Consortium leader: MIRJA PUOLAKKAINEN,
Haartman Institute, Department of Virology, POB 21, 00014 University of Helsinki and KTL, Department of Viral Diseases and Immunology, Mannerheimintie 166, 00300 Helsinki. mirja.puolakkainen@helsinki.fi, mirja.puolakkainen@ktl.fi

Partner of the consortium:
Professor Riitta Lahesmaa, Turke Centre for Biotechnology, PO Box 123, 20521 Turku, riitta.lahesmaa@btk.utu.fi

Researchers:
Anu Haveri, KTL, Helsinki
Juha Korhonen, Turku Centre for Biotechnology, Turku
Minna Kyläniemi, Turku Centre for Biotechnology, Turku
Ali Mahar, Haartman Institute, Department of Virology, University of Helsinki
Laura Mannonen, Haartman Institute, Department of Virology, University of Helsinki
Tuula Penttilä, Haartman Institute, Department of Virology, University of Helsinki
Anne Tammiruusu, KTL, Helsinki

Key words:Host-microbe interaction, persistent infection, Chlamydia pneumoniae, gene expression, DNA microarray, proteomics

Results 
One of our initial objectives was to elucidate, what are the critical mechanisms by which Chlamydia pneumoniae induces development of persistent infection in the infected cells. To answer this question, we have analyzed host cell transcriptional responses during acute and interferon-gamma induced persistent Chlamydia pneumoniae infection by cDNA array and RT-PCR. The models of acute and interferon-gamma induced persistent infection developed earlier by us were used. Human respiratory epithelial cells were infected with Chlamydia pneumoniae, and the cells were harvested at various time points after inoculation. Uninfected cells treated with interferon-gamma and cell without any treatment were used as controls. Initially, we identified 250 genes, whose expression was significantly and consistently altered upon infection. When the data was further analyzed and confirmed by quantitative RT-PCR to identify those host cell genes, whose mRNA expression was differentially altered during persistent infection, nine human genes were identified. Currently, we are setting up detection methods for these gene products to confirm that changes on transcription level correspond to changes in the protein profile. The products of these nine genes exert various functions in cells and these results indicate that during persistent infection, C. pneumoniae efficiently reprograms the host transcriptional machinery regulating a variety of cellular processes including apoptosis, transcription, cell cycle regulation, signaling, and inflammatory response, which all may play important roles in the pathogenesis of persistent C. pneumoniae infection, that can manifest itself as enhanced atherosclerosis, asthma, or even lung cancer.

Another objective of our project was to analyze whether immunization with C. pneumoniae gene products could induce immune responses leading towards eradication of infection. A well-established mouse model for pulmonary C. pneumoniae-infection has enabled characterization of the infection kinetics and immune responses. CD8+ T cells have been shown to play an important role in protective immunity against C. pneumoniae infection. We have designed different vaccine constructs and regimens to induce T cell immunity in this model.  Initially, we immunized mice with naked DNA and Semliki Forest viral vectors coding for chlamydial proteins MOMP, Hsp60 and OMp2. The immunizations were able to induce a prominent Th1 type immunity together with a strong local immune response, measured as a specific proliferation activity and/or IFN-_ production of lymphocytes from draining lymph nodes. SFV vaccination, with or without DNA priming, induced antigen-specific humoral and cellular immune responses as well as partial protection against C. pneumoniae pulmonary infection. In addition, immunization with SFV induced specific CTL responses against the very same chlamydial epitopes that were identified after DNA immunization, indicating that both antigen delivery systems can result in similar MHC class I-dependent antigen processing and presentation. However, the immunizations with DNA and SFV alone or SFV with DNA priming were only able to confer partial protection against the acute pulmonary infection. This finding is in strong agreement with findings reported by other research groups and vaccine industry using similar approaches.

