Note: Descriptions are shown in the official language in which they were submitted.
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IMMUNOGENIC COMPOSITIONS FOR CHL~4lYIYI~IA PNEUNOMI~IE
This application claims the benefit of U.S. Provisional Application
60/542,832, filed
March 2, 2004; U.S. Provisional Application 60/643,110, filed January 12,
2005; and
U.S. Provisional Application 60/644,552, filed January 19, 2005, all of which
are
incorporated herein in their entireties.
All documents cited herein are incorporated by reference in their entirety.
Field
The invention is in the field of immunology and vaccinology. In particular, it
relates
to immunogenic compositions comprising combinations of immunogenic molecules
from Clzlanzydia ptzeuznoniae.
Background Art
The bacteria of the genus Chlamydia (and Chlarnydophila, according to the
recently
proposed but still controversial re-classification of Chlamydiaceae (Bush et
al (200I)
Int J Syst Evol Microbiol 51: 203-20; Everett et al (1999) Int J Syst
Bacteriol 49: Pt2
415-40; Schachter et al (2001) Int J Syst Evol Microbiol 51: 249, 251-3) are
obligate
intracellular parasites of eukaryotic cells, which have a unique biphasic life
cycle
involving two pleiomorphic developmental forms: an extracellular,
metabolically inert,
spore-Iike, infectious forni (the elementary bodies, EBs) and an
intracellular, non-
infectious, replicative form (the reticulate bodies, RBs) which remains
contained in a
specialized cytoplasmic compartment (the Chlamydial inclusion). The EBs are
responsible for the initial attachment to host cell surface and the
establishment of the
cytoplasmic inclusion .where EBs can differentiate to RBs and thus initiate
the
replicative stage. Eventually RBs revert to infectious EB forms able to start
new
replicative cycles in neighbouring host cells.
As Chlamydia infection is an intracellular infection, the currently accepted
paradigm is
that effective anti-Chlamydial immunisation would require both an adequate T-
cell
response and lugh serum levels of neutralising antibodies and that "an ideal
vaccine
should induce long lasting (neutralising) antibodies and a cell mediated
immunity that
can quickly respond upon exposure to Chlanzydia". Several sometimes
contradictory
studies have indicated that both CD4+ and CD 8 positive T cells have a role in
Clzlanzydial clearance (Loomis and Starnback (2002) Curr Opin Microbiol 5: 87-
91).
Indeed, there now appears to be a prevailing consensus that specific CD4+ T
cells and
B cells axe critical to the complete clearance of intracellular Chlamydia and
for
mediating recall immunity to Clzlamydia infection (see Igietseme, Black and
Caldwell
(2002) Biodrugs 16: 19-35 and Igietseme et al (1999) Immunology 98: 510-519).
Whilst it is now possible to carry out searches of the whole Clzlamydia
pneumoniae
genome, there is still insuff cient information available on parallel proteome
characterisation. By way of example, while sequence data is available for many
of the
Clzlamydia pneumoniae antigens, there is insufficient characterisation of the
Clzlamydia antigens in terms of their immunological and/or biological
function. By
way of example, whilst applications such as WO 99/28475 and WO 99/27105
disclose sequence information, there is no characterisation of these sequences
in terms
of their immunological and/or biological function. In contrast, WO 02/02404
provides information on the immunogenicity and immunoaccessibility of certain
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Chlamydia proteins and highlights that (i) current genomic annotations and/or
(ii)
predictions based on cellular location and/or cellular function based on iyt-
silico
analyses may not always be accurate.
Applicants have recently engaged in a whole-genome search (Montigiani et al
(2002)
Infection and Immunity 70:368-379) for possible vaccine candidates among
proteins
potentially associated with the outer membrane of C.ptteumoytiae. For this
study,
mouse antisera was prepared against over 100 recombinant His-tagged or
Glutathione-S-transferase (GST) fusion proteins encoded by genes predicted by
in
silico analyses to be peripherally located in the Chlarttydial cell. From this
screening
study, 53 recombinant proteins derived from the genome of Chlamydia
(Chlatttydophila p>zeumortiae (CPn) were described which induced mouse
antibodies,
capable of binding, in a FACS assay, to the surface of purified CPn cells.
The scope of the Montigiani study (ibid) was restricted to checking if
polyclonal
antisera produced in mice against the recombinantly expressed antibodies to
CPn
antigens were capable of binding to the surface of the CPn cells. No studies
were
carried out to test whether antisera against the recombinant FACS positive
antigens
were capable of interfering with EB itz vitro infectivity of host cells - that
is, whether
the murine antibodies raised against the recombinantly expressed antigens
could
inhibit CPn infectivity in vitro to an extent greater than 50%, a property
that common
practice qualifies such antigens as "neutralising".
Indeed, so far, only few C, ptteumo>ziae antigens with 'neutralizing'
properties have
been described in the literature: notably, a protein identified as 76-kDa-
homolog
protein (Perez-Melgosa et al (1994) Infect Immunity 62: 880-6), the surface-
exposed
outer membrane proteins MOMP (Wolf et al (2001) Infect Immun 69: 3082-91),
PorB
(Kawa et al (2002) J Immunol 168 : 5184-91 and Kubo et al (2000) Mol Microbiol
38 : 772-80), and very recently also the Pmp21 member of the Chlatttydia-
specific
polymorphic family of outer membrane proteins (A.Szczepek, personal
comunication). All these proteins were in fact selected in the earlier FAGS-
based
screening study (Montigiani et al (2002) ibid). It can be however noted that
outer
membrane antigens, as it is the case for MOMP and PorB, could possibly present
some kind of practical problems for a recombinant vaccine development project.
For
instance both MOMP and PorB are integral membrane proteins which appear to
require a native conformation to maintain neutralizing epitopes which are
discontinuous and conformation-dependent. The production of such proteins may
require special processing steps (refolding) which could be undesirable in the
preparation of an hypothetical vaccine. Other general problems may arise from
the
extent of allelic variation, and from regulated proteins which are not always
expressed
in all Chlamydial cell or all Chlamydial isolates.
Thus, it is desirable to provide improved compositions capable of eliciting an
immune
response upon exposure to Chlafttydia ptteumotziae proteins. It is also
desirable to
provide improved compositions comprising one or more combinations of two or
more
selected CPn proteins with complementary immunological andlor biological
profiles
capable of providing immunity against Chlamydial induced disease andlor
infection
(such as in prophylactic vaccination) or (b) for the eradication of an
established
chronic Chlatttydial infection (such as in therapeutic vaccination).
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Brief description of the drawings and tables
Figure 1A. Assay of ih vitro neutralization of C.pheumofziae infectivity for
LLC-MK2,
cells by polyclonal mouse antisera to recombinant Clzlamydial proteins.
Figure 1B shows serum titres giving 50% neutralization of infectivity for 10
C.pfZeumoniae recombinant antigens. Each titer was assessed in 3 separate
experiments (SEM values shown).
Figure 2 shows immunoblot analysis of two dimensional electrophoretic maps of
C.pyaeumoyi.iae EBs using the imune sera described in the text.
Figure 3 shows mean numbers of C.pneumoreiae IFU recovered from equivalent
spleen samples from immunized and mock-immunized hamsters following a systemic
challenge.
Figure 4 shows flow cytometric analysis of splenocytes from DNA-irmnunized HLA-
A2 transgenic and non transgenic mice.
Figure 5 shows a flow cytometric analysis of splenocytes from transgenic and
non
transgenic mice infected with C. pheumohiae EBs.
Figure 6 shows an alignment of the proteins in the 7105-7110 protein family.
Figure 7 shows an N-terminal alignment of Cpn0794 - Cpn0799.
Figure 8 shows a protein encoded by Cpn0796 and demonstrates a C-terminal
domain
comprising approximately residues from 1 to 648.
Figure 9 shows an alignment of the C-terminal (beta barrel) domains of the
proteins
encoded by the C.pneumoniae genes Cpn0795 and Cpn0796.
Table I shows a summary of data and properties of the C.pheumoyaiae antigens
described in the text.
Table 2 shows results from hamster mouse model studies for hypothetical
proteins.
Table 3 shows expressed genes of CPn EB selected by microarray.
Table 4 shows C. praeumort.iae selected peptides: protein sources and HLA-A2
stabilization assay.
Table 5 shows ELISPOT assay with CD8+ T cells from DNA immunised HLA-A2
transgenic mice.
Table 6 shows IFN-y production from splenocytes of DNA immunized HLA-A2
transgenic and non transgenic mice.
Summary of the Invention
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The present invention relates to a polypeptide for use as an autotransporter
antigen,
the polypeptide comprising: (a) an amino acid sequence selected from the group
consisting of SEQ ID NO: 54, SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ
ID NO: 78, and SEQ ID NO: 79, (b) an amino acid sequence having at least 50%
sequence identity to an amino acid sequence of (a); or (c) an amino acid
sequence
comprising one or more fragments of at least 7 consecutive amino acids from an
amino acid sequence of (a) or combinations thereof.
The present invention also relates to the use of a polypeptide in the
preparation of a
medicament for the prevention or treatment of a Chlamydia pneumoniae infection
in
an individual. For example, the use of the polypeptide may be as an
autotransporter
protein which immunoreacts with seropositive serum of an individual infected
with
Chlamydia pneumoniae.
The present invention further relates to a method of eliciting an immune
response in
an individual comprising administering to the individual a polypeptide
comprising (a)
an amino acid sequence selected from the group consisting of SEQ ID NO: 54,
SEQ
ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 78, and SEQ ID NO: 79 (b)
an amino acid sequence having at least 50% sequence identity to an amino acid
sequence of (a), or (c) an amino acid sequence comprising one or more fragment
of at
least 1, 2, 3, 4, 5, 6, or 7 amino acids from an amino acid sequence of (a) or
mixtures
thereof.
Also, a method is provided for diagnosing an immune response in an individual
comprising (a) contacting a biological sample obtained from the individual
with a
binding agent that binds to a polypeptide with an autotransporter function,
(b)
detecting in the biological sample the amount of the polypeptide that binds to
the
binding agent; and (c) comparing the amount of the polypeptide to a
predetermined
cut-off value and thereby determining the presence of an immune response in
the
individual.
A composition for eliciting an immune response in a subject comprising two or
more
Chlamydia pneunoniae autotransporter proteins or immunogenic fragments thereof
is
also provided. The composition may further comprise one or more
immunostimulants.
Also provided is a polypeptide for use as an autotransporter antigen
comprising an
amino acid sequence corresponding to SEQ ID NO: 86, an amino acid sequence
having at least 50% sequence identity to SEQ ID NO: 86, or an amino acid
sequence
comprising one or more fragments of at least 7 consecutive amino acids of SEQ
ID
NO: 86.
The present invention relates to a composition comprising a first biological
molecule
from a Chlarnydia p>zeunaoniae bacterium and a second biological molecule from
a
Chlamydia prceurrZOhiae bacterium. The first biological molecule is selected
from the
group consisting of SEQ ID No 1 to SEQ ID No 86, or the group consisting of
SEQ
ID No. 1 to 41.
The composition may also contain the second biological molecule being selected
from
the group consisting of SEQ ID No 1 to SEQ ID No. 86 or SEQ ID No 1 to SEQ ID
No 41.
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The composition may also comprise two or more biological molecules selected
from
the group consisting of SEQ ID Nos 1-41.
The composition may also comprise one or more biological molecules selected
from
the group consisting of SEQ ID Nos 1-41 combined with one or more biological
molecules selected from the group consisting of SEQ ID Nos 42-86.
The composition according to any one of the previous claims further comprising
an
adjuvant such as an ADP-ribosylating exotoxin or a derivative thereof or an
adjuvant
is selected from the group consisting of cholera toxin (CT), Escherichia heat-
labile
exterotoxin (LT) and mutants thereof having adjuvant activity.
A vaccine and use of the vaccine is also provided comprising the composition
of the
present invention. The vaccine may be used in the preparation of a medicament
for
the prevention or treatment of a Chlarnydia infection and may be administered
mucosally, infra-nasally or infra-vaginally, for example.
Further, a method is provided for treating a Clzlamydia infection in a host
subject
wherein the method comprises the administration of a safe and effective amount
of a
vaccine.
In another aspect of the invention, an immunogenic composition is provided
comprising a combination of Chlanzydia przeurnorziae antigens, the combination
comprising at least one Clzlarnydia pneumoniae antigen associated with
elementary
bodies of Chlarnydia pneunzorziae and at least one Chlamydia pneumozziae
antigen
associated with reticulate bodies of Chlarnydia pneurrzorziae.
In another aspect of the invention, an immunogenic composition is provided
comprising a combination of Chlamydia pneunzoniae antigens, the combination
comprising at least one Chlamydia pneumoniae antigen of a first antigen group
and at
least one Chlamydia pneumoniae antigen of a second antigen group, said first
antigen
group comprising a Type III secretion system (TTSS) protein and said second
antigen
group comprising a Type III secretion system (TTSS) effector protein.
In yet another aspect of the invention, an immunogenic composition is provided
comprising a combination of Chlamydia przeurnoniae antigens comprising at
least one
Chlamydia pneurnoniae antigen that is conserved over at least two serovars.
In still another aspect of the invention, an immunogenic composition is
provided
comprising a combination of Clzlanzydia pneurnorziae antigens, the combination
eliciting a Chlamydia przeunzorziae specific THl immune response and a
Chlamydia
pneumoniae specific TH2 immune response.
The present invention further provides a method of monitoring the efficacy of
treatment of a patient infected with Chlarnydia przeumoniae comprising
determining
the level of Chlamydia pneurnozziae specific antibody in the patient after
administration of an immunogenic composition of the present invention to the
patient.
Description of the Invention
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The present invention provides compositions comprising a first biological
molecule
from a Chlamydia pneumoniae bacterium and a second biological molecule from a
Chlamydia pneumoniae bacterium. The term "biological molecule" includes
proteins,
antigens and nucleic acids. The compositions may also comprise further
biological
molecules preferably also from Clalamydia pneumoniae. That is to say, the
compositions may comprise two or more biological molecules (eg. 3, 4, 5, 6, 7,
8 etc.)
at least two of which are from a Chlamydia pneurnozziae bacterium (eg. 3, 4,
5, 6, 7, 8
etc.). Such compositions include those comprising (i) two or more different
Chlamydia pneumozziae proteins; (ii) two or more different Clzlamydia
pneumoniae
nucleic acids, or (iii) mixtures of one or more Chlamydia pzzeumoniae protein
and one
or more Chlamydia pneurnoniae nucleic acid.
In one aspect of the present invention, an immunogenic composition is provided
comprising a combination of at least one antigen that elicits a Chlarnydia
pneumoniae
specific TH1 immune response (such as a cell mediated or cellular immune
response)
and at least one antigen that elicits a Chlamydia pneumoniae specific TH2
response
(such as a humoral or antibody response). The immunogenic composition may
further
comprise a TH1 adjuvant and a TH2 adjuvant.
In another aspect of the present invention, an immunogenic composition is
provided
comprising a combination of Chlamydia pneumoniae antigens comprising at least
one
Chlamydia pzzeumoniae antigen that is conserved over at least two serovars.
In yet another aspect of the present invention, an immunogenic composition is
provided comprising a combination of at least one antigen that elicits a
Chlaznydia
pneumoniae specific THl immune response and at least one antigen that elicits
a
Chlamydia pneunzoniae specific TH2 immune response, the combination comprising
at least one Chlamydia pzzeuznoniae antigen that is conserved over at least
two
serovars.
In another aspect of the present invention, the irmnunogenic composition
comprising
at least one antigen that elicits a Clalamydia pzzeuznoniae specific THl
immune
response and at least one antigen that elicits a Clzlamydia pneuznoniae
specific TH2
immmune response preferably comprises a combination of Chlanzydia pzzeunaoniae
antigens comprising at least one Chlamydia pneuznozziae antigen associated
with the
EB of Clzlamydia pneumoniae and at least one Clzlamydia pneumoniae antigen
associated with the RB of Chlanzydia pneumoniae. Still further such
combinations can
comprise EB and/or RB antigens from one serovar combined with RB and/or EB
antigens from at least one other serovar.
In an additional aspect of the present invention, a leit is provided
comprising a
combination of Chlamydia pneumozziae antigens wherein at least one of the
Chlamydia pzzeumoniae antigens is associated with the EB of Clalaznydia
pneumoniae
and at least one of the Chlanzydia pneumoniae antigens is associated with the
RB of
Chlaznydia pneuznoniae. The leit may further include a TH1 adjuvant, a TH2
adjuvant
and instructions.
The present invention further provides methods of eliciting a Chlamydia
specific
immune response by administering an immunogenic composition of this invention.
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The present invention further provides a method of monitoring the efficacy of
treatment of a subject infected with Chlamydia pneumoniae comprising
determining
the level of Chlamydia specific antibody or Chlamydia specific effector
molecule in
the subject after administration of an immunogenic composition of this
invention.
In one preferred embodiment the first and second biological molecules are from
different Chlamydia praeunaoniae species (for example, from different
Clalamydia
pyaeumoniae serovars) but they may be from the same species. The biological
molecules in the compositions may be from different serogroups or strains of
the
same species. The first biological molecule is preferably selected from the
group
consisting of SEQ ID Nos 1-86. More preferably. it is selected from the group
consisting of SEQ IDs 1-4land/or SEQ ID Nos 42-86. It is preferably a purified
or
isolated biological molecule. The second biological molecule is preferably
selected
from the group consisting of SEQ ID Nos 1-86. More preferably. it is selected
from
the group consisting of SEQ IDs 1-41 and/or SEQ ID Nos 42-86. It is preferably
a
purified or isolated biological molecule. Specific compositions according to
the
invention therefore include those comprising: two or more biological molecules
selected from the group consisting of SEQ ID Nos 1-41; one or more biological
molecules selected from the group consisting of SEQ IDs 1-41 combined with one
or
more biological molecules selected from the group consisting of SEQ IDs 42-86.
One
or both of the first and second biological molecules may be a Chlamydia
pneunaoniae
biological molecule which is not specifically disclosed herein, and which may
not
have been identified, discovered or made available to the public or purified
before this
patent application was filed.
In another embodiment, a combination of Chlamydia pneumoniae antigens is
provided, the combination comprising at least one Type III Secretion System
(TTSS)
protein and at least one Type III Secretion System (TTSS) secreted or effector
protein
or fragment thereof. There are many methods for identifying TTSS proteins
(i.e.,
TTSS proteins associated with the Chlamydial TTSS machinery). TTSS is a
complex
protein secretion and delivery machine or apparatus, which may be located,
either
wholly or partially, on the Elementary Body (EB) and which allows an organism,
such as Chlamydia, to maintain its intracellular niche by injecting proteins,
such as
bacterial effector proteins (which may act as anti-host virulence
determinants) into the
cytosol of a eukaryotic cell in order to establish the bacterial infection and
to
modulate the host cellular functions. TTSS proteins exposed on the EB surface
may
play a role in adhesion and/or uptake into host cells.
By way of background information, the TTSS is a complex protein secretion and
delivery machine or apparatus, which may be located on the Elementary Body
(EB)
and which allows an organism, such as Chlamydia, to maintain its intracellular
niche
by inj ecting proteins, such as bacterial effector proteins (which may act as
anti-host
virulence determinants) into the cytosol of a eukaryotic cell in order to
establish the
bacterial infection and to modulate the host cellular functions. These
injected proteins
(the TTSS effector proteins) can have various effects on the host cell which
include
but are not limited to manipulating actin and other structural proteins and
modification of host cell signal transduction systems. The injected (or
translocated)
proteins or substrates of the TTTS system may also be processed and presented
by
MHC-class I molecules.
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Not all the proteins secreted by a Type III secretion system are delivered
into the host
cell or have effector function. Although the Elementary Body (EB) is regarded
as
"metabolically inert", it has been postulated that the Chlamydial TTSS system
located
on the (EB) is triggered by membrane contact and is capable of releasing pre-
fornled
"payload" proteins. The current hypothesis is that Type Three Secretion System
(TTSS) becomes active during the intracellular phase of the chlamydial
replicative
cycle for the secretion of proteins into the host cell cytoplasm and for the
insertion of
chlamydial proteins (like the Inc set) into the inclusion membrane that
separates the
growing chlamydial microcolony from the host cell cytoplasm (see Montigiani et
al
(2002) Infection and Immunity 70(1); 386-379).
Proteins may be expressed and secreted by 2 hours (early cycle) after
infection while
the expression of other early and mid cycle Type III specific genes are not
detectable
until 6-12 hours (mid cycle). After 16-20 hours, the RBs begin to
differentiate into
EBs, and by 48-72 hours, the EBs predominate within the inclusion. Host cell
lysis
results in the release of the EBs to the extracellular space where they can
infect more
cells. For purposes of this description, an early gene is one that is
expressed (in terms
of mRNA expression) early in infection, an intermediate gene is one that is
expressed
in the mid-cycle after infection and a late gene is one which is expressed
during the
terminal transition of RBs to EBs. There may be a time lag between surface
expression of early, mid and late stage proteins and their transcriptional and
translational profiles because mRNA abundance may not always correlate with
protein abundance.
In one example, the present invention may comprise TTSS effector proteins. The
TTSS effector proteins as described are associated with the RB form of
Chlamydia
pneunaoniae and may be identified, for example, using immunofluorescence
microscopy (see Bannantine et al, Infection and Immunity 66(12); 6017-6021).
Effector antibodies to putative Chlamydial TTSS effector proteins secreted by
the
TTSS machinery may be micro-injected into host cells at specified time points
during
Clzlamydia pneumoniae infection (e.g., early, mid or late cycle). Host cell
reaction to
Chlamydia pneun2oniae (e.g., actin remodeling, inhibition of endosomal
maturation,
host lipid acquisition, and MHC Class I and Class II molecule downregulation)
associated with ClZlamydia pneumoniae entry into host cells is then observed.
Based
on these temporal observations, TTSS effector proteins (RB-associated
Chlamydia
pneumoniae proteins) may be detected.
A specific composition of the present invention may comprise a combination of
Chlamydia pneumoniae antigens, said combination consisting of two, three,
four, five
or all six Chlamydia pneumofaiae antigens of a first antigen group, said first
antigen
group consisting of: (1) pmp2; (2) pmpl0; (3) Enolase; (4) OmpH-like protein;
and
(5) the products of CPn specific genes CPn0759 and CPn0042. These antigens are
referred to herein as the 'first antigen group'.
Preferably, the composition of the invention comprises a combination of
Chlanaydia
pneumoniae antigens, said combination selected from the group consisting of:
(1)
pmp2 and pmpl0; (2) pmp2 and Enolase; (3) pmp2 and OmpH-like protein; (4) pmp2
and CPn0759; (5) pmp2 and CPn0042; (6) pmpl0 and Enolase; (7) pmpl0 and
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OmpH-like protein; (8) pmpl0 and CPn0759; (9) pmpl0 and CPn0042; (10) Enolase
and OmpH-like protein (11) Enolase and CPn0759; (12) Enolase and CPn0042; (13)
OmpH-like protein and CPn0759 (14) OmpH-like protein and CPn0042; (15)
CPn0759 and CPn0042; (16) pmp2 and pmpl0 and Enolase; (17) pmp2 and pmpl0
and OmpH-like protein; ( 18) pmp2 and pmp 10 and CPn0759; ( 19) pmp2 and pmp
10
and CPn0042; (20) pmp2 and Enolase and OmpH-like protein; (21) pmp2 and
Enolase and Cpn0759; (22) pmp2 and Enolase and CPn0042; (23) pmp2 and OmpH-
like protein and CPn0759; (24) pmp2 and OmpH-like protein and CPn0042; (25)
pmp2 and Cpn0759 and CPn0042; and (26) pmpl0 and Enolase and OmpH-like
protein; (27) pmpl0 and Enolase and CPn0759; (28) pmpl0 and Enolase and
CPn0042; (29) Enolase and OmpH-like protein and CPn0759; (30) Enolase and
OmpH-like protein and CPn0042; (31) OmpH-like protein and CPn0759 and
CPn0042.
Preferably, the composition of Chlarnydia prreumoniae antigens consists of
pmp2,
pmpl0, Enolase, OmpH-like protein and CPn0759.
Preferably, the composition of Chlamydia pneurraoniae antigens consists of
pmp2,
pmp 10, Enolase, OmpH-like protein and CPn0042.
Preferably, the composition of Chlamydia pneumoniae antigens consists of pmp2,
pmp 10, Enolase, OmpH-like protein and CPn0759 and CPn0042.
The invention also provides for a slightly larger group of 12 Chlamydia
pneurnorriae
antigens that are particularly suitable for immunisation purposes,
particularly when
used in combinations. (This second antigen group includes the six Clrlamydia
pneunroniae antigens of the first antigen group). These 12 Clalanaydia
pneurnoniae
antigens form a second antigen group of (1) pmp2; (2) pmpl0; (3) Enolase; (4)
OmpH-like protein; (5) CPn0759; (6) CPn0042; (7) ArtJ; (8) HtrA; (9) AtoS;
(10)
OmcA; (11) CPn0498; and (12) CPn0525. These antigens are referred to herein as
the 'second antigen group'.
The invention therefore provides a composition comprising a combination of
Chlarnydia pneurnorriae antigens, said combination selected from the group
consisting
of two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve
Chlamydia
pneumoniae antigens of the second antigen group. Preferably, the combination
is
selected from the group consisting of two, three, four or five Chlanaydia
pneurnoniae
antigens of the second antigen group. Still more preferably, the combination
consists
of six Chlamydia pneurnoraiae antigens of the second antigen group. Each of
the
Chlarraydia pneumorZiae antigens of the first and second antigen group are
described in
more detail below.
(1) PmplO (CPn0449)
One example of a pmpl0 protein is set forth as SEQ ID NO: 1 below (GenBank
Accession No.GI:14195016). Preferred pmpl0 proteins for use with the invention
comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%,
65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more) to SEQ ID NO: 1; andlor (b) which is a fragment of at least ra
consecutive amino acids of SEQ ID NO: 1, wherein ra is 7 or more (e.g. 8, 10,
12, 14,
16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
These
pmp2 proteins include variants (e.g. allelic variants, homologs, orthologs,
paralogs,
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mutants, etc.) of SEQ ID NO: 1. Preferred fragments of (b) comprise an epitope
from
SEQ ID NO: 1. Other preferred fragments lack one or more amino acids (e.g. 1,
2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or
more amino
acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-
terminus of SEQ
ID NO: 1. Other fragments omit one or more domains of the protein (e.g.
omission of
a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an
extracellular domain).
SEQ ID No 1
1 MKSQFSWLVL SSTLACFTSC STVFAATAEN IGPSDSFDGS TNTGTYTPKN TTTGIDYTLT
61 GDITLQNLGD SAALTKGCFS DTTESLSFAG KGYSLSFLNI KSSAEGAALS VTTDKNLSLT
121 GFSSLTFLAA PSSVITTPSG KGAVKCGGDL TFDNNGTILF KQDYCEENGG AISTKNLSLK
181 NSTGSISFEG NKSSATGKKG GAICATGTVD ITNNTAPTLF SNNIAEAAGG AINSTGNCTI
241 TGNTSLVFSE NSVTATAGNG GALSGDADVT ISGNQSVTFS GNQAVANGGA IYAKKLTLAS
301 GGGGGISFSN NIVQGTTAGN GGAISILAAG ECSLSAEAGD ITFNGNAIVA TTPQTTKRNS
361 IDIGSTAKIT NLRAISGHSI FFYDPITANT AADSTDTLNL NKADAGNSTD YSGSIVFSGE
421 KLSEDEAKVA DNLTSTLKQP VTLTAGNLVL KRGVTLDTKG FTQTAGSSVI MDAGTTLKAS
481 TEEVTLTGLS IPVDSLGEGK KWIAASAAS KNVALSGPIL LLDNQGNAYE NHDLGKTQDF
2O 541 SFVQLSALGT ATTTDVPAVP TVATPTHYGY QGTWGMTWVD DTASTPKTKT ATLAWTNTGY
601 LPNPERQGPL VPNSLWGSFS DIQAIQGVIE RSALTLCSDR GFWAAGVANF LDKDKKGEKR
661 KYRHKSGGYA TGGAAQTCSE NLISFAFCQL FGSDKDFLVA KNHTDTYAGA FYIQHITECS
721 GFIGCLLDKL PGSWSHKPLV LEGQLAYSHV SNDLKTKYTA YPEVKGSWGN NAFNMMLGAS
781 SHSYPEYLHC FDTYAPYTKL NLTYIRQDSF SEKGTEGRSF DDSNLFNLSL PIGVKFEKFS
841 DCNDFSYDLT LSYVPDLIRN DPKCTTALVI SGASWETYAN NLARQALQVR AGSHYAFSPM
901 FEVLGQFVFE VRGSSRIYNV DLGGKFQF
(2) Pmp2 = Polynzorphic Outer Menzbraue Protein G Fa~rzily (CPu 0013)
One example of a pmp2 protein is disclosed as SEQ ID NOS: 139 and I40 in WO
02/02606. f GenBank accession number: gi~4376270~gb~AAD18172.1 'CPn0013'; SEQ
ID NO: 2 below . Preferred pmp2 proteins for use with the invention comprise
an
amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)
to SEQ ID NO: 2; and/or (b) which is a fragment of at least zz consecutive
amino acids
of SEQ ID NO: 1, wherein zz is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25,
30, 35, 40,
50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These pmp2 proteins include
variants
(e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ
ID NO: 2.
Preferred fragments of (b) comprise an epitope from SEQ ID NO: 1. Other
preferred
fragments lack one or more amino acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5,
6, 7, 8,
9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 2. Other
fragments
omit one or more domains of the protein (e.g. omission of a signal peptide, of
a
cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
SEQ ID No 2
1 MKIPLRFLLI SLVPTLSMSN LLGAATTEEL SASNSFDGTT STTSFSSKTS
51 SATDGTNYVF KDSWIENVP KTGETQSTSC FKNDAAAGDL NFLGGGFSFT
101 FSNIDATTAS GAAIGSEAAN KTVTLSGFSA LSFLKSPAST VTNGLGAINV
5O 151 KGNLSLLDND KVLIQDNFST GDGGAINCAG SLKIANNKSL SFTGNSSSTR
201 GGAIHTKNLT LSSGGETLFQ GNTAPTAAGK GGAIAIADSG TLSISGDSGD
251 IIFEGNTIGA TGTVSHSAID LGTSAKITAL RAAQGHTIYF YDPITVTGST
301 SVADALNINS PDTGDNKEYT GTIVFSGEKL TEAEAKDEKN RTSKLLQNVA
351 FKNGTVVLKG DVVLSANGFS QDANSKLIMD LGTSLVANTE SIELTNLEIN
401 IDSLRNGKKI KLSAATAQKD IRIDRPWLA ISDESFYQNG FLNEDHSYDG
451 ILELDAGKDI VISADSRSID AVQSPYGYQG KWTINWSTDD KKATVSWAKQ
501 SFNPTAEQEA PLVPNLLWGS FIDVRSFQNF IELGTEGAPY EKRFWVAGIS
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551 NVLHRSGREN QRKFRHVSGG AWGASTRMP GGDTLSLGFA QLFARDKDYF
601 MNTNFAKTYA GSLRLQHDAS LYSWSILLG EGGLREILLP YVSKTLPCSF
651 YGQLSYGHTD HRMKTESLPP PPPTLSTDHT SWGGYVWAGE LGTRVAVENT
701 SGRGFFQEYT PFVKVQAVYA RQDSFVELGA ISRDFSDSHL YNLAIPLGIK
751 LEKRFAEQYY HWAMYSPDV CRSNPKCTTT LLSNQGSWKT KGSNLARQAG
801 IVQASGFRSL GAAAELFGNF GFEWRGSSRS YNVDAGSKIK F*
(3) Enolase (Cpn0800)
One example of an 'Eno' protein is disclosed as SEQ ID NOS: 93 and 94 in WO
02/02606. {GenBank accession number: gi~4377111~gb~AAD18938.1~ ' Cpn0800';
SEQ ID NO: 3 below}.Preferred Eno proteins for use with the invention comprise
an
amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)
to SEQ ID NO: 2; and/or (b) which is a fragment of at least ~ consecutive
amino acids
of SEQ ID NO: 2, wherein h is 7 or more (e.g. 8, 10, 12, I4, I6, 18, 20, 25,
30, 35, 40,
50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These Eno proteins include
variants
(e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ
ID NO: 3.
Preferred fragments of (b) comprise an epitope from SEQ ID NO: 3. Other
preferred
fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5,
6, 7, 8,
9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 3. Other
fragments
omit one or more domains of the protein (e.g. omission of a signal peptide, of
a
cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
SEQ ID No 3
1 MFEAVIADIQ AREILDSRGY PTLHVKVTTS TGSVGEARVP SGASTGKKEA
51 LEFRDTDSPR YQGKGVLQAV KNVKEILFPL VKGCSVYEQS LTDSLMMDSD
101 GSPNKETLGA NAILGVSLAT AHAAAATLRR PLYRYLGGCF ACSLPCPMMN
3O 151 LINGGMHADN GLEFQEE'MIR PIGASSIKEA VNMGADVFHT LKKLLHERGL
201 STGVGDEGGF APNLASNEEA LELLLLAIEK AGFTPGKDIS LALDCAASSF
251 YNVKTGTYDG RHYEEQIAIL SNLCDRYPID SIEDGLAEED YDGWALLTEV
301 LGEKVQIVGD DLFVTNPELI LEGISNGLAN SVLIKPNQIG TLTETVYAIK
351 LAQMAGYTTI ISHRSGETTD TTIADLAVAF NAGQIKTGSL SRSERVAKYN
901 RLMEIEEELG SEAIFTDSNV FSYEDSEE*
(4) OfnpH like outer membrane protein (CPfa0301)
One example of 'OmpH-like' protein is disclosed as SEQ ID NOS: 77 ~ 78 in WO
02/02606. fGenBank accession number: gi~4376577~gb~AAD18450.1~ 'CPn0301';
SEQ ID NO: 4 below}. Preferred OmpH-like proteins for use with the invention
comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%,
65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more) to SEQ ID NO: 4; and/or (b) which is a fragment of at least ya
consecutive amino acids of SEQ ID NO: 3, wherein h is 7 or more (e.g. 8, 10,
12, 14,
16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
These
OmpH-like proteins include variants (e.g. allelic variants, homologs,
orthologs,
paralogs, mutants, etc.) of SEQ ID NO: 4. Preferred fragments of (b) comprise
an
epitope from SEQ ID NO: 4. Other preferred fragments lack one or more amino
acids
(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus
and/or one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more;
preferably 19 or
more, to remove the signal peptide) from the N-terminus of SEQ ID NO: 4. Other
fragments omit one or more domains of the protein (e.g. omission of a signal
peptide
as described above, of a cytoplasmic domain, of a transmembrane domain, or of
an
extracellular domain).
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SEQ ID No 4
1 MKKLI,FSTFL LVLGSTSAAH A_NLGYVNhKR CLEESDLGKK ETEELEAMKQ
51 QFVKNAEKIE EELTSIYNKL QDEDYMESLiS DSASEELRKK FEDLSGEYNA
101 YQSQYYQSTN QSNVKRIQKL IQEVKIAAES VRSKEKLEAI LNEEAVLATA
151 PGTDKTTEII AILNESFKKQ N*
(5) CPh0042 (Hypothetical)
One example of hypothetical protein is set forth as SEQ ID NO: 5 below.
GenBank accession number: gi~4376296~gb~AAD18195.1. Preferred hypothetical
proteins for use with the invention comprise an amino acid sequence: (a)
having 50%
or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 5; and/or (b) which is a
fragment of at least h consecutive amino acids of SEQ ID NO: 5, wherein ra is
7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These Hypothetical proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 5. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: 5. Other preferred fragments lack
one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C-
terminus and/or one or more amino acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more; preferably 19 or more, to remove the signal peptide) from the N-terminus
of
SEQ ID NO: 5 Other fragments omit one or more domains of the protein (e.g.
omission of a signal peptide as described above, of a cytoplasmic domain, of a
transmembrane domain, or of an extracellular domain).
SEQ ID No 5
1 MEEVSEYLQQ VENQLESCSK RLTKMETFAL GVRLEAKEEI ESIILSDVVN RFEVLCRDIE
61 DMLSRVEEIE RMLRMAELPL LPIKEALTKA FVQHNSCKEK LTKVEPYFKE SPAYLTSEER
3O 121 LQSLNQTLQR AYKESQKVSG LESEVRACRE QLKDQVRQFE TQGVSLIKEE TLFVTSTFRT
181 KFSYHSFRLH VPCMRLYEEY YDDIDLERTR ARWMAMSERY RDAFQAFQEM LKEGLVEEAQ
241 ALRETEYWLY REERKSKKKH
(6) CP~Z0795 (HypotIZetical)
One example of hypothetical protein is disclosed as SEQ ID NOS: 63 & 64 in WO
02/02606. {GenBank accession number: gi~4377106~gb~AAD18933.1~ 'CPn0795';
SEQ ID NO: 6 below). As the examples demonstrate, we have shown for the first
time that CPn0795 and related proteins in the group Cpn0794 - Cpn0799 have a
secreted autotransporter function. It has been shown that proteins secreted by
the
autotransporter secretion mechanism possess an overall unifying structure,
including
an amino-terminal leader peptide (for secretion across the inner membrane),
the
secreted mature protein (or passenger domain), and a dedicated C-terminal
domain,
which forms a pore in the outer membrane through which the passenger domain
passes to the cell surface. It is likely that requirements for secretion
across the outer
membrane are contained within a single molecule and secretion is an energy-
independent process. Structural properties of the proteins may be confined by
the size
of the pore considering the biophysical constraints that may be imposed on
secretion.
The autotransporter, or type V, secretion system is a dedicated protein
translocation
mechanism which allows the organism to secrete proteins to and beyond the
bacterial
surface. The secretion mechanism and the ability to develop a new
autotransporter
protein simply by a single recominbation event have presented bacteria with
abundant
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opportunities to increase the efficiency of secretion of proteins that were
developed as
periplasmic or exported virulence factors.
In one model of autotransporter (type V) secretion mechanism, proteins are
exported
by the autotransporter secretion mechanism and are translated as a
polyproptein
possessing domains. The autotransporters possess an overall unifying structure
comprising three functional domains: the amino-termianl leader sequence, the
secreted mature proterin (passenger domain) and a carboxy-terminal (beta-)
domain
that forms a beta-barrel pore to allow secretion of the passenger protein. The
leader
sequence directs secretion via the sec apparatus and is cleaved at the inner
membrane
by a signal peptidase releasing the remaining portion of the molecule into the
periplasm. Once in the periplasm the [3-domain assumes a biophysically favored
state
characterized by a (3-barrel shaped structure which inserts itself into the
outer
membrane to form a pore. After insertion into the outer membrane the passenger
domain is translocated to the bacterial cell surface where it may remain
intact or
undergo processing. A processed protein may be released into the extracellular
milieu
or remain associated with the bacterial cell surface. (Henderson and Nataro,
"Virulence Functions of Autotransporter Proteins", Infection and Immunity,
Vol. 69,
No. 3, March 2001, pages 1231-1243).
Preferred hypothetical proteins for use with the invention comprise an amino
acid
sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID
NO: 6; and/or (b) which is a fragment of at least n consecutive amino acids of
SEQ ID
NO: 6, wherein h is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40,
S0, 60, 70,
80, 90, 100, 150, 200, 250 or more). These Hypothetical proteins include
variants
(e.g. allelic variants, hoinologs, orthologs, paralogs, mutants, etc.) of SEQ
ID NO: 6.
Preferred fragments of (b) comprise an epitope from SEQ ID NO: 6. Other
preferred
fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the C-terminus and/or one or more amino acids (e.g. l, 2, 3, 4, 5,
6, 7, 8,
9, 10, 15, 20, 25 or more; preferably 19 or more, to remove the signal
peptide) from
the N-terminus of SEQ ID NO: 6. Other fragments omit one or more domains of
the
protein (e.g. omission of a signal peptide as described above, of a
cytoplasmic
domain, of a transmembrane domain, or of an extracellular domain). As the
Examples
demonstrate, we have shown for the first time that CPn0795 appears to be
present and
accessible to antibodies on the surface of the infectious EB form which makes
this
protein a good component of an immunogenic composition or vaccine.
Table 1 of this application demonstrates that Cpn0795 (SEQ ID NO: 6) a Cpn
specific
hypothetical protein is a FACS positive protein which demonstrates significant
immunoprotective activity in a hamster spleen model of Chlamydia pneumoniae
infection. We have found evidence to demonstrate that other Cpn proteins in
this
group of Cpn specific hypothetical proteins have now been found to have a
secreted
autotransporter function. These proteins, which are absent from Chlamydia
trachomatis include: gi/4377105 (Cpn0794), gi/4377106 (Cpn0795), gi/4377107
(Cpn0796), gi/4377108 (Cpn0797), gi/4377109 (CPn0798), gi/4377110 (Cpn0799).
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SEQ ID No 6
1 MKDLGTLGGT SSTAKTVSPD GKVIMGRSQT ADGSWHAFMC HTDFSSNNVL
51 FDLDNTYKTL RENGRQLNSI FNLQNMMLQR ASDHEFTEFG RSNIALGAGL
101 YWALQNLPS NLAAQYFGIA YKIRPKYRLG VFLDHNFSSH VPNNFNVSHN
151 RLWMGAFIGW QDSDALGSSV KVSFGYGKQK ATITREQLEN TEAGSGESHF
201 EGVAAQIEGR YGKSLGGHVR VQPFLGLQFV HITRKEYTEN AVQFPVHYDP
251 IDYSTGWYL GIGSHIALVD SLHVGTRMGM EQNFAAHTDR FSGSIASIGN
301 FVFEKLDVTH TRAFAEMRW YELPYLQSLN LILRWQQPL QGVMGFSSDL
351 RYALGF*
(7) ArtJ argirci~ze periplasnaic-bindihg protein (CP~z 0482)
One example of 'ArtJ' protein is disclosed as SEQ ID NOS: 73 & 74 in WO
02102606.
{GenBank accession number: gi~4376767~gb~AAD18622.1~ 'CPn0482'; SEQ ID NO:
7 below} . Preferred ArtJ proteins for use with the invention comprise an
amino acid
sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID
NO: 7; and/or (b) which is a fragment of at least n consecutive amino acids of
SEQ ID
NO: 7, wherein fZ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35,
40, 50, 60, 70,
80, 90, 100, 150, 200, 250 or more). These ArtJ proteins include variants
(e.g. allelic
variants, homologs, orthologs, paralogs, mutants, etc. ) of SEQ ID NO: 7.
Preferred
fragments of (b) comprise an epitope from SEQ ID NO: 7. Other preferred
fragments
lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25
or more) from
the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,
I0, 15, 20,
25 or more) from the N-terminus of SEQ ID NO: 7. Other fragments omit one or
more domains of the protein (e.g. omission of a signal peptide, of a
cytoplasmic
domain, of a transmembrane domain, or of an extracellular domain). The ArtJ
protein
may be bound to a small molecule like arginine or another amino acid.
SEQ ID No 7
1 MIKQIGRFFR AFIFIMPLSL TSCESKIDRN RIWIVGTNAT YPPFEWDAQ
51 GEWGFDIDL AKAISEKLGK QLEVREFAFD ALILNLKKHR IDAILAGMSI
101 TPSRQKEIAL LPYYGDEVQE LMWSKRSLE TPVLPLTQYS SVAVQTGTFQ
151 EHYLLSQPGI CVRSFDSTLE VIMEVRYGKS PVAVLEPSVG RVVLKDFPNL
201 VATRLELPPE CWVLGCGLGV AKDRPEEIQT IQQAITDLKS EGVIQSLTKK
WQLSEVAYE*
(8) HtrA DO Serisze Protease (CPn0979)
One example of an 'HrtA' protein is disclosed as SEQ ID NOS: 111 8Z 112 in WO
02/02606. {GenBank accession number: gi~4377306~gb~AAD19116.1~ 'CPn0979';
SEQ ID NO: 8 below. Preferred HrtA proteins for use with the invention
comprise
an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%,
75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)
to SEQ ID NO: 8; and/or (b) which is a fragment of at least n consecutive
amino acids
of SEQ ID NO: 8, wherein ya is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25,
30, 35, 40,
50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These HrtA proteins include
variants
(e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ
ID NO: 8.
Preferred fragments of (b) comprise an epitope from SEQ ID NO: 8. Other
preferred
fragments lack one or more amino acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5,
6, 7, 8,
9, 10, 15, 20, 25 or more; preferably at least 16 to remove the signal
peptide) from the
N-terminus of SEQ ID NO: 8. Other fragments omit one or more domains of the
protein (e.g. omission of a signal peptide as described above, of a
cytoplasmic
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domain, of a transmembrane domain, or of an extracellular domain). In relation
to
SEQ ID NO: 8, distinct domains are residues: 1-16; 17-497; 128-289; 290-381;
394-
485; and 394-497.
SEQ ID No 8
1 MITKQI~RSWI~ AVLVGSSLLA LPLSGQAVGK KESRVSELPQ DVLLKEISGG
51 FSKVATKATP AVVYTESFPK SQAVTHPSPG RRGPYENPFD YFNDEFFNRF
101 FGLPSQREKP QSKEAVRGTG FLVSPDGYIV TNNHWEDTG KIHVTLHDGQ
151 KYPATVIGLD PKTDLAVIKI KSQNLPYLSF GNSDHLKVGD WAIAIGNPFG
1O 201 LQATVTVGVI SAKGRNQLHI ADFEDFTQTD AAINPGNSGG PLLNIDGQVI
251 GVNTAIVSGS GGYIGIGFAI PSLMANRIID QLIRDGQVTR GFLGVTLQPI
301 DAELAACYKL EKVYGALVTD VVKGSPADKA GLKQEDVIIA YNGKEVDSLS
351 MFRNAVSLMN PDTRIVLI<W REGKVIETPV TVSQAPKEDG MSALQRVGIR
401 VQNLTPETAK KLGIAPETKG ILIISVEPGS VAASSGIAPG QLILAVNRQK
451 VSSIEDLNRT LKDSNNENIL LMVSQGDVIR FIALKPEE*
(9) AtoS two-conipoflent regulatory system sensor histidine kiuase protein
(CPn0584)
One example of 'AtoS' protein is disclosed as SEQ ID NOS: 105 & 106 in WO
02/02606. {GenBank accession number: gi~4376878~gb~AAD18723.1~ 'CPn0584';
SEQ ID NO: 9 below}. Preferred AtoS proteins for use with the invention
comprise
an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%,
75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)
to SEQ ID NO: 9; and/or (b) which is a fragment of at least ra consecutive
amino acids
of SEQ ID NO: 9, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25,
30, 35, 40,
50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These AtoS proteins include
variants
(e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ
ID NO: 9.
Preferred fragments of (b) comprise an epitope from SEQ ID NO: 9. Other
preferred
fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the C-terminus andlor one or more amino acids (e.g. 1, 2, 3, 4, 5,
6, 7, 8,
9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 9. Other
fragments
omit one or more domains of the protein (e.g. omission of a signal peptide, of
a
cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
SEQ ID No 9
1 MNVPDSKNLH PPAYELLEIK ARITQSYKEA SATLTAIPDG ILLLSETGHF
51 LICNSQAREI LGIDENLEIL NRSFTDVLPD TCLGFSIQEA LESLKVPKTL
101 RLSLCKESKE KEVELFIRKN EISGYLFIQI RDRSDYKQLE NAIERYKNIA
151 ELGKMTATLA HEIRNPLSGI VGFASILKKE ISSPRHQRML SSIISGTRSL
4O 201 NNLVSSMLEY TKSQPLNLKI INLQDFFSSL IPLLSVSFPN CKFVREGAQP
251 LFRSIDPDRM NSVWNLVKN AVETGNSPTT LTLHTSGDIS VTNPGTIPSE
301 IMDKLFTPFF TTKREGNGLG LAEAQKTIRL HGGDIQLKTS DSAVSFFIII
351 PELLAALPKE RAAS*
(10) OmcA 9kDa-cysteine-rich lipoprotein(CPn0558)
One example of 'OmcA' protein is disclosed as SEQ ID NOS: 9 & 10 in WO
02/02606. {GenBank accession number: gi~4376850~gb~AAD18698.1~ 'CPn0558',
'OmcA', 'Omp3'; SEQ ID NO: 10 below}. Preferred OmcA proteins for use with the
invention comprise an amino acid sequence: (a) having 50% or more identity
(e.g.
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.5% or more) to SEQ ID NO: 10; and/or (b) which is a fragment of
at
least ra consecutive amino acids of SEQ ID NO: 10, wherein ra is 7 or more
(e.g. 8, 10,
12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or
more).
These OmcA proteins include variants (e.g. allelic variants, homologs,
orthologs,
paralogs, mutants, etc.) of SEQ ID NO: 10. Preferred fragments of (b) comprise
an
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epitope from SEQ ID NO: 10. Other preferred fragments lack one or more amino
acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-
terminus and/or
one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or
more;
preferably 18 or more to remove the signal peptide) from the N-terminus of SEQ
ID
NO: 10. Other fragments omit one or more domains of the protein (e.g. omission
of a
signal peptide as described above, of a cytoplasmic domain, of a transmembrane
domain, or of an extracellular domain). The protein may be lipidated (e.g. by
a N
acyl diglyceride), and may thus have a N-terminal cysteine.
SEQ ID No 10
1 MKICAVLIAAM FCGWSLSSC CRIVDCCFED PCAPSSCNPC EVIRKKERSC
51 GGNACGSYVP SCSNPCGSTE CNSQSPQVKG CTSPDGRCKQ
(1l) CPn0498 (Hypothetical)
One example of a hypothetical protein is set forth as SEQ ID NO: 11 below.
(GenBank Accession No. GI:4376784; AAD18638.1). Preferred hypothetical
proteins for use with the invention comprise an amino acid sequence: (a)
having 50%
or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 11; and/or (b) which is
a
fragment of at least h. consecutive amino acids of SEQ ID NO: 11, wherein h is
7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These Hypothetical proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 11. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: 11. Other preferred fragments lack
one
or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)
from the C-
terminus and/or one or more amino acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more; preferably 18 or more to remove the signal peptide) from the N-terminus
of
SEQ ID NO: 11. Other fragments omit one or more domains of the protein (e.g.
omission of a signal peptide as described above, of a cytoplasmic domain, of a
transmembrane domain, or of an extracellular domain). The protein may be
lipidated
(e.g. by a N acyl diglyceride), and may thus have a N-terminal cysteine.
SEQ ID No 11
1 MNRRKARWVV ALFAMTALIS VGCCPWSQAK SRCSIDKYIP VVNRLLEVCG LPEAENVEDL
61 IESSSAWVLT PEERFSGELV SICQVKDEHA FYNDLSLLHM TQAVPSYSAT YDCAWFGGP
121 LPALRQRLDF LVREWQRGVR FKKIVFLCGE RGRYQSIEEQ EHFFDSRYNP FPTEENWESG
181 NRVTPSSEEE IAKFVWMQML LPRAWRDSTS GVRVTFLLAK PEENRWANR KDTLLLFRSY
4O 242 QEAFPGRVLF VSSQPFIGLD ACRVGQFFKG ESYDLAGPGF AQGVLKYHWA PRICLHTLAE
301 WLKETNGCLN ISEGCFG
(12) CPn 0525 (hypotlaetical)
One example of 'Cpn0525' protein is disclosed as SEQ ID NOs: 117 & 118 in WO
02/02606. {GenBank accession number: gi~4376814~gb~AAD18665.1~ 'CPn0525',
SEQ ID NO: 12 below. Preferred hypothetical proteins for use with the
invention
comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%,
65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more) to SEQ ID NO: 12; and/or (b) which is a fragment of at least h
consecutive amino acids of SEQ ID NO: 12, wherein n is 7 or more (e.g. 8, 10,
12, 14,
16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
These
OmcA proteins include variants (e.g. allelic variants, homologs, orthologs,
paralogs,
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mutants, etc.) of SEQ ID NO: 12. Preferred fragments of (b) comprise an
epitope
from SEQ ID NO: 12. Other preferred fragments lack one or more amino acids
(e.g.
1, 2, 3, 4, S, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or
one or more
amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more;
preferably 18 or more
to remove the signal peptide) from the N-terminus of SEQ ID NO: 12. Other
fragments omit one or more domains of the protein (e.g. omission of a signal
peptide
as described above, of a cytoplasmic domain, of a transmembrane domain, or of
an
extracellular domain).
