see, for example, Chee et al., Science, 274:
610 (1996)).
Thus in another aspect, the present invention relates to a diagnostic kit
which comprises:
(a) a polynucleotide of the present invention, preferably 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
susceptibility to a disease, which results from under-expression, over-
expression or altered
expression of the polynucleotide. Organisms, particularly infectious
organisms, carrying
mutations in such polynucleotide may be detected at the polynucleotide level
by a variety of
techniques, such as those described elsewhere herein.
Cells from an organism carrying mutations or polymorphisms (allelic
variations) in a
polynucleotide and/or polypeptide of the invention may also be detected at the
polynucleotide or polypeptide level by a variety of techniques, to allow for
serotyping, for
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28
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
BASB208 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
BASB208 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 andlor classify the infectious agent.
The invention further provides a process for diagnosing, disease, preferably
bacterial
infections, more preferably infections caused by non typeable H. in, fluenzae,
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 BASB208 polynucleotide
can be
measured using any on of the methods well known in the art for the
quantitation of
polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase
protection,
Northern blotting, spectrometry and other hybridization methods.
In addition, a diagnostic assay in accordance with the invention for detecting
over-
expression of BASB208 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 BASB208 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
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29
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 fox probing, such as using
hybridization
or nucleic acid amplif ration, 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. ir~fluenzae, 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,
particularly
peptide mimotopes, of epitopes within the polypeptide sequence may also be
used as
immunogens to produce antibodies immunospecific for the polypeptide of the
invention.
The term "immunospecific" means that the antibodies have substantially greater
affinity for
the polypeptides of the invention than their affinity for other related
polypeptides in the
prior art.
In certain preferred embodiments of the invention there are provided
antibodies against
BASB208 polypeptides or polynucleotides.
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Antibodies generated against the polypeptides or polynucleotides of the
invention can be
obtained by administering the polypeptides and/or polynucleotides of the
invention, or
epitope-bearing fragments of either or both, analogues of either or both, or
cells expressing
either or both, to an animal, preferably a nonhuman, using routine protocols.
For
5 preparation of monoclonal antibodies, any technique known in the art that
provides
antibodies produced by continuous cell line cultures can be used. Examples
include various
techniques, such as those in Kohler, G. and Milstein, C., Nature 256: 495-497
(1975);
Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in
MONOCLONAL
ANTIBODIESAND CANCER 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-
BASB208 or
from naive libraries (McCafferty, et aL, (1990), Nature 348, 552-554; Marks,
et al.,
(1992) Biotechnology 10, 779-783). The affinity of these antibodies can also
be improved
by, for example, chain shuffling (Clackson et al., (1991) Nature 352: 628).
The above-described antibodies may be employed to isolate or to identify
clones expressing
the polypeptides or polynucleotides of the invention to purify the
polypeptides or
polynucleotides by, for example, affinity chromatography.
Thus, among others, antibodies against BASB208 polypeptide or BASB208
polynucleotide
may be employed to treat infections, particularly bacterial infections.
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31
Polypeptide variants include antigenically, epitopically or immunologically
equivalent
variants form a particular aspect of this invention.
Preferably, the antibody or variant thereof is modified to make it less
immunogenic in the
individual. For example, if the individual is human the antibody may most
preferably be
"humanized," where the complimentarity determining region or regions of the
hybridoma-
derived antibody has been transplanted into a human monoclonal antibody, for
example
as described in Jones et al. (1986), Nature 321, 522-525 or Tempest et al.,
(1991)
Biotechnology 9, 266-273.
Antagonists and Agonists - Assays and Molecules
Polypeptides and polynucleotides of the invention may also be used to assess
the binding of
small molecule substrates and ligands in, for example, cells, cell-free
preparations, chemical
libraries, and natural product mixtures. These substrates and ligands may be
natural
substrates and ligands or may be structural or functional mimetics. See, e.g.,
Coligan et al.,
Current Protocols in Immunology I (2): Chapter 5 (1991).
The screening methods may simply measure the binding of a candidate compound
to the
polypeptide or polynucleotide, or to cells or membranes bearing the
polypeptide or
polynucleotide, or a fusion protein of the polypeptide by means of a label
directly or
indirectly associated with the candidate compound. Alternatively, the
screening method
may involve competition with a labeled competitor. Further, these screening
methods
may test whether the candidate compound results in a signal generated by
activation or
inhibition of the polypeptide or polynucleotide, using detection systems
appropriate to the
cells comprising the polypeptide or polynucleotide: Inhibitors of activation
are generally
assayed in the presence of a known agonist and the effect on activation by the
agonist by
the presence of the candidate compound is observed. Constitutively active
polypeptide
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32
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 BASB208 polypeptide and/or polynucleotide activity in the mixture,
and
comparing the BASB208 polypeptide and/or polynucleotide activity of the
mixture to a
standard. Fusion proteins, such as those made from Fc portion and BASB208
polypeptide, as hereinbefore described, can also be used for high-throughput
screening
assays to identify antagonists of the polypeptide of the present invention, as
well as of
phylogenetically and and/or functionally related polypeptides (see D. Bennett
et al., J Mol
Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16):9459-
9471
(1995)).
The polynucleotides, polypeptides and antibodies that bind to and/or interact
with a
polypeptide of the present invention may also be used to configure screening
methods for
detecting the effect of added compounds on the production of mRNA and/or
polypeptide
in cells. For example, an ELISA assay may be constructed for measuring
secreted or cell
associated levels of polypeptide using monoclonal and polyclonal antibodies by
standard
methods known in the art. This can be used to discover agents which may
inhibit or
enhance the production of polypeptide (also called antagonist or agonist,
respectively)
from suitably manipulated cells or tissues.
The invention also provides a method of screening compounds to identify those
which
enhance (agonist) or block (antagonist) the action of BASB208 polypeptides or
polynucleotides, particularly those compounds that are bacteriostatic and/or
bactericidal.
The method of screening may involve high-throughput techniques. For example,
to screen
for agonists or antagonists, a synthetic reaction mix, a cellular compartment,
such as a
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33
membrane, cell envelope or cell wall, or a preparation of any thereof,
comprising BASB208
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 BASB208 agonist or
antagonist. The
ability of the candidate molecule to agonize or antagonize the BASB208
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
BASB208 polypeptide are most likely to be good antagonists. Molecules that
bind well
and, as the case may be, increase the rate of product production from
substrate, increase
signal transduction, or increase chemical channel activity are agonists.
Detection of the rate
or level of, as the case may be, production of product from substrate, signal
transduction, or
chemical channel activity may be enhanced by using a reporter system. Reporter
systems
that may be useful in this regard include but are not limited to colorimetric,
labeled substrate
converted into product, a reporter gene that is responsive to changes in
BASB208
polynucleotide or polypeptide activity, and binding assays known in the art.
