United States Patent: 8,034,358
Issued: October 11, 2011
Inventors: Peak; Ian
Richard Anselm (St. Lucia, AU), Jennings; Michael Paul (Carina, AU), Moxon;
E. Richard (Oxfordshire, GB)
The University of Queesland (St. Lucia, AT)
Appl. No.: 11/776,709
Filed: July 12, 2007
The invention provides a novel surface
polypeptide from Neisseria meningitidis as well as nucleic acid and
nucleic acid sequence homologues encoding this protein. Pharmaceutical
compositions containing the polypeptide and nucleic acids of the invention
are also disclosed as well as methods useful in the treatment, prevention
and diagnosis of N. meningitidis infection.
Description of the
SUMMARY OF THE INVENTION
The present inventors have discovered a new gene which is present in all
tested strains of N. meningitidis and which encodes a novel polypeptide
having a predicted molecular weight of about 62 kDa. Based upon its
sequence characteristics and homologies, this polypeptide is predicted to
be an adhesin and this, together with experimental data suggests that it
constitutes a surface protein which may be useful for the production of
therapeutic and/or prophylactic vaccines against N. meningitides as
Accordingly, in one aspect of the invention, there is provided an isolated
polypeptide or fragment thereof, or variant or derivative of these, said
polypeptide selected from the group consisting of:-- (a) a polypeptide
according to SEQ ID NO 2; (b) a polypeptide according to SEQ ID NO 5; (c)
a polypeptide according to SEQ ID NO 7; (d) a polypeptide according to SEQ
ID NO 9; (e) a polypeptide according to SEQ ID NO 11; (f) a polypeptide
according to SEQ ID NO 13; (g) a polypeptide according to SEQ ID NO 15;
(h) a polypeptide according to SEQ ID NO 17; (i) a polypeptide according
to SEQ ID NO 19; and (j) a polypeptide according to SEQ ID NO 21.
Preferably, said polypeptide, fragment, variant or derivative elicits an
immune response against one or more members selected from the group
consisting of:-- (i) N. meningitidis; (ii) said polypeptide; (iii) said
fragment; (iv) said variant; and (v) said derivative;
According to another aspect, the invention provides an isolated nucleic
acid sequence encoding a polypeptide or fragment thereof, or variant or
derivative of said fragment or polypeptide, according to the
first-mentioned aspect. Suitably, said sequence is selected from the group
consisting of:-- (1) the nucleotide sequence of SEQ ID NO 1; (2) the
nucleotide sequence of SEQ ID NO 3; (3) the nucleotide sequence of SEQ ID
NO 4; (4) the nucleotide sequence of SEQ ID NO 6; (5) the nucleotide
sequence of SEQ ID NO 8; (6) the nucleotide sequence of SEQ ID NO 10; (7)
the nucleotide sequence of SEQ ID NO 12; (8) the nucleotide sequence of
SEQ ID NO 14; (9) the nucleotide sequence of SEQ ID NO 16; (10) the
nucleotide sequence of SEQ ID NO 18; (11) the nucleotide sequence of SEQ
ID NO 20; (12) a nucleotide sequence fragment of any one of SEQ ID NOS 1,
3, 4, 6, 8, 10, 12, 14, 16, 18 and 20; and (13) a nucleotide sequence
homologue of any of the foregoing sequences
Preferably, said sequences encode a product that elicits an immune
response against one or more members selected from the group consisting
of:-- (i) N. meningitidis; (ii) said polypeptide of the first-mentioned
aspect; (iii) said fragment of said first-mentioned aspect; (iv) said
variant of said first-mentioned aspect; and (v) said derivative of said
In yet another aspect, the invention resides in an expression vector
comprising a nucleic acid sequence according to the second-mentioned
aspect wherein said sequence is operably linked to transcriptional and
translational regulatory nucleic acid.
In a further aspect, the invention provides a host cell containing an
expression vector according to the third-mentioned aspect.
In yet a further aspect of the invention, there is provided a method of
producing a recombinant polypeptide according to the first-mentioned
aspect, said method comprising the steps of:-- (A) culturing a host cell
containing an expression vector according to the third-mentioned aspect
such that said recombinant polypeptide is expressed from said nucleic
acid; and (B) isolating said recombinant polypeptide.
In a still further aspect, the invention provides an antibody or antibody
fragment that binds to one or more members selected from the group
consisting of:-- (1) N. meningitidis; (2) said polypeptide of the
first-mentioned aspect; (3) said fragment of the first-mentioned aspect;
(4) said variant of the first-mentioned aspect; and (5) said derivative of
the first-mentioned aspect.
In yet another aspect, the invention provides a method of detecting N.
meningitidis in a biological sample suspected of containing same, said
method comprising the steps of:-- (A) isolating the biological sample from
a patient; (B) mixing the above-mentioned antibody or antibody fragment
with the biological sample to form a mixture; and (C) detecting
specifically bound antibody or bound antibody fragment in the mixture
which indicates the presence of N. meningitidis.
According to a further aspect, there is provided a method of detecting N.
meningitidis bacteria in a biological sample suspected of containing said
bacteria, said method comprising the steps of:-- (I) isolating the
biological sample from a patient; (II) detecting a nucleic acid sequence
according to the second-mentioned aspect in said sample which indicates
the presence of said bacteria.
The invention further contemplates a method for diagnosing infection of
patients by N. meningitidis, said method comprising the steps of:-- (1)
contacting a biological sample from a patient with a polypeptide,
fragment, variant or derivative of the invention; and (2) determining the
presence or absence of a complex between said polypeptide, fragment,
variant or derivative and N. meningitidis-specific antibodies in said
sample, wherein the presence of said complex is indicative of said
The invention also extends to the use of the polypeptide according to the
first-mentioned aspect, the use of the nucleic acids according to the
second-mentioned aspect or the use of the antibody or antibody fragment
mentioned above in a kit for detecting N. meningitidis bacteria in a
According to a further aspect of the invention, there is provided a
pharmaceutical composition comprising an isolated polypeptide or fragment
thereof, or a variant or derivative of these, according to the first
Preferably, said pharmaceutical composition is a vaccine.
In yet a further aspect, the invention provides a method of preventing
infection of a patient by N. meningitidis, comprising the step of
administrating a pharmaceutically effective amount of the above-mentioned
In a further aspect, the invention provides a method of identifying an
immunoreactive fragment of a polypeptide, variant or derivatives according
to the first mentioned aspect, comprising the steps of:-- (a) producing a
fragment of said polypeptide, variant or derivative; (b) administering
said fragment to a mammal; and (c) detecting an immune response in said
mammal which response includes production of elements which specifically
bind N. meningitidis and/or said polypeptide, variant or derivative,
and/or a protective effect against N. meningitidis infection.