SEQ ID No 12
1 MHDALLSILA IQELDIKMIR LMRVKKEHQK ELAKVQSLKS DIRRKVQEKE
51 LEMENLKTQI RDGENRIQEI SEQINKLENQ QAAVKKMDEF NALTQEMTTA
101 NKERRSLEHQ LSDLMDKQAG GEDLIVSLKE SLASTENSSS VIEKEIFESI
151 KKINEEGKAL LEQRTELKHA TNPELLSIYE RLLNNKKDRV WPIENRVCS
201 GCHIVLTPQH ENLVRKKDRL IFCEHCSRIL YWQESQVNAQ ENSTAKRRRR
251 RAAV*
Third Antigen Group
The immunogenicity of other Chlarnydia pneunaofaiae antigens may be improved
by
combination with two or more ChlanZydia pneumoniae antigens from either the
first
antigen group or the second antigen group. Such other Chlamydia pneunZOniae
antigens include a third antigen group consisting of (1) LcrE, (2) DnaK, (3)
Omp85
homolog, (4) Mip-like; (5) OmcB (6) MurG (7) Cpn0186 and (8) flit. These
antigens are referred to herein as the "third antigen group".
(13) LcrE low calcium respotase E protein (CPn0324)
One example of a 'LcrE' protein is disclosed as SEQ ID NOS: 29 & 30 in WO
02/02606. fGenBank accession number: gi~4376602~gb~AAD18473.1~ 'CPn0324';
SEQ ID NO: 13 below . Preferred LcrE proteins for use with the invention
comprise
an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%,
75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)
to SEQ ID NO: 13; and/or (b) which is a fragment of at least n consecutive
amino
acids of SEQ ID NO: 13, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18,
20, 25, 30,
35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These LcrE proteins
include
variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.)
of SEQ ID
NO: 13. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 13.
Other
preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 15,
20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2,
3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 13.
Other
fragments omit one or more domains of the protein (e.g. omission of a signal
peptide,
of a cytoplasmic domain, of a transmembrane domain, or of an extracellular
domain).
SEQ ID No 13
1 MAASGGTGGL GGTQGVNLAA VEAAAAKADA AEWASQEGS EMNMIQQSQD
51 LTNPAAATRT ICISKEEKFQTL ESRKKGEAGK AEKKSESTEE KPDTDLADKY
101 ASGNSEISGQ ELRGLRDAIG DDASPEDILA LVQEKIKDPA LQSTALDYLV
151 QTTPPSQGKL KEALIQARNT HTEQFGRTAI GAKNILFASQ EYADQLNVSP
201 SGLRSLYLEV TGDTHTCDQL LSMLQDRYTY QDMAIVSSFL MKGMATELKR
5O 251 QGPYVPSAQL QVLMTETRNL QAVLTSYDYF ESRVPILLDS LKAEGIQTPS
301 DLNFVKVAES YHKIINDKFP TASKVEREVR NLIGDDVDSV TGVLNLFFSA
351 LRQTSSRLFS SADKRQQLGA MIANALDAVN INNEDYPKAS DFPKPYPWS*
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(14) DhaK heat-slaock protein 70 (chaperone) (CPia0503)
One example of 'DnaK' protein is disclosed as SEQ ID NOS: 103 & 104 in WO
02/02606. {GenBank accessionnumber: gi~4376790~gb~AAD18643.1~ 'CPnOS03';
SEQ ID NO: 14 below. Preferred DnaK proteins for use with the invention
comprise
an amino acid sequence: (a) having SO% or more identity (e.g. 60%, 6S%, 70%,
7S%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)
to SEQ ID NO: 14; and/or (b) which is a fragment of at least ~ consecutive
amino
acids of SEQ ID NO: I4, wherein h is 7 or more (e.g. 8, 10, 12, I4, 16, I8,
20, 2S, 30,
3S, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These DnaK proteins
include
variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.)
of SEQ ID
NO: 14. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 14.
Other
preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, S, 6, 7, 8,
9, 10, 1S,
20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2,
3, 4, S,
6, 7, 8, 9, 10, 1 S, 20, 25 or more) from the N-terminus of SEQ ID NO: 14.
Other
fragments omit one or more domains of the protein (e.g. omission of a signal
peptide,
of a cytoplasmic domain, of a transmembrane domain, or of an extracellular
domain).
SEQ ID No 14
1 MSEHKKSSKIIGIDLGTTNSCVSVMEGGQAKVITSSEGTRTTPSIVAFKG
51 NEKLVGIPAICRQAVTNPEKTLGSTKRFIGRKYSEVASEIQTVPYTVTSGS
101 KGDAVFEVDGKQYTPEEIGAQILMKMKETAEAYLGETVTEAVITVPAYFN
151 DSQRASTKDAGRIAGLDVKRIIPEPTAAALAYGIDKVGDKKIAVFDLGGG
201 TFDISILEIGDGVFEVLSTNGDTLLGGDDFDEVIIKWMIEEFKKQEGIDL
251 SKDNMALQRLKDAAEKAKIELSGVSSTEINQPFITMDAQGPKHLALTLTR
301 AQFEKLAASLIERTKSPCIKALSDAKLSAKDIDDVLLVGGMSRMPAVQET
351 VKELFGKEPNKGVNPDEVVAIGAAIQGGVLGGEVKDVLLLDVIPLSLGIE
401 TLGGVMTTLVERNTTIPTQKKQIFSTAADNQPAVTIWLQGERPMAKDNK
451 ETGRFDLTDIPPAPRGHPQIEVSFDIDANGIFHVSAKDVASGKEQKIRIE
3O 501 ASSGLQEDEIQRMVRDAEINKEEDKKRREASDAKNEADSMTFRAEKAIKD
551 YKEQIPETLVKEIEERIENVRNALKDDAPIEKIKEVTEDLSKHMQKIGES
601 MQSQSASAAASSAANAKGGPNINTEDLKKHSFSTKPPSNNGSSEDHIEEA
651 DVEIIDNDDK*
(IS) Omp85 hoaaolog (Cph0300)
One example of an OmpBS Homolog protein is disclosed as SEQ ID NOS: 147 & 148
in WO 02/02606. f GenBank accession number: gi~4376S76~gb~AAD18449.1~
'CPn0300'; SEQ ID NO: 1S below. Preferred Omp8S proteins for use with the
invention comprise an amino acid sequence: (a) having SO% or more identity
(e.g.
60%, 65%, 70%, 7S%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 9S%, 96%, 97%,
98%, 99%, 99.5% or more) to SEQ ID NO: 15; and/or (b) which is a fragment of
at
least n consecutive amino acids of SEQ ID NO: 1 S, wherein ya is 7 or more
(e.g. 8, 10,
12, 14, 16, 18, 20, 2S, 30, 35, 40, S0, 60, 70, 80, 90, 100, 150, 200, 2S0 or
more).
These DnaK proteins include variants (e.g. allelic variants, homologs,
orthologs,
paralogs, mutants, etc.) of SEQ ID NO: 1S. Preferred fragments of (b) comprise
an
epitope from SEQ ID NO: 1S. Other preferred fragments lack one or more amino
acids (e.g. I, 2, 3, 4, 5, 6, 7, 8, 9, I0, 15, 20, 2S or more) from the C-
terminus and/or
one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, IS, 20, 25 or
more) from the
N-terminus of SEQ ID NO: 1S. Other fragments omit one or more domains of the
protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a
transmembrane domain, or of an extracellular domain).
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SEQ ID No I S
1 MLIMRNKVILQISILALIQTPLTLFSTEKV TIITEGENAS
KEGHWVDSI
51 NKHPLPKLKTRSGALFSQLDFDEDLRILAKEYDSVEPKVEFSEGKTNIAL
101 HLIAKPSIRNIHISGNQWPEHKILKTLQIYRNDLFEREKFLKGLDDLRT
151 YYLKRGYFASSVDYSLEHNQEKGHIDVLIKINEGPCGKIKQLTFSGISRS
201 EKSDIQEFIQTKQHSTTTSWFTGAGLYHPDIVEQDSLAITNYLHNNGYAD
251 AIVNSHYDLDDKGNILLYMDTDRGSRYTLGHVHIQGFEVLPKRLIEKQSQ
301 VGPNDLYCPDKTWDGAHKIKQTYAKYGYINTNVDVLFIPHATRPIYDVTY
1O 351 EVSEGSPYKVGLIKITGNTHTKSDVILHETSLFPGDTFNRLKLEDTEQRL
401 RNTGYFQSVSVYTVRSQLDPMGNADQYRDIFVEVKETTTGNLGLFLGFSS
451 LDNLFGGIELSESNFDLFGARNIFSKGFRCLRGGGEHLFLKANFGDKVTD
501 YTLKWTKPHFLNTPWILGIELDKSINRALSKDYAVQTYGGNVSTTYILNE
551 HLKYGLFYRGSQTSLHEKRKFLLGPNIDSNKGFVSAAGVNLNYDSVDSPR
601 TPTTGIRGGVTFEVSGLGGTYHFTKLSLNSSIYRKLTRKGILKIKGEAQF
651 IKPYSNTTAEGVPVSERFFLGGETTVRGYKSFIIGPKYSATEPQGGLSSL
701 LISEEFQYPLIRQPNISAFVFLDSGFVGLQEYKISLKDLRSSAGFGLRFD
751 VMNNVPVMLGFGWPFRPTETLNGEKIDVSQRFFFALGGMF
(16) Mip-like FKBP-type peptidyl prolyl cis-traps (CPzz0661)
One example of a Mip-like protein is disclosed as SEQ ID NOS: 55 & S6 in WO
02/02606. {GenBank accession number: gi~4376960~gb~AAD18800.1~ 'CPn0661';
SEQ ID NO: 16 below} . Preferred Mip-like proteins for use with the invention
comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%,
65%, ,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more) to SEQ ID NO: 16; and/or (b) which is a fragment of at least r2
consecutive amino acids of SEQ ID NO: 16, wherein h is 7 or more (e.g. 8, 10,
12, 14,
16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
These mip-
like proteins include variants (e.g. allelic variants, homologs, orthologs,
paralogs,
mutants, etc.) of SEQ ID NO: 16. Preferred fragments of (b) comprise an
epitope
from SEQ ID NO: 16. Other preferred fragments lack one or more amino acids
(e.g.
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or
one or more
amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the
N-terminus
of SEQ ID NO: 16. Other fragments omit one or more domains of the protein
(e.g.
omission of a signal peptide, of a cytoplasmic domain, of a transmembrane
domain, or
of an extracellular domain).
SEQ ID No 16
1 MNRRWNLVLA TVALAIaSVAS CDVRSI<DKDK DQGSLVEYKD NKDTNDIELS
51 DNQKLSRTFG HLLARQLRKS EDMFFDIAEV AKGLQAELVC KSAPLTETEY
101 EEKMAEVQKL VFEKKSKENL SLAEKFLKEN SKNAGWEVQ PSKLQYKIIK
151 EGAGKAISGK PSALLHYKGS FINGQVFSSS EGNNEPILLP LGQTIPGFAL
201 GMQGMKEGET RVLYIHPDLA YGTAGQLPPN SLLIFEINLI QASADEVAAV
251 PQEGNQGE*
(17) OszzcB 60 kDa Cysteifze rich OMP (CPn0557)
One example of an OmcB protein is disclosed as SEQ ID NOS: 47 & 48 in WO
02/02606. {GenBank accession number: gi~4376849~gb~AAD18697.1~ 'CPn0557';
SEQ ID NO: 17 below. Preferred OmcB proteins for use with the invention
comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%,
65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more) to SEQ ID NO: 17; and/or (b) which is a fragment of at least rz
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consecutive amino acids of SEQ ID NO: 17, wherein h is 7 or more (e.g. 8, 10,
12, 14,
16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or moxe).
These
OmcB proteins include variants (e.g. allelic variants, homologs, oxthologs,
paralogs,
mutants, etc.) of SEQ ID NO: 17. Preferred fragments of (b) comprise an
epitope
from SEQ ID NO: 17. Other preferred fragments lack one or more amino acids
(e.g.
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or
one or more
amino acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the
N-terminus
of SEQ ID NO: 17. Other fragments omit one or more domains of the protein
(e.g.
omission of a signal peptide, of a cytoplasmic domain, of a transmembrane
domain, or
of an extracellular domain)..
SEQ ID No 17
1 MSKLIRRWT VLALTSMASC _FASGGIEAAV AESLITKIVA SAETKPAPVP
51 MTAKKVRLVR RNKQPVEQKS RGAFCDKEFY PCEEGRCQPV EAQQESCYGR
101 LYSVKVNDDC NVETCQSVPE YATVGSPYPI EILAIGKKDC VDWITQQLP
151 CEAEFVSSDP ETTPTSDGI<L VWKIDRLGAG DKCKTTVWVK PLKEGCCFTA
201 ATVCACPELR SYTKCGQPAT CIKQEGPDCA CLRCPVCYKI EVVNTGSAIA
251 RNVTVDNPVP DGYSHASGQR VLSFNLGDMR PGDKKVFTVE FCPQRRGQIT
301 NVATVTYCGG HKCSANVTTV VNEPCVQVNI SGADWSYVCK PVEYSTSVSN
2O 351 PGDLVLHDW IQDTLPSGVT VLEAPGGEIC CNKWWRIKE MCPGETLQFK
401 LWKAQVPGR FTNQVAVTSE SNCGTCTSCA ETTTHWKGLA ATHMCVLDTN
451 DPTCVGENTV YRICVTNRGS AEDTNVSLIL KFSKELQPIA SSGPTKGTTS
501 GNTWFDALP KLGSKESVEF SVTLKGIAPG DARGEAILSS DTLTSPVSDT
551 ENTHVY*
(18) MurG peptidoglycan transferase proteih (CPti0904)
One example of a 'MurG' protein is disclosed as SEQ ID NOS: 107 & 108 in WO
02/02606. ~GenBank accession number: gi~4377224~gb~AAD19042.1~ 'CPn0904';
SEQ ID NO: 18 below}. Preferred MurG proteins for use with the invention
comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%,
65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more) to SEQ ID NO: 18; and/or (b) which is a fragment of at least n
consecutive amino acids of SEQ ID NO: 18, wherein ra is 7 or more (e.g. 8, 10,
12, 14,
16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
These
MurG proteins include vaxiants (e.g. allelic variants, homologs, orthologs,
paralogs,
mutants, etc.) of SEQ ID NO: 18. Preferred fragments of (b) comprise an
epitope
from SEQ ID NO: 18. Other preferred fragments lack one or more amino acids
(e.g.
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or
one or more
amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, -9, 10, 15, 20, 25 or more) from the
N-terminus
of SEQ ID NO: 18. Other fragments omit one or more domains of the protein
(e.g.
omission of a signal peptide as described above, of a cytoplasmic domain, of a
transmembrane domain, ox of an extracellular domain). The MurG may be
lipidated
e.g. with undecaprenyl.
SEQ ID No 18
1 MMKKIRKVAL AVGGSGGHIV PALSVKEAFS REGIDVLLLG KGLKNHPSLQ
51 QGISYREIPS GLPTVLNPIK IMSRTLSLCS GYLKARKELK IFDPDLVTGF
5O 101 GSYHSLPVLL AGLSHKTPLF LHEQNLVPGK VNQLFSRYAR GIGVNFSPVT
151 KHFRCPAEEV FLPKRSFSLG SPMMKRCTNH TPTICWGGS QGAQILNTCV
201 PQALVKLVNK YPNMYVHHIV GPKSDVMKVQ HVYNRGEVLC CVKPFEEQLL
251 DVLLAADLVI SRAGATTLEE ILWAKVPGIL IPYPGAYGHQ EVNAKFFVDV
301 LEGGTMILEK ELTEKLLVEK VTFALDSHNR EKQRNSLAAY SQQRSTKTFH
351 AFICECL*
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(19) CPfa0186 (Hypothetical)
One example of a hypothetical protein is set forth as SEQ ID NO: 19 below} .
(GenBank Accession No. GI:4376456; AAD18339.1). Preferred hypothetical
proteins for use with the invention comprise an amino acid sequence: (a)
having 50%
or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 19; and/or (b) which is
a
fragment of at least fa consecutive amino acids of SEQ ID NO: 19, wherein fa
is 7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These hypothetical proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 19. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: 19. Other preferred fragments lack
one
or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)
from the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 19. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 19
1 MSSPVNNTPS APNIPIPAPT TPGIPTTKPR SSFIEKVIIV AKYILFAIAA TSGALGTILG
61 LSGALTPGIG IALLVIFFVS MVLLGLTLKD SISGGEERRL REEVSRFTSE NQRLTVITTT
121 LETEVKDLKA AKDQLTLEIE AFRNENGNLK TTAEDLEEQV SKLSEQLEAL ERINQLIQAN
181 AGDAQETSSE LKKLISGWDS KWEQINTSI QALKVLLGQE WVQEAQTHVK AMQEQIQALQ
241 AEILGMHNQS TALQKSVENL LVQDQALTRV VGELLESENK LSQACSALRQ EIEKLAQHET
301 SLQQRIDAML AQEQNLAEQV TALEKMKQEA QKAESEFIAC VRDRTFGRRE TPPPTTPWE
361 GDESQEEDEG GTPPVSQPSS PVDRATGDGQ
(20) FIiY Glutasnihe Bi~adiug P~oteiu (CPsa0604)
One example of a hypothetical protein is set forth as SEQ ID NOS: 11 & 12 in
WO
02/02606. ~GenBank accession number: gi~4376900~gb~AAD18743.1~ 'CPn0604';
SEQ ID NO: 20 below}. Preferred hypothetical proteins for use with the
invention
comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%,
65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more) to SEQ TD NO: 20; and/or (b) which is a fragment of at Ieast ra
consecutive amino acids of SEQ ID NO: 20, wherein h is 7 or more (e.g. 8, 10,
12, 14,
16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
These
hypothetical proteins include variants (e.g. allelic variants, homologs,
orthologs,
paralogs, mutants, etc.) of SEQ ID NO: 20. Preferred fragments of (b) comprise
an
epitope from SEQ ID NO: 20. Other preferred fragments lack one or more amino
acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-
terminus and/or
one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or
more) from the
N-terminus of SEQ ID NO: 20. Other fragments omit one or more domains of the
protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a
transmembrane domain, or of an extracellular domain).
SEQ ID No 20
1 MKIKFSWKVN FLICLLAVGL IFFGCSRVKR EVLVGRDATW FPKQFGIYTS
5O 51 DTNAFLNDLV SEINYKENLN INIVNQDWVH LFENLDDKKT QGAFTSVLPT
101 LEMLEHYQFS DPILLTGPVL VVAQDSPYQS IEDLKGRLIG VYKFDSSVLV
151 AQNIPDAVIS LYQHVPIALE ALTSNCYDAL LAPVIEVTAL IETAYKGRLK
201 ITSKPLNADG LRLAILKGTN GbLLEGFNAG LVKTRRSGKY DAIKQRYRLP
21
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The immunogenicity of other Clalamydia pneumoniae antigens may be improved by
combination with two or more Chlamydia pneumoniae antigens from either the
first
antigen group or the second antigen group or the third antigen group. Such
other
Chlamydia pneumoniae antigens include a fourth antigen group consisting one or
more members of the PMP family. These antigens are referred to herein as the
"fourth antigen group". Each of the Chlamydia pneumoniae antigens of the
fourth
antigen group is described in more detail below.
Fourth Antigen Group
20
(21) Polytnorphic Me>'tbra>ze Proteins (PMP)
A family of twenty one Chlamydia pneumoniae genes encoding predicted
polymorphic membrane proteins (PMP) have been identified (pmpl to pmp2~.
P~rtpl (CPiz0005)
One example of a Pmpl protein is set forth as SEQ ID NOS: 41 ~Z 42 in WO
02/02606. {GenBank accession number: gi~4376260~gb~AAD18163.1 'CPn0005';
SEQ ID NO: 2I below}.
SEQ ID No 21
1 MRFSLCGFPL LTPEDSFHGDSQNAERSYNV
VFSFTLLSVF
DTSI~SATTIS
51 QAGDVYSLTGDVSISNVDNSALNKACFNVTSGSVTFAGNHHGLYFNNISS
101 GTTKEGAVLCCQDPQATARFSGFSTLSFIQSPGDIKEQGCLYSKNALMLL
151 NNYWRFEQNQSKTKGGAISGANVTTVGNYDSVSFYQNAATFGGAIHSSG
201 PLQIAVNQAEIRFAQNTAKNGSGGALYSDGDIDIDQNAYVLFRENEALTT
251 AIGKGGAVCCLPTSGSSTPVPIVTFSDNKQLVFERNHSIMGGGAIYARKL
301 SISSGGPTLFINNTSYANSQNLGGAIAIDTGGEISLSAEKGTITFQGNRT
3O 351 SLPFLNGIHLLQNAKFLKLQARNGYSIEFYDPTTSEADGSTQLNINGDPK
401 NKEYTGTILFSGEKSLANDPRDFKSTIPQNWLSAGYLVIKEGAEVTVSK
451 FTQSPGSHLVLDLGTKLIASKEDIAITGLAIDIDSLSSSSTAAVIKANTA
501 NKQISVTDSIELISPTGNAYEDLRMRNSQTFPLLSLEPGAGGSVTVTAGD
551 FLPVSPHYGFQGNWKLAWTGTGNKVGEFFWDKINYKPRPEKEGNLVPNIL
601 WGNAVDVRSLMQVQETHASSLQTDRGLWIDGIGNFFHVSASEDNIRYRHN
651 SGGYVLSWNEITPKHYTSMAFSQLFSRDKDYAVSNNEYRMYLGSYLYQY
701 TTSLGNIFRYASRNPNVNVGILSRRFLQNPLMIFHFLCAYGHATNDMKTD
751 YANFPMVKNSWRNNCWAIECGGSMPLLVFENGRLFQGAIPFMKLQLVYAY
801 QGDFKETTADGRRFSNGSLTSISVPLGIRFEKLALSQDVLYDFSFSYIPD
4O 851 IFRKDPSCEAALVISGDSWLVPAAHVSRHAFVGSGTGRYHFNDYTELLCR
901 GSIECRPHARNYNINCGSKFRF*
Pntp 4 (CPu0017)
One example of a Pmp 4 protein is designated SEQ ID NO: 22. The sequence for
pmp4 protein can be found at AE001587.1 GI:4376271.
Pfttp 6 (CP~t 0444)
One example of a Pmp 6 protein is set forth as SEQ ID NOS 31 & 32 in WO
02/02606. {GenBank accession number: gi~4376727~gb~AAD18S88.1~ 'CPn0444';
SEQ ID NO: 23 below}.
SEQ ID No 23
1 MKYSLPWLLT SSALVFSLHP LMAANTDLSS SDNYENGSSG SAAFTAKETS
51 DASGTTYTLT SDVSITNVSA ITPADKSCFT NTGGALSFVG ADHSLVLQTI
22
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101 ALTHDGAAINNTNTALSFSGFSSLLIDSAPATGTSGGKGAICVTNTEGGT
151 ATFTDNASVTLQKNTSEKDGAAVSAYSIDLAKTTTAALLDQNTSTKNGGA
201 LCSTANTTVQGNSGTVTFSSNTATDKGGGIYSKEKDSTLDANTGVVTFKS
251 NTAKTGGAWSSDDNLALTGNTQVLFQENKTTGSAAQANNPEGCGGAICCY
301 LATATDKTGLAISQNQEMSFTSNTTTANGGAIYATKCTLDGNTTLTFDQN
351 TATAGCGGAIYTETEDFSLKGSTGTVTFSTNTAKTGGALYSKGNSSLTGN
401 TNLLFSGNKATGPSNSSANQEGCGGAILAFIDSGSVSDKTGLSIANNQEV
451 SLTSNAATVSGGAIYATKCTLTGNGSLTFDGNTAGTSGGAIYTETEDFTL
501 TGSTGTVTFSTNTAKTGGALYSKGNNSLSGNTNLLFSGNKATGPSNSSAN
551 QEGCGGAILSFLESASVSTKKGLWIEDNENVSLSGNTATVSGGAIYATKC
601 ALHGNTTLTFDGNTAETAGGAIYTETEDFTLTGSTGTVTFSTNTAKTAGA
651 LHTKGNTSFTKNKALVFSGNSATATATTTTDQEGCGGAILCNISESDIAT
701 KSLTLTENESLSFINNTAKRSGGGIYAPKCVISGSESINFDGNTAETSGG
751 AIYSKNLSITANGPVSFTNNSGGKGGAIYIADSGELSLEAIDGDITFSGN
~I5 801 RATEGTSTPNSIHLGAGAKITKLAAAPGHTIYFYDPITMEAPASGGTIEE
851 LVINPVVKAIVPPPQPKNGPIASVPWPVAPANPNTGTIVFSSGKLPSQD
901 ASIPANTTTILNQKINLAGGNWLKEGATLQVYSFTQQPDSTVFMDAGTT
951 LETTTTNNTDGSIDLKNLSVNLDALDGKRMITIAVNSTSGGLKISGDLKF
1001 HNNEGSFYDNPGLKANLNLPFLDLSSTSGTVNLDDFNPIPSSMAAPDYGY
2O 1051 QGSWTLVPKVGAGGKVTLVAEWQALGYTPKPELRATLVPNSLWNAYVNIH
1101 SIQQEIATAMSDAPSHPGIWIGGIGNAFHQDKQKENAGFRLISRGYIVGG
1151 SMTTPQEYTFAVAFSQLFGKSKDYWSDIKSQVYAGSLCAQSSYVIPLHS
1201 SLRRHVLSKVLPELPGETPLVLHGQVSYGRNHHNMTTKLANNTQGKSDWD
1251 SHSFAVEVGGSLPVDLNYRYLTSYSPYVKLQWSVNQKGFQEVAADPRIF
25 1301 DASHLVNVSIPMGLTFKHESAKPPSALLLTLGYAVDAYRDHPHCLTSLTN
1351 GTSWSTFATNLSRQAFFAEASGHLKLLHGLDCFASGSCELRSSSRSYNAN
1401 CGTRYSF*
30 Prrrp 7 (CPtz04457
One example of a Pmp 7 protein is set forth as SEQ ID NOS 153 & 154 in WO
02/02606. {GenBank accession number: gi~4376728~gb~AAD18589.1~ 'CPn0445';
SEQ ID NO: 24 below}.
35 SEQ ID No 24
1 MKSSVSWLFF SSIPLFSSLS IVAAEVTLDS SNNSYDGSNG TTFTVFSTTD
51 AAAGTTYSLL SDVSFQNAGA LGIPLASGCF LEAGGDLTFQ GNQHALKFAF
101 INAGSSAGTV ASTSAADKNL LFNDFSRLSI ISCPSLLLSP TGQCALKSVG
4O 151 NLSLTGNSQI IFTQNFSSDN GGVINTKNFL LSGTSQFASF SRNQAFTGKQ
201 GGWYATGTITIENSPGIVSFSQNLAKGSGGALYSTDNCSITDNFQVIFD
251 GNSAWEAAQAQGGAICCTTTDKTVTLTGNKNLSFTNNTALTYGGAISGLK
301 VSISAGGPTLFQSNISGSSAGQGGGGAINIASAGELALSATSGDITFNNN
351 QVTNGSTSTRNAINIIDTAKVTSIRAATGQSIYFYDPITNPGTAASTDTL
45 401 NLNLADANSEIEYGGAIVFSGEKLSPTEKAIAANVTSTIRQPAVLARGDL
451 VLRDGVTVTFKDLTQSPGSRILMDGGTTLSAKEANLSLNGLAVNLSSLDG
501 TNKAALKTEAADKNISLSGTIALIDTEGSFYENHNLKSASTYPLLELTTA
551 GANGTITLGALSTLTLQEPETHYGYQGNWQLSWANATSSKIGSINWTRTG
601 YIPSPERKSNLPLNSLWGNFIDIRSINQLIETKSSGEPFERELWLSGIAN
5O 651 FFYRDSMPTRHGFRHISGGYALGITATTPAEDQLTFAFCQLFARDRNHIT
701 GKNHGDTYGASLYFHHTEGLFDIANFLWGKATRAPWVLSEISQIIPLSFD
751 AKFSYLHTDNHMKTYYTDNSIIKGSWRNDAFCADLGASLPFVISVPYLLK
801 EVEPFVKVQYIYAHQQDFYERHAEGRAFNKSELINVETPIGVTFERDSKS
851 EKGTYDLTLMYILDAYRRNPKCQTSLIASDANWMAYGTNLARQGFSVRAA
55 901 NHFQVNPHMEIFGQFAFEVRSSSRNYNTNLGSKFCF*
Pmp 8 (CPrr 0446)
One example of a Pmp 8 protein is set forth as SEQ ID NOS 45 ~ 46 in WO
60 02/02606. {GenBank accession number: gi~4376729~gb~.AAD18590.1~ 'CPn0446';
SEQ ID NO: 25 below}.
23
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SEQ ID No 25
1 MKIPLHKLLISSTLVTPILLSTATYGADASLSPTDSFDGAGGSTFTPKST
51 ADANGTNYVLSGNVYINDAGKGTALTGCCFTETTGDLTFTGKGYSFSFNT
101 VDAGSNAGAAASTTADKALTFTGFSNLSFIAAPGTTVASGKSTLSSAGAL
152 NLTDNGTILFSQNVSNEANNNGGAITTKTLSISGNTSSITFTSNSAKKLG
201 GAIYSSAAASISGNTGQLVFMNNKGETGGGALGFEASSSITQNSSLFFSG
251 NTATDAAGKGGAIYCEKTGETPTLTISGNKSLTFAENSSVTQGGAICAHG
301 LDLSAAGPTLFSNNRCGNTAAGKGGAIAIADSGSLSLSANQGDITFLGNT
351 LTSTSAPTSTRNAIYLGSSAKITNLRAAQGQSIYFYDPIASNTTGASDVL
401 TTNQPDSNSPLDYSGTIVFSGEKLSADEAKAADNFTSILKQPLALASGTL
451 ALKGNVELDVNGFTQTEGSTLLMQPGTKLKADTEAISLTKLVVDLSALEG
501 NKSVSIETAGANKTITLTSPLVFQDSSGNFYESHTTNQAFTQPLVVFTAA
551 TAASDIYIDALLTSPVQTPEPHYGYQGHWEATWADTSTAKSGTMTWVTTG
601 YNPNPERRASWPDSLWASFTDIRTLQQIMTSQANSIYQQRGLWASGTAN
651 FFHKDKSGTNQAFRHKSYGYIVGGSAEDFSENIFSVAFCQLFGKDKDLFI
701 VENTSHNYLASLYLQHRAFLGGLPMPSFGSITDMLKDIPLILNAQLSYSY
751 TKNDMDTRYTSYPEAQGSWTNNSGALELGGSLALYLPKEAPFFQGYFPFL
801 KFQAVYSRQQNFKESGAEARAFDDGDLVNCSIPVGIRLEKISEDEKNNFE
2O 851 TSLAYTGDVYRKNPRSRTSLMVSGASWTSLCKNLARQAFLASAGSHLTLS
901 PHVELSGEAAYELRGSAHIYNVDCGLRYSF
Pmp 9 (CPn0447)
One example of a Pmp 9 protein is set forth as SEQ ID NOS 33 & 34 in WO
02/02606. ~GenBank accession number: gi~4376731 ~gb~AAD 18591.1 ~ 'CPn0447';
SEQ ID NO: 26 below}.
SEQ ID No 26
3O 1 MKSSLHWFLISSSLALPLSLNFSAFAAVVEINLGPTNSFSGPGTYTPPAQ
51 TTNADGTIYNLTGDVSITNAGSPTALTASCFKETTGNLSFQGHGYQFLLQ
101 NIDAGANCTFTNTAANKLLSFSGFSYLSLIQTTNATTGTGAIKSTGACSI
151 QSNYSCYFGQNFSNDNGGALQGSSISLSLNPNLTFAKNKATQKGGALYST
201 GGITINNTLNSASFSENTAANNGGAIYTEASSFISSNKAISFINNSVTAT
251 SATGGAIYCSSTSAPKPVLTLSDNGELNFIGNTAITSGGAIYTDNLVLSS
301 GGPTLFKNNSAIDTAAPLGGAIATADSGSLSLSALGGDITFEGNTWKGA
351 SSSQTTTRNSINIGNTNAKIVQLRASQGNTIYFYDPITTSITAALSDALN
401 LNGPDLAGNPAYQGTIVFSGEKLSEAEAAEADNLKSTIQQPLTLAGGQLS
451 LKSGVTLVAKSFSQSPGSTLLMDAGTTLETADGITINNLVLNVDSLKETK
4O 501 KATLKATQASQTVTLSGSLSLVDPSGNVYEDVSWNNPQVFSCLTLTADDP
551 ANIHITDLAADPLEKNPIHWGYQGNWALSWQEDTATKSKAATLTWTKTGY
601 NPNPERRGTLVANTLWGSFVDVRSIQQLVATKVRQSQETRGIWCEGISNF
651 FHKDSTKINKGFRHISAGYVVGATTTLASDNLITAAFCQLFGKDRDHFIN
701 KNRASAYAASLHLQHLATLSSPSLLRYLPGSESEQPVLFDAQISYIYSKN
751 TMKTYYTQAPKGESSWYNDGCALELASSLPHTALSHEGLFHAYFPFIKVE
801 ASYIHQDSFKERNTTLVRSFDSGDLINVSVPIGITFERFSRNERASYEAT
851 VIYVADVYRKNPDCTTALLINNTSWKTTGTNLSRQAGIGRAGIFYAFSPN
901 LEVTSNLSMEIRGSSRSYNADLGGKFQF*
Pnap 1l (CPn0451)
One example of a Pmp 11 protein is set forth as SEQ ID NOS 115 & 116 in WO
02/02606. {GenBank accession number: gi~4376733~gb~AAD18593.1~ 'CPn0451';
SEQ ID NO: 27 below).
SEQ ID No 27
1 MKTSIPWVLV SSVI~AFSCHL QSLANEELLS PDDSFNGNID SGTFTPKTSA
51 TTYSLTGDVF FYEPGKGTPL SDSCFKQTTD NLTFLGNGHS LTFGFIDAGT
101 HAGAAASTTA NKNLTFSGFS LLSFDSSPST TVTTGQGTLS SAGGVNLENI
GO 151 RKLWAGNFS TADGGAIKGA SFLLTGTSGD ALFSNNSSST KGGAIATTAG
201 ARIANNTGYV RFLSNIASTS GGAIDDEGTS ILSNNKFLYF EGNAAKTTGG
251 AICNTKASGS PELIISNNKT LTFASNVAET SGGAIHAKKL ALSSGGFTEF
24
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301 LRNNVSSATP KGGAISIDAS GELSLSAETG NITFVRNTLT TTGSTDTPKR
351 NAINIGSNGK FTELRAAKNH TIFFYDPITS EGTSSDVLKI NNGSAGALNP
401 YQGTILFSGE TLTADELKVA DNLKSSFTQP VSLSGGKLLL QKGVTLESTS
451 FSQEAGSLLG MDSGTTLSTT AGSITITNLG INVDSLGLKQ PVSLTAKGAS
501 NKVIVSGKLN LIDIEGNIYE SHMFSHDQLF SLLKITVDAD VDTNVDISSL
551 IPVPAEDPNS EYGFQGQWNV NWTTDTATNT KEATATWTKT GFVPSPERKS
601 ALVCNTLWGV FTDIRSLQQL VEIGATGMEH KQGFWVSSMT NFLHKTGDEN
651 RKGFRHTSGG WIGGSAHTP KDDLFTFAFC HLFARDKDCF IAHNNSRTYG
701 GTLFFKHSHT LQPQNYLRLG RAKFSESAIE KFPREIPLAL DVQVSFSHSD
1O 751 NRMETHYTSL PESEGSWSNE CIAGGIGLDL PFVLSNPHPL FKTFIPQMKV
801 EMVYVSQNSF FESSSDGRGF STGRLLNLSI PVGAKFVQGD IGDSYTYDLS
851 GFFVSDVYRN NPQSTATLVM SPDSWKIRGG NLSRQAFLLR GSNNYVYNSN
901 CELFGHYAME LRGSSRNYNV DVGTKLRF*
Pfnp 12 (CPn0452)
One example of a Pmp 12 protein is set forth as SEQ ID NOS 51 & 52 in WO
02/02606. {GenBank accession number: gi~4376735~gb~AAD18594.1 'CPn0452';
SEQ ID NO: 28 below).
SEQ ID No 28
1 MTILRNFLTC SALFLALPAA AQWYLHESD GYNGAINNKS LEPKITCYPE
51 GTSYIFLDDV RISNVKHDQE DAGVFINRSG NLFFMGNRCN FTFHNLMTEG
101 FGAAISNRVG DTTLTLSNFS YLAFTSAPLL PQGQGATYSL GSVMIENSEE
151 VTFCGNYSSW SGAAIYTPYL LGSKASRPSV NLSGNRYLVF RDNVSQGYGG
201 AISTHNLTLT TRGPSCFENN HAYHDVNSNG GAIAIAPGGS ISISVKSGDL
251 IFKGNTASQD GNTIHNSIHL QSGAQFKNLR AVSESGVYFY DPISHSESHK
301 ITDLVINAPE GKETYEGTTS FSGLCLDDHE VCAENLTSTI LQDVTLAGGT
351 LSLSDGVTLQ LHSFKQEASS TLTMSPGTTL LCSGDARVQN LHILIEDTDN
3O 401 FVPVRIRAED KDALVSLEKL KVAFEAYWSV YDFPQFKEAF TIPLLELLGP
451 SFDSLLLGET TLERTQVTTE NDAVRGFWSL SWEEYPPSLD KDRRITPTKK
501 TVFLTWNPEI TSTP*
Pnzp 13 (CPh0453)
One example of a Pmp 13 protein is set forth as SEQ ID NOS 3 & 4 in WO
02/02606.
{GenBank accession number: gi~4376736~gb~AAD18595.1 'CPn0453'; SEQ ID NO:
29 below.
SEQ ID No 29
1 MKTSIRKFI,I STT7~APCFAS TAFTVEVIMP SENFDGSSGK IFPYTTLSDP
51 RGTLCIFSGD LYIANLDNAI SRTSSSCFSN RAGALQILGK GGVFSFLNIR
101 SSADGAAISS VITQNPELCP LSFSGFSQMI FDNCESLTSD TSASNVIPHA
151 SAIYATTPML FTNNDSILFQ YNRSAGFGAA IRGTSITIEN TKKSLLFNGN
201 GSISNGGALT GSAAINLINN SAPVIFSTNA TGIYGGAIYL TGGSMLTSGN
251 LSGVLFVNNSSRSGGAIYANGNVTFSNNSDLTFQNNTASPQNSLPAPTPP
301 PTPPAVTPLLGYGGAIFCTPPATPPPTGVSLTISGENSVTFLENIASEQG
351 GALYGKKISIDSNKSTTFLGNTAGKGGAIAIPESGELSLSANQGDILFNK
401 NLSITSGTPTRNSIHFGKDAKFATLGATQGYTLYFYDPITSDDLSAASAA
5O 451 ATWVNPKASADGAYSGTIVFSGETLTATEAATPANATSTLNQKLELEGG
501 TLALRNGATLNVHNFTQDEKSWIMDAGTTLATTNGANNTDGAITLNKLV
551 INLDSLDGTKAAWNVQSTNGALTISGTLGLVKNSQDCCDNHGMFNKDLQ
601 QVPILELKATSNTVTTTDFSLGTNGYQQSPYGYQGTWEFTIDTTTHTVTG
651 NWKKTGYLPHPERLAPLIPNSLWANVIDLRAVSQASAADGEDVPGKQLSI
701 TGITNFFHANHTGDARSYRHMGGGYLINTYTRITPDAALSLGFGQLFTKS
751 I<DYLVGHGHSNVYFATVYSNITKSLFGSSRFFSGGTSRVTYSRSNEKVKT
801 SYTKLPKGRCSWSNNCWLGELEGNLPITLSSRILNLKQIIPFVKAEVAYA
851 THGGIQENTPEGRIFGHGHLLNVAVPVGVRFGKNSHNRPDFYTIIVAYAP
901 DVYRHNPDCDTTLPINGATWTSIGNNLTRSTLLVQASSHTSVNDVLEIFG
GO 951 HCGCDIRRTSRQYTLDIGSKLRF*
Ptzzp 14 (CPiz0454)
CA 02557353 2006-08-24
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One example of a Pmp 14 protein is set forth as SEQ ID NOS 35 ~z 36 in WO
02/02606. {GenBank accession number: gi~4376737~gb~AAD18596.1 'CPn0454';
SEQ ID NO: 30 below.
SEQ ID No 30
1 MPLSFKSSSFCL7~ACLCSASCAFAETRLGGNFVPPITNQGEEILLTSDFV
51 CSNFLGASFSSSFINSSSNLSLLGKGLSLTFTSCQAPTNSNYALLSAAET
101 LTFKNFSSINFTGNQSTGLGGLIYGKDIVFQSIKDLIFTTNRVAYSPASV
151 TTSATPAITTVTTGASALQPTDSLTVENISQSIKFFGNLANFGSAISSSP
201 TAVVKFINNTATMSFSHNFTSSGGGVIYGGSSLLFENNSGCIIFTANSCV
251 NSLKGVTPSSGTYALGSGGAICTPTGTFELKNNQGKCTFSYNGTPNDAGA
301 IYAETCNIVGNQGALLLDSNTAARNGGAICAKVLNIQGRGPIEFSRNRAE
351 KGGAIFIGPSVGDPAKQTSTLTILASEGDIAFQGNMLNTKPGIRNAITVE
401 AGGEIVSLSAQGGSRLVFYDPITHSLPTTSPSNKDITINANGASGSWFT
451 SKGLSSTELLLPANTTTILLGTVKIASGELKITDNAVVNVLGFATQGSGQ
501 LTLGSGGTLGLATPTGAPAAVDFTTGKLAFDPFSFLKRDFVSASVNAGTK
551 NVTLTGALVLDEHDVTDLYDMVSLQTPVAIPIAVFKGATVTKTGFPDGEI
601 ATPSHYGYQGKWSYTWSRPLLIPAPDGGFPGGPSPSANTLYAVWNSDTLV
2O 651 RSTYILDPERYGEIVSNSLWISFLGNQAFSDILQDVLLIDHPGLSITAKA
701 LGAYVEHTPRQGHEGFSGRYGGYQAALSMNYTDHTTLGLSFGQLYGKTNA
751 NPYDSRCSEQMYLLSFFGQFPTVTQKSEALISWKAAYGYSKNHLNTTYLR
801 PDKAPKSQGQWHNNSYYVLISAEHPFLNWCLLTRPLAQAWDLSGFISAEF
851 LGGWQSKFTETGDLQRSFSRGKGYNVSLPIGCSSQWFTPFKKAPSTLTIK
901 LAYKPDIYRVNPHNIVTWSNQESTSISGANLRRHGLFVQIHDWDLTED
951 TQAFLNYTFDGKNGFTNHRVSTGLKSTF*
Pmp I S (CPn 0466)
One example of a Pmp 15 protein is set forth as SEQ ID NOS 5 & 6 in WO
02/02606.
{GenBank accession number: gi~4376751~gb~AAD18608.1 'CPn0466'; SEQ ID NO:
31 below}.
SEQ ID No 31
1 MRFFCFGMLL PFTFVLANEG LQLPLETYIT LSPEYQAAPQ VGFTHNQNQD
51 LAIVGNHNDF ILDYKYYRSN GGALTCKNLL ISENIGNVFF EKNVCPNSGG
101 AIYAAQNCTT SKNQNYAFTT NLVSDNPTAT AGSLLGGALF AINCSITNNL
151 GQGTFVDNLA LNKGGALYTE TNLSIKDNKG PTIIKQNRAL NSDSLGGGTY
201 SGNSLNTEGN SGAIQITSNS SGSGGGIFST QTLTISSNKK LIEISENSAF
4O 251 ANNYGSNFNP GGGGLTTTFC TTLNNREGVL FNNNQSQSNG GATHAKSIII
301 KENGPVYFLN NTATRGGALL NLSAGSGNGS FILSADNGDI IFNNNTASKH
351 ALNPPYRNAI HSTPNMNLQI GARPGYRVLF YDPTEHELPS SFPTLFNFET
901 GHTGTVLFSG EHVHQNFTDE MNFFSYLRNT SELRQGVLAV EDGAGLACYK
451 FFQRGGTLLL GQGAVITTAG TTPTPSSTPT TVGSTITLNH IAIDLPSILS
501 FQAQAPKIWI YPTKTGSTYT EDSNPTITTS GTLTLRNSNN EDPYDSLDLS
551 HSLEKVPLLY IVDVAAQKIN SSQLDLSTLN SGEHYGYQGI WSTYWVETTT
601 ITNPTSLLGA NTKHKLLYAN WSPLGYRPHP ERRGEFITNA LWQSAYTALA
651 GLHSLSSWDE EKGHAASLQG IGLLVHQKDK NGFKGFRSHM TGYSATTEAT
701 SSQSPNFSLG FAQFFSKAKE HESQNSTSSH HYFSGMCIEN TLFKEWIRLS
5O 751 VSLAYMFTSE HTHTMYQGLL EGNSQGSFHN HTLAGALSCV FLPQPHGESL
801 QIYPFITALA IRGNLAAFQE SGDHAREFSL HRPLTDVSLP VGIRASWKNH
851 HRVPLVWLTE ISYRSTLYRQ DPELHSKLLI SQGTWTTQAT PVTYNALGIK
901 VKNTMQVFPK VTLSLDYSAD ISSSTLSHYL NVASRMRF*
Pmp 16 (CPtz0467)
One example of a Pmp 16 protein is set forth as SEQ ID NOS 7 & 8 in WO
02/02606.
~GenBank accession number: gi~4376752~gb~AAD18609.1~ 'CPn0467'; SEQ ID NO:
32 below}.
SEQ ID No 32
1 MFGMTPAVYS LQTDSLEKFA LERDEEFRTS FPLLDSLSTL TGFSPITTFV
26
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51 GNRHNSSQDIVLSNYKSIDNILLLWTSAGGAVSCNNFLLSNVEDHAFFSK
101 NLAIGTGGAIACQGACTITKNRGPLIFFSNRGLNNASTGGETRGGAIACN
151 GDFTISQNQGTFYFVNNSVNNWGGALSTNGHCRIQSNRAPLLFFNNTAPS
201 GGGALRSENTTISDNTRPIYFKNNCGNNGGAIQTSVTVAIKNNSGSVIFN
251 NNTALSGSINSGNGSGGAIYTTNLSIDDNPGTILFNNNYCIRDGGAICTQ
301 FLTIKNSGHVYFTNNQGNWGGALMLLQDSTCLLFAEQGNIAFQNNEVFLT
351 TFGRYNAIHCTPNSNLQLGANKGYTTAFFDPIEHQHPTTNPLTFNPNANH
401 QGTILFSSAYTPEASDYENNFISSSKNTSELRNGVLSIEDRAGWQFYKFT
451 QKGGILKLGHAASIATTANSETPSTSVGSQVITNNLAINLPSILAKGKAP
501 TLWIRPLQSSAPFTEDNNPTITLSGPLTLLNEENRDPYDSIDLSEPLQNI
551 HLLSLSDVTARHINTDNFHPESLNATEHYGYQGIWSPYWVETITTTNNAS
601 IETANTLYRALYANWTPLGYKVNPEYQGDLATTPLWQSFHTMFSLLRSYN
651 RTGDSDIERPFLEIQGIADGLFVHQNSIPGAPGFRIQSTGYSLQASSETS
701 LHQKISLGFAQFFTRTKEIGSSNNVSAHNTVSSLYVELPWFQEAFATSTV
751 LAYGYGDHHLHSLHPSHQEQAEGTCYSHTLAAAIGCSFPWQQKSYLHLSP
801 FVQATAIRSHQTAFEEIGDNPRKFVSQKPFYNLTLPLGIQGKWQSKFHVP
851 TEWTLELSYQPVLYQQNPQIGVTLLASGGSWDILGHNYVRNALGYKVHNQ
901 TALFRSLDLFLDYQGSVSSSTSTHHLQAGSTLKF*
2~
Pnzp 18 (CPn0471)
One example of a Pmp 18 protein is set forth as SEQ ID No 33 below~GenBank
accession number: gi~4376753~gb~AAD18610.1~ 'CPn0471'.
SEQ ID No 33
1 MQNNRSLSKS SFFVGALILG KTTILLNATP LSDYFDNQAN QLTTLFPLID TLTNMTPYSH
61 RATLFGVRDD TNQDIVLDHQ NSIESWFENF SQDGGALSCK SLAITNTKNQ ILFLNSFAIK
121 RAGAMYVNGN FDLSENHGSI TFSGNLSFPN ASNFADTCTG GAVLCSKNVT ISKNQGTAYF
3O 181 INNKAKSSGG AIQAAIINIK DNTGPCLFFN NAAGGTAGGA LFANACRIEN NSQPIYFLNN
241 QSGLGGAIRV HQECILTKNT GSVTFNNNFA MEADISANHS SGGAIYCISC SIKDNPGIAA
301 FDNNTAARDG GAICTQSLTI QDSGPVYFTN NQGTWGGATM LRQDGACTLF ADQGDIIFYN
361 NRHFKDTFSN HVSVNCTRNV SLTVGASQGH SATFYDPILQ RYTIQNSIQK FNPNPEHLGT
421 ILFSSTYIPD TSTSRDDFIS HFRNHIGLYN GTLALEDRAE WKVYKFDQFG GTLRLGSRAV
481 FSTTDEEQSS SSVGSVININ NLAINLPSIL GNRVAPKLWT RPTGSSAPYS EDNNPIINLS
541 GPLSLLDDEN LDPYDTADLA QPIAEVPLLY LLDVTAKHIN TDNFYPEGLN TTQHYGYQGV
601 WSPYWIETIT TSDTSSEDTV NTLHRQLYGD WTPTGYKVNP ENKGDIALSA FWQSFHNLFA
661 TLRYQTQQGQ IAPTASGEAT RLFVHQNSNN DAKGFHMEAT GYSLGTTSNT ASNHSFGVNF
721 SQLFSNLYES HSDNSVASHT TTVALQINNP WLQERFSTSA SLAYSYSNHH IKASGYSGKI
4O 781 QTEGKCYSTT LGAALSCSLS LQWRSRPLHF TPFIQAIAVR SNQTAFQESG DKARKFSVHK
841 PLYNLTVPLG IQSAWESKFR LPTYWNTELA YQPVLYQQNP ETNVSLESSG SSWLLSGTTL
901 ARNAIAFKGR NQIFTFPI<LS VFLDYQGSVS SSTTTHYLHA GTTFKF
Ptnp 19 (CPn0539)
One example of a Pmp 19 protein is set forth as SEQ ID No 34 below {GenBank
accession number: gi~4376829~gb~AAD18679.1 'CPn0539'; SEQ ID NO: 34 below}.
SEQ ID No 34
5O 1 MKQMRLWGFLFLSSFCQVSYLRANDVLLPLSGIHSGEDLE.LFTLRSSSPTKTTYSLRKDF
61 TVCDFAGNSIHKPGAAFLNLKGDLFFINSTPLAALTFKNIHLGARGAGLFSESNVTFKGL
121 HSLVLENNESWGGVLTTSGDLSFINNTSVLCQNNISYGPGGALLLQGRKSKALFFRDNRG
181 TILFLKNKAVNQDESHPGYGGAVSSISPGSPITFADNQEILFQENEGELGGAIYNDQGAT
241 TFENNFQTTSFFSNKASFGGAVYSRYCNLYSQWGDTLFTKNAAAKVGGAIHADYVHIRDC
301 KGSIVFEENSATAGGAIAVNAVCDINAQGPVRFINNSALGLNGGAIYMQATGSILRLHAN
361 QGDIEFCGNKVRSQFHSHINSTSNFTNNAITIQGAPREFSLSANEGHRICFYDPITSATE
421 NYNSLYINHQRLLEAGGAVIFSGARLSPEHKKENI<NKTSIINQPVRLCSGVLSIEGGAIL
481 AVRSFYQEGGLLALGPGSKLTTQGKNSEKDKIVITNLGFNLENLDSSDPAEIRATEKASI
541 EISGVPRVYGHTESFYENHEYASKPYTTSIILSAKKLVTAPSRPEKDIQNLIIAESEYMG
6O 601 YGYQGSWEFSWSPNDTKEKKTITASWTPTGEFSLDPKRRGSFIPTTLWSTFSGLNTASNI
661 VNNNYLNNSEVIPLQHLCVFGGPVYQIMEQNPKQSSNNLLVQHAGHNVGARIPFSFNTIL
721 SAALTQLFSSSSQQNVADKSHAQILIGTVSLNKSWQALSLRSSFSYTEDSQVMKHVFPYK
781 GTSRGSWRNYGWSGSVGMSYAYPKGIRYLKMTPFVDLQYTKLVQNPFVETGYDPRYFSSS
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841 EMTNLSLPIG IALEMRFIGS RSSLFLQVST SYIKDLRRVN PQSSASLVLN HYTWDIQGVP
901 LGKEALNITL NSTIKYKIVT AYMGISSTQR EGSNLSANAH AGLSLSF
As the Examples demonstrate, we and others have demonstrated (Grimwood et al
(2001), Infection and Tmmunity 69(4), 2383-2389) using Flow cytometry (FACS)
analyses and Western Blot analyses that PMP19 does not appear to be surface
exposed. However, high levels of mRNA expression is nevertheless observed in
gene
microarray analysis of pmpl9 (CPn0539).
