U.S. patent application number 10/617835 was filed with the patent office on 2005-06-09 for nucleic acid molecules encoding proteins which impart the adhesion of neisseria cells to human cells.
This patent application is currently assigned to MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN e.V.. Invention is credited to Eickernjager, Sandra, Fischer, Eckhard, Maier, Jurgen, Meyer, Thomas F., Rudel, Thomas, Scheuerpflug, Ina, Schwan, Thomas.
Application Number | 20050124037 10/617835 |
Document ID | / |
Family ID | 7772478 |
Filed Date | 2005-06-09 |
United States Patent
Application |
20050124037 |
Kind Code |
A1 |
Meyer, Thomas F. ; et
al. |
June 9, 2005 |
Nucleic acid molecules encoding proteins which impart the adhesion
of Neisseria cells to human cells
Abstract
Described are nucleic acid molecules encoding proteins mediating
the adhesion of bacteria of the genus Neisseria to human cells.
Also described are the proteins encoded by these nucleic acid
molecules and antibodies directed against them. Furthermore,
pharmaceutical compositions, vaccines and diagnostic compositions
containing the nucleic acid molecules, proteins and/or antibodies
are described.
Inventors: |
Meyer, Thomas F.; (Tubingen,
DE) ; Rudel, Thomas; (La Jolla, CA) ;
Scheuerpflug, Ina; (Berlin, DE) ; Maier, Jurgen;
(Kongen, DE) ; Eickernjager, Sandra; (Berlin,
DE) ; Schwan, Thomas; (Berlin, DE) ; Fischer,
Eckhard; (Tubingen, DE) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
MAX-PLANCK-GESELLSCHAFT ZUR
FORDERUNG DER WISSENSCHAFTEN e.V.
Berlin
DE
|
Family ID: |
7772478 |
Appl. No.: |
10/617835 |
Filed: |
July 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10617835 |
Jul 14, 2003 |
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09043302 |
Jun 8, 1998 |
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6617128 |
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09043302 |
Jun 8, 1998 |
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PCT/EP96/04092 |
Sep 18, 1996 |
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Current U.S.
Class: |
435/69.1 ;
435/252.3; 435/320.1; 514/2.8; 530/359; 536/23.7 |
Current CPC
Class: |
C07K 14/22 20130101;
A61K 2039/51 20130101; A61K 39/00 20130101 |
Class at
Publication: |
435/069.1 ;
435/006; 435/252.3; 435/320.1; 530/359; 536/023.7 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 015/74; C07K 014/22; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 18, 1995 |
DE |
195 34 579.7 |
Claims
1. An isolated nucleic acid molecule encoding a lipoprotein or a
biologically active fragment of said lipoprotein that mediates
adhesion of Neisseria cells to human cells from a bacteria of the
genus Neisseria, selected from the group consisting of (a) a
nucleic acid molecule comprising a nucleotide sequence encoding a
protein comprising SEQ ID NO: 4; (b) a nucleic acid molecule
comprising a nucleotide sequence having 95% sequence identity to a
nucleotide sequence encoding a protein comprising SEQ ID NO:4 due
to the degeneracy of the genetic code; (c) a nucleic acid molecule
comprising a nucleotide sequence that hybridizes under stringent
hybridization conditions of 0.2.times.SSC, 0.1% SDS and 68.degree.
C. to (i) the complement of a nucleotide sequence encoding a
protein comprising SEQ ID NO:4, (ii) the complement of a nucleotide
sequence which is 95% identical to a nucleotide sequence encoding a
protein comprising SEQ ID NO:4.
2. The nucleic acid molecule according to claim 1, wherein the
nucleic acid molecule originates from a pathogenic Neisseria
species.
3. The nucleic acid molecule according to claim 2, wherein the
Neisseria species is Neisseria gonorrhoeae or Neisseria
meningitidis.
4. The nucleic acid molecule according to claim 1, wherein the
lipoprotein or biologically active fragment of said lipoprotein has
the ability to adhere to human cells.
5. A vector comprising the nucleic acid molecule according to claim
1.
6. The vector according to claim 5, wherein the nucleic acid
molecule is operatively linked to at least one regulatory DNA
element allowing the expression of said nucleic acid molecule in a
prokaryotic or an eukaryotic cell.
7. A host cell comprising a vector according to claim 5.
8. A host cell comprising the nucleic acid molecule according to
claim 1.
9. An isolated nucleic acid molecule having a length of at least 12
nucleotides specifically hybridizing under stringent hybridization
conditions of 0.2.times.SSC, 0.1% SDS and 68.degree. C. to a
nucleic acid molecule according to claim 1.
Description
[0001] The present invention relates to nucleic acid molecules from
bacteria of the genus Neisseria encoding proteins mediating the
adhesion of Neisseria cells to human cells. Furthermore, the
present invention relates to the proteins encoded by these nucleic
acid molecules and to antibodies directed against them. The present
invention further relates to pharmaceutical compositions, vaccines
and diagnostic compositions containing said nucleic acid molecules,
proteins and/or antibodies.
[0002] To the genus Neisseria (gram-negative cocci) belong a number
of bacterial species which, being saprophytes, populate the upper
human respiratory tract. Apart from commensal species (e.g.: N.
sicca) and opportunistically pathogenic species (e.g.: N.
lactamica), two Neisseria species are known which clearly possess
human-pathogenic properties. One of the species is N. gonorrhoeae,
the pathogen of the venereal disease gonorrhea, which exclusively
occurs in humans, and N. meningitidis, the pathogen of the
bacterial epidemic meningitis. In both cases the etiology, that is
the causal connection between the development of the clinical
picture and the population by bacteria from said species has
meanwhile been substantiated.
[0003] The purulent meningitis (Meningitidis cerebrospinalis
epidemica) caused by N. meningitidis ("meningococcus"), which
usually is epidemical, is a systemic invasive infection of the
human meninx and spinal meninx. Occasionally, hemorrhagic exanthema
at the trunk or concomitant diseases caused by Herpes simplex can
be observed in addition. The pathogen can appear in the form of
several serotypes, which are distinguishable by means of
agglutination assays with immune sera. The main groups differ
remarkably, and their prevalence differs with regard to when and
where they appear. Meningococcus meningitidis has up to now
occurred in large numbers every 8 to 12 years with the increased
prevalence lasting between 4 to 6 years. While serovar B
meningococci brought on 50% to 55% of the recent diseases to the
civilian population as well as to the military personnel in the
United States, most epidemic diseases in the United States during
the first half of the century were caused by serovar A
meningococci.
