Abstract:
An expression system for production of full-length Chlamydial heat shock protein 60 (CHSP60) and an immunore-active subfragment thereof is described. The expression system is arranged such that the CHSP60 or fragment is initially isolated as a fusion protein which is then cleaved off, leaving purified CHSP60.

Description:
PRIOR APPLICATION  
       [0001]    This application claims priority under 35 USC § 119(e) to U.S. Ser. No. 09/759,272, filed Jan. 16, 2001. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The present invention relates generally to the field of immunoassays. More specifically, the present invention relates to recombinant proteins and their use in immunoassays for detecting the presence of antibodies to strains of Chlamydia.  
         BACKGROUND OF THE INVENTION  
         [0003]    Chlamydiae are obligate intracellular bacterial pathogens responsible for a wide range of infections in animals. The genus Chlamydia is divided into four species:  C. trachomatis, C. pneumoniae, C. psittaci  and  C. pecorum.  Genital  Chlamydia trachomatis  infection is the most prevalent bacterial sexually-transmitted disease in many developed countries, including Canada. Women with cervical chlamydial infections are at risk of developing pelvic inflammatory disease, which can lead to long-term reproductive sequelae, such as chronic pelvic pain, ectopic pregnancy and tubal infertility. Ocular strains of  Chlamydia trachomatis  are the leading infectious cause of blindness and is estimated to affect 500 million people worldwide.  Chlamydia pneumoniae  is responsible for 10-20% of community-acquired pneumonia. Studies from around the world show that 40-60% of adult populations possess antibodies to C. pneumoniae, suggesting that infections and re-infections are quite common. Recent studies have linked persistent  c. pneumoniae  infection to a number of chronic diseases including atherosclerosis, asthma, exacerbation of chronic obstructive pulmonary disease, stroke, Alzheimer&#39;s disease and multiple sclerosis.  
           [0004]    The invasion of chlamydiae into a human host creates a stressful condition in the host as well as a hostile environment for the chlamydiae, as the host mounts an immune response against the presence of the invading microbe. Thus, an up-regulation of the heat shock response frequently occurs in both chlamydiae and the host during an infection. Heat shock proteins are among the most abundant proteins in nature and are highly conserved amongst both eukaryotes and prokaryotes. The heat shock response is an important survival mechanism that safeguards the cell or microbe from conditions of stress. The response is triggered transcriptionally and results in the production of newly synthesized proteins within minutes of the cell or microbe encountering a stressful environment. Heat shock proteins are involved in vital cell functions, such as the assembly and disassembly of macromolecules.  
           [0005]    The association between antibody response to a chlamydial heat shock protein and the development of tubal Infertility was first shown by Brunham et al., who reported that 11 of 13 chlamydia seropositive women with tubal infertility had antibody to a 57 kDa protein compared with 2 of 6 seropositive women with non-tubal causes of infertility and 1 of 11 seropositive pregnant controls (Brunham et al., 1985,  J. Infect. Dis.  152: 1275-1282). This 57 kDa chlamydial protein was subsequently determined to belong to a group of proteins known as heat shock proteins (HSPs), specifically to the GroEL or HSP60 family. This family of proteins has been previously implicated in the pathogenesis of immune disorders. The chlamydial Hsp60 (CHSP60) is constitutively expressed and its transcription is upregulated during conditions of stress. The protein is found in both forms of chlamydiae: the elementary body (EB), which is the extracellular, infectious form, and the reticulate body (RB), which is the intracellular, metabolically active form.  
           [0006]    One of the hallmarks of chlamydial infection is that the symptoms are often mild or absent. Undiagnosed and untreated, the infection can persist in the body leading to chronic inflammation at the site of infection. The pathophysiology of these chronic disease conditions is thought to be immunologically mediated, and the CHSP60 has been implicated as a major antigen that stimulates the immunopathological response. In vitro studies of persistent infection show that CHSP60 is disproportionately expressed compared with other chlamydial proteins, such as the major outer membrane protein (Beatty et al, 1994,  Infect Immun  62: 4059-4062). Studies in an animal model of pelvic inflammatory disease show that the CHSP60 antibody response is associated with the persistence of chlamydia in the fallopian tubes (Peeling et al, 1999,  JID  180: 774-779). Thus, antibody response to CHSP60 is a marker for persistent chlamydial infections and as such can be used to predict the risk of developing long term complications as a result of prior chlamydial infections.  
           [0007]    Studies of human chlamydial infection have shown that antibody response to the 60 kDa chlamydial heat shock protein, CHSP60, is associated with the development of adverse sequelae following ocular and genital chlamydial infection with  C. trachomatis  (Peeling and Mabey, 1999,  Infect Dis. Obstet Gynecol.  7:72-79). More recently, the presence of CHSP60 antibody was reported to be correlated with long-term sequelae of  C. pneumoniae  infection such as atherosclerosis and asthma (Fong et al 2000, manuscript submitted to Journal of Infectious Diseases; Peeling and Hahn, unpublished data). However, the mechanism by which CHSP60 contributes to the immunopathological sequelae associated with chlamydial infection remains unclear. It is speculated that antibody response to CHSP60 may be a marker of persistent chlamydial infection or of an autoimmune response elicited as a result of molecular mimicry with human HSP60. CHSP-60 has been localized in human atheroma (Kol et al, 1998,  Circulation  98: 300-307), and may play a role in atherogenesis by regulation of macrophage tumor necrosis factor-alpha (TNFα) and matrix metalloproteinase expression.  
           [0008]    Since the tubal infertility study by Brunham et al. (Brunham et al., 1985) numerous groups have examined the relationship between antibody to CHSP60 and adverse reproductive sequelae associated with  C. trachomatis  Infection. Wager et al. showed by immunoblot that 6 (31%) of 19 patients with pelvic inflammatory disease and 17 (81%) of 21 ectopic pregnancy patients had antibody to CHSP60 in a Triton X-100 soluble extract (Wager et al., 1990,  J. Infect. Dis.  162: 922-927). However, the presence of a chlamydia structural protein with molecular weight very similar to CHSP60 makes immunoblot reactivity of CHSP60 difficult to interpret, especially for low titer sera. To overcome this problem, Brunham et al. used a sarcosyl-soluble extract of  C. trachomatis  (the structural protein is sarkosyl insoluble) to enrich for CHSP60. Nineteen (91%) of 21 seropositive patients with ectopic pregnancy had antibody to this enriched CHSP60 extract by immunoblot compared to 25% of controls (Brunham et al., 1992,  J. Infect. Dis.  165: 1076-1081).  
