Note: Descriptions are shown in the official language in which they were submitted.
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Novel Compounds
FIELD OF THE INVENTION
This invention relates to polynucleotides, (herein referred to as "BASBO51
polynucleotide(s)", "BASB057 polynucleotide(s)", "BASB060 polynucleotide(s)",
"BASB061 polynucleotide(s)", "BASB063 polynucleotide(s)", "BASB065
polynucleotide(s)", "BASB066 polynucleotide(s)" and "BASB071
polynucleotide(s)"),
polypeptides encoded by them (referred to herein as "BASB051", "BASB057",
"BASB060", "BASB061", "BASB063", "BASB065", "BASB066" and "BASB071"
respectively or "BASBO51 polypeptide(s)", BASB057 polypeptide(s)", BASB060
polypeptide(s)", BASB061 polypeptide(s)", BASB063 polypeptide(s)", BASB065
polypeptide(s)", BASB066 polypeptide(s)" and BASB071 polypeptide(s)"
respectively),
recombinant materials and methods for their production. In another aspect, the
invention
relates to methods for using such polypeptides and polynucleotides, including
vaccines
against bacterial infections. In a further aspect, the invention relates to
diagnostic assays for
detecting infection of certain pathogens.
BACKGROUND OF THE INVENTION
Neisseria meningitidis (meningococcus) is a Gram-negative bacterium frequently
isolated
from the human upper respiratory tract. It occasionally causes invasive
bacterial diseases
such as bacteremia and meningitis. The incidence of meningococcal disease
shows
geographical seasonal and annual differences (Schwartz, B., Moore, P.S.,
Broome, C.V.; Clin.
Microbiol. Rev. 2 (Supplement), S18-524, 1989). Most disease in temperate
countries is due
to strains of serogroup B and varies in incidence from 1-10/100,000/year total
population
sometimes reaching higher values (Kaczmarski, E.B. (1997), Common. Dis. Rep.
Rev. 7:
R55-9, 1995; Scholten, R.J.P.M., Bijlmer, H.A., Poolman, J.T. et al. Clin.
Infect. Dis. 16:
237-246, 1993; Cruz, C., Pavez, G., Aguilar, E., et al. Epidemiol. Infect.
105: 119-126, 1990).
1
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Epidemics dominated by serogroup A meningococci, mostly in central Africa, are
encountered, sometimes reaching levels up to 1000/100.000/year (Schwartz, B.,
Moore, P.S.,
Broome, C.V. Clin. Microbiol. Rev. 2 (Supplement), S18-S24, 1989). Nearly all
cases as a
whole of meningococcal disease are caused by serogroup A, B, C, W-135 and Y
meningococci and a tetravalent A, C, W-135, Y polysaccharide vaccine is
available (Armand,
J., Arminjon, F., Mynard, M.C., Lafaix, C., J. Biol. Stand. 10: 335-339,
1982).
The polysaccharide vaccines are currently being improved by way of chemical
conjugating
them to carrier proteins (Lieberman, J.M., Chiu, S.S., Wong, V.K., et al. JAMA
275 : 1499-
1503, 1996).
A serogroup B vaccine is not available, since the B capsular polysaccharide
was found to be
nonimmunogenic, most likely because it shares structural similarity to host
components
(Wyle, F.A., Artenstein, M.S., Brandt, M.L. et al. J. Infect. Dis. 126: 514-
522, 1972; Finne,
J.M., Leinonen, M., Makela, P.M. Lancet ii.: 35~-357, 1983).
For many years efforts have been initiated and carried out to develop
meningococcal outer
membrane based vaccines (de Moraes, J.C., Perkins, B., Camargo, M.C. et al.
Lancet 340:
1074-1078, 1992; Bjune, G., Hoiby, E.A. Gronnesby, J.K. et al. 338: 1093-1096,
1991). Such
vaccines have demonstrated e~cacies from 57% - 85% in older children (>4
years) and
adolescents.
Many bacterial outer membrane components are present in these vaccines, such
as PorA,
PorB, Rmp, Opc, Opa, FrpB and the contribution of these components to the
observed
protection still needs father definition. Other bacterial outer membrane
components have been
defined by using animal or human antibodies to be potentially relevant to the
induction of
protective immunity, such as TbpB and NspA (Martin, D., Cadieux, N., Hamel,
J., Brodeux,
B.R., J. Exp. Med. 185: 1173-1183, 1997; Lissolo, L., Maitre-Wilmotte, C.,
Dumas, p. et al.,
2
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Inf. Immun. 63: 884-890, 1995). The mechanisms of protective immunity will
involve
antibody mediated bactericidal activity and opsonophagocytosis.
A bacteremia animal model has been used to combine all antibody mediated
mechanisms
(Saukkonen, K., Leinonen, M., Abdillahi, H. Poolman, J. T. Vaccine 7: 325-328,
1989). It is
generally accepted that the late complement component mediated bactericidal
mechanism is
crucial for immunity against meningococcal disease (Ross, S.C., Rosenthal
P.J., Berberic,
H.M., Densen, P. J. Infect. Dis. 155: 1266-1275, 1987).
The frequency of Neisseria meningitides infections has risen dramatically in
the past few
decades. This has been attributed to the emergence of multiply antibiotic
resistant strains and
an increasing population of people with weakened immune systems. It is no
longer
uncommon to isolate Neisseria meningitides strains that are resistant to some
or all of the
standard antibiotics. This phenomenon has created an unmet medical need and
demand for
new anti-microbial agents, vaccines, drug screening methods, and diagnostic
tests for this
organism.
SUMMARY OF THE INVENTION
The present invention relates to BASBO~ 1, BASB057, BASB060, BASB061, BASB063,
BASB065, BASB066, and BASB071, in particular BASBO51, BASB057, BASB060,
BASB061, BASB063, BASB065, BASB066, and BASB071 polypeptides and BASBO51,
BASB057, BASB060, BASB061, BASB063, BASB065, BASB066, and BASB071
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 BASBO51, BASB057, BASB060, BASB061, BASB063,
BASB065, BASB066, and BASB071 polynucleotides or polypeptides.
3
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
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 BASBO51, BASB057, BASB060, BASB061, BASB063, BASB065,
BASB066, and BASB071 polypeptides and polynucleotides as described in greater
detail
below. The invention relates especially to BASBO51, BASB057, BASB060, BASB061,
BASB063, BASB065, BASB066, and BASB071 having the nucleotide and amino acid
sequences set out in SEQ ID N0:1,3,5,7,9,11,13,15 and SEQ ID
N0:2,4,6,8,10,12,14,16
respectively. It is understood that sequences recited in the Sequence Listing
below as
"DNA" represent an exemplification of one embodiment of the invention, since
those of
ordinary skill will recognize that such sequences can be usefully employed in
polynucleotides in general, including ribopolynucleotides.
Polypeptides
In one aspect of the invention there are provided polypeptides of Neisseria
meningitides
referred to herein as "BASBO51", "BASB057", "BASB060", "BASB061", "BASB063",
"BASB065", "BASB066" and "BASB071", and "BASBOS1 polypeptides", "BASB057
polypeptides", "BASB060 polypeptides", "BASB061 polypeptides", "BASB063
polypeptides", "BASB065 polypeptides", "BASB066 polypeptides", and "BASB071
polypeptides" as well as biologically, diagnostically, prophylactically,
clinically or
therapeutically useful variants thereof, and compositions comprising the same.
The present invention further provides for:
4
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) an isolated polypeptide which comprises an amino acid sequence which has
at least
85% identity, more preferably at least 90% identity, yet more preferably at
least 95%
identity, most preferably at least 97-99% or exact identity, to that of SEQ ID
N0:2.
(b) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence which has at least 85% identity, more preferably at least 90%
identity, yet more
preferably at least 95% identity, even more preferably at least 97-99% or
exact identity to
SEQ ID NO:1 over the entire length of SEQ ID NO:1.
(c) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence encoding a polypeptide which has at least 85% identity, more
preferably at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity, to the amino acid sequence of SEQ ID N0:2.
The BASB051 polypeptide provided in SEQ ID N0:2 is the BASB051 polypeptide
from
Neisseria meningitidis strain ATCC 13090.
The invention also provides an immunogenic fragment of a BASB051 polypeptide,
that is,
a contiguous portion of the BASB051 polypeptide which has the same or
substantially the
same immunogenic activity as the polypeptide comprising the amino acid
sequence of SEQ
ID N0:2. That is to say, the fragment (if necessary when coupled to a carrier)
is capable of
raising an immune response which recognises the BASB051 polypeptide. Such an
immunogenic fragment may include, for example, the BASB051 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 BASB051 according to
the
invention comprises substantially all of the extracellular domain of a
polypeptide which has
at least 85% identity, more preferably at least 90% identity, yet more
preferably at least
95% identity, most preferably at least 97-99% identity, to that of SEQ ID N0:2
over the
entire length of SEQ ID N0:2.
The present invention further provides for:
5
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) an isolated polypeptide which comprises an amino acid sequence which has
at least
85% identity, more preferably at least 90% identity, yet more preferably at
least 95%
identity, most preferably at least 97-99% or exact identity, to that of SEQ ID
N0:4.
(b) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence which has at least 85% identity, more preferably at least 90%
identity, yet more
preferably at least 95% identity, even more preferably at least 97-99% or
exact identity to
SEQ ID N0:3 over the entire length of SEQ ID N0:3.
(c) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence encoding a polypeptide which has at least 85% identity, more
preferably at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity, to the amino acid sequence of SEQ ID N0:4
The BASB057 polypeptide provided in SEQ ID N0:4 is the BASB057 polypeptide
from
Neisseria meningitides strain ATCC13090.
The invention also provides an immunogenic fragment of a BASB057 polypeptide,
that is,
a contiguous portion of the BASB057 polypeptide which has the same or
substantially the
same immunogenic activity as the polypeptide comprising the amino acid
sequence of SEQ
ID N0:4. That is to say, the fragment (if necessary when coupled to a carrier)
is capable of
raising an immune response which recognises the BASB057 polypeptide. Such an
immunogenic fragment may include, for example, the BASB057 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 BASB057 according to
the
invention comprises substantially all of the extracellular domain of a
polypeptide which has
at least 85% identity, more preferably at least 90% identity, yet more
preferably at least
95% identity, most preferably at least 97-99% identity, to that of SEQ ID N0:4
over the
entire length of SEQ ID N0:4.
The present invention further provides for:
6
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) an isolated polypeptide which comprises an amino acid sequence which has
at least
85% identity, more preferably at least 90% identity, yet more preferably at
least 95%
identity, most preferably at least 97-99% or exact identity, to that of SEQ ID
N0:6.
(b) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence which has at least 85% identity, more preferably at least 90%
identity, yet more
preferably at least 95% identity, even more preferably at least 97-99% or
exact identity to
SEQ ID NO:S over the entire length of SEQ ID NO:S.
(c) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence encoding a polypeptide which has at least 85% identity, more
preferably at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity, to the amino acid sequence of SEQ ID N0:6
The BASB060 polypeptide provided in SEQ ID N0:6 is the BASB060 polypeptide
from
Neisseria meningitides strain ATCC 13090.
The invention also provides an immunogenic fragment of a BASB060 polypeptide,
that is,
a contiguous portion of the BASB060 polypeptide which has the same or
substantially the
same immunogenic activity as the polypeptide comprising the amino acid
sequence of SEQ
ID N0:6. That is to say, the fragment (if necessary when coupled to a carrier)
is capable of
raising an immune response which recognises the BASB060 polypeptide. Such an
immunogenic fragment may include, for example, the BASB060 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 BASB060 according to
the
invention comprises substantially all of the extracellular domain of a
polypeptide which has
at least 85% identity, more preferably at least 90% identity, yet more
preferably at least
95% identity, most preferably at least 97-99% identity, to that of SEQ ID N0:6
over the
entire length of SEQ ID N0:6.
The present invention further provides for:
7
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) an isolated polypeptide which comprises an amino acid sequence which has
at least
85% identity, more preferably at least 90% identity, yet more preferably at
least 95%
identity, most preferably at least 97-99% or exact identity, to that of SEQ ID
N0:8.
(b) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence which has at least 85% identity, more preferably at least 90%
identity, yet more
preferably at least 95% identity, even more preferably at least 97-99% or
exact identity to
SEQ ID N0:7 over the entire length of SEQ ID N0:7.
(c) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence encoding a polypeptide which has at least 85% identity, more
preferably at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity, to the amino acid sequence of SEQ ID N0:8
The BASB061 polypeptide provided in SEQ ID N0:8 is the BASB061 polypeptide
from
Neisseria meningitides strain ATCC13090.
The invention also provides an immunogenic fragment of a BASB061 polypeptide,
that is,
a contiguous portion of the BASB061 polypeptide which has the same or
substantially the
same immunogenic activity as the polypeptide comprising the amino acid
sequence of SEQ
ID N0:8. That is to say, the fragment (if necessary when coupled to a Garner)
is capable of
raising an immune response which recognises the BASB061 polypeptide. Such an
immunogenic fragment may include, for example, the BASB061 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 BASB061 according to
the
invention comprises substantially all of the extracellular domain of a
polypeptide which has
at least 85% identity, more preferably at least 90% identity, yet more
preferably at least
95% identity, most preferably at least 97-99% identity, to that of SEQ ID N0:8
over the
entire length of SEQ ID N0:8.
The present invention further provides for:
8
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) an isolated polypeptide which comprises an amino acid sequence which has
at least
85% identity, more preferably at least 90% identity, yet more preferably at
least 95%
identity, most preferably at least 97-99% or exact identity, to that of SEQ ID
NO:10.
(b) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence which has at least 85% identity, more preferably at least 90%
identity, yet more
preferably at least 95% identity, even more preferably at least 97-99% or
exact identity to
SEQ ID N0:9 over the entire length of SEQ ID N0:9.
(c) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence encoding a polypeptide which has at least 85% identity, more
preferably at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity, to the amino acid sequence of SEQ ID NO:10
The BASB063 polypeptide provided in SEQ ID NO:10 is the BASB063 polypeptide
from
Neisseria meningitidis strain ATCC 13090.
The invention also provides an immunogenic fragment of a BASB063 polypeptide,
that is,
a contiguous portion of the BASB063 polypeptide which has the same or
substantially the
same immunogenic activity as the polypeptide comprising the amino acid
sequence of SEQ
ID NO:10. That is to say, the fragment (if necessary when coupled to a earner)
is capable
of raising an immune response which recognises the BASB063 polypeptide. Such
an
immunogenic fragment may include, for example, the BASB063 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 BASB063 according to
the
invention comprises substantially all of the extracellular domain of a
polypeptide which has
at least 85% identity, more preferably at least 90% identity, yet more
preferably at least
95% identity, most preferably at least 97-99% identity, to that of SEQ ID
NO:10 over the
entire length of SEQ ID NO:10.
The present invention further provides for:
9
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) an isolated polypeptide which comprises an amino acid sequence which has
at least
85% identity, more preferably at least 90% identity, yet more preferably at
least 95%
identity, most preferably at least 97-99% or exact identity, to that of SEQ ID
N0:12.
(b) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence which has at least 85% identity, more preferably at least 90%
identity, yet more
preferably at least 95% identity, even more preferably at least 97-99% or
exact identity to
SEQ ID NO:11 over the entire length of SEQ ID NO:11.
(c) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence encoding a polypeptide which has at least 85% identity, more
preferably at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity, to the amino acid sequence of SEQ ID N0:12
The BASB065 polypeptide provided in SEQ ID N0:12 is the BASB065 polypeptide
from
Neisseria meningitidis strain ATCC13090.
The invention also provides an immunogenic fragment of a BASB065 polypeptide,
that is,
a contiguous portion of the BASB065 polypeptide which has the same or
substantially the
same immunogenic activity as the polypeptide comprising the amino acid
sequence of SEQ
ID N0:12. That is to say, the fragment (if necessary when coupled to a
carrier) is capable
of raising an immune response which recognises the BASB065 polypeptide. Such
an
immunogenic fragment may include, for example, the BASB065 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 BASB065 according to
the
invention comprises substantially all of the extracellular domain of a
polypeptide which has
at least 85% identity, more preferably at least 90% identity, yet more
preferably at least
95% identity, most preferably at least 97-99% identity, to that of SEQ ID
N0:12 over the
entire length of SEQ ID N0:12.
The present invention further provides for:
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) an isolated polypeptide which comprises an amino acid sequence which has
at least
85% identity, more preferably at least 90% identity, yet more preferably at
least 95%
identity, most preferably at least 97-99% or exact identity, to that of SEQ ID
N0:14.
(b) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence which has at least 85% identity, more preferably at least 90%
identity, yet more
preferably at least 95% identity, even more preferably at least 97-99% or
exact identity to
SEQ ID N0:13 over the entire length of SEQ ID N0:13.
(c) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence encoding a polypeptide which has at least 85% identity, more
preferably at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity, to the amino acid sequence of SEQ ID N0:14
The BASB066 polypeptide provided in SEQ ID N0:14 is the BASB066 polypeptide
from
Neisseria meningitidis strain ATCC13090.
The invention also provides an immunogenic fragment of a BASB066 polypeptide,
that is,
a contiguous portion of the BASB066 polypeptide which has the same or
substantially the
same immunogenic activity as the polypeptide comprising the amino acid
sequence of SEQ
ID N0:14. That is to say, the fragment (if necessary when coupled to a Garner)
is capable
of raising an immune response which recognises the BASB066 polypeptide. Such
an
immunogenic fragment may include, for example, the BASB066 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 BASB066 according to
the
invention comprises substantially all of the extracellular domain of a
polypeptide which has
at least 85% identity, more preferably at least 90% identity, yet more
preferably at least
95% identity, most preferably at least 97-99% identity, to that of SEQ ID
N0:14 over the
entire length of SEQ ID N0:14.
The present invention further provides for:
11
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) an isolated polypeptide which comprises an amino acid sequence which has
at least
85% identity, more preferably at least 90% identity, yet more preferably at
least 95%
identity, most preferably at least 97-99% or exact identity, to that of SEQ ID
N0:16.
(b) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence which has at least 85% identity, more preferably at least 90%
identity, yet more
preferably at least 95% identity, even more preferably at least 97-99% or
exact identity to
SEQ ID NO:15 over the entire length of SEQ ID NO:15.
(c) a polypeptide encoded by an isolated polynucleotide comprising a
polynucleotide
sequence encoding a polypeptide which has at least 85% identity, more
preferably at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity, to the amino acid sequence of SEQ ID N0:16
The BASB071 polypeptide provided in SEQ ID N0:16 is the BASB071 polypeptide
from
Neisseria meningitides strain ATCC13090.
The invention also provides an immunogenic fragment of a BASB071 polypeptide,
that is,
a contiguous portion of the BASB071 polypeptide which has the same or
substantially the
same immunogenic activity as the polypeptide comprising the amino acid
sequence of SEQ
ID N0:16. That is to say, the fragment (if necessary when coupled to a
carrier) is capable
of raising an immune response which recognises the BASB071 polypeptide. Such
an
immunogenic fragment may include, for example, the BASB071 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 BASB071 according to
the
invention comprises substantially all of the extracellular domain of a
polypeptide which has
at least 85% identity, more preferably at least 90% identity, yet more
preferably at least
95% identity, most preferably at least 97-99% identity, to that of SEQ ID
N0:16 over the
entire length of SEQ ID N0:16.
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
12
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
BASBO51, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and
BASB071 polypeptides, fragments may be "free-standing," or comprised within a
larger
polypeptide of which they form a part or region, most preferably as a single
continuous region
in a single larger polypeptide.
Preferred fragments include, for example, truncation polypeptides having a
portion of an
amino acid sequence of SEQ ID N0:2,4,6,8,10,12,14,16 or of variants thereof,
such as a
continuous series of residues that includes an amino- and/or carboxyl-terminal
amino acid
sequence. Degradation forms of the polypeptides of the invention produced by
or in a host
cell, are also preferred. Further preferred are fragments characterized by
structural or
functional attributes such as fragments that comprise alpha-helix and alpha-
helix forming
regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming
regions, coil and
coil-forming regions, hydrophilic regions, hydrophobic regions, alpha
amphipathic regions,
beta amphipathic regions, flexible regions, surface-forming regions, substrate
binding region,
and high antigenic index regions.
Further preferred fragments include an isolated polypeptide comprising an
amino acid
sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from
the amino
acid sequence of SEQ ID N0:2,4,6,8,10,12,14,16 or an isolated polypeptide
comprising an
amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino
acids
truncated or deleted from the amino acid sequence of SEQ ID
N0:2,4,6,8,10,12,14,16.
Fragments of the polypeptides of the invention may be employed for producing
the
corresponding full-length polypeptide by peptide synthesis; therefore, these
fragments may
2~ 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.
13
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
The polypeptides, or immunogenic fragments, of the invention may be in the
form of the
"mature" protein or may be a part of a larger protein such as a precursor or a
fusion
protein. It is often advantageous to include an additional amino acid sequence
which
contains secretory or leader sequences, pro-sequences, sequences which aid in
purification such as multiple histidine residues, or an additional sequence
for stability
during recombinant production. Furthermore, addition of exogenous polypeptide
or lipid
tail or polynucleotide sequences to increase the immunogenic potential of the
final
molecule is also considered.
In one aspect, the invention relates to genetically engineered soluble fusion
proteins
comprising a polypeptide of the present invention, or a fragment thereof, and
various
portions of the constant regions of heavy or light chains of immunoglobulins
of various
subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the
constant part of
the heavy chain of human IgG, particularly IgGI, where fusion takes place at
the hinge
region. In a particular embodiment, the Fc part can be removed simply by
incorporation
of a cleavage sequence which can be cleaved with blood clotting factor Xa.
Furthermore, this invention relates to processes for the preparation of these
fusion
proteins by genetic engineering, and to the use thereof for drug screening,
diagnosis and
therapy. A further aspect of the invention also relates to polynucleotides
encoding such
fusion proteins. Examples of fusion protein technology can be found in
International
Patent Application Nos. W094/29458 and W094/22914.
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.
14
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Fusion partners include protein D from Haemophilus influenzae and the non-
structural
protein from influenzae virus, NS 1 (hemagglutinin). Another fusion partner is
the protein
known as LytA. Preferably the C terminal portion of the molecule is used. LytA
is
derived from Streptococcus pneumoniae which synthesize an N-acetyl-L-alanine
amidase, amidase LytA, (coded by the lytA gene {Gene, 43 (1986) page 26~-272})
an
autolysin that specifically degrades certain bonds in the peptidoglycan
backbone. The C-
terminal domain of the LytA protein is responsible for the affinity to the
choline or to
some choline analogues such as DEAE. This property has been exploited for the
development of E.coli C-LytA expressing plasmids useful for expression of
fusion
proteins. Purification of hybrid proteins containing the C-LytA fragment at
its amino
terminus has been described {Biotechnology: 10, (1992) page 795-798}. It is
possible to
use the repeat portion of the LytA molecule found in the C terminal end
starting at
residue 178, for example residues 188 - 305.
The present invention also includes variants of the aforementioned
polypeptides, that is
polypeptides that vary from the referents by conservative amino acid
substitutions, whereby
a residue is substituted by another with like characteristics. Typical such
substitutions are
among Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp
and Glu;
among Asn and Gln; and among the basic residues Lys and Arg; or aromatic
residue's 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
Neisseria meningitidis,
however, it may preferably be obtained from other organisms of the same
taxonomic genus.
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
A polypeptide of the invention may also be obtained, for example, from
organisms of the
same taxonomic family or order.
Polynucleotides
It is an object of the invention to provide polynucleotides that encode
BASBO51
polypeptides, particularly polynucleotides that encode the polypeptide herein
designated
BASBO51.
In a particularly preferred embodiment of the invention the polynucleotide
comprises a region
encoding BASBOS 1 polypeptides comprising a sequence set out in SEQ ID NO:1
which
includes a full length gene, or a variant thereof.
The BASBO51 polynucleotide provided in SEQ ID NO:1 is the BASBO51
polynucleotide
from Neisseria meningitides strains ATCC13090.
As a further aspect of the invention there are provided isolated nucleic acid
molecules
encoding and/or expressing BASBO51 polypeptides and polynucleotides,
particularly
Neisseria meningitides BASBO51 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 BASBO51 polypeptide having a deduced amino acid
sequence of
SEQ ID N0:2 and polynucleotides closely related thereto and variants thereof.
In another particularly preferred embodiment of the invention there is a
BASBO51
polypeptide from Neisseria meningitides comprising or consisting of an amino
acid
sequence of SEQ ID N0:2 or a variant thereof.