Another example of an assay for BASB208 agonists is a competitive assay that
combines
BASB208 and a potential agonist with BASB208 binding molecules, recombinant
BASB208 binding molecules, natural substrates or ligands, or substrate or
ligand mimetics,
under appropriate conditions for a competitive inhibition assay. BASB208 can
be labeled,
such as by radioactivity or a colorimetric compound, such that the number of
BASB208
molecules bound to a binding molecule or converted to product can be
determined
accurately to assess the effectiveness of the potential antagonist.
Potential antagonists include, among others, small organic molecules,
peptides,
polypeptides and antibodies that bind to a polynucleotide and/or polypeptide
of the
invention and thereby inhibit or extinguish its activity or expression.
Potential antagonists
also may be small organic molecules, a peptide, a polypeptide such as a
closely related
protein or antibody that binds the same sites on a binding molecule, such as a
binding
molecule, without inducing BASB208 induced activities, thereby preventing the
action or
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34
expression of BASB208 polypeptides and/or polynucleotides by excluding BASB208
polypeptides and/or polynucleotides from binding.
Potential antagonists include a small molecule that binds to and occupies the
binding site of
the polypeptide thereby preventing binding to cellular binding molecules, such
that normal
biological activity is prevented. Examples of small molecules include but are
not limited to
small organic molecules, peptides or peptide-like molecules. Other potential
antagonists
include antisense molecules (see Okano, J. Neurochem. 56: 560 (1991);
OLIGODEO~'YNUCLEOTIDES AS ANTISENSE INHIBI'T'ORS OF GENE EXPRESSION,
IO CRC Press, Boca Raton, FL (1988), for a description ofthese molecules).
Preferred
potential antagonists include compounds related to and variants of BASB208.
In a further aspect, the present invention relates to genetically engineered
soluble fusion
proteins comprising a polypeptide of the present invention, or a fragment
thereof, and
various portions of the constant regions of heavy or light chains of
immunoglobulins of
various subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the
constant
part of the heavy chain of human IgG, particularly IgGl, where fusion takes
place at the
hinge region. In a particular embodiment, the Fc part can be removed simply by
incorporation of a cleavage sequence which can be cleaved with blood clotting
factor Xa.
Furthermore, this invention relates to processes for the preparation of these
fusion
proteins by genetic engineering, and to the use thereof for drug screening,
diagnosis and
therapy. A further aspect of the invention also relates to polynucleotides
encoding such
fusion proteins. Examples of fusion piotein technology can be found in
International
Patent Application Nos. WO94/29458 and W094/229.14.
Each of the polynucleotide sequences provided herein may be used in the
discovery and
development of antibacterial compounds: The encoded protein, upon expression,
can be
used as a target for the screening of antibacterial drugs. Additionally, the
polynucleotide
sequences encoding the amino terminal regions of the encoded protein or Shine-
Delgarno
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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
5 antagonist of the invention to interfere with the initial physical
interaction between a
pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible
for
sequelae of infection. In particular, the molecules of the invention may be
used: in the
prevention of adhesion of bacteria, in particular gram positive and/or gram
negative
bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins
on in-
I O dwelling devices or to extracellular matrix proteins in wounds; to block
bacterial adhesion
between eukaryotic, preferably mammalian, extracellular matrix proteins and
bacterial
BASB208 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
BASB208
agonists and antagonists, preferably bacteristatic or bactericidal agonists
and antagonists.
The antagonists and agonists of the invention may be employed, for instance,
to prevent,
inhibit and/or treat diseases.
In a further aspect, the present invention relates to mimotopes of the
polypeptide of the
invention. A mimotope is a peptide sequence, sufficiently similar to the
native peptide
(sequentially or structurally), which is capable of being recognised by
antibodies
which recognise the native peptide; or is capable of raising antibodies which
recognise the native peptide when coupled to a suitable ,carrier.
Peptide mimotopes may be designed for a particular purpose by addition,
deletion or
substitution of elected amino acids. Thus, the peptides may be modified for
the
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36
purposes of ease of conjugation to a protein carrier. Fox example, it may be
desirable
for some chemical conjugation methods to include a terminal cysteine. In
addition it
may be desirable for peptides conjugated to a protein carrier to include a
hydrophobic
terminus distal from the conjugated terminus of the peptide, such that the
free
unconjugated end of the peptide remains associated with the surface of the
carrier
protein. Thereby presenting the peptide in a conformation which most closely
resembles that of the peptide as found in the context of the whole native
molecule.
For example, the peptides may be altered to have an N-terminal cysteine and a
C-
terminal hydrophobic amidated tail. Alternatively, the addition or
substitution of a D-
stereoisomer form of one or more of the amino acids (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
95/24916 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 BASB208 polynucleotide and/or polypeptide, or
a
fragment or variant thereof, adequate to produce antibody and/ or T cell
immune response
to protect said individual from infection, particularly bacterial infection
and most
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37
particularly non typeable H. influeyczae 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 BASB208 polynucleotide and/or polypeptide, or a fragment
or a
variant thereof, for expressing BASB208 polynucleotide and/or polypeptide, or
a
fragment or a variant thereof in vivo in order to induce an immunological
response, such
as, to produce antibody and/ or T cell immune response, including, for
example, cytokine-
producing T cells or cytotoxic T cells, to protect said individual, preferably
a human,
from disease, whether that disease is already established within the
individual or not. One
example of administering the gene is by accelerating it into the desired cells
as a coating
on particles or otherwise. Such nucleic acid vector may comprise DNA, RNA, a
ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or
an
RNA-protein complex.
A further aspect of the invention relates to an immunological composition that
when
introduced into an individual, preferably a human, capable of having induced
within it an
immunological response, induces an immunological response in such individual
to a
BASB208 polynucleotide and/or polypeptide encoded therefrom, wherein the
composition
comprises a recombinant BASB208 polynucleotide and/or polypeptide encoded
therefrom
and/or comprises DNA and/or RNA which encodes and expresses an antigen of said
BASB208 polynucleotide, polypeptide encoded therefrom, or other polypeptide of
the
invention. The immunological response may be used therapeuticahy 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.
BASB208 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
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38
antigenic and/or immunogenic properties, and preferably protective properties.
Thus
fused recombinant protein, preferably further comprises an antigenic co-
protein, such as
lipoprotein D from Haemophilus iufluenzae, Glutathione-S-transferase (GST) or
beta-
galactosidase, or any other relatively large co-protein which solubilizes the
protein and
facilitates production and purification thereof. Moreover, the co-protein may
act as an
adjuvant in the sense of providing a generalized stimulation of the immune
system of the
organism receiving the protein. The co-protein may be attached to either the
amino- or
carboxy-terminus of the first protein.
In a vaccine composition according to the invention, a BASB208 polypeptide
and/or
polynucleotide, or a fragment, or a mimotope, or a variant thereof may be
present in a
vector, such as the live recombinant vectors described above for example live
bacterial
vectors.