           [0009]    These previous studies used whole Chlamydia organisms or enriched semi-purified protein preparations from chlamydia to correlate human immune responses to chlamydial antigens with reproductive sequelae. There are a number of drawbacks associated with the use of such preparations. Specifically, these preparations are not pure, but contain many other contaminating chlamydial proteins in addition to CHSP60. Therefore, serum antibody responses to these preparations can only be analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting, to confirm that the antibody is directed to the CHSP60 and not one of the contaminating proteins or LPS. This method of analyzing antibody response to the CHSP60 is extremely laborious and time-consuming. In addition, pure chlamydial organisms are required for these preparations which requires tissue culture for the growth of the chlamydiae, which is both time-consuming and costly. Hence all these studies on pelvic inflammatory disease, ectopic pregnancy, and tubal infertility were limited by small sample size and adequate seropositive fertile controls.  
           [0010]    To obtain large quantities of CHSP60, Cerrone et al cloned the gene for CHSP60 and expressed the CHSP60 and fragments of CHSP60 as fusion proteins linked, at its N terminal, to a 26 kDa glutathione-S-transferase from  Schistosoma japonicum.  Using this recombinant CHSP60 and sera from 5 women with pelvic inflammatory disease or ectopic pregnancy, Cerrone et al. confirmed that immune response detected in these studies was to this protein (Cerrone et al., 1991,  Infect. Immun.  59: 79-90). Cerrone et al reported that sera from  C. trachomatis  infected patients reacted with the fusion protein containing amino acids 274-402 and 405-544 of CHSP-60, but not with those containing amino acids 1-51, 50-143 and 50-266 of CHSP-60. As will be appreciated by one knowledgeable in the art, this indicates that the immunoreactivity of specific fragments of CHSP60 cannot be predicted in advance, likely due to protein folding affecting presentation of antigenic domains. Furthermore, since the recombinant CHSP60 comprised a large fusion partner (26 kDa glutathione-S-transferase) which is similar in size or larger than fragments of CHSP60, it is also possible that the GST tag is blocking or masking antigenic determinates at the amino terminus of the CHSP60 fusions. It is of note that the GST tag can be cleaved from the fusion protein by treatment with thrombin; however, thrombin also recognizes sites within the peptide of interest (in this case, CHSP60), meaning that removal of the GST tag may result in the entire fusion protein being cleaved at multiple sites.  
           [0011]    In addition, Yuan et al (Yuan et al., 1992,  Infect Immun  60: 2288-2296) describe the construction of lacZ-CHSP60 fusion peptides which were used to generate monoclonal antibodies. The monoclonal antibodies were subsequently mapped to epitopes at amino acids 8-14 and 177-189 of CHSP60. It is of note that these epitopes are present in fusion peptides which failed to react with patient sera when tested by Cerrone et al, as discussed above.  
           [0012]    To further examine the role of CHSP60 in tubal infertility and to facilitate the study of a larger number of samples, we developed an ELISA using a full-length CHSP60 GST fusion peptide as antigen. In a study by Toye et al., this ELISA assay was used to determine the prevalence of antibody to the CHSP60 in women with tubal infertility. Antibody to  C. trachomatis  was present in 32 (72.7%) of 44 of women with tubal infertility compared with 9 (32.1%) of 28 with other causes of infertility and 55 (28.9%) of 190 pregnant women (p&lt;0.001). The CHSP60 ELISA detected Chlamydia-associated tubal infertility in infertile women with a sensitivity of 81.3% and a specificity of 97.5% (Toye et al., 1993,  J. Infect. Dis.  168: 1236-1240). Several other studies have been performed to demonstrate that there is a strong association between antibody response to the CHSP60 and the development of Chlamydia-associated tubal infertility (Peeling and Mabey, 1999,  Dis. Obstet Gynecol.  7: 72-79). It was also concluded that a CHSP60 ELISA may be useful as a predictor for poor fertility outcome (Claman et al., 1996,  Fertil. Steril.  65: 146-149).  
           [0013]    Since the CHSP60 ELISA is highly specific, it may prove useful in the investigation of infertile women as a marker of Chlamydia-associated tubal obstruction and lead to more selective use of invasive procedures (e.g. diagnostic laparosopy). This ELISA assay may also be used as a means of assessing the risk or presence of tubal obstruction in women seeking infertility treatment.  
           [0014]    As described above, the clone used in the previous studies had a fusion partner, a 26 kDa glutathione-S-transferase, which necessitated the use of 2 parallel ELISA assays to assess the reactivity against the entire fusion protein as well as against the fusion partner. The use of the additional assays increases the time and labor required to carry out the ELISA assays, reducing the total number of samples that can be screened in a given period of time. Clearly, there is a need for a more efficient ELISA assay that would allow more samples to be processed and perhaps with higher sensitivity and specificity.  
         SUMMARY OF THE INVENTION  
         [0015]    According to a first aspect of the invention, there is provided a method of detecting anti-CHSP60 antibodies in a sample from a patient comprising:  
           [0016]    providing purified CHSP60 80-277  or purified CHSP60 1-544 ;  
           [0017]    binding the CHSP60 80-277  or CHSP60 1-544  to a support;  
           [0018]    mixing the sample with the bound CHSP60 1-544  or CHSP60 80-277  under conditions such that anti-CHSP60 antibodies within the sample bind to the CHSP60 1-544  or CHSP60 80-277 ;  
           [0019]    washing away unbound sample; and  
           [0020]    detecting antibodies bound to the CHSP60 1-544  or CHSP60 80-217 .  
           [0021]    According to a second aspect of the invention, there is provided a kit comprising purified CHSP60 80-277  or CHSP60 1-544.    
           [0022]    According to a third aspect of the invention, there is provided a substantially purified polypeptide consisting of amino acids 1-544 of SEQ ID No. 1.  
           [0023]    According to a fourth aspect of the invention, there is provided a substantially purified polypeptide consisting of amino acids 80-277 of SEQ ID No. 1.  
           [0024]    According to a fifth aspect of the invention, there is provided an expression system comprising a suitable promoter operably linked to a nucleic acid molecule deduced from either amino acids 1-544 of SEQ ID No. 1 or amino acids 80-277 of SEQ ID No. 1 and a nucleic acid molecule encoding a removable fusion partner. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0025]    [0025]FIG. 1 shows the nucleotide sequence of the primers.  
         [0026]    [0026]FIG. 2 shows the amino acid sequence of CHSP60.  
         [0027]    [0027]FIG. 3 shows the sequence variance across amino acids 80-277 of CHSP60.  
         [0028]    [0028]FIG. 4 is a schematic diagram of the expression system. 
     
