Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Method for identification of proteins from intracellular bacteria
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel combination of methods that
enables identification of proteins secreted from intracellular bacteria
regardless of the secretion pathway. The invention further provides proteins
that are identified by these methods. Secreted proteins are known to be
suitable candidates for inclusion in immunogenic compositions andlor
diagnostic purposes. The invention also provides peptide epitopes (T-cell
epitopes) from the identified secreted proteins, as well as nucleic acid
1 o compounds that encode the proteins. The invention further comprises
various
applications of the proteins or fragments thereof, such as pharmaceutical and
diagnostic applications.
BACKGROUND OF THE INVENTION
The Chlamydia are obligate intracellular bacteria, which multiply inside
eukaryotic host cells and are important human pathogens. The order
Chlamydiales comprises one family (Chlamydiaceae) containing one genus
(Chlamydia), which is divided into the four species: C. trachomatis, C.
2 o pneumoniae, C. psittaci and C. pecorum.
The human pathogenic serovars of C. trachomatis are divided into: A-C
which afflict ocular diseases; and D to K, which are sexually transmitted and
causes urethritis or complications such as salpingitis, epidymitis and ectopic
2 5 pregnancies; and L1 to L3 which cause a severe systemic infection,
lymphogranuloma venereum (LGV). The human pathogen C, pneumoniae is
responsible for respiratory tract infections causing bronchitis and pneumonia
and has recently been associated with the development of atherosclerosis
(Saikku et al, 1988 [1.], Shor et al, 1992 [2.]).
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if left untreated Chlamydia infections may become chronic with severe
complications such as sterility, blindness and potentially thrombosis.
Due to the intracellular developmental cycle persistent Chlamydia infections
may cause an aberrant immune response, which fails to clear the organisms.
Many immunogenic Chiamydia proteins have been considered vaccine
candidates, especially surface exposed proteins such as the major outer
membrane protein (MOMP) that is immunodominant in C. trachomatis [7.],
z o but also stress response proteins such as Hsp60 [8.]. However, none of
these candidates have been proven efficient in vaccine trials.
A likely explanation for the limited humoral response and little protective
immunity is the intracellular nature of the organism. An alternative approach
is therefore to find proteins, which are recognizable by the cell- mediated
immune system, which has been shown to be pivotal in the resolution of
chlamydial infection primarily through the effect of cytotoxic T-lymphocytes
(CTL) (Iguitseme et al 1994 [9.])
2 o Great attention has been drawn to secreted proteins since these may be
processed in the host cell proteasomes and presented as MHC-class I
antigens on the surface of cells and thus represent obvious vaccine targets
(Hess and Kaufmann, 1993 [45]). An example of this was shown for Yersinia
infected cells, which presents an epitope of the YopH effector to MHC
restricted cytotoxic T-lymphocytes (CTL). (Starnbach & Bevan, 1995 [14.])
The interaction between Chlamydia and the host cell is essential for the
intracellular survival and propagation of the bacteria.
Complete and searchable Chlamydia genomes exist for C. trachomatis
3o serovar D (Stephens et al, 1998 [4.]) (comprising 894 predicted open
reading
frames (ORFs)) and C. pneumoniae VR1310 (comprising 1073 ORFs)
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(Kalman et al 1999 [5.]). In addition complete genomes of C. trachomafis
MoPn (Read et al, 2000 [12.]) G, pneumoniae AR39 (Read et al [12.] and C.
pneumoniae J138 (Shirai et al 2000 [13.] are public available. From the
genome sequence it is known that Chlamydia posses genes involved in
secretion mechanisms including several genes with homology to type III
secretion genes from other organisms (Stephens et al., 1998 [4.] and Kalman
et al., 1999 [5.]).
Candidates for secreted effector proteins are likely to be present in Type III
1 o secretion subclusters (Subtil et al., 2000) [10.]. This view was recently
illus-
trated by the discovery of the Type III secretion characteristics of CopN
(Fields & Hackstadt 2000) [11.] Type III secreted proteins, however, lack
recognisable signal peptidase cleavage sites and no consensus sequence for
proteins secreted by this system in Chlamydia has been recognized, such
may be restricted to the particular organism in question. Moreover, secreted
proteins may be a functionally diverse group of proteins located in
unpredictable locations in the genome (Subtil, 2000) [10.].
The present state of knowledge concerning secreted Chlamydia effector
2o proteins is limited to proteins present in the inclusion membrane including
members of the Inc family (Rockey et x/.1995) (15.] [16.], CopN (Fields
&Hackstadt,.2000 [11.]) and CT529 (Fling et al, 2001 ) (37.].
It has been shown that CD8+ T-cells specific for Chlamydia arise during an
infection, meaning that Chlamydia proteins are exposed to the host cell
cytoplasm which is a prerequisite for presentation of MHC class I antigens.
CT529 has been identified from a genomic library by expression in a
eukaryotic cell and recognition by a Chlamydia specific T-cell line (Probst)
[41]. CT529 has been shown to contain an epitope, which in mouse vaccine
3 o experiments provides some protection against infection.
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Expression in eukaryotic cells of a genomic Chlamydia trachomatis serovar
L2 library by transfection with a viral vector and subsepuently screening with
Chlamydia specific T-cells for the detection of proteins comprising MHC class
I restricted epitopes has been described in The International Patent
Application No. WO 00/34483 (Probst) [41]. This method has resulted in the
identification of five positive clones, CT529 was contained in one of these,
another clone contained three open reading frames but the remaining three
clones have not been described further. Drawbacks of such a screening is
the eukaryotic expression of bacterial proteins which may differ from
bacterial
1 o expression in a way that alters the probability of processing in the
proteasome and the very presentation as MHC antigens, and the
maintenance and stimulation of T-cell clones differ from the in vivo situation
and clones recognizing proteins, which are not accessible during a normal
infection may result in false positives. Other approaches in the above patent
application concerns identification of candidates for a vaccine directed
against the humoral immune defence.
When searching for proteins secreted from intracellular bacteria, the
straightforward idea would be to isolate the cytoplasm from infected host
2 o cells and look for bacterial proteins. However, this strategy cannot be
employed for Chlamydia due to the fragility of the chlamydial reticulate body.
Another approach would be to identify pathogenicity factors, which are often
secreted proteins, by transposon analysis. However, it is not possible to
transfect Chlamydia. No strategy exists that can predict which proteins are
secreted and genes encoding effector proteins suitable for vaccine
development may be located in unpredictable locations in the genome.
Thus, there is a need for a reliable system, which can limit the number of
vaccine candidates in a cost efficient way and which involves a minimum of
3 o experimental steps.
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In [18] the effect of IFN-y C. trachomatis A and L2 protein expression was
investigated by means of [35S]-methionine/cysteine labelling of C.
trachomatis proteins in combination with autoradiography following 2D-gel
electrophoresis. IFN-~~added during the infection of HeLa cell cultures with
5 C. frahomatis A resulted in a pronounced down-regulation of several C.
trachomatis A proteins, wheras this effect was not apparent for C.
trachomatis L2. IFN-y=dependent induction of ~30 and ~40 kDa proteins in
both C. trachomatis A and L2 was observed. The induction of these proteins
was antagonized by addition of super-fysiological amounts of L-tryptophan to
1 o the growth medium. This indicated that the IFN-y mediated inducibility of
these C. trachomatis proteins is associated with IFN-y mediated up-regulation
of the tryptophan degrading host cell enzyme indoleamine 2, 3 dioxygenase.
One of the IFN-induced C. trachomatis proteins migrated with a significantly
lower molecular Weight in C. trachomafis A compared to C. trachomatis L2.
In [19] The IFN-y inducible C. trachomatis A and L2 proteins described
previously (Shaw et al. 1999) were further characterized. Using MALDI-T0F
mass spectrometry followed by database search the proteins were identified
as the C. trachomatis tryptophan synthase alpha (TrpA) and beta (TrpB)
2 o subunits from preparative 2D-gels. The proteins were also induced by IFN-
~~in C. trachomatis D and the induction was prevented by addition of super-
fysiological amounts of L-tryptophan in all three serovars. TrpA in C.
trachomatis A migrated with a lower molecular weight in C, frachomatis A
compared to C. trachomatis D and L2. C, trachomatis A and C TrpA are
truncated by ~7.7 kDa compared to C. trachomatis D and L2 TrpA as
revealed by analysis of the trpA gene from these C. trachomatis serovars.
The truncation or absence of tryptophan synthase in the trachoma causing
serovars (C. trachomatis A, B and C) may impair the trytophan synthesizing
ability and render these serovars more susceptible to IFN~y mediated
3o tryptophan depletion. This can explain differences seen in pathogenesis
among human C. trachomatis serovars.
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In [43] the host cell proteasomal degradation of a previously described
secreted protein (p60) of the intracellular bacterium Listeria monocytogenes
was investigated. The general strategy used was based on pulse chase
assays using [35 S]-methioninelcysteine labelling in the presence or absence
of two peptidealdehydes: N-acetyl-Leu-Leu-norleucinal(LLnL) and
(benzyloxycarbonyl)-Leu-Leu-phenylalaninal (Z-LLF), which inhibit the
proteolytic activity of the eukaryotic proteasome. Polyclonal antibody raised
against p60 wsa used to immunoprecipitate p60 from proteosome inhibitor
1 o treated and non-treated lysates of L. monocytogenes-infected J774 cells.
Evaluation of autoradiographs of immunoprecipitated labelled p60 separated
by one-dimensional SDS PAGE suggested that proteasome inhibitors were
able to inhibit proteasome degradation of p60. The number of p60- CTL
epitopes per infected cell decreased upon treatment with LLnL and Z-LLF.
This suggested a link between inhibition of proteasomal degradation of p60
and p60-CTL epitope production.
In [44] mechanisms behind protective immunity and general features of the
cellular immunereponse towards intracellular microorganisms were described
2 o with the focus on development of viable recombinant vaccines against
intracellular microbes. Strategies for developing antigen delivery systems
were discussed with emphasis on Mycobacterium bovis BCG and Salmonella
typhi aroA-. These non-virulent intracellular bacteria can be genetically
modified to deliver antigens, which may serve as targets for a vaccine by
immune recognization. The authors point out the advantages of using
secreted proteins as targets for the development of a vaccine as these
proteins will be processed and presented to the cell-mediated immunesystem
while the bacterium still replicates inside the host cell.
SUMMARY OF THE INVENTION
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The present invention comprises the identification of secreted proteins by a
novel combination of methods. The described combination of methods
constitutes a secretome (the collection of secreted proteins) by subtraction
of
the proteome of intracellular bacterial proteins from the total proteome of
bacterial proteins present in infected cells.
The bacterial proteins are selectively visualized by pulse labelling in the
presence of an inhibitor of eukaryotic protein synthesis followed by two
dimensional electrophoresis and autoradiography. Protein profiles of purified
1 o bacteria are compared to protein profiles of the total lysate of infected
cells
and the protein spots present in the differential image, the secretome, are
identified from gels loaded with total lysate of infected cells by advanced
mass spectrometric methods.
The identified secreted proteins are further analysed by advanced artificial
neural networks to provide peptide sequences that are predicted to be good
T-cell epitopes.
Furthermore, proteins are selected for which the turnover is delayed by
2 o inhibitors of the host cell proteasome since these proteins are especially
likely to be degraded in the host cell proteasome and presented as MHC
class I antigens on the host cell surface.
Compared to other strategies for identification of vaccine candidate proteins
and epitopes, the present invention provides a limitation of the number of
candidates, which can only be obtained by the novel combination of
methods.
The invention is based upon the following observations:
~ Proteins that are secreted from an intracellular bacteria into the host
3 o cell will be absent from purified bacteria but present in whole lysates of
infected cells.
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~ 2D protein profiles of purified bacteria as well as whole lysates of
infected cells can be made visible by two-dimensional electrophoresis
using specific pulse-labelling of bacterial proteins.
~ Subtraction of the 2D protein profile of purified bacteria from the 2D
protein profile of bacterial proteins present in the host cell enables the
identification of secreted proteins by mass spectrometri methods.
~ Proteins that are secreted from an intracellular bacteria, which are
processed by the host cell proteasome are likely to generate MHC
class I antigens, which are capable of activating T-cells.
~ Secreted proteins, which exhibit a prolonged turnover in response to
the addition of inhibitors of the eukaryotic proteasome, are likely to be
presented at the host cell surface. The identification of such proteins is
enabled by analysis of 2D proteins profiles.
~ T-cell epitopes can be predicted by artificial neural networks trained to
recognize peptides that have a high affinity for the MHC class I
complex.
The following definitions are used in connection with the present invention:
2 0 DEFINITIONS
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Secretome
Proteins that are very likely to be secreted from an intracellular bacteria.
(Type III)Secretion subclusters
A cluster of Chlamydia genes which contains genes that have significant
homology to type III secretion gene from other organisms. By the definition of
secretion cluster is meant a collection of ORFs (open reading frames) with
known or unknown function, which are located up to four genes away from
any gene with known homology to genes involved in Type III secretion in
other bacteria (e.g. Salmonella, Shigella, Yersinia.)
Proteasome inhibitor
Any chemical synthesized or naturally occurring compound, which is able to
reversibly or irreversibly inhibit the proteolytic activity of the activated
26S
eukaryotic proteasome. Persons skilled in the art will recognize that
proteolytic activity of the proteasome contains several different activities
(e.g.
chymotrypsin-like activity, which cleaves after large hydrophobic residues,
trypsin-like activity, which cleaves after basic residues, post-glutamyl'
hydrolase activity, which cleaves after acidic residue, BrAAP, which cleaves
preferentially after branched-chain amino acid, SNAAP, which cleaves after
2 o small neutral amino acids of subunits). Several compounds known to inhibit
the proteasome are commercially available and mostly include cell
permeable peptide based inhibitors (e.g. peptide aldehydes, peptide vinol
sufphones). Peptide based inhibitors act as transition state analogs, which
form an adduct with the proteasome's active sites, whereas the naturally
occuring clasto-Lactacystin-~i-lactone exerts a proteasome inhibiting effect
by
means of irreversible modification of the active sites of the proteasome
subunits. These compounds and combinations hereof can potentially be used
to successfully inhibit proteasome function and MHC- class I antigen
processing (e.g. MG115, MG132, MG262, PSI, clasto-Lactacystin-(3-lactone,
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Epoxymycin). The application of proteasome inhibitors may be used at any
time point throughout the developmental cycle of Chlamydia either before,
during or after pulse labelling or chase is performed.
Host cells
5 A host cell is any eukaryotic cell, which can be infected with an
intracellular
bacteria. A person skilled in the art will recognize that a wide range of
immortalized cell lines will be suitable hosts for infection with Chlamydia
including epithelial cell lines (e.g. HeLa, Hep-2, BHK cells) or immortalized
mononuclear cell lines, e.g. U-937. Immortalized host cell may be obtained
1o from naturally occurring carcinoma or by transformation of primary cells
with
virus, which carries oncogenic genes, which results in unlimited cell division
and growth (eg. SV40). The definition of host cells also includes primary
epithelial or endothelial mammalian cell lines, which can be obtained from
living mammals or by autopsy, and propagated for a limited time in vitro, and
organ cell culture.
Genetically modified host cell
A person skilled in the art will acknowledge that appropriate host cell also
includes host cell, Which have been genetically modified to overexpress or
suppress genes, which are relevant in context of chlamydial vaccine
2 o development, e.g. genes encoding proteasome subunits or other genes
encoding functionally important proteins involved in MHC-class I
presentation.
Proteasome
The proteasome is the central enzyme complex of non-lysosomal protein
degradation being an essential component of the ATP-dependent proteolytic
pathway catalyzing the rapid degradation of many rate-limiting enzymes,
transcriptional regulators and critical regulatory proteins. In eukaryots it
is
essential for the rapid elimination of abnormal proteins, aggregated, unfolded
or normal host cell proteins as well as proteins coming from intracellular
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bacteria located in the host ceII.The proteasome in higher eukaryotes is
critically involved in MHC class I antigen processing by degrading proteins to
peptides which are delivered to the host cell surface for presentation as T-
cell
epitopes.
Pulse-labelling
By labelling of proteins is meant incorporation of amino acids containing
radioactive isotopes (e.g. L-[35S]-methionine, L-[methyl 3H]-methionine, L-
[methyl-'4C]-methionine, [35S]- cysteine, [3H]-tryptophane or combinations
hereof) in bacterial proteins in a period of time (eg. 0.5 hours, 1 hours, 2
1 o hours, 4 hours, 6 hours) during which the host cell protein synthesis is
inhibited using a sufficient concentration of inhibitors of eukaryotic protein
synthesis. The labelling can be performed throughout the Chlamydia
developmental cycle. The labelling medium must be sufficiently enriched with
nutrients to allow growth of both the host cell and the pathogen during the
time in which the infected cells grow. The inhibition of host cell protein
synthesis may be accomplished through addition of cyclohexamide or other
inhibitors of host cell protein synthesis (e.g. emetine) during the labelling
period, with the effect of allowing incorporation of the radioactive amino
acid
only in the protein synthesizing intracellular bacterium. Protein degradation
2 o can be prolonged by adding cell-permeable inhibitors of protein
degradation
to the growth medium during the labelling period. It is to be noted that the
present invention is not limited to the use of radioactive labelling.
Pulse-chase
By chasing labelled proteins after there synthesis the turnover time can be
estimated, e.g. the time span after which the amount of protein synthesised
during the labelling period is degraded by preferably more than 75% (e.g.
80%, 90 %, 95 %, 99 %, 100 %). This estimation is performed by measuring
the optical density of a given protein spot in the gel before and after a
chase
period and reveals how long time the protein is present in the infected cell.
3o The chase is performed by replacing the labelling medium with a growth
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medium without the radioactive amino acid and harvesting the infected cells
to different time points after labelling. A chase can be performed for varying
periods after the labelling (e.g. 0.5 hours, 1 hours, 1,5 hours, 2 hours, 6
hours, 12 hours, 24 hours, 72 hours) and after labelling at various time
points
in order to determine how long time the protein is present in the infected
cell.
The mature form of certain proteins may be processed from a propeptide,
and thus accumulate during the chase period instead of decreasing in
amount. Under these circumstances the mature protein may accumulate
before the degradation can be visualized during the chase periods. The time
1 o span, during which the protein is degraded, can be prolonged by adding
cell-
permeable inhibitors of protein degradation to the growth medium during the
chase.
Lysis buffer
A lysis buffer for use in the present invention is a buffer used to lyse
infected
cells and solubilize proteins prior to two-dimensional gel electrophoresis.
The lysis buffer contains 9 M Urea, 4% w/v 3-[(3-
cholamidopropyl)dimethylammonium]-1-propanesulfonate (CHAPS; Roche,
Germany), 40 mM Tris Base, 65 mM DTE and 2% voUvol Pharmalyte 3-10
2 o (Amersham Pharmacia Biotech). For the enrichment of high molecular weight
and hydrophobic proteins the lysis buffer alternatively contains 7 M urea, 2 M
thiourea, 4% w/v 3-[(3-cholamidopropyl)dimethylammonium]-1-pro-
panesulfonate (CHAPS; Boehringer Mannheim, Germany), 40 mM Tris
Base,65 mM dithioerythretiol (DTE) and 2% vol/vol Pharmalyte 3-10
(Amersham Pharmacia Biotech).
