Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CHLAMYDIA ANTIGEN COMPOSITIONS AND USES THEREOF
FIELD OF INVENTION
[0001] The present invention relates to bacterial infections. More
specifically, the invention
provides in part fusion proteins for use against Chlamydia infection.
BACKGROUND OF THE INVENTION
[0002] Chlamydia trachomatis is an intracellular pathogen responsible for over
92 million
sexually transmitted infections and 85 million ocular infections per year
worldwide (Starnbach,
M. N., and N. R. Roan. 2008. Conquering sexually transmitted diseases. Nat Rev
Immunol 8:313-
317.). Sexually transmitted C. trachomatis is a major cause of long-term
disease sequelae in
women such as infertility and ectopic pregnancy (Brunham, R. C., D. J. Zhang,
X. Yang, and G.
M. McClarty. 2000. The potential for vaccine development against chlamydial
infection and
disease. J Infect Dis 181 Suppl 3:S538-543; Igietseme, J. U., C. M. Black, and
H. D. Caldwell.
2002. Chlamydia vaccines: strategies and status. BioDrugs 16:19-35). C.
trachomatis infection
in women often goes unnoticed until severe reproductive damage (infertility,
pelvic
inflammatory disease, ectopic pregnancy) is already underway. In addition,
women infected with
C. trachomatis are at increased risk of contracting HIV following exposure.
[0003] The "seek and treat" programs to prevent and control C. trachomatis
sexually transmitted
infections appear to be failing as case rates and reinfection rates continue
to rise (Brunham, R.
C., B. Pourbohloul, S. Mak, R. White, and M. L. Rekart. 2005. The unexpected
impact of a
Chlamydia trachomatis infection control program on susceptibility to
reinfection. J Infect Dis
192:1836-1844), possibly due to early treatment interfering with the
development of protective
immune responses (Su, H., R. Morrison, R. Messer, W. Whitmire, S. Hughes, and
H. D.
Caldwell. 1999. The effect of doxycycline treatment on the development of
protective immunity
in a murine model of chlamydial genital infection. J Infect Dis 180:1252-
1258).
[0004] Previous attempts to vaccinate against C. trachomatis and C. muridarum
infection in both
human and murine models using dead elementary bodies (EBs), which are non-
replicating
infectious particles released when infected cells rupture, provided limited
protection (Grayston,
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J. T., and S. P. Wang. 1978. The potential for vaccine against infection of
the genital tract with
Chlamydia trachomatis. Sex Transm Dis 5:73-77; Grayston, J. T., S. P. Wang, L.
J. Yeh, and C.
C. Kuo. 1985. Importance of reinfection in the pathogenesis of trachoma. Rev
Infect Dis 7:717-
725; Lu, H., Z. Xing, and R. C. Brunham. 2002. GM-CSF transgene-based adjuvant
allows the
establishment of protective mucosal immunity following vaccination with
inactivated Chlamydia
trachomatis. Jlmmunol 169:6324-6331; Schachter, J., and H. D. Caldwell. 1980.
Chlamydiae.
Annu Rev Microbiol 34:285-309). Mice immunized with live C. muridarum EBs have
however
been shown to generate better protection (Lu, H., Z. Xing, and R. C. Brunham.
2002. GM-CSF
transgene-based adjuvant allows the establishment of protective mucosal
immunity following
vaccination with inactivated Chlamydia trachomatis. Jlmmunol 169:6324-6331;
Su, H., R.
Messer, W. Whitmire, E. Fischer, J. C. Portis, and H. D. Caldwell. 1998.
Vaccination against
chlamydial genital tract infection after immunization with dendritic cells
pulsed ex vivo with
nonviable Chlamydiae. JExp Med 188:809-818).
[0005] Investigation into the mechanism underlying the efficient induction of
immunity provided
by live C. muridarum in comparison to dead organisms suggests that dendritic
cells (DCs)
exposed to live or dead C. muridarum develop into distinct phenotypes. In
particular DCs
exposed to live C. muridarum become mature and stimulated antigen-specific CD4
T cells, while
DCs exposed to dead C. muridarum are inhibited in acquiring a mature
phenotype. Co-
stimulation of DCs with dead EB and CpG oligodeoxynucleotide has been show to
partially
overcome dead EB inhibition of DC maturation (Rey-Ladino, J., K. M.
Koochesfahani, M. L.
Zaharik, C. Shen, and R. C. Brunham. 2005. A live and inactivated Chlamydia
trachomatis
mouse pneumonitis strain induces the maturation of dendritic cells that are
phenotypically and
immunologically distinct. Infect Immun 73:1568-1577). Investigation into the
transcriptional
responses of bone marrow derived DCs following exposure to live and dead C.
muridarum using
GeneChip microarrays revealed marked differences in CXC chemokine profiles in
DCs exposed
to live or dead organism (Zaharik, M. L., T. Nayar, R. White, C. Ma, B. A.
Vallance, N. Straka,
X. Jiang, J. Rey-Ladino, C. Shen, and R. C. Brunham. 2007. Genetic profiling
of dendritic cells
exposed to live- or ultraviolet-irradiated Chlamydia muridarum reveals marked
differences in
CXC chemokine profiles. Immunology 120:160-172). In aggregate, the data
suggest that DCs
exposed to live EBs are phenotypically and functionally distinct from DCs
generated by
exposure to dead EBs.
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[0006] Immunity to C. muridarum infection is thought to be largely cell-
mediated and therefore
dependent on Chlamydia-derived peptides presented to CD4 T cells via WIC
molecules on
antigen presenting cells (Brunham, R. C., and J. Rey-Ladino. 2005. Immunology
of Chlamydia
infection: implications for a Chlamydia trachomatis vaccine. Nat Rev Immunol
5:149-161;
Steinman, R. M., and M. Pope. 2002. Exploiting dendritic cells to improve
vaccine efficacy. J
Clin Invest 109:1519-1526; Su, H., and H. D. Caldwell. 1995. CD4+ T cells play
a significant
role in adoptive immunity to Chlamydia trachomatis infection of the mouse
genital tract. Infect
Immun 63:3302-3308; Morrison, S. G., H. Su, H. D. Caldwell, and R. P.
Morrison. 2000.
Immunity to murine Chlamydia trachomatis genital tract reinfection involves B
cells and CD4(+)
T cells but not CD8(+) T cells. Infect Immun 68:6979-6987; Morrison, R. P.,
and H. D. Caldwell.
2002. Immunity to murine chlamydial genital infection. Infect Immun 70:2741-
2751; Igietseme,
J. U., K. H. Ramsey, D. M. Magee, D. M. Williams, T. J. Kincy, and R. G. Rank.
1993.
Resolution of murine chlamydial genital infection by the adoptive transfer of
a biovar-specific,
Thl lymphocyte clone. Reg Immunol 5:317-324).
[0007] Immunoproteomic approaches (Hunt, D. F., R. A. Henderson, J.
Shabanowitz, K.
Sakaguchi, H. Michel, N. Sevilir, A. L. Cox, E. Appella, and V. H. Engelhard.
1992.
Characterization of peptides bound to the class I WIC molecule BLA-A2.1 by
mass
spectrometry. Science 255:1261-1263; de Jong, A. 1998. Contribution of mass
spectrometry to
contemporary immunology. Mass Spectrom Rev 17:311-335; Olsen, J. V., L. M. de
Godoy, G.
Li, B. Macek, P. Mortensen, R. Pesch, A. Makarov, 0. Lange, S. Horning, and M.
Mann. 2005.
Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass
injection into a
C-trap. Mot Cell Proteomics 4:2010-2021) to identify C. muridarum T cell
antigens, based on
isolating and sequencing of pathogen-derived peptides binding to WIC class II
molecules
presented on the surface of DCs after they were pulsed with live EBs, resulted
in the
identification of a number of C. muridarum peptides derived from 8 novel
epitopes
(Karunakaran, K. P., J. Rey-Ladino, N. Stoynov, K. Berg, C. Shen, X. Jiang, B.
R. Gabel, H. Yu,
L. J. Foster, and R. C. Brunham. 2008. Immunoproteomic discovery of novel T
cell antigens
from the obligate intracellular pathogen Chlamydia. J Immunol 180:2459-2465).
These peptides
were recognized by antigen-specific CD4 T cells in vitro and recombinant
proteins containing
the WIC binding peptides were able to induce partial protection via
immunization against C.
muridarum infection in vivo (Yu, H., X. Jiang, C. Shen, K. P. Karunakaran, and
R. C. Brunham.
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2009. Novel Chlamydia muridarum T cell antigens induce protective immunity
against lung and
genital tract infection in murine models. J Immunol 182:1602-1608).
[0008] Chlamydia sequences (nucleic acid and polypeptide) are described in,
for example, US
6030799, US 6696421, US 6676949, US 6464979, US 6653461, US 6642023, US
6887843 and
US 7459524; or in US Patent Publications 2005/0232941, 2009/0022755, and
2008/0102112.
Specific Chlamydia antigens are described in, for example, PCT Publication No.
WO
2010/085896 and W02013/044398.
SUMMARY OF THE INVENTION
[0009] The present disclosure provides in part fusion proteins derived from
Chlamydia spp. The
present invention also provides in part methods for treating or preventing
Chlamydia infection
using the fusion proteins.
[0010] In one aspect, there is provided an immunogenic composition including a
fusion protein
which includes at least two Chlamydia proteins selected from: Polymorphic
membrane protein G
(PmpG), Polymorphic membrane protein F (PmpF), Polymorphic membrane protein E
(PmpE),
Polymorphic membrane protein H (PmpH), Ribosomal protein L6 (Rp1F), Anti-anti-
sigma factor
(Aasf), Translocated actin-recruiting phosphoprotein (Tarp), hypothetical
protein corresponding
to locus tag CT143/TC0420, metalloprotease, insulinase family (CT806/TC0190),
hypothetical
protein corresponding to locus tag CT538/TC0825, hypothetical protein
corresponding to locus
tag CT017/TCO285, hypothetical protein corresponding to locus tag CT619, or
MOlViP, or an
immunogenic fragment thereof, together with a physiologically acceptable
carrier.
[0011] In alternative embodiments, the fusion protein includes combinations
of: PmpG and
MOMP, PmpG and PmpF, PmpG and PmpH or PmpE and PmpF.
[0012] In alternative embodiments, the composition further includes an
adjuvant, such as
DDA/TDB, DDA/M_MG or DDA/MPL.
[0013] In alternative aspects, there is provided a method for eliciting an
immune response
against a Chlamydia spp., or component thereof, in an animal by administering
to the animal an
effective amount of the composition as described herein, thereby eliciting an
immune response in
the animal. The immune response may be a cellular immune response.
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[0014] In alternative aspects, there is provided a method for treating or
preventing infection by a
Chlamydia spp. in an animal by administering to the animal an effective amount
of the
composition as described herein, thereby treating or preventing infection by
the Chlamydia spp.
in the animal.
[0015] In alternative embodiments, the Chlamydia spp. may be a Chlamydia
trachomatis or a
Chlamydia muridarum.
[0016] In alternative embodiments, the animal may be a human.
[0017] In alternative aspects, there is provided the use of the composition as
described herein,
for eliciting an immune response against a Chlamydia spp., or component
thereof, in an animal.
The immune response may be a cellular immune response.
[0018] In alternative aspects, there is provided the use of the composition as
described herein,
for treating or preventing infection by a Chlamydia spp. or component thereof,
in an animal. The
Chlamydia spp. may be a Chlamydia trachomatis or a Chlamydia muridarum. The
animal may
be a human.
[0019] This summary does not necessarily describe all features of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] These and other features of the disclosure will become more apparent
from the following
description in which reference is made to the appended drawings wherein:
[0021] FIGURES 1A-II show amino acid sequences for Chlamydia muridarum and
Chlamydia
trachomatis proteins, together with the corresponding nucleic acid sequences
(SEQ ID Nos: 1-
35).
[0022] FIGURES 2A-F show amino acid and nucleic acid sequences for PmpE-PmpF &
PmpG-
PmpH fusion proteins (italized sequences indicating the second protein;
underlined sequences
representing alpha helix linkers connecting two protein domains; SEQ ID NOs:
36-41).
