EP1104470A2 - Antigenes de chlamydia, fragments d'adn correspondants et leur utilisation - Google Patents

Antigenes de chlamydia, fragments d'adn correspondants et leur utilisation

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Publication number
EP1104470A2
EP1104470A2 EP99938465A EP99938465A EP1104470A2 EP 1104470 A2 EP1104470 A2 EP 1104470A2 EP 99938465 A EP99938465 A EP 99938465A EP 99938465 A EP99938465 A EP 99938465A EP 1104470 A2 EP1104470 A2 EP 1104470A2
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EP
European Patent Office
Prior art keywords
polypeptide
polynucleotide
cpn
seq
amino acid
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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EP99938465A
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German (de)
English (en)
Inventor
Andrew D. Murdin
Raymond P. Oomen
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Sanofi Pasteur Ltd
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Aventis Pasteur Ltd
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Publication of EP1104470A2 publication Critical patent/EP1104470A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56927—Chlamydia
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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

Definitions

  • the present invention relates to Chlamydia antigens and corresponding DNA molecules, which can be used in methods to prevent and treat disease caused by 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. Chlamydiae are differentiated from other bacteria by their morphology and by a unique developmental cycle. They are obligate intracellular 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.
  • chlamydiae are small and multiply only within susceptible cells they were long thought to be viruses. However, they have many characteristics in common with other bacteria: (1) they contain both DNA and RNA, (2) they divide by binary fission, (3) their cell envelopes resemble those of other Gram-negative bacteria, (4) they contain ribosomes similar to those of other bacteria, and (5) they are susceptible to various antibiotics. Chlamydiae can be seen in the light microscope, and the genome is about one-third the size of the Escherichia coli genome.
  • C. trachomatis has a high degree of host specificity, being almost completely limited to man; it causes ocular and genitourinary infections of widely varying severity.
  • C. psittaci strains are rare in man but are found in a wide range of birds and also in wild, domestic, and laboratory mammals, where they multiply in cells of many organs.
  • C. pneumoniae is a common human pathogen, originally described as the T AR strain of C. psittaci, but subsequently recognized to be a new species.
  • C. pneumoniae is antigenically, genetically, and morphologically distinct from other Chlamydia species (C. trachomatis, C. pecorum and C. psittaci). It shows 10% or less DNA sequence homology with either of C. trachomatis or C. psittaci and so far appears to consist of only a single strain, TWAR.
  • C. pneumoniae is a common cause of community acquired pneumonia, less frequent only than Streptococcus pneumoniae and Mycoplasma pneumoniae. Grayston et al. , J. Infect. Dis. 168: 1231 (1995); Campos et al., Invest. Ophthalmol. Vis. Sci. 36: 1477 (1995), each incorporated herein by reference. It can also cause upper respiratory tract symptoms and disease, including bronchitis and sinusitis. See, e.g., Grayston et al., J. Infect. Dis. 168: 1231 (1995); Campos et al, Invest. Ophthalmol. Vis. Sci. 36: 1477 (1995); Grayston et al. . Infect. Dis.
  • C. pneumoniae infection usually presents as an acute respiratory disease (i.e., cough, sore throat, hoarseness, and fever; abnormal chest sounds on auscultation). For most patients, the cough persists for 2 to 6 weeks, and recovery is slow. In approximately 10% of these cases, upper respiratory tract infection is followed by bronchitis or pneumonia. Furthermore, during a C. pneumoniae epidemic, subsequent co-infection with pneumococcus has been noted in about half of these pneumonia patients, particularly in the infirm and the elderly. As noted above, there is more and more evidence that C. pneumoniae infection is also linked to diseases other than respiratory infections.
  • 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. trachomatis infection does not confer cross-immunity to C. pneumoniae. Infections are easily treated with oral antibiotics, tetracycline or erythromycin (2 g/day, for at least 10 to 14 days). A recently developed drug, azithromycin, is highly effective as a single-dose therapy against chlamydial infections.
  • C. pneumoniae infection is mild and without complications, and up to 90% of infections are subacute or unrecognized.
  • infections have been thought to be rare up to the age of five years, although a recent study has reported that many children in this age group show PCR evidence of infection despite being seronegative, and estimates a prevalence of 17-19% in 2-4 years old. See, Normann et al., Acta Paediatrica, 87: 23-27 (1998).
  • the seroprevalence of C. pneumoniae antibodies among young children is elevated, and there are suspicions that C. pneumoniae may be an important cause of acute lower respiratory tract disease and mortality for infants and children in tropical regions of the world.
  • C. pneumoniae infection usually happens between the ages of 5 and 20 years. 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). During an epidemic, C. pneumonia infection may account for 50 to 60% of the cases of pneumonia. During these periods, also, more episodes of mixed infections with S. pneumoniae have been reported. 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 are conserved across all chlamydiae, but 98kDa, 76 kDa and 54 kDa proteins may be C. pneumoniae-specific. Campos et al, Invest. Ophthalmol. Vis. Sci. 36: 1477 (1995); Marrie, Clin. Infect. Dis. 18: 501 (1993); Wiedmann-Al- Ahmad et al, Clin. Diagn. Lab. Immunol 4: 700-704 (1997). Immunoblotting of isolates with sera from patients does show variation of blotting patterns between isolates, indicating that serotypes C. pneumoniae may exist. Grayston et al, J. Infect. Dis.
