EP1729800A2 - Compositions immunogenes pour chlamydia pneunomiae - Google Patents

Compositions immunogenes pour chlamydia pneunomiae

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Publication number
EP1729800A2
EP1729800A2 EP05724183A EP05724183A EP1729800A2 EP 1729800 A2 EP1729800 A2 EP 1729800A2 EP 05724183 A EP05724183 A EP 05724183A EP 05724183 A EP05724183 A EP 05724183A EP 1729800 A2 EP1729800 A2 EP 1729800A2
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EP
European Patent Office
Prior art keywords
seq
amino acid
acid sequence
protein
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05724183A
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German (de)
English (en)
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EP1729800A4 (fr
Inventor
Guido Grandi
Ratti Giulio
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis Vaccines and Diagnostics Inc
Original Assignee
Chiron Corp
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Publication date
Application filed by Chiron Corp filed Critical Chiron Corp
Publication of EP1729800A2 publication Critical patent/EP1729800A2/fr
Publication of EP1729800A4 publication Critical patent/EP1729800A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/118—Chlamydiaceae, e.g. Chlamydia trachomatis or Chlamydia psittaci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention is in the field of immunology and vaccinology.
  • it relates to immunogenic compositions comprising combinations of immunogenic molecules from Chlamydia pneumoniae.
  • the bacteria of the genus Chlamydia (and Chlamydophila, according to the recently proposed but still controversial re-classification of Chlamydiaceae (Bush et ⁇ l (2001) Int J Syst Evol Microbiol 51: 203-20; Everett et ⁇ l (1999) Int J Syst Bacteriol 49: Pt2 415-40; Schachter et ⁇ l (2001) Int J Syst Evol Microbiol 51: 249, 251-3) are obligate intracellular parasites of eukaryotic cells, which have a unique biphasic life cycle involving two pleiomorphic developmental forms: an extracellular, metabolically inert, spore-like, infectious form (the elementary bodies, EBs) and an intracellular, non- infectious, replicative form (the reticulate bodies, Bs) which remains contained in a specialized cytoplasmic compartment (the Chl ⁇ mydi ⁇ l inclusion).
  • the EBs are responsible for the initial attachment to host cell surface and the establishment of the cytoplasmic inclusion where EBs can differentiate to RBs and thus initiate the replicative stage. Eventually RBs revert to infectious EB forms able to start new replicative cycles in neighbouring host cells.
  • Chlamydia infection is an intracellular infection
  • the currently accepted paradigm is that effective anti-Chlamydial immunisation would require both an adequate T-cell response and high serum levels of neutralising antibodies and that "an ideal vaccine should induce long lasting (neutralising) antibodies and a cell mediated immunity that can quickly respond upon exposure to Chlamydia".
  • WO 02/02404 provides information on the immunogenicity and immunoaccessibility of certain Chlamydia proteins and highlights that (i) current genomic annotations and/or (ii) predictions based on cellular location and/or cellular function based on in-silico analyses may not always be accurate.
  • Applicants have recently engaged in a whole-genome search (Montigiani et al (2002) Infection and Immunity 70:368-379) for possible vaccine candidates among proteins potentially associated with the outer membrane of C.pneumoniae.
  • mice antisera was prepared against over 100 recombinant His-tagged or Glutathione-S-transferase (GST) fusion proteins encoded by genes predicted by in silico analyses to be peripherally located in the Chlamydial cell.
  • GST Glutathione-S-transferase
  • 53 recombinant proteins derived from the genome of Chlamydia Chlamydophila pneumoniae (CPn) were described which induced mouse antibodies, capable of binding, in a FACS assay, to the surface of purified CPn cells.
  • the scope of the Montigiani study (ibid) was restricted to checking if polyclonal antisera produced in mice against the recombinantly expressed antibodies to CPn antigens were capable of binding to the surface of the CPn cells.
  • compositions capable of eliciting an immune response upon exposure to Chlamydia pneumoniae proteins. It is also desirable to provide improved compositions comprising one or more combinations of two or more selected CPn proteins with complementary immunological and/or biological profiles capable of providing immunity against Chlamydial induced disease and/or infection (such as in prophylactic vaccination) or (b) for the eradication of an established chronic Chlamydial infection (such as in therapeutic vaccination).
  • FIG. 1A Assay of in vitro neutralization of C.pneumoniae infectivity for LLC-MK2 cells by polyclonal mouse antisera to recombinant Chlamydial proteins.
  • Figure IB shows serum titres giving 50% neutralization of infectivity for 10 C.pneumoniae recombinant antigens. Each titer was assessed in 3 separate experiments (SEM values shown).
  • Figure 2 shows immunoblot analysis of two dimensional electrophoretic maps of C.pneumoniae EBs using the imune sera described in the text.
  • Figure 3 shows mean numbers of C.pneumoniae IFU recovered from equivalent spleen samples from immunized and mock-immunized hamsters following a systemic challenge.
  • Figure 4 shows flow cytometric analysis of splenocytes from DNA-immunized HLA- A2 transgenic and non transgenic mice.
  • Figure 5 shows a flow cytometric analysis of splenocytes from transgenic and non transgenic mice infected with C. pneumoniae EBs.
  • Figure 6 shows an alignment of the proteins in the 7105-7110 protein family.
  • Figure 7 shows an N-terminal alignment of Cpn0794 - Cpn0799.
  • Figure 8 shows a protein encoded by Cpn0796 and demonstrates a C-terminal domain comprising approximately residues from 1 to 648.
  • Figure 9 shows an alignment of the C-terminal (beta barrel) domains of the proteins encoded by the C.pneumoniae genes Cpn0795 and Cpn0796.
  • Table I shows a summary of data and properties of the C.pneumoniae antigens described in the text.
  • Table 2 shows results from hamster mouse model studies for hypothetical proteins.
  • Table 3 shows expressed genes of CPn EB selected by microarray.
  • Table 4 shows C. pneumoniae selected peptides: protein sources and HLA-A2 stabilization assay.
  • Table 5 shows ELISPOT assay with CD8+ T cells from DNA immunised HLA-A2 transgenic mice.
  • Table 6 shows IFN- ⁇ production from splenocytes of DNA immunized HLA-A2 transgenic and non transgenic mice.
  • the present invention relates to a polypeptide for use as an autotransporter antigen, the polypeptide comprising: (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 78, and SEQ ID NO: 79, (b) an amino acid sequence having at least 50% sequence identity to an amino acid sequence of (a); or (c) an amino acid sequence comprising one or more fragments of at least 7 consecutive amino acids from an amino acid sequence of (a) or combinations thereof.
  • the present invention also relates to the use of a polypeptide in the preparation of a medicament for the prevention or treatment of a Chlamydia pneumoniae infection in an individual.
  • the use of the polypeptide may be as an autotransporter protein which immunoreacts with seropositive serum of an individual infected with Chlamydia pneumoniae.
  • the present invention further relates to a method of eliciting an immune response in an individual comprising administering to the individual a polypeptide comprising (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 78, and SEQ ID NO: 79 (b) an amino acid sequence having at least 50% sequence identity to an amino acid sequence of (a), or (c) an amino acid sequence comprising one or more fragment of at least 1, 2, 3, 4, 5, 6, or 7 amino acids from an amino acid sequence of (a) or mixtures thereof.
  • a method for diagnosing an immune response in an individual comprising (a) contacting a biological sample obtained from the individual with a binding agent that binds to a polypeptide with an autotransporter function, (b) detecting in the biological sample the amount of the polypeptide that binds to the binding agent; and (c) comparing the amount of the polypeptide to a predetermined cut-off value and thereby determining the presence of an immune response in the individual.
  • a composition for eliciting an immune response in a subject comprising two or more Chlamydia pneunoniae autotransporter proteins or immunogenic fragments thereof is also provided.
  • the composition may further comprise one or more immunostimulants.
  • polypeptide for use as an autotransporter antigen comprising an amino acid sequence corresponding to SEQ ID NO: 86, an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 86, or an amino acid sequence comprising one or more fragments of at least 7 consecutive amino acids of SEQ ID NO: 86.
  • the present invention relates to a composition comprising a first biological molecule from a Chlamydia pneumoniae bacterium and a second biological molecule from a Chlamydia pneumoniae bacterium.
  • the first biological molecule is selected from the group consisting of SEQ ID No 1 to SEQ ID No 86, or the group consisting of SEQ ID No. l to 41.
  • composition may also contain the second biological molecule being selected from the group consisting of SEQ ID No 1 to SEQ ID No. 86 or SEQ ID No 1 to SEQ ID
  • composition may also comprise two or more biological molecules selected from the group consisting of SEQ ID Nos 1-41.
  • composition may also comprise one or more biological molecules selected from the group consisting of SEQ ID Nos 1-41 combined with one or more biological molecules selected from the group consisting of SEQ ID Nos 42-86.
  • composition according to any one of the previous claims further comprising an adjuvant such as an ADP-ribosylating exotoxin or a derivative thereof or an adjuvant is selected from the group consisting of cholera toxin (CT), Escherichia heat-labile exterotoxin (LT) and mutants thereof having adjuvant activity.
  • an adjuvant such as an ADP-ribosylating exotoxin or a derivative thereof or an adjuvant is selected from the group consisting of cholera toxin (CT), Escherichia heat-labile exterotoxin (LT) and mutants thereof having adjuvant activity.
  • CT cholera toxin
  • LT heat-labile exterotoxin
  • a vaccine and use of the vaccine is also provided comprising the composition of the present invention.
  • the vaccine may be used in the preparation of a medicament for the prevention or treatment of a Chlamydia infection and may be administered mucosally, intra-nasally or intra-vaginally, for example.
  • a method for treating a Chlamydia infection in a host subject wherein the method comprises the administration of a safe and effective amount of a vaccine.
  • an immunogenic composition comprising a combination of Chlamydia pneumoniae antigens, the combination comprising at least one Chlamydia pneumoniae antigen associated with elementary bodies of Chlamydia pneumoniae and at least one Chlamydia pneumoniae antigen associated with reticulate bodies of Chlamydia pneumoniae.
  • an immunogenic composition comprising a combination of Chlamydia pneumoniae antigens, the combination comprising at least one Chlamydia pneumoniae antigen of a first antigen group and at least one Chlamydia pneumoniae antigen of a second antigen group, said first antigen group comprising a Type III secretion system (TTSS) protein and said second antigen group comprising a Type III secretion system (TTSS) effector protein.
  • TTSS Type III secretion system
  • TTSS Type III secretion system
  • an immunogenic composition comprising a combination of Chlamydia pneumoniae antigens comprising at least one Chlamydia pneumoniae antigen that is conserved over at least two serovars.
  • an immunogenic composition comprising a combination of Chlamydia pneumoniae antigens, the combination eliciting a Chlamydia pneumoniae specific TH1 immune response and a Chlamydia pneumoniae specific TH2 immune response.
  • the present invention further provides a method of monitoring the efficacy of treatment of a patient infected with Chlamydia pneumoniae comprising determining the level of Chlamydia pneumoniae specific antibody in the patient after administration of an immunogenic composition of the present invention to the patient.
  • compositions comprising a first biological molecule from a Chlamydia pneumoniae bacterium and a second biological molecule from a Chlamydia pneumoniae bacterium.
  • biological molecule includes proteins, antigens and nucleic acids.
  • the compositions may also comprise further biological molecules preferably also from Chlamydia pneumoniae. That is to say, the compositions may comprise two or more biological molecules (eg. 3, 4, 5, 6, 7, 8 etc.) at least two of which are from a Chlamydia pneumoniae bacterium (eg. 3, 4, 5, 6, 7, 8 etc.).
  • compositions include those comprising (i) two or more different Chlamydia pneumoniae proteins; (ii) two or more different Chlamydia pneumoniae nucleic acids, or (iii) mixtures of one or more Chlamydia pneumoniae protein and one or more Chlamydia pneumoniae nucleic acid.
  • an immunogenic composition comprising a combination of at least one antigen that elicits a Chlamydia pneumoniae specific THl immune response (such as a cell mediated or cellular immune response) and at least one antigen that elicits a Chlamydia pneumoniae specific TH2 response (such as a humoral or antibody response).
  • the immunogenic composition may further comprise a THl adjuvant and a TH2 adjuvant.
  • an immunogenic composition comprising a combination of Chlamydia pneumoniae antigens comprising at least one Chlamydia pneumoniae antigen that is conserved over at least two serovars.
  • an immunogenic composition comprising a combination of at least one antigen that elicits a Chlamydia pneumoniae specific THl immune response and at least one antigen that elicits a Chlamydia pneumoniae specific TH2 immune response, the combination comprising at least one Chlamydia pneumoniae antigen that is conserved over at least two serovars.
  • the immunogenic composition comprising at least one antigen that elicits a Chlamydia pneumoniae specific THl immune response and at least one antigen that elicits a Chlamydia pneumoniae specific TH2 immmune response preferably comprises a combination of Chlamydia pneumoniae antigens comprising at least one Chlamydia pneumoniae antigen associated with the EB of Chlamydia pneumoniae and at least one Chlamydia pneumoniae antigen associated with the RB of Chlamydia pneumoniae. Still further such combinations can comprise EB and/or RB antigens from one serovar combined with RB and/or EB antigens from at least one other serovar.
  • kits comprising a combination of Chlamydia pneumoniae antigens wherein at least one of the Chlamydia pneumoniae antigens is associated with the EB of Chlamydia pneumoniae and at least one of the Chlamydia pneumoniae antigens is associated with the RB of Chlamydia pneumoniae.
  • the kit may further include a THl adjuvant, a TH2 adjuvant and instructions.
  • the present invention further provides methods of eliciting a Chlamydia specific immune response by administering an immunogenic composition of this invention.
  • the present invention further provides a method of monitoring the efficacy of treatment of a subject infected with Chlamydia pneumoniae comprising determining the level of Chlamydia specific antibody or Chlamydia specific effector molecule in the subject after administration of an immunogenic composition of this invention.
  • the first and second biological molecules are from different Chlamydia pneumoniae species (for example, from different Chlamydia pneumoniae serovars) but they may be from the same species.
  • the biological molecules in the compositions may be from different serogroups or strains of the same species.
  • the first biological molecule is preferably selected from the group consisting of SEQ ID Nos 1-86. More preferably, it is selected from the group consisting of SEQ IDs l-41and/or SEQ ID Nos 42-86. It is preferably a purified or isolated biological molecule.
  • the second biological molecule is preferably selected from the group consisting of SEQ ID Nos 1-86.
  • compositions according to the invention therefore include those comprising: two or more biological molecules selected from the group consisting of SEQ ID Nos 1-41; one or more biological molecules selected from the group consisting of SEQ IDs 1-41 combined with one or more biological molecules selected from the group consisting of SEQ IDs 42-86.
  • One or both of the first and second biological molecules may be a Chlamydia pneumoniae biological molecule which is not specifically disclosed herein, and which may not have been identified, discovered or made available to the public or purified before this patent application was filed.
  • a combination of Chlamydia pneumoniae antigens comprising at least one Type III Secretion System (TTSS) protein and at least one Type III Secretion System (TTSS) secreted or effector protein or fragment thereof.
  • TTSS Type III Secretion System
  • TTSS proteins associated with the Chlamydial TTSS machinery.
  • TTSS is a complex protein secretion and delivery machine or apparatus, which may be located, either wholly or partially, on the Elementary Body (EB) and which allows an organism, such as Chlamydia, to maintain its intracellular niche by injecting proteins, such as bacterial effector proteins (which may act as anti-host virulence determinants) into the cytosol of a eukaryotic cell in order to establish the bacterial infection and to modulate the host cellular functions.
  • TTSS proteins exposed on the EB surface may play a role in adhesion and/or uptake into host cells.
  • the TTSS is a complex protein secretion and delivery machine or apparatus, which may be located on the Elementary Body (EB) and which allows an organism, such as Chlamydia, to maintain its intracellular niche by injecting proteins, such as bacterial effector proteins (which may act as anti-host virulence determinants) into the cytosol of a eukaryotic cell in order to establish the bacterial infection and to modulate the host cellular functions.
  • proteins such as bacterial effector proteins (which may act as anti-host virulence determinants) into the cytosol of a eukaryotic cell in order to establish the bacterial infection and to modulate the host cellular functions.
  • These injected proteins can have various effects on the host cell which include but are not limited to manipulating actin and other structural proteins and modification of host cell signal transduction systems.
  • the injected (or translocated) proteins or substrates of the TTTS system may also be processed and presented by MHC-class I molecules. Not all the proteins secreted by a Type III secretion system are delivered into the host cell or have effector function.
  • the Elementary Body (EB) is regarded as "metabolically inert", it has been postulated that the Chlamydial TTSS system located on the (EB) is triggered by membrane contact and is capable of releasing pre-formed "payload” proteins.
  • Type Three Secretion System becomes active during the intracellular phase of the chlamydial replicative cycle for the secretion of proteins into the host cell cytoplasm and for the insertion of chlamydial proteins (like the Inc set) into the inclusion membrane that separates the growing chlamydial microcolony from the host cell cytoplasm (see Montigiani et al (2002) Infection and Immunity 70(1); 386-379).
  • Proteins may be expressed and secreted by 2 hours (early cycle) after infection while the expression of other early and mid cycle Type III specific genes are not detectable until 6-12 hours (mid cycle). After 16-20 hours, the RBs begin to differentiate into EBs, and by 48-72 hours, the EBs predominate within the inclusion. Host cell lysis results in the release of the EBs to the extracellular space where they can infect more cells.
  • an early gene is one that is expressed (in terms of mRNA expression) early in infection
  • an intermediate gene is one that is expressed in the mid-cycle after infection
  • a late gene is one which is expressed during the terminal transition of RBs to EBs.
  • the present invention may comprise TTSS effector proteins.
  • the TTSS effector proteins as described are associated with the RB form of Chlamydia pneumoniae and may be identified, for example, using immunofluorescence microscopy (see Bannantine et al, Infection and Immunity 66(12); 6017-6021). Effector antibodies to putative Chlamydial TTSS effector proteins secreted by the TTSS machinery may be micro-injected into host cells at specified time points during Chlamydia pneumoniae infection (e.g., early, mid or late cycle).
  • TTSS effector proteins RB-associated Chlamydia pneumoniae proteins
  • a specific composition of the present invention may comprise a combination of Chlamydia pneumoniae antigens, said combination consisting of two, three, four, five or all six Chlamydia pneumoniae antigens of a first antigen group, said first antigen group consisting of: (1) pmp2; (2) pmplO; (3) Enolase; (4) OmpH-like protein; and (5) the products of CPn specific genes CPn0759 and CPn0042.
  • These antigens are referred to herein as the 'first antigen group'.
  • the composition of the invention comprises a combination of Chlamydia pneumoniae antigens, said combination selected from the group consisting of: (1) pmp2 and pmplO; (2) pmp2 and Enolase; (3) pmp2 and OmpH-like protein; (4) pmp2 and CPn0759; (5) pmp2 and CPn0042; (6) pmplO and Enolase; (7) pmplO and OmpH-like protein; (8) pmplO and CPn0759; (9) pmplO and CPn0042; (10) Enolase and OmpH-like protein (11) Enolase and CPn0759; (12) Enolase and CPn0042; (13) OmpH-like protein and CPn0759 (14) OmpH-like protein and CPn0042; (15) CPn0759 and CPn0042; (16) pmp2 and pmplO and Enolase; (17) pmp2 and pmplO and OmpH-like protein; (18) pmp
  • the composition of Chlamydia pneumoniae antigens consists of pmp2, pmplO, Enolase, OmpH-like protein and CPn0042.
  • the composition of Chlamydia pneumoniae antigens consists of pmp2, pmplO, Enolase, OmpH-like protein and CPn0759 and CPn0042.
  • the invention also provides for a slightly larger group of 12 Chlamydia pneumoniae antigens that are particularly suitable for immunisation purposes, particularly when used in combinations.
  • This second antigen group includes the six Chlamydia pneumoniae antigens of the first antigen group).
  • These 12 Chlamydia pneumoniae antigens form a second antigen group of (1) pmp2; (2) pmplO; (3) Enolase; (4) OmpH-like protein; (5) CPn0759; (6) CPn0042; (7) ArtJ; (8) HtrA; (9) AtoS; (10) OmcA; (11) CPn0498; and (12) CPn0525.
  • These antigens are referred to herein as the 'second antigen group'.
  • the invention therefore provides a composition comprising a combination of Chlamydia pneumoniae antigens, said combination selected from the group consisting of two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve Chlamydia pneumoniae antigens of the second antigen group.
  • the combination is selected from the group consisting of two, three, four or five Chlamydia pneumoniae antigens of the second antigen group.
  • the combination consists of six Chlamydia pneumoniae antigens of the second antigen group.
  • Each of the Chlamydia pneumoniae antigens of the first and second antigen group are described in more detail below.
  • pmplO protein is set forth as SEQ ID NO: 1 below (GenBank Accession No.GI: 14195016).
  • Preferred pmplO proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 1; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 1, wherein n is 7 or more (e.g.
  • pmp2 proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 1.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 1.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 1.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Pmp2 Polymorphic Outer Membrane Protein G Family (CPn 0013)
  • pmp2 protein is disclosed as SEQ ID NO : 139 and 140 in WO 02/02606.
  • SEQ ID NO: 139 and 140 are disclosed as SEQ ID NO: 139 and 140 in WO 02/02606.
  • Preferred pmp2 proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 2 SEQ ID NO: 2; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 1, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • These pm ⁇ 2 proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 2.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 1.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 2.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Eno' protein is disclosed as SEQ ID NO s : 93 and 94 in WO 02/02606.
  • SEQ ID NO s 93 and 94 in WO 02/02606.
  • Preferred Eno proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 2 SEQ ID NO: 2; and or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 2, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • Eno proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 3.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 3.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 3.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • OmpH-like' protein is disclosed as SEQ ID NO s : 77 & 78 in WO 02/02606. ⁇ GenBank accession number: gi
  • Preferred OmpH-like proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 4 which is a fragment of at least n consecutive amino acids of SEQ ID NO: 3, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • OmpH-like proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 4.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 4.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more; preferably 19 or more, to remove the signal peptide) from the N-terminus of SEQ ID NO: 4.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide as described above, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQ ID No 4 1 MKKLIiFSTFI.
  • hypothetical protein is set forth as SEQ ID NO: 5 below.
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 5; and or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 5, wherein n is 7 or more (e.g.
  • Hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 5.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 5.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g.
  • fragments omit one or more domains of the protein (e.g. omission of a signal peptide as described above, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • proteins secreted by the autotransporter secretion mechanism possess an overall unifying structure, including an amino-terminal leader peptide (for secretion across the inner membrane), the secreted mature protein (or passenger domain), and a dedicated C-terminal domain, which forms a pore in the outer membrane through which the passenger domain passes to the cell surface. It is likely that requirements for secretion across the outer membrane are contained within a single molecule and secretion is an energy- independent process. Structural properties of the proteins may be confined by the size of the pore considering the biophysical constraints that may be imposed on secretion.