[0023] FIGURES 3A-B are graphs showing fusion protein-elicited protection at
day 6 (A) and
day 12 (B) against Chlamydia genital tract infection.
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[0024] FIGURES 4A-C are graphs showing C. muridarum-specific cytokine
responses after
immunization with PmpE, F, G, H plus MOMP either as individual (mixed) or as
fusion formats
in C57 (A), Balb/c (B), or C3H (C) mice.
[0025] FIGURES 5A-C are graphs showing C. muridarum individual antigen-
specific IFN-y
responses in C57 (A), Balb/c (B), or C3H (C) mice after immunization with
PmpE, F, G, H plus
MOMP either as individual (mixed) or as fusion formats.
[0026] FIGURES 6A-C are graphs showing vaccine-elicited protection against C.
muridarum
genital tract infection in C57 (A), Balb/c (B), or C3H (C) mice after
immunization with PmpE,
F, G, H plus MOMP, either as individual(mixed) or as fusion formats.
[0027] FIGURES 7A-C are graphs showing vaccine-elicited protection against C.
muridarum
genital tract infection in C57 (A), Balb/c (B), or C3H (C) mice after
immunization with with
PmpE, F, G, H plus MOMP, either as individual(mixed) or as fusion formats.
DETAILED DESCRIPTION
[0028] The present disclosure provides, in part, fusion proteins derived from
Chlamydia spp.
proteins. The present disclosure also provides, in part, methods for treating
or preventing
Chlamydia infection using the fusion proteins.
[0029] In some embodiments, these fusion proteins may be useful as vaccines
for use in the
prevention or treatment of Chlamydia spp. infection.
[0030] Chlamydia spp.
[0031] By "Chlamydia spp." is meant a genus of bacteria that are obligate
intracellular parasites.
Chlamydia spp. include C. trachomatis (a human pathogen) and C. muridarum
(pathogenic to
mice and hamsters). As C. muridarum and C. trachomatis are highly orthologous
pathogenic
microbes that have co-evolved with their host species, C. muridarum has been
used as a robust
animal model for studying cellular immunity and vaccine development.
[0032] In some embodiments, a C. trachomatis includes without limitation a C.
trachomatis
serovar D/UW-3/CX, as well as serovars A, B, Ba, C (implicated in trachoma),
serovars D, E, F,
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G, H, I, J K (implicated in urogenital tract infections) and Li, L2, L3
(lymphogranuloma
venereum serovars).
[0033] In some embodiments, a C. muridarum includes a C. muridarum mouse
pneumonitis
(MoPn) strain Nigg.
[0034] The genome sequences of various Chlamydia spp. have been determined.
The genome
sequence of C. trachomatis strain D/UW-3/CX is described for example in
Stephens, R.S. et at.,
1998 (Genome sequence of an obligate intracellular pathogen of humans:
Chlamydia
trachomatis. Science 282 (5389): 754-759) and provided in GenBank Accession
No.
NC 000117.1, GI:15604717; referred to herein as the "C. trachomatis genome
sequence").
[0035] The genome sequence of C. muridarum is described in for example Read,
T., et at., 2000
(Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39
Nucleic
Acids Res. 28 (6): 1397-1406) and provided in GenBank Accession No. NC
002620.2,
GI:29337300; referred to herein as the "C. muridarum genome sequence").
[0036] Chlamydia spp. Fusion Polypeptides and Nucleic Acid Molecules
[0037] Compounds for use in the compositions and methods according to the
disclosure include,
without limitation, a fusion protein including the sequence of two or more of
the Chlamydia
polypeptides described herein, for example, the proteins or polypeptides
listed in Tables 1 or 2,
or in Figures 1A-II, or an immunogenic fragment thereof, as well as a nucleic
acid molecule
encoding such a fusion protein.
Table 1: Homology of C. muridarum-derived source proteins to C. trachomatis,
other bacteria
and human
Peptide Sequence Chlamydia Source Protein C.trachomatis
Other Huma
muridarum Proteins Abbr. Identity and
Bacteria
Locus# Locus # (30% cut (25%
off) cut
off)
AFHLFASPAANYIHTG TCO262 Polymorphic PmpF 61% - CT870
(SEQ ID NO: 42) membrane
protein F
NAKTVFLSNVASPIYVDPA TCO263 Polymorphic PmpG 71% -
CT871
(SEQ ID NO: 43) membrane
ASPIYVDPAAAGGQPPA protein G
(SEQ ID NO: 44)
VKGNEVFVSPAAHIIDRPG TC0801 Ribosomal RpIF 96% - CT514
(SEQ ID NO: 45) protein L6
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Peptide Sequence Chlamydia Source Protein C.trachomatis
Other Huma
muridarum Proteins Abbr. Identity and
Bacteria n
Locus# Locus # (30% cut (25%
off) cut
off)
SPGQTNYAAAKAGIIGFS TC0508 3-oxoacyl-(acyl FabG 90% -
CT237 Y Y
(SEQ ID NO: 46) carrier protein) (44%)
reductase
KLDGVSSPAVQESISE TC0707 Anti-anti-sigma Aasf 96% - CT424 Y
N
(SEQ ID NO: 47) factor
IGQEITEPLANTVIA TC0079 ATP dependent CIpP 92% - CT706 Y
Y
(SEQ ID NO: 48) Clp protease, (56%)
proteolytic
subunit
MTTVHAATATQSVVD TC0792 Glyceraldehyde Gap 95% - CT505 Y
Y
(SEQ ID NO: 49) 3-phosphate (56%)
dehydrogenase
DLNVTGPKIQTDVD TC0420 Hypothetical 75% - CT143 N N
(SEQ ID NO: 50) protein
EGTKIPIGTPIAVFSTEQN TC0518 Pyruvate PdhC 87% - CT247 Y
Y
(SEQ ID NO: 51) dehydrogenase (38%)
SVPSYVYYPSGNRAPVV TC0884 Thiol disulfide DsbD 73% - CT595 Y
N
(SEQ ID NO: 52) interchange
protein
YDHIIVTPGANADIL TC0654 Oxidoreductase, 85% - CT375 Y N
(SEQ ID NO: 53) DadA family
LPLMIVSSPKASESGAA TC0190 Metalloprotease 80% - CT806 N N
(SEQ ID NO: 54) , insulinase
family
GANAIPVHCPIGAESQ TC0721 Translation FusA 97% - CT437 Y
Y
(SEQ ID NO: 55) elongation factor (43%)
VFWLGSKINIIDTPG G
(SEQ ID NO: 56)
ISRALYTPVNSNQSVG TC0050 Translation Tsf 89% - CT679 Y
Y
(SEQ ID NO: 57) elongation factor (31%)
Ts
FEVQLISPVALEEGMR TC0596 Translation Tuf 95% - CT322 Y
Y
(SEQ ID NO:58 ) elongation factor (55%)
GDAAYIEKVRELMQ Tu
(SEQ ID NO: 59)
SRALYAQPMLAISEA TCO261 Polymorphic PmpE 69% - CT869 N
N
(SEQ ID NO: 60) membrane
protein E
KPAEEEAGSIVHNAREQ TC0584 V-type, ATP AtpE 91% - CT310 N
N
(SEQ ID NO: 61) synthase
subunit E
SPQVLTPNVIIPFKGDD TCO264 Polymorphic PmpH 76% - CT872 N
N
(SEQ ID NO: 62) membrane
protein H
SMLIIPALGG TC0895 Nucleoside YggV 81% - CT606 Y
Y
(SEQ ID NO: 63) triphosphatase (33%)
LAAAVMHADSGAILKEK TC0839 D-analyl-D- DacC 76% - CT551 Y
N
(SEQ ID NO: 64) alanine
carboxypeptidas
e
DDPEVIRAYIVPPKEP TC0825 Hypothetical 91% - CT538 N N
(SEQ ID NO: 65) protein
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Peptide Sequence Chlamydia Source Protein C.trachomatis
Other Huma
muridarum Proteins Abbr. Identity and
Bacteria n
Locus# Locus # (30% cut (25%
off) cut
off)
KIFSPAGLLSAFAKNGA TC0755 DNA repair Rec0 85% - CT470 N
N
(SEQ ID NO: 66) protein
DPVDMFQMTKIVSKH TC0745 SWIB (YM74) 86% - CT460 Y Y
(SEQ ID NO:67 ) complex protein (33%)
KLEGIINNNNTPS TC0741 Translocated Tarp 45% -
CT456 N N
(SEQ ID NO: 68) actin-recruiting
phosphoprotein
AVPRTSLIF TC0021 Exodeoxyribonu RecD2 81% -
CT652 Y Y
(SEQ ID NO: 69) clease V, alpha _
subunit
GGAEVILSRSHPEFVKQ TC0372 N utilization NusA 97% -
CT097 Y Y
(SEQ ID NO: 70) substance
protein A
APILARLS TCO285 Hypothetical 82% - CT017 N
N
(SEQ ID NO: 71) protein
Table 2 Chlamydia trachomatis Source Proteins
Chlamydia trachomatis Source Proteins Protein
Locus# Abbreviation
CT559 Yop proteins translocation lipoprotein CdsJ
CT837 Hypothetical protein CT837
CT110 Chaperonin GroEL1 GroEL1
CT144 Hypothetical protein CT144
CT289 Hypothetical protein CT289
CT619 Hypothetical protein CT619
CT561 Type III secretion translocase CdsL
CT681 Major Outer Membrane Protein MOMP
CT664 FHA domain; homology to adenylate cyclase
CT113 Clp Protease ATPase ClpB
CT759 Muramidase (invasin repeat family) N1pD
CT045 Leucyl aminopeptidase PepA
CT420 50S ribosomal protein L21 R121
CT622 CHLPN 76kDa Homolog
CT472 Hypothetical protein CT472
CT842 Polyribonucleotide Nucleotidyltransferase Pnp
CT778 Primosome assembly protein PriA
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[0038] In some embodiments, a compound for use in the compositions and methods
according to
the disclosure includes, without limitation, a C. muridarum- or C. trachomatis-
derived amino
acid sequence, such as a fusion protein including an amino acid sequence
substantially identical
to the sequence of two or more of the polypeptides described herein, for
example, those listed in
Tables 1 or 2, or in Figures 1A-II, or an immunogenic fragment thereof
[0039] In some embodiments, a compound for use in the compositions and methods
according to
the disclosure includes, without limitation, a C. muridarum- or C. trachomatis-
derived nucleic
acid molecule, such as a nucleic acid sequence that encodes a fusion protein
including an amino
acid sequence substantially identical to the sequence of two or more of the
polypeptides
described herein, for example, those listed in Tables 1 or 2, or in Figures
Figures 1A-II, or an
immunogenic fragment thereof
[0040] In some embodiments, a compound for use in the compositions and methods
according to
the disclosure includes, without limitation, a C. muridarum- or C. trachomatis-
derived nucleic
acid molecule, such as a nucleic acid sequence substantially identical to the
nucleic acid
sequence of two or more of the polypeptides described herein, for example,
those listed in Tables
1 or 2, or in Figures 1A-II, or an immunogenic fragment thereof
[0041] In alternative embodiments, a compound for use in the compositions and
methods
according to the disclosure includes, without limitation, a fusion protein
including two or more
of a Chlamydia polypeptide, such as Polymorphic membrane protein G (PmpG),
Polymorphic
membrane protein F (PmpF), Polymorphic membrane protein E (PmpE), Polymorphic
membrane
protein H (PmpH), Ribosomal protein L6 (Rp1F), Anti-anti-sigma factor (Aasf),
Translocated
actin-recruiting phosphoprotein (Tarp), hypothetical protein corresponding to
locus tag
CT143/TC0420, metalloprotease, insulinase family (CT806/TC0190), hypothetical
protein
corresponding to locus tag CT538/TC0825, hypothetical protein corresponding to
locus tag
CT017/TCO285, hypothetical protein corresponding to locus tag CT619, or
MOlViP, or an
immunogenic fragment thereof
[0042] In alternative embodiments, a compound for use in the compositions and
methods
according to the disclosure includes, without limitation, a fusion protein
including two or more
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of a Chlamydia-derived amino acid sequence, such as a fusion protein including
an amino acid
sequence substantially identical to the sequence of two or more of the
following polypeptides:
Polymorphic membrane protein G (PmpG), Polymorphic membrane protein F (PmpF),
Polymorphic membrane protein E (PmpE), Polymorphic membrane protein H (PmpH),
Ribosomal protein L6 (Rp1F), Anti-anti-sigma factor (Aasf), Translocated actin-
recruiting
phosphoprotein (Tarp), hypothetical protein corresponding to locus tag
CT143/TC0420,
metalloprotease, insulinase family (CT806/TC0190), hypothetical protein
corresponding to locus
tag CT538/TC0825, hypothetical protein corresponding to locus tag
CT017/TCO285,
hypothetical protein corresponding to locus tag CT619, or MOMP, or an
immunogenic fragment
thereof
[0043] In alternative embodiments, a compound for use in the compositions and
methods
according to the disclosure includes, without limitation, a fusion protein
encoded by two or more
of a Chlamydia-derived nucleic acid sequence, such as a nucleic acid sequence
that encodes a
fusion protein including an amino acid sequence substantially identical to the
sequence of two or
more of the following polypeptides: Polymorphic membrane protein G (PmpG),
Polymorphic
membrane protein F (PmpF), Polymorphic membrane protein E (PmpE), Polymorphic
membrane
protein H (PmpH), Ribosomal protein L6 (Rp1F), Anti-anti-sigma factor (Aasf),
Translocated
actin-recruiting phosphoprotein (Tarp), hypothetical protein corresponding to
locus tag
CT143/TC0420, metalloprotease, insulinase family (CT806/TC0190), hypothetical
protein
corresponding to locus tag CT538/TC0825, hypothetical protein corresponding to
locus tag
CT017/TCO285, hypothetical protein corresponding to locus tag CT619, or
MOlViP, or an
immunogenic fragment thereof
[0044] In alternative embodiments, a compound for use in the compositions and
methods
according to the disclosure includes, without limitation, a fusion protein
encoded by two or more
of a Chlamydia-derived nucleic acid sequence, such as a nucleic acid sequence
substantially
identical to the nucleic acid sequence encoding two or more of the following
polypeptides:
Polymorphic membrane protein G (PmpG), Polymorphic membrane protein F (PmpF),
Polymorphic membrane protein E (PmpE), Polymorphic membrane protein H (PmpH),
Ribosomal protein L6 (Rp1F), Anti-anti-sigma factor (Aasf), Translocated actin-
recruiting
phosphoprotein (Tarp), hypothetical protein corresponding to locus tag
CT143/TC0420,
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metalloprotease, insulinase family (CT806/TC0190), hypothetical protein
corresponding to locus
tag CT538/TC0825, hypothetical protein corresponding to locus tag
CT017/TCO285,
hypothetical protein corresponding to locus tag CT619, or MOMP, or an
immunogenic fragment
thereof
[0045] In some embodiments, a compound for use in the compositions and methods
according to
the disclosure includes, without limitation, a fusion protein including two or
more of PmpG,
PmpF, PmpE, PmpH, Rp1F, Aasf, Tarp, TC0420, TC0190, TC0825, TCO285, CT619,
MOMP, or
an immunogenic fragment thereof, or a nucleic acid molecule encoding such a
fusion protein.
