Keywords

4.1 Introduction

Obesity is defined as a medical condition of abnormal or excessive fat accumulation that may impair health. The body mass index (BMI) is an estimate of body fat based on height and weight and is used clinically to diagnose obesity. For adults, overweight and obesity are defined as BMI ≥ 25 kg/m2 and ≥ 30 kg/m2, respectively. According to the World Health Organization (WHO) fact sheets (16 February 2018, URL: https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight), in 2016, more than 1.9 billion (39%) adults were overweight and 650 million (13%) were obese worldwide. Among children and adolescents in particular, the prevalence of overweight has increased markedly from 4% in 1975 to over 18% in 2016 and that of obesity has also risen explosively from less than 1% to 7%.

Obesity is associated with high all-cause mortality (Flegal et al. 2013) and increased risk of cardiovascular diseases (Wilson et al. 2002), heart failure (Kenchaiah et al. 2002), end-stage renal disease (Vivante et al. 2012), and some cancers (Renehan et al. 2008). Due to its rising prevalence and increased comorbidity burden, obesity is predicted to cost the US healthcare system $48–$66 billion per year by 2030 (Wang et al. 2011). To combat the economic burden of obesity and its comorbidities, innovative strategies to prevent or treat obesity are needed.

Obesity is a chronic disease that involves interactions between environmental and genetic factors. Because environmental factors that lead to an imbalance between energy intake and expenditure are the main driver for obesity, comprehensive lifestyle modifications such as reduced energy intake and increased physical activity are generally the first approach for weight reduction (Bray et al. 2016). In fact, a comprehensive program of lifestyle modification can produce a 7–10% reduction in body weight at 1 year or more (Wadden et al. 2012). Pharmacotherapy should be considered when patients have difficulty achieving or maintaining sufficient weight reduction through lifestyle modification alone.

Six main drugs are currently approved as anti-obesity medications by the Food and Drug Administration (FDA): phentermine (sympathomimetic amine), orlistat (lipase inhibitor), phentermine/topiramate (sympathomimetic amine and an antiepileptic drug), lorcaserin (5-HT2c receptor agonist), naltrexone/bupropion (opioid antagonist and an aminoketone antidepressant), and liraglutide (glucagon-like peptide-1 [GLP1] receptor agonist) (Srivastava and Apovian 2018). Orlistat decreases the absorption of dietary fat, whereas the other drugs suppress appetite and increase satiety through the stimulation of the central nervous system (Fig. 4.1) (Srivastava and Apovian 2018). However, physicians are often reluctant to prescribe weight-loss medications due to patient dissatisfaction with the amount of weight reduction, lingering concerns about drug safety, and weight regain after discontinuation of the medication (Heymsfield and Wadden 2017).

Fig. 4.1
figure 1

Schematic representation of the pathogenesis of obesity and the mechanisms of action of anti-obesity drugs

POMC: pro-opiomelanocortin

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Therapeutic vaccines have the potential to contribute to obesity therapy because of their prolonged therapeutic effect and low frequency of administration. Over the last two decades, numerous attempts have been made to develop vaccines for the prevention and treatment of obesity (Table 4.1). In this chapter, we will introduce and summarize some of the candidates for innovative therapeutic vaccines against obesity.

Table 4.1 Developmental therapeutic vaccines against obesity
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4.2 Vaccination Targets Against Obesity

4.2.1 Ghrelin

Ghrelin is a 28 amino-acid peptide hormone that possesses a unique O-acylation at the Ser3 residue and has been identified as a ligand for growth-hormone secretagogue receptor (GHSR) (Kojima et al. 1999). Ghrelin is the only known circulating peripheral hormone that promotes body weight gain by stimulating food intake and decreasing energy expenditure (Nakazato et al. 2001; Tschop et al. 2000; Wortley et al. 2004). Ghrelin is mainly produced in the gastric X/A-like cells, but is also expressed in the small intestine, brain, pancreas, and other peripheral organs (Ghelardoni et al. 2006). Peripheral ghrelin modulates the nucleus tractus solitarius via the vagus nerve, which results in an increase in noradrenaline in the arcuate nucleus of the hypothalamus and appetite stimulation (Date et al. 2006). The orexigenic action of ghrelin requires O-acylation at Ser3 with octanoate, an eight-carbon fatty acid, and unacylated ghrelin (also called des-acyl ghrelin) has opposite effects from those of ghrelin (i.e., acylated ghrelin) on body weight in that it decreases appetite and body weight (Asakawa et al. 2005). Ghrelin O-acyltransferase (GOAT) is the enzyme responsible for the octanylation of ghrelin (Yang et al. 2008).