Also suitable are non-live vectors for the BASB208 polypeptide, for example
bacterial
outer-membrane vesicles or "blebs". OM blebs are derived from the outer
membrane of
the two-layer membrane of Gram-negative bacteria and have been documented in
many
Gram-negative bacteria (Zhou, L et al. 1998. FEMS Microbiol. Lett. 163:223-
228)
including C. trachomatis and C. psittaci. A non-exhaustive list of bacterial
pathogens
reported to produce blebs also includes: Bordetella pertussis, Bo~relia
bu~gdorfe~-i,
Brucella melitensis, Brucella ovis, Esherichia coli, Haemophilus influeuzae,
Legio~ella
pneumophila, Moraxella catarrhalis, Neisse~ia gonorrhoeae, Neisseria
meningitidis,
Pseudomonas ae~ugiuosa 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 BASB208 polypeptide,
can be
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39
introduced or upregulated (e.g. by altering the promoter). Instead or in
addition, the
expression of outer-membrane molecules which are either not relevant (e.g.
unprotective
antigens or immunodominant but variable proteins) or detrimental (e.g. toxic
molecules
such as LPS, or potential inducers of an autoimmune response) can be
downregulated.
These approaches are discussed in more detail below.
The non-coding flanking regions of the BASB20~ 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: 11 is the non typeable Haemophilus i~cfluenzae
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
BASB20~ 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
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WO 02/32944 PCT/EPO1/11557
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
5 modification of sequences involved in gene expression can be carried out in
vivo by
random mutagenesis followed by selection for the desired phenotype. Another
approach
consists in isolating the region of interest and modifying it by random
mutagenesis, or
site-directed replacement, insertion or deletion mutagenesis. The modified
region can then
be reintroduced into the bacterial genome by homologous recombination, and the
effect
10 on gene expression can be assessed. In another approach, the sequence
knowledge of the
region of interest can be used to replace or delete all or part of the natural
regulatory
sequences. In this case, the regulatory region targeted is isolated and
modified so as to
contain the regulatory elements from another gene, a combination of regulatory
elements
from different genes, a synthetic regulatory region, or any other regulatory
region, or to
15 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.
me~ihgitidis or N. go~orroheae; ompCD, copB, lbpB, ompE, UspAl; UspA2; TbpB
from
20 M. Catarrhalis; p1, p2, p4, p5, p6, lpD, tbpB, D15, Hia, Hmwl, Hmw2 from H.
influenzae.
In one example, the expression of the gene can be modulated by exchanging its
promoter
with a stronger promoter (through isolating the upstream sequence of the gene,
in vitro
25 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.
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41
In other examples, the described approaches can be used to generate
recombinant
bacterial strains with improved characteristics for vaccine applications.
These can be, but
are not limited to, attenuated strains, strains with increased expression of
selected
antigens, strains with knock-outs (or decreased expression) of genes
interfering with the
immune response, strains with modulated expression of immunodominant proteins,
strains with modulated shedding of outer-membrane vesicles.
Thus, also provided by the invention is a modified upstream region of the
BASB208
gene, which modified upstream region contains a heterologous regulatory
element which
alters the expression level of the BASB208 protein located at the outer
membrane. The
upstream region according to this aspect of the invention includes the
sequence upstream
of the BASB208 gene. The upstream region starts immediately upstream of the
BASB208
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
BASB208
gene, a process for achieving this through homologous recombination (for
instance as
described in WO 01/09350 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 BASB208 polypeptide, in a modified bacterial
bleb. The
invention fiu ther provides modified host cells capable of producing the non-
live membrane-
based bleb vectors. The invention further provides nucleic acid vectors
comprising the
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42
BASB208 gene having a modif ed 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
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.'
ivcfluehzae.
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. influenzae infection, in mammals,
particularly
humans.
The invention also includes a vaccine formulation which comprises an
immunogenic
recombinant polypeptide and/or polynucleotide of the invention together with a
suitable
carrier, such as a pharmaceutically acceptable carrier. Since the polypeptides
and
polynucleotides may be broken down in the stomach, each is preferably
administered
parenterally, including, for example, administration that is subcutaneous,
intramuscular,
intravenous, or intradermal. Formulations suitable for parenteral
administration include
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43
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 carrier immediately prior to use.
The vaccine formulation of the invention may also include adjuvant systems for
enhancing the immunogenicity of the formulation. Preferably the adjuvant
system
raises preferentially a TH1 type of response.
An immune response may be broadly distinguished into two extreme catagories,
being a
humoral or cell mediated immune responses (traditionally characterised by
antibody and
cellular effector mechanisms of protection respectively). These categories of
response
have been termed TH1-type responses (cell-mediated response), and TH2-type
immune
responses (humoral response).
Extreme TH1-type immune responses may be characterised by the generation of
antigen
specific, haplotype restricted cytotoxic T lymphocytes, and natural killer
cell responses.
In mice TH1-type responses are often characterised by the generation of
antibodies of
the IgG2a subtype, whilst in the human these correspond to IgGl type
antibodies. TH2
type immune responses are characterised by the generation of a broad range of
immunoglobulin isotypes including in mice IgGl, IgA, and IgM.
It can be considered that the driving force behind the development of these
two types of
immune responses are cytokines. High levels of TH1-type cytokines tend to
favour the
induction of cell mediated immune responses to the given antigen, whilst high
levels of
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44
TH2-type cytokines tend to favour the induction of humoral immune responses to
the
antigen.
The distinction of THI 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 marine CD4 +ve T cell clones by
Mosmann and
Coffman (Mosmann, T.R. and Coffman, R.L. (199) THI and TH2'cells: different
patterns of lymphokine secretion lead to different functional properties.
Annual Review
IO oflmmunology, 7, p145-173). Traditionally, TH1-type responses are
associated with
the production of the INF-y and IL-2 cytokines by T-lymphocytes. Other
cytokines
often directly associated with the induction of THl-type immune responses are
not
produced by T-cells, such as IL-12. In contrast, TH2- type responses are
associated with
the secretion of IL-4, IL-5, IL-6 and IL-13.
It is known that certain vaccine adjuvants are~particularly suited to the
stimulation of
either TH1 or TH2 - type cytokine responses. Traditionally the best indicators
of the
TH1:TH2 balance of the immune response after a vaccination or infection
includes
direct measurement of the production of THl or TH2 cytokines by T lymphocytes
in
vitro after restimulation with antigen, and/or the measurement of the IgGl
:IgG2a ratio
of antigen specific antibody responses.
Thus, a TH1-type adjuvant is one which preferentially stimulates isolated T-
cell
populations to produce high levels of THl-type cytokines when re-stimulated
with
antigen in vitro, and promotes development of both CD8+ cytotoxic T
lymphocytes and
antigen specific immunoglobulin responses associated with TH1-type isotype.
Adjuvants which are capable of preferential stimulation of the THl cell
response are
described in International Patent Application No. WO 94/00153 and WO 95/17209.
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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
5 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).
10 3D-MPL will be present in the range of 10~,g -100p,g preferably 25-SOp,g
per dose
wherein the antigen will typically be present in a range 2-SO~.g per dose.