  • the present invention provides purified and isolated DNA molecules, which encode Chlamydia that can be used in methods to prevent, treat, and diagnose Chlamydia infection.
  • the present invention provides eight separate preferred DNA molecules, each individually and separately defined by one SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, or 15. Further provided in the present invention are eight separate preferred polypeptides, each individually and separately defined by one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, or 16.
  • the invention also includes DNA molecules that encode mutants, variants, and derivatives of such polypeptides, which result from the addition, deletion, or substitution of non-essential amino acids as described herein.
  • the invention also includes RNA molecules corresponding to the DNA molecules of the invention.
  • the invention includes the corresponding polypeptides and 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.
  • the present invention 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 live vaccine vectors such as viral or bacterial live vaccine vectors, including, pox virus, alphavirus, Salmonella typhimurium, or Vibrio cholerae vector, containing a polynucleotide of the invention, such vaccine vectors being useful for, e.g., preventing and treating Chlamydia infection, in combination with a diluent or carrier, and related pharmaceutical compositions and associated therapeutic and/or prophylactic methods; (iii) a therapeutic and/or prophylactic method involving administration of an 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; (iv) a method for diagnosing the
  • FIG. 1 shows the nucleotide sequence (top sequence) and the deduced amino acid sequence of the full-length (middle sequence) and processed (bottom sequence) CPN 100111 polypeptide from Chlamydia pneumoniae.
  • FIG. 2 shows the restriction enzyme analysis of the nucleotide sequence encoding C. pneumoniae CPN 100111.
  • FIG. 3 shows the nucleotide sequence (top sequence) and the deduced amino acid sequence of the full-length (middle sequence) and processed (bottom sequence) CPN 100224 polypeptide from Chlamydia pneumoniae.
  • FIG. 4 shows the restriction enzyme analysis of the nucleotide sequence encoding the C. pneumoniae CPN 100224.
  • FIG. 5 shows the nucleotide sequence (top sequence) and the deduced amino acid sequence (bottom sequence) of the CPN100230 polypeptide from Chlamydia pneumoniae.
  • FIG. 6 shows the restriction enzyme analysis of the nucleotide sequence encoding the C. pneumoniae CPN100230.
  • FIG. 7 shows the nucleotide sequence (top sequence) and the deduced amino acid sequence
  • FIG. 8 shows the restriction enzyme analysis of the nucleotide sequence encoding the C. pneumoniae CPN 100231.
  • FIG. 9 shows the nucleotide sequence (top sequence) and the deduced amino acid sequence (bottom sequence) of the CPN 100232 polypeptide from Chlamydia pneumoniae.
  • FIG. 10 shows the restriction enzyme analysis of the nucleotide sequence encoding the C. pneumoniae CPN100232.
  • FIG. 11 shows the nucleotide sequence (top sequence) and the deduced amino acid sequence (bottom sequence) of the CPN100233 polypeptide from Chlamydia pneumoniae.
  • FIG. 12 shows the restriction enzyme analysis of the nucleotide sequence encoding the
  • FIG. 13 shows the nucleotide sequence (top sequence) and the deduced amino acid sequence of the full-length (middle sequence) and processed (bottom sequence) CPN 100394 polypeptide from Chlamydia pneumoniae.
  • FIG. 14 shows the restriction enzyme analysis of the nucleotide sequence encoding the
  • FIG. 15 shows the nucleotide sequence (top sequence) and the deduced amino acid sequence (bottom sequence) of the CPN100395 polypeptide from Chlamydia pneumoniae.
  • FIG. 16 shows the restriction enzyme analysis of the nucleotide sequence encoding the C. pneumoniae CPN 100395. DETAILED DESCRIPTION OF THE INVENTION
  • ORFs open reading frames encoding chlamydial polypeptides
  • polypeptides include polypeptides permanently found in the bacterial membrane structure, polypeptides that are present in the external vicinity of the bacterial membrane, polypeptides permanently found in the inclusion membrane structure, polypeptides that are present in the external vicinity of the inclusion membrane, and polypeptides that are released into the cytoplasm of the infected cell.
  • These polypeptides can be used in vaccination methods for preventing and treating Chlamydia infection.
  • isolated polynucleotides encoding the precursor and mature forms of Chlamydia polypeptides.
  • An isolated polynucleotide of the invention encodes a polypeptide having an amino acid sequence that is homologous to a Chlamydia amino acid sequence, the Chlamydia amino acid sequence being selected from the group consisting of the amino acid sequences as shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, or 16.
  • isolated polynucleotide is defined as a polynucleotide removed from the environment in which it naturally occurs. For example, a naturally-occurring DNA molecule present in the genome of the bacteria 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 could be part of a vector or a composition and still be isolated in that such a vector or composition is not part of its natural environment.
  • a polynucleotide of the invention can be in the form of RNA or DNA (e.g., cDNA, genomic DNA, or synthetic DNA), or modifications or combinations thereof.
  • the DNA can be double-stranded or single-stranded, and, if single-stranded, can be the coding strand or the non-coding (anti-sense) strand.