DETAILED DESCRIPTION OF THE INVENTION
Throughout this specification and the appendant claims, unless the context
requires otherwise, the words "comprise", "comprises" and "comprising"
will be understood to imply the inclusion of a stated integer or group of
integers but not the exclusion of any other integer or group of integers.
The present invention provides an isolated polypeptide according to SEQ ID
NOS 2, 5, 7, 9, 11, 13, 15, 17, 19 and 21, or fragment respectively
thereof, or variant or derivative of these. In a preferred embodiment, the
polypeptide, fragments, variants and derivatives of the invention elicit
an immune response against any one member selected from the group
consisting of N. meningitidis, said polypeptide, said fragment, said
variant and said derivative.
SEQ ID NO 2 corresponds to the novel about 62 kDa surface polypeptide of
the hiaNm gene obtained from N. meningitidis strain MC58, as described
more fully hereinafter. SEQ ID NOS 5, 7, 9, 11, 13, 15, 17, 19, and 21
correspond to homologous polypeptides deduced from nucleotide sequences
obtained from N. meningitidis strains BZ10, BZ198, EG327, EG329, H15, H38,
H41, P20, and PMC21, respectively.
For the purposes of this invention, the phrase "elicit(s) an immune
response" refers to the ability of the aforementioned polypeptide,
fragment, variant or derivative to produce an immune response in a mammal
to which it is administered, wherein the response includes the production
of elements which specifically bind N. meningitidis and/or said
polypeptide, fragment, variant or derivative, and/or which provide a
protective effect against N. meningitidis infection.
By "isolated" is meant material that is substantially or essentially free
from components that normally accompany it in its native state.
By "polypeptide" is meant a long chain peptide including a protein.
As used herein, the term "fragment" includes deletion mutants and small
peptides, for example of at least 6, preferably at least 10 and more
preferably at least 20 amino acids in length, which comprise antigenic
determinants or epitopes. Several such fragments may be joined together.
Peptides of this type may be obtained through the application of standard
recombinant nucleic acid techniques or synthesized using conventional
liquid or solid phase synthesis techniques. For example, reference may be
made to solution synthesis or solid phase synthesis as described, for
example, in Chapter 9 entitled "Peptide Synthesis" by Atherton and
Shephard which is included in a publication entitled "Synthetic Vaccines"
edited by Nicholson and published by Blackwell Scientific Publications.
Alternatively, peptides can be produced by digestion of a polypeptide of
the invention with proteinases such as endoLys-C, endoArg-C, endoGlu-C and
staphylococcins V8-protease. The digested fragments can be purified by,
for example, high performance liquid chromatographic (HPLC) techniques.
The term "variant" refers to polypeptides in which one or more amino acids
halve been replaced by different amino acids. It is well understood in the
art that some amino acids may be changed to others with broadly similar
properties without changing the nature of the activity of the polypeptide
(conservative substitutions). Exemplary conservative substitutions in the
polypeptide may be made according to the following table
-- see Original Patent.
Substantial changes in function are made by selecting substitutions that
are less conservative than those shown in TABLE 1 (see Original Patent).
Other replacements would be non-conservative substitutions and relatively
fewer of these may be tolerated. Generally, the substitutions which are
likely to produce the greatest changes in a polypeptide's properties are
those in which (a) a hydrophilic residue (e.g., Ser or Thr) is substituted
for, or by, a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val); (b) a
cysteine or proline is substituted for, or by, any other residue; (c) a
residue having an electropositive side chain (e.g., Arg, His or Lys) is
substituted for, or by, an electronegative residue (e.g., Glu or Asp) or
(d) a residue having a bulky side chain (e.g., Phe or Trp) is substituted
for, or by, one having a smaller side chain (e.g., Ala, Ser) or no side
chain (e.g., Gly).
In general, variants will be at least 75% homologous, more suitably at
least 80%, preferably at least 85%, and most preferably at least 90%
homologous to the basic sequences as for example shown in SEQ ID NOS 2, 5,
7, 9, 11, 13, 15, 17, 19 and 21. Homology is defined as the percentage
number of amino acids that are identical or constitute conservative
substitutions as defined in Table 1. Homology may be determined using
sequence comparison programs such as GAP (Deveraux et al. 1984, Nucleic
Acids Research 12, 387-395) which is incorporated herein by reference. In
this way sequences of a similar or substantially different length to those
cited herein may be compared by insertion of gaps into the alignment, such
gaps being determined, for example, by the comparison algorithm used by
GAP. What constitutes suitable variants may be determined by conventional
techniques. For example, nucleic acids encoding polypeptides according to
SEQ ID NOS 2, 5, 7, 9, 11, 13, 15, 17, 19 and 21 can be mutated using
either random mutagenesis for example using transposon mutagenesis, or
site-directed mutagenesis. The resultant DNA fragments are then cloned
into suitable expression hosts such as E. coli using conventional
technology and clones that retain the desired activity are detected. Where
the clones have been derived using random mutagenesis techniques, positive
clones would have to be sequenced in order to detect the mutation. The
term "variant" also includes naturally occurring allelic variants.
By "derivative" is meant a polypeptide that has been derived from the
basic sequence by modification, for example by conjugation or complexing
with other chemical moieties or by post-translational modification
techniques as would be understood in the art. Such derivatives include
amino acid deletions and/or additions to polypeptides according to SEQ ID
NOS 2, 51 7, 9, 11, 13, 15, 17, 19 and 21 or variants thereof wherein said
derivatives retain activity eliciting an immune response. "Additions" of
amino acids may include fusion of the polypeptides or variants thereof
with other polypeptides or proteins. In this regard, it will be
appreciated that the polypeptides or variants of the invention may be
incorporated into larger polypeptides, and such larger polypeptides may
also be expected to retain immunological activity against, for example, N.
meningitidis. The polypeptides as described above may be fused to a
further protein, for example, which is not derived from N. meningitidis.
The other protein may, by way of example, assist in the purification of
the protein. For instance a polyhistidine tag, or a maltose binding
protein may be used in this respect as described in more detail below.