It is recognized that it is possible to alter the lysis buffer in order to
increase
the solubility of certain proteins (e.g. thiourea will increase the solubility
of
hydrophobic and high molecular weight proteins).
Secreted effector protein
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By the term secreted effector protein is meant any protein, which is
secreted by the bacteria into the host cell cytoplasm or any intracellular
organelle. Secreted effector protein may have great influence on the
host/ pathogen relation and due to its presence in the host cell cytoplasm
may be targeted to the proteasome and presented as MHC-class I
antigens on the surface of the host cell. Secreted effector proteins may
be secreted by one of several Sec-dependent or independent systems
(e.g. Type I, Type II, Type III, Type IV) described in the literature.
Intracellular bacteria
~ Any bacteria, which has the ability to infect and propagate inside a
eukaryotic
host cell (e.g. Chlamydia, Salmonella, Shigella, Listeria, Legionella,
Yersinia).
The definition includes intracellular bacteria, which are obligate
intracellular
meaning that they may only live and propagate using an eukaryotic host cell
or facultative intracellular meaning that they may both survive in an
extracellular as well as an intracellular milieu.
Elementary bod~~EB)
The collection of Chlamydia bacteria purified by ultracentrifugation and
characterized by electron microscopy as being about 300 nm in diameter and
2 o having a condensed nucleus.
Reticulate body (RBA
The collection of Chlamydia bacteria purified by ultracentrifugation and
characterised by electron microscopy as being about 1000 nm in
diameter and having a normal bacterial nucleus.
Analytical ael
Any 2D-PAGE gel, which is loaded with a protein sample amount necessary
to visualize proteins. The amount applied for analytical purposes in the
herein
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described examples is typically 200.000 to 300.000 counts per minutes (cpm)
or >100 pg protein for stained gels (e.g. silver stained, Coomasie stained).
Sianificant~ decreased intensity/amount
A reproducible detectable reduction preferably greater than 10% (e.g. 20%,
35%, 50%, 65%, 80%, 90%, 100%) in the optical density integrated over total
area of a given spot localized on a 2D-PAGE gel.
Significantly increased intensity/amount
A reproducible detectable increase preferably greater than 10 % (e.g. 20%,
30%, 45%, 60%, 75%, 90%, 100%, 150%, 200%,300% or more) in the
optical density integrated over total area of a given spot localized on a 2D-
PAGE gel.
Preparative ael
A 2D-PAGE gel, which is loaded with a protein sample amount necessary to
allow identification of specific protein spots by one of the herein described
identification methods (e.g. MALDI- MS, ESI-Q-TOF, Edman degradation).
Typically >500 pg is applied on gels for preparative purposes depending on
the immobilised pH gradient used. The definition of preparative gels used in
the present invention includes gels with proteins that are unfixed, fixed
using
staining protocols (e.g. silver staining, Coomasie staining) or electroblotted
on to PVDF membranes. It is possible to visualize proteins on preparative
gels by applying a background of labelled proteins to the preparative gels,
which are separated along with the non-labelled proteins. It is also possible
to compare such gels with analytical gels in order to excise the exact protein
of interest.
Vaccine candidate
A protein, which based on results obtained by the methods of the present
invention is potentially secreted from an intracellular bacteria. Secreted
proteins are accessible for degradation by the host cell proteasome and
peptides derived from these proteins may therefore be presented as MHC-
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class I antigens at the surface of the infected cell, thus being recognizable
by
T-cells. Such proteins will therefore be obvious targets for the development
of
a vaccine against the intracellular bacteria. A protein described as a vaccine
candidate may also serve useful as a component of a diagnostic test.
5 Vaccine
In the present invention the term vaccine is to be understood in its broadest
sense as an immunogenic composition, which is able to elicit an adaptive
immune response (humoral or cellular). Vaccines candidates, which are able
to elicit an adaptive immune response may be administered to animal or
1 o human recipient as injectables either in the form of solutions,
suspensions or
as emulsions. The vaccine candidate being the active component of the
immunogenic composition may be mixed with pharmaceutical acceptable
excipients such as water, saline, glycerol and ethanol before injection into
the
recipient. Injection may be carried out in different ways (e.g. subcutaneously
15 or intramuscularly). Vaccine candidates may serve as a vaccine either i) in
its
full length, or ii) as a source for providing immunogenic fragments, e.g. T-
cell
epitopes. It is acknowledged that specific proteins or peptides alone or in
combination with other proteins or peptides may be administered to an
animal or human recipient and serve as a vaccine.
zo
Furthermore a DNA fragment encoding a vaccine candidate protein can be
cloned in a vector, which can be introduced by injection into an animal or a
human recipient. The DNA fragment is taken up by e.g. muscle cells and
expressed under the control of a promoter, which will be active in eukaryots.
In this so-called DNA vaccine the expressed DNA fragment is capable of
stimulating the immune system.
MHC class I antigen
A major histocompatibility class 1 antigen comprises a peptide diverged from
3 o a protein which is exposed to the host cell cytoplasm, which is conjugated
to
a heterodimeric MHC class I molecule in the endoplasmatic reticulum and is
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presented on the surface of the cell bound in the grove of HLA complex (in
humans) where it may serve as a T-cell epitope. The majority of MHC-class (-
presented peptides are diverged from a protein processed in the cytoplasm
of the host cell by the activated 26S-proteasome.
HLA
Human leukocyte antigen, the name for the human major histocompatibility
complex.
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T-cell epitope
A peptide of short length, which bound to a dimeric MHC class I molecule on
the surface of a cell, can be recognized by the receptor of a specific
cytotoxic
T-cell, e.g. consisting of typically 8-10 amino acids.
Whole cell Iysates
Infected host cells harvested directly in lysis buffer, without prior
purification
or fractionation. It is recognized that whole cell lysates may be obtained at
any time point throughout the chlamydial developmental cycle and that the
whole cell lysates will contain a mixture of all proteins present in the
infected
1 o host cell including those coming from the bacteria.
Purified bacteria
Bacteria, which is purified from infected cells. Using Chlamydia as an
example, it is possible to purify both RB and EB as well as intermediate
forms of Chlamydia by density gradient ultra centrifugation methods,
depending on the time point in the developmental cycle at which the
Chlamydia are harvested. The purity can be determined by electron
microscopy. In the present invention the purity is also readily verified on
silverstained 2D gels by estimating the contribution contaminating highly
abundant host cell proteins (e.g. actin, beta-tubulin, alfa-tubulin,
calreticulin)
2 o to the total optical density of all proteins present on the gels.
Identified protein
Names on proteins identified using identification methods based on mass
spectrometry or Edman degradation are indicated following a nomenclature
according to and compatible with the one used for genes in the Chlamydia
Genome Project available http://socrates.berkeley.edu: 4231/ and as
published in
i) Stephens, R.S., Kalman, S., Lammel, C., Fan, J., Marathe, R., Aravind, L.,
3o Mitchell, W., Olinger, L., Tatusov, R.L., Zhao, Q., Koonin, E.V., Davis,
R.W.
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(1998) Genome sequence of an obligate intracellular pathogen of humans:
Chlamydia trachomatis. Science 282: 754-759
ii) Kalman, S., Mitchell, W., Marathe, R., Lammel, C., Fan, J., Hyman, R.W.,
Olinger, L., Grimwood, J., Davis, R.W., Stephens, R.S (1999). Comparative
genomes of Chlamydia pneumoniae and C. trachomatis. Nat Gent. 21: 385-
389
ELISPOT
1 o In the ELISPOT method, T-cells that have been stimulated with antigen in
vitro are incubated in microtitter wells pre-coated with anti-cytokine (e.g.
IFN-
g, 1L-6, TNF-a) antibody. After a period of incubation the local production of
cytokines around activated T-cells can be visualized by adding a secondary
antibody conjugated to an enzyme such as horseradish peroxidase alkaline
peroxidase. An estimation of the production of cytokines is done by finally
adding a substrate that wilt be enzymatically converted into a coloured
product thus allowing cytokine producing cells to be visualized.
Adiuvant
2 o By adjuvant is meant an emulsion, which contains a specific immunogen,
which can elicit an immuneresponse in a mammal recipient (e.g. Freunds
adjuvant).It is recognized that an adjuvant together with the immunogen can
be supplemented with components such as dried bacteria or bacterial
products, which will enhance the immune response in an immunized
mammal. Alternatively, immunomodulating substances such as lymphokines
(e.g., IFNg, IL12) or poly I:C may also be administered together with the
immunogen and adjuvant.
Seroconversion
3 o The development of different classes or subclasses of antibodies in
response
to an antigen.
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Micro immuno fluorescence (MIF or micro-IF)
An assay, which measures antibodies against fixed bacteria or proteins by
immuno fluorescence microscopy.
Immunoaenic
A protein or peptide is immunogenic if it can elicit an-adaptive immune
response upon injection into a person or an animal.
DESCRIPTION OF THE DRAWINGS
Figure 1A shows an example of a gel image of C. trachomatis D proteins
1o from whole cell lysates [35S]- labelled from 22-24 hours post infection
(h.p.i.),
harvested immediately after labelling and separated by 2D-PAGE. Black
arrows mark proteins which intensities are significantly reduced on gels with
proteins from purified EB (elementary bodies) labelled from 22-24 h.p.i. and
purified 72 h.p.i.. The p1 and Mw characteristics of the marked spots (DT1-
77) are listed in Table I.
Figure 1 B-E show examples of identified vaccine candidates and their
presence in C. trachomatis D at different times after synthesis shown by
enlargements of selected areas from Figure 1A. The protein turnover time
2o was estimated by chasing at different times after labelling throughout the
developmental cycle until purification of EB. The upper scale indicates points
in time after labelling starting with zero; the lower scale represents the
time in
hours post infection starting with 24 hours. Figure 1 B: DT1 and DT2 (CT668).
Figure 1 C: DTB. Figure 1 D: DT7 (upper arrow) and DT11 (lower arrow) (both
identified as CT610). Figure 1 E: DT3 (lower arrow, CT783),DT4 (upper
arrow, CT858).
Figure 2A shoves an example of gel image of C, pneumoniae VR1310
proteins from whole cell lysates [35S]-labelled from 55-57 h.p.i.,
3o harvested immediately after labelling and separated by 2D-PAGE. Black
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arrows mark proteins which intensities are significantly reduced on gels
of proteins from EB labelled at two hour periods throughout the
developmental cycle i.e. 6, 12, 24, 36, 42, 48, 54, 60 h.p.i. and purified
72 h.p.i.. Arrows mark proteins which are significantly reduced in
5 intensity on gels from purified EB. The p1 and Mw characteristics of the
marked spots (CP1-91 ) are listed in Table II.
Figure 2B shows an enlarged section of Figure 2A.
1 o Figure 2C shows corresponding enlargement of image of 2D-PAGE
separated EB proteins labelled as described above. The section enlarged
in Figures 2B and 2C gives an example of two proteins CP63 (identified
as CPN1016) and CP65, which are present in whole cell lysates but not
in EB. The encircled spot is present in both EB and whole cell lysates and
15 has been identified as polypeptide deformylase.
Figure 3A shows a peptide mass fingerprint used to identify spot no. DT1 as
the hypothetical protein CT668. Hollow arrows indicate peptides arising from
autodigestion of trypsin. These peaks were used to make an internal
2o calibration of the spectra. Black arrows indicate peptide masses matching
the
CT668 sequence. Doublet arrows indicate peptides originating from a human
contaminating protein.
Fi ure 3B shows results from using the identification software MS-Fit on the
peptide masses obtained from Figure 3A, showing that the highest-ranking C.
2 5 trachomatis protein is CT668.
Figure 4A shows a peptide mass spectrum generated by ESI-Q-TOF MS of
spot DT1 comprising the double charged 1744.9 Da parent ion
(R)KIVDWVSSGEEILNR(A) (black arrow), which matches the CT668 amino
acid sequence. Dotted lines point at the Y-peptide ions that were generated
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by fragmentation of the parent ion. The deduced amino acid sequence is
shown in bold one-letter code.
Figure 4B shows a peptide mass spectrum generated by PSD MALDI MS of
spot CP63. The 1919.8 Da parent ion (K)ELLFGWDLSQQTQQAR(L) was
fragmented and gave rise to several peptides revealing the sequence. Two of
these are exemplified by the 243.35 Da b2-ion (EL) and 356.34 Da b3 -ion
(ELL) that differ in mass by the mass of leucine (113Da).
Figure 5 shows the nucleotide sequence of the novel C. trachomatis specific
1 o vaccine protein candidate DT8 and corresponding amino acid sequence
shown by one-letter code. Bold amino acid sequence was covered by
sequence tags obtained by ESI-Q-TOF MS.
Figure 6 shows pulse chase studies in combination with one the proteasome
inhibitor MG-132. Figure 6.1: Total gel image of a 2D-gel loaded with C.
trachomatis D proteins labelled from 22-24 h.p.i. and chased for additionally
4 hours in the presence of 20 ~,M MG132. Figures 6A, 6B and 6C:
Enlargements of regions containing C. trachomatis D proteins, which has a
prolonged turnover time due to treatment with MG-132, when comparing
2 o chase studies performed with (chased+ MG-132) or without (chased) MG-
132. The first row represents protein profiles of infected cell harvested
immediately after the two-hour labelling period. Note that the intensity of
DT9,
DT10 and DT11 is significantly greater on gels with whole cell lysates which
are labelled and chased in the presence of MG132 compared to gels with
whole cell lysates harvested immediately after labelling without MG132.
Figure 7A shows a total gel image of IMB of whole cell lysates using PAb
245. A1: IMB showing reaction with spot no. DT4 and DT48, which are the C-
terminal and N-terminal fragments of CT858, respectively. A2: Corresponding
3o radio labelled background of the IMB.
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Figure 7B: IMB with PAb241 against YscN (B1 ) and PAb238 against CT668
(spot DT1 and DT2) (B4). Corresponding autoradiography of IMB, showing
co-localization with B2: YscN and B5: CT668. Localization of YscN(B3) and
CT668(B6) on analytical 2D-gels.
Figure 7C shows IMB with PAb255 against CT610 G1: Enlargement of 2D-
gel blot showing that PAb 255 stains to rows of spots, the upper representing
DT7 and the lower representing DT9, 10, 11 and 12. C2: Enlargement
showing the effect of treatment of infected cells with MG132 for 6 hours prior
1o to harvesting of the cells at 30 h.p.i. Note that MG132 treatment results
in an
clear relative increase in the abundance of DT9, DT10 and DT11 compared
to the row representing DT7. C3: Corresponding labelled analytical gel. C4:
SDS-PAGE IMB with PAb255: Lane a and c: 20 ~,g and 10 g.g (respectively)
of whole infected cell lysates harvested at 30 h.p.i. Lane b and d: 10 ~,g and
5 ~.g (respectively) of whole infected cell lysates treated for 6 hours with
50
~,M MG132, prior to harvesting of the cells at 30 h.p.i.
Fi ure 8 shows an indirect immunofluorescence microscopy showing sub-
cellular localization of vaccine candidates. A and B: HeLa 229 cells infected
2 o with C. trachomatis D and fixed with formalin 24 h.p.i. C: HEp-2 cells
infected
with C. pneumoniae and fixed with formalin 72 h.p.i.. Row 1 shows Normasky
images. Row 2 shows reaction with MAb 32.3 against C. trachomatis MOMP
visualized by rhodamine conjugated GAM IgG antibody. Row 3 shows
reaction with rabbit polyclonal antibody specific for the vaccine candidate in
question visualized by FITCH-conjugated GAR IgG antibody. The
investigated vaccine candidates are A; DTB, B; CT858, C: CPN1016 (CT858
homologue in C. pneumoniae). White single headed arrows points at borders
of Chlamydia inclusions in infected cells. Hollow arrows point at uninfected
cells. Note that the CPN1016 shows the same sub-cellular localization and
3 o secreted characteristics as CT858.
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Figure 9 shows a genomic localization of examples of identified vaccine
candidates from C. trachomatis D including identified proteins, which are
located in type (II secretion subc(usters 1,2 and 3.
Figure 10 shows the position of C. trachomatis D secretion candidates in 2D-
gel images from whole lysates of infected cells labelled from 22-24 h.p.i., RB
labelled and purified at the same point in time and EB purified at 72 h.p.i.
All
gels have been made using non-linear pH 3-10 Immobiline Drystrips.
22-24 hpi: enlargements of the gel image shown in Figure 1A of C.
1 o trachomatis D proteins from whole lysates of infected cells [35S]-labelled
from 22-24 h.p.i. harvested immediately after labelling and separated by 2D-
PAGE.
Purified RB: Corresponding regions from a gel image of C. trachomatis D
proteins from bacteria purified as RB at 24 h.p.i immediately after labelling
from 22-24 h.p.i.
Purified EB: Corresponding regions from a gel image of proteins from
bacteria labelled from 22-24 h.p.i. and purified as EB 72 h.p.i.
In rows A-G the secretion candidates DT4, DT48, DT23, DT76, DT77, DT47
and DT75 have been encircled.
Fiaure11 shows the position of C. pneumoniae VR1310 secretion candidates
in 2D-gel images from whole lysates of infected cells labelled from 55-57
h.p.i., purified EB, whole lysates of infected cells labelled at 34-36 h.p.i.
and
RB labelled and purified at the same point in time:
55-57 hpi: enlargements of the gel image shown in Figure2A of C.
pneumoniae VR1310 proteins from whole lysates of cells [35S]-labelled from
55-57 h.p.i., harvested immediately after labelling and separated by 2D-
PAGE using non-linear pH 3-10 Immobiline Drystrip.
Purified EB: Corresponding regions from a gel image (non-linear pH 3-10
3 o Immobiline Drystrip) of proteins from bacteria labelled at two hour
periods
throughout the developmental cycle i.e. 6, 12, 24, 36, 42, 48, 54, 60 h.p.i.
and purified as EB at 72 h.p.i.
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34-36 hpi: Corresponding regions from a gel image (linear pH 4-7 Immobiline
Drystrip) of C. pneumoniae VR1310 proteins from whole lysates of infected
cells labelled at 34-36 h.p.i. and harvested immediately after labelling.
RB 36 hpi: Corresponding regions from a gel image (linear pH 4-7 Immobiline
Drystrip) of C, pneumoniae VR1310 proteins from bacteria purified as RB at
36 h.p.i immediately after labelling from 34-36 h.p.i.
In A-F the secretion candidates CP34, CP37, CP46, CP47, CP52, CP63 and
CP75 have been encircled.
G shows a region from the parent images of the regions in A-F that contains
1 o no secretion candidates.
DETAILED DESCRIPTION OF THE INVENTION
Comparison of 2D-PAGE protein profiles from whole cell Iysates and purified
bacteria
Proteins, which are present in whole infected cells but absent from purified .
bacteria have potentially been secreted from the bacteria, e.g. Chlamydia.
Thus, an initial method of the invention is a comparison of 2D- PAGE protein
profiles of [35S]-labelled Chlamydia proteins from whole cell lysates of
2 o infected cells labelled at different time points of the developmental
cycle to
2D-PAGE protein profiles of purified bacteria [35S]-labelled at corresponding
time points. This method provides the detection of several proteins, which are
clearly present in the protein profile of whole cell lysates, but only faintly
detectable or absent in the protein profile of purified bacteria.