  • the autotransporter, or type V, secretion system is a dedicated protein translocation mechanism which allows the organism to secrete proteins to and beyond the bacterial surface. The secretion mechanism and the ability to develop a new autotransporter protein simply by a single recominbation event have presented bacteria with abundant opportunities to increase the efficiency of secretion of proteins that were developed as periplasmic or exported virulence factors.
  • proteins are exported by the autotransporter secretion mechanism and are translated as a polyproptein possessing domains.
  • the autotransporters possess an overall unifying structure comprising three functional domains: the amino-termianl leader sequence, the secreted mature proterin (passenger domain) and a carboxy-terminal (beta-) domain that forms a beta-barrel pore to allow secretion of the passenger protein.
  • the leader sequence directs secretion via the sec apparatus and is cleaved at the inner membrane by a signal peptidase releasing the remaining portion of the molecule into the periplasm.
  • the ⁇ -domain assumes a biophysically favored state characterized by a ⁇ -barrel shaped structure which inserts itself into the outer membrane to form a pore.
  • the passenger domain is translocated to the bacterial cell surface where it may remain intact or undergo processing.
  • a processed protein may be released into the extracellular milieu or remain associated with the bacterial cell surface.
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 6; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 6, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • Hypothetical proteins include variants (e.g.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 6.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more; preferably 19 or more, to remove the signal peptide) from the N-terminus of SEQ ID NO: 6.
  • Other fragments omit one or more domains of the protein (e.g.
  • Cpn0795 SEQ ID NO: 6
  • a Cpn specific hypotlietical protein is a FACS positive protein which demonstrates significant immunoprotective activity in a hamster spleen model of Chlamydia pneumoniae infection.
  • Chlamydia trachomatis include: gi/4377105 (Cpn0794), gi 4377106 (Cpn0795), gi/4377107 (Cpn0796), gi 4377108 (Cpn0797), gi/4377109 (CPn0798), gi 4377110 (Cpn0799).
  • 'ArtJ' protein is disclosed as SEQ ID NO s : 73 & 74 in WO 02/02606.
  • SEQ ID NO s GenBank accession number: gi
  • Preferred ArtJ proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 7 SEQ ID NO: 7; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 7, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • ArtJ proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 7.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 7.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 7.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • the ArtJ protein may be bound to a small molecule like arginine or another amino acid.
  • HtrA DO Serine Protease (CPn0979)
  • 'HrtA' protein is disclosed as SEQ ID NO s : 111 & 112 in WO 02/02606.
  • SEQ ID NO: 8 GenBank accession number: gi
  • Preferred HrtA proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • HrtA proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 8.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 8.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more; preferably at least 16 to remove the signal peptide) from the N-terminus of SEQ ID NO: 8.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide as described above, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQID N0 8 1 MITKQ RSWIi AVLVGSS IA LP SGQAVGK KESRVSELPQ DVLLKEISGG 51 FSKVATKATP AVVYIESFPK SQAVTHPSPG RRGPYENPFD YFNDEFFNRF 101 FG PSQREKP QSKEAVRGTG FLVSPDGYIV TNNHWEDTG KIHVTLHDGQ 151 KYPATVIGLD PKTD AVIKI KSQNLPY SF GNSDHLKVGD WAIAIGNPFG 201 LQATVTVGVI SAKGRNQLHI ADFEDFIQTD AAINPGNSGG P LNIDGQVI 251 GVNTAIVSGS GGYIGIGFAI PSLMANRIID QLIRDGQVTR GFLGVTLQPI 301 DAEL
  • AtoS two-component regulatory system sensor histidine kinase protein (CPn0584)
  • AtoS proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 9; and/or (b) which is a fragment of at least ..
  • AtoS proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 9.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 9.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and or one or more amino acids (e.g.
  • fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • OmcA' protein is disclosed as SEQ ID NO s : 9 & 10 in WO 02/02606. ⁇ GenBank accession number: gi
  • Preferred OmcA proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 10 SEQ ID NO: 10; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 10, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • OmcA proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 10.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 10.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more; preferably 18 or more to remove the signal peptide) from the N-terminus of SEQ ID NO: 10.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide as described above, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • the protein may be lipidated (e.g.
  • SEQ ID NO: 11 One example of a hypothetical protein is set forth as SEQ ID NO: 11 below.
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 11 ; and or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 11, wherein ra is 7 or more (e.g.
  • Hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 11.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 11.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more; preferably 18 or more to remove the signal peptide) from the N-terminus of SEQ ID NO: 11.
  • fragments omit one or more domains of the protein (e.g. omission of a signal peptide as described above, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • the protein may be lipidated (e.g. by a N-acyl diglyceride), and may thus have a ⁇ -terminal cysteine.
  • 'Cpn0525' protein is disclosed as SEQ ID ⁇ O s : 117 & 118 in WO 02/02606.
  • SEQ ID NO: 12 GenBank accession number: gi
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 12 SEQ ID NO: 12; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 12, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • OmcA proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 12.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 12.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more; preferably 18 or more to remove the signal peptide) from the N-terminus of SEQ ID NO: 12.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide as described above, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Chlamydia pneumoniae antigens may be improved by combination with two or more Chlamydia pneumoniae antigens from either the first antigen group or the second antigen group.
  • Chlamydia pneumoniae antigens include a third antigen group consisting of (1) LcrE, (2) DnaK, (3) Omp85 homolog, (4) Mip-like; (5) OmcB (6) MurG (7) Cpn0186 and (8) fliY. These antigens are referred to herein as the "third antigen group".
  • LcrE' protein is disclosed as SEQ ID NO s : 29 & 30 in WO 02/02606. ⁇ GenBank accession number: gi
  • Preferred LcrE proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • LcrE proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 13.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 13.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 13.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • DnaK' protein is disclosed as SEQ ID NO s : 103 & 104 in WO 02/02606.
  • SEQ ID NO: 14 GenBank accessionnumber: gi
  • Preferred DnaK proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 14 which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 14, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • DnaK proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 14.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 14.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 14.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Omp85 Homolog protein is disclosed as SEQ ID NO s : 147 & 148 in WO 02/02606. ⁇ GenBank accession number: gi
  • Preferred Omp85 proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 15 amino acids of SEQ ID NO: 15; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 15, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • DnaK proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 15.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 15.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 15.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Mip-like protein is disclosed as SEQ ID NO s : 55 & 56 in WO 02/02606. ⁇ GenBank accession number: gi
  • Preferred Mip-like proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 16 amino acids of SEQ ID NO: 16; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 16, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • mip- like proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 16.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 16.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 16.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • OmcB protein is disclosed as SEQ ID NO s : 47 & 48 in WO 02/02606. ⁇ GenBank accession number: gi
  • Preferred OmcB proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • OmcB proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 17.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 17.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 17.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain) .
  • MurG peptidoglycan transferase protein (CPn0904)
  • MurG proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • MurG proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 18.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 18.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 18.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide as described above, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • the MurG may be lipidated e.g. with undecaprenyl.
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 19; and/or (b) which is a fragment of at least « consecutive amino acids of SEQ ID NO: 19, wherein n is 7 or more (e.g.
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 19.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 19.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 19.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • FUY Glutamine Binding Protein (CPn0604)
  • SEQ ID NO s 11 & 12 in WO 02/02606.
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 20 amino acids of SEQ ID NO: 20; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 20, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 20.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 20.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 20.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Chlamydia pneumoniae antigens include a fourth antigen group consisting one or more members of the PMP family. These antigens are referred to herein as the "fourth antigen group”. Each of the Chlamydia pneumoniae antigens of the fourth antigen group is described in more detail below. Fourth Antigen Group
  • Pmpl protein is set forth as SEQ ID NO s : 41 & 42 in WO 02/02606. ⁇ GenBank accession number: gi
  • Pmp 4 (CPn0017)
  • SEQ ID NO: 22 The sequence for pmp4 protein can be found at AE001587.1 GI.4376271.
  • Pmp 6 protein is set forth as SEQ ID NO s 31 & 32 in WO 02/02606. ⁇ GenBank accession number: gi
  • Pmp 7 protein is set forth as SEQ ID NO s 153 & 154 in WO 02/02606. ⁇ GenBank accession number: gi
  • Pmp 8 protein is set forth as SEQ ID NO s 45 & 46 in WO 02/02606. ⁇ GenBank accession number: gi
  • Pmp 9 protein is set forth as SEQ ID NO s 33 & 34 in WO 02/02606. ⁇ GenBank accession number: gi
  • Pmp 11 protein is set forth as SEQ ID NO s 115 & 116 in WO 02/02606. ⁇ GenBank accession number: gi
  • SEQ ID No 27 1 MKTSIPWVT ⁇ .
  • Pmp 12 protein is set forth as SEQ ID NO s 51 & 52 in WO 02/02606. ⁇ GenBank accession number: gi
  • Pmp 13 (CPn0453)
  • CPn0453 One example of a Pmp 13 protein is set forth as SEQ ID NO s 3 & 4 in WO 02/02606. ⁇ GenBank accession number: gi
  • Pmp 14 (CPn0454)
  • CPn0454 One example of a Pmp 14 protein is set forth as SEQ ID NO s 35 & 36 in WO 02/02606. ⁇ GenBank accession number: gi]4376737
  • Pmp 15 protein is set forth as SEQ ID NO s 5 & 6 in WO 02/02606. ⁇ GenBank accession number: gi
  • Pmp 16 protein is set forth as SEQ ID NO s 7 & 8 in WO 02/02606. ⁇ GenBank accession number: gi
  • Pmp 18 protein is set forth as SEQ ID No 33 below ⁇ GenBank accession number: gi
  • SEQ ID No 33 1 MQNNRSLSKS SFFVGALILG KTTILLNATP LSDYFDNQAN QLTTLFPLID TLTNMTPYSH 61 RATLFGVRDD TNQDIVLDHQ NSIES FENF SQDGGALSCK SLAITNTKNQ ILFLNSFAIK 121 RAGAMYVNGN FDLSENHGSI IFSGNLSFPN ASNFADTCTG GAVLCSKNVT ISKNQGTAYF 181 INNKAKSSGG AIQAAIINIK DNTGPCLFFN NAAGGTAGGA LFANACRIEN NSQPIYFLNN 241 QSGLGGAIRV HQECILTKNT GSVIFNNNFA MEADISANHS SGGAIYCISC SIKDNPGIAA 301 FDNNTAARDG GA
  • Pmp 19 (CPn0539)
  • SEQ ID No 34 GenBank accession number: gi
  • Pmp 20 protein is set forth as SEQ ID NO s 119 & 120 in WO 02/02606. ⁇ GenBank accession number: gi
  • Pmp 21 protein is set forth as SEQ ID NO s 83 & 84 in WO 02/02606. ⁇ GenBank accession number: gi
  • PMP proteins include variants (e.g., amino acids, amino acids, amino acids set forth for the pmp proteins above and/or (b) which is a fragment of at least ra consecutive amino acids of one of the polypeptide sequences set forth above wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • PMP proteins include variants (e.g.
  • Preferred fragments of (b) comprise an epitope from one of the polypeptide sequences set forth above.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of one of the polypeptide sequences set forth above.
  • Other fragments omit one or more domains of the protein (e.g.
  • Chlamydia pneumoniae antigens may be improved by combination with two or more Chlamydia pneumoniae antigens from either the first antigen group or the second antigen group or the third antigen group or the fourth antigen group.
  • Such other Chlamydia pneumoniae antigens mclude a fifth antigen group consisting one or more cell surface exposed proteins. These antigens are referred to herein as the "fifth antigen group”.
  • Each of the Chlamydia pneumoniae antigens of the fifth antigen group is described in more detail below.
  • PorB protein is set forth as SEQ ID NO s : 67 & 68 in WO 02/02606. ⁇ GenBank accession number: gi
  • Preferred PorB proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 37 SEQ ID NO: 37; and or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 37, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • PorB proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 37.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 37.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 37.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • 76kDa Protein Homolog (CPn0728)
  • a 76kDa Protein Homolog protein is set forth as SEQ ID NO s : 13 & 14 in WO 02/02606.
  • SEQ ID NO s 13 & 14 in WO 02/02606.
  • Preferred 76kDa proteins homologs for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 38 amino acid sequence corresponding to SEQ ID NO: 38; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 21, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • variants e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 38.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 38.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • OmpA conserved outer membrane protein protein is set forth as SEQ ID NO s : 59 & 60 in WO 02/02606.
  • SEQ ID NO s 59 & 60 in WO 02/02606.
  • Preferred ompA proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 39 SEQ ID NO: 39; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 39, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 39.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 39.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 39.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • PepA protein protein is set forth as SEQ ID NO s : 99 & 100 in WO 02/02606. ⁇ GenBank accession number: gi
  • Preferred PepA proteins for use with the invention comprise an amino acid sequence: (a) having 50%> or more identity (e.g.
  • PepA proteins include variants (e.g.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 40.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 40.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • conserved outer membrane protein protein is set forth as SEQ ID NO: 41 below.
  • GenBank Accession No. GL4376552; AAD18427.1 Preferred conserved outer membrane proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%,, 75%,, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 41; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 41, wherein ra is 7 or more (e.g.
  • conserved outer membrane proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 41.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 41.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 41.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Chlamydia pneumoniae antigens may be improved by combination with two or more Chlamydia pneumoniae antigens from either the first antigen group or the second antigen group or the third antigen group or the fourth antigen group or the fifth antigen group.
  • Chlamydia pneumoniae antigens include a sixth antigen group consisting one or more FACS positive CPn antigens. These antigens are referred to herein as the " sixth antigen group".
  • Each of the Chlamydia pneumoniae antigens of the sixth antigen group is described in more detail below.
  • Omp protein is set forth as SEQ ID NO s : 91 & 92 in WO 02/02606. ⁇ GenBank accession number gi
  • Preferred Omp proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 42 amino acids of SEQ ID NO: 42; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 42, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • Omp proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 42.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 42.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 42.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Omp protein is set forth as SEQ ID NO s : 49 & 50 in WO 02/02606.
  • SEQ ID NO s 49 & 50 in WO 02/02606.
  • Preferred Omp proteins for use with the invention comprise an amino acid sequence: (a) having 50%, or more identity (e.g.
  • SEQ ID NO: 43 SEQ ID NO: 43; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 43, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 43.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 43.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 43.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • oligopeptide binding protein is set forth as SEQ ID NO s : 23 and 24 in WO 02/02606. ⁇ GenBank accession number gi
  • Preferred oligopeptide binding proteins for use with the invention comprise an amino acid sequence: (a) having 50%, or more identity (e.g.
  • SEQ ID NO: 44 amino acids of SEQ ID NO: 44, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 44.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 44.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 44.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • CHLPS protein is set forth as SEQ ID NO: 45 below. GenBank Accession No. GI.4376854; AAD 18702.1.
  • Preferred CHLPS proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 45 98%, 99%, 99.5% or more
  • SEQ ID NO: 45 amino acids which are 98%, 99%, 99.5% or more
  • ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • CHLPS proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 45.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 45.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 45.
  • Other fragments omit one or more domains of the protein (e.g.
  • YscJ protein is set forth as SEQ ID NO s : 109 and 110 in WO 02/02606. ⁇ GenBank accession number gi
  • Preferred YscJ proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 46 SEQ ID NO: 46; and or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 46, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • YscJ proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 46.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 46.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 46.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • a hypothetical protein is set forth as SEQ ID NO s : 101 and 102 in WO 02/02606. ⁇ GenBank accession number gi
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50%) or more identity (e.g.
  • SEQ ID NO: 47 SEQ ID NO: 47; and or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 47, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 47.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 47.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 47.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 48; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 48, wherein n is 7 or more (e.g.
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 48.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 48.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 48.
  • fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQ ID No 48 1 MSNQLQPCIS LGCVSYINSF PLSLQLIKRN DIRCVLAPPA DLLNLLIEGK 51 LDVALTSSLG AISHNLGYVP GFGIAANQRI LSVNLYAAPT
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%o or more) to SEQ ID NO: 49; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 49, wherein ra is 7 or more (e.g.
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 49.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 49.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 49.
  • fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQ ID No 49 1 MKFLLYVPLL VLVSTGCDA KPVSFEPFSG KLSTQRFEPQ HSAEEYFSQG 51 QEFLKKGNFR KALLCFGIIT HHFPRDILRN QAQYLIGVCY FTQDHPDLAD 101 KAFASYLQLP DAEYSEELFQ MKYAIAQRFA QGKRKRICRL EGFPKLMNAD 151 EDALRIYDEI LTAFPSKDLG AQALYSKAAL LIVKNDLTEA TKTLKKLTLQ 201 FPLHILSSEA FVRLSEIYLQ QAKKEPHNLQ YLHFAKLNEE AMKKQHPNHP 251 LNEWSANVG AMREHYARGL YATGRFYEKK KKAEAANI
  • a hypothetical protein is set forth as SEQ ID NO s : 123 and 124 in WO 02/02606. ⁇ GenBank accession number gi
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 50 amino acids of SEQ ID NO: 50, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 50.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 50.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 50.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • hypothetical (CPn0792) One example of a hypothetical protein is set forth as SEQ ID NO s : 61 and 62 in WO 02/02606. ⁇ GenBank accession number gi
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 51 SEQ ID NO: 51; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 51, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 51.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 51.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 51.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • a hypothetical protein is set forth as SEQ ID NO s : 113 and 114 in WO 02/02606. ⁇ GenBank accession number gi
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50%, or more identity (e.g.
  • SEQ ID NO: 52 SEQ ID NO: 52; and or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 52, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 52.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 52.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 52.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQ ID NO: 53 Hypothetical (CPn0126)
  • SEQ ID NO: 53 GenBank Accession No. GL4376390; AAD 18279.1
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 53; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 53, wherein ra is 7 or more (e.g.
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 53.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 53.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 53.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • PROPreferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 54; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 54, wherein n is 7 or more (e.g.
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 54.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 54.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 54.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • PROMAGE Identities are set forth as SEQ ID NO: 55 below.
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50%> or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 55; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 55, wherein ra is 7 or more (e.g.
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 55.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 55.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 55.
  • Cpn0796 may be secreted from C. pneumoniae and is localized in the membrane of Chlamydia in young inclusions whereas an N-terminal part of Cpn0796 is secreted into the host cell cytoplasm at later times.
  • Cpn0796 was proposed to be an autotransporter and it is the first example of secretion into the host cell cytoplasm of a proposed Chlamydia autotrasporter.
  • One preferred protein for use with the invention comprises an ⁇ -terminal peptide of
  • the ⁇ -terminal peptide of Cpn0796 may form a beta-propeller structural conformation.
  • SEQ ID NO: 86 One example of the N-terminal peptide of Cpn0796 is set forth as SEQ ID NO: 86 below.
  • the N-terminal peptide of Cpn0796 for use with the invention may comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 86 amino acids of SEQ ID NO: 86; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 86, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 86.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 86.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 86.
  • PROMEQ ID NO: 56 One example of a hypothetical protein is set forth as SEQ ID NO: 56 below.
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 56; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 56, wherein ra is 7 or more (e.g.
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 56.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 56.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 56.
  • fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQ ID No S ⁇ 1 MSKKIKVLGH LTLCTLFRGV LCAAALSNIG YASTSQESPY QKSIEDWKGY TFTDLELLSK 61 EG SEAHAVS GNGSRIVGAS GAGQGSVTAV I ESHLIKHL GTLGGEASSA EGISKDGEW 121 VGWSDTREGY THAFVFDGRD MKDLGTLGAT YSVARGVSGD GSIIVGVSAT ARGEDYGWQV 181 GVKWEKGKIK QLKLLPQGLW SEANAISEDG TVIVGRGEIS RNHIVAVK N KNAVYSLGTL 241 GGSVASAEAI SANGKVIVGW STTNNGETHA FMHKDETMHD LGTLGGGFSV ATGVSADGRA 301 IVGFSAV
  • oligopeptide binding protein is set forth as SEQ ID NO s : 127 and 128 in WO 02/02606. ⁇ GenBank accession number G 4376467; AAD18349.1 'CPn0196'; SEQ ID NO: 76 below ⁇ .
  • Preferred oligopeptide binding proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 76 amino acids of SEQ ID NO: 76; and or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 76, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO 76.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 76.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 76.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Chlamydia pneumoniae antigens may be improved by combination with two or more Chlamydia pneumoniae antigens from either the first antigen group or the second antigen group or the third antigen group or the fourth antigen group or the fifth antigen group or the sixth antigen group.
  • Such other Chlamydia pneumoniae antigens include a seventh antigen group consisting one or more hypothetical proteins (ie proteins which, for example, have no known cellular location and/or function. These antigens are referred to herein as the "seventh antigen group".
  • Each of the Chlamydia pneumoniae antigens of the seventh antigen group is described in more detail below.
  • polypeptide sequence (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 91%, 98%, 99%, 99.5% or more) to SEQ ID NO: 57; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 57, wherein ra is 7 or more (e.g.
  • SEQ ID No 57 a hypothetical protein that has been modified by SEQ ID No 57.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 57.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 57.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQ ID No 57 a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain.
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 58; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 21, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 58.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 58.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 58.
  • Other fragments omit one or more domains of the protein (e.g.
  • SEQ ID No 58 1 MLQSCKKALL SIWSILAFH PIPGMGVEAK SGFLGKVKG FSKKEIQEEA RILPVKDSLS 61 KRYDYTSSS GFSVEFPGEP DHSGQIVEVP QSEITIRYDT YVTETHPDNT VYWSV EYP 121 EKVDISRPEL NLQEGFSGMM QALPESQVLF MQARQIQGHK ALEF IVCED VYFRGMLISV 181 NHTLYQVFMV YKNKNPQALD KEYEAFSQSF KITKIREPRT IPSSVKKKVS L (59) Hypothetical (CPn0572) One example of a hypothetical protein is set forth as SEQ ID NO: 59 below.
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 59; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 59, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 59.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 59.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 59.
  • Other fragments omit one or more domains of the protein (e.g.
  • Chlamydia pneumoniae antigens may be unproved by combination with two or more Chlamydia pneumoniae antigens from either the first antigen group or the second antigen group of the third antigen group or the fourth antigen group or the fifth or the sixth antigen group or the seventh antigen group.
  • Chlamydia pneumoniae antigens include an eigth antigen group consisting one or more FACS positive CPn antigens. These antigens are referred to herein as the "eight antigen group".
