EP1129202A2 - Chlamydia antigenes and corresponding dna fragments and uses thereof - Google Patents

Chlamydia antigenes and corresponding dna fragments and uses thereof

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Publication number
EP1129202A2
EP1129202A2 EP99955602A EP99955602A EP1129202A2 EP 1129202 A2 EP1129202 A2 EP 1129202A2 EP 99955602 A EP99955602 A EP 99955602A EP 99955602 A EP99955602 A EP 99955602A EP 1129202 A2 EP1129202 A2 EP 1129202A2
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EP
European Patent Office
Prior art keywords
polypeptide
seq
nucleic acid
nos
chlamydia
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.)
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Application number
EP99955602A
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German (de)
English (en)
French (fr)
Inventor
Andrew D. Murdin
Raymond P. Oomen
Joe Wang
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Sanofi Pasteur Ltd
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Aventis Pasteur Ltd
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Publication of EP1129202A2 publication Critical patent/EP1129202A2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/295—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Chlamydiales (O)
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to Chlamydia antigens and corresponding DNA molecules, which can be used to prevent and treat Chlamydia infection in mammals, such as humans.
  • Chlamydiae are prokaryotes. They exhibit morphologic and structural similarities to gram-negative bacteria including a trilaminar outer membrane, which contains lipopolysaccharide and several membrane proteins that are structurally and functionally analogous to proteins found in E coli . They are obligate intra-cellular parasites with a unique biphasic life cycle consisting of a metabolically inactive but infectious extracellular stage and a replicating but non-infectious intracellular stage. The replicative stage of the life-cycle takes place within a membrane-bound inclusion which sequesters the bacteria away from the cytoplasm of the infected host cell. C.
  • C. pneumoniae is a common human pathogen, originally described as the TWAR strain of Chlamydia psi ttaci but subsequently recognised to be a new species.
  • C. pneumoniae is antigenically, genetically and morphologically distinct from other chlamydia species (C. trachoma tis , C. pecorum and C. psi ttaci ) . It shows 10% or less DNA sequence homology with either of C. trachoma tis or C. psi ttaci .
  • C. pneumoniae is a common cause of community acquired pneumonia, only less frequent than Streptococcus pneumoniae and Mycoplasma pneumoniae (Grayston et al . (1995) Journal of
  • C. pneumoniae infection usually presents as an acute respiratory disease (i.e., cough, sore throat, hoarseness, and fever; abnormal chest sounds on auscultation) .
  • cough i.e., cough, sore throat, hoarseness, and fever; abnormal chest sounds on auscultation
  • the cough persists for 2 to 6 weeks, and recovery is slow.
  • upper respiratory tract infection is followed by bronchitis or pneumonia.
  • subsequent co-infection with pneumococcus has been noted in about half of these pneumonia patients, particularly in the infirm and the elderly.
  • C. pneumoniae infection is also linked to diseases other than respiratory infections.
  • the reservoir for the organism is presumably people.
  • C. pneumoniae appears to spread slowly through a population (case- to-case interval averaging 30 days) because infected persons are inefficient transmitters of the organism. Susceptibility to C. pneumoniae is universal. Reinfections occur during adulthood, following the primary infection as a child. C. pneumoniae appears to be an endemic disease throughout the world, noteworthy for superimposed intervals of increased incidence (epidemics) that persist for 2 to 3 years. C.
  • trachoma tis infection does not confer cross-immunity to C. pneumoniae. Infections are easily treated with oral antibiotics, tetracycline or erythromycin (2 g/d, for at least 10 to 14 d) . A recently developed drug, azithromycin, is highly effective as a single-dose therapy against chlamydial infections. In most instances, C. pneumoniae infection is often mild and without complications, and up to 90% of infections are subacute or unrecognized.
  • C. pneumoniae infection usually happens between the ages of 5 and 20 y. In the USA, for example, there are estimated to be 30,000 cases of childhood pneumonia each year caused by C. pneumoniae . Infections may cluster among groups of children or young adults ⁇ e . g. , school pupils or military conscripts) .
  • C. pneumoniae causes 10 to 25% of community-acquired lower respiratory tract infections (as reported from Sweden, Italy, Finland, and the USA) .
  • C. pneumonia infection may account for 50 to 60% of the cases of pneumonia.
  • Reinfection during adulthood is common; the clinical presentation tends to be milder.
  • Based on population seroprevalence studies there tends to be increased exposure with age, which is particularly evident among men.
  • Some investigators have speculated that a persistent, asymptomatic C. pneumoniae infection state is common. In adults of middle age or older, C. pneumoniae infection may progress to chronic bronchitis and sinusitis.
  • a study in the USA revealed that the incidence of pneumonia caused by C. pneumoniae in persons younger than 60 years is 1 case per 1,000 persons per year; but in the elderly, the disease incidence rose three-fold.
  • C. pneumoniae infection rarely leads to hospitalization, except in patients with an underlying illness .
  • mice which have recovered from a lung infection with C. trachoma tis are protected from infertility induced by a subsequent vaginal challenge (Pal et al . (1996) Infection and Immunity.64 : 5341) .
  • sheep immunized with inactivated C. psi ttaci were protected from subsequent chlamydial-induced abortions and stillbirths (Jones et al . (1995) Vaccine 13:715).
  • the gene encoding a 76kDa antigen has been cloned from a single strain of C. pneumoniae. It has no significant similarity with other known chlamydial genes (Marrie (1993) Clinical Infectious Diseases. 18:501) .
  • the present invention provides purified and isolated polynucleotide molecules that encode Chlamydia polypeptides which can be used in methods to prevent, treat, and diagnose Chlamydia infection.
  • the polynucleotide molecules are selected from DNA that encode polypeptides CPN100397 (SEQ ID Nos: 1 and 2), CPN100421 (SEQ ID Nos: 3 and 4), CPN100422 (SEQ ID Nos: 5 and 6), CPN100424 (SEQ ID Nos: 7 and 8), CPN100426 (SEQ ID Nos: 9 and 10), CPN100508 (SEQ ID Nos: 11 and 12), CPN100515 (SEQ ID Nos: 13 and 14), CPN100538 (SEQ ID Nos: 15 and 16), CPN100557 (SEQ ID Nos: 17 and 18), CPN100622 (SEQ ID Nos: 19 and 20), CPN100626 (SEQ ID Nos
  • Another form of the invention provides polypeptides corresponding to the isolated DNA molecules.
  • the amino acid sequences of the corresponding encoded polypeptides are shown for CPN100397 as SEQ ID Nos: 27 and 28, CPN100421 as SEQ ID No: 29, CPN100422 as SEQ ID No: 30, CPN100424 as SEQ ID No: 31, CPN100426 as SEQ ID No: 32, CPN100508 as SEQ ID Nos: 33 and 34, CPN100515 as SEQ ID Nos: 35 and 36, CPN100538 as SEQ ID No: 37, CPN100557 as SEQ ID Nos: 38 and 39, CPN100622 as SEQ ID Nos: 40 and 41, CPN100626 as SEQ ID No: 42, CPN100628 as SEQ ID No: 43 and CPN100630 as SEQ ID Nos: 44 and 45.
  • the invention having provided the polynucleotide sequences encoding Chlamydia polypeptides, also provides polynucleotides encoding fragments derived from such peptides. Moreover, the invention is understood to provide mutants and derivatives of such polypeptides and fragments derived therefrom, which result from the addition, deletion, or substitution of non-essential amino acids as described herein. Those skilled in the art would also readily understand that the invention, having provided the polynucleotide sequences encoding Chlamydia polypeptides, further provides monospecific antibodies that specifically bind to such polypeptides
  • the present invention has wide application and includes expression cassettes, vectors, and cells transformed or transfected with the polynucleotides of the invention. Accordingly, the present invention further provides (i) a method for producing a polypeptide of the invention in a recombinant host system and related expression cassettes, vectors, and transformed or transfected cells; (ii) a vaccine, or a live vaccine vector such as a pox virus, Salmonella typhimurium, or Vijbrio cholerae vector, containing a polynucleotide of the invention, such vaccines and vaccine vectors being useful for, e . g.
  • RNA or DNA molecule of the invention either in a naked form or formulated with a delivery vehicle, a polypeptide or combination of polypeptides, or a monospecific antibody of the invention, and related pharmaceutical compositions
  • a method for diagnosing the presence of Chlamydia in a biological sample which can involve the use of a DNA or RNA molecule, a monospecific antibody, or a polypeptide of the invention
  • a method for purifying a polypeptide of the invention by antibody-based affinity chromatography can involve the use of a DNA or RNA molecule, a monospecific antibody, or a polypeptide of the invention.