From a total of approximately 600 protein spots visualized in whole cell
lysates at 22-24 h.p.i. by high resolution 2D- PAGE (IPG), these studies
elucidated the existence of 77 C, trachomatis D proteins, of which the
intensity is significantly reduced in elementary bodies (EB). Similarly, 91
3 o proteins had significantly reduced intensities in C. pneumoniae VR1310,
when comparing whole cell lysates from labelling 55-57 h.p.i. to purified EB.
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The detected and annotated proteins have the Mw and p1 described for
protein no. DT1 - DT77 for C. trachomatis D, as listed in Table I, and CP1-
CP91 for C, pneumoniae as listed in Table II.
5 This method gives an overview of potentially secreted proteins necessary for
further investigations. The examples shows comparison of whole cell iysates
to purified EB labelled either at i) time points corresponding to labelling of
the
whole cell lysates (C. trachomatis) Figure 1 or 2) at time points throughout
the entire developmental cycle (C, pneumoniae, Figure 2).
Purification of RB allows the discrimination between secreted proteins and
RB-specific proteins. Protein profiles of whole lysates of infected cells can
be
compared to protein profiles of RB purified at the same point in time to
identify secreted proteins. In this approach RB specific proteins will not be
detected as false positives. Proteins synthesized and secreted at the
investigated point in time will be detected. Proteins may be synthesized and
secreted at other points in time.
The method also includes detection of proteins secreted immediately after
2 o infection, which may have been synthesised in the preceding developmental
cycle. These proteins are visualised by infection with EB labelled in the
preceding developmental cycle followed by 2D-PAGE of total cell lysates. At
an early stage of the developmental cycle before EB differentiate to RB, host
cell cytoplasm is obtained by Saponin penetration of the cell membrane [30,
31]. This is only possible because there at this time will be no contamination
of Chlamydia proteins from disrupting RB.
Identification of vaccine candidates
The vaccine candidate proteins cut out from preparative 2D-gels are
identified through advanced mass spectrometric methods.
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The excised spots are digested with an enzyme such as trypsin, which
generates a number of tryptic peptides. By means of MALDI-MS or other
approaches the masses of these peptides are determined with an accuracy
of better than 100 parts per million (ppm). Obtained masses are matched to
theoretical tryptic cleavage products of all proteins present in databases
using the MS-Fit or Peptidesearch software allowing the identification of the
analyzed protein on a statistical basis.
When protein spots are cut out from gels loaded with whole cell lysates,
1 o contaminating host cell proteins may be located at the same positions as
bacteria proteins and as a further complication one spot may contain more
than one bacteria protein. To avoid interference from contaminants that may
lead to unambiguous identifications, ESI-Q-TOF or post source decay (PSD)
may e.g. be used to obtain sequence information of the bacteria proteins) if
necessary.
The method includes proteins, which are identified by mass spectrometry as
indicated by examples from C. trachomatis D or C. pneumonia VR1310 in
Table III (A and B, respectively).
Accordingly, the invention relates in a first aspect to a method for
identifying
proteins secreted from an intracellular bacteria, comprising the following
steps:
1 ) infecting host cells by the intracellular bacteria,
2) labelling the intracellular bacteria present in the infected cells,
3) preparing
a) whole cell lysates of the infected cells
b) purified and lysed bacteria from the
infected cells,
4) comparing 2D-gel electrophoresis protein profiles of i) the
whole cell lysates from step 3a) with ii) the purified and lysed
bacteria from step 3b),
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5) detecting protein spots from step 4) which are present in the
whole cell lysates but absent or present in significantly
reduced amount in the purified bacteria,
6) identifying the proteins in the spots selected in step 5).
Pulse/chase of vaccine candidates
The object of this method is to detect secreted proteins, which are degraded
in the host cell. In order to estimate the time for which the identified
vaccine
candidate proteins are present inside the host cell a series of pulselchase
1 o studies are performed. The turnover time of [35S]-labelled proteins is
monitored on 2D-gels by chasing the proteins for various periods after
labelling. The turnover time provides valuable information on how fast the
proteins are degraded, thus how long they are present inside the infected
cell.
The method provides estimated turnover times of potentially secreted C.
trachomatis proteins as exemplified in Table I.
In this alternative aspect of the invention it relates, accordingly, to a
method
2 o for identifying proteins secreted from an intracellular bacteria,
comprising the
following steps:
1 ) infecting host cells by the intracellular bacteria,
2) pulse labelling of the intracellular bacteria present in the
infected cells,
3) preparing whole cell lysates of the infected cells after different
periods of chase following step 2),
4) comparing 2D-gel electrophoresis protein profiles of the whole
cell lysates prepared after different period of chase from step
3),
5) detecting protein spots from step 4) which are present in
decreasing amount as chasing periods increase in step 3),
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6) identifying the proteins in the spots selected in step 5).
Pulse chase in combination with proteasome inhibitors
In order to limit the number of candidates suitable for a vaccine, the
invention
includes proteasome inhibitor methods in combination with pulse chase
studies. These experiments provide an excellent tool for monitoring the effect
of the host cell proteasome on the turnover time of secreted Chlamydial
proteins.
Immunogenic proteins, which are presented on the surtace of eukaryotic cell
1 o as MHC-class I antigens must be ubiquitinylated and cleaved by proteolysis
in a multi-catalytic protein complex, the proteasome. The proteasome
cleaves immunogenic proteins into peptides of a typical length of 8-10 amino
acids. These peptides are transported to the ER (endoplasmatic reticulum),
where they are bound to a heterodimeric MHC class I molecule, and the
MHC-antigen complex is subsequently transported to the surface of cells. On
the surface of the cell the MHC-antigen complex will be recognizable by
specific receptors on cytotoxic T-lymphocytes (reviewed in Rock and
Goldberg [6.J).
2 o It is possible to inhibit the activity of the eukaryotic proteasome and
prevent
MHC class I presentation by adding cell-permeable proteasome inhibitors
such as peptide aldehydes to cell cultures (Rock et al. 1994) [36.]. Chlamydia
proteins for which the turnover time is prolonged by the addition of
proteasome inhibitors are likely to be secreted from the bacteria and
subsequently processed by the proteasome. In addition, this part of the
invention will allow the detection of Chlamydial proteins, which are degraded
in the proteasome very shortly after their release into the host cell and
therefore only detectable in the presence of proteasome inhibitors.
3 o The invention comprises C. trachomatis D and G. pneumonia VR1310
vaccine candidates, which are affected by proteasome inhibitors.
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The proteins DT1, DT2, DT3, DTS, DT9, DT10, DT11, DT13, DT14, DT36,
DT47, DT59, DT60, DT61, DT62 (as set out in Table IV below) are examples
of C, trachomatis D vaccine candidates for which the turnover time is
prolonged by addition of proteasome inhibitors during the chase period.
The invention also provides a method for purifying RB, which before harvest
are treated with proteasom inhibitors during a labelling period. A comparison
of proteasome treated whole cell lysates labelled at the same time as
1 o proteasome inhibitor treated purified RB, will elucidate further, which
proteins
are secreted to the host cell cytoplasm and degraded in the proteasome. In
addition, the host cell lines in these experiments can be genetically altered
to
overexpress genes, which are pivotal in the processing of MHC class I
restricted T-cell epitopes [38, 39, 40] (Sijts,2000, Van Ha11,2000, Shockett,
1995). By the use of such cell lines the effect of proteasome inhibitors will
be
more pronounced. The invention therefore also comprises the use of
commercially available host cell lines, which has been genetically modified in
genes, which are involved in MHC-class I antigen presentation.
2 o Accordingly, the invention relates in another alternative aspect to a
method
for identifying proteins secreted from an intracellular bacteria, comprising
the
following steps:
1 ) infecting host cells by the intracellular bacteria,
2) cultivating the host cells in the presence and in the absence of
a proteasome inhibitor, respectively,
3) labelling the intracellular bacteria present in the infected cells
cultivated in the presence and in the absence of a proteasome
inhibitor, respectively,
4) preparing whole cell lysates of the infected cells,
5) comparing 2D-gel electrophoresis protein profiles of the whole
cell lysates of the infected cells cultivated in the presence and
in the absence of a proteasome inhibitor, respectively,
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6) detecting protein spots from step 5) which are present in the
whole cell lysates cultivated in the presence of a proteasome
inhibitor, but absent or present in significantly reduced amount
in the whole cell lysates cultivated in the absence of a
5 proteasome inhibitor,
7) identifying the proteins in the spots selected in step 6).
Generation of polyclonal antibodies
The invention provides polyclonal antibodies, which are specific for vaccine
candidates. The gene encoding the vaccine candidate protein is cloned using
1o e.g. the ligation independent cloning (LIC)-system. Expressed fusion
proteins
encompassing the sequence of the vaccine candidate are used to immunize
rabbits in order to obtain sera containing vaccine candidate specific poly-
clonal antibodies (PAbs). The invention uses the PAbs in 2D-PAGE immuno
blotting in order to confirm the correct specificity of the antibody by co-
15 localization or to identify unrecognized isoforms of vaccine candidates.
The
invention provides verification/identification of vaccine candidates by
immunoblotting and co-localization as exemplified in Table III. The invention
uses the PAbs to determine the sub-cellular localization of vaccine
candidates by means of e.g. indirect immunofluorescence microscopy.
The invention, therefore, also provides one of the alternative methods above
which method further comprises the following steps:
1 ) obtaining antibodies against proteins from said intracellular
bacteria, identified according to any of the above methods,
2) 2D-PAGE immunoblotting on whole cell lysates of cells infected
with the bacteria using antibodies obtained in step 1 ),
3) detecting protein spots reacting in step 2),
4) identifying the proteins in the spots selected in step 3).
3 o Combinations of the four alternative methods are also part of the
invention.
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In preferred embodiments of the methods of the invention the labelling of the
intracellular bacteria is done by radioactive means, such as [35S]cysteine,
[35S]methionine, [14C]labelled amino acids or combinations thereof.
The method for identifying the proteins in the selected protein spots may be
based on Edman degradation or any mass spectrometric method, such as
MALDI TOF MS (Matrix-Assisted Laser Desorptionllonisation Time-Of-Flight
Mass Spectrometry), ESI Q-TOF MS (Electrospray Ionisation Quadrupole
Time-Of-Flight Mass Spectrometry), PSD-MALDI MS (Post Source Decay
1 o MALDI Mass Spectrometry) or combinations of such methods. Further, the
G
proteins may, prior to identification, be subjected to cleavage by chemical
methods, such as cyanogen bromide treatment or hydroxylamine treatment,
or by enzymatic methods with any suitable enzymes, such as trypsin,
slymotrypsin, chymotrypsin, or pepsin, or combinations thereof.
The intracellular bacteria may be a facultative intracellular or obligate
intracellular bacteria, and bacteria from the Genus Chlamydia, such as C.
pneumoniae, C. trachomatis, C, psitacci or- C. pecorum, including any
specific serovar or strain of these, are particularly interesting. However,
other
2 o intracellular bacteria, such as Salmonella, Shigella, Yersinia or
Listeria, , are
interesting, too, in connection with the present invention.
The host cells to be used according to the invention, may be common host
cells known in the art, such as an immortalized cell line, e.g. HeLa, Hep2,
McCoy or U937, a primary cell line obtained from mammalian donors or by
autopsy, a genetically modified cell line, or an organ cell culture, or even
other cells wherein the bacteria can grow. The host cells may be treated with
IFN-y prior to or during infection with the intracellular bacteria and/or may
have been genetically modified to over-express or suppress genes which are
3o recognized as being relevant in context of Chlamydial vaccine development.
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When the method of the invention uses one or more proteasome inhibitor,
any known inhibitor, such as MG132, MG262, MG115, epoxymycin, PSI and
clasto-Lactacystin-(3-lactone, are relevant to use.
The methods of the invention are particularly interesting for identification
of
proteins, which either in their full length or as immunogenic fragments
thereof
are suitable for inclusion in immunogenic compositions and/or diagnostic
purposes, especially such proteins, which comprises T-cell epitopes being
candidates for presentation as MHC-class I or II, and more preferable -class
I, restricted antigens suitable for inclusion in immunogenic compositions.
Accordingly, in another important aspect of the invention it relates to a
protein
identifiable by any of the claimed methods or an immunogenic fragment
thereof, and preferable such proteins and fragments, which are applicable for
inclusion in immunogenic compositions and/or diagnostic purposes.
The proteins of the invention may be proteins, which are secreted from C.
trachomatis and G. pneumoniae. Such proteins are e.g. those characterized
as DT1-77 as given in Table I as well as CP1-CP91 as given in Table II,
respectively, having the p1 and Mw values given in the Tables I and II,
2 o respectively, determined with an average error of +/- 10 %, and
immunogenic
fragments thereof.
TABLE I
Protein Pi Mw
spot
DT1 4.45 23.5
DT2 4.55 23.5
DT3 4.55 34.5
DT4 4.75 36.1
DT5 4.83 11.4
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Protein Pi Mw
spot
DT6 9.3 9.27
DT7 4.85-4.9 24.8
DT8 5.1 7.8
DT9 4.73 23.7
DT10 4.8 23.7
DT11 4.85 23.7
DT12 4.93 23.7
DT13 6.05 24.3
DT14 6.2 27.5
DT15 6.1 32.4
DT16 5.98 39
DT17 6.28 55.2
DT18 6.1 41.1
DT19 6.1 47.9
DT20 7.4 37.6
DT21 7.7 34.7
DT22 8.2 22.4
DT23 4.83 30.4
DT24 5 29.5
DT25 5 12.6
DT26 4.7 10.9
DT27 5.15 13.5
DT28 5.7 31.9
DT29 4.97 54.8
DT30 5.86 36
DT31 5.78 36.2
DT32 6.4 10.4
DT33 6.3 13.3
DT34 9.5 32.4
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Protein Pi Mv~r
spot
DT35 8 46.5
DT36 7.49 40.6
DT37 7.15 37.6
DT38 7.24 34.5
DT39 7.44 46.5
DT40 6.4 67.2
DT41 5.04 56.4
DT42 8.5 32.9
DT43 8.5 30.5
DT44 8.66 42.6
DT45 8.85 43.1
DT46 4.4 87.6
DT47 5.4 41
DT48 7.36 24.2
DT49 9.25 47.4
DT50 5.0 94
DT51 5.35 100.5
DT52 5.41 59.7
DT53 5.97 23
DT54 6.12 25.5
DT55 5.34 36.4
DT56 4.88 10.5
DT57 4.87 18.5
DT58 6.14 97
DT59 4.5 19.7
DT60 5.5 40.9
DT61 5.5 39.9
DT62 5.98 41.1
DT63 6.9 46.8
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Protein Pi Mw
spot
DT64 5.5 34.5
DT65 4.5 68.2
DT66 4.35 57.6
DT67 6.13 66.5
DT68 6 62.9
DT69 5.85 65.6
DT70 5.72 70.4
DT71 5.5 44
DT72 5.85 10.2
DT73 4.45 30, 5
DT74 5.02 48.2
DT75 4.37 21.9
DT76 5.14 23.3
DT77 5.64 23.0
List of potentially secreted proteins from C. trachomatis D present in whole
cell lysates at 24 h.p.i., but significantly reduced in EB and their estimated
5 pilMw, +l- 10 % average error,
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TABLE II
Name Pi MW
CP01 5.5 100.4
CP02 6.7 91.0
CP03 5.3 75.2
CP04 5.4 68.7
CP05 5.4 72.8
CP06 5.5 68.6
CP07 5.6 80.5
CP08 5.7 74.1
CP09 6.0 77.8
CP10 6.0 71.2
CP11 6.1 82.6
CP12 6.2 68.4
CP13 6.2 72.0
CP14 5.4 64.8
CP15 5.5 63.7
CP16 5.8 61.1
CP17 5.8 102.4
CP18 6.1 63.6
CP19 6.1 61.0
CP20 6.5 64.0
CP21 6.6 63.7
CP22 5.0 60.9
CP23 5.0 60.5
CP24 5.6 60.4
CP25 5.7 50.2
CP26 6.3 57.0
CP27 6.3 52.8
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Name Pi Mw
CP28 6.4 48.5
CP29 4.9 50.6
CP30 5.0 48.6
CP31 4.9 473
C P 32 4. 7 42.1
CP33 4.9 439
CP34 5.0 39.3
CP35 5.1 42.9
CP36 5.2 42,1
CP37 5.3 40.7
CP38 5.4 40.7
CP39 5.6 40.3
CP40 5.6 41.6
CP41 5.9 41.8
CP42 6.4 38.4
CP43 6.2 44.3
CP44 6.5 45.3
CP45 6,8 45.3
CP46 4.6 38.6
C P47 4.6 37. 8
CP48 5.1 35.8
C P49 5. 3 38. 9
CP50 5,5 38.9
CP51 5.6 33.6
CP52 5.7 33.7
CP53 5.9 34.9
CP54 6.2 34.8
CP55 6.2 34.7
CP56 6.3 34.8
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Name Pi Mw
CP57 8.3 36.0
CP58 8.7 36.1
CP59 4.5 29.7
CP60 4.8 26.0
CP61 5.2 27.6
CP62 5.4 30.6
CP63 6.2 25.2
CP64 6.6 26.3
CP65 5,9 22.8
CP66 4.7 24.2
CP67 4.8 22.4
C P68 5.1 24.1
CP69 5.2 24.3
CP70 5.3 22.3
CP71 5.6 21.4
CP72 6.9 17.8
CP73 4.8 12,0
CP74 5.0 8.9
CP75 5.1 11.9
CP76 6.5 9.3
CP77 7.0 10.5
CP78 7.2 10.4
CP79 8.7 13.0
CP80 5.7 93.3
CP81 6.4 37,2
CP82 6.9 45.0
CP83 7.0 41.6
CP84 7.1 38.6
CP85 6.3 32.2
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Name Pi Mw
CP86 6.4 32,0
CP87 8.8 31.3
CP88 5.0 23.8
CP89 4.7 73.0
CP90 7.4 40.1
CP91 7.8 37.7
List of potentially secreted proteins from C. pneumoniae present in whole cell
lysates at 55 h.p.i but significantly reduced in EB and their estimated pllMw,
+/- 10 % average error.
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More preferred proteins of the inventions are C. trachomatis proteins, such
as those identified by the corresponding gene number in the Chlamydia
Genome project as CT017 (gene name CT017), CT044 (gene name ssp),
CT243 (gene name IpxD), CT263 (gene name CT263), CT265 (gene name
5 accA), CT286 (gene name clpC), CT292 (gene name duty, CT407 (gene
name dksA), CT446 (gene name euo), CT460 (gene name SWIB), CT541
(gene name mip), CT610 (gene name CT610), CT650 (gene name recA),
CT655 (gene name kdsA), CT668 (gene name CT668), CT691 (gene name
CT691 ), CT734 (gene name CT734), CT783 (gene name CT783), CT858
10 (gene name CT858), CT875 (gene name CT875), or ORF5 (gene name
ORFS), or by the protein name DT8 as given in Table IIIA, and C.
pneumoniae proteins, such as those identified by the corresponding gene
number as CPN0152 (gene name CPN0152), CPN0702, CPN0705 (gene
name CPN0705), CPN0711 (gene name CPN0711 ), CPN0998 (gene name
15 ftsH), CPN0104 (gene name CPN0104), CPN0495 (gene name aspC),
CPN0684 (gene name parB), CPN0796 (gene name CPN0796), CPN0414
(gene name accA), CPN1016 (gene name CPN1016), CPN1040 (gene name
CPN1040), CPN0079 (gene name R110), CPN0534 (gene name dksA),
CPN0619 (gene name ndk), CPN0711 (gene name CPN0711 ), CPN0628
20 (gene name rs13), CPN0926 (gene name CPN0926), CPN1063 (gene name
tpiS), or CPN0302 (gene name IpxD) as given in Table IIIB, and
immunogenic fragments thereof.
