DCs are at the centre of the initiation of immune responses by naïve T cells and appear to be particularly important to the development of Chlamydia immunity. DCs capture antigen in the periphery and migrate to regional lymph nodes where they present processed antigen on MHC molecules to naïve T cells. DCs undergo a maturation process during migration to lymph nodes and express new surface molecules that act as co-stimulants to naïve T cells causing activation and polarization of the cytokine secretion pattern. Therefore, in addition to containing appropriate molecular antigens, a successful Chlamydia vaccine will need to activate professional antigen-presenting cells and polarize T-cell responses to the protective type cytokine secretion pattern and generate long-lived T-cell memory. Initial work from our laboratory demonstrated that expression of GM-CSF—a cytokine known to mobilize DCs—in the mouse airway compartment significantly enhanced systemic Th
1 cellular immune responses following immunization with inactivated C. muridarum
This suggested that DCs recruited by GM-CSF contributed to the development of a Th
1 immune response. Further in vitro observations demonstrated that DCs exposed to live versus to UV-irradiated C. muridarum
develop distinct phenotypes, such that DCs exposed to live Chlamydia become mature and effectively present antigen to Chlamydia-specific CD4 T cells, while DCs exposed to UV-irradiated Chlamydia were not immunologically mature.18
Since DCs are essential to induce Chlamydia immunity via presentation of chlamydial antigens to naïve T cells, we hypothesized that Chlamydia peptide antigens presented by the surface MHC molecules of Chlamydia infected DCs could be explored as potential source for the identification of T-cell antigens.
An approach called immunoproteomics, in which peptides presented by MHC molecules are identified by tandem mass spectrometry (MS/MS),19,20
allows genomic information to guide the delineation of the complete T-cell immunoproteome of the organism. These methods have been applied to several immunological problems21
but instrument sensitivity has prevented its applicability to pathogens. Recent advancements in MS/MS technology now provide sensitivity limits near one femtomole (fmol) and are able to measure peptide masses to within one part-per-million accuracy.22
This brings the detection technology into a range compatible with the levels of microbial peptides that can reasonably be purified from MHC molecules presented on the surface of antigen presenting cells such as DCs.
The immunoproteomic approach that we used to identify Chlamydia T-cell antigens involved multiple steps (). First, bone marrow cells isolated from the femurs and tibias of mice are grown in the presence of GM-CSF and IL-4. These bone marrow derived DCs (BM-DCs) are pulsed with Chlamydia. Pulsed BM-DCs are lysed and MHC molecules isolated using allele-specific anti-MHC monoclonal antibody affinity columns. Purified MHC molecules are then washed and peptides eluted with acetic acid and separated from high molecular weight material by ultrafiltration through 5-kDa cut-off membranes. The purified MHC-bound peptides are initially analyzed qualitatively using an LTQ-OrbitrapXL (Thermo Electron) online coupled to a nanoflow HPLC using a nanospray ionization source. The mass spectrometer is set to fragment the five most intense multiply-charged ions per cycle. Fragment spectra are extracted using DTASuperCharge (http://msquant.sourceforge.net
) and searched using the Mascot algorithm against a database comprised of the protein sequences from C. muridarum
. DC adoptive transfer method is employed to deliver peptides that are recognized by Chlamydia specific CD4 T cells to identify those that are protective. The selected peptides are further characterized by cloning the parent proteins of these MHC-binding peptides as vaccine candidates and tested in a genital tract mouse model.
Schematic depiction of the sequence of steps involved in the immunoproteomic approach for Chlamydia T cell vaccine development.
We used the immunoproteomic approach to identify 13 Chlamydia peptides derived from 8 novel epitopes presented by MHC class II molecules from BM-DCs infected with Chlamydia ().23
These MHC class II-bound peptides were recognized in vitro by Chlamydia specific CD4 T cells harvested from immune mice recovered from Chlamydia infection and adoptive transfer of DCs pulsed ex vivo with the peptides partially protected mice against intranasal and genital tract Chlamydia infection. We further investigated these peptides by cloning recombinant proteins corresponding to the MHC binding peptides. Recombinant Chlamydia proteins were also found to be recognized in vitro by immune T cells suggesting that these proteins can be processed to generate the immunologically relevant peptides. These proteins were also able to protect mice against Chlamydia infection in vivo as shown by DC adoptive transfer experiments.24
Based on these results, three of the 8 source proteins (RplF, PmpG and PmpE/F) were deemed suitable for further evaluation. However, only PmpE/F and PmpG were selected for further animal studies as RplF has significant homology to the human homolog and thus may not be suitable as a human vaccine.
Chlamydia T cell antigens identified by immunoproteomic approach
The immunoproteomic approach described above directly identifies T cell epitopes presented by antigen presenting cells resulting in a vast improvement in the positive validation rate. Another advantage in using immunoproteomics is that the peptides identified are the result of physiological processing and presentation pathways and are based on both the affinity for the MHC molecules as well as the frequency of their presentation.
Pathogens contain a large number of possible antigens for immune responses but only a few immunodominant antigens are typically recognized following infection or immunization. The reasons for immunodominance are complex and can be intrinsic to the targets (i.e., differences in affinity for immune receptors) or extrinsic (i.e., competition that suppresses the response to one target in favor of another).25
We hypothesized that the antigens we identified through the immunoproteomic approach are immunodominant and can be recognized in varying MHC backgrounds. We tested whether the peptides (originally identified in C57BL/6) and their source proteins are also recognized by mouse strains other than C57BL/6. As expected, proteins and peptides were readily recognized by T cells from C57BL/6 mice. Proteins, but not peptides, were also recognized by Chlamydia-specific T cells from Balb/c and C3H/HeN mice (unpublished data). These data suggest that there are T cell epitopes within the identified proteins that are presented by MHC haplotypes other than I-Ab
and that these Chlamydia proteins are immunologically recognized in genetically different strains of mice, arguing that they are immunodominant.