[0004] The clinical picture caused by N. gonorrhoeae usually is an
infection localized to the mucous membranes, in most cases of the
urogenital tract (gonorrhea), more rarely of the conjunctiva
(conjunctivis gonorrhoeae, gonoblennorrhoe), which is acquired by
new born children perinatally, by adults usually unilaterally by
smear infection. In very rare cases bacteremia and sepsis occur
after hematogenous dissemination. As a consequence, exanthema with
hemorrhagic pustules, diseases from the rheumatic Formenkreis,
arthritis gonorrhoica and/or endocarditis can occur.
[0005] Usually, the diseases caused by N. gonorrhoeae and N.
meningitidis are treated with antibiotics. More and more, however,
the bacteria are becoming resistant to single or groups of the
antibiotics used so that the therapy method that has nearly
exclusively been used up to now will most likely not be successful
in the long run. Therefore, it is desirable and urgent that
alternative therapy methods, preferably preventive ones, be
developed.
[0006] Neisseria gonorrhoeae and N. meningitidis exclusively occur
in humans. They have adapted to the host organism and show a number
of properties that are able to make the defense mechanisms of the
host ineffective. Therefore, up to now there is no vaccine
available that prevents gonorrhea. This is to a limited extent also
true for meningococcus meningitidis. Even though the disease has
recently been caused mainly by bacteria of the same serovar, group
B, no effective vaccine against meningococci of group B has existed
up to now. Vaccines against other serovars only offer partial
protection and are not unproblematic from an immunological point of
view. The reason for the failure of the immune defense is, inter
alia, the antigen variation of the pathogens, which in the case of
the pathogenic Neisseria is particularly developed. However, a
limitation of the free development of the antigen variation seems
to be necessary where the functional region has to be sterically
maintained in order to guarantee the interaction with conserved and
constant structures of the host receptors. This requirement
especially applies to the adhesins that serve for adhering to the
host cell. Only if the functional region that is involved in the
physical interaction is kept constant, the interaction with the
receptor of the host cell is possible. This region should be
excluded from antigen variation to a large extent and is therefore
a suitable starting-point for the development of a new therapy
method.
[0007] The initial phase of infections usually is the stable
adhesion of the pathogens to the host tissue. By interactions
between structures of the cell surface of the pathogens and the
cell surface of the host cell a mechanically stable linkage is
formed that allows the bacteria to stay on the tissue of the host
(colonization) and to subsequently propagate locally. The adhesion
to the host cell can be divided into two phases with different
structures being involved in the interaction.
[0008] In the first phase of adhesion a contact between host cell
and pathogen is mediated. Often cell appendage organelles, the
so-called pili, are involved in mediating the contact. These cell
organelles, which are also called fimbriae or fibrils, are few to
several fine filamentous rigid or flexible appendages of the
bacterial cell, which can be several times as long as the cell
diameter. Therefore, there is no contact between the cell walls of
pathogen and host cell in the pilus mediated adhesion. The majority
of the known pili are heteropolymeric structures consisting of
several components. The main subunit, which usually is present in
many copies, fulfills the structural function, that is the
framework function, whereas the actual adhesion function is
fulfilled by side components, which usually are present in few
copies.
[0009] A further form of adherence is the adhesion of pathogens to
the host cells without the contribution of pili (pilus independent
adherence, pia). In this case, the pathogen and the host cell are
approaching each other, and finally the cell walls directly touch.
This adhesion and stabilization of the contact between the cells
takes place with the contribution of adhesines that are located in
the bacterial cell wall. As a result of the direct contact between
the cells, a signal is finally transmitted that initiates the
pathogen induced phagocytosis and starts the invasion process into
the target cell. The pia form of adherence can autonomically effect
the adhesion of pathogens, for example in the case of pathogens
lacking pili. It can, however, also act as the second phase of
adhesion, that is as the consecutive reaction after pilus mediated
adhesion, and stabilize the contact between the cells. The
adhesines that are involved in the pilus independent adhesion can
but do not necessarily have to show different binding specifities
from those that are involved in pilus dependent adhesion.
[0010] In the context of the invention the bacterial structures
that are involved in the adhesion will in the following be called
adhesines, those of the host cells will be called receptors. If
there is no contact between adhesin and receptor, "defense
mechanisms" of the host, such as fibrillation of the epithelia,
mucus secretion, mass flow of body fluids and the like, eliminate
the pathogens. The development of an infection is, therefore,
prevented from the very beginning. Thus, a disturbance of the
adhesion of the pathogens by means of inhibiting the interaction
between adhesin and receptor of the target cell represents a very
effective approach for preventing and treating infections. Such
therapeutically effective approaches comprise the production of
antibodies specifically blocking the adhesin function, either by
active immunization (vaccination) or by administration of
antibodies already existing (passive immunization). The adhesin
receptor binding can, in the same way, be inhibited by means of
passive administration of both receptor analogous and adhesin
analogous substances. These substances competitively bind to the
corresponding partner structures, thereby blocking their
involvement in productive interactions. In the context of the
invention such substances are called inhibitors.
[0011] The approaches using pilin, the main component of the pilus
that fulfills the structural function, in order to develop a
broadly effective vaccine effectively blocking the adhesion of
pathogenic Neisseria have failed so far. The reason probably is
that (i) pilin itself has no adhesin function and (ii) pilin
possesses an especially distinct intra- and interstem specific
antigenic variation. Since both limitations, as described above, do
not apply to adhesins, the use of an adhesin as a vaccine is more
promising.
[0012] The technical problem of the present invention therefore is
to provide proteins and DNA molecules encoding them that serve as
adhesion structures for Neisseria species or contribute to the
development of such structures.
[0013] This problem is solved by providing the embodiments
described in the claims.
[0014] Therefore, the present invention relates to nucleic acid
molecules containing the nucleotide sequence described in Seq ID
No. 1 or parts thereof with these nucleic acid molecules comprising
one or more open reading frames encoding proteins or biologically
active fragments thereof from bacteria of the genus Neisseria that
mediate the adhesion of Neisseria cells to human cells. The term
"reading frame" in this context is used synonymously with the term
"coding region".
