Note: Descriptions are shown in the official language in which they were submitted.
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CHLAMYDIA PROTEINS AND THEIR USES
I. FIELD OF THE INVENTION
The present invention relates to the detection of Chlamydia and to the
diagnosis, treatment
and prevention of Chlamydia infections in animals.
II. BACKGROUND
Chlamydiae are obligate intracellular bacterial pathogens with a unique
biphasic life cycle.
They appear as two distinct cellular types, a small dense cell or elementary
body (EB) that is
enclosed in a rigid bacterial cell wall, and a larger metabolically active
reticulate body (RB). The
EB is resistant to physical disruption and is infectious, whereas the RB is
more fragile and only
exists inside cells. The Chlamydia life cycle begins with the attachment of
the EB form to the host
cell which is followed by endocytosis into a nascent vacuole, also called an
"inclusion membrane."
After EB attachment and entry, replication of the EB form produces RB forms
that continue to
grow within the vacuole. By 72 hour post-infection, this growth phase is
terminated when the RBs
condense, and reorganize back to EBs. The lysis of the host cell results in
release of EBs to infect
new host cells. The difficulties in working with Chlamydiae center on the
obligate intracellular
requirement for growth and the fact that no adequate genetic engineering
methods have been
developed for this organism.
The genus Chlarnydia includes two species that are primarily associated with
human
disease: C. trachomatis and C. pneumoniae. C. trachomatis causes trachoma, an
eye disease that
is the leading cause of preventable infectious blindness worldwide with an
estimated 500 million
cases of active trachoma worldwide. C. trachomatis also causes a sexually
transmitted chlamydial
disease which is very common worldwide. C. trachornatis also causes
lymphogranuloma
venereum, a debilitating systemic disease characterized by lymphatic gland
swelling. The most
serious sequelae of chlamydial genital infections of females include
salpingitis, pelvic
inflammatory disease, and ectopic pregnancy. In the US alone, it is estimated
that over 4 million
new sexually transmitted C. trachomatis infections occurred in 1990, leading
to over four billion
dollars in direct and indirect medical expenses. The World Health Organization
estimates that 89
million new cases of genital Chlamydia occurred worldwide in 1995 (Peeling and
Brunham, 1996).
C. pneumoniae causes respiratory diseases including so called walking
pneumonia, a low
grade disease such that the infected person frequently fails to obtain
treatment and remains in the
community as an active, infectious carrier. C. pneumoniae is currently of
interest because of its
strong epidemiological association with coronary artery disease, and there is
also some evidence to
link it with multiple sclerosis.
Of the other disease-causing species of Chlamydia, Chlamydia psittaci and
Chlamydia
pecorum are primarily pathogens of wild and domestic animals, but these
species may infect
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humans accidentally. C. psitraci is acquired through respiratory droplet
infection and is
considered an occupational health hazard for bird fanciers and poultry
workers.
There is tremendous interest in the identification of candidate antigens for
protection
against chlamydial disease. While a prior infection with C. trachomatis will
protect against a
subsequent challenge t>y the same strain, indicating a protective component
that stimulates the host
immune response, most serious chlamydial diseases are exacerbated by an
overaggressive
anti-chlamydial immune response. Antigens recognized in the context of an
infection appear to
elicit a protective response whereas immunization with purified, killed (Ell
form) Chlamydia
results in an immunopathoiogical response. Therefore for the purposes of
vaccine development,
one needs to find epitopes that confer protection, but do not contribute to
pathology. It is an
object of this invention to provide Chlamydia polypeptides for use as vaccines
that induce a
protective immune response without inducing the pathological response caused
by the antigens
associated with the EB form of Chlamydia. Such immunostimulatory peptides will
be useful in the
treatment, as well as in the diagnosis, detection and prevention of Chlamydial
infections.
III. SUMMARY OF THE INVENTION
The present invention includes the use of Chlamydia proteins that show
enhanced
expression in the reticulate body (RB) stage relative to the elementary body
(EB) stage of the
Chlamydia life cycle. These proteins are not present at detectable levels in
the Ell form using
current immunological techniques and are thus said to be "infection-specific."
Certain of these
infection-specific proteins are found in the inclusion membrane of the
infected cell, and so have
been termed "Inc" proteins. These include the IncA, Inca, and IncC proteins of
Chlamydia as
described in the present disclosure. The genes that encode the IncA, Inca and
IncC proteins are
referred to as incA, incB and incC respectively. Other proteins of Chlamydia
described herein
have also been shown by the inventors to be infection-specific, but are not
known to be
incorporated into the inclusion membrane; these include the p242, TroA, and
Troll proteins. The
TroA and Troll proteins have been so named because they resemble the Tro
proteins of
Treponema pallidum, which are thought to form part of an ABC transport system.
The inventors have shown that the infection-specific Chlamydia proteins of the
disclosure
are recognized by convalescent antisera (i.e., antisera taken from an animal
that has recovered
from a Chlamydia infection) but are not recognized by antisera against the
killed Ell form of
Chlarnydia. Thus, the proteins are expressed only during active chlamydial
infection and are
therefore useful as protective antigens. These infection-specific proteins may
be used to confer a
protective immune response without inducing a pathological effect.
Additionally, immuno-
fluorescence microscopy and immunoblotting with antisera demonstrated that the
infection-specific
proteins are present in Chlamydia-infected HeLa cells, but are undetectable in
purified Ells and
absent in uninfected HeLa cells.
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Immunofluorescense microscopy reveals that IncA, Inca and IncC are localized
to the
inclusion membrane of infected HeLa cells. Reverse-transcription polymerase
chain reactions
(RT-PCR), northern hybridization data, and restriction analysis revealed that
the incB and incC
genes are closely linked and transcribed in an operon. RT-PCR, restriction
analysis and sequential
Southern hybridizations of incA then incC to the same filter provided evidence
that incA is
separated from the incB and incC operon by about l 10 kb. The C. trachomatis
Tro genes are not
closely linked with the p242 gene.
The present invention includes the nucleotide and amino acid sequences for
certain
infection-specific proteins from Chlamydia. These proteins are p242, TroA, and
Troll from C.
trachomatis, and the IneB, and IncC proteins from C. psittaci. The scope of
the invention
includes fragments of these proteins that may be used in a vaccine preparation
or that may be used
in a method of detecting Chlarnydia antibodies. Such fragments may be, for
example, 5, 10, 15,
20, 25, or 30 contiguous amino acids in length. They may even encompass the
entire protein.
More specifically, the present invention encompasses the purified infection-
specific
proteins having amino acid sequences as shown in SEQ ID NOS: 2, 4, 6, 10, and
12, amino acid
sequences that differ from such sequences by one or more conservative amino
acid substitutions,
and amino acid sequences that show at least 75 % sequence identity with such
amino acid
sequences.
Then invention also includes isolated nucleic acid molecules that encode a
protein as
described in the above paragraph, including isolated nucleic acid molecules
with nucleotide
sequences as shown in SEQ ID NOS: 1,3, 5, 9, and 11.
The present invention also includes a vaccine or immunostimulatory preparation
directed
against the reticulate body (RB) form of Chlamydia comprising one or more
purified infection-
specific peptides (or portions or fragments thereof, or peptides showing
sequence similarity to a
portion of such a peptide). Such peptide fragments may be, for example, 5, 10,
15, 20, 25, or 30
contiguous amino acids in length, of the sequence shown in SEQ ID NOS: 2, 4,
6, 8, 10, 12, 14,
16, or 18. Peptides used in such a vaccine may even encompass the entire
purified peptide of SEQ
ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, or 18, a peptide that differs from such a
peptide by one or
more conservative amino acid substitutions, or a peptide having at least 75 %
sequence identity
with such a peptide. Such vaccine preparations may contain one or more
pharmaceutically
acceptable excipients, adjuvants or diluents.
The invention additionally encompasses methods for making a vaccine,
comprising
combining a pharmaceutically acceptable excipient with a peptide described
herein. Also included
is a method of vaccination comprising administering a vaccine as described
herein to a mammal.
The present invention also provides a method for the diagnostic use of the
disclosed
purified infection-specific peptides, for instance by use in a diagnostic
assay to detect the presence
of infection-specific antibodies in a medical specimen, in which antibodies
bind to the Chlamydia
peptide and indicate that the subject from which the specimen was removed was
previously
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exposed to Chlamydia. Such a method may comprise: (i) supplying a biological
sample, such as
blood from an animal, that is suspected to contain infection-specific anti-
Chlamydia antibody, (ii)
contacting the sample with at least one infection-specific Chlamydia peptide
described herein, such
that a reaction between the peptide and the infection-specific anti-Chlamydia
antibody gives rise to
a detectable effect, such as a chromogenic conversion; and (iii) detecting
this detectable effect.
The present invention also provides a method of using antibodies that bind
specifically
with the disclosed proteins for detection of infection-specific Chlamydia
antigen, indicating the
presence of Chlamydia in the RB stage as distinct from the Ell stage. For
instance, the relevant
infection-specific antibodies may be used to provide specific binding in an
Enzyme Linked
Immunosorbant Assay (ELISA) or other immunoiogical assay wherein the antibody
F~ portion is
linked to a chromogenic, fluorescent or radioactive molecule and the Fan
portion specifically
interacts with, and binds to, an infection-specific protein. Such a method may
comprise: (i)
supplying a biological sample from an animal suspected to contain an infection-
specific Chlamydia
antigen, and (ii) contacting the sample with at least one infection-specific
anti-Chlamydia antibody,
such that a reaction between the antibody and the infection-specific Chlamydia
protein gives rise to
a detectable effect; and (iii) detecting this detectable effect.
Other aspects of the present invention include the use of probes and primers
derived from
the nucleotide sequences that encode infection-specific peptides, to detect
the presence of
Chlamydia nucleic acids in medical specimens. Such probes and primers may be
nucleotide
fragments, of, for example, 15, 20, 25, 30 or 40 contiguous nucleotides of the
sequence shown in
SEQ ID NOS: 1, 3, S, 7, 9, 11, 13, 15, or 17.
An additional aspect of the invention is a method of treating a Chlamydia
infection by
directing a therapeutic agent against a specific target, where the target is
chosen from an infection
specific protein of Chlamydia, a gene that encodes an infection-specific
protein of Chlamydia, and
an RNA transcript that encodes an infection-specific protein of Chlamydia,
wherein the therapeutic
agent interacts with said target to affect a reduction in pathology.
These and other aspects of the invention will become more apparent from the
following
description.
IV. SEQUENCE LISTING
SEQ ID NO:1 shows a nucleic acid sequence encoding the p242 C. trachomatis
protein,
with deduced primary amino acid sequence also shown.
SEQ ID N0:2 shows the amino acid sequence of the p242 C. trachomatis protein.
SEQ ID N0:3 shows a nucleic acid sequence encoding the TroA C. trachomatis
protein,
with deduced primary amino acid sequence also shown.
SEQ ID N0:4 shows the amino acid sequence of the TroA C. trachomatis protein.
SEQ ID NO:S shows a nucleic acid sequence encoding the Troll C. trachomatis
protein,
with deduced primary amino acid sequence also shown.
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SEQ ID N0:6 shows the amino acid sequence of the Troll C. trachomatis protein.
SEQ ID N0:7 shows a nucleic acid sequence encoding the IncA C. psittaci
protein, with
deduced primary amino acid sequence also shown.
SEQ 1D N0:8 shows the amino acid sequence of the IncA C. psittaci protein.
SEQ ID N0;9 shows a nucleic acid sequence encoding the Inca C. psittaci
protein, with
deduced primary amino acid sequence also shown.
SEQ ID NO:10 shows the amino acid sequence of the lncB C. psittaci protein.
SEQ ID NO:11 shows a nucleic acid sequence encoding the lncC C. psittaci
protein, with
deduced primary amino acid sequence also shown.
SEQ ID N0:12 shows the amino acid sequence of the IncC C. psittaci protein.
SEQ ID N0:13 shows a nucleic acid sequence encoding the IneA C. trachomatis
protein,
with deduced primary amino acid sequence also shown.
SEQ ID N0:14 shows the amino acid sequence of the IncA C. trachomatis protein.
SEQ ID NO:15 shows a nucleic acid sequence encoding the Inca C. trachomatis
protein,
with deduced primary amino acid sequence also shown.
SEQ ID N0:16 shows the amino acid sequence of the Inca C. trachomatis protein.
SEQ ID NO: l7 shows a nucleic acid sequence encoding the IncC C. trachomatis
protein,
with deduced primary amino acid sequence also shown.
SEQ ID NO: l8 shows the amino acid sequence of the IncC C. trachomatis
protein.
SEQ ID N0:19 shows the upstream oligonucleotide used to amplify the C.
psittaci incC
ORF.
SEQ ID N0:20 shows the downstream oligonucleotide used to amplify the C.
psittaci
incC ORF.
SEQ ID NO:'21 shows the upstream oligonucleotide used to amplify the C.
psittaci incB
ORF.
SEQ ID N0:22 shows the downstream oligonucleotide used to amplify the C.
psittaci
incB ORF.
SEQ ID N0:23 shows the upstream oligonucleotide used to amplify the C.
psittaci incA
ORF.
SEQ ID N0:24 shows the downstream oligonucleotide used to amplify the C.
psittaci
incA ORF.
V. DESCRIPTION OF THE INVENTION
A. DEFINITIONS
Particular terms and phrases used herein have the meanings set forth below.
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"EB" refers to the Elementary Body, an environmentally refractile and largely
metabolically dormant form of Chlamydia that is infectious and is presented as
a small dense body
enclosed by a bacterial cell wall.
"RB" refers to the Reticulate Body, a metabolically active form of Chlamydia
that is not
infectious, and exists only within a host cell, being very fragile, often
branched, and appearing
larger and less dense that the EB.
"Infection-specific" refers to a protein that shows enhanced expression in the
RB form of
Chlamydia compared to the EB form. Infection-specific proteins are not
necessarily absent from
the EB form, but they arc significantly more common in the RB form than in the
EB form.
"infection-specific antibody" is an antibody that binds specifically to an
infection-specific
protein.
"Biological sample" refers to any sample of biological origin including, but
not limited to
a blood sample, a plasma sample, a mucosal smear or a tissue sample.