[0046] In some embodiments, a compound for use in the compositions and methods
according to
the disclosure includes, without limitation, a fusion protein including three
or more of PmpG,
PmpF, PmpE, PmpH, Rp1F, Aasf, Tarp, TC0420, TC0190, TC0825, TCO285, CT619,
MOMP, or
an immunogenic fragment thereof, or a nucleic acid molecule encoding such a
fusion protein.
[0047] In some embodiments, a compound for use in the compositions and methods
according to
the disclosure includes, without limitation, a fusion protein including four
or more of PmpG,
PmpF, PmpE, PmpH, Rp1F, Aasf, Tarp, TC0420, TC0190, TC0825, TCO285, CT619,
MOMP, or
an immunogenic fragment thereof, or a nucleic acid molecule encoding such a
fusion protein.
[0048] In some embodiments, a compound for use in the compositions and methods
according to
the disclosure includes, without limitation, a fusion protein including two or
more of the
following Chlamydia proteins/antigens: PmpG, PmpE, PmpF, PmpH and, optionally,
MOMP, or
an immunogenic fragment thereof, or a nucleic acid molecule encoding such a
fusion protein.
[0049] In some embodiments, a compound for use in the compositions and methods
according to
the disclosure includes, without limitation, a fusion protein including two or
more of the
following Chlamydia proteins/antigens: PmpG, PmpE, PmpF and TC0420 and,
optionally,
MOMP, or an immunogenic fragment thereof, or a nucleic acid molecule encoding
such a fusion
protein.
[0050] In some embodiments, a compound for use in the compositions and methods
according to
the disclosure includes, without limitation, a fusion protein including the
following Chlamydia
proteins/antigens: PmpG and MOlViP, or an immunogenic fragment thereof, or a
nucleic acid
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molecule encoding such a fusion protein. In alternative embodiments a fusion
protein including
only the following Chlamydia proteins/antigens: PmpG and MOMP, or an
immunogenic
fragment thereof, or a nucleic acid molecule encoding such a fusion protein.
[0051] In some embodiments, a compound for use in the compositions and methods
according to
the disclosure includes, without limitation, a fusion protein including the
following Chlamydia
proteins/antigens: PmpG and PmpF, or an immunogenic fragment thereof, or a
nucleic acid
molecule encoding such a fusion protein. In alternative embodiments, a
compound for use in the
compositions and methods according to the disclosure includes a fusion protein
including only
the following Chlamydia proteins/antigens: PmpG and PmpF, or an immunogenic
fragment
thereof, or a nucleic acid molecule encoding such a fusion protein.
[0052] In some embodiments, a compound for use in the compositions and methods
according to
the disclosure includes, without limitation, a fusion protein including the
following Chlamydia
proteins/antigens: PmpG and PmpH, or an immunogenic fragment thereof, or a
nucleic acid
molecule encoding such a fusion protein. In alternative embodiments, a
compound for use in the
compositions and methods according to the disclosure includes a fusion protein
including only
the following Chlamydia proteins/antigens: PmpG and PmpH, or an immunogenic
fragment
thereof, or a nucleic acid molecule encoding such a fusion protein.
[0053] In some embodiments, a compound for use in the compositions and methods
according to
the disclosure includes, without limitation, a fusion protein including the
following Chlamydia
proteins/antigens: PmpE and PmpF, or an immunogenic fragment thereof, or a
nucleic acid
molecule encoding such a fusion protein. In alternative embodiments, a
compound for use in the
compositions and methods according to the disclosure includes a fusion protein
including only
the following Chlamydia proteins/antigens: PmpE and PmpF, or an immunogenic
fragment
thereof, or a nucleic acid molecule encoding such a fusion protein.
[0054] In some embodiments, a compound for use in the compositions and methods
according to
the disclosure includes, without limitation, a fusion protein including the
following Chlamydia
proteins/antigens: PmpG and TC0420, or an immunogenic fragment thereof, or a
nucleic acid
molecule encoding such a fusion protein. In alternative embodiments, a
compound for use in the
compositions and methods according to the disclosure includes a fusion protein
including only
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the following Chlamydia proteins/antigens: PmpG and TC0420, or an immunogenic
fragment
thereof, or a nucleic acid molecule encoding such a fusion protein.
[0055] In alternative embodiments, a compound for use in the compositions and
methods
according to the disclosure includes, without limitation, one or more of the
fusion proteins
described in Figures 2A-F.
[0056] It is to be understood that compositions according to the disclosure
can include mixtures
of fusion proteins and individual (non-fusion) proteins, or immunogenic
fragments thereof, as
long as at least one polypeptide in the mixture is a fusion protein.
[0057] In some embodiments, a composition according to the disclosure
includes, without
limitation, a mixture of two or more fusion proteins, and optionally
individual antigens, such as a
mixture of PmpG/PmpH and PmpE/PmpF and optionally, MOMP; and/or PmpG/ TC0420
and
PmpE/PmpF and optionally, MOMP, or an immunogenic fragment thereof
[0058] In alternative embodiments, compositions according to the disclosure
further include,
without limitation, mixtures of fusion proteins, where MOMP, or an immunogenic
fragment
thereof, is part of a fusion protein.
[0059] In alternative embodiments, compounds for use in the compositions and
methods
according to the disclosure include, without limitation, a fusion protein
including two or more of
a C. trachomatis polypeptide, such as Ribosomal protein L6 (RpIF, gi:3328951),
Anti anti sigma
factor (Aasf, gi: 15605151), Polymorphic membrane protein G ( PmpG,
gi:3329346),
Hypothetical protein (TC0420, gi: 15604862), Polymorphic membrane protein F
(PmpF,
gi:3329345), or major outer membrane protein 1 (MOMP) (gi:3329133), or an
immunogenic
fragment or portion thereof Examples of fragments or portions of such C.
trachomatis
polypeptides include, without limitation, amino acids 25 - 512 of PmpG (PmpG25-
512), amino
acids 26-585 of PmpF (PmpF26-585), or amino acids 22-393 of MOMP.
[0060] In alternative embodiments, compounds for use in the compositions and
methods
according to the disclosure further include, without limitation, a fusion
protein including two or
more of a C. muridarum polypeptide, such as Ribosomal protein L6 (RpIF, gi:
15835415), Anti
anti sigma factor (Aasf, gi: 15835322), Polymorphic membrane protein G (PmpG
or PmpG-1, gi:
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15834883), Hypothetical protein TC0420(gi: 15835038), Polymorphic membrane
protein F
(PmpF or PmpE/F, gi: 15834882), or major outer membrane protein 1 (MOMP,
gi7190091), or
an immunogenic fragment or portion thereof Examples of such fragments or
portions of C.
muridarum polypeptides include, without limitation, amino acids 25 - 500 of
PmpG- 1 (PmpG-
125_500), amino acids 25-575 of PmpE/F-2 (PmpE/F-225-575), or amino acids 23 -
387 of MOMP.
[0061] In alternative embodiments, an immunogenic fragment or portion of a
Chlamydia
polypeptide includes the region of the polypeptide that is generally exposed
on the surface of the
polypeptide. In alternative embodiments, such a fragment or portion of a
Chlamydia polypeptide
includes the passenger domain of a Pmp polypeptide e.g., the domain located
between the signal
sequence and the translocation unit.
[0062] In alternative embodiments, an immunogenic fragment or portion of a C.
muridarum
polypeptide includes the passenger domain, or a portion thereof, of a C.
muridarum Pmp
polypeptide, for example, amino acids 18 to 667 of PmpE; amino acids 18 to 575
of PmpE;
amino acids 20 to 722 of PmpF; amino acids 20 to 575 of PmpF; amino acids 25
to 675 of
PmpG; amino acids 25 to 555 of PmpG; amino acids 27 to 653 of PmpH; or amino
acids 27 to
575 of PmpH. In alternative embodiments, an immunogenic fragment or portion of
a C.
trachomatis polypeptide includes the passenger domain, or a portion thereof,
of a C. trachomatis
Pmp polypeptide. In alternative embodiments, an immunogenic fragment or
portion of a
Chlamydia polypeptide includes a peptide sequence as described in Table 1. In
alternative
embodiments, passenger domain fragments can be about 550 amino acids in
length, or about 600
amino acids from the N-terminus of the Pmp polypeptide, or less. In
alternative embodiments, an
immunogenic fragment can be about 25 to about 600 amino acids in length, for
example, about
25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,
425, 450, 475, 500,
525, 550, 575, 600, or any integer within these values.
[0063] In general, it is to be understood that the sequences of polypeptides
and amino acids
referenced herein correspond to those indicated in the locus tags referenced
in the C. trachomatis
genome sequence and/or the C. muridarum genome sequence. It is also to be
understood that the
nucleic acid sequences corresponding to the locus tags can be obtained from
the C. trachomatis
genome sequence and/or the C. muridarum genome sequence.
