Chlamydia pneumoniae genome sequence analysis and identification of HLA-A2-restricted CD8+ T cell epitopes recognized by infection-primed T cells.

In the present study, we performed in silico analysis of Chlamydia pneumoniae genome sequence to identify human HLA-A2-restricted T cell epitopes. Thirty-one Chlamydia-specific protein antigens were selected and peptides were derived thereof using an HLA-A2 epitope predictive algorithm. Firstly, we tested binding of 55 selected 9mer peptides to HLA-A2 in vitro. Next, infection of HLA-A2 transgenic mice with C. pneumoniae elementary bodies and assessment of effector CD8+ T cells allowed us to identify which of the epitopes binding to HLA-A2 in vitro were recognized by C. pneumoniae infection-primed CD8+ T cells. Finally, we could confirm that CD8+ T cells in association with HLA-A2 recognized the most reactive peptides when the corresponding full-length genes were used to DNA-immunize HLA-A2 transgenic mice. By using this approach, a novel HLA-A2-restricted epitope in the outer membrane protein A (OmpA) of C. pneumoniae was identified, which proved to mediate specific lysis of peptide-loaded target cells.

Inhibition of cell proliferation and induction of apoptosis by novel tetravalent peptides inhibiting DNA binding of E2F.

We have isolated several peptides from random peptide phage display libraries that specifically recognize the cell cycle regulatory transcription factor E2F and inhibit DNA binding of E2F/DP heterodimers (E2F-1, E2F-2, E2F-3, E2F-4 or E2F-5, and DP-1). The inhibitory efficiency could be strongly enhanced by generating branched tetravalent molecules. To analyse the biological consequences of peptide-mediated E2F inhibition, we fused two of these branched molecules to a cell-penetrating peptide derived from the HTV-Tat protein. Incubation of human tumor cells with these branched Tat-containing peptides led to an inhibition of cell proliferation and induction of apoptosis. These results provide new insights into the function of E2F and further validate E2F as a potential therapeutic target in proliferative diseases.

Genomic approach for analysis of surface proteins in Chlamydia pneumoniae.

Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomic-proteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.