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TABLE III A
Protein Gene Gene name Methods p1 Mw
spot number* of
nr identification
DT1 CT668 CT668 M, Q, I 4.45 23.5
DT2 CT668 CT668 M, Q, ! 4. 55 23.5
DT3 CT783 CT783 M, Q, I 4.55 34.5
DT4 CT858 CT858 M, Q, I 4. 75 36.1
DT48 CT858 CT858 M, I 7.36 24.2
DT7 CT610 CT610 M, E, I 4. 85-4. 24.8
9
DT9 CT610 CT610 I 4.73 23.7
DT10 CT610 CT610 I 4.8 23.7
DT11 CT610 CT610 I 4.85 23.7
DT12 CT610 CT610 I 4.93 23.7
DT8 None DT8* Q, I 5.1 7.8
DT6 CT460 SWIB M 9.3 9.27
DT14 ORFS ORFS M 6.2 27.5
DT22 CT446 euo Q 8.2 22.4
. DT23 CT541 m i p M 4. 83 30.4
DT24 CT541 mip M 5 29.5
DT25 CT407 dksA M,Q 5 12.6
DT26 CT734 CT734 Q, I 4.7 10.9
DT27 CT292 dut M,Q 5.15 13.5
DT28 CT655 kdsA M, E 5.7 31.9
DT30 CT265 accA M 5.86 36
DT35 CT017 CT017 M 8 46.5
DT39 CT017 CT017 M 7.44 46.5
DT36 CT243 IpxD M 7.49 40.6
DT37 CT650 recA M, E 7.15 37.6
DT57 CT044 ssp M 4.87 18.5
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Protein Gene Gene name Methods p1 Mw
spot number* of
nr identification
DT58 CT286 clpC M 6.14 97
DT69 CT875 CT875 M 5.85 65.6
DT76 CT691 CT691 Q 5.14 23.3
DT77 CT263 CT263 Q 5.64 23.0
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TABLE III B
Protein Gene numberGene name Methods p1 Mw
spot of
nr identification
CP34 CPN1016 CPN1016 I 5.0 39.3
CP37 CPN0998 ftsH M 5.3 40.7
CP42 CPN0104 CPN0104 M ~ 6.4 38.4
CP46 CPN0796 CPN0796 Q 4.6 38.6
CP47 CPN0705 CPN0705 M 4.6 37.8
CP50 CPN0495 aspC M 5.5 38.9
CP52 CPN0152 CPN0152 M 5.7 33.7
CP55 CPN0684 parB M 6.2 34.7
CP56 CPN0414 accA M 6.3 34.8
CP63 CPN1016 CPN1016 M 6.2 25.2
CP71 CPN1040 CPN1040 M 5.6 21.4
CP72 CPN0079 r110 M 4.8 12.0
CP73 CPN0534 dksA M 5.0 8.9
CP75 CPN0619 ndk M 5.1 11.9
CP76 CPN0711 CPN0711 M 6.5 9.3
CP78 CPN0628 rs13 M 7.2 10.4
CP79 CPN0926 CPN0926 M 8.7 13.0
CP88 CPN1063 tpiS M 5.0 23.8
CP91 CPN0302 IpxD M 7.8 37.7
List of examples of identified A: C. trachomatis D and B: C. pneumoniae
vaccine candidates. M: MALDI-MS, Q: ESI-Q-TOF MS, P: PSD- MALDI MS,
I: Western blotting.
*: DT8 represents an expressed protein encoded by a novel open reading
frame, which is not annotated in the C. trachomatis D genome.
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The proteins DT4, DT23, DT47, DT48, DT75, DT76, and DT 77 shown in
Figure 10, as well as the proteins CP34, CP37, CP46, CP47, CP52, CP63,
and CP75 shown in Figure 11 are of particular relevance.
Also preferred proteins of the invention are C. trachomatis proteins, which
are proteins that have a prolonged turnover time in the presence of
proteasome inhibitors, being characterised by having the p1 and Mw
characteristics of one of the proteins DT1, DT2, DT3, DTS, DT9, DT10,
DT11, DT13, DT14, DT17, DT47, DT59, DT60, DT61 or DT62 as given in
Table IV, determined with an average error of +l- 10%. Proteins from C.
1 o pneumoniae, which are regulated by proteasome inhibitors in the same way,
are also preferred embodiments of the invention.
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TABLE IV
Spot Gene p1 Mw AffectedAfFectedAffectedAffectedAffected
nr. name by by by by by B-
MG115 MG132 PSI epoximicinclasto
lactacystin
DT1 CT6684.45 23.5 + +
DT2 CT6684.55 23.5 + +
DT3 CT7834.55 34.5 + +
DT5 4.83 11.4 + +
DT9 CT6104.73 23.7 + +
DT10 CT6104.8 23.7 + +
DT11 CT6104.85 23.7 + +
DT13 6.05 24.3 + + +
DT14 ORF5 6.2 27.5 + +
DT17 6.28 55.2 + +
DT47 5.4 41 + + + +
DT59 4.5 19.7 + +
DT60 5.5 40.9 + + + +
DT61 5.5 39.9 + + + +
DT62 5.98 41.1 + + + +
List of examples of identified C. trachomatis D proteins, which turnover times
5 is prolonged by treatment with different proteasome inhibitors during
labelling
and chase.
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Another preferred protein of the invention is a Chlamydia trachomatis
polypeptide DTB, which comprises the sequence SEQ ID N0:1 as defined in
the claims and immunogenic fragments thereof.
In other preferred embodiments of this aspect of the invention the proteins
have at least 40 % sequence identity, preferable at least 60 %, more
preferable at least 70 %, even more preferable at least 80 %, further more
preferable 90 %, and most preferable at least 95% sequence identity to the
proteins or fragments thereof of the invention, or it comprises at least 7
1 o consecutive amino acids of the proteins of the invention.
In a further aspect of the invention it relates to a nucleic acid compound,
which comprises a sequence that encodes a protein or an immunogenic
fragment thereof according to the invention.
A preferred nucleic acid compound is one which comprises a sequence (SEQ
ID N0:2) that encodes a polypeptide DTB, which comprises the sequence
SEQ ID N0:1.
2 o In yet other aspects the invention relates to a vector comprising a
nucleic
acid compound of the invention, as well as a host cell transformed or
transfected with the vector.
The invention also provides the use of a protein or an immunogenic fragment
thereof of the invention for the production of antibodies against said
protein, a
method for producing an antibody against intracellular bacteria, wherein a
protein or an immunogenic fragment thereof of the invention are administered
to a producing animal, and the antibody is purified there from, as well as an
antibody obtainable by this method.
Further, the invention provides in other aspects a pharmaceutical or
diagnostic composition comprising the protein or fragment thereof of the
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invention, an antibody or a nucleic acid compound of the invention, as well as
the use of the protein or fragment thereof, the antibody or the nucleic. acid
compound in the preparation of a diagnostic reagent.
Identification of T-cell epitopes from vaccine candidates
The invention provides T-cell epitopes, that are likely to be surface
expressed
as MHC class I antigens and have a T-cell stimulating effect, as predicted by
computer based methods from the sequences of the proteins identified in the
present invention, or experimentally determined by assays as further
1 o described in the examples.
Accordingly, another important aspect of the invention is peptide epitopes
that are likely to be surface presented as MHC Class I antigens and have a
T-cell stimulating effect. In accordance with this the invention provides a
method for identification of T-cell epitopes on secreted proteins from
intracellular bacteria, comprising steps, such as computer prediction, MHC
class molecule binding assays andlor ELISPOT assays on a protein or an
immmunogenic fragment thereof identified by the methods of the invention,
as well as the peptide epitopes. As part of this aspect the invention also
2 o provides a nucleic acid compound, which comprises a sequence that
encodes said peptide epitope, as well as a vector comprising the nucleic acid
compound and a host cell transformed with said vector.
Preferred peptide epitopes of the invention comprises 4 to 25 consecutive
amino acids, preferably 6 to 15, and even more preferably 7 to 10 amino
acids of a protein of the invention.
In a more preferred embodiment of the invention the epitopes comprises 7 to
10 consecutive amino acids of a C. trachomatis or a C. pneumoniae protein.
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Another preferred peptide epitope of the invention is an epitope, which
comprises 4 to 25 consecutive amino acids of a polypeptide comprising the
sequence SEQ ID N0:1, more preferably 6 to 15 and most preferably 7 to 10
amino acids.
Chlamydia trachomatis peptide epitopes, which comprises an amino acid
sequence selected form the sequences SEQ ID N0. 3 - SEQ ID NO. 45,
Chlamydia pneumonia peptide epitopes, which comprises an amino acid
sequence selected from the sequences SEQ ID N0. 46 - SEQ ID N0. 121,
1 o Chlamydia pneumonia peptide epitopes, which comprises an amino acid
sequence, selected from the sequences of SEQ ID N0. 122 - SEQ.ID N0.
148, as well as and Chlamydia trachomatis peptide epitopes, which
comprises an amino acid sequence, selected from the sequences of SEQ ID
N0. 149 - SEQ.ID NO. 194 are of particular relevance. The identified
epitopes of the invention are further characterized in Tables V-VIII.
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TABLE V
Protein PositionPeptide sequenceA2 binding
ID
CT263 181 KLAEAIFPI 8
CT263 170 FLKNNKVKL 123
CT263 56 ALSPPPSGY 210
CT263 141 FIAKQASLV 210
CT263 17 TLSLFPFSL 286
CT263 147 SLVACPCSM 332
CT263 6 LIFADPAEA 386
CT263 4 LLLIFADPA 438
CT541 4 ILSWMLMFA 38
CT541 94 KQMAEVQKA 89
CT541 9 LMFAVALPI 122
CT541 135 KLQYRWKE 221
CT541 118 FLKENKEKA 222
CT541 46 KLSRTFGHL 239
CT541 223 SLLIFEVKL 265
CT541 148 VLSGKPTAL 352
CT541 204 VLYIHPDLA 398
CT541 54 LLSRQLSRT 472
CT691 172 LLQRELMKV 9
CT691 25 STINVLFPL 66
CT691 15 PLQAHLELV 114
CT691 6 SLFGQSPFA 194
CT691 212 KLAYRVSMT 251
CT691 194 VLWMQIIKG 284
CT691 29 VLFPLFSAL 298
CT691 122 FLQKTVQSF 468
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CT691 8 FGQSPFAPL 480
CT858 85 VLADFIGGL 33
CT858 177 RMASLGHKV 52
CT858 92 GLNDFHAGV 90
CT858 490 FSCADFFPV 90
CT858 379 MLTDRPLEL 101
CT858 408 LLENVDTNV 121
CT858 391 RMILTQDEV 132
CT858 491 SCADFFPVV 132
CT858 519 FVFNVQFPN 132
CT858 372 YLYALLSML 247
CT858 539 SLAVREHGA 288
CT858 109 YLPYTVQKS 350
CT858 219 ATIAPSIRA 358
CT858 140 LLEVDGAPV 375
CT858 512 RTAGAGGFV 384
CT858 250 SLFYSPMVP 431
Predicted epitopes from identified C. trachomatis proteins
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TABLE VI
Protein PositionPeptide sequenceA2 binding
ID
CPN0152 6 FLVSCLFSV 18
CPN0152 135 YLRDAQTIL 28
CPN0152 237 LLIRIQDHV 48
CPN0152 100 KLGRKFAAV 51
CPN0152 266 LVSRTQQTL 164
CPN0152 10 CLFSVAIGA 190
CPN0152 222 GFGPPPIIV 354
CPN0152 249 SLPTKPYIL 387
CPN0152 240 RIQDHVTAN 408
CPN0152 15 AIGASAAPV 410
CPN0152 156 RLGISGFSL 444
CPN0619 64 FMVSGPVW 31
CPN0619 73 LVLEGANAV 398
CPN0705 164 FVGANLTLV 24
CPN0705 89 CLAENAFAG 114
CPN0705 233 KIEEVQTPL 116
CPN0705 211 ALKGHQLTL 178
CPN0705 190 QMAEAADLV 358
CPN0796 583 FMGAHVFAS 15
CPN0796 419 LLIQHSAKV 31
CPN0796 372 FLCPFQAPS 39
CPN0796 376 FQAPSPAPV 50
CPN0796 211 AMNACVNGI 86
CPN0796 548 FMGIQVLHL 112
CPN0796 74 RHAAQATGV 134
CPN0796 328 FQYADGQMV 148
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CPN0796 618 SVSAMGNFV 212
CPN0796 460 FLSYRSQVH 214
CPN0796 53 FLLTAIPGS 218
CPN0796 38 VLTPWIYRK 219
CPN0796 656 SWMNQQPL 221
CPN0796 408 SLKNSQQQL 279
CPN0796 162 MLPDTLDSV 284
CPN0796 511 ALPYTEQGL 295
CPN0796 523 VLSGFGGQV 399
CPN0998 22 LLFGWFGV 8
CPN0998 174 SLQERYPTL 29
CPN0998 416 MLLKGQNKV 33
CPN0998 379 FTFLPIILV 53
CPN0998 754 FLGDISSGA 56
CPN0998 36 FLAGKKARV 66
CPN0998 824 LLDAAYQRA 66
CPN0998 374 YLGYLFTFL 78
CPN0998 377 YLFTFLPII 109
CPN0998 717 SLGATHFLP 124
CPN0998 96 ELIDQGHRL 134
CPN0998 381 FLPIILVLL 197
CPN0998 386 LVLLFVYLV 219
CPN0998 161 VTGPATPQL 223
CPN0998 319 SLEKQDPEV 224
CPN0998 567 ILMAATNRP 236
CPN0998 230 LTQETDTEA 237
CPN0998 823 MLLDAAYQR 238
CPN0998 639 LLNEAALLA 254
CPN0998 736 ELYDQLAVL 256
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CPN0998 199 LIGKYLSPV 294
CPN0998 454 SLGGRIPKG 303
CPN0998 781 GMSPQLGNV 306
CPN0998 645 LLAARKDRT 315
CPN0998 424 VTFADVAGI 427
CPN0998 154 VLTEPLWT 439
CPN0998 66 KIALNDNLV 470
CPN1016 5 KLGAIVFGL 7
CPN1016 135 YLGDEILEV 34
CPN1016 284 FLPTFGPIL 99
CPN1016 439 SLQNFSQSV 108
CPN1016 414 FTDEQAVAV 145
CPN1016 92 SLNDYHAGI 164
CPN1016 392 RMIFTQDEV 175
CPN1016 64 TQQARLQLV 294
CPN1016 217 SLVAPLIPE 312
CPN1016 255 YMVPYFWEE 358
CPN1016 576 YVEAVKTIV 389
CPN1016 395 FTQDEVSSA 444
CPN1016 516 GAGGFVFQV 491
CPN1016 464 LLGFAQVRP ~ 498
~ ~
Predicted epitopes from identified C. pneumoniae proteins
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TABLE VII
Protein ID PositionPeptide sequenceA2 binding
CPN0412 (CT263)186 RLEEVSQKL 80
CPN0412 (CT263)103 LTTDTPPVL 103
CPN0412 (CT263)147 KLLDMEGYA 167
CPN0412 (CT263)110 VLSEDPPYI 183
CPN0412 (CT263)62 ALQSYCQAY 215
CPN0412 (CT263)193 KLTQTLVEL 248
CPN0412 (CT263)81 FVGACSPEI 267
CPN0412 (CT263)102 NLTTDTPPV 286
CPN0412 (CT263)205 LMERAIPPK 410
CPN0661 (CT541103 KMAEVQKLV 46
)
CPN0661 (CT541199 ALGMQGMKE 221
)
CPN0661 (CT54154 KLSRTFGHL 239
)
CPN0661 (CT541232 LLIFEINLI 334
)
CPN0661 (CT5418 VLATVALAL 391
)
CPN0661 (CT541187 ILLPLGQTI 396
)
CPN0661 (CT541212 VLYIHPDLA 398
)
CPN0661 (CT5417 LVLATVALA 413
)
CPN0681 (CT69129 YMLPIFTAL 40
)
CPN0681 (CT691136 LLHEFNQLL 66
)
CPN0681 (CT691172 VLQRELMQI 91
)
CPN0681 (CT69115 PLQAHLEMV 169
)
CPN0681 (CT6916 RLFGQSPFA 197
)
CPN0681 (CT69173 GLFMPISRA 223
)
CPN0681 (CT691212 KLAHRINMT 229
)
CPN0681 (CT691194 YLWLQVIRR 322
)
CPN0681 (CT691135 TLLHEFNQL 474
)
CPN0681 (CT6918 FGQSPFAPL 480
)
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Predicted epitopes from C, pneumoniae homologs to identified C.
trachomatis proteins
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TABLE VIII
Protein ID PositionPeptide sequenceA2 binding
CT149 (CPN0152)274 FLGAAPAQM 17
CT149 (CPN0152)237 FLGIQDHIL 29
CT149 (CPN0152)101 LLTANGIAV 31
CT149 (CPN0152)248 SLPRRIPVL 86
CT149 (CPN0152)42 GLQEHCRGV 107
CT149 (CPN0152)160 SLGCHTTIH 170
CT149 (CPN0152)307 ILTHFQSNL 181
CT149 (CPN0152)52 VLSCGYNLV 202
CT149 (CPN0152)195 LLKEICATI 248
CT149 (CPN0152)272 RLFLGAAPA 318
CT149 (CPN0152)141 ATVAKYPEV 338
CT149 (CPN0152)11 LLSGSGFAA 343
CT149 (CPN0152)102 LTANGIAVA 373
CT149 (CPN0152)15 SGFAAPVEV 397
CT500 (CPN0619)64 FMISGPVW 20
CT500 (CPN0619)103 ALFGESIGV 121
CT500 (CPN0619)119 SLENAAIEV 212
CT500 (CPN0619)87 LMGATNPKE 313
CT500 (CPN0619)31 RIAAMKMVH 385
CT671 (CPN0705)102 ALVETPMAV 13
CT671 (CPN0705)167 FCGANLTLV 49
CT671 (CPN0705)214 SLKARQLNL 151
CT671 (CPN0705)193 QLTEATQLV 239
CT671 (CPN0705)127 DLQWVEQLV 403
CT671 (CPN0705)155 IVLDNSNTV 423
CT841 (CPN0998)22 LLFGVIFGV 9
CT841 (CPN0998)415 LLAKGQNKV 14
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CT841 (CPN0998)378 FTFMPIILV 29
CT841 (CPN0998)753 FLGDVSSGA 43
CT841 (CPN0998)824 LLDAAYQRA 66
CT841 (CPN0998)780 GMSDHLGTV 110
CT841 (CPN0998)716 SLGATHFLP 124
CT841 (CPN0998)170 NLAALENRV 153
CT841 (CPN0998)376 YLFTFMPII 160
CT841 (CPN0998)15 FPTAFFFLL 167
CT841 (CPN0998)566 ILMAATNRP 236
CT841 (CPN0998)66 KTALNDNLV 244
CT841 (CPN0998)638 LLNEAALLA 254
CT841 (CPN0998)735 ELYDQLAVL 256
CT841 (CPN0998)318 ALEKQDPEV 264
CT841 (CPN0998)453 SLGGRIPKG 303
CT841 (CPN0998)380 FMPIILVLL 314
CT841 (CPN0998)644 LLAARKDRT 315
CT841 (CPN0998)423 VTFADVAGI 427
CT841 (CPN0998)142 YTISPRTDV 467
CT841 (CPN0998)464 LIGAPGTGK 495
Predicted epitopes from C. trachomatis homologs to identified C.
pneumoniae proteins
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The peptide epitope may be part of a fusion protein or coupled to a carrier
moiety.