  • sequence that encodes a polypeptide of the invention as shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 or 16 can be: (a) the coding sequence as shown in either SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 and 15; or (b) a ribonucleotide sequence derived by transcription of (a); or (c) a different coding sequence; this latter, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptides as the DNA molecules of which the nucleotide sequences are illustrated in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, and 15..
  • homologous amino acid sequence is meant an amino acid sequence that differs from an amino acid sequence shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 or 16, only by one or more conservative amino acid substitutions, or by one or more non-conservative amino acid substitutions, deletions, or additions located at positions at which they do not destroy the specific antigenicity of the polypeptide.
  • such a sequence is at least 75%, more preferably 80%, and most preferably 90% identical to an amino acid sequence shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 or 16.
  • homologous amino acid sequences include sequences that are identical or substantially identical to an amino acid sequence as shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 and 16.
  • 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, if at all, by a majority of conservative amino acid substitutions.
  • Conservative amino acid substitutions typically include substitutions among amino acids of the same class. These classes include, for example, (a) amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, and tyrosine; (b) amino acids having basic side chains, such as lysine, arginine, and histidine; (c) amino acids having acidic side chains, such as aspartic acid and glutamic acid; and (d) amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.
  • 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 glut
  • homology is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Similar amino acid sequences are aligned to obtain the maximum degree of homology (i.e., identity). To this end, it may be necessary to introduce gaps into the sequence. Once the optimal alignment has been set up, the degree of homology (i.e., identity) 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. Alternatively, homology can be determined by aligning the candidate sequence and the reference sequence using an alignment tool, such as the dynamic programming algorithm described in Needleman et al, J. Mol Biol.
  • Similarity factors include similar size, shape and electrical charge.
  • One particularly preferred method of determining amino acid similarities is the PAM25O matrix described in Dayhoff et al, 5 ATLAS OF PROTEIN SEQUENCE AND STRUCTURE 345-352 (1978 & Supp.), incorporated by reference herein.
  • a similarity score is first calculated as the sum of the aligned pairwise amino acid similarity scores. Insertions and deletions are ignored for the purposes of percent homology and identity. Accordingly, gap penalties are not used in this calculation.
  • the raw score is then normalized by dividing it by the geometric mean of the scores of the candidate compound and the reference sequence. The geometric mean is the square root of the product of these scores. The normalized raw score is the percent homology.
  • a homologous sequence is one that is at least 45%, more preferably 60%, and most preferably 85% identical to a coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, and l5.
  • NOS: 2, 4, 6, 8, 10, 12, 14 or 16 include naturally-occurring allelic variants, as well as mutants and variants or any other non-naturally-occurring variants that are analogous in terms of antigenicity, to a polypeptide having a sequence as shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 or 16.
  • 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 substantially 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.
  • the biological function is distinct from the antigenic function.
  • a polypeptide can have more than one biological function.
  • Allelic variants are very common in nature. For example, a bacterial species, e.g., C. pneumoniae, is usually represented by a variety of strains that differ from each other by minor allelic variations. Indeed, a polypeptide that fulfills the same biological function in different strains can have an amino acid sequence that is not identical in each of the strains. Such an allelic variation may be equally reflected at the polynucleotide level.
  • allelic variants of polypeptide antigens comes from, e.g., studies of the Chlamydial MOMP antigen.
  • the amino acid sequence of the MOMP varies from strain to strain, yet cross-strain antibody binding plus neutralization of infectivity occurs, indicating that the MOMP, when used as an immunogen, is tolerant of amino acid variations.
  • Polynucleotides, e.g., DNA molecules, encoding allelic variants can easily be 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 can be designed according to the nucleotide sequence information provided in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 and 15. Typically, a primer can consist of 10 to 40, preferably 15 to 25 nucleotides. It may be also advantageous to select primers containing C and G nucleotides in a proportion sufficient to ensure efficient hybridization; e.g., an amount of C and G nucleotides of at least 40%, preferably 50% of the total nucleotide amount.
  • Useful homologs that do not naturally occur can be designed using known methods for identifying regions of an antigen that are likely to be tolerant of amino acid sequence changes and/or deletions. For example, sequences of the antigen from different species can be compared to identify conserved sequences.
  • Polypeptide derivatives that are encoded by polynucleotides of the invention include, e.g., fragments, polypeptides having large internal deletions derived from full-length polypeptides, and fusion proteins.
  • Polypeptide fragments of the invention can be derived from a polypeptide having a sequence homologous to any of the sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 or 16, to the extent that the fragments retain the desired substantial antigenicity of the parent polypeptide
  • Polypeptide derivatives can also be constructed by large internal deletions that remove a substantial part of the parent polypeptide, while retaining the desired specific antigenicity. Generally, polypeptide derivatives should be about at least 12 amino acids in length to maintain the antigenicity. Advantageously, they can be 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.
  • Useful polypeptide derivatives, e.g., polypeptide fragments can be designed using computer-assisted analysis of amino acid sequences in order to identify sites in protein antigens having potential as surface-exposed, antigenic regions. Hughes et al, Infect. Immun. 60: 3497 (1992).
  • Polypeptide fragments and polypeptides having large internal deletions can be used for revealing epitopes that are otherwise masked in the parent polypeptide and that may be of importance for inducing, for example, a protective T cell-dependent immune response. Deletions can also remove immunodominant regions of high variability among strains.
