A vaccine targeted at CETP alleviates high fat and high cholesterol diet-induced atherosclerosis and non-alcoholic steatohepatitis in rabbit

Abstract

Low HDL-C levels are associated with atherosclerosis and non-alcoholic steatohepatitis, and increased levels may reduce the risk of these diseases. Inhibition of cholesteryl ester transfer protein (CETP) activity is considered a promising strategy for increasing HDL-C levels. Since CETP is a self-antigen with low immunogenicity, we developed a novel CETP vaccine (Fc-CETP6) to overcome the low immunogenicity of CETP and for long-term inhibition of CETP activity. The vaccine consists of a rabbit IgG Fc domain for antigen delivery to antigen-presenting cells fused to a linear array of 6 repeats of a CETP epitope to efficiently activate B cells. Rabbits were fed a high fat/cholesterol (HFC) diet to induce atherosclerosis and NASH, and immunized with Fc-CETP6 vaccine. The Fc-CETP6 vaccine successfully elicited anti-CETP antibodies and lowered plasma CETP activity. The levels of plasma HDL-C and ApoA-I were higher, and plasma ox-LDL lower, in the Fc-CETP6-immunized rabbits as compared to the unimmunized HFC diet-fed rabbits. Pathological analyses revealed less lipid accumulation and inflammation in the aorta and liver of the Fc-CETP6-immunized rabbits. These results show that the Fc-CETP6 vaccine efficiently elicited antibodies against CETP and reduced susceptibility to both atherosclerosis and steatohepatitis induced by the HFC diet. Our findings suggest that the Fc-CETP6 vaccine may improve atherosclerosis and NASH and has high potential for clinical use.

Figure 1. The Strategy for produces the Fc-CETP₆ vaccine.

(A) Schematic diagram of Fc-CETP₆. The CETP vaccine contained the Fc domain of rabbit IgG, followed by 6 repeats of the human CETP epitope, and a double-His₆-tag. (B) The products of adapter-PCR. The 200–300 bp products were boxed. M stands for DNA marker. (C) Purity of the His-Bind-purified Fc-CETP₆ immunogen. Lanes 1 to 3 show Coomassie blue staining results for the flow-through fraction, wash fraction, and elutes, respectively, while lane 4 shows Western blot analysis of elutes using anti-rabbit IgG (Fc) antibodies.

Figure 2. Measurement of plasma anti-CETP titers and CETP activity in rabbits immunized with Fc-CETP₆ (experiment 1).

(A) Plasma antibody titers against CETP. Plasma samples were collected as described in material and methods (Control group n = 7; Fc-CETP₆ group n = 8) and diluted 2000-fold and the anti-CETP titer measured by ELISA. (B) Plasma CETP activity. Plasma CETP activity was measured and expressed as a percentage of that in the pre-vaccination sample (Control group n = 7; Fc-CETP₆ group n = 8). Values are the mean ± SEM for the control rabbits fed the HFC diet and the rabbits fed the HFC diet and immunized with Fc-CETP₆. *p<0.05 compared to the control at the same time.

Figure 3. Vaccination with Fc-CETP₆ reduces Aortic lesions and inflammation (experiment 1).

(A) Representative Sudan IV-stained aortic specimens at the end of week 24. (B) Quantification of the aortic lesion area. The bar graphs represent the average (N: normal group n = 4; C: control group n = 7; Fc-CETP₆ group n = 8) for each group with standard errors. *p<0.05 compared to the control. (C) Expression of NF-κB and RAM-11 (magnification, x200) of the aorta from normal rabbits, control rabbits, and Fc-CETP₆ rabbits. (D) Quantification of the RAM-11 and NF-κB positive area of aorta. The bar graphs represent the average (N: normal group n = 4; C: control group n = 7; Fc-CETP₆ group n = 8) for each group with standard errors. The positive-stained area was calculated as the (stained area/total area) x 100. Values are the mean ± SEM. *p<0.05 compared to the control.

Figure 4. Vaccination with Fc-CETP₆ attenuates high HFC diet-induced steatosis and NASH.

(A) Representative liver sections stained with H&E (magnification, x200). Liver section stained with H&E from control rabbits showing NASH characteristics, such as ballooning degeneration, infiltrating inflammatory cells and Mallory hyaline were observed in the control (magnification, x200). (B) Expression of RAM-11 (magnification, upper panel x100 and lower panel x400) of the liver from control and Fc-CETP₆ rabbits. (C) Expression of NF-kB (magnification, upper panel x100 and lower panel x400) of the liver from control and Fc-CETP₆ rabbits. (D) Expression of inflammation related genes in control rabbits (black bars) and Fc-CETP₆ rabbits (white bars). The bar graphs represent the average (C: control group n = 7; Fc-CETP₆ group n = 8) for each group with standard errors. Values are the mean ± SEM. *p<0.05; ***p<0.001 compared to the control.

Figure 5. Vaccination with Fc-CETP₆ reduces inflammation related gene and fibrosis related gene expression profile (experiment 2).

(A) Representative liver sections stained with Masson's trichrome stain and immunohistochemistry stained for α–SMA, a marker of activated stellate cells and myofibroblasts. α–SMA positive cells were expressed near the fibrotic septa (magnification, upper panel x100 and lower panel x100). Three sections from right lobe liver of each rabbit (control group n = 7; Fc-CETP₆ group n = 8) were analyzed and ten random fields (magnification, x200) of each section photographed under a microscope (Olympus, BX60). (B) Expression of fibrosis related genes in control rabbits (black bars) and Fc-CETP₆ rabbits (white bars) *p<0.05; **p<0.01compared to the control.