A frequently used method to predict peptide binding to MHC involves motif
searches. The most elaborate motif search uses entire matrices representing
the extended motif of the MHC. Although the sequence independent
combinatorial specificity may be correct as an average consideration, it is
certainly known to be wrong for individual peptides. Furthermore, crystal
structures have demonstrated that the interactions at one sub-site may affect
1 o interactions at other sub-sites.
Artificial neural networks (ANN) are particularly well suited to handle and
recognize any such non-linear sequence information. Information can be
trained and distributed into a computer network with input layers, hidden
layers and output layer all connected in a certain structure through weighted
connections. Such ANN can be trained to recognize inputs (peptides)
associated with a given output (say MHC binding). Once trained, the network
should recognize the complicated peptide patterns compatible with binding.
Using the ANN approach, the size and quality of the training set becomes of
2 o major importance. This is particularly true for the HLA since only about 1
% of
a random set of peptides will bind to any given HLA.
Thus, to generate as few as 100 examples of peptide binders would, if
random peptides were screened, require the synthesis and testing of about
10,000 peptides. This would be a very resource demanding and laborious
proposition even at this modest number of binders in the training set - and
this has to be repeated for every HLA to be tested.
Accordingly, in connection with the present invention matrix predictions were
3o used to scan the SWISS-PROT (http://www.expasy.ch/sprot/) database for
potentially high affinity binding epitopes. A large number of these have been
synthesized and tested in biochemical binding assays. As predicted, a much
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higher representation of high affinity binders was obtained (about 80%).
These data were subsequently used to train ANN.
For four out of four MHC class I molecules examined, the ANN performed
better than the matrix-driven prediction. The predictions have been generated
in a fashion, which predicts the actual binding IC50 value rather than an
arbitrary classification into "binders" vs. "non-binders". Indeed, it has been
possible to predict binders over a large range leading to the identification
of
high affinity binders as well as binders of lower affinity as well as non-
1 o binders.
The invention further comprises the use of a peptide epitope of the invention
for the preparation of a vaccine, as well as a vaccine comprising a peptide
epitope of the invention, which vaccine optionally contains acceptable
excipients.
In yet another aspect of the invention it relates to the use of a protein of
the
invention, an antibody of the invention, a nucleic acid compound of the
invention or a peptide epitope of the invention in the preparation of a
2 o pharmaceutical composition for treating or preventing infection due to an
intracellular bacteria, such as a Chlamydia infection, or, alternatively, in
the
preparation of a diagnostic reagent for detecting the presence of an
intracellular bacteria, such as Chlamydia, or antibodies raised against the
intracellular bacteria.
The invention further provides a method of inducing an immune response in
a human, which comprises administering to said human an immunological
effective amount of a protein, an antibody, a nucleic acid compound or a
peptide epitope of the invention, and especially such methods for treating or
3 o preventing infection of humans by an intracellular bacteria, such as C.
pneumoniae or C. trachomatis.
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Finally, the invention provides methods of producing a protein or a fragment
thereof of the invention or a peptide epitope of the invention, respectively,
which comprises transforming, transfecting of infecting a host cell with a
vector comprising a nucleic acid compound that encodes said protein or
5 peptide epitope, and culturing the host cell under conditions, which permit
the
expression of said protein or fragment by the host cell.
The invention is further illustrated by the following, non-limiting examples.
Examples
Example 1:
10 Infection of mammalian cell cultures
Semi-confluent HeLa, HEp-2 or McCoy (ATCC, Rockville, MD, USA) cell
monolayers were infected with one inclusion forming unit (IFU) of C.
pneumoniae VR1310, C. trachomatis serovar A (HAR-13), D (UW-3/Cx) or
L2.(434/Bu)(ATCC) as previously described in [19.] and [17.] The infection
15 medium consisted of RPMI 1640, 25 mM HEPES, 10% FCS. 1 % wlv
glutamine, 10 mg/ml gentamycin for C. trachomatis A and D and RPMI 1640,
25 mM HEPES, 5% FCS. 1 % w/ v glutamine, 10 mg/ml gentamycin for C.
trachomatis L2.
Example 2:
2o Pulse labelling/chase
To label chlamydial proteins for two hour periods, infected cell cultures were
incubated in a medium containing RPMI 1640, 10 mg/ml gentamycin, 40
~.glml cycloheximide, 100 ~.Ci/ml [35S]-methionine/cysteine (Promix,
Amersham Pharmacia Biotech, Uppsala, Sweden) as described previously
25 (Shaw et al., 1999,2000) [18.] [19.]. After labelling the labelling medium
was
changed to normal growth medium following two washes in normal growth
medium and the infected cells were harvested at different points in time after
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labelling. Similarly, labelled EB proteins were obtained by allowing the
Chlamydia to grow until 72 h.p.i. after the two hour labelling periods. The
labelled EB were then harvested and purified using two consecutive steps of
density gradient ultracentrifugation essentially as described for C.
trachomatis in (Schacter and Wyrick, 1994) [22.]and for C. pneumoniae
(Knudsen et al. 1999 [17.]). Proteins in the EB preparation and pulse chase
preparation were labelled at the same intervals as proteins from the whole
cell lysate preparation to facilitate correct 2D-PAGE protein profile
comparison.
Example 3:
Sample preparation
Following [35S]-labelling cells were washed twice in PBS and solubilised
in a standard lysis buffer containing 9 M Urea, 4% w/v 3-[(3-
cholamidopropyl)dimethylammonium]-1-propanesulfonate (CHAPS;
Roche, Germany), 40 mM Tris Base, 65 mM DTE and Pharmalyte 3-10
(Amersham Pharmacia Biotech). For the enrichment of high molecular
weight and hydrophobic proteins 7 M urea, 2 M thiourea, 4% w/v 3-[(3-
cholamidopropyl)dimethylammonium]-1-propanesulfonate (CHAPS;
Boehringer Mannheim, Germany), 40 mM Tris Base,65 mM dithioeryth-
retiol (DTE) and 2% vol/vol Pharmalyte 3-10 (Amersham Pharmacia
Biotech) was used essentially according to (Harder et al. 1999 [23.]).
Samples containing whole cell lysates or purified EB were sonicated and
centrifuged at 10 000 ?C g for 10 min. Samples were stored at -70°
until
used.
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Example 4:
Separation of Chlamydia proteins
Chlamydia proteins from whole cell lysates and purified EB were separated
by two-dimensional gel electrophoresis essentially as described in (Shaw et
al., 1999,2000) [18.] [19.].
For isoelectric focusing in the first dimension, 18 cm long pH 3-10 NL (non-
linear), 4-7 L (linear) or 6-11 (lfiniar) immobilized pH-gradient drystrips
(Amersham Pharmacia Biotech) were reswelled with a sample amount of
200.000 counts per minute (cpm) labelled protein in 350 ~I of lysis buffer for
12 hours at 20 °C using the IPGphorTM. Other strips used in the
invention
include ultranarrow IPG strips. The strips are described in Table IX. These
allows us to focus on specific pH intervals containing proteins of interest,
if
necessary. The voltage during isoelectric focusing at 20° G was
programmed
as follows: 1 h at 300 V, 2 hours at 300-500 V (linear increase), 1 h at 1000
V, 1 h at 2000 V, 3 h at 3500 V and 5000 V at 24 h when using 3-10 NL, 4-7
L and 6-11 L drystrips.
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TABLE IX:
IPG strip name LiniarityCovered Strip length
pH
interval
Immobiline Drystrip pH non-lfiniar3-10 18 cm
3-10
Immobiline Drystrip pH lfiniar3-10 18 cm
3-10
Immobiline Drystrip pH lfiniar4-7 18 cm
4-7
Immobiline Drystrip pH lfiniar6-11 18 cm
6-11
Immobiline Drystrip pH lfiniar6-9 18 cm
6-9
Immobiline Drystrip pH lfiniar3.5 -4.5 18 cm
3.5 -4.5
Immobiline Drystrip pH lfiniar4-5 18 cm
4-5
Immobiline Drystrip pH lfiniar4.5-5.5 18 cm
4.5-5.5
Immobiline Drystrip pH lfiniar5-6 18 cm
5-6
Immobiline Drystrip pH lfiniar5.5 -6.7 18 cm
5.5 -6.7
List of examples of commercial available IPG drystrips usefull in the
invention
After the first dimension the drystrips were equilibrated in a buffer
containing
6 M Urea, 30% v/v glycerol, 2% w/v DTE, 2% wlv SDS, 0.05 M Tris-HCI pH
6.8 for 15 min. The strips were then equilibrated for additionally 15 min. in
a
buffer in which DTE was replaced by 2.5% wlv iodacetamide. For the second
1 o dimension, the Protean I I xi Multicell system (Bio-Rad, Richmond, CA,
USA)
was used to separate proteins on 9- 16% linear gradient SDS-PAGE gels(18
cm X 20 cm X 1 mm). Analytical gels were fixed in a solution containing 10%
acetic acid and 25% 2-Propanol for 30 min, and treated with Amplify (Amer-
sham Pharmacia Biotech) for 30 min. Labelled proteins were visualized by
autoradiography after 8- 10 days exposure of Kodak Biomax-MR film
(Amersham Pharmacia Biotech) at -70°C.
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Figure 1 A shows an example of a autoradiography of a high resolution
analytical 3-10 IPG 2D-gel (standard lysis buffer was used) of C. trachomatis
D proteins labelled from 22-24 h.p.i. Figure 2A shows an example on a
autoradiography of a 3-10 IPG 2D-gel (thiourea containing lysis buffer was
used) of C. Pneumoniae proteins labelled from 55-57 h.p.i. A total of
approximately 600 protein spots could be visualized on each gel as
estimated by means of the Melanie II software.
To prepare samples for analysis by mass spectrometry, 2D gels were run
1 o with 500-1000 p,g of whole cell lysates. To visualize proteins on
preparative
gels on X-ray films 2 X 106 cpm of [35S]-protein labelled protein from cells
grown in parallel with the unlabelled samples was run on the same gels.
Preparative gels were washed for 10 min. in ddH20 and dried un-fixed.
Radioactive ink was used to mark anchor spots on the sides of the gels, so
that an exact matching of the dried gel and the corresponding X-ray-film
could be performed after exposure. Proteins of interest were excised and
pooled together from minimum three identical gels. Gels using narrow or
ultra-narrow drystrips (Table IX) were used to increase the separation
distance, if host cell contamination was a problem in the mass spectrometric
2 o identification.
Example 5:
Identification of vaccine candidates usingi MALDI MS, ESI-Q-TOF MS, PSD
MALDI MS and Edman degradation
Protein spots from preparative gels of whole cell lysates representing vaccine
candidates were subjected to in-gel digestion with trypsin. The resulting
peptides were purified using reverse phased columns (Gobom et al, 1999
[20.])or beads (Gevaert et al., 1997 [21.]) consisting of Poros R2 material.
The samples were subsequently analyzed using a Bruker REFLEX MALDI
time of flight mass spectrometer (Bruker-Daltonik, GmbH, Bremen, Germany)
operating in reflection mode. The resulting masses were compared to
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peptide masses generated by a theoretical tryptic cleavage of proteins
present in databases by peptide mapping as described previously (Schev-
chenko 1996 [24.]).
5 An example of an identification of DT1 as CT668 using MALDI-MS is shown
in Figure 3. Figure 3A shows the peptide mass fingerprint obtained by an
MALDI mass spectrometer. Obtained masses were matched to theoretical
tryptic cleavage products of all proteins present in databases using the
Prospector software MS-Fit. If the search was restricted to a pl/Mw area in
1 o proximity to the protein found on the gels the highest-ranking protein was
CT668 (Figure 3B). However, as proteins from the host cell are sometimes
present in the spots from gels with whole cell lysates identification may be
unambiguous. Therefore, tandem mass spectrometry and post source decay
(PSD) analysis was used to verify the results, if necessary (Reviewed in
15 Mann and Wilm,1995 [25.], Gevaert, 1997 [21.]).
Tandem mass spectrometry of peptides generated by in-gel digestion was
performed on an Electrospray Ionization Quadrupole Time-Of-Flight (ESI-Q-
TOF) mass spectrometer (Micromass, Manchester, UK). Using this method a
single ionized peptide can be isolated from the sample. Through
2 o fragmentation of this parent ion by collision with a gaseous atmosphere
several new ions were generated and recorded in a new peptide mass
fingerprint. These new ions were distinguished in size by only one amino
acid, thus providing details of the amino acid sequence of the original
peptide
(Mann and Wilm, 1995) [25.].
25 Figure 4A shows an example of a fragmented peptide parent ion from DT1,
yielding a sequence, which through database search using BLAST or MS-
Tag were found to correspond to fragments of CT668. Sequence tags arising
from the human progesterone binding protein were also identified from the
CT668 sample.
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Peptides from this protein could also be detected in the MALDI MS peptide
mass fingerprint (double headed arrows, Fig 3A) if the search Was restricted
to human proteins. This shows that problematic spots, which contain more
than one protein, can be unambiguously identified by ESI-Q-TOF, thus
conforming problematic MALDI MS identification.
Another approach used in the invention to confirm MALDI results was PSD.
PSD utilizes that peptides undergo metastable decay after ionization
meaning that peptide fragments of the same velocity have different mass and
therefore possess different kinetic energy. The differences in kinetic energy
1 o can be resolved by reflecting the fragments in a magnetic field. High
energy
fragments will penetrate further into the magnetic field than low energy
fragments and thereby be delayed. The spectra resulting from fractionation of
a single peptide can be used to deduce the amino acid sequence of a
peptide sequence tag (PST)(Mann et al, 1993) [28.] as fragmentation
predominantly occurs at the peptide bonds. PSTs can be matched against
protein databases and thereby the protein from which they originate can be
identified (Wilkins et al, 1996 [27.]).
An example of the identification of spot no. CP63 as CPN1016 through PSD
MALDI MS is shown in Fig 4B.From a total of 36 observed masses in the
2 o PSD spectra 16 could be matched within one mass unit to masses originating
from a theoretical fragmentation of the peptide with the sequence of the
1919.80 Da parent ion (R)ELLFGWDLSQQTQQAR(L), which matched the
CPN1016 protein from C. pneumoniae.
Using mass spectrometric approaches, examples of the identification of C.
trachomatis D (Table IIIA) and C. pneumoniae (fable Illb) vaccine
candidates are provided. Mw and Pi values were determined
electrophoretically with an average error of +/- 10%.
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CT858 had a theoretical Mw of 67 kDa, thus indicating that the protein
identified was a processed fragment of a~ larger protein. Indeed all three
peptides from ESI-Q-TOF analysis of DT4 was located in the C-terminal part
of the protein.
Another spot, DT48, located in the basic region of the gel also contained
CT858. All the peptides identifying DT48 as CT858 was matching the N-
terminal part of the protein suggesting that DT48 represents the N-terminal
fragment of CT858.
CT610 was identified from EBs by both MALDI MS and Edman degradation
in both C. trachomatis D and L2. However, the protein was significantly
reduced in EB compared to whole cell lysates and was therefore still
considered a candidate for vaccine. The identification of CT610 by Edman
degradation Was done from C. trachomatis L2 CT610. The N-terminal was
determined to be MNFLDQ, which is different from the MMEVFMNFLDQ
sequence predicted from the Chlamydia Genome Project (Stephens et al
1998b)[35. ].
DT8 was identified based on four sequence tags generated by ESI TOF MS.
These sequences did not correspond to any predicted open reading frame in
the C. trachomatis D genome [35.]. However, by searching the Chlamydia
genome in all 6 reading frames with BLAST significant matches could be
generated for all four sequence tags. Analysis of the DNA sequence
encoding the peptides and their surroundings elucidated a novel open
reading frame including a ribosomal binding site, which comprised a 7.2 kDa
protein. The translated DNA sequence of DT8 is shown in Figure 5. This
finding illustrates how mass spectrometric approaches used in this invention
can identify potentially important ORFs encoding vaccine candidates, which
3o may be neglected in large genome sequencing projects.
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Spot CP63 from C. pneumoniae was identified as an N-terminal fragment of
CPN1016 the C. pneumoniae homologue of CT858 (Figure 2 B and C, Table
IIIB) indicating processing of these proteins in both Chlamydia species.
In the following examples more detailed investigations of the properties of
the
examples on identified proteins will be presented.
Example 6
1 o Comlcarison of whole Iysates of infected cells to purified RB
C, trachomatis or C, pneumoniae infected cells were labelled with [35S]-
methionine/cysteine for a two-hour period in the presence of cycloheximide
as described in Example 2. At the end of the labelling period the infected
cells were either harvested directly in lysis buffer as described in Example 3
or used for immediate purification of chlamydia RB. The purification of RB
was performed by density gradient ultracentrifugation essentially as
described by Schachter and Wyrick, 1994 [22.].
In Figure 10, examples of regions from gel images of C. trachomatis D
2 o proteins from whole lysates of infected HeLa cells labelled from 22-24
h.p.i.
are compared to corresponding regions from gel images of RB and EB
purified from C. trachomatis D infected HeLa cells labelled from 22-24 h.p.i.
Identification by mass spectrometry was obtained for DT4 (C-terminal
fragment of CT858), DT48 (N-terminal fragment of CT858), DT23 (Mip),
DT76 (hypothetical protein CT691 ) and DT77 (hypothetical protein CT263) as
listed in Table III.
In Figure 11, examples of regions from gel images of C. pneumoniae infected
HEp-2 cells labelled at 55-57 h.p.i. and from purified EB labelled at points
in
3 o time throughout the developmental cycle are compared to corresponding
regions from images of C. pneumoniae infected cell cultures labelled at 34-36
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hpi and either harvested as whole lysates of infected cells at 36 h.p.i. or
purified as RB at 36 h.p.i.
The protein spots encircled in Figure 11 were identified as follows. Figure
11A and E show CP34 and CP63, which have been identified as two
fragments of CPN1016. Figure 11 B shows CP37, which has been identified
as CPN0998. Figure 11 C shows CP46 and CP47, which have been identified
as CPN0796 and CPN0705, respectively. Figure 11 D shows CP52 which has
been identified as CPN0152. Figure 11 F shows CP75 which has been
1 o identified as CPN0619.
Example 7:
Detection and identification of proteins located in type III secretion gene
subclusters
Whereas the type III secretion genes in most intracellular bacteria are
located
in one gene cluster as an pathogenesis island, the Chlamydia type III
secretion genes have been identified in three different subclusters located
different places in the genome in both C. trachomatis and C. pneumoniae
(Stephens et al., 1998 [4.]and Kalman et al. 1999 [5.]). As part of a global
proteomic analysis of C. trachomatis A,D and L2 and C. pneumoniae
2 o VR1310, proteins which were present in the Type III secretion clusters
were
identified from gels run with purified EB.
