EP1133572A2 - Sequence genomique de chlamydia pneumoniae - Google Patents

Sequence genomique de chlamydia pneumoniae

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Publication number
EP1133572A2
EP1133572A2 EP99960323A EP99960323A EP1133572A2 EP 1133572 A2 EP1133572 A2 EP 1133572A2 EP 99960323 A EP99960323 A EP 99960323A EP 99960323 A EP99960323 A EP 99960323A EP 1133572 A2 EP1133572 A2 EP 1133572A2
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European Patent Office
Prior art keywords
protein
procein
hypothetical
nucleic acid
hypothencal
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EP99960323A
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German (de)
English (en)
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EP1133572A4 (fr
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Richard Stephens
Wayne Mitchell
Sue Kalman
Ronald Davis
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University of California
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University of California
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Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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

Definitions

  • This invention relates to nucleic acids and polypeptides from Chlamydia pneumoniae and to their use in the diagnosis, prevention and treatment of diseases associated with C. pneumoniae.
  • Chlamydiaceae is a family of obligate intracellular parasite with a tropism for epithelial cells lining the mucus membranes.
  • the bacteria have two morphologically distinct forms, "elementary body” and “reticulate body”.
  • the elementary body is the infectious form, and has a rigid cell wall, primarily of cross-linked outer membrane proteins.
  • the reticulate body is the intracellular, metabohcally active form. A unique developmental cycle between these two forms characterizes Chlamydia growth.
  • C. pneumoniae is a human respiratory pathogen that causes acute respiratory disease, and approximately 10% of community-acquired pneumonia. Antibody prevalence studies have shown that virtually everyone is infected with C. pneumoniae at some time, and that reinfection is common.
  • C. pneumoniae is related to other Chlamydia species, but the level of sequence similarity is relatively low. Very little is known about the biology of this organism, although it appears to be an important human pathogen. Allelic diversity and structural relationships between specific genes of Chlamydial species is described in Kaltenboeck et al. (1993) J Bacteriol 175(2):487-502; Gaydos et al.
  • This invention provides the genomic sequence of Chlamydia pneumoniae.
  • the sequence information is useful for a variety of diagnostic and analytical methods.
  • the genomic sequence may be embodied in a variety of media, including computer readable forms, or as a nucleic acid comprising a selected fragment of the sequence.
  • Such fragments generally consist of an open reading frame, transcriptional or translational control elements, or fragments derived therefrom. Proteins encoded by the open reading frames are useful for diagnostic purposes, as well as for their enzymatic or structural activity.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group., e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the rUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • “Amplification” primers are oligonucleotides comprising either natural or analogue nucleotides that can serve as the basis for the amplification of a select nucleic acid sequence. They include, e.g., polymerase chain reaction primers and ligase chain reaction oligonucleotides.
  • Antibody refers to an immunoglobulin molecule able to bind to a specific epitope on an antigen.
  • Antibodies can be a polyclonal mixture or monoclonal. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies may exist in a variety of forms including, for example, Fv, F aD , and F(ab) , as well as in single chains. Single-chain antibodies, in which genes for a heavy chain and a light chain are combined into a single coding sequence, may also be used.
  • an "antigen” is a molecule that is recognized and bound by an antibody, e.g., peptides, carbohydrates, organic molecules, or more complex molecules such as glycolipids and glycoproteins.
  • the part of the antigen that is the target of antibody binding is an antigenic determinant and a small functional group that corresponds to a single antigenic determinant is called a hapten.
  • Bio sample refers to any sample obtained from a living or dead organism.
  • biological samples include biological fluids and tissue specimens. Such biological samples can be prepared for analysis of the presence of C. pneumoniae nucleic acids, proteins, or antibodies specifically reactive with the proteins.
  • C pneumoniae gene shall be intended to mean the open reading frame encoding specific C. pneumoniae polypeptides, as well as adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression, up to about 2 kb beyond the coding region, but possibly further in either direction.
  • the gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into a host genome.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences.
  • conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res . 19:5081 (1991); Ohtsuka et al., J. Biol. Chem.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid can be modified to yield a functionally identical molecule. Accordingly, each silen: variation of a nucleic acid which encodes a polypeptide is implicit in each describ id sequence.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • This definition also refers to the complement of a test sequence, which has a designated percent sequence or subsequence complementarity when the test sequence has a designated or substantial identity to a reference sequence.
  • a designated amino acid percent identity of 95% refers to sequences or subsequences that have at least about 95% amino acid identity when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences would then be said to have substantial identity, or to be substantially identical to each other.
  • sequences have at least about 70% identity, more preferably 80% identity, more preferably 90-95% identity and above.
  • the percent identity exists over a region of the sequence that is at least about 25 amino acids in length, more preferably over a region that is 50-100 amino acids in length.
  • sequence identity When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
  • a conservative substitution is given a score between zero and 1.
  • the scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).
  • PC/GENE Intelligents, Mountain View, California, USA.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Pefault program parameters can be used, or alternative parameters can be designated.
  • the sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated or default program parameters.
  • a comparison window includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 25 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment.
  • PILEUP uses a simplification of the progressive alignment method of Feng & Ooolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153 (1989).
  • the program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
  • the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences.
  • This cluster is then aligned to the next most related sequence or cluster of aligned sequences.
  • Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences.
  • the final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.
  • PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package, e.g, version 7.0 (Pevereaux et al, Nuc. Acids Res. 12:387-395 (1984).
  • BLAST algorithm Another example of algorithm that is suitable for determining percent sequence identity (i.e., substantial similarity or identity) is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra).
  • HSPs high scoring sequence pairs
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues, always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as default parameters a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat 'I.
  • nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross ,-eactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative suostitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • stringent conditions are sequence dependent and will be different in different circumstances.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Typical stringent conditions for a Southern blot protocol involve hybridizing in a buffer comprising 5x SSC, 1% SPS at 65°C or hybridizing in a buffer containing 5x SSC and 1% SPS at 42°C and washing at 65°C with a 0.2x SSC, 0.1% SPS wash.
  • label is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • nucleic acids containing known nucleotide analogs or modified backbone residues or linkages which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • nucleic acid probe or oligonucleotide is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.).
  • the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
  • probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.
  • probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
  • the probes are preferably directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
  • a labeled nucleic acid probe or oligonucleotide is one that is bound, either covalently, through a linker, or through ionic, van der Waals or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
  • “Pharmaceutically acceptable” means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with a Chlamydia antigen without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition.
  • polypeptide polypeptide
  • peptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the phrase "specifically or selectively hybridizing to,” refers to hybridization between a probe and a target sequence in which the probe binds substantially only to the target sequence, forming a hybridization complex, when the target is in a heterogeneous mixture of polynucleotides and other compounds. Such hybridization is determinative of the presence of the target sequence.
  • the probe may bind other unrelated sequences, at least 90%, preferably 95% or more of the hybridization complexes formed are with the target sequence.
  • recombinant when used with reference to a cell, or nucleic acid, or vector, indicates that the cell, or nucleic acid, or vector, has been modified by the introduction of a heterologous nucleic acid or the alteration of a native nucleic acid, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein and are described in detail below.
  • substantially pure or “isolated” when referring to a Chlamydia peptide or protein means a chemical composition which is free of other subcellular components of the Chlamydia organism.
  • a monomeric protein is substantially pure when at least about 85% or more of a sample exhibits a single polypeptide backbone. Minor variants or chemical modifications may typically share the same polypeptide sequence. Oepending on the purification procedure, purities of 85%, and preferably over 95% pure are possible.
  • Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band on a polyacrylamide gel upon silver staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized.
  • the present invention provides the nucleotide sequence of the C. pneumoniae genome SEQ IP NO: 1 or a representative fragment thereof, in a form which can be readily used, analyzed, and interpreted by a skilled artisan.
  • a "representative fragment" of the nucleotide sequence depicted in SEQ IP NO: 1 refers to any portion which is not presently represented within a publicly available database.
  • Preferred representative fragments of the present invention are open reading frames, expression modulating fragments, uptake modulating fragments, and fragments which can be used to diagnose the presence of C. pneumoniae in sample.
  • nucleic acid hybridization-based assays for the detection of C. pneumoniae. Any of a number of well known techniques for the specific detection of target nucleic acids can be used.
  • Exemplary hybridization-based assays include, but are not limited to, traditional "direct probe” methods such as Southern Blots, dot blots, m situ /zybndization (e g , FISH), PCR, and the like
  • the methods can be used in a wide vanety of formats including, but not limited to substrate- (e g membrane or glass) bound methods or array-based approaches as desc ⁇ bed below
  • this invention also embraces methods for detecting the presence of Chlamydia PNA or RNA m biological samples These sequences can be used to detect Chlamydia in biological samples from patients suspected of being infected
  • a vanety of methods of specific PNA and RNA measurement using nucleic acid hybridization techniques are known to those of skill in the art (see Sambrook et al , supra) In situ h>bndization assays are well known (e g , Angerer (1987) Meth
  • in situ hybridization comprises the following major steps (1) fixation of tissue or biological structure to analyzed, (2) prehyb ⁇ dization treatment of the biological structure t increase accessibility of target PNA, and to reduce nonspecific binding, (3) hybndizati n of the mixture of nucleic acids to the nucleic acid m the biological structure or tissue, (4) post-hyb ⁇ dization washes to remove nucleic acid fragments not bound in the hybridization and (5) detection of the hyb ⁇ dized nucleic acid fragments
  • the reagent used in each of these steps and the conditions for use vary depending on the particular application
  • cells are fixed to a solid support, typically a glass slide If a nucleic acid is to be probed, the cells are typically denatured with heat or alkali The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein
  • the targets e g , cells
  • the targets are then typically washed at a predetermined st ⁇ ngency or at an increasing st ⁇ ngency until an approp ⁇ ate signal to noise ratio is obtained
  • nucleic acids of this invention are particularly well suited to array- based hyb ⁇ dization formats
  • Arrays are a multiplicity of different "probe” or “target” nucleic acids (or other compounds) attached to one or more surfaces (e g , solid, membrane, or gel).
  • the multiplicity of nucleic acids (or other moieties) is attached to a single contiguous surface or to a multiplicity of surfaces juxtaposed to each other
  • mate ⁇ al for the solid surface
  • Illustrative solid surfaces include, e g , nitrocellulose, nylon, glass, quartz, diazotized membranes (paper or nylon), sihcones, polyformaldehyde, cellulose, and cellulose acetate
  • plastics such as polyethylene, polypropylene, polystyrene, and the like can be used.
  • mate ⁇ als which may be employed include paper, ceramics, metals, metalloids, semiconductive mate ⁇ als, cermets or the like
  • substances that form gels can be used
  • Such matenals include, e.g , proteins (e.g , gelatins), hpopolysacchandes, silicates, agarose and polyacrylamides
  • vanous pore sizes may be employed depending upon the nature of the system In prepa ⁇ ng the surface, a plurality of different matenals may be employed, particularly as laminates, to obtain vanous properties.
  • proteins e g , bovme serum albumin
  • macromolecules e g , Oenhardt's solution
  • the surface will usually be polyfunctional or be capable of being polyfunctionalized.