Another preferred adjuvant comprises QS21, an Hplc purified non-toxic fraction
derived from the bark of Quillaja Sapo~caria Molina. Optionally this may be
admixed
15 with 3 De-O-acylated monophosplioryl lipid A (3D-MPL), optionally together
with an
carrier.
The method of production of QS21 is disclosed in US patent No. 5,057,540.
20 Non-reactogenic adjuvant formulations containing QS21 have been described
previously (WO 96/33739). Such formulations comprising QS21 and cholesterol
have
been shown to be successful TH1 stimulating adjuvants when formulated together
with
an antigen.
25 Further adjuvants which are preferential stimulators of THl cell response
include
immunomodulatory oligonucleotides, for example unmethylated CpG sequences as
disclosed in WO 96!02555.
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Combinations of different TH1 stimulating adjuvants, such as those mentioned
hereinabove,~are also contemplated as providing an adjuvant which is a
preferential
stimulator of TH1 cell response. For example, QS21 can be formulated together
with
3D-MPL. The ratio of QS21 : 3D-MPL will typically be in the order of 1 : 10 to
10 : 1;
preferably 1:5 to 5 : 1 and often substantially 1 : 1. The preferred range for
optimal
synergy is 2.5 : 1 to 1 : 1 3D-MPL: QS21.
Preferably a carrier is also present in the vaccine composition according to
the
invention. The carrier may be an oil in water emulsion, or an aluminium salt,
such as
aluminium phosphate or aluminium hydroxide.
A preferred oil-in-water emulsion comprises a metabolisible oil, such as
squalene, alpha
tocopherol and Tween 80. In a particularly preferred aspect the antigens in
the vaccine
composition according to the invention are combined with QS21 and 3D-MPL in
such
an emulsion. Additionally the oil in water emulsion may contain span 85 and/or
lecithin and/or tricaprylin.
Typically for human administration QS21 and 3D-MPL will be present in a
vaccine in
the range of 1 ~,g - 200~,g, such as 10-100~,g, preferably 10~,g - SO~g per
dose.
Typically the oil in water will comprise from 2 to 10% squalene, from 2 to 10%
alpha
tocopherol and from 0.3 to 3% tween 80. Preferably the ratio of squalene:
alpha
tocopherol is equal to or less than 1 as this provides a more stable emulsion.
Span 85
may also be present at a level of 1 %. In some cases it may be advantageous
that the
vaccines of the present invention will further contain a stabiliser.
Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g.
squalane or
squalene, an emulsifier, e.g. Tween 80, in an aqueous carrier. The aqueous
carrier may
be, for example, phosphate buffered saline.
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A particularly potent adjuvant formulation involving QS21, 3D-MPL and
tocopherol
in an oil in water emulsion is described in WO 95/17210.
While the invention has been described with reference to certain BASB208
polypeptides
and polynucleotides, it is to be understood that this covers fragments of the
naturally
occurring polypeptides and polynucleotides, and similar polypeptides and
polynucleotides
with additions, deletions or substitutions which do not substantially affect
the
immunogenic properties of the recombinant polypeptides or polynucleotides.
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 otitis media. Such a polyvalent vaccine composition may
include a TH-1
inducing adjuvant as hereinbefore described.
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) one 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 pneumoniae
infection;
d) one or more fiuther non typeable Haemophilus influehzae 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) and/or c) are preferred. Such
vaccines may be
advantageously used as global otitis media vaccines.
The pneumococcal. capsular polysaccharide antigens are preferably selected
from
serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C,
19A, 19F,
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48
20, 22F, 23F and 33F (most preferably from serotypes l, 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 pneumoniae, 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 pneumo~iae types 1 and
2.",
Mitchell et al. Biochim Biophys Acta 1989 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 99/03884 (NAVA)];
PspA and transmembrane deletion variants thereof (WO 92/14488; WO 99/53940;
LTS
5804193 - Briles et al.); PspC and transmembrane deletion variants thereof (WO
99/53940; WO 97/09994 - Briles et al); PsaA and transmembrane deletion
variants
thereof (Berry & Paton, Infect Immun 1996 Dec;64(12):5255-62 "Sequence
' heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for
virulence of
Streptococcus p~ceumo~ciae"); 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 Microbiol 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 98/18930 and PCT/LTS99/30390.
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Preferred Moraxella catarrhalis protein antigens which can be included in a
combinatiomvaccine (especially for the prevention of otitis media) are: OMP106
[WO
97/41731 (Antex) & WO 96/34960 (PMC)]; OMP21; LbpA &/or LbpB [WO 98155606
(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 (PCT/EP00101468); lipo06 (GB 9917977.2); lipol0 (GB 9918208.1); lipoll
(GB 9918302.2); lipol8 (GB 9918038.2); P6 (PCT/EP99/03038); D15
(PCT/EP99/03822); OmplA1 (PCT/EP99/06781); Hly3 (PCT/EP99/03257); and OmpE.
Preferred further non-typeable Haemophilus iufluenzae 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 [eg LB 1 (f) peptide fusions; US 5843464 (OSU)
or WO
99/64067]; OMP26 [WO 97/01638 (Cortecs)]; P6 [EP 281673 (State University of
New
York)]; protein D (EP 594610); TbpA and/or TbpB; Hia; Hsf; Hin47; Hif; Hmwl;
Hmw2; Hmw3; Hmw4; Hap; D15 (WO 94112641); 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
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In a further aspect of the invention there are provided compositions
comprising a BASB208
polynucleotide and/or a BASB208 polypeptide for administration to a cell or to
a
multicellular organism.
5 The invention also relates to compositions comprising a polynucleotide
and/or a
polypeptides discussed herein or their agonists or antagonists. The
polypeptides and
polynucleotides of the invention may be employed in combination with a non-
sterile or
sterile Garner or Garners for use with cells, tissues or organisms, such as a
pharmaceutical
carrier suitable for administration to an individual. Such compositions
comprise, for
10 instance, a media additive or a therapeutically effective amount of a
polypeptide and/or
polynucleotide of the invention and a pharmaceutically acceptable Garner or
excipient. Such
Garners may include, but are not limited to, saline, buffered saline,
dextrose, water, glycerol,
ethanol and combinations thereof. The formulation should suit the mode of
administration.
The invention further relates to diagnostic and pharmaceutical packs and kits
comprising
15 one or more containers filled with one or more of the ingredients of the
a~oremeritioned
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.
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.
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In a further aspect, the present invention provides for pharmaceutical
compositions
comprising aytherapeutically effective amount of a polypeptide and/or
polynucleotide, such
as the soluble form of a polypeptide and/or polynucleotide of the present
invention, agonist
or antagonist peptide or small molecule compound, in combination with a
pharmaceutically
acceptable Garner or excipient. Such carriers include, but are not limited to,
saline, buffered
saline, dextrose, water, glycerol, ethanol, and combinations thereof. The
invention further
relates to pharmaceutical packs and kits comprising one or more containers
filled with one
or more of the ingredients of the aforementioned compositions of the
invention.
Polypeptides, polynucleotides and other compounds of the present invention may
be
employed alone or in conjunction with other compounds, such as therapeutic
compounds.