    
       [0029]    TABLE 1 shows absorbance readings at 405 nm for-the CHSP60 ELISA assay for serum samples tested against serovar L 2  CHSP60-GST fusion and CHSP60 80-277 .  
         [0030]    TABLE 2 shows effectiveness of CHSP60 80-277  vs CHSP60-GST in asthma cases.  
       DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0031]    Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.  
         [0032]    As used herein, CHSP60 1-544  refers to a purified polypeptide having an amino acid sequence substantially as shown in FIG. 2. CHSP60 1-544  also refers to a purified polypeptide substantially as shown in FIG. 2 including sequence variations, for example, as shown in FIG. 3, which do not significantly alter the immunoreactivity of the peptide as discussed herein.  
         [0033]    As used herein, “removable fusion partner” refers to a compound, for example, a polypeptide, which can be used in purifying a polypeptide of interest and can be separated from the polypeptide of interest using means known in the art without cleaving or otherwise modifying the polypeptide of interest so that the reactivity or functioning of the polypeptide in the intended use is compromised.  
         [0034]    As used herein, “substantially purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated and are at least about 60% free, preferably about 75% free, and most preferably about 90% free from other components with which they are naturally associated.  
         [0035]    Herein described is an expression system for the production of CHSP60 1-544  as described above and consisting of amino acids 1-544 of CHSP60 and a fragment of CHSP60 1-544  consisting of amino acids 80-277 of CHSP60, designated as CHSP60 80-277 . Specifically, the expression system is arranged so that CHSP60 1-544  or CHSP60 80-277  is produced as a fusion protein which can be purified based on the activity or property of the fusion partner, as described below. The fusion protein is then treated such that the fusion partner is cleaved, producing purified, CHSP60 1-555  or CHSP60 80-277 . As a result of this arrangement, the termini of the peptides are not blocked or masked by the fusion partner, meaning that more antigenic epitopes are available. As will be apparent to one knowledgeable in the art, given the high degree of homology between proteins of the GroE family, it is very difficult to isolate purified native CHSP60 using traditional_means, such as, for example, sizing columns, ion exchange columns or even antibody columns. Furthermore, as discussed above, traditional tags used for isolation of recombinant proteins may mask antigenic determinants. As will be appreciated by one knowledgeable in the art, the availability of larger number of epitopes for antibody reactivity may lead to a more sensitive assay. It is also of note that the lack of a fusion partner means that the use of CHSP60 1-544  or CHSP60 80-277  as antigens in the ELISA assays as described herein more closely correspond to those presented in vivo. Furthermore, removal of the fusion partner reduces the number of assays required, allowing many more samples to be screened. Also described are PCR primers for generating a DNA fragment encoding the CHSP60 1-544  protein or CHSP60 80-277  fragment for subsequent cloning into other expression vectors and construction of these expression vectors, as described below.  
         [0036]    In one embodiment, described below, the substantially purified polypeptide consisting of CHSP60 1-544  or CHSP60 80-277  fragment is used in ELISA assays for screening samples from patients suspected of having chlamydial infections. As will be appreciated by one of skill in the art, the presence of antibodies bound to CHSP60 1-544  or CHSP60 80-277  may be detected by any suitable means known in the art. As discussed herein, these may include patients having, suspected of having or at risk of developing diseases or disorders such as, but by no means limited to chronic pelvic pain, pelvic inflammation disease, tubal infertility, chronic inflammation, ectopic pregnancy, atherioscierosis, asthma, stroke, Alzheimer&#39;s disease, multiple sclerosis, urogenital tract infections, pneumonia, respiratory infections or other chlamydia associated autoimmune diseases. Specifically, the presence of anti-CHSP60 antibody indicates persistent, acute or repeated chlamydial infections which in turn indicates that the patient is at risk of developing complications, as discussed herein. As will be appreciated by one knowledgeable in the art, complications of  C. pneumoniae  infections can include, for example, cardiovascular diseases (atheriosclerosis, stroke, abdominal aortic aneurysm, etc), pulmonary diseases for example COPD and asthma, as well as neurodegenerative diseases for example Alzheimer&#39;s disease and multiple sclerosis.  
         [0037]    [0037] Chlamydia pneumoniae  is one of the most common causes of respiratory infection. Seroepidemiologic studies show the 50-80% of adult populations worldwide has evidence of past infection. The infection is typically mild or asymptomatic and therefore remained undiagnosed or not treated. It is now recognized that  C. pneumoniae  can persist in the host leading to long term sequelae of cardiovascular disease, exacerbation of asthma and other chronic illnesses such as multiple sclerosis, Alzheimer&#39;s disease, chronic obstructive pulmonary disease. The genetic and biologic basis for susceptibility to these long term complications of  C. pneumoniae  infection to unknown but it is likely immune-mediated. We have shown that antibody response to a fragment of the  C. pneumoniae  60 kDa heat shock protein is found significantly more frequently in men with atherosclerosis and asthma compared to those without evidence of these chronic diseases.  
         [0038]    Persistent ocular and genital infection with  Chlamydia trachomatis  can lead to binding trachoma, and adverse reproductive sequelae of pelvic inflammatory disease, ectopic pregnancy and tubal infertility, respectively. Antibody to the CHSP60  