Alternatively, it may produce an immune response, which is effective
against N. meningitidis, or it may produce an immune response against
another pathogen. Other possible fusion proteins are those which produce
an immunomodulatory response. Particular examples of such proteins include
Protein A or glutathione S-transferase (GST). In addition, the polypeptide
may be fused to an oligosaccharide based vaccine component where it acts
as a carrier protein.
Other derivatives contemplated by the invention include, but are not
limited to, modification to side chains, incorporation of unnatural amino
acids and/or their derivatives during peptide, polypeptide or protein
synthesis and the use of crosslinkers and other methods which impose
conformational constraints on the polypeptides, fragments and variants of
Examples of side chain modifications contemplated by the present invention
include modifications of amino groups such as by acylation with acetic
anhydride; acylation of amino groups with succinic anhydride and
tetrahydrophthalic anhydride; amidination with methylacetimidate;
carbamoylation of amino groups with cyanate; pyridoxylation of lysine with
pyridoxal-5-phosphate followed by reduction with NaBH.sub.4; reductive
alkylation by reaction with an aldehyde followed by reduction with
NaBH.sub.4; and trinitrobenzylation of amino groups with 2, 4,
6-trinitrobenzene sulphonic acid (TNBS).
The carboxyl group may be modified by carbodiimide activation via O-acylisourea
formation followed by subsequent derivitization, by way of example, to a
The guanidine group of arginine residues may be modified by formation of
heterocyclic condensation products with reagents such as 2,3-butanedione,
phenylglyoxal and glyoxal.
Sulphydryl groups may be modified by methods such as performic acid
oxidation to cysteic acid; formation of mercurial derivatives using
4-chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate;
2-chloromercuri-4-nitrophenol, phenylmercury chloride, and other
mercurials; formation of a mixed disulphides with other thiol compounds;
reaction with maleimide, maleic anhydride or other substituted maleimide;
carboxymethylation with iodoacetic acid or iodoacetamide; and
carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified, for example, by alkylation of the
indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides or
by oxidation with N-bromosuccinimide.
Tyrosine residues may be modified by nitration with tetranitromethane to
form a 3-nitrotyrosine derivative.
The imidazole ring of a histidine residue may be modified by N-carbethoxylation
withdiethylpyrocarbonate or by alkylation with iodoacetic acid
Examples of incorporating unnatural amino acids and derivatives during
peptide synthesis include but are not limited to, use of 4-amino butyric
acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,
4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine,
norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/or
D-isomers of amino acids. A list of unnatural amino acids contemplated by
the present invention is shown in TABLE 2 (see Original Patent).
The invention also contemplates covalently modifying a polypeptide,
fragment or variant of the invention with dinitrophenol, in order to
render it immunogenic in humans
Preferably the invention comprises a polypeptide selected from any one of
the polypeptides according to SEQ ID NOS 2, 5, 7, 9, 11, 13, 15, 17, 19
Polypeptides of the inventions may be prepared by any suitable procedure
known to those of skill in the art. For example, the polypeptides may be
prepared by a procedure including the steps of:
(a) preparing a recombinant nucleic acid containing a nucleotide sequence
encoding a polypeptide according to any one of SEQ ID NOS 2, 5, 7, 9, 11,
13, 15, 17, 19 and 21, or fragment thereof, or variant or derivative of
these, which nucleotide sequence is operably linked to transcriptional and
translational regulatory nucleic acid;
(b) transfecting or transforming a suitable host cell with the recombinant
(c) culturing the host cell to express recombinant polypeptide from said
recombinant nucleic acid; and
(d) isolating the recombinant polypeptide.
Suitably said nucleotide sequence is selected from the group consisting of
SEQ ID NOS 1, 3, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
By "recombinant polypeptide" is meant a polypeptide made using recombinant
techniques, i.e., through the expression of a recombinant nucleic acid.
The term "recombinant nucleic acid" as used herein refers to nucleic acid
formed in vitro by the manipulation of nucleic acid into a form not
normally found in nature. In this regard, the recombinant nucleic acid
preferably comprises an expression vector that may be either a
self-replicating extra-chromosomal vector such as a plasmid, or a vector
that integrates into a host genome. Generally, such expression vectors
include transcriptional and translational regulatory nucleic acid operably
linked to the said nucleotide sequence.
By "operably linked" is meant that the transcriptional and translational
regulatory nucleic acid is positioned relative to the nucleotide sequence
encoding the said polypeptide, fragment, variant or derivative in such a
manner that such transcription is initiatable. The transcriptional and
translational regulatory nucleic acid will generally be appropriate for
the host cell used for expression. Numerous types of appropriate
expression vectors and suitable regulatory sequences are known in the art
for a variety of host cells.
Typically, the transcriptional and translational regulatory nucleic acid
may include, but is not limited to, promoter sequences, leader or signal
sequences, ribosomal binding sites, transcriptional start and stop
sequences, translational start and stop sequences, and enhancer or
Constitutive or inducible promoters as known in the art are contemplated
by the invention. The promoters may be either naturally occurring
promoters, or hybrid promoters that combine elements of more than one
In a preferred embodiment, the expression vector contains a selectable
marker gene to allow the selection of transformed host cells. Selection
genes are well known in the art and will vary with the host cell used.
The expression vector may also include a fusion partner (typically
provided by the expression vector) so that the recombinant polypeptide of
the invention is expressed as a fusion polypeptide with said fusion
partner. The main advantage of fusion partners is that they assist,
identification and/or purification of said fusion polypeptide.
In order to express said fusion polypeptide, it is necessary to ligate a
nucleotide sequence according to the invention into the expression vector
so that the translational reading frames of the fusion partner and the
nucleotide sequence of the invention coincide.
Well known examples of fusion partners include, but are not limited to,
glutathione-S-transferase (GST), Fc potion of human IgG, maltose binding
protein (MBP) and hexahistidine (HIS.sub.6) (SEQ ID NO: 34), which are
particularly useful for isolation of the fusion polypeptide by affinity
chromatography. For the purposes of fusion polypeptide purification by
affinity chromatography, relevant matrices for affinity chromatography are
glutathione-, amylose-, and nickel- or cobalt-conjugated resins
respectively. Many such matrices are available in "kit" form, such as the
QIAexpress.TM. system (Qiagen) useful with (HIS.sub.6) (SEQ ID NO: 34)
Another fusion partner well known in the art is green fluorescent protein
(GFP). This fusion partner serves as a fluorescent "tag" which allows the
fusion polypeptide of the invention to be identified by fluorescence
microscopy or by flow cytometry. The GFP tag is useful when assessing
subcellular localization of the fusion polypeptide of the invention, or
for isolating cells which express the fusion polypeptide of the invention.