"Isolated" An isolated nucleic acid has been substantially separated or
purified away
from other nucleic acid sequences in the cell of the organism in which the
nucleic acid naturally
occurs, i.e., other chromosomal and extrachromosomal DNA and RNA. The term
"isolated" thus
encompasses nucleic acids purified by standard nucleic acid purification
methods. The term also
embraces nucleic acids prepared by recombinant expression in a host cell as
well as chemically
synthesized nucleic acids.
"Probes" and "primers." Nucleic acid probes and primers may readily be
prepared based
on the nucleic acid sequences provided by this invention. A "probe" comprises
an isolated nucleic
acid attached to a detectable label or reporter molecule. Typical labels
include radioactive
isotopes, ligands, chemiluminescent agents, and enzymes.
"Primers" are short nucleic acids, typically DNA oligonucleotides 15
nucleotides or more
in length, which are annealed to a complementary target DNA strand by nucleic
acid hybridization
to form a hybrid between the primer and the target DNA strand, then extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification of a
nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other
nucleic-acid
amplification methods known in the art.
Probes and primers as used in the present invention typically comprise at
least 15
nucleotides of the nucleic acid sequences that are shown to encode infection-
specific proteins. In
order to enhance specificity, longer probes and primers may also be employed,
such as probes and
primers that comprise at least 20, 30 or 40 consecutive nucleotides of the
disclosed nucleic acid
sequences.
Methods for preparing and using probes and primers are well known in the art
and are
described in, for example Sambrook et al. (1989); Ausubel et al., (1987); and
Innis et al., (1990).
PCR primer pairs can be derived from a known sequence, for example, by using
computer
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programs intended for that purpose such as Primer (Version 0.5, 1991,
Whitehead Institute for
Biomedical Research, Cambridge, MA).
"Conservative amino acid substitutions" are those substitutions that, least
interfere with
the properties of the original protein, i.e., the structure and especially the
function of the protein is
conserved and not significantly changed by such substitutions. The table below
shows amino acids
which may be substituted for an original amino acid in a protein and which are
regarded as
conservative substitutions.
Original Residue Conservative Substitution
Ala Ser
Arg Lys
Asn gln, his
Asp Glu
Cys Ser
Gln Asn
Glu Asp
Gly Pro
His asn, gln
Ile leu, val
Leu ile, val
Lys arg, gln, glu
Met leu, ile
Phe met, leu, tyr
Ser Thr
Thr Ser
Trp Tyr
Tyr trp, phe
Val ile, leu
Conservative substitutions generally maintain (a) the structure of the
polypeptide
backbone in the area of the substitution, for example, as a sheet or helical
conformation, (b) the
charge or hydrophobicity of the molecule at the target site, or (c) the bulk
of the side chain.
The substitutions which in general are expected to produce the greatest
changes in protein
properties will be non-conservative, for instance changes in which (a) a
hydrophilic residue, e.g.,
Beryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.,
leucyl, isoleucyl,
phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for
(or by) any other residue;
(c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or
histadyl, is substituted for
(or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a
residue having a bulky side
chain, e.g., phenylalanine, is substituted for (or by) one not having a side
chain, e.g., glycine.
"Sequence identity" The similarity between two nucleic acid sequences, or two
amino
acid sequences is expressed in terms of the level of sequence identity shared
between the
sequences. Sequence identity is typically expressed in terms of percentage
identity; the higher the
percentage, the more similar the two sequences are. Variants of naturally
occurring infection-
specific peptides useful in the present invention are typically characterized
by possession of at least
50% sequence identity counted over the full length alignment with the amino
acid sequence of a
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naturally occurring infection-specific peptide when aligned using BLAST 2Ø1
(Altschul et al.,
1997). For comparisons of amino acid sequences of greater than about 30 amino
acids, the
BLAST 2 analysis is employed using the blastp program set to default
perameters (open gap = 11,
extension gap = 1 penalty, gap x dropoff = 50, expect = 10, word size = 3,
filter on), and using
the default BLOSUMti2 matrix (gap existence cost = 11, per residue gap cost =
1, lambda ratio
= 0.85). When aligning short peptides (fewer than around 30 amino acids), the
alignment should
be performed using the Blast 2 sequences function, employing the PAM30 matrix
(gap existence
cost = 9, per residue gap cost = 1, lambda ratio = 0.87). Proteins with even
greater similarity to
the reference sequences will show increasing percentage identities when
assessed by this method,
I 0 such as at least 60 % , at least 70 % , at least 80 % , at least 85 % , at
least 90 % , or at least 95 %
sequence identity. The NCBI Basic Local Alignment Search Tool (BLAST)
(Altschul et al., 1990)
is available from several sources, including the National Center for
Biotechnology Information
(NCBI, Bethesda, MD) and on the Internet, for use in connection with the
sequence analysis
programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at
http//www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence
identity using
this program is available at http:l/www.ncbi.nlm.nih.gov/BLAST/blast
help.html.
Similarly, when comparing nucleotides, blastn may be used with default
settings (rewards
for match = 1, penalty for mismatch = -2, open gap = 5, extension gap = 2
penalty, gap x
dropoff = 50, expect = 10, word size = I1, filter on), with the default
BLOSUM62 matrix (as
above). Variants of naturally occurring infection-specific nucleic acid
sequences useful in the
present invention are typically characterized by possession of at least 50%
sequence identity
counted over the full length alignment with the nucleic acid sequence of a
naturally occurring
infection-specific ORF when aligned using BLAST 2Ø I . Useful nucleic acids
may show even
greater percentage identity, and may, for example, possess at least 55%, at
least 65%, at least
75 % , at least 80 % , at least 85 % , at least 90 % , or at least 95 %
sequence identity naturally
occurring infection-specific ORF.
"Operably linked" A first nucleic acid sequence is "operably" linked with a
second
nucleic acid sequence when the first nucleic acid sequence is placed in a
functional relationship
with the nucleic acid sequence. For instance, a promoter is operably linked to
a coding sequence
if the promoter affects the transcription or expression of the coding
sequence. Generally, operably
linked DNA sequences are contiguous and, where necessary to join two protein
coding regions, in
the same reading frame.
"Recombinant" A recombinant nucleic acid is one that has a sequence that is
not naturally
occurring or has a sequence that is made by an artificial combination of two
otherwise separated
segments of sequence. This artificial combination is often accomplished by
chemical synthesis or,
more commonly, by the artificial manipulation of isolated segments of nucleic
acids, e.g., by
genetic engineering techniques.
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"Stringent Conditions" Stringent conditions, in the context of nucleic acid
hybridization,
are sequence-dependent and are different under different environmental
parameters. Generally,
stringent conditions are selected to be about 5 degrees to 20 degrees lower
than the thermal
melting point (Tm) for the specific sequence at a defined ionic strength and
pH. The Tm is the
temperature (under defined ionic strength and pH) at which 50% of the target
sequence hybridizes
to a perfectly matched probe. Conditions for nucleic acid hybridization and
calculation of
stringencies can be found in Sambrook et al. ( 1989), pages 9.49-9.55. Typical
high stringency
hybridization conditions (using radioiabeled probes to hybridize to nucleic
acids immobilized on a
nitrocellulose filter) may include, for example, wash conditions of 0.1 X SSC,
0.5 % SDS at a
wash temperature of 68°C.
When referring to a probe or primer, the term "specific for (a target
sequence)" indicates
that the probe or primer hybridizes under high-stringency conditions
substantially only to the target
sequence in a given sample comprising the target sequence.
"Purified" A purified peptide is a peptide that has been extracted from the
cellular
environment and separated from substantially all other cellular peptides. As
used herein, the term
peptide includes peptides, polypeptides and proteins. In certain embodiments,
a purified peptide is
a preparation in which the subject peptide comprises 50% or more of the
protein content of the
preparation. For certain uses, such as vaccine preparations, even greater
purity may be
preferable.
"Immunostimulatory peptide" as used herein refers to a peptide that is capable
of
stimulating a humoral or antibody-mediated immune response when inoculated
into an animal.
"Vaccine" A vaccine is a composition containing at least one immunostimulatory
peptide
which may be inoculated into an animal with the intention of producing a
protective immunological
reaction against a certain antigen. The antigen to be protected against may
be, for instance, an
infectio-specific antigen of Chlamydia.
B. ISOLATION OF INFECTION SPECIFIC CHLAMYDIA POLPEPTIDES
AND IDENTIFICATION OF GENES ENCODING THESE
POLYPEPTIDES
1. ISOLATION OF IncA, Inca AND IncC
Bacterial strains. Chlamydia (C. psittaci strain GPIC or C. trachomatis LGV-
434, ser.
L2) was cultivated in HeLa 229 cells using standard methods (Caldwell et al.,
1981). Purified
Chlarnydiae were obtained using Renografin (E. R. Squibb & Sons, Inc.,
Princeton, N.J.) density
gradient centrifugation. Escherichia colt DHS~ (Bethesda Research
Laboratories, Inc.,
Gaithersburg, Md.) was used as the host strain for transformations with
recombinant DNA. E.
colt XL1-Blue MRF' (Stratagene, La Jolla, Calif.) was used as the host strain
for infection with
lambda ZAPII phage vector. E. colt SOLR (Stratagene) was used as the host
strain for infection
with in vivo excised filamentous lambda ZAPII.
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Antisera. MBP (Maltose Binding Protein)-Inc fusion proteins were used as
antigens for
the production of mono-specific antibody reagents in Hartley strain guinea-
pigs. The protein was
diluted to 100 ug/ml-' sterile saline and mixed with the Ribi Trivalent
Adjuvant (Ribi
Immunochem.). The antigen/adjuvant emulsion was administered to anaesthetized
guinea-pigs
using a procedure provided by the manufacturer. Sera were collected 14 days
after secondary and
tertiary immunizations. Control antisera were produced by immunizing guinea-
pigs with adjuvant
alone, or with adjuvant plus purified maltose-binding protein.
Convalescent guinea-pig antisera, antisera against live EBs, and antisera
against formalin-
fixed EBs were produced using standard methods (Rockey and Rosquist, 1994 and
Rockey et al. ,
1995).
C. psittaei library construction and screening. For the incB and incC genes,
C. psittaci
strain GPIC DNA was extracted using a genomic DNA extraction kit (Qiagen) with
one
modification; dithiothreitol (5mM) was added to the suspension buffer to
assist EB lysis. DNA
was partially digested with Tsp509I and ligated to EcoRI digested 2.-ZAPII
phage arms
(Stratagene). The ligation was packaged in vitro with Gigapack extracts
according to the
manufacturer's instructions (Stratagene). Recombinant phage were plated on E.
coli XL-1 Blue at
densities of approximately 10" PFU/150-mm (diameter) plate. Following a nine
hour incubation to
allow development of the plagues, the plates were sequentially overlaid with
nitrocellulose disks
and the resulting lifts were processed for immunoblotting with convalescent
antisera and antisera to
fixed EBs. Of approximately 8,000 plaques, 18 had reactivity with the
convalescent sera but not
sera generated against. EBs. One of these was subcloned into pBluescript SK(-)
phagmid by in
vitro excision in the E.'. coli SOLR strain (Stratagene) and designated pBS200-
7.
For the incA gene, genomie DNA from C. psittaci strain GPIC was partially
digested
with Sau3A, size-selected (2-8 kb) by electrophoresis through low-melting-
temperature agarose,
and blunt-ended with T4 DNA polymerase. This DNA was ligated to an EcoRllNotl
adapter (Life
Technologies), kinased, and ligated to EcoRl-digested Lambda ZAP II vector
(Stratagene Cloning
Systems). Recombinants were packaged (Lambda Gigapack Gold, Stratagene) and
used to infect
E. coli XL1-Blue (Stratagene). Plaques were allowed to develop for 4 h at
37°C. Nitrocellulose
filters laden with 10 mM IPTG (US Biochemical Corp.) were placed onto the
plaques and
incubated for an additional 4 h at 37°C. These filters were removed and
placed into a blocking
solution consisting of PBS (150 mM NaCI, 10 mM NaPOa, pH7.2) plus 0.1 % Tween-
20 (TPBS)
and 2% BSA-TPBS. Filters were incubated for l h, rinsed twice in TPBS, and
incubated
overnight in convalescent-guinea-pig sera diluted 1:100 in BSA-TPBS. After
three washes in
TPBS, the filters were incubated for I h in '=51-staphylococcal protein A (New
England Nuclear)
diluted to approx. 124 nCiml-' in BSA-TPBS. Filters were again washed three
times in TPBS and
positive plaques were detected by exposure of the dried filters to
autoradiography film overnight at
room temperature. Positive clones were picked and plaque-purified. pBluescript-
SK- plasmids
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containing the chlamydial genes of interest were recovered from the purified
bacteriophage using
ExAssist filamentous bacteriophages (Stratagene).
Identification of antigens recognized by convalescent antisera. Recombinant
plaques
were identified that showed reactivity with convalescent (anti-RB) antisera,
but not with anti-EB
serum. The purified recombinant phage were converted into pBluescriptII SK
plasmid by in vivo
excision and recircularization and these recombinant DNAs were used to
transform E. coli. SDS-
PAGE and immunoblot analysis of lysates of these recombinant E. coli showed
that each expressed
one or more proteins that reacted with convalescent antisera but not with the
EB serum.
DNA Cloning and fusion protein production. The plasmid pJC2 contains a 5.0 kb
EcoRl GPIC genomic fragment cloned into the pZEro2.1 vector (Invitrogen). To
construct pJC2,
the incC ORF sequence was 3'P-radiolabeled using random priming (Gibco-BRL)
and used to
probe EcoRI cut GPIC genomic DNA fragments separated by agarose gel
electrophoresis.
Fragments in the size range of the positive signal were excised from the gel
and purified by Gene-
Clean (Bio101). The gel-purified fragments were used in a ligation along with
EcoRI-digested
pZEro2.1. Kanamycin resistant colonies were screened by colony hybridization
with radiolabeled
incC.
MBP fusions of the five ORFs present in pJC2 were produced using the pMAL-C2
vector
(New England Biolabs). The reading frame of incC, with the exception of the
first four codons,
was amplified using Pwo polymerase (Boehringer Mannheim) and pBS200-7 as the
template. The
upstream and downstream oligonucleotides for this amplification were
5'-AGAACC:GATTTAACTCCAGGCG-3' (SEQ ID NO: I9) and
5'-GCGCGGATCCTTAATG'fCCGGTAGGCCTAG-3' (SEQ ID NO: 20), respectively.