  • Polynucleotides encoding polypeptide fragments and polypeptides having large internal deletions can be constructed using standard methods (see, e.g., Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons Inc. (1994)); for example, by PCR, including inverse PCR, by restriction enzyme treatment of the cloned DNA molecules, or by the method of Kunkel et al. (Proc. Natl Acad. Sci. USA 82: 448 (1985)); biological material available at Stratagene.
  • a polypeptide derivative can also be produced as a fusion polypeptide that contains a polypeptide or a polypeptide derivative of the invention fused, e.g., at the N- or C-terminal end, to any other polypeptide.
  • a first DNA encoding amino acid sequence corresponding to any of the portions of CPN100111, CPN 100224, CPN 100230, CPN 100231, CPN 100232, CPN 100233, CPN 100394, or CPN 100395 nucleotide sequence (SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 or 15) is joined to a second DNA using methods described in, for example, U.S.
  • a product can then be easily obtained by translation of the genetic fusion.
  • Vectors for expressing fusion polypeptides are commercially available, such as the pMal-c2 or pMal-p2 systems of New England Biolabs, in which the fusion peptide is a maltose binding protein, the glutathione-S-transferase system of Pharmacia, or the His-Tag system available from Novagen. These and other expression systems provide convenient means for further purification of polypeptides and derivatives of the invention.
  • fusion polypeptides included in the invention includes a polypeptide or polypeptide derivative of the invention fused to a polypeptide having adjuvant activity, such as, e.g., the subunit B of either cholera toxin or E. coli heat-labile toxin.
  • a polypeptide having adjuvant activity such as, e.g., the subunit B of either cholera toxin or E. coli heat-labile toxin.
  • the polypeptide of the invention can be fused to the N-, or preferably, to the C-terminal end of the polypeptide having adjuvant activity.
  • a polypeptide fragment of the invention can be fused within the amino acid sequence of the polypeptide having adjuvant activity.
  • the polynucleotides of the invention encode Chlamydia polypeptides in precursor or mature form. They can also encode hybrid precursors containing heterologous signal peptides, which can mature into polypeptides of the invention.
  • heterologous signal peptide is meant a signal peptide that is not found in the naturally-occurring precursor of a polypeptide of the invention.
  • a polynucleotide of the invention hybridizes, preferably under stringent conditions, to a polynucleotide having a sequence as shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 and 16. Hybridization procedures are described in, e.g., Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons Inc.
  • Tm 81.5 + 0.5 x (% G+C) + 1.6 log (positive ion concentration) - 0.6 x (% formamide).
  • hybridization temperature (Th) is approximately 20-40°C, 20-25°C or, preferably, 30-40°C below the calculated Tm.
  • optimal temperature and salt conditions can be readily determined empirically in preliminary experiments using conventional procedures. For example, stringent conditions can be achieved, both for pre-hybridizing and hybridizing incubations, (i) within 4-16 hours at 42°C, in 6xSSC containing 50% formamide or (ii) within 4-16 hours at 65°C in an aqueous 6xSSC solution (1 M NaCl, 0.1 M sodium citrate (pH 7.0)).
  • Tm 4 x (G+C) + 2 (A+T).
  • an 18 nucleotide fragment of 50% G+C would have an approximate Tm of 54°C.
  • a polynucleotide molecule of the invention can have various applications.
  • a DNA molecule can be used: (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 overexpress a polynucleotide of the invention or express it in a modified, mutated form, such as a non-toxic form, if appropriate.
  • the proteins and peptides and encoding nucleotides of the present invention for protection against diseases caused by Chlamydia, it is not preferred to use naked DNA encoding the protein or peptides and administering these nucleotides intranasally or intramuscularly.
  • Eukaryotic 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.
  • 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
  • plant cells e.g., a prokaryotic host such as E. coli is used.
  • Bacterial and eukaryotic cells are available from a number of different sources to those skilled in the art, e.g., the American Type Culture Collection (ATCC; Rockville, Maryland).
  • ATCC American Type Culture Collection
  • 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.
  • 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 can be homologous or heterologous to the DNA molecule encoding the mature polypeptide and can be specific to 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, signal peptide encoding regions are widely known and available to those skilled in the art and includes, 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 5,028,530 and in Cagnon et al., (Cagnon et al, Protein Engineering 4: 843 (1991)); the promoter of the gene of bacteriophage T7 encoding RNA polymerase, that is functional in a number of E. coli strains expressing T7 polymerase (described in U.S. Patent 4,952,496); OspA lipidation signal peptide; and RlpB lipidation signal peptide (Takase et al, J. Bad. 169: 5692 (1987)).
  • 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 from those described in Pouwels et al. (CLONING VECTORS: LABORATORY MANUAL, 85, Supp. 1987). They can be purchased from various commercial sources.
  • 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 can then be recovered in a substantially purified form from the cell extract or from the supernatant after centrifugation of the recombinant cell culture.
  • the recombinant polypeptide can be purified by antibody-based affinity purification or by any other method that can be readily adapted by a person skilled in the art, such as by genetic fusion to a small affinity binding domain.
  • Antibody-based affinity purification methods are also available for purifying a polypeptide of the invention extracted from a Chlamydia strain.
  • Antibodies useful for purifying by immunoaffinity the polypeptides of the invention can be obtained as described below.