[0015] The subject matter of the invention also relates to nucleic
acid molecules that basically show the nucleotide sequence
described in Seq ID No. 1 but whereby the nucleotide sequences of
the open reading frames deviate from those described in Seq ID No.
1 due to the degeneration of the genetic code. Preferably, the open
reading frames of those nucleic acid molecules have nucleotide
sequences encoding proteins with one of the amino acid sequences
described in Seq ID No. 1. The subject matter of the invention
further relates to nucleic acid molecules hybridizing to the
nucleic acid molecules described above and comprising coding
regions encoding proteins that mediate the adhesion of Neisseria
cells to human cells.
[0016] In the context of the present invention the term
"hybridization" is used as described in Sambrook et al. (Molecular
Cloning, A Laboratory Manual; Cold Spring Harbor Laboratory Press
(1989), 1.101 to 1.104). Preferably, this term has the meaning of
hybridization under stringent conditions. In particular, it has the
meaning of a hybridization that still shows a positive
hybridization signal after being washed for 1 h with 1.times.SSC
and 0.1% SDS, preferably with 0.2.times.SSC and 0.1% SDS, at
55.degree. C., preferably at 62.degree. C. and most preferably at
68.degree. C.
[0017] In a preferred embodiment the nucleic acid molecule of the
invention originates from a pathogenic Neisseria species, in
particular from Neisseria gonorrhoea or Neisseria meningitidis.
[0018] The term "nucleic acid molecule" as used here according to
the invention relates to the polymeric form of nucleotides of any
length, either as ribonucleotides or as desoxyribonucleotides. The
term only relates to the primary structure of the molecule. In this
sense, it comprises DNA and RNA molecules, in single- or
double-stranded form. The DNA can either be cDNA or genomic DNA.
The term further comprises the non-modified form as well as
scientifically known modifications, e.g., methylation, capping,
base substitution with natural or synthetic analogues,
internucleotide modifications with uncharged compounds (e.g.,
methyl phosphate, phosphoamidate, carbamate, phosphotriester and
the like) or with charged compounds (e.g., phosphorothioate,
phosphorodithioate and the like) or with binding components such as
proteins and peptides (e.g., nucleases, toxins, antibodies,
poly-L-lysine, and the like). The term also comprises forms with
intercalating substances (e.g., acridin, psoralen, and the like),
chelators (e.g., with metals, radioactive metals or oxidizing
metals and the like), with alkylating agents and finally with
modified bonds (e.g., alpha anomeric nucleic acids, and the
like).
[0019] The invention also relates to vectors containing a nucleic
acid molecule of the invention. The vector can be any prokaryotic
or eukaryotic vector. Examples of prokaryotic vectors are
chromosomal vectors, such as bacteriophages (e.g., bacteriophage
lambda, P1), and extrachromosomal vectors, such as plasmids with
circular plasmids being particularly preferred. Suitable
prokaryotic vectors are, for example, described in Sambrook et al.
(see above), chapters 1 to 4. The vector according to the invention
can also be a eukaryotic vector, for example a yeast vector or a
vector suitable for higher cells (e.g., a plasmid vector, a viral
vector, a plant vector, and the like). Examples of such vectors are
also described in Sambrook et al. (see above, chapter 16). A vector
containing a nucleic acid molecule of the invention is, for
example, plasmid pES25 (contained in the E. coli strain H 2560 (DSM
10257)). The E. coli strain H 2560 was deposited on Sep. 18, 1995
with Deutsche Sammlung von Mikroorganismen (DSM) [German collection
of microorganisms] in Brunswick, Federal Republic of Germany, as
international recognized depositary authority in accordance with
the stipulations of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purpose of
Patent Procedure under accession number DSM 10257.
[0020] The invention furthermore relates to host cells containing a
vector as described above or being genetically manipulated with a
nucleic acid molecule as described above. The term "host cell" in
the context of this invention comprises both prokaryotic and
eukaryotic host cells. Prokaryotic cells are preferred,
particularly gram-negative prokaryotic cells, in particular E. coli
cells. Suitable eukaryotic host cells are, for example, fungal
cells (e.g., yeast cells), animal or plant cells.
[0021] The nucleotide sequence described in Seq ID No. 1 comprises
three open reading frames. They represent an operon forming a
functional unity. The three open reading frames called orfl, orfA
and orfB encode three proteins that in the context of this
invention are called OrfI, OrfA and OrfB. These sequences are
responsible for the expression of a protein in Neisseria cells, in
particular of the protein OrfA, which is involved in the adhesion
of Neisseria cells to human cells. The proteins OrfI and OrfB
obviously possess a regulatory function or a function as factors
that are able to influence the functionality of OrfA.
[0022] This nucleic acid molecule therefore represents a region of
the Neisseria genome that encodes proteins having the adhesin
function of Neisseria cells.
[0023] The present invention further relates to nucleic acid
molecules encoding a lipoprotein or biologically active fragments
thereof from bacteria of the genus Neisseria having the amino acid
sequence as described in Seq ID No. 2. In a preferred embodiment
the invention relates to nucleic acid molecules encoding a protein
having the amino acid sequence from the amino acid residue 19 to
the amino acid residue 320 of the amino acid sequence as described
in Seq ID No. 2. Such nucleic acid molecules preferably have the
nucleotide sequence described in Seq ID No. 2, in particular the
nucleotide sequence from nucleotide 189 to nucleotide 1095 of the
sequence described in Seq ID No. 2.
[0024] The subject matter of the invention also relates to nucleic
acid molecules encoding a lipoprotein from bacteria of the genus
Neisseria whereby their nucleotide sequence deviates from the
nucleic acid molecules described above due to the degeneration of
the genetic code.
[0025] Furthermore, the present invention relates to nucleic acid
molecules encoding a lipoprotein from bacteria of the genus
Neisseria and hybridize to one of the nucleic acid molecules
described above (for the definition of the term "hybridization" see
above).