  • Each of the Chlamydia pneumoniae antigens of the eight antigen group is described in more detail below.
  • Low Calcium Response Protein H is set forth as SEQ ID NO: 60 below. Genbank Accession No. GL4377123; AAD18949.1.
  • Preferred low calcium response proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 60; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 60, wherein n is 7 or more (e.g.
  • These low calcium response proteins mclude variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 60.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 60.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 60.
  • fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQ ID No 60 1 mskpsprnan qpqkpsasfn kktrsrlael aaqkkakadd leqvhpvpte eeikkalgni 61 feglsngldl qqilglsdyl leeiytvayt fysqgkynea vglfqllaaa qpqnykymlg 121 lsscyhqlhl yneaafgffl afdaqpdnpi ppyyiadsll klqqpeesrm fldvtmdicg 181 nnpefkilke rcq
  • Yop Proteins Translocation Protein T is set forth as SEQ ID NO: 61 below. Genbank Accession No. G 4377135; AAD18960.1.
  • Preferred Yop proteins for use with the invention comprise an amino acid sequence: (a) having 50%> or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 61; and or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 61, wherein « is 7 or more (e.g.
  • Yop proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 61.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 61.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 61.
  • fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQ ID No 61 1 mgislpelfs nlgsayldyi fq ppayvws vfllllarll pifavapflg aklfpspiki 61 gisls laii fpkvladtqi tnymdnnlfy vllvkemiig ivigfvlafp fyaaqsagsf 121 itnqqgiqgl egatslisie qtsphgilyh yfvtiifwlv gghrivisll lqtlevipih 181 sffpaemmsl sapiwitm ⁇ k mcqlclvmti qlsapaal
  • Yop Proteins Translocation Protein J is set forth as SEQ ID NO: 62 below Genbank Accession No. GL4377140; AAD18965.1.
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 62; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 62, wherein ra is 7 or more (e.g.
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 62.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 62.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 62.
  • fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQ ID No 62 1 mvrrsisfcl fflratllcct scnsrslivh glpgreanei vvllvskgva aqklpqaaaa 61 tagaateqmw diavpsaqit ealailnqag lprmkgtsll dlfakqglvp selqekiryq 121 eglseqmast irkmdgvvda svqisftten ednlpltasv yik rgvldn pnsimvskik 181 rliasavpgl vpenvsvvsd raa
  • OmpA (CPn0695)
  • SEQ ID NO: 63 One example of an OmPA encoded (MOMP) protein is set forth as SEQ ID NO: 63 below Genbank Accession No. GL4376998; AAD 18834.1.
  • Preferred OmpA proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 63; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 63, wherein ra is 7 or more (e.g.
  • OmpA proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 63.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 63.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 63.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Hypothetical Protein is set forth as SEQ ID NO: 64 below Genbank Accession No. G 4376482; AAD 18363.1.
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 64; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 64, wherein ra is 7 or more (e.g.
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 64.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 64.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 64.
  • fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQID No 64 1 mlvelealkr efahlkdqkp tsdqeitsly qcldhlefvl lglgqdkflk atededvlfe 61 sqkaidawna lltkardvlg lgdigaiyqt ieflgaylsk vnrrafcias ei flktair 121 dlnayylldf rwplckieef vdwgndcvei akrklctfek etkelnesll reehamekcs 181 iqdlqrklsd iiielhdvsl f
  • Low Calcium Response Locus Protein H (CPnl 021)
  • SEQ ID NO: 65 One example of a Low Calcium Response Protein H is set forth as SEQ ID NO: 65 below Genbank Accession No. GL4377352; AAD19158.1.
  • Preferred low calcium response proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 65; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 65, wherein ra is 7 or more (e.g.
  • These low calcium response proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 65.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 65
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 65.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • the immunogenicity of other Chlamydia pneumoniae antigens may be improved by combination with two or more Chlamydia pneumoniae antigens from either the first antigen group or the second antigen group or the third antigen group or the fourth antigen group or the fifth antigen group or the sixth antigen group or the seventh antigen group or the eight antigen group.
  • Such other Chlamydia pneumoniae antigens include a ninth antigen group. These antigens are referred to herein as the "ninth antigen group".
  • Each of the Chlamydia pneumoniae antigens of the ninth antigen group is described in more detail below.
  • Low Calcium Response Protein D (CPn0323)
  • SEQ ID NO: 66 One example of a Low Calcium Response Protein D is set forth as SEQ ID NO: 66 below Genbank Accession No. G 4376601; AAD18472.1.
  • Preferred low calcium response proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 66; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 66, wherein ra is 7 or more (e.g.
  • These low calcium response proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 66.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 66.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 66.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • CHLPS 43kDa Protein Homolog-1 (CPn0062)
  • One example of a CHLPS 43kDa Protein Homolog-1 is set forth as SEQ ID NO: 67 below Genbank Accession No. GL4376318; AAD18215.1.
  • Preferred CHLPS proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 67 SEQ ID NO: 67; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 67, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • CHLPS proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 67.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 67.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 67.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • CHLPS 43kDa Protein Homolog-1 is set forth as SEQ ID NO: 68 below Genbank Accession No. G 4376437; AAD18322.1.
  • Preferred CHLPS proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 68; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 68, wherein n is 7 or more (e.g.
  • CHLPS proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 68.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 68.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 68.
  • fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQ ID No 68 1 mknvgsecsq plvmelntqp lrnlcesrlv kitsfviall alvggitlta lagagilsfl 61 pwlvlgivlv vlcalfllfs ykfcpikelg vvyntdsqih qwfqkqrnkd lekatenpel 121 fgenraednn rsarsqvket lrdcdgnvlk kiyernldvl lfmnwvpktr ⁇ ddvdpvs ⁇ ds 181 irtviscykl ikackpefrs lisellrar ⁇ q sgl
  • PmpD family (CPn0963)
  • SEQ ID NO: 69 Genbank Accession No. GL4377287; AAD19099.1.
  • Preferred PmpD proteins for use with the invention comprise an amino acid sequence: (a) having 50%> or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 69; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 69, wherein ra is 7 or more (e.g.
  • PmpD proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 69.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 69.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 69.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • Chlamydia pneumoniae antigens may be improved by combination with two or more Chlamydia pneumoniae antigens from either the first antigen group or the second antigen group or the third antigen group or the fourth antigen group or the fifth antigen group or the sixth antigen group or the seventh antigen group or the eight antigen group or the ninth antigen group.
  • Such other Chlamydia pneumoniae antigens include a tenth antigen group. Each of the Chlamydia pneumoniae antigens of the tenth antigen group is described in more detail below.
  • OmpH-like' protein is disclosed as SEQ ID NO s : 77 & 78 in WO 02/02606.
  • SEQ ID NO s 77 & 78 in WO 02/02606.
  • Preferred OmpH-like proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 4 which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 3, wherein ra is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • OmpH-like proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 4.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 4.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more; preferably 19 or more, to remove the signal peptide) from the N-terminus of SEQ ID NO: 4.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide as described above, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • L7/L12 Ribosomal Protein (CPn0080)
  • CPn0080 One example of an L7/L12 Ribosomal protein is set forth as SEQ ID No 71 below ⁇ GenBank accession number: GL4376338; AAD18233.1 ⁇ . 'CPn0080'; SEQ ID NO: 71 below.
  • Preferred L7/L12 proteins for use with the invention comprise an amino acid sequence: (a) having 50%, or more identity (e.g.
  • L7/L12 ribosomal proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 71.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 71.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more; preferably 19 or more, to remove the signal peptide) from the N-terminus of SEQ ID NO: 71.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide as described above, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • AtoS two-component regulatory system sensor histidine kinase protein (CPn0584)
  • AtoS proteins for use with the invention comprise an amino acid sequence: (a) having 50%> or more identity (e.g.
  • AtoS proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 72.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 72.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 72.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • OmcA' protein is disclosed as SEQ ID NO s : 9 & 10 in WO 02/02606. ⁇ GenBank accession number: gi
  • Preferred OmcA proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g.
  • SEQ ID NO: 73 SEQ ID NO: 73; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 73, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • OmcA proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 73.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 73.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more; preferably 18 or more to remove the signal peptide) from the N-terminus of SEQ ID NO: 73.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide as described above, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • the protein may be lipidated (e.g. by a N-acyl diglyceride), and may thus have a ⁇ -terminal cysteine.
  • hypothetical (CPn0331) One example of a hypothetical protein is set forth as SEQ ID NO: 74 below and SEQ ID No 57 above. Genbank Accession No. GI.4376609; AAD 18480.1.
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 74; and/or (b) which is a fragment of at least n consecutive amino acids of SEQ ID NO: 74, wherein ra is 7 or more (e.g.
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 74.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 74.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 74.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQ ID NO 74 1 mavsggggvq pssdpgk np alqgeqaegp splkesifse tkqassaakq eslvrsgstg 61 myatesqink akyrkaqdrs stspksklkg tfskmrasvq gfmsgfgsra srvsakrasd 121 sgegtsllpt emdvalkkgn rispemqgff ldasg ggss sdisqlslea lkssafsgar 181 slslsssess svasfgsfqk aiepmseekv na tvarlgg emvsslldpn vetsslvrra 241 matgnegmid lsdlgqeevs tamtsprave gkvk
  • PmpD protein is set forth as SEQ ID NO: 75 below Genbank Accession No. GI.4377287; AAD19099.1.
  • Preferred PmpD proteins for use with the invention comprise an amino acid sequence: (a) having 50%) or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%>, 99.5% or more) to SEQ ID NO: 75; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 75, wherein ra is 7 or more (e.g.
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 75.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 75.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 75.
  • fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • SEQ ID No 75 1 mvakktvrsy rssfshsviv ailsagiafe ahslhsseld Igvfnkqfee hsahveeaqt 61 svlkgsdpvn psqkesekvl ytqvpltqgs sgesldlada nflehfqhlf eettvfgidq 121 klvwsdldtr nfsqptqepd tsnavsekis sdtkenrkdl etedpskksg lkevssdlpk 181 spetavaais edleisenis ardp
  • SEQ ID NO: 78 One example of a hypothetical protein is set forth as SEQ ID NO: 78 below.
  • Preferred hypothetical proteins for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,, 99.5% or more) to SEQ ID NO: 78; and/or (b) which is a fragment of at least ra consecutive amino acids of SEQ ID NO: 78, wherein ra is 7 or more (e.g.
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 78.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 78.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 78.
  • Other fragments omit one or rnore domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • These hypothetical proteins include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 79.
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO: 79.
  • Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 79.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • the composition of the invention comprises a combination of CPn antigens selected from the group consisting of: (1) CPn0301 and CPn0080; (2) CPn 0584 and CPn 0558; and (3) CPn 0331 and CPN 0963.
  • the composition comprises a combination of any one or more of groups (1), (2) and (3).
  • the composition of the present invention comprises a combination of CPn antigens selected from the group consisting of (1) CPn0385, CPn0324, CPn 0503, CPn0525 and CPn 0482.
  • CPn0385, CPn0324, CPn 0503, CPn0525 and CPn 0482 Preferably the composition is administered in the presence of alum and/or cPG.
  • the invention thus includes a composition comprising a combination of Chlamydia pneumoniae antigens, said combination selected from the group consisting of two, three, four, five or six Chlamydia pneumoniae antigens of the first antigen group and two, three, four, five, or six Chlamydia pneumoniae antigens of the second antigen group.
  • the combination is selected from the group consisting of three, four, five or six Chlamydia pneumoniae antigens from the first antigen group and three, four, five or six Chlamydia pneumoniae antigens from the second antigen group.
  • the combination consists of six Chlamydia pneumoniae antigens from the first antigen group and three, four, five or six, Chlamydia pneumoniae antigens from the second antigen group.
  • the invention further includes a composition comprising a combination of Chlamydia pneumoniae antigens, said combination selected from the group consisting of two, three, four, five or six, Chlamydia pneumoniae antigens of the second antigen group and two, three, four, five, six, seven or eight Chlamydia pneumoniae antigens of the third antigen group.
  • the combination is selected from the group consisting of three, four, five or six Chlamydia pneumoniae antigens from the second antigen group and three, four, five, six, seven or eight Chlamydia pneumoniae from the third antigen group.
  • the combination consists of six Chlamydia pneumoniae antigens from the second antigen group and three, four, five, six, seven or eight Chlamydia pneumoniae antigens of the third antigen group.
  • the number of Chlamydia pneumoniae antigens which will be in the compositions of the invention.
  • the number of Chlamydia pneumoniae antigens in a composition of the invention is less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, or less than 3.
  • the number of Chlamydia pneumoniae antigens in a composition of the invention is less than 6, less than 5, or less than 4.
  • Chlamydia pneumoniae antigens used in the invention are preferably isolated, i.e., separate and discrete, from the whole organism with which the molecule is found in nature or, when the polynucleotide or polypeptide is not found in nature, is sufficiently free of other biological macromolecules so that the polynucleotide or polypeptide can be used for its intended purpose.
  • the composition comprises one or more Chlamydia pneumoniae antigens from the fourth antigen group which includes porB.
  • the composition comprises one or more Chlamydia pneumoniae antigens from the fourth antigen group which includes porB.
  • Chlamydia pneumoniae antigens from the fourth antigen group includes one or more members of the pmp3 family.
  • references to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of Current Protocols in Molecular Biology (F.M. Ausubel et al, eds., 1987) Supplement 30.
  • a preferred alignment is determined by the Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62.
  • the Smith- Waterman homology search algorithm is disclosed in Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489.
  • the mechanism by which the immune system controls disease includes the induction of neutralising antibodies via humoral immunity and the generation of T-cell responses via cellular immunity.
  • immune response against an antigen refers to the development in a host mammalian subject of a humoral and/or a cellular immune response against that antigen.
  • humoral immune response refers to an immune response mediated by antibody molecules.
  • the antibodies generated by humoral immunity are primarily effective against extracellular infectious agents.
  • SEQ ID Nos 1-86 in the compositions of the invention may be supplemented or substituted with an antibody that binds to the protein.
  • This antibody may be monoclonal or polyclonal.
  • CMI cell mediated immune response
  • T-lymphocytes and/or other white blood cells The CMI immune mechanisms are generally more effective against intracellular infections and disease because the CMI mechanisms prime T cells in a way that, when an antigen appears at a later date, memory T cells are activated to result in a CMI response that destroys target cells that have the corresponding antigen or a portion thereof on their cell surfaces, and thereby the infecting pathogen.
  • the CMI response is focused on the destruction of the source of infection mediated by either effector cells that destroy infected cells of the host by direct cell-to-cell contact and/or by the release of molecules, such as cytokines, that possess anti-viral activity.
  • an immunogenic composition comprising a combination of at least one antigen that elicits a Chlamydia pneumoniae specific Thl immune response (such as a cell mediated or cellular immune response) and at least one antigen that elicits a Chlamydia pneumoniae specific Th2 response (such as a humoral or antibody response).
  • the immunogenic composition may further comprise a Thl adjuvant and a Th2 adjuvant.
  • the invention provides a composition comprising a combination of Chlamydia pneumoniae antigens that elicit at least a Chlamydia pneumoniae specific Thl immune response.
  • the combination of Chlamydia pneumoniae antigens may include at least one antigen associated with reticulate bodies (RBs) of Chlamydia pneumoniae, including but not limited to antigens expressed, exposed on or translocated into, through or across on the inclusion membrane, antigens expressed, secreted, released or translocated into the cytosol of host cells, or antigens processed or degraded in the cytosol of host cells and/or expressed, exposed or presented on the surface of the host cell.
  • RBs reticulate bodies
  • compositions of the invention will preferably elicit both a cell mediated immune response as well as a humoral immune response in order to effectively address a Chlamydia intracellular infection.
  • This immune response will preferably induce long lasting (eg neutralising) antibodies and a cell mediated immunity that can quickly respond upon exposure to Chlamydia.
  • the invention also comprises an immunogenic composition comprising one or more immunoregulatory agents.
  • one or more of the immunoregulatory agents include an adjuvant.
  • the adjuvant may be selected from one or more of the group consisting of a Thl adjuvant and Th2 adjuvant, further discussed below.
  • the adjuvant may be selected from the group consisting of a mineral salt, such as an aluminium salt and an oligonucleotide containing a CpG motif.
  • the immunogenic composition includes both an aluminium salt and an oligonucleotide containing a CpG motif. Use of the combination of a mineral salt, such as an aluminium salt, and an oligonucleotide containing a CpG motif provide for an enhanced immune response.
  • the invention therefore includes an oligonucleotide containing a CpG motif, a mineral salt such as an aluminium salt, and an antigen, such as a Chlamydia pneumoniae antigen.
  • T cells At least two special types of T cells are required to initiate and/or to enhance CMI and and humoral responses.
  • the antigenic receptors on a particular subset of T cells which express a CD4 co-receptor can be T helper (Th) cells or CD4 T cells (herein after called T helper cells) and they recognise antigenic peptides bound to MHC class II molecules.
  • T helper cells T helper cells
  • CD8+ T cells CD8+ T cells
  • Helper T cells or CD4+ cells can be further divided into two functionally distinct subsets: Thl and Th2 which differ in their cytokine and effector function.
  • Thl and Th2 responses have been shown to be regulated not only in a positive but also in a negative way such that Thl cellular responses are augmented by Thl cytokines such as IL-2, IL-12 and IFN-gamma and decreased by Th2 cytokines such as E -4 and IL- 10.
  • Th2 cytokines such as IL-4 and IL-10
  • Thl cytokines such as IFN-gamma and another cytokine IL-12 that enhances IFN-gamma and is produced by monocytes.
  • Thl cytokines such as IFN-gamma, IL-2 and IL-12 can be regarded as immune co-factors that induce an effective inflammatory response.
  • the classic Th2 cytokines such as IL-4 and IL-10 can be regarded as cytokines that will suppress a severe inflammatory response.
  • CD8+ T cells may function in more than one way.
  • the best known function of CD8+ T cells is the killing or lysis of target cells bearing peptide antigen in the context of an MHC class I molecule. Hence the reason why these cells are often termed cytotoxic T lymphocytes (CTL).
  • CTL cytotoxic T lymphocytes
  • CD8+ T cells may secrete interferon gamma (IFN-gamma).
  • IFN-gamma interferon gamma
  • assays of lytic activity and of IFN-gamma release are both of value in measuring CD8+ T cell immune response (eg in an ELISPOT assay as set forth below).
  • ELISPOT assay as set forth below.
  • the present invention concerns methods, processes and compositions capable of enhancing and or modulating the CMI response in a host subject against a target antigen.
  • enhancing encompasses improvements in all aspects of the CMI response which include but are not limited to a stimulation and/or augmentation and/or potentiation and/or up-regulation of the magnitude and/or duration, and/or quality of the CMI response to an antigen or a nucleotide sequence encoding an antigen of interest.
  • the CMI response may be enhanced by either (i) enhancing the activation and/or production and/or proliferation of CD 8+ T cells that recognise a target antigen and/or (ii) shifting the CMI response from a Th2 to a Thl type response.
  • This enhancement of the Thl associated responses is of particular value in responding to intracellular infections because, as explained above, the CMI response is enhanced by activated Thl (such as, for example, IFN- gamma inducing) cells.
  • Such an enhanced immune response may be generally characterized by increased titers of interferon-producing CD4 + and/or CD8 + T lymphocytes, increased antigen- specific CD8+ T cell activity, and a T helper 1-like immune response (Thl) against the antigen of interest (characterized by increased antigen-specific antibody titers of the subclasses typically associated with cellular immunity (such as, for example IgG2a), usually with a concomitant reduction of antibody titers of the subclasses typically associated with humoral immunity (such as, for example IgGl)) instead of a T helper 2-like immune response (Th2).
  • Thl T helper 1-like immune response against the antigen of interest
  • the enhancement of a CMI response may be determined by a number of well-known assays, such as by lymphoproliferation (lymphocyte activation) assays, CD8+ T cell assays, or by assaying for T-lymphocytes specific for the epitope in a sensitized subject (see, for example, Erickson et al. (1993) J. Immunol. 151: 4189-4199; and Doe et al. (1994) Eur. J. Immunol. 24: 2369-2376) or CD8+ T cell ELISPOT assays for measuring Interferon gamma production (Miyahara et al PNAS(USA) (1998) 95: 3954-3959).
  • the term "enhancing a T -cell response” encompasses improvements in all aspects of the T-cell response which include but are not limited to a stimulation and or augmentation and or potentiation and/or up-regulation of the magnitude and/or duration, and/or quality of the T-cell response to an antigen (which may be repeatedly administered) or a nucleotide sequence encoding an antigen.
  • the antigen may be a Chlamydia antigen, preferably a Chlamydia pneumoniae antigen.
  • the T-cell response may be enhanced by either enhancing the activation and/or production and/or distribution and/or proliferation of the induced T-cells and/or longevity of the T-cell response to T-cell inducing/modulating antigen or nucleotide sequence encoding an antigen.
  • the enhancement of the T-cell response in a host subject may be associated with the enhancement and/or modulation of the Thl immune response in the host subject.
  • the enhancement of the T-cell response may be determined by a number of well- known assays, such as by lymphoproliferation (lymphocyte activation) assays, CD8+ T-cell cytotoxic cell assays, or by assaying for T-lymphocytes specific for the epitope in a sensitized subject (see, for example, Erickson et al. (1993) J. Immunol. 151: 4189-4199; and Doe et al. (1994) Eur. J. Immunol. 24: 2369-2376) or CD8+ T-cell ELISPOT assays for measuring Interferon gamma production (Miyahara et al PNAS(USA) (1998) 95: 3954-3959).
  • lymphoproliferation lymphoproliferation
  • CD8+ T-cell cytotoxic cell assays or by assaying for T-lymphocytes specific for the epitope in a sensitized subject (see, for example, Erickson et al
  • Activated Thl cells enhance cellular immunity (including an increase in antigen- specific CTL production) and are therefore of particular value in responding to intracellular infections.
  • Activated Thl cells may secrete one or more of IL-2, IFN- gamma, and TNF-beta.
  • a Thl immune response may result in local inflammatory reactions by activating macrophages, NK (natural killer) cells, and CD8 cytotoxic T cells (CTLs).
  • a Thl immune response may also act to expand the immune response by stimulating growth of B and T cells with IL-12.
  • Thl stimulated B cells may secrete IgG2a.
  • Activated Th2 cells enhance antibody production and are therefore of value in responding to extracellular infections.
  • Activated Th2 cells may secrete one or more of IL-4, IL-5, IL-6, and IL-10.
  • a Th2 immune response may result in the production of IgGl, IgE, IgA and memory B cells for future protection.
  • each disease causing agent or disease state has associated with it an antigen or immunodominant epitope on the antigen which is crucial in immune recognition and ultimate elimination or control of a disease causing agent or disease state in a host.
