Preparation and analysis of immunocompetence of recombinant fusion protein of the immunodominant region in chlamydial protease-like activity factor from Chlamydophila pneumoniae and its application in serodiagnosis

To clone the gene coding the immunodominant region in the chlamydial protease-like activity factor （CPAF） from Chlaroydophila pneumoniae, to analyze immunoeoropetenee of the expressed protein, and to evaluate its value in serodiagnosis, the CPAF immunodominant region gene was amplified, ligated into a pGEX6p-2 vector, and then the expressed recombinant protein was purified with glutathione Stransferase （GST） agarose gel FF after renaturation, then identified by SDS-PAGE and Western blot. A new indirect ELISA was developed with the purified protein as coating antigen. The immunogenicity of the recombinant protein was evaluated by immunization to New Zealand rabbits, and its immunoreactivity was analyzed by reacting with anti-C, pneumoniae antibody. 300 clinical sera samples were respectively detected by mieroimmunofluorescenee （MIF） as reference method and the indirect ELISA, and the difference between the two methods was analyzed. Cross-reactivity against Chlamydia trachomatis was investigated with the indirect ELISA to detect anti-C, trachomatis positive antisera. The results indicated that a 51.3 kDa recombinant protein was obtained. Western blot assay proved that the recombinant protein could merely specifically react with human anti- C. pneurnoniae antisera. The titers of the specific IgG antibodies in the immunized New Zealand rabbits were above 1 ： 16 000. Anti- C. pneumoniae IgG positive and negative reference sera were detected with the indirect ELISA, and the concordance rate of negative and positive results were both 100% （40/40）. The sensitivity and specificity of the indirect ELISA in comparison with MIF were 93.8% （45/48） and 100% （252/252） separately by detecting 300 clinical sera samples, and the concordance rate between the two methods was 99.0%. No cross reaction against C. trachomatis was found with the indirect ELISA to detect anti-C, trachomatis positive antisera. In conclusion, the prepared recombinant protein of the CPAF immunodominant region shows excellent immunocompetence and can be used to develop a new indirect ELISA as a method to detect anti-C, pneumoniae antibody for diagnosis of C. pneumoniae infection.

Involvement of TLR2 and TLR4,Chlamydophila pneumoniae and Mycoplasma pneumoniae in adventitial inflammation of aortic atherosclerotic aneurysm

Aortic atherosclerotic aneurysm (AAA) is associated with adventitial inflammation where infection is suggested to have a role. Co-infection with Chlamydophila pneumoniae (Cp) and Mycoplasma pneumoniae (Mp) was linked with coronary plaque rupture, in association with vessel dilatation and adventitial inflammation. Pathogens are recognized by Toll-like receptors (TLRs) development of the inflammatory process. Objective: Here, we studied whether co-infection by Cp and Mp was involved in the increased inflammation present in AAA and if it could be associated with deficient expression of TLRs. We compared human samples of AAA with non-dilated human aortic atherosclerotic lesions, regarding the amount of Cp and Mp antigens, and expression of TLR2 and TLR4. Methods: Two groups of aorta fragments were analyzed: G1 (n = 13) moderate atherosclerosis and G2 (n = 14) AAA samples, through immunohisto-chemistry and in situ hybridization methods. Results: Mp and Cp antigens in intima/medial layer were greater in G2 than G1, with no difference in adventitia. TLR2 and TLR4 were higher in G2 than G1 adventitia fat. There was a correlation between Mp versus TLR2 and of TLR4 in intima/medial layer and in adventitia of G1, but there was a lack of correlation in G2. In Cp adventitia, the correlation in G1 was high with TLR2 but not with TLR4, and in G2 the correlation was positive for both TLRs. Conclusion: This study favors the concept that symbiotic co-infection by Cp and Mp participates in the pathogenesis of AAA. It also emphasizes that adventitial fat is the initial site for colonization of these bacteria that probably reach the tissue through vasa vasorum. An exacerbated immune reaction is not efficient to control the infection that reaches and proliferates in high levels at the medial and intimal layer, contributing to the development of vessel dilatation.

Development and Evaluation of a MAb-Based ELISA for Detection of Chlamy- dophila pneumoniae Infection with Variable Domain 2 and 3 of the Major Outer Membrane protein

Objective This paper aims to develop a monoclonal antibodies （MAbs）- based ELISA for detecting Chlamydophila pneumoniae （C. pneumonioe） antigens in humans with the variable domains （VD） 2 and 3 of the major outer membrane protein （MOMPvD2-VD~） and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae. Methods MOMPvo2-vo3were overexpressed in Escherichia coil and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I （156 patients with typical respiratory illness clinically confirmed before） and Group II （57 healthy donors）. Results In Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II ＂healthy donors＂, 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively. Conclusion The novel MOMPvD2.VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.

Assessment of polymerase chain reaction and serology for detection of chlamydia pneumoniae in patients with acute respiratory tract infection

OBJECTIVE: To study Chlamydia pneumoniae (C. pneumoniae) infection in 110 patients with respiratory tract infection admitted to our hospital from January to December 1995 in Nanjing. METHODS: Sputum and throat swab specimens were taken and C. pneumoniae DNA was detected by using polymerase chain reaction (PCR) with the HM-1-HR-1 primer pair. At the same time, serum samples were taken and immunoglobulin G and M (IgG and IgM) fractions of antibodies to C. pneumoniae were studied by microimmunofluorescence test. RESULTS: Prevalence of specific IgG was 70% in patients with respiratory tract infection. Seventeen patients (15.5%) were serologically diagnosed as having recent C. pneumoniae infections and 12 patients (10.9%) had positive PCR in sputum and/or swab specimens. The total positive rate was 22.7% (25/110) detected by PCR combined with serological tests. Acute infection of C. pneumoniae was common in patients with asthma (57.1%), pneumonia (35.0%), COPD (25.9%) and bronchitis (25.0%). Clinical features between C. pneumoniae infection and non-C. pneumonia infection showed no significant differences. CONCLUSIONS: Chlamydia pneumoniae is an important pathogen that causes infection of the human respiratory tract and attention should be drawn to this special illness.