16
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Using the information provided herein, such as a polynucleotide sequence set
out in SEQ ID
NO:1 a polynucleotide of the invention encoding BASBOS 1 polypeptide may be
obtained
using standard cloning and screening methods, such as those for cloning and
sequencing
chromosomal DNA fragments from bacteria using Neisseria meningitides cells as
starting
material, followed by obtaining a full length clone. For example, to obtain a
polynucleotide
sequence of the invention, such as a polynucleotide sequence given in SEQ ID
NO:1
typically a library of clones of chromosomal DNA of Neisseria meningitides in
E. coli or
some other suitable host is probed with a radiolabeled oligonucleotide,
preferably a 17-mer
or longer, derived from a partial sequence. Clones carrying DNA identical to
that of the
probe can then be distinguished using stringent hybridization conditions. By
sequencing
the individual clones thus identified by hybridization with sequencing primers
designed
from the original polypeptide or polynucleotide sequence it is then possible
to extend the
polynucleotide sequence in both directions to determine a full length gene
sequence.
Conveniently, such sequencing is performed, for example, using denatured
double stranded
DNA prepared from a plasmid clone. Suitable techniques are described by
Maniatis, T.,
Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL,
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
(1989). (see
in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-
Stranded
DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to
obtain
a full length gene sequence. Illustrative of the invention, each
polynucleotide set out in SEQ
ID NO:1 was discovered in a DNA library derived from Neisseria meningitides.
Moreover, the DNA sequence set out in SEQ ID NO:1 contains an open reading
frame
encoding a protein having about the number of amino acid residues set forth in
SEQ ID N0:2
with a deduced molecular weight that can be calculated using amino acid
residue molecular
weight values well known to those skilled in the art.
17
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
The polynucleotide of SEQ ID NO:l, between the start codon at nucleotide
number 1 and the
stop codon which begins at nucleotide number 802 of SEQ ID NO:1, encodes the
polypeptide
of SEQ ID N0:2.
In a further aspect, the present invention provides for an isolated
polynucleotide comprising
or consisting of:
(a) a polynucleotide sequence which has at least 85% identity, more preferably
at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity to SEQ ID NO:1 over the entire length of SEQ ID NO:1; or
(b) a polynucleotide sequence encoding a polypeptide which has at least 85%
identity,
more preferably at least 90% identity, yet more preferably at least 95%
identity, even
more preferably at least 97-99% or 100% exact, to the amino acid sequence of
SEQ ID
N0:2 over the entire length of SEQ ID N0:2.
A polynucleotide encoding a polypeptide of the present invention, including
homologs and
orthologs from species other than Neisseria meningitidis, may be obtained by a
process which
comprises the steps of screening an appropriate library under stringent
hybridization
conditions (for example, using a temperature in the range of 45 - 65°C
and an SDS
concentration from 0.1 -1%) with a labeled or detectable probe consisting of
or comprising
the sequence of SEQ ID NO: 1 or a fragment thereof; and isolating a full-
length gene and/or
genomic clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire
length to a coding
sequence (open reading frame) in SEQ ID NO: 1. Also provided by the invention
is a coding
sequence for a mature polypeptide or a fragment thereof, by itself as well as
a coding
sequence for a mature polypeptide or a fragment in reading frame with another
coding
sequence, such as a sequence encoding a leader or secretory sequence, a pre-,
or pro- or
prepro-protein sequence. The polynucleotide of the invention may also contain
at least one
non-coding sequence, including for example, but not limited to at least one
non-coding 5' and
3' sequence, such as the transcribed but non-translated sequences, termination
signals (such as
18
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
rho-dependent and rho-independent termination signals), ribosome binding
sites, Kozak
sequences, sequences that stabilize mRNA, introns, and polyadenylation
signals. The
polynucleotide sequence may also comprise additional coding sequence encoding
additional
amino acids. For example, a marker sequence that facilitates purification of
the fused
polypeptide can be encoded. In certain embodiments of the invention, the
marker sequence is
a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and
described in Gentz
et al., Proc. Natl. Acad Sci., USA 86: 821-824 (1989), or an HA peptide tag
(Wilson et al.,
Cell 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 BASBO51 polypeptide of SEQ ID N0:2 may be
identical to the polypeptide encoding sequence contained in nucleotides 1 to
801 of SEQ ID
NO:1. Alternatively it may be a sequence, which as a result of the redundancy
(degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID N0:2.
The term "polynucleotide encoding a polypeptide" as used herein encompasses
polynucleotides that include a sequence encoding a polypeptide of the
invention, particularly
a bacterial polypeptide and more particularly a polypeptide of the Neisseria
meningitidis
BASBO51 having an amino acid sequence set out in SEQ ID N0:2. The term also
encompasses polynucleotides that include a single continuous region or
discontinuous regions
encoding the polypeptide (for example, polynucleotides interrupted by
integrated phage, an
integrated insertion sequence, an integrated vector sequence, an integrated
transposon
sequence, or due to RNA editing or genomic DNA reorganization) together with
additional
regions, that also may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described
herein that encode
variants of a polypeptide having a deduced amino acid sequence of SEQ ID N0:2.
Fragments of polynucleotides of the invention may be used, for example, to
synthesize full-
length polynucleotides of the invention.
19
~
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Further particularly preferred embodiments are polynucleotides encoding
BASBO51 variants,
that have the amino acid sequence of BASBO51 polypeptide of SEQ ID N0:2 in
which
several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are
substituted, modified,
deleted and/or added, in any combination. Especially preferred among these are
silent
substitutions, additions and deletions, that do not alter the properties and
activities of
BASBO51 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at
least 85%
identical over their entire length to a polynucleotide encoding BASBO51
polypeptide having
an amino acid sequence set out in SEQ ID N0:2 and polynucleotides that are
complementary
to such polynucleotides. In this regard, polynucleotides at least 90%
identical over their entire
length to the same are particularly preferred, and among these particularly
preferred
polynucleotides, those with at least 95% are especially preferred.
Furthermore, those with at
least 97% are highly preferred among those with at least 95%, and among these
those with at
least 98% and at least 99% are particularly highly preferred, with at least
99% being the more
preferred.
Preferred embodiments are polynucleotides encoding polypeptides that retain
substantially
the same biological function or activity as the mature polypeptide encoded by
a DNA of SEQ
ID NO:1.
In accordance with certain preferred embodiments of this invention there are
provided
polynucleotides that hybridize, particularly under stringent conditions, to
BASBO51
polynucleotide sequences, such as those polynucleotides in SEQ ID NO:l .
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
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
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,
5x SSC (150mM
NaCI, lSmM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's
solution,
10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm
DNA,
followed by washing the hybridization support in O.lx SSC at about
65°C. Hybridization
and wash conditions are well known and exemplified in Sambrook, et al.,
Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,
(1989),
particularly Chapter 11 therein. Solution hybridization may also be used with
the
polynucleotide sequences provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a
polynucleotide
sequence obtained by screening an appropriate library containing the complete
gene for a
polynucleotide sequence set forth in SEQ ID NO:1 under stringent hybridization
conditions
with a probe having the sequence of said polynucleotide sequence set forth in
SEQ ID
NO:l or a fragment thereof; and isolating said polynucleotide sequence.
Fragments useful
for obtaining such a polynucleotide include, for example, probes and primers
fully
described elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the
invention, for instance,
the polynucleotides of the invention, may be used as a hybridization probe for
RNA, cDNA
and genomic DNA to isolate full-length cDNAs and genomic clones encoding
BASB051 and
to isolate cDNA and genomic clones of other genes that have a high identity,
particularly high
sequence identity, to the BASB051 gene. Such probes generally will comprise at
least 15
nucleotide residues or base pairs. Preferably, such probes will have at least
30 nucleotide
residues or base pairs and may have at least 50 nucleotide residues or base
pairs. Particularly
preferred probes will have at least 20 nucleotide residues or base pairs and
will have less than
nucleotide residues or base pairs.
21
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
A coding region of a BASBO51 gene may be isolated by screening using a DNA
sequence
provided in SEQ ID NO:1 to synthesize an oligonucleotide probe. A labeled
oligonucleotide
having a sequence complementary to that of a gene of the invention is then
used to screen a
library of cDNA, genomic DNA or mRNA to determine which members of the library
the
probe hybridizes to.
22
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
It is an object of the invention to provide polynucleotides that encode
BASB057
polypeptides, particularly polynucleotides that encode the polypeptide herein
designated
BASB057.
S In a particularly preferred embodiment of the invention the polynucleotide
comprises a region
encoding BASB057 polypeptides comprising a sequence set out in SEQ ID N0:3
which
includes a full length gene, or a variant thereof.
The BASB057 polynucleotide provided in SEQ ID N0:3 is the BASB057
polynucleotide
from Neisseria meningitides strains ATCC 13090.
As a further aspect of the invention there are provided isolated nucleic acid
molecules
encoding andlor expressing BASB057 polypeptides and polynucleotides,
particularly
Neisseria meningitides BASB057 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 BASB057 polypeptide having a deduced amino acid
sequence of
SEQ ID N0:4 and polynucleotides closely related thereto and variants thereof.
In another particularly preferred embodiment of the invention there is a
BASB057
polypeptide from Neisseria meningitides comprising or consisting of an amino
acid
sequence of SEQ ID N0:4 or a variant thereof.
Using the information provided herein, such as a polynucleotide sequence set
out in SEQ ID
N0:3 a polynucleotide of the invention encoding BASB057 polypeptide may be
obtained
using standard cloning and screening methods, such as those for cloning and
sequencing
23
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
chromosomal DNA fragments from bacteria using Neisseria meningitidis cells as
starting
material, followed by obtaining a full length clone. For example, to obtain a
polynucleotide
sequence of the invention, such as a polynucleotide sequence given in SEQ ID
N0:3
typically a library of clones of chromosomal DNA of Neisseria meningitidis in
E coli or
some other suitable host is probed with a radiolabeled oligonucleotide,
preferably a 17-mer
or longer, derived from a partial sequence. Clones carrying DNA identical to
that of the
probe can then be distinguished using stringent hybridization conditions. By
sequencing
the individual clones thus identified by hybridization with sequencing primers
designed
from the original polypeptide or polynucleotide sequence it is then possible
to extend the
polynucleotide sequence in both directions to determine a full length gene
sequence.
Conveniently, such sequencing is performed, for example, using denatured
double stranded
DNA prepared from a plasmid clone. Suitable techniques are described by
Maniatis, T.,
Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL,
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
(1989). (see
in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-
Stranded
DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to
obtain
a full length gene sequence. Illustrative of the invention, each
polynucleotide set out in SEQ
ID N0:3 was discovered in a DNA library derived from Neisseria meningitidis.
Moreover, the DNA sequence set out in SEQ ID N0:3 contains an open reading
frame
encoding a protein having about the number of amino acid residues set forth in
SEQ ID N0:4
with a deduced molecular weight that can be calculated using amino acid
residue molecular
weight values well known to those skilled in the art.
The polynucleotide of SEQ ID N0:3, between the start codon at nucleotide
number l and the
stop codon which begins at nucleotide number 1402 of SEQ ID N0:3, encodes the
polypeptide of SEQ ID N0:4.
In a further aspect, the present invention provides for an isolated
polynucleotide comprising
or consisting of:
24
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) a polynucleotide sequence which has at least 85% identity, more preferably
at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity to SEQ ID N0:3 over the entire length of SEQ ID N0:3; or
(b) a polynucleotide sequence encoding a polypeptide which has at least 85%
identity,
more preferably at least 90% identity, yet more preferably at least 95%
identity, even
more preferably at least 97-99% or 100% exact, to the amino acid sequence of
SEQ ID
N0:4 over the entire length of SEQ ID N0:4.
A polynucleotide encoding a polypeptide of the present invention, including
homologs and
orthologs from species other than Neisseria meningitides, may be obtained by a
process which
comprises the steps of screening an appropriate library under stringent
hybridization
conditions (for example, using a temperature in the range of 45 - 65°C
and an SDS
concentration from 0.1 - 1 %) with a labeled or detectable probe consisting of
or comprising
the sequence of SEQ ID N0:3 or a fragment thereof; and isolating a full-length
gene and/or
genomic clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire
length to a coding
sequence (open reading frame) in SEQ ID N0:3. Also provided by the invention
is a coding
sequence for a mature polypeptide or a fragment thereof, by itself as well as
a coding
sequence for a mature polypeptide or a fragment in reading frame with another
coding
sequence, such as a sequence encoding a leader or secretory sequence, a pre-,
or pro- or
prepro-protein sequence. The polynucleotide of the invention may also contain
at least one
non-coding sequence, including for example, but not limited to at least one
non-coding 5' and
3' sequence, such as the transcribed but non-translated sequences, termination
signals (such as
rho-dependent and rho-independent termination signals), ribosome binding
sites, Kozak
sequences, sequences that stabilize mRNA, introns, and polyadenylation
signals. The
polynucleotide sequence may also comprise additional coding sequence encoding
additional
amino acids. For example, a marker sequence that facilitates purification of
the fused
polypeptide can be encoded. In certain embodiments of the invention, the
marker sequence is
a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and
described in Gentz
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
et al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag
(Wilson et al.,
Cell 37: 767 (1984), both of which may be useful in purifying polypeptide
sequence fused to
them. Polynucleotides of the invention also include, but are not limited to,
polynucleotides
comprising a structural gene and its naturally associated sequences that
control gene
expression.
The nucleotide sequence encoding BASB057 polypeptide of SEQ ID N0:4 may be
identical to the polypeptide encoding sequence contained in nucleotides 1 to
1401 of SEQ
ID N0:3. Alternatively it may be a sequence, which as a result of the
redundancy
(degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID N0:4.
The term "polynucleotide encoding a polypeptide" as used herein encompasses
polynucleotides that include a sequence encoding a polypeptide of the
invention, particularly
a bacterial polypeptide and more particularly a polypeptide of the Neisseria
meningitides
BASB057 having an amino acid sequence set out in SEQ ID N0:4. The term also
encompasses polynucleotides that include a single continuous region or
discontinuous regions
encoding the polypeptide (for example, polynucleotides interrupted by
integrated phage, an
integrated insertion sequence, an integrated vector sequence, an integrated
transposon
sequence, or due to RNA editing or genomic DNA reorganization) together with
additional
regions, that also may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described
herein that encode
variants of a polypeptide having a deduced amino acid sequence of SEQ ID N0:4.
Fragments of polynucleotides of the invention may be used, for example, to
synthesize full-
length polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding
BASB057 variants,
that have the amino acid sequence of BASB057 polypeptide of SEQ ID N0:4 in
which
several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are
substituted, modified,
deleted and/or added, in any combination. Especially preferred among these are
silent
26
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
substitutions, additions and deletions, that do not alter the properties and
activities of
BASB057 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at
least 85%
identical over their entire length to a polynucleotide encoding BASB057
polypeptide having
an amino acid sequence set out in SEQ ID N0:4 and polynucleotides that are
complementary
to such polynucleotides. In this regard, polynucleotides at least 90%
identical over their entire
length to the same are particularly preferred, and among these particularly
preferred
polynucleotides, those with at least 95% are especially preferred.
Furthermore, those with at
least 97% are highly preferred among those with at least 95%, and among these
those with at
least 98% and at least 99% are particularly highly preferred, with at least
99% being the more
preferred.
Preferred embodiments are polynucleotides encoding polypeptides that retain
substantially
the same biological function or activity as the mature polypeptide encoded by
a DNA of SEQ
ID N0:3.
In accordance with certain preferred embodiments of this invention there are
provided
polynucleotides that hybridize, particularly under stringent conditions, to
BASB057
polynucleotide sequences, such as those polynucleotides in SEQ ID N0:3.
The invention further relates to polynucleotides that hybridize to the
polynucleotide
sequences provided herein. In this regard, the invention especially relates to
polynucleotides
that hybridize under stringent conditions to the polynucleotides described
herein. As herein
used, the terms "stringent conditions" and "stringent hybridization
conditions" mean
hybridization 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,
27
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
followed by washing the hybridization support in O.lx SSC at about
65°C. Hybridization
and wash conditions are well known and exemplified in Sambrook, et al.,
Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,
(1989),
particularly Chapter 11 therein. Solution hybridization may also be used with
the
polynucleotide sequences provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a
polynucleotide
sequence obtained by screening an appropriate library containing the complete
gene for a
polynucleotide sequence set forth in SEQ ID N0:3 under stringent hybridization
conditions
with a probe having the sequence of said polynucleotide sequence set forth in
SEQ ID
N0:3 or a fragment thereof; and isolating said polynucleotide sequence.
Fragments useful
for obtaining such a polynucleotide include, for example, probes and primers
fully
described elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the
invention, for instance,
the polynucleotides of the invention, may be used as a hybridization probe for
RNA, cDNA
and genomic DNA to isolate full-length cDNAs and genomic clones encoding
BASB057 and
to isolate cDNA and genomic clones of other genes that have a high identity,
particularly high
sequence identity, to the BASB057 gene. Such probes generally will comprise at
least 15
nucleotide residues or base pairs. Preferably, such probes will have at least
30 nucleotide
residues or base pairs and may have at least 50 nucleotide residues or base
pairs. Particularly
preferred probes will have at least 20 nucleotide residues or base pairs and
will have less than
nucleotide residues or base pairs.
25 A coding region of a BASB057 gene may be isolated by screening using a DNA
sequence
provided in SEQ ID N0:3 to synthesize an oligonucleotide probe. A labeled
oligonucleotide
having a sequence complementary to that of a gene of the invention is then
used to screen a
library of cDNA, genomic DNA or mRNA to determine which members of the library
the
probe hybridizes to.
28
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
It is an object of the invention to provide polynucleotides that encode
BASB060
polypeptides, particularly polynucleotides that encode the polypeptide herein
designated
BASB060.
In a particularly preferred embodiment of the invention the polynucleotide
comprises a region
encoding BASB060 polypeptides comprising a sequence set out in SEQ ID NO:S
which
includes a full length gene, or a variant thereof.
The BASB060 polynucleotide provided in SEQ ID NO:S is the BASB060
polynucleotide
from Neisseria meningitides strains ATCC13090.
As a further aspect of the invention there are provided isolated nucleic acid
molecules
encoding and/or expressing BASB060 polypeptides and polynucleotides,
particularly
Neisseria meningitides BASB060 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 BASB060 polypeptide having a deduced amino acid
sequence of
SEQ ID N0:6 and polynucleotides closely related thereto and variants thereof.
In another particularly preferred embodiment of the invention there is a
BASB060
polypeptide from Neisseria meningitides comprising or consisting of an amino
acid
sequence of SEQ ID N0:6 or a variant thereof.
Using the information provided herein, such as a polynucleotide sequence set
out in SEQ ID
NO:~ a polynucleotide of the invention encoding BASB060 polypeptide may be
obtained
using standard cloning and screening methods, such as those for cloning and
sequencing
29
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
chromosomal DNA fragments from bacteria using Neisseria meningitides cells as
starting
material, followed by obtaining a full length clone. For example, to obtain a
polynucleotide
sequence of the invention, such as a polynucleotide sequence given in SEQ ID
N0:5
typically a library of clones of chromosomal DNA of Neisseria meningitides in
E. coli or
some other suitable host is probed with a radiolabeled oligonucleotide,
preferably a 17-mer
or longer, derived from a partial sequence. Clones carrying DNA identical to
that of the
probe can then be distinguished using stringent hybridization conditions. By
sequencing
the individual clones thus identified by hybridization with sequencing primers
designed
from the original polypeptide or polynucleotide sequence it is then possible
to extend the
polynucleotide sequence in both directions to determine a full length gene
sequence.
Conveniently, such sequencing is performed, for example, using denatured
double stranded
DNA prepared from a plasmid clone. Suitable techniques are described by
Maniatis, T.,
Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL,
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
(1989). (see
in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-
Stranded
DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to
obtain
a full length gene sequence. Illustrative of the invention, each
polynucleotide set out in SEQ
ID N0:5 was discovered in a DNA library derived from Neisseria meningitides.
Moreover, the DNA sequence set out in SEQ ID N0:5 contains an open reading
frame
encoding a protein having about the number of amino acid residues set forth in
SEQ ID N0:6
with a deduced molecular weight that can be calculated using amino acid
residue molecular
weight values well known to those skilled in the art.
The polynucleotide of SEQ ID N0:5, between the start codon at nucleotide
number l and the
stop codon which begins at nucleotide number 418 of SEQ ID N0:5, encodes the
polypeptide
of SEQ ID N0:6.
In a further aspect, the present invention provides for an isolated
polynucleotide comprising
or consisting of:
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) a polynucleotide sequence which has at least 85% identity, more preferably
at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity to SEQ ID NO:S over the entire length of SEQ ID NO:S; or
(b) a polynucleotide sequence encoding a polypeptide which has at least 85%
identity,
more preferably at least 90% identity, yet more preferably at least 95%
identity, even
more preferably at least 97-99% or 100% exact, to the amino acid sequence of
SEQ ID
N0:6 over the entire length of SEQ ID N0:6.
A polynucleotide encoding a polypeptide of the present invention, including
homologs and
orthologs from species other than Neisseria meningitidis, 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 4~ - 65°C
and an SDS
concentration from 0.1 -1 %) with a labeled or detectable probe consisting of
or comprising
the sequence of SEQ ID NO:S or a fragment thereof; and isolating a full-length
gene and/or
genomic clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire
length to a coding
sequence (open reading frame) in SEQ ID NO:S. 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
31
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
et al., Proc. Natl. Acad Sci., USA 86: 821-824 (1989), or an HA peptide tag
(Wilson et al.,
Cell 37: 767 (1984), both of which may be useful in purifying polypeptide
sequence fused to
them. Polynucleotides of the invention also include, but are not limited to,
polynucleotides
comprising a structural gene and its naturally associated sequences that
control gene
expression.
The nucleotide sequence encoding BASB060 polypeptide of SEQ ID N0:6 may be
identical to the polypeptide encoding sequence contained in nucleotides 1 to
417 of SEQ ID
NO:S. Alternatively it may be a sequence, which as a result of the redundancy
(degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID N0:6.
The term "polynucleotide encoding a polypeptide" as used herein encompasses
polynucleotides that include a sequence encoding a polypeptide of the
invention, particularly
a bacterial polypeptide and more particularly a polypeptide of the Neisseria
meningitidis
BASB060 having an amino acid sequence set out in SEQ ID N0:6. The term also
encompasses polynucleotides that include a single continuous region or
discontinuous regions
encoding the polypeptide (for example, polynucleotides interrupted by
integrated phage, an
integrated insertion sequence, an integrated vector sequence, an integrated
transposon
sequence, or due to RNA editing or genomic DNA reorganization) together with
additional
regions, that also may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described
herein that encode
variants of a polypeptide having a deduced amino acid sequence of SEQ ID N0:6.
Fragments of polynucleotides of the invention may be used, for example, to
synthesize full-
length polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding
BASB060 variants,
that have the amino acid sequence of BASB060 polypeptide of SEQ ID N0:6 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
32
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
substitutions, additions and deletions that do not alter the properties and
activities of
BASB060 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at
least 85%
identical over their entire length to a polynucleotide encoding BASB060
polypeptide having
an amino acid sequence set out in SEQ ID N0:6 and polynucleotides that are
complementary
to such polynucleotides. In this regard, polynucleotides at least 90%
identical over their entire
length to the same are particularly preferred, and among these particularly
preferred
polynucleotides, those with at least 95% are especially preferred.
Furthermore, those with at
least 97% are highly preferred among those with at least 95%, and among these
those with at
least 98% and at least 99% are particularly highly preferred, with at least
99% being the more
preferred.
Preferred embodiments are polynucleotides encoding polypeptides that retain
substantially
the same biological function or activity as the mature polypeptide encoded by
a DNA of SEQ
ID NO:S.
In accordance with certain preferred embodiments of this invention there are
provided
polynucleotides that hybridize, particularly under stringent conditions, to
BASB060
polynucleotide sequences, such as those polynucleotides in SEQ ID NO:S.
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,
33
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
followed by washing the hybridization support in O.lx SSC at about
65°C. Hybridization
and wash conditions are well known and exemplified in Sambrook, et al.,
Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,
(1989),
particularly Chapter 11 therein. Solution hybridization may also be used with
the
polynucleotide sequences provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a
polynucleotide
sequence obtained by screening an appropriate library containing the complete
gene for a
polynucleotide sequence set forth in SEQ ID NO:S under stringent hybridization
conditions
with a probe having the sequence of said polynucleotide sequence set forth in
SEQ ID
NO:S or a fragment thereof; and isolating said polynucleotide sequence.