  • the host immune system In order to mount a humoral and/or cellular immune response against a particular disease, the host immune system must come in contact with an antigen or an immunodominant epitope on an antigen associated with that disease state.
  • the term "antigen” refers to any agent, generally a macromolecule, which can elicit an immunological response in an individual.
  • the term "antigen" is used interchangeably with the term "immunogen”.
  • the immunological response may be of B- and/or T-lymphocytic cells.
  • antigenic macromolecule may be used to refer to an individual macromolecule or to a homogeneous or heterogeneous population of antigenic macromolecules.
  • antigenic macromolecule is used to refer to a protein molecule or portion thereof which contains one or more antigenic determinants or epitopes.
  • antigen means an immunogenic peptide or protein of interest comprising one or more epitopes capable of inducing a CMI response to an infectious Chlamydia pathogen.
  • the antigen can mclude but is not limited to an auto-antigen, a self-antigen, a cross-reacting antigen, an alloantigen, a tolerogen, an allergen, a hapten, an immunogen or parts thereof as well as any combinations thereof.
  • epitope generally refers to the site on an antigen which is recognised by a T-cell receptor and/or an antibody. Preferably it is a short peptide derived from or as part of a protein antigen. However the term is also intended to include peptides with glycopeptides and carbohydrate epitopes. Several different epitopes may be carried by a single antigenic molecule. The term “epitope” also includes modified sequences of amino acids or carbohydrates which stimulate responses which recognise the whole organism. It is advantageous if the selected epitope is an epitope of an infectious agent, such as a Chlamydia bacterium, which causes the infectious disease.
  • an infectious agent such as a Chlamydia bacterium
  • SEQ ID Nos 1-86 in the compositions of the invention may be supplemented or substituted with molecules comprising fragments of SEQ ID Nos 1-86.
  • Such fragments may comprise at least n consecutive monomers from the molecules and. depending on the particular sequence, n is either (i) 7 or more for protein molecules (eg. 8 18, 20 or more), preferably such that the fragment comprises an epitope from the sequence, or (ii) 10 or more for nucleic acid molecules (eg 15, 18, 20, 25, 30, 35, 40 or more).
  • the epitope can be generated from knowledge the amino acid and corresponding
  • DNA sequences of the peptide or polypeptide as well as from the nature of particular amino acids (e.g., size, charge, etc.) and the codon dictionary, without undue experimentation. See, e.g., Ivan Roitt, Essential Immunoloqy, 1988; Kendrew, supra; Janis Kuby, Immunology, 1992 e.g., pp. 79-81.
  • Some guidelines in determining whether a protein will stimulate a response include: Peptide length — preferably the peptide is about 8 or 9 amino acids long to fit into the MHC class I complex and about 13-25 amino acids long to fit into a class II MHC complex. This length is a minimum for the peptide to bind to the MHC complex.
  • the peptides may be longer than these lengths because cells may cut peptides.
  • the peptide may contain an appropriate anchor motif which will enable it to bind to the various class I or class II molecules with high enough specificity to generate an immune response (See Bocchia, M. et al, Specific Binding of Leukemia Oncogene Fusion Protein Pentides to HLA Class I Molecules, Blood 85:2680-2684; Englehard, NH, Structure of peptides associated with class I and class II MHC molecules Ann. Rev. Immunol. 12:181 (1994)). This can be done, without undue experimentation, by comparing the sequence of the protein of interest with published structures of peptides associated with the MHC molecules. Thus, the skilled artisan can ascertain an epitope of interest by comparing the protein sequence with sequences listed in the protein data base.
  • one or more antigens of the present invention contain one or more T cell epitopes.
  • T cell epitope refers generally to those features of a peptide structure which are capable of inducing a T cell response.
  • T cell epitopes comprise linear peptide determinants that assume extended conformations within the peptide-binding cleft of MHC molecules (Unanue et al. (1987) Science 236: 551-557).
  • a T cell epitope is generally a peptide having at least about 3-5 amino acid residues, and preferably at least 5-10 or more amino acid residues.
  • T cell epitope encompasses any MHC Class I-or MHC Class II restricted peptide.
  • the ability of a particular T cell epitope to stimulate/enhance a CMI response may be dete ⁇ nined by a number of well-known assays, such as by lymphoproliferation (lymphocyte activation) assays, CD8+ T-cell cytotoxic cell assays, or by assaying for T-lymphocytes specific for the epitope in a sensitized subject.
  • lymphoproliferation lymphoproliferation
  • CD8+ T-cell cytotoxic cell assays or by assaying for T-lymphocytes specific for the epitope in a sensitized subject.
  • the antigens of the present invention comprisse CD8+ T-cell inducing epitopes.
  • a CD8+ T-cell -inducing epitope is an epitope capable of stimulating the formation, or increasing the activity, of specific CD 8+ T-cells following its administration to a host subject.
  • the CD8+ T-cell epitopes may be provided in a variety of different forms such as a recombinant string of one or two or more epitopes. CD8+ T-cell epitopes have been identified and can be found in the literature, for many different diseases. It is possible to design epitope strings to generate CD8+ T- cell response against any chosen antigen that contains such CD8+ T-cell epitopes.
  • CD8+ T-cell inducing epitopes may be provided in a string of multiple epitopes which are linked together without intervening sequences so that unnecessary nucleic acid material is avoided.
  • the antigens of the present invention comprise helper T lymphocyte epitopes.
  • helper T lymphocyte epitopes Various methods are available to identify T helper cell epitopes suitable for use in accordance herewith. For example, the amphipathicity of a peptide sequence is known to effect its ability to function as a T helper cell inducer. A full discussion of T helper cell-inducing epitopes is given in U.S. Patent 5,128,319, inco ⁇ orated herein by reference.
  • the antigens of the present invention comprise a mixture of CD8+ T-cell epitopes and B cell epitopes.
  • B cell epitope generally refers to the site on an antigen to which a specific antibody molecule binds. The identification of epitopes which are able to elicit an antibody response is readily accomplished using techniques well known in the art. See, e. g., Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3998-4002 (general method of rapidly synthesizing peptides to determine the location of immunogenic epitopes in a given antigen); U. S. Patent No.
  • the antigen or antigen combination comprises a mixture of a CD8+ T-cell -inducing epitopes and a T helper cell-inducing epitopes.
  • T and B cell inducing epitopes are frequently distinct from each other and can comprise different peptide sequences. Therefore certain regions of a protein's peptide chain can possess either T cell or B cell epitopes. Therefore, in addition to the CD8+ T-cell epitopes, it may be preferable to include one or more epitopes recognised by T helper cells, to augment the immune response generated by the CD8+ T-cell epitopes.
  • T helper cell inducing agents The mechanism of enhancing a CD8+ T-cell induced response in vivo by T helper cell inducing agents is not completely clear. However, without being bound by theory, it is likely that the enhancing agent, by virtue of its ability to induce T helper cells, will result in increased levels of necessary cytokines that assist in the clonal expansion and dissemination of specific CD8+ T-cells. Regardless of the underlying mechanism, it is envisioned that the use of mixtures of helper T cell and CD8+ T-cell -inducing antigen combinations of the present invention will assist in the enhancement of the CMI response.
  • T helper cell epitopes are ones which are active in individuals of different HLA types, for example T helper epitopes from tetanus (against which most individuals will already be primed). It may also be useful to include B cell epitopes for stimulating B cell responses and antibody production. Synthetic nucleotide sequences may also be constructed to produce two types of immune responses: T cell only and T cell combined with a B cell response.
  • an individual is immunized with an antigen or combination of antigens or nucleotide sequence or combinations of nucleotide sequences encoding multiple epitopes of a target antigen
  • the majority of responding T lymphocytes will be specific for one or more linear epitopes from that target antigen and/or a majority of the responding B lymphocytes will be specific for one or more linear or conformational epitopes for the antigen or combination of antigens.
  • epitopes are referred to as "immunodominant epitopes”.
  • a single epitope may be the most dominant in terms of commanding a specific T or B cell response.
  • compositions of the present invention may be administered in conjunction with other immunoregulatory agents.
  • the compositions of the present i .nnvvpe/nnttiinonn m m-avy h bie. a aHdmmi.nni.ssttfe»rrfevd. w wiithh a ann . ai_d.j_u_ _vv.ainntt.
  • an adjuvant may be useful in further enhancing or modulating the CMI response.
  • An adjuvant may enhance the CMI response by enhancing the immunogenicity of a co-administered antigen in an immunized subject, as well inducing a Thl-like immune response against the co- administered antigen which is beneficial in a vaccine product.
  • An immune response and particularly a CMI response may be refined, by the addition of adjuvants to combinations of antigens or nucleotide sequences encoding combinations of antigens which lead to particularly effective compositions for eliciting a long lived and sustained enhanced CMI response.
  • adjuvant refers to any material or composition capable of specifically or non-specifically altering, enhancing, directing, redirecting, potentiating or initiating an antigen-specific immune response.
  • adjuvant includes but is not limited to a bacterial ADP-ribosylating exotoxin, a biologically active factor, immunomodulatory molecule, biological response modifier or immunostimulatory molecule such as a cytokine, an interleukin, a chemokine or a ligand or an epitope (such as a helper T cell epitope) and optimally combinations thereof which, when administered with an antigen, antigen composition or nucleotide sequence encoding such antigens enhances or potentiates or modulates the CMI response relative to the CMI response generated upon administration of the antigen or combination of antigens alone.
  • the adjuvant may be any adjuvant known in the art which is appropriate for human or animal use.
  • Immunomodulatory molecules such as cytokines (TNF-alpha, IL-6, GM-CSF, and IL- 2), and co-stimulatory and accessory molecules (B7-1, B7-2) may be used as adjuvants in a variety of combinations.
  • GM-CSF is not administered to subject before, in or after the administration regimen.
  • Simultaneous production of an immunomodulatory molecule and an antigen of interest at the site of expression of the antigen of interest may enhance the generation of specific effectors which may help to enhance the CMI response.
  • the degree of enhancement of the CMI response may be dependent upon the specific immunostimulatory molecules and/or adjuvants used because different immunostimulatory molecules may elicit different mechanisms for enhancing and or modulating the CMI response.
  • the different effector mechanisms/immunomodulatory molecules include but are not limited to augmentation of help signal (IL-2), recruitment of professional APC (GM-CSF), increase in T cell frequency (IL-2), effect on antigen processing pathway and MHC expression (IFN-gamma and TNF-alpha) and diversion of immune response away from the Thl response and towards a Th2 response (LTB) (see WO 97/02045).
  • IL-2 help signal
  • GM-CSF professional APC
  • IL-2 increase in T cell frequency
  • IFN-gamma and TNF-alpha effect on antigen processing pathway and MHC expression
  • LTB Th2 response
  • Unmethylated CpG containing oligonucleotides are also preferential inducers of a Thl response and are suitable for use in the present invention.
  • an adjuvant is advantageous because the adjuvant may help to enhance the CMI response to the expressed antigen by diverting the Th2 response to a Thl response and/or specific effector associated mechanisms to an expressed epitope with the consequent generation and maintenance of an enhanced CMI response (see, for example, the teachings in WO 97/02045).
  • an adjuvant with an antigen or nucleotide sequence encoding the antigen is also advantageous because it may result in a lower dose or fewer doses of the antigen/antigenic combination being necessary to achieve the desired CMI response in the subject to which the antigen or nucleotide sequence encoding the antigen is administered, or it may result in a qualitatively and/or quantitatively different immune response in the subject.
  • the effectiveness of an adjuvant can be determined by administering the adjuvant with the antigen in parallel with the antigen alone to animals and comparing antibody and/or cellular-mediated immunity in the two groups using standard assays such as radioimmunoassay, ELISAs, CD8+ T-cell assays, and the like, all well known in the art.
  • the adjuvant is a separate moiety from the antigen, although a single molecule (such for example, CTB) can have both adjuvant and antigen properties.
  • the term “genetic adjuvant” refers to an adjuvant encoded by a nucleotide sequence and which, when administered with the antigen enhances the CMI response relative to the CMI response generated upon administration of the antigen alone.
  • Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention.
  • the protein is derived from E. coli (i.e., E. coli heat labile enterotoxin "LT), cholera ("CT”), or pertussis ("PT").
  • the genetic adjuvant is a bacterial ADP-ribosylating exotoxin.
  • ADP-ribosylating bacterial toxins are a family of related bacterial exotoxins and include diphtheria toxin (DT), pertussis toxin (PT), cholera toxin (CT), the E. coli heat-labile toxins (LT1 and LT2), Pseudomonas endotoxin A, Pseudomonas exotoxin S, B. cereus exoenzyme, B. sphaericus toxin, C. botulinum C2 and C3 toxins, C. limosum exoenzyme, as well as toxins from C. perfringens, C. spiriforma and C.
  • DT diphtheria toxin
  • PT pertussis toxin
  • CT cholera toxin
  • LT1 and LT2 the E. coli heat-labile toxins
  • Pseudomonas endotoxin A Pseudomonas exo
  • ADP-ribosylating bacterial toxin mutants such as CRM] 9 , a non-toxic diphtheria toxin mutant (see, e.g., Bixler et al. (1989) Adv. Exp. Med. Biol. 251:175; and Constantino et al. (1992) Vaccine).
  • Most ADP- ribosylating bacterial toxins are organized as an A:B multimer, wherein the A subunit contains the ADP-ribosyltransferase activity, and the B subunit acts as the binding moiety.
  • Preferred ADP-ribosylating bacterial toxins for use in the compositions of the present invention include cholera toxin and the E. coli heat-labile toxins.
  • Cholera toxin (CT) and the related E. coli heat labile enterotoxins (LT) are secretion products of their respective enterotoxic bacterial strains that are potent immunogens and exhibit strong toxicity when administered systemically, orally, or mucosally. Both CT and LT are known to provide adjuvant effects for antigen when administered via the intramuscular or oral routes. These adjuvant effects have been observed at doses below that required for toxicity.
  • the two toxins are extremely similar molecules, and are at least about 70-80%, homologous at the amino acid level.
  • the genetic adjuvant is cholera toxin (CT), enterotoxigenic E. Coli heat- labile toxin (LT), or a derivative, subunit, or fragment of CT or LT which retains adjuvanticity.
  • CT cholera toxin
  • LT enterotoxigenic E. Coli heat- labile toxin
  • the genetic adjuvant is LT.
  • the genetic adjuvant may be CTB or LTB.
  • the entertoxin is a non-toxic enterotoxin.
  • the use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in WO 95/17211 and as parenteral adjuvants in WO 98/42375.
  • the toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits.
  • the A subunit contains a detoxifying mutation; preferably the B subunit is not mutated.
  • the adjuvant is a detoxified LT mutant such as LT-K63, LT- R72, and LTR192G.
  • ADP-ribosylating toxins and detoxified derivaties thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in the following references each of which is specifically inco ⁇ orated by reference herein in their entirety (Beignon, et al. Infection and Immunity (2002) 70(6):3012 - 3019; Pizza, et al., Vaccine (2001) 19:2534 - 2541; Pizza, et al, Int. J. Med. Microbiol (2000) 290(4- 5):455-461; Scharton-Kersten et al. Infection and Immunity (2000) 68(9):5306 - 5313; Ryan et al.
  • At least one of the entertoxin subunit coding regions may be genetically modified to detoxify the subunit peptide encoded thereby, for example wherein the truncated A subunit coding region has been genetically modified to disrupt or inactivate ADP-ribosyl transferase activity in the subunit peptide expression product (see, for example, WO 03/004055).
  • this genetic adjuvant is particularly desirable where an even more enhanced CMI response is desired.
  • Other desirable genetic adjuvants include but are not limited to nucleotide sequences encoding IL-10, IL-12, IL-13, the interferons (IFNs) (for example, IFN-alpha, IFN-ss, and IFN-gamma), and preferred combinations thereof.
  • IFNs interferons
  • Still other such biologically active factors that enhance the CMI response may be readily selected by one of skill in the art, and a suitable plasmid vector containing same constructed by known techniques.
  • Preferred further adjuvants include, but are not limited to, one or more of the following set forth below: Mineral Containing Compositions
  • Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminium salts and calcium salts.
  • the invention includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphoshpates, orthophosphates), sulphates, etc. ⁇ e.g. see chapters 8 & 9 of ref. Bush and Everett (2001) Int J Syst Evol Microbiol 51: 203-220), or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amo ⁇ hous, etc.), and with adso ⁇ tion being preferred.
  • the mineral containing compositions may also be formulated as a particle of metal salt. See WO 00/23105.
  • Aluminum salts may be included in immunogenic compositions and/or vaccines of the invention such that the dose of Al 3+ is between 0.2 and 1.0 mg per dose.
  • the adjuvant is alum, preferably an aluminium salt such as aluminium hydroxide (AIOH) or aluminium phospate or aluminium sulfate. Still more preferably the adjuvant is aluminium hydroxide (AIOH).
  • a mineral salt such as an aluminium salt, is combined with and another adjuvant, such as an oligonucleotide containing a CpG motif or an ADP ribosylating toxin. Still more preferably, the mineral salt is combined with an oligonucleotide containing a CpG motif. Oil-Emulsions
  • Oil-emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 (5%, Squalene, 0.5%> Tween 80, and 0.5%> Span 85, formulated into submicron particles using a microfluidizer). See WO90/14837. See also, Frey et al., "Comparison of the safety, tolerability, and immunogenicity of a MF59-adjuvanted influenza vaccine and a non-adjuvanted influenza vaccine in non-elderly adults", Vaccine (2003) 21:4234-4237. MF59 is used as the adjuvant in the FLUADTM influenza virus bivalent subunit vaccine.
  • Particularly preferred adjuvants for use in the compositions are submicron oil-inwater emulsions.
  • Preferred submicron oil-in-water emulsions for use herein are squalene/water emulsions optionally containing varying amounts of MTP-PE, such as a submicron oil-in-water emulsion containing 4-5%> w/v squalene, 0.25-1.0%) w/v Tween 80 TM (polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0%) Span 85TM (sorbitan trioleate), and, optionally, N-acetylmuramyl-L-alanyl-D-isogluatminyl-L- alanine-2-( -2 , -dipalmitoyl-5 , ra-glycero-3-huydroxyphosphophoryloxy)-ethylamine
  • MTP-PE submicron oil-in-water emulsion
  • MF59 submicron oil-in-water emulsion
  • International Publication No. WO90/14837 US Patent Nos. 6,299,884 and 6,451,325, inco ⁇ orated herein by reference in their entireties; and Ott et al., "MF59 — Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines” in Vaccine Design: The Subunit and Adjuvant Approach (Powell, M.F. and Newman, M.J. eds.) Plenum Press, New York, 1995, pp. 277-296).
  • MF59 contains 4-5% w/v Squalene (e.g.
  • MTP-PE may be present in an amount of about 0-500 ⁇ g/dose, more preferably 0-250 ⁇ g/dose and most preferably, 0-100 ⁇ g/dose.
  • MF59-0 refers to the above submicron oil-in-water emulsion lacking MTP- PE, while the term MF59-MTP denotes a formulation that contains MTP-PE.
  • MF59-100 contains 100 ⁇ g MTP-PE per dose, and so on.
  • MF69 another submicron oil-in-water emulsion for use herein, contains 4.3% w/v squalene, 0.25% w/v Tween 80TM, and 0.75% w/v Span 85TM and optionally MTP-PE.
  • MF75 also known as SAF, containing 10% squalene, 0.4% Tween 80TM, 5% pluronic-blocked polymer L121, and thr-MDP, also microfluidized into a submicron emulsion.
  • MF75-MTP denotes an MF75 formulation that includes MTP, such as from 100-400 ⁇ g MTP-PE per dose.
  • Submicron oil-in-water emulsions, methods of making the same and immunostimulating agents, such as muramyl peptides, for use in the compositions, are described in detail in International Publication No. WO90/14837 and US Patent Nos. 6,299,884 and 6,45 1,325, inco ⁇ orated herein by reference in their entireties.
  • Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IF A) may also be used as adjuvants in the invention.
  • Saponin formulations may also be used as adjuvants in the invention.
  • Saponins are a heterologous group of sterol glycosides and trite ⁇ enoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root).
  • Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs. Saponin compositions have been purified using High Performance Thin Layer Chromatography (HP-LC) and Reversed Phase High Performance Liquid Chromatography (RP-HPLC). Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method of production of QS21 is disclosed in U.S. Patent No. 5,057,540. Saponin formulations may also comprise a sterol, such as cholesterol (see WO 96/33739).
  • a sterol such as cholesterol
  • ISCOMs Combinations of saponins and cholesterols can be used to form unique particles called ___nmunostim.ula.ing Complexs (ISCOMs).
  • ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs.
  • the ISCOM includes one or more of Quil A, QHA and QHC.
  • ISCOMS may be devoid of additional detergent. See WO 00/07621.
  • VLPs Virosomes and Virus Like Particles
  • Virosomes and Virus Like Particles can also be used as adjuvants in the invention.
  • These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non- pathogenic, non-replicating and generally do not contain any of the native viral genome.
  • the viral proteins may be recombinantly produced or isolated from whole viruses.
  • viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and- Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA- phages, Q ⁇ -phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pi).
  • influenza virus such as HA or NA
  • Hepatitis B virus such as core or capsid proteins
  • Hepatitis E virus measles virus
  • Sindbis virus Rotavirus
  • Foot-and- Mouth Disease virus Retrovirus
  • Norwalk virus Norwalk virus
  • human Papilloma virus HIV
  • RNA- phages Q ⁇ -phage (such as coat proteins)
  • GA-phage such as fr-phage,
  • VLPs are discussed further in WO 03/024480, WO 03/024481; Niikura et al Virology (2002) 293:273 - 280; Lenz et al Journal of Immunology (2001) 5246 - 5355; Pinto, et al Journal of Infectious Diseases (2003) 188:327 - 338; and Gerber et al Journal of Virology (2001) 75(10):4752 - 47601; Virosomes are discussed further in, for example, Gluck et al Vaccine (2002) 20:B10 -B16.
  • Bacterial or Microbiol Derivatives are discussed further in, for example, Gluck et al Vaccine (2002) 20:B10 -B16.
  • Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as: Non-toxic derivatives of enterobacterial lipopolysaccharide (LPS)
  • Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL).
  • 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.
  • a preferred "small particle" form of 3 De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454.
  • Such "small particles" of 3dMPL are small enough to be sterile filtered tlirough a 0.22 micron membrane (see EP 0 689 454).
  • Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529. See Johnson et al. (1999) BioorgMed Chem Lett 9:2273-2278.
  • Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM- 174.
  • OM-174 is described for example in Meraldi et al. Vaccine (2003) 21:2485 - 2491; Pajak, et al Vaccine (2003) 21:836 - 842.
  • Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a sequence containing an unmethylated cytosine followed by guanosine and linked by a phosphate bond). Bacterial double stranded RNA or oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.
  • the CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded.
  • the guanosine may be replaced with an analog such as 2'-deoxy-7-deazaguanosine. See Kandimalla, et al Nucleic Acids Research (2003) 31(9): 2393 - 2400; WO 02/26757 and WO 99/62923 for examples of possible analog substitutions.
  • the CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT. See Kalman et al (1999) (Nature Genetics 21: 385-389).
  • the CpG sequence may be specific for inducing a Thl immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN.
  • CpG-A and CpG-B ODNs are discussed in Blackwell, et al J. Immunol. (2003) 170(8):4061 - 4068; Krieg BBRC (2003) 306:948 - 953; and WO 01/95935.
  • the CpG is a CpG-A ODN.
  • the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition.
  • two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers". See, for example, Kandimalla, et al (2003) 31(part 3):664 - 658; Bhagat et al BBRC (2003) 300:853 - 861 and WO 03/035836.
  • the adjuvant is CpG. Even more preferably, the adjuvant is Alum and an oligonucleotide containg a CpG motif or AIOH and an oligonucleotide containing a CpG motif.
  • Human immunomodulators suitable for use as adjuvants in the invention include cytokinfes, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon- ⁇ ), macrophage colony stimulating factor, and tumor necrosis factor.
  • cytokinfes such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon- ⁇ ), macrophage colony stimulating factor, and tumor necrosis factor.
  • ADP-ribosylating toxins and detoxified derivatives thereof.
  • Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention.
  • the protein is derived from E. coli (i.e., E. coli heat labile enterotoxin "LT), cholera ("CT"), or pertussis ("PT").
  • LT E. coli heat labile enterotoxin
  • CT cholera
  • PT pertussis
  • the use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in W095/17211 and as parenteral adjuvants in W098/42375.
  • the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LTR192G.
  • ADP-ribosylating toxins and detoxified derivaties thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in the following references, each of which is specifically inco ⁇ orated by reference herein in their entirety: Beignon, et al., "The LTR72 Mutant of Heat-Labile Enterotoxin of Escherichia coli Enahnces the Ability of Peptide Antigens to Elicit CD4+ T Cells and Secrete Gamma Interferon after Coapplication onto Bare Skin", Infection and Immunity (2002) 70(6):3012-3019; Pizza, et al., "Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants", Vaccine (2001) 19:2534-2541; Pizza, et al., "LTK63 and LTR72, two mucosal adjuvants ready for clinical trials" Int.
  • Vaccine Differential Effects of the Nontoxic AB Complex and Enzyme Activity on Thl and Th2 Cells" Infection and Immunity (1999) 67(12):6270-6280; Partidos et al., "Heat-labile enterotoxin of Escherichia coli and its site-directed mutant LTK63 enhance the proliferative and cytotoxic T-cell responses to intranasally co-immunized synthetic peptides", Immunol. Lett.
  • Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in Domenighini et al., Mol. Microbiol (1995) 15(6): 1165-1167, specifically inco ⁇ orated herein by reference in its entirety.
  • the adjuvant is an ADP-ribosylating toxin and an oligonucleotide containing a CpG motif (see for example, WO 01/34185)
  • the adjuvant is a detoxified ADP-ribosylating toxin and an oligonucleotide containing a CpG motif.
  • the detoxified ADP-ribosylating toxin is LTK63 or LTK72.
  • the adjuvant is LTK63.
  • the adjuvant is LTK72.
  • the adjuvant is LTK63 and an oligonucleotide containing a CpG motif.
  • the adjuvant is LTK72 and an oligonucleotide containing a CpG motif.
  • Bioadhesives and mucoadhesives may also be used as adjuvants in the invention.
  • Suitable bioadhesives include esterified hyaluronic acid microspheres (Singh et al.
  • mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention. See for example, WO99/27960.
  • Microparticles may also be used as adjuvants in the invention.
  • Microparticles i.e. a particle of -lOOnm to ⁇ 150 ⁇ m in diameter, more preferably ⁇ 200nm to ⁇ 30 ⁇ m in diameter, and most preferably ⁇ 500nm to ⁇ 10 ⁇ m in diameter
  • materials that are biodegradable and non-toxic e.g. a poly( ⁇ -hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.
  • a negatively- charged surface e.g. with SDS
  • a positively-charged surface e.g. with a cationic detergent, such as CTAB
  • liposome formulations suitable for use as adjuvants are described in U.S.
  • Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters (W099/52549). Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (WOO 1/21207) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol (WO01/21152).
  • Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35- lauryl ether, and polyoxyethylene-23-lauryl ether.
  • PCPP Polyphosphazene
  • PCPP formulations are described, for example, in Andrianov et al Biomaterials
  • muramyl peptides suitable for use as adjuvants in the invention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L- alanyl-D-isoglutamine (nor-MDP), and N-acetyhnuramyl-L-alanyl-D-isoglutaminyl-L- alanine-2-(r-2 , -dipalmitoyl-5ra-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP- PE).
  • thr-MDP N-acetyl-muramyl-L-threonyl-D-isoglutamine
  • nor-MDP N-acetyl-normuramyl-L- alanyl-D-isoglutamine
  • imidazoquinolone compounds suitable for use adjuvants in the invention include Imiquamod and its homologues, described further in Stanley, “Imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential” Clin Exp Dermatol (2002) 27(7):571 - 577; and Jones, “Resiquimod 3M", Curr Opin Investig Drugs (2003) 4(2):214 - 218.
  • the invention may also comprise combinations of aspects of one or more of the adjuvants identified above.
  • the following adjuvant compositions may be used in the invention:
  • a saponin e.g. QS21
  • 3dMPL + IL-12 optionally + a sterol (W098/57659); combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions (European patent applications 0835318, 0735898 and 0761231).
  • SAF containing 10% Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion.
  • Ribi adjuvant system (RAS), (Ribi Immunochem) containing 2%> Squalene, 5 0.2%) Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM); and (7) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dPML).
  • MPL monophosphorylipid A
  • TDM trehalose dimycolate
  • CWS cell wall skeleton
  • LPS such as 3dPML
  • Bacterial toxins and bioadhesives are preferred adjuvants for use with mucosally-delivered vaccines, such 20 as nasal vaccines.
  • the composition may include an antibiotic.
  • the compositions of the present invention are administered with alum and/or CpG sequences.
  • 25 Nucleic Acid The antigens or epitopes of the present invention may be administered as nucleotide sequences encoding the antigens or epitopes.
  • nucleotide sequence refers to one of more nucleotide sequences which encode one or more 30 epitopes which are used in the compositions or combinations of the present invention.
  • NOI nucleotide sequence
  • polynucleotide or “nucleic acid”.
  • the NOI may be DNA or RNA of genomic or synthetic or of recombinant origin.
  • the NOI may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.
  • the NOI is DNA.
  • the NOI is prepared by use of recombinant DNA techniques (e.g. recombinant DNA).
  • the NOI is cDNA.
  • the NOI may be the same as the naturally occurring form.
  • nucleic acid includes DNA and RNA, and also their analogues, such as those containing modified backbones (e.g. phosphorothioates, etc.), and also peptide nucleic acids (PNA), etc.
  • the invention includes nucleic acid comprising sequences complementary to those described above (e.g. for antisense or probing pu ⁇ oses).
  • Nucleic acid according to the invention can be prepared in many ways (e.g. by chemical synthesis, from genomic or cDNA libraries, from the organism itself, etc.) and can take various forms (e.g. single stranded, double stranded, vectors, probes, etc.). They are preferably prepared in substantially pure form (i.e.
  • the invention provides a process for producing nucleic acid of the invention, comprising the step of amplifying nucleic acid using a primer-based amplification method (e.g. PCR).
  • a process for producing nucleic acid of the invention comprising the step of synthesising at least part of the nucleic acid by chemical means.
  • an antigen or antigenic combination or NOI encoding same is administered directly to a host subject.
  • a vector comprising an NOI is administered to a host subject.
  • the NOI is prepared and/or administered using a genetic vector.
  • a vector is a tool that allows or faciliates the transfer of an entity from one environment to another.
  • some vectors used in recombinant DNA techniques allow entities, such as a segment of DNA (such as a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a host and/or a target cell for the pu ⁇ ose of replicating the vectors comprising the NOI of the present invention and/or expressing the antigens or epitopes of the present invention encoded by the NOI.
  • examples of vectors used in recombinant DNA techniques include but are not limited to plasmids, chromosomes, artificial chromosomes or viruses.
  • the term “vector” includes expression vectors and/or transformation vectors.
  • expression vector means a construct capable of in vivo or in vitro/ex vivo expression.
  • transformation vector means a construct capable of being transferred from one species to another.
  • the vectors comprising the NOI of the present invention may be administered directly as "a naked nucleic acid construct", preferably further comprising flanking sequences homologous to the host cell genome.
  • naked DNA refers to a plasmid comprising the NOI of the present invention together with a short promoter region to control its production. It is called “naked” DNA because the plasmids are not carried in any delivery vehicle.
  • a host cell such as a eukaryotic cell, the proteins it encodes are transcribed and translated within the cell.
  • the vectors comprising the NOI of the present invention may be introduced into suitable host cells using a variety of viral techniques which are known in the art, such as for example infection with recombinant viral vectors such as retroviruses, he ⁇ es simplex viruses and adenoviruses.
  • the vector may be a recombinant viral vectors.
  • Suitable recombinant viral vectors include but are not limited to adeno virus vectors, adeno-associated viral (AAV) vectors, he ⁇ es-virus vectors, a retroviral vector, lentiviral vectors, baculoviral vectors, pox viral vectors or parvovirus vectors (see Kestler et al 1999 Human Gene Ther 10(10): 1619-32).
  • AAV adeno-associated viral
  • he ⁇ es-virus vectors he ⁇ es-virus vectors
  • retroviral vector lentiviral vectors
  • baculoviral vectors pox viral vectors or parvovirus
  • target vector refers to a vector whose ability to infect or transfect or transduce a cell or to be expressed in a host and/or target cell is restricted to certain cell types within the host subject, usually cells having a common or similar phenotype.
  • the NOI of the present invention which is inserted into a vector is operably linked to a control sequence that is capable of providing for the expression of the antigens or epitopes by the host cell, i.e. the vector is an expression vector.
  • the agent produced by a host cell may be secreted or may be contained intracellularly depending on the NOI and/or the vector used.
  • expression vectors containing the NOI can be designed with signal sequences which direct secretion of the EOI through a particular prokaryotic or eukaryotic cell membrane.
  • Chlamydia pneumoniae antigens used in the invention may be present in the composition as individual separate polypeptides, but it is preferred that at least two (i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) of the antigens are expressed as a single polypeptide chain (a 'hybrid' polypeptide).
  • Hybrid polypeptides offer two principal advantages: first, a polypeptide that may be unstable or poorly expressed on its own can be assisted by adding a suitable hybrid partner that overcomes the problem; second, commercial manufacture is simplified as only one expression and purification need be employed in order to produce two polypeptides which are both antigenically useful.
  • the hybrid polypeptide may comprise two or more polypeptide sequences from the first antigen group.
  • the invention includes a composition comprising a first amino acid sequence and a second amino acid sequence, wherein said first and second amino acid sequences are selected from a Chlamydia bactgerium, preferably a Chlamydia pneumoniae antigen or a fragment thereof of the first antigen group.
  • the first and second amino acid sequences in the hybrid polypeptide comprise different epitopes.
  • the hybrid polypeptide may comprise two or more polypeptide sequences from the second antigen group.
  • the invention includes a composition comprising a first amino acid sequence and a second amino acid sequence, wherein said first and second amino acid sequences are selected from a Chlamydia pneumoniae antigen or a fragment thereof of the second antigen group.
  • the first and second amino acid sequences in the hybrid polypeptide comprise difference epitopes.
  • the hybrid polypeptide may comprise one or more polypeptide sequences from the first antigen group and one or more polypeptide sequences from the second antigen group.
  • the invention includes a composition comprising a first amino acid sequence and a second amino acid sequence, said first amino acid sequence selected from a Chlamydia pneumoniae antigen or a fragment thereof from the first antigen group and said second amino acid sequence selected from a Chlamydia bactgerium, preferably a Chlamydia pneumoniae antigen or a fragment thereof from the second antigen group.
  • the first and second amino acid sequences in the hybrid polypeptide comprise difference epitopes.
  • the hybrid polypeptide may comprise one or more polypeptide sequences from the first antigen group and one or more polypeptide sequences from the third antigen group or the fourth antigen group or the fifth antigen group or the sixth antigen group or the seventh antigen group or the eight antigen group or the ninth antigen group or the tenth antigen group.
  • the invention includes a composition comprising a first amino acid sequence and a second amino acid sequence, said first amino acid sequence selected from a Chlamydia pneumoniae antigen or a fragment thereof from the first antigen group and said second amino acid sequence selected from a Chlamydia pneumoniae antigen or a fragment thereof from the third antigen group or the fourth antigen group or the fifth antigen group or the sixth antigen group or the seventh antigen group or the eight antigen group or the ninth antigen group or the tenth antigen group.
  • the first and second amino acid sequences in the hybrid polypeptide comprise difference epitopes.
  • the hybrid polypeptide may comprise one or more polypeptide sequences from the second antigen group and one or more polypeptide sequences from the third antigen group or the fourth antigen group or the fifth antigen group or the sixth antigen group or the seventh antigen group or the eight antigen group or the ninth antigen group or the tenth antigen group.
  • the invention includes a composition comprising a first amino acid sequence and a second amino acid sequence, said first amino acid sequence selected from a Chlamydia pneumoniae antigen or a fragment thereof from the second antigen group and said second amino acid sequence selected from a Chlamydia pneumoniae antigen or a fragment thereof from the third antigen group or the fourth antigen group or the fifth antigen group or the sixth antigen group or the seventh antigen group or the eight antigen group or the ninth antigen group or the tenth antigen group.
  • the first and second amino acid sequences in the hybrid polypeptide comprise difference epitopes.
  • Hybrids consisting of amino acid sequences from two, tliree, four, five, six, seven, eight, nine, or ten Chlamydia pneumoniae antigens are preferred.
  • hybrids consisting of amino acid sequences from two, three, four, or five Chlamydia pneumoniae antigens are preferred.
  • Different hybrid polypeptides may be mixed together in a single formulation.
  • a Chlamydia pneumoniae antigen may be present in more than one hybrid polypeptide and/or as a non-hybrid polypeptide. It is preferred, however, that an antigen is present either as a hybrid or as a non-hybrid, but not as both.
  • Two-antigen hybrids for use in the invention may comprise any one of the combinations disclosed above.
  • Hybrid polypeptides can be represented by the formula NH 2 -A- ⁇ -X-L- ⁇ diligent-B-COOH, wherein: X is an amino acid sequence of a Chlamydia pneumoniae antigen or a fragment thereof from the first antigen group, the second antigen group or the third antigen group or the fourth antigen group or the fifth antigen group or the sixth antigen group or the seventh antigen group or the eight antigen group or the ninth antigen group or the tenth antigen group.; L is an optional linker amino acid sequence; A is an optional N-terminal amino acid sequence; B is an optional C-terminal amino acid sequence; and ra is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • a -X- moiety has a leader peptide sequence in its wild-type form, this may be included or omitted in the hybrid protein.
  • the leader peptides will be deleted except for that of the -X- moiety located at the N-terminus of the hybrid protein i.e. the leader peptide of Xi will be retained, but the leader peptides of X 2 ... X n will be omitted. This is equivalent to deleting all leader peptides and using the leader peptide of Xi as moiety -A-.
  • linker amino acid sequence -L- may be present or absent.
  • the hybrid may be NH 2 -X ⁇ -L ⁇ -X 2 -L 2 -COOH, NH 2 -Xj- X 2 -COOH, NH 2 -X!-L ⁇ -X 2 -COOH, NH 2 -X ! -X 2 -L 2 -COOH, etc.
  • Linker amino acid sequence(s) -L- will typically be short (e.g. 20 or fewer amino acids i.e. 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1).
  • Other suitable linker amino acid sequences will be apparent to those skilled in the art.
  • a useful linker is GSGGGG (SEQ ID No 77), with the Gly-Ser dipeptide being formed from a BamYS. restriction site, thus aiding cloning and manipulation, and the (Gly) 4 tetrapeptide being a typical poly-glycine linker.
  • -A- is an optional N-terminal amino acid sequence.
  • Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art. If Xi lacks its own N-terminus methionine, -A- is preferably an oligopeptide (e.g.
  • the invention also provides nucleic acid encoding hybrid polypeptides of the invention. Furthermore, the invention provides nucleic acid which can hybridise to this nucleic acid, preferably under "high stringency" conditions (e.g. 65°C in a O.lxSSC, 0.5%> SDS solution).
  • "high stringency" conditions e.g. 65°C in a O.lxSSC, 0.5%> SDS solution.
  • the NOI of the present invention may be expressed as a fusion protein comprising an adjuvant and/or a biological response modifier and/or immunomodulator fused to the antigens or epitopes of the present invention to further enhance and/or augment the CMI response obtained.
  • the biological response modifier may act as an adjuvant in the sense of providing a generalised stimulation of the CMI response.
  • the antigens or epitopes may be attached to either the amino or carboxy terminus of the biological response modifier.
  • Polypeptides of the invention can be prepared by various means (e.g. recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various forms (e.g. native, fusions, non-glycosylated, lipidated, etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other Chlamydial or host cell proteins).
  • the invention also provides a process for producing a polypeptide of the invention, comprising the step of culturing a host cell transformed with nucleic acid of the invention under conditions which induce polypeptide expression.
  • the invention provides a process for producing a polypeptide of the invention, comprising the step of synthesising at least part of the polypeptide by chemical means.
  • the invention further provides a process for producing a composition according to the invention comprising the step of bringing one or more of SEQ IDs 1-86 into combination with one or more other of SEQ IDs 1-86
  • Preferred polypeptides of the invention comprise an amino acid sequence found in
  • the individual antigens within the hybrid may be from one or more strains.
  • X 2 may be from the same strain as Xj or from a different strain.
  • heterologous host Whilst expression of the polypeptides of the invention may take place in Chlamydia, the invention preferably utilises a heterologous host.
  • the heterologous host may be prokaryotic (e.g. a bacterium) or eukaryotic. It is preferably E.coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria (e.g. M.tuberculosis), yeasts, etc.
  • compositions include a protein that exists in different nascent and mature forms
  • the mature form of the protein is preferably used.
  • the mature form of the Chlamydia pneumoniae protein lacking the signal peptide may be used
  • compositions of the invention will generally be administered directly to a patient.
  • Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral (e.g. tablet, spray), vaginal, topical, transdermal ⁇ e.g. see
  • the invention may be used to elicit systemic and/or mucosal immunity.
  • the compositions of the present invention may be administered, either alone or as part of a composition, via a variety of different routes. Certain routes may be favoured for certain compositions, as resulting in the generation of a more effective immune response, prefereably a CMI response, or as being less likely to induce side effects, or as being easier for administration.
  • compositions of the present invention may be administered via a systemic route or a mucosal route or a transdermal route or it may be administrered directly into a specific tissue.
  • systemic administration includes but is not limited to any parenteral routes of administration.
  • parenteral administration includes but is not limited to subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular, or intrasternal injection, intravenous, intraarterial, or kidney dialytic infusion techniques.
  • the systemic, parenteral administration is intramuscular injection.
  • the compositions of the present invention are administered via a transdermal route.
  • transdermal administration of a composition may be preferred because it more efficiently activates the cell mediated immune (CMI) arm of the immune system.
  • CMI cell mediated immune
  • transdermal delivery intends intradermal (e.g., into the dermis or epidermis), transdermal (e.g.,”percutaneous") and transmucosal administration, i.e., delivery by passage of an agent into or through skin or mucosal tissue.
  • Transdermal Drug Delivery Developmental Issues and Research Initiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989); Controlled Drug Delivery: Fundamentals and Applications, Robinson and Lee (eds.), Marcel Dekker Inc.,(1987); and Transdermal Delivery of Drugs, Vols. 1-3, Kydonieus and Berner (eds.), CRC Press, (1987).
  • the term encompasses delivery of an agent using a particle delivery device (e.g., a needleless syringe) such as those described in U.S. Patent No. 5,630,796, as well as delivery using particle-mediated delivery devices such as those described in U.S. Patent No. 5,865,796.
  • a particle delivery device e.g., a needleless syringe
  • particle-mediated delivery devices such as those described in U.S. Patent No. 5,865,796.
  • the term “mucosal administration” includes but is not limited to oral, intranasal, intravaginal, intrarectal, intratracheal, intestinal and ophthalmic administration.
  • Mucosal routes particularly intranasal, intratracheal, and ophthalmic are preferred for protection against natural exposure to environmental pathogens such as RSV, flu virus and cold viruses or to allergens such as grass and ragweed pollens and house dust mites.
  • the enhancement of the immune response preferably the CMI response will enhance the protective effect against a subsequently encountered target antigen such as an allergen or microbial agent.
  • the compositions of the present invention may be administered to cells which have been isolated from the host subject.
  • the composition is administered to professional antigen presenting cells (APCs), such as dendritic cells.
  • APCs may be derived from a host subject and modified ex vivo to express an antigen of interest and then transferred back into the host subject to induce an enhanced CMI response.
  • Dendritic cells are believed to be the most potent APCs for stimulating enhanced CMI responses because the expressed epitopes of the antigen of interest must be acquired, processed and presented by professional APCs to T cells (both Thl and Th2 helper cells as well as CD8+ T-cells) in order to induce an enhanced CMI response.
  • Particle-mediated methods for delivering the compositions of the present invention are known in the art.
  • the above-described antigens or NOI encoding same can be coated onto core carrier particles using a variety of techniques known in the art.
  • Carrier particles are selected from materials which have a suitable density in the range of particle sizes typically used for intracellular delivery from a gene gun device. The optimum carrier particle size will, of course, depend on the diameter of the target cells.
  • core carrier is meant a carrier on which a guest antigen or guest nucleic acid (e.g., DNA, RNA) is coated in order to impart a defined particle size as well as a sufficiently high density to achieve the momentum required for cell membrane penetration, such that the guest molecule can be delivered using particle-mediated techniques (see, e.g., U.S. Patent No. 5,100,792).
  • Core carriers typically include materials such as tungsten, gold, platinum, ferrite, polystyrene and latex. See e.g., Particle Bombardment Technology for Gene Transfer, (1994) Yang, N. ed., Oxford University Press, New York, NY pages 10-11. Tungsten and gold particles are preferred.
  • Tungsten particles are readily available in average sizes of 0.5 to 2.0 microns in diameter.