[0026] The subject matter of the invention also relates to
fragments, derivatives and allelic variants of the nucleic acid
molecules described above that encode the lipoprotein described
above. Fragments are understood to be parts of the nucleic acid
molecules that are long enough to encode the protein described. The
term derivative in this context means that the nucleotide sequences
of these molecules differ at one or more positions from the
sequences of the nucleic acid molecules described above and that
they show a high level of homology to these nucleotide sequences.
Homology means a sequence identity of at least 40%, in particular
an identity of at least 60%, preferably of more than 80% and
particularly preferred of more than 90%. The deviations to the
nucleic acid molecules described above can be caused by deletion,
substitution, insertion or recombination.
[0027] Homology further means that there is a functional and/or
structural equivalence between the corresponding nucleic acid
molecules or the proteins encoded by them. The nucleic acid
molecules that are homologous to those described above and that
represent derivatives of these nucleic acid molecules usually are
variants of these molecules displaying modifications that have the
same biological function. They can be naturally occurring variants,
for example sequences from other organisms, or mutations, which
either occur naturally or that have been introduced by means of
specific mutagenesis. Furthermore, the variants can be
synthetically produced sequences.
[0028] The allelic variants can be both naturally occurring
variants or variants that were synthetically produced or that were
produced by recombinant DNA techniques. The proteins encoded by the
various variants of the nucleic acid molecules according to the
invention show certain common characteristics, for example enzyme
activity, molecular weight, immunological reactivity, conformation
etc., as well as physical properties such as the electorphoretic
mobility, chromatographic behavior, sedimentation coefficients,
solubility, spectroscopic properties, stability, pH optimum,
temperature optimum etc.
[0029] Preferably, the proteins encoded by the nucleic acid
molecules according to the invention show a homology of 80%,
particularly preferred of more than 90% to the nucleotide sequence
described in Seq ID No. 2.
[0030] The nucleic acid molecules described above encode a
lipoprotein from bacteria of the genus Neisseria. This protein is
called OrfA in the context of the present invention. This protein
is, according to experimental data, located on the cell surface of
Neisseria cells, in particular on the outer membrane. The protein
preferably has a molecular weight of about 36 kd if it is analyzed
in the T7 expression system.
[0031] Furthermore, this protein possesses a biological activity
that mediates the adhesion of Neisseria cells to human cells. This
is in particularly made because this protein forms a complex with
the protein from Neisseria known as PilC. The adhesion preferably
takes place on human epithelial cells.
[0032] Furthermore, the invention relates to vectors containing
nucleic acid molecules described above. Examples of such vectors
have already been described above.
[0033] In a preferred embodiment the DNA molecules according to the
invention are linked in such vectors with regulatory DNA elements
that make the expression of the protein in prokaryotic or
eukaryotic cells possible. Examples thereof are in the context of
this invention promoters, operators, enhancers and the like.
[0034] Furthermore, the invention relates to host cells that
contain vectors according to the invention described above or that
have been genetically manipulated with the nucleic acid molecules
described above. Genetically manipulated means that such a molecule
has been introduced into the host cell or in a precursor cell by
means of (gene) technological methods. Again, the above-described
host cells are suitable.
[0035] The invention also relates to methods for the production of
the described lipoprotein or a biologically active fragment thereof
whereby the host cells described above are cultivated under
conditions that allow the expression of the protein and the protein
is isolated from the cells and/or the culture supernatant.
[0036] The invention also relates to proteins encoded by one of the
nucleic acid molecules described above, as well as to biologically
active fragments thereof as well as to proteins available by the
method described above. In particular, the invention relates to
proteins having amino acid sequences that immunologically
cross-react with the described proteins. The term "protein"
comprises in the context of the present invention also naturally
occurring variants or modifications or fragments or synthetically
produced modifications, variants or fragments with the
corresponding biological activity. Derived or recombinant proteins
do not necessarily have to be biologically translated from the
nucleotide sequence. They can be produced in any way, including
chemical synthesis, in vitro synthesis by means of an expression
system or by isolation from organisms. Proteins according to the
invention can contain one or more amino acid analogues or amino
acids not naturally occurring. Also, modifications (e.g.,
glycosylation, and the like) or labeling (e.g., biotinylation)
according to the scientific knowledge can be contained.
[0037] The fragments preferably have a length of at least 3 to 5
amino acids, particularly preferred of 8 to 10 amino acids and in
particular preferred of 11 to 15 amino acids. This is also true for
the proteins according to the invention described below.
[0038] The lipoprotein OrfA according to the invention can be
purified, for example, by a method that is based on the interaction
of this protein with the PilC protein from Neisseria gonorrhoeae.
It is preferably purified from homogenates of cells expressing this
protein by means of chromatography matrices containing immobilized
PilC protein. The protein can then be selectively eluted using its
affinity to PilC and produced in essentially pure form.
[0039] The proteins according to the invention or fragments thereof
can be used as immunogens for the production of antibodies.
Therefore, the present invention also relates to antibodies that
are directed against a protein according to the invention or a
fragment thereof. The antibodies can be both polyclonal and
monoclonal. Methods for the production of such antibodies are known
to the skilled person.
[0040] In a preferred embodiment such antibodies are directed
against epitopes of the protein according to the invention or
fragments thereof that are important for the adherence and for the
interaction with PilC. The antibodies according to the invention
can be, for example, produced by introducing the nucleic acid
sequences according to the invention described above into hosts by
in vivo transfection. Thereby, the protein or a fragment thereof is
expressed in the host and the antibodies directed against them are
induced (nucleic acid vaccination). This is also the case with the
antibodies described below.
[0041] The present invention further relates to nucleic acid
molecules having a length of at least 12 nucleotides and
specifically hybridizing to the nucleic acid molecule described
above. Preferably, such nucleic acid molecules have a length of at
least 15 nucleotides, particularly preferably of 20 nucleotides.
Such molecules are, for example, suitable as primers for in vitro
amplification, for example by polymerase chain reaction (PCR), or
suitable for diagnostic purposes, that is for specifically
identifying the nucleic acid molecules of the invention in
samples.
[0042] The invention further relates to pharmaceutical compositions
containing a nucleic acid molecule according to the invention
described above, a protein, a biologically active fragment thereof
and/or an antibody according to the invention described above. In
the context of the present invention such pharmaceutical
compositions can contain the usual pharmaceutical adjuvants,
diluents, additives and/or carriers. The invention also relates to
vaccines containing the nucleic acid molecules described above,
proteins, biologically active fragments thereof and/or
antibodies.