  • a polynucleotide of the invention can also be useful in the vaccine field, e.g., for achieving
  • DNA vaccination There are two major possibilities, 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.
  • live vaccine vector live vaccine vector
  • Therapeutic or prophylactic efficacy of a polynucleotide of the invention can be evaluated as described below.
  • a vaccine vector such as a poxvirus, containing a DNA molecule of the invention, placed under the control of elements required for expression;
  • 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., cats or birds), which involves administering to the mammal an immunogenically effective amount of a vaccine vector of the invention to elicit an immune response, e.g., a protective or therapeutic immune response to Chlamydia; and particularly, (v) a method for preventing and
  • a vaccine vector of the invention can express one or several polypeptides or derivatives of the invention, as well as at least one additional Chlamydia antigen, fragment, homolog, mutant, or derivative thereof.
  • a cytokine such as interleukin-2 (IL-2) or interleukin-12 (IL-12), that enhances the immune response (adjuvant effect).
  • a vaccine vector can include an additional DNA sequence encoding, e.g., a chlamydial antigen , or a cytokine, placed under the control of elements required for expression in a mammalian cell.
  • a composition of the invention can include several vaccine vectors, each of them being capable of expressing a polypeptide or derivative of the invention.
  • a composition can also contain 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.
  • a vaccine vector of the invention can be administered by any conventional route in use in the vaccine field, particularly, to a mucosal (e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral route (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal).
  • a mucosal e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract
  • parenteral route e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal.
  • Preferred routes depend upon the choice of the vaccine vector.
  • the administration can be achieved 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 (weight, age and
  • Live vaccine vectors available in the art include viral vectors such as adenoviruses, alphavirus, and poxviruses as well as bacterial vectors, e.g., Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilie de Calmette-Guerin (BCG), and Streptococcus.
  • viral vectors such as adenoviruses, alphavirus, and poxviruses
  • bacterial vectors e.g., Shigella, Salmonella, Vibrio cholerae, 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 4,920,209.
  • Poxvirus vectors that can be used include, e.g., vaccinia and canary pox virus, described in U.S. Patent 4,722,848 and U.S. Patent 5,364,773, respectively (also see, e.g., Tartaglia et al., Virology 188: 217 (1992)) for a description of a vaccinia virus vector; and Taylor et al, Vaccine 13: 539 (1995) for a reference of a canary pox).
  • Poxvirus vectors capable of expressing a polynucleotide of the invention can be obtained by homologous recombination as described in Kieny et al., Nature 312: 163 (1984) 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 lxl 0 4 to about 1x10", advantageously from about lxlO 7 to about lxlO 10 , preferably of from about lxlO 7 to about lxl 0 9 plaque-forming units per kilogram.
  • viral vectors are administered parenterally, for example, in three doses, four weeks apart.
  • Those skilled in the art recognize that 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 described in Mekalanos et al., Nature 306: 551 (1983) and U.S. Patent 4,882,278 (strain in which a substantial amount of the coding sequence of each of the two ctxA alleles has been deleted so that no functional cholerae toxin is produced); WO 92/11354 (strain in which the irgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations); and WO 94/1533 (deletion mutant lacking functional ctxA and attRSl DNA sequences).
  • 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 can contain, e.g., about lxlO 5 to about lxlO 9 , preferably about lxl 0 6 to about lxlO 8 viable bacteria in an appropriate volume 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 6: 693 (1988) and WO 92/11361.
  • Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.
  • Others bacterial strains useful as vaccine vectors are described in High et al, EMBO 11:
  • polynucleotide of the invention can be inserted into the bacterial genome or can remain in a free state, carried on a plasmid.
  • An adjuvant can also be added to a composition containing a vaccine bacterial vector.
  • a number of adjuvants are known to those skilled in the art. Preferred adjuvants can be selected from the list provided below.
  • a fourth aspect of the invention there is also provided: (i) a composition of matter containing a polynucleotide of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition containing 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 administering to the mammal, an immunogenically effective amount of a polynucleotide of the invention to elicit an immune response, e.g., a protective immune response to Chlamydia; and particularly, (iv) a method for preventing and/or treating a Chlamydia (e.g.,
  • 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.
  • the fourth aspect of the invention preferably includes the use of a DNA molecule placed under conditions for expression in a mammalian cell, e.g., in a plasmid that is unable to replicate in mammalian cells and to substantially integrate in a mammalian genome.
  • Polynucleotides (DNA or RNA) of the invention can also be administered as such to a mammal for vaccine, e.g., therapeutic or prophylactic, purpose.
  • a DNA molecule of the invention can be in the form of a plasmid that is unable to replicate in a mammalian cell and unable to integrate in the mammalian genome.
  • a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell.
  • the promoter can function ubiquitously or tissue-specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (described in U.S.
  • Patent 4,168,062 and the Rous Sarcoma Virus promoter (described in Norton & Coffin, Molec. Cell Biol. 5: 281(1985)).
  • the desmin promoter (Li et al., Gene 78: 243 (1989), Li & Paulin, J. Biol. Chem. 266: 6562 (1991), and Li & Paulin, J. Biol Chem. 268: 10403 (1993)) is tissue-specific and drives expression in muscle cells. More generally, useful vectors are described, La., WO 94/21797 and Hartikka et al, Human Gene Therapy 7: 1205 (1996).