Although ghrelin would appear to be an attractive target for the development of a drug to treat or prevent obesity, as of 2018, there is no clinically available anti-obesity drug that targets ghrelin function, such as a ghrelin inhibitor, GHSR antagonist, or GOAT inhibitor. Animal experiments have suggested that inhibition of ghrelin function is effective against obesity. A few reports indicate that the genetic deletion of ghrelin does not alter food intake and body weight (Wortley et al. 2004), but ghrelin-deficient mice have increased energy expenditure and locomotor activity and are protected from diet-induced obesity after early exposure to a high-fat diet from 3 weeks after weaning (Kushnir et al. 2012). Genetic deletion of the ghrelin receptor GHSR also suppresses weight gain in mice fed a high-fat diet (Zigman et al. 2005). In terms of the drug development, GOAT inhibitors and GHSR antagonists have the potential to attenuate diet-induced obesity (Ambuhl et al. 2007; Maurer et al. 2005); however, none have been approved for clinical use.

The first attempt to prevent weight gain by using a therapeutic anti-ghrelin vaccine was reported in 2006 (Zorrilla et al. 2006). Zorrilla et al. synthesized three different ghrelin antigens (Ghr1, Ghr2, and Ghr3) as candidate vaccine antigens and coupled them to the carrier protein keyhole limpet hemocyanin (KLH) (Zorrilla et al. 2006). Ghr1 spanned N-terminal residues 1–10 and was butanoylated at Ser3, Ghr2 comprised C-terminal residues 13–28, and Ghr3 spanned the whole ghrelin analog (1–28) and contained Ser3-(butanoyl). Rats were immunized with these vaccine candidates conjugated with Ribi adjuvant, an oil-in-water emulsion containing monophosphoryl lipid A, on days 0, 21, and 35, and subsequently immunized with the vaccines conjugated with alum adjuvant on days 56 and 84. Rats that received five immunizations with Ghr1-KLH, Ghr2-KLH, or Ghr3-KLH developed antigen-specific antibodies that did not cross-react with the other antigens. Rats immunized with Ghr1-KLH or Ghr3-KLH developed an antibody that possessed good plasma binding affinity for acylated ghrelin and gained less weight than non-immunized rats without changes in their food consumption. In contrast, vaccination with Ghr2-KLH did not affect weight gain.

Vizcarra et al. also reported on the effectiveness of a peptide vaccine composed of the N-terminal region of ghrelin (Vizcarra et al. 2007). Pigs subcutaneously immunized three times with a 20-day interval with a vaccine comprising the N-terminal residues (1–10) of porcine ghrelin coupled with bovine serum albumin (BSA) and conjugated with Freund’s incomplete adjuvant and diethylaminoethyl-dextran showed a decrease in both daily food intake (15% less than control) and weight gain (10% less than control) (Vizcarra et al. 2007).

Recently, advances in biomedical technology and nanotechnology, such as virus-like particles (VLPs) and nanometer-sized polymer hydrogel (nanogel), have been applied to the development of anti-ghrelin vaccines. VLPs are formed by structural viral proteins that self-assemble and display antigenic epitopes in the correct conformation and in a highly repetitive manner (Kushnir et al. 2012). VLP-based vaccines targeting non-communicable diseases such as nicotine dependence and hypertension were found to be safe and well-tolerated in human clinical trials and to successfully induce antigen-specific antibodies (Ambuhl et al. 2007; Maurer et al. 2005). Andrade et al. conjugated VLPs consisting of tubules of non-structural protein (NS) 1 of Bluetongue virus to ghrelin peptide and then intraperitoneally immunized mice with 75 μg of the ghrelin-NS1 immunoconjugate three times with 2-week intervals (Andrade et al. 2013). The immunized mice raised ghrelin-specific serum IgG antibodies and showed increased energy expenditure and decreased food intake, but no change in body weight (Andrade et al. 2013). The lack of weight reduction despite decreased food intake and increased energy expenditure after vaccination with ghrelin-NS1 may be partially explained by the relatively short follow-up period or the possible activation of compensatory mechanisms of energy homeostasis or both. Interestingly, feedback responses leading to increased ghrelin expression in the stomach did not occur after immunization. Ghrelin-NS1 vaccination also promoted the formation of circulating immune complexes of ghrelin on anti-ghrelin antibodies and resulted in increased fasting plasma ghrelin concentrations (Andrade et al. 2013).