  • Functional groups which may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like. The manner of linking a wide variety of compounds to various surfaces is well known and is amply illustrated in the literature.
  • Target elements of various sizes ranging from 1 mm diameter down to 1 ⁇ m can be used.
  • Smaller target elements containing low amounts of concentrated, fixed probe PNA are used for high complexity comparative hybridizations since the total amount of sample available for binding to each target element will be limited.
  • Such small array target elements are typically used in arrays with densities greater than 10 /cm 2 .
  • Relatively simple approaches capable of quantitative fluorescent imaging of 1 cm 2 areas have been described that permit acquisition of data from a large number of target elements in a single image (see, e.g., Wittrup (1994) Cytometry 16:206-213).
  • Substrates such as glass or fused silica are advantageous in that they provide a very low fluorescence substrate, and a highly efficient hybridization environment.
  • Covalent attachment of the target nucleic acids to glass or synthetic fused silica can be accomplished according to a number of known techniques (described above). Nucleic acids can be conveniently coupled to glass using commercially available reagents.
  • materials for preparation of silanized glass with a number of functional groups are commercially available or can be prepared using standard techniques (see, e.g., Gait (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press, Wash., O.C.). Quartz cover slips, which have at least 10- fold lower auto fluorescence than glass, can also be silanized.
  • probes can also be immobilized on commercially available coated beads or other surfaces.
  • biotin end-labeled nucleic acids can be bound to commercially available avidin-coated beads.
  • Streptavidin or anti-digoxigenin antibody can also be attached to silanized glass slides by protein-mediated coupling using e.g., protein A following standard protocols (see, e.g., Smith (1992) Science 258: 1122- 1126).
  • Biotin or digoxigenin end-labeled nucleic acids can be prepared according to standard techniques. Hybridization to nucleic acids attached to beads is accomplished by suspending them in the hybridization mix, and then depositing them on the glass substrate for analysis after washing.
  • paramagnetic particles such as ferric oxide particles, with or without avidin coating, can be used.
  • nucleic acid hybridization formats are known to those skilled in the art.
  • common formats include sandwich assays and competition or displacement assays.
  • Hybridization techniques are generally described in Hames and Higgins (1985) Nucleic Acid Hybridization, A Practical Approach, IRL Press; Gall and Pardue (1969) Proc. Natl. Acad. Sci. USA 63: 378-383; and John et al. (1969) Nature 223: 582-587.
  • Sandwich assays are commercially useful hybridization assays for detecting or isolating nucleic acid sequences. Such assays utilize a "capture" nucleic acid covalently immobilized to a solid support and a labeled "signal" nucleic acid in solution. The sample will provide the target nucleic acid. The "capture” nucleic acid and “signal” nucleic acid probe hybridize with the target nucleic acid to form a "sandwich” hybridization complex. To be most effective, the signal nucleic acid should not hybridize with the capture nucleic acid. Petection of a hybridization complex may require the binding of a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids.
  • such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal.
  • the sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected. Examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Other methods recently described in the art are the nucleic acid sequence based amplification (NASBAO, Cangene, Mississauga, Ontario) and Q Beta Replicase systems.
  • Nucleic acid hybridization simply involves providing a denatured probe and target nucleic acid under conditions where the probe and its complementary target can form stable hybrid duplexes through complementary base pairing. The nucleic acids that do not form hybrid duplexes are then washed away leaving the hybridized nucleic acids to be detected, typically through detection of an attached detectable label. It is generally recognized that nucleic acids are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids, or in the addition of chemical a ents, or the raising of the pH.
  • hybrid duplexes Under low stringency conditions (e.g., low temperature and/or high salt and/or high target concentration) hybrid duplexes (e.g., ONA:PNA, RNA:RNA, or RNA:PNA) will form even where the annealed sequences are not perfectly complementary. Thus specificity of hybridization is reduced at lower stringency. Conversely, at higher stringency (e.g., higher temperature or lower salt) successful hybridization requires fewer mismatches.
  • hybridization conditions may be selected to provide any degree of stringency. In a preferred embodiment, hybridization is performed at low stringency to ensure hybridization and then subsequent washes are performed at higher stringency to eliminate mismatched hybrid duplexes.
  • Successive washes may be performed at increasingly higher stringency (e.g., down to as low as 0.25 X SSPE-T at 37°C to 70°C) until a desired level of hybridization specificity is obtained.
  • Stringency can also be increased by addition of agents such as formamide.
  • Hybridization specificity may be evaluated by comparison of hybridization to the test probes with hybridization to the various controls that can be present.
  • the wash is performed at the highest stringency that produces consistent results and that provides a signal intensity greater than approximately 10% of the background intensity.
  • the hybridized array may be washed at successively higher stringency solutions and read between each wash. Analysis of the data sets thus produced will reveal a wash stringency above which the hybridization pattern is not appreciably altered and which provides adequate signal for the particular probes of interest.
  • the hybridized nucleic acids are detected by detecting one or more labels attached to the sample or probe nucleic acids.
  • the labels may be incorporated by any of a number of means well known to those of skill in the art.
  • Means of attaching labels to nucleic acids include, for example nick translation or end- labeling (e.g. with a labeled RNA) by kinasing of the nucleic acid and subsequent attachment (ligation) of a nucleic acid linker joining the sample nucleic acid to a label (e.g., a fluorophore).
  • a label e.g., a fluorophore
  • linkers for the attachment of labels to nucleic acids are also known.
  • intercalating dyes and fluorescent nucleotides can also be used.
  • Oetectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., PynabeadsTM), fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like, see, e.g., Molecular Probes, Eugene, Oregon, USA), radiolabels (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold (e.g., gold particles in the 40 -80 nm diameter size range scatter green light with high efficiency) or colored glass or plastic (e.g., polysty
  • a fluorescent label is preferred because it provides a very strong signal with low background. It is also optically detectable at high resolution and sensitivity through a quick scanning procedure.
  • the nucleic acid samples can all be labeled with a single label, e.g., a single fluorescent label.
  • different nucleic acid samples can be simultaneously hybridized where each nucleic acid sample has a different label. For instance, one target could have a green fluorescent label and a second target could have a red fluorescent label. The scanning step will distinguish cites of binding of the red label from those binding the green fluorescent label.
  • Each nucleic acid sample (target nucleic acid) can be analyzed independently from one another.
  • Suitable chromogens which can be employed include those molecules and compounds which absorb light in a distinctive range of wavelengths so that a color can be observed or, alternatively, which emit light when irradiated with radiation of a particular wave length or wave length range, e.g., fluorescers.
  • fluorescers should absorb light above about 300 nm, preferably about 350 nm, and more preferably above about 400 nm, usually emitting at wavelengths greater than about 10 nm higher than the wavelength of the light absorbed. It should be noted that the absorption and emission characteristics of the bound dye can differ from the unbound dye. Therefore, when referring to the various wavelength ranges and characteristics of the dyes, it is intended to indicate the dyes as employed and not the dye which is unconjugated and characterized in an arbitrary solvent.
  • Fluorescers are generally preferred because by irradiating a fluorescer with light, one can obtain a plurality of emissions. Thus, a single label can provide for a plurality of measurable events.
  • Petectable signal can also be provided by chemiluminescent and bioluminescent sources.
  • Chemiluminescent sources include a compound which becomes electronically excited by a chemical reaction and can then emit light which serves as the detectable signal or donates energy to a fluorescent acceptor.
  • luciferins can be used in conjunction with luciferase or lucigenins to provide bioluminescence.
  • Spin labels are provided by reporter molecules with an unpaired electron spin which can be detected by electron spin resonance (ESR) spectroscopy.
  • ESR electron spin resonance
  • Exemplary spin labels include organic free radicals, transitional metal complexes, particularly vanadium, copper, iron, and manganese, and the like.
  • Exemplary spin labels include nitroxide free radicals.
  • the label may be added to the target (sample) nucleic acid(s) prior to, or after the hybridization.
  • direct labels are detectable labels that are directly attached to or incorporated into the target (sample) nucleic acid prior to hybridization.
  • indirect labels are joined to the hybrid duplex after hybridization.
  • the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization.
  • the target nucleic acid may be biotinylated before the hybridization. After hybridization, an avidin-conjugated fluorophore will bind the biotin bearing hybrid duplexes providing a label that is easily detected.
  • Fluorescent labels are easily added during an in vitro transcription reaction.
  • fluorescein labeled UTP and CTP can be inco ⁇ orated into the RNA produced in an in vitro transcription.
  • the labels can be attached directly or through a linker moiety.
  • the site of label or linker-label attachment is not limited to any specific position.
  • a label may be attached to a nucleoside, nucleotide, or analogue thereof at any position that does not interfere with detection or hybridization as desired.
  • certain Label-ON Reagents from Clontech (Palo Alto, C A) provide for labeling interspersed throughout the phosphate backbone of an oligonucleotide and for terminal labeling at the 3' and 5' ends.
  • labels can be attached at positions on the ribose ring or the ribose can be modified and even eliminated as desired.
  • the base moieties of useful labeling reagents can include those that are naturally occurring or modified in a manner that does not interfere with the purpose to which they are put.
  • Modified bases include but are not limited to 7-deaza A and G, 7-deaza-8-aza A and G, and other heterocyclic moieties.
  • fluorescent labels are not to be limited to single species organic molecules, but include inorganic molecules, multi-molecular mixtures of organic and/or inorganic molecules, crystals, heteropolymers, and the like.
  • CdSe-CdS core-shell nanocrystals enclosed in a silica shell can be easily derivatized for coupling to a biological molecule (Bruchez et al (1998) Science, 281: 2013-2016).
  • highly fluorescent quantum dots (zinc sulfide-capped cadmium selenide) have been covalently coupled to biomolecules for use in ultrasensitive biological detection (Warren and Nie (1998) Science, 281: 2016-2018).
  • amplification-based assays can be used to detect nucleic acids.
  • the nucleic acid sequences act as a template in an amplification reaction (e g Polymerase Cham Reaction (PCR) Detailed protocols for quantitative PCR are provided in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
  • ligase chain reaction LCR
  • LCR ligase chain reaction
  • Genomics 4- 560 Landegren et al (1988) Science 241 : 1077
  • Barnnger et al. (1990) Gene 89 117 transcnption amplification
  • transcnption amplification Kwoh et al. (1989) Proc. Natl Acad. Sci USA 86. 1173
  • self- sustamed sequence replication (Guatelh et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874).
  • the nucbic acids of the invention can also be used to C pneumoniae detect gene transcnpts
  • Methods of detecting and/or quantifying gene transcnpts using nucleic acid hybndizatu >n techniques are known to those of skill in the art (see Sambrook et al. supra).
  • a Northern transfer may be used for the detection of the desired mRNA directly.
  • the mRNA is isolated from a given cell sample using, for example, an acid guanidinium-phenol-chloroform extraction method. The mRNA is then electrophoresed to separate the mRNA species and the mRNA is transferred from the gel to a nitrocellulose membrane.
  • labeled probes are used to identify and/or quantify the target mRNA.
