Immunization against active ghrelin using virus-like particles for obesity treatment

Abstract

Ghrelin is a gut hormone that stimulates food intake. In physiological conditions, ghrelin plasma levels rise with fasting and decrease after meals. Obese individuals have low fasting ghrelin levels that rise after food restriction, which is pointed out as a reason for the difficulty in maintaining weight loss. Some bariatric surgery procedures prevent rise in ghrelin levels with weight loss and this has been hypothesised to contribute to the long-term success of the treatment. The main goal of this study was to develop a safe and effective anti-ghrelin vaccine for obesity, through the chemical conjugation of ghrelin with a virus like particle, namely NS1 protein tubules from the Bluetongue Virus (BTV) using a hetero-bifunctional cross linker. Male adult C57BL/6 mice, with a normal weight and with diet-induced obesity (DIO), were randomized into six weight matched groups (n=6/group) and each group of mice received three intra-peritoneal injections with two weeks intervals, containing either 75 μg of ghrelin- NS1 immunoconjugate, 75 μg of NS1 or PBS. Our data show that immunized animals present increasing titres of anti-ghrelin antibodies, while their cumulative food intake significantly decreased and energy expenditure was significantly enhanced, although there were no significative changes in body weight.Vaccinated DIO mice also displayed significant decrease of NPY gene expression in the basal hypothalamus reflecting a decrease in central orexigenic signals. This study suggests that this anti-ghrelin vaccine has a positive impact on energy homeostasis and may be an additional therapeutical tool to be used with diet and exercise for obesity treatment.

Fig. (1)

Silver stained SDS-PAGE showing Low Molecular Weight Markers on lane 1, NS1 protein alone on lane 2 and immunoconjugate on lane 3.

Fig. (2)

Mean daily food intake of normal weight (A) and DIO mice (B). Normal weight mice vaccinated with the immunoconjugate displayed a significant decrease in daily food intake (A); while DIO mice, displayed an initial increase in daily food intake after changing of hypercaloric to standard diet, which was followed by stabilization, there was no significative difference between vaccinated and control mice (B).

Fig. (3)

Graph displaying cumulative food intake in the first 24h after each immunization in normal weight (A) and DIO mice (B). Vaccinated normal weight mice, after the first two inoculations, depicted an acute decrease in food intake when compared to the PBS controls, corresponding to 95.3% and 94.8% of the PBS control, respectively (A). Vaccinated DIO mice also displayed a significant decrease of food intake in the first 24 hours after each inoculation of the immunoconjugate, corresponding to 66.16% (p=0.036), 82.22% (p=0.008) and 50.09 % (p=0.039) of the food intake of the PBS control group, respectively (B).

Fig. (4)

Anti-ghrelin antibody titres of normal weight (*p=0.035) (A) and DIO mice (*p=0.03) (B) after the immunizations and two weeks after the third immunization. Mice inoculated with the immunoconjugate developed specific anti-ghrelin antibodies in increasing titres, reaching a maximum after the third inoculation in comparison with control groups that maintained constant the basal titres.

Fig. (5)

Circulating immune complexes titres and plasma ghrelin levels. There was a positive correlation between ghrelin plasma levels and the titre of circulating immune complexes (r=0.846).