Fragments useful
for obtaining such a polynucleotide include, for example, probes and primers
fully
described elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the
invention, for instance,
the polynucleotides of the invention, may be used as a hybridization probe for
RNA, cDNA
and genomic DNA to isolate full-length cDNAs and genomic clones encoding
BASB060 and
to isolate cDNA and genomic clones of other genes that have a high identity,
particularly high
sequence identity, to the BASB060 gene. Such probes generally will comprise at
least 15
nucleotide residues or base pairs. Preferably, such probes will have at least
30 nucleotide
residues or base pairs and may have at least 50 nucleotide residues or base
pairs. Particularly
preferred probes will have at least 20 nucleotide residues or base pairs and
will have less than
nucleotide residues or base pairs.
25 A coding region of a BASB060 gene may be isolated by screening using a DNA
sequence
provided in SEQ ID NO:S 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.
34
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
It is an object of the invention to provide polynucleotides that encode
BASB061
polypeptides, particularly polynucleotides that encode the polypeptide herein
designated
BASB061.
In a particularly preferred embodiment of the invention the polynucleotide
comprises a region
encoding BASB061 polypeptides comprising a sequence set out in SEQ ID N0:7
which
includes a full length gene, or a variant thereof.
The BASB061 polynucleotide provided in SEQ ID N0:7 is the BASB061
polynucleotide
from Neisseria meningitides strains ATCC13090.
As a further aspect of the invention there are provided isolated nucleic acid
molecules
encoding and/or expressing BASB061 polypeptides and polynucleotides,
particularly
Neisseria meningitides BASB061 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 BASB061 polypeptide having a deduced amino acid
sequence of
SEQ ID N0:8 and polynucleotides closely related thereto and variants thereof.
In another particularly preferred embodiment of the invention there is a
BASB061
polypeptide from Neisseria meningitides comprising or consisting of an amino
acid
sequence of SEQ ID N0:8 or a variant thereof.
Using the information provided herein, such as a polynucleotide sequence set
out in SEQ ID
N0:7 .a polynucleotide of the invention encoding BASB061 polypeptide may be
obtained
using standard cloning and screening methods, such as those for cloning and
sequencing
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
chromosomal DNA fragments from bacteria using Neisseria meningitides cells as
starting
material, followed by obtaining a full length clone. For example, to obtain a
polynucleotide
sequence of the invention, such as a polynucleotide sequence given in SEQ ID
N0:7
typically a library of clones of chromosomal DNA of Neisseria meningitides in
E.coli or
some other suitable host is probed with a radiolabeled oligonucleotide,
preferably a 17-mer
or longer, derived from a partial sequence. Clones carrying DNA identical to
that of the
probe can then be distinguished using stringent hybridization conditions. By
sequencing
the individual clones thus identified by hybridization with sequencing primers
designed
from the original polypeptide or polynucleotide sequence it is then possible
to extend the
polynucleotide sequence in both directions to determine a full length gene
sequence.
Conveniently, such sequencing is performed, for example, using denatured
double stranded
DNA prepared from a plasmid clone. Suitable techniques are described by
Maniatis, T.,
Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL,
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
(1989). (see
in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-
Stranded
DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to
obtain
a full length gene sequence. Illustrative of the invention, each
polynucleotide set out in SEQ
ID N0:7 was discovered in a DNA library derived from Neisseria meningitides.
Moreover, the DNA sequence set out in SEQ ID N0:7 contains an open reading
frame
encoding a protein having about the number of amino acid residues set forth in
SEQ ID N0:8
with a deduced molecular weight that can be calculated using amino acid
residue molecular
weight values well known to those skilled in the art.
The polynucleotide of SEQ ID N0:7, between the start codon at nucleotide
number 1 and the
stop codon which begins at nucleotide number 514 of SEQ ID N0:7, encodes the
polypeptide
of SEQ ID N0:8.
In a further aspect, the present invention provides for an isolated
polynucleotide comprising
or consisting of:
36
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) a polynucleotide sequence which has at least 85% identity, more preferably
at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity to SEQ ID N0:7 over the entire length of SEQ ID N0:7; or
(b) a polynucleotide sequence encoding a polypeptide which has at least 85%
identity,
more preferably at least 90% identity, yet more preferably at least 95%
identity, even
more preferably at least 97-99% or 100% exact, to the amino acid sequence of
SEQ ID
N0:8 over the entire length of SEQ ID N0:8.
A polynucleotide encoding a polypeptide of the present invention, including
homologs and
orthologs from species other than Neisseria meningitidis, may be obtained by a
process which
comprises the steps of screening an appropriate library under stringent
hybridization
conditions (for example, using a temperature in the range of 45 - 65°C
and an SDS
concentration from 0.1 -1 %) with a labeled or detectable probe consisting of
or comprising
the sequence of SEQ ID N0:7 or a fragment thereof; and isolating a full-length
gene and/or
genomic clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire
length to a coding
sequence (open reading frame) in SEQ ID N0:7. 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
37
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
et al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag
(Wilson et al.,
Cell 37: 767 (1984), both of which may be useful in purifying polypeptide
sequence fused to
them. Polynucleotides of the invention also include, but are not limited to,
polynucleotides
comprising a structural gene and its naturally associated sequences that
control gene
S expression.
The nucleotide sequence encoding BASB061 polypeptide of SEQ ID N0:8 may be
identical to the polypeptide encoding sequence contained in nucleotides 1 to
513 of SEQ ID
N0:7. Alternatively it may be a sequence, which as a result of the redundancy
(degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID N0:8.
The term "polynucleotide encoding a polypeptide" as used herein encompasses
polynucleotides that include a sequence encoding a polypeptide of the
invention, particularly
a bacterial polypeptide and more particularly a polypeptide of the Neisseria
meningitidis
BASB061 having an amino acid sequence set out in SEQ ID N0:8. The term also
encompasses polynucleotides that include a single continuous region or
discontinuous regions
encoding the polypeptide (for example, polynucleotides interrupted by
integrated phage, an
integrated insertion sequence, an integrated vector sequence, an integrated
transposon
sequence, or due to RNA editing or genomic DNA reorganization) together with
additional
regions, that also may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described
herein that encode
variants of a polypeptide having a deduced amino acid sequence of SEQ ID N0:8.
Fragments of polynucleotides of the invention may be used, for example, to
synthesize full-
length polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding
BASB061 variants,
that have the amino acid sequence of BASB061 polypeptide of SEQ ID N0:8 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
38
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
substitutions, additions and deletions that do not alter the properties and
activities of
BASB061 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at
least 85%
identical over their entire length to a polynucleotide encoding BASB061
polypeptide having
an amino acid sequence set out in SEQ ID N0:8 and polynucleotides that are
complementary
to such polynucleotides. In this regard, polynucleotides at least 90%
identical over their entire
length to the same are particularly preferred, and among these particularly
preferred
polynucleotides, those with at least 95% are especially preferred.
Furthermore, those with at
least 97% are highly preferred among those with at least 95%, and among these
those with at
least 98% and at least 99% are particularly highly preferred, with at least
99% being the more
preferred.
Preferred embodiments are polynucleotides encoding polypeptides that retain
substantially
the same biological function or activity as the mature polypeptide encoded by
a DNA of SEQ
ID N0:7.
In accordance with certain preferred embodiments of this invention there are
provided
polynucleotides that hybridize, particularly under stringent conditions, to
BASB061
polynucleotide sequences, such as those polynucleotides in SEQ ID N0:7.
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,
~x SSC (150mM
NaCI, lSmM trisodium citrate), 50 mM sodium phosphate (pH'l.6), Sx Denhardt's
solution,
10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm
DNA,
39
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
followed by washing the hybridization support in O.lx SSC at about
65°C. Hybridization
and wash conditions are well known and exemplified in Sambrook, et al.,
Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,
(1989),
particularly Chapter 11 therein. Solution hybridization may also be used with
the
polynucleotide sequences provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a
polynucleotide
sequence obtained by screening an appropriate library containing the complete
gene for a
polynucleotide sequence set forth in SEQ ID N0:7 under stringent hybridization
conditions
with a probe having the sequence of said polynucleotide sequence set forth in
SEQ ID
N0:7 or a fragment thereof; and isolating said polynucleotide sequence.
Fragments useful
for obtaining such a polynucleotide include, for example, probes and primers
fully
described elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the
invention, for instance,
the polynucleotides of the invention, may be used as a hybridization probe for
RNA, cDNA
and genomic DNA to isolate full-length cDNAs and genomic clones encoding
BASB061 and
to isolate cDNA and genomic clones of other genes that have a high identity,
particularly high
sequence identity, to the BASB061 gene. Such probes generally will comprise at
least 15
nucleotide residues or base pairs. Preferably, such probes will have at least
30 nucleotide
residues or base pairs and may have at least 50 nucleotide residues or base
pairs. Particularly
preferred probes will have at least 20 nucleotide residues or base pairs and
will have less than
nucleotide residues or base pairs.
25 A coding region of a BASB061 gene may be isolated by screening using a DNA
sequence
provided in SEQ ID N0:7 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.
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
It is an object of the invention to provide polynucleotides that encode
BASB063
polypeptides, particularly polynucleotides that encode the polypeptide herein
designated
BASB063.
In a particularly preferred embodiment of the invention the polynucleotide
comprises a region
encoding BASB063 polypeptides comprising a sequence set out in SEQ ID N0:9
which
includes a full length gene, or a variant thereof.
The BASB063 polynucleotide provided in SEQ ID N0:9 is the BASB063
polynucleotide
from Neisseria meningitides strains ATCC13090.
As a further aspect of the invention there are provided isolated nucleic acid
molecules
encoding and/or expressing BASB063 polypeptides and polynucleotides,
particularly
Neisseria meningitides BASB063 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 BASB063 polypeptide having a deduced amino acid
sequence of
SEQ ID NO:10 and polynucleotides closely related thereto and variants thereof.
In another particularly preferred embodiment of the invention there is a
BASB063
polypeptide from Neisseria meningitides comprising or consisting of an amino
acid
sequence of SEQ ID NO:10 or a variant thereof.
Using the information provided herein, such as a polynucleotide sequence set
out in SEQ ID
N0:9 a polynucleotide of the invention encoding BASB063 polypeptide may be
obtained
using standard cloning and screening methods, such as those for cloning and
sequencing
41
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
chromosomal DNA fragments from bacteria using Neisseria meningitides cells as
starting
material, followed by obtaining a full length clone. For example, to obtain a
polynucleotide
sequence of the invention, such as a polynucleotide sequence given in SEQ ID
N0:9
typically a library of clones of chromosomal DNA of Neisseria meningitides in
E. coli or
some other suitable host is probed with a radiolabeled oligonucleotide,
preferably a 17-mer
or longer, derived from a partial sequence. Clones carrying DNA identical to
that of the
probe can then be distinguished using stringent hybridization conditions. By
sequencing
the individual clones thus identified by hybridization with sequencing primers
designed
from the original polypeptide or polynucleotide sequence it is then possible
to extend the
polynucleotide sequence in both directions to determine a full length gene
sequence.
Conveniently, such sequencing is performed, for example, using denatured
double stranded
DNA prepared from a plasmid clone. Suitable techniques are described by
Maniatis, T.,
Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL,
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
(1989). (see
in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-
Stranded
DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to
obtain
a full length gene sequence. Illustrative of the invention, each
polynucleotide set out in SEQ
ID N0:9 was discovered in a DNA library derived from Neisseria meningitides.
Moreover, the DNA sequence set out in SEQ ID N0:9 contains an open reading
frame
encoding a protein having about the number of amino acid residues set forth in
SEQ ID
NO:10 with a deduced molecular weight that can be calculated using amino acid
residue
molecular weight values well known to those skilled in the art.
The polynucleotide of SEQ ID N0:9, between the start codon at nucleotide
number l and the
stop codon which begins at nucleotide number 814 of SEQ ID N0:9, encodes the
polypeptide
of SEQ ID NO:10.
In a further aspect, the present invention provides for an isolated
polynucleotide comprising
or consisting of:
42
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) a polynucleotide sequence which has at least 85% identity, more preferably
at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity to SEQ ID N0:9 over the entire length of SEQ ID N0:9; or
(b) a polynucleotide sequence encoding a polypeptide which has at least 85%
identity,
more preferably at least 90% identity, yet more preferably at least 95%
identity, even
more preferably at least 97-99% or 100% exact, to the amino acid sequence of
SEQ ID
NO:10 over the entire length of SEQ ID NO:10.
A polynucleotide encoding a polypeptide of the present invention, including
homologs and
orthologs from species other than Neisseria meningitides, may be obtained by a
process which
comprises the steps of screening an appropriate library under stringent
hybridization
conditions (for example, using a temperature in the range of 45 - 65°C
and an SDS
concentration from 0.1 - 1 %) with a labeled or detectable probe consisting of
or comprising
the sequence of SEQ ID N0:9 or a fragment thereof; and isolating a full-length
gene and/or
genomic clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire
length to a coding
sequence (open reading frame) in SEQ ID N0:9. 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
43
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
et al., Proc. Natl. Acad Sci., USA 86.~ 821-824 (1989), or an HA peptide tag
(Wilson et al.,
Cell 37.~ 767 (1984), both of which may be useful in purifying polypeptide
sequence fused to
them. Polynucleotides of the invention also include, but are not limited to,
polynucleotides
comprising a structural gene and its naturally associated sequences that
control gene
expression.
The nucleotide sequence encoding BASB063 polypeptide of SEQ ID NO:10 may be
identical to the polypeptide encoding sequence contained in nucleotides 1 to
813 of SEQ ID
N0:9. Alternatively it may be a sequence, which as a result of the redundancy
(degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID
NO:10.
The term "polynucleotide encoding a polypeptide" as used herein encompasses
polynucleotides that include a sequence encoding a polypeptide of the
invention, particularly
a bacterial polypeptide and more particularly a polypeptide of the Neisseria
meningitides
BASB063 having an amino acid sequence set out in SEQ ID NO:10. The term also
encompasses polynucleotides that include a single continuous region or
discontinuous regions
encoding the polypeptide (for example, polynucleotides interrupted by
integrated phage, an
integrated insertion sequence, an integrated vector sequence, an integrated
transposon
sequence, or due to RNA editing or genomic DNA reorganization) together with
additional
regions, that also may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described
herein that encode
variants of a polypeptide having a deduced amino acid sequence of SEQ ID
NO:10.
Fragments of polynucleotides of the invention may be used, for example, to
synthesize full-
length polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding
BASB063 variants,
that have the amino acid sequence of BASB063 polypeptide of SEQ ID NO:10 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
44
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
substitutions, additions and deletions that do not alter the properties and
activities of
BASB063 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at
least 85%
identical over their entire length to a polynucleotide encoding BASB063
polypeptide having
an amino acid sequence set out in SEQ ID NO:10 and polynucleotides that are
complementary to such polynucleotides. In this regard, polynucleotides at
least 90% identical
over their entire length to the same are particularly preferred, and among
these particularly
preferred polynucleotides, those with at least 95% are especially preferred.
Furthermore,
those with at least 97% are highly preferred among those with at least 95%,
and among these
those with at least 98% and at least 99% are particularly highly preferred,
with at least 99%
being the more preferred.
Preferred embodiments are polynucleotides encoding polypeptides that retain
substantially
the same biological function or activity as the mature polypeptide encoded by
a DNA of SEQ
ID N0:9.
In accordance with certain preferred embodiments of this invention there are
provided
polynucleotides that hybridize, particularly under stringent conditions, to
BASB063
polynucleotide sequences, such as those polynucleotides in SEQ ID N0:9.
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, 1 ~mM trisodium citrate), ~0 mM sodium phosphate (pH7.6), Sx Denhardt's
solution,
10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm
DNA,
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
followed by washing the hybridization support in O.lx SSC at about
65°C. Hybridization
and wash conditions are well known and exemplified in Sambrook, et al.,
Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,
(1989),
particularly Chapter 11 therein. Solution hybridization may also be used with
the
polynucleotide sequences provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a
polynucleotide
sequence obtained by screening an appropriate library containing the complete
gene for a
polynucleotide sequence set forth in SEQ ID N0:9 under stringent hybridization
conditions
with a probe having the sequence of said polynucleotide sequence set forth in
SEQ ID
N0:9 or a fragment thereof; and isolating said polynucleotide sequence.
Fragments useful
for obtaining such a polynucleotide include, for example, probes and primers
fully
described elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the
invention, for instance,
the polynucleotides of the invention, may be used as a hybridization probe for
RNA, cDNA
and genomic DNA to isolate full-length cDNAs and genomic clones encoding
BASB063 and
to isolate cDNA and genomic clones of other genes that have a high identity,
particularly high
sequence identity, to the BASB063 gene. Such probes generally will comprise at
least 15
nucleotide residues or base pairs. Preferably, such probes will have at least
30 nucleotide
residues or base pairs and may have at least 50 nucleotide residues or base
pairs. Particularly
preferred probes will have at least 20 nucleotide residues or base pairs and
will have less than
nucleotide residues or base pairs.
25 A coding region of a BASB063 gene may be isolated by screening using a DNA
sequence
provided in SEQ ID N0:9 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.
46
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
It is an object of the invention to provide polynucleotides that encode
BASB065
polypeptides, particularly polynucleotides that encode the polypeptide herein
designated
BASB065.
In a particularly preferred embodiment of the invention the polynucleotide
comprises a region
encoding BASB065 polypeptides comprising a sequence set out in SEQ ID NO:11
which
includes a full length gene, or a variant thereof.
The BASB065 polynucleotide provided in SEQ ID NO:11 is the BASB065
polynucleotide from Neisseria meningitides strains ATCC13090.
As a further aspect of the invention there are provided isolated nucleic acid
molecules
encoding and/or expressing BASB065 polypeptides and polynucleotides,
particularly
Neisseria meningitides BASB065 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 BASB065 polypeptide having a deduced amino acid
sequence of
SEQ ID N0:12 and polynucleotides closely related thereto and variants thereof.
In another particularly preferred embodiment of the invention there is a
BASB065
polypeptide from Neisseria meningitides comprising or consisting of an amino
acid
sequence of SEQ ID N0:12 or a variant thereof.
Using the information provided herein, such as a polynucleotide sequence set
out in SEQ ID
NO:11 a polynucleotide of the invention encoding BASB065 polypeptide may be
obtained
using standard cloning and screening methods, such as those for cloning and
sequencing
47
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
chromosomal DNA fragments from bacteria using Neisseria meningitides cells as
starting
material, followed by obtaining a full length clone. For example, to obtain a
polynucleotide
sequence of the invention, such as a polynucleotide sequence given in SEQ ID
NO:11
typically a library of clones of chromosomal DNA of Neisseria meningitides in
E. coli or
some other suitable host is probed with a radiolabeled oligonucleotide,
preferably a 17-mer
or longer, derived from a partial sequence. Clones carrying DNA identical to
that of the
probe can then be distinguished using stringent hybridization conditions. By
sequencing
the individual clones thus identified by hybridization with sequencing primers
designed
from the original polypeptide or polynucleotide sequence it is then possible
to extend the
polynucleotide sequence in both directions to determine a full length gene
sequence.
Conveniently, such sequencing is performed, for example, using denatured
double stranded
DNA prepared from a plasmid clone. Suitable techniques are described by
Maniatis, T.,
Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL,
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
(1989). (see
in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-
Stranded
DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to
obtain
a full length gene sequence. Illustrative of the invention, each
polynucleotide set out in SEQ
ID NO:11 was discovered in a DNA library derived from Neisseria meningitides.
Moreover, the DNA sequence set out in SEQ ID NO:11 contains an open reading
frame
encoding a protein having about the number of amino acid residues set forth in
SEQ ID
N0:12 with a deduced molecular weight that can be calculated using amino acid
residue
molecular weight values well known to those skilled in the art.
The polynucleotide of SEQ ID NO:l 1, between the start codon at nucleotide
number l and
the stop codon which begins at nucleotide number 715 of SEQ ID NO:11, encodes
the
polypeptide of SEQ ID N0:12.
In a further aspect, the present invention provides for an isolated
polynucleotide comprising
or consisting of:
48
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) a polynucleotide sequence which has at least 85% identity, more preferably
at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity to SEQ ID NO:11 over the entire length of SEQ ID NO:11;
or
(b) a polynucleotide sequence encoding a polypeptide which has at least 85%
identity,
more preferably at least 90% identity, yet more preferably at least 95%
identity, even
more preferably at least 97-99% or 100% exact, to the amino acid sequence of
SEQ ID
N0:12 over the entire length of SEQ ID N0:12.
A polynucleotide encoding a polypeptide of the present invention, including
homologs and
orthologs from species other than Neisseria meningitidis, may be obtained by a
process which
comprises the steps of screening an appropriate library under stringent
hybridization
conditions (for example, using a temperature in the range of 45 - 65°C
and an SDS
concentration from 0.1 - 1 %) with a labeled or detectable probe consisting of
or comprising
the sequence of SEQ ID NO:11 or a fragment thereof; and isolating a full-
length gene and/or
genomic clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire
length to a coding
sequence (open reading frame) in SEQ ID NO:11. 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. IWcertain embodiments of the invention, the marker
sequence is
a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and
described in Gentz
49
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
et al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag
(Wilson et al.,
Cell 37: 767 ( 1984), both of which may be useful in purifying polypeptide
sequence fused to
them. Polynucleotides of the invention also include, but are not limited to,
polynucleotides
comprising a structural gene and its naturally associated sequences that
control gene
expression.
The nucleotide sequence encoding BASB065 polypeptide of SEQ ID N0:12 may be
identical to the polypeptide encoding sequence contained in nucleotides 1 to
714 of SEQ ID
NO:11. Alternatively it may be a sequence, which as a result of the redundancy
(degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID
N0:12.
The term "polynucleotide encoding a polypeptide" as used herein encompasses
polynucleotides that include a sequence encoding a polypeptide of the
invention, particularly
a bacterial polypeptide and more particularly a polypeptide of the Neisseria
meningitidis
BASB065 having an amino acid sequence set out in SEQ ID N0:12. The term also
encompasses polynucleotides that include a single continuous region or
discontinuous regions
encoding the polypeptide (for example, polynucleotides interrupted by
integrated phage, an
integrated insertion sequence, an integrated vector sequence, an integrated
transposon
sequence, or due to RNA editing or genomic DNA reorganization) together with
additional
regions, that also may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described
herein that encode
variants of a polypeptide having a deduced amino acid sequence of SEQ ID
N0:12.
Fragments of polynucleotides of the invention may be used, for example, to
synthesize full-
length polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding
BASB06~ variants,
that have the amino acid sequence of BASB065 polypeptide of SEQ ID N0:12 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
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
substitutions, additions and deletions, that do not alter the properties and
activities of
BASB065 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at
least 85%
identical over their entire length to a polynucleotide encoding BASB065
polypeptide having
an amino acid sequence set out in SEQ ID N0:12 and polynucleotides that are
complementary to such polynucleotides. In this regard, polynucleotides at
least 90% identical
over their entire length to the same are particularly preferred, and among
these particularly
preferred polynucleotides, those with at least 95% are especially preferred.
Furthermore,
those with at least 97% are highly preferred among those with at least 95%,
and among these
those with at least 98% and at least 99% are particularly highly preferred,
with at least 99%
being the more preferred.
Preferred embodiments are polynucleotides encoding polypeptides that retain
substantially
the same biological function or activity as the mature polypeptide encoded by
a DNA of SEQ
ID NO:11.
In accordance with certain preferred embodiments of this invention there are
provided
polynucleotides that hybridize, particularly under stringent conditions, to
BASB065
polynucleotide sequences, such as those polynucleotides in SEQ ID NO:11.
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,
51
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
followed by washing the hybridization support in O.lx SSC at about
65°C. Hybridization
and wash conditions are well known and exemplified in Sambrook, et al.,
Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,
(1989),
particularly Chapter 11 therein. Solution hybridization may also be used with
the
polynucleotide sequences provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a
polynucleotide
sequence obtained by screening an appropriate library containing the complete
gene for a
polynucleotide sequence set forth in SEQ ID NO:11 under stringent
hybridization
conditions with a probe having the sequence of said polynucleotide sequence
set forth in
SEQ ID NO:11 or a fragment thereof; and isolating said polynucleotide
sequence.
Fragments useful for obtaining such a polynucleotide include, for example,
probes and
primers fully described elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the
invention, for instance,
the polynucleotides of the invention, may be used as a hybridization probe for
RNA, cDNA
and genomic DNA to isolate full-length cDNAs and genomic clones encoding
BASB065 and
to isolate cDNA and genomic clones of other genes that have a high identity,
particularly high
sequence identity, to the BASB065 gene. Such probes generally will comprise at
least 15
nucleotide residues or base pairs. Preferably, such probes will have at least
30 nucleotide
residues or base pairs and may have at least 50 nucleotide residues or base
pairs. Particularly
preferred probes will have at least 20 nucleotide residues or base pairs and
will have less than
nucleotide residues or base pairs.
25 A coding region of a BASB065 gene may be isolated by screening using a DNA
sequence
provided in SEQ ID NO:11 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.