Flow cytometric methods such as fluorescence activated cell sorting (FACS)
are particularly useful in this latter application.
Preferably, the fusion partners also have protease cleavage sites, such as
for Factor X.sub.a or Thrombin, which allow the relevant protease to
partially digest the fusion polypeptide of the invention and thereby
liberate the recombinant polypeptide of the invention therefrom. The
liberated polypeptide can then be isolated from the fusion partner by
subsequent chromatographic separation.
Fusion partners according to the invention also include within their scope
"epitope tags", which are usually short peptide sequences for which a
specific antibody is available. Well known examples of epitope tags for
which specific monoclonal antibodies are readily available include c-myc,
influenza virus haemagglutinin and FLAG tags.
Recombinant polypeptides of the invention may be produced by culturing a
host cell transformed with an expression vector containing nucleic acid
encoding a polypeptide, fragment, variant or derivative according to the
invention. The conditions appropriate for protein expression will vary
with the choice of expression vector and the host cell. This is easily
ascertained by one skilled in the art through routine experimentation.
Suitable host cells for expression may be prokaryotic or eukaryotic. One
preferred host cell for expression of a polypeptide according to the
invention is a bacterium. The bacterium used may be Escherichia coli.
Alternatively, the host cell may be an insect cell such as, for example,
SF9 cells that may be utilized with a baculovirus expression system.
The recombinant protein may be conveniently prepared by a person skilled
in the art using standard protocols as for example described in Sambrook,
et al., MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbor Press,
1989), incorporated herein by reference, in particular Sections 16 and 17;
Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons,
Inc. 1994-1998), incorporated herein by reference, in particular Chapters
10 and 16; and Coligan et al., CURRENT PROTOCOLS IN PROTEIN SCIENCE (John
Wiley & Sons, Inc. 1995-1997) which is incorporated by reference herein,
in particular Chapters 1, 5 and 6.
The invention further provides a nucleotide sequence that encodes a
polypeptide, fragment, variant or derivative as defined above. Suitably,
said sequence is selected from the group consisting of:--SEQ ID NOS 1, 3,
4, 6, 8, 10, 12, 14, 16, 18 and 20; a nucleotide sequence fragment of any
one of SEQ ID NOS 1, 3, 4, 6, 8, 10, 12, 14, 16, 18 and 20; and a
nucleotide sequence homologue of the foregoing sequences. Suitably, these
sequences encode a product that elicits an immune response as defined
As will be more fully described hereinafter, SEQ ID NO 1 corresponds to
the hiaNm gene obtained from N. meningitidis strain MC58. This gene
encodes the novel 62 kDa (approximately) surface polypeptide of SEQ ID NO
2. SEQ ID NO 3 corresponds to the hiaNim open reading frame sequence of
strain MC58, HiaNm. SEQ ID NOS 4, 6, 8, 10, 12, 14, 16, 18, and 20
correspond to the homologous hiaNm open reading frame sequences obtained
from N. meningitidis strains BZ10, BZ198, EG327, EG329, H15, H38, H41,
P20, and PMC21, respectively.
The term "nucleotide sequence" as used herein designates mRNA, RNA, cRNA,
cDNA or DNA.
The term "nucleotide sequence homologues" generally refers to nucleotide
sequences that hybridize with a wild-type nucleotide sequence according to
the invention under substantially stringent conditions. Suitable
hybridization conditions will be discussed hereinafter.
The nucleotide sequence homologues of the invention may be prepared
according to the following procedure: (i) obtaining a nucleic acid extract
from a suitable host; (ii) creating primers which are optionally
degenerate wherein each comprises a portion of a wild-type nucleotide
sequence of the invention; and (iii) using said primers to amplify, via
nucleic acid amplification techniques, one or more amplification products
from said nucleic acid extract.
Suitably, the host may be a bacterium. Preferably, the host is from the
genus Neisseria, more preferably from N. meningitidis.
Preferably, the primers are selected from the group consisting of
-- see Original Patent..
Suitable nucleic acid amplification techniques are well known to the
skilled addressee, and include polymerase chain reaction (PCR) as for
example described in Ausubel et al. (1994-1998, supra, Chapter 15) which
is incorporated herein by reference; strand displacement amplification (SDA)
as for example described in U.S. Pat. No. 5,422,252 which is incorporated
herein by reference; rolling circle replication (RCR) as for example
described in Liu et al., (1996, J. Am. Chem. Soc. 118:1587-1594 and
International application WO 92/01813) and Lizardi et al., (International
Application WO 97/19193) which are incorporated herein by reference;
nucleic acid sequence-based amplification (NASBA) as for example described
by Sooknanan et al., (1994, Biotechniques 17:1077-1080) which is
incorporated herein by reference; and Q-.beta. replicase amplification as
for example described by Tyagi: et al., (1996, Proc. Natl. Acad. Sci. USA
93:5395-5400) which is incorporated herein by reference.
As used herein, an "amplification product" refers to a nucleic acid
product generated by nucleic acid amplification techniques.
"Hybridize" or "hybridization" is used here to denote the pairing of
complementary bases of distinct nucleotide sequences to produce a DNA-DNA
hybrid, a DNA-RNA hybrid, or an RNA-RNA hybrid according to base-pairing
In DNA, complementary bases are:
(i) A and T; and
(ii) C and G.
In RNA, complementary bases are:
(i) A and U; and
(ii) C and G.
In RNA-DNA hybrids, complementary bases are:
(i) A and U;
(ii) A and T; and
(iii) G and C.
Typically, substantially complementary nucleotide sequences are identified
by blotting techniques that include a step whereby nucleotides are
immobilized on a matrix (preferably a synthetic membrane such as
nitrocellulose), a hybridization step, and a detection step. Southern
blotting is used to identify a complementary DNA sequence; northern
blotting is used to identify a complementary RNA sequence. Dot blotting
and slot blotting can be used to identify complementary DNA/DNA, DNA/RNA
or RNA/RNA polynucleotide sequences. Such techniques are well known by
those skilled in the art, and have been described in Ausubel et al.
(1994-1998, supra) at pages 2.9.1 through 2.9.20.
According to such methods, Southern blotting involves separating DNA
molecules according to size by gel electrophoresis, transferring the
size-separated DNA to a synthetic membrane, and hybridizing the membrane
bound DNA to a complementary nucleotide sequence labeled radioactively,
enzymatically or fluorochromatically. In dot blotting and slot blotting,
DNA samples are directly applied to a synthetic membrane prior to
hybridization as above.