  • the gene transcript can be measured using amplification (e.g PCR) based methods as desc ⁇ bed above for directly assessing copy number of the target sequences
  • nucleic acids disclosed here can be used for recombinant expression of the proteins.
  • the nucleic acids encoding the proteins of interest are introduced into suitable host cells, followed by induction of the cells to produce large amounts of the protein.
  • the invention relies on routine techniques in the field of recombinant genetics, well known to those of ordinary skill in the art. A basic text disclosing the general methods of use in this invention is Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989).
  • Standard transfection methods are used to produce prokaryotic, mammalian, yeast or insect cell lines which express large quantities of the desired polypeptide, which is then purified using standard techniques (see, e.g., Colley et al, J. Biol Chem. 264: 17619-17622, 1989; Guide to Protein Purification, supra).
  • the nucleotide sequences used to transfect the host cells can be modified to yield Chlamydia polypeptides with a variety of desired properties.
  • the polypeptides can vary from the naturally-occurring sequence at the primary structure level by amino acid, insertions, substitutions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.
  • the amino acid sequence variants can be prepared with various objectives in mind, including facilitating purification and preparation of the recombinant polypeptide.
  • the modified polypeptides are also useful for modifying plasma half life, improving therapeutic efficacy, and lessening the severity or occurrence of side effects during therapeutic use.
  • the amino acid sequence variants are usually predetermined variants not found in nature but exhibit the same immunogenic activity as naturally occurring protein.
  • modifications of the sequences encoding the polypeptides may be readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis (see Gillman & Smith, Gene 8:81-97 (1979); Roberts et al, Nature 328:731- 734 (1987)).
  • the effect of many mutations is difficult to predict. Thus, most modifications are evaluated by routine screening in a suitable assay for the desired characteristic. For instance, the effect of various modifications on the ability of the polypeptide to elicit a protective immune response can be easily determined using in vitro assays. For instance, the polypeptides can be tested for their ability to induce lymphoproliferation, T cell cytotoxicity, or cytokine production using standard techniques.
  • the particular procedure used to introduce the genetic material into the host cell for expression of the polypeptide is not particularly critical. Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, spheroplasts, electroporation, liposomes, microinjection, plasmid vectors, viral vectors and any of the other well known methods for introducing cloned genomic PNA, cPNA, synthetic PNA or other foreign genetic material into a host cell (see Sambrook et al. , supra). It is only necessary that the particular procedure utilized be capable of successfully introducing at least one gene into the host cell which is capable of expressing the gene.
  • prokaryotic cells such as E. coli can be used.
  • Eukaryotic cells include, yeast, Chinese hamster ovary (CHO) cells, COS cells, and insect cells.
  • the particular vector used to transport the genetic information into the cell is also not particularly critical.
  • Any of the conventional vectors used for expression of recombinant proteins in prokaryotic and eukaryotic cells may be used.
  • Expression vectors for mammalian cells typically contain regulatory elements from eukaryotic viruses.
  • the expression vector typically contains a transcription unit or expression cassette that contains all the elements required for the expression of the polypeptide PNA in the host cells.
  • a typical expression cassette contains a promoter operably linked to the PNA sequence encoding a polypeptide and signals required for efficient polyadenylation of the transcript.
  • the term "operably linked” as used herein refers to linkage of a promoter upstream from a PNA sequence such that the promoter mediates transcription of the PNA sequence.
  • the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function. Following the growth of the recombinant cells and expression of the polypeptide, the culture medium is harvested for purification of the secreted protein.
  • the media are typically clarified by centrifugation or filtration to remove cells and cell debris and the proteins are concentrated by adsorption to any suitable resin or by use of ammonium sulfate fractionation, polyethylene glycol precipitation, or by ultrafiltration. Other routine means known in the art may be equally suitable.
  • Further purification of the polypeptide can be accomplished by standard techniques, for example, affinity chromatography, ion exchange chromatography, sizing chromatography, His 6 tagging and Ni-agarose chromatography (as described in Oobeli et al., Mol. and Biochem. Parasit. 41:259-268 (1990)), or other protein purification techniques to obtain homogeneity.
  • the purified proteins are then used to produce pharmaceutical compositions, as described below.
  • vaccinia virus is grown in suitable cultured mammalian cells such as the HeLa S3 spinner cells, as described by Mackett et al, in DNA cloning Vol. II: A practical approach, pp. 191-211 (Glover, ed.).
  • the proteins of the present invention can be used to produce antibodies specifically reactive with C pneumoniae antigens. If isolated proteins are used, they may be recombinantly produced or isolated from Chlamydia cultures. Synthetic peptides made using the protein sequences may also be used.
  • an immunogen preferably a purified protein
  • an adjuvant preferably an adjuvant
  • animals are immunized.
  • blood is collected from the animal and antisera is prepared.
  • Polyclonal antisera are used to identify and characterize Chlamydia in the tissues of patients using, for instance, in situ techniques and immunoperoxidase test procedures described in Anderson et al. JA VMA 198:241 (1991) and Barr et al. Vet.
  • Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see Kohler &
  • Monoclonal antibodies produced in such a manner are used, for instance, in ELISA diagnostic tests, immunoperoxidase tests, immunohistochemical tests, for the in vitro evaluation of spirochete invasion, to select candidate antigens for vaccine development, protein isolation, and for screening genomic and cPNA libraries to select appropriate gene sequences.
  • Immunodiagonostic detection of C. pneumoniae infections are used, for instance, in ELISA diagnostic tests, immunoperoxidase tests, immunohistochemical tests, for the in vitro evaluation of spirochete invasion, to select candidate antigens for vaccine development, protein isolation, and for screening genomic and cPNA libraries to select appropriate gene sequences.