         [0039]    CHSP60 80-277  was previously used along with CHSP60-GST fusion described above to test sera of individuals with scarring trachoma for antibodies against CHSP60. Compared to the GST fusion protein, the fragment showed not only an increase in the number of positive responses in the cases, but also in the controls (Peeling et al 1999  Infect Dis. Obstet Gynecol.  7:108-9). Thus, this data indicated that CHSP60 80-277  was of no value in analyzing individuals with scarring trachoma. However, as discussed below and as shown in Tables 1 and 2, CHSP60 80-277  and the CHSP60 1-544  protein show greater sensitivity and lower background compared to the CHSP60-GST fusion in samples of patients with complications of urogenital tract infections or respiratory infections.  
         [0040]    As will be appreciated by one knowledgeable in the art, other suitable fusion partners or expression systems which allow for expression and isolation of the native CHSP60 1-544  or CHSP60 80-277  may also be used. That is, in other embodiments, there are provided expression systems comprising a promoter operably linked to a DNA molecule deduced from the amino acid sequence of CHSP60 1-544  or CHSP60 80-277  and a DNA molecule encoding a suitable (i.e. removable) fusion partner. As will be appreciated by one of skill in the art, the DNA encoding the fusion partner may be either 5′ or 3′ to the DNA encoding the CHSP60 1-544  or CHSP60 80-277 .  
         [0041]    In yet other embodiments of the invention, there are provided host cells transformed with the above-described expression system, as discussed below.  
         [0042]    The invention provides kits for carrying out the methods of the invention. Accordingly, a variety of kits are provided. In some embodiments, the kits include purified CHSP60 1-544  and/or CHSP60 80-277  and/or expression systems for producing same. In some embodiments, the kit may also include ELISA reagents. The kits may be used for detecting antibodies against CHSP60 in patients having, suspected of having or at risk of developing diseases or disorders such as, but by no means limited to, chronic pelvic pain, pelvic inflammation disease, tubal infertility,, chronic inflammation, ectopic pregnancy, atheriosclerosis, asthma, stroke, Alzheimer&#39;s disease, multiple sclerosis, urogenital tract infections, pneumonia, respiratory infections or other chlamydia associated autoimmune diseases. As will be appreciated by one knowledgeable in the art, the kits may also include instructions for purification and/or preparation of CHSP60 1-544  and/or CHSP60 80-277 , as described below.  
         [0043]    The kits of the invention comprise one or more containers comprising substantially purified CHSP60 1-544  and/or CHSP60 80-277  or an expression system for producing same and a set of instructions, generally written instructions although electronic storage media (e.g., magnetic diskette or optical disk) containing instructions are also acceptable, relating to the use intended for the purified peptides or expression system. The containers may contain unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. As will be appreciated by one knowledgeable in the art, samples from a variety of sources as discussed herein may be screen using the described kits.  
         [0044]    The substantially purified CHSP60 80-277  fragment or CHSP60 1-544  of the kit may be packaged in any convenient, appropriate packaging. For example, if there is a freeze-dried formulation, an ampoule with a resilient stopper is normally used, so that the peptide may be easily reconstituted by injecting fluid through the resilient stopper.  
         [0045]    The following Examples are provided to illustrate, but not limit, the invention.  
       EXAMPLE I  
       [0046]    Bacterial Isolates  
         [0047]    The bacterial isolates used in the present invention were from a laboratory collection. All cultures were grown in Minimal Essential Media supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 25 μg/ml gentamycin, 100 μg/ml vancomycin, and 1 μg/ml cycloheximide.  
       EXAMPLE II  
       [0048]    Isolation of Genomic DNA  
         [0049]    High molecular weight genomic DNA was isolated by SDS/proteinase K digestion at 56° C. for 3 hours followed by phenol/chloroform extraction and ethanol precipitation. DNA concentration was determined by UV spectroscopy, at A 260  and purity estimated by the A 260 /A 280  ratio.  
       EXAMPLE III  
       [0050]    PCR Amplification  
         [0051]    The deoxyribonucleotide triphosphates dATP, dCTP, dGTP, dTTP are added to the synthesis mixture, either separately or together with the primers, in adequate amounts and the resulting solution is heated to 95° C. for 5 minutes. After this heating period, the solution is subjected to 35 cycles of 1 minute at 95° C., 1 minute at 55° C., 1.5 minutes at 72° C. Following the final cycle, the solution was held at 72° C. for 10 minutes, and then cooled to 4° C. The agent used for the polymerase chain reaction (PCR) was Taq DNA polymerase, purchased from GIBCO Life Technologies.  
         [0052]    The following PCR conditions were used: 50 mM KCl, 10 mM Tris, pH 8.4, 1.5 mM MgCl 2 , 200 μM of each dNTP (all final concentrations), 50 ng of genomic DNA, 2.5 U of Taq DNA polymerase (GIBCO) and 0.5 μM of each of the degenerate primers MH279 and MH280, and CLH1 and CLH2, described below. A final volume made up to 50 μl with dH 2 O was used.  
         [0053]    The sequences of the primers used to amplify the CHSP60 protein fragment encompassing amino acids 80-277 from  C. trachomatis, C. pneumoniae,  and  C. psittaci  are as follows:  
                                       MH279:               5′ AAA ACT CAT ATG AAA GCW GGV GAY GGA ACY ACA           ACA 3′                       MH280:           5′ CAT AGC TGC TCT TCC GCA WCC RAA VCC WGG AGC           TTT MAC WGC 3′          
 