[0043] In a further aspect the present invention relates to
diagnostic compositions containing the nucleic acid molecules
according to the invention described above, proteins, biologically
active fragments thereof and/or antibodies.
[0044] A further aspect of the present invention relates to
receptors and substances having receptor function, interacting as
ligands with the adhesin according to the invention, the OrfA-PilC
complex. Such substances can be identified as competitive
inhibitors of the adherence function due to their interaction with
the OrfA-PilC complex. They can be surface components of human
cells, particularly preferred surface components of human
epithelial cells or chemical substances of any origin.
[0045] Finally, the present invention relates to inhibitors that
influence the interaction between the OrfA-PilC adhesin complex and
its receptors. Enclosed are all substances according to the
invention that influence the interaction between the OrfA-PilC
adhesin and its cellular receptor and therefore disturb the
adherence. In a particularly preferred embodiment substances that
irreversibly bind to the adhesin complex such as receptor analogues
are encompassed.
[0046] Finally, the present invention relates to pharmaceutical
compositions containing as an agent
[0047] (a) a receptor according to the invention;
[0048] (b) a receptor analogue according to the invention;
and/or
[0049] (c) an inhibitor according to the invention,
[0050] optionally together with the usual pharmaceutical adjuvants,
diluents, additives and carriers.
[0051] The pharmaceutical compositions described in the context of
the present invention can be used for identifying and
characterizing a bacterial sample not yet known as pathogenic
Neisseria spc. and for diagnosing a Neisseria infection.
[0052] On the polynucleotide level, preferably hybridization probes
are used containing the nucleotide sequences of the invention that
are specific for one of the orf-gene regions or nucleotide
sequences of the invention from one of the orf gene regions are
used as primers for the PCR amplification of the genomic DNA region
to be identified that is specific for pathogenic Neisseria.
[0053] On the polypeptide level diagnosis is preferably performed
with the help of antibodies of the invention or, in the case of
antibody screening tests, with the help of immunogenic proteins of
the invention or fragments thereof.
[0054] Receptors, receptor analogous substances and inhibitors of
the interaction between the OrfA of the invention and the
corresponding receptors of the host cells can be used as
therapeutics for infections at an early stage or if an infection is
suspected. By strongly inhibiting the adherence, the adhesion of
the pathogens to the epithelial host cells can be prevented so that
by the usual defense mechanisms, such as ciliary movement of the
epithelial cells, mucus secretion, mass flow of body fluids and the
like, the pathogens can be eliminated.
[0055] Finally, the pharmaceutical compositions of the invention
can be used for preventing or fighting Neisseria infections.
Preferably, for preventive applications the proteins of the
invention or fragments thereof are used for the production of a
vaccine for active immunization, or antibodies of the invention are
used for the production of a passive vaccine applicable as a
therapeutic. The applications described above also apply to the
pharmaceutical compositions and diagnostic compositions described
below.
[0056] The subject matter of the invention further relates to
nucleic acid molecules encoding a protein or a biologically active
fragment thereof from bacteria of the genus Neisseria having the
amino acid sequence described in Seq ID No. 3. Such nucleic acid
molecules preferably have the nucleotide sequence described in Seq
ID No. 3, in particular the one of the described coding region. The
invention also relates to nucleic acid molecules the sequence of
which deviates from the sequences of the molecules mentioned above
due to the degeneration of the genetic code. Also nucleic acid
molecules are the subject matter of the invention that hybridize to
the nucleic acid molecules mentioned above (for the definition of
the term "hybridization" see above). For the possible variants of
the nucleic acid molecules the same is true what has already been
described in connection with the nucleic acid molecules encoding
OrfA.
[0057] The invention also relates to, vectors containing the
described nucleic acid molecules, in particular those in which they
are linked to regulatory DNA elements for the expression in
prokaryotic or eukaryotic cells, as well as to host cells that
contain such vectors or that are genetically manipulated with the
described nucleic acid molecules.
[0058] The invention also relates to proteins encoded by the
nucleic acid molecules described above and to proteins containing
amino acid sequences that immunologically cross-react with the
amino acid sequence depicted in Seq ID No. 3 or fragments thereof.
In the context of this invention they are called OrfI proteins. The
protein from Neisseria gonorrhoeae having the amino acid sequence
depicted in Seq ID No. 3 shows in the T7 expression system an
apparent molecular weight of about 18 kd. A homology to presently
known proteins could not be shown. Experimental data indicate that
the protein is located intracellularly and possibly has a
regulatory function.
[0059] This protein can be produced by a method in which a host
cell described above is cultivated under conditions allowing the
expression of the protein and in which the protein is obtained from
the cells and/or the culture supernatant. Therefore, the invention
also relates to proteins obtainable by such a method.
[0060] The invention also relates to antibodies against a protein
described above or a fragment thereof as well as to nucleic acid
molecules having a length of at least 12 nucleotides and
specifically hybridizing to a nucleic acid molecule described
above. Preferably, the molecules have a length of more than 15
nucleotides and particularly preferably of more than 20
nucleotides.
[0061] The invention further relates to pharmaceutical compositions
containing a nucleic acid molecule, protein, biologically active
fragment thereof and/or an antibody described above and,
optionally, a pharmaceutically acceptable carrier.
[0062] The invention further relates to diagnostic compositions
containing the nucleic acid molecules, proteins, biologically
active fragments thereof and/or antibodies described above.
[0063] The subject matter of the invention further relates to
nucleic acid molecules encoding a protein or a biologically active
fragment thereof from bacteria of the genus Neisseria that has the
amino acid sequence depicted in Seq ID No. 4. Such nucleic acid
molecules preferably have the nucleotide sequence depicted in Seq
ID No. 4, in particular the one of the indicated coding region. The
invention also relates to nucleic acid molecules the sequences of
which deviate from the nucleotide sequence of the above-mentioned
molecules due to the degeneration of the genetic code. Furthermore,
the subject matter of the invention also relates to nucleic acid
molecules hybridizing to the above-mentioned nucleic acid molecules
(for the definition of the term "hybridization" see above). The
same applies to possible variants of the nucleic acid molecules as
has already been described in connection with the nucleic acid
molecules encoding OrfA.