Phap 20 (CP~a0540)
One example of a Pmp 20 protein is set forth as SEQ ID NOS 119 & 120 in WO
02/02606. fGenBank accession number: gi~4376830~gb~AAD18680.1 'CPn0540';
SEQ ID NO: 35 below.
SEQ ID No 35
1 MKWLPATAVF SHTGSGDPTSDAALTGFTQS
AAVLPALTAF
GDPASVEIST
51 STETDGTTYTIVGDITFSTFTNIPVPWTPDANDSSSNSSKGGSSSSGAT
101 SLIRSSNLHSDFDFTKDSVLDLYHLFFPSASNTLNPALLSSSSSGGSSSS
2O 151 SSSSSSGSASAWAADPKGGAAFYSNEANGTLTFTTDSGNPGSLTLQNLK
201 MTGDGAAIYSKGPLVFTGLKNLTFTGNESQKSGGAAYTEGALTTQAIVEA
251 VTFTGNTSAGQGGAIYVKEATLFNALDSLKFEKNTSGQAGGGIYTESTLT
301 ISNITKSIEFISNKASVPAPAPEPTSPAPSSLINSTTIDTSTLQTRAASA
351 TPAVAPVAAVTPTPISTQETAGNGGAIYAKQGISISTFKDLTFKSNSASV
401 DATLTVDSSTIGESGGAIFAADSIQIQQCTGTTLFSGNTANKSGGGIYAV
451 GQVTLEDIANLKMTNNTCKGEGGAIYTKKALTINNGAILTTFSGNTSTDN
501 GGAIFAVGGITLSDLVEVRFSKNKTGNYSAPITKAASNTAPVVSSSTTAA
551 SPAVPAAAAAPVTNAAKGGALYSTEGLTVSGITSILSFENNECQNQGGGA
601 YVTKTFQCSDSHRLQFTSNKAADEGGGLYCGDDVTLTNLTGKTLFQENSS
3O 651 EKHGGGLSLASGKSLTMTSLESFCLNANTAKENGGGANVPENIVLTFTYT
701 PTPNEPAPVQQPVYGEALVTGNTATKSGGGIYTKNAAFSNLSSVTFDQNT
751 SSENGGALLTQKAADKTDCSFTYITNWITNNTATGNGGGIAGGKAHFDR
BO1 IDNLTVQSNQAKKGGGVYLEDALILEKVITGSVSQNTATESGGGIYAKDI
851 QLQALPGSFTITDNKVETSLTTSTNLYGGGIYSSGAVTLTNISGTFGITG
901 NSVINTATSQDADIQGGGIYATTSLSINQCNTPILFSNNSAATKKTSTTK
951 QIAGGAIFSAAVTIENNSQPIIFLNNSAKSEATTAATAGNKDSCGGAIAA
1001 NSVTLTNNPEITFKGNYAETGGAIGCIDLTNGSPPRKVSIADNGSVLFQD
1051 NSALNRGGAIYGETIDISRTGATFIGNSSKHDGSAICCSTALTLAPNSQL
1101 IFENNKVTETTATTKASINNLGAAIYGNNETSDVTISLSAENGSIFFKNN
4O 1151 LCTATNKYCSIAGNVKFTAIEASAGKAISFYDAVNVSTKETNAQELKLNE
1201 KATSTGTILFSGELHENKSYIPQKVTFAHGNLILGKNAELSVVSFTQSPG
1251 TTITMGPGSVLSNHSKEAGGIAINNVIIDFSEIVPTKDNATVAPPTLKLV
1301 SRTNADSKDKIDITGTVTLLDPNGNLYQNSYLGEDRDITLFNIDNSASGA
1351 VTATNVTLQGNLGAKKGYLGTWNLDPNSSGSKIILKWTFDKYLRWPYIPR
1401 DNHFYINSIWGAQNSLVTVKQGILGNMLNNARFEDPAFNNFWASAIGSFL
1451 RKEVSRNSDSFTYHGRGYTAAVDAKPRQEFILGAAFSQVFGHAESEYHLD
1501 NYKHKGSGHSTQASLYAGNIFYFPAIRSRPILFQGVATYGYMQHDTTTYY
1551 PSIEEKNMANWDSIAWLFDLRFSVDLKEPQPHSTARLTFYTEAEYTRIRQ
1601 EKFTELDYDPRSFSACSYGNLAIPTGFSVDGALAWREIILYNKVSAAYLP
5O 1651 VILRNNPKATYEVLSTKEKGNWNVLPTRNAARAEVSSQIYLGSYWTLYG
1701 TYTIDASMNTLVQMANGGIRFVF*
Ptrtp2l (CPn0963)
One example of a Pmp 21 protein is set forth as SEQ ID NOS 83 & 84 in WO
02/02606. {GenBank accession number: gi~4377287~gb~AAD19099.1~ 'CPn0963';
SEQ ID NO: 36 below}.
SEQ ID No 36
F)O 1 MVAKKTVRSY RSSESHSVIV AIIiSAGIAFE AHSLHSSELD LGVFNKQFEE
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51 HSAHVEEAQTSVLKGSDPVNPSQKESEKVLYTQVPLTQGSSGESLDLADA
101 NFLEHFQHLFEETTVFGIDQKLVWSDLDTRNFSQPTQEPDTSNAVSEKIS
151 SDTKENRKDLETEDPSKKSGLKEVSSDLPKSPETAVAAISEDLEISENIS
201 ARDPLQGLAFFYKNTSSQSISEKDSSFQGIIFSGSGANSGLGFENLKAPK
251 SGAAVYSDRDIVFENLVKGLSFISCESLEDGSAAGVNIWTHCGDVTLTD
301 CATGLDLEALRLVKDFSRGGAVFTARNHEVQNNLAGGILSWGNKGAIW
351 EKNSAEKSNGGAFACGSFVYSNNENTALWKENQALSGGAISSASDIDIQG
401 NCSAIEFSGNQSLIALGEHIGLTDFVGGGALAAQGTLTLRNNAWQCVKN
451 TSKTHGGAILAGTVDLNETISEVAFKQNTAALTGGALSANDKVIIANNFG
501 EILFEQNEVRNHGGAIYCGCRSNPKLEQKDSGENINTIGNSGAITFLKNK
551 ASVLEVMTQAEDYAGGGALWGHNVLLDSNSGNIQFIGNTGGSTFWIGEYV
601 GGGAILSTDRVTISNNSGDVVFKGNKGQCLAQKYVAPQETAPVESDASST
651 NKDEKSLNACSHGDHYPPKTVEEEVPPSLLEEHPWSSTDIRGGGAILAQ
701 HIFITDNTGNLRFSGNLGGGEESSTVGDLAIVGGGALLSTNEVNVCSNQN
751 WFSDNVTSNGCDSGGAILAKKVDISANHSVEFVSNGSGKFGGAVCALNE
801 SVNITDNGSAVSFSKNRTRLGGAGVAAPQGSVTICGNQGNIAFKENFVFG
851 SENQRSGGGAIIANSSVNIQDNAGDILFVSNSTGSYGGAIFVGSLVASEG
901 SNPRTLTITGNSGDTLFAKNSTQTAASLSEKDSFGGGAIYTQNLKIVKNA
951 GNVSFYGNRAPSGAGVQIADGGTVCLEAFGGDILFEGNINFDGSFNAIHL
2O 1001 CGNDSKIVELSAVQDKNIIFQDAITYEENTIRGLPDKDVSPLSAPSLIFN
1051 SKPQDDSAQHHEGTIRFSRGVSKIPQIAAIQEGTLALSQNAELWLAGLKQ
1101 ETGSSIVLSAGSILRIFDSQVDSSAPLPTENKEETLVSAGVQINMSSPTP
1151 NKDKAVDTPVLADIISITVDLSSFVPEQDGTLPLPPEIIIPKGTKLHSNA
1201 IDLKIIDPTNVGYENHALLSSHKDTPLISLKTAEGMTGTPTADASLSNIK
1251 IDVSLPSITPATYGHTGVWSESKMEDGRLVVGWQPTGYKLNPEKQGALVL
1301 NNLWSHYTDLRALKQEIFAHHTIAQRMELDFSTNVWGSGLGWEDCQNIG
1351 EFDGFKHHLTGYALGLDTQLVEDFLIGGCFSQFFGKTESQSYKAKNDVKS
1401 YMGAAYAGILAGPWLIKGAFVYGNINNDLTTDYGTLGISTGSWTGKGFTA
1451 GTSIDYRYIVNPRRFISAIVSTWPFVEAEYVRIDLPEISEQGKEVRTFQ
3O 1501 KTRFENVATPFGFALEHAYSRGSRAEVNSVQLAYVFDVYRKGPVSLITLK
1551 DAAYSWKSYGVDIPCKAWKARLSNNTEWNSYLSTYLAFNYEWREDLIAYD
1601 FNGGIRIIF*
Preferred PMP proteins for use with the invention comprise an amino acid
sequence:
(a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to one of the
polypeptide sequences set forth for the pmp proteins above and/or (b) which is
a
fragment of at least n consecutive amino acids of one of the polypeptide
sequences set
forth above wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30,
35, 40, 50,
60, 70, 80, 90, 100, 150, 200, 250 or more). These PMP proteins include
variants (e.g.
allelic variants, homologs, orthologs, paralogs, mutants, etc.) of the
polypeptide
sequences set forth above. Preferred fragments of (b) comprise an epitope from
one
of the polypeptide sequences set forth above. Other preferred fragments lack
one or
more amino acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of one of the polypeptide sequences set forth above.
Other fragments omit one or more domains of the protein (e.g. omission of a
signal
peptide, of a cytoplasmic domain, of a transmembrane domain, or of an
extracellular
domain).
Fifth Ahtigeu Group
The immunogenicity of other Chlamydia pneumohiae antigens may be improved by
combination with two or more Chlamydia pneumoniae antigens from either the
first
antigen group or the second antigen group or the third antigen group or the
fourth
antigen group. Such other Cl~lanaydia pneumohiae antigens include a fifth
antigen
group consisting one or more cell surface exposed proteins. These antigens are
referred to herein as the "fifth antigen group". Each of the Chlamydia
pneumoraiae
antigens of the fifth antigen group is described in more detail below.
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(37) PorB Outer Me~abrane Proteih B (CPu0854)
One example of a PorB protein is set forth as SEQ ID NOS: 67 & 68 in WO
02/02606.
fGenBank accession number: gi~4377170~gb~AAD18992.1~ 'CPn0854'; SEQ ID NO:
37 below}. Preferred PorB proteins for use with the invention comprise an
amino
acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to
SEQ ID NO: 37; andlor (b) which is a fragment of at least r~ consecutive amino
acids
of SEQ ID NO: 37, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25,
30, 35,
40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These PorB proteins
include
variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.)
of SEQ ID
NO: 37. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 37.
Other
preferred fragments lack one or more amino acids (e.g. l, 2, 3, 4, 5, 6, 7, 8,
9, 10, 15,
20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2,
3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 37.
Other
fragments omit one or more domains of the protein (e.g. omission of a signal
peptide,
of a cytoplasmic domain, of a transmembrane domain, or of an extracellular
domain).
SEQ ID No 37
1 MNSKMLKHLR I~ATI~SFSMFF GIVSSPAVYA LGAGNPAAPV LPGVNPEQTG
51 WCAFQLCNSY DLFAALAGSL KFGFYGDYVF SESAHITNVP VITSVTTSGT
101 GTTPTTTSTT KNVDFDLNNS SISSSCVFAT IALQETSPAA IPLLDIAFTA
151 RVGGLKQYYR LPLNAYRDFT SNPLNAESEV TDGLIEVQSD YGIVWGLSLQ
201 KVLWKDGVSF VGVSADYRHG SSPTNYIIVY NKANPEIYFD ATDGNLSYKE
251 WSASTGISTY LNDYVLPYAS VSIGNTSRKA PSDSFTELEK QFTNFKFKIR
301 KITNFDRVNF CFGTTCCISN NFYYSVEGRW GYQRAINITS GLQF*
(38) 76kDa Protein IIonZOlog (CPn0728)
One example of a 76kDa Protein Homolog protein is set forth as SEQ ID NOS: 13
&
14 in WO 02/02606. {GenBank accession number: gi~4377033~gb~AAD18867.1~
'CPn0728'; SEQ ID NO: 38 below}. Preferred 76kDa proteins homologs for use
with
the invention comprise an amino acid sequence: (a) having 50% or more identity
(e.g.
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.5% or more) to SEQ ID NO: 38; andlor (b) which is a fragment of
at
least ~a consecutive amino acids of SEQ ID NO: 21, wherein h is 7 or more
(e.g. 8, 10,
12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or
more).
These 76kDa protein homologs include 'variants (e.g. allelic variants,
homologs,
orthologs, paralogs, mutants, etc.) of SEQ ID NO: 38. Preferred fragments of
(b)
comprise an epitope from SEQ ID NO: 38. Other preferred fragments lack one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C
terminus andlor one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 38. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 38
1 MVNPIGPGPI DETERTPPAD LSAQGLEASA ANKSAEAQRI AGAEAKPKES
5O 51 KTDSVERWSI LRSAVNALMS LADKLGIASS NSSSSTSRSA DVDSTTATAP
101 TPPPPTFDDY KTQAQTAYDT IFTSTSLADI QAALVSLQDA VTNIKDTAAT
151 DEETAIAAEW ETKNADAVKV GAQITELAKY ASDNQAILDS LGKLTSFDLL
201 QAALLQSVAN NNKAAELLKE MQDNPVVPGK TPAIAQSLVD QTDATATQIE
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251 KDGNAIRDAY FAGQNASGAV ENAKSNNSIS NIDSAKAAIA TAKTQIAEAQ
301 KKFPDSPILQ EAEQMVIQAE KDLKNTKPAD GSDVPNPGTT VGGSKQQGSS
351 IGSIRVSMLL DDAENETASI LMSGFRQMIH MFNTENPDSQ AAQQELAAQA
401 RAAKAAGDDS AAAALADAQK ALEAALGKAG QQQGILNALG QIASAAWSA
451 GVPPAAASSI GSSVKQLYKT SKSTGSDYKT QISAGYDAYK SINDAYGRAR
501 NDATRDVINN VSTPALTRSV PRARTEARGP EKTDQALARV ISGNSRTLGD
551 VYSQVSALQS VMQIIQSNPQ ANNEEIRQKL TSAVTKPPQF GYPWQLSND
601 STQKFIAKLE SLFAEGSRTA AEIKALSFET NSLFIQQVLV NIGSLYSGYL
651 Q*
(39) OmpA conserved outer naenabrane protein (CPn0695)
One example of an OmpA conserved outer membrane protein protein is set forth
as
SEQ ID NOS: 59 & 60 in WO 02/02606. {GenBank accession number:
gi~4376998~gb~AAD18834.1~ 'CPn069S'; SEQ ID NO: 39 below}. Preferred ompA
proteins for use with the invention comprise an amino acid sequence: (a)
having SO%
or more identity (e.g. 60%, 6S%, 70%, 7S%, 80%, 8S%, 90%, 91%, 92%, 93%, 94%,
9S%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 39; and/or (b) which is
a
fragment of at least h consecutive amino acids of SEQ ID NO: 39, wherein ya is
7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 2S, 30, 3S, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These hypothetical proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 39. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: 39. Other preferred fragments lack
one
or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1S, 20, 25 or more)
from the C-
terminus and/or one or more amino acids (e.g. l, 2, 3, 4, S, 6, 7, 8, 9, 10,
1S, 20, 2S or
more) from the N-terminus of SEQ ID NO: 39. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ 11) No 39
1 MKKT.LKSALL SAAFAGSVGS I~QALPVGNPS DPSLLIDGTI WEGAAGDPCD
51 PCATWCDAIS LRAGFYGDYV FDRILKVDAP KTFSMGAKPT GSAAANYTTA
101 VDRPNPAYNK HLHDAEWFTN AGFIALNIWD RFDVFCTLGA SNGYIRGNST
151 AFNLVGLFGV KGTTVNANEL PNVSLSNGW ELYTDTSFSW SVGARGALWE
201 CGCATLGAEF QYAQSKPKVE ELNVICNVSQ FSVNKPKGYK GVAFPLPTDA
251 GVATATGTKS ATINYHEWQV GASLSYRLNS LVPYIGVQWS RATFDADNIR
301 IAQPKLPTAV LNLTAWNPSL LGNATALSTT DSFSDFMQIV SCQINKFKSR
351 KACGVTVGAT LVDADKWSLT AEARLINERA AHVSGQFRF*
(40) PepA (CPn0385)
One example of a PepA protein protein is set forth as SEQ ID NOs: 99 & 100 in
WO
02/02606. fGenBank accession number: gi~4376664~gb~AAD18S29.1 'CPn038S';
SEQ ID NO: 40 below}. Preferred PepA proteins for use with the invention
comprise
an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 6S%, 70%,
7S%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)
to SEQ ID NO: 40; and/or (b) which is a fragment of at least h consecutive
amino
acids of SEQ ID NO: 40, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18,
20, 2S, 30,
35, 40, S0, 60, 70, 80, 90, 100, 150, 200, 2S0 or more). These PepA proteins
include
variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.)
of SEQ ID
NO: 40 Preferred fragments of (b) comprise an epitope from SEQ ID NO: 40.
Other
preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, S, 6, 7, 8,
9, I0, 1S,
20, 2S or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2,
3, 4, 5,
6, 7, 8, 9, 10, 1 S, 20, 25 or more) from the N-terminus of SEQ ID NO: 40.
Other
fragments omit one or more domains of the protein (e.g. omission of a signal
peptide,
of a cytoplasmic domain, of a transmembrane domain, or of an extracellular
domain).
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SEA ID No 40
1 MVLFHAQASG RNRVKADAIV LPFWHFKDAK NAASFEAEFE PSYLPALENF
51 QGKTGEIELL YSSPKAKEKR IVLLGLGKNE ELTSDWFQT YATLTRVLRK
101 AKCSTVNTIL PTISELRLSA EEFLVGLSSG ILSLNYDYPR YNKVDRNLET
151 PLSKVTVIGI VPKMADAIFR KEAAIFEGVY LTRDLVNRNA DEITPKKLAE
201 VALNLGKEFP SIDTKVLGKD AIAKEKMGLL LAVSKGSCVD PHFIWRYQG
251 RPKSKDHTVL IGKGVTFDSG GLDLKPGKSM LTMKEDMAGG ATVLGILSAL
1O 301 AVLELPINVT GIIPATENAI DGASYKMGDV YVGMSGLSVE ICSTDAEGRL
351 ILADAITYAL KYCKPTRIID FATLTGAMW SLGEEVAGFF SNNDVLAEDL
401 LEASAETSEP LWRLPLVKKY DKTLHSDIAD MKNLGSNRAG AITAALFLQR
451 FLEESSVAWA HLDIAGTAYH EKEEDRYPKY ASGFGVRSIL YYLENSLSK*
(41) Conserved Outer Membrane Protein (Cpu0278)
One example of a conserved outer membrane protein protein is set forth as SEQ
ID
NO: 41 below. GenBank Accession No. GI:4376552; AAD18427.1. Preferred
conserved outer membrane proteins for use with the invention comprise an amino
acid
sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID
NO: 41; and/or (b) which is a fragment of at least n consecutive amino acids
of SEQ
ID NO: 41, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35,
40, 50,
60, 70, 80, 90, 100, 150, 200, 250 or more). These conserved outer membrane
proteins include variants (e.g. allelic variants, homologs, orthologs,
paralogs, mutants,
etc.) of SEQ ID NO: 41. Preferred fragments of (b) comprise an epitope from
SEQ ID
NO: 41. Other preferred fragments lack one or more amino acids (e.g. l, 2, 3,
4, 5, 6,
7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino
acids
(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus
of SEQ ID
NO: 41. Other fragments omit one or more domains of the protein (e.g. omission
of a
signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an
extracellular domain).
SEA ID No 41
1 MKKKLSLLVG LIFVLSSCHK EDAQNKIRIV ASPTPHAELL ESLQEEAKDL GIKLKILPVD
61 DYRIPNRLLL DKQVDANYFQ HQAFLDDECE RYDCKGELVV IAKVHLEPQA IYSKKHSSLE
121 RLKSQKKLTI AIPVDRTNAQ RALHLLEECG LIVCI<GPANL NMTAKDVCGK ENRSINILEV
181 SAPLLVGSLP DVDAAVIPGN FAIAANLSPK KDSLCLEDLS VSKYTNLVVI RSEDVGSPKM
4O 241 IKLQKLFQSP SVQHFFDTKY HGNILTMTQD NG
Sixth Antigen Group
The immunogenicity of other Chlamydia pneurnoniae antigens may be improved by
combination with two or more Chlamydia pyaeumo~2iae antigens from either the
first
antigen group or the second antigen group or the third antigen group or the
fourth
antigen group or the fifth antigen group. Such other Chlamydia pneumoniae
antigens
include a sixth antigen group consisting one or more FACS positive CPn
antigens.
These antigens are referred to herein as the " sixth antigen group". Each of
the
Chlamydia pneufnoraiae antigens of the sixth antigen group is described in
more detail
below.
(42) Predicted Onap (CPai0020)
One example of a predicted Omp protein is set forth as SEQ ID NOS: 91 & 92 in
WO
02/02606. fGenBank accession number gi~4376272~gb~AAD18173.1: 'CPn0020';
32
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
SEQ ID NO: 42 below). Preferred Omp proteins for use with the invention
comprise
an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%,
75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)
to SEQ ID NO: 42; and/or (b) which is a fragment of at least h consecutive
amino
acids of SEQ ID NO: 42, wherein h is 7 or more (e.g. 8, 10, 12, 14, 16, 18,
20, 25, 30,
35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These Omp proteins
include
variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.)
of SEQ ID
NO: 42. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 42.
Other
preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 15,
20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. l, 2,
3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 42.
Other
fragments omit one or more domains of the protein (e.g. omission of a signal
peptide,
of a cytoplasmic domain, of a transmembrane domain, or of an extracellular
domain).
SEQ ID No 42
1 MKRCFLFLAS LTHQEAVKKKNSYLSHFKSVSGIVTIEDGV
FVLMGSSADA
51 LNIHNNLRIQANKVYVENTVGQSLKLVAHGNVMVNYRAKTLVCDYLEYYE
101 DTDSCLLTNGRFAMYPWFLGGSMITLTPETIVIRKGYISTSEGPKKDLCL
ZO 151 SGDYLEYSSDSLLSIGKTTLRVCRIPILFLPPFSIMPMEIPKPPINFRGG
201 TGGFLGSYLGMSYSPISRKHFSSTFFLDSFFKHGVGMGFNLHCSQKQVPE
251 NVFNMKSYYAHRLAIDMAEAHDRYRLHGDFCFTHKHVNFSGEYHLSDSWE
301 TVADIFPNNFMLKNTGPTRVDCTWNDNYFEGYLTSSVKVNSFQNANQELP
351 YLTLRQYPISIYNTGVYLENIVECGYLNFAFSDHIVGENFSSLRLAARPK
~5 401 LHKTVPLPIGTLSSTLGSSLIYYSDVPEISSRHSQLSAKLQLDYRFLLHK
451 SYIQRRHIIEPFVTFITETRPLAKNEDHYIFSIQDAFHSLNLLKAGIDTS
501 VLSKTNPRFPRIHAKLWTTHILSNTESKPTFPKTACELSLPFGKKNTVSL
551 DAEWIWKKHCWDHMNIRWEWIGNDNVAMTLESLHRSKYSLIKCDRENFIL
601 DVSRPIDQLLDSPLSDHRNLILGKLFVRPHPCWNYRLSLRYGWHRQDTPN
3O 651 YLEYQMILGTKIFEHWQLYGVYERREADSRFFFFLKLDKPKKPPF*
(43) Predicted Omp (CP~Z0021)
One example of a predicted Omp protein is set forth as SEQ ID NOS: 49 & 50 in
WO
02/02606. {GenBank accession numbe gi~4376273~gb~AAD18174.1: 'CPn0021'; SEQ
35 ID NO: 43 below}. Preferred Omp proteins for use with the invention
comprise an
amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)
to SEQ ID NO: 43; and/or (b) which is a fragment of at least h consecutive
amino
acids of SEQ ID NO: 43, wherein h is 7 or more (e.g. 8, 10, 12, 14, 16, 18,
20, 25, 30,
40 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These hypothetical
proteins
include variants (e.g. allelic variants, homologs, orthologs, paralogs,
mutants, etc.) of
SEQ ID NO: 43. Preferred fragments of (b) comprise an epitope from SEQ ID NO:
43. Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,
5, 6, 7, 8,
9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids
(e.g. 1,
45 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ
ID NO: 43.
Other fragments omit one or more domains of the protein (e.g. omission of a
signal
peptide, of a cytoplasmic domain, of a transmembrane domain, or of an
extracellular
domain).
50 SEQ ID No 43
1 MGLFHLTLFG LLLCSLPISL VAKFPESVGH KILYISTQST QQALATYLEA
51 LDAYGDHDFF VLRKIGEDYL KQSIHSSDPQ TRKSTIIGAG LAGSSEALDV
101 LSQAMETADP LQQLLVLSAV SGHLGKTSDD LLFKALASPY PVIRLEAAYR
55 151 LANLKNTKVI DHLHSFIHKL PEEIQCLSAA~TFLRLETEES DAYIRDLLAA
33
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WO 2005/084306 PCT/US2005/006588
201 KKSAIRSATA LQIGEYQQKR FLPTLRNLLT SASPQDQEAI LYALGKLKDG
251 QSYYNIKKQL QKPDVDVTLA AAQALIALGK EEDALPVIKK QALEERPRAL
301 YALRHLPSEI GIPIALPIFL KTKNSEAKLN VALALLELGC DTPKLLEYIT
351 ERLVQPHYNE TLALSFSKGR TLQNWKRVNI IVPQDPQERE RLLSTTRGLE
401 EQILTFLFRL PKEAYLPCIY KLLASQKTQL ATTAISFLSH TSHQEALDLL
451 FQAAKLPGEP IIRAYADLAI YNLTKDPEKK RSLHDYAKKL IQETLLFVDT
501 ENQRPHPSMP YLRYQVTPES RTKLMLDILE TLATSKSSED IRLLIQLMTE
551 GDAKNFPVLA GLLIKIVE*
(44) Oligopeptide Binding Protein Oppa-1 Lipopt~oteih (CPh0195)
One example of an oligopeptide binding protein is set forth as SEQ ID NOS: 23
and
24 in WO 02102606. {GenBank accession number gi~4376466~gb~AAD18348.1:
'CPn0195'; SEQ ID NO: 44 below). Preferred oligopeptide binding' proteins for
use
with the invention comprise an amino acid sequence: (a) having 50% or more
identity
(e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.5% or more) to SEQ ID NO: 44; and/or (b) which is a fragment of
at
least yZ consecutive amino acids of SEQ ID NO: 44, wherein h is 7 or more
(e.g. 8, I0,
12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or
more).
These hypothetical proteins include variants (e.g. allelic variants, homologs,
orthologs, paralogs, mutants, etc.) of SEQ ID NO: 44. Preferred fragments of
(b)
comprise an epitope from SEQ ID NO: 44. Other preferred fragments lack one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C-
terminus and/or one or more amino acids (e.g. I, 2, 3, 4, 5, 6, 7, 8, 9, I0,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 44. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 44
3O 1 MRECISVGICI TILLSLSVVL QGCKESSHSS TSRGELAINI RDEPRSLDPR
51 QVRLLSEISL VKHIYEGLVQ ENNLSGNIEP ALAEDYSLSS DGLTYTFKLK
101 SAFWSNGDPL'TAEDFIESWK QVATQEVSGI YAFALNPIKN VRKIQEGHLS
151 IDHFGVHSPN ESTLVVTLES PTSHFLKLLA LPVFFPVHKS QRTLQSKSLP
201 IASGAFYPKN IKQKQWTKLS KNPHYYNQSQ VETKTITIHF IPDANTAAKL
251 FNQGKLNWQG PPWGERIPQE TLSNLQSKGH LHSFDVAGTS WLTFNINKFP
301 LNNMKLREAL ASALDKEALV STIFLGRAKT ADHLLPTNIH SYPEHQKQEM
351 AQRQAYAKKL FKEALEELQI TAKDLEHLNL IFPVSSSASS LLVQLIREQW
401 KESLGFAIPI VGKEFALLQA DLSSGNFSLA TGGWFADFAD PMAFLTIFAY
451 PSGVPPYAIN HKDFLEILQN IEQEQDHQKR SELVSQASLY LETFHIIEPI
4O 501 YHDAFQFAMN KKLSNLGVSP TGWDFRYAK EN*
(45) CHLPS 43 kDa Pt~otein Hofnologue-1 (CPn0562)
One example of a CHLPS protein is set forth as SEQ ID NO: 45 below. GenBank
Accession No. GI:4376854; AAD18702.1. Preferred CHLPS proteins for use with
the invention comprise an amino acid sequence: (a) having 50% or more identity
(e.g.
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.5% or more) to SEQ ID NO: 45; and/or (b) which is a fragment of
at
least h consecutive amino acids of SEQ ID NO: 45, wherein ya is 7 or more
(e.g. 8, 10,
12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or
more).
These CHLPS proteins include variants (e.g. allelic variants, homologs,
orthologs,
paralogs, mutants, etc.) of SEQ ID NO: 45. Preferred fragments of (b) comprise
an
epitope from SEQ ID NO: 45. Other preferred fragments lack one or more amino
acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, I0, 15, 20, 25 or more) from the C-
terminus and/or
one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or
more) from the
34
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WO 2005/084306 PCT/US2005/006588
N-terminus of SEQ ID NO: 45. Other fragments omit one or more domains of the
protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a
transmembrane domain, or of an extracellular domain).
SEQ ID No 45
1 MSIAIAREQY AAILDMHPKP SIAMFSSEQA RTSWEKRQAH PYLYRLLEII WGWKFLLGL
61 IFFTPLGLFW VLQKICQNFI LLGAGGWIFR PICRDSNLLR QAYAARLFSA SFQDHVSSVR
121 RVCLQYDEVF TDGLELRLPN AKPDRWMLIS NGNSDCLEYR TVLQGEKDWI FRIAEESQSN
1O 181 2LIFNYPGVM KSQGNITRNN WKSYQACVR YLRDEPAGPQ ARQTVAYGYS LGASVQAEAL
241 SKEIADGSDS VRWFWKDRG ARSTGAVAKQ'FIGSLGVWLA NLTHWNINSE KRSKDLHCPE
301 LFIYGKDSQG NLTGDGLFKK ETCFAAPFLD PKNLEECSGK KIPVAQTGLR HDH2LSDDVI
361 KEVAGHIQRH FDN
(4d) YscJ (Yop trahslocatioss Jp~otein) (CPh0828)
One example of a YscJ protein is set forth as SEQ ID NOS: 109 and 110 in WO
02102606, {GenBank accession number gi~4377140~gb~AAD18965.1~ 'CPn0828';
SEQ ID NO: 46 below. Preferred YscJ proteins for use with the invention
comprise
an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%,
75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)
to SEQ ID NO: 46; and/or (b) which is a fragment of at least n consecutive
amino
acids of SEQ ID NO: 46, wherein h is 7 or more (e.g. 8, 10, 12, 14, 16, 18,
20, 25, 30,
35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These YscJ proteins
include
variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.)
of SEQ ID
NO: 46. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 46.
Other
preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 15,
20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2,
3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 46.
Other
fragments omit one or more domains of the protein (e.g. omission of a signal
peptide,
of a cytoplasmic domain, of a transmembrane domain, or of an extracellular
domain).
SEQ ID No 46
1 MVRRSISFCI~ FFIMTLLCCT SCNSRSLIVfi GI~PGREANEI WS~hVSKGVA
51 AQKLPQAAAA TAGAATEQMW D2AVPSAQTT EALATLNQAG LPRMKGTSLL
101 DLFAKQGLVP SELQEKIRYQ EGLSEQMAST IRKMDGWDA SVQISFTTEN
151 EDNLPLTASV YTKHRGVLDN PNSIMVSKIK RLIASAVPGL VPENVSWSD
201 RAAYSD2TIN GPWGLTEEID YVSVWGI2LA KSSLTKFRLI FYVLIL2LFV
4O 251 ISCGLLWVIW KTHTL2MTMG GTKGFFNPTP YTKNALEAKK AEGAAADKEK
301 KEDADSQGES KNAETSDKDS SDKDAPEGSN E2EGA*
(47) Hypothetical (CP~z 0415)
One example of a hypothetical protein is set forth as SEQ ID NOS: 101 and 102
in
WO 02/02606. {GenBank accession number gi~4376696~gb~AAD18559.1~
'CPn0415'; SEQ ID NO: 47 below}. Preferred hypothetical proteins for use with
the
invention comprise an amino acid sequence: (a) having 50% or more identity
(e.g.
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97°t°,
98%, 99%, 99.5% or more) to SEQ ID NO: 47; and/or (b) which is a fragment of
at
least ra consecutive amino acids of SEQ ID NO: 47, wherein ~c is 7 or more
(e.g. 8, 10,
12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or
more).
These hypothetical proteins include variants (e.g. allelic variants, homologs,
orthologs, paralogs, mutants, etc.) of SEQ ID NO: 47, Preferred fragments of
(b)
comprise an epitome from SEQ ID NO: 47. Other preferred fragments lack one or
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C
terminus and/or one or more amino acids (e.g. l, 2, 3, 4, S, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 47. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular.domain).
SEQ ID No 47
1 MTLIFVITIV WCNAFLIKLC VIMGLQSRLQ HCIEVSQNSN FDSQVKQFIY
51 ACQDKTLRQS VLKIFRYHPL LKIHDTARAV YLLMALEEGE DLGLSFLNVQ
101 QYPSGAVELF SCGGFPWKGL PYPAEHAEFG LLLLQTAEFY EESQAYVSKM
7.51 SHFQQALFDH QGSVFPSLWS QENSRLLKEK TTLSQSFLFQ LGMQIHPEYS
201 LEDPALGFWM QRTRSSSAFV AASGCQSSLG AYSSGDVGVI AYGPCSGDIS
251 DCYYFGCCGI AKEFVCQKSH QTTETSFLTS TGKPHPRNTG FSYLRDSYVH
~I5 301 LPIRCKITIS DKQYRVHAAL AEATSAMTFS IFCKGKNCQV VDGPRLRSCS
351 LDSYKGPGND IMILGENDAI NIVSASPYME IFALQGKEKF WNADFLINIP
401 YKEEGVMLIF EKKVTSEKGR FFTKMN*
(48) Hypotlzetical (CP~t0514)
20 One example of a hypothetical protein is set forth as SEQ ID NOS: 87 and 88
in WO
02/02606, fGenBank accession number gi~4376802~gb~AAD18654.1~ 'CPn0514';
SEQ ID NO: 48 below}. Preferred hypothetical proteins for use with the
invention
comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%,
65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
25 99.5% or more) to SEQ ID NO: 48; and/or (b) which is a fragment of at least
r~
consecutive amino acids of SEQ ID NO: 48, wherein h is 7 or more (e.g. 8, 10,
12, 14,
16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
These
hypothetical proteins include variants (e.g. allelic variants, homologs,
orthologs,
paralogs, mutants, etc.) of SEQ ID NO: 48. Preferred fragments of (b) comprise
an
30 epitope from SEQ ID NO: 48. Other preferred fragments lack one or more
amino
acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-
terminus and/or
one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or
more) from the
N-terniinus of SEQ ID NO: 48. Other fragments omit one or more domains of the
protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a
35 transmembrane domain, or of an extracellular domain).
SEQ ID No 48
1 MSNQLQPCIS LGCVSYINSF PLSLQLIKRN DTRCVLAPPA DLLNLLTEGK
4O 51 LDVALTSSLG AISHNLGYVP GFGIAANQRI LSVNLYAAPT FFNSPQPRIA
101 ATLESRSSIG LLKVLCRHLW RIPTPHILRF ITTKVLRQTP ENYDGLLLIG
151 DAALQHPVLP GFVTYDLASG WYDLTKLPFV FALLLHSTSW KEHPLPNLAM
201 EEALQQFESS PEEVLKEAHQ HTGLPPSLLQ EYYALCQYRL GEEHYESFEK
251 FREYYGTLYQ QARL*
(49) Hypothetical (CPn0668)
One example of a hypothetical protein is set forth as SEQ ID NOS: 57 and 58 in
WO
02/02606. ~GenBank accession number gi~4376968~gb~AAD18807.1 'CPn0668'; SEQ
ID NO: 49 below}. Preferred hypothetical proteins for use with the invention
comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%,
65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more) to SEQ ID NO: 49; and/or (b) which is a fragment of at least n
consecutive amino acids of SEQ ID NO: 49, wherein ra is 7 or more (e.g. 8, 10,
12, 14,
16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
These
36
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
hypothetical proteins include variants (e.g. allelic variants, homologs,
orthologs,
paralogs, mutants, etc.) of SEQ ID NO: 49. Preferred fragments of (b) comprise
an
epitope from SEQ ID NO: 49. Other preferred fragments lack one or more amino
acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-
terminus and/or
one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or
more) from the
N-terminus of SEQ ID NO: 49. Other fragments omit one or more domains of the
protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a
transmembrane domain, or of an extracellular domain).
SEQ ID No 49
1 MKFI,LYVPLI, LVI,VSTGCDA KPVSFEPFSG KLSTQRFEPQ HSAEEYFSQG
51 QEFLKKGNFR KALLCFGIIT HHFPRDILRN QAQYLIGVCY FTQDHPDLAD
101 KAFASYLQLP DAEYSEELFQ MKYAIAQRFA QGKRKRICRL EGFPKLMNAD
~5 151 EDALRIYDEI LTAFPSKDLG AQALYSKAAL LIVKNDLTEA TKTLKKLTLQ
201 FPLHILSSEA FVRLSEIYLQ QAKKEPHNLQ YLHFAKLNEE AMKKQHPNHP
251 LNEWSANVG AMREHYARGL YATGRFYEKK KKAEAANIYY RTAITNYPDT
301 LLVAKCQKRL DRISKHTS*
(SO) Hypothetical (CPsa0791)
One example of a hypothetical protein is set forth as SEQ ID NOS: 123 and 124
in
WO 02/02606. {GenBank accession number gi~4377101~gb~AAD18929.1~ 'CPn0791';
SEQ ID NO: 50 below . Preferred hypothetical proteins for use with the
invention
comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%,
65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more) to SEQ ID NO: 50; and/or (b) which is a fragment of at least h
consecutive amino acids of SEQ ID NO: 50, wherein fZ is 7 or more (e.g. 8, 10,
12, 14,
16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
These
hypothetical proteins include variants (e.g. allelic variants, homologs,
orthologs,
paralogs, mutants, etc.) of SEQ ID NO: 50. Preferred fragments of (b) comprise
an
epitope from SEQ ID NO: 50. Other preferred fragments lack one or more amino
acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-
terminus and/or
one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or
more) from the
N-terminus of SEQ ID NO: 50. Other fragments omit one or more domains of the
protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a
transmembrane domain, or of an extracellular domain).
SEQ ID No 50
4O 1 MYSCYSKGISHNYLLHPMSRLDIFVFDSLIANQDQNLLEEIFCSEDTVLF
51 KAYRTTALQSPLAAKNLNIARKVANYILADNGEIDTVKLVEAIHHLSQCT
101 YPLGPHRHNEAQDREHLLKMLKALKENPKLKESIKTLFVPSYSTIQNLIR
151 HTLALNPQTILSTIHVRQAALTALFTYLRQDVGSCFATAPAILIHQEYPE
201 RFLKDLNDLISSGKLSRIVNQREIAVPINLSGCIGELFKPLRILDLYPDP
251 LVKLSSSPGLKKAFSAANLIETLGDSEAQIQQLLSHQYLMQKLQNVHETL
301 TANDIIKSTLLHYYQLQESTVRAIFFKEGLFSKEQVAFSTQHPRELSEIQ
351 RVYHYLHAYEEAKSAFIHDTQNPLLKAWEYTLATLADASQPTISNHIRLA
401 LGWKSEDPHSLVSLVTHFVEEEVENIRILVQQCEQTYHEARSQLEYIEGR
451 MRNPLNNQDSQILTMDHMRFRQELNKALYEWDSAQEKAKKFLHLPEFLLS
5O 501 FYTKQIPLYFRSSYDAFIQEFAHLYANAPAGFRILFTHGRTHPNTWSPIY
551 SINEFIRFLSEFFTSTESELLGKHAVINLEKETSRLVHNITAMLHTDVFQ
601 EALLTRILEAYQLPVPPSILNHLDQLSQTPWVYVSGGTVDTLLLDYFESS
651 EPLTLTEKHPENPHELAAFYADALKDLPTGIKSYLEEGSHSLLSSSPTHV
701 FSIIAGSPLFREAWDNDWYSYTWLRDVWVKQHQDFLQDTILPQLSIYAFT
751 ENFCNKYALQHWHDFHDFCSDHSLTLPELYDKGSRFLSSLFTKDKTVAL
801 IYIRRLLYLMVREVPYVSEQQLPEVLDNVSSYLGISSRITYEKFRSLIEE
851 TIPKMTLLSSADLRHIYKGLLMQSYQKIYTEEDTYLRLTTAMRHHNLAYP
37
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WO 2005/084306 PCT/US2005/006588
901 APLLFADSNW PSIYFGFILN PGTTEIDLWK FNYAGLQGQP LDNIQELFAT
951 SRPWTLYANP IDYGMPPPPG YRSRLPKEFF
(5I) Hypothetical (CPn0792)
One example of a hypothetical protein is set forth as SEQ ID NOS: 61 and 62 in
WO
02/02606. f GenBank accession number gi~4377102~gb~AAD18930.1~ 'CPn0792';
SEQ ID NO: 51 below} . Preferred hypothetical proteins for use with the
invention
comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%,
65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more) to SEQ ID NO: 51; and/or (b) which is a fragment of at least n
consecutive amino acids of SEQ ID NO: 51, wherein ~2 is 7 or more (e.g. 8, 10,
12, 14,
16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
These
hypothetical proteins include variants (e.g. allelic variants, homologs,
orthologs,
paralogs, mutants, etc.) of SEQ ID NO: Sl. Preferred fragments of (b) comprise
an
epitope from SEQ ID NO: 51. Other preferred fragments lack one or more amino
acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-
terminus and/or
one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or
more) from the
N-terminus of SEQ ID NO: 51. Other fragments omit one or more domains of the
protein (e.g. omission of a signal peptide, of a cytoplasmic domain., of a
transmembrane domain, or of an extracellular domain).
SEQ ID No 51
1 MKHTFTKRVL IWLNI~MWGFFSFSAAKANLVQVLHTRATN
FFFFLVIPIP
5l LSIEFEKKLTIHKLFLDRLANTLALKSYASPSAEPYAQAYNEMMALSNTD
101 FSLCLIDPFDGSVRTKNPGDPFIRYLKQHPEMKKKLSAAVGKAFLLTIPG
151 KPLLHYLILVEDVASWDSTTTSGLLVSFYPMSFLQKDLFQSLHITKGNIC
201 LVNKYGEVLFCAQDSESSFVFSLDLPNLPQFQARSPSAIEIEKASGILGG
251 ENLITVSINKKRYLGLVLNKIPIQGTYTLSLVPVSDLIQSALKVPLNICF
3O 301 FYVLAFLLMWWIFSKINTKLNKPLQELTFCMEAAWRGNHNVRFEPQPYGY
351 EFNELGNIFNCTLLLLLNSIEKADIDYHSGEKLQKELGILSSLQSALLSP
401 DFPTFPKVTFSSQHLRRRQLSGHFNGWTVQDGGDTLLGIIGLAGDIGLPS
451 YLYALSARSLFLAYASSDVSLQKISKDTADSFSKTTEGNEAWAMTFIKY
501 VEKDRSLELLSLSEGAPTMFLQRGESFVRLPLETHQALQPGDRLICLTGG
551 EDILKYFSQLPIEELLKDPLNPLNTENLIDSLTMMLNNETEHSADGTLTI
601 LSFS*
,(52) Hypothetical (CPn0820)
One example of a hypothetical protein is set forth as SEQ ID NOS: 113 and 114
in
WO 02/02606. f GenBank accession number gi~4377132~gb~AAD18958.1~
'CPn0820'; SEQ ID NO: 52 below}. Preferred hypothetical proteins for use with
the
invention comprise an amino acid sequence: (a) having 50% or more identity
(e.g.
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.5% or more) to SEQ ID NO: 52; and/or (b) which is a fragment of
at
least yz consecutive amino acids of SEQ ID NO: 52, wherein n is 7 or more
(e.g. 8, 10,
12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or
more).
These hypothetical proteins include variants (e.g. allelic variants, homologs,
orthologs, paralogs, mutants, etc.) of SEQ ID NO: 52. Preferred fragments of
(b)
comprise an epitope from SEQ ID NO: 52. Other preferred fragments lack one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 52. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
38
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WO 2005/084306 PCT/US2005/006588
SEQ ID No 52
1 MCNSIAMKKQ KRGFVT,MELL MSFTLIAI,LL GTLGFWYRKI YTVQKQKERI
51 YNFYIEESRA YKQLRTLFSM SLSSSYEEPG SLFSLIFDRG VYRDPKLAGA
101 VRASLHHDTK DQRLEI,RICN IKDQSYFETQ RLLSHVTHW LSFQRNPDPE
151 KLPETIALTI TREPKAYPPR TLTYQFAVGK*
(53) Hypothetical (CPiZ0126)
One example of a hypothetical protein is set forth as SEQ ID NO: S3 below.
GenBank Accession No. GI:4376390; AAD18279.1 Preferred hypothetical proteins
for use with the invention comprise an amino acid sequence: (a) having SO% or
more
identity (e.g. 60%, 6S%, 70%, 7S%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 9S%,
96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: S3; and/or (b) which is a
fragment of at least h consecutive amino acids of SEQ ID NO: S3, wherein h is
7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These hypothetical proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 53. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: 53. Other preferred fragments lack
one
or moxe amino acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1S, 20, 25 or more)
from the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
1S, 20, 2S or
more) from the N-terminus of SEQ ID NO: S3. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 53
1 MVFSYYCMGL FFFSGAISSC GLLVSLGVGL GLSVLGVLLL LLAGLLLFKI QSMLRF,VPKA
61 PDLLDLEDAS ERLRVKASRS LASLPKEISQ LESYIRSAAN DLNTIKTWPH KDQRLVETVS
3O 121 RKLERLAAAQ NYMISELCEI SEILEEEEHH LTLAQESLEW IGKSLFSTFL DMESFLNLSH
181 LSEVRPYLAV NDPRLLEITE ESWEWSHFI NVTSAFKKAQ ILFKNNEHSR MKKKLESVQE
241 LLETFIYKSL KRSYRELGCL SEKMRIIHDN PIJFPWVQDQQ KYAHAKNEFG EIARCT,EEFE
301 KTFFWLDEEC AISYMDCWDF LNESIQNI<KS RVDRDYISTK KIALKDRART YAKVLT,EENP
361 TTEGKIDLQD AQRAFERQSQ EFYTLEHTET KVRLEALQQC FSDI,REATNV RQVRFTNSEN
421 ANDLKESFEK IDKERVRYQK EQRLYWETID RNEQELREEI GESZRLQNRR KGYRAGYDAG
481 RLKGLLRQWK KNLRDVEAHL EDATMDFEHE VSKSELCSVR ARLEVLEEEL MDMSPKVADI
541 EELLSYEERC ILPIRENLER AYLQYNKCSE ILSIfAKFFFP EDEQLLVSEA NLREVGAQLK
601 QVQGKCQERA QKFAIFEKHI QEQKSLIKEQ VRSFDLAGVG FLKSELLSIA CNLYIKAWK
661 ESIPVDVPCM QLYYSYYEDN EAWRNRLLN MTERYQNFKR SLNSIQFNGD VLLRD$VYQP
4O 721 EGHETRLKER ELQETTLSCK KLKVAQDRLS EI,ESRLSRR
(54) Hypothetical (CPn0794)
One example of a hypothetical protein is set forth as SEQ ID NO: S4 below.
GenBank Accession No. GI:4377105; AAD18932.1. Preferred hypothetical proteins
for use with the invention comprise an amino acid sequence: (a) having SO% or
more
identity (e.g. 60%, 65%, 70%, 7S%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: S4; and/or (b) which is a
fragment of at least n consecutive amino acids of SEQ ID NO: S4, wherein ya is
7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 2S, 30, 3S, 40, S0, 60, 70, 80, 90, 100,
150, 200,
250 or more). These hypothetical proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: S4. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: S4. Other preferred fragments Iack
one
or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)
from the C-
terminus andlor one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 2S or
more) from the N-terminus of SEQ ID NO: S4. Other fragments omit one or more
39
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 54
1 MSLYQKWWNS QLKKSLCYST VAALIFMIPS QESFADSLID LNLGLDPSVE CLSGDGAFSV
61 GYFTKAGSTP VEYQPFKYDV SKKTFTILSV ETANQSGYAY GISYDGTITV GTCSLGAGKY
121 NGAKWSADGT LTPLTGITGG TSHTEARAIS KDTQVIEGFS YDASGQPKAV QWASGATTVT
181 QLADISGGSR SSYAYAISDD GTIIVGSMES TITRKTTAVK WWNVPTYLG TLGGDASTGL
1O 241 YISGDGTVIV GAANTATVTN GNQESHAYMY KDNQMKD
(55) Hypothetical (CPtZ0796)
One example of a hypothetical protein is set forth as SEQ ID NO: 55 below.
GenBank Accession No. GI:4377107; AAD18934.1. Preferred hypothetical proteins
for use with the invention comprise an amino acid sequence: (a) having 50% or
more
identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 55; and/or (b) which is a
fragment of at least ya consecutive amino acids of SEQ ID NO: 55, wherein fz
is 7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These hypothetical proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 55. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: 55. Other preferred fragments lack
one
or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)
from the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 55. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain). Cpn0796 may be
secreted
from C. pyaeumoniae and is localized in the membrane of Chlamydia in young
inclusions whereas an N-terminal part of Cpn0796 is secreted into the host
cell
cytoplasm at later times. Cpn0796 was proposed to be an autotransporter and it
is the
first example of secretion into the host cell cytoplasm of a proposed
Chlamydia
autotrasporter. Te finding in the host cell cytoplasm of Cpn0796 suggests that
an
unknown transport mechanism exists for translocation over the inclusion
membrane
(Vandahl, "Proteome analysis of Chlamydia pneumoniae - proteins at the
Chlamydia
host cell Interface," Abstract of PhD Dissertation, Dan Med Bull 2004:
51:306).
SEQ ID No 55
1 MQPCLNMSIV RNSALPLPCL SRSETFKKVR SHMKFMKVLT PWIYRKDLWV TAFLLTAIPG
4O 61 SFAHTLVDIAGEPRHAAQATGVSGDGKIVIGMKVPDDPFAITVGFQYIDGHLQPLEAVRP
121 QCSVYPNGITPDGTVIVGTNYAIGMGSVAVKWVNGKVSELPMLPDTLDSVASAVSADGRV
181 IGGNRNINLGASVAVICWEDDVITQLPSLPDAMNACVNGISSDGSIIVGTMVDVSWRNTAV
241 QWIGDQLSVIGTLGGTTSVASAISTDGTVIVGGSENADSQTHAYAYKNGVMSDIGTLGGF
301 YSLAHAVSSDGSVIVGVSTNSEHRYHAFQYADGQMVDLGTLGGPESYAQGVSGDGKVIVG
361 RAQVPSGDWHAFLCPFQAPSPAPVHGGSTVVTSQNPRGMVDINATYSSLKNSQQQLQRLL
421 IQHSAKVESVSSGAPSFTSVKGATSKQSPAVQNDVQKGTFLSYRSQVHGNVQNQQLLTGA
481 FMDWKLASAPKCGFKVALHYGSQDALVERAALPYTEQGLGSSVLSGFGGQVQGRYDFNLG
541 ETWLQPFMGIQVLHLSREGYSEKNVRFPVSYDSVAYSAATSFMGAHVFASLSPKMSTAA
601 TLGVERDLNSHIDEFKGSVSAMGNFVLENSTVSVLRPFASLAMYYDVRQQQLVTLSVVMN
661 QQPLTGTLSLVSQSSYNLSF
One preferred protein for use with the invention comprises an N-terminal
peptide of
Cpn0796 that may be secreted to be exposed on the bacterial cell surface and
can also
become detached via a proteolytic event. In one embodiment, the N-terminal
peptide
of Cpn0796 may form a beta-propeller structural conformation. One example of
the
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
N-terminal peptide of Cpn0796 is set forth as SEQ ID NO: 86 below. The N-
terminal
peptide of Cpn0796 for use with the invention may comprise an amino acid
sequence:
(a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 86;
and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID
NO:
86, wherein ~ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40,
50, 60, 70, 80,
90, 100, 150, 200, 250 or more). These hypothetical proteins include variants
(e.g.
allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:
86.