  • Figure 1 shows the nucleotide sequence of the CPN100397 (SEQ ID No: 1 - entire sequence and SEQ ID No: 2 - coding sequence) and the deduced amino acid sequence of the CPN100397 protein from Chlamydia pneumoniae (SEQ ID No: 27 and 28) .
  • Figure 2 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100397 gene.
  • Figure 3 shows the nucleotide sequence of the CPN100421 (SEQ ID No: 3 - entire sequence and SEQ ID No: 4 - coding sequence) and the deduced amino acid sequence of the CPN100421 protein from Chlamydia pneumoniae (SEQ ID No: 29) .
  • Figure 4 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100421 gene.
  • Figure 5 shows the nucleotide sequence of the CPN100422 (SEQ ID No: 5 - entire sequence and SEQ ID No: 6 - coding sequence) and the deduced amino acid sequence of the CPN100422 protein from Chlamydia pneumoniae (SEQ ID No: 30) .
  • Figure 6 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100422 gene.
  • Figure 7 shows the nucleotide sequence of the CPN100424 (SEQ ID No: 7 - entire sequence and SEQ ID No: 8 - coding sequence) and the deduced amino acid sequence of the CPN100424 protein from Chlamydia pneumoniae (SEQ ID No: 31).
  • Figure 8 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100424 gene.
  • Figure 9 shows the nucleotide sequence of the CPN100426 (SEQ ID No: 9 - entire sequence and SEQ ID No: 10 - coding sequence) and the deduced amino acid sequence of the CPN100426 protein from Chlamydia pneumoniae (SEQ ID No: 32) .
  • Figure 10 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100426 gene.
  • Figure 11 shows the nucleotide sequence of the CPN100508 (SEQ ID No: 11 - entire sequence and SEQ ID No: 12 - coding sequence) and the deduced amino acid sequence of the CPN100508protein from Chlamydia pneumoniae (SEQ ID No: 33 - full length sequence and SEQ ID No: 34 - processed sequence) .
  • Figure 12 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100508 gene.
  • Figure 13 shows the nucleotide sequence of the CPN100515 (SEQ ID No: 13 - entire sequence and SEQ ID No: 14 - coding sequence) and the deduced amino acid sequence of the CPN100515 protein from Chlamydia pneumoniae (SEQ ID No: 35 - full length sequence and SEQ ID No: 36 - processed sequence) .
  • Figure 14 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100515 gene.
  • Figure 15 shows the nucleotide sequence of the CPN100538 (SEQ ID No: 15 - entire sequence and SEQ ID No: 16 - coding sequence) and the deduced amino acid sequence of the CPN100538 protein from Chlamydia pneumoniae (SEQ ID No: 37) .
  • Figure 16 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100538 gene.
  • Figure 17 shows the nucleotide sequence of the CPN100557 (SEQ ID No: 17 - entire sequence and SEQ ID No: 18 - coding sequence) and the deduced amino acid sequence of the CPN100557 protein from Chlamydia pneumoniae (SEQ ID No: 38 - full length sequence and SEQ ID No: 39 - processed sequence) .
  • Figure 18 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100557 gene.
  • Figure 19 shows the nucleotide sequence of the CPN100622 (SEQ ID No: 19 - entire sequence and SEQ ID No: 20 - coding sequence) and the deduced amino acid sequence of the CPN100622 protein from Chlamydia pneumoniae (SEQ ID No: 40 - full length sequence and SEQ ID No: 41 - processed sequence) .
  • Figure 20 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100622 gene.
  • Figure 21 shows the nucleotide sequence of the CPN100626 (SEQ ID No: 21 - entire sequence and SEQ ID No: 22 - coding sequence) and the deduced amino acid sequence of the CPN100626 protein from Chlamydia pneumoniae (SEQ ID No: 42) .
  • Figure 22 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100626 gene.
  • Figure 23 shows the nucleotide sequence of the CPN100628 (SEQ ID No: 23 - entire sequence and SEQ ID No: 24 - coding sequence) and the deduced amino acid sequence of the CPN100628 protein from Chlamydia pneumoniae (SEQ ID No: 43) .
  • Figure 24 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100628 gene.
  • Figure 25 shows the nucleotide sequence of the CPN100630 (SEQ ID No: 25 - entire sequence and SEQ ID No: 26 - coding sequence) and the deduced amino acid sequence of the CPN100630 protein from Chlamydia pneumoniae (SEQ ID No: 44 - full length sequence and SEQ ID No: 45 - processed sequence) .
  • Figure 26 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100630 gene.
  • Figures 27 through 39 show an identification of T and B cell epitopes from the amino acid sequences shown in the foregoing figures.
  • ORFs Open reading frames encoding chlamydial polypeptides have been identified from the C. pneumoniae genome. These polypeptides include polypeptides found permanently in the bacterial membrane structure, polypeptides present in the external vicinity of the bacterial membrane, polypeptides found permanently in the inclusion membrane structure, polypeptides present in the external vicinity of the inclusion membrane, and polypeptides released into the cytoplasm of the infected cell. These polypeptides can be used to prevent and treat Chlamydia infection.
  • isolated polynucleotides which encode the precursor and mature forms of Chlamydia polypeptides, whose amino acid sequences are selected from the group consisting of: SEQ ID Nos: 27 to 45.
  • isolated polynucleotide is defined as a polynucleotide removed from the environment in which it naturally occurs.
  • a naturally-occurring DNA molecule present in the genome of a living bacteria or as part of a gene bank is not isolated, but the same molecule separated from the remaining part of the bacterial genome, as a result of, e.g., a cloning event (amplification), is isolated.
  • an isolated DNA molecule is free from DNA regions (e.g., coding regions) with which it is immediately contiguous at the 5' or 3' end, in the naturally occurring genome.
  • Such isolated polynucleotides may be part of a vector or a composition and still be defined as isolated in that such a vector or composition is not part of the natural environment of such polynucleotide .
  • the polynucleotide of the invention is either RNA or DNA
  • DNA cDNA, genomic DNA, or synthetic DNA
  • the DNA is either double-stranded or single-stranded, and, if single-stranded, is either the coding strand or the non-coding (anti-sense) strand.
  • any one of the sequences that encode the polypeptides of the invention as shown in SEQ ID Nos: 1 to 26 is (a) a coding sequence, (b) a ribonucleotide sequence derived from transcription of (a) , or (c) a coding sequence which uses the redundancy or degeneracy of the genetic code to encode the same polypeptides.
  • polypeptide or “protein” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Both terms are used interchangeably in the present application. Consistent with the first aspect of the invention, amino acid sequences are provided which are homologous to any one of SEQ ID Nos: 27 to 45.
  • homologous amino acid sequence is any polypeptide which is encoded, in whole or in part, by a nucleic acid sequence which hybridizes at 25-35°C below critical melting temperature (Tm) , to any portion of the nucleic acid sequences of SEQ ID Nos: 1 to 26.
  • a homologous amino acid sequence is one that differs from an amino acid sequence shown in any one of SEQ ID Nos: 27 to 45 by one or more amino acid substitutions.
  • Such a sequence also encompass serotypic variants (defined below) as well as sequences containing deletions or insertions which retain inherent characteristics of the polypeptide such as immunogenicity.
  • such a sequence is at least 75%, more preferably 80%, and most preferably 90% identical to any one of SEQ ID Nos: 27 to 45.
  • Homologous amino acid sequences include sequences that are identical or substantially identical to SEQ ID Nos: 27 to 45.
  • amino acid sequence substantially identical is meant a sequence that is at least 90%, preferably 95%, more preferably 97%, and most preferably 99% identical to an amino acid sequence of reference and that preferably differs from the sequence of reference by a majority of conservative amino acid substitutions.
  • amino acids having uncharged polar side chains such as asparagine, glutamine, serine, threonine, and tyrosine
  • amino acids having basic side chains such as lysine, arginine, and histidine
  • amino acids having acidic side chains such as aspartic acid and glutamic acid
  • amino acids having nonpolar side chains such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine .
  • sequence analysis software such as Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705. Amino acid sequences are aligned to maximize identity. Gaps may be artificially introduced into the sequence to attain proper alignment. Once the optimal alignment has been set up, the degree of homology is established by recording all of the positions in which the amino acids of both sequences are identical, relative to the total number of positions.