Identified type III secretion proteins from C. trachomatis include the Yop
secretion ATPase (yscN), the Yop translocator proteins L (YscL) and the
2 5 secretion chaperone(SycE) necessary for the transport of proteins from the
bacterial cytoplasm to the secretion machinery. Additionally identified C.
trachomatis D proteins, which have unknown functions but are located in type
III secretion subclusters, include CT560 and the abundant CT577 and
CT579. CT668, is clearly present in whole cell lysates, but absent from
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purified EB, and due to its localization next to YscN, this protein may be
secreted. The genomic location of the proteins is shown in Figure 9.
For C. pneumoniae the type III secretion apparatus proteins LcrE
5 (CPN0324), YscC (CPN0702), YscN (CPN0707) and YscL (CPN0826) have
been identified. YscC (CP89 Figure II, Table II) is absent from purified EB,
probably due to localization in the inclusion membrane, where it is exposed
to the host cell cytoplasm. Two proteins present in a type III cluster located
around YscC (CPN0702) and YscN (CPN0707) were detected in
1 o considerably higher amount in whole cell lysates than in purified EB.
These
were CPN0705 (CP90, Figure II, Table II) and CPN0711 (CP76, Figure II,
Table 1l). These proteins may also be located in the inclusion membrane or
present in the host cell cytoplasm and in both cases these proteins may be
accessible for the proteasome.
15 Example 8:
Pulse chase studies of candidates for secreted proteins
In order to estimate the time in which the identified candidate proteins could
be present inside infected cells, the invention provides a series of pulse
chase studies. In the following examples infected cell cultures were [35S]-
20 labelled from 22-24 hours. After the labelling period the medium was
changed to normal RPMI growth medium without [35S]-methionine and cells
were harvested at different times after labelling. Results from several
independent studies showed minor variation, probably due to small
differences in the host cell density and/or the efficiency of infection.
Figure 1 B,C,D and E provides an example of a pulse/chase experiment. The
intensities of CT668 and DT8 (Figure 1: A, B, respectively) were significantly
decreased 1.5 hours after synthesis and virtually absent from the gels at 4.5
hours and until the EB stage. CT610 and CT783 (Fig 1: C, D) decreased
3o significantly, but was still detectable at 4.5 hours and until the EB
stage. The
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C-terminal fragment of CT858 (D) (and the N-terminal fragment of CT858)
was gradually increased during the first chase periods, but absent from EB,
suggesting that the cleavage product accumulated during the Chlamydial
development. The N-terminal fragment of the C. pneumoniae homologue of
CT858, CPN1016 was also absent from EB (Figure 2)
Example 9:
Pulse chase studies in combination with proteasome inhibitors
This example shows how to determine which of the vaccine candidates that
are processed in the proteasome. Cell permeable proteasome inhibitors are
1 o added to the infected cell cultures during the labelling and chase period
and
the turnover time of proteins compared to that observed for labelling/chase
without proteasome inhibitors added. An example of the importance of this
approach is shown in Fig 6. Proteins were labelled in the presence or
absence of 10-100 ~,M of the proteasome inhibitor MG-132 from 22-24 h.p.i.
The labelling medium was then replaced with growth medium with or without
MG132 after two washes in normal growth medium and chased from 24 h.p.i.
to 28 h.p.i. Cell lysates were run on 2D-PAGE(IPG) and compared to controls
without MG-132. and to gels with proteins harvested immediately after
labelling (Fig 6A, B and C). The invention provides examples of fifteen C.
2 o trachomatis D proteins, which had an increased turnover time due to
treatment with proteasome inhibitors.
The levels of DT9, DT10 and DT11 were actually higher in chased+MG132
gels than in controls harvested immediately after the labelling period. This
indicates, that DT9, DT10 and DT11 are very rapidly degraded by the
proteasome. In contrast the levels of DT7 were not significantly affected by
the addition of proteasome inhibitors.
The invention includes the use of several other proteasome inhibitors (e.g.
3o MG115, MG262, PSI and lactocystein), which inhibit different parts of the
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catalytic activity of the proteasome, which may elucidate other proteins that
are degraded in the proteasome (Table IV).
Example 10:
Using aenetically altered cell lines to assay the effect of proteasome
inhibitor
on the turnover time of Chlamydia proteins.
The invention also provides the use of the commercially available mouse
1o embryonal cell lines MEC-PA28 cell line PW8875. MEC-PA28 is transfected
with the IFN-y inducible PA28 alpha and beta subunit of the proteaosome
and MEC217 cell line is transfected with IFN-y inducible LMP2, LMP7 and
MECL of the proteasome. MEC-PA28 and MEC217 is grown to semi
confluence and infected with C. trachomatis or C. pneumoniae. Control
mouse embryonal cell f fines, which is not transfected with the proteoasome
subunits, were infected in parallel.
As the transfected genes encoding the overexpressed subunits from the cell
lines MEC-PA28 and MEC217 are essential for the processing and
2 o presentation of MHC class I antigens the experimental procedures using
proteasome inhibitors combined with pulse labelling/chase is performed with
these host cell as mentioned previously.
Example 11:
Cloning and expression of open reading frames (ORFs) encoding vaccine
candidates
Cloning and expression of the ORFs encoding vaccine candidates was done
using the pET-30 LIC Vector Kit (Novagen, Madison, USA) in accordance
with the instructions of the manufacturer.
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The primers used for PCR of the genes had the following 5' end and 3'-end
LIC overhangs:
Forward primer: 5' GACGACGACAAGATX- gene specific sequence 3'
Reverse primer: 5' GAGGAGAAGCCCGGT-gene specific sequence 3'.
(X: the first nucleotide of the insert specific sequence)
Either full-length genes or genes without the leader sequence were amplified
by PCR using the ExpandTM High Fidelity PCR (Roche, Germany). Thirty-five
1 o PCR-cycles were performed on a DNA Thermal Cycler (GeneAmp PCR
system 9600, Perkin Elmer) as follows: 15 s at 92°C (denaturation), 15
s at
55°C (primer annealing) and 4 min. at 68°C (extension). The
resulting PCR
products included the LIC-overhang proximal to the gene specific sequence.
The PCR products were Wizard (Promega, Madison, USA) purified and
ligated into the pET-30 vector. The pET-30 vector contains the gene
encoding kanamycine resistance and a Histidine tag upstream of the LIC
cloning site.
pET-30 vectors containing the candidate genes were transformed into
2 o competent E. coli Nova Blue strain. Colonies were selected on kanamycin
agar plates, and control PCR on selected colonies was performed using a
vector specific primer and an insert specific primer. Plasmid DNA was
purified from positive colonies containing the candidate gene specific insert.
The plasmid DNA was subsequently transformed into E. coli(BL21 ). Insert
positive colonies were selected on kanamycin agar plates. Expression of the
fusion protein comprising the gene specific insert including an N-terminal
located histidine tag, was induced in 500 ml LB-medium by addition of 1 mM
IPTG (Apollo Scientific, GB).
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E.coli (BL21 ) expressing recombinant fusion proteins of
CT668, CT858, CT783, CT610, YscN and DT8 from C. trachomatis D and
CPN1016 and YscC from C. pneumoniae VR1310 were generated.
The recombinant fusion proteins were purified from lysed bacteria using a
nickel resin column (High Trap Sepharose, Amersham Pharmacia Biotech)
as previously described. Coomassie stained SDS-PAGE gels of the purified
proteins were run. Coomassie stained bands representing fusion proteins
was unambiguously identified by MALDI MS to verify that the correct fusion
1 o protein had been generated. Sera containing polyclonal antibodies were
obtained by immunizing New Zealand Whiterabbits intramuscularly three
times with 50 p.g of fusion protein dissolved in Freunds adjuvant and
intravenously twice with 50 p.g of fusion protein dissolved in PBS as
described in [17] by Knudsen et al.
Example 12:
Western blotting using PAbs against vaccine candidates
In order to confirm that the PAbs recognized the correct vaccine candidates
and to visualize potential post translational modifications or processing, 2D-
PAGE immunoblotting were performed. 500 pg unlabelled and 2 X 106 cpm
2 0 labelled C. trachomatis D or C, pneumoniae protein from whole cell lysates
was separated by 2D-PAGE, and proteins were electroblotted on to PVDF
membranes. Immunostaining was carried out using a 1/500-1/1000 dilution of
the PAbs in a buffer containing 150 mM NaCI (or in high salt, 400 mM), 20
mM Tris, 0.2% wlv gelatin, 0.05% vlv Tween 20 (Bio-Rad) and 2% v/v normal
goat serum (Dako, Glostrup Denmark).
The secondary antibody used was alkaline phosphatase-conjugated goat
anti-rabbit IgG (Bio-Rad) diluted 1/ 2000 in antibody buffer. Blots were
stained with 5-bromo-4-chloro-3-indolylphosphate toluidium (BCIP)/nitroblue
3o tetrazolium (NBT) (Bio-Rad) until a clear reaction was detected.
Immunoblots
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were computer scanned and subsequently exposed to X-ray films for
approximately 8 days. Melanie II Software was used in order to compare
immunostained proteins with their [35S]-labelled counterparts.
5 In Figure 7A1 and 7A2 an example of a total 2D-PAGE IMB image with
PAb245 against CT858 is shown. PAb 245 reacts reproducible with DT4
(CT858 C-terminal fragment) and DT48 (CT858 N-terminal fragment) (Fig
7A1 ) as indicated from the corresponding labelled background (Fig 7A2).
1o Figure 7B1 and 7B4 shows IMB with CT668 and YscN. Immunoreactive
protein spots on the gel and their localization based on a labelled background
(Fig7B2 and B5) corresponded to the positions of the proteins on analytical
gels (Figure 7 B3 and B6), thus confirming that the PAbs reacted with the
correct proteins.
The invention provides an example of IMB showing that levels of certain
isoforms of CT610 are clearly increased in MG132 treated infected cells
compared to non-treated controls. DT7 was identified as CT610 and was
located just above DT9, DT10, DT11 and DT12(Fig 6A). IMB with PAb255
2o against CT610 on 2D-gel blots (Fig 7C1) of whole cell lysates clearly
reacted
with two rows of spots, one row representing DT7 and the other row
representing DT9, DT10, DT11 and DT12. CT610 is therefore present in
different isoforms presumably due to different posttranslational modification
and processing. The abundance of DT9, DT10 and DT11 was clearly
increased if cells were treated with the proteasome inhibitor MG132 (Fig 7
C2).
IMB with PAb255 was pertormed on ordinary SDS-PAGE PVDF blots with
protein from whole cell lysates treated for six hours in the presence or
3o absence of MG132, respectively. Fig 7 C4, clearly shows an extra band
below the band representing the DT7 row of spots in lanes containing
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proteins from MG132 treated infected cells (lane b and d) compared to
untreated controls (lane a and c). The levels of the upper band, representing
the DT7 row, was not significantly altered by the proteasome inhibitor
treatment.
Example 13:
Expression of candidates in different C, trachomatis serovars and upon
Growth in different host cells
The invention considers serovar/host cell specific differences in expression
levels of potentially secreted proteins, as host cell/Chlamydia interactions
1 o may be different for other serovars or by cultivation in other host cells.
This is
relevant for the choice of the vaccine candidate with greatest potential for a
general C. trachomatis vaccine.
CT668, CT858, CT783 and CT610 were all detected at the same positions in
C. trachomatis A, D and L2. This is in agreement with the very high
conservation of the genes encoding these proteins between C, trachomatis D
and L2. In addition, all of the proteins were expressed when C. trachomatis
A, D and L2 was cultivated in McCoy or Hep-2 cells instead of HeLa cells,
indicating that the expression of these proteins is independent of cell types
2 o used. However, based on gels with Chlamydia protein labelled from 22- 24
h.p.i. or 34-36 h.p.i. DT8 was detectable at p1 5.1 and Mw 7.5 in serovars A
and D. However, in serovar L2 DT8 was detected at p1 6.4 Mw 7.5
presumably due to minor amino acid substitutions which alter the net charge
and isoelectric point of the protein.
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Example 14:
Indirect immunofluorescence microscopy of vaccine candidates
Wells with glass cover slips containing semiconfluent HeLa cell monolayers
were infected with C. pneumoniae or C. trachomatis D. A low titer of
Chlamydia was used so that only approximately 50% of the cells was
infected, thus making it possible to clearly discriminate between infected and
un-infected cells. Cells were washed twice in PBS and fixed in formaldehyde
or methanol at different h.p.i. and subjected to indirect immunofluorescence
microscopy. If necessary PAbs were pre-absorbed by acetone precipitated
1 o HeLa protein in order to obtain minimum cross reaction to human proteins.
In the example shown in Fig 8, a 1/200 dilution of pre-absorbed rabbit PAb
and a 1125 dilution of mouse monoclonal antibody directed against C.
trachomatis MOMP(MAb 32.3) or MAb18.3 against C. pneumoniae was
used. Fluorescein isothiocyanate-conjugated (FITC) goat anti-rabbit (GAR)
IgG antibody and rhodamine conjugated goat anti-mouse (GAM) IgG
antibody (Jackson, Trichem, Denmark) was used as secondary antibodies.
The double immunostaining were performed in order to determine the sub-
cellular localization of the vaccine candidates relative to the Chlamydial
2o inclusion.
PAb 249 directed against DTB, which had a short turnover time, reacted
weakly with RB in the Chlamydial inclusion (Figure 8 A3). No significant
reaction to host cell structures were detected beyond the inclusion despite
minimal cross reaction with HeLa cell proteins.
In contrast, CT858 clearly stained the host cell cytoplasm in infected but not
un-infected cells (Figure 8 B3). In general, the staining was more intense at
the borders of the inclusions. The staining of the host cell cytoplasm could
be
3o visualized from 12 h.p.i to 72 h.p.i. in agreement with the long turnover
time
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predicted by the pulse chase studies.The same characteristics of reaction
were observed when the PAb raised against C. pneumoniae CPN1016 which
is homolog to CT858 was reacted with C. pneumoniae infected Hep-2 cells.
Description of examples on identified proteins
Example 15:
Spot number CT668 (DT1 and DT2)
CT668 was placed immediately upstream of the YscN ATPase in one of the
subclusters containing genes with homology to Type III secretion genes and
did not contain a predicted recognizable signal peptidase cleavage site.
1 o CT668 was only present in C. trachomatis D for approximately 4-6 hours
during which it steadily decreased in abundance. PAbs against CT668 only
seems to react weakly with the RB in the inclusion in IMF, but considering the
short turnover time of this protein, this may be explained by a fast
degradation in the host cell. CT668 has been detected from 12-40 h.p.i.,
which suggests that the protein is produced during most of the intracellular
development. It may therefore be that C. trachomatis is exporting CT668
continuously to the host cell, where it exerts its action and is rapidly
degraded.
2 o Two variants of CT668 were identified. The basic variant was most abundant
on gels with C. trachomatis proteins labelled from 22-24 h.p.i and
subsequently harvested. Interestingly, the intensity of the acidic variant
increased and the basic variant decreased with time in all studies performed.
When CT668 was chased in 30 min. intervals the increase of the acidic
variant was detectable already at one hour (Figure 1 B).
This finding may suggest that the modification is not due to the running
conditions of the gel, which can create modifications such as carbamylation
or amidation. Instead the modification seems exclusively to originate through
3 o an unknown enzyme, which modifies the protein.
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Modification of secreted proteins has been described in C. psitacci, where
IncA is phosphorylated by a host cell Ser /Thr kinase after translocation to
the inclusion membrane (Rockey et al., 1997) [29.]. The turnover time of
CT668 was prolonged upon treatment with proteasome inhibitor, suggesting
that at least limited amounts of the CT668 produced may be processed in the
proteasome.
Example 16:
1 o Spot number DT8(DT8)
DT8 is a novel 7.2 kDa protein, which based on homology search is a C.
trachomatis specific protein. PSORT analysis indicated no recognizable
leader sequence for this protein. The theoretical coordinates p1 5.21 / 7.2
kDa
were in excellent agreement with the experimentally determined. Many of the
features recognized for CT668 were observed for DTB, as well. A short
turnover time of < 6 hours was observed and IMF showed only a weak
reaction with RB.
After the stop codon in DT8 a potential stem-loop region can be predicted
2 o indicating a Rho-independent transcription termination of the protein.
Example 17:
Shot number DT7, DT 9. DT10. DT11, DT12 (CT610)
CT610 was not located near any genes with homology to genes which are
involved in secretion in other organisms. The protein was identified from both
whole cell lysates and EB, but by means of the Melanie II Software was
estimated to be at least 30 times more abundant in whole cell lysates. The
protein was detected in several isoforms represented by two molecular
weight polymorphisms as determined by means of the Pab255 raised against
3 o CT610. These rows of spots represented DT7(upper row) and
DT9,10,11,12(lower row). Both these row had a short turnover time in the
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pulse chase studies. Interestingly, the abundance of DT9, DT10 and DT11
was significantly increased upon treatment with proteasome inhibitor based
on pulse chaselMG132 studies. The pulse chase studies were verified by
SDS PAGE IMB with Pab255.
5
Thus, the invention provides evidence that certain isoforms of CT610 are
secreted and processed in the proteasome. The different fate of the CT610
isoforms shows the relevance of using Pab raised against vaccine
candidates to detect such isoforms.
Example 18:
Spot number DT3 (CT783~
CT783 has been suggested to be a C. trachomatis protein disulfide bond
isomerase. CT783 show homology to thioredoxin disulfide isomerase
(CT780), and to a protein disulfide isomerase from Methanobacterium
thermoautotrophicum. A 33 amino acid leader sequence can be predicted by
PSORT and SignaIP, and the theoretical p1 and molecular weight of the
cleaved protein was in agreement with the one experimentally determined. In
addition polyclonal antibodies generated against CT783 stained the correct
2 o spot in 2D-PAGE IMB using 4-7 L IPG.
Due to the cleavage of the N-terminal leader sequence, this protein is prob-
ably not secreted via the Type I or III systems, but more likely the Type II
system. A weak PSORT prediction suggested a sub-cellular localization in
the bacterial inner membrane, although most PDI are normally located in the
periplasm. CT783 had a very short turnover time and is virtually absent in the
Chlamydia after approximately 4-6 hours chase following synthesis.
The turnover time of CT783 was prolonged by proteasome inhibitors
3 o suggesting a potential processing in the proteasome.
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Example 19:
Spot number DT4 and DT48 (CT858) and CP63 (CPN1016)
CT858 had a cleavable N-terminal leader sequence and was predicted to be
a 67 kDa periplasmic protein, but was identified at two different positions on
the gels. The sequence tags, which identified DT4 as CT858 all matched the
C- terminal part of CT858. In IMB on 2D-PAGE PVDF membrane with whole
cell lysates PAb245 reacts clearly and reproducible with this C-terminal
fragment. In addition Pab245 reacted with the protein spot DT48, which also
has a long turnover time in the pulse chase studies as seen for DT4.
DT48 was also identified as an N-terminal fragment of CT858 by MALDI MS.