         [0054]    It is of note that these degenerative primers have been used previously (Goh et al, 1996,  J Clin Micro  34: 818-823) for generating a DNA fragment used as a species-specific probe for identifying different HSP60 genes.  
         [0055]    The sequences of the primers used to amplify the DNA encoding CHSP60 1-544  protein from  C. trachomatis, C. pneumoniae,  and  C. psittaci  are as follows:  
                               CLH1:   5′ AGM RCA CAT ATG GYM GCK AAA AAY ATT AAA TAY AA 3′                   CLH2:   5′ TWR TWC YGC TCT TCC GCA YTA RTA GTC CAT TCC TGC GCY WG 3′.          
 
         [0056]    It is of note that the 5′ end sequences of MH279 and CLH1 contain an NdeI restriction site, while the 3′ end sequences of MH280 and CLH2 contain a SapI restriction site.  
         [0057]    The amplified PCR products were digested with NdeI and SapI and then ligated into pCYB1. The recombinant plasmids were transformed into competent  E. coli  and screened and selected.  
       EXAMPLE IV  
       [0058]    Plasmid Construction  
         [0059]    [0059] Escherichia coli  JM109 containing the pCYB1 plasmids encoding the CHSP60 1-544  protein and the CHSP60 80-277  protein were generated using the New England Biolabs™ IMPACT I kit. Expression and purification of the CHSP60 proteins were performed according to established protocols for the IMPACT I (Intein Mediated Purification with an Affinity Chitin-binding Tag) kit. The IMPACT I system utilizes a protein splicing element, an intein, from  Saccharomyces cerevisiae  VMA1 gene. The intein has been modified such that it undergoes a self-cleavage reaction at its N-terminus at low temperatures in the presence of thiols such as 1,4-dithiothreitol (DTT), β-mercaptoethanol or cysteine. The gene/nucleic acid encoding the target protein/protein fragment is inserted into a multiple cloning site (mcs) of the pCYB1 vector to create a fusion between the C-terminus of the target gene and the N-terminus of the gene encoding the intein. The DNA encoding a small 5 kDa chitin binding domain (CBD) from  Bacillus circulans  has been added to the C-terminus of the intein for affinity purification of the 3 part fusion, shown schematically in FIG. 4. When crude extracts of cells from an inducible  E. coli  expression system are passed through a chitin column, the fusion protein binds to the chitin column while all other contaminants are washed through the column. The fusion is then induced to undergo an intein-mediated self-cleavage on the column by overnight incubation at 4° C. in the presence of DTT or β-mercaptoethanol. The target protein is released while the intein-chitin binding domain fusion partner remains bound to the column.  
       EXAMPLE V  
       [0060]    CHSP60 Protein Expression and Purification  
         [0061]    A 10 ml culture of LB broth containing 100 μg/ml ampicillin was inoculated with a freshly grown colony of the  E coli  clone to be cultured. The culture was then incubated at 37° C. overnight with shaking. The overnight culture was used to inoculate a 1 L flask of LB, which was then grown at 37° C. with shaking to OD 600  of 0.6-0.8. IPTG was added to the culture to a final concentration of 0.7 mM and the culture was transferred to 30° C. The culture was incubated for a further 3 hours with moderate shaking. The cells were then spun down from the culture at 5000×g for 15 minutes at 4° C. and the supernatant was discarded. The pellet was resuspended in 10 ml of ice-cold Column Buffer (2.42 g Tris-HCl, 29.22 g NaCl, 0.0372 g EDTA, 1 ml Triton X-100 per litre) and the cells were lysed by sonication on ice. The lysed cells were centrifuged at 12,000×g for 30 minutes and the pellet was discarded. The supernatant was loaded onto a chitin column at a rate of approximately 1 drop per second at 4° C. The column was then washed with 200 ml of Column Buffer at a flow rate of one drop per second at 4° C. All traces of the cell extract were washed off the sides of the column. The column was then quickly flushed with 30 ml of Cleavage Buffer (2.42 g Tris-HCl, 2.92 g NaCl, 0.0372 g EDTA per litre) containing 30 mM DTT at 4° C. The column flow was stopped when almost all of the Cleavage Buffer had passed through the column. The column was left at 4° C. overnight. The target protein was eluted from the column using 20 ml of Cleavage Buffer without DTT and 1 ml fractions were collected. The fractions were stored at −20° C. Fractions were analyzed by Bradford assay, SDS-PAGE and Western blotting using anti-CHSP60 antibodies. The eluted fractions were dialyzed against 5 litres of PBS for 4 hours at 4° C. The PBS was replaced and the protein fractions were dialyzed overnight at 4° C.  
       EXAMPLE VI  
       [0062]    Enzyme-Linked Immunosorbant Assay  