Preferred fragments of (b) comprise an epitope from SEQ ID NO: 86. Other
preferred
fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5,
6, 7, 8,
9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 86.
SEQ ID NO: 86
HTLVDIAGEPRHAAQATGVSGDGKIVIGMKVPDDPFAITVGFQYIDGHLQPLEAVRPQCSVYPNG
ITPDGTVIVGTNYAIGMGSVAVKWVNGKVSELPMLPDTLDSVASAVSADGRVIGGNRNINLGASV
AVKWEDDVITQLPSLPDAMNACVNGISSDGSIIVGTMVDVSWRNTAVQWIGDQLSVIGTLGGTTS
VASAISTDGTVIVGGSENADSQTHAYAYKNGVMSDIGTLGGFYSLAHAVSSDGSVIVGVSTNSEH
2O RYHAFQYADGQMVDLGTLGGPESYAQGVSGDGKVIVGRAQVPSGDVdHAFLC
(56) Hypothetical (CPrz0797)
One example of a hypothetical protein is set forth as SEQ ID NO: 56 below.
GenBank Accession No. GI:4377108; AAD18935.1 Preferred hypothetical proteins
for use with the invention comprise an amino acid sequence: (a) having 50% or
more
identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 56; and/or (b) which is a
fragment of at least zz consecutive amino acids of SEQ ID NO: 56, wherein n is
7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These hypothetical proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 56. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: 56. Other preferred fragments lack
one
or more amino acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)
from the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 56. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 56
1 MSKKIKVLGH LTLCTLFRGV LCAAALSNIG YASTSQESPY QKSIEDWKGY TFTDLELLSK
61 EGWSEAHAVS GNGSRIVGAS GAGQGSVTAV IWESHLIKHL GTLGGEASSA EGISKDGEW
121 VGWSDTREGY THAFVFDGRD MKDLGTLGAT YSVARGVSGD GSIIVGVSAT ARGEDYGWQV
181 GVKWEKGKIK QLKLLPQGLW SEANAISEDG TVIVGRGEIS RNHIVAVKWN KNAVYSLGTL
241 GGSVASAEAI SANGKVIVGW STTNNGETHA FMHKDETMHD LGTLGGGFSV ATGVSADGRA
301 TVGFSAVKTG EIHAFYYAEG EMEDLTTLGG EEARVFDISS EGNDIIGSIK TDAGAERAYL
361 FHIHK
(76) Oligopeptide Birzdirzg Proteirz Oppa-2 Lipoproteirz (CPrz0196)
One example of an oligopeptide binding protein is set forth as SEQ ID NOS: 127
and
128 in WO 02/02606. {GenBank accession number GI:4376467; AAD18349.1
41
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
'CPn0196'; SEQ ID NO: 76 below. Preferred oligopeptide binding proteins for
use
with the invention comprise an amino acid sequence: (a) having 50% or more
identity
(e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.5% or more) to SEQ ID NO: 76; and/or (b) which is a fragment of
at
least n consecutive amino acids of SEQ ID NO: 76, wherein n is 7 or more (e.g.
8, 10,
12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or
more).
These hypothetical proteins include variants (e.g. allelic variants, homologs,
orthologs, paralogs, mutants, etc.) of SEQ ID NO 76. Preferred fragments of
(b)
comprise an epitope from SEQ ID NO: 76. Other preferred fragments lack one or
more amino acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 76. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 76 .
1 mlrffavfis tlwlitsgcs psqsskgifv vnmkemprsl dpgktrliad qtlmrhlyeg
61 lveehsqnge ikpalaesyt isedgtrytf kiknilwsng dpltaqdfvs swkeilkeda
121 ssvylyaflp iknaraifdd tespenlgvr aldkrhleiq letpcahflh fltlpiffpv
181 hetlrnysts feempitcga frpvslekgl rlhleknpmy hnksrvklhk iivqfisnan
241 taailfkhkk ldwqgppwge pippeisasl hqddqlfslp gasttwllfn iqkkpwnnak
301 lrkalslaid kdmltkvvyq glaeptdhil hprlypgtyp erkrqneril eaqqlfeeal
361 delqmtredl eketltfstf sfsygricqm lreqwkkvlk ftipivgqef ftiqknfleg
421 nysltvnqwt aafidpmsyl mifanpggis pyhlqdshfq tllikitqeh kkhlrnqlii
481 ealdylehch ileplchpnl rialnknikn fnlfvrrtsd frfiekl
Seventh Antigefa Group
The immunogenicity of other Chlamydia pneumoniae antigens may be improved by
combination with two or more Chlamydia pneumoniae antigens from either the
first
antigen group or the second antigen group or the third antigen group or the
fourth
antigen group or the fifth antigen group or the sixth antigen group. Such
other
Chlamydia pneumoniae antigens include a seventh antigen group consisting one
or
more hypothetical proteins (ie proteins which, for example, have no known
cellular
location and/or function. These antigens are referred to herein as the
"seventh antigen
group". Each of the Clzlamydia pneumoniae antigens of the seventh antigen
group is
described in more detail below.
(57) Hypothetical (CP~a0331)
One example of a hypothetical protein is set forth as SEQ ID NO: 57 below.
GenBank Accession No. GI:4376609; AAD18480.1. Preferred hypothetical proteins
for use with the invention comprise an amino acid sequence: (a) having 50% or
more
identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 57; and/or (b) which is a
fragment of at least n consecutive amino acids of SEQ ID NO: 57, wherein n is
7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, S0, 60, 70, 80, 90, 100,
150, 200,
250 or more). These hypothetical proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 57. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: 57. Other preferred fragments lack
one
or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)
from the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 57. Other fragments omit one or more
42
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 57
1 MAVSGGGGVQ PSSDPGKWNP ALQGEQAEGP SPLKESIFSE TKQASSAAKQ ESLVRSGSTG
61 MYATESQINK AKYRKAQDRS STSPKSKLKG TFSKMRASVQ GFMSGFGSRA SRVSAKRASD
121 SGEGTSLLPT EMDVALKKGN RISPEMQGFF LDASGMGGSS SDISQLSLEA LKSSAFSGAR
181 SLSLSSSESS SVASFGSFQK AIEPMSEEKV NAWTVARLGG EMVSSLLDPN VETSSLVRRA
241 MATGNEGMID LSDLGQEEVS TAMTSPRAVE GKVKVSSSDS PEANPTGIPN SNTLERAEKE
1O 301 AEKQESREQL SEDQMMLARA MAGLLTGAAP QEVLSNSVWS GPSTVFPPPK FSGTLPTQRS
361 GDKSKHKSPG TEKSTNHTNF SPLREGTVKS AEVKSLPHPE SMYRFPKDSI VSREEPEAW
421 KESTAFKNPE NSSQNFLPIA VESVFPKESG TGGALGSDAV SSSYHFLAQR GVSLLAPLPR
481 ATDDYKEKLE AHKGPGGPPD PLIYQYRNVA VEPPIVLRSP QPFSGSSRLS VQGKPEAASV
541 HDDGGGGNSG GFSGDQRRGS SGQKASRQEK KGKKLSTDI
(58) Hypothetical (CPh0234)
One example of a hypothetical protein is set forth as SEQ ID NO: 58 below.
GenBank Accession No. gi~4376508~gb~ AAD18387.1 Preferred hypothetical
proteins
for use with the invention comprise an amino acid sequence: (a) having 50% or
more
identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 58; and/or (b) which is a
fragment of at least h consecutive amino acids of SEQ ID NO: 21, wherein n is
7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These hypothetical proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 58. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: 58. Other preferred fragments lack
one
or more amino acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)
from the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 58. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 58
1 MLQSCKKALL SIWSILAFH PIPGMGVEAK SGFLGKVKGW FSKKEIQEEA RILPVKDSLS
61 WKRYDYTSSS GFSVEFPGEP DHSGQIVEVP QSETTIRYDT YVTETHPDNT VYWSVWEYP
121 EKVDISRPEL NLQEGFSGMM QALPESQVLF MQARQIQGHK ALEFWIVCED VYFRGMLISV
181 NHTLYQVFMV YKNKNPQALD KEYEAFSQSF KITKIREPRT IPSSVKKKVS L
(59) Hypothetical (CPiZ0572)
One example of a hypothetical protein is set forth as SEQ ID NO: 59 below.
Genbank Accession No. gi~4376866~gb~; AAD18712.1. Preferred hypothetical
proteins for use with the invention comprise an amino acid sequence: (a)
having 50%
or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 59; andlor (b) which is
a
fragment of at least f2 consecutive amino acids of SEQ ID NO: 59, wherein n is
7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These hypothetical proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 59. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: 59. Other preferred fragments lack
one
or more amino acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)
from the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 59. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
43
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SEQ ID No 59
1 TTQITQTGQTTTTTTVGSLGEHSVTTTGSG LIADHEMQEI
MAAPINQPST AAAQTSQTVT
61 ASQDGSAVSFSAEHSFSTLPPETGSVGATAQSAQSAGLFSLSGRTQRRDSEISSSSDGSS
121 ISRTSSNASSGETSRAESSPDLGDLDSLSGSERAEGAEGPEGPGGLPESTIPHYDPTDKA
181 SILNFLKNPAVQQKMQTKGGHFVWDEARSSFIFVRNGDWSTAESIKVSNAKTKENITKP
241 ADLEMCIAKFCVGYETIHSDWTGRVKPTMEERSGATGNYNHLMLSMKFKTAWYGPWNAK
301 ESSSGYTPSAWRRGAKVETGPIWDDVGGLKGTNWKTTPAPDFSFINETPGGGAHSTSHTG
361 PGTPVGATWPNVNVNLGGIKVDLGGINLGGITTNVTTEEGGGTNITSTKSTSTDDKVSI
421 TSTGSQSTIEEDTIQFDDPGQGEDDNAIPGTNTPPPPGPPPNLSSSRLLTISNASLNQVL
481 QNVRQHLNTAYDSNGNSVSDLNQDLGQWKNSENGVNFPTVTLPKTTGDTDPSGQATGGV
541 TEGGGHIRNTIQRNTQSTGQSEGATPTPQPTTAKIVTSLRKANVSSSSVLPQPQVATTIT
601 PQARTASTSTTSIGTGTESTSTTSTGTGTGSVSTQSTGVGTPTTTTRSTGTSATTTTSSA
661 STQTPQAPLPSGTRHVATISLVRNAAGRSIVLQQGGRSQSFPIPPSGTGTQNMGAQLWAA
721 ASQVASTLGQVVNQAATAGSQPSSRRSSPTSPRRK
Eiglztlz Antigen Gz~oup
The immunogenicity of other Clzla~nydia pneumoyziae antigens may be unproved
by
combination with two or more Chlanzydia pneumoniae antigens from either the
first
antigen group or the second antigen group of the third antigen group or the
fourth
antigen group or the fifth or the sixth antigen group or the seventh antigen
group.
Such other Chlamydia pneunaoniae antigens include an eigth antigen group
consisting
one or more FACS positive CPn antigens. These antigens are referred to herein
as the
"eight antigen group". Each of the Chlatnydia pr2eumoniae antigens of the
eight
antigen group is described in more detail below.
(60) Low Calcium Response Protei~z H (CP>z0811)
One example of a Low Calcium Response Protein H is set forth as SEQ ID NO: 60
below. Genbank Accession No. GI:4377123; AAD18949.1. Preferred low calcium
response proteins for use with the invention comprise an amino acid sequence:
(a)
having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 60; and/or
(b) which is a fragment of at least n consecutive amino acids of SEQ ID NO:
60,
wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50,
60, 70, 80, 90,
100, 150, 200, 250 or more). These low calcium response proteins include
variants
(e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ
ID NO: 60.
Preferred fragments of (b) comprise an epitope from SEQ ID NO: 60. Other
preferred
fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5,
6, 7, 8,
9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 60. Other
fragments
omit one or more domains of the protein (e.g. omission of a signal peptide, of
a
cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
SEQ ID No 60
1 mskpsprnan qpqkpsasfn kktrsrlael aaqkkakadd leqvhpvpte eeikkalgni
61 feglsngldl qqilglsdyl leeiytvayt fysqgkynea vglfqllaaa qpqnykymlg
121 lsscyhqlhl yneaafgffl afdaqpdnpi ppyyiadsll klqqpeesnn fldvtmdicg
181 nnpefkilke rcqimkqsie kqmagetkka ptkkpagksk tttnkksgkk r
(61) Yop Proteins Tra~zslocation Proteitz T (CPiz0823)
One example of a Yop Proteins Translocation Protein T is set forth as SEQ ID
NO: 61
below. Genbank Accession No. GI:4377135; AAD18960.1. Preferred Yop proteins
44
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WO 2005/084306 PCT/US2005/006588
for use with the invention comprise an amino acid sequence: (a) having 50% or
more
identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 61; and/or (b) which is a
fragment of at least h consecutive amino acids of SEQ ID NO: 61, wherein h is
7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These Yop proteins include variants (e.g. allelic variants,
homologs,
orthologs, paralogs, mutants, etc.) of SEQ ID NO: 61. Preferred fragments of
(b)
comprise an epitope from SEQ ID NO: 61. Other preferred fragments lack one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 61. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 61
1 mgislpelfs nlgsayldyi fqhppayvws vfllllarll pifavapflg aklfpspiki
61 gislswlaii fpkvladtqi tnymdnnlfy vllvkemiig ivigfvlafp fyaaqsagsf
121 itnqqgiqgl egatslisie qtsphgilyh yfvtiifwlv gghrivisll lqtlevipih
181 sffpaemmsl sapiwitmik mcqlclvmti qlsapaalam lmsdlflgii nrmapqvqvi
241 yllsalkafm gllfltlaww fiikqidyft lawfkevpim llgsnpqvl
(62) Yop Proteins Translocation Proteifz J
One example of a Yop Proteins Translocation Protein J is set forth as SEQ ID
NO: 62
below Genbank Accession No. GI:4377140; AAD18965.1. Preferred hypothetical
proteins for use with the invention comprise an amino acid sequence: (a)
having 50%
or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 62; and/or (b) which is
a
fragment of at least h consecutive amino acids of SEQ ID NO: 62, wherein ~z is
7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These hypothetical proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 62. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: 62. Other preferred fragments lack
one
or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)
from the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 62. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 62
1 mvrrsisfcl fflmtllcct scnsrslivh glpgreanei vvllvskgva aqklpqaaaa
61 tagaateqmw diavpsaqit ealailnqag lprmkgtsll dlfakqglvp selqekiryq
121 eglseqmast irkmdgvvda svqisftten ednlpltasv yikhrgvldn pnsimvskik
181 rliasavpgl vpenvsvvsd raaysditin gpwglteeid yvsvwgiila kssltkfrli
241 fyvlililfv iscgllwviw kthtlimtmg gtkgffnptp ytknaleakk aegaaadkek
301 kedadsqges knaetsdkds sdkdapegsn eiega
(63) OmpA (CPst0695)
One example of an OmPA encoded (MOMP) protein is set forth as SEQ ID NO: 63
below Genbank Accession No. GI:4376998; AAD18834.1. Preferred OmpA proteins
for use with the invention comprise an amino acid sequence: (a) having 50% or
more
identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 63; and/or (b) which is a
fragment of at least fa consecutive amino acids of SEQ ID NO: 63, wherein h is
7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These OmpA proteins include variants (e.g. allelic variants,
homologs,
orthologs, paralogs, mutants, etc.) of SEQ ID NO: 63. Preferred fragments of
(b)
comprise an epitope from SEQ ID NO: 63. Other preferred fragments lack one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C-
terminus and/or one or more amino acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 63. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 63
1 mkkllksall saafagsvgs lqalpvgnps dpsllidgti wegaagdpcd pcatwcdais
61 lragfygdyv fdrilkvdap ktfsmgakpt gsaaanytta vdrpnpaynk hlhdaewftn
121 agfialniwd rfdvfctlga sngyirgnst afnlvglfgv kgttvnanel pnvslsngvv
181 elytdtsfsw svgargalwe cgcatlgaef qyaqskpkve elnvicnvsq fsvnkpkgyk
241 gvafplptda gvatatgtks atinyhewqv gaslsyrlns lvpyigvqws ratfdadnir
301 iaqpklptav lnltawnpsl lgnatalstt dsfsdfmqiv scqinkfksr kacgvtvgat
361 lvdadkwslt aearlinera ahvsgqfrf
(64) Hypothetical (CPn0210)
One example of a Hypothetical Protein is set forth as SEQ ID NO: 64 below
Genbank
Accession No. GI:4376482; AAD18363.1. Preferred hypothetical proteins for use
with the invention comprise an amino acid sequence: (a) having 50% or more
identity
(e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.5% or more) to SEQ ID NO: 64; and/or (b) which is a fragment of
at
least n consecutive amino acids of SEQ ID NO: 64, wherein h is 7 or more (e.g.
8, 10,
12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or
more).
These hypothetical proteins include variants (e.g. allelic variants, homologs,
orthologs, paralogs, mutants, etc.) of SEQ ID NO: 64. Preferred fragments of
(b)
comprise an epitope from SEQ ID NO: 64. Other preferred fragments lack one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, S, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 64. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 64
1 mlvelealkr efahlkdqkp tsdqeitsly qcldhlefvl lglgqdkflk atededvlfe
61 sqkaidawna lltkardvlg lgdigaiyqt ieflgaylsk vnrrafcias eihflktair
121 dlnayylldf rwplckieef vdwgndcvei akrklctfek etkelnesll reehamekcs
181 iqdlqrklsd iiielhdvsl fcfsktpsqe eyqkdclyqs rlryllllye ytllcktstd
241 fqeqarakee firekfslle lekgikqtke lefaiakskl ergclvmrky eaaakhslds
301 mfeeetvksp rkdte
(65) Low Calciuut Response Locus Proteifa H (CPfa1021)
One example of a Low Calcium Response Protein H is set forth as SEQ ID NO: 65
below Genbank Accession No. GI:4377352; AAD19158.1. Preferred low calcium
response proteins for use with the invention comprise an amino acid sequence:
(a)
having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
46
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 65; and/or
(b) which is a fragment of at least n consecutive amino acids of SEQ ID NO:
65,
wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50,
60, 70, 80, 90,
100, 150, 200, 250 or more). These low calcium response proteins include
variants
(e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ
ID NO: 65.
Preferred fragments of (b) comprise an epitope from SEQ ID NO: 65 Other
preferred
fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5,
6, 7, 8,
9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 65. Other
fragments
omit one or more domains of the protein (e.g. omission of a signal peptide, of
a
cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
SEQ ID No 65
1 mshlnyllek iaasskedfp fpddlesyle gyvpdknial dtyqkifkis sedlekvyke
61 gyhayldkdy aksitvfrwl vffnpfvskf wfslgaslhm seqysqalha ygvtavlrdk
121 dpyphyyayi cytltnehee aekalemawv raqhkplyne lkeeildirk hk
Ninth Antigen Group
The immunogenicity of other Chlamydia pneumoniae antigens may be improved by
combination with two or more Clalanzydia praeumoniae antigens from either the
first
antigen group or the second antigen group or the third antigen group or the
fourth
antigen group or the fifth antigen group or the sixth antigen group or the
seventh
antigen group or the eight antigen group. Such other Clalanaydia pneunaoniae
antigens
include a ninth antigen group. These antigens are referred to herein as the
"ninth
antigen group". Each of the Chlarnydia p~zemnoniae antigens of the ninth
antigen
group is described in more detail below.
(66) Low Calcium Response Protein D (CPn0323)
One example of a Low Calcium Response Protein D is set forth as SEQ ID NO: 66
below Genbank Accession No. GI:4376601; AAD18472.1. Preferred low calcium
response proteins for use with the invention comprise an amino acid sequence:
(a)
having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 66; and/or
(b) which is a fragment of at least n consecutive amino acids of SEQ ID NO:
66,
wherein rt is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50,
60, 70, 80, 90,
100, 150, 200, 250 or more). These low calcium response proteins include
variants
(e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ
ID NO: 66.
Preferred fragments of (b) comprise an epitope from SEQ ID NO: 66. Other
preferred
fragments lack one or more amino acids (e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the C-terminus and/or one or more amino acids (e.g. l, 2, 3, 4, 5,
6, 7, 8,
9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 66. Other
fragments
omit one or more domains of the protein (e.g. omission of a signal peptide, of
a
cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
SEQ ID No 66
7. mnkllnfvsr tlggdtalnm inkssdlila lwmmgvvlmi iiplpppivd lmitinlsis
61 vfllmvalyi psalqlsvfp slllittmfr lginisssrq illkayaghv iqafgdfvvg
121 gnyvvgfiif liitiiqfiv vtkgaervae vaarfrldam pgkqmaidad lragmidatq
181 ardkraqiqk eselygamdg amkfikgdvi agivislini vggltigvam hgmdlaqaah
241 vytllsigdg lvsqipslli altagivttr vssdkntnlg keistqlvke pralllagaa
47
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WO 2005/084306 PCT/US2005/006588
301 tlgvgffkgf plwsfsilal ifvalgilll tkksaagkkg ggsgasttvg aagdgaatvg
361 dnpddysltl pvilelgkdl skliqhktks gqsfvddmip kmrqalyqdi girypgihvr
421 tdspslegyd ymillnevpy vrgkipphhv ltnevednls rynlpfityk naaglpsawv
481 sedakailek aaikywtple viilhlsyff hkssqeflgi qevrsmiefm ersfpdlvke
541 vtrliplqkl teifkrlvqe qisikdlrti leslsewaqt ekdtvlltey vrsslklyis
601 fkfsqgqsai svylldpeie emirgaikqt sagsylaldp dsvnlilksm rntitptpag
661 gqppvlltai dvrryvrkli etefpdiavi syqeilpeir iqplgriqif
(67) CHLPS 43kDa Protei>z Hontolog-1 (CP>z0062)
One example of a CHLPS 43kDa Protein Homolog-1 is set forth as SEQ ID NO: 67
below Genbank Accession No. GI:4376318; AAD18215.1. Preferred CHLPS
proteins for use with the invention comprise an amino acid sequence: (a)
having 50%
or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 67; and/or (b) which is
a
fragment of at least f~ consecutive amino acids of SEQ ID NO: 67, wherein yt
is 7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These CHLPS proteins include variants (e.g. allelic variants,
homologs,
orthologs, paralogs, mutants, etc.) of SEQ ID NO: 67. Preferred fragments of
(b)
comprise an epitope from SEQ ID NO: 67. Other preferred fragments lack one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C-
terminus and/or one or more amino acids (e.g. l, 2, 3, 4, 5, fj, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 67. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 67
1 mmsskrtski avlsilltft hsigfanans svglgtvyit sevvkkpqkg serkqakkep
61 rarkgylvps srtlsaraqk mknssrkess ggcneisans tprsvklrrn kraeqkaakq
121 gfsafsnltl ksllpklpsk qktsiherek atsrfvnesq lssarkryct pssaapslfl
187. eteivrapve rtkelqdnei hipvvqvqtn pkeqntkttk qlasqasiqq segteqslre
241 laqgaslpvl vrsnpevsvq rqkeellkel vaerrqckrk svrqalears ltkkvarggs
301 vtstlrydpe kaaeiksrrn ckvspeareq kyssckrdar angkqdkttp sedasqeeqq
361 tgaglvrktp ksqvasnaqn fyrnskntni dsyltanqys csseetdwpc sscvskrrth
421 nsisvctmvv tviamivgal iianatesqt tsdptpptpt p
(68) Hypothetical (CPtt0169)
One example of a CHLPS 43kDa Protein Homolog-1 is set forth as SEQ ID NO: 68
below Genbank Accession No. GI:4376437; AAD18322.1. Preferred CHLPS
proteins for use with the invention comprise an amino acid sequence: (a)
having SO%
or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 68; and/or (b) which is
a
fragment of at least n consecutive amino acids of SEQ ID NO: 68, wherein ra is
7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These CHLPS proteins include variants (e.g. allelic variants,
homologs,
orthologs, paralogs, mutants, etc.) of SEQ ID NO: 68. Preferred fragments of
(b)
comprise an epitope from SEQ ID NO: 68. Other preferred fragments lack one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 68. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
48
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
SEQ ID No 68
1 mknvgsecsq plvmelntqp lrnlcesrlv kitsfviall alvggitlta lagagilsfl
61 pwlvlgivlv vlcalfllfs ykfcpikelg vvyntdsqih qwfqkqrnkd lekatenpel
121 fgenraednn rsarsqvket lrdcdgnvlk kiyernldvl lfmnwvpktm ddvdpvseds
181 irtviscykl ikackpefrs lisellramq sglgllsrcs ryqeraktvs hkdaplfcpt
241 hsyyrdgylt plragpryii nrai
(69) PnipD family (CPn0963)
One example ~f a PmpD protein is set forth as SEQ ID NO: 69 below Genbank
Accession No. GI:4377287; AAD19099.1. Preferred PmpD proteins for use with the
invention comprise an amino acid sequence: (a) having 50% or more identity
(e.g.
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.5% or more) to SEQ ID NO: 69; and/or (b) which is a fragment of
at
least n consecutive amino acids of SEQ ID NO: 69, wherein n is 7 or more (e.g.
8, 10,
12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or
more).
These PmpD proteins include variants (e.g. allelic variants, homologs,
orthologs,
paralogs, mutants, etc.) of SEQ ID NO: 69. Preferred fragments of (b) comprise
an
epitope from SEQ ID NO: 69. Other preferred fragments lack one or more amino
acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-
terminus and/or
one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or
more) from the
N-terminus of SEQ ID NO: 69. Other fragments omit one or more domains of the
protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a
transmembrane domain, or of an extracellular domain).
SEQ ID No 69
1 mvakktvrsyrssfshsvivailsagiafeahslhsseldlgvfnkqfeehsahveeaqt
61 svlkgsdpvnpsqkesekvlytqvpltqgssgesldladanflehfqhlfeettvfgidq
121 klvwsdldtrnfsqptqepdtsnavsekissdtkenrkdletedpskksglkevssdlpk
181 spetavaaisedleisenisardplqglaffykntssqsisekdssfqgiifsgsgansg
241 lgfenlkapksgaavysdrdivfenlvkglsfiscesledgsaagvnivvthcgdvtltd
301 catgldlealrlvkdfsrggavftarnhevqnnlaggilsvvgnkgaivveknsaeksng
361 gafacgsfvysnnentalwkenqalsggaissasdidiqgncsaiefsgnqslialgehi
421 gltdfvgggalaaqgtltlrnnavvqcvkntskthggailagtvdlnetisevafkqnta
481 altggalsandkviiannfgeilfeqnevrnhggaiycgcrsnpkleqkdsgeniniign
541 sgaitflknkasvlevmtqaedyagggalwghnvlldsnsgniqfigniggstfwigeyv
601 gggailstdrvtisnnsgdvvfkgnkgqclaqkyvapqetapvesdasstnkdekslnac
661 shgdhyppktveeevppslleehpvvsstdirgggailaqhifitdntgnlrfsgnlggg
721 eesstvgdlaivgggallstnevnvcsnqnvvfsdnvtsngcdsggailakkvdisanhs
781 vefvsngsgkfggavcalnesvnitdngsavsfsknrtrlggagvaapqgsvticgnqgn
841 iafkenfvfgsenqrsgggaiianssvniqdnagdilfvsnstgsyggaifvgslvaseg
901 snprtltitgnsgdilfaknstqtaaslsekdsfgggaiytqnlkivknagnvsfygnra
961 psgagvqiadggtvcleafggdilfegninfdgsfnaihlcgndskivelsavqdkniif
1021 qdaityeentirglpdkdvsplsapslifnskpqddsaqhhegtirfsrgvskipqiaai
1081 qegtlalsqnaelwlaglkqetgssivlsagsilrifdsqvdssaplptenkeetlvsag
1141 vqinmssptpnkdkavdtpvladiisitvdlssfvpeqdgtlplppeiiipkgtklhsna
1201 idlkiidptnvgyenhallsshkdiplislktaegmtgtptadaslsnikidvslpsitp
1261 atyghtgvwseskmedgrlvvgwqptgyklnpekqgalvlnnlwshytdlralkqeifah
1321 htiaqrmeldfstnvwgsglgvvedcqnigefdgfkhhltgyalgldtqlvedfliggcf
1381 sqffgktesqsykakndvksymgaayagilagpwlikgafvygninndlttdygtlgist
1441 gswigkgfiagtsidyryivnprrfisaivstvvpfveaeyvridlpeiseqgkevrtfq
1501 ktrfenvaipfgfalehaysrgsraevnsvqlayvfdvyrkgpvslitlkdaayswksyg
1561 vdipckawkarlsnntewnsylstylafnyewredliaydfnggiriif
Tenth
Antigen
Group
The moniae
immunogenicity antigens
of may
other be improved
Chlanaydia by
pneu
combination or more urnoniae either
with Chlamydia antigens the first
two pne from
49
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
antigen group or the second antigen group or the third antigen group or the
fourth
antigen group or the fifth antigen group or the sixth antigen group or the
seventh
antigen group or the eight antigen group or the ninth antigen group. Such
other
Chlamydia pneumoniae antigens include a tenth antigen group. Each of the
Chlamydia pneumoniae antigens of the tenth antigen group is described in more
detail
below.
(70) OmpH like outer tnefrZbraue protei~z (CPh0301)
One example of 'OmpH-like' protein is disclosed as SEQ ID NOS: 77 ~ 78 in WO
02/02606. ~GenBank accession number: gi~4376577~gb~AAD18450.1~ 'CPn0301';
SEQ ID NO: 70 below and SEQ ID No 4 above}. Preferred OmpH-like proteins for
use with the invention comprise an amino acid sequence: (a) having 50% or more
identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 4; and/or (b) which is a
fragment of at least n consecutive amino acids of SEQ ID NO: 3, wherein n is 7
or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These OmpH-like proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 4. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: 4. Other preferred fragments lack
one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more; preferably 19 or more, to remove the signal peptide) from the N-terminus
of
SEQ ID NO: 4. Other fragments omit one or more domains of the protein (e.g.
omission of a signal peptide as described above, of a cytoplasmic domain, of a
transmembrane domain, or of an extracellular domain).
SEQ ID No 70
1 MKICGI~FSTFL LVI~GSTSAAIi A_NLGYVNLKR CLEESDLGKK ETEELEAMKQ
3O 51 QFVKNAEKIE EELTSIYNKL QDEDYMESLS DSASEELRKK FEDLSGEYNA
101 YQSQYYQSIN QSNVKRIQKL IQEVKIAAES VRSKEKLEAI LNEEAVLAIA
151 PGTDKTTEII AILNESFKKQ N*
(71) L7/L12 Ribosomal Protein (CPh0080)
One example of an L7/L12 Ribosomal protein is set forth as SEQ ID No 71
below{GenBank accession number: GI:4376338; AAD18233.1}. 'CPn0080'; SEQ
ID NO: 71 below. Preferred L7/L12 proteins for use with the invention comprise
an
amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)
to SEQ ID NO: 71; and/or (b) which is a fragment of at least n consecutive
amino
acids of SEQ ID NO: 71, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18,
20, 25, 30,
35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These L7/L12
ribosomal
proteins include variants (e.g. allelic variants, homologs, orthologs,
paralogs, mutants,
etc.) of SEQ ID NO: 71. Preferred fragments of (b) comprise an epitope from
SEQ ID
NO: 71. Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3,
4, 5, 6,
7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino
acids
(e.g. l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more; preferably 19 or
more, to remove
the signal peptide) from the N-terminus of SEQ ID NO: 71. Other fragments omit
one
or more domains of the protein (e.g. omission of a signal peptide as described
above,
of a cytoplasmic domain, of a transmembrane domain, or of an extracellular
domain).
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
SEQ ID No 71
1 mttesletlv eklsnltvle lsqlkkllee kwdvtasapv vavaagggge apvaaeptef
61 avtledvpad kkigvlkvvr evtglalkea kemteglpkt vkektsksda edtvkklqda
121gakasfkgl
(72) AtoS two-co~rtpoaae~zt regulatory systes~a sensor histidi~ze ki~zase
proteih
(CPn0584)
One example of 'AtoS' protein is disclosed as SEQ ID NOS: 105 & 106 in WO
02/02606. {GenBank accession number: gi~4376878~gb~AAD18723.1~ 'CPn0584';
SEQ ID NO: 72 below and SEQ ID No 9 above. Preferred AtoS proteins for use
with the invention comprise an amino acid sequence: (a) having 50% or more
identity
(e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.5% or more) to SEQ ID NO: 72; and/or (b) which is a fragment of
at
least ~ consecutive amino acids of SEQ ID NO: 72, wherein h is 7 or more (e.g.
8, I0,
12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or
more).
These AtoS proteins include variants (e.g. allelic variants, homologs,
orthologs,
paralogs, mutants, etc.) of SEQ ID NO: 72. Preferred fragments of (b) comprise
an
epitope from SEQ ID NO: 72. Other preferred fragments lack one or more amino
acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-
terminus and/or
one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or
more) from the
N-terminus of SEQ ID NO: 72. Other fragments omit one or more domains of the
protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a
transmembrane domain, or of an extracellular domain).
SEQ ID No 72
1 MNVPDSKNLH PPAYELLEIK ARITQSYKEA SAILTAIPDG ILLLSETGHF
51 LICNSQAREI LGIDENLEIL NRSFTDVLPD TCLGFSIQEA LESLKVPKTL
3O 101 RLSLCKESKE KEVELFIRKN EISGYLFIQI RDRSDYKQLE NAIERYKNIA
151 ELGKMTATLA HEIRNPLSGI VGFASILKKE ISSPRHQRML SSIISGTRSL
201 NNLVSSMLEY TKSQPLNLKI INLQDFFSSL IPLLSVSFPN CKFVREGAQP
251 LFRSIDPDRM NSVVWNLVKN AVETGNSPIT LTLHTSGDIS VTNPGTIPSE
301 IMDKLFTPFF TTKREGNGLG LAEAQKIIRL HGGDIQLKTS DSAVSFFIII
351 PELLAALPKE RAAS*
(73) OmcA 9kDa-cysteine-rich lipoprotein(CP~z0558)
One example of 'OmcA' protein is disclosed as SEQ ID NOS: 9 & 10 in WO
02/02606. {GenBank accession number: gi~4376850~gb~AAD18698.1~ 'CPn0558',
'OmcA', 'Omp3'; SEQ ID NO: 73 below and SEQ ID No 10 above. Preferred
OmcA proteins for use with the invention comprise an amino acid sequence: (a)
having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 73; and/or
(b) which is a fragment of at least a consecutive amino acids of SEQ ID NO:
73,
wherein h is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50,
60, 70, 80, 90,
100, 150, 200, 250 or more). These OmcA proteins include variants (e.g.
allelic
variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 73.
Preferred
fragments of (b) comprise an epitope from SEQ ID NO: 73. Other preferred
fragments
lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25
or more) from
the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20,
25 or more; preferably 18 or more to remove the signal peptide) from the N-
terminus
of SEQ ID NO: 73. Other fragments omit one or more domains of the protein
(e.g.
omission of a signal peptide as described above, of a cytoplasmic domain, of a
51
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
transmembrane domain, or of an extracellular domain). The protein may be
lipidated
(e.g. by a N acyl diglyceride), and may thus have a N-terminal cysteine.
SEQ ID No 73
1 MKKAVhIAAM FCGWSLSSC CRIVDCCFED PCAPSSCNPC EVIRKKERSC
51 GGNACGSYVP SCSNPCGSTE CNSQSPQVKG CTSPDGRCKQ
(74) Hypothetical (CPn0331)
7 0 One example of a hypothetical protein is set forth as SEQ ID NO: 74 below
and SEQ
ID No 57 above. Genbank Accession No. GI:4376609; AAD18480.1. Preferred
hypothetical proteins for use with the invention comprise an amino acid
sequence: (a)
having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 74; andlor
(b) which is a fragment of at least a consecutive amino acids of SEQ ID NO:
74,
wherein fZ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50,
60, 70, 80, 90,
100, 150, 200, 250 or more). These hypothetical proteins include variants
(e.g. allelic
variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 74.
Preferred
fragments of (b) comprise an epitope from SEQ ID NO: 74. Other preferred
fragments
lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25
or more) from
the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20,
or more) from the N-terminus of SEQ ID NO: 74. Other fragments omit one or
more domains of the protein (e.g. omission of a signal peptide, of a
cytoplasmic
domain, of a transmembrane domain, or of an extracellular domain).
SEQ ID NO 74
1 mavsggggvq pssdpgkwnp alqgeqaegp splkesifse tkqassaakq eslvrsgstg
61 myatesqink akyrkaqdrs stspksklkg tfskmrasvq gfmsgfgsra srvsakrasd
121 sgegtsllpt emdvalkkgn rispemqgff ldasgmggss sdisqlslea lkssafsgar
181 slslsssess svasfgsfqk aiepmseekv nawtvarlgg emvsslldpn vetsslvrra
241 matgnegmid lsdlgqeevs tamtsprave gkvkvsssds peanptgipn sntleraeke
301 aekqesreql sedqmmlara maglltgaap qevlsnsvws gpstvfpppk fsgtlptqrs
361 gdkskhkspg iekstnhtnf splregtvks aevkslphpe smyrfpkdsi vsreepeavv
421 kestafknpe nssqnflpia vesvfpkesg tggalgsdav sssyhflaqr gvsllaplpr
481 atddykekle ahkgpggppd pliyqyrnva veppivlrsp qpfsgssrls vqgkpeaasv
541 hddggggnsg gfsgdqrrgs sgqkasrqek kgkklstdi
(75) PmpD family (CPiZ0963)
One example of a PmpD protein is set forth as SEQ ID NO: 75 below Genbank
Accession No. GI:4377287; AAD19099.1. Preferred PmpD proteins for use with the
invention comprise an amino acid sequence: (a) having 50% or more identity
(e.g.
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.5% or more) to SEQ ID NO: 75; and/or (b) which is a fragment of
at
Ieast ra consecutive amino acids of SEQ ID NO: 75, wherein n is 7 or more
(e.g. 8, 10,
12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or
more).
These hypothetical proteins include variants (e.g. allelic variants, homologs,
orthologs, paralogs, mutants, etc.) of SEQ ID NO: 75. Preferred fragments of
(b)
comprise an epitope from SEQ ID NO: 75. Other preferred fragments lack one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 75. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
52
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
SEQ ID No 75
1 mvakktvrsyrssfshsvivailsagiafeahslhsseldlgvfnkqfeehsahveeaqt
61 svlkgsdpvnpsqkesekvlytqvpltqgssgesldladanflehfqhlfeettvfgidq
121 klvwsdldtrnfsqptqepdtsnavsekissdtkenrkdletedpskksglkevssdlpk
181 spetavaaisedleisenisardplqglaffykntssqsisekdssfqgiifsgsgansg
241 lgfenlkapksgaavysdrdivfenlvkglsfiscesledgsaagvnivvthcgdvtltd
301 catgldlealrlvkdfsrggavftarnhevqnnlaggilsvvgnkgaivveknsaeksng
361 gafacgsfvysnnentalwkenqalsggaissasdidiqgncsaiefsgnqslialgehi
421 gltdfvgggalaaqgtltlrnnavvqcvkntskthggailagtvdlnetisevafkqnta
481 altggalsandkviiannfgeilfeqnevrnhggaiycgcrsnpkleqkdsgeniniign
541 sgaitflknkasvlevmtqaedyagggalwghnvlldsnsgniqfigniggstfwigeyv
601 gggailstdrvtisnnsgdvvfkgnkgqclaqkyvapqetapvesdasstnkdekslnac
661 shgdhyppktveeevppslleehpvvsstdirgggailaqhifitdntgnlrfsgnlggg
721 eesstvgdlaivgggallstnevnvcsnqnvvfsdnvtsngcdsggailakkvdisanhs
78l vefvsngsgkfggavcalnesvnitdngsavsfsknrtrlggagvaapqgsvticgnqgn
841 iafkenfvfgsenqrsgggaiianssvniqdnagdilfvsnstgsyggaifvgslvaseg
901 snprtltitgnsgdilfaknstqtaaslsekdsfgggaiytqnlkivknagnvsfygnra
961 psgagvqiadggtvcleafggdilfegninfdgsfnaihlcgndskivelsavqdkniif
1021 qdaityeentirglpdkdvsplsapslifnskpqddsaqhhegtirfsrgvskipqiaai
1081 qegtlalsqnaelwlaglkqetgssivlsagsilrifdsqvdssaplptenkeetlvsag
1141 vqinmssptpnkdkavdtpvladiisitvdlssfvpeqdgtlplppeiiipkgtklhsna
1201 idlkiidptnvgyenhallsshkdiplislktaegmtgtptadaslsnikidvslpsitp
1261 atyghtgvwseskmedgrlvvgwqptgyklnpekqgalvlnnlwshytdlralkqeifah
1321 htiaqrmeldfstnvwgsglgvvedcqnigefdgfkhhltgyalgldtqlvedfliggcf
1381 sqffgktesqsykakndvksymgaayagilagpwlikgafvygninndlttdygtlgist
1441 gswigkgfiagtsidyryivnprrfisaivstvvpfveaeyvridlpeiseqgkevrtfq
1501 ktrfenvaipfgfalehaysrgsraevnsvqlayvfdvyrkgpvslitlkdaayswksyg
1561 vdipckawkarlsnntewnsylstylafnyewredliaydfnggiriif
(76) Hypothetical (CPh0798)
One example of a hypothetical protein is set forth as SEQ ID NO: 78 below.
GenBank Accession No. GI:4377109; AAD18936 Preferred hypothetical proteins for
use with the invention comprise an amino acid sequence: (a) having 50% or more
identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 78; and/or (b) which is a
fragment of at least n consecutive amino acids of SEQ ID NO: 78, wherein n is
7 or
more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These hypothetical proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 78. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: 78. Other preferred fragments lack
one
or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)
from the C-
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 78. Other fragments omit one or rr~ore
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 78
1 mkktccqnyr sigvvfsvvl fvlttqtlfa ghfidigtsg lyswargvsg dgrvvvgyeg
61 gnafkyvdge kflleglvpr sealvfkasy dgsviigisd qdpscravkw vngalvdlgi
121 fsegmqsfae gvssdgktiv gclysddtet nfavkwdetg mvvlpnlped rhscawdase
181 dgsvivgdam gseeiakavy wkdgeqhlls nipgakrssa havskdgsfi vgefiseene
241 vhafvyhngv ikdigtlggd ysvatgvsrd gkvivghstr tdgeyrafky vdgrmidlgt
301 lggsasfafg vsddgktivg kfetelgech afiyldd
(77) Hypotlaetical (CPn0799)
53
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
One example of a hypothetical protein is set forth as SEQ ID NO: 79 below.
GenBank Accession No. GI: 15618708; AAD18937 Preferred hypothetical proteins
for use with the invention comprise an amino acid sequence: (a) having 50% or
more
identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 79; and/or (b) which is a
fragment of at least n consecutive amino acids of SEQ ID NO: 79, wherein n is
7 or
more (e.g. 8, 10, 12, I4, I6, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
150, 200,
250 or more). These hypothetical proteins include variants (e.g. allelic
variants,
homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 79. Preferred
fragments
of (b) comprise an epitope from SEQ ID NO: 79. Other preferred fragments lack
one
or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)
from the C
terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 79. Other fragments omit one or more
domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of
a transmembrane domain, or of an extracellular domain).
SEQ ID No 79
1 maaikqilrs mlsqsslwmv lfslyslsgy cyvitdkped dfhsssavkw dhwgkttlsr
61 lsnkkasaka vsgtgattvg fikdtwsrty avrwnywgtk elptsswvkk skatgissdg
121 siiagivene lsqsfavtwk nnemyllpst wavqskaygi ssdgsvivgs akdawsrtfa
181 vkwtgheaqv lpvgwavksv ansvsangsi ivgsvqdasg ilyavkwegn tithlgtlgg
241 ysaiakavsn ngkvivgrse tyygevhafc hkngvmsdlg tlggsysaak gvsatgkviv
301 gmsttangkl hafkyvggrm idlgeyswke acanavsidg eiivgvqse
Preferably the composition of the invention comprises a combination of CPn
antigens
selected from the group consisting of (1) CPn0301 and CPn0080; (2) CPn 0584
and
CPn 0558; and (3) CPn 0331 and CPN 0963. Preferably the composition comprises
a
combination of any one or more of groups ( 1), (2) and (3).
Even more preferably, the composition of the present invention comprises a
combination of CPn antigens selected from the group consisting of (I) CPn0385,
CPn0324, CPn 0503, CPn0525 and CPn 0482. Preferably the composition is
administered in the presence of alum andlor cPG.
The invention thus includes a composition comprising a combination of
Chlamydia
pneumoniae antigens, said combination selected from the group consisting of
two,
three, four, five or six Chlamydia pneumoniae antigens of the first antigen
group and
two, three, four, five, or six Clalan2ydia pneurnoraiae antigens of the second
antigen
group. Preferably, the combination is selected from the group consisting of
three,
four, five or six Chlanaydia pneumoniae antigens from the first antigen group
and
three, four, five or six Chlamydia pneunZOniae antigens from the second
antigen
group. Still more preferably, the combination consists of six Chlanaydia
pneurraoniae
antigens from the first antigen group and three, four, five or six, Chlamydia
pheumoniae antigens from the second antigen group.
The invention further includes a composition comprising a combination of
Clalamydia
pneumoniae antigens, said combination selected from the group consisting of
two,
three, four, five or six, Chlamydia pneumoniae antigens of the second antigen
group
and two, three, four, five, six, seven or eight Chlamydia pneumoniae antigens
of the
third antigen group. Preferably, the combination is selected from the group
consisting
of three, four, five or six Chlamydia pneumoniae antigens from the second
antigen
54
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
group and three, four, five, six, seven or eight Chlamydia pneumoniae from the
third
antigen group. Still more preferably, the combination consists of six
Chlamydia
pneumoniae antigens from the second antigen group and three, four, five, six,
seven
or eight Chlamydia pneumozziae antigens of the third antigen group.
There is an upper limit to the number of Chlanzydia przeumorziae antigens
which will
be in the compositions of the invention. Preferably, the number of Chlamydia
pneunzoniae antigens in a composition of the invention is lass than 20, less
than 19,
less than 18, less than 17, less than 16, less than I5, less than 14, less
than I3, less
than 12, less than 11, less than I0, less than 9, less than 8, less than 7,
less than 6, less
than 5, less than 4, or less than 3. Still more preferably, the number of
Chlamydia
pneumozziae antigens in a composition of the invention is less than 6, less
than 5, or
less than 4. The Chlanzydia pneumoniae antigens used in the invention are
preferably
isolated, i.e., separate and discrete, from the whole organism with which the
molecule
9 5 is found in nature or, when the polynucleotide or polypeptide is not found
in nature, is
sufficiently free of other biological macromolecules so that the
polynucleotide or
polypeptide can be used for its intended purpose.
In either of the above combinations, preferably the composition comprises one
or
more Clzlamydia przeurrzo~ziae antigens from the fourth antigen group which
includes
porB. Or, alternatively, in either of the above combinations, preferably the
Chlamydia pneumoniae antigens from the fourth antigen group includes one or
more
members of the pmp3 family.
Other aspects of the present invention are presented in the accompanying
claims and
in the following description and drawings. These aspects are presented under
separate
section headings. However, it is to be understood that the teachings under
each
section are not necessarily limited to that particular section heading.
Before describing the present invention in detail, it is to be understood that
this
invention is not limited to particularly exemplified molecules or process
parameters as
such may, of course, vary. It is also to be understood that the terminology
used herein
is for the purpose of describing particular embodiments of the invention only,
and is
not intended to be limiting. In addition, the practice of the present
invention will
employ, unless otherwise indicated, conventional methods of virology,
microbiology,
molecular biology, recombinant DNA techniques and immunology all of which are
within the ordinary skill of the art. Such techniques are explained fully in
the
literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory
Manual
(2nd Edition, 1989); DNA Cloning: A Practical Approach, vol. I & II (D.
Glover,
ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); A Practical Guide to
Molecular
Cloning (1984); and Fundamental Virology, 2nd Edition, vol. I ~ II (B.N.
Fields and
D.M. Knipe, eds.).
All publications, patents and patent applications cited herein, whether sup>"a
or inf °a,
are hereby incorporated by reference in their entirety. It must be noted that,
as used in
this specification and the appended claims, the singular forms "a", "an" and
"the"
include plural referents unless the content clearly dictates otherwise. All
scientific
and technical terms used in this application have meanings commonly used in
the art
unless otherwise specified. As used in this application, the following words
or phrases
have the meanings specified.
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The term "comprising" means "including" as well as "consisting" e.g. a
composition
"comprising" X may consist exclusively of X or may include something
additional
e.g. X + Y.
The term "about" in relation to a numerical value x means, for example, x~10%.
References to a percentage sequence identity between two amino acid sequences
means that, when aligned, that percentage of amino acids are the same in
comparing
the two sequences. This alignment and the percent homology or sequence
identity can
be determined using software programs known in the art, for example those
described
in section 7.7.18 of Current Protocols izz Moleculaf~ Biology (F.M. Ausubel et
al.,
eds., 1987) Supplement 30. A preferred alignment is determined by the Smith-
Waterman homology search algorithm using an affine gap search with a gap open
penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-
Waterman homology search algorithm is disclosed in Smith & Waterman (1981)
Adv.
Appl. Math. 2: 482-489.
IMMUNE RESPONSE
The mechanism by which the immune system controls disease includes the
induction
of neutralising antibodies via humoral immunity and the generation of T-cell
responses via cellular immunity. As used herein, the term "immune response"
against
an antigen refers to the development in a host mammalian subject of a humoral
and/or
a cellular immune response against that antigen.
As used herein, the term "humoral immune response" refers to an immune
response
mediated by antibody molecules. The antibodies generated by humoral immunity
are
primarily effective against extracellular infectious agents.
SEQ ID Nos 1-86 in the compositions of the invention may be supplemented or
substituted with an antibody that binds to the protein. This antibody may be
monoclonal or polyclonal.
As used herein, the term "cell mediated immune (CMI) response" is one mediated
by
T-lymphocytes and/or other white blood cells. The CMI immune mechanisms are
generally more effective against intracellular infections and disease because
the CMI
mechanisms prime T cells in a way that, when an antigen appears at a later
date,
memory T cells are activated to result in a CMI response that destroys target
cells that
have the corresponding antigen or a portion thereof on their cell surfaces,
and thereby
the infecting pathogen. The CMI response is focused on the destruction of the
source
of infection mediated by either effector cells that destroy infected cells of
the host by
direct cell-to-cell contact and/or by the release of molecules, such as
cytokines, that
possess anti-viral activity. Thus the CMI response, which is characterised by
a
specific T lymphocyte cellular response, is crucial to produce resistance to
diseases
caused by cancer, viruses, pathogenic and other intracellular microorganisms.
In one aspect of the present invention, an immunogenic composition is provided
comprising a combination of at least one antigen that elicits a Chlanzydia
pneumoniae
specific Thl immune response (such as a cell mediated or cellular immune
response)
and at least one antigen that elicits a Chlanaydia pneumozziae specific Th2
response
(such as a humoral or antibody response). The immunogenic composition may
further
comprise a Thl adjuvant and a Th2 adjuvant.
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In one embodiment, the invention provides a composition comprising a
combination
of Clalamydia pneurnoniae antigens that elicit at least a Chlamydia pneumoniae
specific Thl immune response. As an example, the combination of Chlanaydia
pneumoniae antigens may include at least one antigen associated with
reticulate
bodies (RBs) of Chlamydia pneumoniae, including but not limited to antigens
expressed, exposed on or translocated into, through or across on the inclusion
membrane, antigens expressed, secreted, released or translocated into the
cytosol of
host cells, or antigens processed or degraded in the cytosol of host cells
and/or
expressed, exposed or presented on the surface of the host cell. The
compositions of
the invention will preferably elicit both a cell mediated immune response as
well as a
humoral immune response in order to effectively address a Chlamydia
intracellular
infection. This immune response will preferably induce long lasting (eg
neutralising)
antibodies and a cell mediated immunity that can quickly respond upon exposure
to
Chlamydia.