The composition will be adapted to the route of administration, for instance
by a systemic or
an oral route. Preferred forms of systemic administration include injection,
typically by
intravenous injection. Other injection routes, such as subcutaneous,
intramuscular, or
intraperitoneal, can be used. Alternative means for systemic administration
include
transmucosal and transdermal administration using penetrants such as bile
salts or fusidic
acids or other detergents. In addition, if a polypeptide or other compounds of
the present
invention can be formulated in an enteric or an encapsulated formulation, oral
administration may also be possible. Administration of these compounds may
also be
topical and/or localized, in the form of salves, pastes, gels, solutions,
powders and the like.
For administration to mammals, and particularly humans, it is expected that
the daily
dosage level of the active agent will be from 0.01 mglkg to 10 mg/kg,
typically around 1
mg/kg. The physician in any event will determine the actual dosage which will
be most
suitable for an individual and will vary with the age, weight and response of
the particular
individual. The above dosages are exemplary of the average case. There can, of
course,
be individual instances where higher or lower dosage ranges are merited, and
such are
within the scope of this invention.
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The dosage range required depends on the choice of peptide, the route of
administration, the
nature of theformulation, the nature of the subject's condition, and the
judgment of the
attending practitioner. Suitable dosages, however, are in the range of O.I-I00
uglkg 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
microgram/kg of antigen, and such dose is preferably administered 1-3 tames
and with an
interval of 1-3 weeks. With the indicated dose range, no adverse toxicological
effects will
I 0 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
I S 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.
20 Sequence Databases, Sequences in a Tangible Medium, and Algorithms
Polynucleotide and polypeptide sequences form a valuable information resource
with which
to determine their 2- and 3-dimensional structures as well as to identify
further sequences of
similar homology. These approaches are most easily facilitated by storing the
sequence in a
computer readable medium and then using the stored data in a known
macromolecular
25 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
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53
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 Ieast 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; arid
comparing said first polypeptide sequence to at least one second
polynucleotide or
polypeptide sequence to identify homology.
All publications and references, including but not limited to patents and
patent
applications, cited in this specification are herein incorporated by reference
in their
entirety as if each individual publication or reference were specifically and
individually
indicated to be incorporated by reference herein as being fully set forth. Any
patent
application to which this application claims priority is also incorporated by
reference
herein in its entirety in the manner described above for publications and
references.
DEFINITIONS
"Identity," as known in the art, is a relationship between two or more
polypeptide sequences
or two or more polynucleotide sequences, as the case may be, as determined by
comparing
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54
the sequences. In the art, "identity" also means the degree of sequence
relatedness between
polypeptide or polynucleotide sequences, as the case may be, as determined by
the match
between strings of such sequences. "Identity" can be readily calculated by
known
methods, including but not limited to those described in (Computational
Molecular
Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988;
Biocomputing:
Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York,
1993;
Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G.,
eds.,
Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von
Heine,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and
Devereux, J.,
eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM
J.
Applied Math., 48: 1073 (1988). Methods to determine identity are designed to
give the
largest match between the sequences tested. Moreover, methods to determine
identity are
codified in publicly available computer programs. Computer program methods to
determine identity between two sequences include, but are not limited to, the
GAP
program in the GCG program package (Devereux, J., et' al., Nucleic Aeids
Research 12(1):
387 (1984)), BLASTP, BLASTN (Altschul, S.F. et al., J. Molec. Biol. 215: 403-
410
(1990), and FASTA( Pearson and Lipman Proc. Natl. Acad. Sci. USA 85; 2444-2448
(1988). The BLAST family of programs is publicly available from NCBI and other
sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894;
Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well known Smith
Waterman
algorithm may also be used to determine identity.
Parameters for polypeptide sequence comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,
Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)
Gap Penalty: 8
Gap Length Penalty: 2
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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).
5 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
10 Available as: The "gap" program from Genetics Computer Group, Madison WI.
These
are the default parameters for nucleic acid comparisons.
A preferred meaning for "identity" for polynucleotides and polypeptides, as
the case may
be, are provided in (1) and (2) below.
(1) Polynucleotide embodiments further include an isolated polynucleotide
comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90,
95, 97 or
100% identity to the reference sequence of SEQ ID NO:l, wherein said
polynucleotide
sequence may be identical to the reference sequence of SEQ ID NO:1 or may
include up
to a certain integer number of nucleotide alterations as compared to the
reference
sequence, wherein said alterations are selected from the group consisting of
at least one
nucleotide deletion, substitution, including transition and transversion, or
insertion, and
wherein said alterations may occur at the 5' or 3' terminal positions of the
reference
nucleotide sequence or anywhere between those terminal positions, interspersed
either
individually among the nucleotides in the reference sequence or in one or more
contiguous groups within the reference sequence, and wherein said number of
nucleotide
alterations is determined by multiplying the total number of nucleotides in
SEQ ID NO:1
by the integer defining the percent identity divided by 100 and then
subtracting that
product from said total number of nucleotides in SEQ ID NO:1, or:
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nn ~ xn ' ~xn' Y)
wherein nn is the number of nucleotide alterations, xn is the total number of
nucleotides
in SEQ ID NO:l, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%,
0.85 for
85%, 0.90 for 90%, 0.95 for 95%, 0.97. for 97% or 1.00 for 100%, and ~ is the
symbol for
the multiplication operator, and wherein any non-integer product of xn and y
is rounded
down to the nearest integer prior to subtracting it from xn. Alterations of
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:1, that is it may be 100% identical,
or it may
include up to a certain integer number of nucleic acid alterations as compared
to the
reference sequence such that the percent identity is less than 100% identity.
Such
alterations are selected from the group consisting of at least one nucleic
acid deletion,
substitution, including transition and transversion, or insertion, and wherein
said
alterations may occur at the 5' or 3' terminal positions of the reference
polynucleotide
sequence or anywhere between those terminal positions, interspersed either
individually
among the nucleic acids in the reference sequence or in one or more contiguous
groups
within the reference sequence. The number of nucleic acid alterations for a
given percent
identity is determined by multiplying the total number of nucleic acids in SEQ
ID NO:1
by the integer defining the percent identity divided by 100 and then
subtracting that
product from said total number of nucleic acids in SEQ ID NO:1, or:
nn ~ xn ' ~xn' Y)
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wherein nn is the number of nucleic acid alterations, xn is the total number
of nucleic
acids in SEA ID NO:1, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for
85% etc.,
is the symbol for the multiplication operator, and wherein any non-integer
product of xn
and y is rounded down to the nearest integer prior to subtracting it from xn.
(2) Polypeptide embodiments further include an isolated polypeptide comprising
a
polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity
to 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)
wherein na is the number of amino acid alterations, xa is the total number of
amino acids
in SEQ ID N0:2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%,
0.85 for
85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and ~ is the
symbol for
the multiplication operator, and wherein any non-integer~product of xa and y
is rounded
down to the nearest integer prior to subtracting it from xa.