  • homologous polynucleotide sequences are defined in a similar way.
  • a homologous sequence is one that is at least 45%, more preferably 60%, and most preferably 85% identical to any one of coding sequences SEQ ID Nos: 1 to 26.
  • polypeptides having a sequence homologous to any one of SEQ ID Nos: 27 to 45 include naturally-occurring allelic variants, as well as mutants or any other non-naturally occurring variants that retain the inherent characteristics of the polypeptide of SEQ ID Nos: 27 to 45.
  • an allelic variant is an alternate form of a polypeptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that does not alter the biological function of the polypeptide.
  • biological function is meant the function of the polypeptide in the cells in which it naturally occurs, even if the function is not necessary for the growth or survival of the cells.
  • the biological function of a porin is to allow the entry into cells of compounds present in the extracellular medium.
  • Biological function is distinct from antigenic property.
  • a polypeptide can have more than one biological function.
  • Allelic variants are very common in nature.
  • a bacterial species such as C. pneumoniae
  • C. pneumoniae is usually represented by a variety of strains that differ from each other by minor allelic variations.
  • a polypeptide that fulfills the same biological function in different strains can have an amino acid sequence (and polynucleotide sequence) that are not identical in each of the strains.
  • an immune response directed generally against many allelic variants has been demonstrated.
  • cross-strain antibody binding plus neutralization of infectivity occurs despite amino acid sequence variation of MOMP from strain to strain, indicating that the MOMP, when used as an immunogen, is tolerant of amino acid variations.
  • Polynucleotides encoding homologous polypeptides or allelic variants are retrieved by polymerase chain reaction (PCR) amplification of genomic bacterial DNA extracted by conventional methods. This involves the use of synthetic oligonucleotide primers matching upstream and downstream of the 5' and 3' ends of the encoding domain. Suitable primers are designed according to the nucleotide sequence information provided in SEQ ID Nos : 1 to 26. The procedure is as follows: a primer is selected which consists of 10 to 40, preferably 15 to 25 nucleotides.
  • primers containing C and G nucleotides in a proportion sufficient to ensure efficient hybridization; i.e., an amount of C and G nucleotides of at least 40%, preferably 50% of the total nucleotide content.
  • An alternative method for retrieving polynucleotides encoding homologous polypeptides or allelic variants is by hybridization screening of a DNA or RNA library. Hybridization procedures are well-known in the art and are described in Ausubel et al . , (Ref 41), Silhavy et al . (Ref 43), and Davis et al . (ref 44) .
  • stringent conditions are achieved for both pre-hybridizing and hybridizing incubations (i) within 4-16 hours at 42 °C, in 6 x SSC containing 50% formamide, or (ii) within 4-16 hours at 65°C in an aqueous 6 x SSC solution (1 M NaCl, 0.1 M sodium citrate (pH 7.0)).
  • homologous polypeptides from different species are compared; conserved sequences are identified. The more divergent sequences are the most likely to tolerate sequence changes. Alternatively, sequences are modified such that they become more reactive to T-
  • the screening procedure comprises the steps:
  • Conferring protection is meant that there is a reduction is severity of any of the effects of Chlamydia infection, in comparison with a control animal which was not immunized with the test homolog or fragment.
  • mice are susceptible to intranasal infection with different isolates of C. pneumoniae.
  • Strain AR-39 (Grayston, 1989) was used in Balb/c mice as a challenge infection model to examine the capacity of chlamydia gene products delivered as naked DNA to elicit a protective response against a sublethal C. pneumoniae lung infection.
  • Protective immunity is defined as an accelerated clearance of pulmonary infection.
  • mice Groups of 7 to 9 week old male Balb/c mice (6 to 10 per group) were immunized intramuscularly (i.m.) plus intranasally
  • Lungs were taken from mice at day 9 post-challenge and immediately homogenised in SPG buffer (7.5% sucrose, 5mM glutamate, 12.5mM phosphate pH7.5). The homogenate was stored frozen at -70°C until assay. Dilutions of the homogenate were assayed for the presence of infectious chlamydia by inoculation onto monolayers of susceptible cells. The inoculum was centrifuged onto the cells at 3000rpm for 1 hour, then the cells were incubated for three days at 35°C in the presence of l ⁇ g/ml cycloheximide.
  • polypeptide derivatives are provided that are partial sequences of SEQ ID Nos: 27 to 45, partial sequences of polypeptide sequences homologous to SEQ ID Nos: 27 to 45, polypeptides derived from full-length polypeptides by internal deletion, and fusion proteins. It is an accepted practice in the field of immunology to use fragments and variants of protein immunogens as vaccines, as all that is required to induce an immune response to a protein is a small (e.g., 8 to 10 amino acid) immunogenic region of the protein.
  • polypeptide fragments preferably are at least 12 amino acids in length.
  • polypeptide fragments are at least 20 amino acids, preferably at least 50 amino acids, more preferably at least 75 amino acids, and most preferably at least 100 amino acids in length.
  • Polynucleotides of 30 to 600 nucleotides encoding partial sequences of sequences homologous to SEQ ID Nos: 27 to 45 are retrieved by PCR amplification using the parameters outlined above and using primers matching the sequences upstream and downstream of the 5' and 3' ends of the fragment to be amplified.
  • the template polynucleotide for such amplification is either the full length polynucleotide homologous to one of SEQ ID Nos: 1 to 26, or a polynucleotide contained in a mixture of polynucleotides such as a DNA or RNA library.
  • screening hybridization is carried out under conditions described above and using the formula for calculating Tm.
  • the calculated Tm is corrected by subtracting (600/polynucleotide size in base pairs) and the stringency conditions are defined by a hybridization temperature that is 5 to 10 °C below Tm.
  • Tm 4 x (G+C) + 2 (A+T) .
  • Useful polypeptide derivatives e . g. , polypeptide fragments
  • polypeptide fragments are designed using computer-assisted analysis of amino acid sequences. This identifies probable surface- exposed, antigenic regions (Ref 37).
  • Epitopes which induce a protective T cell-dependent immune response are present throughout the length of the polypeptide. However, some epitopes may be masked by secondary and tertiary structures of the polypeptide. To reveal such masked epitopes large internal deletions are created which remove much of the original protein structure and exposes the masked epitopes. Such internal deletions sometimes effects the additional advantage of removing immunodominant regions of high variability among strains.
  • Polynucleotides encoding polypeptide fragments and polypeptides having large internal deletions are constructed using standard methods (Ref 41) . Such methods include standard PCR, inverse PCR, restriction enzyme treatment of cloned DNA molecules, or the method of Kunkel et al . (Ref 42) . Components for these methods and instructions for their use are readily available from various commercial sources such as Stratagene. Once the deletion mutants have been constructed, they are tested for their ability to prevent or treat Chlamydia infection as described above.
  • a fusion polypeptide is one that contains a polypeptide or a polypeptide derivative of the invention fused at the N- or C-terminal end to any other polypeptide (hereinafter referred to as a peptide tail) .
  • a simple way to obtain such a fusion polypeptide is by translation of an in- frame fusion of the polynucleotide sequences, i.e., a hybrid gene.
  • the hybrid gene encoding the fusion polypeptide is inserted into an expression vector which is used to transform or transfect a host cell.
  • polynucleotide sequence encoding the polypeptide or polypeptide derivative is inserted into an expression vector in which the polynucleotide encoding the peptide tail is already present.
  • vectors and instructions for their use are commercially available, e.g. the pMal-c2 or pMal-p2 system from New England Biolabs, in which the peptide tail is a maltose binding protein, the glutathione-S-transferase system of Pharmacia, or the His-Tag system available from Novagen.
  • a fusion polypeptide is one where the polypeptide or homolog or fragment of the invention is fused to a polypeptide having adjuvant activity, such as subunit B of either cholera toxin or E. coli heat-labile toxin.
  • a polypeptide having adjuvant activity such as subunit B of either cholera toxin or E. coli heat-labile toxin.
  • Another advantageous fusion is one where the polypeptide, homolog or fragment is fused to a strong T-cell epitope or B-cell epitope.
  • Such an epitope may be one known in the art (e.g. the Hepatitis
  • a fusion polypeptide comprising T- or B-cell epitopes from one of SEQ ID Nos: 27 to 45 or its homolog or fragment, wherein the epitopes are derived from multiple variants of said polypeptide or homolog or fragment, each variant differing from another in the location and sequence of its epitope within the polypeptide.