An alternative blotting step is used when identifying complementary
nucleotide sequences in a cDNA or genomic DNA library, such as through the
process of plaque or colony hybridization. A typical example of this
procedure is described in Sambrook et al., (1989, supra) Chapters 8-12.
Typically, the following general procedure can be used to determine
hybridization conditions. Nucleotide sequences are blotted/transferred to
a synthetic membrane, as described above. A wild type nucleotide sequence
of the invention is labeled as described above, and the ability of this
labeled nucleotide sequence to hybridize with an immobilized nucleotide
A skilled addressee will recognize that a number of factors influence
hybridization. The specific activity of radioactively labeled
polynucleotide sequence should typically be greater than or equal to about
10.sup.8 dpm/mg to provide a detectable signal. A radiolabeled nucleotide
sequence of specific activity 10.sup.8 to 10.sup.9 dpm/mg can detect
approximately 0.5 pg of DNA. It is well known in the art that sufficient
DNA must be immobilized on the membrane to permit detection. It is
desirable to have excess immobilized DNA, usually 10 .mu.g. Adding an
inert polymer such as 10% (w/v) dextran sulfate (MW 500,000) or
polyethylene glycol 6000 during hybridization can also increase the
sensitivity of hybridization (see Ausubel supra at 2.10.10).
To achieve meaningful results from hybridization between a nucleotide
sequence immobilized on a membrane and a labeled nucleotide sequence, a
sufficient amount of the labeled nucleotide sequence must be hybridized to
the immobilized nucleotide sequence following washing. Washing ensures
that the labeled nucleotide sequence is hybridized only to the immobilized
nucleotide sequences with a desired degree of complementarity to the
labeled nucleotide sequence.
"Stringency" as used herein, refers to the temperature and ionic strength
conditions, and presence or absence of certain organic solvents, during
hybridization. The higher the stringency, the higher will be the degree of
complementarity between the immobilized nucleotide sequences and the
labeled polynucleotide sequence.
"Stringent conditions" designates those conditions under which only
nucleotide sequences having a high frequency of complementary bases will
Typical stringent conditions include, for example, (1) 0.75 M dibasic
sodium phosphate/0.5 M monobasic sodium phosphate/1 mM disodium EDTA/1%
sarkosyl at about 42.degree. C. for at least 30 minutes; or (2) 6.0 M
urea/0.4% sodium lauryl sulfate/0.1.times.SSC at about 42.degree. C. for
at least 30 minutes; or (3) 0.1.times.SSC/0.1% SDS at about 68.degree. C.
for at least 20 minutes; or (4) 1.times.SSC/0.1% SDS at about 55.degree.
C. for about 60 minutes; or (5) 1.times.SSC/0.1% SDS at about 62.degree.
C. for about 60 minutes; or (6) 1.times.SSC/0.1% SDS at about 68.degree.
C. for about 60 minutes; or (7) 0.2.times.SSC/0.1% SDS at about 55.degree.
C. for about 60 minutes; or (8) 0.2.times.SSC/0.1% SDS at about 62.degree.
C. for about one hour; or (9) 0.2.times.SSC/0.1% SDS at about 68.degree.
C. for about 60 minutes. For a detailed example, see CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY supra at pages 2.10.1 to 2.10.16 and Sambrook et al. in
MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbour Press, 1989)
at sections 1.101 to 1.104, which are hereby incorporated by reference.
While stringent washes are typically carried out at temperatures from
about 42.degree. C. to 68.degree. C., one skilled in the art will
appreciate that other temperatures may be suitable for stringent
conditions. Maximum hybridization typically occurs at about 20.degree. C.
to 25.degree. C. below the T.sub.m for formation of a DNA-DNA hybrid. It
is well known in the art that the T.sub.m is the melting temperature, or
temperature at which two complementary polynucleotide sequences
dissociate. Methods for estimating T.sub.m are well known in the art (see
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY supra at page 2.10.8). Maximum
hybridization typically occurs at about 10.degree. C. to 15.degree. C.
below the T.sub.m for a DNA-RNA hybrid.
Other stringent conditions are well known in the art. A skilled addressee
will recognize that various factors can be manipulated to optimize the
specificity of the hybridization. Optimization of the stringency of the
final washes can serve to ensure a high degree of hybridization.
Methods for detecting labeled nucleotide sequences hybridized to an
immobilized nucleotide sequence are well known to practitioners in the
art. Such methods include autoradiography, chemiluminescent, fluorescent
and colorimetric detection.
The invention also contemplates antibodies against the aforementioned
polypeptides, fragments, variants and derivatives. Such antibodies may
include any suitable antibodies that bind to or conjugate with a
polypeptide, fragment, variant or derivative of the invention. For
example, the antibodies may comprise polyclonal antibodies. Such
antibodies may be prepared for example by injecting a polypeptide,
fragment, variant or derivative of the invention into a production
species, which may include mice or rabbits, to obtain polyclonal antisera.
Methods of producing polyclonal antibodies are well known to those skilled
in the art. Exemplary protocols which may be used are described for
example in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley &
Sons, Inc, 1991) which is incorporated herein by reference, and Ausubel et
al., (1994-1998, supra), in particular Section III of Chapter 1.1.
In lieu of the polyclonal antisera obtained in the production species,
monoclonal antibodies may be produced using the standard method as for
example, described in an article by Kohler and Milstein (1975, Nature 256,
495-497) which is herein incorporated by reference, or by more recent
modifications thereof as for example, described in Coligan et al., (1991,
supra) by immortalizing spleen or other antibody producing cells derived
from a production species which has been inoculated with one or more of
the polypeptides, fragments, variants or derivatives of the invention.
The invention also includes within its scope antibodies which comprise Fc
or Fab fragments of the polyclonal or monoclonal antibodies referred to
above. Alternatively, the antibodies may comprise single chain Fv
antibodies (scFvs) against the peptides of the invention. Such scFvs may
be prepared, for example, in accordance with the methods described
respectively in U.S. Pat. No. 5,091,513, European Patent No 239,400 or the
article by Winter and Milstein (1991, Nature, 349 293) which are
incorporated herein by reference.
The antibodies of the invention may be used for affinity chromatography in
isolating natural or recombinant N. meningitidis polypeptides. For example
reference may be made to immunoaffinity chromatographic procedures
described in Chapter 9.5 of Coligan et al., (1995-1997, supra).