  • the present invention also provides methods for detecting the presence or absence of C. pneumoniae, or antibodies reactive with it, in a biological sample.
  • antibodies specifically reactive with Chlamydia can be detected using either Chlamydia proteins or the isolates described here.
  • the proteins and isolates can also be used to raise specific antibodies (either monoclonal or polyclonal) to detect the antigen in a sample.
  • nucleic acids disclosed and claimed here can be used to detect Chlamydia-specific sequences using standard hybridization techniques.
  • the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); Tijssen, Laboratory Techniques in Biochem. stry and Molecular Biology (1985)).
  • Enzyme Immunoassay Maggio, ed., 1980
  • Tijssen Laboratory Techniques in Biochem. stry and Molecular Biology (1985)
  • the proteins and antibodies disclose i here are conveniently used in ELISA, immunoblot analysis and agglutination assays.
  • immunoassays to measure -Chlamydia antibodies or antigens can be either competitive or noncompetitive binding assays.
  • the sample analyte e.g., zn ⁇ -Chlamydia antibodies
  • a labeled analyte e.g., anti-Chlamydia monoclonal antibody
  • a capture agent e.g., isolated Chlamydia protein
  • Noncompetitive assays are typically sandwich assays, in which the sample analyte is bound between two analyte-specific binding reagents.
  • One of the binding agents is used as a capture agent and is bound to a solid surface.
  • the second binding agent is labelled and is used to measure or detect the resultant complex by visual or instrument means.
  • a number of combinations of capture agent and labelled binding agent can be used.
  • an isolated Chlamydia protein or culture can be used as the capture agent and labelled anti-human antibodies specific for the constant region of human antibodies can be used as the labelled binding agent.
  • Goat, sheep and other non- liuman antibodies specific for human immunoglobulin constant regions e.g., ⁇ or ⁇
  • the anti-human antibodies can be the capture agent and the antigen can be labelled.
  • the assay may be bound to a solid surface.
  • a solid surface may be a membrane (e.g., nitrocellulose), a microtiter dish (e.g., PVC or polystyrene) or a bead.
  • the desired component may be covalently bound or noncovalently attached through nonspecific bonding.
  • the immunoassay may be carried out in liquid phase and a variety of separation methods may be employed to separate the bound labeled component from the unbound labelled components. These methods are known to those of skill in the art and include immunoprecipitation, column chromatography, adsorption, addition of magnetizable particles coated with a binding agent and other similar procedures. An immunoassay may also be carried out in liquid phase without a separation procedure. Various homogeneous immunoassay methods are now being applied to immunoassays for protein analytes. In these methods, the binding of the binding agent to the analyte causes a change in the signal emitted by the label, so that binding may be measured without separating the bound from the unbound labelled component.
  • Western blot (immunoblot) analysis can also be used to detect the presence of antibodies to Chlamydia in the sample.
  • This technique is a reliable method for confirming the presence of antibodies against a particular protein in the sample.
  • the technique generally comprises separating proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the separated proteins. This causes specific target antibodies present in the sample to bind their respective proteins. Target antibodies are then detected using labeled anti- human antibodies.
  • the immunoassay formats described above employ labelled assay components.
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art.
  • a wide variety of labels may be used.
  • the component may be labelled by any one of several methods. Traditionally a radioactive label incorporating 3 H, I25 1, 35 S, 14 C, or 32 P was used.
  • Non-radioactive labels include ligands which bind to labelled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labelled ligand.
  • the choice of label depends on sensitivity required, ease of conjugation with the compound, stability requirements, and available instrumentation.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • an anti-ligand e.g., streptavidin
  • a number of ligands and anti-ligands can be used.
  • a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labelled, naturally occurring anti-ligands.
  • any haptenic or antigenic compound can be used in combination with an antibody.
  • agglutination assays can be used to detect the presence of the target antibodies.
  • antigen-coated particles are agglutinated by samples comprising the target antibodies.
  • none of the components need be labelled and the presence of the target antibody is detected by simple visual inspection.
  • the peptides or antibodies (typically monoclonal antibodies) of the present invention and pharmaceutical compositions thereof are useful for administration to mammals, particularly humans, to treat and/or prevent Chlamydia infections. Suitable formulations are found in Remington 's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).
  • the immunogenic peptides or antibodies of the invention are administered prophylactically or to an individual already suffering from the disease.
  • the peptide compositions are administered to a patient in an amount sufficient to elicit an effective immune response to Chlamydia. An effective immune response is one that inhibits infection.
  • Amount adequate to accomplish this is defined as “therapeutically effective dose” or “immunogenically effective dose.” Amounts effective for this use will depend on, e.g., the peptide composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician, but generally range for the initial immunization (that is for therapeutic or prophylactic administration) from about 0.1 mg to about 1.0 mg per 70 kilogram patient, more commonly from about 0.5 mg to about 0.75 mg per 70 kg of body weight.
  • Boosting dosages are typically from about 0.1 mg to about 0.5 mg of peptide using a boosting regimen over weeks to months depending upon the patient's response and condition. A suitable protocol would include injection at time 0, 4, 2, 6, 10 and 14 weeks, followed by further booster injections at 24 and 28 weeks.
  • Vaccine compositions containing the peptides are administered prophylactically to a patient susceptible to or otherwise at risk of the infection.
  • compositions are intended for parenteral or oral administration.
  • the pharmaceutical compositions are administered parenterally, e.g., subcutaneously, intradermally, or intramuscularly.
  • the invention provides compositions for parenteral administration which comprise a solution of the immunogenic polypeptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
  • an aqueous carrier e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
  • These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered.
  • compositions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • the compositions may also comprise carriers to enhance the immune response.