The molecular coordinates of DT48 were p1 7.3/ Mw 25.8. The N-terminal
part of CT858 yields a peptide with the coordinates agreeable with the one
determined experimentally using the pl/Mw tool on different lengths of the N-
terminal (without the signal peptide). This analysis suggests a cleavage site
around K2ssS234M2as,
The fact that the N-terminal and C-terminal fragment of CT858 can be
detected on 2D-gels all the way up to the EB stage (72 h.p.i.), but not in
2 o purified EB is indicative of a long turnover time in the host cell
cytoplasm.
This was in agreement with the very clear detection of CT858 in the host cell
cytoplasm up to 72 hours by IMF studies. CT858 showed weak homology to
the tail-specific protease (tsp) from E.coli, which has been involved in the
processing of penicillin binding proteins and includes a IRBP domain from
human interphotoreceptor retinoid-binding proteins, which bind hydrophobic
ligands (Silber et x1.,1992) [32.]. Type II secretion has been linked to the
export of degradative proteins in several gram negative bacteria including P.
aeroginosa and Aeromonas hydrophila (Reviewed in Hobbs and
Mattick,1993) [33.] In Aerosiginosa hydrophila a secreted elastase (ahpB)
has recently been suggested to be important for the virulence of the
organism (Cascon et al., 2000) [34.].
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These proteins has similar fates as CT858. They are mostly synthesized as a
propeptide with a cleavable signal peptide and then processed to a mature
protein. Another C. trachomatis protein, which also shows homology to tail-
s specific protease, is the hypothetical protein CT441. It remains to be
determined whether this protein may show the same secretory characteristics
as CT858.
The same sub-cellular localization of CT858 was observed for the C.
1o pneumoniae homolog, CPN1016 with Pab253 against CPN1016, thus
indicating a functional conservation of this gene between the two Chlamydia
species.
Example 20:
15 The secreted C. trachomatis D and C. pneumoniae proteins, which were
identified by their gene number, were analysed by an ANN trained to
recognize peptides with affinity for the human HLA-A2. In Tables V-VIII
peptides selected by the ANN for predicted binding to HLA-A2 are listed and
the affinity (Kd) is given in nM. The lower the value the better. Most
peptides
2 o with Kd below 50 nM are immunogenic. Peptides with Kd below 500 nM (but
above 50 nM) are potentially immunogenic. The binding of a given peptide
may be improved by substitution of a sub optimal amino acid in the anchor
positions P2 or P9 - a strategy that often will retain the specificity
directed
againsfi the natural peptide.
Example 21:
Determination of the ability of a vaccine candidate to Generate specific
cytotoxic CD8+ T- cells in experimental animal models.
In one approach the vaccine candidates are used as full length recombinant
3o proteins to immunize experimental animals (mouse or guinea pig) including
transgenic A2 mice expressing human HLA class I molecules.
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In another approach the vaccine candidates are screened for T-cell epitopes
by computer algorithms and subsequently peptides encompassing these
epitopes are synthesized and used for immunization as described for full
length vaccine candidates.
In a third approach 8-10 amino acids long peptides are synthesized, in an
overlapping way so that they cover the entire sequence of a vaccine
candidate, and used for MHC class I binding assay in competition with radio
labelled intermediate binders. Peptide, which are good binders are used for
immunization as described for full length vaccine candidates.
The vaccine candidates are administrated either as combinations of
peptides/proteins or as single peptides/proteins in adjuvant. The vaccine
candidates can also be administrated as a DNA-vaccine or by a virus
expressing the vaccine candidate.
Peripheral blood mononuclear cells (PBMC) from the immunized animals are
purified by density gradient centrifugation and CD8+ cells are purified by use
2 0 of antibodies bound to magnetic beads or by other methods.
CD8+ T-cell activity is measured by proliferation assays such as ELISPOT
and incorporation of tritiated thymidine and by specific lysis assays (chrome
release).
Purified PBMC or CD8+ cells from immunized animals are plated on
microtiter plates, in limitting dilution, with irradiated antigen presenting
cells,
growth factors and a specific or non-specific stimulator. For specific
stimulation single vaccine candidate proteins or peptides, which has been
predicted as a good T-cell epitopes or found to be a good binders to the MHC
3 o class I molecule are used. For non-specific stimulation Chlamydia infected
cells are used. The cells are cultured for 9-14 days during which antigen
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specific cells proliferate. Generation of specific cytotoxic CD8+ T-cells is
determined by measuring the cytokine secretion from stimulated cells with
the ELISPOT assay. The proliferation of T-cells is measured by incorporation
of tritiated thymidine followed by scintillation counting. The cytotoxicity of
the
proliferated cells is measured using a cytotoxic assay as the chromium-
release assay using Chlamydia infected cells or recombinant cells expressing
the vaccine candidate protein/peptide as target cells [42].
Example 21
1 o Testing of the vaccine candidates for the ability to protect mice and
guinea
bias against Chlamydia infection.
Experimental animals are immunized (as described above) with the vaccine
candidates as single proteine/peptides or with a combination of vaccine
candidates. Following immunization, the animals are experimentally infected
with Chlamydia (intra nasal infection for C, pneumoniae and genital infection
for C. trachomatis. Protection against infection is measured by cultivation of
the Chlamydia, immunohistochemistry, puantitative PCR and by investigation
of seroconversion upon infection.
2 o Example 23:
Determination of the ability of Chlamydia infection to generate vaccine
candidate-specific cytotoxic CD8+ T-cells in humans.
Human serum samples are tested by ELISA (Medac) for the presence of
antibodies to Chlamydia. Sero-positive individuals are selected for the
presence of vaccine candidate-specific cytotoxic CD8+ T-cells.
Peripheral blood mononuclear cells (PBMC) from humans who are tested
antibody positive for Chlamydia are purified by density gradient
centrifugation
and CD8+ cell are purified by use of antibodies and magnetic beads or other
3 o method. CD8+ T-cell activity specifically directed against vaccine
candidate
proteins/peptides is measured by the methods described in example 19.
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Example 24
Using the vaccine candidates for the developments of a ELISA test for
diagnostic purposes
5 As the secreted proteins in the present invention are not present or
significantly reduced in the purified microorganisms, immuno assay based on
purified elementary bodies cannot detect antibodies to such proteins.
Therefore secreted proteins can represent unrecognised major antigens,
which are also involved in the humoral immune response. In addition an
1o ELISA based on secreted Chlamydia proteins may detect persistent infection
with Chlamydia as the secreted proteins are only expressed during the
intracellular stage of Chlamydia development.
1 ) The secreted proteins are produced as recombinant proteins, which are
15 purified. Alternatively overlapping synthetic peptides representing the
secreted proteins are also produced.
2) ELISA plates are coated with purified recombinant proteins representing
secreted proteins (or synthetic peptides originating from the secreted
20 proteins). The ELISA plate is blocked with 15% foetal calf serum to avoid
unspecific binding
3) Patient sera are screened for antibodies against C. Trachomafis or C.
pneumoniae using micro-IF or ELISA (Medac). The positive sera is tested on
25 a ELISA plate coated with the recombinant antigens representing the
secreted proteins
4) For detection of antibody binding anti-human IgG, IgA or IgM is used. As
positive control sera from infected mice are used.
5) The results from micro-IF or ELISA (Medac) are compared to the ELISA
based on recombinant proteins representing the secreted proteins.
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P200100157 WO Sekvensliste final.ST25.txt
SEQUENCE LISTING
<110> Vandahl, Brian Berg
<120> Method for identification of proteins from intracellular ba
cteria
<130> P200100157W0
<150> DK PA 200100581
<151> 2001-04-09
<160> 194
<170> PatentIn version 3.1
<210> 1
<211> 67
<212> PRT
<213> Chlamydia trachomatis
<400> 1
Met Gln His Thr Ile Met Leu Ser Leu Glu Asn Asp Asn Asp Lys Leu
1 5 10 15
Ala Ser Met Met Asp Arg Val Val Ala Ala Ser Ser Ser Ile Leu Ser
20 25 30
Ala Ser Lys Asp Ser Glu Ser Asn Arg Gln Phe Thr Ile Ser Lys Ala
35 40 45
Pro Asp Lys Glu Ala Pro Cys Arg Val Ser Tyr Val Ala Ala Ser Ala
50 55 60
Leu Ser Glu
<210> 2
<211> 204
<212> DNA
<213> Chlamydia trachomatis
<400> 2
atgcaacaca caattatgct gtctttagag aacgataatg ataagcttgc ttctatgatg
gatcgagttg ttgctgcgtc atcaagcatt ctttctgctt ccaaagattc tgagtccaat
Side 1
CA 02443813 2003-10-08
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120
P200100157 WO Sekvensliste final.ST25.txt
agacagttta ctatttctaa agctccggat aaagaagctc cttgcagagt atcttatgta
180
gctgcaagtg cactttcaga atag
204
<210> 3
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT263 immunogenic peptide
<400> 3
Lys Leu Ala Glu Ala Tle Phe Pro Ile
1 5
<210> 4
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT263 immunogenic peptide
<400> 4
Phe Leu Lys Asn Asn Lys Val Lys Leu
1 5
<210> 5
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT263 immunogenic peptide
Side 2
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<400> 5
Ala Leu Ser Pro Pro Pro Ser Gly Tyr
1 5
<210> 6
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT263 immunogenic peptide
<400> 6
Phe Ile Ala Lys Gln Ala Ser Leu Val
1 5
<210> 7
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT263 immunogenic peptide
<400> 7
Thr Leu Ser Leu Phe Pro Phe Ser Leu
1 5
<210> 8
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT263 immunogenic peptide
<400> 8
Side 3
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
Ser Leu Val Ala Cys Pro Cys Ser Met
1 5
<210> 9
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT263 immunogenic peptide
<400> 9
Leu Ile Phe Ala Asp Pro Ala Glu Ala
1 5
<210> 10
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT263 immunogenic peptide
<400> 10
Leu Leu Leu Ile Phe Ala Asp Pro Ala
1 5
<210> 11
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT541 immunogenic peptide
<400> 11
Ile Leu Ser Trp Met Leu Met Phe Ala
Side 4
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
1 5
<210> 12
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT541 immunogenic peptide
<400> 12
Lys Gln Met Ala Glu Val Gln Lys Ala
1 5
<210> 13
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT541 immunogeniC peptide
<400> 13
Leu Met Phe Ala Val Ala Leu Pro Ile
1 5
<210> 14
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT541 immunogenic peptide
<400> 14
Lys Leu Gln Tyr Arg Val Val Lys Glu
1 5
Side 5
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<210> 15
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT541 immunogenic peptide
<400> 15
Phe Leu Lys Glu Asn Lys Glu Lys Ala
1 5
<210> 16
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT541 immunogenic peptide
<400> 16
Lys Leu Ser Arg Thr Phe Gly His Leu
1 5
<210> 17
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT541 immunogenic peptide
<400> 17
Ser Leu Leu Ile Phe Glu Val Lys Leu
1 5
<210> 18
Side 6
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT541 immunogenic peptide
<400> 18
Val Leu Ser Gly Lys Pro Thr Ala Leu
1 5
<210> 19
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BTNDING
<222> (1) . . (9)
<223> CT541 immunogenic peptide
<400> 19
Val Leu Tyr Ile His Pro Asp Leu Ala
1 5
<210> 20
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT541 immunogenic peptide
<400> 20
Leu Leu Ser Arg Gln Leu Ser Arg Thr
1 5
<210> 21
<211> 9
<212> PRT
Side 7
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT691 immunogenic peptide
<400> 21
Leu Leu Gln Arg Glu Leu Met Lys Val
1 5
<210> 22
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT691 immunogenic peptide
<400> 22
Ser Thr Ile Asn Val Leu Phe Pro Leu
1 5
<210> 23
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT691 immunogenic peptide
<400> 23
Pro Leu Gln Ala His Leu Glu Leu Val
1 5
<210> 24
<211> 9
<212> PRT
<213> Chlamydia trachomatis
Side 8
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT691 immunogenic peptide
<400> 24
Ser Leu Phe Gly Gln Ser Pro Phe Ala
1 5
<210> 25
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT691 immunogenic peptide
<400> 25
Lys Leu Ala Tyr Arg Val Ser Met Thr
1 5
<210> 26
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT691 immunogenic peptide
<400> 26
Val Leu Trp Met Gln Ile Ile Lys Gly
1 5
<210> 27
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
Side 9
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<222> (1) . . (9)
<223> CT691 immunogenic peptide
<400> 27
Val Leu Phe Pro Leu Phe Ser A1a Leu
1 5
<210> 28
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT691 immunogenic peptide
<400> 28
Phe Leu Gln Lys Thr Val Gln Ser Phe
1 5
<210> 29
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT691 immunogenic peptide
<400> 29
Phe Gly Gln Ser Pro Phe Ala Pro Leu
1 5
<210> 30
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT858 immunogenic peptide
Side 10
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<400> 30
Val Leu Ala Asp Phe Ile Gly Gly Leu
1 5
<210> 31
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT858 immunogenic peptide
<400> 31
Arg Met Ala Ser Leu Gly His Lys Val
1 5
<210> 32
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT858 immunogenic peptide
<400> 32
Gly Leu Asn Asp Phe His Ala Gly Val
1 5
<210> 33
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT858 immunogenic peptide
Side 11
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO SekVensliste final.ST25.txt
<400> 33
Phe Ser Cys Ala Asp Phe Phe Pro Val
1 5
<210> 34
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT858 immunogenic peptide
<400> 34
Met Leu Thr Asp Arg Pro Leu Glu Leu
1 5
<210> 35
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT858 immunogenic peptide
<400> 35
Leu Leu Glu Asn Val Asp Thr Asn Val
1 5
<210> 36
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT858 immunogeniC peptide
<400> 36
Side 12
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
Arg Met Ile Leu Thr Gln Asp Glu Val
1 5
<210> 37
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT858 immunogeniC peptide
<400> 37
Ser Cys Ala Asp Phe Phe Pro Val Val
1 5
<210> 38
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT858 immunogenic peptide
<400> 38
Phe Va1 Phe Asn Val Gln Phe Pro Asn
1 5
<210> 39
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT858 immunogenic peptide
<400> 39
Tyr Leu Tyr Ala Leu Leu Ser Met Leu
1 5
Side 13
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<210> 40
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT858 immunogenic peptide
<400> 40
Ser Leu A1a Val Arg Glu His Gly Ala
1 5
<210> 41
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT858 immunogenic peptide
<400> 41
Tyr Leu Pro Tyr Thr Val Gln Lys Ser
1 5
<210> 42
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT858 immunogenic peptide
<400> 42
Ala Thr Ile Ala Pro Ser Ile Arg Ala
1 5
Side 14
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.t~t
<210> 43
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT858 immunogenic peptide
<220>
<221> BINDING
<222> (1) . . (9)
<223>
<400> 43
Leu Leu Glu Val Asp Gly Ala Pro Val
1 5
<210> 44
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT858 immunogenic peptide
<400> 44
Arg Thr Ala Gly Ala Gly Gly Phe Val
1 5
<210> 45
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT858 immunogenic peptide
<400> 45
Side 15
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
Ser Leu Phe Tyr Ser Pro Met Val Pro
1 5
<210> 46
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0152 immunogenic peptide
<400> 46
Phe Leu Val Ser Cys Leu Phe Ser Val
1 5
<210> 47
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0152 immunogenic peptide
<400> 47
Tyr Leu Arg Asp Ala Gln Thr Ile Leu
1 5
<210> 48
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0152 immunogenic peptide
<400> 48
Leu Leu Ile Arg Ile Gln Asp His Val
1 5
Side 16
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<210> 49
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0152 immunogenic peptide
<400> 49
Lys Leu Gly Arg Lys Phe Ala Ala Val
1 5
<210> 50
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BTNDING
<222> (1) . . (9)
<223> CPN0152 immunogenic peptide
<400> 50
Leu Val Ser Arg Thr Gln Gln Thr Leu
1 5
<210> 51
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0152 immunogenic peptide
<400> 51
Cys Leu Phe Ser Val Ala Ile Gly Ala
1 5
Side 17
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<210> 52
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0152 immunogenic peptide
<400> 52
Gly Phe Gly Pro Pro Pro Ile Ile Val
1 5
<210> 53
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0152 immunogenic peptide
<400> 53
Ser Leu Pro Thr Lys Pro Tyr Ile Leu
1 5
<210> 54
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BTNDING
<222> (1) . . (9)
<223> CPN0152 immunogenic peptide
<400> 54
Arg Ile Gln A'sp His Val Thr Ala Asn
1 5
<210> 55
<211> 9
Side 18
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0152 immunogenic peptide
<400> 55
Ala Ile Gly Ala Ser Ala Ala Pro Val
1 5
<210> 56
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPNOl52 immunogenic peptide
<400> 56
Arg Leu Gly Ile Ser Gly Phe Ser Leu
1 5
<210> 57
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0619 immunogeniC peptide
<400> 57
Phe Met Val Ser Gly Pro Val Val Val
1 5
<210> 58
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
Side 19
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0619 immunogenic peptide
<400> 58
Leu Val Leu Glu Gly Ala Asn Ala Val
1 5
<210> 59
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0705 immunogenic peptide
<400> 59
Phe Val Gly Ala Asn Leu Thr Leu Val
1 5
<210> 60
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0619 immunogenic peptide
<400> 60
Cys Leu Ala Glu Asn Ala Phe Ala Gly
1 5
<210> 61
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
Side 20
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<221> BINDING
<222> (1) . . (9)
<223> CPN0705 immunogenic peptide
<400> 61
Lys Ile Glu Glu Val Gln Thr Pro Leu
1 5
<210> 62
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0705 immunogeniC peptide
<400> 62
Ala Leu Lys Gly His Gln Leu Thr Leu
1 5
<210> 63
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0705 immunogenic peptide
<400> 63
Gln Met Ala Glu Ala Ala Asp Leu Val
1 5
<210> 64
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) , . (9)
Side 21
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<223> CPN0796 immunogeniC peptide
<400> 64
Phe Met Gly Ala His Val Phe Ala Ser
1 5
<210> 65
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0796 immunogenic peptide
<400> 65
Leu Leu Ile Gln His Ser Ala Lys Val
1 5
<210> 66
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0796 immunogenic peptide
<400> 66
Phe Leu Cys Pro Phe Gln Ala Pro Ser
1 5
<210> 67
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0796 immunogenic peptide
Side 22
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<400> 67
Phe Gln Ala Pro Ser Pro Ala Pro Val
1 5
<210> 68
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0796 immunogenic peptide
<400> 68
Ala Met Asn Ala Cys Val Asn Gly Ile
1 5
<210> 69
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9) ,
<223> CPN0796 immunogeniC peptide
<220>
<221> BINDING
<222> (1) .. (9)
<223>
<400> 69
Phe Met Gly Ile Gln Val Leu His Leu
1 5
<210> 70
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
Side 23
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<221> BINDING
<222> (1) . . (9)
<223> CPN0796 immunogenic peptide
<400> 70
Arg His Ala Ala Gln Ala Thr Gly Val