[0064] In a preferred embodiment the above-described nucleic acid
molecules encode a protein that is able to form a complex with the
protein PilC and therefore shows an ability of adherence to human
cells.
[0065] The invention also relates to vectors containing the
described nucleic acid molecules, in particular those in which they
are linked to regulatory DNA elements for the expression in
prokaryotic or eukaryotic cells, as well as to host cells that
contain such vectors or that have been genetically manipulated with
the above-described nucleic acid molecules.
[0066] The invention also relates to proteins encoded by the
above-described nucleic acid molecules and to proteins containing
the amino acid sequences that immunologically cross-react with the
amino acid sequence depicted in Seq ID No. 4 or parts thereof.
These are called OrfB in the context of the present invention. The
protein from Neisseria gonorrhoeae having the amino acid sequence
depicted in Seq ID No. 4 shows in the T7 expression system an
apparent molecular weight of about 57 kd. A homology to presently
known proteins could not be shown. Experimental data indicate that
the protein is, like OrfA, located at the cell surface and is
accessible from the outside. Furthermore, it obviously also
possesses the ability to form a complex with the protein PilC and
to induce either alone or in combination with OrfA the adhesion to
human cells.
[0067] This protein can be produced by a method in which an
above-described host cell is cultivated under conditions allowing
the expression of the protein and in which the protein is obtained
from the cells and/or the culture supernatant. Therefore, the
invention also relates to proteins obtainable by such a method.
[0068] The invention also relates to antibodies against an
above-described protein or fragment thereof, as well as to nucleic
acid molecules having a length of at least 12 nucleotides and
specifically hybridizing to an above-described nucleic acid
molecule. Preferably, such molecules have a length of more than 15
nucleotides and particularly preferred of more than 20
nucleotides.
[0069] Furthermore, the invention relates to pharmaceutical
compositions containing an above-described nucleic acid molecule,
protein, biologically active fragment thereof and/or antibody and,
optionally, pharmaceutically acceptable carriers.
[0070] The subject matter of the invention further relates to
diagnostic compositions containing the above-described nucleic acid
molecules, proteins, fragments thereof and/or antibodies.
[0071] Illustration of the figures and the sequence protocols:
[0072] FIG. 1 schematically shows the construction of the plasmid
pES25.
[0073] FIG. 2 shows the nucleotide sequence (SEQ ID No. 1) of the
orf gene region, starting from position 1 at the modified BglI
cleavage site and ending with position 3260, the last nucleotide of
the HindIII cleavage site. Restriction cleavage sites, ribosome
binding sites (Shine-Dalgarno sequences) and promoter sequences
(-35 and -10 regions) are labeled.
[0074] SEQ ID No. 1 further shows the amino acid sequences of the
proteins OrfI, OrfA and OrfB encoded by the orf gene region. The
amino acids of the lipoprotein signal sequence of OrfA are written
in italic, the cleavage sites of the lipoprotein signal peptidase
II is labeled with the tip of an arrow. The amino acid cysteine
that represents the amino terminal of the processed OrfA
lipoprotein and is modified to glyceryl cysteine with fatty acid is
marked with a circle. The first seven amino acids of OrfB that are
similar to a typeIV-pilin-signal sequence are written in bold. The
labeling between amino acids 7 and 8 and between 11 and 12
characterize potential cleavage sites analogous to the processing
of the typeIV-pilin.
[0075] Seq ID No. 2 shows the nucleotide sequence of the gene
region encoding OrfA as well as flanking sequences. The amino acid
sequence of OrfA is depicted, too.
[0076] Seq ID No. 3 shows the nucleotide sequence of the gene
region encoding OrfI as well as flanking sequences. The amino acid
sequence of OrfI is depicted, too.
[0077] Seq ID No. 4 shows the nucleotide sequence of the gene
region encoding OrfB as well as flanking sequences. The amino acid
sequence of OrfB is depicted, too.
[0078] The examples illustrate the invention.
EXAMPLES
Example 1
Method for the Isolation of the Lipoprotein Adhesin OrfA
[0079] During the chromatographic purification of the PilC protein
a decisive observation with regard to the identification of the new
adhesin of Neisseria gonorrhoeae of the invention was made. A
recombinant PilC protein was used that was amplified by an
oligo-histidine region with six histidine residues (His.sub.6-tag)
in order to make the chromatographic purification easier (Rudel et
al., Nature 373, 357-359, 1995). The amplification of the protein
by the histidine hexapeptide makes the selective binding to a
nickel-nitrilotriacetate-agarose matrix (Ni-NTA matrix) possible.
After the cell wall fraction produced from cultures of a pilus-free
PilC overexpression strain N560 (Rudel et al., see above) from
Neisseria gonorrhoeae had been extracted, the extract was loaded on
an Ni-NTA chromatography matrix. Usually, for the method that was
developed for the purification of recombinant PilC unspecifically
bound material was removed by extensive washing with a buffer
containing imidazole. However, in the first elution fraction a
protein of 36 kd (OrfA) could be identified together with PilC in
an approximately equimolar ratio.
[0080] For the preparation of the PilC-OrfA protein fraction the
strain N560 from Neisseria gonorrhoeae was plated on 30 GC-agar
plates and incubated in 5% CO.sub.2 at 37.degree. C. for 20 hours.