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[0064] In some embodiments, fusion proteins for use according to the
disclosure consist
essentially of two Chlamydia polypeptides, or an immunogenic fragment thereof,
as described
herein.
[0065] In some embodiments, fusion proteins for use according to the
disclosure consist
essentially of three Chlamydia polypeptides, or an immunogenic fragment
thereof, as described
herein.
[0066] In some embodiments, fusion proteins for use according to the
disclosure consist
essentially of four Chlamydia polypeptides, or an immunogenic fragment
thereof, as described
herein.
[0067] In some embodiments, fusion proteins for use according to the
disclosure include at least
two Chlamydia polypeptides, or an immunogenic fragment thereof, for example,
at least 2, 3, 4,
5, or more.
[0068] By "fusion protein" or "chimeric protein" is meant a recombinant
protein or polypeptide
in which at least two Chlamydia proteins or antigens, as for example,
described herein or set
forth in Tables 1 or 2, or Figures 1A-II, are present in a single, recombinant
polypeptide. It is to
be understood that the individual Chlamydia proteins or antigens that make up
the fusion protein
can be present in the fusion protein in any order or orientation. For example,
in some
embodiments, the individual Chlamydia proteins or antigens can be present in
the fusion protein
in the opposite direction relative to the naturally occurring (i.e. N-terminal
to C-terminal
reversed) direction. In some embodiments, the fusion protein can include full-
length Chlamydia
proteins or antigens. In alternative embodiments, the fusion protein can
include portions or
fragments of Chlamydia proteins or antigens, such as regions of the Chlamydia
proteins or
antigens exposed to the surface ("passenger domains") or including
immunodominant epitopes.
[0069] In some embodiments, a fusion protein may be provided in combination
with a
heterologous peptide or polypeptide, such as an epitope tag.
[0070] In some embodiments, the individual Chlamydia protein or antigen
sequences may be
joined directly to each other.
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[0071] In some embodiments, a fusion protein may be provided in combination
with a
heterologous peptide or polypeptide, such as a linker or spacer that, for
example, enables correct
folding and/or presentation and/or expression of the fusion protein. The
linker or spacer may be
placed between each individual Chlamydia protein or antigen sequence, or may
be placed
between only some of the individual Chlamydia protein or antigen sequences
present in the
fusion protein.
[0072] In alternative embodiments, the linker may be a heterologous linker,
such as a sequence
(e.g., an alpha helical sequence) from another Chlamydia protein or antigen or
from a non-
adjacent location of the Chlamydia proteins or antigens forming the fusion
protein, or may be a
homologous linker, such as as a sequence (e.g., an alpha helical sequence)
from one of the
Chlamydia proteins or antigens forming the fusion protein and adjacent to the
sequence used in
the fusion protein. For example, the passenger domains of the Pmp fusion
partners can be
connected via an alpha-helical linker (shown in underline in Figures 2E-F)
polypeptide. The
linker polypeptides can be derived from polypeptide sequences of one of the
fusion protein
partners. For example, the linker for the PmpE-PmpF fusion protein includes a
sequence from
PmpE and the linker for the PmpG-PmpH fusion protein includes a sequence from
PmpG
(Figures 2E-F).
[0073] It is well known in the art that some modifications and changes can be
made in the
structure of a polypeptide without substantially altering the biological
function of that
polypeptide e.g., its ability to be cleaved into smaller peptides that are
capable of binding to
MiFIC proteins, to obtain a biologically equivalent polypeptide. Accordingly,
it will be
appreciated by a person of skill in the art that the numerical designations of
the positions of
amino acids within a sequence are relative to the specific sequence. Also the
same positions may
be assigned different numerical designations depending on the way in which the
sequence is
numbered and the sequence chosen. Furthermore, sequence variations such as
insertions or
deletions, may change the relative position and subsequently the numerical
designations of
particular amino acids at and around a site.
[0074] A "protein," "peptide," or "polypeptide" is any chain of two or more
amino acids,
including naturally occurring or non-naturally occurring amino acids or amino
acid analogues,
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regardless of post-translational modification (e.g., glycosylation or
phosphorylation). An "amino
acid sequence," "polypeptide," "peptide," or "protein" of the invention may
include peptides or
proteins that have abnormal linkages, cross links and end caps, non-peptidyl
bonds or alternative
modifying groups. Such modified peptides are also within the scope of the
invention. The term
"modifying group" is intended to include structures that are directly attached
to the peptidic
structure (e.g., by covalent coupling), as well as those that are indirectly
attached to the peptidic
structure (e.g., by a stable non-covalent association or by covalent coupling
to additional amino
acid residues, or mimetics, analogues or derivatives thereof, which may flank
the core peptidic
structure). For example, the modifying group can be coupled to the amino-
terminus or carboxy-
terminus of a peptidic structure, or to a peptidic or peptidomimetic region
flanking the core
domain.
[0075] Alternatively, the modifying group can be coupled to a side chain of at
least one amino
acid residue of a peptidic structure, or to a peptidic or peptido- mimetic
region flanking the core
domain (e.g., through the epsilon amino group of a lysyl residue(s), through
the carboxyl group
of an aspartic acid residue(s) or a glutamic acid residue(s), through a
hydroxy group of a tyrosyl
residue(s), a serine residue(s) or a threonine residue(s) or other suitable
reactive group on an
amino acid side chain). Modifying groups covalently coupled to the peptidic
structure can be
attached by means and using methods well known in the art for linking chemical
structures,
including, for example, amide, alkylamino, carbamate or urea bonds.
[0076] In one aspect of the invention, polypeptides of the present invention
also extend to
biologically equivalent peptides or "variants" that differ from a portion of
the sequence of the
polypeptides of the present invention by conservative amino acid
substitutions, or differ by non-
conservative substitutions that do not affect biological function e.g.,
immunogenicity. As used
herein, the term "conserved amino acid substitutions" refers to the
substitution of one amino acid
for another at a given location in the peptide, where the substitution can be
made without
substantial loss of the relevant function. In making such changes,
substitutions of like amino acid
residues can be made on the basis of relative similarity of side-chain
substituents, for example,
their size, charge, hydrophobicity, hydrophilicity, and the like, and such
substitutions may be
assayed for their effect on the function of the peptide by routine testing.
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[0077] As used herein, the term "amino acids" means those L-amino acids
commonly found in
naturally occurring proteins, D-amino acids and such amino acids when they
have been
modified. Accordingly, amino acids of the invention may include, for example:
2-Aminoadipic
acid; 3-Aminoadipic acid; beta-Alanine; beta-Aminopropionic acid; 2-
Aminobutyric acid; 4-
Aminobutyric acid; piperidinic acid; 6-Aminocaproic acid; 2-Aminoheptanoic
acid; 2-
Aminoisobutyric acid; 3- Aminoisobutyric acid; 2-Aminopimelic acid; 2,4
Diaminobutyric acid;
Desmosine; 2,2'-Diaminopimelic acid; 2,3-Diaminopropionic acid; N-
Ethylglycine; N-
Ethylasparagine; Hydroxylysine; allo-Hydroxylysine; 3-Hydroxyproline; 4-
Hydroxyproline;
Isodesmosine; allo-Isoleucine; N-Methylglycine; sarcosine; N-Methylisoleucine;
6-N-
methyllysine; N-Methylvaline; Norvaline; Norleucine; and Ornithine.
[0078] In some embodiments, conserved amino acid substitutions may be made
where an amino
acid residue is substituted for another having a similar hydrophilicity value
(e.g., within a value
of plus or minus 2.0, or plus or minus 1.5, or plus or minus 1.0, or plus or
minus 0.5), where the
following may be an amino acid having a hydropathic index of about -1.6 such
as Tyr (-1.3) or
Pro (-1.6) are assigned to amino acid residues (as detailed in United States
Patent No. 4,554,101,
incorporated herein by reference): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu
(+3.0); Ser (+0.3);
Asn (+0.2); Gin (+0.2); Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-
0.5); Cys (-1.0); Met (-
1.3); Val (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-
3.4).
[0079] In alternative embodiments, conservative amino acid substitutions may
be made where an
amino acid residue is substituted for another having a similar hydropathic
index (e.g., within a
value of plus or minus 2.0, or plus or minus 1.5, or plus or minus 1.0, or
plus or minus 0.5). In
such embodiments, each amino acid residue may be assigned a hydropathic index
on the basis of
its hydrophobicity and charge characteristics, as follows: He (+4.5); Val
(+4.2); Leu (+3.8); Phe
(+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-
0.8); Trp (-0.9); Tyr (-
1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5);
Lys (-3.9); and Arg (-
4.5).
[0080] In alternative embodiments, conservative amino acid substitutions may
be made using
publicly available families of similarity matrices (60, 70, 102, 103, 94, 104,
86) The PAM matrix
is based upon counts derived from an evolutionary model, while the Blosum
matrix uses counts
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derived from highly conserved blocks within an alignment. A similarity score
of above zero in
either of the PAM or Blosum matrices may be used to make conservative amino
acid
substitutions.
[0081] In alternative embodiments, conservative amino acid substitutions may
be made where an
amino acid residue is substituted for another in the same class, where the
amino acids are divided
into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala,
Val, Leu, He, Phe,
Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr,
Cys, Asn, Gln, Tyr.
[0082] Conservative amino acid changes can include the substitution of an L-
amino acid by the
corresponding D-amino acid, by a conservative D-amino acid, or by a naturally-
occurring, non-
genetically encoded form of amino acid, as well as a conservative substitution
of an L-amino
acid. Naturally-occurring non-genetically encoded amino acids include beta-
alanine, 3-amino-
propionic acid, 2,3-diamino propionic acid, alpha-aminoisobutyric acid, 4-
amino-butyric acid, N-
methylglycine (sarcosine), hydroxyproline, ornithine, citrulline, t-
butylalanine, t-butylglycine, N-
methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, norvaline, 2-
napthylalanine,
pyridylalanine, 3-benzothienyl alanine, 4-chlorophenylalanine, 2-
fluorophenylalanine, 3-
fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-
isoquinoline-3-
carboxylix acid, beta-2-thienylalanine, methionine sulfoxide, homoarginine, N-
acetyl lysine, 2-
amino butyric acid, 2-amino butyric acid, 2,4,-diamino butyric acid, p-
aminophenylalanine, N-
methylvaline, homocysteine, homoserine, cysteic acid, epsilon-amino hexanoic
acid, delta-amino
valeric acid, or 2,3-diaminobutyric acid.
[0083] In alternative embodiments, conservative amino acid changes include
changes based on
considerations of hydrophilicity or hydrophobicity, size or volume, or charge.
Amino acids can
be generally characterized as hydrophobic or hydrophilic, depending primarily
on the properties
of the amino acid side chain. A hydrophobic amino acid exhibits a
hydrophobicity of greater than
zero, and a hydrophilic amino acid exhibits a hydrophilicity of less than
zero, based on the
normalized consensus hydrophobicity scale of Eisenberg et at. (Ann. Rev.
Biochem. 53: 595-623,
1984). Genetically encoded hydrophobic amino acids include Gly, Ala, Phe, Val,
Leu, He, Pro,
Met and Trp, and genetically encoded hydrophilic amino acids include Thr, His,
Glu, Gln, Asp,
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Arg, Ser, and Lys. Non-genetically encoded hydrophobic amino acids include t-
butylalanine,
while non-genetically encoded hydrophilic amino acids include citrulline and
homocysteine.
[0084] Hydrophobic or hydrophilic amino acids can be further subdivided based
on the
characteristics of their side chains. For example, an aromatic amino acid is a
hydrophobic amino
acid with a side chain containing at least one aromatic or heteroaromatic
ring, which may contain
one or more substituents such as -OH, -SH, -CN, -F, -CI, -Br, -I, -NO2, -NO, -
NH2, -NHR, -
NRR, -C(0)R, -C(0)0H, -C(0)0R, -C(0)NH2, -C(0)NHR, -C(0)NRR, etc., where R is
independently ( -C6) alkyl, substituted (CI-C6) alkyl, (C C6) alkenyl,
substituted ( -C6) alkenyl,
(Cj-C6) alkynyl, substituted (Ct-C6) alkynyl, (C5-C2o) aryl, substituted (C5-
C20) aryl, (C6-C26)
alkaryl, substituted (C6-C26) alkaryl, 5-20 membered heteroaryl, substituted 5-
20 membered
heteroaryl, 6-26 membered alkheteroaryl or substituted 6-26 membered
alkheteroaryl.
