A Targeted Multiple Antigenic Peptide Vaccine Augments the Immune Response to Self TGF-β1 and Suppresses Ongoing Hepatic Fibrosis

Abstract

Transforming growth factor (TGF)-β1 expression is induced upon liver injury, and plays a critical role in hepatic fibrosis. Antibodies against TGF-β1 represent a novel approach in the treatment of hepatic fibrosis. However, TGF-β1 is not a suitable antigen due to immunological tolerance. In the current study, we synthesized a multiple antigenic peptide (MAP) vaccine against the dominant B-cell epitope of TGF-β1. The immunogenicity and potential therapeutic effects of this vaccine were examined using a rat model of hepatic fibrosis. Dominant B-cell epitopes of TGF-β1 were identified using bioinformatic program. An MAP vaccine corresponding to the 90-98 amino acid domain of TGF-β1 and containing four dendritic arms was synthesized using a 9-fluorenylmethoxycarbonyl solid phase method. Hepatic fibrosis which was induced in male Sprague-Dawley rats received a high-fat diet and ethanol (1.8 g/kg). Starting from the third week, rats were exposed to 40 % carbon tetrachloride (CCl4; 150 μl/100 g body weight twice weekly, initially 200 μl/100 g) treatment for a duration of 8 weeks. Rats received the MAP vaccine (100 μg) or Freund's adjuvant at weeks 1, 3, 5. A group of rats receiving the fibrosis-inducing regimen alone and a group of healthy rats (receiving an olive oil vehicle alone) were included as controls. At the conclusion of the experiment, serum titre of TGF-β1 antibody was measured using ELISA and a standard liver functional test panel was examined. The extent of hepatic fibrosis was determined by measuring hydroxyproline content in the liver as well as hematoxylin-eosin (HE) and van Gieson (VG) staining. The expression of TGF-β1 and alpha-smooth muscle actin (α-SMA) was examined using immunohistochemistry, and presented as positive staining cells. The MAP purity was >90 % upon reverse phase high-performance liquid chromatography, with apparent molecular weight at 4.77 kDa. Serum TGF-β1 antibody titre was 1:256. The fibrosis-inducing treatment produced significant liver damage, as reflected by increases in liver functional test, HE and VG staining. The MAP vaccine attenuated such damage, as reflected by decreased alanine aminotransferase, aspartate aminotransferase, total bilirubin, and hepatic hydroxyproline (116.78 ± 23.76 vs. 282.71 ± 136.94 IU/L; 319.78 ± 82.48 vs. 495.29 ± 137.13 IU/L; 2.02 ± 0.27 vs. 4.01 ± 0.52 μmol/L; 263.67 ± 41.18 vs. 439.14 ± 43.29 μg/g vs. in model rats, respectively; p < 0.01), as well as fibrosis extent by HE and VG staining. The MAP vaccine reduced TGF-β1 and α-SMA expression in rats (0.325 ± 0.059 vs. 0.507 ± 0.044 IOD/area; 0.318 ± 0.058 vs. 0.489 ± 0.029 IOD/area vs. model rats, respectively; p < 0.05). The TGF-β1 MAP vaccine could generate sufficient antibody that suppresses the development of hepatic fibrosis.