  • Gold particles or microcrystalline gold e. g., gold powder A1570, available from Engelhard Co ⁇ ., East Newark, NJ
  • Gold particles provide uniformity in size (available from Alpha Chemicals in particle sizes of 1-3 microns, or available from Degussa, South Plainfield, NJ in a range of particle sizes including 0.95 microns).
  • Microcrystalline gold provides a diverse particle size distribution, typically in the range of 0.5-5 microns. However, the irregular surface area of microcrystalline gold provides for highly efficient coating with nucleic acids. A number of methods are known and have been described for coating or precipitating NOIs onto gold or tungsten particles.
  • Most such methods generally combine a predetermined amount of gold or tungsten with plasmid DNA, CaC12 and spermidine.
  • the resulting solution is vortexed continually during the coating procedure to ensure uniformity of the reaction mixture.
  • the coated particles can be transferred to suitable membranes and allowed to dry prior to use, coated onto surfaces of a sample module or cassette, or loaded into a delivery cassette for use in particular gene gun instruments.
  • the particle compositions or coated particles are administered to the individual in a manner compatible with the dosage formulation, and in an amount that will be effective for the pu ⁇ oses of the invention.
  • the amoimt of the composition to be delivered e. g., about 0.1 mg to 1 mg, more preferably 1 to 50 ug of the antigen or allergen, depends on the individual to be tested. The exact amount necessary will vary depending on the age and general condition of the individual to be treated, and an appropriate effective amount can be readily determined by one of skill in the art upon reading the instant specification.
  • the term "host mammalian subject” means any member of the subphylum cordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
  • the terms do not denote a particular age. Thus, both adult and newborn individuals are intended to be covered.
  • the methods described herein are intended for use in any of the above vertebrate species, since the immune systems of all of these vertebrates operate similarly. If a mammal, the subject will preferably be a human, but may also be a domestic livestock, laboratory subject or pet animal.
  • the mammal is preferably a human.
  • the human is preferably a child (e.g. a toddler or infant) or a teenager; where the vaccine is for therapeutic use, the human is preferably a teenager or an adult.
  • a vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc.
  • the invention also provides the use of the compositions of the invention in the manufacture of a medicament for raising an immune response in a mammal.
  • the medicament is preferably a vaccine and to the preparation of a vaccine to prevent and/or treat an disorder associated with a Chlamydia bacterium. It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment.
  • the administration of antigenic combinations of the present invention or a composition comprising the NOI encoding the antigenic combinations may be for either "prophylactic” or "therapeutic” pu ⁇ ose.
  • therapeutic or “treatment” includes any of following: the prevention of infection or reinfection; the reduction or elimination of symptoms; and the reduction or complete elimination of a pathogen. Treatment may be effected prophylactically (prior to infection) or therapeutically (following infection).
  • Prophylaxis or therapy includes but is not limited to eliciting an effective immune response, preferably a CMI immune response and/or alleviating, reducing, curing or at least partially arresting symptoms and/or complications resulting from a T cell mediated immune disorder.
  • the composition of the present invention When provided prophylactically, the composition of the present invention is typically provided in advance of any symptom.
  • the prophylactic administration of the composition of the present invention is to prevent or ameliorate any subsequent infection or disease.
  • the composition of the present invention When provided therapeutically, the composition of the present invention is typically provided at (or shortly after) the onset of a symptom of infection or disease.
  • the composition of the present invention may be provided either prior to the anticipated exposure to a disease causing agent or disease state or after the initiation of an infection or disease.
  • prophylactic or therapeutic administration is the more appropriate will usually depend upon the nature of the disease.
  • immunotherapeutic composition of the present invention could be used in immunotherapy protocols to actively inducing immunity by vaccination. This latter form of treatment is advantageous because the immunity is prolonged.
  • a vaccine composition will preferably, though not necessarily be used prophylactically to induce an effective CMI response against subsequently encountered antigens or portions thereof (such as epitopes) related to the target antigen.
  • Chlamydia e.g. trachoma, pelvic inflammatory disease, epididymitis, infant pneumonia, artherosclerosis, cardiovascular disease etc.
  • the compositions may also be effective against C.pneumoniae.
  • composition dose administrated to a host subject should be sufficient to effect a beneficial prophylactic or therapeutic immune response, preferably a CMI response in the subject over time.
  • the invention also provides a method for raising an immune response in a mammal comprising the step of administering an effective amount of a composition of the invention.
  • the immune response is preferably protective and preferably involves antibodies and/or cell-mediated immunity.
  • the method may raise a booster response.
  • prophylactically or therapeutically effective dose means a dose in an amount sufficient to elicit an enhanced immune response, preferably a CMI response to one or more antigens or epitopes and/or to alleviate, reduce, cure or at least partially arrest symptoms and/or complications from a T cell mediated immune disorder.
  • Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen(s), as well as any other components, as needed.
  • 'immunologically effective amount' it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • the mammal is preferably a human.
  • the human is preferably a child (e.g. a toddler or infant) or a teenager or an adult; where the vaccine is for therapeutic use, the human is preferably a teenager or an adult.
  • a vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc.
  • the human is a teenager. More preferably, the human is a pre-adolescent teenager. Even more preferably, the human is a pre- adolescent female or male Preferably the pre-adolescent male or female is around 9- 12 years of age.
  • One way of assessing the immunogenicity of the component proteins of the immunogenic compositions of the present invention is to express the proteins recombinantly and to screen patient sera or mucosal secretions by immunoblot or by protein or DNA microarray. A positive reaction between the protein and the patient serum indicates that the patient has previously mounted an immune response to the protein in question- that is, the protein is an immunogen. This method may also be used to identify immunodominant proteins.
  • One way of checking efficacy of therapeutic treatment involves monitoring Chlamydia infection after administration of the composition of the invention.
  • One way of checking efficacy of prophylactic treatment involves monitoring immune responses against the Chlamydia antigen, such as the Chlamydia pneumoniae antigen in the compositions of the invention after administration of the composition.
  • checking efficacy of prophylactic treatment may involve monitoring immune responses both sys temically (such as monitoring the level of IgGl and IgG2a production) and mucosally (such as monitoring the level of IgA production) against the Chlamydia pneumoniae antigens in the compositions of the invention after administration of the composition.
  • serum Chlamydia specific antibody responses are determined post-immunization but pre-challenge whereas mucosal Chlamydia specific antibody body responses are determined post-immunization and post-challenge.
  • Chlamydia pneumoniae e.g. pneumonia, bronchitis, pharyngitis, sinusitis, erythema nodosum, asthma, atherosclerosis, stroke, myocardial infarctions, coronary artery disease, etc.
  • a disease caused by Chlamydia pneumoniae e.g. pneumonia, bronchitis, pharyngitis, sinusitis, erythema nodosum, asthma, atherosclerosis, stroke, myocardial infarctions, coronary artery disease, etc.
  • the vaccine compositions of the present invention can be evaluated in in vitro and in vivo animal models prior to host, e.g., human, administration.
  • in vitro neutralization by Peterson et al (1988) is suitable for testing vaccine compositions directed toward Chlamydia, preferably Chlamydia pneumoniae.
  • Hyper-immune antisera is diluted in PBS containing 5%> guinea pig serum, as a complement source.
  • Chlamydia pneumoniae (10 4 IFU; inclusion forming units) are added to the antisera dilutions.
  • the antigen-antibody mixtures are incubated at 37°C for 45 minutes and inoculated into duplicate confluent Hep-2 or HeLa cell monolayers contained in glass vials (e.g., 15 by 45 mm), which have been washed twice with PBS prior to inoculation.
  • the monolayer cells are infected by centrifugation at 1000X g for 1 hour followed by stationary incubation at 37°C for 1 hour.
  • Infected monolayers are incubated for 48 or 72 hours, fixed and stained with Chlamydia specific antibody, such as anti-MOMP. Inclusion-bearing cells are counted in ten fields at a magnification of 200X. Neutralization titer is assigned on the dilution that gives 50%o inhibition as compared to control monolayers/IFU.
  • the efficacy of immunogenic compositions can also be determined in vivo by challenging animal models of Chlamydia pneumoniae infection, e.g., guinea pigs or mice, with the immunogenic compositions.
  • the immunogenic compositions may or may not be derived from the same serovars as the challenge serovars.
  • the immunogenic compositions are derivable from the same serovars as the challenge serovars.
  • the serovars of the present invention are obtainable from clinical isolates or from culture collections such as the American Tissue Culture Collection (ATCC).
  • ATCC American Tissue Culture Collection
  • In vivo efficacy models include but are not limited to: (i) A murine infection model using human Chlamydia pneumoniae serotypes; (ii) a murine disease model which is a murine model using a mouse-adapted Chlamydia pneumoniae strain, such as the Chlamydia pneumoniae mouse pneumonitis (MoPn) strain also known as Chlamydia muridarum; and (iii) a primate model using human Chlamydia pneumoniae isolates.
  • the MoPn strain is a mouse pathogen while human Chlamydia pneumoniae serotypes are human pathogens (see for example, Brunham et al (2000) J Infect Dis 181 (Suppl 3) S538-S543; Murdin et al (2000) J Infect Dis 181 (Suppl 3) S544-S551 and Read et al (2000) NAR 28(6); 1397-1406).
  • human Chlamydia pneumoniae serotypes can be used in mouse models although they normally require high inocula or pretreatment with progesterone.
  • Progesterone is generally used because it seems to render the epithelium more susceptible to chlamydial infection (see Pal et al 2003 Vaccine 21: 1455-1465).
  • MoPn which was originally isolated from mouse tissues, is thought to be a natural murine pathogen and thus offers an evolutionarily adapted pathogen for analysis of host-pathogen interactions.
  • the MoPn serovar is thought to have a high degree of DNA homology to the human Chlamydia serovars, it may also have some unique properties (see for example, Pal et al (2002) Infection and Immunity 70(9); 4812-4817.
  • mice 7 to 12 weeks of age receive 2.5 mg of depoprovera subcutaneously at 10 and 3 days before vaginal infection.
  • Post-vaccination mice are infected in the genital tract with 1,500 inclusion- forming units of Chlamydia pneumoniae contained in 5ml of sucrose-phosphate- glutamate buffer, pH 7.4.
  • the course of infection is monitored by determining the percentage of inclusion-bearing cells by indirect immunofluorescence with Chlamydia pneumoniae specific antisera, or by a Giemsa-stained smear from a scraping from the genital tract of an infected mouse.
  • the presence of antibody titers in the serum of a mouse is determined by an enzyme-linked immunosorbent assay.
  • the immunogenic compositions of the present invention can be administered using a number of different immunization routes such as but not limited to intra-muscularly (i.m.), intra- peritoneal (i.p.), intra-nasal (i.n.), sub-cutaneous (s.c) or transcutaneous (t.c) routes.
  • the challenge serovars may be administered by a number of different routes.
  • the challenge serovars are administered mucosally, such as but not limited to an intranasal (i.n) challenge.
  • guinea pig models For example, in vivo vaccine composition challenge studies in the guinea pig model of Chlamydia pneumoniae infection can be performed. A description of one example of this type of approach follows. Female guinea pigs weighing 450 - 500 g are housed in an environmentally controlled room with a 12 hour light-dark cycle and immunized with vaccine compositions via a variety of immunization routes. Post-vaccination, guinea pigs are infected in the genital tract with the agent of guinea pig inclusion conjunctivitis (GPIC), which has been grown in HeLa or McCoy cells (Rank et al. (1988)).
  • GPIC guinea pig inclusion conjunctivitis
  • Each animal receives approximately 1.4xl0 7 inclusion forming units (IFU) contained in 0.05 ml of sucrose-phosphate-glutamate buffer, pH 7.4 (Schacter, 1980).
  • IFU inclusion forming units
  • the course of infection monitored by determining the percentage of inclusion-bearing cells by indirect immunofluorescence with GPIC specific antisera, or by Giemsa- stained smear from a scraping from the genital tract (Rank et al 1988).
  • Antibody titers in the serum is determined by an enzyme-linked immunosorbent assay.
  • compositions of the invention will generally be administered directly to a patient.
  • Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or mucosally, such as by rectal, oral (e.g. tablet, spray), vaginal, topical, transdermal (See e.g. W099/27961) or transcutaneous (See e.g. WO02/074244 and WO02/064162), intranasal (See e.g. WO03/028760), ocular, aural, pulmonary or other mucosal administration.
  • parenteral injection e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue
  • mucosally such as by rectal, oral (e.g. tablet, spray), vaginal, topical, transdermal (See e.g. W099
  • Prophylaxis or therapy can be accomplished by a single direct administration at a single time point or multiple time points. Administration can also be delivered to a single or to multiple sites. Some routes of administration, such as mucosal administration via ophthalmic drops may require a higher dose. Those skilled in the art can adjust the dosage and concentration to suit the particular route of delivery.
  • Dosage treatment can be a single dose schedule or a multiple dose schedule, multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule, in a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc.
  • HOMOLOGUES a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc.
  • SEQ IDs 1-86 in the compositions of the invention may be supplemented or substituted with molecules comprising sequences homologous (ie. sharing sequence identitv) to SEQ ID Nos 1-86.
  • Proteins (including protein antigens) as used in the invention may have homology and/or sequence identity with naturally occurring forms. Similarly coding sequences capable of expressing such proteins will generally have homology and/or sequence identity with naturally occurring sequences.
  • Techniques for determining nucleic acid and amino acid "sequence identity" also are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence.
  • identity refers to an exact nucleotide-to-nucleotide or amino acid-to- amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.
  • a preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S.
  • homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease (s), and size determination of the digested fragments.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80%>-85%>, preferably at least about 90%,, and most preferably at least about 95%-98%> sequence identity over a defined length of the molecules, as determined using the methods above.
  • substantially homologous or homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
  • DNA sequences that are substantially homologous or homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system.
  • stringent hybridization conditions can include 50%> formamide, 5x Denhardt's Solution, 5x SSC, 0.1% SDS and 100 pg/ml denatured salmon sperm DNA and the washing conditions can include 2x SSC, 0.1 %> SDS at 37 C followed by lx SSC, 0.1% SDS at 68 C.
  • the degree of identity is preferably greater than 50% (eg. 65%. 80%. 90%. or more) and include mutants and allelic variants.
  • SEQ IDs 1-86 in the compositions of the invention may be supplemented or substituted with nucleic acid which can hybridise to the Chlamydia nucleic acid, preferably underv"high stringency" conditionsv(c. 65 C in an 0.1 x SSC, 0.5%, SDS solution).
  • hypothetical protein refers to a protein which lacks a known cellular location or a known cellular function. Typically, a hypothetical protein lacks significant homologies with known well characterised proteins.
  • the invention also provides the compositions of the invention for use as medicaments (eg. as immunogenic compositions or vaccines) or as diagnostic reagents for detecting a Chylamydia infectioin in a host subject. It also provides the use of the compositions in the manufacture of: (i) a medicament for treating or preventing infection due to Chlamydia pneumoniae bacteria: (ii) a diagnostic reagent for detecting the presence of Chlamydia Pneumonaie bacteria or of antibodies raised against Chlamydia Pneumonaie bacteria; and/or (iii) a reagent which can raise antibodies against Chlamydia pneumonaie bacteria.
  • the invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of a composition according to the invention.
  • the present invention provides compositions that are useful for preventing and/or treating T cell mediated immune disorders.
  • the composition is a pharmaceutical composition.
  • the composition is an immunotherapeutic composition.
  • the composition is a vaccine composition.
  • the composition may also comprise a carrier such as a pharmaceutically or immunologically acceptable carrier.
  • Pharmaceutically acceptable carriers or immunologically acceptable carriers are determined in part by the particular composition being administered as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions or vaccine compositions or immunotherapeutic compositions of the present invention.
  • compositions of the invention are preferably immunogenic compositions, and are more preferably vaccine compositions.
  • the pH of the composition is preferably between 6 and 8, preferably about 7.
  • the pH may be maintained by the use of a buffer.
  • the composition may be sterile and/or pyrogen-free.
  • the composition may be isotonic with respect to humans.
  • Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic.
  • the invention includes a method for the therapeutic or prophylactic treatment of Chlamydia pneumoniae infection in an animal susceptible to Chlamydial infection comprising administering to said animal a therapeutic or prophylactic amount of the immunogenic compositions of the invention.
  • the immunogenic composition comprises a combination of Chlamydia pneumoniae antigens, said combination selected from the group consisting of two, three, four, five or all six Chlamydia pneumoniae antigens of the first antigen group. Still more preferably, the combination consists of all six Chlamydia pneumoniae antigens of the first antigen group.
  • the immunogenic composition comprises a combination of Chlamydia pneumoniae antigens, said combination selected from the group consisting of two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve Chlamydia pneumoniae antigens selected from the first antigen group and the second antigen group.
  • the combination is selected from the group consisting of three, four, or five Chlamydia pneumoniae antigens selected from the second antigen group.
  • the combination consists of five Chlamydia pneumoniae antigens selected from the second antigen group.
  • the immunogenic composition comprises a combination of Chlamydia pneumoniae antigens, said combination consisting of two, three, four, or five Chlamydia pneumoniae antigens of the first antigen group and one, two, three, four, five or six Chlamydia pneumoniae antigens of the third antigen group.
  • the combination consists of three, four or five Chlamydia pneumoniae antigens of the first antigen group and one, two, three, four, five or six Chlamydia pneumoniae antigens of the third antigen group.
  • the immunigenic composition comprises a combination of Chlamydia pneumoniae antigens, said combination consisting of two, three, four, five, six, seven, eight, nine, ten, eleven or twelve Chlamydia pneumoniae antigens of the first antigen group and the second antigen group and one, two, three, four, five or six Chlamydia pneumoniae antigens of the third antigen group.
  • the combination is selected from the group consisting of three, four, or five Chlamydia pneumoniae antigens from the second antigen group and three, four or five Chlamydia pneumoniae from the third antigen group.
  • the combination consists of five Chlamydia pneumoniae antigens from the second antigen group and three, four or five Chlamydia pneumoniae antigens of the third antigen group.
  • the composition comprises molecules from different Chlamydia species.
  • the composition may comprise molecules from different serogroups and/or strains of the same Chlamydia species. Further embodiments comprise mixtures of one or more Chlamydia molecules from different strains.
  • Many proteins are relatively conserved between different species serogroups and strains of Chlamydia trachomatis and Chlamydia pneumoniae. To ensure maximum cross-strain recognition and reactivity, regions of proteins that are conserved between different Chlamydia species, serogroups and strains can be used in the compositions of the present invention. The invention therefore provides proteins which comprise stretches of amino acid sequence that are shared across the majority of Chlamydia strains.
  • the composition comprises a protein comprising a fragment of a Chlamydia pneumoniae protein (preferably a protein from SEQ ID Nos 1-86 or more preferably SEQ ID Nos 1-41 wherein said fragment consists of n consecutive conserved amino acids.
  • compositions of the invention may further comprise antigen derived from one or more sexually transmitted diseases in addition to Chlamydia trachomatis.
  • the antigen is derived from one or more of the following sexually transmitted diseases: N.gonorrhoeae ⁇ e.g. i, ii, iii, iv ⁇ ; human papiloma virus; Treponema pallidum; he ⁇ es simplex virus (HSV-1 or HSV-2); HIV (HIV-1 or HIV-2); and Haemophilus ducreyi.
  • a preferred composition comprises: (1) at least t of the Chlamydia pneumoniae antigens from either the first antigen group or the second antigen group, where t is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, preferably t is five; (2) one or more antigens from another sexually transmitted disease.
  • the sexually transmitted disease is selected from the group consisting of he ⁇ es simplex virus, preferably HSV-1 and/or HSV-2; human papillomavirus; N.gonorrhoeae; Treponema pallidum; and Haemophilus ducreyi.
  • compositions can thus provide protection against the following sexually-transmitted diseases: Chlamydia, genital he ⁇ es, genital warts, gonorrhoea, syphilis and chancroid (see Stephens et al (1998) Science 282: 754-759).
  • a saccharide or carbohydrate antigen is used, it is preferably conjugated to a carrier /protein i order to enhance immunogenicity (For example, Ramsay et al.
  • Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria or tetanus toxoids.
  • the CRM ⁇ 9 diphtheria toxoid is particularly preferred (Research Disclosure, 453077 (Jan 2002).
  • Other carrier polypeptides include the N.meningitidis outer membrane protein EP-A-0372501), synthetic peptides (EP-A-0378881, EP- A-0427347), heat shock proteins (W093/17712, WO94/03208) pertussis proteins (W098/58668, EP-A-0471177) protein D from H.influenzae (WO00/56360) cytokines (WO91/01146), lymphokines, hormones, growth factors, toxin A or B from C.difficile (WO00/61761) iron-uptake proteins WO01/72337) etc.
  • a mixture comprises capsular saccharides from both serogroups A and C
  • the ratio (w/w) of MenA saccharide:MenC saccharide is greater than 1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 or higher).
  • Different saccharides can be conjugated to the same or different type of carrier protein. Any suitable conjugation reaction can be used, with any suitable linker where necessary.
  • Toxic protein antigens may be detoxified where necessary e.g. detoxification of pertussis toxin by chemical and/or genetic means.
  • a diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens.
  • a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens.
  • a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens.
  • Antigens in the composition will typically be present at a concentration of at least 1 ⁇ g/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.
  • nucleic acid encoding the antigen may be used Robinson & Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al.
  • compositions of the invention may thus be replaced by nucleic acid (preferably DNA e.g. in the form of a plasmid) that encodes the protein.
  • nucleic acid preferably DNA e.g. in the form of a plasmid
  • compositions of the present invention- may be used to prevent and/or treat disorders such as but not limited to: pneumonia, cardiovascular diseases, atherosclerosis, bronchitis, pharyngitis, laryngitis, sinusitis, obstructive lung diseases, asthma, chronic obstructive pulmonary disease, reactive arthritis, otitis media, abdominal aortic aneurysm, erythema nodosum, Reiter syndrome, sarcoidosis, Alzheimer's disease, multiple sclerosis, lymphogranuloma venereum, ocular trachoma, pelvic inflammatory disease, inclusion conjunctivitis, genital trachoma, infant pneumonitis, incipient trachoma, keratitis, papillary hypertrophy, corneal infiltration, vulvovaginitis, mucopurulent rhinitis, salpingitis, cervicitis, cervical follicles, prostatitis, proctitis, urethritis
  • compositions of the invention may be prepared in various forms.
  • the compositions may be prepared as injectables, either as liquid solutions or suspensions.
  • Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition).
  • the composition may be prepared for topical administration e.g. as an ointment, cream or powder.
  • the composition may be prepared for oral administration e.g. as a tablet or capsule, as a spray, or as a syrup (optionally flavoured).
  • the composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray.
  • the composition may be prepared as a suppository or pessary.