Genetically encoded aromatic amino acids include Phe, Tyr, and Trp, while non-
genetically
encoded aromatic amino acids include phenylglycine, 2-napthylalanine, beta-2-
thienylalanine, 1
,2,3, 4-tetrahydro-isoquinoline-3 -carboxylic acid, 4-chlorophenylalanine, 2-
fluorophenylalanine3-fluorophenylalanine, and 4-fluorophenylalanine.
[0085] An apolar amino acid is a hydrophobic amino acid with a side chain that
is uncharged at
physiological pH and which has bonds in which a pair of electrons shared in
common by two
atoms is generally held equally by each of the two atoms (i.e., the side chain
is not polar).
Genetically encoded apolar amino acids include Gly, Leu, Val, He, Ala, and
Met, while non-
genetically encoded apolar amino acids include cyclohexylalanine. Apolar amino
acids can be
further subdivided to include aliphatic amino acids, which is a hydrophobic
amino acid having
an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids
include Ala, Leu,
Val, and He, while non-genetically encoded aliphatic amino acids include
norleucine.
[0086] A polar amino acid is a hydrophilic amino acid with a side chain that
is uncharged at
physiological pH, but which has one bond in which the pair of electrons shared
in common by
two atoms is held more closely by one of the atoms.
Genetically encoded polar amino acids include Ser, Thr, Asn, and Gin, while
non-genetically
encoded polar amino acids include citrulline, N-acetyl lysine, and methionine
sulfoxide.
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[0087] An acidic amino acid is a hydrophilic amino acid with a side chain pKa
value of less than
7. Acidic amino acids typically have negatively charged side chains at
physiological pH due to
loss of a hydrogen ion. Genetically encoded acidic amino acids include Asp and
Glu. A basic
amino acid is a hydrophilic amino acid with a side chain pKa value of greater
than 7. Basic
amino acids typically have positively charged side chains at physiological pH
due to association
with hydronium ion.
Genetically encoded basic amino acids include Arg, Lys, and His, while non-
genetically encoded
basic amino acids include the non-cyclic amino acids ornithine, 2,3,-
diaminopropionic acid, 2,4-
diaminobutyric acid, and homoarginine.
It will be appreciated by one skilled in the art that the above
classifications are not absolute and
that an amino acid may be classified in more than one category. In addition,
amino acids can be
classified based on known behaviour and or
characteristic chemical, physical, or biological properties based on specified
assays or as
compared with previously identified amino acids. Amino acids can also include
bifunctional
moieties having amino acid-like side chains.
[0088] Conservative changes can also include the substitution of a chemically
derivatised moiety
for a non-derivatised residue, by for example, reaction of a functional side
group of an amino
acid. Thus, these substitutions can include compounds whose free amino groups
have been
derivatised to amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy
groups, t-
butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Similarly, free
carboxyl groups
can be derivatized to form salts, methyl and ethyl esters or other types of
esters or hydrazides,
and side chains can be derivatized to form 0-acyl or 0-alkyl derivatives for
free hydroxyl groups
or N-im-benzylhistidine for the imidazole nitrogen of histidine.
[0089] Peptides or peptide analogues can be synthesised by standard chemical
techniques, for
example, by automated synthesis using solution or solid phase synthesis
methodology.
Automated peptide synthesisers are commercially available and use techniques
well known in
the art. Peptides and peptide analogues can also be prepared using recombinant
DNA technology
using standard methods such as those described in, for example, Sambrook, et
at. (Molecular
Cloning: A Laboratory Manual. 3( ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor
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Laboratory Press, Cold Spring Harbor, N.Y., 2000) or Ausubel et at. (Current
Protocols in
Molecular Biology, John Wiley & Sons, New York, N.Y., 1987-2012).
[0090] Accordingly, and as discussed herein, compounds for use according to
the disclosure
include nucleic acid molecules encoding the fusion proteins described herein.
[0091] The terms "nucleic acid" or "nucleic acid molecule" encompass both RNA
(plus and
minus strands) and DNA, including cDNA, genomic DNA, and synthetic (e.g.,
chemically
synthesized) DNA. The nucleic acid may be double-stranded or single-stranded.
Where single-
stranded, the nucleic acid may be the sense strand or the antisense strand. A
nucleic acid
molecule may be any chain of two or more covalently bonded nucleotides,
including naturally
occurring or non-naturally occurring nucleotides, or nucleotide analogs or
derivatives. By
"RNA" is meant a sequence of two or more covalently bonded, naturally
occurring or modified
ribonucleotides. One example of a modified RNA included within this term is
phosphorothioate
RNA. By "DNA" is meant a sequence of two or more covalently bonded, naturally
occurring or
modified deoxyribonucleotides. By "cDNA" is meant complementary or copy DNA
produced
from an RNA template by the action of RNA-dependent DNA polymerase (reverse
transcriptase). Thus a "cDNA clone" means a duplex DNA sequence complementary
to an RNA
molecule of interest, carried in a cloning vector. By "complementary" is meant
that two nucleic
acids, e.g., DNA or RNA, contain a sufficient number of nucleotides which are
capable of
forming Watson-Crick base pairs to produce a region of double-strandedness
between the two
nucleic acids. Thus, adenine in one strand of DNA or RNA pairs with thymine in
an opposing
complementary DNA strand or with uracil in an opposing complementary RNA
strand. It will be
understood that each nucleotide in a nucleic acid molecule need not form a
matched Watson-
Crick base pair with a nucleotide in an opposing complementary strand to form
a duplex. A
nucleic acid molecule is "complementary" to another nucleic acid molecule if
it hybridizes,
under conditions of high stringency, with the second nucleic acid molecule.
[0092] A compound is "isolated" when it is separated from the components that
naturally
accompany it. Typically, a compound is isolated when it is at least 50%, or
60%, or more
generally at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% by weight, of the
total material in a
sample. Thus, for example, a polypeptide that is chemically synthesized or
produced by
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recombinant technology will be generally be substantially free from its
naturally associated
components. A nucleic acid molecule will generally be substantially pure or
"isolated" when it is
not immediately contiguous with (i.e., covalently linked to) the coding
sequences with which it is
normally contiguous in the naturally occurring genome of the organism from
which the DNA of
the invention is derived. Therefore, an "isolated" gene or nucleic acid
molecule is intended to
mean a gene or nucleic acid molecule which is not flanked by nucleic acid
molecules which
normally (in nature) flank the gene or nucleic acid molecule (such as in
genomic sequences)
and/or has been completely or partially purified from other transcribed
sequences (as in a cDNA
or RNA library). For example, an isolated nucleic acid of the invention may be
substantially
isolated with respect to the complex cellular milieu in which it naturally
occurs. The term
therefore includes, e.g., a recombinant nucleic acid incorporated into a
vector, such as an
autonomously replicating plasmid or virus; or into the genomic DNA of a
prokaryote or
eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic
DNA fragment
produced by PCR or restriction endonuclease treatment) independent of other
sequences. It also
includes a recombinant nucleic acid which is part of a hybrid gene encoding
additional
polypeptide sequences. Preferably, an isolated nucleic acid comprises at least
about 50, 80 or 90
percent (on a molar basis) of all macromolecular species present. Thus, an
isolated gene or
nucleic acid molecule can include a gene or nucleic acid molecule which is
synthesized
chemically or by recombinant means. Recombinant DNA contained in a vector are
included in
the definition of "isolated" as used herein. Also, isolated nucleic acid
molecules include
recombinant DNA molecules in heterologous host cells, as well as partially or
substantially
purified DNA molecules in solution. In vivo and in vitro RNA transcripts of
the DNA molecules
of the present invention are also encompassed by "isolated" nucleic acid
molecules.
[0093] Various genes and nucleic acid sequences of the invention may be
recombinant sequences. The term "recombinant" means that something has been
recombined, so
that when made in reference to a nucleic acid construct the term refers to a
molecule that is
comprised of nucleic acid sequences that are joined together or produced by
means of molecular
biological techniques. The term "recombinant" when made in reference to a
protein or a
polypeptide refers to a protein or polypeptide molecule which is expressed
using a recombinant
nucleic acid construct created by means of molecular biological techniques.
Recombinant nucleic
acid constructs may include a nucleotide sequence which is ligated to, or is
manipulated to
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become ligated to, a nucleic acid sequence to which it is not ligated in
nature, or to which it is
ligated at a different location in nature. Referring to a nucleic acid
construct as "recombinant"
therefore indicates that the nucleic acid molecule has been manipulated using
genetic
engineering, i.e. by human intervention.
[0094] Recombinant nucleic acid constructs may for example be introduced into
a host cell by
transformation. Such recombinant nucleic acid constructs may include sequences
derived from
the same host cell species or from different host cell species, which have
been isolated and
reintroduced into cells of the host species. Recombinant nucleic acid
construct sequences may
become integrated into a host cell genome, either as a result of the original
transformation of the
host cells, or as the result of subsequent recombination and/or repair events.
[0095] As used herein, "heterologous" in reference to a nucleic acid or
protein is a molecule that
has been manipulated by human intervention so that it is located in a place
other than the place in
which it is naturally found. For example, a nucleic acid sequence from one
species may be
introduced into the genome of another species, or a nucleic acid sequence from
one genomic
locus may be moved to another genomic or extrachromasomal locus in the same
species. A
heterologous protein includes, for example, a protein expressed from a
heterologous coding
sequence or a protein expressed from a recombinant gene in a cell that would
not naturally
express the protein. A heterologous fusion protein may include a protein in a
non-natural
orientation (i.e., N to C) or may include a fragment or portion of a protein
located in a place,
within the protein, other than the place in which it is naturally found.
[0096] A "substantially identical" sequence is an amino acid or nucleotide
sequence that differs
from a reference sequence only by one or more conservative substitutions, as
discussed herein, or
by one or more non-conservative substitutions, deletions, or insertions
located at positions of the
sequence that do not destroy the biological function of the amino acid or
nucleic acid molecule.
Such a sequence can be any integer from 45% to 99%, or more generally at least
45%, 50, 55%
or 60%, or at least 65%, 75%, 80%, 85%, 90%, or 95%, or as much as 96%, 97%,
98%, or 99%
identical at the amino acid or nucleotide level to the sequence used for
comparison using, for
example, the Align Program or FASTA. For polypeptides, the length of
comparison sequences
may be at least 2, 5, 10, or 15 amino acids, or at least 20, 25, or 30 amino
acids. In alternate
CA 02881213 2015-02-05
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embodiments, the length of comparison sequences may be at least 35, 40, or 50
amino acids, or
over 60, 80, or 100 amino acids. For nucleic acid molecules, the length of
comparison sequences
may be at least 5, 10, 15, 20, or 25 nucleotides, or at least 30, 40, or 50
nucleotides. In alternate
embodiments, the length of comparison sequences may be at least 60, 70, 80, or
90 nucleotides,
or over 100, 200, or 500 nucleotides. Sequence identity can be readily
measured using publicly
available sequence analysis software (e.g., Sequence Analysis Software Package
of the Genetics
Computer Group, University of Wisconsin Biotechnology Center, 1710 University
Avenue,
Madison, Wis. 53705, or BLAST software available from the National Library of
Medicine, or
as described herein). Examples of useful software include the programs Pile-up
and PrettyBox.
Such software matches similar sequences by assigning degrees of homology to
various
substitutions, deletions, substitutions, and other modifications.
[0097] Alternatively, or additionally, two nucleic acid sequences may be
"substantially identical" if they hybridize under high stringency conditions.
In some
embodiments, high stringency conditions are, for example, conditions that
allow hybridization
comparable with the hybridization that occurs using a DNA probe of at least
500 nucleotides in
length, in a buffer containing 0.5 M NaHPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1%
BSA
(fraction V), at a temperature of 65 C, or a buffer containing 48% formamide,
4.8x SSC, 0.2 M
Tris-C1, pH 7.6, lx Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at
a temperature of
42 C. (These are typical conditions for high stringency northern or Southern
hybridizations.)