The GC-agar medium (GC agar base, Becton Dickinson, Heidelberg)
contained the usual additional factors necessary for the growth of
Neisseria gonorrhoeae (0.1 mg vitamin B12, 10 mg adenine, 0.3 mg
guanine, 100 mg glutamine, 1 mg cocarboxylase, 0.3 mg thiamine, 259
mg L-cysteine, 11 mg L-cystine, 1.5 mg arginine, 5 mg uracil, 0.2
mg Fe(NO.sub.3).sub.3, 2.5 mg nicotineamide-adenine dinucleotide,
0.13 mg p-aminobenzoic acid and 1 g dextrose per 1 liter of medium)
that were added as a sterile filtrate to the GC basis medium after
heat sterilization. Furthermore, the so supplemented GC agar medium
contained 5 .mu.g/ml tetracycline and 100 .mu.M IPTG. The bacterial
lawns were removed with cotton pads, transferred to 30 ml of
washing buffer (Tris-HCl pH 8.0 with 0.15 M NaCl) and centrifuged
at 4,000 rpm, 4.degree. C. for 15 minutes (Du Pont Sorvall
Centrifuge RC-5B, Rotor SS-34). The cell sediment was again
resuspended in 30 ml of washing buffer, and the bacteria were
broken up by ultrasonic homogenization after lysozyme and 5 mM EDTA
Na.sub.2 had been added. Intact bacteria were separated by
centrifugation at 5,000 rpm at 4.degree. C. for 15 minutes. The
cell coats of the lysed bacteria were sedimented by centrifugation
of the supernatant at 20,000 rpm at 4.degree. C. for 60 minutes and
taken up in 10 ml of washing buffer additionally containing 10%
glycerine, 10 mM MgCl.sub.2 and 2% Triton X-100. After an
incubation of 45 minutes at 37.degree. C. they were centrifuged
again (20,000 rpm, 4.degree. C. for 60 minutes) and the membrane
sediment suspended in 10 ml of washing buffer with 10% glycerine,
10 mM MgCl.sub.2 and 2% LDAO (N,N-Dimethyldodecylamin-N-oxide) and
incubated at 37.degree. C. for 60 minutes. After they were
centrifuged again (20,000 rpm, 4.degree. C. for 60 minutes), the
supernatant containing the biologically active PilC-OrfA complex
was subjected to a nickel-chelate-affinity chromatography for
further purification. For this purpose a Ni-NTA-gel matrix (300 ml
bed volume) was washed with 5 bed volumes of aqua bidest. and
loaded with 10 ml of the supernatant. Unspecifically bound proteins
were removed by elution with 5 column volumes of 50 mM imidazole in
PBS buffer pH 8.0. After the column had been washed again with 5 to
10 bed volumes 20 mM sodium phosphate pH 7.5 with 0.15 M NaCl (PBS
buffer) the biologically active PilC-OrfA complex was eluted with a
citrate/phosphate buffer (10 mM citric acid, 1 M sodium phosphate,
pH 3.5, 10% glycerin, 0.15 M NaCl) in the first elution fraction
and instantly neutralized with a 1 M Na.sub.2HPO.sub.4 solution.
The eluate containing PilC and OrfA was frozen in liquid nitrogen
and strored at -70.degree. C.
Example 2
Isolation of the Polynucleotide Sequence Carrying the Orf-Gene
Region
[0081] To further characterize the 36 kd OrfA protein, mice were
immunized with the PilC-36 kd protein fraction. The 36 kd protein
proved to be very immunogenic. With the antibodies obtained this
way a pBA plasmid gene library of the Neisseria gonorrhoeae MS11
genome in E. coli GC1 was screened for the presence of antigens.
Several clones showing a positive reaction were isolated and clone
H1967 was chosen for further characterization.
[0082] The library plasmid pES25 (FIG. 1) of clone H1967 contained
a genomic fragment of approximately 11 kb, cloned in vector pBA.
Restriction fragments of the total region were subcloned in pUC and
pBluescript KS (+) vectors, respectively. On the basis of the
expression of the derived plasmids in minicells and immunoblotting
analyses subclones were chosen producing the 36 kd protein. The
subclones were used for sequencing. The sequences were determined
by directly sequencing restriction fragments, by sequencing
continuously shortened ExoIII nuclease fragments of the BglI-PstI
fragment (positions 1 to 2560 of Seq ID No. 1), as well as by
sequencing PCR amplified fragments.
[0083] The region depicted in SEQ ID No. 1 starting from the BglI
cleavage site (position 1) to the HindIII cleavage site (position
3260) had three open reading frames with a high coding probability
with each reading frame beginning with the start codon ATG, having
a ribosome binding site that precedes the start codon in a suitable
distance (S.D. sequence) and ending with a stop codon.
[0084] The three reading frames have the same orientation. The
first open reading frame starts at position 136 of the sequence
depicted in SEQ ID No. 1 and ends at position 450 with the stop
codon TAA. The encoded protein was called OrfI and had an apparent
molecular weight of 18 kd in the T7 expression system.
[0085] No significant homologues could be identified by sequence
comparison in the EMBL gene library (Release 43.0 from 6/95) and in
the SwissProt data bank (Release 31.0 from 3/95), neither on a
nucleotide sequence level nor on an amino acid sequence level.
[0086] The second open reading frame starts at position 583 and
ends at position 1545 with the stop codon TGA. It encodes the OrfA
protein having an apparent molecular weight of 36 kd in the T7
expression system. Also to this sequence no significant homologues
could be detected via data base search. The sequence analysis by
means of the protein analysis program "Motifs" (GCG Genetics
Computer Group, Inc., Madison, Wis., USA) showed, however, a
complete homology of the N-terminus of OrfA to lipoprotein specific
signal sequences (position 583 to 636). The characterization of
OrfA as a lipoprotein could be substantiated by experiments (vide
infra).
[0087] The third open reading frame starts at position 1585 and
ends at position 3114 with the stop codon TGA. The protein OrfB
hereby encoded has an apparent molecular weight of 57 kd in the T7
expression system. Also to this reading frame no homologue could be
identified via data base search.
[0088] As a structural peculiarity the amino terminus of the OrfB
sequence displays a signal sequence showing similarities to the
type IV-prepilin signal sequence. At positions 8 and 12 of the
amino acid sequence there is phenylalanine so that there are in
addition two possible cleavage sites for the type IV pilin signal
peptidase. It can be derived herefrom that OrfB presumably is a
secreted protein.
[0089] The molecular weights of all the three gene products
measured in the T7 expression system correspond to the values
theoretically calculated from the sequence. The separation of the
expression products by means of gel electrophoresis showed that the
OrfB-band was significantly weaker than the OrfA-band in all the
cases. This points to a weaker expression of OrfB.
[0090] Two regions showing a sequence homology to the promoter
regions were identified. One of them is located in front of the
orfl gene, the second one in front of the orfA gene, each leaving
an appropriate distance (SEQ ID No. 1). Therefore, it can be
assumed that orfA and orfB form a transcription unity.