  • Useful carriers are well known in the art, and include, e.g., KLH, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly(lysine:glutamic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like.
  • conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed t y inco ⁇ orating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.
  • compositions and methods of administration suitable for maximizing the immune response are preferred.
  • peptides may be introduced into a host, including humans, linked to a carrier or as a homopolymer or heteropolymer of active peptide units from various Chlamydia proteins disclosed here.
  • a "cocktail" of polypeptides can be used.
  • a mixture of more than one polypeptide has the advantage of increased immunological reaction and, where different peptides are used to make up the polymer, the additional ability to induce antibodies to a number of epitopes.
  • compositions also include an adjuvant.
  • adjuvants include incomplete Freund's adjuvant, alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-P-isoglutamine (thr-MPP), N-acetyl-nor-muramyl-L-alanyl-P-isoglutamine (CGP 11637, referred to as nor-MPP), N-acety-muramyl-Lalanyl-P-isoglutaminyl-L-alanine-2-( -2'-dipalmitoyl-sn- glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (M
  • the concentration of immunogenic peptides of the invention in the pharmaceutical formulations can vary widely, i.e. from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • the peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia or fowlpox.
  • This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention.
  • the recombinant vaccinia virus Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits an immune response.
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848.
  • Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351 :456-460 (1991)).
  • the peptides may also be encapsulated, introduced into the lumen of liposomes, prepared as a colloid, or other conventional techniques may be employed which provide an extended serum half-life of the peptides.
  • liposomes as described in, e.g., Szoka et al, Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. ⁇ os. 4, 235,871, 4,501,728 and 4,837,028.
  • Example 1 This example describes comparison of the C. pneumoniae genome disclosed here and the, previously sequenced, C. trachomatis genome (Stephens, et al. Science 282:754-759 (1998)).
  • the previously sequenced C. trachomatis genome contains 1,042,519 nucleotides and 875 likely protein-coding genes. Similarity searching permitted the inferred functional assignment of sequences 636 (60%) genes disclosed here and 251 (23%) are similar to hypothetical genes for other bacterial organisms including those for C. trachomatis. The remaining 186 (17%) genes are not homologous to sequences deposited in GenBank.. Seventy C. trachomatis genes are not represented in the C. pneumoniae genome. These are contained within blocks consisting of 2-17 genes and 19 single genes. Of the 70 C. trachomatis genes without homo logs in C. pneumoniae, 60 are classified as encoding hypothetical proteins. The remaining genes not represented in C.
  • pneumoniae consist of the tryptophan operon (trpA,B,R), trpC, two predicted thiol protease genes, and 4 genes assigned to the phospholipase-O superfamily. It is evident that there is a high level of functional conservation between C. pneumoniae and C. trachomatis as orthologs to C. trachomatis genes were identified for 859 (80%) of the predicted coding sequences for C. pneumoniae. The level of similarity for individual encoded proteins spans a wide spectrum (22-95% amino acid identity) with an average of 62% amino acid identity between orthologs from the two species.
  • the percent amino acid identity between orthologous chlamydial proteins is similar among functional groups with the highest for proteins associated with translation and the lowest for proteins whose function in chlamydiae is uncharacterized and not related to proteins encoded by other organisms.
  • the gene order of the homologous set of genes in C. pneumoniae shows reorganization relative to the genome of C. trachomatis; however, there is a high level of synteny for the gene organization of the two genomes.
  • the distribution of genome reorganization is not evenly distributed on the chromosome as the region between C. pneumoniae coding sequences 0130-0300 contains substantially more reorganization than other areas of the genome. This region coincides with the predicted chromosome replication terminus.
  • trachomatis proteins containing SET and SWTB domains, and a SWTB domain fused to the C-terminus of the chlamydial topoisomerase I are found in C. pneumoniae supporting their possible role in the chromatin condensation-decondensation characteristic of the biologically unique chlamydial developmental cycle.
  • C. pneumoniae has a glycolytic pathway and a linked tricarboxylic acid cycle, although likely functional, is incomplete as genes for citrate synthase, aconitase, and isocitrate dehydrogenase were not identified.
  • C. pneumoniae has a complete glycogen synthesis and degradation system supporting a role for glycogen synthesis and utilization of glucose-derivatives in chlamydial metabolism.
  • C. pneumoniae also contains the V (vacuolar)-type ATPase operon and the two ATP translocases found in C. trachomatis.
  • the type-Ill secretion virulence system required for invasion by several pathogenic bacteria and found in the C. trachomatis genome in three chromosomal locations is also present in the C. pneumoniae genome.
  • Each of the components is conserved and their relative genomic contexts are conserved.
  • Genes such as a predicted serine/threonine protein kinase and other genes physically linked to genes encoding structural components of the type-Ill secretion apparatus, but without identified homologs, are also highly similar between the two species suggesting the functional roles in modifying cellular biology are fundamentally conserved.
  • Chlamydia-encoded proteins that are not found in chlamydial organisms but localized to the intracellular chlamydial inclusion membrane are likely essential for the unique intracellular biology and perhaps differences in inclusion mo ⁇ hology observed between species of Chlamydia.
  • Several such proteins, termed IncA,B&C have been characterized for a C. psittaci strain (-Rockey, et al. Mol. Microbiol. 15:617-626 (1995); Rockey et al. Inject. Immun. 62: 106-112 (1994)).
  • C. pneumoniae and C. trachomatis encode orthologs to C. psittaci IncB and IncC and C. trachomatis also contains an ortholog to icA.
  • C. pneumoniae contains two genes that encode proteins with similarity to IncA (CPn0186 and CPn0585), although the level of homology is low suggesting analogous but possibily altered functions.