  • Such a fusion is effective in the prevention and treatment of Chlamydia infection since it optimizes the T- and B-cell response to the overall polypeptide, homolog or fragment .
  • the polypeptide of the invention is fused to the N-, or preferably, to the C-terminal end of the polypeptide having adjuvant activity or T- or B-cell epitope.
  • a polypeptide fragment of the invention is inserted internally within the amino acid sequence of the polypeptide having adjuvant activity.
  • the T- or B-cell epitope may also be inserted internally within the amino acid sequence of the polypeptide of the invention.
  • the polynucleotides of the invention also encode hybrid precursor polypeptides containing heterologous signal peptides, which mature into polypeptides of the invention.
  • heterologous signal peptide is meant a signal peptide that is not found in naturally- occurring precursors of polypeptides of the invention.
  • a polynucleotide molecule according to the invention including RNA, DNA, or modifications or combinations thereof, have various applications.
  • a DNA molecule is used, for example, (i) in a process for producing the encoded polypeptide in a recombinant host system, (ii) in the construction of vaccine vectors such as poxviruses, which are further used in methods and compositions for preventing and/or treating Chlamydia infection, (iii) as a vaccine agent (as well as an RNA molecule) , in a naked form or formulated with a delivery vehicle and, (iv) in the construction of attenuated Chlamydia strains that can over-express a polynucleotide of the invention or express it in a non-toxic, mutated form.
  • a second aspect of the invention encompasses (i) an expression cassette containing a DNA molecule of the invention placed under the control of the elements required for expression, in particular under the control of an appropriate promoter; (ii) an expression vector containing an expression cassette of the invention; (iii) a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, as well as (iv) a process for producing a polypeptide or polypeptide derivative encoded by a polynucleotide of the invention, which involves culturing a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, under conditions that allow expression of the DNA molecule of the invention and, recovering the encoded polypeptide or polypeptide derivative from the cell culture.
  • a recombinant expression system is selected from procaryotic and eucaryotic hosts.
  • Eucaryotic hosts include yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris) , mammalian cells (e.g., COS1, NIH3T3, or JEG3 cells), arthropods cells (e.g., Spodoptera frugiperda (SF9) cells), and plant cells.
  • a preferred expression system is a procaryotic host such as E. coli .
  • Bacterial and eucaryotic cells are available from a number of different sources including commercial sources to those skilled in the art, e.g., the American Type Culture Collection (ATCC; Rockville, Maryland) . Commercial sources of cells used for recombinant protein expression also provide instructions for usage of the cells.
  • the choice of the expression system depends on the features desired for the expressed polypeptide. For example, it may be useful to produce a polypeptide of the invention in a particular lipidated form or any other form.
  • the host In selecting a vector, the host must be chosen that is compatible with the vector which is to exist and possibly replicate in it. Considerations are made with respect to the vector copy number, the ability to control the copy number, expression of other proteins such as antibiotic resistance.
  • an expression control sequence a number of variables are considered. Among the important variable are the relative strength of the sequence (e.g. the ability to drive expression under various conditions), the ability to control the sequence's function, compatibility between the polynucleotide to be expressed and the control sequence (e.g. secondary structures are considered to avoid hairpin structures which prevent efficient transcription) .
  • unicellular hosts are selected which are compatible with the selected vector, tolerant of any possible toxic effects of the expressed product, able to secrete the expressed product efficiently if such is desired, to be able to express the product in the desired conformation, to be easily scaled up, and to which ease of purification of the final product.
  • an expression cassette includes a promoter that is functional in the selected host system and can be constitutive or inducible; a ribosome binding site; a start codon (ATG) if necessary; a region encoding a signal peptide, e.g., a lipidation signal peptide; a DNA molecule of the invention; a stop codon; and optionally a 3' terminal region (translation and/or transcription terminator) .
  • the signal peptide encoding region is adjacent to the polynucleotide of the invention and placed in proper reading frame.
  • the signal peptide-encoding region is homologous or heterologous to the DNA molecule encoding the mature polypeptide and is compatible with the secretion apparatus of the host used for expression.
  • the open reading frame constituted by the DNA molecule of the invention, solely or together with the signal peptide, is placed under the control of the promoter so that transcription and translation occur in the host system.
  • Promoters and signal peptide encoding regions are widely known and available to those skilled in the art and include, for example, the promoter of Salmonella typhimurium (and derivatives) that is inducible by arabinose (promoter araB) and is functional in Gram-negative bacteria such as E. coli (as described in U.S. Patent No.
  • the expression cassette is typically part of an expression vector, which is selected for its ability to replicate in the chosen expression system.
  • Expression vectors e.g., plasmids or viral vectors
  • plasmids or viral vectors can be chosen, for example, from those described in Pouwels et al . (Cloning Vectors: A Laboratory Manual 1985, Supp. 1987) .
  • Suitable expression vectors can be purchased from various commercial sources. Methods for transforming/transfecting host cells with expression vectors are well-known in the art and depend on the host system selected as described in Ausubel et al . , (Ref 41).
  • a recombinant polypeptide of the invention (or a polypeptide derivative) is produced and remains in the intracellular compartment, is secreted/excreted in the extracellular medium or in the periplasmic space, or is embedded in the cellular membrane.
  • the polypeptide is recovered in a substantially purified form from the cell extract or from the supernatant after centrifugation of the recombinant cell culture.
  • the recombinant polypeptide is purified by antibody-based affinity purification or by other well-known methods that can be readily adapted by a person skilled in the art, such as fusion of the polynucleotide encoding the polypeptide or its derivative to a small affinity binding domain.
  • Antibodies useful for purifying by immunoaffinity the polypeptides of the invention are obtained as described below.
  • a polynucleotide of the invention can also be useful as a vaccine. There are two major routes, either using a viral or bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid. Therapeutic or prophylactic efficacy of a polynucleotide of the invention is evaluated as described below.
  • a third aspect of the invention provides (i) a vaccine vector such as a poxvirus, containing a DNA molecule of the invention, placed under the control of elements required for expression; (ii) a composition of matter comprising a vaccine vector of the invention, together with a diluent or carrier; specifically (iii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a vaccine vector of the invention; (iv) a method for inducing an immune response against Chlamydia in a mammal (e.g., a human; alternatively, the method can be used in veterinary applications for treating or preventing Chlamydia infection of animals, e . g.
  • a vaccine vector such as a poxvirus, containing a DNA molecule of the invention, placed under the control of elements required for expression
  • a composition of matter comprising a vaccine vector of the invention, together with a diluent or carrier
  • a pharmaceutical composition containing a therapeutically or pro
  • a vaccine vector of the invention which involves administering to the mammal an immunogenically effective amount of a vaccine vector of the invention to elicit a protective or therapeutic immune response to Chlamydia ; and particularly, (v) a method for preventing and/or treating a Chlamydia (e . g. , C. trachoma tis , C. psi ttaci , C. pneumonia , C. pecorum) infection, which involves administering a prophylactic or therapeutic amount of a vaccine vector of the invention to an infected individual.
  • the third aspect of the invention encompasses the use of a vaccine vector of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection.
  • a vaccine vector expresses one or several polypeptides or derivatives of the invention, as well as at least one additional Chlamydia antigen (??), fragment, homolog, mutant, or derivative thereof.
  • the vaccine vector may express additionally a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12) , that enhances the immune response (adjuvant effect) .
  • IL-2 interleukin-2
  • IL-12 interleukin-12
  • composition comprising several vaccine vectors, each of them capable of expressing a polypeptide or derivative of the invention.
  • a composition may also comprise a vaccine vector capable of expressing an additional Chlamydia antigen, or a subunit, fragment, homolog, mutant, or derivative thereof; or a cytokine such as IL-2 or IL-12.
  • Vaccination methods for treating or preventing infection in a mammal comprises use of a vaccine vector of the invention to be administered by any conventional route , particularly to a mucosal (e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route.
  • mucosal e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract
  • parenteral e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal
  • Treatment may be effected in a single dose or repeated at intervals. The appropriate dosage depends on various parameters understood by skilled artisans such as the vaccine vector itself, the route of administration or the condition of the mammal to be vaccinated
  • Live vaccine vectors available in the art include viral vectors such as adenoviruses and poxviruses as well as bacterial vectors, e. g. , Shigella , Salmonella, Vibrio cholerae,
  • Lactobacillus Lactobacillus, Bacille bilie de Calmette-Guerin (BCG) , and Streptococcus .