Hybridizations may be carried out over a period of about 20 to 30 minutes, or
about 2 to 6 hours,
or about 10 to 15 hours, or over 24 hours or more. High stringency
hybridization is also relied
upon for the success of numerous techniques routinely performed by molecular
biologists, such
as high stringency PCR, DNA sequencing, single strand conformational
polymorphism analysis,
and in situ hybridization. In contrast to northern and Southern
hybridizations, these techniques
are usually performed with relatively short probes (e.g., usually about 16
nucleotides or longer
for PCR or sequencing and about 40 nucleotides or longer for in situ
hybridization). The high
stringency conditions used in these techniques are well known to those skilled
in the art of
molecular biology (Ausubel et at, Current Protocols in Molecular Biology, John
Wiley & Sons,
New York, N.Y., 1998).
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[0098] Substantially identical sequences may for example be sequences that are
substantially
identical to the Chlamydia spp. sequences described or referenced herein. A
substantially
identical sequence may for example be a sequence that is substantially
identical to the sequence
of any one of SEQ ID NOs: 1-71, or to any one of the sequences indicated by
the locus tags
referenced in the C. trachomatis genome sequence and/or the C. muridarum
genome sequence as
indicated herein, or a fragment or variant thereof In some embodiments, a
substantially
identical sequence may for example be a nucleotide sequence that is
complementary to or
hybridizes with the sequence of any one of the nucleic acid sequences
described herein, or with
the sequence encoding any one of the amino acid sequences described herein, or
to any one of
the sequences indicated by the locus tags referenced in the C. trachomatis
genome sequence
and/or the C. muridarum genome sequence as indicated herein, or a fragment or
variant thereof
In some embodiments, a substantially identical sequence may be derived from a
Chlamydia spp.,
such as a C. trachomatis or a C. muridarum.
[0099] Pharmaceutical & Veterinary Compositions, Dosages, And Administration
[00100] The compounds and compositions as described herein may be used to
prepare
vaccine or other formulations and/or used in the induction of an immune
response to a
Chlamydia antigen or epitope. In some embodiments, the compositions may be
formulated as
admixtures of fusion proteins consisting of two or more Chlamydia proteins or
immunogenic
fragments thereof, as described herein. In alternative embodiments, the
compositions may be
formulated using a single fusion protein. In alternative embodiments, the
compositions may
include MOlViP, either as part of a fusion protein or as an individual protein
in admixture with a
fusion protein as described herein.
[00101] The compounds and compositions can be provided alone or in
combination with
other compounds (for example, nucleic acid molecules, small molecules,
polypeptides, peptides,
or peptide analogues), in the presence of a liposome, an adjuvant, or any
pharmaceutically
acceptable carrier, in a form suitable for administration to an animal
subject, for example, mice,
humans, pigs, etc. If desired, treatment with a compound according to the
invention may be
combined with more traditional and existing therapies for Chlamydia infection.
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[00102] Conventional pharmaceutical practice may be employed to provide
suitable
formulations to administer the compounds or compositions to subjects infected
by a Chlamydia
pathogen. Any appropriate route of administration may be employed, for
example, parenteral,
intravenous, subcutaneous, intramuscular, intracranial, intrathecal,
intraorbital, ophthalmic,
intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal,
intranasal, epidermal,
transdermal, mucosal membrane aerosol, nasal, rectal, vaginal, topical or oral
administration. In
some embodiments, the compounds or compositions described herein may be
applied to
epithelial surfaces. Some epithelial surfaces may comprise a mucosal membrane,
for example
buccal, gingival, nasal, tracheal, bronchial, gastrointestinal, genital,
rectal, urethral, vaginal,
cervical, uterine and the like. Some epithelial surfaces may comprise
keratinized cells, for
example, skin, tongue, gingival, palate or the like. In some embodiments, the
Chlamydia
infection may be in the lung, genital tract or eye and the compounds or
compositions described
herein may be administered intranasally or by injection.
[00103] Formulations may be in the form of liquid solutions or
suspensions; tablets or
capsules; powders, nasal drops, or aerosols. Methods are well known in the art
for making
formulations (Berge et al. 1977. J. Pharm Sci. 66: 1 - 19); Remington-The
Science and Practice
of Pharmacy, 21st edition. Gennaro et al editors. Lippincott Williams &
Wilkins Philadelphia.).
Such excipients may include, for example, salts, buffers, antioxidants,
complexing agents,
tonicity agents, cryoprotectants, lyoprotectants, suspending agents,
emulsifying agents,
antimicrobial agents, preservatives, chelating agents, binding agents,
surfactants, wetting agents,
anti-adherents agents, disentegrants, coatings, glidants, deflocculating
agents, anti-nucleating
agents, surfactants, stabilizing agents, non-aqueous vehicles such as fixed
oils, polymers or
encapsulants for sustained or controlled release, ointment bases, fatty acids,
cream bases,
emollients, emulsifiers, thickeners, preservatives, solubilizing agents,
humectants, water,
alcohols or the like.
[00104] Formulations for parenteral administration may, for example,
contain excipients,
sterile water, or saline, polyalkylene glycols such as polyethylene glycol,
oils of vegetable origin,
or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer,
lactide/glycolide
copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to
control the release
of the compounds or compositions. Other potentially useful parenteral delivery
systems for
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modulatory compounds include ethylene-vinyl acetate copolymer particles,
osmotic pumps,
implantable infusion systems, and liposomes. Formulations for inhalation may
contain
excipients, for example, lactose, or may be aqueous solutions containing, for
example,
polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily
solutions for
administration in the form of nasal drops, or as a gel.
[00105] For therapeutic or prophylactic compositions, the compounds or
compositions are
administered to an animal in an amount effective to stop or slow a Chlamydia
infection.
[00106] An "effective amount" of a compound according to the invention
includes a
therapeutically effective amount or a prophylactically effective amount. A
"therapeutically effective amount" refers to an amount effective, at dosages
and for periods of
time necessary, to achieve the desired therapeutic result, such as reduction
of a Chlamydia
infection or induction of an immune response to a Chlamydia antigen or
epitope. A
therapeutically effective amount of a compound may vary according to factors
such as the
disease state, age, sex, and weight of the subject, and the ability of the
compound to elicit a
desired response in the subject. Dosage regimens may be adjusted to provide
the optimum
therapeutic response. A therapeutically effective amount is also one in which
any toxic or
detrimental effects of the compound are outweighed by the therapeutically
beneficial effects. A
"prophylactically effective amount" refers to an amount effective, at dosages
and for periods of
time necessary, to achieve the desired prophylactic result, such as prevention
of a Chlamydia
infection or induction of an immune response to a Chlamydia antigen or
epitope. Typically, a
prophylactic dose is used in subjects prior to or at an earlier stage of
disease, so that a
prophylactically effective amount may be less than a therapeutically effective
amount. A suitable
range for therapeutically or prophylactically effective amounts of a compound
maybe any integer
from 0.1 nM-0.1M, 0.1 nM-0.05M, 0.05 nM-151.tM or 0.01 nM-10pM.
[00107] In some embodiments, an effective amount may be calculated on a
mass/mass
basis (e.g. micrograms or milligrams per kilogram of subject), or may be
calculated on a
mass/volume basis (e.g. concentration, micrograms or milligrams per
milliliter). Using a
mass/volume unit, one or more peptides or polypeptides may be present at an
amount from about
0.1 ug/ml to about 20 mg/ml, or any amount therebetween, for example 0.1, 0.5,
1, 2, 5, 10, 15,
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20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160 180, 200, 250, 500,
750, 1000, 1500,
2000, 5000, 10000, 20000 ug/ml, or any amount therebetween; or from about 1
ug/ml to about
2000 ug/ml, or any amount therebetween, for example 1.0, 2.0, 5.0, 10.0, 15.0,
20.0, 25.0, 30.0,
35.0, 40.0, 50.0 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160 180, 200, 250,
500, 750, 1000, 1500,
2000, ug/ml or any amount therebetween; or from about 10 ug/ml to about 1000
ug/ml or any
amount therebetween, for example 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0,
50.0 60.0, 70.0, 80.0,
90.0, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000 ug/ml, or any amount
therebetween; or
from about 3Oug/m1 to about 1000ug/m1 or any amount therebetween, for example
30.0, 35.0,
40.0, 50.0 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160 180, 200, 250, 500, 750,
1000 ug/ml.
[00108] Quantities and/or concentrations may be calculated on a mass/mass
basis (e.g.
micrograms or milligrams per kilogram of subject), or may be calculated on a
mass/volume basis
(e.g. concentration, micrograms or milligrams per milliliter). Using a
mass/volume unit, one or
more peptides or polypeptides may be present at an amount from about 0.1 ug/ml
to about 20
mg/ml, or any amount therebetween, for example 0.1, 0.5, 1, 2, 5, 10, 15, 20,
25, 30, 35, 40, 50,
60, 70, 80, 90, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000, 1500, 2000,
5000, 10000,
20000 ug/ml, or any amount therebetween; or from about 1 ug/ml to about 2000
ug/ml, or any
amount therebetween, for example 1.0, 2.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0,
35.0, 40.0, 50.0
60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000,
1500, 2000, ug/ml or
any amount therebetween; or from about 1Oug/m1 to about 100Oug/m1 or any
amount
therebetween, for example 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 50.0 60.0,
70.0, 80.0, 90.0,
100, 120, 140, 160 180, 200, 250, 500, 750, 1000 ug/ml, or any amount
therebetween; or from
about 3Oug/m1 to about 1000ug/m1 or any amount therebetween, for example 30.0,
35.0, 40.0,
50.0 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000
ug/ml.
[00109] Compositions according to various embodiments of the invention,
including
therapeutic compositions, may be administered as a dose comprising an
effective amount of one
or more peptides or polypeptides. The dose may comprise from about 0.1 ug/kg
to about
20mg/kg (based on the mass of the subject), for example 0.1, 0.5, 1,2, 5, 10,
15, 20, 25, 30, 35,
40, 50, 60, 70, 80, 90, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000,
1500, 2000, 5000,
10000, 20000 ug/kg, or any amount therebetween; or from about lug/kg to about
2000ug/kg or
any amount therebetween, for example 1.0, 2.0, 5.0, 10.0, 15.0, 20.0, 25.0,
30.0, 35.0, 40.0, 50.0
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60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000,
1500, 2000 ug/kg, or
any amount therebetween; or from about 10 ug/kg to about 1000 ug/kg or any
amount
therebetween, for example 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 50.0 60.0,
70.0, 80.0, 90.0,
100, 120, 140, 160 180, 200, 250, 500, 750, 1000 ug/kg, or any amount
therebetween; or from
about 3Oug/kg to about 1000ug/kg or any amount therebetween, for example 30.0,
35.0, 40.0,
50.0 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000
ug/kg.
[00110] One of skill in the art will be readily able to interconvert the
units as necessary,
given the mass of the subject, the concentration of the composition,
individual components or
combinations thereof, or volume of the composition, individual components or
combinations
thereof, into a format suitable for the desired application.
[00111] It is to be noted that dosage values may vary with the severity of
the condition to
be alleviated. For any particular subject, specific dosage regimens may be
adjusted over time
according to the individual need and the professional judgment of the person
administering or
supervising the administration of the compositions. Dosage ranges set forth
herein are exemplary
only and do not limit the dosage ranges that may be selected by medical
practitioners. The
amount of active compound in the composition may vary according to factors
such as the disease
state, age, sex, and weight of the individual. Dosage regimens may be adjusted
to provide the
optimum therapeutic response. For example, a single bolus may be administered,
several divided
doses may be administered over time or the dose may be proportionally reduced
or increased as
indicated by the exigencies of the therapeutic situation. It may be
advantageous to formulate
parenteral compositions in dosage unit form for ease of administration and
uniformity of dosage.