The invention also comprises an immunogenic composition comprising one or more
immunoregulatory agents. Preferably, one or more of the immunoregulatory
agents
include an adjuvant. The adjuvant may be selected from one or more of the
group
consisting of a Thl adjuvant and Th2 adjuvant, further discussed below. The
adjuvant
may be selected from the group consisting of a mineral salt, such as an
aluminium salt
and an oligonucleotide containing a CpG motif. Most preferably, the
immunogenic
composition includes both an aluminium salt and an oligonucleotide containing
a
CpG motif. Use of the combination of a mineral salt, such as an aluminium
salt, and
an oligonucleotide containing a CpG motif provide for an enhanced immune
response.
This improved immune response is wholly unexpected and could not be predicted
from the use of either agent alone. The invention therefore includes an
oligonucleotide containing a CpG motif, a mineral salt such as an aluminium
salt, and
an antigen, such as a Clalamydia pneumoniae antigen.
T CELLS IMPLICATED IN THE CMI RESPONSE
At least two special types of T cells are required to initiate and/or to
enhance CMI and
and humoral responses. The antigenic receptors on a particular subset of T
cells
which express a CD4 co-receptor can be T helper (Th) cells or CD4 T cells
(herein
after called T helper cells) and they recognise antigenic peptides bound to
MHC class
II molecules. In contrast, the antigenic receptors on a particular subset of T
cells
which express a CD8 co-receptor are called Cytotoxic T lymphocytes (CTLs) or
CD8+ T cells (hereinafter called CD8+ T cells) and they react with antigens
displayed
on MHC Class I molecules.
HELPER T CELLS
Helper T cells or CD4+ cells can be further divided into two functionally
distinct
subsets: Thl and Th2 which differ in their cytokine and effector function. Thl
and
Th2 responses have been shown to be regulated not only in a positive but also
in a
negative way such that Thl cellular responses are augmented by Thl cytokines
such
as IL-2, IL-12 and IFN-gamma and decreased by Th2 cytokines such as IL-4 and
IL-
10. In contrast, antibody responses are enhanced by Th2 cytokines such as IL-4
and
IL-10 but are downregulated by Thl cytokines such as IFN-gamma and another
cytokine IL-12 that enhances IFN-gamma and is produced by monocytes. Thus,
classic Thl cytokines such as IFN-gamma, IL-2 and IL-12 can be regarded as
immune
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co-factors that induce an effective inflammatory response. In contrast, the
classic Th2
cytokines such as IL-4 and IL-10 can be regarded as cytokines that will
suppress a
severe inflammatory response.
CD8+ T CELLS
CD8+ T cells may function in more than one way. The best known function of
CD8+
T cells is the killing or lysis of target cells bearing peptide antigen in the
context of an
MHC class I molecule. Hence the reason why these cells are often termed
cytotoxic T
lymphocytes (CTL). However, another function, perhaps of greater protective
relevance in certain infections is the ability of CD8+ T cells to secrete
interferon
gamma (IFN-gamma). Thus assays of lytic activity and of IFN-gamma release are
both of value in measuring CD8+ T cell immune response (eg in an ELISPOT assay
as set forth below). In infectious diseases there is evidence to suggest that
CD8+ T
cells can protect by killing an infectious agent comprising an infectious
antigen at the
early stages of a disease before any symptoms of disease are produced.
ENHANCED CMI RESPONSE
The present invention concerns methods, processes and compositions capable of
enhancing and/or modulating the CMI response in a host subject against a
target
antigen. As used herein, the term "enhancing" encompasses improvements in all
aspects of the CMI response which include but are not limited to a stimulation
and/or
augmentation and/or potentiation andlor up-regulation of the magnitude and/or
duration, and/or quality of the CMI response to an antigen or a nucleotide
sequence
encoding an antigen of interest. By way of example, the CMI response may be
enhanced by either (i) enhancing the activation and/or production and/or
proliferation
of CD8+ T cells that recognise a target antigen and/or (ii) shifting the CMI
response
from a Th2 to a Thl type response. This enhancement of the Thl associated
responses
is of particular value in responding to intracellular infections because, as
explained
above, the CMI response is enhanced by activated Thl (such as, for example,
IFN
gamma inducing) cells.
Such an enhanced irmnune response may be generally characterized by increased
titers of interferon-producing CD4+ and/or CD8+ T lymphocytes, increased
antigen-
specific CD8+ T cell activity, and a T helper 1-like immune response (Thl)
against
the antigen of interest (characterized by increased antigen-specific antibody
titers of
the subclasses typically associated with cellular immunity (such as, for
example
IgG2a), usually with a concomitant reduction of antibody titers of the
subclasses
typically associated with humoral immunity (such as, for example IgGI))
instead of a
T helper 2-like immune response (Th2).
The enhancement of a CMI response may be determined by a number of well-known
assays, such as by lymphoproliferation (lymphocyte activation) assays, CD8+ T
cell
assays, or by assaying for T-lymphocytes specific for the epitope in a
sensitized
subject (see, for example, Erickson et al. (1993) J. Immunol. 151: 4189-4199;
and
Doe et al. (1994) Eur. J. Immunol. 24: 2369-2376) or CD8+ T cell ELISPOT
assays
for measuring Interferon gamma production (Miyahara et al PNAS(USA) (1998) 95:
3954-3959).
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ENHANCED T-CELL RESPONSE
As used herein, the term "enhancing a T -cell response" encompasses
improvements
in all aspects of the T-cell response which include but are not limited to a
stimulation
and/or augmentation and/or potentiation and/or up-regulation of the magnitude
and/or
duration, and/or quality of the T-cell response to an antigen (which may be
repeatedly
administered) or a nucleotide sequence encoding an antigen. The antigen may be
a
Chlarnydia antigen, preferably a Clalamydia pneumoniae antigen. By way of
example, the T-cell response may be enhanced by either enhancing the
activation
and/or production andlor distribution and/or proliferation of the induced T-
cells
and/or longevity of the T-cell response to T-cell inducinglmodulating antigen
or
nucleotide sequence encoding an antigen. The enhancement of the T-cell
response in
a host subject may be associated with the enhancement and/or modulation of the
Thl
immune response in the host subject.
The enhancement of the T-cell response may be determined by a number of well-
known assays, such as by lymphoproliferation (lymphocyte activation) assays,
CD8+
T-cell cytotoxic cell assays, or by assaying for T-lymphocytes specific for
the epitope
in a sensitized subject (see, for example, Erickson et al. (1993) J. Immunol.
151:
4189-4199; and Doe et al. (1994) Eur. J. Immunol. 24: 2369-2376) or CD8+ T-
cell
ELISPOT assays for measuring Interferon gamma production (Miyahara et al
PNAS(USA) (1998) 95: 3954-3959).
Activated Thl cells enhance cellular immunity (including an increase in
antigen
specific CTL production) and are therefore of particular value in responding
to
intracellular infections. Activated Thl cells may secrete one or more of IL-2,
IFN
gamma, and TNF-beta. A Thl immune response may result in local inflammatory
reactions by activating macrophages, NIA (natural killer) cells, and CD8
cytotoxic T
cells (CTLs). A Thl immune response may also act to expand the immune response
by stimulating growth of B and T cells with IL-12. Thl stimulated B cells may
secrete
IgG2a.
Activated Th2 cells enhance antibody production and are therefore of value in
responding to extracellular infections. Activated Th2 cells may secrete one or
more of
IL-4, IL-5, IL-6, and IL-10. A Th2 immune response may result in the
production of
IgGl, IgE, IgA and memory B cells for future protection.
ANTIGEN
Each disease causing agent or disease state has associated with it an antigen
or
immunodominant epitope on the antigen which is crucial in immune recognition
and
ultimate elimination or control of a disease causing agent or disease state in
a host. In
order to mount a humoral and/or cellular immune response against a particular
disease, the host immune system must come in contact with an antigen or an
immunodominant epitope on an antigen associated with that disease state.
As used herein, the term "antigen" refers to any agent, generally a
macromolecule,
which can elicit an immunological response in an individual. The term
"antigen" is
used interchangeably with the term "immunogen". The immunological response may
be of B- and/or T-lymphocytic cells. The term may be used to refer to an
individual
macromolecule or to a homogeneous or heterogeneous population of antigenic
macromolecules. As used herein, "antigen" is used to refer to a protein
molecule or
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portion thereof which contains one or more antigenic determinants or epitopes.
As
used herein, the term "antigen" means an immunogenic peptide or protein of
interest
comprising one or more epitopes capable of inducing a CMI response to an
infectious
Chlamydia pathogen. The antigen can include but is not limited to an auto-
antigen, a
self antigen, a cross-reacting antigen, an alloantigen, a tolerogen, an
allergen, a
hapten, an immunogen or parts thereof as well as any combinations thereof.
EPITOPE
As used herein, the terns "epitope" generally refers to the site on an antigen
which is
recognised by a T-cell receptor and/or an antibody. Preferably it is a short
peptide
derived from or as part of a protein antigen. However the term is also
intended to
include peptides with glycopeptides and carbohydrate epitopes. Several
different
epitopes may be carried by a single antigenic molecule. The term "epitope"
also
includes modified sequences of amino acids or carbohydrates which stimulate
responses which recognise the whole organism. It is advantageous if the
selected
epitope is an epitope of an infectious agent, such as a Chlanaydia bacterium,
which
causes the infectious disease'.
SEQ ID Nos 1-86 in the compositions of the invention may be supplemented or
substituted with molecules comprising fragments of SEQ ID Nos 1-86. Such
fragments may comprise at least n consecutive monomers from the molecules and.
depending on the particular sequence. n is either (i) 7 or more for protein
molecules
(eg. 8 18, 20 or more), preferably such that the fragment comprises an epitope
from
the sequence, or (ii) 10 or more for nucleic acid molecules (eg 15, 18, 20,
25, 30, 35,
40 or more).
SOURCE OF EPITOPES
The epitope can be generated from knowledge the amino acid and corresponding
DNA sequences of the peptide or polypeptide, as well as from the nature of
particular
amino acids (e.g., size, charge, etc.) and the codon dictionary, without undue
experimentation. See, e.g., Ivan Roitt, Essential Immunology, 1988; Kendrew,
supra;
Janis Kuby, Immunology, 1992 e.g., pp. 79-81. Some guidelines in determining
whether a protein will stimulate a response, include: Peptide length-
preferably the
peptide is about 8 or 9 amino acids long to fit into the MHC class I complex
and about
13-25 amino acids long to fit into a class II MHC complex. This length is a
minimum
for the peptide to bind to the MHC complex. It is preferred for the peptides
to be
longer than these lengths because cells may cut peptides. The peptide may
contain an
appropriate anchor motif which will enable it to bind to the various class I
or class II
molecules with high enough specificity to generate an immune response (See
Bocchia, M. et al, Specific Binding of Leukemia Oncogene Fusion Protein
Pentides to
HLA Class I Molecules, Blood 85:2680-2684; Englehard, VH, Structure of
peptides
associated with class I and class II MHC molecules Ann. Rev. Immunol. 12:181
(1994)). This can be done, without undue experimentation, by comparing the
sequence of the protein of interest with published structures of peptides
associated
with the MHC molecules. Thus, the skilled artisan can ascertain an epitope of
interest
by comparing the protein sequence with sequences listed in the protein data
base.
T CELL EPITOPES
Preferably one or more antigens of the present invention contain one or more T
cell
epitopes. As used herein, the term "T cell epitope" refers generally to those
features
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of a peptide structure which are capable of inducing a T cell response. In
this regard,
it is accepted in the art that T cell epitopes comprise linear peptide
determinants that
assume extended conformations within the peptide-binding cleft of MHC
molecules
(Unanue et al. (1987) Science 236: 551-557). As used herein, a T cell epitope
is
generally a peptide having at least about 3-5 amino acid residues, and
preferably at
least 5-10 or more amino acid residues. However, as used herein, the term "T
cell
epitope" encompasses any MHC Class I-or MHC Class II restricted peptide. The
ability of a particular T cell epitope to stimulate/enhance a CMI response may
be
determined by a number of well-known assays, such as by lyrnphoproliferation
(lymphocyte activation) assays, CD8+ T-cell cytotoxic cell assays, or by
assaying for
T-lymphocytes specific for the epitope in a sensitized subject. See, e. g.,
Erickson et
al. (1993) J. Immunol. 151: 4189-4199; and Doe et al. (1994) Eur. J. Immunol.
24:
2369-2376 or CD8+ T-cell ELISPOT assays for measuring Interferon gamma
production (Miyahara et al PNAS(USA) (1998) 95: 3954-3959).
CD8+ T-CELL EPITOPES
Preferably the antigens of the present invention comprisse CD8+ T-cell
inducing
epitopes. A CD8+ T-cell -inducing epitope is an epitope capable of stimulating
the
formation, or increasing the activity, of specific CD8+ T-cells following its
administration to a host subject. The CD8+ T-cell epitopes may be provided in
a
variety of different forms such as a recombinant string of one or two or more
epitopes.
CD8+ T-cell epitopes have been identified and can be found in the literature,
for
many different diseases. It is possible to design epitope strings to generate
CD8+ T-
cell response against any chosen antigen that contains such CD8+ T-cell
epitopes.
Advantageously, CD8+ T-cell inducing epitopes may be provided in a string of
multiple epitopes which are linked together without intervening sequences so
that
unnecessary nucleic acid material is avoided.
T HELPER EPITOPES
Preferably the antigens of the present invention comprise helper T lymphocyte
epitopes. Various methods are available to identify T helper cell epitopes
suitable for
use in accordance herewith. For example, the amphipathicity of a peptide
sequence is
known to effect its ability to function as a T helper cell inducer. A full
discussion of
T helper cell-inducing epitopes is given in U.S. Patent 5,128,319,
incorporated herein
by reference.
B CELL EPITOPES
Preferably the antigens of the present invention comprise a mixture of CD8+ T-
cell
epitopes and B cell epitopes. As used herein, the term "B cell epitope"
generally
refers to the site on an antigen to which a specific antibody molecule binds.
The
identification of epitopes which are able to elicit an antibody response is
readily
accomplished using techniques well known in the art. See, e. g., Geysen et al.
(1984)
Proc. Natl. Acad. Sci. USA 81: 3998-4002 (general method of rapidly
synthesizing
peptides to determine the location of immunogenic epitopes in a given
antigen); U. S.
Patent No. 4,708,871 (procedures for identifying and chemically synthesizing
epitopes of antigens); and Geysen et. al.(1986) Molecular hnmunology 23: 709-
715
(technique for identifying peptides with high affinity for a given antibody).
COMBINATION OF EPITOPES
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In a preferred embodiment of the present invention, the antigen or antigen
combination comprises a mixture of a CD8+ T-cell -inducing epitopes and a T
helper
cell-inducing epitopes.
As is well known in the art, T and B cell inducing epitopes are frequently
distinct
from each other and can comprise different peptide sequences. Therefore
certain
regions of a protein's peptide chain can possess either T cell or B cell
epitopes.
Therefore, in addition to the CD8+ T-cell epitopes, it may be preferable to
include one
or more epitopes recognised by T helper cells, to augment the immune response
generated by the CD8+ T-cell epitopes.
The mechanism of enhancing a CD8+ T-cell induced response ih vivo by T helper
cell
inducing agents is not completely clear. However, without being bound by
theory, it
is likely that the enhancing agent, by virtue of its ability to induce T
helper cells, will
result in increased levels of necessary cytokines that assist in the clonal
expansion and
dissemination of specific CD8+ T-cells. Regardless of the underlying
mechanism, it
is envisioned that the use of mixtures of helper T cell and CD8+ T-cell -
inducing
antigen combinations of the present invention will assist in the enhancement
of the
CMI response. Particularly suitable T helper cell epitopes are ones which are
active
in individuals of different HLA types, for example T helper epitopes from
tetanus
(against which most individuals will already be primed). It may also be useful
to
include B cell epitopes for stimulating B cell responses and antibody
production.
Synthetic nucleotide sequences may also be constructed to produce two types of
immune responses: T cell only and T cell combined with a B cell response.
IMMUNODOM1NANT EPITOPE
When an individual is immunized with an antigen or combination of antigens or
nucleotide sequence or combinations of nucleotide sequences encoding multiple
epitopes of a target antigen, in many instances the majority of responding T
lymphocytes will be specific for one or more linear epitopes from that target
antigen
and/or a majority of the responding B lymphocytes will be specific for one or
more
linear or conformational epitopes for the antigen or combination of antigens..
For the
purposes of the present invention, then, such epitopes are referred to as
"immunodominant epitopes". In an antigen having several immunodominant
epitopes, a single epitope may be the most dominant in terms of commanding a
specific T or B cell response.
As the Examples show, at least sixteen peptides of the present invention were
recognised by IFN-gamma positive CD8+ T cell populations which were actually
expanded as a result of bacterial infection.
ADJUVANTS
The compositions of the present invention may be administered in conjunction
with
other immunoregulatory agents. In particular, the compositions of the present
invention may be administered with an adjuvant.
The inclusion of an adjuvant and in particular, a genetic adjuvant may be
useful in
further enhancing or modulating the CMI response. An adjuvant may enhance the
CMI response by enhancing the immunogenicity of a co-administered antigen in
an
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immunized subject, as well inducing a Thl-like immune response against the co-
administered antigen which is beneficial in a vaccine product.
An immune response and particularly a CMI response may be refined, by the
addition
of adjuvants to combinations of antigens or nucleotide sequences encoding
combinations of antigens which lead to particularly effective compositions for
eliciting a long lived and sustained enhanced CMI response.
As used herein, the term "adjuvant" refers to any material or composition
capable of
specifically or non-specifically altering, enhancing, directing, redirecting,
potentiating
or initiating an antigen-specific immune response.
The term "adjuvant" includes but is not limited to a bacterial ADP-
ribosylating
exotoxin, a biologically active factor, immunomodulatory molecule, biological
response modifier or immunostimulatory molecule such as a cytokine, an
interleukin,
a chemokine or a ligand or an epitope (such as a helper T cell epitope) and
optimally
combinations thereof which, when administered with an antigen, antigen
composition
or nucleotide sequence encoding such antigens enhances or potentiates or
modulates
the CMI response relative to the CMI response generated upon administration of
the
antigen or combination of antigens alone. The adjuvant may be any adjuvant
known
in the art which is appropriate for human or animal use.
hnmunomodulatory molecules such as cytokines (TNF-alpha, IL-6, GM-CSF, and IL
2), and co-stimulatory and accessory molecules (B7-1, B7-2) may be used as
adjuvants in a variety of combinations. In one embodiment GM-CSF is not
administered to subject before, in or after the administration regimen.
Simultaneous
production of an immunomodulatory molecule and an antigen of interest at the
site of
expression of the antigen of interest may enhance the generation of specific
effectors
which may help to enhance the CMI response. The degree of enhancement of the
CMI response may be dependent upon the specific immunostimulatory molecules
and/or adjuvants used because different immunostimulatory molecules may elicit
different mechanisms for enhancing and/or modulating the CMI response. By way
of
example, the different effector mechanisms/immunomodulatory molecules include
but
are not limited to augmentation of help signal (IL-2), recruitment of
professional APC
(GM-CSF), increase in T cell frequency (IL-2), effect on antigen processing
pathway
and MHC expression (IFN-gamma and TNF-alpha) and diversion of immune
response away from the Thl response and towards a Th2 response (LTB) (see WO
97/02045). Unmethylated CpG containing oligonucleotides (see W096/02555) are
also preferential inducers of a Thl response and are suitable for use in the
present
invention.
Without being bound by theory, the inclusion of an adjuvant is advantageous
because
the adjuvant may help to enhance the CMI response to the expressed antigen by
diverting the Th2 response to a Thl response and/or specific effector
associated
mechanisms to an expressed epitope with the consequent generation and
maintenance
of an enhanced CMI response (see, for example, the teachings in WO 97/02045).
The inclusion of an adjuvant with an antigen or nucleotide sequence encoding
the
antigen is also advantageous because it may result in a lower dose or fewer
doses of the
antigen/antigenic combination being necessary to achieve the desired CMI
response in
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the subject to which the antigen or nucleotide sequence encoding the antigen
is
administered, or it may result in a qualitatively and/or quantitatively
different immune
response in the subject. The effectiveness of an adjuvant can be determined by
administering the adjuvant with the antigen in parallel with the antigen alone
to animals
and comparing antibody and/or cellular-mediated immunity in the two groups
using
standard assays such as radioimmunoassay, ELISAs, CD8+ T-cell assays, and the
like,
all well known in the art. Typically, the adjuvant is a separate moiety from
the antigen,
although a single molecule (such for example, CTB) can have both adjuvant and
antigen
properties.
As used herein, the term "genetic adjuvant" refers to an adjuvant encoded by a
nucleotide sequence and which, when administered with the antigen enhances the
CMI response relative to the CMI response generated upon administration of the
antigen alone.
Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be
used as
adjuvants in the invention. Preferably, the protein is derived from E. eoli
(i.e., E. coli
heat labile enterotoxin "LT), cholera ("CT"), or periussis ("PT").
In one preferred embodiment, the genetic adjuvant is a bacterial ADP-
ribosylating
exotoxin.
ADP-ribosylating bacterial toxins are a family of related bacterial exotoxins
and
include diphtheria toxin (DT), pertussis toxin (PT), cholera toxin (CT), the
E. coli
heat-labile toxins (LT1 and LT2), Pseudomoraas endotoxin A, Pseudornohas
exotoxin
S, B. cereus exoenzyme, B. splZaericus toxin, C. botulizzuna C2 and C3 toxins,
C.
lirnosurra exoenzyme, as well as toxins from C. perf °ifrge>zs, C.
spirifor~ma and C.
difficile, Staphylococcus aureus ED1N, and ADP-ribosylating bacterial toxin
mutants
such as CRMlg7, a non-toxic diphtheria toxin mutant (see, e.g., Bixler et al.
(1989)
Adv. Exp. Med. Biol. 251:175; and Constantino et al. (1992) Vaccine). Most ADP-
ribosylating bacterial toxins are organized as an A:B multimer, wherein the A
subunit
contains the ADP-ribosyltransferase activity, and the B subunit acts as the
binding
moiety. Preferred ADP-ribosylating bacterial toxins for use in the
compositions of
the present invention include cholera toxin and the E. coli heat-labile
toxins.
Cholera toxin (CT) and the related E. coli heat labile enterotoxins (LT) are
secretion
products of their respective enterotoxic bacterial strains that are potent
immunogens
and exhibit strong toxicity when administered systemically, orally, or
mucosally.
Both CT and LT are known to provide adjuvant effects for antigen when
administered
via the intramuscular or oral routes. These adjuvant effects have been
observed at
doses below that required for toxicity. The two toxins are extremely similar
molecules, and are at least about 70-80% homologous at the amino acid level.
Preferably the genetic adjuvant is cholera toxin (CT), enterotoxigenic E. Coli
heat-
labile toxin (LT), or a derivative, subunit, or fragment of CT or LT which
retains
adjuvanticity. In an even more preferred embodiment, the genetic adjuvant is
LT. In
another preferred embodiment, the genetic adjuvant may be CTB or LTB.
Preferably the entertoxin is a non-toxic enterotoxin.
The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is
described in
WO 95/17211 and as parenteral adjuvants in WO 98/42375. The toxin or toxoid is
64
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preferably in the form of a holotoxin, comprising both A and B subunits.
Preferably,
the A subunit contains a detoxifying mutation; preferably the B subunit is not
mutated. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-
R72, and LTR192G. The use of ADP-ribosylating toxins and detoxified derivaties
thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in the
following
references each of which is specifically incorporated by reference herein in
their
entirety (Beignon, et al. Infection and Immunity (2002) 70(6):3012 - 3019;
Pizza, et
al., Vaccine (2001) 19:2534 - 2541; Pizza, et al., Int. J. Med. Microbiol
(2000) 290(4-
5):455-461; Scharton-Kersten et al. Infection and Immunity (2000) 68(9):5306 -
5313; Ryan et al. Infection and Immunity (1999) 67(12):6270 - 6280; Partidos
et al.
Iminunol. Lett. (1999) 67(3):209 - 216; Peppoloni et al. Vaccines (2003)
2(2):285 -
293; and Pine et al J. Control Release (2002) 85(1-3):263 - 270). Numerical
reference for amino acid substitutions is preferably based on the alignments
of the A
and B subunits of ADP-ribosylating toxins set forth in Domenighini et al.,
Mol.
Microbiol (1995) 15(6):1165 - 1167, specifically incorporated herein by
reference in
its entirety.
By way of further example, at least one of the entertoxin subunit coding
regions may
be genetically modified to detoxify the subunit peptide encoded thereby, for
example
wherein the truncated A subunit coding region has been genetically modified to
disrupt or inactivate ADP-ribosyl transferase activity in the subunit peptide
expression
product (see, for example, WO 03/004055).
Thus, these results demonstrate that this genetic adjuvant is particularly
desirable
where an even more enhanced CMI response is desired. Other desirable genetic
adjuvants include but are not limited to nucleotide sequences encoding IL-10,
IL-12,
IL-13, the interferons (IFNs) (for example, IFN-alpha, IFN-ss, and IFN-gamma),
and
preferred combinations thereof. Still other such biologically active factors
that
enhance the CMI response may be readily selected by one of skill in the art,
and a
suitable plasmid vector containing same constructed by known techniques.
Preferred further adjuvants include, but are not limited to, one or more of
the
following set forth below:
Mihei°al Contaih.ihg Compositions
Mineral containing compositions suitable for use as adjuvants in the invention
include
mineral salts, such as aluminium salts and calcium salts. The invention
includes
mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g.
hydroxyphoshpates, orthophosphates), sulphates, etc. {e.g. see chapters 8 ~ 9
of ref.
Bush and Everett (2001) Int J Syst Evol Microbiol 51: 203-220), or mixtures of
different mineral compounds, with the compounds taking any suitable form (e.g.
gel,
crystalline, amorphous, etc.), and with adsorption being preferred. The
mineral
containing compositions may also be formulated as a particle of metal salt.
See WO
00/23105.
Aluminum salts may be included in immunogenic compositions and/or vaccines of
the invention such that the dose of Al3+ is between 0.2 and 1.0 mg per dose.
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
Preferably the adjuvant is alum, preferably an aluminium salt such as
aluminium
hydroxide (AIOH) or aluminium phospate or aluminium sulfate. Still more
preferably
the adjuvant is aluminium hydroxide (AIOH).
Preferably a mineral salt, such as an aluminium salt, is combined with and
another
adjuvant, such as an oligonucleotide containing a CpG motif or an ADP
ribosylating
toxin. Still more preferably, the mineral salt is combined with an
oligonucleotide
containing a CpG motif.
Oil-Emulsions
Oil-emulsion compositions suitable for use as adjuvants in the invention
include
squalene-water emulsions, such as MF59 (5% Squalene, 0.5% Tween 80, and 0.5%
Span 85, formulated into submicron particles using a microfluidizer). See
WO90/14837. See also, Frey et al., "Comparison of the safety, tolerability,
and
immunogenicity of a MF59-adjuvanted influenza vaccine and a non-adjuvanted
influenza vaccine in non-elderly adults", Vaccine (2003) 21:4234-4237. MF59 is
used
as the adjuvant in the FLUADTM influenza viru"s trivalent subunit vaccine.
Particularly preferred adjuvants for use in the compositions are submicron oil-
inwater
emulsions. Preferred submicron oil-in-water emulsions for use herein are
squalene/water emulsions optionally containing varying amounts of MTP-PE, such
as
a submicron oil-in-water emulsion containing 4-5% w/v squalene, 0.25-1.0% w/v
Tween 80 T"" (polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0% Span
85T"~
(sorbitan trioleate), and, optionally, N-acetylmuramyl-L-alanyl-D-
isogluatminyl-L-
alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-huydroxyphosphophoryloxy)-ethylamine
(MTP-PE), for example, the submicron oil-in-water emulsion known as "MF59"
(International Publication No. WO90/14837; US Patent Nos. 6,299,884 and
6,451,325, incorporated herein by reference in their entireties; and Ott et
al., "MF59 --
Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines" in
Vaccine Design: The Subunit and Adjuvant Approach (Powell, M.F. and Newman,
M.J. eds.) Plenum Press, New York, 1995, pp. 277-296). MF59 contains 4-5% w/v
Squalene (e.g. 4.3%), 0.25-0.5% w/v Tween 80T"~, and 0.5% w/v Span 85T"~ and
optionally contains various amounts of MTP-PE, formulated into submicron
particles
using a microfluidizer such as Model 110Y microfluidizer (Microfluidics,
Newton,
MA). For example, MTP-PE may be present in an amount of about 0-500 ~g/dose,
more preferably 0-250 ~g/dose and most preferably, 0-100 ~,g/dose. As used
herein,
the term "MF59-0" refers to the above submicron oil-in-water emulsion lacking
MTP-
PE, while the term MF59-MTP denotes a formulation that contains MTP-PE. For
instance, "MF59-100" contains 100 ~,g MTP-PE per dose, and so on. MF69,
another
submicron oil-in-water emulsion for use herein, contains 4.3% w/v squalene,
0.25%
w/v Tween 80T"~, and 0.75% w/v Span 85TM and optionally MTP-PE. Yet another
submicron oil-in-water emulsion is MF75, also known as SAF, containing 10%
squalene, 0.4% Tween 80T"~, 5% pluronic-blocked polymer L121, and thr-MDP,
also
microfluidized into a submicron emulsion. MF75-MTP denotes an MF75 formulation
that includes MTP, such as from 100-400 ~,g MTP-PE per dose.
Submicron oil-in-water emulsions, methods of making the same and
immunostimulating agents, such as muramyl peptides, for use in the
compositions, are
described in detail in International Publication No. W090/14837 and US Patent
Nos.
6,299,884 and 6,45 1,325, incorporated herein by reference in their
entireties.
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Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may
also
be used as adjuvants in the invention.
Saporaih Fof°mulatioyas
Saponin formulations, may also be used as adjuvants in the invention. Saponins
are a
heterologous group of sterol glycosides and triterpenoid glycosides that are
found in
the bark, leaves, stems, roots and even flowers of a wide range of plant
species.
Saponin from the bark of the Quillaia saponaria Molina tree have been widely
studied as adjuvants. Saponin can also be commercially obtained from Smilax
orhata
(sarsaprilla), Gypsophilla paniculata (brides veil), and Sapoharia
officianalis (soap
root). Saponin adjuvant formulations include purified formulations, such as
QS21, as
well as lipid formulations, such as ISCOMs. Saponin compositions have been
purified using High Performance Thin Layer Chromatography (HP-LC) and Reversed
Phase High Performance Liquid Chromatography (RP-HPLC). Specific purified
fractions using these techniques have been identified, including QS7, QS17,
QS18,
QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method of
production of QS21 is disclosed in U.S. Patent No. 5,057,540. Saponin
formulations
may also comprise a sterol, such as cholesterol (see WO 96/33739).
Combinations of saponins and cholesterols can be used to form unique particles
called
hnmunostimulating Complexs (ISCOMs). ISCOMs typically also include a
phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any
known
saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of
Quil A, QHA and QHC. ISCOMs are further described in EP 0 109 942, WO
96/11711 and WO 96/33739. Optionally, the ISCOMS may be devoid of additional
detergent. See WO 00/07621.
A review of the development of saponin based adjuvants can be found in Barr et
al
(1998) Advanced Drug Delivery Reviews 32: 247-271 and Sjolander et al (1998)
Advanced Drug Delivery Reviews (1998) 32: 321-338.
Virosof~aes ahd Virus Like Particles (VLPs)
Virosomes and Virus Like Particles (VLPs) can also be used as adjuvants in the
invention. These structures generally contain one or more proteins from a
virus
optionally combined or formulated with a phospholipid. They are generally non-
pathogenic, non-replicating and generally do not contain any of the native
viral
genome. The viral proteins may be recombinantly produced or isolated from
whole
viruses. These viral proteins suitable for use in virosomes or VLPs include
proteins
derived from influenza virus (such as HA or NA), Hepatitis B virus (such as
core or
capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus,
Foot-and-
Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV,
RNA-
phages, Q13-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage,
and Ty
(such as retrotransposon Ty protein p1). VLPs are discussed fiuther in WO
03/024480, WO 03/024481; Niikura et al Virology (2002) 293:273 - 280; Lenz et
al
Journal of Immunology (2001) 5246 - 5355; Pinto, et al Journal of Infectious
Diseases (2003) 188:327 - 338; and Gerber et al Journal of Virology (2001)
75(10):4752 - 47601; Virosomes are discussed further in, for example, Gluck et
al
Vaccine (2002) 20:B 10 -B 16.
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WO 2005/084306 PCT/US2005/006588
Bacterial or Microbial Derivatives
Adjuvants suitable for use in the invention include bacterial or microbial
derivatives
such as:
Nor-toxic derivatives of ehterobacterial lipopolysacclaaride (LPS)
Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylated MPL
(3dMPL). 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4,
5
or 6 acylated chains. A preferred "small particle" form of 3 De-O-acylated
monophosphoryl lipid A is disclosed in EP 0 689 454. Such "small particles" of
3dMPL are small enough to be sterile filtered through a 0.22 micron membrane
(see
EP 0 689 454). Other non-toxic LPS derivatives include monophosphoryl lipid A
mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529.
See
Johnson et al. (1999) Bioofg Med Chem Lett 9:2273-2278.
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WO 2005/084306 PCT/US2005/006588
Lipid A Derivatives
Lipid A derivatives include derivatives of lipid A from Escherichia coli such
as OM-
174. OM-174 is described for example in Meraldi et al. Vaccine (2003) 21:2485 -
2491; Pajak, et al Vaccine (2003) 21:836 - 842.
Immunostirnulatory oligonucleotides
hnmunostimulatory oligonucleotides suitable for use as adjuvants in the
invention
include nucleotide sequences containing a CpG motif (a sequence containing an
unmethylated cytosine followed by guanosine and linked by a phosphate bond).
Bacterial double stranded RNA or oligonucleotides containing palindromic or
poly(dG) sequences have also been shown to be immunostimulatory.
The CpG's can include nucleotide modifications/analogs such as
phosphorothioate
modifications and can be double-stranded or single-stranded. Optionally, the
guanosine may be replaced with an analog such as 2'-deoxy-7-deazaguanosine.
See
Kandimalla, et al Nucleic Acids Research (2003) 31(9): 2393 - 2400; WO
02/26757
and WO 99/62923 for examples of possible analog substitutions. The adjuvant
effect
of CpG oligonucleotides is further discussed in Krieg Nature Medicine (2003)
9(7):
831 - 835; McCluskie, et al FEMS Immunology and Medical Microbiology (2002)
32:179 - 185; WO 98/40100, U.S. Patent No. 6,207,646, U.S. Patent No.
6,239,116,
and U. S. Patent No. 6,429,199.
The CpG sequence may be directed to TLR9, such as the motif GTCGTT or
TTCGTT. See Kalman et al (1999) (Nature Genetics 21: 385-389). The CpG
sequence may be specific for inducing a Thl immune response, such as a CpG-A
ODN, or it may be more specific for inducing a B cell response, such a CpG-B
ODN.
CpG-A and CpG-B ODNs are discussed in Blackwell, et al J. Immunol. (2003)
170(8):4061 - 4068; Krieg BBRC (2003) 306:948 - 953; and WO 01/95935.
Preferably, the CpG is a CpG-A ODN.
Preferably, the CpG oligonucleotide is constructed so that the 5' end is
accessible for
receptor recognition. Optionally, two CpG oligonucleotide sequences may be
attached
at their 3' ends to form "immunomers". See, for example, Kandimalla, et al
(2003)
31(part 3):664 - 658; Bhagat et al BBRC (2003) 300:853 - 861 and WO 03/035836.
Preferably the adjuvant is CpG. Even more preferably, the adjuvant is Alum and
an
oligonucleotide containg a CpG motif or AIOH and an oligonucleotide containing
a
CpG motif.
Human Inamunomodulato~s
Human immunomodulators suitable for use as adjuvants in the invention include
cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-
12, etc.),
interferons (e.g. interferon-y), macrophage colony stimulating factor, and
tumor
necrosis factor.
ADP-f~ibosylating toxins arad detoxified derivatives thef~eof.
Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be
used as
adjuvants in the invention. Preferably, the protein is derived from E. coli
(i.e., E. coli
heat labile enterotoxin "LT), cholera ("CT"), or periussis ("PT"). The use of
detoxified ADP-ribosylating toxins as mucosal adjuvants is described in
W095/17211
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WO 2005/084306 PCT/US2005/006588
and as parenteral adjuvants in W098/42375. Preferably, the adjuvant is a
detoxified
LT mutant such as LT-K63, LT-R72, and LTR192G. The use of ADP-ribosylating
toxins and detoxified derivaties thereof, particularly LT-K63 and LT-R72, as
adjuvants can be found in the following references, each of which is
specifically
incorporated by reference herein in their entirety: Beignon, et al., "The
LTR72 Mutant
of Heat-Labile Enterotoxin of Escherichia coli Enahnces the Ability of Peptide
Antigens to Elicit CD4+ T Cells and Secrete Gamma Interferon after
Coapplication
onto Bare Skin", Infection and Immunity (2002) 70(6):3012-3019; Pizza, et al.,
"Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants",
Vaccine (2001) 19:2534-2541; Pizza, et al., "LTK63 and LTR72, two mucosal
adjuvants ready for clinical trials" Int. J. Med. Microbiol (2000) 290(4-
5):455-461;
Scharton-Kersten et al., "Transcutaneous Immunization with-Bacterial ADP-
Ribosylating Exotoxins, Subunits and Unrelated Adjuvants", Infection and
Immunity
(2000) 68(9):5306-5313; Ryan et al., "Mutants of Escherichia coli Heat-Labile
Toxin
Act as Effective Mucosal Adjuvants for Nasal Delivery of an Acellular
Pertussis
Vaccine: Differential Effects of the Nontoxic AB Complex and Enzyme Activity
on
Thl and Th2 Cells" Infection and Immunity (1999) 67(12):6270-6280; Partidos et
al.,
"Heat-labile enterotoxin of Escherichia coli and -its site-directed mutant
LTK63
enhance the proliferative and cytotoxic T-cell responses to intranasally co-
immunized
synthetic peptides", Immunol. Lett. (1999) 67(3):209-216; Peppoloni et al.,
"Mutants
of the Escherichia coli heat-labile enterotoxin as safe and strong adjuvants
for
intranasal delivery of vaccines", Vaccines (2003) 2(2):285-293; and Pine et
al., (2002)
"Intranasal immunization with influenza vaccine and a detoxified mutant of
heat
labile enterotoxin from Escherichia coli (LTK63)" J. Control Release (2002)
85(1-
3):263-270. Numerical reference for amino acid substitutions is preferably
based on
the alignments of the A and B subunits of ADP-ribosylating toxins set forth in
Domenighini et al., Mol. Microbiol (1995) 15(6):1165-1167, specifically
incorporated
herein by reference in its entirety.
Preferably the adjuvant is an ADP-ribosylating toxin and an oligonucleotide
containing a CpG motif (see for example, WO 01/34185)
Preferably the adjuvant is a detoxified ADP-ribosylating toxin and an
oligonucleotide
containing a CpG motif.
Preferably the detoxified ADP-ribosylating toxin is LTK63 or LTK72.
Preferably the adjuvant is LTK63. Preferably the adjuvant is LTK72.
Preferably the adjuvant is LTK63 and an oligonucleotide containing a CpG
motif.
Preferably the adjuvant is LTK72 and an oligonucleotide containing a CpG
motif.
Bioadlzesives and Mucoadhesives
Bioadhesives and mucoadhesives may also be used as adjuvants in the invention.
Suitable bioadhesives include esterified hyaluronic acid microspheres (Singh
et. al.
(2001) J. Coht. Rele. 70:267-276) or mucoadhesives such as cross-linked
derivatives
of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,
polysaccharides and
carboxymethylcellulose. Chitosan and derivatives thereof may also be used as
adjuvants in the invention. See for example, W099/27960.
Microparticles
Microparticles may also be used as adjuvants in the invention. Microparticles
(i.e. a
particle of ~100nm to ~150~,m in diameter, more preferably ~200nm to ~30~,m in
diameter, and most preferably ~SOOnm to ~lOEun in diameter) formed from
materials
that are biodegradable and non-toxic (e.g. a poly(a-hydroxy acid), a
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a
polycaprolactone, etc.),
with poly(lactide-co-glycolide) are preferred, optionally treated to have a
negatively-
charged surface (e.g. with SDS) or a positively-charged surface (e.g. with a
cationic
detergent, such as CTAB).
Liposomes
Examples of liposome formulations suitable for use as adjuvants are described
in U.S.
Patent No. 6,090,406, U.S. Patent No. 5,916,588, and EP 0 626 169.
Polyoxyethyleyae ether and Polyoxyetlzylehe Estef~ Pof~mulatiohs
Adjuvants suitable for use in the invention include polyoxyethylene ethers and
polyoxyethylene esters (W099/52549). Such formulations further include
polyoxyethylene sorbitan ester surfactants in combination with an octoxynol
(WO01/21207) as well as polyoxyethylene alkyl ethers or ester surfactants in
combination with at least one additional non-ionic surfactant such as an
octoxynol
(W001/21152). Preferred polyoxyethylene ethers are selected from the following
group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl
ether,
polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether,
polyoxyethylene-35-
lauryl ether, and polyoxyethylene-23-lauryl ether.
Polyphosphazeue (PCPP)
PCPP formulations are described, for example, in Andrianov et al Biomaterials
(1998) 19(1 - 3):109 - 115; Payne et al Adv. Drug. Delivery Review (1998)
31(3):185 -196.
Muramyl peptides
Examples of muramyl peptides suitable for use as adjuvants in the invention
include
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-
alanyl-D-isoglutamine (nor-MDP), and N-acetyhnuramyl-z-alanyl-D-isoglutaminyl-
L-
alanine-2-(1'-2'-dipalmitoyl-sic-glycero-3-hydroxyphosphoryloxy)-ethylamine
MTP-
PE).
Imidazoquinolohe Compounds
Examples of imidazoquinolone compounds suitable for use adjuvants in the
invention
include Imiquamod and its homologues, described further in Stanley, "Imiquimod
and
the imidazoquinolones: mechanism of action and therapeutic potential" Clin Exp
Dermatol (2002) 27(7):571 - 577; and Jones, "Resiquimod 3M", Curr Opin
Investig
Drugs (2003) 4(2):214 - 218. The invention may also comprise combinations of
aspects of one or more of the adjuvants identified above. For example, the
following
adjuvant compositions may be used in the invention:
(1) a saponin and an oil-in-water emulsion (W099/11241);
(2) a saponin (e.g.., QS21) + a non-toxic LPS derivative (e.g., 3dMPL) (see WO
94/00153);
(3) a saponin (e.g.., QS21) + a non-toxic LPS derivative (e.g., 3dMPL) + a
cholesterol;
(4) a saponin (e.g. QS21) + 3dMPL + IL-12 (optionally + a sterol (W098/57659);
combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions
(European patent applications 0835318, 0735898 and 0761231).
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(5) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-block polymer
L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed
to
generate a larger particle size emulsion.
(6) Ribi adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene,
0.2% Tween 80, and one or more bacterial cell wall components from the group
consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell
wall skeleton (CWS), preferably MPL + CWS (DetoxTM); and
(7) one or more mineral salts (such as an aluminum salt) + a non-toxic
derivative of LPS
(such as 3dPML).
(7) combinations of 3dMPL with, for example, QS21 and/or oil-in-water
emulsions (See European patent applications 0835318, 0735898 and 0761231);
(8) one or more mineral salts (such as an aluminum salt) + a non-toxic
derivative
of LPS (such as 3dPML); and
(9) one or more mineral salts (such as an aluminum salt) + an
immunostimulatory
oligonucleotide (such as a nucleotide sequence including a CpG motif).
Aluminium salts and MF59 are preferred adjuvants for parenteral immunisation.
Mutant bacterial toxins are preferred mucosal adjuvants. Bacterial toxins and
bioadhesives are preferred adjuvants for use with mucosally-delivered
vaccines, such
as nasal vaccines.
The composition may include an antibiotic.
Preferably the compositions of the present invention are administered with'
alum
and/or CpG sequences.
Nucleic Acid
The antigens or epitopes of the present invention may be administered as
nucleotide
sequences encoding the antigens or epitopes. As used herein, the term
nucleotide
sequence refers to one of more nucleotide sequences which encode one or more
epitopes which are used in the compositions or combinations of the present
invention.
The term "nucleotide sequence (NOI)" is synonymous with the term
"polynucleotide"
or "nucleic acid". The NOI may be DNA or RNA of genomic or synthetic or of
recombinant origin. The NOI may be double-stranded or single-stranded whether
representing the sense or antisense strand or combinations thereof. For some
applications, preferably, the NOI is DNA. For some applications, preferably,
the NOI
is prepared by use of recombinant DNA techniques (e.g. recombinant DNA). For
some applications, preferably, the NOI is cDNA. For some applications,
preferably,
the NOI may be the same as the naturally occurring form.
The term "nucleic acid" includes DNA and RNA, and also their analogues, such
as
those containing modified backbones (e.g. phosphorothioates, etc.), and also
peptide
nucleic acids (PNA), etc. The invention includes nucleic acid comprising
sequences
complementary to those described above (e.g. for antisense or probing
purposes).
Nucleic acid according to the invention can be prepared in many ways (e.g. by
chemical synthesis, from genomic or cDNA libraries, from the organism itself,
etc.)
and can take various forms (e.g. single stranded, double stranded, vectors,
probes,
etc.). They are preferably prepared in substantially pure form (i.e.
substantially free
from other Chlarnydial or host cell nucleic acids).
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The invention provides a process for producing nucleic acid of the invention,
comprising the step of amplifying nucleic acid using a primer-based
amplification
method (e.g. PCR).
The invention provides a process for producing nucleic acid of the invention,
comprising the step of synthesising at least part of the nucleic acid by
chemical
means.
VECTOR
In one embodiment of the present invention, an antigen or antigenic
combination or
NOI encoding same is administered directly to a host subject. In another
embodiment
of the present invention, a vector comprising an NOI is administered to a host
subject.
Preferably the NOI is prepared and/or administered using a genetic vector. As
it is
well known in the art, a vector is a tool that allows or facihiates the
transfer of an
entity from one environment to another. In accordance with the present
invention, and
by way of example, some vectors used in recombinant DNA techniques allow
entities,
such as a segment of DNA (such as a heterologous DNA segment, such as a
heterologous cDNA segment), to be transferred into a host and/or a target cell
for the
purpose of replicating the vectors comprising the NOI of the present invention
and/or
expressing the antigens or epitopes of the present invention encoded by the
NOI.
Examples of vectors used in recombinant DNA techniques include but are not
limited
to phasmids, chromosomes, artificial chromosomes or viruses. The term "vector"
includes expression vectors and/or transformation vectors. The term
"expression
vector" means a construct capable of ih vivo or in vitrolex vivo expression.
The term
"transformation vector" means a construct capable of being transferred from
one
species to another.
NAKED DNA
The vectors comprising the NOI of the present invention may be administered
directly
as "a naked nucleic acid construct", preferably further comprising flanking
sequences
homologous to the host cell genome. As used herein, the term "naked DNA"
refers to
a plasmid comprising the NOI of the present invention together with a short
promoter
region to control its production. If is called "naked" DNA because the
phasmids are
not carried in any delivery vehicle. When such a DNA plasmid enters a host
cell, such
as a eukaryotic cell, the proteins it encodes are transcribed and translated
within the
cell.
VIRAL VECTORS
Alternatively, the vectors comprising the NOI of the present invention may be
introduced into suitable host cells using a variety of viral techniques which
are known in
the art, such as for example infection with recombinant viral vectors such as
retroviruses,
herpes simplex viruses and adenoviruses. The vector may be a recombinant viral
vectors. Suitable recombinant viral vectors include but are not limited to
adenovirus
vectors, adeno-associated viral (AAV) vectors, herpes-virus vectors, a
retroviral vector,
lentiviral vectors, bacuhoviral vectors, pox viral vectors or parvovirus
vectors (see
Kestler et al 1999 Human Gene Ther 10(10):1619-32). In the case of viral
vectors,
administration of the NOI is mediated by viral infection of a target cell.
TARGETED VECTOR
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The term "targeted vector" refers to a vector whose ability to infect or
transfect or
transduce a cell or to be expressed in a host and/or target cell is restricted
to certain cell
types within the host subject, usually cells having a common or similar
phenotype.
EXPRESSION VECTOR
Preferably, the NOI of the present invention which is inserted into a vector
is operably
linked to a control sequence that is capable of providing for the expression
of the
antigens or epitopes by the host cell, i.e. the vector is an expression
vector. The agent
produced by a host cell may be secreted or may be contained intracellularly
depending
on the NOI and/or the vector used. As will be understood by those of skill in
the art, .
expression vectors containing the NOI can be designed with signal sequences
which
direct secretion of the EOI through a particular prokaryotic or eukaryotic
cell membrane.
FUSION PROTEINS
The Chlamydia pneumoniae antigens used in the invention may be present in the
composition as individual separate polypeptides, but it is preferred that at
least two
(i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) of
the antigens are
expressed as a single polypeptide chain (a 'hybrid' polypeptide). Hybrid
polypeptides
offer two principal advantages: first, a polypeptide that may be unstable or
poorly
expressed on its own can be assisted by adding a suitable hybrid partner that
overcomes the problem; second, commercial manufacture is simplified as only
one
expression and purification need be employed in order to produce two
polypeptides
which are both antigenically useful.
The hybrid polypeptide may comprise two or more polypeptide sequences from the
first
antigen group. Accordingly, the invention includes a composition comprising a
first
amino acid sequence and a second amino acid sequence, wherein said first and
second
amino acid sequences are selected from a Clalamydia bactgerium, preferably a
Chlarnydia pneumoniae antigen or a fragment thereof of the first antigen
group.
Preferably, the first and second amino acid sequences in the hybrid
polypeptide comprise
different epitopes.
The hybrid polypeptide may comprise two or more polypeptide sequences from the
second antigen group. Accordingly, the invention includes a composition
comprising
a first amino acid sequence and a second amino acid sequence, wherein said
first and
second amino acid sequences are selected from a Chlamydia pneunaoniae antigen
or a
fragment thereof of the second antigen group. Preferably, the first and second
amino
acid sequences in the hybrid polypeptide comprise difference epitopes.
The hybrid polypeptide may comprise one or more polypeptide sequences from the
first
antigen group and one or more polypeptide sequences from the second antigen
group.
Accordingly, the invention includes a composition comprising a first amino
acid
sequence and a second amino acid sequence, said first amino acid sequence
selected
from a Chlanaydia pneumoniae antigen or a fragment thereof from the first
antigen group
and said second amino acid sequence selected from a Clalamydia bactgerium,
preferably
a Chlamydia pneumoniae antigen or a fragment thereof from the second antigen
group.
Preferably, the first and second amino acid sequences in the hybrid
polypeptide comprise
difference epitopes.
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The hybrid polypeptide may comprise one or more polypeptide sequences from the
first antigen group and one or more polypeptide sequences from the third
antigen
group or the fourth antigen group or the fifth antigen group or the sixth
antigen group
or the seventh antigen group or the eight antigen group or the ninth antigen
group or
the tenth antigen group. Accordingly, the invention includes a composition
comprising a first amino acid sequence and a second amino acid sequence, said
first
amino acid sequence selected from a Clalamydia pheumo~ciae antigen or a
fragment
thereof from the first antigen group and said second amino acid sequence
selected
from a Chlamydia pheumor~iae antigen or a fragment thereof from the third
antigen
group or the fourth antigen group or the fifth antigen group or the sixth
antigen group
or the seventh antigen group or the eight antigen group or the ninth antigen
group or
the tenth antigen group. Preferably, the first and second amino acid sequences
in the
hybrid polypeptide comprise difference epitopes.
The hybrid polypeptide may comprise one or more polypeptide sequences from the
second antigen group and one or more polypeptide sequences from the third
antigen
group or the fourth antigen group or the fifth antigen group or the sixth
antigen group
or the seventh antigen group or the eight antigen group or the ninth antigen
group or
the tenth antigen group. Accordingly, the invention includes a composition
comprising a first amino acid sequence and a second amino acid sequence, said
first
amino acid sequence selected from a Chlamydia p~eumoraiae antigen or a
fragment
thereof from the second antigen group and said second amino acid sequence
selected
from a Clalamydia pheufnoraiae antigen or a fragment thereof from the third
antigen
group or the fourth antigen group or the fifth antigen group or the sixth
antigen group
or the seventh antigen group or the eight antigen group or the ninth antigen
group or
the tenth antigen group. Preferably, the first and second amino acid sequences
in the
hybrid polypeptide comprise difference epitopes.