         [0063]    The enzyme-linked immunosorbant assay (ELISA) was performed as follows. One hundred microliters of CHSP60 protein (10 ng) was added to each well of a 96-well microtiter plate and allowed to adsorb for 3 hours at 37° C. or overnight at 4° C. The unbound antigen was washed from the plate and discarded, and the wells were blocked with 150 μl of 3% bovine serum albumin (BSA) in PBS for 90 minutes. The plates were then washed and 100 μl of patient sera (1:500 dilution in PBS containing 0.5% BSA and 0.2% Tween 20) was added and incubated for 60 minutes at 37° C. The wells were then washed three times with PBS containing 0.2% Tween 20, and then 100 μl of horseradish peroxidase-conjugated goat anti-human immunoglobulin antibody (1:4000) was added to each well and incubated for 60 minutes at 37° C. The wells were then washed three times with PBS containing 0.2% Tween 20 and then bound antibody was detected by the addition of 100 μl of substrate (4 mg of 2,2′-azino-bis[3-ethyl-benz-thiazoline-6-sulfonate]/ml in 0.1 M citric acid buffer, pH 4.2 and 10 μl hydrogen peroxide). The plates were then developed in the dark for 20 minutes and the optical density of each well was read in an ELISA reader at 405 nm. All sera were assayed in duplicate against the CHSP60 protein with one negative control serum and 2 positive control sera included with each plate.  
         [0064]    To determine the threshold for a positive antibody response, all CHSP60 protein preparations were tested against a panel of 50 Chlamydia-seronegative sera. The threshold is defined as the mean of the response of these negative sera plus three standard deviations from the mean. Results are summarized in Table 1.  
         [0065]    Similarly, CHSP60 antibodies in asthma cases and nonasthmatic controls who were Cpn seroreactive were tested for reactivity to CHSP60-GST and CHSP 80-277 , as shown in Table 2.  
       EXAMPLE VII  
       [0066]    CHSP-60 In Atherosclerotic Plaques  
         [0067]    Patients undergoing carotid endarectomy for significant symptomatic stenosis (&gt;60%) were enrolled in the study after informed consent. The demographic features and results of immunohistochemical staining for  C. pneumoniae,  cytomegalovirus and herpes simplex Type I were previously reported (Chiu et al, 1997). Immunohistochemical staining was performed on paraffin embedded sections by the labeled (Strep) avidin-biotin-peroxidase method. The antisera used included  C. pneumoniae  specific RR-402 and CF2 monoclonal antibodies.  
         [0068]    Sera were diluted 1:500 and tested against a purified recombinant fragment of CHSP60 (amino acids 80-277) as antigen in a standard ELISA as described previously (Toye et al, 1993,  J. Infect Dis  168: 1236-1240) and as described above.  
         [0069]    Chlamydia serology was performed by the micro-immunofluorescence (MIF) method to detect IgM and IgG antibodies to purified elementary bodies of chlamydia species of  C. pneumoniae, C. trachomatis  and  C. psittaci.  (Wang, 1999, in  Chlamydia Pneumoniae: The Lung and the Heart,  Allegra and Blasi, eds, Springer-Verlag Italia: Milano) Sera were screened at 1:16 dilution and all positive sera were titered to end point.  
         [0070]    [0070] C. pneumoniae  antigen was detected by immunohistochemical staining in 54 (72%) of 75 carotid atheromatous plaques. Of the 54 patients with detectable  C. pneumoniae  antigen, the mean OD was 0.19±0.15 and in the 21 patients without  C. pneumoniae  antigen the OD was 0.11±0.08, p=0.01 (2 tailed). For CHSP-60 IgG antibody reactivity ≧0.12 OD, 38 (70.4%) of 54 patients with  C. pneumoniae  antigen in atheromas had anti-CHSP-60, versus 5 (23.8%) of 21 patients without  C. pneumoniae  antigen, p&lt;0.001.  
         [0071]    None of the patients had IgM antibodies and 80% of the total cohort had detectable IgG antibodies to  C. pneumoniae  (&gt;1;16), suggesting a history of  C. pneumoniae  infection. There was poor correlation between MIF serology and  C. pneumoniae  antigen detection (previously reported in Chiu et al, 1997). There was also lack of correlation with serology and CHSP-60 antibodies, suggesting that the CHSP60 antibody response is not just a marker of past infection but is uniquely associated with the presence of  C. pneumoniae  in the atheromas, and possibly with the development of atherosclerosis.  
                                                                               C. pneumoniae       C. pneumoniae                 (Ag Positive)   (Ag Negative)           (n = 54)   (n = 21)   p value                                        Anti-CHSP-60   0.19 ± 0.15   0.11 ± 0.08   0.01           (Mean OD ± SD)           OD ≧ 0.12   38 (70.4%)   5 (23.8%)   &lt;0.001           EXAMPLE VIII-           DISCUSSION                      
 