1 5
<210> 71
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0796 immunogenic peptide
<400> 71
Phe Gln Tyr Ala Asp Gly Gln Met Val
1 5
<210> 72
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0796 immunogenic peptide
<400> 72
Ser Val Ser Ala Met Gly Asn Phe Val
1 5
<210> 73
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
Side 24
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<223> CPN0796 immunogenic peptide
<400> 73
Phe Leu Ser Tyr Arg Ser Gln Val His
1 5
<210> 74
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0796 immunogeniC peptide
<400> 74
Phe Leu Leu Thr Ala Ile Pro Gly Ser
1 5
<210> 75
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0796 immunogenic peptide
<400> 75
Val Leu Thr Pro Trp Ile Tyr Arg Lys
1 5
<210> 76
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0796 immunogenic peptide
Side 25
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<400> 76
Ser Val Val Met Asn Gln Gln Pro Leu
1 5
<210> 77
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0796 immunogenic peptide
<400> 77
Ser Leu Lys Asn Ser Gln Gln Gln Leu
1 5
<210> 78
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0796 immunogenic peptide
<400> 78
Met Leu Pro Asp Thr Leu Asp Ser Val
1 5
<210> 79
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0796 immunogenic peptide
<400> 79
Side 26
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
Ala Leu Pro Tyr Thr Glu Gln Gly Leu
1 5
<210> 80
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDTNG
<222> (1) . . (9)
<223> CPN0796 immunogenic peptide
<400> 80
Val Leu Ser Gly Phe Gly G1y Gln Val
1 5
<210> 81
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0998 immunogenic peptide
<400> 81
Leu Leu Phe Gly'Val Val Phe Gly Val
1 5
<210> 82
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0998 immunogenic peptide
<400> 82
Ser Leu Gln Glu Arg Tyr Pro Thr Leu
Side 27
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
1 5
<210> 83
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BTNDING
<222> (1) .. (9)
<223> CPN0998 immunogenic peptide
<400> 83
Met Leu Leu Lys Gly Gln Asn Lys Val
1 5
<210> 84
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0998 immunogeniC peptide
<400> 84
Phe Thr Phe Leu Pro Ile Ile Leu Val
1 5
<210> 85
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDTNG
<222> (1) .. (9)
<223> CPN0998 immunogeniC peptide
<400> 85
Phe Leu Gly Asp Ile Ser Ser Gly Ala
1 5
Side 28
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<210> 86
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0998 immunogenic peptide
<400> 86
Phe Leu Ala Gly Lys Lys Ala Arg Val
1 5
<210> 87
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0998 immunogenic peptide
<400> 87
Leu Leu Asp Ala Ala Tyr Gln Arg Ala
1 5
<210> 88
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0998 immunogeniC peptide
<400> 88
Tyr Leu Gly Tyr Leu Phe Thr Phe Leu
1 5
<210> 89
Side 29
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0998 immunogeniC peptide
<400> 89
Tyr Leu Phe Thr Phe Leu Pro Ile Tle
1 5
<210> 90
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0998 immunogenic peptide
<400> 90
Ser Leu Gly Ala Thr His Phe Leu Pro
1 5
<210> 91
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0998 immunogenic peptide
<400> 91
Glu Leu Ile Asp Gln Gly His Arg Leu
1 5
<210> 92
<211> 9
<212> PRT
Side 30
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0998 immunogenic peptide
<400> 92
Phe Leu Pro Ile Ile Leu Val Leu Leu
1 5
<210> 93
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0998 immunogenic peptide
<400> 93
Leu Val Leu Leu Phe Val Tyr Leu Val
1 5
<210> 94
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BTNDING
<222> (1) . . (9)
<223> CPN0998 immunogenic peptide
<400> 94
Val Thr Gly Pro Ala Thr Pro Gln Leu
1 5
<210> 95
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
Side 31
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0998 immunogenic peptide
<400> 95
Ser Leu Glu Lys Gln Asp Pro Glu Val
1 5
<210> 96
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0998 immunogenic peptide
<400> 96
Ile Leu Met Ala Ala Thr Asn Arg Pro
1 5
<210> 97
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0998 immunogenic peptide
<400> 97
Leu Thr Gln Glu Thr Asp Thr Glu Ala
1 5
<210> 98
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
Side 32
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<222> (1) . . (9)
<223> CPN0998 immunogenic peptide
<400> 98
Met Leu Leu Asp Ala Ala Tyr Gln Arg
1 5
<210> 99
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0998 immunogenic peptide
<400> 99
Leu Leu Asn Glu Ala Ala Leu Leu Ala
1 5
<210> 100
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0998 immunogenic peptide
<400> 100
Glu Leu Tyr Asp Gln Leu Ala Val Leu
1 5
<210> 101
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0998 immunogenic peptide
Side 33
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<400> 101
Leu Ile Gly hys Tyr T~eu Ser Pro Val
1 5
<210> 102
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0998 immunogenic peptide
<400> 102
Ser Zeu Gly Gly Arg Ile Pro T~ys Gly
1 5
<210> 103
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0998 immunogenic peptide
<400> 103
Gly Met Ser Pro Gln Zeu Gly Asn Val
1 5
<210> 104
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0998 immunogenic peptide
Side 34
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<400> 104
Leu Leu Ala Ala Arg Lys Asp Arg Thr
1 5
<210> 105
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0998 immunogenic peptide
<400> 105
Val Thr Phe Ala Asp Val Ala Gly Ile
1 5
<210> 106
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0998 immunogenic peptide
<400> 106
Val Leu Thr Glu Pro Leu Val Val Thr
1 5
<210> 107
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0998 immunogenic peptide
<400> 107
Side 35
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste ~inal.ST25.txt
Lys Ile Ala Leu Asn Asp Asn Leu Val
1 5
<210> 108
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN1016 immunogenic peptide
<400> 108
Lys Leu Gly Ala Ile Val Phe Gly Leu
1 5
<2l0> 109
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN1016 immunogenic peptide
<400> 109
Tyr Leu Gly Asp Glu Ile Leu Glu Val
1 5
<210> 110
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN1016 immunogenic peptide
<400> 110
Phe Leu Pro Thr Phe Gly Pro Ile Leu
1 5
Side 36
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<210> 111
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN1016 immunogenic peptide
<400> 111
Ser Leu Gln Asn Phe Ser Gln Ser Val
1 5
<210> 112
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN1016 immunogenic peptide
<400> 112
Phe Thr Asp Glu Gln Ala Val Ala Val
1 5
<210> 113
<211> 9
<212 > PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN1016 immunogeniC peptide
<400> 113
Ser Leu Asn Asp Tyr His Ala Gly Ile
1 5
Side 37
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<210> 114
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1)..(9)
<223> CPN1016 immunogenic peptide
<400> 114
Arg Met Ile Phe Thr Gln Asp Glu Val
1 5
<210> 115
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1)..(9)
<223> CPN1016 immunogenic peptide
<400> 115
Thr Gln Gln Ala Arg Leu Gln Leu Val
1 5
<210> 116
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN1016 immunogenic peptide
<400> 116
Ser Leu Val Ala Pro Leu Ile Pro Glu
1 5
<210> 117
<211> 9
Side 38
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN1016 immunogenic peptide
<400> 117
Tyr Met Val Pro Tyr Phe Trp Glu Glu
1 5
<210> 118
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1)..(9)
<223> CPN1016 immunogenic peptide
<400> 118
Tyr Val Glu Ala Val Lys Thr Ile Val
1 5
<210> 119
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN1016 immunogenic peptide
<400> 119
Phe Thr Gln Asp Glu Val Ser Ser Ala
1 5
<210> 120
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
Side 39
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN1016 immunogenic peptide
<400> 120
Gly Ala Gly Gly Phe Val Phe Gln Val
1 5
<210> 121
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<22l> BINDING
<222> (1) . . (9)
<223> CPN1016 immunogenic peptide
<400> 121
Leu Leu Gly Phe Ala Gln Val Arg Pro
1 5
<210> 122
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0412 immunogenic peptide
<400> 122
Arg Leu Glu Glu Val Ser Gln Lys Leu
1 5
<210> 123
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
Side 40
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<221> BINDING
<222> (1) . . (9)
<223> CPN0412 immunogenic peptide
<400> 123
Leu Thr Thr Asp Thr Pro Pro Val Leu
1 5
<210> 124
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> ( 1 ) . . ( 9 )
<223> CPN0412 immunogenic peptide
<400> 124
Lys Leu Leu Asp Met Glu Gly Tyr Ala
1 5
<210> 125
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0412 immunogenic peptide
<400> 125
Val Leu Ser Glu Asp Pro Pro Tyr Ile
1 5
<210> 126
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
Side 41
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<223> CPN0412 immunogenic peptide
<400> 126
Ala Leu Gln Ser Tyr Cys Gln Ala Tyr
1 5
<210> 127
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0412 immunogenic peptide
<400> 127
Lys Leu Thr Gln Thr Leu Val Glu Leu
1 5
<210> 128
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0412 immunogenic peptide
<400> 128
Phe Val Gly Ala Cys Ser Pro Glu Ile
1 5
<210> 129
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0412 immunogenic peptide
Side 42
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<400> 129
Asn Leu Thr Thr Asp Thr Pro Pro Val
1 5
<210> 130
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0412 immunogenic peptide
<400> 130
Leu Met Glu Arg Ala Ile Pro Pro Lys
1 5
<210> 131
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> ( 1 ) . . ( 9)
<223> CPN0661 immunogenic peptide
<400> 131
Lys Met Ala Glu Val Gln Lys Leu Val
1 5
<210> 132
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0661 immunogenic peptide
Side 43
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<400> 132
Ala Leu Gly Met Gln Gly Met Lys Glu
1 5
<210> 133
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0661 immunogeniC peptide
<400> 133
Lys Leu Ser Arg Thr Phe Gly His Leu
1 5
<210> 134
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0661 immunogenic peptide
<400> 134
Leu Leu Ile Phe Glu Ile Asn Leu Ile
1 5
<210> 135
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0661 immunogeniC peptide
<400> 135
Side 44
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
Val Leu Ala Thr Val Ala Leu Ala Leu
1 5
<210> 136
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0661 immunogenic peptide
<400> 136
Ile Leu Leu Pro Leu Gly Gln Thr Ile
1 5
<210> 137
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0661 immunogenic peptide
<400> 137
Val Leu Tyr Ile His Pro Asp Leu Ala
1 5
<210> 138
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> HOMOLOGY
<222> (1) .. (9)
<223> Homolog to CT541 immunogenic peptide
<400> 138
Leu Val Leu Ala Thr Val Ala Leu Ala
Side 45
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
1 5
<210> 139
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0681 immunogenic peptide
<400> 139
Tyr Met Leu Pro Ile Phe Thr Ala Leu
1 5
<210> 140
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0681 immunogenic peptide
<400> 140
Leu Leu His Glu Phe Asn Gln Leu Leu
1 5
<210> 141
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0681 immunogenic peptide
<400> 141
Val Leu Gln Arg Glu Leu Met Gln Ile
1 5
Side 46
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<210> 142
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0681 immunogenic peptide
<400> 142
Pro Leu Gln Ala His Leu Glu Met Val
1 5
<210> 143
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> ( 1 ) . . ( 9 )
<223> CPN0681 immunogenic peptide
<400> 143
Arg Leu Phe Gly Gln Ser Pro Phe Ala
1 5
<210> 144
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0681 immunogenic peptide
<400> 144
Gly Leu Phe Met Pro Ile Ser Arg Ala
1 5
<210> 145
Side 47
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0681 immunogenic peptide
<400> 145
Lys Leu Ala His Arg Ile Asn Met Thr
1 5
<210> 146
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) . . (9)
<223> CPN0681 immunogenic peptide
<400> 146
Tyr Leu Trp Leu Gln Val Ile Arg Arg
1 5
<210> 147
<211> 9
<212> PRT
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0681 immunogenic peptide
<400> 147
Thr Leu Leu His Glu Phe Asn Gln Leu
1 5
<210> 148
<211> 9
<212> PRT
Side 48
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<213> Chlamydia pneumoniae
<220>
<221> BINDING
<222> (1) .. (9)
<223> CPN0681 immunogenic peptide
<400> 148
Phe Gly Gln Ser Pro Phe Ala Pro Leu
1 5
<210> 149
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT149 immunogenic peptide
<400> 149
Phe Leu Gly Ala Ala Pro Ala Gln Met
1 5
<210> 150
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT149 immunogenic peptide
<400> 150
Phe Leu Gly Ile Gln Asp His Ile Leu
1 5
<210> 151
<211> 9
<212> PRT
<213> Chlamydia trachomatis
Side 49
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT149 immunogenic peptide
<400> 151
Leu Leu Thr Ala Asn Gly Ile Ala Val
1 5
<210> 152
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT149 immunogenic peptide
<400> 152
Ser Leu Pro Arg Arg Ile Pro Val Leu
1 5
<210> 153
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT149 immunogenic peptide
<400> 153
Gly Leu Gln Glu His Cys Arg Gly Val
1 5
<210> 154
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
Side 50
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<222> (1) .. (9)
<223> CT149 immunogenic peptide
<400> 154
Ser Leu Gly Cys His Thr Thr Ile His
1 5
<210> 155
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT149 immunogenic peptide
<400> 155
Ile Leu Thr His Phe Gln Ser Asn Leu
1 5
<210> 156
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1)..(9)
<223> CT149 immunogenic peptide
<400> 156
Val Leu Ser Cys Gly Tyr Asn Leu Val
1 5
<210> 157
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT149 immunogenic peptide
Side 51
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<400> 157
Leu Leu Lys G1u Ile Cys Ala Thr Ile
1 5
<210> 158
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT149 immunogenic peptide
<400> 158
Arg Leu Phe Leu Gly Ala Ala Pro Ala
1 5
<210> 159
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT149 immunogenic peptide
<400> 159
Ala Thr Val Ala Lys Tyr Pro Glu Val
1 5
<210> 160
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT149 immunogenic peptide
Side 52
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<400> 160
Leu Leu Ser Gly Ser Gly Phe Ala Ala
1 5
<210> 161
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT149 immunogenic peptide
<400> 161
Leu Thr Ala Asn Gly Ile Ala Val Ala
1 5
<210> 162
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT149 immunogenic peptide
<400> 162
Ser Gly Phe Ala Ala Pro Val Glu Val
1 5
<210> 163
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT500 immunogenic peptide
<400> 163
Side 53
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P20010015'7 WO Sekvensliste final.ST25.txt
Phe Met Ile Ser Gly Pro Val Val Val
1 5
<210> 164
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT500 immunogenic peptide
<400> 164
Ala Leu Phe Gly Glu Ser Ile Gly Val
1 5
<210> 165
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT500 immunogenic peptide
<400> 165
Ser Leu Glu Asn Ala Ala Ile Glu Val
1 5
<210> 166
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT500 immunogenic peptide
<400> 166
Side 54
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
Leu Met Gly Ala Thr Asn Pro Lys Glu
1 5
<210> 167
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT500 immunogenic peptide
<400> 167
Arg Ile Ala Ala Met Lys Met Val His
1 5
<210> 168
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT671 immunogenic peptide
<400> 168
Ala Leu Val Glu Thr Pro Met Ala Val
1 5
<210> 169
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT671 immunogenic peptide
<400> 169
Phe Cys Gly Ala Asn Leu Thr Leu Val
1 5
Side 55
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<210> 170
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<22l> BINDING
<222> (1) .. (9)
<223> CT671 immunogenic peptide
<400> 170
Ser Leu Lys Ala Arg Gln Leu Asn Leu
1 5
<210> 171
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT671 immunogenic peptide
<400> 171
Gln Leu Thr Glu Ala Thr Gln Leu Val
1 5
<210> 172
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT671 immunogenic peptide
<400> 172
Asp Leu Gln Trp Val Glu Gln Leu Val
1 5
Side 56
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<210> 173
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1)..(9)
<223> CT671 immunogenic peptide
<400> 173
Ile Val Leu Asp Asn Ser Asn Thr Val
1 5
<210> 174
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1)..(9)
<223> CT841 immunogenic peptide
<400> 174
Leu Leu Phe Gly Val Ile Phe Gly Val
1 5
<210> 175
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1)..(9)
<223> CT841 immunogenic peptide
<400> 175
Leu Leu Ala Lys Gly Gln Asn Lys Val
1 5
<210> 176
<211> 9
Side 57
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1)..(9)
<223> CT841 immunogenic peptide
<400> 176
Phe Thr Phe Met Pro Ile Ile Leu Val
1 5
<210> 177
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT841 immunogenic peptide
<400> 177
Phe Leu Gly Asp Val Ser Ser Gly Ala
1 5
<210> 178
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT841 immunogenic peptide
<400> 178
Leu Leu Asp Ala Ala Tyr Gln Arg Ala
1 5
<210> 179
<211> 9
<212> PRT
<213> Chlamydia trachomatis
Side 58
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<220>
<221> BINDING
<222> (l) . . (9)
<223> CT841 immunogenic peptide
<400> 179
Gly Met Ser Asp His Leu Gly Thr Val
1 5
<210> 180
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT841 immunogenic peptide
<400> 180
Ser Leu Gly Ala Thr His Phe Leu Pro
1 5
<210> 181
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT841 immunogeniC peptide
<400> 181
Asn Leu Ala Ala Leu Glu Asn Arg Val
1 5
<210> 182
<211> 9
<212> PRT
<213> Chlamydia trachomatis
Side 59
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT841 immunogenic peptide
<400> 182
Tyr Leu Phe Thr Phe Met Pro Ile I1e
1 5
<210> 183
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT841 immunogenic peptide
<400> 183
Phe Pro Thr Ala Phe Phe Phe Leu Leu
1 5
<210> 184
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT841 immunogenic peptide
<400> 184
Ile Leu Met Ala Ala Thr Asn Arg Pro
1 5
<210> 185
<211>
<212> PRT
<213> Chlamydia trachomatis
<220>
Side 60
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<221> BINDING
<222> (1) . . (9)
<223> CT841 immunogenic peptide
<400> 185
Lys Thr Ala Leu Asn Asp Asn Leu Val
1 5
<210> 186
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT841 immunogenic peptide
<400> 186
Leu Leu Asn Glu Ala A1a Leu Leu Ala
1 5
<210> 187
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> ( 1 ) . . ( 9 )
<223> CT841 immunogenic peptide
<400> 187
Glu Leu Tyr Asp Gln Leu Ala Val Leu
1 5
<210> 188
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
Side 61
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<223> CT841 immunogenic peptide
<400> 188
Ala Leu Glu Lys Gln Asp Pro Glu Val
1 5
<210> 189
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT841 immunogenic peptide
<400> 189
Ser Leu Gly Gly Arg Ile Pro Lys Gly
1 5
<210> 190
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) .. (9)
<223> CT841 immunogenic peptide
<400> 190
Phe Met Pro Ile Ile Leu Val Leu Leu
1 5
<210> 191
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT841 immunogenic peptide
Side 62
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 w0 Sekvensliste final.ST25.txt
<400> 191
Leu Leu Ala Ala Arg Lys Asp Arg Thr
1 5
<210> 192
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1)..(9)
<223> CT841 immunogenic peptide
<400> 192
Val Thr Phe Ala Asp Val Ala Gly Ile
1 5
<210> 193
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1) . . (9)
<223> CT841 immunogenic peptide
<400> 193
Tyr Thr Ile Ser Pro Arg Thr Asp Val
1 5
<210> 194
<211> 9
<212> PRT
<213> Chlamydia trachomatis
<220>
<221> BINDING
<222> (1)..(9)
<223> CT841 immunogenic peptide
Side 63
CA 02443813 2003-10-08
WO 02/082091 PCT/DK02/00234
P200100157 WO Sekvensliste final.ST25.txt
<400> 194
Leu Ile Gly Ala Pro Gly Thr Gly Lys
1 5
Side 64