Hybrids consisting of amino acid sequences from two, three, four, five, six,
seven, eight,
nine, or ten Chlanaydia pheumohiae antigens are preferred. In particular,
hybrids
consisting of amino acid sequences from two, three, four, or five Clalamydia
pheumo~ziae
antigens are preferred. Different hybrid polypeptides may be mixed together in
a single
formulation. Within such combinations, a Clalamydia pfaeumohiae antigen may be
present in more than one hybrid polypeptide and/or as a non-hybrid
polypeptide. It is
preferred, however, that an antigen is present either as a hybrid or as a non-
hybrid, but
not as both.
Two-antigen hybrids for use in the invention may comprise any one of the
combinations disclosed above.
Hybrid polypeptides can be represented by the formula NHZ-A-{-X-L-~"-B-COOH,
wherein: X is an amino acid sequence of a Clalamydia pneufraoniae antigen or a
fragment thereof from the first antigen group, the second antigen group or the
third
antigen group or the fourth antigen group or the fifth antigen group or the
sixth
antigen group or the seventh antigen group or the eight antigen group or the
ninth
antigen group or the tenth antigen group.; L is an optional linker amino acid
sequence;
A is an optional N-terminal amino acid sequence; B is an optional C-terminal
amino
acid sequence; and h is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
If a -X- moiety has a leader peptide sequence in its wild-type form, this may
be
included or omitted in the hybrid protein. In some embodiments, the leader
peptides
will be deleted except for that of the -X- moiety located at the N-terminus of
the
hybrid protein i. e. the leader peptide of Xl will be retained, but the leader
peptides of
XZ ... X" will be omitted. This is equivalent to deleting all leader peptides
and using
the leader peptide of Xl as moiety -A-.
For each n instances of {-X-L-}, linker amino acid sequence -L- may be present
or
absent. For instance, when h=2 the hybrid may be NHZ-Xl-Ll-XZ-L2-COOH, NH2-Xl
XZ-COOH, NH2-Xl-Ll-XZ-COOH, NH2-XI-X2-L2-COOH, etc. Linker amino acid
sequence(s) -L- will typically be short (e.g. 20 or fewer amino acids i.e. 19,
18, 17, 16,
15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples comprise short
peptide
sequences which facilitate cloning, poly-glycine linkers (i. e. comprising
Gly" where fa =
2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (i.e. His" where fa =
3, 4, 5, 6, 7, 8, 9,
10 or more). Other suitable linker amino acid sequences will be apparent to
those skilled
in the art. A useful linker is GSGGGG (SEQ ID No 77), with the Gly-Ser
dipeptide
being formed from a BamHI restriction site, thus aiding cloning and
manipulation, and
the (Gly)4 tetrapeptide being a typical poly-glycine linker.
-A- is an optional N-terminal amino acid sequence. This will typically be
short (e.g.
40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28,
27, 26, 2S,
24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2, 1).
Examples include leader sequences to direct protein trafficking, or short
peptide
sequences which facilitate cloning or purification (e.g. histidine tags i.e.
His" where h
= 3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable N-terminal amino acid
sequences will
be apparent to those skilled in the art. If Xl lacks its own N-terminus
methionine, -A-
is preferably an oligopeptide (e.g. with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids)
which
provides a N-terminus methionine.
-B- is an optional C-terminal amino acid sequence. This will typically be
short (e.g.
or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27,
26, 25,
24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, S, 4,
3, 2, 1).
Examples include sequences to direct protein trafficking, short peptide
sequences
which facilitate cloning or purification (e.g. comprising histidine tags i.e.
His" where
35 h = 3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences which enhance protein
stability. Other
suitable C-terminal amino acid sequences will be apparent to those skilled in
the art.
Most preferably, fa is 2 or 3.
The invention also provides nucleic acid encoding hybrid polypeptides of the
invention.
40 Furthermore, the invention provides nucleic acid which can hybridise to
this nucleic
acid, preferably under "high stringency" conditions (e.g. 65°C in a
O.IxSSC, 0.5% SDS
solution).
The NOI of the present invention may be expressed as a fusion protein
comprising an
adjuvant and/or a biological response modifier and/or immunomodulator fused to
the
antigens or epitopes of the present invention to further enhance and/or
augment the CMI
response obtained. The biological response modifier may act as an adjuvant in
the sense
of providing a generalised stimulation of the CMI response. The antigens or
epitopes
may be attached to either the amino or carboxy terminus of the biological
response
modifier.
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METHODS OF MAKING
Polypeptides of the invention can be prepared by various means (e.g.
recombinant
expression, purification from cell culture, chemical synthesis, etc.) and in
various forms
(e.g. native, fusions, non-glycosylated, lipidated, etc.). They are preferably
prepared in
substantially pure form (i. e. substantially free from other Chlaznydial or
host cell
proteins).
The invention also provides a process for producing a polypeptide of the
invention,
comprising the step of culturing a host cell transformed with nucleic acid of
the
invention under conditions which induce polypeptide expression. The invention
provides a process for producing a polypeptide of the invention, comprising
the step
of synthesising at least part of the polypeptide by chemical means. The
invention
further provides a process for producing a composition according to the
invention
comprising the step of bringing one or more of SEQ IDs 1-86 into combination
with
one or more other of SEQ IDs 1-86
Strains
Preferred polypeptides of the invention comprise an amino acid sequence found
in
C.pneumoniae serovars, or in one or more of an epidemiologically prevalent
serotype.
Where hybrid polypeptides are used, the individual antigens within the hybrid
(i. e.
individual -X- moieties) may be from one or more strains. Where n=2, for
instance,
XZ may be from the same strain as Xl or from a different strain. Where n=3,
the
strains might be (i) Xl=Xz=X3 (ii) Xl=XZ~X3 (iii) X1~X2=X3 (iv) XI~Xz~X3 or
(v) Xl=X3~Xz, etc.
Heterologous host
Whilst expression of the polypeptides of the invention may take place in
Chlamydia,
the invention preferably utilises a heterologous host. The heterologous host
may be
prokaryotic (e.g. a bacterium) or eukaryotic. It is preferably E.coli, but
other suitable
hosts include Bacillus subtilis, Vibnio cholenae, Salmonella typlzi,
Salznozzella
typhiznuz°ium, lVeisse~ia lactaznica, Neisse>~ia cine>~ea,
Mycobactei"ia (e.g.
M. tuberculosis), yeasts, etc.
Details as to how the molecules which make up the SEQ IDs 1-86 can be produced
and
used can be found from the relevant international applications such as WO
00/37494,
WO 02/02606 and WO 03/049762 and WO 03/068811 and these details need not be
repeated here. Where the composition includes a protein that exists in
different nascent
and mature forms, the mature form of the protein is preferably used. For
example, the
mature form of the Clzlamydia pneunzoniae protein lacking the signal peptide
may be
used
ADMINISTRATION
Compositions of the invention will generally be administered directly to a
patient.
Direct delivery may be accomplished by parenteral injection (e.g.
subcutaneously,
intraperitoneally, intravenously, intramuscularly, or to the interstitial
space of a
tissue), or by rectal, oral (e.g. tablet, spray), vaginal, topical,
transdermal {e.g. see
W099/27961) or transcutaneous {e.g. WO02/074244 and W0021064162 intranasal
{e.g. see W003/028760) ocular, aural, pulmonary or other mucosal
administration.
The invention may be used to elicit systemic and/or mucosal immunity.
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The compositions of the present invention may be administered, either alone or
as part
of a composition, via a variety of different routes. Certain routes may be
favoured for
certain compositions, as resulting in the generation of a more effective
immune
response, prefereably a CMI response, or as being less likely to induce side
effects, or
as being easier for administration.
By way of example, the compositions of the present invention may be
administered
via a systemic route or a mucosal route or a transdermal route or it may be
administrered directly into a specific tissue. As used herein, the term
"systemic
administration" includes but is not limited to any parenteral routes of
administration.
In particular, parenteral administration includes but is not limited to
subcutaneous,
intraperitoneal, intravenous, intraarterial, intramuscular, or intrasternal
injection,
intravenous, intraarterial, or kidney dialytic infusion techniques.
Preferably, the
systemic, parenteral administration is intramuscular injection.
In one preferred embodiment of the method, the compositions of the present
invention
are administered via a transdermal route. While it is believed that any
accepted mode
and route of immunization can be employed and nevertheless achieve some
advantages in accordance herewith, the examples below demonstrate particular
advantages with transdermal NOI administration. In this regard, and without
being
bound by theory, it is believed that transdermal administration of a
composition may
be preferred because it more efficiently activates the cell mediated immune
(CMI)
arm of the immune system.
The term "transdermal" delivery intends intradermal (e.g., into the dermis or
epidermis), transdermal (e.g.,"percutaneous") and transmucosal administration,
i.e.,
delivery by passage of an agent into or through skin or mucosal tissue. See,
e.g.,
Trahsdermczl Drug Delivery: Developnzeratal Issues and Researcla Initiatives,
Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989); Controlled Drug
Delivery:
Furadamehtals a~.d Applications, Robinson and Lee (eds.), Marcel Dekker
Inc.,(1987);
and Ti°a~zsdermal Delivefy of Drugs, Vols. 1-3, Kydonieus and Berner
(eds.), CRC
Press, (1987). Thus, the term encompasses delivery of an agent using a
particle
delivery device (e.g., a needleless syringe) such as those described in U.S.
Patent No.
5,630,796, as well as delivery using particle-mediated delivery devices such
as those
described in U.S. Patent No. 5,865,796.
As used herein, the term "mucosal administration" includes but is not limited
to oral,
intranasal, intravaginal, intrarectal, intratracheal, intestinal and
ophthalmic
administration.
Mucosal routes, particularly intranasal, intratracheal, and ophthalmic are
preferred for
protection against natural exposure to environmental pathogens such as RSV,
flu virus
and cold viruses or to allergens such as grass and ragweed pollens and house
dust
mites. The enhancement of the immune response, preferably the CMI response
will
enhance the protective effect against a subsequently encountered target
antigen such
as an allergen or microbial agent.
In another preferred embodiment of the present invention, the compositions of
the
present invention may be administered to cells which have been isolated from
the host
subject. In this preferred embodiment, preferably the composition is
administered to
professional antigen presenting cells (APCs), such as dendritic cells. APCs
may be
derived from a host subject and modified ex vivo to express an antigen of
interest and
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WO 2005/084306 PCT/US2005/006588
then transferred back into the host subject to induce an enhanced CMI
response.
Dendritic cells are believed to be the most potent APCs for stimulating
enhanced CMI
responses because the expressed epitopes of the antigen of interest must be
acquired,
processed and presented by professional APCs to T cells (both Thl and Th2
helper
cells as well as CDS+ T-cells) in order to induce an enhanced CMI response.
PARTICLE ADMINISTRATION
Particle-mediated methods for delivering the compositions of the present
invention are
known in the art. Thus, once prepared and suitably purified, the above-
described
antigens or NOI encoding same can be coated onto core carrier particles using
a
variety of techniques known in the art. Carrier particles are selected from
materials
which have a suitable density in the range of particle sizes typically used
for
intracellular delivery from a gene gun device. The optimum carrier particle
size will,
of course, depend on the diameter of the target cells.
By "core carrier"" is meant a carrier on which a guest antigen or guest
nucleic acid
(e.g., DNA, RNA) is coated in order to impart a defined particle size as well
as a
sufficiently high density to achieve the momentum required for cell membrane
penetration, such that the guest molecule can be delivered using particle-
mediated
techniques (see, e.g., U.S. Patent No. 5,100,792). Core carriers typically
include
materials such as tungsten, gold, platinum, ferrite, polystyrene and latex.
See e.g.,
Paf°ticle Bonaba~dment. Technology fof° Gene Transfef°,
(1994) Yang, N. ed., Oxford
University Press, New York, NY pages 10-11. Tungsten and gold particles are
preferred. Tungsten particles are readily available in average sizes of 0.5 to
2.0
microns in diameter. Gold particles or microcrystalline gold (e. g., gold
powder
A1570, available from Engelhard Corp., East Newark, NJ) will also fmd use with
the
present invention. Gold particles provide uniformity in size (available from
Alpha
Chemicals in particle sizes of 1-3 microns, or available from Degussa, South
Plainfield, NJ in a range of particle sizes including 0.95 microns).
Microcrystalline
gold provides a diverse particle size distribution, typically in the range of
0.5-5
microns. However, the irregular surface area of microcrystalline gold provides
for
highly efficient coating with nucleic acids. A number of methods are known and
have
been described for coating or precipitating NOIs onto gold or tungsten
particles. Most
such methods generally combine a predetermined amount of gold or tungsten with
plasmid DNA, CaCl2 and spermidine. The resulting solution is vortexed
continually
during the coating procedure to ensure uniformity of the reaction mixture.
After
precipitation of the NOI, the coated particles can be transferred to suitable
membranes
and allowed to dry prior to use, coated onto surfaces of a sample module or
cassette,
or loaded into a delivery cassette for use in particular gene gun instruments.
The particle compositions or coated particles are administered to the
individual in a
manner compatible with the dosage formulation, and in an amount that will be
effective for the purposes of the invention. The amount of the composition to
be
delivered (e. g., about 0.1 mg to 1 mg, more preferably 1 to 50 ug of the
antigen or
allergen, depends on the individual to be tested. The exact amount necessary
will vary
depending on the age and general condition of the individual to be treated,
and an
appropriate effective amount can be readily determined by one of skill in the
art upon
reading the instant specification.
HOST MAMMALIAN SUBJECT
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As used herein, the term "host mammalian subject" means any member of the
subphylum cordata, including, without limitation, humans and other primates,
including non-human primates such as chimpanzees and other apes and monkey
species; farm animals such as cattle, sheep, pigs, goats and horses; domestic
mammals
such as dogs and cats; laboratory animals including rodents such as mice, rats
and
guinea pigs; birds, including domestic, wild and game birds such as chickens,
turkeys
and other gallinaceous birds, ducks, geese, and the like. The terms do not
denote a
particular age. Thus, both adult and newborn individuals are intended to be
covered.
The methods described herein are intended for use in any of the above
vertebrate
species, since the immune systems of all of these vertebrates operate
similarly. If a
mammal, the subject will preferably be a human, but may also be a domestic
livestock, laboratory subject or pet animal.
The mammal is preferably a human. Where the vaccine is for prophylactic use,
the
human is preferably a child (e.g. a toddler or infant) or a teenager; where
the vaccine
is for therapeutic use, the human is preferably a teenager or an adult. A
vaccilie
intended for children may also be administered to adults e.g. to assess
safety, dosage,
immunogenicity, etc.
PREVENT AND/OR TREAT
The invention also provides the use of the compositions of the invention in
the
manufacture of a medicament for raising an immune response in a mammal. The
medicament is preferably a vaccine and to the preparation of a vaccine to
prevent
and/or treat an disorder associated with a Chlafnydia bacterium. It is to be
appreciated that all references herein to treatment include curative,
palliative and
prophylactic treatment.
The administration of antigenic combinations of the present invention or a
composition comprising the NOI encoding the antigenic combinations may be for
either "prophylactic" or "therapeutic" purpose. As used herein, the term
"therapeutic"
or "treatment" includes any of following: the prevention of infection or
reinfection;
the reduction or elimination of symptoms; and the reduction or complete
elimination
of a pathogen. Treatment may be effected prophylactically (prior to infection)
or
therapeutically {following infection).
Prophylaxis or therapy includes but is not limited to eliciting an effective
immune
response, preferably a CMI immune response and/or alleviating, reducing,
curing or at
least partially arresting symptoms and/or complications resulting from a T
cell
mediated immune disorder. When provided prophylactically, the composition of
the
present invention is typically provided in advance of any symptom. The
prophylactic
administration of the composition of the present invention is to prevent or
ameliorate
any subsequent infection or disease. When provided therapeutically, the
composition
of the present invention is typically provided at (or shortly after) the onset
of a
symptom of infection or disease. Thus the composition of the present invention
may
be provided either prior to the anticipated exposure to a disease causing
agent or
disease state or after the initiation of an infection or disease.
Whether prophylactic or therapeutic administration (either alone or as part of
a
composition) is the more appropriate will usually depend upon the nature of
the
disease. By way of example, immunotherapeutic composition of the present
invention could be used in immunotherapy protocols to actively inducing
immunity
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
by vaccination. This latter form of treatment is advantageous because the
immunity is
prolonged. On the other hand a vaccine composition will preferably, though not
necessarily be used prophylactically to induce an effective CMI response
against
subsequently encountered antigens or portions thereof (such as epitopes)
related to the
target antigen.
These uses and methods are preferably for the prevention and/or treatment of a
disease
caused by a Clalamydia (e.g. trachoma, pelvic inflammatory disease,
epididymitis,
infant pneumonia, artherosclerosis, cardiovascular disease etc.). The
compositions
may also be effective against C.pneufnoniae.
PROPHYLACTICALLY OR THERAPEUTICALLY OR IMMUNOLOGICALLY
EFFECTIVE AMOUNT
The composition dose administrated to a host subject, in the context of the
present
invention, should be sufficient to effect a beneficial prophylactic or
therapeutic
immune response, preferably a CMI response in the subject over time.
The invention also provides a method for raising an immune response in a
mammal
comprising the step of administering an effective amount of a composition of
the
invention. The immune response is preferably protective and preferably
involves
antibodies and/or cell-mediated immunity. The method may raise a booster
response.
As used herein, the term ""prophylactically or therapeutically effective dose"
means a
dose in an amount sufficient to elicit an enhanced immune response, preferably
a CMI
response to one or more antigens or epitopes and/or to alleviate, reduce, cure
or at
least partially arrest symptoms and/or complications from a T cell mediated
immune
disorder.
Immunogenic compositions used as vaccines comprise an immuriologically
effective
amount of antigen(s), as well as any other components, as needed. By
'immunologically effective amount', it is meant that the administration of
that amount
to an individual, either in a single dose or as part of a series, is effective
for treatment
or prevention. This amount varies depending upon the health and physical
condition
of the individual to be treated, age, the taxonomic group of individual to be
treated
(e.g. non-human primate, primate, etc.), the capacity of the individual's
immune
system to synthesise antibodies, the degree of protection desired, the
formulation of
the vaccine, the treating doctor's assessment of the medical situation, and
other
relevant factors. It is expected that the amount will fall in a relatively
broad range that
can be determined through routine trials.
The mammal is preferably a human. Where the vaccine is for prophylactic use,
the
human is preferably a child (e.g. a toddler or infant) or a teenager or an
adult; where
the vaccine is for therapeutic use, the human is preferably a teenager or an
adult. A
vaccine intended for children may also be administered to adults e.g. to
assess safety,
dosage, immunogenicity, etc. Preferably, the human is a teenager. More
preferably,
the human is a pre-adolescent teenager. Even more preferably, the human is a
pre-
adolescent female or male Preferably the pre-adolescent male or female is
around 9-
12 years of age.
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One way of assessing the immunogenicity of the component proteins of the
immunogenic compositions of the present invention is to express the proteins
recombinantly and to screen patient sera or mucosal secretions by immunoblot
or by
protein or DNA microarray. A positive reaction between the protein and the
patient
serum indicates that the patient has previously mounted an immune response to
the
protein in question- that is, the protein is an immunogen. This method may
also be
used to identify immunodominant proteins.
One way of checking efficacy of therapeutic treatment involves monitoring
Chlamydia infection after administration of the composition of the invention.
One
way of checking efficacy of prophylactic treatment involves monitoring immune
responses against the ChlanZydia antigen, such as the Chlamydia pneumoniae
antigen
in the compositions of the invention after administration of the composition.
For
example, checking efficacy of prophylactic treatment may involve monitoring
immune responses both systemically (such as monitoring the level of IgGl and
IgG2a
production) and mucosally (such as monitoring the level of IgA production)
against
the Chlamydia praeumoniae antigens in the compositions of the invention after
administration of the composition. Typically, serum Chlamydia specific
antibody
responses are determined post-immunization but pre-challenge whereas mucosal
Chlamydia specific antibody body responses are determined post-immunization
and
post-challenge.
These uses and methods are preferably for the prevention and/or treatment of a
disease caused by Chlanaydia pneumoniae (e.g. pneumonia, bronchitis,
pharyngitis,
sinusitis, erythema nodosum, asthma, atherosclerosis, stroke, myocardial
infarctions,
coronary artery disease, etc.).
The vaccine compositions of the present invention can be evaluated in in vitro
and in
vivo animal models prior to host, e.g., human, administration. For example, in
vitro
neutralization by Peterson et al (1988) is suitable for testing vaccine
compositions
directed toward Clalamydia, preferably Chlamydia pneumoniae.
One example of such an in vitro test is described as follows. Hyper-immune
antisera
is diluted in PBS containing 5% guinea pig serum, as a complement source.
Chlamydia praeumoraiae .(104 IFU; inclusion forming units) are added to the
antisera
dilutions. The antigen-antibody mixtures are incubated at 37°C for 45
minutes and
inoculated into duplicate confluent Hep-2 or HeLa cell monolayers contained in
glass
vials (e.g., 15 by 45 mm), which have been washed twice with PBS prior to
inoculation. The monolayer cells are infected by centrifugation at 1000X g for
1 hour
followed by stationary incubation at 37°C fox 1 hour. Infected
monolayers are
incubated for 48 or 72 hours, fixed and stained with CIZlamydia specific
antibody,
such as anti-MOMP. Inclusion-bearing cells are counted in ten fields at a
magnification of 200X. Neutralization titer is assigned on the dilution that
gives 50%
inhibition as compared to control monolayers/IFU.
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The efficacy of immunogenic compositions can also be determined 'in vivo by
challenging animal models of Chlamydia pneumoniae infection, e.g., guinea pigs
or
mice, with the immunogenic compositions. The immunogenic compositions may or
may not be derived from the same serovars as the challenge serovars.
Preferably the
immunogenic compositions are derivable from the same serovars as the challenge
serovars. More preferably, The serovars of the present invention are
obtainable from
clinical isolates or from culture collections such as the American Tissue
Culture
Collection (ATCC).
In vivo efficacy models include but are not limited to: (i) A marine infection
model
using human Chlamydia pneumoniae serotypes; (ii) a marine disease model which
is
a marine model using a mouse-adapted Chlamydia pneumoniae strain, such as the
Chlamydia pneumoniae mouse pneumonitis (MoPn) strain also known as Chlamydia
muridarum; and (iii) a primate model using human Chlamydia pneumoniae
isolates.
The MoPn strain is a mouse pathogen while human Chlamydia pneumoniae serotypes
are human pathogens (see for example, Brunham et al (2000) J Infect Dis 181
(Suppl
3) 5538-5543; Murdin et al (2000) J Infect Dis 181 (Suppl 3) 5544-5551 and
Read et
al (2000) NAR 28(6); 1397-1406). As the Examples demonstrate, human Chlamydia
pneumoniae serotypes can be used in mouse models although they normally
require
high inocula or pretreatment with progesterone. Progesterone is generally used
because it seems to render the epithelium more susceptible to chlamydial
infection
(see Pal et al 2003 Vaccine 21: 1455-1465). One the other hand, MoPn, which
was
originally isolated from mouse tissues, is thought to be a natural marine
pathogen and
thus offers an evolutionarily adapted pathogen for analysis of host-pathogen
interactions. Although the MoPn serovar is thought to have a high degree of
DNA
homology to the human Chlamydia serovars, it may also have some unique
properties
(see for example, Pal et al (2002) Infection and Immunity 70(9); 4812-4817.
By way of example, in vivo vaccine compositions challenge studies can be
performed
in the marine model of Chlamydia pneumoniae (Morrison et al 1995). A
description
of one example of this type of approach is as follows. Female mice 7 to 12
weeks of
age receive 2.5 mg of depoprovera subcutaneously at 10 and 3 days before
vaginal
infection. Post-vaccination, mice are infected in the genital tract with 1,500
inclusion
forming units of Chlamydia pneumoniae contained in 5m1 of sucrose-phosphate
glutamate buffer, pH 7.4. The course of infection is monitored by determining
the
percentage of inclusion-bearing cells by indirect immunofluorescence with
Chlamydia
pneumoniae specific antisera, or by a Giemsa-stained smear from a scraping
from the
genital tract of an infected mouse. The presence of antibody titers in the
serum of a
mouse is determined by an enzyme-linked immunosorbent assay. The immunogenic
compositions of the present invention can be administered using a number of
different
immunization routes such as but not limited to infra-muscularly (i.m.), intra-
peritoneal (i.p.), infra-nasal (i.n.), sub-cutaneous (s.c) or transcutaneous
(t.c) routes.
Generally, any route of administration can be used provided that the desired
immune
response at the required mucosal surface or surfaces is achieved. Likewise,
the
challenge serovars may be administered by a number of different routes.
Typically,
the challenge serovars are administered mucosally, such as but not limited to
an intra-
nasal (i.n) challenge.
Alternative in-vivo efficacy models include guinea pig models. For example, in
vivo
vaccine composition challenge studies in the guinea pig model of Chlamydia
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pneumoniae infection can be performed. A description of one example of this
type of
approach follows. Female guinea pigs weighing 450 - 500 g are housed in an
environmentally controlled room with a 12 hour light-dark cycle and immunized
with
vaccine compositions via a variety of immunization routes. Post-vaccination,
guinea
pigs are infected in the genital tract with the agent of guinea pig inclusion
conjunctivitis (GPIC), which has been grown in HeLa or McCoy cells (Rank et
al.
(1988)). Each animal receives approximately 1.4x107 inclusion forming units
(IFU)
contained in 0.05 ml of sucrose-phosphate-glutamate buffer, pH 7.4 (Schacter,
1980).
The course of infection monitored by determining the percentage of inclusion-
bearing
cells by indirect immunofluorescence with GPIC specific antisera, or by Giemsa-
stained smear from a scraping from the genital tract (Rank et al 1988).
Antibody titers
in the serum is determined by an enzyme-linked immunosorbent assay.
Compositions of the invention will generally be administered directly to a
patient.
Direct delivery may be accomplished by parenteral injection (e.g.
subcutaneously,
intraperitoneally, intravenously, intramuscularly, or to the interstitial
space of a
tissue), or mucosally, such as by rectal, oral (e.g. tablet, spray), vaginal,
topical,
transdermal (See e.g. WO99/27961) or transcutaneous (See e.g. W002/074244 and
W002/064162), intranasal (See e.g. W003/028760), ocular, aural, pulinonary or
other mucosal administration.
DOSAGE
Prophylaxis or therapy can be accomplished by a single direct administration
at a
single time point or multiple time points. Administration can also be
delivered to a
single or to multiple sites. Some routes of administration, such as mucosal
administration via ophthalmic drops may require a higher dose. Those skilled
in the
art can adjust the dosage and concentration to suit the particular route of
delivery.
Dosage treatment can be a single dose schedule or a multiple dose schedule.
multiple
doses may be used in a primary immunisation schedule and/or in a booster
immunisation schedule. in a multiple dose schedule the various doses may be
given
by the same or different routes e.g. a parenteral prime and mucosal boost, a
mucosal
prime and parenteral boost, etc.
HOMOLOGUES
SEQ IDs 1-86 in the compositions of the invention may be supplemented or
substituted with molecules comprising sequences homologous (ie. sharing
sequence
identity) to SEQ ID Nos 1-86.
Proteins (including protein antigens) as used in the invention (as encoded by
the NOI)
may have homology and/or sequence identity with naturally occurring forms.
Similarly coding sequences capable of expressing such proteins will generally
have
homology and/or sequence identity with naturally occurring sequences.
Techniques
for determining nucleic acid and amino acid "sequence identity" also are known
in the
art. Typically, such techniques include determining the nucleotide sequence of
the
mRNA for a gene and/or determining the amino acid sequence encoded thereby,
and
comparing these sequences to a second nucleotide or amino acid sequence.
In general, "identity" refers to an exact nucleotide-to-nucleotide or amino
acid-to-
amino acid correspondence of two polynucleotides or polypeptide sequences,
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respectively. Two or more sequences (polynucleotide or amino acid) can be
compared
by determining their "percent identity." The percent identity of two
sequences,
whether nucleic acid or amino acid sequences, is the number of exact matches
between two aligned sequences divided by the length of the shorter sequences
and
multiplied by 100.
An approximate alignment for nucleic acid sequences is provided by the local
homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:
482-489 (1981). This algorithm can be applied to amino acid sequences by using
the
scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure,
M.
O. Dayhoff ed., 5 suppl. 3: 353-358, National Biomedical ResearchFoundation,
Washington, D. C., USA, and normalized by Gribskov, Nucl. AcidsRes. 14 (6):
6745-
6763 (1986). An exemplary implementation of this algorithm to deternline
percent
identity of a sequence is provided by the Genetics Computer Group (Madison,
Wl' in
the"BestFit"utility application. The default parameters for this method are
described
in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995)
(available from Genetics Computer Group,
Madison, WI). A preferred method of establishing percent identity in the
context of
the present invention is to use the MPSRCH package of programs copyrighted by
the
University of Edinburgh, developed by John F. Collins and Shane S.
Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From
this suite
of packages the Smith-Waterman algorithm can be employed where default
parameters are used for the scoring table (for example, gap open penalty of
12, gap
extension penalty of one, and a gap of six). From the data generated the
"Match"value
reflects"sequence identity." Other suitable programs for calculating the
percent
identity or similarity between sequences are generally known in the art, for
example,
another alignment program is BLAST, used with default parameters. For example,
BLASTN and BLASTP can be used using the following default parameters: genetic
code = standard; filter = none; strand = both; cutoff-- 60; expect = 10;
Matrix =
BLOSUM62 ; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases =
non-redundant, GenBank +EMBL + DDBJ + PDB + GenBank CDS translations +
Swiss protein + Spupdate + PIR. Details of these programs can be found at the
following Internet address: http://www. ncbi. nlm. gov/cgi-bin/BLAST.
Alternatively, homology can be determined by hybridization of polynucleotides
under
conditions which form stable duplexes between homologous regions, followed by
digestion with single-stranded-specific nuclease (s), and size determination
of the
digested fragments. Two DNA, or two polypeptide sequences are "substantially
homologous" to each other when the sequences exhibit at least about 80%-85%,
preferably at least about 90%, and most preferably at least about 95%-98%
sequence
identity over a defined length of the molecules, as determined using the
methods
above.
As used herein, substantially homologous or homologous also refers to
sequences
showing complete identity to the specified DNA or polypeptide sequence. DNA
sequences that are substantially homologous or homologous can be identified in
a
Southern hybridization experiment under, for example, stringent conditions, as
defined for that particular system. For example, stringent hybridization
conditions can
include 50% formamide, Sx Denhardt's Solution, Sx SSC, 0.1% SDS and 100 pg/ml
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
denatured salmon sperm DNA and the washing conditions can include 2x SSC, 0.1%
SDS at 37 C followed by lx SSC, 0.1% SDS at 68 C. Defining appropriate
hybridization conditions is within the skill of the art.
Preferably the degree of identity is preferably greater than 50% (eg. 65%.
80%. 90%.
or more) and include mutants and allelic variants. Sequence identity between
the
proteins is preferably determined by the Smith-Waterman homology search
algorithm
as implemented in the MPSRCH program (Oxford. Molecular). using an affme gap
search with parameters gap open penalty=12 and gap extension penalty=1.
15
SEQ IDs 1-86 in the compositions of the invention may be supplemented or
substituted with nucleic acid which can hybridise to the Chlamydia nucleic
acid.
preferably underv"high stringency"conditionsv(c. 65 C in an 0.1 x SSC, 0.5%
SDS
solution).
Ilypotlzetical P~~otein
As used herein, the term "hypothetical protein" refers to a protein which
lacks a
known cellular location or a known cellular function. Typically, a
hypothetical
protein lacks significant homologies with known well characterised proteins.
COMPOSITIONS
The invention also provides the compositions of the invention for use as
medicaments
(eg. as immunogenic compositions or vaccines) or as diagnostic reagents for
detecting
a Chylamydia infectioin in a host subject. It also provides the use of the
compositions
in the manufacture of: (i) a medicament for treating or preventing infection
due to
ClZlamydia praeumoniae bacteria: (ii) a diagnostic reagent for detecting the
presence of
Clalarnydia Pneumonaie bacteria or of antibodies raised against Chlamydia
Pneumonaie bacteria; and/or (iii) a reagent which can raise antibodies against
Chlamydia pneunaonaie bacteria.
The invention also provides a method of treating a patient, comprising
administering
to the patient a therapeutically effective amount of a composition according
to the
invention.
The present invention provides compositions that are useful for preventing
and/or
treating T cell mediated immune disorders. In one embodiment, the composition
is a
pharmaceutical composition. In another preferred embodiment, the composition
is an
immunotherapeutic composition. In an even more preferred embodiment, the
composition is a vaccine composition.. The composition may also comprise a
carrier
such as a pharmaceutically or immunologically acceptable carrier.
Pharmaceutically
acceptable carriers or immunologically acceptable carriers are determined in
part by
the particular composition being administered as well as by the particular
method
used to administer the composition. Accordingly, there is a wide variety of
suitable
formulations of pharmaceutical compositions or vaccine compositions or
immunotherapeutic compositions of the present invention.
Immunogeraic conapositions and medicaments
Compositions of the invention are preferably immunogenic compositions, and are
more preferably vaccine compositions. The pH of the composition is preferably
between 6 and 8, preferably about 7. The pH may be maintained by the use of a
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buffer. The composition may be sterile and/or pyrogen-free. The composition
may be
isotonic with respect to humans.
Vaccines according to the invention may either be prophylactic (i. e. to
prevent
infection) or therapeutic (i.e. to treat infection), but will typically be
prophylactic.
Accordingly, the invention includes a method for the therapeutic or
prophylactic
treatment of Chlamydia pneumoniae infection in an animal susceptible to
Chlanaydial
infection comprising administering to said animal a therapeutic or
prophylactic
amount of the immunogenic compositions of the invention. Preferably, the
immunogenic composition comprises a combination of Chlanaydia pneumoraiae
antigens, said combination selected from the group consisting of two, three,
four, five
or all six Chlamydia prreumoniae antigens of the first antigen group. Still
more
preferably, the combination consists of all six Chlanaydia pneumoniae antigens
of the
first antigen group.
Alternatively, the immunogenic composition comprises a combination of
Chlamydia
pneumoniae antigens, said combination selected from the group consisting of
two,
three, four, five, six, seven, eight, nine, ten, eleven, or twelve Chlarnydia
pneumofaiae
antigens selected from the first antigen group and the second antigen group.
Preferably, the combination is selected from the group consisting of three,
four, or
five Chlamydia pneumorriae antigens selected from the second antigen group.
Still
more preferably, the combination consists of five Chlamydia pneurnoniae
antigens
selected from the second antigen group.
Alternatively, the immunogenic composition comprises a combination of
Chlamydia
pneumorriae antigens, said combination consisting of two, three, four, or five
Chlamydia pr~eunaofziae antigens of the first antigen group and one, two,
three, four,
five or six Chlarnydia pneumoniae antigens of the third antigen group.
Preferably, the
combination consists of three, four or five Chlanaydia pneumoniae antigens of
the first
antigen group and one, two, three, four, five or six Chlamydia pneunaoniae
antigens of
the third antigen group.
Alternatively, the immunigenic composition comprises a combination of
Chlamydia
pneumoyriae antigens, said combination consisting of two, three, four, five,
six, seven,
eight, nine, ten, eleven or twelve Cl2lamydia pneunaoniae antigens of the
first antigen
group and the second antigen group and one, two, three, four, five or six
Chlanrydia
pneurnoniae antigens of the third antigen group. Preferably, the combination
is
selected from the group consisting of three, four, or five Chlamydia
prreurnoniae
antigens from the second antigen group and three, four or five Cl2larnydia
pyreurnoniae
from the third antigen group. Still more preferably, the combination consists
of five
Chlarnydia pneumoniae antigens from the second antigen group and three, four
or five
Clalamydia pneumoniae antigens of the third antigen group.
In certain embodiments. the composition comprises molecules from different
Chlarraydia species. In some embodiments. the composition may comprise
molecules
from different serogroups and/or strains of the same Clalanaydia species.
Further
embodiments comprise mixtures of one or more Chlamydia molecules from
different
strains.
Many proteins are relatively conserved between different species serogroups
and
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strains of Chlamydia trachomatis and Clalamydia praeurnoniae. To ensure
maximum
cross-strain recognition and reactivity. regions of proteins that are
conserved between
different Chlamydia species, serogroups and strains can be used in the
compositions
of the present invention. The invention therefore provides proteins which
comprise
stretches of amino acid sequence that are shared across the majority of
Chlamydia
strains. Preferably, therefore, the composition comprises a protein comprising
a
fragment of a Clalanaydia pneumoniae protein (preferably a protein from SEQ ID
Nos
1-86 or more preferably SEQ ID Nos 1-41 wherein said fragment consists of n
consecutive conserved amino acids.
Further antigens
The compositions of the invention may further comprise antigen derived from
one or
more sexually transmitted diseases in addition to Chlamydia trachomatis.
Preferably
the antigen is derived from one or more of the following sexually transmitted
diseases: N.gonorrhoeae {e.g. i, ii, iii, iv}; human papiloma virus;
Treponenaa
pallidunZ; herpes simplex virus (HSV-1 or HSV-2); HIV (HIV-1 or HIV-2); and
Haemophilus ducreyi.
A preferred composition comprises: (1) at least t of the Chlamydia pneumoniae
antigens from either the first antigen group or the second antigen group,
where t is 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, preferably t is five; (2) one or more
antigens from
another sexually transmitted disease. Preferably, the sexually transmitted
disease is
selected from the group consisting of herpes simplex virus, preferably HSV-1
and/or
HSV-2; human papillomavirus; N.gonorrlZOeae; Treponerna pallidum; and
Haemophilus ducreyi. These compositions can thus provide protection against
the
following sexually-transmitted diseases: Chlamydia, genital herpes, genital
warts,
gonorrhoea, syphilis and chancroid (see Stephens et al (1998) Science 282: 754-
759).
Where a saccharide or carbohydrate antigen is used, it is preferably
conjugated to a
carrier ~ protein i11 order to enhance immunogenicity (For example, Ramsay et
al.
(2001) Lancet 357(9251):195-196; Lindberg (1999) Yaccirae 17 Suppl 2:528-36;
Buttery & Moxon (2000) .l R Coll Playsicians Lond 34:163-168; Ahmad & Chapnick
(1999) Infect Dis Clira North Am 13:113-133; Goldblatt (1998) J. Med.
Microbiol.
47:563-567; European patent 0 477 508; US Patent No. 5,306,492; International
patent application WO98/42721; Conjugate Vaccines (eds. Cruse et. al.) ISBN
3805549326, particularly vol. 10:48-114; and Hermanson (1996) Bioconjugate
Techniques ISBN: 0123423368 or 012342335).
Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria
or tetanus
toxoids. The CRM19~ diphtheria toxoid is particularly preferred (Research
Disclosure,
453077 (Jan 2002). Other carrier polypeptides include the N.meningitidis outer
membrane protein EP-A-0372501), synthetic peptides (EP-A-0378881, EP
A-0427347), heat shock proteins (W093/17712, W094/03208) pertussis proteins
(W098/58668, EP-A-0471177) protein D from H.irafluenzae (W000/56360)
cytokines (W091/01146), lymphokines, hormones, growth factors, toxin A or B
from
C.difficile (W000/61761) iron-uptake proteins WO01/72337) etc. Where a mixture
comprises capsular saccharides from both serogroups A and C, it may be
preferred
that the ratio (w/w) of MenA saccharide:MenC saccharide is greater than 1
(e.g. 2:1,
3:1, 4:1, 5:1, 10:1 or higher). Different saccharides can be conjugated to the
same or
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different type of carrier protein. Any suitable conjugation reaction can be
used, with
any suitable linker where necessary.
Toxic protein antigens may be detoxified where necessary e.g. detoxification
of
pertussis toxin by chemical and/or genetic means. Where a diphtheria antigen
is
included in the composition it is preferred also to include tetanus antigen
and periussis
antigens. Similarly, where a tetanus antigen is included it is preferred also
to include
diphtheria and pertussis antigens. Similarly, where a pertussis antigen is
included it is
preferred also to include diphtheria and tetanus antigens.
Antigens in the composition will typically be present at a concentration of at
least
1 p,g/ml each. In general, the concentration of any given antigen will be
sufficient to
elicit an immune response against that antigen. As an alternative to using
protein
antigens in the composition of the invention, nucleic acid encoding the
antigen may
be used Robinson & Torres (1997) Semiyaars in Inamuyaology 9:271-283; Donnelly
et
al. (1997) Ahhu Rev Immuhol 15:617-648; Scott-Taylor & Dalgleish (2000) Expert
Opiu Ihvestig Drugs 9:471-480; Apostolopoulos ~ Plebanski (2000) Cum°
Opih Mol
Ther 2:441-447; Ilan (1999) Curr Opin Mol Ther 1:116-120; Dubensky et al.
(2000)
Mol Med 6:723-732; Robinson & Pertmer (2000) Adv hirus Res 55:1-74; Donnelly
et
al. (2000) Am J Respir Cf°it Care Med 162(4 Pt 2):5190-193 and Davis
(1999) Mt.
Sinai J. Med. 66:84-90. Protein components of the compositions of the
invention may
thus be replaced by nucleic acid (preferably DNA e.g. in the form of a
plasmid) that
encodes the protein.
DISEASE STATES
The compositions of the present invention may be used to prevent and/or treat
disorders such as but not limited to: pneumonia, cardiovascular diseases,
atherosclerosis, bronchitis, pharyngitis, laryngitis, sinusitis, obstructive
lung diseases,
asthma, chronic obstructive pulmonary disease, reactive arthritis, otitis
media,
abdominal aortic aneurysm, erythema nodosum, Reiter syndrome, sarcoidosis,
Alzheimer's disease, multiple sclerosis, lymphogranuloma venereum, ocular
trachoma,
pelvic inflammatory disease, inclusion conjunctivitis, genital trachoma,
infant
pneumonitis, incipient trachoma, keratitis, papillary hypertrophy, corneal
infiltration,
vulvovaginitis, mucopurulent rhinitis, salpingitis, cervicitis, cervical
follicles,
prostatitis, proctitis, urethritis, lymphogranule inguinale, climatic bubo,
tropical bubo,
and/oresthiomene.
FORMULATIONS
Chlamydial infections affect various areas of the body and so the compositions
of the
invention may be prepared in various forms. For example, the compositions may
be
prepared as injectables, either as liquid solutions or suspensions. Solid
forms suitable
for solution in, or suspension in, liquid vehicles prior to injection can also
be prepared
(e.g. a lyophilised composition). The composition may be prepared for topical
administration e.g. as an ointment, cream or powder. The composition may be
prepared for oral administration e.g. as a tablet or capsule, as a spray, or
as a syrup
(optionally flavoured). The composition may be prepared for pulmonary
administration e.g. as an inhaler, using a fine powder or a spray. The
composition may
be prepared as a suppository or pessary. The composition may be prepared for
nasal,
aural or ocular administration e.g. as drops. The composition may be in kit
form,
designed such that a combined composition is reconstituted just prior to
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administration to a patient. Such kits may comprise one or more antigens in
liquid
form and one or more lyophilised antigens.
Further components of the compositiota
The composition of the invention will typically, in addition to the components
mentioned above, comprise one or more 'pharmaceutically acceptable Garners',
which
include any carrier that does not itself induce the production of antibodies
harmful to
the individual receiving the composition. Suitable carriers are typically
large, slowly
metabolised macromolecules such as proteins, polysaccharides, polylactic
acids,
polyglycolic acids, polymeric amino acids, amino acid copolymers, and lipid
aggregates (such as oil droplets or liposomes). Such carriers are well known
to those
of ordinary skill in the art. The vaccines may also contain diluents, such as
water,
saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or
emulsifying agents, pH buffering substances, and the like, may be present. A
thorough
discussion of pharmaceutically acceptable excipients is available in Gemiaro
(2000)
Remiyigtoh: The ScieTZCe afZd Practice of Pharmacy. 20th ed., ISBN:
0683306472.
The biological molecules of the present invention be formulated into a
pharmaceutical
composition or an immunotherapeutic composition or a vaccine composition. Such
formulations comprise biological molecules combined with a pharmaceutically
acceptable carrier, such as sterile water or sterile isotonic saline. Such
formulations
may be prepared, packaged, or sold in a form suitable for bolus administration
or for
continuous administration. Injectable formulations may be prepared, packaged,
or sold
in unit dosage form, such as in ampoules or in mufti-dose containers
containing a
preservative. Formulations include, but are not limited to, suspensions,
solutions,
emulsions in oily or aqueous vehicles, pastes, and implantable sustained-
release or
bi~degradable formulations. Such formulations may further comprise one or more
additional ingredients including, but not limited to, suspending, stabilizing,
or
dispersing agents. In one embodiment of a formulation for parenteral
administration,'
the active ingredient is provided in dry (for eg, a powder or granules) .form
for
reconstitution with a suitable vehicle (e. g., sterile pyrogen-free water)
prior to
parenteral administration of the reconstituted composition.The pharmaceutical
compositions may be prepared, packaged, or sold in the form of a sterile
injectable
aqueous or oily suspension or solution. This suspension or solution may be
formulated
according to the known art, and may comprise, in addition to the active
ingredient,
additional ingredients such as the dispersing agents, wetting agents, or
suspending
agents described herein. Such sterile inj ectable formulations may be prepared
using a
non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-
butane diol,
for example. Other acceptable diluents and solvents include, but are not
limited to,
Ringer's solution, isotonic sodium chloride solution, and fixed oils such as
synthetic
mono-or di-glycerides. Other parentally-administrable formulations which are
useful
include those which comprise the active ingredient in microcrystalline form,
in a
liposomal preparation, or as a component of a biodegradable polymer systems.
Compositions for sustained release or implantation may comprise
pharmaceutically
acceptable polymeric or hydrophobic materials such as an emulsion, an ion
exchange
resin, a sparingly soluble polymer, or a sparingly soluble salt.
KITS
Also included in the invention is a kit for enhancing a CMI response to the
biological
molecules of the present invention. Such a kit may comprise an antigenic
composition
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
or nucleotide sequence encoding same. The kit may also include an adjuvant,
preferably a genetic adjuvant is administered with or as part of the
biological molecule
and instructions for administering the biological molecule. Other preferred
components of the kit include an applicator for administering the biological
molecule.
As used herein, the term "applicator" refers to any device including but not
limited to a
hypodermic syringe, gene gun, particle acceleration device, nebulizer,
dropper,
bronchoscope, suppository, impregnated or coated vaginally-insertable material
such
as a tampon, douche preparation, solution for vaginal irrigation, retention
enema
preparation, suppository, or solution for rectal or colonic irrigation for
applying the
NOI either systemically or mucosally or transdermally to the host subject.
The invention also provides for a kit comprising comprising a combination of
Chlamydia pneumofZiae antigens. The combination of Clalamydia pyaeumoyaiae
antigens may be one or more of the immunogenic compositions of the invention.
The
kit may further include a second component comprising one or more of the
following:
instructions, syringe or other delivery device, adjuvant, or pharmaceutically
acceptable formulating solution. The invention also provides a delivery device
pre-
filled with the immunogenic compositions of the invention.
EXAMPLES
The following invention will now be further described only by way of example
in
which reference is made to the following Figures. The following examples are
presented only to illustrate the present invention and to assist one of
ordinary skill in
making and using the same. The examples are not intended in any way to
otherwise
limit the scope of the invention. Efforts have been made to ensure accuracy
with
respect to numbers used (e.g., amounts, temperatures, etc.), but some
experimental
error and deviation should, of course, be allowed for.
Figure 1A. Assay of iya vitr~ neutralization of C.ph.eunzoyaiae infectivity
for LLC-MK2
cells by polyclonal mouse antisera to recombinant Chlamydial proteins. Results
are
shown as reduction in the number of inclusions obtained when monolayers were
infected
with antiserum-treated infectious EBs, as compared to inclusion numbers given
by
untreated EBs. Percent reduction values are plotted against the reciprocal of
the
corresponding serum dilution. For each dilution inclusion counts were
corrected for
background inhibition of infectivity observed with the corresponding dilution
of the pre-
immune serum. The figure shows results obtained with serial dilutions of
antibodies
raised against a 'neutralizing' antigen (~), a 'non-neutralizing' FACS-
positive antigen
(v), and against the GST polypeptide, used in the fusion constructs, alone
(6).
Figure 1B shows serum titres giving 50% neutralization of infectivity for the
10
C.pneumo~aiae recombinant antigens described in the text. Each titer was
assessed in 3
separate experiments (SEM values shown).
Figure 2 shows immunoblot analysis of two dimensional electrophoretic maps of
C.praeumoniae EBs using the imune sera described in the text. Immunoblots were
obtained from either of two EB gels (panels A and B at the top) covering
different pH
intervals, according to which of the two allowed the best detection of a given
antigen.
The arrows in the HtrA immunoblot show which of the signals had a
corresponding
stained spot in the gel (arrows in panel A) which was subjected to MALDI-TOF
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identification. The two patterns in the HtrA blot are both suggestive of
typical
electrophoretic 'trains' composed of single charge variants of the same
protein.
Figure 3 shows mean numbers of C.pheurnohiae IFLT recovered from equivalent
spleen samples from immunized and mock-immunized hamsters following a systemic
challenge. Standard deviation values are shown above the bars. Antigens which
induced significant protection are highlighted with an asterisk above the
corresponding bar. All antigens were were delivered in Freund's adjuvant. n.i.
= non
immunized controls
Figure 4 shows flow cytometric analysis of splenocytes from DNA-immunized HLA-
A2 transgenic and non transgenic mice. Groups of 4 mice were immunized 3 times
i.m. with 50~g of plasmid DNA expressing C. pheurnofZiae Low Calcium Response
Protein H. IFN-y production from splenocytes was monitored following either a
6h
(ex-vivo) or a 6 day (restimulated) pulse with peptide CH-6 (10~g/ml). Equal
numbers of gated live lymphocyte cells were acquired with a LSRII FACS System
(Becton Dickinson) and percentages of IFN-y producing CD8+ T cells were
calculated
using DIVA Software (Becton Dickinson).
Figure 5 shows a flow cytometric analysis of splenocytes from transgenic and
non
transgenic mice infected with C. pneumoniae EBs. (A) HLA-A2 transgenic mice
were intranasally infected twice with 5x105 C. praeumohiae FB/96 EBs and
splenocytes were stimulated for 6 days in the presence of relevant peptides
before
determining IFN-y production by CD8+ T cells as described in the legend of
Figure 4.
(B) HLA-A2 transgenic and non transgenic mice were infected together with the
same
EBs preparation and CD8+ T cells were subjected to FRCS analysis as reported
in
(A).
Table I shows a summary of data and properties of the C.pyzeumohiae antigens
described in the text. The neutralization titer is reported is as the
reciprocal of the
antiserum dilution causing a 50% reduction in the number of inclusions in the
iya vitro
infectivity assay. For the hamster model data the statistical significance of
the results
was evaluated by a two-tailed Student's t-test: significant data (p< 0.05) are
highlighted with an asterisk. ND = not detected.
Table 2 shows results from hamster mouse model studies for hypothetical
proteins.
Table 3 shows expressed genes of CPn EB selected by microarray.
Table 4 shows C. pheumoniae selected peptides: protein sources and HLA-A2
stabilization assay.
Table 5 shows ELISPOT assay with CD8+ T cells from DNA immunised HLA-A2
transgenic mice.
Table 6 shows IFN-y production from splenocytes of DNA immunized HLA-A2
transgenic and non transgenic mice.
METHODS AND MATERIALS (Examples 1-4) (see Reference Section 1)
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C.pneumoniae EB purification
C.pyaeumohiae FB196, a clinical isolate obtained from a patient with pneumonia
at the
Sant'Orsola Polyclinic, Bologna, Italy, was grown in LLC-MK2 cells seeded in
individual wells of a six-well plastic plate (7). Cells were harvested 72 hr
after
infection with a sterile rubber, sonically disrupted and the elementary bodies
(EB)
purified by gradient centrifugation as described (26). Purified CIZlamydiae
were
resuspended in sucrose-phosphate-glutamic acid (SPG) transport buffer, and
stored in
0.5 ml aliquots, at -80°C until used. When required, prior to storage,
EB infectivity
was heat-inactivated by 3 hour incubation at 56°C.