         [0072]    Atherosclerosis involves a low grade chronic inflammatory process (Ross, 1999; Alexander, 1994; Munro and Cotran, 1988), and circulating markers of inflammation such as CRP, fibrinogen, serum amyloid A protein and serum proinflammatory cytokines are predictors of current cardiovascular disease or future myocardial infarction (Danesh et al, 1998,  JAMA  279: 1477-1482; Ridker, 1999,  Am Intern Med  130: 933-937; Koenig et al, 1999,  Circulation  99: 237-242; Kuller et al, 1996,  Am J Epid  144: 537-547). It has been postulated that these circulating markers of inflammation may possibly be associated with the presence of infectious agent(s), such as  C. pneumoniae,  playing a role in atherogenesis. A recent study of patients with coronary artery disease (N=302) and seropositive for  C. pneumoniae  (≧1:16 IgG titer), demonstrated a reduction in global tests of 4 inflammatory markers (CRP, IL-1, IL-6 and TNFα) 3 months after the completion of a 3 month regimen of azithromycin (Anderson et al, 1999,  Circulation  99: 1538-1539). In a study from Finland, part of the 8.5 year trial in the Helsinki Heart Study, the independent and joint effects of infections and inflammation were studied in a nested case: control design (Roivainen et al, 2000,  Circulation  101: 252-257). Both  C. pneumoniae  and herpes simplex virus-I antibodies were associated with increased risk for coronary artery disease.  
         [0073]    One of the mechanisms by which  C. pneumoniae  may be involved in the pathogenesis of atherosclerosis is through chlamydial heat shock protein. It has been postulated that molecular mimicry of CHSP-60 with human HSP60 may induce an autoimmune reaction, leading to activation of inflammatory pathways and an increase in concentration of inflammatory markers (Mayr et al, 1999,  Circulation  99: 1560-1566). Specifically, the homology between the amino acid sequence of the 80-277 fragment and the corresponding human HSP60 fragment is 50%. Our results suggest that immune response against epitopes within this region of the chlamydial HSP60 may have elicited an autoimmune response due to cross-reactivity to epitopes of the human HSP-60. It is also of note that patients with  C. pneumoniae  antigen in atheromatous plaques have significantly higher levels of CHSP-60 antibodies than those without detectable antigen. Furthermore, CHSP-60 localizes in human atheroma and regulates TNFα and matrix metalloprotease expression (Kol et al, 1998), factors that are considered atherogenic. In addition, CHSP-60 is able to activate human vascular endothelium, smooth muscle cells and macrophages (Kol et al, 1999,  J Clin Invest  103: 571-577), and in vitro it can stimulate low density lipoprotein (LDL) cellular oxidation (Kalayoglu et al, 1999,  J Infect Dis  180: 780-790), the major harmful component of LDL. Recently, it was also been shown that serum antibodies to CHSP-60 cross-react with human HSP-60 and mediate endothelial cytotoxicity, a key event in pathogenesis of atherosclerosis (Mayr et al, 1999).  
         [0074]    In summary, CHSP-60 antibody levels are correlated with the presence of  C. pneumoniae  antigen in atheromas and may play a role in the pathogenesis of atherosclerosis.  
         [0075]    While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.  
                                       TABLE 1                           Absorbance readings at 405 nm for the CHSP60 ELISA assay       comparing the mean ± standard deviation for each serum sample       tested against the  Chlamydia trachomatis  serovar L 2  CHSP60-       GST fusion protein (whole CHSP60 protein) from Richard       Stephens and the CHSP60 protein fragment from serovar D       (Health Canada protein).                Mean ± Standard Deviation                CHSP60-GST   CHSP60 protein           fusion protein   fragment from       Sample   (whole protein)     C. trachomatis  serovar D               PBS (negative control)   0.001 ± 0.001   0.001 ± 0.001       A9302         0.090 ± 0.033   0.072 ± 0.010       (clinical negative control)       7698   0.216 ± 0.019   0.250 ± 0.001       (low positive control)       7710   0.633 ± 0.078   0.908 ± 0.061       (high positive control)       St   0.112 ± 0.005   0.065 ± 0.004         2   0.030 ± 0.002   0.033 ± 0.001         9   0.076 ± 0.003   0.104 ± 0.004        30   0.076 ± 0.023   0.063 ± 0.001        40   0.055 ± 0.002   0.074 ± 0.001        47   0.069 ± 0.016   0.080 ± 0.001        79   0.080 ± 0.003   0.082 ± 0.007        138   0.096 ± 0.009   0.098 ± 0.002        214   0.053 ± 0.008   0.043 ± 0.004        269   0.049 ± 0.002   0.051 ± 0.001        276   0.127 ± 0.008   0.149 ± 0.022        357   0.158 ± 0.018   0.105 ± 0.033        369   0.088 ± 0.008   0.089 ± 0.004        380   0.083 ± 0.007   0.109 ± 0.002        428   0.047 ± 0.004   0.048 ± 0.001        484   0.095 ± 0.001   0.080 ± 0.011        497   0.077 ± 0.016   0.063 ± 0.013        560   0.109 ± 0.007   0.086 ± 0.006        588   0.071 ± 0.012   0.075 ± 0.012        610   0.125 ± 0.016   0.117 ± 0.008        620   0.064 ± 0.001   0.070 ± 0.003        632   0.103 ± 0.001   0.096 ± 0.001        659   0.100 ± 0.014   0.103 ± 0.008        680   0.259 ± 0.024   0.239 ± 0.010        763   0.233 ± 0.008   0.299 ± 0.017        826   0.053 ± 0.001   0.064 ± 0.004        840   0.063 ± 0.004   0.071 ± 0.001        841   0.117 ± 0.030   0.106 ± 0.013        896   0.041 ± 0.012   0.038 ± 0.008        963   0.084 ± 0.001   0.115 ± 0.003        987   0.036 ± 0.007   0.031 ± 0.004       1051   0.086 ± 0.001   0.066 ± 0.004       1057   0.127 ± 0.004   0.160 ± 0.005       1080   0.086 ± 0.006   0.070 ± 0.007       1094   0.050 ± 0.011   0.054 ± 0.007       1102   0.136 ± 0.034   0.116 ± 0.001       1110   0.026 ± 0.005   0.025 ± 0.001       1127   0.115 ± 0.004   0.202 ± 0.014       1160   0.060 ± 0.008   0.069 ± 0.008       1184   0.130 ± 0.033   0.097 ± 0.023       1313   0.086 ± 0.001   0.131 ± 0.006       1350   0.103 ± 0.011   0.117 ± 0.007       1357   0.058 ± 0.001   0.076 ± 0.002       1420   0.079 ± 0.001   0.059 ± 0.012       1427   0.051 ± 0.001   0.060 ± 0.007       1433   0.165 ± 0.003   0.141 ± 0.006       1436   0.240 ± 0.008   0.285 ± 0.001       1454   0.212 ± 0.029   0.270 ± 0.018       1478   0.182 ± 0.001   0.197 ± 0.003       1501   0.126 ± 0.008   0.107 ± 0.012       1505   0.155 ± 0.007   0.139 ± 0.014       1507   0.194 ± 0.025   0.265 ± 0.002       1512   0.122 ± 0.004   0.136 ± 0.000       1514   0.161 ± 0.006   0.135 ± 0.010       1522   0.139 ± 0.013   0.098 ± 0.007       1527   0.112 ± 0.008   0.088 ± 0.025       1530   0.080 ± 0.004   0.097 ± 0.001       1538   0.196 ± 0.017   0.223 ± 0.012       1542   0.167 ± 0.008   0.222 ± 0.004       1547   0.166 ± 0.003   0.153 ± 0.007       1550   0.097 ± 0.008   0.110 ± 0.001       1552   0.408 ± 0.035   0.143 ± 0.019       1559   0.122 ± 0.021   0.147 ± 0.022       1567   0.184 ± 0.024   0.191 ± 0.021       1572   0.131 ± 0.006   0.132 ± 0.001       1576   0.186 ± 0.023   0.203 ± 0.014       1577   0.163 ± 0.013   0.190 ± 0.026       1579   0.348 ± 0.093   0.265 ± 0.013       1581   0.122 ± 0.018   0.140 ± 0.003       1582   0.070 ± 0.005   0.081 ± 0.007       1583   0.108 ± 0.002   0.103 ± 0.005       1587   0.197 ± 0.005   0.204 ± 0.007       1590   0.102 ± 0.001   0.089 ± 0.004       1595   0.179 ± 0.002   0.173 ± 0.000       1598   0.172 ± 0.018   0.182 ± 0.014       1621   0.152 ± 0.004   0.175 ± 0.010       1627   0.256 ± 0.026   0.132 ± 0.004       1632   0.178 ± 0.003   0.123 ± 0.011       1649   0.162 ± 0.021   0.201 ± 0.013       1652   0.267 ± 0.033   0.253 ± 0.028       1672   0.049 ± 0.006   0.036 ± 0.001       1677   0.186 ± 0.006   0.125 ± 0.001       1683   0.183 ± 0.067   0.141 ± 0.008       1687   0.301 ± 0.067   0.415 ± 0.045       1688   0.054 ± 0.004   0.043 ± 0.003       1692   0.065 ± 0.009   0.050 ± 0.008       1693   0.095 ± 0.006   0.098 ± 0.000       1695   0.134 ± 0.006   0.129 ± 0.025                  
 