  • adenovirus vector An example of an adenovirus vector, as well as a method for constructing an adenovirus vector capable of expressing a DNA molecule of the invention, are described in U.S. Patent No. 4,920,209.
  • Poxvirus vectors include vaccinia and canary pox virus, described in U.S. Patent No. 4,722,848 and U.S. Patent No. 5,364,773, respectively. (Also see, e.g., Tartaglia et al .
  • Poxvirus vectors capable of expressing a polynucleotide of the invention are obtained by homologous recombination as described in Kieny et al . , Nature (1984) 312:163 so that the polynucleotide of the invention is inserted in the viral genome under appropriate conditions for expression in mammalian cells.
  • the dose of vaccine viral vector for therapeutic or prophylactic use, can be of from about lxlO 4 to about lxlO 11 , advantageously from about lxlO 7 to about lxlO 10 , preferably of from about lxlO 7 to about lxlO 9 plaque-forming units per kilogram.
  • viral vectors are administered parenterally; for example, in 3 doses, 4 weeks apart. It is preferable to avoid adding a chemical adjuvant to a composition containing a viral vector of the invention and thereby minimizing the immune response to the viral vector itself.
  • Non-toxicogenic Vibrio cholerae mutant strains that are useful as a live oral vaccine are known.
  • Mekalanos et al . Nature (1983) 306:551 and U.S. Patent No. 4,882,278 describe strains which have a substantial amount of the coding sequence of each of the two ctxA alleles deleted so that no functional cholerae toxin is produced.
  • WO 92/11354 describes a strain in which the irgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations.
  • WO 92/11354 describes a strain in which the irgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations.
  • 94/1533 describes a deletion mutant lacking functional ctxA and a ttRSl DNA sequences. These mutant strains are genetically engineered to express heterologous antigens, as described in WO 94/19482.
  • An effective vaccine dose of a Vibrio cholerae strain capable of expressing a polypeptide or polypeptide derivative encoded by a DNA molecule of the invention contains about lxlO 5 to about lxlO 9 , preferably about lxlO 6 to about lxlO 8 , viable bacteria in a volume appropriate for the selected route of administration.
  • Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.
  • Attenuated Salmonella typhimurium strains genetically engineered for recombinant expression of heterologous antigens or not, and their use as oral vaccines are described in Nakayama et al . (Bio/Technology (1988) 6:693) and WO 92/11361.
  • Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.
  • the polynucleotide of the invention is inserted into the bacterial genome or remains in a free state as part of a plasmid.
  • composition comprising a vaccine bacterial vector of the present invention may further contain an adjuvant .
  • adjuvants are known to those skilled in the art. Preferred adjuvants are selected from the list provided below.
  • a fourth aspect of the invention provides (i) a composition of matter comprising a polynucleotide of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a polynucleotide of the invention; (iii) a method for inducing an immune response against Chlamydia in a mammal by administration of an immunogenically effective amount of a polynucleotide of the invention to elicit a protective immune response to Chlamydia; and particularly, (iv) a method for preventing and/or treating a Chlamydia ( e .
  • the fourth aspect of the invention encompasses the use of a polynucleotide of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection.
  • a preferred use includes the use of a DNA molecule placed under conditions for expression in a mammalian cell, especially in a plasmid that is unable to replicate in mammalian cells and to substantially integrate in a mammalian genome.
  • polynucleotides of the invention include their administration to a mammal as a vaccine, for therapeutic or prophylactic purposes.
  • Such polynucleotides are used in the form of DNA as part of a plasmid that is unable to replicate in a mammalian cell and unable to integrate into the mammalian genome.
  • a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell.
  • the promoter functions either ubiquitously or tissue- specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (described in U.S. Patent No.
  • CMV Cytomegalovirus
  • tissue-specific promoter is the desmin promoter which drives expression in muscle cells (Li et al . , Gene (1989) 78:243, Li & Paulin, J. Biol. Chem. (1991) 266:6562 and Li & Paulin, J. Biol. Chem. (1993) 268:10403).
  • Use of promoters is well-known to those skilled in the art. Useful vectors are described in numerous publications, specifically WO 94/21797 and Hartikka et al . , Human Gene Therapy (1996) 7:1205.
  • Polynucleotides of the invention which are used as a vaccine encode either a precursor or a mature form of the corresponding polypeptide.
  • the signal peptide is either homologous or heterologous.
  • a eucaryotic leader sequence such as the leader sequence of the tissue-type plasminogen factor (tPA) is preferred.
  • a composition of the invention contains one or several polynucleotides with optionally at least one additional polynucleotide encoding another Chlamydia antigen such as urease subunit A, B, or both, or a fragment, derivative, mutant, or analog thereof.
  • the composition may also contain an additional polynucleotide encoding a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12) so that the immune response is enhanced.
  • IL-2 interleukin-2
  • IL-12 interleukin-12
  • DNA molecules of the invention and/or additional DNA molecules to be included in the same composition are present in the same plasmid.
  • Standard techniques of molecular biology for preparing and purifying polynucleotides are used in the preparation of polynucleotide therapeutics of the invention.
  • a polynucleotide of the invention is formulated according to various methods outlined below.
  • One method utililizes the polynucleotide in a naked form, free of any delivery vehicles.
  • a polynucleotide is simply diluted in a physiologically acceptable solution such as sterile saline or sterile buffered saline, with or without a carrier.
  • the carrier preferably is isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength, such as provided by a sucrose solution, e.g., a solution containing 20% sucrose.
  • An alternative method utilizes the polynucleotide in association with agents that assist in cellular uptake.
  • agents are (i) chemicals that modify cellular permeability, such as bupivacaine (see, e.g., WO 94/16737), (ii) liposomes for encapsulation of the polynucleotide, or (iii) cationic lipids or silica, gold, or tungsten microparticles which associate themselves with the polynucleotides .
  • Liposomes A Practical Approach, RPC New Ed, IRL press (1990), for a detailed description of methods for making liposomes
  • Cationic lipids are also known in the art and are commonly used for gene delivery.
  • Such lipids include LipofectinTM also known as DOTMA (N-[l-(2,3- dioleyloxy) propyl] -N,N,N-trimethylammonium chloride), DOTAP (1, 2-bis (oleyloxy) -3- (trimethylammonio) propane) , DDAB (dimethyldioctadecylammonium bromide) , DOGS
  • Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine) , as described in WO 90/11092 as an example .
  • DOPE dioleyl phosphatidylethanolamine
  • Formulation containing cationic liposomes may optionally contain other transfection-facilitating compounds.
  • transfection-facilitating compounds include spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO 93/18759) and membrane-permeabilizing compounds such as GALA, Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).
  • Gold or tungsten microparticles are used for gene delivery, as described in WO 91/359, WO 93/17706, and Tang et al . (Nature (1992) 356:152).
  • microparticle-coated polynucleotide is injected via intradermal or intraepidermal routes using a needleless injection device ("gene gun"), such as those described in U.S. Patent No. 4,945,050, U.S. Patent No. 5,015,580, and WO 94/24263.
  • a needleless injection device such as those described in U.S. Patent No. 4,945,050, U.S. Patent No. 5,015,580, and WO 94/24263.
  • the amount of DNA to be used in a vaccine recipient depends, e.g., on the strength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product, the condition of the mammal intended for administration (e.g., the weight, age, and general health of the mammal) , the mode of administration, and the type of formulation.
  • a therapeutically or prophylactically effective dose from about 1 ⁇ g to about 1 mg, preferably, from about 10 ⁇ g to about 800 ⁇ g and, more preferably, from about 25 ⁇ g to about 250 ⁇ g, can be administered to human adults.
  • the administration can be achieved in a single dose or repeated at intervals.
  • the route of administration is any conventional route used in the vaccine field.
  • a polynucleotide of the invention is administered via a mucosal surface, e . g. , an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, and urinary tract surface; or via a parenteral route, e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route.
  • a polynucleotide formulated in association with bupivacaine is advantageously administered into muscles.
  • the formulation can be advantageously injected via intravenous, intranasal (aerosolization) , intramuscular, intradermal, and subcutaneous routes.
  • a polynucleotide in a naked form can advantageously be administered via the intramuscular, intradermal, or sub- cutaneous routes.
  • such a composition can also contain an adjuvant.