[00112] The amount of a composition administered, where it is
administered, the method
of administration and the timeframe over which it is administered may all
contribute to the
observed effect. As an example, a composition may be administered systemically
e.g. by
intravenous administration and have a toxic or undesirable effect, while the
same composition
administered subcutaneously or intranasally may not yield the same undesirable
effect. In some
embodiments, localized stimulation of immune cells in the lymph nodes close to
the site of
subcutaneous injection may be advantageous, while a systemic immune
stimulation may not.
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[00113] In general, compounds or compositions should be used without
causing
substantial toxicity. Toxicity of the compounds of the invention can be
determined using
standard techniques, for example, by testing in cell cultures or experimental
animals and
determining the therapeutic index, i.e., the ratio between the LD50 (the dose
lethal to 50% of the
population) and the LD100 (the dose lethal to 100% of the population). In some
circumstances
however, such as in severe disease conditions, it may be necessary to
administer substantial
excesses of the compositions.
[00114] Compositions according to various embodiments of the invention may
be
provided in a unit dosage form, or in a bulk form suitable for formulation or
dilution at the point
of use. Compositions according to various embodiments of the invention may be
administered to
a subject in a single-dose, or in several doses administered over time. Dosage
schedules may be
dependent on, for example, the subject's condition, age, gender, weight, route
of administration,
formulation, or general health. Dosage schedules may be calculated from
measurements of
adsorption, distribution, metabolism, excretion and toxicity in a subject, or
may be extrapolated
from measurements on an experimental animal, such as a rat or mouse, for use
in a human
subject. Optimization of dosage and treatment regimens are discussed in, for
example, Goodman
& Gilman's The Pharmacological Basis of Therapeutics llth edition. 2006. LL
Brunton, editor.
McGraw-Hill, New York, or Remington- The Science and Practice of Pharmacy,
21st edition.
Gennaro et al editors. Lippincott Williams & Wilkins Philadelphia.
[00115] A "vaccine" is a composition that includes materials that elicit a
desired immune
response. A desired immune response may include protection against infection
by a Chlamydia
spp. pathogen. For example, a desired immune response may include any value
from between
10% to 100%, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
protection against
infection by a Chlamydia spp. pathogen in a vaccinated animal when compared to
a non-
vaccinated animal.
[00116] An "immune response" may generally refer to a response of the
adaptive immune
system, such as a humoral response, and a cell-mediated response. The humoral
response is the
aspect of immunity that is mediated by secreted antibodies, produced in the
cells of the B
lymphocyte lineage (B cell). Secreted antibodies bind to antigens on the
surfaces of invading
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microbes (such as viruses or bacteria), which flags them for destruction.
Humoral immunity is
used generally to refer to antibody production and the processes that
accompany it, as well as the
effector functions of antibodies, including Th2 cell activation and cytokine
production, memory
cell generation, opsonin promotion of phagocytosis, pathogen elimination and
the like. A cell-
mediated response may refer to an immune response that does not involve
antibodies but rather
involves the activation of macrophages, natural killer cells (NK), antigen-
specific cytotoxic T-
lymphocytes, and the release of various cytokines in response to an antigen.
Cell-mediated
immunity may generally refer to some Th cell activation, Tc cell activation
and T-cell mediated
responses.
[00117] Antigen presenting cells (APCs) such as dendritic cells (DCs) take
up
polypeptides and present epitopes of such polypeptides within the context of
the DC MiFIC I and
II complexes to other immune cells including CD4+ and CD8+ cells. An "MiFIC
complex" or
"MiFIC receptor" is a cell-surface receptor encoded by the major
histocompatibility complex of a
subject, with a role in antigen presentation for the immune system. MiFIC
proteins may be found
on several cell types, including antigen presenting cells (APCs) such as
macrophages or dendritic
cells (DCs), or other cells found in a mammal. Epitopes associated with MiFIC
Class I may range
from about 8- 11 amino acids in length, while epitopes associated MiFIC Class
II may be longer,
ranging from about 9-25 amino acids in length.
[00118] Accordingly, an "immune response" includes, but is not limited to,
one or more of
the following responses in a mammal: induction of antibodies, B cells, T cells
(including helper
T cells, suppressor T cells, cytotoxic T cells, y6 T cells) directed
specifically to the antigen(s) in
a composition or vaccine, following administration of the composition or
vaccine. An immune
response to a composition or vaccine thus generally includes the development
in the host
mammal of a cellular and/or antibody-mediated response to the composition or
vaccine of
interest. In general, the immune response will result in prevention or
reduction of infection by a
Chlamydia spp. pathogen. In some embodiments, an immune response refers
specifically to a
cell-mediated response. In some embodiments, an immune response refers
specifically to a cell-
mediated response against a Chlamydia spp. pathogen. In some embodiments, the
compounds
and compositions described herein may be used in the induction of a cell-
mediated immune
response against a Chlamydia spp. pathogen.
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[00119] Vaccines according to the disclosure may include the polypeptides
and nucleic
acid molecules described herein, or immunogenic fragments thereof, and may be
administered
using any form of administration known in the art or described herein.
[00120] An "immunogenic fragment" of a polypeptide or nucleic acid
molecule refers to
an epitope or amino acid or nucleotide sequence that elicits an immune
response. The term
"epitope" refers to an arrangement of amino acids in a protein or
modifications thereon (for
example glycosylation). The amino acids may be arranged in a linear fashion,
such as a primary
sequence of a protein, or may be a secondary or tertiary arrangement of amino
acids in close
proximity once a protein is partially or fully configured. Epitopes may be
specifically bound by
an antibody, antibody fragment, peptide, peptidomimetic or the like, or may be
specifically
bound by a ligand or held within an WIC I or WIC II complex. Epitopes may be
present in a
larger fragment or sequence of a Chlamydia protein as described herein.
[00121] Thus, an immunogenic fragment may include, without limitation, any
portion of
any of the sequences described herein, or a sequence substantially identical
thereto, that includes
one or more epitopes (the site recognized by a specific immune system cell,
such as a T cell). For
example, an immunogenic fragment may include, without limitation, peptides of
any value
between 6 and 60, or over 60, amino acids in length, e.g., peptides of any
value between 10 and
20 amino acids in length, or between 20 and 40 amino acids in length, derived
from any one or
more of the sequences described herein. Such fragments may be identified using
standard
methods known to those of skill in the art, such as epitope mapping techniques
or antigenicity or
hydropathy plots using, for example, the Omiga version 1.0 program from Oxford
Molecular
Group (see, for example, U. S. Patent No. 4,708,871)(76, 77, 81, 92, 73,). An
epitope may have a
range of sizes - for example a linear epitope may be as small as two amino
acids, or may be
larger, from about 3 amino acids to about 20 amino acids. In some embodiments,
an epitope may
be from about 5 amino acids to about 10 or about 15 amino acids in length. An
epitope of
secondary or tertiary arrangements of amino acids may encompass as few as two
amino acids, or
may be larger, from about 3 amino acids to about 20 amino acids. In some
embodiments, a
secondary or tertiary epitope may be from about 5 amino acids to about 10 or
about 15 amino
acids in proximity to some or others within the epitope. In some embodiments,
a fusion protein
as described herein will contain multiple epitopes; in such cases, an
immunogenic fragment may
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include a significant portion of a whole protein that is present in a fusion
protein, as described
herein.
[00122] In some embodiments, a vaccine includes a suitable carrier, such
as an adjuvant,
which is an agent that acts in a non-specific manner to increase the immune
response to a
specific antigen, or to a group of antigens, enabling the reduction of the
quantity of antigen in
any given vaccine dose, or the reduction of the frequency of dosage required
to generate the
desired immune response.
[00123] Exemplary adjuvants include, without limitation, aluminum
hydroxide, alum,
AlhydrogelTM (aluminum trihydrate) or other aluminum-comprising salts,
virosomes, nucleic
acids comprising CpG motifs such as CpG oligodeoxynucleotides (CpG-ODN),
squalene, oils,
1V11 F59 (Novartis), LTK63 (Novartis), QS21, various saponins, virus-like
particles, monomycolyl
glycerol (MMG), monophosphoryl-lipid A (MPL)/trehalose dicorynomycolate, toll-
like receptor
agonists, copolymers such as polyoxypropylene and polyoxyethylene, AbISCO,
ISCOM
(AbISCO-100), montanide ISA 51, Montanide ISA 720 + CpG, etc. or any
combination thereof
In some embodiments, exemplary adjuvants include a cationic lipid delivery
agent such as
dimethyldioctadecylammonium Bromide (DDA) together with a modified
mycobacterial cord
factor trehalose 6,6'-dibehenate (TDB) (DDA/TDB), DDA/M MG or DDA/MPL or any
combination thereof Liposomes with or without incorporated MPL further been
adsorbed to
alum hydroxide may also be useful, see, for example US Patent Nos. 6,093,406
and 6,793,923
B2. In some embodiments, exemplary adjuvants include prokaryotic RNA. In some
embodiments, exemplary adjuvants include those described in for example US
Patent Publication
2006/0286128 In some embodiments, exemplary adjuvants include DDA/TDB, DDA/MMG
or
DDA/MPL and prokaryotic RNA.
[00124] In some embodiments, vaccine compositions include, without
limitation, fusion
proteins as described herein in combination with DDA/TDB, DDA/MMG or DDA/MPL
and,
optionally, prokaryotic RNA.
[00125] In alternative embodiments, vaccine compositions include, without
limitation,
fusion proteins as described herein, in admixture with MOMP, in combination
with DDA/TDB,
DDA/M MG or DDA/MPL and, optionally, prokaryotic RNA.
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[00126] In alternative embodiments, vaccine compositions include, without
limitation,
fusion proteins as described herein, in admixture with MOMP, in combination
with DDA/TDB,
DDA/M MG or DDA/MPL and, optionally, prokaryotic RNA.
[00127] In alternative embodiments, vaccine compositions include a) a
recombinant fusion
protein including the polypeptide sequences of the Chlamydia proteins PmpG,
PmpE, PmpF and
PmpH or immunogenic fragment thereof, b) the adjuvant DDA/MPL and c)
prokaryotic RNA.
[00128] In alternative embodiments, vaccine compositions include a) a
recombinant fusion
protein including the polypeptide sequences of the Chlamydia proteins PmpG,
PmpE, PmpF and
PmpH or immunogenic fragment thereof, b) the adjuvant DDA/TDB and c)
prokaryotic RNA.
[00129] In alternative embodiments, vaccine compositions include a) a
recombinant fusion
protein including the polypeptide sequences of the Chlamydia proteins PmpG,
PmpE, PmpF and
PmpH or immunogenic fragment thereof and b) the adjuvant DDA/MPL.
[00130] In alternative embodiments, vaccine compositions include a) a
recombinant fusion
protein including the polypeptide sequences of the Chlamydia proteins PmpG,
PmpE, PmpF and
PmpH and b) the adjuvant DDA/TDB.
[00131] In alternative embodiments, vaccine compositions include a
formulation
comprising a) a combination (admixture) of two separate fusion proteins, such
as PmpG/PmpH
and PmpE/PmpF respectively, or immunogenic fragments thereof; b) the adjuvant
DDA/MPL
and c) prokaryotic RNA.
[00132] In alternative embodiments, vaccine compositions include a
formulation
comprising a) a combination (admixture) of two separate fusion proteins,
between PmpG/PmpH
and PmpE/PmpF respectively, or immunogenic fragments thereof; b) the adjuvant
DDA/TDB
and c) prokaryotic RNA.
[00133] In alternative embodiments, vaccine compositions include a
formulation
comprising a)a combination (admixture) of two separate fusion proteins,
between PmpG/PmpH
and PmpE/PmpF respectively or immunogenic fragments thereof; and b) the
adjuvant
DDA/1\SPL.
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[00134] In alternative embodiments, vaccine compositions include a
formulation
comprising a) a combination (admixture) of two separate fusion proteins,
between PmpG/PmpH
and PmpE/PmpF respectively or immunogenic fragments thereof; and b) the
adjuvant
DDA/TDB.
[00135] In some embodiments, a composition as described herein may be used
to
inoculate a test subject, for example, an animal model of Chlamydia infection,
such as a mouse.