The vector was digested with XmnI and BamHI, and the amplication product was
digested with
BamHI. Ligation of these products resulted in an in-frame fusion between the
malE gene in the
vector and the incC reading frame from pBS200-7. The stop codon for this
construction is
provided by the insert. Following ligation, the products were transformed into
E.coli strain
HD50. The resulting fusion protein (MBP/IncC) was overexpressed and purified
by maltose
affinity chromatography using an amylose resin supplied by New England
Biolabs.
The same approach was used for production of the MBP/IncB fusion protein. The
sequence encoding the N-terminal 101 amino acids of the Inca ORF was PCR
amplified using the
oligonucleotides
5'-ATGTCAACAAC:ACCAGCATCTTC-3' (SEQ ID NO: 21 ) and
5'-GCGCGGATCC7.'TAAT1'AGTGCCTTCTGGATTAGG-3' (SEQ ID NO: 22).
The purified MBP/IncB and MBP/IncC fusion proteins were used as antigen for
the
production of monospecific antibody in Hartley strain guinea-pigs by standard
methods (Rockey et
al., 1995). Inserts in each construct were confirmed by DNA sequencing.
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For IncA, a maltose-binding protein/IncA fusion protein was produced using the
pMAL-
C2 vector system from New England Biolabs. The reading frame of incA shown in
Fig.l, with the
exception of the initiator ATG, the incA ORF was amplified using Vent DNA
polymerise (New
England Biolabs) and plasmid pGPl7 as template. The upstream and downstream
oligo-
nucleotides for this amplification were
5'-CGCAGTACTGTATCCACAGACAAC-3' (SEQ ID NO: 23) and
5'-GTCGGATCCGAGAAACTCTCCATGCC-3' (SEQ ID NO: 24), respectively. The
vector was digested with Xmnl and BamHl, and the amplification product was
digested with Scal
and BumHl. Ligation of these products resulted in an in-frame fusion between
the malE gene in
the vector and the incA reading frame from pGPl7. The stop codon for this
construction is
provided by the insert. Following ligation, the products were transformed into
E. coli strain
DH50. The resulting fusion protein (MBP/IncA) was overexpressed and purified
by maltose
affinity chromatography using amylose resin (New England Biolabs).
MBP/IncA was used as antigen for the production of mono-specific antibody
reagents in
Hartley strain guinea-pigs.
DNA sequencing and sequence analysis. The pBS200-7 and pJC2 genomic clones as
well as the MBP fusions were sequenced with the Tag DyeDeoxy Terminator Cycle
Sequencing
Kit (Perkin Elmer/Applied Biosystems Division). Several internal primers were
designed to
sequence further into the cloned inserts. Sequence assembly was performed
using AssemblyLIGN
software and sequence analysis was performed with MacVector software
(International
Biotechnologies Incorporated). Hydrophilicity profiles were determined using
the Kyte-Doolittle
scale (Kyte and Doolittle, 1982) with a window of 7. Deduced amino acid
sequences were
compared with the database using the BLAST program (on default settings)
available from the
National Center for Biotechnology Information on the world wide web. The
entire nucleotide
sequence of the pJC2 insert was deposited in the GenBank/EMBL Nucleotide
Sequence Data
Library, under accession number AF017105.
For incA, nucleotide sequencing was conducted using the Sequences system (US
Biochemical) with the M13 forward and reverse primers, and internal primers
synthesized on an
Milligen/Biosearch Cyclone Plus DNA synthesizer. Computer analyses were
conducted using the
MacVector Sequence Analysis Software (International Biotechnologies
Incorporated).
Hydrophilicity profiles were determined using the Kyte-Doolittle scale (Kyte
and Doolittle, 1982)
with a window of 7. Secondary-structure predictions were generated using a
combination of the
Chou-Fasman and Robson-Garnier methods (Robson and Suzuki, 1976; Chou and
Fasman, 1978).
Deduced amino acid sequences were compared with those in the EMBL and GenBank
databases
using the BLASTP program available from the National Center for Biotechnology
Information.
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Electrophoresis and immunoblotting. Polyacrylamide gel electrophoresis (PAGE)
was
conducted using standard methods (Rockey and Rosquist, 1994). Immunoblotting
was performed
using standard methods (Rockey et al., 1995).
Immunofluorescence studies. Chlamydiae grown in HeLa cells on sterile glass
coverslips were fixed for microscopy one of two ways. Cells were either
incubated in methanol
for 5 minutes, or in the combination fixative periodate-lysine-
paraformaldephyde (PLP) for three
hours at room temperature followed by permeabilization with 0.05 % saponin
(Brown and
Farquhar, 1989). Immunostaining of the fixed coverslips was performed
according to standard
methods (Rockey et al., 1995) and visualized under a Nikon Microphot FXA
microscope using the
63x objective and oil immersion.
RT-PCR analysis. RNA for RT-PCR analysis was extracted from approximately 2 x
10'°
C. psittaci-infected cells. A Qiagen column was used for extraction and
purification according to
the manufacturer's instructions (Qiagen). RQI RNase DNase (Promega) was used
to ensure
removal of contaminating genomic DNA. cDNA was prepared by incubating 1.5 ug
of RNA, 2.5
uM of the reverse oligonucleotide primer, and AMV reverse transcriptase
(Promega) for 1 hour at
42°C in sodium pyrophosphate buffer, according to the manufacturer's
instructions. PCR
reactions were carried out using 1 ul of the cDNA reaction, 1.25 uM of each
oligonucleotide
primer, and Pwo polymerase (Boehringer Mannheim). Each RT-PCR reaction was
accompanied
by a positive control reaction that utilized the same primer set and 10 ng of
C. psittaci genomic
DNA, and a negative control reaction in which 1 ul of the same RNA preparation
was used as
template in the PCR reaction. A control primer set located within the incC
gene was also used as
an RT-PCR control.
Identification of incA, incB and incC genes of C. trachomatis. The nucleotide
sequence
information obtained for the incA, incB and incC of C. psittaci (above) was
used, with standard
methods, to identify the inc gene orthologues of C. trachomatis. Probes were
made that
corresponded to the 3' and 5' ends of the C. psittaci inc open reading frames.
Standard PCR
amplification (as above) was used, with the C. trachomatis genome as a
template, to amplify the
corresponding C. trac:homatis nucleotide sequence. The amplified DNA was then
sequenced,
using standard methods.
2. ISOLATION OF p242, TroA AND Troll
Bacterial strains. C. trachomatis LGV-434, serotype L2, was cultivated in HeLa
229
cells using standard methods (Caldwell et al., 1981). Purified chlamydiae were
obtained using
Renografm (E. R. Squibb & Sons, Inc., Princton, N.J.) density gradient
centrifugation (Hackstadt
et al., 1992). Escherichia coli DH50 (Bethesda Research Laboratories, Inc.,
Gaithersburg, Md.)
was used as the host strain for transformations with recombinant DNA. E. coli
XLI-Blue MRF'
(Stratagene, La 3olla, Calif. ) was used as the host strain for infection with
lambda ZAPII phage
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vector. E. coli SOLR (Stratagene) was used as the host strain for infection
with in vivo excised
filamentous lambda ZAPII.
Antisera. Two Cynomolgus monkeys (Macaca fasicularis) were anaesthetized and
infected urethrally with C. trachomatis Ells. Each monkey was infected twice
and allowed to
recover between infections. Symptoms of infection were monitored over time.
Antisera from
infected monkeys were tested for reactivity to Chlamydia by ELISA (Su et al.,
1990).
Sera were collected every two weeks and anti-chlamydial titers were
determined. These
animals showed mild clinical signs of disease which cleared spontaneously. A
second challenge
was then administered. Sera were collected from these animals and used to
probe a C. trachomatis
expression library as discussed below. As a control, Guinea Pigs were
immunized with killed C.
trachomatis of the Ell form. Sera from these animals were obtained and also
used to probe the C.
trachomatis expression library.
C. trachomatis library construction and immunoscreening. A C. trachomatis
genomic
library was constructed with the lambda ZAPII vector as described above for C.
psittaci.
Approximately 15,000 plaques were plated, transferred to nitrocellulose
filters (Schleicher and
Schuell, Keene, N.H.) in duplicate, and probed with the monkey convalescent
antiserum and with
Guinea Pig serum against killed Ells (Bannantine et al., 1998). Plaques that
reacted only with the
monkey convalescent antisera were selected for further study.
Identification of antigens recognized by convalescent antisera. Four positive
recombinant plaques were identified that showed reactivity with convalescent
antisera but not with
anti-Ell serum. The purified recombinant phage were converted into
pBluescriptIl SK plasmid by
in vivo excision and recircularization and these recombinant DNAs (pCtl, pCt2,
pCt3 and pCt4)
were used to transform E. coli. SDS-PAGE and immunoblot analysis of lysates of
these
recombinant E. coli showed that each expressed one or more proteins that
reacted with
convalescent (anti-RB) antisera but not with the anti-Ell antiserum. Two of
the recombinants
clones, pCt2 and pCt3, expressed an identical 19.9 kDa protein (p242). The
pCt4 recombinant
expressed two different proteins of approximately 32 kDa each that are
strongly recognized by
convalescent antisera (TroA and Troll).
C. SEQUENCE ANALYSIS
Sequence analysis of pCtl, 2, and 3 revealed overlapping inserts with only one
open
reading frame (ORF) common in all three. This ORF encodes an approximately
19.9 kDa protein
(p242) that shows no similarity to other known proteins. The nucleotide
sequence encoding C.
trachomatis p242, and the amino acid sequence of the protein are shown in SEQ
ID NOS:1 and 2,
respectively.
The insert in pCt4 contains two complete ORFs which code for two proteins,
each of
approximately 32kDa (TroA and Troll) that show some homology with proteins
from Treponema
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pallidum. The nucleotide sequences encoding the 32 kDa proteins (TroA and
Troll) and the amino
acid sequences of these proteins are shown in SEQ ID NOS: 3, 4, 5, and 6.
D. EMBODIMENTS OF THE INVENTION
The present invention includes the nucleotide and amino acid sequences for
certain
infection-specific proteins from Chlamydia. These proteins are p242, TroA, and
Troll from C.
trachomatis, and the lneB, and IneC proteins from C. psittaci. The scope of
the invention
includes fragments of these proteins that may be used in a vaccine preparation
or that may be used
in a method of detecting Chlamydia antibodies. Such fragments may be, for
example, 5, 10, 15,
20, 25, or 30 contiguous amino acids in length, or may even encompass the
entire protein.
The present invention also encompasses the use of infection-specific proteins
of
Chlamydia, and the use of nucleotides encoding such proteins. Infection-
specific proteins include
the IncA, Inca and IneC proteins of C. psittaci, the IncA, IneB and IneC
proteins of C.
trachomatis, and the TroA, Troll, and p242 proteins of C. trachomatis. The
inventors have shown
that these proteins are infection-specific by using immunological techniques
such as immuno-
fluorescence microscopy and immunoblotting.
The present invention includes a vaccine against chlamydial infections
comprising
infection-specific proteins or fragments of these proteins or proteins that
are homologous or show
substantial sequence similarity to these proteins. In one embodiment, one or
more purified
infection-specific proteins may be mixed with a pharmaceutically acceptable
excipient to produce a
vaccine that stimulates a protective immunological response in an animal. In
one embodiment the
vaccine may be administered infra-muscularly or sub-cutaneously or
intravenously. In another
embodiment, the vaccine may be administered by inoculation into or onto the
mucous membranes
of the subject animal. For example, the vaccine may be administered urethrally
or genitally as a
liquid or in the form of a pessary. In another embodiment, it may be
administered to the mucosa
of the lungs as a spray or vapor suspension.
Since at least three amino acids are required to produce an antigenic epitope,
the vaccine
should comprise at least three consecutive amino acids, preferably at least
five consecutive amino
acids, and may comprise at least 10, 15, 25, 30, 40, or 45 consecutive amino
acids of the
infection-specific proteins as shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,
16, and 18.
The vaccine of the invention may be used to inoculate potential animal targets
of any of
the chlamydial diseases including those caused by C. psittaci, C. trachomatis,
C. pneumoniae or
C. pecorum. Indeed the vaccine of the invention may be used to inoculate
animals against any
disease that shows immunological cross-protection as a result of exposure to
infection-specific
Chlamydia antigen.
Vaccines of the present invention can include effective amounts of
immunological
adjuvants known to enhance an immune response (e.g., alum). The protein or
polypeptide is
present in the vaccine in an amount sufficient to induce a protective immune
response whether
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through humoral or cell mediated pathways or through both. Such a response
protects the
immunized animal against chlamydial infections specifically by raising an
immune response against
the Reticulate Body form of Chlamydia. Protective antibodies may be elicited
by a series of two
or three doses of the antigenic vaccine given about two weeks apart.
The present invention also teaches a method of making a vaccine against
chlamydial
infections. The method of making the vaccine comprises providing a pure (or
substantially pure)
infection-specific chlamydial peptide or portion thereof, and mixing the
peptide with a
pharmacologically acceptable excipient or adjuvant. Adjuvants may include
commonly used
compounds such as alum. Additionally, the vaccines may be formulated using a
peptide according
to the present invention together with a pharmaceutically acceptable excipient
such as water,
saline, dextrose and glycerol. The vaccines may also include auxiliary
substances such as
emulsifying agents and pH buffers. Doses of the vaccine administered will vary
depending on the
antigenicity of the particular peptide or peptide combination employed in the
vaccine and
characteristics of the animal or human patient to be vaccinated.
The infection-specific vaccine of the invention is directed towards not only
C. psittaci, but
against all forms of Chlamydia including C. pneumoniae, C. trachomatis and C.
pecorurn, and the
vaccine may comprise not just peptides derived from C. psittaci, but also
orthologous peptides and
fragments of such orthologous peptides from other species of Chlamydia and
peptides that are
substantially similar to such peptides.
The present invention also teaches a method of vaccination comprising
administering a
vaccine formulated as described above to an animal either intravenously,
intramuscularly,
subcutaneously, by inhalation of a vapor or mist, or by inoculation in the
form of a liquid, spray,
ointment, pessary or pill into or onto the mucous membranes of the mouth,
nose, lungs or
urogenital tract or colon.
The methods of the invention may be practiced equally with human or non-human
animal
subjects.