52
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
It is an object of the invention to provide polynucleotides that encode
BASB066
polypeptides, particularly polynucleotides that encode the polypeptide herein
designated
BASB066.
In a particularly preferred embodiment of the invention the polynucleotide
comprises a region
encoding BASB066 polypeptides comprising a sequence set out in SEQ ID N0:13
which
includes a full length gene, or a variant thereof.
The BASB066 polynucleotide provided in SEQ ID N0:13 is the BASB066
polynucleotide from Neisseria meningitides strains ATCC13090.
As a further aspect of the invention there are provided isolated nucleic acid
molecules
encoding and/or expressing BASB066 polypeptides and polynucleotides,
particularly
Neisseria meningitides BASB066 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 BASB066 polypeptide having a deduced amino acid
sequence of
SEQ ID N0:14 and polynucleotides closely related thereto and variants thereof.
In another particularly preferred embodiment of the invention there is a
BASB066
polypeptide from Neisseria meningitides comprising or consisting of an amino
acid
sequence of SEQ ID N0:14 or a variant thereof.
Using the information provided herein,, such as a polynucleotide sequence set
out in SEQ ID
N0:13 a polynucleotide of the invention encoding BASB066 polypeptide may be
obtained
using standard cloning and screening methods, such as those for cloning and
sequencing
53
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
chromosomal DNA fragments from bacteria using Neisseria meningitides cells as
starting
material, followed by obtaining a full length clone. For example, to obtain a
polynucleotide
sequence of the invention, such as a polynucleotide sequence given in SEQ ID
N0:13
typically a library of clones of chromosomal DNA of Neisseria meningitides in
E. coli or
some other suitable host is probed with a radiolabeled oligonucleotide,
preferably a 17-mer
or longer, derived from a partial sequence. Clones carrying DNA identical to
that of the
probe can then be distinguished using stringent hybridization conditions. By
sequencing
the individual clones thus identified by hybridization with sequencing primers
designed
from the original polypeptide or polynucleotide sequence it is then possible
to extend the
polynucleotide sequence in both directions to determine a full length gene
sequence.
Conveniently, such sequencing is performed, for example, using denatured
double stranded
DNA prepared from a plasmid clone. Suitable techniques are described by
Maniatis, T.,
Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL,
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
(1989). (see
in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-
Stranded
DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to
obtain
a full length gene sequence. Illustrative of the invention, each
polynucleotide set out in SEQ
ID N0:13 was discovered in a DNA library derived from Neisseria meningitides.
Moreover, the DNA sequence set out in SEQ ID N0:13 contains an open reading
frame
encoding a protein having about the number of amino acid residues set forth in
SEQ ID
N0:14 with a deduced molecular weight that can be calculated using amino acid
residue
molecular weight values well known to those skilled in the art.
The polynucleotide of SEQ ID N0:13, between the start codon at nucleotide
number l and
the stop codon which begins at nucleotide number 1174 of SEQ ID N0:13, encodes
the
polypeptide of SEQ ID N0:14.
In a further aspect, the present invention provides for an isolated
polynucleotide comprising
or consisting of:
54
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) a polynucleotide sequence which has at least 85% identity, more preferably
at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity to SEQ ID N0:13 over the entire length of SEQ ID N0:13;
or
(b) a polynucleotide sequence encoding a polypeptide which has at least 85%
identity,
more preferably at least 90% identity, yet more preferably at least 95%
identity, even
more preferably at least 97-99% or 100% exact, to the amino acid sequence of
SEQ ID
N0:14 over the entire length of SEQ ID N0:14.
A polynucleotide encoding a polypeptide of the present invention, including
homologs and
orthologs from species other than Neisseria meningitidis, may be obtained by a
process which
comprises the steps of screening an appropriate library under stringent
hybridization
conditions (for example, using a temperature in the range of 45 - 65°C
and an SDS
concentration from 0.1 - 1 %) with a labeled or detectable probe consisting of
or comprising
the sequence of SEQ ID N0:13 or a fragment thereof; and isolating a full-
length gene and/or
genomic clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire
length to a coding
sequence (open reading frame) in SEQ ID N0:13. 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
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
et al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag
(Wilson et al.,
Cell 37: 767 (1984), both of which may be useful in purifying polypeptide
sequence fused to
them. Polynucleotides of the invention also include, but are not limited to,
polynucleotides
comprising a structural gene and its naturally associated sequences that
control gene
expression.
The nucleotide sequence encoding BASB066 polypeptide of SEQ ID N0:14 may be
identical to the polypeptide encoding sequence contained in nucleotides 1 to
1173 of SEQ
ID N0:13. Alternatively it may be a sequence, which as a result of the
redundancy
(degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID
N0:14.
The term "polynucleotide encoding a polypeptide" as used herein encompasses
polynucleotides that include a sequence encoding a polypeptide of the
invention, particularly
a bacterial polypeptide and more particularly a polypeptide of the Neisseria
meningitidis
BASB066 having an amino acid sequence set out in SEQ ID N0:14. The term also
encompasses polynucleotides that include a single continuous region or
discontinuous regions
encoding the polypeptide (for example, polynucleotides interrupted by
integrated phage, an
integrated insertion sequence, an integrated vector sequence, an integrated
transposon
sequence, or due to RNA editing or genomic DNA reorganization) together with
additional
regions, that also may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described
herein that encode
variants of a polypeptide having a deduced amino acid sequence of SEQ ID
N0:14.
Fragments of polynucleotides of the invention may be used, for example, to
synthesize full-
length polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding
BASB066 variants,
that have the amino acid sequence of BASB066 polypeptide of SEQ ID N0:14 in
which
several, a few, ~ 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
56
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
substitutions, additions and deletions, that do not alter the properties and
activities of
BASB066 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at
least 85%
identical over their entire length to a polynucleotide encoding BASB066
polypeptide having
an amino acid sequence set out in SEQ ID N0:14 and polynucleotides that are
complementary to such polynucleotides. In this regard, polynucleotides at
least 90% identical
over their entire length to the same are particularly preferred, and among
these particularly
preferred polynucleotides, those with at least 95% are especially preferred.
Furthermore,
those with at least 97% are highly preferred among those with at least 95%,
and among these
those with at least 98% and at least 99% are particularly highly preferred,
with at least 99%
being the more preferred.
Preferred embodiments are polynucleotides encoding polypeptides that retain
substantially
the same biological function or activity as the mature polypeptide encoded by
a DNA of SEQ
ID N0:13.
In accordance with certain preferred embodiments of this invention there are
provided
polynucleotides that hybridize, particularly under stringent conditions, to
BASB066
polynucleotide sequences, such as those polynucleotides in SEQ ID N0:13.
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 (I~OmM
NaCI, I ~mM 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,
57
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
followed by washing the hybridization support in O.lx SSC at about
65°C. Hybridization
and wash conditions are well known and exemplified in Sambrook, et al.,
Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,
(1989),
particularly Chapter 11 therein. Solution hybridization may also be used with
the
polynucleotide sequences provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a
polynucleotide
sequence obtained by screening an appropriate library containing the complete
gene for a
polynucleotide sequence set forth in SEQ ID N0:13 under stringent
hybridization
conditions with a probe having the sequence of said polynucleotide sequence
set forth in
SEQ ID N0:13 or a fragment thereof; and isolating said polynucleotide
sequence.
Fragments useful for obtaining such a polynucleotide include, for example,
probes and
primers fully described elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the
invention, for instance,
the polynucleotides of the invention, may be used as a hybridization probe for
RNA, cDNA
and genomic DNA to isolate full-length cDNAs and genomic clones encoding
BASB066 and
to isolate cDNA and genomic clones of other genes that have a high identity,
particularly high
sequence identity, to the BASB066 gene. Such probes generally will comprise at
least 15
nucleotide residues or base pairs. Preferably, such probes will have at least
30 nucleotide
residues or base pairs and may have at least 50 nucleotide residues or base
pairs. Particularly
preferred probes will have at least 20 nucleotide residues or base pairs and
will have less than
nucleotide residues or base pairs.
25 A coding region of a BASB066 gene may be isolated by screening using a DNA
sequence
provided in SEQ ID N0:13 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.
58
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
It is an object of the invention to provide polynucleotides that encode
BASB071
polypeptides, particularly polynucleotides that encode the polypeptide herein
designated
BASB071.
In a particularly preferred embodiment of the invention the polynucleotide
comprises a region
encoding BASB071 polypeptides comprising a sequence set out in SEQ ID NO:15
which
includes a full length gene, or a variant thereof.
The BASB071 polynucleotide provided in SEQ ID NO:1 ~ is the BASB071
polynucleotide from Neisseria meningitides strains ATCC13090.
As a further aspect of the invention there are provided isolated nucleic acid
molecules
encoding and/or expressing BASB071 polypeptides and polynucleotides,
particularly
Neisseria meningitides BASB071 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 BASB071 polypeptide having a deduced amino acid
sequence of
SEQ ID N0:16 and polynucleotides closely related thereto and variants thereof.
In another particularly preferred embodiment of the invention there is a
BASB071
polypeptide from Neisseria meningitides comprising or consisting of an amino
acid
sequence of SEQ ID N0:16 or a variant thereof.
Using the information provided herein, such as a polynucleotide sequence set
out in SEQ ID
NO:15 a polynucleotide of the invention encoding BASB071 polypeptide may be
obtained
using standard cloning and screening methods, such as those for cloning and
sequencing
59
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
chromosomal DNA fragments from bacteria using Neisseria meningitides cells as
starting
material, followed by obtaining a full length clone. For example, to obtain a
polynucleotide
sequence of the invention, such as a polynucleotide sequence given in SEQ ID
NO:15
typically a library of clones of chromosomal DNA of Neisseria meningitides in
E. coli or
some other suitable host is probed with a radiolabeled oligonucleotide,
preferably a 17-mer
or longer, derived from a partial sequence. Clones carrying DNA identical to
that of the
probe can then be distinguished using stringent hybridization conditions. By
sequencing
the individual clones thus identified by hybridization with sequencing primers
designed
from the original polypeptide or polynucleotide sequence it is then possible
to extend the
polynucleotide sequence in both directions to determine a full length gene
sequence.
Conveniently, such sequencing is performed, for example, using denatured
double stranded
DNA prepared from a plasmid clone. Suitable techniques are described by
Maniatis, T
Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL,
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
(1989). (see
in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-
Stranded
DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to
obtain
a full length gene sequence. Illustrative of the invention, each
polynucleotide set out in SEQ
ID NO:15 was discovered in a DNA library derived from Neisseria meningitides.
Moreover, the DNA sequence set out in SEQ ID NO:15 contains an open reading
frame
encoding a protein having about the number of amino acid residues set forth in
SEQ ID
N0:16 with a deduced molecular weight that can be calculated using amino acid
residue
molecular weight values well known to those skilled in the art.
The polynucleotide of SEQ ID NO:15, between the start codon at nucleotide
number 1 and
the stop codon which begins at nucleotide number 805 of SEQ ID NO:15, encodes
the
polypeptide of SEQ ID N0:16.
In a further aspect, the present invention provides for an isolated
polynucleotide comprising
or consisting o~
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
(a) a polynucleotide sequence which has at least 85% identity, more preferably
at least
90% identity, yet more preferably at least 95% identity, even more preferably
at least 97-
99% or exact identity to SEQ ID NO:15 over the entire length of SEQ ID NO:1 ~;
or
(b) a polynucleotide sequence encoding a polypeptide which has at least 85%
identity,
more preferably at least 90% identity, yet more preferably at least 95%
identity, even
more preferably at least 97-99% or 100% exact, to the amino acid sequence of
SEQ ID
N0:16 over the entire length of SEQ ID N0:16.
A polynucleotide encoding a polypeptide of the present invention, including
homologs and
orthologs from species other than Neisseria meningitidis, may be obtained by a
process which
comprises the steps of screening an appropriate library under stringent
hybridization
conditions (for example, using a temperature in the range of 45 - 65°C
and an SDS
concentration from 0.1 - 1 %) with a labeled or detectable probe consisting of
or comprising
the sequence of SEQ ID NO:15 or a fragment thereof; and isolating a full-
length gene and/or
genomic clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire
length to a coding
sequence (open reading frame) in SEQ ID NO:15. 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
61
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
et al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag
(Wilson et al.,
Cell 37: 767 (1984), both of which may be useful in purifying polypeptide
sequence fused to
them. Polynucleotides of the invention also include, but are not limited to,
polynucleotides
comprising a structural gene and its naturally associated sequences that
control gene
expression.
The nucleotide sequence encoding BASB071 polypeptide of SEQ ID N0:16 may be
identical to the polypeptide encoding sequence contained in nucleotides 1 to
804 of SEQ ID
NO:1 ~. Alternatively it may be a sequence, which as a result of the
redundancy
(degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID
N0:16.
The term "polynucleotide encoding a polypeptide" as used herein encompasses
polynucleotides that include a sequence encoding a polypeptide of the
invention, particularly
a bacterial polypeptide and more particularly a polypeptide of the Neisseria
meningitides
BASB071 having an amino acid sequence set out in SEQ ID N0:16. 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 SEQ ID
N0:16.
Fragments of polynucleotides of the invention may be used, for example, to
synthesize full-
length polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding
BASB071 variants,
that have the amino acid sequence of BASB071 polypeptide of SEQ ID N0:16 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
62
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
substitutions, additions and deletions, that do not alter the properties and
activities of
BASB071 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at
least 85%
identical over their entire length to a polynucleotide encoding BASB071
polypeptide having
an amino acid sequence set out in SEQ ID N0:16 and polynucleotides that are
complementary to such polynucleotides. In this regard, polynucleotides at
least 90% identical
over their entire length to the same are particularly preferred, and among
these particularly
preferred polynucleotides, those with at least 95% are especially preferred.
Furthermore,
those with at least 97% are highly preferred among those with at least 95%,
and among these
those with at least 98% and at least 99% are particularly highly preferred,
with at least 99%
being the more preferred.
Preferred embodiments are polynucleotides encoding polypeptides that retain
substantially
the same biological function or activity as the mature polypeptide encoded by
a DNA of SEQ
ID NO:15.
In accordance with certain preferred embodiments of this invention there are
provided
polynucleotides that hybridize, particularly under stringent conditions, to
BASB071
polynucleotide sequences, such as those polynucleotides in SEQ ID NO:15.
The invention further relates to polynucleotides that hybridize to the
polynucleotide
sequences provided herein. In this regard, the invention especially relates to
polynucleotides
that hybridize under stringent conditions to the polynucleotides described
herein. As herein
used, the terms "stringent conditions" and "stringent hybridization
conditions" mean
hybridization occurnng only if there is at least 95% and preferably at least
97% identity
between the sequences. A specific example of stringent hybridization
conditions is
overnight incubation at 42°C in a solution comprising: 50% formamide,
Sx SSC (150mM
NaCI, lSmM trisodium citrate), 50 mM sodium phosphate (pH7.6), ~x Denhardt's
solution,
10% dextran sulfate, and 20 niicrograms/ml of denatured, sheared salmon sperm
DNA,
63
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
followed by washing the hybridization support in O.lx SSC at about
65°C. Hybridization
and wash conditions are well known and exemplified in Sambrook, et al.,
Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,
(1989),
particularly Chapter 11 therein. Solution hybridization may also be used with
the
polynucleotide sequences provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a
polynucleotide
sequence obtained by screening an appropriate library containing the complete
gene for a
polynucleotide sequence set forth in SEQ ID NO:15 under stringent
hybridization
conditions with a probe having the sequence of said polynucleotide sequence
set forth in
SEQ ID NO:15 or a fragment thereof; and isolating said polynucleotide
sequence.
Fragments useful for obtaining such a polynucleotide include, for example,
probes and
primers fully described elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the
invention, for instance,
the polynucleotides of the invention, may be used as a hybridization probe for
RNA, cDNA
and genomic DNA to isolate full-length cDNAs and genomic clones encoding
BASB071 and
to isolate cDNA and genomic clones of other genes that have a high identity,
particularly high
sequence identity, to the BASB071 gene. Such probes generally will comprise at
least 15
nucleotide residues or base pairs. Preferably, such probes will have at least
30 nucleotide
residues or base pairs and may have at least 50 nucleotide residues or base
pairs. Particularly
preferred probes will have at least 20 nucleotide residues or base pairs and
will have less than
nucleotide residues or base pairs.
25 A coding region of a BASB071 gene may be isolated by screening using a DNA
sequence
provided in SEQ ID NO:15 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.
64
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
There are several methods available and well known to those skilled in the art
to obtain
full-length DNAs, or extend short DNAs, for example those based on the method
of
Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al. ,
PNAS
USA 85: 8998-9002, 1988). Recent modifications of the technique, exemplified
by the
MarathonTM technology (Clontech Laboratories Inc.) for example, have
significantly
simplified the search for longer cDNAs. In the MarathonTM technology, cDNAs
have
been prepared from mRNA extracted from a chosen tissue and an ' adaptor'
sequence
ligated onto each end. Nucleic acid amplification (PCR) is then carried out to
amplify
the "missing" 5' end of the DNA using a combination of gene specific and
adaptor
specific oligonucleotide primers. The PCR reaction is then repeated using
"nested"
primers, that is, primers designed to anneal within the amplified product
(typically an
adaptor specific primer that anneals further 3' in the adaptor sequence and a
gene specific
primer that anneals further 5' in the selected gene sequence). The products of
this
reaction can then be analyzed by DNA sequencing and a full-length DNA
constructed
either by joining the product directly to the existing DNA to give a complete
sequence,
or carrying out a separate full-length PCR using the new sequence information
for the
design of the 5' primer.
The polynucleotides and polypeptides of the invention may be employed, for
example, as
research reagents and materials for discovery of treatments of and diagnostics
for diseases,
particularly human diseases, as further discussed herein relating to
polynucleotide assays.
The polynucleotides of the invention that are oligonucleotides derived from a
sequence of
SEQ ID NOS:1 - 16 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.
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
The invention also provides polynucleotides that encode a polypeptide that is
the mature
protein plus additional amino or carboxyl-terminal amino acids, or amino acids
interior to the
mature polypeptide (when the mature form has more than one polypeptide chain,
for
instance). Such sequences may play a role in processing of a protein from
precursor to a
mature form, may allow protein transport, may lengthen or shorten protein half
life or may
facilitate manipulation of a protein for assay or production, among other
things. As generally
is the case in vivo, the additional amino acids may be processed away from the
mature protein
by cellular enzymes.
For each and every polynucleotide of the invention there is provided a
polynucleotide
complementary to it. It is preferred that these complementary polynucleotides
are fully
complementary to each polynucleotide with which they are complementary.
A precursor protein, having a mature form of the polypeptide fused to one or
more
prosequences may be an inactive form of the polypeptide. When prosequences are
removed
such inactive precursors generally are activated. Some or all of the
prosequences may be
removed before activation. Generally, such precursors are called proproteins.
In addition to the standard A, G, C, T/IJ 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
66
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
prosequences, which generally are removed during processing steps that produce
active and
mature forms of the polypeptide.
In accordance with an aspect of the invention, there is provided the use of a
polynucleotide
of the invention for therapeutic or prophylactic purposes, in particular
genetic
immunization.
The use of a polynucleotide of the invention in genetic immunization will
preferably
employ a suitable delivery method such as direct injection of plasmid DNA into
muscles
(Wolff et al., Hum Mol Genet (1992) 1: 363, Manthorpe et al., Hum. Gene Ther.
(1983) 4:
419), delivery of DNA complexed with specific protein carriers (Wu et al.,
JBiol Chem.
(1989) 264: 16985), coprecipitation of DNA with calcium phosphate (Benvenisty
&
Reshef, PNAS USA, ( 1986) 83: 9551 ), encapsulation of DNA in various forms of
liposomes
(Kaneda et al., Science (1989) 243: 375), particle bombardment (Tang et al.,
Nature (1992)
356:152, Eisenbraun et al., DNA Cell Biol (1993) 12: 791) and in vivo
infection using
cloned retroviral vectors (Seeger et al., PNAS USA (1984) 81: 5849).
Vectors, Host Cells, Expression Systems
The invention also relates to vectors that comprise a polynucleotide or
polynucleotides of the
invention, host cells that are genetically engineered with vectors of the
invention and the
production of polypeptides of the invention by recombinant techniques. Cell-
free 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
67
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
are genetically engineered with such expression systems, and to the production
of
polypeptides of the invention by recombinant techniques.
For recombinant production of the polypeptides of the invention, host cells
can be genetically
engineered to incorporate expression systems or portions thereof or
polynucleotides of the
invention. Introduction of a polynucleotide into the host cell can be effected
by methods
described in many standard laboratory manuals, such as Davis, et al., BASIC
METHODS IN
MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULAR CLONING: A
LABORATORYMANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran
mediated
transfection, transvection, microinjection, cationic lipid-mediated
transfection,
electroporation, transduction, scrape loading, ballistic introduction and
infection.
Representative examples of appropriate hosts include bacterial cells, such as
cells of
streptococci, staphylococci, enterococci, E coli, streptomyces, cyanobacteria,
Bacillus
subtilis, Moraxella catarrhalis, Haemophilus influenzae and Neisseria
meningitides; fungal
cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete,
Candida
albicans and Aspergillus; insect cells such as cells of Drosophila S2 and
Spodoptera Sf~;
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
68
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
expression. Generally, any system or vector suitable to maintain, propagate or
express
polynucleotides and/or to express a polypeptide in a host may be used for
expression in this
regard. The appropriate DNA sequence may be inserted into the expression
system by any of
a variety of well-known and routine techniques, such as, for example, those
set forth in
Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, (supra).
In recombinant expression systems in eukaryotes, for secretion of a translated
protein into the
lumen of the endoplasmic reticulum, into the periplasmic space or into the
extracellular
environment, appropriate secretion signals may be incorporated into the
expressed
polypeptide. These signals may be endogenous to the polypeptide or they may be
heterologous signals.
Polypeptides of the present invention can be recovered and purified from
recombinant cell
cultures by well-known methods including ammonium sulfate or ethanol
precipitation, acid
extraction, anion or cation exchange chromatography, phosphocellulose
chromatography,
hydrophobic interaction chromatography, amity chromatography, hydroxylapatite
chromatography and lectin chromatography. Most preferably, ion metal afFnity
chromatography (IMAC) is employed for purification. Well known techniques for
refolding proteins may be employed to regenerate active conformation when the
polypeptide is denatured during intracellular synthesis, isolation and or
purification.
The expression system may also be a recombinant live microorganism, such as a
virus or
bacterium. The gene of interest can be inserted into the genome of a live
recombinant
virus or bacterium. Inoculation and in vivo infection with this live vector
will lead to in
vivo expression of the antigen and induction of immune responses. Viruses and
bacteria
used for this purpose are for instance: poxviruses (e.g; vaccinia, fowlpox,
canarypox),
alphaviruses (Sindbis virus, Semliki Forest Virus, Venezuelian Equine
Encephalitis
Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus,
rhinovirus),
herpesviruses (varicella zoster virus, etc), Listeria, Salmonella , Shigella,
Neisseria, BCG.
69
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
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 BASB051, BASB057, BASB060,
BASB061,
BASB063, BASB065, BASB066 or BASB071 polynucleotides and polypeptides of the
invention for use as diagnostic reagents. Detection of BASB051, BASB057,
BASB060,
BASB061, BASB063, BASB065, BASB066 or BASB071 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
BASB051,
BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 gene or
protein, may be detected at the nucleic acid or amino acid level by a variety
of well known
techniques as well as by methods provided herein.
Polypeptides and polynucleotides for prognosis, diagnosis or other analysis
may be obtained
from a putatively infected and/or infected individual's bodily materials.
Polynucleotides from
any of these sources, particularly DNA or RNA, may be used directly for
detection or may be
amplified enzymatically by using PCR or any other amplification technique
prior to analysis.
RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same
ways.
Using amplification, characterization of the species and strain of infectious
or resident
organism present in an individual, may be made by an analysis of the genotype
of a 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
BASBO51, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071
polynucleotide sequences. Perfectly or significantly matched sequences can be
distinguished
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
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 1 and S 1
protection
assay or a chemical cleavage method. See, for example, Cotton et al., Proc.
Natl. Acad. Sci.,
USA, 8~: 4397-4401 (1985).
In another embodiment, an array of oligonucleotides probes comprising a BASBO~
1,
BASB057, BASB060, BASB061, BASB063, BASB06~, BASB066 or BASB071 nucleotide
sequence or fragments thereof can be constructed to conduct efficient
screening of, for
example, genetic mutations, serotype, taxonomic classification or
identification. Array
technology methods are well known and have general applicability and can be
used to address
a variety of questions in molecular genetics including gene expression,
genetic linkage, and
genetic variability (see, for example, Chee et al., Science, 27=t: 610
(1996)).