We recently developed a new anti-ghrelin therapeutic vaccine that can be administered intranasally (Azegami et al. 2017). Intranasal immunization offers distinct advantages over other injectable delivery methods including decreased cost, less psychological and physiological stress, and no risk of localized skin adverse events (Lamichhane et al. 2014). However, nasal administration of antigen alone often fails to induce sufficient antigen-specific mucosal and systemic immune responses (Azegami et al. 2018). Application of a nanogel, which is a promising vaccine-antigen delivery vehicle, has great potential in vaccine development because nanogels can incorporate various proteins through hydrophobic interactions, which prevents the irreversible aggregation of the incorporated proteins, and subsequently allows their release in their native form (Azegami et al. 2018). Among nanogels, the cationic type of cholesteryl-group-bearing pullulan (cCHP) nanogel is suitable as a intranasal vaccine-delivery system because the cationic property of cCHP nanogels allows efficient adhesion of the nanogels to the negatively charged nasal epithelial layer, leading to effective and continuous delivery of the vaccine antigen to the dendritic cells beneath the nasal epithelial cells (Nochi et al. 2010). We created a new vaccine antigen, ghrelin-PspA (pneumococcal surface protein A), which is a recombinant fusion protein incorporating three repeats of mouse whole ghrelin and PspA as a carrier protein (Azegami et al. 2017). Intranasal immunization of mice with the ghrelin-PspA vaccine, which comprised 5 μg of antigen and 10 μg of cyclic di-GMP adjuvant within a cCHP nanogel, on five occasions at 1-week intervals induced ghrelin-specific serum IgG antibodies and attenuated body weight gain (7% less than control) in diet-induced obese mice (Azegami et al. 2017). This anti-obesity effect was caused by a decrease in both visceral and subcutaneous fat accumulation and an increase in energy expenditure that was partially due to the increased expression of mitochondrial uncoupling protein 1 in brown adipose tissue (Azegami et al. 2017). In addition, intranasal vaccination with ghrelin-PspA decreased the body weight of genetically obese ob/ob mice (control +4.7 g vs. ghrelin-PspA −0.9 g, weight change between pre- and 1 week post-vaccination). The ghrelin-PspA vaccine also promoted the formation of circulating immune complexes of ghrelin on anti-ghrelin antibodies and prolonged the circulating half-life of ghrelin, resulting in increased fasting plasma ghrelin concentrations, similar to the ghrelin-NS1 vaccine (Azegami et al. 2017).

4.3 Glucose-Dependent Insulinotropic Polypeptide

Incretin hormone is secreted from enteroendocrine cells in the intestinal epithelium and regulates glucose metabolism. In humans, GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 are the two main incretin hormones and stimulate pancreatic insulin secretion in a glucose-dependent manner (Sadry and Drucker 2013). In addition to its insulinotropic action, GIP also promotes adipose tissue accretion. GIP receptor-deficient mice exhibit increased energy expenditure and are resistant to diet-induced obesity (Miyawaki et al. 2002). Administration of a GIP receptor antagonist also attenuates diet-induced obesity and improves glucose metabolism in mice (McClean et al. 2007); accordingly, GIP is a strong target candidate for anti-obesity treatment.

To induce a humoral immune response against a self-derived peptide (i.e., GIP), Fulurija et al. coupled the N-terminal residues (1–15) of GIP to the surface of bacteriophage Qβ VLPs (Fulurija et al. 2008). Four subcutaneous immunizations with 100 μg of VLP-based GIP vaccine with 2-week intervals elicited antigen-specific serum IgG antibodies that bound to circulating GIP and reduced diet-induced body weight gain in mice (35% less weight gain than control) without inducing an auto-inflammatory reaction in the intestine or disturbing glucose homeostasis (Fulurija et al. 2008). This anti-obesity effect of the GIP vaccine was derived from suppression of fat accumulation and caused by an increase in energy expenditure due to a high basal metabolic rate (Fulurija et al. 2008).

4.4 Adipocytes

Obesity results from excessive accumulation of adipose tissue, and adipose tissue dysfunction causes metabolic complications in the obese. Therefore, treatment that directly targets adipose tissues is an attractive strategy to overcome obesity and its metabolic comorbidities.

Two unique approaches to the development of an adipocyte-based anti-obesity vaccine were reported in 2010. Lai et al. examined the efficacy of mouse 3T3-L1 adipocytes as a xenogeneic adipocyte vaccine in rats (Lai et al. 2010). Sprague Dawley rats were intraperitoneally immunized with 106 mouse 3T3-L1 adipocytes weekly for 4 weeks and fed a high calorie diet after the final immunization. Xenogeneic adipocyte vaccination dramatically decreased weight gain and induced adipocyte apoptosis (Lai et al. 2010).