The antibodies can be used to screen expression libraries for variant
polypeptides of the invention. The antibodies of the invention can also be
used to detect N. meningitidis infection described hereinafter.
Detection of N. meningitidis
The presence or absence of N. meningitidis in a patient may determined by
isolating a biological sample from a patient, mixing an antibody or
antibody fragment described above with the biological sample to form a
mixture, and detecting specifically bound antibody or bound fragment in
the mixture which indicates the presence of N. meningitidis in the sample.
The term "biological sample" as used herein refers to a sample that may be
extracted, untreated, treated, diluted or concentrated from a patient.
Suitably, the biological sample is selected from the group consisting of
whole blood, serum, plasma, saliva, urine, sweat, ascitic fluid,
peritoneal fluid, synovial fluid, amniotic fluid, cerebrospinal fluid,
skin biopsy, and the like.
Any suitable technique for determining formation of the complex may be
used. For example, an antibody or antibody fragment according to the
invention having a label associated therewith may be utilized in
immunoassays. Such immunoassays may include, but are not limited to,
radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs) and
immunochromatographic techniques (ICTs) which are well known those of
skill in the art. For example, reference may be made to "CURRENT PROTOCOLS
IN IMMUNOLOGY" (1994, supra) which discloses a variety of immunoassays
that may be used in accordance with the present invention. Immunoassays
may include competitive assays as understood in the art.
The label associated with the antibody or antibody fragment may include
the following: i. direct attachment of the label to the antibody or
antibody fragment; ii. indirect attachment of the label to the antibody or
antibody fragment; i.e., attachment of the label to another assay reagent
which subsequently binds to the antibody or antibody fragment; and iii.
attachment to a subsequent reaction product of the antibody, or antibody
The label may be selected from a group including a chromogen, a catalyst,
an enzyme, a fluorophore, a chemiluminescent molecule, a lanthanide ion
such as Europium (Eu.sup.34), a radioisotope and a direct visual label.
In the case of a direct visual label, use may be made of a colloidal
metallic or non-metallic particle, a dye particle, an enzyme or a
substrate, an organic polymer, a latex particle, a liposome, or other
vesicle containing a signal producing substance and the like.
A large number of enzymes suitable for use as labels is disclosed in
United States Patent Specifications U.S. Pat. No. 4,366,241, U.S. Pat. No.
4,843,000, and U.S. Pat. No. 4,849,338, all of which are herein
incorporated by reference. Suitable enzyme labels useful in the present
invention include alkaline phosphatase, horseradish peroxidase, luciferase,
.beta.-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and
the like. The enzyme label may be used alone or in combination with a
second enzyme that is in solution.
Suitably, the fluorophore is selected from a group including fluorescein
isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITL) or R-Phycoerythrin
The invention also extends to a method for detecting infection of patients
by N. meningitidis, said method comprising the steps of contacting a
biological sample from a patient with a polypeptide, fragment, variant or
derivative of the invention, and determining the presence or absence of a
complex between said polypeptide, fragment, variant or derivative and N.
meningitidis-specific antibodies in said serum, wherein the presence of
said complex is indicative of said infection.
In a preferred embodiment, detection of the above complex is effected by
detectably modifying said polypeptide, fragment, variant or derivative
with a suitable label as is well known in the art and using such modified
compound in a suitable immunoassay as for example described above.
In another aspect, the invention provides a method of detecting N.
meningitides bacteria in a biological sample suspected of containing said
bacteria, said method comprising the steps of isolating the biological
sample from a patient, detecting a nucleic acid sequence according to the
invention in said sample which indicates the presence of said bacteria.
Detection of the said nucleic acid sequence may be determined using any
suitable technique. For example, a labeled nucleic acid sequence according
to the invention may be used as a probe in a Southern blot of a nucleic
acid extract obtained from a patient as is well known in the art.
Alternatively, a labeled nucleic acid sequence according to the invention
may be utilized as a probe in a Northern blot of a RNA extract from the
patient. Preferably, a nucleic acid extract from the patient is utilized
in concert with oligonucleotide primers corresponding to sense and
antisense sequences of a nucleic acid sequence according to the invention,
or flanking sequences thereof, in a nucleic acid amplification reaction
such as PCR, or the ligase chain reaction (LCR) as for example described
in International Application WO89/09385 which is incorporated by reference
herein. A variety of automated solid-phase detection techniques are also
appropriate. For example, very large scale immobilized primer arrays (VLSIPS.TM.)
are used for the detection of nucleic acids as for example described by
Fodor et al., (1991, Science 251:767-777) and Kazal et al., (1996, Nature
Medicine 2:753-759). The above generic techniques are well known to
persons skilled in the art.
A further feature of the invention is the use of the polypeptide,
fragment, variant or derivative of the invention ("immunogenic agents") as
actives in a pharmaceutical composition for protecting patients against
infection by N. meningitides. Suitably, the pharmaceutical composition
comprises a pharmaceutically acceptable carrier.
By "pharmaceutically-acceptable carrier" is meant a solid or liquid
filler, diluent or encapsulating substance that may be safely used in
systemic administration. Depending upon the particular route of
administration, a variety of pharmaceutically acceptable carriers, well
known in the art may be used. These carriers may be selected from a group
including sugars, starches, cellulose and its derivatives, malt, gelatine,
talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic
acid, phosphate buffered solutions, emulsifiers, isotonic saline, and
Any suitable route of administration may be employed for providing a
patient with the composition of the invention. For example, oral, rectal,
parenteral, sublingual, buccal, intravenous, intra-articular,
intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular,
intraperitoneal, intracerebroventricular, transdermal and the like may be
employed. Intra-muscular and subcutaneous injection is appropriate, for
example, for administration of immunogenic compositions, vaccines and DNA
Dosage forms include tablets, dispersions, suspensions, injections,
solutions, syrups, troches, capsules, suppositories, aerosols, transdermal
patches and the like. These dosage forms may also include injecting or
implanting controlled releasing devices designed specifically for this
purpose or other forms of implants modified to act additionally in this
fashion. Controlled release of the therapeutic agent may be effected by
coating the same, for example, with hydrophobic polymers including acrylic
resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic
acids and certain cellulose derivatives such as hydroxypropylmethyl
cellulose. In addition, the controlled release may be effected by using
other polymer matrices, liposomes and/or microspheres.