  • the tryptophan biosynthesis operon (trpA, trpB, trpR) and trpC identified in C. trachomatis is conspicuously missing in the C. pneumoniae genome. This represents the entire repertoire of genes associated with tryptophan biosynthesis identified in C. trachomatis. Seventeen genes adjacent to the C. trachomatis tryptophan operon also were not found in the C. pneumoniae genome. This region is the single largest loss of a contiguous genomic segment and includes 4 HKD superfamily encoding genes that encompass a family of proteins related to endonuclease and phospholipase D. These findings may be important for the ability of Chlamydia to persist in their hosts and cause disease by eliciting potent, focal and persistent inflammatory responses thought to be essential for pathogenesis.
  • the C. pneumoniae genome contains 187,711 additional nucleotides compared to the C. trachomatis genome, and the 214 coding sequences not found in C. trachomatis account for most of the increased genome size. Eighty-eight of these genes are found in blocks of >10 genes (11-30 genes/block), 41 are single genes, and the remainder are partnered with at least one other gene. Based upon the observation that -70% of all the C. pneumoniae genes have an identifiable homolog in GenBank, exclusive of C. trachomatis, it would be expected that over 150 of the 214 genes should have a homolog in GenBank, many associated with a function. However, only 28 coding sequences have similarity to genes from other organisms.
  • the major functionally identifiable addition to the C. pneumoniae genome is a large expansion of genes encoding a new family of chlamydial polymo ⁇ hic membrane proteins (Pmp), alone representing 22% of the increased coding capacity. While the C. trachomatis genome has 9 pmp genes, remarkably the C. pneumoniae genome contains 21 pmp genes. Most of these genes appear to be amplified in two regions of the genome with three stand-alone genes. Interestingly one of the stand-alone genes is most closely related to the C. trachomatis pmpD which is the only stand-alone pmp gene in the C. trachomatis genome and it is located with the same relative genomic context, suggesting an essential and conserved function for this paralog.
  • Pmp chlamydial polymo ⁇ hic membrane proteins
  • Aromatic amino acid hyroxlyases include three distinct enzymes that function to receptively oxidize phenylalanine to tyrosine, tyrosine to Popa, and tryptophan to 5-hydroxytryptophan and serotonin.
  • the chlamydial protein is similar to proteins of this family and incrementally more closely related to tryptophan hydroxylase, its specific function could not be confidently predicted. We hypothesize that it may be involved in C. pneumoniae virulence.
  • Tryptophan hydroxylase has not been previously identified in bacteria and the origin of the chlamydial gene appears to be from eukaryotes.
  • the functional role of an aromatic amino acid hydroxylase for C. pneumoniae is linked to the unique intracellular biology of this organism and may represent a key contribution to C. pneumoniae persistence and pathogenesis.
  • Table 1 provides functional assignments of C. pneumoniae nonprotein- encoding genomic sequences.
  • Table 2 provides functional assignments of protein coding sequences.
  • Table 3 provides the amino acid sequences of the proteins corresponding to the coding sequences. TABLE 1 type » SEQ !DNO:1 SEQ ID NO: 1 Gene start position end position
  • Ori 841664 841396 (R) Putative Origin of Repli tmRNA 138493 138074 (R) tmRNA pRNA 607342 607649 Rifaonuclease P RNA rRNA 1000564 1002115 15S rRNA rRNA 1002415 1005278 23S rRNA rRNA 1005393 1005509 5S rRNA tRNA 269070 269142 Ala tRNA_l tRNA 164318 164389 Asn tRNA tRNA 296224 296151 (R) Asp tRNA tRNA 836191 835119 (R) Ala tRNA_2 tRNA 1030533 1030603 Cys tRNA tRNA 784896 784822 (R) Glu tRNA tRNA 781680 781610 (R) Gly tRNA_l tRNA 961536 961607 Gly tRNA_2 tRNA 999949 1000023 His tRNA tRNA
  • CT016 hypothetical protein cpnoioe 135091 136374 F phoH-ATPase- (CT015)
  • CPH0625 718485 718060 R rll7-L17 Ribosomal Procein- (CTS06)
  • CT601 hypothetical protein CPn0780 879205 878591
  • CT601 hypothetical protein

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Abstract

L'invention concerne la séquence génomique de Chlamydia pneumoniae et l'analyse des polypeptides et des ARN codés. Les compositions d'acide nucléique du gène de Chlamydia pneumoniae permettent d'identifier des protéines homologues ou associées ainsi que les séquences d'ADN codant pour ces protéines, de produire des compositions modulant l'expression ou la fonction de la protéine, et d'étudier les mécanismes physiologiques associés. En outre, la modulation de l'activité génique in vivo est utilisée à des fins prophylactiques et thérapeutiques, telles que l'identification du type cellulaire sur la base de l'expression et analogues.
EP99960323A 1998-11-12 1999-11-12 Sequence genomique de chlamydia pneumoniae Withdrawn EP1133572A4 (fr)

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US10827998P 1998-11-12 1998-11-12
US108279P 1998-11-12
US12860699P 1999-04-08 1999-04-08
US128606P 1999-04-08
PCT/US1999/026923 WO2000027994A2 (fr) 1998-11-12 1999-11-12 Sequence genomique de chlamydia pneumoniae

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EP1133572A2 true EP1133572A2 (fr) 2001-09-19
EP1133572A4 EP1133572A4 (fr) 2005-06-15

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AU (1) AU1722300A (fr)
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WO2000027994A3 (fr) 2000-11-23
CA2350775A1 (fr) 2000-05-18
AU1722300A (en) 2000-05-29
JP2002529069A (ja) 2002-09-10
EP1133572A4 (fr) 2005-06-15

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