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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 NO:2, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85%
etc., and ~ is
the symbol for the multiplication operator, and wherein any non-integer
product of xa and
y is rounded down to the nearest integer prior to subtracting it from xa.
"Individual(s)," when used herein with reference to an organism, means a
multicellular
eukaryote, including, but not limited to a metazoan, a mammal, an ovid, a
bovid, a
simian, a primate, and a human.
"Isolated" means altered "by the hand of man" from its natural state, i. e.,
if it occurs in
nature, it has been changed or removed from its original environment, or both.
For example,
a polynucleotide or a polypeptide naturally present in a living organism is
not "isolated," but
the same polynucleotide or polypeptide separated from the coexisting materials
of its natural
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state is "isolated", as the term is employed herein. Moreover, a
polynucleotide or
polypeptide that is introduced into an organism by transformation, genetic
manipulation or
by any other recombinant method is "isolated" even if it is still present in
said organism,
which organism may be living or non-living.
"Polynucleotide(s)" generally refers to any polyribonucleotide or
polydeoxyribonucleotide,
which may be unmodified RNA or DNA or modified RNA or DNA including single and
double-stranded regions.
"Variant" refers to a polynucleotide or polypeptide that differs from a
reference
polynucleotide or polypeptide, but retains essential properties. A typical
variant of a
polynucleotide differs in nucleotide sequence from another, reference
polynucleotide.
Changes in the nucleotide sequence of the variant may or may not alter the
amino acid
sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide
changes may result in amino acid substitutions, additions, deletions, fusions
and
truncations in the polypeptide encoded by the reference sequence, as discussed
below.
A typical variant of a polypeptide differs in amino acid sequence from
another,
reference polypeptide. Generally, differences are limited so that the
sequences of the
reference polypeptide and the variant are closely similar overall and, in many
regions,
identical. A variant and reference polypeptide may differ in amino acid
sequence by
one or more substitutions, additions, deletions in any combination. A
substituted or
inserted amino acid residue may or may not be one encoded by the genetic code.
A
variant of a polynucleotide or polypeptide may be a naturally occurring such
as an
allelic variant, or it may be a variant that is not known to occur naturally.
Non-naturally
occurring variants of polynucleotides and polypeptides may be made by
mutagenesis
techniques or by direct synthesis.
"Disease(s)" means any disease caused by or related to infection by a
bacteria, including,
for example, otitis media in infants and children, pneumonia in elderlies,
sinusitis,
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nosocomial infections and invasive diseases, chronic otitis media with hearing
loss, fluid
accumulation in the middle ear, auditive nerve damage, delayed speech
learning, infection
of the upper respiratory tract and inflammation of the middle ear.
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EXAMPLES:
The examples below are carned out using standard techniques, which are well
known and routine to those of skill in the art, except where otherwise
described in
detail. The examples are illustrative, but do not limit the invention.
Example I: DNA sequencing of the BASB208 gene from Non typable Haemophilus
influenzae strain 3224A.
A: BASB208 in Non typable Haemophilus influenzae strain 3224A.
The DNA sequence of the BASB208 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 BASB208 polynucleotidic sequence is showed in SEQ
ID
N0:2.
'
B: BASB208 in Non typable Haemophilus influenzae strain 3224A.
The sequence of the BASB208 polynucleotide was confirmed in Non Typable
Haemophilus influenzae strain 3224A. For this purpose, plasmid DNA (see
example
3A) containing the gene region encoding BASB208 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 using primers oli 1 ORF1457 (S'-TC ATG AAA AAA TTA
CTT ATT GTA AC-3') [SEQ ID N0:12] and oli 2 ORF1457 (5'-AGA TCT GAA ACT
AAA ACG TAA ACC -3') [SEQ ID N0:13] specific for the BASB208 polynucleotide
and M13 Universal Sequence Primer(5'-GTA AAA CGA CGG CCA GT-3') [SEQ ID
N0:14] and M13 Reverse Sequence Primer (5'-CAG GAA ACA GCT ATG AC-3')
[SEQ ID NO:15] specific for the vector. As a result, the polynucleotide and
deduced
polypeptide sequences, respectively, were obtained. Using the Clustalx I.8
program, the
polynucleotide sequence SEQ ID NO: 3 was aligned with SEQ ID NO:1; a pairwise
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comparison of identities showed that the polynucleotide sequence was 98%
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 NO:2; a pairwise comparison of identities
showed that the polypeptide sequence was 96% identical to SEQ ID N0:2 (FIG 2)
Example 2:
Variability analysis of the BASB208 gene among Non Typable Haemoplzilus
influeuzae strains.
Genomic DNA was extracted from 3 further NT Haemophilus i~fluenzae 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. Genomic DNA was extracted from the pellet of the
NT
Haemophilus i~fluev~zae cells using the QIAGEN genomic DNA extraction kit
(Qiagen
Gmbh). 1 ~,g of this material was submitted to Polymeiase Chain Reaction DNA
amplification using primers MCMOlIB (5'- CTG ATG TAT TTT CAC ACA TTT
AGA G-3') [SEQ ID N0:16] and MCM012 (5'- TGC TCT TGC AAA TAA CTG TTT
CAC-3') [SEQ ID N0:17]. This PCR product was purified using the High Pure PCR
Product Purification Kit (Ruche) , 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 MCMO11B [SEQ ID N0:16] and MCM012 [SEQ ID N0:17] 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:S,
land 9 turned out to be 99 and 100 % 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
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polypeptidic sequences SEQ ID NO: 6, 8 and 10 turned out to be 100 % identical
to
SEQ ID NO:4 (Table 3).
Table 1: Features of the 1VT Haemoplailus influenzae strains used in this
study
Strain isolated from Nucleotidic peptidic
in sequence sequence
3224A USA Otitis mediaSEQ ID N0:3 SEQ ID N0:4
810956 NL Meningitis SEQ ID NO:S SEQ ID N0:6
27W116791N1 DK Cystic FibrosisSEQ ID N0:7 SEQ ID N0:8
A840164 NL Carrier SEQ ID N0:9 SEQ ID NO:10
strain
Table 2: Pairwaise comparison of polynucleiotidic sequences
SEQ ID N0:3SEQ ID SEQ ID SEQ ID
NO:S N0:7 NO:9
SEQ ID 99 100 100
N0:3
sEQ ID 99 99
NO:S
SEQ ID 100
N0:7
SEQ ID
N0:9
Table 3: Pairwaise comparison of polypeptidic sequences
SEQ ID N0:4SEQ ID SEQ ID SEQ ID
N0:6 N0:8 NO:10
sEQ ID 100 100 100
No:4
SEQ ID 100 100
N0:6
SEQ ID 100
N0:8
SEQ ID
NO:10
Example 3 : Construction of Plasmid to Express Recombinant BASB208
A: Cloning of BASB208.