  • a systemic adjuvant that does not require concomitant administration in order to exhibit an adjuvant effect is preferable such as, e.g., QS21, which is described in U.S. Patent No. 5,057,546.
  • a fifth aspect of the invention provides a nucleotide probe or primer having a sequence found in or derived by degeneracy of the genetic code from a sequence shown in any one of SEQ ID Nos:l to 26.
  • probe refers to DNA (preferably single stranded) or RNA molecules (or modifications or combinations thereof) that hybridize under the stringent conditions, as defined above, to nucleic acid molecules having SEQ ID Nos: 1 to 26 or to sequences homologous to SEQ ID Nos:l to 26, or to their complementary or anti-sense sequences.
  • probes are significantly shorter than full-length sequences .
  • Such probes contain from about 5 to about 100, preferably from about 10 to about 80, nucleotides.
  • probes have sequences that are at least 75%, preferably at least 85%, more preferably 95% homologous to a portion of any of SEQ ID Nos : 1 to 26 or that are complementary to such sequences.
  • Probes may contain modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5- deoxyuridine, or diamino-2, 6-purine.
  • Sugar or phosphate residues may also be modified or substituted.
  • a deoxyribose residue may be replaced by a polyamide (Nielsen et al . , Science (1991) 254:1497) and phosphate residues may be replaced by ester groups such as diphosphate, alkyl, arylphosphonate and phosphorothioate esters.
  • the 2 ' -hydroxyl group on ribonucleotides may be modified by including such groups as alkyl groups.
  • Probes of the invention are used in diagnostic tests, as capture or detection probes. Such capture probes are conventionally immobilized on a solid support, directly or indirectly, by covalent means or by passive adsorption.
  • a detection probe is labelled by a detection marker selected from: radioactive isotopes, enzymes such as peroxidase, alkaline phosphatase, and enzymes able to hydrolyze a chromogenic, fluorogenic, or luminescent substrate, compounds that are chromogenic, fluorogenic, or luminescent, nucleotide base analogs, and biotin.
  • Probes of the invention are used in any conventional hybridization technique, such as dot blot (Maniatis et al . , Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), Southern blot (Southern, J. Mol. Biol. (1975) 98:503), northern blot
  • RNA is used as a target
  • sandwich technique Dist et al . , Cell (1977) 12:23.
  • the latter technique involves the use of a specific capture probe and/or a specific detection probe with nucleotide sequences that at least partially differ from each other.
  • a primer is a probe of usually about 10 to about 40 nucleotides that is used to initiate enzymatic polymerization of DNA in an amplification process (e.g., PCR), in an elongation process, or in a reverse transcription method. Primers used in diagnostic methods involving PCR are labeled by methods known in the art.
  • the invention also encompasses (i) a reagent comprising a probe of the invention for detecting and/or identifying the presence of Chlamydia in a biological material; (ii) a method for detecting and/or identifying the presence of Chlamydia in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA or RNA is extracted from the material and denatured, and (c) exposed to a probe of the invention, for example, a capture, detection probe or both, under stringent hybridization conditions, such that hybridization is detected; and (iii) a method for detecting and/or identifying the presence of Chlamydia in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA is extracted therefrom, (c) the extracted DNA is primed with at least one, and preferably two, primers of the invention and amplified by polymerase chain reaction, and (d) the ampl
  • a sixth aspect of the invention features a substantially purified polypeptide or polypeptide derivative having an amino acid sequence encoded by a polynucleotide of the invention.
  • substantially purified polypeptide as used herein is defined as a polypeptide that is separated from the environment in which it naturally occurs and/or that is free of the majority of the polypeptides that are present in the environment in which it was synthesized.
  • a substantially purified polypeptide is free from cytoplasmic polypeptides.
  • the polypeptides of the invention may be purified from a natural source, i.e., a Chlamydia strain, or produced by recombinant means.
  • Consistent with the sixth aspect of the invention are polypeptides, homologs or fragments which are modified or treated to enhance their immunogenicity in the target animal, in whom the polypeptide, homolog or fragments are intended to confer protection against Chlamydia.
  • modifications or treatments include: amino acid substitutions with an amino acid derivative such as 3-methyhistidine, 4-hydroxyproline, 5- hydroxylysine etc., modifications or deletions which are carried out after preparation of the polypeptide, homolog or fragment, such as the modification of free amino, carboxyl or hydroxyl side groups of the amino acids.
  • Identification of homologous polypeptides or polypeptide derivatives encoded by polynucleotides of the invention which have specific antigenicity is achieved by screening for cross- reactivity with an antiserum raised against the polypeptide of reference having an amino acid sequence of any one of SEQ ID Nos: 27 to 45.
  • the procedure is as follows: a monospecific hyperimmune antiserum is raised against a purified reference polypeptide, a fusion polypeptide (for example, an expression product of MBP, GST, or His-tag systems) , or a synthetic peptide predicted to be antigenic. Where an antiserum is raised against a fusion polypeptide, two different fusion systems are employed.
  • Specific antigenicity can be determined according to a number of methods, including Western blot (Towbin et al . , Proc. Natl. Acad. Sci. USA (1979) 76:4350), dot blot, and ELISA, as described below.
  • Western blot assay the product to be screened, either as a purified preparation or a total __.. coli extract, is submitted to SDS-Page electrophoresis as described by Laemmli
  • the material is further incubated with the monospecific hyperimmune antiserum diluted in the range of dilutions from about 1 : 5 to about 1:5000, preferably from about
  • the product to be screened is preferably used as the coating antigen.
  • a purified preparation is preferred, although a whole cell extract can also be used. Briefly, about 100 ⁇ l of a preparation at about 10 ⁇ g protein/ml are distributed into wells of a 96-well polycarbonate ELISA plate. The plate is incubated for 2 hours at 37 °C then overnight at 4°C. The plate is washed with phosphate buffer saline (PBS) containing 0.05% Tween 20 (PBS/Tween buffer). The wells are saturated with 250 ⁇ l PBS containing 1% bovine serum albumin (BSA) to prevent non-specific antibody binding.
  • PBS phosphate buffer saline
  • BSA bovine serum albumin
  • the plate After 1 hour incubation at 37 °C, the plate is washed with PBS/Tween buffer. The antiserum is serially diluted in PBS/Tween buffer containing 0.5% BSA. 100 ⁇ l of dilutions are added per well. The plate is incubated for 90 minutes at 37 °C, washed and evaluated according to standard procedures. For example, a goat anti-rabbit peroxidase conjugate is added to the wells when specific antibodies were raised in rabbits. Incubation is carried out for 90 minutes at 37 °C and the plate is washed. The reaction is developed with the appropriate substrate and the reaction is measured by colorimetry (absorbance measured spectrophotometrically) .
  • a positive reaction is shown by O.D. values greater than a non immune control serum.
  • a dot blot assay a purified product is preferred, although a whole cell extract can also be used. Briefly, a solution of the product at about 100 ⁇ g/ml is serially two-fold diluted in 50 mM Tris-HCl (pH 7.5). 100 ⁇ l of each dilution are applied to a nitrocellulose membrane 0.45 ⁇ m set in a 96-well dot blot apparatus (Biorad) . The buffer is removed by applying vacuum to the system. Wells are washed by addition of 50 mM Tris-HCl (pH 7.5) and the membrane is air-dried.
  • the membrane is saturated in blocking buffer (50 mM Tris-HCl (pH 7.5) 0.15 M NaCl, 10 g/L skim milk) and incubated with an antiserum dilution from about 1:50 to about 1:5000, preferably about 1:500.
  • the reaction is revealed according to standard procedures. For example, a goat anti-rabbit peroxidase conjugate is added to the wells when rabbit antibodies are used. Incubation is carried out 90 minutes at 37 °C and the blot is washed. The reaction is developed with the appropriate substrate and stopped. The reaction is measured visually by the appearance of a colored spot, e.g., by colorimetry. Under the above experimental conditions, a positive reaction is shown once a colored spot is associated with a dilution of at least about 1:5, preferably of at least about 1:500.
  • a seventh aspect of the invention provides (i) a composition of matter comprising a polypeptide of the invention together with a diluent or carrier; specifically (ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polypeptide of the invention; (iii) a method for inducing an immune response against Chlamydia in a mammal, by administering to the mammal an immunogenically effective amount of a polypeptide of the invention to elicit a protective immune response to Chlamydia ; and particularly, (iv) a method for preventing and/or treating a Chlamydia ( e .
  • the seventh aspect of the invention encompasses the use of a polypeptide of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection.