Thus in another aspect, the present invention relates to a diagnostic kit
which comprises:
(a) a polynucleotide of the present invention, preferably the nucleotide
sequence of SEQ ID
N0:1,3,5,7,9,11,13,15 or a fragment thereof ;
(b) a nucleotide sequence complementary to that of (a);
(c) a polypeptide of the present invention, preferably the polypeptide of SEQ
ID
N0:2,4,6, 8,10,12,14,16 or a fragment thereof; or
(d) an antibody to a polypeptide of the present invention, preferably to the
polypeptide of
SEQ ID N0:2,4,6,8,10,12,14,16.
71
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise
a substantial
component. Such a kit will be of use in diagnosing a disease or susceptibility
to a disease,
among others.
This invention also relates to the use of polynucleotides of the present
invention as diagnostic
reagents. Detection of a mutated form of a polynucleotide of the invention,
preferable, SEQ
ID N0:1,3,5,7,9,11,13,15 which is associated with a disease or pathogenicity
will provide a
diagnostic tool that can add to, or define, a diagnosis of a disease, a
prognosis of a course of
disease, a determination of a stage of disease, or a susceptibility to a
disease, which results
from under-expression, over-expression or altered expression of the
polynucleotide.
Organisms, particularly infectious organisms, carrying mutations in such
polynucleotide may
be detected at the polynucleotide level by a variety of techniques, such as
those described
elsewhere herein.
Cells from an organism carrying mutations or polymorphisms (allelic
variations) in a
polynucleotide and/or polypeptide of the invention may also be detected at the
polynucleotide
or polypeptide level by a variety of techniques, to allow for serotyping, for
example. For
example, RT-PCR can be used to detect mutations in the RNA. It is particularly
preferred to
use RT-PCR in conjunction with automated detection systems, such as, for
example,
GeneScan. RNA, cDNA or genomic DNA may also be used for the same purpose, PCR.
As
an example, PCR primers complementary to a polynucleotide encoding BASBO51,
BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071
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 BASBO51,
BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 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
72
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
polynucleotide sequence. In this way, mutations in the polynucleotide sequence
may be
detected and used to diagnose and/or prognose the infection or its stage or
course, or to
serotype and/or classify the infectious agent.
The invention further provides a process for diagnosing disease, preferably
bacterial
infections, more preferably infections caused by Neisseria meningitidis,
comprising
determining from a sample derived from an individual, such as a bodily
material, an
increased level of expression of polynucleotide having a sequence of SEQ ID
N0:1,3,5,7,9,11,13,15. Increased or decreased expression of a BASBO51,
BASB057,
BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 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 BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or
BASB071 polypeptide compared to normal control tissue samples may be used to
detect the
presence of an infection, for example. Assay techniques that can be used to
determine levels
of a BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or
BASB071 polypeptide, in a sample derived from a host, such as a bodily
material, are well-
known to those of skill in the art. Such assay methods include
radioimmunoassays,
competitive-binding assays, Western Blot analysis, antibody sandwich assays,
antibody
detection and ELISA assays.
The polynucleotides of the invention may be used as components of
polynucleotide
arrays, preferably high density arrays or grids. These high density arrays are
particularly
useful for diagnostic and prognostic purposes. For example, a set of spots
each
comprising a different gene, and further comprising a polynucleotide or
polynucleotides
of the invention, may be used for probing, such as using hybridization or
nucleic acid
amplification, using a probe obtained or derived from a bodily sample, to
determine the
73
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
presence of a particular polynucleotide sequence or related sequence in an
individual.
Such a presence may indicate the presence of a pathogen, particularly
Neisseria
meningitidis, and may be useful in diagnosing and/or prognosing disease or a
course of
disease. A grid comprising a number of variants of the polynucleotide sequence
of SEQ
ID N0:1,3,5,7,9,11,13,15 are preferred. Also preferred is a grid comprising a
number of
variants of a polynucleotide sequence encoding the polypeptide sequence of SEQ
ID
N0:2,4,6,8,10,12,14,16.
Antibodies
The polypeptides and polynucleotides of the invention or variants thereof, or
cells expressing
the same can be used as immunogens to produce antibodies immunospecific for
such
polypeptides or polynucleotides respectively.
In certain preferred embodiments of the invention there are provided
antibodies against
BASBO51, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071
polypeptides or polynucleotides.
Antibodies generated against the polypeptides or polynucleotides of the
invention can be
obtained by administering the polypeptides and/or polynucleotides of the
invention, or
epitope-bearing fragments of either or both, analogues of either or both, or
cells expressing
either or both, to an animal, preferably a nonhuman, using routine protocols.
For preparation
of monoclonal antibodies, any technique known in the art that provides
antibodies produced
by continuous cell line cultures can be used. Examples include various
techniques, such as
those in Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et
al., Immunology
Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIESAND CANCER
THERAPY, Alan R. Liss, Inc. (1985).
Techniques for the production of single chain antibodies (U.S. Patent No.
4,946,778) can be
adapted to produce single chain antibodies to polypeptides or polynucleotides
of this
74
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
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-
BASBO51,
BASB0~7, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 or from
naive libraries (McCafferty, et al., (1990), Nature 348, 5~2-554; 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 3.52: 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 BASBO51, BASB057, BASB060, BASB061,
BASB063, BASB065, BASB066 or BASB071 -polypeptide or BASBO51, BASB057,
BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 -polynucleotide may
be employed to treat infections, particularly bacterial infections.
Polypeptide variants include antigenically, epitopically or immunologically
equivalent
variants form a particular aspect of this invention.
Preferably, the antibody or variant thereof is modified to make it less
immunogenic in the
individual. For example, if the individual is human the antibody may most
preferably be
"humanized," where the complimentarity determining region or regions of the
hybridoma-
derived antibody has been transplanted into a human monoclonal antibody, for
example as
described in Jones et al. (1986), Nature 321, 522-525 or Tempest et al.,
(1991)
Biotechnology 9, 266-273.
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Antagonists and Agonists - Assa s 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 1 (2): Chapter 5 (1991).
The screening methods may simply measure the binding of a candidate compound
to the
polypeptide or polynucleotide, or to cells or membranes bearing the
polypeptide or
polynucleotide, or a fusion protein of the polypeptide by means of a label
directly or
indirectly associated with the candidate compound. Alternatively, the
screening method
may involve competition with a labeled competitor. Further, these screening
methods may
test whether the candidate compound results in a signal generated by
activation or
inhibition of the polypeptide or polynucleotide, using detection systems
appropriate to the
cells comprising the polypeptide or polynucleotide. Inhibitors of activation
are generally
assayed in the presence of a known agonist and the effect on activation by the
agonist by
the presence of the candidate compound is observed. Constitutively active
polypeptide
and/or constitutively expressed polypeptides and polynucleotides may be
employed in
screening methods for inverse agonists or inhibitors, in the absence of an
agonist or
inhibitor, by testing whether the candidate compound results in inhibition of
activation of
the polypeptide or polynucleotide, as the case may be. Further, the screening
methods may
simply comprise the steps of mixing a candidate compound with a solution
containing a
polypeptide or polynucleotide of the present invention, to form a mixture,
measuring
BASBO51, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071
polypeptide and/or polynucleotide activity in the mixture, and comparing the
BASBO51,
BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071
polypeptide and/or polynucleotide activity of the mixture to a standard.
Fusion proteins,
such as those made from Fc portion and BASBO51, BASB057, BASB060, BASB061,
76
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
BASB063, BASB065, BASB066 or BASB071 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 BASBO51, BASB057,
BASB060,
BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptides or polynucleotides,
particularly those compounds that are bacteristatic and/or bactericidal. The
method of
screening may involve high-throughput techniques. For example, to screen for
agonists or
antagonists, a synthetic reaction mix, a cellular compartment, such as a
membrane, cell
envelope or cell wall, or a preparation of any thereof, comprising BASBO51,
BASB057,
BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 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 BASBO51, BASB057, BASB060, BASB061, BASB063,
BASB065, BASB066 or BASB071 agonist or antagonist. The ability of the
candidate
molecule to agonize or antagonize the BASBO51, BASB057, BASB060, BASB061,
BASB063, BASB065, BASB066 or BASB071 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 BASBO51, BASB057,
BASB060,
77
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
BASB061, BASB063, BASB065, BASB066 or BASB071 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 BASB051, BASB057, BASB060, BASB061, BASB063, BASB065,
BASB066 or BASB071 polynucleotide or polypeptide activity, and binding assays
known in
the art.
Another example of an assay for BASB051, BASB057, BASB060, BASB061, BASB063,
BASB065, BASB066 or BASB071 agonists is a competitive assay that combines
BASBO51,
BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 and a
potential agonist with BASB051, BASB057, BASB060, BASB061, BASB063, BASB065,
BASB066 or BASB071 -binding molecules, recombinant BASB051, BASB057, BASB060,
BASB061, BASB063, BASB065, BASB066 or BASB071 binding molecules, natural
substrates or ligands, or substrate or ligand mimetics, under appropriate
conditions for a
competitive inhibition assay. BASB051, BASB057, BASB060, BASB061, BASB063,
BASB065, BASB066 or BASB071 can be labeled, such as by radioactivity or a
colorimetric
compound, such that the number of BASB051, BASB057, BASB060, BASB061, BASB063,
BASB065, BASB066 or BASB071 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
. BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071
78
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
-induced activities, thereby preventing the action or expression of BASBO51,
BASB057,
BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptides and/or
polynucleotides by excluding BASBO51, BASB057, BASB060, BASB061, BASB063,
BASB065, BASB066 or BASB071 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. ~ 6: 560 ( 1991 );
OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION,
CRC Press, Boca Raton, FL (1988), for a description of these molecules).
Preferred potential
antagonists include compounds related to and variants of BASBO~ 1, BASB057,
BASB060,
BASB061, BASB063, BASB065, BASB066 or BASB071.
In a further aspect, the present invention relates to genetically engineered
soluble fusion
proteins comprising a polypeptide of the present invention, or a fragment
thereof, and
various portions of the constant regions of heavy or light chains of
immunoglobulins of
various subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the
constant
part of the heavy chain of human IgG, particularly IgGl, where fusion takes
place at the
hinge region. In a particular embodiment, the Fc part can be removed simply by
incorporation of a cleavage sequence which can be cleaved with blood clotting
factor Xa.
Furthermore, this invention relates to processes for the preparation of these
fusion proteins
by genetic engineering, and to the use thereof for drug screening, diagnosis
and therapy. A
further aspect of the invention also relates to polynucleotides encoding such
fusion
proteins. Examples of fusion protein technology can be found in International
Patent
Application Nos. W094/29458 and W094/22914.
Each of the polynucleotide sequences provided herein may be used in the
discovery and
development of antibacterial compounds. The encoded protein, upon expression,
can be
79
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
used as a target for the screening of antibacterial drugs. Additionally, the
polynucleotide
sequences encoding the amino terminal regions of the encoded protein or Shine-
Delgarno
or other translation facilitating sequences of the respective mRNA can be used
to construct
antisense sequences to control the expression of the coding sequence of
interest.
The invention also provides the use of the polypeptide, polynucleotide,
agonist or
antagonist of the invention to interfere with the initial physical interaction
between a
pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible
for
sequelae of infection. In particular, the molecules of the invention may be
used: in the
prevention of adhesion of bacteria, in particular gram positive and/or gram
negative
bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins
on in-dwelling
devices or to extracellular matrix proteins in wounds; to block bacterial
adhesion between
eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial
BASBO51,
BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 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
BASBO51,
BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 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.
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Peptide mimotopes may be designed for a particular purpose by addition,
deletion or
substitution of elected amino acids. Thus, the peptides may be modified for
the purposes
of ease of conjugation to a protein carrier. For example, it may be desirable
for some
chemical conjugation methods to include a terminal cysteine. In addition it
may be
desirable for peptides conjugated to a protein carrier to include a
hydrophobic terminus
distal from the conjugated terminus of the peptide, such that the free
unconjugated end of
the peptide remains associated with the surface of the carrier protein.
Thereby presenting
the peptide in a conformation which most closely resembles that of the peptide
as found
in the context of the whole native molecule. For example, the peptides may be
altered to
have an N-terminal cysteine and a C-terminal hydrophobic amidated tail.
Alternatively,
the addition or substitution of a D-stereoisomer form of one or more of the
amino acids
may be performed to create a beneficial derivative, for example to enhance
stability of the
peptide.
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 B I ). 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 BASBO51, BASB057, BASB060, BASB061, BASB063,
BASB065, BASB066 or BASB071 polynucleotide and/or polypeptide, or a fragment
or
variant thereof, adequate to produce antibody and/ or T cell immune response
to protect
said individual from infection, particularly bacterial infection and most
particularly
81
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Neisseria meningitidis 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 BASBO51, BASB057, BASB060,
BASB061,
BASB063, BASB065, BASB066 or BASB071 polynucleotide and/or polypeptide, or a
fragment or a variant thereof, for expressing BASBO51, BASB057, BASB060,
BASB061,
BASB063, BASB065, BASB066 or BASB071 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
BASBO51, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071
polynucleotide and/or polypeptide encoded therefrom, wherein the composition
comprises
a recombinant BASBO51, BASB057, BASB060, BASB061, BASB063, BASB065,
BASB066 or BASB071 polynucleotide and/or polypeptide encoded therefrom and/or
comprises DNA and/or RNA which encodes and expresses an antigen of said
BASB0~1,
BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071
polynucleotide, polypeptide encoded therefrom, or other polypeptide of the
invention. The
immunological response may be used therapeutically or prophylactically and may
take the
82
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
form of antibody immunity and/or cellular immunity, such as cellular immunity
arising
from CTL or CD4+ T cells.
A BASBO51, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or
BASB071 polypeptide or a fragment thereof may be fused with co-protein or
chemical
moiety which may or may not by itself produce antibodies, but which is capable
of
stabilizing the first protein and producing a fused or modified protein which
will have
antigenic and/or immunogenic properties, and preferably protective properties.
Thus fused
recombinant protein, preferably further comprises an antigenic co-protein,
such as
lipoprotein D from Haemophilus influenzae, Glutathione-S-transferase (GST) or
beta-
galactosidase, or any other relatively large co-protein which solubilizes the
protein and
facilitates production and purification thereof. Moreover, the co-protein may
act as an
adjuvant in the sense of providing a generalized stimulation of the immune
system of the
organism receiving the protein. The co-protein may be attached to either the
amino- or
carboxy-terminus of the first protein.
In a vaccine composition according to the invention, a BASBO51, BASB057,
BASB060,
BASB061, BASB063, BASB065, BASB066 or BASB071 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 BASBO51, BASB057, BASB060, BASB061,
BASB063, BASB065, BASB066 or BASB071 polypeptide, for example bacterial outer-
membrane vesicles or "blebs". OM blebs are derived from the outer membrane of
the two-
layer membrane of Gram-negative bacteria and have been documented in many Gram-
negative bacteria (Zhou, L et al. 1998. FEMS Microbiol. Lett. 163:223-228)
including C.
trachomatis and C. psittaci. A non-exhaustive list of bacterial pathogens
reported to
produce blebs also includes: Bordetella pertussis, Borrelia burgdorferi,
Brucella
melitensis, Brucella ovis, Esherichia coli, Haemophilus influenza, Legionella
pneumophila,
83
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Neisseria gonorrhoeae, Neisseria meningitides, Pseudomonas aeruginosa and
Yersinia
enterocolitica.
Blebs have the advantage of providing outer-membrane proteins in their native
conformation and are thus particularly useful for vaccines. Blebs can also be
improved for
vaccine use by engineering the bacterium so as to modify the expression of one
or more
molecules at the outer membrane. Thus for example the expression of a desired
immunogenic protein at the outer membrane, such as the BASBO51, BASB0~7,
BASB060,
BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide, can be introduced
or
upregulated (e.g. by altering the promoter). Instead or in addition, the
expression of outer-
membrane molecules which are either not relevant (e.g. unprotective antigens
or
immunodominant but variable proteins) or detrimental (e.g. toxic molecules
such as LPS,
or potential inducers of an autoimmune response) can be downregulated. These
approaches
are discussed in more detail below.
The non-coding flanking regions of the BASBO51, BASB057, BASB060, BASB061,
BASB063, BASB065, BASB066 or BASB071 gene contains 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 information allows the modulation of the natural expression of
the
BASBO51, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071
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
84
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
other elements involved. Likewise, downregulation of expression can be
achieved by
similar types of modification. Alternatively, by changing phase variation
sequences, the
expression of the gene can be put under phase variation control, or it may be
uncoupled
from this regulation. In another approach, the expression of the gene can be
put under the
control of one or more inducible elements allowing regulated expression.
Examples of such
regulation include, but are not limited to, induction by temperature shift,
addition of
inductor substrates like selected carbohydrates or their derivatives, trace
elements,
vitamins, co-factors, metal ions, etc.
Such modifications as described above can be introduced by several different
means. The
modification of sequences involved in gene expression can be carried out in
vivo by
random mutagenesis followed by selection for the desired phenotype. Another
approach
consists in isolating the region of interest and modifying it by random
mutagenesis, or site-
directed replacement, insertion or deletion mutagenesis. The modified region
can then be
reintroduced into the bacterial genome by homologous recombination, and the
effect on
gene expression can be assessed. In another approach, the sequence knowledge
of the
region of interest can be used to replace or delete all or part of the natural
regulatory
sequences. In this case, the regulatory region targeted is isolated and
modified so as to
contain the regulatory elements from another gene, a combination of regulatory
elements
from different genes, a synthetic regulatory region, or any other regulatory
region, or to
delete selected parts of the wild-type regulatory sequences. These modified
sequences can
then be reintroduced into the bacterium via homologous recombination into the
genome. A
non-exhaustive list of preferred promoters that could be used for up-
regulation of gene
expression includes the promoters porA, porB, lbpB, tbpB, pl 10, Ist, hpuAB
from N.
meningitides or N. gonorroheae; ompCD, copB, lbpB, ompE, UspAl; UspA2; TbpB
from
M. Catarrhalis; pl, p2, p4, p5, p6, lpD, tbpB, D15, Hia, Hmwl, Hmw2 from H.
influenzae.
In one example, the expression of the gene can be modulated by exchanging its
promoter
with a stronger promoter (through isolating the upstream sequence of the gene,
in vitro
modification of this sequence, and reintroduction into the genome by
homologous
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
recombination). Upregulated expression can be obtained in both the bacterium
as well as in
the outer membrane vesicles shed (or made) from the bacterium.
In other examples, the described approaches can be used to generate
recombinant bacterial
strains with improved characteristics for vaccine applications. These can be,
but are not
limited to, attenuated strains, strains with increased expression of selected
antigens, strains
with knock-outs (or decreased expression) of genes interfering with the immune
response,
strains with modulated expression of immunodominant proteins, strains with
modulated
shedding of outer-membrane vesicles.
Thus, also provided by the invention is a modified upstream region of the
BASBO51,
BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 gene,
which modified upstream region contains a heterologous regulatory element
which alters
the expression level of the BASBO51, BASB057, BASB060, BASB061, BASB063,
BASB065, BASB066 or BASB071 protein located at the outer membrane. The
upstream
region according to this aspect of the invention includes the sequence
upstream of the
BASBO51, BASB057, BASB060, BASB061, BASB063, BASB06~, BASB066 or BASB071
gene. The upstream region starts immediately upstream of the BASBO51, BASB057,
BASB060, BASB061, BASB063, BASB06~, BASB066 or BASB071 gene and continues
usually to a position no more than about 1000 by upstream of the gene from the
ATG start
codon. In the case of a gene located in a polycistronic sequence (operon) the
upstream region
can start immediately preceding the gene of interest, or preceding the first
gene in the operon.
Preferably, a modified upstream region according to this aspect of the
invention contains a
heterologous promotor at a position between 500 and 700 by upstream of the
ATG.
Thus, the invention provides a BASBO51, BASB057, BASB060, BASB061, BASB063,
BASB065, BASB066 and BASB071 polypeptide, in a modified bacterial bleb. The
invention
further provides modified host cells capable of producing the non-live
membrane-based bleb
vectors. The invention further provides nucleic acid vectors comprising the
BASBO51,
86
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 gene
having a modified upstream region containing a heterologous regulatory
element.
Further provided by the invention are processes to prepare the host cells and
bacterial blebs
according to the invention.
Also provided by this invention are compositions, particularly vaccine
compositions, and
methods comprising the polypeptides and/or polynucleotides of the invention
and
immunostimulatory DNA sequences, such as those described in Sato, Y. et al.
Science 273:
352 (1996).
Also, provided by this invention are methods using the described
polynucleotide or
particular fragments thereof, which have been shown to encode non-variable
regions of
bacterial cell surface proteins, in polynucleotide constructs used in such
genetic
immunization experiments in animal models of infection with Neisseria
meningitidis. Such
experiments will be particularly useful for identifying protein epitopes able
to provoke a
prophylactic or therapeutic immune response. It is believed that this approach
will allow
for the subsequent preparation of monoclonal antibodies of particular value,
derived from
the requisite organ of the animal successfully resisting or clearing
infection, for the
development of prophylactic agents or therapeutic treatments of bacterial
infection,
particularly Neisseria mercingitidis infection, in mammals, particularly
humans.
The invention also includes a vaccine formulation which comprises an
immunogenic
recombinant polypeptide and/or polynucleotide of the invention together with a
suitable
carrier, such as a pharmaceutically acceptable carrier. Since the polypeptides
and
polynucleotides may be broken down in the stomach, each is preferably
administered
parenterally, including, for example, administration that is subcutaneous,
intramuscular,
intravenous, or intradermal. Formulations suitable for parenteral
administration include
aqueous and non-aqueous sterile injection solutions which may contain anti-
oxidants,
buffers, bacteristatic compounds and solutes which render the formulation
isotonic with the
87
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
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 TH 1 type of response.
An immune response may be broadly distinguished into two extreme catagories,
being a
humoral or cell mediated immune responses (traditionally characterised by
antibody and
cellular effector mechanisms of protection respectively). These categories of
response
have been termed TH 1-type responses (cell-mediated response), and TH2-type
immune
responses (humoral response).
Extreme TH 1-type immune responses may be characterised by the generation of
antigen
specific, haplotype restricted cytotoxic T lymphocytes, and natural killer
cell responses.
In mice TH1-type responses are often characterised by the generation of
antibodies of the
IgG2a subtype, whilst in the human these correspond to IgGI type antibodies.
TH2-type
immune responses are characterised by the generation of a broad range of
immunoglobulin isotypes including in mice IgGI, IgA, and IgM.
It can be considered that the driving force behind the development of these
two types of
immune responses are cytokines. High levels of THl-type cytokines tend to
favour the
induction of cell mediated immune responses to the given antigen, whilst high
levels of
TH2-type cytokines tend to favour the induction of humoral immune responses to
the
antigen.
88
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
The distinction of TH1 and TH2-type immune responses is not absolute. In
reality an
individual will support an immune response which is described as being
predominantly
TH 1 or predominantly TH2. However, it is often convenient to consider the
families of
cytokines in terms of that described in murine CD4 +ve T cell clones by
Mosmann and
Coffman (Mosmann, T.R. and Coffman, R.L. (1989) THI and TH2 cells: different
patterns of lymphokine secretion lead to different functional properties.
Annual Review of
Immunology, 7, p145-173). Traditionally, THl-type responses are associated
with the
production of the INF-y and IL-2 cytokines by T-lymphocytes. Other cytokines
often
directly associated with the induction of TH1-type immune responses are not
produced by
T-cells, such as IL-12. In contrast, TH2- type responses are associated with
the secretion
of IL-4, IL-5, IL-6 and IL-13.
It is known that certain vaccine adjuvants are particularly suited to the
stimulation of
either TH1 or TH2 - type cytokine responses. Traditionally the best indicators
of the
THl :TH2 balance of the immune response after a vaccination or infection
includes direct
measurement of the production of THl or TH2 cytokines by T lymphocytes in
vitro after
restimulation with antigen, and/or the measurement of the IgGI :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 TH1 cell
response are
described in International Patent Application No. WO 94/00153 and WO 95/17209.
3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant. This is
known from GB 2220211 (Ribi). Chemically it is a mixture of 3 De-O-acylated
monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by
Ribi
89
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Immunochem, Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid
A
is disclosed in European Patent 0 689 454 B 1 (SmithKline Beecham Biologicals
SA).
Preferably, the particles of 3D-MPL are small enough to be sterile filtered
through a
0.22micron membrane (European Patent number 0 689 454).
3D-MPL will be present in the range of 10~g - 100~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 Saponaria Molina. Optionally this may be admixed
with 3 De-
O-acylated monophosphoryl lipid A (3D-MPL), optionally together with a
carrier.