The BspHI and BgIII restriction sites engineered into the oli 1 ORF1457 (5'-TC
ATG
AAA AAA TTA CTT ATT GTA AC-3') [SEQ ID N0:12] forward and oli 2 ORF1457
(5'-AGA TCT GAA ACT' AAA ACG TAA ACC -3') [SEQ ID N0:13] reverse
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amplification primers, respectively, permitted directional cloning of the PCR
product
into the E. coli expression plasmid pQE60 such that BASB208 protein could be
expressed as a fusion protein containing a (His)6 affinity chromatography tag
at the C-
terminus. The BASB208 PCR product was first introduced into the pCRIITOPO
cloning vector (In vitrogen) using ToplO bacterial cells, according to the
manufacturer's
instructions. This intermediate construct was realized to facilitate further
cloning into
an expression vector. Transformants containing the BASB208 DNA insert were
selected
by restriction enzyme analysis. Following digestion, a ~20~1 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 DNA fragments. Plasmid purified from selected
transformants
was then sequentially digested to completion with BspHI and BgIII restriction
enzymes
as recommended by the manufacturer (Life Technologies). The digested DNA
fragment
was then purified using silica geI-based spin columns prior to Iigation 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 BglII. An approximately 5-fold molar excess of
the
digested fragments to the prepared vector was used to program the ligation
reaction. A
standard ~20 ~,l ligation reaction (~16°C, ~16 hours), using methods
well known in the
art, was performed using T4 DNA ligase (~2.0 units / reaction, Life
Technologies). An
aliquot of the ligation (~5 ~,l) was used to transform M15(pREP4) electro-
competent
cells according to methods well known in the art. Following a ~2-3 hour
outgrowth
period at 37°C in ~1.0 ml of LB broth, transformed cells were plated on
LB agar plates
containing ampicillin (100 ~,g/m1) and kanamycin (30~g/ml). Antibiotic was
included
in the selection. Plates were incubated overnight at 37°C for ~16
hours. Individual
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ApR/KanR colonies were picked with sterile toothpicks and used to "patch"
inoculate
fresh LB ApR/KanR plates as well as a ~l .0 ml LB Ap/ Iran 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
S employed to verify that transformants contained the BASB208 DNA insert.
Here, the
~1.0 ml overnight LB Ap/Kan broth culture was transferred to a 1.S ml
polypropylene
tube and the cells collected by centrifugation in a Beckmann microcentrifuge
(~3 min.,
room temperature, 12,000 X g). The cell pellet was suspended in ~200~,1 of
sterile
water and a ~10~,1 aliquot used to program a ~50~,1 final volume PCR reaction
10 containing both BASB208 forward and reverse amplification primers. The
initial 9S°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. 9S°C, 45sec;
SS-S8°C, 4Ssec,
72°C, lmin., were used to amplify the BASB208 fragment from the lysed
transformant
1 S samples. Following thermal amplification, a ~20~1 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 I~b ladder, Life
Technologies) was electrophoresed in parallel with the test samples and was
used to
20 estimate the size of the PCR products. Transformants that produced the
expected size
PCR product were identified as strains containing a BASB208 expression
construct.
Expression plasmid containing strains were then analyzed for the inducible
expression
of recombinant BASB208.
2S 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 ~2S ml of LB Ap/Kan broth and grown at 37
°C with
shaking 0250 rpm) until the culture turbidity reached O.D.600 of ~O.S, i.e.
mid-log
phase (usually about 1.S - 2.0 hours). At this time approximately half of the
culture
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012.5 ml) was transferred to a second 125 ml flask and expression of
recombinant
BASB208 protein induced by the addition of IPTG (1.0 M stock prepared in
sterile
water, Sigma) to a final concentration of 1.0 mM. Incubation of both the IPTG-
induced
and non-induced cultures continued for an additional ~4 hours at 37 °C
with shaking.
Samples (~1.0 ml) of both induced and non-induced cultures were removed after
the
induction period and the cells collected by centrifugation in a
microcentrifuge at room
temperature for ~3 minutes. Individual cell pellets were suspended in ~50~,1
of sterile
water, then mixed with an equal volume of 2X Laemelli SDS-PAGE sample buffer
containing 2-mercaptoethanol, and placed in boiling water bath for ~3 min to
denature
protein. Equal volumes (~15~1) of both the crude IPTG-induced and the non-
induced
cell Iysates were loaded onto duplicate 12% Tris/glycine polyacrylamide gel (1
mm
thick Mini-gels, Novex). The induced and non-induced lysate samples were
electrophoresed together with prestained molecular weight markers (SeeBIue,
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 BASB208 IPTG-inducible
protein(s). The second gel was electroblotted onto a PVDF membrane (0.45
micron pore
size, Novex) for ~2 hrs at 4 °C using a BioRad Mini-Protean II blotting
apparatus and
Towbin's methanol (20 %) transfer buffer. Blocking of the membrane and
antibody
incubations were performed according to methods well known in the art. A
monoclonal
anti-RGS (His)3 antibody, followed by a second rabbit anti-mouse antibody
conjugated
to HRP (QiaGen), was used to confirm the expression and identity of the
BASB208
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 BASB208
Bacterial strain
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A recombinant expression strain of E. coli M15(pREP4) containing a plasmid
(pQE60)
encoding BASB208 from NT haemophilus influenzae was used to produce cell mass
for
purification of recombinant protein. The expression strain was cultivated on
LB agar
plates containing 100~,g/ml ampicillin ("Ap") and 30~,g/ml kanamycin ("Km") to
ensure that pQE60 and pREP4 were maintained. For cryopreservation at -80
°C, the
strain was propagated in LB broth containing the same concentration of
antibiotics then
mixed with an equal volume of LB broth containing 30% (w/v) glycerol.
Media
The growth medium used for the production of recombinant protein consisted of
LB
broth (Difco) containing 100~g/ml Ap and 30 ~,g/ml Km. To induce expression of
the
BASB208 recombinant protein, IPTG (Isopropyl l3-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 SOOmI working volume erlen containing LB broth and both Ap and I~m
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 BASB208 protein in
Esclterichia coli.
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The construction of the pET-BASB208 cloninglexpression vector is described in
Example
1. This vector harbours the BASB208 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 Novablue (DE3)
(Novagen), in which,
the gene for the T7 polymerise is placed under the control of the isopropyl-
beta-D
thiogalactoside (IPTG)-regulatable lac promoter. Liquid cultures (100 ml) of
the
Novablue (DE3) [pET-BASB208] 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 1mM and the culture 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 resuspended during 30 min at
25°C in buffer
A (6M guanidine hydrochloride, O.1M NaH2P04, O.O1M 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.IMNaH2P04, O.O1M Tris, pH 8.0), 40m1 of buffer C (8M
Urea,
O.IMNaH2PO4, O.OlM Tris, pH 6.3). The recombinant protein BASB208/His6 is then
eluted from the column with 30m1 of buffer D (8M Urea, O.IMNaH2P04, O.OlM
Tris, pH
6.3) containing SOOmM of imidazole and 3m1-size fractions are collected.