  • the immunogenic compositions of the invention are administered by conventional routes known the vaccine field, in particular to a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route.
  • a mucosal e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract
  • parenteral e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal
  • the choice of administration route depends upon a number of parameters, such as the adjuvant associated with the polypeptide. If a mucosal adjuvant is used, the intranasal or oral route is preferred. If a lipid formulation or an aluminum compound is used, the parenteral route is preferred with the sub-
  • composition of the invention contains one or several polypeptides or derivatives of the invention.
  • the composition optionally contains at least one additional Chlamydia antigen, or a subunit, fragment, homolog, mutant, or derivative thereof.
  • a polypeptide or derivative thereof is formulated into or with liposomes, preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response.
  • liposomes preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response.
  • liposomes preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response.
  • VLPs virus-like-particles
  • Treatment is achieved in a single dose or repeated as necessary at intervals, as can be determined readily by one skilled in the art. For example, a priming dose is followed by three booster doses at weekly or monthly intervals.
  • An appropriate dose depends on various parameters including the recipient (e.g., adult or infant), the particular vaccine antigen, the route and frequency of administration, the presence/absence or type of adjuvant, and the desired effect (e.g., protection and/or treatment), as can be determined by one skilled in the art.
  • a vaccine antigen of the invention is administered by a mucosal route in an amount from about 10 ⁇ g to about 500 mg, preferably from about 1 mg to about 200 mg.
  • the dose usually does not exceed about 1 mg, preferably about 100 ⁇ g.
  • polynucleotides and polypeptides of the invention may be used sequentially as part of a multistep immunization process.
  • a mammal is initially primed with a vaccine vector of the invention such as a pox virus, e.g., via the parenteral route, and then boosted twice with the polypeptide encoded by the vaccine vector, e . g. , via the mucosal route.
  • liposomes associated with a polypeptide or derivative of the invention is also used for priming, with boosting being carried out mucosally using a soluble polypeptide or derivative of the invention in combination with a mucosal adjuvant (e.g., LT) .
  • a mucosal adjuvant e.g., LT
  • a polypeptide derivative of the invention is also used in accordance with the seventh aspect as a diagnostic reagent for detecting the presence of anti-Chlamydia antibodies, e.g., in a blood sample.
  • Such polypeptides are about 5 to about 80, preferably about 10 to about 50 amino acids in length. They are either labeled or unlabeled, depending upon the diagnostic method. Diagnostic methods involving such a reagent are described below.
  • a polypeptide or polypeptide derivative is produced and purified using known laboratory techniques.
  • the polypeptide or polypeptide derivative may be produced as a fusion protein containing a fused tail that facilitates purification.
  • the fusion product is used to immunize a small mammal, e.g., a mouse or a rabbit, in order to raise antibodies against the polypeptide or polypeptide derivative (monospecific antibodies) .
  • an eighth aspect of the invention provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the invention.
  • monospecific antibody is meant an antibody that is capable of reacting with a unique naturally-occurring Chlamydia polypeptide.
  • An antibody of the invention is either polyclonal or monoclonal.
  • Monospecific antibodies may be recombinant, e.g., chimeric (e.g., constituted by a variable region of murine origin associated with a human constant region) , humanized (a human immunoglobulin constant backbone together with hypervariable region of animal, e.g., murine, origin), and/or single chain.
  • Both polyclonal and monospecific antibodies may also be in the form of immunoglobulin fragments, e.g., F(ab)'2 or Fab fragments.
  • the antibodies of the invention are of any isotype, e.g., IgG or IgA, and polyclonal antibodies are of a single isotype or a mixture of isotypes.
  • Antibodies against the polypeptides, homologs or fragments of the present invention are generated by immunization of a mammal with a composition comprising said polypeptide, homolog or fragment.
  • Scu antibodies may be polyclonal or monoclonal. Methods to produce polyclonal or monoclonal antibodies are well known in the art. For a review, see “Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Eds. E. Harlow and D. Lane (1988), and D.E. Yelton et al., 1981. Ann. Rev. Biochem. 50:657-680. For monoclonal antibodies, see Kohl and Milstein?...
  • the antibodies of the invention which are raised to a polypeptide or polypeptide derivative of the invention, are produced and identified using standard immunological assays, e.g., Western blot analysis, dot blot assay, or ELISA (see, e.g., Coligan et al . , Current Protocols in Immunology (1994) John Wiley & Sons, Inc., New York, NY).
  • the antibodies are used in diagnostic methods to detect the presence of a Chlamydia antigen in a sample, such as a biological sample.
  • the antibodies are also used in affinity chromatography for purifying a polypeptide or polypeptide derivative of the invention. As is discussed further below, such antibodies may be used in prophylactic and therapeutic passive immunization methods.
  • a ninth aspect of the invention provides (i) a reagent for detecting the presence of Chlamydia in a biological sample that contains an antibody, polypeptide, or polypeptide derivative of the invention; and (ii) a diagnostic method for detecting the presence of Chlamydia in a biological sample, by contacting the biological sample with an antibody, a polypeptide, or a polypeptide derivative of the invention, such that an immune complex is formed, and by detecting such complex to indicate the presence of Chlamydia in the sample or the organism from which the sample is derived.
  • the immune complex is formed between a component of the sample and the antibody, polypeptide, or polypeptide derivative, whichever is used, and that any unbound material is removed prior to detecting the complex.
  • a polypeptide reagent is useful for detecting the presence of anti-Chlamydia antibodies in a sample, e. g. , a blood sample, while an antibody of the invention is used for screening a sample, such as a gastric extract or biopsy, for the presence of Chlamydia polypeptides .
  • the reagent i.e., the antibody, polypeptide, or polypeptide derivative of the invention
  • a solid support such as a tube, a bead, or any other conventional support used in the field. Immobilization is achieved using direct or indirect means. Direct means include passive adsorption (non-covalent binding) or covalent binding between the support and the reagent. By “indirect means” is meant that an anti-reagent compound that interacts with a reagent is first attached to the solid support.
  • an antibody that binds to it can serve as an anti-reagent, provided that it binds to an epitope that is not involved in the recognition of antibodies in biological samples.
  • Indirect means may also employ a ligand-receptor system, for example, where a molecule such as a vitamin is grafted onto the polypeptide reagent and the corresponding receptor immobilized on the solid phase. This is illustrated by the biotin- streptavidin system.
  • a peptide tail is added chemically or by genetic engineering to the reagent and the grafted or fused product immobilized by passive adsorption or covalent linkage of the peptide tail.
  • Such diagnostic agents may be included in a kit which also comprises instructions for use.
  • the reagent are labeled with a detection means which allows for the detection of the reagent when it is bound to its target.
  • the detection means may be a fluorescent agent such as fluorescein isocyanate or fluorescein isothiocyanate, or an enzyme such as horse radish peroxidase or luciferase or alkaline phosphatase, or a radioactive element such as 125 I or 51 Cr.
  • a tenth aspect of the invention provides a process for purifying, from a biological sample, a polypeptide or polypeptide derivative of the invention, which involves carrying out antibody-based affinity chromatography with the biological sample, wherein the antibody is a monospecific antibody of the invention.
  • the antibody is either polyclonal or monospecific, and preferably is of the IgG type.
  • Purified IgGs is prepared from an antiserum using standard methods (see, e.g., Coligan et al . , supra ) .
  • Conventional chromatography supports, as well as standard methods for grafting antibodies, are described in, e.g., Antibodies: A Laboratory Manual, D. Lane, E. Harlow, Eds. (1988) and outlined below.
  • a biological sample such as an C. pneumoniae extract preferably in a buffer solution
  • a chromatography material preferably equilibrated with the buffer used to dilute the biological sample so that the polypeptide or polypeptide derivative of the invention (i.e., the antigen) is allowed to adsorb onto the material.
  • the chromatography material such as a gel or a resin coupled to an antibody of the invention, is in either a batch form or a column.
  • the unbound components are washed off and the antigen is then eluted with an appropriate elution buffer, such as a glycine buffer or a buffer containing a chaotropic agent, e.g., guanidine HC1, or high salt concentration (e.g., 3 M MgCl 2 ) .
  • an appropriate elution buffer such as a glycine buffer or a buffer containing a chaotropic agent, e.g., guanidine HC1, or high salt concentration (e.g., 3 M MgCl 2 ) .
  • Eluted fractions are recovered and the presence of the antigen is detected, e.g., by measuring the absorbance at 280 nm.