  • the composition may be prepared for nasal, aural or ocular administration e.g. as drops.
  • the composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a patient.
  • kits may comprise one or more antigens in liquid form and one or more lyophilised antigens.
  • composition of the invention will typically, in addition to the components mentioned above, comprise one or more 'pharmaceutically acceptable carriers', which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • lipid aggregates such as oil droplets or liposomes.
  • the vaccines may also contain diluents, such as water, saline, glycerol, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like.
  • the biological molecules of the present invention be formulated into a pharmaceutical composition or an immunotherapeutic composition or a vaccine composition.
  • Such formulations comprise biological molecules combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline.
  • a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline.
  • Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative.
  • Formulations include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (for eg, a powder or granules) form for reconstitution with a suitable vehicle (e. g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e. g., sterile pyrogen-free water
  • the pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • a non-toxic parenterally-acceptable diluent or solvent such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides.
  • Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems.
  • Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • kits for enhancing a CMI response to the biological molecules of the present invention may comprise an antigenic composition or nucleotide sequence encoding same.
  • the kit may also include an adjuvant, preferably a genetic adjuvant is administered with or as part of the biological molecule and instructions for administering the biological molecule.
  • Other preferred components of the kit include an applicator for administering the biological molecule.
  • the term "applicator” refers to any device including but not limited to a hypodermic syringe, gene gun, particle acceleration device, nebulizer, dropper, bronchoscope, suppository, impregnated or coated vaginally-insertable material such as a tampon, douche preparation, solution for vaginal irrigation, retention enema preparation, suppository, or solution for rectal or colonic irrigation for applying the NOI either systemically or mucosally or transdermally to the host subject.
  • the invention also provides for a kit comprising comprising a combination of Chlamydia pneumoniae antigens.
  • the combination of Chlamydia pneumoniae antigens may be one or more of the immunogenic compositions of the invention.
  • the kit may further include a second component comprising one or more of the following: instructions, syringe or other delivery device, adjuvant, or pharmaceutically acceptable formulating solution.
  • the invention also provides a delivery device pre- filled with the immunogenic compositions of the invention.
  • FIG. 1A Assay of in vitro neutralization of C.pneumoniae infectivity for LLC-MK2 cells by polyclonal mouse antisera to recombinant Chlamydial proteins. Results are shown as reduction in the number of inclusions obtained when monolayers were infected with antiserum-treated infectious EBs, as compared to inclusion numbers given by untreated EBs. Percent reduction values are plotted against the reciprocal of the corresponding serum dilution. For each dilution inclusion counts were corrected for background inhibition of infectivity observed with the corresponding dilution of the pre- immune serum.
  • FIG. 1 shows results obtained with serial dilutions of antibodies raised against a 'neutralizing' antigen (v), a 'non-neutralizing' FACS- positive antigen (v), and against the GST polypeptide, used in the fusion constructs, alone ( ⁇ ).
  • Figure IB shows serum litres giving 50% neutralization of infectivity for the 10 C.pneumoniae recombinant antigens described in the text. Each titer was assessed in 3 separate experiments (SEM values shown).
  • FIG. 2 shows immunoblot analysis of two dimensional electrophoretic maps of C.pneumoniae EBs using the imune sera described in the text. Immunoblots were obtained from either of two EB gels (panels A and B at the top) covering different pH intervals, according to which of the two allowed the best detection of a given antigen.
  • the arrows in the HtrA immunoblot show which of the signals had a corresponding stained spot in the gel (arrows in panel A) which was subjected to MALDI-TOF identification.
  • the two patterns in the HtrA blot are both suggestive of typical electrophoretic 'trains' composed of single charge variants of the same protein.
  • Figure 4 shows flow cytometric analysis of splenocytes from DNA-immunized HLA- A2 transgenic and non transgenic mice.
  • Groups of 4 mice were immunized 3 times i.m. with 50 ⁇ g of plasmid DNA expressing C. pneumoniae Low Calcium Response Protein H.
  • IFN- ⁇ production from splenocytes was monitored following either a 6h (ex-vivo) or a 6 day (restimulated) pulse with peptide CH-6 (lO ⁇ g/ml).
  • Equal numbers of gated live lymphocyte cells were acquired with a LSRII FACS System (Becton Dickinson) and percentages of IFN- ⁇ producing CD8 + T cells were calculated using DIVA Software (Becton Dickinson).
  • Figure 5 shows a flow cytometric analysis of splenocytes from transgenic and non transgenic mice infected with C. pneumoniae EBs.
  • HLA-A2 transgenic mice were intranasally infected twice with 5x10 5 C. pneumoniae FB/96 EBs and splenocytes were stimulated for 6 days in the presence of relevant peptides before determining IFN- ⁇ production by CD8 + T cells as described in the legend of Figure 4.
  • B HLA-A2 transgenic and non transgenic mice were infected together with the same EBs preparation and CD8 + T cells were subjected to FACS analysis as reported in (A). Table I shows a summary of data and properties of the C.pneumoniae antigens described in the text.
  • the neutralization titer is reported is as the reciprocal of the antiserum dilution causing a 50% reduction in the number of inclusions in the in vitro infectivity assay.
  • Table 2 shows results from hamster mouse model studies for hypothetical proteins.
  • Table 3 shows expressed genes of CPn EB selected by microarray.
  • Table 4 shows C. pneumoniae selected peptides: protein sources and HLA-A2 stabilization assay.
  • Table 5 shows ELISPOT assay with CD8+ T cells from DNA immunised HLA-A2 transgenic mice.
  • Table 6 shows IFN- ⁇ production from splenocytes of DNA immunized HLA-A2 transgenic and non transgenic mice.
  • METHODS AND MATERIALS (Examples 1-4) (see Reference Section 1)
  • C.pneumoniae FB/96 a clinical isolate obtained from a patient with pneumonia at the Sant'Orsola Polyclinic, Bologna, Italy, was grown in LLC-MK2 cells seeded in individual wells of a six- well plastic plate (7). Cells were harvested 72 hr after infection with a sterile rubber, sonically disrupted and the elementary bodies (EB) purified by gradient centrifugation as described (26). Purified Chlamydiae were resuspended in sucrose-phosphate-glutamic acid (SPG) transport buffer, and stored in 0.5 ml aliquots, at -80°C until used. When required, prior to storage, EB infectivity was heat-inactivated by 3 hour incubation at 56°C.
  • SPG sucrose-phosphate-glutamic acid
  • ORFs Open reading frames (ORFs), selected from the C. pneumoniae CWL029 genome sequence (16), were PCR-cloned into plasmid expression vectors and purified from E.coli cultures, essentially as previously described (25). Recombinant Chlamydial proteins were obtained as GST fusion proteins by using pGEX-KG derived vectors (12) in E. coli BL21 (Novagen). PCR primers were designed so as to amplify genes without the N-terminal signal peptide coding sequence. When a signal peptide or processing site was not clearly predictable, the ORF sequence was cloned as annotated by Kalman and coworkers (16).
  • Cells were collected by centrifugation ' 3 hr after induction and broken in a French Press (SLM Aminco, Rochester, NY). After centrifugation at 30.000 g, the supernatants were loaded onto Glutathione Sepharose 4B columns (Amersham Pharmacia Biotech) and column bound proteins were eluted with 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0. Protein concentrations in the samples were determined using the Bradford method.
  • mice Groups of four 5/6-week old CD1 female mice (Charles River, Como, Italy) were immunized intraperitoneally at day 1 with 20ug of protein in Complete Freund's adjuvant (CFA) and boosted at day 15 and 28 with 20ug of recombinant protein in Incomplete Freund's adjuvant (IF A).
  • CFA Complete Freund's adjuvant
  • IF A Incomplete Freund's adjuvant
  • Pre-immune and immune sera were prepared from blood samples collected on days 0, 27 and 42. In order to reduce the amount of antibodies possibly elicited by contaminating E. coli antigens, the immune sera were incubated overnight at 4°C with nitrocellulose strips adsorbed with a total protein extract from E . coli BL21.
  • Protein samples (200 or 20 ⁇ g of protein for Coomassie Blue stained reference gels, or gels to be processed for immunoblotting, respectively) were adsorbed overnight on Immobiline DryStrips (7 cm, pH 3-10 NL, or pH 4-7). Electrofocusing was performed in an IPGphor Isoelectric Focusing Unit (Amersham Biosciences, Uppsala, Sweden). The focused strips were equilibrated as described (15) and loaded on linear 9-16.5 % acrylamide gradients (7x 4 cm, 1.5 mm thick), for SDS-PAGE separation in a Mini Protean III Cell (Bio-Rad, Hercules, CA). Gels were stained with colloidal Coomassie Blue (Novex, San Diego, CA) (4) and the protein maps so obtained were scanned with a Personal Densitometer SI (Molecular Dynamics) at 12 bits and 50 mm per pixel.
  • the proteins separated in the 2DE maps were transferred onto nitrocellulose membranes, overnight at 30 Volts, using a Protean III apparatus (BioRad, Hercules, CA). Membranes were stained with a 0.05% (w/v) CPTS (Copper(II) phthalocyanine-3,4',4",4'"-tetrasulfonic acid tetrasodium salt) in 12 mM HCl, and marked peripherally with 8 India-ink dots to provide anchors for subsequent image superimposition and matching.
  • CPTS Copper(II) phthalocyanine-3,4',4",4'"-tetrasulfonic acid tetrasodium salt
  • the membranes were destained with 0,5 M NaHC03, incubated with the mouse sera to be analyzed (either pre-immune or specific immune sera, diluted 1:1000), and then with a peroxidase-conjugated anti-mouse antibody (Amersham Biosciences, Uppsala, Sweden). After washing with PBS, 0.1 %> Tween-20, blots were developed using the Opti-4CN Substrate Kit (Biorad, Hercules, CA), and the images of the immunostained blots again acquired as above. Images were analysed with the computer program Image Master 2D Elite, version 4.01 (Amersham Biosciences, Uppsala, Sweden).
  • Tryptic peptides were desalted and concentrated using Zip-Tip (Millipore, Bedford, MA). Peptides were directly eluted and loaded onto a SCOUT 384 Anchor Chip multiprobe plate (400 ⁇ m, Bruker Daltonics, Bremen, Germany) with a solution of 2-5 dihydroxybenzoic acid (5g/l), in 50% acetonitrile, 0.1% trifluoroacetic acid. Spectra were acquired on a Bruker Biflex III matrix-assisted laser deso ⁇ tion ionization-time of flight (MALDI- TOF) apparatus. Resulting values for monoisotopic peaks were used for database searches using the Mascot software (32), as available at the website http://www.matrixscience.com/.
  • Antibody-EB interaction was allowed to proceed for 30 min at 37°C on a slowly rocking platform.
  • the lOOul of reaction mix of each sample was used to inoculate PBS-washed LLC-MK2 confluent monolayers (in triplicate for each serum dilution), in a 24-well tissue culture plate, and centrifuged at 805 x g for 1 hour at 37°C. After centrifugation Eagle's minimal essential medium containing Earle's salts, 20% fetal bovine serum and lug/ml cycloheximide was added. Infected cultures were incubated at 37°C in 5%>C0 2 for 72 hours.
  • the monolayers were fixed with methanol and the Chlamydial inclusions were detected by staining with mouse a ti-Chlamydia fluorescein-conjugated monoclonal antibody (Merifluor Chlamydia, Meridian Diagnostics, Inc.) and quantified by counting 10 fields per well at a magnification of 40X.
  • the inhibition of infectivity due to EBs interaction with the immune sera was calculated as percentage reduction in mean IFU number as compared to the SPG (buffer only)/EBs control. In this calculation the IFU counts obtained with immune sera were corrected for background inhibition of infection due to the corresponding pre-immune mouse serum.
  • the sera were considered as "neutralizing” if they could cause a 50% > or greater reduction in infectivity.
  • the corresponding neutralizing titer was defined as the serum dilution at which a 50% reduction of infectivity was observed.
  • Experimental variability was evaluated by calculating the standard error of measurement (SEM), from three titration experiments for each recombinant antigen, as shown in Fig. IB.
  • the clarified homogenates (0.2 ml) were inoculated in duplicate onto LLC-MK2 cells seeded in plastic individual well of a 24 well plate, incubated at 37°C for 72 h and fixed in acetone before detection and counting of numbers of Chlamydial inclusions per well by immunofluorescence microscopy.
  • the protocol was approved by the ethical committee of the University of Bologna.
  • pmp 10 and pmp2 encoding two members of the heterogeneous Chlamydial PMP family of polymo ⁇ hic membrane proteins; • artl, encoding a putative extracellular solute (possibly Arginine) binding protein of an aminoacid transport system;
  • htrA encoding a putative chaperone with heat-shock inducible protease activity
  • Cpn0301 "hypothetical" gene encoding a protein homologous to the ompH family of bacterial proteins, some members of which have been shown to be chaperones involved in outer membrane biosynthesis
  • a limit of the hamster model is that, because of the absence of immunological reagents, the relative contribution of humoral and cell-mediated immunity cannot be assessed.
  • Example 4 Two 'hypothetical'proteins 6784 and 6814 (encoded by the ORFs Cpn0498 and Cpn0525) yielded FACS-positive sera which, however, were not able to neutralize host cell infection in vitro. However, these antigens performed remarkably well in the hamster-spleen test. Table 2
  • cytoplasmic inclusions which can be stained with Chlamydia specific fluorescence-labeled monoclonal antibodies and counted with an UV light microscope.
  • cycloheximide which inhibits host cell protein synthesis and favours Chlamydial intracellular growth with the consequent formation of typical cytoplasmic inclusions which can be stained with Chlamydia specific fluorescence-labeled monoclonal antibodies and counted with an UV light microscope.
  • an anti-serum is labelled as 'neutralizing' when the reduction of infectivity is equal or greater than 50%>, and the serum dilution yielding a 50% reduction in infectivity is referred to as the 50% end- point neutralization titer.
  • hamster model Using a recently described in vivo model of systemic infection (hamster model), hamsters immunised with 6 of the in vitro neutralising antigens, when challenged with CPn EBs, showed a greater than 80%> reduction of spleen infection as compared with non-immunised controls.
  • proteins identified by the present work can be divided in 3 groups: • proteins which have an annotation compatible with (could be reasonably expected to have) an expected/predicted exposure on the Chlamydial cell surface and with the possibility that antibodies binding to them may actually interfere with host cell attachment and entry (ie proteins which could possibly induce neutralising antibodies)
  • the first group includes the 2 polymo ⁇ hic outer membrane proteins (Pmp's) Pmp2 and PmplO (10, 11, 14, 30), the outer membrane protein OmpH-like, and OmcA, which is annotated (Chlamydia Genome Project at htkp-J/Chlamydia- www.berkeley.edu:4231/) as "predicted 9-kD cysteine-rich, outer membrane protein, lipoprotein".
  • the Pmp family of Chlamydia-specific proteins is generally thought to comprise probable pathogenicity factors, with an autonomous secretion capacity (autotransporters), important for adhesion to host cells and are generally considered as promising vaccine candidates.
  • autotransporters important for adhesion to host cells and are generally considered as promising vaccine candidates.
  • this apart from very recent unpublished results on Pmp21, this is the first time that antisera to recombinant Pmp's are reported to have neutralizing properties.
  • OmcA 2 polymo ⁇ hic outer membrane proteins
  • OmcA is the product of a gene co-transcribed in the same operon with the 60 kDa OmcB cystein-rich protein which is a major structural component of the Chlamydial outer membrane and a major immunogen in human C. trachomatis infections.
  • OmcB and OmcA are likely to interact in some as yet unknown outer membrane structure, so it is possible that antibodies to OmcA can interfere with EB infectivity.
  • Chlamydial OmpH is probably a member of the OmpH (Skp) family of proteins which have been reported to have chaperonin activities in other bacteria very important for the correct biosynthesis of the outer membrane. These proteins appear to cooperate in this task with HtrA (see below). In fact, in E.coli single KO mutants of either OmpH (Skp) or HtrA (DegP) are still viable, but double mutants do not grow
  • Second Group of Selected Proteins (ArtJ, AtoS, HtrA and Enolase)
  • the second group which represents a somehow smprising finding, includes ArtJ, AtoS, HtrA and Enolase. If the current annotation (justified by analogy with homologous genes in other bacteria) is correct, all these proteins would be expected to have a periplasmic location in gram-negative bacteria, and to be surface-exposed only in a gram-positive bacterium. It is possible that owing to their atypical life cycle, requiring an efficient passage from a dormant spore-like status (the EB) to an active form needing to adapt quickly to host-cell responses to invasion, Chlamydiae in fact display some sensors directly on the outer surface of their infectious form.
  • EB dormant spore-like status
  • HtrA (DegP), which in other bacteria has a complex hexameric structure, has been described as having multiple functions (3, 5, 18, 19, 27, 38) : a chaperonin assisting a correct outer membrane biogenesis, inducible protease for the elimination of misfolded membrane proteins, and also a sensor of 'stress' conditions.
  • a chaperonin assisting a correct outer membrane biogenesis
  • inducible protease for the elimination of misfolded membrane proteins
  • a sensor of 'stress' conditions In Chlamydia none of these properties has been demonstrated yet, however we find that in purified EB HtrA is present in two forms one of which appears to be processed by being deprived of the N-terminal fragment. This fragment, if aligned with the homologous HtrA sequence from Thermologa maritima (18), would comprise a predicted loop acting as a structural lid controlling the access to the protease active.
  • HtrA could have a similar protease activity and the two forms identified on the 2-D map represent the active and inactive species.
  • C. trachomatis HtrA ortholog is recognized by human sera from patients who had a Chlamydial genital infection (35), and a similarly HtrA is one of the antigens in the immunoproteome of Helicobacter pylori (13).
  • the homologue protein in Haemophilus influenzae is a protective antigen in both a passive infant rat model of bacteremia and the active chinchilla model of otitis media (23) .
  • Cpn enolase Also in the second group of proteins expected to be located elsewhere than the cell surface, is Cpn enolase. This protein aligns with the well known family of conserved glycosylases, which are essentially cytoplasmic enzymes, but in Streptococci enolase has been shown to have also a cell surface location, and extracellular matrix binding properties (1, 28, 29)). Interestingly, Gaston and colleagues (8) also showed that in patients with reactive arthritis induced by C. trachomatis, enolase induces specific CD4 + T-cell responses.
  • the third of the 3 groups in which we propose to divide, just for the sake of discussion, the 10 neutralizing antigens above described, comprises two proteins which are still annotated in public Chlamydial databases as the hypothetical products of two CPn-specific genes: Cpn0759 and Cpn0042.
  • the Cpn0759 gene is the second gene in a cluster of 6 Cpn-specific hypothetical genes (from Cpn0794 to Cpn0799) immediately upstream of the enolase gene. With the exception of Cpn0759 the products of all the other genes in the cluster share similarities of 30 to 40% over long stretches of amino acids.
  • the Cpn0042 gene encodes a hypothetical protein, with 4 coiled-coil regions, which has been described as a member of a new family of hypervariable outer membrane proteins (33).
  • the hypervariability of these proteins could be due to a strand-slippage mechanism induced by the presence of a poly(C) stretch within the coding region of the corresponding genes, a mechanism already described in the Pmp's family for the pmpl 0 gene (30).
  • the function of these proteins is still unknown, and our observations provide the first experimental indication of a possible function related to the Chlamydial infection process.
  • Cpn0795 SEQ ID NO: 6
  • a Cpn specific hypothetical protein is a FACS positive protein which demonstrates significant immunoprotective activity in a hamster spleen model of Chlamydia pneumoniae infection.
  • Fig. 6 shows an alignment of the proteins in the 7105-7110 protein family. This
  • Alignment shows a new family of proteins expected to constitute a system of antigens probably delivered on the Cpn surface or secreted by a type V (autotransporter) secretion mechanism. This alignment was generated as follows:
  • 7107 can be predicted to fold in a beta-barrel structure which can form a translocation pore for secretion across the outer membrane.
  • Cpn0795 and Cpn0796 have C terminal ends that may form transmembrane pores (see alignment, FIG. 9).
  • CPn0794, Cpn0797, Cpn0798, and Cpn0799 have N-terminal ends indicating that all proteins have N-terminal and C-terminal ends.
  • Fig. 7 shows alignment of Cpn0794 - Cpn 0799. Proteins encoded by the genes Cpn0794, Cpn0795, Cpn0796, and Cpn0797 have been identified as likely to be exposed on the surface of the chlamydia cell and as possible vaccine candidates. These proteins are shown to be actually expressed by Cpn in vivo (WB data and FACS data). In the case of Cpn0797 we also showed that the level of expression in CPn EBs is high enough to be detected by mass spectrometry analysis on 2DE maps of protein extracts (see Montigiani et al.)
  • Cpn0794, Cpn0796 and Cpn0797 proteins can be aligned according to a set of imperfect repeats present within their aminoacid sequences (see FIG. 7) , whereas the putative product of CPn0795 can be mostly aligned to the C-terminal portion of the Cpn0796 protein.
  • proteins encoded by genes Cpn0798 or Cpn0799 can alse be aligned to the above proteins according to the above mentioned repeated sequence motifs (see FIG. 7). Overall alignment of the 6 genes demonstrates that the genes encode for a family of functionally-related proteins.
  • FIG. 8 illustrates Cpn0796.
  • Cpn0796 forms a beta-barrel structure and is capable of forming a pore across the bacterial outer membrane (OM).
  • OM bacterial outer membrane
  • the molecule may form a pore in the OM through which the N-terminal domain may pass (the 'passenger' domain) to the outside of the bacterial cell.
  • these molecules may either remain anchored to the bacterial surface or undergo a proteolytic cut which releases the 'passenger domain' or a portion of it into the medium surrounding the bacterial cell an example of which is represented in the following sequence:
  • amino acid residues 365-385 represent an alpha helix conformation that spans the beta barrel pore
  • the N-terminal passenger domain may be cleaved via a specific proteolytic action from the membrane-anchored pore structure.
  • a linker domain comprising the peptide sequence PSPAPV (SEQ ID NO: 84) as shown in bold in the following sequence illustrates a site at which cleavage of the N-terminal passenger domain may occur:
  • the N-terminal peptide may be secreted to be exposed on the bacterial cell surface and can also become detached via the proteolytic event described above.
  • the peptide may form a structural conformation known as beta-propellers indicated in the following sequence:
  • N-terminal passenger domain can also possess a specific protease activity, such as a serine protease-like activity.
  • the protease activity may act on the membrane anchored form of the molecule such that the N-terminal passenger domain is cleaved off form the surface of the chlamydial cell.