  • the polynucleotide of the invention can encode a precursor or a mature form.
  • the precursor form can be homologous or heterologous.
  • a eukaryotic leader sequence can be used, such as the leader sequence of the tissue-type plasminogen factor (tPA).
  • a composition of the invention can contain one or several polynucleotides of the invention.
  • DNA molecules of the invention and/or additional DNA molecules to be included in the same composition can be carried in the same plasmid.
  • Standard techniques of molecular biology for preparing and purifying polynucleotides can be used in the preparation of polynucleotide therapeutics of the invention.
  • a polynucleotide of the invention can be formulated according to various methods.
  • a polynucleotide can be used in a naked form, free of any delivery vehicles, such as anionic liposomes, cationic lipids, microparticles, e.g., gold microparticles, precipitating agents, e.g., calcium phosphate, or any other transfection-facilitating agent.
  • the polynucleotide can be 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.
  • a polynucleotide can be associated with agents that assist in cellular uptake. It can be, La., (i) complemented with a chemical agent that modifies the cellular permeability, such as bupivacaine (see, e.g., WO 94/16737), (ii) encapsulated into liposomes, or (iii) associated with cationic lipids or silica, gold, or tungsten microparticles.
  • bupivacaine see, e.g., WO 94/16737
  • encapsulated into liposomes or associated with cationic lipids or silica, gold, or tungsten microparticles.
  • Anionic and neutral liposomes are well-known in the art (see, e.g., LIPOSOMES: A PRACTICAL APPROACH, RPC New Ed, IRL press (1990)), for a detailed description of methods for making liposomes) and are useful for delivering a large range of products, including polynucleotides.
  • 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 (l,2-bis(oleyloxy)-3-(trimethylammonio)propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Chol (3 beta-(N-(N',N'-dimethyl aminomethane)-carbamoyl) cholesterol).
  • DC-Chol beta-(N-(N',N'-dimethyl aminomethane)-carbamoyl) cholesterol
  • Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine), as described in, e.g., WO 90/11092.
  • DOPE dioleyl phosphatidylethanolamine
  • Other transfection-facilitating compounds can be added to a formulation containing cationic liposomes. A number of them are described in, e.g., WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/2397.
  • spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane see, for example, WO 93/18759
  • membrane-permeabilizing compounds such as GALA, Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).
  • Gold or tungsten microparticles can also be used for gene delivery, as described in WO 91/359, WO 93/17706, and Tang et al. (Nature 356: 152 (1992)).
  • the microparticle-coated polynucleotides can be injected via intradermal or intra-epidermal routes using a needleless injection device ("gene gun"), such as those described in U.S. Patents 4,945,050 and 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 can be any conventional route used in the vaccine field.
  • a polynucleotide of the invention can be 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, intra- epidermal, or intramuscular route.
  • the choice of the administration route will depend on, e.g., the formulation that is selected.
  • 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 subcutaneous routes.
  • such a composition can also contain an adjuvant. If so, 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 5,057,546.
  • nucleotide probe or primer having a sequence found in or derived by degeneracy of the genetic code from a sequence shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 and 15.
  • 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 sequences homologous to those shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 and 15, or to a complementary or anti-sense sequence.
  • probes are significantly shorter than full-length sequences shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 and 15; for example, they can 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 a sequence as shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 and 15 or that are complementary to such sequences.
  • Probes can contain modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, or diamino-2, 6-purine. Sugar or phosphate residues can also be modified or substituted.
  • a deoxyribose residue can be replaced by a polyamide (Nielsen et al., Science 254: 1497 (1991)) and phosphate residues can be replaced by ester groups such as diphosphate, alkyl, arylphosphonate and phosphorothioate esters.
  • ester groups such as diphosphate, alkyl, arylphosphonate and phosphorothioate esters.
  • the 2'-hydroxyl group on ribonucleotides can be modified by including, e.g., alkyl groups.
  • Probes of the invention can be used in diagnostic tests, as capture or detection probes. Such capture probes can be conventionally immobilized on a solid support, directly or indirectly, by covalent means or by passive adsorption.
  • a detection probe can be 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 can be 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. 98: 503 (1975)), northern blot (identical to Southern blot to the exception that RNA is used as a target), or the sandwich technique (Dunn et al, Cell 12: 23 (1977)).
  • dot blot Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL (1982) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
  • Southern blot Southern blot
  • northern blot identical to Southern blot to the exception that RNA is used as a target
  • sandwich technique Nordet al, Cell 12: 23 (1977)
  • the latter technique involves the use of a specific capture probe and/or a
  • a primer is usually a probe of 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 can be labelled.
  • the invention also encompasses: (i) a reagent containing 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 amplified DNA
  • polypeptides that can be produced upon expression of the newly identified open reading frames are useful vaccine agents.
  • 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.
  • a "substantially purified polypeptide” 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 can be purified from a natural source, i.e., a Chlamydia strain, or can be produced by recombinant means.