Using the genomic chlamydial sequence data and a computer-based epitope prediction method, we have identified several mouse CD8 epitopes from C. pneumoniae proteins. Nineteen C. pneumoniae-derived peptides were identified as CD8 epitopes by their ability to induce cytotoxic response after peptide immunization. Seven of the identified epitopes were able to induce long-term peptide-specific CTL lines, and three were natural epitopes presented by C. pneumoniae infected cells. This kind of identification of CD8 epitopes may serve as a tool for more specific characterization of CD8+ lymphocyte function and the development of epitope-based prevention methods. Similarly, human CD8 epitopes were identified in transgenic HHD mice expressing only human class I molecule (HLA-A2.1). Immunization of HHD mice with the whole chlamydial protein induced an HLA-A2.1-restricted peptide-specific CTL line, indicating that the classical human class I molecule can support the development of murine CD8+ T cell response against a chlamydial protein. In spite of a lower number of CD8+ T cells in HHD mice, the outcome of C. pneumoniae infection in the transgenic mice resembled that observed in the wild type mice. These findings encourage the use of HHD mouse model for the further characterization of C. pneumoniae infection and the host immunity against it, as well as for identification of human CD8 epitopes from chlamydial antigens.

Chlamydial proteins that are secreted out of the inclusion or associated with the inclusion membrane are potential targets for the host's MHC class I-dependent antigen presentation, thereby representing ideal antigens for CD8+ T cell-based vaccination. Chlamydiae interact actively with the host cell, presumably by exploiting a type three secretion system (TTSS). We demonstrated that an obviously TTSS-secreted chlamydial outer protein N (CopN) can be used as a vaccine. Heat denatured CopN protein with adjuvant LT induced a strong humoral and cellular immune response, detected as antigen-specific antibodies, proliferation and IFN-  production. Most importantly, the CopN immunization induced a significant level of protection against subsequent C. pneumoniae infection which certainly recommends this antigen as a candidate vaccine for further investigation.

Selected publications:

Mannonen L, Kamping E, Penttila T, Puolakkainen M. IFN-_ induced persistent Chlamydia pneumoniae infection in HL and Mono Mac 6 cells: Characterization by real-time quantitative PCR and culture. Microb Pathog, 36:41-50;2004

Penttilä T, Tammiruusu A, Liljeström P, Vuola J, Sarvas M, Mäkelä PH, Puolakkainen M. DNA immunization followed by a viral vector booster induces protective immunity in Chlamydia pneumoniae mouse model. Vaccine, 22(25-26):3386-94;2004.

Tammiruusu A, Haveri A, Pascolo S, Lahesmaa R, Stevanovic S, Rammensee H-G, Sarvas M, Puolakkainen M, Vuola JM. Clearance of  Chlamydia pneumoniae Infection in H-2 class I-/- Human Leukocyte Antigen-A2.1 monochain transgenic mice. Scand J Immunol, 62:131-139;2005.

Penttilä T, Wahlström E, Vuola JM, Sarvas M and Puolakkainen M. Antigen-specific serology in murine Chlamydia pneumoniae infection. Submitted, 2006.

Tammiruusu A, Penttilä T, Sarvas M, Puolakkainen M, Vuola JM. Intranasal administration of Chlamydial outer protein N (CopN) induces partial protection against pulmonary Chlamydia pneumoniae infection in BALB/c mice. Submitted 2006.
 
Mannonen L,  Nikula T, Haveri A, Reinikainen A, Lahesmaa R, Puolakkainen M. Host cell responses to persistent Chlamydia pneumoniae infection by cDNA array. Submitted, 2006.
 
Nurminen TA, Korhonen JT, Penate Medina P, Puolakkainen M, Lahesmaa R, Kinnunen PKJ. Sphingomyelinase activity in Chlamydia pneumoniae bacteria. Submitted 2006.

An abstract of the research plan (January 2003)
Viimeksi muokattu 20.9.2007

Lisätietoja

Englanniksi:

Ohjelmajohtajana toimi Soile Juuti ja ohjelmapäällikkönä Sirpa Nuotio.