Expression and purification of recombinant proteins
Open reading frames (ORFs), selected from the C. pneumo~2iae CWL029 genome
sequence (16), were PCR-cloned into plasmid expression vectors and purified
from
E.coli cultures, essentially as previously described (25). Recombinant
Clalamydial
proteins were obtained as GST fusion proteins by using pGEX-KG derived vectors
(12) in E. coli BL21 (Novagen). PCR primers were designed so as to amplify
genes
without the N-terminal signal peptide coding sequence. When a signal peptide
or
processing site was not clearly predictable, the ORF sequence was cloned as
annotated by Kalman and coworkers (16). Recombinant E.coli cells were grown in
LB medium (500 ml), containing 100 ~,g/ml Ampicillin, and grown at
37°C until
OD6oo = 0.5 , and then induced with 1 mM IPTG. Cells were collected by
centrifugation ' 3 hr after induction and broken in a French Press (SLM
Aminco,
Rochester, NY). After centrifugation at 30.000 g, the supernatants were loaded
onto
Glutathione Sepharose 4B columns (Amersham Pharmacia Biotech) and column
bound proteins were eluted with 50 mM Tris-HCI, 10 mM reduced glutathione, pH
8Ø Protein concentrations in the samples were determined using the Bradford
method.
P_renaration of mouse antisera
Groups of four 5/6-week old CD1 female mice (Charles River, Como, Italy) were
immunized intraperitoneally at day 1 with 20ug of protein in Complete Freund's
adjuvant (CFA) and boosted at day 15 and 28 with 20ug of recombinant protein
in
Incomplete Freund's adjuvant (IFA). Pre-immune and immune sera were prepared
from blood samples collected on days 0, 27 and 42. In order to reduce the
amount of
antibodies possibly elicited by contaminating E. coli antigens, the immune
sera were
incubated overnight at 4°C with nitrocellulose strips adsorbed with a
total protein
extract from E. coli BL21.
Flow cytometr~assays
Analyses were performed essentially as previously described (25). Gradient
purified,
heat-inactivated EBs (2x105 cells) from C.ph.eu»aoyZiae FBl9, resuspended in
. phosphate-saline buffer (PBS), 0.1% bovine serum albumin (BSA), were
incubated
for 30 min. at 4°C with the specific mouse antisera (standard dilution
1:400). After
centrifugation and washing with 200 ~.l of PBS-0.1% BSA, the samples were
incubated for 30 minutes at 4°C with Goat Anti-Mouse IgG, F(ab)'2-
specific,
conjugated with R-Phycoerythrin (Jackson Immunoresearch Laboratories Inc.).
The
samples were washed with PBS-0.1%BSA, resuspended in 150 p1 of PBS-0.1%BSA
and analysed by Flow Cytometry using a FACSCalibur apparatus (Becton
Dickinson,
Mountain View, CA). Control samples were similarly prepared. Positive control
antibodies were: i), a commercial anti-C.pheumoniae specific monoclonal
antibody
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(Argene Biosoft, Varilhes, France) and, ii), a mouse polyclonal serum prepared
by
immunizing mice with gradient purified Gpneumohiae EBs. Background control
sera
were obtained from mice immunized with the purified GST peptide used in the
fusion
constructs (GST-fusions control). FACS data were analysed using the Cell Quest
Software (Becton Dickinson, Mountain View, CA). The shift between the
background
control histogram and the immune serum testing histogram was taken as a
measure of
antibody binding to the EB cell surface. The Kolmorov-Smirnov (K-S) two-sample
test (44) was performed on the two overlapped histograms. The D/s(n) values
(an
index of dissimilarity between the two curves) are reported as "K-S score" in
Table 1.
2D Western Blot analysis of immune sera and Mass Spectrometry
Gradient purified C. pheumorziae EBs were washed with 5 mM Tris-HCl pH 7.5,
0.1
mM EDTA, 10% glycerol, centrifuged 15 min. at 13 000 x g and pellets were
resuspended in reswelling solution (7 M urea, 2 M thiourea, 2% (w/v) CHAPS,
(2%w/v) ASB 14, 2% (v/v) IPG buffer pH 3-10 NL, or pH 4-7, 2 mM TBP, b5 mM
DTT). Protein samples (200 or 20 ~,g of protein for Coomassie Blue stained
reference
gels, or gels to be processed for immunoblotting, respectively) were adsorbed
overnight on Tmmobiline DryStrips (7 cm, pH 3-10 NL, or pH 4-7).
Electrofocusing
was performed in an IPGphor Isoelectric Focusing Unit (Amersham Biosciences,
Uppsala, Sweden). The focused strips were equilibrated as described (15) and
loaded
on linear 9-16.5 % acrylamide gradients (7x 4 cm, 1.5 mm thick), for SDS-PAGE
separation in a Mini Protean III Cell (Bio-Rad, Hercules, CA). Gels were
stained with
colloidal Coomassie Blue (Novex, San Diego, CA) (4) and the protein maps so
obtained were scanned with a Personal Densitometer SI (Molecular Dynamics) at
12
bits and 50 mm per pixel.
For Western Blot analyses, the proteins separated in the 2DE maps were
transferred
onto nitrocellulose membranes, overnight at 30 Volts, using a Protean III
apparatus
(BioRad, Hercules, CA). Membranes were stained with a 0.05% (w/v) CPTS
(Copper(II) phthalocyanine-3,4',4",4"'-tetrasulfonic acid tetrasodium salt) in
12 mM
HCI, and marked peripherally with 8 India-role dots to provide anchors for
subsequent
image superimposition and matching. After scanning and image acquisition, the
membranes were destained with 0,5 M NaHC03, incubated with the mouse sera to
be
analyzed (either pre-immune or specific immune sera, diluted 1:1000), and then
with
a peroxidase-conjugated anti-mouse antibody (Amersham Biosciences, Uppsala,
Sweden). After washing with PBS, 0.1% Tween-20, blots were developed using the
Opti-4CN Substrate Kit (Biorad, Hercules, CA), and the images of the
immunostained
blots again acquired as above. Images were analysed with the computer program
Image Master 2D Elite, version 4.01 (Amersham Biosciences, Uppsala, Sweden).
Superimposition and matches between Western-blot membranes and Coomassie
stained gels were performed as follow. First, the CPTS-stained membrane image
and
the immunostained blot image were superimposed using the peripheral dot marks.
Then, the sum image so obtained was superimposed to the Coomassie stained
protein
map using the CPTS stained CPn proteins as anchors. The areas on the
Coommassie
stained map corresponding to immunostained spots on the blot were excised from
the
preparative gel for protein identification. Protein sample were dried in a
vacuum
centrifuge, and in-gel digested, for 2h at 37°C, with an excess of
porcine Trypsin
(Promega, Madison, WI), in 100 mM ammonium bicarbonate. Tryptic peptides were
desalted and concentrated using Zip-Tip (Millipore, Bedford, MA). Peptides
were
directly eluted and loaded onto a SCOUT 384 Anchor Chip multiprobe plate (400
~,m,
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Broker Daltonics, Bremen, Germany) with a solution of 2-5 dihydroxybenzoic
acid
(5g!1), in 50% acetonitrile, 0.1% trifluoroacetic acid. Spectra were acquired
on a
Broker Biflex III matrix-assisted laser desorption ionization-time of flight
(MALDI-
TOF) apparatus. Resulting values for monoisotopic peaks were used for database
searches using the Mascot software (32), as available at the website
http://www.matrixscience.com/.
In vitro neutralization assays
In vitro neutralization assays were performed on LLC-MK2 (Rhesus monkey
kidney)
epithelial cell cultures. Serial four-fold dilutions of mouse immune and
corresponding
preimmune sera were prepared in sucrose-phosphate--glutamic acid buffer (SPG).
Mouse polyclonal sera to whole EBs were used as positive control of
neutralization,
whereas SPG buffer alone was used as negative control of neutralization
(control of
infection). Purified infectious EBs from the C.pheunaohiae FB/96 were diluted
in
SPG buffer to contain 2.5x107 IFU/ml, and 10u1 of EBs suspension were added to
each serum dilution in a final volume of 100u1. Antibody-EB interaction was
allowed
to proceed for 30 min at 37°C on a slowly rocking platform. The 100u1
of reaction
mix of each sample was used to inoculate PBS-washed LLC-MK2 confluent
monolayers (in triplicate for each serum dilution), in a 24-well tissue
culture plate,
and centrifuged at 805 x g for 1 hour at 37°C. After centrifugation
Eagle's minimal
essential medium containing Earle's salts, 20% fetal bovine serum and lug/ml
cycloheximide was added. Infected cultures were incubated at 37°C in
5%C02 for 72
hours. The monolayers were fixed with methanol and the Chlamydial inclusions
were
detected by staining with mouse anti-Chlanaydia fluorescein-conjugated
monoclonal
antibody (Merifluor ClZlamydia, Meridian Diagnostics, Inc.) and quantified by
counting 10 fields per well at a magnification of 40X. The inhibition of
infectivity due
to EBs interaction with the immune sera was calculated as percentage reduction
in
mean IFU number as compared to the SPG (buffer only)lEBs control. In this
calculation the IFU counts obtained with immune sera were corrected for
background
inhibition of infection due to the corresponding pre-immune mouse serum.
According
to common practice, the sera were considered as "neutralizing" if they could
cause a
50% or greater reduction in infectivity. The corresponding neutralizing titer
was
defined as the serum dilution at which a 50% reduction of infectivity was
observed.
Experimental variability was evaluated by calculating the standard error of
measurement (SEM), from three titration experiments for each recombinant
antigen,
as shown in Fig.lB.
Iya vivo screening
Ifa vivo evaluation was performed using a hamster model of systemic infection,
as
recently described (34). Essentially, adult (10-11 week old) Syrian hamsters
(Morini,
S. Polo D'Enza, Italy), previously immunized with the recombinant vaccine
candidates were challenged systemically with infectious Cpn elementary bodies
(EB).
Protection was assessed by determining the levels of viable EB recovered from
the
spleen, as compared to non-immunized animals. Statistical significance of the
results
was evaluated by a two-tailed Student's t-test.
Groups of 8 hamsters were immunized subcutaneously with recombinant antigens,
or
only injected with buffer for the control group, at days 0, 7, and 21. For
each
immunization 20 ug protein 1:1 diluted with Freund's complete adjuvant (first
dose)
and Freund's incomplete adjuvant (booster doses) was injected. At day 35 post-
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infection the hamsters were anaesthetised with Ketamine and inoculated
intraperitoneally and intranasally with 0.1 ml of C.pheumofaiae EB suspension
(1.0x10$) at each site. Animals were sacrificed seven days after infection.
The spleen
was weighed, and homogenized in a mortar to obtain a 10% (wt/vol) suspension
in
cold SPG buffer. Tissue suspensions were centrifuged at 300 x g for 10 min at
4°C to
remove coarse debris. The clarified homogenates (0.2 ml) were inoculated in
duplicate onto LLC-MK2 cells seeded in plastic individual well of a 24 well
plate,
incubated at 37°C for 72 h and fixed in acetone before detection and
counting of
numbers of Chlamydial inclusions per well by immunofluorescence microscopy.
The
protocol was approved by the ethical committee of the University of Bologna.
Example 1 (i~a vitro studies)
Screeuihg autisera for in vitro ueutralizijzg properties
Following a genome-wide screening for proteins likely to be localized on the
cell
surface of C. pyaeumofziae, we recently reported (25) that antisera to 53
recombinant
Chlamydial antigens were capable to bind in a FAGS assay, the surface of
Clalamydial
cells. In order to check whether some of the FACS positive antigens were
capable of
interfering with EB i~c vitro infectivity, we raised mouse antisera against
the
recombinant FACS positive antigens and assessed the effect of each antiserum
on the
infectivity of purified EBs with respect to monolayers of LLC-MK2 cells.
Infectious
EB were first incubated with the antiserum and then used to infect cell
monolayers in
24-well multititer plates. In parallel, control samples were similarly
processed in
which the EBs were: i), either treated with buffer only, or, ii), treated with
the same
dilutions of the corresponding preimmune mouse sera.
Results I
Using this assay, 10 sera have so far proved to effectively neutralize ih
vitro
infectivity to an extent greater than 50%, a property that common practice
qualifies
such antigens as "neutralising" (Figure 1). These 10 sera were obtained by
mouse
immunization with recombinant proteins derived from the following C.pheumohiae
genes:
~ pmpl0 and pmp2, encoding two members of the heterogeneous Chlamydial PMP
family of polymorphic membrane proteins;
~ artJ, encoding a putative extracellular solute (possibly Arginine) binding
protein
of an aminoacid transport system;
~ eho, encoding a protein homologous to bacterial enolases, glycolytic enzymes
which can be found also on the bacterial surface;
~ htrA, encoding a putative chaperone with heat-shock inducible protease
activity;
~ the Cpn0301 "hypothetical" gene, encoding a protein homologous to the ompH
family of bacterial proteins, some members of which have been shown to be
chaperones involved in outer membrane biosynthesis;
~ two Cpn-specific "hypothetical" genes Cpn0795 and Cpn0042;
~ o»zcA encoding a 7-9 kDa protein annotated as an outer membrane protein; and
~ atoS a putative sensor member of a transport system.
As shown in Figure 1 and summarized in Table I, OmpH, enolase and Cpn0795
appeared to induce the highest neutralizing sera, with titers above 400. By
contrast,
Pmp2, ArtJ and Cpn0042 induced titers equal or less than 100, while the
remaining 4
antigens, PmplO, HixA, AtoS and OmcA showed intermediate titers.
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Example 2 (irz vivo studies)
Evaluation of autisera specificity by 2D irrzrrzurzoblot analysis of Cpu
protein
extracts
In order to investigate if the neutralizing activity observed in the irz vitro
infection of
LLC-MK2 monolayers was actually due to the binding of the antibodies to the
selected C.pneumorziae proteins, rather than to possible cross-rections with
other
antigens, we assessed the specificity of the antisera by immunoblot analysis
of two
dimensional electrophoretic maps of EB proteins.
In particular, this analysis was carried out on six antigens (Pmp2, PmplO,
Eno, ArtJ,
HtrA and OmpH-like) known to be visible in the 2D maps of EB total proteins
(Montigiani et al., 2002 Infection and Immunity 70: 368-379). Total EB
proteins
were resolved by 2D-electrophoresis using two different pH intervals (pH 3-10
non
linear, and pH 4-7, respectively) since previous experiments had shown that
some of
the proteins under study were better detected using one rather that the other
of the
above pH intervals. For each pH interval four gels were run in parallel. One
gel was
stained with Coomassie Blue to visualize the protein spots, while the other
gels were
blotted on nitrocellulose filters and stained with one of the selected sera at
1000-fold
dilution. Subsequently, the images of the immunostained blots (Fig.2, panels c
to h)
were superimposed to the corresponding Coomassie Blue-stained gel to identify
the
spots which had reacted with a given antiserum. The matching protein spots
were
excised and processed for peptide identification by MALDI-TOF analysis.
Results 2
In all six maps the immunoreactive protein species in the excised gel area
were found
to contain peptides from the expected Chlanzydial protein: Even when the serum
reacted with more than one electrophoretic protein species, the mass spectra
of all
spots which could be detected in the COOmassie Blue stains 2DE map were always
consistent with the same polypeptide being present as multiple electrophoretic
species.
Interestingly, the immunoblot obtained with the HtrA antiserum showed two sets
of 4
spots arranged as two typical electrophoretic 'trains' at two different
molecular
weights. On the Coomassie Blue stained gel it was possible to identify 4
corresponding spots, 3 in the upper train and 1 in the lower Mw set. MS
analysis
identified all of them as products of Cpn HtrA gene. Interestingly the lower
Mwt
species missed 3 N-terminal tryptic peptides, detected in the higher Mw spot
series,
and mapping within the first 100 as of the ORF. These results suggest that
HtrA was
present in the EB protein sample both as a full htrA product, and as a
discrete species
missing a short N-terminal peptide, possibly as a result of some post-
translational
processing.
Discussion of Results 2
In the analysis of data which are based on polyclonal antibody reactivity one
should
consider that cross-reactions due to epitope mimickry are always difficult to
exclude.
The problem of antisera specificity was addressed in this work by 2D
immunoblotting
and identification of the reacting electrophoretic species by mass
spectrometry
analysis. This approach was possible for 6 of the 10 antisera, i.e. those
corresponding
to proteins previously identified by mass spectrometry (MALDI-TOF) analysis on
2D
electrophoretic maps of C.pneumorziae EB proteins (25, 42) (Table 1, and
Figure 2).
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The probability of fortuitous cross-reactions between unrelated Chlantydial
protein
species was minimized by the results of the immunoblotting analyses which
showed
that out of ca 300 protein spots in a map, all those reacting with the tested
antisera
were consistent with the expected antiserum specificity. Obviously, since
during 2-D
electrophoresis conformational epitopes are generally lost, structure-
dependent cross-
reactions cannot be ruled out in this type of analysis.
Exatrtple 3
Iu vivo evaluation of the ift vitro neutralizing a~ttigens itz a hamster model
of
systetttic infection
We have recently described a new hamster model of systemic ClZlamydia
pneuntoniae
infection in which replicating Chlantydia disseminate through macrophages and
accumulate in the spleen (34). We therefore asked the question whether the in
vitro
neutralizing antigens we identified would also have protective activity in
vivo using
this model. To this aim, the 10 in vitro neutralizing recombinant antigens
were used
to immunize 8 hamsters with 3 subcutaneous injections over a three-week
period, and
challenged with 2x108 Cpn EBs two weeks later. Spleen infection was assessed 7
days
after challenge. The difference between the mean number of infectious
Chlamydiae
recovered from control animals and the mean number of Chlamydiae recovered
from
animals immunized with the recombinant Chlamydial antigens, was taken as a
measure of protection specifically induced by the putative vaccine candidate.
Results 3
The results of spleen protection observed for the various antigens in repeated
experiments are shown in Figure 3 and reported as percentage values in Table
1. Six
out of ten antigens, Pmp2, PmplO, Enolase, the OmpH-like protein, and the
products
of the C.pneumoniae-specific genes Cpn0759 and Cpn0042, showed a statistically
significant protective activity, with a reduction in IFU recovered from the
spleens of
immunized animals higher than 80% with respect to mock-immunized controls.
A limit of the hamster model is that, because of the absence of immunological
reagents, the relative contribution of humoral and cell-mediated immunity
cannot be
assessed. However, we asked the question whether recombinant antigens could
elicit
also in the hamster neutralizing antibodies with sufficiently high titers.
Therefore we
tested the sera from hamsters immunized with Pmp2 and enolase, two of the most
protective antigens, in the in vitro neutralization assay. Both antigens had a
neutralizing titer of approximately 100 (data not shown).
Sumf~zary of Results 3
In conclusion, a considerable proportion (60%) of the in vitro neutralizing
antigens
were also protective in the hamster in vivo model and our data suggest that
antibody-
mediated neutralization could play a role at least in this model of systemic
infection.
Discussioiz of Results 3
Beside assaying the in vitro neutralization properties of the selected subset
of 10
FACS-positive antigens, we also assessed the performance of these antigens in
protecting against C. pneumoniae infection in an animal model of systemic
infection
recently described in the hamster (34). This evaluation addressed the
capability that
the recombinant antigens would have of inducing a protective response against
naturally replicating Chlarnydiae (rather than EB's purified from in vitro
cultures
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grown under artificial conditions) and in the context of a systemic infection.
In fact
the hamster model we used, while it does not model the typical respiratory
infection
considered to be the predominant route by which C. pheumoyaiae infects humans,
it
nevertheless simulates a situation of systemic invasion which could be
preliminary to
the establishment of C. pheumohiae chronic infection considered by several
authors as
being associated to the development or progression of cardiovascular disease,
and
other chronic degenerative diseases. Notably, a limit of any hamster model is
the
current lack of hamster-specific immunological reagents which would allow the
analysis of cell mediated immune responses. However, in the case of systemic
infections, by common wisdom, neutralizing antibodies are likely to have a
protective
action. The fording that 6 of the 10 'ih vitro neutralizing' antigens had also
a >80%
protective action in vivo, and that a measurable neutralizing activity was
also found in
the sera of immunized hamsters, suggests that a specific antibody mediated
immunity
could at least partially contribute to the animal protection here described.
Example 4
Two 'hypothetical'proteins 6784 and 6814 (encoded by the ORFs Cpn0498 and
Cpn0525) yielded FACS-positive sera which, however, were not able to
neutralize
host cell infection iu vitro. However, these antigens performed remarkably
well in the
hamster-spleen test.
Table 2
Gene/ORFProteinRecombinaAnnotationRecipr% Protection
ID in ID nt Fusion ocal in the
of
CWL029 Type 50% hamster
neutralspleen
test
isation(ref 34)
titre
Cpn0498 4376784GST Hypothetical0 94
protein
CPn0525 4376814GST Hypothetical0 97
protein
(similarity
to
CT398)
CPn0324 HIS Low Calcium Completely
Response protected
8 of
Element 16 animals
(LcrE) and reduced
the infectivity
titres
of the
eight positive
animals
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Discussion of Results 4
Interestingly, whilst antiserum against CPn0525 gave negative in vitro results
(ie no
neutralising activity), the CPn0525 protein gave 97 per cent protection from
spleen
infection in an io vivo hamster immunisation assay (ie a positive i~c vivo
result).
Likewise, whilst antiserum against Cpn0498 gave negative ire vitro results (ie
no
neutralising activity), the CPn0498 protein gave 94 per cent protection from
spleen
infection in an ii2 vivo hamster immunisation assay. Thus a positive signal
obtained in
the FACE assay does not guarantee a corresponding positive in vitf~o
neutralization
activity and conversely a negative neutralization activity does not mean that
a positive
iya vivo result can be excluded.
General Discussion of Results 1-4
Strategy fof~ idehtificatio~z of Chlamydia pheumohiae antigefzs of interest
Our strategy was based on the following experimental steps: 1) analysis of
Chlamydia genome sequence to select putative membrane-associated antigens, 2)
cloning, expression and purification selected antigens, 3) preparation of
antigen
specific sera by mouse immunization with the purified antigens, 4) FACE
analysis of
Chlamydia EBs using the mouse sera to identified surface-exposed antigens, 5)
"in
vita°o neutralization" assay to test whether antibodies elicited by a
given antigen can
interfere with the process of eukaryotic cell infection, and 6) use of
appropriate
animal model to test the capacity of selected antigens to confer protection
against
CIZlanaydia challenge.
As recently described by Montigiani et al ((2002) Infection and Immunity 70:
368-
379) from the initial screening of the C.pheumofiiae genome, a panel of mouse
sera
was prepared against over 170 recombinant His-tagged or GST-fusion proteins
encoded by genes or "open reading frames" somehow predicted to be peripherally
located in the Clalamydial cell. When these antibodies were tested in a FACS
assay
for their ability to bind the surface of purified C.pheumohiae EBs, a list of
53 "FACS-
positive" sera was obtained. The corresponding putative surface antigens were
then
further assessed for their capability of inducing neutralizing antibodies.
This part of
the work involved testing which of the sera contained antibodies capable of
interfering with the process of in vitro infection of epithelial cell
cultures. In the in
vitro "neutralization" assay purified infectious EBs are incubated with
progressive
dilutions of the immune sera and, in parallel, dilutions of the corresponding
pre-
immune sera, and of sera against non Chlamydia control antigens.
Cell cultures are infected in the presence of cycloheximide, which inhibits
host cell
protein synthesis and favours Chlamydial intracellular growth with the
consequent
formation of typical cytoplasmic inclusions which can be stained with
Chlamydia
specific fluorescence-labeled monoclonal antibodies and counted with an UV
light
microscope. Working with appropriate pathogen-to-host cell ratios, it can be
reasonably assumed that the number of detected cytoplasmic inclusion is
proportional
to the number of infectious Chlamydiae in the original sample. So a reduction
in
inclusion numbers caused by the presence of an antigen-specific antiserum, as
compared to the numbers obtained with control sera, gives a measure of the
capability
of a given antigen to elicit antibodies which can inhibit some stage of the
Chlamydial
infection process. According to common convention, an anti-serum is labelled
as
'neutralizing' when the reduction of infectivity is equal or greater than 50%,
and the
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serum dilution yielding a 50% reduction in infectivity is referred to as the
50% end-
point neutralization titer.
Some of the results obtained by screening the panel of recombinant antigens
with the
C.pyzeumoh.iae ih vitro neutralization assay confirm that some of the listed
antigens,
like the members of the family of heterogeneous polymorphic membrane proteins
(PMP), which, on the basis of published literature data, could be reasonably
expected
to be surface-exposed and possibly induce neutralizing antibodies. However,
there
are also proteins which could be considered so far only hypothetical, and
proteins
which just on the basis of their current functional annotation could not be at
all
expected to be found on the bacterial surface. Using an iyz vitz~o
neutralising assay, it
was found that sera to 10 CPn antigens have so far proved to effectively
neutralize in
vitf~o infectivity to an extent greater than 50%, a property that common
practice
qualifies such antigens as "neutralising" (Figure 1). These 10 sera were
obtained by
mouse immunization with recombinant proteins derived from the C.p~zeurrzohiae
genes listed below.
Using a recently described ih vivo model of systemic infection (hamster
model),
hamsters immunised with 6 of the iu vitr~o neutralising antigens, when
challenged with
CPn EBs, showed a greater than 80% reduction of spleen infection as compared
with
non-immunised controls.
CIZaractez~isatiou of 10 CPh proteins
The proteins identified by the present work can be divided in 3 groups:
~ proteins which have an annotation compatible with (could be reasonably
expected
to have) an expected/predicted exposure on the Clzlamydial cell surface and
with the
possibility that antibodies binding to them may actually interfere with host
cell
attachment and entry (ie proteins which could possibly induce neutralising
antibodies)
~ proteins which by homology with other gram-negative bacteria could be
expected
to have a periplasmic exposure (ie would not be expected at all to be found on
the
bacterial cell surface); and
~ proteins which are still labelled as 'hypothetical' (ie cellular location
and/or
cellular function not known)
Group 1
(Psrzp proteins (pnzp2 and pzzzpl0), OrzzcA and O~zzpH)
Pmp proteins (pmp2 afzd pmpl0)
The first group includes the 2 polymorphic outer membrane proteins (Pmp's)
Pmp2
and PmplO (10, 11, 14, 30), the outer membrane protein OmpH-like, and OmcA,
which is annotated (Chlamydia Genome Proj ect at http:llChlarrzydia
www.berkeley.edu:4231~ as "predicted 9-kD cysteine-rich, outer membrane
protein,
lipoprotein". The Pmp family of Chlamydia-specific proteins is generally
thought to
comprise probable pathogenicity factors, with an autonomous secretion capacity
(autotransporters), important for adhesion to host cells and are generally
considered as
promising vaccine candidates. However, apart from very recent unpublished
results
on Pmp2l, this is the first time that antisera to recombinant Pmp's are
reported to
have neutralizing properties.
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OnzcA
OmcA is the product of a gene co-transcribed in the same operon with the 60
kDa
OmcB cystein-rich protein which is a major structural component of the
Chlamydial
outer membrane and a major immunogen in human C. tf~achomatis infections. OmcB
and OmcA are likely to interact in some as yet unknown outer membrane
structure, so
it is possible that antibodies to OmcA can interfere with EB infectivity.
OnipH
Finally, the Chlamydial OmpH is probably a member of the OmpH (Skp) family of
proteins which have been reported to have chaperonin activities in other
bacteria very
important for the correct biosynthesis of the outer membrane. These proteins
appear
to cooperate in this task with HtrA (see below). In fact, in E. coli single KO
mutants of
either OmpH (Skp) or HtrA (DegP) are still viable, but double mutants do not
grow
(37). It should be pointed out that even if the amino acid sequences of the
ompH-like
proteins of ChlanZydia (all C.pneumoniae and C. t~achomatis or G caviae
variants)
line-up very well with the rest of the bacterial OmpH proteins, they are the
only ones
to be acidic, whereas the rest of the family comprises mostly very basic
proteins
(including some with histone like behaviour, at least in vitno). One could
speculate
that if the chaperone activity is maintained also in the ompH like Chlamydial
proteins,
this may be related to some Chlamydial peculiarity.
Secotzd Group of Selected Proteitzs
(ArtJ, AtoS, HtrA atzd Euolase)
The second group, which represents a somehow surprising finding, includes
ArtJ,
AtoS, HtrA and Enolase. If the current annotation (justified by analogy with
homologous genes in other bacteria) is correct, all these proteins would be
expected to
have a periplasmic location in gram-negative bacteria. and to be surface-
exposed only
in a gram-positive bacterium. It is possible that owing to their atypical life
cycle,
requiring an efficient passage from a dormant spore-like status (the EB) to an
active
form needing to adapt quickly to host-cell responses to invasion, Chlamydiae
in fact
display some sensors directly on the outer surface of their infectious form.
Ar~tJ
In the case ArtJ - for which we have data supporting both antigen expression
and
serum specificity - the hypothesis of an atypical situation peculiar to
Chlatnydia is
supported by the anomalous gene set-up resulting from the present analysis of
the
Chlanaydia genomes. ArtJ is so. annotated by analogy with the ART transport
systems
of E.coli wluch has 5 genes organized in two operons (24) : artPIQM and artJ
which
are responsible for the arginine transport. In Cpn however the artPIQM genes
are
absent and therefore it appears that Chlamydial ArtJ operates in a molecular
context
which is different from its E.coli model and must be peculiar to this species.
HtrA
HtrA (DegP), which in other bacteria has a complex hexameric structure, has
been
described as having multiple functions (3, 5, 18, 19, 27, 38) : a chaperonin
assisting a
correct outer membrane biogenesis, inducible protease for the elimination of
misfolded membrane proteins, and also a sensor of 'stress' conditions. In
Chlamydia
none of these properties has been demonstrated yet, however we find that in
purified
EB HtrA is present in two forms one of which appears to be processed by being
deprived of the N-terminal fragment. This fragment, if aligned with the
homologous
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HtrA sequence from Thermologa maritima (18), would comprise a predicted loop
acting as a structural lid controlling the access to the protease active. So
it appears
tempting to speculate that HtrA could have a similar protease activity and the
two
forms identified on the 2-D map represent the active and inactive species.
Interestingly, the C. tnachonzatis HtrA ortholog is recognized by human sera
from
patients who had a Chlamydial genital infection (35), and a similarly HtrA is
one of
the antigens in the immunoproteome of Helicobacter pylori (13). Furthermore
the
homologue protein in Haemophilus influenzae is a protective antigen in both a
passive
infant rat model of bacteremia and the active chinchilla model of otitis media
(23) .
E>zolase
Also in the second group of proteins expected to be located elsewhere than the
cell
surface, is Cpn enolase. This protein aligns with the well known family of
conserved
glycosylases, which are essentially cytoplasmic enzymes, but in Streptococci
enolase
has been shown to have also a cell surface location, and extracellular matrix
binding
properties (1, 28, 29)). Interestingly, Gaston and colleagues (8) also showed
that in
patients with reactive arthritis induced by G trachomatis, enolase induces
specific
CD4+ T-cell responses. Furthermore, a clone responding to the enolase C.
trachomatis
ortholog; responded also to C.pneumoniae EBs, and, since no proliferative
response
could be observed using a fungal or a mammalian enolase, the authors of this
study
concluded that the CD4 T-cell stimulating epitope must be Chlamydia specific.
Third Group of Proteizzs
(mzlifzowzz cellular location azzdlor cellular functio>z, Cp>z0795, CPn0042)
The ~ third of the 3 groups in which we propose to divide, just for the sake
of
discussion, the 10 neutralizing antigens above described, comprises two
proteins
which are still annotated in public Chlanzydial databases as the hypothetical
products
of two CPn-specific genes: Cpn0759 and Cpn0042. The Cpn0759 gene is the second
gene in a cluster of 6 Cpn-specific hypothetical genes (from Cpn0794 to
Cpn0799)
immediately upstream of the enolase gene. With the exception of Cpn0759 the
products of all the other genes in the cluster share similarities of 30 to 40%
over long
stretches of amino acids. The Cpn0042 gene encodes a hypothetical protein,
with 4
coiled-coil regions, which has been described as a member of a new family of
hypervariable outer membrane proteins (33). Interestingly, the
hypervariability of
these proteins could be due to a strand-slippage mechanism induced by the
presence
of a poly(C) stretch within the coding region of the corresponding genes, a
mechanism already described in the Pmp's family for the pmpl0 gene (30).
However,
as indicated by their annotation, the function of these proteins is still
unknown, and
our observations provide the first experimental indication of a possible
function
related to the Chlamydial infection process.
Table 1 of this application demonstrates that Cpn0795 (SEQ ID NO: 6) a Cpn
specific
hypothetical protein is a FAGS positive protein which demonstrates significant
immunoprotective activity in a hamster spleen model of Chlamydia pneumoniae
infection. We have found evidence to demonstrate that other Cpn proteins in
this
group of Cpn specific hypothetical proteins have now been found to have a
secreted
autotransporter function. These proteins, which axe absent from Clzlamydia
trachomatis include: gi/4377105 (Cpn0794), gi/4377106 (Cpn0795), gi/4377107
(Cpn0796), gi/4377108 (Cpn0797), gi/4377109 (CPn0798), gi/4377110 (Cpn0799).
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Fig. 6 shows an alignment of the proteins in the 7105-7110 protein family.
This
Alignment shows a new family of proteins expected to constitute a system of
antigens
probably delivered on the Cpn surface or secreted by a type V
(autotransporter)
secretion mechanism. This alignment was generated as follows:
Imperfect repeats were identified which allowed the alignment of the genes.
Molecular modelling has also demonstrated that the C-terminal ends of 7106 and
7107 can be predicted to fold in a beta-barrel structure which can form a
translocation
pore for secretion across the outer membrane.
Cpn0794 = 7105 = FAGS positive
Cpn0795 = 7106 = FAGS positive
Cpn0796 = 7107 = FACS positive
Cpn0797 = 7108 = FACS positive
Cpn0798 = 7109 = No data available
Cpn0799 = 7110 = No data available
(Reference for FACS positive data = Montigiani et al (2002) Infect Immun 70(1)
368-
79)
Operonl = 0794, 0795, 0796 Operon2 = 0797, 0798
Cpn0795 and Cpn0796 have C terminal ends that may form transmembrane pores
(see
alignment, FIG. 9). CPn0794, Cpn0797, Cpn0798, and Cpn0799 have N-terminal
ends indicating that all proteins have N-terminal and C-terminal ends.
Fig. 7 shows alignment of Cpn0794 - Cpn 0799. Proteins encoded by the genes
Cpn0794, Cpn0795, Cpn0796, and Cpn0797 have been identified as likely to be
exposed on the surface of the chlamydia cell and as possible vaccine
candidates.
These proteins are shown to be actually expressed by Cpn in vivo (WB data and
FACS data). In the case of Cpn0797 we also showed that the level of expression
in
CPn EBs is high enough to be detected by mass spectrometry analysis on 2DE
maps
of protein extracts (see Montigiani et al.)
Following these observations, it is seen that the proteins encoded by Cpn0794,
Cpn0796 and Cpn0797 proteins can be aligned according to a set of imperfect
repeats
present within their aminoacid sequences (see FIG. 7) , whereas the putative
product
of CPn0795 can be mostly aligned to the C-terminal portion of the Cpn0796
protein.
Furthermore, proteins encoded by genes Cpn0798 or Cpn0799 can alse be aligned
to
the above proteins according to the above mentioned repeated sequence motifs
(see
FIG. 7).
Overall alignment of the 6 genes demonstrates that the genes encode for a
family of
functionally-related proteins.
Furthermore, in silico analysis of the protein encoded by Cpn0796, which
encompasses the entire alignment of all the proteins in this family
demonstrates that a
functional precursor with the aminoacid sequence reported below:
SEQ ID NO: 80
MKFMKVLTPWTYRKDLWVTAFLLTAIPGSFAHTLVDIAGEPRHAAQATGVSGDGKIVIGMKVPDDPFA
5O ITVGFQYIDGHLQPLEAVRPQCSVYPNGITPDGTVIVGTNYAIGMGSVAVKWVNGKVSELPMLPDTLD
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SVASAVSADGRVIGGNRNINLGASVAVKWEDDVITQLPSLPDAMNACVNGISSDGSIIVGTMVDVSWR
NTAVQWIGDQLSVIGTLGGTTSVASAISTDGTVIVGGSENADSQTHAYAYKNGVMSDIGTLGGFYSLA
HAVSSDGSVIVGVSTNSEHRYHAFQYADGQMVDLGTLGGPESYAQGVSGDGKVIVGRAQVPSGDWHAF
LCPFQAPSPAPVHGGSTWTSQNPRGMVDINATYSSLKNSQQQLQRLLIQHSAKVESVSSGAPSFTSV
KGAISKQSPAVQNDVQKGTFLSYRSQVHGNVQNQQLLTGAFMDWKLASAPKCGFKVALHYGSQDALVE
RAALPYTEQGLGSSVLSGFGGQVQGRYDFNLGETWLQPFMGIQVLHLSREGYSEKNVRFPVSYDSVA
YSAATSFMGAHVFASLSPKMSTAATLGVERDLNSHIDEFKGSVSAMGNFVLENSTVSVLRPFASLAMY
YDVRQQQLVTLSWMNQQPLTGTLSLVSQSSYNLSF
Processing sites that assiste in the secretion of the polypeptide from the
cytoplasm and
its release into the periplasm are located after aminoacid 31 (based on PSORT
prediction and/or after aminoacid 47 similar to experimentally determined
processing
sites in other bacterial autotransporter molecules (e.g. BrkA from
B.pertussis). Hence,
the mature form of the Cpn0796 product is as follows:
SEQ ID NO: 81
HTLVDIAGEPRHAAQATGVSGDGKIVIGMKVPDDPFAITVGFQYIDGHLQPLEAVRPQCSVYPNGITP
D
2O GTVIVGTNYAIGMGSVAVKWVNGKVSELPMLPDTLDSVASAVSADGRVIGGNRNINLGASVAVKWEDD
VITQLPSLPDAMNACVNGISSDGSIIVGTMVDVSWRNTAVQWIGDQLSVIGTLGGTTSVASAISTDGT
VIVGGSENADSQTHAYAYKNGVMSDIGTLGGFYSLAHAVSSDGSVIVGVSTNSEHRYHAFQYADGQMV
DLGTLGGPESYAQGVSGDGKVIVGRAQVPSGDWHAFLCPFQAPSPAPVHGGSTWTSQNPRGMVDINA
TYSSLKNSQQQLQRLLIQHSAKVESVSSGAPSFTSVKGAISKQSPAVQNDVQKGTFLSYRSQVHGNVQ
~5 NQQLLTGAFMDWKLASAPKCGFKVALHYGSQDALVERAALPYTEQGLGSSVLSGFGGQVQGRYDFNLG
ETWLQPFMGIQVLHLSREGYSEKNVRFPVSYDSVAYSAATSFMGAHVFASLSPKMSTAATLGVERDL
NSHIDEFKGSVSAMGNFVLENSTVSVLRPFASLAMYYDVRQQQLVTLSVVMNQQPLTGTLSLVSQSSY
NLSF
30 Or
SEQ ID NO: 82
TGVSGDGKIVIGMKVPDDPFAITVGFQYIDGHLQPLEAVRPQCSVYPNGITPDGTVIVGTNYAIG
35 MGSVAVKWVNGKVSELPMLPDTLDSVASAVSADGRVIGGNRNINLGASVAVKWEDDVITQLPSLP
DAMNACVNGISSDGSIIVGTMVDVSWRNTAVQWIGDQLSVIGTLGGTTSVASAISTDGTVIVGGS
ENADSQTHAYAYKNGVMSDIGTLGGFYSLAHAVSSDGSVIVGVSTNSEHRYHAFQYADGQMVDLG
TLGGPESYAQGVSGDGKVIVGRAQVPSGDWHAFLCPFQAPSPAPVHGGSTWTSQNPRGMVDINA
TYSSLKNSQQQLQRLLIQHSAKVESVSSGAPSFTSVKGAISKQSPAVQNDVQKGTFLSYRSQVHG
4O NVQNQQLLTGAFMDWKLASAPKCGFKVALHYGSQDALVERAALPYTEQGLGSSVLSGFGGQVQGR
YDFNLGETWLQPFMGIQVLHLSREGYSEKNVRFPVSYDSVAYSAATSFMGAHVFASLSPKMSTA
ATLGVERDLNSHIDEFKGSVSAMGNFVLENSTVSVLRPFASLAMYYDVRQQQLVTLSWMNQQPL
TGTLSLVSQSSYNLSF
45 In silico analysis of the protein encoded by Cpn0796 also demonstrates a C-
terminal
domain comprising approximately residues from 1 to 648. FIG. 8 illustrates
Cpn0796.
As shown in FIG. 8, Cpn0796 forms a beta-barrel structure and is capable of
forming
a pore across the bacterial outer membrane (OM). As is typical of
'autotransporter'
molecules, after being secreted across the bacterial inner membrane into the
periplasm
50 through an N-terminal signal peptide mechanism, the molecule may form a
pore in the
OM through which the N-terminal domain may pass (the 'passenger' domain) to
the
outside of the bacterial cell. Also, these molecules may either remain
anchored to the
bacterial surface or undergo a proteolytic cut which releases the 'passenger
domain'
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or a portion of it into the medium surrounding the bacterial cell an example
of which
is represented in the following sequence:
SEQ ID NO: 83
MKFMKVLTPWIYRKDLWVTAFLLTAIPGSFAHTLVDIAGEPRHAAQATGV
SGDGKIVIGMKVPDDPFAITVGFQYIDGHLQPLEAVRPQCSVYPNGITPD
GTVIVGTNYAIGMGSVAVKWVNGKVSELPMLPDTLDSVASAVSADGRVIG
GNRNINLGASVAVKWEDDVITQLPSLPDAMNACVNGISSDGSIIVGTMVD
1O VSWRNTAVQWIGDQLSVIGTLGGTTSVASAISTDGTVIVGGSENADSQTH
AYAYKNGVMSDIGTLGGFYSLAHAVSSDGSVIVGVSTNSEHRYHAFQYAD
GQMVDLGTLGGPESYAQGVSGDGKVIVGRAQVPSGDWHAFLCPFQAPSPA
PVHGGSTWTSQNPRGMVDINATYSSLKNSQQQLQ
RLLIQHSAKVESVSSGAPSFTSVKGAISKQSPAVQNDVQKGTFLSYRSQVHGNVQNQQLLTGAFM
DWKLASAPKCGFKVALHYGSQDALVERAALPYTEQGLGSSVLSGFGGQVQ
GRYDFNLGETWLQPFMGIQVLHLSREGYSEKNVRFPVSYDSVAYSAATS
FMGAHVFASLSPKMSTAATLGVERDLNSHIDEFKGSVSAMGNFVLENSTV
SVLRPFASLAMYYDVRQQQLVTLSWMNQQPLTGTLSLVSQSSYNLSF
Also shown in FIG. 8, amino acid residues 365-385 represent an alpha helix
conformation that spans the beta barrel pore
The N-terminal passenger domain may be cleaved via a specific proteolytic
action
from the membrane-anchored pore structure. A linker domain comprising the
peptide
sequence PSPAPV (SEQ ID NO: 84) as shown in bold in the following sequence
illustrates a site at which cleavage of the N-terminal passenger domain may
occur:
SEQ ID NO: 85
HTLVDIAGEPRHAAQATGVSGDGKIVIGMKVPDDPFAITVGFQYIDGHLQPLEAVRPQCSVYPNG
ITPDGTVIVGTNYAIGMGSVAVKWVNGKVSELPMLPDTLDSVASAVSADGRVIGGNRNINLGASV
AVKWEDDVITQLPSLPDAMNACVNGISSDGSIIVGTMVDVSWRNTAVQWIGDQLSVIGTLGGTTS
VASAISTDGTVIVGGSENADSQTHAYAYKNGVMSDIGTLGGFYSLAHAVSSDGSVIVGVSTNSEH
RYHAFQYADGQMVDLGTLGGPESYAQGVSGDGKVIVGRAQVPSGDWHAFLCPFQAPSPAPVHGGS
TWTSQNPRGMVDINATYSSLKNSQQQLQRLLIQHSAKVESVSSGAPSFTSVKGAISKQSPAVQN
DVQKGTFLSYRSQVHGNVQNQQLLTGAFMDWKLASAPKCGFKVALHYGSQDALVERAALPYTEQG
LGSSVLSGFGGQVQGRYDFNLGETWLQPFMGIQVLHLSREGYSEKNVRFPVSYDSVAYSAATSF
MGAHVFASLSPKMSTAATLGVERDLNSHIDEFKGSVSAMGNFVLENSTVSVLRPFASLAMYYDVR
4O QQQLVTLSWMNQQPLTGTLSLVSQSSYNLSF
The N-terminal peptide may be secreted to be exposed on the bacterial cell
surface
and can also become detached via the proteolytic event described above. The
peptide
may form a structural conformation known as beta-propellers indicated in the
following sequence:
SEQ ID NO: 86
HTLVDIAGEPRHAAQATGVSGDGKIVIGMKVPDDPFAITVGFQYIDGHLQPLEAVRPQCSVYPNG
5O ITPDGTVIVGTNYAIGMGSVAVKWVNGKVSELPMLPDTLDSVASAVSADGRVIGGNRNINLGASV
AVKWEDDVITQLPSLPDAMNACVNGISSDGSIIVGTMVDVSWRNTAVQWIGDQLSVIGTLGGTTS
VASAISTDGTVIVGGSENADSQTHAYAYKNGVMSDIGTLGGFYSLAHAVSSDGSVIVGVSTNSEH
RYHAFQYADGQMVDLGTLGGPESYAQGVSGDGKVIVGRAQVPSGDWHAFLC
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Furthermore, the N-terminal passenger domain can also possess a specific
protease
activity, such as a serine protease-like activity. In addition to acting on a
variety of
substrates, the protease activity may act on the membrane anchored form of the
molecule such that the N-terminal passenger domain is cleaved off form the
surface of
the chlamydial cell. The serine protease like activity is supported by the
presence of a
consensus serine protease triad of adequately spaced amino acid residues
(namely H,
D and S) which can be located on the virtual structure of the 'passenger'
domain
modelled on a set of experimentally-determined templates, e.g. lnr0 (PDB
identification code)
Based on the above analysis, the gene Cpn0796 gene encodes for a protein which
promotes its own secretion on the EB surface and may also mediate or promote
its
own release into the surrounding medium. The secreted passenger peptide has
several
activities, including:
1. actin binding peptide, part of a chlamydial surface layer, and instrumental
to
the process of establishing the host cell infection
2. specific protease activity within the host cell cytoplasm instrumental to
the
intracellular survival of infecting chlamydiae.
3. specific activity within the host cell cytoplasm to down regulate
expression of
selected genes, either by repressing their transcription andlor by repressing
their translation (m-RNA degradation)
4. cooperation with the products of genes Cpn0794, 0795, 0797, 0798, 0799
5. another function of the above N-terminal beta propeller domain is the
regulation/ modulation of the activity of a cytosolic protease of the host
cell in
order to alter host cell properties in favour of chlamydial development,
survival or persistence. See Fulop V, Bocskei Z, Polgar L. in "Prolyl
oligopeptidase: an unusual beta-propeller domain regulates proteolysis." Cell.
1998 Jul 24;94(2):161-70.
The proteins encoded by Cpn0794, Cpn0797, Cpn0798, Cpn0799 - all comprising
variants of the above described Cpn0796 structure - also provide beta
propeller
structures with activities similar and/or complementary to the ones described
above.
Thus, a family of proteins cooperating to a common function either by
generating -
through events of site specific recombination - new molecules with structures
and
activities similar to the above described Cpn0796 product, OR by independently
contributing to a multi-protein structure requiring a coordinated action of
several
related components.
FIG. 9 illustrates an alignment of the C-terminal domains of the proteins
encoded by
C.pneumoniae genes Cpn0795 and Cpn0796. As seen in FIG. 9, beta barrel domains
of Cpn0795 or Cpn0796 include MKDLGTLGG (SEQ ID NO: 87), SXDGK (SEQ
ID NO: 88) VIVG (SEQ ID NO: 89), VIXG (SEQ ID NO: 90) or HAF (SEQ ID
NO: 91).
Fourth Group of P~~oteius
Cpn0498
So in this case the triple parallel-screening evaluation, with two positive
and one
negative result, brought to the identification of a previously unknown antigen
(ie an
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antigen with unknown biological function) having, according to current views,
just
the desirable basic properties in terms of antigenic function of a vaccine
candidate.
Further characterization of Cpn antigen data is included in Fihco et al.,
"Identification
of New Potential Vaccine Candidates Against Chlamydia pneumoniae by Multiple
Screenings," Vaccine, 23 (2005) 1178-1188, incorporated herein in its
entirety.
Exa~zzple 5
Background
The main stages in the Chlamydial life cycle are: .
(i) the binding to the host cell surface and entry into the cytoplasm through
a
specialised vacuole (the Chlanzydial inclusion) by an extracellular spore-
like infective form, called the elementary body (EB); and
(ii) the conversion of the EB to a non-infective replicative form called a
reticulate body (RB) that replicates by binary fission a number of times
within the inclusion to form a microcolony.
The sets of genes which are expressed in the various stages of the Chlanaydial
life
cycle and the signals that trigger the passage from one stage to the next are
largely
unknown and still need investigation.
Protein microarrays are used for high throughput protein analysis by detecting
proteins and monitoring their expression levels. Through use of protein
microarrays,
complex screening of thousands of proteins and interactions with proteins may
be
performed in parallel. A protein array typically includes a surface, such as
glass,
membrane, microtiter wells, mass spectrometer plates, beads or other
particles, for
binding ligands, proteins, or antibodies. For example, antibodies may be bound
to the
microarray to form a capture array. The capture array may be contacted with a
biological sample to quantify the proteins in the biological sample. Also,
proteins may
be bound to the microarray and contacted with a biological sample to quantify
protein-protein or protein-ligand interactions. Thus, protein microarrays may
also be
used in diagnostics in which multiple immunoassays may be conducted in
parallel
such that levels of proteins in different samples may be quantified and
compared for
applications in the treatment or diagnosis of disease.
For example, in a capture array, antibodies are bound to the microarray and
exposed
to a biological sample. Proteins and ligands that bind to the antibody array
may be
detected by direct labelling of the bound proteins. If a higher sensitivity or
specificity
is desired, a sandwich technique may be employed in which pairs of antibodies
are
directed to the same protein ligand. This technique is particularly useful if
the amount
of protein to be detected is low or if there are modifications to the protein.
In addition,
the use of sandwich assays minimizes the risk of cross-reactivity in highly
multiplexed assays by providing dual level target recognition, i.e. two levels
of
specificity for each locus in the array. Alternatively, the bound proteins may
be
detected via label-free detection methods such as including mass spectrometry,
surface plasmon resonance and atomic force microscopy. This technique is
useful if
modification or alteration of the protein is to be avoided.
Also, Large-scale functional chips containing large numbers of immobilized
purified
proteins may be used to assay a wide range of biochemical functions, such as
protein
interactions with other proteins, drug-target interactions, enzyme-substrates,
etc. Such
proteins may be purified from an expression library, for example, and the
protein
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array can be used to screen libraries to select specific binding partners,
including
antibodies, synthetic scaffolds, peptides and aptamers. In this way, 'library
against
library' screening can be carried out, such as screening of drug candidates in
combinatorial chemical libraries against an array of protein targets
identified from
genome projects.
Protein microarray technology permits analysis of the proteins themselves
rather than
inferring protein function, interactions and characteristics through mRNA
expression.
In many cases, mRNA expression does not correlate accurately with protein
abundance. Furthermore, mRNA expression analysis does not provide sufficient
information on protein-protein interaction or post-translational
modifications. Thus,
direct analysis of proteins via protein microarrays provides an advantage by
providing
more accurate information of proteins and protein-protein interactions that
may not be
readily available through measurment of mRNA expression.
Current DNA microarray techniques permit profiling of gene expression at the
mRNA
level as a function of the cellular state. This can lead to the identification
of genes or
clusters of genes whose up- or down-regulation is associated to a particular
state of
the cell and to the identification of therapeutically relevant targets. Using
this
technology, DNA fragments representing specific portions of all genes
belonging to a
given organism (the fragments can be derived from cDNA libraries or can be
obtained
by PCR amplification and chemical synthesis) are chemically bound to the
surfaces of
solid supports (chips) at high densities and in an ordered manner. Currently
up to 10,
000 DNA fragments or 250, 000 oligonucleotides can be spotted onto a single
squared
centimetre of chip surface. The DNA chips are then utilised to define which of
the
spotted genes are transcriptionally active in a particular cellular
population. To do so,
RNA is prepared, labelled with fluorescent dyes and finally hybridised to the
DNA
fragments fixed to the surface of the chip. By using an appropriate computer-
assisted
fluorescence detector, the fluorescence signals emitted by each spot upon
excitation
with a laser beam is carefully quantified to define the transcription activity
of all the
arrayed genes.
CPn DNA microarrays have been developed to look at the transcriptional events
which occur when a given CPn pathogen gets into contact with the host cells,
both in
in vivo and in vitro settings. DNA chips carrying the entire genome of a
particular
bacterium, such as the CPn bacterium can be prepared in a very short period of
time
so that whole genome expression analysis can be determined.