The present invention also teaches a method of detecting Chlamydia infection-
specific
proteins produced by the Reticulate Body form of the organism. In this
embodiment, antibodies
raised to the infection-specific proteins are used in an immunological assay
such as an Enzyme
Linked Immunosorbant Assay or Biotin-Avidin assay or a radioimmunoassay or any
other assay
wherein specific antibodies are used to recognize a specific protein. Such
assays may be used to
detect both the quantity of proteins present and also the specificity of
binding of such proteins. In
such an assay, antibodies have attached to them, usually at the Fc portion, a
detectable label, such
as an enzyme, fluorescent marker, a radioactive marker or a Biotin-Avidin
system marker that
allows detection. A biological sample is provided from an animal that has been
putatively exposed
to Chlamydia. Such a sample may be, for example, whole blood, serum, tissue,
saliva or a
mucosal secretion. The sample is then contacted with the labeled antibody and
specific binding, if
any, is detected. Other methods of using infection-specific antibodies to
detect infection-specific
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antigens that are present in cells or tissues include immunofluorescense,
indirect-
immunofluorescense and immunohistochemistry. In immunofluorescense, a
fluorescent dye is
bound directly to the antibody. In indirect-immunofluorescence, the dye is
bound to an anti-
immunoglobulin. Specific binding occurs between antigen and bound antibody is
detected by
virtue of flourescent emissions from the dye moiety. This technique would be
particularly useful,
for instance, for detection of Chlamydia antigen present on a urogenital
mucosal smear.
Other techniques, such as competitive inhibition assays may also be used to
assay for
antigen, and one of ordinary skill in the art will readily appreciate that the
precise methods
disclosed may be modified or varied without departing from the subject or
spirit of the invention
taught herein.
The present invention also teaches a method of detection of Chlamydia
infection-specific
antibodies made against the Reticulate Body. In this embodiment a sample is
provided from an
animal putatively exposed to Chlamydia to determine whether the sample
contains infection-
specific antibodies. Such a sample may be, for example, whole blood, serum,
tissue, saliva or a
IS mucosal secretion. This sample is contacted with infection-specific
antigens such that the amount
and specificity of binding of the antibody may be measured by its binding to a
specific antigen.
Many techniques are commonly known in the art for the detection and
quantification of antigen.
Most commonly, the purified antigen will be bound to a substrate, the antibody
of the sample will
bind via its Fab portion to this antigen, the substrate will then be washed
and a second, labeled
antibody will then be added which will bind to the Fc portion of the antibody
that is the subject of
the assay. The second, labeled antibody will be species specific, i.e., if the
serum is from a
human, the second, labeled antibody will be anti-human-IgG antibody. The
specimen will then be
washed and the amount of the second, labeled antibody that has been bound will
be detected and
quantified by standard methods.
The present invention also teaches a method of treating a Chlamydial infection
by
directing a therapeutic. agent against a specific target, such as: (i) an
infection-specific protein of
Chlanrydia, (ii) a gene that encodes an infection-specific protein of
Chlamydia and (iii) an RNA
transcript that encodes an infection-specific protein of Chlamydia, wherein
said therapeutic agent
interacts with said target to affect a reduction in pathology.
For example, the present invention teaches a method of treating chlamydial
infection
wherein antisense technology is used to prevent the expression of infection-
specific genes, thereby
preventing the pathologies associated these proteins and preventing
reproduction of the RB phase
of Chlamydia. In this embodiment, RNA molecules complementary to transcripts
of infection
specific genes are introduced into the host cells that contain Chlamydia, and
by binding to the
mRNA transcripts of the infection-specific genes, prevent translation and
therefore expression of
the infection-specific proteins that are associated with pathogenesis.
The invention may be practiced to produce a vaccine against any species of
Chlamydia,
including C. psittaci, C. pecorum, C. trachomaris and C. pneumoniae.
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The following examples illustrate various embodiments of the invention.
EXAMPLE I: Homologous Sequences
The DNA and protein sequences discussed herein are shown in SEQ ID NOS:1-18.
These sequences refer to infection-specific proteins and to the DNA sequences
that encode these
proteins. Although these sequences are from C. psittaci and C. trachomatis, it
would be equally
possible to substitute in the present invention, the orthologs of these
sequences from other
Chlamydia species such as C. pecorum and C. pneumoniae.
Such orthologous sequences may be obtained from the appropriate organisms by
isolation
of the genome of the organism, digestion with restriction enzymes, separation
of restriction
fragments by electrophoresis and purification of these fragments and selection
of fragments of
appropriate size. Identity of the fragments can be confirmed by dot-blot and
by standard DNA
sequencing techniques. The orthologous sequences in different Chlamydia
species may also be
found by selection of appropriate PCR primers {selected from appropriate
regions flanking the
IS Chlamydia gene of interest), and the use of these primers in a PCR
reaction, using the genome of
the particular species of Chlamydia of interest as a template, to amplify the
ortholog of interest.
Such PCR primers would be selected from the flanking regions to allow specific
amplification of
the target gene. The fragments so obtained could then be run on a gel to check
size and sequenced
and compared against the known sequences to determine sequence identity.
The degree of sequence identity between the infection-specific genes of C.
psittaci or C.
trachomatis and their orthologs from C. pecorum and C. pneumoniae, may be
determined by
comparing sequences using the National Center for Biotechnology Information
(NCBI) Basic Local
Alignment Search Tool (BLAST) as described herein.
Orthologues of interest infection-specific proteins are characterized by
possession of at
least SO% or greater sequence identity counted over the full length alignment
with one of the
disclosed amino acid sequences of the C. psittaci or C. trachomatis infection-
specific proteins
using gapped blastp set to default parameters (described herein).
EXAMPLE 2: Heterologous Expression of Infection-Specific Antigens
Methods for expressing large amounts of protein from a cloned gene introduced
into
Escherichia coli (E. coli) may be utilized for the purification of the
Chlamydia peptides. Methods
and plasmid vectors for producing fusion proteins and intact native proteins
in bacteria are well
known and are described in Sambrook et al. (1989). Such fusion proteins may be
made in large
amounts, are relatively simple to purify, and can be used to produce
antibodies. Native proteins
can be produced in bacteria by placing a strong, regulated promoter and an
efficient ribosome
binding site upstream of the cloned gene. If low levels of protein are
produced, additional steps
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may be taken to increase protein production; if high levels of protein are
produced, purification is
relatively easy.
Often, proteins expressed at high levels are found in insoluble inclusion
bodies. Methods
for extracting proteins from these aggregates are described in chapter 17 of
Sambrook et al.
(1989). Vector systems suitable for the expression of lacZ fusion genes
include the pUC series of
vectors (Ruther et al. (1983)), pEXl-3 (Stanley and Luzio (1984)) and pMR100
(Gray et al.
(1982)). Vectors suitable for the production of intact native proteins include
pKC30 (Shimatake
and Rosenberg (1981);>, pKKI77-3 (Amann and Brosius (1985)) and pET-3 (Studiar
and Moffatt
( 1986)).
Fusion proteins may be isolated from protein gels, lyophilized, ground info a
powder and
used as antigen preparations.
Mammalian or other eukaryotic host cells, such as those of yeast, filamentous
fungi,
plant, insect, amphibian or avian species, may also be used for protein
expression, as is well
known in the art. Examples of commonly used mammalian host cell lines are VERO
and HeLa
cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines,
although it will be
appreciated by the skilled practitioner that other prokaryotic and eukaryotic
cells and cell lines may
be appropriate for a variety of purposes, e.g., to provide higher expression,
post-translational
modification, desirable glycosylation patterns, or other features.
Additionally, peptides, particularly shorter peptides, may be chemically
synthesized,
avoiding the need for purification from cells or culture media. It is known
that peptides as short as
3 amino acids can act as an antigenic determinant and stimulate an immune
response. Such
peptides may be administered as vaccines in ISCOMs (Immune Stimulatory
Complexes) as
described by Janeway & Travers, Immunobiology: The Immune System In Health and
Disease,
13.21 (Garland Publishing, Inc. New York, 1997). Accordingly, one aspect of
the present
invention includes small peptides encoded by the nucleic acid molecules
disclosed herein. Such
peptides include at least 5, and may be at least 10, 15, 20, 25, or 30 or more
contiguous amino
acids of the polypeptide sequences described herein.
EXAMPLE 3: Production of Antibodies Specific for
Infection-Specific Antigens
Antibody against infection-specific antigen is encompassed by the present
invention,
particularly for the detection of Chlamydia infection-specific antigen. Such
antibody may be
produced by inoculation of an animal such as a guinea-pig or a monkey with
infection-specific
antigen produced as described above. Such antigen may be a polypeptide as
disclosed herein, such
as a complete or partial polypeptide from C. psittaci, C. trachomatis, C.
pneumoniae or C.
pecorum. As discussed above, any molecule that can elicit a specific,
protective immune response
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may be used as a vaccine, but since a minimum of three amino acids are
required to do this, a
vaccine should comprise at least three amino acids.
The peptide for use in the vaccine of the invention may be naturally derived
or may be
synthetic such as those synthesized on a commercially available peptide
synthesizer. The peptide
may also comprise a complete or partial peptide derived from the C. pneumoniae
or C. pecorum
infection-specific orthologs of the C. trachomatis or C. psittaci proteins as
set out herein.
In one method of production, a polyclonal antibody is produced by providing a
purified
peptide which is diluted to 100 micrograms per milliliter in sterile saline
and mixed with RiBi
Trivalent Adjuvant (RiBi Immunochem Inc). The antigen/adjuvant emulsion is
then administered
(0 to an anaesthetized guinea pig using a procedure as provided by the
manufacturer. Serum is
collected 14 days after secondary and tertiary immunizations.
Monoclonal antibody to epitopes of the Chlamydia peptides identified and
isolated as
described can be prepared from murine hybridomas according to the classical
method of Kohler
and Milstein (1975) or derivative methods thereof. Briefly, a mouse is
repetitively inoculated with
15 a few micrograms of the selected purified protein over a period of a few
weeks. The mouse is
then sacrificed, and the antibody-producing cells of the spleen isolated. The
spleen cells are fused
by means of polyethylene glycol with mouse myeloma cells, and the excess
unfused cells destroyed
by growth of the system on selective media comprising aminopterin, e.g.,
Hypoxanthene,
Aminopterin and Thymidine (HAT) medium. The successfully fused cells are
diluted and aliquots
20 of the dilution placed in wells of a microtiter plate where growth of the
culture is continued.
Antibody-producing clones are identified by detection of antibody in the
supernatant fluid of the
wells by immunoassay procedures, such as ELISA, as originally described by
Engvall (1980), and
derivative methods thereof. Selected positive clones can be expanded and their
monoclonal
antibody product harvested for use. Detailed procedures for monoclonal
antibody production are
25 described in Harlow and Lane ( 1988).
An alternative approach to raising antibodies against the Chlamydia peptides
is to use
synthetic peptides synthesized on a commercially available peptide synthesizer
based upon the
amino acid sequence of the peptides predicted from nucleotide sequence data.
In another embodiment of the present invention, monoclonal antibodies that
recognize a
30 specific Chlamydia peptide are produced. Optimally, monoclonal antibodies
will be specific to
each peptide, i.e., such antibodies recognize and bind one Chlamydia peptide
and do not
substantially recognize or bind to other proteins, including those found in
uninfected human cells.
The determination that an antibody specifically detects a particular Chlamydia
peptide is
made by any one of a number of standard immunoassay methods; for instance, the
western blotting
35 technique (Sambrook et al., 1989). To determine that a given antibody
preparation (for instance
from a guinea pig) specifically detects one Chlamydia peptide by western
blotting, total cellular
protein is extracted from a sample of blood from an unexposed subject and from
a sample of blood
from an exposed subject. As a positive control, total cellular protein is also
extracted from
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Chlamydia cells grown in vitro. These protein preparations are then
electrophoresed on a sodium
dodecyl sulfate-polyacrylamide gel. Thereafter, the proteins are transferred
to a membrane (for
example, nitrocellulose) by western blotting, and the antibody preparation is
incubated with the
membrane. After washing the membrane to remove non-specifically bound
antibodies, the
presence of specifically bound antibodies is detected by the use of an anti-
guinea pig antibody
conjugated to an enzyme such as alkaline phosphatase; application of the
substrate 5-bromo-4-
chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of
a dense blue
compound by immuno-localized alkaline phosphatase. Antibodies which
specifically detect the
Chlamydia protein will, by this technique, be shown to bind to the Chlamydia-
extracted sample at
a particular protein band (which will be localized at a given position on the
gel determined by its
molecular weight) and to the proteins extracted from the blood of the exposed
subject. No
significant binding will be detected to proteins from the unexposed subject.
EXAMPLE 4: Use of Infection-Specific Sequences
and their Corresponding Peptides and
Antibodies in Diagnostic Assays
Another aspect of the present invention is a method for detecting the presence
of anti-
Chlamydia antibodies that react with infection-specific Chlamydia proteins,
Chlamydia peptides
and Chlamydia nucleic acid sequences in biological samples. These methods
include detection of
antigen and antibody by ELISA and similar techniques, the detection of
proteins in a tissue sample
by immunofluorescence and related techniques and the detection of specific DNA
sequences by
specific hybridization and amplification.
One aspect of the invention is an ELISA that detects anti-Chlamydia antibodies
in a
medical specimen. An immunostimulatory infection-specific Chlamydia peptide of
the present
invention is employed as an antigen and is preferably bound to a solid matrix
such as a crosslinked
dextrin such as SEPHADEX (Pharmacia, Piscataway, NJ), agarose, polystyrene, or
the wells of a
microtiter plate. The polypeptide is admixed with the specimen, such as blood,
and the admixture
is incubated for a sufficient time to allow antibodies present in the sample
to immunoreact with the
polypeptide. The presence of the positive immunoreaction is then determined
using an ELISA
assay, usually involving the use of an enzyme linked to an anti-immunoglobulin
that catalyzes the
conversion of a chromogenic substrate.
In one embodiment, the solid support to which the polypeptide is attached is
the wall of a
microtiter assay plate. After attachment of the polypeptide, any nonspecific
binding sites on the
microtiter well walls are blocked with a protein such as bovine serum albumin.