[0091] The analysis of the Neisseria gonorrhoeae MS11 genome after
ClaI and MluI digestion showed a complex band pattern in Southern
hybridization with plasmid pES-8 as sample. This fact indicates the
existence of several copies of the orf-gene region, probably of
three copies, in the genome of Neisseria gonorrhoeae MS11. If all
these loci are expressed, if they are subjected to antigenic
variations like, for example, the Neisseria genes pilS and opa, and
if the flanking regions of the orf gene region are involved in the
sequence repetitions, is presently not known.
Example 3
Characterization of the Localization of OrfA and OrfB on the Cell
Surface
[0092] In order to experimentally prove the lipoprotein nature of
orfA derivable from the perfect structure homology of the amino
terminus of orfA to lipoprotein signal sequences, both N.
gonorrhoeae and E. coli recombinants transformed with the orf-gene
region were labeled with [.sup.3H] palmitate. The results of the
labeling show that in all the cases, both with N. gonorrhoeae and
with the E. coli recombinants, lipoproteins in the corresponding
molecular weight range could be identified. While with N.
gonorrhoeae several proteins were labeled and the labeled band
could not be precisely assigned since there was no orfA.sup.-
mutant available, the orfA recombinants of E. coli showed in
comparison to the control strain unambiguously only one additional
band having the molecular weight of OrfA. An OrfA fusion protein
that was tested in addition and was amplified at the
carboxy-terminal by a fusion of 3 kd, also had a [.sup.3H]
palmitate labeling and migrated to a position precisely
corresponding to the molecular weight that was, as expected,
increased due to the fusion.
[0093] When prepared cell coats were treated with detergents, OrfA
showed a solubility that is typical of proteins of the outer
membrane. By separating the cell coat by means of density-gradient
centrifugation it could be confirmed by means of marker proteins
that OrfA was located in the outer membrane of N. gonorrhoeae. Also
with orf recombinants of E. coli, OrfA was shown to be a protein
component of the outer membrane by means of said method.
[0094] The accessibility of the cell surface was proven by means of
an immunofluorescence test both for OrfA and OrfB. A defective pilC
mutant of Neisseria gonorrhoeae the two pilC genes of which were
switched off is labeled by the PilC-OrfA antiserum in the same way
as recombinant E. coli strains carrying the orf-gene region. The
non-transformed control strain showed, as was to be expected, a
negative reaction. A positive reaction in the immunofluorescence
test of N. gonorrhoeae and orf recombinant E. coli strains could be
brought about by means of OrfA and OrfB specific antisera using
purified fusion proteins of either OrfA or OrfB. for the production
of these antisera. If antisera were used directed against an OrfI
fusion protein, the immunofluorescence test with N. gonorrhoeae was
negative. From this it can be deduced that OrfA and OrfB are
located on the cell surface and are accessible from the outside,
whereas OrfI probably is located intracellularly.
[0095] The surface localization of OrfA and OrfB could only be
proven in recombinant E. coli strains carrying the whole orf
region.
Example 4
Adhesin Property of the OrfA-PilC Complex
[0096] As mentioned above OrfA could be obtained in pure form by
chromatography on an Ni-NTA-chelate matrix due to its affinity to
PilC. Since the function of PilC as pilus associated adhesin had
been proven and the direct binding of PilC to human ME-180 cells
had been known, it was obvious to test the adherence property of
the PilC-OrfA complex. The experiments were performed with the E.
coli strain HB101 (E141) since it does not possess the mannose
specific typeI pili and shows almost no binding to human ME-180 and
Chang epithelial cells. After the transformation of HB101 with the
plasmid pES25, no adherence, neither to ME-180 nor to Chang cells,
could be mediated. If the same recombinants, however, were
pre-incubated with PilC protein, a strong adherence to Chang
epithelial cells but not to ME-180 cells could be induced (Table
I).
1TABLE I OrfA-dependent modulation of the PiIC mediated adhesin
function Adherence to human epithelial cells ME180 cells Chang
cells N. gonorrhoeae, Orf+ PilCi+, Pili+ +++ + N. gonorrhoeae,
Orf+, PilC+, Pili- + +++ E. coli (E141) - - E. coli (E141) + PilC
(extern) - - E. coil (H2561) - - E. coli (H2561) + PilC (extern) -
- E. coli (H2560) - + E. coli (H2560) + PilC (extern) - +++
[0097] Three independent experiments were evaluated, whereby the
adherence of Neisseria was determined using 500 cells and the
adherence of the E. coli strains was determined per epithelial
cell.
[0098] +++100%, ++50%, +25% adherence.
[0099] E. coli E141=E. coli strain HB101 without plasmid; E. coli
H2561=E. coli strain HB101 with plasmid pBA; E. coli H2560=E. coli
strain HB101 with plasmid pES25 The plasmid pES25 (FIG. 1) is a pBA
vector containing a genomic fragment from Neisseria gonorrhoeae of
approximately 11 kb carrying the coding regions orfA, orfB and
orfI.
[0100] The E. coli strain H2560 was deposited at the Deutsche
Sammiung fur Mikroorganismen (DSM, Braunschweig, Germany) under the
DSM-Accession Number DSM 10257.
[0101] The result obtained is surprising since pilus carrying
Neisseria bind to ME-180 cells with a significantly higher affinity
than to Chang epithelial cells. This result can be put down to the
fact that PilC has different adherence properties depending on its
localization. As an adhesin component in the pilus PilC preferably
binds to receptors of the ME-1800 cell surface, whereas as an
adhesin located on the cell surface in the complex with OrfA PilC
preferably recognizes receptors on Chang epithelial cells. If in
the latter case adhesin properties also can be ascribed to OrfA
and/or OrfB, is presently not known.
[0102] The results obtained for recombinant E. coli strains could
be reproduced with the same result with N. gonorrhoea. If the
pilus-free strain N 300 (P-Opa-), which hardly binds to ME-180 or
Chang cells, is pre-incubated with purified PilC, the adherence to
Chang epithelial cells can be significantly increased.
[0103] The described experimental approaches obviously provide for
a model that is suitable to analyze a mechanism for the modulation
of the adherence properties, how they can in cascade-like order
effect the increasingly strong adherence of the pathogens to the
host cells or how they can be the basis for the tissue tropism.
Sequence CWU 0
0
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