The method of production of QS21 is disclosed in US patent No. 5,057,540.
Non-reactogenic adjuvant formulations containing QS21 have been described
previously
(WO 96/33739). Such formulations comprising QS21 and cholesterol have been
shown
to be successful TH1 stimulating adjuvants when formulated together with an
antigen.
Further adjuvants which are preferential stimulators of THl cell response
include
immunomodulatory oligonucleotides, for example unmethylated CpG sequences as
disclosed in WO 96/02555.
Combinations of different THl stimulating adjuvants, such as those mentioned
hereinabove, are also contemplated as providing an adjuvant which is a
preferential
stimulator of THl 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.
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Preferably a carrier is also present in the vaccine composition according to
the invention.
The carrier may be an oil in water emulsion, or an aluminium salt, such as
aluminium
phosphate or aluminium hydroxide.
A preferred oil-in-water emulsion comprises a metabolisible oil, such as
squalene, alpha
tocopherol and Tween 80. In a particularly~preferred aspect the antigens in
the vaccine
composition according to the invention are combined with QS21 and 3D-MPL in
such an
emulsion. Additionally the oil in water emulsion may contain span 85 and/or
lecithin
and/or tricaprylin.
Typically for human administration QS21 and 3D-MPL will be present in a
vaccine in the
range of lp,g - 200~.g, such as 10-100~g, preferably lOp.g - SOp.g per dose.
Typically the
oil in water will comprise from 2 to 10% squalene, from 2 to 10% alpha
tocopherol and
from 0.3 to 3% tween 80. Preferably the ratio of squalene: alpha tocopherol is
equal to
or less than 1 as this provides a more stable emulsion. Span 85 may also be
present at a
level of 1 %. In some cases it may be advantageous that the vaccines of the
present
invention will further contain a stabiliser.
Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g.
squalane or
squalene, an emulsifier, e.g. Tween 80, in an aqueous carrier. The aqueous
carrier may
be, for example, phosphate buffered saline.
A particularly potent adjuvant formulation involving QS21, 3D-MPL and
tocopherol in
an oil in water emulsion is described in WO 95/17210.
The present invention also provides a polyvalent vaccine composition
comprising a vaccine
formulation of the invention in combination with other antigens, in particular
antigens
useful for treating cancers, autoimmune diseases and related conditions. Such
a polyvalent
vaccine composition may include a TH-1 inducing adjuvant as hereinbefore
described.
91
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
While the invention has been described with reference to certain BASBO51,
BASB057,
BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 polypeptides and
polynucleotides, it is to be understood that this covers fragments of the
naturally occurring
polypeptides and polynucleotides, and similar polypeptides and polynucleotides
with
additions, deletions or substitutions which do not substantially affect the
immunogenic
properties of the recombinant polypeptides or polynucleotides.
The antigen can also be delivered in the form of whole bacteria (dead or
alive) or as
subcellular fractions, these possibilities do include N. meningitidis itself.
Compositions, kits and administration
In a further aspect of the invention there are provided compositions
comprising a BASBO51,
BASB057, BASB060, BASB061, BASB063, BASB06~, BASB066 or BASB071
polynucleotide and/or a BASBO51, BASB057, BASB060, BASB061, BASB063, BASB065,
BASB066 or BASB071 polypeptide for administration to a cell or to a
multicellular
organism.
The invention also relates to compositions comprising a polynucleotide and/or
a polypeptide
discussed herein or their agonists or antagonists. The polypeptides and
polynucleotides of the
invention may be employed in combination with a non-sterile or sterile carrier
or carriers for
use with cells, tissues or organisms, such as a pharmaceutical carrier
suitable for
administration to an individual. Such compositions comprise, for instance, a
media additive
or a therapeutically effective amount of a polypeptide and/or polynucleotide
of the invention
and a pharmaceutically acceptable carrier or excipient. Such Garners may
include, but are not
limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and
combinations thereof.
The formulation should suit the mode of administration. The invention further
relates to
diagnostic and pharmaceutical packs and kits comprising one or more containers
filled with
one or more of the ingredients of the aforementioned compositions of the
invention.
92
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
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.
In a further aspect, the present invention provides for pharmaceutical
compositions
comprising a therapeutically effective amount of a polypeptide and/or
polynucleotide, such as
the soluble form of a polypeptide and/or polynucleotide of the present
invention, agonist or
antagonist peptide or small molecule compound, in combination with a
pharmaceutically
acceptable carrier or excipient. Such carriers include, but are not limited
to, saline, buffered
saline, dextrose, water, glycerol, ethanol, and combinations thereof. The
invention further
relates to pharmaceutical packs and kits comprising one or more containers
filled with one or
more of the ingredients of the aforementioned compositions of the invention.
Polypeptides,
polynucleotides and other compounds of the present invention may be employed
alone or in
conjunction with other compounds, such as therapeutic compounds.
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.
93
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
For administration to mammals, and particularly humans, it is expected that
the daily
dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg,
typically around 1
mg/kg. The physician in any event will determine the actual dosage which will
be most
suitable for an individual and will vary with the age, weight and response of
the particular
individual. The above dosages are exemplary of the average case. There can, of
course, be
individual instances where higher or lower dosage ranges are merited, and such
are within
the scope of this invention.
The dosage range required depends on the choice of peptide, the route of
administration, the
nature of the formulation, the nature of the subject's condition, and the
judgment of the
attending practitioner. Suitable dosages, however, are in the range of 0.1-100
~g/kg of
subj ect.
A vaccine composition is conveniently in injectable form. Conventional
adjuvants may be
employed to enhance the immune response. A suitable unit dose for vaccination
is 0.5-~
microgram/kg of antigen, and such dose is preferably administered 1-3 times
and with an
interval of 1-3 weeks. With the indicated dose range, no adverse toxicological
effects will
be observed with the compounds of the invention which would preclude their
administration to suitable individuals.
Wide variations in the needed dosage, however, are to be expected in view of
the variety of
compounds available and the differing efficiencies of various routes of
administration. For
example, oral administration would be expected to require higher dosages than
administration
by intravenous injection. Variations in these dosage levels can be adjusted
using standard
empirical routines for optimization, as is well understood in the art.
Sequence Databases, Sequences in a Tangible Medium, and Algorithms
94
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Polynucleotide and polypeptide sequences form a valuable information resource
with which
to determine their 2- and 3-dimensional structures as well as to identify
further sequences of
similar homology. These approaches are most easily facilitated by storing the
sequence in a
computer readable medium and then using the stored data in a known
macromolecular
structure program or to search a sequence database using well known searching
tools, such as
the GCG program package.
Also provided by the invention are methods for the analysis of character
sequences or
strings, particularly genetic sequences or encoded protein sequences.
Preferred methods of
sequence analysis include, for example, methods of sequence homology analysis,
such as
identity and similarity analysis, DNA, RNA and protein structure analysis,
sequence
assembly, cladistic analysis, sequence motif analysis, open reading frame
determination,
nucleic acid base calling, codon usage analysis, nucleic acid base trimming,
and sequencing
chromatogram peak analysis.
A computer based method is provided for performing homology identification.
This
method comprises the steps of: providing a first polynucleotide sequence
comprising the
sequence of a polynucleotide of the invention in a computer readable medium;
and
comparing said first polynucleotide sequence to at least one second
polynucleotide or
polypeptide sequence to identify homology.
A computer based method is also provided for performing homology
identification, said
method comprising the steps of: providing a first polypeptide sequence
comprising the
sequence of a polypeptide of the invention in a computer readable medium; and
comparing
said first polypeptide sequence to at least one second polynucleotide or
polypeptide
sequence to identify homology.
All publications and references, including but not limited to patents and
patent applications,
cited in this specification are herein incorporated by reference in their
entirety as if each
individual publication or reference were specifically and individually
indicated to be
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
incorporated by reference herein as being fully set forth. Any patent
application to which
this application claims priority is also incorporated by reference herein in
its entirety in the
manner described above for publications and references.
DEFINITIONS
"Identity," as known in the art, is a relationship between two or more
polypeptide sequences
or two or more polynucleotide sequences, as the case may be, as determined by
comparing
the sequences. In the art, "identity" also means the degree of sequence
relatedness between
polypeptide or polynucleotide sequences, as the case may be, as determined by
the match
between strings of such sequences. "Identity" can be readily calculated by
known methods,
including but not limited to those described in (Computational Molecular
Biology, Lesk,
A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics
and
Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer
Analysis
of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana
Press, New Jersey,
1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press,
1987; and
Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton
Press, New
York, 1991; and Carillo, H., and Lipman, D., SIAM.I. Applied Math., 48: 1073
(1988).
Methods to determine identity are designed to give the largest match between
the sequences
tested. Moreover, methods to determine identity are codified in publicly
available
computer programs. Computer program methods to determine identity between two
sequences include, but are not limited to, the GAP program in the GCG program
package
(Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP,
BLASTN
(Altschul, S.F. et al., J. Mol. Biol. 21 S: 403-410 (1990), and FASTA( Pearson
and Lipman
Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988). The BLAST family of programs
is
publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et
al., NCBI
NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 21~: 403-410
(1990).
The well known Smith Waterman algorithm may also be used to determine
identity.
Parameters for polypeptide sequence comparison include the following:
96
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,
Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)
Gap Penalty: 8
Gap Length Penalty: 2
A program useful with these parameters is publicly available as the "gap"
program from
Genetics Computer Group, Madison WI. The aforementioned parameters are the
default
parameters for peptide comparisons (along with no penalty for end gaps).
Parameters for polynucleotide comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix: matches = +10, mismatch = 0
Gap Penalty: 50
Gap Length Penalty: 3
Available as: The "gap" program from Genetics Computer Group, Madison WI.
These are
the default parameters for nucleic acid comparisons.
A preferred meaning for "identity" for polynucleotides and polypeptides, as
the case may
be, are provided in ( 1 ) and (2) below.
(1) Polynucleotide embodiments further include an isolated polynucleotide
comprising
a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or
100% identity
to the reference sequence of SEQ ID NO:1, wherein said polynucleotide sequence
may be
identical to the reference sequence of SEQ ID NO: l or may include up to a
certain integer
number of nucleotide alterations as compared to the reference 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
97
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
reference sequence, and wherein said number of nucleotide alterations is
determined by
multiplying the total number of nucleotides in SEQ ID NO:1 by the integer
defining the
percent identity divided by 100 and then subtracting that product from said
total number of
nucleotides in SEQ ID NO:1, or:
nn ~ xn' ~xn' Y)
wherein nn is the number of nucleotide alterations, xn is the total number of
nucleotides in
SEQ ID NO:1, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85
for 85%,
0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and ~ is the symbol
for the
multiplication operator, and wherein any non-integer product of xn and y is
rounded down
to the nearest integer prior to subtracting it from xn. Alterations of a
polynucleotide
sequence encoding the polypeptide of SEQ ID N0:2 may create nonsense, missense
or
frameshift mutations in this coding sequence and thereby alter the polypeptide
encoded by
the polynucleotide following such alterations.
By way of example, a polynucleotide sequence of the present invention may be
identical to
the reference sequence of SEQ ID NO:I, 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:
98
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
nn ~ xn ' (xn ' Y)
wherein nn is the number of nucleic acid alterations, xn is the total number
of nucleic acids
in SEQ ID NO:1, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85%
etc., ~ is the
symbol for the multiplication operator, and wherein any non-integer product of
xn and y is
rounded down to the nearest integer prior to subtracting it from xn.
(2) Polypeptide embodiments further include an isolated polypeptide comprising
a
polypeptide having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity
to a
polypeptide reference sequence of SEQ ID N0:2, wherein said polypeptide
sequence may
be identical to the reference sequence of SEQ ID N0:2 or may include up to a
certain
integer number of amino acid alterations as compared to the reference
sequence, wherein
said alterations are selected from the group consisting of at least one amino
acid deletion,
substitution, including conservative and non-conservative substitution, or
insertion, and
wherein said alterations may occur at the amino- or carboxy-terminal positions
of the
reference polypeptide sequence or anywhere between those terminal positions,
interspersed
either individually among the amino acids in the reference sequence or in one
or more
contiguous groups within the reference sequence, and wherein said number of
amino acid
alterations is determined by multiplying the total number of amino acids in
SEQ ID N0:2
by the integer defining the percent identity divided by 100 and then
subtracting that product
from said total number of amino acids in SEQ ID N0:2, or:
na ~ xa ' ~xa' Y)
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.
99
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
By way of example, a polypeptide sequence of the present invention may be
identical to the
reference sequence of SEQ ID N0:2, that is it may be 100% identical, or it may
include up
to a certain integer number of amino acid alterations as compared to the
reference sequence
such that the percent identity is less than 100% identity. Such alterations
are selected from
the group consisting of at least one amino acid deletion, substitution,
including
conservative and non-conservative substitution, or insertion, and wherein said
alterations
may occur at the amino- or carboxy-terminal positions of the reference
polypeptide
sequence or anywhere between those terminal positions, interspersed either
individually
among the amino acids in the reference sequence or in one or more contiguous
groups
within the reference sequence. The number of amino acid alterations for a
given % identity
is determined by multiplying the total number of amino acids in SEQ ID N0:2 by
the
integer defining the percent identity divided by 100 and then subtracting that
product from
said total number of amino acids in SEQ ID N0:2, or:
na ~ xa ' ~xa' Y)
wherein na is the number of amino acid alterations, xa is the total number of
amino acids in
SEQ ID N0:2, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.,
and ~ is the
symbol for the multiplication operator, and wherein any non-integer product of
xa and y is
rounded down to the nearest integer prior to subtracting it from xa.
"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
100
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
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, fasions and
truncations in the
polypeptide encoded by the reference sequence, as discussed below. A typical
variant of
a polypeptide differs in amino acid sequence from another, reference
polypeptide.
Generally, differences are limited so that the sequences of the reference
polypeptide and
the variant are closely similar overall and, in many regions, identical. A
variant and
reference polypeptide may differ in amino acid sequence by one or more
substitutions,
additions, deletions in any combination. A substituted or inserted amino acid
residue
may or may not be one encoded by the genetic code. A variant of a
polynucleotide or
polypeptide may be a naturally occurring such as an allelic variant, or it may
be a variant
that is not known to occur naturally. Non-naturally occurring variants of
polynucleotides
and polypeptides may be made by mutagenesis techniques or by direct synthesis.
"Disease(s)" means any disease caused by or related to infection by a
bacteria, including , for
example, upper respiratory tract infection, invasive bacterial diseases, such
as bacteremia and
meningitis.
101
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
EXAMPLES
The examples below are carried out using standard techniques, which are well
known and
routine to those of skill in the art, except where otherwise described in
detail. The examples
are illustrative, but do not limit the invention.
Example 1
The BASB0~1 gene in N.meningitidis strain ATCC 13090.
The BASBO51 gene of N. meningitidis strain ATCC 13090 is shown in SEQ ID NO:1.
The translation of the BASBO51 polynucleotide sequence, shown in SEQ ID N0:2,
shows significant similarity to Neisseria gonorrhoeae ComL lipoprotein. The
BASBO51
polypeptide contains a leader sequence characteristic of a lipoprotein signal
sequence.
Example 2
The BASB057 gene in N.meningitidis strain ATCC 13090.
The BASB057 gene of N. meningitidis strain ATCC 13090 is shown in SEQ ID N0:3.
The translation of the BASB057 polynucleotide sequence, shown in SEQ ID N0:4,
shows significant similarity to Neisseria gonorrhoeae MtrE outer membrane
lipoprotein. The BASB057 polypeptide conains a leader sequence characteristic
of a
lipoprotein signal sequence.
Example 3
The BASB060 gene in N.meningitidis strain ATCC 13090.
102
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
The BASB060 gene of N. meningitidis strain ATCC 13090 is shown in SEQ ID NO:S.
The translation of the BASB060 polynucleotide sequence, shown in SEQ ID N0:6,
shows no significant similarity to any known protein. However, the BASB060
polypeptide contains a leader sequence which is characteristic of a
lipoprotein signal
sequence and has the characteristics of an outer membrane lipoprotein.
Example 4
The BASB061 gene in N.meningitidis strain ATCC 13090.
The BASB061 gene of N. meningitidis strain ATCC 13090 is shown in SEQ ID N0:7.
The translation of the BASB061 polynucleotide sequence, shown in SEQ ID N0:8,
shows significant similarity to Neisseria meningitidis mlp gene product. The
BASB061
polypeptide contains a leader sequence which is characteristic of a
lipoprotein signal
sequence and has the characteristics of an outer membrane lipoprotein.
Example 5
The BASB063 gene in N.meningitidis strain ATCC 13090.
The BASB063 gene of N. meningitidis strain ATCC 13090 is shown in SEQ ID N0:9.
The translation of the BASB063 polynucleotide sequence, shown in SEQ ID NO:10,
shows significant similarity to any known protein. However, the BASB063
polypeptide
does contain a leader sequence which is characteristic of a lipoprotein signal
sequence
and has the characteristics of an outer membrane lipoprotein.
Example 6
The BASB065 gene in N.meningitidis strain ATCC 13090.
103
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
The BASB065 gene of N. meningitidis strain ATCC 13090 is shown in SEQ ID
NO:11. The translation of the BASB065 polynucleotide sequence, shown in SEQ ID
N0:12, shows significant similarity to any known protein. However, the BASB065
polypeptide does contain a leader sequence which is characteristic of a
lipoprotein
signal sequence and has the characteristics of an outer membrane lipoprotein.
Example 7
The BASB066 gene in N.meningitidis strain ATCC 13090.
The BASB066 gene of N. meningitidis strain ATCC 13090 is shown in SEQ ID
N0:13. The translation of the BASB066 polynucleotide sequence, shown in SEQ ID
N0:14, shows significant similarity to Neisseria meningitidis CtrA protein.
The
BASB066 polypeptide contains a leader sequence which has the characteristics
of a
lipoprotein signal sequence and has the characteristics of a protein located
in the outer
membrane .
Example 8
The BASB071 gene in N.meningitidis strain ATCC 13090.
The BASB071 gene of N. meningitidis strain ATCC 13090 is shown in SEQ ID
NO:15. The translation of the BASB071 polynucleotide sequence, shown in SEQ ID
N0:16, shows significant similarity to Neisseria gonorrhoeae HisJ protein. The
2~ BASB071 polypeptide is contains a leader sequence which has the
characteristics of a
lipoprotein signal sequence.
