Abstract
The role of inflammation in the pathogenesis and progression of atherosclerosis has been conclusively demonstrated over the past two decades. Experimental studies have investigated the use of several immuno-modulatory agents as therapeutic or prophylactic treatments against this disease. The approach in the present study was to use the Chlamydophila pneumoniae major outer membrane protein (MOMP) as an immuno-modulatory treatment. Previous studies have shown that MOMP can attenuate T cell-mediated immune responses, thus prompting this study to assess whether MOMP could have an impact on atherosclerotic plaque development. MOMP was delivered and expressed in animal and tissue culture models using two different vector systems; a heterologous Mycobacterium vaccae expression vector carrying constructs for MOMP expression, with and without signal peptide sequence (M vaccae-rMOMP+SPS or M. vaccae-rMOMP -SPS) and a DNA vaccine delivery system carrying the full length of MOMP gene (pcDNA-MOMP). For characterisation of the immune response, C57B1/6J mice were immunised intranasally with one prime dose and one booster dose following a two week interval. Flow cytometry analysis showed that rMOMP delivered from either M. vaccae (with signal peptide sequence) or from a eukaryotic expression plasmid, was capable of significantly increasing the percentage of CD4+ and CD8+ Treg cells compared to the control group. Additionally, rMOMP delivered by M. vaccae induced increased production of IL-10, while pcDNA-MOMP alone, decreased production of IFN-y secretion; indicating a shift towards an antiinflammatory immune response. The effect of rMOMP on the surface marker expression on mouse peritoneal macrophages and naive CD4+ T cells was also investigated in vitro. The data revealed that rMOMP caused a significant reduction in the percentage of macrophages expressing the CD40 molecule; while increasing Treg numbers as indicated by increasing the number of CD4+ T cells expressing CTLA-4 and CD25 markers. Moreover, pcDNA-MOMP treatment decreased the percentage of CD4+ T cells expressing CD3 receptors and reduced CD4+ T cell proliferation in vitro. Together, these studies suggest that MOMP inhibits the pro-inflammatory signals that promote pro-inflammatory T cell development. Subsequently, the effect of MOMP on atherosclerosis development was examined using the apoE-mouse model of atherosclerosis. Animals received one inoculation and two boosters, three weeks apart. Intranasal administration of M. vaccae-rMOMP-SPS arrested 95% of atherosclerosis development in the brachiocephalic artery and induced smooth muscle cell proliferation in the aortic sinus of apoE-/- mice. Concomitantly, both +SPS and -SPS M. vaccae-rMOMP vaccines enhanced the percentage of Treg cells in the population and the production of the anti-inflammatory cytokines IL-10, TGF-β and IL-4. These two vectors also resulted in a reduction in total and HDL-cholesterol levels. Conversely, DNA immunisation had no effect on atherosclerosis development or the immune response but did induce a reduction in triglycerides and HDL-cholesterol levels. In conclusion, the results of this study suggest that immunisation with rMOMP-based vaccines could promote an athero-protective T-cell response by tempering pro-inflammatory signals. However, it is pertinent to note that the choice of delivery vector and the form of protein are critical in determining the immune response and thus the success of the treatment.