  • the serine protease like activity is supported by the presence of a consensus serine protease triad of adequately spaced amino acid residues (namely H, D and S) which can be located on the virtual structure of the 'passenger' domain modelled on a set of experimentally-determined templates, e.g. InrO (PDB identification code)
  • the gene Cpn0796 gene encodes for a protein which promotes its own secretion on the EB surface and may also mediate or promote its own release into the surrounding medium.
  • the secreted passenger peptide has several activities, including: 1. actin binding peptide, part of a chlamydial surface layer, and instrumental to the process of establishing the host cell infection 2.
  • specific protease activity within the host cell cytoplasm instrumental to the intracellular survival of infecting chlamydiae.
  • beta propeller domain Another function of the above N-terminal beta propeller domain is the regulation modulation of the activity of a cytosolic protease of the host cell in order to alter host cell properties in favour of chlamydial development, survival or persistence. See Fulop V, Bocskei Z, Polgar L. in "Prolyl oligopeptidase: an unusual beta-propeller domain regulates proteolysis.” Cell. 1998 Jul 24;94(2): 161-70.
  • the proteins encoded by Cpn0794, Cpn0797, Cpn0798, Cpn0799 - all comprising variants of the above described Cpn0796 structure - also provide beta propeller structures with activities similar and/or complementary to the ones described above.
  • a family of proteins cooperating to a common function either by generating - through events of site specific recombination - new molecules with structures and activities similar to the above described Cpn0796 product, OR by independently contributing to a multi-protein structure requiring a coordinated action of several related components.
  • FIG. 9 illustrates an alignment of the C-terminal domains of the proteins encoded by C.pneumoniae genes Cpn0795 and Cpn0796.
  • beta barrel domains of Cpn0795 or Cpn0796 include MKDLGTLGG (SEQ ID NO: 87), SXDGK (SEQ ID NO: 88) VIVG (SEQ ID NO: 89), VIXG (SEQ ID NO: 90) or HAF (SEQ ID NO: 91).
  • the main stages in the Chlamydial life cycle are: (i) the binding to the host cell surface and entry into the cytoplasm through a specialised vacuole (the Chlamydial inclusion) by an extracellular sporelike infective form, called the elementary body (EB); and (ii) the conversion of the EB to a non-infective replicative form called a reticulate body (RB) that replicates by binary fission a number of times within the inclusion to form a microcolony.
  • EB extracellular sporelike infective form
  • Protein microarrays are used for high throughput protein analysis by detecting proteins and monitoring their expression levels. Through use of protein microarrays, complex screening of thousands of proteins and interactions with proteins may be performed in parallel.
  • a protein array typically includes a surface, such as glass, membrane, microtiter wells, mass spectrometer plates, beads or other particles, for binding ligands, proteins, or antibodies. For example, antibodies may be bound to the microarray to form a capture array. The capture array may be contacted with a biological sample to quantify the proteins in the biological sample.
  • proteins may be bound to the microarray and contacted with a biological sample to quantify protein-protein or protein-ligand interactions.
  • protein microarrays may also be used in diagnostics in which multiple immunoassays may be conducted in parallel such that levels of proteins in different samples may be quantified and compared for applications in the treatment or diagnosis of disease.
  • a capture array antibodies are bound to the microarray and exposed to a biological sample. Proteins and ligands that bind to the antibody array may be detected by direct labelling of the bound proteins. If a higher sensitivity or specificity is desired, a sandwich technique may be employed in which pairs of antibodies are directed to the same protein ligand. This technique is particularly useful if the amount of protein to be detected is low or if there are modifications to the protein. In addition, the use of sandwich assays minimizes the risk of cross-reactivity in highly multiplexed assays by providing dual level target recognition, i.e. two levels of specificity for each locus in the array. Alternatively, the bound proteins may be detected via label-free detection methods such as including mass spectrometry, surface plasmon resonance and atomic force microscopy. This technique is useful if modification or alteration of the protein is to be avoided.
  • label-free detection methods such as including mass spectrometry, surface plasmon resonance and atomic force microscopy. This technique is useful if modification or alteration of
  • Large-scale functional chips containing large numbers of immobilized purified proteins may be used to assay a wide range of biochemical functions, such as protein interactions with other proteins, drug-target interactions, enzyme-substrates, etc.
  • proteins may be purified from an expression library, for example, and the protein array can be used to screen libraries to select specific binding partners, including antibodies, synthetic scaffolds, peptides and aptamers. In this way, 'library against library' screening can be carried out, such as screening of drug candidates in combinatorial chemical libraries against an array of protein targets identified from genome projects.
  • Protein microarray technology permits analysis of the proteins themselves rather than inferring protein function, interactions and characteristics through mRNA expression. In many cases, mRNA expression does not correlate accurately with protein abundance. Furthermore, mRNA expression analysis does not provide sufficient information on protein-protein interaction or post-translational modifications. Thus, direct analysis of proteins via protein microarrays provides an advantage by providing more accurate information of proteins and protein-protein interactions that may not be readily available through measurment of mRNA expression.
  • DNA microarray techniques permit profiling of gene expression at the mRNA level as a function of the cellular state. This can lead to the identification of genes or clusters of genes whose up- or down-regulation is associated to a particular state of the cell and to the identification of therapeutically relevant targets.
  • DNA fragments representing specific portions of all genes belonging to a given organism are chemically bound to the surfaces of solid supports (chips) at high densities and in an ordered manner.
  • chips solid supports
  • RNA is prepared, labelled with fluorescent dyes and finally hybridised to the DNA fragments fixed to the surface of the chip.
  • fluorescence signals emitted by each spot upon excitation with a laser beam is carefully quantified to define the transcription activity of all the arrayed genes.
  • CPn DNA microarrays have been developed to look at the transcriptional events which occur when a given CPn pathogen gets into contact with the host cells, both in in vivo and in vitro settings.
  • DNA chips carrying the entire genome of a particular bacterium, such as the CPn bacterium can be prepared in a very short period of time so that whole genome expression analysis can be determined.
  • a genomic DNA (open reading frame probes) microarray approach for gene expression in CPn bacteria was adopted.
  • an array was prepared for the analysis of the CPn life cycle on the basis of the published annotation of the complete genome.
  • the Chlamydia DNA chips carry about 1000 PCR-derived DNA fragments, which have an average size of 400-700bp and correspond to internal portions of all CPn annotated genes.
  • Table 3(i)-(xi) shows transcriptional activity for expressed genes for CPn EB selected by microarray.
  • the data in Tables 3(i)-(iv) shows different rnRNAs in order of abundance from cells in their infectious "spore-like" (EB) form.
  • Data in Tables 3(v)- (xi) correlates and summarizes mRNA expression levels of genes for CPn. The cells were used at the end of their cycle where gene expression is likely to be at its highest. As values less than approximately 10000 is likely to be background, the top set of proteins (approx top 30) with more intense signals are likely to be the most interesting proteins.
  • Table 3(v)-(xi) shows the transcriptional activity for expressed genes for CPn EB selected by microarray.
  • the Table shows different mRNA in order of abundance from cells in their infectious "spore-like" (EB) form. The cells were used at the end of the cycle where gene expression is likely to be at its highest.
  • Chlamdydia late gene products have been described more frequently than early gene products. This is primarily because of the presence of late gene products in EBs but not RBs and that it is easier to study EBs rather than RBs.
  • Late gene functions appear to be predominantly those associated with the terminal differentiation of RBs back to EBs (Shaw et al., Mol Microbiology 37(4), 2000, 913-925). Late gene products appear to function in the termination of bacterial cell division and constitute structural components and remodelling activities involved in the formation of the cross-linked outer membrane complex that functions in the attachment and invasion of new host cells.
  • an important aspect of the secondary differentiation process (RB to infectious EB) is the expression of genes that encode proteins that form the highly disulfide cross-linked bacterial outer membrane (OM) complex.
  • Cpn 0384 whose CT equivalent is CT046 (hctB) has been shown to be associated with differentiation from RB to EB (see Belland et al, PNAS (USA) 100(14), 2003, 8478-83). We also found Cpn0384 to have relatively high levels of transcriptional activity (again see top of Table 3(v)-(xi)). Other Cpn antigens thought to be involved in the Type III secretion system were found to have moderate expression levels in terms of transcriptional activity.
  • Chlamydial Pmps The function of Chlamydial Pmps remains unknown, although based on sequence prediction and experimental testing, these Pmps are regarded as surface proteins and thus, likely to be critical for Chlamydial virulence. Like the Inclusion (Inc) Membrane proteins, the Pmp proteins are regarded, at present, as unique to the Chlamydiae family (see Rockey et al (2000) Infection and Immunity 69(10) 5473-5479). The findings disclosed here and by others, such as Grimwood et al, demonstrates that the Chlamydia organism appears to expend a considerable metabolic cost in Pmp transcription, such as Pmp 19 transcription, despite the potential lack of production of a functional Pmp proteins, such as the Pmp 19 protein.
  • T cell epitope prediction was carried out on the genomic sequence of C. pneumoniae CWL029 strain (Accession numbers NC 000922 or AE001363. using the BIMAS algorithm [24]. Synthetic peptides (purity > 80%o) were synthesized by Primm Sri (Milan, Italy), suspended in 100% DMSO and kept at -20° C before use.
  • the T cell lymphoma murine cell line RMA-S stably transfected with HLA-A2 was kindly provided by Dr. Barnaba, Universita degli Studi “La Sapienza", Rome, Italy, and cultured at 37° C in RPMI-1640 (GIBCO) supplemented with heat inactivated 10%) FCS, 100 RJ/ml penicillin/streptomycin, 2 mM Lglutamine (GIBCO) and 5x10-5 M 2-ME (Sigma).
  • H2-b HLA-A2 transgenic mice [35] were housed in a pathogen-free environment and screened for HLA-A2 expression by FCM carried out on total blood samples using the BB7.2 anti-A2 mAb [48]. Only mice with percentages of A2 expressing cells higher than 70-80 %> were used for DNA immunization and C. pneumoniae infection experiments. Animals which showed no HLA-A2 expression were mated in order to obtain an HLA-A2 non transgenic population, to be used as a control in the experiments. Epitope stabilization assay
  • RMA-S/A2 cells (3-5 x 10 5 /well) were seeded in serum-free RPMI medium, supplemented with human ⁇ 2 microglobulin (3 ⁇ g/ml, Sigma), without or with the test peptide (lO ⁇ M). Following overnight incubation at 26°C in humidified 5% C0 2 atmosphere, cells were shifted to 37° C for 2 h before determining the HLA-A2 expression level at the cell surface using the BB7.2 anti-A2 mAb and a PE-conjugated anti-mouse IgG (Jackson ImmunoResearch). Fluorescence intensity on living cells, which did not inco ⁇ orate propidium iodide, was analyzed by FCM. As controls, corresponding samples without peptide and samples with peptide but treated only with the anti-mouse secondary antibody, were used.
  • mice were intranasally infected twice with a month interval, using 5xl0 5
  • C. pneumoniae FB/96 EBs [4] diluted in 50 ⁇ l of PBS.
  • C. pneumoniae antigen coding genes were amplified by PCR using FB/96 genomic DNA, cloned into plasmid pcmvKaSF2120 [49] and verified by DNA sequence analysis.
  • Splenocytes from DNA immunized mice were prepared one week after the third immunization using Cell Strainer (Falcon) filters. Following red blood cells lysis, CD8 + T cells from spleen cells suspensions were enriched by positive selection using magnetic activated cell sorting (MACS-Miltenyi Biotec) with CD8a (Ly-2) microbeads. CD8 + T cells purity was higher than 90%>, as determined by FMC. Multiscreen 96-well nitrocellulose plates (Millipore) were coated with 5 ⁇ g/ml of the anti-mouse IFN- ⁇ antibody (R4-6A2, PharMingen) in 100 ⁇ l of carbonate buffer, pH 9.2.
  • CD8 + 5xl0 4
  • spleen cells from non immunized HLA-A2 transgenic mice as a source of antigen-presenting cells (2xl0 5 /well), 10 ⁇ g/ml of peptide and lOU/ml of human r-IL-2 (Chiron Co ⁇ oration).
  • Splenocytes from infected mice were isolated one week after the second infection with C. pneumoniae Ebs.
  • 2xl0 6 splenocytes were seeded in the presence of the test peptide (lO ⁇ g/ml) and anti-mouse CD28 antibody (1 ⁇ g/ml, PharMingen) as co-stimulus.
  • the test peptide lO ⁇ g/ml
  • anti-mouse CD28 antibody (1 ⁇ g/ml, PharMingen
  • Brefeldin A 10 ⁇ g/ml, Sigma
  • mice For analysis of IFN- ⁇ production by short term T cell lines, 5-10xl0 6 splenocytes from infected mice were cultured for 6 days in the presence of the test peptide (20 ⁇ g/ml), with rIL-2 (10 ⁇ g/ml) being added after the first two days. At the end of the incubation period, cells were washed twice in RPMI, pulsed again for 6 h in the presence of the test peptide (lO ⁇ g/ml), lxlO 5 freshly prepared CD8 depleted antigen presenting cells from HLA-A2 transgenic mice (irradiated at 3000 rad) and anti-mouse CD28 antibody (1 ⁇ g/ml, PharMingen) as co-stimulus. After a two h incubation at 37° C, 5 % C0 2 , Brefeldin A (10 ⁇ g/ml, Sigma) was added, the incubation was extended for 4 additional hours and IFN- ⁇ production was analyzed by FCM.
  • Chlamydial proteins have been reported to induce autoimmune responses [25-28]
  • the predicted binding score of 157.22, obtained for the well characterized HIV-1 pl7 gag epitope 77 SLYNTVATL 85 [29] was taken as an arbitrary cut-off for peptide selection.
  • the capacity of the selected peptides to bind to HLA-A2 was assessed using an in vitro MHC class I stabilization assay, carried out with the murine transporter associated with antigen processing (TAP)-deficient cell line RMA-S/A2, stably transfected with the human class I A2 gene.
  • MHC class I molecules cultured at 37° C, are unstably expressed on the cell surface of TAP-deficient cells [30-32]. Culturing the cells at 37° C in the presence of binding peptides, results in formation of a more stable MHC/peptide complex which can be monitored by flow cytometric analysis.
  • RMA-S/A2 cells were therefore cultured overnight at 26° C in the presence of the test peptides, shifted to 37° C for 2 hours and the surface level of stabilized A2 molecules was quantified by direct staining with an anti-HLA-A2 specific mAb.
  • Two known HLA-A2 restricted CTL epitopes were used as positive controls for binding to A2, the HIN-1 pl7 gag peptide [29] and the influenza matrix Ml protein peptide FluMP58 [33], while the Hepatitis B virus envelope antigen peptide HbenvAgl25 (HepB) was used as a negative control [34].
  • the binding results obtained are shown in Table 4 and allowed the identification of 15 peptides with a net mean fluorescence intensity (Net MFI) higher than 92.3, corresponding to the value obtained with the HIV-1 pl7 gag positive control peptide, 8 peptides with a Net MFI intermediate between the values 92.3 and 63.1, obtained with the two positive control peptides, and 12 peptides with an Net MFI ranging between 29.6 and 63.
  • Net MFI net mean fluorescence intensity
  • HLA-A2 binders are recognized by CD8 + T cells from DNA-immunized transgenic mice
  • the in vitro assay with RMA-S/A2 cells allowed the definition of a set of peptides which were able to bind and stabilize the HLA-A2 molecules on the cell surface.
  • peptide recognition by CD8 + T cells was studied under conditions in which the related complete antigen was intracellularly expressed and presented in vivo.
  • the ORF sequences were selected among those containing either one or more epitopes positive in the in vitro assay or a combination of positive and negative epitopes.
  • the ORF sequence corresponding to the outer membrane protein A (OMPA, CPn 0695) was included in this analysis, since human MHC-I-restricted epitopes have already been reported for this protein in C. trachomatis [18;36].
  • One coding sequence, related to gene CPn 0131 was chosen, which included four epitopes, all negative in the in vitro stabilization assay.
  • transgenic mice were sacrificed, spleen CD8 + T cells were isolated, stimulated for 20 hour with the corresponding peptide and ex vivo IFN- ⁇ production was assessed using an enzyme- linked immunospot (ELISpot) assay,
  • Example 8 To test the capacity of peptides to amplify specific CD8 + T cell populations in vitro, some of these plasmids were used to repeat the DNA immunization experiment and to determine by flow cytometry the intracellular IFN- ⁇ production by CD8 + T cells, both ex vivo and after a 6 day stimulation in the presence of the relevant peptides. In the attempt to establish a direct correlation between IFN- ⁇ production by CD8 + T cells and HLA-A2 specific restriction, the experiment was carried out with both transgenic and non transgenic syngenic mice.
  • the plasmids used contained genes CPn 0695, CPn 0811 and CPn 0823, including peptides CH-13, CH-6 and CH-7 respectively, which were highly positive in the in vitro binding and in the ELISpot assays and gene CPn 0323, including six different peptides, all of them with ELISpot values slightly higher than background
  • CD8 + T cells of transgenic mice infected with C. pneumoniae recognize HLA-A2 binders in vivo It has been recently shown that infection of mice with C. pneumoniae elicits a pathogen-specific murine class I-restricted immune response [22]. Therefore, we asked whether any of the A2 in vitro binders could be recognized by specific CD8 T cells that are clonally selected during the immune response raised against the corresponding native antigen in C. pneumoniae infected cells.
  • HLA-A2 transgenic mice were intranasally infected with a non lethal dose of C. pneumoniae EBs and challenged with an equal dose of bacteria one month later, before being sacrificed to obtain splenocytes that were used to measure IFN- ⁇ production by CD8 + T cells. Since no appreciable IFN- ⁇ -production could be observed if splenocytes from infected mice were tested directly ex vivo (data not shown), spleen cells were cultured with each individual peptide or with the HepB irrelevant peptide for 6 days. The resulting short-term TCLs were then pulsed again for 6 hours with the same peptides and intracellular IFN- ⁇ production by CD8 + T cells was assessed.
  • Fig. 5A The results obtained with 40 tested peptides are shown in Fig. 5A.
  • Sixteen peptides (CH-2, CH-7, CH-8, CH-10, CH-13, CH-15, CH-20, CH-21, CH-28, CH-35, CH-37, CH-45, CH-46, CH-47, CH-50 and CH-55) elicited the strongest CD8 + responses (1 to 7.1 % of IFN- ⁇ -producing CD8 + T cells), while 19 peptides elicited low but consistent responses (percentages of CD8 + /IFN- ⁇ + T cells between 0.3 and 0.9). Five peptides did not induce percentages of IFN- ⁇ -producing CD8 + T cells significantly higher than those observed in response to the HepB control peptide.
  • pneumoniae antigens can indeed reach the cytosol of infected cells and enter the MHC-I presentation pathway, i.e. during remodeling that occurs during Chlamydia replication or following autolysis of developing bacterial particles [22].
  • Kuon et al. [42] recently reported the identification of 11 C. trachomatis-de ⁇ ved HLA-B27-restricted peptides, capable of stimulating CD8 + T cells obtained from patients with Chlamydia-m ⁇ uced reactive arthritis. Importantly, 8 of them overlapped those selected by analyzing splenocytes of HLA-B27 transgenic mice infected with C. trachomatis, indicating that antigen processing can be closely reproduced using the murine animal model, although differences between murine and human antigen processing and T cell repertoires have been hypothesized [43].
  • CH-8 which is the most reactive peptide in the assay with the infected mice, does not seem to be recognized by a specific T cell population when the corresponding antigen is expressed by DNA immunization (Tables 5 and 6). This may be due to different factors, i.e. low in vivo expression level of the injected DNA or altered protein conformation.
  • pneumoniae infected mammalian cells and, possibly, to correlate the identified T cell epitopes with CD8 + T cell populations naturally induced in C. pneumoniae infected patients.
  • results obtained with DNA-mediated expression of distinct antigens can represent an initial step towards the definition of a significant set of C.
  • Example 9 Immunizations with Combinations of the First Antigen Group
  • the five antigens of the first antigen group (OmpH-like protein, pmp 10, pmp2, Enolase, OmpH-like, CPn0042 and CPn00795 were prepared as described in the Materials and Methods Section above for Examples 1-4.
  • the antigens are expressed and purified.
  • Compositions of antigen combinations are then prepared comprising five antigens per composition (and containing 15 ⁇ g of each antigen per composition).
  • CD1 mice are divided into seven groups (5-6 mice per group for groups 1 through 4; 3 to 4 mice for groups 5, 6 and 7), and immunized as follows:
  • mice are immunized at two week intervals. Two weeks after the last immunization, all mice are challenged by intravaginal infection with Chlamydia pneumoniae serovars. Experiment 9 was repeated with another group of CPn antigens. These were: CPn0385 (PepA), CPn0324 (LcrE), CPn0503 (DnaK), CPn0525 (Hypothetical) and CPn0482 (ArtJ). These antigens are combined and administered with and without alum and CpG as described in Experiment 9. Summary
  • CPn proteins with desirable immunological and or biological properties. Specifically, at least twelve CPn proteins have been identified which are capable of inducing the production of antibodies, which can neutralise, in a dose-dependent manner, the infectivity of C. pneumoniae in in vitro cell cultures. The induction of neutralising antibodies is important because it prevents infectious EBs from invading human tissues. Furthermore, at least six of these CPn proteins were also capable of attenuating Chlamydial (C. pneumoniae) infection in a in vivo hamster model. In addition, some of these CPn proteins were also capable of inducing not only adequate T-cell responses but also high serum levels of neutralising antibodies.
  • C. pneumoniae Chlamydial
  • this paper describes a group of recombinant antigens which can induce antibodies inhibiting the infectivity of C. pneumoniae in vitro and have protective effects in vivo.
  • alpha-Enolase of Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface.
  • Chlamydia pneumoniae major outer membrane protein is a surface-exposed antigen that elicits antibodies primarily directed against conformation-dependent determinants.
  • Rappuoli,R. Reverse vaccinology. Current Opinion in Microbiology 2000. 3: 445- 450. 3. Rappuoli,R., Reverse vaccinology, a genome-based approach to vaccine development. Vaccine 2001. 19: 2688-2691.

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Abstract

La présente invention a trait à des polypeptides destinés à être utilisés comme un antigène transporteur autonome. L'invention a également trait à des procédés et des utilisations d'un polypeptide pour une fonction de transporteur autonome dans la préparation d'un médicament pour la prévention ou le traitement d'une infection de Chlamydia pneumoniae ou pour la préparation d'un dosage pour le diagnostic d'une infection de Chlamydia pneumoniae chez un sujet. L'invention a trait en outre à un procédé pour provoquer une réponse immunitaire chez un sujet par l'administration au sujet d'un polypeptide destiné à être utilisé comme un antigène transporteur autonome.
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RU2006134631A (ru) 2008-04-10
WO2005084306A2 (fr) 2005-09-15
BRPI0508365A (pt) 2007-07-24

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