104
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Polynucleotide and Polypeptide Sequences
SEQ ID NO:1
Neisseria meningitides BASBOS 1 polynucleotide sequence from strain ATCC 13090
S ATGAAAAAAATTCTTTTAACGGTTTCATTAGGTTTGGCACTGAGTGCCTGTGCCACTCAA
GGTACGGTCGATAAAGATGCTCAGATTACCCAAGATTGGAGTGTGGAGAAGCTCTATGCC
GAAGCCCAGGACGAATTGAACAGCAGCAATTATACGCGGGCTGTCAAGTTATACGAAATC
TTGGAATCGCGCTTCCCCACCAGCCGCCATGCCCGGCAATCCCAACTGGATACCGCATAC
GCCTATTATAAAGACGATGAAAAAGACAAGGCTCTGGCGGCAATCGAACGCTTCCGCCGC
lO CTCCATCCGCAGCATCCGAATATGGATTACGCGCTGTATCTGCGCGGCTTGGTGCTGTTC
AACGAAGACCAGTCCTTCTTGAACAAACTGGCCTCGCAAGACTGGTCCGACCGCGACCCG
AAAGCCAACCGCGAAGTAACCCAGGCGTTTGCGGAACTCGTCCAACGCTTCCCCAACAGC
AAATACGCCGCCGATGCGACCGCACGCATGGTCAAACTGGTCGATGCACTGGGCGGCAAT
GAAATGTCGGTGGCGCGCTACTACATGAAACGCGGCGCATATATCGCCGCCGCCAACCGC
IS GCCCAAAAAATTATCGGCAGCTACCAAAATACACGCTATGTCGAAGAATCGCTCGCCATC
TTGGAACTTGCCTACCAAAAACTCGGCAAACCACAGCTTGCCGCCGATACGCGCCGCGTG
TTGGAAACCAACTTCCCGAAAAGCCCGTTTTTGACGCACGCTTGGCAGCCCGACGATATG
CCTTGGTGGCGTTACTGGCATTAA
20 SEQ ID N0:2
Neisseria meningitides BASBO51 polypeptide sequence deduced from the
polynucleotide
sequence of SEQ ID NO:1
MKKILLTVSLGLALSACATQGTVDKDAQITQDWSVEKLYAEAQDELNSSNYTRAVKLYEI
LESRFPTSRHARQSQLDTAYAYYKDDEKDKALAAIERFRRLHPQHPNMDYALYLRGLVLF
2S NEDQSFLNKLASQDWSDRDPKANREVTQAFAELVQRFPNSKYAADATARMVKLVDALGGN
EMSVARYYMKRGAYIAAA:~1RAQKIIGSYQNTRYVEESLAILELAYQKLGKPQLAADTRRV
LETNFPKSPFLTHAWQPDDMPWWRYWH
SEQ ID N0:3
30 Neisseria meningitides BASBOS7 polynucleotide sequence from strain ATCC
13090
ATGGATACTACATTGAAAACCACCTTGACTTCTGTTGCAGCAGCCTTCGCATTATCCGCC
TGCACCATGATTCCCCAATACGAGCAGCCCAAAGTCGAAGTTGCCGAAACGTTTAAAAAC
GATACCGCCGACAGCGGCATCCGTGCGGTCGATTTAGGTTGGCATGACTATTTTGCCGAC
CCGCGCCTGCAAAAGCTGATCGACATCGCACTCGAGCGCAATACCAGTTTGCGTACCGCC
3S GTATTGAACAGCGAAATCTACCGCAAACAATACATGATTGAGCGCAACAACCTCCTGCCC
ACGCTTGCCGCCAATGCGAACGGCTCGCGCCAAGGCAGCTTGAGCGGCGGCAATGTCAGC
AGCAGCTACAATGTCGGACTGGGTGCGGCATCTTACGAACTCGACCTGTTCGGACGCGTC
CGCAGCAGCAGCGAAGCAGCACTGCAAGGCTATTTTGCAAGTGTCGCCAACCGCGATGCG
GCACATTTGAGCCTGATTGCCACCGTTGCCAAAGCCTATTTCAACGAACGTTATGCCGAA
4O GAAGCGATGTCTTTGGCGCAGCGTGTTTTGAAAACGCGCGAGGAAACCTACAAGCTGTCC
GAATTACGTTACAAGGCAGGCGTGATTTCCGCCGTCGCCCTACGTCAGCAGGAAGCCCTG
ATCGAATCTGCCAAAGCCGATTATGCCCATGCCGCGCGCAGCCGCGAACAGGCGCGCAAT
GCCTTGGCAACCTTGATTAACCAACCGATACCCGAAGACCTGCCTGCCGGTTTGCCGCTG
GACAAGCAGTTTTTTGTTGAAAAACTGCCGGCCGGTTTGAGTTCCGAAGTATTGCTCGAC
4S CGTCCCGATATCCGTGCTGCCGAACACGCGCTCAAACAGGCAAACGCCAATATCGGTGCG
GCACGCGCCGCCTTTTTCCCATCCATCCGCCTGACCGGAACCGTCGGTACGGGTTCTGCC
lOS
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
GAATTGGGTGGGTTGTTCAAAAGCGGCACGGGCGTTTGGTCGTTCGCGCCGTCTATTACC
CTGCCGATTTTTACCTGGGGTACGAACAAAGCCAACCTTGATGTAGCCAAGCTGCGCCAA
CAGGCACAAATCGTTGCCTATGAAGCCGCCGTCCAATCCGCATTTCAAGACGTGGCAAAC
GCATTGGCGGCGCGCGAGCAGCTGGATAAAGCCTATGACGCTTTAAGCAAACAAAGCCGC
S GCCTCTAAAGAGGCGTTGCGCTTGGTCGGCCTGCGTTACAAGCACGGCGTATCCGGCGCG
CTCGACTTGCTCGATGCGGAACGCAGCAGCTATGCGGCGGAGGGTGCGGCTTTGTCGGCA
CAACTGACCCGCGCCGAAAACCTTGCCGATTTGTACAAGGCACTCGGCGGCGGATTGAAA
CGGGATACCCAAACCGACAAATAA
SEQ ID N0:4
Neisseria meningitides BASBOS7 polypeptide sequence deduced from the
polynucleotide
sequence of SEQ ID N0:3
MDTTLKTTLTSVAAAFALSACTMIPQYEQPKVEVAETFKNDTADSGIRAVDLGWHDYFAD
PRLQKLIDIALERNTSLRTAVLNSEIYRKQYMIERNNLLPTLAANANGSRQGSLSGGNVS
IS SSYNVGLGAASYELDLFGRVRSSSEAALQGYFASVANRDAAHLSLIATVAKAYFNERYAE
EAMSLAQRVLKTREETYKLSELRYKAGVISAVALRQQEALIESAKADYAHAARSREQARN
ALATLINQPIPEDLPAGLPLDKQFFVEKLPAGLSSEVLLDRPDIRAAEHALKQANANIGA
ARAAFFPSIRLTGTVGTGSAELGGLFKSGTGVWSFAPSITLPIFTWGTNKANLDVAKLRQ
QAQIVAYEAAVQSAFQDVANALAAREQLDKAYDALSKQSRASKEALRLVGLRYKHGVSGA
ZO LDLLDAERSSYAAEGAALSAQLTRAENLADLYKALGGGLKRDTQTDK
SEQ ID N0:5
Neisseria meningitides BASB060 polynucleotide sequence from strain ATCC 13090
ZS ATGAAA.AAACTTCTAATGATAACCCTCACCGGTATGCTTGCAGCTTGTGCAACAGGTGTC
AATGTCGGCCGGTTGATGGTTGAAATGCCGCAGGGAGAACGTTCTGTCGTTGTGCAGGTT
CCCGCGACAAATAACCCGCTTTCCGATACGGTAGCTGTCGGAATGATTAAAACATCCGGT
TCGCCTTCGGCATCAAATATGATTGAAATGCTCGGCGCGGACAATATCAACGTCGGCGTG
GTGGGAAGCAGCCAAATGCTTAATAAGGCGACCGCACTTTATTCCTTAAACCATGCAAAG
3O AAAGTCGGAAATAATGTCAGTGTTTATATGATGGGCGACAGCGAAAGTGACAAGGCCGAT
TTGGAAAACGCGGCAAATGCCAAAAATATCAAATTGCATTATTTCTTTAACCAAAAATAA
SEQ ID N0:6
Neisseria meningitides BASB060 polypeptide sequence deduced from the
polynucleotide
3S sequence of SEQ ID NO:S
MKKLLMITLTGMLAACATGVNVGRLMVEMPQGERSWVQVPATNNPLSDTVAVGMIKTSG
SPSASNMIEMLGADNINVGWGSSQMLNKATALYSLNHAKKVGNNVSVYMMGDSESDKAD
LENAANAKNIKLHYFFNQK
40 SEQ ID N0:7
Neisseria meningitides BASB061 polynucleotide sequence from strain ATCC 13090
ATGAAAATCAAACAAATCGTCAAACCGGGCTTGGCAGTATTGGCGGCGGGCGTTCTGTCT
GCCTGCGCAACCAAAAGCAACGTCAAAGCCGACGGAACGACCGACAATCCGGTTTTCCCG
AAACCCTATTCCGTAACGCTCGACAACAATCGCGGTACATTCCCGACCTATGACGAATTG
106
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
GACTTGATGCGTCCCGGTCTGACCAAAGACGACATCTACAAAATCCTGGGTCGTCCGCAT
TACGACGAAGGTATGTACGGCGTGCGCGAATGGGATTATCTGTTCCACTTCCACACCCCG
GGCGTAGGCATCGACCCTGAAAACACTTCCGGCGTAGAAGGCATTACCACCTGTCAATAC
AAAATTATTTTCGATAAAGACAAATTTGCCCGCAGCTTCTACTGGAACCCCGTCTTCCCG
S AAAGATGCCGCCTGTCCGCCGCCCGCACCCAAAGCCGAGCCGCAAgTCATCATCCGCGAA
ATCGTGCCCGCCAAAcCCAAACGCATCCGCCAATAA
SEQ ID N0:8
Neisseria meningitides BASB061 polypeptide sequence deduced from the
polynucleotide
sequence of SEQ ID N0:7
MKIKQIVKPGLAVLAAGVLSACATKSNVKADGTTDNPVFPKPYSVTLDNNRGTFPTYDEL
DLMRPGLTKDDIYKILGRPHYDEGMYGVREWDYLFHFHTPGVGIDPENTSGVEGITTCQY
KIIFDKDKFARSFYWNPVFPKDAACPPPAPKAEPQVIIREIVPAKPKRIRQ
1 S SEQ ID N0:9
Neisseria meningitides BASB063 polynucleotide sequence from strain ATCC 13090
ATGAGACCATATGCTACTACCATTTATCAACTTTTTATTTTGTTTATTGGGAGTGTTTTT
ACTATGACCTCATGTGAACCTGTGAATGAAAAGACAGATCAAAAAGCAGTAAGTGCGCAA
CAGGCTAAAGAACAAACCAGTTTCAACAATCCCGAGCCAATGACAGGATTTGAACATACG
Z.O GTTACATTTGATTTTCAGGGCACCAAAATGGTTATCCCCTATGGCTATCTTGCACGGTAT
ACGCAAGACAATGCCACAAAATGGCTTTCCGACACGCCCGGGCAGGATGCTTACTCCATT
AATTTGATAGAGATTAGCGTCTATTACAAAAAAACCGACCAAGGCTGGGTTCTTGAGCCA
TACAACCAGCAAAACAAAGCACACTTTATCCAATTTCTACGCGACGGTTTGGATAGCGTG
GACGATATTGTTATCCGAAAAGATGCGTGTAGTTTAAGTACGACTATGGGAGAAAGATTG
2S CTTACTTACGGGGTTAAAAAAATGCCATCTGCCTATCCTGAATACGAGGCTTATGAAGAT
AAAAGACATATTCCTGAAAATCCATATTTTCATGAATTTTACTATATTAAAAAAGGAGAA
AATCCGGCGATTATTACTCATCGGAATAATCGAATAAACCAAACTGAAGAAGATAGTTAT
AGCACTAGCGTAGGTTCCTGTATTAACGGTTTCACGGTACAGTATTACCCGTTTATTCGG
GAAAAGCAGCAGCTCACACAGCAGGAGTTGGTAGGTTATCACCAACAAGTAGAGCAATTG
3O GTACAGAGTTTTGTAAACAATTCAAATAAAAAATAA
SEQ ID NO:10
Neisseria meningitides BASB063 polypeptide sequence deduced from the
polynucleotide
sequence of SEQ ID N0:9
3S MRPYATTIYQLFILFIGSVFTMTSCEPVNEKTDQKAVSAQQAKEQTSFNNPEPMTGFEHT
VTFDFQGTKMVIPYGYLARYTQDNATKWLSDTPGQDAYSINLIEISVYYKKTDQGWVLEP
YNQQNKAHFIQFLRDGLDSVDDIVIRKDACSLSTTMGERLLTYGVKKMPSAYPEYEAYED
KRHIPENPYFHEFYYIKKGENPAIITHRNNRINQTEEDSYSTSVGSCINGFTVQYYPFIR
EKQQLTQQELVGYHQQVEQLVQSFVNNSNKK
SEQ ID NO:11
Neisseria meningitides BASB06S polynucleotide sequence from strain ATCC 13090
ATGAAGACCAAATTACCGCTTTTTATCATTTGGCTGTCCGTATCCGCCGCCTGTTCTTCC
107
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
CCTGTTTCCCGCAATATTCAGGATATGCGGCCCGAACCGCAGGCAGAGGCAGGTAGTTCG
GACGCTATTCCCTATCCCGTTCCCACTCTGCAAGACCGTTTGGATTATCTGGAAGGCACA
CTCGTCCGCCTGTCGAACGAAGTGGAAACCTTAAACGGCAAAGTCAAAGCACTGGAGCAT
GCGAAAACACACCCTTCCGGTAGGGCATACGTCCAAAAACTCGACGACCGCAAGTTGAAA
S GAGCATTACCTCAATACCGAAGGCGGCAGCGCATCCGCACATACCGTCGAAACCGCACAA
AACCTCTACAATCAGGCACTCAAACACTATAAAAGCGGCAGGTTTTCTGCCGCAGCCGCC
CTGTTGAAAGGCGCGGACGGAGGCGACGGCGGCAGCATCGCGCAACGCAGTATGTACCTG
TTGCTGCAAAGCAGGGCGCGTATGGGCAACTGCGAATCCGTCATCGAAATCGGAGGGCGT
TACGCCAACCGTTTCAAAGACAGCCCAACCGCGCCCGAAGCCATGTTCAAAATCGGCGAA
lO TGCCAATACAGGTTGCAGCAGAAAGACATTGCAAGGGCAACTTGGCGCAGCCTGATACAG
GCTTACCCGAGCAGCCCGGCGGCAAAACGCGCCGCCGCAGCCGTACGCAAACGATAG
SEQ ID N0:12
Neisseria meningitides BASB06S polypeptide sequence deduced from the
polynucleotide
1S sequence of SEQ ID NO:11
MKTKLPLFIIWLSVSAACSSPVSRNIQDMRPEPQAEAGSSDAIPYPVPTLQDRLDYLEGT
LVRLSNEVETLNGKVKALEHAKTHPSGRAYVQKLDDRKLKEHYLNTEGGSASAHTVETAQ
NLYNQALKHYKSGRFSAAAALLKGADGGDGGSIAQRSMYLLLQSRARMGNCESVIEIGGR
YANRFKDSPTAPEAMFKIGECQYRLQQKDIARATWRSLIQAYPSSPAAKRAAAAVRKR
SEQ ID N0:13
Neisseria meningitides BASB066 polynucleotide sequence from strain ATCC 13090
GTGTTTAAAGTGAAATTTTATATTCGTCACGCAGTATTATTATTGTGTGGAAGTTTAATT
GTAGGATGCTCTGCGATTCCTTCATCAGGCCCCAGCGCAAAAAAAATTGTCTCTTTAGGG
2S CAACAATCTGAAGTTCAAATTCCTGAAGTGGAGCTGATTGATGTGAATCATACGGTTGCT
CAGTTATTATATAAGGCTCAGATAAATCAGTCATTCACTCAGTTTGGCGATGGTTATGCT
TCGGCTGGTACGCTAAATATTGGTGATGTATTGGATATTATGATTTGGGAAGCGCCGCCG
GCAGTATTGTTTGGTGGTGGCCTTTCTTCGATGGGCTCGGGTAGTGCGCATCAAACTAAG
TTGCCAGAGCAGTTGGTCACGGCACGTGGTACGGTTTCTGTGCCGTTTGTTGGCGATATT
3O TCGGTGGTCGGTAAAACGCCTGGTCAGGTTCAGGAAATTATTAAAGGCCGCCTGAAAAAA
ATGGCCAATCAGCCACAAGTGATGGTGCGTTTGGTGCAGAATAATGCGGCGAATGTGTCG
GTGATTCGTGCTGGGAATAGTGTGCGTATGCCGCTGACGGCAGCCGGTGAGCGTGTGTTG
GATGCGGTGGCTGCGGTAGGTGGTTCAACGGCAAATGTGCAGGATACGAATGTGCAGCTG
ACACGTGGCAATGTAGTACGAACTGTTGCCTTGGAAGATTTAGTTGCAAATCCGCGACAA
3S AATATTTTGCTGCGTCGCGGTGATGTGGTTACCATGATTACCAATCCCTATACCTTTACG
TCTATGGGTGCGGTGGGGAGAACACAAGAAATCGGTTTTTCAGCCAGAGGCTTATCGCTT
TCTGAAGCCATTGGCCGTATGGGCGGTTTGCAAGATCGCCGTTCTGATGCGCGTGGTGTG
TTTGTGTTCCGCTATACGCCATTGGTGGAATTGCCGGCAGAACGTCAGGATAAATGGATT
GCTCAAGGTTATGGCAGTGAGGCAGAGATTCCAACGGTATATCGTGTGAATATGGCTGAT
4O GCGCATTCGCTATTTTCTATGCAGCGCTTTCCTGTGAAGAATAAAGATGTATTGTATGTG
TCGAATGCGCCGTTGGCTGAAGTGCAGAAATTCTTGTCGTTTGTGTTCTCGCCGGTTACC
AGTGGCGCGAACAGTATTAATAATTTAACTAATTAA
SEQ ID N0:14
4S Neisseria meningitides BASB066 polypeptide sequence deduced from the
polynucleotide
sequence of SEQ ID N0:13
108
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
MFKVKFYIRHAVLLLCGSLIVGCSAIPSSGPSAKKIVSLGQQSEVQIPEVELIDVNHTVA
QLLYKAQINQSFTQFGDGYASAGTLNIGDVLDIMIWEAPPAVLFGGGLSSMGSGSAHQTK
LPEQLVTARGTVSVPFVGDISWGKTPGQVQEIIKGRLKKMANQPQVMVRLVQNNAANVS
VIRAGNSVRMPLTAAGERVLDAVAAVGGSTANVQDTNVQLTRGNVVRTVALEDLVANPRQ
S NILLRRGDWTMITNPYTFTSMGAVGRTQEIGFSARGLSLSEAIGRMGGLQDRRSDARGV
FVFRYTPLVELPAERQDKWIAQGYGSEAEIPTWRVNMADAHSLFSMQRFPVKNKDVLW
SNAPLAEVQKFLSFVFSPVTSGANSINNLTN
SEQ ID NO:15
Neisseria meningitides BASB071 polynucleotide sequence from strain ATCC 13090
ATGAATATGAAAAAATGGATTGCCGCCGCCCTTGCCTGTTCCGCGCTCGCGCTGTCTGCC
TGCGGCGGTCAGGGCAAAGATGCCGCCGCGCCCGCCGCAAACCCCGACAAAGTGTACCGC
GTGGCTTCCAACGCCGAGTTTGCCCCCTTTGAATCTTTAGACTCGAAAGGCAATGTTGAA
GGTTTCGATGTGGATTTGATGAACGCGATGGCGAAGGCGGGCAATTTTAAAATCGAATTC
IS AAACACCAGCCGTGGGACAGCCTTTTCCCCGCCTTGAACAACGGCGATGCGGACGTTGTG
ATGTCGGGCGTAACCATTACCGACGACCGCAAACAGTCTATGGACTTCAGCGACCCGTAT
TTTGAAATCACCCAAGTCGTCCTCGTTCCGAAAGGCAAAAAAATATCTTCTTCCGAAGAT
TTGAAAAACATGAACAAAGTCGGCGTGGTAACCGGCTACACGGGCGATTTCTCCGTATCC
AAACTCTTGGGCAACGACAACCCGAAAATCGCGCGCTTTGAAAACGTTCCCCTGATTATC
2O AAAGAACTGGAAAACGGCGGCTTGGATTCCGTGGTCAGCGACAGCGCAGTCATCGCCAAT
TATGTGAAAAACAATCCGACCAAAGGGATGGACTTCGTTACCCTGCCCGACTTCACCACC
GAACACTACGGCATCGCGGTACGCAAAGGCGACGAAGCAACCGTCAAAATGCTGAACGAT
GCGTTGAAAAAAGTACGCGAAAGCGGCGAATACGACAAAATCTACGCCAAATATTTTGCA
AAAGAAGACGGACAGGCCGCAAAATAA
2S
SEQ ID N0:16
Neisseria meningitides BASB071 polypeptide sequence deduced from the
polynucleotide
sequence of SEQ ID NO:1 S
MNMKKWIAAALACSALALSACGGQGKDAAAPAANPDKWRVASNAEFAPFESLDSKGNVE
3O GFDVDLMNAMAKAGNFKIEFKHQPWDSLFPALNNGDADVVMSGVTITDDRKQSMDFSDPY
FEITQWLVPKGKKISSSEDLKNMNKVGVVTGYTGDFSVSKLLGNDNPKIARFENVPLII
KELENGGLDSWSDSAVIANWKNNPTKGMDFVTLPDFTTEHYGIAVRKGDEATVKMLND
ALKKVRESGEYDKIYAKYFAKEDGQAAK
109
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Deposited materials
A deposit containing a Neisseria meningitides Serogroup B strain has been
deposited with the
American Type Culture Collection (herein "ATCC") on June 22, 1997 and assigned
deposit
number 13090. The deposit was described as Neisseria meningitides (Albrecht
and Ghon)
and is a freeze-dried, 1.5-2.9 kb insert library constructed from N.
meningitides isolate. The
deposit is described in Int. Bull. Bacteriol. Nomencl. Taxon. 8: 1-15 (1958).
The Neisseria meningitides strain deposit is referred to herein as "the
deposited strain" or as
"the DNA of the deposited strain."
The deposited strain contains the full-length BASBO51, BASB057, BASB060,
BASB061,
BASB063, BASB065, BASB066 and BASB071 genes. 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 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 3~ U.S.C. ~112.
110
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
Applicant's oragent's International application No.
filereference Kp/BM45348
INDICATIONS RELATING TO DEPOSITED MICROORGANISM
OR OTHER BIOLOGICAL MATERIAL
(PCT Rule l3bir)
A. The indications made below
relate to the deposited microorganism
or ocher biological material
referred to in the description
on page 110 , line 2-22
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depositary institution
AMERICAN TYPE CULTURE COLLECTION
Address of depositary institution
(including postal code and country)
10801 UNIVERSITY BLVD, MANASSAS,
VIRGINIA 20110-2209,
UNITED STATES OF AMERICA
Date of deposit Accession Number
22 June 1997 (22.06.97) 13090
C. ADDITIONAL INDICATIONS (leave
blank ijnot applicable) This
information is continued on an
additional sheet
In respect of those designations
where a European Patent is sought,
a sample
of the deposited microorganism
will be made available until
the publication
of the mention of the grant of
the European Patent or until
the date on which
the application has been refused.or
withdrawn, only by issue of such
a sample
to an expert nominated by the
person requesting:.the-sample.
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (ijthe indications
are not jor all designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank ijnot applicable)
The indications listed below will
be submitted to the International
Bureau later (spec~rhegeneral
naa~reofthe indicationse.g..
'accession
Number ojDeposit')
For receiving Office use only For International Bureau use only
Q This sheet was received with the international application a This sheet was
received by the International Bureau on:
Authorized officer ~ ~ Authorized officer
Form PCT/RO/134 (JuIv199R1 - 11I -
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
SEQUENCE LISTING
<110> SmithKline Beecham Biologicals S.A.