Excess bovine
serum albumin is removed by rinsing and the medical specimen is admixed with
the polypeptide in
the microtiter wells. After a sufficient incubation time, the microtiter wells
are rinsed to remove
excess sample and then a solution of a second antibody, capable of detecting
human antibodies is
added to the wells. This second antibody is typically linked to an enzyme such
as peroxidase,
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alkaline phosphatase or glucose oxidase. For example, the second antibody may
be a peroxidase-
labeled goat anti-human antibody. After further incubation, excess amounts of
the second
antibody are removed by rinsing and a solution containing a substrate for the
enzyme label (such
as hydrogen peroxide for the peroxidase enzyme) and a color-forming dye
precursor, such as
o-phenylenediamine is added. The combination of Chlamydia peptide (bound to
the wall of the
well), the human anti-Chlamydia antibodies (from the specimen), the enzyme-
conjugated anti-
human antibody and the color substrate will produce a color that can be read
using an instrument
that determines optical density, such as a spectrophotometer. These readings
can be compared to a
negative control such as a sample known to be free of anti-Chlamydia
antibodies. Positive
readings indicate the presence of anti-Chlamydia antibodies in the specimen,
which in turn indicate
a prior exposure of the patient to Chlamydia.
In another embodiment, antibodies that specifically recognize a Chlamydia
peptide
encoded by the nucleotide sequences disclosed herein are useful in diagnosing
the presence of
infection-specific Chlamydia antigens in <i subject or sample. For example,
detection of infection-
specific antigens that are present in cells or tissues may be done by
immunofluorescence, indirect-
immunofluorescense and immunohistochemistry. In immunofluorescense, a
fluorescent dye is
bound directly to the antibody. In indirect-immunofluorescence, the dye is
bound to an anti-
immunoglobulin. Specific binding occurs between antigen and bound antibody is
detected by
virtue of fluorescent emissions from the dye moiety. This technique may be
particularly useful,
for instance, for detection of Chlamydia antigen present on a urogenital
mucosal smear.
Chlamydia may be present in urogenital mucosa, and a smear on a glass slide
may be fixed and
bathed in a solution containing an antibody specific to the infection-specific
antigen. The slide is
then washed to remove the unbound antibody, and a fluorescent anti-
immunoglobulin antibody is
added. The slide is washed again, and viewed microscopically under an
appropriate wavelength of
light to detect fluorescence. Fluorescence indicates the presence of Chlamydia
antigen.
Alternatively, a urogenital mucosal smear may be taken, the sample cultured
with HeLa cells to
produce large amounts of the RB form, and immunofluorescence may then be used
to detect
infection-specific Chlamydia antibodies.
Another aspect of the invention includes the use of nucleic acid primers to
detect the
presence of Chlamydia nucleic acids that encode infection-specific antigens in
body samples and
thus to diagnose infection. In other embodiments, these oligonucleotide
primers will comprise at
least 15 contiguous nucleotides of a DNA sequence as shown in SEQ ID NOS: 1,
3, 5, 7, 9, 11,
13, 15, or 17. In other embodiments, such oligonucleotides may comprise at
least 20 or at least
25 or more contiguous nucleotides of the aforementioned sequences.
One skilled in the art will appreciate that PCR primers are not required to
exactly match
the target gene sequence to which they anneal. Therefore, in another
embodiment, the
oligonucleotides will comprise a sequence of at least 15 nucleotides and
preferably at least 20
nucleotides, the oligonucleotide sequence being substantially similar to a DNA
sequence set forth
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in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, and 17. Such oligonucleotides may
share at least about
75 % , 85 % , 90 % or greater sequence identity.
The detection of specific nucleic acid sequences in a sample by polymerase
chain reaction
amplification (PCR) is discussed in detail in Innis et al., (1990). PCR
Protocols: A Guide to
Methods and Applications, Academic Press: San Diego, part 4 in particular. To
detect Chlamydia
sequences, primers based on the sequences disclosed herein would be
synthesized, such that PCR
amplification of a sample containing Chlamydia DNA would result in an
amplified fragment of a
predicted size. If necessary, the presence of this fragment following
amplification of the sample
nucleic acid could be detected by dot blot analysis. PCR amplification
employing primers based
on the sequences disclosed herein may also be employed to quantify the amounts
of Chlamydia
nucleic acid present in a particular sample (see chapters 8 and 9 of Innis et
al., (1990)).
Alternatively, probes based on the nucleic acid sequences described herein may
be labeled
with suitable labels (such a P'Z or biotin) and used in hybridization assays
to detect the presence of
Chlamydia nucleic acid in provided samples.
Reverse-transcription PCR using these primers may also be utilized to detect
the presence
of Chlamydia RNA which is indicative of an ongoing infection.
EXAMPLE 5: Production of Chlamydia Vaccines
The purified peptides of the present invention may be used directly as
immunogens for
vaccination. Methods for using purified peptides as vaccines are well known in
the art and are
described in Yang et al. ( 1991 ), Andersen ( 1994) and Jardim et al. ( I
990). As is well known in
the art, adjuvants such as alum, Complete Freund's Adjuvant (CFA) and
Incomplete Freund's
Adjuvant (IFA) may t>e used in formulations of purified peptides as vaccines.
Accordingly, one
embodiment of the present invention is a vaccine comprising one or more
immunostimulatory C.
trachomatis or C. psittaci peptides encoded by nucleotide sequences as shown
in the attached
sequence listing, together with a pharmaceutically acceptable adjuvant.
Additionally a vaccine may comprise a defined fraction of the disclosed
peptide of C.
trachomatis or C. psittaci or may comprise a peptide wherein the gene coding
for the peptide
shows substantial similarity to the DNA sequences disclosed herein, such as
for orthologous genes
of C. pneumoniae or C. pecorum.
Additionally, the vaccines may be formulated using a peptide according to the
present
invention together with a pharmaceutically acceptable excipient such as water,
saline, dextrose and
glycerol. The vaccines may also include auxiliary substances such as
emulsifying agents and pH
buffers.
It will be appreciated by one of skill in the art that vaccines formulated as
described above
may be administered in a number of ways including subcutaneous, infra-muscular
and infra-venous
injection. Doses of the vaccine administered will vary depending on the
antigenicity of the
particular peptide or peptide combination employed in the vaccine, and
characteristics of the
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animal or human patient to be vaccinated. While the determination of
individual doses will be
within the skill of the administering physician, it is anticipated that doses
of between 1 microgram
and 1 milligram will be employed.
As with many vaccines, the vaccines of the present invention may routinely be
administered several times over the course of a number of weeks to ensure that
an effective
immune response is triggered. Where such multiple doses are administered, they
will normally be
administered at from two to twelve week intervals, more usually from three to
five week intervals.
Periodic boosters at intervals of 1-5 years, usually three years, may be
desirable to maintain the
desired levels of protective immunity.
Alternatively, multiple immunostimulatory peptides may also be administered by
expressing the nucleic acids encoding the peptides in a nonpathogenic
microorganism, and using
this transformed nonpathogenic microorganism as a vaccine.
Finally, a recent development in the field of vaccines is the direct injection
of nucleic acid
molecules encoding peptide antigens, as described in Janeway & Travers,
(1997). Thus, plasmids
which include nucleic acid molecules described herein, or which include
nucleic acid sequences
encoding peptides according to the present invention may be utilized in such
DNA vaccination
methods.
The vaccine of the invention may be used to inoculate potential animal targets
of any of
the chlamydial diseases including those caused by C. trachomatis, C. psittaci,
C. pneumoniae or
C. pecorum. Indeed the vaccine of the invention may be used to inoculate
animals against any
disease that shows immunological cross-protection as a result of exposure to
infection-specific
Chlamydia antigen. The protein or polypeptide is present in the vaccine in an
amount sufficient to
induce a protective immune response whether through humoral or cell mediated
pathways or
through both. Such a response protects the immunized animal against chlamydial
infections
specifically by raising an immune response against the Reticulate Body form of
Chlamydia.
The above embodiments are set out only by way of example and are not intended
to be
exclusive, one skilled in the art will understand that the invention may be
practiced in various
additional ways without departing from the subject of the spirit of the
invention.
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REFERENCES
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Blanco, D. R., et al. (1995) J. Bacteriol. 177:3556-3562.
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Sambrook et al. (1989) Molecular Cloning: A Laboratory Manuul, 2nd ed., vol. I-
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Shimatake and Rosenberg (1981). Nature (London) 292:128.
Stanley and Luzio ( 1984). EMBO J. 3:1429.