Highly
enriched BASB208/His6 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 BASB208-His6 protein is solubilized in a solution
devoid of
urea. For this purpose, denatured BASB208-His6 contained in 8M urea is
extensively
dialyzed (2 hours) against buffer R (NaCI 150mM, lOmM NaH2P04, Arginine O.SM
pH6.8) containing successively 6M, 4M, 2M and no urea. Alternatively, this
polypeptide
is purified under non-denaturing conditions ' using protocoles described in
the
Quiexpresssionist booklet (Qiagen Gmbh).
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Example 6: Production of Antisera to Recombinant BASB208
Polyvalent antisera directed against the BASB208 protein are generated by
vaccinating
rabbits with the purified recombinant BASB208 protein. Polyvalent antisera
directed
against the BASB208 protein are also generated by vaccinating mice with the
purified
recombinant BASB208 protein. Animals are bled prior to the first immunization
("pre-
bleed") and after the last immunization.
Anti-BASB208 protein titers are measured by an ELISA using purified
recombinant
BASB208 protein as the coating antigen. The titer is defined as mid-point
titers
calculated by 4-parameter logistic model using the XL Fit software.The
antisera are also
used as the first antibody to identify the protein in a western blot as
described in
eacample 8 below.
Example 7: Immunological characterization: Surface exposure of BASB208
Anti-BASB208 protein titres are determined by an ELISA using formalin-killed
whole
cells of non typable Haemophilus influe~~ae (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 plates
for 24 hours at 36°C and 5% GOz. Several colonies are used to inoculate
Brain Heart
Infusion (BHI) broth supplemented by NAD and hemin, each at 10 ~.g/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% 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.
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PVDF membranes are incubated with preimmune serum or rabbit or mouse immune
serum. PVDF membranes are then washed.
PVDF membranes are incubated with biotin-labeled sheep anti-rabbit or mouse
Ig.
5 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.
Example 9: Immunological characterization: Bactericidal Activity
10 Complement-mediated cytotoxic activity of anti-BASB208 antibodies is
examined to
determine the vaccine potential of BASB208 protein antiserum that is prepared
as
described above. The activities of the pre-immune serum and the anti-BASB208
antiserum in mediating complement killing of NTHi are examined.
15 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-BASB208 sera are deposited into the first well of
a 96-
20 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
25 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).
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Bactericidal activity of rabbit or mice antiserum (50% killing of homologous
strain) is
measured.
Example 10: Presence of Antibody to BASB208 in Human Convalescent Sera
Western blot analysis of purified recombinant BASB208 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 BASB208 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 BASB208 vaccine. After the booster, the mice
are
challenged by instillation of bacterial suspension into the nostril under
anaesthesia.
Mice are killed between 30 minutes and 24 hours after challenge and the lungs
are
removed aseptically and homogenized individually. The 1og10 weighted mean
number
of CFU/lung is determined by counting the colonies grown on agar plates after
plating
of dilutions of the homogenate. The arithmetic mean of the 1og10 weighted mean
number of CFU/lung and the standard deviations are calculated for each group.
Results are analysed statistically.
In this experiment groups of mice are immunized either with BASB208 or with a
killed
whole cells (kwc) preparation of NTHi or sham immunized.
Example 12: Inhibition of NTHi adhesion onto cells by anti-BASB208 antiserum.
This assay measures the capacity of anti BASB208 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-
BASB208 immune serum dilution. This mixture is subsequently added in the wells
of a
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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 BASB208 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, Brunel Bioinformatics Group, Dept. Biological
Sciences,
Brunel University, Uxbridge UB8 3PH, UK) (Fig.S). The antigenic index was
calculated on the basis of the method described by Jameson and Wolf (CABIOS
4:181-
186 [1988]). The parameters used in this program are the antigenic index and
the
minimal length for an antigenic peptide. An antigenic index 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, 11 or 12) amino acids
therefrom or
extensions comprising e. g. 1, 2, 3, 5, 10 further amino acids at either or
both ends from
the native context of BASB208 polypeptide which preserve an effective epitope
which
can elicit an immune response in a host against the BASB208 polypeptide.
Table 4: Potential B-cell epitopes from SEQ ID N0:2
Position Sequence ,
PSFT
55 ~ FDKNF
65 YTNYGKVT
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73
83 ~ LKGKS
110 STNEA
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 BASB208
polypeptide was based on the TEPITOPE method describe by Sturniolo at al.
(Nature
Biotech. 17: SSS-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% identical to the
sequences
below) or truncates comprising 5 or more (e.g. 6, 7, 8, 9, 10, 1 l, 12, 14,
16, 18 or 20)
amino acids therefrom or extensions comprising e. g. 1, 2, 3, 5, 10 further
amino acids
at either or both ends from the native context of BASB208 polypeptide which
preserve
an effective T-helper epitope from BASB208 polypeptide.
Table 5: Potential T-helper cell epitopes from SEQ ID N0:2
PositionSequence
1 MKKLLIVTMLSTLAL
22 YVQSDLGAS
61 LAVDYTNYGKVTA
78 IVDVSLKGKSLGLTGFYDFDL
lOl FKPYVGVRVSTNEAD
142 YKLTDNVAL
156 YNRLASDAS
168 VKAGLRFSF
All identified regions containing epitopes as defined above are in respect of
SEQ ID
NO:2. The corresponding regions in SEQ ID N0:4 as defined by position in table
4&5
with respect to SEQ ID N0:2 and by its corresponding peptide in the alignment
of
figure 2 for SEQ ID N0:4 are also preferred peptides of the invention as
described in
this example.
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74
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 BASB208 polynucleotide sequence.
The sequence of the polynucleotides contained in the deposited strain, as well
as the amino
acid sequence of any polypeptide encoded thereby, are controlling in the event
of any
conflict with any description of sequences herein.
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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INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule l3bis)
A. The indications made below
relate to the microorganism
referred to in the description
on page 74 Lines 1-22.
B. IDENTIFICATION OF DEPOSIT
Further deposits are identified
on an additional sheet
Name of depository institution
AMERICAN TYPE CULTURE COLLECTION
Address of depository institution
(including postal code and country)
10801 UNIVERSITY BLVD, MANASSAS,
VIRGINIA 20110-2209, UNITED
STATES OF
AMERICA
Date of deposit 5 May 2000 Accession Number PTA-1816
C. ADDITIONAL INDICATIONS (leave
blank if not applicable) This
information is continued on
an additional sheet
In respect of those designations
where a European Patent is sought,
a sample of the deposited microorganisms
will be made available until
the publication of the mention
of the grant of the European
Patent or until the
date on which the application
has been refused or withdrawn,
only by issue of such a sample
to an expert
nominated by the person requesting
the sample
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (if the
indications are not for all
designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank if not applicable)
The indications listed below
will be submitted to the International
Bureau later (sped the general
nature of the indications e.g.,
' Accession Number of Deposit'
For receiving Office use only For International Bureau use only
This sheet was received with the international ~ This sheet was received by
the International Bureau
application on:
Authorized officer Authorized officer
~ll~l'I~ ~'G~BS
Form PCT/RO/134 (July 1992)