Bourinbaiar and Jirathitikal conducted a clinical trial to evaluate the efficacy and safety of antigenic adipose tissue on body weight and lipid metabolism (Bourinbaiar and Jirathitikal 2010). In this clinical study, 9 females and 4 males, aged between 22 and 79, received oral tablets containing pig adipose tissue for 3 months. The small sample size and short observation period in this single-arm experiment were clear limitations, and unfortunately it is unclear whether oral vaccination with xenogeneic adipocytes induced a humoral immune reaction against adipose tissue in the immunized subjects. However, oral adipocyte vaccine successfully reduced waist size (−7.6%) and increased serum high-density lipoprotein cholesterol levels (+25.9%) without causing any adverse events in the human subjects, but it did not change body weight, or low-density lipoprotein cholesterol and triglyceride levels.

4.5 Somatostatin

Growth hormone (GH) deficiency is known to cause an increase in fat accumulation, whereas GH replacement decreases body fat in patients with adult-onset GH deficiency (Baum et al. 1996). Moreover, low-dose GH treatment decreases body fat in obese adults who are not GH deficient (Kim et al. 1999). Although GH therapy may be a promising strategy for the attenuation of obesity, its clinical application is limited by its very short half-life, necessitating a daily subcutaneous injection (Faria et al. 1989). As an alternative strategy, the sustained inhibition of somatostatin, an endogenous suppressor of pituitary GH secretion, may be a therapeutic option for GH-mediated anti-obesity treatment.

In 2012, a chimeric polypeptide consisting of somatostatin and the carrier protein chloramphenicol acetyltransferase was developed as an anti-somatostatin vaccine for the inhibition of somatostatin effects (Haffer 2012). To enhance its immunogenicity, chimeric somatostatin antigen was mixed with either JH17 or JH18 adjuvant as well as squalene, Tween 80, and Span 85 with or without tragacanthin and arabinogalactan. Intraperitoneal immunization with the somatostatin vaccine containing 500 μg of antigen elicited chimeric antigen protein (not somatostatin alone)-specific serum IgG antibodies and slightly increased the levels of insulin-like growth factor 1, which is secreted by the liver upon GH stimulation. Surprisingly, a single dose of the somatostatin vaccine immediately caused weight reduction (12.2% and 13.1% for the JH17 and JH18 adjuvants, respectively) compared with PBS controls without a change in food intake in diet-induced obese mice at only 4 days post-immunization.

4.6 Adenovirus 36

Obesity is a multifactorial disease caused by environmental and genetic factors. Among the environmental factors, although food and physical activity play the biggest role in the development of obesity, a link between virus infections such as adenovirus 36 (Ad36) and obesity has also been shown through animal and human studies (Dhurandhar et al. 2002; Lyons et al. 1982). In one human study, 30% of obese subjects were positive for serum Ad36 antibody (i.e., evidence of prior Ad36 infection) compared with 11% of non-obese subjects (Atkinson et al. 2005). Inoculation of Ad36 directly promotes weight gain and fat accumulation in non-human primates (Dhurandhar et al. 2002). Although the pathogenic mechanism responsible for Ad36-induced obesity remains to be fully elucidated, Ad36 enhances the differentiation of pre-adipocytes via E4 open reading frame-1 gene signaling, reduces leptin production, and increases glucose uptake by adipocytes (Rogers et al. 2008; Vangipuram et al. 2004; Vangipuram et al. 2007).

Na and Nam chose Ad36 as the therapeutic target of their novel anti-obesity vaccine (Na and Nam 2014). They used UV-irradiated inactive Ad36 as a vaccine antigen. Two doses of 5 μg of inactivated Ad36 in Freund’s adjuvant were intraperitoneally injected into mice with 2-week intervals. This regimen attenuated body weight gain, fat accumulation, and adipose tissue inflammation in the Ad36-inoculated mice without altering food consumption (Na and Nam 2014).

4.7 Future Perspectives

Obesity is one of the biggest health problems in the world. To overcome the global obesity epidemic, the development of public education programs that raise awareness of obesity and its consequent health risks is essential. In addition, obesity must be tackled at the individual level, encouraging comprehensive lifestyle modification. Because of their prolonged therapeutic effects and low frequency of administration, therapeutic vaccines may have potential as an approach to the prevention and treatment of obesity. Induction of the humoral immune response (i.e., B cell activation) is essential to elicit antigen-specific antibodies to neutralize endogenous targets that promote obesity. To effectively induce a humoral immune response against an endogenous self-derived target, various adjuvants are widely used. However, they may also trigger undesired autoimmune reactions; therefore, careful consideration of not only the therapeutic effect but also the risk for autoimmune reactions must be applied to a therapeutic vaccine for clinical use. To overcome this concern, recent advances in nanotechnology and biotechnology such as VLPs and other delivery vehicles will assist in the development of innovative vaccines.