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Yuan, Y., et al. (1992) Infect Immun 60: 2288-2296.
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SEQUENCE LISTING
<110> Oregon Statc~ University
<120> Methods of use for infection-specific INCA, INCB, and
INCC proteins of Chlamydia
<130> 52297
<140>
<141>
<150> 60/082,588
<151> 1998-04-21
<150> 60/082,438
<151> 1998-04-20
<150> 60/086,450
<151> 1998-05-22
<160> 29
<170> PatentIn Ver. 2.0
<210>
1
<211>
534
<212>
DNA
<213> trachomatis
Chlamydia
<220>
<221>
CDS
<222> )..(534)
(1
<400>
1
atg aagttc ttattactt agctta atgtctttg tcatctcta cct 48
aaa
Met LysPhe LeuLeuLeu SerLeu MetSerLeu SerSerLeu Pro
Lys
1 5 10 15
aca gcaget aattctaca ggcaca attggaatc gttaattta cgt 96
tt:t
Thr AlaAla AsnSerThr GlyThr IleGlyIle ValAsnLeu Arg
Phe
20 25 30
cgc ctagaa gagtctget cttggg aaaaaagaa tctgetgaa ttc 194
tgc
Arg LeuGlu GluSerAla LeuGly LysLysGlu SerAlaGlu Phe
Cys
35 40 45
gaa atgaaa aaccaattc tctaac agcatgggg aagatggag gaa 192
aag
Glu MetLys AsnGlnPhe SerAsn SerMetGly LysMetGlu Glu
Lys
50 55 60
gaa tcttct atctattcc aagctc caagacgac gattacatg gaa 290
ctg
Glu SerSer IleTyrSer LysLeu GlnAspAsp AspTyrMet Glu
Leu
65 70 75 80
ggt tecgag aecgeaget gccgaa ttaagaaaa aaattcgaa gat 288
eta
Gly SerGlu 'ChrAlaAla AlaGlu LeuArgLys LysPheGlu Asp
Leu
85 90 95
eta gcagaa -.acaacaca getcaa gggcagtat taccaaata tta 336
tct
Leu AlaGlu TyrAsnThr AlaGln GlyGlnTyr TyrGlnIle Leu
Ser
1
CA 02326002 2000-10-19
WO 99/53948 PCT/US99/08744
100 105 110
aaccaaagt aatttc aagcgcatg caaaag attatggaa gaagtg aaa 389
AsnGlnSer AsnPhe LysArgMet GlnLys IleMetGlu GluVal Lys
115 120 125
aaagettet gaaact gtgcgtatt caagaa ggettgtca gtcctt ctt 932
LysAlaSer GluThr ValArgIle GlnGlu GlyLeuSer ValLeu Leu
130 135 190
aacgaagat attgtc ttatctatc gatagt tcggcagat aaaacc gat 480
AsnGluAsp IleVal LeuSerIle AspSer SerAlaAsp LysThr Asp
145 150 155 160
getgttatt aaagtt cttgatgtt etttte aaaataatt aacatg ega 528
AlaValIle LysV,alLeuAspVal LeuPhe LysIleIle AsnMet Arg
165 170 175
agctag 534
Ser
<210>
2
<211> 7
17
<212>
PRT
<213> lamydiatrachomatis
Ch
<400>
2
MetLys LysPheLeu LeuLeu SerLeuMet SerLeuSer SerLeuPro
1 5 10 15
ThrPhe AlaAlaA.snSerThr GlyThrIle GlyIleVal AsnLeuArg
20 25 30
ArgCys LeuG1uGlu SerAla LeuGlyLys LysGluSer AlaGluPhe
35 40 45
GluLys MetLysAsn GlnPhe SerAsnSer MetGlyLys MetGluGlu
50 55 60
GluLe~uSerSerI:leTyrSer LysLeuGln AspAspAsp TyrMetGlu
65 70 75 80
GlyLeu SerGluThr AlaAla AlaG1uLeu ArgLysLys PheGluAsp
85 90 95
LeuSer AlaGluTyr AsnThr AlaGlnGly GlnTyrTyr GlnIleLeu
100 105 110
AsnGln SerAsnF?heLysArg MetGlnLys IleMetGlu GluValLys
115 120 125
LysAla SerGluThr ValArg IleGlnGlu GlyLeuSer ValLeuLeu
I30 135 140
AsnGlu AspIleVal LeuSer IleAspSer SerAlaAsp LysThrAsp
145 150 155 160
AlaVal IleLysVal LeuAsp ValLeuPhe LysIleIle AsnMetArg
165 170 175
2
CA 02326002 2000-10-19
WO 99/53948 PCT/US99/08744
Ser
<210> 3
<211> 846
<212> DNA
<213> C;hlamydia trachomatis
<220>
<221> CDS
<222> (1)..(846)
<400> 3
atg aat atgatt tgtgattgc gtgtct cgcataact ggggat cga 48
cgc
Met Asn MetIle CysAspCys ValSer ArgIleThr GlyAsp Arg
Arg
1 5 10 15
gtc aag attgtt ctgattgat ggagcg attgatcct cattca tat 96
aat
Val Lys IleVal LeuIleAsp GlyA:LaIleAspPro HisSer Tyr
Asn
20 25 30
gag atg aagggg gatgaagac cgaatg getatgagc cagctg att 144
gtg
Glu Met LysGly AspGluAsp ArgMet AlaMetSer GlnLeu Ile
Val
35 40 45
ttt tgc ggttta ggtttagag cattca getagttta cgtaaa cat 192
aat
Phe Cys GlyLeu GlyLeuGlu HisSer AlaSerLeu ArgLys His
Asn
50 55 60
cta gag aaccca aaagtcgtt gattta ggtcaacgt ttgctt aac 290
ggt
Leu Glu AsnPro LysValVal AspLeu GlyGlnArg LeuLeu Asn
Gly
65 70 75 80
aaa aac tttgat cttctgagt gaagaa ggattccct gaccca cat 288
tgt
Lys Asn PheAsp LeuLeuSer GluGlu GlyPhePro AspPro His
Cys
85 90 95
atttggacg gatatgaga gtatgg ggtgetget gtaaaagag atgget 336
I1eTrpThr AspMetArg ValT.rpGlyAlaAla ValLysGlu MetAla
100 105 110
gcggcatta attcaacaa tttcct caatatgaa gaagatttt caaaag 384
AlaAlaLeu IleGlnGln PhePro GlnTyrGlu GluAspPhe GlnLys
115 120 125
aatgcggat cagatctta tcagag atggaggaa cttgatcgt tgggca 432
AsnAlaAsp GlnIleLeu SerGlu MetGluGlu LeuAspArg TrpAla
130 135 140
gtgcgttct ctctctacg attcct gaaaaaaat cgctattta gtcaca 480
ValArgSer LeuSerThr IlePro GluLysAsn ArgTyrLeu ValThr
145 150 155 160
ggccacaat gcgttcagt tacttt actcgtcgg tatctatcc tctgat 528
GlyHisAsn AlaPheSer TyrPhe ThrArgArg TyrLeuSer SerAsp
165 170 175
gcggagaga gtgtctggg gaatgg agatcgcgt tgcatttct ccagaa 576
AlaGluArg ValSerGly G1uTrp ArgSerArg CysIleSer ProGlu
180 185 190
3
CA 02326002 2000-10-19
WO 99/53948 PCT/US99/08744
ggg ttg tct cct gag get cag att agt atc cga gat att atg cgt gta 624
G1y Leu Ser Pro Glu Ala Gln I1e Ser Ile Arg Asp Ile Met Arg Val
195 200 205
gtg gag tat atc tct gca aac gat gta gaa gtt gtc ttt tta gag gat 672
Val Glu Tyr Ile Ser Ala Asn Asp VaI Gl.u Val Val Phe Leu Glu Asp
210 215 220
acg tta aat caa gat get ttg aga aag att gtt tct tgc tct aag agc 720
Thr Leu Asn Gln Asp Ala Leu Arg Lys Ile Val Ser Cys Ser Lys Ser
225 230 235 240
gga caa aag att cgt ctc get aag tet ect tta tat agc gat aat gte 768
Gly Gl.n Lys Ile Arg Leu Ala Lys Ser Pro Leu Tyr Ser Asp Asn Val
245 250 255
tgt gat aac tat t:tt agc acg ttc cag cac aat gtt cgc aca att aca 816
Cys Asp Asn Tyr E'he Ser Thr Phe Gln His Asn Val Arg Thr Ile Thr
260 265 270
gaa gaa ttg gga ggg act gtt ctt gaa tag 846
Glu Gl_u Leu Gly Gly Thr Val Leu G1u
275 280
<210> 4
<211> 281
<212> PRT
<213> Chlamydia trachomatis
<900> 4
Met Asn MetIle CysAspCys ValSerArg IleThr GlyAspArg
Arg
1 5 10 15
Val Lys IleVal LeuIleAsp GlyAlaIle AspPro HisSerTyr
Asn
20 25 30
Glu Met LysGly AspGluAsp ArgMetAla MetSer GlnLeuIle
Val
35 40 45
Phe Cys GlyLeu GlyLeuGlu HisSerAla SerLeu ArgLysHis
Asn
50 55 60
Leu G:Lu Gly Asn Pro Lys Val Val Asp Leu Gly Gln Arg Leu Leu Asn
65 70 75 80
Lys Asn Cys Phe Asp Leu Leu Ser Glu Glu Gly Phe Pro Asp Pro His
85 90 95
Ile T.rp Thr Asp Met Arg Val Trp Gly Ala Ala Val Lys Glu Met Ala
100 105 110
Ala Ala Leu Ile Gln Gln Phe Pro Gln Tyr Glu Glu Asp Phe Gln Lys
115 120 125
Asn Ala Asp Gln Ile Leu Ser Glu Met Glu Glu Leu Asp Arg Trp Ala
130 135 140
Val Arg Ser Leu Ser Thr Ile Pro Glu Lys Asn Arg Tyr Leu Val Thr
145 150 155 160
4
CA 02326002 2000-10-19
WO 99/53948 PCT/US99/08744
G1y HisAsnAla PheSer TyrPhe ThrArgArg TyrLeuSer SerAsp
165 170 175
Ala GluArgVal SerGly GluTrp ArgSerArg CysIleSer ProGlu
180 185 190
Gly LeuSerPro GluAla GlnIle SerTleArg AspIleMet ArgVal
195 200 205
Val GluTyrIle SerAla AsnAsp ValGluVal ValPheLeu GluAsp
210 215 220
Thr LeuAsnGln A~~pAla LeuArg LysIleVal SerCysSer LysSer
225 230 235 240
Gly GlnLysIle ArgLeu AlaLys SerProLeu TyrSerAsp AsnVal
245 250 255
Cys AspAsnTyr PheSer ThrPhe GlnHisAsn ValArgThr IleThr
260 265 270
Glu Glu Leu Gly G:_y Thr Val Leu Glu
275 280
<210>
'_i
<211>
861
<212>
DNA
<213> trachomatis
Chlamydia
<220>
<221>
CDS
<222> 861)
(1)..(
<400>
atg gtg ataar_tattttagca cgttccagc acaatg~ttcgcacaa 98
to
Met Val IleThr IleLeuAla ArgSerSer ThrMetPhe AlaGln
Ser
1 5 10 15
tta aag aattgg gagggactg ttcttgaat agagataat gcaatt 96
cag
Leu Lys AsnTrp GluGlyLeu PheLeuAsn ArgAspAsn AlaIle
Gln
20 25 30
get tec gtagag gatctttgt gttaattat gatcactca gacgtc 144
tgg
Ala Ser ValGlu AspLeuCys ValAsnTyr AspHisSer AspVal
Trp
35 40 45
tta cac attact ttttctctg cctgcaggg gcaatgget getatt 192
tgt
Leu His IleThr PheSerLeu ProAlaGly AlaMetAla AlaIle
Cys
50 55 60
att ccg aatgga getggtaaa agtactttg cttaagget tcttta 240
ggg
Ile Pro AsnGly AlaGlyLys SerThrLeu LeuLysAla SerLeu
Gly
65 70 75 80
gga att cgtget tcttctgge caaagettg ttctttggt cagaga 288
etg
Gly Ile ArgAla SerSerGly GlnSerLeu PhePheGly GlnArg
Leu
85 90 95
ttt aag gcacat catagaata gcctatatg cctcaaaga gcgagt 336
tcc
Phe Lys AlaHis HisArgIle AlaTyrMet ProGlnArg AlaSer
Ser
5
CA 02326002 2000-10-19
WO 99/53948 PCT/US99/08744
100 105 110
gtggat:tgggat tt:cccaatg actgttctt gatctc gtgttgatg ggg 384
ValAsp TrpAsp PheProMet ThrValLeu AspLeu ValLeuMet Gly
115 120 125
tgttac ggctat as ggaata tggaatcgt atttcc actgatgat cgt 432
a
CysTyr GlyTyr LysGlyIl.eTrpAsnArg IleSer ThrAspAsp Arg
130 135 190
caggag getatg cgtatttta gagcgggtt ggtttg gaagetttt gca 480
GlnGlu AlaMet ArgIleLeu G.LuArgVal GlyLeu GluAlaPhe Ala
145 150 155 160
aatcgt caaata ggtaagctc tctggagga caacaa cagagaget ttt 528
AsnArg GlnIle GlyLysLeu SerGlyGly GlnGln GlnArgAla Phe
165 170 175
ttagcg cggtca ttaatgcaa aaagcagat ttgtat ctcatggat gag 576
LeuAla ArgSer LeuMetGln LysAlaAsp LeuTyr LeuMetAsp Glu
180 185 190
ctgttc tctgcg atcgatatg gcctcttat cagatg gttgtagat gtt 629
LeuPhe SerAla IleAspMet AlaSerTyr GlnMet.ValValAsp Val
195 200 205
ttgcaa gagctt aaaagcgaa gggaagact attgtg gtcattcat cat 672
LeuGln GluLeu LysSerGlu GlyLysThr IleVal ValIleHis His
210 215 220
gatttg agtaat gtccggaag ctttttgat catgtg attttatta aat 720
AspLeu SerAsn ValArgLys LeuPheAsp HisVal IleLeuLeu Asn
225 230 235 240
aagcat cttgtg tgctctgga agcgtagaa gaatgc ttgactaaa gaa 768
LysHis LeuVal CysSerGly SerValGlu GluCys LeuThrLys Glu
245 250 255
gccatt tttcag gettatggg tgtgacttg agcttt tggattaca cac 816
AlaIl.ePheGln AlaTyrGly CysAspLeu SerPhe TrpIleThr His
260 265 270
tcaaat tgtcta gaggcaagt accaaggat cgtget agatgctga 861
SerA,>nCysLeu GluAlaSer ThrLysAsp ArgAla ArgCys
275 280 285
<210>
6
<211> 86
2
<212> RT
P
<213> hlamydia t:rachomatis
C
<400> 6
Met Ser Val Ile Thr Ile Leu Ala Arg Ser Ser Thr Met Phe Ala Gln
1 5 10 15
Leu Gln Lys Asn Trp Glu Gly Leu Phe Leu Asn Arg Asp Asn Ala Ile
20 25 30
Ala Trp Ser Val Glu Asp Leu Cys Val Asn Tyr Asp His Ser Asp Val
35 40 45
6
CA 02326002 2000-10-19
WO PCT/US99/08744
99/53948
LeuCys HisIleThr PheSerLeu ProA.LaGlyA1aMet AlaAla Ile
50 55 60
IleGly ProAsnG:LyAlaGlyLys SerThr LeuLeuLys AlaSer Leu
65 70 75 80
GlyLeu IleArgAla SerSerG:LyGlnSer LeuPhePhe GlyGln Arg
85 90 95
PheSe:rLysAlaH.isHisArgIle AlaTyr MetProGln ArgAla Ser
100 105 110
ValAsp TrpAspPhe ProMetThr ValLeu AspLeuVal LeuMet Gly
115 120 125
Cys Tyr Gly Tyr Lys Gly Ile Trp Asn Arg Ile Ser Thr Asp Asp Arg
130 135 190
Gln Glu Ala Met Arg Ile Leu Glu Arg Val Gly Leu Glu Ala Phe Ala
145 150 155 160
Asn Arg Gln Ile Gly Lys Leu Ser Gly Gly Gln Gln Gln Arg Ala Phe
165 170 175