Pharmaceutical compositions of the present invention suitable for oral or
parenteral administration may be presented as discrete units such as
capsules, sachets or tablets each containing a pre-determined amount of
one or more therapeutic agents of the invention, as a powder or granules
or as a solution or a suspension in an aqueous liquid, a non-aqueous
liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such
compositions may be prepared by any of the methods of pharmacy but all
methods include the step of bringing into association one or more
immunogenic agents as described above with the carrier which constitutes
one or more necessary ingredients. In general, the compositions are
prepared by uniformly and intimately admixing the immunogenic agents of
the invention with liquid carriers or finely divided solid carriers or
both, and then, if necessary, shaping the product into the desired
The above compositions may be administered in a manner compatible with the
dosage formulation, and in such amount as is immunogenically-effective to
protect patients from N. meningitidis infection. The dose administered to
a patient, in the context of the present invention, should be sufficient
to effect a beneficial response in a patient over time such as a reduction
in the level of N. meningitidis, or to inhibit infection by N.
meningitidis. The quantity of the immunogenic agent(s) to be administered
may depend on the subject to be treated inclusive of the age, sex, weight
and general health condition thereof. In this regard, precise amounts of
the immunogenic agent(s) required to be administered will depend on the
judgement of the practitioner. In determining the effective amount of the
immunogenic agent to be administered in the treatment or prophylaxis
against N. meningitidis, the physician may evaluate circulating plasma
levels, progression of disease, and the production of anti-N. meningitidis
antibodies. In any event, suitable dosages of the immunogenic agents of
the invention may be readily determined by those of skill in the art. Such
dosages may be in the order of nanograms to milligrams of the immunogenic
agents of the invention.
The above compositions may be used as therapeutic or prophylactic
vaccines. Accordingly, the invention extends to the production of vaccines
containing as actives one or more of the immunogenic agents of the
invention. Any suitable procedure is contemplated for producing such
vaccines. Exemplary procedures include, for example, those described in
NEW GENERATION VACCINES (1997, Levine et al., Marcel Dekker, Inc. New
York, Basel Hong Kong), which is incorporated herein by reference.
An immunogenic agent according to the invention can be mixed, conjugated
or fused with other antigens, including B or T cell epitopes of other
antigens. In addition, it can be conjugated to a carrier as described
When an haptenic peptide of the invention is used (i.e., a peptide which
reacts with cognate antibodies, but cannot itself elicit an immune
response), it can be conjugated with an immunogenic carrier. Useful
carriers are well known in the art and include for example: thyroglobulin;
albumins such as human serum albumin; toxins, toxoids or any mutant
crossreactive material (CRM) of the toxin from tetanus, diptheria,
pertussis, Pseudomonas, E. coli, Staphylococcus, and Streprococcus;
polyamino acids such as poly(lysine:glutamic acid); influenza; Rotavirus
VP6, Parvovirus VP1 and VP2; hepatitis B virus core protein; hepatitis B
virus recombinant vaccine and the like. Alternatively, a fragment or
epitope of a carrier protein or other immnogenic protein may be used. For
example, a haptenic peptide of the invention can be coupled to a T cell
epitope of a bacterial toxin, toxoid or CRM. In this regard, reference may
be made to U.S. Pat. No. 5,785,973 which is incorporated herein by
In addition, a polypeptide, fragment, variant or derivative of the
invention may act as a carrier protein in vaccine compositions directed
against Neisseria, or against other bacteria or viruses.
The immunogenic agents of the invention may be administered as multivalent
subunit vaccines in combination with antigens of N. meningitidis, or
antigens of other organisms inclusive of the pathogenic bacteria H.
influenzae, M. catarrhalis, N. gonorrhoeae, E. Coli, S. pneumoniae etc.
Alternatively or additionally, they may be administered in concert with
oligosaccharide or polysaccharide components of N. meningitidis.
The vaccines can also contain a physiologically acceptable diluent or
excipient such as water, phosphate buffered saline and saline.
The vaccines and immunogenic compositions may include an adjuvant as is
well known in the art. Suitable adjuvants include, but are not limited to:
surface active substances such as hexadecylamine, octadecylamine,
octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium
methoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran,
dextransulfate, poly IC carbopol; peptides such as muramyl dipeptide and
derivatives, dimethylglycine, tuftsin; oil emulsions; and mineral gels
such as aluminum phosphate, aluminum hydroxide or alum; lymphokines, QuilA
and immune stimulating complexes (ISCOMS).
The immunogenic agents of the invention may be expressed by attenuated
viral hosts. By "attenuated viral hosts" is meant viral vectors that are
either naturally, or have been rendered, substantially avirulent. A virus
may be rendered substantially avirulent by any suitable physical (e.g.,
heat treatment) or chemical means (e.g., formaldehyde treatment). By
"substantially avirulent" is meant a virus whose infectivity has been
destroyed. Ideally, the infectivity of the virus is destroyed without
affecting the proteins that carry the immunogenicity of the virus. From
the foregoing, it will be appreciated that attenuated viral hosts may
comprise live viruses or inactivated viruses.
Attenuated viral hosts which may be useful in a vaccine according to the
invention may comprise viral vectors inclusive of adenovirus,
cytomegalovirus and preferably pox viruses such as vaccinia (see for
example Paoletti and Panicali, U.S. Pat. No. 4,603,112 which is
incorporated herein by reference) and attenuated Salmonella strains (see
for example Stocker, U.S. Pat. No. 4,550,081 which is herein incorporated
by reference). Live vaccines are particularly advantageous because they
lead to a prolonged stimulus that can confer substantially long-lasting
Multivalent vaccines can be prepared from one or more microorganisms that
express different epitopes of N. meningitidis (e.g., other surface
proteins or epitopes of N. meningitidis). In addition, epitopes of other
pathogenic microorganisms can be incorporated into the vaccine.
In a preferred embodiment, this will involve the construction of a
recombinant vaccinia virus to express a nucleic acid sequence according to
the invention. Upon introduction into a host, the recombinant vaccinia
virus expresses the immunogenic agent, and thereby elicits a host CTL
response. For example, reference may be made to U.S. Pat. No. 4,722,848,
incorporated herein by reference, which describes vaccinia vectors and
methods useful in immunization protocols.
A wide variety of other vectors useful for therapeutic administration or
immunization with the immunogenic agents of the invention will be apparent
to those skilled in the art from the present disclosure.