  • Homologous polypeptides or polypeptide derivatives encoded by polynucleotides of the invention can be screened for specific antigenicity by testing cross-reactivity with an antiserum raised against the polypeptide of reference having an amino acid sequence as shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 and 16. Briefly, a monospecific hyperimmune antiserum can be raised against a purified reference polypeptide as such or as a fusion polypeptide, for example, an expression product of MBP, GST, or His-tag systems or a synthetic peptide predicted to be antigenic. The homologous polypeptide or derivative screened for specific antigenicity can be produced as such or as a fusion polypeptide.
  • the product to be screened is submitted to SDS-Page electrophoresis as described by Laemmli, Nature 227: 680 (1970).
  • SDS-Page electrophoresis as described by Laemmli, Nature 227: 680 (1970).
  • 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 1:100 to about 1 :500.
  • Specific antigenicity is shown once a band corresponding to the product exhibits reactivity at any of the dilutions in the above range.
  • 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). Under the above experimental conditions, a positive reaction is shown by O.D. values greater than a non immune control serum.
  • a purified product is preferred, although a whole cell extract can also be used.
  • 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.
  • Therapeutic or prophylactic efficacy of a polypeptide or derivative of the invention can be evaluated as described below.
  • composition of matter containing a polypeptide of the invention together with a diluent or carrier; in particular,
  • a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polypeptide of the invention containing a therapeutically or prophylactically effective amount of a polypeptide of the invention
  • 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 an immune response, e.g., a protective immune response to Chlamydia
  • an immune response e.g., a protective immune response to Chlamydia
  • a method for preventing and/or treating a Chlamydia e.g., C. trachomatis. C. psittaci, C. pneumoniae. or C.
  • 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 can be administered by any conventional route in use in 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.
  • the choice of the administration route depends upon a number of parameters, such as the adjuvant associated with the polypeptide. For example, if a mucosal adjuvant is used, the intranasal or oral route will be preferred and if a lipid formulation or an aluminum compound is used, the parenteral route will be preferred. In the latter case, the subcutaneous or intramuscular route is most preferred.
  • the choice can also depend upon the nature of the vaccine agent. For example, a polypeptide of the invention fused to CTB or LTB will be best administered to a mucosal surface.
  • a composition of the invention can contain one or several polypeptides or derivatives of the invention. It can also contain at least one additional Chlamydia antigen, or a subunit, fragment, homolog, mutant, or derivative thereof.
  • a polypeptide or derivative thereof can be 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.
  • VLPs virus-like-particles
  • Adjuvants other than liposomes and the like can also be used and are known in the art. An appropriate selection can conventionally be made by those skilled in the art, for example, from the list provided below.
  • Administration can be achieved in a single dose or repeated as necessary at intervals as can be determined by one skilled in the art. For example, a priming dose can be 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 can be 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 should not exceed about 1 mg, preferably about 100 ⁇ g.
  • polynucleotides and polypeptides of the invention can be used sequentially as part of a multistep immunization process.
  • a mammal can be 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 can also be 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 useful as a diagnostic reagent for detecting the presence of an ⁇ -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 and can be labeled or unlabeled, depending upon the diagnostic method. Diagnostic methods involving such a reagent are described below.
  • a polypeptide or polypeptide derivative Upon expression of a DNA molecule of the invention, a polypeptide or polypeptide derivative is produced and can be purified using known laboratory techniques.
  • the polypeptide or polypeptide derivative can be produced as a fusion protein containing a fused tail that facilitates purification.
  • the fusion product can be 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).
  • the eighth aspect of the invention thus provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the invention.
  • Monospecific antibody an antibody that is capable of reacting with a unique naturally-occurring Chlamydia polypeptide.
  • An antibody of the invention can be polyclonal or monoclonal.
  • Monospecific antibodies can 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 can also be in the form of immunoglobulin fragments, e.g., F(ab)'2 or Fab fragments.
  • the antibodies of the invention can be of any isotype, e.g., IgG or IgA, and polyclonal antibodies can be of a single isotype or can contain a mixture of isotypes.
  • the antibodies of the invention which are raised to a polypeptide or polypeptide derivative of the invention, can be 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 can be used in diagnostic methods to detect the presence of a Chlamydia antigen in a sample, such as a biological sample.
  • the antibodies can also be used in affinity chromatography methods for purifying a polypeptide or polypeptide derivative of the invention. As is discussed further below, such antibodies can 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 can be removed prior to detecting the complex.
  • a polypeptide reagent is useful for detecting the presence of mti-Chlamydia antibodies in a sample, e.g., a blood sample, while an antibody of the invention can be 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 can be 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 can also employ a ligand-receptor system, for example, a molecule such as a vitamin can be grafted onto the polypeptide reagent and the corresponding receptor can be immobilized on the solid phase. This is illustrated by the biotin-streptavidin system.
  • indirect means can be used, e.g., by adding to the reagent a peptide tail, chemically or by genetic engineering, and immobilizing the grafted or fused product by passive adsorption or covalent linkage of the peptide tail.
  • 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 can be polyclonal or monospecific, and preferably is of the IgG type.
  • Purified IgGs can be 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 disclosed in, e.g., ANTIBODIES: A LABORATORY MANUAL, D. Lane, E. Harlow, Eds. (1988).
  • 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, can be in batch form or in 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 antibody of the invention can be screened for therapeutic efficacy as described as follows.