Leu Ala Arg Ser Leu Met Gln Lys Ala Asp Leu Tyr Leu Met Asp Glu
180 185 190
Leu Phe Ser Ala Ile Asp Met Ala Ser Tyr Gln Met Val Val Asp Val
195 200 205
Leu Gln Glu Leu Lys Ser Glu Gly Lys Thr Ile Val Val Ile His His
210 215 220
Asp Leu Ser Asn Val Arg Lys Leu Phe Asp His Val Ile Leu Leu Asn
225 230 ~ 235 240
Lys His Leu Val Cys Ser Gly Ser Val Glu Glu Cys Leu Thr Lys Glu
2.95 250 255
Ala Il.e Phe Gln Ala Tyr Gly Cys Asp Leu Ser Phe Trp Ile Thr His
260 265 270
Ser Asn Cys Leu Glu Ala Ser Thr Lys Asp Arg Ala Arg Cys
275 280 285
<210> 7
<211> 1068
<212> DNA
<213> Chlamydia psittaci
<220>
<221> CDS
<222> (1)..(1068)
<400> 7
atg aca gta tcc aca gac aac aca agt cct gta ata tcg aga gcg tcc 48
Met Thr Val 5er Thr Asp Asn Thr Ser Pro Val Ile Ser Arg Ala Ser
1 5 10 15
7
CA 02326002 2000-10-19
WO 99/53948 PCT/US99/08744
tca cct: act ttt gga gat cat ggt aag gat ttc gac aac aat aaa att 96
Ser Pro Thr Phe Gly Asp His Gly Lys Asp Phe Asp Asn Asn Lys Ile
20 25 30
ata ccc att tca ata gaa get cca act tct tca get get get gta ggg 144
Ile Pro Ile Ser I.Le Glu Ala Pro Thr Ser Ser Ala Ala Ala Val Gly
35 40 45
get aaa acg get atc gag cct gaa gga aga agc cca cta ctt caa agg 192
Ala Lys Thr Ala Ile Glu Pro Glu Gly Arg Ser Pro Leu Leu Gln Arg
50 55 60
att tgc tat ctt gtt aaa att atc get gcc atc gcc ctc ttt gtt gtt 240
Ile Cys Tyr Leu Val Lys Ile Ile Ala Ala Ile Ala Leu Phe Val Val
65 70 75 80
ggt atc gca gcc tta gtt tgc tta tat ctc ggt agc gtt atc tca acg 288
Gly Ile Ala Ala Leu Va.1 Cys Leu Tyr Leu Gly Ser Val Ile Ser Thr
85 90 95
cct tct ctt att ctt atg ctt gcg atc atg ctt gta tcc ttt gtg atc 336
Pro Ser Leu Ile Leu Met Leu Ala Ile Met Leu Val Ser Phe Val Ile
100 105 110
gtt att acg gca att cga gat ggc aca ccg tct caa gtg gtc cgt cac 384
Val Ile Thr Ala Ile Arg Asp Gly Thr Pro Ser Gln Val Val Arg His
115 120 125
atg aaa cag caa a.tt cag caa ttt ggc gaa gaa aac acg cgt tta cat 432
Met Lys Gln Gln Ile Gln Gln Phe Gly Glu Glu Asn Thr Arg Leu His
130 135 140
acc gca gta gaa aat cta aaa get gtt aac gtt gag ctc tca gag caa 480
Thr Ala Val Glu Asn Leu Lys Ala Val Asn Val Glu Leu Ser Glu Gln
195 150 155 160
att aac caa ctt aaa caa cta cat act aga tta tcg gat ttt ggt gat 528
Ile Asn Gln Leu Lys Gln Leu His Thr Arg Leu Ser Asp Phe Gly Asp
165 170 175
agg ctt gaa gcg aat acc ggt gat ttt act gca ctt att gcg gat ttc 576
Arg LE:u G1u Ala Asn Thr Gly Asp Phe Thr Ala Leu Ile Ala Asp Phe
180 185 190
caa ctc agt ctg gaa gag ttt aag tct gtt ggt act aaa gtt gaa acc 629
Gln Leu Ser Leu Glu Glu Phe Lys Ser Val Gly Thr Lys Val Glu Thr
195 200 205
atg ctc tct cca ttt gag aaa tta get cag tct ttg aaa gag acc ttt 672
Met L<su Ser Pro Phe Glu Lys Leu Ala Gln Ser Leu Lys Glu Thr Phe
2:L0 215 220
tct caa gaa get gtt cag gca atg atg tcc tct gta act gag tta aga 720
Ser Gln Glu Ala Val Gln Ala Met Met Ser Ser Val Thr Glu Leu Arg
225 230 235 240
acc aat ttg aat gca ttg aaa gag ctt ata aca gag aat aaa acc gta 768
Thr Asn Leu Asn Ala Leu Lys Glu Leu Ile Thr Glu Asn Lys Thr Val
245 250 255
ata gag caa cta aaa get gat get caa ctt aga gaa gag caa gtg cgg 816
8
CA 02326002 2000-10-19
WO 99/53948 PCT/tJS99/08744
Ile GluGln LeuLysAla AspAla GlnLeuArg GluGlu GlnValArg
260 265 270
ttt ttagaa aagcgtaaa caagag ttagaagag gettgt tcaacattg 864
Phe LeuGlu LysArgLys GlnGlu LeuGluGlu AlaCys SerThrLeu
275 280 285
tcc cattca attgcgact ctacag gaatccaca accctt ctaaaggac 912
Ser HisSer IleAlaThr LeuGln GluSerThr ThrLeu LeuLysAsp
290 295 300
tct acaact aactt:acat gcagtt gaaagtcgt cttatc ggtgttatg 960
Ser ThrThr AsnLeuHis AlaVal GluSerArg LeuIle GlyValMet
305 310 315 320
gtt caggat ggtgcagag tcctcc accgtagag gaaget tcacaagat 1008
Val GlnAsp GlyAlaGlu SerSer ThrValGlu GluAla SerGlnAsp
325 330 335
gat agcgcg caaccccaa gatgaa aatcaatet gatget ggagagcat 1056
Asp SerAla GlnProGln AspGlu AsnGlnSer AspAla GlyGluHis
390 345 350
aaa gatagt taa 1068
Lys AspSer
355
<210> 8
<211> 355
<212> PRT
<213> Chlamydia psittaci
<400> 8
Met Thr Val Ser Thr Asp Asn Thr Ser Pro Val Ile Ser Arg Ala Ser
1 5 10 15
Ser Pro Thr Phe Gly Asp His Gly Lys Asp Phe Asp Asn Asn Lys Ile
20 25 30
Ile Pro Ile Ser Ile Glu Ala Pro Thr Ser Ser Ala Ala Ala Val Gly
35 40 45
Ala Lys Thr Ala Ile Glu Pro Glu Gly Arg Ser Pro Leu Leu Gln Arg
50 55 60
Ile Cys Tyr Leu Val Lys I1e Ile Ala Ala Ile Ala Leu Phe Val Val
65 70 75 80
Gly Ile Ala Ala L~eu Val Cys Leu Tyr Leu Gly Ser Val Ile Ser Thr
85 90 95
Pro Ser Leu Ile Leu Met Leu Ala Ile Met Leu Val Ser Phe Val Ile
100 105 110
Val Ile Thr Ala Ile Arg Asp Gly Thr Pro Ser Gln Val Val Arg His
115 120 125
Met Lys Gln Gln I:le Gln Gln Phe Gly Glu Glu Asn Thr Arg Leu His
130 135 140
9
CA 02326002 2000-10-19
WO 99/53948 PCT/US99/08744
Thr Ala Val Glu Asn Leu Lys Ala Val Asn Val Glu Leu Ser Glu Gln
- 145 150 155 160
Ile Asn Gln Leu Lys Gln Leu His Thr Arg Leu Ser Asp Phe Gly Asp
165 170 175
Arg Leu Glu Ala Aan Thr Gly Asp Phe Thr Ala Leu Ile Ala Asp Phe
180 185 190
Gln Leu Ser Leu Glu Glu Phe Lys Ser Val Gly Thr Lys Val Glu Thr
195 200 205
Met Leu Ser Pro Phe Glu Lys Leu Ala Gln Ser Leu Lys Glu Thr Phe
210 215 220
Ser Gln Glu Ala Val Gln Ala Met Met Ser Ser Val Thr Glu Leu Arg
225 230 235 240
Thr Asn Leu Asn Ala Leu Lys Glu Leu Ile Thr Glu Asn Lys Thr Val
245 250 255
Ile Glu Gln Leu Lys Ala Asp Ala Gln Leu Arg Glu Glu Gln Val Arg
260 265 270
Phe Leu Glu Lys Arg Lys Gln Glu Leu Glu Glu A1a Cys Ser Thr Leu
275 280 285
Ser His Ser Ile Ala Thr Leu Gln Glu Ser Thr Thr Leu Leu Lys Asp
290 295 300
Ser Thr Thr Asn Leu His Ala Val Glu Ser Arg Leu Ile Gly Val Met
305 310 315 320
Val Gln Asp Gly Ala Glu Ser Ser Thr Val Glu Glu Ala Ser Gln Asp
325 330 335
Asp Ser Ala Gln Pro Gln Asp Glu Asn Gln Ser Asp Ala G1y Glu His
340 345 350
Lys Asp Ser
355
<210> 9
<211> 597
<212> DNA
<213> Chlamydia psittaci
<220>
<221> CDS
<222> (1)..(597)
<400> 9
atg tca aca aca cca gca tct tca gca agt cga gac gta tta tta gat 98
Met Ser Thr Thr F?ro Ala Ser Ser Ala Ser Arg Asp Val Leu Leu Asp
1 5 10 15
gac gtt tta ata <;ct ttt aat aga aag cta aat ctc gta gaa caa caa 96
Asp V<il Leu Ile Ala Phe Asn Arg Lys Leu Asn Leu Val Glu Gln Gln
20 25 30
CA 02326002 2000-10-19
WO 99/53948 PCT/US99/08744
gcg aaa gaa ctt gaa acg aaa gtc agt ttg gta gac aga aca get act 144
Ala Lys Glu Leu Glu Thr Lys Val Ser Leu Val Asp Arg Thr Ala Thr
35 40 45
tta tca ctt acc act ggc aat aat gta gcc acg gat gta ctc ctt tta 192
Leu Ser Leu Thr Thr Gly Asn Asn Val Ala Thr Asp Val Leu Leu Leu
50 55 60
aaa gat gag gtt gca gaa cta aaa gga tgt ttg tct gca gtt acg gat 290
Lys Asp Glu Val Ala Glu Leu Lys Gly Cys Leu Ser Ala Val Thr Asp
65 70 75 80
cta tta atc cgc tca ggc tca tca aga aca cct ggg ggt get cct aat 288
Leu Leu Ile Arg Ser Gly Ser Ser Arg Thr Pro Gly Gly Ala Pro Asn
85 90 95
cca gaa ggc act aat tac cta ata gga tgc aca cct cct tct ctt tgc 336
Pro Glu Gly Thr Asn Tyr Leu Ile Gly Cys Thr Pro Pro Ser Leu Cys
100 105 110
get aaa ctt aca gcg tta gcg tta aca att ata gcc ctc att get atc 384
Ala Lys Leu Thr Ala Leu Ala Leu Thr Ile Ile Ala Leu Ile Ala Ile
115 120 125
aca gta ctt gtt atc tgt att gtt act gtt tgc ggc ggt ttc ccc cta 432
Thr Val Leu Val I:le Cys Ile Val Thr Val Cys Gly Gly Phe Pro Leu
1 30 135 140
ttt att tcc cta ca c aac atg tac aca gtt ggt get tgt ata tcc tta 480
Phe Ile Ser Leu Leu Asn Met Tyr Thr Val Gly Ala Cys Ile Ser Leu
145 150 155 160
ccg atc att tcg tgt gcc gca gtt tca atg atg att cta tgc tca cat 528
Pro Ile Ile Ser C:ys Ala Ala Val Ser Met Met Ile Leu Cys Ser His
1'_65 170 175
tct at:t aac tct taa tta aga aac agg cct gcg atc tat atg act aac 576
Ser Ile Asn Ser heu Leu Arg Asn Arg Pro Ala Ile Tyr Met Thr Asn
180 1B5 190
aat tia caa aca gaa tct taa 597
Asn Phe Gln Thr Glu Ser
195
<210>
<211>
198
<212>
PRT
<213> psittaci
Chlamydia
<400>
10
Met Ser Thr Pro Ala SerAla SerArg Asp Leu Leu
Thr Ser Val Asp
1 5 10 15
Asp Val Ile .Ala Phe ArgLys LeuAsn Leu Glu Gln
Leu Asn Val Gln
25 30
Ala Lys Leu Glu Thr ValSer LeuVal Asp Thr Ala
Glu Lys Arg Thr
35 90 45
Leu Ser Leu Thr Thr Gly Asn Asn Val Ala Thr Asp Val Leu Leu Leu
11
CA 02326002 2000-10-19
WO 99/53948 PCT/U599/08744
50 55 60
Lys Asp Glu Val Ala Glu Leu Lys Gly Cys Leu Ser Ala Val Thr Asp
65 70 75 80
Leu Leu Ile Arg Ser Gly Ser Ser Arg Thr Pro Gly Gly Ala Pro Asn
~,5 90 95
Pro Glu Gly Thr A:;n Tyr Leu Ile Gly Cys Thr Pro Pro Ser Leu Cys
100 105 110
Ala Lys Leu Thr Ala Leu Ala Leu Thr Ile Ile Ala Leu Ile Ala Ile
115 120 125
Thr Val Leu Val Ile Cys Ile Val Thr Val Cys Gly Gly Phe Pro Leu
130 135 140
Phe Ile Ser Leu Leu Asn Met Tyr Thr Val Gly Ala Cys Ile Ser Leu
145 150 155 160
Pro Ile Ile Ser Cys Ala Ala Val Ser Met Met Ile Leu Cys Ser His
165 170 175
Ser Ile Asn Ser Leu Leu Arg Asn Arg Pro Ala Ile Tyr Met Thr Asn
180 185 190
Asn Phe Gln Thr Glu Ser
195
<210> 11
<211> 561
<212> DNA
<213> Chlamydia psittaci
<220>
<221> CDS
<222> (1)..(561)
<400> 11
atg acc gtaagaacc gatttaact ccaggc gacacctca ctccaa 48
tct
Met Thr ValA.rgThr AspLeuThr ProGly AspThrSer LeuGln
Ser
1 5 10 15
tct tct ttaaatccg agtgatctc acaaca caactatcc aacctc 96
tta
5er Ser LeuAsnPro SerAspLeu ThrThr GlnLeuSer AsnLeu
Leu
20 25 30
cag act ctcgcaggg atacaacaa caacat cctttaaac ggtggt 144
gtt
Gln Thr LeuAlaGly IleGlnGln GlnHis ProLeuAsn GlyGly
Val
35 90 45
tgg ect catc:atcct actggcget gcagat caaaattat cteatg 192
cag
Trp Pro HisHisPro ThrGlyAla AlaAsp GlnAsnTyr LeuMet
Gln
50 55 60
cgt ct:g caat:ctcat atggcaagt accgta tcagcagta tctgaa 240
atg
Arg Leu G1nSerHis MetAlaSer ThrVal SerA1aVal SerGlu
Met
65 70 75 80
tta aga gaagtcact gcaatcaag acaaaa ttgcacggg ctatct 288
acc
12
CA 02326002 2000-10-19
WO 99!53948 PCT1US99/08744
LeuArg_ThrGlu ValThr AlaIle LysThrLys LeuHisGly LeuSer
85 90 95
actccagetaat gt:ttge ageggt ectatgget ctagccget tttett 336
ThrProAlaAsn ValCys SerGly ProMetAla LeuAlaAla PheLeu
100 105 110
ctagct=atatct tt_agtt gcgatt atcatcatt gttttagcc tcctta 384
LeuA1<~IleSer LeuVal AlaIle IleIleIle ValLeuAla SerLeu
115 120 125
ggccttgcaggc atacta ectcaa getgccget atcttagtg aataca 432
GlyLeuAlaGly I.LeLeu ProGln AlaAlaAla IleLeuVal AsnThr
130 135 140
gcaaactctata tggget attgtt agcgettcg atagtcact gttatc 480
AlaAsnSerIle TrpAla IleVal SerAlaSer IleValThr ValIle