  • An eleventh aspect of the invention provides (i) a composition of matter comprising a monospecific antibody of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a monospecific antibody of the invention, and (iii) a method for treating or preventing a Chlamydia ( e . g. , C. trachoma tis , C. psi ttaci , C. pneumoniae or C. pecorum) infection, by administering a therapeutic or prophylactic amount of a monospecific antibody of the invention to an infected individual.
  • a Chlamydia e . g. , C. trachoma tis , C. psi ttaci , C. pneumoniae or C. pecorum
  • the eleventh aspect of the invention encompasses the use of a monospecific antibody of the invention in the preparation of a medicament for treating or preventing Chlamydia infection.
  • the monospecific antibody is either polyclonal or monoclonal, preferably of the IgA isotype (predominantly) .
  • the antibody is administered to a mucosal surface of a mammal, e.g., the gastric mucosa, e.g., orally or intragastrically, advantageously, in the presence of a bicarbonate buffer.
  • systemic administration not requiring a bicarbonate buffer, is carried out.
  • a monospecific antibody of the invention is administered as a single active component or as a mixture with at least one monospecific antibody specific for a different Chlamydia polypeptide.
  • the amount of antibody and the particular regimen used are readily determined by one skilled in the art. For example, daily administration of about 100 to 1,000 mg of antibodies over one week, or three doses per day of about 100 to 1,000 mg of antibodies over two or three days, are effective regimens for most purposes.
  • Therapeutic or prophylactic efficacy are evaluated using standard methods in the art, e.g., by measuring induction of a mucosal immune response or induction of protective and/or therapeutic immunity, using, e.g., the C. pneumoniae mouse model.
  • the C. pneumoniae strain of the model may be replaced with another Chlamydia strain.
  • the efficacy of DNA molecules and polypeptides from C. pneumoniae is preferably evaluated in a mouse model using C. pneumoniae strain. Protection is determined by comparing the degree of Chlamydia infection to that of a control group. Protection is shown when infection is reduced by comparison to the control group.
  • Adjuvants useful in any of the vaccine compositions described above are as follows.
  • Adjuvants for parenteral administration include aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate.
  • the antigen is precipitated with, or adsorbed onto, the aluminum compound according to standard protocols.
  • Other adjuvants such as RIBI (ImmunoChem, Hamilton, MT) , is used in parenteral administration.
  • Adjuvants for mucosal administration include bacterial toxins, e.g., the cholera toxin (CT) , the E. coli heat-labile toxin (LT) , the Clostridium difficile toxin A and the pertussis toxin (PT) , or combinations, subunits, toxoids, or mutants thereof such as a purified preparation of native cholera toxin subunit B (CTB) . Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity. Preferably, a mutant having reduced toxicity is used.
  • CT cholera toxin
  • LT E. coli heat-labile toxin
  • PT pertussis toxin
  • CTB native cholera toxin subunit B
  • Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity.
  • Suitable mutants are described, e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant).
  • Additional LT mutants that are used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69- Gly, Glu-110-Asp, and Glu-112-Asp mutants.
  • Other adjuvants such as a bacterial monophosphoryl lipid A (MPLA) of, e . g. , E.
  • MPLA bacterial monophosphoryl lipid A
  • Adjuvants useful for both mucosal and parenteral administrations include polyphosphazene (WO 95/2415), DC-chol (3 b- (N- (N ', N' -dimethyl aminomethane) -carbamoyl) cholesterol; U.S. Patent No. 5,283,185 and WO 96/14831) and QS-21 (WO 88/9336).
  • composition of the invention containing a polynucleotide, a polypeptide, a polypeptide derivative, or an antibody of the invention, is manufactured in a conventional manner.
  • a pharmaceutically acceptable diluent or carrier e.g., water or a saline solution such as phosphate buffer saline.
  • a diluent or carrier is selected on the basis of the mode and route of administration, and standard pharmaceutical practice.
  • Suitable pharmaceutical carriers or diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations are described in Remington ' s Pharmaceutical Sciences, a standard reference text in this field and in the USP/NF.
  • the invention also includes methods in which Chlamydia infection are treated by oral administration of a Chlamydia polypeptide of the invention and a mucosal adjuvant, in combination with an antibiotic, an antacid, sucralfate, or a combination thereof.
  • antibiotics including, e.g., macrolides, tetracyclines, and derivatives thereof (specific examples of antibiotics that can be used include azithromycin or doxicyclin or immunomodulators such as cytokines or steroids) .
  • compounds containing more than one of the above-listed components coupled together, are used.
  • compositions for carrying out these methods i.e., compositions containing a Chlamydia antigen (or antigens) of the invention, an adjuvant, and one or more of the above-listed compounds, in a pharmaceutically acceptable carrier or diluent.
  • Amounts of the above-listed compounds used in the methods and compositions of the invention are readily determined by one skilled in the art.
  • Treatment/immunization schedules are also known and readily designed by one skilled in the art.
  • the non-vaccine components can be administered on days 1-14, and the vaccine antigen + adjuvant can be administered on days 7, 14, 21, and 28.

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EP99955602A 1998-10-28 1999-10-28 Chlamydia antigenes and corresponding dna fragments and uses thereof Withdrawn EP1129202A2 (en)

Applications Claiming Priority (27)

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US10603498P 1998-10-28 1998-10-28
US10604298P 1998-10-28 1998-10-28
US10604498P 1998-10-28 1998-10-28
US10603998P 1998-10-28 1998-10-28
US106034P 1998-10-28
US106044P 1998-10-28
US106042P 1998-10-28
US106039P 1998-10-28
US10607298P 1998-10-29 1998-10-29
US10608798P 1998-10-29 1998-10-29
US10607398P 1998-10-29 1998-10-29
US10607498P 1998-10-29 1998-10-29
US106074P 1998-10-29
US106072P 1998-10-29
US106073P 1998-10-29
US106087P 1998-10-29
US10703598P 1998-11-02 1998-11-02
US10658798P 1998-11-02 1998-11-02
US10658998P 1998-11-02 1998-11-02
US10658898P 1998-11-02 1998-11-02
US10703498P 1998-11-02 1998-11-02
US107035P 1998-11-02
US106589P 1998-11-02
US106587P 1998-11-02
US107034P 1998-11-02
US106588P 1998-11-02
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AU3790900A (en) 1998-12-01 2000-06-19 Aventis Pasteur Limited Chlamydia antigens and corresponding DNA fragments and uses thereof
US7297341B1 (en) 1998-12-23 2007-11-20 Sanofi Pasteur Limited Chlamydia antigens and corresponding DNA fragments and uses thereof
US6808713B1 (en) 1998-12-28 2004-10-26 Aventis Pasteur Limited Chlamydia antigens and corresponding DNA fragments and uses thereof
JP4832646B2 (ja) 1999-03-12 2011-12-07 サノフィ、パストゥール、リミテッド クラミジア抗原および対応するｄｎａ断片ならびにその使用
EP1177301B1 (en) 1999-05-03 2007-10-10 Sanofi Pasteur Limited Chlamydia antigens and corresponding dna fragments and uses thereof
EP1220925B8 (en) 1999-09-20 2008-04-23 Sanofi Pasteur Limited Chlamydia antigens and corresponding dna fragments and uses thereof
US7807802B2 (en) 2002-11-12 2010-10-05 Abbott Lab Polynucleotides for the amplification and detection of Chlamydia trachomatis and Neisseria gonorrhoeae

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BR9810288A (pt) * 1997-06-23 2000-09-19 Loke Diagnostics Aps Teste diagnóstico especìfico para espécie para identificar infecção de um mamìfero, como um humano, com chlamydia pneumoniae, fragmento de ácido nucleico derivado de chlamydia pneumoniae, proteìna derivada de chlamydia pneumoniae, anticorpo monoespecìfico policlonal, kit diagnóstico para o diagnóstico de infecção de um mamìfero, como um humano, com chlamydia pneumoniae, composição para imunizar um mamìfero comoum humano, contra chlamydia pneumoniae, uso de uma proteìna, e, uso de um fragmento de ácido nucleico.
CN100390283C (zh) * 1997-11-21 2008-05-28 根瑟特公司 肺炎衣原体的基因组序列和其多肽,片段以及其用途特别是用于诊断、预防和治疗感染

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WO2000024765A8 (en) 2001-12-20
WO2000024765A2 (en) 2000-05-04

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