  • a composition of matter containing a monospecific antibody of the invention together with a diluent or carrier;
  • a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a monospecific antibody of the invention and
  • a method for treating or preventing a Chlamydia e.g., C. trachomatis, C. psittaci, 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 can be polyclonal or monoclonal, preferably of the IgA isotype (predominantly).
  • the antibody can be 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, can be carried out.
  • a monospecific antibody of the invention can be 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 can be 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, can be an effective regimens for most purposes.
  • Therapeutic or prophylactic efficacy can be 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 can be replaced with another Chlamydia strain.
  • the efficacy of DNA molecules and polypeptides from C. pneumoniae is preferably evaluated in a mouse model using an C. pneumoniae strain. Protection can be 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.
  • Such an evaluation can be made for polynucleotides, vaccine vectors, polypeptides and derivatives thereof, as well as antibodies of the invention.
  • 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 can be precipitated with, or adsorbed onto, the aluminum compound according to standard protocols.
  • Other adjuvants such as RIBI (ImmunoChem, Hamilton, MT), can be 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.
  • CT cholera toxin
  • LT E. coli heat-labile toxin
  • PT pertussis toxin
  • a purified preparation of native cholera toxin subunit B (CTB) can be of use. Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity.
  • a mutant having reduced toxicity is used.
  • 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 can be 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. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri; saponins, or polylactide glycolide (PLGA) microspheres, can also be used in mucosal administration.
  • MPLA bacterial monophosphoryl lipid A
  • PLGA polylactide glycolide
  • 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 5,283,185 and WO 96/14831) and QS-21 (WO 88/9336).
  • any pharmaceutical composition of the invention containing a polynucleotide, a polypeptide, a polypeptide derivative, or an antibody of the invention, can be manufactured in a conventional manner.
  • it can be formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline solution such as phosphate buffer saline.
  • a diluent or carrier can be 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, can be 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 can readily be determined by one skilled in the art.
  • one skilled in the art can readily design treatment immunization schedules.
  • the non-vaccine components can be administered on days 1-14
  • the vaccine antigen + adjuvant can be administered on days 7, 14, 21, and 28.

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Abstract

La présente invention concerne essentiellement un procédé mettant en oeuvre un acide nucléique, et notamment de l'ADN, pour immuniser un hôte tel qu'un humain contre des affections imputables à une souche de Chlamydia, et plus particulièrement de C. pneumoniae. On utilise à cet effet un vecteur contenant une séquence de nucléotides codant l'un quelconque des polypeptides tels que les CPN 100111, CPN 100224, CPN 100230, CPN 100231, CPN 100232, CPN 100235, CPN 100394 et CPN 100395 d'une souche de Chlamydia pneumoniae. L'invention concerne également un promoteur capable de réaliser l'expression chez l'hôte de l'un quelconque de ces polypeptides. Des modifications restent possible dans les limites des intentions de cette invention.
EP99938465A 1998-08-20 1999-08-18 Antigenes de chlamydia, fragments d'adn correspondants et leur utilisation Withdrawn EP1104470A2 (fr)

Applications Claiming Priority (19)

Application Number Priority Date Filing Date Title
US9719598P 1998-08-20 1998-08-20
US9719798P 1998-08-20 1998-08-20
US9718798P 1998-08-20 1998-08-20
US9719698P 1998-08-20 1998-08-20
US9718998P 1998-08-20 1998-08-20
US9719098P 1998-08-20 1998-08-20
US9718898P 1998-08-20 1998-08-20
US97189P 1998-08-20
US97188P 1998-08-20
US97187P 1998-08-20
US97195P 1998-08-20
US97197P 1998-08-20
US97190P 1998-08-20
US97196P 1998-08-20
US9719198P 1998-08-27 1998-08-27
US97191P 1998-08-27
US37677099A 1999-08-17 1999-08-17
US376770 1999-08-17
PCT/IB1999/001449 WO2000011183A2 (fr) 1998-08-20 1999-08-18 Antigenes de chlamydia, fragments d'adn correspondants et leur utilisation

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WO2000034483A2 (fr) * 1998-12-08 2000-06-15 Corixa Corporation Composes et procedes pour le traitement et le diagnostic d'infections par le chlamydia
GB0203403D0 (en) * 2002-02-13 2002-04-03 Chiron Spa Chlamydia cytotoxic-T cell epitopes
US7807802B2 (en) 2002-11-12 2010-10-05 Abbott Lab Polynucleotides for the amplification and detection of Chlamydia trachomatis and Neisseria gonorrhoeae
GB0709373D0 (en) * 2007-05-16 2007-06-27 Thrombosis Res Inst BCG-based anti-atheroma vaccine

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CN100365017C (zh) * 1994-09-20 2008-01-30 日立化成工业株式会社 肺炎衣原体抗原多肽
CA2263316A1 (fr) * 1996-08-14 1998-02-19 Klara Berencsi Procedes et compositions pour empecher ou pour retarder le developpement de lesions atherosclereuses
BR9814878A (pt) * 1997-11-21 2000-10-03 Genset Sa Sequência genÈmica e polipeptìdeos de chlamydia pneumoniae, fragmentos dos mesmos e usos dos mesmos, em particular para o diagnóstico, a prevenção e o tratamento de infecção

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AU5297399A (en) 2000-03-14
WO2000011183A2 (fr) 2000-03-02

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