In a further embodiment, the nucleotide sequence may be used as a vaccine
in the form of a "naked DNA" vaccine as is known in the art. For example,
an expression vector of the invention may be introduced into a mammal,
where it causes production of a polypeptide in vivo, against which the
host mounts an immune response as for example described in Barry, M. et
al., (1995, Nature, 377:632-635) which is hereby incorporated herein by
The present invention also provides kits for the detection of N.
meningitidis in a biological sample. These will contain one or more
particular agents described above depending upon the nature of the test
method employed. In this regard, the kits may include one or more of a
polypeptide, fragment, variant, derivative, antibody, antibody fragment or
nucleic acid according to the invention. The kits may also optionally
include appropriate reagents for detection of labels, positive and
negative controls, washing solutions, dilution buffers and the like. For
example, a nucleic acid-based detection kit may include (i) a nucleic acid
according to the invention (which may be used as a positive control), (ii)
an oligonucleotide primer according to the invention, and optionally a DNA
polymerase, DNA ligase etc depending on the nucleic acid amplification
Preparation of Immunoreactive Fragments
The invention also extends to a method of identifying an immunoreactive
fragment of a polypeptide, variant or derivatives according to the
invention. This method essentially comprises generating a fragment of the
polypeptide, variant or derivative, administering the fragment to a
mammal; and detecting an immune response in the mammal. Such response will
include production of elements which specifically bind N. meningitidis
and/or said polypeptide, variant or derivative, and/or a protective effect
against N. meningitidis infection.
Prior to testing a particular fragment for immunoreactivity in the above
method, a variety of predictive methods may be used to deduce whether a
particular fragment can be used to obtain an antibody that cross-reacts
with the native antigen. These predictive methods may be based on
amino-terminal or carboxy-terminal sequence as for example described in
Chapter 11.14 of Ausubel et al., (1994-1998, supra.). Alternatively, these
predictive methods may be based on predictions of hydrophilicity as for
example described by Kyte and Doolittle (1982, J. Mol. Biol. 157:105-132)
and Hopp and Woods (1983, Mol. Immunol. 20:483-489) which are incorporated
by reference herein, or predictions of secondary structure as for example
described by Choo and Fasman (1978, Ann. Rev. Biochem. 47:251-276), which
is incorporated herein by reference.
Generally, peptide fragments consisting of 10 to 15 residues provide
optimal results. Peptides as small as 6 or as large as 20 residues have
worked successfully. Such peptide fragments may then be chemically coupled
to a carrier molecule such as keyhole limpet hemocyanin (KLH) or bovine
serum albumin (BSA) as for example described in Sections 11.14 and 11.15
of Ausubel et al., (1994-1998, supra).
The peptides may be used to immunize an animal as for example discussed
above. Antibody titers against the native or parent polypeptide from which
the peptide was selected may then be determined by, for example,
radioimmunoassay or ELISA as for instance described in Sections 11.16 and
114 of Ausubel et al., (1994-1998, supra).
Antibodies may then be purified from a suitable biological fluid of the
animal by ammonium sulfate fractionation or by chromatography as is well
known in the art. Exemplary protocols for antibody purification is given
in Sections 10.11 and 11.13 of Ausubel et al., (1994-1998, supra).
Immunoreactivity of the antibody against the native or parent polypeptide
may be determined by any suitable procedure such as, for example, western
The polypeptides according to SEQ ID NOS 2, 5, 7, 9, 11, 13, 15, 17, 19
and 21 are believed to have adhesin properties. They in fact have some
similarity to adhesins of Haemophilus influenzae, which are surface
antigens. Specifically they are approximately 67% homologous to the Hia
protein of H. influenzae (Barenkamp, S. and St. Geme III, J. 1996
Molecular Microbiology 19: 1215-1233), and 74% homologous to the Hsf
protein of H. influenzae (St. Geme III, J. et al, 1996, Journal of
Bacteriology 178: 6281-6287; and U.S. Pat. No. 5,646,259). For these
comparisons, a gap weight of 3, and length weight of 0.01 was used using
the GAP program (Deveraux, 1984, supra). Aligned sequences of these
proteins are illustrated in FIG. 6 (see Original Patent). Thus,
interruption of the function of these polypeptides would be of significant
therapeutic benefit since they would prevent N. meningitidis bacteria from
adhering to and invading cells. Interruption of the function may be
effected in several ways.
For example, moieties such as chemical reagents or polypeptides which
block receptors on the cell surface which interact with a polypeptides
according to SEQ ID NOS 2, 5, 7, 9, 11, 13, 15, 17, 19 and 21 may be
administered. These compete with the infective organism for receptor
sites. Such moieties may comprise for example polypeptides of the
invention, in particular fragments, or functional equivalents of these as
well as mimetics.
The term "mimetics" is used herein to refer to chemicals that are designed
to resemble particular functional regions of the proteins or peptides.
Anti-idiotypic antibodies raised against the above-described antibodies
which block the binding of the bacteria to a cell surface may also be
used. Alternatively, moieties which interact with the receptor binding
sites in the polypeptides according to SEQ ID NO 2, 5, 7, 9, 11, 13, 15,
17, 19 and 21 may effectively prevent infection of a cell by N.
meningitidis. Such moieties may comprise blocking antibodies, peptides or
other chemical reagents.
All such moieties, pharmaceutical compositions in which they are combined
with pharmaceutically acceptable carriers and methods of treating patients
suffering from N. meningitidis infection by administration of such
moieties or compositions form a further aspect of the invention.
The polypeptides of the invention may be used in the screening of
compounds for their use in the above methods. For example, polypeptides of
the invention may be combined with a label and exposed to a cell culture
in the presence of a reagent under test. The ability of reagent to inhibit
the binding of the labeled polypeptide to the cell surface can then be
observed. In such a screen, the labeled polypeptides may be used directly
on an organism such as E. coli. Alternatively, N. meningitidis itself may
be engineered to express a modified and detectable form of the
polypeptide. The use of engineered N. meningitidis strains in this method
is preferred as it is more likely that the tertiary structure of the
protein will resemble more closely that expressed in wild-type bacteria.
Claim 1 of 9 Claims
1. An isolated polypeptide comprising a
member selected from the group consisting of (a) the amino acid sequence
SEQ ID NO:2 or SEQ ID NO:21; and (b) an immunogenic fragment of at least
15 contiguous amino acids of SEQ ID NO:2 or SEQ ID NO:21; wherein the
immunogenic fragment, when administered to a subject in a suitable
composition which can include an adjuvant, or a suitable carrier coupled
to the polypeptide, induces an antibody or T-cell mediated immune response
that recognizes the isolated polypeptide SEQ ID NO:2 or SEQ ID NO:21.
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