         [0076]    [0076]                                                       TABLE 2                           CHSP60 antibodies versus HSP60 (Cpn Fragment) antibodies in       asthma cases and nonasthmatic controls who were Cpn seroreactive       (MIF IgG 1:16)            STUDY GROUP   No.   Findings                        All subjects   143                   CHSP60 v HSP60 (Cpn)       r = 0.012,   p = .889   Pearson               r = 0.068,   p = .356   Spearman       CHSP60 v FEV1/FVC %       r = −0.17,   p = .13   Pearson       pred       HSP60 (Cpn Fragment) v       r = −.24,    p = .02   Pearson       FEV1/FVC % pred       CHSP60 (Gst fusion)       O.D. × 1000   (Sd)   P-value*       ASTHMA   91   131   (207)       CONTROLS   52   94   (154)   .82       HSP60 (Cpn Frag)       O.D. × 1000   (Sd)   P-value*       ASTHMA   91   156   (115)       CONTROLS   52   114   (57)   .03                            
         [0077]    [0077] 
     
       
       
         1 
         
           
             5  
           
           
             1  
             36  
             DNA  
             chlamydia trachomatis  
           
            1 

aaaactcata tgaaagcwgg vgayggaacy acaaca                               36 

 
           
             2  
             42  
             DNA  
             chlamydia trachomatis  
           
            2 

catagctgct cttccgcawc craavccwgg agctttmacw gc                        42 

 
           
             3  
             35  
             DNA  
             chlamydia trachomatis  
           
            3 

agmrcacata tggymgckaa aaayattaaa tayaa                                35 

 
           
             4  
             41  
             DNA  
             chlamydia trachomatis  
           
            4 

twrtwcygct cttccgcayt artagtccat tcctgcgcyw g                         41 

 
           
             5  
             544  
             PRT  
             chlamydia trachomatis  
             
               MISC_FEATURE  
               (101)..()  
               T or S  
             
           
            5 

Met Val Ala Lys Asn Ile Lys Tyr Asn Glu Glu Ala Arg Lys Lys Ile 
1               5                   10                  15 

Gln Lys Gly Val Lys Thr Leu Ala Glu Ala Val Lys Val Thr Leu Gly 
            20                  25                  30 

Pro Lys Gly Arg His Val Val Ile Asp Lys Ser Phe Gly Ser Pro Gln 
        35                  40                  45 

Val Thr Lys Asp Gly Val Thr Val Ala Lys Glu Val Glu Leu Ala Asp 
    50                  55                  60 

Lys His Glu Asn Met Gly Ala Gln Met Val Lys Glu Val Ala Ser Lys 
65                  70                  75                  80 

Thr Ala Asp Lys Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala 
                85                  90                  95 

Glu Ala Ile Tyr Xaa Glu Gly Leu Arg Asn Val Thr Ala Gly Ala Asn 
            100                 105                 110 

Pro Met Asp Leu Lys Arg Gly Ile Asp Lys Ala Xaa Lys Val Val Val 
        115                 120                 125 

Asp Gln Xaa Xaa Lys Ile Ser Lys Pro Val Gln His His Lys Glu Ile 
    130                 135                 140 

Ala Gln Val Ala Thr Ile Ser Ala Asn Asn Asp Xaa Glu Ile Gly Asn 
145                 150                 155                 160 

Leu Ile Ala Glu Ala Met Glu Lys Val Gly Lys Asn Gly Ser Ile Thr 
                165                 170                 175 

Val Glu Glu Ala Lys Gly Phe Glu Thr Val Leu Asp Xaa Val Xaa Gly 
            180                 185                 190 

Met Asn Phe Asn Arg Gly Tyr Leu Ser Ser Tyr Phe Xaa Thr Asn Pro 
        195                 200                 205 

Glu Thr Gln Glu Cys Val Leu Glu Xaa Ala Leu Xaa Leu Ile Tyr Asp 
    210                 215                 220 

Lys Lys Ile Ser Gly Ile Lys Asp Phe Leu Pro Xaa Leu Gln Gln Val 
225                 230                 235                 240 

Ala Glu Ser Gly Arg Pro Leu Leu Ile Ile Ala Glu Xaa Ile Xaa Gly 
                245                 250                 255 

Glu Ala Leu Ala Thr Leu Val Gly Asn Arg Xaa Arg Xaa Gly Phe Arg 
            260                 265                 270 

Val Cys Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met 
        275                 280                 285 

Phe Glu Asp Ile Ala Ile Leu Thr Gly Gly Gln Leu Ile Ser Glu Glu 
    290                 295                 300 

Leu Gly Met Lys Leu Glu Asn Ala Asn Leu Ala Met Leu Gly Lys Ala 
305                 310                 315                 320 

Lys Lys Val Ile Val Ser Lys Glu Asp Thr Thr Ile Val Glu Gly Met 
                325                 330                 335 

Gly Glu Lys Glu Ala Leu Glu Ala Arg Cys Glu Ser Ile Lys Lys Gln 
            340                 345                 350 

Ile Glu Asp Ser Ser Ser Asp Tyr Asp Lys Glu Lys Leu Gln Glu Arg 
        355                 360                 365 

Leu Ala Lys Leu Ser Gly Gly Val Ala Val Ile Arg Val Gly Ala Ala 
    370                 375                 380 

Thr Glu Ile Glu Met Lys Glu Lys Lys Asp Arg Val Asp Asp Ala Gln 
385                 390                 395                 400 

His Ala Thr Ile Ala Ala Val Glu Glu Gly Ile Leu Pro Gly Gly Gly 
                405                 410                 415 

Thr Ala Leu Ile Arg Cys Ile Pro Thr Leu Glu Ala Phe Leu Pro Met 
            420                 425                 430 

Leu Thr Asn Glu Asp Glu Gln Ile Gly Ala Arg Ile Val Leu Lys Ala 
        435                 440                 445 

Leu Ser Ala Pro Leu Lys Gln Ile Ala Ala Asn Ala Gly Lys Glu Gly 
    450                 455                 460 

Ala Ile Ile Phe Gln Gln Val Met Ser Arg Ser Ala Asn Glu Gly Tyr 
465                 470                 475                 480 

Asp Ala Leu Arg Asp Ala Tyr Thr Asp Met Leu Glu Ala Gly Ile Leu 
                485                 490                 495 

Asp Pro Ala Lys Val Thr Arg Ser Ala Leu Glu Ser Ala Ala Ser Val 
            500                 505                 510 

Ala Gly Leu Leu Leu Thr Thr Glu Ala Leu Ile Ala Glu Ile Pro Glu 
        515                 520                 525 

Glu Lys Pro Ala Ala Ala Pro Ala Met Pro Gly Ala Gly Met Asp Tyr 
    530                 535                 540