Experimental Methodology
Specifically, a genomic DNA (open reading frame probes) microarray approach
for
gene expression in CPn bacteria was adopted. In this respect, an array was
prepared
for the analysis of the CPn life cycle on the basis of the published
annotation of the
complete genome. The Chlamydia DNA chips carry about 1000 PCR-derived DNA
fragments, which have an average size of 400-700bp and correspond to internal
portions of all CPn annotated genes.
Results 5
Table 3(i)-(xi) shows transcriptional activity for expressed genes for CPn EB
selected
by microarray. The data in Tables 3(i)-(iv) shows different mRNAs in order of
abundance from cells in their infectious "spore-like" (EB) form. Data in
Tables 3(v)
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(xi) correlates and summarizes mRNA expression levels of genes for CPn. The
cells
were used at the end of their cycle where gene expression is likely to be at
its highest.
As values less than approximately 10000 is likely to be background, the top
set of
proteins (approx top 30) with mare intense signals are likely to be the most
interesting proteins.
A review of the values for the expressed genes indicates that three of the
FACS
positive CPn antigens (CPn0331 (hypothetical), CPn0234 (hypothetical) and
CPn0572 (hypothetical) are all highly expressed genes.
Table 3(v)-(xi) shows the transcriptional activity for expressed genes for CPn
EB
selected by microarray. The Table shows different mRNA in order of abundance
from
cells in their infectious "spore-like" (EB) form. The cells were used at the
end of the
cycle where gene expression is likely to be at its highest. A review of Table
3(i)-(iv)
and (v)-(xi) indicates that CPn antigens CPn0558 (OmcA), CPn0331
(hypothetical),
CPn0539 (Pmpl9), CPn0234 (Hypothetical) and CPn0572 (Hypothetical) are all
relatively highly expressed genes.
Where possible, attempts were made to place the transcriptional activities
disclosed in
Table 3(v)-(xi) in the context of the Chlamydia developmental cycle In this
respect,
Chlamdydia late gene products have been described more frequently than early
gene
products. This is primarily because of the presence of late gene products in
EBs but
not RBs and that it is easier to study EBs rather than RBs.
In addition, late gene functions appear to be predominantly those associated
with the
terminal differentiation of RBs back to EBs (Shaw et al., Mol Microbiology
37(4),
2000, 913-925). Late gene products appear to function in the termination of
bacterial
cell division and constitute structural components and remodelling activities
involved
in the formation of the cross-linked outer membrane complex that functions in
the
attachment and invasion of new host cells. By way of example, an important
aspect
of the secondary differentiation process (RB to infectious EB) is the
expression of
genes that encode proteins that form the highly disulfide cross-linked
bacterial outer
membrane (OM) complex. It is thought that several late cycle genes encode
proteins
with potential roles in the formation and maturation of the OM complex, a
crucial step
in the development of infectious EBs (see Belland et al., PNAS (USA) 100(14),
2003,
8478-83). The late genes omcA and omcB encode two cysteine-rich OM proteins
that
interact with the major OM protein (OmpA) to form this complex. A key protein
component of the OM complex, the OmcB protein, has been found to undergo post-
translational proteolytic processing. We have found that OmcB and OmcA show
high
levels of transcriptional activity (see top of Table 3(ii)). Cpn 0384 whose CT
equivalent is CT046 (hctB) has been shown to be associated with
differentiation from
RB to EB (see Belland et al., PNAS (USA) 100(14), 2003, 8478-83). We also
found
Cpn0384 to have relatively high levels of transcriptional activity (again see
top of
Table 3(v)-(xi)). Other Cpn antigens thought to be involved in the Type III
secretion
system were found to have moderate expression levels in terms of
transcriptional
activity. This fording appears to be in line with published commentary where
it is
thought that while transcription of the two putative structural components of
the Type
III secretion system (yscJ and yscN (Cpn669)) begins at mid-cycle, export of
effector
molecules may be at a different stage of the developmental cycle.
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Table 3(v)-(xi) indicates that high transcriptional activity was observed for
Cpn0539
(CT412) which corresponds with a 98I~da protein known either as PmpA or Pmpl9.
Even though the Pmpl9 protein demonstrates relatively "high" levels of
transcriptional activity, this result is interesting because mRNA abundance
for pmpl9
does not seem to correlate with protein abundance. In this respect, results
from our
laboratory have shown that (i) Pmpl9 was not detected in either 2D maps,
Western
Blots or FACS analysis studies which suggests that the pmpl9 protein either is
not
surface exposed even though high levels of mRNA are expressed or that (ii)
Pmpl9
protein is expressed but processed or degraded by proteolytic digestion
rendering it
undectable by immunoblot analysis. The results in our laboratory are confirmed
by
others. In this respect, Grimwood et al (2001) Infection and Immunity 69(4)
2383-
2389 have shown that transcriptional profiles were detected for each of the
Chlamydia
pneumoniae 21 Pmp genes demonstrating that all pmp genes are transcribed
during
infection. Since each of the Pmp genes was transcribed, Grimwood et al (2001)
evaluated protein expression by irmnunoblotting of Chlamydia pneumoniae CWL029
EB lysates using peptide-specific antisera. Interestingly, no Pmp-specific
reactivity
was detected for sera from either PmpA (Pmpl9) or PmpB/C and PmpD gene by
immunoblot analysis regardless of high antipeptide reactivity. This result
suggested
that these proteins either are not stable or not translated. These findings
demonstrate
that there appears to be a variation in Pmp expression for the Chlamydia
p~eumoniae
family of 21 polymorphic membrane proteins (Pmps) which are predicted to be
localised to the bacterial outer membrane. The function of Chlamydial Pmps
remains
unknown, although based on sequence prediction and experimental testing, these
Pmps are regarded as surface proteins and thus, likely to be critical for
Chlamydial
virulence. Like the Inclusion (Inc) Membrane proteins, the Pmp proteins are
regarded, at present, as unique to the Chlamydiae family (see Rockey et al
(2000)
Infection and Immunity 69(10) 5473-5479). The findings disclosed here and by
others, such as Grimwood et al, demonstrates that the Chlamydia organism
appears to
expend a considerable metabolic cost in Pmp transcription, such as Pmpl9
transcription, despite the potential lack of production of a functional Pmp
proteins,
such as the Pmp 19 protein.
Materials and Methods (Examples 6-8) (Reference Section II)
T cell Epitope prediction and peptide synthesis
T cell epitope prediction was carried out on the genomic sequence of C.
pneumof2iae
CWL029 strain (Accession numbers NC 000922 or AE001363) using the BIMAS
algorithm [24]. Synthetic peptides (purity > 80%) were synthesized by Primm
Srl
(Milan, Italy), suspended in 100% DMSO and kept at -20° C before use.
RMA-S/A2 cell line and HLA-A2 transgenic and non transgenic mice
The T cell lymphoma marine cell line RMA-S stably transfected with HLA-A2
(RMA-S/A2, H-2b , TAP2-), was kindly provided by Dr. Barnaba, Universita degli
Studi "La Sapienza", Rome, Italy, and cultured at 37° C in RPMI-1640
(GIBCO)
supplemented with heat inactivated 10% FCS, 100 IU/ml penicillin/streptomycin,
2
mM Lglutamine (GIBCO) and 510-5 M 2-ME (Sigma). H2-b HLA-A2 transgenic
mice [35] were housed in a pathogen-free environment and screened for HLA-A2
expression by FCM carried out on total blood samples using the BB7.2 anti-A2
mAb
[48]. Only mice with percentages of A2 expressing cells higher than 70-80 %
were
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used for DNA immunization and C. pneumoniae infection experiments. Animals
which showed no HLA-A2 expression were mated in order to obtain an HLA-A2 non
transgenic population, to be used as a control in the experiments.
Epitope stabilization assay
RMA-S/A2 cells (3-5 x 105/well) were seeded in serum-free RPMI medium,
supplemented with human (32 microglobulin (3 ~,g/ml, Sigma), without or with
the
test peptide (10~M). Following overnight incubation at 26°C in
humidified 5% COz
atmosphere, cells were shifted to 37° C for 2 h before determining the
HLA-A2
expression level at the cell surface using the BB7.2 anti-A2 mAb and a PE-
conjugated
anti-mouse IgG (Jackson ImmunoResearch). Fluorescence intensity on living
cells,
which did not incorporate propidium iodide, was analyzed by FCM. As controls,
corresponding samples without peptide and samples with peptide but treated
only
with the anti-mouse secondary antibody, were used.
Infection and DNA immunization of HLA-A2 transgenic and non transgenic
mice
Transgenic mice were intranasally infected twice with a month interval, using
Sx105
C. pneumoniae FB/96 EBs [4] diluted in 50 ~,l of PBS. C. pneumoniae antigen
coding
genes were amplified by PCR using FB/96 genomic DNA, cloned into plasmid
pcmvKaSF2120 [49] and verified by DNA sequence analysis. Fifty p,g of
endotoxin
free recombinant plasmid DNA, diluted in Dulbecco's phosphate buffer (GIBCO),
were injected into the tibialis muscle of mice at days 0, 21 and 35.
CD8+ T cells isolation and IFN-y determination by ELISpot assay
Splenocytes from DNA immunized mice were prepared one week after the third
immunization using CeII Strainer (Falcon) filters. Following red blood cells
Iysis,
CD8+ T cells from spleen cells suspensions were enriched by positive selection
using
magnetic activated cell sorting (MACS-Miltenyi Biotec) with CDBa (Ly-2)
microbeads. CD8+ T cells purity was higher than 90%, as determined by FMC.
Multiscreen 96-well nitrocellulose plates (Millipore) were coated with 5 ~g/ml
of the
anti-mouse IFN-y antibody (R4-6A2, PharMingen) in 100 p1 of carbonate buffer,
pH
9.2. After overnight incubation at 4°C, plates were saturated at
37°C with 200 ~1 of
BSA (1%) in PBS for 2 h. Purified CD8+ (5x104) were added in a total volume of
200
~,1/well in the presence of irradiated (3,000 rad) spleen cells from non
immunized
HLA-A2 transgenic mice as a source of antigen-presenting cells (2x105/well),
10
~.g/ml of peptide and l0U/ml of human r-IL-2 (Chiron Cozporation). After
incubation
for 20 h at 37° C, 5% COz, plates were washed and developed for bound
IFN-y using
sequential incubations with biotinylated antimouse IFN-y (XMB 1.2,
PharMingen),
ExtrAvidin-alkaline phosphatase and substrate BCIP/NBT (Sigma) dissolved in
water. Spots were enumerated in each well using a dissecting microscope.
Medium
containing an irrelevant peptide or without peptide was used as negative
controls,
while positive controls were represented by CD8+ T cells treated with ConA (5
~,g/ml).
In vitro cultures and flow cytometric analysis of splenocytes from transgenic
and
non transgenic mice infected with C. ptzeufrtoniae
Splenocytes from infected mice were isolated one week after the second
infection
with C. pneunaoniae Ebs. For ex vivo analysis of IFN-y production, 2x106
splenocytes
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were seeded in the presence of the test peptide (10~,g/ml) and anti-mouse CD28
antibody (l~,g/ml, PharMingen) as co-stimulus. After a two h incubation at
37° C, 5
COZ, Brefeldin A (10 ~,g/ml, Sigma) was added and the incubation was extended
for 4 additional hours. Following two washes with PBS, cells were
penneabilized,
fixed and stained both with anti-marine-IFN-y-(PE), anti-marine CD8 (APC) and
anti-marine-CD69 (FITC) and the corresponding isotypes. Positive controls for
cytokine production were performed on cells stimulated with anti-mouse CD3 and
CD28 antibodies (1 ~.g/ml, PharMingen) . Cells cultured either in the absence
of
peptide or pulsed with the HepB negative control peptide were used as negative
controls. All samples were analyzed using a FACS LSRII flow cytometer (Becton
Dickinson). For analysis of IFN-y production by short term T cell lines, 5-
10x106
splenocytes from infected mice were cultured for 6 days in the presence of the
test
peptide (20 ~g/ml), with rIL-2 (10 ~,glml) being added after the first two
days. At the
end of the incubation period, cells were washed twice in RPMI, pulsed again
for 6 h
in the presence of the test peptide (l0~ghn1), 1x105 freshly prepared CD8
depleted
antigen presenting cells from HLA-A2 transgenic mice (irradiated at 3000 rad)
and
anti-mouse CD28 antibody (lp,glml, PharMingen) as co-stimulus. After a two h
incubation at 37° C, 5 % COZ, Brefeldin A (10 ~g/ml, Sigma) was added,
the
incubation was extended for 4 additional hours and IFN-y production was
analyzed by
FCM.
Example 6
In silico analysis of Chlamydia pheutnohiae genome and prediction of HLA-A2 T
cell epitopes
The genome of the Chlamydia pneurnoniae CWL029 strain was used to predict 9mer
peptide sequences with high probability to bind class I HLA-A2 molecules. The
analysis was carried out using the predictive algorithm available at the NIH
Bioinformatics & Molecular Analysis Section Web server
(http://bimas.cit.nih.gov~,
which ranks potential MHC binders according to the predictive half time
dissociation
of, peptide/MHC complexes [24]. Since some Clzlanaydial proteins have been
reported
to induce autoimmune responses [25-28], we restricted our search to a subset
of
proteins, distinctive of the Chlamydia genus, consisting of 13 protein
identified as
members of the type III secretion system, 17 Polymorphic Membrane Proteins
(PMP)
and 19 additional proteins, 5 of which already identified as EB surface
antigens [4].
The predicted binding score of 157.22, obtained for the well characterized HIV-
1 p17
gag epitope 77SLYNTVATL85 [29], was taken as an arbitrary cut-off for peptide
selection. A total of 55 potential C. pneumoniae-derived T cell epitopes,
belonging to
31 different proteins, were identified (Table I), which had predicted binding
scores
ranging from 156.77 to 42,485.263
In vitro binding of peptides to HLA-AZ
The capacity of the selected peptides to bind to HLA-A2 was assessed using an
in
vitro MHC class I stabilization assay, carried out with the marine transporter
associated with antigen processing (TAP)-deficient cell line RMA-S/A2, stably
transfected with the human class I A2 gene. MHC class I molecules, cultured at
37°
C, are unstably expressed on the cell surface of TAP-deficient cells [30-32].
Culturing
the cells at 37° C in the presence of binding peptides, results in
formation of a more
stable MHC/peptide complex which can be monitored by flow cytometric analysis.
RMA-S/A2 cells were therefore cultured overnight at 26° C in the
presence of the
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test peptides, shifted to 37° C for 2 hours and the surface level of
stabilized A2
molecules was quantified by direct staining with an anti-HLA-A2 specific mAb.
Two
known HLA-A2 restricted CTL epitopes were used as positive controls for
binding to
A2, the HIV-1 p17 gag peptide [29] and the influenza matrix M1 protein peptide
FluMP58 [33], while the Hepatitis B virus envelope antigen peptide HbenvAg125
(HepB) was used as a negative control [34].
Results 6
The binding results obtained are shown in Table 4 and allowed the
identification of 15
peptides with a net mean fluorescence intensity (Net MFI) higher than 92.3,
corresponding to the value obtained with the HIV-1 p17 gag positive control
peptide,
8 peptides with a Net MFI intermediate between the values 92.3 and 63.1,
obtained
with the two positive control peptides, and 12 peptides with an Net MFI
ranging
between 29.6 and 63. Fifteen of the in silico predicted peptides (27.2 %) did
not
confer stabilization to the A2 molecules, showing a Net MFI lower than 14,
obtained
with the HepB negative control peptide.
Exanzple 7
Some HLA-A2 binders are recognized by CD8+ T cells from DNA-immunized
transgenic mice
The in vitro assay with RMA-S/A2 cells allowed the definition of a set of
peptides
which were able to bind and stabilize the HLA-A2 molecules on the cell
surface. To
gain information about the possibility that the predicted epitopes could
indeed be
generated by in vivo processing of the antigens from which they were derived,
peptide recognition by CD8+ T cells was studied under conditions in which the
related complete antigen was intracellularly expressed and presented in vivo.
The
full-length ORF sequences coding for 13 Chlanaydial proteins, including a
total of 24
predicted peptides, were cloned into a suitable DNA expression vector and each
recombinant plasmids was used to immunize distinct groups of transgenic mice
expressing a chimeric class I molecule composed of the a,1 and a,2 domains of
HLA-
0201 and the oc3 domains, transmembrane and cytoplasmic, of H-2Kb [35].
The ORF sequences were selected among those containing either one or more
epitopes positive in the in vitro assay or a combination of positive and
negative
epitopes. The ORF sequence corresponding to the outer membrane protein A
(OMPA,
CPn 0695) was included in this analysis, since human MHC-I-restricted epitopes
have
already been reported for this protein in C. traclaornatis [18;36]. One coding
sequence,
related to gene CPn 0131 was chosen, which included four epitopes, all
negative in
the in vitro stabilization assay. After three immunization cycles, transgenic
mice were
sacrificed, spleen CD8+ T cells were isolated, stimulated for 20 hour with the
corresponding peptide and ex vivo IFN-y production was assessed using an
enzyme-
linked immunospot (ELISpot) assay.
Results 7
DNA-mediated expression of the ORFs including peptides CH-6 (CPn 0811), CH-7
(CPn 0623), CH-10 (CPn 0828), CH-13 (CPn 0695, OMPA) and CH-37 (CPn 0210)
were associated with numbers of spot forming cells (SFC) significantly higher
than
those obtained with the HepB unrelated peptide, whereas some peptides related
to
antigens coded by genes CPn 0131, CPn 0323 and CPn 0062 induced SFC values
only 2-3 times higher than the HepB control peptide (Table 5). Peptides
related to
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antigens coded by genes CPn 0132, CPn 0322, CPn 0325, CPn 0415 and CPn 0728
did not induce any IFN-y production (data not shown).
Exaynple 8
To test the capacity of peptides to amplify specific CD8+ T cell populations
in vitro,
some of these plasmids were used to repeat the DNA immunization experiment and
to
determine by flow cytometry the intracellular IFN-y production by CD8+ T
cells, both
ex vivo and after a 6 day stimulation in the presence of the relevant
peptides. In the
attempt to establish a direct correlation between IFN-y production by CD8+ T
cells
and HLA-A2 specific restriction, the experiment was carried out with both
transgenic
and non transgenic syngenic mice. The plasmids used contained genes CPn 0695,
CPn
0811 and CPn 0823, including peptides CH-13, GH-6 and CH-7 respectively, which
were highly positive in the ira vitro binding and in the ELISpot assays and
gene CPn
0323, including six different peptides, all of them with ELISpot values
slightly higher
than background
Results 8
The results of the experiment are summarized in Table 6, while representative
dot
plots from flow cytometric analysis of splenocytes stimulated with peptide CH-
6 are
shown in Fig. 4. When fresh spleen cells of DNA-immunized transgenic mice were
pulsed with the tested peptides, only CH-6 or CH-7 induced relative fold
increase
(RFI) values about 5 times higher than those obtained pulsing the same cells
with the
HepB negative control peptide (Table 6, 4.58 and 5.2 RFI respectively).
When short term T cell lines (TCLs) instead of fresh splenocytes were used, a
larger
panel of peptides were able to trigger a significantly higher IFN-y production
by CD8+
T cells (Table 6). In fact, in addition to peptides CH-6 and CH-7, also
peptides CH-
13, CH-44, CH-45 and CH-46 were recognized by CD8+ T cell populations
significantly larger than those induced by pulsing the same cells with the
HepB
peptide (12FI > 5). Importantly, peptide-induced IFN-y production, appeared to
be
largely HLA-A2-dependent, since when the same experiments were carried out
with
non transgenic mice, the RFI values obtained were reliably lower (Table 6).
The fact
that non transgenic and transgenic spleen cells were both efficiently
activated using
the polyclonal stimulus (anti-CD3/anti-CD28), reinforced the hypothesis that
the
lower CD8+ T cells induction in non transgenic mice was due to the absence of
specific interactions between the peptides and the human HLA-A2 molecules.
CD8+ T cells of transgenic mice infected with C, pheunaoniae recognize HLA-A2
binders i~a vivo
It has been recently shown that infection of mice with C. pneurnoraiae elicits
a
pathogen-specific marine class I-restricted immune response [22]. Therefore,
we
asked whether any of the A2 ira vitro binders could be recognized by specific
CD8+ T
cells that are clonally selected during the immune response raised against the
corresponding native antigen in C. pneumoniae infected cells.
To address this issue, HLA-A2 transgenic mice were intranasally infected with
a non
lethal dose of C'. praeumoniae EBs and challenged with an equal dose of
bacteria one
month later, before being sacrificed to obtain splenocytes that were used to
measure
IFN-y production by CD8+ T cells. Since no appreciable IFN-y-production could
be
observed if splenocytes from infected mice were tested directly ex vivo (data
not
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shown), spleen cells were cultured with each individual peptide or with the
HepB
irrelevant peptide for 6 days. The resulting short-term TCLs were then pulsed
again
for 6 hours with the same peptides and intracellular IFN-y production by CD8+
T cells
was assessed. The results obtained with 40 tested peptides are shown in Fig.
5A.
Sixteen peptides (CH-2, CH-7, CH-8, CH-10, CH-13, CH-15, CH-20, CH-21, CH-28,
CH-35, CH-37, CH-45, CH-46, CH-47, CH-50 and CH-55) elicited the strongest
CD8+ responses (1 to 7.1 % of IFN-y-producing CD8+ T cells), while 19 peptides
elicited low but consistent responses (percentages of CD8~/IFN-y T cells
between
0.3 and 0.9). Five peptides did not induce percentages of IFN-y-producing CD8+
T
cells significantly higher than those observed in response to the HepB control
peptide.
When eight among the most reactive peptides were used again to pulse
splenocytes of
both transgenic and non transgenic mice infected with C. pheunzoyziae, seven
of them
were recognized by specific CD8+/IFN-y+ T cell populations present only in the
transgenic mice, while peptide CH-7 was recognized by CD8+ T cells present in
both
mice groups (Fig. 5B).
General Discussion of Results in Examples 6-8
In this work we have described peptides derived from C. pheumo~iae antigens
identified as putative T cell epitopes recognized by the common human class I
MHC
A2 haplotype.
Understanding C. pneumohiae-specific CD8~ T cell-mediated immune response and
designing protective vaccines rely on the possibility of identifying bacterial
antigens
or epitopes recognized by CD8+ T cells. Whereas the induction of a CTL-
dependent
immune response is predictable in response to pathogens which replicate in the
cellular cytosol, providing antigens which can enter the cellular MHC-I
presentation
pathway, in the case of Clalamydiae it is not immediately obvious which
antigens are
made available to the proteasome and how they reach the cytosol, since these
bacteria
have a stringent intravacuolar localization inside the infected cell.
We have chosen an in vivo system based on HLA-A2 transgenic mice to test which
of
the predicted peptides could be recognized by specific CD8+ T cells following
either
DNA immunization with individual antigen coding genes or infection with C.
pfaeumoniae. Our choice of a murine model is supported by previously published
data.
Wizel et al. [22], recently reported the first evidence that CD8+ T cells
specific for G
pyaeumoniae antigens are induced in infected mice, and identified bacterial-
derived
murine MHC-I-restricted T cell epitopes able to trigger either lysis of C.
pneumoniae
infected cells or irc vitro inhibition of the pathogen intracellular growth.
These
findings seem to confirm that some C. pheumoniae antigens can indeed reach the
cytosol of infected cells and enter the MHC-I presentation pathway, i.e.
during
remodeling that occurs during ClalanZydia replication or following autolysis
of
developing bacterial particles [22].
Furthermore, Kuon et al. [42] recently reported the identification of 11 C.
trachonaatis-derived HLA-B27-restricted peptides, capable of stimulating CD8+
T
cells obtained from patients with Clalarnydia-induced reactive arthritis.
Importantly, 8
of them overlapped those selected by analyzing splenocytes of HLA-B27
transgenic
mice infected with C. trachomatis, indicating that antigen processing can be
closely
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reproduced using the marine animal model, although differences between marine
and
human antigen processing and T cell repertoires have been hypothesized [43].
The experiment which we have performed with C. pneunZOn.iae infected A2
transgenic
mice revealed that at least 16 peptides were recognized by IFN-y-positive CD8+
T cell
populations, which were actually expanded as a consequence of bacterial
infection,
since we could not detect IFN-y production pulsing spleen cells from non
infected
transgenic mice with the same peptides (data not shown). These results suggest
that
the corresponding Chlaniydial antigen may be able to enter the MHC-I
presentation
pathway. The fording that a number of these peptides can also be recognized by
specific CD8+ T cells when the corresponding protein is separately expressed
by DNA
immunization, strongly reinforces the hypothesis that the responses observed
with the
infected mice are indeed specific for the in silico predicted peptide epitopes
and their
corresponding antigens. Importantly, the comparisons of peptide-induced IFN-y-
positive CD8~ T cells in A2 transgenic and non transgenic mice, either
immunized
with DNA or infected with C. pneumon.iae, indicate that this recognition event
is
largely A2-specific.
Though, we cannot rule out the possibility that some of the tested peptides
are also
able to interact with the marine class-I MHC molecules, as suggested by the
result
obtained with CH-7 in infected non transgenic mice (Fig. 5) and by the RFI
values
obtained with CH-7, CH-8 and CH-13 in DNA-immunized non transgenic mice
(Table 6).
Both with DNA immunization and bacterial infection, we were able to show that
the
OMPA-derived CH-13 peptide induces a specific CD8+ T cell response in A2
transgenic mice. These results appear to validate the choice of this animal
model,
since our observation that OMPA can enter the MHC-I presentation pathway
correlates with the previous identification of HLA-A2-restricted and of marine
MHC-I-restricted epitopes in OMPA proteins of C. trachomatis [18] and of C.
pneumoniae [23] respectively. With the exception of CH-13 and CH-17, all the
other
peptides recognized by CD8+ T cells of infected mice belong to C. pneumoniae
antigens for which neither human nor marine T cell epitopes have been
identified
[22;23]. Interestingly, a couple of positively reacting peptides (CH-50 and CH-
55)
belong to the group of polymoiphic outer membrane proteins [44;45], while most
of
the others are part of the group of Type III secretion system-related proteins
[45;46].
Peptides CH-7 and CH-8, both included in protein T of the Yersinia outer
protein
(Yop) system [47] and CH-10, included in protein J, which is part of the same
translocation system, appear to be particularly reactive in the assay with the
infected
mice (Fig. 5A).
This is also true for other peptides included in antigens which are again
related to the
type III secretion system, such as CH-45, CH-46, and CH-47, all present in the
low
calcium response protein D. Intriguingly, CH-8, which is the most reactive
peptide in
the assay with the infected mice, does not seem to be recognized by a specific
T cell
population when the corresponding antigen is expressed by DNA immunization
(Tables 5 and 6). This may be due to different factors, i.e. low ira vivo
expression level
of the injected DNA or altered protein conformation.
On the other hand, we should also consider the possibility that, following
infection of
mice with C. pneumoniae, this peptide is recognized by a CD8+ T cell
population
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which is instead specific for an epitope derived from an unidentified C.
pneunZOniae
antigen having a closely related sequence. Contrarily to CH-8, stimulation of
spleen
cells from infected transgenic mice with peptide CH-6 did not allow the
detection of
IFN-y~/CD8+ T cells (Fig. 5A), but the same peptide was clearly reactive in
the DNA
immunization experiments (Tables 5 and 6). This may suggest that Low Calcium
Response Protein H is not available for the cellular proteasome, but we could
also
assume either that the amount of peptide available to the MHC-presenting
machinery
is not sufficient to induce a cell response which is detectable with our
assay, or that
the reacting CD8+ T cell population does not expand over the detection limit
of our
assay.
On the whole, the results presented here allowed the identification of a
number of
antigens which may be available for proteasome-mediated processing in the
course of
C. przeumoniae infections, proposing them as possible targets for a HLA-A2-
dependent cellular immune response. Further analysis will be required to
verify if the
specifically induced CDB~ T cells are able to recognize and induce lysis of
peptide
pulsed or C. pneurnoniae infected mammalian cells and, possibly, to correlate
the
identified T cell epitopes with CD8+ T cell populations naturally induced in
C.
pneurnoniae infected patients. Importantly, the results obtained with DNA-
mediated
expression of distinct antigens, can represent an initial step towards the
definition of a
significant set of C. pneumoniae HLA-A2-restricted epitopes, which could
eventually
be combined in DNA minigenes in the attempt to induce a CTL-dependent anti-
Chlarnydia protective immune response
Example 9
Immunizations with Combinations of the First Antigen Group
The five antigens of the first antigen group (OmpH-like protein, pmp 10, pmp2,
Enolase, OmpH-like, CPn0042 and CPn00795 were prepared as described in the
Materials and Methods Section above for Examples 1-4. The antigens are
expressed
and purified. Compositions of antigen combinations are then prepared
comprising
five antigens per composition (and containing 15 ~,g of each antigen per
composition).
CD1 mice are divided into seven groups (5-6 mice per group for groups 1
through 4; 3
to 4 mice for groups 5, 6 and 7), and immunized as follows:
Group Immunizing Composition Route of
Delivery
1 Mixture of 5 antigens (15 ~g/each) Intra-peritoneal
+ CFA
2 Mixture of 5 antigens (15 ~,g/each) Intra-peritoneal
+AIOH (200~g)
3 Mixture of 5 antigens (15 ~,g/each) Intra-peritoneal
+ AIOH (200~g) +
C G (10~,g)
4 Complete Freunds Adjuvant (CFA) Intra- eritoneal
5 Mixture of 5 anti ens (5 ~g/each) Intranasal
+ LTK63 (5~,g)
6 AIOH (200~,g) + CpG (10~g) Intra-peritoneal
7 LTK63 (S~,g) Intranasal
Mice are immunized at two week intervals. Two weeks after the last
immunization,
all mice are challenged by intravaginal infection with Chlanaydia pneumoniae
serovars.
Experiment 9 was repeated with another group of CPn antigens. These were:
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CPn0385 (PepA), CPn0324 (LcrE), CPn0503 (DnaK), CPn0525 (Hypothetical) and
CPn0482 (ArtJ). These antigens are combined and administered with and without
alum and CpG as described in Experiment 9.
Summary
Applicants have identified a number of CPn proteins with desirable
immunological
and/or biological properties. Specifically, at least twelve CPn proteins have
been
identified which are capable of inducing the production of antibodies, which
can
neutralise, in a dose-dependent manner, the infectivity of C. pneurnoniae in
in vitro
cell cultures. The induction of neutralising antibodies is important because
it prevents
infectious EBs from invading human tissues. Furthermore, at least six of these
CPn
proteins were also capable of attenuating Chlanaydial (C. pneumoniae)
infection in a
ire vivo hamster model. In addition, some of these CPn proteins were also
capable of
inducing not only adequate T-cell responses but also high serum levels of
neutralising
antibodies.
Apart from very recent unpublished results on pmp2l, this is the first time
that antisera
to recombinant Amps (pmp2 and pmpl0) are reported to have neutralising
properties.
Interestingly, whilst antiserum against CPn0525 gave negative in vitro results
(ie no
neutralising activity), the CPn0525 protein gave 97 per cent protection from
spleen
infection in an in vivo hamster immunisation assay (see Table 2) (ie a
positive in vivo
result). Likewise, whilst antiserum against Cpn0498 gave negative in vitro
results (ie
no neutralising activity), the CPn0498 protein gave 94 per cent protection
from spleen
infection in an in vivo hamster immunisation assay (ie a positive in vivo
result). Thus
a positive signal obtained in the FACS assay does not guarantee a
corresponding
positive in vitro neutralization activity and conversely a negative
neutralization
activity does not mean that a positive ih vivo result can be excluded.
Some of the results obtained by screening the panel of recombinant antigens
with the
C.pneurnoniae ira vitro neutralization assay are shown in Table 2. Just by a
cursory
look at the 'current annotation' column it can be seen that both in Table 1
and 2 are
listed antigens, like the members of the family of heterogeneous polymorphic
membrane proteins (PMP), which, on the basis of published literature data,
could be
reasonably expected to be surface-exposed and possibly induce neutralizing
antibodies, but there are also proteins which could be considered so far only
hypothetical, and proteins which just on the basis of their current functional
annotation
could not be at all expected to be found on the bacterial surface.
The characterisation for the first time of some of these CPn proteins in terms
of not
only neutralising properties but also different score profiles in a panel of
screening
tests is an important contribution to the art because it facilitates the
selective
combination of CPn antigens with particular immunological and biological
properties.
In conclusion, this paper describes a group of recombinant antigens which can
induce
antibodies inhibiting the infectivity of C pneumoniae ira vitro and have
protective
effects in vivo.
All publications mentioned in the above specification are herein incorporated
by
reference. Various modifications and variations of the described methods and
system
of the invention will be apparent to those skilled in the art without
departing from the
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scope and spirit of the invention. Although the invention has been described
in
connection with specific preferred embodiments, it should be understood that
the
invention as claimed should not be unduly limited to such specific
embodiments.
Indeed, various modifications of the described modes for carrying out the
invention
which are obvious to those skilled in molecular biology or related fields are
intended
to be covered by the present invention.
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REFERENCE SECTION II (EXAMPLES 6-8)
1. Pizza,M., Scarlato,V., Masignani,V., Giuliani,M.M., Aricò, ,B.,
Comanducci,M., Jennings,G.T., Baldi,L., Bartolini,E., Capecchi,B.,
Galeotti,C.L., Luzzi,E., Manetti,R., Marchetti,E., Mora,M., Nuti,S., Ratti,G.,
Santini,L., Savino,S., Scarselli,M., Storni,E., Zuo,P., Broeker,M., Hundt,E.,
Knapp,B., Blair,E., Mason,T., Tettelin,H., Hood,D.W., Jeffries,A.C.,
Saunders,N.J., Granoff,D.M., Venter,J.C., Moxon,E.R., Grandi,G., and
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Mariani,M., Manetti,R., Agnusdei,M., Cevenini,R., Donati,M., Nogarotto,R.,
Norais,N., Garaguso,L, Nuti,S., Saletti,G., Rosa,D., Ratti,G., and Grandi,G.,
Genomic Approach for Analysis of Surface Proteins in Chlamydia pneurraoniae.
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5. Ross,B.C., Czajkowski,L., Hocking,D., Margetts,M., Webb,E., Rothel,L.,
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129
CA 02557353 2006-08-24
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134
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
'--' .
Chlamydia Further Filingi_.
Microarray Expressed Gene'lable
15-9-04 and 17-12-04
TABLE 3(v)-(xf)
CPn CT Protein MRNA Stage in ReferenceEB RB
the
FunctionlevelsChlamydial (see Surfacesecreted
developmentallegehd)exposed
c cle
CT081 CHLTR Very Late Nicholson
T2
rotein
0557 CT443 OmcB 43304.22Late Gene BellandEB
Shaw Surface
ex osed
0558 CT444 OmcA 42530.16Late Gene BellandYes
FAGS
pos
In vivo
protective
with
CT467
0695 CT681 MOMP Mid Late FAGS
II
ositive
0384 CT046 hctB Late Gene Belland
0331 CT082 Hypothetical33219.36Late NicholsonFACS
ositive
0811 CT576 LcrH Late Gene Belland
0474 CT365 Hypothetical ImmediatelEarlyBelland
0443 CT005
0808 CT579 Hypothetical Late Gene Belland Type
III
secretion
cluster
(WO
02/082091)
0134 CT110 GroEL Immediate (see FRCS
Early Table
Heat gene 1 of positive
shock
protein Belland
et
(Hsp-60) Midcycle aI (2003)
Midlate Nicholson
I
0499 Cpn
hypothetical
rotein
Yyd
conserved
hypothetical
rotein
CPn specific
rotein
0333 CT080 ltuB Late Gene Belland
0369 CT058
0539 CT412 Pmpl9 19039.90 FACS
ositive
0728 CT622
0809 CT578 Late Gene Belland
0676 CT695
1016 CT858 predicted Secreted
Protease Protein
containing (WO
IRBP 021082091)
E ito
a
135
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
Chlamydia Further Filingf -
Microarcay F,xpressed Gene Table
15-9-04 and 17-12-04
TABLE 3(v)-(ill
0234 CT181 Hypothetical FAGS
ositive
0588 CT469 H othetical
0065 CT2$8
0998 CT841 $pitope
(WO .
021082091)
0369 CT058
0810 CT577 Hypothetical Type
III
secretion
cluster
(WO
02/082091)
0875 CT734 Hypothetical Immediate Belland Secreted
Early
Protein
(WO
02/082091)
0127 CT034
0538 CT814 Late Belland
0482 CT381 ArtJ FAGS
ositive
0329 CT154-CT157 FACS
I58 Phospolipase positive
D
Superfamily
0572 CT456 Hypothetical8664.88 FAGS Cr456
(now positiveis
Tare) s~reted
from
Chiamydia
by
a Type
IIT
secretion
system
(T'TSS)
(Clifton
et al
2004)
and
is
translocated
into
the
cytoplasm
of
the host
cell.
New function
assigned
=
' Tarp
=
translocated
actin-recruiting
pliosphoprotein
CT456
gene
is
transcribed
from
mid
to
late
cycle
in the
Chlamydia
developmental
cycle
CT456
may
vary
across
the serovars
because
it
has tandem
re eats
0876 CT735
136
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WO 2005/084306 PCT/US2005/006588
Chlamydia Further Filing/'> .1
Microarray Expressed Gene Table
15-9-04 and 17-12-04
TABLE 3fv)-fxil
1024CT864
0726CT620
1025CT378
0482CT581
0720CT659 Late Belland
0589CT470
1004CT847 Late Belland
0383CT047
0058CT293
1005CT848
0933CT783 predicted Secreted
disul Protein
fide (likely
bond to be
isomerase a Type
III
secretion
protein)
(WO
02/082091
0520CT431
0538CT814
0466CT869 PmpE FACS
ositive
0707CT669 yscN Type
III
secretion
rotein
0707CT669 yscN Mid-cycle Shaw
OS03CT396 DnaK 6667.15Late NicholsonFRCS
ositive
0564CT448
0504CT397
0453CT871 PmpG Late BellandFACS
ositive
0762CT650 recA Secreted
Protein
(WO
02/082091)
1031CT374
0786CT595
0743CT634
0715CT661
0001CT001 Late Nicholson
1028CT376
_0323CT090
0885CT742
0876CT735
0105CT016 Hypothetical FACS Secreted
positiveProtein
(see
WO
02/48185),
pg 36
(ie secreted
by a
Type
III
a aratus
0854CT713 PorB Midlate NicholsonFACS
II ositive
137
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Chlamydia Further Filing!,, ',,
Microarray Expressed Qen'e able
15-9-04 and 17-12-04
TABLE 31v1-(xt1
0321 CT092
0708 GT668 Hypothetical Secreted
Protein
(WO
02/082091)
OS20 CT432
0521 CT432
_0855CT714
0814 CTS73
0506 CT421
.
0702 CT674
0475 CT866
!0S7 CT3S6
0925 CT779 ' -
0828 CTS59 YscJ FACS
ositive
OS CT42S
12
0560 CT44S - -
0647 CTS28
0704 CT672 mid late Nicholson
I
0497 CT388
0083 CT313
0919 CT773
0406 CT104
1012 CT854
0880 CT739
0963 CT812 PmpD FAGS
ositive
0727 CT619
0437 CT286 CIpC Secreted
Protein
(WO
02/082091
0369 CT058
0134 CT110 Hsp-60 Immediate (see FAGS
(omp2) Early Table positive
Chaperonin gene 1 of
Belland
Midcycle et
Midlate al (2003)
I Nicholson
0672 CTS51
0778 CT603
0500 CT393
0804 CTS83
OS90 CT47I
0822 CT565
0807 CT580
0149 CT146
0335 CT078
138
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
Chtamydia Further Filing/ _ ,)
Microarray Expressed Gene xFible
I S-9-04 and 17-12-04
TABIr,E 3f~1-(~dl
0078 CT318
_ _
0078 CT318
0520 CT431
0626 CT507
0298 CT239 Mid late Nicholson
0550 CT437
0573 CT457
1032 CT373
0624 CT505
0682 CT690
0381 CT326
0374 CT056
0062 CT289
0598 CT479
0733 CT626
0623 CT504
0607 CT489
0742 CT635 Hypothetical FRCS
Positive
0613 CT494 mid late Nicholson
II
0675 CT696
00405CT105
0650 CT531
0136 CT __
112
0665 CT54
4
0705 _ Hypothetical FACE wo o2l4siss
CT671 PositiveCT67I
=
secreted
protein,
gg 36
(ie secreted
by
a Type
TIl
apparatus)
E ito_e_
0874 CT733
0385 CT045 PepA 5044.06Mid late NicholsonFRCS
I Positive
.0832CT558
0514 CT427
0904 CT761 MurG 5005.81See biogenome FAGS
paper 2004 PosltIVe
-
MurG was
consistently
selected
across
the
Serovars
0059 CT292 _ Secreted
dut Protein
(WO
021082091)
139
CA 02557353 2006-08-24
WO 2005/084306 PCT/US2005/006588
'_
Chlatnydia Further Filinp~~_,
Mieroatray Expressed Gene cable
15-9-04 and 17-12-04
TABLE 3fvl-(xil
0999 CT842
0296 CT237
0513 CT426
0758 CT613
0207 CT204
0304 CT245
0698 CT678
0191 CT130
0378 CT054 -
0611 CT492
0584 CT467 AtoS 4877.72Cross- FACS
reactivity Positive
between
CT
and CPn
strains
Ie CT467
and
its CPn
homologue
are
neutralising
for
their own
species
but are
also cross-
rotective
0479 CT380
0970 CT8I8
_
0558 CT444 OmcA Late gene FAGS
(see Table Positive
1 of
Belland
et al
(2003) In viva
protective
effect
with
CT444
and
CT467
0878 CT737
0680 CT692
0 CT322
074
_ _
_ CT557
0833
0793 CT588
0604 CT486
0106 CT0I5
0528 CT401
0359 CT064
0610 CT491 Mid late Nicholson
II
1014 CT856
0466 CT869?
0351 CT065 ADP1ATP
Transclocase
0314 CT099
0484 CT382
0932 CT782
0690 CT686
0427 CT278
4453 CT871 pmpG Lade gene (see FRCS
Table
1 of Positive
Belland
et
140
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Chlamydia Further Filing/_ ~. .~'
Microarray Expressed Gene . able
15-9-04 and 17-12-04
TABLE 3(vl-(xi)
al (2003
0035 CT339
0300 CT241
0741 CT636
056_7CT45I
0803 CT584
0555 CT441
0633 CT514 .
0502 CT395
0916 CT770
0286 CT194
0416 CT267
_0422CT273
0692 CT684
_
0415 CT266 Hypothetical FAGS
Positive
0495 CT390
0392 CT039
0586 CT468
0004 CT004
0827 CT560 Hypothetical Type
protein III
secretion
cluster
(WO
02/082091)
_0734CT627
0122 CT032
0657 CT537
0635 CT516
1062 CT329
0859 CT718
0430 CT281
0414 CT265 AccA Secreted
Protein
(WO
02/082091)
0599 CT480 Oligopeptide Imnnediate(see FRCS
binding early Table Positive
lipoprotein gene 1 Belland
O a et al
2003
0094 CT302
0681 CT691 Hypothetical Secreted
Protein
(WO
02/082091)
E ito
a
0309 CT250
0823 CT564
0641 CT522
141
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Table 4. C. pneumonir~e selected peptides: protein sources and IiLA-A2
stabilization assay
PeptideSequence Protein Group ScoreNet
CPn a~ b~ I MFi
~
HepB '~T'AFHQTLQD'~ Wepatitis B virus envelope ~_~- l4.Ot24.4
antigen
GAG "SLYNNATL~ HIV-1 gag 157.2292.423.8
IMa beGILGFVFTL~ Influenza virus matrix 550,9263.1118.1
Mi
CNi 9'SQLLDEGKEL9~0322Yop proteins translocationType 324.0674.O~r22.6
protein U Iii
CH2 ~~ILLNEVPYV""0323Low calcium response Type 5534.14140.5~36.i
protein D ill
GH3 9'~VLNLFFSAL~'0324Low calcium response Type 262.2040.1123.1
protein E III
CH4 'QLLESLAPL'S0325Secretion chaperone Type 745.35120.1125.2
III
CH5 2"SILELLQFVZ'90702Probable Yop proteins Type 1835.2285.534.4
translocation protein ill
C
CH6 'YLLEEIYTV'0811Low calcium response Type 11162.99148.5138.9
protein H lil
GH7 YMDNNLFYV'0823Yop proteins translocationType 6781.36164.124.3
protein T III
CH8 2'~FLTLAWWFIZ~0823Yop proteins translocationType 3365.36144.i~-22.i
protein T 111
GH10 2'"GLTEEIDYV~0828Yop proteins translocationType 1767.58144.0137.9
protein J III
CH12 'WLVFFNPFV'1021Low calcium response Type 6688.7250.122,2
locus protein H III
CH13 sgYVFDRiLKV"0695Outer membrane protein Ch 976.76139.0138.7
A spec
CH14 ''sVMLIFEKKV4"0415CT2 66 hypothetical Cpn 1200.6474.1120.2
protein spec
CH15 '~'YLTSYSPYV'2'0444Polymorphic outer membranePmp 1759.66138.1123.5
protein G/I family
CH16 '~VQLAYVFDV'6'0963Putative outer membranePmp 591.7048.119.1
protein D family
CH17 aoeILQEAEQMV3'6072876 kDa homolog_i Ch 484.77202.124.2
spec
CH18 "IALLVIFFV'0186Simllarto CT119 IncA Ch 445.8046.1122.3
spec
CH19 '~'LILTLGYAV'~50444Poiymorphfc outer membranePmp 437.4856.i~21.6
protein GII family
CH20 ''~ALMLLNNYV'~0005Potymorphic outer membranePmp 1415.38142.5138.6
protein G family
CH21 s"TLWGSFVDV~0447Polymorphic outer membranePmp 1096.83121.118.0
protein G/I family
CH22 '~INLFDLRFSV'b"0540Pofymorphic membrane 28150.1768.5111.0
protein 8 family Pmp
CH24 'LfQETLLFV'90021Predicted OMP Ch 843.21105.1120.8
spec
CH28 '~RLLEIIWGV~0062CHLPS 43 kDa protein Cpn 18200.5499.5115.0
homolog_i spec
CH29 2YLMQKLQNVz'0791CT 590 hypothetical Ch 2722.68108.5112.1
protein spec
CH30 6~FLQRGESFV520792CT 589 hypothetical Ch 759,66105.118.1
protein spec
CH31 ''WLLRDDWLL"90009hypothetical Cpn 2726.91101.1116.3
spec
CH32 ''KLWEWLGYL'0041hypothetical Cpn 4184.2172.1112.0
spec
CH33 ~LLMLAISLV'0131hypothetical Cpn 1006.20l8.Oti.4
spec
CH34 ~'KLLKDHFDL~90132hypothetical Cpn 1604.5385.14.9
spec
CH35 ssILSFLPWLV0169hypothetical Cpn 886.7890.615.7
spec
CH36 '4LLLIFNNYL'S'0170hypothetical Ch 2808.3241.0119.8
spec
CH37 '~1'LLDFRWPL'~0210hypothetical Cpn 42485,2697.1117.7
spec
CH38 "4NLLKRWQFV3~0352hypothetical Cpn 2406.1564.1113.4
spec
CH39 3'FLLFiHLSSV~0355hypothetical Cpn 2722.6888.116.4
spec
CH41 '~KLSEQLEAL"Oi865imflartoCTli9tncA Ch 345.4851.6127.6
spec
CH42 Z'4KVLLGQEWVz''0186Similar toCT1191ncA Ch 212.39l6.0137.5
spec
CH43 9'6NLAEQVTAL3a0186Similar to CT119 IncA Ch 201.4471.6134.6
spec
CH44 '23YWGFIIFL''0323Low calcium response Type 413.3225.p116.3
protein D ill
CH45 92WMMGWLMI'0323Low calcium response Type 294.958.1118.4
protein D III
CH46 56NLSISVFLL0323Low calcium response Type 284.97i 8.026.2
protein D Ill
CH47 "VIQAFGDFV"e0323Low calcium response Type 166.4923.0132.5
protein D III
CH48 ~YI.ALDPDSV~0323Low calcium response Type 156.7774.6134.4
protein D III
CH49 '-09KMSHFG1QAL'S'0415CT2 66 hypothetical Gpn 205.1929.0133,9
protein spec
CH50 "'SLCAQSSYV"950444Polymorphic outer membranePmp 382.5345.1122.6
protein Gll family
CH51 '9NLSRQAFFA'3'~0444Polymorphic outer membranePmp 158.4725.0134.5
protein G/I family
CH52 ~'SLLEEHPW0963Putative outer membranePmp 432.5943.6121.9
protein D family
CH53 '~zNLWSHYTDL'3'o0963Putative outer membranePmp 265.961.6124.7
protein D family
CN54 9"ALWKENQAL9~0963Putative outer membranePmp i 45.6121.9
protein D family 77.30
CH55 bALWGHNVLLS'0963Putative outer membranePmp 177.3047.6129.0
protein D family
CH56 ~NLAGGILSV'4'0963Putative outer membranePmp 159.9729.6129.0
protein D family
CH57 FVSKFWFSL~1021Low calcium response Type 322.16l6.010,
locus protein H III i
CH58 '3SITVFRWLV'1021Low calcium response Type 272.5530.0120.5
locus protein H fll
CH59 6YLtVFVLTh0131hypothetical Cpn 419.4429,0110.6
spec
CH60 "2VMLFIGLGV~0131hypothetical Cpn 315.9528.012.8
spec
CH61 'VLFLLIRSV~0131hypothetical Cpn 201.2414.02.8
spec
CH62 99'FLFQLGMQh50415CT2 66 hvaotheticai Ch 177.5630,0112.0
protein spec
a~ Gene sequence designation as annotated from the genome sequence of Cpn
strain CWL029 (http:l/chlamydia-
www,berkeley,edu:423i)
b~Type III: type III secretion system; Ch and Cpn spec: Chlamydia and C.
pneumoniae specific; Pmp: Polymorphic membrane
protein
°~ Calculated using the BIMAS algorithm
°~ Mean Fluorescence Intensity of cells with peptide - M Mean
Fluorescence Intensity of cells without peptide f Standard
Deviation calculated on three experiments
142
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Table 5. ELISpot assay with CD8+ T cells from DNA
immunized HLA-A2 transgenic mice
Protein Gene PeptideSFC
~'
Hypothetical CPn medium13
0131
HepB 47
CH 33
33
CH 53
59
CH 80
60
GH 40
61
Hypothetical CPn medium7
0210
HepB 13
CH 120
37
LCR Protein CPn medium27
D 0323
HepB 27
C H 2 93
CH 80
44
CH 87
45
CW 40
46
CH 80
47
CH 60
48
CHLPS 43 kDa CPn medium33
0062
HepB 27
CH 93
28
OMP A CPn medium13
0695
HepB 33
CH 727
l
3
LCR Protein H CPn 081113
medium
. HepB 27
CH 6 213
Yop pt protein T CPn 7
0823 medium
NepB 47
CH 7 493
CH 8 53
Yop pt protein J CPn 20
0828 medium
HepB 60
CH 10 247
at
SFC = Spot Forming Colonies1106 CD8 cells
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Table 6. IFN-y production from splenocytes of DNA immunized HLA-A2 transgenic
and non transgenic mice
Ex vivo stTCLsb~
RFI RFI e~
A~
Protein Gene Peptide A2'~ A2+~ A2'~ A2+>
LCR Protein D CPn 0323 CH 0,05 1,11 0,79 2,57
2
CH 44 3,30 1,78 0,73 6,86
CH 45 1,00 1,56 0,47 4,71
CH 46 0,90 1,44 0,41 9,00
CH 47 1,00 1,78 1,17 1,14
CH 48 1,30 1,67 0,11 1,29
CD3+CD28 134,00 90,55
OMP A CPn 0695 CH 13 3,29 2,54 23,42 209,81
CD3 + CD28 248,71 73,23
LCR Protein H CPn 0811 CH 6 1,00 4,58 1,53 31,56
CD3 + CD28 290,83 96,10
Yop pt Protein T CPn 0823 CH 7 1,20 5,20 11,69 94,57
CH 8 2,00 1,60 16,81 28,21
CD3 + CD28 247,60 91,00
a Relative Fold Increase: ratio between the percentage of IFN-y'"/CD8'' cells
obtained with the tested peptide (or
the CD3/CD28 co-stimulus) and the HepB negative control peptide
b~ Short term T cell lines
°~ HLA-A2 non transgenic and transgenic mine
144