<120> Novel compounds
<130> BM45348
<160> 16
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 804
<212> DNA
<213> Neisseria meningitides
<400>
1
atgaaaaaaattcttttaacggtttcattaggtttggcactgagtgcctgtgccactcaa60
ggtacggtcgataaagatgctcagattacccaagattggagtgtggagaagctctatgcc120
gaagcccaggacgaattgaacagcagcaattatacgcgggctgtcaagttatacgaaatc180
ttggaatcgcgcttccccaccagccgccatgcccggcaatcccaactggataccgcatac240
gcctattataaagacgatgaaaaagacaaggctctggcggcaatcgaacgcttccgccgc300
ctccatccgcagcatccgaatatggattacgcgctgtatctgcgcggcttggtgctgttc360
aacgaagaccagtccttcttgaacaaactggcctcgcaagactggtccgaccgcgacccg420
aaagccaaccgcgaagtaacccaggcgtttgcggaactcgtccaacgcttccccaacagc480
aaatacgccgccgatgcgaccgcacgcatggtcaaactggtcgatgcactgggcggcaat540
gaaatgtcggtggcgcgctactacatgaaacgcggcgcatatatcgccgccgccaaccgc600
gcccaaaaaattatcggcagctaccaaaatacacgctatgtcgaagaatcgctcgccatc660
ttggaacttgcctaccaaaaactcggcaaaccacagcttgccgccgatacgcgccgcgtg720
ttggaaaccaacttcccgaaaagcccgtttttgacgcacgcttggcagcccgacgatatg780
ccttggtggcgttactggcattaa 804
<210> 2
<211> 267
<212> PRT
<213> Neisseria meningitides
<400> 2
Met Lys Lys Ile Leu Leu Thr Val Ser Leu Gly Leu Ala Leu Ser Ala
1 5 10 15
Cys Ala Thr Gln Gly Thr Val Asp Lys Asp Ala Gln Ile Thr Gln Asp
20 25 30
Trp Ser Val Glu Lys Leu Tyr Ala Glu Ala Gln Asp Glu Leu Asn Ser
1
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
35 40 45
Ser Asn Tyr Thr Arg Ala Val Lys Leu Tyr G1u Ile Leu Glu Ser Arg
50 55 60
Phe Pro Thr Ser Arg His Ala Arg Gln Ser Gln Leu Asp Thr Ala Tyr
65 70 75 80
Ala Tyr Tyr Lys Asp Asp Glu Lys Asp Lys Ala Leu Ala Ala Ile Glu
85 90 95
Arg Phe Arg Arg Leu His Pro Gln His Pro Asn Met Asp Tyr Ala Leu
100 105 110
Tyr Leu Arg Gly Leu Val Leu Phe Asn Glu Asp Gln Ser Phe Leu Asn
115 120 125
Lys Leu A1a Ser Gln Asp Trp Ser Asp Arg Asp Pro Lys Ala Asn Arg
130 135 140
Glu Val Thr Gln Ala Phe Ala Glu Leu Val Gln Arg Phe Pro Asn Ser
145 150 155 160
Lys Tyr Ala Ala Asp Ala Thr Ala Arg Met Val Lys Leu Val Asp Ala
165 170 175
Leu Gly Gly Asn Glu Met Ser Val Ala Arg Tyr Tyr Met Lys Arg Gly
180 185 190
Ala Tyr Ile Ala Ala Ala Asn Arg Ala Gln Lys Ile Ile Gly Ser Tyr
195 200 205
Gln Asn Thr Arg Tyr Val Glu Glu Ser Leu Ala Ile Leu Glu Leu Ala
210 215 220
Tyr Gln Lys Leu G1y Lys Pro Gln Leu Ala Ala Asp Thr Arg Arg Val
225 230 235 240
Leu Glu Thr Asn Phe Pro Lys Ser Pro Phe Leu Thr His Ala Trp Gln
245 250 255
Pro Asp Asp Met Pro Trp Trp Arg Tyr Trp His
260 265
<210> 3
<211> 1404
<212> DNA
<213> Neisseria meningitides
<400> 3
atggatactacattgaaaaccaccttgacttctgttgcagcagccttcgcattatccgcc60
tgcaccatgattccccaatacgagcagcccaaagtcgaagttgccgaaacgtttaaaaac120
gataccgccgacagcggcatccgtgcggtcgatttaggttggcatgactattttgccgac180
ccgcgcctgcaaaagctgatcgacatcgcactcgagcgcaataccagtttgcgtaccgcc240
gtattgaacagcgaaatctaccgcaaacaatacatgattgagcgcaacaacctcctgccc300
acgcttgccgccaatgcgaacggctcgcgccaaggcagcttgagcggcggcaatgtcagc360
agcagctacaatgtcggactgggtgcggcatcttacgaactcgacctgttcggacgcgtc420
cgcagcagcagcgaagcagcactgcaaggctattttgcaagtgtcgccaaccgcgatgcg480
gcacatttgagcctgattgccaccgttgccaaagcctatttcaacgaacgttatgccgaa540
gaagcgatgtctttggcgcagcgtgttttgaaaacgcgcgaggaaacctacaagctgtcc600
gaattacgttacaaggcaggcgtgatttccgccgtcgccctacgtcagcaggaagccctg660
2
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
atcgaatctgccaaagccgattatgcccatgccgcgcgcagccgcgaacaggcgcgcaat720
gccttggcaaccttgattaaccaaccgatacccgaagacctgcctgccggtttgccgctg780
gacaagcagttttttgttgaaaaactgccggccggtttgagttccgaagtattgctcgac840
cgtcccgatatccgtgctgccgaacacgcgctcaaacaggcaaacgccaatatcggtgcg900
gcacgcgccgcctttttcccatccatccgcctgaccggaaccgtcggtacgggttctgcc960
gaattgggtgggttgttcaaaagcggcacgggcgtttggtcgttcgcgccgtctattacc1020
ctgccgatttttacctggggtacgaacaaagccaaccttgatgtagccaagctgcgccaa1080
caggcacaaatcgttgcctatgaagccgccgtccaatccgcatttcaagacgtggcaaac1140
gcattggcggcgcgcgagcagctggataaagcctatgacgctttaagcaaacaaagccgc1200
gcctctaaagaggcgttgcgcttggtcggcctgcgttacaagcacggcgtatccggcgcg1260
ctcgacttgctcgatgcggaacgcagcagctatgcggcggagggtgcggctttgtcggca1320
caactgacccgcgccgaaaaccttgccgatttgtacaaggcactcggcggcggattgaaa1380
cgggatacccaaaccgacaaataa 1404
<210> 4
<211> 467
<212> PRT
<213> Neisseria meningitidis
<400> 4
Met Asp Thr Thr Leu Lys Thr Thr Leu Thr Ser Val Ala Ala Ala Phe
1 5 10 15
Ala Leu Ser Ala Cys Thr Met Ile Pro Gln Tyr Glu Gln Pro Lys Val
20 25 30
Glu Val Ala Glu Thr Phe Lys Asn Asp Thr Ala Asp Ser Gly Ile Arg
35 40 45
Ala Val Asp Leu Gly Trp His Asp Tyr Phe Ala Asp Pro Arg Leu Gln
50 55 60
Lys Leu Ile Asp Ile Ala Leu Glu Arg Asn Thr Ser Leu Arg Thr Ala
65 70 75 80
Val Leu Asn Ser Glu Ile Tyr Arg Lys Gln Tyr Met Ile Glu Arg Asn
85 90 95
Asn Leu Leu Pro Thr Leu Ala Ala Asn Ala Asn Gly Ser Arg Gln Gly
100 105 110
Ser Leu Ser Gly Gly Asn Val Ser Ser Ser Tyr Asn Val Gly Leu Gly
115 120 125
Ala Ala Ser Tyr Glu Leu Asp Leu Phe Gly Arg Val Arg Ser Ser Ser
130 135 140
Glu Ala Ala Leu Gln Gly Tyr Phe Ala Ser Val Ala Asn Arg Asp Ala
145 150 155 160
Ala His Leu Ser Leu Ile Ala Thr Val Ala Lys Ala Tyr Phe Asn Glu
165 170 175
Arg Tyr Ala Glu Glu Ala Met Ser Leu Ala Gln Arg Val Leu Lys Thr
180 185 190
Arg Glu Glu Thr Tyr Lys Leu Ser Glu Leu Arg Tyr Lys Ala Gly Val
195 200 205
Ile Ser Ala Val Ala Leu Arg Gln Gln Glu Ala Leu Ile Glu Ser Ala
3
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
210 215 220
Lys Ala Asp Tyr Ala His Ala Ala Arg Ser Arg Glu Gln Ala Arg Asn
225 230 235 240
Ala Leu Ala Thr Leu Ile Asn Gln Pro Ile Pro Glu Asp Leu Pro Ala
245 250 255
Gly Leu Pro Leu Asp Lys Gln Phe Phe Val Glu Lys Leu Pro Ala Gly
260 265 270
Leu Ser Ser Glu Val Leu Leu Asp Arg Pro Asp Ile Arg Ala Ala Glu
275 280 285
His Ala Leu Lys Gln Ala Asn Ala Asn Ile Gly Ala Ala Arg Ala Ala
290 295 300
Phe Phe Pro Ser Ile Arg Leu Thr Gly Thr Val Gly Thr Gly Ser Ala
305 310 315 320
Glu Leu Gly Gly Leu Phe Lys Ser Gly Thr Gly Val Trp Ser Phe Ala
325 330 335
Pro Ser Ile Thr Leu Pro Ile Phe Thr Trp Gly Thr Asn Lys Ala Asn
340 345 350
Leu Asp Val Ala Lys Leu Arg Gln Gln Ala Gln Ile Val Ala Tyr Glu
355 360 365
Ala Ala Val Gln Ser Ala Phe Gln Asp Val Ala Asn Ala Leu Ala Ala
370 375 380
Arg Glu Gln Leu Asp Lys Ala Tyr Asp Ala Leu Ser Lys Gln Ser Arg
385 390 395 400
Ala Ser Lys Glu Ala Leu Arg Leu Val Gly Leu Arg Tyr Lys His Gly
405 410 415
Val Ser Gly Ala Leu Asp Leu Leu Asp Ala Glu Arg Ser Ser Tyr Ala
420 425 430
Ala Glu Gly Ala Ala Leu Ser Ala Gln Leu Thr Arg Ala Glu Asn Leu
435 440 445
Ala Asp Leu Tyr Lys Ala Leu Gly Gly Gly Leu Lys Arg Asp Thr Gln
450 455 460
Thr Asp Lys
465
<210> 5
<211> 420
<212> DNA
<213> Neisseria meningitidis
<400> 5
atgaaaaaacttctaatgataaccctcaccggtatgcttgcagcttgtgcaacaggtgtc60
aatgtcggccggttgatggttgaaatgccgcagggagaacgttctgtcgttgtgcaggtt120
cccgcgacaaataacccgctttccgatacggtagctgtcggaatgattaaaacatccggt180
tcgccttcggcatcaaatatgattgaaatgctcggcgcggacaatatcaacgtcggcgtg240
gtgggaagcagccaaatgcttaataaggcgaccgcactttattccttaaaccatgcaaag300
aaagtcggaaataatgtcagtgtttatatgatgggcgacagcgaaagtgacaaggccgat360
ttggaaaacgcggcaaatgccaaaaatatcaaattgcattatttctttaaccaaaaataa420
4
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
<210> 6
<211> 139
<212> PRT
<213> Neisseria meningitidis
<400> 6
Met Lys Lys Leu Leu Met Ile Thr Leu Thr Gly Met Leu Ala Ala Cys
1 5 10 15
Ala Thr Gly Val Asn Val Gly Arg Leu Met Val Glu Met Pro Gln Gly
20 25 30
Glu Arg Ser Val Val Val Gln Val Pro Ala Thr Asn Asn Pro Leu Ser
35 40 45
Asp Thr Val Ala Val Gly Met Ile Lys Thr Ser G1y Ser Pro Ser Ala
50 55 60
Ser Asn Met Ile Glu Met Leu Gly Ala Asp Asn Ile Asn Val Gly Val
65 70 75 80
Val Gly Ser Ser Gln Met Leu Asn Lys Ala Thr Ala Leu Tyr Ser Leu
85 90 95
Asn His Ala Lys Lys Val Gly Asn Asn Val Ser Val Tyr Met Met Gly
100 105 110
Asp Ser Glu Ser Asp Lys Ala Asp Leu Glu Asn Ala Ala Asn Ala Lys
115 120 125
Asn Ile Lys Leu His Tyr Phe Phe Asn Gln Lys
130 135
<210> 7
<211> 516
<212> DNA
<213> Neisseria meningitidis
<400> 7
atgaaaatcaaacaaatcgtcaaaccgggcttggcagtattggcggcgggcgttctgtct60
gcctgcgcaaccaaaagcaacgtcaaagccgacggaacgaccgacaatccggttttcccg120
aaaccctattccgtaacgctcgacaacaatcgcggtacattcccgacctatgacgaattg180
gacttgatgcgtcccggtctgaccaaagacgacatctacaaaatcctgggtcgtccgcat240
tacgacgaaggtatgtacggcgtgcgcgaatgggattatctgttccacttccacaccccg300
ggcgtaggcatcgaccctgaaaacacttccggcgtagaaggcattaccacctgtcaatac360
aaaattattttcgataaagacaaatttgcccgcagcttctactggaaccccgtcttcccg420
aaagatgccgcctgtccgccgcccgcacccaaagccgagccgcaagtcatcatccgcgaa480
atcgtgcccgccaaacccaaacgcatccgccaataa 516
<210> 8
<211> 171
<212> PRT
<213> Neisseria meningitidis
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
<400> 8
Met Lys Ile Lys Gln Ile Val Lys Pro Gly Leu Ala Val Leu Ala Ala
1 5 10 15
Gly Val Leu Ser Ala Cys Ala Thr Lys Ser Asn Val Lys Ala Asp Gly
20 25 30
Thr Thr Asp Asn Pro Val Phe Pro Lys Pro Tyr Ser Val Thr Leu Asp
35 40 45
Asn Asn Arg Gly Thr Phe Pro Thr Tyr Asp Glu Leu Asp Leu Met Arg
50 55 60
Pro Gly Leu Thr Lys Asp Asp Ile Tyr Lys Ile Leu Gly Arg Pro His
65 70 75 80
Tyr Asp Glu Gly Met Tyr Gly Val Arg Glu Trp Asp Tyr Leu Phe His
85 90 95
Phe His Thr Pro Gly Val Gly Ile Asp Pro Glu Asn Thr Ser Gly Val
100 105 ' 110
Glu Gly Ile Thr Thr Cys Gln Tyr Lys Ile Ile Phe Asp Lys Asp Lys
115 120 125
Phe Ala Arg Ser Phe Tyr Trp Asn Pro Val Phe Pro Lys Asp Ala Ala
130 135 140
Cys Pro Pro Pro Ala Pro Lys Ala Glu Pro Gln Val Ile Ile Arg Glu
145 150 155 160
Ile Val Pro Ala Lys Pro Lys Arg Ile Arg Gln
165 170
<210> 9
<211> 816
<212> DNA
<213> Neisseria meningitidis
<400>
9
atgagaccatatgctactaccatttatcaactttttattttgtttattgggagtgttttt60
actatgacctcatgtgaacctgtgaatgaaaagacagatcaaaaagcagtaagtgcgcaa120
caggctaaagaacaaaccagtttcaacaatcccgagccaatgacaggatttgaacatacg180
gttacatttgattttcagggcaccaaaatggttatcccctatggctatcttgcacggtat240
acgcaagacaatgccacaaaatggctttccgacacgcccgggcaggatgcttactccatt300
aatttgatagagattagcgtctattacaaaaaaaccgaccaaggctgggttcttgagcca360
tacaaccagcaaaacaaagcacactttatccaatttctacgcgacggtttggatagcgtg420
gacgatattgttatccgaaaagatgcgtgtagtttaagtacgactatgggagaaagattg480
cttacttacggggttaaaaaaatgccatctgcctatcctgaatacgaggcttatgaagat540
aaaagacatattcctgaaaatccatattttcatgaattttactatattaaaaaaggagaa600
aatccggcgattattactcatcggaataatcgaataaaccaaactgaagaagatagttat660
agcactagcgtaggttcctgtattaacggtttcacggtacagtattacccgtttattcgg720
gaaaagcagcagctcacacagcaggagttggtaggttatcaccaacaagtagagcaattg780
gtacagagttttgtaaacaattcaaataaaaaataa 816
<210> 10
<211> 271
6
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
<212> PRT
<213> Neisseria meningitides
<400> 10
Met Arg Pro Tyr Ala Thr Thr Ile Tyr Gln Leu Phe Ile Leu Phe Ile
1 5 10 15
Gly Ser Val Phe Thr Met Thr Ser Cys Glu Pro Val Asn Glu Lys Thr
20 25 30
Asp Gln Lys Ala Val Ser Ala Gln Gln Ala Lys Glu Gln Thr Ser Phe
35 40 45
Asn Asn Pro Glu Pro Met Thr Gly Phe Glu His Thr Val Thr Phe Asp
50 55 60
Phe Gln Gly Thr Lys Met Val Ile Pro Tyr Gly Tyr Leu Ala Arg Tyr
65 70 75 80
Thr G1n Asp Asn Ala Thr Lys Trp Leu Ser Asp Thr Pro Gly Gln Asp
85 90 95
Ala Tyr Ser Ile Asn Leu Ile Glu Ile Ser Val Tyr Tyr Lys Lys Thr
100 105 110
Asp Gln Gly Trp Val Leu Glu Pro Tyr Asn Gln Gln Asn Lys Ala His
115 120 125
Phe Ile Gln Phe Leu Arg Asp G1y Leu Asp Ser Val Asp Asp Ile Val
130 135 140
Ile Arg Lys Asp Ala Cys Ser Leu Ser Thr Thr Met Gly Glu Arg Leu
145 150 155 160
Leu Thr Tyr Gly Val Lys Lys Met Pro Ser Ala Tyr Pro Glu Tyr Glu
165 170 175
Ala Tyr Glu Asp Lys Arg His Ile Pro Glu Asn Pro Tyr Phe His Glu
180 185 190
Phe Tyr Tyr Ile Lys Lys Gly Glu Asn Pro Ala Ile Ile Thr His Arg
195 200 205
Asn Asn Arg Ile Asn Gln Thr Glu Glu Asp Ser Tyr Ser Thr Ser Val
210 215 220
Gly Ser Cys Ile Asn Gly Phe Thr Val Gln Tyr Tyr Pro Phe Ile Arg
225 230 235 240
Glu Lys Gln Gln Leu Thr Gln Gln Glu Leu Val Gly Tyr His Gln Gln
245 250 255
Val Glu Gln Leu Val Gln Ser Phe Val Asn Asn Ser Asn Lys Lys
260 265 270
<210> 11
<211> 717
<212> DNA
<213> Neisseria meningitides
<400> 11
atgaagacca aattaccgct ttttatcatt tggctgtccg tatccgccgc ctgttcttcc 60
cctgtttccc gcaatattca ggatatgcgg cccgaaccgc aggcagaggc aggtagttcg 120
7
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
yacgctattccctatcccgttcccactctgcaagaccgtttggattatctggaaggcaca180
ctcgtccgcctgtcgaacgaagtggaaaccttaaacggcaaagtcaaagcactggagcat240
gcgaaaacacacccttccggtagggcatacgtccaaaaactcgacgaccgcaagttgaaa300
gagcattacctcaataccgaaggcggcagcgcatccgcacataccgtcgaaaccgcacaa360
aacctctacaatcaggcactcaaacactataaaagcggcaggttttctgccgcagccgcc420
ctgttgaaaggcgcggacggaggcgacggcggcagcatcgcgcaacgcagtatgtacctg480
ttgctgcaaagcagggcgcgtatgggcaactgcgaatccgtcatcgaaatcggagggcgt540
tacgccaaccgtttcaaagacagcccaaccgcgcccgaagccatgttcaaaatcggcgaa600
tgccaatacaggttgcagcagaaagacattgcaagggcaacttggcgcagcctgatacag660
gcttacccgagcagcccggcggcaaaacgcgccgccgcagccgtacgcaaacgatag 717
<210> 12
<211> 238
<212> PRT
<213> Neisseria meningitidis
<400> 12
Met Lys Thr Lys Leu Pro Leu Phe Ile Ile Trp Leu Ser Val Ser Ala
1 5 10 15
Ala Cys Ser Ser Pro Val Ser Arg Asn Ile Gln Asp Met Arg Pro Glu
20 25 30
Pro Gln Ala Glu Ala Gly Ser Ser Asp Ala Ile Pro Tyr Pro Val Pro
35 40 45
Thr Leu Gln Asp Arg Leu Asp Tyr Leu Glu Gly Thr Leu Val Arg Leu
50 55 60
Ser Asn Glu Val Glu Thr Leu Asn Gly Lys Val Lys Ala Leu Glu His
65 70 75 80
Ala Lys Thr His Pro Ser Gly Arg Ala Tyr Val Gln Lys Leu Asp Asp
85 90 95
Arg Lys Leu Lys Glu His Tyr Leu Asn Thr Glu Gly Gly Ser Ala Ser
100 105 110
Ala His Thr Val Glu Thr Ala Gln Asn Leu Tyr Asn Gln Ala Leu Lys
115 120 125
His Tyr Lys Ser Gly Arg Phe Ser Ala Ala Ala Ala Leu Leu Lys Gly
130 135 140
Ala Asp Gly Gly Asp Gly G1y Ser Ile Ala Gln Arg Ser Met Tyr Leu
145 150 155 160
Leu Leu Gln Ser Arg Ala Arg Met Gly Asn Cys Glu Ser Val Ile Glu
165 170 175
Ile Gly Gly Arg Tyr Ala Asn Arg Phe Lys Asp Ser Pro Thr Ala Pro
180 185 190
Glu Ala Met Phe Lys Ile Gly Glu Cys Gln Tyr Arg Leu Gln Gln Lys
195 200 205
Asp Ile Ala Arg Ala Thr Trp Arg Ser Leu Ile Gln Ala Tyr Pro Ser
210 215 220
Ser Pro Ala Ala Lys Arg Ala Ala Ala Ala Val Arg Lys Arg
225 230 235
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
<210> 13
<211> 1176
<212> DNA
<213> Neisseria meningitides
<400> 13
gtgtttaaagtgaaattttatattcgtcacgcagtattattattgtgtggaagtttaatt60
gtaggatgctctgcgattccttcatcaggccccagcgcaaaaaaaattgtctctttaggg120
caacaatctgaagttcaaattcctgaagtggagctgattgatgtgaatcatacggttgct180
cagttattatataaggctcagataaatcagtcattcactcagtttggcgatggttatgct240
tcggctggtacgctaaatattggtgatgtattggatattatgatttgggaagcgccgccg300
gcagtattgtttggtggtggcctttcttcgatgggctcgggtagtgcgcatcaaactaag360
ttgccagagcagttggtcacggcacgtggtacggtttctgtgccgtttgttggcgatatt420
tcggtggtcggtaaaacgcctggtcaggttcaggaaattattaaaggccgcctgaaaaaa480
atggccaatcagccacaagtgatggtgcgtttggtgcagaataatgcggcgaatgtgtcg540
gtgattcgtgctgggaatagtgtgcgtatgccgctgacggcagccggtgagcgtgtgttg600
gatgcggtggctgcggtaggtggttcaacggcaaatgtgcaggatacgaatgtgcagctg660
acacgtggcaatgtagtacgaactgttgccttggaagatttagttgcaaatccgcgacaa720
aatattttgctgcgtcgcggtgatgtggttaccatgattaccaatccctatacctttacg780
tctatgggtgcggtggggagaacacaagaaatcggtttttcagccagaggcttatcgctt840
tctgaagccattggccgtatgggcggtttgcaagatcgccgttctgatgcgcgtggtgtg900
tttgtgttccgctatacgccattggtggaattgccggcagaacgtcaggataaatggatt960
gctcaaggttatggcagtgaggcagagattccaacggtatatcgtgtgaatatggctgat1020
gcgcattcgctattttctatgcagcgctttcctgtgaagaataaagatgtattgtatgtg1080
tcgaatgcgccgttggctgaagtgcagaaattcttgtcgtttgtgttctcgccggttacc1140
agtggcgcgaacagtattaataatttaactaattaa 1176
<210> 14
<211> 391
<212> PRT
<213> Neisseria meningitides
<400> 14
Met Phe Lys Val Lys Phe Tyr Ile Arg His Ala Val Leu Leu Leu Cys
1 5 10 15
Gly Ser Leu Ile Val Gly Cys Ser Ala Ile Pro Ser Ser Gly Pro Ser
20 25 30
Ala Lys Lys Ile Val Ser Leu Gly Gln Gln Ser Glu Val Gln Ile Pro
35 4p q5
Glu Val Glu Leu Ile Asp Val Asn His Thr Val Ala Gln Leu Leu Tyr
50 55 60
Lys Ala Gln Ile Asn Gln Ser Phe Thr Gln Phe Gly Asp Gly Tyr Ala
65 70 75 80
Ser Ala Gly Thr Leu Asn Ile Gly Asp Val Leu Asp Ile Met Ile Trp
85 90 95
Glu Ala Pro Pro Ala Val Leu Phe Gly Gly Gly Leu Ser Ser Met Gly
9
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
100 105 110
Ser Gly Ser Ala His Gln Thr Lys Leu Pro Glu Gln Leu Val Thr Ala
115 120 125
Arg Gly Thr Val Ser Val Pro Phe Val Gly Asp Ile Ser Val Val Gly
130 135 140
Lys Thr Pro Gly Gln Val Gln Glu Ile Ile Lys Gly Arg Leu Lys Lys
145 150 155 160
Met Ala Asn Gln Pro Gln Val Met Val Arg Leu Val Gln Asn Asn Ala
165 170 175
Ala Asn Val Ser Val Ile Arg Ala Gly Asn Ser Val Arg Met Pro Leu
180 185 190
Thr Ala Ala Gly G1u Arg Val Leu Asp Ala Val Ala Ala Val Gly Gly
195 200 205
Ser Thr Ala Asn Val Gln Asp Thr Asn Val Gln Leu Thr Arg Gly Asn
210 215 220
Val Val Arg Thr Val Ala Leu Glu Asp Leu Val Ala Asn Pro Arg Gln
225 230 235 240
Asn I1e Leu Leu Arg Arg Gly Asp Val Val Thr Met Ile Thr Asn Pro
245 250 255
Tyr Thr Phe Thr Ser Met Gly Ala Val Gly Arg Thr Gln Glu Ile Gly
260 265 270
Phe Ser Ala Arg Gly Leu Ser Leu Ser Glu Ala Ile Gly Arg Met Gly
275 280 285
Gly Leu Gln Asp Arg Arg Ser Asp Ala Arg Gly Val Phe Val Phe Arg
290 295 300
Tyr Thr Pro Leu Val Glu Leu Pro Ala Glu Arg Gln Asp Lys Trp Ile
305 310 315 320
Ala Gln Gly Tyr Gly Ser Glu Ala Glu Ile Pro Thr Val Tyr Arg Val
325 330 335
Asn Met Ala Asp Ala His Ser Leu Phe Ser Met Gln Arg Phe Pro Val
340 345 350
Lys Asn Lys Asp Val Leu Tyr Val Ser Asn Ala Pro Leu Ala Glu Val
355 360 365
Gln Lys Phe Leu Ser Phe Val Phe Ser Pro Val Thr Ser Gly Ala Asn
370 375 380
Ser Ile Asn Asn Leu Thr Asn
385 390
<210> 15
<211> 807
<212> DNA
<213> Neisseria meningitidis
<400> 15
atgaatatga aaaaatggat tgccgccgcc cttgcctgtt ccgcgctcgc gctgtctgcc 60
tgcggcggtc agggcaaaga tgccgccgcg cccgccgcaa accccgacaa agtgtaccgc 120
gtggcttcca acgccgagtt tgcccccttt gaatctttag actcgaaagg caatgttgaa 180
1~
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
yymtcgatgtggatttgatgaacgcgatggcgaaggcgggcaattttaaaatcgaattc240
aaacaccagccgtgggacagccttttccccgccttgaacaacggcgatgcggacgttgtg300
atgtcgggcgtaaccattaccgacgaccgcaaacagtctatggacttcagcgacccgtat360
tttgaaatcacccaagtcgtcctcgttccgaaaggcaaaaaaatatcttcttccgaagat420
ttgaaaaacatgaacaaagtcggcgtggtaaccggctacacgggcgatttctccgtatcc480
aaactcttgggcaacgacaacccgaaaatcgcgcgctttgaaaacgttcccctgattatc540
aaagaactggaaaacggcggcttggattccgtggtcagcgacagcgcagtcatcgccaat600
tatgtgaaaaacaatccgaccaaagggatggacttcgttaccctgcccgacttcaccacc660
gaacactacggcatcgcggtacgcaaaggcgacgaagcaaccgtcaaaatgctgaacgat720
gcgttgaaaaaagtacgcgaaagcggcgaatacgacaaaatctacgccaaatattttgca780
aaagaagacggacaggccgcaaaataa g07
<210> 16
<211> 268
<212> PRT
<213> Neisseria meningitidis
<400> 16
Met Asn Met Lys Lys Trp Ile Ala Ala Ala Leu Ala Cys Ser Ala Leu
1 5 10 15
Ala Leu Ser Ala Cys Gly Gly Gln Gly Lys Asp Ala Ala Ala Pro Ala
20 25 30
Ala Asn Pro Asp Lys Val Tyr Arg Val Ala Ser Asn Ala Glu Phe Ala
35 40 45
Pro Phe Glu Ser Leu Asp Ser Lys Gly Asn Val Glu Gly Phe Asp Val
50 55 60
Asp Leu Met Asn Ala Met Ala Lys Ala Gly Asn Phe Lys Ile Glu Phe
65 70 75 80
Lys His Gln Pro Trp Asp Ser Leu Phe Pro Ala Leu Asn Asn Gly Asp
85 90 95
Ala Asp Val Val Met Ser Gly Val Thr Ile Thr Asp Asp Arg Lys Gln
100 105 110
Ser Met Asp Phe Ser Asp Pro Tyr Phe Glu Ile Thr Gln Val Val Leu
115 120 125
Val Pro Lys Gly Lys Lys Ile Ser Ser Ser Glu Asp Leu Lys Asn Met
130 135 140
Asn Lys Val Gly Val Val Thr Gly Tyr Thr Gly Asp Phe Ser Va1 Ser
145 150 155 160
Lys Leu Leu Gly Asn Asp Asn Pro Lys Ile Ala Arg Phe Glu Asn Val
165 170 175
Pro Leu Ile Ile Lys G1u Leu Glu Asn Gly Gly Leu Asp Ser Val Val
180 185 190
Ser Asp Ser Ala Val I1e Ala Asn Tyr Val Lys Asn Asn Pro Thr Lys
195 200 205
Gly Met Asp Phe Val Thr Leu Pro Asp Phe Thr Thr Glu His Tyr Gly
210 215 220
Ile Ala Val Arg Lys Gly Asp Glu Ala Thr Val Lys Met Leu Asn Asp
11
CA 02359504 2001-07-12
WO 00/42191 PCT/EP00/00135
225 230 235 240
Ala Leu Lys Lys Val Arg Glu Ser Gly Glu Tyr Asp Lys Ile Tyr Ala
245 250 255
Lys Tyr Phe Ala Lys Glu Asp Gly Gln Ala Ala Lys
260 265
12