195 150 155 160
tgcttaattagc gtgcta tgcata acgctaatt cgacaccat aaaccc 528
CysLeuIleSer ValLeu CysIle ThrLeuIle ArgHisHis LysPro
165 170 175
ttacctattgaa actagg cctacc ggacattaa 561
LeuProIleGlu ThrArg ProThr GlyHis
180 185
<210>
12
<211> 6
18
<212> T
PR
<213> lamydia psittaci
Ch
<400>
12
Met SerVal ArgThr AspLeuThr ProGlyAsp ThrSerLeu Gln
Thr
1 5 10 15
Ser LeuLeu AsnPro SerAspLeu ThrThrGln LeuSerAsn Leu
Ser
20 25 30
Gln ValLeu F,laGly IleGlnGln GlnHisPro LeuAsnGly Gly
Th.r
35 40 45
Trp GlnHis F:isPro ThrGlyAla AlaAspGln AsnTyrLeu Met
Pro
50 55 60
Arg MetGln ~~erHis MetAlaSer ThrValSer AlaValSer Glu
Leu
65 70 75 BO
Leu ThrGlu ValThr AlaIleLys ThrLysLeu HisGlyLeu Ser
Arg
85 90 95
Thr AlaAsn ValCys SerGlyPro MetAlaLeu AlaAlaPhe Leu
Pro
100 105 110
Leu IleSer LeuVal AlaIleIle IleIleVal LeuA1aSer Leu
A1a
115 120 125
Gly AlaGly ~leLeu ProGlnAla AlaAlaIle LeuValAsn Thr
Leu
130 135 140
13
CA 02326002 2000-10-19
WO 99/53948 PCT/US99/08744
Ala Asn Ser Ile Trp Ala Ile Val Ser Ala Ser Ile Val Thr Val Ile
145 150 155 160
Cys Leu Ile Ser Val Leu Cys Il.e Thr Leu Ile Arg His His Lys Pro
lfi5 1'70 175
Leu Pro Ile Glu Thr Arg Pro Thr Gly His
180 185
<210>
13
<211> 2
82
<212>
DNA
<213> lamydiatrachomatis
Ch
<220>
<221> S
CD
<222> )..(822)
(1
<400>
13
atgacaacg cctactcta atcgtg attcctcca tctccccct gcacct 98
MetThrThr ProThrLeu IleVal IleProPro SerProPro AlaPro
1 5 10 15
tcctactca gccaatcgc gtacct caaccttct ttgatggac aaaatt 96
SerTyrSer AlaAsnArg ValPro GlnProSer LeuMetAsp LysIle
20 25 30
aagaaaata gcagccatt gcctcc ctaattctt ataggcaca ataggc 144
LysLysIle AlaAlaIle AlaSer LeuIleLeu IleGlyThr IleGly
35 40 45
tttttaget cttttggga catctt gttggcttt ctgatcget ccacaa 192
PheLeuAla LeuLeuGly HisLeu ValGlyPhe LeuIleA1a ProGln
50 55 60
atcactatt gttcttctt gcccta ttcattacc tcattagca gggaat 240
IleThrIle ValLeuLeu AlaLeu PheIleThr SerLeuAla GlyAsn
65 70 75 80
getctttat ctacagaaa accget aatctacat ctataccag gatctg 288
AlaLeuTyr LeuGlnLys ThrAla AsnLeuHis LeuTyrGln AspLeu
85 90 95
caaagagaa gttgggtct ctaaaa gaaattaat ttcatgctg agcgtt 336
GlnArgGlu ValGlySer LeuLys GluIleAsn PheMetLeu SerVal
100 105 110
ctacagaaa gaatttctt cattta tctaaagaa tttgcaacg acatct 384
LeuGl.nLys GluPheLeu HisLeu SerLysGlu PheAlaThr ThrSer
115 120 125
aaagacctc tctgetgta tctcaa gatttttat tcttgtttg caagga 432
LysAspLeu SerAlaVal SerGln AspPheTyr SerCysLeu GlnGly
130 135 140
tttagagat aact:ataaa ggtttt gaatctctt ttggatgag tataaa 480
PheArgAsp AsnTyrLys GlyPhe GluSerLeu LeuAspGlu TyrLys
145 150 155 160
aactcaaca gaagaaatg cgcaaa ctcttttcg caagaaatc atagca 528
14
CA 02326002 2000-10-19
WO 99/53948 PCT/US99/08744
AsnSerThr GluGl.uMetArgLys LeuPheSer GlnGlu IleIleAla
165 170 175
gatcttaaa ggctca gttgcctca ttaagagag gaaatc cgattccta 576
AspLeuLys GlySe:rValAlaSer LeuArgGlu GluIle ArgPheLeu
180 185 190
accccatta gcagaa gaagttcgc cgattagcg cataac caggaatca 624
ThrProLeu AlaGlu GluValArg ArgLeuAla HisAsn GlnGluSer
195 200 205
ttaacageg getat=tgaagaatta aaaacaatt egtgat agettacga 672
LeuThr_Ala A1aI1e GluGluLeu LysThrIle ArgAsp SerLeuArg
210 215 220
gatgaaatt ggaca ctttcacaa ctttctaaa actctt accagtcaa 720
a
AspGluIle GlyG:LnLeuSerGln LeuSerLys ThrLeu ThrSerGln
225 230 235 240
attgcatta caacga aaagagagc tcagatctg tgttcc cagataaga 768
IleAlaLeu GlnArg LysGluSer SerAspLeu CysSer GlnIleArg
245 250 255
gagacgctc tcctcc cccagaaag tctgcatca ccctct acaaaaagc 816
GluThrLeu SerSer ProArgLys SerAlaSer ProSer ThrLysSer
260 265 270
tcctag 822
Ser
<210> 14
<211> 273
<212> PRT
<213> Chlamydia trachomatis
<400> 14
Met Thr Thr Pro Thr Leu Ile Val Ile Pro Pro Ser Pro Pro Ala Pro
1 5 10 15
Ser Tyr Ser Ala Asn Arg Va.1 Pro Gln Pro Ser Leu Met Asp Lys Ile
20 25 30
Lys Lys Ile Ala Ala Ile Ala Ser Leu Ile Leu Ile Gly Thr Ile Gly
35 40 45
Phe Leu Ala Leu Leu Gly His Leu Val Gly Phe Leu Ile Ala Pro Gln
50 55 60
Ile Thr Ile Val Leu Leu Ala Leu Phe Ile Thr Ser Leu Ala Gly Asn
65 70 75 80
Ala Leu Tyr Leu Gln Lys Thr Ala Asn Leu His Leu Tyr Gln Asp Leu
85 90 95
Gln Arg Glu Val Gly Ser Leu Lys Glu Ile Asn Phe Met Leu Ser Val
100 105 110
Leu Gln Lys Glu Phe Leu His Leu Ser Lys Glu Phe Ala Thr Thr Ser
115 120 125
CA 02326002 2000-10-19
WO 99/53948 PCT/US99/08744
Lys Asp Leu Ser A:La Val Ser Gln Asp Phe Tyr Ser Cys Leu Gln Gly
13c) 135 190
Phe Arg Asp Asn T;yr Lys Gly Phe Glu Ser Leu Leu Asp Glu Tyr Lys
145 150 155 160
Asn Se:r Thr Glu Glu Met Arg Lys Leu Phe Ser Gln Glu Ile Ile Ala
165 170 175
Asp Leu Lys G1y Ser Val Ala Ser Leu Arg Glu Glu Ile Arg Phe Leu
180 185 190
Thr Pro Leu Ala Glu Glu Val Arg Arg Leu Ala His Asn Gln Glu Ser
195 200 205
Leu Thr Ala A1a Ile Glu Glu Leu Lys Thr Ile Arg Asp Ser Leu Arg
210 215 220
Asp Glu Ile Gly Gln Leu Ser Gln Leu Ser Lys Thr Leu Thr Ser Gln
225 230 235 240
Ile Ala Leu Gln Arg Lys Glu Ser Ser Asp Leu Cys Ser Gln Ile Arg
245 250 255
Glu Thr Leu 5er Ser Pro Arg Lys Ser Ala Ser Pro Ser Thr Lys Ser
260 265 270
Ser
<210>
15
<211>
348
<212>
DNA
<213> t:rachomatis
Chlamydia
<220>
<221>
CDS
<222>
(1)..(348)
<400>
15
atg gt:t tct gtatac aattcattg getccagaa ggtttt agccaa 48
cat
Met Val Ser ValTyr AsnSerLeu AlaProGlu GlyPhe SerGln
His
1 5 10 15
gtc tct caa cccagt cagattcca accagcaaa aaagta atgatt 96
att
Val Ser Gln ~?roSer GlnIlePro ThrSerLys LysVal MetI1e
Ile
20 25 30
gcg ata act cttttt gcactcaca gccattgca gcaata gtcctt 144
atg
Ala Ile Thr LeuPhe AlaLeuThr AlaIleAla AlaIle ValLeu
Met
35 40 45
tcc atc aca gtttgt ggagggttt ccttttctt cttget gcactt 192
gtt
Ser I:Le Thr ValCys GlyGlyPhe ProPheLeu LeuAla AlaLeu
Val
50 55 60
aac acc act attggt gcatgcgta tccttgccg gtattc acttgc 240
gta
Asn Thr Thr IleGly AlaCysVal SerLeuPro ValPhe ThrCys
Val
65 70 75 80
ata get aca acg tta tta ctt ctt tgt ctc cgt aat atc gaa ctc cta 288
16
CA 02326002 2000-10-19
WO 99/53948 PCT/US99/08744
Ile Ala Thr Thr Leu Leu Leu Leu Cys Leu Arg Asn Ile Glu Leu Leu
85 90 95
gcc aga ccg caa gta ttt acc ctc tcc act caa ttc agc cca aca aaa 336
Ala Arg Pro Gln Val Phe Thr Leu Ser Thr G1n Phe Ser Pro Thr Lys
100 105 110
cct caa gaa tag 348
Pro Gln Glu
115
<210> 16
<211> 115
<212> PRT
<213> Chlamydia trachomatis
<400> 16
Met Val His Ser Val Tyr Asn Ser Leu Ala Pro Glu Gly Phe Ser Gln
1 5 10 15
Val Ser Ile Gln Pro Ser Gln Ile Pro Thr Ser Lys Lys Val Met Ile
20 25 30
Ala Ile Met Thr Leu Phe Ala Leu Thr Ala Ile Ala Ala Ile Val Leu
35 40 45
Ser Ile Val Thr Val Cys G.ly Gly Phe Pro Phe Leu Leu Ala Ala Leu
50 55 60
Asn Thr Val Thr Ile Gly Ala Cys Val Ser Leu Pro Val Phe Thr Cys
65 70 75 80
Ile Ala Thr Thr L~eu Leu Leu Leu Cys heu Arg Asn Ile Glu Leu Leu
85 90 95
Ala Arg Pro Gln V'ai. Phe Thr Leu Ser Thr G1n Phe Ser Pro Thr Lys
100 105 110
Pro Gl.n Glu
115
<210> 17
<211> 537
<212> DNA
<213> Chlamydiat:rachomatis
<220>
<221> CDS
<222> (1)..(537)
<400> 17
atg acg tac atatcc atagcacac aaatct gatatttct aat
tct gat 48
Met Thr Tyr IleSer IleAlaHis LysSer AspIleSer Asn
Ser Asp
1 5 10 15
ccc aeg tct getcca agaaaaega ggatcc tttccccca caa
ccc tca 96
Pro Thr Ser AlaPro ArgLysArg GlySe.rPheProPro Gln
Pro Ser
20 25 30
17
CA 02326002 2000-10-19
"" WO 99/53948 PCT/US99/08744
tct cct tct gcc gt.g ggc tct tta gag gga get aat ttc tct aca tgg 194
Ser Pro Ser Ala Val Gly Ser Leu Glu Gly Ala Asn Phe Ser Thr Trp
35 90 45
ggg cca gge cec tt:c tte act gte cet gtt tat eca caa eaa ctc get 192
Gly Pro Gly Pro Phe Phe Thr Val Pro Val Tyr Pro Gln Gln Leu Ala
50 55 60
gca atg caa aac aac ctt ttt aca ttg caa aca gag gtt tet get ctc 240
Ala Met Gln Asn Asn Leu Phe Thr Leu Gln Thr Glu Val Ser Ala Leu
65 70 75 80
aag aaa aaa tta gi.t cag tct agt cag aca cgc gga tct tta gga ctc 288
Lys Lys Lys Leu V<31 Gln Ser Ser Gln Thr Arg Gly Ser Leu Gly Leu
85 90 95
ggc ccg cag ttt tta gcg gca tgc tta gtt get gcg aca atc ctt gca 336
Gly Pro Gln Phe Le a Ala Ala Cys Leu Val Ala Ala Thr Ile Leu Ala
100 105 110
gta gc= gtt ate gta ctt get tcc tta gga ctt gge ggt gtt ett ect 384
Val Ala Val Ile Val Leu Ala Ser Leu Gly Leu Gly Gly Val Leu Pro
115 120 125
ttt gte ctt gtt tgt ctg get ggg tca act aat gca att tgg get att 932
Phe Val Leu Val Cys Leu Aia Gly Ser Thr Asn Ala Ile Trp Ala Ile
130 135 140
gtg age gcc tee atc act aca ctg att tgt tge gtt tcc atc get tgc 480
Val Ser Ala Ser Ile Thr Thr Leu Ile Cys Cys Val Ser Ile Ala Cys
145 150 155 160
atc ttc tta gca aaa tgt gat aag gga tct gat cct caa act tta tat 528
Ile Phe Leu Ala Lys Cys Asp Lys Gly Ser Asp Pro Gln Thr Leu Tyr
165 170 175
gta agc taa 537
Val Ser
<210> 18
<211> 178
<212> PRT
<213> Chlamydia t.rachomatis
<400> 18
Met Thr Tyr Ser I:le Ser Asp Ile Ala His Lys Ser Asp Ile Ser Asn
1 5 10 15
Pro Thr Ser Pro Ala Pro Ser Arg Lys Arg Gly Ser_ Phe Pro Pro Gln
20 25 30
Ser Pro Ser Ala Val Gly Ser Leu Glu Gly Ala Asn Phe Ser Thr Trp
35 90 45
Gly Pro Gly Pro L'he Phe Thr Val Pro Va1 Tyr Pro Gln G1n Leu Ala
50 55 60
Ala Met Gln Asn Asn Leu Phe Thr Leu Gln Thr Glu Val Ser Ala Leu
65 70 75 80
18
CA 02326002 2000-10-19
WO 99/53948 PCT/US99/08~44
Lys Lys Lys Leu Val Gln Ser Ser Gln Thr Arg Gly Ser Leu Gly Leu
g5 90 95
Gly Pro Gln Phe Le:u Ala Ala Cys Leu Val Ala Ala Thr I1e Leu Ala
100 105 110
Val Ala Val Ile Val Leu Ala Ser Leu Gly Leu G1y Gly Val Leu Pro
115 12.0 125
Phe Val Leu Val Cys Leu Ala Gly Ser Thr Asn Ala Ile Trp Ala Ile
130 135 140
Val Ser Ala Ser Ile Thr Thr Leu Ile Cys Cys Val Ser Ile Ala Cys
145 150 155 160
Ile Phe Leu Ala Lys Cys Asp Lys Gly Ser Asp Pro Gln Thr Leu Tyr
165 170 175
Val Ser
<210> 19
<211> 22
<212> DNA
<213> ,artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 19
agaaccgatt taactccagg cg 22
<210> 20
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 20
gcgcggatcc ttaatgtccg gtaggcctag 30
<210> 21
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<900> 21
atgtcaacaa caccagcatc ttc 23
<210> 22
<211> 34
<212> DNA
<213> Artificial Sequence
19
CA 02326002 2000-10-19
WO 99/53948 PCT/US99/08744
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 22
gcgcggatcc ttaattagtg ccttctggat tagg 34
<210> 23
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 23
cgcagtactg tatccacaga caac 24
<210> 24
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 24
gtcggatccg agaaactctc catgcc 26