Methods of experimentally inoculating experimental animals are known in the
art. For example,
testing a Chlamydia spp. vaccine may involve infecting previously inoculated
mice intranasally
with an inoculum comprising an infectious Chlamydia strain, and assessing for
development of
pneumonia. An exemplary assay is described in, for example Tammiruusu et al
2007. Vaccine
25(2):283-290, or in Rey-Ladino et al 2005. Infection and Immunity 73:1568-
1577. It is within
the ability of one of skill in the art to make any minor modifications to
adapt such an assay to a
particular pathogen model.
[00136] In another example, testing a Chlamydia vaccine may involve
serially inoculating
female mice with a candidate T-cell antigen cloned and expressed as described
above. A series of
inoculations may comprise two, three or more serial inoculations. The
candidate T-cell antigens
may be combined with an adjuvant. About three weeks following the last
inoculation in the
series, mice may be treated subcutaneously with 2.5 mg Depo-Provera and one
week later both
naive and immunized mice may be infected intravaginally with Chlamydia . The
course of
infection may be followed by monitoring the number of organisms shed at 2 to 7
day intervals
for 6 weeks. The amount of organism shed may be determined by counting
Chlamydia inclusion
formation in HeLa cells using appropriately diluted vaginal wash samples.
Immunity may be
measured by the reduction in the amount of organism shed in immunized mice
compared to
naive mice.
[00137] In some embodiments, the present disclosure also provides for a
composition for
inducing an immune response in a subject. Compositions according to various
embodiments of
the invention may be used as a vaccine, or in the preparation of a vaccine.
[00138] In another embodiment, a fusion protein as described herein may be
used in the
preparation of a medicament such as a vaccine composition, for the prevention
or treatment of a
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Chlamydia infection. Treatment or treating includes prevention unless
prevention is specifically
excluded, as in alternative embodiments of the disclosure. Treatment or
treating refers to fully or
partially reducing severity of a Chlamydia infection and/or delaying onset of
a Chlamydia
infection, and/or reducing incidence of one or more symptoms or features of a
Chlamydia
infection, including reducing survival, growth, and/or spread of a Chlamydia
spp., such as C.
muridarum or C. trachomatis. In some embodiments, treatment includes inducing
immunity in
an animal subject. In alternative embodiments, treatment includes inducing
cellular immunity in
an animal subject. Treatment may be administered to a subject who does not
exhibit signs of a
disease, disorder, and/or condition (an asymptomatic subject), and/or to a
subject who exhibits
only early signs of a disease, disorder, and/or condition for the purpose of
decreasing the risk of
developing pathology associated with the disease, disorder, and/or condition.
In some
embodiments, treatment includes delivery of an immunogenic composition (e.g.,
a vaccine) to a
subject.
[00139] The composition or medicament may be used for the prevention or
treatment of a
Chlamydia infection in a subject having, or suspected of having such an
infection. In some
embodiments, the composition or medicament may be used for the prevention or
treatment of
urogenital or ocular conditions. Urogenital conditions include without
limitation urethritis,
cervicitis, pharyngitis, proctitis, epididymitis, and prostatis. Ocular
conditions include without
limitation trachoma and conjunctivitis.
[00140] In some embodiments, a fusion protein described herein, alone or
in combination,
may be used to diagnose the presence of a Chlamydia infection in a subject for
example even in
an asymptomatic subject. Diagnosis may be determine T cell responses and may
be performed
using any technique described herein or known to the skilled person.
[00141] Articles of Manufacture
[00142] Also provided is an article of manufacture, comprising packaging
material and a
composition comprising one or more fusion proteins as provided herein. The
composition
includes a physiologically or pharmaceutically acceptable excipient, and may
further include an
adjuvant, a delivery agent, or an adjuvant and a delivery agent, and the
packaging material may
include a label which indicates the active ingredients of the composition
(e.g. the fusion protein,
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adjuvant or delivery agent as present). The label may further include an
intended use of the
composition, for example as a therapeutic or prophylactic composition to be
used in the manner
described herein.
[00143] Kits
[00144] In another embodiment, a kit for the preparation of a medicament,
comprising a
composition comprising one or more fusion proteins as provided herein, along
with instructions
for its use is provided. The instructions may comprise a series of steps for
the preparation of the
medicament, the medicament being useful for inducing a therapeutic or
prophylactic immune
response in a subject to whom it is administered. The kit may further comprise
instructions for
use of the medicament in treatment for treatment, prevention or amelioration
of one or more
symptoms of a Chlamydia infection, and include, for example, dose
concentrations, dose
intervals, preferred administration methods or the like.
[00145] The present invention will be further illustrated in the following
examples.
EXAMPLES
[00146] EXAMPLE 1
[00147] Molecular cloning, expression and purification of recombinant
fusion
proteins
[00148] PmpE, pmpF, pmpG, and pmpH DNA fragments were generated by PCR
using
genomic DNA isolated from C. muridarum. The DNA fragments generated by PCR
were cloned
stepwise into pET32a expression vector (GE Healthcare) after restriction
enzyme digestion using
standard molecular biology techniques. For all four pmp genes, only the
regions that encode
passenger domain of the Pmp protein were cloned into the vector for
expression. The amino acid
sequences of C. muridarum PmpE, PmpF, PmpG and PmpH proteins are shown in
Figure 1.
Passenger domain portions of PmpE, PmpF, PmpG and PmpH, between amino acid 18
to 575,
20 to 575, 25 to 555, and 27 to 575, respectively, of the whole proteins were
used. A C-terminal
His-tag was introduced to all the fusion proteins. Plasmids containing the pmp
genes were
transformed into the E. coil strain BL21(DE3) (Strategene) where protein
expression was carried
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out by inducing the lac promoter for expression of T7 RNA polymerase using
isopropyl-b-D-
thiogalactoside pyranoside.
[00149] The soluble expressed fusion proteins were purified from E. coil
lysates by
affinity chromatography using glutathione sepharose 4 fastflow purification
system (GE
Healthcare) using the N-terminal GST-tag . Insoluble fusion proteins were
purified by nickel
column using the His bind purification system (Qiagen) using the C-terminal
His-tag and
refolded by removing urea stepwise. LPS removal of these proteins was carried
out by adding
0.1% Triton-114 in one of the wash buffers during purification. The amino acid
sequences of
recombinant fusion proteins between PmpE and PmpF (i.e. PmpE-PmpF) and PmpG
and PmpH
(i.e. PmpG-PmpH) are shown in Figure 2.
[00150] Protection against Chlamydia genital tract infection in mice
immunized with both
individual protein/antigens, as well as in combinations of proteins formulated
with different
adjuvants, was evaluated. More specifically, groups of eight C57BL/6 mice were
immunized 3
times at 2-week interval with Chlamydia proteins PmpG (G), PmpF (F), MOMP (M),
either
mixed or fused formulated with DDA/TDB (D/T) adjuvant. A group of mice
immunized with
phosphatebuffered saline (PBS) was used as a negative control. Another group
of mice infected
once with 1,500 inclusion-forming units (IFU) of live C. muridarum elementary
bodies (EB)
intranasally (in) was used as a positive control. The experimental groups were
as follows: 1)
PBS (negative control), 2) PmpG + PmpF mixed proteins + DDA/TDB, 3) PmpG-PmpF
fusion
protein + DDA/TDB, 4) PmpG + MOMP mixed proteins + DDA/TDB, 5) PmpG-MOMP
fusion
protein + DDA/TDB and 6) Live EB (in) 1500 IFU (positive control). All groups
were
intravaginally challenged with 1,500 IFU of live C. muridarum EBs 4 weeks
after the final
immunization or 8 weeks after infection. Cervicovaginal washes were taken at
day 6 and day 12,
and bacterial titers were measured on HeLa 229 cells.
[00151] The results indicated that PmpG-PmpF and PmpG-MOMP fusion proteins
formulated in the adjuvant DDA/TDB were protective against Chlamydia genital
tract infection
when evaluated at days 6 and 12 (Figures 3A-B).
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[00152] EXAMPLE 2
[00153] To evaluate protective effect against Chlamydia muridarum genital
tract infection,
C57, Balb/c or C3H mice were immunized with PmpE, F, G, H plus MOMP (either
individual
proteins or fusion proteins), as in the following groups.
[00154] C57 mice: (1) PmpE+PmpF+PmpG+PmpH+MOMP (mixed); (2) PmpE-F
fusion+PmpG-H fusion+MOMP (fusion); (3) PBS; (4) Live EB.
[00155] Balb/c mice: (5) PmpE+PmpF+PmpG+PmpH+MOMP (mixed);
(6) PmpE-F fusion+PmpG-H fusion+MOMP (fusion); (7) PBS; (8) Live EB.
[00156] C3H mice: (9) PmpE+PmpF+PmpG+PmpH+MOMP (mixed); (10) PmpE-F
fusion+PmpG-H fusion+MOMP (fusion); (11) PBS; (12) Live EB.
[00157] Mice were immunized 3 times at 2-week intervals with the fused
proteins
formulated with DDA/MPL. PBS was used as the negative control and mice
infected once with
1,500 inclusion-forming units (IFU) of live C. muridarum elementary bodies
(EB), administered
intranasally, were used as positive controls. All groups were intravaginally
challenged with
1,500 IFU of live C. muridarum elementary bodies 4 weeks after the final
immunization or 8
weeks after live Chlamydia infection. Cervicovaginal washes were taken at day
12 and bacterial
titers were measured on HeLa 229 cells to assess protection.
[00158] Two weeks after the final immunization, mouse splenocytes were
harvested and
stimulated with HK-EB (5x105 IFU/ml). IFN-y- or TNF-a-producing CD4 T cells
were analyzed
by multiparameter flow cytometry. The results are expressed as means SEM for
groups of four
mice (Figure 4).
[00159] C. muridarum individual antigen-specific IFN-y responses in C57,
Balb/c, or C3H
mice after immunization with PmpE, F, G, H plus MOMP either as individual
(mixed) or as
fusion formats were determined by ELISPOT assay. Two weeks after the final
immunization,
mouse splenocytes were harvested and stimulated in vitro for 20 h with 5x105
IFU/ml of HK-
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EB, or 1 [tg/m1 of indicated Chlamydia recombinant protein respectively. The
results are
expressed as means SEM for groups of four mice (Figures 5A-C).
[00160] Vaccine-elicited protection against Chlamydia muridarum genital
tract infection
in C57, Balb/c, or C3H mice after immunization with PmpE, F, G, H plus MOMP,
either as
individual(mixed) or as fusion formats, was evaluated. Four weeks after the
final immunization,
mice were challenged intravaginally with 1,500 IFU of C. muridarum.
Cervicovaginal washes
were taken at day 3,day 6, day 13 and day 13 after infection, and bacterial
shedding was
measured on HeLa 229 cells. Mice immunized with PBS were used as a negative
control, and
mice infected with 1,500 IFU of live C. muridarum intravaginally were used as
a positive
control. *** P value <0.001 in comparison with the PBS group (Figures 6A-C).
[00161] Vaccine-elicited protection against Chlamydia muridarum genital
tract infection
in C57 (A), Balb/c (B), or C3H (C) mice after immunization with PmpE, F, G, H
plus MOMP,
either as individual(mixed) or as fusion formats, was evaluated. Four weeks
after the final
immunization, mice were challenged intravaginally with 1,500 IFU of C.
muridarum.
Cervicovaginal washes were taken at day 3,day 6, day 13 and day 13 after
infection, and
bacterial shedding was measured on HeLa 229 cells. Mice immunized with PBS
were used as a
negative control, and mice infected with 1,500 IFU of live C. muridarum
intravaginally were
used as a positive control. The mean Chlamydia IFU SD is indicated (Figures
7A-C).
[00162] Different mouse strains showed equal levels of protective effect
against
Chlamydia muridarum genital tract infection after immunization with PmpE, F,
G, H plus
MOMP either as individual or as fusion formats.
[00163] All citations are hereby incorporated by reference.
[00164] The present invention has been described with regard to one or
more
embodiments. However, it will be apparent to persons skilled in the art that a
number of
variations and modifications can be made without departing from the scope of
the invention as
defined in the claims.
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